It forms an object of the present invention a composition comprising R- Ketorolac, Bumetadine, at least one Cl’ channels blocker and, optionally, Infliximab for use in the treatment of low- and high-grade glioma.

Background

Glioblastoma is an aggressive type of brain tumor belonging to the family of glioma. Glioblastoma may occur in the brain or spinal cord and forms from astrocytes that support nerve cells. Glioblastoma, also known as glioblastoma multiforme (GBM, i.e. grade IV glioma), is very difficult to treat and a cure is not possible. Only few patients survive 2.5 years and less than 5% of patients survive 5 years following diagnosis ¹ , with a median survival of approximately 14 to 15 months from the diagnosis ² . The standard of treatment is surgery, followed by daily radiation and oral chemotherapy. Treatments may slow progression of the cancer and reduce signs and symptoms. No single drug can arrest this deadly progression because of the highly heterogeneity of cancer cells. Furthermore, during the progression of both low- and high-grade glioma, epilepsy occurs in 40-90% of cases ³ .

The malignancy of glioma is due to genetic mutations in tumour suppressors and oncogenes, but also in the signalling molecules contained in extracellular vesicles released by glioma stem cells (GSC) and glioma associated stem cells (GASC), referred to as exosomes ⁴ Moreover, membrane proteins such as ionic channels and transporters were shown to play a major role in cell condensation and uncontrolled replication/infiltration of GBM ⁵ . Several lines of evidence suggest that Chloride (Ch) and K ⁺ channels have a major role in cell volume shrinking and swelling, which are two fundamental steps during mitosis and replication possibly shared by almost all eukaryotic cells.

There is a large variety of Cl’ channels, among them there is the recently discovered family of membrane proteins referred to as TMEM16. Recently, the role of TMEM16A in cancer has been considered and reviewed ⁶ . TMEM16A over-expression in cancerous cells, however, is not homogeneous and TMEM16A is not overexpressed in subtypes of breast cancers and Pancreatic Neuro- Endocrine tumors ⁷ .

We have previously described the existence of Calcium flares in GBM, which can reach levels in the range of 1 -5 pM, suggesting a role for calcium flares and Cl’ channels during GBM mitosis ⁸ .

(R)-ketorolac is a 5-benzoyl-2,3-dihydro-1 H-pyrrolizine-1 -carboxylic acid that has R configuration. Unlike the S-enantiomer, it does not exhibit COX1 and COX2 inhibition, but does exhibit analgesic activity. (R)- ketorolac is used as a potent analgesic for the short-term management of post-operative pain, and in eye drops to relieve the ocular itching associated with seasonal allergic conjunctivitis.

Bumetanide is a potent sulfamoylanthranilic acid derivative belonging to the class of loop diuretics. In the brain, bumetanide may prevent seizures in neonates by blocking the bumetanide-sensitive Na/K/CI cotransporter (NKCC1 ), thereby inhibiting chloride uptake thus, decreasing the internal chloride concentration in neurons and may block the excitatory effect of GABA in neonates. Niflumic acid is an aromatic carboxylic acid and a member of pyridines. It is widely used as an analgesic and anti-inflammatory agent in the treatment of rheumatoid arthritis.

Infliximab is a monoclonal antibody belonging to the family of tumor necrosis factor-a (TNF- a) inhibitors. It is used for the treatment of autoimmune diseases such as Chron’s disease, chronic plaque psoriasis and rheumatoid arthritis.

There is a strong need for an effective treatment of low- and high-grade glioma, and for glioma-related epilepsy.

Description

The authors of the present invention have surprisingly found that a composition comprising R-Ketorolac, which inhibits the dynamics of the cytoskeleton, Bumetanide, centrally blocking the Na-K-CI cotransporter (NKCC) and at least one Cl’ channels blocker significantly reduces motility and replication of a human primary glioblastoma cell line and motility and replication of Glioma Stem Cells from patients affected by Grade IV glioblastoma.

In an embodiment, the composition additionally comprises at least one TMEM16A inhibitor.

The composition has an almost negligible effect on the electrical activity of healthy neurons.

In addition, the authors have found that Infliximab reduces the epileptic discharges induced in neurons by exosomes derived from patients affected by glioma (of all grades).

In an embodiment, the composition comprises R-Ketorolac, Bumetanide, at least one Cl’ channels and Infliximab.

Brief drawings description

Figure 1 : Transwell assay, % of migrated cells evaluated after 72h exposure to the indicated treatment. Figure 2: GBM U87 cell line (A) before and (B) after treatment with a composition comprising R-Ketorolac 100 pM, Bumetanide 100 pM and NFA 100 pM.

Figure 3: Co-culture of neurons, glial cells and GBM U87 cell line. (A), (C), (E) before and (B), (D), (F) 48 hours after treatment with a composition comprising R-Ketorolac 100 pM, Bumetanide 100 pM and NFA 100 pM. (A), (B) Bright field image. (C) and (D) a first fluorescent channel, U87 cells. (E) and (F) a second fluorescent channel, neurons. Scale bar 10 pm.

Figure 4: Microelectrode array (MEA) recordings showing the spontaneous electrical activity of cortical neurons after 3 weeks of culture. The raster plots represent the timing of neuronal spikes for each electrode (for a total of 120 electrodes on the MEA device). (A) control conditions. (B) One hour after the application of the composition comprising R-Ketorolac 100 pM, Bumetanide 100 pM and NFA 100 pM. (C) Six hours after the application of the composition. (D) 24 hours after the application of the composition.

Figure 5: Quantification of the (A) average velocity and (B) distribution of velocities of U87 cells in pm per minute.

Figure 6: Quantification of the (A) average number of mitotic events in the presence of the indicated treatment (black), with reference to the average number of mitosis per cell in U87 cells without any addition (grey) and (B) average area per cell (pm ² ) in U87 cells in the presence of the indicated treatment (black), with reference to the average area per cell of U87 cells without any addition (grey).

Figure 7: (A) Alamar blue viability test on U87 cells in the presence of the indicated treatment for 24 hours versus control and (B) quantification of the average velocity of U87 cells in the presence of the indicated treatment versus control. Figure 8: (A) Quantification of the average velocity and distribution of velocities of patient cells in pm per minute. (B) Distribution of velocity of U87 GBM cells per minute. (C) Average velocity of patient cells per minute in black, in grey standard deviation of the velocities with different additions of drugs. (D) Distribution of velocity of U87 cells per minute.

Figure 9: The effect of exosomes released from glioma of five different patients and from human healthy astrocytes (HA, last column). First raw: examples of intracellular electrical activity from rat hippocampal neurons obtained with intracellular patch pipette in current clamp mode. From left to right: in control conditions, following incubation for 24 hours with 10 ng of glioma exosomes and from HA. The second row collected data relative to the voltage resting membrane potential (RMP) and the third-row reports collected data on the mean firing frequency in the different conditions. All columns refer to data obtained in the same conditions.

Figure 10: Quantification of the average number of mitotic events with reference to the average number of mitosis per cell in U87 cells in control condition or after exposure to compositions according to the invention, where each of the three compound is at 100 pM (A) or at 50 pM (B).

Detailed description

It forms an object of the present invention a composition comprising R- Ketorolac, Bumetanide, and at least one Cl’ channels blocker for use in the treatment of low- and high-grade glioma.

In an embodiment, the composition further comprises at least one TMEM16A inhibitor.

In an embodiment, said at least one Cl’ channels blocker is selected from the group comprising Tamoxifen, 5-nitro-2-(3-phenyl-propylamino) benzoic acid (NPPB, niflumic acid) Niflam™, 3[3-Hydroxy-11 -oxoolean- 12-en-30-oic acid 3-hemisuccinate (carbenoxolone), or salts thereof, preferably it is Niflam™.

In an embodiment, said composition consists of R-Ketorolac, Bumetanide, and at least one Cl’ channels blocker and pharmaceutically acceptable excipients.

In an embodiment, said composition comprises R-Ketorolac, Bumetanide, and at least one Cl’ channels blocker in a ratio 1 : 1 : 1.

In an embodiment, said composition additionally comprises Infliximab.

In an embodiment, said composition comprises R-Ketorolac, Bumetanide, one Cl- channels blocker and Infliximab in an overall ratio 1 : 1 : 1 : 0.1 , respectively.

In an embodiment, said composition comprises:

- 30-100 pM R-Ketorolac

- 30-100 pM Bumetanide

- 30-100 pM Niflam™

In an embodiment, said composition comprises:

- 30-100 pM R-Ketorolac

- 30-100 pM Bumetanide

- 30-100 pM Niflam™

- 1-10 pM Infliximab

In an embodiment, said composition comprises:

- 30-100 pM R-Ketorolac

- 30-100 pM Bumetanide

- 30-100 pM Carbenoxolone or salts thereof

In an embodiment, said composition comprises:

- 30-100 pM R-Ketorolac

- 30-100 pM Bumetanide

- 30-100 pM Carbenoxolone or salts thereof

- 1-10 pM Infliximab In addition, given the known variability observed for chloride channel in tumors, and the Cl’ channels inhibitors available so far, the composition according to the present invention is conveniently employed in a personalized therapy regimen. In this approach, the composition comprises R-Ketorolac, Bumetanide and a Cl’ channel inhibitor, wherein said inhibitor is selected on the basis of the Cl’ channel expressed in tumoral tissue from the specific subject.

It is further described a method to treat low- and high-grade glioma, wherein said method comprises:

- Obtaining a bioptic sample from a subject in need thereof;

- Evaluating the most expressed subtype of Cl’ channel in said sample at the molecular level;

- Selecting the Cl’ channel inhibitor suitable for the Cl’ channel identified in said sample;

- Administering to said subject a composition comprising R- Ketorolac, Bumetanide, the selected Cl’ channel inhibitor and, optionally, Infliximab, preferably in a ratio 1 : 1 : 1 , or 1 : 1 : 1 : 0.1 when in the presence of Infliximab.

Experimental section

Example 1: effect of R-Ketorolac on GBM mobility

R-Ketorolac, at a concentration of 50-100 pM, significantly slows down the ability of U87 GBM cells to migrate as shown by transwell assay reported and described in one of our previous work ⁹ . In this assay, human U87 GBM cells (Sigma-Aldrich®) were seeded on top of an insert with narrow holes (3-4 microns in diameter). The ability of GBM to pass or infiltrate through these narrow holes (as small as the diameter of GBM nucleus) has been evaluated. A chemoattractant solution was placed in the bottom compartment. After 72h incubation with the indicated treatments, GBM cells chemoattracted to the bottom compartment were quantified to estimate their migration.

The results - quantified as the number of cells migrated into the bottom compartment - were observed at day 1 , day 2 and day 3 after exposure. At day 2 and 3, R-Ketorolac 50-100 pM slows down the percentage of migrated cells comparing to control (i.e. blank solution), with maximum decrease observed with R-Ketorolac 100 pM at day 3.

Example 2: effect of R-Ketorolac and Bumetanide on GBM infiltration

The activity of R-Ketorolac and Bumetanide on the ability of GBM to pass or infiltrate through narrow holes as described in Example 1. Results are reported in Figure 1. Both R-Keterolac at 100 pM and Bumetanide at 50 and 100 pM significantly reduce GBM infiltration.

Example 3: effect of the composition according to the invention on GBM motility and replication

A composition comprising:

- 100 pM R-Ketorolac

- 100 pM Bumetanide

- 100 pM Niflam™ was tested on U87 GBM cell lines in vitro. We used live cell imaging, in which a bright field image was taken every 10 minutes in order to follow the effect of the drugs for 2-4 days. As shown in Figure 2, the composition is capable to strongly inhibit GBM motility and to almost completely block GBM replication. The comparison of GBM images at the beginning of the experiment (Figure 2A) and after 3 days (Figure 2B) shows that most GBM could not exit from meta/anaphase and collapsed while attempting to divide. Their cytokinesis (i.e. final division into daughter cells) is blocked.

By acquiring several fields of views - within 5-10 minutes - in one sample, it is possible to follow the motion of several tens of GBM at the same time, to obtain a more precise quantification of the effect of used drugs on GBM motility.

In control conditions, U87 GBM moved at a rate of about 0.3 microns per minute (Figure 5A) and there were GBM with a much higher motility, as high as 1 micron per minute (Figure 5B). The addition of R-Ketorolac slowed down the motion by 20-30 %, however it requires several hours to observe a significant decrease. The addition of Bumetanide further decreased GBM motility and especially that of fast migrating GBM. Following the addition of NFA, GBM stopped moving and appeared almost immobile (Fig.5A and B).

By using the same methodology, the effect of the composition on GBM replication was observed. A reduction around 75% of GBM replication was observed after 3 days of treatment (Figure 6A), as well as of the average area per cell of GBM U87 cells visible in the dish, computed by quantification of the fluorescence of glioma cells stained by m-Cherry that is reduced more than 60% (Figure 6B).

The experiment was repeated by using two different compositions according to the present invention. A first one, using Niflam™ as a Cl’ channels inhibitor, and a second one, using Carbenoxolone disodium salt. Results are reported in Figure 10, panel A by using a composition where the three components are present at 100 pM each one, panel B by using a composition where the three components are present at 50 pM each one.

The activity of the single components of the composition was tested separately, in a comparative experiment.

As shown in Figure 7A, after 1 days of incubation, R-Ketorolac 100 pM and Bumetanide 100 pM did not affect GBM vitality, wherein 100 pM NFA decreased significantly the number of alive and vital GBM. Therefore we looked to the effect of higher concentrations of a single drug NFA on GBM motility and vitality.

As shown in Figure 7B, 300 pM NFA alone did not reduce GBM motility as observed when exposing the same to the composition according to the present invention, comprising 100 pM R-Ket + 100 pM Bumetanide + 100 pM NFA.

Example 4: effect of the composition according to the invention on healthy neurons

Example 3 has surprisingly demonstrated that the composition according to the invention is active on GBM motility and replication. Aim of these experiments was to verify the impact, if any, of the composition on healthy neurons and glia.

The experiment described in Example 3 was therefore repeated on a co-culture consisting of U87 cell lines transfected with m-Cherry and healthy cortical (or hippocampal) neurons. Red fluorescence allows to easily distinguish U87 GBM cells from healthy neurons. We performed several experiments with these co-cultures and we found that - after 2-4 days in the presence of the composition - both the number of mitosis of red U87 GBM and their motion were diminished more than 60% and more than 65%, respectively while neurons kept their usual shape and properties (Figure 3A, B). The composition according to the present invention blocked, in almost 80 % of glioma cells, the usual cycle of rounding-mitosis-spreading underlying glioma replication and indeed, as shown in Figure 3C, D, glioma are not able to spread and remain in the rounded shape (unable to complete mitosis).

Moreover, the experiment demonstrates the composition according to the invention does not impact the shape of healthy cortical of hippocampal neurons (Figure 3E, F). To confirm this, a Multi Electrode Arrays (MEAs) assay has been performed, to verify that the composition does not impair the basal electrical activity of neurons.

Each MEA contains 120 titanium nitrate (TiN) microelectrodes with a diameter of 30 pm and an inter-electrode distance of 100 pm. MEAs’ individual microelectrodes were used to detect extracellular voltage signals from neurons sitting in their close proximity. We employed an electronic multichannel amplifier (MEA-1060-Up-BC, Multi-Channel Systems) with 1-3000 Hz bandwidth and an amplification factor of 1200. Recordings were performed inside of a dry) incubator, at 37°C and 5% CO2 (C150, Binder GmbH, Tuttlingen, Germany). Extracellular raw electrical signals were sampled at 10 kHz/channel and digitized at 16 bits of resolution by an analog-to-digital (A/D) electronic acquisition board (MCCard, MultiChannel Systems).

Healthy neuronal cultures were plated on the MEA. Figure 4 shows the raster plots in control conditions (A) and after 1 (B), 6 (C) and 24 (D) hours of exposure to the composition. The spontaneous electrical activity transiently declined by 30% after 6 hours, but it fully recovered after 1-2 days. Three independent experiments were performed, obtaining similar results.

Example 5: effect of the composition according to the invention on GBM from patients with Grade IV tumors.

We verified the effect of the composition on Glioma Stem Cells (GSM) obtained from patients’ bioptic tissues.

All these patients were affected by Grade IV glioblastoma as verified by histological analysis. The shape of these GSC is similar - but not identical - to that of U87 GBM. In fact, a detailed biophysical analysis of patient GSC reveals two fundamental facts: i) in all GSC from Grade IV glioblastoma replication occurs in the same way, during which the mitotic cell first shrinks and become perfectly round. Following rounding, the GSC divides passing through the usual step of telophase and cytokinesis. ii) the biophysical mechanisms underlying swelling is different in different GSC, because different kind of Chloride channels are involved.

Nonetheless, as shown in Figure 8, the composition according to the invention decreased GBM motility and replication rate.

GSC from patients differ by the properties of their Cl’ channels, and indeed we found that different mixtures of FDA/EMA approved drugs targeting Cl’ channels are more effective, such as Ani9. Adding Ani9 to the composition, indeed, decreased the average velocity of GSC of a further 10%, bringing it close to zero motion (Fig. 8A, C). This effect was not observed in GBM cells’ mobility (Fig. 8B, D).

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