CN118178408A

CN118178408A - Application of PTC124 in preparation of medicine for treating thrombocytopenia

Info

Publication number: CN118178408A
Application number: CN202410334462.0A
Authority: CN
Inventors: 姜江; 谢展利
Original assignee: Nuclear Industry General Hospital
Current assignee: Nuclear Industry General Hospital
Priority date: 2024-03-22
Filing date: 2024-03-22
Publication date: 2024-06-14

Abstract

The invention discloses an application of PTC124 in the biomedical technical field in preparing a medicament for treating thrombocytopenia, aiming at solving the problems that the relative content of full-length protein is increased by nonsense inhibitory medicaments in the prior art, but the medicaments have larger ototoxicity and nephrotoxicity, limit the application and the like, and the invention provides that the PTC124 can be used as an active ingredient in the medicament for treating the thrombocytopenia.

Description

Application of PTC124 in preparation of medicine for treating thrombocytopenia

Technical Field

The invention relates to an application of PTC124 in preparing a medicament for treating thrombocytopenia, and belongs to the technical field of biological medicines.

Background

Thrombocytopenia (Glanzmann's thrombasthenia, GT) is a severe hemorrhagic disease, patients often develop self-bleeding, persisting for life, and can be manifested as colporrhagia, epistaxis, gastrointestinal hemorrhage, luteal rupture hemorrhage, etc., even intracranial hemorrhage occurs, leading to death. The pathological mechanism of the thrombocytopenia is caused by the defect of glycoprotein alpha IIb beta 3 on the surface of platelet membrane due to the mutation of ITGA2B or ITGB3 genes of chromosome 17, and further the platelet aggregation dysfunction. There is currently no effective treatment for thrombocytopenia caused by nonsense mutations.

The database (https:// glanzmann. Mcw.edu /) shows that 67% of the mutation sites leading to GT are located in the ITGA2B gene and 33% are located in the ITGB3 gene. Thus, ITGA2B gene mutation is the main causative agent of thrombocytopenia.

Nonsense mutations that create premature stop codons (Premature termination codon, PTC) can lead to three molecular defects: (1) producing a truncated protein (Truncated protein); (2) degrading the PTC-containing transcript via the NMD pathway; (3) PTC-containing exons are removed by nonsense related alternative splicing (nonsense-associated ALTERED SPLICING, NAS) pathways, altering the way of splicing. All three cause defects in the encoded protein or amount, resulting in a disease phenotype. Wherein NMD plays a key role in the pathological mechanism of nonsense mutations.

NMD is an important post-transcriptional monitoring mechanism formed by eukaryotic cells during evolution. The abnormal mRNA containing the PTC is identified and degraded through various cell components, so that the accumulation of truncated proteins is effectively avoided, and the organism is protected. However, NMD is a double edged sword. Its decreased efficiency can lead to disease development and excessive effects can also exacerbate the disease phenotype. Changes in NMD efficiency may affect the pathological mechanisms of genetic diseases.

Increasing the relative content of full-length proteins by nonsense suppression drugs is a potential therapeutic strategy for diseases caused by nonsense mutations. Aminoglycoside antibiotics, such as gentamicin, G418, and the like, are the nonsense inhibitory drugs reported earlier. However, due to their greater ototoxicity and nephrotoxicity, the clinical application of such drugs is limited.

Disclosure of Invention

The invention aims to overcome the defects in the prior art and provides an application of PTC124 in preparing a medicament for treating the thrombocytopenia, wherein the PTC124 can be used as an active ingredient in the medicament for treating the thrombocytopenia.

In order to achieve the above purpose, the invention is realized by adopting the following technical scheme:

in a first aspect, the present invention provides the use of PTC124 in the manufacture of a medicament for the treatment of thrombocytopenia, said PTC124 being an active ingredient of a medicament for the treatment of thrombocytopenia.

Further, the application concentration of the PTC124 is 700-900 mg/kg.

Further, the optimal application concentration of the PTC124 is 800mg/kg. D.

Further, the PTC124 is non-cytotoxic and acts by increasing the level of cell surface aIIbb3 by increasing the expression of the mutant megakaryocyte line ITGA2B protein.

In a second aspect, the present invention provides a medicament for treating thrombocytopenia, comprising PTC124 and pharmaceutical excipients, wherein PTC124 is used as an active ingredient of the medicament for treating thrombocytopenia.

Optionally, the pharmaceutical excipients comprise a pH regulator, an osmotic pressure regulator, a solubilizer, a cosolvent, an emulsifier, a stabilizer, a preservative, a filler, a disintegrating agent, an adhesive, a flavoring agent and a lubricant.

Optionally, the pharmaceutical dosage form for treating the thrombocytopenia comprises the form of powder, granules, tablets, capsules, pills, syrup, powder, solution, oral liquid, suspension or injection.

Optionally, the pharmaceutical formulation for treating the thrombocytopenia is injection.

Alternatively, the injection is administered by injection, nasal administration, pulmonary administration, transdermal administration or oral administration.

Compared with the prior art, the invention has the beneficial effects that:

The invention provides application of PTC124 in preparing medicament for treating or preventing platelet weakness caused by nonsense mutation, PTC124 can be used as an independent active ingredient of medicine effect, and can be combined with the rest nonsense inhibitory medicament, PTC124 has no cytotoxicity in preparing medicament for treating platelet weakness, ITGA2B protein expression of platelets can be increased after the PTC124 is injected intraperitoneally, platelet aggregation function can be improved, and bleeding phenotype is improved.

Drawings

FIG. 1 is a schematic diagram of a cell model for constructing nonsense mutations in human megakaryocyte Meg01 cells ITGA2B by CRISPR/Cas9 technology in example 1 of the present invention;

FIG. 2 is a graph showing cytotoxicity results of the CCK8 kit of example 1 of the present invention after 24 hours of PTC124 at different concentrations (0, 0.625, 1.25, 2.5, 5, 10uM concentration gradients);

FIG. 3 is a schematic diagram showing a flow cytometry test for detecting the expression of ITGA2B protein in cells in example 1 of the present invention;

FIG. 4 is a schematic diagram of a test for detecting the content of alpha IIb beta 3 in cells by using SZ22 monoclonal antibody Western blot in the embodiment 1 of the invention;

FIG. 5 is a schematic representation of sequencing and identification of positive clones from different mouse models in example 2 of the present invention;

FIG. 6 is a graph showing the comparison of platelet morphology and number of different mouse models in example 2 of the present invention, wherein (i) is a graph showing the comparison of platelet morphology of different mouse models with wild-type mice, and (ii) is a graph showing the comparison of platelet number of different mouse models with wild-type mice;

FIG. 7 is a schematic diagram showing platelet aggregation function test of mutant mouse model and wild mouse model under different induction in example 2 of the present invention;

FIG. 8 is a graph showing the results of the platelet aggregation function test of the PTC124 acting on KT mice in example 3 of the present invention;

FIG. 9 is a graph showing the results of platelet bleeding time measurement of the wild-type mice and the KT mice after PTC124 was applied in example 3 of the present invention;

FIG. 10 is a graph showing the results of detecting the content of alpha IIb beta 3 in platelet cells of the wild type mice and the KT mice after the PTC124 was applied in example 3 of the present invention.

Detailed Description

The invention is further described below with reference to the accompanying drawings. The following examples are only for more clearly illustrating the technical aspects of the present invention, and are not intended to limit the scope of the present invention.

The embodiment of the present application provides an application of PTC124 in preparing a medicament for treating thrombocytopenia, and for better explaining the purpose, technical scheme and advantages of the present application, the present application will be further described with reference to the accompanying drawings and specific examples. The experimental methods in the application, unless otherwise specified, all adopt the general methods in the art, and the experimental results of the application can be obtained through limited tests on the basis of the data disclosed by the embodiment of the application and combining the known techniques in the art.

The PTC124 was prepared into solutions of different concentration gradients using a mixed solution of 10% dmso and 90% sbe- β -CD physiological saline as a solvent, and the mixed solution without PTC124 was used as a comparative example for experimental tests.

EXAMPLE 1 influence of PTC124 on megakaryocytes

1. The experimental method comprises the following steps:

1.1 construction of ITGA2 Bc.2671C > T (p.Q891X) human megakaryocyte Meg01 cell model combinations (mutant, wild-type, PTC 124-mutant)

The CRISPR/Cas9 technology is used for constructing an ITGA2B c.2671C > T (p.Q891X) humanized megakaryocyte Meg01 cell model (mutant), and the steps are as follows:

gRNA was designed and synthesized for specific nonsense mutation sites.

The pX458-GFP plasmid was used to construct CRISPR/Cas9 plasmids containing grnas.

The CRISPR/Cas9 plasmid is transfected into Meg01 and DAMI cells, GFP+ monoclonal cells are subjected to flow sorting, visible clone extraction genes are selected, and the CRISPR/Cas9 plasmid with targeted cleavage activity is screened out through PCR sequencing.

Template plasmids were constructed by PCR using patient DNA.

The screened CRISPR/Cas9 plasmid with targeted cleavage activity and the template plasmid were co-transfected into Meg01 cells in the appropriate ratio.

And (3) carrying out flow sorting on GFP+ monoclonal cells, culturing and passaging to 96-well plates, picking 50% of extracted genes of visible clones, carrying out PCR amplification, and sequencing to verify whether construction is successful, so as to obtain an ITGA2Bc.2671C > T (p.Q891X) humanized megakaryocyte Meg01 cell model (hereinafter referred to as mutant cell model) after construction is successful, as shown in figure 1.

The wild cell model is obtained by direct acquisition, the gene structure is stable, and no gene mutation occurs. PTC124 (0, 10, 25, 50, 100, 200 ug/ml) with different concentrations is added into the wild type cell model and the mutant cell model respectively, and cells are incubated with PTC124 for 24 hours to obtain a PTC 124-wild type cell model and a PTC 124-mutant cell model respectively.

1.2, PTC124 cytotoxicity, ITGA2B protein expression, and detection of alpha IIb beta 3 content in cells

PTC124 cytotoxicity assay:

The cytotoxicity of PTC124 (concentration gradients 0, 10, 25, 50, 100, 200 ug/ml) on megakaryocytes after 24 hours was measured using CCK8 kit.

ITGA2B protein expression detection:

PTC124 (0, 50, 100 ug/ml) was added at various concentrations and CD41 (2 ug/ml) antibody was added to the mutant cell model.

CD41 (2. Mu.g/ml) antibody was added to the wild-type cell model as a control, incubated at room temperature for 20min in the absence of light, washed once with MTB, and detected on-machine by flow cytometry.

Detection of αiibβ3 content in cells:

PTC124 (concentration gradient of 0, 100, 200 ug/ml) with different concentrations was added to the mutant cell model, and the content of alpha IIb beta 3 in the cells was detected by using SZ22 monoclonal antibody row Westernblot as a comparative example.

2. Experimental results

As shown in fig. 2, fig. 2 is a schematic diagram showing the measurement results of the CCK8 kit of cytotoxicity after PTC124 of different concentrations acts on megakaryocytes for 24 hours, and it can be seen from the figure that there is no statistical difference between the cell viability of PTC124 added with different concentration gradients and PTC124 not treated, which indicates that PTC124 has no cytotoxicity and PTC124 has no influence on megakaryocyte viability.

As shown in fig. 3, fig. 3 is a schematic diagram of a flow cytometry test for detecting the protein expression of ITGA2B in a cell, and it is obvious from the figure that after PTC124 is added in a mutant cell model, the protein expression of ITGA2B in the cell is improved compared with a wild cell model, and the protein expression of ITGA2B is also improved along with the increasing concentration of PTC124, which indicates that PTC124 can effectively improve the protein expression of ITGA2B in Meg01 cells, that is, the translation reading function.

As shown in fig. 4, fig. 4 is a schematic diagram of a test for detecting the content of αiib beta 3 in cells by using SZ22 monoclonal antibody line Westernblot, and it is known that the content of αiib beta 3 in a mutant cell model without adding PTC124 is smaller than that in a wild cell model, when the concentration of PTC124 reaches 100ug/ml, the content of αiib beta 3 in the mutant cell model is almost equal to that in the wild cell model, and when the concentration of PTC124 reaches 200ug/ml, the content of αiib beta 3 in the mutant cell model is significantly higher than that in the wild cell model, so that PTC124 can significantly increase the content of αiib beta 3 in the mutant cell, i.e., the protein expression of ITGA2B is enhanced (αiib beta 3 is the protein name of ITGA2B, and ga2B is the gene name).

Example 2 construction of transgenic GT mouse models

1. The experimental method comprises the following steps:

1.1 construction of the C57BL/6 mouse model of ITGA2 Bc.2659C > T (p.Q887X)

The mouse ITGA2B gene (NM-010575.2; ensembl: ENSMUSG00000034664) is located on mouse chromosome 11. The gene has 30 exons in total, the start codon ATG is positioned in exon 1, the stop codon TGA is positioned in exon 30, c.2659C is positioned in exon 26, and exon 26 is the target point of gene editing.

Design of gRNA and template oligonucleotides:

1) gRNA targeting sequence 5'-ATGCCTGTCTGCGCTCACGCTGG-3'

2) Template sequence 5' -GGTGGACTGGAAACTATCCACGCCCAGCCCTTCT

TCCATTCCCCGTCCATCACCAACGTGAGCGCAGATAGGCATTCCTGCA G GGGCCCAAGCCAGGGCAGCAGGACCCAGTTCTGGTGGTGAGAAGGCTC-3'

Construction of gRNA vector links:

gRNA:https://en.vectorbuilder.com/vector/VB190829-1209qve.html

the mutation site c.2659c > T (CAG to TAG) of the template introduces exon 26 by homology directed repair.

Cas9 mRNA, in vitro transcribed gRNA and template oligonucleotide were simultaneously injected into fertilized eggs for mutant mouse production, resulting in a C57BL/6 mouse model of ITGA2B c.2659C > T (p.Q887X), i.e., KI mouse (KI stands for mutant), also called transgenic mouse model.

And (3) identifying positive mice by PCR identification and sequencing, and carrying out seed conservation and passage, wherein two mice with allelic mutation are homozygous KI mice, and one mice with allelic mutation is heterozygous KI mice.

Wherein, PCR identifies sequencing primers:

Mouse ITGA2B(Q887X)-F:CCCTCGGATCTGCTCTACATCCTG

Mouse ITGA2B(Q887X)-R:AGCGACACACACAGAGAACCTACGTG 2

The wild mouse model can be directly obtained, and has stable gene structure and no gene mutation. The results of sequencing the positive clones from the different mouse models are shown in FIG. 5.

1.2 Platelet morphology and quantitative detection in different mouse models

Vital signs, adverse reactions and survival conditions of each mouse model were monitored at different times, clinical manifestations of the mice were observed, and the morphology and number of platelets thereof were detected.

1.3 Platelet aggregation function detection in different mouse models

Platelet aggregation functions of wild-type and mutant mouse models were tested under the induction of 0.1U/ml thrombin, 10. Mu.MADP and 2. Mu.g/ml collagen, respectively.

2. Experimental results

By monitoring vital signs, adverse reactions and survival conditions of each mouse model at different times, it is found that mutation of the ITGA2B gene does not affect normal physiological states of mice and has no adverse reactions.

As shown in fig. 6, fig. 6 (i) is a schematic view of platelet status of three mouse models, and fig. 6 (ii) is a schematic view of platelet count of three mouse models, wherein it can be seen that the platelet status and count of both mutant mouse model and wild mouse model are not significantly different.

As shown in fig. 7, fig. 7 is a schematic diagram of platelet aggregation function detection of a mutant mouse model and a wild-type mouse model, and it is known that the platelet aggregation function of the mutant mouse model is significantly worse than that of the wild-type mouse model under different inducers, and the phenotype identification result shows that the mutant mouse model accords with the platelet weakness (GT) phenotype.

Example 3 PTC124 acts on KI mice

1. Experimental materials:

c57BL/6 mice, weighing 20-25g, purchased from Nanjing university model animal center. PTC124 (99.82% purity) was purchased from MCM.

2. Experimental method and results:

2.1PTC124 determination of optimal drug concentration and onset time

The mice were fed PTC124 and water at concentration gradients of 0, 200, 400, 800, 1600mg/kg.d, respectively, for 2 weeks without affecting normal physiological function of the mice, and the bleeding time of the mice' tails was measured and the results are shown in table 1:

Table 1: effect of PTC124 on bleeding time in mice with different concentration gradients

As can be seen from Table 1, the bleeding time from the tail of the KI mice was significantly reduced when the PTC124 concentration was 800mg/kg.d, and thus the optimum dosage concentration of PTC124 was 800mg/kg.d.

Mice treated with 800mg/kg. D PTC124 were tested for rat tail bleeding time before and after 30min, 2h, 8h, 24h, 1w, 2w, respectively, and the results are shown in Table 2:

Table 2: comparison of mouse tail bleeding time before and after different administration times

As can be seen from table 2, KI mice showed significantly reduced rat tail bleeding time when PTC124 was treated with 1w, thus determining that the onset time of PTC124 in KI mice was 1 week each.

Platelet aggregation function assay following 2.2PTC124 action on KI mice

Platelet aggregation was tested by change in transmittance using a two-channel platelet aggregation instrument (product of Chrono-Log company, usa). The temperature of the platelet aggregation meter was set to 37℃and the stirring speed was set to 1200rpm. The washed mouse platelet suspension was taken in 250ul (2X 10 ⁸/ml) and added to the aggregation tube, 1mM CaCl ₂ was added, and the stimulator was added to detect blood cell aggregation. Platelet aggregation function was examined in KI mice 1 week after PTC124 was applied to the mice under the induction of 0.1U/ml thrombin, 10. Mu.MADP, and 2. Mu.g/ml collagen, respectively.

As shown in FIG. 8, comparing the results of FIG. 8 with those of FIG. 7, it is evident that the platelet aggregation function is improved after PTC124 acts on the KI mouse.

Bleeding time detection after 2.3PTC124 acting on GT mice

The mice were anesthetized with 5% chloral hydrate solution, the tail was cut off at a distance of 3mm from the tail end of the mice, the cut tail was immersed in a 15ml centrifuge tube containing normal saline, timing was started when red blood flow occurred at the tail wound, timing was stopped when red blood flow disappeared, and this time interval was recorded as bleeding time. The upper limit of bleeding time was set to 20min.

After 1 week of PTC124 administration to the KI mice, the bleeding time of PTC124 administration to the KI mice and the bleeding time of wild-type mice were measured.

As a result, as shown in FIG. 9, the bleeding time of the KI mouse was reduced after the PTC124 was applied to the mouse, as compared with the case where the PTC124 was not applied.

Detection of alpha IIb beta 3 content in platelets after 2.4PTC124 acts on KI mice

Collecting peripheral blood of a mouse by adopting an orbital blood sampling method, separating the platelet, adding a cell lysate to lyse the platelet, using a BCA protein quantitative kit to measure protein concentration, performing SDS-PAGE gel electrophoresis on a prepared protein sample, transferring the protein sample onto a PVDF membrane after electrophoresis, using a rabbit anti-mouse alpha IIb beta 3 antibody (Ab 1) as a primary antibody, using a sheep anti-rabbit IgG marked by IRDye800 as a secondary antibody, and detecting whether alpha IIb beta 3 protein expression in the platelet is enhanced.

After 1 week of PTC124 action on mice, cells were assayed for αIIbβ3 content using a Westernblot.

As a result, as shown in FIG. 10, the αIIbβ3 content of the platelet cells of the KI mouse was increased, i.e., the ITGA2B protein expression was enhanced, after the PTC124 was applied to the KI mouse, as compared with that of the non-applied PTC 124.

These results all demonstrate that PTC124 can improve platelet aggregation by enhancing the ITGA2B protein expression of platelets, thereby alleviating bleeding and ultimately achieving the effect of treating platelet weakness.

The foregoing is merely a preferred embodiment of the present invention, and it should be noted that modifications and variations could be made by those skilled in the art without departing from the technical principles of the present invention, and such modifications and variations should also be regarded as being within the scope of the invention.

Claims

Use of PTC124 in the manufacture of a medicament for the treatment of thrombocytopenia, characterized in that PTC124 is an active ingredient of a medicament for the treatment of thrombocytopenia.
2. The use of PTC124 according to claim 1 for the preparation of a medicament for the treatment of thrombocytopenia, wherein the PTC124 is administered at a concentration of 700-900 mg/kg.
3. Use of PTC124 according to claim 2 for the preparation of a medicament for the treatment of thrombocytopenia, wherein the PTC124 is optimally administered at a concentration of 800mg/kg.
4. The use of PTC124 according to claim 1 for the preparation of a medicament for the treatment of thrombocytopenia, wherein PTC124 is non-cytotoxic and acts by increasing the level of cell surface aIIbb by increasing the expression of the mutant megakaryocyte line ITGA2B protein.
5. A medicament for treating thrombocytopenia, which is characterized by comprising PTC124 and medicament auxiliary materials, wherein the PTC124 is used as an active ingredient of the medicament for treating thrombocytopenia.
6. The drug for treating thrombocytopenia according to claim 5, wherein the pharmaceutical excipients comprise pH adjusting agents, osmotic pressure adjusting agents, solubilizing agents, cosolvents, emulsifying agents, stabilizing agents, preserving agents, fillers, disintegrating agents, binders, flavoring agents, smelling agents, lubricants.
7. The drug for treating thrombocytopenia according to claim 5, wherein the drug for treating thrombocytopenia is in the form of powder, granule, tablet, capsule, pill, syrup, powder, solution, oral liquid, suspension or injection.
8. The drug for treating thrombocytopenia according to claim 7, wherein the drug for treating thrombocytopenia is in the form of an injection.
9. The medicament for treating thrombocytopenia according to claim 8, wherein the injection is administered by injection, nasal administration, pulmonary administration, transdermal administration or oral administration.