Patent Application
20240287134
The present invention pertains to the medical field. Precisely, the present invention refers to senotherapeutic agents comprising a peptide selected from the list consisting of or comprising: SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 3. In a preferred embodiment the present invention is directed to the prevention or treatment of osteoarthritis or aging-associated diseases caused by the persistence and accumulation of senescent cells (SnCs).
Osteoarthritis (OA) is the most common form of arthritis and it has been classified as an aging-associated disease [1, 2]. OA is a degenerative joint disorder characterised by progressive degeneration of synovial joints that lead to limited mobility and pain [3]. This disease is the major cause of disability with a major socio-economic impact, which affects an increasing number of the ageing population [4, 5]. Articular cartilage damage is the most typical sign of OA. Yet, this condition also affects the whole joint including bone, synovium, muscles, tendons and ligaments. Articular cartilage from patients with OA shows an accumulation of dedifferentiated and SnCs together with an increase in the production of pro-inflammatory cytokines such as IL-6 and IL-1β and catabolic enzymes that lead to the breakdown of cartilage extracellular matrix (ECM) and joint degeneration. The prevalence of OA is rising worldwide and the aetiology is still under study, with new insights into molecular mechanisms involved in OA progression beginning to open new possibilities to restore phenotypic stability of articular chondrocytes and to develop new approaches in order to promote cartilage repair and restore normal joint function in OA patients [6-8].
Accumulation of SnCs has been involved in the progression of different age-related diseases such as osteoarthritis (OA) [7, 9-12]. Cellular senescence consists in a cell-cycle arrest mechanism through sustained activation of the p16INK4A and/or p53-p21 pathways, which is involved in different biological processes such as development and tissue repair. Senescence cells cease dividing but are metabolic active cells, secreting factors refereed as senescence-associated secretory phenotype (SASP), which include cytokines, chemokines, growth factors and proteases [13]. This secretome is very complex and its products are mainly associated with inflammation, reprogramming, dedifferentiation and proliferation of surrounding cells together with changes in the synthesis and degradation of the extracellular matrix. The accumulation of SnCs in a chronic condition negatively impact on tissue structure and function. Examples of aging-associated diseases, which have been associated with accumulation of SnCs, are (non-exhaustive list): obesity, sarcopenia, fibrotic disease, atherosclerosis, cardiovascular disease, cancer, OA, arthritis, cataracts, osteoporosis, diabetes, Alzheimer's disease, hair loss, hypertension, inflammatory disease, dementia, kidney disease, muscular atrophy, neurological disease, pulmonary disease, vertebral disc degeneration and alopecia. The incidence of all of these diseases increases exponentially with age [9].
SnCs accumulate with age and contribute to the development of age-associated diseases. As a result, different strategies have emerged lately in order to therapeutically target SnCs. The therapeutic approach that takes advantage of compounds that target SnCs (senotherapeutics) is commonly described as senotherapy. Senotherapeutic agents comprise senolytics (selectively kill SnCs) and senomorphics/senostatics (compounds that modulate SASP). The clearance of SnCs and/or targeting of pathways involved in SASP production and secretion have been reported to alleviate the symptoms or evolution of some age-related diseases, including in OA. However, there is still a clear medical need of effective senotherapeutic agents with regenerative capacity able to selectively induce death of SnCs and/or reduce their accumulation in aged tissue in order to restore regeneration and tissue normal function.
The transmembrane protein connexin43 (Cx43) has been reported to reinforce senescence and dedifferentiation in articular chondrocytes from patients with OA and to contribute to disease progression [6]. However, Cx43 is a complex channel protein implicated in multiples biological processes. The different and diverse functions of Cx43 are specifically mediated by small domains throughout the protein sequence [14, 16-18]. Actually, several compounds including different peptide compounds have been designed and tested to modulate a specific function of the protein without affecting other functions [15, 19, 20]. Pharmacological inhibitors that specifically target certain activities of Cx43 such as therapeutic peptides are emerging as promising drugs for the treatment of different disorders such as cancer or neurodegeneration by targeting protein-protein interactions [15, 20, 21].
In fact, targeted modulation protein-protein interactions by therapeutic peptides has represented a unique and potent class of pharmaceutical compounds which is providing life-saving medicines since first half of the 20th century. Over 60 peptide drugs targeting different protein domains have been already approved and over 150 new peptides are in active clinical development [22-24].
The present invention refers to peptides which can be used as senotherapeutic agents to restore regeneration for the prevention or treatment of OA and aging-associated diseases caused by the persistence and accumulation of dedifferentiated and senescent cells, particularly senescent chondrocyte and dedifferentiated cells in articular cartilage and synovial joints.
The present invention refers to senotherapeutic agents comprising the following peptides, and their use in the prevention or treatment of OA or other aging-associated diseases caused by the persistence and accumulation of senescent cells:
In a preferred embodiment, the peptides SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 3 have been selected from a region of the protein Cx43 that is comprised between residues 232 to 266.
In a preferred embodiment the above cited peptides can comprise a tail of 8 arginines at the N-terminal domain to facilitate the internalization of the peptides within the cell, as well as other tag such as TAT or other molecules that facilitate their internalization.
In a preferred embodiment the above cited peptides can comprise a fluorophore like tetramethylrhodamine (TAMRA) linked to aminohexanoic acid (Ahx), or a fluorophore like tetramethylrhodamine (TAMRA) linked to a tail of 8 arginines and also to the aminohexanoic acid (Ahx).
OA is assayed in the present invention as an illustrative example of aging-associated diseases caused by the persistence and accumulation of senescent cells. Such as it is explained below, the peptides of the invention are able to i) reduce the accumulation of chondrocyte senescent cells for example by inducing their death via apoptosis or inhibition of the SASP and/or ii) trigger the re-differentiation of chondrocytes once they have been de-differentiated.
Such as can be seen in the results provided by the present invention:
Thus, these peptides are useful for the treatment of OA or other aging-associated diseases that progress with accumulation of senescent cells. OA can used in the present invention as an illustrative example to prove the suitability of the peptides of the invention to i) reduce the accumulation of senescent cells, for example by inducing their death via apoptosis or inhibition of the SASP and/or ii) trigger the re-differentiation of cells once they have been de-differentiated. Nevertheless, this means that the peptides of the invention could be used for the prevention and treatment not only of OA, but of any other aging-associated disease associated with the persistence and accumulation of senescent cells for example (non-exhaustive list): obesity, renal disease, sarcopenia, fibrotic disease, atherosclerosis, cardiovascular disease, cancer, arthritis, cataracts, osteoporosis, type 2 diabetes or Alzheimer's disease. The incidence of all of these diseases increases exponentially with age [9].
Therefore, the first embodiment of the present invention refers to a senotherapeutic agent comprising at least one peptide selected from the list consisting of SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 3, or any combination thereof. In a preferred embodiment, the peptides comprise a N-terminal tail with 8 arginines.
The second embodiment of the invention refers to a pharmaceutical composition comprising the above mentioned senotherapeutic agent and, optionally, pharmaceutically acceptable excipients or carriers. In a preferred embodiment the peptides or the pharmaceutical composition are administered orally or by means of an intraarticular, subcutaneous, intra-venous or muscular injection.
The third embodiment of the present invention refers to a peptide selected from the list consisting of SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 3, or any combination thereof (or a pharmaceutical composition comprising said peptides), for use as a senotherapeutic agent, preferably in the prevention or treatment of osteoarthritis. In a preferred embodiment, the prevention or treatment of osteoarthritis is achieved by reducing the accumulation of chondrocyte senescent cells. In a preferred embodiment the prevention or treatment of osteoarthritis is achieved by triggering the re-differentiation of osteoarthritic chondrocytes once they have been de-differentiated.
On the other hand, this embodiment refers to a method for the prevention or treatment of osteoarthritis which comprises the administration of a therapeutically effective amount of a peptide selected from the list consisting of SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 3, or any combination thereof (or a pharmaceutical composition comprising said peptides). In a preferred embodiment, the prevention or treatment of osteoarthritis is achieved by reducing the accumulation of chondrocyte senescent cells. In a preferred embodiment the prevention or treatment of osteoarthritis is achieved by triggering the re-differentiation of osteoarthritic chondrocytes once they have been de-differentiated.
The fourth embodiment of the invention refers to a peptide selected from the list consisting of: SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 3, or any combination thereof (or a pharmaceutical composition comprising said peptides), for use as a senotherapeutic agent, preferably in the induction of senescent cells death or modulation of SASP, particularly in the prevention and treatment of an age-related disease. In a preferred embodiment the age-related disease comprises at least one of obesity, sarcopenia, fibrotic disease, atherosclerosis, cardiovascular disease, cancer, OA, arthritis, cataracts, osteoporosis, diabetes, Alzheimer's disease, hair loss, hypertension, inflammatory disease, dementia, kidney disease, muscular atrophy, neurological disease including mild cognitive impairment; motor neuron dysfunction; Alzheimer's disease; Parkinson's disease; and macular degeneration, idiopathic pulmonary fibrosis or chronic obstructive pulmonary disease, vertebral disc degeneration, alopecia. diabetes, metabolic syndrome, and obesity. In a preferred embodiment the aging-associated diseases are caused by the persistence and accumulation of senescent cells.
On the other hand, this embodiment refers to a method for the prevention and treatment of an age-related disease which comprises the administration of a therapeutically effective amount of a peptide selected from the list consisting of SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 3, or any combination thereof (or a pharmaceutical composition comprising said peptides). In a preferred embodiment the age-related disease comprises at least one of obesity, sarcopenia, fibrotic disease, atherosclerosis, cardiovascular disease, cancer, OA, arthritis, cataracts, osteoporosis, diabetes, Alzheimer's disease, hair loss, hypertension, inflammatory disease, dementia, kidney disease, muscular atrophy, neurological disease including mild cognitive impairment; motor neuron dysfunction; Alzheimer's disease; Parkinson's disease; and macular degeneration, idiopathic pulmonary fibrosis or chronic obstructive pulmonary disease, vertebral disc degeneration, alopecia. diabetes, metabolic syndrome, and obesity. In a preferred embodiment the aging-associated diseases are caused by the persistence and accumulation of senescent cells.
For the purpose of the present invention the following definitions are provided:
The primary cultures of chondrocytes were obtained from articular cartilage of the hip and knee from patients with OA and healthy individuals obtained in the Complexo Hospitalario Universitario de Santiago de Compostela (CHUS-XXIS) under informed consent and approved by the Institutional Ethics Committee within the private collection of biological samples of the group (C.0003333, 2012/094 and 2015/029). With the help of a scalpel, the cartilage was separated from the bone and divided into small fragments of 1 mm. The fragments were then digested enzymatically with trypsin for 10 min at 37° C. under stirring, followed by digestion with collagenase IV at 37° C. under stirring for 16 hours. Subsequently, the sample was passed through a 100 μM filter and the cells were seeded in Dulbecco's modified Eagle medium medium (DMEM, Lonza) supplemented with 10% foetal bovine serum (SFB, Gibco, Thermo Scientific) and 1% Penicillin (100 U/ml)/Streptomycin (100 μg/ml) (Gibco, Thermo Scientific) and kept in culture in an incubator at 37° C., 5% CO2 and humidity saturation.
The tested peptides were synthesized in Centro Singular de Investigación en Quimica Biolóxica e Materiais Moleculares (CIQUS) and Centro de Investigacións Cientificas Avanzadas (CICA). The YSA1 peptide sequence corresponds to part of the C-terminal domain of Cx43 and contains a phosphorylation site of the Src kinase (Y247) and another phosphorylation site (S255) of the mitogen-activated protein kinase (Mitogen-Activated Protein Kinase, MAPK). A new peptide of similar sequence to YSA1 was synthesized but mutated in two positions and designated as YTP1 (
After completing the synthesis of the peptide sequences, their N-terminus were deprotected by treatment with 20% 4-methylpiperidine in N,N-dimethylformamide (DMF). Then, the fluorophore was attached to the N-terminus of the peptides by treatment of the deprotected resins with 6-Carboxytetramethylrhodamine succinimidyl ester in presence of N,N-diisopropylethylamine (DIEA) and DMF. Once the fluorophore was coupled to the peptides, those were cleaved from the resin and the corresponding residues were purified by reversed-phase HPLC, and the fractions identified as the desired products were lyophilized.
In a preferred embodiment the peptides can comprise a fluorophore like tetramethylrhodamine (TAMRA) linked to aminohexanoic acid (Ahx), or a fluorophore like tetramethylrhodamine (TAMRA) linked to a tail of 8 arginines and also to the aminohexanoic acid (Ahx).
An initial number of cells (5,000-10,000) were seeded in a 96-well culture plate and treated with different concentrations of the peptides. After the treatment, the medium was removed from each well and 10 μL of the MTT reagent was added to 100 μL of fresh medium and incubated at 37° C. for 4 hours. To solubilize the formazan crystals of the MTT reagent, it was incubated with DMSO at room temperature and under stirring for 30 minutes. Absorbance was measured at 570 nm in a plate reader (Nanoquant Infinite M200, TECAN).
Primary cultures of chondrocytes at a confluence of 70-80% were treated with the corresponding peptide. After 16 hours, the subcellular localization of the TMR-labeled peptides was visualized in vivo in a fluorescence inverted microscope (Inverted Research Microscope Eclipse Ti, Nikon).
Primary cultures of chondrocytes at a confluence of 70-80% were treated with mimetic peptides of Cx43, and fixed with 2-4% paraformaldehyde (PFA, Sigma-Aldrich) in phosphate buffered saline (PBS, MP Biomedicals) for 10 minutes at room temperature. The PFA was removed and cells were incubated in 0.1 M glycine (Sigma-Aldrich) for 10 minutes to remove autofluorescence from the PFA, and the cell membranes were permeabilized with 0.2% Triton X-100 (Sigma-Aldrich) in PBS for 10 minutes. The non-specific binding sites were blocked with 1% bovine serum albumin (BSA, Sigma-Aldrich) in PBS with Tween-20 (PBS-T, Sigma-Aldrich), for 30 minutes. Subsequently, the cells were incubated with the primary anti-Cx43 antibody (C6219, Sigma-Aldrich) for 1 hour, at room temperature and washed with PBS for 10 minutes to remove excess antibody. It was incubated with the secondary antibody labelled with FITC (anti-rabbit F2765, Thermo Fisher) for 1 hour at room temperature, in the dark. Excess antibody was removed, and the nuclei were stained with 4′, 6-diamino-2-phenylindole (DAPI, Sigma-Aldrich) for 4 minutes. Finally, preparations were mounted with a drop of glycergel aqueous mounting medium (Dako) and visualized in an Olympus BX61 fluorescence microscope using a DP71 digital camera (Olympus).
The cell pellet isolated from patients with OA and treated with the peptides or untreated were mechanically and chemically lysed using a needle of 29 gauges (BD Medical) in the presence of a cold lysis buffer (50 mM Tris HCl pH 7.5; 50 mM EDTA pH 8, 0.5% NP-40, 1% Triton X-100, 150 mM NaCl). Once the cell extracts were obtained, they were diluted in loading buffer (10% SDS, 200 mM Tris HCl pH 6.8, 50% glycerol, 0.1% Bromophenol Blue, 10% 0-Mercaptoethanol). Equal amounts of the cell extracts were separated on a 10% Acrylamide/Bisacrylamide (Acr/Bis) gel with SDS. The proteins were subsequently transferred to a polyvinylidene fluoride membrane (PVDF, Merck) in a Trans-Blot® SD Semi-Dry Transfer Cell (Bio-Rad). It was verified that the transfer was correct by staining the membrane with ATX Ponceau S (Sigma-Aldrich). To block possible nonspecific binding with the membrane, 1-hour incubation was performed at room temperature with a 5% milk solution in a TTBS buffer (20 mM Tris, 150 mM NaCl, 0.05% Tween-20). The membrane was incubated with the primary antibody at 4° C. under stirring for 16 hours. The excess antibody was removed with successive washes with TTBS and then incubated with the secondary antibody at room temperature under stirring for 1 hour. Excess antibody was removed, and the signal was developed with the commercial kit of Pierce™ ECL Western Blotting Substrate (Thermo Fisher Scientific) in an Amersham Imager 600 developing chamber (GE Healthcare). The primary antibodies used were anti-Cx43 (C6219, Sigma-Aldrich) and anti-α-tubulin (T9026 Sigma-Aldrich). The secondary antibodies used were a goat anti-rabbit (A6154 Sigma-Aldrich) and a sheep anti-mouse (NA-931 Sigma-Aldrich), respectively.
Cells were seeded in culture plates to an approximate 90% confluence. After treating the cells with the peptides, the medium was removed and washed twice with PBS. The fluorescent compound Lucifer Yellow (LY, Cell Projects Ltd © Kent) 0.4% in PBS was added and two cuts were made, one with a scalpel and the other with a needle on the cell monolayer. The cells were incubated for 5-7 minutes at 37° C. to allow the transfer of the LY between undamaged cells in contact, after which the LY was removed, and cells were washed with PBS. The cells were fixed with 4% formaldehyde for 10 minutes. The visualization was performed in an inverted fluorescence microscope (Inverted Research Microscope Eclipse Ti, Nikon).
After treatments with the peptides, the cells were trypsinized and the pellets were lysed in TRI Reagent (MRC), following the manufacturer's instructions until obtaining an RNA pellet. Subsequently, a treatment with 1 U/μl of DNAases was performed to eliminate contaminating DNA. The quality and quantity of RNA isolated was quantified in a Nanodrop ND-1000 spectrophotometer (Thermo Fisher Scientific). For retro-transcription of the RNA and obtaining the complementary DNA, 1 μg of RNA and the Superscript® VILO™ Master Mix commercial kit (Invitrogen) were used in a Veriti thermocycler (Applied Biosystems) with the following program: a 10-minute denaturation 65° C., followed by 10 minutes at 25° C., an incubation of 60 minutes at 42° C. and 5 minutes at 85° C. The complementary DNA was diluted in water free of proteases, DNAases and RNAases (Sigma-Aldrich). Finally, quantitative real-time PCR (qPCR) was carried out using the PowerUp™ SYBR® Green Master Mix (Thermo Fisher Scientific) in the LightCycler® 480 Instrument (Roche, Applied Science) with the following program: 10 minutes incubation at 95° C., followed by amplification of 30-40 cycles from 10 to 95° C., 30 to 60° C. and 12 to 72° C. HPRT-1 was used as a housekeeping gene, and the levels of gene expression were analysed with respect to the control cells without treatment. The primers used were the following:
Primary chondrocytes isolated from donors with osteoarthritis were cultured as three-dimensional micromasses. For this, 500,000 cells were seeded in polystyrene tubes and centrifuged at 1500 rpm for 10 minutes, forming a cell pellet. Then, the medium was replaced by the corresponding treatment: untreated control (DMEM 10% FBS), or chondrogenic medium alone or supplemented with 5 μM 8Arg-YSA1 or 8Arg-TUB1. The medium was changed every 2-3 days, and after 14 days, the micromasses were frozen in Tissue-Tek® O.C.T. (Sakura Finetek).
For the detection of the senescence-associated β-galactosidase activity we have used the Senescence Cells Histochemical Staining kit (Sigma-Aldrich). Cells were rinsed with warm PBS and fixed for 7 minutes at room temperature with a buffer composed by 2% paraformaldehyde and 0.2% glutaraldehyde. After three washes with PBS, cells were incubated 16 h at 37° C. without CO2 in a staining solution containing X-gal, which is cleaved by the β-galactosidase enzyme producing an intense blue insoluble product.
Such as it is explained in
The different concentrations studied did not affect cell viability with respect to the same untreated (UT) cells.
Chondrocytes at a confluence of 70-80% were treated for 16 hours with the peptides labelled with TMR for visualization at the cellular level at a concentration of 150 μM (YSA1, YTP1 and YSA1-NO2) (see
Patients with OA have significantly elevated levels of Cx43 in the articular cartilage activating cellular dedifferentiation via EMT by activation of Twist-1 and cellular senescence via p53/p16.
Such as it is shown in
Such as it is shown in
A Scrape Loading/Dye Transfer (SL/DT) test was performed in which the transfer of Lucifer Yellow fluorophore (LY) between contact chondrocytes through GJs was studied.
OA chondrocytes treated with peptide YSA1 (150 μM) for 16 hours showed a decrease in cell coupling compared to untreated (UT) cells (
OA chondrocytes show increased Cx43 levels that lead to EMT and senescence via Twist-1 and p53/p21/p16, respectively. In addition, senescent cell accumulation is involved in OA progression. The treatment with the 8Arg-YSA1 peptide (5 μM) for 16 hours lead to reduced expression Twist-1, a transcription factor that activate cell dedifferentiation via EMT, and the senescence-activation factors p16 and p21 (
Chondrocytes isolated from OA donors were cultured as 3D micromasses for 14 days in normal growth medium or in chondrogenic medium (CM) alone or supplemented with 5 μM of peptides 8Arg-YSA1 or 8Arg-TUB1. Hematoxylin-Eosin stain showed that micromasses treated with these peptides lead to a better ECM structure. In addition, blue toluidine stain showed an increased deposition of proteoglycans in the ECM of micromasses treated with the peptides 8Arg-YSA1 or 8Arg-TUB1, as detected by the increased violet dye (see
| Number | Date | Country | Kind |
|---|---|---|---|
| P201930708 | Jul 2019 | ES | national |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/EP2020/071242 | 7/28/2020 | WO |
