Glutaminase, methods and uses thereof

Abstract

The present disclosure relates to a glutaminase for use in the treatment of a condition susceptible of being improved or prevented by a decrease of systemic glutamine bioavailability, in particular for use in the treatment of a glioma. The present disclosure also describes a pharmaceutical composition comprising a glutaminase, preferably a human-recombinant glutaminase (GLS1).

Description

D E S C R I P T I O NG LUTAMINASE, METHODS AND USES THEREOFTECH NICAL FIELD

[0001] The present disclosure relates to a therapy for conditions susceptible of being improved or prevented by a decrease of glutamine bioavailability, such as cancer, in particular brain cancer.BACKGROUND

[0002] Gliomas stand as the prevailing primary tumors within the brain, accounting for 24% of all central nervous system (CNS) tumors [1], Among the wide spectrum of gliomas, glioblastomas (GBMs), classified as high-grade tumors (WHO grade IV) account for 14.2% of all CNS tumors and 50.1% of all malignant CNS tumors [1], GBM exhibit a remarkably aggressive nature due to their challenging cerebral location, high proliferation rate, increased heterogeneity, and infiltrative / invasive capacity [2], According to the current glioma classification (WHO 2021), GBMs correspond to IDH- wild-type diffuse and astrocytic gliomas that present at least one of the following features: microvascular proliferation, TERT promoter mutation, EGFR gene amplification or 7 + / 10 - chromosome copy number changes [3], With the updated classification, GBM patients have a median overall survival of only 8 months [1,4], The current clinical approach involves a multidisciplinary strategy, involving maximal tumor resection, followed by adjuvant radiotherapy and chemotherapy (temozolomide, TMZ) [5], However, complete surgical removal is not possible because of the high invasiveness degree, leading to later disease progression or recurrence [6], Therefore, the search for new biomarkers urges, in order to find specific targets and predictors of patient outcomes and therapy response.

[0003] In the last years, cancer metabolism has acquired a central position in oncology research, since metabolic adaptation is crucial to sustain tumor growth [7], In the case of malignant gliomas, the glutaminergic cycle suffers a remodeling due to the abrogation of glutamine synthetase [8,9] and overexpression of glutamine transporters ASCT2

[0010] and SNAT3

[0011] , Thus, these neoplasms present a phenotype of high glutamine dependence, since they are unable to produce it, acting as 'glutamine traps' by importing glutamine from the tumor microenvironment and blood stream [2,12], Nevertheless, these tumors are able to fully metabolize glutamine, since they express glutaminase isoform 1 (GLS1)

[0013] , using this amino acid as a carbon (C) and nitrogen (N) source [7,14,15], These adaptations may reflect in the use of glutamine for bioenergetics, acting as the main glucose substitute [7],

[0004] Even though the amount of knowledge generated by cancer metabolism research is increasing, its application to cancer therapy has been scarce. Different strategies have been addressed such as the development of inhibitors to impair glutamine and glutamate transport and the inhibition of some key enzymes, as glutaminase and glutamine synthetase

[0017] , However, the abovementioned tactics are challenging to apply clinically. Moreover, brain cancer treatment presents a major anatomical and molecular obstacle, the blood brain barrier (BBB)

[0018] ,

[0005] Document US 8.465,736 B2

[0031] discloses a glutamine targeting approach for use in the treatment of colorectal cancer, but not in GBM, aiming a systemic application of a mix enzyme exhibiting glutaminase and asparaginase activities. However, the clinical success of this approach was not confirmed, and it may be mainly due to the fact that normal cells use asparaginase as a precursor of glutamine

[0032] , therefore this approach represents a huge aggression also to normal body cells.

[0006] These facts are disclosed in order to illustrate the technical problem addressed by the present disclosure.GENERAL DESCRI PTION

[0007] The present disclosure relates to a glutaminase for use in the treatment of a condition susceptible of being improved or prevented by a decrease of glutamine bioavailability, preferably a decrease of systemic glutamine bioavailability, preferably wherein the condition is a glioma. In a more preferred embodiment, the glutaminase is glutaminase GLS1.

[0008] GLS1 Glutaminase (Glutaminase kidney isoform, mitochondrial) is better identified by the primary accession number = 094925 and is available in UniProt database094925 and entry name: GLSK_HUMAN.

[0009] GLS1 Glutaminase (Glutaminase kidney isoform, mitochondrial) is also identified by the Secondary accession numbers: Q9UL05, Q9UL06, Q9UL07 and Q9UN40.

[0010] . In an embodiment, the glioma is a glioblastoma.

[0011] In particular, the present disclosure relates to a composition comprising a glutaminase, preferably GLS1, for use in the treatment of a glioma, more in particular a glioblastoma, preferably wherein the glutaminase is systemically administered. Surprisingly, the systemic treatment with GLS1 decreases the glutamine bioavailability, affecting the GBM cells' metabolism and survival, with reduced impact on healthy cells. Since the other cells in the human body retain the ability to synthesize glutamine, this strategy affects preferentially the GBM cells. Moreover, this strategy overcomes the blood-brain barrier (BBB) hurdle, since it is a systemic treatment but able to impact brain cancer cells. Until now the majority of therapies targeting the glutamine / glutamate metabolism in GBM focus on inhibition of glutaminolysis enzymes [22-26], overexpression or inhibition of glutamate transporters [27-29], but none was focused on decreasing the systemic glutamine availability. Furthermore, these metabolism- targeted strategies have been challenging to apply clinically so far, due to drugs' adverse effects (e.g., neurotoxicity) and difficulties in overlapping the BBB hurdle [18,30],

[0012] The composition of the present disclosure showed to be effective in affecting the GBM cells' metabolism and survival, even without the co-administration of cytotoxic and / or cytostatic compounds.

[0013] In an embodiment for better results, the glutaminase is a human-recombinant glutaminase, preferably glutaminase GLS1.

[0014] In an aspect, the present disclosure relates to a pharmaceutical composition comprising a glutaminase and a pharmaceutically acceptable carrier for use in the treatment of a condition susceptible of being improved or prevented by a decrease ofsystemic glutamine bioavailability, wherein the condition is a glioma, and preferably the glutaminase is glutaminase GLS1

[0015] In an embodiment, the pharmaceutical composition is free of cytotoxic and / or cytostatic compounds.

[0016] In an embodiment, the dosage amount of the pharmaceutical composition ranges from 1 pg / g - 50 pg / g; preferably 3 pg / g -30 pg / g; more preferably 5 pg / g-20 pg / g; even more preferably 7 pg / g-14 pg / g.

[0017] In an embodiment, the pharmaceutical composition is in unit dosage form to provide a dosage (drug dosage per weight of patient) ranging from 1 pg / g - 50 pg / g (weight glutaminase / weight patient); preferably 3 pg / g -30 pg / g; more preferably 5 pg / g-20 pg / g; even more preferably 7 pg / g-14 pg / g.

[0018] In an embodiment, the pharmaceutical composition is administered every 72 hours; preferably every 48 hours; more preferably every 24 hours.

[0019] In an embodiment, the composition may be administrated in a single dose. Preferably, the daily form consists of an intravenous dosage, intraperitoneal dosage, oral dosage, intrathecal dosage, or combinations thereof), preferably comprising a definitive amount of glutaminase (preferably GLS1), the whole of which is intended to be administered as a single dose.

[0020] In an embodiment, the condition is a cancer. In a further embodiment, the cancer is a brain cancer. In a yet further embodiment, the brain cancer is a glioma. In a preferred embodiment, the glioma is a glioblastoma.

[0021] In an embodiment, the concentration of glutaminase ranges from 0.05 to 0.40 ng / pL, preferably from 0.14 ng / pL to 0.28 ng / pL.

[0022] In an embodiment, the composition is systemically administered.

[0023] In a further embodiment the composition is administered by intravenous administration, intraperitoneal administration, oral administration, intrathecal administration, or combinations thereof.

[0024] In an embodiment for better results, the composition is administered by intravenous administration or intraperitoneal administration.

[0025] In an embodiment, the pharmaceutical composition further comprises an active ingredient, in particular a chemotherapy agent, a radiotherapy agent, an imaging agent, or combinations thereof.

[0026] An aspect of the present disclosure relates to the use of a glutaminase for the manufacture of a medicament for the treatment of cancer.

[0027] In an aspect, the disclosure relates to a method for treating a cancer in a subject, the method comprising administering a glutaminase. In an embodiment, the glutaminase is administered systemically. In a further embodiment, the administration is intravenous administration, oral administration, intrathecal administration, or combinations thereof.BRI EF DESCRI PTION OF THE DRAWI NGS

[0028] The following figures provide preferred embodiments for illustrating the disclosure and should not be seen as limiting the scope of invention.

[0029] Figure 1A-1B: Embodiment of results of in vitro cell treatment with GLS1. Cell death was assessed in GBM cell lines U251 (A) and U- 87MG (B) after treatment; Cell proliferation in U251 (C) and U-87MG (D) after being exposed to GLS1. Invasion of U251 (E) and U-87MG (F) after treatment with different concentrations of GLS1. All data are represented as mean ± SD. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

[0030] Figure 2A-2B: Embodiment of results of the effect of GLSl-therapy in overall survival and slows body weight loss (cachexia) in a GBM intracranial mice model. (A) Kaplan-Meier overall survival curves of mice (p = 0.0391; Log-rank test). (B) Weight curves after tumor implantation. The dot lines represent the treatment days, i.e., days when GLS1 was administered. (C) Tumor volume in mm3. (D) Multivariate analysis (PLS- DA) of the 1H-NMR spectra acquired from the blood sera (Euthanasia of control group at day 30 and GLSl-treated group at day 41).

[0031] Figure 3: Embodiment of results of hematoxylin and eosin staining of liver and kidney sections collected from animals without treatment and GLSl-treated animals. Kidney sections presenting cortex and medullar regions (magnification 40 x), amplification of cortex region (magnification 100 x). Liver sections presenting hepaticparenchyma and functional structures (magnification 40 x), amplification of hepatic lobules, highlighting centrolobular veins and portal structures (magnification 100 x).

[0032] Figure 4A-4C. GLSl-therapy increases overall survival (OS) in a dose-depending manner and slows body weight loss (cachexia) in a GBM intracranial mice model. U- 87MG cells were orthotopically injected in the brain of NSG mice: n=5 mice for control group, n=5 mice for dose 1 (0.14 ng / pL) (which corresponds to a dosage in the mice of 7 pg / g (weight glutaminase / weight mice)) and n=7 mice for dose 2 (0.28 ng / pL) per group) (which corresponds to a dosage in the mice of 14 pg / g). (A) Kaplan-Meier OS curves of mice (p = 0.0212; Log-rank test). Control median OS: 27 days; dose 1 median OS: 34.5 days; and dose 2 median OS: 48.5 days. (B) Weight curves after tumor implantation. The dot lines represent the treatment days. Blood collection was performed at four distinct time points: week -1 (one week before surgery), week 1 (6h after the first treatment and 1 week after tumor implantation), week 4 (halfway through the treatment) and euthanasia. (C) Multivariate analysis (PCA) of the 1H-NMR spectra acquired from the blood sera. (D) PLS-DA analysis of the 1H-NMR spectra acquired from the blood sera.DETAILED DESCRI PTION

[0033] The present disclosure relates to a glutaminase for use in the treatment of a condition susceptible of being improved or prevented by a decrease of glutamine bioavailability, in particular for use in the treatment of a cancer, more particularly a glioma. The present disclosure also describes a pharmaceutical composition comprising a glutaminase, preferably a human-recombinant glutaminase (GLS1).

[0034] It was surprisingly found that the systemic administration of glutaminase (GLS1) deplete glutamine bioavailability in the peripheral blood and leads to starvation for GBM cells. It was also observed that this approach is conceptually safe for healthy cells as they can produce glutamine. Additionally, since the therapy is applied to the peripheral blood, there is no need to cross the BBB. Unlike small-molecule drugs that have failed in clinical trials by targeting intracellular glutamine metabolism, GSL1 biologies do not needto enter cells, thus avoiding the major anatomical hurdle in brain therapy, posed by the BBB.

[0035] The present disclosure relates to a systemic treatment where the biologic drug acts in the bloodstream, depleting the bioavailability of glutamine and ultimately affecting the viability and survival capacity of tumor cells. Normal cells will be protected from the impact of this therapy as they retain the ability to synthesize glutamine. This makes the present strategy safer than previous solutions more relevant. By impacting the glutamine bioavailability in the blood, the present disclosure overcomes the need for drugs capable of crossing the BBB, a main hurdle in brain tumors' clinical management.

[0036] The present disclosure represents a novel therapeutic strategy to combat GBM based on systemic limitation of glutamine bioavailability without need to cross bloodbrain barrier (BBB). This therapeutics strategy results in starvation of GBM cancer cells, limiting its growth and is achieved by systemic administration of Glutaminase (GSL1). The rational strategy is based on the non-dependence of normal healthy cells from glutamine since they can synthesize it. Additionally, acting systemically in bloodstream, this approach bypasses the need to cross BBB, the main anatomical hurdle in brain tumour therapy.EXAMPLE 1

[0037] In an embodiment, decreased glutamine bioavailability by a systemic treatment with glutaminase, preferably GLS1, reduces GBM cells survival and proliferative capacity, in vitro.

[0038] GBM are characterized by glutamine dependence, as previously confirmed

[0016] , Therefore, the effect on cell features of a decreased glutamine bioavailability, upon GLS1 treatment, was assessed. Two concentrations of GLS1 were tested, 0.07 and 0.14 ng / pL. The treatment with GLS1 induced cell death in the two different GBM cell lines tested: U251 displayed significantly increased cell death at 48 h of exposure at both concentrations (Figure 1 A), while the highest GLS1 concentration affected U-87MGviability at 24 and 48 h (Figure 1 B). In an embodiment, cell death was assessed by annexin V / propidium iodide (PI) staining by flow cytometry in U251 (Figure 1 A) and U- 87MG (Figure 1 B) exposed to GLS1 (0.07 and 0.14 ng / pL). Regarding cell proliferation (Figure 1 C and D) only U-87MG cell line was affected, with decreased cell proliferation upon exposure to 0.14 ng / pL GLS1 at 48 and 96 h (with a tendency at 72 h) (Figure 1 D). In an embodiment, cell proliferation was assessed by the WST-1 colorimetric assay in U251 and U-87MG cells exposed to GLS1 using the indicated concentrations. Moreover, when U-87MG cells were exposed to 0.14 ng / pL of GLS1 there was an increased migration to the bottom well, due to the low glutamine availability in the upper well after hydrolysis by GLS1 (Figure 1 F). This is clearer when cells were cultured with less glutamine (500 pM), which was more quickly consumed, inducing cell migration to the bottom well. In the case of U251 cell line, there are no significant differences (Figure 1 E). In an embodiment, Invasion was evaluated in a transwell assay.

[0039] Surprisingly, decreased glutamine bioavailability by a systemic GLS1 therapy significantly increases the overall survival (OS) and decreases cachexia in an in vivo murine model. In an embodiment, a GBM orthotopic NSG mice model was established, and GLSl-therapy (0.14 ng / pL of glutaminase) was intraperitoneal administered two times per week (every 3 days). A significant increase in overall survival was observed in mice treated with GLS1 (median OS: 41 days), comparing to the control mice (median OS: 30 days) (Figure 2 A). It was also observed a slower body weight loss, due to cancer cachexia, in mice treated with GLS1 (Figure 2 B). There is also a tendency to decrease tumour volume in the mice treated group (Figure 2 C). It is important to emphasize that mice were sacrificed when they reached >20% of body weight loss, and GLSl-treated mice lived more than control mice.

[0040] In an embodiment, tumour volume in mm3was calculated using the formula: Tumour volume = (length x width2) / 2. Longer (length) and shorter (width) axis of the tumours were measured in the coronal plane of the brain, in the middle region of the tumour.

[0041] Moreover, therapy-response can be evaluated through NMR, since there were statistically different metabolic profiles midst treatment. In an embodiment, blood serum was collected during the experiment and1H-NMR spectra were obtained. Duringthe experiment it is possible to observe a separation of both mice populations, sera from control mice vs sera from GLSl-treated mice, mainly at the midst of the treatment, at week 4 (Figure 2 D), indicating a difference in the metabolic profiles of both groups. At euthanasia time point, the metabolic profiles were similar, however, the OS of GLSl- treated group is longer, as the control mice were sacrificed around 30 days after GBM induction while GLS1 treated mice were sacrificed around 41 days after induction.

[0042] Surprisingly, mainly the malignant glioma cells were affected by the systemic treatment using GLS. Importantly, no morphological alterations were observed in the liver and kidney of mice (Figure 3), suggesting that GLS1 therapy has reduced systemic toxic effects. In an embodiment, liver and kidney representative sections (4 pm) were stained with hematoxylin and eosin (H&E) from Control and GLSl-treated mice to evaluate the morphological alterations.

[0043] Surprisingly, a systemic GLS1 therapy, besides taking advantage of glutamine addiction and deadly disturb GBM cells, it also protects normal cells from cytotoxicity since these cells retain the ability of synthesizing glutamine, and importantly it overcomes BBB, the main anatomical and molecular obstacle to access brain.

[0044] In an embodiment, the impact of GLS1 in cell features was evaluated to understand the efficacy of the disclosed treatment, including in cell death and proliferation. The in vitro results showed that GLS1 induced increased cell death and decreased cell proliferation (Figure 1). Moreover, there was increased migration of U- 87MG when exposed to the highest GLS1 concentration, due to quick consume of glutamine (Figure 1). The orthotopic mouse model of GBM tested showed an increase in OS with the GLS1 treatment, decreased tumor volume, along with slower cachexia (Figure 2). Moreover, during the treatment it is possible to observe different metabolic profiles of peripheral blood serum between control vs GLSl-treated populations. This indicates that blood serum analysis by NMR is a way to monitor the therapy-response to GLSl-therapy. NMR has been pointed out as a relevant analytical technique, which can constitute a powerful tool in patients monitoring during follow up to evaluate therapy response and anticipate the disease advance, allowing more timely interventions

[0012] ,

[0045] The systemic use of GLS1 is the first attempt to affect GBM cancer cells and protect normal cells, by targeting GBM metabolic needs for glutamine and taking advantage of their inefficacy of synthesizing glutamine.

[0046] In an embodiment, GLS1 systemic individual conditioning reduces glutamine bioavailability. On its turn, reduced glutamine levels decrease GBM cells survival and proliferation.

[0047] In an embodiment, systemic reduced glutamine levels increase overall survival and decrease cachexia.

[0048] In an embodiment, systemic individual conditioning overcomes BBB difficulties for drug delivery, since the desired effect is in the peripheral blood the natural carrier of nutrients including glutamine to the brain tumor. Importantly, the approach presents reduced effects on normal cells.

[0049] In an embodiment of cell culture, two commercial GBM cell lines, U-87 MG (HTB- 14, American Type Culture Collection (ATCC)) and U251 (09063001, European Collection of Authenticated Cell Cultures (ECACC)), were used and cultured in Dulbecco's modified Eagle medium F-12 (DMEM / F-12; 11330-032, Gibco, Life Technologies), supplemented with 10% (v / v) fetal bovine serum (FBS; P40-37500, PAN Biotech), 1% (v / v) Antibiotic- Antimycotic (AA; P06-07300, PAN Biotech) and 50 pg / mLgentamicin (15750-060, Gibco, Life Technologies). All cells were maintained in a humidified environment of 5% CO2 at 37°C. Cells were cultured until an optical confluence of 75-100% and then detached with 0.05% (v / v) trypsin-EDTA lx (25300-054, Invitrogen, Thermo Fisher Scientific), at 37°C for approximately 5 min, and split to new plates according to the experimental procedures. Before in vitro assays, cells were washed with phosphate buffer saline (PBS) lx and synchronized under starvation (FBS-free culture medium) overnight. During exposure to experimental conditions, cells were maintained in DMEM / F-12 with 1% (v / v) FBS. Experimental conditions comprised two GLS-1 (71102, BPS Biosciences) concentrations: 0.07 ng / pL and 0.14 ng / pL.

[0050] In an embodiment, cell death was analyzed by flow cytometry. To analyze the effects of the experimental conditions on cell viability, GBM cell lines were seeded in 24- well plates (1 x 105cells / well). After a 24 h exposure to experimental conditions (i.e.,two distinct GLS-loncentrations: 0.07 ng / pL and 0.14 ng / pL), the conditioned culture medium (supernatant) was collected, and adherent cells were detached with 0.05% (v / v) trypsin-EDTA. Cells and supernatant were collected in the same tube and centrifuged at 155 x g for 3 min. Then, cell pellets were incubated with 0.5 pL annexin V-fluorescein isothiocyanate labeled (annexin V-FITC; 640906, BioLegend), in annexin V binding buffer lx (10mM HEPES (pH 7.4; 391333, Millipore), 140 mM sodium chloride (NaCI; 106404, Merck), 2.5 mM calcium chloride (CaC ; 449709, Sigma-Aldrich) for 15 min in the dark, at room temperature. Cells were resuspended in 200 pL PBS lx / 0.1% (v / w) bovine serum albumin (BSA; A9647, Sigma-Aldrich) and centrifuged at 155 x g for 2 min. The remaining pellets were resuspended in annexin V binding buffer lx and 2.5 pL of 50 pg / mL propidium iodide (PI; P4170, Sigma-Aldrich). Samples were analyzed by flow cytometry in a FACScalibur (Becton Dickinson) and data were treated using FlowJo X 10.0.7 (http: / / flowjo.com) software.

[0051] For the scope and interpretation of the present disclosure, "room temperature" relates to a temperature ranging from 20 to 25°C, preferably 21-22°C.

[0052] In an embodiment, the WST-1 based colorimetric assay (11 644807 001, Roche) was performed to assess cell proliferation. Cells were seeded in 96-well clear bottom tissue culture plates and treated with GLS1 for several timepoints: 0, 4, 8, 10, 24, 32, 48, 72, 96 and 120 h. Cells were incubated with WST-1 reagent for 2 h at 37°C with 5% CO2, according to the manufacturer's instructions. In actively metabolizing cells, the tetrazolium salt WST-1 is cleaved to formazan by cellular enzymes. The presence of formazan was assessed through spectrophotometric quantification (450 nm, with a reference wavelength > 600 nm) in a Bio-Rad Laboratories iMark™ Microplate Absorbance Reader (1681130).

[0053] In an embodiment, GBM cells were under starvation using serum-free DMEM / F- 12, overnight were used for the invasion assay. After hydration of the transwells' membrane (PI8P01250, Millicell) for lh30 with starvation medium at 37°C, geltrex (A1413202, Gibco) was added to each transwell and incubated 30 min at 37°C. Cells were harvested (U251: 1 x 105cells / insert; U-87MG: 5 x 104cells / insert) and resuspended in medium with 1% (v / v) FBS (without glutamine, with 500 pM or 2500 pM of glutamine) and experimental conditions were added: 0.07 and 0.14 ng / pL of GLS1. Mediasupplemented with 10% (v / v) FBS and 20ng / pL of EGF was added to the lower well, used as chemoattractant (with the corresponding glutamine concentration: 0, 500 or 2500 pM. After 24h incubation, cells on the upper transwell surface were removed with a cotton swab and the invading GBM cells were fixed with cold methanol. Slides were mounted in VECTASHIELD media with DAPI and examined by standard fluorescence microscopy using an Axio Imager.Zl microscope (Zeiss, Oberkochen, Germany). Images were acquired with CytoVision software (Leica). Three fields in each well were counted (20x magnification) using the ImageJ software (imagej.nih.gov / ij / ).

[0054] In an embodiment of orthotopic xenografts, NSG (NOD. Cg-Prkdcscid H2rgtmlWjl / Szi) immunocompromised mice aged 1.5 months obtained from Charles River Laboratory were anesthetized by inhalation of 2-3% sevoflurane in oxygen (500 mL / min) and subcutaneously injected with a mixture of butorphanol (5 mg / kg) for its analgesic properties and saline (10 mL / kg) for hydration. The animals were placed into a stereotaxic head frame (digital 3-axis, Stoelting), a small incision in the skin was made and a burr hole was drilled in the skull. A total of 2 x 105U-87MG transfected cells (resuspended in 5 pL PBS lx) were injected into the brain striatum (1.8 mm medial- lateral right, 0.1 mm anterior-posterior, and 2.5 mm dorsal-ventral from the bregma) using a 10 pL Hamilton syringe (point style 4 beveled 26s-gauge needle) at the velocity of 1.7 pL / min, as previously described [19,20], After injection, the needle was left in place for 2 min to avoid any backflow from the needle tract. Mice were randomly assigned to control or treatment groups (8 mice per group), ensuring no major body weight and age differences between groups, one week after injection. GLSl-therapy was intraperitoneally administered, by injection of 1.5 pL of a solution with 0.14 ng / pL of GLS1, diluted in saline solution (making up a final dose of 0.21 pg per mice), every 3-4 days (2 times per week), and started one week after U-87MG cells implantation. Mice were maintained under standard laboratory conditions, which included an artificial 12 h light / dark cycle (all experiments performed during daylight), controlled ambient temperature (21 ± 1°C) and a relative humidity of 50-60%. Mice had available ad libitum irradiated food and autoclaved water and were housed in groups of 3-5 per cage to allow social interaction. Cages were also enriched with paper to promote nest building and mice hiding. The confirmation of specified pathogen-free health status of sentinelmice maintained within the same animal room was performed according to FELASA guidelines. Experimental mice were always manipulated in a flow hood chamber, except during the surgery. Mice's body weight was evaluated 3 times per week, while general behavior and symptomatology were registered daily. Blood collection was performed at four distinct timepoints: week -1 (one week prior to surgery), week 1 (6h after the first treatment), week 4 (halfway through the treatment) and euthanasia (at sacrifice, by cardiac puncture). Sacrifice at the pre-established humane endpoint was performed when mice presented severe weight loss (> 20% of maximum body weight). Mice were sacrificed with a lethal dose of anesthesia injected intraperitoneally. Animals were perfused with saline solution followed by 4% paraformaldehyde (PFA) and brains were collected immediately and stored in buffered formalin until embedding in paraffin. All brains were collected for morphological analysis in 4 pm sections under hematoxylin and eosin (H&E) counterstaining, as well as the kidneys and liver to address morphological alterations due to therapy toxic effects. The tumor volume was calculated using the formula: tumor volume = (length x width2) / . All animal experiments were approved by the national ethical committee (Dire^ao Geral de Alimenta^ao e Veterinaria, Portugal) and were performed in accordance with the European Union Directive 2010 / 63 / EU.

[0055] In an embodiment, the serum from the blood collected during the experiment was evaluated by 1H-NMR spectroscopy. Sera were diluted in a buffer of 100 mM potassium phosphate (KPi, pH 7) with trimethylsilylpropanoic acid (TSP, 0.16mM) and spectra were acquired in a 500 MHz magnetic field in the 500 UltraShieldTM Spectrometer (Bruker) in 2.5 mm Bruker tubes. Spectra were processed using NMRProcFlow 1.4 software (nmrprocflow.org) and the buckets assigned for each condition were then analyzed by MetaboAnalyst 5.0.

[0056] In an embodiment of realization of immunohistochemistry studies, paraffin sections (4 pm) from paraffin-embedded tissue blocks were deparaffinized, rehydrated in a rinsing sequence of ethanol (90-10%) and finally water rinse, and antigen recovery was performed using citrate buffer. Slides were rinsed with lx Tris Buffered Saline (TBS) followed by the blockage of endogenous peroxidase (K4011, Dako) for 10 min. For nuclear staining, slides were immersed in Mayer's Hematoxylin (RE7164, Leica) for 5min. Slides were dehydrated and mounted with Entellan (1079610500, Merck) and digitalized using Aperio ImageScope-Pathology Slide Viewing software version 12.4 (Leica, Wetzlar, Germany).

[0057] All in vitro data were analyzed using student's t-test, one-way ANOVA, or two- way ANOVA in GraphPad Prism v9 software (www.graphpad.com / ). The assays were performed with at least 3 biological replicates per condition and the differences were determined statistically significant at p-value < 0.05. The effect of GLS1 in the overall survival of orthotopic GBM xenografted mice was represented by Kaplan-Meier survival curves, and the differences evaluated by the univariate Log-rank test. MetaboAnalyst 5.0 online platform was used for NMR bioinformatic analyses. Multivariate analysis involved partial least squares-discriminant analysis (PLS-DA), a supervised method to identify separation between sample groups.EXAMPLE 2

[0058] In example 2, a GBM orthotopic NSG mice model was established and two doses of GLS1, dose 1 - 0.14 ng / pL and dose 2 - 0.28 ng / pL, were intraperitoneally administered twice a week (every 3 days). A significant increase in OS was observed in mice treated with dose 1 of GLS1 (median OS: 34.5 days) and dose 2 of GLS1 (median OS: 48.5 days) comparing to control untreated mice (median OS: 27 days) (Error! Reference source not found. Figure 4 A). As observed in the first experiment, the weight loss due to cancer-related cachexia was proportional slower in the two doses of GLS1 treated mice (Figure 4 B).

[0059] Mice blood sera were also evaluated by1H-NMR spectroscopy (Figure 4 C - D). PCA analysis did not show the separation at week 4 (Figure 4 C). However, in PLS-DA a separation of the metabolic profiles from control untreated mice, dose 1 and dose 2 GLS1 treated mice was observed (Figure 4 D).

[0060] According to the results obtained, it was observed an increase in OS dependent on the GLS1 dose, and slower cachexia.Methods

[0061] Orthotopic xenografts: All animal experiments were approved by the national ethical committee (Diregao Geral de Alimentagao e Veterinaria, Portugal; ref 008516) and were performed in accordance with the European Union Directive 2010 / 63 / EU. NSG (NOD. Cg-PrkdcscldH2rgtmlw l / Sz ) immunocompromised male mice aged 7 weeks obtained from Charles River Laboratory were anesthetized by inhalation of 2-3% sevoflurane in oxygen (500 mL / min) and subcutaneously injected with a mixture of butorphanol (5 mg / kg) for its analgesic properties and saline solution (10 mL / kg) for hydration. The animals were placed into a stereotaxic head frame (digital 3-axis, Stoelting), a small incision in the skin was made and a burr hole was drilled in the skull. U-87MG were previously cultured in DMEM (Sigma-Aldrich) supplemented with 10% FBS (Sigma-Aldrich) and were verified as mycoplasma-free (kit Venor®GeM qOneStep, Minerva Biolabs). A total of 2 x 105U-87MG cells (resuspended in 5 pL PBS lx) were injected into the brain striatum (+1.8 mm mediolateral, +0.1 mm anteroposterior, and - 2.5 mm dorsoventral from bregma) using a 10 pL Hamilton syringe (point style 4 beveled 26s-gauge needle) at the velocity of 1.7 pL / min, as previously described [533-535], After injection, the needle was left in place for 2 min to avoid any backflow from the needle tract. One week after tumor implantation, mice were randomly assigned to control or treatment groups (8 mice per group), ensuring no major body weight and age differences between groups. At this time, GLSl-therapy was intraperitoneally administered (dose 1: 0.14 ng / pL and dose 2: 0.28 ng / pL) diluted in saline solution (100 pL), every 3-4 days (2 times per week). Mice were maintained under standard laboratory conditions, which included an artificial 12 h light / dark cycle (all experiments performed during daylight), controlled ambient temperature (21 ± 1 °C) and relative humidity of 50-60%. Mice had available ad libitum irradiated food and autoclaved water and were housed in groups of 3-5 per cage to allow social interaction. Cages were also enriched with paper to promote nest building and mice hiding. The confirmation of the absence of specific pathogens was assessed by the health status of sentinel mice maintained within the same animal room as recommended by the Federation of European Laboratory Animal Science Associations (FELASA) guidelines. Experimental mice were always manipulated in a flow hood chamber, except during the surgery. Mice's body weight was evaluated at least 3 times per week, while general behavior and symptomatology were registered daily. Blood collection was performed at four distincttime points: week -1 (one week before surgery); week 1 (6 h after the first treatment at 1 week after tumor implantation); week 4 (halfway through the treatment), and euthanasia. Blood was collected by mandibular puncture or, at euthanasia, by cardiac puncture. Sacrifice at the pre-established humane endpoint was performed when mice presented severe weight loss (> 20% of maximum body weight). Mice were sacrificed with a lethal dose of anesthesia injected intraperitoneally. Animals were perfused with saline solution followed by formaldehyde, and skulls, kidneys and liver were collected immediately and stored in buffered 10 % formalin until embedding in paraffin.

[0062] Mice blood serum evaluation by1H-NMR: The blood serum collected during the experiment was evaluated by1H-NMR spectroscopy. Sera were diluted in a buffer of 100 mM KPi (pH 7) with TSP (0.16 mM) and transferred to NMR match tubes of 2.5 mm (16545, Bruker), in the case of sera from euthanasia mice (70 pl sera + 70 pl KPi buffer), or 1.7 mm (29138, Bruker), for the other time-points (25 pl of mice's pool sera + 25 pL KPi buffer). Spectra were acquired in a 500 MHz magnetic field in a 500 UltraShield™ Spectrometer (Bruker) equipped with a 5 mm TCI C / N Prodigy Cryo probe at 37 °C. For each sample three1D 1H-NMR spectra were acquired: one with a noesyprld pulse sequence as a sum of 64 free induction decays (FIDs) (48K of Time-Domain (TD), 11.76 ppm of spectral window (SW), 4 s relaxation delay and 10 ms of mixing time); other with a cpmgprld as a sum of 64 FIDs (65K TD, 20 ppm SW, 4 s relaxation delay, an echo time of 0.2 ms and 300 repetitions of the echo time per scan); and diffusion edited pulse experience Jedbpgppr2sld) as a sum of 64 FIDS (65 k TD, 30 ppm SW, 4 s relaxation delay and a diffusion time of 120ms and a diffusion time of 0.2 ms). Spectra were acquired and processed using Topspin 3.6 software (Bruker). NMRProcFlow 1.4 software (nmrprocflow.org) was used for further spectra process and to define the spectra buckets that were analyzed by MetaboAnalyst 6.0 (accessed on 26 February 2024). All data obtained from NMRProcFlow was normalized through Constant Sum Normalization.

[0063] Statistical Analysis: The effect of GLS1 in the OS of orthotopic GBM xenografted mice was represented by Kaplan-Meier survival curves and the differences were evaluated by the univariate Log-rank test.

[0064] MetaboloAnalyst 6.0 (http: / / www.metaboanalyst.ca / ) online platform was used for NMR bioinformatic analyses. Cell extracts samples were normalized by sample weight. The multivariate analyses involved principal component analysis (PCA) and partial least squares-discriminant analysis (PLS-DA).

[0065] The term "comprising" whenever used in this document is intended to indicate the presence of stated features, integers, steps, components, but not to preclude the presence or addition of one or more other features, integers, steps, components or groups thereof.

[0066] The disclosure should not be seen in any way restricted to the embodiments described and a person with ordinary skill in the art will foresee many possibilities to modifications thereof. The above-described embodiments are combinable.

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Claims

C L A I M S1. A glutaminase for use in the treatment of a condition susceptible of being improved or prevented by a decrease of systemic glutamine bioavailability, wherein the condition is a glioma; wherein the glutaminase is glutaminase GLS1.

2. The glutaminase for use according to the previous claim wherein the glioma is a glioblastoma.

3. A pharmaceutical composition comprising a glutaminase and a pharmaceutically acceptable carrier for use in the treatment of a condition susceptible of being improved or prevented by a decrease of systemic glutamine bioavailability, wherein the condition is a glioma, wherein the glutaminase is glutaminase GLS1.

4. The pharmaceutical composition according to the previous claim wherein the composition is systemically administered.

5. The pharmaceutical composition according to the previous claim wherein the composition is administered by intravenous administration, intraperitoneal administration, oral administration, intrathecal administration, or combinations thereof.

6. The pharmaceutical composition according to any of the previous claims 3-5 wherein the composition is administered by intravenous administration or intraperitoneal administration.

7. The pharmaceutical composition according to any of the previous claims 3-6 wherein the composition is free of cytotoxic and / or cytostatic compounds.

8. The pharmaceutical composition according to any of the previous claims 3-7 wherein the dosage amount ranges from 1 pg / g - 50 pg / g; preferably 3 pg / g -30 pg / g; more preferably 5 pg / g-20 pg / g; even more preferably 7 pg / g- 14 pg / g.

9. The pharmaceutical composition according to any of the previous claims 3-8 for administration every 72 hours; preferably every 48 hours; more preferably every 24 hours.

10. The pharmaceutical composition according to any of the previous claims 3-9 for administration as a single dose.

11. The pharmaceutical composition according to any of the previous claims 3-10 further comprising an active ingredient, in particular a chemotherapy agent a radiotherapy agent, an imaging agent, or combinations thereof.

12. The use of a glutaminase for the manufacture of a medicament for the treatment of cancer, wherein said cancer is a glioma, wherein the glutaminase is glutaminase GLS1.

13. A method for treating or preventing a cancer, preferably a glioma, in a subject, the method comprising administering glutaminase GLS1 to the subject.

14. The method according to the previous claim wherein the glutaminase is administered systemically.

15. The method according to the previous claim wherein the administration is intravenous administration, intraperitoneal administration, oral administration, intrathecal administration, or combinations thereof.

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