WO2012085608A2 - Use of trifluoro phthalimides for the treatment of cancerous diseases - Google Patents

Use of trifluoro phthalimides for the treatment of cancerous diseases Download PDF

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Publication number
WO2012085608A2
WO2012085608A2 PCT/HU2011/000130 HU2011000130W WO2012085608A2 WO 2012085608 A2 WO2012085608 A2 WO 2012085608A2 HU 2011000130 W HU2011000130 W HU 2011000130W WO 2012085608 A2 WO2012085608 A2 WO 2012085608A2
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WIPO (PCT)
Prior art keywords
compound
tumour
treatment
cells
group
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PCT/HU2011/000130
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English (en)
French (fr)
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WO2012085608A3 (en
Inventor
László PUSKÁS
Iván KANIZSAI
Márió GYURIS
Ramóna MADÁCSI
Béla ÓZSVÁRI
Liliána FEHÉR
Gabriella FÁBIÁN
Klára KITAJKA
Original Assignee
"Avidin" Kutató, Fejlesztö És Kereskedelmi Korlátolt
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Publication of WO2012085608A2 publication Critical patent/WO2012085608A2/en
Publication of WO2012085608A3 publication Critical patent/WO2012085608A3/en

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Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/40—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with one nitrogen as the only ring hetero atom, e.g. sulpiride, succinimide, tolmetin, buflomedil
    • A61K31/403—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with one nitrogen as the only ring hetero atom, e.g. sulpiride, succinimide, tolmetin, buflomedil condensed with carbocyclic rings, e.g. carbazole
    • A61K31/4035—Isoindoles, e.g. phthalimide
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A61P35/02—Antineoplastic agents specific for leukemia
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A61P35/04—Antineoplastic agents specific for metastasis

Definitions

  • the present invention relates to compounds that are used for the treatment of cancerous diseases and induce oxidative stress of the endoplasmic reticulum by influencing formation and/or functioning of cellular vesicular systems, especially lipid droplets resulting in destruction of cancerous cells. Furthermore, the present invention also relates to therapeutic compositions containing the compounds listed and to. their use for the treatment of cancerous diseases.
  • the compounds listed in the present invention are able to destroy various tumour cells with surprisingly high efficiency in comparison to the already known compounds described in the Hungarian Patent Application No. P0700433, therefore these can be used for targeted treatment of cancerous diseases.
  • the compounds described in the present patent application proved to be effective in in vivo animal experiments in the treatment of glioma (cerebral tumour) , liver carcinoma, breast tumour and leukemia. Furthermore, the compounds described in the present patent application proved to be effective in the treatment of melanoma in in vivo animal experiments.
  • Protein disulphide isomerase is a multifunctional enzyme playing a role in several known cell processes, including protection of cells against oxidative stress and as a chaperone (Bassuk JA, Berg RA: Protein disulphide isomerase, a multifunctional endoplasmic reticulum protein. Matrix 1989; 9:244-258.) Overfunction of protein disulphide isomerase enzyme in endoplasmic stress has been described and it has been brought into connection with hypoxia, which often occurs in tumour cells (Graven KK, MolvarC, Roncarati JS, KlahnBD, Lowrey S, FarberHW: Identification of protein disulphide isomerase as an endothelial hypoxic stress protein. Am J Physiol Lung Cell Mol Physiol 2002; 282:L996- L1003.) . We assume that the compounds described by us exert their anti-tumour effect in part via the inhibition of protein disulphide isomerase.
  • Fig. 1 a table containing EC50 values of various compounds on tumour cells is shown;
  • Fig. 2 a diagram showing the liver mass index of samples of hepatocellular carcinoma induced with DEN in mouse Matrilin-2 KO model treated with the compound of Example 1 and the compound of Example 4, respectively, and those of the control samples can be seen;
  • Fig. 3 a diagram showing the tumour count of samples of hepatocellular carcinoma induced with DEN in mouse Matrilin-2 KO model treated with the compound of Example 1, and the compound of Example 4, respectively, and those of the control samples can be seen;
  • Fig. 4 a diagram showing the anti-cancer effect of the compound of Example 3 and that of the compound of Example 4 on subcutanous implanted MCF7 human breast tumour can be seen in the case of SCID immunodeficient mice;
  • Fig. 5 a survival curve showing the anti-cancer effect of the compound of Example 4 can be seen in the case of HT168 human melanoma implanted in the spleen of SCID immunodeficient mice;
  • a survival curve showing the anti-cancer effect of the compound of Example 4 can be seen in the case of U87 human glioblastoma implanted in the brain of nude immunodeficient rats;
  • Example 1 a diagram showing the inhibitory effect of the compound of Example 4 on protein disulphide isomerase activity is illustrated.
  • the compound is selected from the following compounds: Example 1 :
  • HT168 human, melanoma
  • HepG2 and Hep3B human hepatocellular carcinoma
  • MCF7 Human breast adenocarcinoma
  • PC3 human prostate cancer
  • A549 human lung carcinoma
  • HT29 human colon tumour
  • K562 and HL60 human leukemia
  • GBMl human glioma
  • HT168 Minimum Essential Medium Eagle (Sigma, St. Louis, MO, USA) , Na-piruvate, Glutamine, Non-essential amino acids, penicillin (50 IU/ml ) -streptomycin (50 mg/ml), 10% foetal cattle serum
  • D-MEM Dulbecco's Modified Eagle Medium
  • high glucose Gibco BRL, Carlsbad, CA, USA
  • penicillin 50 IU/ml
  • -streptomycin 50 mg/ml
  • D- MEM Dulbecco's Modified Eagle Medium
  • Gibco BRL high glucose
  • Nutrient Mixture F-12 Ham Sigma, St. Louis, MO, USA
  • penicillin 50 IU/ml
  • -streptomycin 50 mg/ml
  • 10% foetal cattle serum Dulbecco's Modified Eagle Medium
  • the cells were grown at- 37 °C, in 5% C0 2 atmosphere, and divided into 60 mm, 100 mm plates with trypsin/EDTA (Sigma- Aldrich, St. Louis, MO) treatment every second day. When the cell count sufficient for the experiment planned (app. 10 6 ) , was reached, 50.000 cells were placed in each well of a 24-well plate ( Costar® 24 Well TC-Treated Microplates) with trypsin/EDTA ( Sigma-Aldrich, St. Louis, MO) treatment and allowed them to get adhered by the following day.
  • trypsin/EDTA Sigma- Aldrich, St. Louis, MO
  • the substance was added as follows: the medium was sucked off the cells adhered and a medium containing the compound according to the invention at the proper concentration and mixed with it well was pipetted on the cells (2 ml / well) .
  • the substances to be examined were dissolved in 100% DMSO thus an amount of DMSO corresponding to the highest concentration of the substance used in the particular experiment was added to the medium of the control samples.
  • the treatment time was 48 h.
  • CellTiter® 96 Aqueous Non-radioactive Cell Proliferation Assay (Promega, Madison, Wi, USA) was performed in order to determine the amount of viable cells in the treated samples and the control samples, respectively.
  • the assay contains:
  • MTS 3- (4, 5-dimethylthiazol-2-yl) -5- (3- carboxymethoxyphenyl ) -2- ( 4-sulfophenyl ) -2H-tetrazolium) (Promega, Madison, Wi, USA)
  • MTS is converted to formazan by viable cells which product can be found dissolved in the medium; quantity / absorbance of the product can be detected at 490 ran.
  • CellTiter 96® AQueous Assay applies to 96-well plates where 10,000 cells per well are incubated with the various test substances then 20 ⁇ of MTS and 1 ⁇ of PMS are added to 100 ⁇ of medium to determine the ratio of viable cells.
  • the enzyme reaction takes place at 37 °C, in 5% C0 2 atmosphere in 1-4 h incubation time, then the quantity of formazan produced by the cells to be found in a 96-well plate is detected using an ELISA plate reader, from which the quantity of viable cells can be concluded indirectly.
  • the solutions of the 24-well plate were distributed on a 96-well plate (Micromethod plates 96 wells flat form) in such a way that 400 ⁇ of the solution in a well were dispensed into four wells, 100 ⁇ in each, thus any dispensing inaccuracy could be screened easily.
  • 4 wells were used for control samples; such control samples were dispensed three times, 100 ⁇ . in each case, in the 96-well plate (control samples were placed in altogether 12 wells out of the 96 wells) so 4 wells on each plate were used for measuring the background. In each of those wells 100 ⁇ of the mixture of the medium, MTS and PMS described above were placed.
  • liver carcinogenesis model in which enhanced tumour formation can be observed in the liver of Matn2-/- transgenic mice on the effect of diethylnitrosamine (DEN, Sigma-Aldrich, Hungary) .
  • the homozygote male descendants of controlled genotype were injected at the age of 15 days (then the average weight of mice: 7.3 g) intraperitoneally with 10 ⁇ / (g) body weight of DEN.
  • the livers of mice were examined 8 and 10 months after inoculation. 8 months after the treatment the average tumour count in the liver of Matn2 -/- mice was 6.8; after 10 months the average tumour count increased to 22.8 +6 and the tumour incidence was 100 %. Percentage ratio of liver weight per body weight was measured. In Matn2-/- animals, it increased from 5.43 ⁇ 0.4 to 8.46 ⁇ 2.9 on the effect of DEN tumorigenesis.
  • the tumour formation inhibiting effect of the compound of example 1 and the compound of Example 4 was examined in Matn2 -/- transgenic mice after DEN tumour induction.
  • the experiment was carried out with male mice of inbred transgenic mouse lines from crossing of 129Sv x C57B1/6. 4 months after the DEN injection the test animals were divided into three groups.
  • One group was injected with 0.75 mg of the compound of example 1 (0.1 mL, dissolved in 20% DMSO (Sigma), 10% Solutol (BASF) and 70% PBS solution) (16 mice), the second group was intraperitoneally injected with 0.5 mg of the compound of Example 4 (10 mice), while the third group received only the carrier (13 mice).
  • the treatment was carried out twice a week (Monday, Friday) for 3 months; in the case of the compound of Example 4, the treatment was carried out twice a week (Monday, Friday) for 2 months.
  • 10 months after adding DEN mice were dissected and the liver tumour counts and liver weight indices were compared in the groups. 10 months after the DEN tumour induction the percentage ratio of the liver weight per body weight of mice was average 9.7 %, while the same parameter in mice also received the compound of Example 1 was 5.5 % and in mice also received the compound of Example 4 it was 4 % (Fig. 2) .
  • the average tumour count in the mouse group received only DEN was 32.6; it decreased to 13 on the effect of the compound of Example 1 and to 1.6 in the case of the compound of Example 4 (Fig. 3) .
  • mice The breast tumour test was carried out on SCID immunodeficient mouse model subcutaneously injected with MCF7 human cells (ATCC) (3 million cells/animal) .
  • Three groups of randomly selected mice were formed with 8 animals in each group.
  • Group 1: control group received only the carrier (0.1 mL, 20% DMSO (Sigma), 10% Solutol (BASF) and 70% PBS solution) ;
  • Group 3 group treated with the compound of Example 4, this group received 3 mg/kg of the compound of Example 4 (0.1 mL, dissolved in 20% DMSO (Sigma), 10% Solutol (BASF) and 70% PBS solution) intravenously.
  • the treatments were carried out from the third day, three times a week (Monday, Wednesday, Friday) for 4 weeks. From the 20 th day, the size of the increasing tumours was determined on every second day in the case of every animal and the average of each group was plotted (Fig. 4). It can be seen that both the treatment with the compound of Example 3 and the treatment with the compound of Example 4 reduced the size of the increasing breast tumour.
  • mice The melanoma test was carried out on SCID immunodeficient mouse model injected with HT168 human cells (ATCC) ( 100 , 000 cells/animal) into the spleen.
  • Three groups of randomly selected mice were formed with 10 animals in each group.
  • Group 1: control group received only the carrier (100% PBS solution) intravenously ;
  • Group 3 group treated with the compound of Example 4, received 6 mg/kg of the compound of Example.4 (0.075 mL, suspended in 100% PBS solution, a 1 mg/ml solution) intravenously.
  • the suspension of the compound of Example 4 was made as follows: the compound of Example 4 was dissolved in 60 C ethanol (100 mg in 1 ml of ethanol) and the solution was added dropwise, continuously within one minute to 100 ml of PBS solution continuously mixed with an ultrasonic mixer (Ultrasonic Processor HIELSCHER UP200S) .
  • an ultrasonic mixer Ultrasonic Processor HIELSCHER UP200S
  • Example 9 The treatments were carried out from the third day, three times a week (Monday, Wednesday, Friday) . As time went on, the number of surviving animals was determined every day and the average of the group was plotted (Fig. 5) . It can be seen that both the 3 mg/kg dose and the 6 mg/kg dose were effective; the treatment with the compound of Example 4 increased the survival of animals.
  • Example 9 Example 9:
  • glioblastoma The glioblastoma test was carried out on nude immunodeficient rat model injected with U87 human cells (ATCC) into the brain (1,000,000 cells/animal). Two groups were formed from randomly selected mice with 8 animals in both groups. Group 1 : control group, received only the carrier (100% PBS solution) intravenously; Group 2: group treated with the compound of Example 4, the group received 3 mg/kg of the compound of Example 4 (0.075 mL, suspended in 100% PBS solution, resulting in a 2 mg/ml solution) intravenously.
  • the suspension of the compound of Example 4 was made as follows: the compound of Example 4 was dissolved in 60 C ethanol (200 mg in 2 ml of ethanol) and the solution was added dropwise, continuously within one minute to 100 ml of PBS solution continuously mixed with an ultrasonic mixer (Ultrasonic Processor HIELSCHER UP200S) . The treatments were carried out from the sixth day, three times a week (Tuesday, Wednesday, Thursday) for 4 weeks. As time went on, the number of surviving animals was determined every day and the average of the group was plotted (Fig. 6) . It can be seen that the treatment with 3 mg/kg dose of the compound of Example 4 was effective, it increased the survival of animals.
  • the compound of Example 4 is an inhibitor of protein disulphide isomerase (PDI)
  • PDI protein disulphide isomerase
  • the measurement was executed on positive and negative controls as well.
  • the positive control contains 1 ⁇ of DMSO instead of 1 ⁇ of inhibitor in the composition of the reaction described above.
  • the negative control contains 1 ⁇ of assay buffer instead of 1 ⁇ of PDI compared to the positive control.
  • the final concentrations of the compound of Example 4 in the reactions were: 20 ⁇ , 10 ⁇ , 5 ⁇ , 2.5 ⁇ and 1 ⁇ .

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  • Health & Medical Sciences (AREA)
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  • Chemical & Material Sciences (AREA)
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  • Pharmacology & Pharmacy (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Animal Behavior & Ethology (AREA)
  • General Health & Medical Sciences (AREA)
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  • Chemical Kinetics & Catalysis (AREA)
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PCT/HU2011/000130 2010-12-23 2011-12-23 Use of trifluoro phthalimides for the treatment of cancerous diseases WO2012085608A2 (en)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
HU1000688A HUP1000688A2 (en) 2010-12-23 2010-12-23 Use of trifluoro-phthalimides in the treatment of cancer
HUP1000688 2010-12-23

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WO2012085608A2 true WO2012085608A2 (en) 2012-06-28
WO2012085608A3 WO2012085608A3 (en) 2012-09-07

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Family Cites Families (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
HUP0700433A2 (en) * 2007-06-21 2009-03-30 Avidin Kutato Compounds influencing development or functioning of cellular vesicular systems particularly lipid droplets, pharmaceutical compositions containing these compounds and use them for treatment of illnesses
HUP0700432A2 (en) * 2007-06-21 2009-03-30 Avicor Kft Compounds for detecting of lipid droplets, compositions for detecting of lipid droplets and method for visualization of cells or cell components

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
BASSUK JA; BERG RA: "Protein disulphide isomerase, a multifunctional endoplasmic reticulum protein", MATRIX, vol. 9, 1989, pages 244 - 258
GRAVEN KK; MOLVARC; RONCARATI JS; KLAHNBD; LOWREY S; FARBERHW: "Identification of protein disulphide isomerase as an endothelial hypoxic stress protein", AM J PHYSIOL LUNG CELL MOL PHYSIOL, vol. 282, 2002, pages L996 - L1003

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HUP1000688A2 (en) 2012-08-28
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