CA2195082A1

CA2195082A1 - Electrically hydrolyzed salines as in vivo microbicides for the treatment of cardiomyopathy and multiple sclerosis

Info

Publication number: CA2195082A1
Application number: CA002195082A
Authority: CA
Inventors: Robert E. Morrow
Original assignee: Individual
Current assignee: Global Clean Energy Holdings Inc
Priority date: 1994-07-15
Filing date: 1994-07-22
Publication date: 1996-02-01
Also published as: US5731008A; WO1996002271A1; AU7371294A; US5622848A; JP3187841B2; JPH09511752A; US5674537A; EP0767677A1

Abstract

A microbiocidal solution for in vivo and in vitro treatment of microbial infections comprises an electrolyzed saline containing regulated amounts of ozone and active chlorine species wherein the ozone content is between about 5 and 100 mg/L and the active chlorine species content of between about 5 and 300 ppm. The active chlorine species comprises free chlorine, hypochlorous acid and the hypochlorite ion as measured by a chlorine selective electrode.
The solution is prepared by subjecting a 1 % or less saline to electrolysis under conditions sufficient to produce the desired active ingredients. The solution is preferably utilized at an isotonic saline concentration and may be adjusted with hypertonic saline. The solution may be used for the in vitro treatment of infected whole blood, blood cells or plasma to reduce contamination and is effective in treatment of fluids infected with HIV, hepatitis and other viral, bacterial and fungal agents. The solution may also be administered to warm blooded animals, including humans, by intravenous injection or other modes for similar purposes. If desired, neutralizing agents, such as antioxidants, may be administered in correlation with the solution.

Description

~ W O 96/02271 ' ~ ~ 2 ~ q ~ 0 8 2 PC~RN S94/08280 ElEK~rRlCALLY RrYDROLYZED SALnN_S AS DN VIVO ~r~-v~r~nPc ROR Ille TR_AT,~n3Nr OF C~v m ~'r~rATHY ANnD ~rULllPLE SCIIEROSIS

BACKGROUND OF THE INVENTION
This application is a cnnt;mlAtion-in-part of pending patent application Serial N.umber 07/527,321 filed ~ay 23, 1990.
This application relates to the in vitro and in vivo antimicrobial use of electrically hydrolyzed salines. More particularly, this invention relates to the in vitro treatment of pathogen cnntAmin~tpd fluids and the in vivo treatment of microbial (including viral) infections or conditions in warm blooded animals.
The use of ozone (03) for the treatment of viral infections has been do~ tP~ for over thirty years.
Typical publications illustrating antiviral activity include Wehrli, ~ransactions of the VI Congress of the ~uropean Society of Haematology, 1:318 (1957); Wolff, vjm Neidelberg 2. Auflage (1982); Rilling, vjm ~eidelberg, 2 Auflage (1986); Mattasi et al., Medical Applications of Ozone, ed. LaRaus J. Norwalk, TntPrn~t;nn~l Ozone Association, pp. 134-137 (1985);
Konrad, ~edical Applications of Ozone, ed. LaRaus J.
Norwalk, International Ozone Association, pp. 140-146 (1985) and Jacobs, Ozonachrichten, 1-5 (1986).
Stephens, et al, Science, 231:589-594 (1986) reports the use of ozone in the treatment of e~uine infectious anemia, a viral infection analogous to HIV in horses.
Chlorine, in the form of chlorinated lime was used successfully as early as 1846 by Semmelweiss to prevent and fight puerperal fever. By 1911 the United States purified as much as 800,000,000 gallons of water through the chlorination process. Wide use of chlorine as a 0.05% sodium hypochlorite solution ~Dakins Solution) for open and ;n~ectP~ wounds began in 1915. Dakins Solution was a standard product up to 1963 listed in the British Pharmacopeia. = .
Both ozone and chlorine have demonstrated in~vitro anti-XIV activity as shown by Carpenaale, Antiviral W096/0227~ 95082 PCT/US94108280 Research, 16:281-292, ~1991) and Martin et al., ~.
In~ect. Dis., 152:400-403 (19~5).
As reported by Wilk et al., International Congress on Technology and Techno70gy ~xchange, First Euro-American Sympo5ium, Paris, France (1992) and Science, Total ~nvironment, 63 :191-197 (1987), certain combinations of ozone and chlorine have significantly greater activity than either used separately against a variety of bacteria including Sta~hylococrllc a~rell~ and Pseudn~rn~R aeruqinosa. ~an~ hic~nq was also reported to be effect:ively killed by a combination of ozone and chlorine.
In warm-blooded animals, there is a natural defense mechanism which produce6 in vivo naturally occurring free radicals in order to respond to antigens or other infectious pathogens.
Phagocytic cells (neutrophils, monocytes, eosinophils, macrophages), and large granular lymphocytes (collecti~ely called "killer cells") give off superoxide in what is called the "respiratory burst," which has an ~mtimicrobial action and, if not properly controlled, can also cause tissue damage. The superoxide radical itself may not be directly responsible for the microbicidal action. Rather, this activity and any resultant tissue damaye may be attributed to superoxide derivatives such as hydrogen peroxide, hydroxyl raaical and possibly, singlet oxygen.
Polymorphonuclear neutrophils and macrophages not only give off superoxide, leading to the production of hydrogen peroxide and hydroxyl free radicals, but also generate hypohalous acids and N-chloroamines as one of their mechanisms which also destroy bacteria. These leukocytes consume oxygen, which is transformed by membranous reduced nicotinamide adenine dinucleotide phosphate (NADPH) oxidase to superoxide.
The "respiratory burst" is observed as a dramatic increase in the consumption of oxygen and the activation , . .. , .. . . . ~

~ WO96/0227~ 5 a 8 2 PCT~S94/08280 ~ 3 of a membrane-associated NADPH oxidase. This oxidase reduces molecular oxygen to superoxide anions, which in turn dismutates to hydrogen peroxide. Superoxide and hydrogen peroxide can interact to give rise to the hydroxyl radical and possibly also to singlet oxygen.
The superoxide anion, hydrogen peroxide, hydroxide radicals and si~glet oxygen, all possess antimicrobial activity and are ~uite unstable. The respiratory burst cnnt;n~lF,c during phagocytosis by polymorphonuclear leukocytes until engulfment is complete. The respiratory burst may also occur in leukocytes under various chemical ;nfln~nr~c in addition to phagocytosis.
The respiratory burst, although intimately connected with phagocytosis, is not an essential ac~, ~ im~nt to phagocytosis. Recent evidence suggests that free tissue macrophages and newly recruited monocytes, as distinguished from fixed tissue macrophages, can respond to lyl hnk;n~R and phagocytic stimuli by mounting a respiratory burst. The failure of fixed ti~sue macrophages, such as ~upffer cells, to produce active metabolites of oxygen may be important in protecting tissues from damage during the scavenger functions of the macrophage. Many soluble agents, inrln~;ng antigen/antibody complexes, C5a, ionophores and tumor promoters, can trigger the respiratory burst without phagocytosis. The respiratory burst can also be triggered by opsonized particles or surfaces when phagocytosis is frustrated by the use of a drug such as cytorhAl~c;n B. In addition to the reactive species of oxygen referred to above, i.e. superoxide anions, hydrogen peroxide, hydroxyl radicals, and singlet oxygen, there are a number of other potential micrnh;r;~l mPrh~n;r~c in macrophages, many of which are oxygen dependent. A major oxygen flrrPn~nt system is mediated by myeloperoxidase (MPO), which catalyzes oxidation of a number o~ substances to hydrogen peroxide. MPO is the oxidase of neutrophils, and the 2 1 ~ 5 0 8 2 WO96/02271 ~ PCT~S94/08280 green color of pus is due to its presence. A co~actor in the MPO system is the iodide ion from the thyroid ht ~, thyroxine or triiodothyronine. However, this microbicidal system sometimes also utilizes other halide ions such as bromide Dr chloride as cofactors in the place of iodide.
It is well ~tt~llm~ntea that two free~radicals of superoxide combine with hydrogen to form normal oxygen and hydlo~en peroxide~ This is known as the dismutation reaction with superoxide tlir~-lt~ce (SOD) acting as the catalyst. Unless hydrogen peroxide is denatured promptly with catala,ses:or peroxidases, there ls an interaction between superoxide and hydrogen peroxide leading to the production o~ the highly reactive hydroxyl radicals via pathways known as the Haber-Weiss or Fenton's reactions. Singlet oxygen is also generated by the removal o~ the unpaired electrons of the superoxide radical.
~eukocytes, in vivo, use the formation of ~u~el~ide, hydrogen percxide, hydroxide radicals, singlet oxygen and halogenated products such as hypochlorous acid to destroy bacteria, fungi and viruses and perhaps also tumor cells. Other oxygen-~Pp~n~t~nt antimicrobial systems, unrelated to MPO, are also believed to rely on the production of hydroyen peroxide, superoxide anion, the hydroxyl radical and/or singlet oxygen to do the micrQbial killing in vivo. Some of these systems are not well documented but it is known that when such systems shut down or operate ine~iciently, severe infections results.
There may be problems involved with over production or an excess o~ these radicals within the cells of the host. Hence, the body has provided means for mediating or neutralizing these products once they have performed their antimicrobial functions.
As previously mentioned SOD is effective in scavenging superoxide radicals (each ct~nt~;n;ng an ~ WO 96/02271 ' I ~ 2 1 9 5 ~ 8 2 PCT/US94/08280 . 5 unpaired electron) in a simultaneous oxidation-reduction reaction with l-ydlu~n called dismutation. Two ~u~uuLu~ide radicals combine with two hydrogen atoms to form hYdLU~11 peroxide and oxygen. ~ydrogen peroxide is reduced by the enzymes ~t=l=~, glutathione peroxidase and MPO into oxygen and water.
~ence, in a normal functioning host, such as in a human or other warm blooded animal, there is an intra vivo interaction and balance ~-int=;n~ between respiratory bursts brought on by the presence of an invading foreign substance such as bacteria, virus or fungi ~rc ~ by the formation of superoxide, hydr U~Ull peroxide, hydroxyl radicals, singlet oxygen, hypohalous acids, and hypochlorite ions [collectively referred to as free_ r~ ] with their accompanying antimicrobial actions and the ~o~;st;ng or neutralizing action of the enzymes SOD, MPO, glnt=th;~ne, glutathione peroxidase, catalase, ascorbic acid and its salts and perhaps others.
There are situations, both in vivo and in vitro, when there are not sufficient free radicals present to ~ l;sh their desired antimicrobial tasks. There are numerous bacterial, viral and fungal related syndromes and immunological disorders ~herein it would be bPn~f; ~ to have free radicals such as ozone and active chlorine species available to cells and/or fluids for the short period required ior their antimicrobial action followed, if necessary, by mediation and/or neutr~l;7=t;~n of the free radicals. Examples of such syndromes and/or immunological ~;~or~nS for which either in vitro or in vivo treatment could be beneficial are Epstein-Barr virus, hepatitis A, B and C, rhinovirus, rubeola, rubella, parvovirus, papilloma ~ virus, ;nflll~n7~ and parainfluenza viruses, enteroviruses; ~erpes simplex viruses; Varicella-zoster viruses, Adenoviruses, respiratory syncytial viruses, alphaviruses, flaviviruses, retroviruses ~including AIDS

WO96/02271 ~ IJ ~ 21 9 5 0~ 2 PCT~S94108280 and AIDS related syndromes), bacteremia, septicemia, fungal infections, parasitic infections ~nematodes, trematodes, protozoal [e.g., Cryptosporldium]
h~lm;nth;c), mycobacterial infections, bacterial Gram positive and Gram negative superficial and systemic infections and other viral, bacterial and/or fungal associated ~ A~S. Many of these are diseases which are affected by a slow, latent or temperate organism, (i.e. virus, bacterium or fungus) which may have long incubation periods ancl, in some cases, have a low ratio of reported cases to infections. Many of these are also diseases for which there i6 no known cure and usually slowly progress until they, or a concurrent ~ Lullistic infection, results in the death of the host.
An infected host or patient may be treated by a variety of regimens which may alleviate the symptoms for a time. ~owever, the immune system eventually is weakened to the point that it can no longer adec~uately contend with the invading or autoimmune related infections and the natural biocidal action in the cells ceases to function prcperly.
There are also situations where fluids can be beneficially treated in vitro, to purify, decontaminate, or otherwise render such fluid acceptable for administration to a warm-blooded host. For e~ample, the blood supply taken from donors at blood banks has been found on occasion to be ~nt~ml nAtPd by the ~IV ~irus and other organisms such as hepatitis A, B and C
viruses, CMV (cytomegalovirus), and bacteria (such as Yersinia). Any treatment of whole blood, plasma or cell isolates to render them benign from infectious organisms without destroying the therapeutic characteristics of such fluids would be very b~n~f;~;Al.
In pending patent application =Serial Number 07/527,321 it is shown that an electrolyzed saline solution, properly made a~d administered in vivo, is ~WO96/02271 ' ~' \ 2~950~2 PCT/US94/08280 effective in the t~atm~nt of various infections brought on by invading antigens and particularly viral infections resulting in carfl;~ dthy, multiple sclerosis and AIDS. In that application, a restriction requirement was issued such that the issued claims in that application are drawn to the treatment of cardiomyopathy and multiple sclerosis. The present invention is directed to embodiments not the subject of the claims allowed in the original application and data developed in support thereof.

0~3JECTS AND SUMMA~Y OF T~E INVENTION
It is an object of the present invention to provide a method of treating microbe related infections wherein an electrolyzed saline solution containing regulated amounts of ozone and active chlorine species is injected into the body of a warm blooded animal which mimics or enhances the naturally occurring free radicals produced by respiratory bursts in the cells in responding to such infections.
It is also an object of this invention to provide a method of treating microbe related infections by injecting an electrolyzed saline solution cnnt~;n;ng regulated amounts of ozone and active chlorine species into the bloodstream of a warm blooded animal along with the administration of mnfl~t;ng or neutralizing agents which enables the body to utilize such chemicals as microbiocides in the same manner as it does during i~
vivo respiratory bursts.
A still further object of this invention is to provide a method of trea~ing antigenic related infections by the co~flm;n;~tration of electrolyzed saline solutions ~nnt~;n;ng free radicals and colchicine - into the bloodstream of a warm blooded animal along with the administration of moderating agents to enhance the ability of the body to utilize the free radicals as microbiocides.

C~ ~ 2~95082 W096/02271 ~ PCT~S94/08280 Yet another object of this invention i8 to provide a method of treating microbial infections by the administration of a precisely regulated electrolyzed saline.
A yet different object of this lnvention is to provlde a method for the in vitro derontAm;n~tion or treatment of mlcrobially cnntAm;nAted solutlon by an electrolyzed saline ~nntAining amounts of ozone and active chlorine species.
Another object of thls invention is to provide an electrolyzed saline ~nntAin;ng regulated amounts of ozone and active chlorine specles in ~nnc~ntrations sufficient to bring about the desired disinfection, antimicroblal or dernntAminAtion properties when utillzed for the deslred in vitro or in vivo purposes.
These and other objects may ~e arC~ hP~ by means of first preparlng a dllute sallne solution, subjecting this solution to electrical hydrolysis wlth adequate voltage, amperage and time to produce an electrolyzed solution ~nntAining ozone and actlve chlorine specles ln designated concentrations and also ~nntA;n;ng other products of the electrolysis reaction including members elected from the group consisting of hydrogen, sodium and hydroxide ions; The interaction of the electrolysis products results in a solution cnntA;r;ng bioactive atoms, radicals or ions selected from the group conslsting of chlorlne, ozone, hydroxlde, ~ hypochlorous acld, hypochlorlte, peroxlde, oxygen and perhaps others along with correspondlng amounts of molecular hydrogen and sodium and hydroyen lons.
Preferably the finished solution wlll have an ozone ~nnc~ntration of about 5 to 100 mg/liter and an active chlorine species concentration of between about 5 and 30D ppm. By active chlorine species is meant the total chlorine concentration attributable to chlorine co~tent detectable by a chlorine ion selective electrode and will be selected from the group Consisting of chlorine, 1 9 5 ~ 8 2 W O 96102271 ~ PC~I~US94/08280 hypochlorous acid and hypochlorite ions or moieties.
The pX of the solution is preferably between about 7.2 and 7.6 and, when used for intravenous administration, most preferably between about 7.35 and 7.45 which is the pH range of human blood. Preferably the ozone content will be between about 5 to 30 mg/L and the active chlorine species content will be between about 10 and 100 ppm. Most preferably, the ozone content will be between about 9 to 15 mg/L and the active chlorine species content will be between about 10 and 80 ppm.
The injecting of effective amounts of the regulated electrolyzed solution intravenously into a warm blooded animal affected by an infectious agent results in a microbicidal action which mimics or QnhAncQA action of the free radicals produced in vivo as a result of respiratory bursts.
If nPrQqsAry, the regulated electrolyzed saline solution may be injected along with the administration of moderating and/or nQntrAli7;ng amounts of Antin~;flAnt~ or rQflnn;ng agents such as catalase, superoxide dismutase, MP0 or other suitable peroxidase, glutathione, gllltAth;one peroxidase, ascorbic acid or other suitablc agents. The moderating antioxidants and/or neutralizing agents may be administered just prior to, concurrent with or shortly following the administration of the electrolyzed saline solution.
Also, the Art;n~iflAntq or neutralizing agents may be administered either orally, intravenously or parQntQrAlly. Additionally, the micrnh;~;flAl effects of the.electrolyzed solution may be enhanced by the cnAflm;n;~tration of effective amounts of colchicine and perhaps other QnhAnc;ng agents. Xowever, it is not always necessary, or even desirable, to administer moderating agents due to the fact that the active ingredie~ts of the electrolyzed saline dissipate rapidly into innocuous products and the dosage administered is ~ 2 1 9 5 0 8 2 WO96/0227~ ~ PCTNS94/08280 ~ufficiently regulated to prevent unwarranted side effects and/or damage to tissues in the host.
When used for the in vitro treatment of fluids for antimicrobial purposes the active ayents are neutralized or converted into inert products in a time frame without harmful exposure to r~l 1 nl ar or organ systems, alleviating the need for the use of neutralizing agents.

DETAIhED DESCRIPTION OF THE INVENTION
The products resulting from the electrolysis of saline solutions has long been known as has the fact that such solutions are in vitro microbicides for hard surfaces. Themy, U.S. Patents 4, 236,992 and g,316,787 are drawn to a novel electrode, method and apparatus for electrolyzing dilute saline solutions to produce effective amounts of disinfecting agents such as chlorine, ozone and hydroxide ions. One apparatus for producing electrolyzed saline solutions was previously available under the tradename Ster-O-hizer. ~aboratory reports and other data available from testing of electrolyzed saline solutions from various Ster-O-Lizer models have shown that it is effective in keeping water free of pathogenic organisms. Tests conducted in vitro further show that certain microorganisms, inclusive of ~sen~nm~n~f ae~u~;nnsal Escherirhi~ coli, St~nhylococcus , cAn~;~a alhinall~, and S~lmnn~lla tY~hi, are non-infectious after e~osure to electrolyzed saline solutions. However, it has not heretoforezbeen shown that physiological fluids trea~ed by electrolyzed saline containing finite amounts of ozone and active chlorine species to destroy microbes is safe for administration to warm blooded animals.
Although it is krown that electrolyzed saline solutions possess in vitro microbicidal activity it has long been thought that cnmpnn~ntq ir. the electrolyzed solution, such as ozone and chlorine, are to ic to warm blooded animals and should not be utilized for in vivo .. . . . . _ _ . _ . . . ... . _ . _ _ _ _ .

t~
~ wos6lo~27l - 2 I q5~82 PcrluS94108280 purposes. It has now been found, however, that saline solutions, which have been subjected to electrolysis to produce finite amounts of ozone and active chlorine products, can be injected into the vascular system to create a reaction to assist in the removal, passivation or destruction of a toxin. When desired, to mimic or enhance the physiological action in immuno-logical/cellular "respiratory bursts" to the generated microbicide, one or more of several modulating chemicals may be added to the complete treatment. These r~ ting chemicals are administered before, concurrent with or af ter the electrolyzed saline and may be administered intravenously, parenterally or, in some cases, orally.
In rop~n~;ng application Serial No. 07/527,321 filed May 23, 1990, it was considered that 100-300 ppm of rhl Qr; rl.o was the desired, nn~nt along with other reaction products of the electrolysis. At the time that application was filed the inventor did not have in his possession such equipment as could accurately determine ozone or active rl~l nr; nP species content . The term "chloride" as used in that application meant the active chlorine content of the solution and not the chloride ion per se. The use of superior electrodes for the electrolysis reaction and more sensitive detection or analytical er~uipment has shown that the electrolyzed solutions can have an ozone content which can vary between about 5 to 100 mg/liter and an active chlorine species conc~ntr~t;~n of between about 5 and 300 ppm.
The pH of the solution is preferably between about 7.2 and 7 . 6 and, when used for intravenous administration, most preferably between about 7.35 and 7.45, which is the normal pH range of blood. PrPf~r~hly the ozone content will be between about 5 to 3 0 mg/L and the active chlorine species content will be between about 10 and 100 ppm. Most preferably, the ozone content will be between about 9 to 15 mg/L and the active chlorine .

~ r,Lf~ 1 9 5 0 8 2 WO96/02271 '~ ' PCT~S94/08280 species will be present in amounts ranging between about lO and 80 ppm. While the chloride content as stated in the copending application is within the ranges considered as operative, if it had been accurately been measured by a chlorine selective electrode, it would most likely have beeI1 lower and comparable with the solution ~ljfi~ in ~xample I which follows.
When the electrolyzed saline is injected into the vascular system of a warm-blooded animal the active agents are tL~ puLLed rapidly throughout the system and pass intracellularly into cells affected by invading microorganisms. The rrmp~nrnts of the solution pass readily through cell walls and function in the manner described above for free radicals. Chlorine is thought to be present primarily as free=chlorine or as a hypochlorite ion. However, the primary microbicidal action of chlorine and its compounds comes through the formation of hypochlorous acid. This acid is formed upon the crm~;ning of free chlorine and water.
Hypochlorites undergo hydrolysis with the formation of hypochlorsus acid. The hypochlorous acid then decomposes to form hydrochloric acid and nascent oxygen.
Nascent oxygen is a strong oxidizing agent having microbicidal action. Chlorine also interacts directly with intracellular substances as a microbicide. The hypochlorite ion is also microbicidal. Sodium hypochlorite has long been used as an antiseptic, disinfectant and sterilant. It has found use in dilute form, about 0.5~ concentration, in surgery and in dissolving and deodorizing ne5rotic tissue. It has also been used to irrigate ragged or dirty wounds and as an antiseptic in Certain peritoneal dialysis sy6tems.
~or purposes of this invention, the term active chlorine agent or species, shall mean any active form of chlorine resulting from the subjecting of a saline solution to electrolysis which can be measured by a chlorine selective electrode. These species will be ~ 2 1 9~08~
WOg6/0227l 13 PCT~S94/08280 . !
primarily free chlorine, hypochlorous acid and the hypochlorite ion_ The intracellular actions of the hydroxide ion have previously been described.
In certain situations where it may be desired to utilize higher nnnrPntrations of chlorine and oxygen active agents produced from the electrolysis of a saline solution to accomplish their microbiocidal purposes, it may be desirable to concurrently administer in vivo or subject a solution in vitro to r-- l~t;ng or moderating chemicals. As used herein the terms "moderating", ~ At;ng~ and ~neutralizing" agents may be used interchangeably.
The modulating chemicals are enzymes or reducing agents which interact with and reduce the active microbicidal agents to innocuous compounds. The enzymes are inclusive of, but not limited to, the superoxide dismutases ~SOD), catalase and glutathione peroxidase.
As previously stated, they function to remove the superoxides, peroxides and hydroxides that are formed in the cells. Otherwise oxygen toxicity results. These oxygen radicals are converted to 11YdLU~ peroxide by Cu/Zn activated superoxide dismutases (SOD) in the cells. In a properly functioning system the hydrogen peroxide ~is then converted to oxygen and water by a catalase. I~ the hydrogen peroxide and the superoxide radical are allowed to combine, the more deadly hydroxide radical is formed.
The primary activated SOD in warm-blooded animals is Cu, Zn-superoxide ~;, t~qP This metalloenzyme undergoes a reduction-oxidation cycle with the superoxide radical with the net result of dismutation of the superoxide radical to hydrogen peroxide and oxygen.
- The metals required for this activity are copper and zinc. Other forms, i.e. Mn-SOD and ~e-SOD, are also known but occur primarily in bacteria and cellular miton~nn~r;~. Without the presence of copper, the SOD

' ! ~ 2 ~ 9 5 0 8 2 W096/02271 = PCTtUS94tO8280 enzyme is virtually inactive in the animal. The activity of the Cu, Zn-SOD enzyme can be suppressed by the too rapid accumulation of hydLOy~ll peroxide.
Therefore, it i8 es~ential that other enzymes which deplete hydrogen peroxide be functional within the cell to m~;ntA;n SOD activity.
Catalase is a large molecular weight enzyme that ~nntA;ns four heme groups per molecule. Catalase is the primary enzyme necesEary for the breakdown of hydrogen peroxide in the cell to oxygen and water and is found in all cells of the body that utilize oxygen.
Glutathione peroxidase (GS~-Px) has a selenium ~PrPn~Pnt form which contains four moles of selenium per mole of=the enzyme. The oxidative role of this enzyme is similar to catalase in that it converts hydrogen peroxide to water and oxygen. ~thenever catalase or glutath;~nP peroxidase activity is impaired there can be a toxic build-up of peroxides. This, in turn7 can lead to a build-up of the hydroxide radical. The non-selenium glutathione peroxidase (GSH-P) plays a role in controlling lipid peroxidation. The primary form of glutathione peroxidase within the red blood cell is the selenium dependent form.
Glutathione and ascorbic acid are both reducing agents involved in biological systems of oxidation.
Gl~ltAth;onP is a tripeptide of cysteine, glutamic acid and glycine. It is most often isolated from animal ti8sues in the form of its cuprous salt. The oxidized form is readily reduced by tissues to the sulfhydryl form. The latter form, in the presence of traces of copper gives up its hydrogen to molecular oxygen, becoming oxidized in tur~_ In other words, in the oxidized form it acts in the cells as a hydrogen acceptor and in the reduced form, as a hydrogen donor.
The n~;~;7ed form is reduced by glutAth~nP Teductase.
Glutathione appears to be a ubiquitous reducing agent involved in many intracellular redox reactions.

I q 5 0 8 2 WO96/02271 PCT~594/08280 Ascorbic acid (Vitamin C) functions in a number of biorhPm;rAl rPAct;nn~, mostly involving n~i~Atinn. It is a reducing agent associated with the regeneration and maintenance of the cnrnPctive tissue. Vitamin C has been shown to be an effective stimulator to the immune system. As a strong reducing agent it is used as an Antjn~;~Ant to nPntrAl;7e the nY;~;7;ng chemicals in the electrolyzed saline solution. Ascorbic acid is also a coenzyme for the oxidation of glutathione. Ascorbic acid is readlly absorbed from the intestine. It is present in the plasma and is ubicluitously distributed in the cells of the body. ~ence, it may be orally administered. ~owever, intramuscular or intravenous injections of either ascorbic acid or sodium or calcium ascorbate may also be utilized when faster action is preferred Timely administration of one or more of these modulating agents prevents the toxic effects where and when excess amounts of n~;~;7;rg agents are present following administration of the electrolyzed saline solution.
The sterile saline solution that is to be subjected to treatment in the electrolysis unit has an initial rnnrPntrAtion of about 0.25 to 1.0~ NaCl which is about one-fourth to full strength of normal or isotonic saline solution. Aecording to Ta~er's Cyclopedic Medical Dictionary, E.A. Davis, Co. 1985 Ed., an "isotonic saline" is defined as a 0.16 M NaCl solution or one rnntA;n;ng approximately 0.95~ NaCl; a "physiological ~ salt solution" is defined as a sterile solution r~ntA;n;ng 0.85~ NaCl and is cnn~;~Pred isotonic to body fluids and a "normal saline solution" a 0.9~ NaCl solution which is considered isotonic to the body.
- There~ore, for purposes of this disclosure, the term "isotonic", "normal saline", "balanced saline" or ~physiological fluid" is considered to be a saline solution cnntA;n;ng between about 0.85 and 0.95~ NaCl.

; ' ~ fi ~ ; 2 1 9 50 8 2 WO 96/02271 PCT/US9~/08280 The saline solution may be subjected to electrolysis at cr,nr~ntrations between about 0.15 and 1.0~. Preferably the solution will be diluted with sterile distilled water to the desired rnnrPntration, preferably between about 0.15 to 0.35~, and subjected to electrolysis at sufficient voltage, amperage and time to produce an electrolyzed solution. The electrolysis reaction is carried out at ambient temperatures. Obviously, the voltage and amperage :to be used and the time of electrolysis is subject to many variables, i.e. the size and composition of the electrodes, the volume and/or concentration of saline being electrolyzed. For large electrodes or saline volumes or higher rnncrntrations cf saline solutions the voltage, amperage or time may be higher and/or longer. It is the generation o~ the desired concentration of ozone and active chlorine species which is important. According to Faraday's laws o~ electrolysis, the amount of chemical change produced by a current is proportional to the quantity of electricity passed. Also, the amounts of different substances liberated by a yiven quantity of electricity are proportional to the chemical equivalent weights of those substances. Therefore, to generate an electrolyzed saline having the desired concentrations of 2~ ozone and active rhl nr; n~ species from saline solutions having a saline concentration of less than about 1.0~, voltage, ampera~ne and time parameters appropriate to the electrodes and solution are required to produce an electrolyzed solution containing between about 5 to 100 mg/L of ozone and a free:chlorine'content of between about 5 to 300 ppm. For in vitro use these solutions can be utilized without further modification or they can be adjusted as desired with saline or other solutions.
Prior to i~ vivo use, this solution may be adjusted or bAl~nrP~ to an isotonic saline cnnr~ntr~t; nn with sufficient hypertonic saline, e.g. ~ hypertonic saline solution.

~ ' 21 95Q82 WO96/02271 PCT~S94/08280 Generally speaking, such microbiocidal solutions will have an ozone content of=between about 5 and lO0 mg/L and an active chlorine species content of between about 5 and 300 ppm. Preferably the ozone content will be between about 5 to 30 mg/L and the active chlorine species content will be between about lO and lO0 ppm.
Most preferably the ozone content will range between about 9 to 15 mg/L and the active species content will be between about lO and 80 ppm. An effective amount of this b~l~nr~ microbiocidal saline solution is then administered by appropriate modes, e.g. intravenously, orally, vaginally or rectally and may vary greatly ~rr~r~;ng to the mode of administration, condition being treated, the size of the warm-blooded animal, etc. For human beings to be injected intravenously the dosage of this b~l~nr~ electrolyzed solution may vary from between about 0.25 to 4 ml/kg/day body weight with ranges of 0.5 to 3.0 ml/kg/day being preferred. The doses can be divided into smaller doses and administered two or more times per day or may be administered in a single dose. Also, the regimen may vary Arrr,r~;ng to the indication being treated. For ~IV treatments, for example, it may be adv~nt~geo~1~ to administer the microbiocidal solution for several days followed by a rest period and then repeating the cycle for as long as necessary or as indicated by the test results, e.g. of Western Blot, T cell subsets, chem profile (SMAC 20), CBC, p24 antigen and ~I~ mRNA quantitation. A typical regimen might be five days of treatment followed by two days rest and the cycle repeated for two months.
Depe~ding on clinical status or laboratory tests, this regimen may be reduced to, e.g. three days of treatment per week for six weeks. These regimens are exemplary ~ only and are not meant to be limiting as any number or variation might be dictated according to circumstances.

'J 1~ 2 t ~ 5 ~ 8 2 WO96/0227l ~cT~ss4lo828n When utilized, the amount of moderating agent to be administered will depend somewhat upon the method and time of administration. Dosages of moderating agents administered orally will be somewhat higher than if injected intrave~ously. Also, if the m~nlAtlng agent is administered before injection of the electrolyzed saline, there must be sufficient time allowed for the modulating agent to be absorbed and carried into the bloodstream to the site where it can reduce the ., ;n;ng free radicals from the electrolyzed saline after the solution has accomplished its microbicidal function.
The dosage of modulating: agent or agents to administer is not ne~essarily stoichiometric with the free radicals of the electrolyzed saline and may initially have to be det~rm;n~d empirically. There should be sufficient modulating agent in the system to prevent the free radical comp~n~nt~ of the electrolyzed saline from causing irreparable tissue damage. For ~hat reason, it may be beneficlal to administer modulating agents such as the superoxide dismutase, catalase, L-glut~th;~n~, glutathione peroxidase, MP0 and ascorbic acid orally for a period of time prior to the injection of thb electrolyzed saline to provide the availability of adequate amounts of these agents in the cells at the time the electrolyzed saline is injected. However, it is equally important that the free radical components be available to perform their desired microbicidal function before being suppressed or deactivated by the modulating agents. As a generalization only, oral dosages of superoxide ~;cmnt~e varying from about 5,000 to 60,000 units per day may be administered. Catalase, MPO and glutathione peroxidase dosages may vary between about 10,000 to 120,000 units per day. Glutathione may be administered in amounts ranging from about 10 to 120 mg per day. Ascorbic acid or~its sodium or calcium salts may be administered over a wide range of about 50 to PCTIVS 94/082~30 2 1 9 50 a 2 IPEA/US I 6 SEP~

20,000 mg per day. Preferably, the ascorbic acid is administered intravenously shortly after the injection of the electrolyzed saline to make sure that no unreacted oxidative components of the saline are reduced and/or neutralized. Based on the above guidelines, one skilled in the art can readily determine what i9 an effective amount of modulating agent.
By injecting the electrolyzed saline intravenously and administering the modulating agent in the manner describcd above, there is created in the cells the same elements as are created ~aturally in the body to fight infectïons.~ In other~ words, the electrolyzed saline solution mimics the action of the free radicals produced during the respiratory burst from the macrophages and monocytes. Similarly, the moduIating agents mimic the action of the enzymes produced by macrophages and monocytes as reducing agents to neutralize the oxidants.
This results in a straight forward attack on the microorganisms within the host cell by the injected chemicals.
The ~ m;n;~tration of co~chicine with the electrolyzed saline may also prove beneficial as an adjunct in preventing replication of the invading microorganisms. Colchicine, [N-(5,6,7,9-Tetrahydro-1,2,3,10-tetramethoxy-9-oxobenzo[a]heptalen-7-yl)acetamide] C22H2sNO6, is a major alkaloid of Colchicum autn~n~le. It is an anti-inflammatory agent used primarily as a gout suppressant and in the treatment of Familial Mediterranean Fever, scleroderma and psoriasis.
It functions by inhibiting the migration of granulocytes into an inflamed area, reducing the release of lactic acid and proinflammatory enzymes that occur during phagocytosis, thereby breaking the cycle that lead to the infl~ tory response. The neutrophils and leukocytes produce glycoproteins which bind cells and may be a cause of acute inflammation. Colchicine prevents either the production by or release from ~ ED SHEET

~ '3~'~ 2 1 9 ~ 0 ~ 2 W096/02271 PCT~S94/08280 leukocytes of glycoproteins. It also produces a temporary leukopenia that is soon replaced by a leukocytosis, sometimes due to a striking increase in the number of basophilic granulocytes and may have the same action in increasing lymphocyte prn~n~t; nn . The site of~action is apparently directly on the bone marrow. Moreover, colchicine is an antipyretic, lowering body temperature. It also increases the sensitivity of~ the body to CNS depressants, depresses the respiratory center, ~n~i~nr~ the response to sympathomimetic agents, constricts blood vessels and induces hypertension by central vasomotor stimulation.
It is well tnl~ri~t~ in moderate dosage and, although not a cort;rnsteroid, acts much like cortisone in suppressing the immune system without the attendant high risk and side effects of corticosteroids. In low dosages, colchicine may work as an immune system st; llAnt helping to relieve an overworked immunosystem.
While not known for a certainty, it is believed that colchicine functions in the present invention primarily in preventing the release of glycoproteins which bind cells and breaks the cycle of ;nfl; tory response. Other secondary effects may be that it functions as an antimitotic, antiviral agent, as a mild immunosuppressant and produces a leukocytosis by stimulating the bone marrow.
To understand why the use of colchicine to prevent the release of glycoproteins may be an important adjunct to the present invention, the following information regardi~g ~IV and AIDS is brn~f;rii~l. This extreme viral infection is popularly referred to as AIDS
(acquired immune deficiency synarome). However, it is more d~ Liately an HIV Ihuman ;mmllnn~;r;~ncy virus) infection leading to AIDS. This disease proceeds : through various stages from HIV exposure to HIV
infection to devrlopm~nt of AIDS. These stages are classified by Redfield, et al. in an article entitled - ~ ; 2 1 9 5 ~ 82 W 0 96/02271 ~ t ~ PC~rN S94/08280 "The Walter Reed Staging Classification for HTLV-III/LAV
Infection" pnhliRh~ in the New England Journal of Medicine, Volume 314, Page 131, January, 1986, and are referred to as the Walter Reed (WR) classification.
They are thus referred to as WR0 through WR6. The WR0 classification means there has been exposure to the HIV
virus although there are no symptomatic indications.
WR1 means there is a positive HIV antibody and/or virus det~rm;r~t;nn but no other symptoms. A WR2 classification is characterized by chronic lymrh~nnp~thy or swollen lymph nodes in addition to positive HIV antibody and/or virus ~t~rm;n~t;on A WR3 classification is reached when the T4-cell count drops and remains below 400 cells per cubic millimeter of blood. The normal T4-cell count is about 800. There may or may not be chronic lyrrhA~nopathy in WR3 through WR6 classifications but the T4-cell count stays below 400. A patient moves to the WR4 stage after partial sub-clinical ~asymptomatic) defects are ~ound in delayed hypersensitivity, i.e. the ability to react to skin tests that are a barometer of immune functioning. The line into WR5 is crossed when the patient c ~let~ly fails to respond to the skin test or when thrush (a fungal disease of the mouth) develops. Lym7h~rnpathy and abnormalities of the T4-cell and skin tests must persist for at least three months to serve as valid criteria. Patients enter into the WR6 stage and are said to have AIDS when uuuoLLullistic infections, which occur because the immune system has broken down, develop elsewhere in the body. Typical opportunistic infections include Kaposi's sarcoma, cryptococcal meningitis, cytomegalovirus (causing hl ;n~ln~ s) and classic Pneumocystis carinii ~r~ ; A, The HIV virus is a retrovirus which does not per se cause death of its host. However, the presence of the HIV virus contributes to the decline of T4-cells in the body. The T4 lymphocytes, or T4-cells, recognize WO96/0227~ 9 5 0 8 2 PCT~Sg4/08280 foreign antigens or infected cells. Upon recognition, the T4-cells help activate another set of white blood cells called B-lymphoc:ytes. These B-cells then multiply and produce specific antibodies that bind to the infected cells and other organisms cnnt~;n;ng the antigen. The binding of the ~ntihofl;es to the antigen r~nt~;n;ng cells OI oryanisms inactivates and/or destroys those cells or organisms.
The T4-cells have other functions as well. They orchestrate cell-r~ tecl and humoral immunity by killing infected cells or infecting microbes with antibodies and cytotoxic cells suc~ as T8 lymphocytes and white cells known as killer cells. The T4-cells also influence mobile scavenger cells known as monocytes and macrocytes. These scavengers engulf infected cells and foreign particles and secrete a variety of cytokines. The cytokines are small but highly potent proteins that modulate the activity of many cell types, ;nr~ ing T and B cells. The T4-cells also secrete cytokines on their own which stimulate the proliferation of T and B cells in the body.
From the above, it is apparent that the loss of T4-cells can seriously ilmpair the body's ability to fight microbe-caused diseases and viral infections in particular. The eradication of these invading microbes rec~uires a highly-orchestrated cell-r~~1~t~ response.
Without T4-cells this immune response does not function satisfactorily.
According to Redfield~et al., dHIV Infection: The Clinical Picture," Scientific American, 259:90, October, 1988, there is a balance of power between the HIV virus and the immune system arranged hy the T4-cells. From the WR0 (exposure stage~ to the WR1 stage the HIV virus increases rapidly at which point the immune system begins to respond. By the time the WR2 stage is reached the viable virus in t:he body has dropped dramatically with the concomitant rise in scavengers, macrophages, T-2 I q~O82 ~ WO96102271 ~ t ~ PCT~S94108280 ; ~ 23 cells, ~3-cells, ~nt;h~ c and other immune system -.-m~nn.-nt_, The immune system remains somewhat in control throughout the WR2 and into the WR3 stages although there is a gradual rise in HIV. However, by the time the WR4 stage is reached the HIV has begun to overwhelm the immune system and the T4-cells become EO
depleted that the balance of power switches, and, from that point on, the HIV replicates wildly, overwhelming the r,-~-;n;ng T4-cells and any vestiges of immune defense.
How the XIV virus infects and kills T4 cells raises many ~uestions leading to certain theories and/or conclusions. Infection begins as a protein, gpl20, on the viral envelope binds tightly to a protein known as the CD4 receptor on the cell surface. The virus then merges with the T4 cell and reverse transcribes its RNA
genome into double-stra~d DNA. The viral DNA becomes incorporated into the genetic material in the nucleus of the cell and directs the production of new viral RNA and viral proteins which combine to form new virus particles. These particles bud from the cell and infect other cells.
Under certain circumstances the HIV virus can multiply prodigiously in the helper T cells and kill them, suggesting that viral replication is the main cause of cell destruction. In particular, it has been found that HIV replication and cell deaths increase when infected helper T cells become activated, as they do when they take part in an immune response to other infections. Thus, the very immunological process that should defeat the XIV virus has the opposite effect of increasing the proliferation of the virus.
Purther investigation reveals an apparent paradox, i.e. HIV replication could be demonstrated in only a small fraction of T4 cells collected from HIV infected patients. The cells killed by replication alone might hamper the immune system somewhat, but that would not WO96/02271 ''~ 9 5 0 8 2 PCT~S94108280 cause the severe immune ~Pf; r; Pnry seen in AIDS.
However, another mechanism for T4 cell destruction, one which is compatible w:ith the present invention, may be P~plA;nP~ by the formation of syncytia or massive bodies consisting of many merged cells having multinuclei.
Syncytia develops after a single cell becomes infected with HIV and produces viral proteins, including gpl20, which is displayed on the surface of the infected cell.
Because gpl20 and the CD4 receptors of the T4 cells have a high affinity for each other, uninfected T4 cells can agglomerate and/or bind to the infected cell and merge with it. The resulting syncytium cannot function anZ
dies. The original infected cell is killed, but 80 are myriad uninfected T4 cells that could otherwise be used to attack and kill the HIV virus.
Furthermore, in a process that is unique to the HIV
infection, free viral gpl20 protein may circulate in the blood and the lymph system and bind to the CD4 receptors of uninfected helper T cells, making them susceptible to attack by the immune system. Regardless of how helper T cells are killed by HIV, the decline in number of cells leads to a more general decline in immune functioning leading through the six stages of the disease progression rPfPrrP~ to above.
It is believed that colchicine blocks the release of~glycoproteins, i.e. gpl20, which promote adhesion between the cells as described above. In the~
development o~ syncytia, the T4 cells are bound together to create megacells of in~ected _and uninfected leukocytes which cannot carry out their~immune function.
It is believed that the rrlrh;r;nP dissolves and/or prevents the glycoprotein bond. This action prevents the T4 cells from agglomerating and releases~ the unin~ected le~kocytes (T-cells~ to be active in an immune response and prevents their death and eventual depletion.

WO96/02271 f ~ ~9 5 0 ~ 2 PCT~S94/08280 There is also believed to be a synergistic effect in that the liberation of infected T4 cells from the glycoprotein also renders them more available, and hence susceptible, to the microbicidal action of the free radical type components of the electrolyzed saline solution. Moreover, colchicine is a mild immunost;mnlAnt which may slow the replication of the virus lying dormant inside T4 cells. This dormant virus is waiting for an outside infection to stimulate an immune response which will activate viral replication.
When used, the dosage of colchicine may vary between about l.0 to 3.0 mg, with about l.5 mg being considered optimal for adults. It is preferably administered intravenously just prior to or concurrent with the administration of the electrolyzed saline solution.
As noted above, colchicine is used as an adjunct to the administration of the electrolyzed saline. The most advantageous treatment of any disease is the use of the minimal amount of any active agent needed to accomplish the desired results. In many instances the use of colchicine may not be indicated or even desired.
If desired in order to conclude the treatment, about 500 to 5000 mgs, and preferably about lO00 to 4000 mgs of ascorbic acid, or its sodium or calcium salt, is administered about two to twenty minutes after the injection of the electrolyzed saline. This reducing agent neutralizes the rPm~;n;n~ unreacted active components of the electrolyzed saline.
While moderating agents and/or colchicine may supplement the administration of electrolyzed saline ~nnt~;n;ng quantitated amounts of ozone and active chlorine species, it is the quantitated saline and its uses to which this invention is particularly drawn.
There~ore, the administration of moderating agents and/or colchicine are corsidered optional.

WO96/02271 qS O 8 2 PCT~S94/08280 The following ~ ~lP~ are illustrative of the invention and its use. The electrolyzed saline solution used in Example X was obtained by subjecting about a 0.33~ (about one third physiologically normal) saline solution to electrolyE;is for about 5 to 15 minutes. The voltage between the electrodes was m~;nt~;nP~ in the range of about I0 to 20 volts at a current in the range of about 5 to 20 amps. The freshly prepared electrolyzed 8aline when b~l~nnr~ or normalized with sterile 5~ saline rnntA;nrd about 200 ppm of active chlorine species along with about 5 to 30 mg/L of ozone and corresponding amounts of molecular 1LYdr U~1L and 80dium and hydrogen ions. Precise measurements were not made.
Later solutions, i.e., all except in Example X, rnnt~;n;ng more precisely regulated amounts of ozone and active chlorine species were obtained using improved electrodes with closely contrQlled parameters of voltage, current, time and saline concentration. The following Example I delineates the preparation of a preferred electrolyzed saline for use in the present invention.
EXAMPLE I
To 300 ml of sterile distilled water was added 100 ml of sterile 0.9~ saline resulting in a 0.225~ saline solution. This solution was placed in a plastic chamber rnnt~;n;ng novel titanium and platinum electrodes. The=
0.225~ saline was th.en subjected to a current of 3 amperes at 20 volts ~DC) for a period of three minutes.
~ 17 ml portion of :this electrolyzed solution was aseptically diluted with 3 mls of a sterile 5~ saline resulting in a finished isotonic electrolyzed saline having an active ozone content of 12i2 mg/L and an active chlorine species content of 60i4 ppm at a pX of 7 4. The chlorine conc~ntr~tlnn was determined using an Orion chlorine ion s~elective electrode and the ozone concentration was measured by a potassium indigo ~ 21 q5Q82 W096/0227l ~ PCTt~S94tO8280 trisulfonate method acrnr~1n~ to the pLUOedUL~ of ~oigno et al. Nater Research, 5:449-456 (1981).

EXAMPLE II
The stability of the solution prepared in Example I was determined over a 24 hour period by making periodic ozone meabuL, s as described by ~oigno et al. in Example I. The results are listed in Table I as follows:

Table I
Time (hours) Ozone ~nnrrnt ration (mg/L) 0 12.35iO.8 1 ll.90iO.7 2 12.64iO.7 3 11.70iO.8 21 11.78~0.7 22 11.26~0.7 23 11.67~0.8 EXAMPLE III
The stability of electrolyzed solutions were further detrrm; nr~ over an r~trn~ period of time at 4~C. Two separate isotonic solutions (Solution A and Solution B prepared using separate electrodes) were measured for stability. Each solution was electrolyzed and rendered isotonic using the same procedure as in Example I. The solutions were m~rt~;nrd at a temperature of 4~C ~or a period of 200 hours and periodic mea~uL t~ were made to determine active chlorine species (C12) and ozone (03) rnntrntfi. The results are shown in Table II as follows:

2 1 .q ~ ~ 8 2 WO96/02271 PCT~S94/08280 TABLE II
Stability of C12 and 03 Over Time at 4~ C.
Cl2 ~3 Concentration Concentrati (ppm) (mg/Ml) Hours A B A

0 61.6 61.7 13.7 12.4 1 61.5 61.0 13.5 12.9 24 60.5 71.6 13.9 13.1 42 60.7 NT' 14.0 NT' 72 61.0 67.3 13.7 12.9 124 NT- 65.3 NT' 13.4 176 60.8 NT- 12.8 NT~
200 60.0 61.7 12.0 12.~/
NT' = Not Tested ~ =

It is evident from the above that the solu~ions are stable in maintaining rela~ively constant the concentrations of the actiYe chlorine species and ozone.
EXAMPLE IV
To show tha~ the electrolysis reaction car. be carried out effec-ively in salilie solutions UE) to~about 1~ in concentration, the electrslysis reaction was carried out at saline concentrations of 0.3, 0.6 and 0.9~ respectively. The active chlorine species (C13) ar.d ozone (03) contents were measured and are given in Tabsle III as follows: --~ 2 1 9 5 0 8 2 W O 96/02271 PC~ri~S94/08280 . .

~ Table III
Cl2 and O3 Content from Salines 5at Varying Concentrations Saline Con- Cl2 ~3 centration ~onrGrtration ~n~n~ration (%NaCl) (ppm) (mg/mL) 0.3 129 21.8 0.6 161 26.6 C.9 168 28.0 As can be seen, the active ingredients are well within the parameters required in the invention. The final active ingredient concentration can be adjusted by saline and/or water to provide a final active ingredient concentration as desired.
EXAMPLE V
The in vitro toxicity of the electrolyzed saline of Example I is illustrated by adding it to human lymphocytes at varying ozone and active chlorine agent concentrations and exposure intervals as shown in Table IV. Briefly, a 0.3% Trypan blue solution was prepared by combining 3 parts of a 1% Trypan blue solution ~1 gram Trypan blue powder placed in a 100 ml volumetric flask and dissolved in water to a volume of 100 mls anc then filtered prior to use) with 7 parts of RPMI 1640 media containing 10~o FBS. A 1 mL sample of lymphocytes (e.g. 10~ live cellsl was sedimented, the medium decanted, and the lymphocytes resuspended in an electrolyzed saline solution where,in the ozone and ~ active chlorine species concentrations were modified by ma]cing selected dilutions with normalized saline. The - lymphocytes were incubated for a selected time, then the lymphocytes were washed by sedimentation and resuspension in fresh medium An aliquot of l~mphocytes was then mixed with 0.3% Trypan blue solution and o~served microscopically. One hundred cells were ~.{~i .''. ~ 2 1 ~5082 W096/02271 PCT~S94/08280 screened and the number of cells excluding Trypan blue was deemed as the percentage of viable cells.

Tabl~ IV
Sample Dilution oi Expoeure Percent Blectrolyzed Period Via~ilityb Sali.ne a~min,) A 1:1 1.0 100 B 1:1 2.5 100 C 1:1 5.0 70 D 1:1 10.0 50 E 1:5 1.0 100 F 1:5 2.5 100 G 1:5 5.0 70 H 1:5 10.0 50 I1:].0 1.0 100 J1:~.0 2.5 100 K1:10 5.0 100 L1:10 10.0 80 M1:100 1.0 100 N1:100 2.5 100 O1:100 5.0 100 P1: 100 10 . O 100 Q1:1000 1.0 100 R1:1000 2.5 100 S1:1000 5.0 100 T1:1000 10.0 100 uc o 0 100 Vd C 0 100 a Parts electrolyzed saline r~nt~;nP~ in total parts saline solution, (1:1 = 100~ electrolyzed saline of Example 1).
b Percent viability is a relative percentage with the percent viable versus the control ad~usted to e~ual 100 percent.
c Titration control.
d Test control.

~ , j't~ 2 ~ ~5082 WO96/02271 PCT~S94108280 ~ 31 As do, ~P~ in Table IV, 100~ of cells exposed for 2.5 minutes to undiluted concentrations of electrolyzed saline were viable. At 5 and 10 minutes some toxicity to cells were noted at both 1:1 and 1:5 dilutions and at 1:10 minimal toxicity was noted at 10 minutes. For lesser times at the 1:10 dilution and at higher dilutions there was no toxicity noted up to 10 minutes.

EXAMPLE VI
The in vitro mutagenicity of the electrolyzed saline of Example I is illustrated by adding it to bacterial cells accordi~g to the ~A7mnnP7 7A reversc mutation assay (Ames Test). This test was conducted by an independent testing laboratory in accordance with ~SFDA Good Laboratory Practices RegnlAt;nnc [21 C.F.R.
Part 58].
The Ames tests employ several strains of S~7mnn~77~
typhimurium which have been selected based on their sensitivity to , tAt;nn. The Ames tests were performed by mixing the electrolyzed saline of Example I with the test organism in a soft agar solution that cnntA;nC only small amounts of histidine. The histidine permits the inoculated test organism to undergo a limited number of divisions, but is insufficient to permit normal growth.
The tester strains require histidine for growth, due to a mutation in the gene that controls production of histidine. If, however, the strain undergoes a reverse mutation (spnntAnpnllc or induced by the test substance or a positive control material) the organism no longer requires histidine to grow and can produce a visible cQlony or revertant. Only mutations to the test organism in the region of the histidine gene will cause the test organism to undergo a reverse mutation to an organism that then no longer requires histidine. The tester strains were selected to detect various types of ~ g ~ 2 1 9 5 0 8 2 W O 96/02271 PC~riUS94/08280 ~ntAg~nA. The tester strains employed were TA97A, TA98, TA100, TA102, and TA1535.
The conclusion oi- the in~p~n~nt laboratory as to the mutagenicity of the electrolyzed saline solution is that the solution "tested against the i-ive strains did not meet the criteria for a potential mutagen."

EXAMPLE VII
The in vivo toxicity of the electrolyzed saline of Example I is illustrated by injecting it into the tail vein of Harlan Sprague Dawley:ICR mice at varying rnnrGntrationS, i.e. 2, 4, 6 and 8 mL/kg body weight.
These tests were conducted by an independent testing laboratory in adherence with Good Laboratory Procedure reg~lat;nn~ (21 C.F.R. Part 58). The conclusion of these tests was that "the electrolyzed saline solution of Example I was non-toxic at a single intravenous dose of at least 8 mL/kg b.w." The dose of 8 mL/kg b.w. is four times what was given to five human pAt;~nts treated with electrolyzed saline solutions as will be described in Example XI.
EXAMPLE VIII
The in vitro activity of rl;n;~Al isolates (field isolates) of ~IV infected human lymphocytes was tested with the electrolyzed saline of Bxample I according to the method of Ho et al. N. Eng. J. ~ed. 321:1621-1625 (1989). The TCID (Tissue Culture Infectious Dose) per 106 lymphocytes [PBMC (Peripheral Blood ~nnnm~ Ar Cells)] was 5000 for each isolate. The p24 antigen was checked weekly for five weeks. The results at the end of five weeks are shown in Table V.

~ t~ 2 1 9 ~ 0 8 2 W096/OZ271 ;!~ PCTNS94/08280 Table V
~ 5 ExposureDetectable p24 HIV
TimeAnrigen (min)Isolate #1 Isolate # 2 1.0None None 2.5None None 5.0None None 10.0None None As shown in Table V, there was a complete killing of HIV, as evidenced by the absence of detectable p24 antigen, after only one minute exposure. This provides evidence of the in vitro antiviral effectiveness of the electrolyzed saline in the treating of infected blood cells, whole blood, or any other fluid.
EXAMPLE IX
To further demonstrate the in vitro activity of the electrolyzed saline of Example I against HIV infected lymphocytes, additional testing was completed using the HIV infected laboratory isolate HB-2. The TCID (Tissue Culture Infectious Dose) ranged from 108 to 10~ at an ~O~U1~ time ranging from 1-10 minutes at dilutions of 1:1, 1:5 and 1:10. The results are given in Table VI.

Table VI

Dilution Exposure HB-2 Tiss~e Cul~ure Infect-.ous ~ose(TCID) (min) 108 107 106 105 10 4 103 1o2 10 1: 1 1 . O O a O O O O O O O ,~
1:1 2.5 0 0 0 0 0 0 0 0 1:5 1.0 +b + o 0 0 0 0 0 ~.
1:5 2.5 + + 0 0 0 0 0 0 1:5 5.o + o 0 0 0 0 0 0 1:5 10.0 + 0 0 0 0 0 0 0 1:10 1.0 + + + + + +8~c 34 1:10 2.5 + + + + + +20~ 47 1:10 5.0 + + + + + +28~ 0 ~n 1:10 10 + + + + + + d 0 co a No~detection of p24 antigen in cultures after 5 weeks incubation.
b Greater than 380 pg/ml p24 antigen in cultures after 5 weeks incubation.
c Percent reduction of p24 antigen calculated as: sample well p24 antigen level divided by control well p24 antigen level minus 1 multiplied by 100.
.

. ~

~ j 2 1 ~'5~8~
Wo96102271 ' PCT~S94/08280 ~ 35 It is evident from these results that, at an end point rnnrPntr~tion of lO'? infected cells, complete killing occurred at a l:l dilution (full strenyth) after a one minute exposure. A 1:5 ~;lnt;nn of the saline was sufficient to kill a 107 TCID of HIV after a 5 minute incubation period but not at l or 2 minute incubatione. No killing of HIV occurred at a 1:10 dilution except at the lower 102 and 101 TCID concentrations.
As in Example VIII it is shown that the electrolyzed saline is effective in the ln vitro killing of HIV in HB-2 isolates.

~XAMPLE X
This examples focuses on treatment of an AIDS patient in the last stages (i.e. WR6) of this disease. The patient was a male, age 53, who had tested positive several years earlier as being infected by the HIV virus. He had been hospitalized numerous times and had contracted pneumonia.
He was extremely fatigued, had thrush in his mouth along with other usual AIDS related symptoms. This patient, realizing that his death was near, volunteered for treatment with electrolyzed saline rnnt~1nlng 60i4 ppm of active chlorine species.
The patient was injected intravenously, first with 1.5 mg of colchicine followed by 30 cc of the b-?l~nred saline [26.25 cc of electrolyzed saline blended with 3.75 cc of 5~
hypertonic saline] over a perlod of approximately 15 minutes followed about five minutes later by intravenous injecFion of 1000 mg of ascorbic acid. The patient was treated daily for five consecutive days with the same injections. There was no evidence of any abnormal side effects during the treatment period. The patient was monitored with continuous monitoring of a cardiogram. At = the beginning of the ~irst injection, the patient WO96/022~1 tt~ C 2 1 9 5 0 8 ~ pCT~S94,08280 demonstrated a very irregular heart rhythm. At the end of the series of injectiQns he showed marked illl~L~V~ t but still had a small amount of irregularity.
Subjectively, the patient stated that he felt better after each injection. His energy level increased daily and he was able to sleep better. The thrush in his mouth was improved. He was able to eat better and the pain associated with his disease was lessened.
Blood tests were conducted each day. There was no decrease in t?~e red blood-count and the blood showed no abnormalities or hemolysis. Repeat white blood counts revealed that the patient started with a leukocyte count of about 2000 with lO~ lymphocytes. At the end of the fifth day the leukocyte count was 2625 with 20~ lymphocytes.
This means that the patient has a total lymphocyte count of 220 cells/mm3 at first and a total of 525 cells/mm3 at the end of five days.

EXAMP_E XI
As added evidence that the specification is enabling relative to the tr~' t of patients suffering fr~om AIDS
there follows a summary of testin~ of AIDS patients completed outside the United States.
Tests, using five (5) HIV positive male patients, were conducted in a foreign country under a protocol established and observed by the inventor and conducted under the supervision of licensed physicians in that country. The five patients, noted herein si~iply as A~LA, BBB, CCC, DDD
and EEE, were select~d because they had been HIV positive for many years and haa accepted AIDS syndrome as characterized by the history of having had opportunistic diseases. They were treated monthly for~four series of treatments. Four of the patients were treated with a fifth series of treatments. Patient CCC terminated the test following the fourth series of treatmer,t.

W O 96/02271 ' ; ' ~ 9 ~ 0 8 Z P~IAUS94/08280 T ~ t~l y following the colchicine there was administered, also intravenously, 120 cc of the isotonic electrolyzed saline solution nnnt~;n;ng 60+4 ppm chloride ion and about 12~2 mg/mL ozone. Ascorbic acid, 20,000 mg diluted in 45 cc of normal saline was intravenously administered with a minor amount being given between the colchicine and electrolyzed saline and the . ;n~P~ following the electrolyzed saline administration. The four patients were followed with laboratory tests for an additional four months for a total of ten (10) months. The patients were not charged for the tr~A t fi and each siyned a consent form for investigational study including the right to use and publish laboratory results. Each was informed that the treatment was experimental only and was not d~Luv~d by the United States Food and Drug Administration.
Blood samples were drawn prior to the beginniny of the test, on each day of the administration of the solutions and at periodic intervals between the trr~tmPnt~ and throughout the follow-up period subsequent to the tr~t~nts. The analysis of the blood samples was performed in the ULited States by major clinical laboratories. Complete laboratory test results were obtained. However/ for purposes of this response, only the T4 cell absolute count is reported. As the AIDS disease progresses, there is typically a consistent decrease in the helper T4 cell count. It is accepted as a positive sign in the treatment of AIDS patients if an increase in the helper T4 lymphocytes can be 3 0 demonstrated.
A summary of the T4 cell results are given in the following table.

Table VII
Absolute CD4 Counts Pe- Milliliter a Patient Initial 2 months 6 months 10 months 24 monthsb A~A 79 132 (~796~ 177 (12490)192 (14396) NAc BBB 94~ 1199 (2596) 1042 (1096)1088 (1596)801 (-169r) ,."
CCC 193 344 (7496) 278 (4096) NA NA "r;, DDD 44 47 (796) 48 (996)54 (2396) NA
EEE 278 391 (4196) 355 (2896)355 (2896) 297 (796) w a Cour.ts represent ar. average of four determinations taken during each month for the O
interval described The percentages in parentheses represent the percent of change ~
from the initial counts. I~) b Patients did rot receive electrolyzed saline therapy for one year after the ten month count was taken.
c l~esults not available.
d Five month average because CCC removed himself from the testing program.

~ W O 96/02271 ~ ' 2 1 9 5 0 8 ~ P~rrUS94/08280 The test results indicate that the T4 cell absolute count ; ~ uv~d and stayed improved over the ten month follow up period in all four patients who completed their trP~ - The T4 count in patient CCC also showed i~ uv, t during the period he was in the program.
While subjective in nature, Patient CCC, who disrnrt;nnPd his treatment, reported verbally that he continues to feel better and is working at his occupation. While also subjective in nature, the other patients reported they have felt improved health, have less fatigue, more energy, are able to work more, have less depression and possess a more positive attitude.
Only minor side effects from the tluai".~ were noted, i.e. some superficial phlebitis in the forearm with discomfort due to the IV injections was noted and some gastrointestinal cramping was experienced.
It is i~uite clear from the above results that even those patients who did not show an immediate ii..~LUV.
following the first treatment, experienced a positive T4 cell count over the ten month period.
Other positive results were also noted in the study. Although consistent tests were not made, it was observed that there was an increase in the anti-p24 antibody titer. A decrease in anti-p24 antibody titer is generally seen with progression qf the AIDS disease, therefore, any increase in anti-p24 antibody titer is an indication of an i ,'LUVI t resulting from the treatment. Late in the study, the p24 antigen core quantitative tests were negative in all four patients completing the testing, which is desirable. The HIV
virus culture also showed "no virus isolated" for HIV
growth in the four patients who completed the study. It is reported by the laboratory conducting the virus culture study, that it was able to recover the virus in 80-90~ of the HIV positive p~t;~ntS tested and the WO96i0227l 2 ~ 9 ~ 0 8 2 PCTNSg4~08280 culture is a sensitive test of infectiousness of the blood EXAMPLE XII
The in vivo toxicity of electrolyzed saline to the liver of a human patient is illustrated by normal levels of enzymes indicative of liver function in all five patients of Example XI. During the 10 month course of electrolyzed saline therapy, the only period for which data are available, the following enzyme levels were observed: aspartate aminotransferase (AST 1-45 U/L{SGOT}), alanine aminotransferase (ALT 1-35 U/L~SGPT~), and lactate dehydrogenase (LDH) 100-225 U/L.
EXAMPLE XIII
The stability of antibody to the p24 core antigen to detection by Western blot analysis ~~ ; n~d positive during the course of electrolyzed saline therapy for patients AAA, BBB, DDD, and EEE. No data were available for patient CCC. This result is significant in that reports have d~ that ~nt;hn~ to the p24 antigen tend to become undetectable with the onset of clinical symptoms. J. Esteban et al., 2 Lancet 1083 (1985); J. Goudsmit et al., 155 J. Infect. Dis. 558 (1987).
EXAMPLE XIV
Increases in serum IgM levels for patients receiving electrolyzed saline therapy according to Example XI are illustrated in Table VIII. Four of the five patients showed an average increase in their serum IgM levels after four monEhs of therapy, whereas one patient, CCC, displayed a 3~ decrease. Normal ranges of serum IgM levels in adults are in the range pf about 40-260 mg/L.

21 9~082 WO96/02271 PC~S94108280 Table VIII
: Serum IgM Levels (mg/L) and Patient~ Change ~ Initial 4 months AAA 96 330 (71~) BLL318 364 (13~) CCC151 147 (-3~) DDD177 440 (60~) EEE394 442 (11~) EXAMPLE XV
The stability of serum IgG levels of patients receiving electrolyzed saline therapy is illustrated in that all of the patients of Example XI remained within the normal reference range of 700-1950 mg/dL.

EXAMPLE XVI
A male (FFF) who was HIV positive had an initial CD-4 count of 244 and a p24 antigen count of 114. This patient was treated with the solution of Example 1 for a series of 21 treatments over a one month period. No supplements such as moderating agents or colchicine were administered The isotonic electrolyzed saline dosage was approximately 2mg/kg body weight. Following treatments the energy level of the patient increased and the p24 antigen was negative. Follow up testing on FFF
is rrnt;n1ling After approximately 3.5 months from the date treatment was begun, his CD-4 count is 360 and his overall health r~nt;nnP~ to improve.
~ 30 EXAMPLE XVII
A patient, MV, was enrolled in a NIH sponsored clinical trial for chronic, symptomatic p~t;Pnt~ with Hepatitis C infections. MV was treated with interferon and ribavirin for a six month period. Tests of liver w096/02271 ~ ~ , 2 1 95082 PCT~Sg4/08280 function, including AST, ALT, and LDX rrrtjnllP~ to increase to levels P~rP~fl;nJ 400 for AST and ALT and 700 for LDX. Because no clinical or laboratory impLuv -t was seen by MV or attending physicians following the conclusion of the NlH clinical trial, MV elected to receive therapy using the electrolyzed saline of Example I. Following intravenous treatment at a dosage of 2 mg/kg body weight for five consecutive days a dL t;c drop in the AST, ALT and IDH levels were observed. one month after receiving the electrolyzed saline therapy, ALT, AST and LDH values were reest~h11 sh~ within normal ranges and the overall health of MV was stated to being comparable to be~ore becoming symptomatic for Hepa~itis C. MV r~nt;nll~s to demonstrate good health and laboratory findings show measured parameters to be within normal ranges.
The above examples show there i~s evidence that the in vitro and in vivo use of electrolyzed saline in treating physiological solutions a~d patients in Arcrr~nre with the invention resulted in der~nt~;n~tion of solutions and marked illlVLVV~ in patiPnts with no visible toxic side ef~ects.

Claims

43

1. A microbiocidal solution for in vitro treatment of a microbially contaminated fluid selected from the group consisting of whole blood, blood cells, blood plasma, and mixtures thereof and for treatment of microbial infections in warm blooded animals comprising a sterile electrolyzed saline containing ozone and active chlorine species wherein the ozone content is in the range of about 5 and 100 mg/L and the active chlorine species content is in the range of about 5 and 300 ppm.

2. A microbiocidal solution according to Claim 1 which has an isotonic saline concentration.

3. A microbiocidal solution according to Claim 1 wherein the ozone content is in the range of about 8 to 30 mg/L and the active chlorine species content is in the range of about 10 and 100 ppm.

4. A microbiocidal solution according to Claim 3 which has an isotonic saline concentration.

5. A microbiocidal solution according to Claim 1 wherein the ozone content is in the range of about 9 and 15 mg/L and the active chlorine species content is between about 10 and 80 ppm.

6. A microbiocidal solution according to Claim 5 which has an isotonic saline concentration.

7. A method of preparing a microbiocidal solution containing regulated amounts of ozone and active chlorine species which comprises (a) subjecting, in a sterile atmosphere, a sterile saline solution having a saline content in the range of about 0.15 and 1.0% to an electric current at a voltage, amperage and time sufficient to provide an electrolyzed solution containing regulated amounts of ozone and active chlorine species; and (b) adjusting said electrolyzed solution, if necessary, with sterile hypertonic saline, to provide a microbiocidal solution wherein the ozone content is in the range of about 5 and 100 mg/L and the active chlorine species content is in the range of about 5 and 300 ppm.

8. The method of Claim 7 wherein said electrolyzed solution is adjusted with hypertonic saline to an isotonic saline concentration.

9. The microbiocidal solution prepared according to the method of Claim 7.

10. The microbiocidal solution prepared according to the method of Claim 8.

11. The microbiocidal solution prepared according to Claim 8 wherein the ozone content is in the range of about 8 and 30 mg/L and the active chlorine species content is in the range of about 10 and 100 ppm.

12. The microbiocidal solution prepared according to Claim 8 wherein the ozone content is in the range of about 9 and 15 mg/L and the active chlorine species content is in the range of about 10 and 80 ppm.

13. A method for the in vitro treatment of a microbially contaminated fluid selected from the group consisting of whole blood, blood cells, blood plasma, and mixtures thereof which comprises contacting said fluid with an effective amount of a microbiocidal solution comprising an electrolyzed saline containing ozone and active chlorine species wherein the ozone content is in the range of about 5 and 100 mg/L and the active chlorine species content is in the range of about 5 and 300 ppm for a time sufficient to reduce the microbial contamination in said fluid while maintaining therapeutic characteristics thereof.

14. The method of Claim 13 wherein said microbially contaminated fluid is contaminated by an agent selected from the group consisting of Epstein-Barr virus, hepatitis A, B or C virus, rhinovirus, rubeola virus, rubella virus, parvovirus, papilloma virus, influenza viruses, parainfluenza viruses, enteroviruses, Herpes simplex viruses, Varicella-zoster viruses, Adenoviruses, respiratory syncytial viruses, alphaviruses, flaviviruses, retroviruses, and agents of bacteremia, septicemia, fungal infections, parasitic infections, mycobacterial infections, Bacterial Gram positive infections, Bacterial Gram negative infections, superficial and systemic infections.

15. The method of Claim 14 wherein said microbiocidal solution is an isotonic saline solution.

16. The method of Claim 15 wherein said microbially contaminated fluid is whole blood.

17. The method of Claim 15 wherein said microbially contaminated fluid is blood cells.

18. The method of Claim 15 wherein said microbially contaminated fluid is blood plasma.

19. The method of Claim 15 wherein said microbially contaminated fluid is retrovirus contaminated.

20. The method of Claim 19 wherein said retrovirus is HIV.

21. The method of Claim 20 wherein said microbially contaminated fluid is whole blood.

22. The method of Claim 20 wherein said microbially contaminated fluid is blood cells.

23. The method of Claim 20 wherein said microbially contaminated fluid is blood plasma.

24. The method of Claim 15 wherein said microbially contaminated fluid is contaminated by a hepatitis virus.

25. A method for the treatment of microbial infections in warm blooded animals which comprises administering to said warm blooded animal an effective amount of a microbiocidal solution comprising an electrolyzed saline containing regulated amounts of ozone and active chlorine species wherein the ozone content is in the range of about 5 and 100 mg/L and the active chlorine species content is in the range of about 5 and 300 ppm.

26. The method of Claim 25 wherein said microbial infection is induced, at least in part, by a member selected from the group consisting of Epstein-Barr virus, hepatitis A, B or C virus, rhinovirus, rubeola virus, rubella virus, parvovirus, papilloma virus, influenza viruses, parainfluenza viruses, enteroviruses, Herpes simplex viruses, Varicella-zoster viruses, Adenoviruses, respiratory syncytial viruses, alphaviruses, flaviviruses, retroviruses, and agents of bacteremia, septicemia, fungal infections, parasitic infections, mycobacterial infections, Bacterial Gram positive infections, Bacterial Gram negative infections, superficial and systemic infections.

27. The method of Claim 26 wherein said microbiocidal solution is an isotonic saline solution.

28. The method of Claim 27 wherein said microbiocidal solution is administered intravenously.

29. The method of Claim 28 wherein said warm blooded animal is a human.

30. The method of Claim 29 wherein the microbial infection is induced by a retrovirus.

31. The method of Claim 30 wherein said retrovirus is HIV.

32. The method of Claim 31 wherein said microbiocidal solution is administered at a daily dosage in the range of about 0.25 to 4 ml/kg body weight.

33. The method of Claim 32 wherein, in correlation with the administration of said microbiocidal solution, an effective amount of one or more modulating agents selected from the group consisting of superoxide dismutase, myeloperoxidase, glutathione peroxidase, glutahione, catalase, ascorbic acid, sodium ascorbate and calcium ascorbate is also administered.

34. The method of Claim 32 wherein, in correlation with the administration of said microbiocidal solution, an effective amount of colchicine is also administered intravenously.

35. The method of Claim 29 wherein the microbial infection is induced by a hepatitis virus.