Pharmaceutical composition for treating parkinsonism

Abstract

The present invention belongs to the technical field of bio-pharmaceuticals, and particularly relates to a pharmaceutical composition for treating parkinsonism. Research in the present invention shows that Antarctic fish proteins and multi-domain derivatives thereof have the functions of alleviating PD pathology and protecting dopaminergic neurons.

Description

A pharmaceutical composition for treating parkinsonism TECHNICAL FIELD

[0001] The present application belongs to the technical field of biological medicine, and particularly relates to a pharmaceutical composition for treating parkinsonism. BACKGROUND

[0002] Parkinson disease (PD) is the second most common central nervous system degenerative disease after Alzheimer's disease, which mainly occurs in the elderly. With the aging of the population, the incidence of PD is increasing. At present, the etiology of these diseases is unknown and cannot be cured, which poses a serious threat to human health and causes huge economic burden. Its pathological feature is the aggregation of alpha-synuclein in multiple nervous systems and peripheral systems, but its distribution and type are different, and it may also be affected by other pathologies or cause similar manifestations. Before this, the disease was mainly considered as a movement disorder, and the typical characteristics include static tremor, motor retardation, posture instability, and rigidity of neck, trunk and limbs, etc. However, PD is now considered as a disease with significant neuroinflammation and immune dysfunction. Almost all parkinsonism is affected by genetic factors. More than 100 genes or genetic loci have been found, and most cases may be caused by the interaction of multiple common and rare genetic variations. These genetic variations involve pathogenic genes of early-onset and late-onset parkinsonism, such as PARKIN, PINK1, DJ-1, SNCA and LRRK2, and some susceptibility genes, such as GBA, TMEM175, GAK and HTRA2, etc.

[0003] Genetic studies have revealed some unified biological themes, including synapses, lysosomes, mitochondria and immune-mediated mechanisms. There are interactions and regulations between these mechanisms, which affect the function and survival of neurons. For example, alpha-synuclein can affect synaptic release, mitochondrial respiration, lysosomal degradation and inflammatory response, while LRRK2 can regulate lysosomal function, mitochondrial dynamics, autophagy and immune signals, etc. Due to the heterogeneity of parkinsonism, more biomarkers need to be developed to diagnose, stratify, monitor and predict the progression and treatment response of the disease. At present, some biomarkers have been used in clinical or research, such as alpha-synuclein in cerebrospinal fluid, dopamine transporter and neuron-specific enolase in neuroimaging, etc. In addition, some strategies for targeted treatment are also being developed, such as inhibiting the aggregation of alpha-synuclein, improving mitochondrial function, activating lysosomal enzymes, regulating the activity of LRRK2, etc.

[0004] Many studies have shown that the antifreeze proteins of Antarctic fish can protect plants and animals from low temperature stress, including protecting the integrity of cell membranes, regulating ion balance (including calcium and potassium ions) and regulating ROS levels, etc. Studies have shown that the antifreeze proteins of Antarctic fish can also maintain intracellular ATP levels and improve cell viability. ATP metabolism is also closely related to the rate of brain activity, and lower neuronal activity is associated with lower energy consumption. The inhibition of ion currents by antifreeze proteins has an impact on neuronal function, such as the generation of action potentials, synaptic transmission, and potential neuroprotection under conditions of excessive ion movement, such as cerebral hypoxia / ischemia and cold / re-warming injury. There are also studies showing that AFPI has the ability to reversibly inhibit neural network activity without affecting the ability of neurons to survive, which can play a protective role in the process of re-warming of hippocampal neurons at low temperature. The protective effect on cells may be related to the inhibitory effect of AFPI, which may also provide a neuroprotective mechanism - due to the lack of energy consumption, intracellular ATP levels are maintained, and neurons may be able to better survive the cold and re-warming process.

[0005] In our previous studies, we found that the pathogenic gene α-syn gene of PD is adjacent to the AFPIII gene in the genome of Antarctic fish, indicating that AFPIII and α-syn may have a certain regulatory relationship.

[0006] SUMMARY

[0007] The present application studies show that Antarctic fish proteins and their multi-domain derivatives have the function of reducing PD pathology and protecting dopaminergic neurons, thereby completing the present application.

[0008] In a first aspect, the present application provides a pharmaceutical composition for treating Parkinson's syndrome, the pharmaceutical composition containing an Antarctic fish protein and its derivative, the Antarctic fish protein being selected from one or more of CYN-1, CYN-2, CYN-3 and CYN-4 proteins, the nucleotide sequences of the CYN-1, CYN-2, CYN-3 and CYN-4 proteins being respectively as shown in SEQ ID NO: 1 to SEQ ID NO: 4, and the amino acid sequences thereof being respectively as shown in SEQ ID NO: 5 to SEQ ID NO: 8, wherein;

[0009] The nucleotide sequences of CYN-1 to CYN-4 are:

[0010] The amino acid sequences of CYN-1 to CYN-4 are:

[0011] The derivative of the CYN-1, CYN-2, CYN-3 and CYN-4 proteins refers to having at least 80% homology with the amino acid sequence of SEQ ID NO: 5-8 of the proteins, further preferably having at least 85% homology; further preferably having at least 90% homology; further preferably having at least 95% homology; further preferably having at least 97% homology; further preferably having at least 98% homology; further preferably having at least 99% homology.

[0012] Further, the derivative of the CYN-1, CYN-2, CYN-3 and CYN-4 proteins refers to having 1-50 amino acids deleted, added and / or mutated in the amino acid sequence of SEQ ID NO: 5-8 of the proteins, and the deletion, addition and / or mutation site is continuous or discontinuous. Further, the deleted, added and / or mutated amino acids are 1-40, 1-35, 1-30, 1-25, 1-20, 1-15, 1-10, 1-5, 2, 3, 4 or 5.

[0013] Further, the Antarctic fish protein is preferably a CYN-4 protein.

[0014] Further, the pharmaceutical composition further contains other active ingredients, which are drugs different from the Antarctic fish protein and capable of promoting the improvement of the body and / or treating the symptoms of Parkinson's syndrome.

[0015] Further, the pharmaceutical composition can be prepared into sugar-coated tablets, film-coated tablets, enteric-coated tablets, capsules, hard capsules, soft capsules, oral liquids, oral tablets, granules, instant powders, pills, beads, suspensions, powders, wine, preparations, drops, injection solutions, powder injections, creams, sustained-release preparations, targeted preparations, etc.

[0016] Further, the administration mode of the pharmaceutical composition includes oral administration, injection, implantation, external use, spraying or inhalation.

[0017] In the second aspect, the application provides an application of an Antarctic fish protein in the preparation of a drug for treating Parkinson's syndrome, wherein the Antarctic fish protein is selected from one or more of CYN-1, CYN-2, CYN-3 and CYN-4 proteins, and the nucleotide sequence of the CYN-1, CYN-2, CYN-3 and CYN-4 proteins is shown in SEQ ID NO: 1-SEQ ID NO: 4, and the amino acid sequence is shown in SEQ ID NO: 5-SEQ ID NO: 8.

[0018] The derivatives of the CYN-1, CYN-2, CYN-3 and CYN-4 proteins refer to at least 80% homology with the amino acid sequences of SEQ ID NO: 5-8 of the proteins, further preferably at least 85% homology; further preferably at least 90% homology; further preferably at least 95% homology; further preferably at least 97% homology; further preferably at least 98% homology; further preferably at least 99% homology.

[0019] Further, the derivatives of the CYN-1, CYN-2, CYN-3 and CYN-4 proteins refer to 1-50 amino acids of deletion, addition and / or mutation in the amino acid sequences of SEQ ID NO: 5-8 of the proteins, and the positions of the deletion, addition and / or mutation are continuous or discontinuous. Further, the deleted, added and / or mutated amino acids are 1-40, 1-35, 1-30, 1-25, 1-20, 1-15, 1-10, 1-5, 2, 3, 4 or 5.

[0020] Further, the proteins and their derivatives of the Antarctic fish can reduce the phosphorylation level and aggregation of alpha-Syn in Parkinson's syndrome patients.

[0021] Further, the proteins and their derivatives of the Antarctic fish can reduce the TH cell damage in Parkinson's syndrome patients. BRIEF DESCRIPTION OF DRAWINGS

[0022] Figure 1: Antarctic fish CYN protein reduces the phosphorylation level and aggregation of alpha-Syn in A53T cells (A expression plasmid map of Antarctic fish CYN protein; B pSyn immunofluorescence staining in A53T cells overexpressing Antarctic fish CYN; C quantitative statistics of pSyn aggregation points in cells);

[0023] Figure 2: Antarctic fish CYN-1 and CYN-4 proteins can improve the motor ability of alpha-Syn PFF-induced mouse PD model (A construction mechanism of transgenic mice, B anti-freeze protein CYN-1 improves the motor ability of alpha-Syn PFF-injected mice, C anti-freeze protein CYN-4 improves the motor ability of alpha-Syn PFF-injected mice);

[0024] Figure 3: Antarctic fish CYN-1 protein reduces the over-phosphorylation level and aggregation of alpha-Syn in different brain regions of mice (A p_Syn immunostaining of brain sections after unilateral injection of PFF in striatum for 3 months; B quantitative determination of p_Syn intensity in each brain region after immunofluorescence staining of brain sections of mice after unilateral injection of PFF in striatum for 3 months (N=3-4 mice per group));

[0025] Figure 4: Antarctic fish CYN-4 protein reduces the over-phosphorylation level and aggregation of a-Syn in different brain regions of mice (A p_Syn immunostaining of brain sections after unilateral injection of PFF in the striatum for 3 months, B quantification of p_Syn intensity in each brain region after immunofluorescence staining of brain sections of mice after unilateral injection of PFF in the striatum for 3 months (N=3-4 per group));

[0026] Figure 5: Antarctic fish CYN protein reduces TH cell damage induced by a-Syn PFF (A TH immunofluorescence staining of SN-DA neurons after a-Syn PFF injection in WT, CYN-1 and CYN-4 mice for 3 months; B TH positive neuron cell count). DETAILED DESCRIPTION

[0027] The Parkinson's syndrome involved in the present application includes "resting tremor", which is often the first symptom and usually starts in the distal part of the upper limb on one side, appears in a resting position or is more obvious. When the disease occurs, the thumb and the flexed index finger form a "marble rolling" action.

[0028] The "muscle rigidity" refers to the state of the patient's limbs similar to a curved soft lead pipe, which is called "lead pipe rigidity"; in patients with resting tremor, intermittent and intermittent tremor can occur, like rotating gears, which is called "gear rigidity". In severe cases, patients can have special flexion positions or postures, and even cannot take care of themselves.

[0029] The "bradykinesia" refers to the slow movement of the patient's fingers in early stage, such as unbuttoning or buttoning, tying shoelaces, etc., which is particularly obvious.

[0030] The "postural balance disorder" refers to the appearance in the late stage of the disease, which is manifested as difficulty in standing up and easy to fall backward. Sometimes, after taking a step, the patient walks faster and faster with very small steps, and cannot stop in time, which is called "forward gait" or "flurry gait".

[0031] The "sensory disorder" refers to the possible reduction of olfaction in the early stage of the patient, and the numbness and pain of the limbs in the middle and late stages of the disease.

[0032] The "sleep disorder" refers to the patient's frequent dreams at night, accompanied by loud shouting and limb dancing.

[0033] The "autonomic nervous dysfunction" refers to the possible constipation, excessive sweating, urination disorder, postural hypotension, etc. of the patient.

[0034] The "mental disorder" refers to about 50% of patients with depression, and often accompanied by anxiety. In the late stage of the disease, the patient has cognitive impairment, even dementia, and visual hallucinations, i.e. hallucinations.

[0035] In order to make the objectives, technical solutions and advantages of the present application clearer, further detailed description will be made to the present application in combination with specific examples, but it should not be understood that the present application is limited to the following examples only.

[0036] The experimental methods in the following examples are all conventional methods, and the experimental materials used in the following examples are all commercially available, unless otherwise specified.

[0037] Example 1 Expression of Antarctic fish CYN protein in A53T cells and pathological detection

[0038] A53T is a point mutation of alpha-syn, which is closely related to the pathogenesis and related dysfunction of Parkinson's disease. A53T cell line is a stable hereditary cell line created by transgenic technology, which overexpresses human alpha-synuclein with Parkinson's disease related mutation (A53T).

[0039] The A53T cell line was cultured with DMEM medium, and the Antarctic fish CYN protein expression plasmid was transfected into the A53T cells with lipo3000 transfection reagent (Invitrogen). After 12h of transfection, in vitro synthesized a-SynPFFs (5μg / mL) were added to the cells, and treated for 48h. Cell immunofluorescence was used to detect the level of alpha-syn phosphorylation (AB_2564891, Abeam) and quantitative analysis was performed.

[0040] Firstly, the cells were washed with PBS for 3 times, then fixed with 4% PFA for 15 minutes, and washed with PBS for 2 times. Next, 0.5% Triton was used for 15 minutes, and 2% BSA was used for blocking for 1 hour. Alpha-syn phosphorylation antibody (AB_2564891, Abeam) was added and incubated at room temperature for 1 hour, and washed with PBS for 3 times, then incubated with secondary antibody (Alexa Fluor 594, Invitrogen) for 1 hour and hoechst for 5 minutes. Finally, after washing with PBS for 3 times, the slide was mounted and observed. Fluorescence images were taken using confocal scanning microscope (LSM710, Carl Zeiss). All images were processed by Carl Zeiss Zen software (Carl Zeiss), and the fluorescence intensity level was evaluated by ImageJ.

[0041] Results are shown in Figure 1. The CYN protein of Antarctic fish is obtained by tandem repeat of different numbers of AFPIII domains (see Figure 1A). The CYN protein expression plasmid of Antarctic fish was transfected into A53T cells using a chemical transfection reagent, and treated with PFF for 48 h. The phosphorylation level and aggregation of a-Syn were detected. The results showed that the expression of Antarctic fish CYN can significantly reduce the phosphorylation level and aggregation of a-Syn (Figures 1B-C), indicating that the CYN protein can reduce the phosphorylation level of a-Syn and inhibit the aggregation of a-Syn in vitro.

[0042] Example 2 Establishment of Antarctic fish CYN gene transgenic mice and analysis of motor ability and pathological detection

[0043] To obtain mice expressing Antarctic fish CYN protein, the CYN gene was inserted into the H11 site of WT (C57BL6) mice by microinjection technology. These mice were cultured according to the standard feeding scheme. At 12 weeks of age, in vitro expressed a-syn PFF was injected into the dorsal striatum of WT (C57BL6) and CYN gene transgenic heterozygous mice (AP+0.8mm, ML+2.0mm, DV-4.5mm) by stereotactic brain injection. After unilateral single injection of a-syn PFF in the striatum, the motor function of WT and transgenic mice (N=7 and 10 mice) was evaluated at 3 months (3mpi), including the rotarod fatigue test and the hanging test. The results were plotted and analyzed using GraphPad Prism 9.

[0044] Results: To further verify the effect of Antarctic fish CYN on Parkinson's disease in vivo, transgenic mice with Antarctic fish CYN gene inserted into the H11 site were constructed (see Figure 2A). Three-month-old transgenic heterozygous mice were injected with exogenous PFF by stereotactic brain injection into the striatum, and their motor function was evaluated after continuous feeding for 3 months, including the rotarod fatigue test and the hanging test. The results showed that mice expressing Antarctic fish CYN-1 and CYN-4 proteins had significantly stronger fatigue resistance and hanging ability compared to WT mice (Figures 2B-C). This indicates that Antarctic fish CYN protein has a protective function for PD model mice.

[0045] Example 3 Establishment of Antarctic fish CYN gene transgenic mice and analysis of motor ability and pathological transformation detection

[0046] To confirm the pathological conversion, over-phosphorylated a-syn (pSyn) deposits, a marker of Lewy bodies, were detected after a single injection of 3 mpi of PFFs. Briefly, whole brains were perfused and fixed with 4% PFA for 24 h, and then dehydrated with 30% sucrose solution. Immunofluorescence (IF) detection was performed on 30 pm-thick serial brain cryosections. Primary and working dilutions: p-a-syn ser129 (AB_2564891, Abeam) 1:500; Th (ab_10077691, Abeam) 1:1000. For immunofluorescence studies, primary antibody-labeled sections were incubated with Alexa-fluor 488 or 594-conjugated secondary antibodies (Invitrogen). Fluorescence images were taken using a confocal scanning microscope (LSM710, Carl Zeiss). All images were processed by Carl Zeiss Zen software. ImageJ was used to evaluate TH-neurons total number and over-phosphorylated a-Syn (pSyn) deposits levels.

[0047] Results: CYN protein from Antarctic fish reduces a-Syn phosphorylation and aggregation in different brain regions of mice. After 3 months of unilateral injection of PFFs in the striatum, pathological conversion was detected by immunofluorescence of a-Syn phosphorylation. The results showed that in the different brain regions of WT mice, a-Syn over-phosphorylation pathology appeared, and dense Lewy body-like aggregates were observed (Figure 3A). However, the a-Syn over-phosphorylation pathology was lighter and the Lewy body-like aggregates were fewer in CYN-1 mice (Figure 3A-B), and the a-Syn over-phosphorylation pathology was even lighter in CYN-4 mice (Figure 4A-B). The above results indicate that CYN protein from Antarctic fish reduces a-Syn phosphorylation and aggregation in different brain regions of mice, thereby alleviating the pathological symptoms of PD.

[0048] Results: CYN protein from Antarctic fish reduces TH cell damage induced by a-Syn PFFs. PFF injection in the striatum leads to a-Syn pathology in dopamine (SN_DA) neurons in the substantia nigra region. In WT mice, a-Syn pathology in the substantia nigra region was accompanied by loss of TH-positive neurons (Figure 5A), indicating that the production of a-Syn aggregates within neurons leads to DA neuron loss as previously reported. In contrast, a-Syn pathology and loss of TH-positive neurons were well alleviated in CYN-1 and CYN-4 mice. At the same time, CYN-4 showed stronger protection of SN_DA neurons in mice than CYN-1 mice (Figure 5B).

[0049] The above results indicate that CYN protein from Antarctic fish has a protective effect on TH-positive neuron loss caused by PFF injection in the striatum.

Claims

1. A pharmaceutical composition for treating Parkinson's syndrome, comprising an Antarctic fish protein selected from one or more of CYN-1, CYN-2, CYN-3 and CYN-4 proteins, the nucleotide sequences of which are shown in SEQ ID NO: 1-4, respectively, and the amino acid sequences of which are shown in SEQ ID NO: 5-8, respectively.

2. The pharmaceutical composition of claim 1, wherein, The derivative of the CYN-1, CYN-2, CYN-3 and CYN-4 proteins refers to having at least 80% homology, further preferably at least 85% homology; further preferably at least 90% homology; further preferably at least 95% homology; further preferably at least 97% homology; further preferably at least 98% homology; further preferably at least 99% homology with the amino acid sequences of SEQ ID NO: 5-8.

3. The pharmaceutical composition of claim 1 or 2, wherein, The derivative of the CYN-1, CYN-2, CYN-3 and CYN-4 proteins refers to having 1-50 amino acids deleted, added and / or mutated in the amino acid sequences of SEQ ID NO: 5-8, the positions of the deletion, addition and / or mutation being continuous or discontinuous; further, the number of the deleted, added and / or mutated amino acids is 1-40, 1-35, 1-30, 1-25, 1-20, 1-15, 1-10, 1-5, 2, 3, 4 or 5.

4. The pharmaceutical composition of any one of claims 1-3, wherein the Antarctic fish protein is preferably CYN-4 protein.

5. The pharmaceutical composition of any one of claims 1-3, further comprising other active ingredients, which are drugs different from the Antarctic fish protein and capable of promoting the improvement of the body and / or treating the symptoms of Parkinson's syndrome.

6. Use of an Antarctic fish protein in the preparation of a drug for treating Parkinson's syndrome, the Antarctic fish protein being selected from one or more of CYN-1, CYN-2, CYN-3 and CYN-4 proteins, the nucleotide sequences of which are shown in SEQ ID NO: 1-4, respectively, and the amino acid sequences of which are shown in SEQ ID NO: 5-8, respectively.

7. Use according to claim 6, wherein, Derivatives of the CYN-1, CYN-2, CYN-3 and CYN-4 proteins mean at least 80% homology to the amino acid sequence SEQ ID NO: 5-8 of the proteins, further preferably at least 85% homology; further preferably at least 90% homology; further preferably at least 95% homology; further preferably at least 97% homology; further preferably at least 98% homology; further preferably at least 99% homology.

8. Use according to claim 6 or 7, wherein, Derivatives of the CYN-1, CYN-2, CYN-3 and CYN-4 proteins mean 1-50 amino acids are deleted, added and / or mutated in the amino acid sequence SEQ ID NO: 5-8 of the proteins, the deletion, addition and / or mutation site is continuous or discontinuous; further, the deleted, added and / or mutated amino acids are 1-40, 1-35, 1-30, 1-25, 1-20, 1-15, 1-10, 1-5, 2, 3, 4 or 5.

9. Use according to any one of claims 6-8, wherein the Antarctic fish protein is preferably a CYN-4 protein.

10. Use according to any one of claims 6-8, wherein the Antarctic fish protein and derivatives thereof can reduce the phosphorylation level and aggregation of alpha-Syn in Parkinson's syndrome patients, reduce TH cell damage.

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