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WO2011054399A1 - Amélioration du fonctionnement des récepteurs aux glucocorticoïdes chez les asthmatiques - Google Patents

Amélioration du fonctionnement des récepteurs aux glucocorticoïdes chez les asthmatiques Download PDF

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WO2011054399A1
WO2011054399A1 PCT/EP2009/064784 EP2009064784W WO2011054399A1 WO 2011054399 A1 WO2011054399 A1 WO 2011054399A1 EP 2009064784 W EP2009064784 W EP 2009064784W WO 2011054399 A1 WO2011054399 A1 WO 2011054399A1
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fkbp51
agents
patients
epithelial cells
asthma
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Sabrina Mattoli
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AVAIL GmbH
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/4353—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom ortho- or peri-condensed with heterocyclic ring systems
    • A61K31/436—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom ortho- or peri-condensed with heterocyclic ring systems the heterocyclic ring system containing a six-membered ring having oxygen as a ring hetero atom, e.g. rapamycin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/44—Non condensed pyridines; Hydrogenated derivatives thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/56—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids
    • A61K31/57—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids substituted in position 17 beta by a chain of two carbon atoms, e.g. pregnane or progesterone
    • A61K31/573—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids substituted in position 17 beta by a chain of two carbon atoms, e.g. pregnane or progesterone substituted in position 21, e.g. cortisone, dexamethasone, prednisone or aldosterone
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P11/00—Drugs for disorders of the respiratory system
    • A61P11/06—Antiasthmatics

Definitions

  • This invention relates to the use of agents that improve the glucocorticoid receptor function in bronchial epithelial cells and that are useful to reduce the risk of treatment failure in a medical field.
  • Asthma is a common inflammatory disease of the airways, and inhaled glucocorticoids (GCs) are recommended as the first-line anti- inflammatory treatment option for this condition (Bateman ED et al . Eur Respir J 31: 143-178, 2008) because their broad range of antiinflammatory activities is necessary to block the multiple proinflammatory events that are involved in the pathogenesis of the disease (Barnes PJ, Adcock IM. Ann Intern Med 139: 359-370, 2003) .
  • a number of patients with asthma fail to respond satisfactorily to inhaled GCs and some of them show a poor or absent response even to treatment with oral GCs (Chung KF et al .
  • the patients with GC-hyporesponsive or resistant asthma differ from patients with familial GC resistance (Kino T, Chrousos GP . Essays Biochem 40: 137-155, 2004; Hurley DM et al . J Clin Invest 87: 680-686, 1991) because they are not cortisol-deficient and do not have hormonal abnormalities consistent with GC receptor (GCR) mutations (Carmichael J et al. BMJ 282: 1419-1422, 1981) .
  • GCR GC receptor
  • bronchial epithelial cells in asthma are structurally and functionally different from normal bronchial epithelial cells (Davies DE . Curr Allergy Asthma Rep 1: 127-133, 2001; Swindle EJ, et al . J Allergy Clin Immunol 124: 23-34, 2009) . They produce multiple cytokines and chemokines capable of promoting the recruitment and local activation of eosinophils, monocytes and T lymphocytes in the bronchial mucosa (Soloperto M, et al. Am J Physiol (Lung Cell Mol Physiol) 260: L530-538, 1991; Vittori E, et al.
  • GC-hyporesponsive or resistant asthma Patients with GC-hyporesponsive or resistant asthma are those with more severe disease and such individuals also show a marked increase in the proliferation of bronchial epithelial cells (Cohen L, et al . Am J Respir Crit Care Med 176: 138-145, 2007) .
  • the present invention is based on the hypothesis that the increased proliferation of epithelial cells in asthma may be associated with a persistent increase in the expression of the chaperon protein FKBP51, an immunophilin preferentially over-expressed in mitotically active cells (Yeh W-C, et al. Proc Natl Acad Sci USA 92: 11081-1185, 1995), which can potentially reduce GCR function through various mechanisms (Wochnik GM, et al.
  • the present invention aims at restoring the impaired GCR function in the bronchial epithelium of asthmatic patients by reducing the detrimental effects of the aberrant persistent over-expression of FKBP51 on the efficient receptor signalling in these cells.
  • Figure 1 shows the increased proliferation rate (A) and the concomitant increase in the expression of FKBP51 messenger ribonucleic acid (mRNA) (B) in the bronchial epithelial cells from GC-resistant asthma, as compared to the bronchial epithelial cells of GC-responsive asthma.
  • A the increased proliferation rate
  • B messenger ribonucleic acid
  • Each data point is a mean of duplicate observations obtained for each individual donor and the horizontal line is the median value for each group.
  • FIG 2 shows the differences in the intracellular distribution of the FKBP51 protein in the epithelial cells from the two groups of patients and the effects of an agent which reduces the intracellular availability of functionally active FKBP51, the FKBP51-ligand FK506.
  • Figure 3 shows the effects of pre-incubation with the FKBP51-ligand FK506 on the intracellular distribution of GCR .
  • FKBP51-ligand FK506 FKBP51-ligand FK506
  • Figure 3 shows the effects of pre-incubation with the FKBP51-ligand FK506 on the intracellular distribution of GCR .
  • Western blot analysis was performed by using cytoplasmic and nuclear extracts of pooled epithelial cell samples from two donors in each group of patients.
  • Figure 4 shows the effects of pre-incubation with the FKBP51-ligand FK506 on the nuclear translocation of the GCR upon subsequent treatment with the GC dexamethasone in bronchial epithelial cells from GC-responsive and GC-resistant asthmatics.
  • Western blot analysis was performed by using cytoplasmic and nuclear extracts of pooled epithelial cell samples from three donors in each group of patients.
  • Figure 5 shows the effects of pre-incubation with the FKBP51-ligand FK506 on the efficiency of the GCR function in bronchial epithelial cells from GC-resistant or GC responsive asthmatics, as assessed by analysis of the GC-induced inhibition of the expression of the messenger ribonucleic acid (mRNA) for MCP-1/CCL2 and eotaxin/CCLll .
  • the GC used in these experiments was dexamethasone. Each data point is a mean of duplicate observations obtained for each individual donor and the horizontal line is the median value for each group.
  • Figure 6 shows the effects of pre-incubation with the FKBP51-ligand rapamycin on the efficiency of the GCR function in bronchial epithelial cells from GC-resistant or GC responsive asthmatics, as assessed by analysis of the GC-induced inhibition of the expression of the mRNA for MCP-1/CCL2 and eotaxin/CCLll .
  • the GC used in these experiments was dexamethasone . Each data point is a mean of duplicate observations obtained for each individual donor.
  • GCR receptor function Improvement of the GCR receptor function in bronchial epithelial cells of GC-hyporesponsive or resistant asthmatic patients is achieved by treating epithelial cells with an agent that reduces the intracellular availability of functionally active FKBP51 before GC exposure.
  • an agent that reduces the intracellular availability of functionally active FKBP51 in comparison with the cells of patients with GC-responsive asthma ( Figures 1 and 2), such procedure reduces the nuclear accumulation of the isoform beta of the GCR (GCR ) ( Figure 3), which acts as a nuclear competitor and dominant negative regulator of the functionally active isoform alpha of the GCR (GCR ) (Charmandari E, et al . Mol Endocrinol 19: 52-64, 2005; Oakley RH, et al.
  • the agents used to reduce the intracellular availability of functionally active FKBP51 are FK506 (tacrolimus) and rapamycin (sirolimus), two compounds that bind FKBP51 with high affinity (Weiwad M, et al . Biochemistry 45: 15776-15784, 2006; Baughman G, et al . Mol Cell Biol 15: 43-95-4402, 1995; Yeh WC, et al . Proc Natl Acad Sci USA 92: 11086-11090, 1995) .
  • FK506 derivatives or other immunophilin-ligands such as pimecrolimus (Edlich F, Fischer G. Handb Exp Pharmacol 172: 359-404, 2006), antibodies against FKBP51, compounds that function as antagonists of FKBP51, and constructs that reduce the synthesis of FKBP51 by inducing FKBP51 gene silencing or by inhibiting FKBP51 gene translation (Weiwad M, et al . Biochemistry 45: 15776-15784, 2006; Avellino R, et al . Blood 106: 1400-1406, 2005) .
  • agents that reduce the intracellular availability of functionally active FKBP51 to patients with GC- hyporesponsive or resistant asthma can increase responsiveness to a concomitant treatment with inhaled GCs in such individuals and reduce the risk of side effects associated with the use of excessive doses of inhaled GCs or oral GCs.
  • the preferred but not exclusive route of administration is by inhalation, orally or intranasally .
  • the solution provided by the present invention for improving the impaired GCR function in patients with GC-hyporesponsive or resistant asthma is an improvement over the prior art.
  • excessive levels of FKBP51 can induce GC resistance in certain mammalian cells and cell lines (Wochnik GM, et al . J Biol Chem 280: 4609-4616, 2005; Westberry JM, et al . J Steroid Biochem Mol Biol 100: 34-41, 2006)
  • the possibility that bronchial epithelial from certain asthmatic patients exhibit an impaired GCR function and that this alteration can be corrected by reducing the intracellular availability of excessive amounts of functionally active FKBP51 has never been evaluated.
  • FK506 The anti-inflammatory and immunosuppressant activities of FK506 are selectively linked to its ability to function as a potent calcineurin inhibitor by binding the small immunophilin FKBP12 highly expressed in T lymphocytes (Liu J, et al . Cell 66: 807-815, 1991; O'Keefe SJ, et al. Nature 357: 692-694, 1992; Clipstone NA, Crabtree GR. Nature 357: 695-697, 1992; Jain J, et al . Nature 365: 352-355, 1993; McCaffrey PG, et al. J Biol Chem 268: 3747-3752, 1993; Xu X, et al .
  • FK506 inhibits the expansion of various T lymphocyte subsets and the release of pro-inflammatory lymphokines like interleukin (IL)-2 and IL-5 from these cells.
  • NFs nuclear factors
  • IL interleukin
  • the previously known use of FK506 and FK506 derivatives in asthma is related to the well-known ability of FK506 to function as a calcineurin inhibitor in T lymphocytes and is supported by studies in animal models of asthma (Morishita Y, et al .
  • the present invention does not relate to this antiinflammatory/immunosuppressive property of FK506, and treatment of bronchial epithelial cells from asthmatic patients with FK506 alone does not have any effect on the expression of pro-inflammatory chemokines in these cells.
  • Figure 5 shows that treatment of bronchial epithelial cells with the FKBP51-ligand FK506 without subsequent stimulation with GCs does not significantly change the level of expression of MPC-1/CCL2 and eotaxin/CCLl 1 messenger ribonucleic acid (mRNA) (p > 0.05 in comparison with no treatment) .
  • mRNA messenger ribonucleic acid
  • Rapamycin is a drug approved for the prevention of allograft rejection, which affects T cell proliferation and survival by targeting the serine-threonine kinase mTOR (Abraham RT, Wiederrecht GJ. Annu Rev Immunol 14: 483-510, 1996; Schmelzle T, Hall MN. Cell 103: 253-262, 2000) . It also binds FKBP51 with high affinity (Weiwad M, et al . Biochemistry 45: 15776-15784, 2006) and it has been proposed that this property may be exploited at the clinical level to decrease the chemoresistance of malignant cells (Avellino R, et al . Blood 106: 1400-1406, 2005) .
  • This invention provides agents that reduce the intracellular availability of functionally active FKBP51 in bronchial epithelial cells and, respectively, a method which comprises administering agents that reduce the intracellular availability of functionally active FKBP51 in bronchial epithelial cells and, respectively, the use of agents that reduce the intracellular availability of functionally active FKBP51 in bronchial epithelial cells for manufacturing a medicament, said agents and, respectively, method and, respectively, medicament increasing the responsiveness to treatment with GCs in patients with GC-hyporesponsive or resistant asthma.
  • the agents that can reduce the intracellular availability of functionally active FKBP51 either exist and are used as alternative anti-inflammatory medicaments or can be developed on the basis of existing information (Nair SC, et al . Mol Cell Biol 17: 594-603, 1997; Wu B, et al . Proc Natl Acad Sci USA 101: 8348-8353, 2004) .
  • the present invention relates to new medicaments for improving the response to GC treatment in asthmatic individuals who show incomplete or absent clinical improvement after GC treatment, either temporarily or on a long-term basis .
  • the best mode for carrying out the invention is described in detail .
  • GC response of patients with asthma is classified on the basis of the improvement in pulmonary function following treatment with 40 mg daily of oral prednisone for at least 1 week (Barnes PJ, et al . Am J Respir Crit Care Med 152: 125S-140S, 1995; Goleva E, et al . Am J Respir Crit Care Med 173: 607-616, 2006) .
  • patients are defined as GC-resistant/insensitive if they show less than 15% increase in the pre-bronchodilator morning forced expiratory volume in one second (FEVi) in comparison with the pre- treatment FEVi .
  • Patients are defined as GC-responsive/sensitive if they have a post-treatment increase in FEVi greater that 20% in comparison with the pre-treatment FEVi.
  • Bronchial epithelial cells are obtained by brushing the bronchial mucosa of the second and third generation bronchi, from the distal part of each sub- segment to the proximal part, with a sterile cytology brush (for example the BC 9C-26101 cytology brush from Olympus, Tokyo, Japan) .
  • a sterile cytology brush for example the BC 9C-26101 cytology brush from Olympus, Tokyo, Japan
  • the preferable medium is composed of a 1:1 mixture of Dulbecco' s modified Eagle's medium and F- 12 (DMEM/F12 ) devoid of phenol red (Invitrogen, Carlsbad, California, USA) and is supplemented with antibiotics (100 U/ml penicillin and 100 g/ml streptomycin, both from Invitrogen) and 10% heat-inactivated, charcoal- and dextran-stripped, GC-free foetal bovine serum (FBS)
  • the samples are then centrifuged at 150 x g for 5 minutes and the pelleted cells are re-suspended in the same medium for further processing.
  • the total number of epithelial cells that can be isolated from each patient by performing 10 to 14 brushings is usually in the range between 1.5 x 10 s and 4 x 10 s .
  • the viability is greater than 90%, as assessed by the standard trypan blue exclusion method.
  • the purity of epithelial cells can be confirmed on cytocentrifuge preparations by immunocytochemistry (Bucchieri F, et al .
  • mRNA messenger ribonucleic acid
  • MCP- 1/CCL2 and eotaxin/CCLll in epithelial cells can be evaluated by the reverse transcription-polymerase chain reaction procedure (RT-PCR) , using specific primers for human FKBP51 (Periyasami S, et al . Endocrinology 148: 4716-4726, 2007), MCP-1/CCL2 (Tran MT, et al . Invest Ophthalmol Vis Sci 37: 987-996, 1996) and eotaxin/CCLll (Jahnsen FL, et al . J Immunol 163: 1545-1551, 1999) .
  • RT-PCR reverse transcription-polymerase chain reaction procedure
  • Total RNA is isolated by lysing epithelial cells in Trizol (Invitrogen) , according to the manufacturer's instructions. Genomic deoxyribonucleic acid (DNA) is removed from RNA samples by treatment with DNase-free DNA Treatment and Removal Agent (Ambion, Austin, USA) . Quantification of total RNA is then performed with the RiboGreen RNA quantification assay (Invitrogen) and total RNA integrity is analysed by agarose gel electrophoresis. Total RNA (50 ng) is reverse-transcribed by using the iScript cDNA Synthesis Kit (Bio-Rad, Hercules, CA, USA), according to the manufacturer's instructions, and the resulting cDNA is subjected to amplification by PCR.
  • PCR is performed using the HotStarTaq DNA polymerase (Qiagen, Hilden, Germany) and the FKBP51 gene-specific set of primers in a thermal cycler.
  • the housekeeping gene can be ubiquitin C (Willems E, et al . Int J Dev Biol 50: 627-635, 2006) .
  • PCR is run for up to 35 cycles of denaturation at 94°C for 45 s, annealing at 55-62°C for 1 min, and extension at 72°C for 1 min, followed by a final elongation step at 72°C for 10 min.
  • Optimal annealing temperature and cycle number can be determined for each primer set by using the cDNAs prepared from human universal RNA ( Stratagene/Agilent Technologies, Santa Clara, CA, USA) . Reactions conducted without the reverse-transcription step should be run in parallel to confirm the absence of genomic DNA contamination. The PCR products are resolved by electrophoresis on 2% agarose gel and visualized by staining with ethidium bromide. For each RNA sample, the signal for each target gene can be normalised to the signal for ubiquitin C by densitometric analysis .
  • bronchial epithelial cells are seeded onto 6-well culture plates in DMEM/F12 , devoid of phenol red and antibiotics, at a density of 5 x 10 5 cells /ml and incubated for 4 hours at 37°C in a humidified atmosphere with 5% C0 2 in air.
  • Replicate cells samples are then pre-treated with the agent that reduces the intracellular availability of functionally active FKBP51 or an appropriate control for the required period of time, depending on the nature and mode of action of each agent, and then exposed to a GC or its vehicle for 30 minutes.
  • FKBP51-ligand FK506 the agent that reduces the intracellular availability of functionally active FKBP51
  • FK506 the agent that reduces the intracellular availability of functionally active FKBP51
  • cells are pre-incubated for up to 2 hours at 37°C with 1 ⁇ FK506 (Sigma-Aldrich, St. Louis, Missouri, USA) or its vehicle and then treated with the GC dexamethasone (100 nM) (Sigma-Aldrich) or its vehicle for 30 minutes at 37°C.
  • Analysis of the intracellular localisation of FKBP51 and GCR and evaluation of the nuclear translocation of GCR are then performed on cytoplasmic and nuclear extracts by Western blot, according to a method well known to persons skilled in the art (Goleva E, et al .
  • Cytoplasmic and nuclear extracts from cells can be prepared with commercially available kits such as the NE-PER nuclear and cytoplasmic extraction reagent kit from Pierce Biotechnology/Thermo Fisher Scientific (Rockford, Illinois, USA) (Goleva E, et al . Am J Respir Crit Care Med 173: 607-616, 2006) .
  • Samples are normalised for protein concentration, for example by using Pierce BCA Protein Assay (Pierce Biotechnology/Thermo Fisher Scientific) .
  • Ten g of each cytosolic and nuclear extract sample are subjected to a gradient of sodium dodecyl sulfate polyacrilamide gel electrophoresis and Western blotted using rabbit polyclonal antibodies against human FKBP51 (Affinity Bioreagents, Golden, Colorado, USA) , GCR (Abeam) or GCRp (Affinity Bioreagents) .
  • the blotting membranes are stripped and re-probed with a mouse monoclonal antibody against the cytoplasmic marker ⁇ -tubulin (Abeam) or the nuclear marker NF-1 (Santa Cruz Biotechnology, Santa Cruz, California, USA) .
  • the association of FKBP51 with GCR in the cytoplasmic and nuclear compartments of the cells is also evaluated by co-immunoprecipitation followed by Western blotting (Goleva E, et al . J Immunol 169: 5934-5940, 2002; Wochnik GM, et al . J Biol Chem 280: 4609-4616, 2005; Zhang X, et al .
  • Invest Ophthalmol Vis Sci 49: 1037- 1047, 2008 Immunoprecipitation of 50-100 g of cytoplasmic or nuclear fractions is performed with 2-4 g of a mouse monoclonal antibody against human FKBP51 (BD-Transduction Laboratories, Franklin Lakes, New Jersey, USA) and after detection of the co-precipitated GCR by using the polyclonal antibody mentioned above, the blotting membrane is treated with stripping buffer (Pierce Biotechnology/Thermo Fisher Scientific) and re-probed with the rabbit polyclonal antibody against human FKBP51 to detect the precipitated protein.
  • bronchial epithelial cells are seeded onto 6-well culture plates in DMEM/F12 , devoid of phenol red and antibiotics, at a density of 5 x 10 5 cells /ml and incubated for 4 hours at 37°C in a humidified atmosphere with 5% C0 2 in air.
  • Replicate cell samples are then pre-treated with the agent that reduces the intracellular availability of functionally active FKBP51 or an appropriate control for the required period of time, depending on the nature and mode of action of each agent, and then exposed to a GC or its vehicle for 6 hours at 37°C in a humidified atmosphere with 5% C0 2 in air.
  • the agent that reduces the intracellular availability of functionally active FKBP51 is the FKBP51-ligand FK506, cells are pre- incubated for up to 2 hours at 37°C with 1 ⁇ FK506 (Sigma-Aldrich, St.
  • Bronchial epithelial cells were isolated from 5 patients with GC- resistant asthma and 5 patients with GC-responsive asthma by brushing upon fiberoptic bronchoscopy. All patients stopped any treatment with inhaled and oral GCs 72 hours before the procedure. On the average, 2.4 x 10 s (standard deviation: 0.5 x 10 s ) and 2.9 x 10 s (standard deviation: 0.4 x 10 s ) viable bronchial cells were recovered from the GC-resistant asthmatics and the GC-responsive patients, respectively. More than 97% of these cells were epithelial cells, as demonstrated by staining the cytocentrifuge preparations with the pan-cytokeratin monoclonal antibody (clone AE1/AE3, Abeam) .
  • the percentage of proliferating cells was significantly higher in the epithelial cell samples from patients with GC-resistant asthma than in the epithelial cell samples from patients with GC responsive asthma, as assessed by staining the cytocentrifuge preparations with the antibody against the proliferation marker Ki-67 (Dako) ( Figure 1A) .
  • the epithelial cells from patients with GC-resistant asthma showed increased expression of FKBP51 mRNA in comparison with the cells from patients with GC-responsive asthma ( Figure IB) .
  • the intracellular distribution of the FKBP51 protein also differed markedly in the epithelial cells from the two groups of patients, as assessed by Western blot.
  • NFKBP51 was detected only in the cytoplasmic extracts of cells from GC-responsive asthmatics while this protein was present both in the cytoplasmic and nuclear extracts of cells from GC- resistant asthmatics ( Figure 2) .
  • bronchial epithelial cells were seeded onto 6-well culture plates in DMEM/F12 , devoid of phenol red and antibiotics, at a density of 5 x 10 5 cells /ml and left to settle down for 4 hours at 37°C in a humidified atmosphere with 5% C0 2 in air.
  • bronchial epithelial cells were seeded onto 6-well culture plates in DMEM/F12, devoid of phenol red and antibiotics, at a density of 5 x 10 5 cells /ml and incubated for 4 hours at 37°C in a humidified atmosphere with 5% C0 2 in air.
  • Replicate cell samples were then pre-incubated for 2 hours with 1 ⁇ of the FKBP51-ligand FK506 or its vehicle at 37°C and treated with 100 nM of the GC dexamethasone or its vehicle for additional 6 hours at 37°C in a humidified atmosphere with 5% C0 2 in air.
  • Bronchial epithelial cells were isolated from two patients with GC- resistant asthma and two patients with GC-responsive asthma by brushing upon fiberoptic bronchoscopy as described in the Example 1. Bronchial epithelial cells were seeded onto 6-well culture plates in DMEM/F12 , devoid of phenol red and antibiotics, at a density of 5 x 10 5 cells/ml and incubated for 4 hours at 37°C in a humidified atmosphere with 5% C0 2 in air.
  • Replicate cells samples were then pre-incubated for 2 hours with 10 ⁇ of the FKBP51-ligand rapamycin ( Sigma-Aldrich or Santa Cruz Biotechnology) or its vehicle at 37°C and treated with 100 nM of the GC dexamethasone or its vehicle for additional 6 hours at 37°C in a humidified atmosphere with 5% C0 2 in air.
  • Analysis of the dexamethasone-induced inhibition of the expression of MCP-1/CCL2 and eotaxin/CCLll genes was then performed by RT-PCR.

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Abstract

La présente invention concerne un dysfonctionnement des récepteurs aux glucocorticoïdes dans les cellules épithéliales bronchiques de certains patients asthmatiques, des agents capables de restaurer une signalisation efficace des récepteurs aux glucocorticoïdes dans ces cellules, avec pour résultat une inhibition efficace de l'expression des cytokines et des chimiokines pro-inflammatoires, ainsi que des procédés associés d'utilisation desdits agents et l'utilisation desdits agents dans le cadre de la fabrication de médicaments correspondants. L'invention concerne, en particulier, des agents capables de faire baisser la disponibilité intracellulaire de l'immunophiline FKBP51 fonctionnellement active, ainsi qu'un procédé d'utilisation desdits agents et l'utilisation desdits agents pour la fabrication de médicaments correspondants, lesdits agents, procédés et médicaments se révélant capables de renforcer la réponse à un traitement anti-inflammatoire à base de glucocorticoïdes chez des patients souffrant d'un asthme résistant, ou réagissant mal, aux glucocorticoïdes.
PCT/EP2009/064784 2009-11-06 2009-11-06 Amélioration du fonctionnement des récepteurs aux glucocorticoïdes chez les asthmatiques Ceased WO2011054399A1 (fr)

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Cited By (2)

* Cited by examiner, † Cited by third party
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EP2690102A1 (fr) 2012-07-24 2014-01-29 Max-Planck-Gesellschaft zur Förderung der Wissenschaften e.V. Aza-amides bicycliques pour le traitement de troubles psychiatriques
WO2017003469A1 (fr) * 2015-06-30 2017-01-05 University Of South Florida Inhibiteurs de la protéine fkbp51 à partir d'un criblage de médicaments à rendement élevé et procédés d'utilisation

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EP2690102A1 (fr) 2012-07-24 2014-01-29 Max-Planck-Gesellschaft zur Förderung der Wissenschaften e.V. Aza-amides bicycliques pour le traitement de troubles psychiatriques
WO2017003469A1 (fr) * 2015-06-30 2017-01-05 University Of South Florida Inhibiteurs de la protéine fkbp51 à partir d'un criblage de médicaments à rendement élevé et procédés d'utilisation

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