Specific tetradentate copper chelators for use in treatment of wilson's disease

Abstract

To provide a more effective method for the treatment of Wilson's disease with reduced side effects.SOLUTION: A method for treating Wilson's disease comprises administering an effective amount of a specific copper chelator of the TDMQ tetradentate copper chelators series (structural formula shown below). In the formula, Y represents a group of the formula: :(CH2)n-NH-(CH2)m-N(CH3)2, wherein n and m are each independently 1, 2, or 3. R5, R6 and R7 are the same or different and each represent a hydrogen atom, chlorine atom, fluorine atom, or trifluoromethyl group.SELECTED DRAWING: Figure 1

Description

[Technical field]

[0001] The present invention relates to the field of Wilson's disease. More specifically, the present invention provides a method for treating Wilson's disease. [Background technology]

[0002] Wilson's disease (WD) is a genetic disorder caused by mutations in the Atp7b gene, which encodes a copper transport protein responsible for excreting excess copper in the liver from the body. Worldwide, this autosomal recessive disorder of copper metabolism affects 1 in 30,000 people. The ATP7B protein transports excess copper from the liver to the bile and then to the feces. When this copper transport protein is deficient, excess copper is released into the blood and slowly excreted from the body through the urine, resulting in the accumulation of copper ions, which leads to chronic hepatitis and cirrhosis. Finally, excess copper is found in the brain, causing neurological disorders (A. Lucena-Valera et al., World J. Hepatol.,13 (6) 634-649, 2021). If untreated, WD can cause death.

[0003] Current treatments include the use of copper chelators to increase copper excretion. Currently, the first-line treatment for WD is D-penicillamine (DPA), which is administered orally to adults at doses of 750–1500 mg daily. Over time, several side effects of DPA have been noted, including nephrotoxicity, regenerative disorders, retinitis, and neurological decline.

[0004] The second drug used to treat WD is trientine (triethylenetetramine), with an oral dose of 900–2700 mg / day for adults. Over time, serious side effects have been noted, including bone marrow suppression, anemia, rash, and hemorrhagic gastritis.

[0005] In the pharmaceutical field, there is a dire need for specific copper chelators that can effectively regulate copper overload at lower doses than DPA and trientine. The main drawback of DPA and trientine is their lack of metal selectivity, as they have similar chelating abilities for both copper and zinc ions. Our research group designed a novel tetradentate ligand compound based on an 8-aminoquinoline scaffold, named TDMQ, which has high selectivity for copper compared with other metal ions such as zinc and has been confirmed as a potential drug for treating Alzheimer's disease (for patents, see Y. Liu et al., China (GCI), 2009). Liu et al., China (GDUT-CNRS), May 27, 2016, CN201610369550.X, Patent No. US10,807,957B2. Granted October 20, 2020. Canada, Patent No. 3025406, Granted June 1, 2021. Japan, Patent No. 6889825, granted May 26, 2021; Europe, Patent No. EP3466931B1, granted January 10, 2024). Because these TDMQ ligands are highly bioavailable (L. Huang et al., Pharmaceutics, 14, 2691, 2022), we decided to evaluate their activity as potential therapeutic agents for Wilson's disease. It should also be noted that these TDMQ ligands have a weak affinity for zinc, so there is good compatibility when these molecules are combined with zinc salts. Zinc salts are also used in the treatment of Wilson's disease to reduce copper absorption in the intestine (see A. Lucena-Valera et al. cited above).

[0006] The chemical synthesis of TDMQ ligands is reported in the above patent (see article W. Zhang et al., ChemMedChem, 13, 864-704, 2018). Among various TDMQ ligands, TDMQ20 was selected as the best drug candidate because it showed suitable pharmacological properties in AD model mice (see J. Zhao et al., ACS Chem.Neurosci.). JPEG2025071035000002.jpg28170General formula of TDMQ compound For TDMQ20, R = 5,7-dichloro - , n=m=2. Summary of the Invention

[0007] In view of the demand in the medical field for more effective copper chelating agents with fewer side effects for the treatment of Wilson's disease, the present invention relates to a specific tetradentate copper chelating agent that provides an effective internal treatment by oral route in Wilson's disease model mice.

[0008] The present invention relates to the treatment of Wilson's disease with a tetradentate specific copper chelator called TDMQ. The present invention provides evidence that TDMQ20 has superior pharmacological activity when administered orally at lower doses compared to D-penicillamine (DPA), the drug currently used to treat Wilson's disease.

[0009] TDMQ20 is superior to D-penicillamine in improving normal / nonpathological copper excretion pathways.

[0010] After treatment with three different doses of TDMQ20, the copper concentration in the liver of mice decreased with increasing drug dose, indicating that the pharmacological action of TDMQ20 is dose-dependent. TDMQ20 (TDMQ-H) reduced the copper concentration in the liver more effectively at a dose of 50 mg / kg / day than D-penicillamine (DPA) at 200 mg / kg / day, the latter currently considered one of the best treatments for WD.

[0011] The fecal copper content of mice treated with TDMQ20 (25 or 50 mg / kg / day) was significantly higher than that of untreated WD mice or mice treated with DPA (200 mg / kg / day), indicating that the excretion of copper in the feces of mice was increased after TDMQ20 treatment. This effect of TDMQ20 was dose-dependent.

[0012] The copper concentration in the serum of WD mice treated with TDMQ20 increased with increasing doses of TDMQ20. The highest dose of TDMQ20 (TDMQ20-H, 50 mg / kg / day) corresponded to the highest copper concentration in serum, confirming the ability of this drug to extract copper from the liver. The copper concentration in the serum of WD mice treated with DPA (200 mg / kg / day) was not significantly elevated compared to untreated WD mice and was much lower than that in the serum of mice treated with TDMQ20 (50 mg / kg). The copper concentration in the urine of WD mice was obviously higher than that of the control group. After treatment with TDMQ20 at a dose of 50 mg / kg, the copper concentration in the urine significantly increased by 1.8-fold compared to WD mice, indicating that the excretion status of copper in the urine was significantly improved.

[0013] The copper concentration in the brains of the mice was also measured. The data showed that the copper concentration in the brains of control mice and mice with WD was similar, and the copper concentration in the brains of WD mice treated with TDMQ20 did not increase, regardless of the dose used. The finding that TDMQ20 does not allow copper to enter the brain is very significant, since WD patients often develop neurological disorders.

[0014] Ceruloplasmin (CP) concentrations were measured in the serum of WD mice after treatment, and CP contains most of the copper in the blood. As expected, CP in WD mice was significantly lower than in control mice. Mice treated with TDMQ20 at 25 or 50 mg / kg (TDMQ20-M and TDMQ20-H) had significantly increased CP levels compared to WD mice. CP levels were comparable (25 mg / kg) or higher than control mice (50 mg / kg). As CP levels are considered an important parameter in the clinical management of Wilson's disease in humans, the reversal of CP damage after TDMQ20 treatment supports the promise of this drug as a potential therapeutic agent for Wilson's disease.

[0015] Screening results of other differentially expressed proteins (DEPs) in the WD mice treatment group compared with the control group showed p<0.05. Compared with the control group, 14 proteins were down-regulated in the liver of WD mice, while 29 liver proteins were up-regulated. In the serum of WD mice, 5 proteins were down-regulated and 5 were up-regulated. After treatment with 25 or 50 mg / kg TDMQ20 (TDMQ20-M group and TDMQ20-H group, respectively), these serum and liver DEPs all returned to normal values. [Brief description of the drawings]

[0016] [Figure 1] Figure 1 shows the effect of oral administration of three different doses of TDMQ20 (12.5 mg / kg, 25 mg / kg, or 50 mg / kg, denoted as L, M, and H, respectively) or DPA (200 mg / kg) on ​​copper concentrations in liver (a), feces (b), brain (c), serum (d), kidney (e), and urine (f) in control, TX (WD) and TX mice. Data analysis was performed using SPSS Statistics v.26. p>0.05 was considered not significant, *p<0.05, **p<0.01, and ***p<0.001, n=6. Graphs were generated using Prism Graph Pad 8.0.2 software. [Diagram 2] The effect of different concentrations of TMT-labeled ceruloplasmin on serum ceruloplasmin detection by LC-MS / MS is shown. *p<0.05, **p<0.01, ***p<0.001. Definitions: WD is the abbreviation for Wilson's disease. DPA is the abbreviation for D-penicillamine, a metal chelator currently used to treat Wilson's disease. TDMQ refers to a series of compounds of formula (I). The term "specific tetradentate copper chelators" refers to a series of compounds of formula (I). TDMQ-L, TDMQ-M, and TDMQ-H represent different doses of TDMQ (12.5, 25.0, and 50.0 mg / kg), respectively. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0017] The technical solutions of the present invention will be described clearly and completely below with reference to the embodiments of the present invention, but it is obvious that the described embodiments are only a part of the embodiments of the present invention, and are not all of the embodiments. Based on the embodiments of the present invention, all other embodiments that can be obtained by those skilled in the art without creative labor fall within the protection scope of the present invention.

[0018] The test methods used in the examples of the present invention are conventional methods unless otherwise specified. The materials, reagents, etc. used are commercially available reagents and materials unless otherwise specified. Example 1: Study of the pharmacological activity of TDMQ compounds on Wilson's disease model mice

[0019] The selected drug candidate TDMQ20 has been evaluated in the homozygous toxic neonatal mouse model (TX model), a conventional mouse model of Wilson's disease suitable for evaluating drug candidates (for an overview, see E. Reed et al., J. Neurochem., 146, 356-373, 2018). TX mice have a naturally occurring point mutation in the ATP7B gene, which changes methionine to valine and causes the loss of copper transport function of the protein. C57BL / 6 mice have no mutation in ATP7B and were used as control mice during TDMQ20 evaluation. TX mice were provided by Sun Yat-sen University (Guangzhou), and C57BL / 6 mice were provided by Guangdong Provincial Laboratory Animal Center (Guangzhou), and all experiments were performed according to a protocol approved by the Guangdong University of Technology Laboratory Animal Welfare Committee (approval number: GDUTXS2022083, approval date: August 3, 2022) and in accordance with the 3R principles.

[0020] Eight-week-old TX mice (WD) and eight-week-old C57BL / 6 mice (control group) were used in the experiment. The TX mice (WD, TDMQ-L, TDMQ-M, and TDMQ-H groups, respectively) were given the drug TDMQ20 (0, 12.5, 25.0, and 50.0 mg / kg, added to 0.9% sodium chloride) by oral gavage twice daily for 14 consecutive days. The C57BL / 6 mice (control group) were administered 0.9% sodium chloride in the same manner. For comparison, WD mice were treated with DPA (200 mg / kg) (DPA group). There were six groups, with six mice in each group. The animal groups and treatments are shown in Table 1. JPEG2025071035000003.jpg88170

[0021] *Definition of six groups of mice (A-F) that received different drug treatments. D-penicillamine dosage was according to JWZhang et al., Biochem. Biochem. Res. Commun., 458, 82-85, 2015. Half the dose shown in the table was administered at 12-hour intervals for forced intragastric administration twice a day. *DPA stands for D-penicillamine.

[0022] After the end of treatment, mice (10 weeks old) were placed in metabolic cages for 12 hours to collect urine and feces. After 12 hours, mice were anesthetized with 2.5% Avertin in 0.9% sodium chloride solution at a dose of 125 ml / 10 g body weight and sacrificed. Cardiac blood was collected and left at room temperature for 30 minutes, and then centrifuged at 4°C for 10 minutes at a rotation speed of 3000 r / min to obtain serum samples. During this 30 minutes, samples of liver, kidney, brain, and serum were also collected. Copper concentrations in urine, feces, serum, liver, kidney, and brain were measured using inductively coupled plasma mass spectrometry (ICP-MS). Data analysis was performed using SPSS statistical software (version 26). Data were expressed as mean ± standard error of the mean (SEM). One-way analysis of variance was used for statistical analysis of liver, feces, and urine, and nonparametric tests were used for statistical analysis of serum, kidney, and brain. Differences p>0.05 were considered not significant (ns), *p<0.05, **p<0.01, ***p<0.001, n = 6. Graphs were drawn with Prism GraphPad 8.0.2.

[0023] The pharmacological results are shown in Table 2 and FIG. JPEG2025071035000004.jpg74170

[0024] The copper concentrations (mean ± SEM of six mice in each group) in the control group mice, TX mice (WD), and TX mice treated with TDMQ20 or DPA were calculated using SPSS statistical software version 26. The results for solids (liver, feces, kidney, brain) are in mg / kg, and those for liquids (urine, serum) are in mg / L.

[0025] Evaluation of the results 1) The copper concentration in the liver of mice treated with three different doses of TDMQ20 (Figure 1a) decreased with increasing drug dose, indicating that the pharmacological action of TDMQ20 is dose-dependent. At 50 mg / kg / day, TDMQ20 (TDMQ-H) was more effective in reducing copper concentration in the liver than D-penicillamine (DPA) at 200 mg / kg / day, which is considered the optimal treatment for WD at present. In particular, it is noteworthy that the copper concentration in the liver of mice treated with 25 mg / kg / day (256 mg / kg) TDMQ20 is close to that of mice treated with 200 mg / kg / day (265 mg / kg) DPA. For similar results, the dose of DPA was 8 times that of TDMQ20, indicating the effect of TDMQ20 is better.

[0026] 2) Mice treated with 25 or 50 mg / kg / day TDMQ20 had significantly higher fecal copper content than untreated WD mice or mice treated with 200 mg / kg / day DPA (Figure 1b). This indicates that the fecal copper excretion of mice was increased after treatment with TDMQ20. This effect of TDMQ20 was dose-dependent.

[0027] 3) The copper concentration in the serum of WD mice treated with TDMQ20 increased with increasing doses of TDMQ20 (Fig. 1d). The highest copper concentration in serum corresponded to the highest dose of TDMQ20 (TDMQ20-H, 50 mg / kg / day), confirming that the drug could extract copper from the liver. The copper concentration in the serum of WD mice treated with DPA (200 mg / kg / day) was not significantly higher than that of untreated WD mice and was much lower than that of mice treated with TDMQ20 (50 mg / kg). These data support that TDMQ20 can transport copper ions to endogenous copper carriers in vivo.

[0028] 4) After treatment with 50 mg / kg TDMQ20, the copper concentration in the kidneys of WD mice was 1.64-fold higher compared to WD mice (Figure 1e). This ratio is very similar to the TDMQ20-H / WD concentration ratio of 1.75 observed in urine (see Figure 1f and data in Table 2). These data indicate that copper ions are excreted through the kidney when WD mice are treated with TDMQ20. Furthermore, this excretion mediated by TDMQ20 was significantly higher than the excretion observed when WD mice were treated with 200 mg / kg DPA.

[0029] 5) As expected, the copper concentration in the urine of WD mice was significantly higher than that of the control group (Figure 1f). After treatment with 50 mg / kg TDMQ20, the copper concentration in the urine of mice was significantly increased by 1.8-fold compared to WD mice, indicating that the copper excretion in the urine of mice was significantly improved.

[0030] 6) We also measured the copper concentration in the brains of the mice (Figure 1c). The data revealed that the copper concentration in the brains of control and WD mice was similar, and that the copper concentration in the brains of WD mice treated with TDMQ20 did not increase regardless of the drug dose. Because WD patients often develop neurological disorders, the finding that TDMQ20 does not bring copper into the brain is very significant.

[0031] Conclusion: Compared with WD mice, treatment of Wilson's disease model TX mice with TDMQ20 can significantly reduce copper concentrations in the liver and significantly increase copper excretion in the feces. This dose-dependent effect indicates that treatment with TDMQ20 can significantly improve normal / non-pathological copper excretion pathways, but this effect was not observed in WD mice treated with 200 mg / kg of the reference drug DPA. Example 2: Differentially expressed proteins in liver and serum of WD mice returned to normal levels after TDMQ20 administration.

[0032] To confirm the positive effect of TDMQ20 in treating TX mice, a proteomics experiment was performed. Table 1 shows samples from three mice in each group. The samples were treated with TMT (tandem mass spectrometry tag) and then quantitatively measured by LC-MS / MS. The data were then analyzed with ProteomeDiscoverer 2.4.1.15 (ThermoFisher Scientific). Reliable proteins were screened based on the criteria that the Sequest program score was greater than 0 and the number of unique peptides was greater than 1, and blank values ​​were removed.

[0033] Serum ceruloplasmin Using TMT LC-MS / MS, we measured the concentration of ceruloplasmin (CP) in the serum of WD mice, which contained most of the blood copper. As expected, it was significantly lower than that of control mice (p<0.05, Figure 2). Mice treated with 25 or 50 mg / kg (TDMQ20-M and TDMQ20-H) of TDMQ20 had significantly increased CP levels compared to WD mice. CP levels were comparable (25 mg / kg) or higher than those of control mice (50 mg / kg). As CP levels are considered an important parameter for the clinical control of Wilson's disease, the recovery of CP damage after TDMQ20 treatment supports the promise of this agent as a candidate for the treatment of Wilson's disease.

[0034] Other proteins Other differentially expressed proteins (DEPs) with p<0.05 were screened in the WD mice group compared to the control group. The expression of 14 proteins was downregulated and the expression of 29 proteins was upregulated in the liver of WD mice compared to the control group. The expression of 5 proteins was downregulated and the expression of 5 proteins was upregulated in the WD mice serum. All these serum and liver DEPs returned to normal values ​​after treatment with TDMQ20 at 25 or 50 mg / kg (TDMQ20-M group and TDMQ20-H group, respectively), and the TDMQ20-H / control ratio approached 1. The results of liver protein and serum protein are shown in Table 3 and Table 4, respectively. JPEG2025071035000005.jpg237170JPEG2025071035000006.jpg81170

[0035] After treatment with TDMQ20 at 0, 25 or 50 mg / kg (WD, TDMQ20-M and TDMQ20-H groups), the list of hepatic DEPs was restored: a) DEPs were down-regulated in WD, and b) DEPs were up-regulated in WD. JPEG2025071035000007.jpg91170

[0036] *After treatment with TDMQ20 at 0, 25 or 50 mg / kg (WD, TDMQ20-M and TDMQ20-H groups), the serum DEPs list was restored. a) DEPs were down-regulated in WD, b) DEPs were up-regulated in WD.

[0037] These results indicate that after administration of 25 or 50 mg / kg of TDMQ20, both up- and down-regulated proteins in the liver and serum of WD mice were restored to normal levels.

[0038] Obviously, the above specific embodiments are merely to further explain the objectives, technical solutions and beneficial effects of the present invention, and it should be understood that the above are merely specific examples of the present invention and do not limit the present invention, and any modifications, equivalent replacements, improvements, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. A method for treating Wilson's disease, comprising administering an effective amount of compound (I). Compound (I) In the molecular formula, Y represents a group of the formula: (CH2) ｎ -NH-(CH2) ｍ -N(CH3) ２ , n and m each represent 1, 2 or 3. R5, R6 and R7 are the same or different and each represent a hydrogen atom, a chlorine atom, a fluorine atom or a trifluoromethyl group.

2. 2. The method of claim 1, wherein n=m=2, R5=R7=Cl, R6=H is the TDMQ molecule of first choice for the synthesis of Wilson's disease drugs.

3. 2. The method according to claim 1, characterized in that the treatment of Wilson's disease with compound (I) can significantly reduce copper concentration in the liver and significantly increase copper excretion in feces and urine.

4. 2. The method of claim 1, wherein the compound (I) does not increase copper levels in the brain during treatment.

5. 2. The method of claim 1, wherein liver and serum proteins can be restored to normal levels.

6. 2. The method according to claim 1, characterized in that after treatment with compound (I), the serum ceruloplasmin level can be restored to normal values ​​in the control group.

7. 13. The method of claim 1, which may be used as a monotherapy or as part of a combination drug therapy, including in combination with a zinc salt.

8. Therapeutic use of a compound according to claim 1 or 2, wherein the drug comprises an acceptable salt of the TDMQ series, the hydrochloride form being the first choice.

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