PROPHYLACTIC AND THERAPEUTIC APPLICATIONS OF PEROXIDASES

Abstract: Prophylactic and therapeutic applications of peroxidases for the manufacture of medicaments for the prevention and treatment of enveloped virus infections and, in particular, of herpes simplex and human immunodeficiency virus infections. The medicaments include a peroxidase, a substrate and a peroxide in a pharmaceutically acceptable carrier. Peroxidases of the medicaments include lactoperoxidase and myeloperoxidase. The medicaments are formulated with pharmaceutically acceptable carriers for topical, oral and injectable administration to individuals in need thereof.

Description: PROPHYLACTIC AND TΗERAPEUTIC APPLICATIONS OF PEROXIDASES Field Or" The Inven on The present invention relates to prophylactic and therapeutic applications or " peroxidases and methods for the prevention and treatment of viral infections and, in particular, to prophylactic and therapeutic applications of peroxidase medicaments, and methods for utilising such peroxidase medicaments for the prevention and treatment of infections of enveloped viruses, εucn as the Hemes Simplex Viruses and the Human Immunodeficiency Viruses. Background Of The Invention The development of effective prophylactic and therapeutic medicaments for preventing and inhibiting the cytotoxic potential of infections of enveloped viruses, and in particular of Herpes Simplex Viruses <HSV ε) and Human I munodeficiency Viruses (HIV's), have proven problematic. Herpes Simplex Viruses <such as HSV-1 and HSV-2) sire widespread. Prophylac ic and therapeu ic medicaments and methods developed for the prevention and treatment of _ infections of herpes simplex viruses have, m general, only been partially successful. Secretions of human milk have long been known to exhibit antiviral activity Cεee Matthews et al., Lancet, ≤: 1388-1390 (1976); Micheals, R. H. , J. Immunol., 94: 2££-£7i (1964); Laegried et al., ftcta Paedπatz Scand. , 75: 696-701 (1986); and Isaacs et al., J. Infect. Dis. , 154: 969-971 (1986)3. In particular, whole human breast milk has "in vitro" been noted to exhibit antiviral neutralizing activity against herpes simplex virus ≤. CLopez et al. , Arch. Fr. Pediatr. , 46: 263-265 (1989)3. While the origins of this antiviral activity has been attributed to several varying sources, it has never been able to be defini ively characte ized. The primary source of the antiviral activity of milk has been attributed to the presence of immuno lobinε (IgG'ε) therein. Other sources that have been suggested as the origin of this antiviral activity includes a non-lipid macro olecule that is relatively stable to heat (Matthews et al. , and Micheaiε, both supra) and/or a molecule having a molecular weight of 400,000 daltonε (Laeπried et al., supra) and/or a component of the lipid layer that effects only encapsulated viruses (Isεacε et al., supra). This inability to definitely characterize the antiviral origin(ε) of milk limits the use thereof, or of components or systems thereof, for antiviral purposes. Human saliva has also long been known to be active against a number of viruses, including herpes simplex virus 1. Csee Gyεelink, et al., J. Infect. Diε. , 137: 583-586 (1978)3. Unfortunately, the origin of the anti—viral activity of human saliva has not been definitively characterized, being ascribed variously to glycoproteins CLearner, et al., J. Immunol. 96: 59—63 (1966)3, immunoglobulin ft CTomasi, J. clin. Invest. 42: 1552-1560 (1963)3 or immuno lobulin G (Byεelink, et al., Eupra), More recently, it has further been suggested that the antiviral activity may be more of a eel1-protective activity than a virus neutralizing activity — that is to say, the saliva directly affects the oral epithelial cells, protecting them against infection Csee Heinernan, H. S. , and M.S. Greenberg, Pirchε Oral Biol. 225: 257-261 (1980)3. Unfortunately, the origin of such activity still remains unknown. No medicament has been successful in preventing and inhibiting infections of, and the cytotoxic potential of, herpes simplex viruses in all stages. The human immunodeficiency viruses (HIV'E) are fatal and widespread viruses that have only relatively recently been identified. The biochemistry and physiology of these HIV s are poorly known and understood. It has been reported that "in vitro" contact, for at least one-half hour or more, with whole human saliva inhibits the ability of human immunodeficiency virus (HIV) to infect phytohaemagglutlnin- stimulated lymphocytes. CFultz, Lancet, 2:1215 (1986)3. However, shorter periods of incubation have failed to demonstrate an impressive antiseptic effect (see Fultz, supra). Moreover, not all of the saliva samples reported can insure a 10054 inhibition of HIV-1 infectivity Csee Fox et al., JftDfi, 118: 709-711 (1989)3. fts yet, no medicaments or methods of which we are aware have proven to be consistently successful for preventing and treating infections of, and the cytotoxic potential, of the HIV ε. Other enveloped viruses that are particularly troublesome to effectively prevent and treat include other herpes viruses (such as Varicella-zoster virus, cytomegalovirus, Epstein-Barr virus and human herpes virus- 6), the paramyxoviruεeε (such as human parainfluenza viruses), the family of orthomyxoviruses (such as the influenza type viruses ft and B) , rotaviruseε, coronaviruses and retroviruses (such as Human T-cell leukemia virus-1, bovine leukemia virus and simian immunodeficiency virus). It is well known that natural antimicrobial agents are contained in most natural external mammalian secretions. In particular**, the natural ly-occurmg antimicrobi l thi cyanate peroxιdase/H a O( ≥ systems present m saliva and in milk have deen extensively studied. In saliva, an antimicrobi l pero idase-dependent system has been described which can generate hypothiocyamte (OSCN- , as follows: pero idases HaOc ΞCN- -> OSCN- Hs COrarn and Reiter, Biochem. J. , 100:373-381 (1966) ; Hogg and Jago, Biochem. J. , 117: 779-790 (1970) ; and Carlεεon et al., Infect. Irn un. , 44: 581-586 (1984)3. The peroxidases thought to be present in saliva that oxidize thiocyanate in this system include salivary peroxidase and lactoperoxidase. ft similar antimicrobial lactopero idase— ependent system has also been identified in milk. Orarn and Reiter, Biochem. J. , 100:382 (1966)3. Indeed, it has been suggested that the same peroxidaεe/thiocyanate/hydrogen peroxide system that operates in saliva also operates in milk. Csee Klebanoff, S. J. , et al., J. Dent Res. (supp. ) p.86 (1965).3 The antimicrobi l efficiency of the thiocyanate/peroκidaεe/hydrogen peroxide system has been demonstrated "in vitro" against several bacteria known to be responsible for frequent destruction of teeth and/or periodontlum CSee Carlsεon, supra, and Courtoiε et al., J. Dent. Res. 68 (spec, issue) : 1002 (1989)3. The anti icrobial efficiency of this system was also demonstrated "in vivo" in caεeε of aphtous lesions of the buccal mucoεa CHooπendoorn and PiesseriΞ. J. Oral Pathol. , 16: 425-427 (1987)3. Unfortunately, the precise anti icrobal mechanism of the thi cyanate/pero idase/HβOe system has not been defini ely characterized. However, it is believed that at pπyεiologicai pH, hypothiocyanite (generated by this system) mediates the oxidation of essential proteins and enzymes sulfhydrylε groups of the bacteria, resulting in microbial inhibition, ftdditionally, it has been suggested that lactopero idase may □e responsible for the rormation of higner oxyaciαs of the thiocyanate ion, sucn as cyanoεulfυrouε and cyanosulfuric acids, which may also be responsible for antimicrobial inhibition. CBjoerck, L. , and 0. Claesson, J. Dairy Sci. 63:919-921 (1980) ; Hogg, et al., supra; and Prυitt, et al. , Biochemistry 21:562-567 (1982)3. It is also known to provide orally-activa ed antimicrobial dentifrices that contain a thiocyanate/peroxidase/hydrogen peroxide system. Upon oral administration, the enzyme-dependent systems in these _ an lmicr ni l dentifrices are ac ivated by various components ( such as oxygen and/or water) of the natural chemical environment of the oral cavity. In particular, United States Letters Patent No. 4,564,519 issued to Pellico et al., ( hereinafter sometimes referred to as Pellico '519) discloses a di-enzy atlc chewable orally-activated antimicrobial dentifrice that includes a thiocyanate salt and lactoperoxidase which, through interaction with hydrogen peroxide formed by another enzymatic system in the dentifrice, produces a bacterial inhibitor m the form of a negative, monovalent hypothiocyanite ion (OSCN *- ) whiri exists in solution in acid-base equilibrium with hypothiocyanouε acid (HOSCN). — * ->— Also, in United States Letters Patent No. 4,576,817 issued to Montgomery et al. , it was disclosed to provide antimicrobial enzymatic bandages and pads for disinfection purposes. These pads include a serum-activa ed oxidoreductase enzyme for producing hydrogen peroxide upon contact of tne enzymatic materials with serum. In one embodiment, these antimicrobial bandages are formulated to also include a peroxidatic peroxidaee, sucn as l ctoperoxidase. In the journal BIDFUTUR (February, 1990, at page 52), a system is disclosed having two enzymes that, in tandem, generates toxic radicals that may be useful for the treatment of various infections. This system includes lucose o idase which, in the presence of glucose, generates H e 0e. This system also includes a peroxidase which, with the H a 0 e , generates iodides that are highly toxic for the cell. Unfor nately, the precise mechanism of this toxicity is not known. It was further reported therein that this glucose oxidase/ eroxidaεe system has been coupled to a monoclonal antibody against Candida albicans and has been found effective for protecting against such infections in mice. This glucose oxidase/peroxidaεe system has also been coupled to a monoclonal antibody for the epitope of the gp 120 fraction of HIV and has been found to be effective against infections of Sacharomvces expressing t-his epitope. In the journal CLINICAL RESEftRCH, vol. 36, No. 5 (1988) at 309ft, a lactoperoxidaεe-halide-hydrogen peroxide (LHHP) system was reported to be effective, in vitro, on reepirator εyncytial virus (RSV) replication. It was also suggested that a myeloperoxidase-halide-peroxidase system (that plays an important role in host defense mechaniε ε against phagocytoεed bacteria) may also have a role--in host defense against RSV. Finally, in PCT patent application no. WO 8912457, the incorporation of myel pero idase with a cAt~τ~ιt-'r is disclosed for administration to humans for reinf rcing the natural antibody activity thereof at the macrophage level. In this disclosure, purified myeloperoxidase is linked with a carri that has an affinity f r the macrophage, so that the carrie will transport the myeloperoxidase to where it may be captured and utilized oy the macrophage for antiDody defens Carriers disclosed include antibodies, or fragments thereof that have an affinity for the acrophages. Other suggested carriers include particular liposomes and human εerum albumin. Preferably, the myel pero idase and the linked carrier are formed utilizing recombinant DNfi technologies. Such a composition is provided to aid, augment and reinforc the bodies natural antibody defenses and is believed to be useful in combatting various infections, including HIV. Despite the long-standing coexistence of the knowledge of the properties of the peroxidases and the thiocyanate/peroxidaεe/hydrogen peroxide system, as well as the need for prophylatIC and therapeutic medicaments for th prevention and treatment of infections of enveloped viruses (and in particular of herpes simplex and human immunodeficiency viruses), to the best of our knowledge no one has utilized medicaments incorporating εuch peroxidases f 4 peroxide systems for the prevention or treatment of infections of enveloped viruses and, in particular, for the prevention and treatment of herpes simplex and human immunodeficiency virus infections. _,Thus, it can be εeen that there remains a need for prophylactic and therapeutic peroxidase medicaments which prevent and/or treat infections of enveloped viruses, including HSV and HIV, and for prophylactic and therapeutic- applications of peroxidases in a medicament that may be administered to an individual in need thereof without depending upon naturally-occurring concentra ions of substrate, oxygen donors or peroxidases for their inhibitory effect. Finally, there remains a need for methods for the prevention and treatment of infections of enveloped viruses, including HSV s and HIV 2, by the administration of prophylactic and therapeutic effective amounts of such peroxidase medicaments to an individual in need thereof. Summary Of The Invention It is a primary object of the present invention to provide uses (applications) for peroxidases in the formula ion (manufact re) of medicaments for the prevention and treatment of enveloped viruses. It is another object of the present invention to provide prophylactic and therapeutic applications of peroxidases in medicaments for the prevention and treatment of infections of enveloped viruses, including herpes simplex and human immunodeficiency viruses. It is still another primary object of the present invention to provide prophylactic and therapeutic methods fo viruses, by the administration of prophylactic and therapeutic effective amounts of peroxidaεe medicaments to individuals in need thereof. ftε utilized herein, the term "prophylactic" refers variously to medicaments, amounts or quantities, methods, useε and effects, etc., that prevent and/or aid in preventing infections of enveloped viruses and, in particular of the HSV ε and the HIV ε. ftε utilized herein, the term "therapeutic" refers variously to medicaments, amounts or quantities, methods, uses and effects, etc., that ameliorate infections of enveloped viruses and, in particular of the HSV s and the HIV s. In accordance with the teachings of the present invention, there are disclosed herein prophylactic and therapeutic applications of peroxidases in medicaments, and methods for the administration of the peroxidase medicaments, for the prevention and treatment of infections of enveloped viruses, including herpes simplex and human immunodeficiency viruses. The preferred peroxidase medicament (hereinafter sometimes referred to as the "peroxidase composition" or simply as the "composition") of the present invention, is a substantially self-contained antiviral system that may operate without depending upon naturally-occuπng " vivo" compounds, or concentra ions thereof, of the user thereof. This medicament includes: an oxygen donor, which is preferrably a glucose- lucose oxidase enzymatic system that generates hydrogen peroxide; a peroxidase, such as lactoperoxidase; and a substrate, chosen from a group consisting of halogens and pseudo-halogens. These peroxidas medicaments are formulated, such that comparatively short- term administration thereof is effective for the prevention and treatment of enveloped viruses, such as the HSV ε and HIV s. Preferably, the concentrati ns and/or formulations o these various components have been chosen to maximize oxygen donor and pero idase-generated compound production, while simultaneously maintaining the oxygen donor concentra ions at leveiε which do not impede with the activity of the peroxidaεe. It is noted that it is preferred to formulate the peroxidase medicaments of the present invention, so as to include various components of, and mimic somewhat, the salivary thiocyanate—peroxidase system. Such a Formulation is preferred since the components thereof would be genuine comDonentε of human external secretions. This formulation i further preferred since the antiviral activity thereof can possibly be further enhanced through the absorption of foods enriched with any of the various components thereof. Accordingly, the present invention most preferably provides substantially self-corr amed hypothiocyanite- generating, prophylactic and therapeutic peroxidase/thiocyanate/hydrogen peroxide medicaments that ma be applied and utilized without depending upon the users naturally-occurmg "in vivo" oral concentrati ns thereof, fo the prevention and treatment of infections of enveloped viruses. Other *1 enveloped viruses to be prevented and/or treated by the peroxidaεe medicaments of the present invention include the paramyxoviruεes (such as human parainf1uenza viruses) , the family of " orthomyxoviruses (such as the influenza type viruses ft and B) , rotaviruseε, coronaviruses, herpes viruses (such as Varicella-zoster virus, cytomegalovirus, Epstein-Barr virus and HHV6) and retroviruses (such as Human T—cell leukemia viruε-1, bovine leukemia virus and SIV) . In still further accordance with the teachings of the present invention, there is disclosed a method for the prevention and treatment of infections of enveloped viruses-, such as herpes simplex viruses and human immunode iciency ziruseε. This method .involves the administra on of prophylactic and therapeutic effective amounts of the peroxidaεe medicaments of the present invention t an individual in need thereof. These and other objects of the present invention will become apparent from the following specification, •• ■ hen taken in conjunction with the enclosed figures. Brier " Description Of The Drawings Figure 1 is a bar chart diagrarπrnatically representing the results obtained after 20, 30 and 120 minutes of pretreatment of HSV-1 with the peroxidase/thiocyanate/hydrogen peroxide system of the medicaments of the present invention. Figure 2 is a line chart iagrarnmatically representing the results obtained of growth of HIV-generated p24 m lymphocyte cultures in the supernatant after treatment with the peroxidase/thiocyanate/hydrogen peroxide system of the medicaments of the present invention. Figure 3 is a line chart diagrarnmat ically illustrating the results obtained of intracel lular growth of p24 per 10* cells after treatmen with the peroxidaεe/thiocyanate/hydrogen peroxide system of the medicaments of the present invention. Figure 4 is a line chart diagrarnmatical ly illustrating the results of lactoperoxidase and myelopero idase asεayε. Descr pti n Of The I vent1on The peroxidaεe medicaments of the present invention include a peroxidase. Preferably, the peroxidaεe medicaments include peroxidase/oxidizable substrate/oxygen donor system that exhibits antiviral properties against enveloped viruses, including HSV ε and HIV ε. In these antiviral medicaments, peroxidaεe catalyzeε oxidation of the substrate '.a halogen or pseudo-halogen) by the oxygen donor (a peroxide) to form negatively—charged, monovalent oxidizing compounds. The medicaments may be formulated for prophylactic and/or therapeutic purposes, as desired and needed, for permitting _ the administration of prophylactic and/or therapeutic- effective amounts thereof to an individual in need thereof for preventing and/or treating infections of enveloped viruses, such aε the HSV ε and the HIV s. The peroxidaεe medicaments of the present invention ar- also useful for prophylactic and therapeutic applications against infections of enveloped viruses, such aε paramyxoviruses, the family of ortho yxoviruseε, rotaviruseε, coronaviruεeε, herpes viruses (such aε varicella-zoster virus, cytomegalovirus, Epstein-Barr virus and human herpes virus—6) and retrovirυseε (such aε human T—cell virus—1, bovine leukemia virus and simian immunodeficiency virus). The substrate of the medicaments of the present invention is chosen from a group consisting of negatively- cnarged halogens, and their derivatives, and negatively- charged pseudo-halogens, and their derivatives. The term "halogens" refers to certain of those elements, in their negatively-charged monovalent states, that belong to Group VII of tj-ιe Periodic Table of Elements and, as is well known to those skilled in the art, includes bromide, chloride and iodide. The term "pseudo-halogens" refers to certain negatively-cnarged ions and ionic compounds that are onovalent. The "pseudo-halogens" of the present invention include the thiocyanate salts, such as sodium thiocyanate, potassium thiocyanate, ammonium thiocyanate, ferric thiocyanate and mixtures thereof. These substrates and their deriva ives are able to be extracted (isolated) from natural material (for example, saliva and human milk) or produced by natural or chemical methods, all of which are well known to those skilled in the art. The peroxidaεeε in the medicamentε of the present invention catalyze the oxidation of the substrate by the oxygen donor for generating the oxidizing compounds. These peroxidaseε include plant (vegetable) peroxidases, such aε horεeradiεh peroxidase, and mammalian peroxidases, such as salivary peroxidases, lactoperoxidases, rnyeloperoxidases and eoεinophil peroxidase. The peroxidases for the medicaments of the present invention may be extracted, by methods and techniques well known to those skilled in the art, from natural milieu, such aε those peroxidases extracted from horseradish and saliva Cfor example, aε described in Mansεon- Rahemtulla et al., Biochemistry, 27:233-239 (1988)3, as well as the lactoperoxidases extracted from milk derivatives ( i.e., whey) and rnyeloperoxidases produced by leukocytes. These peroxidaεeε alεo include those peroxidases (including rnyeloperoxidases) that are produced by reco binant DNft techniques, also well known to those skilled in the art. Cftε utilized herein, the term "International Uni (ε) " identifieε that amount of the enzyme that will effect catalyeiε of 1 micro ole of substrate per minute at pH 7 and 25°C. Enzymes are supplied in dry or liquid form with the label specifying the concentration in IU' s on a per gram or per mil li liter basis, as appropiate. •14- Lactoperoxidase is a glycoprotein which, in one commercial embodiment iε a lyophilized powder derived from milk. Thiε commercial peroxidaεe has an activity of 80 International Units (hereinafter sometimes referred to as IU' s) and a projected molecular weight of 93,000 for L— Tyroεine Iodination. The phyεical-chemical properties reported for lactoperoxidase includes: a molecular weight of 78,000; partial specific volume 0.74; and heme/mole 1.0. Salivary pero idase iε a glycoprotein which may be derived from the saliva or the acini of the parotid glands. The chemical characteristics of salivary peroxidaεe are not well known. However, the physical-chemical properties reported for salivary peroxidase includes a molecular weight ranging from appro imately 80,000-100,000. Myeloperoxidase is also a glycoprotein. In one commercial embodiment (from SIGMft corp. , St. Louis, Missouri, U. S. ft. ) , myeloperoxidase may be obtained from human leukocytes, being lyophilized from 0.02 M sodium acetate buffer. Thiε commercial embodiment has an activity of 40—100 I.U. ' ε. Horεeradiεh peroxidase is a glycoprotein. In one commercial embodiment (SIGMft, corp., St. Louis, Mo., U.S.A.), it iε an essentially salt free powder. Thiε commercial embodiment has an activity of 250-330 units- per rng. solid. Preliminary studies of thiε embodiment have indicated the presence of two basic and no acidic isoenzymeε therein. Cflε utilized herein, the term "unit" refers- to that amount of the horseradish peroxidase that will form 1.0 mg purpurogal1in in 20 sec. at pH 6.0 at 20' :* C. This purpurogal 1in (20 sec. ) unit- is equivalent to approx. 18 uM units per m . at 25°C.3 Exa pleε of the preferred peroxidase/suDstrate combinations to utilize in the medicament of the present invention are set forth below in Table Ift: TABLE Ift . Peroxidase . Substrates ._ (1) Salivary peroxidase thiocyanate, iodide (2) Lactoperoxidase thiocyanate, iodide (3) Myeloperoxidase chloride, iodide, thiocyanate (4) Horseradish peroxidaεe chloride, iodide (5) Plant peroxidaεe chloride, iodide, Dro ide The reactions of representative enzyme systems from Table IA (in the presence of a peroxide -which for purposes of illustration herein, will be hydrogen peroxide- from the oxygen donor) to produce either a hypohalite or hypo¬ thiocyanite compound, are set forth in Table IB, aε follows; . TABLE IB ._ (la) Salivary peroxidase catalyzes the interaction of thiocyanate and hydrogen peroxide to produce hypothiocyanite and water; (lb) Salivary peroxidase catalyzes the interaction of iodide and hydrogen peroxide to produce hypoiodite and water; (2a) Lactopero idaεe catalyzeε the interaction of thiocyanate and hydrogen peroxide to produce hypothiocyanite and water; (2b) Lactopero idase catalyzes the interaction of iodide and hydrogen peroxide to produce hypoiodite and water; (3a) Myeloperoxidase catalyzes the interaction of chloride and hydrogen peroxide to produce hypochlorite and water; (3b) Myeloperoxidase catalyzeε the interaction of iodide and hydrogen peroxide to produce hypoiodite and water; (3c) Myeloperoxidase catalyzeε the interaction of thiocyanate and hydrogen peroxide to produce hypothiocyanite and water; (4a) Horεeradiεh peroxidaεe catalyzes the interaction of chloride and hydrogen peroxide to produce hypochlorite and water; ■ ib- Table IB continued (4b) Horεeradiεh peroxidaεe catalyzes the interaction of iodide and hydrogen peroxide to produce hypoiodite and water; (5a) Plant peroxidaseε catalyze the interaction of chloride and hydrogen peroxide to produce hypochlorite and water: (5b) Plant peroxidaseε catalyze the interaction of iodide and hydrogen peroxide to produce hypoiodite and water; and (5c) Plant peroxidaseε catalyze the interaction of bromide and πydrogen peroxide to produce hypobro ite and water. The oxygen donor of the present invention provides (supplies) the peroxide (for example, hydrogen peroxide) in the medicament necessary for oxidation of the substrate. Preferably, the oxygen donor iε an enzymatic system including a substrate, an enzyme specific to such substrate -and other necessary reactantε, such aε water and/or oxygen and/or hydrogen. Alternatively, microorganiε s, such aε the Steptococci and Lactobacilli that are commonly referred to aε lactic acid bacteria may be utilized in the medicaments of the present invention to supply the peroxide (in the form of hydrogen peroxide). Specific examples of such lactic acid bacteria include Lactobacci1luε caεei and Streptococcus faecaliε and Streptococcus rnutans. Uεe of such microorganisms (microbes) is especially preferred in the medicaments formulated for use aε a vaginal cream for topical application. It ie alεo contemplated herein that inorganic peroxideε ( such as sodium peroxide and magnesium peroxide) or organic peroxides (such as benzyl peroxide and urea peroxide) may be utilized. Also, chemicals that, upon reaction, produce hydrogen peroxide may be utilized. Indeed, even hydrogen peroxide itself may be utilized aε the oxygen donor. The preciεe oxygen donor to be utilized will vary depending upon εeveral factors, including the formulation into which the medicament iε to be made for administration. -1 ' Most preferably, the oxygen donor iε an enzymatic system including an oxidizable substrate, an oxidoreductase enzyme specific to such substrate and other necessary reac n ε, such aε oxygen and/or water. Exa pleε of such oxidizable substrates, and oxidoreductase enzymes specific therefor, include those enumerated in United States Patent No. 4,564,519 issued to Pellico et al. (hereinafter referred to as Pellico '519). Such examples are set forth below in Table IIA: TABLE lift Oxidizable Oxidoreductase Other Substrate Enzyme Reactants . ■ .a) B-D-glucose 1ucose oxidase Water, Oxygen (b) D-galactose galactose o idase Oxygen (c) urate urate oxidase Water, Oxygen <d) ch line choline oxidase Oxygen (e) D-amino acids 1 D-amino acid oxidase Water, Oxygen <f) D- l utamate D-glutamate oxidase Water, Oxygen (g) glycine glycine oxidase Water, Oxygen (h) glycoll te glycollate oxidase Water, Oxygen (i) L-sorbose L-sorbose oxidase <j> primary 1coho1 alcohol oxidase (k) primary a ine amine oxidase (1) NAD(P)H NAD(P)H oxidase 1 D-amino acids includes D iso erε of proline, ethi nine, isoleucine, alanme, vaiine and phenylalanine. The reactions of representative enzyme systems from Table IIA to produce hydrogen peroxide are set forth in Table IIB. . TABLE IIB j_ (a) Glucose oxidase catalyzeε the interaction of Beta-ϋ- glucose, water and oxygen to produce hydrogen peroxide and luconic acid; (b) Galactoee oxidaεe catalyzes the interaction of D- TflBLE I IB continued galactoεe and oxygen to produce hydrogen peroxide and E>— galacto-hexo-dialdoεe; (c) Urate oxidase catalyzeε the interaction of urate, water and oxygen to produce hydrogen peroxide, allantoin and carbon dioxide; (d) Choline oxidase catalyzes- the interaction of cholme and oxygen to produce hydrogen peroxide and betame aldehyde; (e) D-arnmo acid oxidase catalyzeε the interaction of D—ami o acids, such as the D-iεomers of proline, methionine, isoleucine, alanme, vaiine and phenylalanine together with water and oxygen to produce hydrogen peroxide, ammonia and the corresponding alpha-keto acids; (f) D—gluta ate oxidase catalyzes the interaction of D- glutamate, water anύ oxygen to produce hydrogen peroxide, a mon1a and 2—o g1utarate; and (g) Glycine oxidase catalyzeε the interaction of glycine, water and oxygen to produce hydrogen peroxide, ammonia and glyoxyiic acid. The characteristicε of representative oxidoreductase enzymes identified in Table IIA, from specific sources, are recited in Pellico '519, which recitations relating to these characteristicε are hereby incorporated herein aε part hereof. Moεt preferably, the peroxidaee me icamentε of the present invention include either lactoperoxidaεe or myeloperoxidase in combination with a thiocyanate (SCN~) substrate and of a glucose/glucose oxidase enzymatic system oxygen donor. It is preferred that the above-menti ned peroxidase/εubεtrate/peroxide systems be formulated into the prophylactic and therapeutic medicaments for "in vivo" use as- a substantially self-contained system that may be applied or* used substantially without depending upon the users- naturally-occurring "in vivo" concentrations of substrate, oxygen donors, peroxidases or other ingredients. It is noted that the effectiveness of the peroxidase medicaments of the present invention may be effected by the naturally-occurmg environment in which the medicament is to be administered. For example, in the human mouth, the concentration of hydrogen peroxide varies as a direct function of biological production and salivary flow. When salivary flow is at a diminished level, either as a natural event or as an event arising out of certain types of medical treatment, the oral concentrations of various elements, such aε potassium thiocyanate and peroxidase, will be correspondingly reduced. Thiε, turn, may be a limiting factor the prophylactic or therapeutic effectiveness of the medicament when it is orally administered. Moreover, when the oral concentration of peroxidase is εuppresεed through diminished salivary flow, oral concentrati ns of hydrogen peroxide may increase to a threshold level, wherein the hydrogen peroxide can impede the ef ectiveness of peroxidase of the medicament. Accordingly, it can be seen that the concentra ions of the substrate, oxygen donor and peroxidaεe m the medicamentε described above εhould be adjusted and controlled to harmonize hydrogen peroxide and peroxidase concentrations, so as to limit the hydrogen peroxide concentrations to levels which do not impede with the activity of the peroxidase. CAs utilized herein, the term milli ole identifieε that quantity in grams corresponding to the molecular weight of the medicament divided by one thousand.3 When the oxygen donor is hydrogen peroxide itself, it iε generally present in the medicament of the present invention in an amount from about 2 to about 300 millimole per gram or per milliliter of medicament and, preferably, from about 3 to --t - aoout 30 millimole per gra or per rπilliliter of medicament. In the event the oxygen donor is an oxidizable substrate and an oxidoreductase enzyme specific to the substrate, then the oxidizable substrate is generally present in the peroxidase medicamen in an amount from about 0.015 to about 0.6 millimole per gra or per milliliter of medicament and, preferably, from about 0.025 to about 0.1 millimole per gram or per milliliter of medicament while the oxidoreductase iε generally present m the medicament in an amount from aoout 0.5 to about 500 IU' s per gram or per rπilliliter of the medicament and, preferably, from about 1.0 to about 40 IU per gram or per milliliter of the medicament. In the event the oxygen donor iε an organic or inorganic peroxide, then such peroxide iε generally present in the medicament in an amount from about 0.000006 to about 0.6 millimole per gram or per milliliter of medicament and, preferably, from about 0.00006 to about 0.06 millimole per gram or per milliliter of medicament. ^ _ The substrate is generally present in the medicament in an amount ranging from about 0.0000008 to about 0.01 millimole per gram or per milliliter of medicamen and, preferably, from about 0. OOOOOS to about 0.006 millimole per gram or per milliliter o -medicament. — In the event that the substrate iε a thiocyanate salt (a pseudo-halogen), then it is generally present in the medicament in an amount from about 0.0001 to about 0.01 millimole per gram or per milliliter of medicament and, preferably, from about 0.001 to about 0.006 millimole per gram or per milliliter of medicament. Care should be taken in formulating the medicament, so aε to avoid the use of :ι- rπetal compounds which inhibit or impair the effectiveness of the enzymes. In the event that the substrate is a halogen, then it iε generally present in the medicament in an amount from about 0.0000008 to about 0.008 millimole per gram or milliliter of medicament and, preferably, from about 0.000008 to about 0.004 millimole per gram or per rπilliliter of medicament. The peroxidase is generally present in the medicaments in an amount from about 0.01 to about 50 IU per gram or per milliliter of medicament and, preferably, in an amount from about 0.2 to about 4.0 IU per gram or per rπilliliter of me icament. It is noted that, if desired, the peroxidase medicament may be formulated for "in vivo" use as a system that relies upon certain naturally—occurring "in vivo" concentra ions of any one or combination of compounds of the system for obtaining the peroxidase—generated antiviral compound. The antiviral prophylactic and therapeutic qualities of the peroxidaεe medicaments of the present invention may be dependent on the concentra ion of compounds that are produced by the formulation of the medicament of the present invention. The produced concentrations of these compounds may vary between 1 micro molar and 100 millirπolar, with concentrations of between 5 micro molar and 1 mi11imolar being preferred. For achieving thiε, the concentration of peroxidase units relative to the concentrations of the oxygen donor and/or of the substrate iε able to be varied ovc- a large range. The presence of water promotes the oxidation/reduction reactions of the peroxidaεe medicaments of this invention. It also is a reactant in certain reactions. Thus, preferably, the use of water in formulating the said medicaments should be at a relatively low concentration levels in order to impart maximum stability and shelf life thereto. Where the products of the activated enzyme systems in the medicaments include a weak organic acid, it ie advantageous to formulate the medicament with a buffering agent to neutralize the organic acid. ft suitable buffering agent iε sodium bicarbonate. In thiε regard, it iε preferred that the peroxidase medicaments of the present invention should be formulated, so as to have a pH that substantially approximates physiological pH. In particular, it iε preferred that the medicaments of the present invention have a pH ranging from 4.5 to 6.5, with a pH of from 6 to 6.5 being especially preferred. It iε to be understood that to be formulated as a self- contained active medicament, the ingredients of the medicament must be diεpoεed together in the formulation with at least some of the ingredients thereof being maintained chemically-separated from one another until the use thereof. For example, the peroxidaεe, oxygen donors (the oxygen donor enzymatic systems or the microorganisms) may be immobilized or microencapεulated, so that until the use thereof, they will not react with one another. If none of the substances of the systems described above are destroyed or inhibited by any ingredientε, the medicament will have activity against enveloped viruses, including herpes simplex viruses and HIV ε. The peroxidase medicaments of the present invention may iύ— be formulated with ~ . phar aceutical ly-acceptabie carrier in any εui able manner desired for administration in the particular situation. For example, the medicaments may be formulated as a dentifrice (such aε a chewing gum, mouthwash, toothpaεte, spray, lozenge or edible oonbon) ^ oral administration m the treatment of mouth infections. Alternatively, the medicaments may be formulated in a topical formulation '.such as a spray, gel, cream, eye drops, shampoo, etc. ) and/or incorporated in a bandage or a pad for topical administ ation to the skin, eyes, hair, etc., ;>f I,ιdα ziαuals in need thereof. Finally, the medicaments mav also oe formulated aε an injectable solution for internal application. Formulations, equipment and processing techniques have been well developed and are well known in the art for preparing and packaging the medicaments of the present invention as either topical, oral or injectable formulations. The peroxidase/substrate/peroxide systems m the medicaments of this invention are adapted to be incorporated into these formulations. However, the enzymes described herein are subject to degradation and act1vation under conditions such as high shear and elevated temperatures. Accordingly, processing conditions should be controlled during the time span that the enzymes are being admixed with tne other ingredients of the Formulation of the medicaments and converted into finished products, so that the temperature does not rise above 55' : 'C. for any extended period of time. In order to enhance shelf stability, the admixture used in the preparation of the f rmulations of the peroxidaεe medicaments of the present invention should be substantially free of unbound water and the finished product should be packaged a manner, so aε to minimize exposure to air and moisture. — i-\— The peroxidaεe medicaments, of the present invention will setter understood by reference to the following examples, which are illustrative only and are not meant to be limiting m any manner: EXAMPLE I Illustrative base formulations for pharmaceu ically— cceptaole carriers for the peroxidase medicaments to ae formulated with as a dentifrice for oral administration, rucπ aε a chewing gum and chewable tabletε and lozenges are εet forth in Table III, aε follows: Sorbitol, crystalline 98 28 Corn sugar Gum base F1avor Color- Buffer Saccharin, sodium In Table III, formulations (a) and (b) illustrate phav-maceutically-acceptable carriers in the form of chewing gum compositions while formulations (c) and (d) illustrate pharmaceutically-acceptable carriers the form of tablet and lozenge compositions. ftspartame can be substituted for sodium saccharin m these formulations. The following examples show varying ingredients and concentration levels which can be used in the preparation of dentifrices for providing the prophylactic and therapeutic effective amounts for oral administration according to the present invent1 n: Che ing 6urn Sorbitol, Crystalline 70 70 70 Sum base Glycerol F1avor Co1or Sodiurn Bicarbonate Pero idase/Subs rate/Peroxide System (per lOOq. chewing gum) ._ Glucose oxidase 40,000 IU B-D g1ucose 1.0 g Choline oxidase 8,000 IU D-glutamate oxidase 2,500 IU D-g1utamate 0.1 g Lactoperoxidaεe 4,000 IU 1,500 IU 1,000 IU Potassium thiocyanate 0.01 g 0. 05 g Sodium thiocyanate 0.01 g Chewing Gum Sorbitol, Cryεt. 43 Gum Base 20 Glycerol 25 Flavor 1 Color 0. 5 Sod l urn B i car bonat e 0. 5 100. 0 TABLE V continued Peroxidaεe/Subεtrate/Peroxide Syεtem (per 100 g chewing gum) ._ D-Amino acid oxid εe D-Alanine Glucose oxidase B-D-Glucose Lactopero idase Potassium thiocyanate Sodium thiocyanate Sodiurn ascorbate 0.01 a Lozenge Sorbitol, Crystalline Glycerol F1avor Color Sodium Bicarbonate Peroxidase/Subεtrate/Peroxide System (per lOOg. lozenge) ._ Glucose o idaεe 10,000 IU B-D g1ucose 1.0 g Choline oxidase 2,000 IU Choline 0.5 g Urate o idase 10,000 IU Urate 0.75 g Lactoperoxidase 200 IU 200 IU 1,500 IU Potassium thiocyanate — 0.01 g Sodium thiocyanate 0.05 g 0.0Θ g — Lozenge Sorbitol, Crystalline Corn sugar F1avor Color Sodium Bicarbonate Peroxidaεe/Subεtrate/Peroxide Syεterπ '.per QOg. lozenge) ._ Glucose oxidase B-D glucose D-glutamate oxidaεe D- lutamate Lactoperoxidase Potaeεium thiocyanate Sodium thiocyanate EXAMPLE II Illustrative base formulations for pharrπaceutically- acceptable carriers for the peroxidase medicaments of the present invention to be formulated aε a topical medicament for topical administration, such as a cream, a gel or to be incorporated in a bandage or pad, are set forth in Table VIII, aε followε: Deionized water 19.02 20.0 Corn Starch 1 38.04 Lubrajel DV* 5 * 38.04 Aloe vera 0.000021 Watroεol 250 M 3 0.1 Xylitol 4.76 Cirarni N. I *** * * Sunflower Oil Vit min E Tenεami 4/07 * * * Tenεarπi 1/05 * * Bronopol"* Myacide SP * * * Propylene Glycol 1 An example of such a corn starch is the hydrogenated εtarch εolution marketed under the name HYSTAR TPF by Alban Muller International, Montreuil, France. -- Lubragel DV iε a Glycerine and acrylic εolution marketed by Alban Muller International, Montreuil, France. 3 Natroεol 250 M iε a hydroxyethycelluloee marketed by Aqualon, Inc., of Hopewell, Virgina, U.S.A. ■ * ■ Cirarni N.1, Tensa i 4/07, Tensarπi 1/05, Bronopoi and Myacide SP are all marketed by Alban Muller International, Montreuil, France. In Table VIII, formulation (a) illustrates pharnaceutically-acceptable carriers in the form of a gel, and (b) illustrates pharrnaceutically-acceptable carriers the form of a cream. The following Tables show varying ingredients and prophylactic and therapeutic effective amounts (quantities) which can be used in the preparation of topical peroxidaεe medicamente, according to the preεent invention: Gel Deionized water 19.03 19.03 Corn Starch 38.054 33.054 Lubrajel DV 38.054 38.054 Aloe vera 0.001 0. O01 Natrosol 250 M 0.1 0.1 Xylitol 4.76 4.76 100.00 100. 0 Peroxidase/SuDstrate/Peroxide Syεtem (per 100 g Gel) ._ Glucose oxidase B-D Glucose Choline oxidase Choime Lacteroperoxidase Potassium thiocyanate Sodiurn thiocyanate 0.05 g Cream Deionized Water Cira i N. Sunflower Oil Vitamin E Tensarπi 4/07 Tensa i 1/05 Bronopol Myacide SP Propylene Glycol Peroxidase/Substrate/Peroxide System (per 100 g Cream) Glucose oxidase 5,000 IU B-D g1ucose 0.5 g D-Amino acid oxidase 5,000 IU TABLE X con inued D- l anme 0.1 g Urate o idase 10, 000 Urate 0.75 Lactoperoxidase 2, 0 0 500 IU 200 Pot ass l urn t h 1 ocy anat e 0.005 □ Sodium thiocyanate 0.01 g 0.0S EXAMPLE III Illustrati e formulations for pharrnaceutical ly- acceptable carriers for the peroxidase medi aments of the present invention to be formulated aε an eye waεh solution- for topical administration as an eye drop or an eye wash, are set forth in Table XI, as follows: Sorbic Acid 0.0025"/- 0.0002 Purified Wa er 99.4 98.1 Boric acid 0.018 0.0176 Sodium Borate (Hydrated 10 H ffi 0) 0.0015 0.0013 Sodium ch1 ride 0.0025 Benzalkoniurn chloride* 0.0001 Edetate disodium - O.0 1 - Benzalkoniurn chloride and Edetate diεodiurπ are added aε preεervativee. The following Table εhowε the varying ingredients and the prophylactic and therapeutic effective amounts ( quantities) which can be uεed in the preparation of eye waεh medicamentε, according to the present invention: TABLE X I I I gredient Amoun s per ml Eve Wasn 1 Glucose oxidase 2,500 u its"* * * Gl cose 20 milligrams Lactopero i ase 150,000 ABTS units 3 Potassium thiocyanate 200 rπicrograrπε The eye wash solution is a 5 ml solution of: 90 milligrams of Boric acid; 6.6 milligrams or hyαrated sodium borate (10 HeO) ; 2500 units Vitamin A and 0.125 ug of sorbl acid .0025"/. As utilized in this example, the term "unit" of Glucose oxidaεe identifies that amount of Glucose oxidaεe that o idizes 3. 0 milligram glucose to giuconic aciα in one minute at pH 5.10 and 37°C. The assay conditions are set forth m Asεay method FS 250 of Finnish Sugar Co. Ltd., of Finland. In this Example, 1 milligram of glucose oxidase has an activity of 100-120 units at 37*C at pH 5. As utilized herein, the term "ABTS units" identifies that amount of lactoperoxidase that catalyzeε the oxidation of ImM of the ABTS substrate C2, ' -Azino-biε (3- Ethylbenzthiazolιne-6-Sulfomc Aeιd>3 one minute at pH 5 and 37 **** C. The assay conditions are set forth by Mansεon-Raherπtulla, B. , et al., Biochemis y. Vol. 27, at pages 233-239 (1988). In this Example, 1 milligram of lactoperoxidaεe hae an activity of 600 ABTS units at 37*C and 5 pH. The composition is formulated εeparately in two parts, whicn, before application, are combined and shaken to dissolve and mix the two parts. The firεt part lε a mixture of the lactoperoxidaεe and the glucose oxidase. The second part is a 5 ml solution of the boric acid, hydrated sodium borate ',10 H a ϋ) , vitamin H, 0.0025"/- εorbic acid, potassium thiocyanate, water and glucose. The 5 ml solution (the second part) is mixed with the first part and shaken to dissolve the powder. Administration may be made aε normal eye drops. EXAMPLE I V An illuεtrative baee orrnu1ation f r a pharrnaceutica11y- acceptable carrier for the peroxidaεe medicaments to be formulated as an injectable composition (solution ) for internal (injectable) administration. The base composition iε a buffer solution (pH 7) of 0.15 molar sodium chloride and 60 rπillimolar sodium phosphate. To thiε composition 30 units of myeloperoxidase is added and the solution iε mixed to form the medicament. CAs utilized in thiε Example, a unit refers to that quantity of the enzyme necessary for catalyzing the increase of i unit of absorbance at 470 nra in one minute at room temperature using auto-αianiεidine, eee Krawic , et al. , Eastroentero1ogy. vol. 87, ppε. 1344-1350 (1984). In thiε context, i rπicrograrπ equals 1 unit 2. A prophylatic or therapeutic effective amount (for example, approximately 0.5 ml) of this medicament is then administered to a patient in need thereof. Preferably, thiε adminiεtration will be by intramuscular injection. It iε noted that this same solution may be used as a spray .when nebulized aε normally authorized. EXAMPLE V Thiε example shows the effectiveness of the peroxidase/subεtrate/peroxide εyεtern of the peroxidaεe nedicamente of the present invention against an enveloped virus and, in particular, against herpes simplex virus-1. The peroxidaεe/εubεtrate/peroxide eyεterπ of the peroxidase medicaments was prepared having the formulation set forward in Table XIII below: -jώ- TABLE X I I I Ingreαient Amounts per 100 ml buffer solution 1 Glucose oxidaεe 0.02 l11igrams Glucose 1.20 i 11imoleε SCN- 0.06 mi11 i o1eε Lactoperoxidase 4.00 mi11igrams - The buffer solution lε a Hank' ε-Balanced Salt Solution that is calcium, magnesium and glucose free. The pH of the buffer solution is 7.4. The weights of the contents of the HBSS buffer solution is as follows, for every 1000 ml of diεtilled water: 3 grans Nad; 0.4 grams KC1 ; 0.06 grams Na s HP0 * .; and 0.06 grams of KHβPQ . Four (4) εtrains of HSV-1 viruses were collected from exsudateε of herpetic lesions on tongue, nasopharyngeal cavity and vulva. These samples were pooled then typed as HSV-1 using immunof1uoreεcence. They were subsequently allowed to multiply in MRC5 cells and growth medium. Separation from cells and cell debris was performed by centπfugation. Viruses were then stocked in aliquotε in liquid nitrogen. Samples from the HSV-1 pool were then diluted from ten to ten fold down to the antepenultimate cytotoxic titre which was taken aε baεe line for each experiment or control. The peroxidaεe formulation set forth above Table XV waε then mixed with an equal volume of the HSV-1 pool suspension at the baεe line titre (l l/lml). These virus and the peroxidase formulation rπixtureε were allowed 30, 60 or 120 minutes incubation at 37 r -'C They were -ώ-t- then diluted from 10 to 10 fold to obtain suspensions at 5 exponentially decreasing concentra ions. Of each of these suspensions, 50 microliterε were sampled to inoculate a layer of fibroblaεtε grown "in vitro". After inoculation, the cell cultures were examined daily up to seven days. Marks of cytotoxity were semiquantitatively quoted, as follows: 1+ being from 0 to 25%; 2+ being from 25 to 50%; 3+ being from 50 to 75;--; 4+ being from 75 to 100"/.. Controls were settled by substituting the oxidizing moiety with an equal volume of the HBSS buffer. On the contrary, blanks held the complete oxidative system of the formulations but the virus moiety was replaced by the growth rπediurn. The cytotoxity of pretreated virus was compared with that of suspensions which had not been in contact with the peroxidaεe formulation. Thiε comparison allowed to express the resultε in terms of: 1. no effect: that iε, no difference of cytotoxity between experimentε and controlε; 2. delaying effect: when a minimum 24 hour delay lengthened the lag phaee before the onset of cytotoxity; 3. inhibiting effect: when a complete inhibition of the virus cytotoxicity was noticed. Twenty samples of the HSV-1 pool, were dispersed each into five (5) consecutive dilutions (from 10-" **•■ to 10- θ which were treated by mixing in the peroxidase formulations. These tests were compared with the equivalent- number of controls which yield the results plotted in Figure 1. HS can be seen by reference to Figure i, a one πundred and twenty (120) minute incubation period the presence of the peroxidaεe formulation induced complete li-.nibitιz>n of HSV-1 cytotoxic potential. Sixty (60) and thirty '.30) minutes of incubation yield 2/3 and 1/3 of complete inhibition, 1/3 and 1/6 delaying effect, respec ively. However, 1/2 of the samples which had sustained thirty (30) minutes incubation with the oxidase formulation, showed no loss of cytotoxicity. A direct toxicity on the fibroblast layers, due to the oxidative moiety, could not be avoided in few cases, when assaying the highest concentration (H) of the mixtures. This toxicity, however, was no more found while using the ensuing dilutions (that is from H x 10 -1 ), so that it never disturbed the reading of viral toxicity itself. The results nonetheless disclose an obvious weakening of the viruses cytotoxicity power. They appear to be time dependent. Example VI Thiε example shows the effectiveness of the peroxidase/εubεtrate/pero ide syεtem of the peroxidase medicaments of the present invention against an enveloped virus and, particular, against human immunodeficiency virus. The peroxidase/substrate/peroxide system of the peroxidaεe medicaments was prepared having the formulation set forward in Table XIV below: TABLE X IV I gredient Amounts per 100 ml buffer solution 1 Glucose oxidase 0.02 rπi11igrams Glucose 1.20 mill i oles ΞC - * 0.06 mi11i o1es Lactopero idase 4.00 milligrams 1 The buffer solution iε a Hank' ε-Balanced Salt Solution that iε calcium, magnesium and glucose free. The pH of the buffer εolution iε 7.4. The weightε of the contents of the HBSS buffer solution is aε follows, for every 1000 ml of diεtilled water: a grams NaCl ; 0.4 gra s KCl; 0.06 grams eHPO-c; and 0.06 grams of KHePC . HIV aliquotε were obtained from a supernatant of ARV-4 cell line. The aliquots were then extemporaneouεly mingled with an equal volume of the peroxidase formulation eet forth in Table XIV and incubated from 1 hour down to 2 minutes at 37*C. HIV plus the peroxidase formulation was then inoculated to phytohaemagglutinin—εtirπulated human lymphocyte cultures. Final dilution of the aliquots was 1:20, 1:100 and 1:200. Controls were obtained by preincubating the virus the buffer alone. The cultures were supplied again with fresh lymphocytes on day 11 (arrows in figure 2). Virus growth was monitored with an ELISA detecting p24 either intra-cel1ular (per 10 s cells) or in the supernatant. In control experiments experiments, the virus produced early intracel1ular p24 when inoculated to human lymphocytes, at final dilutions- 1:20 and 1:100. Dilution 1:200 however yielded both delayed and lower amounts of p24. By contrast, virus treated with the peroxidase formulation only produced low amounts of p24 at dilution 1:20. The results of these experiments are summarized and can be seen with reference to Fi ures 2 and 3. Fifteen (15) days lymphocyte culture yielded 90 pg of p24 per 10* in controls inoculated with 1:200 diluted virus, while 25 pg only were detected after innoculation of virus treated within the peroxidase formulation for one (1) hour and diluted 1:20. With higher dilutions, no p24 was detected 10 s cells. But the whole culture cf 1 " 7 cells had nevertheleεε been contaminated oy infectious particles since p24 had been shed later on into the supernatant. Cytopathic effect brought forth by the virus in control experiments was not observed after treatment of lymphocytes with the peroxide formulation alone. By contrast, leaving SCN- out the mixture (and therefor allowing H β 0β accumulation), proved to be cytotoxic at the lowest dilution (1:20). Kinetic experiments were preformed with pre cubation at 2, 10, 20, 30 and 60 minutes. These showed that 2 minutes contact with the undiluted peroxidase formulation was enough to reduce the HIV infectivity exhibited in the sampleε. Example VII Thiε example aleo shows the effectiveness of the peroxidase/substrate/peroxide system of the peroxidaεe medicaments of the present invention against an enveloped virus and, particular, against human immunode iciency virus. The peroxidaεe utilized in thiε example lε purified hunian reco o ant myeloperoxidase. An MPO-MIX waε prepared. This- MPO-MIX included 500 ul of culture rned1urn (RPMI, Gibco and 5% fetal calf εerurn, Seralab), supplemented with sodium thiocyanate (20 ug/rnl), glucose 1%, glucose oxidase (6 mU/rπl) and from 10 to 40 ug/ml of purified human reco binant rπyeloperoxidaεe. Thiε human recombinant myelopero idase was produced utilizing the method described in patent application no. PCT/EPΘ9/00668. However, it iε to be understood that thiε myeloperoxidase may be obtained from any suitable source. A 60 ul viral suspension of HTLVIIIB virus, derived from infected Molt3 cells, i.e., 1200 TCIDs (Tcells infectious dosiε 50%) iε prepared. Finally, 2.10* reporter Sup Tl cellε are obtained. In particular, supTl cellε, derived from a human lymphoma (J. Hoaxie, Univ. of Penns-1-yvania, Philadelphia, Pennεlyvania, U.S.A. ) were utilized. The standard procedure was performed aε follows: The HTLVIIIB viral suspension (60 ul) waε added to the MPO-MIX (500 ul) and the reeulting mixture waε incubated for 15 minutes at 37°C. The mixture waε then transferred onto a Sup Tl cell pellet (containing 2.10 e cellε) and further incubated for 30 minutes at 37' : 'C with gentle stirring. Cells were then washed twice with culture medium RPMI and fetal calf serum), pelleted and reεuεpended in 10 l of the same culture medium, i.e., at a cell density of 2.10 s cells/ml. These resuεpended cellε were then cultivated at 37*C for ten (10) days. Microεcopic exa ina i n '.monitoring of the cultures was done at days 3, 5 and 7 to record cytopathic effects, such aε the formation of syncitia. On day 10, 450 ul of the cell culture was collected. This 450 ul of the cell culture was then mixed with 50 ul of buffered saline containing 10% Triton X-100 and stored at -20 ,: 'C before use. The samples were subsequently analyzed by ELISA to quantify the p24 HTLVIIIB antigen (the viral progeny). More precisely, the chosen ELISA measures the amount of HTLVIIIB p24 protein and uses as primary antibody * a rnurine monoclonal antibody raised against p24 (Dupont. U.S.A.; and, as secondary antibody, hu an antl HTL 111B lmmunog1obu11ns iabe11ed with biotin. Specific complexes were revealed using a εtreptavidin-norse radish pero lαaεe conjugate (A erεham) and the 0PDA chromogenic substrate (Sigma). Optical densities were read at 490 nrn. The results of these experiments are set forth below in Table XV. These results show that the peroxidaεe medicament of the present invention containing human recombinant myeloperoxidase at concentrations ranging from 10 to 40 ug/ml, corπletely " inhibits the replication of tne HTLVIIIB virus. TABLE XV Days of Culture Following exposure of virus to rMPD Test 0 3 5 7 10 . Virus HTLVIIIB + CPE 1 nd nd MPO-MI ELISA-* nd 18 19 13 (10 ug/ml MPQ ) T BLE XV ntinued CPE (Cytopathic effectε) : - means no syncitia waε observed. + means a few small syncitia waε observed. + —> ++ means an increase in the number and size of syncitia waε observed. ELISA: are expressed in rnilli units 0D^, βo As can be from Table XV, in samples 1, 2 and 3 no εyncitia waε observed and inhibition of viral replication was noted. Sample 4 exhibited positive co-itrol: both syncitia and viral replication were observed. Sample 5 exhibited negative control: no virus in the assay. In Sample 6 the MPO enzyme lacked one of its- substrates (H a 0a). Thus, no effect on iral replication was noted. Finally, sample 7, since there was no MPO in the assay, no effect on viral replication waε observed. The sum total of the above assays is to demonstrate that use of myeloperoxidase, in appropiate concentrations, the peroxidase εyεtem of the medicament of the present invention completely inhibits the replication of HTLVIIIB virus. Example VIII Thiε example demonstrates the anti-viral effec iveness of the lactoperoxidaεe/εubεtrate/peroxide system and of the rπyeioperoxidase/εubstrate/peroxide system against genital herpes m the genital herpes guinea pig model (the intravaginal guinea pig model). 20 female Hartley guinea pigs received an intravaginal i nocul tion of 10= pfu of HSV2 MS virus. Begining with day ■ H and thereafter continuing daily until αay 24, these guinea pigs were monitored for the appearance and development of herpes lesions (based on a scale from 0 to 4) and were treated by use of one of the three following gels: 1. A control gel (f r a group of four guinea pigs; prepared having the formulation set forth for the gel only in example 9A of Table IX; 2. A gel containing a lactoperoxidaεe/εubεtrate/ peroxide εyεterπ (for a group of eight guinea pigε) prepared having the formulation set forth in example 9A of Table IX, except that 88 IU of lactoperoxidaεe (per lOOg of gel) ,-ιas utilized and not 200 IU of lactoperoxidase; 3. H gei contai ing a myeloperoxidase/suDstrate/ peroxiαe system '.for a group of eight guinea pigs. ) prepared having the formulation set forth in example A of Table IX, except that 70.8 IU of myelopero idase '.per lOOg of gel) waε utilized and not 200 IU of l ctoperoxidase; The developmen of herpes lesions occurs m two successive phasee: the first phase (primary infection; is due to the mnoculated virus; and the second phase (recurrences; is due to the reac ivation, more or lees frequent, of the virus present- in t e nerve cellε m a latent form. The treatment- consisted of applying 0.6 grams of gel on the herpes lesions appearing around the external genital organs. The results of these experiments are summarized in Table XVI (wherein the effect of the treatments on the primary infection are summarized) and in Table XVII (wherein the effect of the treatments on the recurrences- are summarized) and can be seen with reference to the graph set forth in figure 4. Table XVI Average Average Average Duration Gel Severity 1 Maximal Score of Primary I fection 12.7 "7 c -J 2 7.4 p 0.02 6* * 1.8 p 0.03 e 3 8.4 p 0.02- 2 1.9 p O.Ol- ≥ 1 Severity = The sum of the scores from day 4 to day .- . ■a Significant according to the Student's Test ■ -> j>— " ab l e XV I Average Number Average Duration Gel ~f Recurrences 1 of Λ Recurrence i αavs; . 1 1.5 •-+. *-> 2 1.4 N. S. ■ = ■ 3 N. S. ■** - 3 1.9 N. S. ε 3.3 N. S. β - These is a recurrence if one measures, during two successive days, a score equal to 0.5 (erythma; or, during one day, at least one score equal to one '.vesic ie . A recurrence ι= preceeαed and followed by a day witnout ieεi nε. 13 Not significant according to the Students Test. As can be seen from Table XVI and from figure 4, lesions from the primary infections were generally severe (maximal score 2.5-3) and persiεted from day 4 to daye 12-14, while lesions from the recurrences were relatively benign (maximal score 0.5-1) and disappeared after 3-4 days on average. The results of these treatments clearly show that the gelε containing the lactopero idase or the myelopero idase significantly reduce the severity, the maximal scores and the duration of the primary infection. Key To Figures 2 and 3 Figure 2 is illustrative of the growth of HIV-generated p24 in lymphocyte cultures by measurement of the ρ24 in the supernatant. Figure 3 is illustrative of the growth of HIV- generated p24 in lymphocyte cultures by measurement or " intracel 1ular p24 per 10 s cellε. Symbols utilized in figures 2 and 3 are as follows: Solid lines (———> : preincubation 1 hour, buffer alone (controls) ; Stppled lines ( ): preincubation 1 hour, in oxidizmg complex. Final dilutions of HIV initial pool: 1:20 (•O) ; 1:100 (i0 ) ; and 1:200 \ΛΔ) . In view of the foregoing description and examples, it will become apparent to those of crαinary skill in the art that equivalent modifications thereof may be made without departing from the spirit and scope of thiε invention.

Claims: CLAIMS 1. The use of a peroxidaεe for the manufacture of a medicament for the prevention or treatment of enveloped virus infections. 2. The use of claim 1, further chacterized in that a oxygen donor and an oxidizable subεtrate for whicn the peroxidaεe is specific are also uεed for the manufacture of a medicament for the prevention or treatment of enveloped virus infections. 3. The use of claim 1, further characterized that the peroxidase iε lactoperoxidase. 4. The use of claim 1, further characterized in that the enveloped virus is a herpes simplex virus. 5. The use of claim 1, further characterized in that the enveloped virus iε a human immunodeficiency virus. 6. The use of claim 2, further characterized in that the oxygen donor lε hydrogen peroxide. 7. The use of claim 2, further characterized in that the oxygen donor is an enzymatic system including a substrate and an enzyme specific for the subεtrate, such that hydrogen pero ide is formed thereby. 8. The use of claim 7, further characterized in that the εubεtrate of the enzymatic εystem iε glucoεe, and the enzyme of the enzymatic εyεtern iε glucose oxidaεe. 9. The use or " claim 2, further characterized m that the oxygen donor is an inorganic peroxide. 10. The use of claim 2, further characterized in that the oxygen donor is an organic peroxide. 11. The use of claim 2, further characterized in that the oxygen donor is a microorganism. 12. The use or " claim 2, further characterized in that the oxidizable substrate is a thiocyanate salt. 13. The use of claim 2, further characterized in that the oxidizable subεtrate is a halide chosen from the group consisting of chloride, bromide and iodide. 14. The use of claim 1, further characterized in that the peroxidase iε a mammalian peroxidase. 15. The use of claim 1, further characterized in that the peroxidaee is myeloperoxidase. 16. The use of claim 1, further characterized in that the peroxidase is a plant peroxidase. 17. The use of claim 2, further characterized in that the εubstrate is a thiocyanate salt and the peroxidaεe iε a mammalian peroxidaεe. IS. The use of claim 2, further characterized in that the substrate is a halide chosen from the group consiεting of chloride, bromide and iodide and the peroxidaεe lε a plant peroxidase or rnyeloperoxidaεe. 19. The use of claim 2, further characterized in that the oxidizable substrate is a thiocyanate salt, the oxygen donor is an enzymatic system including a glucose subεtrate and an glucoεe oxidaεe and the peroxidaεe is lactoperoxidase. 20. The use of claim 2, further characterized in that the oxidizable εubstrate is a thiocyanate salt, the oxygen donor le an enzymatic system including a glucoεe subεtrate and an glucoεe oxidase and the peroxidaεe is myeloDeroxidase. 21. The use of claim 1, further characteri ed i that the medicament is a topical medicament. 22. The use of claim 1, further characterized in that the medicament is an oral dentifrice. 23. The use of claim 1, further characterized in that the medicament is an injectable composition. 24. A method for the preparation of a medicament for the prevention or treatment of enveloped viruses characterized in that the composition of claims 1 or 2 is combined with a pharmaceutically acceptable carrier. 25. A method for the prevention or treatment of enveloped viruses characterized in that a therapeutic or prophylatic effective amount of the medicament of clairnε 1 or 2 iε administered to a patient in need thereof. 26. The method of claim 25, further characterized in that the enveloped virus is a herpes simplex virus. 27. The method of claim 25, further characterized in that the enveloped virus is a human immunodeficiency virus. 28. The method of claim 25, further characterized in that the medicament iε topically administered. 29. The method of claim 25, further characterized in that the medicament iε orally administered. 30. The method of claim 25, further characterized in that the medicament is injectably administered.

Inventors: BOLLEN ALEX, MOGUILEVSKY NICOLE, POURTOIS MICHEL, BOLLEN, ALEX, MOGUILEVSKY, NICOLE, POURTOIS, MICHEL
Applicants: UNIV BRUXELLES, UNIVERSITE LIBRE DE BRUXELLES
Application year: 1991
Main classification: A61K7
IPC codes: A61K8, A61K9, A61K38, A61P31, A61Q11