EP2534139A1 - Neue aminoalkyl-oxazol- und aminoalkyl-thiazolcarbonsäureamide als regenerationsfördernde substanzen für sinnesorgane und postmitotische gewebe - Google Patents

Neue aminoalkyl-oxazol- und aminoalkyl-thiazolcarbonsäureamide als regenerationsfördernde substanzen für sinnesorgane und postmitotische gewebe

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Publication number
EP2534139A1
EP2534139A1 EP11703394A EP11703394A EP2534139A1 EP 2534139 A1 EP2534139 A1 EP 2534139A1 EP 11703394 A EP11703394 A EP 11703394A EP 11703394 A EP11703394 A EP 11703394A EP 2534139 A1 EP2534139 A1 EP 2534139A1
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Prior art keywords
cells
regeneration
aminoalkyl
oxazole
organ
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EP11703394A
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German (de)
English (en)
French (fr)
Inventor
Holger Eickhoff
Hubert LÖWENHEIM
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EMC Microcollections GmbH
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EMC Microcollections GmbH
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D413/00—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and oxygen atoms as the only ring hetero atoms
    • C07D413/02—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and oxygen atoms as the only ring hetero atoms containing two hetero rings
    • C07D413/06—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and oxygen atoms as the only ring hetero atoms containing two hetero rings linked by a carbon chain containing only aliphatic carbon atoms
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P21/00—Drugs for disorders of the muscular or neuromuscular system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P27/00—Drugs for disorders of the senses
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P27/00—Drugs for disorders of the senses
    • A61P27/16—Otologicals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D263/00—Heterocyclic compounds containing 1,3-oxazole or hydrogenated 1,3-oxazole rings
    • C07D263/02—Heterocyclic compounds containing 1,3-oxazole or hydrogenated 1,3-oxazole rings not condensed with other rings
    • C07D263/30—Heterocyclic compounds containing 1,3-oxazole or hydrogenated 1,3-oxazole rings not condensed with other rings having two or three double bonds between ring members or between ring members and non-ring members
    • C07D263/34—Heterocyclic compounds containing 1,3-oxazole or hydrogenated 1,3-oxazole rings not condensed with other rings having two or three double bonds between ring members or between ring members and non-ring members with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals, directly attached to ring carbon atoms
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D413/00—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and oxygen atoms as the only ring hetero atoms
    • C07D413/02—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and oxygen atoms as the only ring hetero atoms containing two hetero rings
    • C07D413/12—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and oxygen atoms as the only ring hetero atoms containing two hetero rings linked by a chain containing hetero atoms as chain links
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D417/00—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and sulfur atoms as the only ring hetero atoms, not provided for by group C07D415/00
    • C07D417/02—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and sulfur atoms as the only ring hetero atoms, not provided for by group C07D415/00 containing two hetero rings
    • C07D417/04—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and sulfur atoms as the only ring hetero atoms, not provided for by group C07D415/00 containing two hetero rings directly linked by a ring-member-to-ring-member bond
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D417/00—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and sulfur atoms as the only ring hetero atoms, not provided for by group C07D415/00
    • C07D417/02—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and sulfur atoms as the only ring hetero atoms, not provided for by group C07D415/00 containing two hetero rings
    • C07D417/12—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and sulfur atoms as the only ring hetero atoms, not provided for by group C07D415/00 containing two hetero rings linked by a chain containing hetero atoms as chain links

Definitions

  • the present invention relates to novel aminoalkyl-oxazole and aminoalkyl-thiazolecarboxamides which stimulate the endogenous regeneration of terminally differentiated cells in highly specialized organs, tissues and sensory epithelia in the mammal in situ.
  • the claimed low molecular weight compounds are capable of inducing appropriate cell biological alterations such as dedifferentiation, proliferation and subsequent terminal redifferentiation of normally postmitotic tissue cells.
  • the invention relates to compounds with which a de novo formation of hair cells in the Corti organ can be achieved by induction of cell division of support cells of the inner ear and hearing after hair cell loss can be restored.
  • the invention furthermore relates to processes for the preparation of the compounds according to the invention, to their formulation as pharmaceutical preparations and to their use for the production of medicaments for regenerative medicine.
  • the ability to regenerate after injury is limited or absent.
  • organs and tissues such as the liver, bone, or skin, have the capacity for spontaneous regeneration through cell regeneration throughout the life of the organism.
  • the corresponding cells in the highly specialized organs and tissues e.g., heart, brain, skeletal muscle, or the sensory epithelia of the eye and inner ear
  • these tissues have also lost their ability to spontaneously regenerate upon the occurrence of harmful events. This leads accordingly to irreversible functional deficits.
  • a heart attack for the heart or a stroke for the brain often cause irreversible damage to the affected tissue components with correspondingly permanent loss of function.
  • tissue and organs as shown by the examples of retina or limbs, which, although terminally differentiated, are nevertheless capable of spontaneous in vivo regeneration (Tsonis, 2000, Tsonis, 2002 ).
  • the central cell biological event in these examples consists of cellular dedifferentiation, which allows the formation of multipotent progenitor cells, from which proliferation and redifferentiation can give rise to regenerated cells.
  • Dedifferentiation thus plays the decisive role in the regeneration of the terminally differentiated amphibian tissue.
  • other vertebrates show lower regeneration abilities.
  • the basic biological mechanism for hair cell regeneration in the avian cochlea has been described as the cell division of supporting cells adjacent to the destroyed hair cells (Corwin and Cotanche, 1988, Ryals and Rubel, 1988), producing a population of undifferentiated cells are to redifferentiate to newly formed hair cells and support cells. This results in almost complete morphological and functional recovery of sensory epithelium in birds (Cotanche, 1999, Smolders, 1999).
  • the regenerated tissues of amphibians can be used to extract an extract capable of inducing dedifferentiation in mammalian cells as well (McGann et al., 2001).
  • the dedifferentiation-based mechanism for the regeneration of terminally differentiated cells can be transferred from the amphibians to the mammal (Odelberg, 2002).
  • the regeneration extracts mentioned are "protein cocktails" whose composition is not known in detail.
  • it has also been possible to achieve a corresponding effect on muscle cells of the mammal with a defined low-molecular compound Choen et al., 2004).
  • these compounds are said to facilitate the restoration of hearing in the mammal by de novo formation of hair cells in the adult organ of Corti.
  • a causal treatment of inner ear hearing loss on the basis of a regenerative biologically active drug should be made possible for the first time.
  • the essential feature of the present invention is that with the compounds according to the invention structurally defined chemical agents are present for the first time, which can bring about a regeneration of terminally differentiated cells in the mammal, in particular hair cells in the inner ear of the mammal.
  • the present invention has to do with the identification of novel, previously unknown low molecular weight compounds that are able to stimulate regeneration biologically relevant processes such as dedifferentiation, proliferation and the resulting regeneration of cells from normally postmitotic tissues Unique selling point.
  • the invention further includes the use of the regeneration-promoting properties of the compounds according to the invention for the causative treatment of inner ear hearing loss after damage and loss of hair cells in the organ of Corti up to the complete restoration of hearing in humans and animals.
  • Target cells are the support cells that are directly adjacent to the damaged cells and can serve as potential precursors for the regeneration of hair cells.
  • the present invention therefore relates to aminoalkyl-oxazole and aminoalkyl-thiazolcarbon- acid amides according to the formulas (1) and (2)
  • Y is C or N, where both atoms must be different
  • R2 is hydrogen or acyl
  • R 1 and R 3 which may be the same or different, represent a substituent selected from the following groups:
  • alkylcycloalkyl groups alkylaryl groups, cycloalkyl groups, cycloalkylaryl groups, aryl groups and arylcycloalkyl groups,
  • R 1 is preferably (1H-indol-3-yl) -ethyl and R 3 is preferably cyclohexyl.
  • a preferred embodiment relates to aminoalkyl-oxazole and amino-alkyl-thiazolcarbonklaamide according to the invention according to the following formulas (3) to (8).
  • the potential of the low molecular weight aminoalkyl-oxazole and aminoalkyl-thiazolecarboxamides according to the invention to dedifferentiate already differentiated cells was determined in a cell culture assay on stem cells isolated from the mouse's inner ear with otic development potential, which in cell culture belong to the original sensory epithelium Epithelial islands, as different as possible, were detected.
  • the proportion of Sox2-expressing cells a stem cell marker which is also expressed during the ontogenesis of the organ of Corti and is regarded as a sign of cellular dedifferentiation, could more than be doubled by administration of the compounds according to the invention.
  • BrdU bromodeoxyuridine
  • the immunocytochemical analysis of the stem cell markers on the dedifferentiated epithelium confirmed the assumption that the compounds according to the invention after their accumulation in the cell nucleus first induce a dedifferentiation of differentiated cells in the sense of a reprogramming in order subsequently to enable their proliferation.
  • the concentration range for an effective activity with regard to proliferation within the range of 0.1 ⁇ to 100 ⁇ , preferably from 1 ⁇ to 3 ⁇ substance in the cell culture medium was defined by a dose-response analysis for the compounds according to the invention. Comparable effects as in the cell culture assay could also be achieved in the in vitro organ culture of a mouse's natural organ, ie in the complex, cellular association of the organ of Corti.
  • the application of the compounds of the invention for in situ proliferation of various support cells in the Corti'schen organ such as lying in the outer hair cell laterally oriented Deiterszellen (outer phalanxes) and outer border cells, in the area the inner hair cells lying inner phalangeal cells and inner border cells and other supporting cells, which are considered as potential precursor cells for the regeneration of inner hair cells.
  • individually labeled nuclei could be detected in different stages of mitosis up to complete cell division with duplicate cell nuclei.
  • the immunohistochemical findings also showed a regeneration of hair cells induced by the compounds of the invention on the basis of cell divisions of support cells, preferably in the damaged by noise trauma areas of the organ of Corti. In appropriate control animals and control organs without application of the compounds showed no spontaneously regenerated hair cells.
  • the invention further provides the preparation of the aminoalkyl oxazole and aminoalkylthiazolecarboxamides according to the invention, which are used according to the invention as active ingredients for stimulating the endogenous in situ regeneration of terminally differentiated cells in highly specialized organs and tissues, preferably the brain, the heart, skeletal muscle, and most preferably of sensory epithelia, can be used.
  • One possible route is the synthesis of the compounds of the invention on a solid phase starting from an amino acid amide.
  • the amino acid whose side chain represents the substituent R1
  • the cyclization of the amino acid amide to the oxazole or of the amino acid thioamide to the thiazole is carried out with bromobutyric acid and ⁇ , ⁇ -dimethylaniline.
  • Brombrenztraubenklathylester the resulting ester group must be subsequently saponified.
  • the amino acid amides are converted into the corresponding thioamides before the cyclization with Lawesson's reagent.
  • the resulting amino acid oxazole / thiazolecarboxylic acids are subsequently reacted with an amine which represents the substituent R3.
  • the acidic cleavage of the amino acid oxazole / thiazolecarboxamides from the synthetic resin and the subsequent chromatographic purification are carried out.
  • the synthesis of the compounds according to the invention is carried out in solution.
  • the synthesis is carried out using the protective group chemistry starting from a Boc-protected amino acid with the side chain R1.
  • the synthesis steps are in principle adequate to those described for the solid phase.
  • the acidic cleavage of the Boc protective group and the subsequent chromatographic purification are carried out.
  • salts of the compounds of the invention pharmaceutically acceptable salts are preferred.
  • examples of such salts are salts of inorganic or organic acids, salts of inorganic or organic bases and salts of basic or acidic amino acids.
  • the compounds of the invention may also be modified as prodrug compounds, preferably as prodrug esters or prodrug peptides, or the like.
  • coupling of cell penetration promoting molecules such as e.g. Biotin or maleimidopropionic, optionally via suitable spacer molecules, to the primary amino group or the acylation of this amino group, corresponding to the substituent R2, the bioavailability and thus the effectiveness of the compounds of the invention can be improved.
  • Another object of the invention is the use of Aminoalkyl- oxazole- and aminoalkyl-thiazolcarbonklaamide invention for the preparation of pharmaceutical preparations for the treatment of diseases in the mammal, which are associated with damaged postmitotic tissues, in particular for the treatment of damage and loss of Haarsinneszellen in the organ of Corti, caused by inner ear hearing loss
  • Mammal Mammal. These preparations, containing at least one active ingredient according to the invention alone or in combination, optionally contain further pharmaceutically suitable auxiliaries and additives, such as excipients, preservatives, stabilizers, emulsifiers, detergents, solvents, solubilizers, salts for regulating the osmotic pressure and buffer salts. In addition, they may contain other therapeutically relevant active ingredients, adjuvants and regeneration-promoting substances such as growth factors or anti-inflammatories. Pharmaceutically acceptable materials are the substances known to be useful in the pharmaceutical and food technology industries and in adjacent fields, in particular those listed in the relevant pharmacopoeias whose properties do not preclude physiological application.
  • auxiliaries and additives such as excipients, preservatives, stabilizers, emulsifiers, detergents, solvents, solubilizers, salts for regulating the osmotic pressure and buffer salts.
  • auxiliaries and additives such as excipients, preservatives
  • Suitable administration forms for the pharmaceutical preparations according to the invention containing at least one active compound of the formulas (1) and (2) may be, for example, solutions, suspensions, sprays, gels, hydrogels, lotions, emulsions, pastes, ointments or creams.
  • the preparation of the pharmaceutical preparations according to the invention is carried out in the usual way by methods known to those skilled in the art, as described in the relevant pharmacopoeias, e.g. by mixing, granulating or coating methods.
  • the pharmaceutical preparations according to the invention can additionally be sterilized.
  • the invention also relates to the use of the aminoalkyl-oxazole and aminoalkyl-thiazolcarbonklaamide invention as regeneration-promoting agents for the treatment of diseases in humans and animals associated with damaged postmitotic tissues, preferably for restoring hearing after loss or damage to the hair cells in the inner ear ,
  • a person or animal in need of such a treatment is administered a therapeutically effective amount of a preparation according to the invention which promotes regeneration, comprising at least one compound according to the invention of the formulas (1) and (2).
  • the active substance or the pharmaceutical preparation according to the invention are applied directly or indirectly (including systemically), preferably locally, directly on / to the damaged tissue structures.
  • the application to or in the inner ear for example, transtympanally by injection into the middle ear, by applying to the round or oval window of the inner ear or by injection into the inner ear.
  • Various systems for drug administration can be used.
  • the compounds according to the invention and pharmaceutical preparations can be used alone, in combination with other compounds according to the invention or in combination with one or more active substances which are relevant for the respective described therapeutic indication of the compounds according to the invention.
  • the temporal sequence of the application is not restricted.
  • a compound according to the invention can be administered either simultaneously, before or after the other active substances, as a separate drug or combination preparation and on the same or different administration routes.
  • the exact therapeutically effective amount for the treatment of sensorineural hearing loss in a subject will depend on various factors, including the extent of damage to the tissue, size, stature, age and health of the patient, route and form of administration, the particular compound used, and, where appropriate of other medicines used. Thus it does not make sense to specify the exact quantity at this time.
  • compositions according to the invention can in principle of a multiple application of the pharmaceutical compositions according to the invention over a period of up to 8 weeks at intervals of one to seven days to a continuous application by systems for drug dosing, which have a mechanism for permanent delivery "sustained release"
  • the amount of active ingredient used should be in the range from 0.5 pg to 1, 0 mg per inner ear and application.
  • Regeneration-biologically relevant compounds that are able to induce corresponding cell biological changes such as dedifferentiation, proliferation or terminal redifferentiation, and their formulations represent a new form of active ingredients, since it is a completely new therapeutic concept.
  • aminoalkyl-oxazole and aminoalkyl-thiazolecarboxamides according to the invention, it has been possible for the first time to detect a cell division of otic supporting cells induced by low molecular weight compounds and the resulting regeneration of hair cells in the highly differentiated postmitotic tissue of the Corti organ in the mammal. To date, no comparable methods have been described for the in vivo regeneration of hair cells based on pharmaceutical active ingredients.
  • the described syntheses allow the preparation of the compounds in stereochemically pure form. They are obtained either in free form or as a salt, if salt-forming groups are included.
  • N-Boc-D-Tryptophan (9) (1 equiv, 2.75 mmol, 1.00 g), hydroxybenzotriazolammonium salt (2 equiv, 5.50 mmol, 924 mg) and diisopropylcarbodiimide (DIC) (1.1 equiv , 3.03 mmol, 382 mg, 454 pL) were dissolved in molecular sieve-dried dimethylformamide (DMF) and stirred at room temperature for 18 hours. The solvent was then removed on a rotary evaporator under reduced pressure and the residue taken up in ethyl acetate.
  • DMF molecular sieve-dried dimethylformamide
  • the crude product (10) was taken up in a solution of 50% trifluoroacetic acid (TFA) in dichloromethane (DCM) and stirred at room temperature for 3 h. After evaporation of the solvents, the product (11) was recrystallized.
  • TFA trifluoroacetic acid
  • DCM dichloromethane
  • Chloro (2'-chloro) trityl-polystyrene resin (12) (Rapp Polymere H 10033, loading 1.31 mmol / g) (1 equiv, 655 pmol, 500 mg) was washed twice with DMF (via molecular sieve) , a solution of the amino acid amide hydrochloride (13) (2 equiv; 1, 31 mmol) and diisopropylethylamine (DIPEA) (5 equiv, 3.28 mmol, 424 mg) in DMF and shaken for 18 h at room temperature , To cap the resin, methanol (MeOH) was then added to the suspension and shaken for another 15 minutes at room temperature. The reaction solution was filtered off with suction, the loaded resin (14) was washed (5 ⁇ DMF, 3 ⁇ MeOH, tetrahydrofuran (THF), diethyl ether) and dried in an oil pump vacuum.
  • DIPEA diisopropy
  • Amino acid amide resin (14) (1 equiv, 655 pmol, 500 mg) was added to a solution of Lawesson's reagent (3 equiv, 1.97 mmol, 797 mg) in dimethoxyethane (DME) (10 mL) and 18 h shaken at room temperature. The reaction solution was filtered off with suction, the resin (16) was washed (6 ⁇ DMF, 3 ⁇ MeOH, THF, DCM, diethyl ether each time) and dried in an evacuated desiccator.
  • DME dimethoxyethane
  • the amino acid-oxazole (15) or amino acid-thiazolecarboxylic acid ethyl ester resin (17) (1 equiv; 655 ⁇ ; 500 mg) was preswollen in THF (2.5 mL) and washed with a solution of lithium hydroxide monohydrate (5 equiv. 3.28 mmol, 137 mg) in water (1.25 mL) and MeOH (1.25 mL). After 3 h at room temperature, the reaction solution was filtered off with suction, the resin (18) or (19) was washed (3 ⁇ water, water / DMF 1: 1, DMF, dioxane, THF, DCM, diethyl ether) and dried in an evacuated desiccator.
  • amino acid-oxazole (18) or amino acid-thiazolecarboxylic acid resin (19) (1 equiv, 655 mol, 500 mg) was added a solution of 1-hydroxybenzotriazole (HOBt) (5 equiv, 3.28 mmol; 443 mg) and DIC (5 equiv, 3.28 mmol, 413 mg) in dry DMF (5 mL). After shaking at room temperature for 30 min, the R3 amine was added (10 equiv, 6.55 mmol) and the suspension further shaken for 16 h at room temperature. After aspirating the reaction solution, the resin was washed (3 times each DMF, MeOH, THF, DCM, diethyl ether) and sucked dry in a stream of air.
  • HOBt 1-hydroxybenzotriazole
  • DIC equiv, 3.28 mmol, 413 mg
  • the product (20) or (21) was cleaved from the resin with a solution of 5% TFA in DCM for 1 h at room temperature.
  • the crude products can be recrystallized from ethyl acetate or ethyl acetate / petroleum ether or purified by flash chromatography on silica gel.
  • Boc-amino acid-thiazolecarboxylic acid (24) (1 equiv, 0.80 mmol) was treated with (benzotriazol-1-yloxy) tripyrrolidinophosphonium hexafluorophosphate (PyBOP) (1:05 mmol, 0.84 mmol;
  • reaction solution was evaporated and the crude product was chromatographed on silica gel using a DCM-MeOH gradient.
  • the cultured otospheres were differentiated under adherent culture conditions on an ornithine / fibronectin surface to, the original sensory epithelium of similar single-layered epithelial islets.
  • Figure 2 shows a comparison of the situation in the native organ (in vivo) and in the differentiated epithelial islets of the cell culture (in vitro) using these markers.
  • the compound 2- [1-amino-2- (1H-indol-3-yl) -ethyl] -oxazole-4-carboxylic acid cyclohexylamide (3) according to the invention achieved both in terms of Sox2 expression and SrcfLZ incorporation. significantly positive results. Thus, it was demonstrated that it is capable of inducing the dedifferentiation and subsequent proliferation of otic supporting cells.
  • the optimal concentration range was determined, in which this compound unfolds its maximum regenerative biological effect in the cell culture.
  • the SroiLZ incorporation was detected in the stem cell-based in vitro cell culture assay at 4 concentrations between 0.3 ⁇ and 10 ⁇ in the culture medium. It was found that for the concentration 0.3 ⁇ no significant effect can be achieved. The mean at a concentration of 1 ⁇ but already increased. At a concentration of 3 ⁇ , the saturation level of approximately 9% BrdL / -positive cells is already reached ( Figure 5).
  • a form of organ culture was used in which the tissue to be cultured, in this case the entire inner ear of the mouse, in a rotating cylinder filled with culture medium (Hahn et al., 2008).
  • the Cochlea was previously opened basal and apical in the area of the scala tympani. This allowed the influence of gravity to be minimized, while at the same time achieving optimal gas and nutrient exchange between the tissue of the organ of Corti and the culture medium. Under these conditions, the explant could be maintained longer in culture compared to a stationary culture.
  • the support cells remaining after hair cell loss in the organ of Corti differentiate into progenitor cells and subsequently proliferate, a situation analogous to a hearing-impaired ear was created.
  • the ototoxic aminoglycoside antibiotic neomycin (1 mM) were cell death within 24 hours, 2/3 of all hair cells. Only in the apical areas of the cochlea survived some hair cells, whose number decreased in the course of the experiment by the initial damage on. After removing the neomycin from the medium, the remaining support cells could be further cultured.
  • Brdil was simultaneously administered with the compounds of the invention (5 ⁇ ) in the culture medium and the proliferation quantified by the SrdL-incorporation after 4 days.
  • the different morphology of the ßrdLZ-positive nuclei indicates different stages of mitosis, including complete cell division. Chromatin condensation was observed on different nuclei ( Figures 6D, E). At the same time, ßrdl / -positive nuclei were detected in immediate, relative proximity to each other ( Figures 6E, F).
  • the regeneration-biological activity of the compounds according to the invention was also demonstrated in the in vivo model of the adult guinea pig.
  • Impulse noise caused acute noise damage, resulting in loss of hair cells of the organ of Corti, especially in the outer hair cells.
  • the continuous topical application of the compound 2- [1-amino-2- (1H-indol-3-yl) -ethyl] -oxazole-4-carboxylic acid cyclohexylamide (3) according to the invention was carried out from a subcutaneously implanted miniosmotic pump (Alzet ® ) via cholestomy directly into the scala tympani of the cochlea.
  • the substance in the pump was dosed correspondingly higher with 200 ⁇ , since a dilution effect in the perilymph was to be expected.
  • BrdU was administered via drinking water or the miniosmotic pump to label proliferating cells in the organ of Corti.
  • Brc-labeled support cells and hair cells could be detected preferentially in the areas damaged by the noise trauma of the organ of Corti.
  • Example E Toxicity In the cell culture, organ culture and in vivo investigations, no evidence for a toxic effect of the aminoalkyl-oxazole and aminoalkyl-thiazolecarboxamides according to the invention was found for the investigated concentration range from 0.3 ⁇ to 200 ⁇ . pictures
  • Figure 1 Isolation and culture of stem cells from the mouse organ of Corti.
  • Stem cells from the inner ear can be obtained by isolating the cells of the postnatal Corti organ, isolated the stem cells still contained and cultured under selective conditions in a suspension culture. Under these conditions, spheres are formed that express the stem cell marker Sox2 ( ⁇ ' ), which is believed to play a major role in the pluripotency of embryonic stem cells and in the induction of pluripotent stem cells from differentiated cells (Takahashi and Yamanaka, 2006). In addition, the nuclei in the spheres incorporate BrdU (A " ), which indicates the proliferation of the cells.
  • Figure 2 In vivo and in vitro protein expression profile of hair and support cell markers.
  • p27 Kip1 is expressed in all the supporting cells of the organ of Corti. Neither inner nor outer hair cells are p27 K ' ; p, -positive. Within the epithelial islands a p27 K * "expression could be detected ( ⁇ ' ).
  • E-cadherin marks all support cells oriented laterally to the pillar cells. E-cadherin is also found in the membranes of the epithelial islands (C). Hair-sense cell markers such as myosin VIIA (D), myosin VI (E) and calretinin (F) in vivo reliably label inner and outer hair cells. All markers are also detectable in individual cells within the in vitro culture (D ' , E ' , F ' ).
  • the stars mark the outer 3 hair cells and the inner hair cell (always on the right).
  • Figure 4 Relative number of 8rdL positive cells in the screening assay.
  • the Brc / L / -positive cell nuclei were in different stages of mitosis or completely through cell division (D, E, F).
  • the BrdU label indicates regeneration based on cell division.
  • Tsonis PA (2002) Regenerative biology: the emerging field of tissue repair and restoration.

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  • Orthopedic Medicine & Surgery (AREA)
  • Physical Education & Sports Medicine (AREA)
  • Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
  • Heterocyclic Carbon Compounds Containing A Hetero Ring Having Nitrogen And Oxygen As The Only Ring Hetero Atoms (AREA)
  • Plural Heterocyclic Compounds (AREA)
  • Thiazole And Isothizaole Compounds (AREA)
  • Medicinal Preparation (AREA)
EP11703394A 2010-02-08 2011-02-04 Neue aminoalkyl-oxazol- und aminoalkyl-thiazolcarbonsäureamide als regenerationsfördernde substanzen für sinnesorgane und postmitotische gewebe Withdrawn EP2534139A1 (de)

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DE102010007281A DE102010007281A1 (de) 2010-02-08 2010-02-08 Neue Aminoalkyl-oxazol- und Aminoalkyl-thiazolcarbonsäureamide und ihre Anwendung zur Stimulation der endogenen situ Regeneration von Haarsinneszellen im Corti'schen Organ des Innenohres beim Säuger
PCT/EP2011/000502 WO2011095338A1 (de) 2010-02-08 2011-02-04 Neue aminoalkyl-oxazol- und aminoalkyl-thiazolcarbonsäureamide als regenerationsfördernde substanzen für sinnesorgane und postmitotische gewebe

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CA (1) CA2789013A1 (https=)
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EP2871181B1 (en) 2013-11-12 2016-10-05 Acousia Therapeutics GmbH Novel Compounds for Regeneration of Terminally-Differentiated Cells and tissues
EP3366683A1 (en) * 2017-02-28 2018-08-29 Acousia Therapeutics GmbH Cyclic amides, acteamides and ureas useful as potassium channel openers

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US5378803A (en) * 1987-12-11 1995-01-03 Sterling Winthrop Inc. Azole-fused peptides and processes for preparation thereof
DE19807426A1 (de) * 1998-02-23 1999-10-14 Otogene Biotechnologische Fors Verfahren zur Behandlung von Erkrankungen oder Störungen des Innenohrs
EP1126855B1 (en) * 1998-09-25 2007-05-09 Cephalon, Inc. Use of fused pyrrolocarbazoles for preventing/treating damage to sensory hair cells and cochlear neurons
TW502019B (en) * 1999-03-26 2002-09-11 Cocensys Inc Aryl substituted pyrazoles, imidazoles, oxazoles, thiazoles and pyrroles, and the use thereof
CN1441841B (zh) * 2000-07-11 2013-03-20 桑得医药品公司 对内耳的细胞再生和分化的刺激
UA89035C2 (ru) * 2003-12-03 2009-12-25 Лео Фарма А/С Эфиры гидроксамовых кислот и их фармацевтическое применение
JP2008502595A (ja) * 2004-03-31 2008-01-31 エグゼリクシス， インコーポレイテッド 未分化リンパ腫キナーゼモジュレータおよびその使用方法
JP5182088B2 (ja) * 2006-04-19 2013-04-10 アステラス製薬株式会社 アゾールカルボキサミド誘導体
GB0614538D0 (en) * 2006-07-21 2006-08-30 Univ Cambridge Tech Therapeutic Compounds And Their Use

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US8889723B2 (en) 2014-11-18
CN102947281A (zh) 2013-02-27
CA2789013A1 (en) 2011-08-11
DE102010007281A1 (de) 2011-08-11
WO2011095338A1 (de) 2011-08-11
CN102947281B (zh) 2015-11-25
US20140288135A1 (en) 2014-09-25
JP2013518917A (ja) 2013-05-23
US20130012514A1 (en) 2013-01-10

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