U.S. patent application number 10/204675 was filed with the patent office on 2003-02-20 for immune enhancement compositions and use thereof.
Invention is credited to Ito, Shinobu, Ogata, Eiji.
Application Number | 20030035812 10/204675 |
Document ID | / |
Family ID | 18576205 |
Filed Date | 2003-02-20 |
United States Patent
Application |
20030035812 |
Kind Code |
A1 |
Ito, Shinobu ; et
al. |
February 20, 2003 |
Immune enhancement compositions and use thereof
Abstract
Immune enhancement compositions for vaccines for virus, bacteria
and/or infectious pathogens which contain stable activity-type
antioxidant provitamins; a method of enhancing the immunity of
vaccines for virus, bacteria and/or infectious pathogens with use
of these compositions; and use of the above composition. As the
stable activity-type antioxident provitamins, at least one compound
selected from among L-ascorbic acid derivatives and
.alpha.-tocopherly phophates is used.
Inventors: |
Ito, Shinobu; (Tokyo,
JP) ; Ogata, Eiji; (Kanagawa, JP) |
Correspondence
Address: |
SUGHRUE MION, PLLC
2100 PENNSYLVANIA AVENUE, N.W.
WASHINGTON
DC
20037
US
|
Family ID: |
18576205 |
Appl. No.: |
10/204675 |
Filed: |
August 23, 2002 |
PCT Filed: |
February 28, 2001 |
PCT NO: |
PCT/JP01/01512 |
Current U.S.
Class: |
424/204.1 ;
424/234.1; 514/100; 514/474 |
Current CPC
Class: |
A61K 39/39 20130101;
A61K 47/22 20130101; A61K 47/24 20130101; A61K 31/665 20130101;
A61K 2039/55511 20130101; A61K 31/375 20130101; A61K 45/06
20130101; A61K 9/0019 20130101 |
Class at
Publication: |
424/204.1 ;
424/234.1; 514/100; 514/474 |
International
Class: |
A61K 031/665; A61K
039/12; A61K 039/02; A61K 031/375 |
Foreign Application Data
Date |
Code |
Application Number |
Feb 29, 2000 |
JP |
2000-55019 |
Claims
What is claimed is:
1.(amended) A composition for enhancing the immunological effects
of a vaccine for viruses, bacteria and/or infectious disease
pathogens, comprising at least one compound selected from
L-ascorbic acid derivatives, .alpha.-tocopheryl phosphates and
salts thereof as a stable activity-type antioxidant provitamin.
2. (cancelled)
3. (amended) The composition for enhancing the immunological
effects as claimed in claim 1, wherein the antioxidant provitamin
is selected from an alkaline metal salt and an alkaline earth metal
salt.
4. The composition for enhancing the immunological effects as
claimed in claim 3, wherein the antioxidant provitamin is selected
from a sodium salt, potassium salt, magnesium salt and calcium
salt
5. The composition for enhancing the immunological effects as
claimed in any one of claims 1 to 4, wherein the immunological
effects of a vaccine is increased to 2 times or more in terms of
the serum antibody unit.
6. The composition for enhancing the immunological effects as
claimed in claim 5, wherein the serum antibody unit is an antibody
unit determined by hemagglutination inhibition (HI) reaction or
passive hemagglutination (PHA) reaction.
7. The composition for enhancing the immunological effects as
claimed in any one of claims 1 to 4, wherein enhancing the
immunological effects includes enhancing macrophage activity.
8. The composition for enhancing the immunological effects as
claimed in any one of claims 1 to 4, wherein enhancing the
immunological effects includes enhancing induction of secretory IgA
in the respiratory tract.
9. The composition for enhancing the immunological effects as
claimed in any one of claims 1 to 8, which is perorally
administered.
10. The composition for enhancing the immunological effects as
claimed in any one of claims 1 to 8, which is administered
intramuscularly, intradermally, intraveneously, subcutaneously,
intranasally or externally.
11. The composition for enhancing the immunological effects as
claimed in any one of claims 1 to 10, which is used for mammals
including human, birds or fishes.
12. The composition for enhancing the immunological effects as
claimed in claim 11, which is used for at least one animal under
high-density feeding, selected from from the group consisting of
cattle, pig, fowl, horse, poultry, pet, ornamental animal, breeding
marine animal and laboratory animal.
13. A method for enhancing the immunological effects of a vaccine
for viruses, bacteria and/or infectious disease pathogens, which
uses the composition for enhancing the immunological effects
described in any one of claims 1 to 12.
14. Feed, premix or drink comprising the composition for enhancing
the immunological effects described in any one of claims 1 to
12.
15. A method for using a composition for enhancing the
immunological effects, comprising previously mixing the composition
for enhancing the immunological effects described in any one of
claims 1 to 12 with a vaccine and then administering the vaccine.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application is based on the provisions of 35 U.S.C.
Article 111(a) with claiming the benefit of filing dates of U.S.
provisional application Serial No. 60/253,744 filed on Nov. 29,
2000 under the provisions of 35 U.S.C. 111(b), pursuant to 35
U.S.C. Article 119 (e) (1).
TECHNICAL FIELD
[0002] The present invention relates to a composition for enhancing
the immunological effects of a vaccine for viruses, bacteria and/or
infectious disease pathogens, containing at least one compound
selected from L-ascorbic acid derivatives and .alpha.-tocopheryl
phosphates, which are stable activity-type antioxidant provitamins
(hereinafter referred to as "stable activity-type provitamin") as
an active ingredient; a method for enhancing the immunological
effects of a vaccine for viruses, bacteria and/or infectious
disease pathogens by using the composition; and a use of the
composition.
BACKGROUND OF THE INVENTION
[0003] By virtue of the development of a vaccine or an antibiotic,
human and animals under feeding, such as cattle, pet and bleeding
marine animal, have been prevented from infectious diseases via
various viruses or bacteria. In other words, antibiotics specific
for bacterial infectious diseases have already succeeded in greatly
mitigating the fear for bacterial diseases.
[0004] However, antibiotics or chemotherapics effective for viral
infectious diseases are small in the number and the predominating
prophylaxis is vaccine inoculation represented by poliomyelitis and
vaccination.
[0005] The vaccine in general includes an "inactivated vaccine" by
the inactivation of a pathogenesis virus and a "live vaccine" by
the attenuation thereof. Examples of the vaccine practically used
for human at present include vaccines of poliomyelitis, measles,
rubella, mumps, influenza, Japanese encephalitis, varicella,
icterus and hepatitis B. Among these, vaccines of influenza,
Japanese encephalitis and hepatitis B belong to the "inactivated
vaccine" and others belong to the "live vaccine".
[0006] As compared with the inactivated vaccine, the live vaccine
is generally advantageous in that the immunity acquisition is close
to that by the natural infection and the immunological effects is
high. However, the live vaccine is said to have problems such that
the toxicity may recover or the quality is unstable. On the other
hand, the inactivated vaccine lower in the toxicity than the live
vaccine is not completely free of toxicity, and aggressive studies
are being made with an attempt to develop more safe and effective
component vaccines using genetic engineering means. However, these
are also pointed out to have some problems in the practical
use.
[0007] As described above, many vaccines are being used in
practice, however, their phylactic effect is not sufficiently high
and various methods for enhancing the immunological effects of
vaccine are being studied. For example, use of an adjuvant is one
of means for enhancing the effect of vaccine and a long known
synthetic Freund's adjuvant is also attempted but this adjuvant
disadvantageously causes a strong adverse reaction, that is, has a
problem in the safety.
[0008] In general, when a vaccine is inoculated to an organism, the
immunological effect thereof can be known by measuring the level of
the specific antibody in the blood (circulating antibody).
[0009] The circulating antibody level can be measured by using
hemagglutination inhibition (HI) reaction when the antigen has a
hemagglutination activity such as in the case of influenza virus.
Specifically, 2-fold step dilution series of a target virus
solution are prepared first, a constant amount of erythrocytes are
added to each dilution and after the passing of a predetermined
time, the agglutination images are examined. The HA unit (HAU) is
the reciprocal of the highest dilution number in a test tube
showing agglutination positive. Next, 2-fold step dilution series
of a serum whose antibody value is to be measured are prepared, the
same amount of a virus solution diluted to 4 HAU are added to each
of the dilution series and the mixed solution is allowed to stand
for a predetermined time. A constant amount of erythrocytes is
added to each of these solutions and after the passing of a
predetermined time, the agglutination images are examined. The
antibody value of the serum (HI unit, HIU) is the reciprocal of the
highest dilution number in a test tube in which the agglutination
of the erythrocytes is completely inhibited.
[0010] When the antigen does not have a hemagglutination activity,
the amount of a specific antibody in the blood can be measured by
using a passive hemagglutination (PHA) and the like. This method
uses erythrocytes to which an antigen has been bound by tannic acid
or chromium chloride, etc. and the antibody value of an antiserum
is determined therewith as HA unit.
[0011] As described above, the live vaccine has a fear of
occurrence of mutation of the attenuated toxicity strain in vivo
into a strongly toxic strain and the inactivated vaccine has a
latent danger in essence such that the antigenicity is distorted
during the inactivation operation and this gives rise to an
unexpected adverse reaction.
[0012] Apart from this, group administration of a vaccine is being
performed at present for the low ages and therefore, in order to
prevent the adverse reaction ascribable to the dispersion of the
sensitivity in the host side, a safer and more effective vaccine is
being demanded.
[0013] Furthermore, among various viruses, in the case of a virus
which causes infection and proliferates on the surface of
respiratory tract, such as influenza virus, unless the local
secretory antibody (IgA antibody) of the respiratory tract is
present in a sufficiently high concentration, the virus infection
cannot be always prevented even if the antibody concentration in
blood is high. At present, subcutaneous inoculation of an
inactivated vaccine is employed for the prophylaxis of influenza.
However, the IgA antibody will not be increased, although this
method is effective for increasing the antibody in blood.
Accordingly, for preventing the infection not only by the influenza
but also by viruses which cause infection and proliferate on the
surface of respiratory tract or on the mucosa of digestive tract,
the matter of importance is to increase the IgA antibody in
addition to the antibody in blood.
DISCLOSURE OF THE INVENTION
[0014] In order to solve the above-described problems, the present
inventors have made extensive investigations on a method for
enhancing the immunological effects of a vaccine based on the
estimation that when the immunological effects of a vaccine is
enhanced, the dose can be reduced and in turn, the frequency in
generation of an adverse reaction can be reduced. As a result, it
has been found that the above-described problems can be overcome by
using a specific antioxidant provitamin in combination with a
vaccine. The present invention has been accomplished based on this
finding.
[0015] More specifically, the present invention relates to the
following composition and method for enhancing the immunological
effects and use thereof.
[0016] 1. A composition for enhancing the immunological effects of
a vaccine for viruses, bacteria and/or infectious disease
pathogens, comprising a stable activity-type antioxidant
provitamin.
[0017] 2. The composition for enhancing the immunological effects
as described in the above item 1, wherein the antioxidant
provitamin is at least one compound selected from L-ascorbic acid
derivatives, .alpha.-tocopheryl phosphates and salts thereof.
[0018] 3. The composition for enhancing the immunological effects
as described in the above items 1 or 2, wherein the antioxidant
provitamin is selected from an alkaline metal salt and an alkaline
earth metal salt.
[0019] 4. The composition for enhancing the immunological effects
as described in the above item 3, wherein the antioxidant
provitamin is selected from a sodium salt, potassium salt,
magnesium salt and calcium salt.
[0020] 5. The composition for enhancing the immunological effects
as described in any one of the above items 1 to 4, wherein the
immunological effects of a vaccine is increased to 2 times or more
in terms of the serum antibody unit.
[0021] 6. The composition for enhancing the immunological effects
as described in the above item 5, wherein the serum antibody unit
is an antibody unit determined by hemagglutination inhibition (HI)
reaction or passive hemagglutination (PHA) reaction.
[0022] 7. The composition for enhancing the immunological effects
as described in any one of the above items 1 to 4, wherein
enhancing the immunological effects includes enhancing macrophage
activity.
[0023] 8. The composition for enhancing the immunological effects
as described in any one of the above items 1 to 4, wherein
enhancing the immunological effects includes enhancing induction of
secretory IgA in the respiratory tract.
[0024] 9. The composition for enhancing the immunological effects
as described in any one of the above items 1 to 8, which is
perorally administered.
[0025] 10. The composition for enhancing the immunological effects
as described in any one of the above items 1 to 8, which is
administered intramuscularly, intradermally, intraveneously,
subcutaneously, intranasally or externally.
[0026] 11. The composition for enhancing the immunological effects
as described in any one of the above items 1 to 10, which is used
for mammals including human, birds or fishes.
[0027] 12. The composition for enhancing the immunological effects
as described in the above item 11, which is used for at least one
animal under high-density feeding, selected from the group
consisting of cattle, pig, fowl, horse, poultry, pet, ornamental
animal, breeding marine animal and laboratory animal.
[0028] 13. A method for enhancing the immunological effects of a
vaccine for viruses, bacteria and/or infectious disease pathogens,
which uses the composition for enhancing the immunological effects
described in any one of the above items 1 to 12.
[0029] 14. Feed, premix or drink comprising the composition for
enhancing the immunological effects described in any one of the
above items 1 to 12.
[0030] 15. A method for using a composition for enhancing the
immunological effects, comprising previously mixing the composition
for enhancing the immunological effects described in any one of the
above items 1 to 12 above with a vaccine and then administering the
vaccine.
DETAILED DESCRIPTION OF THE INVENTION
[0031] The present invention will be hereinafter described in
detail.
[0032] By taking account of the fact that the stable activity-type
provitamin exhibits excellent immunopotentiative activity in the
host when injection administered into a body and also that
L-ascorbic acid phosphates as one of the provitamin concerned
exhibit antiviral activity based on the immunopotentiative activity
against virus diseases, the present inventors have considered that
the provitamin concerned must be effective in enhancing the
phylactic effect of a vaccine based on the sthenia of circulating
antibody or cell-mediated immunity, and have made investigations to
use a vaccine and the provitamin concerned in combination.
[0033] Other than provitamins, some substances exhibit the
above-described immunopotentiative activity. For example, a natural
substance contained in the fruit body of Grifola erondosa exhibits,
when perorally administered, immunopotentiative activity (see,
International Journal of Immunopharmacology, Vol. 12, No. 6,
675-684 (1990)) and also fluctuates, when perorally administered,
the lymphocyte subset in blood (see, Syokaki to Meneki (Digestive
Apparatus and Immunity), No. 20, pp. 78-82 (1988)). As such, the
effect by the combination of an immunopotentiator perorally
administered and a vaccine is already known. Also, an agent for
enhancing the immunological effect of a vaccine for viruses and
bacterial, comprising a glucan having a .beta.-1,3-glycoside bond
as a main chain and originated from a mushroom was disclosed (see,
JP-A-6-172217) . However, the effect by the combination of a
provitamin derivative and a vaccine is not yet known at
present.
[0034] As a result of investigations by the present inventors, it
is recognized that when a vaccine and the provitamin concerned are
administered in combination, the circulating antibody and the
production of the IgA antibody both are enhanced as compared with
the administration of a vaccine alone, and also the macrophage
activity as one index for the cell-mediated immunity is enhanced.
Furthermore, it is verified that even when the amount of a vaccine
used is greatly reduced, the phylactic effect obtained is
sufficiently high. By these, the above-described problems all can
be overcome.
[0035] The term "provitamin" as used in the present invention means
a substance which is physically stable and inactive but under the
action of an intercellular enzyme of the objective animal,
expresses satisfactory vitamin activity.
[0036] Specifically, L-ascorbic acid derivatives and tocopheryl
phosphates can be used as the provitamin of the present invention,
however, among L-ascorbic acid derivatives, L-ascorbic
acid-2-sulfate and the like are known to fail in exerting
satisfactory activity in organisms because the organism does not
have a hydrolase therefor, and therefore, these cannot be used in
the present invention.
[0037] The L-ascorbic acid derivatives or the salts thereof for use
in the present invention are represented by the following formula
(1): 1
[0038] wherein R.sup.1, R.sup.2, R.sup.3 and R.sup.4 independently
represent a hydroxyl group; an ester group of such a hydroxyl group
with an inorganic acid or an organic acid; a glycoside group of
such a hydroxyl group with a sugar; a ketal group of such a
hydroxyl group with a ketone; or an acetal group of such a hydroxyl
group with an aldehyde provided that R.sup.1 and R.sup.2 are not a
hydroxyl group at the same time.
[0039] Specific examples of preferred L-ascorbic acid derivatives
or the salts thereof include salts of L-ascorbic acid-2-phosphate
or salts thereof, L-ascorbic acid-2-glycoside,
6-alkylcarbonyloxy-L-ascorbic acid-2-phosphate or salts thereof,
etc. R.sup.4 is --OC(O)R in 6-alkylcarbonyloxy-L-ascorbic
acid-2-phosphate or salts thereof, wherein R is preferably an alkyl
group having 9 to 21 carbon atoms.
[0040] More specifically, 6-dodecanoyloxy-L-ascorbic
acid-2-phosphate, 6-tetradecanoyloxy-L-ascorbic acid-2-phosphate,
6-palmytoyloxy-L-ascorbic acid-2-phosphate, 6-stearyloxy-L-ascorbic
acid-2-phosphate, 6-(cis-9-octadecenoyloxy)-L-ascorbic
acid-2-phosphate, 6-linoloxy-L-ascorbic acid-2-phosphate,
6-linoloxy-L-ascorbic acid-2-phosphate, 6-linolenyloxy-L-ascorbic
acid-2-phosphate, 6-arachidonyloxy-L-ascorbic acid-2-phosphate,
5,6-O-benzylidene-L-ascorbi- c acid-2-phosphate or salts thereof.
Monophosphate is a preferred 2-phosphate.
[0041] Metallic salts of L-ascorbic acid-2-phosphate for use in the
present invention can be prepared by conventional methods.
[0042] As for L-ascorbic acid-2-glycoside, preferred are glucose
glycoside such as L-ascorbic acid-2-glucoside
2-O-.alpha.-D-glucopyranosyl-L-ascorb- ic acid) and the like.
[0043] Examples of the salts of ascorbic acid derivatives include
those with a cation such as ammonium, sodium, potassium, magnesium,
calcium, strontium, barium, aluminum, iron, zinc, bismuth and
organic amines. That is, the salt may be a salt with at least one
of these cations. The ascorbic acid derivative and the salt thereof
may be also a water adduct or hydrorate thereof.
[0044] Among the ascorbic acid derivatives and salts thereof usable
in the present invention, those having an excellent effect include
sodium salt, potassium salt and zinc salt of L-ascorbic
acid-2-monophosphoric acid and L-ascorbic acid-2-glucoside.
Particularly, sodium salt of L-ascorbic acid-2-monophosporic acid
is preferred. Magnesium and calcium salts of L-ascorbic
acid-2-monophosporic acid also have some effect, which, however, is
inferior to that of the sodium salt.
[0045] The tocopherol in the tocopheryl phosphate and salts thereof
for use in the present invention is represented by the following
formula (2): 2
[0046] wherein R.sup.5, R.sup.6 and R.sup.7 represent hydrogen atom
or methyl group, and R represents a long chain alkyl group: 3
[0047] According to the substituents R.sup.5, R.sup.6 and R.sup.7,
the tocopherol represented by the formula (2) is known as
.alpha.-tocopherol (R.sup.5, R.sup.6 and R.sup.7: CH.sub.3),
.beta.-tocopherol (R.sup.5 and R.sup.7: CH.sub.3, R.sup.6: H),
.gamma.-tocopherol (R.sup.6 and R.sup.7: CH.sub.3, R.sup.5: H),
.delta.-tocopherol (R.sup.7: CH.sub.3, R.sup.5 and R.sup.6: H),
.zeta.2-tocopherol (R.sup.5 and R.sup.6: CH.sub.3, R.sup.7: H) or
.eta.-tocopherol (R.sup.5 and R.sup.7: CH.sub.3, R.sup.6: H). Among
these, .alpha.-tocopherol is particularly preferred.
[0048] In addition, .zeta.1-tocopherol and .epsilon.-tocopherol
where the long chain alkyl group represented by R which bonds to
the carbon atom adjacent to the O atom in the benzopyrane structure
of .alpha.-tocopherol or .beta.-tocopherol is displaced by the
following formula (3): 4
[0049] are known and either one can be a constituent element of the
tocopheryl phosphate and salts thereof for use in the present
invention.
[0050] Metallic salts of tocopheryl phosphate for use in the
present invention can be prepared by conventional methods.
[0051] Examples of the salt of tocopheryl phosphate, which can be
used in the present invention, include alkali metals and alkaline
earth metals such as sodium, potassium, calcium, magnesium,
aluminum, zinc and lithium. Among these, sodium salt is preferred
because the solubility in water is high and the toxicity is
low.
[0052] In order to effectively exert the immunopotentiative
activity, the stable activity-type provitamin concerned is
preferably administered by mixing it with feed or drinking water or
by a syringe. If the case is so, the IgA antibody secreted on the
respiratory airway or digestive tract mucosa shows no sthenia of
production and therefore, the immunopotentiation of a influenza
vaccine is not so much enhanced because the IgA antibody plays an
important role in the phylaxis.
[0053] In the case where the provitamin concerned is perorally
administered, it is recognized that the provitamin concerned
appropriately contacts with the respiratory airway or digestive
tract mucosa to stimulate the mucosa and thereby promote the
secretion of the IgA antibody and by virtue of these three
mechanisms of circulating antibody, cell-mediated immunity and IgA
antibody, the living body is prevented from the virus infection.
This effective immunopotentiative effect is estimated to occur
also, for example, on the influenza vaccine. In fact, the present
inventors have confirmed that when the provitamin is perorally
administered, the IgA antibody shows significant enhancement of the
production accompanying the administration of a Sendai virus
vaccine closely related to the influenza virus and the phylactic
effect is enhanced.
[0054] In the present invention, the activity of enhancing the
immunological effects of a vaccine by the provitamin concerned is
considered to result because the provitamin concerned
non-specifically activates the prophylactic mechanism of a host.
The provitamin concerned is found to have capability of
accelerating the immunological effects of any kind of vaccines and
to be widely applicable also to bacteria vaccines irrespective of
the kind of the virus vaccine. Furthermore, this effectiveness is
expected to extend even to protozoan diseases such as malaria.
[0055] In the composition of the present invention, the
immunological effects of a vaccine is as high as 2 times or more,
preferably 4 times or over in terms of the serum antibody unit. An
immunological effects of less than 2 times the normal serum
antibody unit is disadvantageously insufficient as the
immunological effects of the composition of the present
invention.
[0056] The provitamin concerned containing a stable activity-type
provitamin may be administered into a body by injection
administration, peroral administration or the like. The
administration method may be appropriately selected according to
the kind of vaccines, the condition of the host and the like.
However, in view of the accelerated production of IgA antibody and
the safety and easiness of administration, the peroral
administration is preferred than the injection administration. The
provitamin concerned may be previously mixed with a vaccine and
then perorally administered.
[0057] The peroral administration is further advantageous in that
the provitamin concerned needs not be purified to a high purity and
for example, the bacteria which produce the provitamin concerned
can be used in the form of a cell as it is or as a mixture thereof,
so that the production cost can be reduced.
[0058] The composition of the present invention can be in the form
of a solid composition and a liquid composition for peroral
administration or a liquid composition for parental administration
(parental injection).
[0059] Examples of the solid composition for peroral administration
include tablet, capsule, powder medicine, granule or the like.
[0060] Capsules include a hard capsule and s soft capsule.
[0061] Such a solid composition comprises one or more active
ingredients admixed with at least one inert diluent such as
lactose, mannitol, mannit, glucose, hydroxypropylcellose,
hydroxypropylcellose phthalate, microcrystalline cellulose, starch,
polyvinylpyrrolidone, magnesium aluminometasilicate.
[0062] The composition can also comprise additives other than
diluent in accordance with conventional methods, for examples,
lubricants such as magnesium stearate, disintegrating agents such
calcium carboxymethylcellulose, solubilizer and the like. Tablets
may be optionally covered with a film of white sugar, gellatine,
hydroxypropylcellose, hydroxypropylcellose phthalate and the like.
Furthermore, capsules of digestible substances such as gellatine
can be also used.
[0063] The liquid compositions for peroral administration include
emulsion and solution, etc. Such a liquid composition comprises one
or more active ingredients in a commonly used inert diluent such as
purified water and ethanol. The compositions can also comprise
additives such as humectants and elusifiers, sweetners, flavors,
aroma and anticeptics.
[0064] The liquid compositions for parental injection include
sterile aqueous and non-aqueous solvents, suspensions and
emulsions. Examples of aqueous solvents and suspensions can be form
of distilled water for injection and physiological saline. Examples
of non-aqueous solvents and suspensions can be form of propylene
glycol, polyethylene glycol, vegetable oils such as olive oils, and
alcohols such as ethanol and the like.
[0065] The composition may also comprise other additives such as
antiseptics, humectants, emulsifiers, dispersants, stabilizers,
solubility aids (e.g. glutamic acid, aspartic acid). These
additives may be sterilized by way of filtering with a
bacteria-trapping filter, addition of a germicide or radiation.
They can be formed as a sterilized solid composition and solved in
a sterilized or aseptic distilled water for injection or the like
solvent prior to use.
[0066] In the present invention, the effective dose of the
provitamin which is an active ingredient is from 0.5 to 1,000 mg/kg
per day for the peroral administration, and from 0.05 to 150 mg/kg
for the injection administration. Preferably, the dose is from 5 to
500 mg/kg for the peroral administration and from 0.5 to 120 mg/kg
for the injection administration, and more preferably from 10 to
100 mg/kg for the peroral administration and from 1 to 100 mg/kg
for the injection administration.
[0067] Needless to say, the dose may vary depending on the various
conditions, and therefore, the dose less than the above mentioned
value may be suffice and the dose more than the above mentioned
value may be required.
[0068] A vaccine is inoculated in a usual amount within a range of
assuring the biological safety and the effect. Usually, an
attenuated virus strain is used and this strain may be administered
by a method commonly used in the vaccine administration and may be
inoculated perorally or using a nebulizer or syringe. Depending on
the kind of viruses, the amount inoculated varies but usually, a
vaccine is suitably inoculated within the range from 10 to 10,000
CIU (cell infectious unit) With respect to the administration
method, any method commonly used for the vaccine inoculation may be
used and examples thereof include contacting with any mucosal
tissue, such as oral, nasal or branchial mucosa, administration by
nebulization, and injection such as intradermal injection.
[0069] The composition of the present invention can be blended with
feed, premix or drink according to the above-described dose.
[0070] The composition for enhancing the vaccine effect of the
present invention may be fed to an animal by blending it with a
general feed such as a protein feed, carbohydrate feed, a feed
additive or a feed ingredient. The optimal feed amount varies
depending on the kind of animals, stage or eucrasia and therefore,
may be appropriately determined according to the conditions where
the composition is applied.
[0071] The composition for enhancing immunological effects of a
vaccine of the present invention is effective for mammals including
human, birds and fishes and particularly effective for animals
under high-density feeding such as cattle, pig, fowl, horse,
poultry, pet, breeding marine animal and laboratory animal. In
general, the feeding environment of the animal under high-density
feeding is restricted and therefore, the animal suffers from stress
to readily cause deterioration of the immunological function and is
prone to viral infection. Thus, it is revealed that the composition
of the present invention has an effect of preventing viral
infection which gives rise to health deterioration of a useful
animal, and ameliorating the eucrasia.
BEST MODE OF THE INVENTION
[0072] The present invention is described in greater detail below
by referring to the Examples, however, the present invention is not
limited to these Examples.
[0073] Hereinafter, L-ascorbic acid-2-monophosphate is referred to
as "A2P" and .alpha.-tocopheryl phosphate is referred to as "VEP".
For example, A2P-Mg indicates a magnesium salt of L-ascorbic
acid-2-monophosphate and d1-VEP-Na indicates an Na salt of
d1-.alpha.-tocopheryl phosphate.
EXAMPLE 1
Effect of Provitamins on the Circulating Antibody Unit (HIU) After
the Vaccine Administration
[0074] The provitamins prepared to have a composition in the
following Formulation Examples 1 to 4 were individually or in
combination (hereinafter referred to as "provitamin") dissolved in
a phosphate buffered saline (PBS) to a concentration of 0.5% and
then continuously administered perorally or peritoneally to a mouse
(ICR, 3 weeks old, male) from two weeks before the administration
of a vaccine. The sample was administered such that APM per day was
80 mg/kg-body weight in the case of peroral administration and the
dose per day was 40 mg/kg-body weight in the case of injection.
[0075] The vaccine used here was TR-5 strain which is an attenuated
strain of Sendai virus, and 500 CIU (cell infectious unit) thereof
was nasally administered to a mouse under ether anesthesia. That
is, a stock solution (10.sup.6 CIU/ml) of TR-5 strain was diluted
with PBS and 50 .mu.l of each sample was nasally administered.
After the administration of vaccine, the serum was sampled in aging
(inoculation day, after 7 days, after 14 days and after 21 days)
and the antiviral antibody unit in the serum was measured by the
hemagglutination inhibition (HI) reaction which inhibits the
hemagglutination (HA) reaction.
[0076] The HA reaction is a reaction where a virus particle or an
HA antigen adsorbs to an erythrocyte and combines the erythrocytes
with each other to cause hemagglutination. In the measurement of
the HA unit, 2-fold step dilution series of a virus solution are
prepared, a constant amount of erythrocytes is added to each
dilution and after the passing of a predetermined time, the
agglutination image is examined. The HA unit (HAU) is the
reciprocal of the highest dilution number in a test tube showing
agglutination positive. The HA reaction is inhibited by previously
adding an antiviral antibody to the virus solution. This is the HI
reaction.
[0077] The HI antibody unit (HIU) is the reciprocal of the highest
dilution number of a serum which completely inhibits the HA
reaction attributable to 4 HAU virus antigen.
1 TABLE 1 Composition (%) Formulation Formulation Formulation
Formulation Example 1 Example 2 Example 3 Example 4 A2P-Mg 100% 40%
30% A2P-Na 100% 30% 10% A2P-Ca 10% A2P-K 10% d1-VEP-Mg 10%
d1-VEP-Na 10% d1-VEP-Ca 30% 10% d1-VEP-K 10%
[0078] Test Method
[0079] To 25 .mu.l of a serum subjected to 2-fold step dilution, 25
.mu.l of a virus solution adjusted to 16 HAU was added and allowed
to stand at room temperature for 1 hour. Subsequently, 50 .mu.l of
a 0.5% chicken erythrocyte suspension was added and allowed to
stand at 4.degree. C. for 1 hour. Thereafter, the HI unit was
determined. The results are shown in Table 2.
2TABLE 2 HIU (Peroral (Peritoneal Days Passed After Administration)
Administration) Infection 0 7 14 21 0 7 14 21 Vaccine-free segment
<16 <16 <16 <16 <16 <16 <16 <16 500 CIU
vaccine <16 <16 <16 16 <16 <16 16 32 500 CIU vaccine
+ <16 <16 16 64 <16 16 32 64 Formulation Example 1 500 CIU
vaccine + <16 <16 16 64 <16 <16 32 64 Formulation
Example 2 500 CIU vaccine + <16 <16 64 64 Formulation Example
3 500 CIU vaccine + <16 16 64 64 Formulation Example 4
[0080] By using a vaccine and a provitamin, the circulating
antibody unit was significantly increased as compared with the case
of using the vaccine alone.
EXAMPLE 2
Effect of Provitamin and Vaccine on the Activity of the
Macrophage
[0081] The administration of provitamin and inoculation of a
vaccine (500 CIU) were performed in the same manner as in Example
1. 14 Days after the inoculation of the vaccine, macrophages were
sampled from the abdominal cavity of the mouse and the number of
macrophages was counted through a microscope. Also, the activity of
the macrophage was examined as follows. Fibrosarcoma SMT-5 (target
cell) and the sampled macrophages (effector cell) were mixed at a
ratio of 1:7 and cultured in CO.sub.2 medium for 24 hours. 8 Hours
before the completion of culture, .sup.3H-thymidine was added and
the amount of .sup.3H-thymidine taken up into the residual target
cells was measured by a liquid scintillation counter. From the
value obtained, the activity of macrophage was determined. The
amount of .sup.3H-thymidine in the control group was the amount of
.sup.3H-thymidine taken up into the target cells where the
macrophages were not added.
[0082] The results are shown in Table 3.
3TABLE 3 Administration Macrophage/ Macrophage Sample Route Body
(.times. 10.sup.6) Activity 500 CIU vaccine Peritoneal 3.9 9.5 500
CIU vaccine + Peritoneal 9.1 22.1 Formulation Example 1 500 CIU
vaccine + Peroral 6.8 20.5 Formulation Example 1 500 CIU vaccine +
Peroral 6.5 20.2 Formulation Example 2 500 CIU vaccine + Peroral
6.3 21.3 Formulation Example 3 500 CIU vaccine + Peroral 7.1 23.7
Formulation Example 4
[0083] By using a vaccine and a provitamin in combination, the
production of macrophage was enhanced and the activation was
revealed, as compared with the case of using the vaccine alone.
EXAMPLE 3
Effect of Rovitamin on Secretory IgA Induction by Sendai Virus Live
Vaccine
[0084] The administraion of provitamin and inoculation of a vaccine
were performed in the same manner as in Example 1. 14 Days after
the inoculation of vaccine, the mouse was sacrificed, the trachea
and bronchia were twice washed with 1 ml of PBS (phosphate buffered
saline), and IgA recovered in PBS was measured by the enzyme linked
immunosorbent assay (ELISA).
[0085] ELISA
[0086] Sendai virus was fixed on a plate and reacted with a test
solution. Thereafter, peroxidase marker anti-mouse IgA
immunoglobulin was reacted and then phenylenediamine 2HCl was added
to cause coloration. OD 492 was measured and from the calibration
curve obtained based on the standard IgA, the IgA amount in the
test solution was determined.
[0087] The results are shown together in Table 4.
4 TABLE 4 Administration Sample Route IgA (U/.mu.g) Control group
(not treated) 14 500 CIU vaccine Peritoneal 19 500 CIU vaccine +
Peritoneal 69 Formulation Example 1 500 CIU vaccine + Peroral 51
Formulation Example 1 500 CIU vaccine + Peroral 69 Formulation
Example 2 500 CIU vaccine + Peroral 68 Formulation Example 3 500
CIU vaccine + Peroral 70 Formulation Example 4
[0088] By using a vaccine and a provitamin in combination, the
induction of secretory IgA was enhanced, as compared with the case
of using the vaccine alone.
EXAMPLE 4
[0089] The administration of provitamin and inoculation of a
vaccine were performed in the same manner as in Example 1, and 14
days after the inoculation of attenuated vaccine, Sendai virus
strongly toxic strain (1.7.times.10.sup.7 CIU/ml) was subjected to
15-hold dilution in PBS. Subsequently, 70 .mu.l of the dilution
(about 8.times.10.sup.4 CIU:15 LD.sub.50) was nasally infected to a
mouse under ether anesthesia and 20 days after the infection with
the strongly toxic strain, the survival rate of mice was examined.
The results are shown in Table 5.
[0090] Measurement of LD.sub.50
[0091] The test solution was step-diluted and each dilution was
used for a group of animals. The life and death were examined and
the point of dilution degree where 50% of death was recognized was
designated as LD.sub.50.
5 TABLE 5 Survival Rate (number of survival mice/ number of mice
tested) Amount of Vaccine Administration Inoculated (CIU) Sample
Group 0 50 500 Control group Peritoneal 0/10 0/10 2/10 (PBS
administration) Control group Peroral 0/10 0/10 1/10 (PBS
administration) Formulation Peritoneal 0/10 8/10 8/10 Example 1
Formulation Peroral 0/10 9/10 9/10 Example 1 Formulation Peroral
1/10 9/10 10/10 Example 2 Formulation Peroral 1/10 8/10 10/10
Example 3
[0092] By using a vaccine and a provitamin, the survival rate was
increased as compared with the case of using the vaccine alone.
[0093] INDUSTRIAL APPLICABILTTY
[0094] According to the composition for enhancing immunological
effects of the present invention which contains at least one
compound selected from L-ascorbic acid derivatives and
.alpha.-tocopheryl phosphates, the provitamins are resistant to the
decomposition by a digestive enzyme in vivo, very low in the
toxicity, almost free of an adverse reaction even by the injection
administration. Accordingly, the composition is, as prominent
characteristic features, completely nulled in the toxicity in the
case of peroral administration. In addition, the provitamin of the
present invention is converted into a natural nutrient and since
the toxicity is low, the provitamin may be taken as food or animal
feed and even in this case, a sufficiently high vaccine enhancing
effect can be expected. In the case of using the provitamin
concerned as food or feed, purification to a high purity is not
necessary but the coarse product or a culture dry product in the
provitamin production can provide a sufficiently high expected
effect as it is and therefore, the composition for enhancing the
immunological effects of a vaccine for viruses, bacteria and/or
infectious disease pathogens is useful over a wide range.
* * * * *