WO2023170107A1 - Pi3k/akt/mtor inhibitor for improving the cellular uptake of a radiopharmaceutical - Google Patents

Pi3k/akt/mtor inhibitor for improving the cellular uptake of a radiopharmaceutical Download PDF

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WO2023170107A1
WO2023170107A1 PCT/EP2023/055815 EP2023055815W WO2023170107A1 WO 2023170107 A1 WO2023170107 A1 WO 2023170107A1 EP 2023055815 W EP2023055815 W EP 2023055815W WO 2023170107 A1 WO2023170107 A1 WO 2023170107A1
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pi3k
akt
mtor
composition
kit
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PCT/EP2023/055815
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French (fr)
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Karim ABID
Eric Grouzmann
Melpomeni Fani
Rosalba Mansi
Joana GRAND-GUILLAUME-PERRENOUD
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Centre Hospitalier Universitaire Vaudois (Chuv)
Universität Basel
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Publication of WO2023170107A1 publication Critical patent/WO2023170107A1/en

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/13—Amines
    • A61K31/155—Amidines (), e.g. guanidine (H2N—C(=NH)—NH2), isourea (N=C(OH)—NH2), isothiourea (—N=C(SH)—NH2)
    • 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
    • A61K31/4353—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 ortho- or peri-condensed with heterocyclic ring systems
    • A61K31/436—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 ortho- or peri-condensed with heterocyclic ring systems the heterocyclic ring system containing a six-membered ring having oxygen as a ring hetero atom, e.g. rapamycin
    • 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
    • A61K31/47—Quinolines; Isoquinolines
    • A61K31/4738—Quinolines; Isoquinolines ortho- or peri-condensed with heterocyclic ring systems
    • 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
    • A61K51/00—Preparations containing radioactive substances for use in therapy or testing in vivo
    • A61K51/02—Preparations containing radioactive substances for use in therapy or testing in vivo characterised by the carrier, i.e. characterised by the agent or material covalently linked or complexing the radioactive nucleus
    • A61K51/04—Organic compounds

Definitions

  • the present invention relates to compositions comprising a PI3K/Akt/mTOR inhibitor, and a compound, in particular a radiopharmaceutical, whose cellular uptake is upregulated upon inhibition of PI3K/Akt/mTOR, as well as to pharmaceutical compositions and kits of parts comprising the same.
  • Instant compositions, pharmaceutical compositions and kits of parts are particularly useful in therapy and diagnosis, in particular of neuroendocrine tumours.
  • the present invention further relates to PI3K/Akt/mTOR inhibitors for use in diagnosis and therapy.
  • Cancer is a disease characterised by uncontrollable cell division and growth, usually as a result of genetic alterations in specific genes.
  • Several important signalling pathways have been identified as frequently genetically altered in cancer, including phosphoinositide 3-kinase and/or protein B kinase and/or mammalian target of rapamycin (PI3K/Akt/mTOR) signalling. These pathways play a crucial role in cell-cycle progression, apoptosis and cell growth.
  • Metastatic cancers can exhibit fast progression and may be difficult to diagnose due to a lack of severe symptoms.
  • the metastatic cancers that are particularly challenging to treat, are neuroendocrine tumours which often go undiagnosed or misdiagnosed for several years.
  • Neuroendocrine tumours are neoplasms originating from the hormone-producing cells of the endocrine and nervous systems.
  • NBs neuroblastomas
  • Pheochromocytomas (PHEOs) and paragangliomas (PGLs) are neuroendocrine tumours found in the adrenal medulla and parasympathetic ganglia respectively, 10% of those presenting with benign PHEO/PGL develop malignancy.
  • NB and PHEO/PGL cells are characterized by an excessive production and secretion of catecholamines (norepinephrine, dopamine and epinephrine).
  • catecholamine analogues are an attractive diagnostic and therapeutic target.
  • This strategy has been employed in a recent phase II clinical study with Vorinostat, a histone deacetylase (HDAC) inhibitor, which upregulates the expression of norepinephrine transporters (NETs), thereby increasing the uptake of norepinephrine and its analogues, including the radiotherapeutic agent 131 1- metaiodobenzylguanine ( 131 l-mlBG).
  • HDAC histone deacetylase
  • Document W02020/021456 describes the therapeutic concept of the combination of a peptide receptor radionuclide therapeutic (PRRT) agent and immuno-oncology (l-O) therapy.
  • PRRT peptide receptor radionuclide therapeutic
  • l-O immuno-oncology
  • This combination provides, in particular, combinations with l-O therapeutic agents that inhibit LAG-3, TIM-3, GITR, TGF-[3, IL15/IL-15RA and PD-1 pathways.
  • the PRRT agent comprises a peptide linked to a chelating agent and radionuclide.
  • the present invention provides novel combinations comprising a PI3K/Akt/mTOR inhibitor, with optionally, a dual or independent HDAC inhibitor, which upon inhibition increases the cellular uptake of compounds regulated by variously impacted transmembrane proteins such as norepinephrine transporters, dopamine transporters and somatostatin receptors.
  • Such compounds include radioisotopes of meta-iodobenzylguanidine (mIBG), which may be used for the diagnosis or treatment of neuroendocrine tumours.
  • mIBG meta-iodobenzylguanidine
  • Document WO2018009638 describes compounds based on the N-(2,3- difluorophenyl)-2-fluoro-4-(iodo/alkynyl)aniline moiety which display inhibitory activity against at least two of the PI3K/mTOR/MEK proteins, effecting tumour initiation via the KRAS-pathway.
  • Document WO2010062571 describes derivatives of 3,4-dihydropyrazino[2,3- b]pyranzine-2-(1 H)-ones and their use as inhibitors of kinase pathways, especially mTOR, PI3K or Akt, for the treatment of various conditions including cancer, inflammation and neurodegenerative diseases.
  • Document DE10234201 describes the treatment of neuroendocrine or gastrointestinal tumours that express monoamine transporters, using agents taken up by a monoamine transporter or benzodiazepine receptor. This document does not disclose any inhibitors which facilitate the uptake of molecules by monoamine transporters and is therefore distinct from the present invention.
  • the objective technical problem of the present invention is the provision of methods and compositions for improving the uptake of therapeutic and diagnostic agents in tumours, in particular neuroendocrine tumours.
  • the objective technical problem is solved by the embodiments described herein and as characterized in the claims.
  • the present inventors have found that, unexpectedly, compounds that inhibit PI3K/Akt/mTOR significantly increase the internalization of certain compounds, including mIBG and its radionuclide-bearing analogue, into tumour cells.
  • the use of PI3K/Akt/mTOR inhibitors was unexpectedly found to upregulate monoamine transporters, particularly norepinephrine and dopamine transporters.
  • the inhibition of PI3K/Akt/mTOR was found to increase mRNA production of somatostatin receptors SST1 , 2 and 3 thereby likely increasing the uptake of somatostatin or its analogue.
  • the present invention provides means and methods for utilizing this mechanism for increasing the uptake of radiopharmaceuticals based on catecholamines and somatostatin and their derivatives comprising a radionuclide for therapeutic and diagnostic purposes.
  • Example 1 and Figure 1 show that the use of CUDC-907, a dual PI3K/Akt/mTOR and HDAC inhibitor, increases the expression of norepinephrine and dopamine transporters (NET and DAT, respectively) in NB8, a well characterised human neuroblastoma cell line.
  • CUDC-907 a dual PI3K/Akt/mTOR and HDAC inhibitor
  • Example 2 and 3, and Figure 2 show that NET is the main transporter involved in the uptake of mIBG, whereas DAT plays a minor role.
  • Example 3 and 4 and Figure 2 confirm that the inhibition PI3K is the major pathway involved in increased mIBG uptake as compared to HDAC/Akt/mTOR.
  • SST somatostatin receptors
  • Example 7 compares the treatment of a cohort of NB8 xenograft mice with CUDC-907 and an administered dose of 0.5 MBq of 131 l-mlBG with that of a cohort of the same mice receiving only a vehicle (10% DMSO in corn oil).
  • the biodistribution of 131 l-mlBG in NB8 xenografts was confirmed 4h p.i. and 24h p.i.
  • compositions comprising inhibitors of PI3K/Akt/mTOR and of HDAC (either as separate molecules or as, e.g., a single molecule that is capable of inhibiting both PI3K/Akt/mTOR and HDAC) are particularly efficacious in the uptake of radionuclides.
  • the present invention relates to a composition
  • a composition comprising a) a PI3K/Akt/mTOR inhibitor, and b) a compound whose cellular uptake is upregulated upon inhibition of PI3K/Akt/mTOR, wherein the compound is a radiopharmaceutical.
  • the present invention relates to a pharmaceutical composition
  • a pharmaceutical composition comprising a) a PI3K/Akt/mTOR inhibitor, b) a compound whose cellular uptake is upregulated upon inhibition of PI3K/Akt/mTOR, wherein the compound is a radiopharmaceutical, and a pharmaceutically acceptable carrier.
  • the present invention relates to a kit of parts comprising a) a PI3K/Akt/mTOR inhibitor and a pharmaceutically acceptable carrier, and b) a compound whose cellular uptake is upregulated upon inhibition of PI3K/Akt/mTOR, wherein the compound is a radiopharmaceutical, and a pharmaceutically acceptable carrier.
  • the present invention relates to the composition of the present invention, the pharmaceutical composition of the present invention, or the kit of parts of the present invention, for use in diagnosis or therapy.
  • the present invention relates to the composition of the present invention, the pharmaceutical composition of the present invention, or the kit of parts of the present invention, for use in diagnosis or therapy of a neuroendocrine tumour.
  • the present invention relates to the use of a PI3K/Akt/mTOR inhibitor in a method of upregulating the expression of a norepinephrine transporter (NET) and/or dopamine transporter (DAT).
  • NET norepinephrine transporter
  • DAT dopamine transporter
  • the present invention relates to a PI3K/Akt/mTOR inhibitor for use in the method of upregulating the expression of a somatostatin receptor in a subject, preferably wherein the somatostatin receptor is SST2.
  • Figure 1 presents the effects of CUDC-907 on NB8 cells: NET and DAT mRNA expression and protein levels as described in Example 1.
  • A) NET and DAT mRNA expression levels in NB8 cells incubated with CUDC-907 0.1 pM.
  • Figure 2 presents the cellular mIBG internalization in NB8 cells treated with CUDC- 907 and several classes of inhibitors.
  • Example 3 Comparison of mIBG internalization in NB8 cells following CUDC-907 or BGT226 for 48 hours as described in Example 3.
  • E mIBG internalization following NET and/or DAT knockdown and BGT226 treatment as described in Example 2 and 3.
  • F mIBG internalization in NB8 cells incubated with different PI3K, Akt and mTOR inhibitors for 48 hours as described in Example 4.
  • Figure 3 presents the biodistribution data and SPECT/CT images of 123 l-mlBG in NB8 xenografts at 4 hours and 24 hours post injection (p.i.) as described in Example 5.
  • a representative mouse for each group (vehicle, 5 mg/kg and 10 mg/kg) are reported.
  • the white arrows indicate the gallbladder and adrenal glands which show high uptake at 4 and 24 hours p.i. respectively.
  • the grey arrows indicate that the tumours were better visualized at 24 hours p.i. due to the excellent tumour to background ratio.
  • Figure 4 presents the upregulation of SST mRNA and proteins in prostate cancer cell lines treated with HDAC inhibitors (HDACi) and dual HDACi/PI3K/Akt/mTOR inhibitors as described in Example 6.
  • HDACi HDAC inhibitors
  • FIG. 4 presents the upregulation of SST mRNA and proteins in prostate cancer cell lines treated with HDAC inhibitors (HDACi) and dual HDACi/PI3K/Akt/mTOR inhibitors as described in Example 6.
  • HDACi HDAC inhibitors
  • FIG. 1 SST gene upregulation (as the mRNA fold change) after 48-hour treatment with the indicated HDACi compared with untreated PC3 cells (control).
  • the present invention relates to a composition
  • a composition comprising a) a PI3K/Akt/mTOR inhibitor, and b) a compound whose cellular uptake is upregulated upon inhibition of PI3K/Akt/mTOR, wherein the compound is a radiopharmaceutical.
  • composition of the present invention comprises a PI3K/Akt/mTOR inhibitor. It is meant to be understood that this term refers preferably to at least one PI3K/Akt/mTOR inhibitor. Thus, more than one inhibitor of PI3K/Akt/mTOR inhibitor, for example 2, 3, or 4 inhibitors, are also encompassed by the term.
  • the term “inhibition” relates to a reduction in a certain parameter, e.g., an activity of an enzyme or of a particular pathway.
  • inhibition of a particular enzyme may relate to reduction of its activity of at least 5%, 10%, 20%, 30%, 40% or more is included by this term.
  • the inhibition does need not to be 100% reduction of, e.g., said activity.
  • inhibitor is a molecule capable of causing inhibition, e.g., an immune checkpoint inhibitor, or PI3K/Akt/mTOR inhibitor.
  • PI3K/Akt/mTOR inhibitor preferably relates to a compound that is capable of interfering with the function of phosphoinositide 3-kinase (PI3K) and/or protein-B kinase (Akt) and/or mammalian target of rapamycin (mTOR) in a subject, upon administration to said subject.
  • PI3K phosphoinositide 3-kinase
  • Akt protein-B kinase
  • mTOR mammalian target of rapamycin
  • said interference with certain functions of a protein may refer to its catalytic function.
  • inhibitors of PI3K/Akt/mTOR are substrate-competitive inhibitors or allosteric inhibitors of said proteins. These may be covalent (reversible or irreversible) or non-covalent.
  • inhibition may be measured by inhibition constant, K.
  • an inhibitor of PI3K/Akt/mTOR is capable of inhibiting at least one of these proteins with a Ki not exceeding 10 pM, preferably not exceeding 1 pM, more preferably not exceeding 100 nM, even more preferably not exceeding 10 nM.
  • phosphatidylinositol 3-kinase preferably refers to all class IA PI3K isoforms, which are a part of the PI3K family of kinases.
  • the PI3K enzyme family is divided into four categories: class I, class II, class III, and class IV, which differ in structure and substrate specificity.
  • Class I PI3Ks are heterodimeric molecules composed of a regulatory and a catalytic subunit; they are further divided into IA and IB subsets on sequence similarity.
  • Class IA PI3Ks are composed of a heterodimer between a p110 catalytic subunit and a p85 regulatory subunit.
  • the PI3K family of enzymes are involved in cellular functions such as cell growth, proliferation, differentiation, motility, survival and intracellular trafficking, which in turn are involved in cancer.
  • PI3K p110 subunits have shown the involvement of the catalytic domain of the class IA PI3K p110 subunits in various tumour types (Liu, P. et al. Nat. Rev. Drug Discov. 2009; 8:627-644, Fruman, D. A., and Rommel, C., Nat. Rev. Drug Discov. 2014; 13:140-156).
  • mammalian target of rapamycin (also referred to as the mechanistic target of rapamycin, and sometimes called FK506-binding protein 12- rapamycin-associated protein 1 (FRAP1 )), refers to a kinase which belongs to the phosphatidylinositol 3-kinase-related kinase (PIKK) family.
  • mTOR complex 1 mTORCI
  • mTOR complex 2 mT0RC2
  • mTORCI is a key mediator of transcription and cell growth (via its substrates p70S6 kinase and 4E-BP1 ) and promotes cell survival via the serum and glucocorticoid- activated kinase SGK, whereas mT0RC2 promotes activation of the pro-survival kinase AKT.
  • the catalytic domain of mTOR is homologous to that of PI3K.
  • Dysregulation of PI3K signalling is a common function of tumour cells.
  • mTOR inhibition may be considered as a strategy in many of the tumour types in which PI3K signalling is implicated, and the use of mTOR inhibitors for the treatment of various cancers is known in the art.
  • protein B kinase refers to a family of three serine/threonine-specific protein kinases which play a role in multiple cellular processes including survival pathways via the inhibition of apoptosis, signalling and protein synthesis pathways.
  • Akt may be activated by mT0RC2 via phosphorylation which results in the activation or deactivation of various substrates.
  • Akt is also a downstream effector of PI3K, therefore the rate of its activation directly influences PI3K activity.
  • the therapeutic use of Akt inhibitors in the treatment of various cancers is described in the art.
  • the PI3K/Akt/mTOR inhibitor as referred to herein is selected from BEZ235, BKM120, Everolimus, MK-2206 dichloride, Pictilisib, LY294002, CAL-101 , PI-3065, HS-173, PI-103.
  • BEZ235 is a compound according to formula: or a pharmaceutically acceptable salt thereof.
  • BKM120 is a compound according to formula: or a pharmaceutically acceptable salt thereof.
  • Everolimus is a compound according to formula: or a pharmaceutically acceptable salt thereof.
  • MK-2206 is a compound according to formula: or a pharmaceutically acceptable salt thereof.
  • Pictilisib is a compound according to formula: or a pharmaceutically acceptable salt thereof.
  • LY294002 is a compound according to formula: or a pharmaceutically acceptable salt thereof.
  • CAL-101 is a compound according to formula: or a pharmaceutically acceptable salt thereof.
  • PI-3065 is a compound according to formula: or a pharmaceutically acceptable salt thereof.
  • HS-173 is a compound according to formula: or a pharmaceutically acceptable salt thereof.
  • PI-103 is a compound according to formula: or a pharmaceutically acceptable salt thereof.
  • NU7441 is a compound according to formula: or a pharmaceutically acceptable salt thereof.
  • TGX-221 is a compound according to formula: or a pharmaceutically acceptable salt thereof.
  • IC-87114 is a compound according to formula: or a pharmaceutically acceptable salt thereof.
  • Wortmannin is a compound according to formula: or a pharmaceutically acceptable salt thereof.
  • XL147 is a compound according to formula: or a pharmaceutically acceptable salt thereof.
  • ZSTK474 is a compound according to formula: or a pharmaceutically acceptable salt thereof.
  • BYL719 is a compound according to formula: or a pharmaceutically acceptable salt thereof.
  • AS-605240 is a compound according to formula: or a pharmaceutically acceptable salt thereof.
  • PIK-75 is a compound according to formula: or a pharmaceutically acceptable salt thereof.
  • 3-methyladenine is a compound according to formula: or a pharmaceutically acceptable salt thereof.
  • A66 is a compound according to formula: or a pharmaceutically acceptable salt thereof.
  • SAR245409 is a compound according to formula: or a pharmaceutically acceptable salt thereof.
  • PIK-93 is a compound according to formula: or a pharmaceutically acceptable salt thereof.
  • GSK2126458 is a compound according to formula: or a pharmaceutically acceptable salt thereof.
  • PIK-90 is a compound according to formula: or a pharmaceutically acceptable salt thereof.
  • PF-04691502 is a compound according to formula: or a pharmaceutically acceptable salt thereof.
  • AZD6482 is a compound according to formula: or a pharmaceutically acceptable salt thereof.
  • GDC-0980 is a compound according to formula: or a pharmaceutically acceptable salt thereof.
  • GSK1059615 is a compound according to formula: or a pharmaceutically acceptable salt thereof.
  • Duvelisib is a compound according to formula: or a pharmaceutically acceptable salt thereof.
  • Gedatolisib (PKI-587), is a compound according to formula: or a pharmaceutically acceptable salt thereof.
  • TG100-115 is a compound according to formula: or a pharmaceutically acceptable salt thereof.
  • AS-252424 is a compound according to formula: or a pharmaceutically acceptable salt thereof.
  • BGT226, is a compound according to formula: or a pharmaceutically acceptable salt thereof.
  • CUDC-907 is a compound according to formula: or a pharmaceutically acceptable salt thereof.
  • PIK-294 is a compound according to formula:
  • AS-604850 is a compound according to formula: or a pharmaceutically acceptable salt thereof.
  • GSK2636771 is a compound according to formula: or a pharmaceutically acceptable salt thereof.
  • BAY80-6946 is a compound according to formula:
  • YM201636 is a compound according to formula: or a pharmaceutically acceptable salt thereof.
  • CH5132799 is a compound according to formula: or a pharmaceutically acceptable salt thereof.
  • CAY10505 is a compound according to formula: or a pharmaceutically acceptable salt thereof.
  • Rapamycin is a compound according to formula: or a pharmaceutically acceptable salt thereof.
  • PIK-293 is a compound according to formula: or a pharmaceutically acceptable salt thereof.
  • “pharmaceutically acceptable salt” forms of the inhibitors of the present invention may be formed, e.g., by protonation of an atom carrying an electron lone pair which is susceptible to protonation, such as an amino group, with an inorganic or organic acid, or as a salt of an acid group (such as a carboxylic acid group) with a physiologically acceptable cation.
  • Exemplary base addition salts comprise, for example: alkali metal salts such as sodium or potassium salts; alkaline earth metal salts such as calcium or magnesium salts; zinc salts; ammonium salts; aliphatic amine salts such as trimethylamine, triethylamine, dicyclohexylamine, ethanolamine, diethanolamine, triethanolamine, procaine salts, meglumine salts, ethylenediamine salts, or choline salts; aralkyl amine salts such as N,N-dibenzylethylenediamine salts, benzathine salts, benethamine salts; heterocyclic aromatic amine salts such aspyridine salts, picoline salts, quinoline salts or isoquinoline salts; quaternary ammonium salts such as tetramethylammonium salts, tetraethylammonium salts, benzyltrimethylammonium salts, benzyltriethylammoni
  • Exemplary acid addition salts comprise, for example: mineral acid salts such as hydrochloride, hydrobromide, hydroiodide, sulfate salts (such as, e.g., sulfate or hydrogensulfate salts), nitrate salts, phosphate salts (such as, e.g., phosphate, hydrogenphosphate, or dihydrogenphosphate salts), carbonate salts, hydrogencarbonate salts, perchlorate salts, borate salts, or thiocyanate salts; organic acid salts such as acetate, propionate, butyrate, pentanoate, hexanoate, heptanoate, octanoate, cyclopentanepropionate, decanoate, undecanoate, oleate, stearate, lactate, maleate, oxalate, fumarate, tartrate, malate, citrate, succinate, adipate, gluconate, glycolate, nic
  • Preferred pharmaceutically acceptable salts of the compounds described in the present invention include a hydrochloride salt, a hydrobromide salt, a mesylate salt, a sulfate salt, a tartrate salt, a fumarate salt, an acetate salt, a citrate salt, and a phosphate salt.
  • a particularly preferred pharmaceutically acceptable salt of the compounds as described herein is a hydrochloride or an acetate salt.
  • composition of the present invention comprises CUDC-907 as (a).
  • the composition of the present invention further comprises a radiopharmaceutical whose cellular uptake is upregulated upon inhibition of PI3K/Akt/mTOR.
  • the present inventors have demonstrated that a number of proteins that partake in transport through the cellular membrane, in other words in uptake of certain compounds, are upregulated upon inhibition of PI3K/Akt/mTOR.
  • monoamine transporters are upregulated upon inhibition of PI3K/Akt/mTOR.
  • norepinephrine and dopamine transporters are upregulated upon inhibition of PI3K/Akt/mTOR.
  • radiopharmaceuticals whose cellular uptake is upregulated upon inhibition of PI3K/Akt/mTOR, preferably selected from compounds that are transported by monoamine transporters, in particular by norepinephrine and/or dopamine transporters.
  • radiopharmaceutical refers to a pharmaceutical compound comprising a radionuclide which may be used in diagnostics and/or treatment of a disease. Radionuclides as part of the radiopharmaceuticals of the present invention may emit alpha, beta or gamma radiation.
  • the radionuclide comprised in the radiopharmaceutical of the present invention includes 123 l, 124 l, 125 l, 131 l, 225 Ac, 213 Bi, " m Tc, 111 in, i 77 Lu, 67 Ga, 68 Ga, 90 Y, 64 Cu, 67 Cu, 61 Cu, 43 Sc, 44 Sc, 47 Sc, 212 Pb, 203 Pb, 161 Tb, 152 Tb, 155 Tb and 149 Tb, but is not limited to these radionuclides.
  • upregulated preferably refers to the process of increasing the response to a stimulus, for example an increase in a cellular response to a molecular stimulus due to increase in the number of receptors on the cell surface.
  • the term upregulated relates to increased expression level or increased concentration of a particular transporter or receptor in the organism or in the cell.
  • the compound, in particular the radiopharmaceutical, whose cellular uptake is upregulated upon inhibition of PI3K/Akt/mTOR is a compound that is transported by monoamine transporters, in particular, by NET and/or DAT respectively.
  • a compound that is transported by a particular transporter is preferably meant to mean a compound that can be transported by said transporter, e.g., transported into the cell.
  • the term “monoamine transporter” refers to integral membrane proteins that act to transport monoamines - particularly neurotransmitters such as dopamine, norepinephrine, epinephrine, serotonin and histamine - from the cellular cytosol into synaptic vesicles.
  • NET neuronorepinephrine transporter
  • the NET gene is encoded by 14 exons, which span 45 kb from the start to the stop codon.
  • the nucleotide and deduced amino acid sequence of the transporter predict a protein of 617 amino acids, containing 12 membrane-spanning domains.
  • the organization of the protein is highly homologous to that of other neurotransmitter transporters including those transporting dopamine, epinephrine, serotonin and gamma-aminobutyric acid (GABA), which are members of a family of sodium- and chloride-dependent transport proteins in the plasma membranes of neurons and glial cells.
  • GABA gamma-aminobutyric acid
  • Analysis of the NET gene and protein has facilitated the investigation of its potential role in psychiatric and other neuronal disorders. At least 13 genetic variants of NET have been identified so far by methods such as singlestranded conformational polymorphism analysis.
  • NET is a protein comprising the polypeptide sequence according to any one of SEQ ID NO: 1 to 7.
  • DAT dopamine transporter
  • hDAT human DAT gene
  • the hDAT gene spans over 64 kb, consisting of 15 exons separated by 14 introns.
  • the intron-exon structure of the hDAT gene is most similar to that of the human NET gene.
  • Promoter sequence analysis demonstrated a ‘TATA’-less, ‘CAT’-less and G+C-rich structure.
  • the DAT gene encodes for a 620-amino acid protein with a calculated molecular weight of 68,517 (Giros et al., 1992).
  • DAT is a protein comprising the polypeptide sequence according to SEQ ID NO: 8.
  • the compound, in particular the radiopharmaceutical, whose cellular uptake is upregulated upon inhibition of PI3K/Akt/mTOR can be a radioactive derivative of metaiodobenzylguanidine (mIBG).
  • mIBG is a compound of the formula: or a pharmaceutically acceptable salt thereof.
  • the radionuclide derivative of mIBG is 123 l-meta-iodobenzylguanidine, 124 l- meta-iodobenzylguanidine, 125 l-meta-iodobenzylguanidine or 131 l-meta- iodobenzylguanidine, preferably 123 l-meta-iodobenzylguanidine or 131 l-meta- iodobenzylguanidine.
  • a compound in particular a radiopharmaceutical whose cellular uptake is upregulated upon inhibition of PI3K/Akt/mTOR can be a radioactive derivative of somatostatin or its analogue.
  • somatostatin is a compound of the formula:
  • composition of the present invention comprises a radioactive derivative of somatostatin or its analogue as (b).
  • Analogue of somatostatin is herein defined as a compound, (preferably a peptide compound) capable of binding to somatostatin receptor(s), preferably with a dissociation constant of not more than 1 pM, more preferably not more than 100 nM, even more preferably not more than 10 nM.
  • an analogue of somatostatin has a structure similar to somatostatin, preferably wherein at least 80% of atoms remain unchanged between somatostatin and its analogue, more preferably wherein at least 90% of atoms remain unchanged between somatostatin and its analogue.
  • Preferred analogues of somatostatin include octreotide (and derivatives thereof), TOC, TATE, NOC, AM3, pasireotide, and lanreotide.
  • somatostatin or its analogue is selected from octreotide, TOC, TATE, NOC, AM3, pasireotide and lanreotide. More preferably, somatostatin or its analogue is selected from TATE, AM3 and pasireotide.
  • octreotide is a compound according to formula:
  • TATE is a compound according to formula: or a pharmaceutically acceptable salt thereof.
  • pasireotide is a compound according to formula:
  • lanreotide is a compound according to formula: or a pharmaceutically acceptable salt thereof.
  • AM3 is a compound according to formula:
  • NOC is a compound according to formula: or a pharmaceutically acceptable salt thereof.
  • somatostatin receptors are receptors for the ligand somatostatin, a small neuropeptide associated with neural signalling, particularly in the post-synaptic response to NMDA receptor co-stimulation/activation.
  • SST1 , SST2, SST3, SST4 and SST5 which are G protein-coupled seven transmembrane receptors. All five SSTs are commonly expressed in neuroendocrine tumours, in most cases, SST2 is dominantly expressed followed by SST5, SST3, SST1 and SST4, however this may vary depending on cell type.
  • SSTs in general, are involved in signalling cascades that supress tumour cell proliferation, survival and angiogenesis.
  • SSTs all five SST subtypes are targeted for diagnostic and therapeutic purposes. Stimulation of SSTs can inhibit hormone release from tumours as well as tumour cell growth. SSTs are also involved in neuroendocrine tumour cell apoptosis. The use of somatostatin and its analogues which target somatostatin receptors for the treatment of neuroendocrine tumours is described in the art.
  • the radioactive derivative of somatostatin or its analogue is preferably conjugated to a chelator moiety, optionally loaded with a radionuclide, preferably selected from 225 Ac, 213 Bi, 99m Tc, 11 1 In, 177 Lu, 67 Ga, 68 Ga, 90 Y, 64 Cu, 67 Cu, 61 Cu, 43 Sc, 44 Sc, 47 Sc, 212 Pb, 203 Pb, 161 Tb, 152 Tb, 155 Tb and 149 Tb.
  • a radionuclide preferably selected from 225 Ac, 213 Bi, 99m Tc, 11 1 In, 177 Lu, 67 Ga, 68 Ga, 90 Y, 64 Cu, 67 Cu, 61 Cu, 43 Sc, 44 Sc, 47 Sc, 212 Pb, 203 Pb, 161 Tb, 152 Tb, 155 Tb and 149 Tb.
  • Chelators that may be conjugated to somatostatin or its analogue of the invention include, but are not limited to, 1 ,4,7,10-tetraazacyclododecane-1 ,4,7, 10-tetraacetic acid (DOTA), diethylenetriaminepentaacetic (DTPA), desferrioxamine (DFO) and triethylenetetramine (TETA), 1 ,4,8,1 1 -tetraazabicyclo[6.6.2]hexadecane-4, 1 1 -diacetic acid (CB-TE2A); ethylenediaminetetraacetic acid (EDTA); ethylene glycolbis(2- aminoethyl)-N,N,N',N'-tetraacetic acid (EGTA); 1 ,4,8, 1 1 -tetraazacyclotetradecane- 1 ,4,8, 1 1 -tetraacetic acid (TETA); ethylenebis-(2-4 hydroxy-phenylglycine) (EH
  • the payload may comprise more than one chelator.
  • Other preferred chelators can be selected from the group consisting of cyclic DTPA (diethylene triaminepentaacetic acid) anhydride, ethylenediaminetetraacetic acid (EDTA), DOTA (1 ,4,7,10- tetraazacyclododecane-1 ,4,7,10- tetraacetic acid), and OTA (1 ,4,7-triazonane-l,4,7- triacetic acid).
  • EDTA ethylenediaminetetraacetic acid
  • DOTA 1,4,7,10- tetraazacyclododecane-1 ,4,7,10- tetraacetic acid
  • OTA 1,4,7-triazonane-l,4,7- triacetic acid
  • somatostatin or its analogue is AM3
  • somatostatin or its analogue conjugated to a chelator moiety is a compound according to formula or a pharmaceutically acceptable salt thereof.
  • the chelator moiety is DOTA.
  • somatostatin or its analogue is NOC
  • somatostatin or its analogue conjugated to a chelator moiety is a compound according to formula: or a pharmaceutically acceptable salt thereof.
  • the chelator moiety is DOTA.
  • a compound, in particular a radiopharmaceutical whose cellular uptake is upregulated upon inhibition of PI3K/Akt/mTOR can be a compound/radiopharmaceutical that is transported by a serotonin transporter (SERT), gastrin-releasing peptide receptors (BB2/GRPR), cholecystokinin B (CCK2) receptor, glucagon-like peptide 1 (GLP-1 ) receptor, glucosedependent insulinotropic polypeptide (GIP) receptor and/or neuropeptide Y (Y1 ) receptor.
  • SERT serotonin transporter
  • BB2/GRPR gastrin-releasing peptide receptors
  • CCK2 cholecystokinin B
  • GLP-1 glucagon-like peptide 1
  • GIP glucosedependent insulinotropic polypeptide
  • Y1 neuropeptide Y
  • the Table below lists the targeting systems, i.e., peptide compounds and their target receptors typically used in nuclear oncology.
  • composition of the present invention as described herein further comprises a HDAC inhibitor.
  • HDAC inhibitor preferably relates to a compound that is capable of interfering with the function of histone deacetylases (i.e. a class of enzymes that remove acetyl groups from a side chain N-acetyl lysine residue on a histone) in a subject, upon administration to said subject.
  • histone deacetylases i.e. a class of enzymes that remove acetyl groups from a side chain N-acetyl lysine residue on a histone
  • said interference with certain functions of a protein may refer to its catalytic function.
  • inhibitors of HDAC are substrate-competitive inhibitors or allosteric inhibitors of said proteins. These may be covalent (reversible or irreversible) or non-covalent. As known to the skilled person, inhibition may be measured by inhibition constant, Ki.
  • an inhibitor of HDAC is capable of inhibiting at least one of histone deacetylases with a Ki not exceeding 10 pM, preferably not exceeding 1 pM, more preferably not exceeding 100 nM, even more preferably not exceeding 10 nM.
  • histone deacetylases comprise HDAC1 , HDAC2, HDAC3, HDAC4, HDAC5, HDAC6, HDAC7, HDAC8, HDAC9, HDAC10, HDAC11 , SIRT1 , SIRT2, SIRT3, SIRT4. SIRT5, SIRT6, and SIRT7.
  • HDAC inhibitor is selected from Vorinostat (SAHA), Romidepsin (FK288), Chidamide, Panobinostat (LBH589), Belinostat (PXD101 ), Valproic acid, Tacedinaline, Mocetinostat, Abexinostat (PCI24781 ), MS275-SNDX- 275, Pracinostat (SB939), Resminostat (4SC201 ), Givinostat (IFT2357), Quisinostat (JNJ-26481585), HBI-8000, Kevetrin, CUDC-101 , AR42, Tefinostat (CHR-2845), CHR-3996, 4SC202, CG200745, Rocilinostat (ACY1215), and ME-344.
  • SAHA Vorinostat
  • Romidepsin FK288)
  • Chidamide Chidamide
  • Panobinostat LH589)
  • Belinostat PXD101
  • Valproic acid Valproic
  • the present invention relates to a pharmaceutical composition
  • a pharmaceutical composition comprising a) a PI3K/Akt/mTOR inhibitor, b) a compound/radiopharmaceutical whose cellular uptake is upregulated upon inhibition of PI3K/Akt/mTOR, and a pharmaceutically acceptable carrier.
  • the pharmaceutical composition of the present invention comprises a PI3K/Akt/mTOR inhibitor.
  • the a PI3K/Akt/mTOR inhibitor is as described hereinabove.
  • the PI3K/Akt/mTOR inhibitor as referred to herein is selected from BEZ235, BKM120, Everolimus, MK-2206 dichloride, Pictilisib, LY294002, CAL-101 , PI-3065, HS-173, PI- 103 , NU7441 , TGX-221 , IC-87114, Wortmannin, XL147, ZSTK474, BYL719, AS- 605240, PIK-75, 3-methyladenine, A66, SAR245409, PIK-93, GSK2126458, PIK-90, PF-04691502, AZD6482, Apitolisib, GSK1059615, Duvelisib, Gedatolisib, TG100-115, AS-252424, BGT226, CUDC-907, PIK-294, AS-604850, GSK2636771 , BAY80-6946
  • Particularly preferred PI3K/Akt/mTOR inhibitor is CUDC-907.
  • the pharmaceutical composition of the present invention comprises CUDC-907 as (a).
  • the pharmaceutical composition of the present invention further comprises a radiopharmaceutical/compound whose cellular uptake is upregulated upon inhibition of PI3K/Akt/mTOR.
  • the radiopharmaceutical/compound whose cellular uptake is upregulated upon inhibition of PI3K/Akt/mTOR is as described hereinabove.
  • the compound whose cellular uptake is upregulated upon inhibition of PI3K/Akt/mTOR is selected from radiopharmaceuticals/compounds that are transported by monoamine transporters, in particular by norepinephrine and/or dopamine transporters.
  • the radiopharmaceutical/compound whose cellular uptake is upregulated upon inhibition of PI3K/Akt/mTOR can be a radioactive derivative of metaiodobenzylguanidine (mIBG).
  • mIBG metaiodobenzylguanidine
  • the radionuclide derivative of mIBG is 123 l-meta-iodobenzylguanidine, 124 l- meta-iodobenzylguanidine, 125 l-meta-iodobenzylguanidine or 131 l-meta- iodobenzylguanidine, preferably 123 l-meta-iodobenzylguanidine or 131 l-meta- iodobenzylguanidine.
  • the pharmaceutical composition of the present invention comprises 123 l-meta-iodobenzylguanidine, 124 l-meta- iodobenzylguanidine, 125 l-meta-iodobenzylguanidine or 131 l-meta- iodobenzylguanidine, preferably 123 l-meta-iodobenzylguanidine or 131 l-meta- iodobenzylguanidine, more preferably 131 l-meta-iodobenzylguanidine.
  • the radiopharmaceutical/compound whose cellular uptake is upregulated upon inhibition of PI3K/Akt/mTOR can be a radioactive derivative of somatostatin or its analogue.
  • Preferred analogues of somatostatin include octreotide (and derivatives thereof), TOC, TATE, NOC, AM3, pasireotide, and lanreotide.
  • somatostatin or its analogue is selected from octreotide, TOC, TATE, NOC, AM3, pasireotide and lanreotide. More preferably, somatostatin or its analogue is selected from TATE, AM3 and pasireotide.
  • the radioactive derivative of somatostatin or its analogue is preferably conjugated to a chelator moiety, optionally loaded with a radionuclide, preferably selected from 225 Ac, 213 Bi, 99m Tc, 1 11 1n, 177 Lu, 67 Ga, 68 Ga, 90 Y, 64 Cu, 67 Cu, 61 Cu, 43 Sc, 44 Sc, 47 Sc, 212 Pb, 203 Pb, 161 Tb, 152 Tb, 155 Tb and 149 Tb.
  • a radionuclide preferably selected from 225 Ac, 213 Bi, 99m Tc, 1 11 1n, 177 Lu, 67 Ga, 68 Ga, 90 Y, 64 Cu, 67 Cu, 61 Cu, 43 Sc, 44 Sc, 47 Sc, 212 Pb, 203 Pb, 161 Tb, 152 Tb, 155 Tb and 149 Tb.
  • Chelators that may be conjugated to somatostatin or its analogue of the invention include, but are not limited to, 1 ,4,7,10-tetraazacyclododecane-1 ,4,7, 10-tetraacetic acid (DOTA), diethylenetriaminepentaacetic (DTPA), desfemoxamine (DFO) and triethylenetetramine (TETA), 1 ,4,8,1 1 -tetraazabicyclo[6.6.2]hexadecane-4, 1 1 -diacetic acid (CB-TE2A); ethylenediaminetetraacetic acid (EDTA); ethylene glycolbis(2- aminoethyl)-N,N,N',N'-tetraacetic acid (EGTA); 1 ,4,8, 1 1 -tetraazacyclotetradecane- 1 ,4,8, 1 1 -tetraacetic acid (TETA); ethylenebis-(2-4 hydroxy-phenylglycine) (E
  • the payload may comprise more than one chelator.
  • Other preferred chelators can be selected from the group consisting of cyclic DTPA (diethylene triaminepentaacetic acid) anhydride, ethylenediaminetetraacetic acid (EDTA), DOTA (1 ,4,7, 10- tetraazacyclododecane-1 ,4,7, 10- tetraacetic acid), and OTA (1 ,4,7-triazonane-l,4,7- triacetic acid).
  • EDTA ethylenediaminetetraacetic acid
  • OTA 1,4,7-triazonane-l,4,7- triacetic acid
  • the compound/radiopharmaceutical whose cellular uptake is upregulated upon inhibition of PI3K/Akt/mTOR can be a compound that is transported by a serotonin transporter (SERT), gastrin-releasing peptide receptors (BB2/GRPR), cholecystokinin B (CCK2) receptor, glucagon-like peptide 1 (GLP-1 ) receptor, glucose-dependent insulinotropic polypeptide (GIP) receptor and/or neuropeptide Y (Y1 ) receptor.
  • SERT serotonin transporter
  • BB2/GRPR gastrin-releasing peptide receptors
  • CCK2 cholecystokinin B
  • GLP-1 glucagon-like peptide 1
  • GIP glucose-dependent insulinotropic polypeptide
  • Y1 neuropeptide Y
  • the pharmaceutical composition of the present invention further comprises a pharmaceutically acceptable carrier.
  • the compounds provided herein may be administered as compounds per se or may be formulated as medicaments.
  • the medicaments/pharmaceutical compositions may optionally comprise one or more pharmaceutically acceptable carriers, such as diluents, fillers, disintegrants, lubricating agents, binders, colorants, pigments, stabilizers, preservatives, antioxidants, and/or solubility enhancers.
  • compositions of the invention may comprise one or more solubility enhancers, such as, e.g., polyethylene glycol), including poly(ethylene glycol) having a molecular weight in the range of about 200 to about 5,000 Da (e.g., PEG 200, PEG 300, PEG 400, or PEG 600), ethylene glycol, propylene glycol, glycerol, a non-ionic surfactant, tyloxapol, polysorbate 80, macrogol-15-hydroxystearate (e.g., Kolliphor® HS 15, CAS 70142-34-6), a phospholipid, lecithin, dimyristoyl phosphatidylcholine, dipalmitoyl phosphatidylcholine, distearoyl phosphatidylcholine, a cyclodextrin, a- cyclodextrin, [3-cyclodextrin, y-cyclodextrin, hydroxyethyl-[3-cyclo
  • compositions of the invention may also comprise one or more preservatives, particularly one or more antimicrobial preservatives, such as, e.g., benzyl alcohol, chlorobutanol, 2 ethoxyethanol, m cresol, chlorocresol (e.g., 2-chloro- 3-methyl-phenol or 4-chloro-3-methyl-phenol), benzalkonium chloride, benzethonium chloride, benzoic acid (or a pharmaceutically acceptable salt thereof), sorbic acid (or a pharmaceutically acceptable salt thereof), chlorhexidine, thimerosal, or any combination thereof.
  • preservatives particularly one or more antimicrobial preservatives, such as, e.g., benzyl alcohol, chlorobutanol, 2 ethoxyethanol, m cresol, chlorocresol (e.g., 2-chloro- 3-methyl-phenol or 4-chloro-3-methyl-phenol), benzalkonium chloride, benzethonium chloride, benzo
  • compositions can be formulated by techniques known to the person skilled in the art, such as the techniques published in “Remington: The Science and Practice of Pharmacy”, Pharmaceutical Press, 22nd edition.
  • the pharmaceutical compositions can be formulated as dosage forms for oral, parenteral, such as intramuscular, intravenous, subcutaneous, intradermal, intraarterial, intracardial, rectal, nasal, topical, aerosol or vaginal administration.
  • Dosage forms for oral administration include coated and uncoated tablets, soft gelatin capsules, hard gelatin capsules, lozenges, troches, solutions, emulsions, suspensions, syrups, elixirs, powders and granules for reconstitution, dispersible powders and granules, medicated gums, chewing tablets and effervescent tablets.
  • Dosage forms for parenteral administration include solutions, emulsions, suspensions, dispersions and powders and granules for reconstitution. Emulsions are a preferred dosage form for parenteral administration.
  • Dosage forms for rectal and vaginal administration include suppositories and ovula.
  • Dosage forms for nasal administration can be administered via inhalation and insufflation, for example by a metered inhaler.
  • Dosage forms for topical administration include creams, gels, ointments, salves, patches and transdermal delivery systems.
  • the pharmaceutical composition of the present invention as described herein further comprises a HDAC inhibitor.
  • the HDAC inhibitor in the pharmaceutical composition of the present invention is selected from Vorinostat (SAHA), Romidepsin (FK288), Chidamide, Panobinostat (LBH589), Belinostat (PXD101 ), Valproic acid, Tacedinaline, Mocetinostat, Abexinostat (PCI24781 ), MS275-SNDX-275, Pracinostat (SB939), Resminostat (4SC201 ), Givinostat (IFT2357), Quisinostat (JNJ-26481585), HBI-8000, Kevetrin, CUDC-101 , AR42, Tefinostat (CHR-2845), CHR-3996, 4SC202, CG200745, Rocilinostat (ACY1215), and ME-344.
  • the present invention relates to a kit of parts comprising a) a PI3K/Akt/mTOR inhibitor and a pharmaceutically acceptable carrier, and b) a radiopharmaceutical/compound whose cellular uptake is upregulated upon inhibition of PI3K/Akt/mTOR and a pharmaceutically acceptable carrier.
  • the kit of parts of the present invention comprises the PI3K/Akt/mTOR inhibitor and a pharmaceutically acceptable carrier.
  • the PI3K/Akt/mTOR inhibitor is as described hereinabove.
  • the PI3K/Akt/mTOR inhibitor as referred to herein is selected from BEZ235, BKM120, Everolimus, MK-2206 dichloride, Pictilisib, LY294002, CAL-101 , PI-3065, HS-173, PI-103.
  • a Particularly preferred PI3K/Akt/mTOR inhibitor is CUDC-907.
  • the kit of parts of the present invention comprises CUDC-907 as PI3K/Akt/mTOR inhibitor.
  • the pharmaceutically acceptable carrier is known to the skilled person and is preferably as described hereinabove.
  • the kit of parts of the present invention further comprises a compound whose cellular uptake is upregulated upon inhibition of PI3K/Akt/mTOR and a pharmaceutically acceptable carrier.
  • the radiopharmaceutical/compound whose cellular uptake is upregulated upon inhibition of PI3K/Akt/mTOR is as described hereinabove.
  • the radiopharmaceutical/ whose cellular uptake is upregulated upon inhibition of PI3K/Akt/mTOR is selected from compounds that are transported by monoamine transporters, in particular by norepinephrine and/or dopamine transporters.
  • the radiopharmaceutical/ whose cellular uptake is upregulated upon inhibition of PI3K/Akt/mTOR is a radioactive derivative of meta-iodobenzylguanidine (mIBG).
  • the radionuclide derivative of mIBG is 123 l-meta-iodobenzylguanidine, 124 l- meta-iodobenzylguanidine, 125 l-meta-iodobenzylguanidine or 131 l-meta- iodobenzylguanidine, preferably 123 l-meta-iodobenzylguanidine or 131 l-meta- iodobenzylguanidine.
  • the kit of parts of the present invention comprises 123 l-meta-iodobenzylguanidine, 124 l-meta-iodobenzylguanidine, 125 l-meta- iodobenzylguanidine or 131 l-meta-iodobenzylguanidine, preferably 123 l-meta- iodobenzylguanidine or 131 l-meta-iodobenzylguanidine, more preferably 131 l-meta- iodobenzylguanidine.
  • the radiopharmaceutical/compound whose cellular uptake is upregulated upon inhibition of PI3K/Akt/mTOR can be a radioactive derivative of somatostatin or its analogue.
  • Preferred analogues of somatostatin include octreotide (and derivatives thereof), TOC, TATE, NOC, AM3, pasireotide, and lanreotide.
  • somatostatin or its analogue is selected from octreotide, TOC, TATE, NOC, AM3, pasireotide and lanreotide. More preferably, somatostatin or its analogue is selected from TATE, AM3 and pasireotide.
  • the radioactive derivative of somatostatin or its analogue is preferably conjugated to a chelator moiety, optionally loaded with a radionuclide, preferably selected 225 Ac, 213 Bi, 99m Tc, 1 11 In, 177 Lu, 67 Ga, 68 Ga, 90 Y, 64 Cu, 67 Cu, 61 Cu, 43 Sc, 44 Sc, 47 Sc, 212 Pb, 203 Pb, 161 Tb, 152 Tb, 155 Tb and 149 Tb.
  • a radionuclide preferably selected 225 Ac, 213 Bi, 99m Tc, 1 11 In, 177 Lu, 67 Ga, 68 Ga, 90 Y, 64 Cu, 67 Cu, 61 Cu, 43 Sc, 44 Sc, 47 Sc, 212 Pb, 203 Pb, 161 Tb, 152 Tb, 155 Tb and 149 Tb.
  • Chelators that may be conjugated to somatostatin or its analogue of the invention include, but are not limited to, 1 ,4,7,10-tetraazacyclododecane-1 ,4,7,10-tetraacetic acid (DOTA), diethylenetriaminepentaacetic (DTPA), desferrioxamine (DFO) and triethylenetetramine (TETA), 1 ,4,8,11-tetraazabicyclo[6.6.2]hexadecane-4,11 -diacetic acid (CB-TE2A); ethylenediaminetetraacetic acid (EDTA); ethylene glycolbis(2- aminoethyl)-N,N,N',N'-tetraacetic acid (EGTA); 1 ,4,8,11-tetraazacyclotetradecane- 1 ,4,8,11 -tetraacetic acid (TETA); ethylenebis-(2-4 hydroxy-phenylglycine) (EHPG); 5- CI
  • the payload may comprise more than one chelator.
  • Other preferred chelators can be selected from the group consisting of cyclic DTPA (diethylene triaminepentaacetic acid) anhydride, ethylenediaminetetraacetic acid (EDTA), DOTA (1 ,4,7,10- tetraazacyclododecane-1 ,4,7,10- tetraacetic acid), and OTA (1 ,4,7-triazonane-l,4,7- triacetic acid).
  • EDTA ethylenediaminetetraacetic acid
  • DOTA 1,4,7,10- tetraazacyclododecane-1 ,4,7,10- tetraacetic acid
  • OTA 1,4,7-triazonane-l,4,7- triacetic acid
  • the radiopharmaceuticals of the present invention preferably target specific cells, such as cancer cells such that a diagnostic and/or therapeutic outcome is achieved. Targeting of cells is typically achieved via an interaction between the radiopharmaceutical and its target.
  • mIBG may be used for targeting cancer cells, in particular neuroendocrine cells, as it is structurally similar to norepinephrine which is transported by NET. Neuroendocrine cells express NET, thus enabling tumor-selective imaging and therapy.
  • the compound whose cellular uptake is upregulated upon inhibition of PI3K/Akt/mTOR can be a compound that is transported by a serotonin transporter (SERT), gastrin-releasing peptide receptors (BB2/GRPR), cholecystokinin B (CCK2) receptor, glucagon-like peptide 1 (GLP-1 ) receptor, glucose-dependent insulinotropic polypeptide (GIP) receptor and/or neuropeptide Y (Y1 ) receptor.
  • SERT serotonin transporter
  • BB2/GRPR gastrin-releasing peptide receptors
  • CCK2 cholecystokinin B
  • GLP-1 glucagon-like peptide 1
  • GIP glucose-dependent insulinotropic polypeptide
  • Y1 neuropeptide Y
  • the pharmaceutically acceptable carrier is known to the skilled person and preferably is as described hereinabove.
  • kit of parts of the present invention as described herein further comprises an HDAC inhibitor.
  • HDAC inhibitor in the pharmaceutical composition of the present invention is selected from Vorinostat (SAHA), Romidepsin (FK288), Chidamide, Panobinostat (LBH589), Belinostat (PXD101 ), Valproic acid, Tacedinaline, Mocetinostat, Abexinostat (PCI24781 ), MS275-SNDX-275, Pracinostat (SB939), Resminostat (4SC201 ), Givinostat (IFT2357), Quisinostat (JNJ-26481585), HBI-8000, Kevetrin, CUDC-101 , AR42, Tefinostat (CHR-2845), CHR-3996, 4SC202, CG200745, Rocilinostat (ACY1215), and ME-344.
  • compositions of the present invention are useful in diagnosis or therapy.
  • the present invention relates to the composition of the present invention, the pharmaceutical composition of the present invention or the kit of parts of the present invention for use in diagnosis or therapy.
  • kits of parts of the present invention it is to be understood that the parts of the kit of parts of the present invention can be administered to a subject simultaneously, separately or sequentially.
  • therapy relates, preferably, to treatment of prevention of a disease.
  • treatment of a disorder or disease, as used herein, is well known in the art.
  • Treatment of a disorder or disease implies that a disorder or disease is suspected or has been diagnosed in a patient/subject.
  • a patient/subject suspected of suffering from a disorder or disease typically shows specific clinical and/or pathological symptoms which a skilled person can easily attribute to a specific pathological condition (i.e., diagnose a disorder or disease).
  • the “treatment” of a disorder or disease may, for example, lead to a halt in the progression of the disorder or disease (e.g., no deterioration of symptoms) or a delay in the progression of the disorder or disease (in case the halt in progression is of a transient nature only).
  • the “treatment” of a disorder or disease may also lead to a partial response (e.g., amelioration of symptoms) or complete response (e.g., disappearance of symptoms) of the subject/patient suffering from the disorder or disease.
  • the “treatment” of a disorder or disease may also refer to an amelioration of the disorder or disease, which may, e.g., lead to a halt in the progression of the disorder or disease or a delay in the progression of the disorder or disease.
  • Such a partial or complete response may be followed by a relapse.
  • a subject/patient may experience a broad range of responses to a treatment (such as the exemplary responses as described herein above).
  • the treatment of a disorder or disease may, inter alia, comprise curative treatment (preferably leading to a complete response and eventually to healing of the disorder or disease) and palliative treatment (including symptomatic relief).
  • prevention of a disorder or disease is also well known in the art.
  • a patient/subject suspected of being prone to suffer from a disorder or disease may particularly benefit from a prevention of the disorder or disease.
  • the subject/patient may have a susceptibility or predisposition for a disorder or disease, including but not limited to hereditary predisposition.
  • Such a predisposition can be determined by standard methods or assays, using, e.g., genetic markers or phenotypic indicators.
  • a disorder or disease to be prevented in accordance with the present invention has not been diagnosed or cannot be diagnosed in the patient/subject (for example, the patient/subject does not show any clinical or pathological symptoms).
  • the term “prevention” comprises the use of a compound of the present invention before any clinical and/or pathological symptoms are diagnosed or determined or can be diagnosed or determined by the attending physician.
  • diagnosis relates to a process of determining whether a subject is suffering from any particular disease or disorder or a process of determining which disease or condition explains symptoms and sign exhibited by said subject.
  • Successful diagnosis that said subject is suffering from a particular disease allows its targeted treatment.
  • Modem diagnosis methods involve often the use of diagnostic agents, i.e. compounds and composition that are administered to a subject in order to aid the process of diagnosis.
  • the diagnostic agents may be suitable for easy identification and quantification, for example, in computed tomography (CT), single photon emission computed tomography (SPECT), magnetic resonance imaging (MRI), positron emission tomography (PET), ultrasound imaging or via in vivo optical detection of fluorophores in the far red/near IR spectral range.
  • diagnosis or therapy relates herein to the diagnosis or therapy of a neuroendocrine tumour.
  • Neuroendocrine tumour as encompassed in the present invention is not particularly limited.
  • neuroendocrine tumour is a neuroendocrine tumour characterized by increased expression of monoamine transporters, somatostatin receptors, serotonin transporter (SERT), gastrin-releasing peptide receptors (BB2/GRPR), cholecystokinin B (CCK2) receptor, glucagon-like peptide 1 (GLP-1 ) receptor, glucose-dependent insulinotropic polypeptide (GIP) receptor and/or neuropeptide Y (Y1 ) receptor.
  • monoamine transporters somatostatin receptors
  • SERT serotonin transporter
  • BB2/GRPR gastrin-releasing peptide receptors
  • CCK2 cholecystokinin B
  • GLP-1 glucagon-like peptide 1
  • GIP glucose-dependent insulinotropic polypeptide
  • Y1 neuro
  • the neuroendocrine tumour is characterized by increased expression of monoamine transporters and/or somatostatin receptors. Even more preferably, the neuroendocrine tumour is characterized by increased expression of monoamine transporters. Particularly preferred neuroendocrine tumours are neuroblastoma, pheochromocytoma and paraganglioma.
  • the present invention relates to the composition of the present invention, the pharmaceutical composition of the present invention or the kit of parts of the present invention for use in diagnosis.
  • a particularly preferred radionuclide analogue of mIBG for use in diagnosis is 123 l-meta-iodobenzylguanidine.
  • the present invention relates to the composition of the present invention, the pharmaceutical composition of the present invention or the kit of parts of the present invention for use in diagnosis wherein the compound whose cellular uptake is upregulated upon inhibition of PI3K/Akt/mTOR is 123 l-meta-iodobenzylguanidine.
  • the present invention relates to the composition of the present invention, the pharmaceutical composition of the present invention or the kit of parts of the present invention for use in therapy.
  • a particularly preferred radionuclide analogue of mIBG for use in therapy is 131 l-meta-iodobenzylguanidine.
  • the present invention relates to the composition of the present invention, the pharmaceutical composition of the present invention or the kit of parts of the present invention for use in therapy, wherein the compound whose cellular uptake is upregulated upon inhibition of PI3K/Akt/mTOR is 131 l-meta-iodobenzylguanidine.
  • compositions, pharmaceutical compositions or parts of the kit of parts may be administered to a subject by any convenient route of administration, whether systemically/peripherally or at the site of desired action, including but not limited to one or more of: oral (e.g., as a tablet, capsule, or as an ingestible solution), topical (e.g., transdermal, intranasal, ocular, buccal, and sublingual), parenteral (e.g., using injection techniques or infusion techniques, and including, for example, by injection, e.g., subcutaneous, intradermal, intramuscular, intravenous, intraarterial, intracardiac, intrathecal, intraspinal, intracapsular, subcapsular, intraorbital, intraperitoneal, intratracheal, subcuticular, intraarticular, subarachnoid, or intrasternal by, e.g., implant of a depot, for example, subcutaneously or intramuscularly), pulmonary (e.g., by inhalation or
  • compositions or parts of said kit of parts are administered parenterally, then examples of such administration include one or more of: intravenously, intraarterially, intraperitoneally, intrathecally, intraventricularly, intraurethrally, intrasternally, intracardially, intracranially, intramuscularly or subcutaneously administering the compounds or pharmaceutical compositions, and/or by using infusion techniques.
  • parenteral administration the compounds, the compositions are best used in the form of a sterile aqueous solution which may contain other substances, for example, enough salts or glucose to make the solution isotonic with blood.
  • the aqueous solutions should be suitably buffered (preferably to a pH of from 3 to 9), if necessary.
  • the preparation of suitable parenteral formulations under sterile conditions is readily accomplished by standard pharmaceutical techniques well known to those skilled in the art.
  • compositions, said pharmaceutical compositions or parts of said kit of parts can also be administered orally in the form of tablets, capsules, ovules, elixirs, solutions or suspensions, which may contain flavoring or coloring agents, for immediate-, delayed- , modified-, sustained-, pulsed- or controlled-release applications.
  • the tablets may contain excipients such as microcrystalline cellulose, lactose, sodium citrate, calcium carbonate, dibasic calcium phosphate and glycine, disintegrants such as starch (preferably corn, potato or tapioca starch), sodium starch glycolate, croscarmellose sodium and certain complex silicates, and granulation binders such as polyvinylpyrrolidone, hydroxypropylmethylcellulose (HPMC), hydroxypropylcellulose (HPC), sucrose, gelatin and acacia. Additionally, lubricating agents such as magnesium stearate, stearic acid, glyceryl behenate and talc may be included. Solid compositions of a similar type may also be employed as fillers in gelatin capsules.
  • excipients such as microcrystalline cellulose, lactose, sodium citrate, calcium carbonate, dibasic calcium phosphate and glycine
  • disintegrants such as starch (preferably corn, potato or tapioca starch), sodium starch glyco
  • Preferred excipients in this regard include lactose, starch, a cellulose, or high molecular weight polyethylene glycols.
  • the agent may be combined with various sweetening or flavoring agents, coloring matter or dyes, with emulsifying and/or suspending agents and with diluents such as water, ethanol, propylene glycol and glycerin, and combinations thereof.
  • compositions, the pharmaceutical compositions or parts of the kit of parts are preferably administered by oral ingestion, particularly by swallowing.
  • the compositions, the pharmaceutical compositions or the parts of the kit of parts can thus be administered to pass through the mouth into the gastrointestinal tract, which can also be referred to as “oral-gastrointestinal” administration.
  • compositions, pharmaceutical compositions or parts of the kit of parts can be administered in the form of a suppository or pessary, or may be applied topically in the form of a gel, hydrogel, lotion, solution, cream, ointment or dusting powder.
  • the compositions, pharmaceutical compositions or parts of the kit of parts of the present invention may also be dermally or trans-dermally administered, for example, by the use of a skin patch.
  • sustained-release compositions include semi permeable polymer matrices in the form of shaped articles, e.g., films, or microcapsules.
  • Sustained-release matrices include, e.g., polylactides, copolymers of L glutamic acid and gamma-ethyl-L-glutamate, poly(2- hydroxyethyl methacrylate), ethylene vinyl acetate, or poly D (-)-3-hydroxybutyric acid.
  • Sustained-release pharmaceutical compositions also include liposomally entrapped compounds. The present invention thus also relates to liposomes containing compounds comprised in the composition, pharmaceutical composition or parts of the kit of the invention.
  • compositions, pharmaceutical compositions or parts of the kit of parts may also be administered by the pulmonary route, rectal routes, or the ocular route.
  • they can be formulated as micronized suspensions in isotonic, pH adjusted, sterile saline, or, preferably, as solutions in isotonic, pH adjusted, sterile saline, optionally in combination with a preservative such as a benzalkonium chloride.
  • they may be formulated in an ointment such as petrolatum.
  • dry powder formulations of the compositions, pharmaceutical compositions or parts of the kit of parts for pulmonary administration, particularly inhalation may be prepared by spray drying under conditions which result in a substantially amorphous glassy or a substantially crystalline bioactive powder. Accordingly, dry powders of the compounds of the present invention can be made according to an emulsification/spray drying process.
  • compositions, pharmaceutical compositions or parts of the kit of parts can be formulated as a suitable ointment containing the active compound suspended or dissolved in, for example, a mixture with one or more of the following: mineral oil, liquid petrolatum, white petrolatum, propylene glycol, emulsifying wax and water.
  • suitable lotion or cream suspended or dissolved in, for example, a mixture of one or more of the following: mineral oil, sorbitan monostearate, a polyethylene glycol, liquid paraffin, polysorbate 60, cetyl esters wax, 2-octyldodecanol, benzyl alcohol and water.
  • the present invention thus relates to the compositions, pharmaceutical compositions or parts of the kit of parts provided herein that are to be administered by any one of: an oral route; topical route, including by transdermal, intranasal, ocular, buccal, or sublingual route; parenteral route using injection techniques or infusion techniques, including by subcutaneous, intradermal, intramuscular, intravenous, intraarterial, intracardiac, intrathecal, intraspinal, intracapsular, subcapsular, intraorbital, intraperitoneal, intratracheal, subcuticular, intraarticular, subarachnoid, intrasternal, intraventricular, intraurethral, or intracranial route; pulmonary route, including by inhalation or insufflation therapy; gastrointestinal route; intrauterine route; intraocular route; subcutaneous route; ophthalmic route, including by intravitreal, or intracameral route; rectal route; or vaginal route.
  • Preferred routes of administration are oral administration or parenteral administration.
  • the present invention relates to a PI3K/Akt/mT0R inhibitor for use in diagnosis.
  • the present inventors have surprisingly found that a PI3K/Akt/mT0R upon administration of a subject is useful in combination with diagnostic agents, for example for a combination with a radioactive derivative of mIBG as it increases its cellular uptake, thereby increasing the sensitivity of the diagnostic methods.
  • said PI3K/Akt/mT0R inhibitor is selected from BEZ235, BKM120, Everolimus, MK-2206 dichloride, Pictilisib, LY294002, CAL-101 , PI-3065, HS-173, PI- 103.
  • Particularly preferred PI3K/Akt/mTOR inhibitor for use in diagnosis is CUDC-907.
  • the present invention relates to the use of a PI3K/Akt/mTOR inhibitor in the method of upregulating the expression of norepinephrine transporter (NET) and/or dopamine transporter (DAT).
  • NET norepinephrine transporter
  • DAT dopamine transporter
  • the present invention relates to the use of a PI3K/Akt/mT0R inhibitor in the method of upregulating the expression of a somatostatin receptor in a subject.
  • a PI3K/Akt/mT0R inhibitor in the method of upregulating the expression of a somatostatin receptor in a subject.
  • the somatostatin receptor is SST2.
  • the present invention preferably relates to the use of a PI3K/Akt/mT0R inhibitor as described hereinabove, wherein the PI3K/Akt/mT0R inhibitor is selected from BEZ235, BKM120, Everolimus, MK-2206 dichloride, Pictilisib, LY294002, CAL-101 , PI- 3065, HS-173, PI-103.
  • the present invention further relates to a method for treating a neuroendocrine tumour in a subject, the method comprising the steps of: a) administering a PI3K inhibitor to a subject in need thereof, followed by b) administering a compound, in particular a radiopharmaceutical whose cellular uptake is upregulated upon inhibition of PI3K/Akt/mTOR to a subject in need thereof.
  • a therapeutically effective amounts of the PI3K inhibitor and of the radiopharmaceutical/compound whose cellular uptake is upregulated upon inhibition of PI3K/Akt/mT0R is to be administered in accordance with the method.
  • the PI3K inhibitor as well as radiopharmaceutical/compound whose cellular uptake is upregulated upon inhibition of PI3K/Akt/mTOR can be administered to a subject in the need thereof in accordance with the detailed description of the means and method for administration of compositions, pharmaceutical compositions and parts of the kit of parts of the present invention, as discussed hereinabove.
  • the neuroendocrine tumour is a neuroendocrine tumour expressing monoamine transporters, somatostatin receptors, serotonin transporters (SERT), gastrin-releasing peptide receptors (BB2/GRPR), cholecystokinin B (CCK2) receptor, glucagon-like peptide 1 (GLP-1 ) receptor, glucose-dependent insulinotropic polypeptide (GIP) receptor and/or neuropeptide Y (Y1 ) receptor.
  • monoamine transporters somatostatin receptors, serotonin transporters (SERT), gastrin-releasing peptide receptors (BB2/GRPR), cholecystokinin B (CCK2) receptor, glucagon-like peptide 1 (GLP-1 ) receptor, glucose-dependent insulinotropic polypeptide (GIP) receptor and/or neuropeptide Y (Y1 ) receptor.
  • SERT serotonin transporters
  • BB2/GRPR gastrin-releasing peptide receptors
  • CCK2
  • the neuroendocrine tumour is a neuroendocrine tumour expressing monoamine transporters and/or somatostatin receptors.
  • the neuroendocrine tumour is a neuroendocrine tumour expressing monoamine transporters.
  • the neuroendocrine tumour is selected from neuroblastoma, pheochromocytoma and paraganglioma.
  • the PI3K/Akt/mTOR inhibitor in the method for treating a neuroendocrine tumour in a subject of the present invention is as described hereinabove.
  • the PI3K/Akt/mTOR inhibitor is selected from BEZ235, BKM120, Everolimus, MK-2206 dichloride, Pictilisib, LY294002, CAL-101 , PI-3065, HS-173, PI- 103.
  • Particularly preferred PI3K/Akt/mTOR inhibitor is CUDC-907.
  • the radiopharmaceutical/compound whose cellular uptake is upregulated upon inhibition of PI3K/Akt/mTOR is as described hereinabove.
  • the compound whose cellular uptake is upregulated upon inhibition of PI3K/Akt/mTOR is a catecholamine or its analogue comprising a radionuclide.
  • a preferred radioactive derivative of catecholamine or its analogue is 123 l- meta-iodobenzylguanidine, 124 l-meta-iodobenzylguanidine, 125 l-meta- iodobenzylguanidine or 131 l-meta-iodobenzylguanidine.
  • metaiodobenzylguanidine or the radionuclide derivative thereof is 123 l-meta- iodobenzylguanidine or 131 l-meta-iodobenzylguanidine.
  • the radiopharmaceutical/compound whose cellular uptake is upregulated upon inhibition of PI3K/Akt/mTOR is a radioactive derivative of somatostatin or its analogue.
  • the somatostatin or its analogue is selected from TATE, AM3 and pasireotide.
  • the somatostatin or its analogue is conjugated to a chelator moiety, optionally loaded with a radionuclide.
  • Said radionuclide is preferably selected from 225 Ac, 213 Bi, 99m Tc, 11 1 1n, 177 Lu, 67 Ga, 68 Ga, 90 Y, 64 Cu, 67 Cu, 61 Cu, 43 Sc, 44 Sc, 47 Sc, 212 Pb, 203 Pb, 161 Tb, 152 Tb, 155 Tb and 149 Tb.
  • said method further comprises the step of administering an HDAC inhibitor to a subject in need thereof.
  • said HDAC inhibitor is preferably to be administered before the administration of the compound whose cellular uptake is upregulated upon inhibition of PI3K/Akt/mTOR.
  • the HDAC inhibitor is selected from Vorinostat (SAHA), Romidepsin (FK288), Chidamide, Panobinostat (LBH589), Belinostat (PXD101 ), Valproic acid, Tacedinaline, Mocetinostat, Abexinostat (PCI24781 ), MS275-SNDX-275, Pracinostat (SB939), Resminostat (4SC201 ), Givinostat (IFT2357), Quisinostat (JNJ-26481585), HBI-8000, Kevetrin, CUDC-101 , AR42, Tefinostat (CHR-2845), CHR-3996, 4SC202, CG200745, Rocilinostat (ACY1215), and ME-344. It is conceivable to the skilled person that increasing dopamine transporter expression may also be beneficial for dopaminergic cortical degeneration occurring in subjects suffering from Parkinson’s disease.
  • the present invention relates to a PI3K/Akt/mTOR inhibitor for use in the treatment or prevention of Parkinson’s disease.
  • PI3K/Akt/mT0R inhibitor for use in the treatment or prevention of Parkinson’s disease is as defined hereinabove,
  • said PI3K/Akt/mT0R inhibitor is selected from BEZ235, BKM120, Everolimus, MK-2206 dichloride, Pictilisib, LY294002, CAL-101 , PI-3065, HS-173, PI- 103.
  • Particularly preferred PI3K/Akt/mTOR inhibitor for use in the treatment or prevention of Parkinson’s disease is CUDC-907.
  • Intracellular mIBG was extracted by solid-phase extraction performed on Waters Oasis WCX pElution 96-well plates (Waters), preconditioned with 200 pL of methanol and equilibrated with 200 pL of PBS. A total of 30 pL or 40 pL of an internal standard solution at 2 nM (deuterated mIBG), and 30 pL or 40 pL of each sample were loaded and washed three times for HEK cell and NB8 cell experiments, respectively. A first wash with 200 pL of water, then with 200 pL of methanol and finally with 200 pL of 0.2% formic acid in acetonitrile.
  • the analytes were eluted with a solution containing 2% formic acid in acetonitrile:water (95:5) in 350 pL 96-well plates or in conical 700 pL 96-well plates for HEK cell experiments and NB8 cell experiments, respectively.
  • Separations were performed in HILIC mode on a Waters Acquity LIPLC l-class system (Waters) where 2 pL (HEK cell experiments) or 10 pL (NB8 cell experiments) of sample were injected on a silica column (Interchim Uptisphere Strategy 100 A HILIC, 100 mm x 2.1 mm, 2.2 pm) (Alsachim).
  • the mobile phases consisted of 100% acetonitrile (A) and 100 mM ammonium formate (B).
  • the gradient and flow rates are described on Supplementary Table 4.
  • a solution containing 50%, 95% and 5% of acetonitrile was used for the strong, weak and seal washes, respectively.
  • the temperatures of the autosampler and the column were 10°C and 25°C, respectively.
  • a Waters Xevo TQ-S triple quadrupole mass spectrometer equipped with an electrospray interface was coupled to the LC system, and the analyses were performed in a positive ionization mode.
  • the MRM transitions used for quantification were 275.97 (m/z) and 89.93 (m/z) for the precursor ion and product ion, respectively, with cone voltage at 34 V and collision energy at 20 V.
  • the ESI conditions were set as follows: capillary voltage 0.60 kV, desolvation temperature 600°C, source temperature 150°C, desolvation gas flow 900 L/h, cone gas flow 150 L/h, nebulizer gas 7.0 bar, and collision gas flow 0.25 mL/min. At the beginning of each series, a calibration curve was injected, and three quality controls samples were randomly injected. Data was processed using the TargetLynx module.
  • Example 1 Study of the expression of NET and DAT upon treatment with CU DC- 907
  • PCR was performed by using the SYBR Green Master Mix (Roche) for NET, DAT, PMAT, OCT1-3, glyceraldehyde 3- phosphate dehydrogenase (GAPDH) and eukaryotic translation elongation factor 1 alpha 1 (EEIF1A1 ).
  • the primers were designed using the “Primer Blast” tool from the NCBI website.
  • Table 2 - Primers used for RT-qPCR of NB8 cells incubated or not with different HDAC inhibitors. Reactions were performed in a QuantStudio 6 Real-Time PCR System, and amplification was carried out in a 384-well reaction plate as follows: 95°C for 10 minutes, followed by 40 cycles of 95°C for 15 seconds and 60°C for 1 minute. The limit of quantification values was set at 33 cycles, and values above this threshold were considered as 34 cycles. Each sample was analyzed in duplicate, and a negative control was prepared by using the same amount of total RNA without adding the enzyme reverse transcriptase.
  • NET and DAT transcripts were calculated relative to the level of the housekeeping genes GAPDH and EEIF1A1 using the AACt method (Rao, X., et al., Biostat Bioinforma Biomath, 2013; 3(3):71 -85.).
  • Immunofluorescence detection was used and an increase of NET and DAT protein fluorescence in treated cells observed ( Figure 1 B and 1 C, respectively).
  • Example 2 Study on the effect of CUDC-907 on mIBG internalization through NET upregulation
  • DMI and GBR12935 inhibitors were used. DMI and GBR12935 specifically inhibit NET and DAT respectively. This example demonstrated that NET is the main transporter involved in mIBG internalization.
  • DMI and GBR12935 toward NET and DAT were determined by increasing concentrations of inhibitors (from 0 to 10 pM) at 37°C, 30 minutes before mIBG incubation.
  • inhibitors from 0 to 10 pM
  • CUDC-907 0.1 pM for 48 hours following DMI and/or GBR12935 treatment at different concentrations (from 0 to 1 pM) for 30 minutes prior to mIBG incubation (10 nM for 10 minutes).
  • DMI reduced mIBG internalization by nearly 60% at the lowest concentration used while at an increased concentration (up to 1 pM) it nearly eliminated mIBG internalization.
  • GBR12935 0.5 pM was added to the cells in the presence of CUDC-907, it inhibited nearly 50% of mIBG internalization, and the combination of DMI and GBR12935 once again was similar to the profile of inhibition observed with DMI alone.
  • NB8 cells were plated in a 24-well cell culture plate (9 x 104 cells) for 24 hours. Cells were then transfected with the following 25 nM siRNA mixtures: ON-TARGETplus SMARTpool siRNA mixtures targeting NET (L-007602-00), DAT (L-007603-00), nontargeting control siRNA (D-001810-10) and GAPDH control siRNA (D-001830-10) from Horizon. After a 24 hour incubation period, cells were treated with CUDC-907 0.1 pM or BGT226 0.05 pM for a further 48 hours and then incubated with 10 nM mIBG at 37°C for 10 minutes before mIBG extraction and quantification (Figure 2B and 2E).
  • Example 3 Study of PI3K inhibition on mIBG internalization in NB8 cells through NET
  • CUDC-907 is a dual inhibitor of HDAC and PI3K, to identify which inhibition pathway is prevalent in the mIBG internalization process, the role of each transducing pathway was assessed separately. This example demonstrated that the inhibition of PI3K increases mIBG internalization.
  • BGT226, a specific PI3K inhibitor, targeting subunits a, [3 and y was tested on NB8 cells and was unexpectedly shown to increase mIBG internalization in a dosedependent manner (Figure 2C).
  • Example 4 Study of the PI3K/Akt/mTOR signalling pathway: effect on mIBG internalization
  • PI3K is a component of the Akt/mTOR signalling pathway, to identify the key component(s) contributing to mIBG uptake, Akt and mTOR inhibitors were studied. This study demonstrated that mTOR inhibition facilitates mIBG uptake, with Akt having a negative effect on uptake.
  • Example 5 Study of CUDC-907 treatment and 123 l-mlBG uptake in NB8 xenografts in mice
  • mice All animal experiments were carried out with female athymic nude- Foxn1 n 7Foxn1 + mice (Envigo). The mice (4-6 weeks old) were subcutaneously implanted with 2 x 10 6 NB8 cells/mouse in DMEM/Matrigel (1/1 v/v, 200 pL) and monitored twice weekly. After one month the mice were used for the study once the tumours reached a size of 120-200 mm 3 .
  • CUDC-907 was administered for 5 days, via oral gavage, at the indicated doses and after 2 days drug-free the mice were injected with 123 l-mlBG (2-4 MBq/100 pL). Quantitative biodistribution studies were performed 4 hours and 24 hours post-injection (p.i.) of 123 l-mlBG via the tail vein. All mice were administered intravenously with sodium perchlorate (100 pL Irenat, 120 mg/kg) 5 minutes before 123 l-mlBG injection for blocking the uptake of free radioiodine in iodine-avid organs.
  • SPECT/CT images were acquired at 4 hours and 24 hours p.i. of 123 l-mlBG (13-17 MBq/100 pL) using a small animal scanner (Nano-SPECT/CTTM Bioscan Inc.). The SPECT/CT images 4 hours p.i. were acquired for 90 minutes whilst mice were under anaesthesia. After one day the mice were euthanized and SPECT/CT images of the same mice were acquired at 24 hours p.i. for 170 minutes.
  • NB8 xenografted mice were randomly divided into three groups (A: vehicle; B: 5 mg/kg; C: 10 mg/kg). CUDC-907 given orally to the mice for 5 days, was well tolerated with no side effects. After 2 days’ drug-free, biodistribution studies of 123 l-mlBG were performed at 4 hours and 24 hours p.i. ( Figure 3A). At 4 hours p.i., the treated groups showed no significant differences in the accumulation of 123 l-mlBG in most of the organs and tumours, except for the gallbladder (8.37 ⁇ 2.19% of injected activity/g of tissue [%IA/g] vs.
  • tumour uptake was similar between 4 and 24 hours p.i.).
  • the tumour uptake remained significantly higher in the treated groups compared to the untreated group (2.71 ⁇ 0.64%IA/g vs 1 .42 ⁇ 0.44%IA/g, for group B and A, respectively, p ⁇ 0.05).
  • p values ⁇ 0.05 are considered statistically significant.
  • PC3 prostate cancer cell lines were used to study the effect of several HDAC/PI3K/Akt/mT0R inhibitors on the expression of somatostatin receptor genes (Figure 4A). The use of these inhibitors was found to significantly upregulate SST genes (as the mRNAfold change) after 48 hour-treatment with various HDAC inhibitors compared with untreated PC3 cells (control).
  • LnCAP prostate cancer cell lines were used to investigate SST2 and SST5 protein levels upon treatment with HDAC/PI3K/Akt/mTOR inhibitors ( Figure 4B and C). The use of various inhibitors showed an increase in these protein levels upon treatment. LnCAP cells were incubated with HDAC inhibitors for 48 hours and quisinostat, sodium-4-phenylbutyrate, decitabine and romidepsin increased SST5 protein expression, while tucidinostat increased SST2 protein expression.
  • Example 7 Study of the CUDC-907 treatment and 131 l-mlBG radiation dose in NB8 xenografts in mice
  • mice were injected with 131 l-mlBG (0.5 MBq/100 pL). Quantitative biodistribution studies were performed 4 hours and 24 hours post-injection (p.i.) of 131 1- mlBG via the tail vein. All mice were administered intravenously with sodium perchlorate (100 pL Irenat, 120 mg/kg) 5 minutes before 131 l-mlBG injection for blocking the uptake of free radioiodine in iodine-avid organs.
  • sodium perchlorate 100 pL Irenat, 120 mg/kg
  • the absorbed tumour radiation dose in the pre-treated mice was compared with the absorbed tumour radiation dose in the untreated mice in order to illustrate the enhancement of the tumour dose, and thus enhanced therapeutic effect obtained upon the CUDC-907 treatment.
  • I-MIBG NB8 xenografts
  • a composition comprising a) a PI3K/Akt/mTOR inhibitor, and b) a radiopharmaceutical whose cellular uptake is upregulated upon inhibition of PI3K/Akt/mTOR.
  • a pharmaceutical composition comprising a) a PI3K/Akt/mTOR inhibitor, b) a radiopharmaceutical whose cellular uptake is upregulated upon inhibition of PI3K/Akt/mTOR, and a pharmaceutically acceptable carrier.
  • a kit of parts comprising a) a PI3K/Akt/mTOR inhibitor and a pharmaceutically acceptable carrier, and b) a radiopharmaceutical whose cellular uptake is upregulated upon inhibition of PI3K/Akt/mTOR and a pharmaceutically acceptable carrier.
  • composition of item 1 the pharmaceutical composition of item 2 or the kit of parts of item 3, wherein the PI3K/Akt/mTOR inhibitor is selected from BEZ235, BKM120, Everolimus, MK-2206 dichloride, Pictilisib, LY294002, CAL-101 , PI- 3065, HS-173, PI-103.
  • the PI3K/Akt/mTOR inhibitor is selected from BEZ235, BKM120, Everolimus, MK-2206 dichloride, Pictilisib, LY294002, CAL-101 , PI- 3065, HS-173, PI-103.
  • composition of item 1 or 4 the pharmaceutical composition of item 2 or 4, or the kit of parts of item 3 or 4, wherein the composition, the pharmaceutical composition or the kit of parts comprises CUDC-907.
  • composition, the pharmaceutical composition or the kit of parts of item 6, wherein the catecholamine or its analogue comprising a radionuclide is is 123 l- meta-iodobenzylguanidine, 124 l-meta-iodobenzylguanidine, 125 l-meta- iodobenzylguanidine or 131 l-meta-iodobenzylguanidine, preferably 123 l-meta- iodobenzylguanidine or 131 l-meta-iodobenzylguanidine.
  • a radionuclide preferably selected from 225 Ac, 213 Bi, 99m Tc, 11 1 1n, 177 Lu, 67 Ga, 68 Ga, 90 Y, 64 Cu, 67 Cu, 61 Cu, 43 Sc, 44 Sc, 47 Sc,
  • composition for use or the pharmaceutical composition for use or the kit of parts for use of item 12 wherein the neuroendocrine tumour is a neuroendocrine tumour expressing monoamine transporters and/or somatostatin receptors, preferably selected from neuroblastoma, pheochromocytoma and paraganglioma.
  • the composition for use or the pharmaceutical composition for use or the kit of parts for use of any one of items 11 to 13, for use in diagnosis.
  • the composition for use or the pharmaceutical composition for use or the kit of parts for use of item 14 wherein the radiopharmaceutical whose cellular uptake is upregulated upon inhibition of PI3K/Akt/mTOR is 123 l-meta- iodobenzylguanidine.
  • composition for use or the pharmaceutical composition for use or the kit of parts for use of any one of items 11 to 13, for use in therapy.
  • the composition for use or the pharmaceutical composition for use or the kit of parts for use of item 16 wherein the radiopharmaceutical whose cellular uptake is upregulated upon inhibition of PI3K/Akt/mT0R is 131 l-meta- iodobenzylguanidine.
  • NET norepinephrine transporter
  • DAT dopamine transporter
  • PI3K/Akt/mT0R inhibitor in a method of upregulating the expression of a somatostatin receptor in a subject, preferably wherein the somatostatin receptor is SST2.
  • PI3K/Akt/mTOR inhibitor is selected from BEZ235, BKM120, Everolimus, MK-2206 dichloride, Pictilisib, LY294002, CAL- 101 , PI-3065, HS-173, PI-103.
  • any one of items 18 to 20, wherein said PI3K/Akt/mTOR inhibitor is CUDC-907.
  • a method for treating a neuroendocrine tumour in a subject comprising the steps of: a) administering a PI3K inhibitor to a subject in need thereof, followed by b) administering a radiopharmaceutical whose cellular uptake is upregulated upon inhibition of PI3K/Akt/mTOR to a subject in need thereof.
  • the neuroendocrine tumour is a neuroendocrine tumour expressing monoamine transporters and/or somatostatin receptors, preferably selected from neuroblastoma, pheochromocytoma and paraganglioma.
  • PI3K/Akt/mTOR inhibitor is selected from BEZ235, BKM120, Everolimus, MK-2206 dichloride, Pictilisib, LY294002, CAL- 101 , PI-3065, HS-173, PI-103.
  • the method of item 23, wherein the PI3K/Akt/mTOR inhibitor is CUDC-907.
  • the radiopharmaceutical whose cellular uptake is upregulated upon inhibition of PI3K/Akt/mTOR is a catecholamine or its analogue comprising a radionuclide.
  • the catecholamine or its analogue comprising a radionuclide is 123 l-meta-iodobenzylguanidine, 124 l-meta-iodobenzylguanidine, 125 l-meta-iodobenzylguanidine or 131 l-meta-iodobenzylguanidine, preferably 123 l- meta-iodobenzylguanidine or 131 l-meta-iodobenzylguanidine.
  • radiopharmaceutical whose cellular uptake is upregulated upon inhibition of PI3K/Akt/mTOR is somatostatin or its analogue, preferably wherein the somatostatin or its analogue is selected from TATE, AM3 and pasireotide.

Abstract

The present invention relates to compositions comprising a P13K/Akt/mTOR inhibitor, and a radiopharmaceutical, whose cellular uptake is upregulated upon inhibition of P13K/Akt/mTOR, as well as to pharmaceutical compositions and kits of parts comprising the same. Instant compositions, pharmaceutical compositions and kits of parts are particularly useful in therapy and diagnosis, in particular of neuroendocrine tumours. The present invention further relates to P13K/Akt/mTOR inhibitors for use in diagnosis and therapy.

Description

PI3K/AKT/MTOR INHIBITOR FOR IMPROVING THE CELLULAR UPTAKE OF A RADIOPHARMACEUTICAL
The present invention relates to compositions comprising a PI3K/Akt/mTOR inhibitor, and a compound, in particular a radiopharmaceutical, whose cellular uptake is upregulated upon inhibition of PI3K/Akt/mTOR, as well as to pharmaceutical compositions and kits of parts comprising the same. Instant compositions, pharmaceutical compositions and kits of parts are particularly useful in therapy and diagnosis, in particular of neuroendocrine tumours. The present invention further relates to PI3K/Akt/mTOR inhibitors for use in diagnosis and therapy.
Background of invention
Cancer is a disease characterised by uncontrollable cell division and growth, usually as a result of genetic alterations in specific genes. Several important signalling pathways have been identified as frequently genetically altered in cancer, including phosphoinositide 3-kinase and/or protein B kinase and/or mammalian target of rapamycin (PI3K/Akt/mTOR) signalling. These pathways play a crucial role in cell-cycle progression, apoptosis and cell growth. Metastatic cancers can exhibit fast progression and may be difficult to diagnose due to a lack of severe symptoms. Among the metastatic cancers that are particularly challenging to treat, are neuroendocrine tumours which often go undiagnosed or misdiagnosed for several years.
Neuroendocrine tumours are neoplasms originating from the hormone-producing cells of the endocrine and nervous systems. Among the most aggressive neuroendocrine tumours are neuroblastomas (NBs), which have a high propensity to metastasize and represent 12% of paediatric cancer fatalities. Pheochromocytomas (PHEOs) and paragangliomas (PGLs) are neuroendocrine tumours found in the adrenal medulla and parasympathetic ganglia respectively, 10% of those presenting with benign PHEO/PGL develop malignancy. NB and PHEO/PGL cells are characterized by an excessive production and secretion of catecholamines (norepinephrine, dopamine and epinephrine). Upon release, a portion of these catecholamines are taken up by cells through monoamine membrane transporters that are present on chromaffin and tumour cells. One of the greatest challenges with NB and PHEO/PGL is to assess the presence of metastases and find efficient therapies, since current treatments have proven to be insufficient in terms of outcome. The first line of treatment for PHEO and PGL is surgery, however when the tumour is malignant and has metastasised, surgery is precluded, leaving chemotherapy and radiotherapy as the only options. The lack of efficacious diagnostic and therapeutic treatments leads to poor patient outcomes, for example in PHEO patients with metastasis, a half-life of 50 to 60% at 5 years is observed.
Due to the high production, secretion and subsequent uptake of catecholamines by NBs, PHEOs and PGLs, catecholamine analogues are an attractive diagnostic and therapeutic target. This strategy has been employed in a recent phase II clinical study with Vorinostat, a histone deacetylase (HDAC) inhibitor, which upregulates the expression of norepinephrine transporters (NETs), thereby increasing the uptake of norepinephrine and its analogues, including the radiotherapeutic agent 1311- metaiodobenzylguanine (131 l-mlBG). (DuBois, S. G., et al., J Clin Oncol, 2021 ; 39(31 ):3506-3514). In a further study however, in the context of another type of neuroendocrine carcinoma, the use of a combination of Vorinostat and mIBG in patients presenting liver metastases, showed no significant increase in mIBG uptake (Pollard J. H. et al., Cancer Biother. Radiopharm. 2021 ; 36(8):632-641 ). Thus, this result has highlighted an ongoing need in the art for more efficacious therapeutic and diagnostic tools capable of reliably increasing the uptake of diagnostic and therapeutic agents.
Document W02020/021456 describes the therapeutic concept of the combination of a peptide receptor radionuclide therapeutic (PRRT) agent and immuno-oncology (l-O) therapy. This combination provides, in particular, combinations with l-O therapeutic agents that inhibit LAG-3, TIM-3, GITR, TGF-[3, IL15/IL-15RA and PD-1 pathways. The PRRT agent comprises a peptide linked to a chelating agent and radionuclide. The present invention provides novel combinations comprising a PI3K/Akt/mTOR inhibitor, with optionally, a dual or independent HDAC inhibitor, which upon inhibition increases the cellular uptake of compounds regulated by variously impacted transmembrane proteins such as norepinephrine transporters, dopamine transporters and somatostatin receptors. Such compounds include radioisotopes of meta-iodobenzylguanidine (mIBG), which may be used for the diagnosis or treatment of neuroendocrine tumours. The combinations described herein and the targeted pathway is therefore distinct from those described in W02020/021456.
Document WO2018009638 describes compounds based on the N-(2,3- difluorophenyl)-2-fluoro-4-(iodo/alkynyl)aniline moiety which display inhibitory activity against at least two of the PI3K/mTOR/MEK proteins, effecting tumour initiation via the KRAS-pathway.
Document WO2010062571 describes derivatives of 3,4-dihydropyrazino[2,3- b]pyranzine-2-(1 H)-ones and their use as inhibitors of kinase pathways, especially mTOR, PI3K or Akt, for the treatment of various conditions including cancer, inflammation and neurodegenerative diseases.
Document DE10234201 describes the treatment of neuroendocrine or gastrointestinal tumours that express monoamine transporters, using agents taken up by a monoamine transporter or benzodiazepine receptor. This document does not disclose any inhibitors which facilitate the uptake of molecules by monoamine transporters and is therefore distinct from the present invention.
Summary of the invention
It was an object of the present invention to improve the efficacy of therapeutic and diagnostic tools for tumours, particularly for neuroendocrine tumours.
Thus, the objective technical problem of the present invention is the provision of methods and compositions for improving the uptake of therapeutic and diagnostic agents in tumours, in particular neuroendocrine tumours.
The objective technical problem is solved by the embodiments described herein and as characterized in the claims. The present inventors have found that, unexpectedly, compounds that inhibit PI3K/Akt/mTOR significantly increase the internalization of certain compounds, including mIBG and its radionuclide-bearing analogue, into tumour cells. The use of PI3K/Akt/mTOR inhibitors was unexpectedly found to upregulate monoamine transporters, particularly norepinephrine and dopamine transporters. Furthermore, the inhibition of PI3K/Akt/mTOR was found to increase mRNA production of somatostatin receptors SST1 , 2 and 3 thereby likely increasing the uptake of somatostatin or its analogue. Accordingly, the present invention provides means and methods for utilizing this mechanism for increasing the uptake of radiopharmaceuticals based on catecholamines and somatostatin and their derivatives comprising a radionuclide for therapeutic and diagnostic purposes.
Example 1 and Figure 1 show that the use of CUDC-907, a dual PI3K/Akt/mTOR and HDAC inhibitor, increases the expression of norepinephrine and dopamine transporters (NET and DAT, respectively) in NB8, a well characterised human neuroblastoma cell line.
Example 2 and 3, and Figure 2, show that NET is the main transporter involved in the uptake of mIBG, whereas DAT plays a minor role.
Example 3 and 4 and Figure 2 confirm that the inhibition PI3K is the major pathway involved in increased mIBG uptake as compared to HDAC/Akt/mTOR.
SPECT/CT imaging studies on NB8 xenograft mice showed a significant increase in 123l-mlBG uptake upon treatment with CUDC-907, as described in Example 5 and Figure 3.
The effect of dual HDAC and PI3K/Akt/mTOR inhibition on the expression of somatostatin receptors (SST) was investigated by the present inventors, as described in Example 6 and Figure 4. SST gene expression was increased upon inhibition of HDAC/PI3K/Akt/mTOR signalling.
Example 7 compares the treatment of a cohort of NB8 xenograft mice with CUDC-907 and an administered dose of 0.5 MBq of 131 l-mlBG with that of a cohort of the same mice receiving only a vehicle (10% DMSO in corn oil). The biodistribution of 131 l-mlBG in NB8 xenografts was confirmed 4h p.i. and 24h p.i.
The present inventors have further demonstrated that the compositions comprising inhibitors of PI3K/Akt/mTOR and of HDAC (either as separate molecules or as, e.g., a single molecule that is capable of inhibiting both PI3K/Akt/mTOR and HDAC) are particularly efficacious in the uptake of radionuclides.
The present invention will be summarized in the following embodiments.
In one embodiment, the present invention relates to a composition comprising a) a PI3K/Akt/mTOR inhibitor, and b) a compound whose cellular uptake is upregulated upon inhibition of PI3K/Akt/mTOR, wherein the compound is a radiopharmaceutical.
In a further embodiment, the present invention relates to a pharmaceutical composition comprising a) a PI3K/Akt/mTOR inhibitor, b) a compound whose cellular uptake is upregulated upon inhibition of PI3K/Akt/mTOR, wherein the compound is a radiopharmaceutical, and a pharmaceutically acceptable carrier.
In again a further embodiment, the present invention relates to a kit of parts comprising a) a PI3K/Akt/mTOR inhibitor and a pharmaceutically acceptable carrier, and b) a compound whose cellular uptake is upregulated upon inhibition of PI3K/Akt/mTOR, wherein the compound is a radiopharmaceutical, and a pharmaceutically acceptable carrier.
In again a further embodiment, the present invention relates to the composition of the present invention, the pharmaceutical composition of the present invention, or the kit of parts of the present invention, for use in diagnosis or therapy.
In again a further embodiment, the present invention relates to the composition of the present invention, the pharmaceutical composition of the present invention, or the kit of parts of the present invention, for use in diagnosis or therapy of a neuroendocrine tumour. In again a further embodiment, the present invention relates to the use of a PI3K/Akt/mTOR inhibitor in a method of upregulating the expression of a norepinephrine transporter (NET) and/or dopamine transporter (DAT).
In again a further embodiment, the present invention relates to a PI3K/Akt/mTOR inhibitor for use in the method of upregulating the expression of a somatostatin receptor in a subject, preferably wherein the somatostatin receptor is SST2.
Brief description of drawings
Figure 1 presents the effects of CUDC-907 on NB8 cells: NET and DAT mRNA expression and protein levels as described in Example 1. A) NET and DAT mRNA expression levels in NB8 cells incubated with CUDC-907 0.1 pM. B) Immunofluorescence analysis of NET protein expression in NB8 cells incubated with CUDC-907 0.1 pM, left panels: positive controls. C) Immunofluorescence analysis of DAT protein expression in NB8 cells incubated with CUDC-907 0.1 pM, left panels: positive controls.
Figure 2 presents the cellular mIBG internalization in NB8 cells treated with CUDC- 907 and several classes of inhibitors. A) mIBG internalization in NB8 cells incubated with DMI, GBR12935 or a combination of both inhibitors with different concentrations (0.01 , 0.05, 0.1 , 0.5 and 1 pM) as described in Example 2. B) mIBG internalization following NET and/or DAT knockdown and CUDC-907 treatment as described in Example 2. C) mIBG internalization in NB8 cells incubated with BGT226 in a dose-dependent manner for 48 hours as described in Example 3. D) Comparison of mIBG internalization in NB8 cells following CUDC-907 or BGT226 for 48 hours as described in Example 3. E) mIBG internalization following NET and/or DAT knockdown and BGT226 treatment as described in Example 2 and 3. F) mIBG internalization in NB8 cells incubated with different PI3K, Akt and mTOR inhibitors for 48 hours as described in Example 4.
Figure 3 presents the biodistribution data and SPECT/CT images of 123l-mlBG in NB8 xenografts at 4 hours and 24 hours post injection (p.i.) as described in Example 5. A) Biodistribution data of 123l-mlBG in NB8 xenografts at 4 hours and 24 hours p.i. Values are expressed as % of injected activity/gram (%IA/g) of tissue. NB8 values are in bold. B) and C) present SPECT/CT images after injection of 123l-mlBG (13-17 MBq/100 pL) at 4 hours and 24 hours p.i.. A representative mouse for each group (vehicle, 5 mg/kg and 10 mg/kg) are reported. The white arrows indicate the gallbladder and adrenal glands which show high uptake at 4 and 24 hours p.i. respectively. The grey arrows indicate that the tumours were better visualized at 24 hours p.i. due to the excellent tumour to background ratio.
Figure 4 presents the upregulation of SST mRNA and proteins in prostate cancer cell lines treated with HDAC inhibitors (HDACi) and dual HDACi/PI3K/Akt/mTOR inhibitors as described in Example 6. A) SST gene upregulation (as the mRNA fold change) after 48-hour treatment with the indicated HDACi compared with untreated PC3 cells (control). The most efficacious HDACi were: for SST1 , LMK235 (which induced a fold change (FC) of 1.92 compared with control (p = 0.0079)); for SST2, quisinostat (induced a FC of 3.58 (p = 0.0006), CUDC907 induced a FC of 1.96 (p = 0.0006)) and vorinostat induced a FC of 1.95 (p = 0.0022); for SST3, CUDC907 (induced a FC of 2.80 (p = 0.0079)). No p value could be calculated for the HDACi CUDC101 , valproic acid, ACY, tucidinostat and PC24781. B) and C) In LnCAP cells incubated with HDACi for 48 hours, quisinostat, sodium-4-phenylbutyrate, decitabine and romidepsin increased SST5 protein expression, while tucidinostat increased SST2 protein expression.
Detailed description of the invention
Compounds, compositions, kits of parts and methods of the invention will be described in the following. It is to be understood that all the combinations of features are envisaged.
In a first embodiment, the present invention relates to a composition comprising a) a PI3K/Akt/mTOR inhibitor, and b) a compound whose cellular uptake is upregulated upon inhibition of PI3K/Akt/mTOR, wherein the compound is a radiopharmaceutical.
The composition of the present invention comprises a PI3K/Akt/mTOR inhibitor. It is meant to be understood that this term refers preferably to at least one PI3K/Akt/mTOR inhibitor. Thus, more than one inhibitor of PI3K/Akt/mTOR inhibitor, for example 2, 3, or 4 inhibitors, are also encompassed by the term.
As understood herein, the term “inhibition” relates to a reduction in a certain parameter, e.g., an activity of an enzyme or of a particular pathway. For example, inhibition of a particular enzyme may relate to reduction of its activity of at least 5%, 10%, 20%, 30%, 40% or more is included by this term. Thus, it is to be understood that the inhibition does need not to be 100% reduction of, e.g., said activity.
As understood herein, the term “inhibitor” is a molecule capable of causing inhibition, e.g., an immune checkpoint inhibitor, or PI3K/Akt/mTOR inhibitor.
As understood herein, the term “PI3K/Akt/mTOR inhibitor” preferably relates to a compound that is capable of interfering with the function of phosphoinositide 3-kinase (PI3K) and/or protein-B kinase (Akt) and/or mammalian target of rapamycin (mTOR) in a subject, upon administration to said subject. It is to be understood that, preferably, said interference with certain functions of a protein may refer to its catalytic function. Thus, preferably, inhibitors of PI3K/Akt/mTOR are substrate-competitive inhibitors or allosteric inhibitors of said proteins. These may be covalent (reversible or irreversible) or non-covalent. As known to the skilled person, inhibition may be measured by inhibition constant, K. Preferably, an inhibitor of PI3K/Akt/mTOR is capable of inhibiting at least one of these proteins with a Ki not exceeding 10 pM, preferably not exceeding 1 pM, more preferably not exceeding 100 nM, even more preferably not exceeding 10 nM.
As understood herein, phosphatidylinositol 3-kinase (PI3K), preferably refers to all class IA PI3K isoforms, which are a part of the PI3K family of kinases. The PI3K enzyme family is divided into four categories: class I, class II, class III, and class IV, which differ in structure and substrate specificity. Class I PI3Ks are heterodimeric molecules composed of a regulatory and a catalytic subunit; they are further divided into IA and IB subsets on sequence similarity. Class IA PI3Ks are composed of a heterodimer between a p110 catalytic subunit and a p85 regulatory subunit. The PI3K family of enzymes are involved in cellular functions such as cell growth, proliferation, differentiation, motility, survival and intracellular trafficking, which in turn are involved in cancer. Several studies have shown the involvement of the catalytic domain of the class IA PI3K p110 subunits in various tumour types (Liu, P. et al. Nat. Rev. Drug Discov. 2009; 8:627-644, Fruman, D. A., and Rommel, C., Nat. Rev. Drug Discov. 2014; 13:140-156).
As understood herein, mammalian target of rapamycin (mTOR) (also referred to as the mechanistic target of rapamycin, and sometimes called FK506-binding protein 12- rapamycin-associated protein 1 (FRAP1 )), refers to a kinase which belongs to the phosphatidylinositol 3-kinase-related kinase (PIKK) family. Two distinct forms of mTOR complexes exist: mTOR complex 1 (mTORCI ) and mTOR complex 2 (mT0RC2). mTORCI is a key mediator of transcription and cell growth (via its substrates p70S6 kinase and 4E-BP1 ) and promotes cell survival via the serum and glucocorticoid- activated kinase SGK, whereas mT0RC2 promotes activation of the pro-survival kinase AKT. The catalytic domain of mTOR is homologous to that of PI3K. Dysregulation of PI3K signalling is a common function of tumour cells. In general, mTOR inhibition may be considered as a strategy in many of the tumour types in which PI3K signalling is implicated, and the use of mTOR inhibitors for the treatment of various cancers is known in the art.
As understood herein, protein B kinase (Akt), refers to a family of three serine/threonine-specific protein kinases which play a role in multiple cellular processes including survival pathways via the inhibition of apoptosis, signalling and protein synthesis pathways. Akt may be activated by mT0RC2 via phosphorylation which results in the activation or deactivation of various substrates. Akt is also a downstream effector of PI3K, therefore the rate of its activation directly influences PI3K activity. The therapeutic use of Akt inhibitors in the treatment of various cancers is described in the art.
Preferably, the PI3K/Akt/mTOR inhibitor as referred to herein is selected from BEZ235, BKM120, Everolimus, MK-2206 dichloride, Pictilisib, LY294002, CAL-101 , PI-3065, HS-173, PI-103. , NU7441 , TGX-221 , 10-87114, Wortmannin, XL147, ZSTK474, BYL719, AS-605240, PIK-75, 3-methyladenine, A66, SAR245409, PIK-93, GSK2126458, PIK-90, PF-04691502, AZD6482, Apitolisib, GSK1059615, Duvelisib, Gedatolisib, TG100-1 15, AS-252424, BGT226, CUDC-907, PIK-294, AS-604850, GSK2636771 , BAY80-6946, YM201636, CH5132799, CAY10505, rapamycin and PIK- 293.
As preferably understood herein, BEZ235 is a compound according to formula:
Figure imgf000011_0001
or a pharmaceutically acceptable salt thereof.
As preferably understood herein, BKM120 is a compound according to formula:
Figure imgf000011_0002
or a pharmaceutically acceptable salt thereof.
As preferably understood herein, Everolimus (RAD001 ), is a compound according to formula:
Figure imgf000012_0001
or a pharmaceutically acceptable salt thereof.
As preferably understood herein, MK-2206, is a compound according to formula:
Figure imgf000012_0002
or a pharmaceutically acceptable salt thereof.
As preferably understood herein, Pictilisib, is a compound according to formula:
Figure imgf000012_0003
or a pharmaceutically acceptable salt thereof.
As preferably understood herein, LY294002, is a compound according to formula:
Figure imgf000013_0001
or a pharmaceutically acceptable salt thereof.
As preferably understood herein, CAL-101 , is a compound according to formula:
Figure imgf000013_0002
or a pharmaceutically acceptable salt thereof.
As preferably understood herein, PI-3065, is a compound according to formula:
Figure imgf000013_0003
or a pharmaceutically acceptable salt thereof.
As preferably understood herein, HS-173, is a compound according to formula:
Figure imgf000014_0001
or a pharmaceutically acceptable salt thereof.
As preferably understood herein, PI-103, is a compound according to formula:
Figure imgf000014_0002
or a pharmaceutically acceptable salt thereof.
As preferably understood herein, NU7441 , is a compound according to formula:
Figure imgf000014_0003
or a pharmaceutically acceptable salt thereof.
As preferably understood herein, TGX-221 , is a compound according to formula:
Figure imgf000014_0004
or a pharmaceutically acceptable salt thereof.
As preferably understood herein, IC-87114, is a compound according to formula:
Figure imgf000015_0001
or a pharmaceutically acceptable salt thereof.
As preferably understood herein, Wortmannin, is a compound according to formula:
Figure imgf000015_0002
or a pharmaceutically acceptable salt thereof.
As preferably understood herein, XL147, is a compound according to formula:
Figure imgf000015_0003
or a pharmaceutically acceptable salt thereof.
As preferably understood herein, ZSTK474, is a compound according to formula:
Figure imgf000016_0001
or a pharmaceutically acceptable salt thereof.
As preferably understood herein, BYL719 is a compound according to formula:
Figure imgf000016_0002
or a pharmaceutically acceptable salt thereof.
As preferably understood herein, AS-605240, is a compound according to formula:
Figure imgf000016_0003
or a pharmaceutically acceptable salt thereof.
As preferably understood herein, PIK-75, is a compound according to formula:
Figure imgf000016_0004
or a pharmaceutically acceptable salt thereof.
As preferably understood herein, 3-methyladenine, is a compound according to formula:
Figure imgf000017_0001
or a pharmaceutically acceptable salt thereof.
As preferably understood herein, A66, is a compound according to formula:
Figure imgf000017_0002
or a pharmaceutically acceptable salt thereof.
As preferably understood herein, SAR245409, is a compound according to formula:
Figure imgf000017_0003
or a pharmaceutically acceptable salt thereof.
As preferably understood herein, PIK-93, is a compound according to formula:
Figure imgf000018_0001
or a pharmaceutically acceptable salt thereof.
As preferably understood herein, GSK2126458, is a compound according to formula:
Figure imgf000018_0002
or a pharmaceutically acceptable salt thereof.
As preferably understood herein, PIK-90, is a compound according to formula:
Figure imgf000018_0003
or a pharmaceutically acceptable salt thereof.
As preferably understood herein, PF-04691502, is a compound according to formula:
Figure imgf000018_0004
or a pharmaceutically acceptable salt thereof.
As preferably understood herein, AZD6482, is a compound according to formula:
Figure imgf000019_0001
or a pharmaceutically acceptable salt thereof.
As preferably understood herein, GDC-0980, is a compound according to formula:
Figure imgf000019_0002
or a pharmaceutically acceptable salt thereof.
As preferably understood herein, GSK1059615, is a compound according to formula:
Figure imgf000019_0003
or a pharmaceutically acceptable salt thereof.
As preferably understood herein, Duvelisib (IPI-145), is a compound according to formula:
Figure imgf000020_0001
or a pharmaceutically acceptable salt thereof.
As preferably understood herein, Gedatolisib (PKI-587), is a compound according to formula:
Figure imgf000020_0002
or a pharmaceutically acceptable salt thereof.
As preferably understood herein, TG100-115, is a compound according to formula:
Figure imgf000020_0003
or a pharmaceutically acceptable salt thereof.
As preferably understood herein, AS-252424, is a compound according to formula:
Figure imgf000021_0001
or a pharmaceutically acceptable salt thereof.
As preferably understood herein, BGT226, is a compound according to formula:
Figure imgf000021_0002
or a pharmaceutically acceptable salt thereof.
As preferably understood herein, CUDC-907, is a compound according to formula:
Figure imgf000021_0003
or a pharmaceutically acceptable salt thereof.
As preferably understood herein, PIK-294, is a compound according to formula:
Figure imgf000022_0001
or a pharmaceutically acceptable salt thereof.
As preferably understood herein, AS-604850, is a compound according to formula:
Figure imgf000022_0002
or a pharmaceutically acceptable salt thereof.
As preferably understood herein, GSK2636771 , is a compound according to formula:
Figure imgf000022_0003
or a pharmaceutically acceptable salt thereof.
As preferably understood herein, BAY80-6946, is a compound according to formula:
Figure imgf000023_0001
or a pharmaceutically acceptable salt thereof.
As preferably understood herein, YM201636, is a compound according to formula:
Figure imgf000023_0002
or a pharmaceutically acceptable salt thereof.
As preferably understood herein, CH5132799, is a compound according to formula:
Figure imgf000023_0003
or a pharmaceutically acceptable salt thereof.
As preferably understood herein, CAY10505, is a compound according to formula:
Figure imgf000024_0001
or a pharmaceutically acceptable salt thereof.
As preferably understood herein, Rapamycin (AY-22989), is a compound according to formula:
Figure imgf000024_0002
or a pharmaceutically acceptable salt thereof.
As preferably understood herein, PIK-293, is a compound according to formula:
Figure imgf000024_0003
or a pharmaceutically acceptable salt thereof.
As understood herein, “pharmaceutically acceptable salt” forms of the inhibitors of the present invention may be formed, e.g., by protonation of an atom carrying an electron lone pair which is susceptible to protonation, such as an amino group, with an inorganic or organic acid, or as a salt of an acid group (such as a carboxylic acid group) with a physiologically acceptable cation. Exemplary base addition salts comprise, for example: alkali metal salts such as sodium or potassium salts; alkaline earth metal salts such as calcium or magnesium salts; zinc salts; ammonium salts; aliphatic amine salts such as trimethylamine, triethylamine, dicyclohexylamine, ethanolamine, diethanolamine, triethanolamine, procaine salts, meglumine salts, ethylenediamine salts, or choline salts; aralkyl amine salts such as N,N-dibenzylethylenediamine salts, benzathine salts, benethamine salts; heterocyclic aromatic amine salts such aspyridine salts, picoline salts, quinoline salts or isoquinoline salts; quaternary ammonium salts such as tetramethylammonium salts, tetraethylammonium salts, benzyltrimethylammonium salts, benzyltriethylammonium salts, benzyltributylammonium salts, methyltrioctylammonium salts or tetrabutylammonium salts; and basic amino acid salts such as arginine salts, lysine salts, or histidine salts. Exemplary acid addition salts comprise, for example: mineral acid salts such as hydrochloride, hydrobromide, hydroiodide, sulfate salts (such as, e.g., sulfate or hydrogensulfate salts), nitrate salts, phosphate salts (such as, e.g., phosphate, hydrogenphosphate, or dihydrogenphosphate salts), carbonate salts, hydrogencarbonate salts, perchlorate salts, borate salts, or thiocyanate salts; organic acid salts such as acetate, propionate, butyrate, pentanoate, hexanoate, heptanoate, octanoate, cyclopentanepropionate, decanoate, undecanoate, oleate, stearate, lactate, maleate, oxalate, fumarate, tartrate, malate, citrate, succinate, adipate, gluconate, glycolate, nicotinate, benzoate, salicylate, ascorbate, pamoate (embonate), camphorate, glucoheptanoate, or pivalate salts; sulfonate salts such as methanesulfonate (mesylate), ethanesulfonate (esylate), 2-hydroxyethanesulfonate (isethionate), benzenesulfonate (besylate), p-toluenesulfonate (tosylate), 2 naphthalenesulfonate (napsylate), 3 phenylsulfonate, or camphorsulfonate salts; glycerophosphate salts; and acidic amino acid salts such as aspartate or glutamate salts. Preferred pharmaceutically acceptable salts of the compounds described in the present invention include a hydrochloride salt, a hydrobromide salt, a mesylate salt, a sulfate salt, a tartrate salt, a fumarate salt, an acetate salt, a citrate salt, and a phosphate salt. A particularly preferred pharmaceutically acceptable salt of the compounds as described herein is a hydrochloride or an acetate salt.
Particularly preferred PI3K/Akt/mTOR inhibitor is CUDC-907. Thus, preferably the composition of the present invention comprises CUDC-907 as (a). The composition of the present invention further comprises a radiopharmaceutical whose cellular uptake is upregulated upon inhibition of PI3K/Akt/mTOR. The present inventors have demonstrated that a number of proteins that partake in transport through the cellular membrane, in other words in uptake of certain compounds, are upregulated upon inhibition of PI3K/Akt/mTOR. For example, monoamine transporters are upregulated upon inhibition of PI3K/Akt/mTOR. In particular, norepinephrine and dopamine transporters are upregulated upon inhibition of PI3K/Akt/mTOR. Thus, encompassed by the present invention are the radiopharmaceuticals whose cellular uptake is upregulated upon inhibition of PI3K/Akt/mTOR, preferably selected from compounds that are transported by monoamine transporters, in particular by norepinephrine and/or dopamine transporters.
The term “radiopharmaceutical” as used herein refers to a pharmaceutical compound comprising a radionuclide which may be used in diagnostics and/or treatment of a disease. Radionuclides as part of the radiopharmaceuticals of the present invention may emit alpha, beta or gamma radiation. The radionuclide comprised in the radiopharmaceutical of the present invention includes 123l, 124l, 125l, 131 l, 225Ac, 213Bi, "mTc, 111 in, i77Lu, 67Ga, 68Ga, 90Y, 64Cu, 67Cu, 61 Cu, 43Sc, 44Sc, 47Sc, 212Pb, 203Pb, 161Tb, 152Tb, 155Tb and 149Tb, but is not limited to these radionuclides.
The term “upregulated” as used herein preferably refers to the process of increasing the response to a stimulus, for example an increase in a cellular response to a molecular stimulus due to increase in the number of receptors on the cell surface. Preferably, with regard to a particular transporter or receptor, the term upregulated relates to increased expression level or increased concentration of a particular transporter or receptor in the organism or in the cell.
Preferably the compound, in particular the radiopharmaceutical, whose cellular uptake is upregulated upon inhibition of PI3K/Akt/mTOR is a compound that is transported by monoamine transporters, in particular, by NET and/or DAT respectively. A compound that is transported by a particular transporter is preferably meant to mean a compound that can be transported by said transporter, e.g., transported into the cell. The term “monoamine transporter” refers to integral membrane proteins that act to transport monoamines - particularly neurotransmitters such as dopamine, norepinephrine, epinephrine, serotonin and histamine - from the cellular cytosol into synaptic vesicles.
The term “norepinephrine transporter (NET)” refers to a member of a large family of Na+ and Cl" dependent transporters, exhibits a sub-millimolar substrate potency and can concentrate norepinephrine against its concentration gradient. NET accumulates norepinephrine by coupling the substrate and co-transported ions at a proposed stoichiometry of 1 NE/1 Na2+/1 CI". Approximately 70-90% of the norepinephrine released into synapses is estimated to be cleared using NET. NET was isolated by expression cloning in 1991 , and the gene was found to be located on human chromosome 16q 12.2. The NET gene is encoded by 14 exons, which span 45 kb from the start to the stop codon. The nucleotide and deduced amino acid sequence of the transporter predict a protein of 617 amino acids, containing 12 membrane-spanning domains. The organization of the protein is highly homologous to that of other neurotransmitter transporters including those transporting dopamine, epinephrine, serotonin and gamma-aminobutyric acid (GABA), which are members of a family of sodium- and chloride-dependent transport proteins in the plasma membranes of neurons and glial cells. Analysis of the NET gene and protein has facilitated the investigation of its potential role in psychiatric and other neuronal disorders. At least 13 genetic variants of NET have been identified so far by methods such as singlestranded conformational polymorphism analysis.
As referred to herein, preferably NET is a protein comprising the polypeptide sequence according to any one of SEQ ID NO: 1 to 7.
The term “dopamine transporter (DAT)” refers to a member of the subfamily of monoamine transporters with numerous common topological structures and significant amino acid sequence homology. DAT has been identified as located on the distal end of chromosome 5 (5p15.3), isolated and characterized the human DAT gene (hDAT) including about 1 kb of 5'-flanking region. The hDAT gene spans over 64 kb, consisting of 15 exons separated by 14 introns. The intron-exon structure of the hDAT gene is most similar to that of the human NET gene. Promoter sequence analysis demonstrated a ‘TATA’-less, ‘CAT’-less and G+C-rich structure. Two E box and several Sp-1 -binding sites exist in the promoter region. These structural features are similar to that of the human D1 A dopamine receptor gene and the human monoamine oxidase A gene. The DAT gene encodes for a 620-amino acid protein with a calculated molecular weight of 68,517 (Giros et al., 1992).
As referred to herein, preferably DAT is a protein comprising the polypeptide sequence according to SEQ ID NO: 8.
In a further embodiment, according to the present invention, the compound, in particular the radiopharmaceutical, whose cellular uptake is upregulated upon inhibition of PI3K/Akt/mTOR can be a radioactive derivative of metaiodobenzylguanidine (mIBG). As preferably understood here, mIBG is a compound of the formula:
Figure imgf000028_0001
or a pharmaceutically acceptable salt thereof.
Preferably the radionuclide derivative of mIBG is 123l-meta-iodobenzylguanidine, 124l- meta-iodobenzylguanidine, 125l-meta-iodobenzylguanidine or 131 l-meta- iodobenzylguanidine, preferably 123l-meta-iodobenzylguanidine or 131 l-meta- iodobenzylguanidine.
In a further embodiment, according to the present invention, a compound, in particular a radiopharmaceutical whose cellular uptake is upregulated upon inhibition of PI3K/Akt/mTOR can be a radioactive derivative of somatostatin or its analogue. As preferably understood here, somatostatin is a compound of the formula:
Figure imgf000029_0001
or a pharmaceutically acceptable salt thereof.
Thus, the composition of the present invention comprises a radioactive derivative of somatostatin or its analogue as (b).
Analogue of somatostatin is herein defined as a compound, (preferably a peptide compound) capable of binding to somatostatin receptor(s), preferably with a dissociation constant of not more than 1 pM, more preferably not more than 100 nM, even more preferably not more than 10 nM. Preferably, an analogue of somatostatin has a structure similar to somatostatin, preferably wherein at least 80% of atoms remain unchanged between somatostatin and its analogue, more preferably wherein at least 90% of atoms remain unchanged between somatostatin and its analogue. Preferred analogues of somatostatin include octreotide (and derivatives thereof), TOC, TATE, NOC, AM3, pasireotide, and lanreotide. Thus preferably, somatostatin or its analogue is selected from octreotide, TOC, TATE, NOC, AM3, pasireotide and lanreotide. More preferably, somatostatin or its analogue is selected from TATE, AM3 and pasireotide.
As preferably understood herein, octreotide is a compound according to formula:
Figure imgf000030_0001
or a pharmaceutically acceptable salt thereof.
As preferably understood herein, TATE is a compound according to formula:
Figure imgf000030_0002
or a pharmaceutically acceptable salt thereof.
As preferably understood herein, pasireotide is a compound according to formula:
Figure imgf000031_0001
or a pharmaceutically acceptable salt thereof.
As preferably understood herein, lanreotide is a compound according to formula:
Figure imgf000031_0002
or a pharmaceutically acceptable salt thereof.
As preferably understood herein, AM3 is a compound according to formula:
Figure imgf000032_0001
or a pharmaceutically acceptable salt thereof.
As preferably understood herein, NOC is a compound according to formula:
Figure imgf000032_0002
or a pharmaceutically acceptable salt thereof.
As understood herein, somatostatin receptors (SST) are receptors for the ligand somatostatin, a small neuropeptide associated with neural signalling, particularly in the post-synaptic response to NMDA receptor co-stimulation/activation. There are five known somatostatin receptors: SST1 , SST2, SST3, SST4 and SST5, which are G protein-coupled seven transmembrane receptors. All five SSTs are commonly expressed in neuroendocrine tumours, in most cases, SST2 is dominantly expressed followed by SST5, SST3, SST1 and SST4, however this may vary depending on cell type. SSTs, in general, are involved in signalling cascades that supress tumour cell proliferation, survival and angiogenesis. Therefore, all five SST subtypes are targeted for diagnostic and therapeutic purposes. Stimulation of SSTs can inhibit hormone release from tumours as well as tumour cell growth. SSTs are also involved in neuroendocrine tumour cell apoptosis. The use of somatostatin and its analogues which target somatostatin receptors for the treatment of neuroendocrine tumours is described in the art.
In a further embodiment, according to the present invention, the radioactive derivative of somatostatin or its analogue is preferably conjugated to a chelator moiety, optionally loaded with a radionuclide, preferably selected from 225Ac, 213Bi, 99mTc, 11 1 In, 177Lu, 67Ga, 68Ga, 90Y, 64Cu, 67Cu, 61 Cu, 43Sc, 44Sc, 47Sc, 212Pb, 203Pb, 161Tb, 152Tb, 155Tb and 149Tb.
Chelators that may be conjugated to somatostatin or its analogue of the invention include, but are not limited to, 1 ,4,7,10-tetraazacyclododecane-1 ,4,7, 10-tetraacetic acid (DOTA), diethylenetriaminepentaacetic (DTPA), desferrioxamine (DFO) and triethylenetetramine (TETA), 1 ,4,8,1 1 -tetraazabicyclo[6.6.2]hexadecane-4, 1 1 -diacetic acid (CB-TE2A); ethylenediaminetetraacetic acid (EDTA); ethylene glycolbis(2- aminoethyl)-N,N,N',N'-tetraacetic acid (EGTA); 1 ,4,8, 1 1 -tetraazacyclotetradecane- 1 ,4,8, 1 1 -tetraacetic acid (TETA); ethylenebis-(2-4 hydroxy-phenylglycine) (EHPG); 5- CI-EHPG; 5BrEHPG; 5-Me-EHPG; 5t-Bu-EHPG; 5-sec-Bu-EHPG; benzodiethylenetriamine pentaacetic acid (benzo-DTPA); dibenzo-DTPA; phenyl- DTPA, diphenyl-DTPA; benzyl-DTPA; dibenzyl-DTPA; bis-2(hydroxybenzyl)-ethylene- diaminediacetic acid (HBED) and derivatives thereof; Ac-DOTA; benzo-DOTA; dibenzo-DOTA; 1 ,4,7-triazacyclononane N,N',N"-triacetic acid (NOTA); benzo-NOTA; 1 ,4,7-triazacyclononane N,N'-diacetic acid N”-glutamic acid (NODAGA), benzo-TETA, benzo-DOTMA, where DOTMA is 1 ,4,7, 10-tetraazacyclotetradecane-1 ,4,7, 10- tetra(methyl tetraacetic acid), benzo-TETMA, where TETMA is 1 ,4,8, 1 1 - tetraazacyclotetradecane-1 ,4,8, 1 1 -(methyl tetraacetic acid); derivatives of 1 ,3- propylenediaminetetraacetic acid (PDTA); triethylenetetraaminehexaacetic acid (TTHA); derivatives of 1 ,5, 10-N, N', N"-tris(2,3- dihydroxybenzoyl)-tricatecholate (LICAM); and 1 ,3,5-N,N',N"-tris(2,3- dihydroxybenzoyl)aminomethylbenzene (ME CAM), 6-carboxy-1 ,4,8, 1 1 -tetraazaundecane (N4), and 6-hydrazinonicotinic acid (HYNIC) or other metal chelators. As encompassed by the present invention, the payload may comprise more than one chelator. Other preferred chelators can be selected from the group consisting of cyclic DTPA (diethylene triaminepentaacetic acid) anhydride, ethylenediaminetetraacetic acid (EDTA), DOTA (1 ,4,7,10- tetraazacyclododecane-1 ,4,7,10- tetraacetic acid), and OTA (1 ,4,7-triazonane-l,4,7- triacetic acid). The skilled person will understand that in certain cases additional chemical moiety or moieties may have to be added to the chelator in order to attach it (conjugate it) to the somatostatin or its analogue.
For example, if somatostatin or its analogue is AM3, then somatostatin or its analogue conjugated to a chelator moiety is a compound according to formula
Figure imgf000034_0001
or a pharmaceutically acceptable salt thereof. Herein the chelator moiety is DOTA.
For example, if somatostatin or its analogue is NOC, then somatostatin or its analogue conjugated to a chelator moiety is a compound according to formula:
Figure imgf000034_0002
or a pharmaceutically acceptable salt thereof. Herein, the chelator moiety is DOTA. In a further embodiment, according to the present invention, a compound, in particular a radiopharmaceutical whose cellular uptake is upregulated upon inhibition of PI3K/Akt/mTOR can be a compound/radiopharmaceutical that is transported by a serotonin transporter (SERT), gastrin-releasing peptide receptors (BB2/GRPR), cholecystokinin B (CCK2) receptor, glucagon-like peptide 1 (GLP-1 ) receptor, glucosedependent insulinotropic polypeptide (GIP) receptor and/or neuropeptide Y (Y1 ) receptor.
The Table below lists the targeting systems, i.e., peptide compounds and their target receptors typically used in nuclear oncology.
Table 1
Figure imgf000035_0001
In a further embodiment of the present invention, the composition of the present invention as described herein further comprises a HDAC inhibitor.
As understood herein, the term “HDAC inhibitor” preferably relates to a compound that is capable of interfering with the function of histone deacetylases (i.e. a class of enzymes that remove acetyl groups from a side chain N-acetyl lysine residue on a histone) in a subject, upon administration to said subject. It is to be understood that, preferably, said interference with certain functions of a protein may refer to its catalytic function. Thus, preferably, inhibitors of HDAC are substrate-competitive inhibitors or allosteric inhibitors of said proteins. These may be covalent (reversible or irreversible) or non-covalent. As known to the skilled person, inhibition may be measured by inhibition constant, Ki. Preferably, an inhibitor of HDAC is capable of inhibiting at least one of histone deacetylases with a Ki not exceeding 10 pM, preferably not exceeding 1 pM, more preferably not exceeding 100 nM, even more preferably not exceeding 10 nM. As understood to the skilled person, histone deacetylases comprise HDAC1 , HDAC2, HDAC3, HDAC4, HDAC5, HDAC6, HDAC7, HDAC8, HDAC9, HDAC10, HDAC11 , SIRT1 , SIRT2, SIRT3, SIRT4. SIRT5, SIRT6, and SIRT7.
Preferably, as understood herein, HDAC inhibitor is selected from Vorinostat (SAHA), Romidepsin (FK288), Chidamide, Panobinostat (LBH589), Belinostat (PXD101 ), Valproic acid, Tacedinaline, Mocetinostat, Abexinostat (PCI24781 ), MS275-SNDX- 275, Pracinostat (SB939), Resminostat (4SC201 ), Givinostat (IFT2357), Quisinostat (JNJ-26481585), HBI-8000, Kevetrin, CUDC-101 , AR42, Tefinostat (CHR-2845), CHR-3996, 4SC202, CG200745, Rocilinostat (ACY1215), and ME-344.
In a further embodiment, the present invention relates to a pharmaceutical composition comprising a) a PI3K/Akt/mTOR inhibitor, b) a compound/radiopharmaceutical whose cellular uptake is upregulated upon inhibition of PI3K/Akt/mTOR, and a pharmaceutically acceptable carrier.
The pharmaceutical composition of the present invention comprises a PI3K/Akt/mTOR inhibitor. The a PI3K/Akt/mTOR inhibitor is as described hereinabove.
Preferably, in the pharmaceutical composition of the present invention, the PI3K/Akt/mTOR inhibitor as referred to herein is selected from BEZ235, BKM120, Everolimus, MK-2206 dichloride, Pictilisib, LY294002, CAL-101 , PI-3065, HS-173, PI- 103 , NU7441 , TGX-221 , IC-87114, Wortmannin, XL147, ZSTK474, BYL719, AS- 605240, PIK-75, 3-methyladenine, A66, SAR245409, PIK-93, GSK2126458, PIK-90, PF-04691502, AZD6482, Apitolisib, GSK1059615, Duvelisib, Gedatolisib, TG100-115, AS-252424, BGT226, CUDC-907, PIK-294, AS-604850, GSK2636771 , BAY80-6946, YM201636, CH5132799, CAY10505, rapamycin and PIK-293.
Particularly preferred PI3K/Akt/mTOR inhibitor is CUDC-907. Thus, preferably the pharmaceutical composition of the present invention comprises CUDC-907 as (a).
The pharmaceutical composition of the present invention further comprises a radiopharmaceutical/compound whose cellular uptake is upregulated upon inhibition of PI3K/Akt/mTOR. The radiopharmaceutical/compound whose cellular uptake is upregulated upon inhibition of PI3K/Akt/mTOR is as described hereinabove.
Thus preferably, in the pharmaceutical composition of the present invention the compound whose cellular uptake is upregulated upon inhibition of PI3K/Akt/mTOR is selected from radiopharmaceuticals/compounds that are transported by monoamine transporters, in particular by norepinephrine and/or dopamine transporters.
Preferably, the radiopharmaceutical/compound whose cellular uptake is upregulated upon inhibition of PI3K/Akt/mTOR can be a radioactive derivative of metaiodobenzylguanidine (mIBG).
Preferably the radionuclide derivative of mIBG is 123l-meta-iodobenzylguanidine, 124l- meta-iodobenzylguanidine, 125l-meta-iodobenzylguanidine or 131 l-meta- iodobenzylguanidine, preferably 123l-meta-iodobenzylguanidine or 131 l-meta- iodobenzylguanidine. Thus, in one embodiment, the pharmaceutical composition of the present invention comprises 123l-meta-iodobenzylguanidine, 124l-meta- iodobenzylguanidine, 125l-meta-iodobenzylguanidine or 131 l-meta- iodobenzylguanidine, preferably 123l-meta-iodobenzylguanidine or 131 l-meta- iodobenzylguanidine, more preferably 131 l-meta-iodobenzylguanidine.
In a further embodiment, according to the present invention, in the pharmaceutical composition of the present invention the radiopharmaceutical/compound whose cellular uptake is upregulated upon inhibition of PI3K/Akt/mTOR can be a radioactive derivative of somatostatin or its analogue. Preferred analogues of somatostatin include octreotide (and derivatives thereof), TOC, TATE, NOC, AM3, pasireotide, and lanreotide. Thus preferably, somatostatin or its analogue is selected from octreotide, TOC, TATE, NOC, AM3, pasireotide and lanreotide. More preferably, somatostatin or its analogue is selected from TATE, AM3 and pasireotide.
In a further embodiment, according to the present invention, in the pharmaceutical composition of the present invention the radioactive derivative of somatostatin or its analogue is preferably conjugated to a chelator moiety, optionally loaded with a radionuclide, preferably selected from 225Ac, 213Bi, 99mTc, 1 111n, 177Lu, 67Ga, 68Ga, 90Y, 64Cu, 67Cu, 61Cu, 43Sc, 44Sc, 47Sc, 212Pb, 203Pb, 161Tb, 152Tb, 155Tb and 149Tb.
Chelators that may be conjugated to somatostatin or its analogue of the invention include, but are not limited to, 1 ,4,7,10-tetraazacyclododecane-1 ,4,7, 10-tetraacetic acid (DOTA), diethylenetriaminepentaacetic (DTPA), desfemoxamine (DFO) and triethylenetetramine (TETA), 1 ,4,8,1 1 -tetraazabicyclo[6.6.2]hexadecane-4, 1 1 -diacetic acid (CB-TE2A); ethylenediaminetetraacetic acid (EDTA); ethylene glycolbis(2- aminoethyl)-N,N,N',N'-tetraacetic acid (EGTA); 1 ,4,8, 1 1 -tetraazacyclotetradecane- 1 ,4,8, 1 1 -tetraacetic acid (TETA); ethylenebis-(2-4 hydroxy-phenylglycine) (EHPG); 5- CI-EHPG; 5BrEHPG; 5-Me-EHPG; 5t-Bu-EHPG; 5-sec-Bu-EHPG; benzodiethylenetriamine pentaacetic acid (benzo-DTPA); dibenzo-DTPA; phenyl- DTPA, diphenyl-DTPA; benzyl-DTPA; dibenzyl-DTPA; bis-2(hydroxybenzyl)-ethylene- diaminediacetic acid (HBED) and derivatives thereof; Ac-DOTA; benzo-DOTA; dibenzo-DOTA; 1 ,4,7-triazacyclononane N,N',N"-triacetic acid (NOTA); benzo-NOTA; 1 ,4,7-triazacyclononane N,N'-diacetic acid N”-glutamic acid (NODAGA), benzo-TETA, benzo-DOTMA, where DOTMA is 1 ,4,7, 10-tetraazacyclotetradecane-1 ,4,7, 10- tetra(methyl tetraacetic acid), benzo-TETMA, where TETMA is 1 ,4,8, 1 1 - tetraazacyclotetradecane-1 ,4,8, 1 1 -(methyl tetraacetic acid); derivatives of 1 ,3- propylenediaminetetraacetic acid (PDTA); triethylenetetraaminehexaacetic acid (TTHA); derivatives of 1 ,5, 10-N, N', N"-tris(2,3- dihydroxybenzoyl)-tricatecholate (LICAM); and 1 ,3,5-N,N',N"-tris(2,3- dihydroxybenzoyl)aminomethylbenzene (ME CAM), 6-carboxy-1 ,4,8, 1 1 -tetraazaundecane (N4), and 6-hydrazinonicotinic acid (HYNIC) or other metal chelators. As encompassed by the present invention, the payload may comprise more than one chelator. Other preferred chelators can be selected from the group consisting of cyclic DTPA (diethylene triaminepentaacetic acid) anhydride, ethylenediaminetetraacetic acid (EDTA), DOTA (1 ,4,7, 10- tetraazacyclododecane-1 ,4,7, 10- tetraacetic acid), and OTA (1 ,4,7-triazonane-l,4,7- triacetic acid). The skilled person will understand that in certain cases additional chemical moiety or moieties may have to be added to the chelator in order to attach it (conjugate it) to the somatostatin or its analogue.
In a further embodiment, according to the present invention, in the pharmaceutical composition of the present invention, the compound/radiopharmaceutical whose cellular uptake is upregulated upon inhibition of PI3K/Akt/mTOR can be a compound that is transported by a serotonin transporter (SERT), gastrin-releasing peptide receptors (BB2/GRPR), cholecystokinin B (CCK2) receptor, glucagon-like peptide 1 (GLP-1 ) receptor, glucose-dependent insulinotropic polypeptide (GIP) receptor and/or neuropeptide Y (Y1 ) receptor.
The pharmaceutical composition of the present invention further comprises a pharmaceutically acceptable carrier. The compounds provided herein may be administered as compounds per se or may be formulated as medicaments. The medicaments/pharmaceutical compositions may optionally comprise one or more pharmaceutically acceptable carriers, such as diluents, fillers, disintegrants, lubricating agents, binders, colorants, pigments, stabilizers, preservatives, antioxidants, and/or solubility enhancers.
The pharmaceutical compositions of the invention may comprise one or more solubility enhancers, such as, e.g., polyethylene glycol), including poly(ethylene glycol) having a molecular weight in the range of about 200 to about 5,000 Da (e.g., PEG 200, PEG 300, PEG 400, or PEG 600), ethylene glycol, propylene glycol, glycerol, a non-ionic surfactant, tyloxapol, polysorbate 80, macrogol-15-hydroxystearate (e.g., Kolliphor® HS 15, CAS 70142-34-6), a phospholipid, lecithin, dimyristoyl phosphatidylcholine, dipalmitoyl phosphatidylcholine, distearoyl phosphatidylcholine, a cyclodextrin, a- cyclodextrin, [3-cyclodextrin, y-cyclodextrin, hydroxyethyl-[3-cyclodextrin, hydroxypropyl-[3-cyclodextrin, hydroxyethyl-y-cyclodextrin, hydroxypropyl-y- cyclodextrin, dihydroxypropyl-[3-cyclodextrin, sulfobutylether-[3-cyclodextrin, sulfobutylether-y-cyclodextrin, glucosyl-a-cyclodextrin, glucosyl-[3-cyclodextrin, diglucosyl-[3-cyclodextrin, maltosyl-a-cyclodextrin, maltosyl-[3-cyclodextrin, maltosyl-y- cyclodextrin, maltotriosyl-[3-cyclodextrin, maltotriosyl-y-cyclodextrin, dimaltosyl-|3- cyclodextrin, methyl-[3-cyclodextrin, a carboxyalkyl thioether, hydroxypropyl methylcellulose, hydroxypropylcellulose, polyvinylpyrrolidone, a vinyl acetate copolymer, vinyl pyrrolidone, sodium lauryl sulfate, dioctyl sodium sulfosuccinate, or any combination thereof.
The pharmaceutical compositions of the invention may also comprise one or more preservatives, particularly one or more antimicrobial preservatives, such as, e.g., benzyl alcohol, chlorobutanol, 2 ethoxyethanol, m cresol, chlorocresol (e.g., 2-chloro- 3-methyl-phenol or 4-chloro-3-methyl-phenol), benzalkonium chloride, benzethonium chloride, benzoic acid (or a pharmaceutically acceptable salt thereof), sorbic acid (or a pharmaceutically acceptable salt thereof), chlorhexidine, thimerosal, or any combination thereof.
The pharmaceutical compositions can be formulated by techniques known to the person skilled in the art, such as the techniques published in “Remington: The Science and Practice of Pharmacy”, Pharmaceutical Press, 22nd edition. The pharmaceutical compositions can be formulated as dosage forms for oral, parenteral, such as intramuscular, intravenous, subcutaneous, intradermal, intraarterial, intracardial, rectal, nasal, topical, aerosol or vaginal administration. Dosage forms for oral administration include coated and uncoated tablets, soft gelatin capsules, hard gelatin capsules, lozenges, troches, solutions, emulsions, suspensions, syrups, elixirs, powders and granules for reconstitution, dispersible powders and granules, medicated gums, chewing tablets and effervescent tablets. Dosage forms for parenteral administration include solutions, emulsions, suspensions, dispersions and powders and granules for reconstitution. Emulsions are a preferred dosage form for parenteral administration. Dosage forms for rectal and vaginal administration include suppositories and ovula. Dosage forms for nasal administration can be administered via inhalation and insufflation, for example by a metered inhaler. Dosage forms for topical administration include creams, gels, ointments, salves, patches and transdermal delivery systems.
In a further embodiment of the present invention, the pharmaceutical composition of the present invention as described herein further comprises a HDAC inhibitor.
Preferably, as understood herein, the HDAC inhibitor in the pharmaceutical composition of the present invention is selected from Vorinostat (SAHA), Romidepsin (FK288), Chidamide, Panobinostat (LBH589), Belinostat (PXD101 ), Valproic acid, Tacedinaline, Mocetinostat, Abexinostat (PCI24781 ), MS275-SNDX-275, Pracinostat (SB939), Resminostat (4SC201 ), Givinostat (IFT2357), Quisinostat (JNJ-26481585), HBI-8000, Kevetrin, CUDC-101 , AR42, Tefinostat (CHR-2845), CHR-3996, 4SC202, CG200745, Rocilinostat (ACY1215), and ME-344. In again a further embodiment, the present invention relates to a kit of parts comprising a) a PI3K/Akt/mTOR inhibitor and a pharmaceutically acceptable carrier, and b) a radiopharmaceutical/compound whose cellular uptake is upregulated upon inhibition of PI3K/Akt/mTOR and a pharmaceutically acceptable carrier.
The kit of parts of the present invention comprises the PI3K/Akt/mTOR inhibitor and a pharmaceutically acceptable carrier.
The PI3K/Akt/mTOR inhibitor is as described hereinabove.
Preferably, in the kit of parts of the present invention, the PI3K/Akt/mTOR inhibitor as referred to herein is selected from BEZ235, BKM120, Everolimus, MK-2206 dichloride, Pictilisib, LY294002, CAL-101 , PI-3065, HS-173, PI-103. , NU7441 , TGX-221 , IC- 87114, Wortmannin, XL147, ZSTK474, BYL719, AS-605240, PIK-75, 3- methyladenine, A66, SAR245409, PIK-93, GSK2126458, PIK-90, PF-04691502, AZD6482, Apitolisib, GSK1059615, Duvelisib, Gedatolisib, TG100-1 15, AS-252424, BGT226, CUDC-907, PIK-294, AS-604850, GSK2636771 , BAY80-6946, YM201636, CH5132799, CAY10505, rapamycin and PIK-293.
A Particularly preferred PI3K/Akt/mTOR inhibitor is CUDC-907. Thus preferably, the kit of parts of the present invention comprises CUDC-907 as PI3K/Akt/mTOR inhibitor.
The pharmaceutically acceptable carrier is known to the skilled person and is preferably as described hereinabove.
The kit of parts of the present invention further comprises a compound whose cellular uptake is upregulated upon inhibition of PI3K/Akt/mTOR and a pharmaceutically acceptable carrier.
In the kit of parts of the present invention, the radiopharmaceutical/compound whose cellular uptake is upregulated upon inhibition of PI3K/Akt/mTOR is as described hereinabove. Thus, preferably in the kit of parts composition of the present invention the radiopharmaceutical/ whose cellular uptake is upregulated upon inhibition of PI3K/Akt/mTOR is selected from compounds that are transported by monoamine transporters, in particular by norepinephrine and/or dopamine transporters.
Preferably, the radiopharmaceutical/ whose cellular uptake is upregulated upon inhibition of PI3K/Akt/mTOR is a radioactive derivative of meta-iodobenzylguanidine (mIBG).
Preferably the radionuclide derivative of mIBG is 123l-meta-iodobenzylguanidine, 124l- meta-iodobenzylguanidine, 125l-meta-iodobenzylguanidine or 131 l-meta- iodobenzylguanidine, preferably 123l-meta-iodobenzylguanidine or 131 l-meta- iodobenzylguanidine. Thus, in one embodiment, the kit of parts of the present invention comprises 123l-meta-iodobenzylguanidine, 124l-meta-iodobenzylguanidine, 125l-meta- iodobenzylguanidine or 131 l-meta-iodobenzylguanidine, preferably 123l-meta- iodobenzylguanidine or 131 l-meta-iodobenzylguanidine, more preferably 131 l-meta- iodobenzylguanidine.
In a further embodiment, according to the present invention, in the kit of parts of the present invention the radiopharmaceutical/compound whose cellular uptake is upregulated upon inhibition of PI3K/Akt/mTOR can be a radioactive derivative of somatostatin or its analogue. Preferred analogues of somatostatin include octreotide (and derivatives thereof), TOC, TATE, NOC, AM3, pasireotide, and lanreotide. Thus preferably, somatostatin or its analogue is selected from octreotide, TOC, TATE, NOC, AM3, pasireotide and lanreotide. More preferably, somatostatin or its analogue is selected from TATE, AM3 and pasireotide.
In a further embodiment, according to the present invention, in the kit of parts of the present invention the radioactive derivative of somatostatin or its analogue is preferably conjugated to a chelator moiety, optionally loaded with a radionuclide, preferably selected 225Ac, 213Bi, 99mTc, 1 11 In, 177Lu, 67Ga, 68Ga, 90Y, 64Cu, 67Cu, 61Cu, 43Sc, 44Sc, 47Sc, 212Pb, 203Pb, 161Tb, 152Tb, 155Tb and 149Tb.
Chelators that may be conjugated to somatostatin or its analogue of the invention include, but are not limited to, 1 ,4,7,10-tetraazacyclododecane-1 ,4,7,10-tetraacetic acid (DOTA), diethylenetriaminepentaacetic (DTPA), desferrioxamine (DFO) and triethylenetetramine (TETA), 1 ,4,8,11-tetraazabicyclo[6.6.2]hexadecane-4,11 -diacetic acid (CB-TE2A); ethylenediaminetetraacetic acid (EDTA); ethylene glycolbis(2- aminoethyl)-N,N,N',N'-tetraacetic acid (EGTA); 1 ,4,8,11-tetraazacyclotetradecane- 1 ,4,8,11 -tetraacetic acid (TETA); ethylenebis-(2-4 hydroxy-phenylglycine) (EHPG); 5- CI-EHPG; 5BrEHPG; 5-Me-EHPG; 5t-Bu-EHPG; 5-sec-Bu-EHPG; benzodiethylenetriamine pentaacetic acid (benzo-DTPA); dibenzo-DTPA; phenyl- DTPA, diphenyl-DTPA; benzyl-DTPA; dibenzyl-DTPA; bis-2(hydroxybenzyl)-ethylene- diaminediacetic acid (HBED) and derivatives thereof; Ac-DOTA; benzo-DOTA; dibenzo-DOTA; 1 ,4,7-triazacyclononane N,N',N"-triacetic acid (NOTA); benzo-NOTA; 1 ,4,7-triazacyclononane N,N'-diacetic acid N”-glutamic acid (NODAGA), benzo-TETA, benzo-DOTMA, where DOTMA is 1 ,4,7,10-tetraazacyclotetradecane-1 ,4,7,10- tetra(methyl tetraacetic acid), benzo-TETMA, where TETMA is 1 ,4,8,11- tetraazacyclotetradecane-1 ,4,8,11 -(methyl tetraacetic acid); derivatives of 1 ,3- propylenediaminetetraacetic acid (PDTA); triethylenetetraaminehexaacetic acid (TTHA); derivatives of 1 ,5, 10-N, N', N"-tris(2,3- dihydroxybenzoyl)-tricatecholate (LICAM); and 1 ,3,5-N,N',N"-tris(2,3- dihydroxybenzoyl)aminomethylbenzene (ME CAM), 6-carboxy-1 ,4,8,11 -tetraazaundecane (N4), and 6-hydrazinonicotinic acid (HYNIC) or other metal chelators. As encompassed by the present invention, the payload may comprise more than one chelator. Other preferred chelators can be selected from the group consisting of cyclic DTPA (diethylene triaminepentaacetic acid) anhydride, ethylenediaminetetraacetic acid (EDTA), DOTA (1 ,4,7,10- tetraazacyclododecane-1 ,4,7,10- tetraacetic acid), and OTA (1 ,4,7-triazonane-l,4,7- triacetic acid). The skilled person will understand that in certain cases additional chemical moiety or moieties may have to be added to the chelator in order to attach it (conjugate it) to the somatostatin or its analogue.
The radiopharmaceuticals of the present invention preferably target specific cells, such as cancer cells such that a diagnostic and/or therapeutic outcome is achieved. Targeting of cells is typically achieved via an interaction between the radiopharmaceutical and its target. For example, in the present invention, mIBG may be used for targeting cancer cells, in particular neuroendocrine cells, as it is structurally similar to norepinephrine which is transported by NET. Neuroendocrine cells express NET, thus enabling tumor-selective imaging and therapy.
In a further embodiment, according to the present invention, in the kit of parts of the present invention the compound whose cellular uptake is upregulated upon inhibition of PI3K/Akt/mTOR can be a compound that is transported by a serotonin transporter (SERT), gastrin-releasing peptide receptors (BB2/GRPR), cholecystokinin B (CCK2) receptor, glucagon-like peptide 1 (GLP-1 ) receptor, glucose-dependent insulinotropic polypeptide (GIP) receptor and/or neuropeptide Y (Y1 ) receptor.
The pharmaceutically acceptable carrier is known to the skilled person and preferably is as described hereinabove.
In a further embodiment of the present invention, the kit of parts of the present invention as described herein further comprises an HDAC inhibitor.
Preferably, as understood herein, HDAC inhibitor in the pharmaceutical composition of the present invention is selected from Vorinostat (SAHA), Romidepsin (FK288), Chidamide, Panobinostat (LBH589), Belinostat (PXD101 ), Valproic acid, Tacedinaline, Mocetinostat, Abexinostat (PCI24781 ), MS275-SNDX-275, Pracinostat (SB939), Resminostat (4SC201 ), Givinostat (IFT2357), Quisinostat (JNJ-26481585), HBI-8000, Kevetrin, CUDC-101 , AR42, Tefinostat (CHR-2845), CHR-3996, 4SC202, CG200745, Rocilinostat (ACY1215), and ME-344.
The compositions of the present invention, the pharmaceutical compositions of the present invention and the kits of parts of the present invention are useful in diagnosis or therapy. Thus, the present invention relates to the composition of the present invention, the pharmaceutical composition of the present invention or the kit of parts of the present invention for use in diagnosis or therapy.
In case of the kit of parts of the present invention, it is to be understood that the parts of the kit of parts of the present invention can be administered to a subject simultaneously, separately or sequentially.
The term therapy relates, preferably, to treatment of prevention of a disease. The term “treatment” of a disorder or disease, as used herein, is well known in the art. “Treatment” of a disorder or disease implies that a disorder or disease is suspected or has been diagnosed in a patient/subject. A patient/subject suspected of suffering from a disorder or disease typically shows specific clinical and/or pathological symptoms which a skilled person can easily attribute to a specific pathological condition (i.e., diagnose a disorder or disease). The “treatment” of a disorder or disease may, for example, lead to a halt in the progression of the disorder or disease (e.g., no deterioration of symptoms) or a delay in the progression of the disorder or disease (in case the halt in progression is of a transient nature only). The “treatment” of a disorder or disease may also lead to a partial response (e.g., amelioration of symptoms) or complete response (e.g., disappearance of symptoms) of the subject/patient suffering from the disorder or disease. Accordingly, the “treatment” of a disorder or disease may also refer to an amelioration of the disorder or disease, which may, e.g., lead to a halt in the progression of the disorder or disease or a delay in the progression of the disorder or disease. Such a partial or complete response may be followed by a relapse. It is to be understood that a subject/patient may experience a broad range of responses to a treatment (such as the exemplary responses as described herein above). The treatment of a disorder or disease may, inter alia, comprise curative treatment (preferably leading to a complete response and eventually to healing of the disorder or disease) and palliative treatment (including symptomatic relief).
The term “prevention” of a disorder or disease, as used herein, is also well known in the art. For example, a patient/subject suspected of being prone to suffer from a disorder or disease may particularly benefit from a prevention of the disorder or disease. The subject/patient may have a susceptibility or predisposition for a disorder or disease, including but not limited to hereditary predisposition. Such a predisposition can be determined by standard methods or assays, using, e.g., genetic markers or phenotypic indicators. It is to be understood that a disorder or disease to be prevented in accordance with the present invention has not been diagnosed or cannot be diagnosed in the patient/subject (for example, the patient/subject does not show any clinical or pathological symptoms). Thus, the term “prevention” comprises the use of a compound of the present invention before any clinical and/or pathological symptoms are diagnosed or determined or can be diagnosed or determined by the attending physician.
The term “diagnosis” as used herein relates to a process of determining whether a subject is suffering from any particular disease or disorder or a process of determining which disease or condition explains symptoms and sign exhibited by said subject. Successful diagnosis that said subject is suffering from a particular disease allows its targeted treatment. Modem diagnosis methods involve often the use of diagnostic agents, i.e. compounds and composition that are administered to a subject in order to aid the process of diagnosis. The diagnostic agents may be suitable for easy identification and quantification, for example, in computed tomography (CT), single photon emission computed tomography (SPECT), magnetic resonance imaging (MRI), positron emission tomography (PET), ultrasound imaging or via in vivo optical detection of fluorophores in the far red/near IR spectral range.
Preferably, diagnosis or therapy relates herein to the diagnosis or therapy of a neuroendocrine tumour. Neuroendocrine tumour, as encompassed in the present invention is not particularly limited. In particularly preferred embodiments, neuroendocrine tumour is a neuroendocrine tumour characterized by increased expression of monoamine transporters, somatostatin receptors, serotonin transporter (SERT), gastrin-releasing peptide receptors (BB2/GRPR), cholecystokinin B (CCK2) receptor, glucagon-like peptide 1 (GLP-1 ) receptor, glucose-dependent insulinotropic polypeptide (GIP) receptor and/or neuropeptide Y (Y1 ) receptor. More preferably, the neuroendocrine tumour is characterized by increased expression of monoamine transporters and/or somatostatin receptors. Even more preferably, the neuroendocrine tumour is characterized by increased expression of monoamine transporters. Particularly preferred neuroendocrine tumours are neuroblastoma, pheochromocytoma and paraganglioma.
In one specific embodiment, the present invention relates to the composition of the present invention, the pharmaceutical composition of the present invention or the kit of parts of the present invention for use in diagnosis. A particularly preferred radionuclide analogue of mIBG for use in diagnosis is 123l-meta-iodobenzylguanidine. Thus preferably, the present invention relates to the composition of the present invention, the pharmaceutical composition of the present invention or the kit of parts of the present invention for use in diagnosis wherein the compound whose cellular uptake is upregulated upon inhibition of PI3K/Akt/mTOR is 123l-meta-iodobenzylguanidine.
In one specific embodiment, the present invention relates to the composition of the present invention, the pharmaceutical composition of the present invention or the kit of parts of the present invention for use in therapy. A particularly preferred radionuclide analogue of mIBG for use in therapy is 131 l-meta-iodobenzylguanidine. Thus, preferably, the present invention relates to the composition of the present invention, the pharmaceutical composition of the present invention or the kit of parts of the present invention for use in therapy, wherein the compound whose cellular uptake is upregulated upon inhibition of PI3K/Akt/mTOR is 131 l-meta-iodobenzylguanidine.
The above described compositions, pharmaceutical compositions or parts of the kit of parts may be administered to a subject by any convenient route of administration, whether systemically/peripherally or at the site of desired action, including but not limited to one or more of: oral (e.g., as a tablet, capsule, or as an ingestible solution), topical (e.g., transdermal, intranasal, ocular, buccal, and sublingual), parenteral (e.g., using injection techniques or infusion techniques, and including, for example, by injection, e.g., subcutaneous, intradermal, intramuscular, intravenous, intraarterial, intracardiac, intrathecal, intraspinal, intracapsular, subcapsular, intraorbital, intraperitoneal, intratracheal, subcuticular, intraarticular, subarachnoid, or intrasternal by, e.g., implant of a depot, for example, subcutaneously or intramuscularly), pulmonary (e.g., by inhalation or insufflation therapy using, e.g., an aerosol, e.g., through mouth or nose), gastrointestinal, intrauterine, intraocular, subcutaneous, ophthalmic (including intravitreal or intracameral), rectal, or vaginal administration.
If said compounds, said pharmaceutical compositions or parts of said kit of parts are administered parenterally, then examples of such administration include one or more of: intravenously, intraarterially, intraperitoneally, intrathecally, intraventricularly, intraurethrally, intrasternally, intracardially, intracranially, intramuscularly or subcutaneously administering the compounds or pharmaceutical compositions, and/or by using infusion techniques. For parenteral administration, the compounds, the compositions are best used in the form of a sterile aqueous solution which may contain other substances, for example, enough salts or glucose to make the solution isotonic with blood. The aqueous solutions should be suitably buffered (preferably to a pH of from 3 to 9), if necessary. The preparation of suitable parenteral formulations under sterile conditions is readily accomplished by standard pharmaceutical techniques well known to those skilled in the art.
Said compositions, said pharmaceutical compositions or parts of said kit of parts can also be administered orally in the form of tablets, capsules, ovules, elixirs, solutions or suspensions, which may contain flavoring or coloring agents, for immediate-, delayed- , modified-, sustained-, pulsed- or controlled-release applications. The tablets may contain excipients such as microcrystalline cellulose, lactose, sodium citrate, calcium carbonate, dibasic calcium phosphate and glycine, disintegrants such as starch (preferably corn, potato or tapioca starch), sodium starch glycolate, croscarmellose sodium and certain complex silicates, and granulation binders such as polyvinylpyrrolidone, hydroxypropylmethylcellulose (HPMC), hydroxypropylcellulose (HPC), sucrose, gelatin and acacia. Additionally, lubricating agents such as magnesium stearate, stearic acid, glyceryl behenate and talc may be included. Solid compositions of a similar type may also be employed as fillers in gelatin capsules. Preferred excipients in this regard include lactose, starch, a cellulose, or high molecular weight polyethylene glycols. For aqueous suspensions and/or elixirs, the agent may be combined with various sweetening or flavoring agents, coloring matter or dyes, with emulsifying and/or suspending agents and with diluents such as water, ethanol, propylene glycol and glycerin, and combinations thereof.
For oral administration, the compositions, the pharmaceutical compositions or parts of the kit of parts are preferably administered by oral ingestion, particularly by swallowing. The compositions, the pharmaceutical compositions or the parts of the kit of parts can thus be administered to pass through the mouth into the gastrointestinal tract, which can also be referred to as “oral-gastrointestinal” administration.
Alternatively, said compositions, pharmaceutical compositions or parts of the kit of parts can be administered in the form of a suppository or pessary, or may be applied topically in the form of a gel, hydrogel, lotion, solution, cream, ointment or dusting powder. The compositions, pharmaceutical compositions or parts of the kit of parts of the present invention may also be dermally or trans-dermally administered, for example, by the use of a skin patch.
Said compositions, pharmaceutical compositions or parts of the kit of parts may also be administered by sustained release systems. Suitable examples of sustained- release compositions include semi permeable polymer matrices in the form of shaped articles, e.g., films, or microcapsules. Sustained-release matrices include, e.g., polylactides, copolymers of L glutamic acid and gamma-ethyl-L-glutamate, poly(2- hydroxyethyl methacrylate), ethylene vinyl acetate, or poly D (-)-3-hydroxybutyric acid. Sustained-release pharmaceutical compositions also include liposomally entrapped compounds. The present invention thus also relates to liposomes containing compounds comprised in the composition, pharmaceutical composition or parts of the kit of the invention.
Said compositions, pharmaceutical compositions or parts of the kit of parts may also be administered by the pulmonary route, rectal routes, or the ocular route. For ophthalmic use, they can be formulated as micronized suspensions in isotonic, pH adjusted, sterile saline, or, preferably, as solutions in isotonic, pH adjusted, sterile saline, optionally in combination with a preservative such as a benzalkonium chloride. Alternatively, they may be formulated in an ointment such as petrolatum.
It is also envisaged to prepare dry powder formulations of the compositions, pharmaceutical compositions or parts of the kit of parts for pulmonary administration, particularly inhalation. Such dry powders may be prepared by spray drying under conditions which result in a substantially amorphous glassy or a substantially crystalline bioactive powder. Accordingly, dry powders of the compounds of the present invention can be made according to an emulsification/spray drying process.
For topical application to the skin, said compositions, pharmaceutical compositions or parts of the kit of parts can be formulated as a suitable ointment containing the active compound suspended or dissolved in, for example, a mixture with one or more of the following: mineral oil, liquid petrolatum, white petrolatum, propylene glycol, emulsifying wax and water. Alternatively, they can be formulated as a suitable lotion or cream, suspended or dissolved in, for example, a mixture of one or more of the following: mineral oil, sorbitan monostearate, a polyethylene glycol, liquid paraffin, polysorbate 60, cetyl esters wax, 2-octyldodecanol, benzyl alcohol and water.
The present invention thus relates to the compositions, pharmaceutical compositions or parts of the kit of parts provided herein that are to be administered by any one of: an oral route; topical route, including by transdermal, intranasal, ocular, buccal, or sublingual route; parenteral route using injection techniques or infusion techniques, including by subcutaneous, intradermal, intramuscular, intravenous, intraarterial, intracardiac, intrathecal, intraspinal, intracapsular, subcapsular, intraorbital, intraperitoneal, intratracheal, subcuticular, intraarticular, subarachnoid, intrasternal, intraventricular, intraurethral, or intracranial route; pulmonary route, including by inhalation or insufflation therapy; gastrointestinal route; intrauterine route; intraocular route; subcutaneous route; ophthalmic route, including by intravitreal, or intracameral route; rectal route; or vaginal route. Preferred routes of administration are oral administration or parenteral administration.
In a further embodiment, the present invention relates to a PI3K/Akt/mT0R inhibitor for use in diagnosis. The present inventors have surprisingly found that a PI3K/Akt/mT0R upon administration of a subject is useful in combination with diagnostic agents, for example for a combination with a radioactive derivative of mIBG as it increases its cellular uptake, thereby increasing the sensitivity of the diagnostic methods.
Hence preferably, said PI3K/Akt/mT0R inhibitor is selected from BEZ235, BKM120, Everolimus, MK-2206 dichloride, Pictilisib, LY294002, CAL-101 , PI-3065, HS-173, PI- 103. , NU7441 , TGX-221 , IC-87114, Wortmannin, XL147, ZSTK474, BYL719, AS- 605240, PIK-75, 3-methyladenine, A66, SAR245409, PIK-93, GSK2126458, PIK-90, PF-04691502, AZD6482, Apitolisib, GSK1059615, Duvelisib, Gedatolisib, TG100-115, AS-252424, BGT226, CUDC-907, PIK-294, AS-604850, GSK2636771 , BAY80-6946, YM201636, CH5132799, CAY10505, rapamycin and PIK-293.
Particularly preferred PI3K/Akt/mTOR inhibitor for use in diagnosis is CUDC-907.
In a further embodiment, the present invention relates to the use of a PI3K/Akt/mTOR inhibitor in the method of upregulating the expression of norepinephrine transporter (NET) and/or dopamine transporter (DAT).
In again a further embodiment, the present invention relates to the use of a PI3K/Akt/mT0R inhibitor in the method of upregulating the expression of a somatostatin receptor in a subject. Particularly preferred is an embodiment, wherein the somatostatin receptor is SST2.
Thus the present invention preferably relates to the use of a PI3K/Akt/mT0R inhibitor as described hereinabove, wherein the PI3K/Akt/mT0R inhibitor is selected from BEZ235, BKM120, Everolimus, MK-2206 dichloride, Pictilisib, LY294002, CAL-101 , PI- 3065, HS-173, PI-103. , NU7441 , TGX-221 , IC-87114, Wortmannin, XL147, ZSTK474, BYL719, AS-605240, PIK-75, 3-methyladenine, A66, SAR245409, PIK-93, GSK2126458, PIK-90, PF-04691502, AZD6482, Apitolisib, GSK1059615, Duvelisib, Gedatolisib, TG100-1 15, AS-252424, BGT226, CUDC-907, PIK-294, AS-604850, GSK2636771 , BAY80-6946, YM201636, CH5132799, CAY10505, rapamycin and PIK- 293. Particularly preferred is CUDC-907.
The present invention further relates to a method for treating a neuroendocrine tumour in a subject, the method comprising the steps of: a) administering a PI3K inhibitor to a subject in need thereof, followed by b) administering a compound, in particular a radiopharmaceutical whose cellular uptake is upregulated upon inhibition of PI3K/Akt/mTOR to a subject in need thereof.
It is to be understood that a therapeutically effective amounts of the PI3K inhibitor and of the radiopharmaceutical/compound whose cellular uptake is upregulated upon inhibition of PI3K/Akt/mT0R is to be administered in accordance with the method. The PI3K inhibitor as well as radiopharmaceutical/compound whose cellular uptake is upregulated upon inhibition of PI3K/Akt/mTOR can be administered to a subject in the need thereof in accordance with the detailed description of the means and method for administration of compositions, pharmaceutical compositions and parts of the kit of parts of the present invention, as discussed hereinabove. Preferably, in the method for treating a neuroendocrine tumour in a subject of the present invention, the neuroendocrine tumour is a neuroendocrine tumour expressing monoamine transporters, somatostatin receptors, serotonin transporters (SERT), gastrin-releasing peptide receptors (BB2/GRPR), cholecystokinin B (CCK2) receptor, glucagon-like peptide 1 (GLP-1 ) receptor, glucose-dependent insulinotropic polypeptide (GIP) receptor and/or neuropeptide Y (Y1 ) receptor.
More preferably, in the method for treating a neuroendocrine tumour in a subject of the present invention, the neuroendocrine tumour is a neuroendocrine tumour expressing monoamine transporters and/or somatostatin receptors.
More preferably, in the method for treating a neuroendocrine tumour in a subject of the present invention, the neuroendocrine tumour is a neuroendocrine tumour expressing monoamine transporters.
Preferably, in the method for treating a neuroendocrine tumour in a subject of the present invention, the neuroendocrine tumour is selected from neuroblastoma, pheochromocytoma and paraganglioma.
The PI3K/Akt/mTOR inhibitor in the method for treating a neuroendocrine tumour in a subject of the present invention is as described hereinabove.
Thus preferably, in the method for treating a neuroendocrine tumour in a subject of the present invention the PI3K/Akt/mTOR inhibitor is selected from BEZ235, BKM120, Everolimus, MK-2206 dichloride, Pictilisib, LY294002, CAL-101 , PI-3065, HS-173, PI- 103. , NU7441 , TGX-221 , IC-87114, Wortmannin, XL147, ZSTK474, BYL719, AS- 605240, PIK-75, 3-methyladenine, A66, SAR245409, PIK-93, GSK2126458, PIK-90, PF-04691502, AZD6482, Apitolisib, GSK1059615, Duvelisib, Gedatolisib, TG100-115, AS-252424, BGT226, CUDC-907, PIK-294, AS-604850, GSK2636771 , BAY80-6946, YM201636, CH5132799, CAY10505, rapamycin and PIK-293.
Particularly preferred PI3K/Akt/mTOR inhibitor is CUDC-907.
In the method for treating a neuroendocrine tumour in a subject of the present invention the radiopharmaceutical/compound whose cellular uptake is upregulated upon inhibition of PI3K/Akt/mTOR is as described hereinabove.
Thus, in one embodiment of the method for treating a neuroendocrine tumour in a subject of the present invention, the compound whose cellular uptake is upregulated upon inhibition of PI3K/Akt/mTOR is a catecholamine or its analogue comprising a radionuclide. A preferred radioactive derivative of catecholamine or its analogue is 123l- meta-iodobenzylguanidine, 124l-meta-iodobenzylguanidine, 125l-meta- iodobenzylguanidine or 131 l-meta-iodobenzylguanidine. Even more preferably, metaiodobenzylguanidine or the radionuclide derivative thereof is 123l-meta- iodobenzylguanidine or 131 l-meta-iodobenzylguanidine.
In another embodiment of the method for treating a neuroendocrine tumour in a subject of the present invention, the radiopharmaceutical/compound whose cellular uptake is upregulated upon inhibition of PI3K/Akt/mTOR is a radioactive derivative of somatostatin or its analogue. Preferably, the somatostatin or its analogue is selected from TATE, AM3 and pasireotide. Preferably, the somatostatin or its analogue is conjugated to a chelator moiety, optionally loaded with a radionuclide. Said radionuclide is preferably selected from 225Ac, 213Bi, 99mTc, 11 11n, 177Lu, 67Ga, 68Ga, 90Y, 64Cu, 67Cu, 61Cu, 43Sc, 44Sc, 47Sc, 212Pb, 203Pb, 161Tb, 152Tb, 155Tb and 149Tb.
In one embodiment of the method for treating a neuroendocrine tumour in a subject of the present invention, said method further comprises the step of administering an HDAC inhibitor to a subject in need thereof. It is to be understood that said HDAC inhibitor is preferably to be administered before the administration of the compound whose cellular uptake is upregulated upon inhibition of PI3K/Akt/mTOR.
Preferably, the HDAC inhibitor is selected from Vorinostat (SAHA), Romidepsin (FK288), Chidamide, Panobinostat (LBH589), Belinostat (PXD101 ), Valproic acid, Tacedinaline, Mocetinostat, Abexinostat (PCI24781 ), MS275-SNDX-275, Pracinostat (SB939), Resminostat (4SC201 ), Givinostat (IFT2357), Quisinostat (JNJ-26481585), HBI-8000, Kevetrin, CUDC-101 , AR42, Tefinostat (CHR-2845), CHR-3996, 4SC202, CG200745, Rocilinostat (ACY1215), and ME-344. It is conceivable to the skilled person that increasing dopamine transporter expression may also be beneficial for dopaminergic cortical degeneration occurring in subjects suffering from Parkinson’s disease.
Thus, in a further embodiment, the present invention relates to a PI3K/Akt/mTOR inhibitor for use in the treatment or prevention of Parkinson’s disease.
The PI3K/Akt/mT0R inhibitor for use in the treatment or prevention of Parkinson’s disease is as defined hereinabove,
Thus preferably, said PI3K/Akt/mT0R inhibitor is selected from BEZ235, BKM120, Everolimus, MK-2206 dichloride, Pictilisib, LY294002, CAL-101 , PI-3065, HS-173, PI- 103. , NU7441 , TGX-221 , IC-87114, Wortmannin, XL147, ZSTK474, BYL719, AS- 605240, PIK-75, 3-methyladenine, A66, SAR245409, PIK-93, GSK2126458, PIK-90, PF-04691502, AZD6482, Apitolisib, GSK1059615, Duvelisib, Gedatolisib, TG100-115, AS-252424, BGT226, CUDC-907, PIK-294, AS-604850, GSK2636771 , BAY80-6946, YM201636, CH5132799, CAY10505, rapamycin and PIK-293.
Particularly preferred PI3K/Akt/mTOR inhibitor for use in the treatment or prevention of Parkinson’s disease is CUDC-907.
The invention will be illustrated by the following examples, which however are not to be construed as limiting.
Examples
Quantification of mIBG in the following studies was achieved by solid-phase extraction from cells and media, followed by LC-MS/MS analysis. mIBG extraction and LC-MS/MS
Intracellular mIBG was extracted by solid-phase extraction performed on Waters Oasis WCX pElution 96-well plates (Waters), preconditioned with 200 pL of methanol and equilibrated with 200 pL of PBS. A total of 30 pL or 40 pL of an internal standard solution at 2 nM (deuterated mIBG), and 30 pL or 40 pL of each sample were loaded and washed three times for HEK cell and NB8 cell experiments, respectively. A first wash with 200 pL of water, then with 200 pL of methanol and finally with 200 pL of 0.2% formic acid in acetonitrile. Then, the analytes were eluted with a solution containing 2% formic acid in acetonitrile:water (95:5) in 350 pL 96-well plates or in conical 700 pL 96-well plates for HEK cell experiments and NB8 cell experiments, respectively. Separations were performed in HILIC mode on a Waters Acquity LIPLC l-class system (Waters) where 2 pL (HEK cell experiments) or 10 pL (NB8 cell experiments) of sample were injected on a silica column (Interchim Uptisphere Strategy 100 A HILIC, 100 mm x 2.1 mm, 2.2 pm) (Alsachim). The mobile phases consisted of 100% acetonitrile (A) and 100 mM ammonium formate (B). The gradient and flow rates are described on Supplementary Table 4. A solution containing 50%, 95% and 5% of acetonitrile was used for the strong, weak and seal washes, respectively. The temperatures of the autosampler and the column were 10°C and 25°C, respectively. A Waters Xevo TQ-S triple quadrupole mass spectrometer equipped with an electrospray interface was coupled to the LC system, and the analyses were performed in a positive ionization mode. The MRM transitions used for quantification were 275.97 (m/z) and 89.93 (m/z) for the precursor ion and product ion, respectively, with cone voltage at 34 V and collision energy at 20 V. The ESI conditions were set as follows: capillary voltage 0.60 kV, desolvation temperature 600°C, source temperature 150°C, desolvation gas flow 900 L/h, cone gas flow 150 L/h, nebulizer gas 7.0 bar, and collision gas flow 0.25 mL/min. At the beginning of each series, a calibration curve was injected, and three quality controls samples were randomly injected. Data was processed using the TargetLynx module.
Example 1 : Study of the expression of NET and DAT upon treatment with CU DC- 907
The effect of the CUDC-907 on NET and DAT expression was studied by Quantitative reverse transcription PCR (RT-qPCR) (Figure 1A). This example demonstrated that NET expression is significantly increased, and DAT expression marginally increased upon treatment with CUDC-907. RNA was extracted from biopsies of PHEO/PGL samples prepared devoid of remaining healthy tissue by a surgeon or pathologist, or from NB8 cells using Trizol (Invitrogen, Luzern, Switzerland). cDNA synthesis was performed with the PrimeScript Reverse Transcriptase Kit (Takara Bio Inc). PCR was performed by using the SYBR Green Master Mix (Roche) for NET, DAT, PMAT, OCT1-3, glyceraldehyde 3- phosphate dehydrogenase (GAPDH) and eukaryotic translation elongation factor 1 alpha 1 (EEIF1A1 ). The primers were designed using the “Primer Blast” tool from the NCBI website.
Table 2 - Primers used for RT-qPCR of NB8 cells incubated or not with different HDAC inhibitors.
Figure imgf000056_0001
Reactions were performed in a QuantStudio 6 Real-Time PCR System, and amplification was carried out in a 384-well reaction plate as follows: 95°C for 10 minutes, followed by 40 cycles of 95°C for 15 seconds and 60°C for 1 minute. The limit of quantification values was set at 33 cycles, and values above this threshold were considered as 34 cycles. Each sample was analyzed in duplicate, and a negative control was prepared by using the same amount of total RNA without adding the enzyme reverse transcriptase. Expression levels of NET and DAT transcripts were calculated relative to the level of the housekeeping genes GAPDH and EEIF1A1 using the AACt method (Rao, X., et al., Biostat Bioinforma Biomath, 2013; 3(3):71 -85.).
NET mRNA expression was significantly increased in CUDC-907-treated cells compared with control cells (fold change = 1.76 ± 0.36, p <0.01 ). DAT mRNA expression levels were also increased in treated cells but the results were statistically insignificant (fold change = 1.61 ± 0.51 , p = 0.0527). Immunofluorescence detection was used and an increase of NET and DAT protein fluorescence in treated cells observed (Figure 1 B and 1 C, respectively).
Example 2: Study on the effect of CUDC-907 on mIBG internalization through NET upregulation
To understand the respective roles of NET and DAT in mIBG internalization in NB8 cells following treatment with CUDC-907, DMI and GBR12935 inhibitors were used. DMI and GBR12935 specifically inhibit NET and DAT respectively. This example demonstrated that NET is the main transporter involved in mIBG internalization.
Specificity of DMI and GBR12935 toward NET and DAT, respectively, was determined by increasing concentrations of inhibitors (from 0 to 10 pM) at 37°C, 30 minutes before mIBG incubation. To study the effect of NET and DAT inhibition on mIBG internalization in CUDC-907-treated cells, cells were incubated with CUDC-907 0.1 pM for 48 hours following DMI and/or GBR12935 treatment at different concentrations (from 0 to 1 pM) for 30 minutes prior to mIBG incubation (10 nM for 10 minutes). The addition of CUDC-907 0.1 pM alone increased mIBG internalization 3-fold (fold change = 3.00 ± 0.76, p <0.0001 ) when compared to the control. The addition of DMI 1 pM to CUDC-907 nearly eliminated mIBG internalization when compared with control cells (no inhibitors) (fold change = 0.05 ± 0.01 , p <0.0001 ) (Figure 2A), while the inhibition of GBR12935 at 1 pM was around 70% (fold change = 0.31 ± 0.14, p <0.0001 ). When both inhibitors were added at the same time at high concentrations (0.5 pM and 1 pM, respectively), we observed a similar inhibition profile as when the cells were treated with DMI alone. DMI reduced mIBG internalization by nearly 60% at the lowest concentration used while at an increased concentration (up to 1 pM) it nearly eliminated mIBG internalization. When GBR12935 0.5 pM was added to the cells in the presence of CUDC-907, it inhibited nearly 50% of mIBG internalization, and the combination of DMI and GBR12935 once again was similar to the profile of inhibition observed with DMI alone. These results suggest that mIBG internalization is mainly performed by NET, and DAT is involved to a lesser extent.
To further confirm NET as the main transporter for mIBG internalization, siRNA technology to knockdown NET and/or DAT expression was used (Figure 2B).
NB8 cells were plated in a 24-well cell culture plate (9 x 104 cells) for 24 hours. Cells were then transfected with the following 25 nM siRNA mixtures: ON-TARGETplus SMARTpool siRNA mixtures targeting NET (L-007602-00), DAT (L-007603-00), nontargeting control siRNA (D-001810-10) and GAPDH control siRNA (D-001830-10) from Horizon. After a 24 hour incubation period, cells were treated with CUDC-907 0.1 pM or BGT226 0.05 pM for a further 48 hours and then incubated with 10 nM mIBG at 37°C for 10 minutes before mIBG extraction and quantification (Figure 2B and 2E).
The knockdown of NET led to a decrease of mIBG internalization compared with control cells (no siRNA) (fold change = 0.58 ± 0.18; p <0.05), while the knockdown of DAT showed no effect following treatment with CUDC-907 when compared with control cells (fold change = 0.96 ± 0.19, p = 0.798). Finally, when both NET and DAT were silenced, the profile was similar to the case where only NET expression was inhibited, even though this difference was statistically insignificant (p = 0.0964).
Example 3: Study of PI3K inhibition on mIBG internalization in NB8 cells through NET
CUDC-907 is a dual inhibitor of HDAC and PI3K, to identify which inhibition pathway is prevalent in the mIBG internalization process, the role of each transducing pathway was assessed separately. This example demonstrated that the inhibition of PI3K increases mIBG internalization.
To test HDAC and PI3K/Akt/mT0R inhibition effects, cells were treated for 48 hours at 37°C and incubated with mIBG 10 nM for 10 minutes.
BGT226, a specific PI3K inhibitor, targeting subunits a, [3 and y was tested on NB8 cells and was unexpectedly shown to increase mIBG internalization in a dosedependent manner (Figure 2C). At 50 nM, BGT226 increased mIBG internalization 2.5- fold when compared with non-treated cells (fold change = 2.48 ± 0.47, p <0.01 ).
Treatment with CUDC-907 and BGT226 exhibited the same potency for mIBG uptake, suggesting that CUDC-907 effects might preferentially involve PI3K inhibition (Figure 2D). To further evaluate the effect of BGT226 on NET and DAT, knockdown studies of these transporters were conducted separately and simultaneously (Figure 2E). The knockdown of NET led to a decrease in mIBG internalization after treatment with BGT226 (fold change = 0.54 ± 0.13; p <0.01 ). Conversely, DAT silencing did not have a significant effect on mIBG internalization. Lastly, silencing of both NET and DAT resulted in the same profile as the NET knockdown (fold change = 0.50 ± 0.09; p <0.01 ).
Example 4: Study of the PI3K/Akt/mTOR signalling pathway: effect on mIBG internalization
PI3K is a component of the Akt/mTOR signalling pathway, to identify the key component(s) contributing to mIBG uptake, Akt and mTOR inhibitors were studied. This study demonstrated that mTOR inhibition facilitates mIBG uptake, with Akt having a negative effect on uptake.
VS-5584, a PI3K/mT0R inhibitor and rapamycin, a mTOR inhibitor, significantly increased mIBG internalization when compared to non-treated cells (fold change = 1 .51 ± 0.11 , p <0.001 and 1 .34 ± 0.10, p <0.001 , respectively) (Figure 2F). On the other hand, MK2206, an Akt inhibitor, significantly decreased mIBG internalization when compared to control cells (fold change = 0.72 ± 0.13, p <0.01 ) (Figure 2F).
Example 5: Study of CUDC-907 treatment and 123l-mlBG uptake in NB8 xenografts in mice
The effect of CUDC-907 treatment and mIBG uptake was studied on xenograft mice. Biodistribution data and SPECT/CT imaging showed an increase in 123l-mlBG uptake.
Animal studies
All animal experiments were carried out with female athymic nude- Foxn1 n7Foxn1 +mice (Envigo). The mice (4-6 weeks old) were subcutaneously implanted with 2 x 106 NB8 cells/mouse in DMEM/Matrigel (1/1 v/v, 200 pL) and monitored twice weekly. After one month the mice were used for the study once the tumours reached a size of 120-200 mm3.
Treatment and biodistribution studies of 123l-mlBG
Animals were randomly divided into three groups: group A (n = 7) received the vehicle (10% DMSO in corn oil), group B (n = 9) received 5mg/Kg of CUDC-907 and group C (n = 13) received 10 mg/Kg of CUDC-907. CUDC-907 was administered for 5 days, via oral gavage, at the indicated doses and after 2 days drug-free the mice were injected with 123l-mlBG (2-4 MBq/100 pL). Quantitative biodistribution studies were performed 4 hours and 24 hours post-injection (p.i.) of 123l-mlBG via the tail vein. All mice were administered intravenously with sodium perchlorate (100 pL Irenat, 120 mg/kg) 5 minutes before 123l-mlBG injection for blocking the uptake of free radioiodine in iodine-avid organs.
Single-photon emission computed tomography/Computed tomography (SPECT/CT) imaging studies
SPECT/CT images were acquired at 4 hours and 24 hours p.i. of 123l-mlBG (13-17 MBq/100 pL) using a small animal scanner (Nano-SPECT/CT™ Bioscan Inc.). The SPECT/CT images 4 hours p.i. were acquired for 90 minutes whilst mice were under anaesthesia. After one day the mice were euthanized and SPECT/CT images of the same mice were acquired at 24 hours p.i. for 170 minutes.
NB8 xenografted mice were randomly divided into three groups (A: vehicle; B: 5 mg/kg; C: 10 mg/kg). CUDC-907 given orally to the mice for 5 days, was well tolerated with no side effects. After 2 days’ drug-free, biodistribution studies of 123l-mlBG were performed at 4 hours and 24 hours p.i. (Figure 3A). At 4 hours p.i., the treated groups showed no significant differences in the accumulation of 123l-mlBG in most of the organs and tumours, except for the gallbladder (8.37 ± 2.19% of injected activity/g of tissue [%IA/g] vs. 18.22 ± 5.49%IA/g for group B and C, respectively, p <0.05), due to the physiological excretion. The administration of the two different doses of CUDC-907 in groups B and C did not significantly impact the tumour uptake of 123l-mlBG (2.24 ± 0.40%IA/g vs. 3.19 ± 1.20%IA/g for group B and C, respectively, p = 0.1 ). The tumour uptake of both groups was significantly higher when compared to the uptake in the vehicle group (2.24 ± 0.40%IA/g vs. 0.98 ± 0.35%IA/g, for group B and A, respectively, p <0.05). At 24 hours p.i., a remarkable background clearance and washout from all organs was observed, except for the adrenal glands (Figure 3A). Interestingly, no washout from the tumours was observed (tumour uptake was similar between 4 and 24 hours p.i.). The tumour uptake remained significantly higher in the treated groups compared to the untreated group (2.71 ± 0.64%IA/g vs 1 .42 ± 0.44%IA/g, for group B and A, respectively, p <0.05). p values <0.05 are considered statistically significant.
Representative SPECT/CT images from each group (A, B and C) were acquired 4 and 24 hours (Figure 3B and C, respectively) after the injection of 123l-mlBG. The same mouse per group was imaged at both time points, demonstrating the significant improvement in the tumour to background contrast, and consequently clear tumour visualization and delineation, with 123l-mlBG over time. At 4 hours p.i. 123l-mlBG accumulation in the gallbladder and in the intestine 20 was observed, independently of the CUDC-907 treatment regimen (5 mg/Kg or 10 mg/Kg). At 24 hours p.i. there was a complete background clearance, except for the adrenal glands and the tumours, confirming the quantitative biodistribution data (Figure 3C). Example 6: Study of the upregulation of somatostatin receptor (SST) mRNA and proteins in prostate cancer cell lines treated with HDAC inhibitors and dual HDAC/PI3K/Akt/mTOR inhibitors
PC3 prostate cancer cell lines were used to study the effect of several HDAC/PI3K/Akt/mT0R inhibitors on the expression of somatostatin receptor genes (Figure 4A). The use of these inhibitors was found to significantly upregulate SST genes (as the mRNAfold change) after 48 hour-treatment with various HDAC inhibitors compared with untreated PC3 cells (control). The most efficacious HDAC inhibitors for SST1 were: LMK235 which induced a fold change (FC) of 1.92 compared with control (p = 0.0079); for SST2, quisinostat induced a FC of 3.58 (p = 0.0006), CUDC907 induced a FC of 1.96 (p = 0.0006) and vorinostat induced a FC of 1.95 (p = 0.0022); for SST3, CUDC907 induced a FC of 2.80 (p = 0.0079).
LnCAP prostate cancer cell lines were used to investigate SST2 and SST5 protein levels upon treatment with HDAC/PI3K/Akt/mTOR inhibitors (Figure 4B and C). The use of various inhibitors showed an increase in these protein levels upon treatment. LnCAP cells were incubated with HDAC inhibitors for 48 hours and quisinostat, sodium-4-phenylbutyrate, decitabine and romidepsin increased SST5 protein expression, while tucidinostat increased SST2 protein expression.
Example 7: Study of the CUDC-907 treatment and 131l-mlBG radiation dose in NB8 xenografts in mice
The effect of CUDC-907 treatment on the 131 l-mlBG uptake on xenograft mice was studied.
Animal studies
The animal studies were carried out with female athymic nude-Foxn1 n Foxn1 +mice (Envigo). The mice were subcutaneously implanted with 2 x 106 NB8 cells/mouse in DMEM/Matrigel (1/1 v/v, 200 pL) and monitored twice weekly. Once the implanted tumours reach a certain size and were visible (e.g. 100-150 mm3), the mice were used for the in vivo study. The mice were randomly divided into two groups: group A (n = 4) received the vehicle (10% DMSO in corn oil), group B (n = 5) received 10 mg/Kg of CUDC-907. The treatment was performed for 5 days, via oral gavage and after 2 days drug-free the mice were injected with 131 l-mlBG (0.5 MBq/100 pL). Quantitative biodistribution studies were performed 4 hours and 24 hours post-injection (p.i.) of 1311- mlBG via the tail vein. All mice were administered intravenously with sodium perchlorate (100 pL Irenat, 120 mg/kg) 5 minutes before 131 l-mlBG injection for blocking the uptake of free radioiodine in iodine-avid organs.
The absorbed tumour radiation dose in the pre-treated mice was compared with the absorbed tumour radiation dose in the untreated mice in order to illustrate the enhancement of the tumour dose, and thus enhanced therapeutic effect obtained upon the CUDC-907 treatment.
Table 3 - Biodistribution data of 131 I-MIBG in NB8 xenografts.
131I-MIBG (NB8 xenografts)
4h p.i. 24h p.i.
Organs Vehicle CUDC-907 Vehicle CUDC-907
(n=4) 10 mg/Kg (n=4) 10 mg/Kg
(n=5) (n=5)
Blood 0.51±0.13 0.53±0.07 0.12±0.07 0.12±0.01
Heart 5.23±1.19 5.07±0.49 1.02±0.48 1.29±0.20
Lung 2.71±0.71 3.13±0.36 0.44±0.26 0.45±0.09
Liver 2.99±0.59 2.99±0.16 0.49±0.21 0.55±0.06
Pancreas 2.13±0.38 2.34±0.19 0.38±0.15 0.42±0.05
Spleen 2.18±0.58 2.21±0.27 0.66±0.16 0.74±0.15
Stomach 2.73±0.70 2.91±0.33 1.33±0.56 1.46±0.14
Intestine 4.76±1.25 4.85±0.97 0.85±0.43 1.21±0.06
Adrenal 7.94±2.31 8.25±0.89 5.05±2.44 6.73±2.11
Kidney 1.82±0.34 1.88±0.10 0.46±0.17 0.54±0.06
Muscle 1.16±0.45 0.95±0.24 0.17±0.11 0.18±0.05
Bone 1.07±0.32 1.08±0.15 0.17±0.10 0.21±0.03
NB8 2.71±0.91 3.42±0.83 1.32±0.91 2.21±0.47
Salivary 7.80±1.82 8.15±0.77 1.47±0.65 1.64±0.14 gland
Further embodiments and/or examples of the invention are described in the following numbered items.
1 . A composition comprising a) a PI3K/Akt/mTOR inhibitor, and b) a radiopharmaceutical whose cellular uptake is upregulated upon inhibition of PI3K/Akt/mTOR. A pharmaceutical composition comprising a) a PI3K/Akt/mTOR inhibitor, b) a radiopharmaceutical whose cellular uptake is upregulated upon inhibition of PI3K/Akt/mTOR, and a pharmaceutically acceptable carrier. A kit of parts comprising a) a PI3K/Akt/mTOR inhibitor and a pharmaceutically acceptable carrier, and b) a radiopharmaceutical whose cellular uptake is upregulated upon inhibition of PI3K/Akt/mTOR and a pharmaceutically acceptable carrier. The composition of item 1 , the pharmaceutical composition of item 2 or the kit of parts of item 3, wherein the PI3K/Akt/mTOR inhibitor is selected from BEZ235, BKM120, Everolimus, MK-2206 dichloride, Pictilisib, LY294002, CAL-101 , PI- 3065, HS-173, PI-103. , NU7441 , TGX-221 , IC-87114, Wortmannin, XL147, ZSTK474, BYL719, AS-605240, PIK-75, 3-methyladenine, A66, SAR245409, PIK-93, GSK2126458, PIK-90, PF-04691502, AZD6482, Apitolisib,
GSK1059615, Duvelisib, Gedatolisib, TG100-115, AS-252424, BGT226, CUDC- 907, PIK-294, AS-604850, GSK2636771 , BAY80-6946, YM201636, CH5132799, CAY10505, rapamycin and PIK-293. The composition of item 1 or 4, the pharmaceutical composition of item 2 or 4, or the kit of parts of item 3 or 4, wherein the composition, the pharmaceutical composition or the kit of parts comprises CUDC-907. The composition of any one of items 1 , 4 or 5, the pharmaceutical composition of any one of items 2, 4 or 5, or the kit of parts of any one of items 3 to 5, wherein the radiopharmaceutical whose cellular uptake is upregulated upon inhibition of PI3K/Akt/mTOR is a catecholamine or its analogue comprising a radionuclide. The composition, the pharmaceutical composition or the kit of parts of item 6, wherein the catecholamine or its analogue comprising a radionuclide is is 123l- meta-iodobenzylguanidine, 124l-meta-iodobenzylguanidine, 125l-meta- iodobenzylguanidine or 131 l-meta-iodobenzylguanidine, preferably 123l-meta- iodobenzylguanidine or 131 l-meta-iodobenzylguanidine. The composition of any one of items 1 , 4 or 5, the pharmaceutical composition of any one of items 2, 4 or 5, or the kit of parts of any one of items 3 to 5, wherein the radiopharmaceutical whose cellular uptake is upregulated upon inhibition of PI3K/Akt/mTOR is somatostatin or its analogue. The composition, the pharmaceutical composition or the kit of parts of item 8, wherein the somatostatin or its analogue is preferably selected from TATE, AM3 and pasireotide, more preferably wherein the somatostatin or its analogue is conjugated to a chelator moiety, optionally loaded with a radionuclide preferably selected from 225Ac, 213Bi, 99mTc, 11 11n, 177Lu, 67Ga, 68Ga, 90Y, 64Cu, 67Cu, 61 Cu, 43Sc, 44Sc, 47Sc, 212Pb, 203Pb, 161Tb, 152Tb, 155Tb and 149Tb, and/or wherein the somatostatin or its analogue. The composition of any one of items 1 or 4 to 9, the pharmaceutical composition of any one of items 2 or 4 to 9, or the kit of parts of any one of items 3 to 9, further comprising a HDAC inhibitor. The composition of any one of items 1 or 4 to 10, the pharmaceutical composition of any one of items 2 or 4 to 10, or the kit of parts of any one of items 3 to 10, for use in diagnosis or therapy. The composition of any one of items 1 or 4 to 10, the pharmaceutical composition of any one of items 2 or 4 to 10, or the kit of parts of any one of items 3 to 10, for use in diagnosis or therapy of a neuroendocrine tumour. The composition for use or the pharmaceutical composition for use or the kit of parts for use of item 12, wherein the neuroendocrine tumour is a neuroendocrine tumour expressing monoamine transporters and/or somatostatin receptors, preferably selected from neuroblastoma, pheochromocytoma and paraganglioma. The composition for use or the pharmaceutical composition for use or the kit of parts for use of any one of items 11 to 13, for use in diagnosis. The composition for use or the pharmaceutical composition for use or the kit of parts for use of item 14, wherein the radiopharmaceutical whose cellular uptake is upregulated upon inhibition of PI3K/Akt/mTOR is 123l-meta- iodobenzylguanidine. The composition for use or the pharmaceutical composition for use or the kit of parts for use of any one of items 11 to 13, for use in therapy. The composition for use or the pharmaceutical composition for use or the kit of parts for use of item 16, wherein the radiopharmaceutical whose cellular uptake is upregulated upon inhibition of PI3K/Akt/mT0R is 131 l-meta- iodobenzylguanidine. Use of a PI3K/Akt/mT0R inhibitor in a method of upregulating the expression of norepinephrine transporter (NET) and/or dopamine transporter (DAT) in a subject. Use of a PI3K/Akt/mT0R inhibitor in a method of upregulating the expression of a somatostatin receptor in a subject, preferably wherein the somatostatin receptor is SST2. The use of item 18 or 19, wherein said PI3K/Akt/mTOR inhibitor is selected from BEZ235, BKM120, Everolimus, MK-2206 dichloride, Pictilisib, LY294002, CAL- 101 , PI-3065, HS-173, PI-103. , NU7441 , TGX-221 , 10-87114, Wortmannin, XL147, ZSTK474, BYL719, AS-605240, PIK-75, 3-methyladenine, A66, SAR245409, PIK-93, GSK2126458, PIK-90, PF-04691502, AZD6482, Apitolisib, GSK1059615, Duvelisib, Gedatolisib, TG100-115, AS-252424, BGT226, CUDC- 907, PIK-294, AS-604850, GSK2636771 , BAY80-6946, YM201636, CH5132799, CAY10505, rapamycin and PIK-293. The use of any one of items 18 to 20, wherein said PI3K/Akt/mTOR inhibitor is CUDC-907. A method for treating a neuroendocrine tumour in a subject, the method comprising the steps of: a) administering a PI3K inhibitor to a subject in need thereof, followed by b) administering a radiopharmaceutical whose cellular uptake is upregulated upon inhibition of PI3K/Akt/mTOR to a subject in need thereof. The method of item 23, wherein the neuroendocrine tumour is a neuroendocrine tumour expressing monoamine transporters and/or somatostatin receptors, preferably selected from neuroblastoma, pheochromocytoma and paraganglioma. The method of item 23, wherein the PI3K/Akt/mTOR inhibitor is selected from BEZ235, BKM120, Everolimus, MK-2206 dichloride, Pictilisib, LY294002, CAL- 101 , PI-3065, HS-173, PI-103. , NU7441 , TGX-221 , IC-87114, Wortmannin, XL147, ZSTK474, BYL719, AS-605240, PIK-75, 3-methyladenine, A66, SAR245409, PIK-93, GSK2126458, PIK-90, PF-04691502, AZD6482, Apitolisib, GSK1059615, Duvelisib, Gedatolisib, TG100-115, AS-252424, BGT226, CUDC- 907, PIK-294, AS-604850, GSK2636771 , BAY80-6946, YM201636, CH5132799, CAY10505, rapamycin and PIK-293. The method of item 23, wherein the PI3K/Akt/mTOR inhibitor is CUDC-907. The method of item 23, wherein the radiopharmaceutical whose cellular uptake is upregulated upon inhibition of PI3K/Akt/mTOR is a catecholamine or its analogue comprising a radionuclide. The method of item 27, wherein the catecholamine or its analogue comprising a radionuclide is 123l-meta-iodobenzylguanidine, 124l-meta-iodobenzylguanidine, 125l-meta-iodobenzylguanidine or 131 l-meta-iodobenzylguanidine, preferably 123l- meta-iodobenzylguanidine or 131 l-meta-iodobenzylguanidine. The method of item 23, wherein the radiopharmaceutical whose cellular uptake is upregulated upon inhibition of PI3K/Akt/mTOR is somatostatin or its analogue, preferably wherein the somatostatin or its analogue is selected from TATE, AM3 and pasireotide. The method of item 29, wherein the somatostatin or its analogue is conjugated to a chelator moiety, optionally loaded with a radionuclide preferably selected from 225Ac, 213Bi, 99mTc, 1 11 In, 177Lu, 67Ga, 68Ga, 90Y, 64Cu, 67Cu, 61Cu, 43Sc, 44Sc, 47Sc, 212Pb, 203Pb, 161Tb, 152Tb, 155Tb and 149Tb. The method of item 23, further comprising the step of administering an HDAC inhibitor to a subject in need thereof.

Claims

CLAIMS A composition comprising a) a PI3K/Akt/mTOR inhibitor, and b) a radiopharmaceutical whose cellular uptake is upregulated upon inhibition of PI3K/Akt/mTOR. A pharmaceutical composition comprising a) a PI3K/Akt/mTOR inhibitor, b) a radiopharmaceutical whose cellular uptake is upregulated upon inhibition of PI3K/Akt/mTOR, and a pharmaceutically acceptable carrier. A kit of parts comprising a) a PI3K/Akt/mTOR inhibitor and a pharmaceutically acceptable carrier, and b) a radiopharmaceutical whose cellular uptake is upregulated upon inhibition of PI3K/Akt/mTOR and a pharmaceutically acceptable carrier. The composition of claim 1 , the pharmaceutical composition of item 2 or the kit of parts of claim 3, wherein the PI3K/Akt/mTOR inhibitor is selected from BEZ235, BKM120, Everolimus, MK-2206 dichloride, Pictilisib, LY294002, CAL- 101 , PI-3065, HS-173, PI-103. , NU7441 , TGX-221 , 10-87114, Wortmannin, XL147, ZSTK474, BYL719, AS-605240, PIK-75, 3-methyladenine, A66, SAR245409, PIK-93, GSK2126458, PIK-90, PF-04691502, AZD6482, Apitolisib, GSK1059615, Duvelisib, Gedatolisib, TG100-1 15, AS-252424, BGT226, CUDC-907, PIK-294, AS-604850, GSK2636771 , BAY80-6946, YM201636, CH5132799, CAY10505, rapamycin and PIK-293. The composition of claim 1 or 4, the pharmaceutical composition of claim 2 or 4, or the kit of parts of claim 3 or 4, wherein the composition, the pharmaceutical
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RECTIFIED SHEET (RULE 91 ) ISA/EP composition or the kit of parts comprises CUDC-907. The composition of any one of claims 1 , 4 or 5, the pharmaceutical composition of any one of claims 2, 4 or 5, or the kit of parts of any one of claims 3 to 5, wherein the radiopharmaceutical whose cellular uptake is upregulated upon inhibition of PI3K/Akt/mTOR is a catecholamine or its analogue comprising a radionuclide. The composition, the pharmaceutical composition or the kit of parts of claim 6, wherein the catecholamine or its analogue comprising a radionuclide is 123l- meta-iodobenzylguanidine, 124l-meta-iodobenzylguanidine, 125l-meta- iodobenzylguanidine or 131 l-meta-iodobenzylguanidine, preferably 123l-meta- iodobenzylguanidine or 131 l-meta-iodobenzylguanidine. The composition of any one of claims 1 , 4 or 5, the pharmaceutical composition of any one of claims 2, 4 or 5, or the kit of parts of any one of claims 3 to 5, wherein the radiopharmaceutical whose cellular uptake is upregulated upon inhibition of PI3K/Akt/mTOR is somatostatin or its analogue. The composition, the pharmaceutical composition or the kit of parts of claim 8, wherein the somatostatin or its analogue is preferably selected from TATE, AM3 and pasireotide, more preferably wherein the somatostatin or its analogue is conjugated to a chelator moiety, optionally loaded with a radionuclide preferably selected from 225Ac, 213Bi, 99mTc, 11 11n, 177Lu, 67Ga, 68Ga, 90Y, 64Cu, 67Cu, 61Cu, 43Sc, 44Sc, 47Sc, 212Pb, 203Pb, 161Tb, 152Tb, 155Tb and 149Tb, and/or wherein the somatostatin or its analogue. The composition of any one of claims 1 or 4 to 9, the pharmaceutical composition of any one of claims 2 or 4 to 9, or the kit of parts of any one of claims 3 to 9, further comprising a HDAC inhibitor. The composition of any one of claims 1 or 4 to 10, the pharmaceutical
69
RECTIFIED SHEET (RULE 91 ) ISA/EP composition of any one of claims 2 or 4 to 10, or the kit of parts of any one of claims 3 to 10, for use in diagnosis or therapy. The composition of any one of claims 1 or 4 to 10, the pharmaceutical composition of any one of claims 2 or 4 to 10, or the kit of parts of any one of claims 3 to 10, for use in diagnosis or therapy of a neuroendocrine tumour. The composition for use or the pharmaceutical composition for use or the kit of parts for use of claim 12, wherein the neuroendocrine tumour is a neuroendocrine tumour expressing monoamine transporters and/or somatostatin receptors, preferably selected from neuroblastoma, pheochromocytoma and paraganglioma. The composition for use or the pharmaceutical composition for use or the kit of parts for use of any one of claims 11 to 13, for use in diagnosis. The composition for use or the pharmaceutical composition for use or the kit of parts for use of claim 14, wherein the radiopharmaceutical whose cellular uptake is upregulated upon inhibition of PI3K/Akt/mTOR is 123l-meta- iodobenzylguanidine. The composition for use or the pharmaceutical composition for use or the kit of parts for use of any one of claims 11 to 13, for use in therapy. The composition for use or the pharmaceutical composition for use or the kit of parts for use of claim 16, wherein the radiopharmaceutical whose cellular uptake is upregulated upon inhibition of PI3K/Akt/mTOR is 131l-meta- iodobenzylguanidine. Use of a PI3K/Akt/mTOR inhibitor in a method of upregulating the expression of norepinephrine transporter (NET) and/or dopamine transporter (DAT) in a
70
RECTIFIED SHEET (RULE 91 ) ISA/EP subject. Use of a PI3K/Akt/mT0R inhibitor in a method of upregulating the expression of a somatostatin receptor in a subject, preferably wherein the somatostatin receptor is SST2. The use of claim 18 or 19, wherein said PI3K/Akt/mTOR inhibitor is selected from BEZ235, BKM120, Everolimus, MK-2206 dichloride, Pictilisib, LY294002, CAL-101 , PI-3065, HS-173, PI-103. , NU7441 , TGX-221 , 10-87114, Wortmannin, XL147, ZSTK474, BYL719, AS-605240, PIK-75, 3-methyladenine, A66, SAR245409, PIK-93, GSK2126458, PIK-90, PF-04691502, AZD6482, Apitolisib, GSK1059615, Duvelisib, Gedatolisib, TG100-1 15, AS-252424, BGT226, CUDC-907, PIK-294, AS-604850, GSK2636771 , BAY80-6946, YM201636, CH5132799, CAY10505, rapamycin and PIK-293. The use of any one of claims 18 to 20, wherein said PI3K/Akt/mTOR inhibitor is CUDC-907. A method for treating a neuroendocrine tumour in a subject, the method comprising the steps of: a) administering a PI3K inhibitor to a subject in need thereof, followed by b) administering a radiopharmaceutical whose cellular uptake is upregulated upon inhibition of PI3K/Akt/mTOR to a subject in need thereof. The method of claim 22, wherein the neuroendocrine tumour is a neuroendocrine tumour expressing monoamine transporters and/or somatostatin receptors, preferably selected from neuroblastoma, pheochromocytoma and paraganglioma. The method of claim 22, wherein the PI3K/Akt/mTOR inhibitor is selected from BEZ235, BKM120, Everolimus, MK-2206 dichloride, Pictilisib, LY294002, CAL- 101 , PI-3065, HS-173, PI-103. , NU7441 , TGX-221 , IC-87114, Wortmannin,
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RECTIFIED SHEET (RULE 91 ) ISA/EP XL147, ZSTK474, BYL719, AS-605240, PIK-75, 3-methyladenine, A66, SAR245409, PIK-93, GSK2126458, PIK-90, PF-04691502, AZD6482, Apitolisib, GSK1059615, Duvelisib, Gedatolisib, TG100-1 15, AS-252424, BGT226, CUDC-907, PIK-294, AS-604850, GSK2636771 , BAY80-6946, YM201636, CH5132799, CAY10505, rapamycin and PIK-293. The method of claim 22, wherein the PI3K/Akt/mT0R inhibitor is CUDC-907. The method of claim 22, wherein the radiopharmaceutical whose cellular uptake is upregulated upon inhibition of PI3K/Akt/mT0R is a catecholamine or its analogue comprising a radionuclide. The method of claim 26, wherein the catecholamine or its analogue comprising a radionuclide is 123l-meta-iodobenzylguanidine, 124l-meta-iodobenzylguanidine, 125l-meta-iodobenzylguanidine or 131 l-meta-iodobenzylguanidine, preferably 123l-meta-iodobenzylguanidine or 131 l-meta-iodobenzylguanidine. The method of claim 22, wherein the radiopharmaceutical whose cellular uptake is upregulated upon inhibition of PI3K/Akt/mTOR is somatostatin or its analogue, preferably wherein the somatostatin or its analogue is selected from TATE, AM3 and pasireotide. The method of claim 28, wherein the somatostatin or its analogue is conjugated to a chelator moiety, optionally loaded with a radionuclide preferably selected from 225Ac, 213Bi, 99mTc, 11 11n, 177Lu, 67Ga, 68Ga, 90Y, 64Cu, 67Cu, 61Cu, 43Sc, 44Sc, 47Sc, 212Pb, 203Pb, 161Tb, 152Tb, 155Tb and 149Tb. The method of claim 22, further comprising the step of administering an HDAC inhibitor to a subject in need thereof.
RECTIFIED SHEET (RULE 91 ) ISA/EP
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