Use of niclosamide in the preparation of a medicament for the treatment of upper respiratory tract papillomas

Abstract

The application belongs to the field of biological medicine, and particularly relates to the use of niclosamide in the preparation of a drug for treating upper respiratory tract papilloma. The application provides the use of niclosamide in the preparation of a drug for treating upper respiratory tract papilloma, and the drug is characterized in that niclosamide is the only active ingredient. The experimental results of the application show that niclosamide can achieve long-term control of upper respiratory tract papilloma, reduce the risk of recurrence of upper respiratory tract papilloma to a certain extent, and improve the life quality of patients with upper respiratory tract papilloma, and has important scientific value and clinical application prospect in the field of upper respiratory tract papilloma treatment.

Description

Technical Field

[0001] This invention belongs to the field of biomedicine, specifically relating to the use of niclosamide in the preparation of drugs for treating upper respiratory tract papilloma. Background Technology

[0002] Upper respiratory tract papilloma (URP) is a non-invasive benign tumor originating from the squamous epithelium of the upper respiratory tract, characterized by recurrent papillary hyperplasia. URP has an insidious onset, and its clinical manifestations are closely related to the tumor's growth pattern, location, and size, lacking specificity. It can affect multiple sites such as the larynx, trachea, and bronchi, causing symptoms such as cough, hoarseness, and difficulty speaking. In severe cases, it can lead to airway obstruction.

[0003] The occurrence of upper respiratory tract papillomas is closely related to human papillomavirus (HPV) infection. Epidemiological data show that the disease is most common in the oropharynx, followed by the larynx, and least common in the nasopharynx. This may be related to the direct chronic irritation of the oropharynx from the oral cavity (e.g., smoking, alcohol consumption, and poor oral hygiene). Although upper respiratory tract papillomas are histologically benign lesions, they are characterized by high invasiveness and high recurrence rate. If the disease progresses without effective intervention, it can still cause serious complications, such as airway obstruction, voice dysfunction, and even malignant transformation into squamous cell carcinoma, posing a significant threat to the patient's quality of life and life safety.

[0004] Upper respiratory tract papillomas can occur at any age, but are generally classified into juvenile and adult URPs, with 18 years of age as the dividing line. Juvenile URP is mainly transmitted vertically from mother to child and is closely related to maternal HPV infection in the genital tract, with a peak incidence before the age of 5. Its course is more aggressive, with a higher recurrence rate, often affecting the vocal cords, ventricular folds, epiglottis, and laryngeal surface, and can even spread to the subglottic region, hence the main clinical manifestation is hoarseness. If the lesion spreads down the airways with airflow, it can implant and grow in the trachea, bronchi, and lungs, further aggravating the condition. Adult URP is mostly associated with HPV infection routes such as smoking, sexual contact, or mucosal exposure, and is more common in men than women, with a peak incidence between 30 and 40 years of age. Its progression is relatively slow, but the rate of malignant transformation is higher than that of juvenile URP. Lesions are often located at the glottic level, and the main symptoms are difficulty speaking, progressive hoarseness, stridor, and difficulty breathing; in severe cases, it can be life-threatening. Airway obstruction, malignant tumor progression, and secondary chronic lung disease are the leading causes of death in URP patients.

[0005] Although upper respiratory tract papillomas (URPs) are associated with chronic inflammatory stimuli such as viral infections and smoking, their pathogenesis is not fully understood, therefore, there are currently no targeted and effective treatments. The standardized treatment for URPs is primarily microscopic laryngoscopic resection, encompassing techniques such as CO2 laser ablation, low-temperature plasma knife resection, and mechanical stripping. These surgical methods, by directly removing the proliferating tissue, can improve airway patency and restore some vocal function in the short term. However, because the underlying cause cannot be cured, the recurrence rate of URP remains consistently high (approximately 60%-80%), often requiring multiple surgical interventions, leading to problems such as laryngeal structural damage, accumulated anesthesia risks, and a significant increase in medical costs. Specifically, juvenile URP is particularly difficult to treat due to its high recurrence rate and rapid growth, requiring repeated surgeries to relieve pressure. Statistics show that children with URP in the United States undergo an average of 4.4 surgeries per year, potentially more than 40 surgeries in their lifetime. If the tumor tissue obstructs the airway, it can cause breathing difficulties, necessitating a tracheotomy in emergencies. Such procedures further damage the airway mucosa, causing squamous metaplasia of the respiratory mucosa and forming new fibrosquamous junctions, providing a suitable environment for HPV colonization and downward airway dissemination. In addition, most adult URP patients undergoing surgery will also face complications such as postoperative adhesions, glottic stenosis, hoarseness, dyspnea, and scar formation. At the same time, the recurrence of lesions and the potential risk of malignant transformation after surgery bring enormous psychological stress to patients.

[0006] Although upper respiratory tract papillomas have a certain degree of recurrence and local invasiveness, they are still classified as benign lesions (benign tumors) in tumor classification due to their lack of distant metastasis, low genomic instability, and limited overall risk of malignancy. Currently, clinical trials have explored various treatment methods for upper respiratory tract papillomas, including antiviral drugs (such as interferon-alpha, cidofovir, acyclovir, and 5-fluorouracil), HPV vaccines, photodynamic therapy, traditional Chinese medicine, and even targeted therapies for malignant tumors (such as bevacizumab). However, the treatment effects have been limited, and these methods are currently only used as adjunctive therapies to surgical treatment.

[0007] In conclusion, there is an urgent need to provide drugs that can specifically treat upper respiratory tract papillomas in order to alleviate the current shortcomings in the treatment of upper respiratory tract papillomas. Summary of the Invention

[0008] This invention provides the use of niclosamide in the preparation of medicaments for treating upper respiratory tract papilloma.

[0009] In some embodiments, the drug uses niclosamide as the sole active ingredient.

[0010] In some embodiments, the upper respiratory tract papilloma is HPV positive or HPV negative.

[0011] In some embodiments, the upper respiratory tract papilloma is HPV positive.

[0012] In some embodiments, the upper respiratory tract papilloma includes at least one of HPV6 positive, HPV11 positive, HPV16 positive, and HPV18 positive.

[0013] In some embodiments, the upper respiratory tract papilloma is HPV negative.

[0014] In some embodiments, the upper respiratory tract papilloma is a juvenile upper respiratory tract papilloma or an adult upper respiratory tract papilloma.

[0015] In some embodiments, the upper respiratory tract papilloma occurs in the nose, pharynx, and / or larynx.

[0016] In some embodiments, the drug is used to inhibit the proliferation and / or growth of the upper respiratory tract papilloma.

[0017] In some embodiments, the half-maximal effective concentration of the drug is ≤5 μM.

[0018] In some embodiments, the dosage form of the drug includes injections, oral solutions, and / or tablets.

[0019] In some embodiments, the medicament further includes a pharmaceutically acceptable carrier and / or adjuvant.

[0020] In some embodiments, the drug is used to treat subjects with upper respiratory tract papilloma who are sensitive to the drug.

[0021] In some embodiments, the subject's sensitivity to the drug can be assessed based on upper respiratory tract papilloma organoids derived from the subject. Organoids are three-dimensional microorganism models derived from stem cells or tissue-specific progenitor cells, which self-organize under three-dimensional culture conditions and possess a high degree of structural and functional similarity to the original tissue. Compared to traditional two-dimensional cell lines, organoids can more realistically reproduce the in vivo microenvironment in terms of spatial structure, cellular heterogeneity, cell polarity, and cell-cell / cell-matrix interactions. Simultaneously, organoids exhibit a high degree of consistency with in vivo tissues in terms of gene expression profiles, signaling pathway activation patterns, and drug metabolism pathways, making them more representative and reliable in predicting drug response and toxicity. They can more accurately reflect the efficacy and safety of candidate drugs in vivo, and the experimental results have high clinical relevance.

[0022] In some embodiments, the subject's sensitivity to the drug can be assessed by the half-maximal effective concentration (IC50) of the drug on an upper respiratory tract papillary organoid derived from the subject. Specifically, the assessment method may be:

[0023] In some embodiments, a subject is assessed as sensitive to the drug when the half-maximal effective concentration of the drug for an upper respiratory tract papillary organoid derived from the subject is ≤5 μM.

[0024] In some embodiments, a subject is assessed as insensitive to the drug when the half-maximal effective concentration of the drug for an upper respiratory tract papillary organoid derived from the subject is >5 μM.

[0025] In some embodiments, the half-maximal effective concentration (EC50) of the drug for organoids 50 EC refers to the drug concentration required to produce a 50% inhibitory effect on a certain indicator (such as cell viability) of organoids under specific culture conditions. 50 The value can be obtained by constructing a drug dose-response curve and performing nonlinear fitting. Because organoids retain the spatial structure, gene expression profile, and microenvironment characteristics of the source tissue, this index can realistically reflect the individual's biological response to a specific drug in vitro. Therefore, the obtained EC... 50 The value can serve as a key indicator for assessing a subject's sensitivity to a specific drug.

[0026] In some embodiments, the subject's sensitivity to the drug can also be assessed by the cell viability of upper respiratory tract papilloma organoids derived from the subject after treatment with a first concentration of the drug. Specifically, the assessment method may also be:

[0027] In some embodiments, the subject is assessed as sensitive to the drug when the cell viability of the upper respiratory tract papillary organoids derived from the subject after treatment with a first concentration of the drug is ≤ a preset cell viability threshold.

[0028] In some embodiments, the subject is assessed as insensitive to the drug when the cell viability of the upper respiratory tract papillary organoids derived from the subject is greater than a preset cell viability threshold at a first concentration of the drug.

[0029] In some embodiments, the preset cell viability threshold may be 50%.

[0030] In some embodiments, the first concentration may be adjusted according to the actual requirements of individualized treatment for subjects with upper respiratory tract papilloma. In some embodiments, the first concentration may be set to 0.7–4.8 µM. In some embodiments, the first concentration may also be set to 0.80–2.60 µM, for example 2.50 µM (this value is based on…). Figure 3 EC of the four URP organoids shown 50 The value was calculated by averaging the values) and 0.84 µM (this value is based on...). Figure 3 ECGs of the two URP organoids, LA-010-T and LA-016-T, are shown. 50 The values ​​are calculated by averaging the values ​​to select subjects with upper respiratory tract papillary tumors who are more sensitive to the drug (i.e., niclosamide).

[0031] In some embodiments, the upper respiratory tract papilloma organoids are in the form of organoid suspensions.

[0032] In some embodiments, the method for constructing the upper respiratory tract papilloma organoid includes the following steps:

[0033] S101. Obtain upper respiratory tract papillary tumor samples from the subject;

[0034] S102. The upper respiratory tract papillary tumor sample is single-celled to obtain a single-celled upper respiratory tract papillary tumor.

[0035] S103. The single-celled upper respiratory tract papilloma is cultured to obtain upper respiratory tract papilloma organoids.

[0036] In some embodiments, the culture method in S103 includes: seeding the single-celled upper respiratory tract papilloma into Matrigel and then culturing it in conditioned medium for a first time.

[0037] In some embodiments, the conditioned medium includes: adDMEM / F12+++ medium.

[0038] In some embodiments, the conditioned medium may further include at least one of the following components: B27 supplement, N-acetyl-L-cysteine, nicotinamide, hEGF, hFGF-10, hFGF-2, A83-01, PGE2, CHIR-99021, salivain, RSPO3-Fc fusion protein, and Noggin-Fc fusion protein.

[0039] In some embodiments, the conditioned medium comprises: adDMEM / F12+++ medium, 1× B27 supplement, 1.25 mM N-acetyl-L-cysteine, 10 mM nicotinamide, 50 ng / mL hEGF, 10 ng / mL hFGF-10, 5 ng / mL hFGF-2, 500 nM A83-01, 1 µM PGE2, 3 µM CHIR-99021, 1 µM pharyngeal glycoside, 4% RSPO3-Fc fusion protein conditioned medium, and 4% Noggin-Fc fusion protein conditioned medium.

[0040] In some embodiments, the conditioned medium may further include at least one of the following components: ROCK inhibitor, primoxane, and caspofungin.

[0041] In some embodiments, the conditioned medium may further comprise: 10 µM ROCK inhibitor, 100 µM primomocin, and 0.5 µM caspofungin.

[0042] In some embodiments, the first time period includes 10-20 days.

[0043] Compared with the prior art, the beneficial effects of the present invention include at least the following aspects:

[0044] Niclosamide is an anti-tapeworm drug with the molecular formula C0. 13 H8Cl2N2O4. Although there are some reports of using niclosamide to treat malignant tumors, the results show that niclosamide alone has limited efficacy in treating malignant tumors.

[0045] Surprisingly, this invention found that, compared to erlotinib and gefitinib, which have been reported to treat recurrent respiratory tract papilloma (RRP, a clinically recurrent type of upper respiratory tract papilloma), niclosamide exhibits a targeted therapeutic ability to effectively inhibit the proliferation and growth of this benign tumor. Nicosamide treatment alone showed a stronger inhibitory effect on the proliferation and growth of upper respiratory tract papilloma cells, approximately four times that of erlotinib and approximately 14 times that of gefitinib. In other words, the experimental results of this invention demonstrate that niclosamide can achieve long-term control of upper respiratory tract papilloma and reduce the risk of recurrence to some extent, thus improving the quality of life for patients with upper respiratory tract papilloma. This invention has significant scientific value and clinical application prospects in the field of upper respiratory tract papilloma treatment.

[0046] Furthermore, considering that different subjects may respond differently to the same drug in the individualized treatment of upper respiratory tract papilloma, this invention can also assess the subject's sensitivity to niclosamide based on the upper respiratory tract papilloma organoids derived from the subject, thereby selecting subjects with upper respiratory tract papilloma who are more sensitive to niclosamide (which can also be understood as subjects who may be more suitable for niclosamide treatment), in order to further improve the efficacy of niclosamide for upper respiratory tract papilloma, and thus achieve more precise individualized and targeted treatment of upper respiratory tract papilloma. Attached Figure Description

[0047] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. In all the drawings, similar elements or parts are generally identified by similar reference numerals. The elements or parts in the drawings are not necessarily drawn to scale. Obviously, the drawings described below are some embodiments of the present invention, and those skilled in the art can obtain other drawings based on these drawings without any creative effort.

[0048] Figure 1 Representative bright-field images of papilloma organoids in the upper respiratory tract before and after niclosamide treatment;

[0049] Figure 2 A comparison of the inhibitory effects of niclosamide and other drugs on papillary organoids of the upper respiratory tract;

[0050] Figure 3 The figure shows the survival rate of upper respiratory tract papilloma organoid cells under different concentrations of niclosamide treatment. Detailed Implementation

[0051] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some, not all, of the embodiments of the present invention. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are within the scope of protection of the present invention.

[0052] In this document, "and / or" includes any and all combinations of one or more of the listed related items.

[0053] In this article, "multiple" means two or more, that is, it includes two, three, four, five, etc.

[0054] As used in this specification, the term "about" typically means + / -5% of the value, more typically + / -4% of the value, more typically + / -3% of the value, more typically + / -2% of the value, even more typically + / -1% of the value, and even more typically + / -0.5% of the value.

[0055] In this specification, certain embodiments may be disclosed in a range-bound format. It should be understood that this "range-bound" description is merely for convenience and brevity and should not be construed as a rigid limitation on the disclosed range. Therefore, the description of a range should be considered as having specifically disclosed all possible subranges and the individual numerical values ​​within those ranges. For example, a description of the range 1-6 should be considered as having specifically disclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6, etc., and the individual numbers within those ranges, such as 1, 2, 3, 4, 5, and 6. This rule applies regardless of the breadth of the range.

[0056] Example 1

[0057] (a) Materials and reagents

[0058] Human upper respiratory tract papilloma tissue, 48-well cell culture plates, 96-well cell culture plates, CellTiter-Glo reagent kit, Multidrop Combi reagent dispenser, Tecan D300e digital dispenser, Matrigel, centrifuge tubes (15 mL, 50 mL), 6 cm cell culture dishes, pipette tips (1000 µL, 200 µL, 100 µL, 20 µL...), cell culture incubator, biosafety cabinet, optical microscope, centrifuge, constant temperature water bath, pipettes, ultra-low temperature freezer.

[0059] Reagents: B27 supplement, N-acetyl-L-cysteine, nicotinamide, hEGF (human epidermal growth factor), hFGF-10 (human fibroblast growth factor-10), hFGF-2 (human fibroblast growth factor-2), A83-01, PGE2 (prostaglandin E2), CHIR-99021, linalool, RSPO3-Fc fusion protein conditioned medium, Noggin-Fc fusion protein conditioned medium, Y-27632 (ROCK inhibitor), Caspofungin, Penicillin, Primocin, adDMEM / F12+++ (Advanced DMEM / F12 basal medium), Tween-20, PBS, TrypLE™ Express enzyme (TrypLE), Collagenase, Dispase, DNase I (deoxyribonuclease I), niclosamide, gefitinib, erlotinib.

[0060] (II) Experimental Methods

[0061] (1) Generation of patient-derived upper respiratory tract papilloma organoids (URP organoids):

[0062] 1) Sample Collection and Storage: All surgically removed upper respiratory tract papilloma samples were obtained from West China Hospital of Sichuan University, with written informed consent and approval from the ethics committee. After obtaining the samples surgically, the tissue blocks were immediately placed in 50 mL conical tubes, and 45 mL of ice-cold adDMEM / F12+++ culture medium was added. The tissue samples were stored at 4°C to maintain tissue viability until the tissue separation process began.

[0063] 2) Tissue digestion and cell separation: Examine the tissue block to ensure it consists only of epithelial tissue, and cut the tissue into 1-3 mm pieces in a 6 cm cell culture dish. 3 Small tissue fragments were resuspended in an appropriate amount of trypsin and digested for 30-60 minutes, shaking vigorously every 10-15 minutes and using a P1000 pipette to agitate the mixture up and down to promote digestion. The digestion progress was checked regularly. Once the tissue fragments were sufficiently dissociated, PBS or adDMEM / F12+++ medium was added at a 1:5 ratio to terminate digestion. The cells were filtered through a 70µM cell sieve to remove larger fragments. After centrifugation at 300g for 10 minutes, the supernatant was removed to obtain the cell pellet.

[0064] 3) Organoid seeding: Resuspend the cell pellet in Matrigel to obtain a Matrigel suspension containing organoids, and seed the Matrigel suspension containing organoids onto the bottom of a preheated 48-well cell culture plate. Place the culture plate in a cell culture incubator at 37°C and 5% CO2 and incubate for 20-30 minutes to allow the Matrigel to solidify. 10 µM ROCK inhibitor, 100 µM Primocin, and 0.5 µM caspofungin were added to the conditioned medium (adDMEM / F12+++ medium containing 1× B27 supplement, 1.25 mM N-acetyl-L-cysteine, 10 mM nicotinamide, 50 ng / mL hEGF, 10 ng / mL hFGF-10, 5 ng / mL hFGF-2, 500 nM A83-01, 1 µM PGE2, 3 µM CHIR-99021, 1 µM laryngin, 4% RSPO3-Fc fusion protein conditioned medium, and 4% Noggin-Fc fusion protein conditioned medium), and the medium was preheated in a 37°C water bath. Then, 200 µL of organoid medium was slowly added to each well.

[0065] (2) Organoid culture and passage:

[0066] Incubate the culture plates at 37°C in a 5% CO2 cell culture incubator, changing the medium every 2-3 days to maintain organoid growth and stability. Passage the organoids after 12-14 days of culture. After aspirating the medium, add 500 µL TrypLE, gently disrupt the Matrigel with a pipette tip, and transfer the organoid suspension to a 15 mL centrifuge tube. Mix thoroughly by pipetting up and down. Incubate at 37°C until organoid dissociation occurs, pipetting up and down ≥10 times every 3-5 minutes to promote dissociation. After processing, terminate digestion with 10 mL adDMEM / F12+++ medium, centrifuge at 300 g for 10 minutes, discard the supernatant, and collect the cell pellet. Passage at a ratio of 1:2 to 1:5, resuspend the cell pellet in an appropriate amount of Matrigel, and seed evenly into new 48-well cell culture plates. The culture plates were incubated in a 37°C, 5% CO2 cell culture incubator for 20-30 minutes until Matrigel solidified. Preheated organoid culture medium was then slowly added. The culture plates were continued to be cultured in a 37°C, 5% CO2 cell culture incubator until the organoids reached the conditions required for drug screening experiments. During the first two passages, ROCK inhibitors, Primocin, and Caspofungin were continuously added to the culture medium to improve the success rate of organoid culture and maintain its biological characteristics.

[0067] (3) Organoid drug treatment:

[0068] 1) Organoid suspension preparation: Before screening, confirm that the organoid cells are in good condition and suitable for the experiment. Add 500 µL TrypLE to each well, gently disrupt the Matrigel structure with a pipette tip, and transfer the resulting organoid suspension to a 15 mL centrifuge tube. Mix thoroughly by pipetting to ensure uniform suspension of the organoids. Digest the organoids at 37°C until most organoids are observed to have been digested into single cells under a microscope. Add 10 mL of DMEM medium to stop digestion, centrifuge at 300 g for 10 minutes, discard the supernatant to obtain the cell pellet; resuspend the cell pellet in 1 mL of medium, filter through a 70 µM cell sieve, centrifuge, resuspend the cell pellet in Matrigel, and seed approximately 3000 cells per well into a 96-well plate. Incubate the plate at 37°C for 20-30 minutes, add 50 µL of organoid culture medium to each well, and incubate for 48 h.

[0069] 2) Drug treatment: After 48 hours of culture, the organoid culture medium was replaced with a medium containing 10 µM of drugs: niclosamide, gefitinib, and erlotinib were used as treatment drugs, with a negative control (0.1% DMSO) and a blank control group also included. Cell viability was assessed 72 hours after drug treatment.

[0070] 3) Cell viability assay for drug screening plates: Organoid viability was assessed using the ATP assay (CellTiter-Glo, Promega). CellTiter-Glo 3D reagent was thawed overnight at 4°C and placed in a 22°C water bath for approximately 30 minutes before use to equilibrate to room temperature. Organoid morphology was observed under a microscope before adding the cell viability assay reagent. An equal volume of CellTiter-Glo 3D reagent to each well was added and thoroughly mixed for 5 minutes to induce cell lysis. The culture plate was incubated at room temperature for another 25 minutes, and the fluorescence intensity was recorded using a detection instrument. Subsequently, fluorescence signal data were collected, and the relative fluorescence intensity of each treatment group was measured using a multi-well plate luminometer. The percentage of cell viability (or cell viability%) was calculated using the negative control group as a baseline. Statistical analysis was performed using GraphPad Prism to plot the results and analyze intergroup significance.

[0071] (4) Validation of the efficacy of niclosamide at concentration gradients:

[0072] 1) Drug treatment: The culture medium was replaced with different concentrations of niclosamide for drug treatment. The initial drug concentration was 100 µM, and seven concentration gradients were set up at a 3-fold dilution ratio (i.e., 100 µM; 33.3 µM; 11.1 µM; 3.7 µM; 1.23 µM; 0.41 µM; 0.13 µM). A negative control (0.1% DMSO) and a blank control group were also set up. Cell viability was measured after 72 h of drug treatment.

[0073] 2) Cell viability assay: Organoid viability was assessed using the ATP assay (CellTiter-Glo, Promega). CellTiter-Glo 3D reagent was thawed overnight at 4°C and placed in a 22°C water bath for approximately 30 minutes before use to equilibrate to room temperature. Organoid status was observed under a microscope before adding the cell viability assay reagent. An equal volume of CellTiter-Glo 3D reagent to each well was added and thoroughly mixed for 5 minutes to induce cell lysis. After incubation at room temperature for another 25 minutes, raw luminescence data were collected, and the calculation results were imported into GraphPad Prism to fit EC values. 50 They also estimated the survival rate of URP organoid cells under different concentrations of niclosamide treatment.

[0074] Among them, URP organoid LA-010-T was derived from adult nasal papilloma (HPV16+) samples, URP organoid LA-014-T was derived from pediatric laryngeal papilloma (HPV11+) samples, and URP organoids LA-015-T and LA-015-T were derived from adult pharyngeal papilloma (HPV-) samples.

[0075] Example 2

[0076] Niclosamide has a therapeutic effect on upper respiratory tract papillomas.

[0077] The results of this embodiment show that the growth of all four patient-derived URP organoids was inhibited in bright-field observation after treatment with niclosamide (10 μM). Representative images of URP organoids (taking organoid LA-010-T as an example) before and after niclosamide treatment are shown below. Figure 1 As shown in the figure. The above results indicate that the growth of URP organoids was significantly restricted after treatment with niclosamide, and that niclosamide has a therapeutic effect on upper respiratory tract papillomas.

[0078] Example 3

[0079] Preliminary drug screening results

[0080] This embodiment selected niclosamide, gefitinib, and erlotinib to treat URP organoids, and compared the viability of organoid cells after treatment with these three drugs. Gefitinib and erlotinib, through reversible competitive binding to the ATP-binding site of the EGFR kinase domain, inhibited its phosphorylation and activation of downstream signaling pathways (e.g., MAPK, PI3K / AKT). Furthermore, gefitinib and erlotinib have been reported (J., K., et al., Recurrent Respiratory Papillomatosis Successfully Treated with Gefitinib: A Case Study. AmericanJournal of Medical Case Reports, 2015.3(11): p. 352-358.; Limsukon, A., etal., Regression of recurrent respiratory papillomatosis with celecoxib and erlotinib combination therapy. Chest, 2009, 136(3): p. 924-926.) has a certain therapeutic effect on recurrent respiratory papilloma (RRP, a type of upper respiratory tract papilloma that clinically presents as recurrence). Niclosamide is an anti-tapeworm drug, mainly used to treat tapeworm infection.

[0081] The results are as follows Figure 2 As shown, the viability of URP organoid cells in the niclosamide-treated group was less than 20%, significantly lower than that in the erlotinib and gefitinib groups, with a high statistical difference (p<0.0001). The above results indicate that, compared with erlotinib and gefitinib, niclosamide treatment alone has a stronger inhibitory effect on the growth of upper respiratory tract papilloma cells, approximately 4 times that of erlotinib and approximately 14 times that of gefitinib. The reason for these results may be that this invention found that niclosamide may exert its inhibitory effect on upper respiratory tract papilloma growth through the calcium ion pathway, which is different from the reported antitumor pathways of niclosamide (e.g., the Wnt / β-catenin pathway).

[0082] Based on this, subsequent experiments selected niclosamide for concentration gradient efficacy verification in order to further evaluate its dose-dependent effect on URP organoid growth.

[0083] Example 4

[0084] Concentration gradient efficacy verification of niclosamide

[0085] In this embodiment, the initial concentration of niclosamide was 100 µM, and seven concentration gradients were set at a 3-fold dilution ratio. Cell viability was detected after 72 hours of drug treatment. Raw luminescence value data were collected, and the survival rate of upper respiratory tract papilloma organoid cells under different drug concentrations was calculated. The calculation results were imported into GraphPad Prism to fit EC5 values. 50 .

[0086] The results are as follows Figure 3 As shown, different concentrations of niclosamide can significantly reduce the survival rate of upper respiratory tract papilloma organoid cells and effectively inhibit the growth of upper respiratory tract papilloma organoid cells. Moreover, the cell survival rate decreases in a dose-dependent manner with the increase of niclosamide concentration.

[0087] Dose-response curves showed that EC50 of upper respiratory tract papilloma organoid cells from different patient sources... 50 The values ​​varied slightly, but the overall trend was consistent and all were low (all below 5 μM, and some even reached around 0.7 μM), demonstrating the targeted therapeutic ability of niclosamide for upper respiratory tract papillomas (whether HPV negative or HPV positive, whether adults or children, and whether nasal, pharyngeal or larynx).

[0088] It should be noted that, in this document, the terms "comprising," "including," or any other variations thereof are intended to cover non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements includes not only those elements but also other elements not expressly listed, or elements inherent to such a process, method, article, or apparatus. Unless otherwise specified, an element defined by the phrase "comprising one..." does not exclude the presence of other identical elements in the process, method, article, or apparatus that includes that element.

[0089] The embodiments of the present invention have been described above with reference to the accompanying drawings. However, the present invention is not limited to the specific embodiments described above. The specific embodiments described above are merely illustrative and not restrictive. Those skilled in the art can make many other forms under the guidance of the present invention without departing from the spirit and scope of the claims. All of these forms are within the protection scope of the present invention.

Claims

1. The use of niclosamide in the preparation of medicaments for treating upper respiratory tract papilloma, characterized in that, The drug has niclosamide as its sole active ingredient; the upper respiratory tract papillary tumor is HPV negative; and the upper respiratory tract papillary tumor is a benign tumor.

2. The use as described in claim 1, characterized in that, The upper respiratory tract papillomas occur in the nose, pharynx, and / or larynx.

Images