PANICEIN COMPOUNDS, COMPOSITIONS AND USES THEREOF
20170326076 · 2017-11-16
Assignee
- Centre National De La Recherche Scientifique (Paris, FR)
- UNIVERSITE NICE SOPHIA ANTIPOLIS (NICE CEDEX 2, FR)
Inventors
- ISABELLE MUS-VETEAU (GRASSE, FR)
- Olivier Thomas (Galway, IE)
- Marie-Aude Tribalat (Les Arcs, FR)
- STÉPHANE AZOULAY (NICE, FR)
Cpc classification
A61P35/00
HUMAN NECESSITIES
International classification
Abstract
The present invention relates to the fields of medicine and cancer treatment. The invention more specifically relates to the use of a panicein or a derivative thereof, to decrease or inhibit, in vitro or ex vivo, the Patched receptor drug efflux activity, in particular the chemotherapeutic drug efflux activity and chemotherapy resistance. The present disclosure further relates to uses of such compounds, in particular to prepare a pharmaceutical composition to allow or improve the efficiency of a therapy of cancer in a subject in need thereof. The compound of the invention can indeed be advantageously used, in combination with at least one chemotherapeutic drug, for treating cancer, for preventing cancer metastasis and/or for preventing cancer recurrence in a subject. The invention also discloses methods for preventing or treating cancer, cancer metastasis and/or cancer recurrence in a subject, as well as kits suitable for preparing a composition according to the present invention and/or for implementing the herein described methods.
Claims
1-16. (canceled)
17. A method for decreasing or inhibiting, in vitro or ex vivo, the Patched receptor drug efflux activity comprising a step of contacting a cancer cell with a panicein compound, or of a derivative or analogue thereof.
18. The method according to claim 17, wherein the panicein compound is a compound of formula (I) ##STR00023## wherein: R.sub.1 is H, C.sub.1-C.sub.6 alkyl, C.sub.1-C.sub.6 aminoalkyl, C.sub.1-C.sub.6 hydroxyalkyl, C.sub.1-C.sub.6 halogenoalkyl or —OR.sub.4, R.sub.2 is H or C.sub.1-C.sub.6 alkyl, R.sub.3 is C.sub.1-C.sub.6 alkyl, —CH.sub.2OR.sub.4, —C(═O)R.sub.4, —C(═O)OR.sub.4, —C(═O)NHR.sub.4, or —CH.sub.2NHR.sub.4, each R.sub.4 is independently —H, or C.sub.1-C.sub.6 alkyl, R is selected from the group consisting of: ##STR00024## wherein: bonds (1) and (2) are independently of each other a single bound or a double bond, R.sub.5 is present when bond (1) is a single bond, and represents —H, or —OR.sub.6, and when present, each R.sub.6 is independently H or a C.sub.1-C.sub.6 alkyl; or a pharmaceutically acceptable salt thereof.
19. The method according to claim 18, wherein the compound of formula (I) is such that R is selected in the group consisting of: ##STR00025## when present.
20. The method according to claim 18, wherein the compound of formula (I) comprises one or several of the following features: i) R.sub.1 is H, C.sub.1-C.sub.6 alkyl, or —OR.sub.4, and/or ii) R.sub.2 is H or C.sub.1-C.sub.6 alkyl, and/or iii) R.sub.3 is C.sub.1-C.sub.6 alkyl, —CH.sub.2OR.sub.4 or, —C(═O)R.sub.4, and/or iv) each R.sub.4 is independently —H, or a C.sub.1-C.sub.6 alkyl or a C.sub.1-C.sub.3 alkyl.
21. The method according to claim 18, wherein the compound of formula (I) is selected from Panicein C, Panicein B3, Panicein B2 and Panicein A hydroquinone.
22. The method according to claim 21, wherein the compound of formula (I) is selected from Panicein A hydroquinone and a stereoisomer, derivative or analog thereof.
23. The method according to claim 17, wherein the drug is a chemotherapeutic drug.
24. The method according to claim 18, wherein bonds (1) and (2) are not simultaneously double bonds.
25. The method according to claim 19, wherein R.sub.6 is H and R.sub.5 is H or OH.
26. A method for treating cancer, for reducing cancer metastasis and/or cancer recurrence in a subject comprising a step of administering a panicein compound in combination with at least one chemotherapeutic drug in the subject.
27. The method according to claim 26, wherein the cancer cell expresses the Patched receptor.
28. The method according to claim 27, wherein cancer is selected from a melanoma, a breast cancer, a thyroid cancer, a prostate cancer, a colon cancer, a rectal cancer, an oesophagus cancer, a gastric cancer, an ovarian cancer, a lung cancer, a pancreatic cancer, a glioma, an adrenocortical carcinoma, a pediatric solid malignant tumor, a leukaemia, a multiple myeloma or a sarcoma.
29. The method according to claim 26, wherein the at least one chemotherapeutic drug is selected from cisplatin, doxorubicin, methotrexate, temozolomide, 5-FU, dacarbazine and vemurafenib.
30. The method according to claim 26, wherein the subject is a mammal.
31. The method according to claim 30, wherein the subject is a human being suffering of a cancer and resistant to chemotherapy.
32. A composition comprising at least one panicein compound and at least one chemotherapeutic drug.
33. A method for treating cancer, for reducing cancer metastasis and/or cancer recurrence in a subject comprising a step of administering the composition according to claim 32 in the subject.
34. A kit comprising at least one panicein compound and at least one chemotherapeutic drug in distinct containers.
35. A method for preparing a composition comprising at least one panicein compound and at least one chemotherapeutic drug comprising mixing a composition comprising at least one panicein compound and at least one chemotherapeutic drug.
Description
LEGENDS TO THE FIGURES
[0149]
[0150] Schematic representation of the activity of efflux of chemotherapeutic agent of Patched. Dxr: doxorubicin, CSC: cancer stem cell
[0151]
[0152] A. Haliclona mucosa sponge. B. Haliclona mucosa methanolic fraction contains inhibitors of the resistance of Patched-expressing yeasts to doxorubicin. Yeast S. cerevisiae expressing hPtc were grown in 96 well plates in the presence of 10 μg/mL of the methanolic fraction of Haliclona mucosa crude extract dissolved in DMSO and in presence or not of 10 μM of doxorubicin (dxr). The growth of yeasts was measured by absorbance at 600 nm and the results shown are the mean of three independent experiments. C. Purification profile of Haliclona mucosa methanolic fraction. A preparative phenyl-hexyl HPLC column was used with a gradient starting at (47 H20: 53 ACN: 0.1 TFA) to (0 H20: 100 ACN: 0.1 TFA), flow rate: 10.0 mL/min, injection volume: 75 μl at 100 mg/mL. D. Five of the compounds purified from Haliclona mucosa methanolic fraction strongly inhibit the resistance of hPtc-expressing yeasts to drx. Purified compounds dissolved in DMSO were added (at 10 μg/mL) to the growth medium containing or not 10 μM dxr. The growth of yeasts was measured by absorbance at 600 nm.
[0153]
[0154]
[0155] A. Western-blotting with rabbit anti-Patched antibodies (1/1000) on total extracts from three different melanoma cell lines (MDA-MB-435, MeWo and A375). Patched is expected at 150 KDa. 13 tubulin was used as loading control. B. Immuno-labeling of MeWo and A375 using Patched antibodies (1/200) and rhodamine-anti-rabbit antibodies. Patched labeling (in red) was superimposed to DAPI (in blue) and image of the cells in visible light. Rhodamine-anti-rabbit antibodies alone did not give any signal on these cells (not shown).
[0156]
[0157] Cells were grown in 96 well plates in complete DMEM medium to achieve 60% to 70% confluence. Medium was then removed and replaced with 100 μt/well of complete DMEM medium containing paniceins at 10 μM (or at increasing concentrations for EC50 measurement) or DMSO as control. After 2 hours, 100 μL of complete DMEM medium containing doxorubicin was added in half wells to obtain the final concentration of 2 μM for MDA-MB-435 and MeWo, and 1.5 μM for A375. The cell viability was measured after 24 hours. Effect of compounds P2, P3, P4 and P5 on cell viability, and dose-response for compound P5 are shown for MDA-MB-435 (A), MeWo (B) and A375 (C). D. Enhancement of dxr cytotoxicity by paniceins is represented. The means (+/−sem) of at least three independent experiments are reported.
[0158]
[0159] A. Compound P5 inhibits doxorubicin efflux from melanoma cells MeWo and A375. Cells were grown on cover-slips, incubated for 2 hour at 37° C. with 10 μM doxorubicin and quickly rinsed with phosphate buffer (pH 7.4). One cover-slip of each cell line was immediately fixed for doxorubicin charge control. The other cover-slips were incubated with buffer supplemented with DMSO or 10 μM of compound P5 30 min under gentle shaking and immediately fixed. Dxr intracellular fluorescence was visualized by epifluorescence and analyzed using Image J software on more than 30 cells of 3 different fields for each condition. The results were analyzed using the Student t test in which significance is attained at P<0.05 (**: P<0.005, ***: P<0.0005). B. Paniceins inhibit doxorubicin efflux from Patched-expressing yeasts. Yeasts expressing wild-type Patched (hPtc, in black), mutant Patched G509VD513Y (hPtcVXXXY, in grey), and control yeasts (in white) were incubated in buffer supplemented with 5 mM of 2-deoxy-D-glucose and 10 μM dxr for 2 hours at 4° C. After centrifugation and supernatant removing, one sample was immediately fixed for charge control and the other samples were resuspended in buffer containing 5 mM of 2-deoxy-D-glucose supplemented with DMSO or 10 μM of paniceins 10 minutes at 25° C. with gentle shaking. After centrifugation and supernatant removing, samples were fixed and deposited on coverslips for epifluorescence microscopy observation. Dxr intracellular fluorescence quantification was carried out using Image J software on more than 30 yeasts on 3 different fields for each condition. The results were analyzed using the Student t test in which significance is attained at P<0.05 (*).
[0160]
[0161] A model of Patched structure has been done on the bases of the crystal structure of AcrB, the principal multidrug efflux transporter from the RND family in Escherichia coli describes with and without doxorubicin by Murakami et al (Nature 2006). The AcrB-drug complex consists of three protomers. Dxr was found in the periplasmic domain of one of the three protomers. Docking of Panicein A hydroquinone into the model structure of Patched shows a strong probability of binding in a cluster close to the dxr binding site.
[0162]
[0163] Paniceins were added at a final concentration of 10 μM to the yeast growth medium containing 2 mM of dacarbazine (A), 50 μM of cisplatine (B), or 10 μM of vemurafenib (C). The growth of hPtc-expressing-yeasts was measured by absorbance at 600 nm. (D) Viability test using neutral red shows that panicein A hydroquinone (10 μM) strongly increases vemurafenib cytotoxicity for A375 melanoma cells.
[0164]
[0165] (a) Patched protein is expressed in different cancers (IHC on tissue, data extracted from the Human Protein Atlas web site). (b) Patched mRNAs in 154 melanoma samples (data extracted from ONCOMINE web site).
[0166]
[0167] Cells were sampled after 24 h treatment with DMSO, paniceins and/or dxr, and apoptosis determined via AnnexinV and DAPI co-staining Cells in early apoptosis are AnnexinV positive and DAPI negative, and cells in late apoptosis are AnnexinV and DAPI double positive. Histograms represent the mean percentage (+/−SEM) of cells in late apoptosis from three independent experiments and were analyzed using the Student t-test in which significance is attained at P<0.05 (*) (**: P<0.005).
[0168]
[0169] Western-blotting on total extracts from MEWO and A375 cells after 24 h treatment with 10 μM of panicein A hydroquinone (P5) or with DMSO.
[0170]
EXAMPLES
[0171] Biological Material
[0172] Specimens of Haliclona mucosa were collected in February-May 2013 by hand using SCUBA diving in the rade de Villefranche-sur-Mer (France), at depths ranging from 20 or 40 m in obscure cavities or caves, and kept frozen until used.
[0173] Human melanoma cell lines Mewo and A375 were purchased from ATCC and MDA-MB-435 were obtained from C. Vandier, initially purchased from the ATCC. The three cell lines were grown in DMEM medium supplemented with 10% FBS, 100 U/mL penicillin, and 100 mg/mL streptomycin, at 37° C. in a 5% CO.sub.2/95% air water-saturated atmosphere.
[0174] K699 Saccharomyces cerevisiae yeast strain (Mata, ura3, and leu 2-3, kindly donated by R. Arkowitz) were transformed with pYEP-hPtc-MAP (human Patched over-expression), pYEP-mMyo-MAP (control), or pYEP-hPtcG509VD513Y-MAP (mutant Patched expression) expression vector and grown as described (Bidet et al. 2011) at 18° C. until an OD at 600 nm between 5 and 7.
[0175] Screening Test 1: Effect of Sponge Extracts on the Resistance of Yeast-Expressing Patched to Doxorubicin.
[0176] S. cerevisiae expressing human Patched were grown in 10 mL of minimal medium (supplemented with 2% of glucose and amino acids cocktail without leucine) at 30° C. When the exponential phase was obtained (OD600=5-7), yeasts were precultured in the same medium to OD600=1-2. Yeasts were then diluted in rich medium containing 2% of glucose in 96-well plates. Methanolic or purified fractions (at 10 μg/mL final concentration) were added in all wells, and doxorubicin (10 μM final concentration) was added in half of the wells. Plates were incubated at 18° C. on a shaker at 1250 rpm (microtitre plate shaker SSL5 Stuart) and absorbance at 600 nm was recorded for about 72 hours.
[0177] Screening Test 2: Effect of Sponges Fractions on Doxorubicin Cytotoxicity.
[0178] Melanoma cells MDA-MB-435, Mewo and A375 were seeded on 96-well plates and grown 48 hours in complete DMEM medium to achieve 60% to 70% confluence. Medium was then removed and replaced with 100 μL/well of complete DMEM medium containing the compounds of interest at defined concentration or DMSO as control. After 2 hours, 100 μL of complete DMEM medium containing doxorubicin was added in half wells to obtain 2 μM doxorubicin. Plates were incubated at 37° C. in a 5% CO2/95% air water-saturated atmosphere. After 24 hours, microplates were incubated 3 hours at 37° C. with 100 μL/wells neutral red (NR) solution (50 μg/mL in DMEM). After a rapid wash with PBS at 4° C., microplates were gently tapped several times on absorbent paper. Cells were solubilized with 100 μL of a solution containing 1% acetic acid, 49% H.sub.2O, 50% ethanol by vortexing 3 minutes at 700 rpm and the absorbance at 600 nm was measured. EC50 were calculated using Regressi software.
[0179] Protein Quantification
[0180] Protein concentrations were determined by the Bradford method using a Bio-Rad kit.
[0181] SDS-PAGE and Western Blotting
[0182] Total extracts from melanoma cells were prepared. Samples were separated on 8% SDS-PAGE and transferred to nitrocellulose membranes (Amersham) using standard techniques. After 1 hour at room temperature in blocking buffer (20 mmol/L Tris-HCl pH 7.5, 450 mmol/L NaCl, 0.1% Tween-20, and 4% non-fat milk), nitrocellulose membranes were incubated overnight at 4° C. with rabbit anti-Patched antiserum (Ab130.6 1:1000 generous gift from M. Ruat or Ab39266 from Abcam 1/1000) and monoclonal mouse anti-βtubulin antibody (Sigma; 1/1000). After 3 washes, membranes were incubated 45 min with anti-mouse (1:5000) or anti-rabbit (1:3000) immunoglobulin coupled to horseradish peroxidase (Dako). Detection was carried out with an ECL kit (Millipore) on a Las3000 (Fuji).
[0183] Drug Efflux Measurements
[0184] On melanoma cells: For doxorubicin incorporation in melanoma cells, the protocol was adapted from Bidet et al. (2012). Cells were seeded on coverslips in 12-well plates and allowed to grow to 80% confluence. Coverslips were incubated 2 hours at 37° C. with doxorubicin (10 μM) in physiological buffer (140 mM NaCl, 5 mM KCl, 1 mM CaCl2, 1 mM MgSO4, 5 mM glucose, 20 mM HEPES, pH 7.4) and quickly rinsed with phosphate buffer (pH 7.4). One coverslip of each cell line was immediately fixed 10 minutes with 4% paraformaldehyde (Sigma) for doxorubicin charge control. The other coverslips were incubated with physiological buffer supplemented with DMSO or 10 μM of paniceins 30 min under gentle shaking at 37° C., and immediately fixed with 4% paraformaldehyde. Coverslips were observed by epifluorescence microscopy using an objective 40× and filters for Alexa 594 probe. Quantification of doxorubicin intracellular fluorescence was carried out using Image J software on more than 30 cells of 3 different fields for each condition. The results were analyzed using the Student t test in which significance is attained at P<0.05.
[0185] On yeasts: Yeasts expressing Patched (hPtc), mutant Patched (hPtcG509VD513Y), or control yeasts were washed with cold water, resuspended at an OD600 of 10 in Hepes-NaOH buffer (pH 7.0) supplemented with 5 mM of 2-deoxy-D-glucose, and incubated with 10 μM doxorubicin for 2 hours at 4° C. in the cold room on a rotating wheel protected from light. Yeasts were centrifuged and the supernatant was removed. One sample was immediately fixed with 4% paraformaldehyde for doxorubicin loading control. The other samples were resuspended in Hepes-NaOH buffer (pH 7.0) containing 5 mM of 2-deoxy-D-glucose supplemented with DMSO or 1004 of paniceins, and incubated 10 minutes at 20° C. with gentle shaking in a Benchmark Multi-therm shake protected from light. Samples were centrifuged for 1 minute at 18,000 g, supernatants were removed and yeasts were fixed with 4% paraformaldehyde. 10 μL of each sample were deposited on a coverslip and observed by epifluorescence microscopy using an objective 63× and filters for Alexa 594. Quantification of doxorubicin intracellular fluorescence was carried out using Image J software on more than 30 yeasts on 3 different fields for each condition. The results were analyzed using the Student t test in which significance is attained at P<0.05.
[0186] Results
[0187] Haliclona mucosa Methanolic Fraction Contains Inhibitors of the Resistance of Patched-Expressing Yeasts to Doxorubicin.
[0188] Inventors cultured yeast S. cerevisiae expressing human Patched in their plasma membrane as previously described (Bidet et al., 2011, 2012) in 96 well plates. Methanolic fraction of Haliclona mucosa crude extracts was prepared, dissolved in DMSO at 10 mg/mL and added at a final concentration of 10 μg/mL to the yeast culture medium supplemented or not with doxorubicin (dxr). As previously reported (Bidet et al., 2012), human Patched confers a resistance to growth inhibition by dxr, an alkylating agents used to treat a wide range of cancers (
[0189] The methanolic fraction from Haliclona mucosa was then purified by HPLC on preparative C18 reversed phase to yield 9 compounds (P1 to P9) (
[0190] The 1H NMR spectra allowed the identification of four of these compounds and confirmed their purity. These compounds are members of the panicein family: Panicein-C (P2), Panicein-B3 (P3), Panicein-B2 (P4) and Panicein-A-hydroquinone (P5) (
[0191] Paniceins Purified from Haliclona mucosa Increase the Cytotoxicity of Doxorubicin for Melanoma Cells.
[0192] Three melanoma cell lines have been chosen to evaluate the effect of paniceins purified from Haliclona mucosa extract on the dxr cytotoxicity. MDA-MB-435 cells are derived from the M14 melanoma cell line and used to study cancer metastasis (Rae et al., 2007). MeWo cell line derived from melanoma metastatic site (lymph node tissue) and A375 cell line derived from a human malignant melanoma and carries the BRAF V600E mutation. These three cell lines over express the Patched protein as shown by western-blotting (
[0193] Cells were treated with paniceins and with or without dxr during 24 hours before cell viability measurement (
[0194] Western blot analysis performed on MEWO or A375 cells treated during 24 hours with panicein A hydroquinone or with DMSO indicated panicein A hydroquinone had no effect on Patched expression or degradation in these cells (
[0195] Paniceins Inhibits Doxorubicin Efflux.
[0196] Melanoma cells grown on cover-slips were loaded with dxr, fixed (for loading control) or incubated with efflux buffer containing DMSO (efflux control) or paniceins, fixed and analyzed using cell imaging (
[0197] In order to see if Paniceins inhibit dxr efflux through Patched inhibition, inventors compared dxr efflux on yeasts over-expressing human Patched and control yeasts. After loading with dxr, yeasts were centrifuged and fixed for dxr loading control (LC), or resuspended in buffer supplemented with DMSO or paniceins for efflux measurements. After 10 min a 20° C., yeasts were collected, fixed and deposited on cover-slips. 2-deoxy-D-glucose was added in buffer during loading and efflux in order to inhibit ATP-binding cassette (ABC) transporters. Dxr fluorescence intensity of at least 30 yeasts was measured for each experiment.
TABLE-US-00002 TABLE 2 Inhibition of dxr efflux activity of Patched by paniceins Efflux (%) Panicein Panicein Panicein Panicein DMSO P2 P3 P4 P5 WT-hPtc- 65 51 46 46 40 yeasts hPtc-YXXXG- 30 39 32 38 42 yeasts Control-yeasts 25 20 25 29 39
[0198] Panicein a Hydroquinone Presents a Strong Docking Cluster Close to the Doxorubin Binding Site in Both AcrB Structure and Patched Structural Model.
[0199] The crystal structure of AcrB, the principal multidrug efflux transporter from the RND family in Escherichia coli, was describes with and without doxorubicin by Murakami et al. (2006). The AcrB-drug complex consists of three protomers, each of which has a different conformation corresponding to one of the three functional states of the transport cycle. Bound substrate was found in the periplasmic domain of one of the three protomers. The voluminous binding pocket is aromatic and allows multi-site binding.
[0200] Isabelle Broutin (Laboratoire de Cristallographie et RMN Biologiques, UMR 8015 CNRS, Faculte de Pharmacie Paris V France) carried out the docking of compounds P2 and P5 into the AcrB-dxr structure. The analysis shows many clusters of low probability for P2 binding, and more interestingly, a strong probability of binding of P5 in a cluster close to the dxr binding site. Isabelle Broutin then realized a model of the structure of Patched from the AcrB structure and carried out the docking with compounds P2 and P5 with the same results as those reported for the docking on AcrB structure (
[0201] This analysis shows that panicein A hydroquinone could bind close to the dxr-binding site of Patched and prevent dxr binding. This is in good agreement with the inhibition of dxr efflux observed in presence of panicein A hydroquinone.
[0202] Paniceins Also Inhibit Resistance to Dacarbazine and Cisplatine Conferred to Yeasts by Patched Over-Expression.
[0203] The expression of human Patched in yeasts confers to these yeasts the ability to grow in presence of other chemotherapeutic agents currently used to treat melanoma such as dacarbazine or cisplatine. Inventors' results demonstrate that the presence of compounds P2, P3 or P5 in the growth medium inhibit the resistance of yeasts to dacarbazine, cisplatine and vemurafenib (
CONCLUSION
[0204] Inventors' results show for the first time that some paniceins purified from the Mediterranean sponge Haliclona mucosa, in particular panicein A hydroquinone, are able to enhance doxorubicin cytotoxicity for melanoma cells in vitro. Efflux measurements strongly suggest that paniceins inhibit the dxr efflux activity of Patched. This hypothesis is supported by the docking realized on the structure of the E. coli multidrug transporter AcrB and on the structural model of Patched which shows that panicein A hydroquinone has a strong probability of binding close to the dxr binding site. This demonstrates that binding of panicein A hydroquinone to Patched prevent dxr efflux. These results also show that paniceins are able to inhibit the resistance conferred by Patched to other chemotherapeutic agents currently used to treat melanoma such as dacarbazine, cisplatine and vemurafenib.
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