WO2021226033A1 - Hyaluronic acid drug conjugates - Google Patents

Abstract

Described herein are polymer-drug conjugates relating to combinations of hyaluronic acid, doxorubicin, and camptothecin. Further described herein are methods of using such conjugates in methods of treating precancerous skin lesions or cancer, e.g,. a skin cancer, blood cancer, glioblastoma, or intestinal cancer.

Description

HYALURONIC ACID DRUG CONJUGATES

CROSS-REFERENCE TO RELATED APPLICATIONS [0001] This application claims benefit under 35 U.S.C. § 119(e) of U.S. Provisional Application No. 63/021,422 filed May 7, 2020, the contents of which are incorporated herein by reference in their entirety.

TECHNICAL FIELD

[0002] The technology described herein relates to polymer-drug conjugates.

BACKGROUND

[0003] Improved methods for delivery and dosing control of pharmaceutical agents, e.g., for the treatment of cancer can improve therapeutic outcomes. Drugs with limited individual efficacy can often provide superior results when the proper combinatorial approaches are used. However, many drug combinations are senstitive to relative dosing amounts.

SUMMARY

[0004] Described herein are conjugates of hyaluronic acid with doxorubin and/or camptothecin which provide surprising efficacy. These conjugates use hyaluronic acid (HA) as a backbone, providing nanosized polymer-drug conjugates of doxorubicin and camptothecin which are referred to herein as, DOxorubicin and Camptothecin Tailored at Optimal Ratios (DOCTOR). These conjugates provided surprising delivery kinetics - penetration and accumulating in particular tissues in ways not previously described, thereby permitting efficacious treatment of new therapeutic indications. Additionally, the relative amounts of doxorubicin and camptothecin which provide the best results for the conditions described herein differ from the compositions and correlations described in the prior art.

[0005] In one aspect of any of the embodiments, described herein is a polymer-drug conjugate comprising a hyaluronic acid molecule conjugated to: a. at least one doxorubicin molecule; and b. at least one camptothecin molecule.

In some embodiments of any of the aspects, the molar ratio of doxorubicin : camptothecin is 2:1 or a greater relative amount of doxorubicin. In some embodiments of any of the aspects, the molar ratio of doxorubicin : camptothecin is 2:1. In some embodiments of any of the aspects, the molar ratio of doxorubicin : camptothecin is 5: 1 or a greater relative amount of doxorubicin. In some embodiments of any of the aspects, the molar ratio of doxorubicin : camptothecin is 5 : 1. In some embodiments of any of the aspects, the molar ratio of doxorubicin : camptothecin is 15: 1 or a greater relative amount of doxorubicin. In some embodiments of any of the aspects, the molar ratio of doxorubicin : camptothecin is 15: 1. In some embodiments of any of the aspects, the molar ratio of doxorubicin : camptothecin is 11:1 or a greater relative amount of doxorubicin. In some embodiments of any of the aspects, the molar ratio of doxorubicin : camptothecin is 11 : 1 to 20: 1. In some embodiments of any of the aspects, the molar ratio of doxorubicin : camptothecin is 14: 1 to 16:1.

[0006] In some embodiments of any of the aspects, the camptothecin is conjugated to the hyaluronic acid via an ester bond. In some embodiments of any of the aspects, the doxorubicin is conjugated to the hyaluronic acid via an amide bond.

[0007] In some embodiments of any of the aspects, the conjugate has a combination index (C.I.) value with cancerous cells of less than 0.2, less than 0.6, or from 0.6 to 0.1.

[0008] In one aspect of any of the embodiments, described herein is a composition comprising a polymer-drug conjugate as described herein and a salt. In one aspect of any of the embodiments, described herein is a composition comprising micelles, each micelle comprising one or more of the polymer-drug conjugates as described herein and a salt. In some embodiments of any of the aspects, the composition further comprises a salt. In some embodiments of any of the aspects, the salt comprises NaCl or PBS.

[0009] In one aspect of any of the embodiments, provided herein is a composition or polymer- drug conjugate as described herein, formulated for topical application. In one aspect of any of the embodiments, provided herein is a composition or polymer-drug conjugate as described herein, formulated for parenteral application.

[0010] In one aspect of any of the embodiments, provided herein is a composition or polymer- drug conjugate as described herein, for use in a method of treating a skin cancer or precancerous skin lesion, blood cancer, glioblastoma, or intestinal cancer.

[0011] In one aspect of any of the embodiments, provided herein is a method of treating skin cancer or a precancerous skin lesion in a subject in need thereof, the method comprising administering a conjugate or composition as described herein to the subject. In some embodiments of any of the aspects, the conjugate or composition is administered topically. In some embodiments of any of the aspects, is a non-melanoma skin cancer (NMSC). In some embodiments of any of the aspects, is basal cell carcinoma (BCC) or cutaneous squamous cell carcinoma (cSCC). In some embodiments of any of the aspects, the precancerous skin lesion is actinic keratosis (AK). In some embodiments of any of the aspects, the skin cancer and/or precancerous skin lesion is not superficial. In some embodiments of any of the aspects, the skin cancer and/or precancerous skin lesion is not only superficial. In some embodiments of any of the aspects, the skin cancer and/or precancerous skin lesion is present in the dermis.

[0012] In one aspect of any of the embodiments, provided herein is a method of treating blood cancer in a subject in need thereof, the method comprising administering a conjugate or composition as described herein to the subject. In some embodiments of any of the aspects, the blood cancer is acute myeloid leukemia or a T-cell lymphoma.

[0013] In one aspect of any of the embodiments, provided herein is a method of treating glioblastoma in a subject in need thereof, the method comprising administering a conjugate or composition as described herein to the subject. In some embodiments of any of the aspects, the molar ratio of doxorubicin : camptothecin is 2: 1. In some embodiments of any of the aspects, the molar ratio of doxorubicin : camptothecin is 15:1.

[0014] In one aspect of any of the embodiments, provided herein is a method of treating intestinal cancer in a subject in need thereof, the method comprising administering: a. a polymer-drug conjugate comprising a hyaluronic acid molecule conjugated to at least one doxorubicin molecule; or b. a conjugate or composition as described herein; to the subject by intravenous injection. In some embodiments of any of the aspects, the intestinal cancer is gastrointestinal cancer, colon cancer, and/or colorectal cancer.

BRIEF DESCRIPTION OF THE DRAWINGS [0015] Figs. 1A-1D demonstrate the physical characterization of DOCTOR. (Fig. 1A) Differences in FTIR spectra of native HA and DOCTOR confirmed the covalent linkage of DOX and CPT to the HA polymer backbone. (Fig. IB) TEM imaging revealed the transition from fibrillar morphology for native HA to a micellar appearance for DOCTOR (scale bar, 500 nm). (Fig. 1C) Topographic AFM images, 5 pm ^c 5 pm in size (inset: 1 pm ^c 1 pm) of (Top Row) native HA, DOCTOR R2 and (Bottom Row) DOCTOR R5, R15 revealed a fibrillar morphology for native HA and a particulate structure with average mean sizes of 146 nm, 63.5 nm and 72 nm for R2, R5 and R15 respectively. The adjacent color scale represents the height (z-value) for the 5 pm ^c 5 pm frames, respectively. (Fig. ID) SAXS curves for DOCTOR in DI water and PBS obtained via SAXS analysis. Weak or negligible scattering was observed for the conjugates in water (red trace). The scattering intensity is almost flat at the intermediate and high q values, and shows a rise fitted by the model of poly disperse hard spheres at low values of scattering vector q <0.05 A- 1. In PBS (blue trace), the scattering intensity increased by two orders of magnitude and is fitted by worm-like chain or generalized Gaussian coil model described in the Materials Section of Example 1.

[0016] Figs. 2A-2D demonstrate that DOCTOR has increased synergistic potency against precancerous (HaCaT) and cancerous (A431) human keratinocytes with less toxicity to the healthy cells. (Fig. 2A) When treated with HaCaT, the IC50 values of DOCTOR was approximately 19 to 98- fold lower compared to DOX alone and 6 to 8-fold lower for CPT. (Fig. 2B) With A431, the IC50 values of DOCTOR was approximately 17 to 140-fold less compared to DOX alone and 10 to 13-fold less for CPT. (Fig. 2C) With HEKa, the IC50 values of DOCTOR was approximately 30-fold and 7.5 fold higher than that for HaCaT and A431 respectively. (Fig. 2D) DOCTOR R15 induced about 60- 70% of precancerous and cancerous cell death, while more than 7(U80% of healthy keratinocytes still remained viable.

[0017] Figs. 3A-3D demonstrate that DOCTOR delivers the drug pair inside the tissue without causing skin inflammation. (Fig. 3A) Confocal microscopy imaging and (Fig. 3B) quantification via tape-stripping revealed penetration across the stratum comeum and deposition of DOX (red channel) and CPT (blue channel) within the epidermis and dermis of porcine skin ex vivo. (Figs. 3C, 3D) DOCTOR induced negligible inflammation on human skin. Levels of (Fig. 3C, top panel) IL-la and (Fig. 3D, top panel) TNF- a were assessed on a MatTek EpiDerm human skin equivalent model. PBS and 5% SDS were used as negative and positive controls, respectively. Error bars represent mean ± SEM (n=3). Significantly different compared to the negative control: * p < 0.05, ** p < 0.01, and *** p < 0.001. DOCTOR induced negligible skin inflammation in vivo. Levels of (Fig. 3C, bottom panel) IL-la and (Fig. 3D, bottom panel) TNF- a were assessed in mice during a 2-week repeat dose study with DOCTOR. Eftidex® was used as the clinical comparator and PBS as the negative control. Error bars represent mean ± SEM (n=5). Significantly different compared to the negative control: * p <

0.05, ** p < 0.01, and *** p < 0.0001.

[0018] Figs. 4A-4D demonstrate in vivo efficacy. (Figs. 4A-4C) DOCTOR prevents progression of cSCC lesions and enhanced survival by 65% (p<0.0001) at extremely low doses (5.4 mM or 0.015 mg/kg DOX) of the drug. Error bars represent mean ± SEM (n=9). (Fig. 4D) cSCC tumors showing nuclear and cytoplasmic immunostaining (golden brown) for cleaved caspase-3 depicting apoptotic tumor cell death on treatment with DOCTOR R15.

[0019] Figs. 5A-5B demonstrate safety and efficacy in patient-derived primary cells and live- skin explants. (Fig. 5A) When tested on primary cultures of patient-derived cells, DOCTOR induced a maximum of 70% cSCC cell death while more than 80-90% of adjacent non-cancerous skin cells still remained viable. (Fig. 5B) When tested with live cSCC skin and adjacent non-cancerous skin explants, DOCTOR demonstrated a high relative safety to efficacy ratio over the clinical comparator, 5-FU (Eftidex®). The proliferation activity of cSCC and non-cancerous cells were measured by Ki67 staining (red). The number of Ki67-positive cells in the treated explant was normalized to the number of cells in the untreated control for each patient tissue respectively. Quantitative image analysis was performed using ImageJ software on three separate tissue sections per sample after “threshold”-based reduction of background noise. Positive “red” signal was quantified in the entire tissue section for the cSCC samples and in the epidermis only for the matched normal tissues. All samples were stained at the same time under the same conditions to keep the background signal (false positive counts) similar across all samples. An increase in cSCC proliferation activity was observed for untreated and 5-FU treated skin explants. Further, the proliferation of matched healthy skin tissue: cSCC skin tissues was calculated based on quantified proliferation activity for adjacent non-cancerous tissue over the matching cSCC tissue. [0020] Fig. 6 dpeicts a reaction scheme and conditions for HA polymer-drug conjugates at different ratios.

[0021] Fig. 7 depicts FTIR spectra of native HA, free CPT, free DOX, HA-CPT and HA-DOX respectively.

[0022] Fig. 8 depicts FTIR spectra of HA, free CPT, HA-CPT with and without EDC respectively.

[0023] Fig. 9 depicts FTIR spectra of HA, free DOX, HA-DOX with and without EDC respectively.

[0024] Fig. 10 depicts in vitro release rates for DOX-HA-CPT in PBS at 37°C. Each value represents the mean ± SEM (n = 3)

[0025] Figs. 11A-11C depict topographic AFM images, 5 pm ^c 5 pm in size (inset: 1 pm ^c 1 pm) of (Fig. 11A) native HA, (Fig. 1 IB) HA-CPT and (Fig. 11C) HA-DOX revealed a particulate structure with average mean sizes of 115.5 nm and 116.5 nm respectively.

[0026] Fig. 12 depicts NTA measurements for all formulations diluted in PBS. Each value represents the mean ± SEM (n = 3)

[0027] Figs 13A-13C depict scattering functions for HA-CPT, HA-DOX and DOCTOR R5 in DI water (red) and PBS (blue).

[0028] Fig. 14 depicts dose-response curves and IC50 values for free DOX and free CPT treatment with HaCaT and A431.

[0029] Fig. 15 depicts confocal microscopy imaging revealed co-localization of DOX and CPT (cyan, white arrow - merge) in cells indicating enhanced potency and synergy is due to the simultaneous uptake and accumulation of the drug pair.

[0030] Fig. 16 demonstrates that less than 1% of the applied CPT dose was detected in the plasma following continuous ‘ten’ topical application of the conjugates. PBS was used as the negative control. Error bars represent mean ± SEM (n=5).

[0031] Fig. 17 demonstrates that in vivo efficacy studies revealed that DOCTOR could reduce or eliminate a cSCC tumor at extremely low doses (5.4 mM or 0.016 mg/kg DOX) of chemotherapy. [0032] Figs. 18A-18G depict Safety and Efficacy in primary human cells from patients. (Figs. 18A, 18B & 18C) DOCTOR induced a maximum of 70% cSCC lesion cell death while more than 80- 90% of matched normal cells still remained viable. (Figs. 18D, 18E & 18F). DOCTOR revealed a highly synergistic potency with safety against cSCC when compared to the individual drug conjugates. (Fig. 18G) DOCTOR was more potent than 5FU, the clinical comparator in treating cSCC.

[0033] Figs. 19A-19D depict physical characterization of DOCTOR conjugates. “HDC” = DOCTOR. [0034] Fig. 20 depicts graphs of AML cell line viability after treatment with the indicated compositions. “HDC” = DOCTOR.

[0035] Fig. 21 depicts charts of AML cell line viability, IC50, and Cl after treatment with the indicated compositions. “HDC” = DOCTOR.

[0036] Fig. 22 depicts graphs of T-cell lymphoma cell line viability after treatment with the indicated compositions. “HDC” = DOCTOR.

[0037] Fig. 23 depicts charts of T-cell lymphoma cell line viability, IC50, and Cl after treatment with the indicated compositions. “HDC” = DOCTOR.

[0038] Fig. 24 depicts a graph and chart of the efficacy of the indicated DOCTOR compositions in the killing of Glioblastoma GL261-luc2 cells at 48 hours. “HDC” = DOCTOR.

[0039] Figs. 25A-25B depict characterization of HA-Dox: (Fig. 25A) Representative TEM images and (Fig. 25B) size distribution of HA-Dox in PBS determined by DLS. Thick white line represents scale bar of lpm (Fig. 25A, left panel) and 200nm (Fig. 25A, right panel)

[0040] Fig. 26 depicts the pharmacokinetics of HA-Dox: Pharmacokinetics both free Dox and HA-Dox represented as %ID in blood at 0.08h, 0.5h, 6h, and 24h. Each data point represent means ± SEM (n=3).*, P< 0.05; non paired two-tailed t-test.

[0041] Figs. 27A-27C demonstrate the binding and Biocompatibility of HA-Dox on RBC: (Fig. 27A) Representative confocal image, scale bar = 5pm, and (Fig. 27B) flow cytometry histogram displaying percentage of RBC with HA-Dox bound to their surface in the presence of 55% serum. (Fig. 27C) Representative plot displaying percentages of phosphatidylserine exposing RBC of HA- Dox attached onto RBC. Polystyrene beads were used as a positive control. Values are means (n>10)

± SEM, Inset Representative agglutination image, visualized using a U-shaped bottom plate. (1)

Naive RBC; (2) RBC:HA-Dox; (3) RBCPolystyrene beads.

[0042] Figs. 28A-28B depict the biodistribution of HA-Dox: Biodistribution of Dox and HA- Dox. Bar graph represents percentages of injected dose (%ID) for free Dox and HA-Dox following IV injection in mice at (Fig. 28A) 0.5h and (Fig. 28B) 24h. Each data point represent means ± SEM (n=3).*, P< 0.05; non paired, two-tailed t-test.

[0043] Figs. 29A-29D demonstrate the accumulation of HA-Dox in Intestines: Kinetics of HA- Dox represented as percentages of injected dose (%ID) for free Dox and HA-Dox following I.V. injection in mice at (Fig. 29A) 0.083h (Fig. 29B) 0.5h (Fig. 29C) 6h and (Fig. 29D) 24h in different part of murine intestines. The amount of HA-Dox at 24h Each data point represent means ± SEM (n=3).*, P< 0.05; non paired, two-tailed t-test.

[0044] Figs. 30A-30B demonstrate the preventive effect of HA-Dox on inflammation-linked carcinogenesis in AOM/DSS mouse models: ((Fig. 30A) representative intestines and (Fig. 30B) stomachs are shown. Healthy mice were not injected with AOM and received distilled water only. Healthy; n=4 mice; saline, Dox, and HA-Dox, n=6-7 mice; Error bars, SEM. ###, P < 0.001 vs healthy; ***, P <0.001 vs saline. Each data point represent means ± SEM (n=3).*, P< 0.05; non paired, two-tailed t-test.

[0045] Figs. 31A-3 ID depict Histological Analysis of Murine Intestinal Tissues Induced with Chemical Induced Colon Cancer: Histology of Intestinal Tissues (Fig. 31 A) Duodenum, (Fig. 3 IB) Jejunum; (Fig. 31C) Ileum, and (Fig. 3 ID) Colon treated with azoxymethane and dextran sulfate treated with saline, Free Dox, and HA-Dox, determined by H and E staining; Representative images were taken at lOOx (top) and 400x (bottom).

[0046] Fig. 32 depicts cell proliferation analysis of Murine Intestinal Tissues Induced with Chemical Induced Colon Cancer: Immunohistochemistry of Intestinal Tissues treated with azoxymethane and dextran sulfate treated with saline, Free Dox, and HA-Dox, determined by Ki67. Representative images were taken at 400x. Red scale bar: 50nm

[0047] Fig. 33 depicts the accumulation of HA-Dox in Perfused Intestines:. The amount of HA- Dox represented as percentage of injected dose (%ID) for free Dox and HA-Dox following I.V. injection in mice at 24h after perfusion compared to no perfusion remaining in different parts of murine intestine. Each data point represent means ± SEM (n=3).*, P< 0.05; non paired, two-tailed t- test.

[0048] Figs. 34A-34C demonstrate the accumulation of HA-Dox in Intestines: Representative flow cytometry histograms portraying the presence of HA-Dox following I.V. injection in mice at 24h in (Fig. 34A) Duodenum and Jejunum (Fig. 34B) Ileum and Colon and (Fig. 34C) Cecum homogenates. Dox spiked in was used as a positive control.

[0049] Fig. 35 depicts the accumulation of HA-Dox in Intestines: Representative confocal images portraying the presence of HA-Dox represented by dots following I.V. injection in mice at 0.083h and 6h in Duodenum and Jejunum Ileum and Colon and Cecum. Images were taken with a 40x objective

[0050] Figs. 36A-36B depict the biodistribution of HA-647 in Intestines: Representative ex vivo fluorescence image obtained with IVIS of GI organs (Fig. 36A) 0.08h and (Fig. 36B) 0.5h after I.V. administration of HA conjugated with alexafluor 647. Organs: (1) Stomach; (2) Duodenum; (3) Jejunum; (4) Ileum; (5) Cecum; and (6) Colon. A scale of the radiance efficiency is presented to the right of excised mouse organ image.

[0051] Figs. 37A-37E depict the biodistribution of HA-Dox-647 in Intestine: Representative ex vivo fluorescence image obtained with IVIS of GI organs (Fig. 37A) 0.08h and (Fig. 37B) 0.5h after I.V. administration of HA-Dox conjugated with Alexafluor 647. Organs (1) Stomach; (2) Duodenum; (3) Jejunum; (4) Ileum; (5) Cecum; (6) Colon. A scale of the radiance efficiency is presented to the right of excised mouse organ image. Representative flow cytometric analysis of Dox presence in (Fig. 37C) Duodenum and Jejunum (Fig. 37D) Ileum and Colon (Fig. 37E) Cecum homogenates 0.08h and 0.5h after I.V. administration [0052] Figs. 38A-38B demonstrate inflammation analysis of Murine Intestinal Tissues Induced with Chemical Induced Colon Cancer: Immunohistochemistry of Intestinal Tissues treated with azoxymethane and dextran sulfate treated with saline, Dox, and HA-Dox, determined by (Fig. 38 A) Cox-2 and (Fig. 38B) iNOS staining. Representative images were taken at 400x. Red scale bar: 50nm [0053] Figs. 39A-39B demonstrate apoptotic analysis of Murine Intestinal Tissues Induced with Chemical Induced Colon Cancer: Immunohistochemistry of Intestinal Tissues treated with azoxymethane and dextran sulfate treated with saline, Dox, and HA-Dox, determined by (Fig. 39A) Caspase-3 and (Fig. 39B) Bax staining. Representative images were taken at 400x. Red scale bar: 50nm

DETAILED DESCRIPTION

[0054] Described herein are polymer-drug conjugates which have demonstrated surprising efficacy. In one aspect of any of the embodiments, described herein is a polymer-drug conjugate comprising a hyaluronic acid molecule conjugated to: a. at least one doxorubicin molecule; and b. at least one camptothecin molecule.

[0055] As used herein “polymer-drug conjugate” refers to a molecule comprising at least one polymer and at least one drug conjugated to each other. The term “conjugate,” as it relates to polymer- drug conjugate refers to two or more molecular structures that are linked by a direct or indirect covalent or non-covalent bond. Non-covalent interactions include, but are not limited to, electrostatic interactions, hydrogen bonding interactions, van der Waals interactions, dipole-dipole interactions, p- p stacking, magnetic interactions, and metal coordination. Preferably, the conjugation is via covalent bonds.

[0056] The drugs are conjugated directly or indirectly, via linkers, to the backbone or side chains of one or more polymers. In a polymer-drug conjugate comprising two or more drugs, the strengths of the bonds involved in direct conjugation, and the strengths of the bonds in the linkers, may be the same, different, or a combination thereof (i.e. some of the bond strengths are the same while others are different). In some embodiments, the side chains have the same or different chemical moieties. The bond strengths of the bonds, directly conjugating the two or more drugs to the polymers are the same, different, or a combination thereof (i.e. some of the bond strengths are the same while others are different). The bond strengths of the bonds indirectly conjugating the two or more drugs via linkers are the same, different, or a combination thereof (i.e. some of the bond strengths are the same while others are different). For example, when the bonds or linkers have the same bond strength, each of the drugs is connected to the polymer via the same bond or the same linker. When the bonds or linkers have different bond strengths, each of the drugs is connected to the polymer via a different bond or different linker. [0057] The linkers (or a portion thereof) or one or more of the bonds between a drug and a linker may be cleaved at the same rate or a different rate as the cleavage of another linker or one or more of the bonds between a different drug and another linker in the pharmaceutical composition. Cleavage can occur by any suitable mechanism, such as via hydrolysis, enzymatic cleavage, the application of thermal energy, photoenergy, or a combination thereof.

[0058] The linkers may be homo-bif mctional or hetero-bifuctional. In some instances, combinations of homo-bifunctional linkers and hetero-bif mctional linkers are used.

[0059] Examples of homo-bifimctional linkers include, but are not limited to adipic acid dihydrazide, amino acids such as glycine, aldehydes such as ethanedial, pyruvaldehyde, 2-formyl- malonaldehyde, glutaraldehyde, adipaldehyde, heptanedial, octanedial; di-glycidyl ether, diols such as 1,2-ethanediol, 1,3 -propanediol, 1,4-butanediol, 2,3-butanediol, 1,5-pentanediol, benzene- 1,4-diol, 1,6-hexanediol, tetra(ethylene glycol) diol), PEG, di-thiols such as 1,2-ethanedithiol, 1,3- propane dithiol , 1,4-butanedithiol, 2,3-butanedithiol, 1,5-pentanedithiol, benzene- 1,4-dithiol, 1,6- hexanedithiol, tetra(ethylene glycol) dithiol), di -amine such as ethylene diamine, propane- 1,2- diamine, propane-1, 3-diamine, N-methylethylenediamine, N,N'-dimethylethylenediamine, pentane- 1, 5-diamine, hexane- 1,6-diamine, spermine and spermidine, divinyladipate, divinylsebacate, diamine- terminated PEG, double-ester PEG-N-hydroxysuccinimide, and di-isocyanate-terminated PEG. In a preferred embodiment, the homo-bifunctional linker is adipic acid dihydrazide.

[0060] Examples of hetero-bifimctional linkers include, but are not limited to, epichlorohydrin, S-acetylthioglycolic acid N-hydroxysuccinimide ester, 5-azido-2-nitrobenzoic acid N- hydroxysuccinimide ester, 4-azidophenacyl bromide, bromoacetic acid N-hydroxysuccinimide ester, N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide, Iodoacetic acid N-hydroxysuccinimide ester, 4-(N- mMaleimido)benzophenone 3-(2-pyridyldithio)propionic acid N-hydroxysuccinimide ester 3- maleimidobenzoic acid N-hydroxysuccinimide ester, N,N’-cystamine-bis-acrylamide, N,N’- methylene-bis-acrylamide and N,N’-ethylene-bis-acrylamide.

[0061] In some embodiments of any of the aspects, the camptothecin is conjugated to the hyaluronic acid via an ester bond. In some embodiments of any of the aspects, the doxorubicin is conjugated to the hyaluronic acid via an amide bond.

[0062] Polymer-drug conjugates allow one to modify the ratio between drugs using stoichiometry and/or to modify the schedule of delivery (i.e. release of the drug from the polymer- drug conjugate). A desired schedule of delivery for each drug in the pharmaceutical composition can be achieved by varying the bond strength of the bond between the drug and the polymer or the bond strength of the bond in a linker used to conjugate the drug to the polymer to achieve a synergistic effect when two or more drugs are delivered.

[0063] In some embodiments of any of the aspects, the polymer is hyaluronic acid. [0064] In some embodiments of any of the aspects, the at least one drug of a polymer-drug conjugate comprises, consists of, or consists essentially of doxorubicin. In some embodiments of any of the aspects, the at least one drug of a polymer-drug conjugate comprises, consists of, or consists essentially of camptothecin. In some embodiments of any of the aspects, the at least one drug of a polymer-drug conjugate comprises, consists of, or consists essentially of the combination of doxorubicin and camptothecin. In some embodiments of any of the aspects, a polymer-drug conjugate comprises at least one doxorubicin molecule. In some embodiments of any of the aspects, a polymer-drug conjugate comprises at least one camptothecin molecule. In some embodiments of any of the aspects, a polymer-drug conjugate comprises at least one doxorubicin molecule and at least one camptothecin molecule.

[0065] When the polymer-drug conjugate comprises both doxorubicin and camptothecin, the relative amounts of the two drugs in the conjugate can determine the efficacy of the conjugate. For example, conjugates with particular ratios of doxorubin and camptothecin are shown to work particularly well via certain routes of administration, or to treat particular diseases. In particular, USSN 15/556,798 describes the use of doxorubin and camptothecin for treatment of breast cancer and demonstrates that camptothecin should be present at double the amount of doxorubicin for any positive effect of the combination. However, the instant application demonstrates that polymer-drug conjugates comprising more doxorubin than camptothecin demonstrate surprising efficacy when administered topically or to treat cancers other than breast cancer. This is directly opposite of the effect expected from the data presented in USSN 15/556,798.

[0066] In some embodiments of any of the aspects, the molar ratio of doxorubicin : camptothecin is about 2: 1 or a greater relative amount of doxorubicin. In some embodiments of any of the aspects, the molar ratio of doxorubicin : camptothecin is 2:1 or a greater relative amount of doxorubicin. In some embodiments of any of the aspects, the molar ratio of doxorubicin : camptothecin is about 2:1.

In some embodiments of any of the aspects, the molar ratio of doxorubicin : camptothecin is 2: 1. In some embodiments of any of the aspects, the molar ratio of doxorubicin : camptothecin is about 5 : 1 or a greater relative amount of doxorubicin. In some embodiments of any of the aspects, the molar ratio of doxorubicin : camptothecin is 5: 1 or a greater relative amount of doxorubicin. In some embodiments of any of the aspects, the molar ratio of doxorubicin : camptothecin is about 5 : 1. In some embodiments of any of the aspects, the molar ratio of doxorubicin : camptothecin is 5 : 1. In some embodiments of any of the aspects, the molar ratio of doxorubicin : camptothecin is about 15:1 or a greater relative amount of doxorubicin. In some embodiments of any of the aspects, the molar ratio of doxorubicin : camptothecin is 15:1 or a greater relative amount of doxorubicin. In some embodiments of any of the aspects, the molar ratio of doxorubicin : camptothecin is about 15:1. In some embodiments of any of the aspects, the molar ratio of doxorubicin : camptothecin is 15:1. [0067] In some embodiments of any of the aspects, the molar ratio of doxorubicin : camptothecin is is about 11 : 1 or a greater relative amount of doxorubicin. In some embodiments of any of the aspects, the molar ratio of doxorubicin : camptothecin is is 11 : 1 or a greater relative amount of doxorubicin.

[0068] In some embodiments of any of the aspects, the molar ratio of doxorubicin : camptothecin is is about 11 : 1 to about 20: 1. In some embodiments of any of the aspects, the molar ratio of doxorubicin : camptothecin is is 11 : 1 to 20: 1. In some embodiments of any of the aspects, the molar ratio of doxorubicin : camptothecin is is about 14: 1 to about 16: 1. In some embodiments of any of the aspects, the molar ratio of doxorubicin : camptothecin is is 14: 1 to 16:1.

[0069] As described herein, polymer-drug conjugates comprising two or more drugs provide a synergistic effect, e.g., a synergism not observed when the two or more drugs are administered without being part of a polymer-drug conjugate. Such synergism can be measured by a combination index (C.I.) as described in the Examples herein. In some embodiments of any of the aspects, a polymer drug conjugate described herein has a combination index (C.I.) value with cancerous cells of less than 0.6.In some embodiments of any of the aspects, a polymer drug conjugate described herein has a combination index (C.I.) value with cancerous cells of less than 0.2. In some embodiments of any of the aspects, a polymer drug conjugate described herein has a combination index (C.I.) value with cancerous cells of less than 0.1. In some embodiments of any of the aspects, a polymer drug conjugate described herein has a combination index (C.I.) value with cancerous cells of from about 0.6 to about 0.1. In some embodiments of any of the aspects, a polymer drug conjugate described herein has a combination index (C.I.) value with cancerous cells of from 0.6 to 0.1.

[0070] It is further described herein that when polymer-drug conjugates described herein are combined with a salt, they form micelles wheich are contemplated herein to improve penetration and drug delivery, e.g, particularly when administered topically. Accordingly, in one aspect of any of the embodiments, described herein is a composition comprising a polymer-drug conjugate described herein and a salt. In one aspect of any of the embodiments, described herein is a composition comprising micelles, each micelle comprising one or more of the polymer-drug conjugates described herein and optionally further comprising a salt. Suitable salts can include, but are not limited to NaCl or PBS.

[0071] In some embodiments of any of the aspects, a component or element of a composition/conjugate/ is present at the stated concentration or within 5% thereof, thereby accounting for minor errors, inaccuracies, or deviations in measurement, mixing, and/or solubility. In some embodiments of any of the aspects, a component or element of a composition/conjugate is present at the stated concentration or within 1% thereof, thereby accounting for minor errors, inaccuracies, or deviations in measurement, mixing, and/or solubility. [0072] In some embodiments, the technology described herein relates to a pharmaceutical composition comprising a composition and/or conjugate as described herein, and optionally a pharmaceutically acceptable carrier. In some embodiments, the active ingredients of the pharmaceutical composition comprise a composition and/or conjugate as described herein. In some embodiments, the active ingredients of the pharmaceutical composition consist essentially of a composition and/or conjugate as described herein. In some embodiments, the active ingredients of the pharmaceutical composition consist of a composition and/or conjugate as described herein. Pharmaceutically acceptable carriers and diluents include saline, aqueous buffer solutions, solvents and/or dispersion media. The use of such carriers and diluents is well known in the art. Some non limiting examples of materials which can serve as pharmaceutically-acceptable carriers include: (1) sugars, such as lactose, glucose and sucrose; (2) starches, such as com starch and potato starch; (3) cellulose, and its derivatives, such as sodium carboxymethyl cellulose, methylcellulose, ethyl cellulose, microcrystalline cellulose and cellulose acetate; (4) powdered tragacanth; (5) malt; (6) gelatin; (7) lubricating agents, such as magnesium stearate, sodium lauryl sulfate and talc; (8) excipients, such as cocoa butter and suppository waxes; (9) oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, com oil and soybean oil; (10) glycols, such as propylene glycol;

(11) polyols, such as glycerin, sorbitol, mannitol and polyethylene glycol (PEG); (12) esters, such as ethyl oleate and ethyl laurate; (13) agar; (14) buffering agents, such as magnesium hydroxide and aluminum hydroxide; (15) alginic acid; (16) pyrogen-free water; (17) isotonic saline; (18) Ringer's solution; (19) ethyl alcohol; (20) pH buffered solutions; (21) polyesters, polycarbonates and/or polyanhydrides; (22) bulking agents, such as polypeptides and amino acids (23) serum component, such as semm albumin, HDL and LDL; (22) C2-C12 alcohols, such as ethanol; and (23) other non toxic compatible substances employed in pharmaceutical formulations. Wetting agents, coloring agents, release agents, coating agents, sweetening agents, flavoring agents, perfuming agents, preservative and antioxidants can also be present in the formulation. The terms such as "excipient", "carrier", "pharmaceutically acceptable carrier" or the like are used interchangeably herein. In some embodiments, the carrier inhibits the degradation of the active agent as described herein.

[0073] In some embodiments of any of the aspects, the compositions and/or conjugates described herein are formulated for topical administration. In some embodiments, the compositions and/or conjugates are formulated for topical delivery as a powder, ointment or salve, aerosol, gel, emulsion, foam, cream or lotion. Appropriate doses and formulations of a composition and/or gel for topical application or for a transdermal administration can be determined by an ordinary skilled artisan according to known methods, such as those discussed in Hesse link, 2016 Topical Analgesics: Critical Issues Related to Formulation and Concentration. J Pain Relief 5:274, or Casale R, et ah, Topical Treatments for Localized Neuropathic Pain. Curr Pain Headache Rep. 2017 Mar;21(3): 15, which ares incorporated herein in their entirety by reference. [0074] In some embodiments, a composition and/or conjugates for use in the compositions, kits, and methods as disclosed herein are for topical application and may include a cosmetically-acceptable topical carrier. A cosmetically-acceptable topical carrier may contain ingredients commonly used, such as water, monoalcohols (such as ethanol and isopropanol); glycols and polyols (such as glycerin, propylene glycol, propanediol, 1,4-butanediol, 1,3-butanediol, 1,2-butanediol, hydroxy ethyl urea, sorbitol, sorbitan, xylitol and polyglycerols); glycerin, and combinations thereof. According to certain embodiments, a carrier can includes water. The amount of cosmetically-acceptable topical carrier in the composition may range from about 30% to about 99%, such as from about 40% to about 95%, such as from about 50% to about 95%, such as from about 60% to about 90% by weight of the composition.

[0075] In some embodiments, a composition and/or conjugates is in the form of a concentrate.

[0076] In some embodiments, a composition and/or conjugates may include additional ingredients, e.g., those commonly used in topical compositions. Examples of additional ingredients include but are not limited to surfactants/emulsifiers (cationic, anionic, non-ionic, and zwitterionic), humectants, emollients and hydrophobic compounds, conditioning agents, opacifying agents, chelating agents, conditioning agents, additional preservatives, skin benefit agents, fragrances, water- soluble or dispersible polymers, and active ingredients (e.g., sunscreens, anti-aging actives, anti-acne actives, antibiotics, antimicrobial agents, and the like).

[0077] In some embodiments, a composition and/or conjugates is aqueous and the pH of the composition is about 6.5 or greater, such as from about 6.5 to about 8.5, such as from about 7.5 to about 8.5.

[0078] In particular embodiments, a composition and/or conjugates is topically applied to, or within close proximity to a tumor, lesion, or cancer in the subject, and is not rinsed from the tumor, lesion, or cancer. In some embodiments, topical administration of a composition and/or conjugate as disclosed herein relates to applying or administering the composition and/or conjugate to a specific area on, or in the body, for example, a composition and/or conjugate is applied on the skin surrounding a tumor, lesion, or cancer, or at, or near a target site, e.g., at or in close proximity to tumor, lesion, or cancer, for example, on the exposed surface of the tumor, lesion, or cancer. In some embodiments, the composition and/or conjugate may be contained within or be in fluid communication with an applicator that is suitable for dispensing it.

[0079] The composition and/or conjugate as disclosed herein can take the form of solutions, suspensions, emulsions, pellets, multiparticulates, capsules, capsules containing liquids, powders, sustained-release formulations, aerosols, sprays, ointments, gels, salves, plasters, transdermal patches, suspensions, or any other topical form suitable for use. In one embodiment, the composition and/or conjuate is in the form of a capsule (see e.g., U.S. Patent No. 5,698,155). Other examples of suitable pharmaceutical excipients are described by Radebough et al, "Preformulation," pp. 1447-1676 in Remington's Pharmaceutical Sciences Vol. 2 (Gennaro, ed., 19^th ed., Mack Publishing, Easton, PA, 1995), incorporated herein by reference.

[0080] In some embodiments, a composition and/or conjugate is applied in a transdermal formulation. In alternative embodiments, the composition and/or conjugate is formulated for non- transdermal application. Dosages of a composition and/or conjugate for topical administration or administration via a transdermal patch can be determined by one of ordinary skill in the art.

[0081] Dosage forms for topical administration of a composition and/or conjugate as disclosed herein powders, sprays, ointments and in some embodiments, inhalants. In some embodiments, a composition and/or conjugate may be mixed under sterile conditions with a pharmaceutically acceptable carrier and any needed preservatives, buffers or propellants which may be required.

[0082] In another embodiment, a composition and/or conjugate as disclosed herein, or a pharmaceutically acceptable salt or solvate thereof can be topically administered in a vesicle, in particular a liposome (see Langer, "New Methods of Drug Delivery," Science 249: 1527-1533 (1990); Lopez-Berestein, "Treatment of Systemic Fungal Infections with Liposomal-Amphotericin B," Liposomes in the Therapy of Infectious Disease and Cancer, pp. 317-327 (1989); and Treat et al, "Liposome encapsulated doxorubicin - preliminary results of phase I and phase II trials" Liposomes in the Therapy of Infectious Disease and Cancer, pp. 353-365 (1989). Compositions and/or conjugates as disclosed herein invention can also be administered in the form of liposomes. As is known in the art, liposomes are generally derived from phospholipids or other lipid substances. Liposomes are formed by mono- or multi-lamellar hydrated liquid crystals which are dispersed in an aqueous medium. Any non-toxic, physiologically acceptable and metabolizable lipid capable of forming liposomes can be used. The present compositions in liposome form can contain, in addition to a compound of the present invention, stabilizers, preservatives, excipients and the like. The preferred lipids are natural and synthetic phospholipids and phosphatidyl cholines (lecithins) used separately or together.

Methods to form liposomes are known in the art. See, for example, Prescott, Ed., Methods in Cell Biology, Volume XIV, Academic Press, New York, N.Y. (1976), p. 33 et seq.

[0083] In some embodiments, a composition and/or conjugate as disclosed herein, or a pharmaceutically acceptable salt or solvate thereof can be topically administered via a nanoparticle or microparticle, e.g., nanoparticles and microparticles formulated for skin drug delivery, as disclosed in Prow et at al, Nanoparticles and microparticles for skin drug delivery, Advanced Drug Delivery Reviews, 2011, 63(6), 470-491, which is incorporated herein in its entirety by reference.

[0084] A composition and/or conjugate as disclosed herein can optionally comprise a suitable amount of a pharmaceutically acceptable excipient so as to provide the form for proper topical administration to the animal. Such a pharmaceutical excipient can be a diluent, suspending agent, solubilizer, binder, disintegrant, preservative, coloring agent, lubricant, and the like. The pharmaceutical excipient can be a liquid, such as water or an oil, including those of petroleum, animal, vegetable, or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil, and the like. In some embodiments, a pharmaceutical excipient can be saline, gum acacia, gelatin, starch paste, talc, keratin, colloidal silica, urea, and the like. In addition, auxiliary, stabilizing, thickening, lubricating, and coloring agents can be used. In one embodiment, the pharmaceutically acceptable excipient is sterile when administered to a subject or animal. For topical application, saline solutions and aqueous dextrose and glycerol solutions can also be employed as liquid excipients. Suitable pharmaceutical excipients also include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene glycol, water, ethanol, and the like. The compositions, if desired, can also contain wetting or emulsifying agents, or pH buffering agents.

[0085] In one embodiment, a composition and/or conjugate as disclosed herein for topical administration comprise sterile isotonic aqueous buffer. Where necessary, the compositions can also include a solubilizing agent.

[0086] In some embodiments, a composition and/or conjugate as disclosed herein and a pharmaceutically acceptable salt and solvate thereof can be administered by controlled-release or sustained-release means or by delivery devices that are known to those of ordinary skill in the art. Examples include, but are not limited to, those described in U.S. Patent Nos.: 3,845,770; 3,916,899; 3,536,809; 3,598,123; 4,008,719; 5,674,533; 5,059,595; 5,591,767; 5,120,548; 5,073,543; 5,639,476; 5,354,556; and 5,733,566, each of which is incorporated herein by reference. Such dosage forms can be used to provide controlled- or sustained-release of one or more active ingredients using, for example, hydropropylmethyl cellulose, ethylcellulose, other polymer matrices, gels, permeable membranes, osmoptic systems, multilayer coatings, microparticles, liposomes, microspheres, or a combination thereof to provide the desired release profile in varying proportions. Suitable controlled- or sustained-release formulations known to those of ordinary skill in the art, including those described herein, can be readily selected for use with the composition and/or gel as disclosed herein. Therefore, also encompassed herein are single unit dosage forms of a composition and/or gel for topical administration such as, but not limited to, dermal patches, matrix, dressing, sponge, dermal patch, salve, cream, lotion, foam, emulsion, ointment, powder or gel that are adapted for controlled- or sustained-release.

[0087] Controlled- or sustained-release pharmaceutical compositions can have a common goal of improving drug therapy over that achieved by their non-controlled or non-sustained release counterparts. In one embodiment, a controlled- or sustained-release composition comprises a minimal amount of a composition and/or conjugate as disclosed herein or a pharmaceutically acceptable salt or solvate thereof to cure or control the condition in a minimum amount of time. Advantages of controlled- or sustained-release compositions include extended activity of the drug, reduced dosage frequency, and increased patient compliance. In addition, controlled- or sustained-release compositions can favorably affect the time of onset of action or other characteristics, such as blood or skin levels of a composition and/or gel as disclosed herein and can thus reduce the occurrence of adverse side effects.

[0088] Controlled- or sustained-release compositions can be designed to immediately release an amount of a composition and/or conjugate as disclosed herein or a pharmaceutically acceptable salt or solvate thereof that promptly produces the desired therapeutic or prophylactic effect, and gradually and continually release other amounts of a composition and/or conjugate as disclosed herein, or a second therapeutic agent or a pharmaceutically acceptable salt or solvate thereof to maintain this level of therapeutic or prophylactic effect over an extended period of time. To maintain a constant level of a composition and/or conjugate as disclosed herein at the site of the wart, diseased area, tumor, or cancer, the composition and/or conjugate or a pharmaceutically acceptable salt or solvate thereof can be released from the dosage form at a rate that will replace the amount of composition and/or conjugate being metabolized or broken down or excreted from the body. Controlled- or sustained- release of an active ingredient can be stimulated by various conditions, including but not limited to, changes in pH, changes in temperature, concentration or availability of enzymes, concentration or availability of water, or other physiological conditions or compounds.

[0089] In yet another embodiment, a composition and/or conjugate as disclosed herein or a pharmaceutically acceptable salt or solvate thereof can be delivered in a controlled-release system or sustained-release system (see, e.g., Goodson, "Dental Applications," pp. 1 15-138 in Medical Applications of Controlled Release, Vol. 2, Applications and Evaluation, Langer and Wise, eds., CRC Press (1984), hereafter "Goodson"). Other controlled- or sustained-release systems discussed in the review by Langer, Science 249: 1527-1533 (1990) can be used. In one embodiment, a pump can be used (Langer, Science 249:1527-1533 (1990); Sefton, "Implantable Pumps," in CRC Crit. Rev. Biomed. Eng. 1_4(3):201 -240 (1987); Buchwald et al, "Long-term, Continuous Intravenous Heparin Administration by an Implantable Infusion Pump in Ambulatory Patients with Recurrent Venous Thrombosis," Surgery 88:507-516 (1980); and Saudek et al., "A Preliminary Trial of the Programmable Implantable Medication System for Insulin Delivery," New Engl. J. Med. 321 :574- 579 (1989)). In another embodiment, polymeric materials can be used (see Goodson; Smolen et al, "Drug Product Design and Performance," Controlled Drug Bioavailability Vol. 1, John Wiley & Sons, New York (1984); Langer et al, "Chemical and Physical Structure of Polymers as Carriers for Controlled Release of Bioactive Agents: A Review," J. Macromol. Sci. Rev. Macromol. Chem. C23(l):61-126 (1983); Levy et al, "Inhibition of Calcification of Bioprosthetic Heart Valves by Local Controlled-Release Diphosphonate," Science 228: 190-192 (1985); During et al, "Controlled Release of Dopamine from a Polymeric Brain Implant: In Vivo Characterization," Ann. Neurol. 25:351-356 (1989); and Howard et al, "Intracerebral drug delivery in rats with lesion-induced memory deficits," J. Neurosurg. 71: 105 (1989)). In yet another embodiment, a controlled- or sustained-release system comprising a composition and/or cojugate as disclosed herein can be placed in proximity of the lesion, tumor, or cancer, thus requiring only a fraction of the systemic dose.

[0090] In some embodiments, compositions as disclosed herein comprising a composition and/or conjugate may also contain adjuvants such as preservatives, wetting agents, emulsifying agents and dispersing agents. Prevention of the action of microorganisms can be ensured by the inclusion of various antibacterial and antifungal agents, for example, paraben, chlorobutanol, phenol sorbic acid and the like. It may also be desirable to include isotonic agents such as sugars, sodium chloride and the like. Prolonged absorption of the topical formulation of a composition and/or gel can be brought about by the inclusion of agents which delay absorption such as aluminum monostearate and gelatin. [0091] In some embodiments, a composition and/or conjugate as disclosed herein is topically administered alone, or in combination with one or more compositions and/or conjugate as disclosed herein, or in combination (i.e. co-administered) with one or more additional treatments (i.e., a second treatment). In some emboidments of any of the aspects, the methods described herein can further comprise administering a second agent and/or treatment to the subject, e.g. as part of a combinatorial therapy. Non-limiting examples of a second agent and/or treatment can include radiation therapy, surgery, gemcitabine, cisplastin, paclitaxel, carboplatin, bortezomib, AMG479, vorinostat, rituximab, temozolomide, rapamycin, ABT-737, PI-103; alkylating agents such as thiotepa and CYTOXAN® cyclosphosphamide; alkyl sulfonates such as busulfan, improsulfan and piposulfan; aziridines such as benzodopa, carboquone, meturedopa, and uredopa; ethylenimines and methylamelamines including altretamine, triethylenemelamine, trietylenephosphoramide, triethiylenethiophosphoramide and trimethylolomelamine; acetogenins (especially bullatacin and bullatacinone); a camptothecin (including the synthetic analogue topotecan); bryostatin; callystatin; CC-1065 (including its adozelesin, carzelesin and bizelesin synthetic analogues); cryptophycins (particularly cryptophycin 1 and cryptophycin 8); dolastatin; duocarmycin (including the synthetic analogues, KW-2189 and CB1- TM1); eleutherobin; pancratistatin; a sarcodictyin; spongistatin; nitrogen mustards such as chlorambucil, chlomaphazine, cholophosphamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, novembichin, phenesterine, prednimustine, trofosfamide, uracil mustard; nitrosureas such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine, and ranimnustine; antibiotics such as the enediyne antibiotics (e.g., calicheamicin, especially calicheamicin gammall and calicheamicin omegall (see, e.g., Agnew, Chem. Inti. Ed. Engl., 33: 183-186 (1994)); dynemicin, including dynemicin A; bisphosphonates, such as clodronate; an esperamicin; as well as neocarzinostatin chromophore and related chromoprotein enediyne antiobiotic chromophores), aclacinomysins, actinomycin, authramycin, azaserine, bleomycins, cactinomycin, carabicin, caminomycin, carzinophilin, chromomycinis, dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, ADRIAMYCIN® doxorubicin (including morpholino- doxorubicin, cyanomorpholino-doxorubicin, 2-pyrrolino-doxorubicin and deoxydoxorubicin), epirubicin, esorubicin, idarubicin, marcellomycin, mitomycins such as mitomycin C, mycophenolic acid, nogalamycin, olivomycins, peplomycin, potfiromycin, puromycin, quelamycin, rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex, zinostatin, zorubicin; anti-metabolites such as methotrexate and 5-fluorouracil (5-FU); folic acid analogues such as denopterin, methotrexate, pteropterin, trimetrexate; purine analogs such as fludarabine, 6-mercaptopurine, thiamiprine, thioguanine; pyrimidine analogs such as ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine; androgens such as calusterone, dromostanolone propionate, epitiostanol, mepitiostane, testolactone; anti-adrenals such as aminoglutethimide, mitotane, trilostane; folic acid replenisher such as frolinic acid; aceglatone; aldophosphamide glycoside; aminolevulinic acid; eniluracil; amsacrine; bestrabucil; bisantrene; edatraxate; defofamine; demecolcine; diaziquone; elformithine; elliptinium acetate; an epothilone; etoglucid; gallium nitrate; hydroxyurea; lentinan; lonidainine; maytansinoids such as maytansine and ansamitocins; mitoguazone; mitoxantrone; mopidanmol; nitraerine; pentostatin; phenamet; pirarubicin; losoxantrone; podophyllinic acid; 2-ethylhydrazide; procarbazine; PSK® polysaccharide complex (JHS Natural Products, Eugene, Oreg.); razoxane; rhizoxin; sizofuran; spirogermanium; tenuazonic acid; triaziquone; 2,2',2"-trichlorotriethylamine; trichothecenes (especially T-2 toxin, verracurin A, roridin A and anguidine); urethan; vindesine; dacarbazine; mannomustine; mitobronitol; mitolactol; pipobroman; gacytosine; arabinoside ("Ara-C"); cyclophosphamide; thiotepa; taxoids, e.g., TAXOL® paclitaxel (Bristol-Myers Squibb Oncology, Princeton, N.J.), ABRAXANE® Cremophor- firee, albumin-engineered nanoparticle formulation of paclitaxel (American Pharmaceutical Partners, Schaumberg, Ill.), and TAXOTERE® doxetaxel (Rhone -Poulenc Rorer, Antony, France); chloranbucil; GEMZAR® gemcitabine; 6-thioguanine; mercaptopurine; methotrexate; platinum analogs such as cisplatin, oxaliplatin and carboplatin; vinblastine; platinum; etoposide (VP-16); ifosfamide; mitoxantrone; vincristine; NAVELBINE.RTM. vinorelbine; novantrone; teniposide; edatrexate; daunomycin; aminopterin; xeloda; ibandronate; irinotecan (Camptosar, CPT-11)

(including the treatment regimen of irinotecan with 5-FU and leucovorin); topoisomerase inhibitor RFS 2000; difluoromethylomithine (DMFO); retinoids such as retinoic acid; capecitabine; combretastatin; leucovorin (LV); oxaliplatin, including the oxaliplatin treatment regimen (FOLFOX); lapatinib (Tykerb.RTM.); inhibitors of PKC-alpha, Raf, H-Ras, EGFR (e.g., erlotinib (Tarceva®)) and VEGF-A that reduce cell proliferation and pharmaceutically acceptable salts, acids or derivatives of any of the above.

[0092] In addition, the methods of treatment can further include the use of radiation or radiation therapy. Further, the methods of treatment can further include the use of surgical treatments.

[0093] In some embodiments, the effective amount of the second therapeutic agent when used with a composition and/or conjugateis less than the effective amount of the second therapeutic agent when used alone (or not in the presence or in combination with a composition and/or conjugate). In some embodiments, a composition and/or conjugate and second therapeutic agent are synergistic, in that they work together such that one agent increases the effectiveness of the other. For example, the topical application of a composition and/or conjugate increases the effectiveness of the topical application of the secondary therapeutic agent, such that the effective dose of a topically applied secondary therapeutic agent is lower in the presence of the composition and/or conjugate, (and higher in the absence of the composition and/or conjugate). In some embodiments, the combined effect of a topically applied composition and/or conjugate and topically applied second therapeutic agent is greater than when each of these agents are used alone. Synergistic effects are typically detected when the composition and/or conjugate and second therapeutic agent work by different mechanisms so together they form a stronger effect than then they are used individually. In some embodiments, a composition and/or conjugate and second therapeutic agent are additive, in that combined effect of a topically applied composition and/or conjugate and topically applied second therapeutic agent is equal to the sum of the effect when these two agents are used alone. Additive effects are typically detected when the composition and/or conjugateand second therapeutic agent work by the same or similar mechanism.

[0094] Combination therapy includes administration of a single pharmaceutical dosage formulation containing one or more composition and/or conjugate as disclosed herein and one or more additional pharmaceutical agents, as well as administration of a composition and/or conjugateas disclosed herein and each additional pharmaceutical agent, in its own separate pharmaceutical dosage formulation. For example, a composition and/or conjugate as disclosed herein and one or more additional pharmaceutical agents, may be administered to the patient together, in a single topical dosage composition having a fixed ratio of each active ingredient, such as an ointment, lotion, gel, spray, aerosol etc. or each agent may be administered in separate topical dosage formulations.

[0095] In some embodiments, the pharmaceutical composition comprising a composition and/or conjugate as described herein can be a parenteral dose form. Since administration of parenteral dosage forms typically bypasses the patient's natural defenses against contaminants, parenteral dosage forms are preferably sterile or capable of being sterilized prior to administration to a patient.

Examples of parenteral dosage forms include, but are not limited to, solutions ready for injection, dry products ready to be dissolved or suspended in a pharmaceutically acceptable vehicle for injection, suspensions ready for injection, and emulsions. In addition, controlled-re lease parenteral dosage forms can be prepared for administration of a patient, including, but not limited to, DUROS^®-type dosage forms and dose-dumping.

[0096] Suitable vehicles that can be used to provide parenteral dosage forms are well known to those skilled in the art. Examples include, without limitation: sterile water; water for injection USP; saline solution; glucose solution; aqueous vehicles such as but not limited to, sodium chloride injection, Ringer's injection, dextrose Injection, dextrose and sodium chloride injection, and lactated Ringer's injection; water-miscible vehicles such as, but not limited to, ethyl alcohol, polyethylene glycol, and propylene glycol; and non-aqueous vehicles such as, but not limited to, com oil, cottonseed oil, peanut oil, sesame oil, ethyl oleate, isopropyl myristate, and benzyl benzoate. Compounds that alter or modify the solubility of a pharmaceutically acceptable salt of an ingredient described herein can also be incorporated into the parenteral dosage forms of the disclosure, including conventional and controlled-release parenteral dosage forms.

[0097] Pharmaceutical compositions comprising a composition and/or conjugate as described herein can also be formulated to be suitable for oral administration, for example as discrete dosage forms, such as, but not limited to, tablets (including without limitation scored or coated tablets), pills, caplets, capsules, chewable tablets, powder packets, cachets, troches, wafers, aerosol sprays, or liquids, such as but not limited to, syrups, elixirs, solutions or suspensions in an aqueous liquid, a non- aqueous liquid, an oil-in-water emulsion, or a water-in-oil emulsion. Such compositions contain a predetermined amount of the pharmaceutically acceptable salt of the disclosed compounds, and may be prepared by methods of pharmacy well known to those skilled in the art. See generally,

Remington: The Science and Practice of Pharmacy, 21st Ed., Lippincott, Williams, and Wilkins, Philadelphia PA. (2005).

[0098] In some embodiments of any of the aspects, the composition and/or conjugate described herein is administered as a monotherapy, e.g., another treatment for the cancer is not administered to the subject.

[0099] In some embodiments of any of the aspects, the polymer-drug conjguates and/or compositions described herein can be formulated for topical application.

[00100] Provided herein are methods of treating cancers and/or precancerous lesions in a subject, e.g., by administering a polymer-drug conjugate as described herein. In some embodiments, the methods described herein relate to treating a subject having or diagnosed as having, e.g., cancer with a conjugate and/or composition as described herein. Subjects having cancer can be identified by a physician using current methods of diagnosing cancer. Symptoms and/or complications of cancer which characterize these conditions and aid in diagnosis are well known in the art and include but are not limited to, fevers, weight loss, bumps or tumors. Tests that may aid in a diagnosis of, e.g. cancer include, but are not limited to, biopsy and imaging exams. A family history of cancer, or exposure to risk factors for cancer can also aid in determining if a subject is likely to have cancer or in making a diagnosis of cancer.

[00101] As used herein, the term “cancer” relates generally to a class of diseases or conditions in which abnormal cells divide without control and can invade nearby tissues. Cancer cells can also spread to other parts of the body through the blood and lymph systems. There are several main types of cancer. Carcinoma is a cancer that begins in the skin or in tissues that line or cover internal organs. Sarcoma is a cancer that begins in bone, cartilage, fat, muscle, blood vessels, or other connective or supportive tissue. Leukemia is a cancer that starts in blood-forming tissue such as the bone marrow, and causes large numbers of abnormal blood cells to be produced and enter the blood. Lymphoma and multiple myeloma are cancers that begin in the cells of the immune system. Central nervous system cancers are cancers that begin in the tissues of the brain and spinal cord.

[00102] In some embodiments of any of the aspects, the cancer is a primary cancer. In some embodiments of any of the aspects, the cancer is a malignant cancer. As used herein, the term “malignant” refers to a cancer in which a group of tumor cells display one or more of uncontrolled growth (i.e., division beyond normal limits), invasion (i.e.. intrusion on and destruction of adjacent tissues), and metastasis (i.e. , spread to other locations in the body via lymph or blood). As used herein, the term “metastasize” refers to the spread of cancer from one part of the body to another. A tumor formed by cells that have spread is called a “metastatic tumor” or a “metastasis.” The metastatic tumor contains cells that are like those in the original (primary) tumor. As used herein, the term “benign” or “non-malignant” refers to tumors that may grow larger but do not spread to other parts of the body. Benign tumors are self-limited and typically do not invade or metastasize.

[00103] A “cancer cell” or “tumor cell” refers to an individual cell of a cancerous growth or tissue. A tumor refers generally to a swelling or lesion formed by an abnormal growth of cells, which may be benign, pre -malignant, or malignant. Most cancer cells form tumors, but some, e.g., leukemia, do not necessarily form tumors. For those cancer cells that form tumors, the terms cancer (cell) and tumor (cell) are used interchangeably.

[00104] As used herein the term "neoplasm" refers to any new and abnormal growth of tissue, e.g., an abnormal mass of tissue, the growth of which exceeds and is uncoordinated with that of the normal tissues. Thus, a neoplasm can be a benign neoplasm, premalignant neoplasm, or a malignant neoplasm.

[00105] A subject that has a cancer or a tumor is a subject having objectively measurable cancer cells present in the subject’s body. Included in this definition are malignant, actively proliferative cancers, as well as potentially dormant tumors or micrometastatses. Cancers which migrate from their original location and seed other vital organs can eventually lead to the death of the subject through the functional deterioration of the affected organs.

[00106] Examples of cancer include but are not limited to, carcinoma, lymphoma, blastoma, sarcoma, leukemia, basal cell carcinoma, biliary tract cancer; bladder cancer; bone cancer; brain and CNS cancer; breast cancer; cancer of the peritoneum; cervical cancer; choriocarcinoma; colon and rectum cancer; connective tissue cancer; cancer of the digestive system; endometrial cancer; esophageal cancer; eye cancer; cancer of the head and neck; gastric cancer (including gastrointestinal cancer); glioblastoma (GBM); hepatic carcinoma; hepatoma; intra-epithelial neoplasm.; kidney or renal cancer; larynx cancer; leukemia; liver cancer; lung cancer (e.g., small-cell lung cancer, non small cell lung cancer, adenocarcinoma of the lung, and squamous carcinoma of the lung); lymphoma including Hodgkin’s and non-Hodgkin’s lymphoma; melanoma; myeloma; neuroblastoma; oral cavity cancer (e.g., lip, tongue, mouth, and pharynx); ovarian cancer; pancreatic cancer; prostate cancer; retinoblastoma; rhabdomyosarcoma; rectal cancer; cancer of the respiratory system; salivary gland carcinoma; sarcoma; skin cancer; squamous cell cancer; stomach cancer; testicular cancer; thyroid cancer; uterine or endometrial cancer; cancer of the urinary system; vulval cancer; as well as other carcinomas and sarcomas; as well as B-cell lymphoma (including low grade/follicular non-Hodgkin’s lymphoma (NHL); small lymphocytic (SL) NHL; intermediate grade/follicular NHL; intermediate grade diffuse NHL; high grade immunoblastic NHL; high grade lymphoblastic NHL; high grade small non-cleaved cell NHL; bulky disease NHL; mantle cell lymphoma; AIDS-related lymphoma; and Waldenstrom’s Macroglobulinemia); chronic lymphocytic leukemia (CLL); acute lymphoblastic leukemia (ALL); Hairy cell leukemia; chronic myeloblastic leukemia; and post-transplant lymphoproliferative disorder (PTLD), as well as abnormal vascular proliferation associated with phakomatoses, edema (such as that associated with brain tumors), and Meigs’ syndrome [00107] A “cancer cell” is a cancerous, pre-cancerous, or transformed cell, either in vivo, ex vivo, or in tissue culture, that has spontaneous or induced phenotypic changes that do not necessarily involve the uptake of new genetic material. Although transformation can arise from infection with a transforming virus and incorporation of new genomic nucleic acid, or uptake of exogenous nucleic acid, it can also arise spontaneously or following exposure to a carcinogen, thereby mutating an endogenous gene. Transformation/cancer is associated with, e.g., morphological changes, immortalization of cells, aberrant growth control, foci formation, anchorage independence, malignancy, loss of contact inhibition and density limitation of growth, growth factor or serum independence, tumor specific markers, invasiveness or metastasis, and tumor growth in suitable animal hosts such as nude mice.

[00108] The compositions and conjugates described herein can be administered to a subject having or diagnosed as having a condition described herein. In some embodiments, the methods described herein comprise administering an effective amount of compositions/conjugates described herein to a subject in order to alleviate a symptom of a condition. As used herein, "alleviating a symptom of a condition" is ameliorating any symptom associated with the condition. As compared with an equivalent untreated control, such reduction is by at least 5%, 10%, 20%, 40%, 50%, 60%, 80%, 90%, 95%, 99% or more as measured by any standard technique. A variety of means for administering the compositions/conjugates described herein to subjects are known to those of skill in the art. Such methods can include, but are not limited to oral, parenteral, intravenous, intramuscular, subcutaneous, transdermal, airway (aerosol), pulmonary, cutaneous, topical, injection, or intratumoral administration. Administration can be local or systemic. In some embodiments of any of the aspects, the administration is topical. [00109] The term “effective amount" as used herein refers to the amount of a composition and/or conjugate needed to alleviate at least one or more symptom of the disease or disorder, and relates to a sufficient amount of pharmacological composition to provide the desired effect. The term "therapeutically effective amount" therefore refers to an amount of a composition and/or conjugate that is sufficient to provide a particular effect when administered to a typical subject. An effective amount as used herein, in various contexts, would also include an amount sufficient to delay the development of a symptom of the disease, alter the course of a symptom disease (for example but not limited to, slowing the progression of a symptom of the disease), or reverse a symptom of the disease. Thus, it is not generally practicable to specify an exact “effective amount" . However, for any given case, an appropriate “effective amount" can be determined by one of ordinary skill in the art using only routine experimentation.

[00110] Effective amounts, toxicity, and therapeutic efficacy can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., for determining the LD50 (the dose lethal to 50% of the population) and the ED50 (the dose therapeutically effective in 50% of the population). The dosage can vary depending upon the dosage form employed and the route of administration utilized. The dose ratio between toxic and therapeutic effects is the therapeutic index and can be expressed as the ratio LD50/ED50. Compositions and methods that exhibit large therapeutic indices are preferred. A therapeutically effective dose can be estimated initially from cell culture assays. Also, a dose can be formulated in animal models to achieve a circulating plasma concentration range that includes the IC50 (i.e.. the concentration of the active ingredient which achieves a half-maximal inhibition of symptoms) as determined in cell culture, or in an appropriate animal model. Levels in plasma can be measured, for example, by high performance liquid chromatography. The effects of any particular dosage can be monitored by a suitable bioassay, e.g., assay for cancer cell growth, among others. The dosage can be determined by a physician and adjusted, as necessary, to suit observed effects of the treatment.

[00111] In one aspect of any of the embodiments, described herein is a method of treating skin cancer or a precancerous skin lesion in a subject in need thereof, the method comprising administering a composition or conjugate as described herein, e.g., a polymer-drug conjugate comprising a hyaluronic acid molecule conjugated to: a) at least one doxorubicin molecule; and b) at least one camptothecin molecule, to the subject. In some embodiments of any of the aspects, the conjugate or composition is administered topically.

[00112] As used herein “skin cancer” refers to a cancer arising in the skin and/or from skin cells. Skin cancers include basal cell skin cancer, squamouse cell skin cancer, and melanoma. In some embodiments of any of the aspects, the skin cancer is a non-melanoma skin cancer (NMSC). In some embodiments of any of the aspects, the skin cancer is basal cell carcinoma (BCC) or cutaneous squamous cell carcinoma (cSCC). In some embodiments of any of the aspects, the precancerous skin lesion is actinic keratosis (AK). In some embodiments of any of the aspects the skin cancer and/or precancerous skin lesion is not superficial or is not only superficial. In some embodiments of any of the aspects, the skin cancer and/or precancerous skin lesion is present in the dermis.

[00113] In one aspect of any of the embodiments, described herein is a method of treating blood cancer in a subject in need thereof, the method comprising administering a composition or conjugate as described herein, e.g., a polymer-drug conjugate comprising a hyaluronic acid molecule conjugated to: a) at least one doxorubicin molecule; and b) at least one camptothecin molecule, to the subject. As used herein, “blood cancer” or “hematopoietic cancer” refers to cancers of the hematopoietic and lymphoid tissues. Blood cancers include myeloproliferative and lymphoproliferative cancers. In some embodiments of any of the aspects, the blood cancer or hematopoietic cancer is a leukemia or a lymphoma. In some embodiments of any of the aspects, the blood cancer or hematopoietic cancer is acute myeloid leukemia or a T-cell lymphoma.

[00114] In one aspect of any of the embodiments, described herein is a method of treating glioblastoma in a subject in need thereof, the method comprising administering a composition or conjugate as described herein, e.g., a polymer-drug conjugate comprising a hyaluronic acid molecule conjugated to: a) at least one doxorubicin molecule; and b) at least one camptothecin molecule, to the subject.

[00115] In one aspect of any of the embodiments, described herein is a method of treating intestinal cancer in a subject in need thereof, the method comprising administering a polymer-drug conjugate comprising a hyaluronic acid molecule conjugated to at least one doxorubicin molecule or a composition or conjugate as described herein, e.g., a polymer-drug conjugate comprising a hyaluronic acid molecule conjugated to: a) at least one doxorubicin molecule; and b) at least one camptothecin molecule, to the subject, e.g., by intravenous injection. As used herein, “intestinal cancer” refers to of or arising at least partially in the instestines, e.g., large instestines, small intestines, colon, and/or rectum. In some embodiments of any of the aspects, the intestinal cancer is gastrointestinal cancer, colon cancer, and/or colorectal cancer.

[00116] In certain embodiments, an effective dose of a composition and/or conjugate as described herein can be administered to a patient once. In certain embodiments, an effective dose of a composition and/or conjugate can be administered to a patient repeatedly. In some embodiments, after an initial treatment regimen, the treatments can be administered on a less frequent basis. For example, after treatment biweekly for three months, treatment can be repeated once per month, for six months or a year or longer. Treatment according to the methods described herein can reduce levels of a marker or symptom of a condition, e.g. cancer growth or size by at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80 % or at least 90% or more. [00117] The dosage of a composition/conjguate as described herein can be determined by a physician and adjusted, as necessary, to suit observed effects of the treatment. With respect to duration and frequency of treatment, it is typical for skilled clinicians to monitor subjects in order to determine when the treatment is providing therapeutic benefit, and to determine whether to increase or decrease dosage, increase or decrease administration frequency, discontinue treatment, resume treatment, or make other alterations to the treatment regimen. The dosing schedule can vary from once a week to daily depending on a number of clinical factors, such as the subject's sensitivity to the active ingredient(s). The desired dose or amount of activation can be administered at one time or divided into subdoses, e.g., 2-4 subdoses and administered over a period of time, e.g., at appropriate intervals through the day or other appropriate schedule. In some embodiments, administration can be chronic, e.g., one or more doses and/or treatments daily over a period of weeks or months. Examples of dosing and/or treatment schedules are administration daily, twice daily, three times daily or four or more times daily over a period of 1 week, 2 weeks, 3 weeks, 4 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, or 6 months, or more. A composition and/or gel can be administered over a period of time, such as over a 5 minute, 10 minute, 15 minute, 20 minute, or 25 minute period.

[00118] The dosage ranges for the administration of a composition and/or conjguate, according to the methods described herein depend upon, for example, the form of the composition and/or conjugate, its potency, and the extent to which symptoms, markers, or indicators of a condition described herein are desired to be reduced, for example the percentage reduction desired for cancer cell death. The dosage should not be so large as to cause adverse side effects. Generally, the dosage will vary with the age, condition, and sex of the patient and can be determined by one of skill in the art. The dosage can also be adjusted by the individual physician in the event of any complication. [00119] The efficacy of a composition and/or gel in, e.g. the treatment of a condition described herein, or to induce a response as described herein can be determined by the skilled clinician. However, a treatment is considered “effective treatment," as the term is used herein, if one or more of the signs or symptoms of a condition described herein are altered in a beneficial manner, other clinically accepted symptoms are improved, or even ameliorated, or a desired response is induced e.g., by at least 10% following treatment according to the methods described herein. Efficacy can be assessed, for example, by measuring a marker, indicator, symptom, and/or the incidence of a condition treated according to the methods described herein or any other measurable parameter appropriate, e.g. cancer cell growth/survival. Efficacy can also be measured by a failure of an individual to worsen as assessed by hospitalization, or need for medical interventions (i.e., progression of the disease is halted). Methods of measuring these indicators are known to those of skill in the art and/or are described herein. Treatment includes any treatment of a disease in an individual or an animal (some non-limiting examples include a human or an animal) and includes: (1) inhibiting the disease, e.g., preventing a worsening of symptoms; or (2) relieving the severity of the disease, e.g., causing regression of symptoms. An effective amount for the treatment of a disease means that amount which, when administered to a subject in need thereof, is sufficient to result in effective treatment as that term is defined herein, for that disease. Efficacy of an agent can be determined by assessing physical indicators of a condition or desired response. It is well within the ability of one skilled in the art to monitor efficacy of administration and/or treatment by measuring any one of such parameters, or any combination of parameters. Efficacy can be assessed in animal models of a condition described herein, for example treatment of a mouse model of the conditions described herein. When using an experimental animal model, efficacy of treatment is evidenced when a statistically significant change in a marker is observed, e.g. cancer cell growth/survival.

[00120] Suitable effective dosage amounts, however, will, in one embodiment, range from about 0.01 mg/kg of body weight to about 2500 mg/kg of body weight. In another embodiment, effective dosage amounts will be about 100 mg/kg of body weight or less. In one embodiment, the effective dosage amount ranges from about 0.01 mg/kg of body weight to about 100 mg/kg of body weight of a compound of formula (I) or a pharmaceutically acceptable salt or solvate thereof; in another embodiment, about 0.02 mg/kg of body weight to about 50 mg/kg of body weight; and in another embodiment, about 0.025 mg/kg of body weight to about 20 mg/kg of body weight. In some embodiments, a composition comprising a composition and/or conjugate as disclosed herein is topically administered in an effective amount, e.g., a therapeutically effective amount. For systemic administration, subjects can be administered a therapeutic amount of a composition or conjugate, such as, e.g. 0.1 mg/kg, 0.5 mg/kg, 1.0 mg/kg, 2.0 mg/kg, 2.5 mg/kg, 5 mg/kg, 10 mg/kg, 15 mg/kg, 20 mg/kg, 25 mg/kg, 30 mg/kg, 40 mg/kg, 50 mg/kg, or more.

[00121] For convenience, the meaning of some terms and phrases used in the specification, examples, and appended claims, are provided below. Unless stated otherwise, or implicit from context, the following terms and phrases include the meanings provided below. The definitions are provided to aid in describing particular embodiments, and are not intended to limit the claimed invention, because the scope of the invention is limited only by the claims. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. If there is an apparent discrepancy between the usage of a term in the art and its definition provided herein, the definition provided within the specification shall prevail.

[00122] For convenience, certain terms employed herein, in the specification, examples and appended claims are collected here.

[00123] The terms “decrease”, “reduced”, “reduction”, or “inhibit” are all used herein to mean a decrease by a statistically significant amount. In some embodiments, “reduce,” “reduction" or “decrease" or “inhibit” typically means a decrease by at least 10% as compared to a reference level (e.g. the absence of a given treatment or agent) and can include, for example, a decrease by at least about 10%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99% , or more. As used herein, “reduction” or “inhibition” does not encompass a complete inhibition or reduction as compared to a reference level. “Complete inhibition” is a 100% inhibition as compared to a reference level. A decrease can be preferably down to a level accepted as within the range of normal for an individual without a given disorder.

[00124] The terms “increased”, “increase”, “enhance”, or “activate” are all used herein to mean an increase by a statically significant amount. In some embodiments, the terms “increased”, “increase”, “enhance”, or “activate” can mean an increase of at least 10% as compared to a reference level, for example an increase of at least about 20%, or at least about 30%, or at least about 40%, or at least about 50%, or at least about 60%, or at least about 70%, or at least about 80%, or at least about 90% or up to and including a 100% increase or any increase between 10-100% as compared to a reference level, or at least about a 2-fold, or at least about a 3 -fold, or at least about a 4-fold, or at least about a 5-fold or at least about a 10-fold increase, or any increase between 2-fold and 10-fold or greater as compared to a reference level. In the context of a marker or symptom, a “increase” is a statistically significant increase in such level.

[00125] As used herein, a "subject" means a human or animal. Usually the animal is a vertebrate such as a primate, rodent, domestic animal or game animal. Primates include chimpanzees, cynomologous monkeys, spider monkeys, and macaques, e.g., Rhesus. Rodents include mice, rats, woodchucks, ferrets, rabbits and hamsters. Domestic and game animals include cows, horses, pigs, deer, bison, buffalo, feline species, e.g., domestic cat, canine species, e.g., dog, fox, wolf, avian species, e.g., chicken, emu, ostrich, and fish, e.g., trout, catfish and salmon. In some embodiments, the subject is a mammal, e.g., a primate, e.g., a human. The terms, “individual,” “patient” and “subject” are used interchangeably herein.

[00126] Preferably, the subject is a mammal. The mammal can be a human, non-human primate, mouse, rat, dog, cat, horse, or cow, but is not limited to these examples. Mammals other than humans can be advantageously used as subjects that represent animal models of a condition described herein, e.g., cancer. A subject can be male or female.

[00127] A subject can be one who has been previously diagnosed with or identified as suffering from or having a condition in need of treatment (e.g. cancer) or one or more complications related to such a condition, and optionally, have already undergone treatment for the condition or the one or more complications related to the condition. Alternatively, a subject can also be one who has not been previously diagnosed as having the condition or one or more complications related to the condition. For example, a subject can be one who exhibits one or more risk factors for the condition or one or more complications related to the condition or a subject who does not exhibit risk factors. [00128] A “subject in need” of treatment for a particular condition can be a subject having that condition, diagnosed as having that condition, or at risk of developing that condition.

[00129] As used herein, the terms "treat,” "treatment," "treating,” or “amelioration” refer to therapeutic treatments, wherein the object is to reverse, alleviate, ameliorate, inhibit, slow down or stop the progression or severity of a condition associated with a disease or disorder, e.g. cancer. The term “treating" includes reducing or alleviating at least one adverse effect or symptom of a condition, disease or disorder associated with a cancer. Treatment is generally “effective" if one or more symptoms or clinical markers are reduced. Alternatively, treatment is “effective" if the progression of a disease is reduced or halted. That is, “treatment" includes not just the improvement of symptoms or markers, but also a cessation of, or at least slowing of, progress or worsening of symptoms compared to what would be expected in the absence of treatment. Beneficial or desired clinical results include, but are not limited to, alleviation of one or more symptom(s), diminishment of extent of disease, stabilized (/. e. , not worsening) state of disease, delay or slowing of disease progression, amelioration or palliation of the disease state, remission (whether partial or total), and/or decreased mortality, whether detectable or undetectable. The term "treatment" of a disease also includes providing relief from the symptoms or side-effects of the disease (including palliative treatment).

[00130] In some embodiments of any of the aspects, described herein is a prophylactic method of treatment. As used herein “prophylactic” refers to the timing and intent of a treatment relative to a disease or symptom, that is, the treatment is administered prior to clinical detection or diagnosis of that particular disease or symptom in order to protect the patient from the disease or symptom. Prophylactic treatment can encompass a reduction in the severity or speed of onset of the disease or symptom, or contribute to faster recovery from the disease or symptom. Accordingly, the methods described herein can be prophylactic relative to development of a cancer from a precancerous lesion. In some embodiments of any of the aspects, prophylactic treatment is not prevention of all symptoms or signs of a disease.

[00131] As used herein, the term “pharmaceutical composition” refers to the active agent in combination with a pharmaceutically acceptable carrier e.g. a carrier commonly used in the pharmaceutical industry. The phrase "pharmaceutically acceptable" is employed herein to refer to those compounds, materials, compositions, and/or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit/risk ratio. In some embodiments of any of the aspects, a pharmaceutically acceptable carrier can be a carrier other than water. In some embodiments of any of the aspects, a pharmaceutically acceptable carrier can be a cream, emulsion, gel, liposome, nanoparticle, and/or ointment. In some embodiments of any of the aspects, a pharmaceutically acceptable carrier can be an artificial or engineered carrier, e.g., a carrier that the active ingredient would not be found to occur in in nature.

[00132] As used herein, the term "administering," refers to the placement of a compound as disclosed herein into a subject by a method or route which results in at least partial delivery of the agent at a desired site. Pharmaceutical compositions comprising the compounds disclosed herein can be administered by any appropriate route which results in an effective treatment in the subject. In some embodiments, administration comprises physical human activity, e.g., an injection, act of ingestion, an act of application, and/or manipulation of a delivery device or machine. Such activity can be performed, e.g., by a medical professional and/or the subject being treated.

[00133] As used herein, “contacting" refers to any suitable means for delivering, or exposing, an agent to at least one cell. Exemplary delivery methods include, but are not limited to, direct delivery to cell culture medium, perfusion, injection, or other delivery method well known to one skilled in the art. In some embodiments, contacting comprises physical human activity, e.g., an injection; an act of dispensing, mixing, and/or decanting; and/or manipulation of a delivery device or machine.

[00134] The term “statistically significant" or “significantly" refers to statistical significance and generally means a two standard deviation (2SD) or greater difference.

[00135] Other than in the operating examples, or where otherwise indicated, all numbers expressing quantities of ingredients or reaction conditions used herein should be understood as modified in all instances by the term “about.” The term “about” when used in connection with percentages can mean ±1%.

[00136] As used herein, the term “comprising” means that other elements can also be present in addition to the defined elements presented. The use of “comprising” indicates inclusion rather than limitation.

[00137] The term "consisting of' refers to compositions, methods, and respective components thereof as described herein, which are exclusive of any element not recited in that description of the embodiment.

[00138] As used herein the term "consisting essentially of' refers to those elements required for a given embodiment. The term permits the presence of additional elements that do not materially affect the basic and novel or functional characteristic(s) of that embodiment of the invention.

[00139] The singular terms "a," "an," and "the" include plural referents unless context clearly indicates otherwise. Similarly, the word "or" is intended to include "and" unless the context clearly indicates otherwise. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of this disclosure, suitable methods and materials are described below. The abbreviation, "e.g." is derived from the Latin exempli gratia, and is used herein to indicate a non-limiting example. Thus, the abbreviation "e.g." is synonymous with the term "for example." [00140] Groupings of alternative elements or embodiments of the invention disclosed herein are not to be construed as limitations. Each group member can be referred to and claimed individually or in any combination with other members of the group or other elements found herein. One or more members of a group can be included in, or deleted from, a group for reasons of convenience and/or patentability. When any such inclusion or deletion occurs, the specification is herein deemed to contain the group as modified thus fulfilling the written description of all Markush groups used in the appended claims.

[00141] Unless otherwise defined herein, scientific and technical terms used in connection with the present application shall have the meanings that are commonly understood by those of ordinary skill in the art to which this disclosure belongs. It should be understood that this invention is not limited to the particular methodology, protocols, and reagents, etc., described herein and as such can vary. The terminology used herein is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the present invention, which is defined solely by the claims. Definitions of common terms in immunology and molecular biology can be found in The Merck Manual of Diagnosis and Therapy, 20th Edition, published by Merck Sharp & Dohme Corp., 2018 (ISBN 0911910190, 978-0911910421); Robert S. Porter et al. (eds.), The Encyclopedia of Molecular Cell Biology and Molecular Medicine, published by Blackwell Science Ltd., 1999-2012 (ISBN 9783527600908); and Robert A. Meyers (ed.), Molecular Biology and Biotechnology: a Comprehensive Desk Reference, published by VCH Publishers, Inc., 1995 (ISBN 1-56081-569-8); Immunology by Wemer Luttmann, published by Elsevier, 2006; Janeway's Immunobiology, Kenneth Murphy, Allan Mowat, Casey Weaver (eds.), W. W. Norton & Company, 2016 (ISBN 0815345054, 978-0815345053); Lewin's Genes XI, published by Jones & Bartlett Publishers, 2014 (ISBN- 1449659055); Michael Richard Green and Joseph Sambrook, Molecular Cloning: A Laboratory Manual, 4th ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., USA (2012) (ISBN 1936113414); Davis et al., Basic Methods in Molecular Biology, Elsevier Science Publishing, Inc., New York, USA (2012) (ISBN 044460149X); Laboratory Methods in Enzymology: DNA, Jon Lorsch (ed.) Elsevier, 2013 (ISBN 0124199542); Current Protocols in Molecular Biology (CPMB), Frederick M. Ausubel (ed.), John Wiley and Sons, 2014 (ISBN 047150338X, 9780471503385), Current Protocols in Protein Science (CPPS), John E. Coligan (ed.), John Wiley and Sons, Inc., 2005; and Current Protocols in Immunology (CPI) (John E. Coligan, ADA M Kruisbeek, David H Margulies, Ethan M Shevach, Warren Strobe, (eds.) John Wiley and Sons, Inc., 2003 (ISBN 0471142735, 9780471142737), the contents of which are all incorporated by reference herein in their entireties. [00142] Other terms are defined herein within the description of the various aspects of the invention.

[00143] All patents and other publications; including literature references, issued patents, published patent applications, and co-pending patent applications; cited throughout this application are expressly incorporated herein by reference for the purpose of describing and disclosing, for example, the methodologies described in such publications that might be used in connection with the technology described herein. These publications are provided solely for their disclosure prior to the fding date of the present application. Nothing in this regard should be construed as an admission that the inventors are not entitled to antedate such disclosure by virtue of prior invention or for any other reason. All statements as to the date or representation as to the contents of these documents is based on the information available to the applicants and does not constitute any admission as to the correctness of the dates or contents of these documents.

[00144] The description of embodiments of the disclosure is not intended to be exhaustive or to limit the disclosure to the precise form disclosed. While specific embodiments of, and examples for, the disclosure are described herein for illustrative purposes, various equivalent modifications are possible within the scope of the disclosure, as those skilled in the relevant art will recognize. For example, while method steps or functions are presented in a given order, alternative embodiments may perform functions in a different order, or functions may be performed substantially concurrently. The teachings of the disclosure provided herein can be applied to other procedures or methods as appropriate. The various embodiments described herein can be combined to provide further embodiments. Aspects of the disclosure can be modified, if necessary, to employ the compositions, functions and concepts of the above references and application to provide yet further embodiments of the disclosure. These and other changes can be made to the disclosure in light of the detailed description. All such modifications are intended to be included within the scope of the appended claims.

[00145] Specific elements of any of the foregoing embodiments can be combined or substituted for elements in other embodiments. Furthermore, while advantages associated with certain embodiments of the disclosure have been described in the context of these embodiments, other embodiments may also exhibit such advantages, and not all embodiments need necessarily exhibit such advantages to fall within the scope of the disclosure.

[00146] The technology described herein is further illustrated by the following examples which in no way should be construed as being further limiting.

[00147] In some embodiments, the present technology may be defined in any of the following numbered paragraphs:

1. A polymer-drug conjugate comprising a hyaluronic acid molecule conjugated to: a. at least one doxorubicin molecule; and b. at least one camptothecin molecule.

2. The conjugate of any of the preceding paragraphs, wherein the molar ratio of doxorubicin : camptothecin is 2:1 or a greater relative amount of doxorubicin. The conjugate of any of the preceding paragraphs, wherein the molar ratio of doxorubicin : camptothecin is 2: 1. The conjugate of any of the preceding paragraphs, wherein the molar ratio of doxorubicin : camptothecin is 5: 1 or a greater relative amount of doxorubicin. The conjugate of any of the preceding paragraphs, wherein the molar ratio of doxorubicin : camptothecin is 5: 1. The conjugate of any of the preceding paragraphs, wherein the molar ratio of doxorubicin : camptothecin is 15:1 or a greater relative amount of doxorubicin. The conjugate of any of the preceding paragraphs, wherein the molar ratio of doxorubicin : camptothecin is 15: 1. The conjugate of any of the preceding paragraphs, wherein the molar ratio of doxorubicin : camptothecin is 11:1 or a greater relative amount of doxorubicin. The conjugate of any of the preceding paragraphs, wherein the molar ratio of doxorubicin : camptothecin is 11 : 1 to 20: 1. The conjugate of any of the preceding paragraphs, wherein the molar ratio of doxorubicin : camptothecin is 14: 1 to 16: 1. The conjugate of any of the preceding paragraphs, wherein the camptothecin is conjugated to the hyaluronic acid via an ester bond. The conjugate of any of the preceding paragraphs, wherein the doxorubicin is conjugated to the hyaluronic acid via an amide bond. The conjugate of any of the preceding paragraphs, having a combination index (C.I.) value with cancerous cells of less than 0.2. The conjugate of any of the preceding paragraphs, having a C.I. value with cancerous cells of less than 0.6. The conjugate of any of the preceding paragraphs, having a C.I. value with cancerous cells of from 0.6 to 0.1. A composition comprising the conjugate of any of the preceding paragraphs and a salt. A composition comprising micelles, each micelle comprising one or more of the polymer- drug conjugates of paragraphs 1-15. The composition of paragraph 16, further comprising a salt. The composition of paragraph 16 or 18, wherein the salt comprises NaCl or PBS. The conjugate or composition of any of the preceding paragraphs, formulated for topical application. The conjugate or composition of any of the preceding paragraphs, for use in a method of treating a skin cancer or precancerous skin lesion. 22. A method of treating skin cancer or a precancerous skin lesion in a subject in need thereof, the method comprising administering a conjugate or composition of any of paragraphs 1-20 to the subject.

23. The method of paragraph 22, wherein the conjugate or composition is administered topically.

24. The conjugate, composition, or method of any of paragraphs 22-23, wherein the skin cancer is a non-melanoma skin cancer (NMSC).

25. The conjugate, composition, or method of any of paragraphs 22-24, wherein the skin cancer is basal cell carcinoma (BCC) or cutaneous squamous cell carcinoma (cSCC).

26. The conjugate, composition, or method of any of paragraphs 22-25, wherein the precancerous skin lesion is actinic keratosis (AK).

27. The conjugate, composition, or method of any of paragraphs 22-26, wherein the skin cancer and/or precancerous skin lesion is not superficial.

28. The conjugate, composition, or method of any of paragraphs 22-27, wherein the skin cancer and/or precancerous skin lesion is not only superficial.

29. The conjugate, composition, or method of any of paragraphs 22-28, wherein the skin cancer and/or precancerous skin lesion is present in the dermis.

30. A method of treating blood cancer in a subject in need thereof, the method comprising administering a conjugate or composition of any of paragraphs 1-19 to the subject.

31. The method of paragraph 30, wherein the blood cancer is acute myeloid leukemia or a T-cell lymphoma.

32. A method of treating glioblastoma in a subject in need thereof, the method comprising administering a conjugate or composition of any of paragraphs 1-19 to the subject.

33. The method of paragraph 32, wherein the molar ratio of doxorubicin : camptothecin is 2: 1.

34. The method of paragraph 32, wherein the molar ratio of doxorubicin : camptothecin is 15:1.

35. A method of treating intestinal cancer in a subject in need thereof, the method comprising administering: a. a polymer-drug conjugate comprising a hyaluronic acid molecule conjugated to at least one doxorubicin molecule; or b. a conjugate or composition of any of paragraphs 1-19; to the subject by intravenous injection.

36. The method of paragraph 35, wherein the intestinal cancer is gastrointestinal cancer, colon cancer, and/or colorectal cancer.

[00148] In some embodiments, the present technology may be defined in any of the following numbered paragraphs: A polymer-drug conjugate comprising a hyaluronic acid molecule conjugated to: a) at least one doxorubicin molecule; and b) at least one camptothecin molecule. The conjugate of any of the preceding paragraphs, wherein the molar ratio of doxorubicin : camptothecin is 2:1 or a greater relative amount of doxorubicin. The conjugate of any of the preceding paragraphs, wherein the molar ratio of doxorubicin : camptothecin is 2: 1. The conjugate of any of the preceding paragraphs, wherein the molar ratio of doxorubicin : camptothecin is 5: 1 or a greater relative amount of doxorubicin. The conjugate of any of the preceding paragraphs, wherein the molar ratio of doxorubicin : camptothecin is 5: 1. The conjugate of any of the preceding paragraphs, wherein the molar ratio of doxorubicin : camptothecin is 15:1 or a greater relative amount of doxorubicin. The conjugate of any of the preceding paragraphs, wherein the molar ratio of doxorubicin : camptothecin is 15: 1. The conjugate of any of the preceding paragraphs, wherein the molar ratio of doxorubicin : camptothecin is 11:1 or a greater relative amount of doxorubicin. The conjugate of any of the preceding paragraphs, wherein the molar ratio of doxorubicin : camptothecin is 11 : 1 to 20: 1. The conjugate of any of the preceding paragraphs, wherein the molar ratio of doxorubicin : camptothecin is 14: 1 to 16: 1. The conjugate of any of the preceding paragraphs, wherein the camptothecin is conjugated to the hyaluronic acid via an ester bond. The conjugate of any of the preceding paragraphs, wherein the doxorubicin is conjugated to the hyaluronic acid via an amide bond. The conjugate of any of the preceding paragraphs, having a combination index (C.I.) value with cancerous cells of less than 0.2. The conjugate of any of the preceding paragraphs, having a C.I. value with cancerous cells of less than 0.6. The conjugate of any of the preceding paragraphs, having a C.I. value with cancerous cells of from 0.6 to 0.1. A composition comprising the conjugate of any of the preceding paragraphs and a salt. A composition comprising micelles, each micelle comprising one or more of the polymer- drug conjugates of paragraphs 1-15. The composition of paragraph 16, further comprising a salt. The composition of paragraph 16 or 18, wherein the salt comprises NaCl or PBS. The conjugate or composition of any of the preceding paragraphs, formulated for topical application. The conjugate or composition of any of the preceding paragraphs, for use in a method of treating a skin cancer or precancerous skin lesion. A method of treating skin cancer or a precancerous skin lesion in a subject in need thereof, the method comprising administering a conjugate or composition of any of paragraphs 1-20 to the subject. The method of paragraph 22, wherein the conjugate or composition is administered topically. The conjugate, composition, or method of any of paragraphs 22-23, wherein the skin cancer is a non-melanoma skin cancer (NMSC). The conjugate, composition, or method of any of paragraphs 22-24, wherein the skin cancer is basal cell carcinoma (BCC) or cutaneous squamous cell carcinoma (cSCC). The conjugate, composition, or method of any of paragraphs 22-25, wherein the precancerous skin lesion is actinic keratosis (AK). The conjugate, composition, or method of any of paragraphs 22-26, wherein the skin cancer and/or precancerous skin lesion is not superficial. The conjugate, composition, or method of any of paragraphs 22-27, wherein the skin cancer and/or precancerous skin lesion is not only superficial. The conjugate, composition, or method of any of paragraphs 22-28, wherein the skin cancer and/or precancerous skin lesion is present in the dermis. A method of treating blood cancer in a subject in need thereof, the method comprising administering a conjugate or composition of any of paragraphs 1-19 to the subject. The method of paragraph 30, wherein the blood cancer is acute myeloid leukemia or a T-cell lymphoma. A method of treating glioblastoma in a subject in need thereof, the method comprising administering a conjugate or composition of any of paragraphs 1-19 to the subject. The method of paragraph 32, wherein the molar ratio of doxorubicin : camptothecin is 2: 1. The method of paragraph 32, wherein the molar ratio of doxorubicin : camptothecin is 15:1. A method of treating intestinal cancer in a subject in need thereof, the method comprising administering: a) a polymer-drug conjugate comprising a hyaluronic acid molecule conjugated to at least one doxorubicin molecule; or b) a conjugate or composition of any of paragraphs 1-19; to the subject by intravenous injection. The method of paragraph 35, wherein the intestinal cancer is gastrointestinal cancer, colon cancer, and/or colorectal cancer. EXAMPLES

[00149] Example 1: Hyaluronic acid-Drug Conjugates for Topical Treatment of Skin Cancer and Precancerous Lesions

[00150] More patients in the US are diagnosed with skin cancer than all other cancers combined. Topical products offer an effective therapeutic option, especially for actinic keratosis where surgery is not an option. However, current treatment options suffer from severe side effects including irritation, light sensitivity, burning, scaling and inflammation. Using hyaluronic acid (HA) as a backbone, we engineered nanosized polymer-drug conjugates of doxorubicin and camptothecin termed,

DOxorubicin and Camptothecin Tailored at Optimal Ratios (DOCTOR). When applied topically, DOCTOR penetrated across intact skin and accumulated deep within the dermis. DOCTOR exhibited high selectivity towards cancerous and pre-cancerous cells over healthy skin cells and a better safety profile compared to current clinical comparator, Efudex®. Pharmacokinetic studies confirmed that DOCTOR was retained in the skin and not absorbed systemically. In vivo studies on UV-induced spontaneous tumors confirmed the efficacy of DOCTOR in treating cancer lesions. Further, efficacy of DOCTOR was tested on human patient-derived primary cell cultures as well as skin explant tissues. In human samples, DOCTOR induced selective killing of cancer cells with as high as 21-fold selectivity over healthy skin tissue from the same patient. Collectively, DOCTOR provides a safe and potent option for treating cancerous and pre-cancerous skin lesions in the clinic.

[00151] INTRODUCTION

[00152] In the United States, about 5.4 million people are diagnosed with non-melanoma skin cancers (NMSCs) every year.| 1. 2] This overall incidence is 2.5 times more than all other cancers combined and is expected to rise above 6 million by 2020. [3] Increasing trends in its incidence in North America, Europe, Australia, and the Asia-Pacific region indicate the growing scope of this global epidemic. [4, 5] Basal Cell Carcinoma (BCC) and cutaneous Squamous Cell Carcinoma (cSCC) constitute 99% of all NMSCs. Precancerous skin lesions such as actinic keratosis (AK) also pose a major challenge, with 58 million people affected in the US. [6] NMSCs and AKs are typically removed by surgical excision, cryotherapy, curettage, and electrodesiccation, or Moh’s surgery. [7] While effective for highly localized cancers, these techniques are not suitable for multiple lesions or those located at anatomically sensitive areas. Extended healing time of surgical wounds is also a challenge. [7]

[00153] Current topical products for treating NMSCs or AKs include treatments such as cryotherapy and photodynamic therapy or topical chemotherapeutics such as 5-fluorouracil (Efudex®). Procedures are limited by cost, complexity and in case of photodynamic therapy, light sensitivity. [8, 9] Topical products such as Efudex® are limited by inflammation, swelling and scaling. Further, current topical products are effective in treating superficial skin lesions, but not the deeper regions. At currently prescribed doses, many patients opt out of topical treatment due to side effects, thus leading to disease mismanagement.

[00154] Described herein is a new topical treatment based on a drug combination specifically designed to improve selectivity towards a number of skin cancers. This combination, DOxorubicin and Camptothecin Tailored at Optimal Ratios (DOCTOR) was delivered via topical applications using a hyaluronic acid (HA) conjugate. Conjugation of doxorubicin and camptothecin to HA led to the formation of worm-like micelles that penetrated intact skin and deposited both the drugs deep into the skin. Studies performed in multiple cell lines in vitro, UV -induced mouse tumor models in vivo, and patient-derived healthy and cSCC biopsies confirm the efficacy and safety of DOCTOR in treating skin cancer.

[00155] RESULTS

[00156] Synthesis of DOCTOR. Doxorubicin (DOX) and Camptothecin (CPT) were chemically conjugated to HA at various molar ratios (R2, R5 and R15, R = molar ratio of DOX:CPT in the conjugate). Native HA is a linear hydrophilic polymer, which exists with an extended confirmation in the solution. Conjugation of DOX and CPT imparts hydrophobicity to HA thus leading to its self- assembly into micelles (Figs. IB and 1C). The amount of drug incorporated in DOCTOR was quantified using fluorescence spectra specific to each molecule. Further, the covalent conjugation of each drug to HA was confirmed via the formation of amide and ester bonds using Fourier Transform Infrared Spectroscopy (FTIR) (Fig. 1A, Figs. 7, 8, and 9).

[00157] The FTIR spectra for DOCTOR shows the presence of signature peaks ‘a’ (-OH and -NH groups at 3309 cm-1 from HA), ‘b and c’ (asymmetric vibration of COO- at 1613 cm-1 and 1400 cm- 1 from HA), and ‘d’ (C-O-C hemiacetalic saccharide linkages at 1030 cm-1 from HA) indicating the intact polysaccharide structure of HA backbone (Fig. 1A, Fig. 7). On conjugation of CPT with HA, the characteristic peaks (C-C(=0)-0 stretching at 1150 cm-1) and ‘j’ (contribution from the four adjacent hydrogen bonds on hetero-aromatic nucleus at 767 cm-1) remain unaffected (Fig. 1A, Fig.

7). Further, the formation of ester bond between HA and CPT is evident from the shift of the carbonyl peak at 1613 cm-1 (b) to 1663.8 cm-1 (b’) and the disappearance of peak (-OH groups on CPT,

Fig. 7). As a control, physically mixed CPT and HA yielded just the signature peaks a, b, c, d from the polymer, with no peaks from CPT in the FTIR spectrum of purified product (Fig. 8). The FTIR spectrum of HA-DOX conjugate revealed close to complete disappearance of the peak corresponding to primary amines at 1730 cm-1 (k) indicating its reaction with carboxyl groups of HA to form amide bonds (SI Fig. 2). For the control sample made via the physical mixing of HA and DOX, the peak ‘k’ still existed for the final purified product. This is likely due to the presence of small amounts of residual DOX physically attached to HA (Fig. 9). The FTIR spectrum for DOX-HA-CPT confirmed the presence of signatures for bonds, namely the amide and ester bonds formed following the conjugation of HA to CPT and DOX (Fig. 1A). [00158] The formation of different covalent bonds for DOX and CPT is further supported by the differences in in vitro release rates for both drugs. While a slow and steady release was observed for DOX (10.93 ± 0.33 wt% DOX), approximately 70.17 ± 2 wt% CPT was released at the end of 5 days in physiological buffer conditions at 37°C (Fig. 10).

[00159] Morphology and size of DOCTOR was further confirmed by Transmission Electron Microscopy (TEM), Atomic Force Microscopy (AFM) and Nanoparticle Tracking Analysis (NTA) (Figs. IB and 1C, Fig. 12). Native HA possessed a fibrillar morphology whereas DOCTOR exhibited a particulate structure. The average mean sizes of DOCTOR were approximately 146 nm, 63.5 nm and 72 nm for R2, R5 and R15 respectively (Table 2). These numbers also matched those measured via NTA (Fig. 12). Further, Small-angle X-ray Scattering (SAXS) was performed to determine the structure of DOCTOR in solution. Results revealed a weak or negligible scattering in water (Fig. ID, red trace). However, the scattering intensity increased by two orders of magnitude in PBS indicating a particulate shape for DOCTOR (Fig. ID, blue trace). SAXS revealed a similar trend for the single drug conjugates as well (Fig. 13A-13C)) The zeta potential values changed from highly negative for blank HA to within the neutral range for DOCTOR (Table 4).

[00160] Table 2: Mean size for all formulations measured with AFM.

[00161] Table 4: Zeta potential values for HA polymer-drug conjugates

[00162] Efficacy of DOCTOR against precancerous and cancerous keratinocytes: Role of Synergy.

[00163] DOCTOR was highly effective against precancerous human keratinocytes (HaCaT) with IC50 values approximately 19 to 98-fold lower compared to DOX alone and 6 to 8-fold lower than CPT alone (Figure 2A and Table 5). Similarly, in the human SCC cell line (A431), the IC50 values were approximately 17 to 140-fold lower compared to DOX alone and 10 to 13-fold lower than CPT alone (Figure 2B and Table 6). Further, a synergistic interaction was confirmed for DOCTOR at all ratios based on combination indices (Cl) estimated from the IC50 values of single and dual-drug treatments. A value of Cl < 1 indicates synergism; Cl = 1 suggests an additive effect; and Cl > 1 antagonism. Since the Cl values for DOCTOR at all ratios were «1, they indicated a highly synergistic interaction between the drug pair when incorporated on to HA (Tables 5 and 6). [00164] Table 5: Summary of IC50 and C.I. values for DOCTOR when treating precancerous human keratinocytes (HaCaT). DOCTOR achieves a very high synergy with C.I values «1.

[00165] Table 6: Summary of IC50 anc C.I. values for DOCTOR when treating cancerous human keratinocytes (A431). DOCTOR achieves a very high synergy with C.I values «1.

[00166] DOCTOR is well tolerated by healthy human keratinocytes

[00167] DOCTOR was less toxic to healthy human epidermal keratinocytes (HEKa), compared to precancerous (HaCaT) and cancerous (A431) human keratinocytes (Figs. 2C, 2D). The IC50 values of DOCTOR were approximately 3 to 7.5-fold higher in HEKa compared to A431 and >30-fold higher compared to HaCaT (Table 7). Among the three different ratios tested, the molar ratio of DOX:CPT ‘R15’ was identified to be the least toxic to HEKa cells. At concentrations of 1 mM and 10 mM DOX, DOCTOR R15 induced about 60-70% of precancerous and cancerous cell death, while more than 70- 80% healthy keratinocytes still remained viable. Hence, DOCTOR R15 was chosen as the lead candidate and evaluated extensively in subsequent studies (Fig. 2D and Table 7).

[00168] Table 7 : Summary of IC50 for DOCTOR when treated with normal (HEKa), precancerous (HaCaT), and cancerous human keratinocytes (A431). DOCTOR is less toxic towards normal human keratinocytes compared to its effect on pre-cancerous and cancerous human keratinocytes.

[00169] DOCTOR delivers DOX and CPT inside the skin

[00170] The ability of DOCTOR to deliver DOX and CPT into the skin was assessed ex vivo using porcine skin as an animal model. DOCTOR R15 was used for these studies at a DOX equivalent concentration of 0.5 mg/ml. Confocal microscopy imaging revealed that DOCTOR R15 penetrates well across the strartum comeum (SC) and deposits drugs within the epidermis and dermis (Fig. 3A). Approximately 18 ± 12.2% of the applied DOX and 8 ± 4.9% of the applied CPT penetrated into the epidermis and approximately 2 ± 1.1% of the DOX and 2 ± 1.35% of the applied CPT entered the dermis (Fig. 3B). Separately in an in vitro cell uptake study, confocal imaging revealed that DOCTOR enabled simultaneous internalization of DOX and CPT in both HaCaT and A431 cells (Fig. 15). Intracellular colocalization of the drug -pair was confirmed by the cyan signal (white arrow). This indicates that DOCTOR is able to deliver both the drugs deep into the skin and further inside the cells to induce synergy as observed earlier (Tables 5 and 6).

[00171] Tolerability of DOCTOR

[00172] Using reconstructed human epidermis (EpiDerm) as a model, the tolerability of DOCTOR was assessed. 5% SDS was used as a positive irritation control. [10] Tolerability was assessed via release of inflammatory cytokines (IL-la and TNF-a).[l 1] Native HA, DOCTOR R2 and DOCTOR R15 induced IL-la and TNF-a at levels similar to that of the PBS (Figs. 3C and 3D). Further, the in vivo tolerability of DOCTOR was tested in immunocompetent SKHl-E hairless mice over a 2-week repeat dose study (five times a week for two weeks). PBS was used as a negative control and 160 pg/week of Efiidex® (5% Fluorouracil) was the positive control. DOCTOR R2 and DOCTOR R15 were applied at DOX equivalent concentrations of 0.015 mg/kg. The dose for Efudex®, the clinical comparator, was determined based off a previous study. [12] Efiidex® induced significantly high levels of IL-la and TNF-a compared to the PBS negative control whereas DOCTOR R2 and DOCTOR R15 induced cytokine production comparable to PBS, suggesting that the DOCTOR formulations cause negligible skin inflammation compared to Efudex® (Figs. 3C and 3D, bottom panels). Further, systemic absorption of DOX and CPT after topical application of DOCTOR was tested. No DOX was detected and less than 1% of the applied CPT dose was detected in the plasma at the end of ten topical applications over 2 weeks (Fig. 16).

[00173] In vivo efficacy

[00174] In vivo efficacy of DOCTOR was tested in a UV-induced model of spontaneous cSCC in immunocompetent SKH1-E mice. Mice were treated with DOCTOR R15 (0.015 mg/kg DOX equivalent 3x a week over a period of 90 days). Compared to the group treated with blank HA, a significant delay in tumor progression was observed in mice when treated with DOCTOR R15 (Figs. 4A and 4C). In addition, treatment with DOCTOR significantly increased mean survival by 65% (p<0.0001) compared to the HA control group (Fig. 4B). Skin biopsies from mice treated with DOCTOR were assessed. A positive nuclear and cytoplasmic immunostaining was observed for cleaved caspase-3 in DOCTOR R15 treated cSCC tumor biopsies indicative of apoptotic cell death (Fig 4D). No signals were observed in biopsy samples obtained from mice treated with the HA control. (Fig. 4D).

[00175] Safety and Efficacy in patient-derived primary cells and live skin explants.

[00176] The potential clinical efficacy of DOCTOR in treating cSCC was further tested on skin tissues obtained from patients diagnosed with cSCC. While more than 80-90% viability was observed for adjacent normal skin, DOCTOR reduced the viability of primary cSCC cells to as low as 30% based on the individual patient response to the treatment (Fig. 5A, Figs. 18A, 18B, and 18C). For instance, patient donor 3 displayed high sensitivity to the treatment (Fig. 18C) at half the dose (5 mM DOX) deemed highly effective for the other samples (10 pM DOX). Such variations are critical and underscore the inter-individual differences in response to therapy in the clinic. The variations could possibly be attributed to factors that are inherited or acquired. Furthermore, DOCTOR revealed an increasingly high potency and safety when compared against the single drug conjugates (Figs. 18D, 18E and 18F). This highlights the synergistic interaction between DOX and CPT, thereby making the case for clinical use of DOCTOR against cutaneous cancers. Patient derived skin explants from normal and lesional skin have the ability to stay viable and preserve its metabolic and proliferative capacity for several days in culture. When DOCTOR was tested on patient-derived tissue explants, the proliferative activity of matched normal skin to cSCC tissue explants was approximately 3.6-fold higher for Patient 4, 21-fold higher for Patient 5 and 5.4-fold higher for Patient 6 over 5-FU or Efudex®, the clinical comparator (Fig. 5B). Together, these data indicate the therapeutic ability of DOCTOR in eliminating cSCC lesions without harming the healthy skin tissue.

[00177] DISCUSSION [00178] This study demonstrates the advantage of using HA to topically deliver a synergistic combination chemotherapy for the prevention and treatment of precancerous and cancerous skin lesions. Camptothecin’s clinical utility has previously been limited by poor aqueous solubility and systemic toxicity. [18, 19] Topical application of CPT, where systemic exposure is limited, is a potential application that has not been explored extensively before.

[00179] The in vitro toxicity results where Cl values were calculated to be much less than 1 on account of increased potency for DOCTOR compared to the single drug conjugate (HA-DOX or HA- CPT) (Figs. 2A, 2B, Tables 5 and 6) demonstrate the pair’s synergistic activity.

[00180] Conjugation to HA increased each drug’s individual IC50 compared to their non- conjugated forms (Fig. 14, Tables 5 and 6). Without wishing to be limited by theory, it is contemplated herein that this to the differences in drug availability due to bond hydrolysis and probable differences in its uptake mechanism. While free drugs permeate the membrane directly, the conjugates are likely to be internalized via endocytosis. This was confirmed via confocal microscopy analysis, which revealed intracellular co-localization of the drug-pair (white arrow, Fig. 15). FTIR spectroscopy analysis revealed that CPT was chemically conjugated to HA via an ester bond, while DOX was incorporated with an amide bond (Figure 1A, Figs. 7, 8, 9). Thus, the differences in drug availability is supported by the fact that CPT release was faster on account of ester hydrolysis from HA while DOX release was slower due to amide hydrolysis from HA Fig. 10). However, the differences in release rates did not compromise its synergistic anticancer activity as suggested by the Cl values being increasingly less than 1 (Tables 5 and 6).

[00181] DOCTOR achieves a synergistic interaction between DOX and CPT for skin cancers at all ratios with minimal toxicity to human skin cells both in vitro (HEKa, Figs. 2C, 2D and 2E, Table 7) and in patient-derived biopsies (Figs. 5A-5F). The lack of toxicity to healthy cells was also evident from the absence of skin inflammation when tested with 3D human EpiDerm model and in vivo (Figs. 3C-3D). Interleukin-1 alpha (IL-la) is known to induce a significant pro-inflammatory effect in the skin. When stimulated by large amounts of tissue necrosis factor alpha (TNF- a), the keratinocytes secrete IL-la. An imbalance in the levels of IL-la results in onset of skin inflammation. [20-22] Although treatment with DOCTOR did stimulate the production of TNF- a (Fig 3D), its levels were not sufficient enough to increase the production of IL-la, the endogenous marker for skin inflammation (Fig. 3C). Thus, the safety of DOCTOR was validated across multiple systems including cultured healthy epidermal keratinocytes in vitro, immunocompetent SKH1-E mice in vivo and with patient skin biopsies ex vivo. Without wishing to be bound by theory, it is contemplated herein that this specific anticancer activity is due to CPT, a topoisomerase 1 enzyme inhibitor. The levels of topoisomerase 1 enzyme are relatively high in cancer tissues including squamous cell carcinomas compared to its adjacent healthy tissue. This renders ‘Top G as a highly specific and attractive anticancer target for its inhibitors such as CPT. [23-25] In addition, HA specifically binds to CD44, a cell-surface receptor that has elevated expression in many cancers including the skin, thereby promoting a highly specific uptake for the conjugates. [26-28]

[00182] TEM, AFM and NTA (Figs. IB, 1C and Fig. 12) revealed a polydisperse spherical morphology for DOCTOR in PBS with a mean size ranging from ~60 to 140 nm (Table 2). However, SAXS revealed a weak or negligible scattering from the conjugates when dissolved in water at 10 mg/ml (Fig. ID, red trace). While the scattering intensity is almost flat in the intermediate and at increasing ‘q’ values, the upturn at the low q values (>0.05 A) indicate the presence of a small fraction of aggregates in the range of 1-100 nm (Table 3). Thus, DOCTOR exists as monomers or molecularly dissolved short moieties that coexist with a small fraction of the hundred A sized aggregates in water. To simulate the skin’s internal environment that the conjugates would be exposed following penetration, the samples were further diluted by 10 times in PBS. Interestingly, the scattering intensity unexpectedly rose by two orders of magnitude (Fig. ID and Fig. 13, blue trace). This implied an enhanced aggregation of the molecularly dissolved monomers in the presence of salt. [00183] Table 3: Summary of radius of the spherical particles and the radius of gyration derived from the fitting with the model of polydisperse hard spheres, and the contour length F, the Kuhn length A, and the diameter d derived from the fitting with the Worm Chain Model.

[00184] In water, the electrostatic repulsion and the hydrophilic nature of HA enables DOCTOR to exist as molecularly dissolved entities. Salt mitigates these interactions by shielding the charges and promotes self-assembly via the hydrophobic drugs covalently linked on these polymers. SAXS data analysis revealed the nanoparticle diameter and Kuhn length to be at a maximum value of 3.6 A and 115 A, respectively for DOCTOR R15 (Table 3). Thus SAXS confirms the existence of self- assembled HA polymeric nanoparticles supported by the interplay of three important molecular forces - electrostatic repulsion, hydrophilicity and hydrophobicity.[29, 30] Thus based on SAXS, the small size and the inherently dynamic nature of these conjugates in the presence and absence of salt make it possible to cross an intact skin, enter the epidermis and accumulate gradually within the dermis. [00185] Results from ex vivo porcine skin permeation studies revealed that approximately 20% of DOX and 10% of CPT applied topically penetrated into the skin layers. This was evident from the distinct fluorescent signals for each molecule observed with confocal microscopy imaging of the tissue cross-section and quantification via the tape -stripping method (Figs. 3A and 3B). Generally, nanoparticles are severely restricted in moving across an intact barrier owing to a high MW. Movement of molecules across the stratum comeum barrier has been typically proposed along three different routes - transcellular, intracellular and transfollicular pathways. Transit across the comeocytes (transcellular) depends entirely on the physicochemical aspects (size, shape, charge, MW, surface properties and elasticity) of the particles themselves. This in turn affects the cell uptake and translocalization rates of the particles. On the other hand, movement between the comeocytes involves navigation via the ~100 nm intercellular channels filled with multi- layered lipids. Previous studies have suggested that nanoparticles either accumulate at the surface of the stratum comeum without penetrating further, or cluster and concentrate within the hair follicles via the transfollicular pathway. [31-33] In the case of DOCTOR, we hypothesize that the presence of hydrophobic dmgs on the HA backbone induces micellization. This enables DOCTOR to navigate the intracellular lipids of the stratum comeum possibly by unfolding in the presence of lipids. It is also possible that HA binds to CD44 receptors present in abundance within the epidermis and dermis, which then promotes enhanced deposition inside the skin.

[00186] When tested in Ultraviolet B (UVB) exposed SKHl-E hairless mice - a highly aggressive and immunocompetent model of cSCC, topically applied DOCTOR reduced tumor growth and improved survival significantly (p<0.0001) with no irritation or inflammation. In certain instances, DOCTOR eliminated full-size cSCC tumors in mice by inducing apoptotic tumor cell death (Fig. 17). It is quite remarkable that this efficacy was achieved at a low dose of 5.4 mM or 0.016 mg/kg DOX. By directly administering dmgs to the pathological site, DOCTOR can avoid potential adverse effects associated with systemic toxicity. It also has a significantly increased benefit-risk ratio against current clinical comparator, Efudex®. Thus, conjugating a potent drug pair such as DOX and CPT to the HA backbone at precise molar ratios ensures topical delivery and accumulation of both the dmgs at the target for enhanced therapeutic efficacy and improved prognosis in localized and metastatic skin cancer. It eliminates painful surgery, non-specific systemic injections and highly invasive treatment methods for prevention and treatment of skin cancers. This improves patient compliance significantly. These polymer drug conjugates open new opportunities for treating skin neoplasms and precancerous lesions.

[00187] MATERIALS

[00188] Camptothecin (CPT) and 4-(dimethylamino)pyridine (DMAP) were purchased from

Sigma-Aldrich (St. Louis, MO, USA). N-(3-Dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride (EDC) was purchased from Life Technologies, USA. Doxorubicin Hydrochloride (DOX-Hcl) was obtained from LC Laboratories (Woburn, MA, USA). Hyaluronic acid (HA) of 50 kDa MW was purchased from Creative PEGWorks (Winston Salem, NC, USA). Cell Titer Blue and Hoechst were purchased from Uife Technologies, USA. Epidermoid or Squamous carcinoma cells carcinoma cells (A431, established from an 85 year old female) and primary adult epidermal keratinocytes (HEKa) were purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA). HaCaT cells (established from a 62 year old male) was purchased from AddexBio (San Diego, CA). A431 and HaCaT cells were maintained in Dulbecco's Modified Eagle Medium (DMEM, Life Technologies) supplemented with 10% fetal bovine serum (FBS). HEKa cells were maintained in Dermal Cell Basal Medium and components of the keratinocyte growth kits purchased from ATCC. The cells were cultured according to instructions provided by ATCC. All cells were maintained at 37 °C under a humidified atmosphere of 95% air and 5% C02. Sephadex G-25 PD- 10 columns were obtained from GE Healthcare Life Sciences (Piscataway, NJ, USA), and dialysis cassettes with 3500 MWCO were obtained from Life Technologies, USA. All other chemicals used for this study were obtained from Fisher Scientific and were the highest possible grade commercially available.

[00189] METHODS.

[00190] Synthesis of DOCTOR

[00191] Doxorubicin (DOX), and Camptothecin (CPT) were conjugated to Hyaluronic acid (HA) polymer via nucleophilic acyl substitution reactions. Briefly, 100 mg of 50 kDa MW HA was dissolved in a 2 ml mixture of 1: 1 DI water/dimethyl sulfoxide (DMSO) at 40°C. To this mixture, DMAP and EDC were added at a molar ratio of 1 : 1 relative to the HA monomers. The activation was allowed to continue on for 30 min under stirring. For the synthesis of DOX-HA or CPT-HA, the drugs were added dropwise into the reaction mixture at molar ratios of 0.4: 1 and 0.2: 1, respectively. For the dual-drug conjugates, DOX was initially added to the polymer at varying amounts and durations depending on the ratio synthesized, followed by reacting CPT in a similar manner. All conditions have been summarized in Table 1. Subsequently, the drug-incorporated particles were purified by size exclusion chromatography via Sephadex G-25 PD- 10 desalting columns (5000 M.W. exclusion limit) followed by overnight dialysis (3500 MWCO) against DI water. The samples were then lyophilized, stored at 4°C and reconstituted in PBS before use. Concentrations of DOX and CPT were measured using their respective fluorescence spectra at Ex/Em 470/590 and 370/448 nm respectively.

[00192] Table 1 : The drugs were incorporated onto HA in a series of molar ratios by varying the amount of drugs added and reaction time.

[00193] Physical characterization [00194] FTIR characterization

[00195] Infrared spectra of DOCTOR was collected using a Nicolet™ iSTMlO FTIR spectrometer (Thermo Fisher Scientific, Waltham, MA) in the range 400-4000 cm-1 with a spectral resolution of 4 cm-1. The lyophilized samples were placed on the crystal surface of a single reflection diamond attenuated total reflection (ATR) device and a 32-scan interferogram was recorded for each of them. The absorbance spectra were processed for baseline, atmospheric and ATR correction using Thermo Scientific™ OMNIC™ Specta software, before analyzing the peaks.

[00196] In vitro release studies

[00197] Freshly formulated DOCTOR, was resuspended in PBS buffer (pH 7.4 or 5.0) was incubated in Slide-A-Lyzer MINI™ dialysis devices (10,000 MWCO) and inserted into microcentrifuge tubes with 1 ml PBS for up to 3 days. At the indicated time-points, release medium in the microcentrifuge tubes was collected and analyzed by reading the drugs’ concentration via fluorescence using the TECAN plate reader. The cumulative release was calculated by dividing the amount of drug released each day with the total mass initially loaded. All measurements were carried out in triplicate, and the results were indicated as the mean ± SE.

[00198] Size and surface charge measurement

[00199] The size of HA-DOX, HA-CPT and DOCTOR was measured using the NanoSight™ LM10 system (NanoSight, Amesbury, UK) supplemented with a fast video capture and Nanoparticle Tracking Analysis (NTA 2.3) software. The samples were measured at room temperature by capturing videos set at a recoding time of 30 s each with manual shutter and gain adjustments. The images were then processed using the NTA 2.3 software and size was recorded. Measurements were made in triplicates for each sample following instrument re-calibration. Zeta potential of the particles was measured using the Zetasizer nanoZS™ (Malvern Instruments, Westborough, MA).

[00200] Atomic Force Microscopy (AFM) characterization

[00201] Sample preparation

[00202] HA-DOX, HA-CPT and DOCTOR were diluted to 2 pg/mL concentration using deionized water. The dilute solutions were stirred using a tube revolver for 30 minutes at room temperature, with intermittent vortexing to ensure mixing at the molecular scale. Just prior to imaging, 4 pL of the solution was dropped on a freshly cleaved mica surface and allowed to dry for 15 minutes at room temperature. This procedure was used to prepare all samples.

[00203] Atomic Force Microscopy (AFM) measurements

[00204] Structure of the polymer conjugates was studied with AFM using Cypher microscope (Asylum Research, Santa Barbara, CA), operated in tapping mode at ambient conditions. Silicon cantilevers having chromium/gold (Cr/Au) coating, with resonance frequencies between 44-95 kHz, and spring constant in the range of 0.3 - 4.8 N/m and 9 ± 2 mm uncoated silicon tip (AC240TSA-R3, Asylum Research, Santa Barbara, CA) were used for the dry imaging in air. Depending on the frame size, the scan rate was set in the 0.7-1.0 Hz range. The images were processed and analyzed using Gwyddion 2.47 software (n = 5).

[00205] SAXS analysis

[00206] SAXS experiments help to shed light on the nanoparticles’ architecture and dimensions.

A flow-cell setup was used for SAXS measurements at the LiX- 16-ID beamline at the National Synchrotron Light Source II of the Brookhaven National Laboratory (Upton, NY). A series of scattering images were recorded each with a 1-sec exposure for both polymer and buffer samples. All scattering curves were radially averaged and inspected for possible radiation damage. Final scattering intensity was reported after proper buffer subtraction. The X-ray energy was 13 KeV.

[00207] Form factors used depend on the sample and solvent and are described below. Due to low concentration of nanoparticles in solution interparticle interactions are neglected (structure factor S(q)=l). The scattered intensity curves were fitted using SASFit software. [34]

[00208] Polydisperse hard sphere model

The spherical shell form factor has the following form,

Where R is the radius of sphere, ASLD is the difference in scattering length densities (SLD) between particle and solvent.

[00209] To account for nanoparticles polydispersity, a Schulz-Zimm distribution of R with polydispersity parameter s was included in the following way,

where Z r— 1.

Since the polydispersity parameter s and radius of sphere are correlated parameters, the s value was set to 0.3 for all fitting procedures.

[00210] Generalized Gaussian coil model

[00211] The form factor of Generalized Gaussian coil has the following form:

The fitting parameters for this model are Rg -gyration radius, and u - Flory exponent.

[00212] Worm-like chain model:

[00213] The form factor of a worm-like chain with contour length L, Kuhn length A, and diameter d has been described previously. [35]

[00214] In vitro toxicity analysis

[00215] In vitro toxicity of free drugs, HA-DOX, HA-CPT and DOCTOR (R2, R5 and R15) was evaluated with precancerous (HaCaT) or cancerous (A431) keratinocytes. The cells were seeded in a 96-well cell culture plate and allowed to adhere overnight at 37°C in 5% C02 atmosphere. Media was then replaced with fresh media containing the drugs and cells were subjected to each treatment for 48 hours. Cell viability was measured by Cell Titer-Blue® Viability Assay and expressed as the percentage of viable cells relative to the survival of untreated cells (defined as the maximum cell viability). The combination index (Cl) was then estimated from the dose-response data of single and dual-drug conjugates drug treatments. A value of Cl less than 1 indicates synergism; Cl = 1 indicates additive effect; and Cl > 1 indicates antagonism. The further a Cl value is from 1, the more pronounced is the drug interaction i.e. synergism or antagonism.

[00216] In a separate study, an in vitro toxicity assay was set up to evaluate the effect of DOCTOR on healthy keratinocytes vs the precancerous or cancerous keratinocytes. The assay was set up as described above and cells were exposed to each treatment for 18 hours. Subsequently, the cells were washed with fresh media at least two times and left for further incubation up to 48 hours. Cell viability was once again measured by Cell Titer-Blue® viability assay and was expressed as the percentage of viable cells relative to the survival of untreated cells defined as the maximum cell viability.

[00217] Ex vivo porcine skin permeation study [00218] Visualizing dermal delivery

[00219] Ex vivo porcine skin permeation studies were performed as reported previously. Briefly, full thickness porcine skin samples (Lampire Biological Laboratories, Pipersville, PA) stored at -80°C were defrosted and hair was trimmed prior to use. The pieces were washed with PBS (pH 7.4) and resistivity was measured to ensure that samples with an intact barrier were used for the study. The permeation rates were assessed in Franz diffusion cells (FDCs, 1.77 cm2 penetration area, 12.0 mL receptor volume) as described previously. [36] The receptor compartment was filled with PBS (pH 7.4) and skin samples were mounted with the SC facing up. Care was taken to ensure that the donor compartment was dry and left open to air for 30 minutes. Extra caution was taken to remove air bubbles between the skin’s base and the receptor solution. 200 pi of DOCTOR R15 at a DOX equivalent concentration of 0.5 mg/ml was topically applied onto the skin surface inside the donor compartment and incubated for 24 hours at 37°C under occlusive conditions with moderate stirring in the acceptor compartment. The controls and treatments for the study were assessed in triplicates. At 24 h, the skin surface was washed at least 5 times with PBS (pH 7.4) to remove any excess drugs. The skin samples were then cryosectioned and prepared for confocal microscopy analysis.

[00220] Measuring dermal delivery.

[00221] For quantifying skin permeation of the drugs applied, the samples retrieved after washing at the end of 24 hours were subjected to the tape -stripping method as reported previously. [36] For this, an adhesive tape (Scotch® Transparent Tape, 3M Corporate, St. Paul, MN) was used to separate the stratum comeum (SC) from the epidermis. Ten consecutive tape strips were performed and the strips were placed in glass vials in the following order: Strip 1 = SCI and Strips 2-10 = SC2. This ensured that a majority of the SC was isolated from the epidermis. Subsequently, the epidermal layer was separated from the dermis with a sterile surgical scalpel. Both the epidermis and the dermis were then cut into small pieces and placed in different glass vials. For drug extraction, each separated layer of the skin was incubated with 3 ml of 50% methanol/PBS mixture. To determine the amount of drug that passed completely through the length of the skin, 3 ml of the acceptor chamber solution was mixed with 3 ml of the methanol/PBS. All vials were shaken overnight at room temperature. The solutions were then centrifuged to isolate the skin remnants and the supernatants collected were used to measure the drug concentration at its respective fluorescence spectra as described earlier.

[00222] Cell uptake study

[00223] For visualizing DOX (479Ex/590Em) and CPT (370Ex/450Em) uptake with its nuclear colocalization, the cells were seeded at 300 cells/mΐ and allowed to adhere overnight. Media was then replaced with fresh media containing DOCTOR at DOX equivalent concentrations of 100 mM for 3 hours. Following incubation, the cells were washed three times with warm PBS and nucleus was stained with 5 uM solution of SYTO 17 (621Ex/634Em) for 10 minutes at 37°C and 5% C02. The cells were washed once again with warm PBS and fixed with ice-cold methanol for 10 minutes. The fixed cells were imaged immediately under Cell Discover and z-stacks of 10 gm were captured and averaged.

[00224] Testing for inflammation with EpiDerm human skin model

[00225] The irritation or inflammation potential of DOCTOR was assessed on a MatTek EpiDerm human skin model (MatTek Corporation) by measuring the release of inflammatory cytokines, including IL-la and TNF-a. In brief, EpiDerm tissue inserts were incubated overnight in medium.

The tissue inserts were then transferred into new 6-well plates with fresh medium. 100 pL of PBS (negative control), 5% SDS (positive control), blank HA, and DOCTOR (R2, R5 and R15) at DOX equivalent concentrations of 5 mM were dosed on the top of the inserts. At predetermined time points (2, 6, and 12 hours), 200 pL of medium was collected and equivalent volume of medium was added. Cytokine concentrations were measured by ELISA according to protocols provided by the manufacturer.

[00226] Evaluating tolerability and systemic absorption in vivo

[00227] Female SKH1-E hairless mice (4-6 weeks of age; 5 per group) obtained from Charles River Laboratories received topically 20 mg/week of blank HA control in PBS; 160 pg/week of Eftidex® (5% Fluorouracil); DOCTOR R2 and DOCTOR R15 at DOX equivalent concentrations of 3 pg/week in PBS. The treatments were evenly applied with a cotton applicator to the back skin of the mouse five times a week over 2 weeks and allowed to dry following each application. Any change in the body weight of mice in all treatment groups was monitored throughout the study. Animals that exhibit any signs of anaphylaxis or drug toxicity, including but not limited to respiratory distress, pale or cyanotic skin, lack of grooming, sluggishness, ruffled fur and hunched posture and acute weight loss greater than 10%, were euthanized in accordance with Harvard University’s IACUC-approved protocol. At the end of the study, blood was collected via cardiac puncture in heparin-coated collection tubes. Following centrifugation, plasma was obtained and drugs were extracted following a previous procedure to measure drug (DOX and CPT) and cytokine concentrations. [37] Plasma levels of DOX and CPT were measured at their respective fluorescence spectra as described earlier.

Cytokine concentrations in the plasma were measured using ELISA according to protocols provided by the manufacturer.

[00228] Evaluating cSCC therapeutic efficacy of DOCTOR in vivo

[00229] Female SKH1-E hairless mice (12 weeks of age; 9 per group) were obtained from Charles River Laboratories. As previously described, the mice were exposed to 12.5 kJ/m2 UVB weekly (total divided over three doses Mon, Wed, and Fri) for 90 days using an Oriel solar simulator (Newport). [38, 39] Once the mice developed single cSCC lesions that measured 4mm in diameter, they were randomly enrolled to receive 20mg/week of blank HA control or DOCTOR R15 at DOX equivalent of 5.4 mM in PBS. 100 pL of each treatment was evenly applied with a cotton applicator to the animal’s back skin three times a week and allowed to dry following application. Mice were euthanized once the tumor reached its endpoint size or until completion of the treatment for three months. All animal studies were conducted under the guidelines of University of South Florida IACUC-approved protocol (IACUC IS00002374).

[00230] Assessing safety and efficacy of DOCTOR with patient-derived skin tissues.

[00231] Biopsies with healthy skin and lesional skin with diffuse cSCCs were taken from patients and placed dermal side down onto membranes of transwell inserts (Costar) for culturing in growth medium DMEM containing 1% L-Arginine, 10% human serum (Gemini Bio Products) and antibiotics/antimycotics. The lesion surface was kept in contact with the air and treated with 500 mM of 5-FU (Efiidex®) or DOCTOR for 48h. To ensure topical application only on the lesion surface, they were sealed with semisoft agar. For quantification of apoptosis, DNA fragmentation/TUNEL was performed using In Situ Cell Death Detection Kit, TMR red (Roche) as described by manufacturer.

For precise counting of cells, Image J was used and the viability percentage was calculated on basis of the apoptotic cell number relative to the total number of cells.

[00232] Statistical analysis.

[00233] All experiments were carried out in triplicates, and results are indicated as the mean ± SE unless otherwise indicated. All graphs have been generated and analyzed using Prism nonlinear regression software (GraphPad™ Software). A p value < 0.05 was considered significant.

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Example 2 - HA-DOX-CPT conjugates for treatment of leukemia and lymphoma [00235] DOCTOR conjugates, with a ratio of DOX:CPT of 0.8, 1.5, 12, and 21 were prepared and characterized (Figs. 19A-19C). The efficacy of the DOCTOR preparations was tested using multiple AMF cell lines (Figs. 20, 21) and T-cell lymphoma cell lines (Figs. 22, 23).

Example 3 - HA-DOX-CPT conjugates for treatment of glioblastoma [00236] DOCTOR conjugates, with a ratio of DOX:CPT of 2, 5, and 15 were prepared their efficacy in the killing of glioblastoma GF261-luc2 cells at 48 hours was tested (Fig. 24). DOCTOR R2 and DOCTOR R15 demonstrated synergism.

Example 4 - Hyaluronic Acid-Doxorubicin Nanoparticles for Targeted Treatment of Colorectal Cancer

[00237] Colorectal cancer, common in both men and women, occurs when tumors form in the linings of the colon. Common treatments of colorectal cancer include surgery, chemotherapy, and radiation therapy; however, many colorectal cancer treatments often damage healthy tissues and cells, inducing severe side effects. Conventional chemotherapeutic agents such as doxorubicin (Dox) can be potentially used for the treatment of colorectal cancer, however, they suffer from limited targeting and lack of selectivity. As described herein, doxorubicin complexed to hyaluronic acid (HA) (HA-Dox) exhibits an unusual behavior of high accumulation in the intestines for at least 24h when injected intravenously. Intravenous administrations of HA-Dox effectively preserved the mucosal epithelial intestinal integrity in a chemical induced colon cancer model in mice. Moreover, treatment with HA- Dox decreased the expression of intestinal apoptotic and inflammatory markers. The results indicate that HA-Dox can effectively inhibit the development of colorectal cancer in a safe manner.

[00238] Introduction

[00239] Colorectal cancer is the third leading cause of cancer-related deaths in men and in women. In 2019, over 100,000 new cases of colon cancer have been reported; it is expected to cause about 50,000 deaths. However, early diagnosis, often by screenings, resulting in the removal of colorectal pulps before they develop into cancers, and conventional therapies have resulted significant decrease in morality [1,2] Chemotherapies such as 5-Fluorouracil (5-FU), Triflurdine and Tipiracil (LONSURF), Capecitabine (XELODA), Irinotecan (CAMPTOSAR), and Oxaliplatin (ELOXATIN) are frequently used in chemotherapeutic treatment of gastrointestinal (GI) cancers, including colorectal cancer [3-19] In most cases, two or more of these drugs are combined, resulting in greater efficacy [20-28] Several new chemotherapy drugs have recently been used to treat colorectal cancer, including panitumumab (VECTIBIX), cetuximab (ERBITUX), bevaxizumab (AVASTIN), ramucirumab (CYRAMZA) and aflibercept (ZALTRAP). All of these are usually administrated along with 5-FU, irinotechan or oxaliplatin [29-51] Most of these chemotherapeutic drugs, along with Regorafenib (STIVARGA) [52-53], are given either intravenously and orally. Despite their efficacy, the use of these drugs is hindered by their severe side effects. For example, oxaliplatin may cause neuropathy (nerve damage) as well affecting heart rhythm in which heart muscles take longer than normal to recharge between beats. Irinotecan also induces neutropenia and diarrhea [54-56]

[00240] Encapsulation of drugs in liposomes offers a potential means to reduce toxicity. Towards that, efforts have been made to design liposomal formulations for the treatment of colorectal cancer. MM-398 and IHL-305, a liposomal formulation of Irinotecan, were designed to augment tumor efficacy while minimizing toxicity. Subcutaneous injection of MM-398 showed improved efficacy while reducing toxicity in nude mice with colorectal cancer, however patients receiving one dose of MM-398 experienced neutropenia, diarrhea, as well as vomiting and abdomen pain [57-69]

[00241] Adriamycin, commonly known as Doxorubicin (Dox), has been used, in combinations with other drugs, to treat many different types of cancers, such as breast, lung, ovarian, and bladder cancers, as well as neuroblastoma, leukemia, Hodgkin’s and non-Hodgkin’s lymphoma. Doxorubicin encapsulated in a liposome (Doxil), administrated intravenously, has been used to treat breast cancer, ovarian cancer, Kaposi’s sarcoma, and other solid tumors [70-74] However, administration of Doxil induces low blood counts and increasing risk for anemia. To our knowledge, there have been no reports of free Dox, injected intravenously, treating colorectal cancers.

[00242] In this study, we assessed whether intravenous injection of Dox, complexed to hyaluronic acid (HA), a naturally found polymer found in the body, can target the intestines without any affinity moieties. Our data show that HA -complexed Dox (HA-Dox) exhibits surprisingly high accumulation in the intestine and inhibits the progression of AOM/DSS induced colon cancer as well as provides a potential therapeutic effect in a safe manner.

[00243] Methods [00244] Synthesis

[00245] The drug Doxorubicin (Dox), was chemically linked to 50 KDa Hyaluronic acid (HA) polymer via nucleophilic acyl substitution reactions. Briefly, the polymer was dissolved in a mixture of DI water and DMSO at a ratio of 1 : 1 volume. The catalyst DMAP and the activator EDC were added to the solvent mixture at a molar ratio of 1 : 1 relative to the HA monomers. Following 30 min of activation, DOX was added into the reaction mixture at molar ratios 0.4: 1 relative to the HA monomer and left stirring for 24h. Following reaction, the product was purified by size exclusion chromatography via Sephadex G-25 PD-10 desalting columns (5000 MW exclusion limit) followed by overnight dialysis (3500 MWCO) against DI water. The samples were then lyophilized, stored at 4°C and reconstituted in PBS before use. The amount of DOX incorporated was measured using its respective fluorescence spectra at Ex/Em 470/590.

[00246] Characterization

[00247] The size and morphology of HA-Dox were examined by transmission electron microscopy. Briefly, 2.0 pL of HA-Dox suspensions were allowed to air-dry on Formvar carbon- coated cupper grids. Transmission electron microscopy (TEM) was performed on a JEOL JEM- 1400 TEM instrument, operating at a voltage of 100 kV (JEOL USA, Inc.). Particle zeta potential was measured by dynamic light scattering (DLS) on Malvern Zetasizer (Malvern, U.S.A.). The mean particle size of HA-Dox was estimated with a NanoSight NS3000 (Malvern Panalytical Inc., Westborough, MA).

[00248] Animal Studies

[00249] All animal studies were carried out in strict accordance with the Guide for the Care and Use of Laboratory Animals as adopted by National Institute of Health, approved by Harvard University IACUC. Mice were housed in cages with free access to water and food, located in a well- ventilated temperature-controlled room between 18-23°C with relative humidity ranging from 40-60% under a 12-hour light/dark period).

[00250] Serum and RBC collection

[00251] Healthy female balb/c mice (50-56 days) were purchased from the Charles River Laboratory (Wilmington, MA). Whole blood, collected in EDTA coated tubes (BD Microtainer) or serum collecting tubes (BD Microtainer), was spun at lOOOg for lOmin at 4°C. Plasma and well as the huffy layer containing white blood cells and platelets, was removed; serum was stored at 4°C for lh. Isolated erythrocytes (RBCs) were washed by adding ice cold lx Dulbecco’s-Phosphate-Buffered- Saline (DPBS) pH 7.4 up to 12mL total volume and pipetting gently up and down to mix RBC extensively. RBC suspensions were centrifuged at 600g, 15min, 4C; supernatant was removed and this wash step was repeated three times.

[00252] Agglutination of RBC by HA-Dox

[00253] HA-Dox was adsorbed onto murine RBC at RT in 55% serum. Washed naive RBC and washed RBC: HA-Dox suspensions (1% Hematocrit) were dispensed onto a 96 U-shaped plate and visually accessed after 24h at room temperature after RBC suspension had fully sedimented. Carboxylated polystyrene beads was used as a positive control. [00254] Biodistrubtion

[00255] Dox, HA-Dox_, HA-AlexaFluro647, as well as HA-Dox-AlexaFluro647 were intravenously injected into healthy female Balb/c mice weighing between ~18-20g. The biodistribution studies of Dox and HA-Dox were performed after 0.08h, 0.5h, 6h, 24h post-injection. At these time intervals, the blood was collected from inferior vena cava (IVC) and organs were isolated. Organs were weighed, homogenized in 10% water (w/v) and centrifuged 20,000g for 20min. Homogenates (supernatant) were collected; drug was then extracted with 6x methanol, incubated for 30min with gentle vortex, and centrifuged 20,000g for 20min. Supernatant was read on a SpectraMax 13 plate reader was interpreted as a percentage of injected dose/tissue. The biodistribution studies of HA-AlexaFluro647, as well as HA-Dox-AlexaFluro647 were performed after 0.08h and 0.5h post injection. Intestines were harvested and far red fluorescence signals were imaged using Perkin Elmer IVIS small animal imaging system.

[00256] Flow Cytometry

[00257] HA-Dox was adsorbed onto murine RBC at RT in 55% serum. Washed naive RBC and washed RBC:HA-Dox suspensions (5 pL) at 10% Hematocrit were added to 995 pL of PBS, gently vortexed, and ran on a BD LSRFortessa cell analyzer (BD Biosciences, San Jose, CA USA), gated at 10,000 events. Data were analyzed using FCS Express Version 6 (DeNovo Software, Pasadena, CA USA). Washed RBC and HA-Dox suspensions at 10% Hemocrit were incubated at room temperature with annexin V-647 in buffer containing 2mM CaCF for 15 min. After incubation, an aliquot was aspirated into a BD LSRFortessa cell analyzer (BD Biosciences, San Jose, CA USA) for analysis, gated at 10,000 events. Results were expressed a percentage of Annexin V positive RBCs. Data were analyzed using FCS Express Version 6 (DeNovo Software, Pasadena, CA USA). Homogenates (lOOpL) from intestinal organs (duodenum, jejunum, ileum, cecum, colon), diluted in PBS (900pL) were gently vortexed, and directly aspirated into a BD LSRFortessa cell analyzer (BD Biosciences, San Jose, CA USA) for analysis, gated at 10,000 events.

[00258] Induction of colorectal cancer

[00259] All experiments involving naive and colon cancer mice were carried out in strict accordance with Guide for the Care and Use of Laboratory Animals, adopted by National Institutes of Health, approved by Harvard University IACUC. Briefly, 50-56 days old female balb/c mice were maintained in a temperature and humidity -controlled facility under a 12h light/dark cycle. Water and standard diet were given ad libitum. After one week of acclamation, animals were subcutaneously injected once with azoxymethane (AOM) (20mg/kg body weight). After 7 days, 2.0% Dextran Sulfate Sodium (DSS) (Sigma Aldrich) was given in drinking water over 7 days followed by regular drinking water for 1 week. Saline, 105pg Dox and 105pg HA-Dox was injected i.v. three times post DSS feeding period. After one week, mice were euthanized and intestines were harvested. [00260] Histology: H&E, Immunohistochemistry (IHC) of cell proliferation, inflammation, and apoptosis

[00261] Immediately after mice were euthanized, intestines were harvested in and placed in 10% formalin overnight and then processed for embedding in paraffin. Paraffin-embedded sections (5 pm) were treated with xylene; rehydrated in decreased ethanol series, and stained with hematoxylin and eosin (H and E) or immunohistochemistry and examined under a light microscope. To access the severity of AOM-DSS induced colon carcinogenesis the intensity of apoptotic and inflammatory markers was determined using IHC analysis. Colon and other intestinal tissues were immune stained with Caspase-3 (Abeam, Cambridge MA, USA), Bax (Novus, Littleton, CO, USA), iNOS (Abeam, Cambridge, MA, USA) and COX-2 (Abeam, Cambridge, MA, USA) as previously described and examined under a light microscope. Colon and other intestinal tissues were also stained with 1:200 dilution of anti-KI67 (Abeam, Cambridge, MA, USA) and later with 1:200 dilution of anti-rabbit secondary antibody (Abeam, Cambridge, MA, USA). The sections were then counterstained with hematoxylin and finally dehydrated and covered with coverslips.

[00262] Results

[00263] Characterization ofHA-Dox

[00264] HA-Dox was synthesized by conjugation of doxorubicin to HA through nucleophilic acyl substitution reaction chemistry. The morphology ofHA-Dox was examined by transmission electron microscopy (TEM). HA-Dox are spherical particles possessing a core-shell morphology (Fig. 25A). Typical core size of these “nanocomplexes” was around 50nm. Given the differences in hydrophobicities of HA and Dox, it is likely that Dox forms a hydrophobic core, surrounded by the extended shell of HA shell. Dynamic Light Scattering (DLS) measurements showed that these HA- Dox possessed a hydrodynamic size of approximately 175nm (Fig. 25B) and a zeta potential of -4.8 ± 1.06 mV, which is slightly negative charged. The negative zeta potential was likely due to the protonation of the hydroxyl groups of HA shells, which is favorable for dispersion of nanocomplexes in a biological-relevant environment and the efficient intracellular translocation of therapeutic compounds.

[00265] Pharmacokinetics of HA-Dox

[00266] Pharmacokinetics ofHA-Dox was evaluated in healthy Balb/c mice (105 pg of either free Dox or HA-Dox). HA-Dox exhibited longer circulation compared to free Dox. Dox and HA-Dox exhibited classical elimination behavior, however, a significant difference in the elimination half-life was found. While only 20% ID of free Dox was detected in blood 0.08 hours after injection, 40% ID ofHA-Dox could be detected in the blood at the time point (Fig. 26). The difference between blood concentrations of free Dox and HA-Dox persisted across all time points studied after 24 hours. The amount ofHA-Dox and free Dox circulating in the blood decreased 24 hours after administration (3% ID and 2% ID respectively). [00267] The mechanisms of enhanced persistence of HA-Dox was studied. Hyaluronic acid by itself has a blood half-life of 3 to 5 min in the blood. Hence, simple association of dox with HA is not expected to render long circulation. It was assessed whether association of dox-HA with red blood cells (RBCs) may be responsible for extended circulation. Prior literature has shown that RBC- association enhances the circulation of polymeric nanoparticles [75-77] HA-Dox bound to murine RBC with high efficiency; nearly 100% of RBC exhibited attachment HA-Dox on the surface in the presence of 55% serum (Fig. 27A and 27B).

[00268] Adsorption of HA-Dox onto RBCs did not induce agglutination (Fig. 27C, inset), implying that HA-Dox may not be detrimental to RBC. Furthermore, we investigated whether HA- Dox induces exposure to phosphatidylserine, a signal released from senesced and damaged RBC that facilitates their clearance from the blood circulation. The adsorption of HA-Dox did not induce an increase in RBCs expressing phosphatidylserine (0.41%±0.2) compared to naive (0.46%±0.3) (Fig. 27C).

[00269] Biodistribution of HA-Dox

[00270] Biodistribution of Dox and HA-Dox in mice was assessed. Significant accumulation of HA-Dox was found in intestine (-45% ID), while only -7% ID was found in the liver 0.5h after administration (Fig 28A). Minimal amounts were detected in the heart, lung, spleen, kidney, brain, stomach, pancreas and the spleen. Free Dox accumulated in the liver (8% ID) 0.5h after administration and -30% ID of free Dox accumulated in the intestine. -5% ID of free Dox remained in circulation. There was a significant increase in HA-Dox accumulation in the intestine compared to its free Dox counterpart (-45 % ID vs -30% ID respectively). The amount of HA-Dox in the intestine persisted at -45% ID for 24 hours. Similar trend was found for free dox except that the magnitude of accumulation was at 15% ID. (Fig. 28B). There was nearly a 3-fold increase in the intestine: blood ratio of HA-Dox compared to free Dox.

[00271] Within the intestines, most HA-Dox as well as free Dox was found to accumulate in the duodenum and jejunum after 0.08h. Unlike free Dox, a significant accumulation of HA-Dox was also observed in the ileum, colon, and cecum (Fig. 29A). Compared to 0.08h, the amount of HA-Dox in the duodenum and jejunum remained fairly constant after 0.5h (Fig. 29B), 6h (Fig. 29C), and 24h (Fig. 29D). This trend was also observed in the ileum and colon. The accumulation of HA-Dox in the cecum decreased over time. After perfusion at 24h, the amount of HA-Dox significantly decreased in all parts of the intestines (Fig. 33), suggesting that the majority of HA-Dox accumulates inside the blood vessels of the intestine.

[00272] There have been no prior reports of free Dox or any Dox complexes accumulating in the intestines via intravenous injection. To confirm HA-Dox accumulates in the intestines, duodenum and jejunum, ileum and colon, and cecum homogenate were obtained from mice 24h after IV administration and analyzed using a BD LSRFortessa cell analyzer. For each intestinal homogenate, a mean fluorescence intensity (MFI) shift in the Dox channel (488nm-582/42) compared to the non- injected mice was observed (Fig. 34). Furthermore, HA-Dox accumulation (displayed as bright green dots) in each intestinal tissue, 0.05h and 6h after intravenous administration, was also confirmed using confocal microscopy Fig. 35).

[00273] To assess the role of HA in targeting of HA-Dox in the intestine, localization of AlexaFluro647-labeled HA was measured. Strong signal intensities were observed in the stomach 0.08h after administration. Some signal was observed in all parts of the intestine (duodenum, jejunum, ileum, and the colon), suggesting that HA accumulates in the intestines (Fig. 36A). Although at a reduced level, signal could still be observed in the duodenum, colon, and different parts of the jejunum and ileum 0.5 h post injection (Fig. 36B). Similar to HA-AlexaFluro647, strong signal was observed in the stomach for HA-Dox- AlexaFluro647 and low signal were observed in the cecum and colon at both time points. No signal was detected in the duodenum, jejunum, ileum (Fig. 37A). At 0.5h post injection, however, stronger signal was detected in the duodenum and the beginning of the jejunum and no signal was detected in the ileum, cecum, or colon (Fig. 37B). The presence of Dox was verified using a BD LSRFortessa cell analyzer. For each duodenum and jejunum intestinal homogenate, there was a mean fluorescence intensity (MFI) shift in the Dox channel (488nm-582/42) compared to non-injected mice (Fig. 37C). Ileum, colon, and cecum homogenates displayed a small shift (Fig. 37D, 37E) in the Dox channel. These results indicate that both HA and Dox are responsible for the intestinal accumulation of HA-Dox.

[00274] Effect of Dox and HA-Dox on early signs of chemical induced colon cancer

[00275] The therapeutic potential of HA-Dox was tested using AOM-DDS induced colon cancer. Exposure to AOM-DDS induced colon cancer-like symptoms including significant shortening of the colon and intestines. Treatment of mice with saline alone did not revert the length reduction (Fig. 30). In contrast, dox-treated or HA-dox treated mice exhibited lengths comparable to healthy mice. Saline treated AOM-DDS mice also exhibited significantly larger stomachs (Fig. 30), compared to their free Dox and HA-Dox counterparts. This swelling is most likely due ascites, a pathological fluid accumulation within the peritoneal cavity that associated with colon cancer.

[00276] To investigate the effect of HA-Dox in chemical-induced colon cancer, histological analysis of AOM/DSS mice intestinal tissues was performed. Microscopic evaluation of H and E stained small intestine and colon sections of AOM/DSS treated mice displayed increased intestinal injuries compared to healthy mice (Fig. 31). Saline injected AOM/DSS mice exhibited substantial damaged intestinal villi as well as villi atrophy, which is exhibited by the erosion of the villi, resulting in a flat surface. The degree of villi atrophy is graduated. AOM/DSS mice injected with free Dox displayed a milder form of villi atrophy, while the small intestine of the HA-Dox treated AOM/DSS mice were covered with long villi that were almost the same length as the villi observed in healthy mice. Furthermore, villi of AOM/DSS mice also displayed large tears, compared to the healthy counterparts. Similar to villi atrophy, the length of these tears is graduated. The villi in saline injected AOM/DSS mice displayed large tears while villi in mice treated with free Dox displayed smaller tears. HA -Dox treated AOM/DSS mice had villi that had very small tears and appear to have the lining of the villi almost as intact as that of healthy mice.

[00277] The effect of Dox and HA-Dox on intestinal excessive cell proliferation of mice with chemical induced colon cancer

[00278] To evaluate the effect of HA-Dox on the proliferation and differentiation ability of intestinal crypt cells, Ki67 were assessed by immunohistochemistry staining (Fig. 32). The expression of Ki67 is strongly associated with tumor cell proliferation and growth. Many studies have suggested that the overexpression of Ki67 or the loss of proliferation control appear to be linked to colon cancer. Overall, the number of Ki67 positive cells (stained brown) in duodenum, jejunum, ileum, and colon of AOM/DSS mice injected with HA-Dox were markedly lower compared to the intestinal tissues in AOM/DSS mice injected with either saline and Dox, although the Ki67 expression levels in AOM/DSS mice injected with Dox was lower than AOM/DSS injected with saline. The number of Ki67 positive cells in intestinal tissues of AOM/DSS mice injected with HA-Dox was very comparable to levels of Ki67 positive cells in the intestinal tissues of healthy mice. It is important to note that there is a baseline level of cell proliferation associated with the constantly regenerating epithelial layer of the intestinal villi. These results indicate that HA-Dox might be helpful in maintaining differentiation and proliferation ability of intestinal crypt cells in colon cancer.

[00279] The effect of Dox and HA-Dox on intestinal inflammation in mice with chemical induced colon cancer

[00280] The effect of HA-Dox on the inflammation in AOM/DSS mice was also assessed in terms of (cyclooxygenase -2) COX-2 and inducible nitric oxide synthase (iNOS) expression levels by immunohistochemistry (Fig. 38). Although there were Cox-2 positive cells in healthy/normal intestinal tissues, there was a dramatic increase in COX-2 positive cells, stained brown, in AOM/DSS mice intestinal tissues compared to healthy intestinal tissues. Intestinal tissues of the healthy group had normal architecture, although there were cyclooxygenase-2 positive cells exhibited in the mucosa, particularly prevalent in the epithelial lining encapsulating the villi. AOM/DSS mice injected with HA-Dox had a lower expression of Cox-2 in duodenum, jejunum, ileum, and colon compared to the AOM/DSS mice injected with either saline and free Dox. A higher rate of cell proliferation tended to have an increased expression of COX-2. These findings are significant in HA-Dox and may play a role in the management of COX-2 expression, suggesting it might have COX-2 inhibitor properties. Similarly, iNOS positive cells were also predominantly prevalent in the epithelial lining surrounding the villi. Interestingly, there was no significant difference in the expression of iNOS in all intestinal tissues in AOM/DSS mice injected with saline, free Dox, and HA-Dox. All three of these groups were comparable to iNOS expression levels in the intestines present in the healthy mice. [00281] The effect of Dox and HA-Dox on intestinal apoptosis in mice with chemical induced colon cancer

[00282] There have been studies that have shown that Dox induces apoptosis by activation of caspase 3 in cultured cardiomyocytes in vitro and rat cardiac ventricles in vivo. It is unclear whether administration of Dox can cause the increased activation of caspase-3 in the intestines with the concomitant apoptosis and shedding of villus in healthy or sick mice. To specifically address the type of cell death responsible for intestinal epithelial cell (IEC) shedding and loss from the villus, we performed IHC for active caspase-3 as well as Bax2 in healthy and AOM/DSS mice (Fig. 39). Interestingly, caspase-3 -activation in the villi of the duodenum, jejunum, and ileum was strongly reduced after injection with either Dox or HA-Dox compared to saline in AOM/DSS mice so that the level of caspase-3 -activation in treated mice was comparable to levels in healthy mice. In contrast, there was no significant difference in the levels of caspase -3 -activation in the colon between saline, Dox, and HA-Dox treated AOM/DSS mice though caspase -3 -activation was lower in the colon of healthy mice. Interestingly, there was no significant difference in Bax-2 levels between saline, Dox, and HA-Dox treated AOM/DSS mice and healthy mice, although Bax-2 levels were elevated in the duodenum of HA-Dox treated AOM/DSS.

[00283] Discussion

[00284] Doxorubicin has been used to treat cancers such as leukemia, lymphoma, as well as breast cancer, lung cancer, and thyroid cancer. However, there are no reports of the use of doxorubicin treating intestinal/colon cancer. To our knowledge, this is the first report that intravenous injections of doxorubicin variants travel to the intestines. There are many studies have shown that drugs taken orally are absorbed systemically from the small intestine. Oral administration of doxorubicin remains a challenge for several reasons. Doxorubicin undergoes acid hydrolysis in the stomach and any remaining doxorubicin reaching the intestine is discharged by P-glycoprotein, found in the epithelial cells lining the small intestine and colon. Thus, doxorubicin exhibits low oral bioavailability and therefore there are no oral formulations for Dox on the market yet. It is only available on the market as intravenous formulations, often laden with cardiotoxicity. However, the presently-described variant of doxorubicin conjugated to hyaluronic acid is biocompatible.

[00285] The mechanism that enables HA-Dox to target and remain in the intestine after being intravenously injected is unclear, however, the presence of CD44 and RHAMM in the intestine may offer insight into the phenomenon. HA-Dox, a 150nm nanoparticle -like complex, was observed 0.08h (5min) after administration in the blood, however, over time, HA-Dox was cleared from the bloodstream. HA-Dox is able to absorb onto RBC in the presence of serum, however, it detaches quickly. Unlike most nanoparticles that are able to adsorb onto RBC [77,78], HA-Dox does not accumulate in the lungs, rather it accumulates in the intestines. Interestingly, a large amount of HA- Dox was found in the small intestine at 0.08h (5min) post injection, in particular, the duodenum and the jejunum. Furthermore, approximately the same amount of HA-Dox was still present after 24h post injection. Previous studies have shown that CD44 (a membrane glycoprotein) and RHAMM (receptor for hyalurone-mediated motility) two receptors in which hyaluronic acid (HA) can bind to are found in the intestines. CD44 is normally expressed in lower crypt epithelium of the intestinal mucosa, localized to the basolateral membrane of the cells. This may play a role in the generation and turnover of epithelial cells. In addition, CD44 was found in the intestinal lamina propria, in particular on stromal cells, macrophages, and lymphocytes. Comparable to the results found in mice, CD44 is overexpressed in human colorectal cancer [79-83] Akin to CD44, there have been studies that have shown that RHAMM protein expression is found in organs with high cell turnover, such as the small intestine and colon, in particular the base of the intestinal crypts [84-90] Taken together, the presence of CD44 and RHAMM offers a potential supposition as to why the HA-Dox targets and remains in the small intestine.

[00286] It was therefore analyzed whether intravenous injection of HA-Dox in AOM/DSS mice could be used to prevent colorectal cancer progression. Intriguingly, after multiple injections of HA- Dox in AOM/DSS mice, the length of the small intestine and colon as well as the size of the stomach were similar to those of the healthy mice. These data revealed that cell proliferation in the small intestine and colon crypts, inflammation, and apoptosis in the villi were reduced in the HA-Dox AOM/DSS treated mice, compared to the saline treated AOM/DSS mice, indicating the importance of HA-Dox as a prophylactic therapeutic capable of curbing the spread of cancer in the intestine.

[00287] There is evidence that a link between inflammation and colorectal cancer exists [91-101] Many studies have shown that pro-inflammatory mediators such as cyclooxygenase -2 (COX-2) and lipoxygenase pathways may lead to tumor cell proliferation, growth, thus promoting colorectal cancers. Anti-inflammatory agents such as COX2 inhibitors, as well as iNOS inhibitors, suppress colorectal cancer by inhibiting inflammatory pathways [102-118] Cox-2 inhibitors (e.g. rofexoxib, celecoxib, and valdecoxib), subclass of nonsteroidal anti-inflammatory drugs, reduces the production of prostaglandins, chemical that promotes inflammation [119-121] By providing anti-inflammatory benefits it allows the Cox-1 enzyme to retain its gastroprotectivity functions. Our findings indicate that HA-Dox functions similarly to COX2 inhibitors in this regard.

[00288] There are many reports that show that chemotherapy causes extensive damage to the DNA present in the intestinal cell wall. Researchers have been looking into reducing the damage done to the intestinal cell walls from chemotherapy, as it would render the treatment more bearable and allow for a higher rate of implementation of chemotherapy [122-131] Considering this, in our study, it is unclear whether HA-Dox induces apoptosis in the intestine due to the continuous shedding of the intestinal epithelium. As this is the most rapidly renewing tissue in the body, undergoing almost complete cellular turnover in as little as a few days, it is difficult to distinguish between normal cell death and apoptosis that is symptomatic of intestinal cancer. In inflammatory bowel disease (IBD), excessive cell death and apoptosis is observed in the colon and ileum epithelium. The development of IBD-related colorectal cancer (CRC), chronic inflammation is a major risk factor for gastrointestinal malignancies development in IBD patients. There has been evidence over the decade that show a link between chronic intestinal inflammation and CRC thorough a series of events; from the development of early dysplasia to low grade dysplasia to high-grade dysplasia to eventually converting to invasive adenocarcinoma [132-135] The development of extra-intestinal malignancies has also been shown in IBD patients. Currently, therapies to treat IBD diminish the mucosal inflammatory response [133]

Our findings suggest HA-Dox lowers inflammation levels in all parts of the intestine, stopping the further development of CRC.

[00289] In conclusion, thi study revealed that HA-Dox is an effective therapeutic agent to prevent or reduce the risk of colorectal cancer development, particularly for people who already suffer from inflammatory bowel disease.

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Claims

What is claimed herein is:

1. A polymer-drug conjugate comprising a hyaluronic acid molecule conjugated to: a) at least one doxorubicin molecule; and b) at least one camptothecin molecule.

2. The conjugate of any of the preceding claims, wherein the molar ratio of doxorubicin : camptothecin is 2:1 or a greater relative amount of doxorubicin.

3. The conjugate of any of the preceding claims, wherein the molar ratio of doxorubicin : camptothecin is 2: 1.

4. The conjugate of any of the preceding claims, wherein the molar ratio of doxorubicin : camptothecin is 5: 1 or a greater relative amount of doxorubicin.

5. The conjugate of any of the preceding claims, wherein the molar ratio of doxorubicin : camptothecin is 5: 1.

6. The conjugate of any of the preceding claims, wherein the molar ratio of doxorubicin : camptothecin is 15:1 or a greater relative amount of doxorubicin.

7. The conjugate of any of the preceding claims, wherein the molar ratio of doxorubicin : camptothecin is 15: 1.

8. The conjugate of any of the preceding claims, wherein the molar ratio of doxorubicin : camptothecin is 11:1 or a greater relative amount of doxorubicin.

9. The conjugate of any of the preceding claims, wherein the molar ratio of doxorubicin : camptothecin is 11 : 1 to 20: 1.

10. The conjugate of any of the preceding claims, wherein the molar ratio of doxorubicin : camptothecin is 14: 1 to 16: 1.

11. The conjugate of any of the preceding claims, wherein the camptothecin is conjugated to the hyaluronic acid via an ester bond.

12. The conjugate of any of the preceding claims, wherein the doxorubicin is conjugated to the hyaluronic acid via an amide bond.

13. The conjugate of any of the preceding claims, having a combination index (C.I.) value with cancerous cells of less than 0.2.

14. The conjugate of any of the preceding claims, having a C.I. value with cancerous cells of less than 0.6.

15. The conjugate of any of the preceding claims, having a C.I. value with cancerous cells of from 0.6 to 0.1.

16. A composition comprising the conjugate of any of the preceding claims and a salt.

17. A composition comprising micelles, each micelle comprising one or more of the polymer- drug conjugates of claims 1-15.

18. The composition of claim 16, further comprising a salt.

19. The composition of claim 16 or 18, wherein the salt comprises NaCl or PBS.

20. The conjugate or composition of any of the preceding claims, formulated for topical application.

21. The conjugate or composition of any of the preceding claims, for use in a method of treating a skin cancer or precancerous skin lesion.

22. A method of treating skin cancer or a precancerous skin lesion in a subject in need thereof, the method comprising administering a conjugate or composition of any of claims 1-20 to the subject.

23. The method of claim 22, wherein the conjugate or composition is administered topically.

24. The conjugate, composition, or method of any of claims 22-23, wherein the skin cancer is a non-melanoma skin cancer (NMSC).

25. The conjugate, composition, or method of any of claims 22-24, wherein the skin cancer is basal cell carcinoma (BCC) or cutaneous squamous cell carcinoma (cSCC).

26. The conjugate, composition, or method of any of claims 22-25, wherein the precancerous skin lesion is actinic keratosis (AK).

27. The conjugate, composition, or method of any of claims 22-26, wherein the skin cancer and/or precancerous skin lesion is not superficial.

28. The conjugate, composition, or method of any of claims 22-27, wherein the skin cancer and/or precancerous skin lesion is not only superficial.

29. The conjugate, composition, or method of any of claims 22-28, wherein the skin cancer and/or precancerous skin lesion is present in the dermis.

30. A method of treating blood cancer in a subject in need thereof, the method comprising administering a conjugate or composition of any of claims 1-19 to the subject.

31. The method of claim 30, wherein the blood cancer is acute myeloid leukemia or a T-cell lymphoma.

32. A method of treating glioblastoma in a subject in need thereof, the method comprising administering a conjugate or composition of any of claims 1-19 to the subject.

33. The method of claim 32, wherein the molar ratio of doxorubicin : camptothecin is 2: 1.

34. The method of claim 32, wherein the molar ratio of doxorubicin : camptothecin is 15:1.

35. A method of treating intestinal cancer in a subject in need thereof, the method comprising administering: a) a polymer-drug conjugate comprising a hyaluronic acid molecule conjugated to at least one doxorubicin molecule; or b) a conjugate or composition of any of claims 1-19; to the subject by intravenous injection.

36. The method of claim 35, wherein the intestinal cancer is gastrointestinal cancer, colon cancer, and/or colorectal cancer.