JP4024810B2 - Dried product and production method thereof - Google Patents

Description

The present invention relates to a dried product of an inflammatory skin extract of a rabbit inoculated with vaccinia virus, and a method for producing the dried product.

The inflammatory skin extract of rabbit inoculated with vaccinia virus (hereinafter also referred to as this extract) contains a non-protein active substance extracted and separated from the inflamed skin tissue of rabbit inoculated with vaccinia virus.

Vaccinia virus-inoculated rabbit inflammation skin extract formulation (product name: Neurotropin), a drug containing this extract as an active ingredient, is a medical drug Japan Pharmaceutical Collection [2004 (27th edition), Japan Pharmaceutical Information Center, Stock As published on pages 2499-2501 of Company Jiho], low back pain, cervical-shoulder-arm syndrome, symptomatic neuralgia, shoulder periarthritis, osteoarthritis, skin diseases (eczema, dermatitis, urticaria) It is a very unique formulation with a wide range of indications such as pruritus associated with it, allergic rhinitis, cold feeling / abnormal perception / pain of SMON aftereffects, and neuralgia after herpes zoster, for subcutaneous, intramuscular and intravenous injections Injectables and tablets are commercially available as prescription drugs.

This extract is derived from a living body, and no single active ingredient has been identified. Therefore, the active ingredient is quantified by assaying its biological activity (titer), specifically, SART stress (repeated cold load), which is a chronic stress animal whose pain threshold is lower than that of a normal animal. A biological test method for obtaining an analgesic coefficient using a mouse has been used (“Nichi Pharmacology”, Vol. 72, No. 5, pp. 573-584, 1976). That is, an analgesic test is performed using a SART stress mouse according to a modified Randall-Selitto method to obtain an analgesic coefficient, and a neurotropin unit (NU) is defined by an ED ₅₀ value obtained from the analgesic coefficient for a standard product. is doing. A neurotropin injection contains 1.2 units as a neurotropin unit per mL, and a neurotropin tablet contains 4.0 neurotropin units per tablet.

  In addition to the above-mentioned quantitative analgesic activity, the extract has a kallikrein-like substance production inhibitory activity (hereinafter also referred to as KPI activity) above a specified level in Japan and other countries where pharmaceutical preparations containing this extract as an active ingredient are sold. By conducting a titer test method to confirm that it has, the quality and effectiveness as a drug are more strictly guaranteed.

  Kallikrein is a proteolytic enzyme widely present in plasma and tissues of various animals, and an enzyme system called kallikrein / kinin system is known. In plasma, inactive prekallikrein is converted to active plasma kallikrein through the activation of blood coagulation factor XII. The chemical mediator bradykinin is released. Bradykinin has various effects such as powerful pain caused by sensory nerve stimulation, blood pressure lowering due to vasodilation, and edema due to increased permeability of capillaries, and plays an important role in pain, inflammation and blood flow regulation. It is considered. Therefore, it has been shown that a drug having a bradykinin release inhibitory action exhibits various medicinal effects such as analgesia, anti-inflammatory, anti-edema and the like.

  This extract was shown to have an inhibitory action on bradykinin release (Eur. J. Pharmacol., Vol.157, No.1, p93-99, 1988). This pharmacological activity is an inhibitory action on the production of kallikrein-like substances. It was shown that it is based on. Then, a method has been developed that can quantitatively measure the ability of drugs to inhibit the production of plasma kallikrein-like substances in vitro ("Basic and Clinical", Vol. 20, No. 17, 8889-8895, 1986).

  The present invention relates to a dried product obtained in an intermediate stage for producing a final product having a KPI activity defined in the specifications and test methods of oral preparations containing an inflammatory skin extract of a rabbit inoculated with vaccinia virus as an active ingredient. The present invention relates to a method for producing a dried product. With respect to the dried product of this extract, it is only described in the following Patent Document 1 that it is solidified under reduced pressure. For producing an oral preparation from this extract, a dried product having KPI activity is used. There is no prior art that discloses a specific method for manufacturing.

JP-A-53-101515

When this extract is formulated as an oral solid preparation such as a tablet, it is necessary to dry the extract. However, since the KPI activity is not observed in the dried product of the present extract obtained by usual concentration drying and the like, a solid formulation such as a tablet having KPI activity could not be produced as the final formulation.

The applicant company was forced to make long-term research and development into the oral formulation of the neurotropin injection that was manufactured and sold, and one of the difficulties at that time was obtaining a final formulation having KPI activity. Although it has been empirically found that KPI activity exists in a final preparation produced by a certain method, and development of a neurotropin tablet has been accomplished, the applicant has possessed this as know-how. As a result of systematic research on a drying method for obtaining a dried product having KPI activity with respect to this extract, the present inventors added and mixed ascorbic acid before drying this extract. The inventors have found that a dried product of the present extract having KPI activity can be obtained by drying, and completed the present invention.

The present invention provides a dried product having a kallikrein-like substance production inhibitory activity of an inflammatory skin extract of a rabbit inoculated with vaccinia virus, and this dried product finally has tablets, granules, powders having KPI activity. It can be used as a raw material for producing solid preparations such as fine granules.

The present invention relates to a dried product having a kallikrein-like substance production inhibitory activity of a inflammatory skin extract of a rabbit inoculated with vaccinia virus, and a method for producing the dried product. Specifically, the present invention relates to a method for producing a dried product of the present extract, characterized in that ascorbic acid is added as an additive and mixed before drying to dry out the present extract. By this production method, a dried product of the present extract having KPI activity can be obtained, and finally, a solid preparation such as a tablet retaining KPI activity can be produced using this.

  This extract such as vaccinia virus-inoculated rabbit inflammation skin extract can be produced by the method described later, but when the obtained extract is dried, the extract is dried. It is characterized in that ascorbic acid is added and mixed before drying.

  The amount of ascorbic acid added can be appropriately set depending on the type of additive used, the concentration of the extract, and the like, but in the case of the test extract of Example 1 below, it is preferably 0.1% by weight or more, and is reproduced. In order to obtain a dried product of the present extract having good and high KPI activity, it is more preferable to add 0.5% by weight or more.

  As a drying means, concentrated drying used in usual preparation of pharmaceutical preparations can be used. For example, as a means for concentrating under mild conditions, there may be mentioned reduced-pressure drying in which the water is removed by distilling off the pressure by digestion (35 to 45 ° C.) without heating to a very high temperature. Moreover, after adding the excipient | filler containing the said additive etc. to this extract or its concentrate, and knead | mixing, it can also carry out granulation drying and can also obtain this granular dried product of this invention. The concentration of the extract is preferably carried out at a pH of 10 or less, and in order to obtain a dried product of the extract having a high KPI activity, the pH is preferably adjusted to 8.5 to 9.7. preferable.

  Addition of ascorbic acid in the present invention may be added to the present extract from the beginning or may be added after being concentrated to some extent, but is added after dryness as in the case of normal solid preparation production. By doing so, KPI activity cannot be obtained in the dry product finally produced. When a solid preparation such as a tablet is produced according to the present invention, it can be formulated using various additives. For example, as described in the General Formulation of Japanese Pharmacopoeia (14th revision), ascorbine The dried extract of this extract, which has been concentrated and dried with the addition of an acid, is granulated as it is or with an excipient, a binder, a disintegrant or other suitable additive and mixed evenly. After forming into a shape, a method of compression molding by adding a lubricant or the like, or a method of direct compression molding of a blended material can be mentioned. When the extract is concentrated to a certain extent, additives such as excipients containing ascorbic acid, binders, disintegrants and other additives are added, mixed and kneaded, granulated and dried by an appropriate method. The extract can also be tableted by a method in which the product is compression molded by adding a lubricant or the like. And a coloring agent, a corrigent, etc. can be added as needed, and a coating can also be given with a suitable coating agent.

The present extract used for the production of the dried product of the present invention is to inoculate rabbit skin with vaccinia virus, crush the irritated skin tissue, add the extraction solvent to remove the tissue fragment, and then remove the protein. Can be obtained by adsorbing to an adsorbent and then eluting the active ingredient.

Here, the rabbit encompasses everything belonging to the order of the rabbit eye. That is, the rabbit may be, for example, a rabbit (chira rabbit), a hare (Japanese hare), a rabbit, a snow rabbit, etc., and a rabbit that has been bred from the past and used as a domestic animal or a laboratory animal in Japan ( What is called a rabbit is easy to use.

This extract is manufactured by the following processes, for example.
(A) Rabbit skin tissue inoculated with vaccinia virus is collected, the germ tissue is crushed, and an extraction solvent such as water, phenol water, physiological saline or phenol-added glycerin water is added, followed by filtration. Alternatively, an extract (filtrate or supernatant) is obtained by centrifugation.
(B) The extract is adjusted to an acidic pH, heated and deproteinized. Next, the deproteinized solution is adjusted to be alkaline and heated, followed by filtration or centrifugation.
(C) The obtained filtrate or supernatant is acidified and adsorbed on an adsorbent such as activated carbon or kaolin.
(D) An extract from inflamed tissue inoculated with vaccinia virus can be obtained by adding an extraction solvent such as water to the adsorbent, adjusting to an alkaline pH, and eluting the adsorbed components. Thereafter, it can be appropriately adjusted to a pH near neutral to obtain a drug substance for preparation.
The above steps are described in further detail as follows.

About (a) Inflammatory skin tissue inoculated with rabbits such as rabbits with vaccinia virus is collected and crushed, and 1 to 5 times the amount of extraction solvent is added to obtain an emulsified suspension. As the extraction solvent, distilled water, physiological saline, weakly acidic to weakly basic buffer, etc. can be used. Stabilizers such as glycerin, bactericidal / preservatives such as phenol, sodium chloride, potassium chloride, chloride Salts such as magnesium may be added as appropriate. At this time, extraction can be facilitated by disrupting the cell tissue by treatment with freeze-thaw, ultrasound, cell membrane lytic enzyme, surfactant or the like.

The milky extract obtained in (b) is subjected to protein removal treatment after removing tissue fragments by filtration or centrifugation. The deproteinization operation can be carried out by a commonly known method, and includes heat treatment, treatment with a protein denaturant such as an organic solvent such as acid, base, urea, guanidine, acetone, isoelectric precipitation, salting out. Etc. can be applied. Subsequently, the insoluble protein precipitated by a usual method for removing insoluble matters, for example, filtration using filter paper (cellulose, nitrocellulose, etc.), glass filter, celite, zeit filtration plate, etc., ultrafiltration, centrifugation, etc. Remove.

The active ingredient-containing extract thus obtained for (c) is adjusted to acidity, preferably pH 3.5 to 5.5, using an acid such as hydrochloric acid, sulfuric acid, hydrobromic acid, etc., and the adsorption operation to the adsorbent is performed. . Examples of usable adsorbents include activated carbon, kaolin, and the like. Add the adsorbent to the extract and stir, or pass the extract through an adsorbent-filled column to add the active ingredient to the adsorbent. Can be adsorbed. When an adsorbent is added to the extract, the adsorbent can be obtained by adsorbing the active ingredient by removing the solution by filtration or centrifugation.

In order to elute (desorb) the active ingredient from the adsorbent with respect to (d), an elution solvent is added to the adsorbent, and it is eluted at room temperature or with appropriate heating or stirring, followed by normal methods such as filtration and centrifugation Can be achieved by removing the adsorbent. As an elution solvent to be used, a basic solvent, for example, water adjusted to a basic pH, methanol, ethanol, isopropanol or the like, or an appropriate mixed solution thereof can be used, preferably water adjusted to pH 9 to 12. Can be used.

The extract (eluate) thus obtained can be appropriately prepared in a form preferable as a drug substance or pharmaceutical preparation. For example, the drug substance can be prepared by adjusting the pH of the solution to near neutral or an appropriate pH.

Below, the example of the manufacturing method of this extract is shown.
Reference example 1
The skin of healthy mature rabbits was inoculated with vaccinia virus, and the sprouted skin was exfoliated, crushed and added with phenol water. Next, this was filtered under pressure, and the resulting filtrate was adjusted to pH 5 with hydrochloric acid and then heat-treated at 90-100 ° C. for 30 minutes. The protein was removed by filtration, adjusted to pH 9 with sodium hydroxide, further heated at 90-100 ° C. for 15 minutes, and then filtered. The filtrate was adjusted to about pH 4 with hydrochloric acid, added with 2% activated carbon, stirred for 2 hours, and then centrifuged. Water was added to the collected activated carbon, pH was adjusted to 10 with sodium hydroxide, and the mixture was stirred at 60 ° C. for 1.5 hours, and then centrifuged to obtain a supernatant. Water was added again to the activated carbon precipitated by centrifugation, the pH was adjusted to 11 with sodium hydroxide, the mixture was stirred at 60 ° C. for 1.5 hours, and then centrifuged to obtain a supernatant. Both supernatants were combined and neutralized with hydrochloric acid to obtain the present extract.

Reference example 2
After vaccinia virus was inoculated and infected on the skin of healthy mature rabbits, the wrinkled skin was aseptically peeled and cut into pieces, added with glycerin-phenol water, and ground with a homogenizer to obtain a milky form. Next, this was filtered, and the obtained filtrate was adjusted to weak acidity (pH 4.5 to 5.5) with hydrochloric acid, and then heated at 100 ° C. and filtered. The filtrate was made weakly alkaline (pH 8.5 to 10.0) with sodium hydroxide, further heated at 100 ° C. and filtered. The filtrate was adjusted to about pH 4.5 with hydrochloric acid, added with about 1.5% activated carbon, stirred for 1 to 5 hours and then filtered. Water was added to the filtered activated carbon, the pH was adjusted to 9.4 to 10 with sodium hydroxide, the mixture was stirred for 3 to 5 hours, and then filtered. The filtrate was neutralized to near neutrality with hydrochloric acid.

Reference example 3
After inoculating and activating vaccinia virus on the skin of healthy mature rabbits, the activated skin is aseptically peeled off, chopped into water, added with water, and ground with a homogenizer to obtain a milky product . Next, this was filtered under pressure, and the resulting filtrate was adjusted to pH 5.0 with hydrochloric acid and then heat-treated at 100 ° C. under flowing steam. The protein was removed by filtration, adjusted to pH 9.1 with sodium hydroxide, further heat-treated at 100 ° C., and then filtered. The filtrate was adjusted to pH 4.1 with hydrochloric acid, added with 2% activated carbon, stirred for 2 hours and then filtered. The filtrate was further filtered with 5.5% activated carbon and stirred for 2 hours. Water was added to the first activated carbon collected by filtration, adjusted to pH 9.9 with sodium hydroxide, stirred at 60 ° C. for 1.5 hours, and then filtered. Water was added to the first activated carbon and the next activated carbon, adjusted to pH 10.9 with sodium hydroxide, stirred at 60 ° C. for 1.5 hours, and then filtered. The filtrates were combined and neutralized with hydrochloric acid, and then desalted by electrodialysis using a membrane having a molecular weight of 100.

Method for measuring KPI activity The inhibitory activity (KPI activity) of a test substance on plasma kallikrein-like substance production was measured according to a method described in the literature ("Basic and Clinical", Vol. 20, No. 17, 8889-8895). Page, 1986). That is, as described in detail on page 8890 of the same document, a test solution and human normal plasma diluted with physiological saline are mixed, and a kaolin suspension is added thereto to start a plasma kallikrein production reaction. After a certain time, a specific inhibitor against activated blood coagulation factor XII such as lima bean trypsin inhibitor (LBTI) was added to stop the kallikrein production reaction, and then the produced kallikrein was converted into a chromogenic synthetic substrate (S-2302, Quantogenix). Since the synthetic substrate S-2302 liberates chromogenic p-nitroaniline by kallikrein, the amount (activity) of kallikrein produced can be measured by measuring the amount of released p-nitroaniline by absorbance at 405 nm. . The KPI activity of the test substance can be determined by determining the difference in absorbance between the group not added with the test substance (control) and the test substance addition group.

The criteria for determining whether or not the product has KPI activity can be set as appropriate. However, in the case of a neurotropin preparation, the standard is that this difference in absorbance is 0.1 or more. Is used.

Example 1
The dry matter weight of the rabbit skin inoculated vaccinia virus inoculated vaccinia virus produced according to the above Reference Example 1 was measured, and a test extract was prepared to 1 mg / mL and pH 9.5. 100 mL of the test extract was weighed and concentrated and dried under reduced pressure under digestion at about 40 ° C. Water was added to the dried product and dissolved to give a test solution of 1 mg / mL. 0.2 mL of this test solution and 0.2 mL of 0.5 M sodium chloride solution were mixed, and a measurement test was carried out according to the test procedure of the KPI activity measurement method described above. Table 1 shows the test results when the test extract was concentrated and dried as it was (no additive added) and when the test extract prepared by adding ascorbic acid to 1 wt% each was concentrated and dried (n = 2 ) Is an example. The control is water (hereinafter the same).

  The above test system is a system in which the amount of p-nitroaniline released from the chromogenic synthetic substrate by the enzymatic activity of the produced kallikrein is measured by absorbance. In the control, a certain amount of kallikrein is produced, and the absorbance as shown in the upper part of Table 1 is measured. However, if there is a test substance that inhibits kallikrein production in the reaction system, it is measured as the production of kallikrein decreases. Absorbance is low. That is, the larger the absorbance difference from the control, the higher the KPI activity of the test substance. As shown in Table 1, the absorbance of the test solution concentrated and dried without adding ascorbic acid was not different from the control, and no KPI activity was observed. On the other hand, as shown in Table 1, in the test solution to which ascorbic acid was added and concentrated and dried, clear KPI activity was measured. Therefore, the addition of ascorbic acid added the present extract having KPI activity. It was shown that a dry product can be produced.

However, when excipients such as calcium hydrogen phosphate, crystalline cellulose, and corn starch were used (all added at 1% by weight), KPI activity was not observed as shown in Table 2.

The dried product of the present invention can be appropriately processed into oral solid preparations such as tablets, powders, granules and capsules.

The present invention provides a dried product having a kallikrein-like substance production inhibitory activity of an inflammatory skin extract of a rabbit inoculated with vaccinia virus, and the dried product can be used for formulating a solid preparation such as a tablet. it can. The method for producing a dried product of this extract having KPI activity is essentially important in the production of an oral solid preparation containing this extract as an active ingredient. The present invention is an epoch-making invention that makes it possible to provide an oral solid preparation having the extract as an active ingredient and having KPI activity. On the other hand, ascorbic acid is added under predetermined conditions before the extract is dried. In addition, it can be carried out by a simple operation of mixing and drying, and this is an extremely useful method economically without the need for special additives.

Claims (17)

A method for producing a dried product having an activity of inhibiting kallikrein-like substance production of an extract obtained by adding ascorbic acid to a inflammatory skin extract of a rabbit inoculated with vaccinia virus, mixing the mixture, and drying the concentrated solution. Concentrated extract of inflammatory skin of rabbit inoculated with vaccinia virus, mixed with ascorbic acid before the extract is dried, and then drying the dried product having kallikrein-like substance production inhibitory activity Manufacturing method. A method for producing a dried product having a kallikrein-like substance production inhibitory activity, comprising concentrating and drying an extract of inflammation skin of a rabbit inoculated with vaccinia virus in the presence of ascorbic acid. The method for producing a dried product according to any one of claims 1 to 3 , wherein the extract is concentrated and dried at a pH of 10 or less. The method for producing a dried product according to any one of claims 1 to 3, wherein the extract is concentrated and dried at pH 8.5 to 9.7. A dried product having kallikrein-like substance production inhibitory activity of the extract obtained by adding and mixing ascorbic acid to an inflammatory skin extract of a rabbit inoculated with vaccinia virus, and concentrating and drying the extract. Concentrate the inflammatory skin extract of rabbits inoculated with vaccinia virus, add ascorbic acid and mix before the extract is dried, and dry the resulting extract to inhibit kallikrein-like substance production inhibitory activity. Having dry matter. A dried product having kallikrein-like substance production inhibitory activity obtained by concentrating and drying a inflammatory skin extract of a rabbit inoculated with vaccinia virus in the presence of ascorbic acid. The dried product according to any one of claims 6 to 8, wherein the extract is concentrated and dried at a pH of 10 or less. The dried product according to any one of claims 6 to 8, wherein the extract is concentrated and dried at pH 8.5 to 9.7.   The extract obtained by concentrating the inflammatory skin extract of a rabbit inoculated with vaccinia virus, kneading and adding an excipient containing ascorbic acid before the extract is dried, and granulating and drying the extract Dry granule having activity of inhibiting the production of kallikrein-like substance.   Concentrate the inflammatory skin extract of the rabbit inoculated with vaccinia virus, add the excipient containing ascorbic acid and knead before the extract is dried, granulate and dry the resulting granules A solid preparation for oral use having a kallikrein-like substance production inhibitory activity produced by using the extract as an active ingredient.   The oral solid preparation according to claim 12, which is a tablet.   Dry granule obtained by concentrating inflammatory skin extract of rabbit inoculated with vaccinia virus, kneading and adding excipient containing ascorbic acid before the extract is dried A method for producing an oral solid preparation having a kallikrein-like substance production-inhibiting activity containing the extract as an active ingredient, by using   Kallikrein-like obtained by concentrating inflammatory skin extract of rabbits inoculated with vaccinia virus, kneading and adding excipients containing ascorbic acid before the extract dries A dried product having a substance production inhibitory activity.   Kallikrein-like obtained by concentrating inflammatory skin extract of rabbits inoculated with vaccinia virus, kneading and adding excipients containing ascorbic acid before the extract dries Dry granules with substance production inhibitory activity.   Dry granule obtained by concentrating inflammatory skin extract of rabbit inoculated with vaccinia virus, kneading and adding excipient containing ascorbic acid before the extract is dried An oral solid preparation having an activity of inhibiting the production of a kallikrein-like substance containing the extract as an active ingredient.