WO2013182519A1

WO2013182519A1 - Combination of lysosomotropic or autophagy modulating agents and a gsk-3 inhibitor for treatment of cancer

Info

Publication number: WO2013182519A1
Application number: PCT/EP2013/061380
Authority: WO
Inventors: Serdar KORUR; Florent Beaufils; Matthias Wymann
Original assignee: Universitaet Basel
Priority date: 2012-06-04
Filing date: 2013-06-03
Publication date: 2013-12-12

Abstract

The invention relates to combinations oflysosomotropic agent or agents modulating autophagy, such as chloroquine, 3-methyladenine or bafilomycin A1, and a GSK-3 inhibitor, such as (2'Z,3'E)-6-bromoindirubin-3'-acetoxime, TDZD-8 or L803-mts. These compositions are useful in the treatment of cancer, proliferative inflammatory diseases, degenerative diseases and some infectious diseases, preferably cancer. The tumor cell survival is particularly dependent on proper endoplasmic reticulumf unction.

Description

Combinations of lysosomotropic or autophagy modulating agents and a GSK-3 inhibitor for treatment of cancer

Field of the Invention

The invention relates to combinations of lysosomotropic agent or agents modulating autophagy and a GSK-3 inhibitor, useful in the treatment of cancer, proliferative inflammatory diseases, degenerative diseases and infectious diseases. Background Art

Malignant gliomas remain one of the deadliest of all cancers despite maximal therapy efforts. They present unique challenges to therapy with a median survival of 12 months. Simultaneous activation of several growth promoting and anti-apoptotic pathways represents the reason for the failure of monotherapies against this disease. Highly mutating phenotypes of GBM cells enabled them to gain alterations in several growth promoting pathways and there is an obvious cross interaction between several signaling pathways. Thus, in order to efficiently induce cell death in GBM cells, a combination of two or more drugs is required.

The rationale in combination therapy is to apply drugs that work via different mechanisms in order to decrease the probability of developing drug-resistant cancer cells. Combination therapy also allows, for certain drug combinations, an optimal combined dose to minimize side effects. This is crucial as standard chemotherapeutic agents target essential cellular process such as DNA replication, cell division or induce DNA damage and thus have a general cytotoxic effect. Finally, combination treatment of two compounds may uncover unanticipated synergisms and trigger effects not induced by a single compound. In recent years, drugs are also used in a neoadjuvant setting, i.e. prior to surgery, to reduce the tumor mass or to improve long-term survival.

Among the 518 protein kinases, which constitute the human kinome, the glycogen synthase 3-kinase (GSK-3) members stand out as a particularly interesting and well- studied family of serine/threonine kinases. There are two GSK-3 forms (GSK-3alfa and GSK-3beta), which share extensive similarity (84% overall identity, 98% within the catalytic domain). GSK-3 are highly conserved protein kinases present from unicellular parasites to yeast up to mammals. These kinases are involved in numerous critical physiological events such as WNT and Hedgehog signaling, embryonic development (pattern specification and axial orientation), transcription, insulin action, cell division cycle, cell death, cell survival, differentiation, multiple neuronal functions, circadian rhythm regulation, and stem cell differentiation. In addition GSK-3 are implicated in a large diversity of human diseases, including nervous system disorders such as Alzheimer's disease, schizophrenia, bipolar disorder, diabetes, heart hypertrophy, renal diseases, shock and inflammation, cancers, and the like. There is thus a strong rationale supporting the search for potent and selective GSK-3 inhibitors for use as pharmacological tools in basic research, as potential drugs for the treatment of specific diseases and for the maintenance of pluripotent stem cells in the absence of feeder cells. Numerous GSK-3 inhibitory scaffolds have been described.

Interestingly many of these inhibitors also interact with cyclin-dependent kinases (CDKs), another family of well-studied key regulatory enzymes.

GSK-3 is known for phosphorylating and thus inactivating glycogen synthase. However, GSK-3 works in the WNT signaling pathway to phosphorylate beta-catenin.

Phosphorylation leads to ubiquitination and degradation by cellular proteases, preventing it from entering the nucleus and activating transcription factors. When a protein called Disheveled is activated by WNT signaling, GSK-3 is inactivated, allowing beta-catenin to accumulate and effect transcription of WNT target genes. GSK-3 also phosphorylates Ci in the Hedgehog (Hh) pathway, targeting it for proteolysis to an inactive form. GSK-3 is unusual among the kinases in that it usually requires a "priming kinase" to first phosphorylate a substrate, and then GSK-3 additionally phosphorylates the substrate. The consequence of GSK-3 phosphorylation is usually inhibition of the substrate. For example, when GSK-3 phosphorylates another of its substrates, the NFAT family of transcription factors, these transcription factors cannot translocate to the nucleus and are therefore inhibited.

It has been shown that the activity of GSK can specifically be inhibited by substances referred to herein as "inhibitor of GSK-3" or "GSK-3 inhibitor". As detailed above, the inhibition of GSK-3 leads to activation of the WNT signaling pathway.

A well-known inhibitor of GSK-3 is lithium, e.g. as lithium chloride. The mode of inhibition is through competition for Mg²⁺. The bivalent form of zinc, which mimics insulin action, has also been shown to inhibit GSK-3. In vitro, another metal ion, beryllium, inhibits GSK-3. Several new GSK-3 inhibitors have recently been developed, most of which are ATP competitive. Summary of the Invention

The invention relates to pharmaceutical compositions comprising a lysosomotropic agent or agent modulating autophagy and a GSK-3 (glycogen synthase kinase 3) inhibitor, useful in the treatment of cancer, proliferative inflammatory diseases, degenerative diseases and infectious diseases including malaria, hepatitis A to C, HIV, African trypanosomiasis, cryptosporidiosis, Dengue fever, leishmaniasis, tuberculosis and schistosomiasis, in particular cancer.

Furthermore the invention relates to a method of treatment of cancer, proliferative inflammatory diseases, degenerative diseases and infectious diseases including malaria, hepatitis A to C, HIV, African trypanosomiasis, cryptosporidiosis, Dengue fever, leishmaniasis, tuberculosis and schistosomiasis, in particular cancer.

Brief Description of the Figures

Exemplary embodiments are illustrated in referenced figures. It is intended that the embodiments and figures disclosed herein are to be considered illustrative rather than restrictive. Figure 1: 6-bromoindirubin-3'-acetoxime (BlO-acetoxime) induces an endoplasmic reticulum (ER) stress response

U373 cells treated with 3 μΜ BlO-acetoxime for 16 h and subjected to cDNA microarray analysis.

A: Comparison of the GSK3 inhibitor treated data set with curated gene sets derived from literature revealed that (GSEA analysis) genes upregulated in response to GSK-3 inhibitor treatment were significantly overrepresented in gene sets related to metabolic deprivation (top panel), endoplasmic reticulum stress (middle panel) and ER stress inducing agents (bottom panel). The Y-axis indicates the enrichment score (ES), X-axis indicates the gene rank in the ordered dataset.

B: Responses to metabolic deprivation and/or ER stress response pathways both are known to be regulated through activation of ATF4. The analyses of a set of known ATF4 target genes showed activation of ATF4 transcriptional program. (1 ) indicates the BlO- acetoxime group, (2) indicates the control group.

C: Activation of ATF4 target genes upon treatment with three different GSK-3 inhibitors. Quantitative PCR (qPCR) analysis of the control group (1 ), the 6 μΜ BlO-acetoxime treated group (2), the 20 μΜ SB-216763 treated group (3) and the 50 μΜ L803-mts treated group (4) confirmed ATF4-dependent target gene induction. X-axis indicates fold induction of indicated genes relative to the control.

Figure 2: 6-bromoindirubin-3'-acetoxime (BlO-acetoxime) and chloroquine synergize to kill LN18 glioblastoma cells in vitro.

Y axis indicates cell death, X axis indicates treatment group (Control (1 ), BlO-acetoxime 6 μΜ (2), chloroquine 25 μΜ (3), BlO-acetoxime 6 μΜ and chloroquine 25 μΜ (4)). Cell death was quantified as the % of sub-G1 population 3 days after the addition of the drugs by flow cytometry using propidium iodide staining. Cell death resulting from the

combination of two drugs was analyzed using CalcuSyn Software (Biosoft) to generate a combination index (CI). The CI value is 0.003. A CI value <0.1 indicates a very strong synergism.

Figure 3: Different inhibitors of lysosomal degradation and GSK-3 synergize to kill LN18 and U373 glioblastoma cells in vitro.

Y axis indicates cell death, X axis indicates treatment group.

A and D: Control (1 ), BlO-acetoxime 6 μΜ (2), chloroquine 25 μΜ (3), BlO-acetoxime 6 μΜ and chloroquine 25 μΜ (4).

B and E: Control (1 ), BlO-acetoxime 6 μΜ (2), 3-methyladenine 5 mM (3), BlO-acetoxime 6 μΜ and 3-methyladenine 5 mM (4).

C and F: Control (1 ), BlO-acetoxime 6 μΜ (2), bafilomycin A1 2 nM (in C) or 5 nM (in F) (3), BlO-acetoxime 6 μΜ and bafilomycin A1 2 nM (in C) or 5 nM (in F) (4).

A, B, C represent LN18 and D, E, F represent U373 cells. Cell death was quantified as in Figure 1 .

Figure 4: Different GSK-3 inhibitors and chloroquine synergize to kill LN18 glioblastoma cells in vitro.

Y axis indicates cell death, X axis indicates treatment group.

Control (1 ), chloroquine 25 μΜ (2),TDZD-8 40 μΜ (3), TDZD-8 40 μΜ and chloroquine 25 mM (4), L803-mts at 100 μΜ (5), L803-mts at 100 μΜ and chloroquine 25 mM (6). Cell death was quantified as in Figure 1 . Figure 5: Combination of a GSK-3 inhibitor and chloroquine shows superior efficacy to kill LN18 glioblastoma cells in vitro.

Y axis indicates cell death, X axis indicates treatment group.

A: Combination of BEZ235 (a dual PI3K and mTOR inhibitor) and chloroquine. Control (1 ), BEZ235 1 μΜ (2), chloroquine 25 μΜ (3), BEZ235 1 μΜ and chloroquine 25 μΜ (4).

B: Combination of BlO-acetoxime and chloroquine. Control (1 ), BlO-acetoxime 6 μΜ (2), chloroquine 25 μΜ (3), BlO-acetoxime 6 μΜ and chloroquine 25 μΜ (4).

Cell death was quantified as in Figure 1.

Detailed Description of the Invention

"GSK-3 inhibitors" as understood in the present invention comprise compounds which bind to GSK-3 and decrease its kinase activity. The binding of an inhibitor hinders a substrate from entering the enzyme's active site and/or prevent the enzyme from catalysing its reaction. Inhibitor binding is either reversible or irreversible. Irreversible inhibitors usually react with the enzyme and change it chemically. These inhibitors modify key amino acid residues needed for enzymatic activity. In contrast, reversible inhibitors bind non-covalently and different types of inhibition are produced depending on whether these inhibitors bind the enzyme, the enzyme-substrate complex, or both.

A large number of drugs are enzyme inhibitors, so their discovery and improvement is an active area of research in biochemistry and pharmacology. An enzyme inhibitor used as a pharmaceutical is often judged by its specificity (its lack of binding to other proteins) and its potency (its dissociation constant, which indicates the concentration needed to inhibit the enzyme). A high specificity and potency are desirable in order to minimize side effects and toxicity. Inhibition as understood in the context of the present invention is based on a specific interaction between the inhibitor and GSK-3 and not just based on non-specific mechanisms (e.g. denaturation of the enzyme).

GSK-3 inhibitors according to the invention comprise lithium, e.g. as lithium chloride, zinc, e.g. as a Zn²⁺ salt, and beryllium, e.g. as Be²⁺ salt.

Examples of inhibitors of GSK-3 include aloisines (such as aloisine A and aloisine B), hymenialdisine (such as dibromohymenialdisine), indirubins (such as 5,5'-dibromo- indirubin), maleimides, in particular macrocyclic bisindolylmaleimides (such as Ro 31 - 8220, SB-216763, SB-415286, or 3F8), and muscarinic agonists (such as AFI02B and AFI50).

In another embodiment, the GSK-3 inhibitor comprises flavopiridol, kenpaullone, alsterpaullone, azakenpaullone, pyrazolopyridine, CHIR98014, CHIR99021 , CHIR-637, CT20026, SU9516, ARA014418, and staurosporine.

Preferred examples of the inhibitor are selected from the group consisting of Li⁺, (2'Z,3'E)- 6-bromoindirubin-3'-oxime (BIO), (2Z,3'EJ-6-bromoindirubin-3'-acetoxime (BlO-acet- oxime), SB-216763 (3-(2,4-dichlorophenyl)-4-(1 -methyl-1 H-indol-3-yl)-1 H-pyrrole-2,5- dione), SB-415286 (3-[(3-chloro-4-hydroxyphenyl)amino]-4-(2-nitrophenyl)-1 H-pyrrol-2,5- dione), enzastaurin (3-(1 -methylindol-3-yl)-4-[1 -[1 -(pyridin-2-ylmethyl)piperidin-4-yl]indol- 3-yl]pyrrole-2,5-dione), L803-mts (Myr-N-Gly-Lys-Glu-Ala-Pro-Pro-Ala-Pro-Pro-Gln- pSer(P0₃H)-Pro-NH₂), NP031 1 12 (4-benzyl-2-naphtalen-1 -yl-1 ,2,4-thiadiazolidine-3,5- dione), paliperidone palmitate (3-(2-(4-(6-fluoro-1 ,2-benzisoxazol-3-yl)-1 -piperidinyl)ethyl)- 6,7,8,9-tetrahydro-2-methyl-4-oxo-4H-pyrido(1 ,2-a)pyrimidin-9-yl ester), valproic acid (2- propylpentanoic acid), TDZD-8 (4-benzyl-2-methyl-1 ,2,4-thiadiazolidine-3,5-dione), and 9- hydroxyrisperidone (6,7,8,9-tetrahydro-3-(2-(4-(6-fluoro-1 ,2-benzisoxazol-3-yl)-1 - piperidinyl)ethyl)-9-hydroxy-2-methyl-4H-pyrido[2,1 -a]pyrimidin-4-one).

Even more preferred examples of inhibitors of GSK-3 include lithium such as lithium chloride (LiCI), TWS 1 19 (3-[[6-(3-aminophenyl)-7H-pyrrolo[2,3-d]pyrimidin-4-yl]oxyphenol di-trifluoroacetate) and 3F8 (5-ethyl-7,8-dimethoxy-1 H-pyrrolo[3,4-c]-isoquinoline-1 ,3- (2H)-dione).

Among GSK-3 inhibitors, derivatives of the bis-indole indirubin (collectively referred to as indirubins) appear as a class of original and promising tools and agents. Their moderate selectivity might be inconvenient when used as a research reagent, but their combined effects on several disease-relevant targets (in particular CDKs and GSK-3) may constitute an advantage for potential therapeutic applications. Among many indirubins, 6-bromo- indirubin-3'-oxime (BIO) has been widely used to investigate the physiological role of GSK-3 in various cellular settings and to alter the fate of embryonic stem cells.

In one embodiment, the GSK-3 inhibitor is a compound of formula (I):

wherein X¹ and X², independently of each other, are O, S, N-OR³, N(Z¹), or two groups independently selected from H, F, CI, Br, I, N0₂, phenyl, and (CrC₆)alkyl, and wherein R³ is hydrogen, (C C₆)alkyl, or (C C₆)alkyl-C(0)-;

each Y, independently of each other, is H, (C C₆)alkyl, (C C₆)alkyl-C(0)-, (C C₆)alkyl- C(0)0-, phenyl, N(Z¹)(Z²), sulfonyl, phosphonyl, F, CI, Br, or I;

Z¹ and Z², independently of each other, are H, (d-C₆)alkyl, phenyl, benzyl, or Z¹ and Z² together with the nitrogen to which they are attached represent a 5, 6, or 7-membered heterocyclyl;

n and m, independently of each other, are 0, 1 , 2, 3, or 4;

R¹ and R², independently of each other, are H, (CrC₆)alkyl, (CrC₆)alkyl-C(0)-, phenyl, benzyl, or benzoyl;

and wherein alkyi is branched or straight-chain alkyi, optionally substituted with 1 , 2, 3, 4, or 5 OH, N(Z¹)(Z²), (CrC₆)alkyl, phenyl, benzyl, F, CI, Br, or I; and wherein any phenyl, benzyl, or benzoyl is optionally substituted with 1 , 2, 3, 4, or 5 OH, N(Z¹)(Z²), (C C₆)alkyl, F, CI, Br, or I;

or a salt thereof. In one embodiment, X¹ is O and X² is N-OH, or X¹ is N-OH and X² is O. In another embodiment, one Y is Br. In another embodiment, one Y is Br at the 6'-position. In another embodiment, n is 0 and m is 1 , or n is 1 and m is 0. In another embodiment R¹ and R² are

H. one embodiment, the GSK-3 inhibitor comprises 6-bromoindirubin-3'-oxime of formula

or a salt thereof. In one embodiment, the GSK-3 inhibitor comprises 6-bromoindirubin-3'-acetoxime of formula (III)

The term "(Ci-C₆)alkyl" as used herein refers to a saturated linear or branched-chain monovalent hydrocarbon radical of one to six carbon atoms (Ci-C₆). Preferably, (C C₆)alkyl has one to four carbon atoms (C1-C4). Examples of alkyl groups include, but are not limited to, methyl, ethyl, n-propyl, iso-propyl, n-butyl, iso-butyl, sec-butyl, tert-butyl, n- pentyl, iso-pentyl, and n-hexyl. Such (d-C₆)alkyl may be substituted as indicated.

In the present context, the term "heterocyclyl" refers to a saturated or a partially unsaturated (i.e., having one or more double bonds within the ring) carbocyclic radical of 3 to 20 ring atoms, preferably 5, 6 or 7 ring atoms, in which at least one ring atom is a heteroatom selected from nitrogen, and in which further ring atoms may be a heteroatom selected from nitrogen, oxygen, and sulphur, the remaining ring atoms being carbon atoms, where one or more ring atoms are optionally substituted independently with one or more substituents described below. A heterocycle may be a monocycle having 3 to 7 ring members (2 to 6 carbon atoms, 1 nitrogen atom and 0 to 3 heteroatoms selected from N, O, and S) or a bicycle having 7 to 10 ring members (4 to 9 carbon atoms, 1 nitrogen atom and 0 to 6 heteroatoms selected from N, O, and S), for example, a bicyclo [4,5], [5,5], [5,6], or [6,6] system. "Heterocyclyl" also includes radicals wherein heterocyclic radicals are fused with a saturated or partially unsaturated ring, or aromatic carbocyclic or heterocyclic ring. Examples of heterocyclic rings include, but are not limited to, pyrrolidino, piperidino, morpholino, thiomorpholino, piperazinyl, homopiperazinyl, azetidinyl, homopiperidinyl, oxazepinyl, diazepinyl, thiazepinyl, indolinyl, pyrazolidinyl, pyrazolidinyl- imidazolinyl, and imidazolidinyl. The heterocyclic groups herein are optionally substituted independently with one or more substituents selected from (CrC₆)alkyl, OH, N(Z¹)(Z²), phenyl, benzyl, F, CI, Br, I, or oxo. Examples of substituted heterocyclic group are 2- oxopyrrolidinyl, 2-oxopiperidinyl, 4-methyl-piperazin-1 -yl, and 4-benzyl-piperazin-1 -yl. Most preferred are the compounds used in the Examples, in particular 6-bromoindirubin- 3'-oxime, 6-bromoindirubin-3'-acetoxime (BlO-acetoxime), TDZD-8, and L803-mts, and also lithium carbonate, valproic acid, lithium, divalproex, tideglusib (NP031 1 12), and enzastaurin hydrochloride (LY317615).

A "lysosomotropic agent" is defined herein as a compound, which diffuses into cellular lysosomes, may cause a decrease in the lysosome transmembrane proton gradient, and may increase the pH inside the organelle. Lysosomotropic agents are known to increase the internal pH of acidic vesicles and include the compounds ammonium chloride, monensin, nigericin, methylamine and ethylamine.

For the purpose of this invention, ammonium chloride, methylamine and ethylamine are less suitable as components of the inventive combinations with a GSK-3 inhibitor, and are not part of the preferred combinations.

The lysosomotropic agent is selected from chlorpromazine, LysoTracker® green (7-(3- (2-(dimethylamino)ethylamino)-3-oxopropyl)-5,5-difluoro-1 ,3-dimethyl-2,5-dihydro-1 H- dipyrrolo[1 ,2-c:1 ',2'-f][1 ,3,2]diazaborinin-4-ium-5-uide), LysoTracker® red (7-(3-(2- (dimethylamino)ethylamino)-3-oxopropyl)-5,5-difluoro-3-(1 H-pyrrol-2-yl)-2,5-dihydro-1 H- dipyrrolo[1 ,2-c:1 ',2'-f][1 ,3,2]diazaborinin-4-ium-5-uide), amantadine, 4-aminoquinoline, amiodarone, amodiaquine, azithromycin, chloroquine, clindamycin, N-(3-[(2,4-dinitro- phenyl)-amino]-propyl)-N-(3-aminopropyl)-N-methylamine dihydrochloride (DAMP), Ν,Ν'- bis(2,3-butadienyl)-1 ,4-butanediamine dihydrochloride, imipramine, 3-methyladenine, methylamine, monensin, monodansylcadaverine, NH₄CI, perhexilene, phenylalanine methyl ester, primaquine, quinacrine, suramin, thioridazine, tilorone, tributylamine, and ketotifen fumarate.

In one embodiment the lysosomotropic agent is selected from chlorpromazine,

LysoTracker® green (7-(3-(2-(dimethylamino)ethylamino)-3-oxopropyl)-5,5-difluoro-1 ,3- dimethyl-2,5-dihydro-1 H-dipyrrolo[1 ,2-c:1 ',2'-f][1 ,3,2]diazaborinin-4-ium-5-uide),

LysoTracker® red (7-(3-(2-(dimethylamino)ethylamino)-3-oxopropyl)-5,5-difluoro-3-(1 H- pyrrol-2-yl)-2,5-dihydro-1 H-dipyrrolo[1 ,2-c:1 ',2'-f][1 ,3,2]diazaborinin-4-ium-5-uide), amantadine, 4-aminoquinoline, amiodarone, amodiaquine, azithromycin, chloroquine, clindamycin, N-(3-[(2,4-dinitrophenyl)-amino]-propyl)-N-(3-aminopropyl)-N-methylamine dihydrochloride (DAMP), N,N'-bis(2,3-butadienyl)-1 ,4-butanediamine dihydrochloride, imipramine, 3-methyladenine, monensin, monodansylcadaverine, perhexilene, phenylalanine methyl ester, primaquine, quinacrine, suramin, thioridazine, tilorone, tributylamine, and ketotifen fumarate.

In certain embodiments the lysosomotropic agent is selected from:

Acridine orange (N,N,N',N'-tetramethylacridine-3,6-diamine; CAS Registry No. 494-38-2), N,N-dimethyladamantan-1 -amine (CAS Registry No. 768-94-5), amantadine (adamantan- 1 -amine; CAS Registry No. 665-66-7), 4-aminoquinoline (CAS Registry No. 578-68-7), amiodarone (2-butylbenzofuran-3-yl)-[4-(2-diethylaminoethoxy)-3,5-diiodophenyl]- methanone; CAS Registry No. 1951 -25-3), amodiaquine (4-[(7-chloroquinolin-4-yl)amino]- 2-(diethylaminomethyl)phenol; CAS Registry No. 86-42-0), azithromycin {(2R,3R,4R,5R, 8R, 10R, 11R, 13S, 14R)A 1 -[^2S,3 ?,4S,6 ? -4-dimethylamino-3-hydroxy-6-methyloxan-2- yl]oxy-2-ethyl-3,4,10-trihydroxy-13-[^2S,4 ?,5S,6S 5-hydroxy^-methoxy^,6 limethyl- oxan-2-yl]oxy-3,5,6,8, 10,12,14-heptamethyl-1 -oxa-6-azacyclopentadecan-15-one; CAS Registry No. 1 17772-70-0), 4-benzoyl-oxi-2-azetidinove ((4-oxoazetidin-2-yl) benzoate; CAS Registry No. 28562-58-5), chloroquine (N'-(7-chloroquinolin-4-yl)-N,N-diethyl- pentane-1 ,4-diamine; CAS Registry No. 54-05-7), chlorpromazine (3-(2-chloro-10H- phenothiazin-10-yl)-N,N-dimethyl-propan-1 -amine; CAS Registry No. 50-53-3), citalopram (1 -(3-dimethylaminopropyl)-1 -(4-fluorophenyl)-1 ,3-dihydroisobenzofuran-5-carbonitrile; CAS Registry No. 59729-33-8), clindamycin {(2S,4R)-N-[2-c \oroA -[(2R,3S,4S,5R,6R)- 3,4,5-trihydroxy-6-methylsulfanyloxan-2-yl]propyl]-1 -methyl-4-propyl-pyrrolidine-2- carboxamide; CAS Registry No. 24696-19-3), clomipramine (3-(3-chloro-10,1 1 -dihydro- 5H-dibenzo[b,f]azepin-5-yl)-N,N-dimethylpropan-1-amine; CAS Registry No. 303-49-1 ), DAMP (N-(3-[(2,4-dinitrophenyl)-amino]-propyl)-N-(3-aminopropyl)-N-methyl-amine dihydrochloride, fluoxetine (N-methyl-3-phenyl-3-[4-(trifluoromethyl)phenoxy]-propan-1 - amine; CAS 54910-89-3), flupentixol (2-[4-[3-[2-(trifluoromethyl)thioxanthen-9-ylidene]- propyl]piperazin-1 -yl]ethanol; CAS Registry No. 2709-56-0), fluphenazine (2-[4-[3-[2- (trifluoromethyl)-10H-phenothiazin-10-yl]propyl]-piperazin-1 -yl]ethanol; CAS Registry No. 69-23-8), haloperidol (4-[4-(4-chlorophenyl)-4-hydroxy -1 -piperidyl]-1 -(4-fluorophenyl)- butan-1 -one; CAS Registry No. 52-86-8), hydroxizine (2-[2-[4-[(4-chlorophenyl)-phenyl- methyl]piperazin-1 -yl]ethoxy]ethanol; CAS Registry No. 68-88-2), hydroxychloroquine (2-[{ 4-[(7-chloroquinolin-4-yl)amino]pentyl}(ethyl)amino]ethanol; CAS Registry No. 1 18- 42-3), ilmipramine (3-(5,6-dihydrobenzo[b][1]benzazepin-1 1 -yl)-N,N-dimethylpropan-1 - amine; CAS Registry No. 50-49-7), ketotifen fumarate (4-(1 -methylpiperidin-4-ylidene)- 4,9-dihydro-10H-benzo[4,5]cyclohepta[1 ,2b]thiophen-10-one; CAS Registry No. 34580- 14-8), levomepromazine (('2/?)-3-(2-methoxyphenothiazine-10-yl-)-N,N,2-trimethyl- propanamine; CAS Registry No. 60-99-1 ), LysoTracker® green (7-(3-(2-(dimethylamino)- ethylamino)-3-oxopropyl)-5,5-difluoro-1 ,3-dimethyl-2,5-dihydro-1 H-dipyrrolo[1 ,2-c:1 ',2'- f][1 ,3,2]diazaborinin-4-ium-5-uide; CAS Registry No. 220524-71 -0), LysoTracker® red: 7- (3-(2-(dimethylamino)ethylamino)-3-oxopropyl)-5,5-difluoro-3-(1 H-pyrrol-2-yl)-2,5-dihydro- 1 H-dipyrrolo[1 ,2-c:1 ',2'-f][1 ,3 ,2]diazaborinin-4-ium-5-uide; CAS-No. 231946-72-8), meclofenoxate (2-dimethylaminoethyl 2-(4-chlorophenoxy)acetate; CAS Registry No. 51 - 68-3), mefloquine (2,8-bis(trifluoromethyl)quinolin-4-yl](piperidin-2-yl)methanol; CAS Registry No. 53230-10-7), mesoridazine (10-{2-[1 -methylpiperidin-2-yl]ethyl}-2-methyl- sulfinyl-10H-phenothiazine; CAS Registry No. 5588-33-0), 3-methyladenine (CAS Registry No. 5142-23-4), methylamine (CAS Registry No. 74-89-5), monensin (4-[2-[5-ethyl-5-[5-[6- hydroxy-6-(hydroxymethyl)-3,5-dimethyl-oxan-2-yl]-3-methyl-oxolan-2-yl]oxolan-2-yl]-9- hydroxy-2,8-dimethyl-1 ,6-dioxaspiro[4.5]dec-7-yl]-3-methoxy-2-methyl-pentanoic acid; CAS Registry No. 22373-78-0), monodansyl-cadaverine (N-(5-aminopentyl)-5-(dimethyl- amino)naphthalene-1 -sulfonamide; CAS Registry No. 10121 -91 -2), N,N'-bis(2,3- butadienyl)-1 ,4-butanediamine dihydrochloride (CAS Registry No. 93565-01 -6), ammonium chloride (NH₄CI; CAS Registry No. 12125-02-9), Nile Blue A ([9-(diethyl- amino)benzo[a]phenoxazin-5-ylidene]azanium sulfate; CAS Registry No. 3625-57-8), perhexilene (2-(2,2-dicyclohexylethyl)piperidine; CAS Registry No. 6621 -47-2), perphenazine (2-[4-[3-(2-chloro-10H-phenothiazin-10-yl)propyl]piperazin-1 -yl]ethanol; CAS Registry No. 58-39-9), phenylalanine methyl ester (methyl (^S^-amino-S-phenyl- propanoate hydrochloride; CAS Registry No. 2577-90-4), piperazine (CAS Registry No. 1 10-85-0), primaquine (N-(6-methoxyquinolin-8-yl)pentane-1 ,4-diamine; CAS Registry No. 491 -92-9), prochlorperazine (2-chloro-10-[3-(4-methyl-1 -piperazinyl)propyl]-10H-pheno- thiazine; CAS Registry No. 58-38-8), promazine (N,N-dimethyl-3-(10H-phenothiazin-10- yl)propan-1 -amine; CAS Registry No. 58-40-2), propranolol (1 -(isopropylamino)-3- (naphthalen-1 -yloxy)propan-2-ol; CAS Registry No. 525-66-6), suramin (8-[[4-methyl-3-[[3- [[3-[[2-methyl-5-[(4,6,8-trisulfonaphthalen-1 -yl)-carbamoyl]-phenyl]carbamoyl]-phenyl]- carbamoylamino]benzoyl]amino]benzoyl]amino]naphthalene-1 ,3,5-trisulfonic acid; CAS Registry No. 145-63-1 ), thioridazine (10-{2-[1 -methylpiperidin-2-yl]ethyl}-2-methylsulfanyl- phenothiazine; CAS Registry No. 50-52-2), thioridazine (10-{2-[1 -methylpiperidin-2-yl]- ethyl}-2-methylsulfanyl-phenothiazine; CAS Registry No. 50-52-2), tributylamine (CAS

Registry No. 6309-30-4), trifluoperazine (10-[3-(4-methylpiperazin-1 -yl)propyl]-2-(trifluoro- methyl)-10H-phenothiazine; CAS Registry No. 1 17-89-5), and triflupromazine (N,N-di- methyl-3-[2-(trifluoromethyl)-10H-phenothiazin-10-yl]-propan-1 -amine; CAS Registry No. 146-54-3). As mentioned before, methylamine listed in the preceding paragraph is not considered suitable as a combination partner with GSK-3 inhibitors.

Most preferred are the lysosomotropic agents of the Examples, in particular chloroquine, hydroxychloroquine, 3-methyladenine, quinacrine, mefloquine, monensin and bafilomycin A1 .

Autophagy-lysosome pathway is one of the major route for protein and organelle clearance in eukaryotic cells. Mammalian lysosomes can degrade substrates like protein complexes and organelles. The bulk degradation of cytoplasmic proteins or organelles is largely mediated by macroautophagy, generally referred to as autophagy (Klionsky et al., Science 290:1717-20, 2000). Autophagy is the process by which cells cannibalize cellular elements (e.g., proteins, organelles). Autophagy is the cell's major regulated mechanism for degrading long- lived proteins and the only known pathway for degrading organelles. Downstream of TOR kinase, there are approximately seventeen gene products essential for autophagy and related pathways in yeast. Most of these genes have orthologs in higher eukaryotes (for a review, see Levine et al., Dev. Cell. 6:463-477, 2004). Given the importance of autophagy in cell survival and cell death, the modulation of autophagy and/or the TOR pathway is useful in treating diseases such as cancer, proliferative diseases, protein misfolding disorders, infectious diseases, and neurodegenerative diseases.

As described in patent WO 2008/122038, the following compounds are late inhibitors of autophagosome-lysosome fusion: cefamandole, monensin, astemizole, spiramycin, ('7S,9R)-beta-hydrastine, carnitine, tomatine, K252A, atranorin, tetrandrine, amlodipine, benzyl isothiocyanate, pristimerin, homochlorcyclizine (e.g., homochlorcyclizine dihydrochloride), fluoxetine (e.g., fluoxetine hydrochloride), bafilomycin A 1 , wiskostatin, monensin, quinacrine, nocodazole, vinblastine, colchicine, puromycin, bepridil, spiramycin, migericin, 2-methylcinngel, amiprilose, carnitine, tyrphostin 9, salinomycin, PP 1 , lavendustin A, ZL3VS, astemizole, 006976, RWJ-60475-(AM)3, D609, mefenamic acid, cytochalasin D, E6 berbamine, beta-peltatin, aesculin, GF-109203D, benzyl isothiocyanate, monensin, podophyllotoxin, thimerosal, maprotiline hydrochloride, norethindrone, and gramacidin are also considered as inhibitors of autophagy. The kinase inhibitors sunitinib, UCN01 , PKC412, and ruboxistaurin are likewise considered as inhibitors of autophagy. However, vinblastine is excluded from the present definition of inhibitors of autophagy in combination with a GSK-3 inhibitor due to its multiple mechanism of action.

Based on the mentioned patent WO 2008/122038 and a screen of the Prestwick collection of off-patent FDA-approved drugs, three compounds with a bis-indolyl maleimide core are identified as preferred inhibitors of autophagy: K252A, Go6976, and GF-109203X. The following compounds are also identified as inducer of autophagy: pimozide, trifluoperazine and loperamide. Other modulators (i.e., both promoters and inhibitors) of autophagy include LY-83583, rapamycin, hydroxychloroquine, pimozide, gramicidin, manoalide, doxorubicin (e.g., doxorubicin hydrochloride), daunorubicin (e.g., daunorubicin hydrochloride), rhodo- myrtoxin B, isogedunin, solanine alpha (solanidine), ellipticine, amiprilose, gentian violet, wiskostatin, manumycin A, tetrandrine, trimethobenzamide, tamoxifen, (e.g., tamoxifen citrate), RWJ-60475-(AM)3, amphotericin B, hexetidine, maprotiline (e.g., maprotiline hydrochloride), D609, G06976, nigericin, methyl benzethonium chloride, nocodazole, GF- 109203X, FK-506, PP1 , strophanthidinic acid lactone, mitoxantrone (e.g., mitoxantrone dihydrochloride), tyrothricin, puromycin, chukrasin, tyrphostin 9, norethindrone, colchicine, vinblastine, metixene (e.g., metixene hydrochloride), clemastine (e.g., clemastine fumarate), thioridazine (e.g., thioridazine hydrochloride), creatinine, phorbol 12-myristate 13-acetate, ZL3VS, and triflupromazine (e.g., triflupromazine hydrochloride).

From these, tamoxifen and vinblastine are excluded from the present definition of inhibitors of autophagy in combination with a GSK-3. Preferred modulators therefore include LY-83583, rapamycin, hydroxychloroquine, pimozide, gramicidin, manoalide, doxorubicin (e.g., doxorubicin hydrochloride), daunorubicin (e.g., daunorubicin

hydrochloride), rhodomyrtoxin B, isogedunin, solanine alpha (solanidine), ellipticine, amiprilose, gentian violet, wiskostatin, manumycin A, tetrandrine, trimethobenzamide, RWJ-60475-(AM)3, amphotericin B, hexetidine, maprotiline (e.g., maprotiline

hydrochloride), D609, G06976, nigericin, methyl benzethonium chloride, nocodazole, GF- 109203X, FK-506, PP1 , strophanthidinic acid lactone, mitoxantrone (e.g., mitoxantrone dihydrochloride), tyrothricin, puromycin, chukrasin, tyrphostin 9, norethindrone, colchicine, metixene (e.g., metixene hydrochloride), clemastine (e.g., clemastine fumarate), thioridazine (e.g., thioridazine hydrochloride), creatinine, phorbol 12-myristate 13-acetate, ZL3VS, and triflupromazine (e.g., triflupromazine hydrochloride). Most preferred are the inhibitors of autophagy of the Examples, in particular

hydroxychloroquine and bafilomycin A1 . Pharmaceutical compositions according to the invention are, for example, compositions for enteral administration, such as nasal, buccal, rectal or, especially, oral administration, and for parenteral administration, such as intravenous, intramuscular or subcutaneous administration. The compositions comprise a lysosomotropic agent or agents modulating autophagy and a GKS-3 inhibitor, alone or, preferably, together with a pharmaceutically acceptable carrier. The dosage of the combination of a lysosomotropic agent or agents modulating autophagy and the GSK-3 inhibitor depends upon the disease to be treated and upon the species, its age, weight, and individual condition, the individual

pharmacokinetic data, and the mode of administration. The pharmaceutical compositions comprise from approximately 1 % to approximately 90% of the combination of a lysosomotropic agent or agents modulating autophagy and a GSK- 3 inhibitor.

The pharmaceutical compositions of the present invention are prepared in a manner known per se, for example by means of conventional mixing, granulating, coating, dissolving or lyophilizing processes.

Preference is given to the use of solutions of the combination of a lysosomotropic agent or agents modulating autophagy and a GSK-3 inhibitor, and also suspensions or dispersions, especially isotonic aqueous solutions, dispersions or suspensions which, for example in the case of lyophilized compositions comprising the combination of a lysosomotropic agent or agents modulating autophagy and a GSK-3 inhibitor, alone or together with a carrier, can be made up before use. The pharmaceutical compositions may be sterilized and/or may comprise excipients, for example preservatives, stabilizers, wetting agents and/or emulsifiers, solubilizers, salts for regulating osmotic pressure and/or buffers and are prepared in a manner known per se, for example by means of conventional dissolving and lyophilizing processes. The said solutions or suspensions may comprise viscosity- increasing agents, typically sodium carboxymethylcellulose, carboxymethylcellulose, dextran, polyvinylpyrrolidone, or gelatins, or also solubilizers, e.g. Tween 80^® (polyoxy- ethylene(20)sorbitan mono-oleate). Suspensions in oil comprise as the oil component the vegetable, synthetic, or semisynthetic oils customary for injection purposes. In respect of such, special mention may be made of liquid fatty acid esters that contain as the acid component a long-chained fatty acid having from 8 to 22, especially from 12 to 22, carbon atoms. The alcohol component of these fatty acid esters has a maximum of 6 carbon atoms and is a monovalent or polyvalent, for example a mono-, di- or trivalent, alcohol, especially glycol and glycerol. As mixtures of fatty acid esters, vegetable oils such as cottonseed oil, almond oil, olive oil, castor oil, sesame oil, soybean oil and groundnut oil are especially useful. The manufacture of injectable preparations is usually carried out under sterile conditions, as is the filling, for example, into ampoules or vials, and the sealing of the containers.

Suitable carriers for preferred solid oral dosage forms are especially fillers, such as sugars, for example lactose, saccharose, mannitol or sorbitol, cellulose preparations, and/or calcium phosphates, for example tricalcium phosphate or calcium hydrogen phosphate, and also binders, such as starches, for example corn, wheat, rice or potato starch, methylcellulose, hydroxypropyl methylcellulose, sodium carboxymethylcellulose, and/or polyvinylpyrrolidone, and/or, if desired, disintegrators, such as the above- mentioned starches, also carboxymethyl starch, crosslinked polyvinylpyrrolidone, alginic acid or a salt thereof, such as sodium alginate. Additional excipients are especially flow conditioners and lubricants, for example silicic acid, talc, stearic acid or salts thereof, such as magnesium or calcium stearate, and/or polyethylene glycol, or derivatives thereof.

Tablet cores can be provided with suitable, optionally enteric, coatings through the use of, inter alia, concentrated sugar solutions which may comprise gum arabic, talc, polyvinylpyrrolidone, polyethylene glycol and/or titanium dioxide, or coating solutions in suitable organic solvents or solvent mixtures, or, for the preparation of enteric coatings, solutions of suitable cellulose preparations, such as acetylcellulose phthalate or hydroxypropyl- methylcellulose phthalate. Dyes or pigments may be added to the tablets or tablet coatings, for example for identification purposes or to indicate different doses of the combination of a lysosomotropic agent or agents modulating autophagy and GSK-3 inhibitor.

Pharmaceutical compositions for oral administration also include hard capsules consisting of gelatin, and also soft, sealed capsules consisting of gelatin and a plasticizer, such as glycerol or sorbitol. The hard capsules may contain the combination of a lysosomotropic agent or agents modulating autophagy and GSK-3 inhibitor in the form of granules, for example in admixture with fillers, such as corn starch, binders, and/or glidants, such as talc or magnesium stearate, and optionally stabilizers. In soft capsules, the combination of a lysosomotropic agent or agents modulating autophagy and GSK-3 inhibitor is preferably dissolved or suspended in suitable liquid excipients, such as fatty oils, paraffin oil or liquid polyethylene glycols or fatty acid esters of ethylene or propylene glycol, to which stabilizers and detergents, for example of the polyoxyethylene sorbitan fatty acid ester type, may also be added. Pharmaceutical compositions suitable for rectal administration are, for example, suppositories that consist of a combination of a lysosomotropic agent or agents modulating autophagy and a GSK-3 inhibitor, and a suppository base. Suitable

suppository bases are, for example, natural or synthetic triglycerides, paraffin

hydrocarbons, polyethylene glycols or higher alkanols.

For parenteral administration, aqueous solutions of a combination of a lysosomotropic agent or agents modulating autophagy and a GSK-3 inhibitor, or aqueous injection suspensions that contain viscosity-increasing substances, for example sodium

carboxymethylcellulose, sorbitol and/or dextran, and, if desired, stabilizers, are especially suitable. The combination of a lysosomotropic agent or agents modulating autophagy and GSK-3 inhibitor, optionally together with excipients, can also be in the form of a lyophilizate and can be made into a solution before parenteral administration by the addition of suitable solvents. Solutions such as are used, for example, for parenteral administration can also be employed as infusion solutions.

Preferred preservatives are, for example, antioxidants, such as ascorbic acid, or microbicides, such as sorbic acid or benzoic acid.

On the basis of the studies described in more detail below, a pharmaceutical compositions comprising a GSK-3 inhibitor and a lysosomotropic agent or agents modulating autophagy according to the invention show therapeutic efficacy against different types of cancer including carcinomas, sarcomas, gliomas, leukemias, lymphomas, e.g. epithelial neoplasms, squamous cell neoplasms, basal cell neoplasms, transitional cell papillomas and carcinomas, adenomas and adenocarcinomas, adnexal and skin appendage neoplasms, mucoepidermoid neoplasms, cystic neoplasms, mucinous and serous neoplasms, ductal-, lobular and medullary neoplasms, acinar cell neoplasms, complex epithelial neoplasms, specialized gonadal neoplasms, paragangliomas and glomus tumors, naevi and melanomas, soft tissue tumors including sarcomas, fibromatous neoplasms, myxomatous neoplasms, lipomatous neoplasms, myomatous neoplasms, complex mixed and stromal neoplasms, fibroepithelial neoplasms, synovial like neoplasms, mesothelial neoplasms, germ cell neoplasms, trophoblastic neoplasms, mesonephromas, blood vessel tumors, lymphatic vessel tumors, osseous and

chondromatous neoplasms, giant cell tumors, miscellaneous bone tumors, gliomas, glioblastomas, oligodendrogliomas, neuroepitheliomatous neoplasms, meningiomas, nerve sheath tumors, granular cell tumors and alveolar soft part sarcomas, Hodgkin^'s and non-Hodgkin^"s lymphomas, other lymphoreticular neoplasms, plasma cell tumors, mast cell tumors, immunoproliferative diseases, leukemias including acute and chronic leukemias, miscellaneous myeloproliferative disorders, lymphoproliferative disorders and myelodysplastic syndromes. On the basis of the studies described, the combination of a GSK-3 inhibitor and a lysosomotropic agent or agents modulating autophagy show therapeutic efficacy against proliferative inflammatory diseases, degenerative diseases and infectious diseases including malaria, hepatitis A to C, HIV, African trypanosomiasis, cryptosporidiosis, Dengue fever, leishmaniasis, tuberculosis and schistosomiasis.

The combination of a GSK-3 inhibitor and a lysosomotropic agent or agents modulating autophagy, and pharmaceutical compositions comprising a lysosomotropic agent or agents modulating autophagy and a GSK-3 inhibitor according to the invention may be applied in the form of fixed combinations. Such fixed combination may contain a lysosomotropic agent or agents modulating autophagy and a GSK-3 inhibitor in a relative molecular amount (Mol per Mol) of between 50 to 1 and 1 to 50, preferably between 5 to 1 and 1 to 5, such as a combination of 1 to 1.

Alternatively, the combination of a lysosomotropic agent or agents modulating autophagy and a GSK-3 inhibitor may be applied in two different, separate pharmaceutical compositions, optionally being provided together in a kit. The administration of a lysosomotropic agent or agents modulating autophagy and a GSK-3 inhibitor may also be staggered, or the compounds may be given independently of one another within a reasonable time window. The combination of a lysosomotropic agent or agents modulating autophagy and GSK-3 inhibitors and pharmaceutical compositions comprising a lysosomotropic agent or agents modulating autophagy and GSK-3 inhibitors may be administered especially for cancer therapy in combination with radiotherapy, immunotherapy, surgical intervention, or a combination of these. Long-term therapy is equally possible as is adjuvant therapy in the context of other treatment strategies or neo-adjuvant therapy in combination with surgery. Other possible treatments are therapy to maintain the patient's status after tumor regression, or chemopreventive therapy, for example in patients at risk. The present invention relates furthermore to a method for the treatment of cancer which comprises administering a combination of a lysosomotropic agent or agents modulating autophagy and a GSK-3 inhibitor in a quantity effective against said disease, to a warmblooded animal requiring such treatment. The combination of a lysosomotropic agent or agents modulating autophagy and GSK-3 inhibitors can be administered as such or especially in the form of pharmaceutical compositions, prophylactically or therapeutically, preferably in an amount effective against the said diseases, to a warm-blooded animal, for example a human, requiring such treatment. In the case of an individual having a bodyweight of about 70 kg the daily dose administered is from approximately 0.1 g to approximately 4 g, preferably from approximately 0.2 g to approximately 2 g, of a combination of the present invention.

The invention relates to the use of a combination of a lysosomotropic agent or agents modulating autophagy and a GSK-3 inhibitor and of pharmaceutical compositions comprising a lysosomotropic agent or agents modulating autophagy and a GSK-3 inhibitor for the treatment of cancer, in particular for the treatment of the particular cancers mentioned above. More specifically, the invention relates to the use of a combination of a lysosomotropic agent or agents modulating autophagy and a GSK-3 inhibitor and of pharmaceutical compositions comprising a lysosomotropic agent or agents modulating autophagy and a GSK-3 inhibitor for the treatment of carcinomas, sarcomas, leukemias, myelomas, lymphomas, and cancers of the nervous system. Furthermore, the invention relates to the use of a combination of a lysosomotropic agent or agents modulating autophagy and a GSK-3 inhibitor and of pharmaceutical compositions comprising a lysosomotropic agent or agents modulating autophagy and a GSK-3 inhibitor for achieving treatment of proliferative inflammatory diseases, degenerative diseases and infectious diseases including malaria, hepatitis A to C, HIV, African trypanosomiasis,

cryptosporidiosis, Dengue fever, leishmaniasis, tuberculosis and schistosomiasis. The preferred relative amount of a lysosomotropic agent or agents modulating autophagy and GSK-3 inhibitor, dose quantity and kind of pharmaceutical composition, which are to be used in each case, depend on the type of cancer or other disease, the severity and progress of the disease, and the particular condition of the patient to be treated, and has to be determined accordingly by the medical doctor responsible for the treatment.

The invention further relates to the use of a combination of a lysosomotropic agent or agents modulating autophagy and a GSK-3 inhibitor for the preparation of a

pharmaceutical composition for the treatment of cancer, proliferative inflammatory diseases, degenerative diseases and infectious diseases including malaria, hepatitis A to C, HIV, African trypanosomiasis, cryptosporidiosis, Dengue fever, leishmaniasis, tuberculosis and schistosomiasis, as explained above. Especially, the invention provides a method for treatment of cancer, proliferative inflammatory diseases, degenerative diseases and infectious diseases including malaria, hepatitis A to C, HIV, African trypanosomiasis, cryptosporidiosis, Dengue fever, leishmaniasis, tuberculosis and schistosomiasis, which comprises administering a combination of a lysosomotropic agent or agents modulating autophagy and a GSK-3 inhibitor or of a pharmaceutical composition comprising a lysosomotropic agent or agents modulating autophagy and a GSK-3 inhibitor, in a quantity effective against said disease, to a warm-blooded animal requiring such treatment.

Examples

Cell lines tested

Sensitivity to the

Human Cancer Cell Malignancy combination

treatment

U373 Brain cancer... YES

LN18 Brain cancer... YES

LN229 Brain cancer YES

U87 Brain cancer... YES

BS287 ( ex vivo) Brain cancer YES

B16 Melanoma... YES

A2058 Melanoma YES Cell culture and reagents

Glioblastoma and melanoma cell lines were grown at 37°C in a 5% C0₂ atmosphere in DMEM (U87, LN18, U373, LN229, A2058, B16) supplemented with 10% heat-inactivated FCS, 1 x alanyl-glutamine, and 1 x penicillin-streptomycin (all from Sigma) and 1 x nonessential amino acids (from AMI MED). BS287 ex vivo glioblastoma cells were cultured in Neurobasal medium (Invitrogen) supplemented with basic fibroblast growth factor (20 ng/ml, Invitrogen), epidermal growth factor (20 ng/ml, R&D Systems), B27 (1x) and N2 supplement (0.5x) (Invitrogen). BlO-acetoxime (from TOCRIS) is dissolved in DMSO at 24 mM, BEZ235 (from Novartis) is dissolved in DMSO at 10 mM and stored at -20°C, further dilutions were performed in cell culture media freshly before each experiment. Drug concentrations used are indicated in the figure legends. Other compounds used were bafilomycin A1 , chloroquine and 3-methyladenine (Sigma), L803mts (Genemed USA), TDZD-8 (EMD Millipore), and BEZ235 (from Novartis).

Determination of cell death

Cells at the logarithmic phase of growth were trypsinized and plated at a concentration of 15x10³ cells/cm². Drugs were added 24 hours later. After 3 days of exposure to the compounds cells were trypsinized and prepared for cell cycle and apoptosis analysis. For cell death evaluation, cells were fixed and permeabilized in 70% cold ethanol for 60 minutes at 4°C, and subsequently washed with PBS. The pellet was resuspended in PBS containing 50 μg mL propidium iodide and 10 μg mL DNase-free RNase and incubated for 8 h at 4°C before fluorescence-activated cell sorting data acquisition (FACSCalibur, Becton Dickinson) and % of cells in sub-G1 cell determined.

Combinatorial index calculation

Apoptosis resulting from the combination of two drugs was analyzed for each cell line using CalcuSyn Software (Biosoft) to generate a combination index (CI). A CI greater than one indicates antagonism. A CI of one indicates an additive effect. A CI less than one indicates synergism. Specifically, a value between 0.85 and 0.90 indicate slight synergism, between 0.70 and 0.85 indicates a moderate synergism, between 0.30 and 0.70 indicates synergism, between 0.10 and 0.30 indicates strong synergism, and <0.1 indicates a very strong synergism.

Claims

1 . A pharmaceutical composition comprising a lysosomotropic agent or agent modulating autophagy and a glycogen synthase kinase-3 (GSK-3) inhibitor.

2. The composition of claim 1 for use in the treatment of cancer, proliferative

inflammatory diseases, degenerative diseases and infectious diseases selected from the group consisting of malaria, hepatitis A to C, HIV, African trypanosomiasis,

cryptosporidiosis, Dengue fever, leishmaniasis, tuberculosis and schistosomiasis.

3. The composition of claim 1 for use in the treatment of cancer.

4. The composition of claim 1 wherein the GSK-3 inhibitor is selected from the group consisting of lithium chloride, a zinc Zn²⁺ salt, a beryllium Be²⁺ salt, aloisine,

hymenialdisine, indirubin, macrocyclic bisindolylmaleimide, a muscarinic agonist, flavopiridol, kenpaullone, alsterpaullone, azakenpaullone, pyrazolopyridine, CHIR98014, CHIR99021 , CHIR-637, CT20026, SU9516, ARA014418, and staurosporine.

5. The composition of claim 1 wherein the GSK-3 inhibitor is selected from the group consisting of Li⁺, (2Z,3E)-6-bromoindirubin-3'-oxime, (2'Z,3'£H3-bromoindirubin-3'- acetoxime, SB-216763, SB-415286, enzastaurin, L803-mts, NP031 1 12, paliperidone palmitate, valproic acid, TDZD-8, 9-hydroxyrisperidone, (3-[[6-(3-aminophenyl)-7H- pyrrolo[2,3-d]pyrimidin-4-yl]oxyphenol ditrifluoroacetate), and 5-ethyl-7,8-dimethoxy-1 H- pyrrolo[3,4-c]-isoquinoline-1 ,3-(2H)-dione.

6. The composition of claim 4 wherein the GSK-3 inhibitor is indirubin. the GSK-3 inhibitor is a compound of formula (I):

wherein X¹ and X², independently of each other, are O, S, N-OR³, N(Z¹), or two groups independently selected from H, F, CI, Br, I, N0₂, phenyl, and (CrC₆)alkyl, and wherein R³ is hydrogen, (C C₆)alkyl, or (C C₆)alkyl-C(0)-; each Y, independently of each other, is H, (C C₆)alkyl, (C C₆)alkyl-C(0)-, (C C₆)alkyl- C(0)0-, phenyl, N(Z¹)(Z²), sulfonyl, phosphonyl, F, CI, Br, or I;

Z¹ and Z², independently of each other, are H, (CrC₆)alkyl, phenyl, benzyl, or Z¹ and Z² together with the nitrogen to which they are attached represent a 5, 6, or 7-membered heterocyclyl;

n and m, independently of each other, are 0, 1 , 2, 3, or 4;

and wherein alkyl is branched or straight-chain, optionally substituted with 1 , 2, 3, 4, or 5 OH, N(Z¹)(Z²), (CrC₆)alkyl, phenyl, benzyl, F, CI, Br, or I; and wherein any phenyl, benzyl, or benzoyl is optionally substituted with 1 , 2, 3, 4, or 5 OH, N(Z¹)(Z²), (C C₆)alkyl, F, CI, Br, or I;

or a salt thereof. 8. The composition of claim 1 wherein the GSK-3 inhibitor is 6-bromoindirubin-3'-oxime of formula (II)

or a salt thereof.

9. The composition of claim 1 wherein the GSK-3 inhibitor is 6-bromoindirubin-3'- acetoxime of formula (III)

or a salt thereof. 10. The composition according to any of claims 1 to 9 wherein the lysosomotropic agent or agent modulating autophagy is selected from the group consisting of chlorpromazine, LysoTracker® green (7-(3-(2(dimethylamino)ethylamin)-3-oxopropyl)-5,5-difluoro-1 ,3- dimethyl-2,5-dihydro-1 H-dipyrrolo[1 ,2-c:1 ',2'-f][1 ,3,2]diazaborinin-4-ium-5-uide), LysoTracker® red (7-(3-(2-(dimethylamino)ethylamino)-3-oxopropyl)-5,5-difluoro-3-(1 H- pyrrol-2-yl)-2,5-dihydro-1 H-dipyrrolo[1 _!2-c:1 \2'-^[1 _!3,2]diazaborinin-4-ium-5-uide)_! amantadine, 4-aminoquinoline, amiodarone, amodiaquine, azithromycin, chloroquine, clindamycin, N-(3-[(2,4-dinitrophenyl)-amino]-propyl)-N-(3-aminopropyl)-N-methylamine dihydrochloride (DAMP), N,N'-bis(2,3-butadienyl)-1 ,4-butanediamine dihydrochloride, imipramine, 3-methyladenine, monensin, monodansylcadaverine, perhexilene, phenylalanine methyl ester, primaquine, quinacrine, suramin, thioridazine, tilorone, tributylamine, and ketotifen fumarate. 1 1 . The composition according to any of claims 1 to 9 wherein the lysosomotropic agent or agent modulating autophagy is selected from the group consisting of acridine orange, N,N-dimethyladamantan-1 -amine, amantadine, 4-aminoquinoline, amiodarone, amodiaquine, azithromycin, 4-oxoazetidin-2-yl benzoate, chloroquine, chlorpromazine, citalopram, clindamycin, clomipramine, N-(3-[(2,4-dinitrophenyl)-amino]-propyl)-N-(3- aminopropyl)-N-methyl-amine dihydrochloride, fluoxetine, flupentixol, fluphenazine, haloperidol, hydroxizine, hydroxychloriquine sulfate, ilmipramine, ketotifen fumarate, levomepromazine, 7-(3-(2-(dimethylamino)ethylamino)-3-oxopropyl)-5,5-difluoro-1 ,3- dimethyl-2,5-dihydro-1 H-dipyrrolo[1 ,2-c:1 ',2'-f][1 ,3,2]diazaborinin-4-ium-5-uide, 7-(3-(2- (dimethylamino)ethylamino)-3-oxopropyl)-5,5-difluoro-3-(1 H-pyrrol-2-yl)-2,5-dihydro-1 H- dipyrrolo[1 ,2-c:1 ',2'-f][1 ,3 ,2]diazaborinin-4-ium-5-uide, meclofenoxate, mesoridazine, 3- methyladenine, monensin, monodansylcadaverine, N,N'-bis(2,3-butadienyl)-1 ,4- butanediamine dihydrochloride, ammonium chloride, [9-(diethylamino)benzo[a]- phenoxazin-5-ylidene]azanium sulfate, perhexilene, perphenazine, phenylalanine methyl ester, piperazine, primaquine, prochlorperazine, promazine, propranolol, suramin, thioridazine, thioridazine, tributylamine, trifluoperazine, and triflupromazine.

12. The composition according to any of claims 1 to 9 wherein the lysosomotropic agent or agent modulating autophagy is selected from the group consisting of cefamandole, monensin, astemizole, spiramycin, ('7S,9R)-beta-hydrastine, carnitine, tomatine, K252A, atranorin, tetrandrine, amlodipine, benzyl isothiocyanate, pristimerin, homochlorcyclizine, fluoxetine, bafilomycin A 1 , wiskostatin, monensin, quinacrine, nocodazole, colchicine, puromycin, bepridil, spiramycin, migericin, 2-methylcinngel, amiprilose, carnitine, tyrphostin 9, salinomycin, PP 1 , lavendustin A, ZL3VS, astemizole, 006976, RWJ-60475- (AM)3, D609, mefenamic acid, cytochalasin D, E6 berbamine, beta-peltatin, aesculin, GF- 109203D, benzyl isothiocyanate, monensin, podophyllotoxin, thimerosal, maprotiline hydrochloride, norethindrone, gramicidin, sunitinib, UCN01 , PKC412, and ruboxistaurin.

13. The composition according to any of claims 1 to 9 wherein the lysosomotropic agent or agent modulating autophagy is selected from the group consisting of K252A, Go6976, and GF-109203X

14. The composition according to any of claims 1 to 9 wherein the lysosomotropic agent or agent modulating autophagy is selected from the group consisting of LY-83583, rapamycin, hydroxychloroquine, pimozide, gramicidin, manoalide, doxorubicin, daunorubicin, rhodomyrtoxin B, isogedunin, solanine alpha, ellipticine, amiprilose, gentian violet, wiskostatin, manumycin A, tetrandrine, trimethobenzamide, RWJ-60475-(AM)3, amphotericin B, hexetidine, maprotiline, D609, G06976, nigericin, methyl benzethonium chloride, nocodazole, GF-109203X, FK-506, PP1 , strophanthidinic acid lactone, mitoxantrone, tyrothricin, puromycin, chukrasin, tyrphostin 9, norethindrone, colchicine, metixene, clemastine, thioridazine, creatinine, phorbol 12-myristate 13-acetate, ZL3VS, and triflupromazine.

15. The composition according to any of claims 1 to 9 wherein the lysosomotropic agent or agent modulating autophagy is selected from the group consisting of chloroquine, 3- methyladenine and bafilomycin A1 .

16. The composition of claim 15 wherein the lysosomotropic agent is chloroquine and the GSK-3 inhibitor is (2'Z,3'£^6-bromoindirubin-3'-acetoxime).

17. A method for treatment of cancer, a proliferative inflammatory disease, a

degenerative disease and an infectious disease, which comprises administering a composition according to any of claims 1 to 16 in a quantity effective against said disease, to a warm-blooded animal requiring such treatment.

18. A method for treatment of cancer which comprises administering a composition according to any of claims 1 to 16 in a quantity effective against said disease, to a warmblooded animal requiring such treatment.