Inhibitor of rho gtpase cdc42 for use in the prevention and / or treatment of parkinson's disease of the human or animal body

Abstract

The invention relates to an inhibitor of Rho GTPase Cdc42 for use in the prevention and / or treatment of Parkinson's disease of the human or animal body. In a mouse model in which the mice were genetically modified to accumulate α-synuclein and develop Parkinson's disease, it has been shown that the administration of a Cdc42 inhibitor in the diseased mice reduces the accumulation of α-synuclein, improves the symptoms of Parkinson's disease, increases the average life expectancy, and prolongs the total life span. Only minimal to no side effects occurred in the diseased mice.

Description

[0001] Rho GTPase Cdc42 Inhibitor for Use in the Prevention and / or Treatment of Parkinson's Disease in Humans or Animals. An inhibitor of the Rho GTPase Cdc42 is proposed for use in the prevention and / or treatment of Parkinson's disease in humans or animals. In a mouse model where the mice were genetically modified to accumulate α-synuclein and develop Parkinson's disease, it was shown that administration of a Cdc42 inhibitor to the diseased mice reduced α-synuclein accumulation, improved the symptoms of Parkinson's disease, increased mean lifespan, and extended overall lifespan. Minimal to no side effects were observed in the diseased mice. It is known in the prior art that Rho GTPases, such as Cdc42, inhibit cellular processes (e.g.,the reorganization of the cytoskeleton, cell polarity, cell growth and cell structuring) regulate (Bement, WM et al., Nat. Rev. Mol. Cell. Biol., Vol. 25, No. 4, pp. 290-308, 2024).

[0002] Furthermore, various inhibitors of the Rho GTPase Cdc42 are known in the state of the art (Guiler, W. et al., Frontiers in Cellular Neuroscience, Vol. 15, No. 661612, 2021; Medina, JI et al., Cancer Research Communications, 2(12):1711-1726, 2022).

[0003] The prior art proposes the use of Cdc42 inhibitors for the treatment of cancer (del Mar Maldonado, M. & Dharmawardhane, S., Cancer Research, Vol. 78, No. 12, pp. 3101-3111, 2018). The specific Cdc42 inhibitor 2,2-[(2,3,4,9-tetrahydro-6-phenyl-1H-carbazol-1-yl)amino]ethanol (“CASIN”) is also known to be suitable for the rejuvenation and mobilization of hematopoietic stem cells (WO 2013 / 166043 Al and Liu, W. et al., Leukemia, Vol. 33, No. 3, pp. 749-761, 2019).

[0004] Parkinson's disease typically manifests in later life, around the age of 60, and is the second most common neurodegenerative, chronic, progressive brain disease after Alzheimer's. In 2016, 6.1 million people worldwide suffered from Parkinson's disease (approximately 400,000 of whom were living in Germany). The progression of the disease can vary considerably from person to person. Symptoms can therefore differ, but typically include impaired motor control, manifesting as stiff muscles, slowed movements, and tremors. Parkinson's disease is thought to be triggered by the death of nerve cells that produce the neurotransmitter dopamine. This leads to a disruption in dopamine levels (dopamine deficiency) and causes the characteristic symptoms of Parkinson's disease. However, the exact cause of the death of these dopamine-producing nerve cells remains unclear.Deposits in the brains of affected individuals are suspected to result from the aggregation of the protein α-synuclein (Ca la bresi, P. et al., Brain, Vol. 146, No. 9, pp. 3587-3597). Current treatment for Parkinson's disease involves administering a dopamine precursor (e.g., levodopa) and / or an inhibitor of catechol-O-methyltransferase (e.g., tolcapone). While these measures can alleviate the symptoms of Parkinson's disease, they are sometimes associated with severe side effects (Thanvi, BR & Lo. TCN, Postgraduate Medical Journal, Vol. 80, pp. 452-458, 2004 and Kaakkola, S., International Review of Neurobiology, Vol. 95, pp. 207-225, 2010).

[0005] To date, there is no therapeutic approach for treating Parkinson's disease that addresses the cause of the disease and has only minor side effects.

[0006] Based on this, the object of the present invention was to provide a new therapeutic agent for the prevention and / or treatment of Parkinson's disease that targets the underlying cause of Parkinson's disease and, in particular, has few to no side effects. This object is achieved by the inhibitor with the features of claim 1. The dependent claims describe advantageous embodiments.

[0007] According to the invention, it is proposed to use an inhibitor of the Rho GTPase Cdc42 for use in the prevention and / or treatment of Parkinson's disease in the human or animal body.

[0008] Using an inhibitor of the Rho GTPase Cdc42 for the prevention and / or treatment of Parkinson's disease in humans or animals has the advantage that Parkinson's disease can be prevented and treated with few to no side effects.

[0009] The Rho GTPase Cdc42 inhibitor can contain or consist of an inhibitor capable of reducing the cellular concentration of the GDP-bound form of Rho GTPase Cdc42. The advantage here is that the inhibitor counteracts an imbalance or disorder in biological cells caused by an elevated concentration of the GDP-bound form of Rho GTPase Cdc42 in these cells, i.e., it reduces or even eliminates the imbalance or disorder. The Rho GTPase Cdc42 inhibitor is preferably selected from the group consisting of CASIN, ZCL278, ZCL367, MBQ-167, MBQ-168, AZA197, Secramine, ML141, and combinations thereof. All of these inhibitors have the property of reducing the concentration of the GDP-bound form of Rho GTPase Cdc42 in biological cells.

[0010] The Rho GTPase inhibitor Cdc42 may contain or consist of a compound of the following formula (I): where

[0011] Y is selected from the group consisting of -OR7, -NRsRg and -NNRsRg;

[0012] R7 is selected from the group consisting of Ci-6-alkyl, -(CH2) u C3-7-cycloal-kyl with u = 0, 1, 2, 3 or 4, C2-6-alkenyl, Ci-6-alkoxy, hydroxy-Ci-6-alkyl, phenyl, optionally Ci-6-alkyl substituted with at least one fluorine or and C1-6-Al-koxy, wherein the Ci-6-alkyl, -(CH2) u C3-7-Cycloalkyl 4, C2-6-Alkenyl, Ci-6-Alkoxy, Hydroxy-Ci-6-Alkyl, Phenyl optionally substituted with at least one substituent selected from the group consisting of Halogen, -CN, -OH, Ci-6-Alkoxyl, Heteroaryl, Rig and -OR24;

[0013] Rig Aryl is optionally substituted with at least one substituent, wherein the at least one substituent is selected from the group consisting of halogen, cyano, nitro, hydroxy, Ci-6-alkyl, Ci-6-alkoxy, Ci-6-alkyl substituted with at least one fluorine and Ci-6-alkoxy substituted with at least one fluorine;

[0014] R24 is hydrogen or aryl, optionally substituted with at least one substituent, wherein the at least one substituent is selected from the group consisting of halogen, cyano, nitro, hydroxy, Ci-6-alkyl, Ci-6-alkoxy, Ci-6-alkyl substituted with at least one fluorine, and Ci-6-alkoxy substituted with at least one fluorine; Rs is a hydrogen atom, R20 is or a Ci-3-alkyl connecting Rs and R2 via a ring, wherein Rs optionally forms indolinyl, pyrrolidinyl, piperidinyl, piperazinyl, or morpholinyl together with the nitrogen to which it is bonded, each of which is optionally substituted with at least one substituent, wherein the at least one substituent is selected from the group consisting of halo, cyano, nitro, hydroxy, Ci-6-alkyl, (CH2) uC3-7-cycloalkyl with u = 0, 1, 2, 3 or 4, C2-6-alkenyl, Ci-6-alkoxy, hydroxy-Ci-6-alkyl, phenyl, Ci-6-alkyl substituted with at least one fluorine and Ci-6-alkoxy substituted with at least one fluorine;

[0015] R9 is a hydrogen atom or R20, wherein R9 optionally forms indolinyl, pyrrolidinyl, piperidinyl, piperazinyl or morpholinyl together with the nitrogen to which it is bonded, each of which is optionally substituted with at least one substituent, wherein the at least one substituent is selected from the group consisting of Halo, Cyano, Nitro, Hydroxy, C1-6-Al kyl, (CH2) U C3-7-cycloalkyl with u = 0, 1, 2, 3 or 4, C2-6-alkenyl, Ci-6-alkoxy, hydroxy-Ci-6-alkyl, phenyl, Ci-6-alkyl substituted with at least one fluorine and Ci-6-alkoxy substituted with at least one fluorine;

[0016] R20 is selected from the group consisting of C1-6-alkyl, C3-7-cycloalkyl and phenyl, wherein the C1-6-Al kyl, C3-7-cycloalkyl and phenyl are optionally substituted with at least one substituent, wherein the at least one substituent is selected from the group consisting of R21 and R22;

[0017] R21 is selected from the group consisting of halogen, cyano, nitro and hydroxy;

[0018] R22 is selected from the group consisting of, optionally substituted with at least one substituent, Ci-6-alkyl, Ci-6-alkoxy-(CH2) u C3-7-cycloalkyl with u = 0, 1, 2, 3 or 4, C2-6-alkenyl, hydroxy-Ci-6-alkyl, R19 and -OR24, wherein the at least one substituent is selected from the group consisting of halogen, cyano, nitro, hydroxy, Ci-6-alkyl and Ci-6-alkoxy;

[0019] R2 is a hydrogen atom, a C1-3 alkyl connecting R2 and Rs via a ring, or is selected from the group consisting of, optionally with at least one substituent, Ci-Ce-alkyl, C3-C7-cycloalkyl and phenyl, wherein the at least one substituent is preferably selected from the group consisting of halogen, cyano, nitro, hydroxy, Ci-Ce-alkyl, - (CH2) u C3-7-cycloalkyl with u = 0, 1, 2, 3 or 4, C2-6-alkenyl, Ci-6-alkoxy, hydroxy-Ci-6-alkyl, phenyl, Ci-6-alkyl substituted with at least one fluorine, Ci-6-alkoxy substituted with at least one fluorine, and -O(CH2) u Phenyl with u = 0, 1, 2, 3 or 4, where -O(CH2) uPhenyl is optionally substituted with at least one substituent, preferably halogen, cyano, nitro, hydroxy, Ci-6-alkyl and Ci-6-Al-koxy, or forms a ring with R2; Ci-3-alkyl is linked; R3, R4, Rs and Re are each independently selected from the group consisting of hydrogen, halogen, cyano, nitro, hydroxy, Ci-e-Al-koxy, (CH2) u C3-7-cycloalkyl with u = 0, 1, 2, 3 or 4, -O(CH2) u C3-7-cycloalkyl with u = 0, 1, 2, 3 or 4, C2-6-alkenyl, Ci-e-alkoxy, hydroxy-Ci-e-alkyl, phenyl, Ci-e-Alkyl substituted with at least one fluorine, and Ci-6-alkoxy substituted with at least one fluorine, wherein the Ci-6-alkyl, (CH2) u C3-7-Cycloalkyl, -O(CH2) u C3-7- cycloalkyl, C2-6-alkenyl, Ci-6-alkoxy, hydroxy-Ci-6-alkyl, phenyl optionally substituted with at least one substituent R23;

[0020] R23 is selected from the group consisting of halogen, cyano, nitro, hydroxy, Ci-6-alkyl, -(CH2) uC3-7-cycloalkyl with u = 0, 1, 2, 3 or 4, C2-6-alkenyl, C1-6-alkoxy, hydroxy-Ci-6-alkyl, phenyl, C1-6-Al-kyl substituted with at least one fluorine and Ci-6-alkoxy substituted with at least one fluorine, wherein the phenyl is optionally substituted with a substituent selected from the group consisting of halogen, cyano, nitro, hydroxy, C1-6-Al-kyl, -(CH2) U C3-7-cycloalkyl with u = 0, 1, 2, 3 or 4, C2-6-alkenyl, Ci-6-alkoxy, hydroxy-Ci-6-alkyl, phenyl, with at least one fluorine-substituted C1-6-Alkyl and with at least one fluorine-substituted Ci-6-alkoxy; provided that if Rs and R2 is ring-linking Ci-3-alkyl, R4 is not hydroxy-substituted.

[0021] In a preferred embodiment, R4 of formula (I) is selected from the group consisting of C1-6-AI kyl, (CH2) u C3-7-cycloalkyl with u = 0, 1, 2, 3 or 4, -O(CH2) uC3-7-cycloalkyl with u = 0, 1, 2, 3 or 4, C2-6-alkenyl, Ci-6-alkoxy, phenyl, Ci-6-alkyl substituted with at least one fluorine, and Ci-6-alkoxy substituted with at least one fluorine, wherein the Ci-6-alkyl, (CH2) u C3-7-Cycloalkyl, -O(CH2) u C3-7-cycloalkyl, C2-6-alkenyl, Ci-6-alkoxy and phenyl is optionally substituted with at least one substituent selected from the group consisting of halogen, Ci-6-alkyl, -(CH2) uC3-7-cycloalkyl with u = 0, 1, 2, 3 or 4, C2-6-alkenyl, Ci-6-alkoxy, Hydroxy-Ci-6-alkyl, Phenyl, C1-6-Alkyl substituted with at least one fluorine and Ci-6-alkoxy substituted with at least one fluorine. In a further preferred embodiment, F of formula (I) is selected from the group consisting of Ci-6-alkyl, C3-7-cycloalkyl, -O(CH2)C3-7-cycloalkyl, phenyl, Ci-6-alkyl substituted with at least one fluorine, and Ci-6-alkoxy substituted with at least one fluorine, wherein the phenyl is optionally substituted with at least one substituent selected from the group consisting of halogen, Ci-6-alkyl, Ci-6-alkoxy, Ci-6-alkyl substituted with at least one fluorine, and Ci-6-alkoxy substituted with at least one fluorine.

[0022] R2 of formula (I) can be hydrogen, preferably Rs is also hydrogen.

[0023] Alternatively, R2 of formula (I) may be selected from the group consisting of Ci-Ce-alkyl, C3-C7-cycloalkyl and phenyl, wherein Ci-Ce-alkyl, C3-C7-cycloalkyl and / or phenyl is optionally with at least one halogen.

[0024] Alternatively, R2 and Rs of formula (I) can be C1-3-AI kyl connecting via a ring and Y can be -NRsRg.

[0025] In another preferred embodiment, for formula (I) the following applies:

[0026] Y -NRsRg is;

[0027] Rs hydrogen is;

[0028] Rg Ci-6-alkyl is, wherein the Ci-6-alkyl is optionally substituted with at least one substituent, wherein the at least one substituent is selected from the group consisting of Hydroxy, Rig and -OR24;

[0029] Rig Phenyl is optionally substituted with at least one substituent, wherein the at least one substituent is selected from the group consisting of halogen, cyano, Ci-6-alkyl, Ci-6-alkoxy, Ci-6-alkyl substituted with at least one fluorine, and C1-6-alkoxy substituted with at least one fluorine; and

[0030] R24 is selected from the group consisting of hydrogen and phenyl, wherein the phenyl is optionally substituted with at least one substituent, the at least one substituent being selected from the group consisting of halogen, cyano, nitro, hydroxy, Ci-6-alkyl, Ci-6-alkoxy, Ci-6-alkyl substituted with at least one fluorine, and Ci-6-alkoxy substituted with at least one fluorine. Furthermore, for formula (I), it can be the case that

[0031] Ri9 is phenyl, which is optionally substituted with at least one substituent, wherein the at least one substituent is selected from the group consisting of halogen, Ci-6-alkyl and Ci-6-alkoxy; and

[0032] R24 Phenyl is optionally substituted with at least one substituent, wherein the at least one substituent is selected from the group consisting of halogen, Ci-6-alkyl and Ci-6-alkoxy.

[0033] Apart from that, the following can be held for formula (I):

[0034] R9 is hydrogen or R9 is Ci-6-alkyl, wherein the Ci-6-alkyl is optionally substituted with at least one substituent, wherein the at least one substituent is selected from the group consisting of hydroxy, R19 and -OR24, R19 is phenyl, which is optionally substituted with at least one substituent, wherein the at least one substituent is selected from the group consisting of halogen, cyano, Ci-6-alkyl, Ci-6-alkoxy, Ci-6-alkyl substituted with at least one fluorine and C1-6-alkoxy substituted with at least one fluorine, and

[0035] R20 is hydrogen or phenyl, which is optionally substituted with at least one substituent, wherein the at least one substituent is selected from the group consisting of halogen, cyano, nitro, hydroxy, Ci-6-alkyl, C1-6-Al-koxy, C1-6-Al-kyl substituted with at least one fluorine and Ci-6-alkoxy substituted with at least one fluorine.

[0036] In a particularly preferred embodiment, the Rho GTPase inhibitor Cdc42 contains or consists of a compound having one of the following formulas:

[0037]

[0038] The Rho GTPase Cdc42 inhibitor for use in the prevention and / or treatment of Parkinson's disease in humans or animals can be administered to the human or animal body for a period of at least two consecutive days, preferably at least three consecutive days, particularly preferably at least four consecutive days, most preferably at least five consecutive days, particularly at least six consecutive days, optionally at least seven consecutive days.

[0039] Furthermore, the Rho GTPase inhibitor Cdc42 can be administered to the human or animal body at a dosage of 5 to 100 mg / kg body weight, preferably 10 to 80 mg / kg body weight, particularly preferably 15 to 60 mg / kg body weight, most preferably 20 to 40 mg / kg body weight, and particularly preferably 25 to 30 mg / kg body weight, for use in the prevention and / or treatment of Parkinson's disease in humans or animals. The dosage can refer to a daily dosage, i.e., one dose per day. In addition, the Rho GTPase inhibitor Cdc42 can be administered to the human or animal body as a single enantiomer, as a mixture of enantiomers, as a pharmaceutically acceptable salt, as a solvate, or as a polymorph.

[0040] The following figures and examples are intended to explain the subject matter of the invention in more detail, without limiting it to the specific embodiments shown here.

[0041] Figure 1 shows the results of the "Accelerating Rotarod" behavioral experiment from Example 2 for the first age group (mice aged between 12 and 16 months, with the experiment beginning at 12 months). Starting at 13 months of age, the treatment group received injections of a CASIN solution. This was administered once daily for four days, followed by a one-week break, and then again once daily for four days, resulting in a total of eight CASIN injections at a dose of 25 mg CASIN / kg. The control group received the same injections of the solution without CASIN.

[0042] Figure 2 shows the results of the "Accelerating Rotarod" behavioral experiment from Example 2 for the second age group (mice aged between 20 and 24 months, with the experiment beginning at 20 months). Starting at 21 months of age, the treatment group received injections of a CASIN solution. This was administered once daily for four days, followed by a one-week break, and then again once daily for four days, resulting in a total of eight CASIN injections at a dose of 25 mg CASIN / kg. The control group received the same injections of the solution without CASIN.

[0043] Figure 3 shows the results of the experiment from Example 3. It can be seen that the administration of the Cdc42 inhibitor CASIN to the mice of the mouse model (see “synAN24+CASIN”) alters, i.e., reduces, the accumulation of α-synuclein compared to the mice of the mouse model that did not receive CASIN (see “synAN24”), as evidenced by the lower normalized luciferase activity of the gel chromatography fractions of the whole brain lysate of these mice.

[0044] Example 1 - Provision of mice of a mouse model for Parkinson's disease

[0045] To study the symptoms and progression of Parkinson's disease, a mouse model was developed in which the mice are genetically modified to accumulate oligomers of the protein α-synuclein. These mice develop Parkinson's disease over the course of their lives. They are referred to as S1 / S2 mice. The method for generating these mice is described in the publication by Kiechle, M. et al. (Cell. Rep., 29(9):2862-2874, e9).

[0046] Kiechle, M. et al. examined mice of this mouse model over a period of four months. They observed that, from a certain age, the nerve cells of these mice are particularly susceptible to the accumulation of α-synuclein. Specifically, they demonstrated an age-dependent accumulation of a particular α-synuclein subtype, which is associated with a loss of nerve cells and impaired motor function in these mice. This observation confirms that these mice were susceptible to developing Parkinson's disease.

[0047] Example 2 - Effect of a Rho GTPase Cdv42 inhibitor on mice of a mouse model for Parkinson's disease

[0048] To assess the motor skills and coordination of the S1 / S2 mice in Example 1, the "Accelerating Rotarod" behavioral experiment (five-lane rotarod, Med Associates) was performed. Animals from two different age groups were examined. The first age group consisted of mice aged 12 to 16 months, which were 12 months old at the start of the experiment, and the second age group consisted of mice aged 20 to 24 months, which were 20 months old at the start of the experiment. The Cdc42 inhibitor CASIN (Xcessbio M60040) was available as a freeze-dried powder. Only a single batch was used for the entire study.To prepare a CASIN solution, CASIN was first dissolved in DMSO to a concentration of 100 mM and then diluted in (2-hydroxypropyl)-beta-cyclodextrin solution (Sigma H5784), thus being present in a solvent consisting of the 2-hydroxypropyl)-beta-cyclodextrin solution and a certain proportion of DMSO. The CASIN solution was freshly prepared for each injection.

[0049] The time spent on the rota rod in mice of each age group was measured at weekly intervals over a period of approximately four months. On each test day, each mouse had to complete three consecutive trials with a five-minute break in between. In one trial, the rod's rotation speed was increased from 4 to 40 rpm over a period of 300 seconds. The latency time until the mouse fell was recorded using software (Rota rod 1.2.0 Software, Med Associates). Mean values ​​were used for analysis.

[0050] Starting approximately one month after the beginning of the behavioral experiment (i.e., with 13-month-old mice in the first age group and 21-month-old mice in the second age group), a first subset of mice in each age group (treatment group) received an intraperitoneal injection of freshly prepared CASIN solution (dose: 25 mg / kg). This was administered on four consecutive days (around midday each day), followed by a one-week break, and then again on four consecutive days (around midday each day). A second subset of mice in each age group (control group) received the same amount of solvent without CASIN, following the same schedule.

[0051] In the mouse model, α-synuclein is switched on ("synAN") and off ("synOFF") by a molecular switch. This means that in the "synAN" condition, α-synuclein oligomers are formed, leading to Parkinson's symptoms, while in the "synOFF" condition, no α-synuclein oligomers are formed, and the animals remain healthy (i.e., do not develop Parkinson's symptoms). Experiments with this mouse model showed that in the affected mice, the symptoms of Parkinson's disease (specifically, motor function) improved significantly upon administration of the CdC42 inhibitor CASIN (see Figures 1 and 2).

[0052] Furthermore, it was found that administering the Cdc42 inhibitor CASIN to the sick mice resulted in few to minimal side effects. This was evident in the animals' unremarkable behavior: The mice treated with CASIN were active and moved regularly within their cages. They explored their surroundings, ran around, and interacted with objects in their cages. They exhibited normal social behavior, interacting with each other, sniffing one another, and playing together. They were curious, ate and drank regularly. They also displayed regular nesting behavior and enjoyed exploring their environment. They typically slept during the day and became active at night.

[0053] Furthermore, it was observed that administration altered the accumulation of α-synuclein in mice of the mouse model that exhibited α-synuclein accumulation due to genetic modification (see Figure 3). Since the CDC42 inhibitor CASIN can alter or reduce α-synuclein accumulation in diseased mice, CDC42 inhibitors (such as CASIN) represent therapeutics that target the underlying cause of Parkinson's disease.

[0054] The experimental data demonstrate that Cdc42 inhibitors not only allow the treatment of Parkinson's disease symptoms (as with known therapeutics), but also, in principle, prevent the onset of Parkinson's disease and, in cases where Parkinson's disease has already occurred, prevent its progression.

[0055] Example 3 - Further effect of a Rho GTPase Cdv42 inhibitor on mice of a mouse model for Parkinson's disease

[0056] It was also investigated whether administration of the Cdc42 inhibitor CASIN alters the accumulation of α-synuclein in the brains of the mice in the mouse model from Example 1. To prepare a whole-brain lysate from the mice in the mouse model, one hemisphere per brain was prepared in PBS at a ratio of 1 g of tissue per

[0057] 10 ml of homogenized PBS. Homogenization was performed using a Tissue Lyser.

[0058] 11 (Qiagen) for 2 x 2 minutes at 25 Hz. Subsequently, the homogenates were centrifuged at 20,800 g for 30 min at 4 °C, and the supernatant was used for further experiments after determination of the protein concentration by BCA assay (Thermo Fisher Scientific).

[0059] The final whole-brain lysates were used for size exclusion chromatography (SEC) performed on the Superdex 200 10 / 300 GL column (Cytiva). The column was connected to an Äkta Pure system (Cytiva) and was equilibrated with two column volumes (CV) of filtered (0.22 pm filter) and PBS prior to use. A 1-ml loop was used for automated sample injection, and 600 pl of each lysate were loaded onto the column. A PBS flow rate of 0.75 ml / min was used to elute the proteins from the column. The maximum pressure was set to 5 MPa, and the eluted proteins were monitored by UV absorption at 280 and 215 nm for chromatogram analysis. The eluate was collected in 500 pl fractions in deep-well plates for later analysis.The molecular mass of the eluted proteins was determined using the Cytiva gel filtration calibration kit with the following standard proteins: conalbumin (75 kDa), ovalbumin (44 kDa), carbonic anhydrase (29 kDa), ribonuclease A (13.7 kDa), and aprotinin (6.5 kDa). The void volume (vo) was determined using blue dextran.

[0060] 200 pl of each protein-containing fraction were used for a duplicate measurement of luciferase activity; that is, duplicates of 100 pl of each protein-containing fraction from size-exclusion chromatography were used for the measurement of luciferase activity. Aggregated α-synuclein with luciferase halves reconstitutes total Gaussia luciferase, which oxidatively decarboxylates the substrate coelenterazine. This results in light emission. Coelenterazine (PJK) was prepared prior to the experiments at a final concentration of 1 mg / ml in methanol and stored at -80 °C. The working solution of coelenterazine was prepared in Opti-MEM (40 pM) and incubated at room temperature (25 °C) for 25 minutes without light. 100 pl of the cell-permeable substrate were added to each sample immediately before measurement using the automatic dispenser module of the plate reader (Victor X3 microplate reader, Perkin-Elmer).The luminescence was then measured at 480 nm with a signal integration time of 1 s.

[0061] The luminescence signal obtained was then normalized to the protein concentration of the respective whole-brain lysates used for size exclusion chromatography (SEC). The protein concentrations of the whole-brain lysates were determined using the colorimetric two-component Pierce BCA Protein Assay Kit (Thermo Fisher Scientific), as specified in the manufacturer's instructions. The lysates were diluted in ratios of 1:5, 1:10, and 1:20, and PBS was used as a blank. The concentrations of the protein standards used (bovine serum albumin) ranged from 0 to 2 mg / ml (0, 125, 250, 500, 750, 1000, 1500, 2000 pg / ml). After mixing the fluorescent dye with the standard samples, lysates and blank, everything was incubated for 30 minutes at 37 °C, and the fluorescence intensity was measured in duplicate using a microplate reader (SPECTROstar Nano) at a wavelength of 562 nm.

[0062] The result of the normalized luminescence signal as a function of the respective protein-containing fraction for the respective whole brain lysates is shown in Figure 3.

Claims

Patent claims 1. Rho GTPase Cdc42 inhibitor for use in the prevention and / or treatment of Parkinson's disease in humans or animals.

2. Inhibitor for use according to the preceding claim, characterized in that the Rho GTPase Cdc42 inhibitor contains or consists of an inhibitor suitable for reducing a cellular concentration of a GDP-bound form of Rho GTPase Cdc42, wherein the Rho GTPase Cdc42 inhibitor is preferably selected from the group consisting of CASIN, ZCL278, ZCL367, MBQ-167, MBQ-168, AZA197, Secramine, ML141 and combinations thereof.

3. Inhibitor for use according to any of the preceding claims, characterized in that the inhibitor of Rho GTPase Cdc42 is a compound of the following formula (I) contains or consists of, wherein Y is selected from the group consisting of -OR?, -NRsRg and -NNR8R9; R7 is selected from the group consisting of Ci-6-alkyl, -(CH2) u C3-7-cycloalkyl with u = 0, 1, 2, 3 or 4, C2-6-alkenyl, Ci-6-alkoxy, hydroxy-Ci- 6-Alkyl, Phenyl, optionally Ci-6-Alkyl or Ci-6-Alkoxy substituted with at least one Fluorine, wherein the Ci-6-Alkyl, -(CH2)uC3-7-Cycloalkyl 4, C2-6-Alkenyl, Ci-6-Alkoxy, Hydroxy-Ci-6-Alkyl, Phenyl is optionally substituted with at least one substituent selected from the group consisting of Halogen, -CN, -OH, Ci-6-Alkoxyl, Heteroaryl, Ri9 and -OR24; R19 Aryl is optionally substituted with at least one substituent, wherein the at least one substituent is selected from the group consisting of halogen, cyano, nitro, hydroxy, Ci-6-alkyl, C1-6-alkoxy, Ci-6-alkyl substituted with at least one fluorine and Ci-6-alkoxy substituted with at least one fluorine; R24 is hydrogen or aryl, which is optionally substituted with at least one substituent, wherein the at least one substituent is selected from the group consisting of halogen, cyano, nitro, hydroxy, C1-6-AI kyl, Ci-6-alkoxy, C1-6-AI kyl substituted with at least one fluorine and Ci-6-alkoxy substituted with at least one fluorine; Rs is a hydrogen atom, R20 is or is a Ci-3-alkyl connecting Rs and R2 via a ring, wherein Rs optionally forms indolinyl, pyrrolidinyl, piperidinyl, piperazinyl or morpholinyl together with the nitrogen to which it is bonded, each of which is optionally substituted with at least one substituent, wherein the at least one substituent is selected from the group consisting of halo, cyano, nitro, hydroxy, Ci-6-alkyl, (CH2)uC3-7-cycloalkyl with u = 0, 1, 2, 3 or 4, C2-6-alkenyl, Ci-6-alkoxy, hydroxy-Ci-6-alkyl, phenyl, Ci-6-alkyl substituted with at least one fluorine and Ci-6-alkoxy substituted with at least one fluorine; R9 is a hydrogen atom or R20 is, wherein R9 optionally forms indolinyl, pyrrolidinyl, piperidinyl, piperazinyl or morpholinyl together with the nitrogen to which it is bonded, each of which is optionally substituted with at least one substituent, wherein the at least one substituent is selected from the group consisting of halo, cyano, nitro, hydroxy, Ci-6-alkyl, (CH2)uC3-7-cycloalkyl with u = 0, 1, 2, 3 or 4, C2-6-alkenyl, Ci-6-alkoxy, hydroxy-Ci-6-alkyl, phenyl, with Ci-e-alkyl substituted with at least one fluorine and Ci-6-alkoxy substituted with at least one fluorine; R20 is selected from the group consisting of C1-6-AI kyl, C3-7-cycloalkyl and phenyl, wherein the C1-6-AI kyl, C3-7-cycloalkyl and phenyl are optionally substituted with at least one substituent, wherein the at least one substituent is selected from the group consisting of R21 and R22; R21 is selected from the group consisting of halogen, cyano, nitro and hydroxy; R22 is selected from the group consisting of, optionally substituted with at least one substituent, C1-6-AI kyl, Ci-6-Alkoxy-(CH2) u C3-7-Cycloa I kyl with u = 0, 1, 2, 3 or 4, C2-6-Alkenyl, Hydroxy-Ci-6-Alkyl, R19 and -OR24, wherein the at least one substituent is selected from the group consisting of halogen, cyano, nitro, hydroxy, Ci-e-alkyl and Ci-6-alkoxy; R2 is a hydrogen atom, a C1-3-Al alkyl connecting R2 and Rs via a ring, or is selected from the group consisting of, optionally with at least one substituent, Ci-Ce alkyl, C3-C7 cycloalkyl and phenyl, wherein the at least one substituent is preferably selected from the group consisting of halogen, cyano, nitro, hydroxy, Ci-Ce alkyl, -(CH2) uC3-7-cycloalkyl with u = 0, 1, 2, 3 or 4, C2-6-alkenyl, Ci-6-alkoxy, hydroxy-Ci-6-alkyl, phenyl, C1-6-Al-kyl substituted with at least one fluorine, Ci-6-alkoxy substituted with at least one fluorine, and -O(CH2) u Phenyl with u = 0, 1, 2, 3 or 4, where -O(CH2) u Phenyl is optionally substituted with at least one substituent, preferably halogen, cyano, nitro, hydroxy, Ci-e-alkyl and Ci-6-alkoxy, or forms a ring with R2 C1-3-AI kyl; R3, R4, Rs and Re are each independently selected from the group consisting of hydrogen, halogen, cyano, nitro, hydroxy, Ci-e-alkyl, (CH2) u C3-7-cycloalkyl with u = 0, 1, 2, 3 or 4, - O(CH2) u C3-7-cycloalkyl with u = 0, 1, 2, 3 or 4, C2-6-alkenyl, Ci-e-alkoxy, hydroxy-Ci-e-alkyl, phenyl, substituted with at least one fluorine CI-6-Al cyl, and Ci-6-alkoxy substituted with at least one fluorine, wherein the Ci-6 alkyl, (CH2)u C3-7-Cycloalkyl, -O(CH2) u C3-7-Cycloalkyl, C2-6-Alkenyl, Ci-6-Alkoxy, Hydroxy-Ci-6-Alkyl, Phenyl optionally substituted with at least one substituent R23; R23 is selected from the group consisting of halogen, cyano, nitro, hydroxy, Ci-6-alkyl, -(CH2) u C3-7-cycloalkyl with u = 0, 1, 2, 3 or 4, C2-6-alkenyl, Ci-6-alkoxy, hydroxy-Ci-6-alkyl, phenyl, Ci-6-alkyl substituted with at least one fluorine and Ci-6-alkoxy substituted with at least one fluorine, wherein the phenyl is optionally substituted with a substituent selected from the group consisting of halogen, cyano, nitro, hydroxy, Ci-6-alkyl, -(CH2) uC3-7-cycloalkyl with u = 0, 1, 2, 3 or 4, C2-6-alkenyl, Ci-6-alkoxy, hydroxy-Ci-6-alkyl, phenyl, with at least one fluorine-substituted Ci-6-alkyl and with at least one fluorine-substituted Ci-6-alkoxy; provided that if Rs and R2 is C1-3-Al kyl connecting via a ring, R4 is not substituted with hydroxy.

4. Inhibitor for use according to claim 3, characterized in that R4 is selected from the group consisting of C1-6-AI kyl, (CH2) u C3-7-cycloalkyl with u = 0, 1, 2, 3 or 4, -O(CH2) u C3-7-cycloalkyl with u = 0, 1, 2, 3 or 4, C2-6-alkenyl, Ci-6-alkoxy, phenyl, Ci-6-alkyl substituted with at least one fluorine, and Ci-6-alkoxy substituted with at least one fluorine, wherein the Ci-6-alkyl, (CH2) u C3-7-Cycloal-kyl, -O(CH2) uC3-7-cycloalkyl, C2-6-alkenyl, Ci-6-alkoxy and phenyl is optionally substituted with at least one substituent selected from the group consisting of halogen, Ci-6-alkyl, -(CH2) u C3-7-Cycloa I kyl with u = 0, 1, 2, 3 or 4, C2-6-Alkenyl, Ci-6-Alkoxy, Hydroxy-Ci-6-Alkyl, Phenyl, Ci-6-Alkyl substituted with at least one fluorine and Ci-6-Alkoxy substituted with at least one fluorine.

5. Inhibitor for use according to one of claims 3 or 4, characterized in that R4 is selected from the group consisting of C1-6-Al kyll, C3-7-cycloalkyl, -O(CH2)C3-7-cycloalkyl, phenyl, C1-6-Al kyll substituted with at least one fluorine, and Ci-6-alkoxy substituted with at least one fluorine, wherein the phenyl is optionally supplemented with is substituted with at least one substituent selected from the group consisting of halogen, Ci-6-alkyl, Ci-6-alkoxy, Ci-6-alkyl substituted with at least one fluorine and Ci-6-alkoxy substituted with at least one fluorine.

6. Inhibitor for use according to any one of claims 3 to 5, characterized in that R2 is hydrogen, wherein preferably Rs is also hydrogen.

7. Inhibitor for use according to any one of claims 3 to 5, characterized in that R2 is selected from the group consisting of Ci-Ce-alkyl, Cs-Cy-cycloalkyl and phenyl, wherein Ci-Ce-alkyl, Cs-Cy-cycloalkyl and / or phenyl is optionally with at least one halogen.

8. Inhibitor for use according to any one of claims 3 to 5, characterized in that R2 and Rs are Ci-3 alkyl connecting via a ring and Y is -NRsRg.

9. Inhibitor for use according to any one of claims 3 to 8, characterized in that Y -NRsRg is; Rs hydrogen is; Rg C1-6-AI kyl is, wherein the Ci-6-alkyl is optionally substituted with at least one substituent, wherein the at least one substituent is selected from the group consisting of Hydroxy, Rig and -OR24; Rig Phenyl is optionally substituted with at least one substituent, wherein the at least one substituent is selected from the group consisting of halogen, cyano, Ci-6-alkyl, Ci-6-alkoxy, Ci-6-alkyl substituted with at least one fluorine, and Ci-6-alkoxy substituted with at least one fluorine; and R24 is selected from the group consisting of hydrogen and phenyl, wherein the phenyl is optionally substituted with at least one substituent, wherein the at least one substituent is selected from the group consisting of halogen, cyano, nitro, hydroxy, Ci-6-alkyl, Ci-6-alkoxy, with at least one fluorine-substituted Ci-6-alkyl and with at least one fluorine-substituted Ci-6-alkoxy.

10. Inhibitor for use according to any one of claims 3 to 9, characterized in that Ri9 is phenyl, which is optionally substituted with at least one substituent, wherein the at least one substituent is selected from the group consisting of halogen, Ci-6-alkyl and Ci-6-alkoxy; and R24 Phenyl is optionally substituted with at least one substituent, wherein the at least one substituent is selected from the group consisting of halogen, Ci-6-alkyl and Ci-6-alkoxy.

11. Inhibitor for use according to any one of claims 3 to 10, characterized in that R9 is hydrogen or R9 is C1-6-AI kyl, wherein the C1-6-AI kyl is optionally substituted with at least one substituent, wherein the at least one substituent is selected from the group consisting of hydroxy, R19 and -OR24, R19 Phenyl is optionally substituted with at least one substituent, wherein the at least one substituent is selected from the group consisting of halogen, cyano, C1-6-Al alkyl, Ci-6-alkoxy, Ci-6-alkyl substituted with at least one fluorine, and Ci-6-alkoxy substituted with at least one fluorine. R20 is hydrogen or phenyl, which is optionally substituted with at least one substituent, wherein the at least one substituent is selected from the group consisting of halogen, cyano, nitro, hydroxy, C1-6-Al kyl, Ci-6-alkoxy, Ci-6-alkyl substituted with at least one fluorine and C1-6-Al-koxy substituted with at least one fluorine.

12. Inhibitor for use according to any of the preceding claims, characterized in that the Rho GTPase Cdc42 inhibitor contains or consists of a compound with one of the following formulas:

13. Inhibitor for use according to one of the preceding claims, characterized in that the inhibitor is administered to the human or animal body for a period of at least two consecutive days, preferably at least three consecutive days, particularly preferably at least four consecutive days, most preferably at least five consecutive days, in particular at least six consecutive days, optionally at least seven consecutive days.

14. Inhibitor for use according to one of the preceding claims, characterized in that the inhibitor is administered to the human or animal body at a dosage of 5 to 100 mg / kg body weight, preferably 10 to 80 mg / kg body weight, particularly preferably 15 to 60 mg / kg body weight, most preferably 20 to 40 mg / kg body weight, and in particular 25 to 30 mg / kg body weight.

15. Inhibitor for use according to any of the preceding claims, characterized in that the inhibitor is administered to the human or animal body as a single enantiomer, as a mixture of enantiomers, as a pharmaceutically acceptable salt, as a solvate or as a polymorph.

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