DESCRIPTION

The present invention relates to the use of CXCR4 receptor inhibitors, in particular of Plerixafor, and compositions including them, as senolytic drugs. In particular it relates to the use of Plerixafor senolytic drug in a method of treatment of the tumour or other conditions associated to the cellular senescence.

STATE OF PRIOR ART

Senescence is a state wherein the cell, in response to precise stimuli, loses the replicative capability even if it maintains metabolically active. It carries out physiological important roles during the ageing processes and it results to be crucial in contrasting the uncontrolled growth of the tumour cells. Notwithstanding the senescent cells are in a not proliferative state, they however remain metabolically active and start to secrete a wide number of cytokines and pro-inflammatory factors, thus assuming a secretory phenotype (senescence-associated secretory phenotype, SASP). This“secretome” is capable of activating the antitumour immune response and promoting the removal of the senescent cells themselves (“senescence surveillance”). However, there are cases wherein the senescent cells avoid the protective action of the immune system by remaining in the cancerous site and by carrying out, through the secretome, a pro tumour action capable of promoting angiogenesis, migration, invasion and metastasis. In this context then, the senolytic therapy, that is the specific removal of the senescent cells, appears as a new clinical strategy apt to improve the effectiveness of the canonical chemotherapies.

Nowadays, the number of senolytic drugs on the market results to be poor and most part thereof is directed against Bcl-2, a known survival regulator typically over expressed by the senescent cells. The effectiveness of these compounds however is variable and strictly connected with the genetics of the senescent cells themselves, consequently it varies depending upon the different inter and intra-tumour features. Moreover, the senolytic drugs can be used in the prevention and/or treatment of all pathologies associated to the cellular senescence.

The object of the present invention is to provide new substances with senolytic activity for use in the treatment of tumours and other pathologies associated to the cellular senescence.

SUMMARY OF THE INVENTION

The present invention is based upon the finding that the CXCR4 receptor inhibition promotes the removal of senescent cells, such finding is supported by the experiments shown in detail in the related experimental section of the present description. In sum, the experiments show that Plerixafor is known as antagonist drug of CXCR4 currently used in clinic with the purpose of mobilizing the haemopoietic stem cells in the blood flow of patients. Plerixafor was selected as senolytic agent after a screening of about 1650 compounds. The experimental data shown in the present patent application show that Plerixafor has a senolytic activity comparable to compound ABT-263 ( gold standard) in terms of removal of senescent cells (PICS), but it results to be more selective. Additional in vitro data on tumour cell lines and in vivo data on murine models shown that Plerixafor is a powerful drug capable of eliminating selectively the senescent cells induced by chemotherapy or by CDK4/6 inhibitors, by determining a slowing-down of growth in most tumours. Moreover, it was demonstrated in vivo on a murine model that Plerixafor is capable of eliminating selectively, at systemic level, the senescent cells produced after a chemotherapeutic treatment.

Therefore, the present invention firstly relates to CXCR4 receptor inhibitors, in particular Plerixafor, as senolytic drugs, in particular to be used as senolytic drugs in a method for the treatment of a tumour or any pathology associated to the cellular senescence. Secondly, it relates to compositions comprising CXCR4 receptor inhibitors, in particular Plerixafor, for such use.

Additional advantages, as well as the features and the use modes of the present invention will result evident from the following detailed description of some preferred embodiments.

DETAILED DESCRIPTION OF THE FIGURES

Figure 1. Platform of senolytic screening in PICS. (A) The circular graph represents the composition, in percentage, of the libraries used in this screening. (B) Screening experimental drawing. MEFs Pten-/- cells were treated for 48 hours with Docetaxel at the concentration of 750 nM with the purpose of inducing senescence. The screening starts when the cell proliferation has stopped and 80% of cells shows a senescent phenotype. The compounds were tested at the single dose of 10 mM. (C) Representation of the different passages thereof the screening is composed. In first place there is the validation of the compounds in cells wherein Pten is deleted, subsequently the selectivity of these compounds is investigated by inserting even MEF wt, and at last IC50 is evaluated. The compounds satisfying these filters will be then tested on cancer cell lines, and at last on in vivo preclinical models.

Figure 2. Identification of Plerixafor as senolytic drug. (A) The senolytic screening performed in PICS determined the identification of Plerixafor as powerful senolytic drug. This compound shows a strong capability in killing the PICS cells, with respect to ABT-263 considered as positive check. Plerixafor shows a marked capability in removing the senescent cells even if the non-senescent, NS, cells are kept vital. (B) Plerixafor is capable of removing the cells in replicative senescence (RS) caused by ageing, without killing the non-senescent (NS) cells as demonstrated by the assays of Crystal violet and SA^-Gal. Once having demonstrated the senolytic activity of this compound in primary lines, the activity in cell lines of prostate cancers, such as TrampCI (C) and PC3 (D), was tested. Our data demonstrate Plerixafor’s capability of removing the senescent cells in both cell lines.

Figure 3 Plerixafor shows in vivo senolytic effectiveness. (A) Scheme of the experimental design. 2x10 ⁶ PC3 were injected to the mice subcutaneously. When the tumour reached a volume of 100mm ³ the mice were treated with Palbociclib. After about three weeks a stop in the tumour growth was observed. At this point the treatment with Plabociclib was suspended and the mice were divided into two groups, control group (Palbo_PBS) and experimental group (Plabo_Pleri). The senolytic treatment was prolonged for 33 days, in which we recorded a clear slowing down in the tumour growth of the treated mice with respect to the controls. The transcriptional profile of known senescence markers such as PAI-1 and p16 were analysed to confirm the senolytic activity of Plerixafor. (C) The graphs show that the tumours treated with Plerixafor (Plabo_Pleri) have a clear decrease in these markers with respect to the control tumours (Palbo_PBS).

Figure 4 Senolytic activity of Plerixafor in in-vivo model p16-Luciferase. (A)

Scheme of the experimental design. The mice were treated with Doxorubicin (10mg/kg) with the purpose of activating the transcription of the senescence marker p16 and at the same time the transcription of luciferin. The first measurement of luciferin intensity was performed after 7 days of treatment with Doxorubicin. The mice treated with Doxorubicin were then randomized into two sub-groups, control group (Doxo) and experimental group (Doxo_Pleri). (B) The graphs show that after 7 days of treatment with Doxorubicin (Day 0 of treatment with Plerixafor), only the mice treated with the drug showed an increase in luminescence. On day 5 and 7 after treatment with Plerixafor these mice (Doxo_Pleri) showed a strong decrease in luminescence intensity with respect to the control mice (Doxo). On the seventh day of treatment the experimental group showed a total removal of light intensity, comparable to the signal recorded in mice not treated with Doxorubicin, as it can be observed in the representative images (C).

DETAILED DESCRIPTION OF THE INVENTION

The present invention is constituted by the following aspects, described in detail hereinafter.

A first aspect of the present invention relates to the use of CXCR4 inhibitors as inductors of the selective death of senescent cells.

Examples of CXCR4 inhibitors are TG-0054 (CAS:1191450-19-7), AMD070 (CAS : 558447 -26-0) , AMD3100 (CAS 155148-31-5), BL-8040 (CAS664334-36-5), LY2510924 (CAS: 1088715-84-7).

According to a preferred embodiment the inhibitor used as senolytic drug is Plerixafor. The structure formula of Plerixafor is shown hereinafter:

In the present description under the term “senolytic drug” a substance is meant capable of removing selectively the senescent cells.

Plerixafor as senolytic drug could be used in a method of prevention and/or treatment of any pathology associated to the cellular senescence. In the present description under the term “pathology associated to the cellular senescence” any pathological condition is meant due to the accumulation of senescent cells or wherein the accumulation of senescent cells determines a worsening of the patient condition. The selective removal of senescent cells aims at bringing about a therapeutic effect, such as for example in the pathologies related to ageing and in tumour diseases.

A CXCR4 receptor inhibitor, in particular Plerixafor, could be used as senolytic in treating patients suffering from cancer, preferably in association to one or more chemotherapeutic agents, for example Docetaxel, Palbociclib, Cisplatin and Doxorubicin.

The treated tumour could be selected for example among prostate, mammary gland, lung, pancreas and colorectal tumour.

A CXCR4 receptor inhibitor, in particular Plerixafor as senolytic drug could be used even for the treatment of other pathologies associated to the cellular senescence such as for example osteoarthritis, atherosclerosis, senile dementia, Alzheimer and other geriatric diseases. In the present description under the expression diseases associated to the cellular senescence even diseases are meant the treatment thereof involves a cell ageing as side effect, such as for example the treatment of tumour diseases with X rays.

The present invention further relates to a pharmaceutical composition comprising a CXCR4 receptor inhibitor and a carrier and/or a diluent for use as a senolytic drug in a method for the treatment of anyone of the herein described conditions to be treated. The composition could be oral, parenteral, rectal, transcutaneous, topic or suitable for other administration route. The compositions for use according to the present invention could be administered by any conventional means available for use together with drugs, or as individual therapeutic agents or in a combination of therapeutic agents. They can be administered alone, but generally administered with a pharmaceutical carrier selected upon the chosen administration route and the standard pharmaceutical practice. The administered dosage, obviously, will vary depending upon known factors, such as the pharmacodynamic features of the particular carrier and upon the administration mode and route thereof; age, health and weight of the recipient; nature and level of symptoms, type of concomitant treatment; frequency of treatment; and wished effect. It can be provided that a daily dosage of active ingredient is about 0.001 up to 1000 milligrams (mg) per kilogramme (kg) of body weight, the preferred dose being 0.1 until about 30 mg/kg. Forms of dosage (compositions suitable for administration) typically include about 1 mg to about 100 mg of active ingredient per dosage unit. In these pharmaceutical compositions, the active ingredient will be usually present in an amount of about 0.1-95% by weight based upon the total weight of the composition.

The composition can be administered by oral route in solid dosage forms, such as capsules, tablets and powders, or in liquid dosage forms, such as elixirs, syrups and suspensions. It can be administered even by parental route, in sterile liquid dosage forms. Capsules of gelatine include the active ingredient and pulverised carriers, such as lactose, starch, cellulose derivatives, magnesium stearate, stearic acid and the like. Similar diluents can be used to prepare compressed tablets. Both tablets and capsules can be manufactured as prolonged-release products to provide continuous release of medicament for a period of few hours. Compressed tablets can be coated with sugar or coated with film to cover any unpleasant taste and protect the tablet from atmosphere, or coated with enteric coating for the selective disintegration in the gastro-intestinal tract. Liquid dosage forms for oral administration can include dyes and flavours to favour acceptance by the patient. Generally, water, a suitable oil, saline solution, aqueous dextrose (glucose), and solutions of connected sugars and glycols such as propylene glycol or polyethylene glycols are carriers suitable for parental solutions. Solutions for parental administration preferably include a salt soluble in water of the active ingredient, suitable stabilizing agents and, if required, buffering substances. Anti-oxidant agents such as sodium bisulphite, sodium sulphite, or ascorbic acid, alone or combined, are suitable stabilizing agents. Even citric acid and salts thereof and sodium EDTA are used. Moreover, parental solutions can include preservatives, such as benzalkonium chloride, methyl or propyl-paraben, and chlorobutanol. The pharmaceutical compositions according to the present invention apart from a CXCR4 receptor inhibitor could include one or more active ingredients, that is pharmacologically active substances, in particular chemotherapeutic agents.

A CXCR4 receptor inhibitor, in particular Plerixafor, could also be used as cosmetic to prevent and/or slow down the cellular senescence not linked to pathological conditions, for example for the improvement and/or prevention of signs of skin ageing such as wrinkles, decrease in softness and/or luminosity of skin and the like.

Herein, the methods are further described for the therapeutic or cosmetic treatment of the above-mentioned pathological conditions comprising a passage of administering a CXCR4 receptor inhibitor or compositions including it. The method could be for the treatment of the tumour and it could include a passage preceding the inhibitor administration wherein a patient’s sample is analysed to detect the presence or absence of senescent cells.

Herebelow examples are shown having the aim of better illustrating the methods detected in the present description, such examples in no way are to be considered as a limitation of the preceding description and of the following claims.

EXPERIMENTS

With the purpose of selecting effectively senolytic drugs the platform was further implemented to identify compounds capable of promoting the senescence previously developed in the inventors’ laboratory (see Kalathur, M. et al. A chemogenomic screening identifies CK2 as a target for pro-senescence therapy in PTEN-deficient tumour Nat. Commun. 6, 1221 (2015)).

With the purpose of being able to identify new compounds which could be quickly translated to the clinic, without facing problems of safety, toxicity and pharmacokinetics, drugs were selected already approved for use in oncological field. Our study started with a library of 150 drugs, including inhibitory molecules at the preclinical development stage or currently used in the oncological therapy. Subsequently, the screening was extended to a wider range of 1500 compounds including:

(1) new chemical entities;

(2) compounds at advanced development stage;

(3) drugs already used in clinic for the treatment of pathologies of not oncological nature (Neurology, HIV, virology, etc.);

(4) new drugs at development stage (LOPAC) and (5) compounds of natural origin (Figure 1. A).

The platform which was devised in the inventors’ laboratory is capable of identifying the senolytic compounds based upon two parameters: (1) the inhibition of the cell viability and (2) the removal of the positive cells to the assay of b-Galactosidase (SA- b -Gal, senescence marker). As positive control of the compounds at the test stage, the compound ABT-263 was included, previously identified as senolytic compound (see Chang, J. et al. Clearance of senescent cells by ABT263 rejuvenates aged hematopoietic stem cells in mice. Nat. Med. 22, 78-83 (2015)). At last the senolytic potentiality of these compounds is tested on murine embryonic fibroblasts (MEF) Pten ^_/ and wild type (WT).

Technically the screening was performed in three passages;

Before treatment, to the culture medium of MEF (DMEM, 10%FBS, 5% P/S) Docetaxel is added, at the concentration of 750 nM for 48 hours. This drug is capable of increasing to 80% the senescence of MEF Pten ^A , without altering the cell cycle of MEF WT (Figure 1 B). - Afterwards, in the cells the culture medium was changed and they were left to grow for 24 hours. The cells were seeded in a 96-well plate at a density of 5000 cells per well. In the more external wells PBS was added, so as to be able to maintain the correct humidity inside the plate.

- At last, 24 hours after the seeding of cells, the compounds of our study were added to the cells for 48 hours at the concentration of 10 mM.

The senolytic screening allows to filter the compounds based upon three properties:

Effectiveness: the drug/tested substance in triplicate, at one single concentration (10 mM), in MEF Pten ^A has to be capable of determining a huge decrease in the cell viability.

- Specificity: the drug/tested substance in triplicate, at one single concentration (10 pM), in MEF Pten ^_/ and wt has to be capable of determining a huge decrease in the viability of cells Pten ^A without impacting negatively on MEFs wt. Dose-Response: the tested drug in duplicate in the assay of dose-response, both in MEF Pten ^_A and wt has to allow the calculation of IC50. The data of each plate were normalized on the respective negative control, depending upon the cell population, Ptenfl/fl and Pten ^A (Figure 1C).

As final result of this 1650-compound screening Plerixafor (trade name Mozobil) was identified. Our data show that at 10pM Plerixafor has an effectiveness comparable to ABT-263, in terms of removal of senescent cells (PICS), but it results to be more selective by impacting less seriously on MEF wt (NS).

The senolytic activity of Plerixafor was validated in the context of the replicative senescence: assay which mimes the natural process of cell ageing due to the shortening of telomers. The MEFs were seeded in 10cm2-plates (3x105 cells/plate). Every 3 days these cells were detached by means of trypsin, counted with Trypan Blu and plated in the same number. This process was performed for 8 passages before starting the senolytic treatment. At passages 8 and 9 the senescence of these cells was tested by means of assay of SA^-Gal (about 78% of senescent cells). At passage 8, the non-senescent cells (NS) and cells in replicative senescence (RS) were seeded in a 96-well plate (5000 cells/well) and treated at different concentrations of Plerixafor. At the concentration of 2.5 mM, Plerixafor was capable of removing the senescent cells without impacting on the cells NS, as demonstrated by the assays of Crystal Violet and BA-b-Gal (Figure 2B).

Subsequently, we tested the effects of Plerixafor on cell lines of cancer pre-treated with compounds which activate senescence. In the specific case TrampCI and PC3, two cell lines of cancer of prostate, murine and human respectively, were used. With the purpose of inducing senescence in these cells, Palbociclib was used, a specific inhibitor of cyclins 4/6 currently used in clinic in the treatment of breast cancer. Once the cells started senescence, Plerixafor was added to the culture medium at the concentration of 5 mM for 48 hours. Our data demonstrate that Plerixaor is capable of removing successfully the senescent cells as it clear from the assays of Crystal Violet and SA^-Gal both in TrampCI and PC3 (Figure 2C, D).

We tested the senolytic capability of Plerixafor in vivo. We injected subcutaneously 2x10 ⁶ PC3 in athymic nude 7-week-old male mice. Once the tumour xenograft has reached a volume of 100mm ³ we started the treatment with Palbociclib (4mg/mouse by means of oral gavage, twice a week). In the three weeks after the treatment, a growth slowing down in most tumour cells is noted (Figure 3A).

Subsequently, the mice were divided into two groups, a control group (Palbo_PBS) and an experimental group (Palbo_Pleri) treated daily with Plerixafor 150 pg/mouse by means of IP. The treatment, which lasted more than one month (33 days), allowed to notice a strong reduction in the tumour growth in the group treated with Plerixafor (Figure 3B). The tumour samples were collected at different treatment time to study in detail the treatment effects on these tumours. Thanks to the technique of RT-qPCR we analysed the expression of some genes known for having a role in senescence. The analysed genes show a strong reduction in the senescence markers in the group treated with Plerixafor with respect to the control group.

We tested the senolytic capability of in vivo Plerixafor even in a second murine model. To this purpose we used the murine model p16 Luc. This model is engineered so that the luciferase protein is produced in the whole body of the animal when the endogenous gene p16, one of the senescence markers, is expressed. One week after chemotherapy with doxurubicin (10mg/kg) in single administration, the mice p16 Luc suffer from systemic senescence detectable from the increase in the light intensity of luciferase in different tissues. As recently demonstrated the presence of these senescent cells is responsible for side effects such as fatigue, accelerated ageing and greater susceptibility to pathologies correlated to ageing (Figure 4A). The removal of these cells is associated to an improvement in fatigue due to chemotherapy and symptoms associated to accelerated ageing.

The mice p16 Luc were divided into two groups, a control group (PBS) and an experimental group (Doxo_Pleri) treated daily with Plerixafor 150 pg/mouse through IP. During the treatment the luminescence of these mice was measured with respect to a group which received only the single treatment of Doxorubicin and the control group (Untr) which did not receive any treatment (Figure 4B). Our analyses demonstrate that 5 and 7 days of treatment with Plerixafor are sufficient to remove selectively, at systemic level, the senescent cells expressing the gene p16 and the luciferase reporter. This phenomenon can be noticed from the reduction in the luminescence levels only in the mice treated with Plerixafor (Figures 4B and C).

In sum, our in vitro and in vivo data show that Plerixafor is a powerful drug capable of removing selectively the senescent cells.