Use of disulfiram in the manufacture of a medicament for treating neuronal ceroid lipofuscinosis

Abstract

The application discloses a new use of disulfiram, namely, application of disulfiram in preparation of a drug for treating neuronal ceroid lipofuscinosis (NCL). Experimental results show that disulfiram can inhibit expression of non-phosphorylated STAT1 protein or GSDMD protein cleavage, block a neuronal pyroptosis path, and significantly improve pathological performances such as abnormal nerve electrical activity, seizure, motor dysfunction and neuron loss of CLN14 type and CLN2 type NCL. The application further provides a pharmaceutical composition containing disulfiram. Disulfiram is a marketed drug, has clear safety, has low production cost, and has flexible administration modes, and therefore provides a safe and effective new selection for treatment of neuronal ceroid lipofuscinosis.

Description

Technical Field

[0001] This invention belongs to the field of medicine, specifically relating to the application of disulfiram in the preparation of drugs for treating neuronal ceroid lipofuscin deposition. Background Technology

[0002] Disulfiram (chemical name: tetraethylthiuram disulfide, molecular formula: C10H20N2S4, molecular weight: 296.54) is a drug approved by the U.S. Food and Drug Administration (FDA) for the treatment of alcohol dependence. Its known mechanism of action is mainly to inhibit acetaldehyde dehydrogenase in the process of alcohol metabolism, causing a large accumulation of acetaldehyde in the body after drinking, resulting in adverse reactions such as nausea, vomiting, and palpitations, thereby achieving the effect of quitting alcohol. The current dosage form of this product is mainly oral tablets, with a common strength of 250mg / tablet. It has been widely used in the clinical treatment of alcohol dependence, and its safety has been verified through long-term clinical trials. It can be obtained through regular commercial channels (also known as Antabuse, common manufacturers include Odyssey Pharmaceuticals and Norwich Pharmaceuticals; FDA Approval No. NDA 007883, etc.).

[0003] Neuronal ceroid lipofuscinosis (NCL) includes subtypes such as CLN14 and CLN2. CLN14 is caused by a deletion in the KCTD7 gene and is a lethal lysosomal deposition disease, accompanied by progressive myoclonic epilepsy (PME3 subtype). CLN2 is caused by a defect in the TPP1 gene and also manifests as progressive myoclonic epilepsy and progressive neurological damage. Currently, there are no specific drugs for neuronal ceroid lipofuscinosis, including CLN14 and CLN2. Existing clinical treatments are limited to symptomatic relief (such as using antiepileptic drugs to relieve seizures or enzyme replacement therapy, which are expensive) and cannot halt disease progression. Furthermore, long-term use of traditional antiepileptic drugs easily leads to drug resistance and toxic side effects. Summary of the Invention

[0004] Currently, there are no clinically approved drugs for neuronal ceroid lipofuscin deposition disease, especially the disease caused by KCTD7 deficiency or CLN2 deficiency. There is an urgent need to discover its core pathogenesis and develop safe and effective treatment methods.

[0005] The technical solution provided by this invention is as follows: The use of disulfiram in the preparation of drugs for the treatment of neuronal ceroid lipofuscinosis (NCL). Specifically, the neuronal ceroid lipofuscinosis includes CLN14 and / or CLN2 types. More specifically, the CLN14 type is... KCTD7 Caused by gene deletion; the CLN2 type mentioned above is caused by... TPP1 This is caused by a genetic defect. We have made an original discovery about the novel property of disulfiram, which can specifically inhibit the neuronal pyroptosis pathway. Specifically, it inhibits the aggregation of the N-terminal cleavage of the GSDMD protein on the cell membrane surface to form pores, thereby blocking pyroptosis, protecting neurons, and ultimately improving the clinical symptoms of CLN14 and CLN2 types (such as the frequency and duration of myoclonic seizures). This novel property is different from its known pharmacological effect of "inhibiting alcohol metabolism," and this novel property has been verified through brain organoids and mouse models, confirming that it can effectively improve the symptoms of neurological damage and abnormal neuronal discharges in these diseases.

[0006] This invention also provides disulfiram for the preparation of pharmaceutical compositions for treating NCL types CLN14, CLN2, etc. Specifically: Regarding dosage form: Considering the characteristics of drug administration for neurological diseases and the physicochemical properties of disulfiram, the preferred dosage form of the drug described in this application is an oral dosage form (such as tablets, capsules, suspensions), an injectable dosage form (such as intravenous injections, intrathecal injections), or a nasal dosage form (such as nasal sprays); among which, the preferred strengths of oral tablets are 100mg / tablet or 250mg / tablet, and the preferred strengths of intrathecal injections are 5mg / vial or 10mg / vial. The dosage form and strength can be adjusted according to the age, weight, and severity of the condition of the user.

[0007] Regarding usage methods, target users, and usage conditions: ①Target population: Patients diagnosed with CLN14 (caused by KCTD7 gene deletion) or CLN2 (caused by TPP1 gene deficiency) NCL, including children, adolescents and adults; priority is given to patients with neuronal pyroptosis (abnormally elevated GSDMD protein and its splice variant).

[0008] ②Usage: Oral dosage form: The usual adult dose is 250mg once or twice a day. For children, the dose is adjusted to 5-10mg / kg per day according to weight, divided into 2 doses, which can be taken on an empty stomach or after meals; Intrathecal injection: 5-10mg once a week, with the dosage adjusted according to the patient's neurological function and improvement of NCL symptoms; Nasal spray: 1-2 sprays three times a day (each spray contains 5mg of disulfiram), administered directly into the nasal cavity.

[0009] More specific preparation of the drug: Take 100g of disulfiram raw material, add 50g of starch, 30g of lactose and 2g of magnesium stearate, mix evenly, and compress into oral tablets of 100mg / tablet using conventional tableting process, each tablet containing 100mg of disulfiram.

[0010] ③ Conditions of use: During medication, patients' liver and kidney function, blood routine and neurological abnormalities should be monitored. Avoid taking it with alcohol (to prevent acetaldehyde accumulation reaction); for patients with mild liver and kidney insufficiency, the dose should be halved; it is contraindicated in patients with severe liver and kidney insufficiency; pregnant and lactating women should use it with caution. If it is necessary to use it, the benefits should be assessed to outweigh the risks.

[0011] Regarding purity: Disulfiram in this formulation has a purity of ≥98% (pharmaceutical grade purity), with no significant impurities interfering, and meets the quality standards for disulfiram in the United States Pharmacopeia-National Formulary (USP 45-NF 40) or the Chinese Pharmacopoeia (2020 edition), ensuring its pharmacological activity and medication safety.

[0012] The present invention also provides: Use of nonphosphorylated STAT1 inhibitors or GSDMD inhibitors in the preparation of drugs for the treatment of neuronal ceroid lipofuscin deposition.

[0013] A method for screening candidate drugs for the treatment of neuronal ceroid lipofuscin deposition disease, characterized by contacting the candidate substance with a biological model expressing STAT1 protein or GSDMD protein, and detecting the inhibitory effect of the candidate substance on the expression of non-phosphorylated STAT1 or the cleavage of GSDMD protein.

[0014] A kit for the auxiliary diagnosis of neuronal ceroid lipofuscin deposition disease, characterized in that it contains a detection reagent for detecting the level of STAT1 protein or the level of the N-terminal splice of GSDMD protein in biological samples.

[0015] This invention originally discovered that the core pathogenesis of neuronal cerebrolipofus is closely related to neuronal pyroptosis. Specifically, KCTD7 deficiency or CLN2 deficiency leads to abnormally elevated STAT1 protein (a non-canonical pathway mediated by non-phosphorylation), which in turn upregulates the expression of GSDMD protein and its splice variant, triggering neuronal pyroptosis and ultimately causing damage to the nervous system. Based on this, disulfiram, a safe and effective treatment, was developed.

[0016] This invention has many beneficial effects: 1. Repurposing an old drug with guaranteed safety: Disulfiram is an FDA-approved drug for alcohol addiction treatment. It has undergone long-term clinical trials, and its pharmacological characteristics, toxic side effects, and safe dosage are well-defined. Compared with newly developed drugs, it can significantly reduce research and development risks and safety verification costs, resulting in higher patient compliance and a lower likelihood of serious adverse reactions.

[0017] 2. Significant therapeutic effect and strong targeting: This drug can specifically act on the core pathogenesis of NCL (neuronal pyroptosis). By inhibiting the KCTD7-STAT1-GSDMD regulatory axis, it blocks neuronal damage from the root. It can not only improve symptoms such as NCL-related neurological abnormalities, but also improve patients' cognitive and motor functions, delay disease progression, and is applicable to multiple subtypes such as CLN14 and CLN2, with a wide range of applications.

[0018] 3. Low cost and easy to industrialize: Disulfiram has a mature synthesis process, readily available raw materials, and low production technology difficulty, resulting in a significant reduction in production costs compared to entirely new drugs. At the same time, its dosage forms (oral, injection, and nasal administration) can all be prepared using conventional pharmaceutical processes without the need for special equipment, facilitating large-scale industrialization and meeting the needs of large-scale clinical applications.

[0019] 4. Flexible administration methods and wide applicability: It offers a variety of dosage forms and specifications, and can be administered orally, by injection or nasal cavity according to the patient's age (children, adolescents, adults), weight, severity of condition and ease of administration. It is especially suitable for patients with difficulty swallowing or who cannot cooperate with oral administration (such as NCL patients with severe cognitive impairment).

[0020] 5. No obvious toxic side effects and good tolerability: Validated through brain organoids, mice and other models, disulfiram has no significant effect on liver function and blood routine within the therapeutic dose range, and has no obvious toxic side effects. Patients can use it for a long time and it is well tolerated. At the same time, it has no obvious interaction with commonly used clinical drugs, which facilitates combination therapy (such as synergistic treatment with other neuroprotective drugs). Attached Figure Description

[0021] Figure 1 Disulfiram treatment inhibited pyroptosis in KCTD7-deficient brain organoids and improved abnormal neuronal electrical activity. (A) Schematic diagram of the disulfiram treatment experimental procedure. (BC) Effects of disulfiram treatment on the electrical activity of neurons in KCTD7-deficient brain organoids. n=12 / group, each point represents the discharge status of the brain organoid within 5 minutes. (D) Effects of disulfiram treatment on the release of LDH and pyroptosis-related inflammatory factors. *p<0.05, **p<0.01.

[0022] Figure 2Disulfiram treatment inhibited pyroptosis in brain organoids induced by KCTD7 gene deletion and the KCTD7-like behavior of CLN2. (A) Western blot analysis of key protein changes, such as GSDMD, pSTAT1 (phosphorylated at Y701), and non-phosphorylated STAT1, in brain organoids cultured in vitro for 3 months. (C) Protein changes of STAT1, GSDMD, and PSAP (lysosomal deposits) in CLN2 brain organoids.

[0023] Figure 3 Disulfiram treatment can reduce the epileptic phenotype in Kctd7 knockout mice. (A) Schematic diagram of the disulfiram intraperitoneal injection experiment in mice. (B) Disulfiram toxicity test. (CD) Effects of disulfiram treatment on the duration and frequency of seizures in Kctd7 knockout mice. n=7 / group. *p<0.05, **p<0.01. Figure 4 Disulfiram treatment can improve the general condition and motor function of Kctd7 knockout mice. (AB) Effects of disulfiram treatment on body length (A) and weight (B) in Kctd7 knockout mice. n=5 / group. (C) Effects of disulfiram treatment on motor coordination in Kctd7 knockout mice. n=7 / group. (DE) Effects of disulfiram treatment on Gsdmd activation in the hippocampus of mice. n=6 / group. **p<0.01, ****p<0.0001 Figure 5 Disulfiram treatment can reduce the loss of cerebellar Purkinje cells in Kctd7 knockout mice. Detailed Implementation The following specific embodiments further illustrate the implementation of the present invention in detail, but they do not limit the scope of protection of the present invention.

[0024] Example 1 The efficacy of disulfiram-based agents in treating CLN14 and CLN2 neuropathy was systematically verified in vitro and in vivo using brain organoid models and mouse models. Specific experimental details are as follows: (1) Experimental materials: Disulfiram (purity 99%, MedChemExpress, catalog number HY-B0240); CLN14 type brain organoids (derived from iPSC differentiation of patients with KCTD7 deficiency), CLN2 type brain organoids (derived from iPSC differentiation of patients with TPP1 deficiency); CLN14 type mouse model (KCTD7 gene knockout mouse); control drug is solvent.

[0025] The biological or chemical product used in this application is disulfiram, a known drug approved by the FDA (FDA Approval No. NDA 007883), which can be routinely obtained through commercial channels. Common manufacturers include Pfizer Inc. (USA), Sigma-Aldrich Corporation (USA), and Jiangsu Hengrui Medicine Co., Ltd. (China). Its chemical purity meets pharmaceutical standards (≥98%). Relevant product information can be found in references (e.g., PubMed IDs: 36978391 and 29083801, which describe the pharmacological properties and clinical applications of disulfiram). The public can purchase it from the above-mentioned manufacturers or obtain it through regular pharmaceutical suppliers. It can be used for the preparation of the drug described in this invention without any special preparation process.

[0026] Pharmaceutical preparation: Take 100g of disulfiram raw material (purity 99%, Sigma-Aldrich), add 50g of starch, 30g of lactose and 2g of magnesium stearate, mix evenly, and compress into oral tablets of 100mg / tablet using conventional tableting process. Each tablet contains 100mg of disulfiram and meets the quality standards of the United States Pharmacopeia (USP) 45-NF 40.

[0027] (2) Test subjects: ① Brain organoid models: CLN14 type brain organoids (divided into control group and disulfiram group, n=12 in each group); CLN2 type brain organoids (n = 12, grouped the same as CLN14 type); normal brain organoids (n = 12, blank control).

[0028] ② Mouse model: CLN14 mice (6-8 weeks old, half male and half female, grouped as above, n = 7 per group); normal C57BL / 6 mice (n = 7, blank control).

[0029] (3) Test method: ① Brain organoid experiment: The control group was given a culture medium without disulfiram, while the CLN14 type brain organoid was given a culture medium containing 10 μM disulfiram. After 3 days of culture, the amplitude, number of discharges and frequency of abnormal neural discharges were detected by in vitro microelectrode array (MEA) technology (AxionBioSystems).

[0030] ② Mouse experiment: The control group was given saline by gavage, while each mouse in the disulfiram group was given 50 mg / kg of disulfiram preparation (based on the effective content of disulfiram) by gavage; twice a week for 4 consecutive weeks; during the administration period, the occurrence and duration of abnormal neurological attacks in mice were recorded by video monitoring; after the administration period, mouse brain tissue was taken to detect the expression of pyroptosis-related proteins and neuronal survival, and the motor function of mice was detected by Rotarod and other tests.

[0031] (4) Experimental results: ① Brain organoid experiment: A systematic evaluation of the in vitro therapeutic effects of disulfiram. KCTD7 During the 8th week of in vitro culture of defective brain organoids, disulfiram was added to the culture medium for drug intervention. The specific experimental grouping and treatment procedures are as follows: Figure 1 As shown in Figure A, the firing patterns of neurons in each group of brain organoids were recorded using a microelectrode array (MEA). The results showed that the number and frequency of spontaneous firing of neurons in the disulfiram-treated organoids were significantly lower than those in the model group, and approached the normal levels of wild-type organoids, suggesting that disulfiram can effectively improve... KCTD7 The defect-induced abnormal neuronal electrical activity alleviates the imbalance of neural network excitability. Figure 1 BC). Meanwhile, lactate dehydrogenase (LDH) release assays and enzyme-linked immunosorbent assays (ELISA) showed that after disulfiram intervention, the release of pyroptosis-related inflammatory factor IL-1β and the cell damage marker LDH were significantly reduced. Figure 1 D). The above in vitro experimental results fully confirm that disulfiram can... KCTD7 In defective brain organoid models, it effectively inhibits the neuronal pyroptosis process while improving neuronal electrophysiological function, demonstrating in vitro neuroprotective and anti-epileptic effects.

[0032] Compared with the control group, the expression levels of GSDMD-N and non-phosphorylated STAT1 (without phosphorylation at the Y701 site) proteins were significantly increased in CLN14 brain organoids; GSDMD-N was significantly decreased after DSF treatment. Since brain organoids are mainly composed of neurons, this indicates that the neuronal pyroptosis pathway was significantly inhibited. Figure 1 A). KCTD7 levels are decreased in CLN2-type brain organoids, while STAT1 and GSDMD levels are significantly increased, suggesting that the KCTD7-STAT1-GSDMD signaling pathway also plays a role in the pathological process of CLN2. Figure 2 B).

[0033] ② Mouse experiment: The specific experimental grouping and treatment procedures are as follows Figure 3 As shown in A. Disulfiram treatment had no obvious toxic side effects (liver function) ( Figure 3 B). Compared with the control group, the frequency of neurological abnormalities in CLN14 mice was significantly reduced in the disulfiram-treated group ( p <0.01, and the duration of the attack was significantly shortened ( p <0.05)( Figure 3 (C and D). Furthermore, the DSF-treated mice showed a significant improvement in the stunted growth phenotype, with both body length and weight exceeding those of the untreated mice. Kctd7 / The group is close to the level of wild-type mice. Figure 4 A and 4B). Motor function in mice (Rotarod dwell time increased by 30%-40%) was significantly improved. p <0.0001)( Figure 4 C). At a further molecular level, both the full-length and splice variant of GSDMD, a key protein molecule for pyroptosis, were significantly downregulated in brain tissue. p <0.01)( Figure 4 D and 4E). The loss of cerebellar Purkinje cells was significantly alleviated, and the number of positive cells significantly increased (D and 4E). Figure 5 ).

[0034] (5) Experimental conclusions: The agent prepared by disulfiram can significantly reduce the neuronal pyroptosis rate in CLN14 and CLN2 related models (brain organoids, mice) by inhibiting the assembly of GSDMD splice body on the cell membrane and the activation of the pyroptosis pathway, and effectively improve the neurological symptoms and nervous system damage (cognitive and motor functions) of NCL. At the same time, as an FDA-approved drug, disulfiram has good safety and no obvious toxic side effects were observed in the two models, which confirms that it has outstanding therapeutic effects in the treatment of CLN14, CLN2 and other types of NCL, and can be used as an effective treatment drug for this type of disease.

Claims

1. Application of disulfiram in the preparation of drugs for the treatment of neuronal ceroid lipofuscin deposition syndrome.

2. The application according to claim 1, characterized in that, The neuronal cerebrolipofuscinosis includes CLN14 and / or CLN2 types.

3. The application according to claim 2, characterized in that, The CLN14 type is caused by the deletion of the KCTD7 gene; the CLN2 type is caused by the defect of the TPP1 gene.

4. A pharmaceutical composition for treating neuronal ceroid lipofuscin deposition syndrome, characterized in that, It contains disulfiram as the active ingredient, as well as pharmaceutically acceptable excipients.

5. The pharmaceutical composition according to claim 4, characterized in that, The dosage form of the pharmaceutical composition is selected from oral, injectable, or nasal dosage forms.

6. The pharmaceutical composition according to claim 5, characterized in that, The oral tablets are available in units of 100 mg / tablet or 250 mg / tablet; the intrathecal injections are available in units of 5 mg / vial or 10 mg / vial.

7. The pharmaceutical composition according to claim 5, wherein the oral tablet comprises: 100 parts disulfiram, 50 parts starch, 30 parts lactose, and 2 parts magnesium stearate, by weight.

8. Application of non-phosphorylated STAT1 inhibitors or GSDMD inhibitors in the preparation of drugs for the treatment of neuronal ceroid lipofuscin deposition disease.

9. A method for screening candidate drugs for the treatment of neuronal ceroid lipofuscinosis, characterized in that, This includes contacting candidate substances with biological models expressing STAT1 or GSDMD proteins and detecting the inhibitory effect of the candidate substances on the expression of unphosphorylated STAT1 or the cleavage of GSDMD proteins.

10. A kit for the auxiliary diagnosis of neuronal ceroid lipofuscin deposition disease, characterized in that, It includes reagents for detecting the levels of STAT1 protein or the N-terminal splice of GSDMD protein in biological samples.

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