Application of L-DOPA in preparation of medicament for evaluating RDN operation curative effect

Abstract

The invention provides an application of L-DOPA in preparation of a medicament for evaluating an RDN operation curative effect. The structural general formula of the L-DOPA is # imgabs0 #, wherein R1, R2 and R3 are respectively or simultaneously hydrogen atoms, alkyl groups, halogen, nitro groups or hydroxyl groups; a is a non-metal radioactive element. The invention finds that L-DOPA can reflect the activity of renal efferent nerves to a certain extent, and can be used as an effective index for observing the curative effect of an RDN operation.

Description

Technical Field

[0001] The present invention relates to application of L-DOPA in preparing a medicament for evaluating the efficacy of RDN surgery. Background Art

[0002] Renal denervation (RDN) is a new hypertension treatment technique that has been developed recently. It uses radiofrequency ablation catheters or ultrasound catheters to degenerate the nerves around the renal artery, block the activity of the renal nerves, and reduce the effects of the efferent nerves on renal vascular resistance, renin release, and sodium reabsorption, thereby lowering blood pressure. It has good prospects in the field of treating essential hypertension and has been confirmed by many clinical studies. However, at present, there is no effective method to evaluate the success of renal efferent nerve ablation after RDN surgery. Only indirect methods can be used, that is, monitoring the changes in the patient's systemic blood volume after surgery. It is impossible to judge whether the renal efferent nerve ablation is successful in patients with increased systemic blood volume receiving RDN treatment before, after, or during surgery.

[0003] Currently, several sympathetic nerve imaging probes have been developed for clinical use. Imaging of cardiac sympathetic innervation can be performed using radioactive tracers that are absorbed in the presynaptic nerve endings of the sympathetic nerves. Commonly used radioactive tracers 123 I-metaiodobenzylguanidine ( 123 I-mIBG) for planar and single photon emission computed tomography (SPECT) imaging and 11C-hydroxyephedrine ( 11 C-HED) is used for positron emission tomography (PET). Sympathetic innervation imaging has been used to assess prognosis, response to treatment, risk of ventricular arrhythmias and sudden death, and to predict response to cardiac resynchronization therapy in patients with heart failure (HF). 11 C-epinephrine (EPI) has also been shown to be highly selective for neuronal uptake in the heart, providing excellent image quality. However, currently, no effective probes are available for the assessment of renal efferent nerve activity.

[0004] L-DOPA is a precursor of the neurotransmitters dopamine, norepinephrine, and epinephrine. 18 F labeling at the sixth carbon atom of L-DOPA can form a radiolabeled L-DOPA analogue that can be imaged by PET. 18 F-DOPA PET imaging was first used to diagnose Parkinson's disease and can show the degeneration of dopaminergic neurons in the substantia nigra and striatum. In recent years, it has become a new diagnostic tool for imaging neuroendocrine tumors and is widely used in clinical practice, including carcinoid, insulinoma, pheochromocytoma, paraganglioma, medullary thyroid cancer, hyperinsulinemia, etc. However, there is no 18 F-DOPA PET imaging is used to evaluate renal efferent nerve activity.

[0005] The kidney is another organ outside the central nervous system that can use a large amount of circulating L-DOPA to produce dopamine. The kidney takes up circulating L-DOPA through the sodium-dependent system and the sodium-independent system; in the sodium-independent system, this function is mainly exerted through the type 2 L-type amino acid transporter; and the kidney has abundant blood flow. Therefore, we wondered whether we could use 18 F-DOPA was used to show renal efferent nerve activity. Summary of the invention

[0006] The purpose of the present invention is to provide the use of L-DOPA in preparing a medicament for evaluating the efficacy of RDN surgery.

[0007] The general structural formula of L-DOPA is:

[0008]

[0009] wherein R1, R2 and R3 are independently or simultaneously hydrogen atoms, alkyl groups, halogen groups, nitro groups or hydroxyl groups;

[0010] A is a non-metallic radioactive element.

[0011] Preferably, R1 is a hydroxyl group, R2 is a hydroxyl group or a nitro group, and R3 is a hydrogen atom or a methyl group.

[0012] Preferably, said A is 18 F.

[0013] Preferably, the structural formula of the L-DOPA is any one of the following:

[0014]

[0015]

[0016] The main indication for RDN surgery is refractory hypertension that is not well treated with medication. Recent studies have shown that RDN can also improve left ventricular dysfunction in patients with chronic kidney disease and heart failure. However, the main indication is still refractory hypertension.

[0017] The present invention finds that L-DOPA can reflect the activity of renal efferent nerves to a certain extent and can be used as an effective indicator for observing the efficacy of RDN surgery. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 , Normal Spontaneously Hypertensive Rats (SHR) rats injected 18PET-CT images after 15 min, 1 h, and 3 h of F-DOPA. From left to right in the figure, they are 15 min, 1 h, and 3 h.

[0019] Figure 2 Uptake of the whole kidney, cortex, and medulla of the left and right kidneys before RDN in Wistar-Kyoto Rats (WKY) and SHR rats 18 F-DOPA SUV statistics; Figure A is the right kidney, and Figure B is the left kidney. (*P<0.05, ANOVA, data are expressed as mean ± SD.)

[0020] Figure 3 The results of the baseline blood pressure measurement and continuous monitoring of blood pressure after RDN for two weeks in WKY and SHR rats were shown in Figure 2. Figure 3 A: Monitoring of systemic blood volume pressure before and two weeks after surgery in the sham and RDN groups of WKY rats. 3B: Monitoring of systemic blood volume pressure before and two weeks after surgery in the Sham and RDN groups of SHR rats. Systolic blood pressure (SBP) and mean arterial pressure (MAP) of SHR rats decreased significantly within two weeks after RDN. (*P<0.05, ****P<0.0001, ANOVA, data are expressed as mean ± SD.)

[0021] Figure 4 , WKY and SHR rats after RDN whole kidney, renal cortex, and partial uptake of renal medulla 18 F-DOPA SUV Statistics. Figure 4 A: The whole kidney, renal medulla and renal cortex of WKY and SHR rats before and after RDN surgery 18 Differences in mean renal uptake of F-DOPA. 4B: Left kidney, renal medulla, and renal cortex in WKY and SHR rats before and after RDN surgery 18 Compared with the same rats before surgery, the uptake of F-DOPA in each part of the kidney of SHR rats after RDN was significantly higher than that in the same rats before RDN. 18 F-DOPA was significantly reduced. (*P<0.05, **P<0.01, ANOVA, data are expressed as mean ± SD.)

[0022] Figure 5 , 18 Representative PET-CT images of F-DOPA uptake differences in the kidneys of SHR rats before and two weeks after RDN surgery, with the left image being before surgery and the right image being after surgery.

[0023] Figure 6 Serum renal function test of WKY and SHR rats after RDN. There was no difference between the creatinine (CREA) (left figure) and urea nitrogen (BUN) (right figure) of the RDN group rats and the Sham group rats. (ANOVA, data are expressed as mean ± SD)

[0024] Figure 7 , Immunohistochemical staining of tyrosine hydroxylase (TH) of the efferent nerves around the renal artery, proving the inactivation of the efferent nerves around the renal artery. The left picture shows the positive immunohistochemical staining of TH in the efferent nerves around the renal artery of rats in the Sham group, and the right picture shows the reduction of the positive area of ​​TH immunohistochemical staining of the efferent nerves around the renal artery of rats in the RDN group.

[0025] Figure 8 , Western Blot detection of TH protein expression in kidney tissue, TH expression in the kidney of SHR rats after RDN surgery was significantly reduced. The upper figure is the Western Blot protein development image of kidney tissue, and the lower figure is the statistical result obtained by using Imagej to calculate the gray value of the band. (*P<0.05, ANOVA, data are expressed as mean ± SD) 。 DETAILED DESCRIPTION

[0026] In order to further explain the technical means and effects adopted by the present invention to achieve the predetermined invention purpose, the specific implementation methods, structures, features and effects of the present invention are described in detail below in conjunction with the accompanying drawings and preferred embodiments. The experimental methods used in the following examples are conventional methods unless otherwise specified. The materials, reagents, etc. used in the following examples, unless otherwise specified, can all be obtained from commercial channels.

[0027] Example 1

[0028] If R1 and R2 are substituted by hydroxyl groups separately or simultaneously, the following synthesis method is followed, and the synthesis of precursor 5a is taken as an example to introduce its synthesis method.

[0029] 1. Synthesis of intermediate 2a

[0030] Under nitrogen protection, DIPEA and DMAP were added to a DCM (8 mL) solution of intermediate 1a at 0°C. Stirred for 10 minutes under an ice bath, MOMCl was added dropwise. The resulting mixture was then heated to reflux for 20 hours. After cooling to room temperature, water was added and the aqueous phase was extracted with DCM. The combined organic layer was washed with 10% K2CO3 aqueous solution, dried over anhydrous sodium sulfate, and purified by column chromatography to obtain a colorless oily intermediate 2a.

[0031] 2. Synthesis of intermediate 3a

[0032] Under nitrogen protection, [bis(trifluoroacetyloxy)iodo]benzene and iodine were added to an ultra-dry DCM solution of intermediate 2a at 0°C. The reaction mixture was stirred for 3 hours and the reaction temperature was allowed to rise to room temperature. After the reaction was completed, the reaction was quenched with an aqueous solution of Na2SO3. The organic layer was separated and the aqueous phase was extracted with DCM. The combined organic layer was dried over anhydrous sodium sulfate and purified by column chromatography to obtain a colorless oily intermediate 3a.

[0033] 3. Synthesis of intermediate 4a

[0034] Under argon atmosphere, add intermediate 3a to a solution of pinacol diboronate, Pd(dppf)Cl2.CH2Cl2 and potassium acetate in DMF (anhydrous, 20 mL) at room temperature. Heat the reaction system to 80°C and stir for 18 hours. After cooling to room temperature, add saturated saline solution, and extract the aqueous phase with ethyl acetate (50 mL×3). Then dry the combined organic layer with anhydrous sodium sulfate, and purify by column chromatography to obtain colorless oily intermediate 4a.

[0035] 4. Synthesis of Intermediate 5a

[0036] Under argon atmosphere, di-tert-butyl dicarbonate, DMAP and triethylamine were added to the ultra-dry acetonitrile solution of intermediate 4a at room temperature. The reaction system was heated to 40°C and stirred for 24 hours. After cooling to room temperature, the solvent was removed by rotary evaporation, and ethyl acetate (100 mL) was added. The organic phase was then washed with saturated aqueous ammonium chloride solution, dried over anhydrous sodium sulfate, and purified by column chromatography to obtain yellow oily intermediate 5a. The specific reaction formula is as follows:

[0037]

[0038] If R1 or R2 is substituted by alkyl or halogen respectively or simultaneously, the following synthesis method is followed. Taking the synthesis of compound 5b as an example, the synthesis method is introduced.

[0039] 5. Synthesis of Intermediate 2b

[0040] Under nitrogen protection, DIPEA and DMAP were added to a DCM (8 mL) solution of intermediate 1a at 0°C. Stirred for 10 minutes under an ice bath, MOMCl was added dropwise. The resulting mixture was then heated to reflux for 20 hours. After cooling to room temperature, water was added and the aqueous phase was extracted with DCM. The combined organic layer was washed with 10% K2CO3 aqueous solution, dried over anhydrous sodium sulfate, and purified by column chromatography to obtain a colorless oily intermediate 2b.

[0041] 6. Synthesis of Intermediate 3b

[0042] Under nitrogen protection, [bis(trifluoroacetyloxy)iodo]benzene and iodine were added to an ultra-dry DCM solution of intermediate 2b at 0°C. The reaction mixture was stirred for 3 hours and the reaction temperature was allowed to rise to room temperature. After the reaction was completed, the reaction was quenched with an aqueous solution of Na2SO3. The organic layer was separated and the aqueous phase was extracted with DCM. The combined organic layer was dried over anhydrous sodium sulfate and purified by column chromatography to obtain a colorless oily intermediate 3b.

[0043] 7. Synthesis of Intermediate 4b

[0044] Under argon atmosphere, intermediate 3b was added to a solution of pinacol diboronate, Pd(dppf)Cl2·CH2Cl2 and potassium acetate in DMF (anhydrous, 20 mL) at room temperature. The reaction system was heated to 80°C and stirred for 18 hours. After cooling to room temperature, saturated saline solution was added, and the aqueous phase was extracted with ethyl acetate (50 mL×3). The combined organic layer was then dried over anhydrous sodium sulfate and purified by column chromatography to obtain colorless oily intermediate 4b.

[0045] 8. Synthesis of Intermediate 5b

[0046] Under argon atmosphere, di-tert-butyl dicarbonate, DMAP and triethylamine were added to the ultra-dry acetonitrile solution of intermediate 4b at room temperature. The reaction system was heated to 40°C and stirred for 24 hours. After cooling to room temperature, the solvent was removed by rotary evaporation, and ethyl acetate (100 mL) was added. The organic phase was then washed with saturated aqueous ammonium chloride solution, dried over anhydrous sodium sulfate, and purified by column chromatography to obtain yellow oily intermediate 5b. The specific reaction formula is as follows:

[0047]

[0048] 9. Radiolabeling of Precursors

[0049] Wherein, no matter R1 or R2 is substituted by hydrogen atom, alkyl group, halogen, nitro group or hydroxyl group, the radiolabeling method is similar. The radiolabeling method is introduced by taking the radiolabeling of precursor 5a as an example.

[0050] Use enriched H2 18 O, carried out on a cyclotron 18 O(p,n) 18 F nuclear reaction to obtain non-carrier added [ 18 F] fluoride. The QMA kit was modified with Kryptofix 222and potassium carbonate aqueous solution into a reaction vessel, and azeotropically dry the solvent at 110°C under nitrogen. Add acetonitrile (anhydrous, 2 mL) and dry at 110°C under nitrogen. Add a DMF solution of intermediate 5a and copper catalyst, heat the mixture to 120°C, and react for 20 minutes. After cooling to room temperature, purify on a C18 column, elute with acetone, and then heat to 110°C under a nitrogen atmosphere to remove volatile solvents. Then add HCl (6M aqueous solution), heat the reaction solution to 120°C and react for 20 minutes. Then cool the reaction mixture, dilute with a 0.1M NaOH aqueous solution, and purify with a C18 column using water containing 0.1% acetic acid / sodium acetate and 0.2g / L ascorbic acid as an eluent, and obtain the target product 6a after purification by HPLC. The specific reaction formula is as follows:

[0051]

[0052] Example 2

[0053] Male SHR and WKY rats were used in this experiment. Ten-week-old WKY and SHR rats were randomly divided into the Sham group and the RDN group, respectively. The basal blood volume pressure of the animals was non-invasively measured through the rat tail artery using the BP2000 measurement and analysis system one week before surgery, and was continuously monitored for two weeks after surgery. 18 F-DOPA PET-CT imaging, a dose of 1 mCi of the prepared in Example 1 was injected through the tail vein 18 F-DOPA, PET-CT scan was performed 1 hour after injection. PET-CT imaging results are as follows Figure 1 As shown, the analysis data is as follows Figure 2 As shown in the figure, L-DOPA is a hydrophilic small molecule amino acid, which is generally rapidly excreted into the bladder through the kidneys and ureters. Figure 1 It can be seen that 18 F-DOPA reached a metabolic stable state in the kidneys 1 hour after injection, with no obvious retention in the ureters, but still remained in the kidneys 3 hours later, proving that 18 F-DOPA has a rapid blood clearance rate and is well retained and utilized by the kidneys. Figure 2 It can be seen that before RDN, SHR had significant differences in mean radioactivity uptake value (SUV) only in the right renal medulla compared with WKY. There were no significant differences in the whole right kidney, right renal cortex, whole left kidney, left renal medulla, and left renal cortex.

[0054] Basal state of WKY and SHR rats 18After F-DOPA PET-CT imaging and measurement of systemic blood volume pressure, bilateral RDN surgery was performed. All rats were fasted for 12 hours before surgery, and then the abdomen was opened under anesthesia with isoflurane. The fat around the renal arteries and veins and the fat in the renal sinus of the rats in the RDN surgery group were peeled off, the arteries and veins were separated, and the visible nerves were peeled off. 10% phenol was applied to the renal artery and renal sinus for nerve ablation. In the Sham group, only the fat around the renal arteries and veins and the fat in the renal sinus were peeled off, and no nerve peeling and ablation were performed. After surgery, the animals were placed on a warming pad to maintain body temperature, and the animal status was observed until the animals woke up. The systemic blood volume pressure of rats in the RDN group and the Sham group was continuously monitored for two weeks after surgery. The results are as follows: Figure 3 As shown, SHRSBP and MAP continued to decrease compared with the Sham group two weeks after surgery, while there was no significant change in SBP and MAP of rats in the WKY group.

[0055] Two weeks after surgery, the rats in the RDN group were scanned again under the same conditions as the baseline state. 18 F-DOPA PET-CT scan. The scan results are as follows Figure 5 As shown, the whole right kidney, right renal medulla, right renal cortex, the whole left kidney, left renal medulla and left renal cortex of SHR rats were 18 The SUV of F-DOPA decreased significantly. The SUV of each part of the kidney of WKY rats also showed a significant downward trend, but it was not statistically significant. Figure 4 For the Sutra Figure 5 The statistical results of SUV obtained from the rat scanning results were compared with the data of the same rats before surgery. All rats were numbered during the experiment.

[0056] After anesthesia, the rats were killed and their serum was taken for renal function test. The results were as follows Figure 6 As shown, RDN surgery did not affect the renal function of the rats in the RDN group, proving that imaging was performed before and after RDN surgery in rats without abnormal renal function.

[0057] The bilateral renal artery tissues were taken for TH immunohistochemical staining, and the staining results were as follows: Figure 7 As shown, it can be seen that RDN surgery effectively reduces renal efferent nerve activity.

[0058] The results of Western Blot of TH in kidney tissue are as follows Figure 8 As shown in Figure 3, the expression level of TH in renal tissue was reduced after RDN surgery.

[0059] The above results show that 18 F-DOPA can reflect the activity of renal efferent nerves to a certain extent and can be used as an effective indicator to observe the efficacy of RDN surgery.

[0060] The above description is only a preferred embodiment of the present invention and does not limit the present invention in any form. Although the present invention has been disclosed as a preferred embodiment as above, it is not used to limit the present invention. Any technical personnel in this field can make some changes or modify the technical contents disclosed above into equivalent embodiments without departing from the scope of the technical solution of the present invention. However, any brief modifications, equivalent changes and modifications made to the above embodiments based on the technical essence of the present invention without departing from the content of the technical solution of the present invention are still within the scope of the technical solution of the present invention.

Claims

1. Application of L-DOPA in the preparation of a medicament for evaluating the efficacy of RDN surgery, wherein the general structural formula of L-DOPA is: wherein R1, R2 and R3 are independently or simultaneously hydrogen atoms, alkyl groups, halogen groups, nitro groups or hydroxyl groups; A is a non-metallic radioactive element.

2. The use according to claim 1, characterized in that: R1 is a hydroxyl group, R2 is a hydroxyl group or a nitro group, and R3 is a hydrogen atom or a methyl group.

3. The use according to claim 1 or 2, characterized in that: The A is 18 F.

4. The use according to claim 1, characterized in that: The structural formula of the L-DOPA is any one of the following:

5. The use according to claim 1, characterized in that: Indications for RDN surgery include refractory hypertension, chronic kidney disease, and left ventricular dysfunction in patients with heart failure.

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