Pre-carbon monoxide saturated hemoglobin oxygen carrier preparation, preparation method thereof and application of pre-carbon monoxide saturated hemoglobin oxygen carrier preparation in pulmonary fibrosis resistance

Abstract

The invention relates to the technical field of medicines, in particular to a pre-carbon monoxide saturated hemoglobin oxygen carrier preparation as well as a preparation method and application thereof in resisting pulmonary fibrosis. The preparation method of the pre-carbon monoxide saturated hemoglobin oxygen carrier preparation comprises the following steps: carrying out carbon monoxide saturation treatment on the hemoglobin oxygen carrier preparation; the carbon monoxide saturation treatment comprises the step of introducing carbon monoxide for 15-25 minutes at the temperature of 2-6 DEG C and the speed of 0.5-1.5 L / min. The pre-carbon monoxide saturated hemoglobin oxygen carrier preparation can significantly reduce bleomycin induced pulmonary edema and injury, improve the pulmonary hypoxia state and inhibit the pulmonary fibrosis process, and provides a brand new treatment strategy for clinical treatment of pulmonary fibrosis.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of medicine, in particular to a pre-carbon monoxide saturated hemoglobin oxygen carrier preparation, a preparation method thereof and application thereof in anti-pulmonary fibrosis. BACKGROUND

[0002] The prevalence and incidence of pulmonary fibrosis have significantly increased in the past few decades, becoming an issue that cannot be ignored in lung diseases. Pulmonary fibrosis is the end-stage change of many chronic interstitial lung diseases and lung cancer, which causes irreversible pulmonary fibrosis lesions due to the excessive deposition of extracellular matrix and collagen, which destroys the normal lung tissue structure. A significant clinical feature of pulmonary fibrosis is dyspnea and progressive hypoxemia, and the end-stage patients die of progressive respiratory distress and respiratory failure.

[0003] So far, the pathophysiology of pulmonary fibrosis has not been fully elucidated, and there is a large gap in clinical medication. Some approved drugs on the market cannot effectively delay the fibrosis process of lung lesions, and have no obvious improvement on the symptoms of hypoxemia or dyspnea caused by pulmonary fibrosis.

[0004] Therefore, the present application is proposed. SUMMARY

[0005] The present application aims to provide a pre-carbon monoxide saturated hemoglobin oxygen carrier preparation that precisely matches the "internal hypoxia-oxidative stress" double pathological characteristics of pulmonary fibrosis, and simultaneously discloses a preparation method thereof and application thereof in anti-pulmonary fibrosis.

[0006] In order to achieve the above-mentioned purpose of the present application, the first aspect of the present application provides a preparation method of a pre-carbon monoxide saturated hemoglobin oxygen carrier preparation, comprising the following steps: performing carbon monoxide saturation treatment on the hemoglobin oxygen carrier preparation; the carbon monoxide saturation treatment comprises: passing carbon monoxide at a speed of 0.5-1.5 L / min for 15-25 min at 2-6℃.

[0007] In the specific embodiment of the present application, the hemoglobin oxygen carrier in the hemoglobin oxygen carrier preparation is a cross-linked bovine hemoglobin.

[0008] In the specific embodiment of the present application, the hemoglobin oxygen carrier preparation further comprises a solvent and an additive. Further, the hemoglobin oxygen carrier preparation comprises the following components based on 1 L of water for injection as the solvent: Hemoglobin oxygen carrier: 100-150 g Antioxidant: 1-2 g Sodium chloride: 4.0-4.5 g Potassium chloride: 0.25-0.35 g Calcium gluconate: 0.45-0.55 g Sodium bicarbonate: 1.0-1.5 g Sodium hydroxide. The amount of sodium hydroxide is limited to adjust the pH of the preparation to 7.3-7.4.

[0009] In the detailed description of the present application, the antioxidant includes at least one of ascorbic acid, superoxide dismutase, catalase, vitamin E and glutathione.

[0010] In the detailed description of the present application, the carbon monoxide saturation treatment is performed in a saturation device. Further, the carbon monoxide saturation treatment includes: introducing nitrogen gas to the saturation device to remove air, and then placing the hemoglobin oxygen carrier preparation in the saturation device, and introducing carbon monoxide at a speed of 0.5-1.5 L / min for 15-25 min under stirring at 2-6℃.

[0011] In the detailed description of the present application, the amount of the hemoglobin oxygen carrier preparation in the carbon monoxide saturation treatment is 80-120 mL.

[0012] The second aspect of the present application provides a pre-carbon monoxide saturated hemoglobin oxygen carrier preparation, which is prepared by any one of the preparation methods provided in the first aspect of the present application.

[0013] In the detailed description of the present application, in the pre-carbon monoxide saturated hemoglobin oxygen carrier preparation, the concentration of hemoglobin is 10-15 gHb / dL, the carbon monoxide saturation is 90%-95%, and the content of methemoglobin (MetHb) is <5%.

[0014] The third aspect of the present application provides the use of the pre-carbon monoxide saturated hemoglobin oxygen carrier preparation of the first aspect of the present application in the preparation of a product for preventing and / or treating pulmonary fibrosis.

[0015] In the detailed description of the present application, the product has at least one of the following uses: (1) preventing and / or treating bleomycin-induced pulmonary edema; (2) preventing and / or treating bleomycin-induced lung injury; (3) preventing and / or treating bleomycin-induced pulmonary fibrosis collagen deposition; (4) preventing and / or treating bleomycin-induced pulmonary hypoxia and circulatory disorder of reoxygenation.

[0016] In the detailed description of the present application, the product is used for inhibiting the expression of N-cadherin in the lung tissue of an animal with bleomycin-induced pulmonary fibrosis; the product is used for promoting the expression of E-cadherin in the lung tissue of an animal with bleomycin-induced pulmonary fibrosis.

[0017] In the detailed description of the present application, the product is used for inhibiting the oxidative stress injury induced by hypoxia in an animal with bleomycin-induced pulmonary fibrosis. Further, the product is used for increasing the content of superoxide dismutase in the lung tissue of the animal; the product is used for decreasing the content of malondialdehyde in the lung tissue of the animal.

[0018] In the detailed description of the present application, the product is used for improving the exercise tolerance of an animal with bleomycin-induced pulmonary fibrosis.

[0019] In the detailed description of the present application, the product is used for increasing the arterial partial pressure of oxygen of an animal with bleomycin-induced pulmonary fibrosis.

[0020] The fourth aspect of the present application further provides a pharmaceutical composition comprising the pre-carbon monoxide saturated hemoglobin oxygen carrier preparation provided by the second aspect of the present application. Further, the pharmaceutical composition further comprises a pharmaceutical excipient.

[0021] In the detailed description of the present application, the pharmaceutical composition comprises at least one of a liquid preparation, a tablet, a capsule and a lyophilized powder.

[0022] Compared with the prior art, the present application has the following beneficial effects: The present application can be used for preventing and / or treating pulmonary fibrosis by specifically saturating the hemoglobin oxygen carrier preparation with carbon monoxide, and provides a brand-new treatment strategy for the clinical treatment of pulmonary fibrosis. BRIEF DESCRIPTION OF DRAWINGS

[0023] In order to more clearly illustrate the technical solutions in the embodiments of the present application or the prior art, the drawings needed in the description of the embodiments or the prior art will be briefly introduced. Obviously, the drawings in the following description are some embodiments of the present application, and other drawings can be obtained by those skilled in the art without creative labor.

[0024] Figure 1 The oxygen release conditions of hemoglobin and hemoglobin oxygen carriers provided by the embodiments of the present application are compared; wherein (A) is the oxygen release capacity test result, and (B) is the oxygen release speed test result; Figure 2Test results of the pre-carbon monoxide saturated hemoglobin oxygen carrier preparation provided by the embodiments of the present application in reducing bleomycin-induced pulmonary edema and lung injury in rats; wherein (A) is rat double lung morphology and pulmonary edema analysis, (B) is rat lung HE, Masson staining analysis; Figure 3 The pre-carbon monoxide saturated hemoglobin oxygen carrier preparation provided by the embodiments of the present application inhibits the pathological process of bleomycin-induced pulmonary fibrosis in rats; wherein (A) is the content analysis of MDA and SOD in rat lung, (B) is the mRNA expression of N-cadherin and E-cadherin in rat lung; Figure 4 The pre-carbon monoxide saturated hemoglobin oxygen carrier preparation provided by the embodiments of the present application has an effect on the motor function of bleomycin-induced pulmonary fibrosis rats; Figure 5 The pre-carbon monoxide saturated hemoglobin oxygen carrier preparation provided by the embodiments of the present application has an effect on the pulmonary oxygenation of bleomycin-induced pulmonary fibrosis rats; wherein (A) is the analysis of rat lung ventilation function, (B) is the analysis of arterial blood gas and oxygen. DETAILED DESCRIPTION

[0025] The technical solutions of the present application will be described clearly and completely in combination with the drawings and specific embodiments, but those skilled in the art will understand that the following described embodiments are part of the embodiments of the present application, not all the embodiments, and are only used to illustrate the present application, and should not be regarded as limiting the scope of the present application. Based on the embodiments in the present application, all other embodiments obtained by those of ordinary skill in the art without creative labor are within the scope of protection of the present application. The specific conditions not mentioned in the embodiments are carried out according to the conventional conditions or the conditions recommended by the manufacturer. The reagents or instruments not mentioned by the manufacturer are all conventional products that can be purchased on the market.

[0026] Currently, the clinical treatment options for pulmonary fibrosis are limited, especially for the symptoms of hypoxemia and dyspnea caused by the disease, and the existing drugs still have unsatisfactory improvement effect. Hypoxemia caused by pulmonary fibrosis is manifested as a decrease in arterial oxygen partial pressure, which is due to the impaired oxygen binding and release function of hemoglobin in fibrotic lung tissue. The destruction of lung tissue and alveolar structure in patients with pulmonary fibrosis leads to a significant decrease in local oxygen partial pressure (PO2) in the lung (the PO2 in the lesion area is often as low as 30-60 mmHg), and the normal human hemoglobin is prone to "premature oxygen release" or "insufficient oxygen binding capacity" in a low-oxygen environment, which cannot effectively transport oxygen to the hypoxic lesion; at the same time, the oxidative stress damage (excess active oxygen) of the lesion further damages the oxygen-carrying structure of hemoglobin, exacerbating hypoxemia. In view of this pathological feature, improving the oxygen affinity of hemoglobin and optimizing its oxygen-carrying capacity, while having anti-inflammatory and antioxidant functions, can help alleviate the respiratory distress, limited activity, and low quality of life of patients with pulmonary fibrosis caused by lung tissue edema and damage.

[0027] In addition, the occurrence and development of many lung diseases are often accompanied by local or systemic hypoxia, so the development of efficient oxygen carriers to achieve specific oxygen delivery to the lesion area has become a promising treatment direction. Hemoglobin (Hb) as the main oxygen transport protein in red blood cells, with the ability of reversible binding of oxygen molecules by iron ions in the heme group, plays a core role in oxygen transport in the body. Natural hemoglobin is a tetrameric protein of about 64 kilodaltons, composed of two pairs of αβ dimers. However, free hemoglobin is easily dissociated into dimers in the plasma, with a short half-life of only a few hours. Through cross-linking, polymerization or molecular modification strategies, its stability, circulation half-life and biological safety can be significantly enhanced.

[0028] This study focuses on hemoglobin-based oxygen carriers (HBOC), which have high oxygen binding and release capacity and can be used as a correction agent for tissue hypoxia. Further pre-carbon monoxide (CO) saturation treatment of HBOC can integrate the anti-inflammatory and antioxidant effects of CO, thereby synergistically regulating the inflammatory response and oxidative stress level on the basis of improving oxygenation, providing a dual mechanism strategy for the intervention of pulmonary fibrosis. Previously, the oxygen delivery system based on HBOC has shown potential applications in diseases such as traumatic hemorrhagic shock, hemolytic anemia, myocardial infarction, and peripheral ischemia. However, there have been no systematic reports on its application in the prevention and treatment of pulmonary fibrosis.

[0029] The present application aims at the current unsatisfied clinical needs, especially the oxygen supply disorder, hypoxemia, insufficient exercise ability and other problems caused by pulmonary edema and lung injury of patients with pulmonary fibrosis, and provides a new pre-CO saturated hemoglobin oxygen carrier preparation and a preparation method thereof.

[0030] Reversible binding mechanism: through the directional release of pre-saturated CO in the lung lesion area, the role of anti-oxidative stress and inhibition of inflammatory damage (blocking the starting link of fibrosis) is played, and in the high-oxygen environment in the lung, CO is competitively dissociated to vacate the oxygen binding sites of the hemoglobin oxygen carrier. The conflict between the anti-inflammatory component and the oxygen carrying function is fundamentally solved, and the dynamic synergy of "carbon monoxide anti-inflammatory" and "hemoglobin oxygen carrier oxygen supply" is realized. Targeted adaptation to the high-oxygen affinity design of the hypoxic lesion: the hemoglobin oxygen carrier itself has a higher oxygen affinity than natural hemoglobin, can stably bind oxygen in the pulmonary fibrosis lesion (due to the sudden drop of oxygen partial pressure in the lung caused by collagen deposition and edema), and slowly and continuously releases oxygen to the hypoxic tissue to provide long-acting oxygen supply support for the lesion. Synergistic effect of double mechanisms: "CO anti-inflammatory protection" and "hemoglobin oxygen carrier high-efficiency oxygen supply" form a closed loop. CO reduces lung damage and improves the integrity of alveolar capillary structure, which clears the physical obstacles for oxygen transport of hemoglobin oxygen carrier; hemoglobin oxygen carrier improves the hypoxic state of the lesion and relieves hypoxia-induced fibrosis progression. The two can effectively relieve pulmonary edema and tissue damage induced by bleomycin and reverse the pathological process of pulmonary fibrosis.

[0031] The present application provides a preparation method of pre-CO saturated hemoglobin oxygen carrier preparation, comprising the following steps: CO saturation treatment is performed on the hemoglobin oxygen carrier preparation; the CO saturation treatment comprises: passing CO gas at a speed of 0.5-1.5 L / min for 15-25 min at 2-6℃.

[0032] The present application performs specific CO saturation treatment on HBOC, and explores the saturation treatment conditions. The CO saturation treatment involves the influence of multiple variables (CO gas flow rate, saturation time, saturation temperature) on saturation efficiency (%COHb) and protein stability (%MetHb). The hemoglobin oxygen carrier preparation not only has excellent oxygen affinity, but also improves the efficiency of hemoglobin oxygen delivery, so that its oxygen release function can provide effective oxygen transfer for hypoxic tissues, while having anti-inflammatory and anti-oxidative stress effects. The requirements for treating pulmonary fibrosis are met.

[0033] As in different embodiments, the flow rate of CO gas in the CO saturation treatment can be 0.5 L / min, 0.8 L / min, 1.0 L / min, 1.2 L / min, 1.5 L / min, or a range consisting of any two of them; the time for passing the CO gas can be 15 min, 18 min, 20 min, 22 min, 25 min, or a range consisting of any two of them; the temperature can be 2℃, 3℃, 4℃, 5℃, 6℃, or a range consisting of any two of them. Regulating the flow rate of CO gas, the time for passing the gas, and the temperature in the saturation treatment within the above ranges can effectively balance the CO content of the preparation after the saturation treatment and the treatment cost, etc. In a preferred embodiment, in the saturation treatment, the CO gas is passed at a speed of 1 L / min for 20 min at 4℃ under stirring conditions.

[0034] HBOC is a red blood cell substitute with oxygen carrying capacity, which is prepared by chemical modification such as cross-linking, polymerization, coupling, or encapsulation in an artificially synthesized membrane, using Hb as raw material. The type or preparation method of HBOC is not specifically limited in the present application, and any existing HBOC with oxygen carrying capacity in the art can be used to implement the technical solutions of the present application. In the specific embodiments of the present application, the hemoglobin oxygen carrier in the hemoglobin oxygen carrier preparation is bovine hemoglobin treated by cross-linking, wherein the cross-linking agent used in the cross-linking treatment includes but is not limited to glutaraldehyde, succinimidyl ester cross-linking agent, polyethylene glycol diglycidyl ether, etc. In some embodiments, the hemoglobin oxygen carrier preparation includes but is not limited to glutaraldehyde cross-linked bovine hemoglobin.

[0035] In the specific embodiments of the present application, the hemoglobin oxygen carrier preparation further includes a solvent and an additive. Further, the hemoglobin oxygen carrier preparation includes, based on 1 L of water for injection as the solvent: 100-150 g of hemoglobin oxygen carrier, 1-2 g of antioxidant, 4.0-4.5 g of sodium chloride, 0.25-0.35 g of potassium chloride, 0.45-0.55 g of calcium gluconate, 1.0-1.5 g of sodium bicarbonate, and an appropriate amount of sodium hydroxide. The amount of sodium hydroxide is limited to adjusting the pH of the preparation to 7.3-7.4.

[0036] In the hemoglobin oxygen carrier preparation of the present application, the introduction of part of the antioxidant can reduce the autoxidation phenomenon of HBOC exposed to the circulatory system, so that the preparation achieves a suitable circulatory persistence to allow effective oxygenation of tissues and provide effective oxygen delivery to hypoxic tissues.

[0037] In the specific embodiments of the present application, the antioxidant includes at least one of ascorbic acid, superoxide dismutase, catalase, vitamin E, and glutathione.

[0038] In the specific embodiment of the present application, the CO saturation treatment comprises: introducing nitrogen gas to the saturation device at a ventilation rate of 0.5-1 L / min to remove air, and then placing the hemoglobin oxygen carrier preparation in the saturation device, and introducing CO gas at a rate of 0.5-1.5 L / min at 2-6℃ under stirring for 15-25 min.

[0039] In actual operation, the stirring speed and degree are adjusted according to the state of the hemoglobin oxygen carrier preparation, for example, a clear and stable vortex can be formed in the hemoglobin oxygen carrier preparation in the saturation device, but it is not limited thereto.

[0040] In the specific embodiment of the present application, the amount of the hemoglobin oxygen carrier preparation used in the CO saturation treatment is 80-120 mL.

[0041] The second aspect of the present application provides a pre-CO saturated hemoglobin oxygen carrier preparation prepared by any one of the preparation methods provided in the first aspect of the present application.

[0042] In the specific embodiment of the present application, in the pre-CO saturated hemoglobin oxygen carrier preparation, the concentration of hemoglobin is 10-15 g Hb / dL, such as 10 g Hb / dL, 12 g Hb / dL, 13 g Hb / dL, 15 g Hb / dL, or a range consisting of any two thereof; the CO saturation degree is 90%-95%, such as 90%, 92%, 93%, 95%, or a range consisting of any two thereof; and the content of methemoglobin (MetHb) is <5%.

[0043] The third aspect of the present application provides the use of the pre-CO saturated hemoglobin oxygen carrier preparation of the first aspect of the present application in the preparation of a product for preventing and / or treating pulmonary fibrosis.

[0044] In the specific embodiment of the present application, the product has at least one of the following uses: (1) preventing and / or treating bleomycin-induced pulmonary edema; (2) preventing and / or treating bleomycin-induced lung injury; (3) preventing and / or treating bleomycin-induced pulmonary fibrosis collagen deposition; (4) preventing and / or treating bleomycin-induced pulmonary hypoxia and circulatory disorder of reoxygenation.

[0045] Bleomycin (BLM) is the most widely used drug for inducing animal pulmonary fibrosis models. The manifestations of pulmonary edema, pneumonia, collagen fiber deposition caused by the lung toxicity of BLM are similar to the pathological manifestations of pulmonary fibrosis in clinical patients.

[0046] In the detailed description of the present application, the product is used to inhibit the expression of N-cadherin in the lung tissue of BLM-induced pulmonary fibrosis animal; the product is used to promote the expression of E-cadherin in the lung tissue of BLM-induced pulmonary fibrosis animal.

[0047] In the detailed description of the present application, the product is used to inhibit the oxidative stress injury induced by hypoxia in the animal of BLM-induced pulmonary fibrosis. Further, the product is used to increase the content of superoxide dismutase in the lung tissue of the animal, and to reduce the content of malondialdehyde in the lung tissue of the animal.

[0048] In the detailed description of the present application, the product is used to improve the exercise tolerance of the animal of BLM-induced pulmonary fibrosis.

[0049] In the detailed description of the present application, the product is used to increase the arterial partial pressure of oxygen of the animal of BLM-induced pulmonary fibrosis.

[0050] In the present application, the animal is exemplified by a rat, but is not limited thereto.

[0051] The fourth aspect of the present application further provides a pharmaceutical composition comprising the pre-CO saturated hemoglobin oxygen carrier preparation provided by the second aspect of the present application. Further, the pharmaceutical composition further comprises a pharmaceutical excipient.

[0052] In the detailed description of the present application, the pharmaceutical composition comprises at least one of a liquid preparation, a tablet, a capsule and a lyophilized powder.

[0053] Example 1 Evaluation of oxygen carrying / carrying capacity of HBOC and Hb-based oxygen carrier 1. Sample preparation: The preparation of the hemoglobin oxygen carrier preparation comprises: dissolving 130.0 g of HBOC (Biopure), 1.5 g of ascorbic acid, 4.2 g of sodium chloride, 0.30 g of potassium chloride, 0.50 g of calcium gluconate, 1.2 g of sodium bicarbonate in about 900 mL of water for injection. An appropriate amount of sodium hydroxide solution (such as 1M NaOH) is used to adjust the pH to 7.3-7.4 (such as 7.4), and finally the volume is adjusted to 1 L, and then stirred uniformly and filtered through a 0.22 μm filter. The osmotic pressure is controlled at 280-320 mOsm / kg.

[0054] The preparation of the control group formulation includes: dissolving Hb (purified from bovine origin hemoglobin) 130.0 g, ascorbic acid 1.5 g, sodium chloride 4.2 g, potassium chloride 0.30 g, calcium gluconate 0.50 g, sodium bicarbonate 1.2 g in about 900 mL of water for injection. Adjust the pH to 7.3-7.4 (such as 7.4) using an appropriate amount of sodium hydroxide solution (such as 1M NaOH), and finally adjust the volume to 1L, stir uniformly, and then filter sterilize through a 0.22 μm filter membrane. The osmotic pressure is controlled at 280-320 mOsm / kg.

[0055] 2. Sample detection ① Physical and chemical property detection The concentration of the sample was determined by ABL800FLEX blood gas analyzer of Radiometer Company; the pH value was determined by pH meter of Mettler Toledo Company; the osmotic pressure was determined using a freezing point osmometer; the methemoglobin content was determined by a commercial kit (Nanjing Jiancheng Biological Engineering Institute).

[0056] ② Oxygen release capacity and oxygen release rate detection BLOODOX-2018Analyser was used to measure and depict the oxygen dissociation curve of the sample, analyze the theoretical oxygen release capacity of the sample, and calculate the Hill coefficient. The spectral characteristic peaks of hemoglobin (Soret and Q wave bands, 400-450 nm and 500-600 nm, respectively) can be used to measure the deoxyhemoglobin content or the oxygenated hemoglobin content produced during the deoxygenation process. In a 96-well plate, add the sample (containing human hemoglobin 1.5 mg / mL, 100 μL / well, 3 replicates per group). The enzyme marker detection chamber is balanced with nitrogen gas in an oxygen environment, set to 1.0% oxygen, and the sample is deoxygenated at 37°C for 2 h. During this period, the ultraviolet spectrophotometry of the enzyme marker was used to detect the absorbance values of each sample at 540 nm, 560 nm, 567 nm, 630 nm, and 700 nm every 2 min, and the oxygenated hemoglobin content of the sample at each detection was calculated.

[0057] ③ Data processing SPSS 26.0 statistical software was used for data processing, and the experimental results were expressed as mean ± standard deviation. Mann-Whitney U test was used for pairwise comparison between groups, p <0.05 was considered statistically significant.

[0058] 3. Detection results ① According to Figure 1The results in (A) show that the P50 values ​​displayed by OECs reflect the oxygen affinity of the HBOC formulation. The P50 values ​​for the HBOC formulation and the Hb control were 20.11 ± 1.18 mmHg and 28.23 ± 0.56 mmHg, respectively. p <0.001), indicating that the HBOC formulation has a lower P50 and stronger oxygen affinity.

[0059] ②According to Figure 1 (B) The test results showed that after full oxygenation, both the HBOC and Hb preparations were deoxygenated in a hypoxic environment (1.0% oxygen) for 2 hours, and the percentage of oxyhemoglobin was periodically and quantitatively measured during the deoxygenation process. The results showed that after 2 hours, the oxygen-carrying capacity of oxyhemoglobin in the Hb preparation decreased by more than 50%. However, approximately 60% of the oxyhemoglobin in the HBOC preparation remained in an oxygenated state, significantly higher than that in the Hb preparation. p <0.05).

[0060] 4. Conclusion The test results above show that HBOC has better oxygen carrying / releasing capacity and can increase the relative high oxygen partial pressure state of hypoxic tissues.

[0061] Example 2 Investigation of saturation conditions for pre-CO-saturated hemoglobin oxygen carrier formulations 1. Sample preparation: (1) Preparation of hemoglobin oxygen carrier preparation includes: dissolving 130.0 g of HBOC, 1.5 g of ascorbic acid, 4.2 g of sodium chloride, 0.30 g of potassium chloride, 0.50 g of calcium gluconate, and 1.2 g of sodium bicarbonate in about 900 mL of water for injection. Adjust the pH to 7.3-7.4 (e.g., 7.4) with an appropriate amount of sodium hydroxide solution (e.g., 1M NaOH), and finally bring the volume to 1 L. After stirring evenly, filter the solution through a 0.22 μm filter membrane for sterilization.

[0062] (2) Setting CO saturation treatment conditions Experiment 1: Investigating the effect of gas mass transfer efficiency on saturation velocity. Too low a flow rate results in slow saturation; too high a flow rate may lead to waste, protein foaming, or shear denaturation.

[0063] Procedure: To the closed CO saturation apparatus, nitrogen gas was introduced at a rate of 1 L / min for 10 min to completely remove the air in the apparatus. 100 mL of the hemoglobin-based oxygen carrier preparation described above was placed in the saturation apparatus. Under the condition of 4°C ice bath and stirring to ensure that the liquid formed a clear vortex, medical grade CO was introduced into the solution at a rate of 1.0 L / min, and the aeration was maintained for 25 min. After reaching the predetermined time, the CO was stopped, and nitrogen gas was continued to be introduced to blow off the residual CO, and the timing was 5 min. The CO-saturated hemoglobin-based oxygen carrier preparation product of each pre-experiment 1 was obtained.

[0064] Experiment 2: Determine the shortest time required to reach saturation equilibrium to improve process efficiency.

[0065] Procedure: To the closed CO saturation apparatus, nitrogen gas was introduced at a rate of 1 L / min for 10 min to completely remove the air in the apparatus. 100 mL of the hemoglobin-based oxygen carrier preparation described above was placed in the saturation apparatus. Under the condition of 4°C ice bath and stirring to ensure that the liquid formed a clear vortex, medical grade CO was introduced into the solution at a rate of 1.0 L / min, and the aeration was maintained for 25 min. After reaching the predetermined time, the CO was stopped, and nitrogen gas was continued to be introduced to blow off the residual CO, and the timing was 5 min. The CO-saturated hemoglobin-based oxygen carrier preparation product of each pre-experiment 1 was obtained.

[0066] Experiment 3: Explore the effect of temperature on the binding rate of CO and hemoglobin and the stability of the protein.

[0067] Procedure: To the closed CO saturation apparatus, nitrogen gas was introduced at a rate of 1 L / min for 10 min to completely remove the air in the apparatus. 100 mL of the hemoglobin-based oxygen carrier preparation described above was placed in the saturation apparatus. Under the condition of 4°C ice bath and stirring to ensure that the liquid formed a clear vortex, medical grade CO was introduced into the solution at a rate of 1.0 L / min, and the aeration was maintained for 25 min. After reaching the predetermined time, the CO was stopped, and nitrogen gas was continued to be introduced to blow off the residual CO, and the timing was 5 min. The CO-saturated hemoglobin-based oxygen carrier preparation product of each pre-experiment 1 was obtained.

[0068] 2. Sampling points and detection items of each group From the beginning of stirring until there is a clear vortex, 1 mL was sampled at 0 min, 5 min, 10 min, 15 min, 20 min, 25 min (in experiment 2, only at the predetermined time). The sample was detected by the ABL800FLEX blood gas analyzer of Radiometer Company for %COHb, %MetHb, and recorded, with 6 repeats in each group. Under the optimal process conditions, %COHb should be in the range of 90-95%, and %MetHb should be <5%.

[0069] 3、Detection results Table 1 CO gas flow rate influence of experiment 1 (control variable: 4°C, 25 min)

[0070] The results of experiment 1 (Table 1) show that the CO saturation rate is significantly improved as the CO gas flow rate increases from 0.5 L / min to 1.0 L / min. However, the improvement is small when the flow rate continues to increase from 1.0 L / min, and tends to be flat. At the same time, a high flow rate (2.0 L / min) leads to %MetHb close to or reaching the critical value of 5% at 25 min, increasing the risk.

[0071] Table 2 Saturation time influence of experiment 2 (control variable: 4°C, 1.0 L / min)

[0072] The results of experiment 2 (Table 2) show that the saturation degree is insufficient (82.3%) at 10 min. It reaches 90.8% at 15 min, just entering the lower limit of the target range. It reaches 93.2% at 20 min, already meeting the requirements. It reaches 94.8% at 25 min, which is not significant compared to the change at 20 min. From the perspective of process efficiency, 20 min is the shortest effective time.

[0073] Table 3 Temperature influence of experiment 3 (control variable: 1.0 L / min, 25 min)

[0074] The results of experiment 3 (Table 3) show that the increase in temperature significantly accelerates the CO binding rate. However, the increase in temperature also dramatically accelerates the oxidation and denaturation of hemoglobin. The %MetHb under the treatment conditions of 25°C and 37°C far exceeds the limit of 5%, and the product is unqualified. Only under the condition of 4°C, both the high CO saturation degree (94.8%) and the production of MetHb (4.1%) can be controlled below 5%.

[0075] 4、Conclusion Based on the above three sets of experimental data, the optimal CO saturation process conditions are: Optimal CO gas flow rate: 1.0 L / min. The efficiency is significantly higher compared to 0.5 L / min; compared to 1.5 L / min and 2.0 L / min, there is no significant disadvantage in terms of saturation and time, and it can better control the risks of foam, gas waste, and protein shear denaturation (manifested as a milder increase in %MetHb).

[0076] Optimal saturation time: 20 min. 20 min has reached the standard (93.2%), and 25 min (94.8%) can provide more sufficient saturation guarantee and process tolerance space to ensure batch - to - batch consistency.

[0077] Optimal saturation temperature: 4°C. This is a decisive factor in controlling oxidative denaturation (%MetHb). 4°C is the condition that can simultaneously meet the two key quality indicators of %COHb > 90% and %MetHb < 5%.

[0078] In summary, to ensure the durability and scalability of the process, and to provide flexibility for subsequent production standard operating procedures; excellent saturation treatment can be obtained under the optimal process conditions: nitrogen is introduced into the saturation device at a ventilation rate of 0.5 - 1 L / min to remove air, then the hemoglobin oxygen carrier preparation is placed in the saturation device, and under stirring conditions at 2 - 6°C, the CO ventilation is maintained at a rate of 0.5 - 1.5 L / min for 15 - 25 min. Subsequently, nitrogen is purged at 1.0 L / min for 5 min.

[0079] Example 3 Pharmacodynamic experiment of pre - CO - saturated hemoglobin oxygen carrier preparation 1. Experimental materials The animals used in the experiment were SD rats (gender not limited, 6 - 8 weeks old, body weight 200 ± 20 g), with a clean - grade of specific - pathogen - free, purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd., and the animal certificate number was SCXK(Beijing)2019 - 0003. The animals were housed in the BioWork Yizhuang Animal Center platform, with constant temperature and humidity, and free access to food and water. Ethical approval number: 20230162.

[0080] The bleomycin (BLM) used in the experiment was purchased from Nippon Kayaku Co., Ltd. (batch number 450260). <000022_3>

[0081] 2. Experimental methods (1) Experiment implementation After the rats were anesthetized with isoflurane (induction: ventilation flow rate of 3 L / min, anesthetic concentration of 3 L / min; maintenance: ventilation flow rate of 0.5 L / min, anesthetic concentration of 2 L / min), they were fixed on a tracheal administration operation board, and the rats were in a supine position, and the glottis was exposed. The right hand was inserted into the rat in a horizontal manner at the throat, and when the needle tip reached the throat of the rat, the needle tail was pressed down, and the needle head was raised to 15 to 30 degrees of the original angle. And slowly insert the needle tube, until the rat trachea is about 3 / 4 (5 cm) in length, start injecting BLM, after injection, pull out the needle tube. After intratracheal administration of BLM, the rats were quickly erected and rotated to evenly distribute the BLM solution in the lungs. The sham operation group was intratracheally instilled with the same amount of normal saline for injection.

[0082] (2) Experimental grouping Day 1: BLM modeling of rats, day 7: pulmonary edema, pulmonary fibrosis and early pathological changes; day 8 to day 28: according to the grouping (see Table 4) to give HBOC and CO-HBOC, after weighing the body weight, the dosage is 200 μL / 20 g. After the end of administration, the animals were sacrificed on day 29, and samples were taken for biochemical and pathological index detection (see Table 5).

[0083] Among them, the hemoglobin oxygen carrier preparation (hereinafter referred to as "HBOC") is prepared according to the sample preparation step (1) of Example 2; Among them, the pre-CO saturated hemoglobin oxygen carrier preparation (hereinafter referred to as "CO-HBOC") is prepared according to the sample preparation step (1) of Example 2, and CO saturation is carried out under the following conditions: 10 min of nitrogen gas at a speed of 1.0 L / min is introduced into a closed CO saturation device to completely remove the air in the device; 100 mL of the above hemoglobin oxygen carrier preparation is placed in the saturation device, and the solution is stirred at a speed of 1.0 L / min under the condition of 4°C ice bath to form a vortex., introduce medical grade CO into the solution at a speed of 1.0 L / min, maintain ventilation for 20 min. After reaching the predetermined time, stop the CO, continue to blow the residual CO with 1.0 L / min of nitrogen gas, and count for 5 min.

[0084] Table 4 Administration grouping

[0085] Table 5 Sampling and detection

[0086] (3) Data processing SPSS 26.0 statistical software was used for data processing, and the experimental results were expressed as mean ± standard deviation. Mann-Whitney U test was used for pairwise comparison between groups,p <0.05 indicates a statistically significant difference.

[0087] 3. Experimental Results In this embodiment, SD rats were injected with BLM (6 mg / kg) via trachea. The drug intervention was carried out on the 7th day of BLM treatment, which is the early stage of pulmonary fibrosis. The effects of HBOC and CO-HBOC on alleviating BLM-induced lung damage and edema and inhibiting the pathological process of pulmonary fibrosis were detected.

[0088] (1) Gross morphology of the rat's lungs as follows Figure 2 As shown in Figure (A), the lungs of rats in the Control group were of normal size and volume, with a smooth, rosy surface and no obvious local congestion, and the lung surface moisture was moderate. Rats in the BLM group showed fibrotic deposition lesions in both lungs. Compared to the BLM group, the lung morphology of the BLM+HBOC group and the BLM+CO-HBOC group was slightly more normal. After observing the condition of the rats' lungs, the water content of the lungs was measured, and the results are as follows... Figure 2 As shown in (A)ii. The lung water content of the BLM group, BLM+HBOC group, and BLM+HBOC group was significantly higher than that of the Control group, and all differences were statistically significant. p <0.05. The lung water content in the BLM+HBOC group and the BLM+CO-HBOC group was lower than that in the BLM group, and the difference was statistically significant. p <0.05). This suggests that intervention with HBOC and CO-HBOC can significantly alleviate the pathological progression of BLM-induced pulmonary edema and pulmonary fibrosis in rats.

[0089] (2) Histopathological results as follows Figure 2 As shown in (B). HE staining is used to assess the degree of inflammation and damage in lung tissue. Figure 2 (B)i), HE results showed that: in the Control group, the alveolar structure of the lung tissue was uniform and fine, the alveolar septa were clear and intact, and there was no obvious congestion, edema, or inflammatory cell infiltration in the lung tissue; in the BLM group, the alveolar structure collapsed, the alveolar septa thickened, and there was inflammatory cell infiltration; compared with the BLM group, the alveolar structure of the BLM+HBOC group and the BLM+CO-HBOC group was significantly improved, the lung tissue structure was clear, and the inflammatory cell infiltration was reduced. Masson staining was used to assess the degree of fibrotic deposition in lung tissue ( Figure 2 (B)ii), Masson's results showed that compared with the Control group, the BLM group showed interstitial fibrosis, that is, the interstitial lung was replaced by collagen fibers and fibroblasts; the collagen fiber deposition in mice in the BLM+HBOC group and the BLM+CO-HBOC group was reduced compared with the BLM group, and the BLM+CO-HBOC group showed more significant improvement than the BLM+HBOC group.

[0090] The degree of pulmonary fibrosis was quantified according to the Ashcroft scoring system (see Table 6), and the score of the BLM group was significantly higher than that of the Control group p <0.05); compared with the BLM group, the scores of the BLM+HBOC group p <0.05) and the BLM+CO-HBOC group p <0.01) were significantly reduced.

[0091] Table 6 Comparison of the degree of fibrosis in lung tissue pathological sections in each group (mean ± standard deviation)

[0092] Note: compared with the Control group, * p <0.05,** p <0.01; compared with the BLM group, # p <0.05,## p <0.01.

[0093] (3) Pulmonary fibrosis causes insufficient oxygen supply to the tissue, and hypoxia leads to inhibition of mitochondrial oxidative phosphorylation, producing a large amount of reactive oxygen species (ROS), which can attack cell membranes, proteins and DNA, leading to cell damage and even death. There is an antioxidant and oxidative balance between superoxide dismutase (SOD) and malondialdehyde (MDA) in cells. Under hypoxic conditions, the activity of these antioxidant enzymes decreases, while the oxidation reaction is enhanced. This leads to a decrease in the body's ability to resist ROS and oxidative stress damage to cells. The oxygen supply state of lung tissue was determined by detecting the activity of antioxidant enzymes, and the results are shown in Figure 3 Figure A. HBOC and CO-HBOC can increase the SOD content of BLM-induced lung tissue and reduce the MDA content of BLM-induced lung tissue, and the difference is statistically significant.

[0094] (4) During the formation of pulmonary fibrosis, there is a loss or decrease in epithelial cell marker (E-cadherin), and an increase in interstitial cell marker (N-cadherin) due to lung interstitial damage and fibrosis deposition. The mRNA expression content of E-cadherin and N-cadherin was detected by qRT-PCR, and the results are shown in Figure 3 Figure B. HBOC and CO-HBOC can reduce the mRNA expression content of N-cadherin in BLM-induced lung tissue, and the difference is statistically significant. In addition, the mRNA expression content of E-cadherin is increased, and the difference is statistically significant.

[0095] 4. Conclusion In the present application, both hemoglobin-oxygen carrier preparation and pre-CO saturated hemoglobin-oxygen carrier preparation can significantly reduce lung inflammation and pulmonary edema caused by BLM in the early stage of pulmonary fibrosis; and the pre-CO saturated hemoglobin-oxygen carrier preparation can more significantly inhibit the pathological process of pulmonary fibrosis.

[0096] Example 4 Exploration of exercise benefits of pre-CO saturated hemoglobin-oxygen carrier preparation on pulmonary fibrosis rat model 1. Experimental materials The same as Example 3 2. Experimental method (1) Experimental implementation The same as Example 3 (2) Experimental grouping Day 1: BLM modeling was performed on rats, and significant fibrosis pathological changes occurred on day 21; from day 21 to day 41: according to the grouping (see Table 7), the rats were administered for 20 consecutive days, and the administration was performed after weighing the body weight, with the administration amount being 200 μL / 20 g. On day 42, after completing the lung function test, the animals were sacrificed, and blood was collected from the abdominal aorta for blood gas analysis.

[0097] Table 7 Administration grouping

[0098] (3) Exercise tolerance determination The exercise tolerance of rats was evaluated on day 0 (baseline determination), day 20 (after modeling), and day 41 (after administration), respectively. The determination method was weight-loaded swimming, and the specific steps were as follows: The rats were put into a swimming pool (length x width x height: 40 x 40 x 25 cm) for weight-loaded swimming, and the timing was started when the animals entered the water. During the swimming process of the rats, appropriate intervention should be made when the rats floated on the water surface until they started to swim. The weight-loaded swimming exhaustion time of the rats, i.e., the time of 5 s after the nose of the rat submerged under the water surface, was recorded. After recording the exhaustion time, the rats were quickly rescued to prevent the rats from being choked with water.

[0099] (4) Lung function test The animals were routinely anesthetized, the skin on the neck of the rats was cut, the trachea was bluntly separated, and a "T" type incision was cut for tracheal intubation. After the above operations, the rats were placed into a lung function test box, the tracheal intubation of the rats was connected with the detection gas path in the test box, and the following parameters were set: breathing ratio = 20:10, breathing frequency = 65 times / min, tidal volume = 8 mL / kg, and volume control = 4 x tidal volume. After the gas path was stabilized, the data collection was started, and the total lung resistance (RL) and forced vital capacity (FVC) were recorded.

[0100] (5) Data processing SPSS 26.0 statistical software was used to process the data, and the experimental results were expressed as mean ± standard deviation. Mann-Whitney U test was used for pairwise comparison between groups, p <0.05 was considered statistically significant.

[0101] 3. Experimental results In this example, SD rats were injected with BLM (6 mg / kg) through the trachea, and drug treatment was performed at the end of pulmonary fibrosis formation on the 21st day after BLM treatment. However, once pulmonary fibrosis is formed, it is difficult to reverse, and the lung is in a state of hypoxia for a long time, and the symptoms are more obvious when moving. How to further improve the quality of life of patients with pulmonary fibrosis and improve the exercise tolerance of patients has always been the focus of research. Therefore, this example aims to evaluate the effect of non-pre-saturated CO hemoglobin oxygen carrier preparation and pre-saturated CO hemoglobin oxygen carrier preparation on relieving gas exchange disorders caused by BLM-induced pulmonary fibrosis.

[0102] (1) Exercise tolerance such as Figure 4 The results showed that: at the baseline measurement on day 0, there was no statistical significance between the rats in each group; from day 1 BLM modeling to day 20 weight-bearing swimming measurement, compared with the Control group, the exhaustive time of rats in the BLM group, BLM+HBOC group and BLM+CO-HBOC group was significantly shortened, indicating that the pulmonary fibrosis modeling was successful; from day 21 after drug administration to the cutoff point of day 41, compared with the BLM group, the exhaustive time of rats in the BLM+HBOC group and BLM+CO-HBOC group was significantly increased, and the increase in the exhaustive time of rats in the BLM+CO-HBOC group was greater p <0.001). The above results show that tail vein infusion of HBOC and CO-HBOC can improve the exercise tolerance of rats with pulmonary fibrosis, and the improvement of CO-HBOC is more significant.

[0103] (2) Due to the fibrotic lesions in the lung, the normal alveolar structure disappears, the lung volume decreases and the compliance decreases, and restrictive ventilation dysfunction occurs. Specifically, the compliance of the lung decreases, the resistance increases (RL increases); in order to maintain normal oxygen supply, lung ventilation dysfunction (FVC decreases) will occur. The lung compliance test results are as follows Figure 5 (A) i: Compared with the Control group of rats, the RL values of the BLM group, BLM+HBOC group and BLM+CO-HBOC group were significantly increased, and the difference was statistically significant, that is, the three groups of rats all had irreversible pulmonary fibrosis characteristics. The lung ventilation function test results are as follows Figure 5In the middle (A) ii, compared with the BLM group, the FVC of the BLM+CO-HBOC group is improved to a certain extent, and the difference is statistically significant, that is, the ventilation dysfunction of the pulmonary fibrosis rats can be relieved by the internal oxygen supply mode through the infusion of the pre-CO saturated hemoglobin oxygen carrier preparation.

[0104] (3) After the end of the experiment, the arterial blood of the rats was obtained for blood gas analysis, and the experimental results were as follows Figure 5 In the middle (B), the arterial oxygen saturation (SaO2) of the rats in each group of the pulmonary fibrosis rats modelled by BLM was significantly lower than that of the Control group, and the SaO2 of the BLM+HBOC group and the BLM+CO-HBOC group was higher than that of the BLM group, and the difference between each group was statistically significant. At the same time, the arterial oxygen partial pressure (PaO2) of the rats in each group of the pulmonary fibrosis rats modelled by BLM was significantly lower than that of the Control group, and the PaO2 of the BLM+HBOC group and the BLM+CO-HBOC group was higher than that of the BLM group, and the difference between each group was statistically significant. Among them, the CO-HBOC has a better effect on the lung oxygenation of the pulmonary fibrosis rats p <0.001>.

[0105] 4. Conclusion In the present application, the pre-CO saturated hemoglobin oxygen carrier preparation makes the whole pulmonary fibrosis rat model exercise benefit and reduces the lung use overload compared with the non-pre-CO saturated hemoglobin oxygen carrier preparation.

[0106] Finally, it should be noted that: the above embodiments are only used to illustrate the technical solutions of the present application, and not to limit them; although the present application has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that: it can still modify the technical solutions recorded in the foregoing embodiments, or make equivalent replacement to part or all of the technical features; and these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the scope of the technical solutions of the embodiments of the present application.

Claims

1. A method for the preparation of a pre-carboxy-saturated hemoglobin oxygen carrier preparation, characterized in that, The method comprises the following steps: subjecting the hemoglobin oxygen carrier preparation to carbon monoxide saturation treatment; the carbon monoxide saturation treatment comprises: introducing carbon monoxide into the saturation device at a speed of 0.5-1.5 L / min for 15-25 min under stirring at 2-6℃.

2. The production method according to claim 1, characterized by, The hemoglobin oxygen carrier in the hemoglobin oxygen carrier preparation is a cross-linked bovine hemoglobin.

3. The preparation method according to claim 1, characterized in that, The carbon monoxide treatment is performed in a saturation device. The carbon monoxide saturation treatment comprises: introducing nitrogen into the saturation device to remove air, and then placing the hemoglobin oxygen carrier preparation in the saturation device, introducing carbon monoxide at a speed of 0.5-1.5 L / min under stirring at 2-6℃ for 15-25 min. In the carbon monoxide saturation treatment, the amount of the hemoglobin oxygen carrier preparation is 80-120 mL.

4. A pre-carbon monoxide saturated hemoglobin-based oxygen carrier formulation, characterized in that, The hemoglobin oxygen carrier preparation is prepared by the method of any one of claims 1-3. In the pre-carbon monoxide saturated hemoglobin oxygen carrier preparation, the concentration of hemoglobin is 10-15 gHb / dL, the carbon monoxide saturation degree is 90%-95%, and the content of methemoglobin is less than 5%.

5. Use of the pre-carbon monoxide saturated hemoglobin oxygen carrier preparation of claim 4 in the preparation of a product for preventing and / or treating pulmonary fibrosis.

6. Use according to claim 5, characterized in that, The product has at least one of the following uses: (1) preventing and / or treating bleomycin-induced pulmonary edema; (2) preventing and / or treating bleomycin-induced lung injury; (3) preventing and / or treating bleomycin-induced pulmonary fibrosis collagen deposition; (4) preventing and / or treating bleomycin-induced pulmonary hypoxia and circulatory disorder of reoxygenation.

7. Use according to claim 5, characterized in that, The product is used for inhibiting the expression of N-cadherin in the lung tissue of an animal with bleomycin-induced pulmonary fibrosis; and the product is used for promoting the expression of E-cadherin in the lung tissue of an animal with bleomycin-induced pulmonary fibrosis.

8. Use according to claim 5, characterized in that, The product has at least one of the following characteristics: (1) the product is used for inhibiting the oxidative stress injury induced by hypoxia in an animal with bleomycin-induced pulmonary fibrosis; (2) the product is used for increasing the content of superoxide dismutase in the lung tissue of the animal; (3) the product is used for reducing the content of malondialdehyde in the lung tissue of the animal.

9. Use according to claim 5, characterized in that, The product is used for improving the exercise tolerance of an animal with bleomycin-induced pulmonary fibrosis; and the product is used for increasing the arterial oxygen partial pressure of an animal with bleomycin-induced pulmonary fibrosis.

10. A pharmaceutical composition, characterized by, The product comprises the pre-carbon monoxide saturated hemoglobin oxygen carrier preparation of claim 4.

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