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Antiviral methods and compositions US2006264510

FIELD OF !E I"#E" IO" [0001] The field of the invention is antiviral compositions. $A%&'(OU"D OF !E I"#E" IO" [0002] Viral infections are relatively common infectious diseases, and various methods of treating a viral infection available to a practitioner. In one method of treating a viral infection, the immune response of an immune system is stimulated. or e!ample, in some instances the Th1 response of the patient can be increased relative to the Th2 response. "n increase in the Th1 response is thought to be beneficial because many viral infections are associated #ith a shift in the cyto$ine profile to#ard a Th2 response, and a bias to#ards a Th1 response is $no#n to be facilitated by several approaches. [000%] In one approach, cyto$ines are administered to modulate the Th1&Th2 balance to#ards a Th1'type response. or e!ample, (night et al. postulate that treatment #ith I)'12 *Interleu$ine'12+, a cyto$ine that promotes the development of Th1 cells, may be used as a treatment for "I,- since I)'12 administration has been sho#n to be effective at restoring cell'mediated immunity in mice infected #ith mouse "I,- virus or #ith .)V [(night, -. /. and 0atterson, -., "nnu. .ev. Immunol. 1112. 134 31%' 513]. In another e!ample, 6racie, 7. ". et al., demonstrated that administration of I)' 18 to mice e!hibited pleiotropic activities critical to the development of Th1 responses. [6racie et al. 7 /lin Invest 1111 9ov 13: 102*10+41%1%'1201]. "lthough the administration of cyto$ines typically results in relatively specific increases in desired Th1 cyto$ines, prolonged administration of cyto$ines may be problematic for various reasons. or e!ample, the production of recombinant cyto$ines is relatively e!pensive, and isolation of non'recombinant cyto$ines from natural sources is generally difficult due to the very lo# concentration of cyto$ines in natural sources. ;oreover, depending on the nature of the cyto$ine, cyto$ines may not be #ell tolerated in patients. [0002] In another approach, immunomodulatory substances other than cyto$ines may be employed to increase the Th1 response. or e!ample, -prietsma 7. <. suggests that =inc ions *>n?2@A + and nitric o!ide *9B+, together #ith glutathione *6-C+ and its o!idi=ed form, 6--6, may help to regulate an immune response to antigens [-prietsma 7. <: ;ed Cypotheses 1111 7uly: 3%*1+45'15]. The author reports in more detail that deficiencies of >n?2@A , 9B and&or 6-C shift the Th1&Th2 balance

to#ards Th2, and that replenishment #ith >n?2@A , 9B and&or 6-C may shift the Th1&Th2 balance to#ards Th1. "dministration of >n?2@A or 6-C&6--6 is especially advantageous, since these substances are non'to!ic at even elevated concentrations, and ine!pensive to produce. urthermore, >n?2@A and 6-C&6--6 preparations may be orally administered, and therefore significantly reduce the ris$ of allergic reactions, especially #hen the preparations are not ultrapure. Co#ever, the administration of >n?2@A and&or 6-C&6--6 seems to be beneficial only to restore a Th1&Th2 balance from a Th2 dominated state, #hereas it is unclear if administration of >n?2@A and&or 6-C&6--6 may increase a Th1 response from a normal Th1&Th2 balance. [0003] In another method of treating a viral infection, the virus is directly targeted #ith an appropriate anti'viral drug. or e!ample, patients infected #ith the CIV virus often receive a coc$tail of drugs to bloc$ virus propagation, and various classes for direct anti'viral treatment are $no#n in the art. -ome direct anti'viral drugs bloc$ the reverse transcriptase of a retrovirus. .everse transcriptase *.T+ inhibitors are typically nucleoside analogs such as ">T, %T/, or ddI. "lternatively, non'nucleoside .T inhibitors, including Duercetin may be employed. In vitro, .T inhibitors are typically potent anti'viral drugs. Co#ever, in vivo, and especially during a period of relatively fast viral replication, the generation of .T inhibitor resistant virus mutants is problematic. ;oreover, many .T inhibitors also e!hibit undesirable activity on ,9" replication in the host organism and significant cytoto!icity at elevated concentrations, thereby limiting the concentration that may be administered #ithout severe side effects. [0005] "mong other direct anti'viral drugs are the protease inhibitors, #hich bloc$ or interfere #ith virus protein processing. 0rotease inhibitors are typically highly specific to#ards the virusesE proteolytic en=ymes, ho#ever, due to their mostly hydrophobic nature, administration at desirable concentrations often becomes problematic. "nother problem is that development of cross'resistance and severe side effects freDuently occur. In order to reduce the development of multidrug resistant virus strains, mi!tures of .T inhibitors and protease inhibitors may be prescribed. "lthough such mi!tures are presently employed relatively successfully, the relatively high occurrence of adverse side effects and the potential of generating multidrug resistant virus strains persist. [000F] To circumvent at least some of the problems associated #ith side effects and relatively high costs of antiviral drugs, Gennett et al. describe in H.-. 0at. 9o. 3,502,180 the use of <,T" comple!es in a suppository. The use of chelating agents, including <,T", has been found to promote disintegration of retroviruses [Iunderlich, V. and -ydo#, 6. "rch. Virol. 1182, F%41F1'18%]. GennettEs suppositories contain disodium <,T" and controlled'release agents, #hich release

the disodium <,T" over a period of about three to four hours after rectal placement of the suppository. Co#ever, GennettEs suppositories are limited to disodium <,T" that e!hibits relatively moderate selectivity bet#een ;g?2@A and /a?2@A . [0008] "lthough various antiviral compositions and antiviral treatments are $no#n in the art, all or almost all of them have one or more disadvantages. Therefore, there is a need to provide improved methods and compositions for treatment of viral infections. SU))A(* OF !E I"#E" IO" [0001] The present invention is directed to an antiviral composition having a supply of chelating agent that chelates an al$aline earth metal ion, #herein the chelating agent is formulated in a rectal deposition formulation, and #herein the supply of chelating agent has an immediate bioavailability. Ihen rectally administered to a subJect in an effective dose in vivo, contemplated agents promote disintegration of a virus. [0010] In one aspect of the inventive subJect matter, generally preferred chelating agents are various chelators other than ethylenediamine'9,9,9E,9E'tetraacetic acid *<,T"+, chelate /a?2@A and&or ;g?2@A , and include at least three carbo!ylic acid groups. Ihile particularly preferred chelating agents include at least three acetic acid groups, especially contemplated chelating agents are 1,2'Gis*2'aminopheno!y+ethane' 9,9,9E,9E'tetraacetic acid *G"0T"+, <thylenebis*o!yethylenenitrilo+tetraacetic acid *<6T"+, 1,2'bis*2'aminopheno!y+ethane'9,9,9E,9E'tetraacetic acid tetra$is*aceto!ymethyl ester+ *G"0T"'";+, diethylenetriamine'pentaacetic acid *,T0"+, trimethylaminetricarbo!ylic acid *9T"+, trans'1,2'diaminocyclohe!ane' tetraacetic acid */,T"+, poly*aspartic acid+, and poly*glutamic acid+. [0011] In another aspect of the inventive subJect matter, contemplated viruses include a retrovirus, and especially contemplated retroviruses include the CIV virus. 0referred rectal deposition formulations are a liDuid or a solid, and #here the rectal deposition formulation is a solid and administered to the colon of a subJect, substantially of the supply of chelating agent is present in the colon in a readily absorbable form in less than 2 hours, preferably less than 1 hour, and more preferably less than %0 minutes. Iith respect to the effective dose in a rectal administration, it is contemplated that the chelating agent is employed in an amount of 300 mg, and more preferably 1300 mg. [0012] In yet another aspect of the inventive subJect matter, chelating agents other than <,T" may also be employed for purposes other than antiviral treatment, including heavy metal deto!ification, and reduction of atherosclerotic plaDues, #herein the chelating agent may be orally or parenterally administered.

[001%] Various obJects, features, aspects and advantages of the present invention #ill become more apparent from the follo#ing detailed description of preferred embodiments of the invention. DE AILED DES%(I+ IO" [0012] "s used herein, the term Kchelating agentK refers to a molecule that binds a metal ion and&or an al$aline earth metal ion via a non'covalent bond, most commonly a coordinate bond, #ith a (, of less than 10?'% A mol?'1A , #herein the chelating agent may be in acid form, base form or a salt form. or e!ample, <6T" in protonated or sodium salt form is considered a free chelating agent, because <6T" binds ;g?2@A and /a?2@A #ith a (, of less than 10?'% A mol?'1A . [0013] "s also used herein, the term Kimmediate bioavailabilityK means that a composition or molecule is present in an active form in a formulation such that a substantial portion of a dose of the composition or molecule e!hibits some systemic chelating effect #ithin minutes, preferably #ithin less than 13 min, more preferably #ithin less than 10 min, and most preferably #ithin less than 3 min. or e!ample, a molecule that is dissolved in a carrier solution is regarded to have immediate bioavailability. [0015] It is $no#n that retroviruses can be disintegrated by chelating agents, especially by agents that chelate ;g?2@A and&or /a?2@A , and that chelating agents may further reduce infectivity of certain viruses [Iunderlich, V. and -ydo#, 6. "rch. Virol. 1182, F%41F1'18%]. Thus, it is contemplated that an antiviral composition generally has a supply of a chelating agent that chelates an al$aline earth metal ion, and it is particularly contemplated that the chelating agent in the antiviral composition is formulated in a rectal deposition formulation, #herein the supply of chelating agent has an immediate bioavailability. [001F] It should be appreciated that many chelating agents are $no#n in the art, and that all of the $no#n chelating agents are contemplated for use herein. It is generally preferred that contemplated chelating agents include at least three carbo!ylic acid groups, all of #hich are preferably acetic acid groups. "lthough not e!cluded, it is further contemplated that appropriate chelating agents are chelating agents other than <,T". The choice of the particular chelating agent is predominantly determined by the desired physicochemical properties and tolerability of the chelating agent. or e!ample, #here a relatively high solubility *e.g., 1;+ is desired, <6T", /,T" or 9T" may advantageously be employed. Ihere a more pronounced selectivity of chelation to#ards /a?2@A is desirable, G"0T" may be utili=ed, and G"0T"'";

may be particularly suitable #here seDuestration of /a?2@A #ithin a cell is desired. "lternatively, contemplated chelating agents may include ,T0", 9T", and polymeric forms of aspartic acid, glutamic acid, and any reasonable combination thereof. [0018] Iith respect to viruses that can be disintegrated and&or reduced in infectivity, virus particles that reDuire /a?2@A and&or ;g?2@A for structural integrity of their envelope are generally contemplated and include ,9" and .9" viruses. 0articularly contemplated .9" viruses are retroviruses in general, and CIV in particular. urther especially contemplated viruses include the hepatitis / and hepatitis , virus. /ontemplated ,9" viruses include polyomaviruses, CGV, etc. Co#ever, many more viruses are also contemplated, and a collection of appropriate viruses are listed in ields Virology, Third <dition *)ippincot Iilliams L Iil$ins+, pages 20'21, 32, and 1F5F'182F, and "rch. Virol. 1182, F%41F1'18%, both of #hich are incorporated by reference herein. [0011] It is still further contemplated that chelating agents are preferably formulated in a rectal deposition formulation, #hich may be in solid or liDuid form. Ihere the formulation is in a solid form, it is further contemplated that appropriate forms include dissolvable carriers such as #ages, fatty acids and oils #ith melting points of about %0[deg.]'%3[deg.] /. <specially preferred formulations are formulations $no#n in the art that are employed in the fabrication of rectal suppositories, so long as such formulations allo# an immediate bioavailability. Thus, #here a supply of chelating agent is administered into the colon of a subJect in a solid form, it is particularly contemplated that substantially all of the supply of chelating agent is present in the colon in a readily absorbable form in less than 2 hours, more preferably less than 1 hour, and most preferably less %0 minutes after the administration of the formulation. "vailability of the chelating agent or a portion of the chelating agent in less that 2 hrs, less than 1 hr or less than %0 min may be achieved by a variety of time release formulations, and contemplated time release formulations may include formulations #ith a melting point of less than %F[deg.] /., en=ymatically degradable carriers, dissolving or s#ellable carriers, etc. Thus, it is contemplated that an entire dose of chelating agent may be available *or released from the time release formulation+ in less than 2 hours, preferably less than 1 hour, and even more preferably in less than %0 min. " particular advantage of such time release formulations is that relatively high dosages may be administered that might other#ise pose a potential ris$ if administered #ithout a time release formulation. Co#ever, it should be appreciated that administrations #ithout time release may safely be administered by employing smaller dosages at multiple administrations. [0020] Ihere the formulation is in a liDuid form, it is contemplated that appropriate

liDuid forms may include buffered and unbuffered solutions, solutions #ith relatively high viscosity such as gels, creams, foams and ointments, #hich may or may not have a decreased viscosity at elevated temperatures. )iDuid forms are particularly advantageous, since the delivery of the chelating agent is almost instantaneous. Ihere the solutions are buffered, it is contemplated that the buffers have an al$aline pC, and a preferred pC range is a range bet#een 8.0 and 10.0. [0021] "lternatively, the chelating agent may also be administered in various alternative routes, and it is especially contemplated that #here the chelating agent is an agent other than <,T" that appropriate routes include oral and parenteral administration. or e!ample, /,T" may be orally administered in form of an acid resistant caplet or capsule. Co#ever, oral administration need not be limited to a caplet or capsule, and alternative oral administrations include syrups, po#ders, tablets, etc. In another e!ample, <6T" may be parenterally administered by intravenous inJection. It is contemplated, ho#ever, that alternative parenteral administrations may also include inhalation, transdermal delivery, inJections into sites other than a vein, etc. [0022] In a particularly contemplated aspect of the inventive subJect matter, it is preferred that the administration of the chelating agent is accompanied by *preferably oral+ administration of a nutritional supplement. 0referred nutritional supplements include supplements that help replenish calcium levels and particularly preferred supplements include aragonite calcium carbonate from fossil coral minerals. Bther contemplated supplements that include herbal products *e.g., adaptogenic formulations #ith no apparent cytoto!icity+ are contemplated to assist in inhibition of viral replication *e.g., by inhibiting the production of reverse transcriptase+. It is further contemplated that such supplements may also help boost the immune system and potentially improve overall vitality and stamina. It is further contemplated that such adaptogenic supplements are considered to have tumor preventive and radio' protective properties, and may help increase the functioning of the immune system by increasing the T'cell population. <!emplary compositions for contemplated nutritional supplements are sho#n in Tables 1 and 2. A$LE 1 Ingredient "mount *mg&tablet+ "rcticum lappa 20 mg *1041 concentrate+ Viola yedoensis 20 mg *1041 concentrate+

"ndrographis paniculata 20 mg *1041 concentrate+ )onicera erythrorhi=on 20 mg *1041 concentrate+ <pimedium saggittatum 20 mg *1041 concentrate+ [002%] A$LE 2 Ingredient "mount *mg&tablet+ "rcticum lappa 10 mg *1041 concentrate+ Viola yedoensis 10 mg *1041 concentrate+ "ndrographis paniculata 10 mg *1041 concentrate+ )onicera erythrorhi=on 10 mg *1041 concentrate+ "ltemanthera philoeroides 10 mg *1041 concentrate+ It should be appreciated, ho#ever, that various additional ingredients may be added to the supplement depicted in Table 1 and 2 to either enhance or modulate the activity of the herbal components. [0022] Iith respect to the amount of chelating agent it is contemplated, that the chelating agent is administered to a subJect in vivo in a dose effective to promote disintegration of a virus in the subJect. The actual dose of the chelating agent may thereby vary among individual subJect and may further be determined by the particular virus that is to be disintegrated. Therefore, an effective dose may comprises rectal administration of the chelating agent bet#een about 3 mg'2300 mg, and generally contemplated doses include rectal administration of 300 mg or 1300 mg of the chelating agent. Co#ever, #here even higher dosages of the chelating agent are reDuired, or #here it is preferred to maintain relatively high dosages over an e!tended period of time, multiple dosages are also contemplated. [0023] It should further be appreciated that appropriate formulations may further comprise active and&or inactive ingredients. or e!ample, active ingredients may include compositions to stimulate the immune system, an immunomodulating composition, a coral mineral product, compositions to facilitate upta$e of the chelating agent into the blood stream, or direct antiviral compounds such as

nucleoside analogs, etc. The term Kimmunomodulating compositionK as used herein refers to a composition that enhances at least one of a humoral and cellular response to#ards a challenge. or e!ample, an immunomodulating composition may increase an antibody titer against a challenge, or an activity of cytoto!ic T'lymphocytes. Inactive ingredients may include fillers, coloring agents, thi!otropic compositions, and foam building agents. [0025] In an e!emplary use, a person diagnosed #ith an CIV infection receives t#ice daily an enema of 20 ml of a 30 mg&ml solution of <6T" in 10 m; sodium phosphate buffer pC8.2 for at least %0 consecutive days. It should be recogni=ed, ho#ever, that the e!emplary use need not be limited to the specified amounts and times, but treatment schedules may vary considerably. or e!ample, #here the person already receives an antiviral medication *e.g., protease inhibitor coc$tail, .T' inhibitor, etc.+, lo#er dosages or less freDuent administrations are contemplated, #hile in cases #here the person does not receive another antiviral treatment, higher dosages and more freDuent administrations are contemplated. It is also contemplated that the antiviral composition may be employed in a preventative fashion, i.e., the antiviral composition may be employed in a person that is not infected #ith a virus. [002F] It is still further contemplated that the compositions according to the inventive subJect matter may have advantageous properties and uses in therapeutic applications other than antiviral activity, especially #here the chelating agent is a substance other than <,T", and particularly contemplated uses include heavy metal deto!ification in animal and human, and reduction of atherosclerotic plaDue. [0028] Iith respect to heavy metal deto!ification in animal and human, it is $no#n in the art that upon oral administration or inJection <,T" comple!es various metals and heavy metals other than /a?2@A , and oral administration or inJection of <,T" has therefore found #idespread use in deto!ifycation of some heavy metal poisonings. Various alternative oral or inJectable chelation agents for heavy metals have also been described [e.g., )lobet, 7. ;. et al. "rch. <nviron. /ontam. To!icol. 1110, 11*2+4 183' 1: Treatment of acute lead into!ication. " Duantitative comparison of a number of chelating agents. )lobet, 7. ;. et al. "rch. To!icol. 1188, 51*2+4%21'%: "ntidotes for =inc into!ication in mice] and include oral and inJectable forms of penicillamine, 2,%' dimercaptosuccinic acid, and 2,%'dimercapto'1'propanesulfonate. Co#ever, it is not $no#n to the inventors that chelators other than <,T" have been used for deto!ification of heavy metals in animal and human via rectal administration. .ectal administration is particularly advantageous for various reasons. or e!ample, suppositories can be self'administered by almost all patients. urthermore, rectal administration inflicts only relatively lo# discomfort to the patient. ;oreover, rectal administration bypasses the stomach, a highly acidic environment that may lead to at

least partial destruction of some of the chelating agents. [0021] Therefore, it is contemplated that rectal administration of chelating agents may also be employed in a method to reduce a heavy metal concentration in a subJect, #herein in one step a chelating agent is provided that chelates a metal ion, #herein the chelating agent is formulated in a rectal deposition formulation and #herein the supply of chelating agent has an immediate bioavailability. "lternatively, the rectal deposition formulation may further comprise a time release agent to release the chelating agent in a period of bet#een 0'%0 min, %0'50 min, 50'120 min, 120'180 min, or longer. In another step, the chelating agent is rectally administered to the subJect in a concentration effective to reduce the heavy metal ion concentration. [00%0] It is generally contemplated that the heavy metal may be in elemental or ionic form, and particularly contemplated heavy metals include mercury, >n?2@A , /u?@A , /d?2@A , and /o?2@A . Co#ever, various alternative metals and their ionic forms are also contemplated, including nic$el, arsenic, selenium, iron, mercury, chromium, antimony, beryllium, thallium, silver, scandium, titanium, vanadium, chromium, manganese, etc. Ihile it is generally contemplated that all $no#n chelating agents may be suitable for reduction of heavy metals in a subJect, it is particularly preferred that the chelating agent comprises a plurality of carbo!ylic acid groups and it is even more preferred that the chelating agent is <,T", <6T", /,T", or ,T0". Iith respect to the rectal deposition formulation the same considerations as already described above apply. [00%1] "n e!emplary method of reducing a heavy metal concentration in a subJect may therefore comprise a single rectal administration of 20 ml of a 10 mg&ml buffered aDueous solution of /,T" three times daily over a period of about 13'20 days. It should be recogni=ed, ho#ever, that depending on the particular heavy metal, the site of accumulation, and the concentration of the heavy metal in the subJect many treatment schedules other than a single rectal administration of 20 ml of a 10 mg&ml buffered aDueous solution of /,T" three times daily over a period of about 13'20 days are also appropriate. [00%2] or e!ample, #here treatment is prophylactic or necessitated by relatively lo# concentrations of a heavy metal, total daily dosages of less than 500 mg are contemplated, including total daily dosages of 200'500 mg, 30'200 mg, and less that 30 mg. )i$e#ise, #here acute and&or severe heavy metal into!ications are to be treated by a method according to the inventive subJect matter, higher total daily dosages of more than 500 mg are contemplated, including total daily dosages of 500' 1300 mg, 1300'2300 mg, and more than 2300 mg. Iith respect to the formulation it should be appreciated that numerous alternative formulations are also appropriate, and

contemplated alternative formulations include the formulations already described above. -imilarly, it should be appreciated that various alternative administration periods other than a period of about 13'20 days are also appropriate, including single administrations in cases #here treatment is prophylactic, or administration over a period of less than 13 days, #here the heavy metal concentration is relatively lo#. Bn the other hand, #here the heavy metal is predominantly is tissues that bind the heavy metal relatively firmly *e.g. lipophilic tissue+ administrations of 2'5 #ee$s and longer are contemplated. [00%%] Iith respect atherosclerotic plaDues it is contemplated that rectal administration of chelating agents may also be employed in a method to reduce a atherosclerotic plaDues in a subJect, #herein in one step a chelating agent is provided that chelates an al$aline earth metal ion, #herein the chelating agent is formulated in a rectal deposition formulation and #herein the supply of chelating agent has an immediate bioavailability. In another step, the chelating agent is rectally administered to the subJect in a concentration effective to reduce the atherosclerotic plaDue in a subJect. "s used herein, the term Kreducing the atherosclerotic plaDueK refers to a gross reduction in si=e and&or volume of one or more atherosclerotic plaDues, #hich may also include complete disappearance of the atherosclerotic plaDue or plaDues. [00%2] In an e!emplary method of reducing atherosclerotic plaDue, a person diagnosed #ith atherosclerotic plaDues receives once daily an enema of 10 ml of a 30 mg&ml solution of <6T" in 10 m; sodium phosphate buffer pC8.2 for a period of about 12 #ee$s. Co#ever, it should be appreciated that the e!emplary method need not be limited to the specified amounts and times, and formulation and treatment schedules may vary considerably. or e!ample, #here the person already under#ent a vasodilation procedure, lo#er dosages or less freDuent administrations are contemplated, #hile in cases #here the person did not receive previous treatment to reduce the atherosclerotic plaDues, higher dosages and more freDuent administrations are contemplated. [00%3] )i$e#ise, the chelating agent need not be limited to <6T", but may be various alternative chelating agents including <,T", /,T", and ,T0", #herein the choice of the chelating agent #ill predominantly depend on the desired specificity of the chelator and the tolerability at a particular concentration. urthermore, the formulation of the chelating agent need not be restricted to 10 ml of a 30 mg&ml solution of <6T" in 10 m; sodium phosphate buffer pC8.2. or e!ample, alternative formulations may be employed to achieve a larger distribution, faster absorbption, etc., and appropriate formulations include those already described above. [00%5] Thus, specific embodiments and applications of antiviral compositions have

been disclosed. It should be apparent, ho#ever, to those s$illed in the art that many more modifications besides those already described are possible #ithout departing from the inventive concepts herein. or e!ample, the route of administration need not necessarily be restricted to a rectal administration of the chelating agent, but may also include vaginal administration. The inventive subJect matter, therefore, is not to be restricted e!cept in the spirit of the appended claims. ;oreover, in interpreting both the specification and the claims, all terms should be interpreted in the broadest possible manner consistent #ith the conte!t. In particular, the terms KcomprisesK and KcomprisingK should be interpreted as referring to elements, components, or steps in a non'e!clusive manner, indicating that the referenced elements, components, or steps may be present, or utili=ed, or combined #ith other elements, components, or steps that are not e!pressly referenced.

%omposition havin, reverse transcriptase inhi-itor activit. US200/00/226


[0001] This application claims the benefit of H.-. provisional application 9o. 50&212,280 filed ;ay %0, 2001, incorporated herein by reference in its entirety. FIELD OF !E I"#E" IO" [0002] The field of the invention is antiviral compositions. $A%&'(OU"D OF !E I"#E" IO" [000%] Treatment of viral infections is freDuently limited by the availability, tolerability, and cost of $no#n and approved pharmacological agents. ;oreover, even if one or more antiviral agents are relatively #ell tolerated *physically as #ell as financially+, resistance to such agents tends to develop rather Duic$ly. Therefore, there is a continuing need for novel antiviral compositions that are #ell tolerated and relatively ine!pensive. DE AILED DES%(I+ IO" [0002] The inventors recogni=ed that various plant e!tracts e!hibit significant antiviral activity and that reverse transcriptase inhibitors *.TI+ may be isolated for such plant e!tracts. ;oreover, the inventors contemplate that such .TIs can be characteri=ed and&or synthesi=ed de novo. [0003] 0articularly contemplated plants include "bies #ebbiana, "cacia spec., "cacia

"rabia, "grimonia eupatoria, "Juga decumbens, "llium cepa, "llium sativum, "loe vera, "ltemanthera philo!eroides or sessiles, "mmi maius, "ndographis paniculata, "pium graveolens, "pium leptophyllum, "rachis hypogaea, "rctium lappa, "mebia euhcroma, "sparagus racemosus, "stragalus spinosus, "stragalus lentingosis s#ainsonine, Guchenavia capita, Gryonia cretica ssp. ,ioica, Gryonia angustifolia, /amellia theifera, /amellia sinensis, /edrela toona, /hrysanthemum morifolium, /offea arabica, /optis chinesis, /optis teetoides, /optis Japonica, /oraria nepalensis, /oriandrum sativum, /urcuma longa, ,atura metel syn alba, ,aucus carota, <chinacea angustiflora and purpurea, <chinacea simulata, <chinacea pallida, <pimedium grandiflorum, <pimedium sagittatum, <pimedium sinense, <pilobium angustifolium, <rigeron /anadensis, <ugenia or -y=igium claviflorum, agara !antho!, oeniculum vulgarel, 6ardenia coronaria, 6aultheria trichophylla, 6lycine ma!, 6lycyrrhi=a labra, 6ossypium herbaceum, Ceracleum sphondylium, Cypericum perforatum, Cypericum Japonicum, Cyssopus officinalis, 7asminum officinale, )ithospermum erythrorhi=on, )onicera Japonica, )uffa luffa, )ycopus europaeus, ;agnolia officinalis, ;allotus repandus, ;allotus philippinesis, ;atricaria chamomil, ;atricaria recutitia, ;elissa parviflora, ;elissa officinalis, ;omordica balsamina, ;omordica charantia, 9arcissus ta=etta, 9arcissus pseudonarcissus, Benthera rosea, 0aeonia spec., 0apaver somniferum, 0erilla frutescens, 0hyllanthus niruri, 0inus $oraicenis, 0inus parviflora, 0iper nirgum, 0lumeria rubra, 0olyantha suberosa, 0runella vulgaris, 0runus ba$ariensis, 0runus amygdalus, 0soralea corylifolia, .andia dunatorum, .aphanus sativus, .heum palmatum, .hus coriaria, .hus chinesis, .icinus communis, .osmarinus officinalis, -alvia miltiorhi=a and officinalis, -ambucus ebulus, -aussurea lappa, -cilla griffithii, -cutellaria baicalensis baiealein, -edum sediforme, -enecio scandens, -enecio aereus, -$immia laureola, -olarium niporum, -#ertia franchetiana, Terminalia chebula, Terminalia catappa, Terminalia alata, Thula occidentalis, Trapalaponica spec., Trichosanthes dioica, Trichosanthes $irilo#ii, Hrtica dioica, Viola yeodensis, Ioodfordia fruticosa, Iood#ardia spec., and >ano!ylum nitidum. Co#ever, in alternative aspects many plants other than the above'listed plants are also contemplated. In fact, all plants are contemplated that e!hibit antiviral activity. [0005] Iith respect to the identification of an .TI in contemplated plants, it should be appreciated that numerous assays are $no#n in the art, and can readily be adapted to a screening process in #hich a fractions of a plant e!tract are screened for .TI activity. or e!ample, H.-. 0at. 9o. 5,1%0,0%5 to )oeb et al. describes a high throughput assay system in #hich positive selective pressure is employed to select and&or identify an .TI. Bnce a fraction has been identified as having .TI activity, it is contemplated that further separation of the components in that fraction #ill eventually lead to an isolated *single or comple!+ compound.

[000F] It is still further contemplated that such isolated compounds may then be characteri=ed using various forms of mass spectroscopy *e.g., <-;-, "G';-, 6/' ;-, etc.+, HV', I.', and VI-'spectroscopy, atom absorption spectroscopy, various forms of 9;. *?1AC'9;., ?1%A/'9;., 9B<'9;., etc.+, or other analytical method. Ihile not limiting to the inventive subJect matter, it is preferred that such characteri=ation methods #ill lead to a chemical structure of the .TI, #hich may be employed to synthesi=e the .TI de novo, or to modify the structure to arrive at an .TI #ith improved or altered physico'chemical properties. [0008] 0articularly contemplated modifications of isolated and characteri=ed .TIs include increased specificity to#ards the viral polymerase over non'specific interactions #ith non'reverse transcriptase molecules in a cell or biological system, higher affinity of the modified .TI to#ards the reverse transcriptase, reduced to!icity, increased solubility, etc. [0001] /onseDuently, it is contemplated that pharmacological composition comprises a synthetic reverse transcriptase inhibitor having a structure of a molecule that is present in a plant e!tract demonstrated to have an antiviral effect, #herein the molecule produces at least in part of the antiviral effect. [0010] Thus, specific embodiments and applications of compositions having reverse transcriptase inhibitor activity have been disclosed. It should be apparent, ho#ever, to those s$illed in the art that many more modifications besides those already described are possible #ithout departing from the inventive concepts herein. The inventive subJect matter, therefore, is not to be restricted e!cept in the spirit of the appended contemplated claims. ;oreover, in interpreting both the specification and the contemplated claims, all terms should be interpreted in the broadest possible manner consistent #ith the conte!t. In particular, the terms KcomprisesK and KcomprisingK should be interpreted as referring to elements, components, or steps in a non'e!clusive manner, indicating that the referenced elements, components, or steps may be present, or utili=ed, or combined #ith other elements, components, or steps that are not e!pressly referenced.

reatment o1 vir2s 2sin, chelator and antiviral a,ent US2002102223


[0001] This application claims the benefit of H.-. provisional application 9o. 50&212,281 filed ;ay %0, 2001, incorporated herein by reference in its entirety. FIELD OF !E I"#E" IO"

[0002] The field of the invention is antiviral compositions. $A%&'(OU"D OF !E I"#E" IO" [000%] Viral infections are unfortunately an almost unavoidable challenge to the health of most human and other mammals, and #hile many viral infections are successfully cleared by the immune system of the infected individual before substantial damage arises, some viral infections lead to severe damage or even death. There are many $no#n antiviral drugs, ho#ever, all or almost all of them suffer from one or more disadvantages, most notably adverse side'effects, built'up of viral resistance, complicated administration schedules, and often high cost. Therefore, there is a need for simple and effective antiviral compositions that are #ell tolerated, simple to administer, and relatively ine!pensive. DE AILED DES%(I+ IO" [0002] The inventors discovered that treatment of a viral infection can be significantly improved by coadministration of an antiviral agent #ith a chelator. ;ore specifically, the inventors contemplate that particularly suitable chelators deplete the viral environment sufficiently to promote disintegration of the viral envelope. [0003] /onseDuently, the inventors contemplate a pharmacological composition that includes an antiviral agent and a chelator in a Duantity sufficient to reduce a serum concentration of a bivalent metal in an amount of at least 23M. [0005] -uitable antiviral agents particularly include direct antiviral agents and indirect antiviral agents. "s used herein, the term Kdirect antiviral drugK refers to an agent that directly interferes #ith one or more viral components. or e!ample, virus protein specific antibodies, reverse transcriptase inhibitors or protease inhibitors are considered direct antiviral agents, because such compounds directly bind and to and&or reduce the activity of their respective viral target structures. "s also used herein, the term Kindirect antiviral drugK refers to a compound that indirectly interferes #ith a replication or propagation of a virus, and particularly include immunomodulatory agents *e.g., cyto$ines, various nucleoside analogs, and&or >n?2A@+. Co#ever, it should be appreciated that chelators are e!plicitly e!cluded from the definitions of direct and indirect antiviral compounds. [000F] <specially preferred antiviral compounds include plant e!tracts and&or one or more isolated compounds *isolated from the plant or synthesi=ed de novo+ that are present in a plant e!tract demonstrated to have an antiviral effect. 0articularly suitable

plants for contemplated e!tracts and isolated compounds include "bies #ebbiana: "cacia spec. "cacia "rabia: "grimonia eupatoria: "Juga decumbens: "llium cepa: "llium sativum: "loe vera: "ltemanthera philo!eroides or sessiles: "mmi maius: "ndographis paniculata: "pium graveolens: "pium leptophyllum: "rachis hypogaea: "rctium lappa: "mebia euhcroma: "sparagus racemosus: "stragalus spinosus: "stragalus lentingosis s#ainsonine: Guchenavia capita: Gryonia cretica ssp. ,ioica: Gryonia angustifolia: /amellia theifera: /amellia sinensis: /edrela toona: /hrysanthemum morifolium: /offea arabica: /optis chinesis: /optis teetoides: /optis Japonica: /oraria nepalensis: /oriandrum sativum: /urcuma longa: ,atura metel syn alba: ,aucus carota: <chinacea angustiflora and purpurea: <chinacea simulata: <chinacea pallida: <pimedium grandiflorum: <pimedium sagittatum: <pimedium sinense: <pilobium angustifolium: <rigeron /anadensis: <ugenia or -y=igium claviflorum: agara !antho!: oeniculum vulgarel: 6ardenia coronaria: 6aultheria trichophylla: 6lycine ma!: 6lycyrrhi=a labra: 6ossypium herbaceum: Ceracleum sphondylium: Cypericum perforatum: Cypericum Japonicum: Cyssopus officinalis: 7asminum officinale: )ithospermum erythrorhi=on: )onicera Japonica: )uffa luffa: )ycopus europaeus: ;agnolia officinalis: ;allotus repandus: ;allotus philippinesis: ;atricaria chamomil: ;atricaria recutitia: ;elissa parviflora: ;elissa officinalis: ;omordica balsamina: ;omordica charantia: 9arcissus ta=etta: 9arcissus pseudonarcissus: Benthera rosea: 0aeonia spec.: 0apaver somniferum: 0erilla frutescens: 0hyllanthus niruri: 0inus $oraicenis: 0inus parviflora: 0iper nirgum: 0lumeria rubra: 0olyantha suberosa: 0runella vulgaris: 0runus ba$ariensis: 0runus amygdalus: 0soralea corylifolia: .andia dunatorum: .aphanus sativus: .heum palmatum: .hus coriaria: .hus chinesis: .icinus communis: .osmarinus officinalis: -alvia miltiorhi=a and officinalis: -ambucus ebulus: -aussurea lappa: -cilla griffithii: -cutellaria baicalensis baiealein: -edum sediforme: -enecio scandens: -enecio aereus: -$immia laureola: -olarium niporum: -#ertia franchetiana: Terminalia chebula: Terminalia catappa: Terminalia alata: Thula occidentalis: Trapalaponica spec.: Trichosanthes dioica: Trichosanthes $irilo#ii: Hrtica dioica: Viola yeodensis: Ioodfordia fruticosa: Iood#ardia spec. >ano!ylum nitidum: [0008] Iith respect to the chelator it is generally contemplated that all chelating agents are suitable for use in conJunction #ith the teachings presented herein so long as such chelators *a+ reduce serum concentration of a bivalent metal *e.g., /a?2@A and ;g?2@A+ in an amount of at least 23M, and *b+ are at least partially effective to promote viral disintegration at an administered dosage. 0articularly contemplated bivalent metals include /a?2@A and ;g?2@A. 0articularly preferred chelators include 1,2'Gis*2'aminopheno!y+ethane'9,9,9E,9E'tetraacetic acid, <thylenebis*o!yethylenenitrilo+tetraacetic acid, 1,2'bis*2'aminopheno!y+ethane' 9,9,9E,9E'tetraacetic acid tetra$is*aceto!ymethyl ester+, trans1,2' diaminocyclohe!ane'tetraacetic acid, and diethyllenetriamine'pentaacetic acid,

trimethylaminetricarbo!ylic acid, poly*aspartic acid+, and poly*glutamic acid+, ethylenediamine'9,9,9E,9E'tetraacetic acid, and <6T". [0001] It is further contemplated that suitable compositions #ill reduce the viral serum titer of a virus in an amount of at least 10M *e.g., as determined by .T'0/.+, and especially contemplated viruses include retroviruses *e.g., CIV, C/V+, ds,9" and ss,9" viruses. [0010] Iith respect to the administration of contemplated compositions, it should be recogni=ed that various protocols are suitable, and especially contemplated protocols include substantially simultaneous administration of the chelator *e.g., coadministration in a single tablet+, or administration of the chelator *or antiviral agent+ #hile there is a measurable concentration of the antiviral agent *or chelator+ in the patient. or e!ample, it is contemplated that suitable antiviral agents may be orally administered, #hile the chelator is parenterally administered *e.g., via inJection or mucosal presentation+. [0011] /onseDuently, the dosage and formulation of contemplated antiviral agents and chelators may vary substantially, ho#ever, it is preferred that the antiviral agent is administered in approved and&or $no#n dosages and formulations. -imilarly, it is preferred that dosages and formulations of appropriate chelators are identical or similar to those $no#n in the art. [0012] Thus, specific embodiments and applications of antiviral treatments using a chelator and an antiviral agent have been disclosed. It should be apparent, ho#ever, to those s$illed in the art that many more modifications besides those already described are possible #ithout departing from the inventive concepts herein. The inventive subJect matter, therefore, is not to be restricted e!cept in the spirit of the appended contemplated claims. ;oreover, in interpreting both the specification and the contemplated claims, all terms should be interpreted in the broadest possible manner consistent #ith the conte!t. In particular, the terms KcomprisesK and KcomprisingK should be interpreted as referring to elements, components, or steps in a non'e!clusive manner, indicating that the referenced elements, components, or steps may be present, or utili=ed, or combined #ith other elements, components, or steps that are not e!pressly referenced.

ime release chelators US2002102213


[0001] This application claims the benefit of H.-. provisional application 9o.

50&212,2F8 filed ;ay %0, 2001, incorporated herein by reference in its entirety. FIELD OF !E I"#E" IO" [0002] The field of the invention is antiviral compositions. $A%&'(OU"D OF !E I"#E" IO" [000%] 9umerous antiviral drugs are $no#n in the art, ho#ever, all or almost all of them suffer from one or more disadvantages. 0articularly problematic in the administration is of such drugs is their relatively lo# solubility and&or comparably short serum half'life time. /onseDuently, many patients need to follo# a strict regimen to maintain effective serum concentration of such drugs, freDuently resulting in repeated disruptions of an other#ise productive lifestyle. Therefore, there is a need for improved antiviral compositions that are #ell tolerated, simple to administer, and maintain a relatively long serum half'life. DE AILED DES%(I+ IO" [0002] The inventors contemplate that treatment of a viral infection can be significantly improved by administration of a chelator in a time'release formulation. urthermore, the inventors contemplate that the chelator is co'administered in a time' release formulation #ith a second or further agent #ith antiviral effect *#hich may be administered follo#ing a conventional protocol or in a second time release formulation+. /ontemplated viruses include retroviruses *e.g., CIV, C/V+, ss,9" and ds,9". [0003] /onseDuently, the inventors contemplate a pharmacological composition that includes an a chelator in a time release formulation in a concentration such that a single administration of the chelator reduces the serum concentration of a bivalent metal in an amount of at least 20M for a period of at least 8 hours, more preferably at least %0M for a period of at least 10 hours, and most preferably at least 20M for a period of at least 12 hours. [0005] Iith respect to the chelator it is generally contemplated that all chelating agents are suitable for use in conJunction #ith the teachings presented herein so long as such chelators *a+ reduce serum concentration of a bivalent metal *e.g., /a?2@A and ;g?2@A+ in an amount of at least 23M, and *b+ are at least partially effective to promote viral disintegration at an administered dosage. 0articularly contemplated bivalent metals include /a?2@A and ;g?2@A. 0articularly preferred chelators include 1,2'Gis*2'aminopheno!y+ethane'9,9,9E,9E'tetraacetic acid,

<thylenebis*o!yethylenenitrilo+tetraacetic acid, 1,2'bis*2'aminopheno!y+ethane' 9,9,9E,9E'tetraacetic acid tetra$is*aceto!ymethyl ester+, trans'1,2' diaminocyclohe!ane'tetraacetic acid, and diethyllenetriamine'pentaacetic acid, tri' methylaminetricarbo!ylic acid, poly*aspartic acid+, and poly*glutamic acid+, ethylenediamine'9,9,9E,9E'tetraacetic acid, and <6T". [000F] There are numerous methods of preparing a time release formulation $no#n in the art, all of #hich are contemplated suitable for use in conJunction #ith the teachings herein. Co#ever, particularly contemplated time release formulations include ion e!change resins, encapsulations #ith acid or base resistant coatings, compacting the formulation to control solvation, slo#'melting carriers, en=yme' degradable carriers, etc. [0008] ,epending on the amount chelator in contemplated compositions, it is contemplated that the viral titer in the serum of a patient infected #ith the virus #ill decrease at least 10M for at least 2 hours, more preferably at least 23M for at least 5 hours, and most preferably at least 20M for at least 8 hours after administration of a single dose of contemplated compounds. [0001] It should further be appreciated that contemplated compositions may further comprise direct antiviral agents and&or indirect antiviral agents. "s used herein, the term Kdirect antiviral agentK refers to an agent that directly interferes #ith one or more viral components. or e!ample, virus protein specific antibodies, reverse transcriptase inhibitors or protease inhibitors are considered direct antiviral agents, because such compounds directly bind and to and&or reduce the activity of their respective viral target structures. "s also used herein, the term Kindirect antiviral agentK refers to a compound that indirectly interferes #ith a replication or propagation of a virus, and particularly include immunomodulatory agents *e.g., cyto$ines, various nucleoside analogs, and&or >n?2@A+. Co#ever, it should be appreciated that chelators are e!plicitly e!cluded from the definitions of direct and indirect antiviral compounds. [0010] Iith respect to the administration of contemplated compositions, it should be recogni=ed that various protocols are suitable, and especially contemplated protocols include oral and parenteral administration *e.g., via a tablet, syrup, inJection, suppository, topical administration transcutaneous administration, etc.+. /onseDuently, the dosage and formulation of contemplated compositions may vary substantially. Co#ever, it is generally preferred that a single dosage is #ithin the range of about 10 mg to about %000 mg. [0011] Thus, specific embodiments and applications of chelators in time release format have been disclosed. It should be apparent, ho#ever, to those s$illed in the art

that many more modifications besides those already described are possible #ithout departing from the inventive concepts herein. The inventive subJect matter, therefore, is not to be restricted e!cept in the spirit of the appended contemplated claims. ;oreover, in interpreting both the specification and the contemplated claims, all terms should be interpreted in the broadest possible manner consistent #ith the conte!t. In particular, the terms KcomprisesK and KcomprisingK should be interpreted as referring to elements, components, or steps in a non'e!clusive manner, indicating that the referenced elements, components, or steps may be present, or utili=ed, or combined #ith other elements, components, or steps that are not e!pressly referenced.

)ethods o1 treatment o1 !I#4associated conditions US2002102232


[0001] This application claims the benefit of H.-. provisional application 9o. 50&212,2F1 filed ;ay %0, 2001, incorporated herein by reference in its entirety. FIELD OF !E I"#E" IO" [0002] The field of the invention is treatment of CIV'associated conditions. $A%&'(OU"D OF !E I"#E" IO" [000%] ;ost patients infected #ith the CIV virus #ill develop "I,-, reflecting a brea$do#n in their immune systemEs capability to #ard off foreign and KselfK' generated antigens. or e!ample, (aposi sarcoma, and numerous bacterial and yeast infections are relatively common diseases associated #ith "I,-. Typically, these secondary diseases are treated #ith drugs that specifically target the etiologic agent *e.g., sarcoma cell, bacterium, or virus+ of those diseases, thereby often increasing an already long list of undesired side effects brought on by attempts to control the propagation of the CIV virus. DE AILED DES%(I+ IO" [0002] The inventors contemplate that treatment of CIV related conditions can be significantly improved by administration of a composition that comprises at least one of a chelator and an antiviral agent, #herein the antiviral agent comprises a plant e!tract, or a synthetic or isolated compound from a plant that is demonstrated to have an antiviral effect. [0003] The term KCIV related conditionK as used herein refers to intrinsic and

e!trinsic challenges to an immune system that may develop into an apparent *i.e., detectable by diagnotic tools+ disease #hile the patient has a detectable CIV serum virus titer. 0articularly contemplated conditions include bacterial infections *e.g., pneumocystis carnii, tuberculosis, salmonellosis, mycobacterium avium comple!, etc.+, viral infections *e.g., cytomegalovirus, herpes simple!, hepatitis, varicella =oster, <pstein'barr, etc.+, fungal infections *e.g., candidiasis, cryptococcal meningitis, histoplasmosis, etc.+, parasite infections *e.g., to!oplasmosis, cryptosporidiosis, etc.+, and (aposi sarcoma. [0005] -uitable compositions are described in copending provisional patent applications #ith the title KTreatment of Virus Hsing /helator and "ntiviral "gentK by Gruce Calstead et al., filed on or about ;ay %0, 2001, KTime .elease /helatorsK by Gruce Calstead et al., filed on or about ;ay %0, 2001, and KTime .elease reverse transcriptase inhibitorsK by Gruce Calstead et al., filed on or about ;ay %0, 2001, all of #hich are incorporated by reference herein. [000F] In a preferred aspect of the inventive subJect matter, a method of treating a patient comprises one step in #hich a patient infected #ith CIV is diagnosed #ith an CIV related condition. In a further step, a composition is administered to the patient that comprises at least one of a chelator and an antiviral agent, #herein the antiviral agent comprises a plant e!tract or a synthetic or isolated compound from a plant that is demonstrated to have an antiviral effect. It should be recogni=ed that all patients infected #ith an CIV virus may be treated using contemplated methods, ho#ever, patients #ith a /,2?@A count of less than 200 are particularly contemplated. /onseDuently, especially preferred patients include patients #ith developing or fully developed "I,-, #herein such patients may or may not receive pharmacological treatment. [0008] Iith respect to the administration of contemplated compounds, it should be appreciated that a particular dosage and regimen #ill typically depend on the particular CIV'related condition. It is generally contemplated that the dosage, route and formulation is substantially identical or similar to those described in the copending provisional applications. Co#ever, #here appropriate, alternative dosages, routes, and formulations may be employed, and in fact all dosages formulations and routes are contemplated that result in a positive response of the patient to the administration. [0001] Thus, specific embodiments and applications of treatment of CIV'related conditions have been disclosed. It should be apparent, ho#ever, to those s$illed in the art that many more modifications besides those already described are possible #ithout departing from the inventive concepts herein. The inventive subJect matter, therefore,

is not to be restricted e!cept in the spirit of the appended contemplated claims. ;oreover, in interpreting both the specification and the contemplated claims, all terms should be interpreted in the broadest possible manner consistent #ith the conte!t. In particular, the terms KcomprisesK and KcomprisingK should be interpreted as referring to elements, components, or steps in a non'e!clusive manner, indicating that the referenced elements, components, or steps may be present, or utili=ed, or combined #ith other elements, components, or steps that are not e!pressly referenced

ime release reverse transcriptase inhi-itors US2002103553


[0001] This application claims the benefit of H.-. provisional application 9o. 50&2122FF filed ;ay %0, 2001, incorporated herein by reference in its entirety. FIELD OF !E I"#E" IO" [0002] The field of the invention is antiviral compositions. $A%&'(OU"D OF !E I"#E" IO" [000%] 9umerous antiviral drugs are $no#n in the art, ho#ever, all or almost all of them suffer from one or more disadvantages. 0articularly problematic in the administration is of such drugs is their relatively lo# solubility and&or comparably short serum half'life time. /onseDuently, many patients need to follo# a strict regimen to maintain effective serum concentration of such drugs, freDuently resulting in repeated disruptions of an other#ise productive lifestyle. Therefore, there is a need for improved antiviral compositions that are #ell tolerated, simple to administer, and maintain a relatively long serum half'life. DE AILED DES%(I+ IO" [0002] The inventors contemplate that treatment of a viral infection can be significantly improved by administration of an antiviral agent in a time'release formulation. ;ore specifically, the inventors contemplate that a reverse transcriptase inhibitor in a time'release formulation is administered to a patient suffering from a viral infection. 0articularly contemplated viruses include retroviruses *e.g., CIV, C/V+, ss,9" and ds,9". [0003] In an especially preferred aspect, the reverse transcriptase inhibitor *.TI+ is an e!tract from a plant that is $no#n to have an antiviral effect, or an isolated or

synthetically prepared compound that can be found in a plant $no#n to have an antiviral effect. <specially contemplated plants include "bies #ebbiana: "cacia spec. "cacia "rabia: "grimonia eupatoria: "Juga decumbens: "llium cepa: "llium sativum: "loe vera: "ltemanthera philo!eroides or sessiles: "mmi maius: "ndographis paniculata: "pium graveolens: "pium leptophyllum: "rachis hypogaea: "rctium lappa: "mebia euhcroma: "sparagus racemosus: "stragalus spinosus: "stragalus lentingosis s#ainsonine: Guchenavia capita: Gryonia cretica ssp. ,ioica: Gryonia angustifolia: /amellia theifera: /amellia sinensis: /edrela toona: /hrysanthemum morifolium: /offea arabica: /optis chinesis: /optis teetoides: /optis Japonica: /oraria nepalensis: /oriandrum sativum: /urcuma longa: ,atura metel syn alba: ,aucus carota: <chinacea angustiflora and purpurea: <chinacea simulata: <chinacea pallida: <pimedium grandiflorum: <pimedium sagittatum: <pimedium sinense: <pilobium angustifolium: <rigeron /anadensis: <ugenia or -y=igium claviflorum: agara !antho!: oeniculum vulgarel: 6ardenia coronaria: 6aultheria trichophylla: 6lycine ma!: 6lycyrrhi=a labra: 6ossypium herbaceum: Ceracleum sphondylium: Cypericum perforatum: Cypericum Japonicum: Cyssopus officinalis: 7asminum officinale: )ithospermum erythrorhi=on: )oniceraJaponica: )uffa luffa: )ycopus europaeus: ;agnolia officinalis: ;allotus repandus: ;allotus philippinesis: ;atricaria chamomil: ;atricaria recutitia: ;elissa parviflora: ;elissa officinalis: ;omordica balsamina: ;omordica charantia: 9arcissus ta=etta: 9arcissus pseudonarcissus: Benthera rosea: 0aeonia spec.: 0apaver somniferum: 0erilla frutescens: 0hyllanthus niruri: 0inus $oraicenis: 0inus parviflora: 0iper nirgum: 0lumeria rubra: 0olyantha suberosa: 0runella vulgaris: 0runus ba$ariensis: 0runus amygdalus: 0soralea corylifolia: .andia dunatorum: .aphanus sativus: .heum palmatum: .hus coriaria: .hus chinesis: .icinus communis: .osmarinus officinalis: -alvia miltiorhi=a and officinalis: -ambucus ebulus: -aussurea lappa: -cilla griffithii: -cutellaria baicalensis baiealein: -edum sediforme: -enecio scandens: -enecio aereus: -$immia laureola: -olarium niporum: -#ertia franchetiana: Terminalia chebula: Terminalia catappa: Terminalia alata: Thula occidentalis: Trapalaponica spec.: Trichosanthes dioica: Trichosanthes $irilo#ii: Hrtica dioica: Viola yeodensis: Ioodfordia fruticosa: Iood#ardia spec. >ano!ylum nitidum. [0005] "lternatively it should be appreciated that .TIs other than plant e!tracts are also appropriate, and such agents particularly include $no#n and commercially available .TIs as indicated in A$LE 1 ,rug 6eneric 9ame Grand 9ame "nalogue %T/ lamivudine <pivir&%T/ cytidine

"G/ abacavir >iagen guanosine ">T =idovudine .etrovir thymidine dd/ =alcitabine CIVI, cytidine ddI didanosine Vide! adenosine d2T stavudine >erit thymidine 'dd" lodenosine adenosine

T/ emtricitabine /oviracil cytidine 0;<" adefovir dipivo!il 0reveon adenosine 0;0" tenofovir disopro!il adenosine [000F] In further especially preferred aspects, contemplated antiviral agents may include a chelating agent that chelates a bivalent metal ion, preferably ;g?2@ Aand&or /a?2@A. <specially preferred chelating agents include <,T", <6T", 1,2'Gis*2' aminopheno!y+ethane'9,9,9E,9E'tetraacetic acid, <thylenebis*o!yethylenenitrilo+tetraacetic acid, 1,2'bis*2'aminopheno!y+ethane' 9,9,9E,9E'tetraacetic acid tetra$is*aceto!ymethyl ester+, trans'1,2' diaminocyclohe!ane'tetraacetic acid, and diethyllenetriamine'pentaacetic acid, trimethylaminetricarbo!ylic acid, poly*aspartic acid+, and poly*glutamic acid+. [0008] There are numerous $no#n methods of preparing a time'release formulation, and all of the $no#n methods are contemplated suitable for use in conJunction #ith the teachings herein. 0articularly contemplated time release formulations include ion e!change resins, encapsulations #ith acid or base resistant coatings, compacting the formulation to control solvation, slo#'melting carriers, en=yme'degradable carriers, etc. [0001] Iith respect to the dosage of contemplated compositions, it is contemplated that the .TI is present in a single dose in a concentration such that the viral titer is reduced at least 20M over a period of at least 5 hours, more at least %0M over a period of at least 8 hours, and most preferably at least 23M over a period of at least 12 hours. urthermore, it is contemplated that #here contemplated compositions further comprise a chelating agent, the chelating agent is present in a single dose in a

concentration such that the serum ;g?2@ Aand&or /a?2@ Aconcentration is reduced at least 20M over a period of at least 5 hours, more preferably at least %3M over a period of at least 12 hours, and most preferably at least 23M over a period of at least 12 hours. [0010] Iith respect to the administration of contemplated compositions, it should be recogni=ed that various protocols are suitable, and especially contemplated protocols include oral, topical, and parenteral administration. /onseDuently, the formulation of contemplated compositions may vary substantially, ho#ever, it is preferred that the .TI is administered in approved and&or $no#n formulations. [0011] Thus, specific embodiments and applications of time release .TIs have been disclosed. It should be apparent, ho#ever, to those s$illed in the art that many more modifications besides those already described are possible #ithout departing from the inventive concepts herein. The inventive subJect matter, therefore, is not to be restricted e!cept in the spirit of the appended contemplated claims. ;oreover, in interpreting both the specification and the contemplated claims, all terms should be interpreted in the broadest possible manner consistent #ith the conte!t. In particular, the terms KcomprisesK and KcomprisingK should be interpreted as referring to elements, components, or steps in a non'e!clusive manner, indicating that the referenced elements, components, or steps may be present, or utili=ed, or combined #ith other elements, components, or steps that are not e!pressly referenced.

)ethods 1or testin, o6idative stress US6541263


[0002] This is a continuation'in'part of allo#ed H.-. application -er. 9o. 01&23%,22%, filed eb. 11, 1111, no# H.-. 0at. 9o. 5,153,F1F #hich is incorporated by reference herein. FIELD OF !E I"#E" IO" [000%] The field of the invention is detection and Duantification of o!idative stress in a subJect. $A%&'(OU"D [0002] It is by no# common $no#ledge that stress in mammalian subJects develops directly or indirectly into a display of o!ygenated species, #hich tends to change the usually reduced state of the body to a hypero!ygenated state. This hypero!ygenated

state includes generation and reaction of hydro!ides, pero!ides and free radical species, #hich are thought to be implicated in physiological imbalance and actual physical damage. 0hysical damage can produce pathological states, #hich for e!ample, may lead to atherosclerotic plaDues. -uch plaDues often result in the deposition of lipids and may further lead to bloc$age of arteries that can cause a cessation of blood flo# to the heart #ith a resulting heart attac$. This is one of many human disease states that are thought to be caused by free radical attac$ from the hypero!ygenated state caused by stress. ,espite the relatively large body of information lin$ing o!idative stress to various diseases and&or disease states, there is still an appreciable need for suitable mar$ers and test systems to determine the level of o!idative stress in a simple and ine!pensive manner. [0003] ;alondialdehyde is a component of normal urine, and its presence can be determined using relatively e!pensive and typically stationary eDuipment such as spectrophotometers, fluorometers, high performance liDuid chromatographs and gas chromatograph mass spectrometers. -uch eDuipment typically enables an operator to determine not only the Duantity of a particular aldehyde, but also to determine the chemical nature of a particular molecule #ith an aldehyde function. Hnfortunately, the operator of such eDuipment needs to be highly trained, and the #eight and si=e of the eDuipment is generally prohibitive for point'of'care tests. [0005] "lternatively, a broad spectrum of chemically distinct aldehydes may be detected by mi!ing a drop of sample solution *#hich may contain the aldehyde+ #ith 2 ml of F2 percent sulfuric acid in a test tube *disclosed at page %13 in KNualitative "nalysis by -pot TestsK, Third <dition, authored by . eigl and published by <lsevier 0ublishing /ompany, Inc.+. " small amount of solid chromatropic acid *1,8' dihydro!ynapthlanene'%,5'disulfate+ is added to the mi!ture, and the test tube is heated in a 50[deg.] /. #ater bath for about ten minutes. If an aldehyde is present, a bright violet color appears in the test solution. Ihile the test is relatively non'specific for a particular aldehyde, the sensitivity of the test is reportedly about % ppm. Co#ever, the reaction mi!ture typically reDuires vigorous heating for at least 10 minutes to provide an at least semi'Duantitative and reliable test result. [000F] In yet another method generally applicable to aldehydes, described at pages %%1'%20 of the eigl publication, a drop of aDueous *or alcoholic+ solution suspected of containing an aldehyde is treated on a spot plate #ith a drop of sulfurous acid and a drop of fuchsin&sulfuric acid and allo#ed to react on the plate. " red to blue color appears #ithin about t#o to thirty minutes, according to the amount of aldehyde present in the test solution being tested. -uch test is reportedly sensitive to about one microgram of formaldehyde in the drop of solution being tested. "lthough the fuchsin&sulfuric acid reaction can advantageously be performed at room temperature,

the test results tend to vary depending on the time allo#ed for the reaction. [0008] "lthough various Duantitative and Dualitative tests for aldehydes are $no#n in the art, all or almost all of them suffer from one or more disadvantage. ;oreover, despite the e!istence of $no#n tests, it has never been appreciated that such tests can be applied to malondialdehyde in urine to detect o!idative stress. Thus, there is still a need to provide methods and apparatus for detecting o!idative stress in subJects. SU))A(* OF !E I"#E" IO" [0001] It has been discovered that the o!idative stress state of a person can be measured from the release into the urine of an aldehyde, and particularly malondialdehyde, and that an aldehyde'reactive chromogen based calorimetric test can measure the released aldehyde in a rapid, easily performed test. [0010] In particular, a method of determining o!idative stress in a subJect has one step in #hich presence of an aldehyde in a biological fluid of a subJect is correlated #ith an o!idative stress in the subJect. In another step, a test reagent comprising a pC regulator, a reducing agent, and an aldehyde'reactive chromogen is provided, and the test reagent is combined #ith the biological fluid to produce an aldehyde'modified chromogen. In yet another step, a color of the aldehyde'modified chromogen is correlated #ith the o!idative stress. [0011] In one aspect of the inventive subJect matter, any biological fluid is considered suitable for use #ith the test, and especially preferred fluids include saliva, serum, plasma, and spinal fluid, most preferably urine. It is further contemplated that such fluids are derived from a mammalian system *e.g., human, live stoc$, pet, or cell culture+. [0012] In a further aspect of the inventive subJect matter, the aldehyde comprises a dialdehyde, and especially contemplated dialdehydes include malondialdehyde. 0articularly preferred pC regulators comprise a buffer or an acid, such as phosphoric acid and&or glacial acetic acid, and reducing agents typically have a sulfur *e.g., sodium metabisulfide+ and&or phosphorous atom *e.g., T/<0+. urther preferred aldehyde'reactive chromogens *e.g., fuchsin+ include a reactive group that selectively reacts #ith an aldehyde and thereby shift their absorption ma!imum to#ards higher or lo#er #avelength in a concentration dependent manner. DE AILED DES%(I+ IO" [001%] " test $it for determination of o!idative stress in a subJect generally comprises

a test reagent #ith a pC regulator, a reducing agent, and an aldehyde'reactive chromogen, #herein the aldehyde'reactive chromogen in the test reagent reacts #ith an aldehyde from a biological fluid to form an aldehyde'modified chromogen, and #herein the aldehyde'modified chromogen has a color intensity that correlates #ith the o!idative stress in the subJect. [0012] /onseDuently, a method of determining o!idative stress in a subJect has a step in #hich the presence of an aldehyde in a biological fluid of a subJect is correlated #ith an o!idative stress in the subJect. In another step, a test reagent comprising a pC regulator, a reducing agent, and an aldehyde'reactive chromogen is provided, and the test reagent is combined #ith the biological fluid to produce an aldehyde'modified chromogen. In a yet further step, a color of the aldehyde'modified chromogen is correlated #ith the o!idative stress. [0013] In a particularly preferred aspect of the inventive subJect matter, a testing solution or reagent for testing for the presence of aldehyde in an aDueous solution comprises a solution of acetic acid, preferably about 20M acetic acid, and t#o additional ingredients designated herein as KIngredient "K and KIngredient GK. Ingredient " consists essentially of sodium metabisulfite, phosphoric acid, and deioni=ed #ater. The preferred proportions of the elements of ingredient " are about 18'22 grams sodium metabisulfite, 1'11 ml of concentrated phosphoric acid, and about 230'330 ml deioni=ed #ater. ;ost preferably, the proportions are 20 grams sodium metabisulfite, 10 ml phosphoric acid, and about 300 ml deioni=ed #ater. Ingredient G consists essentially of a mi!ture of basic fuchsin *certified grade+ and Ingredient " in the preferred proportions of about 0.23'0.33 grams basic fuchsin in about 10'110 ml of Ingredient ". ;ost preferably, the proportions are about 0.30 grams of basic fuchsin in about 100 ml of Ingredient ". [0015] The components of the reagent are mi!ed in the proportion of about 10 to 110 parts of 20M acetic acid, 1%.3'15.3 parts Ingredient ", and about 2.3'3.3 parts Ingredient G. "n alternative method of ma$ing the reagent is as follo#s. irst, dissolve 2 grams of sodium metabisulfite in 80 ml of deioni=ed #ater. Then, add 2 ml of concentrated phosphoric acid, and dilute the mi!ture #ith a Duantity of deioni=ed #ater sufficient to ma$e 100 ml of dilute mi!ture. Then add 0.3 gram of basic fuchsin, and about 10 grams of bone charcoal to decolori=e the mi!ture. .emove the charcoal by centrifuging and filtering the mi!ture. Then, to 100 ml of the decolori=ed solution, add 100 ml of 20M'20M glacial acetic acid, and finally, add 100 ml of deioni=ed #ater. The active components are present in the reagent made this #ay in about the same proportion as in the method previously described. [001F] The testing solution described above is preferably stored in individual, sealed

test'si=e ampoules or vials of conventional medical solution type. Ihen pac$aged in such a manner and stored in a cool, dry place, the sealed bottles or vials have an e!pected shelf storage life of at least 12 months. "ssurance of active testing solution may be achieved, as described belo#, by positive aldehyde test procedures. [0018] " test for the presence of malondialdehyde in an aDueous solution is then made by mi!ing about 1 ml of test solution *containing traces of aldehyde+ into about 0.2' 0.5 ml of testing solution formulated as above. If the mi!ture of the test sample and testing solution remains colorless after a #aiting period of about 2'3 minutes, the test is negative and the test sample therefore contains less than about 2 ppm aldehyde. "ny color change of the mi!ture indicates presence of aldehyde in the test solution in a concentration greater than about 2 ppm. " positive malondialdehyde test is preferably by Duality control techniDues made before testing the test samples to assure that the testing solution is properly formulated or that, for e!ample, the reagent bottles have not been replaced #ith other bottles containing non'testing solutions. [0011] The positive malondialdehyde test is preferably performed by inJecting 1 ml of available K0ositive "ldehyde Test -olution *-tandard+K into a bottle containing about 0.2'0.5 ml of the test solution. In appro!imately 2'3 minutes, the solution in the bottle should develop a pin$ish'purple color provided the bottle contains properly formulated aldehyde testing solution. Bther#ise, the bottle of Ktesting solutionK from #hich the test bottle #as selected should be discarded. The above'described positive test for aldehyde is sensitive to 10 ppm or more of aldehyde. or a 3 ppm, a positive test for aldehyde, 0.3 ml of deioni=ed #ater is used. " color less intense than that of the 10 ppm aldehyde test is obtained for the 3 ppm aldehyde test. [0020] Gasic fuchsin is a purple po#der #hich reacts #ith aldehydes in the s$in, urine or blood plasma. Iith lo# or no aldehydes present, there is no color development. Iith moderate or high levels of aldehydes, color gradations are roughly dependent on the level of aldehydes present. The amino group of the fuchsin couples #ith the aldehyde to produce the pin$ to purple color appro!imately dependent on the amount of aldehyde present in the blood or urine. " 20M glacial acetic acid solution gives ma!imum color development for the fuchsin reaction. -odium metabisulfite ties up free o!ygen so that only the aldehydes react #ith the fuchsin group. Gasic fuchsin changes color in an acidic solution, relative to the amount of aldehyde present in the urine samples. The color developed depends on the pC, #hich is controlled by the amount of acid present. ;etabisulfite is used to stop the interference of o!ygen from air. <stablishing a nitrogen blan$et over the reagent mi!ture gives greater shelf life of the reagent to stop any o!ygen reaction #ith the reagent. The phosphoric acid stabili=es the pC in a rough adJustment and the acetic acid gives the fine acid pC stabili=ation.

[0021] In alternative aspects of the inventive subJect matter, it should be appreciated that the order, composition, and relative molar ratios of the reagents may vary substantially, and numerous modifications are contemplated so long as the test reagent comprises a pC regulator, a reducing agent *#hich may even be optional+, and an aldehyde'reactive chromogen. [0022] or e!ample, the pC regulator need not necessarily be limited to phosphoric acid and glacial acetic acid, and alternative pC regulators may include a buffer, an organic, an inorganic acid, or any reasonable combination thereof. or e!ample, depending on the desired pC or pC range, suitable pC regulators may include a glycin'C/) buffer, a citrate buffer, a phosphate buffer, an acetate buffer, etc., and appropriate acids may include nitric acid, sulfuric acid, hydrochloric acid, and so forth. -till further, it should be appreciated that #here the reaction bet#een the aldehyde and the aldehyde'reactive chromogen is base'facilitated or base'cataly=ed, organic or inorganic bases may be employed, and contemplated bases include sodium hydro!ide, potassium hydro!ide, deprotonated #ea$ organic acids, and any reasonable combination thereof. [002%] Iith respect to the reducing agent, it is contemplated that many alternative reducing agents are also appropriate, and alternative reducing agents include agents #ith a sulfur and&or phosphorous atom. or e!ample, #here cost effectiveness is especially desirable mercaptoethanol, dithioerythrol *,T<+ or dithiothreitol *,TT+ may be utili=ed. Bn the other hand, #here the obJectionable odor of sulfur'based reagents is to be circumvented, phosphorous based reducing agents such as tris*2' carbo!yethyl+phosphine *T/<0+ may be employed. Ihile the use of a reducing agent is generally preferred, it is also contemplated that no reducing agent may be necessary at all, especially #here the remaining reagents&fluids have been purged *e.g., #ith argon+ and&or have been $ept under nitrogen or other o!ygen free atmosphere. [0022] In yet further contemplated aspects, the aldehyde'reactive chromogen need not be limited to fuchsin, and various alternative aldehyde'reactive chromogens are contemplated. It is generally contemplated that suitable aldehyde'reactive chromogens comprise an aromatic system #hich may further be conJugated #ith at least another double' or triple bond containing system, and it is especially preferred that such aldehyde'reactive chromogens #ill have an absortion ma!imum of bet#een about 220 nm to appro!imately 100 nm. Iith respect to the molar e!tinction coefficient, it is generally preferred that the molar e!tinction coefficient if the aldehyde'modified chromogen is bet#een 100'100000, more preferably bet#een 1000 and 30000, and most preferably bet#een 10000 and %3000. It is further contemplated that suitable aldehyde'reactive chromogens have at least one reactive group that specifically reacts

#ith an aldehyde, and particularly contemplated reactive groups include nucleophilic groups such as '9C, '9C2, '-C, 'BC, etc. -uitable aldehyde'reactive chromogens are contemplated to have an absorption ma!imum and a reactive group that selectively reacts #ith the aldehyde, #herein the absorption ma!imum e!hibits a hyperchromatic or hypochromatic shift #hen the reactive group reacts #ith the aldehyde. "lternatively, the ma!imum may be unaffected by the reaction of the reactive group, and it is then contemplated that the aldehyde'modified chromogen has *or looses+ an additional ma!imum #hen compared to the aldehyde'reactive chromogen. [0023] It is generally contemplated that the concentration of aldehyde'modified chromogen can be visually *i.e., in an non'automated manner+ determined, for e!ample, by employing a reference chart #hich may be part of a test $it. /ontemplated reference charts may thereby include a relative or arbitrary readout, or a semi'Duantitative or Duantitative readout. "lternatively, it is contemplated that the determination of the aldehyde may include an at least partially automated routine, and particularly contemplated routines may include a spectrophotometer *single or multiple #ave length+. [0025] Iith respect to molar proportions of alternative components, it should be appreciated that a particular composition #ill typically dictate particular molar proportions of the components, ho#ever, only such molar proportions are contemplated that #ill result in an observable and&or Duantifiable change in light absorption *typically HV&VI-+ #hen the aldehyde'reactive chromogen reacts #ith the aldehyde. Ihile it is generally contemplated that the change in absorption has a substantially linear dependence on the concentration of the aldehyde'modified chromogen *i.e., follo#s the )ambert'Geer la#+, non'linear dependence is also contemplated. or e!ample, #here the aldehyde generates a catalytic intermediate species, logarithmic or pseudo'logarithmic dependence may occur. [002F] It is generally contemplated that malondialdehyde *;,"+ and other related aldehydes are released from the brea$do#n of long chain polyunsaturated fatty acids by free radical attac$. Interestingly, high levels of ;," and related aldehydes are found in a variety of diseases and disease states other than o!idative stress. Therefore, it should be especially appreciated that the methods and compositions according to the inventive subJect matter may also be useful in detecting and&or confirming abnormal metabolism states, including coronary artery disease, type'1 and type'2 diabetes, and 0ar$inson disease. [0028] Thus, specific embodiments and applications of tests for o!idative stress have been disclosed. It should be apparent, ho#ever, to those s$illed in the art that many more modifications besides those already described are possible #ithout departing

from the inventive concepts herein. The inventive subJect matter, therefore, is not to be restricted e!cept in the spirit of the appended claims. ;oreover, in interpreting both the specification and the claims, all terms should be interpreted in the broadest possible manner consistent #ith the conte!t. In particular, the terms KcomprisesK and KcomprisingK should be interpreted as referring to elements, components, or steps in a non'e!clusive manner, indicating that the referenced elements, components, or steps may be present, or utili=ed, or combined #ith other elements, components, or steps that are not e!pressly referenced.

A" I#I(AL )E !ODS A"D %O)+OSI IO"S 7O0105545


Field o1 he Invention The field of the invention is antiviral compositions. $ac8,ro2nd o1 he Invention Viral infections are relatively common infectious diseases, and various methods of treating a viral infection available to a practitioner. In one method of treating a viral infection, the immune response of an immune system is stimulated. or e!ample, in some instances the Thl response of the patient can be increased relative to the Th2 response. "n increase in the Thl response is thought to be beneficial because many viral infections are associated #ith a shift in the cyto$ine profile to#ard a Th2 response, and a bias to#ards a Thl response is $no#n to be facilitated by several approaches. In one approach, cyto$ines are administered to modulate the Thl&Th2 balance to#ards a Thl'type response. or e!ample, (night et al. postulate that treatment #ith I)'12 *Interleu$ine'12+, a cyto$ine that promotes the development of Thl cells, may be used as a treatment for "I,- since I) 12 administration has been sho#n to be effective at restoring cell'mediated immunity in mice infected #ith mouse "I,- virus or #ith .)V [(night, -. /. and 0atterson, -., "nnu. .ev. Immunol. 1112.134 31%' 513]. In another e!ample, 6racie, 7. ". et al., demonstrated that administration of I)'18 to mice e!hibited pleiotropic activities critical to the development of Thl responses. [6racie et al. 7 /lin Invest 1111 9ov 13: 102 *10+4 1%1%'1201]. "lthough the administration of cyto$ines typically results in relatively specific increases in desired Thl cyto$ines, prolonged administration of cyto$ines may be problematic for various

reasons. or e!ample, the production of recombinant cyto$ines is relatively e!pensive, and isolation of non'recombinant cyto$ines from natural sources is generally difficult due to the very lo# concentration of cyto$ines in natural sources. ;oreover, depending on the nature of the cyto$ine, cyto$ines may not be #ell tolerated in patients. In another approach, immunomodulatory substances other than cyto$ines may be employed to increase the Thl response. or e!ample, -prietsma 7. <. suggests that =inc ions *un2+ and nitric o!ide *9B+, together #ith glutathione *6-C+ and its o!idi=ed form, 6--6, may help to regulate an immune response to antigens [-prietsma 7. <: ;ed Cypotheses 1111 7u1 : 3% *1+ 4 5'15]. The author reports in more detail that deficiencies of >n2@, 9B and&or 6-C shift the Thl&Th2 balance to#ards Th2, and that replenishment #ith >n2@, 9B and&or 6-C may shift the Thl&Th2 balance to#ards Thl. "dministration of >n2@ or 6-C&6--6 is especially advantageous, since these substances are non'to!ic at even elevated concentrations, and ine!pensive to produce. urthermore, >n2@ and 6-C&6--6 preparations may be orally administered, and therefore significantly reduce the ris$ of allergic reactions, especially #hen the preparations are not ultrapure. Co#ever, the administration of >n2@ and&or 6-C&6--6 seems to be beneficial only to restore a Thl&Th2 balance from a Th2 dominated state, #hereas it is unclear if administration of >n2@ and&or 6-C&6--6 may increase a Thl response from a normal Thl&Th2 balance. In another method of treating a viral infection, the virus is directly targeted #ith an appropriate anti'viral drug. or e!ample, patients infected #ith the CIV virus often receive a coc$tail of drugs to bloc$ virus propagation, and various classes for direct anti'viral treatment are $no#n in the art. -ome direct anti'viral drugs bloc$ the reverse transcriptase of a retrovirus. .everse transcriptase *.T+ inhibitors are typically nucleoside analogs such as ">T, %T/, or ddI. "lternatively, non'nucleoside .T inhibitors, including Duercetin may be employed. In vitro, .T inhibitors are typically potent antiviral drugs. Co#ever, in vivo, and especially during a period of relatively fast viral replication, the generation of .T inhibitor resistant virus mutants is problematic. ;oreover, many .T inhibitors also e!hibit undesirable activity on ,9" replication in the host organism and significant cytoto!icity at elevated concentrations, thereby limiting the concentration that may be administered #ithout severe side effects. "mong other direct anti'viral drugs are the protease inhibitors, #hich bloc$ or interfere #ith virus protein processing. 0rotease inhibitors are typically highly specific to#ards the virusesEproteolytic en=ymes, ho#ever, due to their mostly hydrophobic

nature, administration at desirable concentrations often becomes problematic. "nother problem is that development of cross'resistance and severe side effects freDuently occur. In order to reduce the development of multidrug resistant virus strains, mi!tures of .T inhibitors and protease inhibitors may be prescribed. "lthough such mi!tures are presently employed relatively successfully, the relatively high occurrence of adverse side effects and the potential of generating multidrug resistant virus strains persist. To circumvent at least some of the problems associated #ith side effects and relatively high costs of antiviral drugs, Gennett et al. describe in H. -. 0at. 9o. 3,502,180 the use of <,T" comple!es in a suppository. The use of chelating agents, including <,T", has been found to promote disintegration of retroviruses [Iunderlich, V. and -ydo#, 6. "rch. Virol. 1182,F%4 1F1'18%]. GennettEs suppositories contain disodium <,T" and controlled'release agents, #hich release the disodium <,T" over a period of about three to four hours after rectal placement of the suppository. Co#ever, GennettEs suppositories are limited to disodium <,T" that e!hibits relatively moderate selectivity bet#een ;g2@ and /a2@. "lthough various antiviral compositions and antiviral treatments are $no#n in the art, all or almost all of them have one or more disadvantages. Therefore, there is a need to provide improved methods and compositions for treatment of viral infections. S2mmar. o1 the Invention The present invention is directed to an antiviral composition having a supply of chelating agent that chelates an al$aline earth metal ion, #herein the chelating agent is formulated in a rectal deposition formulation, and #herein the supply of chelating agent has an immediate bioavailability. Ihen rectally administered to a subJect in an effective dose in vivo, contemplated agents promote disintegration of a virus. In one aspect of the inventive subJect matter, generally preferred chelating agents are various chelators other than ethylenediamine'9, 9, 9E, 9E'tetraacetic acid *<,T"+, chelate /a2@ and&or ;g2@, and include at least three carbo!ylic acid groups. Ihile particularly preferred chelating agents include at least three acetic acid groups, especially contemplated chelating agents are 1, 2'Gis *2'aminopheno!y+ ethane 9, 9, 9E, 9E'tetraacetic acid *G"0T"+, <thylenebis *o!yethylenenitrilo+ tetraacetic acid *<6T"+, 1, 2'bis *2'aminopheno!y+ ethane'9, 9, 9E, 9E'tetraacetic acid tetra$is *aceto!ymethyl ester+ *G"0T"'";+, diethylenetriamine'pentaacetic acid *,T0"+, trimethylaminetricarbo!ylic acid *9T"+, trans'1, 2'diaminocyclohe!anetetraacetic

acid */,T"+, poly *aspartic acid+, and poly *glutamic acid+. In another aspect of the inventive subJect matter, contemplated viruses include a retrovirus, and especially contemplated retroviruses include the CIV virus. 0referred rectal deposition formulations are a liDuid or a solid, and #here the rectal deposition formulation is a solid and administered to the colon of a subJect, substantially of the supply of chelating agent is present in the colon in a readily absorbable form in less than 2 hours, preferably less than 1 hour, and more preferably less than %0 minutes. Iith respect to the effective dose in a rectal administration, it is contemplated that the chelating agent is employed in an amount of 300mg, and more preferably 1300mg. In yet another aspect of the inventive subJect matter, chelating agents other than <,T" may also be employed for purposes other than antiviral treatment, including heavy metal deto!ification, and reduction of atherosclerotic plaDues, #herein the chelating agent may be orally or parenterally administered. Various obJects, features, aspects and advantages of the present invention #ill become more apparent from the follo#ing detailed description of preferred embodiments of the invention. Detailed Description "s used herein, the termKchelating agentKrefers to a molecule that binds a metal ion and&or an al$aline earth metal ion via a non'covalent bond, most commonly a coordinate bond, #ith a (, of less than 10'% molE1, #herein the chelating agent may be in acid form, base form or a salt form. or e!ample, <6T" in protonated or sodium salt form is considered a free chelating agent, because <6T" binds ;g2@ and /a2@ #ith a (, of less than 10'% mol'1. "s also used herein, the termKimmediate bioavailabilityKmeans that a composition or molecule is present in an active form in a formulation such that a substantial portion of a dose of the composition or molecule e!hibits some systemic chelating effect #ithin minutes, preferably #ithin less than 13min, more preferably #ithin less than 10 min, and most preferably #ithin less than 3min. or e!ample, a molecule that is dissolved in a carrier solution is regarded to have immediate bioavailability. It is $no#n that retroviruses can be disintegrated by chelating agents, especially by agents that chelate ;g2@ and&or /a2@, and that chelating agents may further reduce infectivity of certain viruses [Iunderlich, V. and -ydo#, 6. "rch. Virol. 1182, F%4 1F118%]. Thus, it is contemplated that an antiviral composition generally has a supply

of a chelating agent that chelates an al$aline earth metal ion, and it is particularly contemplated that the chelating agent in the antiviral composition is formulated in a rectal deposition formulation, #herein the supply of chelating agent has an immediate bioavailability. It should be appreciated that many chelating agents are $no#n in the art, and that all of the $no#n chelating agents are contemplated for use herein. It is generally preferred that contemplated chelating agents include at least three carbo!ylic acid groups, all of #hich are preferably acetic acid groups. "lthough not e!cluded, it is further contemplated that appropriate chelating agents are chelating agents other than <,T". The choice of the particular chelating agent is predominantly determined by the desired physicochemical properties and tolerability of the chelating agent. or e!ample, #here a relatively high solubility *e. g., 1;+ is desired, <6T", /,T" or 9T" may advantageously be employed. Ihere a more pronounced selectivity of chelation to#ards /a2@ is desirable, G"0T" may be utili=ed, and G"0T"'"; may be particularly suitable #here seDuestration of /a2@ #ithin a cell is desired. "lternatively, contemplated chelating agents may include ,T0", 9T", and polymeric forms of aspartic acid, glutamic acid, and any reasonable combination thereof. Iith respect to viruses that can be disintegrated and&or reduced in infectivity, virus particles that reDuire /a2@ and&or ;g2@ for structural integrity of their envelope are generally contemplated and include ,9" and .9" viruses. 0articularly contemplated .9" viruses are retroviruses in general, and CIV in particular. urther especially contemplated viruses include the hepatitis / and hepatitis , virus. /ontemplated ,9" viruses include polyomaviruses, CGV, etc. Co#ever, many more viruses are also contemplated, and a collection of appropriate viruses are listed in ields Virology, Third <dition *)ippincot Iilliams L Iil$ins+, pages 20'21,32, and 1F5F' 182F, and "rch. Virol. 1182,F%4 1F1'18%, both of #hich are incorporated by reference herein. It is still further contemplated that chelating agents are preferably formulated in a rectal deposition formulation, #hich may be in solid or liDuid form. Ihere the formu' lation is in a solid form, it is further contemplated that appropriate forms include dissolvable carriers such as #a!es, fatty acids and oils #ith melting points of about %0 ' %3 /. <specially preferred formulations are formulations $no#n in the art that are employed in the fabrication of rectal suppositories, so long as such formulations allo# an immediate bioavailability. Thus, #here a supply of chelating agent is administered into the colon of a subJect in a solid form, it is particularly contemplated that substantially all of the supply of chelating agent is present in the colon in a readily absorbable form in less than 2 hours, more preferably less than 1 hour, and most

preferably less %0 minutes after the administration of the formulation. "vailability of the chelating agent or a portion of the chelating agent in less that 2hrs, less than lhr or less than %0min may be achieved by a variety of time release formulations, and contemplated time release formulations may include formulations #ith a melting point of less than %F /, en=ymatically degradable carriers, dissolving or s#ellable carriers, etc. Thus, it is contemplated that an entire dose of chelating agent may be available *or released from the time release formulation+ in less than 2 hours, preferably less than lhour, and even more pref' erably in less than %0min. " particular advantage of such time release formulations is that relatively high dosages may be administered that might other#ise pose a potential ris$ if administered #ithout a time release formulation. Co#ever, it should be appreciated that administrations #ithout time release may safely be administered by employing smaller dosages at multiple administrations. Ihere the formulation is in a liDuid form, it is contemplated that appropriate liDuid forms may include buffered and unbuffered solutions, solutions #ith relatively high viscosity such as gels, creams, foams and ointments, #hich may or may not have a decreased viscosity at elevated temperatures. )iDuid forms are particularly advantageous, since the delivery of the chelating agent is almost instantaneous. Ihere the solutions are buffered, it is contemplated that the buffers have an al$aline pC, and a preferred pC range is a range bet#een 8.0 and 10.0. "lternatively, the chelating agent may also be administered in various alternative routes, and it is especially contemplated that #here the chelating agent is an agent other than <,T" that appropriate routes include oral and parenteral administration. or e!ample, /,T" may be orally administered in form of an acid resistant caplet or capsule. Co#ever, oral administration need not be limited to a caplet or capsule, and alternative oral administrations include syrups, po#ders, tablets, etc. In another e!ample, <6T" may be parenterally administered by intravenous inJection. It is contemplated, ho#ever, that alternative parenteral administrations may also include inhalation, transdermal delivery, inJections into sites other than a vein, etc. In a particularly contemplated aspect of the inventive subJect matter, it is preferred that the administration of the chelating agent is accompanied by *preferably oral+ administration of a nutritional supplement. 0referred nutritional supplements include supplements that help replenish calcium levels and particularly preferred supplements include aragonite calcium carbonate from fossil coral minerals. Bther contemplated supplements that include herbal products *e. g., adaptogenic formulations #ith no apparent cytoto!icity+ are contemplated to assist in inhibition of viral replication *e. g., by inhibiting the production of reverse transcriptase+. It is further contemplated that such supplements may also help boost the immune system and potentially

improve overall vitality and stamina. It is further contemplated that such adaptogenic supplements are considered to have tumor preventive and radio'protective properties, and may help increase the functioning of the immune system by increasing the T'cell population. <!emplary compositions for contemplated nutritional supplements are sho#n in Tables 1 and 2. Ingredient "mount *m&tablet+ "rcticum lappa 20mg *104 1 concentrate+ Viola yedoensis 20mg *104 1 concentrate+ "ndrographis paniculata 20mg *104 1 concentrate+ )onicera erythrorhi=on 20mg *104 1 concentrate+ <pimedium med um a<tOum Table 1 Ingredient "mount *mg&tablet+ "rcticum lappa 1 Bmg *104 1 concentrate+ Viola yedoensis 1 Bmg *104 1 concentrate+ "ndrographis paniculata 1 Bmg *104 1 concentrate+ )onicera erythrorhi=on 1 Bmg *104 1 concentrate+ "ltemanthera philoeroides 1 Bmg *10 4 1 concentrate+ Table 2 It should be appreciated, ho#ever, that various additional ingredients may be added to the supplement depicted in Table 1 and 2 to either enhance or modulate the activity of the herbal components. Iith respect to the amount of chelating agent it is contemplated, that the chelating agent is administered to a subJect in vivo in a dose effective to promote disintegration of a virus in the subJect. The actual dose of the chelating agent may thereby vary among individual subJect and may further be determined by the particular virus that is to be disintegrated. Therefore, an effective dose may comprises rectal administration of the chelating agent bet#een about 3mg'2300mg, and generally contemplated doses include rectal administration of 300mg or 1300mg of the chelating agent. Co#ever, #here even higher dosages of the chelating agent are reDuired, or #here it is preferred to maintain relatively high dosages over an e!tended period of time, multiple dosages are also contemplated. It should further be appreciated that appropriate formulations may further comprise active and&or inactive ingredients. or e!ample, active ingredients may include compositions to stimulate the immune system, an immunomodulating composition, a coral mineral product, compositions to facilitate upta$e of the chelating agent into the blood stream, or direct antiviral compounds such as nucleoside analogs, etc. The term Kimmunomodulating compositionKas used herein refers to a composition that enhances at least one of a humoral and cellular response to#ards a challenge. or e!ample, an

immunomodulating composition may increase an antibody titer against a challenge, or an activity of cytoto!ic T'lymphocytes. Inactive ingredients may include fillers, coloring agents, thi!otropic compositions, and foam building agents. In an e!emplary use, a person diagnosed #ith an CIV infection receives t#ice daily an enema of 20ml of a 30mg&ml solution of <6T" in 10m; sodium phosphate buffer pC8.2 for at least %0 consecutive days. It should be recogni=ed, ho#ever, that the e!emplary use need not be limited to the specified amounts and times, but treatment schedules may vary considerably. or e!ample, #here the person already receives an antiviral medication *e. g., protease inhibitor coc$tail, .T'inhibitor, etc.+, lo#er dosages or less freDuent administrations are contemplated, #hile in cases #here the person does not receive another antiviral treatment, higher dosages and more freDuent administrations are contemplated. It is also contemplated that the antiviral composition may be employed in a preventative fashion, i. e., the antiviral composition may be employed in a person that is not infected #ith a virus. It is still further contemplated that the compositions according to the inventive subJect matter may have advantageous properties and uses in therapeutic applications other than antiviral activity, especially #here the chelating agent is a substance other than <,T", and particularly contemplated uses include heavy metal deto!ification in animal and human, and reduction of atherosclerotic plaDue. Iith respect to heavy metal deto!ification in animal and human, it is $no#n in the art that upon oral administration or inJection <,T" comple!es various metals and heavy metals other than /a2@, and oral administration or inJection of <,T" has therefore found #idespread use in deto!ifycation of some heavy metal poisonings. Various alternative oral or inJectable chelation agents for heavy metals have also been described [e. g., )lobet, 7. ;. et al. "rch. <nviron /ontam. To!icol. 1110,11 *2+4 183' 1: Treatment of acute lead into!ication. " Duantitative comparison of a number of chelating agents. )lobet, 7. ;. et al. "rch. To!icol. 1188,51 *2+4 %21'%: "ntidotes for =inc into!ication in mice] and include oral and inJectable forms of penicillamine, 2,%' dimercaptosuccinic acid, and 2,%'dimercapto'1'propanesulfonate. Co#ever, it is not $no#n to the inventors that chelators other than <,T" have been used for deto!ification of heavy metals in animal and human via rectal administration. .ectal administration is particularly advantageous for various reasons. or e!ample, suppositories can be self'administered by al most all patients. urthermore, rectal administration inflicts only relatively lo# discomfort to the patient. ;oreover, rectal administration bypasses the stomach, a highly acidic environment that may lead to at least partial destruction of some of the chelating agents.

Therefore, it is contemplated that rectal administration of chelating agents may also be employed in a method to reduce a heavy metal concentration in a subJect, #herein in one step a chelating agent is provided that chelates a metal ion, #herein the chelating agent is formulated in a rectal deposition formulation and #herein the supply of chelating agent has an immediate bioavailability. "lternatively, the rectal deposition formulation may further comprise a time release agent to release the chelating agent in a period of bet#een 0'%0min, %0'50min, 50'120min, 120'180min, or longer. In another step, the chelating agent is rectally administered to the subJect in a concentration effective to reduce the heavy metal ion concentration. It is generally contemplated that the heavy metal may be in elemental or ionic form, and particularly contemplated heavy metals include mercury, >n2@, /u@, /d2@, and /o2@. Co#ever, various alternative metals and their ionic forms are also contemplated, including nic$el, arsenic, selenium, iron, mercury, chromium, antimony, beryllium, thallium, silver, scandium, titanium, vanadium, chromium, manganese, etc. Ihile it is generally contemplated that all $no#n chelating agents may be suitable for reduction of heavy metals in a subJect, it is particularly preferred that the chelating agent comprises a plurality of carbo!ylic acid groups and it is even more preferred that the chelating agent is <,T", <6T", /,T", or ,T0". Iith respect to the rectal deposition formulation the same considerations as already described above apply. "n e!emplary method of reducing a heavy metal concentration in a subJect may therefore comprise a single rectal administration of 20ml of a l Bmg&ml buffered aDueous solution of /,T" three times daily over a period of about 13'20 days. It should be recogni=ed, ho#ever, that depending on the particular heavy metal, the site of accumulation, and the concentration of the heavy metal in the subJect many treatment schedules other than a single rectal administration of 20ml of a l Bmg&ml buffered aDueous solu' tion of /,T" three times daily over a period of about 13'20 days are also appropriate. or e!ample, #here treatment is prophylactic or necessitated by relatively lo# concentrations of a heavy metal, total daily dosages of less than 500mg are contemplated, including total daily dosages of 200'500mg, 30'200mg, and less that 30mg. )i$e#ise, #here acute and&or severe heavy metal into!ications are to be treated by a method according to the inventive subJect matter, higher total daily dosages of more than 500mg are contemplated, including total daily dosages of 500'1300mg, 13002300mg, and more than 2300mg. Iith respect to the formulation it should be

appreciated that numerous alternative formulations are also appropriate, and contemplated alternative formulations include the formulations already described above. -imilarly, it should be appreciated that various alternative administration periods other than a period of about 13'20 days are also appropriate, including single administrations in cases #here treatment is prophylactic, or administration over a period of less than 13 days, #here the heavy metal concentration is relatively lo#. Bn the other hand, #here the heavy metal is predominantly is tissues that bind the heavy metal relatively firmly *e. g. lipophilic tissue+ administrations of 2'5 #ee$s and longer are contemplated. Iith respect atherosclerotic plaDues it is contemplated that rectal administration of chelating agents may also be employed in a method to reduce a atherosclerotic plaDues in a subJect, #herein in one step a chelating agent is provided that chelates an al$aline earth metal ion, #herein the chelating agent is formulated in a rectal deposition formulation and #herein the supply of chelating agent has an immediate bioavailability. In another step, the chelating agent is rectally administered to the subJect in a concentration effective to reduce the atherosclerotic plaDue in a subJect. "s used herein, the term Kreducing the atherosclerotic plaDueKrefers to a gross reduction in si=e and&or volume of one or more atherosclerotic plaDues, #hich may also include complete disappearance of the atherosclerotic plaDue or plaDues. In an e!emplary method of reducing atherosclerotic plaDue, a person diagnosed #ith atherosclerotic plaDues receives once daily an enema of 10ml of a 30mg&ml solution of <6T" in 1 Bm; sodium phosphate buffer pC8.2 for a period of about 12 #ee$s. Co#ever, it should be appreciated that the e!emplary method need not be limited to the specified amounts and times, and formulation and treatment schedules may vary considerably. or e!ample, #here the person already under#ent a vasodilation procedure, lo#er dosages or less freDuent administrations are contemplated, #hile in cases #here the person did not receive previous treatment to reduce the atherosclerotic plaDues, higher dosages and more freDuent administrations are contemplated. )i$e#ise, the chelating agent need not be limited to <6T", but may be various alternative chelating agents including <,T", /,T", and ,T0", #herein the choice of the chelating agent #ill predominantly depend on the desired specificity of the chelator and the tolerability at a particular concentration. urthermore, the formulation of the chelating agent need not be restricted to 1 Bml of a 30mg&ml solution of <6T" in 1 Bm; sodium phosphate buffer pC8.2. or e!ample, alternative formulations may be employed to achieve a larger distribution, faster absorbption, etc., and appropriate

formulations include those already described above. Thus, specific embodiments and applications of antiviral compositions have been disclosed. It should be apparent, ho#ever, to those s$illed in the art that many more modifications besides those already described are possible #ithout departing from the inventive concepts herein. or e!ample, the route of administration need not necessarily be restricted to a rectal administration of the chelating agent, but may also include vaginal administration. The inventive subJect matter, therefore, is not to be restricted e!cept in the spirit of the appended claims. ;oreover, in interpreting both the specification and the claims, all terms should be interpreted in the broadest possible manner consistent #ith the conte!t. In particular, the termsKcomprisesKandKcomprisingKshould be interpreted as referring to elements, components, or steps in a non'e!clusive manner, indicating that the referenced elements, components, or steps may be present, or utili=ed, or combined #ith other elements, components, or steps that are not e!pressly referenced.