WO2025180907A1 - Specific galactoside inhibitor of galectin-1 - Google Patents

Abstract

The present invention relates to a compound of formula (I) for use in a method for treatment of a cancer having a higher expression of galectin-1 compared to normal tissue.

Description

SPECIFIC GALACTOSIDE INHIBITOR OF GALECTIN-1

Technical field

The present invention relates to a compound of formula (I)

(i); for use in a method for treatment of a cancer having a higher expression of galectin-1 compared to normal tissue. Further, an oral composition is covered. The present invention also concerns a method of treating a cancer.

Background Art

Galectins are a family of lectins that mediate numerous biological functions in both health and disease. Due to their solubility, galectins are present in the extracellular space, cytosol and nucleus, and can act both intra- and extracellularly. Galectins bind beta-galactoside sugars, a common glycan modification on many cell surface receptors, modulating cellular functions by altering receptor-mediated signal transduction pathways (Liu, 2005; Rabinovich and Toscano, 2009). Galectins can also bind intracellular interactors in a carbohydrate-independent manner, with binding partners varying between different galectin family members (Liu and Stowell, 2023). The 15 galectin family members (11 in humans) are classified by the number and arrangement of their carbohydrate recognition domains (CRD). Prototypical galectins have one, and are found as monomers or homodimers (galectin-1, -2, -5, -7, -10, -11, -13, -14 and - 15); the chimeric galectin-3 has one CRD and an amino-terminal domain which can form oligomers; and the tandem repeat galectins (galectin-4, -6, -8, -9 and -12) contain two distinct CRDs connected by an amino acid linker, and are present as monomers or dimers (Cummings et al., 2009; Sciacchitano et al., 2018; Seyrek, Richter and Lavrik, 2019; Liu and Stowell, 2023). Galectin-1 regulates both physiological and disease-related processes, including tissue growth, cellular proliferation, apoptosis, vascularisation, inflammation and carcinogenesis (Perillo et al., 1995; Cousin and Cloninger, 2016; Yu et al., 2023). In inflammation, galectin-1 is generally considered immunosuppressive due to its ability to induce apoptosis of effector T cells, antagonise T cell receptor signalling, block T cell-extracellular matrix interactions, and activate regulatory T cell populations (Perillo et al., 1995; Rabinovich G A et aL, 1999; Liu et al., 2009; Cutine et al., 2021). Galectin-1 also polarises macrophages to an anti-inflammatory “M2” profile, and can restrict reactive oxygen species generation in neutrophils (Correa et al., 2003; Rodrigues et al., 2019). In a tumour microenvironment, this suppression of T cells in particular may contribute to the persistence of tumours. Galectin-1 is implicated in other hallmarks of cancer, including the promotion of tumour angiogenesis, desmoplasia and hypoxia (Ito et al., 2012; Griffioen and Thijssen, 2014; Kuo and Le, 2014; Martinez-Bosch et al., 2014; Stanley, 2014; van Beijnum et al., 2016).

Several preclinical and clinical studies using gal ectin-3 inhibitors for cancer and chronic inflammatory diseases are underway, of which some have been shown or are proposed to bind to galectin-1 (Laderach and Compagno, 2023). However, the extent of therapeutic effect from the additional inhibition of galectin-1 with these molecules is difficult to ascertain due to their weak affinity for galectin-1 vs. galectin-3, and the lack of solid pharmacological characterisation. The role of galectin-1 specifically in cancer is less well studied, although galectin-1 expression is known to vary in different cancer types, and several analyses have demonstrated correlations between cancers with high galectin-1 expression and overall survival, such as in pancreatic cancer and gastric cancer (Thijssen etal., 2015; Liu et al., 2020). Several studies indicate the potential efficacy of galectin-1 inhibitors in pre-clinical settings, including human bone and lung cancer cells in vitro, and nerve sheath tumour xenograft models in vivo (Goud et aL, 2019, 2020; Shih et aL, 2019). Blockade of galectin-1 using a monoclonal antibody resulted in reduced tumour size in immunocompetent mice bearing lung carcinoma or melanoma xenograft tumours (Croci etal., 2014). In murine oral carcinoma models, antibody blockade of galectin-1 reduced tumour volume and increased T cell infiltration into the tumour (Nambiar et a/., 2019). Summary of the invention

The present invention relates in a first aspect to a compound of formula (I)

(i); for use in a method for treatment of a cancer having a higher expression of galectin-1 compared to normal tissue. Here normal tissue means mammalian tissue, such as human tissue, in a subject that does not have cancer.

In an embodiment the cancer is a solid tumor and the solid tumor is reduced by said treatment. In a further embodiment the compound of formula I further inhibits immune-suppressive cytokines, wherein the cytokines comprise at least IL- 17, IL- 10 and IL-6.

In a still further embodiment the compound of formula (I) is 3,4-Dichlorophenyl 3-deoxy- 3-[4-(2-hydroxythiazol-4-yl)-17/-l,2,3-triazol-l-yl]-2-O-methyl-l-thio-a-D-galactopyranoside.

In a further embodiment the cancer is selected from sarcoma, mesothelioma, glioblastoma, carcinosarcoma, adenocarcinoma, adrenocortical cancer, carcinoma and melanoma.

In a still further embodiment the cancer is melanoma.

In a further embodiment the cancer is carcinoma.

In a still further embodiment the cancer is breast invasive carcinoma.

In a further embodiment the cancer is skin cutaneous melanoma.

In a still further embodiment the treatment is by oral administration of the compound of formula (I). Typically, the compound of formula I is administered in a daily dose from lOmg to 2000mg to a human subject to a human subject. In another embodiment the compound of formula I is administered twice daily in a unit dose from 5mg to lOOOmg. In a further embodiment, the compound of formula I is administered in a daily dose from 50mg to 600mg to a human subject. In a still further embodiment the compound of formula I is administered twice daily in a unit dose from 25mg to 300mg. In a second aspect the present invention relates to an oral pharmaceutical composition comprising the compound of formula I (3,4-Dichlorophenyl 3-deoxy-3-[4-(2-hydroxythiazol-4- yl)-17/-l,2,3-triazol-l-yl]-2-O-methyl-l-thio-a-D-galactopyranoside), and optionally a pharmaceutically acceptable additive.

In a further embodiment the oral pharmaceutical composition of the second aspect is selected from a tablet, a pill, a capsule, granules, a solution, or a suspension.

In a third aspect the present invention relates to a method for treatment of a cancer having a high expression of galectin-1 comprising administering a therapeutically effective amount of a compound of formula (I)

(i); to a mammal, such as a human, in need hereof.

Description of Figures 1-5

Figure 1. GB1908 is a galectin-1 inhibitor that reduces cell surface galectin-1 expression on mouse macrophages. Selectivity of GB1908 for human and mouse galectin-1, galectin-3, galec- tin-9N and galectin-9C (A). Extracellular galectin-1 (B) and galectin-3 (C) were measured by flow cytometry on mouse bone-marrow-derived macrophages, after 2 hours incubation in vitro with a range of doses of GB1908 (B). Data shown as percentage change in mean fluorescence intensity (MFI) from control sample. Each dot represents one mouse; bars show mean +/- SEM. Stars indicate significance using one-way repeated measures ANOVA.

Figure 2. Galectin-1 expression is associated with lower survival rates in breast cancer and melanoma, and inhibition of galectin-1 in vivo reduces tumour size. Cancers with the highest galectin-1 expression according to TCGA; n=57-1062 biopsies per cancer type (A). High galec- tin-1 (LGALS1) expression correlates with lower survival rates in patients with breast invasive carcinoma (B) and skin cutaneous melanoma (metastatic) (C). Syngeneic mouse models of breast carcinoma (D) and melanoma (E) were used to assess the effect of GB1908 on tumour volume. Stars in C and D indicate statistical significance at each timepoint, using multiple corrected unpaired t tests. Thick lines with error bars show mean +/-SEM; thin lines show data from each mouse.

Figure 3. Immunomodulatory profiles of galectin-1 and galectin-3 inhibitors GB1908 and GB1211, and Pembrolizumab, in a stromal NSCLC tumour microenvironment model in vitro. Primary human immune cells and fibroblasts, and a NSCLC cell line were co-cultured and treated with a dose range of GB1211 (A), GB1908 (B), or 25 pg/mL Pembrolizumab (C, black line; red line is 10 pM GB1908 for comparison). Biomarkers listed on the x axes; changes in each biomarker are shown as a log-transformed ratio for the drug treated sample (n=3 per dose) in comparison to the control treatment group (grey “envelope”, n=6). Annotated biomarkers indicate at least one concentration of the drug has an effect size >20% and a p value < 0.01 compared to the control group. Prefix “s” indicates soluble biomarker, measured in cell culture supernatant.

Figure 4. GB1908 reduces IL- 17 and IFN gamma production in stimulated mouse and human T cells. T lymphocytes isolated from human whole blood (A, C, E) or mouse spleen (B,D, F) were stimulated with anti-CD3 and anti-CD28, along with a dose range of GB1908 (left column). IL- 17 (A-B), IFN gamma (C-D) and TNF alpha (E-F) measured in supernatants after 48 hours. Bars show mean +/- SEM, each dot representing one donor (n=3). Data shown as % change from control (DMSO) treated cells for each donor. Stars indicate significance using repeated measured one-way ANOVA.

Figure 5. GB1908 reduces cytokine production in the mouse concanavalin-A model of acute inflammation. Summary of the study (A). IL-17A, IFN gamma, IL-6 and TNF alpha were measured in terminal blood samples by LEGENDPlex assay (B-E). Bars show mean +/- SEM, each dot represents one mouse. Stars indicate significance using unpaired t test (vehicle vs GB1908).

Detailed description

The compound of formula I is a galectin-1 specific inhibitor. This inhibitor is highly selective for galectin-1 over galectin-3, gal ectin- 9N and galectin-9C (Figure 1A) and has similar affinities for mouse and human gal ectins (Table 1).

Table 1. Binding affinities of compounds for human and mouse ga- lectins. KD values shown (nM) with SEM if available.

An oral formulation was developed for administration to mice in in vivo studies, making it a useful compound for pre-clinical investigations. Target engagement of galectin-1 was investigated by assessing a reduction in galectin-1 expression on mouse bone marrow-derived macrophages. Treatment with the compound of formula I resulted in a dose-dependent reduction in cell-surface galectin-1 as measured by flow cytometry (Figure IB). Galectin-3 remained unchanged, showing the specificity of this compound (Figure 1C).

Galectin-1 expression is known to vary across different cancer types (Cousin and Clon- inger, 2016). The Cancer Genome Atlas (TCGA) dataset contains gene expression data from cancers and normal tissue samples from a cohort of 11,000 patients, encompassing 33 different tumour types. To investigate which cancers would benefit from galectin-1 inhibitor therapy, TCGA was mined to identify cancers with the highest expression of galectin-1 (LGALS1) (Figure 2A). Metadata in TCGA also includes overall and progression-free survival. Correlations between galectin-1 expression and progression-free survival was analysed for two cancer types: breast invasive carcinoma and skin cutaneous melanoma (metastatic). For both cancers, high galectin-1 expression was associated with lower survival probability (Figure 2B, C).

The involvement of galectin-1 in tumour progression, along with its association with reduced survival probability, indicates that inhibiting galectin-1 could potentially provide therapeutic advantages in treating these types of cancers. To test this hypothesis, the compound of formula I was used in two syngeneic mouse models: breast carcinoma and melanoma. The mouse breast cancer cell line 4T1, or the mouse melanoma adenocarcinoma cell line B16-F10, were cultured and implanted into the mammary fat pads of mice. After 24 hours, mice were treated with either the compound of formula I (30 mg/kg) or vehicle, and tumour size monitored with callipers. In both the breast carcinoma model (Figure 2D) and in the melanoma model (Figure 2E), treatment with compound of formula I reduced tumour volume.

In addition to directly impacting tumour progression mechanisms, galectin-1 is a known regulator of the inflammatory response, particularly T cells, and may also alter the immune response to the tumour. The mechanisms of action of the compound of formula I in the context of the tumour immune microenvironment were investigated using the in vitro BioMAP system (Eurofins Discovery, CA, USA). Co-cultures of human peripheral blood mononuclear cells, human primary dermal fibroblasts and the H1299 non-small cell lung cancer (NSCLC) cell line were stimulated with T cell receptor ligands, to create a model of the stromal tumour microenvironment (“StroNSCLC”). Changes in biomarkers were assessed after 48-hour treatment with the compound of formula I. Treatment with GB1211, a galectin-3 inhibitor currently in trials for NSCLC (Aparisi et al., 2023), and pembrolizumab, a PD-1 inhibitor used for the treatment of a variety of cancers, were also assessed for comparison. The galectin-3 inhibitor GB1211 (Figure 3 A) upregulated several biomarkers including IL-6 and IFN gamma (IFNg) at 10 pM. The compound of formula I however had a different immunomodulatory profile, inhibiting the expression of numerous immune-suppressive proteins, notably IL- 17, IL- 10 and IL-6 at 10 pM (Figure 3B). The compound of formula I also has a distinct profile in comparison to pembroli- zumab (Figure 3C), suggesting a differing mechanism of action that may act as a complimentary therapy. Prior to in vivo studies, further in vitro investigations were used to determine whether galectin inhibitors result in similar changes in cytokine production in mouse and human T cells. T cells cultured from human whole blood and mouse spleen were stimulated with anti-CD3 and anti-CD28 to induce cytokine production, and co-cultured with a dose range of the compound of formula I for 48 hours. The compound of formula I inhibited IL-17 (Figure 4A-B), IFN gamma (Figure 4C-D) and TNF alpha (Figure 4E-F) in a dose-dependent manner in both mouse and human T cells. This data also suggests that the reduction in cytokines observed using the BioMAP system is at least partially due to the effects of the compound of formula I on T cells.

As the compound of formula I showed similar effects at inhibiting cytokines in activated T cells from both mouse and human, an in vivo mouse model of T cell-mediated inflammation was utilized. Concanavalin-A (con- A) induces acute liver injury by directly activating T lymphocytes, which are recruited to the liver (Heymann et al., 2015). Elevated inflammatory cytokines from the resulting inflammatory response can be measured in circulation. The effects of galectin- 1 inhibition on cytokines were assessed in this model by pre-treating mice orally with the compound of formula I or vehicle (control), one hour prior to con- A injection (Figure 5 A). Terminal blood samples were collected for analysis 8 hours after con-A injection. All cytokines measured were upregulated in con-A treated mice in comparison to naive mice that did not receive con-A. IL-17A, IFN gamma, IL-6 and TNF alpha were all reduced in the compound of formula I treated mice in comparison to vehicle treated mice (Figure 5B-E).

The ability of the compound of formula I, a high affinity and selective galectin- 1 inhibitor, to reduce tumour growth in mouse models of cancer, and downregulate several immune- suppressive cytokines across in vitro and in vivo studies have been clearly demonstrated. Data mining of literature was used to generate a comprehensive overview of pathways regulated by galectin- 1, in tumour cells and the surrounding stroma, that promote cancer progression. Galectin- 1 in tumour-associated endothelial cells upregulated angiogenesis; in cancer-associated fibroblasts, galectin- 1 promoted desmoplasia. In tumour cells, galectin- 1 regulated pathways involved in epithelial-to-mesenchymal transition, cancer cell migration and invasion, and metastasis. Several of the signalling pathways regulated by galectin-1 also have downstream effects on the inflammatory response. In both cancer cells and endothelial cells, galectin-1 induces ERK1/2 signalling, which some studies indicate leads to upregulation of NF-KB activity (Dhawan and Richmond, 2002; Huang et al., 2014; Chen etal., 2016). As IL-6 and TNF alpha are upregu- lated by NF-KB, the reduction in these cytokines observed in our in vitro experiments and mouse studies may be due to the suppression of this pathway by the compound of formula I. Galectin-1 also induces SDF-1 production from tumour stroma cells such as pancreatic stellate cells; literature suggests SDF-1 signalling through CXCR4 upregulates IL-6 production (Tang et al., 2008; Lu et al., 2009; Chen et al., 2011; Thomas et al., 2015). The activation of tissue plasminogen activator (tPA) by galectin-1 upregulates plasminogen conversion to plasmin (Roda et al., 2009).

It has been shown that several cytokines known to be immune-suppressive in a tumour microenvironment were reduced with compound of formula I treatment. IL- 17 is implicated in tumour progression in a wide range of cancer types through various mechanisms, including upregulation of cancer cell migration and metastasis, recruitment of myeloid-derived suppressor cells to induce angiogenesis and suppress antitumour immunity, and increase PD-L1 expression (Zhao etal., 2019; Cui et al., 2021; Liao et al., 2023). Increased PD-L1 promotes resistance to immune checkpoint inhibitor therapy, and blocking IL-17A improved efficacy of anti-PD-1 therapy in mouse models of colorectal cancer (Liu et al., 2021). In murine myeloma transplant models, pharmacological inhibition or genetic ablation of IL-17A improves outcomes (Vucko- vic et al., 2019). In breast cancer, IL-17 expression by gamma-delta T cells results in the recruitment of a neutrophil population that supresses cytotoxic-T cells, promoting metastasis (Coffelt et al., 2015; Wu et al., 2020).

Similarly, IL-6 is also associated with poorer outcomes in many cancers. IL-6 drives the JAK/STAT3 signalling pathway, which suppresses tumour-killing NK cells, effector T cells and dendritic cells, whilst upregulating regulatory T cells and myeloid-derived suppressor cells to create an immunosuppressive environment (Johnson, O’Keefe and Grandis, 2018). High IL- 6 also correlates with poor response to atezolizumab (anti-PD-Ll antibody) in advanced breast, kidney and bladder cancers in patients, while in mouse models a combined blockade of PD-L1 and IL-6 receptor improved outcomes compared to atezolizumab alone (Huseni etal., 2023). IL-6 production in tumour cells and associated fibroblasts, endothelial cells and dendritic cells is upregulated by IL- 17, and in murine models an IL-6 blockade reduced Thl7 cells and improved outcomes (Wang et al., 2009; Hailemichael et al. , 2022).

The blockade of immune-suppressive biomarkers by the compound of formula I differs from the galectin-3 inhibitor GB1211, which is showing promise in clinical trials of non-small cell lung cancer (NSCLC) (Aparisi et al., 2023). Galectin-3 expression is highly correlated with poor response to checkpoint inhibitor therapy in NSCLC (Capalbo et al., 2019). Galectin-3 strengthens the interactions between PD-1/PD-L1, reducing the affinity of the immune checkpoint inhibitors pembrolizumab and atezolizumab, an effect reversed by GB1211 (Mabbitt et al., 2023). This unique mechanism of GB1211 in overcoming resistance to checkpoint inhibitor therapy appears specific to galectin-3 inhibition. Additionally, expression of gal ectin- 1 and galectin-3, and correlation with survival, varies widely across different cancers. Research identified breast carcinoma and metastatic skin cutaneous melanoma as cancers with both high galec- tin-1 expression, and a correlation between gal ectin- 1 and poorer survival outcomes, suggesting these cancers may benefit from galectin-1 inhibitor therapy. Consideration of the cancer type in which these drugs are tested may improve the likelihood of successful clinical trials.

The term “treatment” and “treating” as used herein means the management and care of a patient for the purpose of combating a condition, such as a disease or a disorder. The term is intended to include the full spectrum of treatments for a given condition from which the patient is suffering, such as administration of the active compound to alleviate the symptoms or complications, to delay the progression of the disease, disorder or condition, to alleviate or relief the symptoms and complications, and/or to cure or eliminate the disease, disorder or condition as well as to prevent the condition, wherein prevention is to be understood as the management and care of a patient for the purpose of combating the disease, condition, or disorder and includes the administration of the active compounds to prevent the onset of the symptoms or complications. The treatment may either be performed in an acute or in a chronic way. The patient to be treated is preferably a mammal; in particular, a human being, but it may also include animals, such as dogs, cats, cows, sheep and pigs. The term "a therapeutically effective amount" of a compound of formula (I) of the present invention as used herein means an amount sufficient to cure, alleviate or partially arrest the clinical manifestations of a given disease and its complications. An amount adequate to accomplish this is defined as "therapeutically effective amount". Effective amounts for each purpose will depend on the severity of the disease or injury as well as the weight and general state of the subject. It will be understood that determining an appropriate dosage may be achieved using routine experimentation, by constructing a matrix of values and testing different points in the matrix, which is all within the ordinary skills of a trained physician or veterinary.

In a still further aspect, the present invention relates to a pharmaceutical composition comprising the compound of formula (I) and optionally a pharmaceutically acceptable additive, such as a carrier or an excipient.

As used herein “pharmaceutically acceptable additive” is intended without limitation to include carriers, excipients, diluents, adjuvant, colorings, aroma, preservatives etc. that the skilled person would consider using when formulating a compound of the present invention in order to make a pharmaceutical composition.

The adjuvants, diluents, excipients and/or carriers that may be used in the composition of the invention must be pharmaceutically acceptable in the sense of being compatible with the compound of formula (I) and the other ingredients of the pharmaceutical composition, and not deleterious to the recipient thereof. It is preferred that the compositions shall not contain any material that may cause an adverse reaction, such as an allergic reaction. The adjuvants, diluents, excipients and carriers that may be used in the pharmaceutical composition of the invention are well known to a person skilled within the art.

As mentioned above, the compositions and particularly pharmaceutical compositions as herein disclosed may, in addition to the compounds herein disclosed, further comprise at least one pharmaceutically acceptable adjuvant, diluent, excipient and/or carrier. In some embodiments, the pharmaceutical compositions comprise from 1 to 99 % by weight of said at least one pharmaceutically acceptable adjuvant, diluent, excipient and/or carrier and from 1 to 99 % by weight of a compound as herein disclosed. The combined amount of the active ingredient and of the pharmaceutically acceptable adjuvant, diluent, excipient and/or carrier may not constitute more than 100% by weight of the composition, particularly the pharmaceutical composition.

In some embodiments, only one compound as herein disclosed is used for the purposes discussed above.

In some embodiments, two or more of the compounds as herein disclosed are used in combination for the purposes discussed above.

The composition, particularly pharmaceutical composition comprising a compound set forth herein may be adapted for oral, intravenous, topical, intraperitoneal, nasal, buccal, sublingual, or subcutaneous administration, or for administration via the respiratory tract in the form of, for example, an aerosol or an air-suspended fine powder. Therefore, the pharmaceutical composition may be in the form of, for example, tablets, capsules, powders, nanoparticles, crystals, amorphous substances, solutions, transdermal patches or suppositories.

Further embodiments of the process are described in the experimental section herein, and each individual process as well as each starting material constitutes embodiments that may form part of embodiments.

The above embodiments should be seen as referring to any one of the aspects (such as ‘method for treatment’, ‘pharmaceutical composition’, ‘compound for use as a medicament’, or ‘compound for use in a method’) described herein as well as any one of the embodiments described herein unless it is specified that an embodiment relates to a certain aspect or aspects of the present invention.

All references, including publications, patent applications and patents, cited herein are hereby incorporated by reference to the same extent as if each reference was individually and specifically indicated to be incorporated by reference and was set forth in its entirety herein.

All headings and sub-headings are used herein for convenience only and should not be construed as limiting the invention in any way.

Any combination of the above-described elements in all possible variations thereof is encompassed by the invention unless otherwise indicated herein or otherwise clearly contradicted by context. The terms “a” and “an” and “the” and similar referents as used in the context of describing the invention are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context. The terms “a” and “an” means “one or more” and is interchangeable with “at least one”.

Recitation of ranges of values herein are merely intended to serve as a shorthand method of referring individually to each separate value falling within the range, unless otherwise indicated herein, and each separate value is incorporated into the specification as if it were individually recited herein. Unless otherwise stated, all exact values provided herein are representative of corresponding approximate values (e.g., all exact exemplary values provided with respect to a particular factor or measurement can be considered to also pro-vide a corresponding approximate measurement, modified by "about," where appropriate).

All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context.

The use of any and all examples, or exemplary language (e.g., “such as”) provided herein, is intended merely to better illuminate the invention and does not pose a limitation on the scope of the invention unless otherwise indicated. No language in the specification should be construed as indicating any element is essential to the practice of the invention unless as much is explicitly stated.

The citation and incorporation of patent documents herein is done for convenience only and does not reflect any view of the validity, patentability and/or enforceability of such patent documents.

The term “and/or” as used herein is intended to mean both alternatives as well as each of the alternatives individually. For instance, the expression “xxx and/or yyy” means “xxx and yyy”; “xxx”; or “yyy”, all three alternatives are subject to individual embodiments.

The description herein of any aspect or embodiment of the invention using terms such as “comprising”, “having”, “including” or “containing” with reference to an element or elements is intended to provide support for a similar aspect or embodiment of the invention that “consists of’, “consists essentially of’, or “substantially comprises” that particular element or elements, unless otherwise stated or clearly contradicted by context (e.g., a composition described herein as comprising a particular element should be understood as also describing a composition consisting of that element, unless otherwise stated or clearly contradicted by context). This invention includes all modifications and equivalents of the subject matter recited in the aspects or claims presented herein to the maximum extent permitted by applicable law.

The present invention is further illustrated by the following examples that, however, are not to be construed as limiting the scope of protection. The features disclosed in the foregoing description and in the following examples may, both separately and in any combination thereof, be material for realizing the invention in diverse forms thereof.

EXPERIMENTAL

The compound of formula I as described herein may be prepared as disclosed in W00221/001528 example 9. Herein the compound of formula I is also termed GB1908.

Another compound 5-Bromopyri din-3 -yl 3-deoxy-3-[4-(3,4,5-trifluorophenyl)-lH- l,2,3-triazol-l-yl]-l-thio-a-D-galactopyranoside and having the structure may be prepared as disclosed in WO2021/122719. Herein this compound is also termed GB1211.

C57BL/6 mice were purchased from Charles River. C57BL/6 mice were euthanised by approved methods, and femurs and tibias dissected and cleaned. Epiphyses were removed from each bone in turn, and bone marrow cells collected by flushing each bone with a needle and syringe filled with sterile PBS, pooling all marrow cells from each mouse. Cells were filtered through a 100 pm cell strainer and cultured in 6-well cell culture plates in media (DMEM + 10% FBS + 1% penicillin-streptomycin + 2mM L-glutamine + 20 ng/mL M-CSF) at 37 °C with 5% C0₂. On days 4 and 6 after isolation, cells were washed twice with sterile PBS and fresh media added.

Experiments were carried out on cells after 7 days of culture. Cells were washed and detached from wells using trypsin-EDTA and a cell scraper and resuspended in DMEM-only media. A viable cell count was performed using Trypan Blue, and media volume adjusted to generate cell suspensions of 1 million viable cells per mL. Cells were transferred to 2 mL Eppen- dorfs (500 pL per tube) containing the compound of formula I or diluted DMSO (control). Cells were incubated at 37 °C for 2 hours, with gentle shaking (300 rpm).

After incubation, cells were stained with Zombie-UV viability dye (1 : 1000 in PBS) and incubated at room temperature in the dark for 30 minutes. Cells were centrifuged at 300 x g for 5 minutes and supernatants discarded. Into the residual volume of the tube, 2 pL FC-block was added, and cells gently resuspended and incubated at room temperature for 5 minutes. Directly into the residual volume/Fc block, 1 pL of each antibody was added: CD1 lb (Brilliant Violet 605), F4/80 (AlexaFluor 700), Galectin-3 (FITC), Galectin-1 (PE). Samples were incubated at 4°C for 20 minutes. 1 mL FACS cell staining buffer was added to each sample, and samples centrifuged at 300 x g for 5 minutes. Samples were resuspended in 300 pL FACS cell staining buffer and stored on ice until analysis. Samples were analysed using a BD LSR Fortessa (5 laser) analyser with BD FACSDiva™ software.

Syngeneic mouse cancer models

Mice were housed in individually ventilated cages at 22°C with relative humidity 45- 65%, in 12-hour light/dark cycles.

The murine breast carcinoma cell line 4T1 was cultured in the medium RPMI-1640, 10% fetal calf serum, 1% penicillin/streptomycin, with incubation at 37°C and 5% CO2. The murine melanoma adenocarcinoma cell line B16-F10 was cultured in DMEM high Glutamax 1, 10% fetal calf serum, 1% penicillin/streptomycin, with incubation at 37°C and 10% CO2. When confluent, 10,000 4T1 cells in 100 pL PBS were implanted into the left mammary fat pad of female BALB/c mice (n=16), or 20,000 B16-F10 cells were implanted into female C57BL/6N mice (n=16). Starting one day after tumour cell implantation, mice in both tumour models were treated according to the same dosing schedule. Vehicle group (n=8) received 5 mL/kg PBS by intraperitoneal dosing, three times, 3-4 days apart. GB1908 group (n=8) received 30 mg/kg compound of formula I in PEG 300 + Solutol HS15 + Tween 20 (ratio 84/15/1) at 10 mL/kg volume by oral gavage, twice daily. Tumour size was assessed twice weekly by calliper measurements and calculated as so: width² x length/2.

BioMAP StroNSCLC

The non-small cell lung cancer line NC1-H1299 and human neonatal dermal fibroblasts were cultured in 96-well plates until confluent. Primary human peripheral blood mononuclear cells (PBMCs) were pooled from multiple donors (n=3-6) and added to the plates. GB1211 and the compound of formula I were prepared in DMSO and added to wells. Diluted DMSO-only conditions were used as controls. Each compound was tested in triplicate. After 1 hour, sub-mitotic levels of super antigens were added to wells. These antigens act via the T cell receptor to prime T cells but not drive proliferation. Cells were incubated for 48 hours. Direct ELISA was used to measure cell-associated and cell membrane biomarkers. Soluble biomarkers were measured using HTRF© detection, bead-based multiplex immunoassay or capture ELISA.

Human and mouse T cell assays

For human assays, T cells were isolated from whole blood obtained from donors from the Sygnature Blood Donor panel, using EasySep™ Human T cell Isolation Kit. For mouse assays, T cells were isolated from the spleens of 6-12-week-old male BALB/c mice, sourced from Charles River UK, using the EasySep™ Mouse T cell Isolation Kit. Isolated cells were resuspended in media (RPMI 1640 with 10% inactivated fetal bovine serum, 1% penicillinstreptomycin, 1% GlutaMax, and 55 pM beta-mercaptoethanol in mouse cell media only). 96- well plates were coated with 5 pg/mL anti-CD3 antibody. GB1211 and the compound of formula I were prepared in DMSO and added to wells at desired concentrations. Cells were added to wells at a concentration of 100,000 cells/well. Cells were incubated for 30 minutes (37°C, 5% CO2) before anti-CD28 antibody was added to a final concentration of 1 pg/mL. Cells were incubated for a further 48 hours. Supernatants were harvested and stored at -80°C. Human and mouse 5-plex U-Plex kits for analysis of TNFa, IL-2, IL-6, IL-17A and IFNy were obtained from Meso Scale Discovery for cytokine analysis, and plates read using the MESO QuickPlex SQ120MM instrument. Data was analysed using the MSD Workbench software.

Concanavalin-A mouse models Male C57BL/6 mice received the compound of formula I (30 mg/kg) or GB1211 (10 mg/kg) dissolved in vehicle, or vehicle only (84% PEG + 15% Solutol HS15 + 1% Tween-20) via oral gavage at 10 mL/kg. After 1 hour, mice received con-A (Sigma, C5275; 9 mg/kg, dissolved at 1.5 mg/mL in sterile saline) intravenously via the peripheral vein. Mice were monitored throughout the study and were euthanised prior to the end of the study if moderate clinical signs were breached. At 8 hours post-con- A injection, mice were culled by anaesthetic overdose (intraperitoneal), and blood collected from the vena cava. Blood samples were centrifuged at 400 x g with no brake for 10 minutes, and plasma removed and stored at -20 °C until analysis. Plasma cytokines were measured using BioLegend® LEGENDPlex™ assay (Mouse Inflammation Panel), as per the manufacturer’s protocols. Data was acquired using a BD LSR Fortessa (5 laser) analyser. After acquisition, data was analysed using BioLegend® Qognit software.

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Claims

WE CLAIM:

1. A compound of formula (I)

(i); for use in a method for treatment of a cancer having a higher expression of galectin-1 compared to normal tissue.

2. The compound for use of claim 1 wherein the cancer is a solid tumor and the solid tumor is reduced by said treatment.

3. The compound for use of claim 2 wherein the compound of formula I further inhibits immune- suppressive cytokines, wherein the cytokines comprise at least IL- 17, IL- 10 and IL-6.

4. The compound for use of any one of claims 1-3 wherein the compound of formula (I) is 3,4- Dichloropheny 1 3 -deoxy-3 - [4-(2-hydroxythiazol-4-yl)- 1 H- 1 ,2,3 -triazol - 1 -yl] -2-O-methyl- 1 - thio-a-D-galactopyranoside.

5. The compound for use of any one of claims 1-4 wherein the cancer is selected from sarcoma, mesothelioma, glioblastoma, carcinosarcoma, adenocarcinoma, adrenocortical cancer, carcinoma and melanoma.

6. The compound for use of any one of claims 1-5 wherein the cancer is selected from carcinoma and melanoma.

7. The compound for use of any one of claims 1-6 wherein the cancer is selected from breast invasive carcinoma and skin cutaneous melanoma.

8. The compound for use of any one of claims 1-7 wherein the treatment is by oral administration of the compound of formula (I).

9. The compound for use of claim 8 wherein the compound of formula I is administered in a daily dose from lOmg to 2000mg to a human subject.

10. The compound for use of claim 8 or 9 wherein the compound of formula I is administered in twice daily in a unit dose from 5mg to lOOOmg.

11. An oral pharmaceutical composition comprising the compound of formula I of claim 1, and optionally a pharmaceutically acceptable additive.

12. The oral pharmaceutical composition of claim 10 selected from a tablet, a pill, a capsule, granules, a solution, or a suspension.

13. A method for treatment of a cancer having a high expression of galectin-1 comprising administering a therapeutically effective amount of a compound of formula (I)

(i); to a mammal, such as a human, in need hereof.