WO2018024818A1 - Compound for treating a disease associated with a deregulation of the alternative complement pathway - Google Patents
Compound for treating a disease associated with a deregulation of the alternative complement pathway Download PDFInfo
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- WO2018024818A1 WO2018024818A1 PCT/EP2017/069633 EP2017069633W WO2018024818A1 WO 2018024818 A1 WO2018024818 A1 WO 2018024818A1 EP 2017069633 W EP2017069633 W EP 2017069633W WO 2018024818 A1 WO2018024818 A1 WO 2018024818A1
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- compound according
- compound
- substituted
- procfd
- following structure
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- 150000001875 compounds Chemical class 0.000 title claims abstract description 69
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- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 title claims abstract description 23
- 230000024203 complement activation Effects 0.000 title claims abstract description 20
- 230000003831 deregulation Effects 0.000 title claims abstract description 11
- 230000004913 activation Effects 0.000 claims abstract description 22
- 238000011282 treatment Methods 0.000 claims abstract description 14
- 239000012472 biological sample Substances 0.000 claims abstract description 11
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- 238000000338 in vitro Methods 0.000 claims abstract description 8
- 230000037417 hyperactivation Effects 0.000 claims abstract description 7
- 230000002401 inhibitory effect Effects 0.000 claims abstract description 3
- YBJHBAHKTGYVGT-ZKWXMUAHSA-N (+)-Biotin Chemical group N1C(=O)N[C@@H]2[C@H](CCCCC(=O)O)SC[C@@H]21 YBJHBAHKTGYVGT-ZKWXMUAHSA-N 0.000 claims description 10
- 125000003118 aryl group Chemical group 0.000 claims description 10
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Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/34—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase
- C12Q1/37—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase involving peptidase or proteinase
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P13/00—Drugs for disorders of the urinary system
- A61P13/12—Drugs for disorders of the urinary system of the kidneys
-
- 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
- A61P27/02—Ophthalmic agents
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P37/00—Drugs for immunological or allergic disorders
- A61P37/02—Immunomodulators
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P37/00—Drugs for immunological or allergic disorders
- A61P37/02—Immunomodulators
- A61P37/06—Immunosuppressants, e.g. drugs for graft rejection
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07F—ACYCLIC, CARBOCYCLIC OR HETEROCYCLIC COMPOUNDS CONTAINING ELEMENTS OTHER THAN CARBON, HYDROGEN, HALOGEN, OXYGEN, NITROGEN, SULFUR, SELENIUM OR TELLURIUM
- C07F9/00—Compounds containing elements of Groups 5 or 15 of the Periodic Table
- C07F9/02—Phosphorus compounds
- C07F9/547—Heterocyclic compounds, e.g. containing phosphorus as a ring hetero atom
- C07F9/6561—Heterocyclic compounds, e.g. containing phosphorus as a ring hetero atom containing systems of two or more relevant hetero rings condensed among themselves or condensed with a common carbocyclic ring or ring system, with or without other non-condensed hetero rings
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/81—Protease inhibitors
- C07K14/8107—Endopeptidase (E.C. 3.4.21-99) inhibitors
- C07K14/811—Serine protease (E.C. 3.4.21) inhibitors
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/90—Enzymes; Proenzymes
- G01N2333/914—Hydrolases (3)
- G01N2333/948—Hydrolases (3) acting on peptide bonds (3.4)
- G01N2333/95—Proteinases, i.e. endopeptidases (3.4.21-3.4.99)
- G01N2333/964—Proteinases, i.e. endopeptidases (3.4.21-3.4.99) derived from animal tissue
- G01N2333/96425—Proteinases, i.e. endopeptidases (3.4.21-3.4.99) derived from animal tissue from mammals
- G01N2333/96427—Proteinases, i.e. endopeptidases (3.4.21-3.4.99) derived from animal tissue from mammals in general
- G01N2333/9643—Proteinases, i.e. endopeptidases (3.4.21-3.4.99) derived from animal tissue from mammals in general with EC number
- G01N2333/96433—Serine endopeptidases (3.4.21)
Definitions
- a compound for the treatment of a disease associated with a desegregation of the alternative complement pathway A compound for the treatment of a disease associated with a desegregation of the alternative complement pathway
- the present invention relates to a novel compound for the treatment and / or prophylaxis and / or diagnosis of a disease associated with a deregulation, preferably a dysregulated activation, more preferably a hyperactivation of the alternative complement pathway. It also relates to the use of the novel compound for complexing and / or isolating and / or localizing and / or quantifying proCFD-activating compounds in / from a biological sample.
- the complement system is a system of plasma proteins that is used in the course of
- Immune response can be activated. It is part of the innate immune system.
- the human complement system consists of more than 30 proteins, which are dissolved in the blood plasma or cell-bound. They defend against micro-organisms, but also have strong cell-destroying properties and may, if they are unregulated, be responsible for tissue damage in the course of many diseases.
- CFD complement factor D
- adipsin or C3-proactivator convertase CFD
- CFD complement factor D
- adipsin or C3-proactivator convertase CFD is a serine protease that is predominantly synthesized in adipose tissue and secreted into the bloodstream.
- CFD can be found in various tissues.
- CFD cleaves the complement factor B.
- CFD arises from a protein precursor with an N-terminal signal peptide.
- This signal peptide is cleaved during the secretory pathway and the enzyme is formed as an inactive proenzyme (proCFD) or Zymogen.
- proCFD is converted to the mature and active form, ie the CFD, by the removal of six N-terminal amino acids.
- MASPs mannose-binding lectin serine peptidases
- CFD complement factor B
- CFD has elastase activity.
- the plasma concentrations of CFD are very low and are at 1 -2 ⁇ g ml, making CFD the lowest concentration complement factor, significantly lower than that of complement C3, which is 1-2 mg / ml, or complement factor B , which is 200 ⁇ g ml.
- CFD is therefore the rate-limiting enzyme in the alternative pathway of the complement system.
- Adipsin is an adipokine that better cell function in diabetes, Cell, 158 (1): 41-53, describe a connection between the alternative pathway of the complement system and the development of diabetes mellitus type 2.
- CFD CFD
- Complement pathway can lead to autoimmune diseases, for example, to age-related macular degeneration (AMD); see. Weber et al. (2014) The system of age-related macular degeneration. Dtsch Cardioebl Int 1 1 1, pp. 133-138, on renal diseases manifesting as atypical hemolytic uremic syndrome (aHUS); see. Wong et al. (2013), Complement therapy in atypical haemolytic uraemic syndrome (AHUS). Mol Immunol 56, p. 199-212, or else to C3 glomerulopathy; see. Noris et al.
- Lampalizumab Roche / Genentech, is an antigen binding fragment of a humanized monoclonal antibody that binds CFD. He is currently being tested for the dry form of AMD and is in clinical phase 3. It has been shown that lampalizumab does not discriminate between the active and inactive (pro) forms of CFD. and thus eliminates both forms. This suggests that the use of lampalizumab in humans will be associated with a variety of side effects.
- - n is an integer from 0 to 5;
- R1 is selected from the group consisting of: H, detectable marker;
- R2 is selected from the group consisting of: substituted or unsubstituted aryl each having a MW of ⁇ 200 Da, substituted or unsubstituted aryl each having a MW of ⁇ 200 Da, a side chain of a natural or synthetic amino acid;
- R3 is selected from the group consisting of: substituted or unsubstituted alkyl each having an MW of ⁇ 300 Da, substituted unsubstituted
- tes aryl each having a MW of ⁇ 300 Da, preferably a side chain of a natural or synthetic amino acid
- - R4 and R5 are independently selected from the group consisting of: substituted or unsubstituted aryl each having a MW> 70 kDa and ⁇ 300 Da, substituted or unsubstituted alkyl each having a MW> 70 kDa and ⁇ 300 Da.
- this compound corrects the corrected
- the inactive pool of potential proCFD activators remains unchanged. Because of this high selectivity, the compound of the invention can be specifically intervened in disease-causing processes. The complement system is still functional and is available for alien defense. The compound of the invention therefore has a particularly favorable side effect profile.
- the compound of the invention therefore represents a promising new approach to the treatment of diseases associated with the deregulation of the alternative complement pathway.
- the provision of a detectable marker leads to the obtaining of a compound by means of which the proCFD activation as well as the activating enzymes can be observed by means of imaging methods.
- the use of the compound with a detectable marker according to the invention is possible both in vitro and in vivo, ie in the living organism.
- the compound according to the invention furthermore represents a tool for isolating the proCFD-activating enzymes.
- the compound according to the invention after sufficient incubation in a biological sample which contains or contains proCFD and the activating enzymes, for example blood plasma, from Sample removed. This can be done by methods known in the art, for example.
- a catcher molecule which is directed against a portion of the compound of the invention.
- proCFD-activating enzymes are present in complex with proCFD. They can be removed from the complex and investigated by methods known to those skilled in the art. Thus, a quantitative protease profile of the biological samples can be generated, which can be of prognostic and diagnostic value.
- the spacer may preferably consist of natural or synthetic amino acids or amino acid derivatives.
- Preferred spacers have the following structure, wherein the one-letter code is used:
- Abu-RG Abu-PRG and Abu-PPRG, where "Abu” is a non-natural amino acid with an ethyl side chain, preferably homo-alanine;
- the compound according to the invention has the following structure:
- - Ar is a substituted or unsubstituted aryl each having a MW> 70 kDa and ⁇ 300 Da.
- R6, R7 and R8 are each independently a side chain of a natural or synthetic amino acid.
- this embodiment leads to particular
- the compound R6 is a proline radical
- / or R7 is an arginine radical
- / or R8 is a glycine radical
- the detectable marker is selected from the group consisting of: biotin, biotinyl residue, radioactive marker, fluorescent marker, PET tracer.
- the compound has the following structure:
- the compound developed by the inventors has the following structure:
- the inventors have carried out their experiments. They found that this compound is well suited for the regulation and in particular inhibition of proCFD activation.
- the biotin residue provided at one end here: left
- the compound can be well detected both in vitro and in vivo in the biological sample.
- Another object of the present invention relates to the compound of the invention or its use for the treatment and / or prophylaxis and / or diagnosis of a disease associated with a deregulation, preferably a dysregulated activation, more preferably a hyperactivation of the alternative complement pathway.
- the compound of the invention interacts with the proCFD-activating enzymes and - preferably - inhibits them.
- the compound of the invention interacts or preferably inhibits the processing of proCFD in a mechanistic manner. This is advantageous since it is to be expected that proCFD will be activated not only by a single complement factor or a single enzyme but by various factors and by different pathways.
- the compound according to the invention can interact with, and preferably inhibit, several or possibly all proCFD-activating enzymes.
- deregulation is understood to mean a dysregulation of the cascade of the alternative complement pathway.
- dysregulated activation is meant an impaired activation of the alternative complement pathway, in particular a disturbed activation of proCFD in CFD.
- hyperactivation is meant, according to the invention, an activation of the alternative complement path that goes beyond the normal state.
- the disease is an autoimmune disease.
- the disease is further preferably selected from the group consisting of: age-related macular degeneration (AMD), kidney disease, in particular atypical hemolytic anemic syndrome (aHUS) and C3 glomerulopathy.
- AMD age-related macular degeneration
- aHUS atypical hemolytic anemic syndrome
- C3 glomerulopathy C3 glomerulopathy
- Another object of the present invention relates to a pharmaceutical or diagnostic composition having the compound of the invention in a pharmaceutically or diagnostically effective concentration and a pharmaceutically or diagnostically acceptable formulation.
- compositions are well known in the art. For example, reference is made to the paper by Kibbe A. (2003), Handbook of Pharmaceutical Excipients, 4th Edition, American Pharmaceutical Association and Pharmaceutical Press. The properties, advantages, further developments and embodiments of the compound according to the invention apply correspondingly to the pharmaceutical and diagnostic composition according to the invention.
- Another object of the present invention relates to the use of the compound according to the invention in vitro for inhibiting the activation of proCFD in a biological sample.
- a “biological sample” may be any composition that includes or is believed to have the elements and factors of the alternative complement pathway, such as complement factors, cofactors, etc.
- a preferred biological sample is a blood plasma sample of an individual, for example of any Mammalier, including a human.
- the compound according to the invention is used for the complexation and / or isolation and / or localization and / or quantification of proCFD-activating compounds in / from a biological sample.
- Another object of the present invention relates to a method for the treatment and / or prophylaxis and / or diagnosis of a disease associated with a desegulation, preferably a dysregulated activation, more preferably a Hyperoxidie- tion of the alternative complement pathway, the administration of the compound of the invention and / or the composition of the invention in an individual, for example any mammal, including a human.
- FIG. 1 Chemical structure of an example of an activity-based probe according to the invention, oriented on the activator (s) of proCFD.
- FIG. 2 The inactive proenzyme of the human complement factor D (proCFD) is activated by the cleavage of the six N-terminal amino acids. Several serine proteases are able to catalyze this step in vitro. The difference in the isoelectric points of the active versus inactive forms allows the detection of the activation process by means of isoelectric focusing. Inactive proCFD was incubated with human blood plasma. The physiological activator is present in the plasma, therefore proCFD is activated rapidly (lane # 6, see the band towards the anode (+)).
- proCFD human complement factor D
- Fig. 1 is the structure of an example of an activity-based probe
- the empirical formula is C 5 4H82N 15 0iiPS.
- the molecular weight is 1 179.5777 daltons. It has a targeting element that corresponds to the sequence of the N-terminal pro-peptide (APPRGR) of CFD. Shorter fragments, such as PRGR, have been found to be sufficient to achieve complete inhibition of the proCFD activator (s).
- the C-terminal arginine (R) has been replaced by a functional group often referred to as
- Warhead (engl., “Warhead”) is called. This arginine mimetic, a diphenylphosphonate, covalently modifies target enzymes in an activity-dependent manner.
- the biotin tag attached to the probe can be used in a variety of applications, such as in visualizing the probe in histochemical studies or using streptavidin as a biotin-directed capture molecule in a covalently-affinity-purified affinity purification Enzymes from different organic samples. It is also possible to identify and quantify the amount of bound protein using standard biochemical methods.
- proCFD By transient transfection of adherently growing HEK-293 cells (human embryonic kidney cells) proCFD was recovered. 20 ⁇ M EDTA plasma from healthy blood donors were mixed with 1 ⁇ of the compound of the invention in different Concentrations (1, 6 mM, 0.16 mM, 0.016 mM, 0.0016 mM) preincubated for 10 minutes at 37 ° C, then added to proCFD and incubated for a further 6 hours. The reaction was stopped by acetone precipitation and the activation of proCFD visualized by means of isoelectric focusing and subsequent detection on an immunoblot using a CFD antibody; see Figure 2.
- the compound of the present invention is useful for the treatment, prophylaxis and diagnosis of a disease associated with desegregulation, dysregulated activation and hyperactivation of the alternative complement pathway.
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Abstract
The present invention relates to a compound for the treatment and/or prophylaxis and/or diagnosis of a disease which is associated with a deregulation, preferably a dysregulated activation, more preferably a hyperactivation of the alternative complement pathway. The invention also relates to a pharmaceutical and diagnostic composition comprising the compound according to the invention. The invention further relates to the in vitro use of the compound according to the invention for inhibiting the activation of proCFD in a biological sample.
Description
Verbindung zur Behandlung einer mit einer Desregulierung des alternativen Komplementweges assoziierten Erkrankung A compound for the treatment of a disease associated with a desegregation of the alternative complement pathway
Die vorliegende Erfindung betrifft eine neue Verbindung zur Behandlung und/oder Prophylaxe und/oder Diagnose einer Krankheit, die mit einer Deregulierung, vorzugsweise einer fehlregulierten Aktivierung, weiter vorzugsweise einer Hyperaktivierung des alternativen Komplementweges assoziiert ist. Sie betrifft außerdem die Verwendung der neuen Verbindung zur Komplexierung und/oder Isolierung und/oder Lokalisierung und/oder Quantifizierung von proCFD-aktivierenden Verbindungen in/aus einer biologischen Probe. The present invention relates to a novel compound for the treatment and / or prophylaxis and / or diagnosis of a disease associated with a deregulation, preferably a dysregulated activation, more preferably a hyperactivation of the alternative complement pathway. It also relates to the use of the novel compound for complexing and / or isolating and / or localizing and / or quantifying proCFD-activating compounds in / from a biological sample.
Das Komplementsystem ist ein System von Plasmaproteinen, das im Zuge der The complement system is a system of plasma proteins that is used in the course of
Immunantwort aktiviert werden kann. Es ist Teil des angeborenen Immunsystems. Das menschliche Komplementsystem besteht aus mehr als 30 Proteinen, die im Blutplasma gelöst oder zellgebunden vorliegen. Sie dienen der Abwehr von Mikroorganismen, haben jedoch auch stark zellzerstörende Eigenschaften und können, wenn sie unreguliert sind, im Laufe vieler Krankheiten für Gewebeschäden verantwortlich sein. Immune response can be activated. It is part of the innate immune system. The human complement system consists of more than 30 proteins, which are dissolved in the blood plasma or cell-bound. They defend against micro-organisms, but also have strong cell-destroying properties and may, if they are unregulated, be responsible for tissue damage in the course of many diseases.
Man unterscheidet drei Wege, durch die das Komplementsystem aktiviert wird, nämlich den meist über Antikörper induzierten klassischen Weg, den über das Mannose bindende Lektin aktivierten Lektinweg, und den spontanen und Antikörper-unabhängigen alternativen Weg. A distinction is made between three ways by which the complement system is activated, namely the classical pathway that is mostly induced by antibodies, the lectin pathway activated via the mannose-binding lectin, and the spontaneous and antibody-independent alternative route.
Ein Enzym des alternativen Wegs ist der sogenannte Komplementfaktor D (CFD; EC 3.4.21.46), der auch als Adipsin oder C3-Proactivator-Konvertase, bezeichnet wird. CFD ist eine Serinprotease, die vorwiegend im Fettgewebe synthetisiert und in den Blutkreislauf sezerniert wird. CFD lässt sich darüber hinaus in verschiedenen Geweben finden. CFD spaltet den Komplementfaktor B. An alternative pathway enzyme is the so-called complement factor D (CFD; EC 3.4.21.46), also referred to as adipsin or C3-proactivator convertase. CFD is a serine protease that is predominantly synthesized in adipose tissue and secreted into the bloodstream. In addition, CFD can be found in various tissues. CFD cleaves the complement factor B.
Wie bei anderen sezernierten Serinproteasen entsteht CFD aus einer Proteinvorstufe mit einem N-terminalen Signalpeptid. Dieses Signalpeptid wird während des sekretorischen Wegs abgespalten und es entsteht das Enzym als inaktives Proenzym (proCFD) oder
Zymogen. Dieses proCFD wird durch die Abspaltung von sechs N-terminalen Aminosäuren in die reife und aktive Form, d.h. das CFD, überführt. As with other secreted serine proteases, CFD arises from a protein precursor with an N-terminal signal peptide. This signal peptide is cleaved during the secretory pathway and the enzyme is formed as an inactive proenzyme (proCFD) or Zymogen. This proCFD is converted to the mature and active form, ie the CFD, by the removal of six N-terminal amino acids.
Der physiologische Aktivator von proCFD im ruhenden Blut ist weitgehend unbekannt. Ein Kandidat sind die sogenannten Mannose bindenden Lektin-Serin-Peptidasen (MASPs), deren Funktion als Vermittler zwischen dem alternativen und dem Lektinweg hinreichend beschrieben ist. The physiological activator of proCFD in quiescent blood is largely unknown. One candidate is the so-called mannose-binding lectin serine peptidases (MASPs), whose function as mediators between the alternative and the lectin pathway is sufficiently described.
Reifes CFD hat eine einzigartige enge Substratspezifitat, die auf den Komplementfaktor B (CFB) beschränkt ist. CFD weist Elastaseaktivität auf. Die Plasma-Konzentrationen von CFD sind sehr gering und liegen bei 1 -2 μg ml, womit CFD der Komplementfaktor mit der niedrigsten Konzentration ist, deutlich niedriger als die von Komplementfaktor C3, die bei 1 -2 mg/ml liegt, oder von Komplementfaktor B, die bei 200 μg ml liegt. CFD ist deshalb das geschwindigkeitsbestimmende Enzym im alternativen Weg des Komplementsystems. Mature CFD has a unique tight substrate specificity limited to the complement factor B (CFB). CFD has elastase activity. The plasma concentrations of CFD are very low and are at 1 -2 μg ml, making CFD the lowest concentration complement factor, significantly lower than that of complement C3, which is 1-2 mg / ml, or complement factor B , which is 200 μg ml. CFD is therefore the rate-limiting enzyme in the alternative pathway of the complement system.
In jüngster Zeit sind solche Krankheiten in den Mittelpunkt des Interesses gerückt, die über den alternativen Weg des Komplementsystems vermittelt werden. Diese sind zu- sammengefasst in Holers (2008), The Spectrum of Complement Alternative Pathway- Mediated Diseases, Immunological Reviews, Vol. 223, Seiten 300 bis 316. Recently, the focus has shifted to those diseases that are mediated through the alternative pathway of the complement system. These are summarized in Holer's (2008), The Spectrum of Complementary Alternative Pathway-Mediated Diseases, Immunological Reviews, Vol. 223, pages 300-166.
Lo et al. (2014), Adipsin is an adipokine that improves better cell function in diabetes, Cell, 158(1 ): 41 -53, beschreiben einen Zusammenhang zwischen dem alternativen Weg des Komplementsystems und der Entstehung von Diabetes mellitus Typ 2. In einer Subgruppe von untersuchten Patienten wurden erniedrigte Spiegel von CFD nachgewiesen, die mit einem erhöhten Risiko für einen Funktionsausfall der ß-Zellen korrelierten. Lo et al. (2014), Adipsin is an adipokine that better cell function in diabetes, Cell, 158 (1): 41-53, describe a connection between the alternative pathway of the complement system and the development of diabetes mellitus type 2. In a subgroup of studied Patients were found to have decreased levels of CFD, which correlated with an increased risk of functional failure of the β-cells.
Weiter ist beschrieben worden, dass eine Deregulierung des alternativen It has also been described that deregulation of the alternative
Komplementwegs zu Autoimmunerkrankungen führen kann, bspw. zur altersbedingten Makuladegeneration (AMD); vgl. Weber et al.(2014), The role of the complement System in age-related macular degeneration. Dtsch Arztebl Int 1 1 1 , S. 133-138, zu Nierenerkrankungen, die sich als atypisches hämolytisches urämisches Syndrom (aHUS) manifestieren; vgl. Wong et al. (2013), Complement therapy in atypical haemolytic uraemic Syndrom
(aHUS). Mol Immunol 56, S. 199-212, oder aber zu C3-Glomerulopathie; vgl. Noris et al. (2015), Glomerular diseases dependent on complement activation, including atypical hemolytic uremic Syndrome, membranoproliferative glomerulonephritis, and C3 glomeru- lopathy: core curriculum 2015. Am J Kidney Dis 66, S. 359-375. Complement pathway can lead to autoimmune diseases, for example, to age-related macular degeneration (AMD); see. Weber et al. (2014) The system of age-related macular degeneration. Dtsch Arztebl Int 1 1 1, pp. 133-138, on renal diseases manifesting as atypical hemolytic uremic syndrome (aHUS); see. Wong et al. (2013), Complement therapy in atypical haemolytic uraemic syndrome (AHUS). Mol Immunol 56, p. 199-212, or else to C3 glomerulopathy; see. Noris et al. (2015), Glomerular diseases dependent on complement activation, including atypical hemolytic uremic syndrome, membranoproliferative glomerulonephritis, and C3 glomerulopathy: core curriculum 2015. Am J Kidney Dis 66, pp. 359-375.
[0011] Im Stand der Technik stehen verschiedene Methoden zur Behandlung von Erkrankungen, die durch Veränderungen des alternativen Komplementwegs verursacht werden, zur Verfügung. Dabei kommen vorwiegend Antikörper oder Antikörperfragmente zum Einsatz, die gegen Komplementfaktoren gerichtet sind; vgl. Mamidi et al. (2015), Neutralization of membrane complement regulators improves complement-dependent effector functions of therapeutic anticancer antibodies targeting leukemic cells. Oncoimmunology 4, e979688; Melis et al. (2015), Complement in therapy and disease: Regulating the complement System with antibody-based therapeutics. Various methods of treating diseases caused by alterations of the alternative complement pathway are available in the art. In this case, predominantly antibodies or antibody fragments are used which are directed against complement factors; see. Mamidi et al. (2015), Neutralization of membrane complement regulators, complement-dependent effector functions of therapeutic anticancer antibodies targeting leukemic cells. Oncoimmunology 4, e979688; Melis et al. (2015), Complement in therapy and disease: Regulating the complement system with antibody-based therapeutics.
[0012] Mol Immune 67, S. 1 17-130; Rogers et al (2014), Complement in monoclonal antibody therapy of Cancer. Immunol Res 59, S. 203-210. Einige der Antikörper werden derzeit in klinischen Studien getestet, wie bspw. Eculizumab, Mubodina, Ergidina, LFG-316, Bikaci- omab und TNT-009. Mol Immune 67, p. 1 17-130; Rogers et al (2014), Complement in monoclonal antibody therapy of Cancer. Immunol Res 59, pp. 203-210. Some of the antibodies are currently being tested in clinical trials, such as eculizumab, Mubodina, Ergidina, LFG-316, Bikacomab and TNT-009.
[0013] Allerdings gehen mit den derzeit verfügbaren therapeutischen Antikörper eine Vielzahl von Nachteilen einher. Diese betreffen z.B. ihre unzureichenden pharmakokinetischen Eigenschaften. Auch die Gewebezugänglichkeit der Antikörper ist bislang stark eingeschränkt. Ferner wurden ungewünschte Interaktionen mit dem Immunsystem beschrieben. Üblicherweise binden die therapeutischen Antikörper einen einzigen Komplementfaktor und richten sich hingegen nicht gegen den biologischen Prozess der Komplementaktivierung, was einen weiteren Nachteil bei der Behandlung der in Rede stehenden Krankheiten darstellen kann. However, there are a number of disadvantages associated with currently available therapeutic antibodies. These concern e.g. their inadequate pharmacokinetic properties. Also, the tissue accessibility of the antibodies has hitherto been severely limited. Furthermore, undesirable interactions with the immune system have been described. Usually, the therapeutic antibodies bind a single complement factor and are not directed against the biological process of complement activation, which may be a further disadvantage in the treatment of the diseases in question.
[0014] Lampalizumab, Roche/Genentech, ist ein Antigenbindefragment eines humanisierten monoklonalen Antikörpers, der CFD bindet. Er wird derzeit gegen die trockene Form von AMD getestet und befindet sich in der klinischen Phase 3. Dabei hat sich gezeigt, dass Lampalizumab nicht zwischen den aktiven und inaktiven (pro-) Formen von CFD unter-
scheiden kann und folglich beide Formen eliminiert. Dies lässt Experten davon ausgehen, dass der Einsatz von Lampalizumab im Menschen mit einer Vielzahl von Nebenwirkungen assoziiert sein wird. Lampalizumab, Roche / Genentech, is an antigen binding fragment of a humanized monoclonal antibody that binds CFD. He is currently being tested for the dry form of AMD and is in clinical phase 3. It has been shown that lampalizumab does not discriminate between the active and inactive (pro) forms of CFD. and thus eliminates both forms. This suggests that the use of lampalizumab in humans will be associated with a variety of side effects.
[0015] Vor diesem Hintergrund ist es eine der Erfindung zugrundeliegende Aufgabe, eine Against this background, it is an object underlying the invention, a
verbesserte Verbindung zur Behandlung und/oder Prophylaxe und/oder Diagnose einer Krankheit bereitzustellen, die mit einer Deregulierung des alternativen Komplementweges assoziiert ist. to provide an improved compound for the treatment and / or prophylaxis and / or diagnosis of a disease associated with deregulation of the alternative complement pathway.
[0016] Diese Aufgabe wird durch die Bereitstellung einer Verbindung mit folgender Struktur This object is achieved by providing a compound having the following structure
gelöst: solved:
wobei in which
- n eine ganze Zahl von 0 bis 5 ist; - n is an integer from 0 to 5;
- R1 ausgewählt ist aus der Gruppe bestehend aus: H, detektierbarer Marker; R1 is selected from the group consisting of: H, detectable marker;
- R2 ausgewählt ist aus der Gruppe bestehend aus: substituiertes oder unsubstitu- iertes Aryl mit jeweils einem MW von < 200 Da, substituiertes oder unsubstituier- tes Aryl mit jeweils einem MW von < 200 Da, eine Seitenkette einer natürlichen oder synthetischen Aminosäure; - R2 is selected from the group consisting of: substituted or unsubstituted aryl each having a MW of <200 Da, substituted or unsubstituted aryl each having a MW of <200 Da, a side chain of a natural or synthetic amino acid;
- R3 ausgewählt ist aus der Gruppe bestehend aus: substituiertes oder unsubstitu- iertes Alkyl mit jeweils einem MW von < 300 Da, substituiert unsubstituier- R3 is selected from the group consisting of: substituted or unsubstituted alkyl each having an MW of <300 Da, substituted unsubstituted
tes Aryl mit jeweils einem MW von < 300 Da, vorzugsweise
, eine Sei- tenkette einer natürlichen oder synthetischen Aminosäure;
- R4 und R5 unabhängig ausgewählt sind aus der Gruppe bestehend aus: substituiertes oder unsubstituiertes Aryl mit jeweils einem MW > 70 kDa und < 300 Da, substituiertes oder unsubstituiertes Alkyl mit jeweils einem MW > 70 kDa und < 300 Da. tes aryl, each having a MW of <300 Da, preferably a side chain of a natural or synthetic amino acid; - R4 and R5 are independently selected from the group consisting of: substituted or unsubstituted aryl each having a MW> 70 kDa and <300 Da, substituted or unsubstituted alkyl each having a MW> 70 kDa and <300 Da.
[0017] Wie die Erfinder überraschenderwiese feststellen konnten, korrigiert diese Verbindung die As the inventors surprisingly found out, this compound corrects the corrected
Deregulierung des alternativen Komplementweges, die zur Entstehung der eingangs genannten Krankheiten führen kann. Die Verbindung setzt dabei an der Aktivierung von proCFD an und inhibiert die Überführung in reifes CFD. Dadurch wird ausschließlich die Aktivierung, im engeren Sinne die enzymatische Spaltung, von proCFD inhibiert. Somit werden selektiv solche Proteasen inhibiert, die diesen Schritt katalysieren können, wie bspw. Thrombin. Die Inhibition kommt nur dann zustande, wenn das aktivierende Enzym selbst in aktiver Form vorhanden ist. Viele Proteasen werden als inaktives Proenzym sezerniert, wie bspw. Thrombin als Prothrombin, und/oder liegen in einem Komplex mit einem Inhibitor vor, wie bspw. Thrombin mit Antithrombin. Da die Interaktion der erfindungsgemäßen Verbindung und der Zielproteasen deren Aktivität voraussetzt, bleibt der inaktive Pool von potenziellen proCFD-Aktivatoren unverändert. Aufgrund dieser hohen Selektivität kann die erfindungsgemäße Verbindung gezielt in krankheitsverursachende Prozesse eingegriffen werden. Das Komplementsystem ist aber weiterhin funktionsfähig und steht zur Fremdabwehr zur Verfügung. Die erfindungsgemäße Verbindung weist deshalb ein besonders günstiges Nebenwirkungsprofil auf. Deregulation of the alternative complement pathway, which can lead to the emergence of the aforementioned diseases. The compound initiates the activation of proCFD and inhibits the conversion to mature CFD. As a result, only the activation, in the narrower sense the enzymatic cleavage, of proCFD is inhibited. Thus, such proteases are selectively inhibited that can catalyze this step, such as thrombin. The inhibition occurs only when the activating enzyme itself is present in active form. Many proteases are secreted as an inactive proenzyme, such as thrombin as prothrombin, and / or are in a complex with an inhibitor, such as thrombin with antithrombin. Since the interaction of the compound of the invention and the target proteases requires their activity, the inactive pool of potential proCFD activators remains unchanged. Because of this high selectivity, the compound of the invention can be specifically intervened in disease-causing processes. The complement system is still functional and is available for alien defense. The compound of the invention therefore has a particularly favorable side effect profile.
[0018] Die erfindungsgemäße Verbindung stellt deshalb einen vielversprechenden neuen Ansatz zur Behandlung von Erkrankungen dar, die mit der Deregulierung des alternativen Komplementweges assoziiert sind. The compound of the invention therefore represents a promising new approach to the treatment of diseases associated with the deregulation of the alternative complement pathway.
[0019] Das Vorsehen eines detektierbaren Markers führt zum Erhalt einer Verbindung, über die sich die proCFD-Aktivierung sowie die aktivierenden Enzyme mittels bildgebender Verfahren beobachten lassen. Die Verwendung der Verbindung mit einem detektierbaren Marker ist erfindungsgemäß sowohl in vitro als auch in vivo, also im lebenden Organismus, möglich.
[0020] Die erfindungsgemäße Verbindung stellt ferner ein Werkzeug zur Isolierung der proCFD- aktivieren Enzyme dar. Dazu wird die erfindungsgemäße Verbindung nach ausreichender Inkubation in einer biologischen Probe, die proCFD und die aktivierenden Enzyme enthält bzw. enthalten kann, bspw. Blutplasma, aus der Probe entfernt. Dies kann über im Stand der Technik bekannte Verfahren erfolgen, bspw. mittels eines Fängermoleküls, das gegen einen Abschnitt der erfindungsgemäßen Verbindung gerichtet ist. Die proCFD- aktivierenden Enzyme liegen dabei im Komplex mit proCFD vor. Sie lassen sich aus dem Komplex entfernen und mittels dem Fachmann bekannter Verfahren untersuchen. So lässt sich auch ein quantitatives Proteaseprofil der biologischen Proben erstellen, das von prognostischem und diagnostischem Wert sein kann. The provision of a detectable marker leads to the obtaining of a compound by means of which the proCFD activation as well as the activating enzymes can be observed by means of imaging methods. The use of the compound with a detectable marker according to the invention is possible both in vitro and in vivo, ie in the living organism. The compound according to the invention furthermore represents a tool for isolating the proCFD-activating enzymes. For this purpose, the compound according to the invention, after sufficient incubation in a biological sample which contains or contains proCFD and the activating enzymes, for example blood plasma, from Sample removed. This can be done by methods known in the art, for example. By means of a catcher molecule, which is directed against a portion of the compound of the invention. The proCFD-activating enzymes are present in complex with proCFD. They can be removed from the complex and investigated by methods known to those skilled in the art. Thus, a quantitative protease profile of the biological samples can be generated, which can be of prognostic and diagnostic value.
[0021] Die erfindungsgemäße Verbindung ist durch einen Spacer mit folgender Struktur The compound of the invention is characterized by a spacer having the following structure
gekennzeichnet: characterized:
Der Spacer kann vorzugsweise aus natürlichen oder synthetischen Aminosäuren oder Aminosäurederivaten bestehen. Bevorzugte Spacer haben folgende Struktur, wobei der Ein-Buchstabencode Verwendung findet: The spacer may preferably consist of natural or synthetic amino acids or amino acid derivatives. Preferred spacers have the following structure, wherein the one-letter code is used:
- PRG; - PRG;
- PRA; - PRA;
- PPRG; - PPRG;
- APPRG; - APPRG;
- VPPRG; - VPPRG;
- Abu-RG, Abu-PRG und Abu-PPRG, wobei "Abu" eine nicht-natürliche Aminosäure mit einer Ethylseitenkette ist, vorzugsweise Homo-Alanin; Abu-RG, Abu-PRG and Abu-PPRG, where "Abu" is a non-natural amino acid with an ethyl side chain, preferably homo-alanine;
- Pip-PRG und P-Pip-RG, wobei "Pip" ein Piperidin-Rest ist; - Pip-PRG and P-Pip-RG, where "pip" is a piperidine residue;
- PP-hR-G, wobei "hR" Homo-Arginin ist.
Nach einer bevorzugten Ausführungsform weist die erfindungsgemäße Verbindung folgende Struktur auf: - PP-hR-G, where "hR" is homo-arginine. According to a preferred embodiment, the compound according to the invention has the following structure:
wobei in which
- Ar ein substituiertes oder unsubstituiertes Aryl mit jeweils einem MW > 70 kDa und < 300 Da ist. - Ar is a substituted or unsubstituted aryl each having a MW> 70 kDa and <300 Da.
Die Erfinder haben festgestellt, dass das Vorsehen von Arylresten im angegebenen Molekulargewichtsbereich am Phosphatende zu einer besonders geeigneten Verbindung führt. The inventors have found that the provision of aryl radicals in the stated molecular weight range at the phosphate end results in a particularly suitable compound.
Nach einer Weiterentwicklung der erfindungsgemäßen Verbindung weist diese folgend Struktur auf: After a further development of the compound according to the invention, it has the following structure:
- R6, R7 und R8 jeweils unabhängig eine Seitenkette einer natürlichen oder synthetischen Aminosäure sind. R6, R7 and R8 are each independently a side chain of a natural or synthetic amino acid.
Nach den Erkenntnissen der Erfinder führt diese Ausgestaltung zu besonders According to the findings of the inventors, this embodiment leads to particular
vorteilhaften Eigenschaften und eine solche Verbindung ist zur Lösung der der Erfindung zugrundeliegenden Aufgabe sehr gut geeignet.
Nach einer Weiterentwicklung der Erfindung ist bei der Verbindung R6 ein Prolin-Rest, und/oder R7 ein Arginin-Rest, und/oder R8 ein Glyzin-Rest. advantageous properties and such a compound is very well suited to the solution of the problem underlying the invention. According to a further development of the invention, the compound R6 is a proline radical, and / or R7 is an arginine radical, and / or R8 is a glycine radical.
Das Vorsehen dieser Aminosäurereste hat sich nach den Erkenntnissen der Erfinder ais besonders vorteilhaft herausgestellt. The provision of these amino acid residues has turned out to be particularly advantageous according to the discoveries of the inventors.
In einer Ausführungsform der Erfindung ist bei der Verbindung der detektierbare Marker ausgewählt aus der Gruppe bestehend aus: Biotin, Biotinyl-Rest, radioaktiver Marker, fluoreszierender Marker, PET-Tracer. In one embodiment of the invention, in the compound, the detectable marker is selected from the group consisting of: biotin, biotinyl residue, radioactive marker, fluorescent marker, PET tracer.
Das Vorsehen diese Marker liefert eine erfindungsgemäße Verbindung, die sich mittels üblicher bildgebender Verfahren in vitro aber auch in vivo gut darstellen lässt. The provision of these markers provides a compound of the invention which can be readily visualized by conventional imaging techniques in vitro as well as in vivo.
Nach einer Weiterbildung der Erfindung weist die Verbindung folgende Struktur auf: According to a development of the invention, the compound has the following structure:
[0032] Die Erfinder haben festgestellt, dass sich diese Struktur besonders eignet und gute The inventors have found that this structure is particularly suitable and good
Ergebnisse liefert. Delivers results.
[0033] Nach einer bevorzugten Ausführungsform der Erfindung weist die von den Erfindern entwickelte Verbindung folgende Struktur aus:
According to a preferred embodiment of the invention, the compound developed by the inventors has the following structure:
Mit dieser Verbindung, stellvertretend für sämtliche unter die allgemeine With this connection, representing all under the general
erfindungsgemäße Struktur fallenden Verbindungen, haben die Erfinder ihre Experimente durchgeführt. Sie konnten feststellen, dass sich diese Verbindung hervorragend zur Regulierung und insbesondere Inhibierung der proCFD-Aktivierung eignet. Über den am einen (hier: linken) Ende vorgesehenen Biotinrest lässt sich die Verbindung sowohl in vitro als auch in vivo in der biologischen Probe gut detektieren. According to the structure of the invention, the inventors have carried out their experiments. They found that this compound is well suited for the regulation and in particular inhibition of proCFD activation. By means of the biotin residue provided at one end (here: left), the compound can be well detected both in vitro and in vivo in the biological sample.
Ein weiterer Gegenstand der vorliegenden Erfindung betrifft die erfindungsgemäße Verbindung bzw. deren Verwendung zur Behandlung und/oder Prophylaxe und/oder Diagnose einer Krankheit, die mit einer Deregulierung, vorzugsweise einer fehlregulierten Aktivierung, weiter vorzugsweise einer Hyperaktivierung des alternativen Komplementweges assoziiert ist. Another object of the present invention relates to the compound of the invention or its use for the treatment and / or prophylaxis and / or diagnosis of a disease associated with a deregulation, preferably a dysregulated activation, more preferably a hyperactivation of the alternative complement pathway.
Die Erfinder haben festgestellt, dass die Verbindung sowohl zur Behandlung also auch zur Prophylaxe sowie Diagnose geeignet ist. Dabei interagiert die erfindungsgemäße Verbindung mit den proCFD-aktivierenden Enzymen und - vorzugsweise - inhibiert diese. Im Gegensatz zu den im Stand der Technik bekannten Verbindungen, die lediglich mit einem einzigen Komplementfaktor interagieren, interagiert bzw. vorzugsweise inhibiert die erfindungsgemäße Verbindung das Prozessieren von proCFD auf mechanistische Art und Weise. Dies ist deshalb von Vorteil, da zu vermuten ist, dass proCFD nicht nur durch einen einzelnen Komplementfaktor oder ein einzelnes Enzym aktiviert wird sondern durch verschiedene Faktoren und über verschiedene Wege. Nach Erkenntnissen der Erfinder kann die erfindungsgemäße Verbindung mit mehreren oder ggf. allen proCFD- aktivierenden Enzymen interagieren und diese vorzugsweise hemmen.
Erfindungsgemäß wird unter "Deregulierung" eine Fehlregulierung der Kaskade des alternativen Komplementwegs verstanden. Unter "fehlregulierter Aktivierung" wird eine gestörte Aktivierung des alternativen Komplementwegs, insbesondere eine gestörte Aktivierung von proCFD in CFD, verstanden. Unter "Hyperaktivierung" wird erfindungsgemäß eine über den Normalzustand hinausgehende Aktivierung des alternativen Komplementwegs verstanden. The inventors have found that the compound is suitable both for the treatment and for the prophylaxis and diagnosis. In this case, the compound of the invention interacts with the proCFD-activating enzymes and - preferably - inhibits them. In contrast to the prior art compounds which interact with only a single complement factor, the compound of the invention interacts or preferably inhibits the processing of proCFD in a mechanistic manner. This is advantageous since it is to be expected that proCFD will be activated not only by a single complement factor or a single enzyme but by various factors and by different pathways. According to the inventors, the compound according to the invention can interact with, and preferably inhibit, several or possibly all proCFD-activating enzymes. According to the invention, "deregulation" is understood to mean a dysregulation of the cascade of the alternative complement pathway. By "dysregulated activation" is meant an impaired activation of the alternative complement pathway, in particular a disturbed activation of proCFD in CFD. By "hyperactivation" is meant, according to the invention, an activation of the alternative complement path that goes beyond the normal state.
Die Eigenschaften, Vorteile, Weiterentwicklungen und Ausführungsformen der erfindungsgemäßen Verbindung gelten für die erfindungsgemäße Verwendung entsprechend. The properties, advantages, further developments and embodiments of the compound according to the invention apply correspondingly to the use according to the invention.
Dabei ist es bevorzugt, wenn die Krankheit eine Autoimmunerkrankung ist. Die Krankheit ist weiter vorzugsweise ausgewählt aus der Gruppe bestehend aus: altersbedingte Makuladegeneration (AMD), Nierenerkrankung, insbesondere atypisches hämolytischurämisches Syndrom (aHUS) und C3 Glomerulopathie. It is preferred if the disease is an autoimmune disease. The disease is further preferably selected from the group consisting of: age-related macular degeneration (AMD), kidney disease, in particular atypical hemolytic anemic syndrome (aHUS) and C3 glomerulopathy.
Mit dieser Maßnahme stellen die Erfinder eine Verbindung bereit, mit der eine With this measure, the inventors provide a compound with which a
Behandlung und/oder Diagnose solcher Erkrankungen möglich wird, die mit einer Desre- gulierung des alternativen Komplementweges assoziiert sind und für die derzeit keine zufriedenstellenden Therapiemöglichkeiten zur Verfügung stehen. Treatment and / or diagnosis of such diseases associated with a desegregation of the alternative complement pathway for which there are currently no satisfactory treatment options available.
Vor diesem Hintergrund betrifft ein weiterer Gegenstand der vorliegenden Erfindung eine pharmazeutische oder diagnostische Zusammensetzung, die die erfindungsgemäße Verbindung in einer pharmazeutisch oder diagnostisch wirksamen Konzentration sowie eine pharmazeutisch oder diagnostisch akzeptable Formulierung aufweist. Against this background, another object of the present invention relates to a pharmaceutical or diagnostic composition having the compound of the invention in a pharmaceutically or diagnostically effective concentration and a pharmaceutically or diagnostically acceptable formulation.
Pharmazeutisch akzeptable Träger sind im Stand der Technik hinreichend bekannt. Beispielhaft wird auf die Abhandlung von Kibbe A. (2003), Handbook of Pharmaceutical Excipients, 4. Auflage, American Pharmaceutical Association and Pharmaceutical Press, verwiesen.
Die Eigenschaften, Vorteile, Weiterentwicklungen und Ausführungsformen der erfindungsgemäßen Verbindung gelten für die erfindungsgemäße pharmazeutische und diagnostische Zusammensetzung entsprechend. Pharmaceutically acceptable carriers are well known in the art. For example, reference is made to the paper by Kibbe A. (2003), Handbook of Pharmaceutical Excipients, 4th Edition, American Pharmaceutical Association and Pharmaceutical Press. The properties, advantages, further developments and embodiments of the compound according to the invention apply correspondingly to the pharmaceutical and diagnostic composition according to the invention.
Ein weiterer Gegenstand der vorliegenden Erfindung betrifft die Verwendung der erfindungsgemäßen Verbindung in vitro zur Inhibition der Aktivierung von proCFD in einer biologischen Probe. Another object of the present invention relates to the use of the compound according to the invention in vitro for inhibiting the activation of proCFD in a biological sample.
Bei einer "biologischen Probe" kann es sich dabei um eine beliebige Zusammensetzung handeln, die die Elemente und Faktoren des alternativen Komplementweges aufweist oder für die vermutet wird, dass sie diese aufweist, wie bspw. Komplementfaktoren, Co- Faktoren etc. Eine bevorzugte biologische Probe ist eine Blutplasmaprobe eines Individuums, bspw. eines beliebigen Mammaliers, einschließlich eines Menschen. A "biological sample" may be any composition that includes or is believed to have the elements and factors of the alternative complement pathway, such as complement factors, cofactors, etc. A preferred biological sample is a blood plasma sample of an individual, for example of any Mammalier, including a human.
Dabei ist es bevorzugt, wenn die erfindungsgemäße Verbindung zur Komplexierung und/oder Isolierung und/oder Lokalisierung und/oder Quantifizierung von proCFD- aktivierenden Verbindungen in/aus einer biologischen Probe verwendet wird. In this case, it is preferred if the compound according to the invention is used for the complexation and / or isolation and / or localization and / or quantification of proCFD-activating compounds in / from a biological sample.
Die Eigenschaften, Vorteile, Weiterentwicklungen und Ausführungsformen der erfindungsgemäßen Verbindung gelten für die erfindungsgemäße Verwendung entsprechend. The properties, advantages, further developments and embodiments of the compound according to the invention apply correspondingly to the use according to the invention.
Ein weiterer Gegenstand der vorliegenden Erfindung betrifft ein Verfahren zur Behandlung und/oder Prophylaxe und/oder Diagnose einer Krankheit, die mit einer Desregulierung, vorzugsweise einer fehlregulierten Aktivierung, weiter vorzugsweise einer Hyperaktivie- rung des alternativen Komplementweges assoziiert ist, das die Verabreichung der erfindungsgemäßen Verbindung und/oder der erfindungsgemäßen Zusammensetzung in ein Individuum, bspw. einen beliebigen Mammalier, einschließlich einen Menschen umfasst. Another object of the present invention relates to a method for the treatment and / or prophylaxis and / or diagnosis of a disease associated with a desegulation, preferably a dysregulated activation, more preferably a Hyperaktivie- tion of the alternative complement pathway, the administration of the compound of the invention and / or the composition of the invention in an individual, for example any mammal, including a human.
Die Eigenschaften, Vorteile, Weiterentwicklungen und Ausführungsformen der erfindungsgemäßen Verbindung gelten für das erfindungsgemäße Verfahren entsprechend.
Es versteht sich, dass die vorstehend genannten und die nachstehend noch zu The properties, advantages, further developments and embodiments of the compound according to the invention apply correspondingly to the process according to the invention. It is understood that the above and the still to follow
erläuternden Merkmale nicht nur in der jeweils angegebenen Kombination, sondern auch in anderen Kombinationen oder in Alleinstellung verwendbar sind, ohne den Rahmen der vorliegenden Erfindung zu verlassen. explanatory features are usable not only in the combination given, but also in other combinations or alone, without departing from the scope of the present invention.
Die vorliegende Erfindung wird nun anhand von Ausführungsbeispielen näher erläutert, aus denen sich weitere Merkmale, Eigenschaften und Vorteile der Erfindung ergeben. Die Ausführungsbeispiele sind dabei nicht einschränkend. The present invention will now be explained in more detail by means of exemplary embodiments from which further features, properties and advantages of the invention result. The embodiments are not limiting.
Es versteht sich außerdem, dass einzelne Merkmale, die in den Ausführungsbeispielen offenbart sind, nicht nur im Kontext der jeweiligen spezifischen Ausführungsform sondern in einer Allgemeingültigkeit offenbart sind und für sich genommenen einen eigenen Beitrag zur Erfindung liefern. Der Fachmann kann deshalb diese Merkmale frei mit anderen Merkmalen der Erfindung kombinieren. It should also be understood that individual features disclosed in the exemplary embodiments are disclosed not only in the context of the specific embodiment but in generality and in themselves provide a separate contribution to the invention. The person skilled in the art can therefore freely combine these features with other features of the invention.
Dabei wird Bezug genommen auf die beigefügten Abbildungen, in denen Folgendes dargestellt ist. Reference is made to the accompanying drawings, in which the following is shown.
Fig. 1 : Chemische Struktur eines erfindungsgemäßen Beispiels für eine aktivitäts- basierenden Sonde, ausgerichtet auf den/die Aktivator(en) von proCFD. FIG. 1: Chemical structure of an example of an activity-based probe according to the invention, oriented on the activator (s) of proCFD.
Fig. 2: Das inaktive Proenzym des humanen Komplementfaktors D (proCFD) wird durch die Abspaltung der sechs N-terminalen Aminosäuren aktiviert. Mehrere Serinproteasen sind in der Lage, diesen Schritt in vitro zu katalysieren. Der Unterschied in den isoelektrischen Punkten der aktiven gegenüber inaktiven Formen ermöglicht die Detektion des Aktivierungsprozesses mit Hilfe der isoelektrischen Fokussierung. Inaktiver proCFD wurde mit humanem Blutplasma inkubiert. Der physiologische Aktivator liegt im Plasma vor, daher wird proCFD schnell aktiviert (Spur # 6, siehe die Bande in Richtung der Anode (+)). In Gegenwart der erfindungsgemäßen Verbindung in unterschiedlichen Konzentrationen (Spuren # 2-5, jeweils 1 ,6 mM, 0,16 mM, 0,016 mM, 0,0016 mM) ist die Aktivierung von proCFD vollständig blockiert.
Die Sonde wurde in DMSO gelöst eingesetzt, dennoch hat dieses Lösungsmittel allein keine Wirkung auf die Aktivierung von proCFD (Spur # 1 ). FIG. 2: The inactive proenzyme of the human complement factor D (proCFD) is activated by the cleavage of the six N-terminal amino acids. Several serine proteases are able to catalyze this step in vitro. The difference in the isoelectric points of the active versus inactive forms allows the detection of the activation process by means of isoelectric focusing. Inactive proCFD was incubated with human blood plasma. The physiological activator is present in the plasma, therefore proCFD is activated rapidly (lane # 6, see the band towards the anode (+)). In the presence of the compound of the invention at different concentrations (lanes # 2-5, 1, 6mM, 0.16mM, 0.016mM, 0.0016mM, respectively), activation of proCFD is completely blocked. The probe was used dissolved in DMSO, yet this solvent alone has no effect on the activation of proCFD (lane # 1).
Ausführungsbeispiele embodiments
1 . Struktur eine beispielhaften erfindungsgemäßen Verbindung 1 . Structure of an exemplary compound of the invention
[0054] In der Fig. 1 ist die Struktur eines Beispiels für eine aktivitätsbasierenden Sonde Fig. 1 is the structure of an example of an activity-based probe
dargestellt, die auf den/die Aktivator(en) von proCFD ausgerichtet ist. Die Summenformel lautet C54H82N150iiPS. Das Molekulargewicht beträgt 1 179,5777 Dalton. Es weist ein Zielfindungselement auf, das der Sequenz des N-terminalen Pro-Peptids (APPRGR) von CFD entspricht. Kürzere Fragmente, bspw. PRGR, haben sich als ausreichend herausgestellt, um eine vollständige Inhibierung der/des proCFD-Aktivator(en/s) zu erreichen. which is aligned with the activator (s) of proCFD. The empirical formula is C 5 4H82N 15 0iiPS. The molecular weight is 1 179.5777 daltons. It has a targeting element that corresponds to the sequence of the N-terminal pro-peptide (APPRGR) of CFD. Shorter fragments, such as PRGR, have been found to be sufficient to achieve complete inhibition of the proCFD activator (s).
[0055] Das C-terminale Arginin (R) wurde durch eine funktionale Gruppe ersetzt, die häufig als The C-terminal arginine (R) has been replaced by a functional group often referred to as
"Sprengkopf" (engl, "warhead") bezeichnet wird. Dieses Arginin-Mimetikum, ein Diphe- nylphosphonat, modifiziert kovalent Zielenzyme in einer aktivitätsabhängigen Art und Weise. "Warhead" (engl., "Warhead") is called. This arginine mimetic, a diphenylphosphonate, covalently modifies target enzymes in an activity-dependent manner.
[0056] Der Biotin-Tag, der an der Sonde angebracht ist, kann in verschiedenen Anwendungen zum Einsatz kommen, wie bspw. in der Visualisierung der Sonde in histochemischen Untersuchungen oder unter Verwendung von Streptavidin als gegen Biotin gerichtetes Fängermolekül in einer Affinitätsreinigung von kovalent gebundenen Enzymen aus verschiedenen Bioproben. Es ist ferner möglich, die Menge des gebundenen Proteins mittels standardisierter biochemischer Methoden zu identifizieren und quantifizieren. The biotin tag attached to the probe can be used in a variety of applications, such as in visualizing the probe in histochemical studies or using streptavidin as a biotin-directed capture molecule in a covalently-affinity-purified affinity purification Enzymes from different organic samples. It is also possible to identify and quantify the amount of bound protein using standard biochemical methods.
2. Hemmung der Aktivierung von proCFD in CFD durch die erfindungsgemäße 2. Inhibition of the activation of proCFD in CFD by the invention
Verbindung connection
[0057] Durch transiente Transfektion von adhärent wachsenden HEK-293 Zellen (menschliche embryonale Nierenzellen) wurde proCFD gewonnen. 20 μΙ EDTA-Plasma von gesunden Blutspendern wurden mit 1 μΙ der erfindungsgemäßen Verbindung in unterschiedlichen
Konzentrationen (1 ,6 mM, 0,16 mM, 0,016 mM, 0,0016 mM) für 10 Minuten bei 37 °C vorinkubiert, dann zu proCFD gegeben und weitere 6 Stunden inkubiert. Die Umsetzung wurde durch Acetonfällung gestoppt und die Aktivierung von proCFD mittels isoelektrischer Fokussierung und anschließender Detektion auf einem Immunoblot mit Hilfe eines CFD-Antikörpers visualisiert; siehe Fig. 2. Diese Ergebnisse zeigen, dass die erfindungsgemäße Verbindung die vorhandene Aktivität von proCFD-Aktivator(en) im Plasma während der Vorinkubation vollkommen blockiert hatte, so konnte die Katalyse nicht stattfinden. Bei der nur mit Lösungsmittel (DMSO) vorgenommenen Kontrolle wurde proCFD effizient aktiviert, so dass der Effekt der erfindungsgemäßen Verbindung eindeutig auf die Hemmung der Aktivasen zurückgeführt wird. By transient transfection of adherently growing HEK-293 cells (human embryonic kidney cells) proCFD was recovered. 20 μM EDTA plasma from healthy blood donors were mixed with 1 μΙ of the compound of the invention in different Concentrations (1, 6 mM, 0.16 mM, 0.016 mM, 0.0016 mM) preincubated for 10 minutes at 37 ° C, then added to proCFD and incubated for a further 6 hours. The reaction was stopped by acetone precipitation and the activation of proCFD visualized by means of isoelectric focusing and subsequent detection on an immunoblot using a CFD antibody; see Figure 2. These results show that the compound of the present invention had completely blocked the presence of plasma proCFD activator (s) during preincubation, so catalysis could not occur. In the control carried out only with solvent (DMSO), proCFD was efficiently activated, so that the effect of the compound according to the invention is clearly attributed to the inhibition of the activases.
3. Fazit 3. Conclusion
Die Erfinder konnten anhand einer exemplarischen Verbindung nachweisen, dass die erfindungsgemäße Verbindung zur Behandlung, Prophylaxe und Diagnose einer Krankheit geeignet ist, die mit einer Desregulierung, einer fehlregulierten Aktivierung und einer Hyperaktivierung des alternativen Komplementweges assoziiert ist.
The inventors have demonstrated, by way of example, that the compound of the present invention is useful for the treatment, prophylaxis and diagnosis of a disease associated with desegregulation, dysregulated activation and hyperactivation of the alternative complement pathway.
Claims
Patentansprüche Verbindung mit folgender Struktur: Claims connection with the following structure:
wobei in which
- n eine ganze Zahl von 0 bis 5 ist; - n is an integer from 0 to 5;
- R1 ausgewählt ist aus der Gruppe bestehend aus: H, detektierbarer Marker; R1 is selected from the group consisting of: H, detectable marker;
- R2 ausgewählt ist aus der Gruppe bestehend aus: substituiertes oder unsubstituiertes Aryl mit jeweils einem MW von < 200 Da, substituiertes oder unsubstituiertes Aryl mit jeweils einem MW von < 200 Da, eine Seitenkette einer natürlichen oder synthetischen Aminosäure; - R2 is selected from the group consisting of: substituted or unsubstituted aryl each having an MW of <200 Da, substituted or unsubstituted aryl each having an MW of <200 Da, a side chain of a natural or synthetic amino acid;
- R3 ausgewählt ist aus der Gruppe bestehend aus: substituiertes oder unsubstituiertes Alkyl mit jeweils einem MW von < 300 Da, substituiert unsubstituier- R3 is selected from the group consisting of: substituted or unsubstituted alkyl each having an MW of <300 Da, substituted unsubstituted
tes Aryl mit jeweils einem MW von < 300 Da, vorzugsweise
, eine Sei- tenkette einer natürlichen oder synthetischen Aminosäure; tes aryl, each having a MW of <300 Da, preferably a side chain of a natural or synthetic amino acid;
- R4 und R5 unabhängig ausgewählt sind aus der Gruppe bestehend aus: substituiertes oder unsubstituiertes Aryl mit jeweils einem MW > 70 kDa und < 300 Da, substituiertes oder unsubstituiertes Alkyl mit jeweils einem MW > 70 kDa und < 300 Da.
- R4 and R5 are independently selected from the group consisting of: substituted or unsubstituted aryl each having a MW> 70 kDa and <300 Da, substituted or unsubstituted alkyl each having a MW> 70 kDa and <300 Da.
2. Verbindung nach Anspruch 1 , mit folgender Struktur: 2. A compound according to claim 1, having the following structure:
wobei in which
- Ar ein substituiertes oder unsubstituiertes Aryl mit jeweils einem MW > 70 kDa und < 300 Da ist. - Ar is a substituted or unsubstituted aryl each having a MW> 70 kDa and <300 Da.
3. Verbindung nach Anspruch 1 oder 2, mit folgender Struktur: o 8 o 3. A compound according to claim 1 or 2, having the following structure: o 8 o
° R ° R ° R ° R
wobei in which
- R6, R7 und R8 jeweils unabhängig eine Seitenkette einer natürlichen oder synthetischen Aminosäure sind. R6, R7 and R8 are each independently a side chain of a natural or synthetic amino acid.
4. Verbindung nach Anspruch 3, dadurch gekennzeichnet, dass 4. A compound according to claim 3, characterized in that
- R6 ein Prolin-Rest, und/oder R6 is a proline radical, and / or
- R7 ein Arginin-Rest, und/oder R7 is an arginine residue, and / or
- R8 ein Glyzin-Rest ist.
- R8 is a glycine residue.
5. Verbindung nach Anspruch 3 oder 4, mit folgender Struktur: 5. A compound according to claim 3 or 4, having the following structure:
wobei in which
- Ph ein Phenyl-Rest ist. - Ph is a phenyl radical.
Verbindung nach einem der vorherigen Ansprüche, dadurch gekennzeichnet, dass der detektierbare Marker ausgewählt ist aus der Gruppe bestehend aus: Biotin, Bio- tinyl-Rest, radioaktiver Marker, fluoreszierender Marker, PET-Tracer. Compound according to one of the preceding claims, characterized in that the detectable marker is selected from the group consisting of: biotin, biotin residue, radioactive marker, fluorescent marker, PET tracer.
Verbindung nach einem der vorherigen Ansprüche, mit folgender Struktur: A compound according to any one of the preceding claims, having the following structure:
8. Verbindung nach einem der vorherigen Ansprüche, mit folgender Struktur: 8. A compound according to any one of the preceding claims, having the following structure:
9. Verbindung nach einem der vorherigen Ansprüche zur Behandlung und/oder Prophylaxe und/oder Diagnose einer Krankheit, die mit einer Deregulierung, vorzugsweise einer fehlregulierten Aktivierung, weiter vorzugsweise einer Hyperaktivie- rung des alternativen Komplementweges assoziiert ist. 9. A compound according to any one of the preceding claims for the treatment and / or prophylaxis and / or diagnosis of a disease associated with deregulation, preferably dysregulated activation, more preferably hyperactivation of the alternative complement pathway.
10. Verbindung nach Anspruch 9, dadurch gekennzeichnet, dass die Krankheit eine Autoimmunerkrankung ist, vorzugsweise ausgewählt ist aus der Gruppe bestehend aus: altersbedingte Makuladegeneration (AMD), Nierenerkrankung, insbesondere atypisches hämolytisch-urämisches Syndrom (aHUS) und C3 Glomerulopathie. 10. A compound according to claim 9, characterized in that the disease is an autoimmune disease, preferably selected from the group consisting of: age-related macular degeneration (AMD), kidney disease, in particular atypical hemolytic uremic syndrome (aHUS) and C3 glomerulopathy.
1 1 . Pharmazeutische oder diagnostische Zusammensetzung, die die Verbindung nach einem der Ansprüche 1 bis 10 in einer pharmazeutisch oder diagnostisch wirksamen Konzentration sowie eine pharmazeutisch oder diagnostisch akzeptable Formulierung aufweist. 1 1. A pharmaceutical or diagnostic composition comprising the compound of any one of claims 1 to 10 in a pharmaceutically or diagnostically effective concentration and a pharmaceutically or diagnostically acceptable formulation.
12. Verwendung der Verbindung nach einem der Ansprüche 1 bis 8 in vitro zur Inhibition der Aktivierung von proCFD in einer biologischen Probe. 12. Use of the compound according to any one of claims 1 to 8 in vitro for inhibiting the activation of proCFD in a biological sample.
13. Verwendung der Verbindung nach einem der Ansprüche 1 bis 8 in vitro zur Komple- xierung und/oder Isolierung und/oder Lokalisierung und/oder Quantifizierung von proCFD-aktivierenden Verbindungen in/aus einer biologischen Probe.
13. Use of the compound according to any one of claims 1 to 8 in vitro for complexing and / or isolation and / or localization and / or quantification of proCFD-activating compounds in / from a biological sample.
14. Verfahren zur Behandlung und/oder Prophylaxe und/oder Diagnose einer Krankheit, die mit einer Deregulierung, vorzugsweise einer fehlregulierten Aktivierung, weiter vorzugsweise einer Hyperaktivierung des alternativen Komplementweges assoziiert ist, das die Verabreichung der Verbindung nach einem der Ansprüche 1 bis 8 und/oder der Zusammensetzung nach Anspruch 1 1 in ein Individuum umfasst.
A method for the treatment and / or prophylaxis and / or diagnosis of a disease associated with a deregulation, preferably a dysregulated activation, more preferably a hyperactivation of the alternative complement pathway, which comprises administration of the compound according to any one of claims 1 to 8 and / or the composition of claim 1-1 in an individual.
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| DE102016114392.8A DE102016114392A1 (en) | 2016-08-03 | 2016-08-03 | A compound for the treatment of a disease associated with a desegregation of the alternative complement pathway |
| DE102016114392.8 | 2016-08-03 |
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| EP2676962A1 (en) * | 2012-06-21 | 2013-12-25 | Universität Ulm | Derivatives of 1-aminoalkylphosphonate diaryl esters, method of 1-aminoalkylphosphonate diaryl ester derivatives preparation and their application |
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| WO2015130806A1 (en) * | 2014-02-25 | 2015-09-03 | Achillion Pharmaceuticals, Inc. | Amino compounds for treatment of complement mediated disorders |
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