Abstrict It is intended to provide a blood bag system comprising one or
more of containers capable of holding a liquid, and a connecting
tube connected liquid-tightly to the container, characterized in
that at least one of the containers holds an inactivator that inactivates
a pathogenic microorganism contained in blood and/or an anticoagulant;
and the inactivator contains as a main component a platinum compound
capable of binding to nucleic acids of the microorganism or an aquo
complex of the platinum compound. According to the present invention,
an effect of inactivating pathogenic microorganisms is increased
and an excellent sustained inactivation effect is provided. Furthermore,
transfusion of inactivated blood after neutralization treatment
enables supply of microbiologically and toxicologically safe blood
preparations.
Claims 1. A blood bag system comprising a container holding an inactivator
that inactivates a microorganism contained in blood, a container
holding an anticoagulant and a connecting tube connected liquid-tightly
to the container, wherein the inactivator contains as a main component
a platinum compound capable of binding to nucleic acid of the microorganism
or an aquo complex of the platinum compound; and a tube for introducing
a neutralizing agent to neutralize the inactivator is connected
with the container holding the inactivator.
2. A blood bag system according to claim 1 wherein the platinum
compound is at least one selected from the group consisting of cisplatin,
carboplatin, and nedaplatin.
3. A blood bag system according to claim 1 wherein the aquo complex
of the platinum compound is at least one selected from the group
consisting of a monoaquo complex, a diaquo complex, a monoaquomonohydroxo
complex, and a dihydroxo complex.
4. A blood bag system according to claim 1 wherein the pathogenic
microorganism is at least one selected from the group consisting
of DNA type viruses, RNA type enveloped viruses, and bacteria.
5. A blood bag system according to claim 1 wherein the neutralizing
agent contains as a main component an amino acid compound or a thiosulfate.
6. A blood bag system according to claim 1 wherein a container
holding the neutralizing agent to neutralize the inactivator is
connected with the tube for introducing the neutralizing agent
7. A method of inactivating a pathogenic microorganism in blood,
comprising: adding a microorganism inactivator containing as a main
component a platinum compound capable of binding to nucleic acids
of the microorganism or an aquo complex of the platinum compound
to a blood bag that holds blood collected in advance; thereafter,
adding a neutralizing agent containing as a main component an amino
acid compound or a thiosulfate to neutralize the inactivator.
8. A method of inactivating a pathogenic microorganism according
to claim 7 wherein the microorganism inactivator is added so that
a concentration becomes 0.07 mM (.mu.mol/mL) to inactivate 1 log.sub.10
or more of the pathogenic microorganism in the blood held in the
blood bag.
9. A method of inactivating a pathogenic microorganism according
to claim 7 wherein the neutralizing agent is methionine or sodium
thiosulfate.
10. A method of inactivating a pathogenic microorganism according
to claim 7 wherein the neutralizing agent is added so that a concentration
becomes 10 to 500 times a concentration of the microorganism inactivator.
Description TECHNICAL FIELD
[0001] The present invention relates to a method of inactivating
pathogenic microorganisms in blood and to a blood bag system for
supplying microbiologically and toxicologically safe blood preparations.
BACKGROUND ART
[0002] Blood transfusion is indispensable in modern medicine. Blood
transfusion had caused 9000 people to be infected with HIV until
the American National Red Cross commenced screening to remove blood
contaminated with HIV (human immunodeficiency virus) in 1985. Thereafter,
blood preparations were screened for virus infection and infected
blood preparations were discarded. As a result, the number of infected
patients by transfusion was reduced to mere 41. Even so, however,
it is impossible by the present screening technology to detect pathogenic
microorganisms that cause infectious diseases by transfusion such
as viruses and bacteria out of inspection items, and thus, the danger
of being infected with new virus or the like cannot be excluded.
Accordingly, treatment for inactivating pathogenic microorganisms
becomes necessary for blood preparations.
[0003] Known inactivation technologies for pathogenic microorganisms
such as viruses and bacteria thus far developed include Solvent/Detergent
(SD) treatment for blood preparations. The treatment has a disadvantage
in that it is ineffective to nonenveloped viruses, and safe blood
preparations are not necessarily supplied. For this reason, in recent
years, inactivators against pathogenic microorganisms are being
energetically developed in order to supply safe blood.
[0004] For example, virus inactivators containing an ethyleneimine
oligomer has been proposed (International Publication Nos. WO97/07674
and WO99/17802). The ethylene oligomer is excellent for inactivation
of viruses but has an unstable structure so that it evaporates at
ambient temperature and explodes. Accordingly, use of the ethyleneimine
oligomer for the inactivation of pathogenic microorganisms in blood
preparations involves a high risk of infection of the operator.
[0005] Also, production and storage of the ethyleneimine oligomer
requires maintenance at low temperatures, so that the inactivators
for pathogenic microorganisms containing an ethyleneimine oligomer
not only require much cost for the production facility as well as
transportation and storage, but also require further attention for
safety management of the inactivators against pathogenic microorganisms
containing an ethyleneimine oligomer when used at medical sites.
[0006] Therefore, it is an object of the present invention to provide
a method of inactivating pathogenic microorganisms in blood free
of various problems caused by the conventional inactivators against
pathogenic microorganisms and a blood bag system for blood preparations
in order to supply microbiologically and toxicologically safe blood
preparations. In particular, an object of the present invention
is to provide a blood bag system for blood preparations in which
pathogenic microorganisms in blood are efficaciously inactivated
and the inactivation effect is sustained over a long period of time
and which is stable under environmental conditions such as light
and temperature.
DISCLOSURE OF THE INVENTION
[0007] The above objects may be attained by the present invention
described below.
[0008] The present invention provides a blood bag system including
one or more of containers capable of holding a liquid, and a connecting
tube connected liquid-tightly to the container, characterized in
that: at least one of the containers holds an inactivator that inactivates
a pathogenic microorganism contained in blood and/or an anticoagulant;
and the inactivator contains as a main component a platinum compound
capable of binding to nucleic acids of the microorganism or an aquo
complex of the platinum compound.
[0009] According to the blood bag system of the present invention,
two or more of the containers are provided, and the inactivator
and the anticoagulant are held in different containers, respectively.
The container holding the inactivator and the container holding
the anticoagulant may be connected by the connecting tube.
[0010] Further, according to the blood bag system of the present
invention, the inactivator and the anticoagulant may be held in
the same container.
[0011] Therefore, the blood bag system of the present invention
is a blood bag system including a blood bag holding an anticoagulant
and a container holding an inactivator that inactivates a microorganism
contained in blood, connected to each other, in which the inactivator
contains as a main component a platinum compound capable of binding
to nucleic acid of the microorganism or an aquo complex of the platinum
compound; or a blood bag system holding an anticoagulant and an
inactivator that inactivates a microorganism contained in blood,
in which the inactivator contains as a main component a platinum
compound capable of binding to nucleic acid of the microorganism
or an aquo complex of the platinum compound.
[0012] According to the blood bag system of the present invention,
the platinum compound is preferably at least one selected from the
group consisting of cisplatin, carboplatin, and nedaplatin.
[0013] According to the blood bag system of the present invention,
the aquo complex of the platinum compound is at least one selected
from the group consisting of a monoaquo complex (for example, cis-monochloromonoaqua
diammineplatinous(II)chloride), a diaquo complex (for example, cis-diaquodiammineplatinous(II)dinitrate),
a monoaquomonohydroxo complex (for example, cis-monohydroxymonoaqua
diammineplatinous(II)chloride), and a dihydroxo complex (for example,
cis-dihydroxydiammineplatinum(II)).
[0014] According to the blood bag system of the present invention,
the pathogenic microorganism is preferably at least one selected
from the group consisting of DNA type viruses, RNA type enveloped
viruses, and bacteria.
[0015] The present invention also provides a method of inactivating
a pathogenic microorganism in blood, including: adding a microorganism
inactivator containing as a main component a platinum compound capable
of binding to nucleic acids of the microorganism or an aquo complex
of the platinum compound to a blood bag that holds blood collected
in advance.
[0016] According to the method of inactivating a pathogenic microorganism
of the present invention, it is preferred that the microorganism
inactivator is added so that a concentration becomes 0.07 mM (.mu.mol/mL)
to inactivate 1 log.sub.10 or more of the pathogenic microorganism
in the blood held in the blood bag.
[0017] The method of inactivating a pathogenic microorganism of
the present invention further preferably includes: adding a neutralizing
agent containing as a main component an amino acid compound or a
thiosulfate to neutralize the inactivator, after adding the microorganism
inactivator.
[0018] According to the method of inactivating a pathogenic microorganism
of the present invention, the neutralizing agent is preferably methionine
or sodium thiosulfate.
[0019] According to the method of inactivating a pathogenic microorganism
of the present invention, the neutralizing agent is preferably added
so that a concentration becomes 10 to 500 times a concentration
of the microorganism inactivator.
[0020] Further, the present invention provides an inactivator that
inactivates a microorganism contained in blood that is collected
in advance containing as an active component a platinum compound
or an aquo complex of the platinum compound.
[0021] According to the inactivator of the present invention, the
platinum compound is preferably at least one selected from the group
consisting of cisplatin, carboplatin, and nedaplatin.
[0022] Further, the present invention provides a method of using
a platinum compound or an aquo complex of the platinum compound
as an inactivator for a microorganism in blood that is collected
in advance.
BRIEF DESCRIPTION OF DRAWINGS
[0023] FIG. 1 is a schematic diagram illustrating a blood bag system
according to one construction example of the present invention;
[0024] FIG. 2 is a schematic diagram illustrating a blood bag system
according to another construction example of the present invention;
[0025] FIG. 3 is a graph illustrating SV-40 killing effect of a
platinum compound in Example 1 with the SV-40 killing effect being
shown as inactivation effect (logarithm (log.sub.10)
[0026] FIG. 4 is a graph illustrating SV-40 killing effect of a
platinum compound in Example 2 with the SV-40 killing effect being
shown as inactivation effect (logarithm (log.sub.10));
[0027] FIG. 5 is a graph illustrating an effect of cisplatin to
inactivate Yersinia enterocolitica;
[0028] FIG. 6 is a graph illustrating an effect of neutralizing
cisplatin in Example 6 with methionine; and
[0029] FIG. 7 is a graph illustrating an effect of neutralizing
cisplatin in Example 6 with sodium thio sulfate.
BEST MODE FOR CARRYING OUT THE INVENTION
[0030] Hereinafter, the present invention will be described in
detail.
[0031] A blood bag system of the present invention includes at
least one container capable of holding a liquid, and a connecting
tube liquid-tightly connected to the container capable of holding
the liquid for use in charging and discharging the liquid held in
the container. At least one of the containers constituting the system
holds an anticoagulant in order to hold collected blood. Hereinafter,
the container holding the anticoagulant is referred to as a "blood
bag". The blood bag system may have, in addition to the blood
bag, one or more other containers for use in holding drugs and blood
components such as erythrocytes, leukocytes, platelets, or plasma
after separation.
[0032] Where the blood bag system includes a plurality of containers,
the containers are connected to each other with connecting tubes,
through which the liquid held in the containers, for example, blood
is charged or discharged. Further, the blood bag system may include
a connecting tube that is connected to the container at only one
end of the connecting tube. Such a tube that is connected to the
container at only one end thereof is used mainly for charging and
discharging blood between the inside and the outside of the blood
bag system. Specific example of such a tube includes a blood collection
tube equipped with a needle at an end opposite to the end at which
the container is connected.
[0033] FIG. 1 is a schematic diagram illustrating a blood bag system
according to one construction example of the present invention.
In the blood bag system illustrated in FIG. 1 a blood bag 1 a
container 2 holding an inactivator that inactivates a microorganism
contained in blood, and a plurality of (two in FIG. 1) blood component
bags 3 and 4 as the other containers are connected through connecting
tubes 5. To the blood bag 1 is connected a blood collecting tube
6 equipped with a blood collecting needle 7 at the tip of the tube.
The connecting tubes 5 can be sealed by pressing with an aluminum
ring or by heat fusion using a heat sealer. Further, the blood component
bags 3 and 4 for holding blood components after separation are designed
so as to be readily detached separately.
[0034] FIG. 2 is a schematic diagram illustrating a blood bag system
according to another construction example of the present invention.
In the blood bag system shown in FIG. 2 the blood bag 1 and the
container 2 holding an inactivator that inactivates a microorganism
contained in blood are connected through the connecting tube 5.
To prevent migration of the contents between the blood bag 1 and
the container 2 unless necessary, the connecting tube 5 is provide
with a valve 51 as an opening and closing means for the connecting
tube 5. Note that though not shown, in the blood bag system illustrated
in FIG. 1 it is preferable that the connecting tubes 5 that connect
the blood bag 1 the container 2 and the blood component bags 3
and 4 are provided with a klemme, which is an opening and closing
means.
[0035] To the blood bag 1 is connected the blood collecting tube
6 equipped with the blood collecting needle 7 at the tip of the
tube. Further, the blood bag 1 is provided with an air-bleeding
tube 10. When the collected blood from the blood collecting tube
6 or the inactivator from the container 2 is introduced into the
blood bag 1 air-bleeding of the air in the inside the blood bag
1 through a tube 10 facilitates the introduction of the blood or
inactivator into the blood bag 1.
[0036] Each constituent element of the blood bag systems shown
in FIGS. 1 and 2 are preferably moldings of a thermoplastic resin
such as a vinyl chloride resin containing a plasticizer, taking
safety into consideration.
[0037] In the illustrated blood bag system, the blood bag 1 holds
an anticoagulant, so that the coagulation of the collected blood
can be prevented. The anticoagulant held in the blood bag 1 may
be selected from a wide variety of known anticoagulants. Specific
examples of the anticoagulant that can be used include citric acid,
heparin, low molecular weight heparin, nafamostat mesilate, gabexate
mesilate, and EDTA. The anticoagulants are usually diluted with
physiological saline or an aqueous glucose solution and is held
in such an amount that the ratio of the anticoagulant liquid to
the blood to be held is an ordinary ratio, specifically an amount
of, for example, about (1:2 to 1:20). The anticoagulants may constitute
a part of a blood preserving liquid. Examples of such a blood preserving
liquid include physiological solution such as ACD liquid and CPD
liquid that contains one or more of adenine, mannitol, sorbitol,
guanosine, and the like.
[0038] The container 2 holds an inactivator that inactivates pathogenic
microorganisms contained in blood. The inactivator as necessary
is introduced into the blood bag 1 through the connecting tube 5
by opening preferably the valve 51 which is an opening and closing
means. This inactivates the pathogenic microorganisms contained
in the blood in the blood bag 1.
[0039] Note that the illustrated blood bag system assumes a construction
in which the anticoagulant is held in the blood bag 1 and the inactivator
is held in the container 2 that is, a construction in which the
anticoagulant and the inactivator are held in different containers.
However, the anticoagulant and the inactivator may be held in the
same container. That is, in the illustrated construction, the inactivator
may be held in advance in the blood bag 1. In this case, inactivation
of the pathogenic microorganisms contained in blood is performed
as soon as the collected blood is introduced in to the blood bag
1.
[0040] However, in the case where a blood preserving liquid containing
adenine and guanosine is used, mixing a platinum compound with adenine
and guanosine before inactivation of the pathogenic microorganisms
must be avoided since adenine and guanosine react with the platinum
compound.
[0041] According to the present invention, the pathogenic microorganisms
contained in blood and that need to be inactivated include DNA-type
viruses, RNA enveloped viruses, and bacteria. Viruses are genetically
classified into DNA type and RNA type. Structurally, the viruses
are classified based on whether or not a viral membrane (envelope)
is present. HIV and HCV (human hepatitis C virus) currently causing
transfusion infection problems are RNA type enveloped viruses. HBV
(human hepatitis B virus) is a DNA type enveloped virus.
[0042] Further, bacteria causing transfusion infection problems
include Yersinia enterocolitica and Serratia marcescens. Blood microorganism
level of patients infected with these bacteria is 1.times.10.sup.1
to 1.times.10.sup.2 CFU/mL. Blood collection using a blood bag containing
no inactivator results in an increase to 1.times.10.sup.6 to 1.times.10.sup.8
CFU/mL in about three weeks. Transfusion of blood having such a
microorganism level may sometimes lead to death due to an endotoxin
shock.
[0043] The present invention is characterized by using a platinum
compound known as an anticancer agent, more specifically a platinum
compound capable of binding to the nucleic acids of pathogenic microorganisms
and/or an aquo complex of the platinum compound (both may be sometimes
referred to simply as platinum compound) as a drug that inactivates
the pathogenic microorganisms in blood for transfusion (hereinafter,
referred to as "inactivator"). When the platinum compound
is used as an inactivator, binding of the platinum compound to the
nucleic acids of the pathogenic microorganisms is presumed to inactivate
the pathogenic microorganisms.
[0044] The platinum compound used in the present invention may
bind to the nucleic acids of the pathogenic microorganisms. Specific
examples thereof include cisplatin, carboplatin, and nedaplatin.
Two or more of these may be used in combination.
[0045] Further, in the present invention, aquo complexes of the
platinum compounds, that is, aquo complexes of cisplatin, carboplatin,
nedaplatin, or the like may be used. Preferable aquo complexes include
a monoaquo complex such as cis-monochloromonoaqua(II) chloride,
a diaquo complex such as cis-diaquodiammineplatinous(II) dinitrate,
a monoaquomonohydroxo complex such as cis-monohydroxymonoaqua diammineplatinous(II)
chloride, and a dihydroxo complex such as cis-dihydroxydiammineplatinum(II).
Two or more of these may be used in combination. Further, these
may be used in combination with the above-mentioned platinum compounds.
[0046] It is preferable that the platinum compound is dissolved
in physiological saline or an aqueous glucose solution and held
in the container 2 in a liquid state having an osmotic pressure
ratio of about 1 so that it can be introduced from the container
2 to the blood bag 1 through the connecting tube 5 preferably by
opening the valve 51 as an opening and closing means.
[0047] Further, since the platinum compound is decomposed with
light or its stability is changed depending on ion concentration,
it is preferable that a light-shielded container is used as the
container 2 holding the inactivator or that the ion concentration
of the inactivator is adjusted with sodium chloride or the like
before the inactivator is held in the container 2.
[0048] The method of inactivating pathogenic microorganisms is
to inactivate pathogenic microorganisms contained in blood by addition
of an inactivator containing as a main component the above-mentioned
platinum compound or an aquo complex of the platinum compound to
the blood bag holding blood in advance. The method is preferably
practiced by using the blood bag system of the present invention
as mentioned above. Hereinafter, the method of inactivating pathogenic
microorganisms according to the present invention will be described
by using the blood bag system illustrated in FIG. 2.
[0049] Specifically, in the blood bag system of FIG. 2 the inactivator
is introduced from the container 2 into the blood bag 1 that holds
blood collected through the blood collecting needle 7 and the blood
collecting tube 6 by opening the valve 51 attached to the connecting
tube 5 thus practicing the method of inactivating pathogenic microorganisms
according to the present invention.
[0050] According to the method of the present invention, the amount
of platinum compound to be used may vary depending on time, temperature,
composition and concentration of blood components and so on. At
the time of inactivation, the pathogenic microorganisms in blood
held in the blood bag can be inactivated even when a final concentration
of the platinum compound in the blood bag 1 is about 0.02 mM (.mu.mol/mL),
specifically 0.0209 mM. However, the final concentration of the
platinum compound in the blood bag 1 is preferably 0.07 to 100 mM,
more preferably 1 to 10 mM. If the final concentration of the platinum
compound within the above-mentioned concentration range, the effect
of inactivating the pathogenic microorganisms in blood is high and
specifically the pathogenic microorganisms in blood can be inactivated
by 1 log.sub.10 or more. If the final concentration of the platinum
compound is above 100 mM, the platinum compound cannot be readily
dissolved depending on the solvent so that it is difficult to prepare
a solution.
[0051] Note that the term "final concentration of platinum
compound" as used herein refers to a concentration (mM) of
a platinum compound at the time when substantially all of the platinum
compound to be used is introduced into the blood bag 1 upon the
inactivation treatment.
[0052] Specifically showing the amount of the platinum compound
to be used in the method of the present invention, when the platinum
compound is cisplatin and the pathogenic microorganisms in a concentrated
erythrocyte preparation (RC-MAP) are to be inactivated, the platinum
compound is used in an amount so that a final concentration becomes
21 .mu.g/mL to 30 mg/mL, preferably 0.3 mg/mL to 3 mg/mL.
[0053] Note that the effective concentration of cisplatin against
cancer cells is 0.25 .mu.g/mL (antiviral activity (IC.sub.50) on
initial culture cells of Ehrlich cancer), and the above-mentioned
final concentration of cisplatin of 21 .mu.g/mL is as high as 84
times. That is, in the case of blood preparations, cisplatin is
used in concentrations much higher than the concentration in which
it is used as an anticancer agent to humans.
[0054] According to the method of the present invention, the temperature
at which the platinum compound is added to blood for a reaction
between the platinum compound and the nucleic acids of pathogenic
microorganisms only needs to be 0.degree. C. or higher but is preferably
4 to 30.degree. C. in consideration of influences on blood preparations.
[0055] Also, according to the present invention, upon addition
of the platinum compound to blood, the reaction between the platinum
compound and the nucleic acids of pathogenic microorganisms starts
at once. The reaction is preferably continued for 3 hours or more
in order to effect the inactivation of the pathogenic microorganisms
more completely.
[0056] Since the platinum compound used in the method of the present
invention belongs to poison in toxicology and since the platinum
compound are used in very high concentration as mentioned above,
neutralization treatment is preferably performed after the inactivation
treatment in order to alleviate the toxicity of the platinum compound.
In such neutralization treatment, a neutralizing agent that is usually
used in converting the toxicity of anticancer agents containing
the platinum compounds to toxicologically safe is preferably added
to blood preparations containing the platinum compound after inactivation.
Also, the neutralization treatment may be performed by irradiation
of light. Note that the neutralization treatment also involves decreasing
the reactivity of the platinum compound with the nucleic acids of
the pathogenic microorganisms, and thus, can serve to terminate
the inactivation reaction.
[0057] To add the neutralizing agent to blood preparations containing
the platinum compound after inactivation reaction for a desired
period of time, for example, a container containing a neutralizing
agent is connected in advance to the blood bag 1 through a connecting
tube having a valve as an opening and closing means and the valve
is opened as necessary to introduce the neutralizing agent into
the blood bag 1. Also, a neutralizing agent may be introduced into
the blood bag 1 from the air-bleeding tube 10 by using a syringe
or the like.
[0058] A reaction of the neutralizing agent with the platinum compound,
as a microorganism inactivator, in a concentration 42 times or higher
the concentration of the platinum compound can result in complete
neutralization treatment. Usually, the neutralizing agent is added
in a concentration 10 to 500 times, preferably 25 to 100 times the
concentration of the platinum compound.
[0059] Although the neutralizing treatment is effective at a temperature
of 0.degree. C. or higher, the reaction is preferably performed
at a temperature of 4 to 30.degree. C. in consideration of influences
on the function of blood preparations.
[0060] Further, although the reaction starts immediately after
addition of the platinum compound, the reaction is preferably continued
for about 30 minutes to about 1 hour in order to ensure the neutralization
treatment.
[0061] Examples of the neutralizing agent for cisplatin include
thiosulfates such as sodium thiosulfate, amino acids such as methionine
and cysteine, and thiol compounds such as glutathione and coenzyme
A, with sodium thiosulfate and methionine being preferable. To conveniently
introduce the neutralizing agent into the blood bag 1 the neutralizing
agent is preferably liquid added in a state having an osmotic pressure
ratio of about 1 after dissolving the neutralizing agent in physiological
saline or an aqueous glucose solution.
[0062] Note that photodegradable neutralizing agent such as methionine
is stored preferably in a light-shielding container.
[0063] The method of inactivating pathogenic microorganisms according
to the present invention by using the blood bag systems illustrated
in FIGS. 1 and 2 is described above, the method of the present invention
is not limited thereto. For example, in the case where the blood
bag system of the present invention holds an anticoagulant and an
inactivator in the blood bag in advance, the inactivation reaction
of the pathogenic microorganisms in blood starts at the time when
the collected blood is introduced into the blood bag. Further, the
method of the present invention may be performed by introducing
the inactivator into the blood bag that holds blood in advance by
using a syringe or the like.
[0064] Therefore, the present invention also includes the inactivator
for pathogenic microorganisms in blood collected in advance containing
as active components the above-mentioned platinum compound and aquo
complex of the platinum compound, and a method of using the above-mentioned
platinum compound and aquo complex of the platinum compound as inactivators
for pathogenic microorganisms in blood collected in advance.
EXAMPLES
[0065] Hereinafter, the present invention will be described in
detail by examples. However, the present invention should not be
considered as being limited thereto.
Example 1
[0066] [Virus Inactivation for Simian Virus 40 (SV-40) which is
a DNA Type Nonenveloped Virus]
[0067] In a 400-mL CPDA blood bag in the blood bag system shown
in FIG. 1 RC-MAP (concentrated erythrocyte preparation) was prepared
from blood collected from healthy humans. Thereafter, 2 mL of RC-MAP
was sampled in a 15-mL centrifuging tube aseptically, and 2 mL of
10.sup.8 TCID.sub.50/mL (50% infection end point) SV-40 was added
thereto, followed by gentle stirring. To the resultant was added
2 mL of a cisplatin solution containing 1.67 mM cisplatin in an
aqueous solution containing sodium chloride and hydrochloric acid
(Briplatin (registered trademark)) as an inactivator, followed by
gently stirring and a reaction at room temperature (in the dark)
for 7 hours to effect inactivation. After completion of the inactivation,
the inactivated solution was centrifuged at 3000 rpm for 1 minute
and the supernatant was used for the evaluation of virus inactivation.
[0068] Further, inactivation was similarly performed using, instead
of the cisplatin solution, a 3.5 mM carboplatin solution having
dissolved carboplatin (Paraplatin (registered trademark)) in a phosphate
buffer and a 3.5 mM nedaplatin solution having dissolved nedaplatin
(Aqupla (registered trademark) in a phosphate buffer as inactivators
and the supernatants of centrifuged inactivated solutions were used
for the evaluation of virus inactivation.
[0069] Evaluation of virus inactivation was performed by TCID.sub.50
using a 96-well microplate. In a 25-cm.sup.2 cell culture flask,
a CV-1 cell (simian kidney derived cell), which is a proliferating
cell of SV-40 was cultivated in a 10% fetal bovine serum-added
MEM medium for 3 days for proliferation. Thereafter, the cells were
floated with 0.5 mL of 0.05% trypsin-EDTA, to prepare a cell suspension
in 25 mL of the 10% fetal bovine serum-added MEM medium.
[0070] After 90 .mu.L of the cell suspension was added to each
well of the 96-well microplate, 10 .mu.L of the reaction mixture
(the above-mentioned supernatant) was added to each well of the
first column of the 96-well microplate and pipetting was performed
to stir the resultant. Further, 10 .mu.L of the solution in each
well of the first column was added to each well of the second column
of the microplate to perform 10-fold gradual dilution operation.
This operation was repeated up to the wells of the twelfth column
of the microplate. The microplate was incubated in a 5% carbon dioxide
gas incubator at 37.degree. C. for 10 days. Then, a cytopathic effect
(CPE) by SV-40 was observed using an optical microscope, and TCID.sub.50
were calculated as logarithmic values (log.sub.10).
[0071] At the same time, TCID.sub.50 of a positive control, in
which a phosphate buffer was added instead of the platinum compound,
was calculated as a logarithmic value (log.sub.10). A value obtained
by subtracting TCID.sub.50 of the inactivated solution from TCID.sub.50
of the positive control was defined as a killing effect.
[0072] The results are shown in FIG. 3. In FIG. 3 the inactivation
effect means a value obtained by subtracting TCID.sub.50 of the
inactivated solution (logarithmic value (log.sub.10)) from TCID.sub.50
of the above-mentioned positive control (logarithmic value (log.sub.10)).
[0073] Here, TCID.sub.50 of the positive control was 6.5 log.sub.10
and TCID.sub.50 of the inactivated solutions obtained by reacting
with the cisplatin solution, carboplatin solution, and nedaplatin
solution, respectively, as the inactivators were 1.25 log.sub.10
5.50 log.sub.10 and 2.25 log.sub.10 respectively. The results
obtained by subtracting the latter from the former confirmed that
the 1.67 mM cisplatin solution had an inactivation effect of 5.25
log.sub.10 the 3.5 mM carboplatin solution had an inactivation
effect of 1.00 log.sub.10 and the 3.5 mM nedaplatin solution had
an inactivation effect of 4.25 log.sub.10.
Example 2
[0074] [Virus Inactivation for Simian Virus 40 (SV-40) which is
a DNA Type Nonenveloped Virus using a Blood Bag System of the Present
Invention]
[0075] Blood was collected from healthy humans and RC-MAP (concentrated
erythrocyte preparation) was prepared, which was introduced into
the blood bag 1 (400-mL CPDA blood bag) in the blood bag system
shown in FIG. 2. Thereafter, 10.sup.8 TCID.sub.50/mL (50% infection
endpoint) of SV-40 per 2 mL of RC-MAP was added to the blood bag
1 from the air-bleeding tube 10 by using a syringe, and the blood
bag 1 was gently stirred.
[0076] Then, by opening the valve 51 a cisplatin solution containing
1.67 mM (.mu.mol/mL) cisplatin in an aqueous solution containing
sodium chloride and saline (Briplatin (registered trademark), available
from Bristol Myers Squibb company) was introduced into the blood
bag 1 in an amount of 2 mL per 2 mL of RC-MAP. Thereafter, the blood
bag was stirred gently, and the resultant was allowed to react at
room temperature (in the dark) for 7 hours to effect inactivation.
After completion of the inactivation, 4 mL of the inactivated solution
was sampled from the blood bag 1 and centrifuged at 3000 rpm for
1 minute. The supernatant was used for the evaluation of virus inactivation.
[0077] Further, inactivation was similarly performed using, instead
of the cisplatin solution, a 3.5 mM carboplatin solution having
dissolved carboplatin (Paraplatin (registered trademark), Bristol
Pharmaceuticals K.K.) in a phosphate buffer and a 3.5 mM nedaplatin
solution having dissolved nedaplatin (Aqupla (registered trademark),
Shionogi & Co., Ltd.) in a phosphate buffer as inactivators
and the supernatants of centrifuged inactivated sampled from the
blood bag 1 were used for the evaluation of virus inactivation.
[0078] Evaluation of virus inactivation was performed by TCID.sub.50
using a 96-well microplate. In a 25-cm.sup.2 cell culture flask,
a CV-1 cell (simian kidney derived cell), which is a proliferating
cell of SV-40 was cultivated in a 10% fetal bovine serum-added
MEM medium for 3 days for proliferation. Thereafter, the cells were
floated with 0.5 mL of 0.05% trypsin-EDTA, to prepare a cell suspension
in 25 mL of the 10% fetal bovine serum-added MEM medium.
[0079] After 90 .mu.L of the cell suspension was added to each
well of the 96-well microplate, 10 .mu.L of the reaction mixture
(the above-mentioned supernatant) was added to each well of the
first column of the 96-well microplate and pipetting was performed
to stir the resultant. Further, 10 .mu.L of the solution in each
well of the first column was added to each well of the second column
of the microplate to perform 10-fold gradual dilution operation.
This operation was repeated up to the wells of the twelfth column
of the microplate. The microplate was incubated in a 5% carbon dioxide
gas incubator at 37.degree. C. for 10 days. Then, a cytopathic effect
(CPE) by SV-40 was observed using an optical microscope, and TCID.sub.50
were calculated as logarithmic values (log.sub.10).
[0080] At the same time, TCID.sub.50 of a positive control, in
which a phosphate buffer was added instead of the platinum compound,
was calculated as a logarithmic value (log.sub.10). A value obtained
by subtracting TCID.sub.50 of the inactivated solution from TCID.sub.50
of the positive control was defined as a killing effect (inactivation
effect).
[0081] The results are shown in FIG. 4. In FIG. 4 the inactivation
effect means a value obtained by subtracting TCID.sub.50 of the
inactivated solution (logarithmic value (log.sub.10)) from TCID.sub.50
of the above-mentioned positive control (logarithmic value (log.sub.10)).
[0082] Here, TCID.sub.50 of the positive control was 6.0 log.sub.10
and TCID.sub.50 of the inactivated solutions obtained by reacting
with the cisplatin solution, carboplatin solution, and nedaplatin
solution, respectively, as the inactivators were 1.0 log.sub.10
4.5 log.sub.10 and 2.5 log.sub.10 respectively. As the results
obtained by subtracting the latter from the former, FIG. 4 shows
that the 1.67 mM cisplatin solution had an inactivation effect of
5.0 log.sub.10 the 3.5 mM carboplatin solution had an inactivation
effect of 1.5 log.sub.10 and the 3.5 mM nedaplatin solution had
an inactivation effect of 3.5 log.sub.10.
Example 3
[0083] [Virus Inactivation for Human Cytomegalovirus (HCMV) which
is a DNA Type Enveloped Virus]
[0084] In the blood bag 1 (a 400-mL CPDA blood bag) in the blood
bag system shown in FIG. 1 RC-MAP (concentrated erythrocyte preparation)
was prepared from blood collected from healthy humans. Thereafter,
2 mL of RC-MAP was sampled in a 15-mL centrifuging tube aseptically,
and 2 mL of 10.sup.7 TCID.sub.50/mL HCMV was added thereto, followed
by gentle stirring. To the resultant was added 2 mL of a cisplatin
solution containing 1.67 mM cisplatin in an aqueous solution containing
sodium chloride and hydrochloric acid (Briplatin (registered trademark))
as an inactivator, followed by gently stirring and a reaction at
room temperature (in the dark) for 7 hours to effect inactivation.
After completion of the inactivation, the inactivated solution was
centrifuged at 3000 rpm for 1 minute and the supernatant was used
for the evaluation of virus inactivation.
[0085] Evaluation of virus inactivation was performed by TCID.sub.50
using a 96-well microplate. In a 25-cm.sup.2 cell culture flask,
a HEL cell (primary human embryonic lung fibroblasts cell), which
is a proliferating cell of HCMV, was cultivated in a 10% fetal bovine
serum-added MEM medium for 3 days for proliferation. Thereafter,
the cells were floated with 0.5 mL of 0.05% trypsin-EDTA, to prepare
a cell suspension in 25 mL of the 10% fetal bovine serum-added MEM
medium.
[0086] After 90 .mu.L of this cell suspension was added to each
well of the 96-well microplate, 10 .mu.L of the solution (the above-mentioned
supernatant) was added to each well of the first column of the 96-well
microplate and pipetting was performed to stir the resultant. Further,
10 .mu.L of the solution in each well of the first column was added
to each well of the second column of the microplate to perform 10-fold
gradual dilution operation. This operation was repeated up to the
wells of the twelfth column of the microplate. The microplate was
incubated in a 5% carbon dioxide gas incubator at 37.degree. C.
for 14 days. Then, a cytopathic effect (CPE) by HCMV was observed
using an optical microscope, and TCID.sub.50 was calculated as a
logarithmic value (log.sub.10) . At the same time, TCID.sub.50 of
a positive control, in which a phosphate buffer was added instead
of the platinum compound, was calculated as a logarithmic value
(log.sub.10). A value obtained by subtracting TCID.sub.50 of the
inactivated solution from TCID.sub.50 of the positive control was
defined as an anti-HCMV effect of cisplatin (inactivation effect).
[0087] Here, TCID.sub.50 of the positive control was 5.25 log.sub.10
and TCID.sub.50 of the inactivated solution obtained by reacting
with the cisplatin solution was 1.25 log.sub.10. The results obtained
by subtracting the latter from the former confirmed that the 1.67
mM cisplatin solution killed 4.00 log.sub.10 of HCMV in RC-MAP.
Example 4
[0088] [Virus Inactivation for Mouse Hepatitis Virus (MHV) which
is an RNA Type Enveloped Virus]
[0089] In the blood bag 1 (a 400-mL CPDA blood bag) in the blood
bag system shown in FIG. 1 RC-MAP (concentrated erythrocyte preparation)
was prepared from blood collected from healthy humans. Thereafter,
2 mL of RC-MAP was sampled in a 15-mL centrifuging tube aseptically,
and 2 mL of 10.sup.7 TCID.sub.50/mL MHV was added thereto, followed
by gentle stirring. To the resultant was added 2 mL of a cisplatin
solution containing 1.67 mM cisplatin in an aqueous solution containing
sodium chloride and hydrochloric acid (Briplatin (registered trademark))
as an inactivator, followed by gently stirring and a reaction at
room temperature (in the dark) for 7 hours to effect inactivation.
After completion of the inactivation, the inactivated solution was
centrifuged at 3000 rpm for 1 minute and the supernatant was used
for the evaluation of virus inactivation.
[0090] Evaluation of virus inactivation was performed by TCID.sub.50
using a 96-well microplate. In a 25-cm.sup.2 cell culture flask,
a DBT cell, which is a proliferating cell of MHV, was cultivated
in a 5% fetal bovine serum-added MEM medium for 3 days for proliferation.
Thereafter, the cells were floated with 0.5 mL of 0.05% trypsin-EDTA,
to prepare a cell suspension in 25 mL of the 10% fetal bovine serum-added
MEM medium.
[0091] After 90 .mu.L of this cell suspension was added to each
well of the 96-well microplate, 10 .mu.L of the solution was added
to each well of the first column of the 96-well microplate and pipetting
was performed to stir the resultant. Further, 10 .mu.L of the solution
in each well of the first column was added to each well of the second
column of the microplate to perform 10-fold gradual dilution operation.
This operation was repeated up to the wells of the twelfth column
of the microplate. The microplate was incubated in a 5% carbon dioxide
gas incubator at 37.degree. C. for 14 days. Then, a cytopathic effect
(CPE) by MHV was observed using an optical microscope, and TCID.sub.50
was calculated as a logarithmic value (log.sub.10). At the same
time, TCID.sub.50 of a positive control, in which a phosphate buffer
was added instead of the platinum compound, was calculated as a
logarithmic value (log.sub.10). A value obtained by subtracting
TCID.sub.50 of the inactivated solution from TCID.sub.50 of the
positive control was defined as an MHV inactivation effect.
[0092] Here, TCID.sub.50 of the positive control was 5.50 log.sub.10
and TCID.sub.50 of the inactivated solution obtained by reacting
with the cisplatin solution was 1.25 log.sub.10. The result obtained
by subtracting the latter from the former confirmed that the 1.67
mM cisplatin solution killed 4.00 log.sub.10 of MHV in RC-MAP.
Example 5
[0093] [Bacteria Inactivation of Yersinia enterocolitica (Yersinia
Strain)]
[0094] In the blood bag 1 (400-mL CPDA blood bag) of the blood
bag system shown in FIG. 1 RC-MAP (concentrated erythrocyte preparation)
was prepared from blood collected from healthy humans. Thereafter,
2 mL of RC-MAP was aseptically sampled in a 15-mL centrifuging tube,
and 2 mL of 10.sup.8 CFU/mL Yersinia enterocolitica was added thereto,
followed by gently stirring. To the resultant was added 2 mL of
a cisplatin solution having dissolved cisplatin (available from
Wako Pure Chemical Industries, Ltd.) in physiological saline and
adjusted to a concentration of 4.00 mM, as an inactivator. After
gentle stirring, the resultant was allowed to react at room temperature
(in the dark) for 7 hours to effect inactivation. After completion
of the inactivation, the inactivated solution was centrifuged at
3000 rpm for 1 minute and the. supernatant was used for the evaluation
of virus inactivation.
[0095] Similarly, cisplatin solutions having dissolved cisplatin
(available from Wako Pure Chemical Industries, Ltd.) in physiological
saline and adjusted to concentrations of 2.00 mM, 1.00 mM, 0.50
mM, 0.25 mM, 0.125 mM, and 0.0625 mM were used as inactivators to
effect inactivation, and the supernatants were used for the evaluation
of bacteria inactivation.
[0096] Evaluation of antibacterial activity was performed on 10-fold
gradually diluted solutions obtained by adding 1 mL of the inactivated
solution to 9 mL of sterilized physiological saline, stirring the
mixture by using a touch mixer, and further adding 1 mL of the resultant
to 9 mL of physiological saline for dilution. 1 mL or 100 .mu.L
of the diluted solution in each stage was mixed in a petri dish
having a diameter of 9 cm and containing 15 mL soybean casein digest
agar medium. This was cultivated at 31.degree. C. for 2 days. The
number of colonies emerged was defined as number of survival bacteria.
[0097] Note that solution to which no cisplatin was added was defined
as a positive control. A bacteria inactivation effect was defined
by a value obtained by subtracting the number of survival bacteria
(logarithmic value (log.sub.10)) in cisplatin-added inactivated
solution from the number of survival bacteria (logarithmic value
(log.sub.10)) in the positive control.
[0098] The results are shown in FIG. 5. Here, the number of survival
bacteria was 5.00 log.sub.10 and the numbers of survival bacteria
in the inactivated solutions inactivated by the cisplatin solutions
are as follows.
1 Concentration of Number of Cisplatin survival bacteria 1.33 mM
0.06 log.sub.10 0.67 mM 1.48 log.sub.10 0.33 mM 2.27 log.sub.10
0.17 mM 2.80 log.sub.10 0.083 mM 3.79 log.sub.10 0.042 mM 4.52 log.sub.10
0.0209 mM 4.82 log.sub.10
[0099] As a result of subtracting the latter from the former, the
bacteria killing effect of each inactivator was as follows.
2 Concentration of Bacteria cisplatin in inactivator killing effect
1.33 mM 4.94 log.sub.10 0.67 mM 3.52 log.sub.10 0.33 mM 2.73 log.sub.10
0.17 mM 2.20 log.sub.10 0.083 mM 1.21 log.sub.10 0.042 mM 0.48 log.sub.10
0.0209 mM 0.18 log.sub.10
Example 6
[0100] [Detoxication of Cisplatin/anti-Yersinia enterocolitica
Activity after Reaction of Cisplatin with Sodium Thiosulfate or
Methionine]
[0101] In the blood bag 1 (400-mL CPDA blood bag) of the blood
bag system shown in FIG. 1 RC-MAP (concentrated erythrocyte preparation)
was prepared from blood collected from healthy humans. Thereafter,
2 mL of RC-MAP was aseptically sampled in a 15-mL centrifuging tube
and 2 mL of a cisplatin solution having dissolved cisplatin (available
Wako Pure Chemical Industries, (Ltd.)) in physiological saline and
adjusted to a concentration of 4.00 mM as an inactivation was added,
followed by gentle stirring. To the resultant was added 2 mL of
a 168 mM L-methionine solution having dissolved L-methionine in
physiological saline, for a reaction at room temperature for 30
minutes.
[0102] Similarly, as neutralizing agents, L-methionine solutions
having concentrations of 84 mM, 42 mM, 21 mM, 10.5 mM, 5.25 mM,
2.63 mM, and 1.31 mM, respectively, obtained by dissolving L-methionine
in physiological saline or sodium thiosulfate solutions having concentrations
of 168 mM, 84 mM, 42 mM, 21 mM, 10.5 mM, 5.25 mM, 2.63 mM, and 1.31
mM, respectively, obtained by dissolving sodium thiosulfate in physiological
saline were used to effect a reaction. In addition, solutions to
which a phosphate buffer was added instead of the inactivator and
neutralizing agent (without cisplatin) were prepared as controls,
and the reaction was performed in the similar manner. Thereafter,
10 .mu.L of 10.sup.8 CFU/mL Yersinia enterocolitica was added to
each solution, and the number of bacterial cells after stirring
was measured.
[0103] Measurement of bacterial cells was conducted on solutions
obtained by repeating 3 times the 10-fold gradual dilution operation
consisting of adding 1 mL of the inactivated solution to 9 mL of
sterilized physiological saline, stirring the mixture by using a
touch mixer, and further adding 1 mL of the resultant to 9 mL of
physiological saline for dilution. 10 mL, 1 mL or 100 .mu.L of the
diluted solution in each stage was mixed in a petri dish having
a diameter of 9 cm and containing 15 mL soybean casein digest agar
medium. This was cultivated at 31.degree. C. for 2 days. The number
of colonies emerged was defined as number of survival bacteria,
and a logarithmic value (log.sub.10) thereof was defined as a neutralization
effect.
[0104] The results of neutralization treatment with methionine
as a neutralizing agent are shown in FIG. 6. The results confirmed
that in the case where a neutralizing agent having a methionine
concentration of 56 mM showed the same result as that obtained without
cisplatin (number of survival bacteria of 5.1 log.sub.10) and Yersinia
enterocolitica were not killed at all. In other cases where the
neutralizing agent was used in different concentrations, the numbers
of survival bacteria were as follows.
3 Concentration of methionine Number of in neutralizing agent survival
bacteria 28 mM 3.36 log.sub.10 14 mM 1.87 log.sub.10 7 mM 1.43 log.sub.10
3.5 mM 1.22 log.sub.10 1.75 mM 0.89 log.sub.10 0.88 mM 0.73 log.sub.10
0.44 mM 0.34 log.sub.10
[0105] On the other hand, the results of the neutralization treatment
with sodium thiosulfate as a neutralizing agent are shown in FIG.
7. The results confirmed that in the case where a neutralizing agent
having a sodium thiosulfate concentration of 56 mM showed the same
result as that obtained without cisplatin (number of survival bacteria
of 5.1 log.sub.10) and Yersinia enterocolitica were not killed at
all. In other cases where the neutralizing agent was used in different
concentrations, the numbers of survival bacteria were as follows.
4 Concentration of Number of sodium thiosulfate survival in neutralizing
agent bacteria 28 mM 4.22 log.sub.10 14 mM 2.35 log.sub.10 7 mM
1.98 log.sub.10 3.5 mM 1.51 log.sub.10 1.75 mM 1.28 log.sub.10 0.88
mM 0.86 log.sub.10 0.44 mM 0.44 log.sub.10
Example 7
[0106] [Neutralization Treatment of Cisplatin/anti-SV-40 Activity
after Reaction of Cisplatin with Sodium Thiosulfate or Methionine]
[0107] In the blood bag 1 (400 mL CPDA blood bag) of the blood
bag system shown in FIG. 1 RC-MAP (concentrated erythrocyte preparation)
was prepared from blood collected from healthy humans. Thereafter,
1 mL of RC-MAP was sampled in a 15-mL centrifuging tube aseptically
and 1 mL of a cisplatin solution having dissolved cisplatin (available
from Wako Pure Chemical Industries, Ltd.) in physiological saline
and adjusted to a concentration of 3.5 mM cisplatin was added thereto
as an inactivator, followed by gentle stirring. To the resultant
was added 1 mL of a 175 mM sodium thiosulfate solution having dissolved
sodium thiosulfate in physiological saline, for a reaction at room
temperature for 30 minutes. Thereafter, 0.5 mL of 10.sup.8 TCID.sub.50/mL
SV-40 was added and the resultant was stirred gently and then allowed
to react in an incubator (in the dark) at room temperature for 30
minutes. After completion of the reaction, the resultant mixture
was centrifuged at 3000 rpm for 1 minute and the supernatant was
used for the evaluation of virus inactivation.
[0108] Evaluation of virus inactivation was performed by TCID.sub.50
using a 96-well microplate. In a 25-cm.sup.2 cell culture flask,
a CV-1 cell (simian kidney derived cell), which is a proliferating
cell of SV-40 was cultivated in a 10% fetal bovine serum-added
MEM medium for 3 days for proliferation. Thereafter, the cells were
floated with 0.5 mL of 0.05% trypsin-EDTA, to prepare a cell suspension
in 25 mL of the 10% fetal bovine serum-added MEM medium.
[0109] After 90 .mu.L of the cell suspension was added to each
well of the 96-well microplate, 10 .mu.L of the reaction mixture
(the above-mentioned supernatant) was added to each well of the
first column of the 96-well microplate and pipetting was performed
to stir the resultant. Further, 10 .mu.L of the solution in each
well of the first column was added to each well of the second column
of the microplate to perform 10-fold gradual dilution operation.
This operation was repeated up to the wells of the twelfth column
of the microplate. The microplate was incubated in a 5% carbon dioxide
gas incubator at 37.degree. C. for 10 days. Then, a cytopathic effect
(CPE) by SV-40 was observed using an optical microscope, and TCID.sub.50
was calculated as a logarithmic value (log.sub.10) . At the same
time, TCID.sub.50 of a positive control, in which a phosphate buffer
was added instead of the platinum compound was calculated as a logarithmic
value (log.sub.10). A value obtained by subtracting TCID.sub.50
of the inactivated solution from TCID.sub.50 of the positive control
was defined as a killing effect.
[0110] As a result, subtraction of TCID.sub.50 (6.00 log.sub.10)
of the case where a cisplatin solution having a concentration of
3.5 mM as an inactivator and then a sodium thiosulfate solution
having a concentration of 175 mM as a neutralizing agent were added
from TCID.sub.50 (6.00 log.sub.10) of the positive control indicates
no killing effect, that is, inactivation effect of SV-40. This means
that the inactivator containing cisplatin in a concentration of
3.5 mM was neutralized by addition of the neutralizing agent containing
sodium thiosulfate in a concentration of 175 mM.
Example 8
[0111] [Hemolysis Toxicity of Cisplatin on Erythrocyte Preparation]
[0112] In the blood bag 1 (400-mL CPDA blood bag) of the blood
bag system shown in FIG. 1 RC-MAP (concentrated erythrocyte preparation)
was prepared from blood collected from healthy humans. Thereafter,
1 mL of RC-MAP was aseptically sampled in a 15-mL centrifuging tube,
and 1 mL of a cisplatin solution having dissolved cisplatin (Briplatin
(registered trademark)) in physiological saline in a concentration
of 1.67 mM and 1 mL of physiological saline were added thereto while
gently mixing. The resultant was allowed to react at room temperature
(in the dark) for 7 hours. Thereafter, the resultant mixture was
centrifuged at 2000 rpm for 5 minutes. Drabkin's reagent was added
to the supernatant for a reaction and absorbance at 540 nm was measured
to determine hemolysis rate by a cyanmethemoglobin method. As a
result, the hemolysis rate of cisplatin was found to be 0.1% or
less, confirming that cisplatin has low hemolysis toxicity on erythrocyte
preparations.
INDUSTRIAL APPLICABILITY
[0113] The present invention employs a platinum compound that is
used as an anticancer agent and whose characteristics are widely
known as an inactivator for pathogenic microorganisms in blood,
and thus, the present invention is safe and the inactivator is easily
available. Further, since there is no need to newly develop inactivators,
the present invention is excellent in view of cost.
[0114] Further, use of the platinum compound in the present invention
in a concentration as high as 0.07 mM or more, which is much higher
than the amount of the platinum compound used as original use of
anticancer agent, increases an effect of inactivating pathogenic
microorganisms and provides an excellent sustained inactivation
effect.
[0115] In the present invention, transfusion of inactivated blood
after neutralization treatment of the anticancer agent and washing
enables supply of microbiologically and toxicologically safe blood
preparations unlike the original usage of anticancer agents used
for the treatment of cancers.
[0116] In the present invention, blood or blood components containing
inactivated pathogenic microorganisms and preferably further neutralized
are held in bags, which are separated into individual bags in use
for transfusion. Upon storage and transportation, methods and means
that are applied to conventional bags can be applied as they are. |