CN108837144A - The new opplication of Triptorelin - Google Patents

Abstract

The application belongs to the field of medicine and discloses a new application of triptorelin, especially the application of triptorelin in the preparation of medicaments for treating depression. Experiments have shown that in the forced swimming test, triptorelin can significantly reduce the immobility time of depression model mice induced by chronic restrictive stress, lipopolysaccharide, plantar injection of CFA and early social isolation, indicating that it has a positive effect on depression model mice. Obvious improvement. At the same time, triptorelin can significantly increase the expression of chronic restrictive stress-induced mouse hippocampal BDNF and neuronal synaptic plasticity-related genes c-fos, egr1, arc, npas4. Therefore, triptorelin has significant antidepressant activity, can be used to prepare medicines for treating depression, and has high clinical application value and development prospect.

Description

New Application of Triptorelin

technical field

The invention belongs to the field of medicine, and in particular relates to a new application of triptorelin, in particular to an application of triptorelin in the preparation of medicaments for treating depression.

Background technique

Depression is an affective disorder mental disease that seriously affects physical and mental health. It is a group of mood disorders, affective disorders and cognitive disorders caused by various reasons with depression as the main symptom. Depression is characterized by depressed mood, self Situation-centered clinical symptoms are related to biological, psychological and social factors in their occurrence, development and outcome. Depression has a high prevalence rate, high recurrence rate, high disease burden and high suicide mortality rate. According to the Global Burden of Disease Survey, it is estimated that by 2020, depression will become the second largest disease after cardiovascular disease in China. Research on the pathogenesis, prevention and treatment of depression has great medical significance and practical needs.

Medication is the main treatment for moderate to severe depressive episodes. The application of traditional tricyclic and tetracyclic antidepressants and monoamine oxidase inhibitors has been significantly reduced due to relatively large adverse reactions. At present, the clinical first-line antidepressants mainly include selective serotonin reuptake inhibitors, serotonin and norepinephrine reuptake inhibitors, etc., but the onset of these drugs is slow and the action spectrum is narrow. Easy to relapse. At present, the entire industry is developing more effective therapeutic drugs with quicker results, fewer side effects. Studies have shown that multiple levels of physiological systems are related to the pathogenesis of depression, but the current drug therapy for depression still completely relies on drugs that act on the monoamine transmitter system. However, according to relevant statistical reports, such drugs not only have limited effects in the treatment of patients with depression, but also have side effects that cannot be ignored.

For the pathogenesis of depression, the academic circles put forward a new viewpoint in the 21st century, the neurotrophic hypothesis, which holds that depression is closely related to central brain-derived neurotrophic factor (BDNF) (Duman RS, Monteggia LM.Aneurotrophic model for stress-related mood disorders. Biol Psychiatry. 2006, 59, 1116-1127). BDNF is widely distributed in the brain and regulates important physiological processes in vivo such as nerve growth and development and synaptic plasticity. It is known that BDNF can bind to its receptor tyrosine kinase B (TrkB), through downstream mitogen-activated protein kinase (MAPK)/extracellular regulated kinase (ERK), phosphatidylinositol-3-kinase (PI-3K) / protein kinase B (AKT) and Ca ²⁺ / calmodulin kinase (CaM) several signaling pathways to activate the nuclear cyclic adenosine monophosphate response element binding protein (CREB), thereby promoting synaptic plasticity (Qi X, Lin W , Wang D, et al. A role for the extracellular signal-regulated kinase signal pathway in depressive-like behavior. Behav Brain Res. 2009, 199: 203-209). A number of research reports have confirmed that the expression of BDNF protein in the serum and hippocampus of depressed patients and animal models of depression is significantly lower than that of normal individuals; long-term use of clinical antidepressant drugs and electroconvulsive therapy (ECT) can reverse these changes ( Molteni R, Calabrese F, Bedogni F, et al. Chronic treatment with fluoxetine up-regulates cellular BDNF mRNA expression in rat dopaminergic regions. Int J Neuropsychopharmacol. 2006, 9, 307-317). Therefore, considering the importance of BDNF and neuronal synaptic plasticity in depression, it may be possible to treat depression by up-regulating BDNF and promoting neuronal synaptic plasticity.

Triptorelin is a GnRH agonist, its chemical structure is as follows, its molecular formula is C ₆₄ H ₈₂ N ₁₈ O ₁₃ ; its molecular weight is 1311.45; its CAS number is 57773-63-4, and it is a white powder. Its chemical formula is:

Triptorelin is generally used to treat patients who need to reduce steroid hormones such as androgens and estrogens to low levels. At present, there is no report in the literature that triptorelin can treat depression, and there is no relevant research on the treatment of central nervous system diseases based on this.

Contents of the invention

In view of this, the present invention provides a new application of the GnRH agonist triptorelin, that is, the application of triptorelin in the preparation of medicaments for treating depression.

In one embodiment, the real-time fluorescence quantitative PCR method was used to investigate the effect of triptorelin on the expression of BDNF in the hippocampus of depression model mice induced by chronic restrictive stress and the expression of genes c-fos, egr1, arc, and npas4 related to neuronal synaptic plasticity. The results showed that chronic restrictive stress down-regulated the expression of hippocampal BDNF and neuronal synaptic plasticity-related genes c-fos, egr1, arc, npas4, and triptorelin could reverse the up-regulation of hippocampal BDNF and neuronal synaptic plasticity-related genes c- Expression of fos, egr1, arc, npas4. Therefore, the present invention provides the application of triptorelin in the preparation of drugs for promoting the expression of brain-derived neurotrophic factor BDNF and the application of triptorelin in the preparation of promoting the expression of neuron synaptic plasticity-related genes c-fos, egr1, arc, npas4 application in medicines.

Further, in one embodiment, the present invention uses triptorelin as the research object. First, lipopolysaccharide is used to establish a mouse depression model, and the mice are intraperitoneally injected with triptorelin, a GnRH agonist, and evaluated by forced swimming test. Antidepressant effects of the GnRH agonist triptorelin. The results showed that intraperitoneal injection of LPS could significantly increase the immobility time of mice in the forced swimming test, and pretreatment with triptorelin (0.2 mg/kg) could significantly reduce the immobility time of mice in the forced swimming test. Show that triptorelin can significantly improve LPS-induced depression-like behavior in mice.

In one embodiment, the present invention uses triptorelin as the research object, adopts chronic restraint stress (CRS) to establish mouse depression model, gives mouse intraperitoneal injection of GnRH agonist triptorelin, through forced swimming test To evaluate the antidepressant effect of the GnRH agonist triptorelin. The results showed that chronic restrictive stress can significantly increase the immobility time of mice in the forced swimming test. Triptorelin (0.2mg/kg) pretreatment can significantly reduce the immobility time in the forced swimming test of mice. It shows that the GnRH agonist triptorelin can significantly improve the depression-like behavior of mice induced by chronic restrictive stress.

In one embodiment, the present invention takes triptorelin as the research object, adopts plantar injection of CFA to establish a mouse depression model, injects GnRH agonist triptorelin into mice intraperitoneally, and evaluates GnRH agonism by forced swimming test Antidepressant effect of triptorelin. The results showed that plantar injection of CFA could significantly increase the immobility time of mice in the forced swimming test. Triptorelin (0.2mg/kg) pretreatment can significantly reduce the immobility time in the forced swimming test of mice. It shows that triptorelin can significantly improve the depression-like behavior of mice induced by plantar injection of CFA.

In one embodiment, the present invention takes triptorelin as the research object, adopts early social isolation to establish a mouse depression model, injects the GnRH agonist triptorelin intraperitoneally to mice, and evaluates the GnRH agonist by forced swimming test Antidepressant effect of triptorelin. The results showed that early social isolation can significantly increase the immobility time of mice in the forced swimming test. Triptorelin (0.2mg/kg) pretreatment can significantly reduce the immobility time in the forced swimming test of mice. It shows that triptorelin can significantly improve the early social isolation-induced depression-like behavior in mice.

In summary, triptorelin has a significant improvement effect on depression model mice. At the same time, triptorelin can significantly increase the expressions of BDNF and neuronal synaptic plasticity-related genes c-fos, egr1, arc, npas4 induced by chronic restrictive stress in mouse hippocampus. It shows that triptorelin can be used to prepare medicine for treating depression. Therefore, the present invention provides the application of triptorelin in the preparation of medicaments for treating depression.

Wherein, the depression is lipopolysaccharide (LPS)-induced depression, chronic restrictive stress-induced depression, plantar injection of CFA-induced depression, and early social isolation-induced depression.

Preferably, the medicament of the present invention includes an effective dose of triptorelin and pharmaceutically acceptable auxiliary materials.

Those skilled in the art can directly or indirectly add various commonly used pharmaceutically acceptable excipients required for preparing different dosage forms of the triptorelin, and use conventional pharmaceutical preparation methods to prepare commonly used injection preparations.

Preferably, the injection preparation is injection or powder injection.

It can be known from the above technical solutions that the present invention provides a new application of triptorelin, especially the application of triptorelin in the preparation of medicaments for treating depression. Experiments have shown that in the forced swimming test, triptorelin can significantly reduce the immobility time of depression model mice induced by chronic restrictive stress, lipopolysaccharide, plantar injection of CFA and early social isolation, indicating that it has a positive effect on depression model mice. Obvious improvement. At the same time, triptorelin can significantly increase the expressions of BDNF and neuronal synaptic plasticity-related genes c-fos, egr1, arc, npas4 induced by chronic restrictive stress in mouse hippocampus. Therefore, triptorelin has significant antidepressant activity, can be used to prepare medicines for treating depression, and has high clinical application value and development prospect.

Description of drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings that are required in the description of the embodiments or the prior art.

Fig. 1 shows the statistical result diagram of the effect of triptorelin in embodiment 1 on the behavior of the depression model mice induced by lipopolysaccharide (LPS);

Fig. 2 shows the statistical result diagram of the influence of triptorelin in embodiment 2 on the behavior of depression model mice induced by chronic restrictive stress;

Fig. 3 shows the statistical result figure of embodiment 3 triptorelin on the behavior of depression model mice induced by plantar injection of CFA;

Fig. 4 shows the statistical result diagram of the influence of triptorelin in embodiment 4 on the behavior of early social isolation-induced depression model mice;

Fig. 5 is a graph showing the statistical results of the effects of triptorelin in Example 5 on the expression of BDNF in the hippocampus of depression model mice induced by chronic restrictive stress and neuronal synaptic plasticity-related genes c-fos, egr1, arc, and npas4.

Detailed ways

The invention discloses a new application of triptorelin. Those skilled in the art can refer to the content of this article to appropriately improve the process parameters to achieve. In particular, it should be pointed out that all similar replacements and modifications are obvious to those skilled in the art, and they are all considered to be included in the present invention. The present invention has been described through preferred embodiments, and it is obvious that relevant personnel can make changes or appropriate changes and combinations to the methods described herein without departing from the content, spirit and scope of the present invention to realize and apply the technology of the present invention.

In order to further understand the present invention, the technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only some of the embodiments of the present invention, not all of them. . Based on the embodiments of the present invention, all other embodiments obtained by persons of ordinary skill in the art without making creative efforts belong to the protection scope of the present invention.

Unless otherwise specified, the reagents involved in the examples of the present invention are all commercially available products, which can be purchased through commercial channels.

Embodiment 1: The effect of triptorelin on the behavior of LPS-induced depression model mice

Experimental animals: 36 male C57 mice, 3 months old, weighing about 25g. Animals were kept at room temperature (22±2)°C, humidity (45%-65%) and light and dark (12h:12h) environment, free to eat and drink. Animal grouping and treatment Mice were divided into 3 groups according to random number table method: normal control group (NS, n=12), lipopolysaccharide model group (LPS, n=12), triptorelin (LPS+TRIP, n=12) 12), intraperitoneal injection (i.p) once a day, a total of 1 week of treatment, 1h after the last drug injection, LPS group and LPS+TRIP group intraperitoneal injection of 0.83mg/kg LPS, open field test and Forced to swim for experiments. The result is shown in Figure 1.

Since the shortening of the animal's immobility time in the classic animal model of depression may be caused by the central excitatory effect of the drug, the present invention also carried out the mouse open field test (Open field test) simultaneously to check the autonomous activity of the mouse , to avoid the interference of central stimulants. Mice forced to swim have been used to screen many antidepressant drugs, and most clinically effective antidepressants have also been shown to be effective in reducing immobility time in the forced swim test. The so-called immobility refers to "the animal stops struggling in the water, or is in a floating state, only exposing the nostrils to keep breathing, and only small limb movements to keep the head floating on the water." Desperation in animal behavior due to the animal being unable to escape the harsh environment by swimming in a compulsive state. This model method is simple and reliable, and has been widely used in the evaluation of antidepressants.

The results showed that in the open field test, the activity of the mice in each group had no change; intraperitoneal injection of LPS could significantly increase the immobility time of the mice in the forced swimming test. Triptorelin (0.2mg/kg) pretreatment can significantly reduce the immobility time in the forced swimming test of mice. The experimental results showed that triptorelin could significantly improve LPS-induced depression-like behavior in mice.

Embodiment 2: the influence of triptorelin on the behavior of depression model mice induced by chronic restraint stress (CRS)

Experimental animals: 36 male C57 mice, 3 months old, weighing about 25g. Animals were kept at room temperature (22±2)°C, humidity (45%-65%) and light and dark (12h:12h) environment, free to eat and drink. Animal grouping and treatment Mice were divided into 3 groups according to random number table method: normal control group (Control, n=12), chronic restraint stress model group (CRS, n=12), triptorelin (CRS+TRIP, n=12), chronic restraint stress mice were subjected to 6h restraint stress every day, and the stress was continuous for 3 weeks; The drug was injected 1 hour before the last stress, and the open field test and the forced swimming test were performed 24 hours after the last stress. The result is shown in Figure 2.

The results showed that: in the open field experiment, the activity of the mice in the CRS group was significantly reduced, and pretreatment with triptorelin (0.2 mg/kg) could significantly increase the activity of the model mice; The immobility time increased significantly in the swimming test. Triptorelin (0.2mg/kg) pretreatment can significantly reduce the immobility time in the forced swimming test of mice. The experimental results show that the GnRH agonist triptorelin can significantly improve the depression-like behavior of mice induced by chronic restrictive stress.

Embodiment 3: The effect of triptorelin on the behavior of depression model mice induced by plantar injection of CFA

Experimental animals: 24 male C57 mice, 3 months old, weighing about 25g. Animals were kept at room temperature (22±2)°C, humidity (45%-65%) and light and dark (12h:12h) environment, free to eat and drink. Animal grouping and treatment The mice were divided into 3 groups according to the random number table method: normal control group (Control, n=8), plantar injection of CFA group (CFA, n=8), triptorelin (CFA+TRIP, n =8), plantar injection of CFA is a classic model of chronic inflammatory pain and depressive behavior; triptorelin once a day i.p, a total of 3 weeks of treatment, open field test and forced swimming began 24 hours after the last drug experiment. The result is shown in Figure 3.

The results showed that in the open field test, there was no difference in the activity of the mice in each group; the plantar injection of CFA could significantly increase the immobility time of the mice in the forced swimming test. Triptorelin (0.2mg/kg) pretreatment can significantly reduce the immobility time in the forced swimming test of mice. The experimental results showed that triptorelin could significantly improve the depression-like behavior of mice induced by plantar injection of CFA.

Example 4: Effects of Triptorelin on the Behavior of Depression Model Mice Induced by Early Social Isolation

Animals: 30 male C57 mice, 3 weeks old, animal grouping and treatment The mice were divided into 3 groups according to the random number table method: normal control group (Control, n=10), early social isolation group (SI, n=10 ), triptorelin (SI+TRIP, n=10), early social isolation of mice at 3 weeks after birth is a classic model of depressive behavior; from the 8th week, SI+TRIP group was treated with triptorelin, 1 i.p. a day for 3 weeks, open field test and forced swimming test started 24 hours after the last dose of medicine. The result is shown in Figure 4.

The results showed that in the open field test, there was no difference in the activity of the mice in each group; early social isolation could significantly increase the immobility time of the mice in the forced swimming test. Triptorelin (0.2mg/kg) pretreatment can significantly reduce the immobility time in the forced swimming test of mice. The experimental results showed that triptorelin could significantly improve the early social isolation-induced depression-like behavior in mice.

Example 5: The effect of triptorelin on the expression of BDNF and neuron synaptic plasticity-related genes c-fos, egr1, arc, npas4 in the hippocampus of depression model mice induced by chronic restrictive stress

Experimental animals: 24 male C57 mice, 3 months old, weighing about 25g. Animals were kept at room temperature (22±2)°C, humidity (45%-65%) and light and dark (12h:12h) environment, free to eat and drink. Animal grouping and treatment Mice were divided into 3 groups according to random number table method: normal control group (Control, n=8), chronic restraint stress model group (CRS, n=8), triptorelin (CRS+TRIP, n=8), mice with chronic restraint stress were subjected to restraint stress for 6 hours a day, and the stress was continuous for 3 weeks; triptorelin was treated once a day i.p. for a total of 3 weeks, and the drug was injected 1 hour before each restraint stress .

24 hours after the last stress, the mice were anesthetized by intraperitoneal injection of 4% chloral hydrate. After the anesthesia was complete, hippocampal tissue was taken for RNA extraction. Total tissue RNA was extracted, reverse transcription, and real-time fluorescent quantitative PCR were used to detect the effects of triptorelin on the expression of BDNF and neuronal synaptic plasticity-related genes c-fos, egr1, arc, and npas4 in the hippocampus of depression model mice induced by chronic restrictive stress Impact.

Among them, the methods of RNA extraction and reverse transcription are as follows:

(a) Trizol extraction of mRNA and reverse transcription into cDNA: add 0.8ml Trizol to fully homogenize the tissue, let it stand in the EP tube for 10 minutes; centrifuge at 12,000g for 15 minutes, discard the precipitate; add 300μl chloroform, mix well, and place at room temperature for 15 minutes; 4°C, 12,000g, centrifuge for 15min, absorb the upper aqueous phase into another centrifuge tube, do not absorb the middle layer; add an equal volume of isopropanol to mix, and let stand at room temperature for 5-10min; 4°C, 12,000g, centrifuge for 10min , discard the supernatant, and the RNA sinks to the bottom of the tube; add 1ml of 75% ethanol to suspend the RNA pellet; centrifuge at 8000g at 4°C for 5min, discard the supernatant; dry at room temperature or vacuum for 5-10min; add 20μl DEPC water, and place in a 60°C water bath Dissolve; reverse transcribe into cDNA according to the ReverAid TM First Strand cDNA Synthesis Kit kit.

(b) RT-PCR: Reaction system: SYBR Green Mix 10μl; upstream and downstream primers (2μM) each 2μl; cDNA 1μl; ddH ₂ O 5μl (total system 20μl)

Reaction program: Step 1: 5min at 95°C; Step 2: 95°C for 15s, 60°C for 15s, 72°C for 45s, 40 cycles; Step 3: Melting curve analysis

The amplification of the target gene was normalized with the internal reference GAPDH, and the relative expression of the target gene was calculated by the 2-ddct method.

The Q-PCR primers used are listed in Table 1.

Table 1: Q-PCR Primers

The real-time fluorescence quantitative PCR method was used to detect the expression of triptorelin on hippocampus BDNF and neuronal synaptic plasticity-related genes c-fos, egr1, arc, and npas4 in chronic restrictive stress-induced depression model mice. The results are shown in Figure 5.

The results showed that the expression of BDNF, c-fos, egr1, arc, and npas4 in the hippocampus was restricted by chronic restrictive stress, and triptorelin could reverse the expression.

The above descriptions are only preferred embodiments of the present invention, and are not intended to limit the present invention. Any modifications, equivalent replacements and improvements made within the spirit and principles of the present invention should be included in the protection of the present invention. within range.

Claims (6)

1. application of the Triptorelin in the drug that preparation promotes brain-derived neurotrophic factor BDNF expression.

2. Triptorelin promotes neuronal synaptic plasticity related gene c-fos, egr1, arc, the medicine of npas4 expression in preparation Application in object.

3. application of the Triptorelin in the drug of preparation treatment depression.

4. application according to claim 3, the depression is answered for the depression of lipopolysaccharides (LPS) induction, chronic limitation Swash the depression of induction, the depression of subplantar injection CFA induction, the depression of early stage society isolation induction.

5. being applied described in -4 any one according to claim 1, which is characterized in that the drug includes the Qu Purui of effective dose Woods and pharmaceutically acceptable auxiliary material.

6. applying according to claim 5, which is characterized in that the drug is ejection preparation.