WO2009104167A1 - A substance or composition for the treatment of cancer - Google Patents

A substance or composition for the treatment of cancer Download PDF

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Publication number
WO2009104167A1
WO2009104167A1 PCT/IB2009/050712 IB2009050712W WO2009104167A1 WO 2009104167 A1 WO2009104167 A1 WO 2009104167A1 IB 2009050712 W IB2009050712 W IB 2009050712W WO 2009104167 A1 WO2009104167 A1 WO 2009104167A1
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cells
dppe
cancer
pyridyl
phase
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PCT/IB2009/050712
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French (fr)
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Constance E Medlen
Patricia W Gitari
Judy Coates
Richard J Bowen
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Mintek
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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
    • C07F15/0066—Palladium compounds without a metal-carbon linkage
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • 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
    • C07F15/0086—Platinum compounds
    • C07F15/0093—Platinum compounds without a metal-carbon linkage

Definitions

  • THIS INVENTION relates to the treatment of cancer.
  • the invention relates to a phosphine complex, to a substance or composition for use in the treatment of cancer, to a pharmaceutical composition, to the use of a phosphine complex in the manufacture of a medicament for use in the treatment of cancer, to a process for the preparation of a phosphine complex, and to a method of treating cancer.
  • Cancer is not a single disease but a broad group of cell mutations characterised by malignant cells that are clearly distinguished from normal cells by uncontrolled growth.
  • Cell mutations are caused by abnormalities in the sequence and expression of critical genes, most notably oncogenes and tumour suppressor genes.
  • the language of cancer is to be found in the resulting deregulation of crucial biochemical pathways that control proliferation, the cell cycle, survival/apoptosis, angiogenesis, invasion and metastasis.
  • Aberrant cellular growth is a primary cause in the development of malignant tumours, but additional events take place, which enable tumour cells to invade tissue barriers and metastasise to distant sites. These events include detachment of cells from the primary tumour, the crossing of tissue boundaries, entrance and exit from the circulatory system, the infiltration of distant organs, and the formation of metastatic implants.
  • Herceptin (trade name), for the treatment of breast cancer) and the tumour vasculature
  • DNA-interactive drugs in clinical use represent one of the most important drug classes in cancer therapy. There are three major types of these clinically important drugs: ⁇ The intercalators, which insert between the base pairs of the double helix and determine a significant change of DNA conformation being accompanied by unwinding and elongation of the duplex.
  • the alkylators which react covalently with DNA bases.
  • the DNA strand breakers which generate reactive radicals that produce cleavage of the polynucleotide strands.
  • cytotoxic agents that exert their effects on all proliferating cells, both normal and cancerous. Since cytotoxic agents have a selectively 'antiproliferative' action rather than selective 'anti-cancer' properties, the therapeutic window for tumour vs. normal tissue is modest at best and toxic side effects are the norm. Most tumour types are resistant to current chemotherapy or become resistant during treatment. There is thus a need for new anticancer drugs. However, the clinical application of new anti-tumour drugs has been hindered by their low therapeutic index and lack of efficacy in humans. Thus, an approach to improve old and new anti-cancer drugs has been to manipulate their pharmacokinetic properties. Four interrelated factors determine pharmacokinetic behaviour of a drug: absorption, distribution, metabolism and excretion.
  • Metal ions and metal coordination compounds are known to affect cellular processes in a dramatic way. This metal effect influences not only natural processes, such as cell division and gene expression, but also non-natural processes, such as toxicity, carcinogenicity, and anti-tumour chemistry. However, the mechanisms of action of metal-based drugs are often not well understood.
  • Pd(II) salts form very stable complexes with tertiary phosphines and arsines. Based on the structural analogy between Pt(II) and Pd(II) complexes, some studies on Pd(II) compounds as suitable drugs have been carried out. However, advances in this area have been scarce probably due to kinetic reasons; it is well known that comparable Pt(II) compounds always react more slowly by a factor of 10 5 than corresponding Pd(II) complexes.
  • Pd(II) and Pd(IV) complexes have been investigated for their cytotoxic anti-tumour activity.
  • amine neutral ligands such as ethylenediamine, diaminocyclohexane, ammonia, pyridine and pyrimidine derivatives, alkylaminophosphine oxides, mercapto-imidazoles and pyrimidines.
  • Promising anti-proliferative activity was found in Pd complexes with chelating ligands or alkyl- or aryl-thiosemicarbazones.
  • dmnp 2,6-dimethyl-4-nitropyridine
  • M is Pt or Pd;
  • X is hexafluorophosphate, halogen or pseudo-halogen.
  • a substance or composition for use in the treatment of cancer including a phosphine complex of the general formula (I) as hereinbefore described.
  • a pharmaceutical composition which includes a phosphine complex of the general formula (I) as hereinbefore described, or a pharmaceutically acceptable salt thereof.
  • the invention extends to a method of treating cancer, which includes administering to a patient a therapeutically effective amount of a phosphine complex of the general formula (I) as hereinbefore described, or a pharmaceutically acceptable salt thereof.
  • the phosphine complex may be selected from the group consisting of
  • the phosphine complex is selected from the group consisting of
  • the phosphine complex is selected from the group consisting of
  • M is Pt or Pd;
  • X is hexafluorophosphate, halogen or pseudo-halogen, the process including
  • the process may include reacting the intermediate compound of formula (I) with a hexafluorophosphate salt to produce the compound of formula (I) in which X " is PF 6 " .
  • M 1 is Na
  • M 2 is Pd
  • Hal is Cl so that IvT 2 M 2 HaI 4 is Na 2 PdCI 4 .
  • R 1 and R 2 are both 2-pyridyl.
  • the process includes reacting M 1 2 M 2 HaI 4 in which M 2 is Pd with
  • the hexafluorophosphate salt may be NH 4 PF 6 .
  • Figure 1 shows a synthetic route in accordance with the invention for the preparation of bis(1 ,2-bis(di-2-pyhdyl)phosphino)ethane-P,P')-palladium(ll)
  • Figure 2 shows an Ortep representation of bis(1 ,2-bis(di-2- pyridyl)phosphino)ethane-P,P')-palladium(ll) (hexafluorophosphate), i.e. Pg 8, with PF 6 " ions omitted for clarity.
  • Figure 3 shows graphs of percentage of cells undergoing apoptosis and necrosis after treatment with compounds of the invention and reference gold compounds for 18 (a), 24 (b) and 48 hours (c), and also for cells incubated with camptothecin for 6 hours (d).
  • the data is presented as a mean of three experiments.
  • Figure 4 shows contour diagrams of FITC-Annexin V/PI flow cytometry of Jurkat cells cultured for 48 hours with Pg 8 of the invention, a gold reference compound and camptothecin.
  • Diagram (a) is untreated, diagram (b) is Pg 8 (0.71 1 ⁇ M), diagram (c) is Pg 8 (1 .422 ⁇ M), diagram (d) is [Au(dppe)2]CI (0.131 ⁇ M), diagram (e) is [Au(dppe)2]CI (0.262 ⁇ M) and diagram (f) is camptothecin (1 ⁇ M).
  • Figure 5 shows representative histograms and graphs showing changes in the cell cycle profiles (G 0 /Gi , S and G 2 /M phases) after 18, 24 and 48 hours for untreated Jurkat cells.
  • Figure 6 shows representative histograms and graphs showing changes in the cell cycle profiles (G 0 /Gi, S and G 2 /M phases) after 18, 24 and 48 hours for 0.71 1 ⁇ M of Pg 8 on cell cycle of Jurkat cells.
  • Figure 7 shows representative histograms and graphs showing changes in the cell cycle profiles (G 0 /Gi, S and G 2 /M phases) after 18, 24 and 48 hours for 1 .422 ⁇ M of Pg 8 on cell cycle of Jurkat cells.
  • Figure 8 shows representative histograms and graphs showing changes in the cell cycle profiles (G 0 /Gi, S and G 2 /M phases) after 18, 24 and 48 hours for 0.131 ⁇ M of [Au(dppe) 2 ]CI on cell cycle of Jurkat cells.
  • Figure 9 shows representative histograms and graphs showing changes in the cell cycle profiles (G 0 /Gi, S and G 2 /M phases) after 18, 24 and 48 hours for 0.262 ⁇ M of [Au(dppe) 2 ]CI on cell cycle in Jurkat cells.
  • Figure 10 shows a comparison of cell cycle progression of untreated Jurkat cells and those treated with Pg 8 (0.71 1 and 1.422 M) and [Au(dppe) 2 ]CI (0.131 and 0.262 ⁇ M) after 18, 24 and 48 h. Differences in G 0 /Gi , S and G 2 /M phases of untreated and treated cells were compared by 2-way ANOVA. Significance was established at P ⁇ 0.05.
  • Figure 1 1 shows graphs of mean body weight changes of control and treated mice in phase 1 of Study 4.
  • Figure 12 shows graphs of mean body weight changes of control and treated mice in phase 2 of Study 4.
  • Figure 13 shows a graph of AST levels of untreated mice and mice treated with [Au(dppe) 2 ]CI (3 ⁇ mol/kg) and Pg 8 (3 and 6 ⁇ mol/kg) in Phase 1 of Study 4.
  • Figure 14 shows a graph of AST levels of untreated mice and mice treated with
  • Figure 15 shows GGT levels of untreated mice and mice treated with [Au(dppe) 2 ]CI (3 ⁇ mol/kg) and Pg 8 (3 and 6 ⁇ mol/kg) in Phase 1 of Study 4.
  • Figure 16 shows GGT levels of untreated mice and mice treated with [Au(dppe) 2 ]CI (6 ⁇ mol/kg) and Pg 8 (12 and 15 ⁇ mol/kg) in Phase 2 of Study 4.
  • Figure 17 shows Creatinine levels of untreated mice and mice treated with [Au(dppe) 2 ]CI (3 ⁇ mol/kg) and Pg 8 (3 and 6 ⁇ mol/kg) in Phase 1 of Study 4.
  • Figure 18 shows Creatinine levels of untreated mice and mice treated with [Au(dppe) 2 ]CI (6 ⁇ mol/kg) and Pg 8 (12 and 15 ⁇ mol/kg) in Phase 1 of Study 4.
  • Figure 19 shows comparison of distribution of [ 198 Au(dppe) 2 ]CI (5 and 10 ⁇ M) and [ 103 Pd(d2pyrpe) 2 ][PF 6 ] 2 (5 and 10 ⁇ M) in Jurkat cells after exposure for 1 h. The results shown here were obtained from an average of 8 different cell populations and the values are means ⁇ SEM.
  • Figure 20 shows the biodistribution of selected organs of adult male Sprague Dawley rats as expressed as a percentage of injected dose per gram of [ 198 Au(dppe) 2 ]CI and [ 103 Pd(d2pyrpe) 2 ][PF 6 ] 2 .at 6 h. Illustrated values are means ⁇ S.E.M.
  • Elemental analysis (empirical formulae shown) was determined by the Institute for Soil, climate and Water, Pretoria. Fast atomic bombardment mass spectra were recorded with a VG70SEQ by micromass at the University of the Witwatersrand. Melting points were recorded in unsealed capillaries and are uncorrected.
  • Pg 8 was prepared according to the synthetic route shown in Figure 1. The first step was carried out under an argon atmosphere, using standard Schlenk techniques as the ligand is known to decompose slowly in the presence of oxygen. Pg 8 was prepared from the intermediate (bis(1 ,2-bis(di-2-pyhdylphosphino)ethane palladium) dichloride, [Pd(d2pyrpe) 2 ].2CI.
  • 1a signifies peaks for the mono-chelated compound while b shows those peaks that belong to the bischelated compound.
  • Pg 8 crystallises in the non-centrosymetric space group Pnna with half a molecule of the palladium complex and two ions of PF 6 in the asymmetric unit.
  • the two PF 6 ions are disordered.
  • One has only the equilateral fluoride atoms disordered in a general position while the second has both axial and equilateral fluoride atoms disordered also in a general position but resulting in distorted octahedral geometry.
  • Connection of molecules in the crystal is mainly through C-H... F intermolecular interactions.
  • Packing in the crystal is such that the solvate molecules are sitting in the voids between the palladium complex molecules with the less disordered ions of PF 6 being on the same axis as Pd atoms while the more disordered PF 6 ion is positioned between the Pd complexes.
  • the conformations of the ligands are such that the pyridyl rings get positioned more above and below the coordination plane. Two pyridyl nitrogens, one on each side of the coordination plane are just over 3A from the Pd.
  • Table 1 Crystal Data for [Pd(d2pyrpe) 2 ][PF 6 ] 2
  • Supplemented cell culture medium Cell culture medium with 10% foetal calf serum (FCS).
  • FCS foetal calf serum
  • PBS Phosphate buffered saline
  • PHA Phytohaemagglutinin
  • Test compounds (6 and [Au(dppe) 2 ]CI) - dissolved in DMSO (20 mM) and diluted in cell culture medium.
  • DMSO DMSO
  • FCS heat- inactivated serum
  • EACC ovarian cancer-cisplatin resistant
  • RPMI ⁇ MCF-7- human breast cancer
  • DMEM DMEM
  • RPMI ⁇ DU 145- human prostate cancer
  • Human lymphocytes (resting and PHA stimulated)- fresh venous blood was obtained from healthy volunteers followed by isolation of lymphocytes.
  • Chicken embryo fibroblasts- cells were isolated from chicken embryos.
  • MTT is a pale yellow substance that is metabolised to dark blue formazan crystals by metabolically active cells. The amount of formazan produced is directly proportional to the amount of cells over a wide range.
  • 80 ⁇ l of medium (60 ⁇ l in the case of the lymphocytes earmarked to be stimulated) was dispensed into each well of a 96 well tissue culture plate (micro-titer plate).
  • 100 ⁇ l of cell suspension (2 x 10 4 - 2 x 10 6 cells/ml/well depending on the cell type) was added and then allowed to incubate for 1 hour at 37 Q C in an atmosphere of 5% CO 2
  • 20 ⁇ l of the experimental drug (eight varying concentrations) was added in triplicate to the wells.
  • control wells received 20 ⁇ l of growth medium instead of the experimental drug.
  • Lymphocytes earmarked to be stimulated received 20 ⁇ l PHA 5 minutes after the addition of the drug.
  • the cells were washed with 150 ⁇ l PBS (per well) and centrifuged for 10 min at 2000 rpm (800 g) (Beckman TJ 6 centrifuge). The supernatant was removed and plates were left to dry in the dark. 100 ⁇ l DMSO was added to each well to solubilise the formazan crystals and the plates were shaken for 2-4 hours. Culture plates were read on a plate reader using a wavelength of 570 nm and a reference wavelength of 630 nm.
  • IC 5 O the concentration ( ⁇ M) of the experimental compound inducing a 50% decrease in cell growth).
  • IC 5 O value calculations were performed with the GraphpadTM program and are tabulated below in Table 2.
  • Table 2 IC 50 values ( ⁇ M) and S. E. M ( ⁇ ) of 7 compounds on various cancer cell lines as well as normal cells. The values are presented as a mean of three experiments carried out in triplicate.
  • Pg 1 the least toxic compound followed by Pg 6 and Pg 5 ⁇ Table 2).
  • Pg 8 was the most toxic of the palladium complexes followed by Pg 4a and Pg 3.
  • prostate cancer (DU-145) was the most sensitive cell line (IC 5 O values of 0.253-9.380 ⁇ M) while ovarian cancer-cisplatin resistant cell line (A2780-cis) was the most resistant (IC 5 O values of 2.029-> 50 ⁇ M).
  • Human ovarian cancer cell line- cisplatin sensitive (A2780) was sensitive to all the palladium complexes with IC 50 values of 0.617 ⁇ M (Pg 3), 0.136 ⁇ M (Pg 4a) and 0.974 ⁇ M (Pg 8). Overall, Pg 8 was the most toxic (over the whole range of cells) of the novel compounds followed by Pg 4a which showed comparative activity against Jurkat and MCF-7 cell lines.
  • Pg 8 was selected for further tests in an effort to elucidate its mechanism of action. It was selected due the fact that it showed greater toxicity to a wider range of cancer lines than the rest of the invention compounds.
  • [Au(dppe) 2 ]CI was included in these investigations as a standard. Jurkat cells were used for the remainder of the assays as they could be compared to lymphocytes.
  • the aim of this assay was to evaluate the induction of apoptosis and/or necrosis by [Au(dppe) 2 ]CI and Pg 8 on Jurkat cells with the Annexin V binding assay. Untreated and treated cells were evaluated for apoptosis by flow cytometry. Camptothecin (1 ⁇ M), a topoisomerase I inhibitor that efficiently induces apoptosis in Jurkat cells was used as a positive control.
  • PBS Phosphate buffered saline
  • Binding buffer (238 mg Hepes, 876 mg NaCI, 37.3 mg KCI, 26.5 mg CaCI 2 , 9.5 mg MgCI 2 in de-ionised water, at pH 7.4) ⁇ Annexin V- FITC ( BD Biosciences Pharmingen)
  • the human Jurkat cell line was cultured in RPMI 1640 supplemented with 10% v/v heat-inactivated FCS and 1 % penicillin-streptomycin. Cells were maintained in a humidified atmosphere of 5% carbon dioxide at 37 °C.
  • Cells (1 x 10 5 cells/ml) were treated with [Au(dppe) 2 ]CI and Pg 8 for 18, 24 and 48 h in cell culture flasks. After the incubation period, cells were decanted from flasks and centrifuged for 5 min at 200 g. The cell pellet was washed with PBS (1 % FCS) and resuspended in 1 ml binding buffer. 100 ⁇ l of cell suspension was transferred to flow cytometer tubes. 5 ⁇ l of Annexin V-FITC and 10 ⁇ l propidium iodide were added to some tubes (unstained samples were also prepared). The cell suspensions were mixed gently and incubated for 15 min in the dark at room temperature (25 °C). 400 ⁇ l of binding buffer was then added to each tube and analysis was carried out within an hour with a flow cytometer (Beckman Coulter FC 500). Statistical methods
  • Figure 3 shows graphs indicating the percentages of cells undergoing various stages of apoptosis.
  • Figure 4 shows actual histograms of treated and untreated cells undergoing apoptosis (48 h). After incubation of Jurkat cells with the compounds for 18 and 24 h, no significant differences were noted between the untreated and the treated groups as between 85 and 90% of cells were viable. However, after exposure of the cells for 48 h, significant differences were observed. Untreated cells had an average of 85% of cells in a viable state and only 8% at the early apoptotic stage.
  • Pg 8 (0.71 1 and 1 .422 ⁇ M) significantly induced apoptosis in 25% of the cells while 14% of cells had undergone necrosis. Only 50% of the cells were viable in this group.
  • camptothecin (1 ⁇ M) induced apoptosis in Jurkat cells within a short time. After incubation for 6h, about 40% of the cells were in the late apoptotic stage. Viable cells were just over 50% while only 2% of the cells were necrotic.
  • DNA topoisomerase I and Il inhibitors induce apoptosis in various cell lines and this is due to DNA-protein complex formation stabilised by DNA topoisomerase I inhibitors that ultimately signal the onset of apoptosis.
  • Study 2 has shown that Pg 8 and [Au(dppe) 2 ]CI caused Jurkat cells to undergo apoptosis after exposure for 48 h.
  • the aim of this study was to determine if cell death was as a result of cell cycle arrest by these compounds.
  • Cell cycle analysis was performed using flow cytometric evaluation of DNA content.
  • PBS Phosphate buffered saline
  • the human Jurkat cell line was cultured in RPMI 1640 supplemented with 10% v/v heat-inactivated FCS and 1 % penicillin-streptomycin. Cells were incubated with the experimental compounds for 18, 24 and 48 hours at 37 °C and 5% carbon dioxide.
  • Cells (1 x 10 5 cells/ml) were treated with [Au(dppe) 2 ]CI and Pg 8 for 18, 24 and 48h in cell culture flasks. After the incubation period, they were decanted from flasks and centrifuged for 5 min at 200 g. The cell pellet was re-suspended in 500 ⁇ l PBS and chilled on ice. The cold cell suspension was then added rapidly to flow cytometer tubes containing 500 ⁇ l of ice cold ethanol. Mixing was carried out by forcing air bubbles through the suspension and then kept on ice for 15 minutes.
  • RNAse 2mg/ml 1 .12% w/v sodium citrate
  • Pl 125 ⁇ l of Pl was added to each tube and mixed well. Samples were allowed to stand for 30 minutes at room temperature before analysis by flow cytometry (Beckman Coulter FC 500). DNA histograms were collected and estimation of the percentages of cells in Gi , S and G 2 /M was performed with a computer software program (Multicycle, Phoenix Flow systems, San Diego, CA).
  • the DNA histogram yields the relative number of cells in G 1 ZG 0 , S, and G 2 /M phases of the cell cycle. Although some information about cell cycle progression can be deduced by following changes in the cell cycle phases with time, it gives static information. For example, although it is possible to estimate the percentage of cells in S phase, the measurement does not directly tell whether those cells are still moving through the S phase. A very distinct connection has been forged between the cell cycle clock apparatus and apoptosis. The paclitaxel-induced apoptosis of human breast cancer cells has been found to depend upon the induction of the cdc2 kinase at the G 2 /M phase transition of the cell cycle.
  • Inbred female Balb/C mice of 6-8 weeks were used and housed in standard mouse cages in rooms with controlled environmental conditions. The animals were fed normal pellets (EPOL (trade name)) and water ad libitum.
  • mice The study was carried out in two phases with 4 groups of 6 mice each being used per phase (total of 48 mice).
  • the MTD of [Au(dppe) 2 ]CI was previously shown to be 3.0 ⁇ mol/kg/day for 5 days and hence this concentration was used as a guideline.
  • the weight of the mice was determined to adapt the dosages according to their body weight.
  • the experimental compound Pg 8 and [Au(dppe) 2 ]CI (standard) were dissolved in analytical quality ethanol, with subsequent addition of water to make up a final dose volume of 0.5 ml and a concentration of 5% ethanol.
  • the dosages were prepared immediately prior to each ip. administration.
  • mice were injected ip. each day for 5 days.
  • phase 1 the ethanol-water solution and the first and second dosages of 3 ⁇ mol/kg and 6 ⁇ mol/kg were administered each day from day 1 to 5.
  • Phase 2 followed whereby the ethanol-water solution and the third and fourth dosages of 12 ⁇ mol/kg and 15 ⁇ mol/kg were administered each day from day 1 to 5.
  • the mice were weighed followed by anaesthetisation via isoflurane inhalation. While they were at the surgical plane of anaesthesia, maximum blood was drawn via cardiac puncture. The animals were then further exposed to isoflurane until death occurred.
  • Mice in phase 2 were dissected and major organs (liver, heart and kidneys) were weighed.
  • Body weights were recorded immediately prior to dosing (on day 1 ) so that an objective monitoring of weight could be done, indicating food intake, which is a good measure for animal well-being.
  • the animals were also monitored for pain and stress (behavioural changes) immediately after the injection.
  • GGT levels shown in Figure 15 were also slightly elevated (P ⁇ 0.05) in the group treated with [Au(dppe) 2 ]CI (3 ⁇ mol/kg) when compared to the untreated mice. However, the levels did not differ significantly with the mice treated with Pg 8 (12 and 15 ⁇ mol/kg). In Phase 2 of the study, GGT levels did not show significant differences in all the groups (untreated and treated). Notably, the mice treated with 12 ⁇ mol/kg of Pg 8 exhibited higher levels of GGT than the highest dose (15 ⁇ mol/kg) ( Figure 16). However, this difference was not significant.
  • Creatinine levels ( Figure 17 and 18) in all the groups did not vary significantly. This is an indication of lack of toxicity to the kidneys.
  • [Au(dppe) 2 ]CI showed greater in vitro activity against cancer cells than Pg 8. It was also very toxic to the normal cells such as lymphocytes, chicken embryo fibroblasts and hepatocytes. This lack of selectivity is exhibited in vivo as the acute exposure to mice caused reduced body weight. Measurement of biochemical parameters revealed that AST levels were greatly elevated which may imply damage to organs. As an intracellular enzyme, AST is released into the blood in proportion to the number of damaged cells. While the former drug may show potency against cancer cell lines, its lack of selectivity is dose-limiting. As mentioned earlier, it was also active in tumour implanted mice.
  • Pg 8 was more tolerable although the group treated with the highest dose (15 ⁇ mol/kg) showed slightly reduced weight gain as compared to the rest (3, 6 and 12 ⁇ mol/kg).
  • the desired outcome is that the compound shows in vivo anti-tumour activity especially in distal tumours. This would be determined by good bio-availability which is influenced by factors such as stability against metabolic, pathways, adsorption, plasma level concentrations and excretion rates. Study 5
  • PBS Phosphate buffered saline
  • Human T-cell lines (Jurkat) were cultured in RPMI 1640 supplemented with 10% v/v heat-inactivated FCS and 1 % penicillin-streptomycin. The cell culture was maintained at 37 0 C and 5% CO 2 .
  • the animals were anaesthetised by an intraperitoneal (ip.) injection of a 6 % sodium pentobarbitone solution at a dose of 1 ml/kg.
  • ip. intraperitoneal
  • a 24 G jelco was inserted into the tail vein of the animals to administer the radio- labelled compounds.
  • Two animals per group were scanned in parallel with an Elscint Gamma Camera at the Diagnostic Imaging unit at Onderstepoort in order to obtain the radionuclide imaging.
  • Two minute static studies were performed every half an hour up to 6 hours, lsoflurane was used to immobilize the animals for the 2 minute static studies.
  • the animals were sacrificed using an lsoflurane overdose after a 6 hour period.
  • the organs were separated and counted in a well type counter at NECSA. From the organ counts as well as the reference activity in a syringe the %ID/g (Injected Dose/gram) was calculated.
  • the biodistribution ( Figure 20) showed predominantly high reticulo- endothelial uptake for all compounds.
  • the largest concentration of [Au(dppe) 2 ]CI was found in the lungs followed by the spleen.
  • the highest concentration of Pg 8 was found in the spleen followed by the liver.
  • Hydrophilic drugs are expected to have a limited biodistribution compared to lipophilic drugs. This might imply a more selective tumour uptake.

Abstract

The invention provides a phosphine complex of the general formula (I) in which M is Pt or Pd; R 1 and R 2 are independently Ph, 2-pyridyl, 3-pyridyl or 4-pyridyl; the two Y substituents are independently CH 2 CH 2 or CH=CH; and X is hexafluorophosphate, halogen or pseudo-halogen. The phosphine complex is useful in the treatment of cancer.

Description

A SUBSTANCE OR COMPOSITION FOR THE TREATMENT OF CANCER
THIS INVENTION relates to the treatment of cancer. In particular, the invention relates to a phosphine complex, to a substance or composition for use in the treatment of cancer, to a pharmaceutical composition, to the use of a phosphine complex in the manufacture of a medicament for use in the treatment of cancer, to a process for the preparation of a phosphine complex, and to a method of treating cancer.
Cancer is not a single disease but a broad group of cell mutations characterised by malignant cells that are clearly distinguished from normal cells by uncontrolled growth. Cell mutations are caused by abnormalities in the sequence and expression of critical genes, most notably oncogenes and tumour suppressor genes. The language of cancer is to be found in the resulting deregulation of crucial biochemical pathways that control proliferation, the cell cycle, survival/apoptosis, angiogenesis, invasion and metastasis. Aberrant cellular growth is a primary cause in the development of malignant tumours, but additional events take place, which enable tumour cells to invade tissue barriers and metastasise to distant sites. These events include detachment of cells from the primary tumour, the crossing of tissue boundaries, entrance and exit from the circulatory system, the infiltration of distant organs, and the formation of metastatic implants.
The treatment of cancer has traditionally used agents that interfere with the cell division process. More recently, research and novel therapies have targeted the growth signals that drive the proliferation and survival of cancer cells (for example,
Herceptin (trade name), for the treatment of breast cancer) and the tumour vasculature
(for example, anti-bodies against vascular endothelial growth factor).
DNA-interactive drugs in clinical use represent one of the most important drug classes in cancer therapy. There are three major types of these clinically important drugs: ■ The intercalators, which insert between the base pairs of the double helix and determine a significant change of DNA conformation being accompanied by unwinding and elongation of the duplex.
■ The alkylators, which react covalently with DNA bases. ■ The DNA strand breakers, which generate reactive radicals that produce cleavage of the polynucleotide strands.
Increased cure rates have been achieved in childhood cancer, testicular cancer, leukaemia and lymphoma and survival improvements have been obtained with adjuvant drug treatment of breast and ovarian cancer. However, the goal of routine care or long term management of cancer as a chronic disease remains frustratingly elusive and the development of preventative agents is even more embryonic and challenging.
The majority of current anti-cancer drugs are cytotoxic agents that exert their effects on all proliferating cells, both normal and cancerous. Since cytotoxic agents have a selectively 'antiproliferative' action rather than selective 'anti-cancer' properties, the therapeutic window for tumour vs. normal tissue is modest at best and toxic side effects are the norm. Most tumour types are resistant to current chemotherapy or become resistant during treatment. There is thus a need for new anticancer drugs. However, the clinical application of new anti-tumour drugs has been hindered by their low therapeutic index and lack of efficacy in humans. Thus, an approach to improve old and new anti-cancer drugs has been to manipulate their pharmacokinetic properties. Four interrelated factors determine pharmacokinetic behaviour of a drug: absorption, distribution, metabolism and excretion.
Metal ions and metal coordination compounds are known to affect cellular processes in a dramatic way. This metal effect influences not only natural processes, such as cell division and gene expression, but also non-natural processes, such as toxicity, carcinogenicity, and anti-tumour chemistry. However, the mechanisms of action of metal-based drugs are often not well understood.
The interactions of heavy metals such as platinum (Pt) and gold (Au) with N, S donor atoms have been recognised for their anti-cancer properties with the potential to develop metal-based drugs. The successful use of metal complexes as therapeutic and diagnostic agents depends on the control of their kinetic and thermodynamic properties through appropriate choice of oxidation state, types and numbers of bound ligands, and coordination geometry.
Pd(II) salts form very stable complexes with tertiary phosphines and arsines. Based on the structural analogy between Pt(II) and Pd(II) complexes, some studies on Pd(II) compounds as suitable drugs have been carried out. However, advances in this area have been scarce probably due to kinetic reasons; it is well known that comparable Pt(II) compounds always react more slowly by a factor of 105 than corresponding Pd(II) complexes.
Relatively few Pd(II) and Pd(IV) complexes have been investigated for their cytotoxic anti-tumour activity. Among them were complexes with amine neutral ligands such as ethylenediamine, diaminocyclohexane, ammonia, pyridine and pyrimidine derivatives, alkylaminophosphine oxides, mercapto-imidazoles and pyrimidines. Promising anti-proliferative activity was found in Pd complexes with chelating ligands or alkyl- or aryl-thiosemicarbazones. Two kinds of Pd complexes with 2,6-dimethyl-4-nitropyridine (dmnp) were prepared and tested for cytotoxicity in vitro against 4 human cancer cells: SW707 (adenocarcinoma of the rectum), T47D (breast cancer), HCV (bladder cancer) and A549 (non-small cell lung carcinoma). Free ligand and [Pd2(dmnp)2CI4] showed moderate activity while the [Pd(dmnp)2CI2] was strongly active against all 4 cell lines. The greatest activity was observed against T47D, which is the line known as poor responsive (resistant) on platinum-based drugs. The ID50 (concentration of the compound required to kill 50% of tumour cells) values for the most potent compound ranged from 0.46-8.4 μg/ml.
The bis-chelated 1 :2; Au(l):diphosphine complex, [Au(dppe)2]CI (dppe = Ph2PCH2CH2PPh2) shown below has been investigated as a cancer drug.
Figure imgf000005_0001
[Au{dppe)?]CI
This complex exhibits anti-tumour activity against a range of tumour models in mice and structure activity relationships have been evaluated for a wide range of diphosphine ligands and their metal complexes. Preclinical development of [Au(dppe)2]+ was abandoned after the identification of severe hepatotoxicity in dogs attributed to alterations in mitochondrial function. [Au(dppe)2]+ is extremely lipophilic (containing 8 hydrophobic phenyl substituents) and consequently non-selectively targets mitochondria in all cells.
Pd analogues of [Au(dppe)2]CI have previously being prepared and tested for anti-tumour activity. These complexes, [Pd(dppe)2]CI2 and [Pd(dppen)2]CI2 (dppen = 1 ,2-bis(diphenylphosphino)ethylene), were tested for cytotoxicity against murine and human tumour cell lines. The results showed low potency. They were also tested for anti-tumour activity on intraperitoneal (ip.)-implanted tumours and both of them showed inactivity and were not well tolerated.
According to one aspect of the invention, there is provided a phosphine complex of the general formula (I)
Figure imgf000005_0002
in which
M is Pt or Pd; R1 and R2 are independently Ph, 2-pyridyl, 3-pyridyl or 4-pyridyl; the two Y substituents are independently CH2CH2 or CH=CH; and X is hexafluorophosphate, halogen or pseudo-halogen.
According to another aspect of the invention, there is provided a substance or composition for use in the treatment of cancer, the substance or composition including a phosphine complex of the general formula (I) as hereinbefore described.
According to a further aspect of the invention, there is provided a pharmaceutical composition which includes a phosphine complex of the general formula (I) as hereinbefore described, or a pharmaceutically acceptable salt thereof.
According to yet another aspect of the invention there is provided the use of a phosphine complex of the general formula (I) as hereinbefore described in the manufacture of a medicament for use in the treatment of cancer.
The invention extends to a method of treating cancer, which includes administering to a patient a therapeutically effective amount of a phosphine complex of the general formula (I) as hereinbefore described, or a pharmaceutically acceptable salt thereof.
The phosphine complex may be selected from the group consisting of
Figure imgf000006_0001
Figure imgf000006_0002
Figure imgf000007_0001
Figure imgf000007_0002
Figure imgf000007_0003
Figure imgf000007_0004
Figure imgf000007_0005
10
Figure imgf000008_0001
In one embodiment of the invention, the phosphine complex is selected from the group consisting of
Figure imgf000008_0002
Figure imgf000008_0003
Figure imgf000008_0004
Figure imgf000008_0005
In another embodiment of the invention the phosphine complex is selected from the group consisting of
Figure imgf000009_0001
Figure imgf000009_0002
Figure imgf000009_0003
Figure imgf000009_0004
In yet another embodiment of the invention, the phosphine complex is selected from the group consisting of
Figure imgf000009_0005
Figure imgf000010_0001
Preferably, the phosphine complex is
Figure imgf000010_0003
According to yet a further aspect of the invention, there is provided a process for the preparation of a phosphine complex of the general formula (I)
Figure imgf000010_0002
in which
M is Pt or Pd; R1 and R2 are independently Ph, 2-pyridyl, 3-pyridyl or 4-pyridyl; the two Y constituents are independently CH2CH2 or CH=CH; and X is hexafluorophosphate, halogen or pseudo-halogen, the process including
reacting
Figure imgf000010_0004
in which
R1 and R2 are independently Ph, 2-pyridyl, 3-pyridyl or 4-pyridyl, and Y is CH2CH2 Or CH=CH, with M 1 2 M2 HaI4 in which M1 is an alkali metal, M2 is Pd or Pt and Hal is Cl or Br to produce an intermediate compound of formula (I) in which X" is Cl" or Br".
The process may include reacting the intermediate compound of formula (I) with a hexafluorophosphate salt to produce the compound of formula (I) in which X" is PF6 ".
In one embodiment of the invention, M1 is Na, M2 is Pd and Hal is Cl so that IvT2M2HaI4 is Na2PdCI4.
Preferably R1 and R2 are both 2-pyridyl.
Preferably, the process includes reacting M1 2 M2 HaI4 in which M2 is Pd with
Figure imgf000011_0001
in which Y is CH2CH2.
The hexafluorophosphate salt may be NH4PF6.
The invention will be further described in more detail by way of the following drawings, Example and Studies. In the drawings:
Figure 1 shows a synthetic route in accordance with the invention for the preparation of bis(1 ,2-bis(di-2-pyhdyl)phosphino)ethane-P,P')-palladium(ll)
(hexafluorophosphate), i.e. [Pd(d2pyrpe)2][PF6]2 or Pg 8.
Figure 2 shows an Ortep representation of bis(1 ,2-bis(di-2- pyridyl)phosphino)ethane-P,P')-palladium(ll) (hexafluorophosphate), i.e. Pg 8, with PF6 " ions omitted for clarity.
Figure 3 shows graphs of percentage of cells undergoing apoptosis and necrosis after treatment with compounds of the invention and reference gold compounds for 18 (a), 24 (b) and 48 hours (c), and also for cells incubated with camptothecin for 6 hours (d). The data is presented as a mean of three experiments.
Figure 4 shows contour diagrams of FITC-Annexin V/PI flow cytometry of Jurkat cells cultured for 48 hours with Pg 8 of the invention, a gold reference compound and camptothecin. One representative experiment is shown. Diagram (a) is untreated, diagram (b) is Pg 8 (0.71 1 μM), diagram (c) is Pg 8 (1 .422 μM), diagram (d) is [Au(dppe)2]CI (0.131 μM), diagram (e) is [Au(dppe)2]CI (0.262 μM) and diagram (f) is camptothecin (1 μM).
Figure 5 shows representative histograms and graphs showing changes in the cell cycle profiles (G0/Gi , S and G2/M phases) after 18, 24 and 48 hours for untreated Jurkat cells.
Figure 6 shows representative histograms and graphs showing changes in the cell cycle profiles (G0/Gi, S and G2/M phases) after 18, 24 and 48 hours for 0.71 1 μM of Pg 8 on cell cycle of Jurkat cells.
Figure 7 shows representative histograms and graphs showing changes in the cell cycle profiles (G0/Gi, S and G2/M phases) after 18, 24 and 48 hours for 1 .422 μM of Pg 8 on cell cycle of Jurkat cells. Figure 8 shows representative histograms and graphs showing changes in the cell cycle profiles (G0/Gi, S and G2/M phases) after 18, 24 and 48 hours for 0.131 μM of [Au(dppe)2]CI on cell cycle of Jurkat cells.
Figure 9 shows representative histograms and graphs showing changes in the cell cycle profiles (G0/Gi, S and G2/M phases) after 18, 24 and 48 hours for 0.262 μM of [Au(dppe)2]CI on cell cycle in Jurkat cells.
Figure 10 shows a comparison of cell cycle progression of untreated Jurkat cells and those treated with Pg 8 (0.71 1 and 1.422 M) and [Au(dppe)2]CI (0.131 and 0.262 μM) after 18, 24 and 48 h. Differences in G0/Gi , S and G2/M phases of untreated and treated cells were compared by 2-way ANOVA. Significance was established at P<0.05.
Figure 1 1 shows graphs of mean body weight changes of control and treated mice in phase 1 of Study 4.
Figure 12 shows graphs of mean body weight changes of control and treated mice in phase 2 of Study 4.
Figure 13 shows a graph of AST levels of untreated mice and mice treated with [Au(dppe)2]CI (3 μmol/kg) and Pg 8 (3 and 6 μmol/kg) in Phase 1 of Study 4.
Figure 14 shows a graph of AST levels of untreated mice and mice treated with
[Au(dppe)2]CI (6 μmol/kg) and Pg 8 (12 and 15 μmol/kg) in Phase 2 of Study 4.
Figure 15 shows GGT levels of untreated mice and mice treated with [Au(dppe)2]CI (3 μmol/kg) and Pg 8 (3 and 6 μmol/kg) in Phase 1 of Study 4.
Figure 16 shows GGT levels of untreated mice and mice treated with [Au(dppe)2]CI (6 μmol/kg) and Pg 8 (12 and 15 μmol/kg) in Phase 2 of Study 4. Figure 17 shows Creatinine levels of untreated mice and mice treated with [Au(dppe)2]CI (3 μmol/kg) and Pg 8 (3 and 6 μmol/kg) in Phase 1 of Study 4.
Figure 18 shows Creatinine levels of untreated mice and mice treated with [Au(dppe)2]CI (6 μmol/kg) and Pg 8 (12 and 15 μmol/kg) in Phase 1 of Study 4.
Figure 19 shows comparison of distribution of [198Au(dppe)2]CI (5 and 10 μM) and [103Pd(d2pyrpe)2][PF6]2 (5 and 10 μM) in Jurkat cells after exposure for 1 h. The results shown here were obtained from an average of 8 different cell populations and the values are means ±SEM.
Figure 20 shows the biodistribution of selected organs of adult male Sprague Dawley rats as expressed as a percentage of injected dose per gram of [198Au(dppe)2]CI and [103Pd(d2pyrpe)2][PF6]2.at 6 h. Illustrated values are means ±S.E.M.
Example
Solvents were distilled from sodium/benzophenone ketyl or calcium hydride and degassed. NMR spectra were recorded in c/-dimethylsulphoxide at 298 K using the following Bruker instruments; AVANCE 300 and ARX 300 (1 H, 300.1 MHz; 13C, 75.5 MHz; 31 P, 121 .5 MHz) and referenced internally to residual solvent resonances (data in δ) in the case of 1H and 13C NMR spectra. The 31P spectra were referenced externally to 85% H3PO4. 13C and 31P NMR spectra were proton decoupled. First order analysis was used to assign the spectra. Elemental analysis (empirical formulae shown) was determined by the Institute for Soil, Climate and Water, Pretoria. Fast atomic bombardment mass spectra were recorded with a VG70SEQ by micromass at the University of the Witwatersrand. Melting points were recorded in unsealed capillaries and are uncorrected.
Pg 8 was prepared according to the synthetic route shown in Figure 1. The first step was carried out under an argon atmosphere, using standard Schlenk techniques as the ligand is known to decompose slowly in the presence of oxygen. Pg 8 was prepared from the intermediate (bis(1 ,2-bis(di-2-pyhdylphosphino)ethane palladium) dichloride, [Pd(d2pyrpe)2].2CI. 1 ,2-bis(di-2-pyridylphosphino)ethane (540 mg, 1 .342 mmol) was added to a solution of disodium palladium tetrachloride, Na2PdCI4, (200 mg, 0.680 mmol) in tetrahydrofuran. The reddish-brown solution turned to deep yellow after 20 minutes of stirring. The resultant mixture was stirred for 12 hours at room temperature to yield a yellow precipitate. Filtration was carried out and the remaining solid was dried in vacuo. Yield (crude): 430 mg.
1H NMR (dimethylsulphoxide, 300 MHz): δ la2.93 [broad d (2JPH= 21 .35 Hz), bridging
CH2, 4H], b3.01 (broad s, bridging CH2, 8H), b7.53 (m, Ph, 16H), a7.61 (m, Ph, 4H), b7.93 (m, Ph, 8H), a7.99 (m, Ph,
4H), a8.1 1 (m, Ph, 4H), b8.33 (m, Ph, 8H), a8.76 (m, Ph, 4H)
In the aforegoing, 1a signifies peaks for the mono-chelated compound while b shows those peaks that belong to the bischelated compound.
31P NMR (dimethylsulphoxide, 121 .5 MHz): δ bischelate complex (64.0, s) and monochelated complex (70.4, s).
A solution of ammonium hexafluorophosphate, NH4PF6, (70 mg, 0.429 mmol) in acetone (5 ml) was added to a stirred solution of [Pd(d2pyrpe)2]CI2 (220 mg, -0.224 mmol) in acetone (40ml). On addition of ammonium salt, the cloudy mixture dissolved slightly and immediately turned from yellow to white. The mixture was stirred for 1 hour followed by filtration over celite 545. The colourless filtrate was reduced in vacuo to ~3ml and ether (20ml) was added to facilitate further precipitation. The pale yellow precipitate was washed with ether (3 x 5ml). It was then dried in vacuo. Yield: 100 mg (37 %)
1H NMR (dimethylsulphoxide, 300 MHz): δ 2.85 (broad s, bridging CH2, 8H), 7.46 (m, Ph, 8H), 7.67 (m, Ph, 8H), 7.74 (m, Ph, 8H), 8.42 (m, Ph,
8H). 31P NMR (dimethylsulphoxide, 121 .5 MHz): δ 64.0, s, -143 (spt, 1 JPF = 71 1 Hz, PF6 "). 13C NMR (dimethylsulphoxide, 75.5 MHz): δ 25.8 (s, bridging CH2, 4C), 126.5 (p-pyr,
8C), 132.1 (d, o-pyr, 8C), 137.2 (m-pyr, 8C), 151 .0 (CN, 8C). Ipso-C not observed.
Analysis calculated for C44H40N8P6Fi2Pd: C, 44.00; N, 9.33; H, 3.36. Analysis found: C, 44.26; N, 9.32; H, 3.31 %.
MS-FAB: 1054 M+- PF6; 910 M+-2PF6.
Colourless single crystals of Pg 8 were obtained from DMF solution by slow diffusion of ether. The molecular structure of the compound is illustrated in Figure 2 and crystal data is presented in Table 1 .
Pg 8 crystallises in the non-centrosymetric space group Pnna with half a molecule of the palladium complex and two ions of PF6 in the asymmetric unit. The two PF6 ions are disordered. One has only the equilateral fluoride atoms disordered in a general position while the second has both axial and equilateral fluoride atoms disordered also in a general position but resulting in distorted octahedral geometry. Connection of molecules in the crystal is mainly through C-H... F intermolecular interactions.
Packing in the crystal is such that the solvate molecules are sitting in the voids between the palladium complex molecules with the less disordered ions of PF6 being on the same axis as Pd atoms while the more disordered PF6 ion is positioned between the Pd complexes. In compound Pg 8, the conformations of the ligands are such that the pyridyl rings get positioned more above and below the coordination plane. Two pyridyl nitrogens, one on each side of the coordination plane are just over 3A from the Pd. Table 1 : Crystal Data for [Pd(d2pyrpe)2][PF6]2
Figure imgf000017_0001
Study 1
The in vitro cytotoxic potency of 6 compounds in accordance with the invention and [Au(dppe)2]CI were investigated on various cells.
The following reagents were used:
■ Supplemented cell culture medium - Cell culture medium with 10% foetal calf serum (FCS). ■ Phosphate buffered saline (PBS).
■ DMSO (Sigma-Aldrich, Germany)
■ MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide] (Sigma Diagnostics Inc)
■ Phytohaemagglutinin (PHA) ■ Test compounds (6 and [Au(dppe)2]CI) - dissolved in DMSO (20 mM) and diluted in cell culture medium. Various human and murine cancer cell lines (sources are indicated in parentheses) were grown as monolayer cultures at 37 °C in 5% CO2 in appropriate tissue culture medium (indicated in parentheses) supplemented with 10% v/v heat- inactivated serum (FCS) and 1% penicillin-streptomycin.
The following cancerous cell lines were used:
■ HeLa- human adenocarcinoma of the cervix (ATCC) (EMEM)
■ A2780- human ovarian cancer (EACC) (RPMI)
■ A2780-cis- human ovarian cancer-cisplatin resistant (EACC) (RPMI) ■ MCF-7- human breast cancer (ATCC) (DMEM)
■ CoLo 320 DM- human colon cancer (ATCC) (RPMI)
■ DU 145- human prostate cancer (ATCC) (RPMI)
■ Jurkat- human T-cell line (NRBM) (RPMI)
■ Novikoff- rat hepatocellular cancer (DKFZ) (RPMI) ■ B16- mouse melanoma (ATCC) (RPMI)
Normal cells included:
■ Human lymphocytes (resting and PHA stimulated)- fresh venous blood was obtained from healthy volunteers followed by isolation of lymphocytes. ■ Chicken embryo fibroblasts- cells were isolated from chicken embryos.
■ Porcine hepatocytes- obtained from the Bio-artificial liver project, Department of Internal Medicine, University of Pretoria.
Sample preparation
All the complexes were dissolved in DMSO to give a stock concentration of 20 mM (stored at -70°C). Immediately before the cell experiments, the stock solution was diluted in appropriate growth medium (containing 10% FCS) to give final DMSO concentrations not exceeding 0.5% and drug concentrations of 0.003-100 μM.
General procedure
The assays were performed using a metabolic assay based on the reactivity of MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide]. MTT is a pale yellow substance that is metabolised to dark blue formazan crystals by metabolically active cells. The amount of formazan produced is directly proportional to the amount of cells over a wide range.
80 μl of medium (60 μl in the case of the lymphocytes earmarked to be stimulated) was dispensed into each well of a 96 well tissue culture plate (micro-titer plate). 100 μl of cell suspension (2 x 104 - 2 x 106 cells/ml/well depending on the cell type) was added and then allowed to incubate for 1 hour at 37QC in an atmosphere of 5% CO2 20 μl of the experimental drug (eight varying concentrations) was added in triplicate to the wells. However, control wells received 20 μl of growth medium instead of the experimental drug. Lymphocytes earmarked to be stimulated received 20 μl PHA 5 minutes after the addition of the drug.
After the incubation period (3 and 7 days for lymphocytes and cancerous cells respectively), 20 μl MTT (5 mg/ml) was added to each well and cultures were incubated for 4 hours. Cells were then centrifuged for 10 min at 2000 rpm (800 g)
(Beckman TJ 6 centrifuge) and the supernatant removed without disturbing the pellet.
The cells were washed with 150 μl PBS (per well) and centrifuged for 10 min at 2000 rpm (800 g) (Beckman TJ 6 centrifuge). The supernatant was removed and plates were left to dry in the dark. 100 μl DMSO was added to each well to solubilise the formazan crystals and the plates were shaken for 2-4 hours. Culture plates were read on a plate reader using a wavelength of 570 nm and a reference wavelength of 630 nm.
Statistical analysis
Percentage survival (percentage of the relevant untreated control systems) was calculated and this value was used to determine the IC5O value (IC5O = the concentration (μM) of the experimental compound inducing a 50% decrease in cell growth).
The IC5O value calculations were performed with the Graphpad™ program and are tabulated below in Table 2. Table 2: IC50 values (μM) and S. E. M (±) of 7 compounds on various cancer cell lines as well as normal cells. The values are presented as a mean of three experiments carried out in triplicate.
Figure imgf000020_0001
In general the platinum complexes were less toxic than the palladium ones with Pg 1 being the least toxic compound followed by Pg 6 and Pg 5 { Table 2). Pg 8 was the most toxic of the palladium complexes followed by Pg 4a and Pg 3. With regard to all the novel complexes, prostate cancer (DU-145) was the most sensitive cell line (IC5O values of 0.253-9.380 μM) while ovarian cancer-cisplatin resistant cell line (A2780-cis) was the most resistant (IC5O values of 2.029-> 50 μM). Human ovarian cancer cell line- cisplatin sensitive (A2780) was sensitive to all the palladium complexes with IC50 values of 0.617 μM (Pg 3), 0.136 μM (Pg 4a) and 0.974 μM (Pg 8). Overall, Pg 8 was the most toxic (over the whole range of cells) of the novel compounds followed by Pg 4a which showed comparative activity against Jurkat and MCF-7 cell lines.
[Au(dppe)2]CI was more toxic than the novel compounds { Table 2). However, it exhibited non-selectivity as it was also toxic to both resting (IC5O = 0.903 μM) and stimulated lymphocytes (IC50 = 0.182 μM) while the experimental compounds were not toxic even at a concentration of 100 μM.
As indicated in Table 2, [Au(dppe)2]CI showed high potency, but non- selectivity. Pg 4a was the most selective with IC50 values of porcine hepatocytes and chicken embryo fibroblasts of 31 .08 and 2.379 μM respectively. Pg 8 gave comparable IC50 values among the cells with the lowest being 0.654 (Novikoff) and the highest being 1.755 (porcine hepatocytes).
Pg 8 was selected for further tests in an effort to elucidate its mechanism of action. It was selected due the fact that it showed greater toxicity to a wider range of cancer lines than the rest of the invention compounds. [Au(dppe)2]CI was included in these investigations as a standard. Jurkat cells were used for the remainder of the assays as they could be compared to lymphocytes.
Study 2
The aim of this assay was to evaluate the induction of apoptosis and/or necrosis by [Au(dppe)2]CI and Pg 8 on Jurkat cells with the Annexin V binding assay. Untreated and treated cells were evaluated for apoptosis by flow cytometry. Camptothecin (1 μM), a topoisomerase I inhibitor that efficiently induces apoptosis in Jurkat cells was used as a positive control.
The following reagents were used:
■ RPMI and Foetal Calf Serum.
■ Phosphate buffered saline (PBS).
■ Binding buffer (238 mg Hepes, 876 mg NaCI, 37.3 mg KCI, 26.5 mg CaCI2, 9.5 mg MgCI2 in de-ionised water, at pH 7.4) ■ Annexin V- FITC ( BD Biosciences Pharmingen)
■ Propidium Iodide (Pl) (Sigma-Aldrich)
■ Camptothecin (1 μM) ( Sigma-Aldrich)
■ Pg 8 (0.71 1 and 1 .422 μM)
■ [Au(dppe)2]CI (0.131 and 0.262 μM)
The human Jurkat cell line was cultured in RPMI 1640 supplemented with 10% v/v heat-inactivated FCS and 1 % penicillin-streptomycin. Cells were maintained in a humidified atmosphere of 5% carbon dioxide at 37 °C.
Flow cytometric analysis of apoptosis
Cells (1 x 105 cells/ml) were treated with [Au(dppe)2]CI and Pg 8 for 18, 24 and 48 h in cell culture flasks. After the incubation period, cells were decanted from flasks and centrifuged for 5 min at 200 g. The cell pellet was washed with PBS (1 % FCS) and resuspended in 1 ml binding buffer. 100 μl of cell suspension was transferred to flow cytometer tubes. 5 μl of Annexin V-FITC and 10 μl propidium iodide were added to some tubes (unstained samples were also prepared). The cell suspensions were mixed gently and incubated for 15 min in the dark at room temperature (25 °C). 400 μl of binding buffer was then added to each tube and analysis was carried out within an hour with a flow cytometer (Beckman Coulter FC 500). Statistical methods
All assays were performed at least three times and results are presented as ± S. E. M. The statistical evaluation of the results was performed by two way ANOVA followed by Bonferroni's Multiple Comparison Test. Significance was established at P<0.05.
Figure 3 shows graphs indicating the percentages of cells undergoing various stages of apoptosis. Figure 4 shows actual histograms of treated and untreated cells undergoing apoptosis (48 h). After incubation of Jurkat cells with the compounds for 18 and 24 h, no significant differences were noted between the untreated and the treated groups as between 85 and 90% of cells were viable. However, after exposure of the cells for 48 h, significant differences were observed. Untreated cells had an average of 85% of cells in a viable state and only 8% at the early apoptotic stage. [Au(dppe)2]CI (0.131 and 0.262 μM) induced apoptosis in 12% of the cells (9% in early apoptosis and 3% in the stage of late apoptosis) while 77% of the cells were still viable.
In contrast, Pg 8 (0.71 1 and 1 .422 μM) significantly induced apoptosis in 25% of the cells while 14% of cells had undergone necrosis. Only 50% of the cells were viable in this group. Unlike Pg 8 and [Au(dppe)2]CI, camptothecin (1 μM) induced apoptosis in Jurkat cells within a short time. After incubation for 6h, about 40% of the cells were in the late apoptotic stage. Viable cells were just over 50% while only 2% of the cells were necrotic. DNA topoisomerase I and Il inhibitors induce apoptosis in various cell lines and this is due to DNA-protein complex formation stabilised by DNA topoisomerase I inhibitors that ultimately signal the onset of apoptosis.
The two compounds behaved differently as observed from the histograms (Figure 4). [Au(dppe)2]CI treated group had more viable cells (77%) and very few in the necrotic stage (~4 %) while Pg 8 had a lower percentage of viable cells (50%) and most notably a much higher percentage of them in the necrotic stage (14%). This difference is significant as cytotoxicity assays showed that the former drug was more toxic (IC50 = 0.131 μM) while Pg 8 was at least 5 times less toxic (IC5O = 0.71 1 μM) to Jurkat cells. The mode of cell killing differs as [Au(dppe)2]CI mainly induced apoptosis while Pg 8 seemed to induce both apoptosis and necrosis. While the former compound was toxic to lymphocytes (IC50 = 0.903 μM), Pg 8 was not cytotoxic even at 100 μM. This selectivity for cancer cells by the latter compound may be due to this difference in induction of cell death (necrosis vs. apoptosis). However, it is not clear which factors play a role in triggering an apoptotic or necrotic pathway in the different cells (lymphocytes vs. Jurkat cells).
Study 3
Study 2 has shown that Pg 8 and [Au(dppe)2]CI caused Jurkat cells to undergo apoptosis after exposure for 48 h. The aim of this study was to determine if cell death was as a result of cell cycle arrest by these compounds. Cell cycle analysis was performed using flow cytometric evaluation of DNA content.
The following reagents were used: ■ RPMI and foetal calf serum
■ RNase ( Sigma-Aldrich)
■ Propidium Iodide (Pl) ( Sigma-Aldrich)
■ Phosphate buffered saline (PBS)
■ Ethanol (100%) (Sigma-Aldrich) ■ Pg 8 (0.71 1 and 1 .422 μM)
■ [Au(dppe)2]CI (0.131 and 0.262 μM)
The human Jurkat cell line was cultured in RPMI 1640 supplemented with 10% v/v heat-inactivated FCS and 1 % penicillin-streptomycin. Cells were incubated with the experimental compounds for 18, 24 and 48 hours at 37 °C and 5% carbon dioxide.
Flow cytometric analysis of cell cycle progression
Cells (1 x 105 cells/ml) were treated with [Au(dppe)2]CI and Pg 8 for 18, 24 and 48h in cell culture flasks. After the incubation period, they were decanted from flasks and centrifuged for 5 min at 200 g. The cell pellet was re-suspended in 500 μl PBS and chilled on ice. The cold cell suspension was then added rapidly to flow cytometer tubes containing 500 μl of ice cold ethanol. Mixing was carried out by forcing air bubbles through the suspension and then kept on ice for 15 minutes. The cells were then centrifuged for 3 minutes at 300 g followed by suspension of the cell pellet in 125 μl of RNAse (2mg/ml 1 .12% w/v sodium citrate). After incubation for 15 minutes at 37 °C in a water bath, 125 μl of Pl was added to each tube and mixed well. Samples were allowed to stand for 30 minutes at room temperature before analysis by flow cytometry (Beckman Coulter FC 500). DNA histograms were collected and estimation of the percentages of cells in Gi , S and G2/M was performed with a computer software program (Multicycle, Phoenix Flow systems, San Diego, CA).
Statistical methods
All assays were performed at least five times and all data are presented as ± S. E. M. Differences in the cell cycle profiles (G0/G1, S and G2/M phases) of untreated and treated cells were also compared by ANOVA (2-way) followed by Bonferroni's Multiple Comparison Test. Significance was established at P<0.05.
Representative histograms and graphs showing changes in the cell cycle profiles (G0/Gi, S and G2/M phases) after 18, 24 and 48 hours with or without exposure to the experimental compounds are shown in Figures 5 to 9.
At 24 h, there was a decrease in the number of cells in Gi phase (from 63 to 58%), which was complimented by a slight increase in the number of cells in the S- phase (from 29 to 38 %). At 48 h, there was a decrease in the S-phase (from 38 % to 35 %) and a marked increase in the G2 phase (from 4 to 1 1 %). The observed changes in the cell cycle progression of untreated cells were used to determine if any changes had occurred in the treated samples (Figures 6-9).
Cells exposed to 0.71 1 μM of Pg 8 for 18 and 24 h did not exhibit any differences in cell cycle progression (Figure 6). After 48 h, the number of cells in Gi was significantly reduced (from 61 to 47%) with the subsequent accumulation of cells in
S-phase (from 36 to 57%). Due to this blockade of the S-phase, G2 phase contained virtually no cells (1 %). Cells exposed to 1.422 μM of Pg 8 (Figure 7) showed the same behaviour as the ones exposed to half the concentration. After 48 h, the number of cells in Gi was significantly reduced (from 62 to 47 %) with the subsequent accumulation of cells in S- phase (from 36 to 58%). There were no cells in the G2 phase.
After exposure of Jurkat cells to [Au(dppe)2]CI (0.131 μM) for 18 h and 24 h, no significant changes to progression of cell cycle were observed (Figure 8). However, significant changes were observed after 48h. The number of cells in Gi decreased from
68 to 61 % while there was an increase in S-phase (from 28 to 34%). The number of cells in G2 phase increased insignificantly (from 4 to 5%).
Cells exposed to 0.262 μM of [Au(dppe)2]CI showed the same pattern as those exposed to half the concentration (Figure 9). At 48 h, the number of cells in Gi decreased from 69 to 64 % while S-phase increased from 28 to 34%. G2 phase showed an insignificant decrease (from 4 to 3%).
The DNA histogram yields the relative number of cells in G1ZG0, S, and G2/M phases of the cell cycle. Although some information about cell cycle progression can be deduced by following changes in the cell cycle phases with time, it gives static information. For example, although it is possible to estimate the percentage of cells in S phase, the measurement does not directly tell whether those cells are still moving through the S phase. A very distinct connection has been forged between the cell cycle clock apparatus and apoptosis. The paclitaxel-induced apoptosis of human breast cancer cells has been found to depend upon the induction of the cdc2 kinase at the G2/M phase transition of the cell cycle.
Comparison of untreated and treated cells at different time intervals showed significant changes (Figure 10). At 18 h, cells treated with Pg 8 (0.71 1 and 1 .422 μM) showed an increase of cells (P<0.05) in the G0/Gi phase but there was no complimentary decrease in the number of cells in either the S or G2 phase. After 24 h, only cells treated with [Au(dppe)2]CI (0.130 and 0.262 μM) showed any changes in cell cycle progression. There was a significant increase (P<0.01 ) in the number of cells in the G0/G1 phase with a concomitant decrease in the number of cells in the S-phase. After 48 h, the treated cells showed significant differences in the cell cycle sequence. Cells treated with [Au(dppe)2]CI (0.262 μM) had significantly larger number (P<0.05) of cells in G0/Gi phase than the untreated cells. However, this difference was not as large as that observed at 24 h. It is probable that [Au(dppe)2]CI induced apoptosis was not necessarily as a result of cell cycle arrest.
In contrast, cell cycle progression at 48 h was greatly perturbed by Pg 8. The number of cells in Gi phase was reduced by 15% while those in the S-phase increased by 22% at both concentrations (P<0.01 ). There were no cells detected in the G2 phase due to this blockade in the S-phase. Taken together, these results indicate that Pg 8 inhibited cellular proliferation of Jurkat cells via an S-phase arrest of the cell cycle following exposure for 48 h.
Study 4
This study was carried out in order to establish the safe dosage or maximum tolerated dose (MTD) of Pg 8 in mice.
Ethical approval by the AUCC (Animal Use and Care Committee) was obtained (Protocol No. H1706). The study was conducted at the University of Pretoria Biomedical Research Centre (UPBRC) from 24-29 August 2006 and 22-27 September 2006.
Inbred female Balb/C mice of 6-8 weeks were used and housed in standard mouse cages in rooms with controlled environmental conditions. The animals were fed normal pellets (EPOL (trade name)) and water ad libitum.
The study was carried out in two phases with 4 groups of 6 mice each being used per phase (total of 48 mice). The MTD of [Au(dppe)2]CI was previously shown to be 3.0 μmol/kg/day for 5 days and hence this concentration was used as a guideline. The weight of the mice was determined to adapt the dosages according to their body weight. The experimental compound Pg 8 and [Au(dppe)2]CI (standard) were dissolved in analytical quality ethanol, with subsequent addition of water to make up a final dose volume of 0.5 ml and a concentration of 5% ethanol. The dosages were prepared immediately prior to each ip. administration. [Au(dppe)2]CI was soluble in ethanol, while Pg 8 (15 μmol/kg) was sparingly soluble. Solubility was achieved by sonic bath solvation for 15 minutes. Both compounds precipitated out slightly and were mixed prior to injection.
Tables 3 and 4 below summarise the number of animals and dosages used in both phase 1 and phase 2.
Figure imgf000028_0001
Figure imgf000028_0002
The total duration of the study was 10 days and each group of mice was injected ip. each day for 5 days. For phase 1 , the ethanol-water solution and the first and second dosages of 3 μmol/kg and 6 μmol/kg were administered each day from day 1 to 5. Phase 2 followed whereby the ethanol-water solution and the third and fourth dosages of 12 μmol/kg and 15 μmol/kg were administered each day from day 1 to 5. At the end of the study, the mice were weighed followed by anaesthetisation via isoflurane inhalation. While they were at the surgical plane of anaesthesia, maximum blood was drawn via cardiac puncture. The animals were then further exposed to isoflurane until death occurred. Mice in phase 2 were dissected and major organs (liver, heart and kidneys) were weighed.
Body weights were recorded immediately prior to dosing (on day 1 ) so that an objective monitoring of weight could be done, indicating food intake, which is a good measure for animal well-being. The animals were also monitored for pain and stress (behavioural changes) immediately after the injection.
A whole blood profile was done on all the blood samples by the Department of Clinical Pathology at the Faculty of Veterinary Science (University of Pretoria). Standard liver enzymes (Aspartate aminotransferase (AST) and Gamma glutamyltransferase (GGT)) were also analysed, as well as levels of serum creatinine. Toxicity was to be determined if adverse effects were observed on the experimental animals or with elevated liver enzymes.
Statistical analysis on levels of AST, GGT and creatinine was done on
Graphpad™ by One Way Analysis of Variance (ANOVA) followed by Bonferroni's Multiple Comparison Test. Significance was indicated if P < 0.05.
The acute toxicities of [Au(dppe)2]CI and Pg 8 were examined in mice after ip. administration for 5 days. On the second day of the study (in both phases), mice injected with the former compound made squeaking sounds upon injection. This sign of discomfort was not observed for the remainder of the study. The most significant difference between the groups treated with [Au(dppe)2]CI and the rest of the groups was that the former batch had a greatly reduced weight gain. They lost an average of 7.6% of their total body weight in phase 1 and 12.4% in phase 2. The groups treated with Pg 8 showed similar weight gain to the control group in phase 1 (average of 2.9%) but a reduced weight gain in phase 2 (average of 1 % vs. 4.7 % in control group). Figures 1 1 and 12 show the weight changes in phase 1 and 2 respectively.
Immediately after completion of phase 2, the major organs were surgically removed and weighed. The results are tabulated in Table 5.
Figure imgf000030_0001
No significant gross pathologic differences were observed between the control and treated groups. The average heart and kidney weights did not differ significantly among all the groups but the mean liver weights of the mice exposed to [Au(dppe)2]CI were greater than the rest. This could be as a result of highly reduced body weight gain. No significant difference in full blood counts was observed as haematological parameters between the untreated and treated groups did not vary.
Analysis of liver enzyme AST, showed that there were no significant differences in their levels in all the groups (untreated and treated) in Phase 1 (Figure 13). However, in Phase 2, the mice treated with [Au(dppe)2]CI (6 μmol/kg) had significantly elevated levels of AST (P<0.001 ). These levels of AST were significantly higher than those found in the untreated and Pg 8 (12 and 15 μmol/kg) treated mice (Figure 14).
GGT levels shown in Figure 15 were also slightly elevated (P <0.05) in the group treated with [Au(dppe)2]CI (3 μmol/kg) when compared to the untreated mice. However, the levels did not differ significantly with the mice treated with Pg 8 (12 and 15 μmol/kg). In Phase 2 of the study, GGT levels did not show significant differences in all the groups (untreated and treated). Notably, the mice treated with 12 μmol/kg of Pg 8 exhibited higher levels of GGT than the highest dose (15 μmol/kg) (Figure 16). However, this difference was not significant.
Creatinine levels (Figure 17 and 18) in all the groups did not vary significantly. This is an indication of lack of toxicity to the kidneys.
No significant differences in full blood counts were observed as haematological parameters between the untreated and treated groups did not vary.
In conclusion, [Au(dppe)2]CI showed greater in vitro activity against cancer cells than Pg 8. It was also very toxic to the normal cells such as lymphocytes, chicken embryo fibroblasts and hepatocytes. This lack of selectivity is exhibited in vivo as the acute exposure to mice caused reduced body weight. Measurement of biochemical parameters revealed that AST levels were greatly elevated which may imply damage to organs. As an intracellular enzyme, AST is released into the blood in proportion to the number of damaged cells. While the former drug may show potency against cancer cell lines, its lack of selectivity is dose-limiting. As mentioned earlier, it was also active in tumour implanted mice.
Pg 8 was more tolerable although the group treated with the highest dose (15 μmol/kg) showed slightly reduced weight gain as compared to the rest (3, 6 and 12 μmol/kg). The desired outcome is that the compound shows in vivo anti-tumour activity especially in distal tumours. This would be determined by good bio-availability which is influenced by factors such as stability against metabolic, pathways, adsorption, plasma level concentrations and excretion rates. Study 5
Uptake of [103Pd(d2pyrpe)2][PF6]2 and [198Au(dppe)2]CI by Jurkat cells
Reagents
■ RPMI and foetal calf serum
■ Phosphate buffered saline (PBS)
■ 103Pd labelled [Pd(d2pyrpe)2][PF6]2 (Pg 8)- 5 and 10 μM in DMSO ■ 198Au labelled [Au(dppe)2]CI- 5 and 10 μM in DMSO
Cell lines and culture
Human T-cell lines (Jurkat) were cultured in RPMI 1640 supplemented with 10% v/v heat-inactivated FCS and 1 % penicillin-streptomycin. The cell culture was maintained at 37 0C and 5% CO2.
Experimental procedure
200 μl of radiolabeled Pg 8 and [Au(dppe)2]CI (both at a concentration of 5 and 10 μM) were added to test tubes containing a suspension (1 ,800 μl) of Jurkat cells (5 x 106 cells/ml). The mixture was incubated while mixing at 37 °C (+ 5% CO2) for 1 hr. After incubation, the cells were centrifuged at 800 g (2000 rpm TJ 6) for 10 min. The supernatant was poured into new, clean, marked 5 ml test tubes (kept separately). The pellet was resuspended in PBS (3 ml) and centrifuged as before. The PBS was poured into new, clean, marked 5 ml test tubes and keep separately. The tubes were counted separately using 1261 multigamma manual gamma counter (LKB Wallac).
Statistical methods
Cells from eight different cell culture flasks were used for these assays. The activity that was obtained from the experiments was determined from counts per minute (CPM). Hence this represented the amount of compound distributed either in the supernactant or cell. Statistics was carried out on Graphpad™ and One Way Analysis of Variance (ANOVA) followed by Bonferroni's Multiple Comparison Test to determine significance between the two experimental compounds in the cells and/or supernactant. P< 0.001 was considered significant.
In general, there was a significant difference (P< 0.001 ) between the amounts of compound taken up into the cells when compared to the amount left in the supernactant for both complexes (Figure 19). After incubation for 1 h, [198Au(dppe)2]CI (5 and 10 μM) was taken up into the cells at significantly larger amounts (P< 0.001 ) than [103Pd(d2pyrpe)2][PF6]2 (5 and 10 μM). This large difference in uptake may be as a consequence of lipophilicity. The former compound was found to be more lipophilic (log P= 0.362) than the former (log P= -0.093) and hence able to pass through the lipid layers of cells more rapidly.
In both compounds, the higher concentration (10 μM) was found to accumulate in larger amounts (P< 0.001 ) than the lower concentration (5 μM). Uptake studies of [14C][Au(dppe)2]CI by isolated rat hepatocytes showed that rapid maximal uptake took place within 30 min. The amount of radiolabeled drug associated with hepatocytes was also concentration-dependent. The authors proposed that the mechanism by which [Au(dppe)2]CI gained access to the intracellular compartments was most likely linked to its lipid solubility. In other experiments, NMR studies showed that nearly half of the [Au(dppe)2]CI added to plasma was transferred into cells.
Experiments on red cell ghosts confirmed that the complex can bind intact in the membrane.
Study 6
Biodistribution of [103Pd(d2pyrpe)2][PF6]2 and [198Au(dppe)2]CI in rats
This study was carried out by NECSA at the University of Pretoria Biomedical Research Centre (UPBRC). Animal experimentation was done according to the National
Code for the Handling and Use of Animals in Research, Education, Diagnosis and
Testing of Drugs and Related Substances in South Africa. Ethical approval by the
AUCC (Animal Use and Care Committee) was obtained (Protocol No. H1206) and the study was done according to the rules and regulations laid down by the International Controlling Body for Radioisotope Studies.
Adult male Sprague Dawley rats were used to follow the biodistribution on a gamma camera. Animals were screened from time to time to see when the biodistribution reaches a stable state.
Six rats per compound were obtained from the Onderstepoort facility in South Africa. The animals were kept in separate cages. The animals were fed a balanced diet and water was available ad libitum.
Six rats were injected each with a extremely low, non-toxic dosage of radio- labelled compound (198Au labelled [Au(dppe)2]CI, 103Pd labelled [Pd(d2pyrpe)2][PF6]2). The rats were terminated at the end of each day by an lsoflurane overdose, and the organs counted in a well type gamma counter.
The experimental procedure used was as follows:
140μl dimethylsulphoxide, 800μl EtOH and 3200μl H2O were added to 75 mg Pg 8. The filtered 4000μl fraction had an activity of 3μCi (9:00; 30 November
2006). An injection volume of 450μl and an activity of 0.3μCi was used (7.5X10"4 μCi/μl).
1200μl EtOH and 7200μl H2O was added to 1 1 .43mg [Au(dppe)2]CI. A 3000μl fraction was filtered (Millex GP 0.22 μm). An injection volume of
500μl and an activity of 25μCi was used (5.0X10"2μCi/μl).
On the day of the experiment, the animals were anaesthetised by an intraperitoneal (ip.) injection of a 6 % sodium pentobarbitone solution at a dose of 1 ml/kg. A 24 G jelco was inserted into the tail vein of the animals to administer the radio- labelled compounds. Two animals per group were scanned in parallel with an Elscint Gamma Camera at the Diagnostic Imaging unit at Onderstepoort in order to obtain the radionuclide imaging. Two minute static studies were performed every half an hour up to 6 hours, lsoflurane was used to immobilize the animals for the 2 minute static studies.
The animals were sacrificed using an lsoflurane overdose after a 6 hour period. The organs were separated and counted in a well type counter at NECSA. From the organ counts as well as the reference activity in a syringe the %ID/g (Injected Dose/gram) was calculated.
The biodistribution (Figure 20) showed predominantly high reticulo- endothelial uptake for all compounds. The largest concentration of [Au(dppe)2]CI was found in the lungs followed by the spleen. The highest concentration of Pg 8 was found in the spleen followed by the liver. Hydrophilic drugs are expected to have a limited biodistribution compared to lipophilic drugs. This might imply a more selective tumour uptake.

Claims

CLAIMS:
1. A phosphine complex of the general formula (I)
Figure imgf000036_0001
in which M is Pt or Pd;
R1 and R2 are independently Ph, 2-pyridyl, 3-pyridyl or 4-pyridyl; the two Y substituents are independently CH2CH2 or CH=CH; and X is hexafluorophosphate, halogen or pseudo-halogen.
2. The phosphine complex as claimed in claim 1 , which is selected from the group consisting of
Figure imgf000036_0002
Figure imgf000036_0003
Figure imgf000037_0001
Figure imgf000037_0002
Figure imgf000037_0003
Figure imgf000037_0004
Figure imgf000037_0005
Figure imgf000038_0001
3. The phosphine complex as claimed in claim 2, which is selected from the group consisting of
Figure imgf000038_0002
Figure imgf000038_0003
Figure imgf000038_0004
Figure imgf000038_0005
4. The phosphine complex as claimed in claim 2, which is selected from the group consisting of
Figure imgf000039_0001
Figure imgf000039_0002
Figure imgf000039_0003
Figure imgf000039_0004
5. The phosphine complex as claimed in claim 2, which is selected from the group consisting of
Figure imgf000040_0001
Figure imgf000040_0002
6. The phosphine complex as claimed in claim 2, which is
Figure imgf000040_0003
7. A substance or composition for use in the treatment of cancer, the substance or composition including a phosphine complex as claimed in any of claims 1 to 6 inclusive.
8. A pharmaceutical composition which includes a phosphine complex as claimed in any of claims 1 to 6 inclusive, or a pharmaceutically acceptable salt thereof.
9. The use of a phosphine complex as claimed in any of claims 1 to 6 inclusive in the manufacture of a medicament for use in the treatment of cancer.
10. A process for the preparation of a phosphine complex of the general formula (I)
Figure imgf000041_0001
in which M is Pt or Pd;
R1 and R2 are independently Ph, 2-pyridyl, 3-pyridyl or 4-pyridyl; the two Y constituents are independently CH2CH2 or CH=CH; and
X is hexafluorophosphate, halogen or pseudo-halogen, the process including
reacting
Figure imgf000041_0002
in which R1 and R2 are independently Ph, 2-pyridyl, 3-pyridyl or 4-pyridyl, and Y is
CH2CH2 Or CH=CH, with M 1 2 M2 HaI4 in which M1 is an alkali metal, M2 is Pd or Pt and Hal is Cl or Br to produce an intermediate compound of formula (I) in which X" is Cl" or Br".
1 1 . The process as claimed in claim 10, which includes reacting the intermediate compound of formula (I) with a hexafluorophosphate salt to produce the compound of formula (I) in which X" is PF6 ".
12. The process as claimed in claim 10 or claim 1 1 , in which M1 is Na, M2 is
Pd and Hal is Cl so that M1 2 M2 HaI4 is Na2PdCI4.
13. The process as claimed in any of claims 10 to 12 inclusive, in which R1 and R2 are both 2-pyridyl.
14. The process as claimed in any of claims 10 to 13 inclusive, which includes rreeaadcting M 1 2 M2 HaI4 in which M2 is Pd with
Figure imgf000042_0001
in which Y is CH2CH2
15. The process as claimed in claim 1 1 , in which the hexafluorophosphate salt is NH4PF6.
PCT/IB2009/050712 2008-02-22 2009-02-20 A substance or composition for the treatment of cancer WO2009104167A1 (en)

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EP0227127A2 (en) * 1985-10-25 1987-07-01 Nederlandse Organisatie voor toegepast-natuurwetenschappelijk onderzoek TNO Pharmaceutically active phosphino-hydrocarbon-group VIII-metal complexes, antitumor compositions containing these complexes, and a process for preparing said compounds or antitumor compositions
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