Synthesis and characterization of polymer-drug conjugates by click chemistry reactions

Abstract

A method of preparing polymer-drug conjugates (PDCs) including reacting a drug-bearing compound such as monomer / oligomer / polymer terminated at both ends by "clickable" functional groups with other monomer / oligomer / polymer bearing complementary "clickable" functional group using suitable ratios of each monomer to form PDCs by click chemistry such as strain-promoted [3+2] azide-alkyne cycloaddition (SPAAC)-mediated step-growth polymerization, and PDCs prepared therefrom. Also disclosed is a method for treating cancers using the PDCs.

Description

SYNTHESIS AND CHARACTERIZATION OF POLYMER-DRUG CONJUGATES BY CLICK CHEMISTRY REACTIONS TECHNICAL FIELD

[0001] The present disclosure relates to synthesis of polymer-drug conjugates (PDCs) using click chemistry such as strain-promoted [3+2] azide-alkyne cycloaddition (SPAAC)- mediated step-growth polymerization and characterizations of PDCs, and use of PDCs for treatment of cancers such as ovarian cancer (OC). BACKGROUND OF THE DISCLOSURE

[0002] A major problem of traditional cancer chemotherapy is the lack of selective toxicity resulting in unwanted systemic toxicity and serious adverse effects (Adesina, et al, “Polylactide-based paclitaxel-loaded nanoparticles fabricated by dispersion polymerization: characterization, evaluation in cancer cell lines, and preliminary biodistribution studies”, Journal of Pharmaceutical Sciences, 2014, 103(8), 2546-2555; Ponziani, et al, “Antibody-drug conjugates: the new frontier of chemotherapy”, International Journal of Molecular Sciences, 2020, 21(15), 5510).

[0003] Ovarian cancer (OC) has the highest fatality rate among all gynecological cancers globally. Most patients have advanced OC at the time of diagnosis due to a lack of routine screening tests and non-specific symptoms that are difficult to identify from less significant abdominal symptoms, making treatment very challenging. Combination chemotherapy is the mainstay for the treatment of advanced OC, with the first line being the 3-weekly 24-hour cisplatin / paclitaxel intravenous co-infusion over six cycles.

[0004] Chemotherapeutic drugs are effective against cancer cells, but they also kill healthy cells, limiting the maximum dose that can be safely administered to an individual patient (Larson, et al, “Biodegradable multiblock poly (N-2-hydroxypropyl) methacrylamide gemcitabine and paclitaxel conjugates for ovarian cancer cell combination treatment”, International Journal of Pharmaceutics, 2013, 454(1), 435-443). Effective strategies are thus needed to maximize the therapeutic effects and minimize the non-specific toxicity of existing chemotherapeutic agents.

[0005] Drug delivery approaches that have the potential to improve the efficacy and decrease the toxicity of current chemotherapeutic drugs are, therefore, required. One such approach is the delivery of chemotherapeutic agents as polymer-drug conjugates (PDCs). PDCs are drug delivery systems where active pharmaceutical agents are covalently attached to polymeric chains through stimuli-sensitive linkers (Javia, et al, “Polymer-drug conjugates: Design principles, emerging synthetic strategies and clinical overview”, International Journal of Pharmaceutics, 2022, 121863; Wang, et al, “Rethinking nanoparticulate polymer–drug conjugates for cancer theranostics”, Wiley Interdisciplinary Reviews: Nanomedicine and Nanobiotechnology, 2022, e1828; Ogundipe, et al, “Targeted drug conjugate systems for ovarian cancer chemotherapy”, Biomedicine & Pharmacotherapy, 2023, 165, 115151).

[0006] PDCs offer opportunities such as high drug loading (Feng, et al, “Anticancer nanoparticulate polymer‐drug conjugate”, Bioengineering & translational medicine, 2016, 1(3), 277-296) and modification of the pharmacokinetics of the conjugated drugs to prevent unwanted distribution to healthy tissues. Based on their macromolecular size, PDCs can preferentially accumulate in cancer tissues as a result of the tumor’s leaky vasculature and impaired lymphaticdrainage via the enhanced permeability and retention (EPR) effect (Ejigah, et al, “Approaches to improve macromolecule and nanoparticle accumulation in the tumor microenvironment by the enhanced permeability and retention effect”, Polymers, 2022, 14(13), 2601). This phenomenon is called the enhanced permeability and retention (EPR) effect and is the basis for passive targeting of cancer.

[0007] Additionally, the site-specific expression or overexpression of certain enzymes in cancers with negligible or no expression in healthy tissues increases the targeting specificity of PDCs. A common example of such enzymes is cathepsin B, a lysosomal enzyme that is highly overexpressed and secreted in many solid tumors (Zhang, et al, “Overexpression of cysteine cathepsin L is a marker of invasion and metastasis in ovarian cancer”, Oncology Reports, 2014, 31(3), 1334–1342; Pechar, et al, “Polymer nanomedicines with enzymatically triggered activation: A comparative study of in vitro and in vivo anti-cancer efficacy related to the spacer structure”, Nanomedicine: Nanotechnology, Biology and Medicine, 2022, 46, 102597). Different types of cathepsins, such as cathepsin A, B, C, F, D, and L, perform many functions such as proteolytic degradation of cellular proteins, intracellular housekeeping, and apoptotic signal transduction, in cells (Dheer, et al, “Cathepsin-sensitive nanoscale drug delivery systems for cancer therapy and other diseases”, Advanced Drug Delivery Reviews, 2019, 151, 130–151; Sun, et al, “Recent trends in in situ enzyme-activatable prodrugs for targeted cancer therapy”, Bioconjugate Chemistry, 2020, 31(4), 1012-1024). Specific cathepsins (such as cathepsins B and L) are often upregulated in various cancers and have been implicated in cancer progression and metastasis. Dipeptides (such as valine-citrulline and valine-alanine) and tetrapeptides (such as glycine-phenylalanine-leucine-glycine and glycine-glycine-leucine-glycine) have served ascathepsin B-cleavable linkers in many cancer-targeted drug conjugate systems (Díaz-Rodríguez, et al, “Novel ADCs and strategies to overcome resistance to anti-HER2 ADCs”, Cancers, 2021, 14(1), 154; Balamkundu, et al, “Lysosomal-Cleavable Peptide Linkers in Antibody–Drug Conjugates”, Biomedicines, 2023, 11(11), 3080).

[0008] Despite these efforts, no polymer-drug conjugate is yet approved for cancer treatment. Major factors that limit the clinical translation of PDCs include polymer safety and toxicity, short plasma circulation, structure complexity, and batch-to-batch synthetic reproducibility. Ideal PDCs should have high drug loading, circulate in the blood to sufficiently accumulate in the tumor tissue by the EPR effect, be internalized by the tumor cells, and efficiently release the drug. Also, the polymer carrier must be fully eliminated from the body after drug release.

[0009] To achieve this in the synthesis of drug-coupled polymer conjugates, click- chemistry has been used, which offers the advantage of easy and fast synthesis of polymers with more precise control over the architecture and functional groups.

[0010] Click chemistry refers to certain reactions that are characterized by their rapidity, versatility, high product yields, high specificity, easy synthesis, and easy isolation of the synthesized product. There are typically no byproducts produced by most click reactions, which involve carbon-heteroatom bond (mostly N, O, and S) formation (Finn, et al, “Click chemistry: function follows form”, Chemical Society Reviews, 2010, 39(4), 1231-1232). SUMMARY OF THE DISCLOSURE

[0011] The present inventors have conducted an extensive research and have discovered novel method of synthesis of PDCs using SPAAC-mediated step-growth polymerization.SPAAC-mediated polymerization enables an easy and rapid synthesis of polymers with more precise control over the architecture and functional groups under mild conditions and without a catalyst or initiator. SPAAC is a highly versatile copper-free click reaction that takes place as a result of a fast, spontaneous, and highly specific reaction between azides and cycloalkynes such as bicyclononyne (BCN), dibenzocyclooctyne, and dibenzoazacyclooctyne (DBCO) (Dommerholt, et al, “Strain-promoted 1, 3-dipolar cycloaddition of cycloalkynes and organic azides”, Cycloadditions in Bioorthogonal Chemistry, 2016, 57-76). It is also amenable to a variety of functionalized alkyne- and azide-based monomers (Billiet, et al, “Step-growth polymerization and ‘click’ chemistry”, The oldest polymers rejuvenated. Polymer, 2009, 50(16), 3877-3886).

[0012] Polymer-drug conjugates (PDCs) modify the biodistribution of small-molecule anticancer agents to prevent undesired off-target adverse effects. The advantages of the approach compared to other reported methods for the synthesis of PDCs for anti-cancer drug delivery include modularity, absence of an initiator or catalyst, rapid progression under mild conditions, the ease of synthesis of the monomers, being amenable to different spacers and drugs, and the use of highly specific copper-free click chemistry. Another advantage of the approach in addition to ease of synthesis, modularity, rapid progression under mild temperature, and absence of an initiator or catalyst, is high drug loading.

[0013] The present disclosure includes the following exemplary embodiments but not limited thereto.

[0014] [1] A method of preparing a polymer-drug conjugate (PDC) comprising reacting a drug-bearing compound terminated at both ends by a functional group with other compoundbearing a complementary functional group using suitable ratios of each compound to form the PDC by click chemistry.

[0015] [2] The method according to [1], wherein the PDC is prepared by strain- promoted [3+2] azide-alkyne cycloaddition (SPAAC)-mediated step-growth polymerization.

[0016] [3] The method according to [1], wherein the prepared PDC has a molecular weight of 10 kDa – 2,000 kDa.

[0017] [4] The method according to [1], wherein the prepared PDC has a molecular weight greater than 40 kDa.

[0018] [5] The method according to [1], wherein the prepared PDC has a drug loading ranging from 0.25% - 65% by weight of the PDC for each drug in single drug PDC or the combination drug PDC.

[0019] [6] The method according to [1], wherein the drug is used for disease management or treatment of disease such as an anticancer drug or combination of anticancer agents.

[0020] [7] The method according to [1], wherein the drug-bearing compound and the other compound, which may be drug-bearing or non-drug bearing, are each independently a small molecule, monomer, an oligomer, or a polymer.

[0021] [8] The method according to [1], wherein the functional group and the complementary functional group are each independently selected from the group consisting of azide, thiol, DBCO, BCN, maleimide, tetrazine, trans cyclooctene (TCO), and strained or linear alkynes or any other complementary click-chemistry reaction specific combination.

[0022] [9] The method according to [1], wherein each terminal of the drug bearing compound bears the same or different functional group.

[0023]

[0010] The method according to [1], wherein each terminal of the other compound bears the same or different functional group.

[0024]

[0011] The method according to [1], wherein single or multiple drugs are coupled to a compound via physical or chemical means or reactions.

[0025]

[0012] The method according to [1], wherein the drug-bearing compound is (azidoPEG5)2-EDTA-(GFLG-Drug)2, and the other compound is DBCO-PEG6-DBCO.

[0026]

[0013] The method according to [1], comprising the reaction shown in the following scheme 1:Scheme 1.

[0027]

[0014] The method according to [1], comprising the reaction shown in the following scheme 2:Scheme 2.

[0028]

[0015] A polymer-drug conjugate (PDC) prepared by the method of [1].

[0029]

[0016] A method of treating cancer, comprising administering an effective amount of the polymer-drug conjugate (PDC) or a mixture of effective amounts of PDCs according to

[0015] to a subject in need thereof.

[0030]

[0017] The method according to

[0016] , wherein the cancer is a solid cancer such as colorectal, bladder, endometrial, kidney, liver, prostate, breast, ovarian, brain, lung, skin, pancreatic, and head and neck cancers. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] The application file contains at least one drawing executed in color. Copies of this patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.

[0032] Any figures contained herein are provided only by way of example and not by way of limitation.

[0033] Fig. 1: Plots of the change in refractive index (dn) versus the change in concentration (dc) used for dn / dc determination.

[0034] Fig. 2: Gemcitabine HCl calibration curve based on its UV-Vis absorbance intensity at 268 nm.

[0035] Fig. 3: Doxorubicin HCl calibration curve on the emission intensity at 595 nm (470 nm excitation).

[0036] Fig. 4: Overlaid FT-IR spectra of DBCO-PEG6-DBCO, (azidoPEG5)2-EDTA- (GFLG-Gem)2(3), and p-Gem.

[0037] Fig. 5: Overlaid FT-IR spectra of DBCO-PEG6-DBCO, (azidoPEG5)2-EDTA- (GFLG-Dox)2(4), and p-Dox.

[0038] Fig. 6A: SEC chromatograms of the polymer conjugates of gemcitabine showing the light scattering (continuous green line) and differential refractive index (dotted blue line) following SEC with MALS and RI detection.

[0039] Fig. 6B: SEC chromatograms of the polymer conjugates of doxorubicin showing the light scattering (continuous green line) and differential refractive index (dotted blue line) following SEC with MALS and RI detection.

[0040] Fig. 7: Percent drug loading (% Wt.) in the polymer-drug conjugates as determined by UV-vis (for gemcitabine) and fluorescence spectroscopy (for doxorubicin) (n = 3).

[0041] Fig. 8A: Cathepsin B-catalyzed release of gemcitabine (Gem) from the gemcitabine-coupled polymer (p-Gem) in the presence (blue line) and absence (red line) of exogenous cathepsin B at pH 5.0.

[0042] Fig. 8B: Cathepsin B-catalyzed release of a doxorubicin fragment from the doxorubicin-coupled polymer (p-Dox) in the presence (blue line) and absence (red line) of exogenous cathepsin B at pH 5.0.

[0043] Fig. 9: Overlaid FT-IR spectra of DBCO-PEG6-DBCO, (azidoPEG5)2-EDTA- (GFLG-Gem)2(3), (azidoPEG5)2-EDTA-(GFLG-Dox)2(4) and p-Gem / Dox.

[0044] Fig. 10: SEC chromatogram of p-Gem / Dox showing the light scattering (LS, green line), differential refractive index (dRI, blue line), and molecular weight distribution (red).

[0045] Fig. 11: Cathepsin B-catalyzed release of gemcitabine and a doxorubicin fragment from p-Gem / Dox in the presence (blue and red lines, respectively) and absence (green and purple lines, respectively) of exogenous cathepsin B at pH 5.0.

[0046] Fig. 12. Hydrolytic release of gemcitabine and doxorubicin from p-Gem / Dox in PBS (pH 7.4).

[0047] Fig. 13. Percent viability of OVCAR-3 cells at 72 h post-treatment with p-Dox and DOX at different concentrations. Controls represent cell culture media and DMSO in media. Data represent mean ± SD (n = 4).

[0048] Fig. 14: Percent viability of OVCAR-3 cells at 72 h post-treatment with p-Gem and GEM at different concentrations. Controls include cell culture media and DMSO in media. Data represent mean ± SD (n = 4).

[0049] Fig. 15: Percent viability of OVCAR-3 cells at 72 h post-treatment with p-Dox + p-Gem admixture (50:50) and GEM + DOX admixture (50:50) at different concentrations. Controls represent cell culture media and DMSO in media. Data represent mean ± SD (n = 4).

[0050] Fig. 16: Percent viability of OVCAR-3 cells at 72 h post-treatment with p- Gem / Dox and GEM + DOX admixture (36.6:7) at different concentrations. Controls represent cell culture media and DMSO in media. Data represent mean ± SD (n = 4).

[0051] Fig. 17: Combination index (CI) analysis of GEM / DOX combinations at ratios 50:50 and 36.6:7, respectively for GEM and DOX. DETAILED DESCRIPTION OF THE DISCLOSURE

[0052] The method and polymer-drug conjugates (PDCs) of the present disclosure are specifically described as follows and in reference to the drawings. However, the present invention should not be construed as being limited thereto.

[0053] The advantages of SPAAC-mediated step-growth polymerization compared to other methods of PDC synthesis include modularity, absence of an initiator or catalyst, rapid progression under mild conditions, the ease of synthesis of the monomers, being amenable to different / multiple spacers and drugs, and the use of highly specific copper-free click chemistry.There has been no report of the preparation of flexible, linear polymer-drug conjugate using SPAAC. The present inventors are the first to prepare first generation PDCs using SPAAC.

[0054] The novel design of the PDCs may facilitate selective delivery and passive targeting of cancer by the EPR effect and active targeting of cathepsin B enzyme overexpressed selectively in many solid cancers, namely carcinomas, melanomas and sarcomas. Examples of solid cancers include colorectal, bladder, endometrial, kidney, liver, prostate, breast, ovarian, brain, lung, skin, pancreatic, and head and neck cancers. The prepared PDCs generally have high molecular weights from 10 kDa – 2000 kDa, from 20 kDa – 1900 kDa, or from 30 kDa – 1800 kDa, and specifically molecular weights greater than 40 kDa (renal filtration threshold) such as 40 kDa – 1800 kDa, 50 kDa – 1700 kDa, 60 kDa -1600 kDa, 70 kDa – 1500 kDa, or 80 kDa – 1400 kDa, etc. The prepared PDCs also have high percent drug loadings ranging from 0.25% - 65%, from 0.5% - 60%, from 1% - 55%, from 2% - 50%, from 3% - 45%, from 4% - 45%, from 5% - 40%, from 6% - 38%, or from 7% – 36.6% by weight of the prepared PDCs for each drug, when compared to other known approaches. Thus, they are suitable for passive targeting of cancer by the EPR effect and selective release of higher loads of cytotoxic drugs in the tumor microenvironment. The PDCs also showed cathepsin B-catalyzed cleavage and drug release at acidic – neutral pH values, and no drug release in the absence of cathepsin B within the period evaluated (24h).

[0055] The method disclosed herein permits the coupling of single or several drugs and / or diagnostic agents via a simple reproducible approach yielding PDCs with high molecular weights and high drug or diagnostic agent content. The possibility of coupling diagnostic agents such as dyes or other therapeutic agents make the method versatile and useful not only fortherapeutic purposes, but for diagnostic or theranostic purposes. Advantages of the disclosed method include ease of synthesis, modularity, rapid progression at room temperature or under mild temperature, absence of an initiator or catalyst, and high drug or diagnostic agent loading.

[0056] Using the disclosed method, first generation PDCs are designed to have high molecular weights and capable of passive targeting to tumors via the enhanced permeability and retention effect. They are also by design developed to be flexible and hydrophilic through the use of water-soluble, polyethylene glycol spacers. These attributes enable the PDCs to extravasate and accumulate in primary tumors and at metastatic sites. In addition, after selective drug release or activation in the tumor environment via tumor- or other stimuli-sensitive linkers, the molecular weight of the PDC is expected to fall below the renal threshold thereby facilitating renal excretion preventing accumulation in the body which otherwise may lead to toxicities or other adverse effects.

[0057] The disclosed method involves the synthesis of drug-bearing compounds such as monomers / oligomers / polymers terminated at both ends by “clickable” functional groups such as azide, thiol, DBCO, BCN, maleimide, tetrazine, trans cyclooctene (TCO), and strained or linear alkynes or any other complementary click-chemistry reaction specific combination. Each terminal of the compounds such as monomer / oligomer / polymer may also bear a different “clickable” functional group. All the compounds may be drug-bearing or only one of the compounds may bear drug(s). Single or multiple drugs may be coupled to a compound via physical or chemical means such as covalent bonds.

[0058] To prepare the PDC, the drug-bearing compounds such as monomers / oligomers / polymers terminated at both ends by “clickable” functional groups arereacted with other monomers / oligomers / polymers bearing complementary “clickable” functional groups using suitable ratios of each monomer to form PDCs via click chemistry. As non-limiting examples, complementary “clickable” pairs include azide-alkyne and trans-cyclooctene (TCO)- tetrazine (Tz). Additionally, non-limiting examples of click chemistry include the Staudinger ligation, copper catalyzed azide-alkyne cycloaddition (CuAAC), strain-promoted [3 + 2] azide– alkyne cycloaddition (SPAAC) and inverse-electron demand Diels-Alder (IEDDA) reaction.

[0059] As a general example, PDCs bearing a single drug may be prepared using the disclosed method, as shown in the following Scheme 1.Scheme 1. Single drug PDC synthesis from drug-bearing monomer and non-drug bearing monomer by click chemistry

[0060] Any anticancer drug or combination of drugs with reactive functional groups may be coupled to the PDC. Such drugs include doxorubicin, emetine, gemcitabine, brusatol, SN-38 used as single agents to develop single-drug PDCs or used in different combinations to develop combination PDCs suitable for the treatment of different cancers or disease state etc. The drugrelease mechanisms may be cleavable linker peptides such as Glycine-Phenylalanine-Leucine- Glycine, Valine-Alanine, Valine-Citrulline, Histidine-Serine-Serine-Lysine-Leucine-Glutamine or non-cleavable peptides. Other drug release mechanisms include pH sensitive linkers and pH sensitive chemical bonds, and other stimuli-sensitive linkers. Monomers / oligomers / polymers may be any compounds that have multiple reactive functional groups such as citric acid, or EDTA dianhydride or the monomers may be custom-synthesized to have specific functional groups such as (azidoPEG5)2-EDTA-(GFLG-Drug)2including (azidoPEG5)2-EDTA-(GFLG- Dox)2and (azidoPEG5)2-EDTA-(GFLG-Gem)2capable of further polymerization to form polymers such as PDCs..Examples of drugs include gemcitabine, doxorubicin, daunorubicin, emetine, SN-38, brusatol or any cytotoxic, therapeutic or chemotherapeutic agent. Instead of drugs, diagnostic agents or a combination of therapeutic or diagnostic agents may be used. The custom synthesized monomers can be designed to have different chemical attributes and may have small molecular weights or higher molecular weights. The monomers / oligomers / polymers may be designed to be hydrophilic or hydrophobic. The reaction conditions include all modalities / types of click reactions / click chemistry such as but not limited to SPAAC, copper- catalyzed click reactions, IEDDA, thiol-maleimide reactions etc.

[0061] As a specific, non-limiting example, DBCO-PEG6-DBCO (non-drug bearing monomer) and a gemcitabine-bearing monomer, (azidoPEG5)2-EDTA-(GFLG-Gem)2are polymerized via click chemistry (SPAAC), as discussed in detail in the Examples.

[0062] As another specific, non-limiting example, DBCO-PEG6-DBCO (non-drug bearing monomer) and a doxorubicin-bearing monomer, (azidoPEG5)2-EDTA-(GFLG-Dox)2are polymerized via click chemistry (SPAAC), as discussed in detail in the Examples.

[0063] Similarly, as a general example, PDCs bearing combination drugs may be prepared using the disclosed method, as shown in the following Scheme 2.Scheme 2. Combination drug PDC synthesis from different drug-bearing monomers and non-drug bearing monomer by click chemistry.

[0064] Any anticancer drug or combination of drugs with reactive functional groups may be coupled to the PDC. Such drugs include doxorubicin, emetine, gemcitabine, brusatol, SN-38 used as single agents to develop single-drug PDCs or used in different combinations to develop combination PDCs suitable for the treatment of different cancers or disease state etc. The drug release mechanisms may be cleavable linker peptides such as Glycine-Phenylalanine-Leucine- Glycine, Valine-Alanine, Valine-Citrulline, Histidine-Serine-Serine-Lysine-Leucine-Glutamine or non-cleavable peptides. Other drug release mechanisms include pH sensitive linkers and pH sensitive chemical bonds, and other stimuli-sensitive linkers. Monomers / oligomers / polymers may be any compounds that have multiple reactive functional groups such as citric acid, or EDTA dianhydride or the monomers may be custom-synthesized to have specific functional groups such as (azidoPEG5)2-EDTA-(GFLG-Drug)2including (azidoPEG5)2-EDTA-(GFLG- Dox)2and (azidoPEG5)2-EDTA-(GFLG-Gem)2capable of further polymerization to form polymers such as PDCs. Examples of drugs include gemcitabine, doxorubicin, daunorubicin, emetine, SN-38, brusatol or any cytotoxic, therapeutic or chemotherapeutic agent. Instead of drugs, diagnostic agents or a combination of therapeutic or diagnostic agents may be used. The custom synthesized monomers can be designed to have different chemical attributes and may have small molecular weights or higher molecular weights. The monomers / oligomers / polymers may be designed to be hydrophilic or hydrophobic. The reaction conditions include all modalities / types of click reactions / click chemistry such as but not limited to SPAAC, copper- catalyzed click reactions, IEDDA, thiol-maleimide reactions etc.

[0065] As a specific, non-limiting example, DBCO-PEG6-DBCO (non-drug bearing monomer) and a gemcitabine-bearing monomer (azidoPEG5)2-EDTA-(GFLG-Gem)2, and adoxorubicin-bearing monomer (azidoPEG5)2-EDTA-(GFLG-Dox)2, are polymerized via click chemistry (SPAAC) to prepare PDC, as discussed in detail in the Examples.

[0066] The use of SPAAC-mediated polymerization for the syntheses of tumor-targeted PDCs of gemcitabine and doxorubicin as model drugs will facilitate the accumulation of these potent anticancer agents in tumors leading to greater cytotoxicity in the tumor compared with free drugs and reduced toxicity to healthy cells.

[0067] Click chemistry reactions such as the SPAAC-mediated step-growth polymerization do not require the use of a catalyst or high temperatures and it allows the rapid synthesis of high molecular-weight drug-loaded PDCs with Mw of from 10 kDa – 2,000 kDa, from 20 kDa – 1900 kDz, or from 30 kDa – 1800 kDa, and specifically molecular weights greater than 40 kDa (renal filtration threshold) such as 40 kDa – 1800 kDa, 50 kDa – 1700 kDa, 60 kDa -1600 kDa, 70 kDa – 1500 kDa, or 80 kDa – 1400 kDa, etc. The prepared PDCs also have high percent drug loadings ranging from 0.25% - 65%, from 0.5% - 60%, from 1% - 55%, from 2% - 50%, from 3% - 45%, from 4% - 45%, from 5% - 40%, from 6% - 38%, or from 7% – 36.6% by weight of each drug with respect to the prepared PDCs. For example, high molecular- weight PDCs may be prepared containing gemcitabine (Mw ~ 40.18 kDa) and doxorubicin (Mw ~ 1800 kDa) with suitable molecular weight distribution and high drug loading of 29.2 %wt. gemcitabine and 10.3 %wt. doxorubicin.

[0068] α-ω-Bis-azide-terminated bifunctional gemcitabine-coupled and doxorubicin- coupled monomers, with drug linkage via Gly-Phe-Leu-Gly (GFLG), a cathepsin B-sensitive peptide linker, may be separately synthesized and polymerized using a dibenzoazacyclooctyne bifunctional polyethylene glycol monomer. For example, α-ω-bis-azide-terminated bifunctionalgemcitabine-coupled monomer and α-ω-bis-azide-terminated bifunctional doxorubicin-coupled monomer may be separately polymerized with homo-bifunctional DBCO-PEG6-DBCO monomer to generate high molecular weight PDCs of gemcitabine and doxorubicin, respectively, via copper-free click chemistry. The α-ω-bis-azide-terminated bifunctional drug-coupled monomers were prepared from a linear hydrophilic EDTA derivative via simple synthetic steps and copolymerized with a DBCO-PEG6-DBCO homo-bifunctional monomer using SPAAC- mediated step-growth polymerization. DBCO reacts with azide groups in a highly specific copper-free click chemistry, thus, the resulting PDCs were easily isolated following solvent removal by rotary evaporation with no by-product. In addition, the SPAAC-mediated step- growth polymerization method of synthesis of the PDCs did not require the use of high temperature, initiator, or catalyst. The PDCs demonstrate cathepsin B-catalyzed release of the drugs in vitro at acidic to neutral pH, such as pH 5.0 and pH 6.0. The applied method for the syntheses of the PDCs enables the selective delivery of potent anticancer agents.

[0069] Glycine-phenylalanine-leucine-glycine (GFLG) is advantageous over cathepsin B dipeptide substrates because the use of oligopeptide linkers minimizes steric hindrance effects that may obstruct the formation of enzyme-substrate complexes. GFLG is more specific for cathepsin B and is stable in the plasma.

[0070] The present disclosure also provides the synthesis of gemcitabine / doxorubicin- combination polymer-drug conjugate (p-Gem / Dox) by the SPAAC-mediated step-growth polymerization. The dual delivery of both drugs on a single polymer backbone would result in the passive targeting of a high concentration of drugs to OC via the EPR effect leading toincreased antitumor activity, reduction in adverse effects, and lower the likelihood of chemoresistance development.

[0071] EXAMPLES Hereinafter, Examples of the present disclosure will be specifically described. Note that the present invention is not limited to these Examples.

[0072] I. PREPARATION OF SINGLE OR COMBINATION POLYMER- DRUG CONJUGATES (PDCS) USING CLICK CHEMISTRY

[0073] Materials and Method Materials Dimethyl sulfoxide (DMSO), N, N′-diisopropylethylamine (DIPEA), acetonitrile, trifluoroacetic acid (TFA), dichloromethane (DCM), methanol, N, N′-dimethylformamide (DMF), ethylenediaminetetraacetic acid (EDTA) dianhydride, 4-ethyl morpholine, isobutyl 1,2- dihydro-2-isobutoxy-1-quinolinecarboxylate (IIDQ), lithium chloride (LiCl), sodium phosphate monobasic monohydrate, EDTA disodium salt dihydrate, sodium hydroxide pellets, and 1,4- dithiothreitol (DTT) were purchased from Sigma-Aldrich (Burlington, MA, USA). Piperidine, trifluoro acetic acid (TFA), Fmoc-L-leucine, Fmoc-L-phenylalanine, Fmoc-L-glycine, and hexafluorophosphate azabenzotriazole tetramethyl uronium (HATU) were purchased from Chem-Impex International (Wood Dale, IL, USA). Azido-PEG5-amine was purchased from BroadPharm (San Diego, CA, USA). Glycyl-2-chloro-trityl resin was purchased from AAPPTEC (Louisville, KY, USA). Dibenzoazacyclooctyne-hexa (ethylene glycol)-dibenzoazacyclooctyne (DBCO-PEG6-DBCO) was purchased from AxisPharm (San Diego, CA, USA). Purified native human cathepsin B was purchased from Athens Research and Technology (Athens, GA, USA).Gemcitabine hydrochloride and doxorubicin hydrochloride were purchased from Biosynth Limited (Compton, United Kingdom). All materials were used as received.

[0074] Synthesis of α-ω-bis-azide-terminated bifunctional drug-coupled monomers α-ω-bis-azide-terminated bifunctional drug-coupled monomers containing gemcitabine or doxorubicin were synthesized via amide bond formation reactions between carboxylic acid and amino groups. Amide bonds are commonly used in the synthesis of chemical molecules because of their great stability. These reactions often require the activation of the carboxylic acid either through the use of chemically reactive moieties like acid anhydrides or ester or by adding a coupling agent with the ability to activate the carboxylic acid in situ. The synthesis of the two α- ω-bis-azide-terminated bifunctional drug-coupled monomers was carried out in three steps: i. Synthesis of (azido-PEG5)2-EDTA acid (1) ii. Synthesis of GFLG-bearing polymer backbone intermediate, (azidoPEG5)2-EDTA- (GFLG)2(2) by solid phase peptide synthesis (SPPS) method iii. Synthesis of α-ω-bis-azide-terminated bifunctional gemcitabine-coupled monomer, (azidoPEG5)2-EDTA-(GFLG-Gem)2(3), or iv. Synthesis of α-ω-bis-azide-terminated bifunctional doxorubicin-coupled monomer, (azidoPEG5)2-EDTA-(GFLG-Dox)2(4)

[0075] Synthesis of (azido-PEG5)2-EDTA acid (1) (Azido-PEG5)2-EDTA acid (1) is a α-ω-bis-azide-terminated, linear hydrophilic derivative of ethylenediaminetetraacetic acid bearing two carboxylic acid groups. It was synthesized by a single-step amide bond formation between azido-PEG5-amine and EDTA dianhydride. Briefly, azido-PEG5-amine (6.2218 g, 0.02030 mol) was dissolved in anhydrousDMSO in a round-bottomed flask. DIPEA (5.4 mL, 0.03124 mol) was added, and EDTA dianhydride (2 g, 0.00781 mol) was added. The mixture was stirred for three hours at room temperature. The resultant solution was concentrated by rotary evaporation (Rotavapor R-300, Buchi, Switzerland) at 37°C and purified by preparative high-performance liquid chromatography (HPLC). Pure HPLC fractions were concentrated by rotary evaporation followed by drying over sodium sulfate to isolate the desired compound as a light-cream-colored viscous liquid (4.5 g, 66.3%). Analytical HPLC: single peak at retention time 5.036 min; Fourier-transform infrared (FT-IR) spectroscopy: azide asymmetric stretching frequency at 2103.45 cm-1; proton nuclear magnetic resonance (1H NMR) spectroscopy, 400 MHz, DMSO-d6, δ (ppm): 8.2 – 8.4 (carboxylic acid -OH), 7.7 – 7.9 (secondary -NH), 3.1 – 3.9 (m, repeating - OCH2), and 1.1 – 1.4 (-CH2N3); electrospray ionization mass spectrometry (ESI-MS): m / z calculated for C34H65N10O16[M + H]+= 869.5, found 869.7.

[0076] Synthesis of GFLG-bearing polymer backbone intermediate (AzidoPEG5)2-EDTA-(GFLG)2(2) was synthesized by the solid-phase peptide synthesis (SPPS) method. The cathepsin B-cleavable tetrapeptide linker, GFLG, with a free terminal amino group, was synthesized on glycyl-2-chlorotrityl resin (12.5 g, 0.787 mmol / g resin substitution) using 9-fluorenyl-methoxycarbonyl (Fmoc)-solid phase peptide synthesis. (AzidoPEG5)2-EDTA acid (1) was reacted with an excess of the resin-bound GFLG (0.4: 1) to form the desired compound. Resin cleavage followed by precipitation into cold ether was done, and the compound was purified by preparative HPLC to obtain the pure compound (2) as a light- yellow colored viscous semi-solid (1.62 g, 26.8%). FT-IR: azide asymmetric stretching frequency at 2106.98 cm-1;1H NMR, 400 MHz, DMSO-d6, δ (ppm): 0.83 – 0.89 (dd, terminal -CH3, Leu), 1.1 – 1.35 (-CH2, Leu), 3.4 – 3.9 (m, repeating -OCH2, (azido-PEG5)2-EDTA acid), 7.14 – 7.24 (aromatic -CH, Phe), 8.1 – 8.3 (secondary amide -NH, Leu and Phe), and 8.25 – 8.6 (secondary amide -NH, Gly); ESI-MS: m / z calculated for C72H118N18O24[M + H]+= 1618.9, found 1618.2.

[0077] Synthesis of α-ω-bis-azide-terminated bifunctional gemcitabine-coupled monomer Gemcitabine was coupled to (azidoPEG5)2-EDTA-(GFLG)2(2) using HATU to obtain (azidoPEG5)2-EDTA-(GFLG-Gem)2(3), an azide-terminated homo-bifunctional gemcitabine- coupled monomer. To liberate gemcitabine base from its hydrochloride salt, DIPEA (0.86 mL, 4.9141 mmol) was added to gemcitabine HCl (0.73628 g, 2.4571 mmol) before adding DMF as solvent. (AzidoPEG5)2-EDTA-(GFLG)2(2) (0.99377 g, 0.61426 mmol) was dissolved in anhydrous DMF and added to the clear solution of gemcitabine, followed by the addition of HATU solution in DMF (0.70068 g, 1.84279 mmol). The reaction was monitored with mass spectrometry. After 165 min, the solvent was removed by rotary evaporation and the product was purified by preparative HPLC. The pure fraction was lyophilized to obtain pure compound (3) as a white solid (0.4569 g, 35.3%). Analytical HPLC: single peak at retention time 6.640 min; FT- IR: azide asymmetric stretching frequency at 2101.26 cm-1;1H NMR, 400 MHz, DMSO-d6, δ (ppm): 0.83 – 0.89 (dd, terminal -CH3, Leu), 1.1 – 1.35 (-CH2, Leu), 3.4 – 3.9 (m, repeating - OCH2, (azidoPEG5)2-EDTA-(GFLG)2), 6.15 – 6.25 (t, -CH, gemcitabine furan ring), 6.3 – 6.4 (d, -CH, gemcitabine pyrimidine ring), 7.14 – 7.24 (aromatic -CH, Phe), and 11.05 (d, -CH, gemcitabine pyrimidine ring); ESI-MS: m / z calculated for C90H137F4N24O30[M + H]+= 2110.0, found 2109.3 and 1055.2 [M + 2H]2+.

[0078] Synthesis of α-ω-bis-azide-terminated bifunctional doxorubicin-coupled monomer The second drug-coupled monomer, (azidoPEG5)2-EDTA-(GFLG-Dox)2(4), was synthesized by coupling doxorubicin to (azidoPEG5)2-EDTA-(GFLG)2(2) using IIDQ. To prevent the rapid degradation of doxorubicin in the presence of a strong base, morpholine (less basic than DIPEA) was used to liberate doxorubicin from its hydrochloride salt using the method of Van Heeswijk et al (Van Heeswijk, et al, “The synthesis and characterization of polypeptide- adriamycin conjugates and its complexes with adriamycin”, Part I. Journal of Controlled Release, 1985, 1(4), 301-315, the content of which is incorporated herein by reference) with slight modification. Briefly, compound (2) (200 mg, 0.12362 mmol) was dissolved in DMF. In a separate vial, 4-ethyl morpholine (62.58 μL, 0.49449 mmol) was added to doxorubicin HCl in DMF (0.2868 g, 0.49449 mmol), and transferred into the reaction vial containing compound (2). After stirring for 1h, IIDQ solution in DMF (0.188 g, 0.618115 mmol) was added to the reaction, and the reaction was left to continue stirring in the dark for 42 h and purified by preparative HPLC. The pure fraction was concentrated by rotary evaporation, lyophilized, and obtained as a bright-red solid (0.109 g, 33.1%). Analytical HPLC: single peak at retention time 8.164 min; FT-IR (azide asymmetric stretching frequency at 2101.26 cm-1);1H NMR, 400 MHz, DMSO-d6, δ (ppm): 0.75 – 0.81 (dd, terminal -CH3, Leu), 1.11 – 1.13 (dd, -CH3, doxorubicin amino sugar), 3.2 – 3.7 (repeating -OCH2, (azidoPEG5)2- EDTA-(GFLG)2), 4.6 (O-CH3, doxorubicin), 7.15 – 7.25 (aromatic -CH, Phe), 7.45 – 7.9, 7.8 – 7.9 (aromatic -CH, doxorubicin), 13.23 and 13.99 (aromatic -OH, doxorubicin); ESI-MS: m / z calculated for C126H173N20O44[M + H+] = 2670.2, found 2670.6.

[0079] General Purification and Characterization Techniques The synthesized compounds (1 – 4) were purified by preparative HPLC (Agilent 1290 Infinity II Preparative LC System, Agilent Technologies, Santa Clara, CA; solvent gradient: 10% v / v acetonitrile increased to 90% v / v acetonitrile; flow rate: 50 mL / min; detection wavelength: 280 nm; run time: 18 minutes. The mobile phase contained 0.1% v / v TFA). They were characterized by analytical HPLC (Agilent 1100 Series LC, Agilent Technologies, Santa Clara, CA), FT-IR spectroscopy (Perkin Elmer Spectrum 100 FT-IR spectrometer, Perkin Elmer, Waltham, MA),1H NMR spectroscopy (Bruker AVANCE 400 MHz NMR spectrophotometer, Bruker, Billerica, MA), and positive mode electrospray ionization mass spectrometry (Agilent Accurate-Mass TOF LC / MS, Agilent Technologies, Santa Clara, CA).

[0080] Synthesis of gemcitabine and doxorubicin PDCs by SPAAC-mediated step- growth polymerization For the synthesis of gemcitabine-coupled polymer conjugate (p-Gem), solutions of (azidoPEG5)2-EDTA-(GFLG-Gem)2(3) (0.03224 g; 38.2 mM in 0.4 mL of methanol) and DBCO-PEG6-DBCO (0.01375 g; 38.2 mM in 0.4 mL of DCM) were separately prepared. DBCO-PEG6-DBCO solution (0.7 equivalent; 0.28 mL) was added to the solution of (azidoPEG5)2-EDTA-(GFLG-Gem)2(3) and vortexed before removing the solvent mixture by rotary evaporation (37°C, 60 rpm). The remaining 0.3 equivalent of the DBCO-PEG6-DBCO solution was added in two equal portions (0.06 mL each time) to the diluted solution of the product (0.8 mL DCM / methanol 50 / 50 solvent mixture was used each time) followed by the removal of the solvent by rotary evaporation. The addition of solvent and rotary evaporation was repeated ten times to allow for complete reaction. Doxorubicin-coupled polymer conjugate (p-Dox) was prepared similarly by reacting (azidoPEG5)2-EDTA-(GFLG-Dox)2(4) (0.03718 g; 34.8 mM in 0.4 mL of methanol) with DBCO-PEG6-DBCO (0.01252 g; 34.8 mM in 0.4 mL of DCM).

[0081] Molecular weight analysis The molecular weights of the drug-coupled polymer conjugates (p-Gem and p-Dox) were determined by size-exclusion chromatography (SEC) equipped with multiple angle light scattering and refractive index (concentration) detectors. The chromatographic system consisted of an isocratic pump (Agilent G1310B, Palo Alto, CA), well-plate autosampler (Agilent G1329A), and TSKgel Guard Alpha (18345; 6.0 mm ID x 4.0 cm, 13 μm) and TSKgel Alpha- 3000 (18340; 7.8 mm ID x 30 cm, 7 μm) columns (Tosoh Bioscience LLC, King of Prussia, PA). The size exclusion column was connected in-line to a light scattering detector (DAWN HELEOS-II, 690 nm laser, Wyatt Technology, Santa Barbara, CA) and a refractive index detector (Optilab T-rEX, 658 nm laser, Wyatt Technology, Santa Barbara, CA). The isocratic mobile phase was DMF with 10 mM LiCl at a flow rate of 1 mL / min. Solutions of the drug- coupled polymer conjugates (100 μL; 20 mg / mL prepared in DMF with 10 mM LiCl) were injected into the chromatographic system.

[0082] The specific refractive index increment (dn / dc) values, representing the change in the refractive index in response to a change in concentration, were measured using calibration standards (0.1, 0.2, 0.4, 0.8, and 1 mg / mL) prepared from 20 mg / mL stock solutions of each polymer-drug conjugate. Pure solvent (1 mL, DMF with 10 mM LiCl) was injected first to produce the pure solvent baseline. Next, 800 μL of each sample (starting with the lowest concentration) was injected. After all samples had been injected, pure solvent was injected for asecond baseline determination. ASTRA (v6.1.2.84, Wyatt Technology) was used to calculate dn / dc (Fig. 1). Multiple angle light scattering normalization constants were determined using a 30 kDa polystyrene standard (PS 80317, Pressure Chemical Co., Pittsburgh, PA) prepared at 4 mg / mL in 10 mM LiCl in DMF.

[0083] Drug loading studies

[0084] Evaluation of gemcitabine content in p-Gem Gemcitabine loading in p-Gem was measured by ultraviolet-visible (UV-vis) spectroscopy. UV-Vis spectra of 100 μg / mL solutions of p-Gem (prepared using 90% v / v DMF in water) were recorded using Horiba Duetta spectrometer (HORIBA Scientific, Piscataway, NJ). The spectra were collected from 200–400 nm at a 2 nm step increment, 0.05 second integration time, and 5 nm band pass against 90% v / v DMF in water as the reference. All samples were measured in quartz cuvettes (path length b = 10 mm, Hellma, Plainview, NY). Calibration standards (1 to 75 μg / mL) prepared from a stock solution of gemcitabine HCl (1 mg / mL in 90% v / v DMF in water) were used to obtain the calibration curve used for the determination of gemcitabine content in p-Gem based on the intensity counts at 268 nm (Fig. 2).

[0085] Evaluation of doxorubicin content in p-Dox Doxorubicin loading in p-Dox was measured by fluorescence spectroscopy using Horiba Duetta spectrometer. The emission spectra of 100 μg / mL solutions of p-Dox (prepared using 90% v / v DMF in water) at 470 nm excitation wavelength were collected from 550 to 700 nm at an emission increment of 0.5 nm, 0.05 second integration time, and 5 nm excitation / emission band pass against 90% v / v DMF in water as the reference. Calibration standards (0.5 to 20 μg / mL) prepared from a stock solution of doxorubicin HCl (1 mg / mL in 90% v / v DMF in water)were used to obtain the calibration curve used for the determination of doxorubicin content in p- Dox based on the emission intensity counts at 595 nm (470 nm excitation) (Fig. 3).

[0086] Evaluation of cathepsin B-catalyzed cleavage Cathepsin B-catalyzed cleavage of p-Gem was assessed in vitro by incubation of p-Gem solution with exogenous cathepsin-B following the method of Jin et al (Jin, et al, “Folate receptor targeting and cathepsin B-sensitive drug delivery system for selective cancer cell death and imaging”, ACS medicinal chemistry letters, 2020, 11(8), 1514-1520, the content of which is incorporated herein by reference) with slight modifications. Freshly prepared solutions of 1,4- dithiothreitol (DTT) (30 mM; 24 μL) and EDTA disodium salt dihydrate (15 mM; 12 μL) were added to cathepsin B enzyme solution (0.114 nM; 10 μL), and incubated at 37°C for 10 minutes to activate the enzyme. The cleavage medium was prepared by adding Tween 20®(0.5% v / v) and DMSO (1% v / v) to phosphate buffer (pH 5.0). p-Gem (0.33 mg / mL) was incubated with the activated enzyme mix at 37°C with continuous 360ºrotation at 10 rpm using a fixed angle tube rotator (Thermo Fisher Scientific, Pittsburgh, PA). A similar reaction without the enzyme mix was set up as a control. Samples (100 μL) were withdrawn from the test and control reactions at intervals (0 – 24 h), diluted with acetonitrile (400 μL) to precipitate the enzyme, and analyzed by HPLC (Agilent 1100 Series LC C-18 column (250 x 4.6 mm, 5 mm particle size); diode array detector; mobile phase gradient (acetonitrile: water): 2% v / v acetonitrile increased to 95% v / v acetonitrile; flow rate: 1 mL / min; run time: 15 minutes). The appearance of free gemcitabine and the disappearance of p-Gem was determined by monitoring the HPLC chromatogram peak areas at the characteristic retention times of the pure drug and p-Gem.

[0087] A similar approach was used for the doxorubicin-coupled polymer conjugate (p- Dox) by incubating p-Dox at a concentration of 0.68 mg / mL with the enzyme mix at 37°C. Samples (200 μL) were withdrawn from the test and control reactions at intervals (0 – 24 h), diluted with acetonitrile (400 μL) and analyzed by HPLC. The appearance of the free drug and the disappearance of p-Dox was determined by monitoring the HPLC chromatogram peak areas at the characteristic retention times of the pure drug and conjugate.

[0088] Synthesis of α-ω-bis-azide-terminated bifunctional drug-coupled monomers Two α-ω-bis-azide-terminated drug-coupled monomers each containing gemcitabine and doxorubicin were synthesized, respectively, using an EDTA derivative, (azido-PEG5)2-EDTA acid (1), as the monomer starting material. The choice of using an EDTA derivative for the synthesis of PDC was informed by relevant background information. EDTA is FDA-approved chelating agent for the treatment of metal poisoning. It is usually converted into its dianhydride, EDTA dianhydride, for the synthesis of other organic compounds like polymers and hydrogels. EDTA dianhydride is biodegradable and it is used to introduce carboxylic acid groups in a chemical reaction and cross-linking polymers.

[0089] The schematic of the synthesis of (azido-PEG5)2-EDTA acid (1) is shown in Scheme 3. (Azido-PEG5)2-EDTA acid (1) provided the azide homo-bifunctional groups needed for SPAAC-mediated step-growth polymerization for PDC synthesis in the presence of DBCO- PEG6-DBCO, a dibenzoazacyclooctyne (DBCO) homo-bifunctional monomer. It also provided the pendant carboxylic groups essential for further amide bond formation with other building units of the drug-coupled monomers.Scheme 3. Synthesis of (azido-PEG5)2-EDTA acid (1) by one-step amide bond formation between azido-PEG5-amine and EDTA dianhydride.

[0090] GFLG, a cathepsin B-sensitive tetrapeptide linker, was incorporated in the monomer starting material by SPPS (Scheme 4). SPPS was employed for the synthesis of (azidoPEG5)2-EDTA-(GFLG)2(2), the GFLG-bearing polymer backbone intermediate, because of the ease of synthesis facilitated by SPPS. The FT-IR spectrum of (azidoPEG5)2-EDTA- (GFLG)2(2) showed azide asymmetric stretching frequency at 2106.98 cm-1, indicating that the terminal azide groups present on (azido-PEG5)2-EDTA acid (1) remained intact after its coupling to GFLG.

[0091] The azide-terminated homo-bifunctional gemcitabine-coupled monomer (azidoPEG5)2-EDTA-(GFLG-Gem)2(3) was synthesized from gemcitabine HCl and (azidoPEG5)2-EDTA-(GFLG)2(2) using HATU as the coupling reagent (Scheme 5). Similarly, (azidoPEG5)2-EDTA-(GFLG-Dox)2(4), the second azide-terminated homo-bifunctional drug- coupled monomer, was synthesized from doxorubicin HCl and (azidoPEG5)2-EDTA-(GFLG)2(2) using IIDQ as the coupling reagent (Scheme 6). Although the HATU coupling reaction occurs more rapidly, IIDQ was selected as the coupling reagent for doxorubicin coupling because IIDQ forms only amide bonds and can be used with unprotected hydroxy groups, minimizing side reactions with the multiple hydroxyl groups on doxorubicin.

[0092] The analytical HPLC chromatogram of (azidoPEG5)2-EDTA-(GFLG-Gem)2(3) showed a single peak. A single peak was similarly seen on the analytical HPLC chromatogram of (azidoPEG5)2-EDTA-(GFLG-Dox)2(4). This indicates that the pure compounds were isolated. The terminal azide groups present on (azidoPEG5)2-EDTA-(GFLG)2(2) also remained intact following coupling with either gemcitabine or doxorubicin, demonstrating the robustness of the polymer backbone intermediate.monomer, (azidoPEG5)2-EDTA-(GFLG-Gem)2(3).

[0093] monomer, (azidoPEG5)2-EDTA-(GFLG-Dox)2(4).

[0094] The α-ω-bis-azide-terminated bifunctional drug-coupled monomers (compounds 3 and 4) can undergo self-accelerating step-growth polymerization in the presence of a double- strained alkyne monomer to generate high molecular weight PDCs. Additionally, the resulting PDCs are expected to have high drug loading because the drug is already incorporated in the starting polymer precursor. By predetermining the feed ratio of drug-coupled monomers, more controlled drug conjugation to the polymer backbone has been achieved with high drug loading.

[0095] Synthesis of gemcitabine- and doxorubicin-coupled polymer conjugates The α-ω-bis-azide-terminated bifunctional drug-coupled monomers underwent SPAAC- mediated step-growth polymerization in the presence of homo-bifunctional DBCO-PEG6-DBCO monomer to generate high molecular weight PDCs via click chemistry (Schemes 7 and 8). Stock solutions of DBCO-PEG6-DBCO was added in aliquots to stock solutions of (azidoPEG5)2- EDTA-(GFLG-Gem)2(3) and (azidoPEG5)2-EDTA-(GFLG-Dox)2(4) in different reactions to synthesize gemcitabine- and doxorubicin-loaded polymers, respectively. Solvent evaporationafter the addition of each aliquot resulted in rapid polymerization as a result of an increase in the concentrations of the reactants as the solvent evaporates.

[0096] reaction between the homo-bifunctional DBCO-PEG6-DBCO and (azidoPEG5)2-EDTA-(GFLG- Gem)2(3) monomers.

[0097] reaction between the homo-bifunctional DBCO-PEG6-DBCO and (azidoPEG5)2-EDTA-(GFLG- Dox)2(4) monomers.

[0098] FT-IR analysis showed that the azide functional group of (azidoPEG5)2-EDTA- (GFLG-Gem)2(3) (azide asymmetric stretching frequency at 2111.21 cm-1) was no longer present in p-Gem (Fig. 4). The absence of azide asymmetric stretching frequency at 2111.21 cm-1in Fig. 4 showed that the azide-containing starting monomer (compound 3) had been used up. Similarly, the azide functional group of (azidoPEG5)2-EDTA-(GFLG-Dox)2(4) (azide asymmetric stretching frequency at 2106.97 cm-1) was not present in p-Dox (Fig. 5). Theabsence of the azide functional groups indicated that all the azide-containing monomers (compounds 3 and 4) had been used up in the formation of higher molecular weight molecules.

[0099] Molecular weight analysis The use of size exclusion chromatography (SEC) with multiple detectors which allows continuous monitoring of multiple angle light scattering (MALS) and refractive index (RI) provides a more accurate analysis of polymer molecular weight, independent of their retention time during SEC. The molecular weights of the synthesized gemcitabine and doxorubicin PDCs were measured using SEC coupled with MALS and RI detectors (SEC / MALS-RI). A summary of the measured molecular weights (Mw, Mnand Mp) and polydispersity indices (Mw / Mn) of the PDCs are given in Table 1. Table 1. Molecular weight analysis of synthesized PDCs by SEC-MALS / RI PDCs Mw(kDa) Mn(kDa) PdI Mp(kDa) p-Gem 40.18 29.33 1.37 40.37 p-Dox 1800 1782.18 1.01 1920 Mw= weight-average molecular weight; Mn = number-average molecular weight; PdI = polydispersity index = Mw / Mn; Mp= molecular weight of the most abundant species based on the RI signal.

[0100] The doxorubicin-coupled polymer (p-Dox) had very large molecular weights (~ 1800 kDa), whereas the gemcitabine-coupled polymer (p-Gem) had much lower molecular weights (~ 40 kDa) (Figs. 4A and 4B). Fig. 6A shows SEC chromatograms of the polymer conjugates of gemcitabine showing the light scattering (continuous green line) and differential refractive index (dotted blue line) following SEC with MALS and RI detection. Fig. 6B shows SEC chromatograms of the polymer conjugates of doxorubicin (right) showing the light scattering(continuous green line) and differential refractive index (dotted blue line) following SEC with MALS and RI detection. The molecular weight distribution is shown in red and was calculated using the measured dn / dc values. The reason for the differences in molecular weights obtained for p-Dox compared to p-Gem is not known, but differences in the molecular weights of the bis-azide monomers as a result of coupling to drugs of different molecular weights and different physicochemical characteristics could have contributed to the varying molecular weights.

[0101] The polydispersity index (PdI), defined as the ratio of weight-average molecular weight (Mw) to number-average molecular weight (Mn) (i.e. Mw / Mn), describes the molecular weight distribution of polymers. PdI values < 1.5 were obtained for the PDCs, indicating a narrow molecular weight distribution. The molecular weights of the most abundant species (Mp) of PDCs using the RI signal, a concentration detector, were also determined. The data show that the Mpvalues are close to the Mwvalues obtained for the PDCs (Table 1). This suggests that the polymerization method used herein results in PDCs with relatively narrow molecular weight distribution.

[0102] Percent drug loading Gemcitabine and doxorubicin loading in the PDCs were measured by UV-vis and fluorescence spectroscopy, respectively, using triplicate sample solutions of the PDCs in 90% v / v DMF in water. The drug loading was calculated for each sample using calibration standards obtained from each pure drug and normalized to the total PDC weight to yield the weight percent of each drug in the PDCs. The UV-vis spectra of pure gemcitabine HCl and p-Gem showed similar maximum wavelength of absorption at ~270 nm, indicating gemcitabine coupling to polymer. Similarly, the fluorescence spectra of pure doxorubicin HCl and p-Dox showedidentical spectra with maximum emission intensity at ~590 nm when excited at 470 nm, indicating doxorubicin conjugation in the PDC. Fig. 7 summarizes the weight percent of drugs in each PDC sample. Gemcitabine loading in the polymer conjugates was greater than doxorubicin loading. This could be due to the smaller molecular size of gemcitabine (263 Da) compared with doxorubicin (543 Da).

[0103] Another advantage of the disclosed method in addition to ease of synthesis, modularity, rapid progression under mild temperature, and absence of an initiator or catalyst, is high drug loading. For instance, the loading of gemcitabine and doxorubicin in the PDCs was higher than that obtained in other studies that used poly (2-ethyl-2-oxazoline) (<7 % wt. doxorubicin) (Sedlacek, et al, “Poly (2-ethyl-2-oxazoline) conjugates with doxorubicin for cancer therapy: In vitro and in vivo evaluation and direct comparison to poly [N-(2- hydroxypropyl) methacrylamide] analogues”, Biomaterials, 2017, 146, 1-12), HPMA copolymers (6.5 % wt. doxorubicin) (Lammers, et al, “Effect of Intratumoral Injection on the Biodistribution, the Therapeutic Potential of HPMA Copolymer-Based Drug Delivery Systems”, Neoplasia, 2006, 8(10), 788–795); (7.7 % wt. gemcitabine) (Larson, et al, “Biodegradable multiblock poly (N-2-hydroxypropyl) methacrylamide gemcitabine and paclitaxel conjugates for ovarian cancer cell combination treatment”, International journal of pharmaceutics, 2013, 454(1), 435-443); (<8 % wt. gemcitabine) (Duangjai, et al, “Combination cytotoxicity of backbone degradable HPMA copolymer gemcitabine and platinum conjugates toward human ovarian carcinoma cells”, European Journal of Pharmaceutics and Biopharmaceutics, 2014, 87(1), 187- 196); (<13 % wt. gemcitabine) (Yang, et al, “Backbone degradable N-(2-hydroxypropyl) methacrylamide copolymer conjugates with gemcitabine and paclitaxel: Impact of molecularweight on activity toward human ovarian carcinoma xenografts”, Molecular pharmaceutics, 2017, 14(5), 1384-1394), and PEG (<8.5 % wt. doxorubicin) (Veronese, et al, “PEG−doxorubicin conjugates: influence of polymer structure on drug release, in vitro cytotoxicity, biodistribution, and antitumor activity”, Bioconjugate chemistry, 2005, 16(4), 775- 784) as the polymer backbone.

[0104] In vitro cathepsin B-catalyzed cleavage and drug release Drug release from the PDCs is essential for eliciting the pharmacological effect of the coupled chemotherapeutic drugs. GFLG was selected as the linker between the drugs and the polymer backbone because it is widely used as a substrate for cathepsin B, which is highly overexpressed in many solid tumors. It is also stable in the plasma and selective for cathepsin B. The cathepsin B-catalyzed cleavage of p-Gem and p-Dox was assessed by monitoring the HPLC chromatogram peak areas at the characteristic retention times of the pure drugs and the PDCs following incubation of the PDCs in phosphate buffer (pH 5.0) at 37°C in the presence of exogenous cathepsin B. pH 5.0 was used to simulate acidic tumor pH. In addition, cathepsin B is enzymatically active at acidic pH.

[0105] Fig. 8A shows Cathepsin B-catalyzed release of gemcitabine (Gem) from the gemcitabine-coupled polymer (p-Gem) in the presence (blue line) and absence (red line) of exogenous cathepsin B at pH 5.0. The percent gemcitabine released at the different time points was calculated from the peaks of free gemcitabine versus polymer-bound gemcitabine monitored by a diode array detector at 275 nm. Fig. 8B shows Cathepsin B-catalyzed release of a doxorubicin fragment from the doxorubicin-coupled polymer (p-Dox) in the presence (blue line) and absence (red line) of exogenous cathepsin B at pH 5.0. The percent doxorubicin fragmentreleased at the different time points were calculated from the peaks of the doxorubicin fragment versus polymer-bound drug monitored by a diode array detector at 275 nm.

[0106] Evaluation of gemcitabine cleavage from the gemcitabine-coupled polymer conjugate (p-Gem) in the presence of exogenous cathepsin B, showed an initial rapid release of gemcitabine within 2 hurs, followed by a gradual release over 24 hours (Fig. 8A). In addition, analysis of the cleavage test solution by mass spectrometry showed that free gemcitabine was released. A peak at m / z 526.3, corresponding to [2M + H]+(calculated 527.2), indicating the formation of a non-covalent dimer in the gas phase, was found. The analytical HPLC chromatogram of p-Gem that was incubated without cathepsin B (control) showed a peak retention time 7.3 min but was absent in the test, which showed a new peak retention time 2.9 min, which may correspond to gemcitabine). This showed that the released gemcitabine observed in the cleavage experiment was due to cathepsin B-catalyzed cleavage of p-Gem.

[0107] For the doxorubicin-coupled polymer conjugate (p-Dox) in the presence of exogenous cathepsin B, a red film was observed on the wall of the glass vial containing the cleavage reaction mixture at 3 hours. In addition, a new peak (retention time 3.8 min) was observed when the cleavage test solution was monitored at 275 nm wavelength at 3 hours but was absent in the control with no cathepsin B (. It was also observed that the percent release of this compound (retention time 3.8 min) increased over 24 hours (Fig. 8B).

[0108] Doxorubicin is reported to form a precipitate in buffers, such as PBS, as a result of the formation of doxorubicin dimers (Yamada, “Dimerization of doxorubicin causes its precipitation. ACS omega, 2020, 5(51), 33235-33241). Also, the presence of a hydrophobic anthraquinone ring and a hydrophilic amino sugar moiety in the structure of doxorubicin,suggests that doxorubicin is amphiphilic. The red film could be due to the precipitation of the hydrophobic doxorubicin aglycone moiety while the peak at 3.8 min may be due to the hydrophilic amino sugar moiety following hydrolysis of the released doxorubicin in PBS. To support this hypothesis, acetonitrile was added to the cleavage test solution at 30 hour to re- dissolve the red film that had formed on the container wall and the sample was subjected to mass spectrometry analysis. The ESI+-MS spectrum of re-dissolved p-Dox cleavage test solution at 30 hour showed mass corresponding to the non-covalent dimer of the hydrophilic doxorubicin amino sugar moiety (m / z calculated for C12H25N2O6[2M + H]+= 293.2, found 293.1). The m / z at 481.3 and 499.3 also corresponded to the molecular weight of the doxorubicin aglycone fragmentation products (Hong, et al, “Distinct Fragmentation Pathways of Anticancer Drugs Induced by Charge-Carrying Cations in the Gas Phase”, Journal of The American Society for Mass Spectrometry, 2016, 28(4), 628-637).

[0109] As discussed above, GFLG-containing α-ω-bis-azide-terminated bifunctional gemcitabine- and doxorubicin-coupled monomers were synthesized for the development of cathepsin-B activatable PDCs of both anticancer drugs. The α-ω-bis-azide-terminated bifunctional drug-coupled monomers were prepared from a linear hydrophilic EDTA derivative via simple synthetic steps and copolymerized with a DBCO-PEG6-DBCO homo-bifunctional monomer using SPAAC-mediated step-growth polymerization. DBCO reacts with azide groups in a highly specific copper-free click chemistry, thus, the resulting PDCs were easily isolated following solvent removal by rotary evaporation with no by-product. In addition, the SPAAC- mediated step-growth polymerization method of synthesis of the PDCs did not require the use of high temperature, initiator, or catalyst.

[0110] The use of SPAAC-mediated polymerization for the syntheses of tumor-targeted PDCs of gemcitabine and doxorubicin as model drugs will facilitate the accumulation of these potent anticancer agents in tumors leading to greater cytotoxicity in the tumor compared with free drugs and reduced toxicity to healthy cells.

[0111] II. SYNTHESIS OF GEMCITABINE / DOXORUBICIN- COMBINATION POLYMER-DRUG CONJUGATE, AND CHARACTERIZATION AND EVALUATION IN OVCAR-3 HUMAN OVARIAN CANCER CELL-LINE

[0112] Synthesis of gemcitabine / doxorubicin-combination polymer-drug conjugate (p- Gem / Dox) by SPAAC-mediated step-growth polymerization is provided below. The method does not need high temperatures or catalysts and affords an easy and rapid synthesis of high molecular weight PDCs. The dual delivery of both drugs on a single polymer backbone would result in the passive targeting of a high concentration of drugs to OC via the EPR effect leading to increased antitumor activity, reduction in adverse effects, and lower the likelihood of chemoresistance development. To compare efficacy, the cytotoxic effects of the individual and combination PDCs were evaluated in vitro in OVCAR-3 human OC cells using CellTiter-Glo®2.0 assay, and compared with the free drugs at equivalent concentrations.

[0113] Materials and Method Materials Dimethyl sulfoxide (DMSO), dichloromethane (DCM), methanol, N, N′- dimethylformamide (DMF), lithium chloride (LiCl), sodium phosphate monobasic monohydrate, EDTA disodium salt dihydrate, sodium hydroxide pellets, and 1,4-dithiothreitol (DTT) were obtained from Sigma-Aldrich (Burlington, MA, USA). Dibenzoazacyclooctyne-hexa (ethyleneglycol)-dibenzoazacyclooctyne (DBCO-PEG6-DBCO) was obtained from AxisPharm (San Diego, CA, USA). Polystyrene standard (PS80317) was obtained from Pressure Chemical Co. (Pittsburgh, PA, USA). Purified native human cathepsin B was obtained from Athens Research and Technology (Athens, GA, USA). Human ovarian carcinoma cell line (NIH: OVCAR-3, ATCC® HTB-161) was obtained from the American Type Culture Collection (ATCC) (Manassas, VA, USA). RPMI-1640 medium was purchased from Invitrogen (Carlsbad, CA). Fetal bovine serum was obtained from Fisher Scientific (Pittsburgh, PA, USA). Penicillin / streptomycin was obtained from Cellgro (Manassas, VA, USA). CellTiter-Glo® 2.0 Cell Viability Assay was obtained from Promega (Madison, WI, USA). Gemcitabine hydrochloride (HCl) and doxorubicin HCl were obtained from Biosynth Limited (United Kingdom). All materials were used as received.

[0114] Synthesis of azide-terminated homo-bifunctional drug-coupled monomers and single PDCs The azide-terminated homo-bifunctional doxorubicin-coupled monomer ((azidoPEG5)2- EDTA-(GFLG-Dox)2) and gemcitabine-coupled monomer ((azidoPEG5)2-EDTA-(GFLG-Gem)2) used for the synthesis of the combination polymer-drug conjugate (PDC) were synthesized as discussed above. In addition, the single gemcitabine PDC (p-Gem) and the single doxorubicin PDC (p-Dox) used for the in vitro cytotoxicity evaluation were synthesized and characterized as discussed above.

[0115] Synthesis of gemcitabine / doxorubicin combination PDC The azide-terminated homo-bifunctional drug-coupled monomers were copolymerized with DBCO-PEG6-DBCO, a dibenzoazacyclooctyne-terminated homo-bifunctional polyethyleneglycol (PEG) monomer, via SPAAC-mediated polymerization to prepare the combination PDC (p-Gem / Dox) bearing both doxorubicin and gemcitabine on the same polymer backbone (Scheme 9). Separate solutions of (azidoPEG5)2-EDTA-(GFLG-Gem)2(0.02514 g; 29.8 mM in 400 μL of methanol), (azidoPEG5)2-EDTA-(GFLG-Dox)2(0.03182 g; 29.8 mM in 400 μL of methanol), and DBCO-PEG6-DBCO (0.02144 g; 29.8 mM in 800 μL of DCM) were prepared. The prepared solutions of (azidoPEG5)2-EDTA-(GFLG-Dox)2and (azidoPEG5)2-EDTA-(GFLG- Gem)2were combined in a glass scintillation vial, and vortexed. DBCO-PEG6-DBCO solution (0.7 equivalent; 560 μL) was added to the mixture of the monomers and vortexed before removing the solvent mixture by rotary evaporation (Rotavapor R-300, Buchi, Switzerland, 37 ºC, 60 rpm). The remaining 0.3 equivalent of the DBCO-PEG6-DBCO solution was added in two equal portions (120 μL each time) to the diluted solution of the product (1600 μL DCM / methanol 50 / 50 solvent mixture was used each time) followed by the removal of the solvent by rotary evaporation. The addition of solvent and rotary evaporation was repeated ten times to allow a complete reaction. The PDC product was analyzed by Fourier-transform infrared (FT-IR) spectroscopy (Perkin Elmer Spectrum 100 FT-IR spectrometer, Perkin Elmer, Waltham, MA) to monitor the disappearance of the azide functional groups of the azide-terminated monomers.. y g p sing SPAAC reaction between azide-terminated homo-bifunctional (azidoPEG5)2-EDTA-(GFLG- Dox)2and (azidoPEG5)2-EDTA-(GFLG-Gem)2drug-coupled monomers, and DBCO-PEG6- DBCO, a dibenzoazacyclooctyne-terminated homo-bifunctional PEG monomer.

[0116] Molecular weight characterization The molecular weight and molecular weight distribution of p-Gem / Dox were determined by size-exclusion chromatography (SEC) using high-performance liquid chromatography (HPLC) system (Agilent, Palo Alto, CA) equipped with TSKgel Guard Alpha (6 mm ID x 4 cm, 13 μm) and TSKgel Alpha-3000 (7.8 mm ID x 30 cm, 7 μm) columns (Tosoh Bioscience LLC, King of Prussia, PA). DAWN HELEOS-II multi-angle laser light scattering (MALS) and OptilabT-rEX refractive index (RI) detectors (Wyatt Technology, Santa Barbara, CA) were employed; and DMF with 10 mM LiCl (1 mL / min flow rate) was used as the isocratic mobile phase. A sample solution of p-Gem / Dox (20 mg / mL) was prepared in DMF with 10 mM LiCl, and 100 μL was injected into the chromatographic system. RI change was measured differentially at 660 nm laser wavelength using the Optilab T-rEX RI detector, and ASTRA software (v6.1.2.84, Wyatt Technology) was used to calculate the specific RI value of p-Gem / Dox. Polystyrene standard (PS 80317, 30kDa) prepared at 4 mg / mL in DMF with 10 mM LiCl was used for MALS normalization.

[0117] Drug content evaluation

[0118] Gemcitabine and doxorubicin contents in p-Gem / Dox were determined by ultraviolet-visible (UV-vis) and fluorescence spectroscopy, respectively, on Horiba Duetta spectrometer (HORIBA Scientific, Piscataway, NJ). Triplicate solutions of p-Gem / Dox (100 μg / mL), prepared using 90% v / v DMF in water, were used for each analysis. Gemcitabine absorption spectra were collected from 200–400 nm at a 2 nm step increment, 0.05 second integration time, and 5 nm bandpass against 90 % v / v DMF in water as the reference. For doxorubicin, the fluorescence emission spectra at an excitation wavelength of 470 nm were collected from 550 to 700 nm at an emission increment of 0.5 nm, 0.05 second integration time, and 5 nm excitation / emission bandpass against 90 % v / v DMF in water as the reference. The calibration curves for gemcitabine HCl at 268 nm and doxorubicin HCl at 595 nm emission / 470 nm excitation were used to calculate the loading of each drug (%wt.) in p-Gem / Dox.

[0119] In vitro cathepsin B-catalyzed cleavageCathepsin B-catalyzed cleavage of p-Gem / Dox was assessed in vitro for the release of the conjugated drugs. The conjugate (1.5 mg / mL) in cleavage buffer (pH 5.0; PBS containing 30 mM 1,4-dithiothreitol, 15 mM EDTA disodium salt dihydrate, 0.114 nM cathepsin B enzyme solution, 0.5% v / v Tween 20^, and 1% v / v DMSO) was incubated at 37 °C with continuous 360º rotation at 10 rpm using a fixed angle tube rotator (Thermo Fisher Scientific, Pittsburgh, PA). A similar experiment without cathepsin B was set up as a control. Samples (200 μL) were withdrawn from the test and control at intervals (0 – 24 h), diluted with acetonitrile (400 μL) to precipitate the enzyme, and analyzed by reversed-phase HPLC (RP-HPLC).

[0120] In vitro hydrolytic stability Hydrolytic stability of p-Gem / Dox at 37 °C was evaluated in phosphate-buffered saline (PBS) (pH 7.4) to simulate plasma pH. 0.37 mg / mL solution of p-Gem / Dox was prepared in PBS buffer to which Tween 20®(0.5% v / v) and DMSO (1% v / v) were added and incubated at 37 °C with continuous 360° rotation at 10 rpm using a fixed angle tube rotator for 24 h. At different time points, samples were withdrawn for HPLC analysis as described above. The experiment was performed in triplicate.

[0121] In vitro cytotoxicity evaluation in OVCAR-3 cells

[0122] Cell culture OVCAR-3 human OC cells were maintained in RPMI-1640 medium supplemented with 20% fetal bovine serum and 1% penicillin / streptomycin at 37 ºC in a humid atmosphere with 5% carbon dioxide. The growth medium was replaced every 48 h.

[0123] Cytotoxicity evaluation of the single agentsThe cytotoxicity of p-Dox and p-Gem against OVCAR-3 human OC cells was evaluated in vitro using CellTiter-Glo^2.0 assay (Promega, Madison, WI). Cultured OVCAR-3 cells were seeded in 96-well plates at 10,000 cells / well (100 μL) and allowed to attach for 24 h. For direct comparison with the free drugs, the amount of p-Dox containing the same amount of doxorubicin HCl (DOX) in solution and the amount of p-Gem containing the same amount of gemcitabine HCl (GEM) in solution were used to treat the cells at different concentrations (0.005, 0.05, 0.5, 5, and 50 μg / mL). Cells that were treated with the culture media and DMSO in media were used as controls. Four replicates of each treatment were used. At 72 h post-treatment, 100 μL of CellTiter-Glo^2.0 assay reagent was added to each well and mixed for 2 min on an orbital shaker to induce cell lysis. The plates were incubated in the dark at room temperature for 10 min, and luminescence was recorded using PromegaTM GloMax^plate reader (Promega, Madison, WI). The percent viability was determined by normalizing the average luminescence of treated cells against untreated cells in media. Percent cell viability as a function of drug concentration was plotted and non-linear least-squares regression analysis was used to calculate the concentrations that inhibit the growth of 50% of the cells (IC50 values) using GraphPad Prism 10.1.1 (270) software.

[0124] Cytotoxicity evaluation of the combination agents and combination index analysis The cytotoxicity of p-Dox + p-Gem admixture (50: 50), GEM + DOX admixture (50: 50), p-Gem / Dox, and GEM + DOX admixture (36.6: 7) against OVCAR-3 cells were evaluated in vitro. As with the single agents, the amounts of the conjugates containing equivalent amounts of the free drugs in solution were used to treat the cells at concentrations 0.005, 0.05, 0.5, 5, and50 μg / mL. Cells that were treated with the culture media and DMSO in media were used as controls. At 72 h post-treatment, the viability assay was carried out using CellTiter-Glo^2.0 assay per manufacturer protocol as described earlier. The cytotoxic effect of GEM + DOX interactions in OVCAR-3 cells was determined by calculating the combination index (CI) using COMPUSYN software (version 1.0) based on the Chou–Talalay method (Chou, “Theoretical basis, experimental design, and computerized simulation of synergism and antagonism in drug combination studies”, Pharmacological reviews, 2006, 58(3), 621-681.).

[0125] Results and discussion

[0126] Synthesis and characterization of p-Gem / Dox The homo-bifunctional monomers, DBCO-PEG6-DBCO, (azidoPEG5)2-EDTA-(GFLG- Gem)2, and (azidoPEG5)2-EDTA-(GFLG-Dox)2, were co-polymerized using a 1: 1: 1 stoichiometric ratio, respectively, to obtain gemcitabine / doxorubicin combination PDC (p- Gem / Dox). DBCO-PEG6-DBCO solution was added in aliquots to the mixture of (azidoPEG5)2- EDTA-(GFLG-Gem)2and (azidoPEG5)2-EDTA-(GFLG-Dox)2. Following the addition of each aliquot, the solvent was removed by rotary evaporation. This increased the concentrations of the reactants and caused the polymerization to occur rapidly and more efficiently. Similar to the single PDCs, FT-IR analysis of p-Gem / Dox showed the disappearance of azide stretch at around 2100 cm-1present in the azide-terminated homo-bifunctional (azidoPEG5)2-EDTA-(GFLG-Dox)2and (azidoPEG5)2-EDTA-(GFLG-Gem)2drug-coupled monomers (Fig. 9). The disappearance of the azide peak at around 2100 cm-1showed that the azide-terminated starting monomers had been used up. This confirms that all the azide-terminated drug-coupled monomer molecules had been used up with the formation of higher molecular weight molecules.

[0127] Table 1. Physical characteristics of the PDCs PDCs Mw(kDa) Mp(kDa) Mw / Mn% weight % weight doxorubicin gemcitabine

[0128] The gemcitabine / doxorubicin-combination PDC (p-Gem / Dox) had very large molecular weights (> 1000 kDa) with narrow molecular weight distribution (Mw / Mn= 1.01) (Fig. 10). The large molecular weight and narrow molecular weight distribution obtained were similar to the values obtained for the doxorubicin PDC (p-Dox) (Table 1). Gemcitabine loading (36.6% weight) was greater than doxorubicin loading (7% weight) in p-Gem / Dox, similar to the corresponding single drug-coupled polymers. This could be due to the smaller molecular weight of gemcitabine (263 Da) compared with doxorubicin (543 Da). Repeat synthesis and characterization show that the SPAAC-mediated polymerization mechanism results in reproducible drug loading and molecular weight distributions.

[0129] The molecular weight distribution of PDCs is a very important factor in the passive targeting of cancer by the EPR effect. Macromolecules with molecular weights that are above the renal filtration threshold (40 kDa) can preferentially accumulate within tumor tissues due to the leaky vasculature and impaired lymphatic drainage system present in tumor tissues. It is possible for PDCs with a wider molecular weight distribution to be cleared from the bloodstream more quickly because they contain a variety of molecules with molecular weightsthat may be lower than the renal filtration threshold. Compared with other reported studies, the prepared p-Gem / Dox using the SPAAC approach, had better molecular weight distribution and drug loading. For instance, a poly (N-(2-hydroxypropyl) methacrylamide) (HPMA) copolymer bound to gemcitabine and doxorubicin via GFLG had Mw= 23.5 kDa, Mw / Mn=1.6, 6.4% weight gemcitabine and 5.7% weight doxorubicin (Lammers, et al, “Simultaneous delivery of doxorubicin and gemcitabine to tumors in vivo using prototypic polymeric drug carriers”, Biomaterials, 2009, 30(20), 3466-3475.). Another reported gemcitabine / doxorubicin combination PDC with hyaluronic acid as the polymer backbone had ≤ 3.6 % weight and ≤ 5.0% weight loading for doxorubicin and gemcitabine, respectively (Vogus, et al, “A hyaluronic acid conjugate engineered to synergistically and sequentially deliver gemcitabine and doxorubicin to treat triple negative breast cancer”, Journal of Controlled Release, 2017, 267, 191-202).

[0130] In vitro cathepsin B-catalyzed cleavage of p-Gem / Dox The cathepsin B-catalyzed cleavage of p-Gem / Dox was done to evaluate the release rate of gemcitabine and doxorubicin from the polymer conjugate. The release rate of gemcitabine from the polymer following enzyme cleavage of p-Gem / Dox showed a rapid initial release within 2 h, followed by a gradual release over the 24 h evaluation period (Fig. 11). The percent released drugs at the different time points were calculated from the peaks of released gemcitabine and doxorubicin fragment versus the polymer-bound drug monitored by a diode- array detector at 275 nm. With the evaluation of doxorubicin release rate, no free doxorubicin was detected at the characteristic doxorubicin wavelength of absorption (480 nm). A similar release pattern was observed for individual PDCs of gemcitabine and doxorubicin. A new peak (retention time 3.8 min) that was absent in the control without cathepsin B, was observed in thetest at 3 h with detection at 275 nm. The release of this compound, which started appearing at 3 h in the test also increased gradually when monitored at 275 nm over 24 h. This was similar to the observation with p-Dox, which released a doxorubicin fragment (retention time 3.84 min) when monitored at 275 nm.

[0131] The structure of doxorubicin consists of a hydrophobic anthraquinone ring and a hydrophilic amino sugar moiety, indicating that doxorubicin is amphiphilic. The peak at retention time 3.8 min was attributed to the hydrophilic amino sugar moiety of doxorubicin, which does not absorb at 480 nm. At the end of the cleavage experiment, a sample of the conjugate solution in the cleavage buffer was dissolved in acetonitrile and was analyzed by mass spectrometry. The mass analysis showed masses (m / z) corresponding to free gemcitabine (m / z = 262.9578 [M]+, calculated 263), the hydrophilic doxorubicin amino sugar moiety (m / z = 437.2501 [(M × 3) + H]+, calculated 146.0817), and a doxorubicin aglycone fragmentation product (m / z = 481.2937 [M + H]+, calculated 482).

[0132] The slower release of doxorubicin compared with gemcitabine may be due to hydrophobic interactions between doxorubicin molecules, hindering the enzyme’s access to GFLG and hence, reduced rate of enzyme-catalyzed cleavage. In addition, the drug release pattern from the p-Gem / Dox correlated with the result obtained from a similar p-Gem / Dox that was formed using HPMA copolymer (Lammers et al., 2009, supra). The faster release of gemcitabine than doxorubicin was attributed to the small size of gemcitabine which causes less steric hindrance (Lammers et al., 2009, supra).

[0133] Hydrolytic stability of p-Gem / DoxThe in vitro stability of p-Gem / Dox was carried out in PBS (pH 7.4) to mimic physiological pH. p-Gem / Dox demonstrated good stability in PBS (pH 7.4) (Fig. 12), indicating that it could maintain good stability in the body with efficient drug release in the tumor when cleaved by cathepsin B. The decrease in the peak corresponding to polymer-bound gemcitabine / doxorubicin was monitored by a diode-array detector at 480 nm and the released drugs were monitored at 275 nm (n = 3). The stability of p-Gem / Dox at pH 7.4 may be attributed to the high stability of GFLG and that of the amide bond which was used for the conjugation of the drugs to the polymer backbone. GFLG is more specific for cathepsin B than cathepsin B dipeptide substrates like Val-Ala or Val-Cit, and it is stable in plasma. In addition, amide bonds have the propensity to form resonant structures, which confer great stability on amide bonds.

[0134] In vitro cytotoxicity evaluation The cytotoxicity of the single-drug PDCs (p-Dox and p-Gem), p-Dox and p-Gem admixture (50:50), and p-Gem / Dox (dual-drug PDC) to OVCAR-3 OC cells was evaluated. The cell line is known to express cathepsin B. Also, the most prevalent and aggressive histotype of OC, high-grade serous OC, is well represented by the OVCAR-3 cell line. The cells were exposed to solutions of the PDCs (0.005, 0.05, 0.5, 5, and 50 μg / mL PDC equivalents of gemcitabine and doxorubicin) for 72 h. Gemcitabine HCl (GEM) and doxorubicin HCl (DOX) were used to evaluate the cytotoxicity of the free drugs. CellTiter-Glo^2.0 assay was used for determining the percent viability of OVCAR-3 cells at 72 h post-treatment. The assay gives a valid indication of cell viability by quantifying the amount of intracellular adenosine triphosphate as luminescent signals generated by metabolically active cells present in culture.

[0135] Cytotoxicity evaluation of p-DoxDOX is approved for the treatment of metastatic OC. For direct comparison with DOX, the amount of p-Dox containing the same amount of DOX as DOX in solution was used. The percent viability of OVCAR-3 cells that were treated with p-Dox and DOX relative to untreated controls were determined. The results showed a concentration-dependent inhibition of cell growth, with the free drug being more cytotoxic than the polymer-bound drug (Fig. 13). The IC50value of p-Dox (1.79 ± 0.17 μg / mL) was about ten times higher than the IC50value of DOX (0.17 ± 0.03 μg / mL). Compared with small molecule drugs, which diffuse freely into the cells, PDCs may enter the cells by micropinocytosis which is a slower process than diffusion. To match the cytotoxicity of the small molecule DOX, effective cell internalization followed by cathepsin B-catalyzed drug release is required for p-Dox (Pechar, et al, “Polymer nanomedicines with enzymatically triggered activation: A comparative study of in vitro and in vivo anti-cancer efficacy related to the spacer structure”, Nanomedicine: Nanotechnology, Biology and Medicine, 2022, 46, 102597.).

[0136] Cytotoxicity evaluation of p-Gem GEM, used with carboplatin, is approved for the treatment of advanced OC that is resistant to first-line chemotherapy. The percent viability of OVCAR-3 cells that were treated with solutions of p-Gem and GEM, relative to untreated control cells, were determined. The data show no difference in the inhibition of proliferation of cells between cells treated with the polymer-bound gemcitabine compared to free gemcitabine (Fig. 14). This observation can be explained based on the release pattern of gemcitabine from p-Gem. The cleavage of p-Gem by cathepsin B was very fast. Within 72 h, almost all the conjugated gemcitabine would have been released due to the rapid cathepsin B-catalyzed cleavage of p-Gem. Since an equivalent drugconcentration based on gemcitabine content in the p-Gem was used, at 72 h post-treatment, it is expected that the cells would be exposed to similar gemcitabine concentrations in solution after PDC cleavage and gemcitabine release by cathepsin B. Compared with p-Dox, however, both p- Gem and GEM were less potent against OVCAR-3 cells at the tested concentrations, and thus, had large IC50values (>50 µg / mL) (Table 2).

[0137] Table 2. Calculated IC50values (mean ± SD) of the free drugs and the PDC equivalents in OVCAR-3 cells. Drug treatment IC50(µg / mL) Single p-Dox 1.79 ± 0.17 Doxorubicin HCl (DOX) 0.17 ± 0.03 p-Gem >50 Gemcitabine HCl (GEM) >50 Combination p-Gem + p-Dox admixture (50: 50) 3.87 ± 1.22 GEM + DOX admixture (50: 50) 0.24 ± 0.12 p-Gem / Dox 0.99 ± 0.06 GEM + DOX admixture (36.6: 7) 0.11 ± 0.04

[0138] Cytotoxicity evaluation of the combination PDCs Combination chemotherapy is preferred for the treatment of cancer for enhanced therapeutic efficacy. The use of multiple chemotherapeutic agents with different mechanisms of action also stands a chance to treat cancer more effectively than monotherapy. Gemcitabine inhibits the synthesis of deoxyribonucleic acid (DNA) and induces cell cycle arrest in the synthesis phase. Doxorubicin causes cytotoxicity by intercalating between DNA bases and altering the DNA structure. It also traps topoisomerase II in the double-strand cleaved form, introducing toxic double-strand breaks in the cell.

[0139] The percent viability of OVCAR-3 cells 72 h after treatment with the 50:50 admixtures of p-Dox + p-Gem and GEM + DOX, relative to untreated control cells, were determined and compared. Growth inhibition in the cells increased with increasing concentrations of the treatments (Fig. 15). The 50:50 admixture of p-Dox + p-Gem (IC50value = 3.87 ± 1.22 μg / mL) was less cytotoxic to the cells than the 50:50 admixture of GEM + DOX (IC50value = 0.24 ± 0.12 μg / mL). This higher cytotoxicity of the free drug combination when compared to the combination of p-Dox + p-Gem could be due to the ability of the free drugs to enter into the cells more freely and rapidly compared to the PDCs. In addition, conjugated drugs are not expected to be active until release is triggered by cathepsin B cleavage of GFLG.

[0140] A higher cytotoxicity was observed when the cells were treated with p-Gem and p-Dox as a 50:50 admixture (IC50value = 3.87 ± 1.22 µg / mL) compared to p-Gem alone (IC50value > 50 µg / mL). This observation can be explained by the addition of doxorubicin, which is more potent than gemcitabine. Gemcitabine is a deoxycytidine analog that is converted to its active metabolites, gemcitabine diphosphate and gemcitabine triphosphate, by deoxycytidine kinase. Gemcitabine diphosphate irreversibly inhibits ribonucleotide reductase, which produces deoxyribonucleotides (including deoxycytidine triphosphate) necessary for DNA synthesis, and gemcitabine triphosphate competes with deoxycytidine triphosphate for incorporation in an elongating DNA chain. Treatment of cells with gemcitabine rapidly depletes deoxycytidine triphosphate pools, inhibiting DNA synthesis and inducing cell cycle arrest in the synthesis phase. However, the ‘arrested’ cells can recover without further treatment. It was shown that the cleavage of p-Gem by cathepsin B was faster than p-Dox. The release of gemcitabine from p-Gem / Dox was also faster than doxorubicin. Hence, the fast release of gemcitabine from p-Gemin the p-Dox + p-Gem admixture (50:50) will arrest the cells in the DNA synthesis phase, and the release of doxorubicin later will exploit the vulnerable state of the cells before they adequately recover.

[0141] Additionally, the percent viability of OVCAR-3 cells that were treated with solutions of p-Gem / Dox (where gemcitabine and doxorubicin were coupled to the same polymer backbone) and GEM + DOX admixture (36.6:7), relative to untreated control cells, were also determined. GEM + DOX admixture (36.6:7) was used as the free drug control for p-Gem / Dox based on the drug content in p-Gem / Dox (36.6% weight and 7% weight for gemcitabine and doxorubicin, respectively). Growth inhibition in the cells was also concentration-dependent (Fig. 16), and p-Gem / Dox (IC50value = 0.99 ± 0.06 µg / mL), being a conjugate, was less cytotoxic to the cells compared to the free drug admixture (IC50value = 0.11 ± 0.04 µg / mL). The IC50value of p-Gem / Dox was about four times lower than the IC50value of the 50:50 p-Dox + p-Gem admixture (3.87 ± 1.22 µg / mL). This indicates that the conjugation of doxorubicin and gemcitabine to the same polymer backbone confers an increase in potency against OVCAR-3 cells, compared to the physical mixture of the individual conjugates of each drug (i.e. 50:50 p- Dox + p-Gem admixture). This can be explained by the ability of a polymer conjugate bearing two different drugs on the same polymer backbone to share the same biodistribution and enzyme cleavage kinetics, compared to the individual conjugates which may exhibit distinct kinetics.

[0142] Combination index analysis Combination chemotherapy has demonstrated efficacy in the treatment of OC. When developing a new combination therapy, it is important to determine the overall effect of such drug combination, which may be an additive effect, synergism, or antagonism. The methoddeveloped by Chou and Talalay is widely utilized for evaluating the effect or interaction of combination treatments (Chou, “Theoretical basis, experimental design, and computerized simulation of synergism and antagonism in drug combination studies”, Pharmacological reviews, 2006,58(3), 621-681). The Combination Index (CI) is determined through the assessment of the effects of individual drugs as well as their combined effects. “CI values = 1, < 1, and > 1 are regarded as an additive effect, synergism, and antagonism, respectively” (Chou, 2006, supra).

[0143] The percent viability of OVCAR-3 cells at 72 h post-treatment with GEM + DOX admixture (50:50) and GEM + DOX admixture (36.6:7) were analyzed using COMPUSYN software (version 1.0) to determine the CI values. The effects of free drug combinations at ratios 50:50 and 36.6:7, respectively for GEM and DOX were analyzed and compared. For the GEM + DOX admixture (36.6:7), the effect was synergistic (CI values < 1), and the synergism decreased with increasing drug concentrations (Fig. 17). This was the same for GEM + DOX admixture (50:50), except at the highest concentration (50 µg / mL), where the effect was antagonistic. The antagonism at 50 µg / mL concentration can be explained by the IC50 values obtained for GEM (>50 µg / mL) and DOX (0.17 ± 0.03 µg / mL). The effect of doxorubicin alone had probably overshadowed the effect of gemcitabine, such that the effect of gemcitabine in the GEM + DOX admixture (50:50) at such a high concentration would be insignificant.

[0144] The CI values for GEM + DOX admixture (36.6:7) and GEM + DOX admixture (50:50) at 50% relative viability (EC50) were also compared. At EC50, GEM + DOX admixture (36.6:7) showed very strong synergism (CI value = 0.02731) while GEM + DOX admixture (50:50) showed strong synergism (CI value = 0.19325). This analysis supported the higher potency of p-Gem / Dox (the dual-drug PDC containing 36.6% weight gemcitabine and 7%weight doxorubicin) against OVCAR-3 cells than the p-Dox + p-Gem admixture (50:50) as discussed above. Synergistic effects typically arise when chemotherapeutic agents work through distinct mechanisms of action, or when the impact of one agent enhances the sensitivity of cells to another agent's effects. The data supports the combination of both drugs for the treatment of OC.

[0145] As discussed above, the SPAAC-mediated step-growth polymerization method was used to prepare gemcitabine / doxorubicin-combination PDC (p-Gem / Dox) with high molecular weight, high drug loading, and narrow polydispersity. Single PDCs containing gemcitabine and doxorubicin had been prepared using the same method. p-Gem / Dox had similar drug loading and cathepsin B-catalyzed drug release pattern as the single PDCs. The combination drug PDC was also more toxic to OC cells compared to the combination of individual PDCs of gemcitabine and doxorubicin. p-Gem / Dox holds the potential to reduce chemoresistance as a result of the delivery of combination therapeutics at high concentrations to the tumor. In addition, sequential passive-active tumor targeting facilitated by large molecular- weight PDCs via the EPR effect and the use of GFLG as a selective cathepsin B substrate and mechanism of drug release respectively, will improve therapeutic efficacy and eliminate non- specific toxicity to healthy cells. This approach has the potential to cure OC, which is typically diagnosed at an advanced stage.

[0146] The contents of literature documents cited herein are incorporate herein by reference.

[0147] While the subject matter disclosed herein has been described in connection with what is presently considered to be practical example embodiments, it is to be understood that thepresent disclosure is not limited to the disclosed embodiments, and covers various modifications and equivalent arrangements included within the spirit and scope of the appended claims.

Claims

WHAT IS CLAIMED IS:

1. A method of preparing a polymer-drug conjugate (PDC) comprising reacting a drug-bearing compound terminated at both ends by a functional group with another compound, which may be drug-bearing or non-drug bearing, having a complementary functional group using suitable ratios of each compound to form a single-drug or combination drug PDC by click chemistry.

2. The method according to claim 1, wherein the PDC is prepared by strain- promoted [3+2] azide-alkyne cycloaddition (SPAAC)-mediated step-growth polymerization.

3. The method according to claim 1, wherein the prepared PDC has a molecular weight of 10 kDa – 2,000 kDa.

4. The method according to claim 1, wherein the prepared PDC has a molecular weight greater than 40 kDa.

5. The method according to claim 1, wherein the prepared PDC has a drug loading ranging from 0.25% - 65% by weight of the PDC for each drug in single drug PDC or the combination drug PDC.

6. The method according to claim 1, wherein the drug is used for disease management or treatment of disease such as an anticancer drug or combination of anticancer agents.

7. The method according to claim 1, wherein the drug-bearing compound and the other compound, which may be drug-bearing or non-drug bearing, are each independently a small molecule, monomer, an oligomer, or a polymer.

8. The method according to claim 1, wherein the functional group and the complementary functional group are each independently selected from the group consisting of azide, thiol, DBCO, BCN, maleimide, tetrazine, trans cyclooctene (TCO), and strained or linear alkynes or any other complementary click-chemistry reaction specific combination.

9. The method according to claim 1, wherein each terminal of the drug bearing compound bears the same or different functional group.

10. The method according to claim 1, wherein each terminal of the other compound bears the same or different functional group.

11. The method according to claim 1, wherein single or multiple drugs are coupled to a compound via physical or chemical means or reactions.

12. The method according to claim 1, wherein the drug-bearing compound is (azidoPEG5)2-EDTA-(GFLG-Drug)2, and the other compound is DBCO-PEG6-DBCO.

13. The method according to claim 1, comprising the reaction shown in the following scheme 1:Scheme 1.

14. The method according to claim 1, comprising the reaction shown in the following scheme 2:Scheme 2.

15. A polymer-drug conjugate (PDC) prepared by the method of claim 1.

16. A method of treating cancer, comprising administering an effective amount of the polymer-drug conjugate (PDC) or a mixture of effective amounts of PDCs according to claim 15 to a subject in need thereof.

17. The method according to claim 16, wherein the cancer is a solid cancer such as colorectal, bladder, endometrial, kidney, liver, prostate, breast, ovarian, brain, lung, skin, pancreatic, and head and neck cancers.

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