Alc1 inhibitors alc1i-1 and alc1i-2 for use in treating pancreatic cancer by potentiating the effect of irinotecan

Abstract

The present invention relates to the use of two allosteric inhibitors of ALC1 (CHD1L), namely ALC1i-1 of Formula (I) and ALC1i-2 of Formula (II), that show a potentiating and synergistic effect in the treatment of pancreatic cancer when combined with irinotecan. Thus, the invention relates to the two ALC1i for use in treating or ameliorating pancreatic cancer, wherein the treatment comprises administration of irinotecan, or for use in enhancing efficacy of irinotecan in treating or ameliorating pancreatic cancer, respectively. Furthermore, the invention relates to irinotecan for use in treating or ameliorating pancreatic cancer, wherein the treatment comprises administration of the two ALC1i, as well as a pharmaceutical composition and a kit, respectively, comprising the two ALC1i and irinotecan.

Description

[0001] ALC1 INHIBITORS ALCII-1 AND ALCII-2 FOR USE IN TREATING PANCREATIC CANCER BY POTENTIATING THE EFFECT OF IRINOTECAN

[0002] The present invention relates to the use of two allosteric inhibitors of ALC1 (CHD1L), namely ALCli-1 of Formula (I) and ALCli-2 of Formula (II), that show a potentiating and synergistic effect in the treatment of pancreatic cancer when combined with irinotecan. Thus, the invention relates to the two ALCli for use in treating or ameliorating pancreatic cancer, wherein the treatment comprises administration of irinotecan, or for use in enhancing efficacy of irinotecan in treating or ameliorating pancreatic cancer, respectively. Furthermore, the invention relates to irinotecan for use in treating or ameliorating pancreatic cancer, wherein the treatment comprises administration of the two ALCli, as well as a pharmaceutical composition and a kit, respectively, comprising the two ALCli and irinotecan.

[0003] Background of the Invention

[0004] Generally, cancers are classified into two categories: blood cancer and solid cancer. Solid cancers occur in almost every part of the body such as pancreatic cancer, breast cancer, oral cancer, liver cancer, uterine cancer, esophageal cancer, and skin cancer among others. Although some methods of treatment include targeted drugs such as Gleevec or Herceptin, the majority of cancers are still treated by chemotherapy or radiation therapy. Since chemotherapies are not targeted therapies, the biggest problems of conventional chemotherapeutic agents are the side effects caused by cytotoxicity on one hand and the development of drug resistance on the other. The latter is the main factor that eventually causes the treatment to fail despite an initial successful response to the chemotherapeutic agents. Therefore, in order to overcome the limitations of such chemotherapeutic agents, it is necessary develop new targeted therapeutic agents that specifically interfere with cancer cell proliferation.

[0005] Among solid cancers, pancreatic cancer is the seventh leading cause of cancer death worldwide. Pancreatic cancer is an aggressive disease compared to other cancers, such as uterine cancer, breast cancer, rectal cancer, colon cancer, skin cancer, lung cancer, or liver cancer and has the additional drawback that it is difficult to diagnose. Pancreatic cancer is also one of the most serious malignancies with acute onset, late diagnosis and low survival. Due to the difficulty in treating pancreatic cancer, treatment regimens often comprise combination therapies. While combination therapies might allow delaying development of drug resistance and in some cases provide additive or even synergistic efficacy, there are still only few therapies available.

[0006] Therefore, there is a high medical need to develop novel combination therapies in which chemotherapeutic agents with established effectiveness in the treatment of pancreatic cancers are combined with novel anticancer drugs that potentiate the effect of the established treatment regimens.

[0007] The present inventors considered the rationale that ALC1 inhibitors as a known class of cancer drugs could be combined with irinotecan for the treatment of pancreatic cancer. However, combination of irinotecan with known ALC1 inhibitors did not show synergism. The present invention is based on the surprising finding that specific ALC1 inhibitors, namely those of Formula (I) and of Formula (II), possess synergistic, anti-proliferative activity when being combined with irinotecan for the treatment of pancreatic cancer.

[0008] The rationale that, at least for specific combinations, ALC1 inhibitors in combination with other cancer drugs may leads to more than the expected additive efficacy is as follows. Activation of the DNA damage pathway leads to the recruitment of many proteins, notably also specific chromatin remodeling enzymes, including the macrodomain-containing nucleosome remodeler ALC1 (CHD1L) (Ahel et al., 2009; Gottschalk et al., 2009; Lehmann et al., 2017; Singh et al., 2017). Macrodomains generally bind ADP-ribose, oligo- ADP-ribose and poly-ADP-ribose (PAR) (Karras et al., 2005), thus proteins containing macrodomains respond and recruit to PARP activation sites on the genome, including during DNA damage and with relevance for cancer. Importantly, PAR or oligo-ADP-ribose binding to the macrodomains of ALC1 robustly turns on chromatin remodeling activity (Ahel et al., 2009; Gottschalk et al., 2009; Lehmann et al., 2017; Singh et al., 2017), revealing ALC1 as an allosterically-regulated chromatin remodeling enzyme, the first of its kind, and one of the very few enzymes whose catalytic activity is directly regulated by PAR. Additionally, ALC1 is a validated oncogene and is often genetically amplified together with PARP1 in BRCAl / 2-deficient ovarian and breast cancer samples.

[0009] Without wishing to be bound by any theory, the present inventors considered that ALCli possess anti-proliferative activity in pancreatic cancers since irinotecan induces a transient phenotype similar to an acquired homologous recombination deficiency, e.g. through BRCA1 or BRCA2 deficiency. Disruption of the chromatin remodeling forces of ALC1 through ALCli enables a highly selective therapy of pancreatic cancer. Via inhibition of the ALC1 enzymatic activity, ALCli may potentiate the cancer cell killing properties of irinotecan, in particular enabling therapeutic approaches where irinotecan is already used as part of the standard of care. Irinotecan appears to induce a transient “BRCAness” or a deficiency in homologous recombination, which renders the tumors selectively responsive to ALC1 inhibitors.

[0010] Few ALCli are described in the art e.g. by Abbott et al. in 2020 (Abbott et al., 2020). This document discloses inhibitors of CHD1L and their in vitro antitumor activity. Two of the most active ALCli described, namely “Compound 1” and “Compound 3”, have the following chemical structures:

[0011] Compound 1 Compound 3

[0012] WO 2022 / 117782 Al discloses specific ALC1 inhibitors. In a BRCA negative breast cancer cell line, an at least additive effect is shown when combined with a PARP inhibitor.

[0013] The present inventors tested known ALC1 inhibitors, namely Compounds 1 and 3 as shown above, in combination with other cancer drugs, i.e. the known commercially available ATR inhibitor, namely elimusertib, and with the known and commercially available PARP inhibitor olaparib. Contrary to the above rationale, only about additive effects were achieved in vitro against cancer cell lines. However, extensive further studies then revealed that specific ALC1 inhibitors, with structures according to Formula (I) and Formula (II), and which inhibit the ATPase function and / or nucleosome remodeling function of ALC1, show synergistic effects in combination with irinotecan in vitro against pancreatic cell lines.

[0014] That is, two ALC1 inhibitors, ALCli-1 with a structure according to Formula (I) and ALCli-2 with a structure according to Formula (II), have been identified that inhibit the ATPase function and or nucleosome remodeling function of ALC1, and thus potentiate the effects of irinotecan. Without wishing to be bound by any theory, the present inventors consider that ALC1 inhibition , e.g. by ALCli-1 and ALCli-2, inhibits efficient DNA repair by rendering chromatin less accessible to DNA repair enzymes. This leads to enhanced cancer cell killing and / or reduce off-target effects of the chemotherapeutic agent even if used at lower doses and thus lessen cellular toxicity in non-cancer cells. The sensitivity of cancer cells to DNA damaging agents is generally accepted to be a cornerstone of anti-cancer therapy, therefore it could be hypothesized that ALCli-1 and ALCli-2 may mediate sensitization to irinotecan. Specifically, it is hypothesized that inhibition of ALC1 by ALCli-1 and ALCli-2 may promote DNA damage accumulation and cancer cell killing in particular when co-dosed with irinotecan, which is known to induce DNA double stand breaks as a result of the prevention of strand re-ligation in the ternary topoisomerase I:DNA complex (Hsiang et al., 1985, Liu et al, 2000). Presence of this inhibited complex in cancer cells is expected to lead to replication fork collapse and cell death. The present inventors therefore relate the potentiating effect of ALCli-1 and ALCli-2 and irinotecan in part also to the ability of ALCli-1 and ALCli-2 to induce a similar, ternary ALCLnucleosome complex in the chromatin region that contains the DNA damage. Hence, inhibition of topoisomerase 1 through irinotecan and inhibition of ALC1 through ALCli-1 and ALCli-2 seems to result in potentiation, and in additive or even synergistic killing of pancreatic cancer cells.

[0015] Irinotecan-based regimens are the standard of care for treatment of pancreatic cancer in both first and second line settings. In first line treatment, in the FOLFIRINOX combination chemotherapy, irinotecan is co-administered with the agents folinic acid, fluorouracil and oxaliplatin. Although this type of treatment leads to a significant improvement in survival compared to monotherapy with gemcitabine, duration of treatment is limited due to lack of sustained tumor response and / or severe side-effects (Conroy et al., 2011). In second-line therapy, liposomal irinotecan (Nal-IRI; Onivyde®) in combination with folinic acid and fluorouracil is the only approved treatment regimen based on a randomized phase III trial (Wang-Gillam et al, Lancet 2016). Recently, Nal-IRI, has been reported to be superior to gemcitabine / nab-paclitaxel in first line when used as a NALIRIFOX combination (News release. Ipsen. November 9, 2022. Accessed November 9, 2022. https: / / bit.ly / 3TsnOuZ).

[0016] Despite the significantly improved overall survival of FILFIRINOX combination chemotherapy compared to gemcitabine, there remain concerns about the toxicity in the often old and fragile patients with pancreatic cancer and thus of high clinical relevance (NCCN Guidelines Version 1.2022). In the adjuvant PRODIGE-24 phase III trial, the dose of irinotecan was reduced from 180 mg / m2in the original protocol to 150 mg / m2due to toxicities in accordance with a protocol-specific safety analysis (PMID: 30575490). Multiple mostly retrospective studies have provided additional data showing that modified irinotecan FOLFIRI regimens using reduced doses are non-inferior in outcome and thus, dose adaption at the physicians choice in modified protocols are widely used as described in guidelines (NCCN Guidelines Version 1.2022; Seufferlein et al, Z Gastroenterol 2022, PMID 35671996). Nal-IRI in second line is dose- adjusted according to the UGT1A1 genotype (SmPC Onivyde®).

[0017] The present inventors hypothesized that manipulation of ALC1 activity via ALCli-1 and ALCli-2 could induce strong anti-proliferative effects and in addition be sufficient to bypass an acquired resistance to irinotecan containing chemotherapy. Thus, combined use of ALCli-1 and ALCli-2, respectively, and irinotecan treatment can be used for pancreatic cancer therapies in oncology, including in a relapse condition and when there is progression in advanced clinical progression stages.

[0018] This hypothesis led to a part of the present invention that ALC1 inhibition via the specific inhibitors ALCli-1 and ALCli-2 impacts the response to DNA damage specifically induced by irinotecan. ALCli-1 and ALCli-2, which inhibit the enzymatic activity of the ATP-dependent chromatin remodeler ALC1 (CHD1L), will potentiate accumulation of DNA damage and thus mediate synthetic lethality in the specific combination with irinotecan. Based on preclinical and clinical evidence, ALC1 inhibition via ALCli-1 and ALCli-2 serves as an additional therapeutic approach for pancreatic cancers where irinotecan containing chemotherapy is indicated and a dose reduction is necessary or efficacy has become limiting, hence improving patient outcomes through reduced side-effects and improved therapeutic efficacy.

[0019] Specific subtypes of pancreatic cancer are described to be more amenable to irinotecan treatment. In particular, defects in the DNA damage response pathways seem to confer sensitivity to irinotecan treatment, in combination with the chemotherapeutic agent 5-FU (Rau et al., 2020). This could serve as a biomarker to select patients that could benefit most from a combined treatment with ALCli-1 and ALCli-2, further corroborated observations in a clinical trial that utilized irinotecan containing chemotherapy (Chiorean et al., 2021).

[0020] Given the above described relevance of ALCli-1 and ALCli-2 for pancreatic cancers, in particular those that are amenable to irinotecan treatment, the present invention provides a novel class of compounds to treat or ameliorate pancreatic cancer and in particular tumor diseases characterized by loss of sensitivity to irinotecan containing chemotherapy (Rau et al., 2020).

[0021] Furthermore, the present inventors determined that by using a combination of irinotecan and ALCli-1 and / or ALCli-2, the effect of irinotecan can surprisingly be enhanced by several fold. Thus, the use of ALCli-1 and ALCli-2 in combination with irinotecan provides inter alia (i) an efficient therapy of pancreatic cancers that are amenable to irinotecan containing chemotherapeutic regimens, (ii) mediate irinotecan sensitization, (iii) bypass irinotecan resistance, (iv) and / or allow the reduction of the amount of irinotecan that is administered.

[0022] Summary of the Invention

[0023] In a first aspect, the present invention is directed at an inhibitor of ALC1 (ALCli) according to formula (I) and / or according to formula (II)

[0024] Formula II (ALCli-2) or isomers, pharmaceutically acceptable salts, solvates, chemically protected forms, and prodrugs of ALCli-1 and / or ALCli-2, for use in treating or ameliorating pancreatic cancer, wherein the treating and ameliorating of the pancreatic cancer comprises the administration of said ALCli and the administration of irinotecan, or SN-38, or isomers, pharmaceutically acceptable salts, solvates, chemically protected forms, conjugates and prodrugs thereof.

[0025] In a second aspect, the present invention is directed to ALCli-1 and / or ALCli-2 or isomers, pharmaceutically acceptable salts, solvates, chemically protected forms, and prodrugs of ALCli-1 and / or ALCli-2 for use enhancing efficacy of irinotecan, or SN-38, or isomers, pharmaceutically acceptable salts, solvates, chemically protected forms, conjugates or prodrugs thereof in treating or ameliorating pancreatic cancer. Enhancing efficacy preferably covers providing (i) a more efficient therapy of pancreatic cancers that are amenable to irinotecan containing chemotherapeutic regimens, (ii) mediate irinotecan sensitization, (iii) bypass irinotecan resistance, and / or (iv) allow the reduction of the amount of irinotecan that is administered.

[0026] In a third aspect, the present invention is directed to irinotecan, or SN-38, or isomers, pharmaceutically acceptable salts, solvates, chemically protected forms, conjugates and prodrugs thereof for use in treating or ameliorating pancreatic cancer, wherein the treating and ameliorating of the pancreatic cancer comprises the administration of said irinotecan and the administration of ALCli-1 and / or ALCli-2 or isomers, pharmaceutically acceptable salts, solvates, chemically protected forms, and prodrugs of ALCli-1 and / or ALCli-2. In preferred embodiments of the first, second and third aspects, irinotecan and ALCli are administered simultaneously or subsequently. In a fourth aspect, the present invention is directed to a pharmaceutical composition comprising (i) ALCli-1 and / or ALCli-2 or isomers, pharmaceutically acceptable salts, solvates, chemically protected forms, and prodrugs of ALCli-1 and / or ALCli-2 and (ii) irinotecan or SN-38 or isomers, pharmaceutically acceptable salts, solvates, chemically protected forms, conjugates and prodrugs thereof.

[0027] In a fifth aspect, the present invention relates to kit of parts comprising (i) ALCli-1 and / or ALCli- 2 or isomers, pharmaceutically acceptable salts, solvates, chemically protected forms, and prodrugs of ALCli-1 and / or ALCli-2 with instructions to combine it with irinotecan or SN-38 or isomers, pharmaceutically acceptable salts, solvates, chemically protected forms, conjugates and prodrugs thereof, or (ii) comprising irinotecan with instructions to combine it with ALCli-1 and / or ALCli-2 or isomers, pharmaceutically acceptable salts, solvates, chemically protected forms, and prodrugs of ALCli-1 and / or ALCli-2, or (iii) comprising separately packaged irinotecan or SN-38 or isomers, pharmaceutically acceptable salts, solvates, chemically protected forms, conjugates and prodrugs thereof and ALCli-1 and / or ALCli-2 or isomers, pharmaceutically acceptable salts, solvates, chemically protected forms, and prodrugs of ALCli-1 and / or ALCli-2, for use in treating or ameliorating of pancreatic cancer.

[0028] Description of the Figures

[0029] In the following, the content of the Figures comprised in this specification is described. In this context, it is also referred to the detailed description of the invention above and / or below.

[0030] Figure 1: 96 hour SRB assay of pancreatic cancer cell line treated with ALCli. PSN1 cells were seeded into 96- well plates and treated with titrations of ALCli-1 and ALCli-2. The cells were cultured at 37°C, CO2 5% for 4 days, fixed with 10%TCA and stained with sulforhodamine staining to analyze cell survival. The data was normalized to DMSO controls indicating 100 % survival. Inhibitor vs. response curves were fitted with the Synergy Finder using the curve fitting parameter “LL4”. The curves show an average fit of 3 technical replicates.

[0031] Figure 2: Irinotecan co-treatment Cell proliferation assay of pancreatic cancer cells. PSN1 cells were seeded into 96-well plates and treated with a 2-D titration of SN-38 vs. ALCli-1 and ALCli-2. The cells were cultured at 37°C, CO25% for 4 days, fixed with 10%TCA and stained with sulforhodamine staining to analyze cell survival. The synergy score is calculated using the Synergy Finder. Treatment with ALCli-1 or ALCli-2 in combination with SN-38 show highly synergistic area scores of above 10 respectively. ZIP synergy scores over 10 are an indication of strong synergy.

[0032] Figure 3: Increased yH2AX protein levels in co-treatment of ALCli with SN-38. A) PSN1 cells were seeded into 6- well plates. For SN-38 monotherapy, cells were treated with SN-38 (0.1 pM) 4 h and then incubated with DMSO for another 24 h. For ALCli-1 monotherapy, cells were treated with ALCli-1 (1 and 5 pM) for 28 h. For combination therapy, cells were treated with SN-38 and ALCli-1 for 4 h and then ALCli-1 for another 24 h. B) The same regimen was applied as above, but ALCli-2 instead of ALCli-1 was used. Cells were harvested for protein lysates and analyzed by immunoblot. Figure 4: Increased apoptosis in co-treatment of ALCli with SN-38. PSN1 cells were seeded into 6- well plates. For SN-38 monotherapy, cells were treated with SN-38 (0.1 pM) 4 h and then incubated with DMSO for another 72 h. For ALCli-2 monotherapy, cells were treated with ALCli-2 (5 pM) for 76 h. For combination therapy, cells were treated with SN-38 and ALCli-2for 4 h and then ALCli-2 for another 72 h. In panel A, cells were harvested for cell cycle analysis by flow cytometry (A). Sub-Gl region, as an indication of cell death, was quantitated. In panel B, cells were harvested for immunoblot analysis and cleaved caspase 3 was stained. Beta actin was used as a loading control. In panel C, colony formation assay was performed by crystal violet staining.

[0033] Figure 5: SN-38 sensitization by ALCli in patient-derived organoid (PDO). PDOs (PDO ID: 52113) were seeded in 96-well plates for 2 days. PDOs were then treated with SN-38 and ALCli-2alone (A & B) or combined (C). 72 h post-treatment, PDOs viability was determined by CellTiter-Glo 3D Cell Viability Assay.

[0034] Figure 6: Efficacy of co-treatment of ALCli with irinotecan in a PSNl-xenografted mouse model. PSN1 cells were subcutaneously transplanted into the right flanks of NMRI nude mice. When tumors’ size reached to 150 mm3, mice were treated with ALCli and irinotecan alone or combined. ALCli-1 (500 mg / kg) and ALCli-2 (250 mg / kg) were administrated daily via oral gavage. Irinotecan (15 mg / kg) was administrated three time per week via intraperitoneal injection. Tumor size was measured every three days.

[0035] Detailed Description of the Invention

[0036] Before the present invention is described in detail below, it is to be understood that this invention is not limited to the particular methodology, protocols and reagents described herein as these may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the scope of the present invention which will be limited only by the appended claims. Unless defined otherwise, all technical and scientific terms used herein have the same meanings as commonly understood by one of ordinary skill in the art.

[0037] Several documents are cited throughout the text of this specification. Each of the documents cited herein (including all patents, patent applications, scientific publications, manufacturer’s specifications, instructions etc.), whether supra or infra, is hereby incorporated by reference in its entirety. Nothing herein is to be construed as an admission that the invention is not entitled to antedate such disclosure by virtue of prior invention. Some of the documents cited herein are characterized as being “incorporated by reference”. In the event of a conflict between the definitions or teachings of such incorporated references and definitions or teachings recited in the present specification, the text of the present specification takes precedence.

[0038] In the following, the elements of the present invention will be described. These elements are listed with specific embodiments, however, it should be understood that they may be combined in any manner and in any number to create additional embodiments. The variously described examples and preferred embodiments should not be construed to limit the present invention to only the explicitly described embodiments. This description should be understood to support and encompass embodiments, which combine the explicitly described embodiments with any number of the disclosed and / or preferred elements. Furthermore, any permutations and combinations of all described elements in this application should be considered disclosed by the description of the present application unless the context indicates otherwise.

[0039] Definitions

[0040] To practice the present invention, unless otherwise indicated, conventional methods of chemistry, biochemistry, and recombinant DNA techniques are employed which are explained in the literature in the field (cf., e.g., Molecular Cloning: A Laboratory Manual, 2nd Edition, J. Sambrook et al. eds., Cold Spring Harbor Laboratory Press, Cold Spring Harbor 1989).

[0041] In the following, some definitions of terms frequently used in this specification are provided. These terms will, in each instance of its use, in the remainder of the specification have the respectively defined meaning and preferred meanings.

[0042] As used in this specification and the appended claims, the singular forms “a”, “an”, and “the” include plural referents, unless the content clearly dictates otherwise.

[0043] The term “Chromodomain-helicase-DNA-binding protein 1-like” abbreviated CHD1L refers to a protein that is also termed ALC1. The amino acid sequence of human ALC1 is as known in the art, e.g. as specified in WO2022 / 117782 Al . The 897 amino acid residues long protein consists of an N-terminal Snf2- like DNA dependent ATPase domain spanning amino acid residues 40 to 513, which contains the conserved helicase motifs critical for catalysis (Flaus et al., 2006). This domain is composed of two Rec A like lobes ranging from amino acid residues 48 to 261 and 351 to 513, respectively. The allosteric binding pocket is spatially separated from that part of ALC1 involved in binding ATP. The ATPase domain is followed by a linker region ranging from amino acid residues 514 to 703, which contains a putative coiled-coil region (amino acid residues 638 to 675), and a C-terminal macrodomain (amino acid residues 704 to 897). The macrodomain has been shown to directly interact with the ATPase domain, thereby inhibiting its catalytic function (Lehmann et al., 2017; Singh et al., 2017). This interaction is released upon poly(ADP-ribose) binding to the macrodomain, leading to an activation of the chromatin remodelling enzyme.

[0044] "Pharmaceutically acceptable" means approved by a regulatory agency of the Federal or a state government or listed in the U.S. Pharmacopeia (United States Pharmacopeia-33 / National Formulary-28 Reissue, published by the United States Pharmacopeia Convention, Inc., Rockville Md., publication date: April 2010) or other generally recognized pharmacopeia for use in animals, and more particularly in humans.

[0045] The term "pharmaceutically acceptable salt" refers to a salt of ALCli-1, ALCli-2, or irinotecan or SN-38, respectively. Suitable pharmaceutically acceptable salts, in particular of ALCli-1 of ALCli-2, include acid addition salts which may, for example, be formed by mixing a solution (e.g. of ALCli-1 of ALCli-2) or a positively or negatively charged derivative thereof with a solution of a pharmaceutically acceptable acid such as hydrochloric acid, sulfuric acid, fumaric acid, maleic acid, succinic acid, acetic acid, benzoic acid, citric acid, tartaric acid, carbonic acid or phosphoric acid. ALCli-1 carries an acidic moiety and ALCli-2 may be derivatized to carry an acidic moiety, thus suitable pharmaceutically acceptable salts thereof may include alkali metal salts (e.g., sodium or potassium salts); alkaline earth metal salts (e.g., calcium or magnesium salts); and salts formed with suitable organic ligands (e.g., ammonium, quaternary ammonium and amine cations formed using counteranions such as halide, hydroxide, carboxylate, sulfate, phosphate, nitrate, alkyl sulfonate and aryl sulfonate). Illustrative examples of pharmaceutically acceptable salts include but are not limited to: acetate, adipate, alginate, ascorbate, aspartate, benzenesulfonate, benzoate, bicarbonate, bisulfate, bitartrate, borate, bromide, butyrate, calcium edetate, camphorate, camphorsulfonate, camsylate, carbonate, chloride, citrate, clavulanate, cyclopentanepropionate, digluconate, dihydrochloride, dodecylsulfate, edetate, edisylate, estolate, esylate, ethanesulfonate, formate, fumarate, gluceptate, glucoheptonate, gluconate, glutamate, glycerophosphate, glycolylarsanilate, hemisulfate, heptanoate, hexanoate, hexylresorcinate, hydrabamine, hydrobromide, hydrochloride, hydroiodide, 2-hydroxyethanesulfonate, hydroxynaphthoate, iodide, isothionate, lactate, lactobionate, laurate, lauryl sulfate, malate, maleate, malonate, mandelate, mesylate, methanesulfonate, methylsulfate, mucate, 2-naphthalenesulf onate, napsylate, nicotinate, nitrate, N-methylglucamine ammonium salt, oleate, oxalate, pamoate (embonate), palmitate, pantothenate, pectinate, persulfate, 3- phenylpropionate, phosphate / diphosphate, picrate, pivalate, polygalacturonate, propionate, salicylate, stearate, sulfate, subacetate, succinate, tannate, tartrate, teoclate, tosylate, triethiodide, undecanoate, valerate, and the like (see, for example, Berge, S. M., et al, "Pharmaceutical Salts", Journal of Pharmaceutical Science, 1977, 66, 1-19). Certain specific compounds of the present invention contain both basic and acidic functionalities that allow the compounds to be converted into either base or acid addition salts.

[0046] The neutral forms of ALCli-1 or ALCli-2 may be regenerated by contacting the salt with a base or acid and isolating the parent compound in the conventional manner. The parent form of the compound differs from the various salt forms in certain physical properties, such as solubility in polar solvents, but otherwise the salts are equivalent to the parent form of the compound for the purposes of the present invention.

[0047] In addition to salt forms, ALCli-1, ALCli-2, irinotecan, or SN-38 can be used in a prodrug form. Prodrugs of ALCli-1, ALCli-2 , irinotecan, or SN-38 are those compounds that readily undergo chemical changes under physiological conditions to provide ALCli-1, ALCli-2, irinotecan, or SN-38, respectively. A prodrug is an active or inactive compound that is modified chemically through in vivo physiological action, such as hydrolysis, metabolism and the like, into a compound of this invention following administration of the prodrug to a patient. Additionally, prodrugs can be converted to the compounds used in the present invention by chemical or biochemical methods in an ex vivo environment. For example, prodrugs can be slowly converted to the compounds used in the present invention when placed in a transdermal patch reservoir with a suitable enzyme. The suitability and techniques involved in making and using prodrugs are well known by those skilled in the art. For a general discussion of prodrugs involving esters, see Svensson L.A. and Tunek A. (1988) Drug Metabolism Reviews 19(2): 165-194 and Bundgaard H. “Design of Prodrugs”, Elsevier Science Ltd. (1985). Examples of a masked carboxylate anion include a variety of esters, such as alkyl (for example, methyl, ethyl), cycloalkyl (for example, cyclohexyl), aralkyl (for example, benzyl, p-methoxybenzyl), and alkylcarbonyloxyalkyl (for example, pivaloyloxymethyl). Amines have been masked as arylcarbonyloxymethyl substituted derivatives, which are cleaved by esterases in vivo releasing the free drug and formaldehyde (Bundgaard H. et al. (1989) J. Med. Chem. 32(12): 2503-2507). Also, drugs containing an acidic NH group, such as imidazole, imide, indole and the like, have been masked with N-acyloxymethyl groups (Bundgaard H. “Design of Prodrugs”, Elsevier Science Ltd. (1985)). Hydroxy groups have been masked as esters and ethers. EP 0 039 051 A2 discloses Mannich-base hydroxamic acid prodrugs, their preparation and use.

[0048] ALCli-1 or ALCli-2 may also contain unnatural proportions of atomic isotopes at one or more of the atoms that constitute such compounds. For example, the compounds may be radiolabeled with radioactive isotopes, such as for example tritium (3H), iodine-125 (125I) or carbon-14 (14C). All isotopic variations of ALCli-1 and ALC11-2, whether radioactive or not, are intended to be encompassed within the scope of the present invention.

[0049] As used herein, a “patient” means any mammal or bird that may benefit from a treatment with the compounds described herein. Preferably, a “patient” is selected from the group consisting of laboratory animals, domestic animals, or primates including chimpanzees and human beings. It is particularly preferred that the “patient” is a human being.

[0050] As used herein, "treat", "treating" or “treatment”, or ameliorating, respectively, of a disease or disorder means accomplishing one or more of the following: (a) reducing the severity of the disorder; (b) limiting or preventing development of symptoms characteristic of the disorder(s) being treated; (c) inhibiting worsening of symptoms characteristic of the disorder(s) being treated; (d) limiting or preventing recurrence of the disorder(s) in patients that have previously had the disorder(s); and (e) limiting or preventing recurrence of symptoms in patients that were previously symptomatic for the disorder(s).

[0051] As used herein, “prevent”, “preventing”, “prevention”, or “prophylaxis” of a disease or disorder means preventing that a disorder occurs in a subject for a certain amount of time. For example, if a compound described herein is administered to a subject with the aim of preventing a disease or disorder, said disease or disorder is prevented from occurring at least on the day of administration and preferably also on one or more days (e.g. on 1 to 30 days; or on 2 to 28 days; or on 3 to 21 days; or on 4 to 14 days; or on 5 to 10 days) following the day of administration.

[0052] A “pharmaceutical composition” according to the invention may be present in the form of a composition, wherein the different active ingredients and diluents and / or carriers are admixed with each other, or may take the form of a combined preparation, where the active ingredients are present in partially or totally distinct form. An example for such a combination or combined preparation is a kit-of-parts.

[0053] An “effective amount” is an amount of a therapeutic agent sufficient to achieve the intended purpose. The effective amount of a given therapeutic agent will vary with factors such as the nature of the agent, the route of administration, the size and species of the animal to receive the therapeutic agent, and the purpose of the administration. The effective amount in each individual case may be determined empirically by a skilled artisan according to established methods in the art.

[0054] The term “carrier”, as used herein, refers to a diluent, adjuvant, excipient, or vehicle with which the therapeutic agent is administered. Such pharmaceutical carriers can be sterile liquids, such as saline solutions in water and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like. A saline solution is a preferred carrier when the pharmaceutical composition is administered intravenously. Saline solutions and aqueous dextrose and glycerol solutions can also be employed as liquid carriers, particularly for injectable solutions. Suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, gelatine, malt, rice flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene glycol, water, ethanol and the like. The composition, if desired, can also contain minor amounts of wetting or emulsifying agents, or pH buffering agents. These compositions can take the form of solutions, suspensions, emulsions, tablets, pills, capsules, powders, sustained-release formulations and the like. The composition can be formulated as a suppository, with traditional binders and carriers such as triglycerides. The compounds of the invention can be formulated as neutral or salt forms. Pharmaceutically acceptable salts include those formed with free amino groups such as those derived from hydrochloric, phosphoric, acetic, oxalic, tartaric acids, etc., and those formed with free carboxyl groups such as those derived from sodium, potassium, ammonium, calcium, ferric hydroxides, isopropylamine, triethylamine, 2-ethylamino ethanol, histidine, procaine, etc. Examples of suitable pharmaceutical carriers are described in "Remington's Pharmaceutical Sciences" by E. W. Martin. Such compositions will contain a therapeutically effective amount of the compound, preferably in purified form, together with a suitable amount of carrier so as to provide the form for proper administration to the patient. The formulation should suit the mode of administration.

[0055] Embodiments of the Invention

[0056] The present inventors have identified and characterized the ALC1 inhibitors ALCli-1 and ALCli-2 that appear to be involved in allosteric regulation of the nucleosome sliding activity of ALC1. These compounds specifically bind to an allosteric pocket and are capable of inhibiting activity of ALC1. ALCli- 1 and ALCli-2 were capable to kill pancreatic cancer cell lines. These compounds have been tested for their ability to kill different tumor cell lines one of which was BRCA deficient.

[0057] The present inventors considered the rationale that ALC1 inhibitors as a known class of cancer drugs could be combined with another class of known cancer drugs, i.e. an inhibitor of Topoisomerase I, namely irinotecan. While tests with known ALCli showed not even over-additive efficacies against cancer cell lines when combined with other classes of known cancer drugs, the present invention is based on the surprising finding that specific ALC1 inhibitors, namely those of Formula (I) and of Formula (II), possess synergistic anti-proliferative activity when combined with irinotecan against pancreatic cancer cell lines. Thus, these combinations are expected to be suitable in the treatment of pancreatic cancer. The ALC1 inhibitors characterized by Formula (I) and Formula (II), the latter being known from and obtainable as described in the art (WO 2022 / 117782 Al), which are used in accordance with the present invention, appear to be involved in allosteric regulation of the nucleosome sliding activity of ALC1. These compounds specifically bind to an allosteric pocket and are capable of inhibiting activity of ALC1. Compounds that bind to the ATPase site of ALC1 and block the ATPase activity have to compete with ATP for binding to the ATPase site. Since the cellular ATP concentration is in the range of 1 to 10 mM depending on the cellular compartment, very high binding affinities in the low nanomolar range are required to successfully prevent ATP from binding to the ATPase site of ALC1. The allosteric inhibitors of ALC1 that are characterized by Formula (I) and Formula (II) do not have this limitation since they do not have to prevent ATP from binding but inhibit ALCl’s activity through a different mechanism. The present inventors have identified ALC1 inhibitors that are capable of specifically binding to an allosteric pocket of ALC1. The question remained and has been not answered by the art yet, whether ALC1 inhibitors would provide synergistic efficacy when being combined with other classes of cancer drugs. As shown herein, this is the case for the specific ALC1 inhibitors as described herein.

[0058] In a first aspect, the present invention is directed at an inhibitor of ALC1 (ALCli) according to formula (I) and / or according to formula (II)

[0059] Formula II (ALCli-2) or isomers, pharmaceutically acceptable salts, solvates, chemically protected forms, and prodrugs of ALCli-1 and / or ALCli-2, for use in treating or ameliorating pancreatic cancer, wherein the treating and ameliorating of the pancreatic cancer comprises the administration of said ALCli and the administration of irinotecan, or SN-38, or isomers, pharmaceutically acceptable salts, solvates, chemically protected forms, conjugates or prodrugs thereof. Irinotecan is 7-ethyl-10-[4-(l-piperidino)-l-piperidino] carbonyloxycampothecin, IUPAC name (S)- 4,l l-diethyl-3,4,12,14-tetrahydro-4-hydroxy-3,14-dioxolH-pyrano[3',4':6,7]-indolizino[l,2-b]quinolin-9- yl-[l,4'bipiperidine]-l'-carboxylate. Irinotecan is a topoisomerase I inhibitor class of drugs and is a semisynthetic and water soluble analog of the naturally-occurring alkaloid, camptothecin. Also known as CPT- 11, irinotecan is currently marketed formulated as an aqueous solution as Camptosar® (irinotecan hydrochloride injection). Irinotecan arrests uncontrolled cell growth by inhibiting the unwinding of DNA and thereby preventing DNA replication.

[0060] The pharmacology of irinotecan is complex, with extensive metabolic conversions involved in the activation, inactivation, and elimination of the drug. Irinotecan is a prodrug that is converted by nonspecific carboxylesterases into a 100-1000 fold more active metabolite, SN-38. SN-38 is 7-Ethyl-10- hydroxycamptothecin, IUPAC name (4S)-4,l l-Diethyl-4,9-dihydroxy-l,4-dihydro-3H,14H- pyrano[3',4':6,7]indolizino[l,2-b]quinoline-3, 14-dione. SN-38 is not recognized by P-glycoprotein, a drug transporter that plays an important role in acquired drug resistance by pumping certain drugs out of cells, so irinotecan is likely to be active in tumors resistant to other standard chemotherapies. In the body, SN-38 is cleared via glucuronidation, for which major pharmacogenetic variability has been described, and biliary excretion. These drug properties contribute to the marked heterogeneities in efficacy and toxicity observed clinically with irinotecan. Irinotecan hydrochloride injection is approved in the United States for treatment of metastatic colon or renal cancer and is also used to treat colorectal, gastric, lung, uterine cervical and ovarian cancers.

[0061] Irinotecan is also used and admitted as a liposomal formulation. Thus, the irinotecan for use of the present invention may also be provided as liposomal formulation. Different liposomal irinotecan formulations are known and commercially available. In the context of the present invention, all of these formulations can be used. Exemplary irinotecan liposomal formulations comprise irinotecan sucrose octasulfate salt liposome, which is also referred to as MM-398.

[0062] Irinotecan may also be provided as a conjugate, such as with a protein, preferably with an antibody targeting pancreatic cancer, e.g. by specifically binding to the extracellular part of a protein preferentially expressed on pancreatic cancer cells. An example of such a targeted conjugate is Sacituzumab govitecan in which irinotecan is covalently conjugated to an antibody specifically binding to Tumor-associated calcium signal transducer 2.

[0063] In a second aspect, the present invention is directed to ALCli-1 and / or ALCli-2 or isomers, pharmaceutically acceptable salts, solvates, chemically protected forms, and prodrugs of ALCli-1 and / or ALCli-2 for use enhancing efficacy of irinotecan, or SN-38, or isomers, pharmaceutically acceptable salts, solvates, chemically protected forms, conjugates or prodrugs thereof in treating or ameliorating pancreatic cancer. Enhancing efficacy preferably covers providing (i) a more efficient therapy of pancreatic cancers that are amenable to irinotecan containing chemotherapeutic regimens, (ii) mediate irinotecan sensitization, (iii) bypass irinotecan resistance, and / or (iv) allow the reduction of the amount of irinotecan that is administered. The preferred embodiments of the second aspect correspond to those of the first aspect. In a third aspect, the present invention relates to irinotecan, or SN-38, or isomers, pharmaceutically acceptable salts, solvates, chemically protected forms, conjugates and prodrugs for use in treating or ameliorating pancreatic cancer, wherein the treating or ameliorating of pancreatic cancer comprises the administration of said irinotecan and the administration of ALCli-1 and / or ALCli-2 or isomers, pharmaceutically acceptable salts, solvates, chemically protected forms, and prodrugs of ALCli-1 and / or ALCli-2. In preferred embodiments of the first, second and third aspects, irinotecan and ALCli are administered simultaneously or subsequently.

[0064] The first, second and third aspect of the invention thus preferably relate to a combination treatment in which ALCli-1 and / or ALCli-2 and irinotecan are administered. The timing of administration of ALCli-

[0065] 1 and / or ALCli-2 on one hand and of irinotecan, or SN-38, on the other hand can be at the same time or consecutively. ALCli-1 and / or ALCli-2 can be administered prior to or after administration of irinotecan, or SN-38, respectively.

[0066] Irinotecan, or SN-38 or isomers, pharmaceutically acceptable salts, solvates, chemically protected forms, conjugates or prodrugs thereof is typically administered orally or intravenously (iv), the latter being preferred, and is more preferably administered as a liposomal formulation or as an IV bolus. Three schedules of irinotecan administration are currently in clinical use, namely weekly, bi-weekly and triweekly schedules, among which administration once every 3 weeks and typically a weekly 90-min infusion are the ones most commonly used and suitable for the present invention.

[0067] ALCli-1 or isomers, pharmaceutically acceptable salts, solvates, chemically protected forms, and prodrugs thereof is typically administered orally, preferably as a tablet or capsule.

[0068] ALCli-2 or isomers, pharmaceutically acceptable salts, solvates, chemically protected forms, and prodrugs thereof is typically administered orally, preferably as a tablet or capsule.

[0069] In a fourth aspect, the present invention relates to a pharmaceutical composition comprising (i) irinotecan or SN-38 or isomers, pharmaceutically acceptable salts, solvates, chemically protected forms, conjugates or prodrugs thereof and (ii) ALCli-1 and / or ALCli-2 as described above for use in treating or ameliorating pancreatic cancer. In this aspect, the irinotecan is in admixture with ALCli-1 and / or ALCli- 2. Preferably, the composition is in the form of a solution or a solid dosage form. The pharmaceutical composition may additionally comprise a carrier. The pharmaceutical composition may be for oral or intravenous (iv) administration, preferably iv.

[0070] In a fifth aspect, the present invention relates to kit of parts comprising (i) ALCli-1 and / or ALCli-

[0071] 2 or isomers, pharmaceutically acceptable salts, solvates, chemically protected forms, and prodrugs of ALCli-1 and / or ALCli-2 with instructions to combine it with irinotecan or SN-38, or isomers, pharmaceutically acceptable salts, solvates, chemically protected forms, conjugates and prodrugs thereof, or (ii) comprising irinotecan with instructions to combine it with ALCli-1 and / or ALCli-2 or isomers, pharmaceutically acceptable salts, solvates, chemically protected forms, and prodrugs of ALCli-1 and / or ALCli-2, or (iii) comprising separately packaged irinotecan or SN-38 or isomers, pharmaceutically acceptable salts, solvates, chemically protected forms, conjugates and prodrugs thereof and ALCli-1 and / or ALCli-2 or isomers, pharmaceutically acceptable salts, solvates, chemically protected forms, and prodrugs of ALCli-1 and / or ALCli-2. The kit is for use in treating or ameliorating pancreatic cancer.

[0072] In one embodiment of all aspects described herein, the pancreatic cancer is selected from exocrine pancreatic cancer including adenocarcinoma (e.g. pancreatic ductal adenocarcinoma), which is preferred, squamous cell carcinoma, adenosquamous carcinoma, and colloid carcinoma, and neuroendocrine pancreatic cancer. The combination therapy according to the first to fifth aspect of the invention is particular suitable to treat metastatic pancreatic cancer, and in particular metastatic adenocarcinoma of the pancreas.

[0073] BRCA1 and BRCA2 proteins are involved in both promoting homologous recombination (HR)- mediated DNA repair and also controlling the stability of stalled replication forks. Many tumor types, including, e.g., breast cancer, ovarian cancer, prostate cancer, pancreatic carcinomas, fallopian tube cancer, peritoneal cancer, acute myeloid leukemia, and uveal melanoma often have underlying defects in BRCA1 or BRCA2 activity. These defects are often due to germline or somatic mutations in the BRCA1 or BRCA2 genes. These tumors, with underlying defects in HR repair, are thus particularly expected to be sensitive to ALCli treatment. Surprisingly, the combination of the invention is synergistic irrespectively of whether the pancreatic cancer have underlying defects in BRCA1 or BRCA2 activity. Thus, in one embodiment of all aspects of the invention, the pancreatic cancer cells of the cancer to be treated show defects in BRCA1 or BRCA2 activity or normal BRCA1 and / or BRCA2 activity, the latter being preferred.

[0074] Preferably, the pancreatic cancer to be treated according to the first to fifth aspect of the invention has one or more underlying defects in DNA damage repair, like HR repair, i.e. is a HR deficient pancreatic cancer. In one embodiment, the cancer cells have mutations, e.g. deletions and / or insertions, in a specific DNA repair gene, namely ATM. The gene ataxia-telangiectasia-mutated (ATM) has been recently identified as a pancreatic cancer susceptibility gene and to undergo somatic mutation in some pancreatic ductal adenocarcinomas. Tumoral inactivation of ATM influences chemosensitivity responses (see e.g. Kim et al, Clin Cancer Res. 2014 Apr 1; 20(7): 1865-1872). In one embodiment, the cancer to be treated shown loss of ATM and / or the cancer patients to be treated are selected based on the presence of said tumor marker ATM, i.e. tumoral loss of ATM.

[0075] Preferably, the pancreatic cancer to be treated according to the first to fifth aspect of the invention has relapsed or progressed, e.g. after treatment with standard-of-care chemotherapy, such as with a combination of irinotecan, folinic acid, fluorouracil and oxaliplatin (FOLFIRINOX), a combination of irinotecan, oxaliplatin, 5 -fluorouracil and leucovorin (NALIRIFOX), or with gemcitabine. Preferably, the first to fifth aspect are thus as second line therapy, more preferably after treatment with standard-of-care chemotherapy, such as with a combination of irinotecan, folinic acid, fluorouracil and oxaliplatin (FOLFIRINOX), a combination of irinotecan, oxaliplatin, 5 -fluorouracil and leucovorin (NALIRIFOX), or with gemcitabine . In one embodiment of the invention, the pancreatic cancer is at stage III (locally advanced) or IV (metastatic) and / or the pancreatic cancer is sensitive to irinotecan containing chemotherapy or has lost sensitivity to irinotecan containing chemotherapy.

[0076] According to the present invention, the efficacy of irinotecan or its derivatives is enhanced by combining it with the ALCli as defined herein. This allows reduction of the dosages of irinotecan to be used, in particular to below what is normally used. Typical normal dosages are 150 mg / m2of irinotecan in a combination with folinic acid, fluorouracil and oxaliplatin (FOLFIRINOX), 70 mg / m2as liposomal formulation in combination with fluorouracil (nal-Iri / 5-FU), or 50 mg / m2in a combination with oxaliplatin, 5 -fluorouracil and leucovorin (NALIRIFOX). With regard to the dosage regimen, normal doses are about at least 100 mg / m2, e.g. 120 to 150 mg / m2, in a single agent regimen or a combination agent regimen comprising administration on days 1, 8, 15, and 22 followed by a 2-week rest, at least 180 mg / m2in a single agent regimen comprising administration every two weeks, or at least 240 mg / m2, e.g. about 300 mg / m2to about 350 mg / m2, in a combination agent regimen comprising administration every three weeks. Preferably, in accordance with the present invention, doses lower than these dosages are used, more preferably equal to or lower than 80 %, most preferably equal to or lower than 50% of these dosages, in the respective dosage regimens.

[0077] In some embodiments, the patient treated with the combination therapy according to the first to fifth embodiment of the invention is additionally treated with one or more chemotherapeutics comprising oxaliplatin, 5 -fluorouracil, and / or folinic acid.

[0078] Experimental Section

[0079] Cell Lines and PDO used

[0080] As a pancreatic cancer cell line, PSN1 cells were used. The human cell line was derived from pancreatic adenocarcinoma tissue. It harbors an amplification of c-myc and activated c-Ki-ras and a loss of one of the two p53 alleles. The cell line is available from numerous sources including MERCK (94060601). This cell line was very sensitive to PARP-inhibitors as well as the ALC1 inhibitors described herein. PDO was established from the ascites of a PDAC (pancreatic ductal adenocarcinoma) patient (POD ID: 52113) and reserved in the biobank of University Hospital Essen.

[0081] Inhibitor vs. Response (cell survival) assay

[0082] For validation of the small molecule ALC1 inhibitors, the pancreatic cell line PSN1 is used. Cells were seeded in 96- well plates (2000 cells / well) and treated with titrations of ALC1 inhibitors in the pM range. As a control for “no- treatment”, DMSO was added to the cells. The cells were cultured at 37°C, CO2 5 % for 5 days until they were fixed with 10% TCA for Ih and stained with sulforhodamine dye for 30 minutes. After washing the cells with 1% Acetic Acid, 10 rnM Tris (pH 10.5) solution was used to solubilize the stained cells. The absorbance was measured at 492 nm using the SUNRISE TECAN, the data were normalized to 100 % survival (=DMSO control). Inhibitor vs. response curves were fitted with the Synergy Finder using the curve fitting parameter “LL4”. The curves show an average fit of 3 technical replicates (see Figure 1).

[0083] Synergy with Irinotecan in cellular assays

[0084] For determination of the ZIP synergy score, a 2-D titration of two compounds is added to cells in the 96h-SRB-survival (described above) assay format. The score is calculated by adding the readout of the survival data from the SRB assay of at least 3 replicate plates to an open-source program called Synergy Finder.

[0085] “SynergyFinder (https: / / synergyfinder.fimm.fi) is a stand-alone web-application for interactive analysis and visualization of drug combination screening data. Since its first release in 2017, SynergyFinder has become a widely used web-tool both for the discovery of novel synergistic drug combinations in pre- clinical model systems (e.g. cell lines or primary patient-derived cells), and for better understanding of mechanisms of combination treatment efficacy or resistance” (lanevski et al. 2020).

[0086] In the Zero interaction potency (ZIP) model, the drug interaction relationship is determined by comparison of the change in potency of the dose-dependent curves between individual drugs and their combination (https: / / synergyfinder.fimm.fi / synergy / synfin_docs / ). The model is further described in https : / / www. ncbi. nlm. nih. gov / pmc / articles / PMC4759128 / .

[0087] SynergyFinder uses an cNMF algorithm to detect and replace outlier measurements. The Version used to obtain the data as shown herein are Version 2.0 and 3.0.

[0088] The summary synergy score is averaged over all dose combinations from the 2-D titration matrix. The most synergistic area (MSA) shows the most synergistic 3-by-3 dose-window in the dose-response matrix.

[0089] For interpretation of the synergy scores calculated in this program, see table 1 :

[0090] Table 1:

[0091] Results for treatment with ALC1 inhibitors together with the active metabolite of Irinotecan, SN-38 (termed “SN38”), are shown in Figure 2. The combinations of the invention show synergistic efficacy in the treatment of pancreatic cancer cells.

[0092] Immunoblot analysis of phospho-Histone H2A.X and cleaved caspase-3

[0093] Cells were lysed in RIPA buffer containing protease inhibitor cocktail. Protein extracts were separated on SDS-PAGE, transferred to nitrocellulose membrane and incubated with antibodies dissolved in TBS buffer containing 5% BS A and Tween 20 (0.1 %). Primary antibodies were detected by a peroxidase- coupled secondary antibody and chemiluminescence.

[0094] Flow cytometry analysis of cell cycle distribution Cells were harvested and fixed in 75% ethanol at 4°C overnight. Cells were washed once with PBS, and resuspended in PBS containing 0.5 mg / ml RNase A. Cells were incubated at 37°C for 30 min and then propidium iodide was added (final concentration: 30 pg / ml). The cellular DNA contents were analyzed by the Guava Easycyto System.

[0095] Colony formation assay by crystal violet staining

[0096] Cells were washed once with PBS and fixed with methanol at 4°C for 10 min. Then, methanol was removed and cells were incubated with crystal violet solution for at least 30 min at room temperature. Cells were washed twice with H2O and dried overnight.

[0097] PDO viability assay

[0098] PDOs were seeded in 96-well plates and cultured at 37°C, CO2 5 % for 2 days. Then PDOs were treated with inhibitors. 3 days post-treatment, add CellTiter-Glo 3D reagent, mix by shaking for 5 min and incubate for 20 min. Luminescent signals were read by the Spark Multimode Microplate Reader (Tecan). The values of luminescent signals were normalized to DMSO control wells and presented as percentage of cell viability.

[0099] PSN1 xenografted mouse model

[0100] Suspended PSN1 cells (2 x 105cells) were mixed with equal volume of matrigel and incubated on ice. Cells (2 x 105cells in 100 pl) were then transplanted into the right flank of the NMRI immunodeficient mouse. When tumor volume reached to 150 mm3, mice were treated with inhibitors and tumor volumes were measured every 3 days. The tumor volume = (length x width x width) / 2.

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Claims

Claims1. An inhibitor of ALC1 (ALCli) according to formula (I)Formula I (ALCli-1) or isomers, pharmaceutically acceptable salts, solvates, chemically protected forms, or prodrugs thereof and / or according to formula (II)Formula II (ALCli-2) or isomers, pharmaceutically acceptable salts, solvates, chemically protected forms, or prodrugs thereof for use in treating or ameliorating pancreatic cancer, wherein the treating or ameliorating of pancreatic cancer comprises the administration of said ALCli-1 and / or ALCli-2 and the administration of irinotecan, or SN-38, or isomers, pharmaceutically acceptable salts, solvates, chemically protected forms, conjugates or prodrugs thereof.An inhibitor of ALC1 (ALCli) according to formula (I)Formula I (ALCli-1)or isomers, pharmaceutically acceptable salts, solvates, chemically protected forms, or prodrugs thereof and / or according to formula (II)Formula II (ALCli-2) or isomers, pharmaceutically acceptable salts, solvates, chemically protected forms, or prodrugs thereof for use in enhancing efficacy of irinotecan, or SN-38, or isomers, pharmaceutically acceptable salts, solvates, chemically protected forms, conjugates or prodrugs thereof in treating or ameliorating pancreatic cancer.

3. Irinotecan, or SN-38, or isomers, pharmaceutically acceptable salts, solvates, chemically protected forms, conjugates or prodrugs thereof for use in treating or ameliorating pancreatic cancer, wherein the treating or ameliorating of pancreatic cancer comprises the administration of said irinotecan and the administration of ALCli-1 and / or ALCli-2 as defined in claim 1 or isomers, pharmaceutically acceptable salts, solvates, chemically protected forms, or prodrugs thereof.

4. The ALCli for use of claim 1 or 2 or the irinotecan, or SN-38 for use of claim 3, wherein irinotecan and ALCli are administered simultaneously or subsequently.

5. A pharmaceutical composition comprising irinotecan, or SN-38 or isomers, pharmaceutically acceptable salts, solvates, chemically protected forms, conjugates or prodrugs thereof and ALCli-1 and / or ALCli-2 as defined in claim 1 or isomers, pharmaceutically acceptable salts, solvates, chemically protected forms, or prodrugs thereof for use in treating or ameliorating pancreatic cancer.

6. A kit of parts comprising(i) ALCli-1 and / or ALCli-2 as defined in claim 1 or isomers, pharmaceutically acceptable salts, solvates, chemically protected forms, or prodrugs thereof with instructions to combine it with irinotecan, or SN-38 or isomers, pharmaceutically acceptable salts, solvates, chemically protected forms, conjugates or prodrugs thereof, or(ii) comprising irinotecan, or SN-38 or SN-38 or isomers, pharmaceutically acceptable salts, solvates, chemically protected forms, conjugates or prodrugs thereof with instructions to combine it with ALCli-1 and / or ALCli-2 as defined in claim 1 or isomers,pharmaceutically acceptable salts, solvates, chemically protected forms, or prodrugs thereof, or(iii) comprising separately packaged irinotecan or SN-38 or isomers, pharmaceutically acceptable salts, solvates, chemically protected forms, conjugates or prodrugs thereof and ALCli-1 and / or ALCli-2 as defined in claim 1 or isomers, pharmaceutically acceptable salts, solvates, chemically protected forms, or prodrugs thereof for use in treating or ameliorating pancreatic cancer.

7. The ALCli for use of claim 1, 2 or 4, or the irinotecan, or SN-38 for use of claim 3 or 4, the pharmaceutical composition of claim 5, or the kit of parts of claim 6, wherein the pancreatic cancer has relapsed or progressed, preferably for use as second line therapy, more preferably for use after treatment with standard-of-care chemotherapy, such as with a combination of irinotecan, folinic acid, fluorouracil and oxaliplatin (FOLFIRINOX), a combination of irinotecan, oxaliplatin, 5 -fluorouracil and leucovorin (NALIRIFOX), or with gemcitabine.

8. The ALCli for use of claim 1, 2, 4 or 7, or the irinotecan, or SN-38 for use of claim 3, 4 or 7, the pharmaceutical composition of claim 5 or 7, or the kit of parts of claim 6 or 7, wherein the pancreatic cancer is at stage III (locally advanced) or IV (metastatic), and / or wherein the pancreatic cancer is sensitive to irinotecan containing chemotherapy or has lost sensitivity to irinotecan containing chemotherapy.

9. The ALCli for use of claim 1, 2, 4, 7 or 8, or the irinotecan, or SN-38 for use of claim 3, 4, 7 or 8, the pharmaceutical composition of claim 5, 7 or 8, or the kit of parts of claim 6, 7 or 8, wherein the dose of irinotecan or liposomal irinotecan has been reduced, preferably to dosages- below 100 mg / m2in a single agent regimen or a combination agent regimen comprising administration on days 1, 8, 15, and 22 followed by a 2-week rest,- below 180 mg / m2in a single agent regimen comprising administration every two weeks, and / or- below 300 mg / m2in a combination agent regimen comprising administration every tree weeks.

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