WO2020015731A1

WO2020015731A1 - Uses of sulfoxide compound for preventing radiation-induced oral mucositis and related radiotherapy complications

Info

Publication number: WO2020015731A1
Application number: PCT/CN2019/096739
Authority: WO
Inventors: 从玉文; 善亚军
Original assignee: 北京蔚蓝之源医药科技有限公司
Priority date: 2018-07-20
Filing date: 2019-07-19
Publication date: 2020-01-23
Also published as: WO2020015732A1; CN112437673A; CN110731954A; CN112584866A

Abstract

Provided are the uses of a sulfoxide compound for preparing a preventive drug for head and neck cancers to be administered to humans receiving radiotherapy so as to avoid radiation-induced oral mucositis and related radiotherapy complications. The uses of said sulfoxide compound for preventing humans having head and neck cancers to suffer from radiation-induced oral mucositis and related radiotherapy complications exhibits an excellent effect.

Description

Use of sulfoxide compounds in preventing radiation oral mucositis and related radiotherapy complications

This application claims priority from Chinese application 201810800390.9, filed on July 20, 2018, and entitled "Use of Dimethyl Sulfoxide in the Prevention of Radiation Oral Mucositis", the contents of which are incorporated herein by reference.

Technical field

The invention relates to the field of medicine, in particular to the use of sulfoxide compounds, in particular dimethyl sulfoxide, for preventing radiation oral mucositis and related radiotherapy complications.

Background technique

Tumor radiation therapy is a local treatment method using radiation to treat tumors. While killing tumor cells, radiation also has a killing effect on normal cells, resulting in damage to the irradiated tissues and organs and various complications. Radiation oral mucositis (ROM) is a serious side effect that often occurs in patients with head and neck tumors undergoing radiotherapy. Severe ROM restricts patients' food and water intake, which leads to weight loss and malnutrition. Loss of the epithelial and basement membrane barriers of mucous membranes can easily cause bacterial infections, which may induce bacteremia, pose a serious threat to life, and even interrupt the treatment of the primary disease of the tumor. Every year, tens of thousands of cancer patients suffer from mouth ulcer pain, loss of taste function, and local and systemic infections as a result of receiving radiation therapy. It has been reported that the incidence of ROM in patients with head and neck cancer is as high as 80%, while that of patients receiving hyperfractionated radiotherapy (2 or more radiation doses per day) has an incidence of 100%. Side effects of oral mucositis caused by radiation therapy include oral pain in 69% of patients, dysphagia in 56% of patients, opioids in 53% of patients, and nasal feeding and hospitalization in 5% of patients. 11% -16%.

Oral mucositis caused by tumor treatment has challenged oncologists in many aspects, such as the adjustment of radiation dose restrictions and the negative impact on patients' quality of life. The drugs and measures currently used to prevent or treat ROM either have narrow indications or limited efficacy. For example, benzylamine hydrochloride was an earlier drug used as a mouthwash for the intervention of oral mucositis, but later a large number of clinical trials have proved that benzylamine hydrochloride is not effective for ROM. In 2004, Parivimine (Kepivance) was approved by the US FDA to prevent oral mucositis caused by pretreatment of hematopoietic stem cell transplantation in hematological malignancies. Due to the pro-proliferative effect of tumor cells derived from epithelial tissue, palipimin is not suitable for patients with non-hematological tumors. Amifostine (WR2721) is a drug approved by the US FDA for the prevention of xerostomia after radiation therapy in patients with head and neck tumors. However, amifostine has large side effects and the clinical effect of preventing ROM is not ideal.

Considering these shortcomings of the above methods, there is still a need to develop new drugs that can prevent the occurrence of radiation oral mucositis and related radiotherapy complications, especially to effectively prevent radiation oral mucositis in head and neck tumor subjects who will receive radiation therapy. And related radiotherapy complications.

Summary of the invention

In one aspect, the present invention provides the use of a sulfoxide compound in the preparation of a pharmaceutical composition for preventing radiation oral mucositis and related radiotherapy complications in a tumor-bearing subject who will receive radiation therapy.

In another aspect, the present invention provides a sulfoxide compound or a pharmaceutical composition containing the sulfoxide compound, which is used to prevent radiation-induced oral mucositis and related radiotherapy complications in a tumor subject who will receive radiation therapy.

In yet another aspect, the present invention provides a method for preventing radiation oral mucositis and related radiotherapy complications using a sulfoxide compound or a pharmaceutical composition containing the sulfoxide compound, including the following steps: administering to a subject suffering from a tumor receiving radiation therapy A sulfoxide compound or a drug or a pharmaceutical composition comprising the sulfoxide compound.

In one embodiment, the sulfoxide compound is selected from one or more of the following: dimethyl sulfoxide (DMSO), tetramethylene sulfoxide (tetramethylene sulfoxide), diethyl sulfoxide, methyl Ethyl sulfoxide, propyl sulfoxide, diisopropyl sulfoxide, n-butyl sulfoxide, diisobutyl sulfoxide, diisopentyl sulfoxide, diphenyl sulfoxide, and dibenzyl sulfoxide.

In one embodiment, the sulfoxide compound is DMSO or tetramethylene sulfoxide, preferably DMSO.

In one embodiment, the sulfoxide compound is DMSO.

In one embodiment, the related radiotherapy complications are selected from the group consisting of: esophageal mucosal damage, taste dysfunction, salivary gland secretion, radioactive thyroid disease, radioactive xerostomia, radioactive taste disorders, reduced number of tongue taste bud cells after radiation, radioactivity Dental caries, radiation esophagitis, and radiation esophageal stricture.

In one embodiment, the radioactive thyroid disease is selected from one or more of the following: radioactive hypothyroidism, chronic radioactive thyroiditis, acute radioactive thyroiditis, and radioactive benign nodules.

In one embodiment, the subject with a tumor may be a subject who will receive a combination of radiation and chemotherapy.

In one embodiment, the subject suffering from a tumor is a subject suffering from a tumor of the head and neck, preferably a human suffering from a tumor of the head and neck.

In one embodiment, the head and neck tumor comprises one or more selected from the group consisting of a neck tumor, an ENT tumor, and an oral and maxillofacial tumor. In one embodiment, the neck tumor is a thyroid tumor. In one embodiment, the ENT tumor is nasopharyngeal cancer, laryngeal cancer, or paranasal sinus cancer. In one embodiment, the oral and maxillofacial tumor is oral cancer, such as tongue cancer, gingival cancer, buccal cancer, maxillary sinus cancer, and tonsil cancer.

In one embodiment, the subject with a tumor is a head and neck tumor subject who will receive radiation therapy within 8 hours, preferably within 4 hours, more preferably within 2 hours, and most preferably within 1 hour.

In one embodiment, the subject with a tumor is a head and neck tumor subject who will receive chemotherapy within 8 hours, preferably within 4 hours, more preferably within 2 hours, and most preferably within 1 hour.

In one embodiment, the pharmaceutical composition is a dosage form suitable for an administration mode selected from the group consisting of intravenous administration, oral mucosal administration, transdermal administration, and oral administration.

In one embodiment, the pharmaceutical composition is a dosage form selected from the group consisting of an infusion, injection, mouthwash, film, aerosol, tablet, adhesive, patch, sublingual , Disintegrants, sticks, powders, ointments, gels, aqueous solutions, suspensions and capsules.

In one embodiment, the pharmaceutical composition is administered at a dose of 0.1-10.0 g of sulfoxide compounds, preferably 0.2-5.0 g of sulfoxide compounds, more preferably 0.4-2.0 g of sulfoxide compounds, and most preferably 1.0 g of sulfoxide compounds. Sulfone compounds per kg of body weight.

In one embodiment, the pharmaceutical composition optionally includes one or more compounds selected from the group consisting of WR2721, PrC-210 (aminothiol), WR-1065, WR-3689, WR-151327, WR-638 , WR-77913, WR-44923, cysteamine, AET, lipoic acid, N, N-diethyllipoamide, Mn-SOD transgenic preparation, Cu.Zn-SOD preparation, Fe-SOD preparation, GC4419 (M40403 ), MnBuOE, AEOL10150, MnTnHex-2-PyP5 + (hexyl), MnTE-2-PyP, EUK-207, EUK-189, EUK-134, melatonin, vitamin E and its derivatives, pentoxifylline, glutamate Aminoamide, Tempol (4-hydroxy-2,2,6,6-tetramethylpiperidine-N-oxyl), Edaravone, Vitamin C and its derivatives, Vitamin A and its derivatives, D-methionine, Acetylcysteine, glutathione, 3,3'-diindolylmethane, Oltipraz, Tetrahydrobiopterin, Metformin (metformin), β-carotene, FTY720, RPC1063, BAF312, ACT-128800, KRP203, MT-1303, CBLB502, CBLB612, CBLB613, R-Spondin1, Tat-Smad7, rapamycin, siro Sirolimus, Everolimus (RAD001), ABI-009, Tobramycin, Tetracyclines, Chlorhexidine, Amphotericin B, Nystatin ( nystatin, Zovirax, brilacidin, Triamcinolone acetonide, betamethasone, prednisone, hydrocortisone acetate, etc., Benzydamine, Indomethacin, misoprostol, COX-2 inhibitor, prostaglandin E1, prostaglandin E2, salicylic acid derivatives, Azelastine, diphenhydramine ), Morphine, fentanyl,

(delta-9-tetrahydrocannabinol and cannabidiol), doxepin, lidocaine, dacronine hydrochloride, benzocaine, propantheline, palefermin, Granulocyte colony-stimulating factor (GM-CSF), keratinocyte growth factor (KGF), fibroblast growth factor (FGF), epidermal growth factor (EGF), transforming growth factor (TGF-β), granulocyte colony-stimulating factor (G-CSF), IZN-6N4,

Traumeel, Glycyrrhizic acid, Glycyrrhetinic acid and capsaicin.

In one embodiment, the radiation therapy includes treatment using a radiation selected from the group consisting of alpha rays, beta rays, gamma rays, x-rays, neutrons, electron beams, proton beams, particle beams, and combinations thereof.

In one embodiment, the radiation therapy includes external radiation therapy and internal radiation therapy.

In one embodiment, the radiation therapy method includes conventional segmented radiation therapy and a high-dose radiation therapy.

In one embodiment, a single irradiation dose of the radiotherapy is 1-50 Gy, more preferably 1-8 Gy, and most preferably 2 Gy.

In one embodiment, the radiation therapy includes the use of a radiosensitizer.

In one embodiment, the radiosensitizer is a hypoxia cell radiosensitizer and a non-hypoxia cell radiosensitizer,

The hypoxic cell radiosensitizer is selected from one or more of the following: Nitroimidazoles, such as metronidazole, imidazole, MISO (Ro-07-0582), RSU-1096, Tirapazamine, etc .; and biological reducing agents, such as sodium glycinazole, nitrogen oxides, quinones, and nicotinamide and its derivatives;

The non-hypoxic cell radiosensitizer is selected from one or more of the following: platinum drugs, such as cisplatin, carboplatin, oxaliplatin, etc .; anti-metabolic drugs, such as 5-FU, capecitabine , Gemcitabine, etc .; topoisomerase inhibitors, such as camptothecin, irinotecan, topotecan, CPT417, BMS-286309, etoposide (VP-16), etc .; Microtubule-stabilizing drugs, such as paclitaxel, docetaxel, patupilone, PBOX-15, etc .; second mitochondrial-derived cystinase activator (SMAC) analogs, such as Tat-SMACN7, SM- 164, LCL161, etc .; tumor targeted therapy drugs, such as Sorafenib, Erlotinib, Cetuximab, Bevacizumab, Trastuzumab (trastuzumab), etc .; photosensitizers, such as 5-aminoketovaleric acid, hematoporphyrin monomethyl ether, hematoporphyrin derivative (HPD), dimonium porphyrin ether (DHE), Photofrin II, etc .; DNA alkylating agents, such as Temozolomide, etc .; and traditional Chinese medicine extracts, such as purpurin.

The present invention confirms that sulfoxide compounds, especially DMSO, have a good preventive effect on other related complications of radiation therapy for patients with head and neck tumors.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1a-c: Comparison of the preventive effects of different doses of DMSO on radiation oral mucositis;

Figure 2a-c: Comparison of the preventive effects of DMSO on radiation oral mucositis at different times before irradiation;

Figures 3a-c: Post-dose administration of DMSO has no therapeutic effect on radiation oral mucositis;

Figure 4a-c: Comparison of the preventive effects of DMSO, rhKGF and WR2721 on radiation oral mucositis;

Figures 5a-b: Comparison of DMSO, rhKGF, and WR2721 improvement in body weight and survival of high-dose head and neck irradiation mice;

Figure 6a-c: Preventive effect of DMSO on oral mucositis caused by split head and neck irradiation in mice;

Figure 7a-d: Keratinized epithelial stem, progenitor cell changes and DMSO protection at different time points after head and neck irradiation in mice;

Figure 8a-d: The proliferation of keratinized epithelial cells and the protective effect of DMSO at different time points after head and neck irradiation in mice;

9a-b: DMSO promotes repair of DNA damage of tongue keratinized epithelial stem cells after head and neck irradiation in mice;

Figure 10a-c: Protective effect of dimethyl sulfide, dimethyl sulfone and tetramethylene sulfoxide on radiation-induced oral mucositis in head and neck irradiated mice;

Figure 11a-d: DMSO has no radioprotective effect on head and neck tumor cells of tumor-bearing mice;

Figure 12a-b: Effect of DMSO on body weight and survival of mice treated with chemotherapy combined with head and neck irradiation; and

Figure 13: Preventive effect of DMSO on esophageal mucosal damage in head and neck irradiated mice.

detailed description

When the present inventors screened a new type of protective agent for radiation oral mucositis, it was unexpectedly discovered that in a mouse model of radiation oral mucositis, only mice in the control group irradiated with dimethylsulfoxide (DMSO) solvent were exposed to radiation damage The degree is far lower than that of the control group. This unexpected discovery led the inventors to carry out in-depth research on the role of DMSO in the treatment and prevention of radiation injury, and found that intraperitoneal or oral administration of DMSO can effectively prevent radiation oral mucositis. The invention further confirms that DMSO also has a good preventive effect on other related complications of radiation therapy for head and neck tumor subjects, and its protective effect is related to the sulfoxide group it contains. Further research confirms that DMSO can also prevent the occurrence of radiation oral mucositis in subjects with head and neck tumors who will receive radiation and chemotherapy.

In a first aspect, the present invention provides the use of a sulfoxide compound in the preparation of a pharmaceutical composition for the prevention of radiation oral mucositis and related radiotherapy complications in a tumor-bearing subject who will receive radiation therapy.

The term "sulfoxide compounds" is a class of organic compounds containing thionyl (> S = O) functional groups, and its general formula is RS = O-R ', where R and R' are organic groups, for example, optionally Substituted methyl, optionally substituted ethyl, optionally substituted propyl, optionally substituted butyl, optionally substituted phenyl, optionally substituted allyl, and the like. The "sulfoxide compound" in the present invention refers to a sulfoxide compound containing a sulfoxide group.

The term "dimethyl sulfoxide" (DMSO) refers to compounds of the following structure

DMSO is a common solvent. It is a colorless, odorless, transparent liquid at room temperature with good thermal stability and low toxicity.

In one embodiment, the sulfoxide compound is selected from one or more of the following: dimethyl sulfoxide (DMSO), tetramethylene sulfoxide (tetramethylene sulfoxide), diethyl sulfoxide, methyl Ethyl sulfoxide, propyl sulfoxide, diisopropyl sulfoxide, n-butyl sulfoxide, diisobutyl sulfoxide, diisopentyl sulfoxide, diphenyl sulfoxide, and dibenzyl sulfoxide. In one embodiment, the sulfoxide compound is DMSO or tetramethylene sulfoxide. In one embodiment, the sulfoxide compound is DMSO.

The term "subject" refers to mammals including humans and non-humans, including but not limited to humans, pigs, dogs, rabbits, monkeys, cats, and the like. The subject of the present invention is selected from subjects having a tumor. In one embodiment, the subject is a human. Herein, the terms "subject," "patient," and "subject" are interchangeable unless specifically stated otherwise.

The term "tumor" refers to the neoplasm formed by the proliferation of local tissue cells under the action of various tumorigenic factors. The definition and classification of the tumor can be found in the second edition (1989-2000) of the histological classification of tumors as formulated by the World Health Organization (WHO). In one embodiment, the subject suffering from a tumor is a subject suffering from a head and neck tumor.

The term "head and neck tumor" includes neck tumor, otolaryngology tumor, and oral and maxillofacial tumor. Neck tumors include, for example, thyroid tumors; ENT tumors include, for example, laryngeal cancer, paranasal sinus cancer, and the like; oral and maxillofacial tumors include, for example, oral cancers, such as tongue cancer, gum cancer, cheek cancer, and the like. Due to the anatomical position of the oral mucosa in the human body, radiation may cause loss of the oral mucosa when treating head and neck tumors. In one embodiment, the head and neck tumor comprises one or more selected from the group consisting of a neck tumor, an ENT tumor, and an oral and maxillofacial tumor. In one embodiment, the neck tumor is a thyroid tumor. In one embodiment, the ENT tumor is nasopharyngeal cancer, laryngeal cancer, or paranasal sinus cancer. In one embodiment, the oral and maxillofacial tumor is oral cancer, such as tongue cancer, gingival cancer, buccal cancer, maxillary sinus cancer, and tonsil cancer.

The term "radiation-induced oral mucositis (ROM)" refers to oral mucosal damage caused by ionizing radiation. It is mainly seen in patients with head and neck malignant tumors. Radiation kills tumor cells and also affects normal cells. The killing effect causes congestion, erosion and ulcers in the oral mucosa. There is a certain time-dependent relationship between ROM and tumor radiotherapy progress. In the present invention, "radiational oral mucositis" is different from general oral mucosal inflammation in terms of causes and the like. The term "oral mucositis" or oral ulcer, commonly known as "aphthous ulcer", occurs mostly between the ages of 20 and 50 and is caused by a viral infection. In the present invention, the terms "radial oral mucositis" and "oral mucositis" refer to different diseases. In one embodiment, radiation oral mucositis includes only oral mucositis caused by radiation therapy.

The term "mucosa" or "mucosa" refers to a thin film in the oral cavity, organs, stomach, intestines, urethra and other organs, which contains blood vessels and nerves, and can secrete mucus. Its basic tissue structure includes the epithelial layer, lamina propria, Submucosa and basement membrane. In the present invention, "mucosa" and "skin" are different biological tissues. The term "skin" refers to the tissue that is wrapped on the surface of the body outside the muscle. It consists of three layers: epidermis, dermis, and subcutaneous tissue. Unlike the mucous membrane, it is mainly responsible for protecting the body, perspiration, feeling hot and cold, and stress. In the present invention, damage to the oral mucosa is different from skin damage. In one embodiment, the oral mucosal damage does not include skin damage. In this document, the terms "mucosa" and "mucosa" are interchangeable unless specifically stated otherwise.

The term "related radiotherapy complications" refers to some complications related to the location, mechanism, and other aspects of radiation oral mucositis that arise from tumor patients receiving radiation therapy or combined radiation and chemotherapy. The radiotherapy complications related to radiation oral mucositis of the present invention include esophageal mucosal damage, taste dysfunction, salivary gland secretion, hypothyroidism, radioactive thyroid disease, radioactive dry mouth disease, radioactive taste abnormality, and post-radiation caused by radiotherapy. Reduced number of tongue taste bud cells, radiation caries, radiation esophagitis, and radiation esophageal stricture.

The term "esophageal mucosal injury" refers to esophageal injury caused by radiation therapy.

The term "taste dysfunction" refers to abnormal manifestations of food taste that are dull, diminished, or even disappeared due to radiotherapy.

The term "hypofunction of salivary glands" refers to a decrease in saliva secretion due to radiation therapy.

The term "decreased number of tongue taste bud cells after radiation" refers to the death of taste bud cells caused by radiation therapy, which reduces the number of taste buds on the tongue, thereby causing abnormal taste function.

The term "radioactive thyroid disease" is a dysfunction that occurs after exposure to the thyroid gland. Radiation thyroid disease includes radiation hypothyroidism, chronic radiation thyroiditis, acute radiation thyroiditis, and benign nodules of radiation thyroid.

The term "radiating hypothyroidism" is a common complication after radiotherapy for patients with head and neck tumors and refers to hypothyroidism that occurs after exposure to the thyroid gland. Radiation hypothyroidism can be manifested by a decrease in serum triiodothyronine and serum thyroxine, and an increase in thyroid-stimulating hormone.

The term "chronic radiation thyroiditis" refers to autoimmune thyroid damage caused by thyroid irradiation. Chronic radiation thyroiditis is manifested as positive thyroid microsomal antibodies and / or thyroglobulin antibodies, and increases thyroid-stimulating hormone, which may be accompanied by hypothyroidism.

The term "acute radioactive thyroiditis" refers to localized thyroid damage and hyperthyroidism caused by high-dose acute exposure to the thyroid for a short period of time.

The term "radioactive benign thyroid nodules" refers to nodular lesions that appear after irradiation of the thyroid gland.

The term "radiation xerostomia" refers to radiation-induced salivary glands that are damaged by radiation and cannot produce enough saliva. Saliva becomes less and sticky, which leads to symptoms such as dry mouth, salivary gland inflammation, and even swallowing disorders and difficulty in opening mouth. .

The term "radioactive abnormal taste" refers to a taste disorder caused by impaired taste receptors caused by radiotherapy.

The term "radioactive caries" is a serious complication common to radiotherapy of head and neck tumors, especially nasopharyngeal cancer, sinus cancer, oral cancer, oropharyngeal cancer. Dental enamel is irritated due to radiation, salivary gland secretion is significantly reduced after radiation damage, oral self-cleaning is lost, gum atrophy, exposed neck, and dental caries are more easily broken.

The term "radioesophageitis" is a radiation esophageal injury that occurs during radiotherapy.

The term "radioesophageal stenosis" means that the tumor lesion disappears after radiotherapy, but due to scar formation, the esophageal lumen is narrowed and the wall is stiff at the original lesion and within the irradiation range, which affects the intake of water.

The term "prevention" includes reducing the risk of having, infecting, or experiencing a disease, disorder, condition, or sign, its development and / or progression, and / or its symptoms. The effect of prevention may include partial or complete prevention of the underlying disease. In the present invention, the term prevention does not mean that the disease is completely avoided. In the present invention, the term "prevention" does not include the treatment of a disease. In the present invention, a sulfoxide compound (preferably DMSO) and a drug containing the same need to be administered before the onset of the disease, specifically before the subject receives radiation therapy. In one embodiment, the subject receiving the sulfoxide compound (preferably DMSO) before receiving the radiation treatment optionally includes the subject receiving both the radiation therapy and the sulfoxide compound (preferably DMSO). The present invention confirms that by administering a sulfoxide compound (preferably DMSO) to a subject with a tumor before radiation therapy, the oral mucositis of the subject with a tumor can be effectively prevented after receiving the radiation therapy.

In one embodiment, the subject with a tumor is a head and neck tumor subject who will receive radiation treatment within 8 hours to 1 hour. In one embodiment, the subject with a tumor is a head and neck tumor subject who will receive radiation treatment within 6 hours. In one embodiment, the subject with a tumor is a head and neck tumor subject who will receive radiation treatment within 5 hours. In one embodiment, the subject with a tumor is a head and neck tumor subject who will receive radiation therapy within 4 hours. In one embodiment, the subject with a tumor is a head and neck tumor subject who will receive radiation treatment within 3 hours. In one embodiment, the subject with a tumor is a head and neck tumor subject who will receive radiation treatment within 2 hours. In one embodiment, the subject with a tumor is a head and neck tumor subject who will receive radiation treatment within 1 hour. In one embodiment, the subject with a tumor is a head and neck tumor subject who will receive radiation treatment within 30 minutes.

The term "dose" is based on the amount of a sulfoxide compound (preferably DMSO) or a sulfoxide compound (preferably DMSO) contained in a pharmaceutical / pharmaceutical composition administered to a subject. The dose may vary depending on factors known in the art such as the disease state, age, sex, and weight of the person to be prevented.

In one embodiment, the dosage of the sulfoxide compound, preferably DMSO, is determined based on the weight of the subject. In one embodiment, the sulfoxide compound (preferably DMSO) is administered at a dose of 0.1-10.0 g / kg body weight. In one embodiment, the DMSO is administered at a dose of 0.2-5.0 g / kg body weight. In one embodiment, the sulfoxide compound (preferably DMSO) is administered at a dose of 0.4-2.0 g / kg body weight. In one embodiment, the sulfoxide compound (preferably DMSO) is administered at a dose of 0.5 g / kg body weight. In one embodiment, the sulfoxide compound (preferably DMSO) is administered at a dose of 1.0 g / kg body weight.

The term "radiation therapy" includes irradiating tumor tissue with radiation, which can directly act on targets such as cell DNA or interact with water molecules in the cell through radiation to generate free radicals that indirectly act on targets such as cell DNA, leading to tumor cell death. Cancer treatment. The radiation used in radiotherapy includes α, β, γ rays produced by radioisotopes and x-rays, electron beams, proton beams and other particle beams produced by various types of x-ray therapy machines or accelerators. Radiotherapy works differently than chemotherapy. In this document, the terms "radiotherapy", "radiotherapy" and "radiotherapy" are interchangeable unless specifically stated. In one embodiment, radiation therapy includes treatment using radiation selected from the group consisting of alpha rays, beta rays, gamma rays, x-rays, neutrons, electron beams, proton beams, particle beams, and combinations thereof.

Radiation therapy is divided into external and internal radiation. External radiation therapy refers to the radiation from outside the body through various tissues of the body into the body, which produces a radiation effect in the tumor target area and kills the tumor. Conventional external radiation radiotherapy is a segmented radiation therapy (2Gy 5 times per week) established based on clinical practice experience and is considered to be the standard method for tumor radiation therapy. Internal radiation therapy is also called brachytherapy and includes radioactive particle implantation and radionuclide-targeted tumor treatment.

According to the different radiation doses, radiotherapy subjects can be divided into radiation-sensitive tumors, radiation-sensitive tumors, and radiation-sensitive tumors. In one embodiment, the single dose of the radiation therapy is 1-50 Gy. In one embodiment, the radiation dose of the radiation therapy is 1-8 Gy. In one embodiment, the radiation dose of the radiation therapy is 2Gy.

The term "chemotherapy" is also referred to as "chemotherapy" or "chemotherapy", which refers to the means of using chemotherapy drugs to kill cancer cells to achieve therapeutic purposes. The term "combined radiation and chemotherapy" refers to a combination of radiation and chemotherapy to treat tumors. Radiotherapy and chemotherapy combined therapy is one of the most widely used treatments in clinical tumor treatment. Radiotherapy and chemotherapy combined treatment is to use radiation therapy to directly combat the tumor's focus, and chemotherapy treatment to eliminate cancer cells in patients, so as to achieve the effect of treatment. In the present invention, the subject to be treated is expected to receive radiation therapy and chemotherapy at the same time, or receive radiation therapy and then chemotherapy, or receive chemotherapy and then radiation therapy; provided that the above treatments are being received The sulfoxide compound (preferably DMSO) of the present invention is performed before.

In the present invention, chemotherapeutic drugs that can be used in combination therapy of radiation and chemotherapy include, but are not limited to: alkylating agents or platinum compounds that directly act on DNA, such as melphalan, cyclophosphamide, oxygen Oxazaphosphorine, cisplatin, carboplatin, oxaliplatin, satraplatin, tetraplatin, iproplatin, silk Mitomycin, streptozocin, carmustine (BCNU), lomustine (CCNU), busulfan, ifosfamide , Streptozocin, thiotepa, chlorambucil; nitrogen mustard, such as dichloromethyldiethylamine (mechlorethamine); ethyleneimine compounds; alkyl sulfonates ( alkylsulfonate); antimetabolites that interfere with DNA and RNA synthesis, such as cytarabine, 5-fluorouracil (5-FU), pemetrexed, tegafur, uracil (uracil), uracilmustard, fludarabine, gemcitabine, card Capecitabine, mercaptopurine, cladribine, thioguanine, methotrexate, pentostatin, hydroxyurea, or folic acid Antitumor antibiotics that interfere with DNA by inhibiting the action of enzymes and mitosis or altering cell membranes, phleomycin, bleomycin or its derivatives or their salts, CHPP, BZPP, MTPP, BAPP, Benzomycin (liblomycin), rifamycin, actinomycin, adramycin, doxorubicin, epirubicin, pyramycin, daunorubicin And mitomycin; plant-based anticancer drugs derived from animal and plant components: such as paclitaxel [taxol], docetaxel or taxotere; vinca alkaloids , Such as navelbine, vinblastin, vincristin, vindesine, or vinorelbine; tropone alkaloids, such as colchicine or Its derivatives; macrolides, such as maytansine; stalks Ansamitocin or rhizoxin; anti-mitotic peptides, such as pomipsin or dolastatin; epipodophyllotoxin or podophyllotoxin derivatives, such as etoposide Etoposide or teniposide; steganacin; anti-mitotic carbamate derivatives, such as combetastatin or amphetinile; procarbazine ); Camptothecin, such as irinotecan (iritocan hydrochloride and sorbitol injection camptosar) or topotecan; hormone antineoplastic drugs, such as progesterone or estrogen, such as Estrogen mustard (T-66) or megestrol; antiandrogens such as flutamide, casodex, anandron or cyproterone acetate); aromatase inhibitors, such as aminogluthetimide, anastrozole, formestan, or letrozole; GHrH analogs, such as leuprorelin, Buserelin, goserelin or triptore lin); anti-estrogens, such as tamoxifen or its citrate, droloxifene, trioxifene, raloxifene, or cinoxifene (zindoxifene); 17β-estradiol derivatives, such as ICI 164,384 or ICI 182,780, aminoglutethimide, formestan, fadrozole, finasteride ), Ketoconazole (ketoconazole); LH-RH antagonists, such as leuprolide acetate; steroids, such as prednisone, prednisolone, methylprednisolone ), Dexamethasone, budenoside, fluocortolone, or triamcinolone; other heterogeneous antitumor drugs, such as proteasome inhibitors, such as potentan Bortezomib; enzymes, such as asparaginase, pegylated asparaginase (pegaspargase) or thymidine phosphorylase inhibitor; interferon, such as interferon beta; interleukin, such as IL-10 or IL-12; anti-TNFα antibodies, such as etanercept; and targeted antitumor Substances, such as small molecule antagonists of VEGF receptors, such as vatalanib, SU-5416, SU-6668, SU-11248, SU-14813, AZD-6474, EGFR / HER2 antagonists, such as CI-1033 Or GW-2016; EGFR antagonists, such as iressa [gefitineb ZD-1839], tarceva, PKI-166, EKB-569, HKI-272 or Hersey Herceptin; and antibodies that target cancer cell surface molecules, such as apolizumab or 1D09C3.

In the present invention, the sulfoxide compound (preferably DMSO) may be further administered in combination with other compounds known to be prophylactic and / or therapeutically effective or possibly effective for radiation oral mucositis. This compound includes one or more compounds selected from: WR2721, PrC-210 (aminothiol), WR-1065, WR-3689, WR-151327, WR-638, WR-77913, WR-44923, cysteamine , AET, lipoic acid, N, N-diethyllipoamide, Mn-SOD transgenic preparation, Cu.Zn-SOD preparation, Fe-SOD preparation, GC4419 (M40403), MnBuOE, AEOL10150, MnTnHex-2-PyP5 + ( hexyl), MnTE-2-PyP, EUK-207, EUK-189, EUK-134, melatonin, vitamin E and its derivatives, pentoxifylline, glutamine, Tempol (4-hydroxy-2,2 , 6,6-tetramethylpiperidine-N-oxyl), edaravone, vitamin C and its derivatives, vitamin A and its derivatives, D-methionine, Acetylcysteine, glutathione Peptide, 3,3'-diindolylmethane, Oltipraz, Tetrahydrobiopterin, metformin, β-carotene, FTY720, RPC1063, BAF312, ACT-128800, KRP203, MT-1303, CBLB502, CBLB612, CBLB613, R-Spondin1, Tat-Smad7, rapamycin, sirolimus, Everolimus (RAD001), ABI -009, Tobramycin, Tetracyclines, Chlorhexidine, Amphotericin B, Nystatin, Zovirax, Brilacidin , Triamcinolone acetonide, betamethasone, prednisone, hydrocortisone acetate, etc., Bendydamine, Indomethacin, Misoprostol, COX- 2 inhibitors, prostaglandin E1, prostaglandin E2, salicylic acid derivatives, Azelastine, diphenhydramine, morphine, fentanyl,

Traumeel, Glycyrrhizic acid, Glycyrrhetinic acid and capsaicin.

In the present invention, sulfoxide compounds (preferably DMSO) can be prepared into pharmaceutical dosage forms suitable for different modes of administration. In one embodiment, the medicament is a dosage form selected from one or more of the following administration modes: intravenous administration, oral mucosal administration, transdermal administration, and oral administration.

The term "intravenous dosage form" is also referred to as an infusion solution, and refers to a large-dose injection solution injected into a human body by intravenous drip.

In one embodiment, the medicament is administered by intravenous injection. In one embodiment, the sulfoxide compound (preferably DMSO) is administered intravenously in a concentration range of 1% to 100%, preferably 5% to 50%, still more preferably 10 to 20%, and most preferably 10%. In one embodiment, the intravenous dosage form comprising a sulfoxide compound (preferably DMSO) may further include an injection diluent, which may be selected from the group consisting of sodium bicarbonate injection, sodium chloride injection, glucose injection, Sodium citrate injection, sodium glycerophosphate injection, preferably 1.4% sodium bicarbonate injection. In one embodiment, an intravenous dosage form comprising a sulfoxide compound, preferably DMSO, may further comprise an injection additive, which may be selected from an osmotic pressure regulator (glucose, sodium chloride, phosphate, or citron Acid salts, etc.); pH adjusters (hydrochloric acid, sulfuric acid, citric acid, sodium (potassium hydroxide), sodium bicarbonate, disodium hydrogen phosphate, sodium dihydrogen phosphate, etc.); suspending agents (propylene glycol, glycerin, Tween -80, Span-85, Profluni F-68, carboxymethyl cellulose, sodium alginate, polyvinylpyrrolidone, gelatin, mannitol, sorbitol, aluminum monostearate, silicone oil, etc.); and analgesics Agents (benzyl alcohol (about 1%), procaine hydrochloride (0.5% -2.0%), lidocaine hydrochloride (0.2% -1.0%), trichloro-t-butanol (0.3% -0.5%)).

The term "oral mucosal dosage form" refers to a dosage form in which a drug directly enters the circulatory system after being absorbed by the oral mucosa. The drug can be administered through the oral mucosa to exert local and systemic therapeutic effects.

In the present invention, the dosage forms suitable for oral mucosal administration include local treatment dosage forms: mouthwash, film, aerosol and tablet, preferably mouthwash and film; systemic treatment dosage forms: adhesive tablets, plasters , Sublingual tablets and orally disintegrating tablets, preferably adhesive tablets and patches.

In one embodiment, the dosage form suitable for oral mucosal administration is selected from the group consisting of mouthwashes, films, aerosols, tablets, adhesive tablets, patches, sublingual tablets, and orally disintegrating tablets.

In one embodiment, the medicament is one or more dosage forms selected from the group consisting of a mouthwash (Mouthwash, or Oral Rinse), a film, an aerosol, a tablet, an adhesive tablet, a plaster, a tongue Lower tablets, orally disintegrating tablets, sticks, powders, ointments, suspensions, capsules, gels, transmucosal patches, transmucosal pectin, films, sprays , Colloid, Emulsion, Ointment, Rubber plaster, Sponge, Cream, Ointment, Paste, Coating Agents and Foams.

In one embodiment, the oral mucosa is administered at a concentration ranging from 1% to 100%, preferably 5% to 90%, still more preferably 10 to 70%, and most preferably 10 to 50%.

The term "transdermal drug delivery" (TDD) uses the skin as the route of administration to continuously deliver drugs to the surface of the skin or the circulatory system, thereby exerting the effect of local or systemic treatment of the skin.

The term "transdermal drug delivery system" (TDDS) refers to a dosage form that is administered on the surface of the skin and allows the drug to pass through various layers of the skin at a constant speed (or near a constant speed) into the systemic circulation to produce systemic or local therapeutic effects.

Dosage forms for transdermal drug delivery systems include, but are not limited to, plasters, tinctures, film coatings, cataplasms, matrix-type patches, membrane-controlled patches, microemulsions, liposomes, inclusion compounds, prodrugs, and These drugs can be applied to thinner parts of the skin, such as behind the ears, inside the arms, anterior chest areas, and scrotum.

In one embodiment, the dosage form suitable for transdermal administration may be selected from one or more of the following: plasters, tinctures, film coating agents, babies, skeletal patches, membrane-controlled patches, microemulsions , Liposomes and inclusion complexes.

In one embodiment, a dosage form suitable for transdermal administration can be applied to thinner parts of the skin, such as behind the ear, inside the arm, anterior chest area, scrotum, and the like.

The transdermal drug delivery system is preferably a nanocarrier-based passive transdermal drug delivery system. In one embodiment, the nanocarrier-based passive transdermal drug delivery system may be selected from one or more of the following: nanovesicles (Vesicles), lipid nanoparticles (Lipid nanoparticles), microemulsions (Microemulsion), and polymers Nanoparticles (Polymeric nanoparticles).

There are chemical and physical methods to promote the transdermal penetration of drugs. Chemical methods are mainly transdermal absorption enhancers, and physical methods include ultrasonic methods, iontophoresis methods, and electroporation methods.

Permeation enhancers refer to substances that can help drugs diffuse through the stratum corneum and epidermis of the skin, or are called transdermal enhancers, absorption enhancers, and the like.

In one embodiment, the drug containing a sulfoxide compound (preferably DMSO) may further include a penetration enhancer. Permeation enhancers can be divided into lipophilic lysozymes, surfactants, two-component and multi-component systems. In one embodiment, the penetration enhancer is selected from one or more of the following: Azone, linoleic acid, urea, α-pyrrolidone, propylene glycol, butanediol, dimethylformamide, Oleic acid (OA), dodecanol (LA), etc .; Chinese traditional medicine repair aids include peppermint oil, eucalyptus oil, borneol, turpentine, menthol, camphor, menthol, etc .; N, N-dimethylaminoisopropanoic acid Dialkyl esters (DDAIP) and N, N-dimethylaminoacetic acid dodecyl ester (DDAA).

In one embodiment, the sulfoxide compound (preferably DMSO) is administered at a concentration ranging from 1% to 100%, preferably from 5% to 90%, still more preferably from 25 to 70%, and most preferably 60%.

The term "oral dosage form" includes aqueous solutions, aqueous suspensions, tablets and capsules. In one embodiment, the oral administration dosage form containing a sulfoxide compound (preferably DMSO) may further include an additional agent such as a flavoring agent and a bacteriostatic agent. The term "flavoring agent" refers to pharmaceutical excipients used in pharmaceuticals to improve or shield the bad odor and taste of drugs, making it difficult for patients to perceive the strong bitter taste of the drug (or other offensive odors such as spicy, irritating, etc.), and generally includes sweeteners. , Fragrance, glue and effervescent.

In one embodiment, the sulfoxide compound (preferably DMSO) is orally administered at a concentration ranging from 1% to 100%, preferably from 10% to 90%, still more preferably from 20 to 70%, and most preferably 50%.

In one embodiment, the drug is administered at a dose of 0.1-10.0 g DMSO / kg body weight, preferably 0.2-5.0 g sulfoxide (preferably DMSO) / kg body weight, more preferably 0.4-2.0 g sulfoxide ( Preferably DMSO) / kg body weight, most preferably 0.5-1.0 g of sulfoxide compounds (preferably DMSO) / kg body weight.

In the present invention, the drug containing a sulfoxide compound (preferably DMSO) may further include a pharmaceutically acceptable excipient. In one embodiment, the pharmaceutically acceptable excipient is selected from one or more of the following: D-expanthenol, sodium alginate, povidone iodine, sodium hyaluronate, Pectin, sticky xylan and sucralfate. Herein, the terms "drug" and "pharmaceutical composition" are interchangeable unless specifically stated otherwise.

In one embodiment, the drug containing a sulfoxide compound, preferably DMSO, may be further administered concurrently with a radiosensitizer. The term "radiosensitizer" is a chemical or pharmaceutical preparation that, when applied concurrently with radiation therapy, can change the responsiveness of tumor cells to radiation, thereby increasing the killing effect on tumor cells, including hypoxic cell radiosensitizers and Non-hypoxic cell radiosensitizer.

In one embodiment, the radiosensitizer is a hypoxic cell radiosensitizer or a non-hypoxic cell radiosensitizer. In one embodiment, the hypoxic cell radiosensitizer is selected from the following: Nitroimidazoles, such as metronidazole, midazolam, MISO (Ro-07-0582), RSU-1096, tira Tirapazamine, etc .; and biological reducing agents, such as sodium glycinazole, nitrogen oxides, quinones, and nicotinamide and its derivatives. In one embodiment, the non-hypoxic cell radiosensitizer is selected from the following: platinum drugs, such as cisplatin, carboplatin, oxaliplatin, etc .; anti-metabolic drugs, such as 5-FU, capecitabine, gemcitabine Etc; topoisomerase inhibitors, such as camptothecin, irinotecan, topotecan, CPT417, BMS-286309, etoposide (VP-16), etc .; microtubules Stabilizing drugs, such as paclitaxel, docetaxel, patupilone, PBOX-15, etc .; second mitochondrial-derived cystinase activator (SMAC) analogs, such as Tat-SMACN7, SM-164, LCL161, etc .; Tumor-targeted therapies, such as Sorafenib, Erlotinib, Cetuximab, Bevacizumab, and trastuzumab ), Etc .; Photosensitizers, such as 5-aminoketovaleric acid, hematoporphyrin monomethyl ether, hematoporphyrin derivatives (HPD), d-hemiporphyrin ether (DHE), Photofrin II, etc .; DNA alkylating agents, such as temozolomide, etc. ; And traditional Chinese medicine extracts, such as purpurin.

The present invention confirms that sulfoxide compounds (preferably DMSO) can effectively prevent radiation oral mucositis and related radiotherapy complications, and its prevention effect on radiation oral mucositis and related radiation therapy complications is better than those currently used in clinical practice such as rhKGF and WR2721 And other protective drugs. In addition, DMSO has a wide application time window for prevention, and it has been proven that application of DMSO 6 hours before irradiation can have a preventive effect. In addition, DMSO has low potential toxic and side effects in a range of effective doses for prophylactic administration.

Examples

The present invention will be described in more detail with reference to the following examples. However, the following examples are provided only to explain the present invention and should not be construed as limiting the scope and spirit of the present invention.

Materials and Method:

1.1 Materials, reagents and instruments

1.1.1 Experimental animals

SPF C57BL / 6J male mice, weighing 22 to 24 g, were purchased from Beijing Huafukang Biotechnology Co., Ltd., animal quality certificate number: SCXK (Jing) 2009-0004. All mice were transported to SPF barrier animal rooms of the Experimental Animal Center of the Academy of Military Medical Sciences in clean transport boxes and reared in cages of 5 animals each. Sterilized water and maintenance feed were provided for free feeding. Keep the lights on for 12 hours and turn off the lights for 12 hours every day. The certificate number of the animal breeding facility: SYXK- (Army) -2007-004. Mice were subjected to adaptive rearing grouping experiments for about 7 days.

1.1.2 Main reagents

(1) DMSO (Nanjing Kangmanlin Pharmaceutical Technology Co., Ltd.): 0.9% medical saline is dissolved to a specific concentration, and it is used now.

(2) Recombinant human keratinocyte growth factor rhKGF (Beijing Braunwell Technology Co., Ltd., article number BITP1191).

(3) Amifostine WR2721 (Beijing Braunwell Technology Co., Ltd., article number A576810).

(4) 1% toluidine blue solution (Beijing Regan Biotechnology Co., Ltd., article number DA0057).

(5) Formaldehyde solution (Academy of Military Medical Sciences): Take 80ml formaldehyde in a graduated cylinder and add it to 720ml distilled water to prepare a 10% formaldehyde solution.

(6) 5% goat serum blocking solution: 0.5ml of normal goat serum stock solution (Beijing Zhongshan Jinqiao Biotechnology Co., Ltd., article number ZLI-9021), add 1 × TBST solution 9.5mL, mix thoroughly, and store at -20 ° C.

(7) Rabbit anti-Ki67 (Cell Signaling Technology, Inc., USA, product number 12022s).

(8) Murine anti-P63 (Abcam, Inc., US, No. 550278).

(9) Goat anti-rabbit labeled with horseradish peroxidase (HRP) (Beijing Zhongshan Jinqiao Biotechnology Co., Ltd., product number PV-6001).

(10) Goat anti-mouse labeled with horseradish peroxidase (HRP) (Beijing Zhongshan Jinqiao Biotechnology Co., Ltd., product number PV-6002).

(11) DAB kit 20 × (Beijing Zhongshan Jinqiao Biotechnology Co., Ltd., article number ZLI-9018).

(12) EDTA antigen recovery solution pH 9.0 (Beijing Zhongshan Jinqiao Biotechnology Co., Ltd., article number ZLI-9068): diluted 1:50 in distilled water when in use.

(13) 10% goat serum blocking solution: 1 ml of normal goat serum stock solution (Beijing Zhongshan Jinqiao Biotechnology Co., Ltd., article number ZLI-9021) was added, 9 mL of 1 × TBST solution was added, and they were thoroughly mixed and stored at -20 ° C.

(14) Rabbit p63 monoclonal antibody [EPR5701] (Abcam, Inc., USA, article number ab124762).

(15) Mouse anti-human γ-H2AX antibody (Millipore, USA, Cat. No. 05-636).

(16) Alexa

488-labeled goat anti-rabbit IgG (H + L) (affinity purification) (Beijing Zhongshan Jinqiao Biotechnology Co., Ltd., article number ZF-0511).

(17) Alexa

594-labeled goat anti-mouse IgG (H + L) (affinity purified) (Beijing Zhongshan Jinqiao Biotechnology Co., Ltd., article number ZF0513).

(18) Hoechst 33342 staining solution (Beijing Biyuntian Biotechnology Co., Ltd., C1025)

(19) Mounting Medium anti-quenching sealing tablets (Beijing Zhongshan Jinqiao Biotechnology Co., Ltd., ZLI-9556).

1.1.3 Instruments and Consumables

X-ray machine (Rad-Source Technologies, Model RS-2000, USA).

Biological tissue automatic dehydrator (Zhejiang Kedi Instrument Equipment Co., Ltd., model KD-TS1A).

Biological tissue freezing and embedding machine (Jinhua Cody Instrument Equipment Co., Ltd., Zhejiang Province, model KD-BM, BL).

Computer biological tissue spreading machine (Jinhua City, Zhejiang Cody Instrument Equipment Co., Ltd., model KD-P).

Computer biological tissue drying machine (Zhejiang Jindi Kedi Instrument Equipment Co., Ltd., model KD-P).

Rotary slicer (Leica, Germany, model RM2235).

Upright fluorescence microscope (Leica, Germany, model DB4000).

Digital camera (Canon, Japan, model).

1.2 Experimental methods

1.2.1 Preparation before irradiation

One week prior to irradiation, the animals were housed five per in SPF Laboratory Animal Center, a standard diet ^{for 60} emitted after irradiation sterilization COγ feeding. Two days before the irradiation, the experimental animals were weighed and randomly divided into groups according to the experimental design.

1.2.2 Irradiation conditions

Before the irradiation, the mice were anesthetized with 1% sodium pentobarbital (0.2ml / head, intraperitoneal injection), and the rats were placed in a self-made irradiation box. The lead plate was placed below the neck of the mouse to completely shield the other parts of the mouse. Outside the pulling outlet, the irradiation conditions are: voltage 220kv, current 25mA, dose rate 1.379Gy / min, one irradiation dose is 16.5Gy, or 8Gy once a day for 3 consecutive days. Seven mice were irradiated each time.

1.2.3 Histopathological observation of tongue mucosa

(1) pathological materials

The mice were sacrificed 1d, 3d, 5d, 7d, 9d, 11d, and 14d after the irradiation, and the tongue tissue was removed.

(2) 1% toluidine blue staining

The mouse tongue tissue was completely removed, and the 1% acetic acid solution was applied to the surface of the tongue mucosa. After the surface mucus was dissolved, the 1% toluidine blue solution was applied to the tongue surface. After 40-60s, the toluidine was again applied with 1% acetic acid. The blue staining solution attachment site, the coloring point without dissolution of acetic acid was a positive manifestation, and the results were photographed and observed.

(3) Production of paraffin sections

Tissue fixation: All stained specimens were placed in labeled embedding boxes, and all were immersed in 10% formaldehyde solution for more than 24 hours.

Tissue dehydration, transparency and preparation: After the tissue is fixed, rinse with tap water and put it in the dehydrator: 70% alcohol 25min, 80% alcohol 18min, 90% alcohol 18min, 95% alcohol 18min, 95% alcohol 18min, 100% Alcohol 18min, 100% alcohol 18min, xylene (I) 10min, xylene (II) 10min, paraffin (I) 10min, paraffin (II) 10min, paraffin (III) 2h.

Embedding: Tissue sections are affixed to an embedding box and embedded at 60 ° C wax temperature.

Slicing: thickness 5μm, set the spread sheet at 38 ° C and roasted sheet at 56 ° C.

(4) HE staining

Dewaxing: xylene 10min × 3, 100% alcohol 10min × 2, 95% alcohol 5min × 2, 90% alcohol 5min, 85% alcohol 5min, 70% alcohol 5min, and distilled water for 1min.

Hematoxylin staining: 5min.

Differentiation returned to blue: Hydrochloric acid ethanol was differentiated for 2s, and then immersed in tap water for 5min.

Eosin staining: 40s, rinse under running water.

Dehydrated and transparent: 8% alcohol for 2min, 90% alcohol for 1min, 100% alcohol for 2min × 2, xylene for 5min.

Mounting: Neutral quick-drying adhesive.

The Leica DB4000 microscope was used to observe and take pictures. LAS-X software was used to measure the ulcer length and epithelial thickness (10 fields of view were randomly taken from each tongue and two points were measured from each field) for quantitative analysis.

(5) Immunohistochemistry

Antigen repair: 99 ℃ sodium citrate repair solution for 30min

PBS rinse: 5min × 3 times.

Inactivate endogenous peroxidase activity: 3% H2O2 deionized water and incubate for 15-20min.

PBS rinse: 5min × 3 times.

Serum blocking: TBST containing 5% goat serum was blocked for 1 h.

Incubate primary antibody: ki-67 antibody 1: 200, p63 antibody 1: 100, all antibodies overnight at 4 ° C.

Return to room temperature and rinse in PBS for 5 min x 3 times.

Incubate secondary antibodies: goat anti-rabbit secondary antibody (ki-67), goat anti-mouse secondary antibody (p63) and incubate for 1 h at room temperature.

DAB color development: 1:20 dilution.

Dehydrated, transparent, and mounted (same as experimental HE staining).

The Leica DB4000B microscope was used to observe and take pictures. Leica LAS-X software was used to measure the ulcer length and epithelial thickness (10 fields of view were randomly taken from each tongue, and two points were measured from each field) for quantitative analysis.

(6) Making frozen slices

Tissue fixation: All halves of the tongue tissue were fixed in 4% paraformaldehyde for 4 hours.

Tissue dehydration: Rinse in tap water for 10 min after fixation, 30% sucrose solution for 10 h.

Embedding: Place in the embedding box with the cut side down, embed in OCT, and store in -80 ℃ refrigerator.

Sectioning: cross-section along the long axis of the wax block, thickness 6-7 μm, microtome temperature -20 ° C.

Re-fixing: After the OCT is dried, fix the sections with 1: 1 acetone and methanol for 20 minutes, dry them, wash them 3 times with pbs, 5 minX3, dry them, and store them at -20 degrees Celsius.

(7) Immunofluorescence

Antigen retrieval: Put in EDTA repair solution, heat in a 99 ℃ water bath for 30min, and cool to room temperature.

PBS rinse: 5min × 3 times.

Serum blocking: TBST containing 10% goat serum was blocked for 1 h.

Incubate primary antibodies: All antibodies are diluted with antibody dilutions, p63 and γ-H2AX antibody are incubated together at 1: 100 and overnight at 4 ° C.

Return to room temperature and rinse in PBS for 5 min x 3 times.

Incubate secondary antibodies: goat anti-rabbit secondary antibody Alex-488 and goat anti-mouse secondary antibody Alex-594 1: 200, incubate for 1 h at room temperature.

PBS rinse: 5min × 3 times.

Hoechst 33342 staining nucleus: pbs 1: 800 dilution, 3min.

PBS rinse: 5min × 3 times.

Mounting Medium.

The slides were analyzed by Nikon Ti-A1 laser confocal microscope and imaging system.

1.2.4 Mouse Sucrose Preference Test

Rodents are interested in sweet foods or solutions. The sucrose preference test is one of the commonly used methods to determine taste sensitivity in mice. Before the test, the mice were paired in a squirrel cage with two bearing straw bottles for 3 days, and both drink bottles were filled with ordinary drinking water. During the test, one of the beverage bottles was filled with 100 millimoles of sucrose water and the other was still ordinary drinking water; after 24 hours, the positions of the two bottles were switched once to measure the daily intake of ordinary water and sucrose water, respectively. Sucrose preference is calculated as the percentage of sucrose intake relative to the total volume of fluid intake and averaged over the 2 day test.

Sucrose preference = V (sucrose water) / [V (sucrose water) + V (water)] × 100%.

1.2.5 Determination of saliva secretion in mice

Mice were tested for saliva secretion 8 weeks after irradiation. Mice were anesthetized with phenobarbital sodium, subcutaneously injected with pilocarpine 0.5mg / kg (stimulating salivary gland secretion) 2 minutes, the mice were fixed at an angle, the hematocrit measuring tube was placed at the corner of the mouth of the mouse, and saliva was collected for 10 minutes , Transfer the collected saliva into a pre-weighed 05ml centrifuge tube, weigh and calculate the amount of saliva.

1.2.6 Measurement of thyroid function in mice

Mice were tested for thyroid function 12 weeks after irradiation. Mice were anesthetized with phenobarbital sodium, blood was obtained by cardiac puncture, and the serum was stored at -20 ° C after centrifugation. The ELISA method was used to determine the contents of serum free thyroxine (FT4), serum free triiodothyronine (FT3), and serum thyroid stimulating hormone (TSH) according to the instructions of the kit.

1.2.7 Statistical processing

The data results are expressed by x ± s, and graphPad Prism6 software package is used for mapping. The comparison of ulcer size, epithelial thickness, and keratinocyte proliferation was performed using one-way ANOVA, and statistical software SPSS17.0 (SPSS Inc., Chicago) was used for statistical analysis. P <0.05 considered the difference to be statistically significant. significance.

Example 1. Comparison of the Preventive Effects of Different Dose of DMSO on Radiation Oral Mucositis in Mice

1. Experimental grouping:

Sixteen male C57 mice were randomly selected and divided into the following 4 groups:

Non-irradiated control group (Non-IR): 4;

Irradiation control group (vehicle): intraperitoneal injection of 0.2% 0.9% saline, 4;

DMSO group 1: intraperitoneal injection of DMSO at a dose of 4 g / kg, 4 animals, administered 1 hour before irradiation;

DMSO group 2: Intraperitoneal injection of DMSO at a dose of 6 g / kg, 4 animals, administered 1 hour before irradiation.

Irradiation dose: 16.5Gy local head and neck irradiation.

2. Experimental method:

See section 1.2 in the Materials and Methods

3. Experimental results:

Under the two doses of DMSO 4g / kg and 6g / kg, observe whether the preventive effects of different doses of DMSO on radiation oral mucositis are different.

The toluidine blue staining of the tongue tissue was performed 7 days after the irradiation, and the back of the tongue of all 4 mice in the control group were blue stained. One of the mice in the DMSO 4g / kg group was slightly stained. No staining was observed. Tongue tissue sections were observed under microscope after H.E. staining. It was found that all the tongue tissues in the control group showed ulcers. One animal in the DMSO group had a small ulcer, and the DMSO group had no ulcers in the 6g / kg group (Figure 1a). The analysis of ulcer area and the measurement of epithelial thickness showed that the above two dose groups were statistically different from the control group (Figure 1b-1c). The results showed that DMSO 4g / kg and 6g / kg had the protective effect on radiation oral mucositis in mice, and 6g / kg had the best effect.

Example 2. Comparison of the preventive effect of DMSO on radiation oral mucositis in mice

1. Experimental grouping:

Twenty male C57 mice were randomly selected and divided into the following 5 groups:

Non-irradiated control group (Non-IR): 4;

Irradiation control group (Vehicle): intraperitoneal injection of solvent 0.9% physiological saline 0.2ml, 4 animals;

DMSO group 2: Intraperitoneal injection of DMSO at a dose of 6 g / kg, 4 animals, administered 3 hours before irradiation;

DMSO group 3: Intraperitoneal injection of DMSO at a dose of 6 g / kg, 4 animals, administered 6 hours before irradiation.

Irradiation dose: 16.5Gy local head and neck irradiation.

2. Experimental method:

See section 1.2 in the Materials and Methods

3. Experimental results:

DMSO 6g / kg was injected intraperitoneally at three time points: 1h, 3h, and 6h before the observation. Observe whether there is a difference in the preventive effect of DMSO on radiation oral mucositis.

The toluidine blue staining of the tongue tissue was performed 7 days after the irradiation. DMSO was administered 1 h and 3 h before, and the toluidine blue staining was negative. In the 6 h before administration group, one mouse showed a lightly stained spot of toluidine blue on the back of the tongue (Figure 2a). Tongue tissue sections were observed microscopically after H.E. staining. There were sporadic ulcers in the epithelial layer of the back of the tongue in the 3h and 6h groups, and there were no ulcers in the epithelial layer in the 1h group (Figure 1a). The analysis of ulcer area and the measurement of epithelial thickness showed that there were statistical differences between the three groups of DMSO before administration and the control group (Figure 2b-2c). The above results indicate that DMSO once-administered at 1 h, 3 h, and 6 h before the administration of radiation oral mucositis in mice has an effective protective effect, and the best effect at 1 h before administration.

Example 3. Post-administration of DMSO has no therapeutic effect on radiation oral mucositis in mice

1. Experimental grouping:

Non-irradiated control group (Non-IR): 4;

0min group: DMSO was administered immediately after the irradiation, the dose was 6g / kg, 4 rats;

30min group: DMSO was administered 30min after irradiation, the dose was 6g / kg, 4 rats;

1h group: DMSO was administered 1h after the irradiation, the dose was 6g / kg, 4 rats;

Irradiation dose: 16.5Gy local head and neck irradiation.

2. Experimental method:

See section 1.2 in the Materials and Methods

3. Experimental results:

The toluidine blue staining of the tongue tissue was performed 7 days after the irradiation, and the back of the tongue of the mice in the control group and each of the DMSO treatment groups was found to be blue stained. There was no significant difference between the groups (Figure 3a). Tongue tissue sections were observed microscopically after H.E. staining, and obvious ulcers appeared in the epithelial layer of the back of the tongue in each group of mice. The ulcer area analysis and epithelial thickness measurement showed that there was no statistical difference between the DMSO administration group and the irradiation control group after irradiation (Figures 3b-3c). The results show that DMSO has no therapeutic effect on radiation oral mucositis in mice.

Example 4. Comparison of the preventive effects of DMSO, rhKGF and WR2721 on radiation oral mucositis

1. Experimental grouping:

Non-irradiated control group (Non-IR): 4;

DMSO group: intraperitoneal injection of DMSO at a dose of 6g / kg, 4 animals, administered 1 hour before irradiation;

rhKGF group: intraperitoneal injection of rhKGF at a dose of 6.25mg / kg, 4 animals, continuous administration for 3d before irradiation and continuous administration for 3d after 24h, once a day;

WR2721 group: intraperitoneal injection of WR2721, a dose of 200mg / kg, 4 animals, administered 30 minutes before irradiation.

Irradiation dose: 16.5Gy local head and neck irradiation.

2. Experimental method:

See section 1.2 in the Materials and Methods

3. Experimental results:

Toluidine blue staining of the tongue tissues was performed 7 days after the irradiation, and all the mice in the control group were irradiated with blue staining on the back of the tongue. The tongue body of DMSO group, rhKGF group and WR2721 group was pink, the surface was moist, and the toluidine blue staining was negative. Tongue tissue sections were observed microscopically after H.E. staining. The epithelial layer of the back of the tongue of the 4 mice in the DMSO group remained intact, but individual mice in the rhKGF and WR2721 groups showed scattered microulcer spots (Figure 4a). Analysis of the ulcer area, the three treatment groups were significantly better than the irradiation control group (Figure 4b). Epithelial thickness measurement results showed that the epithelial thickness of the three treatment groups was also significantly higher than that of the irradiated control group, and the DMSO group was better than the rhKGF and WR2721 groups (Figure 4c).

Example 5. Comparison of Improving Body Weight and Survival Rate of High-Dose Head and Neck Irradiated Mice by DMSO, rhKGF and WR2721

1. Experimental grouping:

Irradiation control group (Vehicle): intraperitoneal injection of 0.2% 0.9% saline, 7;

DMSO group: intraperitoneal injection of DMSO at a dose of 6g / kg, 7 animals, administered 1 hour before irradiation;

rhKGF group: intraperitoneal injection of rhKGF at a dose of 6.25mg / kg, 7 animals, continuous administration for 3 days before irradiation and continuous administration for 3 days at 24 hours after irradiation, once a day;

WR2721 group: intraperitoneal injection of WR2721 at a dose of 200 mg / kg, 7 animals, administered 30 minutes before irradiation.

Irradiation dose: 16.5Gy local head and neck irradiation.

2. Experimental method:

See section 1.2 in the Materials and Methods

3. Experimental results:

16.5Gy Locally irradiated mice with mouth ulcers caused difficulty in eating due to oral ulcers. The body weight had decreased significantly after 5 days of irradiation, and became worse with the extension of the time after irradiation, and decreased to the lowest value 10 days after irradiation, and animals appeared. Death, and all mice in the control group died within 15 days after irradiation. The mice in the DMSO group also exhibited weight loss early in the post-photograph period, and their body weight began to increase in 9 days post-photograph period. The body weight of 17 days post-photograph period was close to the level before the photoperiod, and no animal death occurred within 30 days after the photoperiod. The mice in the rhKGF and WR2721 groups lost weight early after irradiation, and began to gain weight 15 days after irradiation. The body weight was close to the level before irradiation 23 days after irradiation, and two mice died within 30 days after irradiation (Figure 5a-5b). Table 1 lists the 30-day survival rate of each group of mice (n = 7).

Table 1

Example 6. Preventive effect of DMSO on oral mucositis caused by split head and neck irradiation in mice

1. Experimental grouping:

Non-irradiated control group (Non-IR): 4;

Irradiation control group (Vehicle): 0.2ml 0.9% physiological saline was injected intraperitoneally before each irradiation, 4 animals;

DMSO group: intraperitoneal injection of DMSO at a dose of 4g / kg, 4 animals, administered 1 hour before each irradiation;

Radiation dose: The mice were exposed to 8Gy once a day for 3 consecutive days.

2. Experimental method:

See section 1.2 in the Materials and Methods

3. Experimental results:

Segmented radiation therapy (2Gy 5 times a week) established based on clinical practice experience is considered to be the standard method for tumor radiotherapy. To simulate the clinical radiation treatment of tumors, mice were irradiated with 8Gy of head and neck daily for 3 consecutive days, and an animal model of oral mucositis induced by segmental irradiation was successfully established. Toluidine blue staining of the tongue tissues was performed 7 days after the irradiation, and all the mice in the control group were irradiated with blue staining on the back of the tongue. In the DMSO group, the tongue was pink, the surface was moist, and the toluidine blue staining was negative (Figure 6a). Tongue tissue sections were observed microscopically after H.E. staining. The ulcers on the back of the tongue in the model group showed ulcers, while the epithelial layer on the back of the tongue in the DMSO group remained intact, and no ulcers occurred. Both ulcer area analysis and epithelium thickness measurement showed that the DMSO administration group was statistically different from the irradiation control group (Figure 6b-6c), indicating that DMSO also has a good preventive effect on radiation-induced oral mucositis caused by segmental irradiation.

Example 7. Effects of DMSO on head and neck irradiated mice

1. Experimental grouping:

Non-irradiated control group (Non-IR): 4;

Irradiation control group (Vehicle): 0.2ml 0.9% physiological saline was injected intraperitoneally before irradiation; 4 at each detection point, a total of 28;

DMSO group: intraperitoneal injection of DMSO at a dose of 6g / kg, administered 1 hour before irradiation; 4 at each detection point, a total of 28;

Irradiation dose: 16.5Gy head and neck irradiation.

2. Experimental method:

See section 1.2 in the Materials and Methods

3. Experimental results:

The mucosal layer of the tongue tissue includes the basal layer, the spinous layer, the granular layer, and the keratinized layer. The basal layer contains keratinized epithelial stem and progenitor cells; the increase in the number of keratinized epithelial stem and progenitor cells and their functional reconstruction after irradiation prevent the occurrence of tongue ulcers Or repair the cytological basis of ulcer tissue. p63 is one of the markers of keratinized epithelial stem and progenitor cells. The p63 immunohistochemical technique was used to observe the changes in the number of keratinized epithelial stem and progenitor cells and the protective effect of DMSO on the tongue tissue of mice at different times after irradiation. As shown in Figures 7a-d, the number of p63-positive cells in the tongue tip and back of the tongue progressively decreased within 11 days after irradiation, until they disappeared. The number of p63 positive cells in the tongue and abdomen tissue also decreased after irradiation, and the number was reduced by about half. In the DMSO prevention group, the number of ki-67 positive cells in the tip of the tongue and the back of the tongue also gradually decreased after irradiation, reaching the lowest value on the 7th and 5th days after the irradiation, and then began to recover quickly; the number of p63-positive cells increased after the irradiation Compared with the control group, there were statistical differences at each observation point.

Ki67 is an antigen that marks the state of cell proliferation; keratinized epithelial stem of the basal layer of the tongue tissue, and progenitor cells are in a proliferative state under physiological conditions, and Ki67 is positively labeled. The ki-67 immunohistochemical technique was used to observe the changes of keratinized epithelial proliferation and the protective effect of DMSO on the tongue tissue of mice at different time after irradiation. As shown in Figures 8a-d, the number of ki-67 positive cells in the tip of the tongue and the back of the tongue decreased significantly on the first day after irradiation, and the decrease was more significant with the extension of the time after irradiation, reaching a minimum on the seventh day after irradiation. Value, then the animals that did not administer began to die, and all died after 11 days. The number of ki-67 positive cells in the tongue and abdomen tissues also decreased to varying degrees after irradiation, but it was not significant. Analysis is one of the reasons why ulcers often appear on the back of the tongue and on the tip of the tongue, but not on the abdomen. The number of ki-67 positive cells in the tip and back of the DMSO prevention group also decreased after 1 to 3 days after irradiation, but it was not significant. Compared with the control group at the corresponding time point, there were statistical differences; on the 5th day after irradiation The number of ki-67 positive cells decreased in the tongue tip and back of the tongue to varying degrees, and began to recover at 7 and 5 days after irradiation, respectively, compared with the control group at the corresponding time points. No animals in the DMSO prevention group died of radiation mucositis during the experiment.

Example 8. DMSO promotes repair of DNA damage of tongue keratinized epithelial stem cells after head and neck irradiation in mice

1. Experimental grouping:

Non-irradiated control group (Non-IR): 4;

Irradiation control group (Vehicle): 0.2% 0.9% saline was injected intraperitoneally before irradiation; 4 at each detection point, a total of 12;

DMSO group: intraperitoneal injection of DMSO at a dose of 6g / kg, administered 1 hour before irradiation; 4 at each detection point, a total of 12;

Irradiation dose: 16.5Gy head and neck irradiation.

2. Experimental method:

See section 1.2 in the Materials and Methods

3. Experimental results:

DNA double-strand breaks (DSBs) are the most serious type of DNA damage in cells. DSBs can activate the DNA damage response (DDR) mechanism of cells, thereby causing the rapid phosphorylation of histone H2AX (phosphorylated H2AX histone is called γ-H2AX). Subsequently, γ-H2AX aggregates at the double-strand break, forming a foci that is aggregated by a large number of γ-H2AX. Detecting the number of γ-H2AX foci in p63-positive cells of the tongue mucosa at different time points early after irradiation can understand the DNA damage and repair process of tongue keratinized epithelial stem cells after irradiation.

The results of the study showed (Fig. 9a-b) that no γ-H2AX foci were seen in p63-positive cells in the tongue tissue of unirradiated mice. In the control group of mice, there were more γ-H2AX focal spots in p63-positive cells of the tongue mucosa 2 hours after irradiation, which was most significant 6 hours after irradiation, and disappeared 24 hours after irradiation, reflecting the early keratinized epithelial stem cell DNA after irradiation. The process from damage to repair. The study found that in the DMSO group, the number of γ-H2AX foci in p63-positive cells of the tongue mucosa was not significantly different from that in the control group at 2 hours after irradiation, but the number of γ-H2AX foci did not increase at 6 hours after irradiation. High, but lower than the number of focal points 2 hours after the comparison, indicating that DMSO has no protective effect on the DNA damage of keratinized epithelial stem cells induced by radiation, but can significantly promote the repair process of keratinized epithelial stem cells after DNA damage.

Example 9. Comparison of the Preventive Effects of Structural Analogs of DMSO on Radiation Oral Mucositis

1. Experimental grouping:

Non-irradiated control group (Non-IR): 4;

DMSO group: intraperitoneal injection of DMSO at a dose of 4g / kg, 4 animals, administered 1 hour before irradiation;

Dimethyl sulfide group: intraperitoneal injection of dimethyl sulfide at a dose of 4 g / kg, 4 animals, administered 1 hour before irradiation;

Dimethyl sulfone group: intraperitoneal injection of dimethyl sulfone at a dose of 4 g / kg, 4 animals, administered 1 hour before irradiation;

Tetramethylene sulfoxide group: intraperitoneal injection of tetramethylene sulfoxide, a dose of 4g / kg, 4 animals, administered 1 hour before irradiation;

Irradiation dose: 16.5Gy local head and neck irradiation.

2. Experimental method:

See section 1.2 in the Materials and Methods

3. Experimental results:

In order to reveal the pharmacophores of DMSO against radiation mucositis, we used DMSO as a positive control, and compared the three structural analogs of DMSO with dimethyl sulfide, dimethyl sulfone, and tetramethylene sulfoxide to mouse oral mucosa Protective effect of inflammation.

The respective structures of the corresponding compounds are as follows:

The head and neck of the mice were irradiated with 16.5 Gy locally, and the tongue tissue was taken 7 days after the irradiation and stained with 1% toluidine blue. The results showed that all the back of the tongue of the irradiation control mice were blue stained; the tongue mucosa of the DMSO group and the tetramethylene sulfoxide group was ruddy without obvious staining, and the toluidine blue staining was negative; The lesions on the back of the tongue were all stained positive, and there was no significant difference from the irradiation control group (Figure 10a). Observation of the tongue tissue section by HE staining, the ulcer on the back of the tongue in the dimethyl sulfide group and dimethyl sulfone group showed obvious ulcers, the epithelium became thin, and a large number of inflammatory cells were attached to the surface; No ulceration was seen in the epithelium, even taste bud cells, thickened basal cell layers, and squamous epithelial hyperplasia (Figure 10a). Quantitative analysis showed that from the ulcer size, the incidence of ulcers in the tetramethylene sulfoxide group and the DMSO group was 0, which was significantly different from the irradiation control group (Figure 10b-c). In terms of epithelial thickness, the tetramethylene sulfoxide group and the DMSO group were also significantly better than the irradiation control group, while the dimethyl sulfide and dimethyl sulfone were not different from the irradiation control group.

These results show that the sulfoxide group in DMSO and tetramethylene sulfoxide is the pharmacophore for its radioprotective effect, and when the sulfoxide group in DMSO is oxidized to sulfone, that is, dimethyl sulfone, or reduced to Ether is dimethyl sulfide, and its radioprotective effect disappears.

Example 10. DMSO has no radioprotective effect on head and neck tumor cells

1 Materials and methods

1.1 Materials and reagents

1.1.1 Experimental animals

Male BALB / C nude mice, weighing 22 to 24 g, were purchased from Beijing Huafukang Biotechnology Co., Ltd., animal quality certificate number: SCXK (Jing) 2009-0004. All mice were sent to SPF barrier animal rooms of the Experimental Animal Center of the Academy of Military Medical Sciences in clean transport boxes and reared in cages, 5 per cage, sterilized water and maintained feed for free feeding. Keep the lights on for 12 hours and turn off the lights for 12 hours every day. The certificate number of the animal breeding facility: SYXK- (Army) -2007-004. Mice were subjected to adaptive rearing grouping experiments for about 7 days.

1.1.2 Cell line: Cal27 cells were purchased from China Type Culture Collection Center (Cell Bank), School of Life Sciences, Wuhan University, and kept in this laboratory.

1.1.3 Reagents and related reagents for cell culture: DMSO (China Innochem Chemical and Biological Company, article number D3855), MEM medium (Beijing Zhongke Maichen Company, article number 10010), fetal bovine serum (FBS) (FBS) ( China ExCell Bio company, article number FSP500), 0.25% trypsin digestion solution (China ExCell Bio company, article number 03.13005A).

1.2 Method

The configured human tongue squamous cell carcinoma Cal27 cell suspension was inoculated into the right neck of nude mice with a 1 mL syringe, and each was injected with 0.2 ml (cell density 1 × 10 ⁶ / mL), and a total of 20 nude mice were inoculated.

When the tumor diameter of the nude mice reaches 5mm, the tumor-bearing mice are given DMSO (6g / kg) or an equal amount of normal saline intraperitoneally, and the head and neck are shielded and irradiated with 12Gy for 1 hour. The other two groups of tumor-bearing mice did not receive irradiation after being given the same amount of DMSO (6g / kg) or saline. The tumor size is recorded once every 3 days, and the tumor volume is measured with a vernier caliper. The volume calculation formula is V = (d ₁ × d ₁ × d ₂ ) / 2 (d1 is the short diameter of the tumor, and d2 is the longest diameter perpendicular to d1) Calculate the tumor volume, draw a tumor growth curve, and weigh the nude mice with an electronic balance. At 30 days after the irradiation, nude mice were sacrificed by cervical dislocation, and the tumor tissue was stripped with surgical instruments such as ophthalmic scissors and forceps, and weighed by an electronic balance.

2 results

The weight of non-irradiated tumor-bearing mice decreased slowly with tumor growth, and there was no significant difference between the DMSO group and the normal saline control group. The two groups of tumor-bearing mice receiving 12Gy head and neck irradiation had a significant decrease in body weight within 6 days after irradiation, and the weight of the control group had been maintained at a low level. The body weight had increased slightly after 25 days of irradiation, and there was one mouse. Died, and the weight of the DMSO group increased significantly after 12 days of irradiation, which is significantly different from that of the control group, suggesting that DMSO also has a protective effect on oral mucositis of tumor-bearing mice caused by radiation (Figure 11a).

It can be seen from Figs. 11b-d that after DMSO was administered to non-irradiated tumor-bearing mice, tumor volume and tumor weight 30 days after administration were not significantly different from those in the control group. 12Gy head and neck local irradiation can significantly inhibit tumor growth. Compared with non-irradiated two groups of tumor-bearing mice, the tumor volume and tumor weight after 30 days of irradiation were significantly reduced, but the DMSO prevention group was still compared with the control group. No significant difference. These results indicate that DMSO has no effect on the efficacy of radiation therapy for tumors while preventing radiation oral mucositis in mice.

Example 11. Preventive effect of DMSO on oral mucositis caused by head and neck irradiation combined with chemotherapy in mice

1. Experimental grouping:

Radiochemotherapy control group (vehicle): intraperitoneal injection of 0.2% 0.9% saline, 8;

DMSO group: intraperitoneal injection of DMSO at a dose of 6g / kg, 8 animals, administered 1 hour before irradiation;

Exposure dose: 16.0Gy local head and neck irradiation.

Chemotherapy drugs: Cisplatin (10mg / kg) was given by intraperitoneal injection 30 minutes before.

2. Experimental method:

See section 1.2 in the Materials and Methods

3. Experimental results:

Due to the side effects of chemotherapeutic drugs, radiotherapy and chemotherapy mice had a significant decrease in body weight at 1 day after irradiation, and increased with the time after irradiation, and subsequently suffered from eating difficulties due to oral ulcers. All died. The mice in the DMSO group also showed weight loss early in the post-photograph period, reaching a minimum value at 11 days post-photograph, and started to recover weight at 13 days post-photograph, and their body weight was close to the previous level at 21 days post-photograph. Death within 10 days (Figures 12a-12b). Table 2 lists the 30-day survival rate of each group of mice (n = 8).

Table 2

Example 12. Preventive effect of DMSO on esophageal mucosal injury in mice with head and neck irradiation

1. Experimental grouping:

Non-irradiated control group (Non-IR): 4;

Irradiation control group (Vehicle): 0.2ml 0.9% saline was intraperitoneally injected before irradiation; 4 rats;

DMSO 5g / kg group: 50% DMSO was administered intragastrically, the dose was 5g / kg, 4 hours before administration, 4 animals;

DMSO 10g / kg group: 50% DMSO was administered intragastrically, the dose was 10g / kg, 4 hours before administration, 4 animals;

Irradiation dose: 12.0Gy head and neck irradiation.

2. Experimental method:

See section 1.2 in the Materials and Methods

3. Experimental results:

The results showed that 5 days after 12Gy head-irradiated mice, the number of esophageal mucosal epithelial cells was reduced compared with the non-irradiated group, which was manifested by deep staining of hematoxylin-eosin staining (H & E) cells and reduction of Ki67-positive cells. The number of BrdU-positive cells increased; compared with the control group, the number of esophageal mucosal epithelial cells in DMSO 5g / kg and 10g / kg groups was significantly increased, and the number of Ki67 and BrdU-positive cells increased significantly, with a certain Dose effect (see Table 3 and Figure 13).

table 3

** Indicates p <0.01.

Example 13. Preventive effect of DMSO on taste dysfunction in head and neck irradiation mice

1. Experimental grouping:

Non-irradiated control group (Non-IR): 6;

Irradiation control group (Vehicle): 0.2ml 0.9% physiological saline was injected intraperitoneally before irradiation; 6 animals;

DMSO group: 50% DMSO was administered intragastrically, the doses were 10g / kg, 6 animals were administered 1 hour before irradiation;

Irradiation dose: 15.0Gy head and neck irradiation.

2. Experimental method:

See section 1.2 in the Materials and Methods

3. Experimental results:

Radiation can directly damage the peripheral receptors of taste, and taste dysfunction appears. Taste sensitivity was measured using sucrose preference experiments. Mice were subjected to sucrose preference experiment 9 to 10 days after irradiation. It was found that the sucrose preference rate of irradiated mice was significantly lower than that of non-irradiated control mice. The drug can prevent taste dysfunction in mice caused by head and neck irradiation (Table 4).

Table 4

** Indicates p <0.01.

Example 14. Preventive effect of DMSO on salivary gland secretion in mice irradiated with head and neck

1. Experimental grouping:

Non-irradiated control group (Non-IR): 6;

Irradiation dose: 15.0Gy head and neck irradiation.

2. Experimental method:

See section 1.2 in the Materials and Methods

3. Experimental results:

High-dose irradiation on the head and neck can cause salivary gland damage, resulting in a decrease in salivary gland secretion function, and then a significant decrease in saliva flow rate and subsequent dry mouth, intraoral mucositis, dental caries, pain, and difficulty swallowing. As shown in Table 5, the saliva secretion of mice in each experimental group was measured 2 months after the irradiation, and it was found that the saliva secretion of the 15.0Gy head and neck irradiated mice was significantly lower than that of the non-irradiated control group, indicating that the salivary glands appeared in the irradiated mice. Secretory function decreased; salivary levels of mice were significantly higher than those of the control group after DMSO was administered before the irradiation, indicating that administration of DMSO before the irradiation had a clear preventive effect on salivary hypofunction of the salivary glands after irradiation of the head and neck mice.

table 5

** Indicates p <0.01.

Example 15. Preventive effect of DMSO on head and neck irradiation in mice

Free thyroxine includes thyroxine (FT4), free triiodothyronine (FT3) and thyroid stimulating hormone (TSH) are routine indicators of thyroid function. The study found that the serum FT4 and FT3 levels of 12Gy head and neck irradiated mice were significantly lower than those of the non-irradiated control group at 3 months after irradiation, and the TSH levels were significantly increased, indicating that the irradiated mice had radioactive hypothyroidism; mice After administration of DMSO as before, the levels of serum FT4 and FT3 were significantly higher than those of the irradiation control group, TSH levels were significantly reduced, and thyroid function was significantly improved after irradiation (Table 6). These results indicate that DMSO has a clear preventive effect on hypothyroidism in mice exposed to head and neck irradiation.

Table 6

** Indicates p <0.01.

Although the preferred embodiments of the present invention have been disclosed for illustrative purposes, those skilled in the art will appreciate that various changes, additions and substitutions may be made without departing from the invention as disclosed in the appended claims. range.

Claims

Use of a sulfoxide compound in the preparation of a pharmaceutical composition for preventing radiation oral mucositis and related radiotherapy complications in a tumor-bearing subject who will receive radiation therapy.
The use according to claim 1, wherein the sulfoxide compound is selected from one or more of the following: dimethyl sulfoxide (DMSO), tetramethylene sulfoxide, diethyl sulfoxide, Methyl ethyl sulfoxide, propyl sulfoxide, diisopropyl sulfoxide, n-butyl sulfoxide, diisobutyl sulfoxide, diisopentyl sulfoxide, diphenyl sulfoxide, and dibenzyl sulfoxide Preferably, the sulfoxide compound is selected from DMSO and tetramethylene sulfoxide; more preferably, the sulfoxide compound is DMSO.
The use according to claim 1 or 2, wherein the subject suffering from a tumor is a subject suffering from a tumor of the head and neck, preferably a human suffering from a tumor of the head and neck.
The use according to any one of claims 1-3, wherein the head and neck tumor is selected from one or more of the following: a neck tumor (such as a thyroid tumor), an otolaryngology tumor (such as a nasopharyngeal cancer, a larynx) Cancer or paranasal sinus cancer) and oral and maxillofacial tumors (such as oral cancer, including tongue cancer, gum cancer, cheek cancer, maxillary sinus cancer, and tonsil cancer).
The use according to any one of claims 1 to 4, wherein the related radiotherapy complications are selected from the group consisting of: esophageal mucosal damage, taste dysfunction, salivary gland secretion, radiation thyroid disease (radiothyroidism, chronic radiation thyroiditis) , Acute radioactive thyroiditis and benign nodules of radioactive thyroid), radioactive xerostomia, radioactive abnormal taste, radioactive dental caries, radioactive esophagitis, and radioactive esophageal stricture.
The use according to any one of claims 1-5, wherein the subject suffering from a tumor can be a subject who will receive a combination of radiation and chemotherapy.
The use according to any one of claims 1-6, wherein the subject suffering from a tumor is a head and neck tumor that will receive radiation treatment within 8 hours, preferably within 4 hours, more preferably within 2 hours, and most preferably within 1 hour. Object.
The use according to any one of claims 1 to 7, wherein the pharmaceutical composition is in a dosage form suitable for an administration mode selected from the group consisting of intravenous administration, oral mucosal administration, transdermal administration, and oral administration Dosing.
The use according to any one of claims 1 to 7, wherein the pharmaceutical composition is in a dosage form selected from the group consisting of an infusion, an injection, a mouthwash, a film, an aerosol, a tablet, an adhesive Preparations, patches, sublinguals, disintegrating agents, sticks, powders, ointments, gels, aqueous solutions, suspensions and capsules.
The use according to any one of claims 1 to 9, wherein the pharmaceutical composition is administered at a dose of 0.1 to 10.0 g of sulfoxide compounds, preferably 0.2 to 5.0 g of sulfoxide compounds, and more preferably 0.4 to 2.0 g of sulfoxide compounds. Compound, most preferably 0.5-1.0 g of sulfoxide compound, per kg of body weight.
The use according to any one of claims 1 to 10, wherein said radiation therapy comprises treatment using a radiation selected from the group consisting of alpha rays, beta rays, gamma rays, x-rays, neutrons, electron beams, proton beams, particle beams And its combination; or the radiation therapy includes external radiation therapy and internal radiation therapy; or the radiation therapy method includes conventional segmented radiation therapy and one high-dose radiation therapy
The use according to any one of claims 1-11, wherein the single dose of the radiation therapy is 1-50 Gy, more preferably 1-8 Gy, and most preferably 2 Gy.
The use according to any one of claims 1-15, wherein said radiation therapy comprises the use of a radiosensitizer.