WO2022151335A2 - Méthode de traitement d'une tumeur au moyen d'oxygène hyperbare associé à un médicament immunologique - Google Patents

Méthode de traitement d'une tumeur au moyen d'oxygène hyperbare associé à un médicament immunologique 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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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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PCT/CN2021/072094 2021-01-15 2021-01-15 Méthode de traitement d'une tumeur au moyen d'oxygène hyperbare associé à un médicament immunologique WO2022151335A2 (fr)

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