A medicine for antagonizing thyroid interference toxicity of perfluorinated compounds and application thereof

Abstract

The application discloses a medicine for antagonizing thyroid interference toxicity of perfluorinated compounds and application thereof. The medicine component is amiodarone, and the medicine can be prepared into a sterile injection. PFOA, PFOS and PFOSA can be combined with and activated thyroid hormone receptors TRalpha and TRbeta, disturb thyroid hormone homeostasis, and cause body development abnormalities, heart rate disorders and other damages. Cell experiments, zebrafish embryo experiments and molecular docking results show that amiodarone in the medicine can competitively block the combination of perfluorinated compounds with TRalpha and TRbeta. The medicine can effectively reverse thyroid damage caused by perfluorinated compounds. The medicine has clear action targets and stable curative effect, can be used for preventing and treating thyroid function disorders caused by perfluorinated compound exposure, and has a wide application prospect.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical preparation technology, specifically relating to a drug that antagonizes the thyroid-interference toxicity of perfluorinated compounds and its application. Background Technology

[0002] Perfluorooctanoic acid (PFOA) and perfluorooctane sulfonyl (PFOS) are typical persistent organic pollutants. Due to their high stability, strong bioaccumulation, and significant endocrine toxicity, they have been included in the Stockholm Convention on Persistent Organic Pollutants for control. Perfluorooctyl sulfonamide (PFOSA), as a precursor of PFOS, is widely detected in humans and other organisms. Current research shows that PFOA, PFOS, and PFOSA all have significant thyroid-disrupting effects. These substances can bind to thyroid hormone receptors TRα and TRβ and abnormally activate related signaling pathways, disrupting the secretion, transport, and metabolism of thyroid hormones in the body, damaging the normal function of the hypothalamus-pituitary-thyroid axis, and ultimately causing various adverse symptoms such as thyroid dysfunction, growth retardation, and abnormal heart rate.

[0003] Currently, clinical interventions for thyroid damage caused by perfluorinated compound exposure are limited to symptomatic hormone replacement therapy. These approaches cannot block the activation of thyroid receptors by exogenous compounds at the molecular level, resulting in limited efficacy and significant drawbacks with long-term use. Therefore, developing novel drugs with clearly defined targets and significant synergistic effects, along with related applications, is a pressing clinical challenge. Summary of the Invention

[0004] To address the shortcomings of existing technologies, this invention provides a drug that antagonizes the thyroid-interfering toxicity of perfluorinated compounds and its application. This drug can block the abnormal activation of TRα and TRβ by three types of perfluorinated compounds and improve developmental abnormalities, heart rate disorders, and thyroid dysfunction caused by exposure to perfluorinated / polyfluoroalkyl compounds (PFASs).

[0005] To achieve the above objectives, the present invention adopts the following technical solution:

[0006] The application of a drug that antagonizes the thyroid-interfering toxicity of perfluorinated compounds, wherein the perfluorinated compounds are PFOA, PFOS, or PFOSA.

[0007] The active ingredient of the drug is amiodarone.

[0008] The drug inhibits the abnormal activation of the TRα and TRβ signaling pathways by competitively binding to the ligand-binding domains of the thyroid hormone receptors (TRα and TRβ) with amiodarone, thereby blocking the binding of PFOA, PFOS, and PFOSA to the receptors.

[0009] The drug can dose-dependently inhibit the abnormal proliferation of rat pituitary tumor cells (GH3 cells) induced by PFOA, PFOS, and PFOSA, thus blocking the toxic effects of perfluorinated compounds at the cellular level.

[0010] Furthermore, the drug may be supplemented with pharmaceutically acceptable excipients to prepare it as an injectable formulation.

[0011] The drug described in this invention is used to prepare a medication for preventing and treating thyroid hormone homeostasis disorders, hypothalamic-pituitary-thyroid axis dysfunction, growth retardation, and abnormal heart rate caused by exposure to perfluorinated compounds. When used, an effective dose of the drug is administered to mammals exposed to perfluorinated compounds to achieve both prevention and treatment of thyroid damage.

[0012] The beneficial effects of this invention are as follows:

[0013] (1) This invention is the first to demonstrate that PFOA, PFOS, and PFOSA can bind to the ligand-binding domains (LBD) of thyroid hormone receptors TRα and TRβ, and their binding sites overlap with the natural ligand T3, thereby abnormally activating the signaling pathway and producing toxicity. This invention utilizes amiodarone to competitively occupy the receptor binding site, blocking the effects of pollutants at the source, thus forming a novel intervention system.

[0014] (2) Definite therapeutic effect: Experiments have confirmed that the drug of the present invention can effectively inhibit the abnormal proliferation of thyroid cells and improve developmental abnormalities and heart rate disorders caused by perfluorinated compounds.

[0015] (3) Great industrialization potential: It can be prepared into a variety of commonly used clinical dosage forms, adapted to different administration scenarios, and is suitable for the prevention and treatment of thyroid injury in people with occupational exposure and environmental exposure. It has broad application prospects. Attached Figure Description

[0016] Figure 1 Effects of total triiodothyronine (T3) and amiodarone on GH3 cell activity after 24 hours of exposure to GH3 cells in a thyroid hormone receptor (TR) positive control group: (A) T3, (B) amiodarone, *p<0.05 indicates a significant difference between the exposure group and the control group;

[0017] Figure 2 Effects of TR positive control T3 and amiodarone on GH3 cell proliferation after 72 hours of exposure: (A) T3, (B) amiodarone, *p<0.05, **p<0.01, ***p<0.001 indicate significant differences between the exposure group and the control group;

[0018] Figure 3Effects of PFOA, PFOS, and PFOSA on GH3 cell proliferation after 72 hours of exposure. (A) PFOA, (B) PFOS, (C) PFOSA. *p<0.05, **p<0.01, ***p<0.001 indicate significant differences between the exposure group and the control group.

[0019] Figure 4 Effects of PFOA, PFOS, and PFOSA on GH3 cell proliferation after 72 hours of exposure to a series of concentrations of amiodarone. (A) PFOA, (B) PFOS, (C) PFOSA. *p<0.05, **p<0.01, ***p<0.001 indicate significant differences between the exposure group and the control group.

[0020] Figure 5 Effects of PFOA, PFOS and PFOSA on zebrafish embryonic development: (A) survival rate, (B) malformation rate, (C) body length, (D) heart rate. *p< 0.05, **p< 0.01 indicates a significant difference between the exposed group and the control group.

[0021] Figure 6 Zebrafish embryos were exposed to 0.1, 1, and 10 μg / L PFOA, PFOS, and PFOSA for 5 days. The levels of thyroid-related hormones were measured: (A) total triiodothyronine (TT3), (B) total thyroxine (TT4), (C) free triiodothyronine (FT3), (D) free thyroxine (FT4), and (E) TT3 / TT4 ratio. *p < 0.05, **p < 0.01, and ***p < 0.001 indicate a significant difference between the exposed group and the control group.

[0022] Figure 7 After zebrafish embryos were exposed to 0.1, 1, and 10 μg / L PFOA, PFOS, and PFOSA for 5 days, the expression of TR-related genes changed. (A) TRα, (B) TRβ, (C) TR, (D) thyrotropin-releasing hormone (TRH), (E) thyroid peroxidase (TPO). *p < 0.05, **p < 0.01, ***p < 0.001 indicate a significant difference between the exposure group and the control group.

[0023] Figure 8 Structures of compounds formed by PFOA, PFOS, and PFOSA with the TRα ligand binding domain; purple indicates human TRα; yellow dashed lines indicate hydrogen bonds between the target compound and amino acid residues; black labels are abbreviations for amino acid residues: (A) PFOA; (B) PFOS; (C) PFOSA.

[0024] Figure 9The structures of PFOA, PFOS, and PFOSA conjugates with TRβ LBD are shown. Purple indicates human TRβ. Yellow dashed lines indicate hydrogen bonds between the target compound and amino acid residues. Black labels are abbreviations of amino acid residues. (A) T3; (B) PFOA; (C) PFOS; (D) PFOSA. Detailed Implementation

[0025] To make the above-mentioned objectives, features, and advantages of the present invention more apparent and understandable, the specific embodiments of the present invention will be described in detail below with reference to examples. The following content is merely illustrative and explanatory of the concept of the present invention. Those skilled in the art can make various modifications or additions to the described specific embodiments or use similar methods to replace them, as long as they do not deviate from the inventive concept, all of which should fall within the protection scope of the present invention. The preparation method of the present invention will be described below through specific embodiments.

[0026] Example

[0027] S1, Experimental Materials

[0028] S11. Reagents: Amiodarone, PFOA, PFOS, PFOSA, DMEM medium, Dual-luciferase reporter gene assay kit, thyroid hormone enzyme-linked immunosorbent assay (ELISA) kit, quantitative polymerase chain reaction (PCR) assay reagent.

[0029] S12. Experimental cells: rat pituitary tumor (GH3) cells and human embryonic kidney cells (HEK293T).

[0030] S13. Experimental animals: Wild-type AB strain zebrafish embryos.

[0031] S14. Instruments: ELISA reader, fluorescence detector, real-time quantitative PCR instrument, laser confocal microscope, and microscopic imaging system.

[0032] S2 and GH3 cell culture and proliferation experiments

[0033] GH3 cells were seeded into culture plates and cultured until adherence. Then, different concentrations of PFOA, PFOS, and PFOSA were added to establish a toxicity model. Simultaneously, different concentrations of amiodarone were added for intervention treatment, and the cells were cultured for 72 h. Cell proliferation activity was detected using the MTT assay to observe the inhibitory effect of the drugs on perfluorinated compound-induced abnormal cell proliferation.

[0034] Experimental results show that PFOA, PFOS, and PFOSA can all significantly promote the abnormal proliferation of GH3 cells. The drug of this invention can reverse this phenomenon in a dose-dependent manner and effectively inhibit the abnormal activation of thyroid cells.

[0035] S3, thyroid receptor transcriptional activity assay

[0036] Plasmids carrying human TRα and TRβ genes were transfected into HEK293T cells. After transfection, perfluorinated compounds and the drug of this invention were added. After culturing for 24 h, the transcriptional activity of TRα and TRβ was detected using a dual-luciferase reporter gene system.

[0037] The results showed that PFOA, PFOS, and PFOSA could significantly activate the TRα and TRβ signaling pathways, and the drugs could effectively block this activation and inhibit abnormal receptor expression.

[0038] S4. Zebrafish embryo pharmacological experiment

[0039] Normally developing zebrafish embryos were selected and subjected to continuous exposure to PFOA, PFOS, and PFOSA to establish a thyroid injury model. After the exposure cycle, embryo survival rate, body length, and heart rate were recorded. The levels of TT3, TT4, FT3, and FT4 hormones in the embryos were measured, and the expression levels of thyroid-related genes such as TRα, TRβ, TPO, TTR, and TRH were detected using qPCR.

[0040] S5. Pharmaceutical Formulation Preparation

[0041] (1) Sterile injection

[0042] Amiodarone was added to sterile water for injection and a solubilizer, stirred and dissolved, and the pH and osmotic pressure of the solution were adjusted. After sterile filtration, filling and sterilization, the injection was obtained.

[0043] Figure 1 Effects of TR positive control T3 and amiodarone on GH3 cell viability after 24 hours of exposure: (A) T3, (B) amiodarone; This experiment first verified the cytotoxicity of the reagents. The results showed that within the concentration range used in the experiment, neither the TR agonist T3 nor amiodarone produced significant toxicity to GH3 cells, proving that the concentration settings in subsequent experiments were safe and effective.

[0044] Figure 2 Effects of TR positive control T3 and amiodarone on GH3 cell proliferation after 72 hours of exposure: (A) T3, (B) amiodarone; The experimental results show that at non-cytotoxic concentrations, T3 can significantly promote GH3 cell proliferation, while amiodarone can significantly inhibit GH3 cell proliferation. This result is consistent with existing literature reports, clarifying the role of the reagents in thyroid receptor function experiments and laying the foundation for the study of the drug action mechanism of this invention.

[0045] Figure 3The effects of PFOA, PFOS, and PFOSA on GH3 cell proliferation after 72 hours of exposure were investigated. Results showed that PFOA, PFOS, and PFOSA all promoted abnormal GH3 cell proliferation in a concentration-dependent manner. The minimum effective concentrations and proliferative effects of the three compounds differed, with the proliferative potency ranking as PFOSA > PFOS > PFOA. The concentration of PFOA in occupationally exposed individuals was on the same order of magnitude as the minimum effective concentration in this experiment, suggesting that this type of perfluorinated compound poses a high health risk and confirming the necessity of the drug of this invention.

[0046] Figure 4 The effects of PFOA, PFOS, and PFOSA combined with various concentrations of amiodarone on GH3 cell proliferation after 72 hours were investigated. GH3 cells were treated with different concentrations of amiodarone in combination with the three perfluorinated compounds. The results showed that amiodarone could reverse the abnormal cell proliferation induced by PFOA, PFOS, and PFOSA in a dose-dependent manner. This result directly demonstrates that the three perfluorinated compounds mediate proliferative effects by activating thyroid receptors, and that amiodarone can effectively block this process, making it the core component responsible for the efficacy of this compound drug.

[0047] Figure 5 Effects of PFOA, PFOS, and PFOSA on zebrafish embryonic development: (A) survival rate, (B) malformation rate, (C) body length, (D) heart rate. *p < 0.05, **p < 0.01 indicates a significant difference between the exposed and control groups. Survival rate, malformation rate, body length, and heart rate are core indicators for evaluating the developmental status of zebrafish embryos. The experiment found that embryo survival rates were significantly reduced in the medium- and high-dose PFOA groups and the high-dose PFOSA group; no significant changes in malformation rate were observed in any of the treatment groups; significant growth inhibition was observed in the medium-dose PFOA group, all experimental PFOS groups, and the medium- and high-dose PFOSA groups; and heart rate disturbances occurred in most exposed embryos, with heart rates exceeding the normal range. These results confirm that PFOA, PFOS, and PFOSA significantly interfere with the normal development of aquatic organisms, and abnormal heart rate is directly related to dysfunction of the thyroid receptor TRα.

[0048] Figure 6Zebrafish embryos were exposed to 0.1, 1, and 10 μg / L PFOA, PFOS, and PFOSA for 5 days. The levels of thyroid-related hormones were measured: (A) TT3, (B) TT4, (C) FT3, (D) FT4, and (E) TT3 / TT4 ratio. Thyroid hormones are key substances regulating embryonic growth and development. The results showed that the three perfluorinated compounds had little effect on free thyroid hormones TT3 and FT4, but significantly inhibited the level of total thyroid hormone TT4. Only the medium-dose PFOA group and the low- and medium-dose PFOSA groups significantly downregulated FT4 levels. The high-dose PFOSA group showed a significantly increased TT3 / TT4 ratio, indicating that the organism was in a compensatory regulatory state. Epidemiological and multiple basic studies have confirmed that PFOA causes a decrease in thyroid hormone levels in organisms, disrupting hormonal homeostasis. TT4 can be converted into the more active TT3 by deiodinases; these experimental results also fully reveal the characteristics of perfluorinated compounds interfering with thyroid hormone metabolism.

[0049] Figure 7 Zebrafish embryos exposed to 0.1, 1, and 10 μg / L PFOA, PFOS, and PFOSA for 5 days showed changes in thyroid-related gene expression: (A) TRα, (B) TRβ, (C) TTR, (D) TRH, and (E) TPO. TRα and TRβ encode thyroid hormone receptors, while TRH, TPO, and TTR are key functional genes of the hypothalamic-pituitary-thyroid axis. The results showed that PFOA, PFOS, and PFOSA significantly inhibited TRα and TRβ gene expression, and the downregulation of TRα expression was highly consistent with abnormal embryonic heart rate. Furthermore, the three compounds showed different sensitivities to TRα and TRβ, with TRα being more sensitive. The TTR gene, encoding the key transporter thyroxine transporter protein responsible for the transport and distribution of thyroid hormones in the circulatory system, was significantly downregulated in all PFAS treatment groups. The inhibition of TTR expression is consistent with the synchronous decrease in TT4 levels observed in this study, indicating that PFOA, PFOS, and PFOSA may impair the peripheral transport capacity and circulating reserves of thyroid hormones in the blood. This suggests that PFOA, PFOS, and PFOSA interfere with thyroid hormone homeostasis through different molecular pathways, and the drug of this invention can comprehensively repair the abnormal expression of the above genes and restore the normal regulatory function of the hypothalamus-pituitary-thyroid axis.

[0050] Figure 8The structures of PFOA, PFOS, and PFOSA bound to TRα LBD are shown. The green band represents human TRα. The yellow dashed lines represent hydrogen bonds between the target compound and amino acid residues. The black labels are abbreviations for the amino acid residues. (A) PFOA; (B) PFOS; (C) PFOSA. Molecular docking results show that the natural ligand T3 can form stable hydrogen bonds with the human TRα ligand binding domain. PFOA, PFOS, and PFOSA can all bind to the ligand pocket of TRα, with binding sites highly overlapping with T3, forming stable hydrogen bond structures. This demonstrates that the three classes of perfluorinated compounds occupy the binding site as receptor agonists, continuously activating the downstream signaling pathway of TRα, thereby producing thyroid toxicity. Amiodarone in this invention can competitively occupy this binding region, blocking the binding of perfluorinated compounds to TRα.

[0051] Figure 9 The structures of PFOA, PFOS, and PFOSA conjugates with TRβ LBD are shown. The green band represents human TRα. The yellow dashed lines represent hydrogen bonds between the target compounds and amino acid residues. The black labels are abbreviations for the amino acid residues. (A) PFOA; (B) PFOS; (C) PFOSA. Consistent with the TRα results, T3 can form hydrogen bonds with the TRβ ligand-binding domain. PFOA, PFOS, and PFOSA can also bind to the TRβ ligand-binding domain, with the binding site coinciding with the natural ligand T3, forming a stable interaction. This result confirms that all three perfluorinated compounds can mediate thyroid interference effects by activating both TRα and TRβ receptor subtypes, fully elucidating the toxic mechanism of perfluorinated compounds and the antagonistic mechanism of the drugs of this invention at the molecular level.

[0052] Based on comprehensive experimental results, from multiple dimensions including molecular docking, cell function, animal phenotype, hormone and gene expression, it is confirmed that PFOA, PFOS, and PFOSA disrupt thyroid hormone homeostasis and impair normal development by binding to and activating the TRα and TRβ signaling pathways. This invention utilizes amiodarone, which competitively blocks receptor binding. This technical approach has a clearly defined target, is supported by amiodarone experimental data, and demonstrates stable efficacy, possessing significant innovativeness and clinical and environmental exposure protection value.

Claims

1. The application of a drug that antagonizes the thyroid-interfering toxicity of perfluorinated compounds, characterized in that, This application refers to the use of the drug in the preparation of a drug for preventing and treating thyroid hormone homeostasis disorders caused by exposure to perfluorinated compounds; The perfluorinated compounds are PFOA, PFOS, and PFOSA; The active ingredient of the drug is amiodarone.

2. The application according to claim 1, characterized in that, The drug blocks the binding of PFOA, PFOS, and PFOSA to thyroid hormone receptors TRα and TRβ by competitively binding to these receptors via amiodarone.

3. The application according to claim 1, characterized in that, The drug also contains pharmaceutically acceptable excipients.

4. The application according to claim 1, characterized in that, The drug is in the form of an injectable preparation.

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