JP5861215B2 - Neuropathic pain relieving drug and antidepressant drug using neuropeptide - Google Patents

Description

The present invention relates to a novel medical use of neuropeptides. More specifically, the present invention relates to a neuropathic pain reducing drug and an antidepressant drug for mammals utilizing such medical use.

  A neuropeptide is a molecule (peptide) in which a plurality of amino acids are bonded via peptide bonds, and has a role of transmitting information at synapses and neuromuscular junctions.

Such neuropeptides have long been isolated from tissue extracts of various animal species, and many findings have been obtained. Reflecting changes in the amount of accumulated knowledge, certain views on neuropeptides have also changed over time.
Currently, as a certain view of neuropeptides, the structure and activity properties of the neuropeptides change as the animal's affinity diverges, and there are usually large differences in the properties of neuropeptide groups belonging to different animal gates. (Non-patent document 1, Non-patent document 2). This suggests that many highly homologous neuropeptides are distributed within the same animal phylum, but the structure and properties of these neuropeptide groups vary greatly depending on the animal phylum.

As described above, the activity and structure of neuropeptides are usually different due to the difference in animal phylums and the distance of related relationships. An exception is that mollusc neuropeptides are active in annelids, which are different animal kingdoms, and that their neuropeptides are similar in structure as if they belonged to the same animal kingdom. (Non-patent document 1, Non-patent document 2).
With some exceptions, it is very rare for neuropeptides to be active or structurally similar in different phylums. Therefore, for example, even in the mollusc neuropeptides that have been studied extensively, there are known reports that the same effect as in the mollusc has been confirmed in mammals that are different animal gates and distantly related. There are no known examples of human medical use.

On the other hand, neuropathic pain (Neuropathic Pain, Neuralgia) refers to a type of chronic pain disease caused by peripheral and central nerve disorders or functional disorders. Representative examples of neuropathic pain include diabetic pain, cancer pain, neuralgia and the like. As characteristic pain symptoms, hyperalgesia that feels pain stimulation more strongly, and allodynia that feels pain and tactile sensation that does not become pain stimulation originally are shown.
Pain caused by such neuropathic pain has already lost its physiological role of warning of tissue damage inherent in pain, and pain itself is an obstacle. Therefore, removing the pain caused by neuropathic pain is the therapeutic purpose of patients, and analgesics are used as drug treatment.

  As analgesics, narcotic analgesics such as morphine and nonsteroidal anti-inflammatory drugs (NSAIDs) such as indomethacin have been known. However, these analgesics generally have little effect on neuropathic pain (Patent Document 1, Non-Patent Document 3, Non-Patent Document 4). In particular, narcotic analgesics have little effect, and the lack of analgesic effect of narcotic analgesics is a major feature of neuropathic pain, which is used to diagnose neuropathic pain (Non-Patent Document 5).

  In this way, since conventional analgesics do not have therapeutic effects on neuropathic pain, selective serotonin reuptake inhibition, which is now called a third-generation antidepressant, is one of the analgesics for neuropathic pain. An agent (hereinafter referred to as SSRI) is used (Patent Document 2). However, SSRIs may require cardiovascular side effects due to psychotic symptoms, hypersensitivity, and other concomitant medications, and require the guidance of specialists because of the risk of developing side effects called serotonin syndrome It is a very difficult drug to handle. The fourth-generation serotonin / norepinephrine reuptake inhibitor (SNRI), which has the least side effects, is a drug that requires careful handling because it interacts with concomitant drugs in the same way as SSRI. (Patent Document 1).

As described above, in neuropathic pain, existing analgesics are ineffective, or even currently used analgesics are difficult to handle and are not necessarily safe. is there. Against this background, there is currently no sufficient therapeutic effect on neuropathic pain.

JP 2007-332030 A Special Table 2004-513916

Yojiro Sooka, Chemistry and Biology, vol.36, No.3, 1998 Y Muneoka et al, Perspectives in Comparative Endocrinology, 1994, pp.109-118 TheLancet, vol.353, 1999, p.1959-1966 History of Medicine, vol.203, No.1, 2002, p.65-6 History of Medicine, vol.189, No.10, 1999, p.751-755

  As described above, there are no drugs that satisfy the treatment of neuropathic pain, and development of such drugs is strongly desired. In view of such circumstances, an object of the present application is to develop an excellent neuropathic pain reducing drug with few side effects.

  As a result of earnest research, the present inventors have surprisingly found that APGWamide, a neuropeptide derived from a mollusk that is considered to have no effect on mammals as a current technical common sense, is a mammal. It was found to act on diabetic model rats and significantly reduce allodynia. In addition, the C-terminal peptides PGWamide and GWamide were found to exhibit the same action. We also found that APGWamide increases serotonin levels in the anterior cingulate gyrus of rats.

  Based on this finding, the inventors have made further studies and completed an invention relating to a neuropathic pain relieving drug and an antidepressant drug for mammals containing a peptide having GWamide as a basic skeleton as an active ingredient. .

That is, the present invention has the following configuration.
The first configuration of the present invention is a neuropathic pain reducing drug for mammals or an antidepressant drug for mammals, characterized by comprising a peptide represented by the following formula (1) as an active ingredient.
xy-Gly-Trp-NH ₂ (1)
(Both x and y are 0 or 1 amino acid selected from Pro, Gly, Ala, Val, Leu, Ile, Met, Phe and Trp)

The second configuration of the present invention is the neuropathic pain alleviating drug for mammals according to the first configuration, wherein x and y are 0 or 1 amino acid selected from Ala or Pro Or it is an antidepressant for mammals.
According to a third configuration of the present invention, the compound of the formula (1) is selected from any of Ala-Pro-Gly-Trp-NH ₂ , Pro-Gly-Trp-NH ₂ , and Gly-Trp-NH ₂ It is a neuropathic pain alleviating drug for mammals.
The fourth configuration of the present invention is a prodrug of the peptide described in the first to third configurations.

Since the drug according to the present invention reduces neuropathic pain in mammals, it can be expected to treat neuropathic pain such as diabetic pain, cancer pain, and neuralgia in humans. In addition, since it increases the amount of serotonin in mammals, it can also be expected as an antidepressant. Furthermore, APGWamide and the like exhibit these effects at low concentrations compared to existing drugs, and thus can be expected as excellent drugs that are inexpensive and have few side effects.

Diagram showing allodynia in diabetic model rats The figure which shows the allodynia reduction effect of APGWamide which is a compound concerning this invention The figure which shows the allodynia reduction action of PGWamide which is the compound concerning this invention The figure which shows GWamide which is a compound concerning this invention has an allodynia reduction effect The figure which shows the allodynia reduction action by the fluvoxamine which is a comparative example The figure which shows the serotonin amount increase effect | action of APGWamide which is a compound concerning this invention The figure which APGWamide which is the compound concerning this invention does not show an analgesic effect with respect to the thermal pain sensation of a normal rat Immunostaining diagram showing that APGWamide, a compound according to the present invention, reduces cFos protein-expressing cells The figure which compared the influence on the cFos protein expression cell of APGWamide which is the compound concerning this invention

  The mammalian neuropathic pain reducing drug and mammalian antidepressant drug according to the present invention will be described.

The neuropathic pain reducing drug for mammals or the antidepressant drug for mammals according to the present invention comprises a peptide represented by the following formula (1) as an active ingredient.
xy-Gly-Trp-NH ₂ (1)
(Both x and y are 0 or 1 amino acid selected from Pro, Gly, Ala, Val, Leu, Ile, Met, Phe and Trp)

In addition, the description of Formula (1) is a normal peptide description. That is, the left is the N-terminus of the peptide, the right is the C-terminus of the peptide, and each amino acid is represented in three letters. In formula (1), NH ₂ shown at the right end means that the C-terminal is amidated. Unless specifically limited, amino acids include both D-form and L-form.

An active ingredient according to the present invention the peptide, Gly - Trp - the NH ₂ exert antidepressant effect for a mammalian neuropathic pain relief action or mammal by a basic skeleton, N-terminus of the backbone It may further have one or two hydrophobic amino acid sequences (x, y) on the side. These hydrophobic amino acids x, y include Pro, Gly, Ala, Val, Leu, Ile, Met, Phe and Trp. A more preferred amino acid sequence is that x and y are peptides selected from Pro or Ala. Most preferably, the peptide is Ala-Pro-Gly-Trp-NH ₂ (where x is Ala and y is Pro). , Pro-Gly-Trp-NH ₂ (no x, y is Pro) _or Gly-Trp-NH ₂ (none of x and y) is selected.
In this specification, Ala-Pro-Gly-Trp-NH ₂ may be expressed as APGWamide, Pro-Gly-Trp-NH ₂ as PGWamide _{, and} Gly-Trp-NH ₂ as GWamide. These APGWamide, PGWamide, and GWamide are also expressed by ordinary peptide notation, and A, P, G, and W are one-letter amino acids, A is alanine, P is proline, G is glycine, and W is tryptophan. . The left side shows the N-terminus of the peptide, and the right side shows the C-terminus of the peptide. In addition, amide indicates that the C-terminal is amidated, and amino acids include both D-form and L-form unless otherwise specified.

  The peptide which is an active ingredient according to the present invention can be synthesized by a commonly used method such as a peptide synthesizer. Of the active ingredients according to the present invention, APGWamide, PGWamide, and GWamide may be extracted and isolated from mollusks. Preferably, those synthesized with a peptide synthesizer are used. Thereby, the peptide which is an active ingredient concerning this invention is compoundable simply and cheaply.

  Moreover, the peptide synthesize | combined by the above can be made into a prodrug body. A prodrug is defined as a compound that is converted into the peptide of the formula (1) by a physiological reaction with an enzyme, gastric acid or the like under physiological conditions in vivo. Examples of the physiological reaction include enzymatic oxidation and reduction, hydrolysis with gastric acid, and the like.

  Examples of the method of using the peptide represented by the formula (1) as a prodrug include a method of modifying an amino group or a carboxyl group of the peptide. For example, acylation or alkylation of an amino group, esterification or amidation of a carboxyl group. This prodrug form can be synthesized and produced from the peptide represented by the formula (1) by a known method.

  Moreover, the peptide which is an active ingredient concerning this invention may be contained as a peptide simple substance or a prodrug body simple substance as a pharmaceutical active ingredient, and may be contained as a salt. Although it is not necessary to specifically limit the salt, it is preferable to select a salt having low toxicity to the human body such as hydrochloride or acetate.

  In the drug according to the present invention, one active ingredient peptide or prodrug may be used, or a plurality of peptides or prodrugs may be selected as active ingredients. Preferably, any one peptide or prodrug form is used as an active ingredient. Thereby, the manufacturing method of a chemical | medical agent can be simplified.

  In the present invention, a neuropathic pain alleviating drug is a drug used for the treatment of neuropathic pain and is defined as a drug having a function of reducing or alleviating pain symptoms. Neuropathic pain is defined as a kind of chronic pain disorder caused by peripheral or central nerve disorders or functional disorders. Specific symptoms include intolerable spontaneous pain, allodynia, hyperalgesia or hypersensitivity. Representative diseases include diabetic pain, cancer pain, neuralgia and the like.

  In the present invention, an antidepressant drug is a drug used for the treatment of depression, and is defined as a drug having a function of alleviating depressive symptoms. Depression is a type of mood disorder and is defined as a mental disorder characterized by depressed mood, anxiety / irritability, decreased mental activity, decreased appetite, and insomnia.

  In the drug according to the present invention, additives usually used for pharmaceuticals can be used. For example, stabilizers, solubilizers and excipients.

  Moreover, in the chemical | medical agent concerning this invention, it can be set as all the dosage forms used for a pharmaceutical. For example, injections, tablets, suppositories, etc.

The dose of the neuropathic pain-reducing drug or antidepressant drug of the present invention is not particularly limited, depending on various conditions such as pain and depression symptoms, patient age, route of administration, purpose of treatment, presence or absence of concomitant drugs, etc. It is possible to select an appropriate dose. Preferably, the amount of the active ingredient can be 28.6 pmol or more per unit weight (kg) of the mammal. Thereby, it can be set as the chemical | medical agent which exhibits a pharmacological effect and has few side effects. In addition, although it is not necessary to specifically limit the upper limit of the amount of the active ingredient from the viewpoint of pharmacological effect, it is necessary to set the upper limit from the viewpoint of safety. Such an upper limit can be set according to common technical knowledge of those skilled in the art. That is, it is possible to set the upper limit of the amount that does not exhibit toxicity and sufficiently exerts the pharmacological effect by the safety test of pharmaceuticals, for example, carcinogenicity test, genotoxicity test, and reproductive and developmental toxicity test.

Hereinafter, the present invention will be specifically described with reference to examples. However, the present invention is not limited to the contents of the examples.

<< Experimental example, confirmation of allodynia effect in diabetic model rats >>
In order to confirm whether it is appropriate as an experimental system for evaluating neuropathic pain reduction, a diabetes model with reference to the method described in the literature (T Ikeda et al., Neurosci. Res, vol. 63, p. 42-46, 2009) An experimental system using rats was constructed and confirmed.

1. experimental method
(1) Preparation method of diabetes model rat Diabetes model rat was prepared by drug administration of streptozotocin (STZ: Sigma). That is, it was prepared by single administration (50 mg / kg) of STZ drug solution from the tail vein of normal rats (SD rats, male, weight 250-350 g).
(2) How to confirm the allodynia effect
The allodynia effect was confirmed by the von Frey test. In other words, using the von Frey type sensory measurement kit (North Coast Medical Co., Ltd.), using several types of filaments with different strengths, stimulating the plantar of diabetic model rats and confirming the threshold value causing the paw withdrawal reflex. It was.

2． result
(1) The results are shown in FIG.
a. Horizontal axis: Elapsed time from STZ administration in days. “Pre” indicates immediately before administration.
b. Left vertical axis: Indicates the filament amount threshold value that caused the retraction of the foot in units of g and a broken line in the figure. It shows that the smaller the g number at the threshold, the more the reflex is caused by less stimulation, which is worse for allodynia.
c. Right vertical axis: The blood glucose level of diabetic model rats is shown in mg / dL, and the bar graph in the figure. This shows that the higher the blood glucose level, the more prominent the pathological condition of diabetes model rats is as diabetes.
(2) The blood glucose level of diabetic model rats markedly increased 1 day after STZ administration, and then increased slightly. Along with this, the threshold of the von Frey test decreased significantly (p ≦ 0.05) one day after STZ administration. From these results, it was confirmed that allodynia was observed in diabetic model rats one day after STZ administration, and it was confirmed that it was suitable as an experimental system for confirming the allodynia effect.

<< Example 1, confirmation of allodynia-reducing action of neuropeptide >>
In order to confirm the allodynia mitigating effect of the neuropeptide according to the present invention, an experiment similar to the experimental example was performed using a comparative example.

1. experimental method
(1) APGWamide, PGWamide, and GWamide were synthesized using L-form amino acids with a peptide synthesizer (Applied Biosystems). Those prepared to a predetermined concentration using physiological saline were used as administration solutions. As a comparative example, SSRI, an antidepressant drug, fluvoxamine (manufactured by Solvay Pharmaceutical Co., Ltd.) was used.
(2) Preparation of diabetic model rats and confirmation of allodynia effect were performed in the same manner as described above (Experimental example, 1- (1), (2)).
(3) With reference to the aforementioned literature (T Ikeda et al., Neurosci. Res, vol. 63, p. 42-46, 2009), the drug solution was administered by intrathecal spinal cord administration. That is, a heat-treated polyethylene tube was used to fabricate a thinly stretched catheter. Cut the dura mater between the occipital bone and the first cervical vertebra of the above-mentioned diabetic model rat, so that the tip of the catheter comes from the medullary cavity below the arachnoid to the position of the 4th lumbar spinal cord to the 5th lumbar spinal cord The catheter was gently inserted, the other end of the catheter was taken out of the body, and the wound was sutured. All drugs prepared at the predetermined concentrations shown in FIGS. 2 to 5 were administered from this catheter at 10 μL, and flushed with the same amount of physiological saline.

2. result
(1) The results are shown in FIGS.
a. Horizontal axis: Time elapsed from administration of each drug is shown in minutes.
b. Left vertical axis: Indicates the filament amount threshold value that caused the retraction of the foot in units of g and a broken line in the figure.
(2) APGWamide (Fig. 2) and PGWamide (Fig. 3) are 10 ^-6M or higher, GWamide (Fig. 4) is 10 ^-5M or higher, and allogeneia is significantly reduced ( ^p≤0.05 ). The neuropeptide showed the maximum effect 60 minutes after administration. In addition, a significant difference was observed in both neuropeptides at 30 minutes after administration and 120 minutes after administration.
(3) On the other hand, fluvoxamine (Fig. 5), which is a comparative example, significantly reduced allodynia at a concentration of 10 ^{-4 M} or higher and showed a maximum effect 60 minutes after administration. However, there was no significant difference at 30 minutes after administration and 120 minutes after administration, in which APGWamide was significantly different.
(4) These results indicate that compared to fluvoxamine, APGWamide and PGWamide reduce allodynia at a low drug concentration of 1/100 and GWamide 1/10. In addition, both neuropeptides were shown to reduce allodynia more quickly and continuously than fluvoxamine.
(5) From the above, it has been shown that allodynia mitigation effects can be expected even with GWamide as the basic skeleton and one or two hydrophobic amino acids such as A and P.
(6) Since all drug solutions, including fluvoxamine, were administered at 10 μL, APGWamide and PGWamide were 10 ^-5 μmol and GWamide was 10 ^-4 μmol. . Since the weight of the rat is 250-350g, APGWamide and PGWamide are 28.6-40.0pmol / kg and GWamide is the minimum amount of 286-400pmol / kg.

<< Example 2, verification of pharmacological action mechanism of APGWamide >>
In order to investigate the mechanism by which APGWamide reduces the allodynia effect, we focused on the amount of serotonin as one possibility.

1. experimental method
(1) The APGWamide drug solution was prepared by the same method as described above (Examples 1, 1- (1)). As animals, normal rats (SD rats, males, weight 250-350 g) were used.
(2) Serotonin amount was measured by a microdialysis method (for example, Ishida Y et al., Neurosci. Lett., 253, p.45-48, 1998), which is a known method. In other words, a microdialysis probe with a drug administration guide tube was implanted in the anterior cingulate of the rat cerebrum, and 2 μL of APGWamide was administered at a predetermined concentration, and at the same time, the dialysate from the anterior cingulate was collected over time. The amount of serotonin was analyzed using an electrochemical detector.

2. result
(1) The results are shown in Fig. 6.
a. Horizontal axis: The elapsed time from the start of drug administration is shown in minutes.
b. Vertical axis: Indicates the rate of change in serotonin after administration of drug in%, based on the amount of serotonin before administration of drug. In other words, it is calculated as (the amount of serotonin at a predetermined elapsed time) / (the amount of serotonin in -60 minutes before the start of drug administration) × 100. Based on 100%, the amount of serotonin increases beyond that , Below that, it indicates that the amount of serotonin is decreasing.
(2) APGWamide showed a significant (p ≦ 0.05) increase in serotonin levels depending on its administration. That is, 20 minutes 40 minutes after the administration at a concentration of 2 × 10 ^-3 M, at a concentration of 2 × ^10-4M, it showed increased significantly (p ≦ 0.05) 20 minutes after administration. The peak was observed 20 minutes after administration at any concentration.
(3) APGWamide was shown to increase serotonin levels in the anterior cingulate gyrus in normal rats. Utilizing this property, an effect as an antidepressant is expected by increasing the amount of serotonin in the brain. Moreover, the similar effect can be exhibited also about PGWamide and GWamide which showed the effect similar to APGWamide in Example 1 from the similarity of the structure. This effect can be confirmed by the same experimental method as APGWamide.

<< Confirmation of selectivity of analgesic effect of Example 3, APGWamide >>
In order to investigate whether the neuropeptide according to the present invention is selective in neuropathic pain, it was confirmed using an experimental system that stimulates thermal pain sensation with different pain mechanisms.

1. experimental method
(1) The animal was a normal rat (SD rat, male, weight 250-350 g), and the drug solution was prepared by the same method as described above (Example 1, 1- (1)) using APGWamide. The drug solution was administered by intrathecal spinal cord in the same manner as described above (Examples 1, 1- (3)).
(2) It was carried out by a known method Hargreaves test (for example, Endo D et al., Neurosci Lett, 392, p. 114-117, 2006). In other words, the thermal pain sensation by infrared rays is given from the bottom of the glass plate to the sole of the foot, and the time (latency) from when the stimulus is applied until the foot is retracted to avoid the pain (foot retraction reflex) occurs. The effects of drugs on painful stimuli were examined by measuring and comparing the latency after drug administration with controls.

2. result
(1) The results are shown in FIG.
a. Horizontal axis: The time from the application of thermal pain sensation to the withdrawal of the foot is shown in seconds.
b. Vertical axis: Time after administration of each concentration of drug solution.
(2) The latency of paw withdrawal reflex after intrathecal administration of APGWamide was not significantly different from that after physiological saline administration. Therefore, APGWamide did not affect the transmission of “normal pain” stimuli in normal rats, that is, it exhibited a selective analgesic effect on neuropathic pain. Furthermore, similar selectivity can be exhibited for PGWamide and GWamide due to the similarity in structure. Such selectivity can be confirmed by the same experimental method as APGWamide.

<< Example 4, Effect of APGWamide on c-Fos protein expression in diabetes model rats >>
We examined whether APGWamide affects the expression of cFos protein, which is used as a marker of neural excitation.

1. experimental method
(1) Thermal stimulation was applied to the left hind limb of the rat in a thermostatic bath at 46 ° C for 2 minutes (stimulation that did not cause burns), and perfusion fixation was performed 2 hours after the stimulation. After perfusion fixation, the spinal cord was removed, frozen sections with a thickness of 50 micrometers were prepared, and immunostaining for cFos was performed.
(2) Of the rats, Group A, Group B, and Group C are STB (50 mg / ml) administered from the tail vein and are diabetic model rats that show allodynia. Saline in Group A, APGWamide in Group B, and Fluvoxamine in Group C were administered intrathecally 1 hour before thermal stimulation. Group D is a normal rat that does not show diabetes, as a diabetic model control, in which only citrate buffer, the solvent of STZ solution, is intravenously injected. As in Groups A, B, and C, physiological saline was administered intrathecally one hour before the thermal stimulation.

2-1. Results of immunostaining
(1) The results of immunostaining are shown in FIG. In the figure, black dots are cFos positive cells.
(2) Diabetic model rats administered with physiological saline showed cFos positive cells in the dorsal horn I / II layer (Fig. 8, A). Compared with this, the number of cFos-positive cells was clearly reduced in the diabetes model rats administered with APGWamide (FIG. 8, B). Fluvoxamine, a positive control for antidepressants, also reduced the number of cFos positive cells to the same extent as APGWamide (Fig. 8, C). Although cFos positive cells were confirmed in D normal rats, there were clearly fewer compared to group A diabetic model rats administered with physiological saline (Fig. 8, D).

2-2. Results of immunostaining analysis
(1) FIG. 9 shows a graph in which the number of cFos positive cells was measured for each layer of the dorsal horn of the spinal cord. For measurement, the average of 10 consecutive sections with the highest number of expressed cFos positive cells per mouse in each group was calculated, and 6 cells were statistically processed. The vertical axis shows the number of cFos positive cells per section, and the horizontal axis shows the results of Group A, Group B, Group C, and Group D from the left.
(2) In FIG. 9, the graph which measured the number of cFos positive cells of spinal cord dorsal horn I / II layer, III / IV layer, V / VI layer, X layer from the top is shown. Differences in the number of cFos positive cells in the I / II layer were observed between groups (* p <0.05).
(3) In diabetic model rats, APGWamide significantly decreased cFos positive cells. The same effect was observed with Fluvoxamine.
(4) Normal rats intravenously injected with Vehicle instead of STZ had significantly fewer cFos-positive cells than diabetic model rats, which was almost the same as the group that received APGWamide in diabetic model rats.
(5) From these results, it was shown that APGWamide has an effect of reducing neuropathic pain in diabetic model rats even by immunohistochemical method using cFos.

Claims (1)

A neuropathic pain-reducing drug for mammals or a mammal containing a peptide selected from Ala-Pro-Gly-Trp-NH ₂ , Pro-Gly-Trp-NH _{2, and} Gly-Trp-NH ₂ as an active ingredient Animal antidepressant.

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