Orally-delivered platinum pro-drug against cisplatin-resistant ovarian cancer

Abstract

The invention belongs to the field of medicinal chemistry, and particularly relates to a novel Pt(IV) complex, nanoparticles and compositions comprising same, methods and uses 5 thereof for the treatment of cancer. More particularly, the Pt(IV) complex comprises formula (I): wherein R1 and R2 are selected from the group comprising a mitochondria-targeting ligand and a chemosensitizer ligand, wherein R1 and R2 are not both mitochondria-targeting 10 ligands or chemosensitizer ligands, and may be used to treat ovarian cancer, preferably cisplatin-resistant ovarian cancer.

Description

[0001] ORALLY-DELIVERED PLATINUM PRO-DRUG AGAINST CISPLATIN-RESISTANT OVARIAN CANCER

[0002] FIELD OF THE INVENTION

[0003] The invention belongs to the field of medicinal chemistry, and particularly relates to a novel Pt(IV) complex, nanoparticles and compositions comprising same, methods and uses thereof for the treatment of cancer. More particularly, the Pt(IV) complex comprises a chemosensitizer ligand and a mitochondria-targeting ligand and may be used to treat ovarian cancer, preferably cisplatin-resistant ovarian cancer.

[0004] BACKGROUND OF THE INVENTION

[0005] Historically, the development of metal-based chemotherapeutics has been focusing on Pt drugs based on Pt(ll) species having DNA damaging properties, with cisplatin, carboplatin, oxaliplatin, nedaplatin, lobaplatin and haptaplatin being the only FDA-approved drugs. Cisplatin and oxaliplatin are the prime bivalent platinum (Pt (II)) examples with much of their use directed to ovarian cancer (OC). However, severe toxicities arising from their reactivity and non-specific interaction with off targets (e.g. blood proteins and / or healthy cells) have limited their effectiveness. These adverse effects, which mainly include nephrotoxicity, ototoxicity and peripheral neuropathy, can lead clinicians to reduce the drug dosage and / or to opt for non-neurotoxic agents, with the risk of limiting the effective clinical outcome. In addition, resistance against front-line Pt(ll) drugs such as cisplatin may develop during repeated treatment cycles due to decreased uptake, failure of apoptosis and accelerated DNA repair, which further limits the treatment effects. Further, all currently approved platinum chemotherapeutics are administered intravenously, and there are no available oral formulations.

[0006] Thus, there is a need for novel platinum-based drugs that can at least ameliorate one or more of the above-identified problems.

[0007] SUMMARY OF THE INVENTION

[0008] The present invention provides a new Pt(IV) complex which has shown the ability to overcome cisplatin resistance in an animal model of ovarian cancer and, differently from current Pt(ll) species (which require slow intravenous infusion), has the potential to be used as an oral drug. While resembling satraplatin, the Pt(IV) complex of the invention clearly differs in a few aspects. More particularly, the Pt(IV) complex comprises a chemosensitizer ligand and a mitochondria-targeting ligand and is resistant to strong acidic conditions. Since these Pt(IV) complexes reconstitute the bioactive Pt(ll) inside cells, the mechanism of action of the drugs is not modified. Rather, the specificity and the intracellular targeting is improved. In addition, the Pt(IV) complex of the invention is found to be effective in inhibiting the growth of liver cancer cells.

[0009] In a first aspect of the invention, there is provided a Pt(IV) complex of formula (I):

[0010] Formula 0) wherein Ri and R2 are selected from the group comprising a mitochondria-targeting ligand and a chemosensitizer ligand, wherein R1 and R2are not both mitochondria-targeting ligands or chemosensitizer ligands.

[0011] In some embodiments, the mitochondria-targeting ligand is selected from the group comprising delocalized cationic molecules such as rhodamine (for example, rhodamine 19, rhodamine 123), triphenylphosphonium (TPP), guanidinium, (E)-4-(1 H-indol-3-ylvinyl)-N- methylpyridinium iodide (F16) and dequalinium (DQA); mitochondria targeting / penetrating peptides (MTPs / MPPs) such as (L-cyclohexyl alanine-D-arginine)3;carbonyl cyanide m- chlorophenyl hydrazine (CCCP), triethylammonium, 3-phenylsulfonylfuroxan, pyridinium and 2,3-dimethylbenzothiazolium iodide.

[0012] In some embodiments, the chemosensitizer ligand is selected from the group comprising calcium channel blockers such as amlodipine, diltiazem, felodipine, isradipine, nicardipine, nifedipine, nimodipine, nisoldipine and verapamil; sodium channel blockers such as dichloro acetate (DCA), disopyramide, flecainide, mexiletine, propafenone and quinidine; steroids and steroid-like compounds such as tamoxifen; cyclic peptides such as cyclosporin A; phytochemicals such as flavonoids, alkaloids and polyphenols; P-gp inhibitors such as curcumin, metformin, resveratrol and elacridar; histone deacetylase inhibitor such as phenyl butyrate (PhB), vorinostat, romidepsin and belinostat. In some embodiments, the mitochondria-targeting ligand is a delocalized cationic molecule selected from the group comprising rhodamine (for example, rhodamine 19, rhodamine 123), TPP, guanidinium, F16 and DQA, and the mitochondria-sensitizer ligand is a sodium channel blocker selected from the group comprising dichloro acetate (DCA), disopyramide, flecainide, mexiletine, propafenone and quinidine, or a histone deacetylase inhibitor selected from the group comprising phenyl butyrate (PhB).

[0013] In some embodiments, the mitochondria-targeting ligand is triphenylphosphonium (TPP) and the mitochondria-sensitizer ligand is dichloroacetate (DCA) or phenylbutyrate (PhB).

[0014] In some embodiments the Pt(IV) complex further comprises a second anticancer drug complexed to it.

[0015] In a second aspect of the invention, there is provided a nanoparticle comprising the Pt(IV) complex of the first aspect of the present disclosure.

[0016] In a third aspect of the invention, there is provided a composition comprising the Pt(IV) complex or the nanoparticle of the present disclosure, and a pharmaceutically acceptable excipient.

[0017] In some embodiments, the composition is in the form of an oral formulation. Preferably, the composition is in a solid dosage form such as tablets, capsules, granules, powders, or in a liquid dosage form such as solutions or suspensions.

[0018] In some embodiments, the composition is used for treating a cancer. In some embodiments, the cancer is selected from the group comprising ovarian cancer, lung cancer, prostate cancer, testicular cancer, liver cancer and colorectal cancer. Preferably, the cancer is an ovarian cancer. More preferably, the cancer is a cisplatin-resistant ovarian cancer.

[0019] In a fourth aspect of the invention, there is provided a method of treating cancer in a subject, comprising administering to the subject an effective amount of the complex of the first aspect, the nanoparticle of the second aspect, or the composition of the third aspect of the invention.

[0020] In some embodiments, said method further comprises administering a second anticancer therapy. Preferably, the second anticancer therapy is selected from a radiotherapy, chemotherapy, hormone therapy or immunotherapy. The second anticancer therapy may be administered concurrently or sequentially with the complex of the first aspect, the nanoparticle of the second aspect, or the composition of the third aspect of the invention. Said second anticancer therapy may be a drug that is complexed with the Pt(IV) complex.

[0021] In a fifth aspect of the invention, there is provided a use of the complex of the first aspect, the nanoparticle of the second aspect, or the composition of the third aspect in the manufacture of a medicament for treating a cancer.

[0022] In some embodiments, the cancer is selected from the group comprising ovarian cancer, lung cancer, prostate cancer, testicular cancer, liver cancer, and colorectal cancer. Preferably, the cancer is an ovarian cancer. More preferably, the cancer is a cisplatin-resistant ovarian cancer.

[0023] In a sixth aspect of the invention, there is provided a method of preparing the complex of the invention, comprising the following steps:

[0024] (a) reacting platinum compound JM149 with a mitochondria-targeting ligand to obtain a

[0025] Pt(IV) monoester wherein JM 149 i

[0026] (b) reacting Pt(IV) monoester obtained in step (a) with a chemosensitizer ligand to obtain the Pt(IV) complex.

[0027] In some embodiments, the order of adding the mitochondria-targeting ligand and the chemosensitizer may be changed. Thus, it is possible to react JM149 with a chemosensitizer first, and then with a mitochondria-targeting ligand to obtain the Pt(IV) complex of the present invention. However, the order of addition of the mitochondria-targeting ligand and the chemosensitizer may affect the yield of the final product.

[0028] In a seventh aspect of the invention, there is provided a method of producing a cell vesicle comprising the Pt(IV) complex of the invention, comprising the following steps:

[0029] (a) mixing lipids and the Pt(IV) complex of the invention in a solvent,

[0030] (b) forming a thin film by removing the solvent,

[0031] (c) hydrating the thin film with a solution comprising cell ghosts, and

[0032] (d) subjecting the hydrated thin film to shearing, extrusion, or freeze thawing and sonication to obtain the cell vesicle. Alternatively, a thin film comprising lipids may be produced and then hydrated in a suspension of cell ghosts and Pt(IV) complex of the invention, before step (d). Optionally, a second anticancer drug may be included in the seventh aspect.

[0033] In this aspect, the obtained cell vesicle consists of cell ghosts fused with lipids, and encapsulates the complex of the invention, and the optionally second anticancer drug.

[0034] In some embodiments, the lipids are selected from a group consisting of 1 ,2-dioleoyl-3- trimethylammonium-propane (DOTAP), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), L-a-phosphatidylcholine (HSPC), (N-(1-(2, 3-dioleoyloxy)propyl)-N,N,N-trimethylamine) (DOTMA) and (dimethyl-dioctadecyl-ammonium-bromide (DDAB), 1,2-dioleoyl-sn-glycero-3- phosphoethanolamine (DOPE), distearophosphoethanolamine polyethyleneglycol 2000 (DSPE-PEG2000), 1 ,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[amino(polyethylene glycol)-2000] (DSPE-PEG2000 Amine) and 1,2-distearoyl-sn-glycero-3- phosphoethanolamine-N-[biotinyl(polyethyleneglycol)-2000] (DSPE-PEG2000 Biotin), dioleoylphosphatidylcholine (DOPC), cholesterol or combinations thereof.

[0035] In some embodiments, the solvent is an organic solvent. Preferably, the solvent is selected from a group comprising methanol, dichloromethane, ethanol, and chloroform.

[0036] BRIEF DESCRIPTION OF THE DRAWINGS

[0037] Certain embodiments of the present disclosure are described more fully hereinafter with reference to the accompanying drawings.

[0038] Figure 1 shows a representative scheme for synthesizing platinum compound JM149 from platinum compound JM118.

[0039] Figure 2 shows a representative scheme for synthesizing platinum compound JM118 from platinum compound K2Pt&4.

[0040] Figure 3 shows a scheme for synthesizing the Pt(IV) complex of the present invention used in Example 1.

[0041] Figure 4 illustrates the effects of the Pt(IV) complex in murine models treated with saline, cisplatin and the Pt(IV) complex. A: I VIS image of tumor growth of mice in week 1 and week 2. B: tumour dry weight of mice on Day 24. C: images of tumors from representative mice on Day 24. D: Tumor-associated luminescence determined by an I VIS imaging system.

[0042] Figure 5 illustrates the toxicity of the Pt(IV) complex in murine models treated with saline, cisplatin and the Pt(IV) complex. A: body weight of treated mice over time. B: dry weight and representative images of spleens of treated mice. C: dry weight of lungs, heart and liver of treated mice. D: dry weight of kidney tissues. E-G: Haematoxylin and eosin staining results on kidney tissues from mice treated with saline (E), cisplatin (F, black arrows indicate that the architecture of renal tubules was affected, while glomeruli were unaffected in the kidneys) and the Pt(IV) complex (G).

[0043] Figure 6 illustrates the stability of the Pt(IV) complex under HCI, PBS and 3 mM ascorbic acid conditions which mimic the strong acidic conditions of the stomach, physiological conditions and the intracellular environment, respectively.

[0044] Figure 7 shows the ICso values at 24h (A) and 48 h (B) for cisplatin, 4Cis, Satraplatin, JM118 and the Pt(IV) complex in HepG2 liver cancer cells (n=3). * Indicates the statistical significance at P<0.05 and ** indicates statistical significance at P<0.01.

[0045] Figure 8 shows flow cytometry results of apoptosis induced by different compounds at 24 hours (A) and 48 hours (B) after treatment. The bottom left quadrant indicates healthy cells, bottom right quadrant indicates early apoptotic cells, top right quadrant indicates late apoptotic cells, and top left quadrant indicates DNA fragmented cells.

[0046] Figure 9 shows representative images of cells under tomocube upon drug treatment after 24h (A) and 48h (B), indicating the morphological changes induced by apoptosis when compared to normal cells. (C) Zoomed image of untreated cells (left) and drug-treated cells (right) after 48 h. Circles indicate cells having undergone apoptosis.

[0047] Figure 10 shows nanoparticle tracking analysis of Pt (IV) complex loaded in nCVTs during the hydration step (A) and during lipid thin film formation (B).

[0048] Figure 11 shows radiant efficiency produced by the labelled nCVTs (left) and Pt(IV) complex- loaded nCVTs (right). Figure 12 shows radiant efficiency produced by the labelled nCVTs (left) and the Pt(IV) complex-loaded nCVTs (right) in different organs. 1. Lungs, 2. Heart, 3. Stomach, 4. Kidney, 5. Spleen, 6. Liver.

[0049] DETAILED DESCRIPTION OF THE INVENTION

[0050] Further details of the invention will now be described with reference to the following nonlimiting examples. Unless otherwise defined herein, technical and scientific terms used in the present description have the meanings that are commonly understood by those of ordinary skill in the art.

[0051] A. Definitions

[0052] For convenience, certain terms employed in the specification, examples and appended claims are collected here.

[0053] As used herein, the singular forms “a,” “an,” and “the” include plural referents unless the context clearly dictates otherwise.

[0054] As used herein, the term “comprising” may include the embodiments “consisting of” and “consisting essentially of”. The terms “comprise(s),” “include(s),” “having,” “has,” “can,” “contain(s),” and variants thereof, as used herein, are intended to be open-ended transitional phrases that require the presence of the named ingredients / steps and permit the presence of other ingredients / steps. However, such description should be construed as also describing compositions, mixtures, or processes as “consisting of” and “consisting essentially of” the enumerated ingredients / steps, which allows the presence of only the named ingredients / steps, along with any impurities that might result therefrom, and excludes other ingredients / steps.

[0055] As used herein, the term “Pt(IV) complex(es)” refers to prodrugs generally having a central platinum atom attached with two axial ligands, which render the whole complex inert outside cancer cells. It is generally agreed that Pt(IV) prodrugs must be reduced to the active Pt(ll) forms (such as cisplatin, carboplatin and oxaliplatin) to exert their antitumor activity. After cellular entrance, Pt(IV) complexes are subsequently activated by biological reducing agents, such as ascorbic acid, cysteine (Cys), glutathione (GSH), and other macromolecules. This reduction results in the release of two axial ligands, which provide a means to fine-tune the properties of the complexes including lipophilicity, redox potential, target specificity and fluorescence. The two axial ligands can also be utilized to enhance the activity of platinum for example by potentiating the anti-proliferative properties and overcome resistance.

[0056] As used herein, the term “mitochondria-targeting ligand(s)” refers to any molecule that can direct the drug to which said ligand attached or conjugated to mitochondria and facilitates its accumulation within the mitochondrial matrix. While mitochondria, similar to the nucleus, contain DNA (mitochondrial (mt)DNA), they lack the Nuclear Excision Repair (NER) mechanisms and thus possess less efficacious recovery features. The lack of repair mechanisms makes mitochondria an attractive target for Pt drugs to overcome resistance of cancer cells to Pt treatment and to induce apoptosis.

[0057] As used herein, the term “chemosensitizer ligand” refers to any molecule or compound that is attached or conjugated to the platinum drug and makes cancer cells more sensitive to the effects of chemotherapy. Chemotherapy is the standard of care in the treatment of many cancers, combining cytotoxic drugs such as anthracyclines, taxanes, cyclophosphamide and platinum, all of which could induce cell death through various mechanisms. However, resistance to chemotherapy may develop over time during repeated treatment, which has become one of the most pressing major dilemmas in cancer therapy. Chemotherapy resistance can arise due to various factors, such as gene amplification, escape and mutation of cancer cells survived from the primary treatment, pump-out and reduced uptake of drugs, enhanced DNA repair ability, and inactivation of drugs. Various solutions have been developed to address this problem, for example, by using combination chemotherapy, using drugs that could prevent the emergence of multidrug resistance, modifying chemotherapy regimen, using drugs that work in different ways, or using chemosensitizers to enhance the sensitivity of cancer cells. In the present disclosure, a chemosensitizer ligand is used to overcome the resistance of platinum-based drugs (such as cisplatin) and to sensitize the cancer cells.

[0058] The phrase "pharmaceutically acceptable", as used in connection with compositions described herein, refers to molecular entities and other ingredients of such compositions that are physiologically tolerable and do not typically produce untoward reactions when administered to a subject (e.g., a human).

[0059] As used herein, “treatment” or “treating” is an approach for obtaining beneficial or desired results including clinical results. For purposes of this invention, beneficial or desired clinical results include, but are not limited to, one or more of the following: alleviating one or more symptoms resulting from the disease, diminishing the extent of the disease, stabilizing the disease (e g., preventing or delaying the worsening of the disease), preventing or delaying the spread (e.g., metastasis) of the disease, preventing or delaying the recurrence of the disease, delay or slowing the progression of the disease, ameliorating the disease state, providing a remission (partial or total) of the disease, decreasing the dose of one or more other medications required to treat the disease, delaying the progression of the disease, increasing the quality of life, and / or prolonging survival. Also encompassed by “treatment” is a reduction of pathological consequence of the disease. The methods of the present disclosure contemplate any one or more of these aspects of treatment.

[0060] As used herein, the term “effective amount” refers to an amount of an agent sufficient to treat a specified disorder, condition or disease such as ameliorate, palliate, lessen, and / or delay one or more of its symptoms. In reference to cancer, an effective amount comprises an amount sufficient to cause a tumor to shrink and / or to decrease the growth rate of the tumor (such as to suppress tumor growth) or to prevent or delay other unwanted cell proliferation. In some embodiments, an effective amount is an amount sufficient to delay development. In some embodiments, an effective amount is an amount sufficient to prevent or delay recurrence. An effective amount can be administered in one or more administrations. 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.

[0061] As used herein, an “individual” or a “subject” refers to a mammal, including, but not limited to, human, bovine, horse, feline, canine, rodent, or primate. In some embodiments, the individual is a human.

[0062] B. The Pt(IV) complex and its delivery

[0063] In a first aspect of the invention, there is provided a Pt(IV) complex having the following formula (I): wherein Ri and R2are selected from the group comprising a mitochondria-targeting ligand and a chemosensitizer ligand, wherein Ri and 2 are not both mitochondria-targeting ligands or chemosensitizer ligands.

[0064] The presence of both the mitochondria-targeting ligand and the chemosensitizer ligand in the Pt(IV) complex shows synergistic effects, with decrease in mitochondrial potential, abrogation of mitochondrial respiratory activity and re-sensitization of cancer cells, thereby enhancing the therapeutic activity of the complex compared to the corresponding Pt(ll) drug used alone, especially when treating cancer cells that have already developed chemotherapy resistance.

[0065] Examples of mitochondria-targeting ligands that can be used in the present disclosure may include, but not limited to, delocalized cationic molecules such as rhodamine (for example, rhodamine 19, rhodamine 123), triphenylphosphonium (TPP), guanidinium, (E)-4-(1 H-indol-3- ylvinyl)-N-methylpyridinium iodide (F16) and dequalinium (DQA); mitochondria targeting / penetrating peptides (MTPs / MPPs) such as (L-cyclohexyl alanine-D-arginine)3; carbonyl cyanide m-chlorophenyl hydrazine (CCCP), triethylammonium, phenylsulfonylfuroxan, pyriudinium, 2,3-dimethylbenzothiazolium iodide. Other mitochondria- targeting ligands that can be used in the present disclosure are discussed in Carmine Pasquale Cerrato & Ulo Langel (2022), 19:2, 133-146; Xiao Q et al., 2021 , 27(47):12207- 12214; Battogtokh G et al., Acta Pharm Sin B. 2018 Oct, 8(6):862-880; each of which is incorporated herein by reference in its entirety to the extent that it does not conflict with the present disclosure.

[0066] Preferably, the mitochondria-targeting ligand is a delocalized cationic molecule selected from the group comprising rhodamine (for example, rhodamine 19, rhodamine 123), TPP, guanidinium, F16 and DQA. More preferably, the mitochondria-targeting ligand is TPP.

[0067] Examples of chemosensitizer ligands that can be used in the present disclosure may include, but not limited to, calcium channel blockers such as amlodipine, diltiazem, felodipine, isradipine, nicardipine, nifedipine, nimodipine, nisoldipine and verapamil; sodium channel blockers such as dichloro acetate (DCA), disopyramide, flecainide, mexiletine, propafenone and quinidine; steroids and steroid-like compounds such as tamoxifen; cyclic peptides such as cyclosporin A; phytochemicals such as flavonoids, alkaloids and polyphenols; P-gp inhibitors such as curcumin, metformin, resveratrol and elacridar; histone deacetylase inhibitors such as phenyl butyrate (PhB), vorinostat, romidepsin and belinostat. Preferably, the chemosensitizer ligand is a sodium channel blocker selected from the group comprising dichloro acetate (DCA), disopyramide, flecainide, mexiletine, propafenone and quinidine, or a histone deacetylase inhibitor selected from the group comprising phenyl butyrate (PhB). More preferably, the chemosensitizer ligand is DCA or PhB. Most preferably, the chemosensitizer ligand is PhB.

[0068] In some embodiments, the mitochondria-targeting ligand is TPP, and the chemosensitizer ligand is DCA or PhB. Preferably, the mitochondria-targeting ligand is TPP, and the chemosensitizer ligand is PhB.

[0069] Both the mitochondria-targeting ligand and the chemosensitizer ligand can be conjugated to the platinum core through various linkers according to the present disclosure. Any suitable linkers known in the art may be used. Examples of linkers that can be used in the present disclosure include, but not limited to, alkyl linkers, hydroxypropyl linker, ether linkers, amide linkers, ester linkers, carbamate linkers, carbonate linkers, 4-aminobenzyl alcohol (4ABA) linker.

[0070] The Pt(IV) complexes of the present disclosure can be delivered by nanoparticles. Thus, in one embodiment, there is also provided a nanoparticle comprising the Pt(IV) complexes of the present disclosure. Various types of nanoparticles have been utilized to deliver platinum drugs, including carbon nanotubes, gold nanoparticles, quantum dots, up-conversion nanoparticles, porous silicon nanoparticles, liposomes, cell vesicles, metal-organic frameworks, and polymeric micelles. Most nanoparticles showed enhanced cytotoxicity due to many advantages such as enhanced permeability and retention (EPR) effects, fewer adverse reactions, and controlled drug release.

[0071] In one embodiment, the nanoparticle includes a hydrophobic core, a hydrophilic layer surrounding the core, and the Pt(IV) complexes of the present disclosure. In some embodiments, the Pt(IV) complexes are contained or embedded within the core. The Pt(IV) complexes are preferably released from the core at a desired rate. In one embodiment, the core is biodegradable and releases the Pt(IV) complexes as the core is degraded or eroded. The mitochondria-targeting ligand of the Pt(IV) complexes preferably extend outwardly from the core so that they are available for interaction with cellular components, which will favour preferential distribution to the mitochondria.

[0072] The core of the nanoparticle may be formed from any suitable synthetic or natural bioabsorbable polymers. Such polymers are recognizable and identifiable by one or ordinary skill in the art. Non-limiting examples of such polymers include poly(amides) such as poly(amino acids) and poly(peptides); poly(esters) such as poly(lactic acid), poly(glycolic acid), poly(lactic-co-glycolic acid) (PLGA), and poly(caprolactone); poly(anhydrides); poly(orthoesters); poly(carbonates); and chemical derivatives thereof (substitutions, additions of chemical groups, for example, alkyl, alkylene, hydroxylations, oxidations, and other modifications routinely made by those skilled in the art), fibrin, fibrinogen, cellulose, starch, collagen, and hyaluronic acid, copolymers and mixtures thereof. Preferably, the hydrophobic core comprises PLGA, such as PLGA-COOH or PLGA-OH.

[0073] The hydrophilic layer or portion thereof may be formed from any suitable hydrophilic polymer or hydrophilic portion of an amphiphilic polymer. The hydrophilic polymer or hydrophilic portion of a polymer may be a linear or branched or dendritic polymer. Examples of suitable hydrophilic polymers include polysaccharides, dextran, chitosan, hyaluronic acid, polyethylene glycol, polymethylene oxide, and the like. In one embodiment, a hydrophilic portion of a block copolymer comprises polyethylene glycol (PEG). In one embodiment, a block copolymer comprises a hydrophobic portion comprising PLGA and a hydrophilic portion comprising PEG.

[0074] In some embodiments, the nanoparticle is a cell vesicle, preferably a cell vesicle consisting of cell ghost membrane components in a hybrid mixture with lipids (i.e., nano-cell vesicle technology (nCVT) of the present invention). In such embodiments, the nCVT may be obtained by fusing cell ghosts with suitable lipids using any known method such as the hydration method or lipid thin film method, as described in Example 6. A “cell ghost” used herein is a cell which has been emptied of all or substantially all of the cytosolic contents of the cell including the nucleus and nuclear contents. Cell ghosts resemble the cell from which they are derived but are non-viable and have a much reduced cytoplasmic content. The cell membrane of a cell ghost includes biomolecules such as lipids and proteins and generally retains the characteristics of the parent cell. For example, cell ghosts derived from monocyte cell lines retain the intrinsic targeting properties of monocytes towards inflammation sites. More specifically, cell ghosts may be derived from a cell line selected from the group comprising RAW264.7 (mouse macrophage), HaCaT (human keratinocyte), HDF (human dermal fibroblast) and, more particularly, U937 (human monocytes), Jurkat (human T cells), CT26 (mouse colon carcinoma), HeLa (human cervical cancer), HEK293 (human kidney epithelial), 3T3-L1 (mouse preadipocytes), ADSC (Adipose-derived stem cells) and HMSC (human mesenchymal stem cells). Examples of suitable lipids that may be used to prepare nCVTs of the present invention include but are not limited to 1 ,2-dioleoyl-3-trimethylammonium-propane (DOTAP), 1 ,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), L-a-phosphatidylcholine (HSPC), (N-(1-(2, 3-dioleoyloxy)propyl)-N,N,N-trimethylamine) (DOTMA) and (dimethyl- dioctadecyl-ammonium-bromide (DDAB), 1 ,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), distearophosphoethanolamine polyethyleneglycol 2000 (DSPE-PEG2000), 1 ,2- distearoyl-sn-glycero-3-phosphoethanolamine-N-[amino(polyethylene glycol)-2000] (DSPE- PEG2000 Amine) and 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N- [biotinyl(polyethyleneglycol)-2000] (DSPE-PEG2000 Biotin), dioleoylphosphatidylcholine (DOPC), cholesterol or combinations thereof.

[0075] C. Method for preparing the Pt(IV) complex

[0076] In another aspect of the invention, there is provided a method for preparing the Pt(IV) complex of the present disclosure, comprising the following steps:

[0077] (a) reacting compound JM149 with a mitochondria-targeting ligand to obtain a Pt(IV) monoester;

[0078] (b) reacting Pt(IV) monoester obtained in step (a) with a chemosensitizer to obtain the Pt(IV) complex.

[0079] Compound JM149 is known in the art, and can be obtained by hydroxylation of JM118 using excess H2O2 (Figure 1). Both JM149 and JM118 are intermediate compounds during the synthesis of satraplatin (see Pu, Shao Ping, et al. Advanced Materials Research, vol. 233- 235, Trans Tech Publications, Ltd., May 2011 , pp. 1192-1197).

[0080] JM118 is commercially available, for example, from Bristol-Myers Squibb. JM118 can also be synthesized by known methods such as the method as shown in Figure 2 (see Johnstone TC, Lippard SJ. Inorganica ChimActa. 2015; 424:254- 259). Briefly, K2PtCl4 is reacted with excess KI first and then with cyclohexyl amine to produce a tetra-iodo platinum complex Pt(Am)2l2, wherein Am represents cyclohexyl amine. Pt(Am)2l2is then reacted with HBF4, and subjected to cleavage with ammonia to give an iodo-bridged platinum complex Pt(NH3)(Am)l2, which is further reacted with AgNOs and HCI to obtain JM118.

[0081] In one embodiment, step (a) is performed by mixing JM149 with a mitochondria-targeting ligand in a suitable solvent to obtain a Pt(IV) monoester. One advantage of this method is that the obtained Pt(IV) monoester can be used directly in the next step (b) without any purification. Examples of the mitochondria-targeting ligand are as defined above. Methods for conjugating the mitochondria-targeting ligand to the Pt(ll) core are known in the art, and can be selected based on the specific types of reactants used in the synthesis. In one embodiment, the mitochondria-targeting ligand is a delocalized cationic molecule selected from the group comprising rhodamine (for example, rhodamine 19, rhodamine 123), TPP, guanidinium, F16 and DQA, preferably is TPP. In one embodiment, step (a) comprises mixing JM149 with TPP- NHS ester in DMSO, wherein DMSO is subsequently removed. Removal of DMSO may be carried out by various means, for example, by excess ether treatment. Preferably, the slurry obtained after DMSO removal is re-dissolved using methanol and re-precipitated using excess ether. Pt(IV) monoester is then obtained by drying the precipitate, which monoester is directly used for the next step (b) without any further purification.

[0082] In one embodiment, step (b) is performed by adding a chemosensitizer ligand in a suitable solvent into the Pt (IV) monoester obtained in step (a). Examples of chemosensitizer ligands are as defined above. Methods for conjugating the chemosensitizer ligand to the Pt(ll) core are known in the art, and can be selected based on the specific types of reactants used in the synthesis. In one embodiment, the chemosensitizer ligand is a sodium channel blocker selected from the group comprising dichloro acetate (DCA), disopyramide, flecainide, mexiletine, propafenone and quinidine, or a histone deacetylase inhibitor selected from the group comprising phenyl butyrate (PhB). More preferably, the chemosensitizer ligand is DCA or PhB. Most preferably, the chemosensitizer ligand is PhB. In one embodiment, step (b) comprises adding PhB NHS ester in DMF in the Pt (IV) monoester obtained in step (a), wherein DMF is subsequently removed for example, by evaporation, to yield a sticky crude reaction mass. Preferably, said reaction mass is precipitated using methanol and excess ether, and then purified to yield the Pt(IV) complex of the present disclosure as an off-white solid.

[0083] D. Composition comprising the Pt(IV) complex

[0084] In another aspect of the invention, there is provided a composition comprising the Pt(IV) complex of the invention, or the nanoparticle comprising the Pt(IV) complex of the invention as the active ingredient and at least one pharmaceutically acceptable excipient.

[0085] It is surprisingly found that the Pt(IV) complex of the present disclosure is able to withstand strong acidic conditions and exhibit comparable activity with the one in physiological conditions. Thus, Pt(IV) complex is suitable to be developed as a promising orally administered anticancer drug. Accordingly, in one embodiment, the composition is in the form of an oral formulation. Preferably, the composition is in a solid dosage form such as tablets, capsules, granules, pills, cachets, lozenges, powders, or in a liquid dosage form such as solutions or suspensions.

[0086] The term “excipient” as used herein broadly refers to any component other than the active therapeutic ingredient. The excipient may be an inert substance, an inactive substance, and / or a not medicinally active substance. The excipient may serve various purposes, e.g. as a carrier, vehicle, filler, binder, lubricant, glidant, disintegrant, flow control agents, crystallization retarders, solubilizers, stabilizer, colouring agent, flavouring agent, surfactant, enzyme inhibitors, basifiers, acidifiers, emulsifier and / or to improve administration, and / or absorption of the active substance, tablet coating agents to control the dissolution rates of the solid dosage form according to the pH in the Gl tract. A person skilled in the art may select one or more of the aforementioned excipients with respect to the particular desired properties of the oral dosage form by routine experimentation and without any undue burden. The amount of each excipient used may vary within ranges conventional in the art. Techniques and excipients which may be used to formulate oral dosage forms are described in Handbook of Pharmaceutical Excipients, 6th edition, Rowe et al., Eds., American Pharmaceuticals Association and the Pharmaceutical Press, publications department of the Royal Pharmaceutical Society of Great Britain (2009) ; and Remington: the Science and Practice of Pharmacy, 21th edition, Gennaro, Ed., Lippincott Williams & Wilkins (2005) .

[0087] In one embodiment, the pharmaceutical composition comprises the Pt(IV) complex or the nanoparticle of the present disclosure, and one or more pharmaceutically acceptable excipients selected from the group comprising of binders / fillers, wetting agents, suspending agents / thickening agents, preservatives, buffering agents, sweetening agents, flavouring agents, colouring agents, pH control agents, surfactants / emulsifiers or combinations thereof.

[0088] In some embodiments, the excipients may include one or more of the following:

[0089] (a) a binder or a filler, which is selected from acacia, agar, alginic acid, carmellose sodium, dextrin, veegum or gel white, gellan gum, sodium alginate, hydroxypropyl starch, maltodextrin, modified starch, pectin, potassium alginate, polyvinyl pyrrolidone, carboxymethyl cellulose or an alkali metal salt thereof, microcrystalline cellulose, bentonite, col loidal silicon dioxide, microcrystalline cellulose / sodium carboxy methylcellulose;

[0090] (b) a wetting agent, which is selected from alcohol, glycerin, propylene glycol, polyethylene glycol, mineral oil, benzalkonium chloride, benzethonium chloride, cetylpyridinium chloride, docusate sodium, nonoxynol 9, octoxynol, poloxamer, poloxamer 124, poloxamer 188, 237, 338, 407, polyoxyl 35 castor oil, polyoxyl 40;

[0091] (c) a suspending agent (sometimes also known as thickening agents), which is selected from gelatin, crosslinked polyacrylic acid, polymethacrylic acid, polyhydroxyethyl methacrylic acid, hydroxypropyl methyl cellulose, polyethylene glycol, sodium carboxymethyl cellulose, hyaluronic acid, chitosan, polycarbophil, pectin, copolymers of dextran, polyacrylamide, acacia, copolymer of caprolactone and ethylene oxide, carbopol 934, tragacanth, eudragit, polyvinyl pyrrolidone, polyacrylate and polyacrylate copolymer resins, celluloses and cellulose derivatives for example methyl-, ethyl- and propyl celluloses; hydroxyalkyl-celluloses, hydroxyl propyl celluloses, hydroxylpropylalkyl celluloses and the like including xanthan gum, polyvinyl resins, polyethylene glycol, polyethylene oxide, sorbitol, sucrose, xylitol, dextrose, fructose, maltitol, sugar, sodium alginate;

[0092] (d) a buffering agent, which is selected from acetate, amino acids, ammonium sulfate, benzoate, bicarbonate, borate, citrate, citric acid monohydrate, disodium hydrogen phosphate, glutamate, lactate, meglumine, potassium citrate, sodium acetate, sodium citrate, sodium phosphate, sulfate, tartrate, triethanolamine, TRIS, trisodium citrate dehydrate;

[0093] (e) a preservative, which is selected from benzyl alcohol, chloro-butanol, chloro-cresol, alkyl esters of paraben, phenol, phenyl ethanol, benzoic acid, potassium sorbate, sodium benzoate and antimicrobial solvents like propylene glycol, chloroform;

[0094] (f) a sweetening agent, which is selected from sucralose, sucrose, liquid glucose, glycerol, sorbitol, maltitol, saccharin sodium and aspartame;

[0095] (g) a flavouring agent, which is selected from essential oils including peppermint oil, orange oil, and lemon oil or can be selected from fruit flavour, e.g. peppermint flavour, strawberry flavour, tutti fruit flavour;

[0096] (h) colouring agents, including dyes and pigments such as Iron Oxide Red or Yellow, titanium dioxide, talc;

[0097] (i) pH control agents such as citric acid, tartaric acid, fumaric acid, sodium citrate, dibasic calcium phosphate, dibasic sodium phosphate; and

[0098] (j) surfactants or emulsifiers such as Pluronic, polyethylene glycols, sodium carboxymethyl cellulose, polyethoxylated and hydrogenated castor oil.

[0099] E. Therapeutic use

[0100] In one aspect of the invention, there is provided a method of treating cancer in a subject, comprising administering to the subject an effective amount of the Pt(IV) complex, the nanoparticle or the composition of the present disclosure.

[0101] In another aspect of the invention, there is provided a use of the Pt(IV) complex, the nanoparticle or the composition of the present disclosure in the manufacture of a medicament for the treatment of cancer. As the Pt(IV) complex of the present disclosure does not modify the action mechanism of the corresponding active Pt(ll) drug in reduced form, theoretically it can be used for treating any cancer that is suitable for Pt(ll) treatment.

[0102] Any suitable type of cancer can be treated by administering an effective amount of a Pt(IV) complex or a nanoparticle including a Pt(IV) complex to a subject in need thereof. Cancers that can be treated by administering an effective amount of Pt(IV) complex or a nanoparticle including a Pt(IV) complex to a subject in need thereof include, but are not limited to, human sarcomas and carcinomas, e.g., fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, chordoma, angiosarcoma, endotheliosarcoma, lymphangiosarcoma, lymphangioendotheliosarcoma, synovioma, mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, colon carcinoma, colorectal cancer, anal carcinoma, esophageal cancer, gastric cancer, hepatocellular cancer, bladder cancer, endometrial cancer, pancreatic cancer, breast cancer, ovarian cancer, prostate cancer, liver cancer, stomach cancer, atrial myxomas, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma, thyroid and parathyroid neoplasms, papillary carcinoma, papillary adenocarcinomas, cystadenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma, hepatoma, bile duct carcinoma, choriocarcinoma, seminoma, embryonal carcinoma, Wilms' tumor, cervical cancer, testicular cancer, lung cancer (such as small cell lung carcinoma, non-small-cell lung cancer), bladder carcinoma, epithelial carcinoma, glioma, pituitary neoplasms, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, schwannomas, oligodendroglioma, meningioma, spinal cord tumors, melanoma, neuroblastoma, pheochromocytoma, Types 1-3 endocrine neoplasia, retinoblastoma; leukemias, e.g., acute lymphocytic leukemia and acute myelocytic leukemia (myeloblastic, promyelocytic, myelomonocytic, monocytic and erythroleukemia); chronic leukemia (chronic myelocytic (granulocytic) leukemia and chronic lymphocytic leukemia); and polycythemia vera, lymphoma (Hodgkin's disease and non-Hodgkin's disease), multiple myeloma, and Waldenstrobm's macroglobulinemia. It is believed that a Pt(IV) complex or a nanoparticle including a Pt(IV) complex may be particularly effective in treating a subject having ovarian cancer, lung cancer, prostate cancer, testicular cancer, liver cancer, or colorectal cancer. In one embodiment, an effective amount of a Pt(IV) complex or a nanoparticle including a Pt(IV) complex is administered to a subject to treat cisplatin-resistant ovarian cancer.

[0103] In addition to being used as a monotherapy, the Pt(IV) complex or nanoparticle of the present disclosure may also find use in combination therapies. Effective combination therapy may be achieved with a single composition or pharmacological formulation that includes both agents, or with two distinct compositions or formulations, administered at the same time, wherein one composition includes the Pt(IV) complex or nanoparticle of the present disclosure, and the other includes the second agent(s). The other therapeutic agent may be administered before, concurrently with, or following administration of the Pt(IV) complex or nanoparticle of the present disclosure. The therapy using the Pt(IV) complex or nanoparticle of the present disclosure may precede or follow administration of the other agent(s) by intervals ranging from minutes to weeks. In embodiments where the other agent and the Pt(IV) complex or nanoparticle of the present disclosure are administered separately, one would generally ensure that a significant period of time did not expire between the time of each delivery, such that each agent would still be able to exert an advantageously combined effect.

[0104] In some embodiments, said method further comprises administering a second anticancer therapy. In some embodiments, the Pt(IV) complex, the nanoparticle or the composition of the present disclosure is used in combination with a second anticancer therapy. In one embodiment, the second anticancer therapy is a radiotherapy, chemotherapy, hormone therapy or immunotherapy. Said second anticancer therapy may be a drug that is complexed with the Pt(IV) complex.

[0105] In one embodiment, the second anticancer therapy is a radiotherapy. Radiotherapy, also called radiation therapy, is the treatment of cancer and other diseases with ionizing radiation. Ionizing radiation deposits energy that injures or destroys cells in the area being treated by damaging their genetic material, making it impossible for these cells to continue to grow. Radiation therapy used according to the present invention may include, but is not limited to, the use of y-rays, X-rays, and / or the directed delivery of radioisotopes to tumor cells. Other forms of DNA damaging factors are also contemplated such as microwaves and UV-irradiation.

[0106] In some embodiments, the second anticancer therapy is a chemotherapy. It is contemplated that the Pt(IV) complex or nanoparticle described herein may be used in combination with a chemotherapeutic agent, which may be selected from the group comprising Alkylating agents such as Altretamine, Bendamustine, Busulfan, Carboplatin, Carmustine, Chlorambucil, Cisplatin, Cyclophosphamide, Dacarbazine, Ifosfamide, Lomustine. Mechlorethamine, Melphalan, Oxaliplatin, Temozolomide, Thiotepa and Trabectedin; Antimetabolites such as Azacitidine, 5-fluorouracil (5-FU), 6-mercaptopurine (6-MP), Capecitabine (Xeloda), Cladribine, Clofarabine, Cytarabine (Ara-C), Decitabine, Floxuridine, Fludarabine, Gemcitabine (Gemzar), Hydroxyurea, Methotrexate, Nelarabine, Pemetrexed (Alimta), Pentostatin, Pralatrexate and Thioguanine; Anti-tumor antibiotics such as Bleomycin, Dactinomycin, Mitomycin-C, Mitoxantrone, Daunorubicin, Doxorubicin (Adriamycin), Doxorubicin liposomal, Epirubicin, Idarubicin and Valrubicin; Topoisomerase inhibitors such as Etoposide (VP-16), Mitoxantrone, Teniposide, Irinotecan, Irinotecan liposomal and Topotecan;Mitotic inhibitors such as taxanes (paclitaxel, docetaxel, cabazitaxel, Nab- paclitaxel) and vinca alkaloids (vinblastine, vincristine, vindesine, and vinorelbine);Corticosteroids such as prednisone, Methylprednisolone, Dexamethasone; and other chemotherapy drugs such as all-trans-retinoic acid, Arsenic trioxide, Asparaginase, Eribulin, Hydroxyurea, Ixabepione, Mitotane, Omacetaxine, Pegaspargase, Procarbazine, Romidepsin, Vorinostat.

[0107] In some embodiments, the second anticancer therapy is a hormone therapy. Hormone therapy is considered a systemic treatment as it targets hormones that circulate in the body. Examples of hormone therapy include but not limited to luteinizing-hormone-releasing hormone (LHRH) agonists such as goserelin, triptorlin, degarelix and leuprolide; selective estrogen receptor modulators (SERMs) such as tamoxifen and raloxifene; aromatase inhibitors such as letrozole, anastrozole and exemestane; estrogen receptor antagonists such as fulvestrant and toremifene; anti-androgens such as apalutamide, enzalutamide, darolutamide, bicalutamide, flutamide, and nilutamide; adrenolytics such as mitotane; progestins such as medroxyprogesterone acetate or megestrol acetate; CYP17 inhibitors such as abiraterone and ketoconazole.

[0108] In some embodiments, the second anticancer therapy is an immunotherapy. Well-known immunotherapy includes CAR-T therapy, cancer vaccines, cytokines, immunomodulators, and immune checkpoint inhibitors. Examples of cancer vaccines that may be used in combination with the present disclosure include but not limited to Sipuleucel-T (Provenge). Examples of cytokines that may be used in combination with the present disclosure include but not limited to interleukins such as IL2, IL7, IL12 and il_21 , and interferons such as IFN-alfa, IFN-beta and IFN-gamma. Examples of immunomodulators that may be used in combination with the present disclosure include but not limited to thalidomide, lenalidomide, pomalidomide, bacillus calmette-Guerin (BCG) and imiquimod. Examples of immune checkpoint inhibitors that may be used in combination with the present disclosure are antibodies targeting immune check points such as PD1 , PDL2, PDL1, CD47, Indoleamine 2,3-dioxygenase (IDO), 4-1-BB, GITR, TIM3, LAG-3, TIGIT, LIGHT and CTLA-4.

[0109] It should be understood that any and all embodiments of the present disclosure can be combined with technical features in any other embodiment or multiple other embodiments to obtain additional embodiments under the premise of no conflict. The invention includes such combinations resulting in further embodiments.

[0110] Examples

[0111] The examples and exemplary embodiments below are intended to be purely exemplary of the invention and should therefore not be considered to limit the invention in any way.

[0112] Example 1 : Synthesis of the Pt(IV) complex

[0113] The Pt(IV) complex of the present invention was synthesized according to Figure 3.

[0114] Specifically, JM149 was synthesized using a method known in the art (see Johnstone TC, Lippard SJ. Inorganica Chim Acta. 2015; 424:254- 259).. In brief, K2PtCU was reacted with excess KI first and then with cyclohexyl amine to produce a tetra-iodo platinum complex Pt(Am)2l2, wherein Am represents cyclohexyl amine. Pt(Am)2l2 is then reacted with HBF4, and subjected to cleavage with ammonia to give an iodo-bridged platinum complex Pt(NH3)(Am)l2, which is further reacted with AgNO3and HCI to produced JM118. Hydroxylation of JM118 using excess H2O2 produced JM149.

[0115] JM149 (1 mol equiv) was added to the TPP-NHS ester (1.1 mol equiv) in DMSO. The solution was stirred at 36 °C for 72 h until a yellowish clear solution was formed. DMSO solvent was removed using excess ether treatment by repeated vigorous vortex and centrifugation. Once the DMSO is removed (to the lowest possible amount), the obtained slurry was re-dissolved using methanol and re-precipitated using excess ether. The solid was obtained after centrifugation and dried using compressed air. This mono ester of Pt(IV) was directly used for the next step without any further purification.195Pt NMR (DMSO): 966 ppm.

[0116] In the next step, the obtained Pt (IV) monoester was treated with excess PhB NHS ester (4-5 equivalents) in DMF and stir the reaction at 36 °C for 48 hours. Then DMF was evaporated under reduced pressure to yield a sticky crude reaction mass. This reaction mass was precipitated using methanol and excess ether. The precipitate was collected and dried under air. The product was finally purified using preparative HPLC (using RP-HPLC column and water with 0.1 % formic acid and methanol) to yield pure Pt(IV) complex as an off-white solid.

[0117] 195Pt NMR (MeOD): -1170 ppm.1H NMR (MeOD) 6 7.98-7.64 (m, 15H), 7.34-.08 (m, 5H), 3.50-3.40 (m, 2H), 2.92-2.89 (m, 1 H), 2.72-2.60 (m, 2H), 2.44-2.31 (m, 4H), 2.17-2.05 (m, 2H), 1.98-1.58 (m, 9H), 1.42-1.13 (m, 5H). ESI-MS for CsgHsoC ^C PPt; calculated 906.2, found [M+H]+907.3. LC-QTOF-HRMS measured is 906.2528, found 906.2538.

[0118] Example 2: Effects and toxicity of Pt(IV) complex in a murine model

[0119] Pt(IV) complex was evaluated for its anti-neoplastic effect against cisplatin on A2780cis-luc OC xenograft model on CrTac:NCr-Foxn1nu nude mice. Specifically, 5-8 week old immunocompromised athymic nude mice (CrTac:NCr-Foxn1nu) were intraperitoneally injected with cisplatin resistant ovarian cancer cells A2780cis-luc on Day 0. The A2780cis-luc cell line expressing luciferase protein was established by transducing a lentiviral plasmid (pHAGE PGK-GFP-IRES-LUC-W, addgene #46793) expressing luciferase to a commercially available A2780 cisplatin-resistant cell line (A2780cis, ECACC catalogue no. 93112517). After sorting and selection, the clones carrying high level of luciferase were expanded and used for xenograft mouse studies. 10 days later, mice were treated 3 times per week with saline, 3 mg / kg cisplatin or 8.99 mg / kg Pt(IV) complex (both corresponding to 1 .94 mg / kg of Pt) by intraperitoneal administration (the date of first treatment was set as Day 11).

[0120] Effects of Pt (IV) complex

[0121] Tumor growth of the mice was monitored over time using an I VIS imaging system. It was observed that Pt(IV) complex controlled the tumor growth very efficiently, while in the saline control group and cisplatin-treated group, the tumor grew and spread quickly in the mice (Figure 4A).

[0122] On Day 24, 3 mice of each group were sacrificed to determine the tumor dry weight. As shown in Figure 4B, while mice treated with cisplatin did not show a great decrease in tumor dry weight compared to the saline control group, the Pt(IV) complex treatment group exhibited significant effects in decreasing the tumor dry weight.

[0123] Tumor-associated luminescence was determined by an IVIS imaging system. As shown in Figure 4D, although cisplatin treatment had certain effects in controlling the tumor growth, Pt(IV) complex treatment showed a superior efficacy. The effect of Pt(IV) complex was more obvious in terms of the tumor size in representative mice sacrificed at Day 24 (Figure 4C).

[0124] In general, compared to cisplatin treatment, Pt(IV) complex treatment was able to overcome the cisplatin resistance and showed more remarkable effects in controlling the tumor growth and in decreasing the tumor size. Toxicity of Pt(IV) complex

[0125] The body weight of mice was monitored over time to determine the toxicity of Pt(IV) complex. The dry weight of different organs spleen, liver, heart, lungs and kidney of sacrificed mice was also measured up to Day 23. Healthy mice without tumor cells were used as a control (see first column in Figure 5B-D).

[0126] Mice treated with cisplatin lost >20% of their body weight due to the drug’s high toxicity. The remaining mice showed remarkably lower weight loss, suggesting much less toxicity from the Pt(IV) complex (Figure 5A). This was confirmed by the normal size of the spleen in the Pt(IV) complex-treated group, in comparison to cisplatin (Figure 5B) (p< 0.001). No obvious toxicity in other organs (liver, heart, lungs and even kidneys) was observed in this study (Figure 5C), as sub-nephrotoxic concentrations of platinum drugs were intentionally used. The H & E staining was performed using the haematoxylin and eosin dyes obtained from Merck Pte. Ltd., Singapore. The tissues were fixed immediately after harvesting in the 10% neutral buffered formalin and embedded in paraffin later. The paraffin-embedded tissues were sectioned using a microtome of about 5 pm thickness and stained with haematoxylin to visualize nuclei and with eosin to visualize cytoplasm. The stained sections were imaged using an inverted light microscope. The H & E staining on kidney tissues showed that degenerative changes were quite high in cisplatin-treated mice (Figure 5F) when compared to controls (Figure 5E), while minimal changes were observed in Pt(IV) complex-treated mice (Figure 5G).

[0127] Example 3: Stability of Pt(IV) complex

[0128] Pt(IV) complex sample analysis was performed using Nexera UHPLC (Shimadzu, Kyoto, Japan). The sample injection volume was 50 pL, and the analytes were separated with Luna C18 5 pm 250 mm x 4.6 mm column. Detection of analytes in samples was made through diode array detection (DAD) at 254 nm based upon comparison with the retention times.

[0129] Solvent A is 0.1% formic acid in Milli-Q water while solvent B is methanol (MeOH). The gradient program for the Pt(IV) complex was as follows: linear gradient from 40% to 100% B (0.01-13 min), isocratic at 100% B (13-14 min), linear gradient from 100% to 40% B (14. QI- 16.0 min) and isocratic at 40% B (16.0-18.0 min). To prevent compound accumulation on the needle, 50% water in ACN was used as needle wash for 20s per sample. Limit of detection and quantification (LoD and LoQ): The aliquots of the standard solution (diluted with Milli-Q water) were injected at various concentrations. The detection and quantification limit for all the detected analytes were calculated on the basis of signal-to-noise ratios (S / N) of 3 and 10, respectively, for corresponding standards.

[0130] LoD = 0.776 pg / ml and LoQ = 2.352 pg / ml

[0131] Stability of Pt(IV) complex was assessed in different conditions. 3 different buffer solutions were prepared:

[0132] - pH 1 .2 prepared with HCI

[0133] -- pH 7.4 prepared with PBS

[0134] - pH 3 prepared with ascorbic acid.

[0135] 100 pg / ml of Pt(IV) complex stock solution in MilliQ water was prepared and diluted with 3 different solutions (1 :4). 25 pg / ml of Pt(IV) complex theoretical concentrations were obtained for each pH solution. Their stability analysis was conducted over time (hours) till 20h.

[0136] Time point zero AUCs were considered as 100% of the calculated concentration of Pt(IV) complex based upon comparison for each pH solutions.

[0137] Pt(IV) complex was shown to withstand the strong acidic conditions of the stomach, exhibiting a stability profile comparable to the one in physiological conditions (PBS) (Figure 6). On the other hand, when treated with 3 mM ascorbic acid, which simulates the intracellular reducing environment, our Pt(IV) complex was reduced to 16.12%, releasing its Pt(ll) species. These data suggest that Pt(IV) complex is suitable to be developed as a promising orally administered anticancer drug.

[0138] Example 4. Effects of Pt(IV) complex in liver cancer cells

[0139] A total of 1 x 104HepG2 liver cancer cells (100 pL) were seeded in a 96-well plate. The plates were incubated overnight in a humidified incubator at 37 °C and 5% CO2. After aspirating the Dulbecco's Modified Eagle's Medium (DM EM) from the wells, drug solution in media (100 pL) was added to each well at varying concentrations (0, 3.125, 6.25, 12.5, 25, 50, 100 and 200 pM). The drug loaded plates were incubated for 24 and 48 h in a humidified incubator at 37 °C and 5% CO2. After 24 and 48 h, the medium was aspirated and 0.5 mg / mL 3-(4,5- dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT, 100 pL) solution was added to each well and incubated for 1 h. The MTT solution was removed and replaced with dimethyl sulfoxide (DMSO, 100 pL) to fully dissolve the formazan crystals. Absorbance values at 570 nm wavelength were measured using a Hidex Sense microplate reader (Hidex, Turku, Finland). The MTT assay was conducted in triplicate on cisplatin, 4Cis (Babak, M. V. et al., Angew. Chem. Int. Ed. 2019, 58 (24), 8109-8114 ), Satraplatin, JM118 and Pt(IV) complex of the invention).

[0140] Experiments were performed in triplicate for at least 3 independent batches. Cytotoxicity was evaluated with reference to the ICso value, which was defined as the concentration of the agent required to inhibit cell viability by 50%, calculated from the dose response curves using GraphPad Prism (Version 8.0, GraphPad Software).

[0141] As shown in Figure 7, at 24 h the Pt(IV) complex of the invention demonstrated a statistically significant stronger anticancer effect as compared to JM118 (P<0.01) than 4 Cis and Satraplatin platinum derivatives. It also showed a comparable effect to cisplatin. At 48 h, the Pt(IV) complex of the invention was the only compound that had an ICso significantly lower than JM118 (P<0.05).

[0142] Taken together, the results demonstrate that Pt(IV) complex of the invention is effective in inhibiting the growth of liver cancer cells, especially compared with known platinum drugs, suggesting its potential to be used as an anticancer drug.

[0143] Example 5. Apoptosis test of Pt(IV) complex on HepG2 cells

[0144] A total of 3 x 105HepG2 liver cancer cells (2 ml_) were seeded in a 6-well plate. The plates were incubated overnight in a humidified incubator at 37 °C and 5% CO2. After aspirating the Dulbecco's Modified Eagle's Medium (DMEM) from the wells, drug (i.e., Pt (IV) complex of the invention) solution in media (2 mL) was added to each well at a concentration of 10 pM. The drug loaded plates were incubated for 24 and 48 h in a humidified incubator at 37 °C and 5% CO2. After 24 and 48 h, the medium was collected and washed with PBS. After washing, 500 pL of trypsin was added to each well and incubated for 5 min. Then 1.5 mL of media was added to each well and the whole solution was collected and added to initially collected medium comprising detached cells and cells were pelleted out. The cell pellet was resuspended in 500 pL of binding buffer (0.02 pm sterile filtered 0.1M Hepes (pH 7.4), 0.14 M NaCI, and 2.5 mM CaCI2 solution), and 100 pL of cell suspension was transferred to a new tube. Then 5 pl of FITC Annexin and 5pl of propidium iodide was added and incubated in the dark. After 15 min of incubation, 400 pL of binding buffer was added, and the mixture was analysed immediately using a BD LSR Fortessa flow cytometer. Data were processed using Flowjo software (Version 10.6, Ashland, Oregon, USA). Further investigation on the mechanism that induced cell death clearly showed that Pt(IV) complex induced both early and late apoptosis (determined based on live, propidium iodide and annexin control) to a higher extent than other platinum species, indicating that this Pt(IV) prodrug is quickly activated once inside cancer cells and induces programmed cell death. Around 60% of apoptosis was observed at both 24 and 48 hours (Figure 8).

[0145] The apoptosis of HepG2 cells was also detected by holotomography. It is a laser technique measuring the 3D refractive index of cells and provides label-free quantitative imaging. Specifically, a total of 1 x 105HepG2 liver cancer cells (2 mL) were seeded in a 12-well plate. The plates were incubated overnight in a humidified incubator at 37 °C and 5% CO2. After aspirating the medium from the wells, drug (i.e., Pt (IV) complex of the invention) solution in media (2 mL) was added to each well at a concentration of 10 |JM. The drug loaded plates were incubated for 24 h and 48 h in Tomocube HT-X1 incubator (Tomocube, Inc, Republic of Korea) at 37 °C and 5% CO2. The plates were imaged every 4 h for up to 48 h to visualise the changes in cell morphology.

[0146] It can be seen from Figure 9 that the Pt(IV) complex caused a significant change in cell morphology upon treatment at both 24 (top panel) and 48 hours (bottom panel), as the effect can be more clearly seen in the zoomed image (Figure 9B) compared to the control untreated group. These data also confirmed the extent of apoptosis obtained through flow cytometry analyses.

[0147] Example 6. Preparation of a hybrid system comprising Pt(IV) complex

[0148] Biohybrid drug delivery systems, termed nano-Cell Vesicle Technologies (nCVTs), were prepared by fusing cellular membranes (ghosts) of cells and synthetic lipids. The cell ghosts were prepared by the method described below. Briefly, 1 x 107cells / mL U937 monocytes were centrifuged at 500G for 10 minutes. The pellet was then washed with PBS and re-centrifuged at 500G for 10 minutes. The resultant pellet was resuspended in 0.25X PBS (with a protease inhibitor cocktail (Abeam Limited, Cat No. AB201111)) and left under slight rotation on a rotating plate overnight. The cells were then collected and centrifuged at 3000G for 10 minutes. The cells were then resuspended in 1X PBS (with protease inhibitor) and left under slight rotation on a rotating plate overnight. The resultant cells were collected and centrifuged at 3000G for 10 minutes. The pellet (i.e., the cell ghosts) were resuspended in PBS (with protease inhibitor) and stored at 4 °C.

[0149] Hydration method

[0150] A lipid thin film was formed by mixing dioleoylphosphatidylcholine (DOPC) and cholesterol at a molar ratio of 70:30. The lipids (1 .63 mg of DOPC and 0.37 mg of cholesterol) were dissolved in 2 ml_ of chloroform and added to a round bottom flask. The solvent was removed by rotary evaporation to form the lipid film.

[0151] Cell ghosts, obtained from U937 monocytes, were centrifuged at 3000G for 10 minutes. The pellet was resuspended in 0.5 mg / mL of Pt(IV) complex in PBS. The cell ghosts were extruded through a 5-micron membrane filter. The resultant suspension was used to hydrate the thin film. The thin film was sonicated for 30 minutes under ice-cold water conditions. The suspension was then extruded through a 0.4 pm membrane filter once and 0.2-micron membrane filter twice.

[0152] Thin film method

[0153] A lipid thin film was formed by mixing DOPC and Cholesterol at a molar ratio of 70:30. The lipids were dissolved in chloroform and added to a round bottom flask. 1 mg of Pt(IV) complex dissolved in 0.5 ml of chloroform was also added to the round bottom flask. The solvent was removed by rotary evaporation to form the lipid film.

[0154] Cell ghosts, obtained from U937 monocytes, were centrifuged at 3000G for 10 minutes. The pellet was resuspended in PBS. The cell ghosts were extruded through a 5-micron membrane filter. The resultant suspension was used to hydrate the thin film. The thin film was sonicated for 30 minutes under cold conditions. The suspension was then extruded through a 0.4 pm membrane filter once and 0.2-micron membrane filter twice.

[0155] The Pt(IV) complexes were loaded by the above 2 methods: loading during hydration and loading onto thin film. The formulations were washed using ultrafiltration, and then characterized by Nanoparticle Tracking Analysis (NanoSight Pro, Malvern Panalytical) in terms of size (Figure 10), and Ultra-Performance Liquid Chromatography (Shimadzu) to determine the encapsulation efficiencies.

[0156] The hydration method had an encapsulation efficiency of 14.1%, while the thin film method had an encapsulation efficiency of 31.2%. The thin film method displayed a higher encapsulation efficiency compared to the hydration method.

[0157] Example 7. Bio-distribution of the hybrid system containing Pt(IV) complex

[0158] The female CrTac:NCr-Foxn1nu mice were purchased from InVivos (Singapore) which were 8 week-old and weighed around 20 g. Animals were housed in Comparative Medicine (CM)- holding units at the National University of Singapore in a pathogen-free environment at constant temperature in a 12 / 12-hour light / dark cycle environment. The mice were acclimatized for 3 days and were given access to food and water ad libitum and were controlled for pain and / or distress development. Free nCVTs and Pt(IV) complex loaded nCVTs prepared by the thin film method were injected into animals via the intraperitoneal route at a dose of 2 E+11 particles (100 pL). After 24 h, the mice were sacrificed, and the organs were harvested for imaging under the in vivo imaging system (I VIS). The harvested organs were imaged using Perkin Elmer I VIS spectrum imaging system and images were analysed using Living Image software (Version 4.5).

[0159] The labelled nCVTs and labelled Pt(IV) com pl ex- loaded nCVTs were first imaged under the I VIS spectrum before they were injected into the animals to confirm the signals in vitro (Figure

[0160] 11).

[0161] Both control nCVTs and Pt(IV) complex-loaded nCVTs were able to reach the liver (Figure

[0162] 12). This is quite expected as nanoparticles accumulate in the liver without any specific targeting. The cell ghosts, being derived from U937 monocytes, would be useful to target the Pt(IV) complex-loaded nCVTs to inflammatory cells like tumor / cancer cells.

[0163] For one skilled in the art, various modifications and changes may be made to the present disclosure. Those skilled in the art should understand that any amendments, equivalent replacements, improvements, and so on, made within the spirit and principle of the present disclosure, should be covered within the scope of protection of the present disclosure.

Claims

Claims1. A Pt(IV) complex having the following formula (I):wherein Ri and R2are selected from the group comprising a mitochondria-targeting ligand and a chemosensitizer ligand, wherein Ri and R2are not both mitochondria- targeting ligands or chemosensitizer ligands.

2. The Pt(IV) complex of claim 1 , wherein the mitochondria-targeting ligand is selected from the group comprising delocalized cationic molecules such as triphenylphosphonium (TPP), rhodamine, preferably rhodamine 19 or rhodamine 123, guanidinium, (E)-4-(1 H-indol-3-ylvinyl)-N-methylpyridinium iodide (F16) and dequalinium (DQA); mitochondria targeting / penetrating peptides (MTPs / MPPs), preferably (L-cyclohexyl alanine-D-arginine)3; carbonyl cyanide m-chlorophenyl hydrazine (CCCP), triethylammonium, phenylsulfonylfuroxan, pyriudinium and 2,3- dimethylbenzothiazolium iodide; and wherein the chemosensitizer ligand is selected from the group comprising sodium channel blockers, preferably dichloro acetate (DCA), disopyramide, flecainide, mexiletine, propafenone and quinidine; histone deacetylase inhibitors, preferably phenyl butyrate (PhB), vorinostat, romidepsin; calcium channel blockers, preferably amlodipine, diltiazem, felodipine, isradipine, nicardipine, nifedipine, nimodipine, nisoldipine and verapamil; steroids and steroid-like compounds, preferably tamoxifen; cyclic peptides, preferably cyclosporin A; phytochemicals, preferably flavonoids, alkaloids and polyphenols; P-gp inhibitors, preferably curcumin, metformin, resveratrol and elacridar and bell nostat.

3. The Pt(l V) complex of claim 1 or 2, wherein the mitochondria-targeting ligand is selected from the group comprising TPP, rhodamine 19, rhodamine 123, guanidinium, F16 and DQA; andwherein the chemosensitizer ligand is selected from the group comprising dichloro acetate (DCA), phenyl butyrate (PhB), disopyramide, flecainide, mexiletine, propafenone and quinidine.

4. The Pt(IV) complex of any one of claims 1-3, wherein the mitochondria-targeting ligand is triphenylphosphonium (TPP), and the chemosensitizer ligand is selected from dichloroacetate (DCA) and phenyl butyrate (PhB).

5. The Pt(IV) complex of any one of claims 1-4, wherein a second anticancer drug is complexed to it.

6. A nanoparticle comprising the Pt(IV) complex of any one of claims 1-5.

7. The nanoparticle of claim 6, wherein the nanoparticle is selected from a group consisting of a carbon nanotube, gold nanoparticle, quantum dot, up-conversion nanoparticle, porous silicon nanoparticle, liposome, cell vesicle, metal-organic framework, and polymeric micelle.

8. The nanoparticle of claim 7, wherein the nanoparticle is a cell vesicle consisting of cell ghosts fused with lipids.

9. The nanoparticle of claim 8, wherein the lipids are selected from a group consisting of1 ,2-dioleoyl-3-trimethylammonium-propane (DOTAP), 1 ,2-dipalmitoyl-sn-glycero-3- phosphocholine (DPPC), L-a-phosphatidylcholine (HSPC), (N-(1-(2, 3- dioleoyloxy)propyl)-N,N,N-trimethylamine) (DOTMA) and (dimethyl-dioctadecyl- ammonium-bromide (DDAB), 1 ,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), distearophosphoethanolamine polyethyleneglycol 2000 (DSPE-PEG2000), 1,2- distearoyl-sn-glycero-3-phosphoethanolamine-N-[amino(polyethylene glycol)-2000] (DSPE-PEG2000 Amine) and 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N- [biotinyl(polyethyleneglycol)-2000] (DSPE-PEG2000 Biotin), dioleoylphosphatidylcholine (DOPC), cholesterol or combinations thereof.

10. A composition comprising the Pt(IV) complex of any one of claims 1-5, or the nanoparticle of any one of claims 6-9, and a pharmaceutically acceptable excipient.11 . The composition of claim 10, where said composition is in the form of an oral formulation.

12. The composition of claim 10 or 11 , wherein said composition is in a solid dosage form, preferably as a tablet, capsule, granule, powder, or in a liquid dosage form, preferably as a solution or suspension.

13. The composition of any one of claims 10 to 12 for use in treating a cancer.

14. The composition of claim 13, wherein the cancer is selected from the group comprising ovarian cancer, preferably cisplatin-resistant ovarian cancer, lung cancer, prostate cancer, testicular cancer, liver cancer and colorectal cancer.

15. A method of treating cancer in a subject, comprising administering to the subject an effective amount of the complex of any one of claims 1-5, the nanoparticle of any one of claims 6-9, or the composition of any one of claims 10-14.

16. The method of claim 15, wherein the cancer is selected from the group comprising ovarian cancer, preferably cisplatin-resistant ovarian cancer, lung cancer, prostate cancer, testicular cancer, liver cancer and colorectal cancer.

17. The method of claim 16, wherein the ovarian cancer is cisplatin-resistant ovarian cancer.

18. The method of any one of claims 15 to 17, further comprising administering a radiotherapy, a chemotherapy, a hormone therapy or an immunotherapy.

19. Use of the complex of any one of claims 1-5, or the nanoparticle of any one of claims 6- 9, or the composition of any one of claims 10-14 in the manufacture of a medicament for treating a cancer.

20. The use of claim 19, wherein the cancer is selected from the group comprising ovarian cancer, lung cancer, prostate cancer, testicular cancer, liver cancer and colorectal cancer.21 . The use of claim 20, wherein the ovarian cancer is cisplatin-resistant ovarian cancer.

22. A method of producing the complex of any one of claims 1-5, comprising the following steps:(a) reacting platinum compound JM149 with a mitochondria-targeting ligand to obtain aPt(IV) monoester, wherein platinum compound(b) reacting Pt(IV) monoester obtained in step (a) with a chemosensitizer to obtain the Pt(IV) complex.

23. A method of producing a cell vesicle comprising the Pt(IV) complex of any one of claims 1-5, comprising the following steps:(a) mixing lipids and the Pt(IV) complex of any one of claims 1-5 in a solvent,(b) forming a thin film by removing the solvent,(c) hydrating the thin film with a solution comprising cell ghosts, and(d) subjecting the hydrated thin film to shearing, extrusion, or freeze thawing and sonication to obtain the cell vesicle.

24. The method of claim 23, wherein a thin film comprising lipids is produced in steps (a) and(b) and then hydrated in a suspension of cell ghosts and Pt(IV) complex of the invention in step (c).

25. The method of claim 23 or 24, wherein a second anticancer drug is included in the cell vesicle.

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