Application of compound in preparation of medicine for treating papilloma of upper respiratory tract

Abstract

The invention belongs to the field of biological medicines, and particularly relates to application of a compound to preparation of a medicine for treating papilloma of the upper respiratory tract. The invention provides an application of a compound in preparation of a medicine for treating and / or preventing papilloma of the upper respiratory tract, and is characterized in that the compound comprises one or more of asperisib, crizotinib, lapatinib, osimertinib and bortezomib. Experimental results show that the asperisib, crizotinib, lapatinib, osimertinib and bortezomib can realize long-term control on the papilloma of the upper respiratory tract and reduce the recurrence risk of the papilloma of the upper respiratory tract to a certain extent, so that the life quality of patients with the papilloma of the upper respiratory tract can be improved; the invention has important scientific value and clinical application prospect in the field of upper respiratory papilloma treatment.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of biological medicine, and particularly relates to a use of a compound in the preparation of a drug for treating upper respiratory papillomatosis. BACKGROUND

[0002] Upper respiratory papillomatosis (URP) is a benign tumor of squamous epithelium origin in the upper respiratory tract, characterized by recurrent papillary hyperplasia. Upper respiratory papillomatosis has an insidious onset, and its clinical manifestations are closely related to the growth pattern, location and size of the tumor, and lack of specificity. It can cause multiple sites such as larynx, trachea and bronchus to be involved, and cause symptoms such as cough, hoarseness and voice difficulty, and can cause respiratory obstruction in severe cases.

[0003] The occurrence of upper respiratory papillomatosis is closely related to human papillomavirus (HPV) infection. Epidemiological data shows that the disease is most common in the oropharynx, followed by the larynx, and least common in the nasopharynx, which may be related to the fact that the oropharynx is directly subjected to chronic irritation from the oral cavity (such as smoking, drinking and poor oral hygiene). Although upper respiratory papillomatosis is a benign lesion histologically, it has the characteristics of 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 squamous cell carcinoma, posing a major threat to the quality of life and life safety of patients.

[0004] Upper respiratory papillomatosis can occur at any age, and is usually divided into juvenile and adult types based on the age of 18 years. Juvenile URP is mainly transmitted vertically from mother to child and is closely related to maternal genital HPV infection, with a peak incidence before the age of 5. Its course is more invasive, with a higher recurrence rate, often involving the vocal cords, ventricular bands, and laryngeal surface of the epiglottis, and even spreading to the subglottic region, so the main clinical manifestation is hoarseness. If the lesion spreads downward along the airway, it can implant and grow in the trachea, bronchus and lung, further aggravating the disease. Adult URP is more common in men than in women and is associated with HPV infection routes such as smoking, sexual contact or mucosal exposure, with a peak incidence of 30-40 years old. Its progression is relatively slow, but the rate of malignant transformation is higher than that of juvenile type, and the lesion is often located at the glottic level, with the main symptoms of voice difficulty, progressive hoarseness, laryngeal stridor and dyspnea, and severe cases can be life-threatening. Airway obstruction, tumor malignant progression and secondary chronic lung disease are the main 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 a compound in the preparation of a medicament for the treatment and / or prevention of upper respiratory tract papilloma.

[0009] In some embodiments, the compound includes one or more of apelips, crizotinib, lapatinib, osimertinib, and bortezomib.

[0010] In some embodiments, the drug uses one or more of apelips, crizotinib, lapatinib, osimertinib, and bortezomib as its active ingredient.

[0011] In some embodiments, the drug uses apelips, crizotinib, lapatinib, osimertinib, or bortezomib as the sole active ingredient.

[0012] In some embodiments, the upper respiratory tract papilloma is HPV positive or HPV negative. In some embodiments, the upper respiratory tract papilloma is HPV positive.

[0013] In some embodiments, the upper respiratory tract papilloma includes at least one of HPV6 positive, HPV11 positive, HPV16 positive, and HPV18 positive.

[0014] In some embodiments, the upper respiratory tract papilloma is HPV negative.

[0015] In some embodiments, the upper respiratory tract papilloma is a juvenile upper respiratory tract papilloma or an adult upper respiratory tract papilloma.

[0016] In some embodiments, the upper respiratory tract papilloma occurs in the nose, pharynx, and / or larynx.

[0017] In some embodiments, the drug is used to inhibit the proliferation and / or growth of the upper respiratory tract papilloma.

[0018] In some embodiments, the half-maximal effective concentration of the drug is ≤3 μM.

[0019] In some embodiments, the dosage form of the drug includes injections, oral solutions, and / or tablets.

[0020] In some embodiments, the medicament further includes a pharmaceutically acceptable carrier and / or adjuvant.

[0021] In some embodiments, the drug is used to treat subjects with upper respiratory tract papilloma who are sensitive to the drug.

[0022] 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.

[0023] In some embodiments, the subject's sensitivity to the drug can be assessed by the half-maximal effective concentration (IC50) of the drug in an upper respiratory tract papillary organoid derived from the subject. Specifically, the assessment method may be: 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 ≤3 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 >3 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: 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.

[0027] 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.

[0028] In some embodiments, the first concentration may be adjusted according to the specific drug and the actual requirements of individualized treatment for subjects with upper respiratory tract papilloma.

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

[0030] In some embodiments, when the drug is apelips, the first concentration may be set to 0.5–2.3 µM. In some embodiments, when the drug is apelips, the first concentration may also be set to 0.70–1.20 µM, for example 1.15 µM (this value depends on…). Figure 2 EC of the four URP organoids shown 50 The value was calculated by averaging the values) and 0.79 µM (this value is calculated by averaging the values). Figure 2 EC shown 50 The EC values ​​after removing outliers are included. 50 The average value was used to select subjects with upper respiratory tract papillary tumors who were more sensitive to the drug (i.e., apeliximab).

[0031] In some embodiments, when the drug is crizotinib, the first concentration may be set to 0.4–1.3 µM. In some embodiments, when the drug is crizotinib, the first concentration may also be set to 0.60–0.90 µM, for example 0.81 µM (this value depends on…). Figure 4 EC of the four URP organoids shown 50 The value was calculated by averaging the values) and 0.67 µM (this value is calculated by averaging the values). Figure 4 EC shown 50 The EC values ​​after removing outliers are included. 50 The average value was used to select subjects with upper respiratory tract papillary tumors who were more sensitive to the drug (i.e., crizotinib).

[0032] In some embodiments, when the drug is lapatinib, the first concentration may be set to 0.6–2.1 µM. In some embodiments, when the drug is lapatinib, the first concentration may also be set to 0.90–1.30 µM, for example 0.26 µM (this value is based on…). Figure 6 EC of the four URP organoids shown 50 The value was calculated by averaging the values) and 0.98 µM (this value is calculated by averaging the values). Figure 6 EC shown 50 The EC values ​​after removing outliers are included. 50 The average value was used to select subjects with upper respiratory tract papillary tumors who were more sensitive to the drug (i.e., lapatinib).

[0033] In some embodiments, when the drug is osimertinib, the first concentration may be set to 0.4–2.9 µM. In some embodiments, when the drug is osimertinib, the first concentration may also be set to 0.40–1.20 µM, for example 1.17 µM (this value is based on…). Figure 8 EC of the four URP organoids shown 50 The value was calculated by averaging the values) and 0.45 µM (this value is calculated by averaging the values). Figure 8 EC shown 50 The EC values ​​after removing outliers are included. 50 The average value was used to select subjects with upper respiratory tract papillary tumors who were more sensitive to the drug (i.e., osimertinib).

[0034] In some embodiments, when the drug is bortezomib, the first concentration may be set to 0.03–2.03 µM. In some embodiments, when the drug is bortezomib, the first concentration may also be set to 0.04–0.60 µM, for example 0.54 µM (this value depends on…). Figure 10 EC of the four URP organoids shown 50 The value was calculated by averaging the values) and 0.05 µM (this value is calculated by averaging the values). Figure 10 EC shown 50 In the middle, EC after removing outliers 50 The average value was used to select subjects with upper respiratory tract papillary tumors who were more sensitive to the drug (i.e., bortezomib).

[0035] In some embodiments, the upper respiratory tract papilloma organoids are in the form of organoid suspensions.

[0036] In some embodiments, the method for constructing the upper respiratory tract papilloma organoid includes the following steps: S101. Obtain upper respiratory tract papillary tumor samples from the subject; S102. The upper respiratory tract papillary tumor sample is single-celled to obtain a single-celled upper respiratory tract papillary tumor. S103. The single-celled upper respiratory tract papilloma is cultured to obtain upper respiratory tract papilloma organoids.

[0037] 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.

[0038] In some embodiments, the conditioned medium includes: adDMEM / F12+++ medium.

[0039] 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.

[0040] 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.

[0041] In some embodiments, the conditioned medium may further include at least one of the following components: ROCK inhibitor, primoxane, and caspofungin.

[0042] In some embodiments, the conditioned medium may further comprise: 10 µM ROCK inhibitor, 100 µM primomocin, and 0.5 µM caspofungin.

[0043] In some embodiments, the first time period includes 10-20 days.

[0044] Compared with the prior art, the beneficial effects of the present invention include at least the following aspects: Although upper respiratory tract papillomas have a certain degree of recurrence and local invasiveness, they are classified as benign lesions (benign tumors) in tumor classification. Benign tumors and malignant tumors differ significantly in their biological behavior and molecular mechanisms, and targeted drugs for malignant tumors may be difficult to effectively treat upper respiratory tract papillomas.

[0045] The experimental results of this invention also confirm that antitumor drugs capable of treating malignant tumors have very limited inhibitory effects on the proliferation and / or growth of upper respiratory tract papillomas. Only apelixir, crizotinib, lapatinib, osimertinib, and bortezomib have shown the ability to effectively inhibit the proliferation and growth of this benign tumor. In other words, the experimental results of this invention indicate that apelixir, crizotinib, lapatinib, osimertinib, and bortezomib can achieve long-term control of upper respiratory tract papillomas and reduce the risk of recurrence to some extent, thus improving the quality of life for patients with upper respiratory tract papillomas. This has significant scientific value and clinical application prospects in the treatment of upper respiratory tract papillomas.

[0046] Furthermore, considering that in the individualized treatment of upper respiratory tract papilloma, different subjects may have different responses to the same drug and the same subject may have different responses to different drugs, the present invention can also assess the subject's sensitivity to the above-mentioned drugs based on the upper respiratory tract papilloma organoids derived from the subject, select the sensitive drugs for the target subject, 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 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 A comparison of the inhibitory effects of apelelis and other drugs on upper respiratory tract papillary organoids; Figure 2 The results of cell survival of upper respiratory tract papilloma organoids under different concentrations of apelips are shown in the figure. Figure 3 A comparison of the inhibitory effects of crizotinib and other drugs on upper respiratory tract papillary organoids; Figure 4The figure shows the survival rate of upper respiratory tract papilloma organoid cells after treatment with different concentrations of crizotinib. Figure 5 A comparison of the inhibitory effects of lapatinib and other drugs on upper respiratory tract papillary organoids; Figure 6 The results of lapatinib treatment on the survival rate of upper respiratory tract papilloma organoid cells are shown in the figure. Figure 7 A comparison of the inhibitory effects of osimertinib and other drugs on upper respiratory tract papillary organoids; Figure 8 The results of osimertinib treatment on the survival rate of upper respiratory tract papilloma organoid cells are shown in the figure. Figure 9 A comparison of the inhibitory effects of bortezomib and other drugs on upper respiratory tract papillary organoids; Figure 10 The figure shows the survival rate of upper respiratory tract papilloma organoid cells under different concentrations of bortezomib treatment. Detailed Implementation

[0049] 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.

[0050] In this document, "and / or" includes any and all combinations of one or more of the listed related items.

[0051] In this article, "multiple" means two or more, that is, it includes two, three, four, five, etc.

[0052] 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.

[0053] 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.

[0054] Example 1 Use of apelips in the preparation of medicines for the treatment and / or prevention of upper respiratory tract papilloma The materials, reagents, and methods involved in the following experiments in this embodiment are as follows: I. Materials and Reagents 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.

[0055] 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, saliva extract, 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, Apelixir, Everolimus.

[0056] II. Experimental Methods (1) Generation of patient-derived upper respiratory tract papilloma organoids (URP organoids): 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.

[0057] 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.

[0058] 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.

[0059] (2) Organoid culture and passage: 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.

[0060] (3) Organoid drug treatment: 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.

[0061] 2) Drug treatment: After 48 hours of culture, the organoid culture medium was replaced with a medium containing 10 µM of drugs: the treatment drugs were apelips and everolimus, and a negative control (0.1% DMSO) and a blank control group were set up. Cell viability was measured 72 hours after drug treatment.

[0062] 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.

[0063] (4) Validation of the efficacy of apelelis at concentration gradients: 1) Drug treatment: The culture medium was replaced with different concentrations of apelelis 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.

[0064] 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 The survival rate of URP organoids treated with different concentrations of apelips was estimated.

[0065] 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.

[0066] III. Experimental Results Preliminary drug screening results This embodiment selected apelisib and everolimus, both capable of acting on the PI3K-AKT-mTOR pathway, to treat URP organoids, and compared the cell viability of organoids treated with apelisib and everolimus (average values ​​of URP organoids LA-010-T, LA-014-T, and LA-015-T, hereinafter the same). Apelisib is a small molecule drug that selectively inhibits PI3Kα kinase activity, blocking upstream signaling in the PI3K-AKT-mTOR pathway; while everolimus is a small molecule drug that selectively inhibits mTORC1 kinase activity, blocking downstream signaling in the PI3K-AKT-mTOR pathway.

[0067] In this embodiment (the same below), the initial screening standard for drugs is set at 20%, that is, drugs with cell viability ≤ 20% are considered to have therapeutic potential and are subsequently screened for drugs at concentration gradients.

[0068] The results are as follows Figure 1 As shown, the cell viability in the apelisi-treated group was approximately 20%, significantly lower than the 60% in the everolimus-treated group, with a highly statistically significant difference between the two groups (p<0.0001). These results indicate that apelisi, acting upstream of the PI3K-AKT-mTOR pathway, significantly inhibits the growth of upper respiratory tract papilloma organoids, with a much stronger inhibitory effect than everolimus, which acts downstream of the PI3K-AKT-mTOR pathway.

[0069] Based on this, subsequent experiments in this embodiment selected apelelis for concentration gradient efficacy verification in order to further evaluate its dose-dependent effect on the growth of upper respiratory tract papilloma organoids.

[0070] Pharmacodynamic validation of apelips at concentration gradients In this embodiment, the initial concentration of apelelis was 100 µM, and seven concentration gradients were set at a 3-fold dilution ratio. Cell viability was measured 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 .

[0071] The results are as follows Figure 2 As shown, different concentrations of apelips significantly reduced the survival rate of upper respiratory tract papillary organoid cells and effectively inhibited their growth. Furthermore, the cell survival rate decreased in a dose-dependent manner with increasing apelips concentration.

[0072] 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 2.5 μM, and some even reached around 0.5 μM), demonstrating the targeted therapeutic ability of apelelis for upper respiratory tract papilloma (whether HPV negative or HPV positive, whether adult or child, whether nasal, pharyngeal or larynx).

[0073] Example 2 Use of crizotinib in the preparation of medicines for the treatment and / or prevention of upper respiratory tract papilloma I. Materials and Reagents The materials used in this embodiment are the same as in Embodiment 1. For the reagents, apelips and everolimus are replaced with crizotinib, pazopanib, and lenvatinib, while the other reagents remain the same.

[0074] II. Experimental Method: Same as Example 1 III. Experimental Results Preliminary drug screening results This embodiment selected crizotinib, pazopanib, and lenvatinib, all tyrosine kinase inhibitors, to treat URP organoids and compared the cell viability of organoids treated with these three drugs. Crizotinib inhibits the activity of the ALK / ROS1 fusion protein, blocking downstream signaling pathways (such as the PI3K / AKT / mTOR and MAPK pathways); pazotinib's main targets are VEGFR-1 / 2 / 3, PDGFR-α / β, and c-Kit, blocking VEGF and PDGF-mediated angiogenesis signaling pathways; lenvatinib's main targets are VEGFR-1 / 2 / 3, FGFR-1 / 2 / 3 / 4, PDGFR-α, RET, and c-Kit, and it blocks multi-pathway signal transduction through a dual mechanism of inhibiting angiogenesis (VEGFR / FGFR) and tumor cell proliferation (RET / c-Kit).

[0075] In other words, all three drugs are tyrosine kinase inhibitors, but the number of targets differs: (1) Crizotinib has the fewest targets, mainly ALK / ROS1; (2) Pazotinib has a moderate number of targets, mainly anti-angiogenic; and (3) Lenvatinib has the most targets, mainly anti-angiogenic and cell proliferation inhibition.

[0076] The results are as follows Figure 3As shown, the cell viability in the crizotinib treatment group was approximately 15%, significantly lower than the 55% in the pazotinib group and the 70% in the lenvatinib group, indicating a high statistical difference among the three groups. These results suggest that among different tyrosine kinase inhibitors, compared to pazotinib and lenvatinib, which have more targets and are anti-angiogenic, crizotinib, which has fewer targets and is not anti-angiogenic, has a stronger inhibitory effect on the growth of upper respiratory tract papilloma cells.

[0077] Based on this, subsequent experiments in this embodiment selected crizotinib for concentration gradient efficacy verification in order to further evaluate its dose-dependent effect on the growth of upper respiratory tract papilloma organoids.

[0078] Concentration gradient efficacy validation of crizotinib In this embodiment, the initial concentration of crizotinib was 100 µM, and seven concentration gradients were set at a 3-fold dilution ratio. Cell viability was measured 72 h after 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 the EC50 values. 50 .

[0079] The results are as follows Figure 4 As shown, different concentrations of crizotinib can significantly reduce the survival rate of upper respiratory tract papillary organoid cells and effectively inhibit the growth of upper respiratory tract papillary organoid cells. Moreover, the cell survival rate decreases in a dose-dependent manner with the increase of crizotinib concentration.

[0080] 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 1.5 μM, and some even reached around 0.5 μM), demonstrating crizotinib's targeted therapeutic ability for upper respiratory tract papilloma (whether HPV negative or HPV positive, whether adult or child, whether nasal, pharyngeal or larynx).

[0081] Example 3 Use of lapatinib in the preparation of drugs for the treatment and / or prevention of upper respiratory tract papilloma I. Materials and Reagents The materials used in this embodiment are the same as in Embodiment 1. For the reagents, apeliximab and everolimus are replaced with lapatinib, gefitinib, and erlotinib, while the other reagents remain the same.

[0082] II. Experimental Method: Same as Example 1 III. Experimental Results Preliminary drug screening results This embodiment selected lapatinib, gefitinib, and erlotinib, all of which are epidermal growth factor receptor tyrosine kinase inhibitors, to treat URP organoids and compared the cell viability of organoids treated with these three drugs. Gefitinib and erlotinib inhibited EGFR phosphorylation and activation of downstream signaling pathways (e.g., MAPK, PI3K / AKT) by reversibly competitively binding to the ATP-binding site of the EGFR kinase domain; while lapatinib inhibited signal transduction by covalently binding to the ATP-binding site of EGFR / HER2.

[0083] The results are as follows Figure 5 As shown, lapatinib exhibited the most significant inhibitory effect on cell viability, with cell viability in the treated group being approximately 5%. In contrast, gefitinib and erlotinib showed weaker or moderate inhibitory effects, with cell viability after treatment at 60% and 23%, respectively, demonstrating a high degree of statistical difference among the three groups. These results indicate that not all epidermal growth factor receptor tyrosine kinase inhibitors are effective in inhibiting upper respiratory tract papillomas, with lapatinib exhibiting a relatively strong inhibitory effect.

[0084] Based on this, subsequent experiments in this embodiment selected lapatinib for concentration gradient efficacy verification in order to further evaluate its dose-dependent effect on the growth of upper respiratory tract papilloma organoids.

[0085] Concentration gradient efficacy validation of lapatinib In this embodiment, the initial concentration of lapatinib was 100 µM, and seven concentration gradients were set at a 3-fold dilution ratio. Cell viability was measured 72 h after 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 the EC50 values. 50 .

[0086] The results are as follows Figure 6 As shown, different concentrations of lapatinib can significantly reduce the survival rate of upper respiratory tract papillary organoid cells and effectively inhibit the growth of upper respiratory tract papillary organoid cells. Moreover, the cell survival rate decreases in a dose-dependent manner with the increase of lapatinib concentration.

[0087] Dose-response curves showed that EC50 of upper respiratory tract papilloma organoid cells from different patient sources... 50While the values ​​varied slightly, the overall trend was consistent and low (all below 2.5 μM, some even reaching around 0.5 μM), demonstrating lapatinib's targeted therapeutic ability against upper respiratory tract papilloma (whether HPV-negative or HPV-positive, adult or child, nasal, pharyngeal, or larynx). The above experimental results show that lapatinib has good inhibitory effect on the proliferation of upper respiratory tract papilloma organoids. EC 50 The values ​​were all at low to medium micromolar levels, suggesting that it can effectively intervene in cell proliferation pathways within a reasonable concentration range. The dose-response curves for various organs all exhibited typical Sigmoid curves, with a significant decrease in cell survival rate in the high concentration range, and differences in the inhibitory slope and onset point for different organ types in the low concentration range.

[0088] Example 4 Use of osimertinib in the preparation of medicines for the treatment and / or prevention of upper respiratory tract papilloma I. Materials and Reagents The materials used in this embodiment are the same as in Embodiment 1. For the reagents, apeliximab and everolimus are replaced with osimertinib, gefitinib, and erlotinib, while the other reagents remain the same.

[0089] II. Experimental Method: Same as Example 1 III. Experimental Results Preliminary drug screening results This embodiment selected osimertinib, gefitinib, and erlotinib, all of which are epidermal growth factor receptor tyrosine kinase inhibitors, to treat URP organoids and compared the cell viability of organoids treated with these three drugs. Gefitinib and erlotinib reversibly competitively bind to the ATP-binding site of the EGFR kinase domain, inhibiting its phosphorylation and activation of downstream signaling pathways (such as MAPK and PI3K / AKT); while osimertinib specifically targets EGFR-sensitive mutations and EGFR T790M resistance mutations, and its binding to EGFR is irreversible and covalent.

[0090] The results are as follows Figure 7 As shown, osimertinib exhibited the most significant inhibitory effect on cell viability, with cell viability in the treated group being approximately 3%. In contrast, gefitinib and erlotinib showed weaker or moderate inhibitory effects, with cell viability after treatment at 60% and 23%, respectively, demonstrating a high degree of statistical difference among the three groups. These results indicate that not all epidermal growth factor receptor tyrosine kinase inhibitors are effective in inhibiting upper respiratory tract papillomas, with osimertinib exhibiting a relatively strong inhibitory effect.

[0091] Based on this, osimertinib was selected for concentration gradient efficacy verification in subsequent experiments of this embodiment, in order to further evaluate its dose-dependent effect on organoid growth.

[0092] Concentration gradient efficacy validation of osimertinib In this embodiment, the initial concentration of osimertinib was 100 µM, and seven concentration gradients were set at a 3-fold dilution ratio. Cell viability was measured 72 h after 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 the EC50 values. 50 .

[0093] The results are as follows Figure 8 As shown, different concentrations of osimertinib can significantly reduce the survival rate of upper respiratory tract papillary organoid cells and effectively inhibit the growth of upper respiratory tract papillary organoid cells. Moreover, with the increase of osimertinib concentration, the cell survival rate decreases in a dose-dependent manner.

[0094] 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 3.0 μM, and some even reached around 0.5 μM), demonstrating osimertinib's targeted therapeutic ability for upper respiratory tract papilloma (whether HPV negative or HPV positive, whether adult or child, whether nasal, pharyngeal or larynx).

[0095] Example 5 Use of bortezomib in the preparation of medicines for the treatment and / or prevention of upper respiratory tract papillomas I. Materials and Reagents The materials used in this embodiment are the same as in Embodiment 1. For the reagents, apelips and everolimus are replaced with bortezomib, enalapril, leflunomide, lorafalicyl, hydroxyphenylxanthine, and ziloutong. The other reagents are the same.

[0096] II. Experimental Method: Same as Example 1 III. Experimental Results Preliminary drug screening results This embodiment selected six inhibitors of key tumor growth enzymes—bortezomib, enalapril, leflunomide, lorafalinib, pentoxifylline, and zileuton—to treat URP organoids, and compared the cell viability of organoids treated with these six drugs. Enalapril acts on the angiotensin system, leflunomide on the pyrimidine synthesis pathway, lorafalinib on the Ras pathway, pentoxifylline and zileuton on inflammatory mediators, while bortezomib is the only drug targeting the proteasome.

[0097] The results are as follows Figure 9 As shown, bortezomib exhibited the most significant inhibitory effect on cell viability, with cell viability in its treatment group being only about 2%, significant organoid apoptosis and structural damage, and a significant decrease in luminescence signal. In contrast, the other drugs showed weaker or moderate inhibitory effects, with cell viability values ​​all greater than 50%. This indicates that not all inhibitors of key tumor growth enzymes are effective against upper respiratory tract papillomas; only bortezomib, which targets the proteasome, showed a strong inhibitory effect on upper respiratory tract papillomas.

[0098] Based on this, subsequent experiments in this embodiment selected bortezomib for concentration gradient efficacy verification in order to further evaluate its dose-dependent effect on organoid growth.

[0099] Concentration gradient efficacy validation of bortezomib In this embodiment, the initial concentration of bortezomib was 100 µM, and seven concentration gradients were set at a 3-fold dilution ratio. Cell viability was measured 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 the EC50 values. 50 .

[0100] The results are as follows Figure 10 As shown, different concentrations of bortezomib 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 bortezomib concentration.

[0101] Dose-response curves showed that EC50 of upper respiratory tract papilloma organoid cells from different patient sources... 50The values ​​varied slightly, but the overall trend was consistent and all were low (all below 2.5 μM, and some even reached around 0.1 μM), demonstrating bortezomib's targeted therapeutic ability for upper respiratory tract papilloma (whether HPV negative or HPV positive, whether adult or child, whether nasal, pharyngeal or larynx).

[0102] 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.

[0103] 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. Use of a compound in the preparation of a medicament for treating and / or preventing upper respiratory tract papilloma, characterized in that, The compounds include one or more of apelipsifaciens, crizotinib, lapatinib, osimertinib, and bortezomib, and the upper respiratory tract papilloma is HPV positive or HPV negative.

2. The use as described in claim 1, characterized in that, The upper respiratory tract papillary tumor was HPV positive.

3. The use as described in claim 1, characterized in that, The upper respiratory tract papillary tumor was HPV negative.

4. The use as described in claim 1, characterized in that, The drug is used to inhibit the proliferation and / or growth of the upper respiratory tract papilloma.

5. The use as described in claim 1, characterized in that, The half-maximal effective concentration of the drug is ≤3 μM.

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

7. The use as described in claim 1, characterized in that, The drug is used to treat subjects with upper respiratory tract papillary tumors that are sensitive to the drug.

8. The use as described in claim 7, characterized in that, The subject's sensitivity to the drug was assessed based on upper respiratory tract papilloma organoids derived from the subject.

9. The use as described in claim 8, characterized in that, The subject is assessed as sensitive to the drug when the half-maximal effective concentration of the drug for upper respiratory tract papillary organoids derived from the subject is ≤3 μM.

10. The use as described in claim 8, characterized in that, 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 the first concentration of the drug is ≤ a preset cell viability threshold; wherein the preset cell viability threshold is 50%; when the drug is apelipsiproxetine, the first concentration is 0.5 – 2.3 µM; when the drug is crizotinib, the first concentration is 0.4 – 1.3 µM; when the drug is lapatinib, the first concentration is 0.6 – 2.1 µM; when the drug is osimertinib, the first concentration is 0.4 – 2.9 µM; when the drug is bortezomib, the first concentration is 0.03 – 2.03 µM.

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