JPH0959178A - Antiviral agent and agent for promoting antiviral action - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain the subject agent containing a superoxide dismutase bonded to lecithin through chemical crosslinking bond and especially having an anti- human immunodeficiency virus action. SOLUTION: The agent contains a superoxide dismutase bonded to lecithin through a chemical crosslinking bond (PC-SOD) as an active component. The PC-SOD is preferably expressed by the formula SOD-[C(O)-(CH2 )n -C(O)-X]n (SOD is a superoxide dismutase; X is a residue produced by removing H of 2-hydroxyl group of a lysolecithin having hydroxyl group at 2-site of glycerol; (m) is an average number of bonds per one SOD molecule and is >=1; (n) is >=2). The raw material for PC-SOD is preferably produced by chemically modifying the 111-site cysteine of a human-originated Cu/Zn SOD containing copper and zinc as active centers. The daily clinical administration dose of the agent is preferably 100-10,000U/kg in terms of PC-SOD.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention belongs to the technical field relating to pharmaceutical use of lecithinized superoxide dismutase. More specifically, antiviral agents such as anti-HIV agents containing lecithinized superoxide dismutase as an active ingredient; lecithinized superoxide dismutase and anti-H
It belongs to a technical field relating to an antiviral agent containing an antiviral substance such as an IV agent as an active ingredient; and an enhancer of action of an antiviral agent containing lecithinized superoxide dismutase as an active ingredient.

[0002]

2. Description of the Related Art Superoxide dismutase (hereinafter, referred to as superoxide dismutase)
Sometimes abbreviated as SOD) is an animal, plant, widely distributed in the living body such as microorganisms, superoxide anion radical is the active oxygen rich in reactivity (hereinafter, simply O ₂ ^-
It is also abbreviated)) is known as an enzyme that decomposes. From the pharmacological point of view, this SOD is used for the treatment of various inflammations caused by radicals and the removal of radicals generated after the use of anti-rheumatic drugs, the use of anti-thrombotic agents used in myocardial infarction and organ transplantation. It is expected to be applied. Recently, application of this SOD to gastric mucosal injury has also been studied, and its therapeutic effect is expected (Lipid Peroxide Research, Vol. 16, p. 74 (1992)). Generally, when administered intravenously, SOD has a low cell affinity and its half-life in blood is only 4 to 6 minutes, and SOD is rapidly excreted in urine. In order to extend the blood half-life of this SOD, SOD was added to Ficoll, polyethylene glycol,
Attempts have been made to modify them into macromolecules by modifying them with rat albumin, dextran, etc.

However, it has been reported that SOD modified with ficoll or polyethylene glycol has a markedly reduced enzymatic activity inherent to SOD and still has a low cell affinity. In addition, it has been reported that rat albumin-modified SOD exhibits antigenicity in the human body. Furthermore, SOD modified with dextran is excellent in that the anti-inflammatory action inherent to SOD is enhanced, but it is also reported that antigenicity in the human body is also recognized. In recent years, S
A substance in which a bioactive protein such as OD is chemically modified with lecithin (phosphatidylcholine: hereinafter sometimes abbreviated as PC) has been reported. It has been reported that this PC-binding modified bioactive protein has markedly increased cell affinity and is significantly different in biodistribution from conventional non-modified bioactive proteins. Further, it is said that enhancement of pharmacological activity of bioactive proteins, reduction of side effects, and promotion of absorption can be expected. It has also been reported that PC SOD (hereinafter sometimes abbreviated as PC-SOD) exhibits an effect of promoting healing of inflammation due to burns (Japanese Patent Laid-Open No. 3-163100).
Japanese Patent Laid-Open No. 3-170438, US Pat. No. 5,109,118). Furthermore, it is said to be useful as an anti-inflammatory agent without side effects such as antigenicity.
JP-A-6-54681 describes the effect of PC-SOD on respiratory resistance by Forssman antiserum. The effect of PC-SOD on ulcerative gastrointestinal disorders has also been reported. Furthermore, the effect of PC-SOD on motor neuron diseases such as amyotrophic lateral sclerosis has also been reported.

[0004]

As described above, further applications of PC-SOD, which can play various roles in vivo, to pharmaceutical applications are expected in the industry. Then, the subject which this invention should solve is establishment of the new medical use of said PC-SOD, and providing this object for medical use.

[0005]

Means for Solving the Problems The present inventor has conducted extensive studies to solve the above problems. As a result, surprisingly, the PC-SOD has an antiviral effect, especially anti-HIV.
It came to acknowledge the action. In addition, in the above-mentioned PC-SOD, P
It has come to be recognized that substances having an antiviral action other than C-SOD enhance the action.

That is, the present application provides the following inventions in each claim. In Claim 1, there is provided an antiviral agent comprising superoxide dismutase bound to lecithin through chemical crosslinking as an active ingredient.

According to a second aspect of the present invention, there is provided an antiviral agent having an anti-HIV activity, which comprises superoxide dismutase bound to lecithin through chemical crosslinking as an active ingredient.

According to a third aspect of the present invention, there is provided an antiviral agent comprising, as an active ingredient, superoxide dismutase bound to lecithin through chemical crosslinking and a substance having an antiviral effect other than this superoxide dismutase.

The present invention provides an antiviral agent having an anti-HIV action, which comprises, as an active ingredient, a superoxide dismutase bound to lecithin through a chemical crosslink and a substance having an anti-HIV action other than the superoxide dismutase. .

In claim 5, in the antiviral agent according to claim 4, the substance having an anti-HIV action other than superoxide dismutase bound to lecithin through chemical cross-linking is an HIV reverse transcriptase inhibitor, HIV.
Provided is an antiviral agent having an anti-HIV action, which is a substance having one or more anti-HIV actions selected from the group consisting of a protease inhibitor and a sulfated polysaccharide.

In claim 6, the HIV reverse transcriptase inhibitor is 3'-azido-2 ', 3'-dideoxythymidine (AZT), 2', 3'-dideoxycytidine (ddC).
And the antiviral agent according to claim 5, which is one or more HIV reverse transcriptase inhibitor selected from the group consisting of 2 ', 3'-dideoxyinosine (ddI).

In claim 7, the HIV protease inhibitor is (R) -N- tert -butyl-3-[(2S, 3
S) -2-Hydroxy-3-N-[(R) -2-N-
(Isoquinolin-5-yloxyacetyl) amino-3
-Methylthiopropanoyl] amino-4-phenylbutanoyl] -5,5-dimethyl-1,3-thiazolidine-
The antiviral agent according to claim 5 or 6, which is 4-carboxamide (KNI-272).

[0013] In the eighth aspect, the antiviral agent according to any one of the fifth to seventh aspects is provided in which the sulfated polysaccharide is dextran sulfate.

In claim 9, the superoxide dismutase bound to lecithin through chemical cross-linking is represented by the following general formula (I), and the antivirus according to any one of claims 1 to 7 Provide the agent. SOD- _{[C (O) - (CH 2} ) n -C (O) -X ] _m (I) (wherein, SOD represents superoxide dismutase, X is lysolecithin having a hydroxyl group at the 2-position of glycerol, Represents a residue excluding the hydrogen atom of the hydroxyl group at the 2-position, m is the average number of bonds to one SOD molecule, represents an integer of 1 or more, and n represents an integer of 2 or more).

A tenth aspect of the present invention provides the antiviral agent according to the ninth aspect, wherein in the general formula (I), the SOD is a human-derived SOD.

In claim 11, SOD is human-derived Cu / Zn superoxide dismutase in which the amino acid at position 111 of the amino acid sequence in the general formula (I) is S- (2-hydroxyethylthio) cysteine. An antiviral agent according to claim 9 is provided.

[0017] In the twelfth aspect, there is provided the antiviral agent according to any one of the ninth to eleventh aspects, wherein in the general formula (I), n is an integer of 2 or more and 10 or less. .

A thirteenth aspect provides the antiviral agent according to the twelfth aspect, wherein n is 3 in the general formula (I).

In a fourteenth aspect, there is provided the antiviral agent according to any one of the ninth to thirteenth aspects, wherein m is a number of 1 or more and 16 or less in the general formula (I).

[0020] In the fifteenth aspect, there is provided the antiviral agent according to the fourteenth aspect, wherein m is 4 in the general formula (I).

In a sixteenth aspect of the present invention, there is provided an antiviral effect enhancer, which comprises, as an active ingredient, superoxide dismutase bound to lecithin through chemical cross-linking.

[0022] In the seventeenth aspect of the invention, there is provided the antiviral activity enhancer according to the sixteenth aspect, wherein the superoxide dismutase bound to lecithin through chemical cross-linking is represented by the following general formula (I). SOD- _{[C (O) - (CH 2} ) n -C (O) -X ] _m (I) (wherein, SOD represents superoxide dismutase, X is lysolecithin having a hydroxyl group at the 2-position of glycerol, Represents a residue excluding the hydrogen atom of the hydroxyl group at the 2-position, m is the average number of bonds to one SOD molecule, represents an integer of 1 or more, and n represents an integer of 2 or more).

[0023] In the eighteenth aspect, there is provided the antiviral action enhancer according to the seventeenth aspect, wherein the SOD in the general formula (I) is a human-derived SOD.

[0024] In claim 19, SOD is human-derived Cu / Zn superoxide dismutase in which SOD is an S- (2-hydroxyethylthio) cysteine in the amino acid at position 111 of the general formula (I). A certain anti-viral effect enhancer according to claim 17 is provided.

[0025] In the twentieth aspect, in the general formula (I), n is an integer of 2 or more and 10 or less, and the antiviral action enhancer according to any one of the seventeenth to nineteenth aspects. provide.

A twenty-first aspect provides the antiviral action-enhancing agent according to the twenty-first aspect, wherein n is 3 in the general formula (I).

[0027] In the twenty-second aspect, there is provided the antiviral action enhancer according to any one of the seventeenth to twenty-first aspects, wherein m is a number of 1 or more and 16 or less in the general formula (I).

A twenty-third aspect of the present invention provides the antiviral action-enhancing agent according to the twenty-second aspect, wherein m is 4 in the general formula (I).

[0029] In the twenty-fourth aspect, the antiviral action enhancer according to any one of the sixteenth to twenty-third aspects, wherein the virus is HIV.

[0030]

Embodiments of the present invention will be described below. <1> The antiviral agent of the present invention and the antiviral action enhancer of the present invention are antiviral agents containing superoxide dismutase bound to lecithin through chemical crosslinking as an active ingredient. In the present specification, "lecithin" includes, in addition to ordinary lecithin which means phosphatidylcholine, "lysolecithin" obtained by removing one molecule of fatty acid bonded to the 1-position or 2-position of glycerol.

Further, in the present specification, "superoxide dismutase (SOD)" means active oxygen O in the living body.
₂ ^- as long as capable of exhibiting its inherent function of the degradation of its origin and the like are not particularly limited, and can be widely used SOD with various animals and plants or microorganisms. However, the present invention is an invention for pharmaceutical use, and considering that it is preferable to reduce the in vivo antigenicity as much as possible, the antiviral agent of the present invention or the antiviral agent of the present invention is administered. It is preferable to select an appropriate SOD depending on the animal species. For example, in the case of targeting a human in whom the antiviral agent of the present invention or the antiviral effect enhancer of the present invention is most required, in order to reduce the antigenicity in the human body as much as possible, a human-derived SOD is used. It is preferably used in the present invention. And among them, human-derived Cu / ZnS
OD (human-derived SOD containing copper and zinc in the active center; hereinafter sometimes abbreviated as human Cu / ZnSOD)
The expression level in cells is high, and the production technology by genetic engineering techniques has been established at the time of completion of the present invention.
It is possible at present to prepare a large amount, and it is particularly preferable.

This human Cu / ZnSOD has a natural human Cu / ZnSOD produced from human tissue; a human Cu / ZnSOD produced by a genetic engineering technique; an amino acid sequence substantially identical to that of natural human Cu / ZnSOD. Recombinant human Cu / ZnSOD and the like are available, and any human Cu / ZnSOD can be used as a material for superoxide dismutase (PC-SOD) bound to lecithin through the chemical crosslinking used in the present invention.

For reference, this natural human Cu / Zn
The primary structure of the SOD protein is shown in FIG. 1 (SEQ ID NO: 1). Actual human Cu / ZnSOD is a dimer of the protein having the primary structure shown in FIG.

As shown in the amino acid sequence of this protein, the 111th amino acid of natural human Cu / ZnSOD is cysteine, but this 111th position was converted to serine by protein engineering techniques such as site-directed mutagenesis. Human Cu / ZnSOD (JP-A-62-130684)
Or chemically modified human C of 111 cysteines
u / ZnSOD (Japanese Patent Laid-Open No. 6-199895) has also been reported, and a PC using these human Cu / ZnSOD as a material.
Any of -SOD can be used as an active ingredient of the antiviral agent or the antiviral effect enhancer of the present invention. Among these human Cu / ZnSODs, the cysteine at position 111, which is uniform in charge and molecular weight and has stable SOD activity, is chemically modified. For example, this cysteine is S- (2-hydroxy). Human Cu / ZnSOD with ethylthio) cysteine was added to the PC-SO.
It is preferably used as the material of D.

In the present specification, human Cu / ZnSOD is obtained by partially converting amino acids by site-directed mutagenesis or the like, or a partial amino acid of human Cu / ZnSOD is chemically modified. Including those obtained by
It is simply called human Cu / ZnSOD. In the PC-SOD used in the present invention, a lecithin derivative obtained by previously binding a chemical cross-linking agent to a residue of lecithin is usually chemically cross-linked to superoxide dismutase (SOD) by the chemical cross-linking agent. It can be obtained by binding one or more lecithins via.

The lecithin bound to SOD is not particularly limited here, but PC-SOD to which the lysolecithin is bound is preferably used in the present invention. The PC-SOD suitable for the present invention is represented by the following formula (I). SOD- _{[C (O) - (CH 2} ) n -C (O) -X ] _m (I) (wherein, SOD represents superoxide dismutase, X is lysolecithin having a hydroxyl group at the 2-position of glycerol, Representing a residue excluding the hydrogen atom of the hydroxyl group at the 2-position, m represents an integer of 1 or more, and n represents an integer of 2 or more)

Here, "2 of glycerol" represented by X
The residue of lysolecithin having a hydroxyl group at the 2-position, excluding the hydrogen atom of the 2-position hydroxyl group, is represented by the following formula (II). _{-O-CH (CH 2 OR)} [CH 2 OP (O) (O -) (OCH 2 CH 2 N + (CH 3) 3)] (II) ( wherein, R fatty acid residue (acyl group) Is)

Particularly, a saturated or unsaturated fatty acid residue having 10 to 28 carbon atoms is preferable as the above R, more preferably myristoyl group, palmitoyl group, stearoyl group, icosanoyl group, docosanoyl group, and other carbon atoms having 14 to 14 carbon atoms.
Among them, there are 22 saturated fatty acid residues, and particularly preferred is a palmitoyl group which is a saturated fatty acid residue having 16 carbon atoms.

In the above formula (I), the residue —C (O) — (CH ₂ ) _n —C (O) — represents a chemical cross-linking residue. Residues of the chemical crosslinking of the formula HO-C (O) - ( CH 2) n -C (O) linear dicarboxylic acid represented by -OH, anhydrides of the dicarboxylic acids, the dicarboxylic acid Hydroxyl groups at both ends of the chemical cross-linking agent molecule such as ester, halide of this dicarboxylic acid or other reactive derivative of this dicarboxylic acid (in the case of the above reactive derivative of dicarboxylic acid, it corresponds to the hydroxyl groups at both ends of this dicarboxylic acid. It is the residue excluding the part). Since this residue can crosslink and bond SOD and lecithin, they are collectively referred to herein as "chemical crosslinks".

The chemical crosslink in the above formula (I) is bound to the above-mentioned lysolecithin residue (II) by an ester bond at one end thereof. The other end of this chemical crosslink is
It is considered that they are directly bonded to the amino group of SOD by an amide bond or the like. In this chemical cross-linking, the group-
(CH _{2) n} - is a bivalent group obtained by removing one by one hydrogen atom from a carbon atom at both ends of a linear alkane, i.e. an alkylene group. n is an integer of 2 or more, preferably n is an integer of 2 to 10, and particularly preferably 3.

Further, m in the above formula (I) represents the average number of lecithin bound to one SOD molecule through the above-mentioned chemical cross-linking. This m is an integer of 1 or more, and m is preferably 1 to 16, and 4 is particularly preferable.
The binding of the lecithin derivative to SOD can be specifically performed based on, for example, the method described in JP-A-6-54681, whereby PC-SO is obtained.
D can be manufactured. Details of the manufacturing process of this PC-SOD will be described in a manufacturing example described later.

The PC-SOD used in the present invention may be a pharmaceutically acceptable salt. The salt that can be used specifically depends on the isoelectric point of PC-SOD used. For example, when the isoelectric point of the PC-SOD molecule is acidic, a pharmaceutically acceptable salt with an alkali can be used. Conversely, when the isoelectric point of the PC-SOD molecule is alkaline, a pharmaceutically acceptable salt with an acid can be used. For example, the isoelectric point of human Cu / ZnSOD obtained by converting the amino acid sequence at position 111 to serine is acidic, and the isoelectric point of PC-SOD obtained by converting this into PC is also acidic. Therefore, this 111-position converted human Cu / Z
As for nSOD, pharmaceutically acceptable salts of salts with alkali, for example, alkali metal salts, alkaline earth metal salts, ammonium salts and the like can be used in the present invention. The thus-produced PC-SOD can be blended as an active ingredient in the dosage form described below to obtain a desired antiviral agent and the antiviral effect enhancer of the present invention.

The diseases to which the present invention is applied are various viral diseases of mammals including humans, and the antiviral agent of the present invention and the antiviral action enhancer of the present invention prevent or treat these viral diseases. Can be administered to the subject to be treated. However, the antiviral agent and the antiviral effect enhancer of the present invention are especially retroviral diseases caused by a retrovirus, which is a virus having a life cycle for converting gene RNA into DNA among RNA viruses, such as human immunodeficiency virus. (HIV)
Human immunodeficiency virus (HI) caused by
V) Effective against sexually transmitted diseases and human T cell leukemia caused by human T cell leukemia virus. Of these retroviral diseases, the former human immunodeficiency virus (HIV) disease is a disease extremely suitable for application of the antiviral agent of the present invention or the antiviral effect enhancer of the present invention. However, the disease names listed here are only examples, and the application range of the antiviral agent and the antiviral effect enhancer of the present invention should not be limited to such exemplified diseases.

<2> In the antiviral agent of the present invention, a substance having an antiviral action other than this superoxide dismutase can be blended as an active ingredient together with superoxide dismutase bound to lecithin through chemical crosslinking. . Further, the antiviral action enhancer of the present invention is administered for the purpose of enhancing the antiviral action which the antiviral agent containing the substance having an antiviral action other than the above-mentioned superoxide dismutase as an active ingredient should intrinsically exhibit. . That is, the antiviral effect enhancer of the present invention,
When used in combination with a substance having an antiviral effect, it means an agent that synergistically enhances the antiviral effect, and an agent that merely additively enhances the antiviral effect is herein referred to as " It is excluded from the "antiviral effect enhancer".

The "substance having an antiviral action other than superoxide dismutase" referred to herein is PC
There is no particular limitation as long as a serious side effect is caused by the combination formulation or combination administration with -SOD, and one substance is not a substance that inhibits the antiviral action originally possessed by the other substance. However, it goes without saying that it is preferable that the combination with PC-SOD is not a mere additive effect but a combination that synergistically improves the antiviral effect as much as possible.

For example, when the target disease is a human immunodeficiency virus (HIV) disease, one or two selected from the group consisting of HIV reverse transcriptase inhibitors, HIV protease inhibitors and sulfated polysaccharides. More than one kind of anti-HI
A substance having a V action can be mentioned as a substance having the above-mentioned antiviral action which exerts a synergistic antiviral action when combined with PC-SOD. Here, these substances having an anti-HIV action which are preferably combined with PC-SOD in the present invention will be described.

First, the HIV reverse transcriptase inhibitor used in the present invention is a substance that literally inhibits the activity of HIV reverse transcriptase, and includes a nucleoside HIV reverse transcriptase inhibitor and a non-nucleoside HIV reverse transcriptase. Both of the inhibitors can be used in the present invention.

Examples of nucleoside HIV reverse transcriptase inhibitors include, for example, 3'-azido-2 ', 3'-dideoxythymidine (AZT), 2', 3'-dideoxyinosine (ddI), 2 ', 3'-dideoxycytidine (dd
C) 2 ′, 3′-didehydro-2 ′, 3′-dideoxythymidine (d4T), 3′-thia-2 ′, 3′-dideoxycytidine (3TC), 2′-β-fluoro-ddC, 3 '-Fluorothymidine (FLT), 9- (2-phosphonyl-methoxyethyl) -adenine (PMEA), 6-Cl-d
dI, 6-Cl-2 ′, 3′-dideoxyguanosine (6
-Cl-ddG) and the like.

Examples of non-nucleoside HIV reverse transcriptase inhibitors include, for example, (+)-S-4,5,6,6.
7-Tetrahydro-5-methyl-6- (3-methyl-2
-Butenyl) imidazo [4,5,1-jk] [1,4]
Benzodiazepine-2 (1H) -thione (R8291
3) etc. tetrahydro-imidazo-benzo-diazepin-one derivative or tetrahydro-imidazo-benzo-diazepine-thione (TIBO) derivative, hydroxyethoxy-methylphenylthiothymine (HEPT) derivative, dipyridodiene such as nevirapine Azepine derivatives; pyridinone derivatives such as L-697,639 and the like.

As these HIV reverse transcriptase inhibitors, which are preferably applied to the present invention, they have already been clinically administered to humans, and in combination with PC-SOD, synergistic antiviral effects are exhibited. To do
3'-azido-2 ', 3'-dideoxythymidine (AZ
T), 2 ', 3'-dideoxyinosine (ddI), 2',
3'-dideoxycytidine (ddC) etc. can be mentioned. Further, among these, particularly preferable to be applied to the present invention is 3'-azido-wherein the anti-HIV action by combination with PC-SOD acts particularly synergistically.
2 ', 3'-dideoxythymidine (AZT) can be mentioned. These HIV reverse transcriptase inhibitors may be commercially available products or those synthesized according to known synthesis methods may be used in the present invention.

Next, the HIV protease inhibitor used in the present invention is a substance which inhibits the protease activity of HIV, and for example, (R) -N- tert -butyl-3-
[(2S, 3S) -2-Hydroxy-3-N-[(R)
-2-N- (isoquinolin-5-yloxyacetyl)
Arophenylnorstatin derivatives such as amino-3-methylthiopropanoyl] amino-4-phenylbutanoyl] -5,5-dimethyl-1,3-thiazolidine-4-carboxamide (KNI-272), N ′-[1 (S)-
Benzyl-3- [4a (S), 8a (S), 3a (S)
-( Tert- Butylcarbamoyl) decahydroisoquinolin-2-yl] -2 (R) -hydroxypropyl]-
N "- (quinolin-2-ylcarbamoyl) -L- asparagine amide (Ro 31-8959) substrate transition state peptidomimetics such as, C ₂ symmetric protease inhibitors such as A-77003, C ₂ etc. ABT-538 Asymmetric protease inhibitors and the like can be mentioned.

KN is preferably used as the HIV protease inhibitor in the present invention.
It is preferable to use an allophenylnorstatin derivative such as I-272. Note that these HIV protease inhibitors can be used in the present invention as commercially available products or those synthesized according to known synthesis methods. For example, the above Ro
31-8959, "J. Med. Chem. Vol.36,
What was synthesize | combined using the synthetic method etc. described in p2300-2310 (1993) "can be used for this invention.

Further, in the present specification, the sulfated polysaccharide means a polysaccharide having at least one sulfo group (-SO ₃ H). In the present invention, any sulfated polysaccharide synthesized, that is, a sulfated polysaccharide (also simply referred to as a synthetic sulfated polysaccharide in the present specification) or a natural sulfated polysaccharide (also referred to as a natural sulfated polysaccharide in the present specification) is used. Can also be used.

Examples of natural sulfated polysaccharides include glycosaminoglycans having at least one sulfo group (-SO ₃ H), specifically heparin, chondroitin sulfate,
Examples thereof include, but are not limited to, dermatan sulfate, heparan sulfate, keratan sulfate, carrageenin, and fucoidin.

Examples of synthetic sulfated polysaccharides include dextran sulfate, alginic acid sulfate, lentinan sulfate, pullulan sulfate, chondroitin polysulfate, dermatan polysulfate,
Examples thereof include heparan polysulfuric acid, synthetic keratan polysulfuric acid, hyaluronic acid sulfuric acid ester, teichronic acid sulfuric acid ester, chitin sulfuric acid ester and chitosan sulfuric acid ester, but are not limited thereto. Among these sulfated polysaccharides, especially dextran sulfate, especially PC-
A synergistic effect due to the combination with SOD is recognized, and it is preferably applied to the present invention.

As the substances having anti-HIV activity and other anti-viral substances listed above, pharmaceutically acceptable salts thereof can be used depending on the properties of the substances or the necessity of formulation. For example, in the case of the above-mentioned sulfated polysaccharide, a salt formed with an inorganic base such as an alkali metal salt (eg sodium salt, potassium salt etc.), an alkaline earth metal salt, an ammonium salt or a diethanolamine salt, cyclohexylamine Among the salts with organic bases such as salts and amino acid salts, pharmaceutically acceptable salts can be used in the present invention.

<3> In the present invention, an arbitrary dosage form may be appropriately selected as the dosage form of the antiviral agent of the present invention or the antiviral effect enhancer of the present invention, depending on the nature and progress of the target disease. You can That is, the antiviral agent and the antiviral agent of the present invention are both orally or parenterally selected depending on the administration method such as injection (intramuscular, subcutaneous, intradermal, intravenous injection), oral, inhalation, etc. can do. These drugs can be formulated as appropriate according to these administration methods.

The dosage form that can be selected is also not particularly limited, and includes, for example, injections (solutions, suspensions, emulsions, solid preparations for dissolution at the time of use), tablets, capsules, granules, powders, liquids, lipo A wide range of agents, gels, external powders, sprays, inhalants, suppositories, etc. can be selected. In preparing these preparations, it is possible to add components used in the preparation of usual pharmaceuticals such as an excipient, a binder, a lubricant, a coloring agent and a disintegrant.

P which is an active ingredient in the antiviral agent of the present invention
The compounding amount of C-SOD and / or the compounding amount of the antiviral substance other than PC-SOD and the dose of the antiviral agent of the present invention are as follows: administration method of the preparation, administration form, intended purpose, specific symptoms of patient, patient It is a matter to be individually determined according to the weight of the person, and is not particularly limited, but PC-SOD
The daily clinical dose is about 100 U / Kg-100
00U / Kg [1U (unit) in this specification]
Means the cytochrome c method at pH 7.8 and 30 ° C. (xanthine-xanthine oxidase-cytochrome c system;
J. Biol. Chem., Vol.244, No.22, 6049-6055 (1969))
Represents the enzyme amount of PC-SOD that inhibits the reduction rate of cytochrome c by 50%, as measured by. Further, the administration interval of the above-mentioned preparation may be once a day, or may be administered in 2 to 4 times a day.

Further, the compounding amount of PC-SOD, which is an active ingredient in the antiviral effect enhancer of the present invention, and the dose of the antiviral agent of the present invention, are the administration method of the preparation, the administration form, the purpose of use, and the specific patient It is a matter that should be individually decided according to the clinical symptoms, the weight of the patient, the type of antiviral agent for which the antiviral effect is to be enhanced, the dosage, etc., but is not particularly limited, but the clinical dosage per day Approximately 100 U / Kg
It is about 10,000 U / Kg. Further, the administration interval of the above-mentioned preparation may be once a day, or may be administered in 2 to 4 times a day. The antiviral effect enhancer of the present invention should be administered in appropriate combination with an antiviral agent that enhances its effect, but the administration method and dosage form of the two may be the same or different. Further, the timing of administration of both may be the same or different.

[0061]

EXAMPLES Examples of the present invention will be specifically described below as Production Examples, Test Examples and Formulation Examples. However, these should not limit the technical scope of the present invention.

<Production Example> (1) Human Cu / ZnSOD derivative [human-derived superoxide dismutase in which the amino acid at position 111 of SOD is S- (2-hydroxyethylthio) cysteine]
Production of this human Cu / ZnSOD derivative
It was manufactured by the method described in JP-A-199895. That is, Escherichia coli 545πHR (pHT351) (Microtechnology Research Institute, No. 9435) was cultured, and the obtained cultured cells were crushed to obtain a crude extract of human Cu / ZnSOD. The crude extract was subjected to ammonium sulphate precipitation, Q-Sepharose FF (Pharmacia) column and ammonium sulphate precipitation in this order for purification, and the filtrate filtered through a 0.2 μm filter was freeze-dried to give a colorless solid (recombinant human apo SOD ) Got. This recombinant human apo SOD was dissolved in 10 mM triethanolamine buffer (pH 7.0), bis (2-hydroxyethyl) disulfide was added, and readjusted to pH 7.0.
The mixture was stirred at 4 ° C for 24 hours. To this, NaCl, 2M acetate buffer (pH 5.0) and 0.1M CuCl ₂ aqueous solution were added, and the mixture was stirred at 4 ° C. for 13 hours. After this stirring, the pH was adjusted to 7.0 and the precipitate formed by ammonium sulfate precipitation (4 ° C., 100% saturation) was collected. The precipitate was desalted by dialysis with 10 mM triethanolamine buffer (pH 7.0). The desalted fraction was passed through a Q-Sepharose FF column and filtered with NaCl.
The concentration was increased stepwise in the order of 5 mM, 10 mM, 20 mM, 50 mM to develop, and the SOD fractions were collected. Then this S
The OD fraction was subjected to ammonium sulfate precipitation (4 ° C, 100% saturation), and the resulting precipitates were collected. This precipitate was used as an aqueous solution,
A human SOD derivative was obtained by dialysis with an aCl aqueous solution and then with purified water, and lyophilization after completion of these dialysis (hereinafter sometimes referred to simply as SOD). The amino acid at position 111 of this human SOD derivative became S- (2-hydroxyethylthio) cysteine.

(2) Production of PC-SOD: Synthesis of active ester form of 2- (4-hydroxycarbonylbutyroyl) lysolecithin (-A) Synthesis of 2- (4-hydroxycarbonylbutyroyl) lysolecithin 2-position of glycerol DM to a chloroform-pyridine (80 ml / 20 ml) suspension of lysolecithin in which is a hydroxyl group.
AP (N, N-dimethylaminopyridine) and glutaric anhydride were added, and the mixture was stirred at 60 ° C for 15 hours. Thereafter, the reaction solution was concentrated under reduced pressure, and chloroform: methanol:
The solvent was dissolved by adding a solvent of water = 4: 5: 1, and the solution was passed through an ion exchange column (Dowex 50W-X8, manufactured by Dow Chemical Company) equilibrated with the same solution. The target compound was fractionated by TLC, the solvent was concentrated under reduced pressure, and the residue was purified by a column packed with ODS (octadecylsilane) to obtain the title compound.

(-B) Synthesis of active ester form of 2- (4-hydroxycarbonylbutyroyl) lysolecithin The carboxylic acid obtained in -A was dissolved in dichloromethane and cooled at 0 ° C., and N-hydroxysuccinimide was added thereto. And tetrazole are added in sequence, then DCC (1,3-
A solution of dicyclohexylcarbodiimide) in dichloromethane was slowly added dropwise, and the mixture was stirred at room temperature for 15 hours. After stirring, the insoluble matter was filtered through Celite to obtain a dichloromethane solution of the active ester compound.

Synthesis of PC-SOD having an average of four lecithin derivatives bound per molecule of SOD: For the SOD prepared according to the above steps dissolved in 50 mM borate buffer (pH 8.5) and all amino groups of this SOD. Then, a 0.8-fold molar amount of the active ester of 2- (4-hydroxycarbonylbutyroyl) lysolecithin was reacted by the following method. Dichloromethane of the active ester solution was distilled off and dissolved in DMF (N, N-dimethylformamide). This was added to 50 mM borate buffer (pH 8.5), the insoluble matter was filtered off, and then 50% of DMF dissolved in the same buffer and cooled to 0 ° C. was added to the above S.
It was added dropwise to the OD solution. After stirring this for 15 hours at 0 ° C., the reaction solution was filtered, applied to a gel filtration column using Sephacryl S-300 (Pharmacia) as a carrier, and eluted with 50 mM borate buffer (pH 8.5), The PC-SOD elution fraction was collected and purified by ion exchange chromatography. This was then concentrated by ultrafiltration and the protein concentration was determined by the Lowry method (Lowry, Oh et al., J. Biol. Chem., 193
Vol., P. 265 (1951)).
S method (trinitrobenzene sulfonate, Goodwin, JF
Et al., Clin. Chem., Vol. 16, p. 24 (1970)), the number of bound lecithin derivatives per SOD molecule was determined to be 4.0 on average. This PC-S
The property of OD is a blue-green to green transparent aqueous solution,
The pH was 7-8. Moreover, when the molecular weight was examined by SDS-polyacrylamide gel electrophoresis, PC
-SOD subunit monomer (as described above, PC
-SOD is a homodimer of subunits).

[Test Example] PC manufactured by the above manufacturing example
-SOD (concentration 30 mg / ml; specific activity 3.07 × 10 ³ U
(/ Mg protein), a toxicity test and a pharmacological test were performed. PC-SOD was added to a 5% mannitol aqueous solution to give a final concentration of 5
Solution dissolved to give mg / ml (hereinafter PC-SOD
(Sometimes abbreviated as a solution) was used. This PC-S
The OD solution was a blue-green to green transparent aqueous solution and had a pH of 6-8. The osmotic pressure ratio with respect to the physiological saline was about 1.

(1) Acute Toxicity Test Acute Toxicity Test Using Rats PC-SOD solution was administered to each of 5 SD male and female rats by tail vein at 100 mg / Kg. For 14 days after the administration, observation of general condition and life and death and measurement of body weight were performed. The animals were necropsied on the 15th day, and the major organs were visually observed. As a result, no dead rat was observed, and no change was observed in any of the general condition, body weight, and autopsy findings. Therefore, the lethal dose of PC-SOD by intravenous administration was estimated to be 100 mg / Kg or more. .

Acute Toxicity Test Using Monkeys PC-SOD solution was administered to two female cynomolgus monkeys at 100 mg / Kg via a vein in the extremities. For 14 days after the administration, observation of general condition and life and death and measurement of body weight were performed. As a result, no dead cynomolgus monkeys were observed, and no change was observed in general condition, body weight, or autopsy findings. Therefore, the lethal dose of PC-SOD by intravenous administration was estimated to be 100 mg / Kg or more. .

(2) Pharmacodynamic test Antiviral activity of PC-SOD HIV-infected MT-4 cells (multiplicity of infection (moi) = 0.
01; hereinafter also referred to as HIV-infected MT-4 cell), or H
IV non-infected MT-4 cells (hereinafter, also referred to as mock-infected MT-4 cells) were added to a 96-well microtiter plate in 3
× 10 ⁴ cells / well (100 µl) were added.
Immediately after that, the PC-SOD manufactured in the above manufacturing example was manufactured.
To various concentrations (final concentration: 0 U / ml, 7
8.13 U / ml, 156.25 U / ml, 312.5 U
/ Ml, 625 U / ml, 1250 U / ml, 2500 U
/ Ml, 5000 U / ml) 100 μl was added, and the cells were cultured at 37 ° C. in a CO ₂ incubator. After 5 days, the number of viable cells was determined by the MTT method (Pauwel et al., J. Virol. Methods, 20, p30.
9-321 (1988)), when PC-SOD was not added to mock-infected MT-4 cells (final concentration: 0 U / m
The cell survival rate of 1) was set as 100%, and each cell survival rate was calculated (FIG. 2).

The test system was the same as the above system except that PC-SOD was replaced with human Cu / ZnSOD which was not modified with lecithin (hereinafter sometimes simply referred to as unmodified SOD). The results of the antiviral activity test are shown in FIG. As a result, mock-infected MT-4 cells were
Even when the C-SOD concentration was increased to 2500 U / ml, the cell viability was maintained at about 100%. From this, PC
-Mock- even if the concentration of SOD is increased to 2500 U / ml-
It was shown to have no effect on the survival of infected MT-4 cells.

In HIV-infected MT-4 cells,
When PC-SOD is not added (final concentration: 0 U / ml)
Cell viability was very close to 0% and was shown to be mostly killed by HIV infection. 2500 U in HIV-infected MT-4 cells with the addition of PC-SOD
/ Ml, the cell viability increased in a dose-dependent manner. PC
When the SOD concentration was 1250 U / ml and 2500 U / ml, the cell viability was restored to about 100%. From this, it became clear that PC-SOD has an excellent anti-HIV effect.

On the other hand, HIV-infected MT-4 cells to which unmodified SOD was added in the same system had a SOD concentration of 5%.
It was found that the cell viability was extremely low even when the dose was increased to 000 U / ml, and that administration of unmodified SOD showed almost no antiviral effect on HIV. In this test, 50% of PC-SOD
The average% effective concentration (EC ₅₀ ) is 718.6 U / ml, 9
The average of 0% effective concentration (EC ₉₀ ) is 1009.9 U / ml.
Met.

Examination of Inhibitory Effect of PC-SOD on Virus Induction by Phorbol Myristate Acetate Phorbol myristate acetate (12-O-tetradecanoylphorbol-13-acetate; hereinafter PM)
(Also referred to as A) is a known virus inducer. The following tests were conducted to examine the effect of PC-SOD on the virus induction by PMA. U1 cells (Folks ™ et al. J. Immunol. , 140 , 1117-1122 (1988))
Or J22HL60 cells (Kitano K et al., Blood, 76 ,
1980-1988 (1990)) was placed in a 24-well microtiter plate at 3 × 10 ⁴ cells / well (1 ml). Immediately thereafter, PMA having a final concentration of 1 ng / ml and the PC-SOD produced in the above Production Example were mixed at various concentrations (U).
For 1 cell: final concentration 50 U / ml, 100 U / ml, 2
00 U / ml; J22HL60 cells: final concentration 12
5 U / ml, 250 U / ml, 500 U / ml, 1000
U / ml was added. As a control, PMA is also P
System without addition of C-SOD (cell control) and P
A test was also conducted on a system to which only MA was added (PMA control).

48 at 37 ° C. in a CO ₂ incubator
After culturing for a time, the number of living cells and dead cells in 1 ml was counted. Further, U1 cells were immunostained with an anti-HIV polyclonal antibody as a primary antibody and a fluorescent-labeled anti-immunoglobulin antibody as a secondary antibody, and the number of cells positive for fluorescence and the number of negative cells were counted, The fluorescence positive cell rate (%) and the fluorescence negative cell rate (%) were calculated.

For J22HL60 cells, the amount of HIV-1 gagp24 antigen in the culture supernatant after culturing was compared with anti-H
It was measured by an ELISA method using an IV polyclonal antibody and a fluorescently labeled anti-immunoglobulin antibody. The results for U1 cells are shown in Table 1 and the results for J22HL60 cells are shown in Table 2.

[0076]

[Table 1] Table 1 According to this Table 1, PC-SOD was found to have P in U1 cells.
It was shown to have no specific inhibitory activity against virus induction by MA.

[0077]

[Table 2] Table 2 From this Table 2, it was shown that the cells treated with PC-SOD did not show a specific decrease in p24 antigen as compared with the control.

From these things, PC-SOD is PM
Virus-infected cells with A (U1 cells and J22HL6
It was shown that the activation of virus (virus induction) from 0 cells) was not affected. However, this result is
It only indicates that SOD does not act at the time of virus induction, and the antiviral effect of PC-SOD cannot be ruled out in any way.

Examination of Synergistic Antiviral Effect by Combination of PC-SOD and Other Antiviral Agent (-A) Combination of PC-SOD and HIV Reverse Transcriptase Inhibitor i) PC-SOD and 3'- Examination of synergistic effect on antiviral action against HIV by combination of azido-2 ′, 3′-dideoxythymidine (AZT) HIV-infected MT-4 cells (multiplicity of infection (moi) = 0.01)
Alternatively, HIV-uninfected MT-4 cells (mock-infected MT-4 cells) were placed in a 96-well microtiter plate at 3 × 10 ⁴ cells / well (100 μl). Immediately thereafter, the PC-SOD and 3'- produced in the above production example were
Azido-2 ', 3'-dideoxythymidine (AZT) (manufactured by Yamasa Shoyu Co., Ltd.) was added at the final concentrations shown in Tables 3 to 5, respectively. After culturing for 5 days in a CO ₂ incubator, the number of viable cells was measured by the MTT method (described above). This test was performed three times independently (triplication) and the average measurement of the absorbance was obtained. The effective value is expressed by the degree of the effect of suppressing the cell damage caused by HIV infection, and the HIV-uninfected MT-
Effective value of the average measured value of the absorbance when 4 cells were cultured without adding a drug was set to 100%, and the average measured value of the absorbance when HIV-infected MT-4 cells were cultured without adding the drug was an effective value. It was expressed on the basis of 0%.

Table 3 shows the results of effective values in the combination of PC-SOD and AZT.

[Table 3] Table 3

The theoretical value, assuming that the antiviral effect of the combination of PC-SOD and AZT is merely additive, is the same as that of PC-SOD only at various concentrations of HIV-infected MT-4. Effective value when added to cells and A
It was calculated as the sum of effective values when ZT alone was added to HIV-infected MT-4 cells at various concentrations. This value is shown in Table 4 as a predicted value.

[0082]

[Table 4] Table 4

Table 5 shows the difference between the effective value and the predicted value obtained by subtracting the predicted value from the above-mentioned effective value. When the difference between the effective value and the predicted value is 0, it indicates that the antiviral effect of the combination of PC-SOD and AZT is additive. Further, when the difference between the predicted value and the effective value is positive (+), it indicates that the antiviral effect of the combination of PC-SOD and AZT is synergistic. The results are shown in the three-dimensional graph in FIG. 4 (PC-SOD and AZ on the plane)
The combined addition amount of T is shown, and the vertical axis shows the difference between the effective value and the predicted value).

[0084]

[Table 5] Table 5 Note that FIG. 5 is a stereograph showing the difference between the effective value and the predicted value when unmodified SOD (human Cu / ZnSOD) was used in place of PC-SOD in the above test system.

In this result, unmodified SOD and AZ
In the system in which T was combined, at most, an additive effect due to the action of both was recognized,
The system using PC-SOD clearly has a synergistic anti-HI
V action was observed. Therefore, PC-SOD and PC-S
Usefulness of the antiviral agent of the present invention containing AZT as an active ingredient other than OD, and P
It was strongly suggested that the antiviral effect enhancer of the present invention containing C-SOD as an active ingredient is useful against HIV when administered together with AZT.

Ii) Examination of synergistic effect on antiviral action against HIV by combination of PC-SOD and 2 ', 3'-dideoxycytidine (ddC) In the test system of the above i), ddC (sigma (sigma ( The same test was performed by using (Sigma). In addition,
Table 6 is an effective value using PC-SOD, Table 7 is the same predicted value, and Tables 8 and 6 are three-dimensional graphs showing the difference between the effective value and the predicted value. Further, FIG. 7 is a table and a three-dimensional graph showing the difference between the effective value and the predicted value when the unmodified SOD is used.

[0087]

[Table 6] Table 6

[0088]

[Table 7] Table 7

[0089]

[Table 8] Table 8

In this result, unmodified SOD and dd
In the system in which C was combined, at best, an additive effect due to the action of both was recognized, but P
In the system using C-SOD, an apparently synergistic anti-HIV
The action was recognized. Therefore, PC-SOD and PC-SO
Usefulness of the antiviral agent of the present invention containing ddC as an active ingredient as a substance having an antiviral action other than D, and PC
It was strongly suggested that the antiviral action enhancer of the present invention containing -SOD as an active ingredient is useful for HIV when administered together with ddC.

Iii) Examination of synergistic effect on antiviral action against HIV by combination of PC-SOD and 2 ', 3'-dideoxyinosine (ddI) In the test system of the above i), ddI (sigma (sigma ( The same test was performed by using (Sigma). In addition,
Table 9 is an effective value using PC-SOD, Table 10 is the same predicted value, and Tables 11 and 8 are a table and a three-dimensional graph showing the difference between the effective value and the predicted value. In addition, FIG. 9 shows unmodified S
It is a three-dimensional graph which shows the difference of an effective value and a predicted value when OD is used.

[0092]

[Table 9] Table 9

[0093]

[Table 10] Table 10

[0094]

[Table 11] Table 11

In this result, unmodified SOD and dd
In the system in which I was combined, at most, an additive effect due to the action of both was recognized.
In the system using C-SOD, an apparently synergistic anti-HIV
The action was recognized. Therefore, PC-SOD and PC-SO
Usefulness of the antiviral agent of the present invention containing ddI as an active ingredient as a substance having an antiviral effect other than D, and PC
It was strongly suggested that the antiviral effect enhancer of the present invention containing -SOD as an active ingredient is useful for HIV when administered together with ddI.

(-B) Combination of PC-SOD and HIV protease inhibitor In the test system of (-A) i), KNI-272 (Japan Energy (Japan Energy), which is an HIV protease inhibitor, is used in place of AZT. (Manufactured by Co.)) was used to perform the same test. Table 12 shows effective values using PC-SOD, Table 13 shows the same predicted values, and Tables 14 and 10
The figure is a table and a three-dimensional graph showing the difference between the effective value and the predicted value.

[0097]

[Table 12] Table 12

[0098]

[Table 13] Table 13

[0099]

[Table 14] Table 14

In this result, PC-SOD and KNI
The -272 combined system clearly demonstrated synergistic anti-H
An IV action was observed. Therefore, PC-SOD and PC-
KNI as a substance having an antiviral effect other than SOD
It is strongly suggested that the usefulness of the antiviral agent of the present invention containing -272 as an active ingredient and the usefulness of the antiviral agent of the present invention containing PC-SOD as an active ingredient with HIV and KNI-272 against HIV are high. It was

(-C) Combination of PC-SOD and sulfated polysaccharide In the test system of (-A) i) above, a similar test was conducted using dextran sulfate which is a sulfated polysaccharide in place of AZT. Note that Table 15 is a valid value using PC-SOD, Table 16 is the same predicted value, and Tables 17 and 11 are a table and a three-dimensional graph showing the difference between the valid value and the predicted value. . Further, FIG. 12 is a stereograph showing the difference between the effective value and the predicted value when the unmodified SOD is used.

[0102]

[Table 15] Table 15

[0103]

[Table 16] Table 16

[0104]

[Table 17] Table 17

In these results, in the system in which unmodified SOD and dextran sulfate were combined, at most, an additive effect due to the action of both was recognized, but in the system using PC-SOD, However, a clear synergistic anti-HIV effect was observed. Therefore, the usefulness of the antiviral agent of the present invention containing dextran sulfate as an active ingredient as a substance having an antiviral effect other than PC-SOD and PC-SOD, and the antiviral effect enhancer of the present invention containing PC-SOD as an active ingredient Was strongly suggested to be useful for HIV when administered with dextran sulfate.

[Formulation Example] (1) Injection Preparation PC-SOD (30 mg /
ml) was dissolved in a 5% mannitol aqueous solution so that the final concentration was 5 mg / ml, and this was aseptically filtered, then dispensed in 2 ml aliquots and sealed to prepare an injection.

PC-SO manufactured in the above manufacturing example
D (30 mg / ml) to a final concentration of 5 mg / ml, 3'-azido-
2 ′, 3′-dideoxythymidine (AZT), 2 ′, 3′-dideoxycytidine (ddC) or 2 ′, 3′-dideoxyinosine (ddI) was added to a final concentration of 4 μM (AZ
T), 200 μM (ddC) and 500 μM (ddI) were dissolved in a 5% mannitol aqueous solution, sterile filtered, and dispensed in 2 ml aliquots and sealed to prepare 3 types of injections.

PC-SO manufactured in the above manufacturing example
D (30 mg / ml) to a final concentration of 5 mg / ml, KNI-272
Was dissolved in a 5% mannitol aqueous solution so as to have a final concentration of 150 μg / ml, and this was aseptically filtered, and then 2 ml each was dispensed into ampoules and sealed to prepare an injection.

PC-SO manufactured in the above manufacturing example
D (30 mg / ml) to a final concentration of 5 mg / ml and dextran sulfate sodium to a final concentration of 400 μg / ml at 5%
After being dissolved in an aqueous mannitol solution and sterile filtered,
An injection was prepared by dispensing 2 ml each into an ampoule and sealing it.

(2) Tablets 100 mg of the freeze-dried product of PC-SOD produced in the above Production Example, 670 mg of lactose, 150 of potato starch
mg, crystalline cellulose 60 mg and light anhydrous silicic acid 50 mg were mixed, and a solution of hydroxypropyl cellulose 30 mg dissolved in methanol (hydroxypropyl cellulose 10% by weight) was added thereto, and kneaded and granulated. Next, this was extruded through a screen having a diameter of 0.8 mm to obtain granules, and after drying, 15 mg of magnesium stearate was added and compression-molded to produce tablets of 200 mg.

PC-SO manufactured in the above manufacturing example
100 mg of freeze-dried product of D, 670 mg of lactose, 150 mg of potato starch, 60 mg of crystalline cellulose and 50 mg of light anhydrous silicic acid were mixed, and further mixed with 3'-azide-2 ',
3'-dideoxythymidine (AZT), 2 ', 3'-dideoxycytidine (ddC) or 2', 3'-dideoxyinosine (ddI) was added to 75 nmol (AZT) and 4 μm, respectively.
Mol (ddC) and 10 μmol (ddI) were mixed, and a solution (30% by weight of hydroxypropyl cellulose) in which 30 mg of hydroxypropyl cellulose was dissolved was added to each mixture and kneaded and granulated. Next, this was extruded with a screen having a diameter of 0.8 mm to form granules, dried, and then added with 15 mg of magnesium stearate and compression-molded to produce three 200 mg tablets.

PC-SO manufactured in the above manufacturing example
100 mg of freeze-dried product of D, 670 mg of lactose, 150 mg of potato starch, 60 mg of crystalline cellulose and 50 mg of light anhydrous silicic acid were mixed, and KNI-272 (3 parts) was further added thereto.
A mixture of 30 mg of hydroxypropyl cellulose in methanol (10% by weight of hydroxypropyl cellulose) was added to this mixture, and the mixture was kneaded and granulated. Next, this was extruded through a screen having a diameter of 0.8 mm to obtain granules, and after drying, 15 mg of magnesium stearate was added and compression-molded to produce tablets of 200 mg.

PC-SO manufactured in the above manufacturing example
100 mg of freeze-dried product of D, 670 mg of lactose, 150 mg of potato starch, 60 mg of crystalline cellulose and 50 mg of light anhydrous silicic acid were mixed, and further 8 mg of sodium dextran sulfate was mixed, and 30 mg of hydroxypropyl cellulose was dissolved in methanol to this mixture. A solution (10% by weight of hydroxypropyl cellulose) was added and kneaded and granulated. Next, this was extruded through a screen having a diameter of 0.8 mm to obtain granules, and after drying, 15 mg of magnesium stearate was added and compression-molded to produce tablets of 200 mg.

(3) Capsule 100 mg of the freeze-dried product of PC-SOD produced in the above Production Example, 150 mg of potato starch, 50 mg of light anhydrous silicic acid, 10 mg of magnesium stearate and 765 mg of lactose were uniformly mixed, and 200 mg of this mixture was mixed. Was collected and filled into hard capsules to produce three types of capsules.

PC-SO manufactured in the above manufacturing example
D freeze-dried product 100 mg, potato starch 150 m
g, 50 mg of light anhydrous silicic acid, 10 mg of magnesium stearate and 765 mg of lactose were uniformly mixed, and further 3'-azido-2 ', 3'-dideoxythymidine (AZ
T) 2 ′, 3′-dideoxycytidine (ddC) or 2 ′, 3′-dideoxyinosine (ddI), 75 nmol (AZT), 4 μmol (ddC), 10 μmol, respectively
(DdI) was added and mixed uniformly, and 200 mg of each mixture was sampled and filled into hard capsules to produce three types of capsules.

PC-SO manufactured in the above manufacturing example
D freeze-dried product 100 mg, potato starch 150 m
g, 50 mg of light anhydrous silicic acid, 10 mg of magnesium stearate and 765 mg of lactose were uniformly mixed, and 3 mg of KNI-272 was further mixed with this mixture, and 200 mg of this mixture was taken and filled into a hard capsule to form a capsule. Was manufactured.

PC-SO manufactured in the above manufacturing example
D freeze-dried product 100 mg, potato starch 150 m
g, 50 mg of light anhydrous silicic acid, 10 mg of magnesium stearate and 765 mg of lactose are uniformly mixed, and further 8 mg of sodium dextran sulfate is further mixed, and 200 mg of this mixture is taken and filled into a hard capsule to form a capsule. Was manufactured.

[0118]

INDUSTRIAL APPLICABILITY The present invention provides a pharmaceutical use invention, that is, an antiviral agent and an antiviral effect enhancer for PC-SOD.

[0119]

[Sequence list]

 SEQ ID NO: 1 Sequence length: 153 Sequence type: Amino acid Topology: Linear Sequence type: Protein Sequence Ala Thr Lys Ala Val Cys Val Leu Lys Gly Asp Gly Pro Val Gln Gly 1 5 10 15 Ile Ile Asn Phe Glu Gln Lys Glu Ser Asn Gly Pro Val Lys Val Trp 20 25 30 Gly Ser Ile Lys Gly Leu Thr Glu Gly Leu His Gly Phe His Val His 35 40 45 Glu Phe Gly Asp Asn Thr Ala Gly Cys Thr Ser Ala Gly Pro His Phe 50 55 60 Asn Pro Leu Ser Arg Lys His Gly Gly Pro Lys Asp Glu Glu Arg His 65 70 75 80 Val Gly Asp Leu Gly Asn Val Thr Ala Asp Lys Asp Gly Val Ala Asp 85 90 95 Val Ser Ile Glu Asp Ser Val Ile Ser Leu Ser Gly Asp His Cys Ile 100 105 110 Ile Gly Arg Thr Leu Val Val His Glu Lys Ala Asp Asp Leu Gly Lys 115 120 125 Gly Gly Asn Glu Glu Ser Thr Lys Thr Gly Asn Ala Gly Ser Arg Leu 130 135 140 Ala Cys Gly Val Ile Gly Ile Ala Gln 145 150

[Brief description of drawings]

FIG. 1 is a drawing showing the primary structure of human Cu / ZnSOD protein.

FIG. 2 HIV-infected MT-4 by addition of PC-SOD
The drawing which showed that the viability of cells was improved.

FIG. 3 is a drawing showing cell viability when unmodified SOD was added to HIV-infected MT-4 cells.

FIG. 4 is a drawing for investigating how the anti-HIV effect is synergistically improved when PC-SOD and 3′-azido-2 ′, 3′-dideoxythymidine (AZT) are combined.

FIG. 5 is a drawing for studying the anti-HIV effect in the case of combining unmodified SOD and 3′-azido-2 ′, 3′-dideoxythymidine (AZT).

FIG. 6 is a drawing for studying the synergistic improvement of the anti-HIV effect when PC-SOD and 2 ′, 3′-dideoxycytidine (ddC) are combined.

FIG. 7 is a drawing for examining the anti-HIV effect in the case of combining unmodified SOD and 2 ′, 3′-dideoxycytidine (ddC).

FIG. 8 is a drawing for investigating how the anti-HIV effect is synergistically improved when PC-SOD and 2 ′, 3′-dideoxyinosine (ddI) are combined.

FIG. 9 is a drawing for examining the anti-HIV effect in the case of combining unmodified SOD and 2 ′, 3′-dideoxyinosine (ddI).

FIG. 10 is a drawing for examining how synergistically the anti-HIV effect is improved when PC-SOD is combined with KNI-272 which is an HIV protease inhibitor.

FIG. 11 is a drawing for investigating how the anti-HIV effect is synergistically improved when PC-SOD and dextran sulfate are combined.

FIG. 12 is a drawing for examining the anti-HIV effect in the case of combining unmodified SOD and dextran sulfate.

Claims (24)

[Claims] 1. An antiviral agent comprising, as an active ingredient, superoxide dismutase bound to lecithin through chemical crosslinking. 2. An anti-HIV comprising superoxide dismutase as an active ingredient, which is bound to lecithin through chemical cross-linking.
Antiviral agent having action. 3. An antiviral agent comprising, as an active ingredient, superoxide dismutase bound to lecithin through chemical crosslinking and a substance having an antiviral effect other than this superoxide dismutase. 4. An antiviral agent having an anti-HIV action, which comprises a superoxide dismutase bound to lecithin through a chemical crosslink and a substance having an anti-HIV action other than the superoxide dismutase as an active ingredient. 5. The antiviral agent according to claim 4, wherein the substance having an anti-HIV activity other than superoxide dismutase bound to lecithin through chemical cross-linking is HI.
An antiviral agent having an anti-HIV action, which is a substance having an anti-HIV action of one or more selected from the group consisting of a V reverse transcriptase inhibitor, an HIV protease inhibitor and a sulfated polysaccharide. 6. The HIV reverse transcriptase inhibitor is 3′-azido-2 ′, 3′-dideoxythymidine (AZT), 2 ′, 3 ′.
The antiviral agent according to claim 5, which is one or more HIV reverse transcriptase inhibitors selected from the group consisting of -dideoxycytidine (ddC) and 2 ', 3'-dideoxyinosine (ddI). 7. An HIV protease inhibitor is (R) -N.
-Tert -butyl-3-[(2S, 3S) -2-hydroxy-3-N-[(R) -2-N- (isoquinoline-5-
Ilooxyacetyl) amino-3-methylthiopropanoyl] amino-4-phenylbutanoyl] -5,5-dimethyl-1,3-thiazolidine-4-carboxamide (KNI-272). The described antiviral agent. 8. The antiviral agent according to any one of claims 5 to 7, wherein the sulfated polysaccharide is dextran sulfate. 9. The antiviral agent according to any one of claims 1 to 7, wherein the superoxide dismutase bound to lecithin through a chemical crosslink is represented by the following general formula (I): SOD- [C (O)-(CH ₂ ) _n- C (O) -X] _m (I) (In the formula, SOD represents superoxide dismutase, and X is lysolecithin having a hydroxyl group at the 2-position of glycerol; Represents a residue excluding the hydrogen atom of the hydroxyl group at the position, m is the average number of bonds to one SOD molecule, represents an integer of 1 or more, and n represents an integer of 2 or more). 10. The antiviral agent according to claim 9, wherein in the general formula (I), SOD is human-derived SOD. 11. In the general formula (I), SOD is human-derived Cu /, in which the amino acid at position 111 of the amino acid sequence is S- (2-hydroxyethylthio) cysteine.
The antiviral agent according to claim 9, which is Zn superoxide dismutase. 12. In the general formula (I), n is 2 or more and 10
The antiviral agent according to any one of claims 9 to 11, which is any of the following integers. 13. The antiviral agent according to claim 12, wherein n is 3 in the general formula (I). 14. In the general formula (I), m is 1 or more and 16
The antiviral agent according to any one of claims 9 to 13, which has the following numbers. 15. The antiviral agent according to claim 14, wherein m is 4 in the general formula (I). 16. An antiviral effect enhancer containing, as an active ingredient, superoxide dismutase bound to lecithin through chemical crosslinking. 17. The antiviral effect enhancer according to claim 16, wherein the superoxide dismutase bound to lecithin through a chemical crosslink is represented by the following general formula (I): SOD- [C (O)-(CH ₂ ) _n -C (O) -X] _m (I) (In the formula, SOD represents superoxide dismutase, and X is lysolecithin having a hydroxyl group at the 2-position of glycerol, except for the hydrogen atom of the hydroxyl group at the 2-position. Represents the residue, m is the average number of bonds to one SOD molecule, represents an integer of 1 or more, and n represents an integer of 2 or more). 18. The antiviral effect enhancer according to claim 17, wherein in the general formula (I), SOD is human-derived SOD. 19. In the general formula (I), SOD is human-derived Cu /, in which the amino acid at position 111 of the amino acid sequence is S- (2-hydroxyethylthio) cysteine.
The antiviral effect enhancer according to claim 17, which is Zn superoxide dismutase. 20. In the general formula (I), n is 2 or more and 10
It is any one of the following integers 17 thru | or 19
The antiviral effect enhancer according to claim 1. 21. The antiviral activity enhancer according to claim 20, wherein n is 3 in the general formula (I). 22. In the general formula (I), m is 1 or more and 16
The antiviral action enhancer according to any one of claims 17 to 21, which has the following numbers. 23. The antiviral activity enhancer according to claim 22, wherein m is 4 in the general formula (I). 24. The antiviral activity enhancer according to any one of claims 16 to 23, wherein the virus is HIV.