U.S. patent application number 11/286644 was filed with the patent office on 2006-06-22 for dried product and a process for manufacturing the product.
Invention is credited to Masaharu Kurohashi, Yoji Shibayama.
Application Number | 20060134139 11/286644 |
Document ID | / |
Family ID | 36010950 |
Filed Date | 2006-06-22 |
United States Patent
Application |
20060134139 |
Kind Code |
A1 |
Kurohashi; Masaharu ; et
al. |
June 22, 2006 |
Dried product and a process for manufacturing the product
Abstract
A dried product of an extract from inflammatory rabbit skin
inoculated with vaccinia virus having an inhibitory activity for
the production of a kallikrein-like substance is produced by
admixing the extract with a saccharide, sugar alcohol or ascorbic
acid before reaching dryness, and then drying the admixture to a
solid form such as granules. The dried product may be employed to
produce a solid preparation for oral administration, such as
tablets, having an inhibitory activity for the production of a
kallikrein-like substance.
Inventors: |
Kurohashi; Masaharu;
(Ono-shi, JP) ; Shibayama; Yoji; (Katoh-gun,
JP) |
Correspondence
Address: |
HOLLANDER LAW FIRM, P.L.C.
SUITE 305
10300 EATON PLACE
FAIRFAX
VA
22030
US
|
Family ID: |
36010950 |
Appl. No.: |
11/286644 |
Filed: |
November 25, 2005 |
Current U.S.
Class: |
424/232.1 ;
424/520 |
Current CPC
Class: |
A61K 9/2018 20130101;
A61K 9/2054 20130101; A61P 29/00 20180101; A61K 35/36 20130101;
A61K 9/1623 20130101 |
Class at
Publication: |
424/232.1 ;
424/520 |
International
Class: |
A61K 39/275 20060101
A61K039/275; A61K 35/12 20060101 A61K035/12 |
Foreign Application Data
Date |
Code |
Application Number |
Dec 1, 2004 |
JP |
348,904/2004 |
Apr 28, 2005 |
JP |
132,070/2005 |
Jun 17, 2005 |
JP |
177,334/2005 |
Oct 13, 2005 |
JP |
298,472/2005 |
Claims
1. A process for the manufacture of a dried product of an extract
from inflammatory rabbit skin inoculated with vaccinia virus
comprising admixing said extract with at least one member selected
from the group consisting of saccharides, sugar alcohols, and
ascorbic acid to obtain a mixture, and then drying the mixture to
obtain a dried product.
2. A process as claimed in claim 1 where the dried product has an
inhibitory activity for the production of a kallikrein-like
substance.
3. A process as claimed in claim 1 wherein a saccharide, sugar
alcohol or ascorbic acid is added to and mixed with an extract from
inflammatory rabbit skin inoculated with vaccinia virus and then
the mixture is concentrated and dried.
4. A process as claimed in claim 1 wherein an extract from
inflammatory rabbit skin inoculated with vaccinia virus is
concentrated and, before the concentrated extract reaches dryness,
a saccharide, sugar alcohol or ascorbic acid is added thereto and
mixed therewith and then the mixture is dried.
5. A process as claimed in claim 3 wherein concentration and drying
of said extract is carried out at a pH of less than or equal to
10.
6. A process as claimed in claim 3 wherein concentration and drying
of said extract is carried out at a pH of 8.5 to 9.7.
7. A process as claimed in claim 1 wherein said at least one member
comprises at least one saccharide selected from the group
consisting of glucose, mannose, arabinose, xylose, galactose,
sorbose, lactose, sucrose, maltose, raffinose, melezitose,
pullulan, dextrin, .beta.-cyclodextrin and dextran.
8. A process as claimed in claim 1 wherein said at least one member
comprises at least one sugar alcohol selected from the group
consisting of mannitol, maltitol, lactitol, palatinit and
sorbitol.
9. A process as claimed in claim 1 wherein the amount of said at
least one member is at least 0.1% by weight, based upon the weight
of the extract and said at least one member.
10. A process as claimed in claim 1 wherein said drying comprises
vacuum drying.
11. A process as claimed in claim 1 wherein said mixture is
granulated and dried to obtain granules.
12. A process as claimed in claim 11 wherein said granules are
formed into tablets.
13. A process as claimed in claim 12 wherein said tablets are spray
coated with a coating solution.
14. A dried product obtained by the process of claim 1.
15. A dried product as claimed in claim 14 where the dried product
has an inhibitory activity for the production of a kallikrein-like
substance.
16. A solid preparation for oral administration comprising a dried
product of claim 14.
17. A dried product having an inhibitory activity for the
production of a kallikrein-like substance comprising a co-dried
admixture of an extract from inflammatory rabbit skin inoculated
with vaccinia virus and at least one member selected from the group
consisting of saccharides, sugar alcohols and ascorbic acid.
18. A dried product as claimed in claim 17 wherein said at least
one member comprises at least one saccharide selected from the
group consisting of glucose, mannose, arabinose, xylose, galactose,
sorbose, lactose, sucrose, maltose, raffinose, melezitose,
pullulan, dextrin, .beta.-cyclodextrin and dextran.
19. A dried product as claimed in claim 17 wherein said at least
one member comprises at least one sugar alcohol selected from the
group consisting of mannitol, maltitol, lactitol, palatinit and
sorbitol.
20. A solid preparation for oral administration comprising granules
of a dried product as claimed in claim 17.
21. A solid preparation for oral administration as claimed in claim
20, wherein the preparation is a tablet.
22. A solid preparation having an inhibitory activity for the
production of a kallikrein-like substance for oral administration
comprising an extract from inflammatory rabbit skin inoculated with
vaccinia virus as an effective ingredient which is manufactured
using dried granules prepared in such a manner that said extract is
concentrated and, before said extract reaches dryness, an excipient
comprising at least one member selected from the group consisting
of saccharides, sugar alcohols and ascorbic acid is added thereto
and kneaded therewith, and then the kneaded mixture is granulated
and dried.
Description
FIELD OF THE INVENTION
[0001] The present invention relates to a dried product of an
extract from inflammatory rabbit skin inoculated with vaccinia
virus and also to a process for manufacturing said dried
product.
BACKGROUND OF THE INVENTION
[0002] An extract from inflammatory rabbit skin inoculated with
vaccinia virus (hereinafter, it may be mentioned as "the present
extract") contains a non-protein active substance which is
extracted and separated from the inflamed skin tissue of rabbit
inoculated with vaccinia virus.
[0003] As mentioned in pages 2499 to 2501 of "List of Japanese
Ethical Drugs" (2004 (27th Edition), edited by the Japan
Pharmaceutical Information Center, published by K. K. Jiho), a
pharmaceutical preparation of an extract from inflammatory rabbit
skin inoculated with vaccinia virus (product name: Neurotropin)
which is a pharmaceutical containing the present extract as an
effective ingredient is a very unique preparation. Namely, broad
indications such as low back pain, neck-shoulder-arm syndromes,
periarthritis scapulohumeralis, osteoarthritis, symptomatic
neuralgia, itching accompanied with skin disorders (such as eczema,
dermatitis and urticaria), allergic rhinitis, sequelae of subacute
myelo-optico-neuropathy (such as coldness, pain and
paresthesia/dysesthesia) and post-herpetic neuralgia have been
allowed and subcutaneous, intramuscular and intravenous injections
and tablets thereof have been approved for manufacture as ethical
drugs and placed into the market.
[0004] The present extract is derived from a living body and no
single effective ingredient has been identified. Accordingly,
quantification of the effective ingredient has been carried out by
testing its biological activity (titer). More specifically, a
biological test method where an analgesic coefficient is determined
using SART stress (repeated cold load) mice in which pain threshold
lowers from normal animals has been used (Nippon Yakurigaku Zasshi,
vol. 72, no. 5, pages 573-584, 1974). Thus, an analgesic
coefficient is determined by conducting an analgesic test in
accordance with a modified Randall-Selitto method using SART mice
and a neurotropin unit (NU) is stipulated by an ED.sub.50 value
calculated from an analgesic coefficient for a standard product. A
Neurotropin injection contains 1.2 units per 1 mL in terms of a
neurotropin unit and Neurotropin tablets contain 4.0 neurotropin
units per tablet.
[0005] With regard to the present extract, in addition to the
aforementioned quantification of analgesic activity, a biological
test method measuring an inhibitory activity for the production of
a kallikrein-like substance (hereinafter, it may be mentioned as
KPI activity) should be carried out in Japan, etc. where
pharmaceutical preparations containing the present extract as an
effective ingredient have been sold. By confirming that a product
has a regulated level or more KPI activity, the quality and the
efficacy as a pharmaceutical agent are strictly guaranteed.
[0006] Kallikrein is a proteinase which is widely present in plasma
and tissues of various animals and an enzyme system called a
kallikrein-kinin system has been known. In plasma, inactive
pre-kallikrein is converted to active plasma kallikrein via
activation of blood coagulation factor XII and the resulting plasma
kallikrein acts on high-molecular weight kininogen in plasma
whereupon bradykinin which is a chemical mediator of nonapeptide is
liberated. Bradykinin has various actions such as a strong
generation of pain by stimulation of sensory nerves, hypotension by
dilation of blood vessels and expression of edema by a rise in
permeability of blood vessels and is thought to play an important
role in pain generation, inflammation and blood flow adjustment.
Accordingly, pharmaceuticals having an inhibiting action for
liberation of bradykinin have been shown to express various
pharmaceutical effects such as analgesic, anti-inflammatory and
anti-edema actions.
[0007] It has been clarified that the present extract has a
suppressive action for liberation of bradykinin (Eur. J.
Pharmacol., vol. 157, no. 1, pages 93-99, 1988) and the
pharmacological action as such is shown to be based on an
inhibitory action for the production of a kallikrein-like
substance. A method quantitatively to measure an inhibitory ability
of a drug for the production of a kallikrein-like substance has
been developed (Kiso to Rinsho, vol. 20, no. 17, pages 8889-8895,
1986).
[0008] The present invention relates to a dried product obtained in
an intermediate step for the manufacture of the final product
having a KPI activity stipulated in approved "Specification and
Testing Methods" of oral preparations containing an extract from
inflammatory rabbit skin inoculated with vaccinia virus as an
effective ingredient. The present invention also relates to a
process for the manufacture of said dried product. In respect of
the dried product of the present extract, for example in Japanese
Patent Laid-Open No. Sho-53/101,515 or the like, there is only
description that it is evaporated to dryness in vacuo. And, there
has been no prior art where a specific process for the manufacture
of a dried product having a KPI activity in the manufacture of oral
preparations from the present extract is disclosed.
[0009] In making the present extract into pharmaceutical
preparations as solid preparations for oral administration such as
tablets, it is necessary to dry said extract. However, in a dried
product of the present extract prepared by commonly-used
concentration, drying, etc., no KPI activity is noted and,
therefore, it has not been possible to manufacture solid
preparations such as tablets having a KPI activity as the final
preparation.
[0010] One of the difficulties encountered for many years by Nippon
Zoki Pharmaceutical Co., the assignee of the present application,
in making oral preparations from Neurotropin injections
manufactured and sold by the company was to prepare a final
preparation having a KPI activity. Although it has been empirically
found that the final product manufactured by a certain process of
Nippon Zoki Pharmaceutical Company has a KPI activity and
development of Neurotropin tablets has been achieved, the assignee
has retained it as know-how. The present inventors have
systematically conducted studies for a drying method of the present
extract for preparing a dried product having a KPI activity. As a
result, it has been found that, when a saccharide, sugar alcohol or
ascorbic acid is added to and mixed with the present extract before
said extract reaches dryness and then it is dried, a dried product
of the present extract having a KPI activity is obtained and the
optimum pH, etc. have been also been found whereupon the present
invention has been achieved.
[0011] The present invention provides a dried product of an extract
from inflammatory rabbit skin inoculated with vaccinia virus having
an inhibitory activity for the production of a kallikrein-like
substance and, in final form, said dried product is able to be used
as a material for the manufacture of solid preparations such as
tablets, granules, diluted powder and fine particles having a KPI
activity.
SUMMARY OF THE INVENTION
[0012] The present invention relates to a dried product of an
extract from inflammatory rabbit skin inoculated with vaccinia
virus having an inhibitory activity for the production of a
kallikrein-like substance and also to a process for the manufacture
of said dried product. To be more specific, it relates to a process
for the manufacture of dried product of the present extract which
is characterized in that, in drying the present extract, a
saccharide, sugar alcohol or ascorbic acid is added thereto and
mixed therewith before said extract reaches dryness and then the
mixture is dried. In accordance with said process for the
manufacture, a dried product of the present extract having a KPI
activity is able to be prepared and it is now possible to
manufacture solid preparations such as tablets finally having a KPI
activity.
DETAILED DESCRIPTION OF THE INVENTION
[0013] Although the present extract such as an extract from
inflammatory rabbit skin inoculated with vaccinia virus is able to
be manufactured by the method which will be mentioned later, a
characteristic feature of the process of the present invention is
that, in drying the present extract, a saccharide, sugar alcohol or
ascorbic acid is added thereto and mixed therewith before said
extract reaches dryness and then the mixture is dried. Examples of
the saccharide are a monosaccharide such as glucose, mannose,
arabinose, xylose, galactose and sorbose, an oligosaccharide such
as lactose, sucrose, maltose, raffinose and melezitose and a
polysaccharide such as pullulan, dextrin, .beta.-dextrin and
dextran. As the sugar alcohol, mannitol, maltitol, lactitol,
palatinit and sorbitol can be used in the present invention. With
regard to the saccharide, sugar alcohol and ascorbic acid to be
added, one of the above-mentioned ones may be used or two or more
thereof may be used in combination. With regard to the
polysaccharide, that which is insoluble in water is not suitable
for the present invention and a polysaccharide which is soluble in
water is able to be used in the present invention. As used herein,
the polysaccharide which is insoluble in water corresponds to
"rarely soluble" in Rule 23 of the Japanese Pharmacopoeia
(Fourteenth Revision) and its examples are crystalline cellulose
and starch.
[0014] The pharmaceutically effective amount of the saccharide,
sugar alcohol or ascorbic acid which may be employed depends upon
the kind of the saccharide or sugar alcohol used, concentration of
the extract, etc. When lactose is used in an extract to be tested
mentioned in the following Example 1, not less than 0.1% by weight
is preferred and, in order to prepare a dried product of the
present extract having a high KPI activity with a good
reproducibility, it is more preferred to add not less than 0.5% by
weight of the additive.
[0015] With regard to a drying means, concentration and drying
which are commonly used in preparing pharmaceutical preparations
may be utilized. For example, as to a means for concentrating under
a mild condition, vacuum drying in which heating at too high
temperature is not conducted but water is removed by evaporating in
vacuo under warm dipping (35.degree. C. to 45.degree. C.) may be
exemplified. In embodiments of the invention an excipient
containing the above-mentioned saccharide, sugar alcohol or
ascorbic acid and the like may be added to the present extract or
to a concentrate thereof, kneaded and granulated, and then dried to
give a granular dried product of the present invention. In
concentrating the present extract, it is preferred to conduct it at
a pH of the solution which is 10 or lower and, in order to prepare
a dried product of the present extract having a high KPI activity,
it is more preferred to conduct it by adjusting the pH to 8.5 to
9.7.
[0016] As to addition of the saccharide, sugar alcohol or ascorbic
acid in the present invention, it may be added to the present
extract in its initial stage or may be added after concentrating to
some extent. However, when it is added after the extract reaches
dryness as in the case of common methods for the manufacture of
solid preparations, no KPI activity is achieved in the finally
manufactured dried product. In the manufacture of a solid
preparation such as tablets according to the present invention, the
preparation may be manufactured using various additives and methods
as shown in General Rules for Pharmaceutical Preparations in the
Japanese Pharmacopoeia (Fourteenth Revision). For example, a dried
product of the present extract prepared by concentration and drying
after addition of the above saccharide, sugar alcohol or ascorbic
acid may be made into granules by an appropriate means either
directly or after uniformly mixing with appropriate additives such
as an excipient, binder, disintegrating agent or others and then
subjected to a compression molding after addition of a lubricant or
the like, and a method where the uniform mixture is directly
subjected to a compression molding. It is also possible to make the
present extract into tablets by a method where, when the present
extract is concentrated to some extent, the above saccharide, sugar
alcohol or ascorbic acid and other additives such as excipient,
binder and disintegrating agent are added, homogeneously mixed,
kneaded and subjected to granulation and drying by an appropriate
means and the resulting dried product is subjected to a compression
molding after addition of a lubricant or the like. If necessary,
coloring agent, corrigent, etc. may be added and it is also
possible to make or apply a coating using an appropriate coating
agent.
[0017] The present extract used for the manufacture of the dried
product of the present invention may be prepared in such manner
that inflammatory rabbit skin tissues inoculated with vaccinia
virus are crushed, an extracting medium is added thereto, tissue
pieces are removed, a treatment for removal of protein
(deproteinization) is conducted, the resulting product is adsorbed
with an adsorbent and the effective ingredient is eluted
therefrom.
[0018] As used herein, rabbit covers all animals belonging to
Lagomorpha. Thus, the rabbit may be any of, for example,
Oryctolagus cuniculus, hare (Japanese hare), mouse hare and
snowshoe rabbit and, in Japan, it is easy to use family rabbit (in
Japanese "Kato") which has been bred since old time and frequently
used as a domestic animal or an experimental animal.
[0019] The present extract is, for example, manufactured by the
following procedures:
[0020] (a) Inflammatory skin tissues of rabbit inoculated with
vaccinia virus are collected, and the finely cut inflammatory
tissues are crushed. An extracting medium such as water, phenol
water, saline or phenol-added glycerin water is added, and then an
extracted fluid (filtrate or supernatant) is obtained by filtration
or centrifugation.
[0021] (b) The pH of the above extracted fluid is made acidic and
it is heated for deproteinization. The deproteinized solution is
subsequently made alkaline and heated, after which it undergoes
filtration or centrifugation.
[0022] (c) The obtained filtrate or supernatant is made acidic,
then adsorbed by an adsorbent such as activated carbon or
kaolin.
[0023] (d) An extracting solvent such as water is added to the
above adsorbent, the pH is made alkaline and the adsorbed component
is eluted, thereby obtaining an extract from inflammatory tissue
inoculated with vaccinia virus. Thereafter, the eluate can be
suitably adjusted to natural pH to make a material for drug
preparation.
[0024] Each step mentioned above is described in detail as
follows:
Step (a):
[0025] Inflammatory skin tissues of rabbit inoculated with vaccinia
virus are collected, ground and made into an emulsified suspension
by adding 1 to 5 times as much extracting solvent thereto. Examples
of the extracting solvent applicable are distilled water,
physiologically saline solution, weakly acidic to weakly basic
buffers, etc. If necessary, stabilizers such as glycerol,
antibacterial/antiseptic agents such as phenol, inorganic salts
such as sodium chloride, potassium chloride, magnesuim chloride,
etc. may be added thereto. At that time, the extraction can be made
easier by subjecting the admixture to a treatment by means of
freezing/melting, ultrasonic wave, cell membrane dissolving enzymes
or surface-active agents.
Step (b):
[0026] The resulting milky extract is filtered or centrifuged to
remove the tissue residue and then proteins are removed therefrom.
Removal of the proteins can be carried out by known methods, for
example, treatments by means of heating, ultrasonic wave, protein
denaturating agents such as acids, bases, urea, guanidine, organic
solvents, surface-active agents, etc., isoelectric precipitation,
salting-out, can be employed. Then the insoluble proteins
precipitated therefrom are removed by means of filtration using
filter paper (cellulose, nitrocellulose, etc.), glass filter,
Celite, Seitz filter, etc. as well as ultrafiltration, gel
filtration, ion exchange resin, centrifugation and the like.
Step (c):
[0027] The resulting extract containing effective components is
adjusted to acidic, preferably to pH 3.5-5.5, by an acid such as
hydrochloric acid, sulfuric acid or hydrobromic acid, and adsorbed
with an adsorbent. Examples of the applicable adsorbent are
activated charcoal, kaolin, etc. The adsorbent is added to the
extract followed by stirring or the extract is passed through a
column filled with the adsorbent whereby the effective component
can be adsorbed.
Step (d):
[0028] In eluting the effective component from the adsorbent, an
extracting solvent is added to the adsorbent, and the elution is
carried out at room temperature or by heating to some extent or
with stirring, and then the adsobent is removed by conventional
means such as filtration whereby the elution can be achieved. The
extracting solvent applicable is a basic solvent, for example,
water, methanol, ethanol, isopropanol or a mixed solution thereof
which is adjusted to basic pH, preferably, water adjusted to pH
9-12 can be utilized.
[0029] The extract (eluted solution) obtained as above can be
prepared to a preferred formulation suitable for drug material and
medical preparations. For example, the solution is prepared to drug
material by adjusting to approximately neutral or appropriate
pH.
EXAMPLES
[0030] The examples of the method for manufacturing the present
extract are as follows:
Referential Example 1
[0031] Skins of healthy adult rabbits were inoculated with vaccinia
virus to cause inflammation. The inflammatory skins were removed,
finely cut and phenol water was added thereto. The mixture was
filtered with pressure, and the resulting filtrate was adjusted to
pH 5 with hydrochloric acid and then heated at 90-100.degree. C.
for 30 minutes. Proteins were removed by filtration, the filtrate
was adjusted to pH 9 with sodium hydroxide, further heated at
90-100.degree. C. for 15 minutes and filtered. The filtrate was
adjusted to about pH 4, stirred for 2 hours after adding 2% of
activated charcoal, and centrifuged. The resulting activated
charcoal was mixed with water, adjusted to pH 10 with sodium
hydroxide, stirred at 60.degree. C. for 1.5 hours and centrifuged
to give a supernatant. The activated charcoals precipitated by
centrifugation were mixed with water, adjusted to pH 11 with sodium
hydroxide, stirred at 60.degree. C. for 1.5 hours and centrifuged
to give a supernatant. Both of the supernatants obtained were
combined and neutralized with hydrochloric acid to give an extract
from inflammatory tissue inoculated with vaccinia virus. In the
following pharmacological studies, the extract was adjusted to
appropriate concentrations to be used.
Referential Example 2
[0032] Skins of healthy adult rabbits were inoculated with vaccinia
virus to cause inflammation. The inflammatory skins were
aseptically removed, finely cut and phenol-added glycerin water was
added thereto. The mixture was ground using a homogenizer to
prepare an emulsion. The emulsion was filtered with centrifugation,
and the resulting filtrate was adjusted to pH 4.8-5.5 with
hydrochloric acid, heated at 100.degree. C. with a steam flow and
then filtered. The filtrate was further filtered with a Seitz
filter, adjusted to pH 9.2 with sodium hydroxide, heated at
100.degree. C. and filtered. The filtrate was adjusted to pH 4.5,
stirred for 1-5 hours after adding 1.5% of activated charcoal, and
filtered. The activated charcoal was mixed with water, adjusted to
pH 9.4-10 with sodium hydroxide, stirred for 3-5 hours and
filtered. The resulting filtrate was neutralized with hydrochloric
acid and dried in vacuo.
Referential Example 3
[0033] Skins of healthy adult rabbits were inoculated with vaccinia
virus to activate or stress the tissues. The activated skins were
aseptically removed, finely cut and water was added thereto. The
mixture was ground using a homogenizer to prepare an emulsion. The
emulsion was filtered with pressure, and the resulting filtrate was
adjusted to pH 5.0 with hydrochloric acid and heated at 100.degree.
C. with a steam flow. Proteins were removed by filtration, the
filtrate was adjusted to pH 9.1 with sodium hydroxide, heated at
100.degree. C. and filtered. The filtrate was adjusted to pH 4.1,
stirred after adding 2% of activated charcoal, and the mixture was
filtered to obtain a filtrate and a first batch of recovered
activated charcoal. To the filtrate was added 5.5% of activated
charcoal and the mixture was stirred for 2 hours, and filtered to
obtain a second batch of recovered activated charcoal. The first
batch of recovered activated charcoal was mixed with water,
adjusted to pH 9.9 with sodium hydroxide, stirred at 60.degree. C.
for 1.5 hours and filtered. Water was then added to the first batch
of the activated charcoal and to the second batch of activated
charcoal. The pH of each batch was then adjusted to pH 10.9 with
sodium hydroxide, and each batch was stirred at 60.degree. C. for
1.5 hours and then filtered. The resulting filtrates were combined,
neutralized with hydrochloric acid, desalted using electrodialysis
with a membrane (molecular weight: 100), and dried in vacuo.
Method for Measuring the KPI Activity
[0034] The inhibitory action for production of plasma
kallikrein-like substances (KPI activity) of the tested drug was
measured according to the method described in the literature (Kiso
to Rinsho, vol. 20, no. 17, pages 8889-8895, 1986). Namely, as
described in detail at page 8890 of the literature, the solution of
the tested drug was mixed with normal human plasma diluted with
saline. A suspension of kaolin was added to the mixture to initiate
the reaction producing plasma kallikrein. After a constant time, a
specific inhibitor of blood coagulation factor XII such as lima
bean trypsin inhibitor (LBTI) was added to terminate the reaction
producing kallikrein, and then the produced kallikrein was
quantitatively determined by using a coloring synthetic substrate
(S-2302, Chromogenix). Since the synthetic substrate S-2302
releases coloring p-nitroaniline by kallikrein action, the amount
(activity) of produced kallikrein can be determined by measuring
the amount of liberated p-nitroaniline by the measurement of
absorbance at 405 nm. The KPI activity of the tested drug can be
evaluated by determining the difference in absorbance between a
control group (a group without the tested drug) and the group to
which the tested drug is added.
[0035] It is able to be appropriately set judging criteria whether
the KPI activity is available and, in Neurotropin preparations,
difference in absorbencies is stipulated to be not less than 0.1
and said stipulation is used in the dried product of the present
invention as well.
Example 1
[0036] An extract for the test was prepared in such a manner that
the weight of dried product of an extract from inflammatory rabbit
skin inoculated with vaccinia virus manufactured according to the
above-mentioned Referential Example 1 was made to be 1 mg/mL where
the pH was 9.5. One hundred mL of said extract for the test was
taken, concentrated and dried in vacuo under warm dipping of about
40.degree. C. Water was added to the dried product to dissolve it
whereupon a test solution of 1 mg/mL was prepared. The test
solution (0.2 mL) and 0.2 mL of 0.5M sodium chloride solution were
mixed and, after that, a measuring test was conducted according to
the test operation in the above-mentioned method for measuring the
KPI activity. Table 1 shows examples of test results (n=2) in the
case where the extract for the test was directly concentrated and
dried and in the case where the extract for the test prepared by
addition of lactose so as to make 1% by weight was then
concentrated and dried. Water was used as a control in this test
and in the following tests as well. TABLE-US-00001 TABLE 1
Absorbance Difference in Observed Mean absorbances Test solution
value value KPI activity Control 0.361 0.361 0.361 Non-addition
0.352 0.363 0.002 of lactose 0.373 Addition of 0.073 0.069 0.292
lactose 0.065
[0037] The above-mentioned test system is a system where the amount
of p-nitroaniline liberated from a coloring synthesis substrate by
enzymatic activity of the produced kallikrein was measured by means
of absorbance. In the control, a certain amount of kallikrein was
produced and the absorbances as mentioned in the upper row of Table
1 were measured. While, when a test substance inhibiting the
kallikrein product was present in the reaction system, the measured
absorbances showed low values as a result of lowering of the
kallikrein production. Thus, it shows that, when the difference in
absorbances from the control is high, the KPI activity of the test
substance is high. As shown in Table 1, the absorbance of the test
solution which was concentrated and dried without addition of
lactose (Non-addition of lactose) was the same as that of the
control whereby no KPI activity was noted at all. On the contrary,
in a test solution concentrated and dried after addition of lactose
(Addition of lactose), an apparent KPI activity was measured
whereby it is shown that a dried product of the present extract
having a KPI activity is able to be manufactured by addition of
lactose.
Example 2
[0038] The same as in Example 1, a comparison was performed between
the case where concentration and drying were conducted after
addition of lactose to the extract to be tested and the case where
lactose was added after concentration and drying of the extract to
be tested. Measurement was also conducted for a lactose solution as
a blank. Examples of the results are shown in Table 2. As shown by
the results in Table 2, only a test solution where lactose was
added before concentrating and drying showed a KPI activity and, in
the case where lactose was added after concentration and drying of
the extract to be tested and in the case of a solution containing
lactose only, no KPI activity was noted at all. TABLE-US-00002
TABLE 2 Absorbance Difference in Observed Mean absorbances Test
solution value value KPI activity Control 0.356 0.364 0.371
Addition of 0.066 0.063 0.301 lactose 0.059 Addition of 0.341 0.342
0.022 lactose after 0.342 drying Lactose 0.365 0.375 0.011 solution
(1 wt %) 0.384
Example 3
[0039] The result where additives other than lactose were added to
an extract to be tested before concentrating and drying in the same
manner as in Example 1 is shown in Table 3-1 below. The cases where
a saccharide such as glucose (monosaccharide) and pullulan
(polysaccharide) is added (1% by weight in each of the cases)
before drying showed a KPI activity but the cases where an
excipient other than a saccharide such as calcium hydrogen
phosphate or a water-insoluble polysaccharide such as crystalline
cellulose or corn starch was used showed no KPI activity.
TABLE-US-00003 TABLE 3-1 Absorbance Difference in Observed Mean
absorbances Test solution value value KPI activity Control 0.330
0.323 0.315 Addition of 0.075 0.069 0.254 glucose 0.063 Addition of
0.283 0.289 0.034 calcium hydrogen 0.294 phosphate Control 0.322
0.325 0.327 Addition of 0.095 0.094 0.231 pullulan 0.093 Addition
of 0.273 0.277 0.048 crystalline 0.280 cellulose Control 0.330
0.324 0.317 Addition of 0.280 0.303 0.021 corn starch 0.326
[0040] In the same manner, mannitol, maltitol, lactitol, palatinit
(monohydrate), sorbitol and ascorbic acid were used (the same
amount is also added) and the results are shown in Table 3-2. In
the tested solution of the dried product where the mannitol,
maltitol, sorbitol, lactitol, palatinit or ascorbic acid was added
to the extract and it was concentrated and dried, the KPI activity
was clearly observed. Therefore, the dried product of the present
extract having KPI activity can be manufactured by addition of
ascorbic acid or a sugar alcohol constituted of at least one
hexose. TABLE-US-00004 TABLE 3-2 Absorbance Difference in Observed
Mean absorbance Test solution value value KPI activity Control
0.368 0.369 0.370 Addition of 0.207 0.223 0.146 sorbitol 0.239
Control 0.346 0.353 0.359 Addition of 0.095 0.090 0.263 mannitol
0.084 Addition of 0.039 0.039 0.314 ascorbic acid 0.038 Control
0.351 0.352 0.352 Addition of 0.042 0.041 0.311 lactitol 0.040
Addition of 0.114 0.115 0.237 palatinit 0.116 Control 0.336 0.343
0.350 Addition of 0.044 0.043 0.300 maltitol 0.041
Example 4
[0041] Results of the cases where the same operation as in Example
1 was conducted using a saccharide other than the saccharides
mentioned in the above Examples is shown in Table 4 (1% by weight
of a saccharide was added to the extract to be tested in each of
the cases). Results of the cases where 0.5% by weight or 0.1% by
weight of lactose was added is also shown in the same table. Table
4 summarizes the results of plural tests (absorbance value of the
control: 0.306 to 0.363) and absorbances of the test solutions and
difference (KPI activity) between those and the control are shown.
TABLE-US-00005 TABLE 4 Difference in Absorbance absorbance from the
Additive to the Mean value of 2 control test solution observed
values KPI activity Arabinose 0.056 0.268 Xylose 0.053 0.271
Mannose 0.049 0.275 Galactose 0.072 0.252 Sorbose 0.056 0.250
Sucrose 0.107 0.249 Maltose 0.060 0.290 Raffinose 0.084 0.264
Melezitose 0.097 0.251 Dextrin 0.104 0.242 .beta.-Cyclodextrin
0.240 0.121 Dextran 0.186 0.177 Lactose 0.5 wt 0.047 0.273 Lactose
0.1 wt 0.143 0.177
Example 5
[0042] Weight of the product of the present extract after being
evaporated to dryness was measured the same as in Example 1 and an
extract to be tested was prepared so as to obtain a concentration
of 1 mg/mL and a pH of 8.8 to 9.3. Ten mL of said extract to be
tested was taken and concentrated in vacuo and temperature control
was performed so that the temperature of the concentrate was
maintained at about 40.degree. C. Concentration was conducted until
200 mL (50 mg/mL), 200 g of lactose and about 160 g of other
excipient, disintegrating agent, etc. were added thereto and
kneaded therewith and the mixture was granulated and dried. The
resulting dried granules prepared as such were able to be
formulated into a solid preparation for oral use. For example,
magnesium stearate or another lubricant was added thereto followed
by subjecting the admixture to compressive molding to manufacture
tablets.
[0043] The above-mentioned granules in an amount calculated to
contain 50 mg of the dried product of the present extract were
taken and 50 mL of a Tris hydrochloride buffer (pH 8.0) was added
thereto and stirred therewith. After that, the mixture was filtered
using a membrane filter, the filtrate was used as a test solution
and its KPI activity was measured the same as in Example 1. Also,
in the above operation before addition of an excipient such as
lactose, a 50 mg/mL concentrated solution of the present extract
was taken and diluted with water to 1 mg/mL and measured as well.
Examples of the results are shown in Table 5. As shown in Table 5,
any of the concentrated solution of the present extract and the
dried granules prepared by adding an excipient such as lactose to
said concentrate followed by kneading granulating and drying showed
a KPI activity. TABLE-US-00006 TABLE 5 Absorbance Difference in
Observed Mean absorbance Test solution value value KPI activity
Control 0.332 0.334 0.335 Dried granules 0.054 0.054 0.280 solution
0.054 Concentrated 0.048 0.047 0.287 solution 0.046
Example 6
[0044] Table 6 shows examples of the results where the extract to
be tested was adjusted to various pH values in concentrating and
drying the extract to be tested. When the pH was 10.5, a KPI
activity was hardly noted and, as the pH was changed from 9.5 to
acidic, a KPI activity of the dried product gradually lowered.
TABLE-US-00007 TABLE 6 Absorbance Difference in Observed Mean
absorbance Test solution value value KPI activity Control 0.338
0.335 0.332 Concentrated 0.178 0.174 0.161 solution (pH6.0) 0.169
Concentrated 0.133 0.128 0.207 solution (pH8.5) 0.122 Concentrated
0.053 0.050 0.285 solution (pH9.5) 0.047 Concentrated 0.241 0.232
0.103 solution (pH10.0) 0.223 Concentrated 0.272 0.272 0.063
solution (pH10.5) 0.272
Formulation Example 1
[0045] According to the same method as mentioned in Example 5,
dried granules of the present extract were manufactured and
subjected to a compressive molding to manufacture tablets. Thus,
the components were kneaded so that each tablet contained 4 mg of
dried product of an extract from inflammatory rabbit skin
inoculated with vaccinia virus, 104 mg of lactose, 40 mg of
crystalline cellulose and 20 mg of carboxymethyl cellulose and then
subjected to granulation and drying. Magnesium stearate (content in
a tablet: 2 mg) was added to and mixed with the dried granules
followed by subjecting the admixture to compressive molding using a
tabletting machine to manufacture tablets.
Formulation Example 2
[0046] According to the same manner as in Formulation Example 1,
tablets were manufactured where each tablet contained 5 mg of dried
product of an extract from inflammatory rabbit skin inoculated with
vaccinia virus, 80 mg of lactose, 20 mg of calcium hydrogen
phosphate, 42 mg of lowly-substituted hydroxypropyl cellulose, 2 mg
of hydroxypropyl cellulose and 1 mg of magnesium stearate. The
resulting crude tablets were subjected to a spray coating with a
coating solution (prepared by mixing 40 g of hydroxypropyl
cellulose, 10 g of Macrogol 6000, 3 g of titanium oxide, 5 g of
talc, 0.5 kg of lake dye and 941.5 g of pure water) to manufacture
film-coated tablets. KPI activity of the above dried granules and
tablet were measured, and both the dried granules and tablet had
KPI activity. Furthermore, when they were formulated by using
D-mannitol instead of lactose, the same results were noted.
[0047] Besides the above, the dried product of the present
invention is appropriately able to be processed into solid
preparations for oral administration such as diluted powder,
granules or capsules.
[0048] The present invention provides a dried product having an
inhibitory activity for the production of a kallikrein-like
substance of an extract from inflammatory rabbit skin inoculated
with vaccinia virus. The dried product can be utilized for the
manufacture of pharmaceutical preparations such as solid
preparations including tablets. A process for the manufacture of
the dried product of the present extract having a KPI activity is
essentially important in the manufacture of solid preparations for
oral administration where the present extract is an effective
ingredient. The present invention is epoch-making whereby it is now
possible to provide solid preparations for oral administration
having a KPI activity and containing the present extract as a
pharmaceutically effective ingredient. The solid preparations
containing a pharmaceutically effective amount of the extract may
be administered orally to patients in need of treatment for
conditions responsive to an inhibiting action for liberation of
bradykinin to provide various pharmaceutical effects such as
analgesic, anti-inflammatory and anti-edema actions. At the same
time, the present invention can be carried out by a simple
operation, that is, before getting the present extract to dryness,
a saccharide, sugar alcohol or ascorbic acid is added thereto and
mixed therewith under a predetermined condition followed by drying.
As such, it is a very useful process in view of economy where no
specific additive or the like is necessary.
* * * * *