FR2987054A1 - In vitro method for screening of drug candidates for treating osteoporosis, comprises detecting an inhibitory activity of the drug candidate on a paracrine regulation between osteoblasts and adipocytes - Google Patents

Abstract

In vitro method for screening of drug candidates, comprises detecting an inhibitory activity of the drug candidate on a paracrine regulation between osteoblasts and adipocytes, where the regulation is derived from osteoblast phenotype to the adipocyte phenotype. ACTIVITY : Osteopathic. MECHANISM OF ACTION : None given.

Description

The present invention relates to an in vitro method for determining the potential activity of a drug candidate in the treatment of osteoporosis. The present invention relates to an in vitro method for determining whether a drug candidate, i.e., for example, a compound or a mixture of compounds, has an effect on paracrine adipocyte-osteoblast regulation involved in the Pathophysiology of osteoporosis. Osteoporosis is a disease characterized by excessive fragility of the skeleton, due to a decrease in bone mass and alteration of bone microarchitecture. Osteoporosis is a common disease in women 10 after menopause (postmenopausal osteoporosis) because bone mass decreases with age and with the deficiency of female hormones (estrogen). Osteoporosis can also occur in men (male osteoporosis) or be secondary to a condition (secondary osteoporosis) which allows to consider the establishment of a prevention of this bone loss. Due to the increase in life expectancy in so-called developed countries, osteoporosis is recognized as a major public health problem. Indeed, an estimated 200 million people suffer from this pathology in the world; and that 30% of postmenopausal women in Europe and the US have osteoporosis. Osteoporosis causes an increased risk of fractures in which the life-threatening condition is sometimes engaged. Currently, few drugs to overcome this bone loss are available on the market and their effects are limited. Thus, the bisphosphonates, reference molecules in the treatment of this pathology reduce the risk of fracture by only about 50%. Other therapeutic approaches target the activating receptor for NF-κB ligand (RANKL) which is involved in the maturation of osteoclasts, or cathepsin K which is involved in osteoclastic resorption. Nevertheless, this pathology remains to date insufficiently treated. An object of the present invention is to provide a novel method of detecting and / or selecting drug candidates for the treatment of osteoporosis. This goal is achieved by means of an in vitro method for screening drug candidates for the treatment of osteoporosis which comprises detecting the possible inhibitory activity of the drug candidate on paracrine regulation between osteoblasts and adipocytes, said regulation generating phenotype drift from osteoblasts to the adipocyte phenotype.

It is known (see article "Human osteoblasts derived from mesenchymal stem cells express adipogenic markers on coculture with bone marrow adipocytes" published in the journal "Differentiation" in 2010) that in the bone marrow of patients with osteoporosis (from either of aging or of another cause) the number of adipocytes increases to the detriment of osteoblastic cells, causing bone fragility.The aforementioned publication further indicates that there is paracrine regulation between adipocytes and osteoblastic cells. Adipocytes appear to cause differentiation of osteoblast cells into adipocytes.These results are evidenced by the assay of gene messenger RNAs coding for specific markers of adipocyte differentiation ("Peroxisome proliferatoractivated receptor gamma" (PPARg), and leptin, in particular) and osteoblastic cells (osteocalcin). Over a period of co-culture, the level of expression of the adipocyte marker genes increases in osteoblastic cells cocultivated with adipocytes: the osteoblastic cells therefore have a phenotype that drifts towards that of the adipocytes. The aforementioned publication suggests that a new therapeutic avenue could be attached to the adipocytes of the bone marrow. On the other hand, the aforementioned publication does not describe an in vitro screening method similar to that of the invention.

According to the invention, the drug candidate can act on the adipocytes and / or on the osteoblasts and / or on the compounds released by the osteoblasts and / or adipocytes so as to slow down or prevent the drift of the osteoblast phenotype. Drug candidates that would inhibit or prevent osteoblast-adipocyte transformation could then be tested in vivo for the preventive and / or curative treatment of osteoporosis. These drug candidates would, in particular, promote the maintenance of bone mass by maintaining the number of osteoblastic cells. For the purposes of the present invention, the term "treatment" is intended to mean curative and / or preventive treatment. The term "treatment" relates to an improvement of at least one of the symptoms of osteoporosis. The drug candidate may be a synthetic chemical compound or a naturally occurring substance. It can also be a mixture of chemical and / or natural substances. The drug candidate may, for example, be a nutrient or a food.

For the purposes of the invention, the term "inhibitory activity" means a complete or partial inhibition of the transformation (drift) of the phenotype of the aforementioned cells into adipocytes or a simple slowing down of the latter. According to a particular embodiment of the screening method of the invention, osteoblastic cells are cultured, during a given test period, in a medium originating from a culture of adipocytes, and possibly containing adipocytes, and comprising said drug candidate; the level of expression of a gene coding for a marker chosen from adipocyte markers and osteoblast markers is then measured; and comparing the measured expression level with a reference value which is obtained by measuring the level of expression of said marker gene in osteoblast cells cultured during said given test period, in a medium derived from an adipocyte culture and comprising a reference product, possibly known for its effectiveness in the treatment of osteoporosis.

If the expression level of a gene coding for an adipocyte marker is measured, the drug candidate is considered to be potentially active if it manages to reduce the expression rate of said gene at least to said reference value, when the reference product is already known for its activity in the treatment of osteoporosis. If the reference product is known to have no efficacy in the treatment of osteoporosis, the drug candidate is considered potentially active if it manages to reduce the expression rate of said gene below the reference value, If the expression level of a gene encoding an osteoblast marker is assayed, the drug candidate compound is considered potentially active if the level of expression of said gene in the presence of said drug candidate is at least equal to said value. reference, when the reference product is already known for its activity in the treatment of osteoporosis. If the reference product is known to have no efficacy in the treatment of osteoporosis, the drug candidate is considered potentially active, if the level of expression of said gene in the presence of said drug candidate is greater than said value of reference. This method is simple to implement, fast and inexpensive in hardware. The level of expression of the gene coding for the marker can be implemented by the assay of the mRNAs specific for the gene coding for the marker which are present in the total RNAs of the osteoblastic cells.

Advantageously, said medium originating from a culture of adipocytes does not comprise adipocytes. Indeed, it is the merit of the Applicant that to have demonstrated that, by using a medium from a culture of adipocytes containing no adipocytes, osteoblasts drift to an adipocyte phenotype. It was not clear that paracrine regulation between osteoblasts and adipocytes may be due to molecules that are secreted by adipocytes and have a long life in the culture medium. The method using a medium without adipocytes, it is simple to implement and easily automated.

Advantageously, said osteoblastic cells and / or adipocytes are obtained by culturing mesenchymal cells in a culture medium promoting their differentiation respectively into osteoblastic cells or adipocytes. The cells come from the species for which the drug candidate must be active. In the case of human osteoporosis, mesenchymal cells are human cells. Advantageously, the mesenchymal cells used for obtaining the osteoblastic cells and for obtaining the adipocytes come from the same donor. This makes it possible to avoid disturbances in the regulation between adipocytes and osteoblasts that could come from the difference between the donors even from the same species. Advantageously, said gene coding for a marker is chosen from the gene coding for leptin, the PPARg gene and the gene coding for osteocalcin. The test duration is greater than or equal to 24 hours, preferably substantially equal to 48 hours. The method of the invention is therefore quick to implement.

According to one embodiment, the medium promoting the differentiation of mesenchymal cells into osteoblasts contains from 0.5% to 1.5% by volume of a streptomycin / penicillin (1: 1 VN) mixture, preferably 1%, of 0.5 to 1.5% and preferably 1% of Glutamine at 200 mM, from 1.5% to 2.5%, preferably 2%, of 10 mM (3-glycerophosphate, from 0.05% to 0%, 15% preferably 0.1% of vitamin D at 10 nM, from 0.0025% to 0.0075%, preferably 0.005% of vitamin C at 50 μM and 10% of fetal calf serum mixed in medium Dulbecco's Modified Eagle Medium containing 4.5 g / l of glucose (DMEM 10%) According to one embodiment which can be combined with the previous one, the medium favoring the differentiation of mesenchymal stem cells into adipocytes contains 0, 5% to 1.5% and preferably 1% of a streptomycin / penicillin (1: 1 VN) mixture, 0.5% to 1.5% and preferably 1% of 200 mM Glutamine, 0.025% at 0.075% of preference e 0.05% of 3-isobutyl-1-methylxanthine (IBMx) at 0.5 mM, 0.025% to 0.075%, preferably 0.05% of dexamethasone at 0.5 μM, 0.05% at 0 15%, preferably 0.1%, 50% indomethacin and 10% fetal calf serum mixed in Dulbecco's Modified Eagle Medium medium (DMEM) containing 4.5 g / l of glucose (DMEM 10%). . According to another embodiment, combinable with one and / or the other of the aforementioned embodiments, said proliferation medium contains 1% streptomycin / penicillin, 1% glutamine 200 mM and 10% of fetal calf serum mixed in Dulbecco's Modified Eagle Medium containing 4.5 g / l of glucose (DMEM 10%). All the above% are in volume. The present invention, its characteristics and the advantages it affords will appear better on reading the following description of a particular embodiment of the invention, given by way of non-limiting example.

Experimental Results The cultured cells are commercial human mesenchymal stem cells (MSCs) (Lonza Walkersville, Inc.). All manipulations (medium change, proliferation and differentiation) are performed in a sterile environment under laminar flow host.

The culture media and the solutions used during this experiment are preheated to 37 ° C. Seeding and Proliferation Thawing of the cells A vial containing 700 000 cells is thawed (3 min in a water bath at 37 ° C). For this, the cell suspension is rapidly placed after thawing in 9 ml of proliferation medium. The solution is then centrifuged for 5 min at 1000 rpm to remove the freezing medium. Following centrifugation, cells form a pellet and thus the medium can be removed. Then, the cell pellet is resuspended in 15 ml of proliferation medium.

Finally, the 15 ml of the cell suspension are placed in a flask of 75 cm 2. The flask is placed in an oven at 37 ° C with an atmosphere enriched with 5% CO2. The proliferation medium is changed 24h after seeding the cells.

MSC proliferation medium (Complete DMEM medium) - 500 ml DMEM medium - 1% 200mM Glutamine - 1% Penicillin (10,000U) and Streptomycin (10mg.mL-1)> 10% calf serum Fetal Proliferation After thawing, a first so-called proliferation step is performed to increase the number of cells contained in the ampoule.

Counting and resuspension of the cells The culture medium is removed, then the cells are rinsed with phosphate buffered saline (137 mM NaCl, 2.7 mM KCl, 4.3 mM Na 2 HPO 4, 1.47 mM KH 2 PO 4, ph = 7.4) to eliminate any traces of culture medium. Then, 1 mL of trypsin is added by T75. Trypsin is a digestive enzyme of the pancreatic juice which makes it possible to detach the cells from the bottom of the flask. To promote the action of the enzyme, the flask is placed in an oven at 37 ° C. After 5 min of incubation, 3 ml of proliferation medium per 1 ml of trypsin are added to neutralize the action thereof. The 6 mL contained in each flask are then removed and placed in a sterile 15 mL tube. A centrifugation step (5 min, 1000 rpm) eliminates the medium containing the trypsin. Finally, the cells are resuspended in the proliferation medium and then seeded in plates of 6 or 12 wells. Differentiation After 4 days of culture, the cells are placed in adipocyte differentiation medium for adipocytes and in osteoblast differentiation medium for osteoblasts. These media differ in the presence of factors favoring the differentiation of MSCs into adipocytes or osteoblasts. During 14 days of culture, 4 changes of medium are made, one change every 3 to 4 days.20 Osteoblastic differentiation medium> Proliferation medium (DMEM 10%) - [3-Glycerophosphate at 10 mM - Vitamin D at 10 nM - Vitamin C at 50 μM Adipose differentiation medium> Proliferation medium (DMEM 10%) - Dexamethasone at 0.5 μM - 3-isobutyl-1-methylxanthine at 0.5 mM> Indomethacin at 50 μM The plate is placed in an oven at 37 ° C with an atmosphere enriched with 5% CO2.

Co-culture The adipocyte differentiation medium is eliminated and replaced by a proliferation medium without FCS. 48 hours later, the culture medium present on the adipocytes is removed and placed in the wells containing the osteoblastic cells and the drug candidate to be tested. The culture is thus extended for 48 hours. It is also possible to add the drug candidate to be tested in the medium present on the adipocytes and to have this mixture on the osteoblasts. It is also possible to add separately to the osteoblast cells the drug candidate to be tested, optionally dissolved or diluted in a solvent, and the medium from the culture of adipocytes.

Analysis of the Effect of the Molecules on the Cell Model Analysis of the Gene Expression of the Osteoblastic Population in Quantitative PCR At the end of coculture, we eliminated the culture medium and extracted the RNAs from the osteoblastic (and adipocyte) cells that find in the wells. RNA Preparation The total RNAs are extracted using a reagent of commercial origin. The cells are lysed directly into the well, after having removed the culture medium, by adding 1.5 ml of Extract'All for the 6-well plates and 0.700 ml for the 12-well plates. A culture well is used for each culture condition which corresponds to 1 tube of 1.5 ml. In each tube are added for 1 ml of Extract'All, 0.3 ml of chloroform followed by vigorous vortexing for 15 seconds. The aqueous phase is transferred to a clean tube and 0.750 mL of isopropanol is added to allow precipitation of the total RNA. This precipitation is done after mixing and centrifugation at 12,000 g for 10 minutes, the RNAs then form a white pellet at the bottom of the tube. The supernatant is removed and the washed RNA pellet is added by adding 1 ml of 75% ethanol, vortexed and then centrifuged at 7,500 g for 5 minutes. The RNA pellet is then dried for 10 minutes at 40 ° C. and then resuspended in 20 μl of ultrapure water. Total RNA is quantified by absorbance reading at 260 nm. For this purpose, 1 μl of RNA is diluted to 1/200 in ultrapure water and placed in a micro-quartz tank for spectrophotometer reading.

Any contamination with deoxyribonucleic acid (DNA) is then eliminated by a treatment with DNase I. 2 μg of RNA are diluted in a final volume of 17 μl of ultrapure water to which 2 μl of 10 × 10% buffer are added. enzyme and 1 μL DNase I diluted 1/4. The digestion was carried out for 30 minutes at 37 ° C., then the reaction was stopped by adding 0.8 μl of 0.2 M EDTA and incubation at 75 ° C. for 10 minutes. Synthesis of the complementary DNAs The synthesis of the complementary DNAs is made on 12.5 μl of RNA previously treated with DNase. RNAs are denatured at 75 ° C for 5 minutes and then placed in ice. The reaction mixture is then added. It consists of 4 μL of 5X enzyme buffer (50 mM Tris-HCl pH8.3, 75 mM KCI and 3 mM MgCl2), 1 μL random primers pdN6 at 100 μM, 1 μL of dNTPs at 10 μM. mM, 1 μL dithiothreitol 100 mM and 0.5 μL Superscript reverse transcriptase enzyme or 100 units. A 10 minute incubation at 20 ° C allows hybridization of the primers and then the samples are placed on the thermocycler for incubation for 30 minutes at 42 ° C followed by 35 minutes at 99 ° C. Then 30 μl of water is added (vf = 50 μl) and the samples are kept in the freezer. To verify the absence of DNA, a similar synthesis but without enzyme addition is performed. Quantitative RT-PCR assay PCR reactions were performed in a volume of 10 μl with 1 μl of cDNA, 4 mM MgCl 2, 0.3 μM of each primer and 1 μl of the FastStart DNA Master SYBR Green I commercial mix containing enzyme, SYBR Green and dNTPs. Samples are analyzed and compared using the RelQuant 1.01 software which is the LightCycler's relative quantization software. The expression profiles of the adipocyte and osteoblastic markers are analyzed.

Table 1 represents the level of expression of the gene coding for leptin obtained using the screening method according to the present invention for a compound (alendronate) known for its use in the treatment of osteoporosis and tested according to US Pat. method of the present invention.

Table 1: Alendronate Leptin expression level ratio +/- standard deviation Control (0 μM) 1 μM 0.14 +/- 0.09 Ethanol is used as a control and as a solvent for alendronate. 20

Claims (10)

REVENDICATIONS1. In vitro method for screening drug candidates for the treatment of osteoporosis comprising detecting an inhibitory activity of the drug candidate on paracrine regulation between osteoblasts and adipocytes, said regulation generating the drift of the phenotype of osteoblasts towards the phenotype of adipocytes. 2. A method according to claim 1, wherein osteoblastic cells are cultured in a medium from a culture of adipocytes, and optionally containing adipocytes, and comprising said drug candidate, during a given test period; and the level of expression of a gene coding for a marker chosen from adipocyte markers and osteoblastic markers is then measured; and - the measured expression level is compared with a reference value which is obtained by measuring the level of expression of said marker gene in osteoblast cells cultured during said given test period in a medium from a culture of adipocytes and comprising a reference product, possibly known for its effectiveness in the treatment of osteoporosis. 3. Method according to claim 2, characterized in that said medium from a culture of adipocytes does not include adipocytes. 20 4. Method according to any one of claims 2 and 3, characterized in that said osteoblastic cells and / or adipocytes are obtained by culturing mesenchymal cells in a culture medium promoting their differentiation respectively osteoblastic cells or in adipocytes. 5. Method according to claim 4, characterized in that said mesenchymal cells are cultured for 14 days to obtain adipocytes or osteoblastic cells. 6. Method according to any one of claims 4 and 5, characterized in that said mesenchymal cells are human cells. 7. Method according to any one of claims 4 to 6, characterized in that the mesenchymal cells used for obtaining osteoblastic cells and for obtaining adipocytes come from the same donor. 8. Method according to any one of claims 2 to 7, characterized in that said gene coding for a marker is chosen from the gene coding for leptin, the PPARg gene and the gene coding for osteocalcin. 9. Method according to any one of claims 2 to 8, characterized in that said test duration is greater than or equal to 24 hours, preferably substantially equal to 48 hours. 10. Method according to any one of claims 4 to 9, characterized in that said medium promoting the differentiation of mesenchymal cells into osteoblasts contains from 0.5% to 1.5% by volume of a streptomycin / penicillin mixture ( 1: 1 VN) preferably 1%, from 0.5 to 1.5% and preferably 1% of Glutamine to 200 mM, from 1.5% to 2.5%, preferably 2% of the (3-glycerophosphate to 10 mM, from 0.05% to 0.15% preferably 0.1% of the vitamin D at 10 nM, from 0.0025% to 0.0075% preferably 0.005%, vitamin C at 50 μM and 10% of fetal calf serum mixed in Dulbecco's Modified Eagle Medium medium containing 4.5 g / l of glucose (DMEM 10%) and in that said medium promoting the differentiation of the mesenchymal stem cells into adipocytes contains 0.5 % to 1.5% and preferably 1% of a mixture streptomycin / penicillin (1: 1 VN), 0.5% to 1.5% and preferably 1% Glutamine 200 mM, 0.025% to 0.075%, preferably 0, 0.5% of 3-isobutyl-1-methylxanthine (IBMx) at 0.5 mM, 0.025% to 0.075% preferably 0.05% dexamethasone at 0.5 μM, 0.05% to 0.15% preferably 0.1% of indomethacin at 50 μM and 10% of fetal calf serum mixed in Dulbecco's Modified Eagle Medium medium (DMEM) containing 4.5 g / l of glucose (DMEM 10%).