WO2022151335A2 - Method for tumor treatment by means of hyperbaric oxygen combined with immune drug - Google Patents

Method for tumor treatment by means of hyperbaric oxygen combined with immune drug Download PDF

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WO2022151335A2
WO2022151335A2 PCT/CN2021/072094 CN2021072094W WO2022151335A2 WO 2022151335 A2 WO2022151335 A2 WO 2022151335A2 CN 2021072094 W CN2021072094 W CN 2021072094W WO 2022151335 A2 WO2022151335 A2 WO 2022151335A2
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inhibitor
tumor
group
hyperbaric oxygen
mice
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PCT/CN2021/072094
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French (fr)
Chinese (zh)
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李子福
杨祥良
刘欣
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华中科技大学
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents

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  • the present invention relates to the field of tumor treatment, and more particularly, to a method for treating tumors with hyperbaric oxygen combined with immune drugs.
  • Tumor immunotherapy especially PD-1 (programmed death-1) antibody immune blockade therapy
  • PD-1 programmed death-1) antibody immune blockade therapy
  • Tc cells cytotoxic T cells
  • PD-1 antibodies have been listed in China, including Bristol-Myers Squibb's Opdivo (Odivo), Merck's Keytruda (Kerida), and domestic Junshi Bio-Toripalimab (Tuoyi), Xinxin Dabio’s sintilimab (Daboshu) and Hengrui’s camrellimumab (Elito).
  • PD-1 antibody drugs like other immune checkpoint blockers, has encountered a huge bottleneck-the overall low response rate.
  • the clinical use of PD-1 antibody drugs in the immunotherapy of some non-solid tumors such as melanoma and small cell lung cancer has good results, it is only limited to some types of tumors in some patients, and the overall response rate is very low. .
  • the effect of immunotherapy greatly limits the effect of immunotherapy, resulting in PD-1 antibody drugs in solid tumors.
  • the overall response rate in tumor immunotherapy is less than 10%.
  • the present invention provides a method of hyperbaric oxygen combined with PD-1 inhibitor for tumor immunotherapy, the purpose of which is to improve hypoxia in tumor tissue of patients through hyperbaric oxygen therapy It can regulate the tumor stromal microenvironment and promote the penetration of PD-1 inhibitors in the tumor site, thereby enhancing the anti-tumor efficacy of PD-1 inhibitors and solving the problem of low response rate of PD-1 inhibitors in clinical practice.
  • the present invention provides a method of hyperbaric oxygen combined with PD-1 inhibitor for tumor immunotherapy, by combining hyperbaric oxygen therapy and PD-1 inhibitor therapy, anti-tumor therapy is performed on tumor patients, Specifically include the following steps:
  • Hyperbaric oxygen therapy is performed on the tumor patient; the hyperbaric oxygen therapy increases the amount of oxygen dissolved in the blood by increasing the air pressure in the environment where the tumor patient is located and the concentration of inhaled oxygen, thereby improving the perfusion of oxygen in each tissue;
  • the technical scheme used for intravenous injection of PD-1 inhibitor to tumor patients is to combine single or multiple administration of hyperbaric oxygen therapy with intravenous injection of PD-1 inhibitor for treatment.
  • hyperbaric oxygen therapy is combined with intravenous injection of PD-1 inhibitor, and anti-tumor immunotherapy is performed on tumor patients.
  • hyperbaric oxygen combined with PD-1 inhibitor can improve the penetration amount and penetration depth of PD-1 in tumors without increasing toxic and side effects.
  • the present invention combines hyperbaric oxygen therapy with intravenous injection of PD-1 inhibitor to carry out anti-tumor immunotherapy for tumor patients.
  • the experiment found that not only has a good inhibitory effect on subcutaneous tumors of liver cancer with a size of 120 mm 3 smaller, but also has a good inhibitory effect on initial With a volume of 500 mm 3 , the subcutaneous tumor of liver cancer, which is difficult to be eliminated with a larger volume, also has a good inhibitory effect, basically inhibiting the growth of the tumor or even eliminating the tumor.
  • the method of hyperbaric oxygen combined with PD-1 inhibitor for tumor immunotherapy proposed by the present invention is simple and easy to operate, safe and reliable, and is beneficial to clinical transformation.
  • Fig. 1 is the tumor volume-time curve of the tumor in the anti-tumor activity experiment carried out in Example 6 of the present invention
  • Fig. 2 is the tumor weight result of mice in the anti-tumor activity experiment carried out in Example 6 of the present invention
  • Fig. 3 is the weight-time curve of mice in the anti-tumor activity experiment carried out in Example 6 of the present invention.
  • Fig. 4 is the result of the blood biochemical index of each experimental group in the embodiment of the present invention 6;
  • Example 5 is a microscopic observation picture of tissue sections of each experimental group in Example 6 of the present invention.
  • Fig. 6 is the tumor volume-time curve of the tumor in the anti-tumor activity experiment carried out in Example 7 of the present invention.
  • Fig. 7 is the tumor weight result of mice in the anti-tumor activity experiment carried out in Example 7 of the present invention.
  • Figure 8 is the body weight-time curve of mice in the anti-tumor activity experiment carried out in Example 7 of the present invention.
  • Figure 9 is the result of the number of lung tumor metastases in mice in the anti-tumor activity experiment carried out in Example 7 of the present invention.
  • Fig. 10 is the result of the blood biochemical index of each experimental group in Example 7 of the present invention.
  • Example 11 is a microscopic observation picture of tissue sections of each experimental group in Example 7 of the present invention.
  • Figure 12 shows the results of the survival period of mice in each group in Example 8 of the present invention.
  • Figure 13 is the tumor weight results of mice in the anti-tumor activity experiment carried out in Example 9 of the present invention.
  • Figure 14 is the body weight-time curve of mice in the anti-tumor activity experiment carried out in Example 9 of the present invention.
  • Figure 15 is the result of the blood biochemical indexes of each experimental group in Example 9 of the present invention.
  • Example 16 is a microscopic observation picture of tissue sections of each experimental group in Example 9 of the present invention.
  • Figure 17 shows the results of the survival period of mice in each group in Example 10 of the present invention.
  • Figure 18 is the tumor weight results of mice in the anti-tumor activity experiment carried out in Example 11 of the present invention.
  • Figure 19 is the body weight-time curve of mice in the anti-tumor activity experiment carried out in Example 11 of the present invention.
  • Figure 20 is the results of the blood biochemical indicators of each experimental group in Example 11 of the present invention.
  • Example 21 is a microscopic observation picture of tissue sections of each experimental group in Example 11 of the present invention.
  • Figure 22 is the tumor volume-time curve of the tumor in the anti-tumor activity experiment performed in Example 12 of the present invention.
  • Figure 23 is the result of tumor weight in mice in the anti-tumor activity experiment carried out in Example 12 of the present invention.
  • FIG. 24 is a fluorescent imaging diagram of in vivo and ex vivo mice in each experimental group of small animals imaging detection in Example 13 of the present invention.
  • Figure 25 is a graph of the enrichment amount-time curve of PD-1 inhibitor in living tumor tissue detected by imaging of small animals in each experimental group in Example 13 of the present invention.
  • 26 is a quantitative diagram of tissue distribution of PD-1 inhibitors detected by imaging in small animals of each experimental group in Example 13 of the present invention.
  • the present invention provides a method for using hyperbaric oxygen combined with PD-1 inhibitor for tumor immunotherapy.
  • hyperbaric oxygen therapy By combining hyperbaric oxygen therapy and PD-1 inhibitor therapy, anti-tumor therapy is performed on tumor patients, which specifically includes the following steps:
  • Hyperbaric oxygen therapy is performed on the tumor patient; the hyperbaric oxygen therapy increases the amount of oxygen dissolved in the blood by increasing the air pressure in the environment where the tumor patient is located and the concentration of inhaled oxygen, thereby improving the perfusion of oxygen in each tissue;
  • the present invention does not limit the order of hyperbaric oxygen therapy and intravenous injection of PD-1 inhibitor therapy.
  • a PD-1 inhibitor is intravenously injected into the tumor patient.
  • hyperbaric oxygen therapy for tumor patients can improve the hypoxic environment of the tumor, especially, it can significantly promote the penetration and enrichment of PD-1 inhibitors in tumor sites, and enhance the anti-tumor effect of PD-1 inhibitors. Efficacy, improve the clinical response rate of PD-1 inhibitors.
  • Hyperbaric oxygen (HBO) therapy refers to the intermittent administration of pure oxygen to patients under conditions of higher than normal atmospheric pressure.
  • the principle of hyperbaric oxygen therapy is to increase the amount of oxygen dissolved in the blood by increasing the air pressure of the patient's environment and the concentration of inhaled oxygen, thereby improving the perfusion of oxygen in various tissues.
  • Hyperbaric oxygen can effectively increase the partial pressure of oxygen in tissues, relieve tissue hypoxia, reduce edema, activate angiogenesis and collagen synthesis, so it is widely used in the prevention and treatment of various diseases, such as CO poisoning, decompression sickness and air pressure injury etc.
  • the present invention combines hyperbaric oxygen with PD-1 antibody, improves tumor tissue hypoxia through hyperbaric oxygen, promotes PD-1 antibody delivery, improves tumor immune microenvironment, and improves anti-tumor effect.
  • the hyperbaric oxygen therapy for tumor patients can be performed according to the conventional method of hyperbaric oxygen therapy in the prior art.
  • the hyperbaric oxygen therapy for tumor patients is specifically: placing the tumor patient in an airtight chamber, and gradually introducing pure Oxygen increases the air pressure in the cabin to 2-5 times atmospheric pressure, and after a period of time, deflation reduces the air pressure in the cabin to atmospheric pressure.
  • PD-1 inhibitor therapy is given by intravenous injection of PD-1 inhibitor.
  • the tumor patient is placed in a closed cabin, and pure oxygen is gradually introduced to increase the pressure in the cabin to 2-5 times atmospheric pressure, preferably 2-2.5 times atmospheric pressure, and maintain for 1-4 hours (preferably 1- After 2 hours), slowly deflate to bring the cabin pressure down to atmospheric pressure.
  • the order of hyperbaric oxygen therapy and PD-1 inhibitor administration is variable, preferably hyperbaric oxygen therapy first, followed by intravenous injection of the PD-1 inhibitor.
  • the interval between the hyperbaric oxygen therapy of the present invention and the intravenous injection of the PD-1 inhibitor is 0-12 hours.
  • the interval time is preferably 1-12 hours, more preferably 1-3 hours.
  • hyperbaric oxygen therapy is administered to tumor patients 1-10 times, and in a preferred embodiment, hyperbaric oxygen therapy is administered 2-5 times.
  • steps (1) and (2) are repeated to complete one treatment, and the treatment is continued for no less than one time.
  • the present invention can adjust the dose of the PD-1 inhibitor according to the actual application.
  • the tumor patient is intravenously injected with the PD-1 inhibitor once every to three weeks, and the single injection dose is 100-300 mg/m 2 .
  • hyperbaric oxygen therapy is performed on tumor patients at least once, and the frequency of hyperbaric oxygen therapy is not limited. , hyperbaric oxygen therapy every two days, every three days, or every four days. In a preferred embodiment, hyperbaric oxygen therapy is administered 1-5 times per day; preferably 1-2 times.
  • the injected dose of PD-1 inhibitor can be based on individual differences, such as intravenous injection every 2 weeks or every 3 weeks, until disease progression or intolerable toxicity.
  • hyperbaric oxygen therapy is performed 1-7 times per week.
  • the PD-1 inhibitor used in the present invention can also be called PD-1 antibody or PD-1 antibody drug, and can be various PD-1 antibody drugs that have been used clinically, such as the currently approved PD-1 antibody Merck's Keytruda, Bristol-Myers Squibb's Opdivo, Sanofi and Regeneron jointly developed Libtayo, and domestically produced Junshi Biotechnology Toripalimab (Tuoyi), Innovent’s sintilimab (Daboshu) and Hengrui’s camrelimab (Elito).
  • PD-1 antibody or PD-1 antibody drug can be various PD-1 antibody drugs that have been used clinically, such as the currently approved PD-1 antibody Merck's Keytruda, Bristol-Myers Squibb's Opdivo, Sanofi and Regeneron jointly developed Libtayo, and domestically produced Junshi Biotechnology Toripalimab (Tuoyi), Innovent’s sintilimab (Daboshu
  • the clinically recommended dose of Dabosu is 200 mg/m 2 , administered once every 3 weeks.
  • the administration method of PD-1 inhibitor, including reconstitution and dilution, etc., can also be the same as the above-mentioned current marketed drug administration method.
  • the tumor immunotherapy method proposed in the present invention can be applied to various tumor types, not only non-solid tumors such as melanoma, small cell lung cancer, etc., but also various solid tumors, including head and neck tumors, thoracic tumors, etc.
  • various tumors including head and neck tumors, thoracic tumors, etc.
  • the combined treatment method proposed by the present invention significantly improves the enrichment of PD-1 inhibitors in tumor sites for various solid tumors such as subcutaneous tumors of liver cancer, in situ tumors of breast cancer, in situ tumors of liver cancer, and pancreatic cancer in situ tumors.
  • it not only has a good inhibitory effect on subcutaneous tumors of liver cancer with a size of 120mm 3 , and 5 tumors in 8 mice were completely eliminated after 17 days; but also for liver cancer with an initial volume of 500mm 3 , which is difficult to eliminate.
  • Subcutaneous tumors also had a good inhibitory effect, and tumor growth was basically inhibited or even eliminated in 4 of the 8 mice.
  • the combination therapy method of the present invention for tumor immunotherapy clinically, the hyperbaric oxygen and the dose of PD-1 inhibitor can be adjusted according to the tumor type and size.
  • the hyperbaric oxygen combined with the PD-1 inhibitor of the present invention significantly improves the response rate of PD-1 treatment without increasing the toxic and side effects, and is beneficial to clinical transformation.
  • the invention provides a new anti-tumor combined treatment mode and expands the new application of hyperbaric oxygen.
  • the PD-1 inhibitor used in specific examples 1 to 4 is Merck's Keytruda
  • the PD-1 inhibitor used in examples 5 to 13 is Xinbosheng's anti -mouse PD-1 antibody.
  • This embodiment provides a method for hyperbaric oxygen combined with PD-1 inhibitor for tumor treatment, the method includes hyperbaric oxygen therapy and PD-1 inhibitor injection.
  • the hyperbaric oxygen therapy method is as follows: place the treated individual in a closed cabin, gradually introduce pure oxygen to increase the pressure in the cabin to 2.5 times the atmospheric pressure, maintain it for 1.5 hours, and slowly deflate to reduce the pressure in the cabin to atmospheric pressure.
  • the above hyperbaric oxygen therapy was performed once a day for 14 consecutive days; PD-1 inhibitor therapy was performed after the end of the hyperbaric oxygen therapy on the 14th day .
  • PD-1 inhibitors were performed once a day for 14 consecutive days; PD-1 inhibitor therapy was performed after the end of the hyperbaric oxygen therapy on the 14th day .
  • This embodiment provides a method for hyperbaric oxygen combined with PD-1 inhibitor for tumor treatment, the method includes hyperbaric oxygen therapy and PD-1 inhibitor injection.
  • the hyperbaric oxygen therapy method is: place the treated individual in a closed cabin, gradually introduce pure oxygen to increase the pressure in the cabin to 2.5 times the atmospheric pressure, maintain it for 1.5 hours, and slowly deflate the cabin to reduce the pressure to the atmospheric pressure.
  • hyperbaric oxygen therapy was administered once a day for three consecutive days, 200 mg/m 2 PD-1 inhibitor was administered intravenously at a two-hour interval after the end of hyperbaric oxygen on the third day. Hyperbaric oxygen was then administered every other day, followed by intravenous injection of 200 mg/m 2 PD-1 inhibitor at two-hour intervals after the end of hyperbaric oxygen treatment two weeks later. Hyperbaric oxygen was then administered every other day, followed by intravenous injection of 200 mg/m 2 PD-1 inhibitor at two-hour intervals after the end of hyperbaric oxygen treatment two weeks later.
  • This embodiment provides a method for hyperbaric oxygen combined with PD-1 inhibitor for tumor treatment, the method includes hyperbaric oxygen therapy and PD-1 inhibitor injection.
  • the hyperbaric oxygen therapy method is as follows: place the treated individual in a closed cabin, gradually introduce pure oxygen to increase the pressure in the cabin to 2 times the atmospheric pressure, and after maintaining for 2 hours, slowly deflate to reduce the pressure in the cabin to atmospheric pressure. Hyperbaric oxygen therapy was performed every two days, and after 3 weeks, PD-1 inhibitor therapy was performed. The specific method was: intravenous injection of 200 mg/m 2 PD-1 inhibitor once every two hours after the end of hyperbaric oxygen therapy.
  • This embodiment provides a method for hyperbaric oxygen combined with PD-1 inhibitor for tumor treatment, the method includes hyperbaric oxygen therapy and PD-1 inhibitor injection.
  • the hyperbaric oxygen therapy method is: place the treated individual in a closed cabin, gradually introduce pure oxygen to increase the pressure in the cabin to 2.5 times the atmospheric pressure, maintain it for 1.5 hours, and slowly deflate the cabin to reduce the pressure to the atmospheric pressure.
  • the PD-1 inhibitor treatment method is as follows: 6.25mg/kg PD-1 inhibitor is intravenously injected at an interval of 12 hours after the end of hyperbaric oxygen therapy.
  • Hyperbaric oxygen therapy was administered every other day, and 200 mg/m 2 PD-1 inhibitor was administered intravenously for the first time at 12-hour intervals after the end of hyperbaric oxygen therapy after 2 weeks. Then, hyperbaric oxygen therapy was performed every other day, and a second dose of 200 mg/m 2 PD-1 inhibitor was administered intravenously at 12-hour intervals after the end of the hyperbaric oxygen therapy after 2 weeks.
  • This embodiment provides a method for hyperbaric oxygen combined with PD-1 inhibitor for tumor treatment, the method includes hyperbaric oxygen therapy and PD-1 inhibitor injection.
  • PD-1 inhibitor therapy is: 6.25mg/kg PD-1 inhibitor intravenously.
  • the hyperbaric oxygen treatment method is: after intravenous injection of PD-1 inhibitor, the treated individual is placed in a closed cabin immediately, and pure oxygen is gradually introduced to increase the pressure in the cabin to 2.5 times the atmospheric pressure. After maintaining for 1.5 hours, slowly deflate Reduce the cabin pressure to atmospheric pressure.
  • a mouse liver cancer H22 subcutaneous tumor model was established.
  • the tumor-bearing mice were randomly divided into 4 groups (PBS group, HBO group, PD-1 inhibitor group and HBO+PD-1 group). Inhibitor group) and recorded as the first day, the mice in the HBO group and the HBO+PD-1 inhibitor group were treated with hyperbaric oxygen on the 1st, 3rd, and 5th days respectively.
  • the treatment method was as follows: the mice were placed in a closed cabin , and gradually introduce pure oxygen to increase the pressure in the cabin to 2.5 times the atmospheric pressure. After maintaining for 1.5 hours, slowly deflate the pressure to reduce the pressure in the cabin to atmospheric pressure.
  • mice in the HBO+PD-1 inhibitor group were given 0.1 mg/0.1 mL of PD-1 inhibitor by tail vein injection at two hours after the end of hyperbaric oxygen therapy.
  • PBS and PD-1 inhibitor were injected into the corresponding experimental group mice through tail vein at doses of 0.1mL/mice and 0.1mg/0.1mL, respectively.
  • the mice in each group were sacrificed on the 17th day, and the subcutaneous tumors were excised and weighed. Heavy. At the same time as tumor volume was measured, the body weight of the mice was measured every two days and recorded.
  • mice were sacrificed, the whole blood was taken out to detect the composition and content of blood cells, and the blood was collected to separate the serum, and the blood biochemical indexes were analyzed.
  • BUN myocardial creatine kinase
  • CK myocardial creatine kinase
  • Table 1 shows the results of tumor volume in each group at different time points (unit: mm 3 )
  • Figure 2 shows the statistical results of the tumor weights taken out of the four groups of mice after treatment.
  • the tumor weights of the PD-1 inhibitor group and the HBO+PD-1 inhibitor group were significantly lower than those of the PBS group.
  • the tumor weight of the treatment group was significantly lower than that of the PD-1 inhibitor treatment group alone, indicating that HBO combined with PD-1 inhibitor can enhance the anti-tumor effect of PD-1 inhibitor.
  • Figure 3 shows the body weight changes of the above 4 groups during the administration period. It can be seen from Figure 3 that compared with the PBS group, the body weight of the mice in the three experimental groups has no obvious decrease or increase trend, which indicates that HBO or PD- 1 Inhibitor treatment, as well as combination treatment, did not affect the health of the mice.
  • Table 2 shows the results of blood cell detection in each group after treatment
  • Figure 4 shows the results of blood biochemical analysis of the PBS group, HBO group, PD-1 inhibitor group and HBO+PD-1 inhibitor group.
  • Alanine aminotransferase, aspartate aminotransferase, myocardial creatine kinase and total urea nitrogen were mainly detected. Elevated alanine aminotransferase and aspartate aminotransferase indicate serious liver toxicity, elevated myocardial creatine kinase value indicates pathological changes in the heart, and elevated total urea nitrogen index indicates renal function problems.
  • the four blood biochemical indexes of the three experimental groups have no significant changes compared with the control group (PBS group). Neither the drug treatment nor the combined treatment will cause damage to the main organs of the mice, and the treatment has good safety.
  • Figure 5 is a structural diagram of a tissue section at a magnification of 200 times. It can be seen from Figure 5 that the structure of the heart, liver, spleen, lung and kidney in the PBS group and the three experimental groups is clear, with no obvious pathological changes, no bleeding and inflammatory infiltration. None of the treatments would cause damage to the main organs of the mice and had good safety.
  • a mouse breast cancer 4T1 orthotopic tumor model was established.
  • the tumor-bearing mice were randomly divided into 4 groups (PBS group, HBO group, PD-1 inhibitor group and HBO+PD- 1 inhibitor group) and recorded as the 1st day, on the 1st, 3rd, and 5th days, the mice in the HBO group and the HBO+PD-1 inhibitor group were treated with hyperbaric oxygen therapy.
  • the treatment method was to place the mice in a closed cabin. In the body, pure oxygen was gradually introduced to increase the air pressure in the cabin to 2.5 times the atmospheric pressure. After maintaining for 1.5 hours, the air pressure was slowly released to reduce the air pressure in the cabin to atmospheric pressure.
  • mice in the HBO+PD-1 inhibitor group were given 0.1 mg/0.1 mL of PD-1 inhibitor by tail vein injection at two hours after the end of hyperbaric oxygen therapy.
  • PBS and PD-1 inhibitor were injected into the corresponding experimental group mice through tail vein at doses of 0.1mL/mice and 0.1mg/0.1mL, respectively.
  • the mice in each group were sacrificed on the 23rd day, and the subcutaneous tumors were excised and weighed. Heavy. At the same time as tumor volume was measured, the body weight of the mice was measured every two days and recorded.
  • mice were sacrificed, the whole blood was taken out to detect the composition and content of blood cells, and the blood was collected to separate the serum, and the blood biochemical indexes were analyzed.
  • BUN myocardial creatine kinase
  • CK myocardial creatine kinase
  • the five main organs of the mouse heart, liver, spleen, lung and kidney were taken out, fixed with 4% paraformaldehyde, then routinely embedded in paraffin, and stained with HE, and the tissue structure was observed under a microscope.
  • Table 3 shows the results of tumor volume in each group at different time points (unit: mm 3 )
  • Figure 7 shows the statistical results of tumor weights taken out of the four groups of mice after treatment. It can be seen from Figure 7 that the tumor weights in the PD-1 inhibitor group and the HBO+PD-1 inhibitor group were significantly lower than those in the PBS group. The tumor weight of the treatment group was significantly lower than that of the PD-1 inhibitor treatment group alone, indicating that HBO combined with PD-1 inhibitor can enhance the anti-tumor effect of PD-1 inhibitor.
  • Figure 8 shows the body weight changes of the above four groups during the administration period. It can be seen from Figure 8 that, compared with the PBS group, the body weight of the mice in the three experimental groups has no obvious decrease or increase trend, which indicates that the use of HBO or PD- 1 Inhibitor treatment, as well as combination treatment, did not affect the health of the mice.
  • Figure 9 shows the statistical results of the number of lung metastases in the four groups of mice after treatment.
  • the PD-1 inhibitor group and the HBO+PD-1 inhibitor group had more The number of nodules was significantly reduced, while the number of pulmonary metastatic nodules in the combination treatment group was significantly less than that in the PD-1 inhibitor treatment group alone, which indicated that HBO combined with PD-1 inhibitor had a significant inhibitory effect on tumor metastasis.
  • Table 4 shows the results of blood cell detection in each group after treatment
  • Figure 10 shows the blood biochemical analysis results of the PBS group, the HBO group, the PD-1 inhibitor group and the HBO+PD-1 inhibitor group.
  • Alanine aminotransferase, aspartate aminotransferase, myocardial creatine kinase and total urea nitrogen were mainly detected. Elevated alanine aminotransferase and aspartate aminotransferase indicate serious liver toxicity, elevated myocardial creatine kinase value indicates pathological changes in the heart, and elevated total urea nitrogen index indicates renal function problems.
  • the four blood biochemical indexes of the three experimental groups have no significant changes compared with the control group (PBS group). The drug treatment and the combined treatment will not cause damage to the main organs of the mice, and the treatment has good safety
  • Figure 11 is a structural diagram of a tissue section at a magnification of 200 times. It can be seen from Figure 11 that the structure of the heart, liver, spleen, lung and kidney in the PBS group and the three experimental groups is clear, with no obvious pathological changes, no bleeding and inflammatory infiltration. The above results show that HBO or PD-1 inhibitor treatment alone, and combined treatment All of them will not cause damage to the main organs of mice, and have good safety.
  • the mouse liver cancer H22 orthotopic tumor model was established and recorded as the first day. Two weeks later, the tumor-bearing mice were randomly divided into 4 groups (PBS group, HBO group, PD-1 inhibitor group and HBO+PD-1 inhibitor group). The mice in the HBO group and the HBO+PD-1 inhibitor group were treated with hyperbaric oxygen on the 14th, 16th, and 18th days, respectively.
  • the treatment method was to place the mice in a closed cabin, and gradually introduce pure oxygen into the cabin.
  • the internal pressure was raised to 2.5 times atmospheric pressure, and after maintaining for 1.5 hours, the air pressure was slowly deflated to reduce the pressure in the cabin to atmospheric pressure.
  • HBO+PD-1 inhibitor group mice were given 0.1mg/0.1mL PD-1 inhibitor by tail vein injection at two hours after the end of hyperbaric oxygen therapy.
  • PBS and PD-1 inhibitor were injected into the corresponding experimental group mice through tail vein at doses of 0.1mL/mice and 0.1mg/0.1mL, respectively. After the treatment, the survival status of the mice was
  • Figure 12 is the Kaplan-Meier chart of the survival period of the mice in each group. It can be seen from the figure that all the mice in the PBS group died on the 39th day after tumor implantation, while all the mice in the HBO group died on the 49th day. Three mice were still alive in the PD-1 inhibitor group, while seven were still alive in the HBO combined PD-1 inhibitor group. The median survival time of the PBS group, HBO group, and PD-1 inhibitor group was 33, 35.5, and 39.5 days, respectively, while more than half of the mice in the HBO combined with PD-1 inhibitor treatment group survived for more than three months. The above analysis of the results showed that HBO combined with PD-1 inhibitor therapy could significantly prolong the survival of H22 hepatocellular carcinoma in situ tumor-bearing mice.
  • the mouse liver cancer H22 orthotopic tumor model was established and recorded as the first day. After two weeks, the tumor-bearing mice were randomly divided into 4 groups (PBS group, HBO group, PD-1 inhibitor group and HBO+PD-1 inhibitor group). On the 14th, 16th, and 18th days, the mice in the HBO group and the HBO+PD-1 inhibitor group were treated with hyperbaric oxygen therapy. The internal pressure was raised to 2.5 times atmospheric pressure, and after maintaining for 1.5 hours, the air pressure was slowly deflated to reduce the pressure in the cabin to atmospheric pressure. The mice in the HBO+PD-1 inhibitor group were given 0.1mg/0.1mL PD-1 inhibitor by tail vein injection at two hours after the end of hyperbaric oxygen therapy.
  • mice were sacrificed, the tumors were stripped and weighed, the whole blood of the mice was taken out to detect the composition and content of blood cells, and the blood was collected to separate the serum, and the blood biochemical indexes were analyzed.
  • AST total urea nitrogen
  • CK cardiac creatine kinase
  • the five main organs of the mouse heart, liver, spleen, lung and kidney were taken out, fixed with 4% paraformaldehyde, then routinely embedded in paraffin, and stained with HE, and the tissue structure was observed under a microscope.
  • Figure 13 shows the statistical results of tumor weights taken out of the four groups of mice after treatment.
  • the tumor weights of the PD-1 inhibitor group and the HBO+PD-1 inhibitor group were significantly lower than those of the PBS group.
  • the tumor weight of the treatment group was significantly lower than that of the PD-1 inhibitor treatment group alone, indicating that HBO combined with PD-1 inhibitor can enhance the anti-tumor effect of PD-1 inhibitor.
  • Figure 14 shows the body weight changes of the above four groups during the administration period. It can be seen from Figure 14 that compared with the PBS group, the body weight of the mice in the three experimental groups has no obvious decrease or increase trend, which indicates that the use of HBO or PD- 1 Inhibitor treatment, as well as combination treatment, did not affect the health of the mice.
  • Table 5 shows the results of blood cell detection in each group after treatment
  • Figure 15 shows the blood biochemical analysis results of PBS group, HBO group, PD-1 inhibitor group and HBO+PD-1 inhibitor group, in which ALT and AST in content A represent alanine aminotransferase and aspartate aminotransferase, respectively. High indicates that the drug has serious liver toxicity.
  • CK in content B is myocardial creatine kinase, and an increase in its value means pathological changes in the heart.
  • BUN in content C is total urea nitrogen, and an increase in this index means kidney function. problem appear.
  • Figure 16 is a structural diagram of a tissue section at a magnification of 200 times. It can be seen from Figure 16 that the structures of the heart, liver, spleen, lung and kidney in the PBS group and the three experimental groups are clear, with no obvious pathological changes, no bleeding and inflammatory infiltration. The above results show that HBO or PD-1 inhibitor treatment alone, and combined treatment All of them will not cause damage to the main organs of mice, and have good safety.
  • the Panc02 orthotopic tumor model of mouse pancreatic cancer was established and recorded as the first day.
  • the tumor-bearing mice were randomly divided into 4 groups (PBS group, HBO group, PD-1 inhibitor group and HBO+PD-1 inhibitor group).
  • the mice in the HBO group and the HBO+PD-1 inhibitor group were treated with hyperbaric oxygen on the 7th, 9th, and 11th days, respectively.
  • the treatment method was to place the mice in a closed cabin, and gradually introduce pure oxygen into the cabin.
  • the internal pressure was raised to 2.5 times atmospheric pressure, and after maintaining for 1.5 hours, the air pressure was slowly deflated to reduce the pressure in the cabin to atmospheric pressure.
  • mice in the HBO+PD-1 inhibitor group were given 0.1mg/0.1mL PD-1 inhibitor by tail vein injection at two hours after the end of hyperbaric oxygen therapy.
  • PBS and PD-1 inhibitor were injected into the corresponding experimental group mice through tail vein at doses of 0.1mL/mice and 0.1mg/0.1mL, respectively. After the treatment, the survival status of the mice was observed and recorded every day.
  • Figure 17 is the Kaplan-Meier chart of the survival period of mice in each group. It can be seen from the figure that the mice in the PBS group, the HBO group and the PD-1 inhibitor group alone were all on the 34th, 37th, and 45th days after tumor implantation, respectively. At this time, there were still 4 survivors in the HBO combined with PD-1 inhibitor group. The median survival time of PBS group, HBO group, and PD-1 inhibitor group were 28.5, 32, and 32 days, respectively, while the median survival time of HBO combined with PD-1 inhibitor treatment group was 43 days. The above analysis of the results showed that HBO combined with PD-1 inhibitor therapy can significantly prolong the survival of Panc02 pancreatic cancer in situ tumor-bearing mice.
  • the Panc02 orthotopic tumor model of mouse pancreatic cancer was established and recorded as the first day.
  • the tumor-bearing mice were randomly divided into 4 groups (PBS group, HBO group, PD-1 inhibitor group and HBO+PD-1 inhibitor group).
  • the mice in the HBO group and the HBO+PD-1 inhibitor group were treated with hyperbaric oxygen on the 7th, 9th, and 11th days, respectively.
  • the treatment method was to place the mice in a closed cabin, and gradually introduce pure oxygen into the cabin.
  • the internal pressure was raised to 2.5 times atmospheric pressure, and after maintaining for 1.5 hours, the air pressure was slowly deflated to reduce the pressure in the cabin to atmospheric pressure.
  • mice in the HBO+PD-1 inhibitor group were given 0.1mg/0.1mL PD-1 inhibitor by tail vein injection at two hours after the end of hyperbaric oxygen therapy.
  • PBS and PD-1 inhibitor were injected into the corresponding experimental group mice through tail vein at doses of 0.1mL/mice and 0.1mg/0.1mL, respectively.
  • the mice were sacrificed, the tumors were stripped and weighed. The whole blood of the mice was taken out to detect the composition and content of blood cells, and the blood was collected to separate the serum.
  • ALT alanine aminotransferase
  • AST aspartate aminotransferase
  • BUN total urea nitrogen
  • CK cardiac creatine kinase
  • Figure 18 shows the statistical results of tumor weights taken out of the four groups of mice after treatment. It can be seen from Figure 18 that the tumor weights of the PD-1 inhibitor group and the HBO+PD-1 inhibitor group were significantly lower than those of the PBS group. The tumor weight of the treatment group was significantly lower than that of the PD-1 inhibitor treatment group alone, indicating that HBO combined with PD-1 inhibitor can enhance the anti-tumor effect of PD-1 inhibitor.
  • Figure 19 is a graph of the body weight changes of the above four groups during the administration period. It can be seen from Figure 19 that compared with the PBS group, the body weight of the mice in the three experimental groups has no obvious decrease or increase trend, which indicates that the use of HBO or PD- 1 Inhibitor treatment, as well as combination treatment, did not affect the health of the mice.
  • Table 6 shows the results of blood cell detection in each group after treatment
  • Figure 20 shows the blood biochemical analysis results of the PBS group, the HBO group, the PD-1 inhibitor group and the HBO+PD-1 inhibitor group.
  • Alanine aminotransferase, aspartate aminotransferase, myocardial creatine kinase and total urea nitrogen were mainly detected. Elevated alanine aminotransferase and aspartate aminotransferase indicate serious liver toxicity, elevated myocardial creatine kinase value indicates pathological changes in the heart, and elevated total urea nitrogen index indicates renal function problems. From the content A, content B and content C of Figure 20, it can be seen that the four blood biochemical indexes of the three experimental groups have no significant changes compared with the control group (PBS group). Neither the drug treatment nor the combined treatment will cause damage to the main organs of the mice, and the treatment has good safety.
  • Figure 21 is a structural diagram of a tissue section at a magnification of 200 times. It can be seen from Figure 21 that the structure of the heart, liver, spleen, lung and kidney in the PBS group and the three experimental groups is clear, with no obvious pathological changes, no bleeding and inflammatory infiltration. The above results show that HBO or PD-1 inhibitor treatment alone, and combined treatment All of them will not cause damage to the main organs of mice, and have good safety.
  • a mouse liver cancer H22 subcutaneous tumor model was established.
  • the tumor-bearing mice were randomly divided into 4 groups (PBS group, HBO group, PD-1 inhibitor group and HBO+PD-1 group). Inhibitor group) and recorded as the first day, the mice in the HBO group and the HBO+PD-1 inhibitor group were treated with hyperbaric oxygen on the 1st, 3rd, and 5th days respectively.
  • the treatment method was as follows: the mice were placed in a closed cabin. , and gradually introduce pure oxygen to increase the pressure in the cabin to 2.5 times the atmospheric pressure. After maintaining for 1.5 hours, slowly deflate the pressure to reduce the pressure in the cabin to atmospheric pressure.
  • mice in the HBO+PD-1 inhibitor group were given 0.1 mg/0.1 mL PD-1 inhibitor by tail vein injection at two hours after the hyperbaric oxygen treatment.
  • PBS and PD-1 inhibitor were injected into the corresponding experimental group mice through tail vein at doses of 0.1mL/mice and 0.1mg/0.1mL, respectively.
  • the mice in each group were sacrificed on the 15th day, and the subcutaneous tumors were excised and weighed. Heavy.
  • Table 7 shows the results of tumor volume in each group at different time points (unit: mm 3 )
  • Figure 23 shows the statistical results of the tumor weights taken out of the four groups of mice after treatment.
  • the tumor weights of the PD-1 inhibitor group and the HBO+PD-1 inhibitor group were significantly lower than those of the PBS group.
  • the tumor weight of the treatment group was significantly lower than that of the PD-1 inhibitor treatment group alone, indicating that HBO combined with PD-1 inhibitor can enhance the anti-tumor effect of PD-1 inhibitor.
  • the 680 rapid fluorescent labeling kit labels the fluorescent molecule Dylight680 on the PD-1 inhibitor, and then dissolves the labeled PD-1 inhibitor in PBS to prepare a solution of 1 mg/mL.
  • a mouse liver cancer H22 subcutaneous tumor model was established.
  • the tumor - bearing mice were randomly divided into two groups (PD-1 inhibitor group and HBO+PD-1 inhibitor group) and administered HBO
  • the mice in the +PD-1 inhibitor group were treated with hyperbaric oxygen.
  • the treatment method was to place the mice in an airtight chamber, and gradually introduce pure oxygen to increase the air pressure in the chamber to 2.5 times the atmospheric pressure. After maintaining for 1.5 hours, the air was slowly deflated. Reduce the cabin pressure to atmospheric pressure.
  • mice were imaged at 0, 0.5, 1, 2, 4, 8, 12, 24, 48, and 72 hours to detect the distribution of PD-1 inhibitors in mice, and at the first Part of the mice were sacrificed at 48 hours, and the heart, liver, spleen, lung, kidney and tumors were separated, and the distribution of PD-1 inhibitors in various tissues of the mice was detected using a small animal imaging system.
  • Figure 24 shows the time-dependent changes in the distribution of PD-1 inhibitors in living mice and the distribution in isolated tissues detected by small animal imaging. It can be seen from the figure that at the 0th hour, there was no background fluorescence signal in the two groups of mice. After the injection of the fluorescently labeled PD-1 inhibitor, different intensities of fluorescence gradually appeared in the mice over time. Signal, the fluorescence signal of PD-1 inhibitor in tumor site of mice in HBO combined with PD-1 inhibitor group was stronger than that in single PD-1 inhibitor group at different time points. The tissue distribution map at 48 hours also showed that the fluorescence signal of PD-1 inhibitor in tumor tissue of HBO combined with PD-1 inhibitor treatment group was stronger than that of PD-1 inhibitor alone group.
  • Figure 25 shows the quantitative results of the time-dependent changes in the distribution of PD-1 inhibitors in the tumor tissue of living mice detected by small animal imaging. It can be seen from the figure that with the prolongation of time, the enrichment of PD-1 inhibitors in the tumor tissues of each group of mice gradually increased. slow down. During the whole process, the enrichment of PD-1 inhibitors in tumor sites in the HBO combined with PD-1 inhibitor treatment group was significantly higher than that in the single PD-1 inhibitor group. The average enrichment was increased by about 35%.
  • Figure 26 shows the quantitative results of tissue distribution of PD-1 inhibitors detected by small animal imaging. It can be seen from the figure that compared with the PD-1 inhibitor group alone, the enrichment of PD-1 inhibitor in the tumor site in the combined treatment group was significantly increased, while the enrichment in the spleen was significantly decreased, and the rest of the organs were enriched There was no significant difference in the amount of accumulation, and the results indicated that HBO could significantly increase the accumulation of PD-1 inhibitors in mouse tumor sites.

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Description

一种高压氧联合免疫药物用于肿瘤治疗的方法A method of hyperbaric oxygen combined with immune drugs for tumor treatment 【技术领域】【Technical field】
本发明涉及肿瘤治疗领域，更具体的，涉及一种高压氧联合免疫药物用于肿瘤治疗的方法。The present invention relates to the field of tumor treatment, and more particularly, to a method for treating tumors with hyperbaric oxygen combined with immune drugs.
【背景技术】【Background technique】
肿瘤免疫治疗，尤其是PD-1(programmed death-1)抗体免疫阻断治疗，近年来受到了广泛关注。PD-1作为T细胞表面受体，通过与肿瘤细胞表面高度表达的PD-L1配体结合可以抑制杀伤T细胞(cytotoxic T cell，Tc细胞)的激活，在肿瘤免疫逃逸中发挥着重要作用。因此，通过使用PD-1抗体阻断PD-1/PD-L1免疫抑制信号通路，恢复Tc细胞对肿瘤细胞的杀伤作用，可以避免肿瘤免疫逃逸从而达到免疫治疗效果。目前国内已上市5款PD-1抗体包括百时美施贵宝的Opdivo(欧狄沃)、默沙东的Keytruda(可瑞达)，以及国产的君实生物特瑞普利单抗(拓益)、信达生物信迪利单抗(达伯舒)和恒瑞生物的卡瑞利达单抗(艾立妥)。Tumor immunotherapy, especially PD-1 (programmed death-1) antibody immune blockade therapy, has received extensive attention in recent years. As a T cell surface receptor, PD-1 can inhibit the activation of killer T cells (cytotoxic T cells, Tc cells) by binding to the highly expressed PD-L1 ligand on the surface of tumor cells, and plays an important role in tumor immune escape. Therefore, by using PD-1 antibody to block the PD-1/PD-L1 immunosuppressive signaling pathway and restore the killing effect of Tc cells on tumor cells, it is possible to avoid tumor immune escape and achieve the effect of immunotherapy. At present, 5 PD-1 antibodies have been listed in China, including Bristol-Myers Squibb's Opdivo (Odivo), Merck's Keytruda (Kerida), and domestic Junshi Bio-Toripalimab (Tuoyi), Xinxin Dabio’s sintilimab (Daboshu) and Hengrui’s camrellimumab (Elito).
然而，这些PD-1抗体药物的临床应用与其它免疫检查点阻断剂一样，遇到了一个巨大瓶颈--总体响应率低。虽然临床上将PD-1抗体药物用于部分非实体瘤比如黑色素瘤、小细胞肺癌等的免疫治疗具有较好的效果，但是也仅限于部分患者部分类型的肿瘤，总体上其响应率很低。尤其在实体瘤中，由于实体瘤典型的乏氧微环境，复杂的生理屏障，多药耐药性和强大的免疫抑制微环境极大地限制了免疫治疗的效果，导致PD-1抗体药物在实体瘤免疫治疗中的总体响应率不到10％。However, the clinical application of these PD-1 antibody drugs, like other immune checkpoint blockers, has encountered a huge bottleneck-the overall low response rate. Although the clinical use of PD-1 antibody drugs in the immunotherapy of some non-solid tumors such as melanoma and small cell lung cancer has good results, it is only limited to some types of tumors in some patients, and the overall response rate is very low. . Especially in solid tumors, due to the typical hypoxic microenvironment of solid tumors, complex physiological barriers, multidrug resistance and strong immunosuppressive microenvironment, the effect of immunotherapy greatly limits the effect of immunotherapy, resulting in PD-1 antibody drugs in solid tumors. The overall response rate in tumor immunotherapy is less than 10%.
【发明内容】[Content of the invention]
针对目前PD-1抑制剂免疫治疗响应率低这一问题，本发明提供一种高压氧联合PD-1抑制剂用于肿瘤免疫治疗的方法，其目的在于通过高压氧治疗改善患者肿瘤组织乏氧状况，调控肿瘤基质微环境，促进PD-1抑制剂在肿瘤部位的穿透，从而增强PD-1抑制剂的抗肿瘤疗效，解决临床上PD-1抑制剂治疗响应率低这一问题。In view of the current low response rate of PD-1 inhibitor immunotherapy, the present invention provides a method of hyperbaric oxygen combined with PD-1 inhibitor for tumor immunotherapy, the purpose of which is to improve hypoxia in tumor tissue of patients through hyperbaric oxygen therapy It can regulate the tumor stromal microenvironment and promote the penetration of PD-1 inhibitors in the tumor site, thereby enhancing the anti-tumor efficacy of PD-1 inhibitors and solving the problem of low response rate of PD-1 inhibitors in clinical practice.
为实现上述目的，本发明提供了一种高压氧联合PD-1抑制剂用于肿瘤免疫治疗的方法，通过将高压氧治疗与PD-1抑制剂治疗相结合，对肿瘤患者进行抗肿瘤治疗，具体包括如下步骤：In order to achieve the above object, the present invention provides a method of hyperbaric oxygen combined with PD-1 inhibitor for tumor immunotherapy, by combining hyperbaric oxygen therapy and PD-1 inhibitor therapy, anti-tumor therapy is performed on tumor patients, Specifically include the following steps:
(1)对肿瘤患者进行高压氧治疗；所述高压氧治疗通过增加肿瘤患者所处环境的气压及吸入氧气的浓度来提高溶解于血液中氧气的量，从而提高各组织中氧的灌注；(1) Hyperbaric oxygen therapy is performed on the tumor patient; the hyperbaric oxygen therapy increases the amount of oxygen dissolved in the blood by increasing the air pressure in the environment where the tumor patient is located and the concentration of inhaled oxygen, thereby improving the perfusion of oxygen in each tissue;
(2)对肿瘤患者静脉注射PD-1抑制剂所采用的技术方案将单次或多次给予患者高压氧治疗与静脉注射PD-1抑制剂相结合进行治疗。(2) The technical scheme used for intravenous injection of PD-1 inhibitor to tumor patients is to combine single or multiple administration of hyperbaric oxygen therapy with intravenous injection of PD-1 inhibitor for treatment.
总体而言，通过本发明所构思的以上技术方案与现有技术相比，能够取得以下有益效果：In general, compared with the prior art, the above technical solutions conceived by the present invention can achieve the following beneficial effects:
(1)本发明将高压氧治疗与静脉注射PD-1抑制剂相结合，对肿瘤患者进行抗肿瘤免疫治疗，实验发现对肿瘤患者进行高压氧治疗能够显著提高PD-1抑制剂在肿瘤包括实体瘤部位的富集，大大提高PD-1抑制剂在肿瘤部位的响应率，并且具有良好的安全性。与单用PD-1抑制剂相比，高压氧联合PD-1抑制剂在不增加毒副作用的同时提高PD-1在肿瘤中的穿透量与穿透深度，优选实施例中其提高倍数分别为2.6倍和2.8倍，并增强PD-1抗体对肿瘤体积和肿瘤重量的抑制效果，减少肿瘤向其余脏器的转移概率，将PD-1抗体治疗的响应率提高62.5％左右，有利于临床转化。(1) In the present invention, hyperbaric oxygen therapy is combined with intravenous injection of PD-1 inhibitor, and anti-tumor immunotherapy is performed on tumor patients. The experiment found that hyperbaric oxygen therapy on tumor patients can significantly increase the level of PD-1 inhibitor in tumors, including solid The enrichment of tumor sites greatly improves the response rate of PD-1 inhibitors in tumor sites, and has good safety. Compared with the single use of PD-1 inhibitor, hyperbaric oxygen combined with PD-1 inhibitor can improve the penetration amount and penetration depth of PD-1 in tumors without increasing toxic and side effects. It is 2.6 times and 2.8 times, and enhances the inhibitory effect of PD-1 antibody on tumor volume and tumor weight, reduces the probability of tumor metastasis to other organs, and increases the response rate of PD-1 antibody treatment by about 62.5%, which is beneficial to clinical practice. transform.
(2)本发明将高压氧治疗与静脉注射PD-1抑制剂相结合，对肿瘤患者进行抗肿瘤免疫治疗，实验发现不仅对于120mm 3尺寸较小肝癌皮下肿瘤具有良好的抑制效果，而且对初始体积为500mm 3，体积较大的很难消除的肝癌皮下肿瘤也具有较好的抑制效果，基本抑制肿瘤的生长甚至消除肿瘤。 (2) The present invention combines hyperbaric oxygen therapy with intravenous injection of PD-1 inhibitor to carry out anti-tumor immunotherapy for tumor patients. The experiment found that not only has a good inhibitory effect on subcutaneous tumors of liver cancer with a size of 120 mm 3 smaller, but also has a good inhibitory effect on initial With a volume of 500 mm 3 , the subcutaneous tumor of liver cancer, which is difficult to be eliminated with a larger volume, also has a good inhibitory effect, basically inhibiting the growth of the tumor or even eliminating the tumor.
(3)实验证明本发明提出的高压氧联合PD-1抑制剂用于肿瘤免疫治疗的方法可以显著抑制肿瘤生长速度，减少肺部肿瘤转移节结数，延长生存期，提高药物在肿瘤部位的富集。(3) Experiments have proved that the method of hyperbaric oxygen combined with PD-1 inhibitor for tumor immunotherapy proposed by the present invention can significantly inhibit tumor growth rate, reduce the number of lung tumor metastasis nodules, prolong survival period, and improve the drug delivery rate at tumor sites. enrichment.
(4)本发明提出的高压氧联合PD-1抑制剂用于肿瘤免疫治疗的方法简单易于操作，安全可靠，有利于临床转化。(4) The method of hyperbaric oxygen combined with PD-1 inhibitor for tumor immunotherapy proposed by the present invention is simple and easy to operate, safe and reliable, and is beneficial to clinical transformation.
【附图说明】【Description of drawings】
图1为本发明实施例6进行的抗肿瘤活性实验中肿瘤的肿瘤体积-时间曲线；Fig. 1 is the tumor volume-time curve of the tumor in the anti-tumor activity experiment carried out in Example 6 of the present invention;
图2为本发明实施例6进行的抗肿瘤活性实验中小鼠的瘤重结果；Fig. 2 is the tumor weight result of mice in the anti-tumor activity experiment carried out in Example 6 of the present invention;
图3为本发明实施例6进行的抗肿瘤活性实验中小鼠的体重-时间曲线；Fig. 3 is the weight-time curve of mice in the anti-tumor activity experiment carried out in Example 6 of the present invention;
图4为本发明实施例6各实验组血生化指标的结果；Fig. 4 is the result of the blood biochemical index of each experimental group in the embodiment of the present invention 6;
图5为本发明实施例6各实验组组织切片的显微观察图片。5 is a microscopic observation picture of tissue sections of each experimental group in Example 6 of the present invention.
图6为本发明实施例7进行的抗肿瘤活性实验中肿瘤的肿瘤体积-时间曲线；Fig. 6 is the tumor volume-time curve of the tumor in the anti-tumor activity experiment carried out in Example 7 of the present invention;
图7为本发明实施例7进行的抗肿瘤活性实验中小鼠的瘤重结果；Fig. 7 is the tumor weight result of mice in the anti-tumor activity experiment carried out in Example 7 of the present invention;
图8为本发明实施例7进行的抗肿瘤活性实验中小鼠的体重-时间曲线；Figure 8 is the body weight-time curve of mice in the anti-tumor activity experiment carried out in Example 7 of the present invention;
图9为本发明实施例7进行的抗肿瘤活性实验中小鼠的肺部肿瘤转移节结数结果；Figure 9 is the result of the number of lung tumor metastases in mice in the anti-tumor activity experiment carried out in Example 7 of the present invention;
图10为本发明实施例7各实验组血生化指标的结果；Fig. 10 is the result of the blood biochemical index of each experimental group in Example 7 of the present invention;
图11为本发明实施例7各实验组组织切片的显微观察图片。11 is a microscopic observation picture of tissue sections of each experimental group in Example 7 of the present invention.
图12为本发明实施例8各组小鼠生存期的结果。Figure 12 shows the results of the survival period of mice in each group in Example 8 of the present invention.
图13为本发明实施例9进行的抗肿瘤活性实验中小鼠的瘤重结果；Figure 13 is the tumor weight results of mice in the anti-tumor activity experiment carried out in Example 9 of the present invention;
图14为本发明实施例9进行的抗肿瘤活性实验中小鼠的体重-时间曲线；Figure 14 is the body weight-time curve of mice in the anti-tumor activity experiment carried out in Example 9 of the present invention;
图15为本发明实施例9各实验组血生化指标的结果；Figure 15 is the result of the blood biochemical indexes of each experimental group in Example 9 of the present invention;
图16为本发明实施例9各实验组组织切片的显微观察图片。16 is a microscopic observation picture of tissue sections of each experimental group in Example 9 of the present invention.
图17为本发明实施例10各组小鼠生存期的结果。Figure 17 shows the results of the survival period of mice in each group in Example 10 of the present invention.
图18为本发明实施例11进行的抗肿瘤活性实验中小鼠的瘤重结果；Figure 18 is the tumor weight results of mice in the anti-tumor activity experiment carried out in Example 11 of the present invention;
图19为本发明实施例11进行的抗肿瘤活性实验中小鼠的体重-时间曲线；Figure 19 is the body weight-time curve of mice in the anti-tumor activity experiment carried out in Example 11 of the present invention;
图20为本发明实施例11各实验组血生化指标的结果；Figure 20 is the results of the blood biochemical indicators of each experimental group in Example 11 of the present invention;
图21为本发明实施例11各实验组组织切片的显微观察图片；21 is a microscopic observation picture of tissue sections of each experimental group in Example 11 of the present invention;
图22为本发明实施例12进行的抗肿瘤活性实验中肿瘤的肿瘤体积-时间曲线；Figure 22 is the tumor volume-time curve of the tumor in the anti-tumor activity experiment performed in Example 12 of the present invention;
图23为本发明实施例12进行的抗肿瘤活性实验中小鼠的瘤重结果；Figure 23 is the result of tumor weight in mice in the anti-tumor activity experiment carried out in Example 12 of the present invention;
图24为本发明实施例13各实验组小动物成像检测小鼠活体与离体荧光成像图；FIG. 24 is a fluorescent imaging diagram of in vivo and ex vivo mice in each experimental group of small animals imaging detection in Example 13 of the present invention;
图25为本发明实施例13各实验组小动物成像检测活体肿瘤组织PD-1抑制剂富集量-时间曲线；Figure 25 is a graph of the enrichment amount-time curve of PD-1 inhibitor in living tumor tissue detected by imaging of small animals in each experimental group in Example 13 of the present invention;
图26为本发明实施例13各实验组小动物成像检测PD-1抑制剂组织分布定量图。26 is a quantitative diagram of tissue distribution of PD-1 inhibitors detected by imaging in small animals of each experimental group in Example 13 of the present invention.
【具体实施方式】【Detailed ways】
下面结合实施例，对本发明的具体实施方式作进一步详细描述。以下实施例用于说明本发明，但不用来限制本发明的范围。The specific embodiments of the present invention will be further described in detail below with reference to the examples. The following examples are intended to illustrate the present invention, but not to limit the scope of the present invention.
本发明提供的一种高压氧联合PD-1抑制剂用于肿瘤免疫治疗的方法，通过将高压氧治疗与PD-1抑制剂治疗相结合，对肿瘤患者进行抗肿瘤治疗，具体包括如下步骤：The present invention provides a method for using hyperbaric oxygen combined with PD-1 inhibitor for tumor immunotherapy. By combining hyperbaric oxygen therapy and PD-1 inhibitor therapy, anti-tumor therapy is performed on tumor patients, which specifically includes the following steps:
(1)对肿瘤患者进行高压氧治疗；所述高压氧治疗通过增加肿瘤患者所处环境的气压及吸入氧气的浓度来提高溶解于血液中氧气的量，从而提高各组织中氧的灌注；(1) Hyperbaric oxygen therapy is performed on the tumor patient; the hyperbaric oxygen therapy increases the amount of oxygen dissolved in the blood by increasing the air pressure in the environment where the tumor patient is located and the concentration of inhaled oxygen, thereby improving the perfusion of oxygen in each tissue;
(2)对肿瘤患者静脉注射PD-1抑制剂。(2) Intravenous injection of PD-1 inhibitor to tumor patients.
原则上本发明不限定高压氧治疗和静脉注射PD-1抑制剂治疗的先后顺序。In principle, the present invention does not limit the order of hyperbaric oxygen therapy and intravenous injection of PD-1 inhibitor therapy.
优选实施例中，对肿瘤患者进行单次或多次高压氧治疗后，再对肿瘤患者静脉注射PD-1抑制剂。实验发现首先对肿瘤患者进行高压氧治疗，可以改善肿瘤的乏氧环境，尤为重要地，能够显著促进PD-1抑制剂在肿瘤部位的穿透和富集，增强PD-1抑制剂的抗肿瘤疗效，提高临床上PD-1抑制剂的治疗响应率。In a preferred embodiment, after a single or multiple hyperbaric oxygen therapy is performed on the tumor patient, a PD-1 inhibitor is intravenously injected into the tumor patient. Experiments have found that hyperbaric oxygen therapy for tumor patients can improve the hypoxic environment of the tumor, especially, it can significantly promote the penetration and enrichment of PD-1 inhibitors in tumor sites, and enhance the anti-tumor effect of PD-1 inhibitors. Efficacy, improve the clinical response rate of PD-1 inhibitors.
高压氧(Hyperbaric oxygen，HBO)治疗是指间歇性地在高于正常大气压条件 下给予病人纯氧的治疗手段。目前通常采用的治疗压力是2-2.5个大气压(2-2.5ATA,1ATA＝101.32KPa)。高压氧治疗的原理是通过增加病人所处环境的气压及吸入氧气的浓度来提高溶解于血液中氧气的量，从而提高各组织中氧的灌注。高压氧能够有效提高组织中的氧分压、缓解组织缺氧、减少水肿、激活血管新生和胶原合成，因此广泛应用于各种疾病的预防与治疗，比如用于CO中毒，减压病和气压伤等。然而本发明将高压氧与PD-1抗体联合，通过高压氧改善肿瘤组织乏氧，促进PD-1抗体递送以及改善肿瘤免疫微环境，提高抗肿瘤效果。Hyperbaric oxygen (HBO) therapy refers to the intermittent administration of pure oxygen to patients under conditions of higher than normal atmospheric pressure. The currently commonly used treatment pressure is 2-2.5 atmospheres (2-2.5ATA, 1ATA=101.32KPa). The principle of hyperbaric oxygen therapy is to increase the amount of oxygen dissolved in the blood by increasing the air pressure of the patient's environment and the concentration of inhaled oxygen, thereby improving the perfusion of oxygen in various tissues. Hyperbaric oxygen can effectively increase the partial pressure of oxygen in tissues, relieve tissue hypoxia, reduce edema, activate angiogenesis and collagen synthesis, so it is widely used in the prevention and treatment of various diseases, such as CO poisoning, decompression sickness and air pressure injury etc. However, the present invention combines hyperbaric oxygen with PD-1 antibody, improves tumor tissue hypoxia through hyperbaric oxygen, promotes PD-1 antibody delivery, improves tumor immune microenvironment, and improves anti-tumor effect.
本发明对肿瘤患者进行高压氧治疗可以按照现有技术高压氧治疗的常规方式进行，一些实施例中，对肿瘤患者进行高压氧治疗具体为：将肿瘤患者置于密闭舱体内，逐渐通入纯氧使舱内气压升高到2-5倍大气压，维持一段时间后，放气使舱内气压降至大气压。PD-1抑制剂治疗方法为：通过静脉注射PD-1抑制剂。According to the present invention, the hyperbaric oxygen therapy for tumor patients can be performed according to the conventional method of hyperbaric oxygen therapy in the prior art. In some embodiments, the hyperbaric oxygen therapy for tumor patients is specifically: placing the tumor patient in an airtight chamber, and gradually introducing pure Oxygen increases the air pressure in the cabin to 2-5 times atmospheric pressure, and after a period of time, deflation reduces the air pressure in the cabin to atmospheric pressure. PD-1 inhibitor therapy is given by intravenous injection of PD-1 inhibitor.
一些实施例中，将肿瘤患者置于密闭舱体内，逐渐通入纯氧使舱内气压升高到2-5倍大气压，优选为2-2.5倍大气压，维持1-4小时(优选为1-2小时)后，缓慢放气使舱内气压降至大气压。In some embodiments, the tumor patient is placed in a closed cabin, and pure oxygen is gradually introduced to increase the pressure in the cabin to 2-5 times atmospheric pressure, preferably 2-2.5 times atmospheric pressure, and maintain for 1-4 hours (preferably 1- After 2 hours), slowly deflate to bring the cabin pressure down to atmospheric pressure.
在一个优选实施方式中，高压氧治疗与PD-1抑制剂给药的先后次序可变，优选为先高压氧治疗，随后静脉注射PD-1抑制剂。In a preferred embodiment, the order of hyperbaric oxygen therapy and PD-1 inhibitor administration is variable, preferably hyperbaric oxygen therapy first, followed by intravenous injection of the PD-1 inhibitor.
本发明所述高压氧治疗与静脉注射PD-1抑制剂之间的间隔时间为0-12小时。优选间隔时间为1-12小时，进一步优选为1-3小时。The interval between the hyperbaric oxygen therapy of the present invention and the intravenous injection of the PD-1 inhibitor is 0-12 hours. The interval time is preferably 1-12 hours, more preferably 1-3 hours.
本发明对肿瘤患者进行静脉注射PD-1抑制剂之前或之后，对肿瘤患者给予1-10次高压氧治疗，优选实施例中给予2-5次。According to the present invention, before or after intravenous injection of PD-1 inhibitor to tumor patients, hyperbaric oxygen therapy is administered to tumor patients 1-10 times, and in a preferred embodiment, hyperbaric oxygen therapy is administered 2-5 times.
本发明一些实施例中，重复步骤(1)和步骤(2)以完成一次治疗，持续治疗不少于1次。In some embodiments of the present invention, steps (1) and (2) are repeated to complete one treatment, and the treatment is continued for no less than one time.
本发明可根据实际应用情况调整PD-1抑制剂的剂量，一些实施例中，每一至三周对肿瘤患者静脉注射PD-1抑制剂一次，其单次注射剂量为100-300mg/m 2。 The present invention can adjust the dose of the PD-1 inhibitor according to the actual application. In some embodiments, the tumor patient is intravenously injected with the PD-1 inhibitor once every to three weeks, and the single injection dose is 100-300 mg/m 2 .
本发明优选实施例中，每一次静脉注射PD-1抑制剂之前，对肿瘤患者进行至少一次的高压氧治疗，其高压氧治疗频率不限，可根据实际需要对肿瘤患者进行每天、每隔一天、每隔两天、每隔三天或每隔四天等的高压氧治疗。在一个优选实施方式中，每天给予高压氧治疗1-5次；优选为1-2次。In a preferred embodiment of the present invention, before each intravenous injection of PD-1 inhibitor, hyperbaric oxygen therapy is performed on tumor patients at least once, and the frequency of hyperbaric oxygen therapy is not limited. , hyperbaric oxygen therapy every two days, every three days, or every four days. In a preferred embodiment, hyperbaric oxygen therapy is administered 1-5 times per day; preferably 1-2 times.
PD-1抑制剂的注射剂量可依据个体差异而定，比如静脉注射每2周或每3周一次，直至疾病进展或出现不可耐受的毒性。一些实施例中，根据确定的PD-1抑制剂的用量和用法，在每一次注射PD-1抑制剂之前，进行每周1-7次的高压氧治疗。The injected dose of PD-1 inhibitor can be based on individual differences, such as intravenous injection every 2 weeks or every 3 weeks, until disease progression or intolerable toxicity. In some embodiments, according to the determined dosage and usage of the PD-1 inhibitor, before each injection of the PD-1 inhibitor, hyperbaric oxygen therapy is performed 1-7 times per week.
本发明采用的PD-1抑制剂也可称之为PD-1抗体或PD-1抗体药物，可以为临床上已在使用的各种PD-1抗体药物，比如目前批准上市的PD-1抗体默沙东 (Merck)的Keytruda(可瑞达)、百时美施贵宝(BMS)的Opdivo(欧狄沃)、赛诺菲(Sanofi)和再生元(Regeneron)联合研发的Libtayo，以及国产的君实生物特瑞普利单抗(拓益)、信达生物信迪利单抗(达伯舒)和恒瑞生物的卡瑞利达单抗(艾立妥)。本发明PD-1抑制剂的静脉注射剂量可参考不同的PD-1抗体药物类型其临床上采用的注射剂量，以及相应的注射频次。比如达伯舒在临床上的推荐剂量为200mg/m 2，每3周给一次药。PD-1抑制剂给药方法，包括复溶和稀释等，也可与上述现行上市药物给药方法相同。 The PD-1 inhibitor used in the present invention can also be called PD-1 antibody or PD-1 antibody drug, and can be various PD-1 antibody drugs that have been used clinically, such as the currently approved PD-1 antibody Merck's Keytruda, Bristol-Myers Squibb's Opdivo, Sanofi and Regeneron jointly developed Libtayo, and domestically produced Junshi Biotechnology Toripalimab (Tuoyi), Innovent’s sintilimab (Daboshu) and Hengrui’s camrelimab (Elito). For the intravenous injection dose of the PD-1 inhibitor of the present invention, reference may be made to the clinically used injection dose of different PD-1 antibody drug types, as well as the corresponding injection frequency. For example, the clinically recommended dose of Dabosu is 200 mg/m 2 , administered once every 3 weeks. The administration method of PD-1 inhibitor, including reconstitution and dilution, etc., can also be the same as the above-mentioned current marketed drug administration method.
本发明提出的肿瘤免疫治疗方法可以适用于各种肿瘤类型，不仅能够适用于诸如黑色素瘤、小细胞肺癌肿瘤等的非实体瘤，同样也适用于各种实体瘤，包括头颈部肿瘤、胸部肿瘤、消化***肿瘤、泌尿生殖***肿瘤、骨肿瘤、中枢神经***肿瘤、软组织肿瘤、皮肤及附件肿瘤等，联合高压氧治疗能够显著提高PD-1抗体药物在实体瘤部位的富集和响应率，显著抑制肿瘤生长速度。The tumor immunotherapy method proposed in the present invention can be applied to various tumor types, not only non-solid tumors such as melanoma, small cell lung cancer, etc., but also various solid tumors, including head and neck tumors, thoracic tumors, etc. For tumors, digestive system tumors, urogenital system tumors, bone tumors, central nervous system tumors, soft tissue tumors, skin and adnexal tumors, etc., combined with hyperbaric oxygen therapy can significantly improve the enrichment and response rate of PD-1 antibody drugs in solid tumor sites , significantly inhibited tumor growth rate.
实验证明本发明提出的联合治疗方法对肝癌皮下瘤、乳腺癌原位瘤、肝癌原位瘤、胰腺癌原位瘤等各种实体瘤均显著提高了PD-1抑制剂在肿瘤部位的富集，实验发现不仅对于120mm 3尺寸较小肝癌皮下肿瘤具有良好的抑制效果，在17天后8只小鼠有5只肿瘤完全消除；而且对初始体积为500mm 3，体积较大的很难消除的肝癌皮下肿瘤也具有较好的抑制效果，8只小鼠中4只小鼠的肿瘤基本被抑制生长甚至消除。在临床上使用本发明联合治疗方法进行肿瘤免疫治疗时，可根据肿瘤类型以及尺寸大小进行高压氧以及PD-1抑制剂剂量的增减调整。 Experiments show that the combined treatment method proposed by the present invention significantly improves the enrichment of PD-1 inhibitors in tumor sites for various solid tumors such as subcutaneous tumors of liver cancer, in situ tumors of breast cancer, in situ tumors of liver cancer, and pancreatic cancer in situ tumors. Experiments found that it not only has a good inhibitory effect on subcutaneous tumors of liver cancer with a size of 120mm 3 , and 5 tumors in 8 mice were completely eliminated after 17 days; but also for liver cancer with an initial volume of 500mm 3 , which is difficult to eliminate. Subcutaneous tumors also had a good inhibitory effect, and tumor growth was basically inhibited or even eliminated in 4 of the 8 mice. When using the combination therapy method of the present invention for tumor immunotherapy clinically, the hyperbaric oxygen and the dose of PD-1 inhibitor can be adjusted according to the tumor type and size.
与单用PD-1抑制剂相比，本发明高压氧联合PD-1抑制剂在不增加毒副作用的同时显著提高提高PD-1治疗的响应率，并且有利于临床转化。本发明提供了一种新的抗肿瘤联合治疗方式，拓展了高压氧的新用途。Compared with the single use of the PD-1 inhibitor, the hyperbaric oxygen combined with the PD-1 inhibitor of the present invention significantly improves the response rate of PD-1 treatment without increasing the toxic and side effects, and is beneficial to clinical transformation. The invention provides a new anti-tumor combined treatment mode and expands the new application of hyperbaric oxygen.
以下为具体实施例，具体实施例1至实施例4中采用的PD-1抑制剂为默沙东(Merck)的Keytruda，实施例5至实施例13采用的PD-1抑制剂为欣博盛的anti-mouse PD-1抗体。The following are specific examples. The PD-1 inhibitor used in specific examples 1 to 4 is Merck's Keytruda, and the PD-1 inhibitor used in examples 5 to 13 is Xinbosheng's anti -mouse PD-1 antibody.
实施例1Example 1
本实施例提供一种高压氧联合PD-1抑制剂用于肿瘤治疗的方法，该方法包括高压氧治疗和PD-1抑制剂注射两部分。高压氧治疗方法为：将治疗个体置于密闭舱体内，逐渐通入纯氧使舱内气压升高到2.5倍大气压，维持1.5小时后，缓慢放气使舱内气压降至大气压。This embodiment provides a method for hyperbaric oxygen combined with PD-1 inhibitor for tumor treatment, the method includes hyperbaric oxygen therapy and PD-1 inhibitor injection. The hyperbaric oxygen therapy method is as follows: place the treated individual in a closed cabin, gradually introduce pure oxygen to increase the pressure in the cabin to 2.5 times the atmospheric pressure, maintain it for 1.5 hours, and slowly deflate to reduce the pressure in the cabin to atmospheric pressure.
每天进行一次上述高压氧治疗，连续14天；在第14天的高压氧治疗结束后进行PD-1抑制剂治疗，具体方法为：高压氧治疗结束后间隔两小时，通过静脉注射200mg/m 2PD-1抑制剂。 The above hyperbaric oxygen therapy was performed once a day for 14 consecutive days; PD-1 inhibitor therapy was performed after the end of the hyperbaric oxygen therapy on the 14th day . PD-1 inhibitors.
实施例2Example 2
本实施例提供一种高压氧联合PD-1抑制剂用于肿瘤治疗的方法，该方法包括高压氧治疗和PD-1抑制剂注射两部分。高压氧治疗方法为：将治疗个体置于密闭舱体内，逐渐通入纯氧使舱内气压升高到2.5倍大气压，维持1.5小时后，缓慢放气使舱内气压降至大气压。This embodiment provides a method for hyperbaric oxygen combined with PD-1 inhibitor for tumor treatment, the method includes hyperbaric oxygen therapy and PD-1 inhibitor injection. The hyperbaric oxygen therapy method is: place the treated individual in a closed cabin, gradually introduce pure oxygen to increase the pressure in the cabin to 2.5 times the atmospheric pressure, maintain it for 1.5 hours, and slowly deflate the cabin to reduce the pressure to the atmospheric pressure.
连续三天每天给予一次高压氧治疗后，在第三天高压氧结束后间隔两小时，通过静脉注射200mg/m 2PD-1抑制剂。随后每间隔一天给予一次高压氧，两周后在高压氧治疗结束后间隔两小时通过静脉注射200mg/m 2PD-1抑制剂。然后每间隔一天给予一次高压氧，两周后在高压氧治疗结束后间隔两小时通过静脉注射200mg/m 2PD-1抑制剂。 After hyperbaric oxygen therapy was administered once a day for three consecutive days, 200 mg/m 2 PD-1 inhibitor was administered intravenously at a two-hour interval after the end of hyperbaric oxygen on the third day. Hyperbaric oxygen was then administered every other day, followed by intravenous injection of 200 mg/m 2 PD-1 inhibitor at two-hour intervals after the end of hyperbaric oxygen treatment two weeks later. Hyperbaric oxygen was then administered every other day, followed by intravenous injection of 200 mg/m 2 PD-1 inhibitor at two-hour intervals after the end of hyperbaric oxygen treatment two weeks later.
实施例3Example 3
本实施例提供一种高压氧联合PD-1抑制剂用于肿瘤治疗的方法，该方法包括高压氧治疗和PD-1抑制剂注射两部分。高压氧治疗方法为：将治疗个体置于密闭舱体内，逐渐通入纯氧使舱内气压升高到2倍大气压，维持2小时后，缓慢放气使舱内气压降至大气压。每隔两天进行一次高压氧治疗，持续3周后，进行PD-1抑制剂治疗，具体方法为：高压氧治疗结束后间隔两小时，通过静脉注射200mg/m 2PD-1抑制剂一次。 This embodiment provides a method for hyperbaric oxygen combined with PD-1 inhibitor for tumor treatment, the method includes hyperbaric oxygen therapy and PD-1 inhibitor injection. The hyperbaric oxygen therapy method is as follows: place the treated individual in a closed cabin, gradually introduce pure oxygen to increase the pressure in the cabin to 2 times the atmospheric pressure, and after maintaining for 2 hours, slowly deflate to reduce the pressure in the cabin to atmospheric pressure. Hyperbaric oxygen therapy was performed every two days, and after 3 weeks, PD-1 inhibitor therapy was performed. The specific method was: intravenous injection of 200 mg/m 2 PD-1 inhibitor once every two hours after the end of hyperbaric oxygen therapy.
实施例4Example 4
本实施例提供一种高压氧联合PD-1抑制剂用于肿瘤治疗的方法，该方法包括高压氧治疗和PD-1抑制剂注射两部分。高压氧治疗方法为：将治疗个体置于密闭舱体内，逐渐通入纯氧使舱内气压升高到2.5倍大气压，维持1.5小时后，缓慢放气使舱内气压降至大气压。PD-1抑制剂治疗方法为：高压氧治疗结束后间隔12小时，通过静脉注射6.25mg/kg PD-1抑制剂。This embodiment provides a method for hyperbaric oxygen combined with PD-1 inhibitor for tumor treatment, the method includes hyperbaric oxygen therapy and PD-1 inhibitor injection. The hyperbaric oxygen therapy method is: place the treated individual in a closed cabin, gradually introduce pure oxygen to increase the pressure in the cabin to 2.5 times the atmospheric pressure, maintain it for 1.5 hours, and slowly deflate the cabin to reduce the pressure to the atmospheric pressure. The PD-1 inhibitor treatment method is as follows: 6.25mg/kg PD-1 inhibitor is intravenously injected at an interval of 12 hours after the end of hyperbaric oxygen therapy.
每间隔一天进行一次高压氧治疗，在2周后的高压氧治疗结束后间隔12小时静脉注射200mg/m 2PD-1抑制剂第一次。然后再每间隔一天进行一次高压氧治疗，在2周后的高压氧治疗结束后间隔12小时静脉注射200mg/m 2PD-1抑制剂第二次。 Hyperbaric oxygen therapy was administered every other day, and 200 mg/m 2 PD-1 inhibitor was administered intravenously for the first time at 12-hour intervals after the end of hyperbaric oxygen therapy after 2 weeks. Then, hyperbaric oxygen therapy was performed every other day, and a second dose of 200 mg/m 2 PD-1 inhibitor was administered intravenously at 12-hour intervals after the end of the hyperbaric oxygen therapy after 2 weeks.
实施例5Example 5
本实施例提供一种高压氧联合PD-1抑制剂用于肿瘤治疗的方法，该方法包括高压氧治疗和PD-1抑制剂注射两部分。PD-1抑制剂治疗方法为：通过静脉注射6.25mg/kg PD-1抑制剂。高压氧治疗方法为：在静脉注射PD-1抑制剂后，立即将治疗个体置于密闭舱体内，逐渐通入纯氧使舱内气压升高到2.5倍大气压，维持1.5小时后，缓慢放气使舱内气压降至大气压。This embodiment provides a method for hyperbaric oxygen combined with PD-1 inhibitor for tumor treatment, the method includes hyperbaric oxygen therapy and PD-1 inhibitor injection. PD-1 inhibitor therapy is: 6.25mg/kg PD-1 inhibitor intravenously. The hyperbaric oxygen treatment method is: after intravenous injection of PD-1 inhibitor, the treated individual is placed in a closed cabin immediately, and pure oxygen is gradually introduced to increase the pressure in the cabin to 2.5 times the atmospheric pressure. After maintaining for 1.5 hours, slowly deflate Reduce the cabin pressure to atmospheric pressure.
实施例6Example 6
基于高压氧联合PD-1抑制剂对荷肝癌皮下瘤小鼠抗肿瘤活性的测试。Based on the test of hyperbaric oxygen combined with PD-1 inhibitor on the antitumor activity of liver cancer subcutaneous tumor-bearing mice.
实验药物的配制：将PD-1抑制剂溶解在PBS中配制成1mg/mL的溶液。Preparation of experimental drugs: The PD-1 inhibitor was dissolved in PBS to prepare a solution of 1 mg/mL.
建立小鼠肝癌H22皮下瘤模型，当皮下瘤长到体积为100mm 3时，随机将荷瘤小鼠分为4个组(PBS组、HBO组、PD-1抑制剂组和HBO+PD-1抑制剂组)并记为第1天，分别在第1、3、5天给予HBO组和HBO+PD-1抑制剂组小鼠高压氧治疗，治疗方式为：将小鼠置于密闭舱体内，逐渐通入纯氧使舱内气压升高到2.5倍大气压，维持1.5小时后，缓慢放气使舱内气压降至大气压。高压氧治疗结束后间隔两小时通过尾静脉注射给予HBO+PD-1抑制剂组小鼠0.1mg/0.1mL PD-1抑制剂。并分别将PBS、PD-1抑制剂以0.1mL/只、0.1mg/0.1mL剂量通过尾静脉注射到对应实验组小鼠。每两天用游标卡尺测量肿瘤的最长处(L)和最宽处(W)，计算肿瘤体积V＝L*W 2/2，在第17天将各组小鼠处死，剥出皮下肿瘤并称重。在检测肿瘤体积的同时，每两天测量小鼠的体重，并记录。 A mouse liver cancer H22 subcutaneous tumor model was established. When the subcutaneous tumor grew to a volume of 100 mm, the tumor-bearing mice were randomly divided into 4 groups (PBS group, HBO group, PD-1 inhibitor group and HBO+PD-1 group). Inhibitor group) and recorded as the first day, the mice in the HBO group and the HBO+PD-1 inhibitor group were treated with hyperbaric oxygen on the 1st, 3rd, and 5th days respectively. The treatment method was as follows: the mice were placed in a closed cabin , and gradually introduce pure oxygen to increase the pressure in the cabin to 2.5 times the atmospheric pressure. After maintaining for 1.5 hours, slowly deflate the pressure to reduce the pressure in the cabin to atmospheric pressure. The mice in the HBO+PD-1 inhibitor group were given 0.1 mg/0.1 mL of PD-1 inhibitor by tail vein injection at two hours after the end of hyperbaric oxygen therapy. PBS and PD-1 inhibitor were injected into the corresponding experimental group mice through tail vein at doses of 0.1mL/mice and 0.1mg/0.1mL, respectively. The longest part (L) and widest part (W) of the tumor were measured with a vernier caliper every two days, and the tumor volume V=L*W 2 /2 was calculated. The mice in each group were sacrificed on the 17th day, and the subcutaneous tumors were excised and weighed. Heavy. At the same time as tumor volume was measured, the body weight of the mice was measured every two days and recorded.
在实验结束时，处死小鼠，取出全血检测血细胞成分及含量，并采血分离出血清，分析其中的血生化指标，共检测谷丙转氨酶(ALT)、谷草转氨酶(AST)、总尿素氮(BUN)和心肌肌酸激酶(CK)四项指标。同时将小鼠心、肝、脾、肺、肾五种主要器官取出，用4％多聚甲醛固定，之后进行常规石蜡包埋切片、HE染色，在显微镜下观察组织结构。At the end of the experiment, the mice were sacrificed, the whole blood was taken out to detect the composition and content of blood cells, and the blood was collected to separate the serum, and the blood biochemical indexes were analyzed. BUN) and myocardial creatine kinase (CK) four indicators. At the same time, the five main organs of the mouse heart, liver, spleen, lung and kidney were taken out, fixed with 4% paraformaldehyde, and then routinely embedded in paraffin and stained with HE, and the tissue structure was observed under a microscope.
表1 为不同时间点各组肿瘤体积结果(单位：mm 3) Table 1 shows the results of tumor volume in each group at different time points (unit: mm 3 )
时间点(天)time (day) PBSPBS HBOHBO PD-1抑制剂PD-1 inhibitors HBO+PD-1抑制剂HBO+PD-1 inhibitor
11 118.03118.03 117.89117.89 117.97117.97 124.02124.02
33 239.58239.58 204.69204.69 180.56180.56 168.63168.63
55 316.89316.89 285.66285.66 219.27219.27 181.07181.07
77 384.64384.64 351.36351.36 273.17273.17 189.50189.50
99 613.63613.63 512.58512.58 357.05357.05 181.62181.62
1111 833.79833.79 689.97689.97 432.94432.94 135.85135.85
1313 1092.751092.75 947.06947.06 449.96449.96 125.76125.76
1515 1380.711380.71 1177.171177.17 523.35523.35 104.93104.93
1717 1739.761739.76 1392.631392.63 526.69526.69 71.6871.68
由表1可知，与PBS组相比，HBO组小鼠肿瘤生长没有明显的抑制，这说明单独使用HBO治疗并没有明显的抑瘤效果。而PD-1抑制剂组和HBO+PD-1抑制剂组相比于PBS组均表现出了抑制肿瘤生长的作用。HBO联合PD-1抑制剂治疗组肿瘤生长速度明显慢于单用PD-1抑制剂治疗组，在第17天，联合治疗组小鼠平均瘤体积为71.68mm 3，显著低于单用PD-1抑制剂组的526.69mm 3。以上分析表明，HBO联合PD-1抑制剂可以显著增强PD-1抑制剂对肿瘤生长的抑制效果。图 1为以上4组实验组的肿瘤生长曲线图。 It can be seen from Table 1 that compared with the PBS group, the tumor growth of the mice in the HBO group was not significantly inhibited, which indicated that the HBO treatment alone had no obvious tumor inhibitory effect. Both the PD-1 inhibitor group and the HBO+PD-1 inhibitor group showed an inhibitory effect on tumor growth compared with the PBS group. The tumor growth rate of the HBO combined with PD- 1 inhibitor treatment group was significantly slower than that of the PD-1 inhibitor treatment group alone. 1 526.69 mm 3 in the inhibitor group. The above analysis shows that HBO combined with PD-1 inhibitor can significantly enhance the inhibitory effect of PD-1 inhibitor on tumor growth. Figure 1 shows the tumor growth curves of the above four experimental groups.
图2为四组小鼠治疗结束后取出肿瘤称重的统计结果，由图2可知，PD-1抑制剂组和HBO+PD-1抑制剂组的肿瘤重量明显低于PBS组，同时，联合治疗组的瘤重显著低于单用PD-1抑制剂治疗组，这表明HBO联合PD-1抑制剂可以增强PD-1抑制剂的抗肿瘤效果。Figure 2 shows the statistical results of the tumor weights taken out of the four groups of mice after treatment. As can be seen from Figure 2, the tumor weights of the PD-1 inhibitor group and the HBO+PD-1 inhibitor group were significantly lower than those of the PBS group. The tumor weight of the treatment group was significantly lower than that of the PD-1 inhibitor treatment group alone, indicating that HBO combined with PD-1 inhibitor can enhance the anti-tumor effect of PD-1 inhibitor.
图3为以上4个组在给药期间的体重变化图，由图3可知，相比与PBS组，三个实验组小鼠体重没有明显的下降或上涨趋势，这表明单独使用HBO或PD-1抑制剂治疗，以及联合治疗均不会影响小鼠的健康状况。Figure 3 shows the body weight changes of the above 4 groups during the administration period. It can be seen from Figure 3 that compared with the PBS group, the body weight of the mice in the three experimental groups has no obvious decrease or increase trend, which indicates that HBO or PD- 1 Inhibitor treatment, as well as combination treatment, did not affect the health of the mice.
表2 为各组治疗结束后血细胞检测结果Table 2 shows the results of blood cell detection in each group after treatment
Figure PCTCN2021072094-appb-000001
Figure PCTCN2021072094-appb-000001
由表2可知，与PBS组相比，三个实验组小鼠在治疗结束后血液中白细胞、红细胞、血小板含量以及平均血红蛋白含量均没有明显差异，这表明单独使用HBO或PD-1抑制剂治疗，以及联合治疗均具有良好的安全性。As can be seen from Table 2, compared with the PBS group, there were no significant differences in the content of white blood cells, red blood cells, platelets and average hemoglobin in the blood of the three experimental groups after treatment, which indicated that HBO or PD-1 inhibitor treatment alone was used. , and combination therapy have good safety.
图4为PBS组、HBO组、PD-1抑制剂组和HBO+PD-1抑制剂组的血生化分析结果，主要检测了谷丙转氨酶、谷草转氨酶、心肌肌酸激酶和总尿素氮，其中谷丙转氨酶和谷草转氨酶的升高代表着药物具有严重的肝脏毒性，心肌肌酸激酶检测值升高意味着心脏发生了病理变化，而总尿素氮指标的升高意味着肾脏功能出现问题。由图4内容A、内容B和内容C可知，三个实验组的四项血生化指标与对照组(PBS组)相比都没有显著性变化，以上结果表明，单独使用HBO或PD-1抑制剂治疗，以及联合治疗均不会对小鼠的主要脏器产生损伤，治疗具有良好的安全性。Figure 4 shows the results of blood biochemical analysis of the PBS group, HBO group, PD-1 inhibitor group and HBO+PD-1 inhibitor group. Alanine aminotransferase, aspartate aminotransferase, myocardial creatine kinase and total urea nitrogen were mainly detected. Elevated alanine aminotransferase and aspartate aminotransferase indicate serious liver toxicity, elevated myocardial creatine kinase value indicates pathological changes in the heart, and elevated total urea nitrogen index indicates renal function problems. It can be seen from the content A, content B and content C of Figure 4 that the four blood biochemical indexes of the three experimental groups have no significant changes compared with the control group (PBS group). Neither the drug treatment nor the combined treatment will cause damage to the main organs of the mice, and the treatment has good safety.
图5为组织切片的结构图，放大倍数为200倍。由图5可知，PBS组与三个实验组的心肝脾肺肾组织结构清晰，没有明显的病理改变，无出血与炎症浸润，以上结果表明，单独使用HBO或PD-1抑制剂治疗，以及联合治疗均不会对小鼠的主要脏器产生损伤，具有良好的安全性。Figure 5 is a structural diagram of a tissue section at a magnification of 200 times. It can be seen from Figure 5 that the structure of the heart, liver, spleen, lung and kidney in the PBS group and the three experimental groups is clear, with no obvious pathological changes, no bleeding and inflammatory infiltration. None of the treatments would cause damage to the main organs of the mice and had good safety.
实施例7Example 7
基于高压氧联合PD-1抑制剂对荷乳腺癌原位瘤小鼠抗肿瘤活性的测试。Based on the test of hyperbaric oxygen combined with PD-1 inhibitor on the antitumor activity of breast cancer in situ tumor-bearing mice.
实验药物的配制：将PD-1抑制剂溶解在PBS中配制成1mg/mL的溶液。Preparation of experimental drugs: The PD-1 inhibitor was dissolved in PBS to prepare a solution of 1 mg/mL.
建立小鼠乳腺癌4T1原位瘤模型，当肿瘤长到体积为100mm 3时，随机将荷瘤 小鼠分为4个组(PBS组、HBO组、PD-1抑制剂组和HBO+PD-1抑制剂组)并记为第1天，分别在第1、3、5天给予HBO组和HBO+PD-1抑制剂组小鼠高压氧治疗，治疗方式为，将小鼠置于密闭舱体内，逐渐通入纯氧使舱内气压升高到2.5倍大气压，维持1.5小时后，缓慢放气使舱内气压降至大气压。高压氧治疗结束后间隔两小时通过尾静脉注射给予HBO+PD-1抑制剂组小鼠0.1mg/0.1mL PD-1抑制剂。并分别将PBS、PD-1抑制剂以0.1mL/只、0.1mg/0.1mL剂量通过尾静脉注射到对应实验组小鼠。每两天用游标卡尺测量肿瘤的最长处(L)和最宽处(W)，计算肿瘤体积V＝L*W 2/2，在第23天将各组小鼠处死，剥出皮下肿瘤并称重。在检测肿瘤体积的同时，每两天测量小鼠的体重，并记录。 A mouse breast cancer 4T1 orthotopic tumor model was established. When the tumor grew to a volume of 100 mm, the tumor-bearing mice were randomly divided into 4 groups (PBS group, HBO group, PD-1 inhibitor group and HBO+PD- 1 inhibitor group) and recorded as the 1st day, on the 1st, 3rd, and 5th days, the mice in the HBO group and the HBO+PD-1 inhibitor group were treated with hyperbaric oxygen therapy. The treatment method was to place the mice in a closed cabin. In the body, pure oxygen was gradually introduced to increase the air pressure in the cabin to 2.5 times the atmospheric pressure. After maintaining for 1.5 hours, the air pressure was slowly released to reduce the air pressure in the cabin to atmospheric pressure. The mice in the HBO+PD-1 inhibitor group were given 0.1 mg/0.1 mL of PD-1 inhibitor by tail vein injection at two hours after the end of hyperbaric oxygen therapy. PBS and PD-1 inhibitor were injected into the corresponding experimental group mice through tail vein at doses of 0.1mL/mice and 0.1mg/0.1mL, respectively. The longest part (L) and widest part (W) of the tumor were measured with a vernier caliper every two days, and the tumor volume V=L*W 2 /2 was calculated. The mice in each group were sacrificed on the 23rd day, and the subcutaneous tumors were excised and weighed. Heavy. At the same time as tumor volume was measured, the body weight of the mice was measured every two days and recorded.
在实验结束时，处死小鼠，取出全血检测血细胞成分及含量，并采血分离出血清，分析其中的血生化指标，共检测谷丙转氨酶(ALT)、谷草转氨酶(AST)、总尿素氮(BUN)和心肌肌酸激酶(CK)四项指标。同时取出小鼠完整的肺，使用Bouin’s染液对其进行固定染色，24小时后用95％乙醇洗涤后，记录肺部肿瘤转移节结数并拍照。同时将小鼠心、肝、脾、肺、肾五种主要脏器取出，用4％多聚甲醛固定，之后进行常规石蜡包埋包埋切片、HE染色，在显微镜下观察组织结构。At the end of the experiment, the mice were sacrificed, the whole blood was taken out to detect the composition and content of blood cells, and the blood was collected to separate the serum, and the blood biochemical indexes were analyzed. BUN) and myocardial creatine kinase (CK) four indicators. At the same time, the intact lungs of the mice were removed, fixed and stained with Bouin's stain, washed with 95% ethanol 24 hours later, and the number of metastatic nodules in the lungs was recorded and photographed. At the same time, the five main organs of the mouse heart, liver, spleen, lung and kidney were taken out, fixed with 4% paraformaldehyde, then routinely embedded in paraffin, and stained with HE, and the tissue structure was observed under a microscope.
表3 为不同时间点各组肿瘤体积结果(单位：mm 3) Table 3 shows the results of tumor volume in each group at different time points (unit: mm 3 )
时间点(天)time (day) PBSPBS HBOHBO PD-1抑制剂PD-1 inhibitors HBO+PD-1抑制剂HBO+PD-1 inhibitor
11 123.17123.17 138.57138.57 131.60131.60 130.70130.70
33 254.08254.08 221.73221.73 183.57183.57 168.37168.37
55 336.68336.68 319.83319.83 238.30238.30 202.47202.47
77 369.69369.69 333.15333.15 245.82245.82 216.42216.42
99 417.63417.63 405.13405.13 261.00261.00 224.79224.79
1111 558.09558.09 471.23471.23 325.35325.35 299.65299.65
1313 755.40755.40 670.67670.67 378.94378.94 326.31326.31
1515 957.99957.99 779.88779.88 468.06468.06 407.05407.05
1717 1091.711091.71 945.52945.52 659.68659.68 520.38520.38
1919 1354.471354.47 1187.981187.98 812.82812.82 635.62635.62
21twenty one 1553.791553.79 1316.031316.03 1008.931008.93 741.26741.26
23twenty three 1941.871941.87 1544.921544.92 1138.231138.23 807.06807.06
由表3可知，与PBS组相比，HBO组小鼠肿瘤生长没有明显的抑制，这说明单独使用HBO治疗并没有明显的抑瘤效果。而PD-1抑制剂组和HBO+PD-1抑制 剂组相比于PBS组均表现出了抑制肿瘤生长的作用。HBO联合PD-1抑制剂治疗组肿瘤生长速度明显慢于单用PD-1抑制剂治疗组，在第23天，联合治疗组小鼠平均瘤体积为807.06mm 3，显著低于单用PD-1抑制剂组的1138.23mm 3。以上分析表明，HBO联合PD-1抑制剂可以显著增强PD-1抑制剂对肿瘤生长的抑制效果。图6为以上4组实验组的肿瘤生长曲线图。 As can be seen from Table 3, compared with the PBS group, the tumor growth of the mice in the HBO group was not significantly inhibited, which indicated that the HBO treatment alone did not have an obvious tumor-inhibiting effect. Both the PD-1 inhibitor group and the HBO+PD-1 inhibitor group showed an inhibitory effect on tumor growth compared with the PBS group. The tumor growth rate of HBO combined with PD- 1 inhibitor treatment group was significantly slower than that of PD-1 inhibitor treatment group alone. 1 1138.23 mm 3 of the inhibitor group. The above analysis shows that HBO combined with PD-1 inhibitor can significantly enhance the inhibitory effect of PD-1 inhibitor on tumor growth. Figure 6 is a graph showing the tumor growth curves of the above four experimental groups.
图7为四组小鼠治疗结束后取出肿瘤称重的统计结果，由图7可知，PD-1抑制剂组和HBO+PD-1抑制剂组的肿瘤重量明显低于PBS组，同时，联合治疗组的瘤重显著低于单用PD-1抑制剂治疗组，这表明HBO联合PD-1抑制剂可以增强PD-1抑制剂的抗肿瘤效果。Figure 7 shows the statistical results of tumor weights taken out of the four groups of mice after treatment. It can be seen from Figure 7 that the tumor weights in the PD-1 inhibitor group and the HBO+PD-1 inhibitor group were significantly lower than those in the PBS group. The tumor weight of the treatment group was significantly lower than that of the PD-1 inhibitor treatment group alone, indicating that HBO combined with PD-1 inhibitor can enhance the anti-tumor effect of PD-1 inhibitor.
图8为以上4个组在给药期间的体重变化图，由图8可知，相比于PBS组，三个实验组小鼠体重没有明显的下降或上涨趋势，这表明单独使用HBO或PD-1抑制剂治疗，以及联合治疗均不会影响小鼠的健康状况。Figure 8 shows the body weight changes of the above four groups during the administration period. It can be seen from Figure 8 that, compared with the PBS group, the body weight of the mice in the three experimental groups has no obvious decrease or increase trend, which indicates that the use of HBO or PD- 1 Inhibitor treatment, as well as combination treatment, did not affect the health of the mice.
图9为四组小鼠治疗结束后肺部转移节结数统计结果，由图9可知，PD-1抑制剂组和HBO+PD-1抑制剂组相比于PBS组，小鼠肺部转移节结数明显减少，而联合治疗组肺部转移节结数显著少于单用PD-1抑制剂治疗组，这表明HBO联合PD-1抑制剂肿瘤转移具有明显的抑制效果。Figure 9 shows the statistical results of the number of lung metastases in the four groups of mice after treatment. As can be seen from Figure 9, compared with the PBS group, the PD-1 inhibitor group and the HBO+PD-1 inhibitor group had more The number of nodules was significantly reduced, while the number of pulmonary metastatic nodules in the combination treatment group was significantly less than that in the PD-1 inhibitor treatment group alone, which indicated that HBO combined with PD-1 inhibitor had a significant inhibitory effect on tumor metastasis.
表4 为各组治疗结束后血细胞检测结果Table 4 shows the results of blood cell detection in each group after treatment
Figure PCTCN2021072094-appb-000002
Figure PCTCN2021072094-appb-000002
由表4可知，与PBS组相比，三个实验组小鼠在治疗结束后血液中白细胞、红细胞、血小板含量以及平均血红蛋白含量均没有明显差异，这表明单独使用HBO或PD-1抑制剂治疗，以及联合治疗均具有良好的安全性。As can be seen from Table 4, compared with the PBS group, there were no significant differences in the content of white blood cells, red blood cells, platelets and average hemoglobin in the blood of the three experimental groups after treatment, which indicated that HBO or PD-1 inhibitor treatment alone was used. , and combination therapy have good safety.
图10为PBS组、HBO组、PD-1抑制剂组和HBO+PD-1抑制剂组的血生化分析结果，主要检测了谷丙转氨酶、谷草转氨酶、心肌肌酸激酶和总尿素氮，其中谷丙转氨酶和谷草转氨酶的升高代表着药物具有严重的肝脏毒性，心肌肌酸激酶检测值升高意味着心脏发生了病理变化，而总尿素氮指标的升高意味着肾脏功能出现问题。由图10内容A、内容B和内容C可知，三个实验组的四项血生化指标与对照组(PBS组)相比都没有显著性变化，以上结果表明，单独使用HBO或PD-1抑制剂治疗，以及联合治疗均不会对小鼠的主要脏器产生损伤，治疗具有良好的安全性Figure 10 shows the blood biochemical analysis results of the PBS group, the HBO group, the PD-1 inhibitor group and the HBO+PD-1 inhibitor group. Alanine aminotransferase, aspartate aminotransferase, myocardial creatine kinase and total urea nitrogen were mainly detected. Elevated alanine aminotransferase and aspartate aminotransferase indicate serious liver toxicity, elevated myocardial creatine kinase value indicates pathological changes in the heart, and elevated total urea nitrogen index indicates renal function problems. It can be seen from the content A, content B and content C of Figure 10 that the four blood biochemical indexes of the three experimental groups have no significant changes compared with the control group (PBS group). The drug treatment and the combined treatment will not cause damage to the main organs of the mice, and the treatment has good safety
图11为组织切片的结构图，放大倍数为200倍。由图11可知，PBS组与三个实验组的心肝脾肺肾结构清晰，没有明显的病理改变，无出血与炎症浸润，以上结果表明，单独使用HBO或PD-1抑制剂治疗，以及联合治疗均不会对小鼠的主要脏器产生损伤，具有良好的安全性。Figure 11 is a structural diagram of a tissue section at a magnification of 200 times. It can be seen from Figure 11 that the structure of the heart, liver, spleen, lung and kidney in the PBS group and the three experimental groups is clear, with no obvious pathological changes, no bleeding and inflammatory infiltration. The above results show that HBO or PD-1 inhibitor treatment alone, and combined treatment All of them will not cause damage to the main organs of mice, and have good safety.
实施例8Example 8
基于高压氧联合PD-1抑制剂对荷肝癌原位瘤小鼠生存期影响的测试。Based on the test of the effect of hyperbaric oxygen combined with PD-1 inhibitor on the survival of mice bearing liver cancer in situ.
实验药物的配制：将PD-1抑制剂溶解在PBS中配制成1mg/mL的溶液。Preparation of experimental drugs: The PD-1 inhibitor was dissolved in PBS to prepare a solution of 1 mg/mL.
建立小鼠肝癌H22原位瘤模型并记为第1天，两周后随机将荷瘤小鼠分为4个组(PBS组、HBO组、PD-1抑制剂组和HBO+PD-1抑制剂组)分别在第14、16、18天给予HBO组和HBO+PD-1抑制剂组小鼠高压氧治疗，治疗方式为，将小鼠置于密闭舱体内，逐渐通入纯氧使舱内气压升高到2.5倍大气压，维持1.5小时后，缓慢放气使舱内气压降至大气压。高压氧治疗结束后间隔两小时通过尾静脉注射给予HBO+PD-1抑制剂组小鼠0.1mg/0.1mL PD-1抑制剂。并分别将PBS、PD-1抑制剂以0.1mL/只、0.1mg/0.1mL剂量通过尾静脉注射到对应实验组小鼠。治疗结束后每天观察小鼠的存活状态，并记录。The mouse liver cancer H22 orthotopic tumor model was established and recorded as the first day. Two weeks later, the tumor-bearing mice were randomly divided into 4 groups (PBS group, HBO group, PD-1 inhibitor group and HBO+PD-1 inhibitor group). The mice in the HBO group and the HBO+PD-1 inhibitor group were treated with hyperbaric oxygen on the 14th, 16th, and 18th days, respectively. The treatment method was to place the mice in a closed cabin, and gradually introduce pure oxygen into the cabin. The internal pressure was raised to 2.5 times atmospheric pressure, and after maintaining for 1.5 hours, the air pressure was slowly deflated to reduce the pressure in the cabin to atmospheric pressure. HBO+PD-1 inhibitor group mice were given 0.1mg/0.1mL PD-1 inhibitor by tail vein injection at two hours after the end of hyperbaric oxygen therapy. PBS and PD-1 inhibitor were injected into the corresponding experimental group mice through tail vein at doses of 0.1mL/mice and 0.1mg/0.1mL, respectively. After the treatment, the survival status of the mice was observed and recorded every day.
图12为各组小鼠生存期Kaplan-Meier图，从图中可以看出，PBS组小鼠在种瘤后第39天全部死亡，而HBO组在第49天全部死亡，此时，单用PD-1抑制剂组仍有3只小鼠存活，而HBO联合PD-1抑制剂组仍有7只存活。PBS组、HBO组、PD-1抑制剂组的中位生存期分别为33、35.5、39.5天，而HBO联合PD-1抑制剂治疗组半数以上小鼠存活时间超过三个月。以上结果分析表明，HBO联合PD-1抑制剂治疗可以显著延长荷H22肝癌原位瘤小鼠生存期。Figure 12 is the Kaplan-Meier chart of the survival period of the mice in each group. It can be seen from the figure that all the mice in the PBS group died on the 39th day after tumor implantation, while all the mice in the HBO group died on the 49th day. Three mice were still alive in the PD-1 inhibitor group, while seven were still alive in the HBO combined PD-1 inhibitor group. The median survival time of the PBS group, HBO group, and PD-1 inhibitor group was 33, 35.5, and 39.5 days, respectively, while more than half of the mice in the HBO combined with PD-1 inhibitor treatment group survived for more than three months. The above analysis of the results showed that HBO combined with PD-1 inhibitor therapy could significantly prolong the survival of H22 hepatocellular carcinoma in situ tumor-bearing mice.
实施例9Example 9
基于高压氧联合PD-1抑制剂对荷肝癌原位瘤小鼠抗肿瘤效果的测试。Based on the test of anti-tumor effect of hyperbaric oxygen combined with PD-1 inhibitor on liver cancer in situ tumor-bearing mice.
实验药物的配制：将PD-1抑制剂溶解在PBS中配制成1mg/mL的溶液。Preparation of experimental drugs: The PD-1 inhibitor was dissolved in PBS to prepare a solution of 1 mg/mL.
建立小鼠肝癌H22原位瘤模型并记为第1天，两周后随机将荷瘤小鼠分为4个组(PBS组、HBO组、PD-1抑制剂组和HBO+PD-1抑制剂组)分别在第14、16、18天给予HBO组和HBO+PD-1抑制剂组小鼠高压氧治疗，治疗方式为，将小鼠置于密闭舱体内，逐渐通入纯氧使舱内气压升高到2.5倍大气压，维持1.5小时后，缓慢放气使舱内气压降至大气压。高压氧治疗结束后间隔两小时通过尾静脉注射给予HBO+PD-1抑制剂组小鼠0.1mg/0.1mL PD-1抑制剂。并分别将PBS、PD-1抑制剂以0.1mL/只、0.1mg/0.1mL剂量通过尾静脉注射到对应实验组小鼠。在第28天处死小鼠，剥离肿瘤并称重，取出小鼠全血检测血细胞成分及含量，并采血分离出血清，分析其中的血生化指标，共检测谷丙转氨酶(ALT)、谷草转氨酶(AST)、总尿素氮(BUN)和心肌肌酸激酶(CK)四项指标。同时将小鼠心、 肝、脾、肺、肾五种主要脏器取出，用4％多聚甲醛固定，之后进行常规石蜡包埋切片、HE染色，在显微镜下观察组织结构。The mouse liver cancer H22 orthotopic tumor model was established and recorded as the first day. After two weeks, the tumor-bearing mice were randomly divided into 4 groups (PBS group, HBO group, PD-1 inhibitor group and HBO+PD-1 inhibitor group). On the 14th, 16th, and 18th days, the mice in the HBO group and the HBO+PD-1 inhibitor group were treated with hyperbaric oxygen therapy. The internal pressure was raised to 2.5 times atmospheric pressure, and after maintaining for 1.5 hours, the air pressure was slowly deflated to reduce the pressure in the cabin to atmospheric pressure. The mice in the HBO+PD-1 inhibitor group were given 0.1mg/0.1mL PD-1 inhibitor by tail vein injection at two hours after the end of hyperbaric oxygen therapy. PBS and PD-1 inhibitor were injected into the corresponding experimental group mice through tail vein at doses of 0.1mL/mice and 0.1mg/0.1mL, respectively. On the 28th day, the mice were sacrificed, the tumors were stripped and weighed, the whole blood of the mice was taken out to detect the composition and content of blood cells, and the blood was collected to separate the serum, and the blood biochemical indexes were analyzed. AST), total urea nitrogen (BUN), and cardiac creatine kinase (CK). At the same time, the five main organs of the mouse heart, liver, spleen, lung and kidney were taken out, fixed with 4% paraformaldehyde, then routinely embedded in paraffin, and stained with HE, and the tissue structure was observed under a microscope.
图13为四组小鼠治疗结束后取出肿瘤称重的统计结果，由图13可知，PD-1抑制剂组和HBO+PD-1抑制剂组的肿瘤重量明显低于PBS组，同时，联合治疗组的瘤重显著低于单用PD-1抑制剂治疗组，这表明HBO联合PD-1抑制剂可以增强PD-1抑制剂的抗肿瘤效果。Figure 13 shows the statistical results of tumor weights taken out of the four groups of mice after treatment. As can be seen from Figure 13, the tumor weights of the PD-1 inhibitor group and the HBO+PD-1 inhibitor group were significantly lower than those of the PBS group. The tumor weight of the treatment group was significantly lower than that of the PD-1 inhibitor treatment group alone, indicating that HBO combined with PD-1 inhibitor can enhance the anti-tumor effect of PD-1 inhibitor.
图14为以上4个组在给药期间的体重变化图，由图14可知，相比于PBS组，三个实验组小鼠体重没有明显的下降或上涨趋势，这表明单独使用HBO或PD-1抑制剂治疗，以及联合治疗均不会影响小鼠的健康状况。Figure 14 shows the body weight changes of the above four groups during the administration period. It can be seen from Figure 14 that compared with the PBS group, the body weight of the mice in the three experimental groups has no obvious decrease or increase trend, which indicates that the use of HBO or PD- 1 Inhibitor treatment, as well as combination treatment, did not affect the health of the mice.
表5 为各组治疗结束后血细胞检测结果Table 5 shows the results of blood cell detection in each group after treatment
Figure PCTCN2021072094-appb-000003
Figure PCTCN2021072094-appb-000003
由表5可知，与PBS组相比，三个实验组小鼠在治疗结束后血液中白细胞、红细胞、血小板含量以及平均血红蛋白含量均没有明显差异，这表明单独使用HBO或PD-1抑制剂治疗，以及联合治疗均具有良好的安全性。It can be seen from Table 5 that compared with the PBS group, there were no significant differences in the content of white blood cells, red blood cells, platelets and average hemoglobin in the blood of the three experimental groups after treatment, which indicated that HBO or PD-1 inhibitor treatment alone was used. , and combination therapy have good safety.
图15为PBS组、HBO组、PD-1抑制剂组和HBO+PD-1抑制剂组的血生化分析结果，其中内容A中ALT与AST分别代表谷丙转氨酶和谷草转氨酶，该指标的升高代表着药物具有严重的肝脏毒性，内容B中CK为心肌肌酸激酶，其值升高意味着心脏发生了病理变化，内容C中BUN为总尿素氮，该指标的升高意味着肾脏功能出现问题。由图15可知，三个实验组的四项血生化指标与对照组(PBS组)相比都没有显著性变化，以上结果表明，单独使用HBO或PD-1抑制剂治疗，以及联合治疗均不会对小鼠的主要脏器产生损伤，治疗具有良好的安全性。Figure 15 shows the blood biochemical analysis results of PBS group, HBO group, PD-1 inhibitor group and HBO+PD-1 inhibitor group, in which ALT and AST in content A represent alanine aminotransferase and aspartate aminotransferase, respectively. High indicates that the drug has serious liver toxicity. CK in content B is myocardial creatine kinase, and an increase in its value means pathological changes in the heart. BUN in content C is total urea nitrogen, and an increase in this index means kidney function. problem appear. As can be seen from Figure 15, there were no significant changes in the four blood biochemical indexes of the three experimental groups compared with the control group (PBS group). It will cause damage to the main organs of mice, and the treatment has good safety.
图16为组织切片的结构图，放大倍数为200倍。由图16可知，PBS组与三个实验组的心肝脾肺肾结构清晰，没有明显的病理改变，无出血与炎症浸润，以上结果表明，单独使用HBO或PD-1抑制剂治疗，以及联合治疗均不会对小鼠的主要脏器产生损伤，具有良好的安全性。Figure 16 is a structural diagram of a tissue section at a magnification of 200 times. It can be seen from Figure 16 that the structures of the heart, liver, spleen, lung and kidney in the PBS group and the three experimental groups are clear, with no obvious pathological changes, no bleeding and inflammatory infiltration. The above results show that HBO or PD-1 inhibitor treatment alone, and combined treatment All of them will not cause damage to the main organs of mice, and have good safety.
实施例10Example 10
基于高压氧联合PD-1抑制剂对荷胰腺癌原位瘤小鼠生存期影响的测试。Based on the test of the effect of hyperbaric oxygen combined with PD-1 inhibitor on the survival of pancreatic cancer in situ tumor-bearing mice.
实验药物的配制：将PD-1抑制剂溶解在PBS中配制成1mg/mL的溶液。Preparation of experimental drugs: The PD-1 inhibitor was dissolved in PBS to prepare a solution of 1 mg/mL.
建立小鼠胰腺癌Panc02原位瘤模型并记为第1天，一周后随机将荷瘤小鼠分 为4个组(PBS组、HBO组、PD-1抑制剂组和HBO+PD-1抑制剂组)分别在第7、9、11天给予HBO组和HBO+PD-1抑制剂组小鼠高压氧治疗，治疗方式为，将小鼠置于密闭舱体内，逐渐通入纯氧使舱内气压升高到2.5倍大气压，维持1.5小时后，缓慢放气使舱内气压降至大气压。高压氧治疗结束后间隔两小时通过尾静脉注射给予HBO+PD-1抑制剂组小鼠0.1mg/0.1mL PD-1抑制剂。并分别将PBS、PD-1抑制剂以0.1mL/只、0.1mg/0.1mL剂量通过尾静脉注射到对应实验组小鼠。治疗结束后每天观察小鼠的存活状态，并记录。The Panc02 orthotopic tumor model of mouse pancreatic cancer was established and recorded as the first day. One week later, the tumor-bearing mice were randomly divided into 4 groups (PBS group, HBO group, PD-1 inhibitor group and HBO+PD-1 inhibitor group). The mice in the HBO group and the HBO+PD-1 inhibitor group were treated with hyperbaric oxygen on the 7th, 9th, and 11th days, respectively. The treatment method was to place the mice in a closed cabin, and gradually introduce pure oxygen into the cabin. The internal pressure was raised to 2.5 times atmospheric pressure, and after maintaining for 1.5 hours, the air pressure was slowly deflated to reduce the pressure in the cabin to atmospheric pressure. The mice in the HBO+PD-1 inhibitor group were given 0.1mg/0.1mL PD-1 inhibitor by tail vein injection at two hours after the end of hyperbaric oxygen therapy. PBS and PD-1 inhibitor were injected into the corresponding experimental group mice through tail vein at doses of 0.1mL/mice and 0.1mg/0.1mL, respectively. After the treatment, the survival status of the mice was observed and recorded every day.
图17为各组小鼠生存期Kaplan-Meier图，从图中可以看出，PBS组、HBO组以及单用PD-1抑制剂组小鼠分别在种瘤后第34、37、45天全部死亡，而此时HBO联合PD-1抑制剂组仍有4只存活。PBS组、HBO组、PD-1抑制剂组的中位生存期分别为28.5、32、32天，而HBO联合PD-1抑制剂治疗组中位生存期为43天。以上结果分析表明，HBO联合PD-1抑制剂治疗可以显著延长荷Panc02胰腺癌原位瘤小鼠生存期。Figure 17 is the Kaplan-Meier chart of the survival period of mice in each group. It can be seen from the figure that the mice in the PBS group, the HBO group and the PD-1 inhibitor group alone were all on the 34th, 37th, and 45th days after tumor implantation, respectively. At this time, there were still 4 survivors in the HBO combined with PD-1 inhibitor group. The median survival time of PBS group, HBO group, and PD-1 inhibitor group were 28.5, 32, and 32 days, respectively, while the median survival time of HBO combined with PD-1 inhibitor treatment group was 43 days. The above analysis of the results showed that HBO combined with PD-1 inhibitor therapy can significantly prolong the survival of Panc02 pancreatic cancer in situ tumor-bearing mice.
实施例11Example 11
基于高压氧联合PD-1抑制剂对荷胰腺癌原位瘤小鼠抗肿瘤效果的测试。Based on the test of the antitumor effect of hyperbaric oxygen combined with PD-1 inhibitor on pancreatic cancer in situ tumor-bearing mice.
实验药物的配制：将PD-1抑制剂溶解在PBS中配制成1mg/mL的溶液。Preparation of experimental drugs: The PD-1 inhibitor was dissolved in PBS to prepare a solution of 1 mg/mL.
建立小鼠胰腺癌Panc02原位瘤模型并记为第1天，一周后随机将荷瘤小鼠分为4个组(PBS组、HBO组、PD-1抑制剂组和HBO+PD-1抑制剂组)分别在第7、9、11天给予HBO组和HBO+PD-1抑制剂组小鼠高压氧治疗，治疗方式为，将小鼠置于密闭舱体内，逐渐通入纯氧使舱内气压升高到2.5倍大气压，维持1.5小时后，缓慢放气使舱内气压降至大气压。高压氧治疗结束后间隔两小时通过尾静脉注射给予HBO+PD-1抑制剂组小鼠0.1mg/0.1mL PD-1抑制剂。并分别将PBS、PD-1抑制剂以0.1mL/只、0.1mg/0.1mL剂量通过尾静脉注射到对应实验组小鼠。在第21天处死小鼠，剥离肿瘤并称重，取出小鼠全血检测血细胞成分及含量，并采血分离出血清，分析其中的血生化指标，共检测谷丙转氨酶(ALT)、谷草转氨酶(AST)、总尿素氮(BUN)和心肌肌酸激酶(CK)四项指标。同时将小鼠心、肝、脾、肺、肾五种主要脏器取出，用4％多聚甲醛固定，之后进行常规石蜡包埋切片、HE染色，在显微镜下观察组织结构。The Panc02 orthotopic tumor model of mouse pancreatic cancer was established and recorded as the first day. One week later, the tumor-bearing mice were randomly divided into 4 groups (PBS group, HBO group, PD-1 inhibitor group and HBO+PD-1 inhibitor group). The mice in the HBO group and the HBO+PD-1 inhibitor group were treated with hyperbaric oxygen on the 7th, 9th, and 11th days, respectively. The treatment method was to place the mice in a closed cabin, and gradually introduce pure oxygen into the cabin. The internal pressure was raised to 2.5 times atmospheric pressure, and after maintaining for 1.5 hours, the air pressure was slowly deflated to reduce the pressure in the cabin to atmospheric pressure. The mice in the HBO+PD-1 inhibitor group were given 0.1mg/0.1mL PD-1 inhibitor by tail vein injection at two hours after the end of hyperbaric oxygen therapy. PBS and PD-1 inhibitor were injected into the corresponding experimental group mice through tail vein at doses of 0.1mL/mice and 0.1mg/0.1mL, respectively. On the 21st day, the mice were sacrificed, the tumors were stripped and weighed. The whole blood of the mice was taken out to detect the composition and content of blood cells, and the blood was collected to separate the serum. The blood biochemical indexes were analyzed, and alanine aminotransferase (ALT), aspartate aminotransferase ( AST), total urea nitrogen (BUN), and cardiac creatine kinase (CK). At the same time, the five main organs of the mouse heart, liver, spleen, lung and kidney were taken out, fixed with 4% paraformaldehyde, then routinely embedded in paraffin, and stained with HE, and the tissue structure was observed under a microscope.
图18为四组小鼠治疗结束后取出肿瘤称重的统计结果，由图18可知，PD-1抑制剂组和HBO+PD-1抑制剂组的肿瘤重量明显低于PBS组，同时，联合治疗组的瘤重显著低于单用PD-1抑制剂治疗组，这表明HBO联合PD-1抑制剂可以增强PD-1抑制剂的抗肿瘤效果。Figure 18 shows the statistical results of tumor weights taken out of the four groups of mice after treatment. It can be seen from Figure 18 that the tumor weights of the PD-1 inhibitor group and the HBO+PD-1 inhibitor group were significantly lower than those of the PBS group. The tumor weight of the treatment group was significantly lower than that of the PD-1 inhibitor treatment group alone, indicating that HBO combined with PD-1 inhibitor can enhance the anti-tumor effect of PD-1 inhibitor.
图19为以上4个组在给药期间的体重变化图，由图19可知，相比于PBS组，三个实验组小鼠体重没有明显的下降或上涨趋势，这表明单独使用HBO或PD-1 抑制剂治疗，以及联合治疗均不会影响小鼠的健康状况。Figure 19 is a graph of the body weight changes of the above four groups during the administration period. It can be seen from Figure 19 that compared with the PBS group, the body weight of the mice in the three experimental groups has no obvious decrease or increase trend, which indicates that the use of HBO or PD- 1 Inhibitor treatment, as well as combination treatment, did not affect the health of the mice.
表6 为各组治疗结束后血细胞检测结果Table 6 shows the results of blood cell detection in each group after treatment
Figure PCTCN2021072094-appb-000004
Figure PCTCN2021072094-appb-000004
由表6可知，与PBS组相比，三个实验组小鼠在治疗结束后血液中白细胞、红细胞、血小板含量以及平均血红蛋白含量均没有明显差异，这表明单独使用HBO或PD-1抑制剂治疗，以及联合治疗均具有良好的安全性。As can be seen from Table 6, compared with the PBS group, there were no significant differences in the content of white blood cells, red blood cells, platelets and average hemoglobin in the blood of the three experimental groups after treatment, which indicated that HBO or PD-1 inhibitor treatment alone was used. , and combination therapy have good safety.
图20为PBS组、HBO组、PD-1抑制剂组和HBO+PD-1抑制剂组的血生化分析结果，主要检测了谷丙转氨酶、谷草转氨酶、心肌肌酸激酶和总尿素氮，其中谷丙转氨酶和谷草转氨酶的升高代表着药物具有严重的肝脏毒性，心肌肌酸激酶检测值升高意味着心脏发生了病理变化，而总尿素氮指标的升高意味着肾脏功能出现问题。由图20内容A、内容B和内容C可知，三个实验组的四项血生化指标与对照组(PBS组)相比都没有显著性变化，以上结果表明，单独使用HBO或PD-1抑制剂治疗，以及联合治疗均不会对小鼠的主要脏器产生损伤，治疗具有良好的安全性。Figure 20 shows the blood biochemical analysis results of the PBS group, the HBO group, the PD-1 inhibitor group and the HBO+PD-1 inhibitor group. Alanine aminotransferase, aspartate aminotransferase, myocardial creatine kinase and total urea nitrogen were mainly detected. Elevated alanine aminotransferase and aspartate aminotransferase indicate serious liver toxicity, elevated myocardial creatine kinase value indicates pathological changes in the heart, and elevated total urea nitrogen index indicates renal function problems. From the content A, content B and content C of Figure 20, it can be seen that the four blood biochemical indexes of the three experimental groups have no significant changes compared with the control group (PBS group). Neither the drug treatment nor the combined treatment will cause damage to the main organs of the mice, and the treatment has good safety.
图21为组织切片的结构图，放大倍数为200倍。由图21可知，PBS组与三个实验组的心肝脾肺肾结构清晰，没有明显的病理改变，无出血与炎症浸润，以上结果表明，单独使用HBO或PD-1抑制剂治疗，以及联合治疗均不会对小鼠的主要脏器产生损伤，具有良好的安全性。Figure 21 is a structural diagram of a tissue section at a magnification of 200 times. It can be seen from Figure 21 that the structure of the heart, liver, spleen, lung and kidney in the PBS group and the three experimental groups is clear, with no obvious pathological changes, no bleeding and inflammatory infiltration. The above results show that HBO or PD-1 inhibitor treatment alone, and combined treatment All of them will not cause damage to the main organs of mice, and have good safety.
实施例12Example 12
基于高压氧联合PD-1抑制剂对荷肝癌皮下瘤小鼠抗肿瘤活性的测试。Based on the test of hyperbaric oxygen combined with PD-1 inhibitor on the antitumor activity of liver cancer subcutaneous tumor-bearing mice.
实验药物的配制：将PD-1抑制剂溶解在PBS中配制成1mg/mL的溶液。Preparation of experimental drugs: The PD-1 inhibitor was dissolved in PBS to prepare a solution of 1 mg/mL.
建立小鼠肝癌H22皮下瘤模型，当皮下瘤长到体积为500mm 3时，随机将荷瘤小鼠分为4个组(PBS组、HBO组、PD-1抑制剂组和HBO+PD-1抑制剂组)并记为第1天，分别在第1、3、5天给予HBO组和HBO+PD-1抑制剂组小鼠高压氧治疗，治疗方式为：将小鼠置于密闭舱体内，逐渐通入纯氧使舱内气压升高到2.5倍大气压，维持1.5小时后，缓慢放气使舱内气压降至大气压。高压氧治疗结束后间隔两小时通过尾静脉注射给予HBO+PD-1抑制剂组小鼠0.1mg/0.1mL PD-1抑制剂。并分别将PBS、PD-1抑制剂以0.1mL/只、0.1mg/0.1mL剂量通过尾静脉注射到对应实验组小鼠。每两天用游标卡尺测量肿瘤的最长处(L)和最宽处(W)， 计算肿瘤体积V＝L*W 2/2，在第15天将各组小鼠处死，剥出皮下肿瘤并称重。 A mouse liver cancer H22 subcutaneous tumor model was established. When the subcutaneous tumor grew to a volume of 500 mm, the tumor-bearing mice were randomly divided into 4 groups (PBS group, HBO group, PD-1 inhibitor group and HBO+PD-1 group). Inhibitor group) and recorded as the first day, the mice in the HBO group and the HBO+PD-1 inhibitor group were treated with hyperbaric oxygen on the 1st, 3rd, and 5th days respectively. The treatment method was as follows: the mice were placed in a closed cabin. , and gradually introduce pure oxygen to increase the pressure in the cabin to 2.5 times the atmospheric pressure. After maintaining for 1.5 hours, slowly deflate the pressure to reduce the pressure in the cabin to atmospheric pressure. The mice in the HBO+PD-1 inhibitor group were given 0.1 mg/0.1 mL PD-1 inhibitor by tail vein injection at two hours after the hyperbaric oxygen treatment. PBS and PD-1 inhibitor were injected into the corresponding experimental group mice through tail vein at doses of 0.1mL/mice and 0.1mg/0.1mL, respectively. The longest part (L) and widest part (W) of the tumor were measured with a vernier caliper every two days, and the tumor volume V=L*W 2 /2 was calculated. The mice in each group were sacrificed on the 15th day, and the subcutaneous tumors were excised and weighed. Heavy.
表7 为不同时间点各组肿瘤体积结果(单位：mm 3) Table 7 shows the results of tumor volume in each group at different time points (unit: mm 3 )
时间点(天)time (day) PBSPBS HBOHBO PD-1抑制剂PD-1 inhibitors HBO+PD-1抑制剂HBO+PD-1 inhibitor
11 523.13523.13 536.42536.42 539.64539.64 484.34484.34
33 679.13679.13 696.90696.90 656.32656.32 549.11549.11
55 856.96856.96 803.96803.96 723.59723.59 572.59572.59
77 1009.621009.62 969.59969.59 753.56753.56 496.52496.52
99 1194.871194.87 1156.071156.07 769.12769.12 428.57428.57
1111 1431.501431.50 1387.111387.11 706.60706.60 286.06286.06
1313 1680.371680.37 1636.561636.56 560.19560.19 158.78158.78
1515 1907.731907.73 1896.561896.56 417.40417.40 41.7341.73
由表1可知，与PBS组相比，HBO组小鼠肿瘤生长没有明显的抑制，这说明单独使用HBO治疗并没有明显的抑瘤效果。而PD-1抑制剂组和HBO+PD-1抑制剂组相比于PBS组均表现出了抑制肿瘤生长的作用。HBO联合PD-1抑制剂治疗组肿瘤生长速度明显慢于单用PD-1抑制剂治疗组，在第17天，联合治疗组小鼠平均瘤体积为41.73mm 3，显著低于单用PD-1抑制剂组的417.40mm 3。以上分析表明，HBO联合PD-1抑制剂可以显著增强PD-1抑制剂对肿瘤生长的抑制效果，并且这种增强效果对初始体积为500mm 3的难治性大肿瘤也有良好的治疗效果。图22为以上4组实验组的肿瘤生长曲线图。 It can be seen from Table 1 that compared with the PBS group, the tumor growth of the mice in the HBO group was not significantly inhibited, which indicated that the HBO treatment alone had no obvious tumor inhibitory effect. Both the PD-1 inhibitor group and the HBO+PD-1 inhibitor group showed an inhibitory effect on tumor growth compared with the PBS group. The tumor growth rate of the HBO combined with PD- 1 inhibitor treatment group was significantly slower than that of the PD-1 inhibitor treatment group alone. 1 417.40 mm 3 of the inhibitor group. The above analysis shows that HBO combined with PD-1 inhibitor can significantly enhance the inhibitory effect of PD-1 inhibitor on tumor growth, and this enhanced effect also has a good therapeutic effect on refractory large tumors with an initial volume of 500 mm. Figure 22 is a graph showing the tumor growth curves of the above four experimental groups.
图23为四组小鼠治疗结束后取出肿瘤称重的统计结果，由图23可知，PD-1抑制剂组和HBO+PD-1抑制剂组的肿瘤重量明显低于PBS组，同时，联合治疗组的瘤重显著低于单用PD-1抑制剂治疗组，这表明HBO联合PD-1抑制剂可以增强PD-1抑制剂的抗肿瘤效果。Figure 23 shows the statistical results of the tumor weights taken out of the four groups of mice after treatment. As can be seen from Figure 23, the tumor weights of the PD-1 inhibitor group and the HBO+PD-1 inhibitor group were significantly lower than those of the PBS group. The tumor weight of the treatment group was significantly lower than that of the PD-1 inhibitor treatment group alone, indicating that HBO combined with PD-1 inhibitor can enhance the anti-tumor effect of PD-1 inhibitor.
实施例13Example 13
基于高压氧联合PD-1抑制剂对荷肝癌皮下瘤小鼠药物递送的测试。Test of drug delivery based on hyperbaric oxygen combined with PD-1 inhibitor in mice bearing liver cancer subcutaneous tumor.
实验药物的配制：使用
Figure PCTCN2021072094-appb-000005
680快速荧光标记试剂盒将荧光分子Dylight680标记到PD-1抑制剂上，再将标记后的PD-1抑制剂溶解在PBS中配制成1mg/mL的溶液。 Preparation of Experimental Drugs: Use
Figure PCTCN2021072094-appb-000005
The 680 rapid fluorescent labeling kit labels the fluorescent molecule Dylight680 on the PD-1 inhibitor, and then dissolves the labeled PD-1 inhibitor in PBS to prepare a solution of 1 mg/mL.
建立小鼠肝癌H22皮下瘤模型，当皮下瘤长到体积为250mm 3时，随机将荷瘤小鼠分为2个组(PD-1抑制剂组和HBO+PD-1抑制剂组)给予HBO+PD-1抑制剂组小鼠高压氧治疗，治疗方式为，将小鼠置于密闭舱体内，逐渐通入纯氧使舱内气压升高到2.5倍大气压，维持1.5小时后，缓慢放气使舱内气压降至大气压。高压氧治疗结束后间隔两小时后记为第0小时，并通过尾静脉注射给予PD-1抑制 剂组和HBO+PD-1抑制剂组小鼠0.1mg/0.1mL Dylight荧光标记PD-1抑制剂。使用小动物成像***在第0、0.5、1、2、4、8、12、24、48、72小时分别对小鼠进行成像，检测PD-1抑制剂在小鼠体内的分布，并在第48小时处死一部分小鼠，分离心肝脾肺肾和肿瘤，使用小动物成像***检测PD-1抑制剂在小鼠各组织中的分布情况。 A mouse liver cancer H22 subcutaneous tumor model was established. When the subcutaneous tumor grew to a volume of 250 mm, the tumor - bearing mice were randomly divided into two groups (PD-1 inhibitor group and HBO+PD-1 inhibitor group) and administered HBO The mice in the +PD-1 inhibitor group were treated with hyperbaric oxygen. The treatment method was to place the mice in an airtight chamber, and gradually introduce pure oxygen to increase the air pressure in the chamber to 2.5 times the atmospheric pressure. After maintaining for 1.5 hours, the air was slowly deflated. Reduce the cabin pressure to atmospheric pressure. The two-hour interval after the end of hyperbaric oxygen therapy was recorded as the 0th hour, and 0.1 mg/0.1 mL of Dylight fluorescently labeled PD-1 inhibitor was administered to the mice in the PD-1 inhibitor group and HBO+PD-1 inhibitor group by tail vein injection. . Using a small animal imaging system, the mice were imaged at 0, 0.5, 1, 2, 4, 8, 12, 24, 48, and 72 hours to detect the distribution of PD-1 inhibitors in mice, and at the first Part of the mice were sacrificed at 48 hours, and the heart, liver, spleen, lung, kidney and tumors were separated, and the distribution of PD-1 inhibitors in various tissues of the mice was detected using a small animal imaging system.
图24为小动物成像检测到PD-1抑制剂在活体小鼠体内分布随时间的变化以及离体组织中的分布情况。从图中可以看出，在第0小时，两个组小鼠均无本底荧光信号，在注射荧光标记的PD-1抑制剂之后，随时间变化，小鼠体内逐渐出现不同强弱的荧光信号，HBO联合PD-1抑制剂组小鼠肿瘤部位PD-1抑制剂的荧光信号在不同时间点均强与单用PD-1抑制剂组。48小时的组织分布图同样显示HBO联合PD-1抑制剂处理组小鼠肿瘤组织中PD-1抑制剂的荧光信号强于单用PD-1抑制剂组。Figure 24 shows the time-dependent changes in the distribution of PD-1 inhibitors in living mice and the distribution in isolated tissues detected by small animal imaging. It can be seen from the figure that at the 0th hour, there was no background fluorescence signal in the two groups of mice. After the injection of the fluorescently labeled PD-1 inhibitor, different intensities of fluorescence gradually appeared in the mice over time. Signal, the fluorescence signal of PD-1 inhibitor in tumor site of mice in HBO combined with PD-1 inhibitor group was stronger than that in single PD-1 inhibitor group at different time points. The tissue distribution map at 48 hours also showed that the fluorescence signal of PD-1 inhibitor in tumor tissue of HBO combined with PD-1 inhibitor treatment group was stronger than that of PD-1 inhibitor alone group.
图25为小动物成像检测到PD-1抑制剂在活体小鼠肿瘤组织分布随时间的变化的定量结果。从图中可以看出，随着时间的延长，PD-1抑制剂在各组小鼠肿瘤组织中的富集逐渐增多，其中，第0到24小时为快速富集阶段，随后富集速度逐渐减慢。而在整个过程中，HBO联合PD-1抑制剂治疗组PD-1抑制剂在肿瘤部位的富集量均显著高于单用PD-1抑制剂组，HBO处理将PD-1抑制剂在肿瘤的平均富集量提高了35％左右。Figure 25 shows the quantitative results of the time-dependent changes in the distribution of PD-1 inhibitors in the tumor tissue of living mice detected by small animal imaging. It can be seen from the figure that with the prolongation of time, the enrichment of PD-1 inhibitors in the tumor tissues of each group of mice gradually increased. slow down. During the whole process, the enrichment of PD-1 inhibitors in tumor sites in the HBO combined with PD-1 inhibitor treatment group was significantly higher than that in the single PD-1 inhibitor group. The average enrichment was increased by about 35%.
图26为小动物成像检测的PD-1抑制剂组织分布定量结果。从图中可以看出，与单用PD-1抑制剂组相比，联合治疗组肿瘤部位PD-1抑制剂的富集量显著增多，而脾脏部位的富集量显著减少，其余各器官富集量无显著性差异，结果表明HBO可以显著提高PD-1抑制剂在小鼠肿瘤部位的富集。Figure 26 shows the quantitative results of tissue distribution of PD-1 inhibitors detected by small animal imaging. It can be seen from the figure that compared with the PD-1 inhibitor group alone, the enrichment of PD-1 inhibitor in the tumor site in the combined treatment group was significantly increased, while the enrichment in the spleen was significantly decreased, and the rest of the organs were enriched There was no significant difference in the amount of accumulation, and the results indicated that HBO could significantly increase the accumulation of PD-1 inhibitors in mouse tumor sites.
结合以上动物实验、血生化分析、组织切片和药物体内递送与分布的实验结果，可以看出，HBO联合PD-1抑制剂可以在不增加毒副作用的同时提高抗肿瘤治疗效果、抑制肿瘤转移、延长小鼠生存期并提高PD-1抑制剂在小鼠肿瘤部位的富集。Combined with the experimental results of the above animal experiments, blood biochemical analysis, tissue sections, and drug delivery and distribution in vivo, it can be seen that HBO combined with PD-1 inhibitors can improve the anti-tumor therapeutic effect, inhibit tumor metastasis, and improve the Prolonged mouse survival and enhanced PD-1 inhibitor enrichment in mouse tumor sites.
最后，本发明的方法仅为较佳的实施方案，并非用于限定本发明的保护范围。凡在本发明的精神和原则之内，所作的任何修改、等同替换、改进等，均应包含在本发明的保护范围之内。Finally, the method of the present invention is only a preferred embodiment, and is not intended to limit the protection scope of the present invention. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included within the protection scope of the present invention.

Claims (10)

  1. 一种高压氧联合PD-1抑制剂用于肿瘤免疫治疗的方法，其特征在于，通过将高压氧治疗与PD-1抑制剂治疗相结合，对肿瘤患者进行抗肿瘤治疗，具体包括如下步骤：A method for using hyperbaric oxygen combined with PD-1 inhibitor for tumor immunotherapy, characterized in that, by combining hyperbaric oxygen therapy and PD-1 inhibitor therapy, anti-tumor therapy is performed on tumor patients, which specifically includes the following steps:
    (1)对肿瘤患者进行高压氧治疗；所述高压氧治疗通过增加肿瘤患者所处环境的气压及吸入氧气的浓度来提高溶解于血液中氧气的量，从而提高各组织中氧的灌注；(1) Hyperbaric oxygen therapy is performed on the tumor patient; the hyperbaric oxygen therapy increases the amount of oxygen dissolved in the blood by increasing the air pressure in the environment where the tumor patient is located and the concentration of inhaled oxygen, thereby improving the perfusion of oxygen in each tissue;
    (2)对肿瘤患者静脉注射PD-1抑制剂。(2) Intravenous injection of PD-1 inhibitor to tumor patients.
  2. 如权利要求1所述的方法，其特征在于，对肿瘤患者进行单次或多次高压氧治疗后，再对肿瘤患者静脉注射PD-1抑制剂。The method according to claim 1, characterized in that after single or multiple hyperbaric oxygen therapy is performed on the tumor patient, the tumor patient is then intravenously injected with a PD-1 inhibitor.
  3. 如权利要求2所述的方法，其特征在于，对肿瘤患者进行高压氧治疗包括如下步骤：将肿瘤患者置于密闭舱体内，逐渐通入纯氧使舱内气压升高到2-5倍大气压，维持一段时间后，放气使舱内气压降至大气压。The method according to claim 2, wherein the hyperbaric oxygen therapy for the tumor patient comprises the following steps: placing the tumor patient in an airtight chamber, gradually introducing pure oxygen to increase the air pressure in the chamber to 2-5 times atmospheric pressure , after maintaining for a period of time, deflate to reduce the pressure in the cabin to atmospheric pressure.
  4. 如权利要求2所述的方法，其特征在于，将肿瘤患者置于密闭舱体内，逐渐通入纯氧使舱体内气压升高到2-5倍大气压，维持1-4小时后，缓慢放气使舱体内气压降至大气压。The method of claim 2, wherein the tumor patient is placed in an airtight chamber, and pure oxygen is gradually introduced to increase the air pressure in the chamber to 2-5 times atmospheric pressure, and after maintaining for 1-4 hours, the air is slowly deflated. Reduce the pressure inside the cabin to atmospheric pressure.
  5. 如权利要求1所述的方法，其特征在于，所述高压氧治疗与静脉注射PD-1抑制剂之间的间隔时间为0-12小时。The method of claim 1, wherein the interval between the hyperbaric oxygen therapy and the intravenous injection of the PD-1 inhibitor is 0-12 hours.
  6. 如权利要求1所述的方法，其特征在于，静脉注射PD-1抑制剂之前或之后，对肿瘤患者给予1-10次高压氧治疗。The method of claim 1, wherein the tumor patient is given 1-10 times of hyperbaric oxygen therapy before or after the intravenous injection of the PD-1 inhibitor.
  7. 如权利要求1所述的方法，其特征在于，每一至三周对肿瘤患者静脉注射PD-1抑制剂一次，其单次注射剂量为100-300mg/m 2。 The method of claim 1, wherein the tumor patient is intravenously injected with the PD-1 inhibitor once every to three weeks, and the single injection dose is 100-300 mg/m 2 .
  8. 如权利要求7所述的方法，其特征在于，重复步骤(1)和步骤(2)以完成一次治疗，持续治疗不少于1次。The method of claim 7, wherein steps (1) and (2) are repeated to complete one treatment, and the treatment is continued for no less than one time.
  9. 如权利要求1所述的方法，其特征在于，所述PD-1抑制剂为Keytruda、Opdivo、Libtayo、拓益、达伯舒或艾立妥。The method of claim 1, wherein the PD-1 inhibitor is Keytruda, Opdivo, Libtayo, Tuoyi, Dabosu or Eritor.
  10. 如权利要求1所述的方法，其特征在于，所述肿瘤为实体瘤。The method of claim 1, wherein the tumor is a solid tumor.
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