WO2022250525A1 - Métallo-médicaments auto-assemblés activés par la lumière - Google Patents

Métallo-médicaments auto-assemblés activés par la lumière Download PDF

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WO2022250525A1
WO2022250525A1 PCT/NL2021/050327 NL2021050327W WO2022250525A1 WO 2022250525 A1 WO2022250525 A1 WO 2022250525A1 NL 2021050327 W NL2021050327 W NL 2021050327W WO 2022250525 A1 WO2022250525 A1 WO 2022250525A1
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group
cyclic
branched
carbon
aromatic
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PCT/NL2021/050327
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Sylvestre Bonnet
Xue-quan ZHOU
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Universiteit Leiden
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07F—ACYCLIC, CARBOCYCLIC OR HETEROCYCLIC COMPOUNDS CONTAINING ELEMENTS OTHER THAN CARBON, HYDROGEN, HALOGEN, OXYGEN, NITROGEN, SULFUR, SELENIUM OR TELLURIUM
    • C07F15/00—Compounds containing elements of Groups 8, 9, 10 or 18 of the Periodic System
    • C07F15/0006—Compounds containing elements of Groups 8, 9, 10 or 18 of the Periodic System compounds of the platinum group
    • C07F15/006—Palladium compounds
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B82—NANOTECHNOLOGY
    • B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
    • B82Y30/00—Nanotechnology for materials or surface science, e.g. nanocomposites
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B82—NANOTECHNOLOGY
    • B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
    • B82Y40/00—Manufacture or treatment of nanostructures

Definitions

  • This invention relates to palladium compounds and pharmaceutical compositions containing palladium compounds adapted for the treatment of tumour cells for use in the treatment of cancer. Methods of treating tumour cells using the palladium compounds and a light source are disclosed.
  • the first is to reduce systemic toxicity whilst retaining cytotoxicity against tumour cells, so as to reduce the side-effects of anti-tumour treatment on the patient.
  • the second is to selectively accumulate the cytotoxic agents and/or activity at the site of the tumour in preference to healthy tissue.
  • PDT Photodynamic therapy
  • Tumour irradiation in vivo is clinically employed during PDT.
  • PDT consists in the administration of a photosensitizer (generally a porphyrin or phthalocyanine compound), followed by the non-invasive irradiation of the tumour(s) with light (Brown, S. B.; Brown, E. A.; Walker,
  • the photosensitizer is non-toxic in the dark, but in presence of oxygen and light it produces a significant amount of highly toxic, short-lived radical species, culminating in cell death via irreversible damage to cellular components such as proteins, lipids, and DNA.
  • PDT exploits chemical agents called photosensitizers with generally low cytotoxicity in the absence of incident light, but with increased cytotoxicity when light is incident on the photosensitizer. In this way, systemic toxicity is generally reduced whilst retaining high cytotoxicity at the site of illumination.
  • PDT has been employed to treat malignant cancers, including oesophagus, head and neck, lung, bladder and in particular the skin. The technology has also been tested for treatment of prostate cancer. It is recognised as a treatment strategy that is both minimally invasive and minimally toxic, hence lessen the need for delicate surgery and lengthy recuperation.
  • PDT is selective and highly effective in certain cancers.
  • the selectivity of PDT is high because: 1 ) the photosensitizer accumulates in tumour tissues; 2) the photochemically generated radical species are highly reactive, which limits oxidative damage to the vicinity of the photosensitizer; and 3) if only the tumour is irradiated, then this physically confines the photodynamic action to tumour tissues. Due to its selectivity, toxicity and patient discomfort are kept to a minimum, while PDT has proven to be highly efficacious in the treatment of various cancer types.
  • Photodynamic therapy requires photosensitisers, which ideally possess the ability to preferentially accumulate in diseased tissue and induce a desired biological effect via the generation of cytotoxic species under illumination of light.
  • Ideal photosensitisers exhibit negligible cytotoxicity in the absence of light and high toxicity under illumination with light of the correct wavelength.
  • the photosensitizer should not be harmful to the target tissue until the treatment beam is applied. Additionally, the photosensitizer should exhibit high chemical stability.
  • the photosensitizer should preferentially accumulate in diseased/target tissue over healthy tissue, exhibit rapid clearance from the body post-procedure, possess high solubility in injectable formulations.
  • the photosensitisers should be obtainable by short and high-yielding synthetic routes, with easy translation into industrial scale production.
  • a major difficulty for use of photosensitisers for photodynamic therapy is to simultaneously achieve high tumour affinity and also retain optimal photophysical and photochemical properties.
  • a second major impediment to the use of most photodynamic sensitizers is that they generally require the presence of molecular oxygen in significant concentrations to be cytotoxic under illumination, particularly so for photosensitizers that primarily act by type-ll PDT. This means that the majority of PDT photosensitizers are not suitable for treating hypoxic cancers. Some of the most aggressive and drug- resistant tumours are typically hypoxic.
  • a long standing bias in the art is that useful PDT photosensitizers must absorb strongly with a high extinction coefficient in the red/near infrared region of the electromagnetic spectrum (600-850 nm). This is based on the assumption that because these wavelengths typically allow deeper penetration of tissue they are beneficial in treatment of tumours.
  • EPR Enhanced Permeability and Retention
  • tumour cells tend to be surrounded by new blood vessels with an abnormal form, typically featuring poorly aligned or otherwise defective endothelial cells with wide fenestrations, lacking a smooth muscle layer or innervation with a wider lumen.
  • nanoparticles can leave the abnormal blood vessel through the abnormally large holes and collect in the tumour tissue.
  • tumour tissues usually lack effective lymphatic drainage the effect is exacerbated. This phenomenon is referred to as the "enhanced permeability and retention (EPR) effect" of nanoparticles in solid tumours.
  • Nanoparticles are 1-1000 nanometer (nm) sized particles that can promote tumour selectivity and aid in delivering low-solubility drugs. Nanoparticles can target tumour tissue passively or actively. Passive targeting exploits the difference between tumour blood vessels and normal blood vessels. As described above, blood vessels in tumours are typically "leaky” with pores of from 200 to 2000 nm, which allow nanoparticles to escape into the tumour. The EPR effect is usually employed to describe passive nanoparticle delivery to cancer tissue. Active targeting uses biological molecules (antibodies, proteins, DNA and receptor ligands) to preferentially target the nanoparticles to the tumour cells. There are many types of nanoparticle delivery systems, such as silica, polymers, liposomes and magnetic particles. Anti-tumour strategies that employ thermal ablation of tumours with gold nanoparticles typically exploit passive accumulation in tumour tissue by the EPR effect.
  • X 1 , X 2 , X 3 and X 4 are selected from carbon atoms (C) or nitrogen atoms (N);
  • Y is identically or differently on each occurrence selected from N and CR 2 , preferably Y
  • Q is identically of differently selected from Y-Y, N or CR 2 ;
  • W is identically or differently selected from C or N, preferably W is C;
  • WX 2 Y 2 Q is identically or differently selected from C or N, preferably W is C;
  • WX 4 Y 2 Q denote aromaticity of the rings
  • Ar is an aromatic or heteroaromatic group with 5-6 aromatic ring atoms
  • R 3 is a linear, branched or cyclic C 1-8 alkyl group, is a linear, branched or cyclic C 2-8 alkylene group comprising of from one to three ethylene moieties in the carbon-carbon chain, or an aromatic or heteroaromatic group with 5-6 aromatic ring atoms, or a pharmaceutically acceptable salt, stereoisomer, solvate or prodrug thereof.
  • the disclose further relates to a process of forming nanoparticles.
  • the disclose further relates to methods of treatment using palladium compounds, preferably treatment of cancer, more preferably melanoma.
  • Figure 1 (a) The theoretical absorbance spectrum of the monomer and the dimer of Compound A according to TDDFT calculations at the TDDFT/PBEO/TZP/COSMO level in methanol. (b)The experimental absorption spectrum (black solid line) and emission spectra of Compound A in pure DMSO solution at different concentrations (black dashed line 10 ⁇ M [bottom]; grey dashed line 100 ⁇ M [middle], light grey dashed line 1000 ⁇ M [top]; excitation 419 nm).
  • Figure 3 Size distribution of Opti-MEM complete medium (black dash line), and its Compound A [denoted PdL] (25 ⁇ M) solution at 0 min (black solid line) or 30 min (grey solid line) according to Dynamic Light Scattering (DLS) analysis at room temperature, (b) DLS derived count rate of Compound A [denoted PdL] in Opti-MEM complete medium for 0 and 30 min. (c) Observation of absorbance spectra of Compound A [denoted PdL] (25 ⁇ M) in Opti-MEM complete medium over 24 h (30 s interval for the first 30 min, 15 min interval for the next 23.5 h).
  • DLS Dynamic Light Scattering
  • Figure 4 (a) EC50 values of Compound A to A375 2D-monolayer and 3D-spheroid cancer cells incubated, either in the dark or upon green light irradiation (13 J/cm 2 ), and in normoxic or hypoxic conditions; statistical significance was set to p ⁇ 0.05 (*). (b) Flow cytometry quantification of healthy, early apoptotic, later apoptotic and necrotic A375 cells after treatment with Compound A (2 ⁇ M) in the dark or with green light irradiation in a time gradient (2 h, 4 h, 24 h). Cisplatin (7.5 ⁇ M, 24 h) was used as positive control.
  • Figure 5 (top) Confocal images of 3D-normoxic A375 spheroids (scale bar 200 ⁇ M) in the dark or green light irradiation, with Hoechst 33342/Propidium iodide double staining after treatment with Compound A (2 ⁇ M) for 72 h. (bottom) H&E and TUNEL staining assay to tumor slices of mice in different groups at day 5.
  • Figure 6 (a) Time evolution of the mice weight 20 days post-treatment, (b) A375 tumour growth inhibition in different mice groups treated by tail intravenous injection, statistical significance was set to p ⁇ 0.01.
  • Light irradiation condition 520 nm, 100 mW/cm 2 , 10 min, 60 J/cm 2 .
  • Dose 2.1 ⁇ mol /kg, 0.9 mg/kg, 420 ⁇ M, 100 ⁇ L injection solution.
  • Figure 7 Cellular uptake in vitro and biodistribution, nanoparticle morphology and Pd content, and tumour accumulation of Compound A in vivo.
  • the palladium content (ICP-MS) of A375 skin melanoma cell monolayers (a) 2 or 24 h after treatment with Compound A (2 ⁇ M), and (b) 2 h after treatment with Compound A (5 ⁇ M) in combination with different uptake inhibitors.
  • Nanoparticles are indicated by red arrows, (d) Biodistribution of palladium (ICP-OES) in different organs of mice at different time points after intravenous tail injection of Compound A. In vivo injection conditions: 420 ⁇ M, 100 ⁇ L injection solution, 0.9 mg/kg.
  • Figure 8 (a) Tumour palladium accumulation efficiency in mice at different time points after intravenous tail injection by Compound A.
  • %ID/g (Pd content of tumour/Pd content of injection solution)x100%/mass of measured organs).
  • Figure 9 (a) Dynamic Light Scattering (DLS) derived count rate in the DMSO/H 2 O or THF/H 2 O system of Compound A (100 ⁇ M) after 30 minutes self-assembly; (b) Size distribution of the DLS analysis in the DMSO/H 2 O or THF/H 2 O system of Compound A (100 ⁇ M) after 30 minutes of self- assembly; TEM images of samples prepared from the DMSO/H 2 O (c) or THF/H 2 O (d) system of Compound A (100 ⁇ M) after 30 minutes self-assembly. Inset picture scale bar: 500 nm.
  • DLS Dynamic Light Scattering
  • Figure 10 Dose-response curves for 2D-monolayer (a) or 3D-spheroid (b) for different human cancer cell lines incubated with Compound A, either in the dark (black data points) or upon green light irradiation (grey data points) under normoxic-2D (520 nm, 20 min, 10.92 mW/cm 2 ,
  • Figure 11 Annexin V/Propidium iodide double staining FACS data for A375 cells after treatment with cisplatin (7.5 ⁇ M) or Compound A [denoted PdL] (0.5 ⁇ M or 2 ⁇ M) in the dark or upon green light irradiation (normoxic 520 nm, 20 min, 10.9 mW/cm 2 , 13 J/cm 2 ) after 2 (a), 4 (b) and 24 h (c).
  • Figure 12 Bright field images (left) and diameter (right, ⁇ M) for A549 (a) and A375 (b) 3D tumor spheroids kept in the dark (black bars) or irradiated with green light (grey bars, 520 nm, 13 J/cm 2 ). Scale bar 500 ⁇ M.
  • Figure 13 The H&E staining of different mice organs after treatment with vehicle control or Compound A, and either without or with green light irradiation (100 mW/cm 2 , 10 min, 60 J/cm 2 ). Scale bar 200 ⁇ M.
  • Figure 14 EM images showing the morphology of nanoparticles found in the blood of mice 12 h after intravenous tail injection of Compound A [denoted PdL] (middle and right images), or in an untreated control mice (left image).
  • Injection dose 2.1 ⁇ mol/kg, 0.9 mg/kg, 420 ⁇ M, 100 ⁇ L injection solution.
  • Figure 15 Dose-response curves for 2D-monolayer and 3D-spheroid for different human uveal melanoma cancer cell lines (92.1, Mel270, OMM2.5) incubated with Compound A [denoted PdL], either in the dark (black data points) or upon green light irradiation (green data points) under normoxic-2D (520 nm, 20 min, 10.9 mW/cm 2 , 13 J/cm 2 ), hypoxic-2D (520 nm, 32 min, 6.9 mW/cm 2 , 13 J/cm 2 ), normoxia-3D spheroid condition (520 nm, 32 min, 6.9 mW/cm 2 , 13 J/cm 2 ), or hypoxia-3D spheroid condition (520 nm, 55 min, 4.0 mW/cm 2 , 13 J/cm 2 ).
  • normoxic-2D 520 nm, 20 min, 10.9
  • Figure 16 Effect of treatment with compound A [indicated as PdL] and green light on 3D tumour spheroids of uveal melanoma cell lines 92.1, Mel270, and OMM2.5 at 0.1, 1, and 5 ⁇ M, under normoxia (21% O 2 ) and hypoxia (1% O 2 ).
  • the invention concerns palladium complexes with the formula 1:
  • X 1 , X 2 , X 3 and X 4 are selected from carbon atoms (C) or nitrogen atoms (N); [0032] wherein two of X 1 , X 2 , X 3 and X 4 are carbon atoms (C) and two of X 1 , X 2 , X 3 and X 4 are nitrogen atoms (N);
  • Y is identically or differently on each occurrence selected from N and CR 2 , preferably Y is CR 2 ;
  • Q is identically of differently selected from Y-Y, N or CR 2 ;
  • W is identically or differently selected from C or N, preferably W is C;
  • Ar is an aromatic or heteroaromatic group with 5-6 aromatic ring atoms
  • R 3 is a linear, branched or cyclic C 1-8 alkyl group, is a linear, branched or cyclic C 2-8 alkylene group comprising of from one to three ethylene moieties in the carbon-carbon chain, or an aromatic or heteroaromatic group with 5-6 aromatic ring atoms, or a pharmaceutically acceptable salt, stereoisomer, solvate or prodrug thereof.
  • R 3 is a linear, branched or cyclic C 1-8 alkyl group, is a linear, branched or cyclic C 2-8 alkylene group comprising of from one to three ethylene moieties in the carbon-carbon chain, or an aromatic or heteroaromatic group with 5-6 aromatic ring atoms, or a pharmaceutically acceptable salt, stereoisomer, solvate or prodrug thereof.
  • an aromatic or heteroaromatic group with 5-6 aromatic ring atoms means either a ring system of 6 C atoms or a ring system of 5 or 6 atoms containing at least one C atom and at least one heteroatom, with the proviso that the sum of C atoms and heteroatoms is 5 or 6.
  • the heteroatoms are preferably selected from N, 0 and/or S.
  • An aryl group or heteroaryl group here is taken to mean either a simple aromatic ring, i.e.
  • benzene or a simple heteroaromatic ring, for example pyridine, pyrimidine, thiophene, etc., or a condensed aryl or heteroaryl group, for example naphthalene, anthracene, phenanthrene, quinoline, isoquinoline, etc.
  • a cyclic alkyl, alkoxy or thioalkoxy group in the sense of this invention is taken to mean a monocyclic, bicyclic or polycyclic group.
  • an alkyl group is taken to mean, for example, the radicals methyl, ethyl, n-propyl, i-propyl, n-butyl, i-butyl, s-butyl, t-butyl, 2-methyl- butyl, n-pentyl, s- pentyl, tert-pentyl, 2-pentyl, cyclopentyl, n-hexyl, s-hexyl, tert-hexyl, 2-hexyl, 3-hexyl, cyclohexyl, 2- methylpentyl, n-heptyl, 2-heptyl, 3-heptyl, 4-heptyl, cycloheptyl, -methylcyclohexyl, n-octyl, 2- ethylhexyl, cyclooctyl, 1-bicyclo
  • An alkylene group is taken to mean, for example, ethenyl, propenyl, butenyl, pentenyl, cyclopentenyl, hexenyl, cyclohexenyl, heptenyl, cycloheptenyl, octenyl, cyclooctenyl or cyclooctadienyl.
  • Ci alkoxy group is taken to mean, for example, methoxy, trifluoromethoxy, ethoxy, n- propoxy, i-propoxy, n-butoxy, i-butoxy, s-butoxy, t-butoxy or 2-methylbutoxy.
  • An aromatic ring system in the sense of this invention contains 6 to 26 C atoms in the ring system.
  • a heteroaromatic ring system in the sense of this invention contains 1 to 25 C atoms and at least one heteroatom in the ring system, with the proviso that the sum of C atoms and heteroatoms is at least 5.
  • the heteroatoms are preferably selected from N, O and/or S.
  • an aromatic or heteroaromatic ring system is intended to be taken to mean a system which does not necessarily contain only aryl or heteroaryl groups, but instead in which, in addition, a plurality of aryl or heteroaryl groups may be interrupted by a non-aromatic unit (preferably less than 10% of the atoms other than H), such as, for example, an sp 3 -hybridised C, N or O atom or a carbonyl group.
  • systems such as 9,9'-spirobifluorene, 9,9-diarylfluorene, triarylamine, diaryl ether, stilbene, etc., are also intended to be taken to mean aromatic ring systems for the purposes of this invention, and likewise systems in which two or more aryl groups are interrupted, for example, by a linear or cyclic alkyl group or by a silyl group.
  • An aromatic or heteroaromatic ring systems with 5 - 25 aromatic ring atoms which may also in each case be substituted by an alkyl group mentioned above and which may be linked to the aromatic or heteroaromatic ring system via any desired positions, is taken to mean, for example, groups derived from benzene, naphthalene, anthracene, benzanthracene, phenanthrene, benzophenanthrene, pyrene, chrysene, perylene, fluoranthene, benzofluoranthene, naphthacene, pentacene, benzopyrene, biphenyl, biphenylene, terphenyl, triphenylene, fluorene, spirobifluorene, dihydrophenanthrene, dihydropyrene, tetrahydropyrene, truxene, isotruxene, furan, benzofuran, isobenzofuran, dibenzo
  • the invention concerns palladium complexes with the formula 2:
  • X 1 , X 2 , X 3 and X 4 are selected from carbon atoms (C) or nitrogen atoms (N);
  • Y is identically or differently on each occurrence selected from N and CR 2 , preferably Y is CR 2 ;
  • Q is identically of differently selected from Y-Y, N or CR 2 ;
  • Ar is an aromatic or heteroaromatic group with 5-6 aromatic ring atoms
  • R 3 is a linear, branched or cyclic C 1-8 alkyl group, is a linear, branched or cyclic C 2-8 alkylene group comprising of from one to three ethylene moieties in the carbon-carbon chain, or an aromatic or heteroaromatic group with 5-6 aromatic ring atoms, or a pharmaceutically acceptable salt, stereoisomer, solvate or prodrug thereof.
  • the invention concerns palladium complexes with the formula 3:
  • X 1 , X 2 , X 3 and X 4 are selected from carbon atoms (C) or nitrogen atoms (N);
  • Y is identically or differently on each occurrence selected from N and CR 2 , preferably Y is CR 2 ;
  • Q is identically of differently selected from Y-Y, N or CR 2 ;
  • Ar is an aromatic or heteroaromatic group with 5-6 aromatic ring atoms
  • R 3 is a linear, branched or cyclic C 1-8 alkyl group, is a linear, branched or cyclic C 2-8 alkylene group comprising of from one to three ethylene moieties in the carbon-carbon chain, or an aromatic or heteroaromatic group with 5-6 aromatic ring atoms, or a pharmaceutically acceptable salt, stereoisomer, solvate or prodrug thereof.
  • the invention concerns palladium complexes with the formula 4:
  • X 1 , X 2 , X 3 and X 4 are selected from carbon atoms (C) or nitrogen atoms (N);
  • Y is identically or differently on each occurrence selected from N and CR 2 , preferably Y is CR 2 ;
  • Ar is an aromatic or heteroaromatic group with 5-6 aromatic ring atoms
  • R 3 is a linear, branched or cyclic C 1-8 alkyl group, is a linear, branched or cyclic C 2-8 alkylene group comprising of from one to three ethylene moieties in the carbon-carbon chain, or an aromatic or heteroaromatic group with 5-6 aromatic ring atoms, or a pharmaceutically acceptable salt, stereoisomer, solvate or prodrug thereof.
  • the invention concerns palladium complexes with the formula 5:
  • X 1 , X 2 , X 3 and X 4 are selected from carbon atoms (C) or nitrogen atoms (N);
  • Ar is an aromatic or heteroaromatic group with 5-6 aromatic ring atoms
  • R 3 is a linear, branched or cyclic C 1-8 alkyl group, is a linear, branched or cyclic C 2-8 alkylene group comprising of from one to three ethylene moieties in the carbon-carbon chain, or an aromatic or heteroaromatic group with 5-6 aromatic ring atoms, or a pharmaceutically acceptable salt, stereoisomer, solvate or prodrug thereof.
  • the invention concerns palladium complexes with the formula 6:
  • X 1 , X 2 , X 3 and X 4 are selected from carbon atoms (C) or nitrogen atoms (N);
  • Ar is an aromatic or heteroaromatic group with 5-6 aromatic ring atoms
  • R 3 is a linear, branched or cyclic C 1-8 alkyl group, is a linear, branched or cyclic C 2-8 alkylene group comprising of from one to three ethylene moieties in the carbon-carbon chain, or an aromatic or heteroaromatic group with 5-6 aromatic ring atoms, or a pharmaceutically acceptable salt, stereoisomer, solvate or prodrug thereof.
  • the invention concerns palladium complexes with the formula 7: (7),
  • X 1 , X 2 , X 3 and X 4 are selected from carbon atoms (C) or nitrogen atoms (N);
  • R 3 is a linear, branched or cyclic C 1-8 alkyl group, is a linear, branched or cyclic C 2-8 alkylene group comprising of from one to three ethylene moieties in the carbon-carbon chain, or an aromatic or heteroaromatic group with 5-6 aromatic ring atoms, or a pharmaceutically acceptable salt, stereoisomer, solvate or prodrug thereof.
  • the invention concerns palladium complexes with the formula 8:
  • X 1 , X 2 , X 3 and X 4 are selected from carbon atoms (C) or nitrogen atoms (N);
  • the palladium compounds of the current invention corresponding to formula 1 surprisingly exhibit high absorption in the visible light spectrum (400 -750 nm).
  • the molar attenuation coefficient is a measurement of how strongly a chemical species attenuates light at a given wavelength. It is an intrinsic property of the species but it might vary depending on the conditions, in particular when self-assembly occurs .
  • the molar attenuation coefficient is also known as the molar extinction coefficient.
  • Compounds suitable for use in photodynamic therapy must possess a molar attenuation coefficient in excess of 50 M -1 ⁇ cm -1 for at least one wavelength of light.
  • the attenuation coefficient For measuring the attenuation coefficient one prepares several solutions with different concentrations of the compound in a given solvent, and one measures the absorption spectra of these solution. Then at one given wavelength a plot of the absorbance of the solution vs. concentration, when linear, provides as described by the Beer-Lambert law, the molar extinction coefficient as the slope of this curve.
  • the palladium compounds of the current invention corresponding to formula 1 surprisingly possess a molar attenuation coefficient in excess of 50 M -1 ⁇ cm -1 for at least one wavelength of light in the visible light spectrum (400 -750 nm), which leads to the generation of radicals.
  • This offers the advantage that compounds of the structure 1 under irradiation with white light absorb light and generate radicals that are toxic to cells.
  • the generation of radicals by the palladium compounds according to structure 1 is a function of both the intensity of incident light [J/cm 2 ] and the molar attenuation coefficient [e] of the palladium compound at the wavelength(s) of incident light.
  • PSD photosensitising agents
  • Light scattering by the irradiated tissue also affects penetration, such as for brain tissue or bone.
  • Wavelength dependence of penetration is also a function of compounds within the irradiated tissue, where the presence of compounds that absorb certain wavelengths of light will affect the degree of penetration (e.g. myoglobin in muscle tissue that has a high molar attenuation coefficient for green wavelengths of light meaning that red light tends to penetrate deeper or melanin of skin).
  • the specific light absorbing properties of palladium compounds of the current invention corresponding to the structure 1 may be modified through routine structural modifications of the ligand framework.
  • the palladium compounds corresponding to structure 1 have a molar attenuation coefficient in excess of 50 M -1 ⁇ cm -1 for at least one wavelength of light in the red light (620-750 nm) region, they are generally well suited as PS agents for treating tumours of greater volume or distance from the irradiated surface of a tissue during PDT.
  • the palladium compounds corresponding to structure 1 have a molar attenuation coefficient in excess of 50 M -1 ⁇ cm -1 for at least one wavelength of light in the red light (620-750 nm) region, they are particularly suited as PS agents for treating advanced and/or deep skin cancers or sarcomas in myoglobin rich tissues, such as Alveolar rhabdomyosarcoma (soft tissue sarcoma of the muscle tissue).
  • the palladium compounds corresponding to structure 1 have a molar attenuation coefficient in excess of 50 M -1 ⁇ cm -1 for at least one wavelength of light in the blue (450-490 nm) to green light (490-565 nm) region, they are generally well suited as PS agents for treating tumours of lesser volume or distance from the irradiated surface of a tissue by PDT.
  • Cancers of this type include skin melanoma, particularly early stage (stage 1, Tla/Tlb/T2a) melanoma in which tumours have a thickness in the range of 0.05-2.0 mm.
  • Palladium compounds corresponding to structure 1 that a molar attenuation coefficient in excess of 50 M -1 ⁇ cm -1 for at least one wavelength of light in the blue to green light (450-565 nm) region confer the advantage of allowing PDT therapy in which damage to underlying tissue is minimized due to lesser penetration of the treated tissue, especially when compared with PDT agents that have a maximum absorption wavelength in the red light (620-750 nm) region.
  • the palladium compounds corresponding to structure 1 have a molar attenuation coefficient in excess of 50 M -1 ⁇ cm -1 for at least one wavelength of light in the green light (490-565 nm) region they are particularly suited as PS agents for the treatment of corneal or conjunctival tumours, squamous cell carcinoma, lymphoma, retinoblastoma, early/shallow skin melanoma, uveal (i.e. eye) melanoma, mucosal (i.e. of the throat, neck, anus or bladder) melanoma, or vaginal melanoma.
  • the palladium compounds of the current invention corresponding to structure 1 were surprisingly found to self-assemble into nanoparticles. These nanoparticles were found to be stable as suspensions in aqueous mediums, such as serum. Without being bound by theory, it is believed that the predominantly planar compounds comprising aromatic rings and the central palladium metal allow for intermolecular metallophilic Pd ⁇ Pd interactions and p-p stacking between adjacent molecules, thermodynamically and/or kinetically favouring aggregation of the molecules, particularly as rod-shaped nanoparticles.
  • Palladium compounds corresponding to formula 1 were found to self-assemble into nanoparticles when introduced to serum or blood. Applicant surprisingly found that unlike for most porphyrin- or phthalocyanin-based PDT sensitizers, such as Photofrin or padeliporfin, the aggregation of the palladium compounds corresponding to structure 1 into nanoparticles did not result in quenching of the excited state and did not result in lower PDT properties. Without being bound by theory, it is believed that palladium compounds corresponding to structure 1 can operate by a Photodynamic therapy (PDT) type-1 mechanism when illuminated with light of the correct wavelength, even when aggregated as nanoparticles.
  • a PDT type-1 mechanism involves electron transfer from the photosensitizer to either dioxygen (to make superoxide, i.e. under normoxic conditions), or to biomolecules such as DNA or proteins (i.e. under hypoxic conditions).
  • Palladium compounds corresponding to structure 1 were surprisingly found to act as PDT agents under illumination of light of the correct wavelength under hypoxic conditions. By correct wavelength, this denotes illuminated with a wavelength at which the palladium compounds have a molar attenuation coefficient in excess of 50 M -1 ⁇ cm -1 . This was specifically demonstrated in that cytotoxicity was conserved in cells cultivated under hypoxia. Without being bound by theory, it is believed that palladium compounds corresponding to structure 1 also operate by a Photodynamic therapy (PDT) type-1 mechanism when illuminated with light of the correct wavelength.
  • PDT type-1 mechanism under hypoxic conditions involves electron transfer from the photosensitizer to biomolecules such as DNA or proteins.
  • Palladium compounds corresponding to structure 1 were found to self-assemble into nanoparticles in serum and blood. Without being bound by theory, the self-assembly into nanoparticles is believed to increase accumulation of palladium compounds corresponding to structure 1 within the tumour tissue due to the enhanced permeability and retention (EPR) effect.
  • An advantage of the self- assembly of nanoparticles comprising palladium compounds corresponding to structure 1 is that accumulation of a PDT active agent at the tumour site can be achieved without a non-PDT active nanoparticle carrier material, thereby reducing cost, as well as reducing regulatory and manufacturing complexity.
  • prodrug represents compounds which are transformed in vivo to the parent compound or other active compound, for example, by hydrolysis in blood.
  • An example of such a prodrug is a pharmaceutically acceptable ester of a carboxylic acid.
  • pharmaceutical formulation includes reference to a formulation comprising at least one active compound and optionally one or more additional pharmaceutically acceptable ingredients, for example a pharmaceutically acceptable carrier. Where a pharmaceutical formulation comprises two or more active compounds, or comprises at least one active compound and one or more additional pharmaceutically acceptable ingredients, the pharmaceutical formulation is also a pharmaceutical composition. Unless the context indicates otherwise, all references to a “formulation” herein are references to a pharmaceutical formulation.
  • product or “product of the invention” as used herein includes reference to any product containing a compound of the present invention.
  • product relates to compositions and formulations containing a compound of the present invention, such as a pharmaceutical composition, for example.
  • terapéuticaally effective amount refers to an amount of a drug, or pharmaceutical agent that, within the scope of sound pharmacological judgment, is calculated to (or will) provide a desired therapeutic response in a mammal (animal or human).
  • the therapeutic response may for example serve to cure, delay the progression of or prevent a disease, disorder or condition.
  • the invention provides compounds of formula I as previously described or a pharmaceutically acceptable salt, stereoisomer, solvate or prodrug thereof.
  • An aspect of the invention relates to a process for formation of nanoparticles comprising compounds according to any of the formulas 1-8, wherein the process comprises the steps of (1) dissolving the compound according to any of the formulas 1-8 in a suitable solvent, and (2) contacting said solution with an aqueous medium comprising proteins.
  • the solvent of the compound according to any of the formulas 1-8 is dissolved in a pharmaceutically acceptable solvent.
  • the protein is selected from any pharmaceutically acceptable protein, more preferably the protein is selected from serum albumin, globulin, plasmin or transferrin, even more preferably the protein is selected from serum albumin, globulin, ceruloplasmin or transferrin, most preferably transferrin.
  • the aqueous medium comprising proteins is serum, wherein the serum preferably comprises a protein selected from serum albumin, globulin, ceruloplasmin or transferrin, most preferably transferrin.
  • the the aqueous medium comprising proteins is blood, preferably the serum comprises a protein selected from serum albumin, globulin, ceruloplasmin or transferrin, most preferably transferrin.
  • the process is an ex vivo process.
  • An aspect of the invention relates to a pharmaceutical composition
  • a pharmaceutical composition comprising a compound with any of the formulas 1-8 or pharmaceutically acceptable salt, solvate, ester, or amide thereof, together with a pharmaceutically acceptable carrier.
  • the compounds or pharmaceutically acceptable salt, solvate, ester, or amide thereof described herein may be presented as a pharmaceutical formulation, comprising the compound, or pharmaceutically acceptable salt, solvate, ester, or amide thereof, together with one or more pharmaceutically acceptable carriers therefor and optionally other therapeutic and/or prophylactic ingredients.
  • Any carrier(s) are acceptable in the sense of being compatible with the other ingredients of the formulation and not deleterious to the recipient thereof.
  • Examples of pharmaceutically acceptable salts of the compounds according to the invention include acid addition salts formed with organic carboxylic acids such as acetic, lactic, tartaric, maleic, citric, pyruvic, oxalic, fumaric, oxaloacetic, isethionic, lactobionic and succinic acids; organic sulfonic acids such as methanesulfonic, ethanesulfonic, benzenesulfonic and p-toluenesulfonic acids and inorganic acids such as hydrochloric, sulfuric, phosphoric and sulfamic acids.
  • organic carboxylic acids such as acetic, lactic, tartaric, maleic, citric, pyruvic, oxalic, fumaric, oxaloacetic, isethionic, lactobionic and succinic acids
  • organic sulfonic acids such as methanesulfonic, ethanesulfonic, benzenes
  • compositions of the present invention may be physiologically functional derivatives of the compounds of the present invention.
  • physiologically functional derivatives may also be referred to as "pro-drugs" or “bio-precursors”.
  • Physiologically functional derivatives of compounds of the present invention include in vivo hydrolysable esters or amides, particularly esters. Determination of suitable pharmaceutically acceptable esters and amides is well within the ability of those skilled in the art.
  • solvate is used herein to refer to a complex of solute, such as a compound or salt of the compound, and a solvent. If the solvent is water, the solvate may be termed a hydrate, for example a mono-hydrate, di- hydrate, tri-hydrate etc, depending on the number of water molecules present per molecule of substrate.
  • the compounds of the present invention may be prepared using reagents and techniques readily available in the art and/or exemplary methods as described hereinafter.
  • compositions include those suitable for oral, topical (including dermal, buccal and sublingual), rectal or parenteral (including subcutaneous, intradermal, intramuscular, intraperitoneal, and intravenous, subcutaneous, epidural, topical, transdermal, parenteral, intrathecal, vaginal, rectal, colorectal, oral, intracranial, retroorbital, intrasternal), nasal and pulmonary administration e.g., by inhalation.
  • the formulation may, where appropriate, be conveniently presented in discrete dosage units and may be prepared by any of the methods well known in the art of pharmacy. Methods typically include the step of bringing into association an active compound with liquid carriers or finely divided solid carriers or both and then, if necessary, shaping the product into the desired formulation.
  • compositions suitable for oral administration wherein the carrier is a solid are most preferably presented as unit dose formulations such as boluses, capsules or tablets each containing a predetermined amount of active compound.
  • a tablet may be made by compression or moulding, optionally with one or more accessory ingredients.
  • Compressed tablets may be prepared by compressing in a suitable machine an active compound in a free-flowing form such as a powder or granules optionally mixed with a binder, lubricant, inert diluent, lubricating agent, surface-active agent or dispersing agent.
  • Moulded tablets may be made by moulding an active compound with an inert liquid diluent.
  • Tablets may be optionally coated and, if uncoated, may optionally be scored.
  • Capsules may be prepared by filling an active compound, either alone or in admixture with one or more accessory ingredients, into the capsule shells and then sealing them in the usual manner. Cachets are analogous to capsules wherein an active compound together with any accessory ingredient(s) is sealed in a rice paper envelope.
  • An active compound may also be formulated as dispersible granules, which may for example be suspended in water before administration, or sprinkled on food. The granules may be packaged, e.g., in a sachet.
  • Formulations suitable for oral administration wherein the carrier is a liquid may be presented as a solution or a suspension in an aqueous or non-aqueous liquid, or as an oil-in-water liquid emulsion.
  • Formulations for oral administration include controlled release dosage forms, e.g., tablets wherein an active compound is formulated in an appropriate release-controlling matrix, or is coated with a suitable release-controlling film. Such formulations may be particularly convenient for prophylactic use.
  • compositions suitable for rectal administration wherein the carrier is a solid are most preferably presented as unit dose suppositories.
  • Suitable carriers include cocoa butter and other materials commonly used in the art.
  • the suppositories may be conveniently formed by admixture of an active compound with the softened or melted carrier(s) followed by chilling and shaping in moulds.
  • compositions suitable for parenteral administration include sterile solutions or suspensions of an active compound in aqueous or oleaginous vehicles.
  • Injectable preparations may be adapted for bolus injection or continuous infusion. Such preparations are conveniently presented in unit dose or multi-dose containers which are sealed after introduction of the formulation until required for use.
  • an active compound may be in powder form which is constituted with a suitable vehicle, such as sterile, pyrogen-free water, before use.
  • An active compound may also be formulated as long-acting depot preparations, which may be administered by intramuscular injection or by implantation, e.g., subcutaneously or intramuscularly.
  • Depot preparations may include, for example, suitable polymeric or hydrophobic materials, or ion- exchange resins. Such long-acting formulations are particularly convenient for prophylactic use.
  • Formulations suitable for pulmonary administration via the buccal cavity are presented such that particles containing an active compound and desirably having a diameter in the range of 0.5 to 7 microns are delivered in the bronchial tree of the recipient.
  • such formulations are in the form of finely comminuted powders which may conveniently be presented either in a pierceable capsule, suitably of, for example, gelatin, for use in an inhalation device, or alternatively as a self- propelling formulation comprising an active compound, a suitable liquid or gaseous propellant and optionally other ingredients such as a surfactant and/or a solid diluent.
  • suitable liquid propellants include propane and the chlorofluorocarbons
  • suitable gaseous propellants include carbon dioxide.
  • Self-propelling formulations may also be employed wherein an active compound is dispensed in the form of droplets of solution or suspension.
  • Such self-propelling formulations are analogous to those known in the art and may be prepared by established procedures. Suitably they are presented in a container provided with either a manually-operable or automatically functioning valve having the desired spray characteristics; advantageously the valve is of a metered type delivering a fixed volume, for example, 25 to 100 microlitres, upon each operation thereof.
  • an active compound may be in the form of a solution or suspension for use in an atomizer or nebuliser whereby an accelerated airstream or ultrasonic agitation is employed to produce a fine droplet mist for inhalation.
  • Formulations suitable for nasal administration include preparations generally similar to those described above for pulmonary administration. When dispensed such formulations should desirably have an atomised or nebulised particle diameter in the range 10 to 200 ⁇ m to enable retention in the nasal cavity; this may be achieved by, as appropriate, use of a powder of a suitable particle size or choice of an appropriate valve. Other suitable formulations include coarse powders having a particle diameter in the range 20 to 500 microns, for administration by rapid inhalation through the nasal passage from a container held close up to the nose, and nasal drops comprising 0.2 to 5% w/v of an active compound in aqueous or oily solution or suspension.
  • the pharmaceutical formulations described above may include, an appropriate one or more additional carrier ingredients such as diluents, buffers, flavouring agents, binders, surface active agents, thickeners, lubricants, preservatives (including anti-oxidants) and the like, and substances included for the purpose of rendering the formulation isotonic with the blood of the intended recipient.
  • additional carrier ingredients such as diluents, buffers, flavouring agents, binders, surface active agents, thickeners, lubricants, preservatives (including anti-oxidants) and the like, and substances included for the purpose of rendering the formulation isotonic with the blood of the intended recipient.
  • Pharmaceutically acceptable carriers are well known to those skilled in the art and include, but are not limited to, 0.1 M and preferably 0.05 M phosphate buffer or 0.8% saline. Additionally, pharmaceutically acceptable carriers may be aqueous or non-aqueous solutions, suspensions, and emulsions. Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate. Aqueous carriers include water, alcoholic/aqueous solutions, emulsions or suspensions, including saline and buffered media.
  • Parenteral vehicles include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's or fixed oils. Preservatives and other additives may also be present, such as, for example, antimicrobials, antioxidants, chelating agents, inert gases and the like.
  • Formulations suitable for topical formulation may be provided for example as gels, creams, or ointments. Such preparations may be applied e.g. to a skin tumour or growth either directly spread upon the surface of the tumour or growth or carried on a suitable support such as a bandage, gauze, mesh or the like which may be applied to and over the area to be treated.
  • Liquid or powder formulations may also be provided which can be sprayed or sprinkled directly onto the site to be treated, e.g. a tumour or growth.
  • a carrier such as a bandage, gauze, mesh or the like can be sprayed or sprinkle with the formulation and then applied to the site to be treated.
  • Therapeutic formulations for veterinary use may conveniently be in either powder or liquid concentrate form.
  • conventional water soluble excipients such as lactose or sucrose, may be incorporated in the powders to improve their physical properties.
  • suitable powders of this invention comprise 50 to 100% w/w and preferably 60 to 80% w/w of the active ingredient(s) and 0 to 50% w/w and preferably 20 to 40% w/w of conventional veterinary excipients.
  • These powders may either be added to animal feedstuffs, for example by way of an intermediate premix, or diluted in animal drinking water.
  • Liquid concentrates of this invention suitably contain the compound or a derivative or salt thereof and may optionally include a veterinarily acceptable water-miscible solvent, for example polyethylene glycol, propylene glycol, glycerol, glycerol formal or such a solvent mixed with up to 30% v/v of ethanol.
  • a veterinarily acceptable water-miscible solvent for example polyethylene glycol, propylene glycol, glycerol, glycerol formal or such a solvent mixed with up to 30% v/v of ethanol.
  • the liquid concentrates may be administered to the drinking water of animals.
  • the pharmaceutical composition is a topical formulation.
  • Advantages of this embodiment include ease of use, manual targeting of cancerous tissue by direct application and that an increased dose of medication may applied where it is needed (as compared to systemic formulations).
  • a further advantage of a topical pharmaceutical composition comprising a compound with a formula according to any of structures 1-8 are reduced toxicity to other organs, such as the liver, as compared to systemic medications.
  • the topical pharmaceutical composition may be a skin cream or a gel, foam or ointment for direct topical application to the skin, which is particularly advantageous for treating skin cancers, such as melanoma.
  • the topical pharmaceutical composition may be provided as a topical solution (e.g. an eye drop liquid or suspension or a douche), which is particularly advantageous for treating corneal cancers, uveal melanoma, mucosal melanoma or vaginal melanoma.
  • the pharmaceutical composition is provided as a composition suitable for intravenous administration.
  • the pharmaceutical composition is provided as a composition suitable for direct injection into tumour tissue.
  • Compositions suitably for direct injection advantageously allow compounds according to any of structures 1-8 to be introduced into hypoxic tumour tissue, which typically has low blood supply.
  • the composition is provided as a composition suitable for systemic administration by transdermal application.
  • An aspect of the invention relates to the use of a compound with any of the formulas 1-8 or a pharmaceutical composition comprising a compound with any of the formulas 1-8 for use as a cytotoxic agent.
  • the compound or composition is used as a cytotoxic agent in the treatment of cancer. More preferably, the compound or composition is used as a cytotoxic agent for use in the treatment of melanoma. Even more preferably, the compound or composition is used as a cytotoxic agent in the treatment of skin, uveal melanoma, mucosal melanoma, vaginal melanoma. Most preferably, the compound or composition is used as a cytotoxic agent in the treatment of uveal melanoma, mucosal melanoma, vaginal melanoma.
  • An aspect of the invention is a method of treatment or prophylaxis of a disease involving cell proliferation, in particular cancer, said method comprising administering a therapeutically or prophylactically useful amount of compound according to any of the formulas 1-8, or pharmaceutically acceptable salt, solvate, ester, or amide thereof, to a subject in need thereof and irradiating the compound with visible light [400-750 nm],
  • the method of treating a disease may extend to non-cancerous diseases such as macular degeneration (an eye condition that can lead to vision loss).
  • the method of prophylaxis includes treatment of actinic keratoses (dry, scaly patches of skin caused by damage from years of sun exposure that could become cancerous if not treated), Barrett's oesophagus (changes in the cells in the lining of the oesophagus that could become cancerous if not treated) and extramammary Paget's disease (a pre-cancerous condition that affects skin in and around the groin).
  • the method of treatment or prophylaxis comprises the following steps:
  • the method of treatment or prophylaxis comprises illumination of the tumour with light of a wavelength at which the palladium compound has a molar attenuation coefficient of 50- 250,000 M -1 ⁇ cm -1 , more preferably, 500-225,000 M -1 ⁇ cm -1 , yet more preferably 1,000-200,000 M -1 ⁇ cm -1 most preferably 2,000- 175,000 M -1 ⁇ cm -1 .
  • the method of treatment or prophylaxis comprises illumination of the tumour with light of a wavelength at which the palladium compound has a molar attenuation coefficient in excess of 50 M -1 ⁇ cm -2 with a total light dose of 2 to 1000 J-cm -2 , more with a total light dose of 5 to 500 J-cm -2 , more preferably a total light dose of 25-300 J-cm -2 and most preferably a total light dose of 50-100 J-cm -2 .
  • the total light dose can be determined by the method of Ribeirode de Souza, A.; LaRochelle, E.; Marra, K; Gunn, J.; Davis, S.; Samkoe, K; Chapman, M.; Maytin, E.; Hasan, T.; Pogue, B., Photodiagnosis and Photodynamic Therapy, 20, December 2017, 227-233.
  • the method of treatment or prophylaxis comprises illumination of the tumour with light of a wavelength at which the palladium compound has a molar attenuation coefficient in excess of 50 M -1 ⁇ cm -1 with a light intensity of 2 to 1,000 mW ⁇ cm -2 , more preferably with a light intensity of 5 to 600 mW ⁇ cm -2 , even more preferably 25 to 150 mW ⁇ cm -2 , most preferably 50-100 mW ⁇ cm -2 .
  • the light intensity can be determined by the method for determining the effective light dose in Ribeirode de Souza, A.; LaRochelle, E.; Marra, K; Gunn, J.; Davis, S.; Samkoe, K; Chapman, M.; Maytin, E.; Hasan, T.; Pogue, B., Photodiagnosis and Photodynamic Therapy, 20, December 2017, 227-233.
  • Suitable light sources include those which are capable of emitting light of the appropriate wavelengths, for example and without limitation, commercially available tungsten lamps (Cole-Parmer), arc lamps, xenon continuous lamps, lasers, e.g. , blue lasers or photo-optic light sources, and light- emitting diodes (LED) or laser diodes.
  • tungsten lamps Cold-Parmer
  • arc lamps arc lamps
  • xenon continuous lamps lasers, e.g. , blue lasers or photo-optic light sources
  • LED light- emitting diodes
  • Such light sources are commercially available (Crysta Laser, Reno, Nevada; Lasever, Jiangdong, Ningbo, China, Lot-Oriel, Modulight, Muller GmbH Elektronik-Optik).
  • Other forms of light, such as sunlight can also be used for the invention (daylight PDT), as necessary or desired.
  • Devices and systems suitable for exposing the photosensitive compounds to visible light further include imaging probes, imaging catheters and fibre optic probes, particularly those containing gradient index, or graded-index, (GRIN) lenses, which are described in U. Utzinger et al., 2003, J. Biomed. Optics, 8 (1): 121-147; and Fujimoto et al., Photoyaic Materials, Devices and Systems-Laser Medicine and Medical Imaging Group, RLE Progress Report 144, pp 27-1 to 27-35, and which are commercially available. (Sp3 plus, UK).
  • imaging probes imaging catheters and fibre optic probes, particularly those containing gradient index, or graded-index, (GRIN) lenses, which are described in U. Utzinger et al., 2003, J. Biomed. Optics, 8 (1): 121-147; and Fujimoto et al., Photoyaic Materials, Devices and Systems-Laser Medicine and Medical Imaging Group, RLE Progress Report 144, pp 27-1 to 27-35
  • the light for exposing the compounds according to the methods of this invention can be sunlight, photo-optic light, or laser light.
  • the light for exposing the compound is other than UV radiation.
  • the light can be green light.
  • the light can be emitted from a variety of sources, including without limitation, a laser light source, a tungsten light source, a photo-optic light source, etc.
  • Another advantage of visible light to expose or irradiate the compounds of the invention relates to the convenience and ability to use a visible light microscope, for example, to view a sample into which a compound is introduced and to microscopically visualize or monitor a tumour during and/or after exposure to visible light.
  • a visible light microscope for example, to view a sample into which a compound is introduced and to microscopically visualize or monitor a tumour during and/or after exposure to visible light.
  • Yet another advantage to the use of visible light is that it is not detrimental to living cells and tissues, making it beneficial for clinical use.
  • the light can be specifically directed to an area where a photosensitive palladium compound is introduced or administered by the use of laser technology, fibres, probes, tubes, and the like.
  • the method comprises irradiating the compound with light with a wavelength of 490 - 700 nm, more preferably 500 - 635 nm, yet more preferably 510 - 590 nm and most preferably 520 - 565 nm.
  • cancers which may be treated include, but are not limited to, a carcinoma, for example a carcinoma of the bladder, breast, colon (e.g. colorectal carcinomas such as colon adenocarcinoma and colon adenoma), kidney, epidermal, liver, lung, for example adenocarcinoma, small cell lung cancer and non-small cell lung carcinomas, oesophagus, gall bladder, ovary, pancreas e.g.
  • a carcinoma for example a carcinoma of the bladder, breast, colon (e.g. colorectal carcinomas such as colon adenocarcinoma and colon adenoma), kidney, epidermal, liver, lung, for example adenocarcinoma, small cell lung cancer and non-small cell lung carcinomas, oesophagus, gall bladder, ovary, pancreas e.g.
  • exocrine pancreatic carcinoma, stomach, cervix, thyroid, prostate, or skin for example - squamous cell carcinoma
  • a hematopoietic tumour of lymphoid lineage for example leukaemia, acute lymphocytic leukaemia, B-cell lymphoma, T-cell lymphoma, Hodgkin's lymphoma, non- Hodgkin's lymphoma, hairy cell lymphoma, or Burkett's lymphoma
  • a hematopoietic tumour of myeloid lineage for example acute and chronic myelogenous leukaemia's, myelodysplastic syndrome, or promyelocytic leukaemia
  • thyroid follicular cancer a tumour of mesenchymal origin, for example fibrosarcoma or habdomyosarcoma
  • a tumour of the central or peripheral nervous system for example astrocytoma, neuroblastoma, glioma or schwannom
  • the compounds of the present invention may find particular application in treating cancer as their administration may be minimally invasive, spatially accurate, and as they can reach places where surgery is impossible. Due to the light activation of the compounds of the present invention, they may be particularly suited for skin, eye, lung, or digestive track cancers, for which a source of light can easily be directed onto the tumour. However, it is also possible to shine light on internal organs using endoscopy, to cure, for example prostate, head and neck, bile duct, or bladder cancers for example. They may also be administered following or during a surgical procedure to remove a tumour, where the compound may be administered to the region of tumour resection and light applied accordingly.
  • Other therapeutic agents may be administered together (whether concurrently or at different time intervals) with the compounds/compositions of the invention.
  • examples of such other therapeutic agents include but are not limited to topoisomerase inhibitors, alkylating agents, antimetabolites, DNA binders and microtubule inhibitors (tubulin target agents), such as cisplatin, cyclophosphamide, doxorubicin, etoposide, irinotecan, fludarabine, 5FU, taxanes or mitomycin C.
  • tubulin target agents such as cisplatin, cyclophosphamide, doxorubicin, etoposide, irinotecan, fludarabine, 5FU, taxanes or mitomycin C.
  • Other therapeutic agents will be evident to those skilled in the art.
  • the two or more treatments may be given in individually varying dose schedules and via different routes.
  • a compound of the invention is administered in combination therapy with one, two, three, four or more, preferably one or two, preferably one other therapeutic agents
  • the compounds can be administered simultaneously or sequentially.
  • they can be administered at closely spaced intervals (for example over a period of 5-10 minutes) or at longer intervals (for example 1, 2, 3, 4 or more hours apart, or even longer period apart where required), the precise dosage regimen being commensurate with the properties of the therapeutic agent(s).
  • the compounds of the invention may also be administered in conjunction with non- chemotherapeutic treatments such as radiotherapy, photodynamic therapy, gene therapy; surgery, and controlled diets.
  • the patient is typically an animal, e.g. a mammal.
  • the patient is an animal selected from humans, cats, dogs, horses, camels, cows, more preferably the patient is a human.
  • a therapeutically or prophylactically effective amount is meant one capable of achieving the desired response, and will be adjudged, typically, by a medical practitioner. The amount required will depend upon one or more of at least the active compound(s) concerned, the patient, the condition it is desired to treat or prevent and the formulation of order of from 1 pg to 1 g of compound per kg of body weight of the patient being treated.
  • Different dosing regiments may likewise be administered, again typically at the discretion of the medical practitioner.
  • the low toxicity of the compounds of the invention allow for at least daily administration although regimes where the compound(s) is (or are) administered more infrequently, e.g. every other day, weekly or fortnightly, for example, are also embraced by the present invention.
  • treatment is meant herein at least an amelioration of a condition suffered by a patient; the treatment need not be curative (i.e. resulting in obviation of the condition) but may be palliative.
  • Analogously references herein to prevention or prophylaxis herein do not indicate or require complete prevention of a condition; its manifestation may instead be reduced or delayed via prophylaxis or prevention according to the present invention.
  • the method of treatment or prophylaxis may optionally involve the step of a drug-to-light interval period. This is a period of time between the first administration of therapeutically or prophylactically useful amount of the compound according to any of the formulas 1-8, or a pharmaceutical composition comprising a compound of any of the formulas 1-8, to a patient and the start of illumination of the area/volume to be treated with light of a wavelength at which the palladium compound has a molar attenuation coefficient in excess of 50 M -1 ⁇ cm -1 .
  • This drug-to-light interval allows the therapeutically or prophylactically useful amount of the compound to penetrate tissue to a greater extent and/or be circulated around the body (e.g. by the blood stream).
  • the method of treatment or prophylaxis comprises the following steps:
  • the method of treatment or prophylaxis comprises an additional step between steps (i) and (ii) of a drug-to-light interval period of 1 minute to 36 hours, more preferably 2 minutes to 24 hours, even more preferably 5 minutes to 6 hours and most preferably 10 to 30 minutes, wherein there is no illumination of the area/volume with light of a wavelength at which the palladium compound has a molar attenuation coefficient in excess of 50 M -1 ⁇ cm -1 .
  • This confers the advantage of greater penetration of the diseased tissue by the compound.
  • the method of treatment or prophylaxis comprises the following steps:
  • a drug-to-light interval period of 1 minute to 36 hours, more preferably 2 minutes to 24 hours, even more preferably 5 minutes to 12 hours, yet more preferably still 10 minutes to 2 hours and most preferably 15 to 30 minutes, wherein there is no illumination of the area/volume with light of a wavelength at which the palladium compound has a molar attenuation coefficient in excess of 50 M -1 ⁇ cm -1 .
  • the drug-to-light interval period allows for passive accumulation in tumour tissue by the Enhanced Permeability and Retention (EPR) effect. It is believed that this advantageously increases the selectivity for damage to tumour tissue over damage of non-tumour tissue during photodynamic therapy.
  • EPR Enhanced Permeability and Retention
  • the method of treatment or prophylaxis comprises the following steps:
  • the method of treatment or prophylaxis comprises an additional step between steps (i) and (ii) of a drug-to-light interval period of 1 minute to 6 hours, more preferably 2 minutes to 3 hours, even more preferably 5 minutes to 1 hours and most preferably 10 to 30 minutes, wherein there is no illumination of the area/volume with light of a wavelength at which the palladium compound has a molar attenuation coefficient in excess of 50 M -1 ⁇ cm 1- . 1 This confers the advantage of greater penetration of the tumour tissue by the compound.
  • the method of treatment or prophylaxis is a method of treating a tumour with hypoxic regions.
  • the methods of treatment or prophylaxis comprises insertion of a fibre optic wire with a light diffusing element into the area to be treated, with an external light source connected to the light diffusing element along the fibre optic cable.
  • the light dose per unit length of the light diffusing element is preferably 50-600 J/cm, more preferably 100-500 J/cm, even more preferably 150-400 J/cm and most preferably 200-300 J/cm.
  • the method preferably comprises insertion of a fibre optic wire with a light diffusing element into the area to be treated, with an external light source connected to the light diffusing element along the fibre optic cable.
  • the invention also relates to a method for the preparation of palladium compounds with the formulas 1-8, wherein a compound with the formula LI is reacted with a palladium (II) source in solution.
  • X 1 , X 2 , X 3 and X 4 are selected from C-H moieties or nitrogen atoms (N); [00188] wherein two of X 1 , X 2 , X 3 and X 4 are C-H moieties and two of X 1 , X 2 , X 3 and X 4 are nitrogen atoms (N);
  • Y is identically or differently on each occurrence selected from N and CR 2 , preferably Y is CR 2 ;
  • Q is identically of differently selected from Y-Y, N or CR 2 ;
  • W is identically or differently selected from C or N, preferably W is C;
  • Ar is an aromatic or heteroaromatic group with 5-6 aromatic ring atoms
  • R 3 is a linear, branched or cyclic C 1-8 alkyl group, is a linear, branched or cyclic C 2-8 alkylene group comprising of from one to three ethylene moieties in the carbon-carbon chain, or an aromatic or heteroaromatic group with 5-6 aromatic ring atoms, or a pharmaceutically acceptable salt, stereoisomer, solvate or prodrug thereof.
  • the method comprises reaction of a compound with the formula L1 is reacted with a palladium (II) source at 100-160 °C for 12-48 hours, more preferably 120-150 °C for 18-36 hours, most preferably 130-140 °C for 20-28 hours.
  • a palladium (II) source at 100-160 °C for 12-48 hours, more preferably 120-150 °C for 18-36 hours, most preferably 130-140 °C for 20-28 hours.
  • the invention also relates to a method for the preparation of nanomaterials comprising palladium complexes of any of the formulas 1-8, wherein the method comprises the step of contacting palladium complexes of any of the formulas 1-8 with protein, preferably a protein that is normally present in blood serum.
  • the invention relates to a method for the preparation of nanostructures comprising palladium complexes of any of the formulas 1-8, wherein the method comprises the step of contacting palladium complexes of any of the formulas 1-8 with protein.
  • These nanostructures comprise palladium complexes of any of the formulas 1-8, a protein. These nanostructures are of such a size that the diameter in any direction as measured by TEM is between 1 and 1000 nm.
  • the invention also relates to the provision of a compound according to any of formulas 1-8, or a composition or material comprising the compound according to any of formulas 1-8, in a container or receptacle impenetrable to incandescent light, preferably entirely insulated from light, or a method employing such a container or receptacle. Without being bound by theory, it is believed that this prevents the compound according to any of formulas 1-8 from photo-generating reactive species before its intended use, thereby preventing damage to the compound or, where present, the additional components of the composition or material according to the claimed invention.
  • the emission spectra and relative phosphorescence quantum yields were measured via an FLS900 Spectrometer from Edinburgh Instruments Ltd.
  • the phosphorescence lifetime of the complexes in water was measured on a LifeSpec-ll spectrometer from Edinburgh Instruments, using as excitation source a 375 nm pulsed diode laser.
  • the singlet oxygen emission spectra were measured on a special custom-built setup as described by X. Q. Zhou et al., Chem. Commun. 55, 4695-4698 (2019).
  • the DFT calculations were carried out using the Amsterdam Density Functional software (ADF2019) from SCM, the PBE0 functional, a triple zeta basis set (TZP), and COSMO to simulate the solvent effect in the water.
  • Human cancer cell lines A549 (lung carcinoma), A431 (skin carcinoma) and A375 (malignant melanoma) were distributed by the European Collection of Cell Cultures (ECACC) and purchased from Sigma Aldrich.
  • Dulbecco's Modified Eagle Medium (DMEM, with and without phenol red, without glutamine), Glutamine-S (GM; 200 mm), tris(hydroxylmethyl)aminomethane (Tris base), trichloroacetic acid (TCA), glacial acetic acid, and sulforhodamine B (SRB) were purchased from Sigma Aldrich.
  • Opti-MEM Reduced Serum Media without phenol red was obtained from Gibco. The measurements of complexes on photocytotoxicity were performed according to the literature.
  • Annexin V/propidium iodide double staining assay was purchased from Bio-Connect BV.
  • the FractionPREPTM Cell Fractionation kit was obtained from BioVision Incorporated.
  • the aqueous layer was separated from the toluene layer.
  • the isolated aqueous layer was extracted with ethylacetate (100 mL) three times.
  • the combined isolated toluene layer and the ethylacetate extraction layers were dried by rotatory evaporation.
  • Example 3 Self-assembly of nanoparticles in various media
  • nanoaggregates such as nanoparticles, nanorods, and/or nanofibers.
  • nanoparticle denotes a particle with a size of 1-1000 nm as measured by Transmission Electron Microscopy (TEM).
  • TEM Transmission Electron Microscopy
  • a spherical nanoparticle has an aspect ratio of 1.
  • a nanorod has an aspect ratio of 1-100.
  • a nanofiber has an aspect ratio of over 100.
  • Opti-MEM complete medium without phenol red was used, supplemented with 2.5% v/v fetal calf serum (FCS), 0.2% v/v penicillin/streptomycin (P/S), and 1% v/v Glutamine).
  • FCS fetal calf serum
  • P/S penicillin/streptomycin
  • Glutamine 1% v/v Glutamine
  • the cells were treated with Palladium Compound A (100 ⁇ L) in a series of concentrations.
  • the cell plates in the light group were irradiated with 520 nm green light with a dose of 13 J/cm 2 (normoxic-2D: 20 min, 10.92 mW/cm 2 ; hypoxic-2D: 32 min, 6.90 mW/cm 2 ), in normoxic (21% O 2 ) or hypoxic (1% O 2 ) conditions, while the dark group was kept in the dark. After irradiation, the cells were incubated in the dark for another 48 h.
  • TCA fixation solutions (10% w/v) were added to the wells, and the plates were kept at 4 °C for 24 h.
  • 3D tumour spheroids viability assay 100 ⁇ L Opti-MEM complete medium suspensions of A549 (500 cells), A431 (500 cells), or A375 (300 cells in normoxic conditions, 1000 cells in hypoxic conditions) cells were seeded into 96-well round-bottom Corning spheroid microplates and split as dark or light groups. Each plate was incubated for 3 days in normoxic or hypoxic conditions, to obtain 3D tumour spheroids. Then, the spheroids were treated with Palladium Compound A (100 ⁇ L Opti-MEM complete medium) in a concentration series (0, 0.05, 0.25, 0.5, 1, 1.25, 2.5, 5, 12.5, 25).
  • the plates of the light group were irradiated with 520 nm green light with a dose of 13 J/cm 2 (normoxia-3D spheroid condition: 32 min, 6.90 mW/cm 2 ; hypoxia-3D spheroid condition: 55 min, 3.99 mW/cm 2 ) and incubated for another 48 h. Then a CellTiter Glo 3D solution (50 ⁇ L/well) was added to each well to stain the 3D tumour spheroids. After 30 min shake on an IKA Vibrax shake at 500 rpm at room temperature, the luminescence in each well was measured by a Tecan Microplate Reader.
  • the cells were pre-treated with different inhibitors for 1 h (NaN3 (1 mg/mL), pitstop 2 (20 ⁇ M), dynasore (80 ⁇ M), nocodazole (40 ⁇ M), and wortmannin (4 ⁇ M)), or incubated at 4 °C for 30 min. Then, the cells were treated with Palladium Compound A (5 ⁇ M) and incubated either in normoxic conditions (37 °C, 5% CO 2 , 21% O 2 , 100% humidity) or at 4 °C (in the air condition) for another 2 h. After that, the cells were harvested, centrifuged and lysed using the same method as in absence of inhibitor.
  • tumour model was established by inoculating 5x10 7 of A375 melanoma cells suspended in 100 ⁇ L of PBS at the right flank region of each mouse, to obtain mouse A375 melanoma implant. 3 weeks later, the tumour volumes were around 100 mm 3 .
  • the mice were then randomly divided into 4 groups (vehicle control, 520 nm light, Palladium Compound A, Palladium Compound A + 520 nm light groups, each group 4 mice).
  • Injection solutions were prepared by dissolving complex A in DMSO to provide a stock solution of 4.2 mM, which was then diluted in the cell culture medium (DMEM+10% FBS+1%P/S) to prepare the 420 ⁇ M Compound A injection solution
  • the mice were treated through tail intravenous injection with saline for vehicle control and 520 nm light groups, or Palladium Compound A (2.1 mitioI/kg, 0.9 mg/kg, 420 ⁇ M,
  • tumour cell damage and apoptosis conditions On day 5, one mouse in each group was sacrificed and the tumour were taken up and fixed with paraformaldehyde (10 % v/v), then sectioned into slices and analysed via Haematoxylin and eosin strain or TUNEL protocols, to evaluate the tumour cell damage and apoptosis conditions.
  • the mice were sacrificed, and the healthy organs were taken up, fixed with paraformaldehyde (10% v/v), then sectioned into slices and analysed via H&E protocol, to determine their side effect after treatment.
  • mice model based on immune-incompetent mice was used.
  • the mice were female BALB/c mice.
  • A375 skin melanoma is known to be a tumour with particular hypoxic character, which was used to evaluate the photodynamic therapy effect of Palladium Compound A under hypoxic conditions.
  • tumour-bearing mouse was treated with Compound A (2.1 ⁇ mol/kg, 0.9 mg/kg, 420 ⁇ M, 100 ⁇ L injection solution) through intravenous tail injection. After 5 min, 1 mL of blood was taken up from the eye socket and diluted to 5 mL by PBS. After centrifugation (1500 rpm, 10 min), the supernatant was collected, and the residual part was washed by PBS (5 mL) and centrifuged (1500 rpm, 10 min) again twice more, to obtain the supernatant PBS solution. These PBS solutions were then combined and centrifuged at a speed of 10000 rpm for 10 min.
  • Compound A 2.1 ⁇ mol/kg, 0.9 mg/kg, 420 ⁇ M, 100 ⁇ L injection solution
  • the prepared samples were then treated with acetone/epon-812 embedding medium in the ratio 1:1 for 2 h, 1:2 for 12 h, and pure epon-812 solution for another 5 h at 37 °C.
  • the tissue-containing embedding medium was filled in the embedding mold for 24 h at 37 °C, and another 60 °C for 48 h.
  • the obtained tissue- containing resin were then sectioned into slices with thickness around 60-80 nm via ultramicrotome (Leica EM UC7), and moved to the copper grid (150 mesh).
  • the obtained grids were stained by 2 % uranyl acetate ethanol solution for 8 min, and 2.6 % lead citrate solution for another 8 min.
  • mice were treated with Compound A (2.1 ⁇ mol/kg, 0.9 mg/kg, 420 ⁇ M, 100 ⁇ L injection solution) through intravenous tail injection. Then, the mice were sacrificed at 2 h, 6 h, 12 h, or 20 h, or 24 h, and their heart, liver, spleen, kidney, lung, and tumour, were taken. Then, around 1 g of each organ were lysed overnight in a mixture solution of 65 % HNO 3 (5 mL) and 30 % H 2 O 2 (2 mL) at 100 °C. Afterward, each sample was evaporated and another 5 mL HNO3 solution (2 %) was added.
  • Compound A 2.1 ⁇ mol/kg, 0.9 mg/kg, 420 ⁇ M, 100 ⁇ L injection solution
  • the palladium content in each organ or tumour was detected via ICP-OES (JY-Horiba ICP-OES Ultima 2).
  • ICP-OES JY-Horiba ICP-OES Ultima 2
  • the complex showed low accumulation (below 0.27 pg/g tissue) in the heart, kidney, and lung, while the liver showed significantly higher accumulation (above 1.0 pg/per gram tissue), as expected considering its role in detoxification and metabolism of exogenous substances.
  • the accumulation level of Compound A in the liver gradually decreased in time, from 3.5 pg/g tissue 2 h after injection to 1.0 pg/g tissue 24 h after injection.
  • tumour tissue showed an increased palladium accumulation from 0.17 to 0.87 pg/g tissue during the first 12 h, and further decreased to 0.17 pg/per gram tissue at 20 h and 24 h.
  • Compound A nanostructures accumulate in parallel in the tumour site and liver, but at different rates. In any case, they show a metabolic cycle around 12-20 h, which highlights the long circulation time of nanoparticles of Compound A.
  • This exceptional drug delivery efficacy which can probably be attributed to the EPR effect, confirms the high in vivo potential of the present invention.
  • the high drug accumulation in the liver is not an issue here, because the liver is not irradiated with light and Compound A is poorly toxic when not irradiated by light.
  • the specific application of drug self-delivery system for the photosensitizer Compound A makes the invention very advantageous, compared to traditional nano-conjugated chemotherapy.
  • Example 9 Photocytotoxicity and apoptosis mechanism determination of Compound A to cancer cells
  • Half-maximal effective concentration (EC 50 in ⁇ M) of Compound A for A549, A5431 and A375 cancer cells in normoxic, hypoxic or 3D-normoxic and 3D-hypoxic spheroids conditions under dark or green light irradiation are shown in Table 2.
  • 95% confidence interval (Cl in ⁇ M) and photoindex (PI EC 50, dark /EC 50, light ) are also indicated.
  • Irradiation condition normoxic 520 nm, 20 min, 10.9 mW/cm 2 , 13 J/cm 2 ; hypoxic 520 nm, 30 min, 7.22 mW/cm 2 , 13 J/cm 2 ; 3D-normoxic 520 nm, 32 min, 6.90 mW/cm 2 , 13 J/cm 2 ; 3D-hypoxic 520 nm, 55 min, 3.99 mW/cm 2 , 13.2 J/cm 2 .
  • Human uveal melanoma cell lines (primary cells: 92.1, Mel270 and metastasis cells: OMM2.5) were obtained from Institut Curie (France). Cells were grown in DMEM/F12 medium (Dulbecco's Modified Eagle Medium/Nutrient Mixture F-12) supplemented with 10% v/v FCS (fetal calf serum), 0.2% v/v P/S (penicillin/streptomycin) and 1% v/v glutamine and were maintained in a humidified atmosphere containing 5% CO 2 at 37 °C.
  • DMEM/F12 medium Dulbecco's Modified Eagle Medium/Nutrient Mixture F-12
  • FCS fetal calf serum
  • P/S penicillin/streptomycin
  • hypoxia cells were grown in the same DMEM/F12 medium but were maintained in a hypoxic incubator (humidified atmosphere containing 1% O 2 and 5% CO 2 at 37 °C). Prior any experiment, hypoxia cells were grown for at least 2 weeks in hypoxic conditions.
  • Opti-MEM medium Minimal Essential Medium
  • phenol red supplemented with 2.5% v/v FCS, 0.2% v/v P/S and 1% v/v glutamine was used instead of DMEM/F12 medium.
  • the working solution of Compound A was typically prepared from 10 mM stock solution in DMSO.
  • Cells were seeded at 1.8 x 10 4 cells/cm 2 in 96-well plates in a volume of 100 ⁇ L of Opti-MEM complete medium and incubated for 24 h under normoxic (21% O 2 ) or hypoxic (1% O 2 ) conditions. Then, cells were treated with 100 ⁇ L of a freshly prepared solution of Compound A in Opti-MEM complete medium at different concentrations.
  • the medium was refreshed, and the irradiation plates were irradiated with green light under normoxic (520 nm, 20 min, 10.9 mW/cm 2 , 13.1 J/cm 2 ) or hypoxic (520 nm, 30 min., 7.22 mW/cm 2 , 13.0 J/cm 2 ) conditions, while the dark plates were kept nonirradiated. After light irradiation, all plates were incubated in the dark for another 48 h under normoxic or hypoxic conditions, respectively.
  • normoxic 520 nm, 20 min, 10.9 mW/cm 2 , 13.1 J/cm 2
  • hypoxic 520 nm, 30 min., 7.22 mW/cm 2 , 13.0 J/cm 2
  • Cells were seeded in a volume of 200 ⁇ L of Opti-MEM complete medium at 600, 810, 1500 cells/cm 2 for normoxic and 900, 1200, 2250 cells/cm 2 for hypoxic conditions for 92.1, Mel270 and OMM2.5 cell lines respectively in 96-well ultra-low attachment Corning spheroid plates. Cells were incubated for 3 days to generate 3D tumour spheroids (300 ⁇ M diameter). Then, 100 ⁇ L of medium was carefully removed from each well, then the spheroids were treated with Compound A in Opti-MEM complete medium at different concentrations. After 24 h of incubation, 150 ⁇ L of medium was carefully removed and 150 ⁇ L of fresh medium was added.
  • the irradiation plates were irradiated with green light under normoxic (520 nm, 32 min, 6.90 mW/cm 2 , 13.2 J/cm 2 ) or hypoxic (520 nm, 55 min, 3.99 mW/cm 2 , 13.2 J/cm 2 ) conditions, while the dark plates were kept nonirradiated.
  • a CellTiter Glo 3D solution 50 ⁇ L/well was added to each well to stain the 3D tumour spheroids. After 30 minutes at room temperature under gently shaking, the luminescence in each well was measured with a bioluminescence plate reader.
  • Half-maximal effective concentration (EC 50 in ⁇ M) of Compound A in uveal melanoma cell lines 92.1, Mel270 (primary tumour) and OMM2.5 (liver metastasis), in normoxic, hypoxic or 3D-normoxic and 3D-hypoxic spheroids conditions under dark or green light irradiation. 95% confidence interval (Cl in ⁇ M) and photoindex (PI EC 50, dark /EC 50, light ) are also indicated.
  • Irradiation condition normoxic 520 nm, 20 min, 10.9 mW/cm2, 13 J/cm 2 ; hypoxic 520 nm, 30 min, 7.22 mW/cm 2 , 13 J/cm 2 ; 3D-normoxic 520 nm, 32 min, 6.90 mW/cm 2 , 13 J/cm 2 ; 3D-hypoxic 520 nm, 55 min, 3.99 mW/cm 2 , 13.2 J/cm 2 .

Abstract

La présente invention concerne des composés contenant du palladium destinés à être utilisés dans un traitement anticancéreux activé par la lumière. De telles molécules sont des complexes de palladium bis-cyclométallatés qui, une fois dans un milieu biologique ou dans le sang, peuvent s'auto-assembler en nanostructures.
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