Application of TPN171H in preparation of medicine for treating spinal cord injury

Abstract

The invention discloses application of TPN171H, an active fragment thereof or a derivative thereof in preparation of a medicine for treating spinal cord injury. The TPN171H shows an excellent treatment effect in spinal cord injury treatment, the levels of apoptosis factors BAX and Caspase-3 cleaved can be remarkably reduced, the level of Bcl-2 is improved, the content of a neuron marker MAP-2 is also remarkably increased, and the number of Tunel bodies and the cavitation-like structure are remarkably reduced. The intervention of the TPN171H also can reduce MNSS, and prompts that the TPN171H has the functions of promoting the spinal cord movement and coordination of the rat with spinal cord injury and improving the sensory system of the rat with spinal cord injury.

Description

Technical Field

[0001] This invention discloses a new medical application for TPN171H, belonging to the field of pharmaceutical preparation technology. Background Technology

[0002] Spinal cord injury (SCI) is one of the major disabilities treated in clinical rehabilitation settings. It is multifactorial, as patients suffer from motor and sensory impairments, as well as many other complications, throughout their lives. Numerous clinical trials have been conducted in recent years focusing on the recovery of the damaged spinal cord. However, only a few drug treatments are used clinically, and their effects on nerve regeneration and spinal cord function recovery remain very limited. Many drugs have been thoroughly investigated in large, multicenter, prospective, randomized controlled trials, including methylprednisolone sodium succinate (MPSS) and follow-up trials. Although MPSS and immunoglobulin G (IgG), chondroitin sulfate ABC (CHABC), etc., have good preclinical animal data, the primary results of these clinical trials have mostly been negative. Drugs with excellent therapeutic effects in the clinical treatment of spinal cord injury are still lacking in this field.

[0003] TPN171H (trade name Smerefi, CAS number 1229018-87-4) is a highly selective PDE5 inhibitor, belonging to the pyrimidinone class of PDE5 inhibitors, and is clinically used to treat erectile dysfunction (ED) in men. The purpose of this invention is to provide a novel use of this drug with significant efficacy in the treatment of spinal cord injury. Summary of the Invention

[0004] Based on the above objectives, the present invention first provides the application of TPN171H, its active fragment or derivative thereof in the preparation of drugs for treating spinal cord injury.

[0005] The TPN171H described in this invention, trade name: Smirnoff, CAS number 1229018-87-4, has the following molecular formula: .

[0006] In a preferred embodiment, the spinal cord injury treatment drug is a drug that inhibits apoptosis of spinal cord neurons.

[0007] In a preferred embodiment, the drug that inhibits neuronal apoptosis is a drug that reduces the level of BAX in the spinal cord.

[0008] In a preferred embodiment, the drug that inhibits spinal cord neuronal apoptosis is a drug that reduces the level of Caspase-3 cleaved in the spinal cord.

[0009] In a preferred embodiment, the drug that inhibits spinal cord neuron apoptosis is a drug that increases Bcl-2 levels.

[0010] In a preferred embodiment, the drug that inhibits spinal cord neuron apoptosis is a drug that increases the level of spinal cord MAP-2.

[0011] In a preferred embodiment, the drug that inhibits spinal cord neuronal apoptosis is a drug that reduces the number of spinal cord tunic bodies.

[0012] In a preferred embodiment, the spinal cord injury treatment drug is a drug that promotes myelin repair. TPN171H has a myelin repair promoting effect in spinal cord injury. Lubilan staining shows an increase in vacuolar structures in the spinal cord after spinal cord injury, and the spinal cord tissue structure is loose, indicating demyelination changes. However, after TPN171H intervention, there are relatively fewer vacuolar structures, suggesting that TPN171H has a myelin repair effect.

[0013] In a preferred embodiment, the spinal cord injury treatment drug is a drug that improves spinal cord motor, coordination, endurance, and / or sensory functions.

[0014] The spinal cord injury described in this invention can be acute or chronic. In a preferred embodiment, the spinal cord injury refers to chronic spinal cord injury. This invention demonstrates excellent efficacy in inhibiting spinal cord neuronal apoptosis, repairing myelin sheath, and improving spinal cord motor, coordination, endurance, and / or sensory functions by initiating TPN171H intervention 2 weeks after the establishment of a spinal cord injury (SCI) model. This demonstrates the therapeutic effect of TPN171H on chronic spinal cord injury.

[0015] The study of this invention shows that TPN171H has the function of inhibiting neuronal apoptosis and protecting neurons in spinal cord injury, especially chronic spinal cord injury. It can significantly reduce the levels of apoptosis factors BAX and Caspase-3 cleaved, increase the level of Bcl-2, significantly increase the content of neuronal marker MAP-2, improve neuronal cell morphology, and significantly reduce the number of Tunel bodies. TPN171H intervention can also significantly reduce vacuolar structures after spinal cord injury, suggesting that TPN171H has a myelin repair effect.

[0016] TPN171H intervention can also reduce MNSS, suggesting that TPN171H can promote spinal cord motor and coordination function in rats with spinal cord injury. In the rotarod test, the TPN171H intervention group also showed better endurance and coordination, and significantly improved the function of the sensory system in rats with spinal cord injury, with a significant increase in the pain threshold of the rats.

[0017] In conclusion, TPN171H shows excellent application prospects in the preparation of drugs for the treatment of spinal cord injury. Attached Figure Description

[0018] Figure 1 The figure shows the Western blot results of the effect of TPN171H intervention on PDE-5 content in spinal cord tissue; Figure 2 This is a quantitative analysis diagram of the effect of TPN171H intervention on PDE-5 content in spinal cord tissue using Western blot. Figure 3 The figure shows the results of Western blot experiments on the effects of TPN171H intervention on neuronal apoptosis-related markers. Figure 4 This is a quantitative analysis diagram of the effects of TPN171H intervention on neuronal apoptosis-related markers using Western blot. Figure 5 To observe the effect of TPN171H intervention on neuronal cell morphology using NISSL staining; Figure 6 The effect of TPN171H intervention on the number of Tunel small bodies; Figure 7 The effect of TPN171H intervention on Caspase-3 cleaved levels; Figure 8 The effect of TPN171H intervention on spinal cord vacuolar structures; Figure 9 The effects of TPN171H intervention on spinal cord motor and coordination function in rats with chronic spinal cord injury; Figure 10 The effects of TPN171H intervention on endurance and coordination in rats with chronic spinal cord injury; Figure 11 The effects of TPN171H on the sensory system of rats with chronic spinal cord injury. Detailed Implementation

[0019] The present invention will be further described below with reference to specific embodiments, and the advantages and features of the present invention will become clearer as a result of the description. However, these embodiments are merely exemplary and do not constitute any limitation on the scope of protection defined by the claims of the present invention.

[0020] Example 1. Effect of TPN171H on PDE-5 content in spinal cord tissue The experiment was conducted in accordance with animal welfare principles. SPF-grade male SD rats (250±20g) were used to establish a spinal cord injury (SCI) model. The rats were acclimatized for 7 days, fasted for 12 hours before surgery, and anesthetized with a 5% isoflurane-oxygen mixture. The concentration was maintained at 1.5-2% during the operation. The patient was fixed in a prone position to a stereotactic frame, and the heating pad was adjusted for warmth. A midline incision was made (T8-T12 level), and the paraspinal muscles were bluntly dissected. A T9-T11 laminectomy was performed using microscopic bone forceps (preserving the integrity of the dura mater), exposing the T10 spinal cord segment. The spinal cord impact head was a 2.5mm stainless steel punch. A 10g hammer was dropped freely from a height of 12.5mm, with a contact time of 100ms (the punch lightly touches the dura mater surface to zero, triggering the impact device to complete the instantaneous impact). The characteristic tail spasm response was observed. After confirming successful injury, the surgical field was rinsed with saline and hemostasis was achieved. The muscles / skin were sutured layer by layer (using 5-0 absorbable sutures). The patient was kept in a single cage until consciousness was restored. 800 units of penicillin G were injected subcutaneously for 3 consecutive days, and artificial urination was performed 4 times a day until spontaneous urination was restored. Nine SD rats from the same batch were used as normal controls. The SCI model rat intervention group (n=12) was administered TPN171H at a dose of 16 mg / kg by gavage starting two weeks after surgery, while the control group (n=9) was administered an equal volume of physiological saline by gavage starting two weeks after surgery. After four weeks of continuous observation, the rats were euthanized, and fresh specimens were collected after cardiac perfusion.

[0021] Phosphodiesterase type 5 (PDE-5) is an intracellular enzyme widely distributed in various tissues and organs. Fresh spinal cord tissue specimens from the above experimental endpoints (normal (n=3), control (n=3), and intervention (n=3) were used for Western blotting to quantitatively analyze PDE-5. The data are shown in Table 1 (PDE-5). The results are as follows: Figure 1 and Figure 2 As shown. Figure 1 The top center shows the results for the internal control (β-actin), and the bottom center shows the results for PDE-5. From left to right, the order of the samples is: normal, control, intervention, normal, model, intervention, normal, model, intervention. The internal control results show that PDE-5 is highly expressed in rat spinal cord tissue after spinal cord injury, while the PDE-5 content in spinal cord tissue decreases after intervention with TPN171H (a PDE-5 inhibitor, PDE-5i).

[0022] Table 1. Calculation of relative protein expression levels

[0023] Example 2. Effect of TPN171H on inhibiting apoptosis-related factors in spinal cord tissue TPN171H has the function of inhibiting neuronal apoptosis and protecting neurons in chronic spinal cord injury. Fresh spinal cord specimens from the above experimental endpoints (normal (n=3), control (n=3), and intervention (n=3) were subjected to Western blotting, NISSL staining of spinal cord sections, and TUNEL staining. Western blotting results showed that in the rat model of spinal cord injury, there were significant changes in the upregulation of pro-apoptotic factors BAX and Caspase-3 and the downregulation of the anti-apoptotic factor Bcl-2 (P<0.05). Changes in the morphology of spinal cord neurons were also observed, including cell collapse and nuclear pyknosis. After TPN171H intervention, the level of BAX molecules decreased (P<0.05), and the level of Bcl-2 molecules increased (P<0.05) in the injury model. Simultaneously, NISSL staining of spinal cord sections showed improved neuronal cell morphology, increased levels of spinal cord neuronal markers (P<0.05), significantly decreased levels of Caspase-3 (an enzyme that initiates programmed cell death) (P<0.05), and significantly increased levels of the neuronal marker MAP-2 (see [link to relevant documentation]). Figure 3 , Figure 4 and Figure 5 (See Table 1 for data).

[0024] Tunel bodies are cellular structures formed during apoptosis, as indicated by immunofluorescence results (see [link]). Figure 6 (Data shown in Table 2) After spinal cord injury in rats, the number of Tunel bodies increased significantly, while after TPN171H intervention, the number of Tunel bodies decreased significantly.

[0025] Table 2. Cell counts under 200×2x microscopy after Tunel staining.

[0026] Spinal cord tissue samples were stained with the apoptosis marker Caspase-3 cleaved for fluorescence staining. Results (see [link to results]). Figure 7 (Data shown in Table 3) indicated that the Caspase-3 cleaved content increased significantly after injury (P < 0.01), and the Caspase-3 cleaved content decreased after TPN171H intervention, with no significant difference from that of normal spinal cord (P > 0.05).

[0027] Table 3. Caspase fluorescence staining (green light) intensity values ​​for each group

[0028] TPN171H promotes myelin repair in chronic spinal cord injury. Luvular blue staining revealed an increase in vacuolar structures and loosening of the spinal cord tissue after injury, suggesting demyelination. However, after TPN171H intervention, the number of vacuolar structures was relatively reduced, indicating that TPN171H has a myelin repair function (see [link to TPN171H]). Figure 8 ).

[0029] Example 3. Improvement of spinal cord motor and coordination function in rats with chronic spinal cord injury by TPN171H 1. MNSS score As previously mentioned, drug intervention was initiated on day 14 after SCI modeling. The MNSS neurological function scores of each group of animals were assessed. Rats were allowed to acclimatize for 30 minutes in a suitable environment, followed by sequential testing of motor function (observation of limb symmetry, gait, and forelimb extension), sensory function (tactile and pain responses), balance and coordination (balance beam and wheel tests), and reflex function (righting reflex and corneal reflex). Performance was scored according to standards, and the scores were tallied; higher total scores indicated more severe neurological impairment. Assessment time points were 0, 3, 7, 14, 21, and 28 days after administration. Spinal cord injury-regulated motor and coordination functions were also affected. The MNSS scores of rats (normal is 0, higher scores indicate poorer function) showed that after administration of TPN171H via gavage to rats following spinal cord injury, the MNSS scores were significantly lower than the control group after day 14, suggesting that TPN171H can promote improvement in spinal cord motor and coordination function in rats with chronic spinal cord injury (see [link to relevant documentation]). Figure 9 (See Table 4 for data).

[0030] Table 4. Total MNSS scores at each time point

[0031] 2. Rotating bar experiment The rotado test is a commonly used behavioral test primarily used to assess motor coordination, balance, and endurance in rats. First, rats are acclimatized to the experimental environment and a stationary rotado. Then, they are trained to walk on the rotado at a low speed (4 rpm). During the formal test, rats are placed on the rotado, and the rotation speed is gradually increased. The latency of falling off the rotado is recorded. Each rat is tested 3-5 times, and the average value is taken. Finally, motor coordination, balance, and endurance are assessed by comparing the latency of different groups. In the rotado test, the TPN171H intervention group also showed better endurance and coordination (intervention vs. control, P < 0.05). (See...) Figure 10 (See Tables 5-6 for data).

[0032] Table 5. Rotation test results for each group of animals (rotation time on the rod, unit: s)

[0033] Table 6. Rotary bar test results for each group of animals (rotary bar drop speed, unit: C / min)

[0034] Example 4. Effects of TPN171H on the sensory system of rats with chronic spinal cord injury The Von Frey Filament Test in rats involves applying a series of Von Frey cilia of varying intensities vertically to the soles of the rats' feet and observing whether they exhibit pain responses such as paw withdrawal or licking, in order to determine the pain threshold. After the rats are placed in a transparent cage to acclimate to their environment, stimulation is gradually applied, starting with the finest cilia. The minimum ciliary intensity that elicits a pain response is recorded, and the test is repeated 3-5 times, with the average value taken. Pain sensitivity is assessed by comparing the pain thresholds of different groups. Following spinal cord injury, the sensory system of rats is also affected. In this chronic spinal cord injury model intervention experiment, the pain threshold of rats was significantly decreased (P < 0.05), while after intervention with TPN171H, the pain threshold of rats significantly increased (P < 0.001), with no statistically significant difference compared to normal rats (P > 0.05). See also Figure 11 The data is shown in Table 7.

[0035] Table 7. Ciliary tingling test (Von Frey, K value) for each group of animals .

Claims

1. Application of TPN171H, its active fragment or derivatives in the preparation of drugs for the treatment of spinal cord injury.

2. The application according to claim 1, characterized in that, The spinal cord injury treatment drug is a drug that inhibits the apoptosis of spinal cord neurons.

3. The application according to claim 2, characterized in that, The drug that inhibits spinal cord neuron apoptosis is a drug that reduces the level of BAX in the spinal cord.

4. The application according to claim 2, characterized in that, The drug that inhibits spinal cord neuron apoptosis is a drug that reduces the level of Caspase-3 cleaved in the spinal cord.

5. The application according to claim 2, characterized in that, The drug that inhibits spinal cord neuron apoptosis is a drug that increases Bcl-2 levels.

6. The application according to claim 2, characterized in that, The drug that inhibits spinal cord neuron apoptosis is a drug that increases the level of MAP-2 in the spinal cord.

7. The application according to claim 2, characterized in that, The drug that inhibits spinal cord neuron apoptosis is a drug that reduces the number of spinal cord tunic bodies.

8. The application according to claim 1, characterized in that, The spinal cord injury treatment drug is a drug that promotes myelin sheath repair.

9. The application according to claim 1, characterized in that, The spinal cord injury treatment drugs are those that improve spinal cord motor, coordination, endurance, and / or sensory functions.

10. The application according to claim 1, characterized in that, The spinal cord injury is a chronic spinal cord injury.

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