Patent Application: US-51085009-A

Abstract:
the formulations have an antimicrobial , antiviral , and anti - pathogenic composition that combines , in various forms , a redox - active polyphenol , an oxidizing agent , and / or a redox - active , transition metal ion and / or electrochemical potential . the composition relates to methods for decreasing or eliminating the infectivity , morbidity , and rate of mortality associated with a variety of pathogenic organisms and viruses . the present invention also relates to methods and compositions for treating herpes simplex and hiv viruses and drug - resistant bacteria , and for decontaminating areas colonized or otherwise infected by pathogenic organisms and viruses . moreover , the present invention relates to methods , compositions , electrochemical devices , and storage containers for decreasing the infectivity of pathogenic organisms in pharmaceuticals , medical devices , personal care products , recreational products , and foodstuffs .

Description:
this invention embodies the ph and redox control of phenolic compounds , with the specific example of egcg , to determine the most efficacious pharmacological activity , namely antiviral and antibacterial properties at different ph &# 39 ; s and eh &# 39 ; s . examples to support this invention include : 1 ) uv - visible absorption spectra ; 2 ) electron spin resonance spectra ; 3 ) electrochemical oxidation reactions ; 4 ) in vitro antiviral assays against herpes simplex 1 and 2 ; 5 ) in vitro antiviral assays against human immunodeficiency virus hiv - 1 ; 6 ) in vitro antibacterial assays against multidrug - resistant staphylococcus aureus ; and in vitro anticancer binding assays against bcl - xl protein . the present invention covers redox - active phenols , polyphenols , or antioxidants , individually or in combination , at use levels between 0 . 2 % and 30 % by weight and preferably between 0 . 5 % and 12 %, or combined with other redox - active phenols , polyphenols or antioxidants to form pro - oxidant systems that exhibit antibacterial , antifungal , antiviral , or anticancer activity . combined redox - active phenols , polyphenols or antioxidants at use levels between 0 . 01 % and 40 . 0 % by weight , preferably between 0 . 5 % and 15 . 0 % by weight , are contemplated to exhibit antibacterial , antifungal , antiviral , or anticancer properties but will vary depending on the composition and its application . concentration of the oxidizing agent should be between 0 . 001 % and 10 . 0 % by weight , preferably between 0 . 05 % and 2 . 0 % by weight . concentration of the redox - active transition metal ion ( catalyst ) should be between 0 . 01 % and 5 . 0 % by weight , and preferably between 0 . 05 % and 1 . 0 % by weight are contemplated . uv - visible absorption spectra vs . ph : egcg solutions undergo an immediate spectral absorption shift as the ph increases from acidic to alkaline , with a change noted at ph 7 when a peak at 320 nm appears and the dominant phenol peak at 272 nm begins to decrease ( fig3 a ). the 320 nm peak likely represents the absorbance of the quinone form of egcg . when produced by simply raising the ph of the solution , the 320 nm oxidation peak is labile under air and disappears within hours ( fig3 b ). however , if the alkaline solution is re - acidified within minutes , the reaction is reversible . it is spontaneous with ph change and does not require a catalyst . epr vs . ph study of egcg : the epr and uv - visible spectra of egcg , stabilized with 0 . 2 m mgcl 2 , were measured as a function of ph ( i . e ., 6 . 03 , 6 . 54 , 7 . 03 , 7 . 57 , 8 . 09 , 8 . 56 , 9 . 10 , 9 . 56 ). a corresponding increase in a peak at ˜ 314 nm and a g = 2 epr spectrum occurred with increasing ph . the addition of mgcl 2 greatly improved the intensity of the epr spectrum . this is in agreement with the known stabilizing effect on semiquinone free radicals by divalent cations like mg + 2 . fig4 shows the semiquinone radical at ph 8 . 5 . basification of the egcg solution resulted in strong semiquinone signals that decayed over 30 minutes , demonstrating radical instability in the absence of electrochemical modulation ( fig5 ). the multiple peaks and valleys in fig5 a and 5 b are the fine structure detail of a high concentration radical solution . epr detects unpaired spinning electrons due to the absorption of microwaves in an applied magnetic field . the peak splitting is due to interaction of the lone electron &# 39 ; s spin magnetic field with environmental magnetic fields . in another experiment at ph 6 where no 320 nm peak is ever observed ( fig1 ), a weak epr signal for the semiquinone was present in the freshly dissolved solution , and the signal increased in intensity as the solution aged to 48 hours . these results demonstrate that the 320 nm peak is not the semiquinone . antiviral activity against herpes simplex 1 and 2 : of the many variant forms of herpes viruses , eight are known to affect more than 90 % of the world &# 39 ; s six billion inhabitants . the three that affect the skin / or mucus membranes are herpes simplex virus 1 ( hsv - 1 , the cold sore virus ), herpes simplex virus 2 ( hsv - 2 , the genital herpes virus ), and the varicella zoster virus ( vzv , the chicken pox and shingles virus ). herpes simplex viral infections are capable of causing life - threatening and lethal herpes encephalitis in otherwise normal people , lethal and blinding infections in newborns , blindness in adults , and serious to life - threatening infections in immuno - compromised patients . currently there is no cure for any of the human herpes viral infections . acyclovir and its derivatives act to interfere with viral reproduction by offering the virus defective nucleoside dna building blocks that are preferentially incorporated into the replicating viral dna chain , thus slowing its continued replication . however , these antiviral medications do not kill the herpes virus . egcg from green tea has been found to inhibit hsv - 1 and hsv - 2 ( lyu et al ., 2005 ; isaacs et al ., 2008 ), influenza virus ( nakayama et al ., 1993 ; song et al ., 2005 ), adenovirus ( weber et al ., 2003 ), epstein - barr virus ( chang et al ., 2003 ), and hiv ( fassina et al ., 2002 ; yamaguchi et al ., 2002 ; kawai et al ., 2003 ). in the studies cited above where the mechanism of activity was addressed , egcg impinged on multiple steps in the life cycle of the virus besides simply blocking absorption . reduced and oxidized states of egcg were not addressed in those studies . several investigations allude to the relative effects of the antioxidant and pro - oxidant forms of polyphenols and related compounds in antiviral assays . koyama et al . ( 2001 ) found a definite correlation between the redox potentials and inhibitory effects on epstein - barr virus activation of azaanthraquinones . those compounds at the lowest redox potentials at ph 7 . 2 had the most potent activity , although all the concentrations were high ( ic 50 s & gt ; 100 μm ). conversely , when the hepatitis c proteinase inhibitor 4 - methyl - 1 -( phenylmethyl )- 2 , 6 - pyridinedione underwent an autooxidation process that resulted in dimer formation , the dimer was found to be a relatively more potent inhibitor of the enzyme ( bennett et al ., 2005 ). another possibility is that the reduced and oxidized forms of egcg have different effects in the same cells . in an anticancer study , the auto - oxidation of egcg led to the inactivation of epidermal growth factor receptor in kyse 150 cells , but the inhibition of cell growth was observed with reduced egcg ( hou et al ., 2005 ). those authors stabilized the egcg in the reduced state in the growth medium by adding superoxide dismutase , with and without catalase . it is notable that pro - oxidant activity of egcg has been recorded in studies of a variety of cell culture lines ( alvarez et al ., 2002 ; tobi et al ., 2002 ; salter et al ., 2004 ; elbling et al ., 2005 ). alvarez et al . ( 2002 ) found egcg acted as a pro - oxidant at low concentrations ( 1 - 10 μm ) and an antioxidant at higher concentrations , but tobi et al . ( 2002 ) and salter et al . ( 2004 ) found the opposite results : at low concentrations egcg was an antioxidant , and at high concentrations it was a pro - oxidant . the inherent ability of egcg and other polyphenols to act as antioxidants or pro - oxidants , undergo redox reactions with stable phenolic radical intermediates , and chelate metals could be why they have variable bioactivity under in vitro assay conditions ( i . e ., the in presence of atmospheric o 2 ). the extent of oxidation ( either auto - oxidation or coupled reactions ) of egcg in vivo , where free o 2 is very low , is largely unknown . sang et al . ( 2007 ) found egcg in the plasma of mice injected with egcg was conjugated as the 4 ″- glucoronide , but no oxidized forms of the polyphenol were detected . egcg is a good model compound to investigate whether it acts as an antioxidant or pro - oxidant as an antiviral drug under a suite of redox states and ph &# 39 ; s . polyphenols such as egcg are a promising class of antiviral agents because they appear to inhibit the viruses at multiple steps in their life cycle . a number of synthetic derivatives of natural polyphenols are also under investigation as possible antiviral drugs ( klocking et al ., 2002 ; savi et al ., 2005 ), and ultimately egcg may not prove to be the most efficacious antiviral polyphenol . tea polyphenols are added to some skin care products and are generally considered to be safe in topical formulas ( thornfeldt , 2005 ; hsu , 2005 ). treatment of green tea polyphenols to the skin has been shown to modulate the biochemical pathways involved in inflammatory responses , cell proliferation , and responses of chemical tumor promoters as well as ultraviolet light - induced markers of skin inflammation ( katiyer and elmets , 2001 ; kapoor et al ., 2004 ; an et al ., 2005 ). stability studies of topical formulations have been conducted ( proniuk et al ., 2002 ) and the pharmacokinetics of topical administration of egcg have been documented ( dvorakova et al ., 1999 ). experimental — hsv - 1 and 2 : herpes simplex viruses were titrated by inoculation of 10 - fold dilutions ( hsv - 1 was inoculated into vero cell cultures , and hsv - 2 was inoculated into cv1 cultures ) in 96 - well microtiter tissue culture plates . a virus dilution ( 0 . 1 ml ) in rpmi 1640 with 1 % fetal bovine serum ( mm ) was inoculated into each well with three wells per dilution . the plates were kept for 2 to 5 days , depending on the virus , and examined daily for cytopathic effect . virus titers were calculated by the method of reed and muench ( 1938 ). about 10 5 50 % tissue culture infective doses ( tcid 50 s ) of virus were mixed with varying concentrations ( 12 . 5 , 25 , 50 , 75 and 100 μm ) of reduced and oxidized egcg in mm and incubated at 37 ° c . for 30 min . the following samples were tested : the reduced form of egcg (# 1 ); the partially oxidized form of egcg (# 2 ); the quinone or fully oxidized form of egcg with the addition of a copper catalyst (# 3 ); and quinone or fully oxidized form of egcg with the addition of a zinc catalyst (# 4 ). in samples # 3 and # 4 , the addition of the copper and zinc catalysts , respectively , raised the ph to 9 . 0 - 9 . 5 . these compounds were totally oxidized , thereby taking a quinone form . these samples polymerized and turned a dark brown color with a heavy amount of precipitate . virus mixed with mm alone was used as a control . after incubation , the infectivity of each mixture was titrated by the serial dilution endpoint method . dilutions ( 10 - fold ) were made in mm . the 10 − 1 to 10 − 5 dilutions were inoculated into monolayers of vero or cv1 cells , and the virus titers were determined as described above . the difference between the titer ( log 10 ) of the control virus and the titers of egcg - virus mixtures , i . e ., the reduction of virus titer , was used as a measure of antiviral activity . as shown in fig6 , the oxidized form of egcg shows the highest efficacy against hsv - 1 and hsv - 2 relative to the reduced form compared to commercial viricides . an important follow - up experiment was performed in light of the hsv - 1 and - 2 data described above . in the antiviral activity assay ( fig7 ) the activated version of egcg completely inactivated hsv - 1 and hsv - 2 viruses . the test involves treating suspensions of viruses with a drug , mixing them with the cells they normally infect , incubating the mixture for a preset time that is generally required for viruses to infect the cells and then determining the number of infected cells . the lower number of infected cells represents a better drug . activated egcg at a very low concentration ( 0 . 1 mm concentration or 40 parts per million ) nearly completely inactivated the viruses as only a few infected cells were observed . other antiviral drugs merely slowed down virus replication but were not able to effectively protect against infection of the test cells . there are plans to test egcg against four members of the herpes family : hsv - 1 , hsv - 2 , human cytomegalovirus , and varicella zoster virus ( chicken pox and shingles ). in on - going experiments , egcg inactivated the herpes family of viruses at very low concentration compare to the concentration that impacted the test cells . the low concentrations that inactivate the viruses , coupled with the high safety level against the host cells make egcg a very promising drug candidate . generally most of the test compounds that work against the viruses are also highly toxic to host cells , as shown in table 1 below . fig8 a and 8b demonstrate that the pro - oxidant forms of egcg are more effective against hsv - 1 and hsv - 2 , respectively , and fig9 shows greater antiviral activity at the more alkaline ph &# 39 ; s when semiquinone , quinone , and oligomeric forms of egcg are present . the concentrations of egcg used in these assays ( 0 - 100 μm ) were not cytotoxic to the host cells ( table 2 ). 1 these assays were performed using the cell titer 96 aqueous cell proliferation assay from promega . it is accomplished by dehydrogenase enzymes in metabolically active cells . 2 each number is the mean ± sd for assays done in triplicate . antiviral activity against hiv - 1 : egcg is a known inhibitor of human immunodeficiency virus hiv - 1 ( fassina et al ., 2002 ; yamaguchi et al ., 2002 ; kawai et al ., 2003 ). the aids epidemic caused by the human immunodeficiency virus hiv - 1 has no cure . antiviral drugs and drug combinations slow viral replication , but don &# 39 ; t eliminate the virus . in the underdeveloped world , the cost of the current drugs is prohibitive , and the death rates among certain populations have changed the fabric of societies . for example , it is estimated worldwide that more than 15 million children under the age of 18 have been orphaned as a result of aids . more than 12 million of these children live in sub - saharan africa , where it is currently estimated that 9 % of all children have lost at least one parent to aids ( http :// www . avert . org / aidsorphans . htm ). as hiv infections become increasingly common among the heterosexual adult population of the region , millions of children will lose parents to aids . by 2010 , it is predicted that there will be around 15 . 7 million aids orphans in sub - saharan africa alone . the technology embodied in this patent can be developed to produce an effective topical viricide that kills hiv - 1 fig1 demonstrates the activity profiles of reduced and oxidized solutions of egcg against hiv - 1 suspensions before infecting cells . the most oxidized formulation was the most effective . inhibition of apoptosis is implicated in virtually every known human malignancy ( reed , 1995 ; johnstone et al ., 2002 ). proteins in the bcl - 2 ( b - cell lymphocyte / leukemia - 2 ) family are critical components of the intrinsic apoptotic pathway , and anti - apoptotic bcl - 2 proteins such as bcl - xl are over - expressed in most human cancer types . inhibition of apoptosis may involve more than one pathway . in mia paca - 2 pancreatic carcinoma cells , egcg invokes bax oligomerization and depolarization of mitochondrial membranes to facilitate cytochrome c release into cytosol and caspase - dependent apoptosis ( qanungo et al ., 2005 ). those authors concluded that egcg induces stress signals by damaging mitochondria and reactive oxygen species - mediated jnk activation in pancreatic cancer cells . nakagawa et al . ( 2004 ) concluded that the generation of hydrogen peroxide by egcg primarily contributes to the induction of fe ( ii )- dependent apoptosis in jurkat cells , indicating a beneficial pro - oxidant mechanism . from a combination of nmr binding assays , fluorescence polarization assays , and computational - docking studies , it is known that the green tea compounds egcg , gallocatechin - 3 gallate ( gcg ), epicatechin - 3 gallate ( ecg ) and catechin - 3 gallate ( cg ) are very potent inhibitors ( k i s in the nanomolar range ) of the anti - apoptotic protein bcl - xl ( kitada et al ., 2003 ). the inherent ability of egcg to act as antioxidant or pro - oxidant , undergo redox reactions with stable phenolic radical intermediates , and chelate metals could be why it is bioactive once it is bound to a target . in this example using the bcl - xl binding assays to support our claims , egcg solutions were the same as those used in some of the antiviral assays ( fig8 a and 8b ), including reduced , partially oxidized , oxidized containing cu 2 + , and oxidized containing zn 2 + under assay conditions outlined by pellechia et al . ( 2002 ) and pellechia and reed ( 2004 ). 1d - 1 h nmr spectra of all the compounds were taken at 1 mm concentration , 1 % d 2 o , 10 % h 2 o , 50 mm phosphate buffer at ph near 7 . 2 . the partially oxidized egcg solution without metal ions showed a two - fold better binding response in the fluorescence polarization displacement assay with a fitc - bad peptide , and the nmr spectrum of bcl - xl had an extra peak at 6 . 1 ppm ( fig1 ). the two other highly oxidized preparations of egcg showed either no difference or less activity than reduced egcg . gossypol , another polyphenol with known anticancer activity , was also tested under similar conditions . the solution of partially oxidized gossypol had similar activity as reduced egcg ( it is normally several times less active than egcg in the same assay ). it produced a similar nmr spectrum as reduced gossypol with a single aromatic peak , but the peak typical of the free aldehyde had a marked decreased intensity . these data demonstrate that oxidized states of egcg and gossypol may be more active agents for binding to the anti - apoptosis protein , and that redox states of activity can be monitored by spectrometric methods . the bcl - xl assay is an appropriate model system to show feasibility and proof of concept that redox control of anticancer drug agents can improve their activity . antibacterial activity : egcg was tested as an antibacterial agent against a multidrug - resistant strain ( 33591 ) of staphylococcus aureus . after pre - incubating the cells with various concentrations of egcg at ph 6 , 7 . 5 , and 8 . 5 , cells were plated and colony - forming units ( cfu ) were counted as a measure of efficacy ( fig1 ). at the lowest concentration tested ( 0 . 08 % egcg ), only the solutions containing pro - oxidant semiquinone and quinone forms ( ph 7 . 5 and 8 . 5 ) were effective at killing nearly all the bacteria . at concentrations of 0 . 4 - 0 . 8 % egcg , the solutions were strongly bactericidal at all three ph &# 39 ; s . at higher concentrations , egcg was less effective . with the burgeoning medical problem of drug - resistant pathogens surviving all available drug therapies , the discovery of highly effective polyphenolic drug candidates whose efficacy can be modulated by redox and ph falls under the claims of this patent . oxidation states modulated and measured electrochemically : the multiple oxidation states of the molecule of interest can be determined electrochemically by cyclic voltammetry . the first phase involves applying an excessive positive potential ( oxidizing potential ) and oxidizing all the species present to one form . then the applied voltage is stepped down in increments while monitoring current . when the mid - point redox potential of a reaction is approached , molecules begin to be reduced , causing current through the circuit . after returning all molecules to the reduced form , the voltage is increased and the redox potentials of the reaction ( s ) are recorded . this technique has been employed by others to determine redox states of egcg ( kilmartin and hsu , 2003 ). additionally , the open circuit potential of the solution versus a standard reference electrode can be measured . this method utilizes two electrodes , working and reference , to determine the electrochemical potential generated by the solution versus a reference electrode , such as ag / agcl . this method will give an indication of the overall degree of oxidation of the formulation , and when combined with cyclic voltammetry , can be used to determine the species present . by knowing the electrochemical potentials that create the semiquinone and quinone species , an external electrical potential can be applied to drive the redox state of the formulation to a predetermined oxidation state . continued application of the electrical potential will maintain the formulation in the desired oxidation state . this electrochemical potential can be supplied from a battery , small circuit , and electrodes designed into a container format ( battery - in - a - bottle ). the reduction potential of egcg at ph 7 is 430 mv versus a normal hydrogen electrode . an electrochemical cell was fabricated using a fine porosity glass frit in one - inch - diameter glass tubing . the electrochemical cell houses the counter electrode and keeps the ag / agcl reference electrode and platinum working electrode separate from the counter electrode . this addition to the system allowed the preparation of bulk solution . the working electrode and reference electrode compartment was continuously stirred using a small stir bar and a magnetic stirrer to minimize gradient effects at the working electrode . the egcg solutions were oxidized using a positive potential to a visual end point . uv - visible spectral data were collected during these experiments , including the absorbance at 270 nm and 320 nm , corresponding to the phenol and quinone forms of egcg , respectively . in addition , the open cell potential was recorded as experiments progressed . the active redox potentials and experimental endpoints were determined based on current and visual color changes to oligomers . due to solution evaporation over time , the ratio of absorbance at 320 nm divided by the absorbance at 270 nm was determined in order to account for overall absorption increases . this value was then tracked throughout the experiments and proved to be a more stable indicator of reaction progress than absorbance alone . in addition , the visual change in solution color from clear to yellow and pink was also monitored as an indicator of degree of oxidation . stirred egcg solutions in 0 . 1 m phosphate buffer with 0 . 1 m kcl at ph 6 . 0 were monitored . the open circuit potential was initially 220 mv . a potential of 800 mv was applied , resulting in approximately 1 microamp of current in the cell . a duplicate solution was left uncovered on the table next to the electrochemical cell as the control . after seven hours , no change in either solution at 270 and 320 nm was found . at this point the experimental potential was increased to 1000 mv for a further 16 hours ( table 3a ). these data demonstrate the increase in 320 nm quinone absorbance and associated decrease in the 270 nm phenol absorbance associated with oxidation of the egcg over 16 hours at 1000 mv . the control sample did not oxidize , which is consistent with the known stability of egcg under acidic conditions . after the combined time of 23 hours ( 7 h plus 16 h ), the potential of the cell was increased to 1500 mv ( table 3b ). at 68 hours , the control sample was still clear , but the experimental solution had a faint yellowish color ( dimerization ). the ph of the solutions remained 6 . 0 throughout the experiment . all data support the forced oxidation of egcg at ph 6 . 0 . solutions were allowed to sit for two more days to observe the stability of the electrochemically - induced changes ( table 3c ). these data indicate that the solutions are stable for over two days without a significant increase or decrease in 320 nm absorbance over time . a summary of these data is presented in fig1 . solutions were ph - modified following electrochemical oxidation to observe stability . the spectra of samples prepared from an egcg solution at ph 6 . 26 under 1500 mv potential for 11 . 4 hours are shown in fig1 . the ph 6 . 26 solutions could not be compared to control samples because control samples at ph 6 . 26 did not oxidize . the figure shows the samples were stable after one day , and in the case of ph 8 . 43 , it was relatively stable after 7 days . these samples were open to air , and no other precautions were taken to prevent oxidation by atmospheric oxygen . the following references are specifically applicable to the examples and to the remainder of the specification . they are incorporated herein by referenced and are identified , where appropriate , by their authors and publication dates as indicated . alvarez e , leiro j , orallo f . 2002 . effect of (−)- epigallocatechin - 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