Application of arginine vasopressin in treatment of slow transit constipation

Abstract

The invention discloses application of arginine vasopressin in treatment of slow transit constipation, and relates to the technical field of medicines for treating slow transit constipation. The arginine vasopressin nose drop is found to have a very good treatment effect on slow transit constipation for the first time, and especially for loperamide-induced slow transit constipation, the arginine vasopressin nose drop can effectively promote intestinal tract movement and improve constipation symptoms. The arginine vasopressin is prepared into nose drops, can bypass a blood brain barrier to enter the brain, directly targets downstream nuclei of AVP neurons in a hypothalamic para-ventricular nucleus PVN nuclei, and intervenes treatment in the nerve center, and the effect is better. The discovery of the invention expands a new application for arginine vasopressin drugs, and also provides a new idea for development of constipation drugs.

Description

Technical Field

[0001] This invention relates to the field of drugs for treating slow-transit constipation, and particularly to the application of arginine vasopressin in the treatment of slow-transit constipation. Background Technology

[0002] With the global trend of population aging, and the continuous changes in dietary structure, lifestyle, mental and psychological stress, and social environmental factors, the incidence of chronic constipation is showing a significant upward trend, affecting all clinical fields, seriously disrupting people's daily lives, and impacting their quality of life. Constipation is defined as having fewer than three bowel movements per week, hard stools, and difficulty in defecation. When constipation lasts for more than 12 weeks, it is called chronic constipation. Currently, functional constipation (FC) is mainly divided into three types: slow transit constipation (STC), outlet obstructive constipation (OOC), and mixed constipation. The prevalence of chronic constipation in adults is 4-6%, and it shows a significant upward trend with population aging, with a prevalence as high as 22% in people over 60 years of age.

[0003] STC is a subtype of functional constipation characterized by colonic transit dysfunction. Clinical symptoms of STC patients are mainly reduced bowel movement frequency (spontaneous bowel movements <3 times / week), straining during defecation, and hard, dry stools. As the disease progresses, STC patients may gradually develop symptoms of incomplete obstruction such as abdominal pain and bloating; severe cases may develop fecal impaction and colonic obstruction.

[0004] Currently, the treatment of constipation is mainly divided into medical treatment and surgical treatment. Medical treatment focuses on individualized symptomatic relief, such as daily life adjustments (supplementing dietary fiber, increasing water intake, etc.), drug therapy (various laxatives, prokinetic agents, and secretagogues, etc.), and gut microbiota-supported therapy, aiming to relieve symptoms, reduce the frequency of attacks, and eliminate patient anxiety. However, when non-surgical measures are ineffective and symptoms worsen, impacting quality of life, surgical treatment may be carefully considered based on a joint decision-making process between the doctor and patient, such as total colectomy with ileorectal anastomosis, subtotal colectomy with anastomosis, or non-colectomy. Current treatments for constipation have some drawbacks, such as: ① drug side effects: long-term use of laxatives may lead to intestinal dysfunction; enema use may cause electrolyte imbalances; and glycerin suppositories may increase bowel movement frequency and affect normal defecation reflexes; ② unstable treatment effects; ③ drug dependence: patients who have used laxatives for a long time may experience worsening constipation after discontinuation.

[0005] Conscious defecation is strictly controlled by neural circuits. When the rectum is full of feces, it stimulates receptors in the intestinal wall, sending impulses to lower defecation centers in the lumbosacral spinal cord and simultaneously transmitting signals to the cerebral cortex, thus generating the urge to defecate. If environmental conditions permit, the cerebral cortex generates impulses, enhancing the excitation of the defecation centers and triggering the defecation reflex, causing contractions of the sigmoid colon and rectum while the anal sphincter relaxes. However, the location and identity of the higher neural centers and specific neurons involved in conscious defecation remain elusive. Based on retrograde tracing, chemogenetics, functional imaging experiments, or electromyography, it has become possible to analyze the higher neural centers that regulate defecation behavior and thus influence the occurrence of constipation.

[0006] Arginine vasopressin (AVP), also known as antidiuretic hormone (ADP), is a nonapeptide synthesized in the hypothalamus. AVP is a major physiological regulator of renal water excretion and blood volume. AVP is synthesized in neurosecretory cells of the supraoptic and paraventricular nuclei of the hypothalamus and stored in neurosecretory granules of the posterior pituitary gland. AVP plays an important role in controlling osmotic homeostasis, blood pressure regulation, sodium homeostasis, and renal function. Clinically, arginine vasopressin is commonly used for intraoperative hypotension, central diabetes insipidus, and postoperative polyuria. Currently, there are no reports of a correlation between arginine vasopressin and slow-transit constipation. Summary of the Invention

[0007] To address the limitations of existing clinical treatments, this invention provides the application of arginine vasopressin nasal drops in the treatment of slow-transit constipation. Compared to previous therapies, this invention innovatively identifies arginine vasopressin neurons in the paraventricular nucleus of the hypothalamus (PVN), a higher neural regulatory center for defecation, elucidates the pathogenesis of slow-transit constipation, and delivers AVP (arginine vasopressin) through nasal drops, bypassing the blood-brain barrier to enter the brain and directly targeting downstream nuclei of the PVN nucleus, thus mimicking the PVN's... AVP Neuronal activation, through intervention in the central nervous system, effectively alleviated slow-transit constipation.

[0008] This invention provides the application of arginine vasopressin in the preparation of a drug for treating slow transit constipation.

[0009] Furthermore, the slow-transit constipation is constipation induced by loperamide hydrochloride.

[0010] Furthermore, in the drug, the dosage of arginine vasopressin is 6.67-13.33 μg / (kg·d).

[0011] Furthermore, in the drug, the dosage of arginine vasopressin is 6.67 μg / (kg·d).

[0012] Furthermore, the medication is a nasal drop.

[0013] Furthermore, the drug also includes pharmaceutically acceptable excipients.

[0014] Furthermore, the pharmaceutically acceptable excipients include at least one of the following: fillers, lubricants, flavoring agents, stabilizers, desiccants, antioxidants, disintegrants, colorants, dispersants, pH adjusters, and coating materials.

[0015] To investigate the pathogenesis of slow-transit constipation, we used pseudorabies virus (PRV) to retrogradely trace its regulatory centers from each part of the intestine. We identified the paraventricular nucleus (PVN) of the hypothalamus as the brain region controlling intestinal peristalsis. Animal experiments confirmed the neural pathway by which the PVN regulates intestinal peristalsis in mice. Exogenous arginine vasopressin effectively mimics the PVN by activating downstream neurons through receptor-positive neurons in the brain. AVP Activation of neurons promotes colonic peristalsis and facilitates defecation, thereby improving slow transit constipation.

[0016] The present invention also provides a drug for treating slow transit constipation, characterized in that the drug comprises arginine vasopressin.

[0017] In addition to traditional oral dosage forms, we have innovatively adopted intranasal administration, a common method in neuroscience, to replace traditional oral or injectable medications, providing a nasal drop solution. Nasal administration enables direct brain-targeting delivery of arginine vasopressin. This nasal administration method minimizes adverse adherence associated with oral or injectable methods and is an effective approach for treating central nervous system diseases or for treating peripheral diseases by targeting the central nervous system.

[0018] Compared with existing technologies, the advantages of this invention are: This invention discovers a new use of arginine vasopressin in the treatment of slow-transit constipation. Further preparation of arginine vasopressin into nasal drops facilitates targeted drug delivery, resulting in better therapeutic effects, faster onset of action, and improved patient compliance. Attached Figure Description

[0019] Figure 1 Immunofluorescence image of the PVN brain region after PRV injection. The image shows neural projections from the PVN brain region to various segments of the colorectal intestine.

[0020] Figure 2 This image shows sections co-stained with PRV-EGFP and fluorescently stained neurons in the brain region. As can be seen from the image, AVP neurons in the PVN brain region are significantly co-stained with PRV.

[0021] Figure 3 To verify PVN AVP The results show how neurons promote defecation by stimulating intestinal peristalsis. Figure A illustrates stereotactic viral injection into the brain. Figure B shows immunofluorescence of a PVN brain section, revealing a high proportion of EGFP+ neurons, confirming the correct viral injection site. Figure C shows fecal samples collected from mice after a 12-hour fast (two weeks post-injection), indicating PVN... AVP Neuronal inhibition significantly reduced defecation in mice. Figure D shows the small intestine harvested 30 minutes after gavage with ink; the ink propulsion rate indicates the PVN (Propulsion Nerve). AVP Inhibition of neurons significantly slowed the small intestinal propulsion rate. Figure E shows the statistical analysis of the number and weight of feces and the small intestinal propulsion rate in the above mice over 4 hours. Figure FG shows that after anesthetizing the mice and exposing the distal colon, electrodes were inserted and continuous recording was performed for 1 hour after the signal stabilized for 30 minutes. It was found that the amplitude and frequency of electromyography in TeNT mice were reduced. Figure H shows that after grinding the distal colon tissue of the mice and extracting the supernatant, the norepinephrine content in the supernatant was detected. It was found that the NE content in TeNT mice was significantly reduced. Figure 3 In this context, EGFP and Tettox represent the experimental group and the control group, respectively.

[0022] Figure 4 To verify optical genetic activation of PVN AVP The results showed that neurons could treat constipation. Two weeks after injecting AVP neuron-specific optogenetic activation virus into the PVN brain region of C57 mice, a slow-transit constipation mouse model (STC mouse model) was created by continuous loperamide gavage for two weeks. Subsequently, PVN was activated by 470 nm light. AVP Neuronal activity was observed for 60 minutes, followed by collection of defecation parameters and measurement of propulsion rate within 4 hours. The results showed that the number and weight of defecation and the propulsion rate of small intestine were significantly improved in STC mice. Figure 4 In B, ChR2 represents the optogenetic activated neuron group.

[0023] Figure 5 A statistical graph showing the defecation (quantity, weight, and water content) in mice after nasal administration of AVP drops. The graph shows that nasal instillation of AVP solution promoted defecation.

[0024] Figure 6 This is a graph showing the blood pressure statistics of mice after nasal administration of AVP. The graph shows that nasal instillation of AVP solution did not significantly increase blood pressure in mice. Detailed Implementation

[0025] The technical solution of the present invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0026] In the following specific embodiments of the present invention, the experimental animals used were 6-8 week old male C57BL / 6 mice from Wuhan Shulaibao Company. All mice were randomly grouped and housed under a 12-hour light / 12-hour dark cycle (ZT7-ZT19, with light on), with free access to food and water, in standard laboratory cages. Subsequently, experiments such as chemogenetics and optogenetics based on neural circuit research methods were conducted.

[0027] Example 1: Study on the neural regulation mechanism of defecation in mice

[0028] 1. Using neural tracing to locate the higher neural regulatory centers for constipation

[0029] We directly injected fluorescent proteins encoding pseudorabies virus (PRV) into the ascending colon wall, transverse colon wall, descending colon wall, and rectal wall of wild-type 6-8 week old male C57BL / 6 mice; 3 mice in each group; and retrograde trans-synaptic tracking upstream neurons.

[0030] Five days after PRV-CAG-EGFP injection, whole-brain sections were prepared. We observed small clusters of EGFP+ neurons on either side of the paraventricular nucleus (PVN) of the hypothalamus in different segments of the intestine, such as... Figure 1 As shown. Figure 1 In the diagram, the ascending colon represents the ascending colon, the transverse colon the transverse colon, the descending colon the descending colon, and the rectum the rectum. These observations suggest that a small group of PVN neurons projects to various segments of the intestine, suggesting that the PVN may be a higher neural center regulating defecation.

[0031] 2. Determination of the exact neuronal type of PRV-infected PVN neurons

[0032] To determine the exact neuronal type of PVN neurons infected with PRV, we injected EGFP-PRV into the colon wall of wild-type 6-8 week old male C57BL / 6 mice. Five days later, the mice were perfused, and the PVN brain region was sectioned and stained using antibodies against corticotropin-releasing hormone neurons (CRH), oxytocin neurons (OXT), and arginine vasopressin neurons (AVP). The results are as follows: Figure 2 As shown, Figure 2 In the image, DAPI represents the cell nucleus, and Merge represents the overlapping image of red neurons and green PRVs. It can be observed that only AVP neurons significantly co-localize with PRVs. These results indicate PVN... AVP Neurons possess neural anatomical structures that extend into the gut.

[0033] 3. Suppress PVN AVP Effects of neurons on mouse defecation

[0034] In order to PVN AVP To regulate neuronal activity, stereotactic manipulation and viral injection were first performed on mice. Wild-type 6-8 week old male C57BL / 6 mice were anesthetized intraperitoneally with 2% chloral hydrate and fixed on a stereotactic apparatus (ZS-FD / W, Beijing Zhongshidichuang Technology Development Co., Ltd.). Using a 1 mm diameter glass pipette, the virus was injected into both brains at a rate of 2 nL / s under the control of a programmable nano-injector (ZS-KES-Ⅱ, Beijing Zhongshidichuang Technology Development Co., Ltd.). After injection, the pipette needle was left in place for 10 minutes before being slowly withdrawn.

[0035] Construct adenovirus AAV2 / 9-mAVP:2.6-Cre-WPRE-pA and AAV2 / 9-CAG-DIO-EGFP-2A-TetTox-pA (i.e., Figure 3 The A in Figure A are AAV-DIO-EGFP-TetTox and AAV2 / 8-hSyn-DIO-mCherry-WPRE-pA.

[0036] The function of adenovirus AAV2 / 9-mAVP:2.6-Cre-WPRE-pA is to target AVP neurons.

[0037] Adenovirus AAV2 / 9-CAG-DIO-EGFP-2A-TetTox-pA can only express TetTox with the assistance of Cre enzyme. The TetTox element is capable of inhibiting PVN. AVP The components that transmit information in neurons.

[0038] Adenovirus AAV2 / 8-hSyn-DIO-mCherry-WPRE-pA is a control virus for TetTox virus.

[0039] 80 nL of AAV2 / 9-mAVP:2.6-Cre-WPRE-pA (titer 1.70 × 10⁻⁶) was added. 12 Viral genome / mL) and AAV2 / 9-CAG-DIO-EGFP-2A-TetTox-pA (titer 1.95×10⁻⁶) 12The viral genome ( / mL) was mixed with 80 nL of AAV2 / 9-mAVP:2.6-Cre-WPRE-pA (titer 1.70 × 10⁻⁶). 12 Viral genome ( / mL) compared with control virus AAV2 / 8-hSyn-DIO-mCherry-WPRE-pA (titer 1.59 × 10⁻⁶) 12 The viral genome ( / mL) was mixed and microinjected into the bilateral PVNs of wild-type 6-8 week old male C57BL / 6 mice.

[0040] Two weeks later, the virus expressed itself, and PVN was activated. AVP Specific inhibition of neurons; collection of defecation-related indicators in mice within 4 hours; subsequent perfusion of mice, and sectioning and immunofluorescence imaging of the PVN brain region.

[0041] The results are as follows Figure 3 As shown in Figure A. Figure A is a schematic diagram of stereotactic injection of the virus into the brain. In Figure B, AAV-DIO-EGFP-TeNT is equivalent to AAV-DIO-EGFP-TetTox. Immunofluorescence of the PVN brain region slices shows a high proportion of EGFP+ neurons, proving that the virus injection site is correct.

[0042] After confirming the correct injection site, we fasted the mice for 12 hours two weeks after viral injection, and then collected feces from the mice within 4 hours. The results of Figure C showed inhibition of PVN. AVP Neurons significantly inhibited defecation in mice. After 30 min of gavage with ink, the small intestine of mice was harvested. The ink propulsion rate in Figure D showed that PVN... AVP After the neurons were inhibited, the small intestinal propulsion rate was significantly slowed down. Figure E shows the inhibition of PVN. AVP Neuronal activity significantly suppressed the quantity and weight of feces in mice, while the water content of the feces remained unchanged.

[0043] After anesthetizing the mice, we exposed the distal colon, inserted electrodes, and recorded data continuously for 1 hour after the signal stabilized for 30 minutes. Electromyography (FG) revealed decreased amplitude and frequency in the TeNT (TetTox) group, indicating suppressed intestinal peristalsis and further demonstrating the inhibition of PVN. AVP Neurons may have inhibited defecation in mice by suppressing intestinal peristalsis.

[0044] Distal colon tissue from mice was ground and the supernatant was extracted. The norepinephrine content in the supernatant was measured. The H-plot revealed a significant decrease in norepinephrine (NE) levels in TeNT mice. This suggests reduced sympathetic nerve activity in the gut, indicating PVN. AVP Neurons may regulate intestinal peristalsis through the sympathetic nervous system.

[0045] These viruses were provided by Shanghai-based Aubo Biotech Ltd. and Wuhan-based BrainVTA. In this study, the effectiveness of these viruses in pharmacogenetic experiments was confirmed. Mice injected incorrectly were excluded from further data analysis for all experiments.

[0046] Example 2: Activating PVN AVP The therapeutic effects of neuronal and intranasal AVP administration on slow-transit constipation

[0047] 1. Activate PVN AVP Neurons can improve slow-transmission constipation

[0048] Based on the above results, we found that activating PVN AVP It can promote bowel movements, therefore we hypothesize that it targets and activates PVN. AVP It may improve constipation.

[0049] To test this hypothesis, we observed optogenetic activation of PVN AVP The effect of neurons on defecation in constipated mice. Figure 4 As shown in A, the specific method is as follows:

[0050] First, an adenovirus AAV2 / 9-hEF1a-DIO-hChR2(H134R)-mCherry-WPRE-pA was constructed. This adenovirus is an optogenetic activation virus that functions only in the presence of Cre enzyme co-expression, and is used for photoactivation of PVN. AVP Neuron.

[0051] We injected AAV2 / 9-mAVP:2.6-Cre-WPRE-pA and AAV2 / 9-hEF1a-DIO-hChR2(H134R)-mCherry-WPRE-pA into wild-type 6-8 week old male C57BL / 6 mice.

[0052] Two weeks later, we induced slow transit constipation in these mice by dissolving loperamide hydrochloride (2 mg / capsule, National Drug Approval Number: H10910085, Xian Janssen Pharmaceutical Co., Ltd.) at a dose of 50 mg / kg in 1 mL of pure water and administering 100 μl by gavage at 7:00 AM and 7:00 PM daily, thus establishing a slow transit constipation mouse model (STC mouse model). The control group was given an equal volume of pure water for 7 days.

[0053] Finally, we used a 470 nm laser to target and activate PVN. AVP Neurons were activated for 60 minutes, and fecal samples were collected within 4 hours after photoactivation. Defecation parameters of mice were collected within 4 hours, and the propulsion rate of the small intestine was measured.

[0054] The results are as follows Figure 4 As shown in Figures B and C, we observed increased fecal particle count, fecal weight, and improved small intestinal propulsion rate in STC mice. This indicates an improvement in slow-transit constipation symptoms and a faster intestinal transit time in STC mice. These results strongly suggest targeting the central PVN. AVP Neurons may be a novel approach to treating slow-transit constipation.

[0055] 2. Study on the effect of intranasal administration of AVP on improving constipation

[0056] Previous studies have reported that nasal administration can result in a significant portion of the drug reaching the brain rather than the peripheral blood. This leads us to speculate whether intranasal AVP could serve as a PVN. AVP Alternatives to neuronal activation are used to treat slow-transit constipation.

[0057] To verify this hypothesis, we first established a slow-transit constipation mouse model using the method described above, and then administered AVP intranasally at a dose of 6.67 μg / (kg·d) twice daily for 2 weeks.

[0058] like Figure 5 As shown, intranasal administration of AVP significantly increased the quantity, weight, and water content of feces in mice, indicating that intranasal administration of AVP can significantly promote intestinal peristalsis and increase defecation in mice with slow-transit constipation. Figure 6 As shown, intranasal administration of AVP does not cause acute or chronic hypertension. These results collectively suggest that intranasal AVP may be a universally applicable, safe, and effective treatment for slow-transit constipation.

[0059] The above detailed embodiments describe the implementation of the present invention; however, the present invention is not limited to the specific details described in the above embodiments. Within the scope of the claims and technical concept of the present invention, various simple modifications and changes can be made to the technical solution of the present invention, and these simple modifications all fall within the protection scope of the present invention.

Claims

1. The application of arginine vasopressin in the preparation of a drug for treating slow transit constipation.

2. The application of arginine vasopressin according to claim 1 in the preparation of a medicament for treating slow-transit constipation, characterized in that, The slow-transit constipation mentioned is constipation induced by loperamide hydrochloride.

3. The application of arginine vasopressin according to claim 1 in the preparation of a medicament for treating slow-transit constipation, characterized in that, In the drug, the dosage of arginine vasopressin is 6.67-13.33 μg / (kg·d).

4. The application of arginine vasopressin according to claim 3 in the preparation of a medicament for treating slow-transit constipation, characterized in that, In the drug, the dosage of arginine vasopressin is 6.67 μg / (kg·d).

5. The application of arginine vasopressin according to claim 1 in the preparation of a medicament for treating slow-transit constipation, characterized in that, The medication is a nasal drop.

6. The application of arginine vasopressin according to claim 1 in the preparation of a medicament for treating slow-transit constipation, characterized in that, The drug also includes pharmaceutically acceptable excipients.

7. The application of arginine vasopressin according to claim 6 in the preparation of a medicament for treating slow-transit constipation, characterized in that, The pharmaceutically acceptable excipients include at least one of the following: fillers, lubricants, flavoring agents, stabilizers, desiccants, antioxidants, disintegrants, colorants, dispersants, pH adjusters, and coating materials.

8. A drug for treating slow-transit constipation, characterized in that, The drug includes arginine vasopressin.

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