Application of polyethylene glycol bovine hemoglobin conjugate

Abstract

The invention relates to the technical field of medical preparations, in particular to application of a polyethylene glycol bovine hemoglobin conjugate. By constructing a traumatic brain injury rat model, it is found that the polyethylene glycol bovine hemoglobin conjugate can effectively improve learning cognition, memory and exercise capabilities, new application of the polyethylene glycol bovine hemoglobin conjugate is expanded, a new thought is provided for research and development of drugs for improving the learning cognition, memory and exercise capabilities, and the application of the polyethylene glycol bovine hemoglobin conjugate is developed. Wide application prospects are realized.

Description

Technical Field

[0001] The invention relates to the technical field of medical preparations, in particular to the application of polyethylene glycol bovine hemoglobin conjugates. Background Art

[0002] Hemoglobin oxygen carriers (HBOCs) are a type of soluble nanoscale oxygen carriers with oxygen carrying and releasing functions that are prepared from hemoglobin through chemical modification and other methods. They can replace red blood cells to transport oxygen to various organs and tissues in the body, and have the characteristics of no blood type and virus transmission risks, long storage time, and immediate availability.

[0003] CN 115317595 A discloses a PEGylated bovine hemoglobin oxygen carrier having a high P 50 value, and can effectively carry and release oxygen. In addition, other chemically modified hemoglobin oxygen carriers have been reported. Usually, researchers only focus on the oxygen carrying and oxygen release capacity of hemoglobin oxygen carriers and use them as blood substitutes. Hu Yucheng et al. found that hemoglobin oxygen carriers can inhibit cell apoptosis after subarachnoid hemorrhage by activating the Akt signaling pathway, thereby alleviating early brain damage.

[0004] Traumatic brain injury (TBI) is a brain injury caused by external force, often accompanied by symptoms such as brain tissue hemorrhage, edema, hypoxia, etc., which can lead to neurological dysfunction or even death in severe cases. In addition, traumatic brain injury can have a serious impact on patients' cognition, memory and motor ability. At present, some researchers have found that hemoglobin oxygen carriers can improve neurological function caused by traumatic brain injury, but so far there is no evidence that hemoglobin oxygen carriers can effectively improve cognition, memory and motor ability. Summary of the invention

[0005] Due to the different chemical modification methods of different hemoglobin oxygen carriers, the oxygen carrying and oxygen release capacities of hemoglobin oxygen carriers prepared by different methods are also very different, which also leads to large differences in the application of hemoglobin oxygen carriers prepared by different methods in different functions.

[0006] The present invention constructs a rat model of traumatic brain injury and finds that polyethylene glycol (PEG) bovine hemoglobin conjugate can effectively improve learning cognition, memory and motor ability. Based on this, the following technical solution is proposed.

[0007] Firstly, the present invention provides the use of polyethylene glycol bovine hemoglobin conjugate in the preparation of medicines, and the medicines are used to improve learning cognition, memory and motor ability.

[0008] Preferably, the drug is used to improve the decline in learning cognition, memory and motor ability caused by brain trauma.

[0009] In some embodiments, the drug improves learning cognition, memory and motor ability by improving cytotoxic edema of brain tissue.

[0010] In some embodiments, the drug improves learning cognition, memory and motor ability by improving brain oxygen levels.

[0011] In some embodiments, the drug improves learning, cognition, memory and motor abilities by increasing neuronal survival rate and improving neural function.

[0012] Preferably, the aldehyde-modified polyethylene glycol in the polyethylene glycol-bovine hemoglobin conjugate selectively modifies the N-terminal α-NH2 and lysine residues of bovine hemoglobin.

[0013] Preferably, the preparation method of the polyethylene glycol bovine hemoglobin conjugate comprises: placing bovine hemoglobin in a Bis-Tris / HAc buffer system, then adding polyethylene glycol and sodium cyanoborohydride to react, purifying with an ultrafiltration membrane after the reaction, and then replacing with a PBS buffer system.

[0014] Preferably, the pH of the Bis-Tris / HAc buffer system and the PBS buffer system is 6.0-8.0 (eg, 6.0, 6.5, 7.4 or 8.0), more preferably 6.2-6.8.

[0015] Preferably, the pH of the Bis-Tris / HAc buffer system is the same as that of the PBS buffer system.

[0016] Preferably, the molar ratio of bovine hemoglobin, polyethylene glycol and sodium cyanoborohydride is 1:(15-25):(500-700), more preferably 1:(18-22):(580-620).

[0017] Preferably, the concentration of the bovine hemoglobin is 18-22 mg / mL.

[0018] Preferably, the concentration of the Bis-Tris / HAc buffer system is 18-22 mmol / L.

[0019] Preferably, the Bis-Tris / HAc buffer system is filtered through a 0.22 micron filter membrane before use.

[0020] Preferably, the bovine hemoglobin is placed in a Bis-Tris / HAc buffer system by dialysis (molecular weight cut-off Mw 50 kDa).

[0021] Preferably, the reaction is carried out at 2-6°C for more than 24 hours.

[0022] Preferably, the pore size of the ultrafiltration membrane is 50 kDa.

[0023] Preferably, the P of the polyethylene glycol bovine hemoglobin conjugate is 50 The value is 7~14mmHg, preferably 7.0~7.2mmHg. 50 The value is the oxygen partial pressure when hemoglobin is 50% saturated or the oxygen partial pressure when the blood oxygen saturation is 50%.

[0024] Preferably, the Hill coefficient of the polyethylene glycol bovine hemoglobin conjugate is 1.1 to 1.4, preferably 1.2 to 1.3.

[0025] Preferably, the polyethylene glycol bovine hemoglobin conjugate has a colloidal osmotic pressure of 27-41 mOsm / kg (preferably 35-41 mOsm / kg) when the protein concentration is 40 mg / mL, a viscosity of 2.8-3.5 mPa·s (preferably 3.4-3.5 mPa·s), and a particle size of 8.2-10.8 nm (preferably 8.2-8.5 nm).

[0026] Compared with the prior art, the present invention has the following beneficial effects: By constructing a rat model of traumatic brain injury, the present invention discovered that the polyethylene glycol bovine hemoglobin conjugate can effectively improve learning cognition, memory and motor ability, thus expanding the new application of the polyethylene glycol bovine hemoglobin conjugate, providing new ideas for the research and development of drugs for improving learning cognition, memory and motor ability, and having broad application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 This is a graph of brain oxygen saturation test results.

[0028] Figure 2 This is the Nissl staining result.

[0029] Figure 3 This is the result of nuclear magnetic resonance imaging.

[0030] Figure 4 This is a picture of the water maze test results.

[0031] Figure 5 This is the mNSS scoring result diagram.

[0032] Reference numerals: Three asterisks indicate statistical differences at the p < 0.001 level, and four asterisks indicate statistical differences at the p < 0.0001 level. DETAILED DESCRIPTION

[0033] In order to make the purpose, technical scheme and advantages of the present invention clearer, the technical scheme in the present invention will be described clearly and completely below. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments in the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention. In the embodiments provided in this specification, if the specific technology or conditions are not indicated, the technology or conditions described in the literature in this field or the product instructions are followed. The reagents or instruments used without indicating the manufacturer are all conventional products that can be purchased through regular channels. The polyethylene glycol bovine hemoglobin conjugate used in the following examples is prepared by referring to the preparation method of pH 6.5 in Section 1.2.1 of "The Effect of pH of the Reaction System on the Properties of Polyethylene Glycol Bovine Hemoglobin Conjugates", and the obtained polyethylene glycol bovine hemoglobin conjugate is named PEG-bHb.

[0034] Example 1. Construction of Traumatic Brain Injury (TBI) Rat Model (1) Twenty male Wistar rats were used for the experimental study after one week of adaptation in the animal room. The rats were first randomly divided into 4 groups: Group A sham operation group (5 rats, Sham), Group B model group (5 rats, TBI), Group C normal saline group (5 rats, TBI+NS), and Group D test drug group (5 rats, TBI+PEG-bHb).

[0035] (2) Except for the normal control group, we first placed Wistar rats on a scale to weigh their body weight, then injected 2% sodium pentobarbital (0.25-0.3 mL / 100 g) intraperitoneally for anesthesia. After the rats were deeply anesthetized, they were fixed in a prone position on the operating table, and routine surgical draping and abdominal skin preparation were performed. The rats were disinfected radially along the midline of the skull with iodine alcohol cotton balls. Ophthalmic scissors were used to cut an opening of about 3 cm in the skin at the midline of the rat's brain to expose the fascia. The fascia was widened to expose the left skull. A 5 mm cranial drill was used to open a window at the midpoint between the anterior bregma and the lambda line. The integrity of the dura mater was ensured during craniotomy (no percussion was performed after craniotomy in the sham operation group). The percussion parameters were set (speed: 5 m / s, percussion depth: 3 mm, time: 1 s). After the percussion, the cranial window was closed with bone wax to prevent the brain contents from bulging. After the operation, the membrane and skin of the fascia were sutured layer by layer with No. 4 thread. After suturing, the wound was disinfected with iodine alcohol cotton balls to prevent wound infection. After surgery, an indwelling needle was inserted into the rat's tail vein (indwelling time was 60 minutes). Except for the sham operation group and the model group, the saline group and the test drug group were intravenously administered with a dose of 12 mL / kg / h. After surgery, the animals were irradiated with a heater or infrared lamp for warming anesthesia recovery, and the rats were given sufficient water and food. After the animals woke up, they were put back in the cage.

[0036] 2. Polyethylene glycol bovine hemoglobin conjugate improves local brain oxygen levels after TBI In order to investigate the effect of polyethylene glycol bovine hemoglobin conjugate on local brain tissue oxygen saturation after TBI, we used PF6000 to monitor the changes in brain oxygen saturation at the impact window. Figure 1 As shown in the figure, from the experimental results, it can be seen that the local brain oxygen of normal rats is maintained at about 90%, and it drops to about 70% instantly after TBI. The brain oxygen saturation of the test drug group can be maintained at about 65-70% after traumatic brain injury, while the brain oxygen saturation of the model group and the normal saline group continues to drop to about 40%. There is a significant difference in brain oxygen saturation between the groups, indicating that the polyethylene glycol bovine hemoglobin conjugate improves the local brain oxygen level after TBI.

[0037] 3. Polyethylene glycol bovine hemoglobin conjugate significantly improves neuronal survival rate In order to investigate the effect of polyethylene glycol bovine hemoglobin conjugate on the survival of brain neurons after brain trauma, Nissl staining was performed on the brain tissues of rats in each group 3 days after brain trauma. Figure 2 As shown in the figure, from the staining results and the Nissl-positive cell counting results, it can be seen that the neurons in the sham operation group are neatly arranged, the outlines are clear, the Nissl bodies are deeply stained, and the number is large. There are significant differences in the neuronal morphology and the number of Nissl bodies in the test drug group compared with the other two groups, indicating that the polyethylene glycol bovine hemoglobin conjugate significantly improves the survival rate of neurons.

[0038] 4. Polyethylene glycol bovine hemoglobin conjugate significantly improves brain tissue cytotoxic edema Three days after brain injury, the brain tissues of rats in each group were scanned by MRI T2 and DWI sequences. Figure 3 As shown in the figure, the T2 sequence scan results of the MRI showed that the brain tissue structure of the test drug group was relatively normal, there was no obvious deviation of the brain midline, and the injury volume was significantly different from the other two groups. The DWI sequence scan results showed that the brain injury area of ​​the test drug group showed a relatively high signal compared with the other two groups, and the ADC value was also relatively increased, indicating that the local brain tissue cytotoxic edema had a trend of improvement.

[0039] 5. Polyethylene glycol bovine hemoglobin conjugate significantly improves learning cognition, memory and motor ability (1) Morris water maze test Morris water maze test was performed 7 days after brain injury. Figure 4 As shown in the figure, the swimming distance, platform-finding time, swimming distance and stay time in the target quadrant, and number of platform crossings in the test drug group were significantly different from those in the other two groups, indicating that the polyethylene glycol bovine hemoglobin conjugate significantly improved the learning cognition, memory and motor ability of rats with traumatic brain injury model.

[0040] (2) mNSS score The mNSS scores of the rats in each group were evaluated 1, 3, and 7 days after TBI. Figure 5 As shown in the figure, from the scoring results, it can be seen that the mNSS scores of the model group and the saline group were at a high level on the first day, and the values ​​of the two groups were similar, indicating that traumatic brain injury and saline treatment did not effectively improve the neurological function of rats. During the observation period of 1-7 days, although the scores of the two groups decreased to a certain extent, they still remained at a high level, indicating that the neurological deficits of rats were more serious. The mNSS score of the test drug group on the first day was significantly lower than that of the model group and the saline group. Within 1-7 days, the score of this group continued to decline and dropped to a lower level on the 7th day, indicating that the polyethylene glycol bovine hemoglobin conjugate has a significant improvement on the neurological function of rats with traumatic brain injury, and can improve the movement, balance, sensation and reflex of rats with traumatic brain injury, thereby improving learning cognition, memory and motor ability.

[0041] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or make equivalent replacements for some of the technical features therein. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. Application of polyethylene glycol bovine hemoglobin conjugate in the preparation of medicines, wherein the medicines are used to improve learning cognition, memory and motor ability.

2. The use according to claim 1, characterized in that: The medicine is used to improve the decline in learning cognition, memory and motor ability caused by brain trauma.

3. The use according to claim 1 or 2, characterized in that: The drug improves learning cognition, memory and motor skills by improving cytotoxic edema of brain tissue.

4. The use according to claim 1 or 2, characterized in that: The drug improves learning cognition, memory and motor skills by improving brain oxygen levels.

5. The use according to claim 1 or 2, characterized in that: The drug improves learning cognition, memory and motor skills by increasing neuron survival rate and improving nerve function.

6. The use according to claim 1, characterized in that: The aldehyde-modified polyethylene glycol in the polyethylene glycol bovine hemoglobin conjugate selectively modifies the N-terminal α-NH2 and lysine residues of the bovine hemoglobin.

7. The use according to claim 1, characterized in that: The preparation method of the polyethylene glycol bovine hemoglobin conjugate comprises: placing the bovine hemoglobin in a Bis-Tris / HAc buffer system, then adding polyethylene glycol and sodium cyanoborohydride to react, purifying with an ultrafiltration membrane after the reaction, and then replacing with a PBS buffer system.

8. The use according to claim 7, characterized in that: The pH of the Bis-Tris / HAc buffer system and the PBS buffer system is 6.0~8.

0.

9. The use according to claim 7, characterized in that: The molar ratio of bovine hemoglobin, polyethylene glycol and sodium cyanoborohydride is 1:(15-25):(500-700); And / or, the concentration of the bovine hemoglobin is 15-25 mg / mL.

10. The use according to claim 7, characterized in that: The P of the polyethylene glycol bovine hemoglobin conjugate 50 The value is 7~14mmHg; And / or, the Hill coefficient of the polyethylene glycol bovine hemoglobin conjugate is 1.1-1.4; And / or, the polyethylene glycol bovine hemoglobin conjugate has a colloid osmotic pressure of 27-41 mOsm / kg, a viscosity of 2.8-3.5 mPa·s, and a particle size of 8.2-10.8 nm when the protein concentration is 40 mg / mL.

Images