WO2019180636A1

WO2019180636A1 - New prosenescence drugs

Info

Publication number: WO2019180636A1
Application number: PCT/IB2019/052258
Authority: WO
Inventors: Ajinkya REVANDKAR; Jingjing Chen; Manuel COLUCCI; Mariantonietta D'AMBROSIO; Andrea Alimonti
Original assignee: Fondazione Per L' Istituto Oncologico Di Ricerca (Ior)
Priority date: 2018-03-21
Filing date: 2019-03-20
Publication date: 2019-09-26
Also published as: IT201800003818A1

Abstract

The present invention relates to the use of inhibitors of the metalloproteinase ADAMTS5 and compositions including it, for use as inducer drug of cellular senescence. In particular they relate to the use of inhibitors of the metalloproteinase ADAMTS5 for use as inducer drug of cellular senescence in a tumour treatment method.

Description

"NEW PROSENESCENCE DRUGS”

DESCRIPTION

The present invention relates to the use of inhibitors of the metalloproteinase ADAMTS5 and compositions including it, for use as inducer drug of cellular senescence. In particular they relate to the use of inhibitors of the metalloproteinase ADAMTS5 for use as an inducer of cellular senescence in a tumour treatment method.

STATE OR PRIOR ART

Senescence is a state wherein the cell, in response to precise stimuli, loses the replicative capacity even if it remains metabolically active. It plays physiologically important roles during the aging processes and it results to be crucial in contrasting the uncontrolled growth of the tumour cells. Even if the senescent cells are in a not proliferative state, they remain however metabolically active and start to secrete a large 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”). The fact of increasing cellular senescence in a tumour context determines the cancer growth arrest and the activation of the immune response, which can be exploited in the oncology therapy. Nowadays there is a limited number of compounds capable of promoting the cellular senescence which show poor specificity for the tumour cells.

The object of the present invention is to provide new substances with pro-senescence activity for use in treating tumours or other associated pathologies wherein the activation of cellular senescence results to be useful.

SUMMARY OF THE INVENTION

The present invention is based upon the finding that the inhibition of ADAMTS5 is capable of activating selectively senescence of tumour cells, such finding is supported by the experiments shown in details in the related experimental section of the present description. The authors of the present invention, in fact, have identified new compounds capable of promoting cellular senescence in a tumour model which provides the deletion of the tumour-suppressor gene Pten. Therefore, the present invention firstly relates to the inhibitors of the metalloproteinase ADAMTS5 for use as an inducer of cellular senescence in a treatment method, in particular in a tumour treatment method.

Secondly, it relates to compositions comprising the metalloproteinase ADAMTS5 for use as an inducer of cellular senescence in a treatment method, in particular in a tumour treatment method.

Additional advantages, such 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 chemogenomic screening for identifying pro-senescence compounds in PICS. (A) Screening experimental drawing. MEFs Pten^_/ and MEF Pten wt cells were treated with one single dose 10 mM in triplicate. Senescence is detected thanks to the inhibition of cell proliferation (Crystal Violet assay) and by the assay from positivity of b-Galactosidase associated to senescence (SA^-Gal). The compounds which are capable of inhibiting proliferation and increasing senescence in MEF Pten null are tested for IC50. Only the compounds which satisfy these filters will be then subjected to subsequent tests to investigate senescence through additional markers, subsequently tested on cancer cell lines, and at least on in-vivo preclinical models. (B). Screening graphic representation. Starting from more than 90,000 compounds, this chemogenomic approach can identify compounds and genes capable of increasing the senescence induced by Pten loss (PICS, Pten loss induced cellular senescence).

Figure 2 Inhibition of ADAMTS5 as pro-senescent strategy. (A) The pro- senescence strategy demonstrated that the inhibition of ADAMTS5 is a strategy for increasing PICS. This was demonstrated by using two different small molecules capable of inhibiting the activity of ADAMTS5. Our data demonstrate a strong decrease in the cell proliferation of MEF Pten null but not in MEF Pten wt. In MEF Pten null the inhibition of ADAMTS5 is capable of increasing PICS over 80%, as demonstrated by the assay of SA^-Gal. These data were validated, by testing the two inhibitors of ADAMTS5 even in human cell lines of prostate cancer, PC3. Our data show a strong inhibition of the cell proliferation and an increase in the percentage of the cells positive to the assay of SA^-Gal with respect to control (DMSO). (B) Chemical structure of the two used inhibitors of ADAMTS5. (C) We validated these results in a wide panel of human cell lines of prostate cancer (22RV1 , LNCaP, Du-145) and murine cell lines of prostate cancer murine (TrampCI and TrampCI Pten null). Moreover, the inhibition of ADAMTS5 was tested even on human cell lines of colon cancer, HTC116 and HTC116 PTEN null. The bar graphs show that in all these cell lines our data demonstrate that the inhibition of ADAMTS5 is capable of decreasing the cell proliferation (C) and at the same time increasing senescence (D).

Figure 3 The pharmacological inhibition and the down-regulation of ADAMTS5 are capable of promoting senescence in cancer cell lines. Analyses of the transcriptional levels of senescence marker genes (p16, p21 , pp27 and PAI-1). (A) The bar graph shows the expression profiles of these genes in TrampCI treated with 2.5 mM of inhibitor of ADAMTS5. A marked increase in all senescence markers in the treated cells (TC1 2.5 pM) is noted with respect to the untreated control (TC1). We subsequently validated these data by decreasing the transcription of ADAMTS5 thanks to the shRNA directed against ADAMTS5. Several sequences were tested both in lines of human, PC3 (B), and murine, TrampCI (C), prostate cancer. Once identified the sequence capable of reducing the transcription of ADAMTS5 more effectively we studied in this context the senescent response caused by the ablation of ADAMTS5. (B) In the cell line PC3 the sh39 sequence is capable of down-regulating ADAMTS5 by 85%. In these cells then we analysed the expression profile of the senescence markers p16, p21 , p27 and PAI-1 by observing a senescence increase in the cells wherein ADAMTS5 is down-regulated (PC3 sh 39) with respect to the parental line (PC3). Analogously, we performed the same experiments in cell lines of murine prostate cancer, TrampCI (C). We identified sh64 as the best one among those tested (down-regulation of ADAMTS5 by 80%). Subsequently in these cells the expression profiles of senescence markers p16, p21 , p27 and PAI-1 were analysed. The bar graph shows a senescence increase in the cells wherein ADAMTS5 is down-regulated (TC1 sh64) with respect to the parental line (TC1).

DETAILED DESCRIPTION OF THE INVENTION

The present invention consists of the following aspects, described in details hereinafter. The present invention firstly relates to the use of inhibitors of the metalloproteinase ADAMTS5 as inducers of cellular senescence in a treatment method, in particular in a tumour treatment method.

The treated tumour could be selected for example among pancreatic, prostate, lung, bladder, mammary gland, colorectal, sarcoma, kidney, thyroid cancer. Other types of cancers can be added, if required.

In the present description under the term“inducer of cellular senescence” a substance capable of promoting the state of cellular senescence is meant.

ADAMTS5 is an enzyme known to the person skilled in the art also as“A disintegrin and metalloproteinase with thrombospondin motifs 5” and it is described in The Journal of Biological Chemistry. 274 (36): 25555-63. The reference of human protein in UNIPROT data bank is Q9UNA0.

Examples of inhibitors of the metalloproteinase ADAMTS5 are the GLPG 1972 small molecule and the CRB0017 therapeutic antibodies and its subsequent therapeutic antibodies developed starting therefrom, for example variants thereof or biosimilar substances. According to an embodiment an inhibiting or degrading anti-ADAMTS5 antibody will be used.

According to an embodiment a genetic inhibitor of the metalloproteinase ADAMTS5 will be used, that is an inhibitor capable of inhibiting the gene expression of ADAMTS5, for example an antagomir, an antisense oligonucleotide and a RNA inhibitor. Specific examples of this type of inhibitor are the short hairpin RNA cloned inside the pLKO vector. In short, with this method the cells are engineered with a constitutively active vector which transcripts nucleotide sequences pairing to the mRNA transcript of ADAMTS5. This will make that ADAMTS5 transcript is degraded, thus obtaining the knock-down of ADAMTS5. The antisense sequences used to make the knock-down of ADAMTS5 are shRNA 39 having SEQ ID NO:1 shown hereinafter: (CCGGACCATT AGAT GT CCGTT AT AGCTCGAGCT AT AACGGACAT CT AAT GGTTTT TTTG) as sequence directed against the human transcript.

Whereas as sequence pairing to the murine transcript we used

shRNA 64 having SEQ ID NO:2 shown hereinafter

(CCGGGCAGAGT AT GAACGAATTT ATCT CGAGAT AAATT CGTT CAT ACT CTGCTTT

TTG).

The inhibitors of the metalloproteinase could be used in treating patients suffering from cancer, preferably in association to one or more chemotherapeutic agents, for example Docetaxel, Palbociclib, Cisplatin and Doxorubicin.

The present invention further relates to a pharmaceutical composition comprising inhibitors of the metalloproteinase ADAMTS5 and a carrier and/or a diluent for use as activator of senescence in a method for treating anyone of the conditions to be treated shown in the present description. The composition comprising the inhibitor of the metalloproteinase ADAMTS5 could be oral, parental, rectal, transdermal, topic or suitable by 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, as individual therapeutic agents or in a combination of therapeutic agents. They can be administered alone, but generally are administered with a pharmaceutical carrier selected based upon the selected 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 agent and upon the administration mode and route thereof; upon age, health and weight of the receiver; upon nature and level of symptoms, upon the type of concomitant treatment; upon the treatment frequency; and upon the wished effect. It can be provided that a daily dosage of active ingredient is about 0.001 up to 1 ,000 milligrams (mg) per kilogram (kg) of body weight, with the preferred dose being 0.1 up to about 30 mg/kg. Dosage forms (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 usually will be present in a quantity of about 0.1-95% by weight based upon the total weight of the composition. The inhibitor of the metalloproteinase ADAMTS5 can be administered by oral route in solid dosage forms, such as capsules, tablets and powders, or in liquid dosage forms, such as elixir, syrups and suspensions. It can be administered even by parental route, in sterile liquid dosage forms. Capsules of gelatine include the active ingredient and pulverized carriers, such as lactose, starch, cellulose derivatives, magnesium stearate, stearic acid, and the like. Similar diluents can be used for preparing pressed tablets. Both tablets and capsules can be manufactured as prolonged-release products to provide continuous release of medicament for a period of some hours. Pressed tablets can be coated with sugar or coated with film to mask any unpleasant taste and protect the tablet from the 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 increase 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 suitable carriers 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. Citric acid and salts thereof and sodium EDTA are also 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 could include one or more active ingredients, that is pharmaceutically active, in particular chemotherapeutic, substances.

Even the methods for therapeutic or cosmetic treatment of the above-mentioned pathological conditions are herein described, comprising a step for administering inhibitors of ADAMTS5 or compositions including them. The method could be for the cancer treatment and include a step preceding the administration of the inhibitor wherein a sample of patient is analysed to detect the senescence state of the cells (this paragraph is only for the possible US patent).

Examples are reported herebelow which have the purpose of better illustrating the methods shown in the present description, such examples in no way are to be considered as a limitation of the previous description and of the following claims.

EXPERIMENTS

With the purpose of selecting effectively 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 tumour” Nat. Commun. 6, 1221 (2015) was implemented.

With the purpose of being able to identify new compounds which could be quickly translated to clinic, without incurring safety, toxicity and pharmacokinetic problems, we selected the pro-senescent capability of drugs already approved in clinic in oncological field and for the treatment of diseases of various nature (500 compounds).

Our study is implemented with a library of 90,000 compounds comprising:

(1) new chemical entity;

(2) compounds in development advanced phase;

(3) drugs already used in clinic for the treatment of pathologies of not oncological nature (Neurology, HIV, virology, etc.) (Figure 1.A).

The platform we devised in our laboratory can identify the compounds capable of activating cellular senescence based upon two parameters: (1) the cell proliferation arrest and (2) the percentage increase in the cells positive to the assay of b- Galactosidase (SA- b -Gal, senescence marker). The pro-senescent potentiality of these compounds is tested on embryonal murine fibroblasts (MEF) Pten-/- and WT (Figure 1.B).

The screening of pro-senescent compounds allows to filter the compounds based upon three properties:

- Effectiveness: the drug tested in triplicate, at one single concentration (10 mM), in MEF Pten-/- has to be capable of determining an enormous decrease in the cell proliferation and increasing the percentage of cells positive to the assay of SA- b -Gal.

Specificity: the drug tested in triplicate, at one single concentration (10 pM), in MEF Pten-/- and wt has to be capable of determining an enormous decrease in the cell proliferation and increasing the percentage of cells positive to the assay of SA- b -Gal in the cells Pten-/- without affecting negatively MEFs wt.

Dose-Response: the drug tested in triplicate in the dose-response assay, both in MEF Pten-/- 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-/- (Figure 1A, B).

As final result of this screening of more than 90,000 compounds, we identified ADAMTS5 as target to be inhibited to create and increase cellular senescence.

ADAMTS5 is a metalloproteinase performing an important function in development and homeostasis of the connective tissue. ADAMTS5 is often studied for its role in the development of inflammation and pathologies connected to osteoarthritis, caused by degradation of cartilage.

Our data show that the selective inhibition of ADAMTS5 with two different inhibitors (the chemical structure thereof is shown in figure 2. B), is capable of arresting the cell proliferation specifically in MEF Pten null, without affecting the proliferation of MEF Pten wt (Figure 2. A). Specularly thereto, the inhibition of ADAMTS5 is capable of increasing the percentage of senescent cells in MEF Pten-/- by 80%, as demonstrated by the assays of SA^-Gal. (Figure 2. A).

This piece of data was subsequently confirmed by testing the effects of the inhibition of ADAMTS5 in PC3, a human cell line of prostate cancer. We tested 3 different inhibitors with different chemical structure (Figure 2. B), which, in both cases, determined an enormous arrest of the proliferation and increase in the SA^-Gal positive cells, with respect to the untreated control (DMSO).

Subsequently we validated the pro-senescent capability of the inhibition of ADAMTS5 on a wide panel of human (22RV1 , LNCaP, Du-145, HTC116 and HTC116 PTEN null) and murine (TrampCI and TrampCI Pten null) cell lines of cancer (Figure 2.C, D). In all these lines we observed an inhibition of the cell proliferation thanks to the Crystal Violet assay (Figure 2.C) and an increase in the percentage of SA^-Gal positive cells, index of cellular senescence. Afterwards, we investigated the gene expression of the senescence markers p16, p21 , p27 and PAI-1 , after the inhibition of ADAMTS5. For this purpose, we used the TrampCI model, a murine cell line of prostate cancer. The cells were treated with the inhibitor of ADAMTS5 at the dose of 2.5mM for 48 hours and subsequently they were collected for the transcriptional expression studies. The cells treated with 2.5mM of inhibitor of ADAMTS5 (TC1 2.5uM) show a strong increase in all senescence markers with respect to the untreated control (TC1) (Figure 3. A).

At last, we studied if, analogously to the pharmacological inhibition, the gene down- regulation of ADAMTS5 was capable of inducing cellular senescence. For this purpose, we used two cell lines of prostate cancer, a human one, PC3, and a murine one, TrampCI . The down-regulation of the mRNA levels was performed by using the technology of the short hairpin RNA (shRNA). We validated several sequences and selected those determining a decrease in mRNA of about l’80% (Figure 3.B, C). Subsequently, we studied the expression profile of the senescence markers p16, p21 , p27 and PAI-1 of the cells wherein we down-regulated ADAMTS5 with respect to the parental cells (wherein ADAMTS5 is not down-regulated).

In the PC3 model, our data show that shRNA 39 is capable of down- regulating the mRNA levels by 85%. These cells were collected to study the transcriptional profiles of p16, p21 , p27 and PAI-1. Our data show that the cells wherein ADAMTS5 is down- regulated (PC3 sh39) have an increase in the expression of these senescence markers with respect to the parental cells (PC3) (Figure 3.B).

Analogously, in the TrampCI model our data show that shRNA 64 is capable of down regulating the mRNA levels by 80% in TrampCI . These cells were collected to study the transcriptional profiles of p16, p21 , p27 and PAI-1. Our data show that the cells wherein ADAMTS5 is down-regulated (TC1 sh64) have an increase in the expression of these senescence markers with respect to the parental cells (TC1) (Figure 3.C).

Our data show that, in both cell lines of prostate cancer the down- regulation of ADAMTS5 is correlated to a strong increase in these senescence markers at transcriptional level. In conclusion our data show that both in primary cell lines and in human and murine cell lines of cancer, the (pharmacological and genetical) inhibition of ADAMTS5 is capable of bringing the cells in senescence.

Claims

1. An inhibitor of the metalloproteinase ADAMTS5 for use as an inducer of cellular senescence in a treatment method.

2. The inhibitor of the metalloproteinase ADAMTS5 for use according to claim 1 wherein said method of treatment is a method of treatment of the tumour.

3. The inhibitor of the metalloproteinase ADAMTS5 for use according to claim 2 wherein said cancer is selected from pancreatic, prostate, lung, bladder, mammary gland, colon-rectum, sarcoma, kidney, thyroid and melanoma cancer.

4. The inhibitor of the metalloproteinase ADAMTS5 for use according to anyone of claims 1 to 3 in association with a chemotherapeutic drug, in particular in association with Docetaxel and/or Palbociclib.

5. The inhibitor of the metalloproteinase ADAMTS5 for use according to anyone of claims 1 to 4 wherein said inhibitor is selected from GLPG1972, shRNA having SEQ ID NO:1 or SEQ ID NO:2, an antagomir, an antisense oligonucleotide, a miRNA, an organic molecule, the CRB0017 antibody or variants thereof.

6. A pharmaceutical composition for use as an inducer of cellular senescence in a treatment method comprising an inhibitor of the metalloproteinase ADAMTS5 and one or more carriers and/or diluents and/or excipients.

7. The pharmaceutical composition for use as an inducer of cellular senescence in a tumour treatment method comprising an inhibitor of the metalloproteinase ADAMTS5 and one or more carriers and/or diluents and/or excipients.

8. The pharmaceutical composition for use according to claim 7 wherein said cancer is selected from pancreatic, prostate, lung, bladder, mammary gland, colorectal, sarcoma, kidney, thyroid cancer.

9. The pharmaceutical composition for use according to anyone of claims 6 to 8 further comprising one or more chemotherapeutic agents.

10. The pharmaceutical composition for use according to anyone of claims 6 to 9 wherein said inhibitor of the metalloproteinase ADAMTS5 is selected from GLPG1972, shRNA having SEQ ID NO:1 or SEQ ID NO:2, an antagomir, an antisense oligonucleotide, a miRNA, an organic molecule, an anti-ADAMTS5 antibody, in particular the CRB0017 antibody or variants thereof.