Application of Gd3 + in preparation of medicine for treating hemolysis and renal injury after pulsed electric field ablation

Abstract

The invention provides application of a Gd < 3 + >-containing compound in preparation of a medicine for treating pulsed electric field ablation induced hemolysis secondary renal injury. Starting from complications in practical application of pulsed electric field ablation, a compound containing Gd < 3 + >, especially a remission effect of gadolinoic acid on hemolysis caused by PFA, is explored in vitro and in vivo levels. Experiments are adopted for the first time to confirm that the Gd3 +-containing compound, especially gadolinolenic acid, can effectively inhibit the occurrence of pulsed electric field ablation induced hemolysis, and then inhibit the complication renal injury.

Description

Technical Field

[0001] The invention belongs to the field of medical technology and relates to a Gd 3+ Application in the preparation of drugs for treating hemolysis and renal injury after pulsed electric field ablation. Background Art

[0002] Atrial fibrillation (AF) is a common arrhythmia that seriously affects the quality of life of patients and increases the morbidity and mortality associated with cardiovascular complications. Pulmonary vein isolation (PVI) is the cornerstone of the current AF ablation strategy. Radiofrequency ablation (RFA) and cryoablation (CBA) still have the limitations of difficulty in forming stable and lasting damage, being greatly affected by the operator's operation, and being prone to adverse events such as pulmonary vein stenosis, esophageal injury, and phrenic nerve injury. In recent years, pulsed field ablation (PFA) has attracted widespread attention as a potential technology to replace traditional thermal ablation methods. PFA uses high-amplitude pulsed electric fields to induce irreversible electroporation in cardiac tissue without causing thermal damage. Its potential safety margin of the ablation site is large, and the operation is faster and has a short learning curve. This method is expected to achieve more precise and safer lesion ablation. The theoretical basis of PFA is derived from electroporation. When cells are exposed to sufficiently strong voltage pulses, lipid oxidation and protein damage will occur in the cell membrane, resulting in nanoscale micropores on the phospholipid bilayer, which increases the permeability of the cell membrane. These micropores are recoverable when the electric field strength is low, which is called reversible electroporation. When the electric field strength reaches a certain level, the micropores on the cell membrane will undergo permanent changes, which is called irreversible electroporation, which can induce cell apoptosis or necrosis. Red blood cells are more sensitive to electric fields than cardiomyocytes, so even in highly selective PFA, red blood cells may become the object of non-target damage. When the electric field reaches a certain intensity, the red blood cell membrane will undergo electroporation and the red blood cells will eventually be destroyed, which will cause hemolysis. Hemolysis releases a large amount of hemoglobin, which can cause renal tubular barrier disorders and oxidative cell damage, leading to acute kidney injury (AKI).

[0003] The MANIFEST-17k study reported that 5 patients with persistent atrial fibrillation (0.03%) developed hemolysis-related renal failure requiring hemodialysis, with an average of 143±27 PFA applications per case. Venier et al. reported 2 cases of severe hemolysis and AKI due to the large-scale application of PFA. Subsequent cohort studies found that approximately 28% of patients had a significant decrease in plasma haptoglobin levels. The number of PFA applications was significantly correlated with the incidence of hemolysis. More than 70 applications significantly increased the risk of hemolysis, while AKI was only observed in patients with more than 100 applications. Jordan et al. compared the incidence of AKI after ablation with different energies. The results showed that the incidence of AKI in the PFA group (1.0%) was significantly lower than that in the radiofrequency group (4.3%) and the cryotherapy group (1.8%). The authors believe that AKI is rare under standardized procedures and without excessive use of PFA. Osmancik et al. compared the postoperative hemolysis in patients undergoing PFA and RFA. The PFA group was applied 52.9±18.4 times on average. The patients' red blood cell microparticle concentration, lactate dehydrogenase and indirect bilirubin increased significantly after surgery, while the haptoglobin level decreased significantly, but recovered within 24 hours. The hemolysis markers of patients in the RFA group changed less. No AKI was observed in either group. In order to deal with AKI caused by hemolysis, Mohanty et al. divided patients who received PFA into a postoperative non-hydration group (28 cases) and a hydration group (NS≥2L) (75 cases). In the former, 75% of patients developed hemoglobinuria with a significant increase in serum creatinine, of which 4 patients reached the AKI grade III standard; no similar situation occurred in the latter. The study believes that adequate postoperative hydration can effectively prevent AKI after PFA, providing a reference for postoperative management.

[0004] In the existing pulsed electric field cardiac ablation scheme, the hemolysis induced by the pulsed electric field increases with the increase of the number of ablations. With the popularization of pulsed electric field ablation technology, the number of ablation discharges is bound to increase in some refractory arrhythmias, and the hemolysis and secondary renal damage caused by it need to be solved urgently. However, there is a lack of an effective therapeutic drug for hemolysis and secondary renal damage caused by arrhythmias in this field, which is a technical problem that needs to be solved urgently in this field. Summary of the invention

[0005] In order to solve the above technical problems, the present invention provides the following technical solutions:

[0006] The present invention provides a Gd-containing 3+ Application of the compound in the preparation of drugs for treating hemolysis and secondary renal damage;

[0007] Furthermore, the hemolysis is hemolysis caused by pulsed electric field stimulation or hemolysis induced by pulsed electric field ablation (PFA);

[0008] Furthermore, the Gd 3+ Compounds include gadopentetate (Gd-DTPA) or gadodiamide; BRIEF DESCRIPTION OF THE DRAWINGS

[0009] Figure 1 This is a result diagram showing that gadopentetate can effectively inhibit the destruction of erythrocytes induced by pulsed electric fields in vitro;

[0010] Figure 2 Gadopentic acid can effectively inhibit hemolysis caused by pulsed electric field ablation in vivo;

[0011] Figure 3 Conclusions: Gadopentetate in vivo can effectively inhibit renal damage caused by high-dose pulsed electric field ablation.

[0012] Beneficial Effects

[0013] Based on the complications in the actual application of pulsed electric field ablation, the present invention explores the effects of Gd-containing 3+ The present invention is the first to demonstrate the alleviating effect of compounds, especially gadopentetate, on PFA-induced hemolysis. The applicant found through in vitro experiments that gadopentetate can effectively reduce PFA-induced erythrocyte destruction; through in vivo experiments, it was found that the early application of gadopentetate can effectively reduce the hemolysis caused by ablation in rats. 3+ Compounds, especially gadopentetate, can effectively inhibit the occurrence of pulsed electric field ablation-induced hemolysis and then inhibit the complication of renal damage. DETAILED DESCRIPTION

[0014] The technical solution of the present invention is further described below in conjunction with the accompanying drawings and specific implementation methods, but these embodiments shall not be used to interpret the limitation of the present invention; the experimental methods in the following embodiments are conventional methods unless otherwise specified, and the test materials used in the following embodiments are commercially available unless otherwise specified; after reading the description of the present invention, various equivalent changes, modifications and modifications made by technical personnel in this field all fall within the scope defined by the claims of the present invention.

[0015] (I) The materials, reagents and drugs involved in the present invention are as follows:

[0016] The rats were wild-type Sprague Dawley rats, with a body weight of about 300 to 400 g. In the in vitro experiment, the pulsed electric field generator was a square wave electroporation system (BTX ECM 830 Square Wave Electroporation System). The electrode cup was purchased from Bio-rad (B165-2088, 0.4 cm). The ELISA kit for detecting free hemoglobin in rats was purchased from Abcam (Ab157733). Gadopentetate was purchased from MedChemExpress (HY-107353). The in vivo pulsed ablation power supply and pulsed ablation catheter were provided by Shanghai Antec Medical Technology Co., Ltd.

[0017] Gadopentetate is one of the intermediates in the manufacture of gadopentetate dimeglumine and can be used for enhanced magnetic resonance imaging (MRI), including enhanced examination of organs and tissues such as the nervous system, myocardium, liver, breast, bone, and kidney. 3+ It has 7 unpaired electrons and is a metal ion with strong paramagnetism. It can significantly shorten the relaxation time of T1 and T2, especially T1. The relaxation time decreases linearly in the concentration range of 0-1mmol / L, thus affecting the signal intensity of MRI. Pharmacokinetics: This product is a chelate of meglumine, and its in vivo process is related to meglumine. After intravenous administration, it quickly diffuses into the extracellular fluid of various tissues in the body, and then is excreted in its original form through glomerular filtration. A small amount is secreted in the gastrointestinal tract and then excreted with feces. This product can enter the diseased tissue through the damaged blood-brain barrier; Indications: Used for magnetic resonance imaging (MRI) enhancement, including: enhanced examination of organs and tissues such as the nervous system, myocardium, liver, breast, bone, and kidney; Usage and dosage: intravenous injection, 0.1mmol / kg. Enhanced imaging is performed 5 minutes after injection, and the enhancement effect can be maintained for 45 minutes; Adverse reactions: The adverse reactions of this product are significantly lower than those of iodine contrast agents. There is a mild transient headache (8.7%), followed by a cold feeling at the injection site (4.8%), nausea, vomiting, numbness, and dizziness (2%); there are also a burning sensation at the injection site, local edema, fatigue, chest tightness, local lymphadenitis, hypotension, abdominal pain, chest pain, salivation, anxiety, convulsions, itchy throat, cough, rash, dry mouth, abnormal taste, sweating, tearing, etc., with an incidence rate of less than 1%; Specification: Injection, 20ml (0.5mol / L).

[0018] The chemical name of gadodiamide is [5,8-bis(carboxymethyl)-11-[2-(methylamino)-2-oxoethyl]-3-oxo-2,5,8,11-tetraazadecane-13-oxo(3-)]gadolinium, and its molecular formula is C 16 H 26 G JZ 5 O 8, with a molecular weight of 573.65600. 1. Gadolinium diamide is mainly used for: Gadolinium diamide is a colorless or light yellow clear liquid, which is an intravenous drug. It is mainly suitable for magnetic resonance imaging of the head, spinal cord and body after intravenous injection. After using gadodiamide injection, it can enhance the contrast and is beneficial for the display of different parts of the body, including the display of abnormal structures of the central nervous system. Gadolinium diamide is similar to gadopentetate, and the active part is also Gd 3+ , therefore, can also play a similar role.

[0019] (II) Specific experimental methods:

[0020] (1) In vivo experiment: We drew 500 μl of fresh anticoagulated blood from rats and placed it in an electrode cup. In the gadopentetate treatment group, the final concentration of gadopentetate was 100 μM and 1000 μM. Gadopentetate was added and mixed before pulsed electric field stimulation, and then placed in a 37°C water bath for 10 minutes. Subsequently, pulsed electric field stimulation was performed, and the pulsed electric field stimulation was generated by an electroporator (ECM 830). The number of pulses was 5 to 20 times. The pulsed electric field intensity was 1800 V / cm, the pulse width was 1 μs, and the frequency was 10 Hz. After stimulation of the blood samples of different groups, the blood samples were centrifuged at 6000 g for 10 minutes, and the supernatant plasma was aspirated. The free hemoglobin concentration in the plasma was detected using an ELISA kit. For the specific method of ELISA, please refer to the relevant instructions.

[0021] (2) In vitro experiment: After male rats (300 g to 400 g) were fully anesthetized, 10 μmol / kg and 100 μmol / kg of gadopentetate were injected into the tail vein, and the control group was injected with an equal amount of saline. After standing for 2 hours, pulsed electric field ablation was performed. An incision was made in the right neck of the rat and the external jugular vein of the rat was freed. Two level 3 catheters were placed in the jugular vein of the rat and entered the heart through the jugular vein. The position was determined and fixed under X-ray fluoroscopy. One electrode was used for recording the intracardiac electrogram, and the other electrode was connected to the ablation device for pulsed electric field ablation. Each pulsed electric field application included 60 pulses, with an electric field strength of 1800 V / cm, a pulse width of 5 μs, and a frequency of 1000 Hz. One ml of blood was collected by cardiac puncture 1 hour after ablation to reduce hemolysis caused by the operation (no rats died). The blood sample was centrifuged at 6000 g for 10 minutes, and the supernatant plasma was aspirated. The free hemoglobin concentration in the plasma was detected using an ELISA kit. The specific method of ELISA is shown in the relevant instructions. Two days after the ablation, the rats in different groups were anesthetized again, and 500 μl of blood was collected from the medial canthal vein to test blood biochemical indicators, including three items of renal function: urea nitrogen, uric acid, and creatinine. The degree of direct renal function damage in different groups was compared.

[0022] Example 1: 100 μM and 1000 μM gadopentetate can effectively inhibit the damage of pulsed electric field to red blood cells in vitro

[0023] The present invention uses an in vitro cell electroporator to release high-frequency high-voltage pulses, and uses anticoagulated fresh rat whole blood and different concentrations of gadopentetate to conduct experiments. In the gadopentetate treatment group, the final concentration of gadopentetate is 100μM and 1000μM. Gadopentetate is added to the fresh blood of rats before pulse electric field stimulation and mixed, and then placed in a 37°C water bath for 10 minutes to simulate the situation in the body under the physiological environment, and a certain time is given to allow gadolinium ions to distribute on the surface of the red blood cell membrane. The control group added an equal amount of physiological saline, mixed it, and placed it in a 37°C water bath for 10 minutes. Subsequently, the three groups were subjected to pulse electric field stimulation, and the pulse electric field stimulation was generated by an electroporator (ECM 830). The number of pulses was 0, 5, 10, 15 and 20, respectively. The pulse electric field strength was 1800V / cm, the pulse width was 1μs, and the frequency was 10Hz. The pulse electric field strength and parameters simulate the clinical application parameter range of the currently available pulse electric field ablation system. After stimulation of different groups of blood samples, blood samples were collected in 1.5 ml centrifuge tubes and centrifuged at 6000 g for 10 min to obtain the supernatant plasma. Figure 1 As shown in A. The supernatant plasma of different groups was quickly placed in a 4-degree refrigerator for storage, and the free hemoglobin concentration in the plasma was simultaneously detected by ELISA kit as soon as possible. The free hemoglobin concentration in the supernatant plasma was as follows Figure 1 As shown in B. We found that whether it is 100μM or 1000μM, gadopentetate can effectively alleviate the acute red blood cell destruction and hemolysis caused by pulsed electric field stimulation. Figure 1 The figure shows the results of gadopentetate inhibiting the destruction of erythrocytes induced by pulsed electric field in vitro. Figure 1 A is a schematic diagram of the color of serum after centrifugation of fresh rat anticoagulated whole blood mixed with different concentrations of gadopentetate and subjected to different numbers of pulsed electric field stimulations. Figure 1 B is a statistical graph of the free hemoglobin concentrations of the supernatant plasma of different groups.

[0024] Example 2: Intravenous administration of gadopentetate can effectively alleviate hemolysis caused by pulsed electric field ablation

[0025] Male rats (300g-400g) were randomly selected. After being fully anesthetized with pentobarbital, the tail vein was injected with 10μmol / kg and 100μmol / kg of gadopentetate. The control group was injected with an equal amount of normal saline. After standing for 2 hours, pulsed electric field ablation was performed. The concentration in the rat body was calculated according to the proportion of the total blood volume of the rat and our previous in vitro experimental data. An incision was made in the right neck of the rat, and the external jugular vein of the rat was freed and threaded for use. Two 3-level pulse ablation electrode catheters were placed in the jugular vein of the rat, and the angle was adjusted to enter the heart through the jugular vein, and the vascular end was fixed. The position was determined and fixed under X-ray fluoroscopy. One electrode was used for recording the intracavitary electrogram of the heart, and the other electrode was connected to the ablation device for pulsed electric field ablation. X-ray fluoroscopy showed Figure 2 B. Each pulsed electric field application includes 60 pulses, with an electric field strength of 1800V / cm, a pulse width of 5μs, and a frequency of 1000Hz. In order to be as close to clinical applications as possible, the pulse parameters also simulate the clinical pulsed electric field ablation system that has been on the market. After 30 days of ablation, significant ablation lesions can be seen in the Masson staining and gross photos of the rat heart, confirming that this field strength can effectively ablate the rat myocardium ( Figure 2 A). During ablation, changes in the morphology of A and V waves can be seen on the intracavitary electrocardiogram ( Figure 2 C) to confirm that the ablation was effective immediately. One hour after ablation, 1 ml of blood was collected by cardiac puncture to reduce the hemolysis caused by the operation. The blood sample was centrifuged at 6000 g for 10 min and the supernatant plasma was aspirated. The free hemoglobin concentration in the supernatant plasma was detected using an ELISA kit. Figure 2 D. The present invention confirms that the in vivo application of gadopentetate can effectively reduce the red blood cell destruction and hemolysis caused by cardiac pulse electric field ablation.

[0026] Example 3: Intravenous administration of gadopentetate can effectively alleviate renal damage caused by high-dose pulsed electric field ablation

[0027] The above-mentioned male rats (300g-400g) were randomly selected, and after being fully anesthetized with pentobarbital, the rats were injected with gadopentetate at doses of 10μmol / kg and 100μmol / kg through the tail vein, and the control group was injected with an equal amount of normal saline. After standing for 2 hours, pulsed electric field ablation was performed on the rats. After the ablation, they were sutured and continued to be raised normally. Two days after the ablation, after the rats were fully anesthetized, a total of 500μl of blood samples were taken from the medial angular vein, and the blood was anticoagulated with heparin and sent to the company to test the blood biochemical indicators of the small animals. Three items of renal function were tested separately: urea nitrogen, uric acid, and creatinine concentration levels, and the degree of direct renal function damage in different groups was compared. It was found that 10 pulsed electric field ablations had no significant effect on renal function, but 20 pulsed electric field ablations could promote the increase of blood urea nitrogen and creatinine ( Figure 3 AC). Intravenous administration of gadopentetate during surgery can significantly reduce the incidence of renal damage after high-dose pulsed electric field ablation ( Figure 3 AC). The present invention confirms that the in vivo application of gadopentetate can effectively reduce the renal function damage caused by cardiac pulse electric field ablation.

[0028] The basic principles, main features and advantages of the present invention are shown and described above, but the present invention is not limited to the embodiments. Various changes and improvements may be made to the present invention without departing from the spirit and scope of the present invention, and these changes and improvements all fall within the scope of the present invention to be protected.

Claims

1. A Gd-containing 3+ The compound is used in the preparation of drugs for treating hemolysis and secondary renal damage.

2. The use according to claim 1, characterized in that: The hemolysis is hemolysis caused by pulse electric field stimulation or hemolysis induced by pulse electric field ablation.

3. The use according to claim 1, characterized in that: The Gd 3+ Compounds include gadopentetate (Gd-DTPA) or gadodiamide.

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