JP2000063279A - New immune function improver - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain a new immune function improver which can increase the immune function in living bodies at the simple composition and is useful in the treatment or the prevention for various infectious diseases including AIDS or immunologic lesion by including active chlorine species. SOLUTION: The immune function improver having preferably isotonic salt concentration is obtained by including active chlorine species (e.g. hypochlorous acid) in the range of 1-1,000 ppm, preferably in addition hydrogen gas. This immune function improver can be administrated in the combination with a reducing relaxation agent, e.g. superoxide dismutase or the like. This immune function improver is preferably obtained by electrolyzing a sterilized salt solution containing a salt content of 0.15-1.0 wt.% at sufficient voltage, ampere and time for giving an electrolyte containing a controlled amount of chlorine species under sterilized atmosphere and, when necessary, adjusting the electrolyte with a sterilized hypertonic salt to the isotonic salt concentration, It is favorable that this immune function improver is adjusted in the pH range of 7.2 to 7.6.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a novel agent for improving immune function. More specifically, the present invention relates to an immune function-improving agent containing an active chlorine species, a therapeutic agent for microbial infection, and a method for producing the same, which can be used for the treatment and prevention of various infectious diseases including AIDS and / or immunological disorders.

[0002]

2. Description of the Related Art Conventionally, active oxygen is a cell that makes up the human body,
It has been reported that the possibility of damaging proteins, genes, etc. is very high. Although the harmful effect of active oxygen has been emphasized in this way, it is recently becoming clear that active oxygen functions as an important regulator of signal transduction in the reaction in the living body. For example,
NF-κB (nuclear factor kappa B) due to hydrogen peroxide and
Regulation of the action of AP-1 (activator protein-1), nitric oxide (N
It is becoming clear that active oxygen is involved in important metabolic control and gene expression such as guanylate cyclase activation by O) and regulation of respiratory enzyme.

As signal transduction, signal transduction through cell membrane, signal transduction by protein kinase, signal transduction by oxidative stress and the like have been reported. Among these, it has recently been clarified that active oxygen and radicals act on the activity of protein phosphatase in a promoting or inhibiting manner. Furthermore, since the gene expression of antioxidant enzymes and various proteins is affected by active oxygen or oxidative stress, it is suggested that active oxygen and the like act as signal transduction.

[0004] active oxygen, singlet oxygen ⁽¹ O _2), superoxide (O ₂ ^-), hydrogen peroxide (H ₂ O _2), but hydroxy radical (HO ·) is included in the basic, In a broad sense, peroxy radicals (ROO), alkoxy radicals (RO), hydroperoxides (ROOH), HOCl, etc. generated by the reaction of the above-mentioned active oxygen with biological components such as unsaturated fatty acids (R) are also active. Considered as oxygen.

[0005] For example, the relationship between the above-mentioned signal transduction and the redox reaction will be described more specifically by taking lymphocytes associated with inflammation and immune reaction as an example. T cells receive antigen stimulation by recognizing the antigen through the T cell antigen receptor (TCR). Antigen stimulation first causes tyrosine phosphorylation. Tyrosine phosphorylation activates multiple signal transduction systems. When recognizing the antigen of TCR, MHC (major histocompatibility co
CD4 and CD8 are known as molecules that bind to the constant region of mplex) and assist antigen recognition. It is possible to transmit signals from these molecules themselves. These signals activate NF-κB and the like via various transmission systems, and as a result, gene transcription is induced.

Transcription factor NF-κB important for regulation of gene expression
Consider (nuclear factor kappa B). It is said that NF-κB forms a complex with an inhibitory protein called I-κB (inhibitor-κB) in resting cells and localizes in the cytoplasm. When various signals such as active oxygen come from outside the cell, NF-κB and I-κB dissociate, and NF-κB moves into the nucleus to become the active form. Activation of NF-κB is carried out in two steps, and it is said that a specific phosphorylase is involved in the first step, and it is reported that the redox reaction acts in the second step. It is known that NF-κB activation is involved in the replication process of AIDS virus in T lymphocytes, and that oxidative stress such as active oxygen promotes the expression of HIV-1. On the other hand, reducing substances are believed to suppress this expression.

Organisms have low-molecular and high-molecular defense systems to protect themselves from the effects of active oxygen.
For example, high molecular compounds such as superoxide dismutase (SOD), catalase, glutathione peroxidase, and low molecular compounds such as ascorbic acid, glutathione (GSH), uric acid, lipoic acid, vitamin E, carotenoids, and bilirubin are active oxygen species. It is thought that the organism itself possesses it as a substance that protects itself from

[0008] In HIV-infected persons and AIDS patients, reduction of intracellular reducing substances such as glutathione and cystine / cysteine has been reported. This is considered to be because the reduction of the reducing substance promotes dissociation of NF-κB and I-κB, and HIV that infects T cells with CD4 as a receptor is efficiently replicated. Further, it is known that T cells are proliferated and activated when glutathione is sufficiently present.

By the way, when tert-butyl hydroxide (tBOOH), H ₂ O ₂ , benzoyl peroxide and the like are administered to animals, glutathione levels in tissues and cells are transiently lowered, but thereafter, they are increased to the initial concentration. It is reported to increase more than the above. Moreover, and hyperoxia, O ₂ ^- When slowly prolonged exposure to such, glutathione levels in a cell without initial drop has been reported to be elevated. (Ohi, T.Arch. Toxicol. 67, 401-410 (1
993)).

[0010] As described above, suppression of HIV expression and recovery of immune function can be expected by setting a reducing atmosphere in cells, but it has not been experimentally proved.

[0011]

As described above, it has been suggested that the redox reaction is involved in the signal transduction system, and by positively utilizing this, the immune function of the living body is enhanced to prevent or treat infectious diseases. It is expected to be used for. An object of the present invention is to provide an inexpensive new immune function-improving agent capable of enhancing the immune function of a living body with a simple composition. In addition, this is to be used as a preventive or therapeutic drug for various infectious diseases.

[0012]

[Means for Solving the Problems] As a result of intensive research to solve the above problems, the present inventors have found that, in addition to the above, an active chlorine species of a specific concentration, particularly an aqueous solution of hypochlorous acid, has an excellent effect of improving immune function. The present invention was completed by discovering that it has.

Active chlorine species such as hypochlorous acid have an oxidative action, and have the same role as in the above-mentioned signal transduction system in that they dissociate NF-κB and I-κB in the same manner as active oxygen, and
It should act in a direction that activates κB, that is, promotes the replication of AIDS virus in T cells.
However, it was revealed that the present invention exerts the opposite effect and improves the immune function.

[0014] Incidentally, the same Japanese Patent Application No. 9-
No. 5,117,52 discloses a microbicidal solution containing ozone and active chlorine species in a specific concentration, but the present invention relates to an immune function improving agent containing no ozone.

That is, the present invention provides an immune function-improving agent containing 1-1000 ppm of an active chlorine species aqueous solution.

The present invention further provides a method for producing the above immune function-improving agent.

The present invention further provides an infectious disease treatment method and therapeutic agent using the above-mentioned immune function-improving agent.

The present invention further provides a method for in vitro decontamination or treatment of a microbially contaminated solution using the above-mentioned immune function-enhancing agent.

[0019]

BEST MODE FOR CARRYING OUT THE INVENTION The active chlorine species contained in the immune function-improving agent of the present invention include free chlorine, hypochlorous acid and hypochlorite ion (hypochlorit ion) measured by a chloride ion selective electrode.
e ion), and hypochlorous acid is particularly preferable.

The active chlorine species concentration contained in the immune function-enhancing agent of the present invention means the total chlorine concentration attributable to the chlorine content detectable by the chloride ion selective electrode, and includes chlorine, hypochlorous acid and hypochlorous acid. This is the total concentration of active chlorine contained in the ions. The concentration of active chlorine species in the immune function improver is 1-1000
ppm, preferably 1-300 ppm, more preferably 1-1
It is in the range of 00 ppm.

The pH of the immune function-improving agent of the present invention is 7.0 to 7.7.
And more preferably 7.2 to 7.6, and when used for intravenous administration, the pH range of human blood is about 7.35 to
More preferably, it is 7.45. When injected into a living body by intravenous administration or the like, it is preferable to adjust the osmotic pressure to prepare a solution having an isotonic salt concentration.

In the agent for improving immune function of the present invention, a reducing substance such as hydrogen can coexist to improve the healing efficiency. As will be described later, when the immune function-improving agent of the present invention is produced by an electrolysis method, hydrogen is added separately because a part of hydrogen gas generated at the cathode of the negative electrode is dissolved in electrolyzed water. do not have to.

Furthermore, if necessary, the immune function-improving agent of the present invention may be used in an appropriate and / or neutralizing amount of superoxide dismutase, myeloperoxidase, glutathione peroxidase, glutathione, catalase, ascorbic acid, ascorbic acid. It can also be administered in combination with one or more antioxidants or reducing agents selected from the group consisting of sodium and calcium ascorbate. The combined use of these reducing emollients can suppress the expression of HIV and promote the recovery of immune function. Therefore, the present invention is an immune function-improving agent containing an active chlorine species in the range of 1-1000 ppm, an effective amount of superoxide dismutase, myeloperoxidase, glutathione peroxidase,
There is provided an immune function-improving agent comprising a combination with one or more reducing emollients selected from the group consisting of glutathione, catalase, ascorbic acid, sodium ascorbate and calcium ascorbate.

Furthermore, the agent for improving immune function of the present invention can be administered in combination with colchicine. Accordingly, the present invention provides an immune function-improving agent comprising a combination of an immune function-improving agent containing an active chlorine species in the range of 1-1000 ppm and an effective amount of colchicine.

To produce the immune function-improving agent of the present invention,
(1) Prepare a thin sterile salt solution first, and then simply dissolve active chlorine, preferably hypochlorous acid, in this solution, and then adjust the pH with sodium hydroxide solution, or (2) dilute salt solution. An element selected from the group consisting of hydrogen ion, sodium ion and hydroxide ion by electrolyzing an aqueous solution at an appropriate voltage, amperage and time to contain active chlorine species such as hypochlorous acid at a specific concentration. This is accomplished by producing an electrolytic solution that also contains other products of the electrolytic reaction, including. The method of (2) by electrolysis is preferable.
By electrolysis, a biologically active atom, radical or ion selected from the group consisting of chlorine, ozone, hydroxide, hypochlorous acid, hypochlorite, peroxide, oxygen, hydrogen and possibly other elements. To produce a solution containing a corresponding amount of molecular hydrogen with sodium and hydrogen ions.
Hereinafter, a solution containing hypochlorous acid may be referred to as an active chlorine aqueous solution regardless of the production method.

When the method of (1) is used to produce the immune function-improving agent of the present invention, the sterile salt solution in which active chlorine is dissolved is about one fourth of the full strength of a normal or isotonic salt solution. It has an initial concentration of 0.25 to 1.0% NaCl. Ta
rber's Cyclopedic Medicaldictionary, EADavis, C
o. 1985.ed. According to, "isotonic salt solution" means 0.16M Na
Cl solution or a solution containing about 0.95% NaCl; "physiological salt solution" is defined as a sterile solution containing 0.85% NaCl and is considered to be isotonic with body fluids. The "normal salt solution" is a 0.9% NaCl solution and is considered to be isotonic with the body. Thus, for the purposes of this disclosure, the terms "isotonic", "normal salt solution", "equilibrium salt solution" or "physiological fluid" include between about 0.85% and 0.95% NaCl. Considered as a salt solution.

In the case of producing the immune function-improving agent of the present invention by using the method (2), that is, in order to produce an active chlorine aqueous solution by electrolysis, electrolysis is performed at a salt concentration of about 0.15% to 1.0%. Call. Preferably the solution is diluted with sterile distilled water to the desired concentration (preferably between 0.15 and 0.35%) and electrolyzed at a voltage, amperage and time sufficient to produce an electrolytic solution. The electrolytic reaction is carried out at room temperature. Here the voltage and amperage and electrolysis time are sensitive to many variables (ie electrode size and composition, capacity and / or concentration of salt solution undergoing electrolysis). For large electrodes or salt solution volumes or higher concentration salt solutions, higher and / or longer voltage, amperage or time may be required. It is important to generate the desired concentration of active chlorine species. According to Faraday's law of electrolysis, the amount of chemical change produced by an electric current is proportional to the amount of electricity passed. Also, the amount of different substances released by a given amount of electricity is proportional to the chemical equivalents of these substances. Therefore, in order to generate an electrolytic salt solution having a desired concentration of ozone and active chlorine species from a salt solution having a salt concentration of less than about 1.0%, an electrolytic salt solution containing a free chlorine content of about 1 to 1000 ppm is required. A suitable voltage, amperage or time parameter is required to produce For in vitro use, these solutions may be used without further modification or they may be prepared and used as desired in salt or other solutions. Prior to in vivo use, this solution may be adjusted or diluted to an isotonic salt concentration with sufficient hypertonic salt solution (eg, 5% hypertonic salt solution).

The immune function-improving agent of the present invention may further contain hydrogen gas. When producing the immune function-improving agent of the present invention by the method (1), hydrogen gas can be added to the salt solution containing active chlorine species containing active chlorine species obtained.

When the immune function-improving agent of the present invention is produced by an electrolysis method, chlorine ions are oxidized at the positive electrode anode, and finally hypochlorous acid is produced. Hydrogen gas is generated at the negative electrode cathode. Some hydrogen gas is dissolved in electrolyzed water. The dissolved hydrogen gas has a low reaction rate with hypochlorous acid and a reaction rate lower than that of other reducing agents, but it remains a reducing agent. Thus, using hypochlorous acid water produced by electrolysis, rather than simply using hypochlorous acid, combined the reducing property of hydrogen gas in addition to the oxidative stress and sterilizing effect of hypochlorous acid. A synergistic effect can be expected.

The immune function-improving agent of the present invention was neither toxic in vitro nor in vivo, as shown in Examples described later. Further, in an in vitro activity test using HIV-infected human lymphocytes, a remarkable antiviral effect was shown.

The immune function-enhancing agent of the present invention contains many bacteria,
It can be used for the prevention and treatment of viral and fungal infections and immunological disorders. Examples of such infectious diseases and / or immunological disorders for which either in vitro or in vivo treatment may be effective are Epstein-Barr virus, A, B and C.
Hepatitis C, rhinovirus, measles, rubella, parvovirus, papilloma virus, influenza and parainfluenza virus, enterovirus, herpes simplex virus, varicella-zoster virus, adenovirus, RS virus (respiratory syncytial virus), alphavirus, Flavivirus, retrovirus (including AIDS
HIV, including bacteremia, sepsis, fungal infections, parasitic infections (nematodes, flukes, protozoa [eg Cryptosp
oridium], helminthic), mycobacterial infections, superficial and systemic infections of Gram-positive and Gram-negative bacteria, and other viral, bacterial and / or fungal infections. Many of these are diseases that are affected by delayed, latent or lysogenic organisms (ie viruses, bacteria or fungi) that have long incubation times. Many of these are also diseases for which no cure is known at present, and usually these diseases or concomitant opportunistic infections progress slowly leading to host death.

Infected hosts or patients can be treated by a variety of regimens that can temporarily alleviate the symptoms. However, the immune system eventually can no longer adequately combat invasive or autoimmune-related infections, eventually terminating in the proper functioning of the natural biocidal effects within the cell, leading to death.

The immune function-improving agent of the present invention is demonstrated to be effective in treating various infectious diseases caused by invading antigens, in particular, viral infections causing AIDS.

The immune function-enhancing agent of the present invention generally contains about
It contains between 1 and 1000 ppm of active chlorine species. Preferably the active chlorine species content will be between about 1 and 300 ppm. More preferably, the active chlorine species content will be between about 1 and 100 ppm. An effective amount of this dilute aqueous solution is then administered in a suitable manner (eg, intravenous, oral, vaginal or rectal), the dosage being the mode of administration, the condition being treated, the size of the warm-blooded animal, etc. Will change significantly. For humans injected intravenously, the dose of active chlorine solution is approximately
Varies between 0.25 and 4 ml / kg / day, 0.5 to 3 m
A range of 1 / kg / day is preferred. The dose can be divided into smaller doses, administered two or more times daily, or may be administered in a single dose. In addition, the administration schedule may be changed depending on the condition. For example, for HIV treatment, several immunoinventive agents of the present invention are administered, followed by a rest period, and then required or, for example, Western blot, T cell subset, chem profile (SMAC20),
The cycle is repeated while dictated by test results such as CBC, p24 antigen and HIV mRNA quantification. A typical dosing regimen is a 5 day treatment followed by a 2 day rest cycle repeated for 2 months. Depending on the clinical condition or laboratory testing, this dosing regimen may be reduced, eg 3 days of treatment for 1 week for 6 weeks. These dosing regimes are exemplary and are not meant to be limited to numbers and changes will be indicated according to the circumstances.

The immune function-improving agent of the present invention can be stored in a closed container such as an ampoule after administration and administered.
Alternatively, an electrolyte can be generated and administered to the patient in situ. For use in a clinical state, it is preferable to administer the electrolyte by intravenous drip or the like immediately after the production of the electrolytic solution (for example, within 2 hours). If the immune function improver is left in a released state for a long time, the concentration of hydrogen gas added or the concentration of hydrogen gas generated by electrolysis and dissolved in the solution may decrease.

When a reducing palliative such as superoxide dismutase, myeloperoxidase, glutathione peroxidase, glutathione, catalase, ascorbic acid, sodium ascorbate and calcium ascorbate is used in combination with the immune function-improving agent of the present invention. The amount of emollient to be administered will depend in part on the mode and timing of administration. The palliative dose administered orally will be somewhat higher than injected intravenously. In addition, if the reducing emollient is administered before injection of the electrolytic salt solution, after the electrolytic salt solution achieves its function, it is reduced to a site capable of reducing free radicals such as active oxygen remaining from the solution. There must be sufficient time to allow the emollient to be absorbed and carried in the bloodstream.

The dose of reducing emollient administered need not be stoichiometric with the active chlorine solution and can be initially determined empirically. Sufficient reducing mitigating agent must be present in the system to prevent free radical components generated from the active chlorine solution from causing irreversible tissue damage. For this reason, superoxide dismutase, catalase, L-glutathione, orally at a time prior to the injection of the active chlorine aqueous solution, so that a suitable amount of the emollient is available in the cells when the electrolytic salt solution is injected, It may be beneficial to administer palliatives such as glutathione peroxidase, MPO and ascorbic acid. However, it is equally important that the free radical component be available to perform the desired function before it is suppressed or inactivated by the mitigating agent. Generally, oral doses of superoxide dismutase in the range of about 5,000 to 60,000 units per day will be administered. Catalase, MPO and glutathione peroxidase doses will vary between about 10,000 and 120,000 units per day. Glutathione is about 10 to 120 mg per day
Will be administered in an amount in the range. About 50 ascorbic acid or its sodium or calcium salt per day
It will be administered in a wide range from 1 to 20,000 mg. To assure that unreacted oxidative components of the salt solution are not reduced and / or neutralized, ascorbic acid is preferably administered intravenously shortly after the electrolytic salt solution injection. Based on the above guidelines, the parties can easily determine what is the effective amount of the emollient.

By injecting the active chlorine aqueous solution intravenously and administering the emollient in the above-mentioned manner, a reducing substance is produced in cells in the living body by oxidative stress, and the immune function is improved. Mitigating agents mimic the action of enzymes produced by macrophages and monocytes as reducing agents in order to make them more reducing. As a result, the injected chemical substance directly attacks the microorganisms in the host cell by the synergistic action of the function of active chlorine and the enhancement of immune function.

Simultaneous administration of an active chlorine aqueous solution and colchicine is more effective for inhibiting the growth of microorganisms and the like in the living body. Colchicine, [N- (5,6,7,9
-Tetrahydro-1,2,3,10-tetramethoxy-
9-oxobenzo [a] heptalene-7-yl) acetamido] C22H25NO6, the Koruhikamu Otsumu
Which is the main alkaloid of Nare (Colchicum autumnale). Colchicine is an anti-inflammatory drug originally used to control gout and to treat familial Mediterranean fever, scleroderma and psoriasis. Colchicine functions by inhibiting the migration of granulocytes into the inflamed area, reducing the release of lactate and proinflammatory enzymes that occur during phagocytosis, thereby breaking the cycle leading to the inflammatory response. Neutrophils and leukocytes bind cells and produce glycoproteins that may be responsible for acute inflammation. Colchicine inhibits both glycoprotein production and leukocyte release. It causes transient leukopenia, sometimes due to a marked increase in the number of basophils and because it has the same effect as increasing lymphopoiesis, after which it is replaced by leukocytosis. Acts directly on the bone marrow. In addition, colchicine is an antipyretic drug that lowers body temperature.

Colchicine also increases the body's sensitivity to central nervous system depressants, suppresses respiratory centers, promotes responses to sympathomimetics, contracts blood vessels, and induces hypertension by vascular central excitation. It is well tolerated at moderate doses and acts like cortisone in suppressing the immune system without the high risk and side effects associated with corticosteroids but not corticosteroids. At low doses, colchicine will act as an immune system stimulant that helps rescue the overworked immune system.

Colchicine is also a weak immunostimulant that will delay replication of the virus latent in T cells. This latent virus awaits external infection that stimulates an immune response that activates viral replication. When using
Colchicine doses vary between about 1.0 and 3.0 mg, with about 1.5 mg considered optimal for adults. Preferably, it is administered intravenously immediately before or simultaneously with the administration of the active chlorine aqueous solution.

As mentioned above, colchicine is used together when the active chlorine aqueous solution is administered. The most advantageous treatment for any disease is to use the minimum amount of active agent required.

Approximately 500 if desired to complete treatment
-5,000 mg, preferably about 1,000-4,000 mg of ascorbic acid or its sodium or calcium salt is administered about 2-20 minutes after the administration of the active chlorine solution. This reducing agent neutralizes the unreacted active components of the remaining active chlorine aqueous solution.

Although the reducing emollient and / or colchicine may be supplemented to administer a predetermined amount of the active chlorine aqueous solution, the predetermined amount of the active chlorine aqueous solution and its use are
The present invention is of particular interest. Therefore, administration of the emollient and / or colchicine is considered optional.

Yet another object of the present invention is to provide a method for in vitro decontamination or treatment of a microbial contaminated solution with an aqueous solution containing active chlorine species. Accordingly, the present invention provides a method for in vitro decontamination or treatment of microbial contaminated solutions with salt solutions containing active chlorine species in the range of 1-1000 ppm.

There are also situations in which liquids can be advantageously treated in vitro to purify, decontaminate, or otherwise make them acceptable for administration to warm-blooded animals. For example, blood supplies obtained from donors in blood banks sometimes include HIV viruses such as hepatitis A, B and C viruses, CMV (cytomegalovirus) and bacteria (eg Yersinia). It has been found to be contaminated by other living organisms.
Treatments that render whole blood, plasma or cell isolates good without infectious organisms without destroying the therapeutic properties of such fluids would be very beneficial.

The method of decontaminating or treating a microbial-contaminated fluid of the present invention in vitro comprises treating the microbial-contaminated fluid with a time in the range of 1-1000 ppm for a time sufficient to reduce microbial contamination in the fluid. Contacting with a salt solution containing the active chlorine species.

Fluids contaminated with microorganisms which can be decontaminated or treated by the above method of the invention include Epstein-Barr virus, hepatitis A, B or C viruses, rhinovirus, measles, rubella, parvovirus, papilloma. Virus, influenza virus, parainfluenza virus, enterovirus, herpes simplex virus, varicella zoster virus, adenovirus, RS
Virus, alphavirus, flavivirus, retrovirus (including HIV including AIDS virus), bacteremia, sepsis, fungal infection, parasitic infection, mycobacterial infection, gram positive bacterial infection, gram negative bacterial infection, shallow Includes fluid that is contaminated with an infectious agent selected from the group consisting of resident and systemic infections.

Fluids contaminated with microorganisms that can be decontaminated or treated by the above method of the present invention include, but are not limited to, whole blood, blood cells, and plasma.

[0050]

Since an active chlorine aqueous solution in which hypochlorous acid or the like is dissolved is a bactericidal agent in vitro, products produced by such electrolysis have been known for a long time. Although it is known that electrolytic salt solutions have bactericidal activity in vitro, the components in such electrolytic solutions are toxic to warm-blooded animals and should be used for in vivo use. Has long been considered. However, it has been found here that a suitable amount of an active chlorine aqueous solution can be injected into the vasculature to produce a reaction that aids in the removal, passivation or degradation of toxins and significantly enhances immune function. It was

The immune function-improving agent of the present invention has no toxicity in in vitro and in vivo use, enhances the immune function of the living body with a simple composition, and can be used as a preventive or therapeutic drug for various infectious diseases. The immune function-improving agent of the present invention can be prepared at low cost, and can be expected to be used as a potent preventive or therapeutic agent for AIDS in Asia, Africa and the like.

[0052]

EXAMPLES The following examples serve to illustrate the invention and its use. The following examples will be described mainly based on the results obtained by using an active chlorine aqueous solution generated by electrolyzing physiological saline. All solutions were obtained using a modified electrode containing a defined amount of active chlorine species and with more precisely controlled parameters of voltage, current, time and salt concentration. Example 1 below details the preparation of a suitable aqueous active chlorine solution for use in the present invention.

Example 1 Preparation of Active Chlorine Aqueous Solution To 300 ml of sterile distilled water was added 100 ml of sterile 0.9% salt (NaCl) solution to obtain a 0.225% salt solution. The solution was placed in a plastic chamber with fresh titanium and platinum electrodes. The 0.225% saline solution was then subjected to a current of 3 amps at 20 volts (DC) for 3 minutes. Aseptically dilute a 17 ml portion of the electrolytic solution with 3 ml of sterile 5% saline solution to obtain an active chlorine species content of 6
A final isotonic electrolytic solution with 0 ± 4 ppm, pH 7.4 was obtained. Chlorine concentration was measured using an Orion chloride ion selective electrode.

Example 2 Stability of Electrolytic Solution The stability of the electrolytic solution was further investigated at 4 ° C. for a long time. The stability of two separate isotonic solutions (Solution A and Solution B prepared with separate electrodes) was measured. By the same procedure as in Example 1, each solution was electrolyzed to make it isotonic. The solution was kept at a temperature of 4 ° C. for 360 minutes, and periodical measurements were made to determine the content of active chlorine species (Cl ₂ ) and hydrogen (H ₂ ). Hydrogen was measured by a potentiostatic electrolysis method.
The results are shown in Table 1 below.

[0055]

[Table 1] It is clear from Table 1 that the active chlorine species are maintained almost constantly and are stable. Furthermore, the dissolved hydrogen concentration is also constant in this measurement range. From these results, it can be seen that active chlorine, which is a type of active oxygen, and hydrogen, which is a type of reducing agent, coexist stably for a certain period of time.

Example 3 Salt Concentration and Electrolysis Reaction To show that the electrolysis reaction can be effectively carried out in a salt solution up to about 1% concentration, 0.3, 0.6 and 0.9 respectively.
The electrolytic reaction was carried out at a salt solution concentration of%. Examine the content of active chlorine species (Cl2) and hydrogen (H _2), shown in Table 2 below.

[0057]

[Table 2] As can be seen from Table 2, the active ingredient is sufficient within the parameters required in the present invention. The final concentration of active ingredient can be adjusted so that saline and / or water gives the desired final concentration of active ingredient.

Example 4 In Vitro Toxicity Test The in vitro toxicity is proved by adding the electrolytic salts of Example 1 to human lymphocytes at various active chlorine agent concentrations and exposure periods as shown in Table 3. That is,
RPMI 1640 medium containing 3 parts of 1% trypan blue solution (1 g of trypan blue powder is placed in a 100 ml volumetric flask, dissolved in water to a volume of 100 ml and then filtered before use) and 10% FBS A 0.3% trypan blue solution was prepared by mixing with 7 parts. 1 ml lymphocyte sample (eg live cells 1
(05 cells) was spun down, the medium was decanted, and the lymphocytes were resuspended in electrolytic saline solution (concentration of active chlorine species changed by constant dilution with saline). Lymphocytes were incubated for a period of time and then washed by sedimentation and resuspension in fresh medium. Aliquots of lymphocytes were then mixed with 0.3% trypan blue solution and observed microscopically. 100 cells were screened and the number of cells that excluded trypan blue was considered as% of viable cells.

The results obtained are shown in Table 3.

[0060]

[Table 3] a The% obtained by diluting this with the electrolytic solution of Example 1 as 100% is shown.

B% viability is 10% viability versus control
Relative% adjusted to 0%.

C Test Control (Saline) As demonstrated in Table 3, 100% at 6 and 10 minutes (no dilution).
There was some toxicity to the cells at both dilutions of 20% and 20%, with a slight toxicity at 10 minutes at 10%. There was no toxicity at 1% for shorter times and at higher dilutions for up to 10 minutes.

Example 5: In vitro mutagenicity The in vitro mutagenicity is demonstrated by adding the electrolytic salts of Example 1 to cells according to the Salmonella reverse mutation assay (Ames test). This test is conducted by an independent laboratory at the USFDA Good Laboratory Prac.
Done according to the tices Regulations [21 CFR Part 58].

The Ames test uses several strains of S. typhimurium selected on the basis of their susceptibility to mutations. The Ames test was carried out by mixing the electrolytic salt of Example 1 with a test living body in a soft agar solution containing only a small amount of histidine. Histidine allows the inoculated test organism to undergo a limited number of divisions, but is insufficient for normal growth. The test strain requires histidine for growth due to a mutation in a gene that suppresses histidine production. However, when the strain undergoes a backmutation (naturally or induced by a test or positive control substance), the organism no longer requires histidine for growth and forms visible colonies or revertants. Only mutations in the histidine gene region to the subject will backmutate the subject to organisms that no longer require histidine. The test strains were selected to detect various types of mutagens. The test strains used were TA97A, TA98, TA100, TA102 and TA1535.

An independent laboratory conclusion on the mutagenicity of electrolytic salt solutions is that "the solutions tested against the five strains did not meet the criteria for potential mutagens". It was

Example 6 In Vivo Toxicity The electrolytic salts of Example 1 were injected into the tail vein of Harlan Sprague Dawley: ICR mice at various concentrations, ie 2, 4, 6 and 8 mL / kg body weight. To demonstrate its in vivo toxicity. These tests were conducted by Good Laborator at an independent laboratory.
y Conducted in accordance with Procedure Regulations [21 CFR Part 58]. The conclusion of these studies was that "the electrolytic salt solution of Example 1 was non-toxic at a single intravenous dose of at least 8 mL / kg body weight".

Example 7: In vitro activity (1) The in vitro activity of clinical isolates (field isolates) of HIV-infected human lymphocytes was determined according to the electrolytic salts of Example 1
Tested according to the method of Ho et al. N. Engl. J. Med. 321: 1621-1625 (1989). TCID (tissue culture infectious dose) per 106 lymphocytes [PBMC (peripheral blood mononuclear cells)] is
It was 5000 for each isolate. p24 antigen weekly
I inspected 5 times a week. The results at the end of 5 weeks are shown in Table 4.

[0068]

[Table 4] As shown in Table 4, H after only 1 minute of exposure as evidenced by the absence of detectable p24 antigen.
The IV is completely dead. This demonstrates that electrolytic salts have an antiviral effect in vitro upon treatment of infected blood cells, whole blood, or any other body fluid.

Example 8: In vitro activity (2) To further demonstrate the in vitro activity of the electrolytic salts of Example 1 on HIV-infected human lymphocytes, HI
Further tests were performed with V-infected laboratory isolate HB-2. HIV-infected human HB-2 cells at a concentration of 2 x 10 ⁶ cells / ml were treated with 100 μg / ml penicillin, 50 μg / ml at 37 ° C.
Were cultured in RPMI medium containing Streptomycin, 2 mM L-glutamic acid, and heat-inactivated bovine serum. The reverse transcriptase activity of these cells was 10,000 to 50,000 cpm per 2 x 10 ⁶ cells. The electrolyte dilution was 100, 20, 10, 1%. HIV p24 antigen was quantified using the EIA assay. The results are shown in Table 5. From these results, it can be seen that the electrolytic solution has antiviral activity.

[0070]

[Table 5]

   ─────────────────────────────────────────────────── ─── Continued front page    (72) Inventor Naoki Yahagi             373-32, Yamanoura, Ryuo-cho, Gamo-gun, Shiga Prefecture F-term (reference) 4C058 AA28 AA30 BB07 CC01 CC10                       DD05 DD07 JJ07 JJ26 JJ30                 4C077 AA25 BB10 GG08 GG10 HH03                       HH12 HH17 JJ03 JJ12 JJ18                       JJ28 KK09 NN14 NN15 PP27                       PP29                 4C084 AA03 DC23 DC24 DC31 NA01                       NA14 ZB091 ZB331 ZB351                       ZB391 ZC282                 4C086 AA01 AA02 BA18 HA09 HA24                       MA01 MA02 MA04 MA10 NA01                       NA14 ZB09 ZB33 ZB35 ZB39                       ZC28

Claims (18)

[Claims] 1. An immune function improving agent containing active chlorine species in the range of 1-1000 ppm. 2. The immune function improving agent according to claim 1, which has an isotonic salt concentration. 3. The immune function improving agent according to claim 1, wherein the content of the active chlorine species is in the range of 1 to 300 ppm. 4. The immune function improving agent according to claim 1, wherein the content of the active chlorine species is in the range of 1-100 ppm. 5. The immune function improving agent according to claim 1, further comprising hydrogen gas. 6. An immune function improver containing an active chlorine species in the range of 1-1000 ppm, and an effective amount of superoxide dismutase, myeloperoxidase, glutathione peroxidase, glutathione, catalase, ascorbic acid,
An immune function improving agent which comprises a combination with one or more reducing emollients selected from the group consisting of sodium ascorbate and calcium ascorbate. 7. An immune function improving agent comprising a combination of an immune function improving agent containing an active chlorine species in the range of 1-1000 ppm and an effective amount of colchicine. 8. A method for producing an immune function-improving agent according to claim 1, which comprises the steps of: (a) controlling a sterile salt solution having a salt content in the range of about 0.15-1.0% in a sterile atmosphere. Subject to electrolysis at a voltage, amperage, and time sufficient to provide an electrolytic solution containing a sufficient amount of chlorine species; and (b) if necessary, adjust the electrolytic solution with sterile hypertonic salt to remove active chlorine species. Content is 1-1000ppm
Immune function improving agent which is in the range of 9. The method according to claim 8, further comprising the step of adjusting the electrolytic solution to a isotonic salt concentration with a hypertonic salt. 10. A microbial-contaminated fluid comprising contacting the microbial-contaminated fluid with the immune function-enhancing agent of claim 1 for a time sufficient to reduce microbial contamination in the fluid. A method for decontaminating or treating in vitro. 11. A microorganism-contaminated fluid is Epstein-Barr virus, hepatitis A, B or C virus, rhinovirus, measles, rubella, parvovirus, papilloma virus, influenza virus, parainfluenza virus, enterovirus, herpes simplex virus. Virus, varicella zoster virus, adenovirus, RS virus, alphavirus, flavivirus, retrovirus (including HIV including AIDS virus), bacteremia,
11. The method of claim 10 contaminated with an infectious agent selected from the group consisting of sepsis, fungal infections, parasitic infections, mycobacterial infections, gram positive bacterial infections, gram negative bacterial infections, superficial and systemic infections. . 12. The method according to claim 10, wherein the fluid contaminated with microorganisms is whole blood, blood cells, or plasma. 13. An active chlorine species in the range of 1-1000 ppm,
A therapeutic agent for microbial infection of warm-blooded animals including humans. 14. A microbial infection is Epstein-Barr virus, hepatitis A, B or C virus, rhinovirus,
Measles, rubella, parvovirus, papillomavirus, influenza virus, parainfluenza virus,
Enterovirus, herpes simplex virus, varicella zoster virus, adenovirus, RS virus, alphavirus, flavivirus, retrovirus (including HIV including AIDS virus), bacteremia, sepsis, fungal infection, parasitic infection, 14. The therapeutic agent according to claim 13, which is at least partially induced by an infectious agent selected from the group consisting of mycobacterial infection, gram positive bacterial infection, gram negative bacterial infection, superficial and systemic infection. 15. The therapeutic agent according to claim 13, which has an isotonic salt concentration. 16. The therapeutic agent according to claim 13, which is administered intravenously. 17. A therapeutic agent for microbial infection containing active chlorine species in the range of 1-1000 ppm, and an effective amount of superoxide dismutase, myeloperoxidase, glutathione peroxidase, glutathione, catalase, ascorbic acid, sodium ascorbate and ascorbic acid. A therapeutic agent for microbial infection, which comprises a combination with one or more reducing emollients selected from the group consisting of calcium. 18. A therapeutic agent for microbial infection, which comprises a therapeutic agent for microbial infection containing active chlorine species in the range of 1-1000 ppm and an effective amount of colchicine.