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WO2018065771A1 - Inhibiteur de ran au niveau transcriptionnel destiné à être utilisé dans le traitement du cancer - Google Patents

Inhibiteur de ran au niveau transcriptionnel destiné à être utilisé dans le traitement du cancer Download PDF

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WO2018065771A1
WO2018065771A1 PCT/GB2017/053009 GB2017053009W WO2018065771A1 WO 2018065771 A1 WO2018065771 A1 WO 2018065771A1 GB 2017053009 W GB2017053009 W GB 2017053009W WO 2018065771 A1 WO2018065771 A1 WO 2018065771A1
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ran
pimozide
cancer
treatment
patient
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Mohamed EL-TANANI
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University of Bradford
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University of Bradford
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Priority claimed from GB1616880.9A external-priority patent/GB2554703A/en
Priority claimed from GBGB1701388.9A external-priority patent/GB201701388D0/en
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    • 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
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/44—Non condensed pyridines; Hydrogenated derivatives thereof
    • A61K31/445—Non condensed piperidines, e.g. piperocaine
    • A61K31/4523—Non condensed piperidines, e.g. piperocaine containing further heterocyclic ring systems
    • A61K31/454—Non condensed piperidines, e.g. piperocaine containing further heterocyclic ring systems containing a five-membered ring with nitrogen as a ring hetero atom, e.g. pimozide, domperidone
    • 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
    • A61K31/535—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with at least one nitrogen and one oxygen as the ring hetero atoms, e.g. 1,2-oxazines
    • A61K31/5375—1,4-Oxazines, e.g. morpholine
    • A61K31/5377—1,4-Oxazines, e.g. morpholine not condensed and containing further heterocyclic rings, e.g. timolol
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/0012—Galenical forms characterised by the site of application
    • A61K9/0019—Injectable compositions; Intramuscular, intravenous, arterial, subcutaneous administration; Compositions to be administered through the skin in an invasive manner
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/48—Preparations in capsules, e.g. of gelatin, of chocolate
    • A61K9/50—Microcapsules having a gas, liquid or semi-solid filling; Solid microparticles or pellets surrounded by a distinct coating layer, e.g. coated microspheres, coated drug crystals
    • A61K9/51—Nanocapsules; Nanoparticles
    • A61K9/5107—Excipients; Inactive ingredients
    • A61K9/513—Organic macromolecular compounds; Dendrimers
    • A61K9/5146—Organic macromolecular compounds; Dendrimers obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyethylene glycol, polyamines, polyanhydrides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/48—Preparations in capsules, e.g. of gelatin, of chocolate
    • A61K9/50—Microcapsules having a gas, liquid or semi-solid filling; Solid microparticles or pellets surrounded by a distinct coating layer, e.g. coated microspheres, coated drug crystals
    • A61K9/51—Nanocapsules; Nanoparticles
    • A61K9/5107—Excipients; Inactive ingredients
    • A61K9/513—Organic macromolecular compounds; Dendrimers
    • A61K9/5146—Organic macromolecular compounds; Dendrimers obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyethylene glycol, polyamines, polyanhydrides
    • A61K9/5153—Polyesters, e.g. poly(lactide-co-glycolide)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents

Definitions

  • the present invention relates to pimozide, or a pharmaceutically acceptable form thereof, for use in the treatment of cancers that overexpress RAN or its protein product Ran, in particular for the treatment of patients with tumours that have been identified as overexpressing RAN or its protein product Ran.
  • the invention also provides novel nanoparticle formulations of pimozide, or a pharmaceutically acceptable form thereof, that are substantially free of dopaminergic effect when administered to a patient.
  • Neoplasm can be benign or malignant (cancerous) and are often referred to as tumours.
  • tumour is used to refer to malignant (cancerous) neoplasms.
  • tumours and the treatment of cancer have the same meaning herein, while reference to breast cancer or breast tumour, and treatment thereof, refers to a cancer or tumour that originates in the breast, or treatment thereof, rather than a tumour that originates in another organ and that has spread to the breast through the process of metastasis.
  • Tumours or cancers that originate in other organs or sites are referred to in a similar manner.
  • Tumours that are derived from the process of metastasis are referred to as metastases or secondary tumours.
  • cancer cells stimulate their own growth; (ii) cancer cells resist inhibitory signals that might otherwise stop their growth; (iii) cancer ceils resist their programmed cell death (they evade apoptosis); (iv) cancer cells can multiply indefinitely; (v) cancer cells stimulate the growth of blood vessels to supply nutrients to tumours (angiogenesis); and (vi) cancer cells invade local tissue and spread to distant sites (metastasis).
  • cancer cells stimulate their own growth;
  • cancer cells resist inhibitory signals that might otherwise stop their growth;
  • cancer ceils resist their programmed cell death (they evade apoptosis);
  • cancer cells can multiply indefinitely; (v) cancer cells stimulate the growth of blood vessels to supply nutrients to tumours (angiogenesis); and (vi) cancer cells invade local tissue and spread to distant sites (metastasis).
  • Cancer cells are addicted to oncogenes or oncogenic pathways (Weinstein, Science 2002;297:83-4), which are usually uniquely present or hyperactivated, and are essential for tumour ceil growth and survival.
  • the two most frequently dysreguiated signaling pathways in cancers are the phosphoinositide 3-kinase (PI3K)/Akt/mTORC1 and Ras/ EK/ERK [mitogen- activated protein/extracellular signal-regulated kinase (ERK; EK)] pathways (Grant, J Clin invest 2008; 1 13:3003-8).
  • Tumours or cancers are commonly referred to by the primary site of their occurrence, i.e. where the tumour first develops, and often also by reference to further characteristics, for example whether their growth is stimulated by hormones or whether they express certain receptors.
  • primary tumours located in the breast are referred to breast cancers while tumours whose growth is stimulated by hormones such as estrogen, progesterone and testosterone are referred to as hormone dependent cancers.
  • breast cancers whose growth is stimulated by the presence of estrogen are known as estrogen receptor positive (ER+ve) breast cancers.
  • ER+ve estrogen receptor positive
  • progesterone positive breast cancers that express progesterone receptors and whose growth is sensitive to the presence of progesterone are referred to as progesterone positive (PR+ve) breast cancers whilst those whose growth is independent of the expression of progesterone receptor are progesterone receptor negative (PR-ve) breast cancers.
  • PR+ve progesterone positive breast cancers
  • PR-ve progesterone receptor negative breast cancers
  • HER2-positive breast cancer a subset of breast cancers that over express human epidermal growth factor 2.
  • HER2 targeted therapies examples include the anti-HER2 antibodies trastuzumab and pertuzumab and the antibody-drug-conjugate trastuzumab emtansine.
  • triple negative breast cancers that are negative for ER, PR and HER2, i.e. those tumour types whose growth is independent of ER, PR or HER2 status, are commonly referred to as triple negative breast cancers. Patients suffering from triple negative breast cancers presently have the worst overall and disease free survival rates. Treatment options for patients with triple negative breast tumours are relatively limited (Ontilo et al, Clin Med Res. 2009 Jun; 7(1-2): 4-13) with the present treatment paradigms usually involving a combination of surgery and radiation as appropriate with cytotoxic chemotherapy with agents such as anthracyclines, taxanes or platinum chemotherapeutic agents, such as carboplatin, unless the patient has a tumour with a BRCA1 mutation.
  • cytotoxic chemotherapy with agents such as anthracyclines, taxanes or platinum chemotherapeutic agents, such as carboplatin, unless the patient has a tumour with a BRCA1 mutation.
  • RAN a member of the RAS Oncogene family
  • RAS Oncogene is a gene that encodes the GTP-binding nuclear protein Ran.
  • Overexpression of RAN gene is observed in a number of cancers and this overexpression has been linked to poor patient prognosis.
  • RAN overexpression has been shown to correlate with increased aggressiveness of cancer cells in vitro and in vivo (Kurisetty et al, Oncogene 2008, 27, 7139-49), i.e. RAN overexpressing cancer cells are seen to grow rapidly and exhibit high metastatic potential.
  • silencing RAN by siRNA or shRNA reduced cell adhesion, migration and invasion in vitro and metastasis in vivo.
  • Ran Ras-related nuclear protein
  • Ran is a G- protein GTPase that cycles between a GDP-bound (RanGDP) and a GTP-bound (RanGTP) state that regulates nucleocytoplasmic transport, mitotic spindle fibre assembly and postmitotic nuclear envelope dynamics.
  • Ran exists in a different conformation depending on whether it is bound to GTP or GDP.
  • Ran in its GTP bound state, Ran is capable of binding karyopherins (importins and exportins) a set of proteins that are involved in importing and exporting molecules between the nucleus and the cytoplasm of a eukaryotic ceil, importins release a molecular cargo upon binding to RanGTP in the nucleus, while exportins must bind RanGTP in the nucleus to form a ternary complex with their export cargo in order to transport the cargo to the cytoplasm.
  • karyopherins importins and exportins
  • the dominant nucleotide binding state of Ran depends on whether it is located in the nucleus (RanGTP) or the cytoplasm (RanGDP), with RanGTP being formed inside the nucleus through interaction of Ran with its specific guanine nucleotide exchange factor (GEF), regulator of chromosome condensation 1 referred to herein as RCC1 , which catalyses the exchange of GDP for GTP on the nucleotide binding pocket of Ran.
  • GEF guanine nucleotide exchange factor
  • RCC1 regulator of chromosome condensation 1
  • Hydrolysis of RanGTP to RanGDP in the cytoplasm by RanGAP and RanBPI releases energy and causes the ternary complex of Ran, exportin and cargo to dissociate thus releasing the cargo exported from the nucleus.
  • Cytoplasmic RanGDP is in turn imported into the nucleus by the small protein NTF2 (Nuclear Transport Factor 2), where RCC1 can then catalyse exchange of GDP for GTP on Ran and
  • the Ran cycle is involved in mitotic spindle assembly and nuclear envelope reassembly after the chromosomes have been separated.
  • the steep gradient in RanGTP-RanGDP ratio at the nuclear pores breaks down as the nuclear envelope becomes leaky and disassembles.
  • RanGTP concentration stays high around the chromosomes as RCC1 stays attached to chromatin as the nucleoporin RanBP2 (Nup358) and Ran GTPase activating protein (RanGAP) move to the kinetochores where they facilitate the attachment of spindle fibres to chromosomes.
  • RanGTP promotes spindle assembly by mechanisms similar to mechanisms of nuclear transport: the activity of spindle assembly factors such as NuMA and TPX2 is inhibited by the binding to importins. By releasing importins, RanGTP activates these factors and therefore promotes the assembly of the mitotic spindle.
  • RanGTP hydrolysis and nucleotide exchange are required for vesicle fusion at the reforming nuclear envelopes of the daughter nuclei.
  • Stable transfection of non-invasive mammary cells with an expression vector for Ran (R37-Ran and MCF-1 OA-Ran) was found to induce an invasive/metastatic phenotype in vitro and the development of metastases in vivo (Kurisetty VV, El-Tanani MK et al. Oncogene. 2008,27(57) .7139-49).
  • Stable transfection of invasive cells MDA-MB-231 , R37-OPN and R37-RAN
  • siRNA directed at Ran specifically inhibits the invasive/metastatic phenotype in vitro and in vivo (Kurisetty VV, El-Tanani MK et al. Oncogene.
  • Ran silencing was found to induce a significant apoptotic response in GR5 c-Met amplified cells in the presence of gefitinib, but not in HCC827 parental cells (p ⁇ 0.01), suggesting that Ran silencing may be used as a modulator of chemosensitivity (Yuen HF, El-Tanani M, et al. J Natl Cancer Inst. 2013; 105(7):475-88).
  • Diseases overexpressing RAN include but are not limited to certain breast, lung, prostate, ovarian, blood, brain and renal cancers and include those cancers currently associated with poor patient prognosis such as triple negative breast cancer.
  • Pimozide or a pharmaceutically acceptable form thereof, for use as an inhibitor of RAN at a transcriptional level.
  • the Pimozide for use as an inhibitor of RAN at a transcriptional level is for use in the treatment of cancer, for example a cancer that overexpresses RAN or its protein product Ran.
  • the Pimozide for use as an inhibitor of RAN at a transcriptional level in the treatment of cancer is for use in patients identified as having a cancer that overexpresses RAN or its protein product Ran.
  • a cancer that overexpresses RAN may overexpress Ran mRNA and/or Ran protein and may be identified, for example, by establishing that elevated levels of Ran mRNA and/or Ran protein are present in the cancer cells.
  • pimozide is used as an inhibitor of RAN in combination with an inhibitor of an epidermal growth factor receptor (EGFR) tyrosine kinase such as the small molecules gefitinib, erlotinib, afatinib, brigatinib, icotinib, or osimertinib or the antibodies cetuximab, panitumumab, zalutumumab, nimotuzumab, or matuzumab.
  • EGFR inhibitors an enzyme that inhibit epidermal growth factor receptor (EGFR) tyrosine kinase.
  • pimozide may be simultaneous, sequential or separate. It has been demonstrated that the combination of pimozide and an EGFR inhibitor has a synergistic effect against cancer cell proliferation. It has also been shown that pimozide can restore sensitivity of gefitinib resistant cancer cells to gefitinib treatment therefore providing a new therapeutic approach for the treatment of patients who have progressed on EGFR inhibitor therapy and, potentially, a new mechanism to prevent development of EGFR inhibitor resistance. Identification of the patients indicated for pimozide treatment, i.e.
  • those having a cancer that overexpresses RAN or its protein product Ran or Ran mRNA may be achieved by obtaining a sample from the candidate patient, for example a tumour biopsy or a blood sample, and analysing the sample for the presence of RAN or a biomarker for RAN expression.
  • the patient identification may involve testing for mutation status of a tyrosine kinase such as EGFR.
  • a tumour biopsy sample can be stained with a stain comprising a RAN selective antibody linked to a visualising means such as an enzyme or fluorophore.
  • the stained tumour biopsy can then be categorised as a RAN overexpressing tumour or a non-RAN overexpressing tumour. This categorisation can be done by a visual or automated scoring.
  • Testing for tyrosine kinase mutation status can be by the methods available in the art, for example the commercially available testing kits produced by Genzyme, QIAGEN and Argenomics S.A. for predicting response to the various tyrosine kinase inhibitors.
  • Analysis of a patient blood sample to determine whether a patient has a RAN overexpressing tumour may be performed by isolating the serum exosome and then carrying out an ELISA or quantitative real time PCR (q RT-PCR) analysis to provide the necessary tumour/patient classification.
  • the patient is a human patient.
  • the patient is a non-human mammal and may be selected from the members of the the Canidae, Felinae, Bovidae, Equidae, Suidae, Camelini and Cervidae families.
  • the pimozide for use is administered in a dosage of from 1 to 5000 mg/day.
  • the pimozide for use is in the form of a nanoparticle formulation, optionally the nanoparticle formulation comprises a poly(lactic-co-glycolic acid) copolymer, for example a poly(lactic-co-glycolic acid) / polyethylene glycol block copolymer.
  • the invention in a second aspect relates to a nanoparticle formulation of Pimozide or a pharmaceutically acceptable form thereof.
  • the nanoparticle formulation comprises a poly(lactic-co-glycolic acid) / polyethylene glycol block copolymer.
  • the invention also relates to a method of treatment of a cancer that overexpresses RAN, or its protein product Ran, comprising a step of administering to a patient in need thereof an effective amount of pimozide or a pharmaceutically acceptable form thereof.
  • This method of treatment may further comprise a step of testing a sample obtained from said patient to determine whether said patient has a RAN overexpressing cancer, e.g. a cancer that overexpresses Ran or Ran mRNA, prior to said administration step.
  • the invention also provides a method of treatment of a cancer that overexpresses RAN, or its protein product Ran, comprising the step of administering to a patient who has been identified as having a cancer that overexpresses RAN, or its protein product Ran, an effective amount of pimozide or a pharmaceutically acceptable form thereof.
  • This method of treatment may further comprise a step of testing a sample obtained from said patient to determine whether said patient has a RAN overexpressing cancer prior to said administration step.
  • the invention also provides a method of treatment of a cancer that overexpresses RAN, or its protein product Ran, comprising the steps of selecting a patient who has a cancer that overexpresses RAN or its protein product Ran, and administering an effective amount of pimozide or a pharmaceutically acceptable form thereof to that patient.
  • This method of treatment may further comprise a step of testing a sample obtained from said patient to determine whether said patient has a RAN overexpressing cancer prior to said administration step.
  • the invention also provides a method of treatment for cancer in a patient diagnosed as having a cancer characterised by an overexpression of RAN or its protein product Ran involving administration to a patient in need thereof an effective amount of pimozide.
  • the cancer characterised by an overexpression of RAN may be triple negative breast cancer.
  • the cancer characterised by an overexpression of RAN may be lung cancer, and in particular lung cancers that have previously been treated with a tyrosine kinase inhibitor, for example wherein the previous treatment involved administration of an tyrosine kinase inhibitor such as an epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor such as gefitinib, erlotinib, afatinib, brigatinib, icotinib or osimertinib to a lung cancer patient and wherein the disease no longer responds to the treatment with the EGFR inhibitor.
  • EGFR epidermal growth factor receptor
  • Such methods may involve coadministration of the previously used tyrosine kinase inhibitor with pimozide.
  • the invention also provides a method of treatment for cancer in a patient diagnosed as having a cancer characterised by an overexpression of a tyrosine kinase, such as a mutated tyrosine kinase, such as epidermal growth factor receptor (EGFR) tyrosine kinase, involving administering pimozide in combination with an inhibitor of the tyrosine kinase that is overexpressed, for example in a cancer with a mutation in the EGFR tyrosine kinase domain, an EGFR inhibitor may be used.
  • the administration of pimozide and an EGFR inhibitor may be simultaneous, sequential or separate.
  • the combination of pimozide and an EGFR inhibitor may prevent or substantially delay the emergence of resistance to the EGFR inhibitor associated with EGFR inhibitor monotherapy.
  • the combination of pimozide and an EGFR inhibitor may resensitize a tumour to treatment with an EGFR inhibitor to which the patient no longer responds as a monotherapy.
  • the invention relates to the use of pimozide, or a pharmaceutically acceptable form thereof, for the manufacture of a medicament for the treatment of cancer, optionally wherein the cancer overexpresses RAN or its protein product Ran or Ran mRNA or wherein the cancer is indicated for tyrosine kinase inhibitor therapy, for example wherein the cancer has a mutated and/or overactive epidermal growth factor receptor.
  • the resultant medicament is provided in a package with instruction for use in the treatment of a cancer that overexpresses RAN or its protein product Ran or Ran mRNA.
  • the invention provides a package comprising pimozide or a pharmaceutically acceptable form thereof and instructions for its use in the treatment of a cancer that overexpresses RAN or its protein product Ran.
  • Figure 1 illustrates the effects of pimozide treatment on the relative Ran mRNA expression in (A) MDA MB-231 human breast cancer cells and (B) A549 lung cancer cells.
  • Figure 2A illustrates the effect of pimozide treatment on RAN transcription in a dual luciferase reporter assay in MDA MB-231 cells.
  • Figure 2B shows the effect of Pimozide treatment on Myc expression in MDA MB-231 cells.
  • Figure 3 shows the effect of pimozide treatment on Ran GTPase activity in MDA MB-231 cells treated with 10 ⁇ and 15 ⁇ of pimozide vs control
  • Figure 4 shows A) the effect on MDA-MB 231 human breast cancer cells, A549 human lung cancer cells and MCF10A mammary epithelial cells of incubation with pimozide for 48 hours and B) the effect of pimozide in provoking DNA damage response (i) control, ii) pimozide treatment and in promoting DNA double strand breaks (iii) control, iv) pimozide treatment).
  • Figure 5 shows A) bioluminescent imaging of tumours and metastases in a murine model in control (Group 2, G2-NT), early treatment (Group 3, G3-ET) and late treatment (Group-4, G4- TL) cohort; B) quantification of tumour volumes at the end of the study in each cohort; & C) number of metastases in each cohort.
  • Figure 6 shows results from histological staining of tumours obtained from the study of figure 5 for haematoxylin & eosin (H & E, row 1), Ki67(row 2); cleaved caspase-3(row 3) and Ran (row 4)) -in untreated animals (UT, first column), early treatment (ET, second column) and late treatment (LT, third column) cohorts respectively.
  • Figure 7 shows histological staining of cells treated 7.5 ⁇ pimozide or control for A) paxillin, actin and merged image in MDA MB-231 ; B) myosin 11 A, actin and merged image in MDA MB- 231 and C) Arp3, actin and merged image in A549 cells.
  • Figure 8 A) RT-PCR of EMT markers Vimentin and Zo-1 24 hours after treatment with pimozide at 7.5 ⁇ and 10 ⁇ , showing a reduction of expression in agarose gel of both vimentin and Zo-1. ⁇ -actin was used as control of expression.
  • C Immunohistochemistry of vimentin expression in untreated mice (PBS) and treated tumor xenograft sections with pimozide (a representative of 10 sections per group).
  • Figure 9 shows cell viability (MDA MB-231 cells) at 24h, 48h, 72h and 96h following treatment with control, empty nanoparticles, free pimozide and pimozide formulated as 5%-PEG-PLGA nanoparticles (NPs).
  • Figure 10 shows flow cytometry data obtained from cells after treatment with A) control, B) FITC dye containing nanoparticles C) FITC dye/pimozide PCL nanoparticles and D) FITC dye/pimozide 5%-PEG-PLGA nanoparticles.
  • Figure 1 1 shows cellular uptake of pimozide labelled nanoparticles by MDA MB-231 cells.
  • Figure 12 shows the effect on HCC827 human cancer cell survival following treatment with A) gefitinib, B) pimozide and C) various concentrations of pimozide in combination with 300 nM gefitinib.
  • Figure 13 shows the effect on HCC827 GR5 (gefitinib resistant) human cancer cell survival following treatment with A) gefitinib B) pimozide and C) various concentrations of pimozide in combination with 300 nM gefitinib.
  • the present invention relates to Pimozide, or a pharmaceutically acceptable form thereof, for use in the treatment of a cancer that overexpresses RAN or its protein product Ran, for example in patients identified as having a cancer that overexpresses RAN or Ran.
  • Ran a protein product of the gene RAN, is a member of the Ras superfamily that regulates nucleocytoplasmic transport, mitotic spindle fibre assembly and post-mitotic nuclear envelope dynamics.
  • Ran acts as a molecular switch through a GTP-GDP cycle in which the conversion between GTP-bound and GDP-bound conformations controls its interaction with different effectors.
  • RanGTP is formed inside the nucleus by interaction of Ran with its specific guanine nucleotide exchange factor (GEF), referred to herein as RCC1 , which catalyses the exchange of GDP for GTP (and vice versa) on the nucleotide binding pocket of Ran.
  • GEF guanine nucleotide exchange factor
  • RAN and its protein product Ran are known to be overexpressed in a number of cancers including those in which the PI3K/Akt/mTORC1 and Ras/MEK/ERK pathways are activated.
  • Diseases overexpressing Ran include but are not limited to certain breast, lung, prostate, ovarian, blood, brain and renal cancers and include those cancers currently associated with poor patient prognosis such as triple negative breast cancer.
  • Pimozide 1-[1-[4,4-bis(4-fluorophenyl)butyl]-4-piperidinyl]-1 ,3-dihydro-2H-benzimidazole-2- one, is an antipsychotic drug of the diphenylbutylpipendine class that acts as an antagonist of dopamine D2 receptors at nanomolar concentrations. Pimozide has also been identified as an antagonist of the ⁇ opioid receptor. There is evidence that the dopamine D2 receptor and ⁇ opioid interactions of pimozide are important factors in delivering antipsychotic activity. In addition, experimental data suggests that pimozide is a weak protein kinase C inhibitor. At present, pimozide is clinically used for the treatment of schizophrenia and chronic psychosis, Tourette's syndrome and resistant tics and is marketed under various trade names such Orap®.
  • pimozide may exert an anti-cancer effect in vitro and in vivo models of cancer (see e.g. StrobI JS et al, Breast Cancer Res Treat 51 :83-95).
  • the effects of pimozide on cancers that overexpress RAN and its protein product Ran were not known.
  • pimozide could inhibit RAN at a transcriptional level.
  • the identification of pimozide as an inhibitor of RAN at a transcriptional level, thereby suppressing the downstream effects of the RAN gene provides a new and advantageous option for therapy of patients with cancers that overexpress RAN or its protein product Ran.
  • the present invention advantageously allows identification of groups of cancer patients that are indicated for pimozide treatment and advantageously allows a therapeutic intervention for such patients that is targeted to the genetic profile of their cancer.
  • the present invention thus provides for improved therapeutic outcomes for cancer patients with cancers that overexpress RAN and its protein product Ran.
  • pimozide examples include prodrugs of pimozide, that is to say compounds which break down and/or are metabolised in vivo to provide an active compound of formula (I).
  • prodrugs include simple esters, and other esters such as mixed carbonate esters, carbamates, glycosides, ethers, acetals and ketals.
  • Prodrug motifs may, for example, be directly introduced at the benzimidazole NH position of pimozide.
  • pimozide examples include metabolites of pimozide, in particular metabolites that retain an inhibitory effect on RAN transcription.
  • a metabolite, as employed herein, is a compound that is produced in vivo from the metabolism of pimozide, such as, without limitation, oxidative metabolites.
  • Pharmaceutically acceptable forms of pimozide also include deuterated forms of pimozide.
  • Deuterated forms of pimozide are derivatives of pimozide in which one or more aliphatic or aromatic hydrogen atom is substituted for a deuterium atom. For example, a hydrogen atom at a site susceptible to metabolism may be replaced by a deuterium atom to reduce metabolism at that site.
  • pimozide examples include formulations of pimozide, for example formulations that exhibit a pharmacological profile that is different to the clinical formulations presently in use for treatment of neurological conditions.
  • the present invention also relates to a pimozide, for use in combination with a tyrosine kinase inhibitor, for example a receptor tyrosine kinase inhibitor such as an inhibitor of EGFR tyrosine kinase or a non-receptor tyrosine kinase inhibitor such as a Bcr-Abl kinase inhibitor, for the treatment of cancer.
  • a tyrosine kinase inhibitor for example a receptor tyrosine kinase inhibitor such as an inhibitor of EGFR tyrosine kinase or a non-receptor tyrosine kinase inhibitor such as a Bcr-Abl kinase inhibitor.
  • Other receptor tyrosine kinases inhibitors that may be used in combination with a pimozide include inhibitors of PDGFR and FGFR kinases.
  • nonreceptor tyrosine kinases inhibitors that may be used in combination with a pimozide include inhibitors of SRC, FAK and JAK.
  • co-administration of pimozide with the receptor tyrosine kinase inhibitor gefitinib or a nonreceptor tyrosine kinase inhibitor dasatinib provides a synergistic, i.e. better than additive, effect against the survival of human lung cancer cells.
  • tyrosine kinase inhibitors that may be used in combination with a Ran inhibitor for the treatment of cancer include afatinib (GiotrifTM), axitinib (InlytaTM), bosutinib (BosulifTM), crizotinib (XalkoriTM), dasatinib (SprycelTM), erlotinib (Tarceva), gefitinib (IressaTM), osimertinib (TagrissoTM), imatinib (GlivecTM), lapatinib (TyverbTM), nilotinib (TasignaTM), pazopanib (VotrientTM), regorafenib (StivargaTM), sorafenib (NexavarTM), sunitinib (SutentTM) and ibrutinib (ImbruvicaTM).
  • pimozide is used as an inhibitor of RAN in combination with an inhibitor of an epidermal growth factor receptor (EGFR) tyrosine kinase such as the small molecules gefitinib, erlotinib, afatinib, brigatinib, icotinib, or osimertinib or the antibodies cetuximab, panitumumab, zalutumumab, nimotuzumab, or matuzumab.
  • EGFR inhibitors an enzyme that inhibit epidermal growth factor receptor (EGFR) tyrosine kinase.
  • pimozide may be simultaneous, sequential or separate. It has been demonstrated that the combination of pimozide and an EGFR inhibitor, namely gefitinib, has a synergistic effect against cancer cell proliferation (see Figures 12 and 13). It has also been shown that pimozide can restore sensitivity of gefitinib resistant cancer cells to gefitinib treatment therefore providing a new therapeutic approach for the treatment of patients who have progressed on EGFR inhibitor therapy and, potentially, a new mechanism to prevent development of EGFR inhibitor resistance (see Figure 13).
  • Identification of the patients indicated for combination therapy with pimozide and a tyrosine kinase inhibitor may involve testing for mutation status of a tyrosine kinase such as EGFR.
  • Testing for tyrosine kinase expression/mutation status can be by the methods available in the art, for example the commercially available testing kits produced by Genzyme, QIAGEN and Argenomics S.A. for predicting response to the various tyrosine kinase inhibitors.
  • pimozide or a pharmaceutically acceptable form thereof, as an inhibitor of RAN transcription for use as a medicament.
  • the present invention provides methods for treatment or alleviation of a cancer over expressing RAN, or its protein product Ran, in the tissue of one or more organs as mentioned herein, comprising the step of administering pimozide or a pharmaceutically acceptable form thereof to a patient in need thereof.
  • the method treatment is for patients with a cancer that have been diagnosed as having a cancer that overexpresses RAN or Ran or Ran mRNA.
  • Such methods according to the present invention in one embodiment comprise one or more steps of administration or release of an effective amount of pimozide or a pharmaceutically acceptable form thereof, or a pharmaceutical composition comprising pimozide or a pharmaceutically acceptable form thereof, to an individual in need thereof.
  • steps of administration or release according to the present invention is simultaneous, sequential or separate.
  • An individual in need as referred to herein is in one embodiment an individual that benefits from the administration of a pimozide or a pharmaceutically acceptable form thereof, in particular an individual with a cancer that overexpresses RAN or its protein product Ran.
  • Such an individual in one embodiment suffers from a malignant neoplasm (tumour) in the tissue of one or more organs.
  • the tumour(s) overexpresses Ran.
  • the cancer is selected from cancers of the breast, lung, ovary or kidney.
  • the cancer is one in which the PI3K/Akt/mTORC1 and/or Ras/MEK/ERK pathways are activated.
  • the cancer is triple negative breast cancer.
  • the individual is in one embodiment any human being, male or female, infant, middle-aged or old.
  • Identification of the patient to be treated in the methods of the invention can be achieved in a number of ways.
  • the identification may involve analysis of a sample obtained from a patient, for example a blood sample or a tumour biopsy sample. Analysis of a patient blood sample may be performed by isolating the serum exosome and then carrying out an ELISA or quantitative real time PCR (q RT-PCR) analysis to provide the necessary tumour/patient classification.
  • q RT-PCR quantitative real time PCR
  • a tumour biopsy sample can be stained with a stain comprising a RAN selective antibody linked to a visualising means such as an enzyme or fluorophore.
  • the stained tumour biopsy can then be categorised as a RAN overexpressing tumour or a non-RAN overexpressing tumour. This categorisation may be performed by a visual or automated scoring.
  • scoring of Ran immunohistochemically stained tumour biopsy sample slides can be performed under a light microscope and that an assessment of the % of tumour cells staining over the whole section provides a method to identify tumours that overexpress RAN.
  • the invention also provides a method of treatment for cancer in a patient diagnosed as having a cancer characterised by an overexpression of RAN or its protein product Ran involving administration to a patient in need thereof an effective amount of pimozide.
  • the cancer characterised by an overexpression of RAN may be triple negative breast cancer.
  • the cancer characterised by an overexpression of RAN may be lung cancer, and in particular lung cancers that have previously been treated with a tyrosine kinase inhibitor, for example wherein the previous treatment involved administration of an tyrosine kinase inhibitor such as an epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor such as gefitinib, erlotinib, afatinib, brigatinib, icotinib or osimertinib to a lung cancer patient and wherein the disease no longer responds to the treatment with the EGFR inhibitor.
  • EGFR epidermal growth factor receptor
  • Such methods may involve coadministration of the previously used tyrosine kinase inhibitor with pimozide.
  • the invention also provides a method of treatment for cancer in a patient diagnosed as having a cancer characterised indicated for tyrosine kinase therapy, by an overexpression of an tyrosine kinase, such as a mutated epidermal growth factor receptor (EGFR) tyrosine kinase, involving administering pimozide in combination with an inhibitor of the tyrosine kinase that is overexpressed, for example in a cancer with a mutation in the EGFR tyrosine kinase domain, an EGFR inhibitor may be used.
  • an EGFR inhibitor may be used.
  • the administration of pimozide and an EGFR inhibitor may be simultaneous, sequential or separate.
  • the combination of pimozide and an EGFR inhibitor may prevent or substantially delay the emergence of resistance to the EGFR inhibitor associated with EGFR inhibitor monotherapy.
  • the combination of pimozide and an EGFR inhibitor may resensitize a tumour to treatment with an EGFR inhibitor to which the patient no longer responds as a monotherapy.
  • treatment refers to the management and care of a patient for the purpose of combating a condition, disease or disorder.
  • the term is intended to include the full spectrum of treatments for a given condition from which the patient is suffering, such as administration of pimozide or a pharmaceutically acceptable form thereof for the purpose of: alleviating or relieving symptoms or complications; delaying the progression of the condition, partially arresting the clinical manifestations, disease or disorder; curing or eliminating the condition, disease or disorder; and/or preventing or reducing the risk of acquiring the condition, disease or disorder, wherein "preventing” or “prevention” is to be understood to refer to the management and care of a patient for the purpose of hindering the development of the condition, disease or disorder, and includes the administration of the active compounds to prevent or reduce the risk of the onset of symptoms or complications.
  • the patient to be treated is preferably a mammal, in particular a human being. Treatment of animals, such as mice, rats, dogs, cats, cows, horses, sheep and pigs, is, however, also within the scope of the present invention.
  • the patients to be treated according to the present invention can be of various ages, for example, adults, children, children under 16, children age 6-16, children age 2-16, children age 2 months to 6 years or children age 2 months to 5 years.
  • the invention is thus, in one embodiment, directed to pimozide or a pharmaceutically acceptable form thereof for use in the treatment of cancer in the tissue of one or more organs of a mammal.
  • said treatment is ameliorative and/or curative.
  • said mammal is a human (homo sapiens).
  • said organ is in one embodiment selected from the group consisting of breast, lung, ovarian, prostate, blood, brain and renal cancers
  • Pimozide or a pharmaceutically acceptable form thereof for use in the treatment of cancer that overexpresses RAN, or its protein product Ran may be provided as a pharmaceutical composition comprising pimozide or a pharmaceutically acceptable form thereof in combination with one or more pharmaceutically acceptable diluents or carriers.
  • the formulation may be a formulation currently used in the clinic for pimozide or may be a new formulation specifically adapted to the purpose of treating RAN overexpressing cancers.
  • Diluents and carriers may include those suitable for parenteral, oral, topical, mucosal and rectal administration.
  • the present invention also provides a process for preparing such a pharmaceutical composition (for example a pharmaceutical composition for parenteral, oral, topical, mucosal or rectal administration), said process comprising mixing the ingredients.
  • compositions may be prepared e.g. for parenteral, subcutaneous, intramuscular or intravenous administration, particularly in the form of liquid solutions or suspensions; for oral administration, particularly in the form of tablets or capsules; for topical e.g. pulmonary or intranasal administration, particularly in the form of powders, nasal drops or aerosols and transdermal administration; for mucosal administration e.g. to buccal, sublingual or vaginal mucosa, and for rectal administration e.g. in the form of a suppository.
  • compositions may conveniently be administered in unit dosage form and may be prepared by any of the methods well-known in the pharmaceutical art, for example as described in Remington's Pharmaceutical Sciences, 17th ed., Mack Publishing Company, Easton, PA., (1985).
  • Formulations for parenteral administration may contain as excipients sterile water or saline, alkylene glycols such as propylene glycol, polyalkylene glycols such as polyethylene glycol, oils of vegetable origin, hydrogenated naphthalenes and the like.
  • Formulations for nasal administration may be solid and may contain excipients, for example, lactose or dextran, or may be aqueous or oily solutions for use in the form of nasal drops or metered sprays.
  • compositions suitable for oral administration may comprise one or more physiologically compatible carriers and/or excipients and may be in solid or liquid form.
  • Tablets and capsules may be prepared with binding agents, for example, syrup, acacia, gelatin, sorbitol, tragacanth, or poly-vinylpyrollidone; fillers, such as lactose, sucrose, corn starch, calcium phosphate, sorbitol, or glycine; lubricants, such as magnesium stearate, talc, polyethylene glycol, or silica; and surfactants, such as sodium lauryl sulfate.
  • binding agents for example, syrup, acacia, gelatin, sorbitol, tragacanth, or poly-vinylpyrollidone
  • fillers such as lactose, sucrose, corn starch, calcium phosphate, sorbitol, or glycine
  • lubricants such as magnesium stearate, talc, polyethylene glycol, or silica
  • surfactants such as sodium lauryl sulfate.
  • Liquid compositions may contain conventional additives such as suspending agents, for example sorbitol syrup, methyl cellulose, sugar syrup, gelatin, carboxymethyl-cellulose, or edible fats; emulsifying agents such as lecithin, or acacia; vegetable oils such as almond oil, coconut oil, cod liver oil, or peanut oil; preservatives such as butylated hydroxyanisole (BHA) and butylated hydroxytoluene (BHT).
  • suspending agents for example sorbitol syrup, methyl cellulose, sugar syrup, gelatin, carboxymethyl-cellulose, or edible fats
  • emulsifying agents such as lecithin, or acacia
  • vegetable oils such as almond oil, coconut oil, cod liver oil, or peanut oil
  • preservatives such as butylated hydroxyanisole (BHA) and butylated hydroxytoluene (BHT).
  • BHA butylated hydroxyanisole
  • BHT butylated hydroxytoluen
  • Solid oral dosage forms include tablets, two-piece hard shell capsules and soft elastic gelatin (SEG) capsules.
  • a dry shell formulation typically comprises of about 40% to 60% w/w concentration of gelatin, about a 20% to 30% concentration of plasticizer (such as glycerin, sorbitol or propylene glycol) and about a 30% to 40% concentration of water.
  • plasticizer such as glycerin, sorbitol or propylene glycol
  • Other materials such as preservatives, dyes, opacifiers and flavours also may be present.
  • the liquid fill material comprises a solid drug that has been dissolved, solubilized or dispersed (with suspending agents such as beeswax, hydrogenated castor oil or polyethylene glycol 4000) or a liquid drug in vehicles or combinations of vehicles such as mineral oil, vegetable oils, triglycerides, glycols, polyols and surface-active agents.
  • suspending agents such as beeswax, hydrogenated castor oil or polyethylene glycol 4000
  • a liquid drug in vehicles or combinations of vehicles such as mineral oil, vegetable oils, triglycerides, glycols, polyols and surface-active agents.
  • Pimozide is a well-known dopamine D2 receptor antagonist and its interaction with the dopamine D2 receptor is commonly thought to underlie its neuropharmacological effects. Pimozide is also known to interact with a range of other receptors including the 5- hydroxytryptamine ( ⁇ - ⁇ ) receptor and hERG.
  • ⁇ - ⁇ 5- hydroxytryptamine
  • hERG 5- hydroxytryptamine
  • the present invention provides a nanoparticle delivery system for pimozide that advantageously reduces, for example essentially avoids, any neuropharmacological effect normally associated with pimozide administration.
  • Pimozide thus include the use of particulate carrier systems such as liposomes, microspheres, micelles and nanoparticles.
  • biodegradable polyester matrices of various forms, features in many innovative formulation approaches evaluated over the past four decades.
  • goserelin acetate peptide incorporated into PLGA copolymer was approved by the FDA in 1998.
  • This delivery system can protect and release the drug slowly into the systemic circulation, where it can reach its target site.
  • NP poly(caprolactone)
  • PEG/PLGA poly(ethylene glycol)/ poly(lactic-co-glycolic acid)
  • the invention relates to poly(lactic-co-glycolic acid) (PLGA) comprising nanoparticle formulations of the inhibitors of the invention.
  • the composition of block copolymers provided herein is by percentage weight of the composition excluding the pimozide for use.
  • PLGA comprising nanoparticle formulations of the inhibitors of the invention are based on PLGA/polyethylene glycol (PEG) block copolymer nanoparticle.
  • the % PEG content of the PLGA/PEG block copolymer nanoparticle is from 1 % to 15%, the remainder being PLGA.
  • the % PEG content of the PLGA/PEG block copolymer nanoparticle is from 2.5% to 10%.
  • the % PEG content of the PLGA/PEG block copolymer nanoparticle is between 5% to 10%. Increasing the PEG content advantageously improves the encapsulation efficiency.
  • the invention relates to nanoparticle formulations of pimozide for example, poly(caprolactone) (PCL) comprising nanoparticle (NP) formulations of pimozide.
  • PCL poly(caprolactone)
  • NP nanoparticle
  • the nanoparticle formulations generally analysed herein have a mean diameter of 201 ⁇ 26 nm a zeta potential of -28.3 ⁇ 2.24 mV.
  • the nanoparticle formulations have a mean polydispersity index of 0.143 ⁇ 0.017.
  • the nanoparticle formulations of the invention are generally fabricated using emulsion solvent evaporation or by solvent displacement techniques.
  • the emulsion solvent evaporation technique is used to form nanoparticle formulations of pimozide.
  • the nanoparticle formulations of the invention advantageously enhance the biophysicochemical properties and achieve controlled cytotoxic and apoptotic efficacy on cancer cells relative to free peptide inhibitors while significantly reducing or eliminating the effects of pimozide on the dopamine receptor and/or hERG.
  • the nanoparticle formulations according to the invention have a mean diameter 100 nm to 400 nm, for example from 150 nm to 352 nm.
  • the nanoparticle formulations according to the invention have a mean diameter between 150 and 300nm. In one embodiment the nanoparticle formulations according to the invention have a zeta potential of from -3 to -21 mV mV. In one embodiment the nanoparticle formulations of the invention have a mean polydispersity index of between 0.14 and 0.23.
  • dosing pimozide as a nanoparticle formulation in PCL increases the absorption of pimozide and also significant reduces or essentially eliminates the neuropharmacological effects that result from dosing of the non-NP form. It is believed that phagocytic transport of the nanoparticle formulations of the invention into cells deliver a selective cytotoxic effect on cancer cells.
  • pimozide, or a pharmaceutically acceptable form thereof, for use or for use in a method of treatment as described herein can be combined with or comprise one or more second active ingredients which are understood as other therapeutic compounds or pharmaceutically acceptable derivatives thereof.
  • Methods for treatment according to the present invention in one embodiment thus further comprise one or more steps of administration of one or more second active ingredients, either concomitantly or sequentially, and in any suitable ratios.
  • second active ingredients is, for example, selected from compounds used to treat or prevent cancer in the tissue of one or more organs or symptoms or used to treat or prevent complications associated with treatment of cancer in the tissue of one or more organs.
  • such a second active ingredients is intended for the treatment of the side effects associated with the primary treatment or is aimed at overcoming or suppressing resistance that may arise in the cancer.
  • the one or more second active ingredients may be directed to the treatment of cancer, the treatment or suppression of emesis, the blockage of drug efflux pumps or potentiation of the anti-cancer effect of pimozide.
  • a preferred class of second active ingredient for use in combination with pimozide are tyrosine kinase inhibitors.
  • the tyrosine kinase inhibitor may be as a small molecule or an antibody tyrosine kinase inhibitor.
  • tyrosine kinase inhibitors that may be used in combination with a Ran inhibitor, for example pimozide, for the treatment of cancer include afatinib (GiotrifTM), axitinib (InlytaTM), bosutinib (BosulifTM), crizotinib (XalkoriTM), dasatinib (SprycelTM), erlotinib (Tarceva), gefitinib (IressaTM), osimertinib (TagrissoTM), imatinib (GlivecTM), lapatinib (TyverbTM), nilotinib (TasignaTM), pazopanib (VotrientTM), regorafenib (StivargaTM), sorafenib (NexavarTM), sunitinib (SutentTM) and ibrutinib (ImbruvicaTM).
  • Methods of treatment according to the present invention in one embodiment include a step wherein pimozide is administered simultaneously, sequentially or separately in combination with one or more second active ingredients.
  • the action of the second therapeutic agent and pimozide is synergistic. Kit of parts
  • kits according to the present invention also provides a kit of parts.
  • a kit of parts according to the present invention in one embodiment comprises pimozide or a pharmaceutically acceptable form thereof or a composition thereof as defined herein for treatment of cancer in the tissue of one or more organs in combination with a further active ingredient as described herein.
  • Kits according to the present invention in one embodiment allows for simultaneous, sequential or separate administration of pimozide and one or more second active ingredients as described herein.
  • the invention provides a package containing pimozide or a pharmaceutically acceptable form thereof and instructions for its use for the treatment of cancer, for example for patients with a cancer that overexpresses RAN or its protein product Ran or Ran mRNA.
  • a composition comprising pimozide or a pharmaceutically acceptable form thereof is in one embodiment administered to a cancer patient having a cancer that overexpresses RAN or its protein product Ran in a pharmaceutically effective dose or a therapeutically effective amount.
  • a therapeutically effective amount of pimozide or a pharmaceutically acceptable form thereof according to the present invention is in one embodiment an amount sufficient to cure, prevent, reduce the risk of, alleviate or partially arrest the clinical manifestations of a cancer that overexpresses RAN or its protein product Ran.
  • the amount that is effective will depend on the severity of the cancer as well as on the weight and general state of the subject. An amount adequate to accomplish this is defined as a "therapeutically effective amount”.
  • the composition is administered in doses of from 1 mg/day to 5000 mg/day.
  • Pimozide may be administered as an oral formulation, for instance in the form of a tablet. If a nanoparticle formulation is to be used, the administration may be in the form a local infusion or injection, or an intravenous or subcutaneous injection depending on the location of the cancer to be treated.
  • the route of administration allows for introducing pimozide, or a pharmaceutically acceptable form thereof, for use into the blood stream to ultimately target the desired site(s) of action.
  • the route of administration is any suitable route, such as an enteral route (including the oral, rectal, nasal, pulmonary, buccal, sublingual, transdermal, intracisternal and intraperitoneal administration), and/or a parenteral route (including subcutaneous, intramuscular, intrathecal, intravenous and intradermal administration).
  • an enteral route including the oral, rectal, nasal, pulmonary, buccal, sublingual, transdermal, intracisternal and intraperitoneal administration
  • a parenteral route including subcutaneous, intramuscular, intrathecal, intravenous and intradermal administration.
  • Appropriate dosage forms for such administration may be prepared by conventional techniques.
  • Parenteral administration is any administration route not being the oral/enteral route whereby the medicament avoids first-pass degradation in the liver. Accordingly, parenteral administration includes any injections and infusions, for example bolus injection or continuous infusion, such as intravenous administration, intramuscular administration or subcutaneous administration. Furthermore, parenteral administration includes inhalations and topical administration. Accordingly, the peptide or composition is in one embodiment administered topically to cross any mucosal membrane of an animal to which the substance or peptide is to be given, e.g.
  • parenteral administration may also include buccal, sublingual, nasal, rectal, vaginal and intraperitoneal administration as well as pulmonal and bronchial administration by inhalation or installation.
  • the peptide is administered topically to cross the skin.
  • the intravenous, subcutaneous and intramuscular forms of parenteral administration are employed.
  • the peptide or composition according to the invention is used as a local treatment, i.e. is introduced directly to the site(s) of action.
  • pimozide may be applied to the skin or mucosa directly, or may be injected into the site of action, for example into the diseased tissue or to an end artery leading directly to the diseased tissue.
  • pimozide or a pharmaceutically acceptable form thereof according to the present invention or pharmaceutically acceptable derivatives thereof are administered alone or in combination with pharmaceutically acceptable carriers or excipients, in either single or multiple doses.
  • the pharmaceutical compositions or compounds according to the invention may be formulated with pharmaceutically acceptable carriers or diluents as well as any other known adjuvants and excipients in accordance with conventional techniques such as those disclosed in Remington: The Science and Practice of Pharmacy, 20th Edition, Gennaro, Ed., Mack Publishing Co., Easton, PA, 2000.
  • pharmaceutically acceptable form in present context includes pharmaceutically acceptable salts, which indicate a salt which is not harmful to the patient.
  • Such salts include pharmaceutically acceptable basic or acid addition salts as well as pharmaceutically acceptable metal salts, ammonium salts and alkylated ammonium salts.
  • a pharmaceutically acceptable derivative further includes esters and prodrugs, or other precursors of a compound which may be biologically metabolized into the active compound, or crystal forms of a compound.
  • the pharmaceutical composition or pharmaceutically acceptable composition may be specifically formulated for administration by any suitable route, such as an enteral route, the oral, rectal, nasal, pulmonary, buccal, sublingual, transdermal, intracisternal, intraperitoneal, and parenteral (including subcutaneous, intramuscular, intrathecal, intravenous and intradermal) route.
  • a suitable route such as an enteral route, the oral, rectal, nasal, pulmonary, buccal, sublingual, transdermal, intracisternal, intraperitoneal, and parenteral (including subcutaneous, intramuscular, intrathecal, intravenous and intradermal) route.
  • compositions or compounds of the present invention are formulated for crossing the blood-brain-barrier.
  • compositions for oral administration include solid dosage forms such as hard or soft capsules, tablets, troches, dragees, pills, lozenges, powders and granules. Where appropriate, they can be prepared with coatings such as enteric coatings, or they can be formulated so as to provide controlled release of the active ingredient, such as sustained or prolonged release, according to methods well known in the art. In the same solid dosage form two active ingredients may be combined so as to provide controlled release of one active ingredient and immediate release of another active ingredient.
  • Liquid dosage forms for oral administration include solutions, emulsions, aqueous or oily suspensions, syrups and elixirs.
  • compositions for parenteral administration include sterile aqueous and nonaqueous injectable solutions, dispersions, suspensions or emulsions, as well as sterile powders to be reconstituted in sterile injectable solutions or dispersions prior to use. Depot injectable formulations are also regarded as being within the scope of the present invention.
  • Suitable administration forms include suppositories, sprays, ointments, creams/lotions, gels, inhalants, dermal patches, implants, etc.
  • pimozide for use according to the present invention is generally utilized as the free substance or as a pharmaceutically derivative such as a pharmaceutically acceptable salt thereof.
  • a pharmaceutically acceptable salt thereof examples of the latter are an acid addition salt of pimozide or a pharmaceutically acceptable form thereof.
  • pharmaceutically acceptable salt refers to a nontoxic salt of a compound for use according to the present invention, which salts are generally prepared by reacting a free base with a suitable organic or inorganic acid. Salts of pimozide derivatives containing a free base functionality can be prepared in a conventional manner by treating a solution or suspension of the compound with a chemical equivalent of a pharmaceutically acceptable acid.
  • Pharmaceutically acceptable acid addition salts include, but are not limited to, hydrochloride, hydrobromide, hydroiodide, nitrate, sulfate, bisulfate, phosphate, acid phosphate, isonicotinate, acetate, lactate, salicylate, citrate, tartrate, pantothenate, bitartrate, ascorbate, succinate, maleate, gentisinate, fumarate, gluconate, glucaronate, saccharate, formate, benzoate, glutamate, methanesulfonate, ethanesulfonate, benzensulfonate, p-toluenesulfonate and pamoate (i.e., 1 , 1 '-methylene-bis-(2-hydroxy-3- naphthoate)) salts.
  • prodrug refers to derivatives of pimozide that are rapidly transformed in vivo to yield the pimozide, for example, by hydrolysis in blood or by metabolism in cells, such as for example the cells of the basal ganglia.
  • prodrugs include pharmaceutically acceptable, non-toxic esters of the compounds of the present invention.
  • Suitable pharmaceutical carriers include inert solid diluents or fillers, sterile aqueous solutions and various organic solvents.
  • solid carriers are lactose, terra alba, sucrose, cyclodextrin, talc, gelatine, agar, pectin, acacia, magnesium stearate, stearic acid and lower alkyl ethers of cellulose.
  • liquid carriers are syrup, peanut oil, olive oil, phospholipids, fatty acids, fatty acid amines, polyoxyethylene and water.
  • the carrier or diluent may include any sustained release material known in the art, such as glyceryl monostearate or glyceryl distearate, alone or mixed with a wax.
  • compositions formed by combining the compounds for use according to the present invention and the pharmaceutically acceptable carriers are then readily administered in a variety of dosage forms suitable for the disclosed routes of administration.
  • the formulations may conveniently be presented in unit dosage form by methods known in the art of pharmacy.
  • Formulations of the present invention suitable for oral administration may be presented as discrete units, such as capsules or tablets, which each contain a predetermined amount of the active ingredient, and which may include a suitable excipient.
  • the orally available formulations may be in the form of a powder or granules, a solution or suspension in an aqueous or non-aqueous liquid, or an oil-in-water or water-in-oil liquid emulsion.
  • compositions intended for oral use may be prepared according to any known method, and such compositions may contain one or more agents selected from the group consisting of sweetening agents, flavouring agents, colouring agents and preserving agents in order to provide pharmaceutically elegant and palatable preparations.
  • Tablets may contain the active ingredient(s) in admixture with non-toxic pharmaceutically acceptable excipients which are suitable for the manufacture of tablets.
  • excipients may, for example, be: inert diluents, such as calcium carbonate, sodium carbonate, lactose, calcium phosphate or sodium phosphate; granulating and disintegrating agents, for example corn starch or alginic acid; binding agents, for example, starch, gelatine or acacia; and lubricating agents, for example magnesium stearate, stearic acid or talc.
  • the tablets may be uncoated or they may be coated by known techniques to delay disintegration and absorption in the gastrointestinal tract and thereby provide a sustained action over a longer period.
  • a time delay material such as glyceryl monostearate or glyceryl distearate may be employed. They may also be coated by the techniques described in U.S. Patent Nos. 4,356, 108; 4, 166,452; and 4,265,874, the contents of which are incorporated herein by reference, to form osmotic therapeutic tablets for controlled release.
  • Oily suspensions may be formulated by suspending the active ingredient in a vegetable oil, for example arachis oil, olive oil, sesame oil or coconut oil, or in a mineral oil such as a liquid paraffin.
  • the oily suspensions may contain a thickening agent, for example beeswax, hard paraffin or cetyl alcohol. Sweetening agents such as those set forth above, and flavouring agents may be added to provide a palatable oral preparation. These compositions may be preserved by the addition of an anti-oxidant such as ascorbic acid.
  • Dispersible powders and granules suitable for preparation of an aqueous suspension by the addition of water provide the active compound in admixture with a dispersing or wetting agent, suspending agent and one or more preservatives.
  • a dispersing or wetting agent e.g., talc, kaolin, kaolin, kaolin, kaolin, kaolin, kaolin, kaolin, kaolin, kaolin, kaolin, kaolin, kaolin, kaolin, kaolin, kaolin, sorbitol, sorbitol, sorbitol, sorbitol, sorbitol, sorbitol, sorbitol, sorbitol, sorbitol, mannitol, mannitol, mannitol, mannitol, mannitol, mannitol, mannitol, mannitol, mannitol, mannitol,
  • Syrups and elixirs may be formulated with sweetening agents, for example glycerol, propylene glycol, sorbitol or sucrose. Such formulations may also contain a demulcent, a preservative and flavouring and colouring agent.
  • the pharmaceutical compositions may be in the form of a sterile injectable aqueous or oleaginous suspension. This suspension may be formulated according to the known methods using suitable dispersing or wetting agents and suspending agents described above.
  • the sterile injectable preparation may also be a sterile injectable solution or suspension in a non-toxic parenterally-acceptable diluent or solvent, for example as a solution in 1 ,3-butanediol.
  • Suitable vehicles and solvents that may be employed are water, Ringer's solution, and isotonic sodium chloride solution.
  • sterile, fixed oils are conveniently employed as solvent or suspending medium.
  • any bland fixed oil may be employed using synthetic mono- or diglycerides.
  • fatty acids such as oleic acid find use in the preparation of injectables.
  • compositions may also be in the form of suppositories for rectal administration of the compounds of the invention.
  • These compositions can be prepared by mixing the compound with a suitable non-irritating excipient which is solid at ordinary temperatures but liquid at the rectal temperature and will thus melt in the rectum to release the drug.
  • suitable non-irritating excipient include, for example, cocoa butter and polyethylene glycols.
  • Pimozide treatment causes a dose dependent reduction in Ran mRNA and Ran protein
  • RT-PCR Real Time PCR
  • MDA MB 231 breast cancer cells Figures 1A and 1 C
  • A549 Figure 1 B
  • lung cancer cells were either untreated (control) or treated with 5 ⁇ to 15 ⁇ concentrations of pimozide.
  • RNA was extracted from the cells after 48h and quantitative RT-PCR was then performed with a ABI PRISM 7500 instrument (Applied Biosystems, Foster City, CA, USA) according to the manufacturer's instructions.
  • the Taqman probe sets for RAN and 18s RNA were used. Gene expression was normalized to 18s RNA. As can be seen a dose dependent decrease in Ran mRNA in treated cells was observed indicating that pimozide decreases the transcriptional activity of RAN.
  • RNA levels in cancer cells quantitative Real Time PCR (RT-PCR) experiments were performed.
  • MDA MB 231 breast cancer cells ( Figure 1 D) were either untreated (control) or treated with 5 ⁇ to 20 ⁇ concentrations of pimozide.
  • RNA was extracted from the cells after 48h and quantitative RT-PCR was then performed with a ABI PRISM 7500 instrument (Applied Biosystems, Foster City, CA, USA) according to the manufacturer's instructions.
  • the Taqman probe sets for RAN and its target genes and 18s RNA were used. Gene expression was normalized to 18s RNA.
  • a dose dependent decrease in Ran C-MET and MMP2 mRNA in treated cells was observed indicating that pimozide decreases the transcriptional activity of RAN down stream targets.
  • Pimozide inhibits RAN at a transcriptional level
  • a dual luciferase reporter assay was developed based on a pGL3 basic reporter construct (Promega, Southampton, UK) into which full length Ran promoter (1400 base pairs upstream of the transcriptional start site, amplified using Phusion Hot Start High-Fidelity DNA polymerase) and partial sequences thereof (770bp, 368 bp and 162 bp segments from the N-terminal region derived by serial deletion) were incorporated.
  • the resultant constructs along with the unmodified construct were then transfected into MDA MB 231 cancer cells. Cells containing the various constructs were then treated with or without pimozide for 24 hours.
  • the firefly luciferase activity was normalized to the internal control Renilla luciferase activity.
  • results from the dual luciferase assay are presented in Figure 2A.
  • the luciferase activity in control and pimozide treated cells was equivalent, i.e. pimozide treatment had no effect.
  • luciferase activity in cells with the pGL3 full length Ran promoter construct and 700bp construct showed essentially no luciferase activity after pimozide treatment indicating that pimozide treatment directly inhibits RAN at a transcriptional level.
  • the constructs with shorter, 368bp and 162bp fragments of the Ran promoter construct had little or no luciferase activity irrespective of pimozide treatment status.
  • FIG 3 shows the results from Western blots of Ran GTPase activity in MDA MB 231 cell in untreated cells and cells treated with 10 ⁇ and 15 ⁇ of pimozide.
  • Ran GTPase activity was measured using the Ran activation assay kit according to the manufacturer's instructions (Cell Biolabs, San Diego, CA). Briefly, GTP-bound Ran was pulled down by using RanBPI PBD Agarose bead slurry, and the pulled-down complex was then dissociated by boiling in 2* reducing sodium dodecyl sulfate polyacrylamide gel electrophoresis sample buffer. The pull-down supernatant was then analyzed by Western blotting. As can be seen pimozide treatment reduces the amount of RanGTP protein compared to the control (DMSO) treated cells.
  • DMSO control
  • Pimozide selectively inhibits the growth of cancer cells relative to non-cancerous cell
  • the relative effect of pimozide treatment on normal mammary epithelial cells and cancer cells is shown in Figure 4 MDA-MB 231 human breast cancer cells, A549 human lung cancer cells and MCF10A mammary epithelial cells were incubated with pimozide for 48 hours.
  • a 12.5 ⁇ concentration caused a ca 50% reduction in cell viability in the cancer cells while the normal mammary epithelial cells were relatively unaffected demonstrating the selective cytotoxicity of pimozide to the RAN expressing cancer cell lines.
  • Pimozide reduces the tumour burden in a nude mice xenograft model, decreases cell proliferation and metastasis incidence in lung.
  • mice Female C57/BL6 nude mice (8-weeks old), kept and handled according to institutional guidelines, complying with UK legislation under 12/12 hours light/dark cycle at a temperature of 22°C, received a standard diet and acidified water ad libitum.
  • MDA-MB-231-Luc cells (2 ⁇ 106) were injected subcutaneously in 100 ⁇ _ phosphate-buffered saline together with 100 ⁇ _ Matrigel basement membrane matrix (Becton Dickinson) into one fat pad of each mouse.
  • mice were randomly assigned to cohorts of ten mice each, and were grouped in four groups.
  • Group-1 (G1), a tumour free control group, were injected with matrigel without MDA MB 231 cell and were not treated with pimozide.
  • Group-2 a tumour bearing control group, received MDA MB 231 cells in matrigel and were not treated with pimozide but were instead injected with an equal volume of vehicle (PBS) in tandem with treatment in group-4 (G2NT);
  • Group-3 the early treatment cohort, received MDA MB 231 cells in matrigel and was treated after 24 hours after implantation with 20 mg/kg i.p.
  • Group-4 the long term dosing cohort, were injected with cells and the tumours were left for 14 days to become established (G4TL). From day 14 onwards, the group Group-4 cohort were treated by i.p. injection with 20mg/kg pimozide in a once daily 5 on, 2 off dosing regime (once daily dosing from Monday to Friday with no dosing on Saturday or Sunday).
  • tumour volume (mm 3 ) was calculated using the formula: (shortest diameter) 2 ⁇ (longest diameter) ⁇ 0.5. Animal body weight and any sign of morbidity were monitored. Drug treatment lasted 4 weeks. Animals were euthanized 24 hours after the last drug administration according to institutional guidelines, and then tumours were carefully removed, weighed and analysed. A total necropsy analysis involving tumours and distinct organs was carried out.
  • Tumour tissue samples obtained at the end of the study were fixed in 4% (w/v) buffered paraformaldehyde and embedded in paraffin. Tissue sections (4-5 ⁇ ) were deparaffinized and hydrated in graded ethanol and distilled water for analysis. Endogenous peroxidase activity was blocked using methanol and 30% H2O2 for 30 minutes.
  • Sections were counterstained with hematoxylin and eosin (H&E, Figure 6 row 1), and then incubated overnight at 4°C with different antibodies: rabbit anti-human Ki67 monoclonal antibody (clone SP6) (1 : 1000 dilution, Abeam, Figure 6 row 2), anti-active human cleaved-caspase-3 (1 :200 dilution for IHC & 1 : 100 for IF, Cell signaling, Figure 6 row 3) rabbit polyclonal antibody that specifically recognized the active ⁇ 20-kDa subunit (Cell signaling) anti-human Ran (1 : 1000, Millipore, Figure 6 row 4), anti-human MMP2 (1 : 100 dilution, Millipore), anti-Aktl , anti-Akt2, pAkt (1 : 1000, Cell Signaling) and anti-human CD31 (1 :50 for IHC and 1 :20 for IF, Abeam) and a-smooth muscle actin (a-SMA)(1 :50
  • tumour volumes were reduced by 65% (p ⁇ 0.05) (Group-3) and by 60% (p ⁇ 0.05) (Group-4).
  • tumor incidence in treated groups were delayed by 55% (P ⁇ 0.05) (Group-3) (data not shown).
  • mice in the untreated cohort showed high degree of metastasis in lung compared to treated animals. Very few focal metastasis were observed in airway epithelium in the treatment group.
  • the level of Ran protein expression in lung metastasis foci was studied by immunohistochemistry (see Fig 6, row 4). This revealed a higher expression of Ran in the lung metastases present in the untreated cohort and lower Ran expression in the lung metastases present in each, early and late, treated tumour cohort. Counting of the metastases in the treated/untreated cohorts showed a significant reduction in metastases in the treated cohorts relative to untreated control (see Figure 5C).
  • Ran protein is known to have a role in carcinogenesis and metastasis. Pimozide treatment was thus evaluated for effects on motility characteristics of different human breast cancer cell lines in vitro. Migration experiments using MDA-MB-231 and MCF7 cell lines in the presence of 7.5 ⁇ pimozide were performed. A wound healing scratch closure assay was performed to determine changes in cellular motility. For this assay, 1x10 5 MCF7 or 6x10 4 MDA-MB-231 human breast cancer cells were seeded into each well of 24-well tissue culture plates and left to grow for 48 hours at which stage they reached 90-95% confluency.
  • the resultant monolayers were then scored with a sterile pipette tip prior, for the treatment cohorts, to addition of 7.5 ⁇ pimozide in normal medium.
  • Wound closure was monitored by collecting digitalized images using the Cell-IQ automated image capture system, (Chip-Man Technologies) on preselected fields using phase contrast microscopy. Analysis of the collected data was performed using the Cell-IQ Analyser Software, to generate percentage of wound closure over time. Quantification of the closed area at the 24 hours' time point were singled out for all conditions and normalized as percentage to the control untreated cells.
  • pimozide treated cells demonstrated clear defects in their abilities to migrate. Quantification of the cells ability to close the gap further demonstrated a reduction by 40% (P ⁇ 0.05) of the wound area in both MDA-MD-231 and MCF7 breast cells in the presence of pimozide.
  • MMP1 and MMP14 at mRNA level were also decreased in MDA-MB- 231 cells treated with pimozide at 7.5 ⁇ after 24 hours compared to control. This indicates that pimozide exerts a negative effect on metalloproteinase activation (i.e. reduces activation of MMPs) by reducing Ran protein expression. Pimozide inhibits migration capacities of both MDA-MB-231 and A549 cancer cells.
  • MDA-MB-231 and A549 were stained for different markers of motility (Figure 7).
  • Paxillin is a component of the focal adhesion, acting as an adaptor protein between integrins and the actin cytoskeleton.
  • Myosin IIA is usually found in the lamella of motile cells, just behind the lamellipodium as well as throughout the cell body, being mainly responsible for the contraction forces require during the organization of actin stress fibers; whilst Arp3 is a component of the Arp2/3 complex that promote actin nucleation and polymerization, mainly located at the leading edge and principally the lamellipodium of migratory cells.
  • Pimozide affects epithelial mesenchymal transition (EMT) markers in MDA-MB-231 cells
  • Twist and Snail are transcription factors that regulate the expression E- cadherin and N-cadherin
  • breast cancer patients with high expression of Twist and Snail have poorer progression-free survival and overall survival, suggesting that cadherin expression impacts survival 45,46.
  • N-cadherin, vimentin and transcription factors Twist, Slug, and Snail expression were reduced both in vitro and in vivo untreated tumours.
  • pimozide appears to have good potential for use in the treatment of tumours with a high metastatic potential, for example as an inhibitor of the Epithelial Mesenchymal Transition, for example in a human patient that has been diagnosed as having a cancer, in particular a cancer that overexpresses RAN or its protein product Ran or Ran mRNA.
  • a polymer nanoparticle formulation of Pimozide can be used to deliver to increase drug absorption and bypass the dopamine receptor: Preparation of pimozide loaded nanoparticles (NP) As pimozide is relatively hydrophobic, two different techniques could be employed for forming pimozide loaded nanoparticle formulations. A modified single emulsion solvent evaporation approach or a modified nanoprecipitation technique could be used. Pimozide (0.5 mg) was mixed with 5% w/v of either PCL or PEG-PLGA block copolymer and dissolved overnight in 2 ml_ of dichloromethane (DCM) or acetone.
  • DCM dichloromethane
  • the resultant organic phase was injected by syringe into 50 ml of a 1.25% aqueous solution of PVA (polyvinyl alcohol) under agitation followed by homogenisation using a Silverson L5T Homogeniser (Silverson Machines, UK) at 10,000 rpm for 7 minutes in case of solvent evaporation technique.
  • the emulsion was stirred by magnetic agitation overnight under vacuum to evaporate the dichloromethane completely and prevent pore formation on the surface of the nanospheres. After the nanospheres had formed, they were centrifuged at 10,000 rpm for 30 min at 4 °C then washed three times with distilled water and 2% w/v sucrose solution and finally freeze-dried. The final product was stored in a desiccator at room temperature.
  • coumarin 6-loaded NP formulations were evaluated using flow cytometry and fluorescence microscopy [J Pharm Pharmacol 64 (2012) 61-67].
  • FACS analysis coumarin 6 tagged NPs were suspended in Optimem ® media and added to MDA-MB-231 cells for 24 hours in 6-well plates. Cells were removed by trypsinisation and resuspended in FACS buffer. Cellular uptake of NP formulations was quantified by gating for positive couramin 6 staining in the FITC channel following control staining with coumarin 6-treated and unstained MDA-MB- 231 cells. Three independent experiments with three replicates were performed for each assay.
  • Cytotoxicity of pimozide NP formulations at 1 , 2.5, 5 and 10 ⁇ was measured by assessing cell viability (MTT assay).
  • MDA-MB-231 cells were seeded in 24-well plates (Nalge Nunc International, Rochester, NY) at a density of 5 x 10 4 cells per well and incubated for 24 hours to allow for 60-70% confluency and sufficient adhesion.
  • the cells were treated with different concentrations of the pimozide-loaded NP, pimozide and blank NP. After 24, 48, 72 and 96 hours, the treated cells were washed with 500 ⁇ PBS, and 500 ⁇ of 15% MTT dye solution in complete media added to each well.
  • the plates were incubated at 37 °C and 5% CO2 for an additional 3 hours. The supernatant was then discarded, 500 ⁇ DMSO added and the solution vigorously mixed. The optical density of each well was measured at 570 nm (reference wavelength 630 nm) in a microplate reader (Fluostar Omega, BMG Lab Tech GMBH, Germany). This experiment was performed in triplicate and repeated three times. Mean values ⁇ SD for each concentration was determined. Percentage cell viability was determined as the ratio of absorbance (570 nm) in treated cells relative to the absorbance in control cells (570 nm). The absorbance of the untreated cells was set at 100%.
  • Figure 13 shows the effects of drug treatment for 24h on the survival of gefitinib resistant HCC827 GR5 cells, a cell line that is derived from normal HCC827 human lung cancer cells (cf Figure 12).
  • treatment with gefitinib in this instance at all of the concentrations evaluated causes no more than a 15% reduction in cell survival.
  • Pimozide treatment (Figure 13B) causes a ca 30% reduction in cell survival at a concentration of 5 ⁇ .
  • the combination of various concentrations of pimozide and gefitinib 300 nM meanwhile proved superior to either agent when they were presented as single agents.

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Abstract

La présente invention concerne des inhibiteurs à petites molécules de RAN au niveau transcriptionnel et leur utilisation dans le traitement du cancer, en particulier de cancers, tels que le cancer du sein triple négatif, qui ont été identifiés comme surexprimant RAN ou son produit protéique Ran. Les inhibiteurs de l'invention sont particulièrement utiles pour le traitement de patients identifiés comme ayant une tumeur qui surexprime RAN ou son produit protéique Ran. L'invention concerne également des formulations de nanoparticules des inhibiteurs à petites molécules de l'invention qui évitent des effets hors cible au niveau du récepteur de la dopamine et qui sont ainsi appropriés pour une thérapie sélective contre le cancer.
PCT/GB2017/053009 2016-10-05 2017-10-05 Inhibiteur de ran au niveau transcriptionnel destiné à être utilisé dans le traitement du cancer Ceased WO2018065771A1 (fr)

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CN114641582A (zh) * 2019-10-28 2022-06-17 布拉德福德大学 用于确定肿瘤的侵袭和/或转移潜力的方法
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