JP4587637B2 - Neuropeptide production / release inhibitor - Google Patents

Description

[0001]
BACKGROUND OF THE INVENTION
The present invention relates to a neuropeptide production or release inhibitor containing hyaluronic acid or a pharmaceutically acceptable salt thereof as an active ingredient. The present invention also relates to a therapeutic agent for diseases caused by neurogenic inflammation comprising hyaluronic acid or a pharmaceutically acceptable salt thereof as an active ingredient.
[0002]
[Prior art]
First, abbreviations used in this specification will be described.
[0003]
CGRP: calcitonin gene-related peptide HA: hyaluronic acid PBS: phosphate buffered saline Sub P: substance P
Patent Document 1 describes a method for reducing inflammation and pain by intravenously administering HA. However, there is no disclosure or suggestion that HA suppresses production or release of neuropeptides. Further, there is no disclosure or suggestion that HA can be used as a therapeutic agent for diseases caused by neurogenic inflammation.
[0004]
[Patent Document 1]
Japanese Patent Laid-Open No. 62-255428
[Problems to be solved by the invention]
An object of the present invention is to provide a neuropeptide production or release inhibitor that contains HA or a pharmaceutically acceptable salt thereof as an active ingredient, is highly safe, and is extremely useful as a medicine. Another object of the present invention is to provide a therapeutic agent for diseases caused by neurogenic inflammation, which has HA or a pharmaceutically acceptable salt thereof as an active ingredient, is highly safe and is clinically extremely useful. To do.
[0006]
[Means for Solving the Problems]
As a result of intensive studies to solve the above problems, the present inventor has shown that HA or a pharmaceutically acceptable salt thereof exhibits an extremely excellent neuropeptide production or release suppressing effect and is also highly safe. I found it. Based on this finding, HA or a pharmaceutically acceptable salt thereof has been applied as a therapeutic agent for diseases caused by neurogenic inflammation that is extremely useful clinically.
[0007]
That is, the present invention provides a neuropeptide production or release inhibitor (hereinafter referred to as “the present invention production / release inhibitor”) comprising HA or a pharmaceutically acceptable salt thereof as an active ingredient.
[0008]
The weight average molecular weight of HA or a pharmaceutically acceptable salt thereof, which is an active ingredient of the production / release inhibitor of the present invention, is preferably 600,000 to 1,200,000. The “neuropeptide” referred to here is preferably Sub P. Further, the production / release inhibitor of the present invention is preferably administered intravenously.
[0009]
The present invention also provides a therapeutic agent for diseases caused by neurogenic inflammation (hereinafter referred to as “the therapeutic agent of the present invention”) comprising HA or a pharmaceutically acceptable salt thereof as an active ingredient.
[0010]
The weight average molecular weight of HA, which is an active ingredient of the treatment agent of the present invention, or a pharmaceutically acceptable salt thereof is preferably 600,000 to 1,200,000. In addition, the treatment agent of the present invention is preferably administered intravenously.
[0011]
DETAILED DESCRIPTION OF THE INVENTION
<1> HA or a pharmaceutically acceptable salt thereof The origin of HA or a pharmaceutically acceptable salt thereof used in the present invention is not particularly limited, and is isolated and purified from a chicken crown, an umbilical cord, a microorganism producing HA, or the like. HA can be used. In particular, those purified to a high purity and substantially free from substances that are not allowed to be mixed as pharmaceuticals are preferred.
[0012]
Examples of pharmaceutically acceptable salts of HA include salts with inorganic bases such as alkali metal salts (sodium salts, lithium salts, potassium salts, etc.), alkaline earth metal salts, ammonium salts, or diethanolamine salts, cyclohexyl. Among salts with organic bases such as amine salts and amino acid salts, pharmaceutically acceptable salts can be used. Of these, HA sodium is preferable.
[0013]
The weight average molecular weight of HA or a pharmaceutically acceptable salt thereof used in the present invention is not particularly limited, but preferably 600,000 to 1,200,000, more preferably 700,000 to 1,100,000, more preferably 800,000 to Those having 1,000,000 are more preferred, those having 850,000-950,000 are extremely preferred, and those having about 900,000 are particularly preferred. The weight average molecular weight of HA used in the present invention or a pharmaceutically acceptable salt thereof is determined by measuring the intrinsic viscosity according to the 13th revised Japanese pharmacopoeia: general test method / viscosity measurement method. (Biochim. Biophys. Acta, 42, 476 (1960)).
[0014]
The intrinsic viscosity of HA having a weight average molecular weight of 600,000 to 1,200,000 or a pharmaceutically acceptable salt thereof is 11.5 to 20 dl / g, and that having 700,000 to 1.1 million is 13 to 18.5 dl / g, 80 10,000 to 1,000,000 is 14.5 to 17.5 dl / g, 850,000 to 950,000 is 15.0 to 16.5 dl / g, and about 900,000 is about 16 dl / g.
[0015]
By using such HA or a pharmaceutically acceptable salt thereof, the production / release inhibitor of the present invention and the treatment agent of the present invention having an extremely excellent pharmacological action can be obtained.
[0016]
It should be noted that the endotoxin concentration in the HA or pharmaceutically acceptable salt thereof used in the present invention is preferably 0.3 EU / mL or less in the case of a solution form agent. The endotoxin concentration can be measured using a conventional endotoxin measurement method well known to those skilled in the art, but the Limulus test method using horseshoe crab, amebocyte, lysate components is preferred. EU (endotoxin unit) can be measured and calculated in accordance with the Japanese Industrial Standard Biochemical Reagent Standards (JIS K8008). The iron content is preferably 20 ppm or less.
<2> Administration Form of the Invention Production / Release Inhibitor and the Treatment Agent of the Present Invention, such as the Dosage Form of the Invention Production / Release Inhibitor, etc. Although it does not specifically limit as long as the effect with respect to the disease which originates is exhibited, For example, administration methods, such as parenteral administration and oral administration, are mentioned. Of these, parenteral administration is preferred, intravascular (intravenous or intraarterial) administration is more preferred, and intravenous administration is particularly preferred.
[0017]
Depending on the administration method, HA or a pharmaceutically acceptable salt thereof can be appropriately formulated into the production / release inhibitor of the present invention or the treatment agent of the present invention. Examples of the dosage form include injections (solutions, suspensions, emulsions, solid agents for dissolution at the time of use), tablets, capsules, liquids and the like. Of these, an injection is preferable.
[0018]
The concentration of HA or a pharmaceutically acceptable salt thereof in the production / release inhibitor of the present invention or the treatment agent of the present invention is not particularly limited, but is 0.05 to 1% (w / v). In particular, when the production / release inhibitor of the present invention or the treatment agent of the present invention is provided as a preparation for parenteral administration, for example, an injection (solution), it is about 0.1 to 0.6% (w / v). Is more preferable, about 0.2 to 0.5% (w / v), more preferably about 0.3 to 0.4% (w / v). When the production / release inhibitor of the present invention or the treatment agent of the present invention is used as a preparation for oral administration, for example, a liquid, it is preferably 0.1% (w / v) or more, preferably 0.2 to 1% More preferably, it is about (w / v).
[0019]
When the production / release inhibitor of the present invention or the treatment agent of the present invention is provided as an injection, the form thereof may be any of a solution, a frozen form, and a lyophilized form. This can be filled and sealed in an appropriate container such as an ampoule, vial, or syringe for injection, and distributed or stored as it is for administration as an injection.
[0020]
Known methods can be used to formulate the production / release inhibitor of the present invention and the treatment agent of the present invention. Further, in the formulation, other pharmaceutically active ingredients, conventional stabilizers, emulsifiers, permeation are used as long as they do not adversely affect HA or its pharmaceutically acceptable salt and do not affect the effects of the present invention. Ingredients usually used in medicine, such as pressure regulators, buffers, isotonic agents, preservatives, soothing agents, coloring agents, excipients, binders, lubricants, disintegrating agents, and the like can be used.
<3> Subjects to be Administered for Production / Release Inhibitors of the Present Invention The animals to be administered with the production / release inhibitor of the present invention and the treatment agents of the present invention are preferably vertebrates, particularly mammals, and particularly preferably humans.
[0021]
The production / release inhibitor of the present invention can be administered to an individual in which production or release of a neuropeptide is desired among these animals for the purpose of suppressing production or release of the neuropeptide. Sub P, CGRP and the like are exemplified as neuropeptides for which production and release are suppressed, but Sub P is preferable.
[0022]
Further, the treatment agent of the present invention is a treatment of a disease caused by neurogenic inflammation, for example, prevention of a disease caused by neurogenic inflammation, against an individual suffering from a disease caused by neurogenic inflammation among the above animals. It can be administered for the purpose of inhibiting progression (preventing deterioration), improving, treating, etc. That is, the treatment agent of the present invention includes ideas such as a prophylactic agent for diseases caused by neurogenic inflammation, a progression inhibitor (aggravation preventing agent), an improving agent, a therapeutic agent, and the like.
[0023]
The production / release inhibitor of the present invention and the treatment agent of the present invention can be widely applied to diseases for which production and release of neuropeptides are desired and diseases caused by neurogenic inflammation. Without limitation, osteoarthritis, rheumatoid arthritis, asthma, migraine, interstitial cystitis, complex regional pain syndrome (CRPS), cedar pollinosis, rhinitis, conjunctivitis, postoperative pain, urinary incontinence It can be preferably applied to depression, atopic dermatitis and the like.
[0024]
The compounding amount of HA or its pharmaceutically acceptable salt in the production / release inhibitor of the present invention or the treatment agent of the present invention, the dose per administration, the administration interval, etc. are the administration method, dosage form and use of these agents. Although it is a matter that should be individually determined according to the patient's specific symptom, age, sex, weight, etc., such as purpose, it is not limited, but the clinical amount of HA or a pharmaceutically acceptable salt thereof is not In the case of oral administration, a dose of about 100 to 500 mg per adult, 200 to 1000 mg per day, and in the case of oral administration, a dose of about 500 to 2500 mg per adult per day and about 1000 to 5000 mg per day is exemplified.
[0025]
Further, the administration interval of the production / release inhibitor of the present invention or the treatment agent of the present invention may be about once a day, or may be divided into 2-3 times a day. Further, it may be administered about once every 1 to 3 days.
[0026]
【Example】
Examples of the present invention will be specifically described below. However, these do not limit the technical scope of the present invention.
<1> Materials, etc. First, test substances used in this example will be described.
(1) Test substance, physiological saline, sodium HA (weight average molecular weight 900,000; limiting viscosity 15.9dl / g)
Sodium HA (in the present example, simply referred to as “HA”) was used by dissolving in PBS so as to be 0.4% (w / v). The endotoxin concentration after dissolution in PBS was 0.3 EU / mL or less, and the iron content was 20 ppm or less.
<2> Medicinal pharmacology test using rat neurogenic inflammation model In order to examine the neuropeptide release inhibitory effect of HA, the following pharmacological test was conducted.
[0027]
Rats (SD (Crj: CD, IGS), SPF) were grouped as follows. PBS (control group) or a PBS solution of HA (HA administration group; 0.4% (w / v)) was used as a test solution.
[0028]
PBS administration group (control group) 5 mice HA 2.5 mg / kg administration group 5 mice HA 5.0 mg / kg administration group 5 mice HA 10 mg / kg administration group 5 mice Each group received halothane anesthesia (introduced at 4.0%, 1.5- The head was fixed in the prone position. A spinal animal was created by cutting the spinal cord of the first cervical vertebra and maintained with a ventilator. The right limb saphenous nerve was exfoliated about 20 mm on the central side of the medial nerve branch, and a paraffin pool was prepared using the surrounding skin. The test solution was administered intravenously, and the detached saphenous nerve was placed on the bipolar silver wire electrode. Thirty minutes after administration of the test solution, electrical stimulation (frequency: 1 Hz, intensity: 0.2 mA, interval: 0.05 milliseconds) was applied for 10 minutes. Immediately after that, the thoracotomy was performed, and a perfusion needle was inserted from the left ventricle into the ascending aorta. After confirming the expansion of the right atrium, an incision was made to secure an outflow path for the perfusate. Then, it perfused for 2 minutes at 120 mmHg using physiological saline.
[0029]
Thereafter, the thigh skin was removed and the wet weight was measured, and then immersed in a 1M aqueous potassium hydroxide solution and incubated at 37 ° C. for 24 hours. After incubation, 9 ml of acetone phosphate solution was added and the solution was centrifuged (3,000 × g, 15 minutes). The Sub P concentration in the supernatant after centrifugation was measured using a Sub P measurement kit (EIA method). The Sub P concentration obtained here reflects the amount of Sub P released to the tissue. The results are shown in FIG. * In the figure indicates that there is a significant difference at P = 0.05 (William's multiple comparison test).
[0030]
As a result, it was shown that the amount of Sub P released into the tissues was significantly decreased in any HA administration group as compared with the control group. From this, it was shown that HA has the effect | action which suppresses discharge | release to the structure | tissue of Sub P.
<3> Pharmacologic pharmacological test using rat electrical stimulation-induced Sub P production model <2> In a system different from HA <2>, the following pharmacological test was conducted in order to examine the production or release inhibitory effect of neuropeptides of HA.
[0031]
Rats (SD (Crj: CD, IGS), SPF) were grouped as follows.
PBS administration group (control group) 1 animal HA 10 mg / kg administration group 1 animal <2> was prepared spinal animals, PBS (control group) or PBS solution of HA (HA administration group; 0.4% (w / v)) was injected as a test solution into the tail vein of each vertebrate.
[0032]
Thereafter, electrical stimulation (same conditions as <2>) was applied to the L5-6 spinal cord cauda equina for 10 minutes. Thereafter, synovial fluid and synovial tissue were collected. The measurement of the Sub P concentration in the collected synovial fluid was performed in the same manner as in <2>. The collected synovial tissue was cultured in Dulbecco's minimum essential medium (DMEM) at 37 ° C. for 24 hours, and the Sub P concentration in the supernatant after the culture was measured in the same manner as in <2>. The former result is shown in FIG. 2, and the latter result is shown in FIG.
[0033]
As a result, in the HA administration group, the amount of Sub P in the synovial fluid and synovial tissue culture supernatant decreased. This suggests that HA not only suppresses the release of Sub P into joint fluid but also suppresses the production of Sub P by synovial cells.
<4> Pharmacologic pharmacological test using rabbit papain-administered arthritis model In order to examine the inhibitory effect of HA on neuropeptide release in systems and animals different from <2> and <3>, the following pharmacological test was conducted.
[0034]
Rabbits (JW strain, male) were grouped as follows.
PBS administration group (control group) 23 mice HA 1.25 mg / kg / day administration group 17 mice HA 2.5 mg / kg / day administration group 17 mice HA 5 mg / kg / day administration group 16 mice 0.5% in the left hind knee joint Was administered to induce arthritis. Immediately after papain administration, the ear of each animal was treated with PBS (control group) or PBS solution of HA (HA administration group; 0.4% (w / v)) three times on day 1 and day 2 in total. It was injected into a vein.
[0035]
The joint fluid was collected on the third day after papain administration. The measurement of the Sub P concentration in the collected synovial fluid was performed in the same manner as in <2>. The amount of synovial fluid and the amount of protein in the synovial fluid were also measured. The amount of protein in synovial fluid was measured by the Lowry method. FIG. 4 shows the measurement result of the Sub P concentration, FIG. 5 shows the measurement result of the joint fluid amount, and FIG. 6 shows the measurement result of the protein amount in the joint fluid. ** in the figure indicates that there is a significant difference at P = 0.01 (Dunnet's multiple comparison test).
[0036]
As a result, it was shown that the amount of Sub P in the joint fluid decreased in the HA administration group even when a system / animal different from the above-described pharmacological test was used. From this, it was confirmed that HA has an action of suppressing the release of Sub P into joint fluid. It was also shown that the amount of synovial fluid and the amount of protein in the synovial fluid are reduced by the administration of HA.
[0037]
Note that HA can be said to be a highly safe material because it is already used as an active ingredient such as a commercially available joint function improving agent or an ophthalmic surgery adjuvant.
[0038]
【The invention's effect】
The production / release inhibitor of the present invention and the treatment agent of the present invention containing HA as an active ingredient exhibit an extremely excellent inhibitory effect on the production and release of neuropeptides, as is apparent from the results of the pharmacological test, And since it is extremely safe, it is extremely useful for the treatment of diseases caused by neurogenic inflammation.
[Brief description of the drawings]
BRIEF DESCRIPTION OF DRAWINGS FIG. 1 is a graph showing the inhibitory effect of Sub P release to tissues by HA administration.
FIG. 2 is a graph showing the inhibitory effect of the release of Sub P into joint fluid by HA administration. In the figure, “control” indicates the control group, and “HA Iv” indicates the HA 10 mg / kg administration group.
FIG. 3 is a graph showing an inhibitory effect on synovial cell Sub P production by HA administration. In the figure, “control” indicates a control group, “HA Iv” indicates a HA 10 mg / kg administration group, and “normal” indicates a normal rat.
FIG. 4 is a diagram showing the inhibitory effect of Sub P release into joint fluid by HA administration. In the figure, “C” indicates a control group, and “N” indicates a normal rabbit.
FIG. 5 is a graph showing a decrease in the amount of joint fluid by HA administration. In the figure, “C” indicates a control group, and “N” indicates a normal rabbit.
FIG. 6 is a graph showing a decrease in the amount of protein in synovial fluid by HA administration.
In the figure, “C” indicates a control group, and “N” indicates a normal rabbit.

Claims (3)

A substance P production or release suppression reagent comprising hyaluronic acid or a pharmaceutically acceptable salt thereof as an active ingredient. The suppression reagent according to claim 1, wherein the weight average molecular weight of hyaluronic acid or a pharmaceutically acceptable salt thereof is 600,000 to 1,200,000. The inhibitory reagent according to claim 1 or 2, which is administered intravenously.

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