WO2016037274A1 - Cellulose-based nanoparticles for drug delivery - Google Patents

Cellulose-based nanoparticles for drug delivery Download PDF

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Publication number
WO2016037274A1
WO2016037274A1 PCT/CA2015/050865 CA2015050865W WO2016037274A1 WO 2016037274 A1 WO2016037274 A1 WO 2016037274A1 CA 2015050865 W CA2015050865 W CA 2015050865W WO 2016037274 A1 WO2016037274 A1 WO 2016037274A1
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peg
ppt
compound
acid
drug
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PCT/CA2015/050865
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French (fr)
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Shyh-Dar Li
Aniruddha ROY
Mark ERNSTING
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Ontario Institute For Cancer Research (Oicr)
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/48—Preparations in capsules, e.g. of gelatin, of chocolate
    • A61K9/50—Microcapsules having a gas, liquid or semi-solid filling; Solid microparticles or pellets surrounded by a distinct coating layer, e.g. coated microspheres, coated drug crystals
    • A61K9/51—Nanocapsules; Nanoparticles
    • A61K9/5107—Excipients; Inactive ingredients
    • A61K9/513—Organic macromolecular compounds; Dendrimers
    • A61K9/5161—Polysaccharides, e.g. alginate, chitosan, cellulose derivatives; Cyclodextrin
    • A—HUMAN NECESSITIES
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    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/56—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule
    • A61K47/59—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyureas or polyurethanes
    • A61K47/60—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyureas or polyurethanes the organic macromolecular compound being a polyoxyalkylene oligomer, polymer or dendrimer, e.g. PEG, PPG, PEO or polyglycerol
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    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
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    • A61K47/61—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule the organic macromolecular compound being a polysaccharide or a derivative thereof
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    • A61K47/6921—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere
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    • A61K47/6929—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere the form being a solid microparticle having no hollow or gas-filled cores the form being a nanoparticle, e.g. an immuno-nanoparticle
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    • A61K47/69—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit
    • A61K47/6921—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere
    • A61K47/6927—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere the form being a solid microparticle having no hollow or gas-filled cores
    • A61K47/6929—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere the form being a solid microparticle having no hollow or gas-filled cores the form being a nanoparticle, e.g. an immuno-nanoparticle
    • A61K47/6931—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere the form being a solid microparticle having no hollow or gas-filled cores the form being a nanoparticle, e.g. an immuno-nanoparticle the material constituting the nanoparticle being a polymer
    • A61K47/6939—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere the form being a solid microparticle having no hollow or gas-filled cores the form being a nanoparticle, e.g. an immuno-nanoparticle the material constituting the nanoparticle being a polymer the polymer being a polysaccharide, e.g. starch, chitosan, chitin, cellulose or pectin
    • A—HUMAN NECESSITIES
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    • A61K9/48—Preparations in capsules, e.g. of gelatin, of chocolate
    • A61K9/50—Microcapsules having a gas, liquid or semi-solid filling; Solid microparticles or pellets surrounded by a distinct coating layer, e.g. coated microspheres, coated drug crystals
    • A61K9/51—Nanocapsules; Nanoparticles
    • A61K9/5107—Excipients; Inactive ingredients
    • A61K9/513—Organic macromolecular compounds; Dendrimers
    • A61K9/5146—Organic macromolecular compounds; Dendrimers obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyethylene glycol, polyamines, polyanhydrides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08B—POLYSACCHARIDES; DERIVATIVES THEREOF
    • C08B11/00—Preparation of cellulose ethers
    • C08B11/02—Alkyl or cycloalkyl ethers
    • C08B11/04—Alkyl or cycloalkyl ethers with substituted hydrocarbon radicals
    • C08B11/10—Alkyl or cycloalkyl ethers with substituted hydrocarbon radicals substituted with acid radicals
    • C08B11/12—Alkyl or cycloalkyl ethers with substituted hydrocarbon radicals substituted with acid radicals substituted with carboxylic radicals, e.g. carboxymethylcellulose [CMC]
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08G—MACROMOLECULAR COMPOUNDS OBTAINED OTHERWISE THAN BY REACTIONS ONLY INVOLVING UNSATURATED CARBON-TO-CARBON BONDS
    • C08G65/00—Macromolecular compounds obtained by reactions forming an ether link in the main chain of the macromolecule
    • C08G65/02—Macromolecular compounds obtained by reactions forming an ether link in the main chain of the macromolecule from cyclic ethers by opening of the heterocyclic ring
    • C08G65/32—Polymers modified by chemical after-treatment
    • C08G65/329—Polymers modified by chemical after-treatment with organic compounds
    • C08G65/331—Polymers modified by chemical after-treatment with organic compounds containing oxygen
    • C08G65/332—Polymers modified by chemical after-treatment with organic compounds containing oxygen containing carboxyl groups, or halides, or esters thereof
    • C—CHEMISTRY; METALLURGY
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    • C08G65/3328—Polymers modified by chemical after-treatment with organic compounds containing oxygen containing carboxyl groups, or halides, or esters thereof heterocyclic
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Definitions

  • This invention relates to the field of nanoparticles and, more specifically, to cellulose- based nanoparticles for the delivery podophyllotoxin and related derivatives.
  • Tubulin a major component of the cellular cytoskeleton, plays an important role in the survival and growth of cells. Its functions extend from cellular transport to cell motility and mitosis. The importance of microtubule in mitosis and cell division makes it an attractive target for anticancer drugs. Chemotherapeutic agents that disrupt the normal function of tubulin are amongst the most potent and broadest spectrum anticancer agents available in the clinic. A structurally diverse class of compounds have been found to antagonize the tubulin function with various tubulin binding sites and different mechanisms of action.
  • Anti -tubulin agents can be divided into two major categories, microtubule-destabilizing agents and microtubule-stabilizing agents, based on their effect on microtubule polymerization.
  • Microtubule-destabilizing agents such as colchicine and the vinca alkaloids, inhibit polymerization and decrease the mass of microtubules.
  • Microtubule- stabilizing agents like taxanes stabilize microtubules, increase microtubule polymer mass, and induce the formation of microtubule bundles in cells.
  • Both classes of anti- microtubule agents function by disrupting the dynamic equilibrium of the microtubules, resulting in arrest of cells in mitosis through blocking cell cycle at the metaphase- anaphase transition and leading to cellular apoptosis. l ' 2
  • MDR multidrug resistance
  • PPT podophyllotoxin
  • Celulose-based nanoparticles for drug delivery are described in Applicant's PCT Patent Publication Nos. 2012/103634 and 2014/015422, which are herein incorporated by reference.
  • a compound comprising an acetylated carboxymethylcellulose (CMC-Ac) covalently linked to: at least one poly(ethylene glycol) (PEG), and at least one hydrophobic drug comprising podophyllotoxin or derivatives thereof, or pharmaceutically acceptable salts, solvates, esters or prodrugs of any of the foregoing.
  • CMC-Ac acetylated carboxymethylcellulose
  • a self-assembling nanoparticle composition comprising the compound described herein.
  • a pharmaceutical composition comprising the self-assembling nanoparticle composition described herein and a pharmaceutically acceptable carrier and/or diluent.
  • a method of treating cancer in a patient in need thereof comprising administering to said patient an effective amount of a self-assembling nanoparticle composition comprising the compound described herein.
  • a process for preparing a self-assembling nanoparticle composition comprising: covalently linking at least one PEG and at least one hydrophobic drug comprising a podophyllotoxin or derivatives thereof, or pharmaceutically acceptable salts, solvates, esters or prodrugs of any of the foregoing, to a CMC-Ac; isolating the product of step (a); dissolving the isolated product of step (b) in a suitable organic solvent, preferably DMF or DMSO and further preferably THF or acetonitrile, to form a solution; precipitating the solution of step (c); resuspending the precipitate of step (d) in an an aqueous solution, and preferably dializing the aqueous solution.
  • R 2 (i)...R 2 (n), R3(i)- ..R3(n), and Rend are each independently
  • -0(HD) represents a hydrophobic drug comprising podophyllotoxin or derivatives thereof, or pharmaceutically acceptable salts, solvates, esters or prodrugs of any of the foregoing, having a point of attachment via a hydroxyl group; or n is an integer.
  • Figure 1 shows an in vitro viability assay of different drugs against PC3 and PC3-RES tumor cells.
  • Figure 2 shows IC50 of different drugs against EMT6 and EMT6 AR1.
  • Figure 3 shows IC50 of different drugs against MDA-MB-231 and MDA-MB-231- RES..
  • Figure 4 shows reaction conditions and 1H-NMR spectra of the PPT-CMC-Ac-PEG, CMC-Ac and PPT.
  • Figure 5 shows the physicochemical characteristics of PPT-CMC-Ac-PEG conjugates (Example 2) prepared with variable PPT/PEG ratios.
  • A Size;
  • B Drug release in serum;
  • C IC50 (nM) against EMT6 AR1.
  • Figure 6 shows intracellular uptake of PPT-NPs (E2-8 particles, 20 nm).
  • Figure 7 shows tumor and liver uptake of Dil labeled PPT-NPs with different PPT/PEG ratios.
  • A Images of tumor and liver uptake at 24 h;
  • B Images of tumor uptake at 24 and 48 h;
  • C Quantitative comparison of tumor and liver uptake;
  • D Quantitative comparison of tumor/liver uptake ratio;
  • E Quantitative comparison of tumor uptake at 24 and 48 h.
  • Figure 10 shows the body weight of EMT6 AR1 tumor bearing mice (BALB/c).
  • Figure 12 shows the body weight of PC3-RES tumor bearing mice (NOD-SCID).
  • Figure 13 shows the composition of analogues prepared in Example 2 and 10.
  • A composition of polymers that produce defined particles from Examples 2 and 10. In Example 2, reactions were fed with excess amounts of reagent (red circles). In Example 10, reactions were generally fed with sub-excess amounts of reagent (blue diamonds).
  • B Reaction model for PEG coupling in the Example 10 syntheses. Blue diamonds represent sub-excess feeds, the red square is an excess feed, and the green triangle is a 1 eqv feed of PEG.
  • C Reaction model for the PPT coupling in the Example 10 syntheses. Blue diamonds represent sub-excess feeds, the red square is a excess feed, and the green triangle is a 1 eqv feed of PPT.
  • the models demonstrate that the reaction chemistry in Example 10 is robust and predictable, enabling design and preparation of all analogues in this invention.
  • PPT can only be used topically for HPV-infected genital warts due to its poor solubility and selectivity.
  • Different derivatives of PPT has been prepared (etoposide, taniposide, ethophos) and is being used clinically for cancer treatment, but these derivatives have different mode of action (topoisomerase inhibitor) and are significantly less potent (100-1000 times) than PPT 16 ' 17 and also substrates for Pgp. 17-19
  • a tumor targeted delivery system for PPT can reduce its off-target toxicities and enhance its efficacy against MDR tumors.
  • CMC was selected as the polymer backbone to synthesize a polymer conjugate of PPT due to its documented safety, biocompatibility and multiple conjugation sites (carboxylate groups) for increased coupling of the drug.
  • CMC like many other biocompatible polymers, is highly polar and only sparingly soluble in organic solvents such as DMF or DMSO. 24
  • PPT is only soluble in organic solvents, and as a result the coupling chemistry could not be performed directly.
  • Doxorubicin-dextran conjugate (DOX-OXD, AD- 70) in Phase I clinical trials induced significant hepatotoxicity, ascribed to increased uptake by the RES.
  • PEGylation has been employed to improve pharmacokinetic of many NPs
  • 36 it has not been extensively used for polymer-drug conjugates, possibly due to the complexities involved in the chemistry.
  • the EDC coupling chemistry employed in this invention enables efficient conjugation of PEG and PPT onto CMC- Ac, with excellent control over reaction outcomes.
  • the PPT and PEG content of the polymers can be adjusted in a predictable manner, enabling the designers to tune the PPT/PEG ratio, creating NPs with a range of size, drug release kinetics and cell killing potency. These parameters are important determinants for in vivo pharmacokinetics, biodistribution, tissue penetration and tumor bioavailability.
  • the conjugate with a low PPT/PEG molar ratio of 2 yielded NPs with a mean diameter of 20 nm and released PPT at ⁇ 5%/day in serum, while conjugates with increased PPT/PEG ratios (5 and 20) produced bigger particles (30 nm and 120 nm respectively) that displayed slower drug release ( ⁇ 2.5%/day and ⁇ l%/day respectively).
  • the 20-nm particles exhibited enhanced cell killing potency, increased tumor delivery and decreased liver uptake compared to the bigger NPs, and the biodistribution in the tumor-bearing mice was highly selective to tumor with a ⁇ 4-fold increase compared to the liver.
  • the 20-nm particles for PPT displayed significantly improved efficacy against two MDR tumor models in mice compared to the standard taxane chemotherapies with minimal toxicity.
  • the ability to control CMC-Ac, PEG and PPT composition parameters enable effective control the conjugates and NPs and an optimal in vivo performance.
  • a compound comprising an acetylated carboxymethylcellulose (CMC-Ac) covalently linked to: at least one poly(ethylene glycol) (PEG), and at least one hydrophobic drug comprising podophyllotoxin or derivatives thereof, or pharmaceutically acceptable salts, solvates, esters or prodrugs of any of the foregoing.
  • CMC-Ac acetylated carboxymethylcellulose
  • the salts of the compounds of this invention refer to non-toxic "pharmaceutically acceptable salts.”
  • Other salts may, however, be useful in the preparation of compounds according to this invention or of their pharmaceutically acceptable salts.
  • Suitable pharmaceutically acceptable salts of the compounds include acid addition salts which may, for example, be formed by mixing a solution of the compound with a solution of a pharmaceutically acceptable acid such as hydrochloric acid, sulfuric acid, fumaric acid, maleic acid, succinic acid, acetic acid, benzoic acid, citric acid, tartaric acid, carbonic acid or phosphoric acid.
  • suitable pharmaceutically acceptable salts thereof may include alkali metal salts, e.g., sodium or potassium salts; alkaline earth metal salts, e.g., calcium or magnesium salts; and salts formed with suitable organic ligands, e.g., quaternary ammonium salts.
  • alkali metal salts e.g., sodium or potassium salts
  • alkaline earth metal salts e.g., calcium or magnesium salts
  • suitable organic ligands e.g., quaternary ammonium salts.
  • representative pharmaceutically acceptable salts include the following: acetate, benzenesulfonate, benzoate, bicarbonate, bisulfate, bitartrate, borate, bromide, calcium edetate, camsylate, carbonate, chloride, clavulanate, citrate, dihydrochloride, edetate, edisylate, estolate, esylate, fumarate, gluceptate, gluconate, glutamate, glycollylarsanilate, hexylresorcinate, hydrabamine, hydrobromide, hydrochloride, hydroxynaphthoate, iodide, isothionate, lactate, lactobionate, laurate, malate, maleate, mandelate, mesylate, methylbromide, methylnitrate, methylsulfate, mucate, napsylate, nitrate, N methylglucamine ammonium salt, oleate,
  • acids and bases which may be used in the preparation of pharmaceutically acceptable salts include the following: acids including acetic acid, 2,2 dichloroactic acid, acylated amino acids, adipic acid, alginic acid, ascorbic acid, L aspartic acid, benzenesulfonic acid, benzoic acid, 4 acetamidobenzoic acid, (+) camphoric acid, camphorsulfonic acid, (+) (IS) camphor 10 sulfonic acid, capric acid, caproic acid, caprylic acid, cinnamic acid, citric acid, cyclamic acid, dodecylsulfuric acid, ethane 1,2 disulfonic acid, ethanesulfonic acid, 2 hydroxy ethanesulfonic acid, formic acid, fumaric acid, galactaric acid, gentisic acid, glucoheptonic acid, D gluconic acid, D glucoronic acid, L glutamic acid, alpha-o
  • the present invention includes within its scope prodrugs of the compounds of this invention.
  • prodrugs will be functional derivatives of the compounds that are readily convertible in vivo into the required compound.
  • the term “administering” shall encompass the treatment of the various disorders described with the compound specifically disclosed or with a compound which may not be specifically disclosed, but which converts to the specified compound in vivo after administration to the patient.
  • Conventional procedures for the selection and preparation of suitable prodrug derivatives are described, for example, in "Design of Prodrugs", ed. H. Bundgaard, Elsevier, 1985.
  • form means, in reference to compounds of the present invention, such may exist as, without limitation, a salt, stereoisomer, tautomer, crystalline, polymorph, amorphous, solvate, hydrate, ester, prodrug or metabolite form.
  • the present invention encompasses all such compound forms and mixtures thereof.
  • isolated form means, in reference to compounds of the present invention, such may exist in an essentially pure state such as, without limitation, an enantiomer, a racemic mixture, a geometric isomer (such as a cis or trans stereoisomer), a mixture of geometric isomers, and the like.
  • the present invention encompasses all such compound forms and mixtures thereof.
  • Certain compounds may exist in various stereoisomeric or tautomeric forms and mixtures thereof.
  • the invention encompasses all such compounds, including active compounds in the form of essentially pure enantiomers, racemic mixtures and tautomers.
  • the covalent linkages are ester linkages.
  • the hydrophobic drug is podophyllotoxin or pharmaceutically acceptable salts, solvates, esters or prodrugs of any of the foregoing.
  • the hydrophobic drug is podophyllotoxin.
  • the hydrophobic drug is selected from the group consisting of etoposide, taniposide, Adva-27a, and NK611r, or pharmaceutically acceptable salts, solvates, esters or prodrugs of any of the foregoing.
  • the hydrophobic drug is:
  • the at least one PEG is poly(ethylene glycol) methyl ether (mPEG) having an average M n of between about 550 and about 10,000, preferably about 2000.
  • the CMC consists of a polymer with a molecular weight of between 4400 and 220,000 Daltons.
  • the wt% of PEG in the nanoparticle is 3 - 50.1 wt%, preferably 10.8-43.0 wt%.
  • the wt% of PPT in the nanoparticle is 1.9-38 wt%, preferably 3.9 - 30.5 wt%, further preferably about 12 wt%.
  • the drug/PEG wt ratio is between about 0.14 to about 5.8, preferably 0.191-3.547, further preferably about 0.4.
  • a self-assembling nanoparticle composition comprising the compound described herein.
  • the nanoparticles have an average diameter of about 20-160 nm, or preferably around 20m.
  • the nanoparticle further comprises at least one hydrophobic agent encapsulated therein, preferably selected from either an imaging agent or a therapeutic agent.
  • a pharmaceutical composition comprising the self- assembling nanoparticle composition described herein and a pharmaceutically acceptable carrier and/or diluent.
  • a method of treating cancer in a patient in need thereof comprising administering to said patient an effective amount of a self-assembling nanoparticle composition comprising the compound described herein.
  • the cancer is a multidrug resistant cancer.
  • Cancers could include Adrenal Cancer, Anal Cancer, Bile Duct Cancer, Bladder Cancer, Bone Cancer, Brain/CNS Tumors, Breast Cancer, Castleman Disease, Cervical Cancer, Colon/Rectum Cancer, Endometrial Cancer, Esophagus Cancer, Ewing Family Of Tumors, Eye Cancer, Gallbladder Cancer, Gastrointestinal Carcinoid Tumors, Gastrointestinal Stromal Tumor (GIST), Gestational Trophoblastic Disease, Hodgkin Disease, Kaposi Sarcoma, Kidney Cancer, Laryngeal and Hypopharyngeal Cancer, Leukemia, Liver Cancer, Lung Cancer, Lung Carcinoid Tumor, Lymphoma, Malignant Mesothelioma, Multiple Myeloma, Myelodysplasia Syndrome, Nasal Cavity and Paranasal Sinus Cancer, Nasopharyngeal Cancer, Neuroblastoma, Non-Hodgkin Lymphoma, Oral Cavity and Oropharyngeal
  • a process for preparing a self-assembling nanoparticle composition comprising: covalently linking at least one PEG and at least one hydrophobic drug comprising a podophyllotoxin or derivatives thereof, or pharmaceutically acceptable salts, solvates, esters or prodrugs of any of the foregoing, to a CMC-Ac; isolating the product of step (a); dissolving the isolated product of step (b) in a suitable organic solvent, preferably DMF or DMSO and further preferably THF or acetonitrile, to form a solution; precipitating the solution of step (c); resuspending the precipitate of step (d) in an an aqueous solution, and preferably dializing the aqueous solution.
  • steps (c) and (d) are repeated one or more times before step (e)
  • R 2 (i)...R 2 (n), R3(i)- ..R3(n), and R-nd are each independently
  • -0(HD) represents a hydrophobic drug comprising podophyllotoxin or derivatives thereof, or pharmaceutically acceptable salts, solvates, esters or prodrugs of any of the foregoing, having a point of attachment via a hydroxyl group; or n is an integer.
  • the PEG is m(PEG).
  • each of the hydrophobic drug and PEG may be covalently linked to the CMC-Ac by a direct linkage between a carboxylic acid residue of the CMC-Ac and a functional group of the hydrophobic drug and PEG (e.g. a hydroxyl group), or by an indirect linkage via one or more bifunctional linkers.
  • Preferred linkers are those that are biodegradable, non-toxic when cleaved from the conjugate, and are relatively stable to hydrolysis in the circulation.
  • Acceptable carriers and diluents would be known to a person skilled in the art. Generally, such carriers should be nontoxic to recipients at the dosages and concentrations employed.
  • Suitable solvents for use in the processes of the present invention include those solvents that are inert under the conditions of the reaction/procedure being described in conjunction therewith.
  • CMC degree of substitution
  • Podophyllotoxin was obtained from Carbosynth (Berkshire, UK). Docetaxel (DTX), Paclitaxel (PTX) and Cabazitaxel (CBZ) were obtained from LC Laboratories (Wobum, MA). Cholchicin (Cho) and Vinblastin (Vin) were purchased from Sigma Aldrich (Oakville, ON). Carboxymethylcellulose (CMC) sodium salt (CEKOL 30000- P) was purchased from CPKelco (Atlanta, GA).
  • Hydrophobic fluorescent dye Dil (l,10-dioctadecyl-3,3,3030- tetramethylindocarbocyanine perchlorate, D-307) was purchased from Invitrogen (Burlington, ON). For all the in vitro studies, free drugs were first dissolved in DMSO.
  • NP Polymer and nanoparticle composition was determined by 3 ⁇ 4- NMR using 2-methyl 5-nitro benzoic acid as an internal standard (IS). A response factor for PPT or PEG relative to the IS was determined in a calibration, and polymer and NP content was estimated using these calibrations.
  • CMCAc or polymer conjugates were measured on a Wyatt HELEOS II multi-angle light scattering system (MALS) coupled with a Waters e2696 pump, a Waters 2414 RI, a Millipore 0.22 ⁇ filter, and a Wyatt high-pressure injection system.
  • MALS multi-angle light scattering system
  • the system was run in batch-mode MALS with acetonitrile flowing at 0.8 mL/min.
  • CMCAc samples were prepared as a range of concentration in acetonitrile (0.25 to 1.6 mg/mL), and injected into the system through a 0.22 ⁇ PTFE filter.
  • the system was validated and normalized using a 20KDa PEG standard. Samples were prepared as 1 mg/mL solution in MeCN, and analyzed in the Astra software using the above dn/dc constants.
  • the CMC-Na polymer is modified with carboxymethyl groups, and was supplied by the manufacturer with an analyzed DS value of 0.82: each glucose monomer unit is specified to contain 0.82 moles carboxylic acid and 2.18 moles hydroxyl.
  • Processing the CMC-Na to a CMC-Ac compound can alter the carboxylic acid content (the DS), and the polymer are titrated to determine functionality.
  • CMC Ac 100 mg is dissolved in MeCN (15 mL) and MeOH (10 mL) in a glass Erlenmeyer flask, and is titrated with KOH (0.1N in methanol), using phenolphthalein (1% alcohol) as an indicator.
  • Polymer and particle samples were analyzed for free drug or PEG by LC/MS analysis.
  • Sample analysis was performed on an LC/MS system consisting of a Waters Acquity UPLC/MS equipped with a PDA and a Xevo QToF MS.
  • samples of polymer were prepared as 1 mg/mL solution in MeCN, and particle solutions prepared as 10X dilutions in MeCN.
  • Samples were injected on an Acquity UPLC BEH CI 8 column (1.7 ⁇ , 2.1 x 50 mm), at a flowrate of 0.4 mL/min, with a gradient program of 95-10% water/acetonitrile over 5 minutes. Calibration of each drug and PEG was performed using the appropriate mass ion.
  • CMC polymer has a high degree of substitution with carboxylic groups.
  • One prerequisite for a candidate drug to be conjugated with the CMC is the presence of, minimum and preferably, one free hydroxyl group in their structure. Free hydroxyl group in the drug as well as in the PEG are conjugated with the free carboxyl group in the polymer using EDC-mediated esterification.
  • An excess amount of DMAP is applied to effect solubilisation of the EDC.HC1 in the organic medium (eg: acetonitrile, DMSO, and to act as a catalyst in the reaction.
  • NHS is introduced into the reaction to control side reactions which increase MW of the polymer conjugation.
  • anhydrous solvents were used, as water retards reaction kinetic (but not does not reduce reaction yields).
  • Nanoparticles were prepared in a controlled nanoprecipitation process using a two- channel microfluidic system (NanoAssemblr, Precision Nanosystems, Canada). Polymer (150 mg) was dissolved in acetonitrile at varying concentrations (4-45 mg/g), fed through one channel and 0.9% NaCl fed through the adjacent channel. Total flow rate was maintained at 12-24 mL/min and the flow ratio of the aqueous to organic stream was 2: 1 to 4: 1. The optimal conditions were typically 30 mg/g polymer in acetonitrile, 18 mL/min flow, and a 3: 1 organic: saline ratio.
  • the outlet stream was immediately diluted in an equal volume of saline, was purified by dialysis, sterile filtered using 0.22 ⁇ Millipore PVDF filters, and concentrated using Vivaspin columns. Particle size and zeta potential were measured with a Zetasizer (Nano-ZS, Malvern Instruments, Malvern, UK). Taxane or podophyllotoxin content were determined by 3 ⁇ 4 NMR with 2-methyl-5-nitrobenzoic acid as an internal standard, and LC analysis was performed to ensure products contained less than 1 wt% free drug or PEG.
  • Fluorescently loaded NP Dil loaded NPs were prepared by dissolving 30 mg of the polymer in MeCN (1 mL) containing 0.1 mg/mL Dil and was precipitated into 3 mL of normal saline in the NanoAssemblr at the flow rate of 18 mL/min. Dil content of the NPs was determined by dissolving the NPs in DMSO and assaying for fluorescence (Excitation filter: 535 nm; Emission Filter 590 nm) and comparing to a calibration curve of fluorescence versus Dil concentration, subtracting the background signal of un-loaded particle fluorescence. Cell culture and animals
  • EMT6/AR1 cells were obtained from the American Type Culture Collection (ATCC). Resistant EMT6/AR1 cells were a gift from Ian Tannock, Princess Margaret Hospital, Toronto. DTX resistant PC3 and MD A-MB-231 cells were generated from the sensitive phenotype by treating them with gradually increasing concentrations of DTX until the cells become fully resistant to 100 nM and 10 nM of DTX respectively. The cells were maintained in Dulbecco's Modified Eagle's Medium (DMEM) supplemented with 10% fetal bovine serum. Female NOD-SCID and BALB/c mice were purchased from Jackson Laboratories (Bar Harbour, ME). All protocols were approved by the Animal Care Committee of the University Health Network.
  • DMEM Dulbecco's Modified Eagle's Medium
  • Cell growth inhibition activity of different drugs or NP were analyzed by measuring cell viability with the XTT assay. Briefly, cells were dislodged and re-suspended at a concentration of 1 ⁇ 10 4 cell/mL, and 100 of cell suspension per well was added to a 96 wellplate. A slight modification (5 ⁇ 10 4 cell/mL) was made for the MD A-MB-231 analysis, as these cells were slow-growing compared to the other lines. The cells were maintained for 24 h in culture (37 °C, 5% C02, humidified) before treatment. After 72 h of treatment, viability was assayed by the XTT assay.
  • EMT6 or EMT6/AR1 cells were plated on sterile glass coverslips placed in a 12-well plate.
  • Cells were treated with Dil-labeled PPT-NPs at the concentration of 0.5 ⁇ g Dil/mL. After 4 h of incubation, the cells were washed with PBS and treated lysotraker green for lh. After that they were washed with PBS and fixed with 10% formalin for 15 min. The fixed cells were washed again with PBS and the glass coverslip was mounted on a glass slide with mounting media containing DAPI. Slides were then scanned on the FluoView A confocal microscope (Olympus). The intensity of the Dil and DAPI signal was measured using ImageScope software with positive pixel count algorithm.
  • Example 1 Selection of PPT from a panel of common chemotherapeutics
  • Anti-tubulin drugs are among the most potent and broadest spectrum chemotherapeutic agents.
  • potency of a panel of six anti-tubulin drugs PTX, DTX, CBZ, PPT, CHO, Vin
  • EMT6, PC3 and MDA-MB- 231) Figure 1-3, Table 1
  • All the tested drugs exhibited significant potency against the native cell lines, with IC50 varied between 0.01 nM and 14 nM.
  • CBZ was found to be the most potent: IC50 varied from 0.01 to 1 nM.
  • PTX, DTX, CBZ, Choi and Vin showed a considerable loss in potency against the MDR lines (EMT6 AR1, PC3-RES and MDA-MB-231-RES).
  • PC3-RES was found to be the most refractive to these drugs: the resistance index (RI, IC50 in the resistant line divided by IC50 in the parent line) of PTX, DTX, CBZ, Choi and Vin for this cell line varied from 100 to 500.
  • composition analogues were conjugated to PEG and PPT via EDC/DMAP coupling chemistry ( Figure 4).
  • CMC-Ac 300 mg was weighed into a 25 mL round bottom flask, and dissolved in a mixture of anhydrous MeCN (9 mL) and DMSO (6 mL).
  • EDC HCl 448 mg, 2.3 mmol
  • DMAP 580 mg, 4.6 mmol
  • the solutions were stirred 24h at room temperature with protection from light.
  • the reaction mixtures were precipitated through 135 mL diethyl ether.
  • the precipitate was dried, re-dissolved in MeCN, and the precipitation process was repeated twice.
  • the product was analyzed by LC-MS for uncoupled PEG and PPT, and washing was repeated if residual reagent was detected: less than 0.5 free drug and PEG was detected.
  • the chemical composition of the polymer conjugate was determined by ⁇ -NMR using 2-methyl 5-nitro benzoic acid as an internal standard (Table 2).
  • Particles were prepared from the polymers by the NanoAssemblr technique (Table 2). Conjugates with >50 wt% PEG loading were water soluble and did not form any measurable particles. Conjugates with ⁇ 50 wt% PEG loading formed distinct nanoparticulate structures in aqueous solution (20-120 nm in diameter). Increased content of PPT in the conjugate led to increased particle size, whereas increased PEG resulted in decreased particle size ( Figure 5A). The conjugates with a PPT/PEG wt ratio of 0.5 assembled into -20 nm particles whereas the particle size of the conjugate with a PPT/PEG ratio of 4.4 was found to be -120 nm.
  • Table 2 Polymers with a range of PPT and PEG content (Example 2).
  • the reaction mixtures were rapidly precipitated through well mixed 45 x 4 mL diethyl ether in conical tubes.
  • the polymer was spun down at 4000 rpm for 5 minutes, and the pellet was dried, re- dissolved in 5 mL MeCN, and the precipitation process was repeated three more times.
  • the product was recovered by lyophylization, analyzed by LC-MS for uncoupled PEG and PPT: less than 0.5 free drug and PEG was detected.
  • the chemical composition of the polymer conjugate was determined by ⁇ -NMR using 2-methyl 5-nitro benzoic acid as an internal standard.
  • E2-1 analogue 120 nm particle: 14.9 wt% PEG, 30.5 wt% PPT.
  • E2-6 analogue (30 nm particle): 25.0 wt% PEG, 31.6 wt% PPT.
  • Polymers were analyzed by MALS for MW: E2-1 : 56.3 kDa, E2-2: 12.2 kDa, E2-3: 48.5 kDa.
  • Table 3 Feeds of PPT and PEG for compounds synthesized in Example 3.
  • Example 3 Particles prepared in Example 3 (the E2-4, 6 and 7 analogues) were analyzed for cytotoxic potency in an EMT-AR1 drug resistant breast cancer line (Table 2). All compounds are active in the nM concentration range.
  • Particles prepared in Example 3 were 1: 1 (v:v) mixed with fetal bovine serum (FBS) at the final concentration of 100 ⁇ g PPT /mL.
  • FBS fetal bovine serum
  • Samples were incubated at 37 °C, and at selected time points triplicate samples were removed and serum protein was precipitated using 600 MeCN containing 1% acetic acid.
  • the samples were centrifuged for 5 min at 10,000 rpm and the supernatant was analyzed for released PPT by HPLC at a flow rate of 0.4 mL/min, with a gradient program of 95/5 to 10/90 water/MeCN over 5 min.
  • a calibration curve for PPT was prepared by spiking known amounts of PPT in a saline/FBS mixture, followed by the same extraction protocol.
  • PPT release profiles in serum from the E2-4, 6 and 7 compounds NPs with different PPT/PEG ratios are depicted in Figure 5B. It was found that NPs with an increased PPT/PEG ratio exhibited a reduced drug release rate. All the NPs exhibited linear drug release kinetics: PPT release rate from the NPs with a PPT/PEG wt ratio of 0.4 was about 5%/day whereas it was ⁇ l%/day from NPs with a PPT/PEG wt ratio of 4.4. Comparing the drug release data with IC50 data in Example 4 (Figure 5C), it can be seen that NPs with a decreased PPT/PEG ratio exhibited increased cytotoxic potency (which may be due to the enhanced drug release).
  • E2-7 particles from Example 3 (20 nm) were co-loaded with Dil, and were incubated with EMT6 and EMT-AR1 cells for 30 min or 6 hours. Uptake by cells was rapid (within 30 minutes, Figure 6), and equivalent between parent and resistant forms of the EMT6 cell line.
  • Example 3 Particles from Example 3 (E2-4, 6 and 7 compounds, which assemble as 120, 30 and 20 particles) were co-loaded with Dil.
  • EMT6-AR1 tumor cells (2* 10 5 ) were s.c. implanted in the right flank of BALB/c mice. When the tumor sizes reached 6-8 mm in diameter, the mice were i.v. injected with 1 ⁇ g Dil/mL dose of Dil loaded NPs. After 24-48 h, animals were sacrificed and different organs and tissues were collected and imaged by the Xenogen system. The Dil signal was quantified in each tissue.
  • the liver accumulation was higher with the 120 nm NPs compared to the others: average signal was 1.62 x 10 s p/s/cm 2 /sr with the 120 nm NPs, 1.13xl0 8 p/s/cm 2 /sr with the 30 nm NPs and 1.07xl0 8 p/s/cm 2 /sr with the 20 nm NPs ( Figures 7A, C).
  • This significant increase in selectivity for tumor accumulation with the 20 nm NPs was reflected in the tumor/liver ratio of the signal: with the 20 nm particles, this ratio was 3.7 compared to 1 with 120 nm particles ( Figure 7D).
  • mice Compounds from Example 3 (E2-4, 6 and 7 compounds, which assemble as 120, 30 and 20 particles) were prepared into particles.
  • the maximum tolerated dose (MTD) of drugs in mice was determined in a dose escalation study, and MTD was identified as the maximum dose of a drug that did not induce animal death or >20% body weight loss.
  • the maximum deliverable dose for an i.v. injection of all 3 PPT-NPs was 180 mg PPT/kg.
  • Example 3 Based on the biodistribution data, the 20 nm particles from Example 3 (E2-7 compound) were selected for efficacy tests. Prior to performing efficacy study in the animal models, the maximum tolerated doses PPT was also determined in a dose escalation study. Native PPT exhibited a steep survival curve. While there was no major weight loss in the animals treated with 20 mg/kg of PPT, 30 mg/kg was found to be lethal. On the other hand, even 180 mg PPT/kg of PPT-NPs was found to be safe without causing any signs of stress or any physiological abnormality in the treated animals. Standard taxane chemotherapies (DTX and CBZ) at their MTDs were included as controls.
  • DTX and CBZ Standard taxane chemotherapies
  • EMT6/AR1 Anticancer efficacy of PPT-NPs was analyzed against two MDR tumor models, EMT6/AR1 and PC-3 RES.
  • EMT6 AR1 cells 0.2 ⁇ 10 6 cells
  • PC-3 RES cells 5 ⁇ 10 6 cells
  • NOD-SCID mice were treated the same way as that for the BALB/c mice except that only 2 doses of CBZ (5 mg/kg; day 0 and 4) and DTX (12 mg/kg; day 0 and 4) could be given to the NOD-SCID mice due to significant body weight loss (-20%).
  • mice treated with DTX and CBZ showed visible signs of distress including piloerection, lethargy, weight loss, and weakness (Figure 10).
  • the PPT-NPs treated mice displayed no body weight loss or any significant sign of toxicity during the therapy.
  • composition analogues CMC- Ac was conjugated to PEG and PPT via EDC/DMAP coupling chemistry.
  • CMC-Ac 100 mg, 1.60 mmol acid/g was weighed into a 25 mL glass vial, and dissolved in anhydrous MeCN (3 mL).
  • EDC HC1 61 mg, 0.32 mmol
  • DMAP 39 mg, 0.32 mmol were added into that solution followed by addition of variable amount of PPT and m-PEG-OH (see Table 4 for feeds). The solutions were stirred for 6h at room temperature with protection from light. The reaction mixtures were precipitated through 50 mL diethyl ether.
  • Table 4 Compositions of PPT-CMCAc-PEG from Examples 10, 12, and 13.
  • the solution was stirred for 6h at room temperature with protection from light.
  • the reaction mixture was precipitated through 50 mL diethyl ether.
  • the precipitate was dried, re-dissolved in MeCN, and the precipitation process was repeated twice.
  • the final precipitate was dried under vacuum, and the fine powder was suspended in water (3x50 mL) and filtered.
  • the product was analyzed by LC-MS for uncoupled PEG and PPT, and washing was repeated if residual reagent was detected: less than 0.5 free drug and PEG was detected.
  • the chemical composition of the polymer conjugate was determined by ⁇ -NMR using 2-methyl 5-nitro benzoic acid as an internal standard. Particles were prepared from the polymer by the NanoAssemblr technique.
  • the El 1-1 conjugate formed defined nanoparticulate structures in aqueous solution (-30 nm in diameter). Polymer molecular weight was determined by MALS (15.1 kDa). The presence of DMF retards the reaction, which increases cross-linking side reactions.
  • a reaction from Example 10 was performed with NHs coupling reagent added: CMC-Ac was conjugated to PEG and PPT via EDC/NHS/DMAP coupling chemistry.
  • CMC -Ac 100 mg, 1.60 mmol acid/g was weighed into a 25 mL glass vial, and dissolved in anhydrous MeCN (3 mL).
  • EDC HC1 61 mg, 0.32 mmol
  • NHS 37 mg, 0.32 mmol
  • DMAP 39 mg, 0.32 mmol
  • the solution was stirred for 24h at room temperature with protection from light.
  • the reaction mixture was precipitated through 50 mL diethyl ether.
  • the precipitate was dried, re-dissolved in MeCN, and the precipitation process was repeated twice.
  • the final precipitate was dried under vacuum, and the fine powder was suspended in water (3x50 mL) and filtered.
  • the product was analyzed by LC-MS for uncoupled PEG and PPT, and washing was repeated if residual reagent was detected: less than 0.5 free drug and PEG was detected.
  • the chemical composition of the polymer conjugate was determined by ⁇ -NMR using 2-methyl 5- nitro benzoic acid as an internal standard. Particles were prepared from the polymer by the NanoAssemblr technique.
  • the E12-1 conjugate formed nanoparticles (-45 nm in diameter).
  • Polymer molecular weight was determined by MALS (10.6 kDa).
  • the presence of NHS produces a polymer of lower molecular weight when compared to a reaction sans NHS.
  • Example 13 Synthesis of polymer with different MW PEGS
  • reactions from Example 10 were carried out with PEG MW analogues: CMC-Ac was conjugated to PEG 550, 5000, and 10,000 Da, separately, and PPT via EDC/DMAP coupling chemistry.
  • CMC- Ac 100 mg, 1.60 mmol acid/g was weighed into a 25 mL glass vial, and dissolved in anhydrous MeCN (3 mL).
  • EDC HC1 61 mg, 0.32 mmol
  • DMAP 39 mg, 0.32 mmol
  • the solution was stirred for 6h at room temperature with protection from light.
  • the reaction mixture was precipitated through 50 mL diethyl ether.
  • the precipitate was dried, re-dissolved in MeCN, and the precipitation process was repeated twice.
  • the final precipitate was dried under vacuum, and the fine powder was suspended in water (3x50 mL) and filtered.
  • the product was analyzed by LC-MS for uncoupled PEG and PPT, and washing was repeated if residual reagent was detected: less than 0.5 free drug and PEG was detected.
  • the chemical composition of the polymer conjugate was determined by ⁇ -NMR using 2-methyl 5-nitro benzoic acid as an internal standard.
  • Particles were prepared from the polymer by the NanoAssemblr technique.
  • the conjugates formed distinct nanoparticulate structures in aqueous solution (314 - 36 nm in diameter.
  • Polymer molecular weights were determined by MALS (10.6 - 30.8 kDa). The coupling of higher molecular weight PEG produces a polymer of higher molecular weight when compared to
  • CMC-Ac analogues were prepared by varying the temperature (40-50°C), time (l-3h), and amount of sulphuric acid (0.12 to 1.2 mL) in the reactor. These CMC-Ac polymers were characterized by 3 ⁇ 4 NMR for composition (% theoretical acetylation) and MW (Table 5, E14-1 to E14-7). These CMC-Ac analogues range from 6200 to 220,000 g/mol.
  • PPT and PEG conjugates were prepared by the same method in Example 10, using the E14-6 and E14-7 CMC-Ac MW analogues described above.
  • CMC-Ac was conjugated to PEG and PPT via EDC/DMAP coupling chemistry.
  • CMC -Ac 100 mg, 1.92 and 2.20 mmol acid/g was weighed into a 25 mL glass vial, and dissolved in anhydrous MeCN (3 mL).
  • EDC HC1 (74 mg, 0.39 mmol and, 84 mg, 0.44 mmol) and DMAP (47 mg, 0.39 mmol and, 54 mg, 0.44 mmol) were added into that solution followed by addition of PPT and m-PEG- OH (see Table 6).
  • the solution was stirred for 6h at room temperature with protection from light.
  • the reaction mixture was precipitated through 50 mL diethyl ether.
  • the precipitate was dried, re-dissolved in MeCN, and the precipitation process was repeated twice.
  • the final precipitate was dried under vacuum, and the fine powder was suspended in water (3x50 mL) and filtered.
  • the product was analyzed by LC-MS for uncoupled PEG and PPT, and washing was repeated if residual reagent was detected: less than 0.5 free drug and PEG was detected.
  • the chemical composition of the polymer conjugate was determined by ⁇ -NMR using 2-methyl 5-nitro benzoic acid as an internal standard. Particles were prepared from the polymer by the NanoAssemblr technique. The conjugates formed distinct nanoparticulate structures in aqueous solution (27 - 47 nm in diameter.
  • the E14-1 CMC-Ac analogue (50°C, 3 h reaction with 1.2 mL acid, used to prepare Example 10 compounds) is preferred along with the E14-6 CMCAc analogue (and all MW in between).
  • CMCAc CMCAc MW analogues up to the E14-6 compound are readily processed and fully soluble in all phases of reaction and processing.
  • the El 4-7 CMCAc (used to prepare El 4-9 polymer) required filtration to remove a sub-population of poorly defined materials, and represents a less preferred composition.
  • NPs with different physicochemical properties size, drug release kinetics, cell killing potency, biodistribution
  • an ultra-small NP formulation E2-8) with a mean diameter of 20 nm was optimized for highly tumor- selective delivery of PPT.
  • PPT could be transformed into an effective and safe therapy for treating MDR tumors.

Abstract

In one aspect, there is provided a compound comprising an acetylated carboxymethylcellulose (CMC-Ac) covalently linked to: at least one poly(ethylene glycol) (PEG), and podophyllotoxin or derivatives thereof. In another aspect, a self- assembling nanoparticle composition comprising such compounds is provided.

Description

CELLULOSE-BASED NANOPARTICLES FOR DRUG DELIVERY
FIELD OF THE INVENTION
This invention relates to the field of nanoparticles and, more specifically, to cellulose- based nanoparticles for the delivery podophyllotoxin and related derivatives.
BACKGROUND OF THE INVENTION
Tubulin, a major component of the cellular cytoskeleton, plays an important role in the survival and growth of cells. Its functions extend from cellular transport to cell motility and mitosis. The importance of microtubule in mitosis and cell division makes it an attractive target for anticancer drugs. Chemotherapeutic agents that disrupt the normal function of tubulin are amongst the most potent and broadest spectrum anticancer agents available in the clinic. A structurally diverse class of compounds have been found to antagonize the tubulin function with various tubulin binding sites and different mechanisms of action.
Anti -tubulin agents can be divided into two major categories, microtubule-destabilizing agents and microtubule-stabilizing agents, based on their effect on microtubule polymerization. Microtubule-destabilizing agents, such as colchicine and the vinca alkaloids, inhibit polymerization and decrease the mass of microtubules. Microtubule- stabilizing agents like taxanes stabilize microtubules, increase microtubule polymer mass, and induce the formation of microtubule bundles in cells. Both classes of anti- microtubule agents function by disrupting the dynamic equilibrium of the microtubules, resulting in arrest of cells in mitosis through blocking cell cycle at the metaphase- anaphase transition and leading to cellular apoptosis. l' 2
However, significant clinical success of these anti-microtubule agents has been compromised by the emergence of drug resistance.3 Often, the resistance renders ineffective a variety of anticancer agents and is termed multidrug resistance (MDR). The therapeutic options after development of MDR are limited. MDR can be induced by various mechanisms, including decreased drug uptake, increased drug efflux, activation of detoxifying systems, activation of DNA repair mechanisms, evasion of drug-induced apoptosis, etc.4 Another shortcoming of these anti -tubulin drugs is their significant side effects, including neutropenia, neurotoxicity and hypersensitivity reactions provoked by the surfactants in the formulation to increase their solubility.5
The goal of the designers was to develop an anti-tubulin drug to treat MDR tumors that are resistant to the standard taxane chemotherapies. We first screened a wide range of tubulin inhibitors against MDR cell lines, and identified that podophyllotoxin (PPT) remained active against those highly resistant lines. However, PPT is water insoluble and exhibits a narrow therapeutic window; and therefore, the drug is only used topically to treat HPV-infected genital warts. We hypothesized that PPT could be targeted to MDR tumors by NPs in a detergent and solvent free formulation to exert significant therapeutic activity with reduced side effects. We first covalently conjugated PPT and polyethylene glycol (PEG) onto acetylated carboxymethyl cellulose via ester linkages. The resultant polymer conjugates self-assembled into NPs of variable sizes (20-160 nm) depending on the PPT-to-PEG ratio. The drug release kinetics, cytotoxic potency and in vivo biodistribution of analogues were analyzed and the optimal formulation was tested for its efficacy and safety in two MDR tumor models in mice in comparison with the standard taxane chemotherapies.
Celulose-based nanoparticles for drug delivery are described in Applicant's PCT Patent Publication Nos. 2012/103634 and 2014/015422, which are herein incorporated by reference.
SUMMARY OF THE INVENTION
In an aspect, there is provided, a compound comprising an acetylated carboxymethylcellulose (CMC-Ac) covalently linked to: at least one poly(ethylene glycol) (PEG), and at least one hydrophobic drug comprising podophyllotoxin or derivatives thereof, or pharmaceutically acceptable salts, solvates, esters or prodrugs of any of the foregoing.
In an aspect, there is provided, a self-assembling nanoparticle composition comprising the compound described herein.
In an aspect, there is provided, a pharmaceutical composition comprising the self- assembling nanoparticle composition described herein and a pharmaceutically acceptable carrier and/or diluent.
In an aspect, there is provided, a method of treating cancer in a patient in need thereof, comprising administering to said patient an effective amount of a self-assembling nanoparticle composition comprising the compound described herein.
In an aspect, there is provided, a process for preparing a self-assembling nanoparticle composition comprising: covalently linking at least one PEG and at least one hydrophobic drug comprising a podophyllotoxin or derivatives thereof, or pharmaceutically acceptable salts, solvates, esters or prodrugs of any of the foregoing, to a CMC-Ac; isolating the product of step (a); dissolving the isolated product of step (b) in a suitable organic solvent, preferably DMF or DMSO and further preferably THF or acetonitrile, to form a solution; precipitating the solution of step (c); resuspending the precipitate of step (d) in an an aqueous solution, and preferably dializing the aqueous solution. In an aspect, there is provided, a compound represented by the formula:
Figure imgf000005_0001
R2(i)...R2(n), R3(i)- ..R3(n), and Rend are each independently
Figure imgf000005_0002
wherein -0(HD) represents a hydrophobic drug comprising podophyllotoxin or derivatives thereof, or pharmaceutically acceptable salts, solvates, esters or prodrugs of any of the foregoing, having a point of attachment via a hydroxyl group; or
Figure imgf000005_0003
n is an integer.
BRIEF DESCRIPTION OF THE DRAWINGS
Embodiments of the invention may best be understood by referring to the following description and accompanying drawings. In the drawings:
Figure 1 shows an in vitro viability assay of different drugs against PC3 and PC3-RES tumor cells. · PC3 Δ PC3-RES. Data = mean ± SEM (n=3).
Figure 2 shows IC50 of different drugs against EMT6 and EMT6 AR1. Figure 3 shows IC50 of different drugs against MDA-MB-231 and MDA-MB-231- RES..
Figure 4 shows reaction conditions and 1H-NMR spectra of the PPT-CMC-Ac-PEG, CMC-Ac and PPT.
Figure 5 shows the physicochemical characteristics of PPT-CMC-Ac-PEG conjugates (Example 2) prepared with variable PPT/PEG ratios. (A) Size; (B) Drug release in serum; (C) IC50 (nM) against EMT6 AR1. Data = mean ± SEM (n = 3) for (B-D).
Figure 6 shows intracellular uptake of PPT-NPs (E2-8 particles, 20 nm).
Figure 7 shows tumor and liver uptake of Dil labeled PPT-NPs with different PPT/PEG ratios. (A) Images of tumor and liver uptake at 24 h; (B) Images of tumor uptake at 24 and 48 h; (C) Quantitative comparison of tumor and liver uptake; (D) Quantitative comparison of tumor/liver uptake ratio; (E) Quantitative comparison of tumor uptake at 24 and 48 h. Data = mean ± SEM (n=3). * indicates p<0.05.
Figure 8 shows the biodistribution analysis of the 20 nm PPT-NPs. Data = mean ± SEM (n=3).
Figure 9 shows an in vivo efficacy study against EMT6 AR1 tumor. Data = mean ± SEM (n=10) * indicates p<0.05.
Figure 10 shows the body weight of EMT6 AR1 tumor bearing mice (BALB/c).
Figure 11 shows an in vivo efficacy study against PC3 RES tumor. Data = mean ± SEM (n=10) * indicates p<0.05.
Figure 12 shows the body weight of PC3-RES tumor bearing mice (NOD-SCID).
Figure 13 shows the composition of analogues prepared in Example 2 and 10. (A) composition of polymers that produce defined particles from Examples 2 and 10. In Example 2, reactions were fed with excess amounts of reagent (red circles). In Example 10, reactions were generally fed with sub-excess amounts of reagent (blue diamonds). (B) Reaction model for PEG coupling in the Example 10 syntheses. Blue diamonds represent sub-excess feeds, the red square is an excess feed, and the green triangle is a 1 eqv feed of PEG. (C) Reaction model for the PPT coupling in the Example 10 syntheses. Blue diamonds represent sub-excess feeds, the red square is a excess feed, and the green triangle is a 1 eqv feed of PPT. The models demonstrate that the reaction chemistry in Example 10 is robust and predictable, enabling design and preparation of all analogues in this invention.
DETAILED DESCRIPTION
In the following description, numerous specific details are set forth to provide a thorough understanding of the invention. However, it is understood that the invention may be practiced without these specific details.
Treatment of MDR cancer is one of the most significant challenges for effective chemotherapy. Drug resistance may occur at different cellular level, but has most often been linked to the overexpression of P -glycoprotein (Pgp) which confers resistance to a wide variety of over 300 compounds.7"9 Even many of the highly potent "new generation" anticancer compounds (e.g., kinase inhibitors) are also substrates for Pgp.10' 11 Different approaches, such as identification of Pgp inhibitors, synthesis and evaluation of more active analogues, synthesis of conjugates or prodrugs as well as combined use with other drugs have been pursued to overcome resistance. Unfortunately, most of them have not produced significant improvements in the clinical use. 12' 13 Hence, there is a significant need for the development of compounds and/or treatment strategies for MDR cancer.
Despite of its significant potency against tumors, clinical use of PPT for systemic cancer treatment has been unsuccessful, mainly due to its poor solubility, low selectivity, narrow therapeutic window and significant toxicity. PPT can only be used topically for HPV-infected genital warts due to its poor solubility and selectivity. Different derivatives of PPT has been prepared (etoposide, taniposide, ethophos) and is being used clinically for cancer treatment, but these derivatives have different mode of action (topoisomerase inhibitor) and are significantly less potent (100-1000 times) than PPT16' 17 and also substrates for Pgp.17-19 We hypothesized that a tumor targeted delivery system for PPT can reduce its off-target toxicities and enhance its efficacy against MDR tumors.
Little research has been done to improve delivery of PPT. Greenwald et al.20 conjugated PPT with PEG to increase the solubility, but failed to demonstrated improved efficacy in animal models. Qin et al. 21 prepared a NP formulation for PPT consisting of layered double hydroxides, which displayed little in vivo efficacy.
Different nanotherapeutic formulations have been developed to provide increased safety and efficacy for cancer chemotherapy.22 Among them, polymer-drug conjugate has received significant attention due to the advantages in increased drug loading capacity, enhanced stability, and prolonged blood circulation.23 In the present study, CMC was selected as the polymer backbone to synthesize a polymer conjugate of PPT due to its documented safety, biocompatibility and multiple conjugation sites (carboxylate groups) for increased coupling of the drug. CMC, like many other biocompatible polymers, is highly polar and only sparingly soluble in organic solvents such as DMF or DMSO.24 However, PPT is only soluble in organic solvents, and as a result the coupling chemistry could not be performed directly. This low solvent solubility hinders their conjugation efficiency with many hydrophobic drugs. Modifications in polysaccharides to increase organic solvent solubility include triethylamine (TEA) or tetrabutylammonium (TBA) complexation, but the coupling efficiency of these polymers remained low (15 - 20 wt%).25' 26 The resolution of the challenge was to acetylate the hydroxyl groups on the CMC (to produce CMC-Ac), which was soluble in DMSO, MeCN and a number of organic solvents. Full acetylation of hydroxyl groups in CMC is also important to prevent polymer cross-linking during the EDC coupling chemistry
Many different polysaccharides including heparin, haluronan, carboxymethyl dextran and chitosan have been used for drug conjugation, but the drug loading is found to be low in most of the studies, ranging from 5 - 20 wt%. However, in this CMC-Ac system increased drug conjugation efficiency > 35 wt%.
Increased drug loading in polymer-conjugates often results in controlled drug release (reduced burst release) and improved pharmacokinetics.28 It has been noted by other investigators as well that polymer-drug conjugates with an increased amount of drug loading display retarded drug release kinetics.29 It has been argued that with increased drug loading in a polymer-drug conjugate, the polymer tend to form complex secondary and tertiary structures in an aqueous medium and can self-assemble into core-shell nanostructures with a stable hydrophobic drug core which can reduce the hydrolytic release of the drug. Conjugates with low drug loading are increasingly hydrated, which exposes the polymer-drug linker to the external medium, facilitating hydrolytic cleavage of the bond for increased drug release.30 In this invention, the PPT/PEG ratio was adjusted to favour a smaller particle size and a larger surface area, to favour an optimal drug release rate.
Although polymer-drug conjugates have been shown to significantly alter the pharmacokinetics of the native drug, in many instances significant reticulo-endothelial system (RES) interaction has been measured, resulting in significant liver and spleen accumulation of the conjugates.31 One of the most advanced polymer-drug conjugate is Opaxio (Poly-glutamic acid-paclitaxel conjugate) which failed to show significant advantage in early phase III clinical trials over native paclitaxel or docetaxel32' 33 and exhibited 8-fold increased liver accumulation and 5-fold increased spleen accumulation compared to tumor accumulation.34 Doxorubicin-dextran conjugate (DOX-OXD, AD- 70) in Phase I clinical trials induced significant hepatotoxicity, ascribed to increased uptake by the RES.35 Although PEGylation has been employed to improve pharmacokinetic of many NPs,36 it has not been extensively used for polymer-drug conjugates, possibly due to the complexities involved in the chemistry. The EDC coupling chemistry employed in this invention enables efficient conjugation of PEG and PPT onto CMC- Ac, with excellent control over reaction outcomes. As the reactions can be modeled, the PPT and PEG content of the polymers can be adjusted in a predictable manner, enabling the designers to tune the PPT/PEG ratio, creating NPs with a range of size, drug release kinetics and cell killing potency. These parameters are important determinants for in vivo pharmacokinetics, biodistribution, tissue penetration and tumor bioavailability.37 In the examples, we demonstrate the interplay of physical parameters: the conjugate with a low PPT/PEG molar ratio of 2 yielded NPs with a mean diameter of 20 nm and released PPT at ~5%/day in serum, while conjugates with increased PPT/PEG ratios (5 and 20) produced bigger particles (30 nm and 120 nm respectively) that displayed slower drug release (~2.5%/day and ~l%/day respectively). The 20-nm particles exhibited enhanced cell killing potency, increased tumor delivery and decreased liver uptake compared to the bigger NPs, and the biodistribution in the tumor-bearing mice was highly selective to tumor with a ~4-fold increase compared to the liver. The 20-nm particles for PPT displayed significantly improved efficacy against two MDR tumor models in mice compared to the standard taxane chemotherapies with minimal toxicity. In short, the ability to control CMC-Ac, PEG and PPT composition parameters enable effective control the conjugates and NPs and an optimal in vivo performance.
In an aspect, there is provided, a compound comprising an acetylated carboxymethylcellulose (CMC-Ac) covalently linked to: at least one poly(ethylene glycol) (PEG), and at least one hydrophobic drug comprising podophyllotoxin or derivatives thereof, or pharmaceutically acceptable salts, solvates, esters or prodrugs of any of the foregoing.
For use in medicine, the salts of the compounds of this invention refer to non-toxic "pharmaceutically acceptable salts." Other salts may, however, be useful in the preparation of compounds according to this invention or of their pharmaceutically acceptable salts. Suitable pharmaceutically acceptable salts of the compounds include acid addition salts which may, for example, be formed by mixing a solution of the compound with a solution of a pharmaceutically acceptable acid such as hydrochloric acid, sulfuric acid, fumaric acid, maleic acid, succinic acid, acetic acid, benzoic acid, citric acid, tartaric acid, carbonic acid or phosphoric acid. Furthermore, where the compounds of the invention carry an acidic moiety, suitable pharmaceutically acceptable salts thereof may include alkali metal salts, e.g., sodium or potassium salts; alkaline earth metal salts, e.g., calcium or magnesium salts; and salts formed with suitable organic ligands, e.g., quaternary ammonium salts. Thus, representative pharmaceutically acceptable salts include the following: acetate, benzenesulfonate, benzoate, bicarbonate, bisulfate, bitartrate, borate, bromide, calcium edetate, camsylate, carbonate, chloride, clavulanate, citrate, dihydrochloride, edetate, edisylate, estolate, esylate, fumarate, gluceptate, gluconate, glutamate, glycollylarsanilate, hexylresorcinate, hydrabamine, hydrobromide, hydrochloride, hydroxynaphthoate, iodide, isothionate, lactate, lactobionate, laurate, malate, maleate, mandelate, mesylate, methylbromide, methylnitrate, methylsulfate, mucate, napsylate, nitrate, N methylglucamine ammonium salt, oleate, pamoate (embonate), palmitate, pantothenate, phosphate/diphosphate, polygalacturonate, salicylate, stearate, sulfate, subacetate, succinate, tannate, tartrate, teoclate, tosylate, triethiodide and valerate.
Representative acids and bases which may be used in the preparation of pharmaceutically acceptable salts include the following: acids including acetic acid, 2,2 dichloroactic acid, acylated amino acids, adipic acid, alginic acid, ascorbic acid, L aspartic acid, benzenesulfonic acid, benzoic acid, 4 acetamidobenzoic acid, (+) camphoric acid, camphorsulfonic acid, (+) (IS) camphor 10 sulfonic acid, capric acid, caproic acid, caprylic acid, cinnamic acid, citric acid, cyclamic acid, dodecylsulfuric acid, ethane 1,2 disulfonic acid, ethanesulfonic acid, 2 hydroxy ethanesulfonic acid, formic acid, fumaric acid, galactaric acid, gentisic acid, glucoheptonic acid, D gluconic acid, D glucoronic acid, L glutamic acid, alpha-oxo glutaric acid, glycolic acid, hippuric acid, hydrobromic acid, hydrochloric acid, (+) L lactic acid, (±) DL lactic acid, lactobionic acid, maleic acid, (-) L malic acid, malonic acid, (±) DL mandelic acid, methanesulfonic acid, naphthalene 2 sulfonic acid, naphthalene 1,5 disulfonic acid, 1 hydroxy 2 naphthoic acid, nicotinic acid, nitric acid, oleic acid, orotic acid, oxalic acid, palmitic acid, pamoic acid, phosphoric acid, L pyroglutamic acid, salicylic acid, 4 amino salicylic acid, sebaic acid, stearic acid, succinic acid, sulfuric acid, tannic acid, (+) L tartaric acid, thiocyanic acid, p toluenesulfonic acid and undecylenic acid; and bases including ammonia, L arginine, benethamine, benzathine, calcium hydroxide, choline, deanol, diethanolamine, diethylamine, 2 (diethylamino) ethanol, ethanolamine, ethylenediamine, N methyl glucamine, hydrabamine, 1H imidazole, L lysine, magnesium hydroxide, 4 (2 hydroxyethyl) morpholine, piperazine, potassium hydroxide, 1 (2 hydroxyethyl) pyrrolidine, secondary amine, sodium hydroxide, triethanolamine, tromethamine and zinc hydroxide.
The present invention includes within its scope prodrugs of the compounds of this invention. In general, such prodrugs will be functional derivatives of the compounds that are readily convertible in vivo into the required compound. Thus, in the methods of treatment of the present invention, the term "administering" shall encompass the treatment of the various disorders described with the compound specifically disclosed or with a compound which may not be specifically disclosed, but which converts to the specified compound in vivo after administration to the patient. Conventional procedures for the selection and preparation of suitable prodrug derivatives are described, for example, in "Design of Prodrugs", ed. H. Bundgaard, Elsevier, 1985.
The term "form" means, in reference to compounds of the present invention, such may exist as, without limitation, a salt, stereoisomer, tautomer, crystalline, polymorph, amorphous, solvate, hydrate, ester, prodrug or metabolite form. The present invention encompasses all such compound forms and mixtures thereof.
The term "isolated form" means, in reference to compounds of the present invention, such may exist in an essentially pure state such as, without limitation, an enantiomer, a racemic mixture, a geometric isomer (such as a cis or trans stereoisomer), a mixture of geometric isomers, and the like. The present invention encompasses all such compound forms and mixtures thereof.
Certain compounds may exist in various stereoisomeric or tautomeric forms and mixtures thereof. The invention encompasses all such compounds, including active compounds in the form of essentially pure enantiomers, racemic mixtures and tautomers.
In some embodiments, the covalent linkages are ester linkages. In some embodiments, the hydrophobic drug is podophyllotoxin or pharmaceutically acceptable salts, solvates, esters or prodrugs of any of the foregoing.
In some embodiments, the hydrophobic drug is podophyllotoxin.
In some embodiments, the hydrophobic drug is selected from the group consisting of etoposide, taniposide, Adva-27a, and NK611r, or pharmaceutically acceptable salts, solvates, esters or prodrugs of any of the foregoing.
In some embodiments, the hydrophobic drug is:
Figure imgf000014_0001
In some embodiments, the at least one PEG is poly(ethylene glycol) methyl ether (mPEG) having an average Mn of between about 550 and about 10,000, preferably about 2000. Preferably, the CMC consists of a polymer with a molecular weight of between 4400 and 220,000 Daltons.
In some embodiments, the wt% of PEG in the nanoparticle is 3 - 50.1 wt%, preferably 10.8-43.0 wt%.
In some embodiments, the wt% of PPT in the nanoparticle is 1.9-38 wt%, preferably 3.9 - 30.5 wt%, further preferably about 12 wt%.
In some embodiments, the drug/PEG wt ratio is between about 0.14 to about 5.8, preferably 0.191-3.547, further preferably about 0.4.
In an aspect, there is provided, a self-assembling nanoparticle composition comprising the compound described herein. In some embodiments, the nanoparticles have an average diameter of about 20-160 nm, or preferably around 20m.
In some embodiments, the nanoparticle further comprises at least one hydrophobic agent encapsulated therein, preferably selected from either an imaging agent or a therapeutic agent.
In an aspect, there is provided, a pharmaceutical composition comprising the self- assembling nanoparticle composition described herein and a pharmaceutically acceptable carrier and/or diluent. In an aspect, there is provided, a method of treating cancer in a patient in need thereof, comprising administering to said patient an effective amount of a self-assembling nanoparticle composition comprising the compound described herein. In some embodiments, the cancer is a multidrug resistant cancer. Cancers could include Adrenal Cancer, Anal Cancer, Bile Duct Cancer, Bladder Cancer, Bone Cancer, Brain/CNS Tumors, Breast Cancer, Castleman Disease, Cervical Cancer, Colon/Rectum Cancer, Endometrial Cancer, Esophagus Cancer, Ewing Family Of Tumors, Eye Cancer, Gallbladder Cancer, Gastrointestinal Carcinoid Tumors, Gastrointestinal Stromal Tumor (GIST), Gestational Trophoblastic Disease, Hodgkin Disease, Kaposi Sarcoma, Kidney Cancer, Laryngeal and Hypopharyngeal Cancer, Leukemia, Liver Cancer, Lung Cancer, Lung Carcinoid Tumor, Lymphoma, Malignant Mesothelioma, Multiple Myeloma, Myelodysplasia Syndrome, Nasal Cavity and Paranasal Sinus Cancer, Nasopharyngeal Cancer, Neuroblastoma, Non-Hodgkin Lymphoma, Oral Cavity and Oropharyngeal Cancer, Osteosarcoma, Ovarian Cancer, Pancreatic Cancer, Penile Cancer, Pituitary Tumors, Prostate Cancer, Retinoblastoma, Rhabdomyosarcoma, Salivary Gland Cancer, Sarcoma, Skin Cancer, Small Intestine Cancer, Stomach Cancer, Testicular Cancer, Thymus Cancer, Thyroid Cancer, Uterine Sarcoma, Vaginal Cancer, Vulvar Cancer, Waldenstrom Macroglobulinemia, and Wilms Tumor.
In an aspect, there is provided, a process for preparing a self-assembling nanoparticle composition comprising: covalently linking at least one PEG and at least one hydrophobic drug comprising a podophyllotoxin or derivatives thereof, or pharmaceutically acceptable salts, solvates, esters or prodrugs of any of the foregoing, to a CMC-Ac; isolating the product of step (a); dissolving the isolated product of step (b) in a suitable organic solvent, preferably DMF or DMSO and further preferably THF or acetonitrile, to form a solution; precipitating the solution of step (c); resuspending the precipitate of step (d) in an an aqueous solution, and preferably dializing the aqueous solution. In some embodiments, steps (c) and (d) are repeated one or more times before step (e) In an aspect, there is provided, a compound represented by the formula:
Figure imgf000016_0001
R2(i)...R2(n), R3(i)- ..R3(n), and R-nd are each independently
Figure imgf000016_0002
wherein -0(HD) represents a hydrophobic drug comprising podophyllotoxin or derivatives thereof, or pharmaceutically acceptable salts, solvates, esters or prodrugs of any of the foregoing, having a point of attachment via a hydroxyl group; or
Figure imgf000016_0003
n is an integer.
In some embodiments, the PEG is m(PEG).
Those of skill in the art will understand that each of the hydrophobic drug and PEG may be covalently linked to the CMC-Ac by a direct linkage between a carboxylic acid residue of the CMC-Ac and a functional group of the hydrophobic drug and PEG (e.g. a hydroxyl group), or by an indirect linkage via one or more bifunctional linkers. Preferred linkers are those that are biodegradable, non-toxic when cleaved from the conjugate, and are relatively stable to hydrolysis in the circulation.
Figure imgf000017_0001
Acceptable carriers and diluents would be known to a person skilled in the art. Generally, such carriers should be nontoxic to recipients at the dosages and concentrations employed.
The processes described herein are generally useful for preparing conjugates of any CMC-Ac with any PEG and any hydrophobic drugs that are appropriately functionalized for linking to the CMC-Ac, as described herein.
Suitable solvents for use in the processes of the present invention include those solvents that are inert under the conditions of the reaction/procedure being described in conjunction therewith.
Commercial sources of CMC vary in degree of substitution (DS). It will be appreciated by those of skill in the art that DS differences influence the maximum PPT and PEG substitution.
It will be appreciated by those of skill in the art that the actual preferred amounts of active agent in a specific case will vary according to the particular formulation and manner of administration. The specific dose for a particular individual depends on age, body weight, the severity of the particular disorder to which the therapy is applied, and other factors. Dosages for a given subject can be determined using conventional considerations.
The following examples are illustrative of various aspects of the invention, and do not limit the broad aspects of the invention as disclosed herein.
EXAMPLES
MATERIALS
Podophyllotoxin (PPT) was obtained from Carbosynth (Berkshire, UK). Docetaxel (DTX), Paclitaxel (PTX) and Cabazitaxel (CBZ) were obtained from LC Laboratories (Wobum, MA). Cholchicin (Cho) and Vinblastin (Vin) were purchased from Sigma Aldrich (Oakville, ON). Carboxymethylcellulose (CMC) sodium salt (CEKOL 30000- P) was purchased from CPKelco (Atlanta, GA). Poly(ethylene glycol) methyl ether (mPEG-OH, MW = 2000), l-ethyl-3-(3-dimethylaminopropyl)-carbodiimide HC1 (EDC.HC1), and 4-dimethylaminopyridine (DMAP) were purchased from Sigma Aldrich (Oakville, ON). Hydrophobic fluorescent dye Dil (l,10-dioctadecyl-3,3,3030- tetramethylindocarbocyanine perchlorate, D-307) was purchased from Invitrogen (Burlington, ON). For all the in vitro studies, free drugs were first dissolved in DMSO.
METHODS
NMR analysis: Polymer and nanoparticle (NP) composition was determined by ¾- NMR using 2-methyl 5-nitro benzoic acid as an internal standard (IS). A response factor for PPT or PEG relative to the IS was determined in a calibration, and polymer and NP content was estimated using these calibrations.
MALS
The molecular weight of the CMCAc or polymer conjugates was measured on a Wyatt HELEOS II multi-angle light scattering system (MALS) coupled with a Waters e2696 pump, a Waters 2414 RI, a Millipore 0.22 μηι filter, and a Wyatt high-pressure injection system. The system was run in batch-mode MALS with acetonitrile flowing at 0.8 mL/min. For calibration of dn/dc constants, CMCAc samples were prepared as a range of concentration in acetonitrile (0.25 to 1.6 mg/mL), and injected into the system through a 0.22 μηι PTFE filter. The system was validated and normalized using a 20KDa PEG standard. Samples were prepared as 1 mg/mL solution in MeCN, and analyzed in the Astra software using the above dn/dc constants. Titration of CMC-Ac polymer
The CMC-Na polymer is modified with carboxymethyl groups, and was supplied by the manufacturer with an analyzed DS value of 0.82: each glucose monomer unit is specified to contain 0.82 moles carboxylic acid and 2.18 moles hydroxyl. Processing the CMC-Na to a CMC-Ac compound can alter the carboxylic acid content (the DS), and the polymer are titrated to determine functionality. Briefly, CMC Ac (100 mg) is dissolved in MeCN (15 mL) and MeOH (10 mL) in a glass Erlenmeyer flask, and is titrated with KOH (0.1N in methanol), using phenolphthalein (1% alcohol) as an indicator.
LC analysis
Polymer and particle samples were analyzed for free drug or PEG by LC/MS analysis. Sample analysis was performed on an LC/MS system consisting of a Waters Acquity UPLC/MS equipped with a PDA and a Xevo QToF MS. In these analyses, samples of polymer were prepared as 1 mg/mL solution in MeCN, and particle solutions prepared as 10X dilutions in MeCN. Samples were injected on an Acquity UPLC BEH CI 8 column (1.7 μηι, 2.1 x 50 mm), at a flowrate of 0.4 mL/min, with a gradient program of 95-10% water/acetonitrile over 5 minutes. Calibration of each drug and PEG was performed using the appropriate mass ion.
Generalized synthesis method
All polymer conjugates are synthesized with an acetylated CMC compound CMC- Ac), which is prepared as described by Namikoshi et al.6 Briefly, sodium CMC was first desalted using 20% sulfuric acid solution, and the free acid was then acetylated with acetic anhydride to yield acetylated CMC (CMC-Ac).
Drug and PEG conjugation with the CMC- Ac polymer were carried out by esterification. CMC polymer has a high degree of substitution with carboxylic groups. One prerequisite for a candidate drug to be conjugated with the CMC is the presence of, minimum and preferably, one free hydroxyl group in their structure. Free hydroxyl group in the drug as well as in the PEG are conjugated with the free carboxyl group in the polymer using EDC-mediated esterification. An excess amount of DMAP is applied to effect solubilisation of the EDC.HC1 in the organic medium (eg: acetonitrile, DMSO, and to act as a catalyst in the reaction. In some embodiments, NHS is introduced into the reaction to control side reactions which increase MW of the polymer conjugation. In all embodiments, anhydrous solvents were used, as water retards reaction kinetic (but not does not reduce reaction yields).
Particle preparation
Nanoparticles were prepared in a controlled nanoprecipitation process using a two- channel microfluidic system (NanoAssemblr, Precision Nanosystems, Canada). Polymer (150 mg) was dissolved in acetonitrile at varying concentrations (4-45 mg/g), fed through one channel and 0.9% NaCl fed through the adjacent channel. Total flow rate was maintained at 12-24 mL/min and the flow ratio of the aqueous to organic stream was 2: 1 to 4: 1. The optimal conditions were typically 30 mg/g polymer in acetonitrile, 18 mL/min flow, and a 3: 1 organic: saline ratio. The outlet stream was immediately diluted in an equal volume of saline, was purified by dialysis, sterile filtered using 0.22 μηι Millipore PVDF filters, and concentrated using Vivaspin columns. Particle size and zeta potential were measured with a Zetasizer (Nano-ZS, Malvern Instruments, Malvern, UK). Taxane or podophyllotoxin content were determined by ¾ NMR with 2-methyl-5-nitrobenzoic acid as an internal standard, and LC analysis was performed to ensure products contained less than 1 wt% free drug or PEG.
Fluorescently loaded NP: Dil loaded NPs were prepared by dissolving 30 mg of the polymer in MeCN (1 mL) containing 0.1 mg/mL Dil and was precipitated into 3 mL of normal saline in the NanoAssemblr at the flow rate of 18 mL/min. Dil content of the NPs was determined by dissolving the NPs in DMSO and assaying for fluorescence (Excitation filter: 535 nm; Emission Filter 590 nm) and comparing to a calibration curve of fluorescence versus Dil concentration, subtracting the background signal of un-loaded particle fluorescence. Cell culture and animals
Human MD A-MB-231 and PC3 and mouse EMT6 breast cancer cell lines were obtained from the American Type Culture Collection (ATCC). Resistant EMT6/AR1 cells were a gift from Ian Tannock, Princess Margaret Hospital, Toronto. DTX resistant PC3 and MD A-MB-231 cells were generated from the sensitive phenotype by treating them with gradually increasing concentrations of DTX until the cells become fully resistant to 100 nM and 10 nM of DTX respectively. The cells were maintained in Dulbecco's Modified Eagle's Medium (DMEM) supplemented with 10% fetal bovine serum. Female NOD-SCID and BALB/c mice were purchased from Jackson Laboratories (Bar Harbour, ME). All protocols were approved by the Animal Care Committee of the University Health Network.
In vitro analysis of inhibition (IC50)
Cell growth inhibition activity of different drugs or NP were analyzed by measuring cell viability with the XTT assay. Briefly, cells were dislodged and re-suspended at a concentration of 1 χ 104 cell/mL, and 100 of cell suspension per well was added to a 96 wellplate. A slight modification (5 χ 104 cell/mL) was made for the MD A-MB-231 analysis, as these cells were slow-growing compared to the other lines. The cells were maintained for 24 h in culture (37 °C, 5% C02, humidified) before treatment. After 72 h of treatment, viability was assayed by the XTT assay. Briefly, a 1 mg/mL solution of XTT reagent and 1.53 mg/mL solution of phenazine methylsulfate in water were prepared, and 5 of phenazine methylsulfate was added to each mL of the XTT solution. Twenty-five of the mixture was added to each well, the culture plate was incubated for 2 h at 37 °C, and absorbance of each well at 480 nm was then measured. Wells treated with media represent 100% viable cultures, and wells containing no cells represent background signal. The viability data were analyzed in GraphPad Prism, and the IC50 was calculated. Nanoparticle uptake in vitro
2 x 105 EMT6 or EMT6/AR1 cells were plated on sterile glass coverslips placed in a 12-well plate. Cells were treated with Dil-labeled PPT-NPs at the concentration of 0.5 μg Dil/mL. After 4 h of incubation, the cells were washed with PBS and treated lysotraker green for lh. After that they were washed with PBS and fixed with 10% formalin for 15 min. The fixed cells were washed again with PBS and the glass coverslip was mounted on a glass slide with mounting media containing DAPI. Slides were then scanned on the FluoView A confocal microscope (Olympus). The intensity of the Dil and DAPI signal was measured using ImageScope software with positive pixel count algorithm.
Example 1: Selection of PPT from a panel of common chemotherapeutics
Although the search of new chemical entities for enhanced treatment of cancer is an ongoing process and has attracted considerable attention, screening of the existing chemotherapeutic candidates for diverse indications can be an alternative. Anti-tubulin drugs are among the most potent and broadest spectrum chemotherapeutic agents. In this study in order to identify candidates for the treatment of MDR tumors, we tested potency of a panel of six anti-tubulin drugs (PTX, DTX, CBZ, PPT, CHO, Vin) against different MDR tumors as well as their native phenotypes (EMT6, PC3 and MDA-MB- 231) (Figure 1-3, Table 1). All the tested drugs exhibited significant potency against the native cell lines, with IC50 varied between 0.01 nM and 14 nM. CBZ was found to be the most potent: IC50 varied from 0.01 to 1 nM. However, PTX, DTX, CBZ, Choi and Vin showed a considerable loss in potency against the MDR lines (EMT6 AR1, PC3-RES and MDA-MB-231-RES). PC3-RES was found to be the most refractive to these drugs: the resistance index (RI, IC50 in the resistant line divided by IC50 in the parent line) of PTX, DTX, CBZ, Choi and Vin for this cell line varied from 100 to 500. The loss of potency because of the MDR phenotype was in the order of PTX (RI: 510) > DTX (450) > Vin (400) > Choi (190) > CBZ (100). A similar trend was also noted in the EMT6 and MDA-MB-231 models (Table 1). However, PPT retained its activity against the MDR cells in all three models (RI = 1-3). Table 1: IC50 and resistance index (RI) of different drugs against different cell lines. Data = mean ± SEM (n= 3). RI = IC50 in the resistant line divided by IC50 in the parent line.
IC50(nM) DTX PTX PPT CBZ Choi Vin
PC3 2 + 1 4±2 13 ±5 0.06 + 0.05 14 ±5 0.2±0.1
PC3-RES 903 ±50 2057 ±120 15 ±3 6.3 ±2 2610 + 140 80 + 5
Resistance .,„
510 1 105 190 400 ndex
E T6 7±2 13±5 12±2 1±2 10 + 5 1±1
EMT6AR1 1091 ±45 6667 ±150 13 ±3 43 ± 5 2375 ±230 80 ±4
Resistance
156 513 1 43 237 80 Index
MDA-MB-231 1 + 2 1±3 6 + 5 0.01 +0.02 2 + 1 0.5 + 0.1
MDA-MB-23
40 + 5 130 + 15 20 + 5 1 + 1 73 + 8 130 + 15 RES
Resistance
40 130 3 100 37 260 Index
Example 2: Synthesis of PPT-CMC-Ac-PEG polymer composition analogues
In a specific embodiment of the general synthesis method, reactions were carried out to prepare composition analogues: CMC-Ac was conjugated to PEG and PPT via EDC/DMAP coupling chemistry (Figure 4). CMC-Ac (300 mg) was weighed into a 25 mL round bottom flask, and dissolved in a mixture of anhydrous MeCN (9 mL) and DMSO (6 mL). EDC HCl (448 mg, 2.3 mmol) and DMAP (580 mg, 4.6 mmol) were added into that solution followed by addition of variable amount of PPT and m-PEG- OH (see Table 2). The solutions were stirred 24h at room temperature with protection from light. The reaction mixtures were precipitated through 135 mL diethyl ether. The precipitate was dried, re-dissolved in MeCN, and the precipitation process was repeated twice. The final precipitates were dried under vacuum, and the fine powders were suspended in water (25 mL) and dialyzed against MilliQ water for 24 h with 3 changes (MW cut-off = 10 kDa). The product was analyzed by LC-MS for uncoupled PEG and PPT, and washing was repeated if residual reagent was detected: less than 0.5 free drug and PEG was detected. The chemical composition of the polymer conjugate was determined by ^-NMR using 2-methyl 5-nitro benzoic acid as an internal standard (Table 2). Particles were prepared from the polymers by the NanoAssemblr technique (Table 2). Conjugates with >50 wt% PEG loading were water soluble and did not form any measurable particles. Conjugates with <50 wt% PEG loading formed distinct nanoparticulate structures in aqueous solution (20-120 nm in diameter). Increased content of PPT in the conjugate led to increased particle size, whereas increased PEG resulted in decreased particle size (Figure 5A). The conjugates with a PPT/PEG wt ratio of 0.5 assembled into -20 nm particles whereas the particle size of the conjugate with a PPT/PEG ratio of 4.4 was found to be -120 nm.
Table 2: Polymers with a range of PPT and PEG content (Example 2).
Mol eqv feeds Product Composition and Characterization
PPT/PEG
PEG PPT PEG ratio
Sample PPT Feed Feed wt% wt% (wt/wt) Size (nm) PDI IC50 (nM)
E2-1 4.6 0.2 35.0 8.0 4.38 120 ± 12 0.1 ± 0.08 463 ± 25
E2-2 1.9 0.2 32.0 13.0 2.46 80 ± 5 0.08 ± 0.05 400 ± 20
E2-3 2.8 0.5 32.0 17.0 1.88 78 ± 5 0.1 ± 0.05 330 ± 20
E2-4 1.6 0.2 30.0 16.0 1.88 95 ± 7 0.08 ± 0.06 420 ± 30
E2-5 0.9 0.2 23.0 23.0 1 40 ± 3 0.06 ± 0.04 200 ± 15
E2-6 1.6 0.5 22.0 22.0 1 30 ± 5 0.07 ± 0.03 250 ± 18
E2-7 1.6 1.2 19.0 38.0 0.5 22 ± 3 0.05 ± 0.02 130 ± 20
E2-8 0.5 0.2 15.0 42.0 0.4 20 ± 2 0.05 ± 0.01 125 ± 20
E2-9 0.5 4.6 3.0 67.0 0.04 - - -
E2-10 0.2 4.6 0.2 76.0 0 - - -
Example 3: Synthesis of PPT-CMC-Ac-PEG polymer composition analogues for biological testing
Of the polymers synthesized in Example 2 and summarized in Table 2, three compositions were selected and synthesized at greater scale for further analysis in vitro and in vivo examples: the E2-4, E2-6, and E2-7 compounds. CMC-Ac (500 mg), EDC HCl (747 mg, 3.9 mmol), DMAP (952 mg, 7.8 mmol), PPT (see Table 3), and PEG (see Table 3) were weighed into a 100 mL round bottom flask, and dried overnight to remove moisture. A mixture of anhydrous MeCN (14 rtiL) and DMSO (6 mL) were added to the reactor to dissolve the compounds, with good stirring. The reaction was stirred 24h at room temperature with protection from light. The reaction mixtures were rapidly precipitated through well mixed 45 x 4 mL diethyl ether in conical tubes. The polymer was spun down at 4000 rpm for 5 minutes, and the pellet was dried, re- dissolved in 5 mL MeCN, and the precipitation process was repeated three more times. The final precipitates were dried under vacuum for 2 h, and the fine powders were suspended in water (15 mL) and dialyzed against MilliQ water for 24 h with 3 changes (MW cut-off = 10 kDa). The product was recovered by lyophylization, analyzed by LC-MS for uncoupled PEG and PPT: less than 0.5 free drug and PEG was detected. The chemical composition of the polymer conjugate was determined by ^-NMR using 2-methyl 5-nitro benzoic acid as an internal standard. E2-1 analogue (120 nm particle): 14.9 wt% PEG, 30.5 wt% PPT. E2-6 analogue (30 nm particle): 25.0 wt% PEG, 31.6 wt% PPT. E2-8 analogue (20 nm particle): 42.8 wt% PEG, 14.1 wt% PPT. Polymers were analyzed by MALS for MW: E2-1 : 56.3 kDa, E2-2: 12.2 kDa, E2-3: 48.5 kDa.
Table 3: Feeds of PPT and PEG for compounds synthesized in Example 3.
Figure imgf000026_0001
Example 4: IC50 analysis
Particles prepared in Example 3 (the E2-4, 6 and 7 analogues) were analyzed for cytotoxic potency in an EMT-AR1 drug resistant breast cancer line (Table 2). All compounds are active in the nM concentration range.
Example 5: In vitro release of PPT
Particles prepared in Example 3 (the E2-4, 6 and 7 analogues) were 1: 1 (v:v) mixed with fetal bovine serum (FBS) at the final concentration of 100 μg PPT /mL. Samples were incubated at 37 °C, and at selected time points triplicate samples were removed and serum protein was precipitated using 600 MeCN containing 1% acetic acid. The samples were centrifuged for 5 min at 10,000 rpm and the supernatant was analyzed for released PPT by HPLC at a flow rate of 0.4 mL/min, with a gradient program of 95/5 to 10/90 water/MeCN over 5 min. A calibration curve for PPT was prepared by spiking known amounts of PPT in a saline/FBS mixture, followed by the same extraction protocol.
Particle size and the PPT/PEG ratio affected the drug release rate and cytotoxic potency of the NPs. PPT release profiles in serum from the E2-4, 6 and 7 compounds NPs with different PPT/PEG ratios are depicted in Figure 5B. It was found that NPs with an increased PPT/PEG ratio exhibited a reduced drug release rate. All the NPs exhibited linear drug release kinetics: PPT release rate from the NPs with a PPT/PEG wt ratio of 0.4 was about 5%/day whereas it was ~l%/day from NPs with a PPT/PEG wt ratio of 4.4. Comparing the drug release data with IC50 data in Example 4 (Figure 5C), it can be seen that NPs with a decreased PPT/PEG ratio exhibited increased cytotoxic potency (which may be due to the enhanced drug release).
Example 6: cellular uptake of PPT-NP
E2-7 particles from Example 3 (20 nm) were co-loaded with Dil, and were incubated with EMT6 and EMT-AR1 cells for 30 min or 6 hours. Uptake by cells was rapid (within 30 minutes, Figure 6), and equivalent between parent and resistant forms of the EMT6 cell line.
Example 7: In vivo biodistribution study of Dil labelled NPs:
Particles from Example 3 (E2-4, 6 and 7 compounds, which assemble as 120, 30 and 20 particles) were co-loaded with Dil. EMT6-AR1 tumor cells (2* 105) were s.c. implanted in the right flank of BALB/c mice. When the tumor sizes reached 6-8 mm in diameter, the mice were i.v. injected with 1 μg Dil/mL dose of Dil loaded NPs. After 24-48 h, animals were sacrificed and different organs and tissues were collected and imaged by the Xenogen system. The Dil signal was quantified in each tissue.
Most of the Dil signal was found in the tumor and liver for all three NPs (Figure 7). When the tumor and liver signal of the three formulations were compared, significantly higher tumor accumulation was found with the 20 nm NPs compared to the 30 and 120 nm particles: average signal was 3.73x l08 p/s/cm2/sr with the 20 nm NPs compared to 2.5xl08 p/s/cm2/sr and 1.78x l08 p/s/cm2/sr with the 30 nm and 120 nm particles, respectively (Figures 7A-C). On the other hand, the liver accumulation was higher with the 120 nm NPs compared to the others: average signal was 1.62 x 10s p/s/cm2/sr with the 120 nm NPs, 1.13xl08 p/s/cm2/sr with the 30 nm NPs and 1.07xl08 p/s/cm2/sr with the 20 nm NPs (Figures 7A, C). This significant increase in selectivity for tumor accumulation with the 20 nm NPs was reflected in the tumor/liver ratio of the signal: with the 20 nm particles, this ratio was 3.7 compared to 1 with 120 nm particles (Figure 7D). Compared between 24 and 48 h post treatment, a slight decrease in tumor signal was noted with all three particles (Figure 7E). One day after the injection of the 20 nm particles, all the major tissues were imaged (Fig 8), and a ~4-fold increased level of Dil signal was detected in the tumor compared to other tissues. No signal of the 20 nm DiI-NPs was detected in the spleen. This may be due to the high degree of PEGylation of the 20 nm PPT-NPs, minimizing immune recognition of the particles. Example 8: Maximum tolerated dose for NP
Compounds from Example 3 (E2-4, 6 and 7 compounds, which assemble as 120, 30 and 20 particles) were prepared into particles. The maximum tolerated dose (MTD) of drugs in mice was determined in a dose escalation study, and MTD was identified as the maximum dose of a drug that did not induce animal death or >20% body weight loss. The maximum deliverable dose for an i.v. injection of all 3 PPT-NPs was 180 mg PPT/kg.
Example 9: In Vivo Antitumor Efficacy in an EMT-AR1 and PC3 Res Model
Based on the biodistribution data, the 20 nm particles from Example 3 (E2-7 compound) were selected for efficacy tests. Prior to performing efficacy study in the animal models, the maximum tolerated doses PPT was also determined in a dose escalation study. Native PPT exhibited a steep survival curve. While there was no major weight loss in the animals treated with 20 mg/kg of PPT, 30 mg/kg was found to be lethal. On the other hand, even 180 mg PPT/kg of PPT-NPs was found to be safe without causing any signs of stress or any physiological abnormality in the treated animals. Standard taxane chemotherapies (DTX and CBZ) at their MTDs were included as controls.
Anticancer efficacy of PPT-NPs was analyzed against two MDR tumor models, EMT6/AR1 and PC-3 RES. EMT6 AR1 cells (0.2 χ 106 cells) and PC-3 RES cells (5 χ 106 cells) were s.c. inoculated into the shaved right lateral flank of female BALB/c and male NOD-SCID mice respectively. When tumors reached 4-5 mm in diameter, the BALB/c mice were treated i.v. with either saline (control), MTD dose of PPT (20mg/kg; single dose), MTD dose of DTX (12 mg/kg; day 0, 4 and 8), MTD dose of CBZ (5 mg/kg; day 0, 4 and 8), maximum deliverable dose of PPT-NPs (180mg PPT/kg; single dose) or repeated dose of PPT-NPs (180 mg PPT/kg; day 0, 4 and 8). The NOD-SCID mice were treated the same way as that for the BALB/c mice except that only 2 doses of CBZ (5 mg/kg; day 0 and 4) and DTX (12 mg/kg; day 0 and 4) could be given to the NOD-SCID mice due to significant body weight loss (-20%). Tumor volume and body weight of the treated animals were monitored. PPT-NPs administered as single dose or multiple dose in the EMT6-ARlmodel exhibited significant antitumor efficacy compared to any other treatments (Figure 9). On day 8, with the single dose PPT-NP treatment, the tumor growth inhibition was more than 95% whereas there was 60% tumor growth inhibition with native PPT treatment. Both DTX and CBZ were found to be much less efficacious: tumor growth inhibition was 38% with CBZ and 13% with DTX treatment at day 8 post treatment. At day 11, no significant difference was found between control and PPT, DTX and CBZ treated tumors (p = 0.2) whereas PPT-NPs treated tumors were significantly smaller than control group (p < 0.05). Mice treated with DTX and CBZ showed visible signs of distress including piloerection, lethargy, weight loss, and weakness (Figure 10). On the other hand, the PPT-NPs treated mice displayed no body weight loss or any significant sign of toxicity during the therapy.
We extended the evaluation of antitumor efficacy the 20 nm particles from Example 3 (E2-7 compound) against PC-3 RES model (Figure 11). Similar to the EMT6 AR1 model, PPT-NPs (both multidose and single dose) exhibited significant tumor growth inhibition. Till day 10 post treatment, single dose PPT-NPs showed very similar efficacy compared to multidose PPT-NPs. Both treatments showed more than 70% tumor growth inhibition at day 10 whereas that for CBZ was only 10%. At day 17, multidose PPT-NPs exhibited 90% tumor growth inhibition compared to 65% with single dose PPT-NPs and 50% with CBZ. Within day 17, animals from all other group had to be sacrificed because of large tumors but multidose PPT-NPs significantly controlled this aggressive growth of the tumor for 21 days. Again, PPT-NPs treatment did not cause any body weight loss in the treated animals (Figure 12), whereas as much as 20% weight loss was recorded in the CBZ treated group.
Example 10: Synthesis of polymer with adjusted reaction chemistry and modeling of reaction
In Examples 2 and 3, feeds of PEG and PPT exceeded carboxylic acid content, and accordingly only fully substituted polymers were formed (a linear trend seen in Figure 13A). In work with feeds of PPT and PEG equal or less than the titrated acid content, a maximum of 70% of titrated acid groups are observed to react with PPT and PEG. When feeds exceed 70% acid content, the reaction model (Figures 13B and 13C) fails, as competition effects between PEG and PPT occur. Below saturation feeds, a wider range of composition is accessible.
In a specific embodiment (see Table 4), sub-saturation reactions were carried out to prepare composition analogues: CMC- Ac was conjugated to PEG and PPT via EDC/DMAP coupling chemistry. CMC-Ac (100 mg, 1.60 mmol acid/g) was weighed into a 25 mL glass vial, and dissolved in anhydrous MeCN (3 mL). EDC HC1 (61 mg, 0.32 mmol) and DMAP (39 mg, 0.32 mmol) were added into that solution followed by addition of variable amount of PPT and m-PEG-OH (see Table 4 for feeds). The solutions were stirred for 6h at room temperature with protection from light. The reaction mixtures were precipitated through 50 mL diethyl ether. The precipitate was dried, re-dissolved in MeCN, and the precipitation process was repeated twice. The final precipitates were dried under vacuum, and the fine powders were suspended in water (3x50 mL) and filtered. E10-2 was dialyzed against MilliQ water for 24 h with 3 changes (MW cut-off = 10 kDa), as it was almost soluble. The products were analyzed by LC-MS for uncoupled PEG and PPT, and washing was repeated if residual reagent was detected: less than 0.5 free drug and PEG was detected. The chemical composition of the polymer conjugate was determined by ^-NMR using 2-methyl 5-nitro benzoic acid as an internal standard (Table 4). Particles were prepared from the polymers by the NanoAssemblr technique (Table 4). Conjugates formed defined nanoparticulate structures in aqueous solution (20-160 nm in diameter). Polymer molecular weights were determined by MALS (Table 4) (8.5 kDa - 16.5 kDa).
Table 4: Compositions of PPT-CMCAc-PEG from Examples 10, 12, and 13.
Mol eqv feeds Product Composition
PPT/PEG
PPT PEG PPT PEG ratio
Sample Feed Feed wt% wt% (wt/wt) Size (nm) PDI MW (kDa)
ElO-1 1 - 32.6 - - ppt - 11.1
E10-2 - 1 - 73.9 - Poorly defined - 41.4
E10-3 0.3 0.01 17.4 3 5.800 159.7 0.138 10.1
E10-4 0.3 0.05 15.3 12.1 1.264 54.8 0.063 10.9
E10-5 0.3 0.1 14.4 19.9 0.724 36.6 0.078 12.0
E10-6 0.3 0.15 13.3 25.7 0.518 36.6 0.219 13.4
E10-7 0.3 0.3 11.3 41.3 0.274 23.3 0.126 16.3
E10-8 0.3 0.4 6.8 50.1 0.136 25.3 0.386 16.5
E10-9 0.3 0.5 5.6 49 0.114 88.5 0.554 16.2
ElO-10 0.01 0.05 1.9 12.7 0.150 100.1 0.122 8.5
ElO-11 0.05 0.05 3.9 13.1 0.298 79.6 0.144 9.0
E10-12 0.1 0.05 5.5 10.8 0.509 71.3 0.077 9.0
E10-13 0.5 0.05 21.6 11.5 1.878 48.0 0.078 11.5
E10-14 0.7 0.05 18.7 7.4 2.527 47.5 0.063 11.5
E10-15 0.8 0.05 30.5 8.6 3.547 59.8 0.100 11.8
Ell-1 0.3 0.15 15.3 25.6 0.598 29.4 0.098 15.1
E12-1 0.3 0.15 14.9 15.9 0.937 44.2 0.033 10.6
E13-1 0.3 0.15 17 9 1.889 313.8 0.281 10.6
E13-2 0.3 0.15 12 40.8 0.294 35.7 0.281 17.4
E13-3 0.3 0.15 8 60 0.235 45.1 0.244 30.8
Ppt = precipitate
Example 11: Synthesis of polymer with a 20% DMF solvent
In a specific embodiment (see Table 4, El l-1) a reaction from Example 10 was carried out with 20% DMF in the reaction solvent: CMC-Ac was conjugated to PEG and PPT via EDC/DMAP coupling chemistry. CMC-Ac (100 mg, 1.60 mmol acid/g) was weighed into a 25 mL glass vial, and dissolved in anhydrous MeCN (2.4 mL) and DMF (0.6 mL). EDC HC1 (61 mg, 0.32 mmol) and DMAP (39 mg, 0.32 mmol) were added into that solution followed by addition of PPT (20.4 mg, 0.05 mmol) and m-PEG-OH (47.3 mg, 0.02 mmol). The solution was stirred for 6h at room temperature with protection from light. The reaction mixture was precipitated through 50 mL diethyl ether. The precipitate was dried, re-dissolved in MeCN, and the precipitation process was repeated twice. The final precipitate was dried under vacuum, and the fine powder was suspended in water (3x50 mL) and filtered. The product was analyzed by LC-MS for uncoupled PEG and PPT, and washing was repeated if residual reagent was detected: less than 0.5 free drug and PEG was detected. The chemical composition of the polymer conjugate was determined by ^-NMR using 2-methyl 5-nitro benzoic acid as an internal standard. Particles were prepared from the polymer by the NanoAssemblr technique. The El 1-1 conjugate formed defined nanoparticulate structures in aqueous solution (-30 nm in diameter). Polymer molecular weight was determined by MALS (15.1 kDa). The presence of DMF retards the reaction, which increases cross-linking side reactions.
Example 12: Synthesis of polymer with NHS reaction chemistry
In a specific embodiment, a reaction from Example 10 was performed with NHs coupling reagent added: CMC-Ac was conjugated to PEG and PPT via EDC/NHS/DMAP coupling chemistry. CMC -Ac (100 mg, 1.60 mmol acid/g) was weighed into a 25 mL glass vial, and dissolved in anhydrous MeCN (3 mL). EDC HC1 (61 mg, 0.32 mmol), NHS (37 mg, 0.32 mmol) and DMAP (39 mg, 0.32 mmol) were added into that solution followed by addition of PPT (21.5 mg, 0.05 mmol) and m- PEG-OH (48.5 mg,0.02 mmol) (see Table 4, E12-1). The solution was stirred for 24h at room temperature with protection from light. The reaction mixture was precipitated through 50 mL diethyl ether. The precipitate was dried, re-dissolved in MeCN, and the precipitation process was repeated twice. The final precipitate was dried under vacuum, and the fine powder was suspended in water (3x50 mL) and filtered. The product was analyzed by LC-MS for uncoupled PEG and PPT, and washing was repeated if residual reagent was detected: less than 0.5 free drug and PEG was detected. The chemical composition of the polymer conjugate was determined by ^-NMR using 2-methyl 5- nitro benzoic acid as an internal standard. Particles were prepared from the polymer by the NanoAssemblr technique. The E12-1 conjugate formed nanoparticles (-45 nm in diameter). Polymer molecular weight was determined by MALS (10.6 kDa). The presence of NHS produces a polymer of lower molecular weight when compared to a reaction sans NHS. Example 13: Synthesis of polymer with different MW PEGS
In a specific embodiment, reactions from Example 10 were carried out with PEG MW analogues: CMC-Ac was conjugated to PEG 550, 5000, and 10,000 Da, separately, and PPT via EDC/DMAP coupling chemistry. CMC- Ac (100 mg, 1.60 mmol acid/g) was weighed into a 25 mL glass vial, and dissolved in anhydrous MeCN (3 mL). EDC HC1 (61 mg, 0.32 mmol) and DMAP (39 mg, 0.32 mmol) were added into that solution followed by addition of PPT and m-PEG-OH (see Table 4, 13-1 to 13-3). The solution was stirred for 6h at room temperature with protection from light. The reaction mixture was precipitated through 50 mL diethyl ether. The precipitate was dried, re-dissolved in MeCN, and the precipitation process was repeated twice. The final precipitate was dried under vacuum, and the fine powder was suspended in water (3x50 mL) and filtered. The product was analyzed by LC-MS for uncoupled PEG and PPT, and washing was repeated if residual reagent was detected: less than 0.5 free drug and PEG was detected. The chemical composition of the polymer conjugate was determined by ^-NMR using 2-methyl 5-nitro benzoic acid as an internal standard. Particles were prepared from the polymer by the NanoAssemblr technique. The conjugates formed distinct nanoparticulate structures in aqueous solution (314 - 36 nm in diameter. Polymer molecular weights were determined by MALS (10.6 - 30.8 kDa). The coupling of higher molecular weight PEG produces a polymer of higher molecular weight when compared to a reaction with a lower M W PEG.
Example 14: CMC-Ac MW analogues
CMC-Ac analogues were prepared by varying the temperature (40-50°C), time (l-3h), and amount of sulphuric acid (0.12 to 1.2 mL) in the reactor. These CMC-Ac polymers were characterized by ¾ NMR for composition (% theoretical acetylation) and MW (Table 5, E14-1 to E14-7). These CMC-Ac analogues range from 6200 to 220,000 g/mol. In general, when reaction time was less than 2 hours, when temperature was less than 45°C, and when acid volume was less that 0.6 mL, the recovery of polymer declined below 65 wt% (average yield in a standard 50°C, reaction with 1.2 mL acid is 65-70%), and acetylation of glucose hydroxyls declined below 100% of theoretical. The titration of the CMC-Ac analogues indicates that acid content varied from 1.76 to 2.20 mmol acid/gram polymer (45-56% of CMC-Na acid content), which indicated that this parameter is not sensitive to reaction conditions.
Table 5: MW Analogues of CMC- Ac
Figure imgf000035_0001
In a specific embodiment (see Table 6, E14-8 and 9), PPT and PEG conjugates were prepared by the same method in Example 10, using the E14-6 and E14-7 CMC-Ac MW analogues described above. CMC-Ac was conjugated to PEG and PPT via EDC/DMAP coupling chemistry. CMC -Ac (100 mg, 1.92 and 2.20 mmol acid/g) was weighed into a 25 mL glass vial, and dissolved in anhydrous MeCN (3 mL). EDC HC1 (74 mg, 0.39 mmol and, 84 mg, 0.44 mmol) and DMAP (47 mg, 0.39 mmol and, 54 mg, 0.44 mmol) were added into that solution followed by addition of PPT and m-PEG- OH (see Table 6). The solution was stirred for 6h at room temperature with protection from light. The reaction mixture was precipitated through 50 mL diethyl ether. The precipitate was dried, re-dissolved in MeCN, and the precipitation process was repeated twice. The final precipitate was dried under vacuum, and the fine powder was suspended in water (3x50 mL) and filtered. The product was analyzed by LC-MS for uncoupled PEG and PPT, and washing was repeated if residual reagent was detected: less than 0.5 free drug and PEG was detected. The chemical composition of the polymer conjugate was determined by ^-NMR using 2-methyl 5-nitro benzoic acid as an internal standard. Particles were prepared from the polymer by the NanoAssemblr technique. The conjugates formed distinct nanoparticulate structures in aqueous solution (27 - 47 nm in diameter. The E14-1 CMC-Ac analogue (50°C, 3 h reaction with 1.2 mL acid, used to prepare Example 10 compounds) is preferred along with the E14-6 CMCAc analogue (and all MW in between). Compounds produced with CMCAc MW analogues up to the E14-6 compound are readily processed and fully soluble in all phases of reaction and processing. The El 4-7 CMCAc (used to prepare El 4-9 polymer) required filtration to remove a sub-population of poorly defined materials, and represents a less preferred composition.
Table 6: PPT-CMCAc-PEG polymers produced with MW variants of CMCAc.
Mol eqv feeds Product Composition
PPT PPT PEG PPT/PEG ratio
Sample Feed PEG Feed wt% wt% (wt/wt) Size (nm) PDI
E14-8 0.3 0.15 15 26.8 0.560 27.1 0.070
E14-9 0.3 0.15 14.2 30.1 0.472 46.6* 0.216
*a sub-population of micron sized particles form, and are filtered off.
In summary, we demonstrated that by varying the PPT/PEG ratio in the CMC-Ac conjugates, NPs with different physicochemical properties (size, drug release kinetics, cell killing potency, biodistribution) could be produced, and an ultra-small NP formulation (E2-8) with a mean diameter of 20 nm was optimized for highly tumor- selective delivery of PPT. With this technology, PPT could be transformed into an effective and safe therapy for treating MDR tumors.
Although preferred embodiments of the invention have been described herein, it will be understood by those skilled in the art that variations may be made thereto without departing from the spirit of the invention or the scope of the appended claims. All references mentioned herein, including in the following list of references, are incorporated by reference in their entirety. Reference List
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Claims

CLAIMS;
1. A compound comprising an acetylated carboxymethylcellulose (CMC-Ac) covalently linked to: at least one poly(ethylene glycol) (PEG), and at least one hydrophobic drug comprising podophyllotoxin or derivatives thereof, or pharmaceutically acceptable salts, solvates, esters or prodrugs of any of the foregoing.
2. The compound of claim 1, wherein the covalent linkages are ester linkages.
3. The compound of claim 1 or 2, wherein the hydrophobic drug is podophyllotoxin or pharmaceutically acceptable salts, solvates, esters or prodrugs of any of the foregoing.
4. The compound of claim 3, wherein the hydrophobic drug is podophyllotoxin.
5. The compound of claim 1 or 2, wherein the hydrophobic drug is selected from the group consisting of etoposide, taniposide, Adva-27a, and NK611r, or pharmaceutically acceptable salts, solvates, esters or prodrugs of any of the foregoing.
6. The compound of claim 5, wherein the hydrophobic drug is:
Figure imgf000042_0001
7. The compound of any one of claims 3-6, wherein the at least one PEG is poly(ethylene glycol) methyl ether (mPEG) having an average Mn of between about 550 and about 10,000, preferably about 2000.
8. The compound of claim 7, wherein the CMC consist of a polymer with a molecular weight of between 4,400 and 220,000 Daltons.
9. The compound of any one of claims 1-8, wherein the wt% of PEG in the polymer is 3 - 59.7 wt%, preferably 10.8-43.0 wt%.
10. The compound of any one of claims 1-9, wherein the wt% of drug is about 1.9- 38 wt %, preferably 3.9-30.5 wt %, further preferably about 12 wt%.
11. The compound of any one of claims 1-10, wherein the drug/PEG wt ratio is between about 0.136 to about 5.800, preferably 0.191-3.547, further preferably 0.4.
12. A self-assembling nanoparticle composition comprising the compound of any one of claims 1-11.
13. The self-assembling nanoparticle composition of claim 12, wherein the nanoparticles have an average diameter of about 20-160 nm, preferably 20-100 nm, and further preferably around 20 nm.
14. The self-assembling nanoparticle composition of any one of claims 12 and 13, further comprising at least one hydrophobic agent encapsulated therein, preferably selected from either an imaging agent or a therapeutic agent.
15. A pharmaceutical composition comprising the self-assembling nanoparticle composition of any one of claims 12-15 and a pharmaceutically acceptable carrier and/or diluent.
16. A method of treating cancer in a patient in need thereof, comprising administering to said patient an effective amount of a self-assembling nanoparticle composition comprising the compound of any one of claims 1-11.
17. The method as defined in claim 16, wherein the cancer is a multidrug resistant cancer.
18. A process for preparing a self-assembling nanoparticle composition comprising: a) covalently linking at least one PEG and at least one hydrophobic drug comprising a podophyllotoxin or derivatives thereof, or pharmaceutically acceptable salts, solvates, esters or prodrugs of any of the foregoing, to a CMC-Ac; b) isolating the product of step (a); c) dissolving the isolated product of step (b) in a suitable organic solvent, preferably DMF or DMSO and further preferably THF or acetonitrile, to form a solution; d) precipitating the solution of step (c); e) resuspending the precipitate of step (d) in an an aqueous solution, and preferably dializing the aqueous solution.
19. The process of claim 18, further comprising repeating steps (c) and (d) one or more times before step (e)
20. A compound represented by the formula:
Figure imgf000044_0001
R2(i)...R2(n), R3(i)...R3(n), and Rend are each independently
Figure imgf000044_0002
wherein -0(HD) represents a hydrophobic drug comprising podophyllotoxin or derivatives thereof, or pharmaceutically acceptable salts, solvates, esters or prodrugs of any of the foregoing, having a point of attachment via a hydroxyl group; or
O
(d) O(PEG); and n is an integer.
21. The compound of claim 22, wherein the PEG is m(PEG).
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