Methods for treating cancers characterized by chondroitin sulfate expression using polynuclear platinum compounds

Abstract

Methods are provided for treating cancers characterized by chondroitin sulfate (CS) expression, particularly chondroitin-4- sulfate (C4S) using polynuclear platinum compounds such as Triplatin tetranitrate. The methods include detecting CS levels in tumor tissue, selecting subjects with elevated CS, and administering an effective amount of the compound to treat cancer. Improved therapeutic selectivity and outcomes are achieved in tumors exhibiting CS enrichment.

Description

[0001] METHODS FOR TREATING CANCERS CHARACTERIZED BY CHONDROITIN

[0002] SULFATE EXPRESSION USING POLYNUCLEAR PLATINUM COMPOUNDS

[0003] CROSS-REFERENCE TO RELATED APPLICATIONS

[0004] This application claims benefit of United States provisional patent application 63 / 658,678 filed June 11, 2024, the contents of which are incorporated herein by reference.

[0005] FIELD OF THE INVENTION

[0006] The invention is generally related to methods for treating cancers characterized by expression of chondroitin sulfate, including chondroitin-4-sulfate, by administering a polynuclear platinum compound (PPC) such as Triplatin tetranitrate (BBR3464).

[0007] BACKGROUND OF THE INVENTION

[0008] Polynuclear platinum compounds, such as Triplatin tetranitrate (BBR3464), differ from classical platinum agents in their ability to interact with negatively charged biomolecules. Chondroitin sulfate (CS), particularly chondroitin-4- sulfate (C4S), is a sulfated glycosaminoglycan that is overexpressed in various solid tumors. Despite recognition of CS alterations in cancer, there has been no established method for stratifying patients based on tumor CS levels for selective treatment with platinum compounds.

[0009] SUMMARY

[0010] Disclosed herein are methods for identifying and treating subjects pre-disposed to respond favorably to treatment with polynuclear platinum compounds. The methods include identifying tumors with elevated CS expression and administering polynuclear platinum compounds compounds to treat these tumors effectively.

[0011] An aspect of the disclosure provides a method for treating cancer characterized by chondroitin sulfate expression in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a polynuclear platinum compound. In some embodiments, the chondroitin sulfate is chondroitin-4- sulfate (C4S) or other sulfated chondroitin-based moiety. In some embodiments, the chondroitin sulfate expression is elevated as compared to a predetermined reference value obtained from healthy tissue or a corresponding reference tumor cohort. Another aspect of the disclosure provides a method for treating cancer characterized by elevated chondroitin sulfate expression in a subject in need thereof, comprising detecting an elevated level of chondroitin sulfate in a biological sample obtained from the subject as compared to a predetermined reference value; and administering to the subject a therapeutically effective amount of a polynuclear platinum compound. In some embodiments, the detecting step is performed using an assay selected from the group consisting of immunohistochemistry (IHC), mass spectrometry-based glycan profiling, and glycan microarray analysis. In some embodiments, the polynuclear platinum compound is selected from the group consisting of Triplatin tetranitrate, BBR3571, TriplatinNC, and pharmaceutically acceptable salts, solvates, or structural analogs thereof. In some embodiments, the cancer is selected from the group consisting of ovarian cancer, breast cancer, pancreatic cancer, colorectal cancer, and lung cancer. In some embodiments, the administering step comprises intravenous administration or intraperitoneal infusion. In some embodiments, the elevated level of chondroitin sulfate expression is above a predetermined reference value obtained from healthy tissue or a corresponding reference tumor cohort. In some embodiments, the administering step provides a reduction in tumor size, inhibition of tumor growth, inhibition of metastasis, or improvement in survival. In some embodiments, the method further comprises co-administering, concurrently or sequentially, an anti-cancer agent selected from the group consisting of immune checkpoint inhibitors, PARP inhibitors, CDK4 / 6 inhibitors, or anti-angiogenic agents.

[0012] In some embodiments, the chondroitin sulfate is detected in a sample selected from the group consisting of tumor tissue, ascites, serum, plasma, or a fine needle aspirate. In some embodiments, the subject has previously failed platinum-based chemotherapy. In some embodiments, the polynuclear platinum compound is administered intraoperatively during a hyperthermic intraperitoneal chemotherapy (HIPEC) procedure. In some embodiments, mRNA or protein expression of a chondroitin sulfate biosynthetic enzyme selected from CHST11, CHST12, CSGALNACT1, or UST is measured to infer elevated CS levels. In some embodiments, expression of a chondroitin sulfate-associated proteoglycan selected from SDC1, VC AN, CD44, or GPC1 is used as a surrogate biomarker. In some embodiments, expression of chondroitin sulfate in tumor-associated stromal cells is used as a surrogate biomarker.

[0013] Another aspect of the disclosure provides a diagnostic kit comprising reagents for detecting elevated chondroitin sulfate in a tumor sample using a method selected from immunohistochemistry, mass spectrometry, or glycan microarray; and instructions for selecting a subject for treatment with a polynuclear platinum compound based on said detection.

[0014] BRIEF DESCRIPTION OF THE DRAWINGS

[0015] FIG. 1A-1D. (1A) General structure of PPCs and specific examples of (IB) Triplatin, (1C) BBR3571, and (ID) TriplatinNC. Counter-ions omitted for clarity but preferentially counter ion is nitrate, NOa’. In the general structure, L may be chloride or other coordinating anion or a formally substitution-inert group such as NH3 or aliphatic amine RNH2 or “dangling amine” as exemplified in ID where L is -H2N(CH2)6NH3+. Further, L may be positioned cis or trans to the bridging group Y.

[0016] FIG. 2A-2B shows cellular platinum uptake in CH0-K1 (control) and CHO-A745 (GAG-deficient) cells treated with 100 pM carboplatin (FIG. 2A) or 10 pM Triplatin (FIG 2B). Platinum content was measured by inductively coupled plasma mass spectroscopy (ICP- MS) and normalized by number of cells. ****p <0.0001, student t-test.

[0017] FIG. 3A-3B shows levels of Pt-DNA adducts in CHO-K1 and CHO-A745 cells treated with 100 p M carboplatin ( FIG. 3A) or 10 p M Triplatin (FIG. 3B). Platinum content in isolated DNA was measured by ICP-MS and normalized DNA quantity. ***p <0.001, ANOVA

[0018] FIG. 4A-4B shows the cytotoxicity of carboplatin (FIG. 4A) and Triplatin (FIG. 4B) in ES2 wt and Xyltl / Xylt2 KO cell lines; Ih treatment. Platinum content was measured by ICP-MS and normalized by number of cells.

[0019] FIG. 5A-5B shows platinum cellular accumulation in ES2 wt and Xyltl / Xylt2 KO cells treated with 10 pM carboplatin (FIG. 5A) or Triplatin (FIG. 5B) for 1, 2, 4, and 8h. Platinum content was measured by ICP-MS and normalized by number of cells.

[0020] FIG. 6A-6B shows platinum-DNA adducts in ES2 wt and Xyltl / Xylt2 KO cells treated with 10 pM carboplatin (FIG. 6A) or Triplatin (FIG. 6B) 4, and 8h.

[0021] FIG. 7A-7C shows that ES2-luc wt and Xyltl / Xylt2 KO tumors were implanted on the left and right flanks mice. (7A) Fold change in WT vs Xylt KO tumors. Tumors were harvested after 24h treatment with (7B) 40 mg / kg i.p Carboplatin or (7C) 0.3 mg / kg i.p. Triplatin. Tumors were digested in nitric acid and platinum measured by ICP-MS.

[0022] FIG. 8A-8C shows quantitative analysis of total GAG levels in ovarian cancer (OC) PDXs using GRIL-MS (FIG. 8A), compositional analysis of chondroitin sulfate in OC PDX using GRIL-MS (FIG. 8B) and compositional analysis of heparan sulfate in OC PDX using GRIL-MS (FIG. 8C).

[0023] FIG. 9A-9D shows representative images of rVAR2-V5 and H&E ovarian cancer PDX staining and Qupath analysis (9A). Qupath segmentation of (9B) rVAR2-V5 (+), (9C) rVAR2- V5 (-), and (9D) necrotic tumor area.

[0024] FIG. 10A-10D shows the sensitivity of ovarian cancer PDX models to Triplatin and carboplatin. PDX models (10A) CTG-1086, (10B) CTG-0964, (IOC) CTG-0791, and (10D) CTG-0258 were treated i.p. with carboplatin (40 mg / kg) or Triplatin (0.3 mg / kg) on days 0, 4 and 8 (arrows). **p<0.01, ****p<0.0001, 2-way ANOVA, Tukey test.

[0025] FIG. 11A-11C shows representative samples of patient OC subtypes (clinical history unknown) and normal tissues stained with rVAR2-V5 peptide from UVA CHTN OC TMA (FIG. 11A). Percentage of sample area staining positively for rVAR2-V5. Values are representative of the mean of 4 cores per patient sample (FIG. 11B). Percentage of TMA samples above the cut-off score (FIG. 11C).

[0026] FIG. 12A-12B shows the cytotoxicity of Triplatin in mouse CHX1990 wt and B4GalT7 (HS / CS deficient) KO PDAC cell lines; Ih treatment. (12A) IC50 value and (12B) percent cell growth are shown.

[0027] FIG. 13A-13B shows levels of Pt-DNA adducts in CHX1990 wt, CHST11 / 12 KO (C4S -deficient), CSGalNAcTl / 2 (CS-deficient) and B4GalT7 (HS / CS deficient) KO PDAC cell lines (FIG. 13A) Platinum cellular accumulation in CHX1990 wt and CHST11 / 12 KO (C4S -deficient) treated with 10 pM Triplatin for 1, 2, 4, and 8h (FIG. 13B). Platinum content was measured by inductively coupled plasma mass spectroscopy (ICP-MS) and normalized by number of cells.

[0028] FIG. 14A-14B show representative images of PDAC PDX tumors with and low rVAR2-V5 staining. The proportional score (PS) was calculated using the QuPath pixel classifier algorithm by segment each the area of each tumor image into regions of positive and negative staining (FIG. 14A) 800 pm and 10pm images of FFPE PDAC PDX tumors stained with GAG binding peptide (rVAR2-V5) (FIG. 14B).

[0029] FIG. 15A-15C shows 800 pm images of FFPE PDAC PDX tumors stained with rVAR2-V5. Qupath analysis calculates the percentage of tumor area staining positive for rVAR2 (FIG. 15A) Survival analysis of s.c. (FIG. 15B) G68 (female) and (FIG. 15C) P90 (male) PDAC PDX models treated with oxaliplatin (4 mg / kg) and Triplatin (0.3 mg / kg) given i.p. on days 1, 5, and 9. Log-rank **p<0.01, ****p<0.001

[0030] DETAILED DESCRIPTION

[0031] Embodiments of the disclosure provide methods for increasing the likelihood of effectiveness of cancer treatment with polynuclear platinum compounds. Embodiments include methods for identifying and treating cancers characterized by expression of chondroitin sulfate through the administration of polynuclear platinum compounds, such as Triplatin tetranitrate (BBR3464).

[0032] “Chondroitin sulfate” (CS) refers to a family of sulfated glycosaminoglycans composed of repeating disaccharide units of N-acetylgalactosamine and glucuronic acid, with sulfation at defined positions. CS is a component of the extracellular matrix that may be upregulated in a variety of solid tumors. Elevated CS levels in the tumor microenvironment contribute to enhanced tumor progression, invasion, and chemoresistance. Cancers exhibiting high CS expression represent an opportunity for selective therapeutic targeting. CS includes chondroitin-4-sulfate, chondroitin-6-sulfate, chondroitin-2,6- sulfate, and chondroitin-4, 6- sulfate. The name for each particular CS identifies the location of sulfation. For example chondroitin-4-sulfate (C4S) refers to CS with sulfation at the 4-position of the N- acetylgalactos amine residue.

[0033] Polynuclear platinum compounds (PPCs) are characterized by having two or more platinum atoms associated through linking groups or ligands and strong electrostatic interactions with polyanionic biomolecules. The multiple platinum centers allows for the formation of distinct DNA adducts which allows for increased potency as compared to conventional platinum drugs such as cisplatin. The generic structure for covalently-binding PPCs is shown in Figure 1A. Exemplary PPCs include Triplatin tetranitrate (BBR3464) (Figured IB) and BBR3571 (Figure 1C). Dinuclear PPCs where the bridging group is formally based on spermidine-based (such as BBR3571) or spermine-based linkers are included in the general structure. An example of a PPC where the linker is spermine-based is BBR3610 where the spermine -based (6-2-6) linker is H2N(CH2)6NH2(CH2 NH2(CH2)6NH2. PPCs may also be substitution-inert (SLPPCs) whereby the Pt-Cl bond is displaced by the substituion-inert NH3 or “dangling” amine H2N-Y-NH3, where Y is generally an aliphatic -(CH2)n- group. An exemplary SLPPC is TriplatinNC (Figure 1C). The PPCs described herein include the pharmaceutically acceptable salts, solvates, or structural analogs thereof.

[0034] The methods disclosed herein include identifying and treating cancers characterized by CS expression. In some embodiments, the cancer is characterized by elevated or increased CS expression. Elevated or increased expression refers to a level of CS in a biological sample that exceeds a predetermined reference value obtained from normal / healthy tissue or a reference tumor cohort (e.g. tumors of the same type as the tumor to be treated). By “healthy”, it is herein intended to mean that the subject is not afflicted by cancer, whether or not the subject is afflicted by another disease.

[0035] In one example, the CS expression is measured by immunohistochemistry (IHC). A common method involves evaluating both the staining intensity and the percentage of positive cells. The intensity is often scored on a scale of 0 to 3 (0: negative, 1: weak, 2: moderate, 3: strong staining), and the percentage of positive cells is scored on a scale of 0 to 3 (0: <1%, 1: 1-10%, 2: 11-50%, 3: 50-100%). A total score is then calculated by multiplying the intensity and percentage scores, which can range from 0 to 9. Elevated or increased expression may be defined as a score of 6 or higher, e.g. a score of 6, 7, 8, or 9.

[0036] Other methods for detecting CS expression include mass spectrometry-based glycan profiling and glycan microarray analysis, which enable quantitative and qualitative assessment of CS abundance and sulfation pattern in tumor tissue. Glycan Reductive Isotope Labeling - Liquid Chromatography / Mass Spectroscopy (GRIL-LC / MS) is used whereby tumor specimens are enzymatically digested using glycosaminoglycan-specific lyases (e.g., chondroitinase ABC) to depolymerize CS chains into characteristic disaccharide units. These disaccharides are then fluorescently labeled and analyzed using liquid chromatography-tandem mass spectrometry. The resulting glycan profile allows for quantification of specific CS disaccharides, based on their unique mass-to-charge ratios and retention times. Elevated C4S content, measured either as absolute abundance or relative proportion of total CS, can be used to stratify tumors likely to respond to Triplatin or related polynuclear platinum agents.

[0037] Alternatively or additionally, glycan-binding specificity and abundance may be assessed using glycan microarray platforms. Tumor lysates, fixed sections, or extracted glycoconjugates are applied to arrays containing immobilized probes such as monoclonal antibodies, lectins, or recombinant binding proteins with defined specificity for chondroitin sulfate motifs. Binding intensity is measured using fluorescence or chemiluminescence detection systems. In a preferred embodiment, a C4S-specific probe is used to assess C4S quantities. Elevated signal intensity, relative to matched normal tissue or standardized reference controls, is indicative of high tumor CS expression.

[0038] The methods described herein may be employed alone or in combination with transcriptomic profiling of CS biosynthetic enzymes (e.g., CHST11, CHST12, CSGALNACT1) and proteoglycan core proteins (e.g., VCAN, SDC1, CD44) to further refine patient selection criteria.

[0039] In some embodiments, mRNA or protein expression of a chondroitin sulfate biosynthetic enzyme selected from CHST11, CHST12, CSGALNACT1, or UST is measured to infer elevated CS levels. In some embodiments, expression of a chondroitin sulfate- associated proteoglycan selected from SDC1, VCAN, CD44, or GPC1 is used as a surrogate biomarker. In some embodiments, expression of chondroitin sulfate in tumor-associated stromal cells is used as a surrogate biomarker. Elevated levels of any of the aforementioned enzymes and surrogate biomarkers as compared to a healthy control or reference tumor cohort is indicative of elevated CS levels.

[0040] In another example, an increase in CS expression can be determined by detection of an increase of at least 2-fold, at least 3-fold, at least 4-fold, or at least 5-fold relative to control values (e.g., expression level in a tissue or cells from a subject without cancer or with a cancer that does not have elevated CS values).

[0041] A reference sample can be used to correlate and compare the results obtained in the methods of the invention from a test sample. Reference samples can be cells (e.g., cell lines, cell pellets) or tissue. The CS levels in the reference sample may be an absolute or relative amount, a range of amount, a minimum and / or maximum amount, a mean amount, and / or a median amount of CS. The methods may involve a comparison between expression levels of CS in a test sample and a reference value. In some embodiments, the reference value is the expression level of the CS in a reference sample. A reference value may be a predetermined value and may also be detennined from reference samples (e.g., control biological samples) tested in parallel with the test samples. A reference value can be a single cut-off value, such as a median or mean or a range of values, such as a confidence interval. Reference values can be established for various subgroups of individuals, such as individuals predisposed to cancer, individuals having early or late stage cancer, male and / or female individuals, or individuals undergoing cancer therapy.

[0042] In some embodiments, the reference sample is a sample from a healthy tissue, in particular a corresponding tissue which is not affected by cancer. These types of reference samples are referred to as negative control samples. In other embodiments, the reference sample is a sample from a tumor tissue that expresses CS. These types of reference samples are referred to as positive control samples. Positive control samples can also be used as a comparative indicator for the uniformity and / or degree of staining intensity, which correlates with the level of CS expression. Appropriate positive and negative reference levels of CS for a particular cancer, may be determined by measuring levels of CS in one or more appropriate subjects, and such reference levels may be tailored to specific populations of subjects (e.g., a reference level may be age-matched so that comparisons may be made between CS levels in samples from subjects of a certain age and reference levels for a particular disease state, phenotype, or lack thereof in a certain age group. Such reference levels may also be tailored to specific techniques that are used to measure levels of CS in biological samples (e.g., immunoassays, etc.), where the levels of CS may differ based on the specific technique that is used.

[0043] In one embodiment, the CS expression in the sample is compared to a negative control sample which demonstrates no or low detectable CS expression. In another embodiment, the CS expression in the sample is compared to a positive control sample having increased CS expression. A score or value for said cancer CS level that is greater than the score for a nominal or low expressing reference sample or a score for said cancer CS level that is equal to or greater than the score for a high CS expressing reference sample identifies said cancer as likely to respond to a polynuclear platinum compound.

[0044] A “sample” or “biological sample” of the present disclosure is of biological origin, in specific embodiments, such as from eukaryotic organisms. In preferred embodiments, the sample is a human sample, but animal samples may also be used. Non-limiting sources of a sample for use include solid tissue, biopsy aspirates, fine needle aspirate, ascites, fluidic extracts, blood, plasma, serum, spinal fluid, lymph fluid, the external sections of the skin, respiratory, intestinal, and genitourinary tracts, tears, saliva, milk, tumors, organs, cell cultures and / or cell culture constituents, for example.

[0045] Further embodiments provide methods of identifying a tumor as sensitive to treatment with a polynuclear platinum compound, said method comprising detecting an elevated level of CS in a biological sample obtained from the subject as compared to a predetermined reference value; and administering to the subject a therapeutically effective amount of a polynuclear platinum compound. By stratifying patients based on CS expression, therapeutic selectivity is enhanced, improving clinical outcomes while minimizing unnecessary toxicity to patients unlikely to benefit from treatment.

[0046] The disclosed methods allow for the selection of subjects that will respond favorably to treatment with PPCs. The term “respond favorably” generally refers to causing a beneficial state in a subject. With respect to cancer treatment, the term refers to providing a therapeutic effect on the subject. Positive therapeutic effects in cancer can be measured in a number of ways (See, W.A. Weber, J. Nucl. Med. 50: IS- IOS (2009)). For example, tumor growth inhibition, molecular marker expression, serum marker expression, and molecular imaging techniques can all be used to assess therapeutic efficacy of an anti-cancer therapeutic. With respect to tumor growth inhibition, according to NCI standards, a TIC < 42% is the minimum level of anti-tumor activity. A TIC <10% is considered a high anti-tumor activity level, with T / C (%) = Median tumor volume of the treated / Median tumor volume of the control x 100. The methods disclosed herein may provide a reduction in tumor size, inhibition of tumor growth, inhibition of metastasis, and / or improvement in survival.

[0047] The disclosed methods are particularly useful for enhancing treatment of cancer in a subject in need thereof. The terms “cancer” and “cancerous” refer to or describe the physiological condition in mammals in which a population of cells are characterized by unregulated cell growth. Examples of cancer include, but are not limited to, carcinoma, lymphoma, blastoma, sarcoma, and leukemia. More particular examples of such cancers include squamous cell cancer, small-cell lung cancer, non- small cell lung cancer, adenocarcinoma of the lung, squamous carcinoma of the lung, cancer of the peritoneum, hepatocellular cancer, gastrointestinal cancer, pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, hepatoma, breast cancer, colon cancer, colorectal cancer, endometrial or uterine carcinoma, salivary gland carcinoma, kidney cancer, liver cancer, prostate cancer, vulval cancer, thyroid cancer, hepatic carcinoma and various types of head and neck cancers. Preferred embodiments encompass treatment of multiple solid tumor types, including but not limited to ovarian, breast, pancreatic, colorectal, and lung cancers.

[0048] “Tumor” and “'neoplasm” refer to any mass of tissue that result from excessive cell growth or proliferation, either benign (noncancerous) or malignant (cancerous) including pre- cancerous lesions. The terms “cancer cell”, “tumor cell”, and grammatical equivalents refer to the total population of cells derived from a tumor or a pre-cancerous lesion, including both non- tumorigenic cells, which comprise the bulk of the tumor cell population, and tumorigenic stem cells (cancer stem cells).

[0049] Embodiments provide methods for treating cancer by the administration of PPCs as described herein. As used herein “treating” or “treatment” means any manner of managing the cancer by medicinal or other therapies, such that the cancer no longer increases in size, metastasizes, or otherwise progresses in severity on a diagnosis scale, such as Duke's classification or any other classification system known. In some embodiments, the treatment ameliorates the disease through a reduction in size or otherwise beneficially improves the severity on a diagnosis scale.

[0050] In certain embodiments, administration of the PPC may be combined with additional anti-cancer therapies, such as immune checkpoint inhibitors or chemotherapeutic agents, to further augment therapeutic efficacy. In some embodiments, the method comprises coadministering, concurrently or sequentially, an anti-cancer agent selected from the group consisting of immune checkpoint inhibitors, PARP inhibitors, CDK4 / 6 inhibitors, or anti- angiogenic agents.

[0051] Exemplary immune checkpoint inhibitors include inhibitors against immune checkpoint molecules such as CD27, CD28, CD40, CD 122, CD96, CD73, CD47, 0X40, GITR, CSF1R, JAK, PI3K delta, PI3K gamma, TAM, arginase, CD137 (also known as 4- 1BB), ICOS, A2AR, B7-H3, B7-H4, BTLA, CTLA-4, LAG3 (e.g., INCAGN2385), TIM3 (e.g., INCB2390), VISTA, PD-1, PD-L1 and PD-L2.

[0052] Examples of chemotherapeutic agents include tyrosine kinase inhibitors, topoisomerase inhibitors, proteasome inhibitors, matrix metalloproteinase inhibitors, alkylating agents, angiogenesis inhibitors, aromatase inhibitors, antimetabolites, anthracyclines, antitumor antibiotics, platinum drugs, radioactive isotopes, radiosensitizing agents, checkpoint inhibitors, PD1 inhibitors, plant alkaloids, glycolytic inhibitors, and prodrugs thereof.

[0053] Representative chemotherapeutic agents include, but are not limited to, cabozantinib, hydroxyl-cabozanitib, camptothecin, amsacrine, bleomycin, busulfan, capecitabine, carboplatin, carmustine, chlorambucil, cisplatin, cladribine, clofarabine, crisantaspase, cyclophosphamide, cytarabine, dacarbazine, dactinomycin, daunorubicin, docetaxel, doxorubicin, epipodophyllotoxins, epirubicin, etoposide, etoposide phosphate, fludarabine, fluorouracil, gemcitabine, hydroxycarb amide, idarubicin, ifosfamide, innotecan, leucovorin, liposomal doxorubicin, liposomal daunorubici, lomustine, mechlorethamine, melphalan, mercaptopurine, mesna, methotrexate, mitomycin, mitoxantrone, oxaliplatin, paclitaxel, pemetrexed, pentostatin, procarbazine, raltitrexed, satraplatin, streptozocin, teniposide, tegafur-uracil, temozolomide, teniposide, thiotepa, tioguanine, topotecan, treosulfan, vinblastine, vincristine, vindesine, vinorelbine, taxol and derivatives thereof, trastuzumab (HERCEPTIN®), cetuximab, and rituximab (RITUXAN® or MABTHERA®), bevacizumab (AVASTIN®), and combinations thereof.

[0054] The compounds described herein may be administered in vivo by any suitable route (e.g. parenterally or enterally) including but not limited to: inoculation or injection (e.g. intravenous, intraperitoneal, intramuscular, subcutaneous, intra-aural, intraarticular, intramammary, and the like), topical application, and by absorption through epithelial or mucocutaneous linings (e.g., nasal, oral, vaginal, rectal, gastrointestinal mucosa, and the like). Other suitable means include but are not limited to: inhalation (e.g. as a mist or spray), orally (e.g. as a pill, capsule, liquid, etc.), intravaginally, intranasally, rectally, by ingestion of a food or probiotic product containing the compound, as eye drops, etc. In preferred embodiments, the mode of administration is via intravenous or intraperitoneal routes, including during surgical procedures such as hyperthermic intraperitoneal chemotherapy (HIPEC). In some embodiments, the treatment described herein is administered with or without radiation therapy. In some embodiments, the polynuclear platinum compound is administered intraoperatively during a hyperthermic intraperitoneal chemotherapy (HIPEC) procedure.

[0055] A patient or subject to be treated by any of the compositions or methods of the present disclosure can mean either a human or a non-human animal including, but not limited to mammals, dogs, horses, cats, rabbits, gerbils, hamsters, rodents, birds, aquatic mammals, cattle, pigs, camelids, and other zoological animals. In some embodiments, the subject has previously failed platinum-based chemotherapy.

[0056] In some embodiments, the formulation or active agent is administered to the subject in a therapeutically effective amount. By a “therapeutically effective amount” or an “effective amount” is meant a sufficient amount to treat the disease or disorder at a reasonable benefit / risk ratio applicable to any medical treatment. It will be understood that the total daily usage of the compositions of the present disclosure will be decided by the attending physician within the scope of sound medical judgment. The specific therapeutically effective dose level for any particular subject will depend upon a variety of factors including the disorder being treated and the severity of the disorder; activity of the specific compound employed; the specific composition employed, the age, body weight, general health, sex and diet of the subject; the time of administration, route of administration, and rate of excretion of the specific compound employed; the duration of the treatment; drugs used in combination or coincidental with the specific active agent employed; and like factors well known in the medical arts. In the case of cancer, the effective amount of the drug or composition may: (i) reduce the number of cancer cells; (ii) reduce tumor size; (iii) inhibit, retard, slow to some extent and preferably stop cancer cell infiltration into peripheral organs; (iv) inhibit (i.e., slow to some extent and preferably stop) tumor metastasis; (v) inhibit tumor growth; (vi) prevent or delay occurrence and / or recurrence of tumor; and / or (vii) relieve to some extent one or more of the symptoms associated with the cancer. It is well within the skill of the art to start doses of the compound at levels or frequencies lower than those required to achieve the desired therapeutic effect and to gradually increase the dosage or frequency until the desired effect is achieved. However, the daily dosage of the active agent may be varied over a wide range from 1 to 1,500 mg per adult per day. In particular, the compositions contain at least or up to 1.0, 2.5, 5.0, 10.0, 15.0, 25.0, 50.0, 100, 250, 500, 750, 1000, 1250, or 1500 mg of the active ingredient for the symptomatic adjustment of the dosage to the subject to be treated. A medicament typically contains from about 0.01 mg to about 1500 mg of the active ingredient, in particular from 1 mg to about 250 mg of the active ingredient. An effective amount of the drug is ordinarily supplied at a dosage level at least or up to 1 mg / kg to 100 mg / kg of body weight per day, e.g. about 5 mg / kg, 10 mg / kg, 15 mg / kg, 20 mg / kg, 30 mg / kg, 40 mg / kg, 50 mg / kg, 60 mg / kg, 70 mg / kg, 80 mg / kg, 90 mg / kg, or 100 mg / kg of body weight per day. Such doses may be administered in a single dose or it may be divided into multiple doses.

[0057] The methods described herein offer a precision medicine approach for polynuclear platinum therapy, leveraging chondroitin sulfate enrichment as a predictive biomarker for patient selection and therapeutic responsiveness.

[0058] Further embodiments provide a method of inhibiting proliferation of CS-expressing cells in vitro or in vivo, comprising contacting the CS-expressing cells with a PPC as described herein.

[0059] Further embodiments provide a diagnostic kit comprising reagents for detecting elevated chondroitin sulfate in a tumor sample using a method selected from immunohistochemistry, mass spectrometry, or glycan microarray; and instructions for selecting a subject for treatment with a polynuclear platinum compound based on said detection.

[0060] Before exemplary embodiments of the present invention are described in greater detail, it is to be understood that this invention is not limited to particular embodiments described, as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the present invention will be limited only by the appended claims.

[0061] Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limit of that range and any other stated or intervening value in that stated range, is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges and are also encompassed within the invention, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the invention.

[0062] Unless defined otherwise, 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. Although any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present invention, representative illustrative methods and materials are now described.

[0063] All publications and patents cited in this specification are herein incorporated by reference as if each individual publication or patent were specifically and individually indicated to be incorporated by reference and are incorporated herein by reference to disclose and describe the methods and / or materials in connection with which the publications are cited. The citation of any publication is for its disclosure prior to the filing date and should not be construed as an admission that the present invention is not entitled to antedate such publication by virtue of prior invention. Further, the dates of publication provided may be different from the actual publication dates which may need to be independently confirmed.

[0064] It is noted that, as used herein and in the appended claims, the singular forms "a", "an", and "the" include plural referents unless the context clearly dictates otherwise. It is further noted that the claims may be drafted to exclude any optional element. As such, this statement is intended to serve as antecedent basis for use of such exclusive terminology as "solely," "only" and the like in connection with the recitation of claim elements, or use of a "negative" limitation.

[0065] As will be apparent to those of skill in the art upon reading this disclosure, each of the individual embodiments described and illustrated herein has discrete components and features which may be readily separated from or combined with the features of any of the other several embodiments without departing from the scope or spirit of the present invention. Any recited method can be carried out in the order of events recited or in any other order which is logically possible.

[0066] The invention is further described by the following non-limiting examples which further illustrate the invention, and are not intended, nor should they be interpreted to, limit the scope of the invention.

[0067] EXAMPLES

[0068] Example 1: Glycosaminoglycan-Dependent Cellular Uptake of Triplatin

[0069] To evaluate whether glycosaminoglycan (GAG) expression affects platinum drug uptake, Chinese hamster ovary cells (CH0-K1, GAG-proficient) and CHO-A745 (GAG- deficient) were treated with 10 pM Triplatin or 100 M carboplatin for 1 hour. Platinum content was quantified by ICP-MS and normalized to cell number. Results showed that Triplatin has significantly higher cellular uptake in CHO-K1 cells compared to CHO-A745 (FIG. 2B), while carboplatin uptake was unaffected by GAG status (FIG. 2A). These data demonstrate that Triplatin uptake is facilitated by GAG expression.

[0070] Example 2: GAG-Dependent DNA Platination by Triplatin

[0071] To determine whether GAG expression influences DNA adduct formation, CHO-K1 and CHO-A745 cells were treated with the same platinum concentrations for 4 hours. DNA was extracted and platinum-DNA adducts were quantified by ICP-MS. Results showed that Triplatin formed significantly more DNA adducts in CHO-K1 versus GAG-deficient CHO- A745 cells (FIG. 3B), whereas carboplatin adduct formation was not dependent on GAG expression (FIG. 3A).

[0072] Example 3: GAGs facilitate Triplatin Activity in Ovarian Cancer Cell Model

[0073] To examine Triplatin sensitivity in ovarian cancer cells, ES2 wild-type and crispR XYET1 / 2 knockout cells were treated with increasing concentrations of Triplatin or carboplatin for 1 hour. Cell viability was assessed 72 hours post-treatment. Results showed that GAG-deficient cells (XYLT1 / 2 KO) exhibited reduced sensitivity to Triplatin (FIG. 4B), with GAG-mediated uptake of Triplatin.

[0074] Example 4: Kinetics of Triplatin Uptake and DNA Binding in GAG-Deficient ES2 Cells

[0075] Time-course experiments (1, 2, 4, 8 hours) were performed using ES2 wild-type and XYLT1 / 2 KO cells exposed to Triplatin (10 pM) or carboplatin (10 pM). Platinum content was measured in whole cells (FIG. 5B) and on DNA (FIG. 6B). Results showed that Triplatin accumulation and DNA adduct formation were consistently lower in GAG-deficient cells at all timepoints, supporting the requirement of GAGs for efficient intracellular delivery.

[0076] Example 5: In Vivo Drug Accumulation in Isogenic Ovarian Cancer Tumors with high or low GAG levels

[0077] To assess tumor- specific platinum accumulation in vivo using matched ES2 GAG- positive and GAG-deficient (XYLT1 / XYLT2 KO) tumors. Nude mice were injected subcutaneously with ES2 wild-type (left flank) and XYLT1 / 2 KO (right flank) cells. Mice were dosed with 0.3 mg / kg Triplatin or 40 mg / kg carboplatin i.p. Tumors were collected after 24 hours and analyzed by ICP-MS. Results showed that Triplatin selectively accumulated in wild-type (GAG+) tumors (FIG. 7C), while carboplatin showed greater accumulation in GAG- deficient tumors (FIG. 7B). Tumor volume differences are shown in FIG. 7A.

[0078] Example 6: Glycomics Profiling of Patient-Derived Xenografts using Glycan Reductive Isotope Labeling Mass Spectrometry (GRIL-MS)

[0079] To identify chondroitin sulfate profiles in ovarian cancer tumors, GRIL-MS was used to quantify total GAG levels and composition in ovarian cancer PDX models. Specific quantification of C4S and heparan sulfate (HS) was performed. Results showed that C4S was the predominant GAG detected in ovarian cancer PDX models (FIG. 8A-8B). HS content was lower and more heterogeneous (FIG. 8C).

[0080] Example 7: C4S Distribution via rVAR2 IHC Staining

[0081] Tumor sections from OC PDXs were stained with rVAR2-V5, a C4S-binding peptide. Digital segmentation using QuPath categorized regions of rVAR2-positive staining, rVAR2- negative staining, and necrosis. Results show clear spatial separation of rVAR2+ and rVAR2- tumor areas (FIG. 9A-9D). These staining patterns were used for determination of the % positive staining area.

[0082] Example 8: Triplatin Response in C4S-High vs. C4S-Low Ovarian Cancer PDXs

[0083] Mice bearing OC PDXs (CTG-1086, CTG-0964, CTG-0791, CTG-0258) were treated i.p. with 0.3 mg / kg Triplatin or 40 mg / kg carboplatin on Days 0, 4, and 8. Tumor volumes were monitored over time. C4S-high PDX models were more sensitive to Triplatin and resistant to carboplatin (FIG. 10A-10D), validating biomarker-guided therapy.

[0084] Example 9: Prevalence of High C4S Expression in Human OC Tissues

[0085] A tissue microarray (TMA) containing ovarian cancer subtypes was stained with rVAR2-V5. Tumor area staining positive for C4S was quantified per core. Results show a substantial portion of patient samples displayed high C4S expression (PS > 40%) (FIG. 11 A- 11C), supporting the relevance of C4S in patient stratification.

[0086] Example 10: Triplatin Activity in C4S-Deficient Pancreatic Cancer Cells

[0087] CHX1990 PDAC cells and CRISPR KO variants (CHST11 / 12 KO, CSGALNACT1 / 2 KO, B4GALT7 KO) were treated with Triplatin. IC50 values and Pt-DNA adducts were measured. Results showed that C4S-deficient (CHST11 / 12 KO) cells had decreased sensitivity to Triplatin (FIG. 12A-12B, FIG. 13A-13B), consistent with findings in ovarian models.

[0088] Example 11: IHC Scoring of PDAC Tumors and Survival Outcome

[0089] FFPE PDAC PDX tumors were stained with rVAR2-V5. Proportional score (PS) was calculated based on rVAR2-positive tumor area using QuPath analysis (FIG. 14A-14B). In vivo survival analysis showed improved outcomes in Triplatin-treated PDAC PDX models (PS >35.5) compared to oxaliplatin-treated controls (FIG. 15A-15C).

[0090] While the invention has been described in terms of its preferred embodiments, those skilled in the art will recognize that the invention can be practiced with modification within the spirit and scope of the appended claims. Accordingly, the present invention should not be limited to the embodiments as described above, but should further include all modifications and equivalents thereof within the spirit and scope of the description provided herein.

Claims

CLAIMSWe claim:

1. A method for treating cancer in a subject in need thereof, comprising: determining that a tumor of the subject exhibits elevated chondroitin sulfate expression relative to a predetermined reference value; and administering to the subject a therapeutically effective amount of a polynuclear platinum compound.

2. The method of claim 1, wherein the chondroitin sulfate is chondroitin-4- sulfate (C4S) or other sulfated chondroitin-based moiety.

3. The method of claim 1, wherein the predetermined reference value is obtained from healthy tissue or a corresponding reference tumor cohort.

4. A method for treating cancer characterized by elevated chondroitin sulfate expression in a subject in need thereof, comprising: detecting an elevated level of chondroitin sulfate in a biological sample obtained from the subject as compared to a predetermined reference value; and administering to the subject a therapeutically effective amount of a polynuclear platinum compound.

5. The method of claim 4, wherein the detecting step is performed using an assay selected from the group consisting of immunohistochemistry (IHC), mass spectrometry-based glycan profiling, and glycan microarray analysis.

6. The method of any of claims 1-5, wherein the polynuclear platinum compound is selected from the group consisting of Triplatin tetranitrate, BBR3571, TriplatinNC, and pharmaceutically acceptable salts, solvates, or structural analogs thereof.

7. The method of any of claims 1-6, wherein the cancer is selected from the group consisting of ovarian cancer, breast cancer, pancreatic cancer, colorectal cancer, and lung cancer.

8. The method of any of claims 1-7, wherein the administering step comprises intravenous administration or intraperitoneal infusion.

9. The method of any of claims 4-7, wherein the elevated level of chondroitin sulfate expression is above a predetermined reference value obtained from healthy tissue or a corresponding reference tumor cohort.

10. The method of any of claims 1-9, wherein the administering step provides a reduction in tumor size, inhibition of tumor growth, inhibition of metastasis, or improvement in survival.

11. The method of any of claims 1-10, further comprising co-administering, concurrently or sequentially, an anti-cancer agent selected from the group consisting of immune checkpoint inhibitors, PARP inhibitors, CDK4 / 6 inhibitors, or anti-angiogenic agents.

12. The method of any of claims 1-11, wherein the chondroitin sulfate is detected in a sample selected from the group consisting of tumor tissue, ascites, serum, plasma, or a fine needle aspirate.

13. The method of any of claims 1-12, wherein the subject has previously failed platinumbased chemotherapy.

14. The method of any of claims 1-12, wherein the polynuclear platinum compound is administered intraoperatively during a hyperthermic intraperitoneal chemotherapy (HIPEC) procedure.

15. The method of any of claims 1-12, wherein mRNA or protein expression of a chondroitin sulfate biosynthetic enzyme selected from CHST11, CHST12, CSGALNACT1, or UST is measured to infer elevated CS levels.

16. The method of any of claims 1-12 wherein expression of a chondroitin sulfate-associated proteoglycan selected from SDC1, VC AN, CD44, or GPC1 is used as a surrogate biomarker.

17. The method of any of claims 1-12 wherein expression of chondroitin sulfate in tumor- associated stromal cells is used as a surrogate biomarker.

18. A diagnostic kit comprising: reagents for detecting elevated chondroitin sulfate in a tumor sample using a method selected from immunohistochemistry, mass spectrometry, or glycan microarray; and instructions for selecting a subject for treatment with a polynuclear platinum compound based on said detection.

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