IT201800005072A1 - NEW PROSENESCENCE DRUGS - Google Patents

Description

"NEW PROSENESCENCE DRUGS"

DESCRIPTION

The present invention refers to the use of retinoid acid receptor activators and compositions that include them, for use as activating drugs of cellular senescence. In particular, they refer to the use of activators of a retinal acid receptor and / or 9-cis-retinoic acid for use as an activator of cellular senescence in a tumor treatment method.

STATE OF THE PRIOR ART

Senescence is a state in which the cell, in response to specific stimuli, loses its replicative capacity while remaining metabolically active. It plays physiologically important roles during the aging process and is crucial in countering the uncontrolled growth of cancer cells. Although senescent cells are in a non-proliferative state, they still remain metabolically active and begin secreting a large number of cytokines and pro-inflammatory factors, thus assuming a senescence-associated secretory phenotype (SASP). This "secretome" is able to activate the antitumor immune response and promote the removal of the senescent cells themselves ("surveillance of senescence"). Increasing cellular senescence in a tumor context leads to an arrest of the growth of the neoplasm and activation of the immune response, which can be exploited in cancer therapy.

To date, there are a limited number of compounds capable of promoting cellular senescence which exhibit poor specificity for cancer cells.

The purpose of the present invention is to provide new substances with pro-senescence activity for use in the treatment of tumors or other associated pathologies in which the activation of cellular senescence is useful.

SUMMARY OF THE INVENTION

The present invention is based on the discovery that the activation of retinoid acid receptors is able to selectively induce the senescence of cancer cells, this discovery is supported by the experiments reported in detail in the relevant experimental section of this description. The authors of the present invention have in fact identified new compounds capable of promoting cellular senescence in a tumor model that involves the deletion of the tumor suppressor gene Pten.

Therefore, a first object of the present invention are activators of a retinal acid and / or 9-cis-retinoic acid receptor for use as an inducer of cellular senescence in a treatment method, in particular in a tumor treatment method.

A second object are compositions comprising activators of a retinal acid and / or 9-cis-retinoic acid receptor for use as an inducer of cellular senescence in a treatment method, in particular in a tumor treatment method.

Further advantages, as well as the characteristics and methods of use of the present invention will become evident from the following detailed description of some preferred embodiments.

DETAILED DESCRIPTION OF THE FIGURES

Figure 1 Chemogenomic screening platform for the identification of pro-senescence compounds in PICS. (A) Experimental design of the screening. MEFs Pten - / - and MEF Pten wt cells were treated at a single dose 10 µM in triplicate. Senescence is detected thanks to the inhibition of cell proliferation (Crystal Violet assay) and the positive β-Galactosidase assay associated with senescence (SA-β-Gal). Compounds that are able to inhibit proliferation and increase senescence in Pten null MEFs are tested for IC50. Only the compounds that satisfy these filters will then be subjected to subsequent tests to investigate senescence using additional markers, subsequently tested on cancer cell lines, and finally on preclinical models in vivo. (B). Graphic representation of the screening. Starting from more than 90,000 compounds, this chemogenomic approach identifies compounds and genes capable of increasing the senescence induced by the loss of Pten (PICS, Pten loss induced cellular senescence).

Figure 2 Activation of RARγ-RXRγ receptors as a pro-senescent strategy. (A) Pro-senescent screening has shown that the activation of retinoid acid receptors (RAR and RXR subunit γ) is a strategy to increase PICS. This is demonstrated using three different small molecules capable of activating the RARγ-RXRγ, Adapalene, Acitracin and Bexarotene receptors. (B) Our data demonstrate a strong decrease in cell proliferation in MEF Pten null but not in MEF Pten wt. In Pten null MEFs, the activation of RARγ-RXRγ receptors is able to increase PICS, as demonstrated by the SA-β-Gal assay. (C) These data were validated by testing the three RARγ-RXRγ receptor activators also in human prostate cancer cell lines, PC3. Our data show a strong inhibition of cell proliferation and an increase in the percentage of SA-β-Gal positive cells compared to the control (DMSO).

Figure 3 Activation of RARγ-RXRγ receptors as a pro-senescent strategy in cancer cell lines. (A) We validated the activation of RARγ-RXRγ receptors in a large panel of human prostate cancer cell lines (22RV1, LNCaP, Du-145), murine prostate cancer cell lines (TrampC1 and TrampC1 Pten null) and human colon cancer cell lines HTC116 and HTC116 PTEN null treated with Acitracin. Our data demonstrate a strong inhibition in cell proliferation and a consequent increase in the percentage of positive cells in the SA-β-Gal (C) assay. These data have been validated by treating these cell lines with Bexarotene, as shown by the data of Crystal Violet which highlight an arrest of cell proliferation (B) and an increase in the percentage of positive cells in the β-Galactosidase assay (D). .

Figure 4 Transcriptional profile of senescence markers following the activation of RARγ-RXRγ receptors. Transcriptional analysis of known genes used as senescence markers (p16, P21, p27, PAI-1). In both human prostate cancer cell lines, PC3 (A) and LNCaP (B), all senescence markers are transcribed more (in gray) than in the untreated control (in black).

DETAILED DESCRIPTION OF THE INVENTION

The pro-senescent screening carried out by the inventors has shown that the activation of retinoid acid receptors (RAR and RXR subunit γ) is able to activate the senescent response. Retinal acid receptors are also known as RAR and 9-cis-retinoic acid as RXR. A first aspect of the present invention therefore relates to the use of activators of a retinal acid and / or 9-cisretinoic acid receptor in a treatment method, in particular in a tumor treatment method. The treated tumor can be chosen, for example, from pancreatic, prostate, lung, bladder, mammary gland, colorectal, sarcomas, kidney, thyroid cancer. In this description, the term "inducer of cellular senescence" means a substance capable of promoting the state of cellular senescence.

Examples of retinoid acid receptor activators are Adapalene, Acitracin and Bexarotene. By way of example, the structural formula of Adapalene is shown below:

The present subject also relates to a pharmaceutical composition comprising one or more activators of the retinoid acid receptors and a carrier and / or a diluent for use as an activator of senescence in a method of treating any of the conditions to be treated reported in the present description. The composition comprising the activator may be oral, parenteral, rectal, transdermal, topical or suitable for other route of administration. The compositions for use according to the present invention may be administered by any conventional means available for use in conjunction 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 based on the chosen route of administration and standard pharmaceutical practice. The dosage administered will obviously vary depending on known factors, such as the pharmacodynamic characteristics of the particular agent and its mode and route of administration; the age, health and weight of the recipient; the nature and degree of symptoms, the type of concomitant treatment; the frequency of treatment; and the desired effect. A daily dosage of active ingredient can be expected to be about 0.001 to 1000 milligrams (mg) per kilogram (kg) of body weight, with the preferred dose being 0.1 to about 30 mg / kg. Dosage forms (compositions suitable for administration) typically contain from about 1 mg to about 100 mg of active ingredient per dosage unit. In these pharmaceutical compositions, the active ingredient will normally be present in an amount of about 0.1-95% by weight based on the total weight of the composition.

The activator, or the composition that includes it, can be administered orally in solid dosage forms, such as capsules, tablets, and powders, or in liquid dosage forms, such as elixirs, syrups, and suspensions. It can also be administered parenterally, in sterile liquid dosage forms. Gelatin capsules contain the active ingredient and pulverized carriers, such as lactose, starch, cellulose derivatives, magnesium stearate, stearic acid, and the like. Similar diluents can be used to prepare pressed tablets. Both tablets and capsules can be manufactured as sustained release products to provide continuous medication release over a period of several hours. Pressed tablets can be sugar-coated or film-coated to cover any unpleasant taste and protect the tablet from the atmosphere, or enteric-coated for selective disintegration in the gastrointestinal tract. Liquid dosage forms for oral administration may contain dyes and flavors to enhance patient acceptance. In general, water, a suitable oil, saline, aqueous dextrose (glucose), and solutions of related sugars and glycols such as propylene glycol or polyethylene glycols are suitable vehicles for parenteral solutions. Solutions for parenteral administration preferably contain a water-soluble salt of the active ingredient, suitable stabilizing agents, and, if necessary, buffering substances. Antioxidant agents such as sodium bisulfite, sodium sulfite, or ascorbic acid, alone or in combination, are suitable stabilizing agents. Citric acid and its salts and sodium EDTA are also used. Furthermore parenteral solutions may contain preservatives, such as benzalkonium chloride, methyl- or propyl-paraben, and chlorobutanol. The pharmaceutical compositions according to the present invention may comprise one or more active ingredients, i.e. pharmacologically active substances, in particular chemotherapeutic substances, such as for example Docetaxel and / or Palbociclib. The chemotherapeutic agents can, for example, be used in the dosages known to those skilled in the art.

The methods of therapeutic or cosmetic treatment of the aforementioned pathological conditions are also described herein, comprising a step for administering the activator or compositions that comprise them.

Also described here are kits of parts comprising a composition containing activators of retinoid acid receptors, such as for example Adapalene, Acitracin or Bexarotene and a composition comprising a chemotherapeutic agent, preferably Docetaxel and / or Palbociclib and their use in the therapeutic treatment of the pathological conditions above said.

Examples are reported below which have the purpose of better illustrating the methodologies disclosed in the present description, these examples are in no way to be considered as a limitation of the previous description and subsequent claims.

EXPERIMENTATION

In order to effectively select substances capable of promoting senescence, the platform developed and described in Kalathur, M. et al. A chemogenomic screening identifies CK2 as a target for pro-senescence therapy in PTEN-deficient tumor Nat. Commun. 6, 7227 (2015).

In order to be able to identify new compounds that can be quickly transferred to the clinic, without encountering safety, toxicity and pharmacokinetic problems, we have chosen to test the pro-senescent ability of drugs already approved in the clinic in the oncology field and for treatment. of various kinds of diseases (500 compounds).

Our study is implemented with a library of 90,000 compounds which includes: (1) new chemical entities;

(2) compounds in an advanced stage of development;

(3) drugs already used in the clinic for the treatment of pathologies of a non-oncological nature (Neurology, HIV, virology, etc.) (Figure 1.A).

The platform that we have developed in our laboratory is able to identify compounds capable of activating cellular senescence on the basis of two parameters: (1) the arrest of cell proliferation and (2) the increase in the percentage of cells positive in the β-Galactosidase assay (SA-β – Gal, marker of senescence). The pro-senescent potential of these compounds is tested on murine embryonic fibroblasts (MEFs) Pten - / - and WT (Figure 1.B).

The screening of pro-senescent compounds allows to filter the compounds on the basis of three properties:

- Efficacy: the drug tested in triplicate, at a single concentration (10 µM), in MEF Pten - / - must be able to bring about a massive decrease in cell proliferation and increase the percentage of positive cells in the SA- β - assay Gal.

- Specificity: the drug tested in triplicate, at a single concentration (10 µM), in the MEF Pten - / - and wt must be able to bring about a massive decrease in cell proliferation and increase the percentage of positive cells in the SA- assay β –Gal in Pten - / - cells without negatively impacting on MEFs wt.

- Dose-Response: the drug tested in duplicate in the dose-response assay, both in the MEF Pten - / - and wt must allow the calculation of the IC50. The data of each plate were normalized on the respective negative control, depending on the cell population, Ptenfl / fl and Pten - / - (Fig 1A, B).

As a final result of this screening of more than 90,000 compounds, we have identified RARγ-RXRγ as a target to be activated to establish and increase cellular senescence.

The retinal acid receptors (known as RAR) and 9-cis-retinoic acid (known as RXR) belong, respectively, to the first and second class of intracellular receptors. Unlike RARs, the characteristic of RXRs is that when activated it does not dimerize with receptors of the same class but with other orphan or first-class receptors. The natural ligand of these receptors is retinoic acid.

Retinoic acid is a metabolite that is involved in the functions of vitamin A (retinol) necessary for growth and development. It is fundamental in embryonic development as it is necessary for the antero-posterior polarization of the embryo.

Our data show that the selective activation of retinoid acid receptors (RAR, RXR γ subunit) is able to activate the senescent response. These data were validated with three different compounds: Adapalene, Acitracin and Bexarotene (Figure 2.A). Treatment with these compounds is able to specifically stop cell proliferation in Pten-null MEFs, without impacting the proliferation of Pten-wt MEFs (Figure 2.B). Mirroring this, the activation of RARγ-RXRγ capable of increasing the percentage of senescent cells in MEF Pten - / - to 80%, as demonstrated by the SA-β-Gal assays. (Figure 2.B).

This finding was subsequently confirmed by testing the effects of activating RARγ-RXRγ in PC3, a human prostate cancer cell line. Also in this case we tested 3 different activators (Acitracin Adapalene, Bexarotene), which led, in all three cases, to a massive arrest of proliferation and increase of SA-β-Gal positive cells, compared to the untreated control. (DMSO) (Figure 2.C)

We then validated the pro-senescent ability of RARγ-RXRγ activation on a large panel of human (22RV1, LNCaP, Du-145, HTC116 and HTC116 PTEN null) and murine (TrampC1 and TrampC1 Pten null) cancer cell lines ( Figure 3.A B). In all these lines, following treatment with Acitracin (Figure 3.A) or Bexarotene (Figure 3.B) we observed an inhibition of cell proliferation detected thanks to the Crystal Violet assay. Our data show an increase in the percentage of SA-β-Gal positive cells, index of cellular senescence in cells treated with either Acitracin (Figure 3.C) or Bexarotene (Figure 3.D) Next, we investigated gene expression of the senescence markers p16, p21, p27 and PAI-1, following activation of RARγ-RXRγ. For this purpose, we used PC3 and LNCaP, two human prostate cancer cell lines as a model. The cells were treated for 48 hours with Adapalene at a dose of 10 µM and 5µM respectively, and were subsequently collected for transcriptional studies. Both cell lines treated with RARγ-RXRγ activators show a strong increase in all senescence markers compared to the untreated control (Figure 4.A and B).

In addition, further experiments were performed for the Chou-Talalay test in order to establish whether the RARγ-RXRγ agonists act additively or synergistically with conventional chemotherapeutic drugs in arresting cell proliferation.

Briefly, this test evaluates the synergy or additive effects obtained from the combination of two drugs at scalar concentrations. As an output of this we obtain the value of "Chou-Talalay index" which is the index of the synergistic strength of the two compounds, the lower this value, the stronger the joint action of the two drugs used (0-0.3 Strong synergism ; 0.3-0.9 Synergism; 0.9-1.1 Additive effect; higher than 1.1 There is no benefit).

RARγ-RXRγ agonist drugs were used in combination with Docetaxel or Palbociclib in this assay. Our data show that in the in vitro model TrampC1 the treatment of Docetaxel and Adapalene has an additive effect (Chou-Talalay Index 1.09), while the combined treatment of Docetaxel and Bexarotene has a synergistic effect (Chou-Talalay Index 0.86). Similarly, the activation of RARγ-RXRγ by Acitracin coupled to treatment with Palbociclib has an additive effect (Chou-Talalay Index 1.1). We also evaluated the Chou Talalay index on the PC3 cell line and we observed that in this model the treatment with Palbociclib and Adapalene has an additive effect (Chou-Talalay Index 1.1).

In conclusion, our data show that both in primary cell lines and in cancer cell lines, both human and murine, the activation of RARγ-RXRγ is able to bring the cells into senescence.

Claims (13)

CLAIMS 1. Activator of a retinal acid and / or 9-cis-retinoic acid receptor for use as an inducer of cellular senescence in a treatment method. 2. Activator for use according to claim 1 wherein said treatment method is a tumor treatment method. 3. Activator for use according to claim 2 wherein said tumor is selected from pancreatic, prostate, lung, bladder, mammary gland, colorectal, sarcomas, kidney, thyroid and melanoma tumors. Activator for use according to any one of claims 1 to 3 in association with a chemotherapeutic drug, in particular in association with Docetaxel and / or Palbociclib. 5. Activator for use according to any one of claims 1 to 4 wherein said receptor is selected between RARγ and RXRγ. 6. Activator for use according to any one of claims 1 to 5 wherein said activator is selected from Adapalene, Acitracin and Bexarotene. Activator for use according to any one of claims 4 to 6 wherein: - said activator is Bexarotene and said chemotherapy is Docetaxel; or - said activator is Acitracin and said chemotherapy is Docetaxel; or - said activator is Bexarotene and said chemotherapy is Palbociclib. 8. Pharmaceutical composition for use as an inducer of cellular senescence in a tumor treatment method comprising a retinal acid and / or 9-cis-retinoic acid receptor activator and one or more carriers and / or diluents and / or excipients. 9. Pharmaceutical composition for use according to claim 8 wherein said tumor is selected from tumor of the pancreas, prostate, lungs, bladder, mammary gland, colorectum, sarcomas, kidney, thyroid. Pharmaceutical composition for use according to any one of claims 8 to 9 further comprising one or more chemotherapeutic agents, in particular Docetaxel or Palbociclib. 11. Pharmaceutical composition for use according to any one of claims 8 to 10 wherein said receptor is selected from RARγ and RXRγ. Pharmaceutical composition for use according to any one of claims 8 to 11 wherein said activator is selected from Adapalene, Acitracin and Bexarotene. 13. Pharmaceutical composition for use according to any one of claims 8 to 12, wherein: - said activator is Bexarotene and said chemotherapy is Docetaxel; or - said activator is Acitracin and said chemotherapy is Docetaxel; or - said activator is Bexarotene and said chemotherapy is Palbociclib.