WO2009024348A1 - Verfahren zur aktivierung regulatorischer t-zellen - Google Patents
Verfahren zur aktivierung regulatorischer t-zellen Download PDFInfo
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- WO2009024348A1 WO2009024348A1 PCT/EP2008/006895 EP2008006895W WO2009024348A1 WO 2009024348 A1 WO2009024348 A1 WO 2009024348A1 EP 2008006895 W EP2008006895 W EP 2008006895W WO 2009024348 A1 WO2009024348 A1 WO 2009024348A1
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- cells
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- 0 *C(CSSB=C(C(N1C(*)C*C1)=O)N)N Chemical compound *C(CSSB=C(C(N1C(*)C*C1)=O)N)N 0.000 description 1
- HJBJGSGWHRZAFR-UHFFFAOYSA-N CCc1cccc(CC)c1N(C(c(cccc1)c1N1)=O)C1=O Chemical compound CCc1cccc(CC)c1N(C(c(cccc1)c1N1)=O)C1=O HJBJGSGWHRZAFR-UHFFFAOYSA-N 0.000 description 1
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Definitions
- the present invention relates to a method for activating regulatory T cells (Treg cells, CD4 + CD25 + cells). More particularly, the invention relates to a method for ex situ activation of regulatory T cells using alanyl aminopeptidase inhibitors (aminopeptidase N; APN; CD13; EC 3.4.11.2) or using inhibitors of enzymes having analogous enzymatic activity. The invention also relates to the use of inhibitors of alanyl aminopeptidase and / or of inhibitors of enzymes with analogous enzymatic action for the activation of regulatory T cells.
- alanyl aminopeptidase inhibitors aminopeptidase N; APN; CD13; EC 3.4.11.2
- the invention also relates to the use of inhibitors of alanyl aminopeptidase and / or of inhibitors of enzymes with analogous enzymatic action for the activation of regulatory T cells.
- Treg natural regulatory T cells
- Treg cells are produced in the thymus [Kawahata K. et al., J. Immunol. 168: 4399-4405, 2002] and accounts for 5 to 10% of T cells in peripheral blood. They have an inhibitory effect on CD4 + T cells of the same antigen specificity via direct cell contact. This inhibitory effect is achieved by a strong expression of TGF-ß1 in / on the Treg.
- TGF- ⁇ 1 is thereby presented on the surface of the Treg and binds to the TG F- ⁇ 1 receptor on autoreactive T cells, which represents a completely new mechanism of action of this strong immunosuppressive cytokine [Nakamura et al., J. Exp. Med. 194: 629-644, 2001].
- Treg cells inhibit autoimmunity more efficiently than the immune response against "foreign" antigens [Romagnoli, P. et al., J. Immunol. 168: 1644-1648, 2002]. Therefore, limitations or losses of the function of Treg cells have particular pathogenetic significance in the development of autoimmune diseases.
- a direct correlation between the number / function of Treg cells and the manifestation of autoimmune diseases is for type I diabetes [Boudaly, S. et al., Eur. Cytokine Netw. 13: 29-37, 2002; Gregori, S. et al., Diabetes 51: 1367-1374, 2002], autoimmune encephalomyelitis (animal model of multiple sclerosis) [Furtado, G.C.
- Treg cells are also responsible for the suppression of intestinal or pulmonary inflammation [Singh, B. et al., Immunol. Rev. 182: 190-200, 2001; Hori, S. et al., Eur. J. Immunol. 32: 1282-1291, 2002]. Equally clearly demonstrated is the role of Treg cells in suppressing rejection episodes after allogeneic (foreign) organ transplantation [Kingsley, Cl. et al., J. Immunol. 168: 1080-1086, 2002; Taylor, PA et al., Blood 99: 3493-3499, 2002; Chiffoleau, E. et al., J. Immunol. 168: 5058-5069, 2002].
- each Treg cell clone is directed against a specific antigen and inhibits autoreactive T cells of the same antigen specificity under normal physiological conditions.
- this function of Treg is lost, and autoreactive T cell clones, as directed against pancreatic beta cell proteins in the case of type I diabetes, lead to the onset of autoimmune disease.
- this antigen specificity can also be utilized therapeutically by increasing or re-increasing the number / function of these cells by targeted "antigen-specific" activation in vivo or ex vivo of Treg cells (or dendritic cells activating these cells) will be produced. Oral administration of "antigens” is also suitable for this [Zhang et al., J. Immunol. 167: 4245-4253, 2001]. However, the production of such antigens is technically extremely time-consuming and expensive and limited to antigen-specific T-cell clones.
- TGF-ß1 in the regulation of immunological hyperreactivity is underscored by two recent publications which show that the overproduction of TGF-ß1 in CD4 + cells caused by genetic manipulation can suppress the disease. Since in the case of asthma Th2 cells play a decisive role in the pathogenesis, the function of pathogenic Th2 cell clones can be effectively inhibited by transgenic overproduction of TGF- ⁇ 1 [Hansen, G. et al., J. Clin. Invest. 105: 61-70, 2000; Thorbecke, GJ et al., Cytokine Growth Factor Rev. 11: 89-96, 2000].
- the document DE-A 102 30 381 relates to the use of one or more inhibitors of alanyl aminopeptidases and / or one or more inhibitors of enzymes of the same substrate specificity for the induction of the production of TGF- ⁇ 1 and expression of TGF- ⁇ 1 in and / or on Treg cells and the use for the prevention and / or treatment of autoimmune diseases, allergies, arteriosclerosis and suppression of transplant rejections.
- Treg cells outside of the human or animal body (ex vivo) by one or more inhibitors of alanyl aminopeptidases and / or by one or more inhibitors of peptidases of the same substrate specificity and by means of the activated Treg cells in the human or animal body to produce tolerance to alloantigens and autoantigens, or even to overcome an excessive immune response in the body.
- the invention therefore relates to a method for activating regulatory T cells (Treg cells) of the human or animal body, comprising a step of bringing the regulatory T cells (Treg cells) into contact in a suitable liquid medium with one or more several inhibitors of alanyl aminopeptidase (aminopeptidase N, APN) and / or with one or more inhibitors of peptidases of the same substrate specificity with the induction of a suppressive effect of the regulatory T cells (Treg cells).
- the invention relates to a method for ex-wVo activation of regulatory T cells (Treg cells) of the human or animal body, comprising the steps:
- the invention also relates to activated regulatory T cells (Treg cells) obtainable by a method as described in detail below.
- the invention further relates to a preparation which comprises activated regulatory T cells (Treg cells), as produced by the process according to the invention, if appropriate together with customary carriers, excipients and / or adjuvants.
- the invention further relates to the use of activated regulatory T cells (Treg cells) according to the following detailed description and / or the use of preparations comprising such regulatory T cells (Treg cells) for the prevention, mitigation or therapy of transplant - rejection reactions, autoimmune diseases, allergies, bronchial asthma and COPD, diseases of chronic inflammatory genesis, including arteriosclerosis, neuronal diseases and cerebral lesions, skin diseases, preferably psoriasis, acne or celloids, and other hyperproliferative conditions, fibrosis, tumors and sepsis.
- Figure 1 is a graph showing quantitatively the activation of human regulatory T cells in the presence of actinonin as an inhibitor of aminopeptidase N;
- Figure 2 is a graph quantitatively showing the activation of human regulatory T cells in the presence of PAQ22 as an inhibitor of cytosolic aminopeptidase (cAAP);
- Figure 3 is a graph quantitatively showing the activation of human regulatory T cells in the presence of IP10.C8 as a dual inhibitor of alanyl aminopeptidase (APN) and dipeptidyl peptidase IV (DPIV);
- API alanyl aminopeptidase
- DPIV dipeptidyl peptidase IV
- Figure 4 is a graph quantitatively showing the activation of murine regulatory T cells in the presence of phebestin as an inhibitor of alanyl aminopeptidase (APN); and Figure 5 is a graph showing quantitatively the effect of ex situ with an inhibitor of APN (Phebestin) activated regulatory T cells (Treg cells) in the mouse colitis model.
- APN alanyl aminopeptidase
- the invention relates to a method for activating regulatory T cells (Treg cells, CD4 + CD25 + cells).
- regulatory T cells are understood to mean those T lymphocytes which have the ability to control pathogenic T cell responses .Treg cells differentiate in the thymus and are then inserted into the thymus
- the main task of Treg cells in the human or animal organism is to block the effector function of autoreactive mature T cells (Sakaguchi, S. et al., J. Immunol., 155: 1151-1164 ( Roncarolo, MG et al., J. Exp. Med. 193 :, F5-F9).
- the Treg cells are activated.
- Activation in the present description and in the claims means that a suppressive effect of the Treg cells is induced, which is expressed in a strong expression of the transforming growth factor ⁇ 1 (TGF- ⁇ 1) and the transcription factor FoxP3.
- TGF- ⁇ 1 transforming growth factor ⁇ 1
- the term “activation” according to the invention also includes a reactivation of Treg cells. This can be done for example after inactivation of Treg cells under inflammatory conditions in vitro as in vivo, eg. B. by prolonged exposure to inflammatory cytokines.
- inhibitor in the present specification and in the claims means those compounds of natural origin, of synthetic origin or of natural origin with synthetic modification which have a regulating effect, in particular an inhibiting effect, on an enzyme or on a group of enzymes
- the regulatory effect can be based on a wide variety of effects, without limiting the broad definition of inhibitor mentioned above.
- Preferred inhibitors according to the invention are inhibitors with an inhibiting effect on enzymes, more preferably groups of certain enzymes, for example inhibitors with inhibiting action on alanyl aminopeptidase N (APN) and on peptidases with alanyl aminopeptidase N-identical substrate specificity or inhibitors with inhibitory action on dipeptidyl peptidase IV (DP IV) and on peptidases with dipeptidyl peptidase IV of the same substrate specificity.
- API alanyl aminopeptidase N
- DP IV dipeptidyl peptidase IV
- a single Inhibitor can be used. Alternatively, several inhibitors may be used. Particularly preferred according to the invention is the use of an inhibitor.
- the inhibitor (s) used in the method of the invention may be one or more inhibitors of alanyl aminopeptidase. Alternatively, the inhibitor (s) used in the method of the invention may be one or more inhibitors of peptidases having the same substrate specificity as the alanyl aminopeptidase.
- the inhibitor (s) used may be one or more inhibitors of both alanyl aminopeptidase and peptidases having the same substrate specificity.
- the inhibitor used or, if more than one inhibitor is used, the inhibitors used may be an inhibitor (as further specified below in preferred embodiments) of alanyl aminopeptidase (aminopeptidase N; APN; CD13; EC 3.4.11.2), or the inhibitor may be an inhibitor of a peptidase having the same substrate specificity as the alanyl aminopeptidase.
- inhibitor of alanyl aminopeptidase refers to those substances which are capable of specifically inhibiting the enzyme activity of APN and other peptidases having the same substrate specificity
- a common feature of APN inhibitors is their affinity for the active site of APN and peptidases with the same substrate specificity, characterized by a Zn 2+ ion, a zinc-binding motif with the sequence HEXXH - (XI 8) -E and the exopeptidase motif GXMEN
- the essential amino acid residues responsible for the binding of all known APN inhibitors in the active site of the APN include E355, H388, E389, H392, E411 and Y477.
- inhibitors of peptidases having the same substrate specificity refers in the sense of the above to inhibitors of such peptidases, the effect of which incorporates high Examples of such peptidases are cytosolic aminopepidase (cAAP, EC 3.4.11.14), aminopeptidase A (APA, EC 3.4.11.7), thyrotrophin-releasing hormone-degrading, and cytosolic aminopeptidase Ectoenzymes (TRH-DE; EC 3.4.19.6), adipocyte-derived leucine aminopeptidase (A-LAP; EC 3.4.11.x), insulin-regulated aminopeptidase (IRAP; EC 3.4.11.3), aminopeptidase B (APB; EC 3.4.11.6), leukotriene A4 hydrolase (LTA4H; EC 3.3.2.6), and leukocyte-derived arginine amino
- inhibitors of alanyl aminopeptidase (aminopeptidase N; APN; CD13; EC 3.4.11.2.) Is particularly preferred.
- the step of activation takes place ex vivo.
- the preferred process according to the invention is not a process performed on the human or animal (living) organism. Rather, the activation step is carried out in vitro with substance taken from the living human or animal body, and the substance treated according to the invention is subsequently returned to the human or animal body in a suitable form.
- activation results of the Treg cells are obtained in this way, which are unexpected to those skilled in the art.
- Ex-wVo method for activating regulatory T cells (Treg cells) of the human or animal body is obtained from at least one, preferably from exactly one, human or animal body at least one body fluid, the extraction of Treg cells can therefore serve, that includes Treg cells.
- a body fluid comprising Treg cells can be obtained from a human or animal body, or body fluids comprising a plurality of Treg cells can be obtained from a human or animal body. This can be done in a way known to the person skilled in the art on or from the living human or animal body and depends on the technique of obtaining the body fluid in question.
- body fluids As a way of obtaining one or more body fluids, it may be a way of delivering the body fluid (s) through the human or animal body (ex., For example, exudates) or a way of removing the bodily fluid (s) (for example, blood) Professional eligible.
- body fluid for example, exudates
- bodily fluid for example, blood
- one or more body fluids selected from blood, fractions of blood, lymph, exudates, or local compartments are obtained from a human or animal body. Practically, one obtains a single body fluid selected from the aforementioned body fluids.
- peripheral blood more preferably intravenous blood.
- pleura or peritoneum can preferably be isolated as local compartments.
- peripheral blood is obtained from a human or animal body, with particular advantage intravenous blood.
- Treg cells are naturally present in concentrations that facilitate the post-successive isolation practically.
- the body fluid (s) obtained in the first step of the method according to the invention preferably from one of the abovementioned body fluids, in particular from one of the abovementioned body fluids obtained from a human or animal body, more particularly from blood and with particular advantage from peripheral blood
- regulatory T cells Treg cells
- This can be done in any conceivable method known to the person skilled in the art for isolating Treg cells, without the invention being subject to any restrictions in this respect.
- separation kits are commercially available for the isolation of Treg cells, with the help of which isolation of Treg cells from one of the aforementioned body fluids can be done reliably.
- cell fractions are separated by suitable separation processes, which also comprise the regulatory T cells (Treg cells).
- suitable separation processes which also comprise the regulatory T cells (Treg cells).
- Treg cells For example, mononuclear cells and T cells enriched therefrom can be obtained in a manner known per se by density gradient centrifugation from peripheral donor blood by various methods which are generally known to the person skilled in the art and comprise Treg cells.
- regulatory T cells can be obtained by separation methods which take account of the properties of the Treg cells, eg. B. (without limitation) using Zeil-typical antibodies that are attached to magnetic particles. According to the invention, a two-stage magnetic separation has proven itself.
- the obtained in the preceding step cell-population of CD4 can "cells are depleted, with CD4 + cells according to the invention remain example, this can be done using commercially available release kits, for example, a CD4.”
- a CD4 - separation kit, as marketed by the company Miltenyi Biotech, Bergisch-Gladbach, Germany.
- CD4 + T cells can already be obtained with a purity of> 95%.
- the second Magnetic column separation step the remaining Zeil population is then treated with anti-CD25 microbeads (Miltenyi Biotech, Bergisch-Gladbach, Germany) and CD25-labeled CD4 + CD25 + T cells (regulatory T cells; Treg Cells) by magnetic column separation.
- Treg cells highly pure regulatory T cells
- the step of isolating the Treg cells is accompanied by a purification of the Treg cells, ie an elimination of other cells, cell components or other materials that oppose, hinder or even prevent the later activation of the Treg cells could.
- the invention is not limited to this merely exemplified process for isolating and purifying Treg cells.
- the thus obtained and purified regulatory T cells in a suitable fluid medium with one or more inhibitors of alanyl aminopeptidase (aminopeptidase N, APN) and / or with one or more inhibitors of peptidases of the same substrate specificity for a time sufficient for activation contacted may be done in any manner known and understood by those skilled in the art without any limitation to the invention.
- Treg cells and one or more inhibitors are contacted according to the following specific description directed to the inhibitors in a suitable fluid medium.
- fluid medium in the present specification and claims it is meant primarily liquid cell culture media as commercially available in various forms with and without protein and serum components, but this is not a limitation of the invention but only a concentration on the practically urgent possibilities is that they should be physiologically acceptable, not only for practical reasons of later reinfundability of the Treg cells into a human or animal body, but also for a naturally-occurring activation process under conditions as close as possible to the conditions existing in vivo come.
- the most preferred media used are thus selected from physiologically acceptable solutions, cell culture media and nutrient media. Even more preferably, these media are selected from the group consisting of physiologically acceptable aqueous solutions, aqueous cell culture media and aqueous nutrient media.
- serum-free AIMV medium is chosen as the medium for the activation process.
- the aforementioned media may be selected singly or in combinations of two or more of them. Particularly preferred is the use of media which contain predominantly water or consist essentially of water.
- customary additives to a fluid medium intended for the activation process in a cell culture and / or cell therapy which a person skilled in the art knows from his specialist knowledge.
- antibiotics, amino acid supplements, vitamin supplements and trace element supplements may be mentioned, which may be used individually or in combination of two or more of the named substance groups in the medium or media provided for the activation process.
- the isolated (and purified) regulatory T cells (Treg cells) as described above are contacted with one or more inhibitors, as described in detail below, for a time sufficient for activation.
- the addition of interleukin-2, preferably 20 to 100 U / ml, to the culture medium is conducive and / or stimulation with the aid of mitogens such as PHA or PWM and / or with the aid of anti-CD3 antibodies is beneficial.
- the incubation period may be from a specialist in the field Frame-oriented experiments for a particular system. It is according to experience in the range of 24 to 48 h, without being limited to this area.
- the regulatory T cells isolated as described above are brought into contact with one or more inhibitors of alanyl aminopeptidase (aminopeptidase N; APN) and / or with one or more inhibitors of peptideases having the same substrate specificity.
- An inhibitor may be used in the method of the invention to activate the Treg cells, or several inhibitors may be used.
- a single inhibitor may be an inhibitor of alanyl aminopeptidase, or a single inhibitor may be an inhibitor of a peptidase having the same substrate specificity. If multiple inhibitors are used, two or more inhibitors may be used in combination.
- These two or more inhibitors may all be inhibitors of alanyl aminopeptidase, or may all be inhibitors of peptidases having the same substrate specificity, or the two or more inhibitors may be inhibitors which are part of the group of inhibitors of alanyl aminopeptidase and part of the group derived from inhibitors of peptidases with the same substrate specificity, or are the inhibitors of alanyl aminopeptidase and at the same time also inhibitors of (one or more) peptidases with the same substrate specificity as alanyl aminopeptidase.
- a single inhibitor of one of the two aforementioned groups is used, and the use of an inhibitor of alanyl aminopeptidase is very particularly preferred.
- one or more known inhibitors from the group actinin, leuhistin, phebestin, amastatin, bestatin are used as the at least one inhibitor of alanyl aminopeptidase and / or as the at least one inhibitor of peptidases having the same substrate specificity.
- alanyl aminopeptidase there may be mentioned the following compounds which can be used alone or in combination of several of them for the activation of Treg cells:
- Arphamenine A 5-amino-8 - ⁇ [amino (imino) methyl] amino ⁇ 2-benzyl-4-oxo-octanoic acid
- Arphamenine B 5-amino-8 - ⁇ [amino (imino) methyl] amino ⁇ 2- (4-hydroxybenzyl) -4-oxooctanoic acid
- Treg cells regulatory T cells
- the at least one inhibitor of alanyl aminopeptidase and / or as the at least one inhibitor of peptidases with the same substrate specificity one or more known inhibitors from the Group ⁇ -ketoamides, ⁇ -aminophosphinic acids, N-phenyl homophthalimides and ⁇ -aminophosphonates to use. If ⁇ -ketoamides are used, one compound from the group of 3-amino-2-oxo-4-phenylbutanoic acid amides can be used with particular preference.
- ⁇ -aminophosphinic acids particular preference is given to using D-Phe- ⁇ [PO (OH) -CH 2 ] -Phe-Phe.
- PAQ-22 can be used with particular preference.
- RB3014 and / or phebestin can be used with particular preference.
- PAQ-22, RB3014 and / or phebestin as the at least one inhibitor of alanyl aminopeptidase and / or as the at least one inhibitor of peptidases having the same substrate specificity.
- the abbreviation RB3014 stands for the substance
- the at least one inhibitor of alanyl aminopeptidase and / or the at least one inhibitor of peptidases having the same substrate specificity is one or more known inhibitors from the group of dual inhibitors of alanyl aminopeptidase or Peptidases having the same substrate specificity and the dipeptidyl peptidase (IV) or peptidases having the same substrate specificity from the group of the compounds of the general formulas (1) and (2)
- X is S 1 O, CH 2 , CH 2 CH 2 , CH 2 O or CH 2 NH and Y is H or CN and * denotes a chiral carbon atom, preferably in the S or L configuration ;
- B and B 1 may be the same or different and is an unsubstituted or substituted, unbranched or branched alkylene radical which contains or does not contain O, N or S, cycloalkylene radical, aralkylene radical, heterocycloalkylene radical, heteroarylalkylene radical , Arylamidoalkylene radical, heretoarylamidoalkylene radical, unsubstituted or mono- or polysubstituted arylene radical or heteroarylene radical having one or more five-, six- or seven-membered ring (s);
- E is the group - CH 2 - CH (NH 2 ) - R 9 or - CH 2 - * CH (NH 2 ) - R 9 , where R 9 is an unsubstituted or substituted O, N or S containing unbranched or branched alkyl radical, cycloalkyl radical, aralkyl radical, heterocycloalkyl radical, heteroarylalkyl radical, arylamidoalkyl radical, heteroarylamidoalkyl radical, unsubstituted or mono- or polysubstituted aryl radical or heteroaryl radical having one or more radicals , six- or seven-membered rings and * denotes a chiral carbon atom, preferably in the S or L configuration;
- inhibitors in the present description and in the claims inhibitors are understood which are inhibitors of both the alanyl aminopeptidase and / or inhibitors of peptidases having the same substrate specificity (as defined above), as well as inhibitors of dipeptidyl peptidase IV (DP IV; CD26; EC 3.4.14.5) and / or inhibitors of peptidases having the same substrate specificity.
- inhibitors of dipeptidyl peptidase IV in the present description and in the patent claims means those substances which are capable of specifically inhibiting the enzyme activity of DP IV and of other peptidases having the same substrate specificity
- a common feature of these inhibitors is their affinity for the active site of DP IV and the peptidases with the same substrate specificity.
- This molecular region of DP IV and other peptidases having the same substrate specificity is characterized by the amino acid groups of DP IV inhibitors. Residues S630, D708, H740 ("catalytic triad"), E205, E206 and Y547.
- the inhibitor-binding amino acid residues include the residues Y666, F357 and R358.
- peptidases which have a substrate specificity identical to dipeptidyl peptidase IV (in an analogous sense to the peptidases already defined above with the APN same substrate specificity) can also be found in the abovementioned document.
- fibroblast-activating protein ⁇ x dipeptidyl peptidase IV ⁇ , dipeptidyl-aminopeptidase-like protein (DPPX), NAALADase (N-acetylated ⁇ -linked acid dipeptidase), QPP (Quiescent Cell Proline Dipeptidase), dipeptide tidyl peptidase II (DP II), attractin (Mahogany protein), dipeptidyl peptidase 8 (DP 8) and dipeptidyl peptidase 9 (DP 9).
- a and A 1 which may be the same or different, are a radical
- X is S, O, CH 2 , CH 2 CH 2 , CH 2 O or CH 2 NH and Y is H or CN and * is a chiral carbon atom.
- Particular preference according to the invention is given to compounds of the general formulas (1) in which A and A 'are identical, and to compounds of the general formulas (1) and (2) in which X in the above radical A represents S, CH 2 or CH 2 CH 2 and / or Y is H or CN.
- such compounds of the general formulas (1) and (2) are prodrugs which are inhibitors which are particularly active in the activation of Treg cells, in which the chiral carbon atom denoted by * denotes an S- or L- Configuration has.
- prodrug in the present specification and in the patent claims naturally occurring or synthetic or naturally occurring, but synthetically modified compounds understood that make other compounds under certain conditions, preferably under physiological or pathological conditions or under conditions of a desired chemical Reaction (such as the activation of Treg cells), chemically derive or derivatize, these other compounds have a chemical or pharmacological activity that qualitatively and / or quantitatively of the starting substance (the "prodrug") different.
- prodrugs compounds of natural or synthetic shear origin or natural, but synthetically modified compounds understood, which may react under physiological or pathological conditions or conditions of a desired chemical reaction, targeted to new substances with inhibitory activity.
- prodrugs as such, even before the conversion into drugs with certain pharmacological (for example, inhibitory) efficacy in a position to develop pharmacological activity (for example, to inhibit one of the two enzymes mentioned above).
- Conditions for converting prodrugs into mammalian or human drugs may be those of the type regularly present in the physiological environment of a mammal, such as a human, or in the body of a mammal, such as a human.
- physiological conditions may be present only under certain conditions, such as a particular physiological condition, such as a disease condition, in a mammal, such as a human, or may be imposed on the organism by external agents, such as, but not limited to, drug intervention of a mammal, such as the human organism, or by creating certain chemical reaction conditions.
- a particular physiological condition such as a disease condition
- external agents such as, but not limited to, drug intervention of a mammal, such as the human organism, or by creating certain chemical reaction conditions.
- B and B ' may be the same or different and represent an O, N or S containing, unsubstituted or substituted, unbranched or branched alkylene radical, cycloalkylene radical Radical, aralkylene radical, heterocycloalkylene radical, heteroarylalkylene radical, arylamidoalkylene radical, heteroarylamidoalkylene radical, unsubstituted or mono- or polysubstituted arylene radical or heteroarylene radical having one or more five-, six- or seven-membered rings.
- alkyl radical is understood as meaning a monovalent straight-chain ("unbranched") or branched radical of carbon atoms bonded together via single bonds and having hydrogen atoms bonded to the carbon atoms. alkyl Residues in the context of the present invention are thus saturated monohydric coal water radicals.
- the alkyl radicals in the compounds of the general formulas (1) and (2) comprise 1 to 18 carbon atoms and are thus selected from the radicals methyl, ethyl, n-propyl, i-propyl and the numerous different straight-chain and branched Isomers of the radicals butyl, pentyl, hexyl, heptyl, octyl, nonyl, decyl, undecyl, dodecyl, tridecyl, tetradecyl, pentadecyl, hexadecyl, heptadecyl and octa-decyl.
- radicals having 1 to 12 carbon atoms Particularly preferred are straight-chain and branched alkyl radicals having 1 to 12 carbon atoms, and straight-chain and branched alkyl radicals having 1 to 6 carbon atoms are even more preferred.
- the radicals are methyl, ethyl, n-propyl, i-propyl, n-butyl, i-butyl, sec-butyl and tert-butyl.
- alkenyl radical and alkynyl radical refer to monovalent straight chain (“unbranched") or branched radicals from each other via single bonds and at least one double bond or triple bond to any one but more defined
- carbon atom bound in the molecule is understood to mean hydrogen atoms bound to the remaining bonds of the carbon atoms and having at least 2 carbon atoms and up to 18 carbon atoms.
- examples of such radicals are preferably vinyl radicals or allyl radicals
- carbon-carbon multiple-bond radicals are not limited to the two aforementioned radicals.
- alkylene radical is understood to mean a divalent straight-chain ("unbranched") or branched radical of carbon atoms bonded together via single bonds with hydrogen atoms bonded to the carbon atoms. Alkylene radicals in the context of the present invention are thus saturated divalent hydrocarbon radicals.
- the alkyl-ene radicals in the compounds of the general formulas (1) and (2) comprise 1 to 18 carbon atoms and are thus selected from the radicals methylene, ethylene, n-propylene, 2,2-propylene, 1, 2-propylene and the numerous different straight-chain and branched isomers of the radicals butylene, pentylene, hexylene, heptylene, octylene, Nonylene, decylene, undecylene, dodecylene, tridecylene, tetradecylene, pentadecylene, hexadecylene, heptadecylene and octadecylene.
- straight-chain and branched alkylene radicals having 1 to 12 carbon atoms Particularly preferred are straight-chain and branched alkylene radicals having 1 to 6 carbon atoms are even more preferred. Most preferred are the radicals methylene, ethylene, n-propylene, 2,2-propylene, 1,2-propylene and the various different butylene positional isomers.
- the chains of carbon atoms can be replaced by -O atoms, -N atoms or S atoms are interrupted;
- one or more group (s) of the group -O-, -NH-, and -S- may be present in the course of the chain instead of one or more -CH 2 group (s), where usually not two of the groups - O-, -NH- and / or -S- follow each other in the chain.
- the one or more group (s) -O-, -NH- or -S- may be introduced anywhere in the molecule.
- such a hetero-group is present, such a group is contained in the molecule.
- Both straight-chain and branched alkyl or alkylene radicals in the compounds of the general formulas (1) and (2) may be substituted according to the invention in another embodiment with one or more substituents, preferably with one substituent.
- the substituent (s) may be at any positions on the backbone formed of carbon atoms, and may preferably be selected from the group consisting of halogen atoms such as fluorine, chlorine, bromine and iodine, without limiting the invention thereto , Particularly preferably chlorine and bromine, alkyl groups having 1 to 6 carbon atoms, particularly preferably alkyl groups having 1 to 4 carbon atoms, alkoxy groups having 1 to 6 carbon atoms in the alkyl radical, preferably with 1 to 3 C atoms in the alkyl radical, unsubstituted or substituted by one or two alkyl radicals each having 1 to 6 carbon atoms, preferably 1 to 3 carbon atoms, substituted amino groups, carbonyl groups and carboxyl groups.
- the latter can also be used in the form of salts or esters with alcohols having 1 to 6 carbon atoms.
- atoms in the alkyl radical are present;
- the substituent alkyl groups are selected from the alkyl groups defined above in detail and are very particularly preferably methyl Groups, ethyl groups, n-propyl groups, i-propyl groups, n-butyl groups, i-butyl groups, sec-butyl groups or tert-butyl groups, alkoxy groups are alkyl groups in the sense defined above, which are bonded via
- amino groups also includes groups of the structure defined above, which are present as quaternized ammonium ions, either by salt formation with organic acids or inorganic acids (ie radicals of the structure R x R y R 2 N + Q ' , wherein R x , R y and R 2 may be the same or different, are preferably the same, and may have the meanings defined above for R x and R y and at least one of the radicals is hydrogen from the quaternization with the organic or inorganic acid and Q is an acid radical of the organic or inorganic acid) or by salt formation with suitable quaternizing reagents known to those skilled in the art, such as, but not limited to, alkyl halides.
- suitable quaternizing reagents known to those skilled in the art, such as, but not limited to, alkyl halides.
- cycloalkyl stands for unsubstituted or substituted monovalent radicals. connected to closed rings -CH 2 groups. According to the invention, these may preferably contain three to eight atoms in the ring and may either consist exclusively of carbon atoms or contain one or more heteroatom (s) selected from -O-, -S- and -NR x -, wherein R x is hydrogen or an (as defined above) alkyl radical having 1 to 6 carbon atoms.
- heteroatoms are incorporated into the rings, they may be the same or different in the case of several heteroatoms.
- a heteroatom is included in the ring.
- Particularly preferred among the purely carbocyclic rings are the radicals cyclopentyl, cyclopentenyl, cyclopentadienyl, cyclohexyl, cyclohexenyl, cyclohexadienyl, cycloheptyl, cycloheptenyl, cycloheptadienyl and cycloheptatrienyl.
- heteroatom-containing cycloalkyl radicals which are also referred to as heterocycloalkyl radicals, in further embodiments of the invention are the radicals tetrahydrofuranyl, pyrrolidinyl, pyrazolidinyl, imidazolidinyl, piperidinyl, piperazinyl and morpholinyl.
- substituents on these carbocyclic or heterocyclic cycloalkyl radicals may preferably, without being limited to the invention, be selected from the above group of substituents for linear alkyl groups.
- Particularly preferred substituents for cycloalkyl groups are the substituents -Cl, -Br, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl or tert-butyl, methoxy, ethoxy, n- Propoxy, i-propoxy; n-butoxy, i-butoxy, sec-butoxy and tert-butoxy, -NH 2 , -NH (CH 3 ), -N (CH 3 ) 2 , -NH (C 2 H 5 ) and -N (C 2 Hs ) 2 , carbonyl and carboxyl.
- cycloalkylene represents unsubstituted or substituted divalent radicals of closed-ring -CH 2 groups which, according to the invention, may preferably contain from three to eight atoms in the ring and may be selected exclusively from carbon atoms or contain one or more heteroatom (s) selected from -O-, -S- and -NR x -, where R x is hydrogen or an alkyl radical (as defined above) 1 to 6 carbon atoms are particularly preferred among the purely carbocyclic rings the radicals cyclopentylene, cyclopentenylene, cyclopentadienylene, cyclohexylene, cyclohexenylene, cyclohexadienylene, cycloheptylene, cycloheptenylene, cyclohep-tadienylene and cycloheptatrienylene.
- heterocyclic groups defined above in the cycloalkyl radicals may occur as divalent radicals in the compounds of the general formulas (1) and (2) as groups "B", and particularly preferred are those cyclic divalent radicals in which a group -O - or -NR x - is integrated in the ring in these cases, both valencies are located at any of C-atoms in the ring preferably, a hetero atom or two hetero atoms are included in the ring, and in particularly preferred embodiments of such groups are the divalent.. Radicals derived from tetrahydrofuran, pyrrolidine, pyrazolidine, imidazolidine, piperidine, piperazine and morpholine.
- Possible substituents on these carbocyclic or heterocyclic cycloalkylene radicals may preferably be selected from the above group of substituents for linear alkyl groups, without limiting the invention thereto.
- Particularly preferred substituents for cycloalkylene groups are the substituents -Cl, -Br, methyl, ethyl, n-propyl, i-propyl, n-butyl, isobutyl, sec-butyl or tert-butyl, methoxy, ethoxy, n- Propoxy, i-propoxy; n-butoxy, i-butoxy, sec-butoxy and tert-butoxy, -NH 2 , -NH (CH 3 ), -N (CH 3 ) 2 , -NH (C 2 H 5 ) and -N (C 2 Hs ) 2 , carbonyl and carboxyl.
- aryl radical is understood as meaning a monovalent hydrocarbon radical derived from a cyclic molecule of aromatic character (4n + 2 delocalized in ring orbitals) which may be unsubstituted or substituted Ring structure of such an aryl radical may be a five-membered, six-membered or seven-membered ring structure with one ring or a structure formed of two or more rings bound together (“annellated”), wherein the fused rings the same or a different number of ring members, in particular of C atoms, may have.
- benzo-fused rings are particularly preferred, ie ring systems in which at least one of the rings aromatic, consisting only of carbon atoms six-membered ring (phenyl ring) is.
- aryl radicals are cyclopentadienyl radicals (C 5 H 5 ' ) (as a five-membered ring), phenyl radicals (as a six-membered ring), cycloheptatrienyl radicals (C 7 H 7 + ) (as seven-membered ring), naphthyl radicals (ring system comprising two annelated six-membered rings), and monovalent radicals (three annealed six-membered rings) derived from anthracene and phenanthrene.
- the most preferred aryl radicals in this invention are phenyl and naphthyl radicals.
- substituents on these carbocyclic aryl radicals may preferably be selected from the above group of substituents for linear alkyl groups, without restricting the invention to these substituents.
- Particularly preferred substituents for aryl groups are the substituents -Cl, -Br, methyl, ethyl, n-propyl, i-propyl, n-butyl, isobutyl, sec-butyl or tert-butyl, methoxy, ethoxy, n- Propoxy, i-propoxy; n-butoxy, i-butoxy, sec-butoxy, tert-butoxy, -NH 2 , -NH (CH 3 ), -N (CH 2 ) 5 , -NH (C 2 H 5 ) and -N (C 2 Hs) 2 , carbonyl and carboxyl.
- arylene radical This is understood to mean a bivalent radical whose basic structure and its choice and its substituent (s) are denoted by above data for the definition of the "aryl radicals” are comparable, except that it is a bivalent radical, whose inclusion can take place at any two carbon atoms of the ring.
- heteroaryl radical is understood to mean an aryl radical (as defined above) in whose ring structure one or more heteroatoms, preferably from the group O, N or S, are present without the aromatic character of the Molecule is lost. Heteroaryl radicals according to the invention may be unsubstituted or substituted.
- the ring structure of such a heteroaryl radical may be a five-membered, six-membered or seven-membered ring structure having one ring or a structure formed from two or more rings joined together ("annulated"), wherein the annulated rings have the same or a different number of ring members
- the heteroatom (s) may be present alone in one or more of the rings of the ring system
- the heteroaryl moieties are preferably one or two rings, and systems consisting of multiple contiguous rings are benzo-fused Rings are particularly preferred, ie ring systems in which at least one of the rings is an aromatic carbocyclic (ie consisting only of C atoms) six-membered ring
- Particularly preferred heteroaryl radicals are selected from furanyl, thio-phenyl, pyridyl, indolyl, coumaronyl, thio naphthenyl, quinolinyl (benzopyridyl), quinazolin
- substituents on these heteroaryl radicals may preferably be selected from the above group of substituents for linear alkyl groups, without restricting the invention to these substituents.
- Particularly preferred substituents for heteroaryl groups are the substituents -Cl, -Br, methyl, ethyl, n-propyl, i-propyl, n-butyl, isobutyl, sec-butyl or tert-butyl, methoxy, ethoxy, n- Propoxy, i-propoxy; n-butoxy, i-butoxy, sec-butoxy, tert-butoxy, -NH 2 , -NH (CH 3 ), -N (CHs) 2 , -NH (C 2 H 5 ) and -N (C 2 H 5 ) 2 , carbonyl and carboxyl.
- heteroarylene radical This is understood to mean a bivalent radical whose basic structure and its selection and its substituent (s) are denoted by the above for the definition of the "heteroaryl radicals" comparable are, except that it is a divalent radical whose integration can be done on any two carbon atoms of the ring or ring system or on a nitrogen atom.
- aralkyl radical means alkyl radicals (or more particularly alkylene radicals) within the meaning of the above general and specific definition, attached to one of their bonds with an aryl radical (according to the above general and specific definition), heteroaryl radical (according to the above general and specific definition), Heterocyclyl radical (according to the above general and specific definition of the heteroatom-substituted cycloalkyl radicals), arylamido radical (according to the following general and specific definition) or heteroarylamido radical (according to the general and specific definitions below) may be unsubstituted or substituted.
- aralkyl radicals are those radicals in which the aryl radical is a phenyl radical, substituted phenyl radical, naphthyl radical or substituted naphthyl radical and the alkyl (en) group is straight-chain or branched, and Has 1 to 6 carbon atoms.
- Possible substituents on the aryl groups of the aralkyl radicals may preferably be selected from the above group of substituents for linear alkyl groups, without restricting the invention to these substituents.
- Particularly preferred substituents for aryl groups of the aralkyl radicals are the substituents -Cl, -Br, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl or tert-butyl, methoxy, Ethoxy, n-propoxy, i -propoxy; n-butoxy, i-butoxy, sec-butoxy, tert-butoxy, -NH 2 , -NH (CH 3 ), -N (CH 3 ) 2 , -NH (C 2 H 5 ) and -N (C 2 Hs) 2 , carbonyl and carboxyl.
- substituent (s) may be the same or different from each other, bonded to an aryl group of an aralkyl radical according to the present invention.
- the substituted ⁇ ) position (s) on the aryl ring (-System) can / can be chosen arbitrarily.
- heteroarylalkyl radicals are those radicals in which the heteroaryl radical of the heteroarylalkyl radicals according to the invention is substituted and the alkylene group is straight-chain or branched and has 1 to 6 carbon atoms.
- the ring structure of such a heteroaryl radical may be a five-membered, six-membered or seven-membered ring structure with one ring or a structure formed from two or more annealed rings, wherein the annulated rings have the same or a different number of ring members
- the heteroatom (s) may be present in one or more of the rings of the ring system alone
- the heteroaryl radicals of the heteroarylalkyl radicals preferably consist of one or two rings, in the case of a plurality of contiguous rings Particular preference is given to benzo-bonded rings, ie ring systems in which at least one of the rings is an aromatic carbocyclic six-membered ring, particularly preferred heteroarylalkyl radicals are selected from furanylmethyl and -ethyl, thiophenyl-methyl and -ethyl, pyridylmethyl and ethyl, indolylmethyl and -ethyl, coumaronyl-methyl
- Possible substituents on these heteroaryl groups of the heteroarylalkyl radicals may preferably be selected from the above group of substituents for linear alkyl groups, without restricting the invention to these substituents.
- Particularly preferred substituents for heteroaryl groups are the substituents -Cl, -Br, methyl, ethyl, n-propyl, i-propyl, n-butyl, isobutyl, sec-butyl or tert-butyl, methoxy, ethoxy, n- Propoxy, i-propoxy; n-butoxy, i-butoxy, sec-butoxy, tert-butoxy, -NH 2 , -NH (CH 3 ), -N (CH 3 ) 2 , -NH (C 2 H 5 ) and -N (C 2 Hs ) 2 , carbonyl and carboxyl.
- heteroarylalkyl radical there may be one or more such substituent (s), which may be the same or different, from a heteroarylalkyl radical be bound according to the present invention.
- the substituted (n) positions) on the heteroaryl ring (-System) can / can be chosen arbitrarily.
- heterocycloalkyl radicals are cycloalkyl radicals according to the above general or specific definition containing one or more heteroatom (s) selected from -O-, -S-, and - NR x - in which R x is hydrogen or an alkyl radical (as defined above) having 1 to 6 carbon atoms, and the alkyl (ene) groups of the heterocycloalkyl radicals are straight-chain or branched and have 1 to 6 carbon atoms ,
- R x is hydrogen or an alkyl radical (as defined above) having 1 to 6 carbon atoms
- the alkyl (ene) groups of the heterocycloalkyl radicals are straight-chain or branched and have 1 to 6 carbon atoms
- a heteroatom is incorporated in the ring.
- heteroatom-containing cycloalkyl radicals which are also referred to as heterocycloalkyl radicals, in further embodiments of the invention are the radicals tetrahydrofuranyl, pyrrolidinyl, pyrazolidinyl, imidazolidinyl, piperidinyl, piperazinyl and morpholinyl.
- substituents on these heterocycloalkyl radicals may preferably be selected from the above group of substituents for linear alkyl groups, without restricting the invention to these substituents.
- Particularly preferred substituents for heteroaryl groups are the substituents -Cl, -Br, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl or tert-butyl, methoxy, ethoxy, n-propoxy, i-propoxy; n-butoxy, i-butoxy, sec-butoxy, tert-butoxy, -NH 2 , -NH (CH 3 ), -N (CH 3 ) 2 , -NH (C 2 H 5 ), and -N (C 2 Hs ) 2 , carbonyl and carboxyl.
- heterocycloalkyl radical It may or may not be several such substituent (s), which may be the same or different from each other, bonded to a heterocycloalkyl radical according to the present invention.
- the substituted position (s) on the heterocycloalkyl ring (system) can be chosen as desired.
- Preferred examples of an arylamidoalkyl radical - without restricting the invention to that extent - are 2-, 3- or 4-benzoic acid amido-n-butyl radicals or 2-nitro-3, 4, 5 or 6-benzoic acid amido-n-butyl radicals; preferred but non-limiting examples of heteroarylamidoalkyl radicals are 2-, 4-, 5- or ⁇ -pyridine-3-carboxylic acid amido-n-butyl radicals.
- substituents on these arylamidoalkyl radicals and heteroarylamidoalkyl radicals may preferably be selected from the above group of substituents for linear alkyl groups, without restricting the invention to these substituents.
- Particularly preferred substituents for aryl groups or heteroaryl groups of the arylamidoalkyl radicals and heteroarylamidoalkyl radicals are the substituents -Cl 1 -Br 1 methyl, ethyl, n-propyl, i-propyl, n-butyl, i-butyl , sec-butyl or tert-butyl, methoxy, ethoxy, n-propoxy, i-propoxy; n-butoxy, i-butoxy, sec-butoxy, tert-butoxy, -NH 2 , -NH (CH 3 ), -N (CH 3 J 2 , -NH (C 2 H 5 ) and -N (C 2 Hs 2
- the radical D stands for - S - S - or - Se - Se -.
- These two S or Se atoms form a bridge between two parts of the molecules of the compounds of the general formulas (1) and (2), which can be cleaved under natural, in particular under reducing conditions.
- Two molecule parts are released which have an inhibiting effect on dipeptidyl peptidase IV (DP IV) and on peptidases with analogous enzymatic action and on alanyl aminopeptidase N (APN) and on peptidases with an analogous enzymatic action.
- DP IV dipeptidyl peptidase IV
- API alanyl aminopeptidase N
- E represents the group - CH 2 - C * H (NH 2) - R 9, wherein R 9 containing an O, N or S or not containing unsubsti- tuted or substituted, unbranched or branched Alkyl radical, cycloalkyl radical, aralkyl radical, heterocycloalkyl radical, heteroarylalkyl radical, arylamidoalkyl radical, heteroarylamidoalkyl radical, unsubstituted or mono- or polysubstituted aryl radical or heteroaryl radical having one or more five -, six- or seven-membered rings.
- alkyl radicals cycloalkyl radicals, aralkyl radicals, heterocycloalkyl radicals, heteroarylalkyl radicals, Arylamidoalkyl- radicals, Heteroarylamidoalkyl radicals, unsubstituted or mono- or polysubstituted aryl radicals or heteroaryl radicals with one or more five-, six- or seven-membered rings and the substituents conceivable and preferred for these radicals, reference may be made to the above definitions of the corresponding radicals and their preferred embodiments; these definitions are equally applicable to the radicals in the general formula (2) which E represents.
- * represents a chiral carbon atom on the amino group-substituted carbon atom.
- Forms of the invention represent such compounds of the general formula (2) prodrugs to particularly effective inhibitors, in which the chiral carbon atom designated * has an S- and L-configuration.
- radicals B and / or B ' are a radical R 1 , which represents a straight-chain or branched alkylene radical having 1 to 6 carbon atoms.
- Particularly preferred compounds of the general formulas (1) and (2) comprise radicals B and / or B 'in the form of one or more of the groups selected from -CH 2 - (methylene), -CH 2 -CH 2 - (ethylene) or ( H 3 C) 2 -C ⁇ (2,2-propylene).
- B and / or B ' are a radical - (CH 2 ) n - R 2 - R 3 - R 4 -, wherein n is an integer from 1 to 5;
- R 4 is an unsubstituted or substituted unbranched or branched alkylene radical containing or not containing O, N or S, cycloalkylene radical, aralkylene radical, heterocycloalkylene radical, heteroarylalkylene radical, unsubstituted or mono- or polysubstituted arylene radical or hetero
- Aryl radical is one or more five-, six- or seven-membered rings.
- R 4 is an amino-substituted alkylene radical, for example an aminoethylene radical, or a unsubstituted or (for example, with a nitro group) substituted phenylene radical or an unsubstituted or substituted pyridyl-2,5-en-residue.
- B and / or B ' are a radical of the formula - R 7 - R 8 -, wherein R 7 is a mono- or polysubstituted benzylene radical and R 8 is a single bond or an O, N or S containing unsubstituted or substituted unbranched or branched alkylene radical, cycloalkylene radical, aralkylene radical, heterocycloalkylene radical or heteroarylalkylene radical which preferably contains one or more amino groups, carbonyl groups or carbonyl Groups may have as functional groups, or is an unsubstituted or mono- or polysubstituted arylene radical or heteroarylene radical having one or more five-, six- or seven-membered rings.
- compounds of the general formulas (1) and (2) are further preferred according to the invention, in which B and B 'may be the same or different and represent a radical - R 7 - R 8 -, wherein R 7 and R 8 in combination stand for a rest
- R 8 and R 6 have the meanings given above, ie wherein R 6 is H 1 NO 2 , CN, halogen or an acyl radical and in which R 8 represents a single bond or an O, N or S-containing unsubstituted or substituted unbranched or branched alkylene radical, cycloalkylene radical, aralkylene radical, heterocycloalkylene radical Rest or heteroarylalkylene radical which may preferably have one or more amino groups, carbonyl groups or carboxyl groups as functional groups, or for an unsubstituted or mono- or polysubstituted arylene radical or heteroaryl radical with one or a plurality of five-, six- or seven-membered ring (s).
- Still more preferred compounds of the general formulas (1) and (2) are those in which B and B 'are the same or different and are independent each other is a radical - R 7 - R 8 - stand, wherein R 7 is a mono- or polysubstituted benzylene radical of the above formula (without R 8 ) and R 8 is NH - or - C r to C 6 alkylene - NH - in combination with
- R 1 has the abovementioned meanings
- the compounds of the general formulas (1) and / or (2) are in the form of neutral molecules and as such are used according to the invention in the activation of Treg cells.
- the compounds of general formulas (1) and / or (2) may also be present in the form of their acid addition salts with inorganic and / or organic acids.
- Such acid addition salts are due to the presence of basic centers (mostly of basic nitrogen atoms) in the molecule by addition of one or more molecules of H-acidic compounds (Bronsted acids), preferably a molecule of an H-acidic compound, and provide for improved solubility of the molecules in polar media, such as in water , The latter property is of particular importance for those compounds which exhibit pharmacological effects.
- the acid addition salts are salts of pharmaceutically acceptable acids and are advantageously (but not limited to the present invention) selected from the group consisting of hydrochlorides, trifluoroacetates, tartrates, succinates, formates and / or citrates of the compounds of the general Formulas (1) or (2).
- Acid addition salts of the compound of the general formula (1a), preferably their acid addition salts with pharmaceutically acceptable inorganic and / or organic acids, in particular with acids from the abovementioned group, with very particular preference the hydrochlorides, trifluoroacetates, tartrates, can be used with particular advantage in the process according to the invention , Succinates, formates and / or citrates of the compounds of the general formula (1a).
- X, Y, R 9 and B have the abovementioned meanings, and their acid addition salts, preferably their acid addition salts with pharmaceutically acceptable inorganic and / or organic acids, preferably from the abovementioned group of pharmaceutically acceptable acids, more preferably the hydrochlorides, trifluoroacetates, Tartrates, succinates, formates and / or citrates of the compounds of general formula (2a).
- the inhibitors are used at a concentration that is equal to or above the IC 50 inhibition value.
- the inhibitor concentration is in the nanomolecular to micromolar range and can be determined without difficulty by the person skilled in a few easy-to-perform standard experiments.
- the cultivation of the Treg cells with the one or more inhibitors according to the above detailed description is preferably carried out at 37 ° C., more preferably in an atmosphere of steam-saturated air having a CO 2 content of, for example, 5%.
- the concentration of Treg cells is adjusted to the total volume and is more preferably from 1 to 5 million cells per ml.
- MBP myelin basic protein
- MOG myelin oligodendrocyte glycoprotein
- MAG myelin-associated glycoprotein
- PLP proteolipid protein
- regulatory T cells are contacted in the medium / media described in detail above in a manner known to those skilled in the art.
- the step of contacting regulatory T cells (Treg cells) with one or more inhibitors of alanyl aminopeptidase (aminopeptidase N APN) and / or with one or more inhibitors of peptidases of the same substrate specificity carried out in conventional, preferably in static or horizontally moving or vertically moving or circularly moving, cell culture vessels.
- These may further preferably be culture dishes, culture plates, cell culture reactors, cell culture bottles, cell culture bags, cell or multi-chamber systems or hollow-fiber reactors or any other containers known to those skilled in the art, such as those for cultivating cells generally used.
- Culturing is particularly preferably carried out in cell culture vessels which have an optionally reaction-promoting surface coating and / or matrix substitutes on part or all of their culture-facing surface.
- the cell culture is preferably carried out in the presence of 5% CO 2 in water-saturated air at 37 ° C.
- the regulatory T cells (Treg cells) activated in the aforementioned way are returned in at least one human or animal body in a suitable medium.
- the Treg cells are regularly supplied to a human or animal body which requires these activated Treg cells to regulate an immune problem.
- the medium is a medium which is pharmaceutically acceptable to the recipient and may be selected in further preferred embodiments of the invention from preferably fluid, more preferably liquid, media from the group of physiologically acceptable solutions, more preferably aqueous solutions which may contain other useful or even beneficial components for the intended use.
- the activated Treg cells are supplied to the organism of the (human or animal) donor body fluid from which the Treg cells were isolated.
- the recycling can be done in any way that is conceivable for a person skilled in the art and fulfills the desired purpose, ie. H. the recipient organism (whether human or animal) re-activates the activated Treg cells.
- Particularly preferred routes of refeeding are administration forms which are selected from the group consisting of intravenous administration, intraarterial administration, intracavitary administration, intrathecal administration and intradermal administration.
- Treg cells regulatory T cells
- the invention also relates to activated regulatory T cells (Treg cells), as described in detail above, with one or more inhibitors of alanyl aminopeptidase and / or with one or more inhibitors of peptidases having the same substrate specificity are available.
- Treg cells activated regulatory T cells
- Such activated Treg cells are still unknown and have a surprisingly high suppressive effect compared to Treg cells, whose activation was carried out in a conventional way.
- the regulatory T cells (Treg cells) activated by way of the method according to the invention can be used to produce tolerance to autoantigens (antigens produced in the own organism) and alloantigens (antigens introduced by external factors).
- ne) in the human and animal body and to overcome an excessive immune response in the human and animal body are used because they surprisingly suppress the immune response of the body.
- the invention also relates to preparations of any kind which comprise or include activated regulatory T cells (Treg cells), which are isolated according to the method described in detail above with one or more inhibitors of alanyl aminopeptidase and / or with one or more Inhibitor (s) of peptidases with the same substrate specificity are activated.
- Such preparations optionally comprise, in addition to the activated regulatory T cells and the carrier or solvent suitable for administration, additionally one or more carriers known to the person skilled in the art, adjuvant (s) and / or adjuvants.
- one or more of the components known to the person skilled in the art as a solvent, carrier, adjuvant and / or adjuvant may fall individually or in combination.
- the invention further relates to the use of activated regulatory T cells (Treg cells) or the use of activated regulatory T cells (Treg cells) comprising preparations for the prevention, mitigation or treatment of numerous diseases and conditions associated with an unbalanced immune response of the human or animal body. With which inhibitor or inhibitors the Treg cells were activated does not play any role in the use according to the invention.
- the activated regulatory T cells (Treg cells) and preparations containing them have proven particularly effective in the prevention, alleviation or therapy of transplant rejections.
- the invention also relates to the use of activated regulatory T cells (Treg cells) or the use of activated regulatory T cells (Treg cells) comprising preparations for the prevention, mitigation or therapy of diseases with excessive immune response and inflammatory genesis, including arteriosclerosis , neuronal diseases, cerebra- lesions, skin diseases such as psoriasis, acne, cellos and other hyperproliferative conditions, fibrosis, tumors and virus-related diseases as well as sepsis and type II diabetes.
- activated regulatory T cells including arteriosclerosis , neuronal diseases, cerebra- lesions, skin diseases such as psoriasis, acne, cellos and other hyperproliferative conditions, fibrosis, tumors and virus-related diseases as well as sepsis and type II diabetes.
- the invention also relates to the use of activated regulatory T cells (Treg cells) or the use of preparations comprising activated regulatory T cells (Treg cells) for the production of a medicament or a cosmetic preparation for the prevention, alleviation or therapy of diseases with excessive immune response and inflammatory genesis, including atherosclerosis, neuronal diseases, cerebral damage, skin diseases such as psoriasis, acne, celloids and other hyperproliferative conditions, fibrosis, tumors and virus-related diseases, as well as type II sepsis and diabetes.
- diseases with excessive immune response and inflammatory genesis including atherosclerosis, neuronal diseases, cerebral damage, skin diseases such as psoriasis, acne, celloids and other hyperproliferative conditions, fibrosis, tumors and virus-related diseases, as well as type II sepsis and diabetes.
- the activated regulatory T cells or of the preparations containing them for the prophylaxis and therapy of diseases such as, for example, multiple sclerosis, Crohn's disease, ulcerative colitis, and other autoimmune diseases as well as inflammatory diseases, bronchial asthma and other allergic diseases, skin and mucous membrane diseases, for example psoriasis, acne and dermatological diseases with hyperproliferation and altered differentiation states of fibroblasts, benign fibrosing and sclerosing skin diseases and malignant fibroblastärer hyperproliferating conditions, acute neuronal diseases, such as ischemia-related cerebral damage after an ischemic or haemorrhagic stroke, traumatic brain injury, cardiac arrest, heart attack or as a result of cardiac surgery, of chronic neuronal Diseases such as Alzheimer's disease, Pick's disease, Progressive Supranuclear Palsy, corticobasal degeneration, frontotemporal dementia, Parkinson's disease, in particular Parkinsonis
- diseases such as, for example, multiple sclerosis, Crohn'
- the activated regulatory T cells (Treg cells) or of the preparations containing them for the prophylaxis and therapy of the rejection of transplanted tissues and cells.
- the use of regulatory T cells (Treg cells) or the use of a preparation comprising Treg cells in allogeneic or xenogeneic transplanted organs, tissues and cells such as kidney, heart, liver , Pancreatic, skin or stem cell transplantation as well as graft versus host diseases.
- the activated regulatory T cells (Treg cells) or the preparations containing them are used for the prophylaxis and therapy of rejection or inflammatory reactions on or by medical devices implanted in an organism ("medical devices"). These may be, for example, stents, joint implants (knee joint implants, hip joint implants), bone implants, heart pacemakers or other implants.
- the Treg cells or composition containing them in the form of a coating or wetting on the Subject or the objects are applied or at least the regulatory T cells (Treg cells) or the compositions containing them material is added to the material of the article / the objects.
- the regulatory T cells in general and the pharmaceutical and cosmetic containing them Compositions alone or in combination of several of them can be used for the preparation of medicaments for the treatment of the above-mentioned diseases or conditions.
- These may comprise the activated regulatory T cells (Treg cells) in the amounts exemplified below, optionally together with known excipients, auxiliaries and / or additives.
- Mononuclear cells were collected by density gradient centrifugation from the peripheral blood of healthy donors.
- the isolation of regulatory T cells was carried out by a two-stage magnetic separation:
- CD4 + T cells were obtained by depletion of all CD4 negative cells [CD4 Separation Kit, Miltenyi Biotech, Bergisch-Gladbach, Germany]. The purity achieved was regularly> 95% CD4 + T cells.
- CD4 + CD25 + CD25 + CD25 + regulatory T cells were again isolated by magnetic column separation from this population using CD25 labeling [anti-CD25 MicroBeads, Miltenyi Biotech].
- the CD4 + CD25 " fraction served as effector cell control.
- the functional capacity of the regulatory T cells was tested in a special coculture. For this purpose, a total of 20,000 effector cells (CD4 + CD25 ' ) and regulatory T cells (CD4 + CD25 + ) were cultured in different quantitative ratios to one another over a period of 120 hours in microplates. As activator of T-cell stimulation, a solid-phase-bound anti-CD3 antibody [UCHT1, 0.25 ⁇ g / well] was used.
- FIG. 1 shows the induction of the suppressive phenotype of regulatory T cells (Treg cells) in the quantitative ratios of 50% (experimentally relevant), 20% (experimentally relevant) and 10% (physiologically relevant) Treg portions in the presence and absence of the APN inhibitor actin-nin.
- Mononuclear cells were collected by density gradient centrifugation from the peripheral blood of healthy donors.
- the isolation of regulatory T cells was carried out by a two-stage magnetic separation:
- CD4 + T cells were obtained by depletion of all CD4 negative cells [CD4 Separation Kit, Miltenyi Biotech, Bergisch-Gladbach, Germany]. The purity achieved was regularly> 95% CD4 + T cells.
- CD4 + CD25 + CD25 + CD25 + regulatory T cells were again isolated by magnetic column separation from this population using CD25 labeling [anti-CD25 MicroBeads, Miltenyi Biotech].
- the CD4 + CD25 " fraction served as effector cell control.
- the functional capacity of the regulatory T cells was tested in a special coculture. For this purpose, a total of 20,000 effector cells (CD4 + CD25 " ) and regulatory T cells (CD4 + CD25 + ) were cultured in different quantitative ratios to each other over a period of 120 hours in microplates. bound anti-CD3 antibody [UCHT1, 0.25 ⁇ g / well].
- FIG. 2 shows the induction of the suppressive phenotype of regulatory T cells (Treg cells) in the physiologically relevant quantitative ratios of 10% and 5% Treg cell portions in the presence and absence of the selective inhibitor of cytosolic aminopeptidase (cAAP ) PAQ22.
- PAQ22 induces the suppressive capacity of regulatory T cells in a concentration-dependent manner after 5 days of co-culture.
- Mononuclear cells were collected by density gradient centrifugation from the peripheral blood of healthy donors. This was followed by T cell separation using nylon wadding adherence.
- the isolation of regulatory T cells was carried out by a two-stage magnetic separation:
- CD4 + T cells were obtained by depletion of all CD4 negative cells [CD4 Separation Kit, Miltenyi Biotech, Bergisch-Gladbach, Germany]. The purity achieved was regularly> 95% CD4 + T cells.
- CD4 + CD25 + CD25 + CD25 + regulatory T cells were again isolated by magnetic column separation from this population using CD25 labeling [anti-CD25 MicroBeads, Miltenyi Biotech].
- the CD4 + CD25 " fraction served as effector cell control.
- FIG. 3 shows the induction of the suppressive phenotype of regulatory T cells (Treg cells) in the quantitative ratios of 50% (experimentally relevant), 20% (experimentally relevant) and 10% (physiologically relevant) Treg cells.
- Treg cells regulatory T cells
- FIG. 3 shows the induction of the suppressive phenotype of regulatory T cells (Treg cells) in the quantitative ratios of 50% (experimentally relevant), 20% (experimentally relevant) and 10% (physiologically relevant) Treg cells.
- MNC Mononuclear cells
- the figure ( Figure 4) shows the induction of the suppressive phenotype of regulatory T cells (Treg cells) in the presence and absence of the APN inhibitor phebestin.
- DSS dextran sodium sulfate solution
- DAI disease activity index
- regulatory T cells from peripheral venous blood were obtained by density gradient centrifugation and CD25 microbeads magnetic separation.
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Priority Applications (7)
Application Number | Priority Date | Filing Date | Title |
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EP08801669A EP2178527A1 (de) | 2007-08-21 | 2008-08-21 | Verfahren zur aktivierung regulatorischer t-zellen |
JP2010521362A JP2010536815A (ja) | 2007-08-21 | 2008-08-21 | 制御性t細胞の活性化方法 |
CN200880025669A CN101808629A (zh) | 2007-08-21 | 2008-08-21 | 激活调节性t细胞的方法 |
EA201070292A EA201070292A1 (ru) | 2007-08-21 | 2008-08-21 | Способ активирования регуляторных т-клеток |
US12/664,263 US20110117069A1 (en) | 2007-08-21 | 2008-08-21 | Method for activating regulatory t-cells |
CA2691368A CA2691368A1 (en) | 2007-08-21 | 2008-08-21 | Method for activating regulatory t-cells |
AU2008290827A AU2008290827B2 (en) | 2007-08-21 | 2008-08-21 | Method for activating regulatory t-cells |
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DE102007039429A DE102007039429A1 (de) | 2007-08-21 | 2007-08-21 | Verfahren zur Aktivierung regulatorischer T-Zellen |
DE102007039429.4 | 2007-08-21 |
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EP (1) | EP2178527A1 (de) |
JP (1) | JP2010536815A (de) |
CN (1) | CN101808629A (de) |
AU (1) | AU2008290827B2 (de) |
CA (1) | CA2691368A1 (de) |
DE (1) | DE102007039429A1 (de) |
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Cited By (4)
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EP2292589A1 (de) * | 2009-09-02 | 2011-03-09 | IMTM GmbH | Neue multifunktionale Peptidaseinhibitoren, insbesondere zur medizinischen Verwendung |
CN102372713A (zh) * | 2010-07-29 | 2012-03-14 | Imtm股份有限公司 | 作为肽酶效应物用于医学用途的新型化合物 |
JP2012527237A (ja) * | 2009-05-18 | 2012-11-08 | セラコス・インコーポレイテッド | 免疫介在性疾患における臨床応用のための、抑制機能の高められた制御性t細胞のエクスビボ増加方法 |
CN104010664A (zh) * | 2011-09-26 | 2014-08-27 | 苏黎世大学数学和自然科学部 | 用于治疗多发性硬化的apc介导的耐受诱导 |
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GB0803076D0 (en) * | 2008-02-20 | 2008-03-26 | Univ Ghent | Mucosal Membrane Receptor and uses thereof |
KR101455930B1 (ko) * | 2012-04-30 | 2014-10-31 | 가톨릭대학교 산학협력단 | 조절 t 세포의 분화 가변성을 억제하는 커큐민의 신규 용도 |
EP3335633B1 (de) | 2016-12-15 | 2019-09-18 | Bühlmann Laboratories AG | Handapplikator |
GB201714777D0 (en) | 2017-09-14 | 2017-11-01 | Univ London Queen Mary | Agent |
AU2019379604A1 (en) * | 2018-11-16 | 2021-06-03 | Rapa Therapeutics, Llc | Als treatment using induced regulatory T (iTREG) cells |
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Cited By (8)
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JP2012527237A (ja) * | 2009-05-18 | 2012-11-08 | セラコス・インコーポレイテッド | 免疫介在性疾患における臨床応用のための、抑制機能の高められた制御性t細胞のエクスビボ増加方法 |
US11186823B2 (en) | 2009-05-18 | 2021-11-30 | Mallinckrodt Pharmaceuticals Ireland Limited | Method for ex-vivo expansion of regulatory T cells with enhanced suppressive function for clinical application in immune mediated diseases |
EP2292589A1 (de) * | 2009-09-02 | 2011-03-09 | IMTM GmbH | Neue multifunktionale Peptidaseinhibitoren, insbesondere zur medizinischen Verwendung |
WO2011026781A1 (en) * | 2009-09-02 | 2011-03-10 | Imtm Gmbh | Novel multifunctional peptidase inhibitors, especially for medical use |
CN102695697A (zh) * | 2009-09-02 | 2012-09-26 | Imtm股份有限公司 | 特别地用于医学用途的新型多功能肽酶抑制剂 |
US20120258960A1 (en) * | 2009-09-02 | 2012-10-11 | Imtm Gmbh | Novel multifunctional peptidase inhibitors, especially for medical use |
CN102372713A (zh) * | 2010-07-29 | 2012-03-14 | Imtm股份有限公司 | 作为肽酶效应物用于医学用途的新型化合物 |
CN104010664A (zh) * | 2011-09-26 | 2014-08-27 | 苏黎世大学数学和自然科学部 | 用于治疗多发性硬化的apc介导的耐受诱导 |
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DE102007039429A1 (de) | 2009-02-26 |
AU2008290827B2 (en) | 2011-06-09 |
US20110117069A1 (en) | 2011-05-19 |
CA2691368A1 (en) | 2009-02-26 |
EP2178527A1 (de) | 2010-04-28 |
AU2008290827A1 (en) | 2009-02-26 |
JP2010536815A (ja) | 2010-12-02 |
CN101808629A (zh) | 2010-08-18 |
EA201070292A1 (ru) | 2010-06-30 |
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