METHODS OF USING SUCROSE OCTASULFATE TO TREAT OR PREVENT ENVELOPED VIRUS INFECTION
TECHNICAL FIELD OF THE INVENTION The present invention relates to the use of sucrose octasulfate and pharmaceutically acceptable salts thereof, including sucralfate, the basic aluminum salt of sucrose octasulfate, to treat or prevent viral infections caused by enveloped viruses.
BACKGROUND OF THE INVENTION It is generally recognized that antiviral therapeutic options are limited in number and scope, with few safe and effective agents approved and available for use [D. Kinchington and S. Goldthorpe Intl. Antiviral News, 4(5), pp. 57-61 (1995); S. Goldthorpe, et al . ibid, 4(7), pp. 92-98 (1995)]. Furthermore, even those approved agents can exhibit limiting toxicities and are susceptible to the development of resistance. As such, both from an individual and a public health perspective, prevention strategies remain the most effective means available for the control of transmission of viral diseases. Anionic, sulfated polysaccharides, such as heparin, heparan sulfate, dermatan sulfate, pentosan sulfate and others have been shown to exhibit antiviral activity [M. Baba et al., Proc. Natl. Acad. Sci. USA. 85, pp. 6132-36 (1988)]. They have also been identified as in vitro inhibitors of enveloped viruses, including the HIV virus [M. Witvrouw et al., Antiviral Chem. Chemother., 5, pp. 345-59 (1994)], herpes simplex type 1 and type 2 viruses [Witvrouw et al., supra; B. Barfield et al., Virology, 208, pp. 531-
539] and others. As a result of these activities, anionic sulfated polysaccharides have been proposed as systemic antiviral agents with broad specificty against the enveloped viruses as a class. Anionic sulfated polysaccharides, however, suffer from poor bioavailablilty following oral ingestion and demonstrate toxicity upon continuous intravenous infusion [Witvrouw et al., supra1. They also exhibit significant anticoagulant activity [R. Nagashima, J. Clin. Gastroenterol. , 3, pp. 103-10 (1981); F. A. Ofosue et al., Brit. J. He atol.. 60, pp. 695-704 (1985)]. Indeed, heparin is a well-recognized and potent anticoagulant drug that is widely used clinically in the control of blood clotting. Increased bleeding in the course of antiviral therapy is an unacceptable side effect, given that enveloped viruses targeted by these agents are transmitted through contact with blood and other bodily fluids. Accordingly, these anionic sulfated polysaccharides have limited use as antiviral therapeutics or prophylactics. None have been approved for use as drugs, either in systemic or in topical form.
The anticoagulant and antiviral activity observed for the anionic sulfated polysaccharides both appear to be critically dependent on the degree of polymerization of those molecules. Anticoagulant and antiviral activity decreases rapidly as the size of the polymer decreases. For example, the anticoagulant activity of disaccharide fragments of heparin is no greater than negative controls [R. Nagashima, supra; R. J. Linhardt et al., J. Biol. Chem L.i./ 257, pp. 7310-13 (1982)], and the antiviral activity of dextran sulfate is obliterated when that compound is digested down to disaccharide units [N. R. Hart an et al., AIDS Res. Hum. Retroviruses, 6, pp. 805-12 (1990)].
One particular anionic sulfated polysaccharide,
sucrose octasulfate, and its well-known basic aluminum salt, sucralfate, have been used therpaeutically. United States Patent 3,432,489 describes these compounds.
Sucralfate has been used for treating gastrointestinal ulcers or inflammed tissue. European patent application EP 0 640 346 refers to the use of sulphated mono- and di-saccharides to prevent inflammation. That application also refers to the use of sulphated saccharides to indirectly prevent viral infection by acting on the cell to be infected. Sucralfate, however has never been suggested to be able to kill virus directly.
Sucralfate is an extremely safe drug, with a remarkably benign side-effects profile. Animal studies have shown that 1 gram/kg of body weight over 24 months is tolerated without toxicity. Sucralfate is one of a handful of drugs that has no known lethal dose.
To date, conventional methods and therapeutic agents have not, in all cases, proved to be effective in the treatment or prevention of infections caused by enveloped viruses, such as HIV, and their accompanying disease states. Moreover, those agents that have demonstrated efficacy against enveloped viral infection also cause undesirable side effects and toxicity at high doses. Accordingly, the need still exists for an anti-viral agent that is both effective and safe even at high doses. In addition, it would also be desirable to have available an antiviral agent that could be used as an effective vaginal microbicide without the obligate contraceptive activity, possessed agents under consideration for such use today.
SUMMARY OF THE INVENTION
The present invention solves the problems set
forth above by providing pharmaceutical combinations and methods utilizing sucrose octasulfate and pharmaceutically acceptable salts thereof, including the basic aluminum salt sucralfate, to treat or prevent viral infection or to reduce or prevent viral contamination. Applicant has discovered, surprisingly and unexpectedly, that sucrose octasulfate and its salts can directly kill enveloped viruses by interacting with the positively charged coat of those viruses. In contrast to larger anionic sulfated polysaccharides, sucrose octasulfate and sucralfate are devoid of anticoagulant activity [R. Nagashima, J. Clin. Gastroenterol, 3, pp. 117-27 (1981) ] . In addition, neither contraceptive, nor teratogenic activity has been demonstrated or reported for sucralfate in the course of the many decades that it has been in use as an anti-ulcerant drug. This property makes sucrose octasulfate ideal for use in preventing sexually transmitted enveloped viral diseases, such as herpes and HIV, even in a pregnant patient. As disclosed herein, the present invention provides methods for treating or preventing enveloped viral infection in a patient using sucrose octasulfate or a pharmaceutically acceptable salt thereof. The methods may also be used to prevent transmission of enveloped virus during casual contact (including touching and kissing), sexual contact or during childbirth. The invention also provides methods for using sucrose octasulfate or its salts to disinfect various devices that come into contact with bodily fluids, such as contraceptive devices (condoms, sponges, diaphragms) , surgical gloves, surgical tools, napkins and tissues.
The invention also provides methods for disinfecting liquid preparations, such as blood, plasma, sperm and ova, as well as laboratory and clinical samples, with sucrose octasulfate and its salts. The invention
further provides methods of preventing transmission of enveloped viral infection in veterinary settings and in the production of animals for food.
The invention also provides pharmaceutical combinations comprising sucrose octasulfate or a pharmaceutically acceptable salt thereof, together with an antiviral agent or a contraceptive agent.
The present invention capitalizes on the favorable properties of sucrose octasulfate, including its ability to be administered in high doses without toxicity or side effects; its affinity for damaged epithelium which is known to be a preferred site for viral entry; and its ability to form non-covalent gels or remain in a liquid state depending upon the particular salt used. This latter property allows a choice between a long-acting, slow release dosage form of the sucrose octasulfate (gel) , and a rapidly cleared, high activity dosage form (liquid) , depending upon the particular anti-viral use desired. Other formulation options are described below.
DETAILED DESCRIPTION OF THE INVENTION
The present invention provides methods utilizing sucrose octasulfate or pharmaceutical salts thereof for the prevention or treatment of an enveloped virus infection through the direct interaction of those compounds with the virus. The sucrose octasulfate salts used in the methods of this invention are characterized by several advantages. They do not disrupt the integrity of the target epithelial surface. Accordingly, they do not increase the risk of viral infection. In addition, they have no known toxic or tumorigenic effects. In addition, they have no anticoagulant activities, contraceptive effects, or other reproductive impairments.
Without being bound by theory, we believe that sucrose octasulfate or its pharmaceutically acceptable salts, exert their therapeutic and prophylactic effects by interacting with the positively-charged regions of viral envelope proteins, such as the V3 region of gpl20 on HIV. By binding to viral proteins, sucrose octasulfate or a pharmaceutically acceptable salt thereof will inhibit the ability of such virus to enter an uninfected cell.
According to one embodiment, the invention provides a pharmaceutical combination for the prophylaxis or treatment of an enveloped viral infection in a patient comprising: an amount of a sucrose octasulfate or a pharmaceutically acceptable salt thereof, sufficient to reduce or prevent viral replication in said patient via direct interaction with said virus; an antiviral agent selected from a reverse transcription inhibitor, a viral protease inhibitor, a viral polymerase inhibitor and an anionic sulfated polysaccharide polymer comprising at least three repeating units; and a pharmaceutically acceptable carrier.
The sucrose octasulfate or pharmaceutically acceptable salt thereof useful in these combinations may be obtained commercially or may be synthesized by conventional techniques (see United States patent 3,432,489, the disclosure of which is incorporated herein by reference) . Advantageously, these compounds may be conveniently synthesized from commercially available starting materials.
Pharmaceutically acceptable salts of sucrose octasulfate useful in the combinations of this invention may include organic or inorganic, acid or base salts.
Preferably, the combinations comprise a base salt of sucrose octasulfate derived from alkaline or alkali earth metal. Most preferably, an aluminum or potassium salt of sucrose octasulfate is employed. The aluminum salt, better known as
sucralfate, forms a gel. The potassium salt is completely soluble.
It should also be understood that sucrose octasulfate or a pharmaceutically acceptable salt thereof as used in the combinations of this invention, may be chemically modified by appending appropriate functionalities to enhance selective biological properties. Such modifications are well recognized in the art and include those which increase topical delivery or binding affinity to epithelial surfaces, or those which are compatible with the chemistry of the target surface.
The antiviral agent present in the combination of this invention may be selected from a wide range of known reverse transcription inhibitors, viral protease inhibitors, viral polymerase inhibitors or anionic sulfated polysaccharide polymers comprising at least three repeating units.
If a viral polymerase inhibitors is employed, it is preferably selected from zidovudine ("AZT") , dianosine, zalcitabine, lamivudine, stavudine, nevirapine, 1592U88, or PMEA. If a viral protease inhibitor is employed in the combinations of this invention, it is preferably selected from saquinavir, ritonavir, indinavir, nelfinavir or VX-478. If a viral polymerase inhibitor is used, it is preferably selected from acyclovir, brivudin, cidofovir, famciclovir, fiacitabine, fialuridine, foscarnet, FTC, ganciclovir, GG- 167, idoxuridine, imiquimod, lobucavir, n-docosanol, netivudine, penciclovir, pirodavir, ribavirin, sorivudine, trifluridine, valaciclovir, dideoxycytidine, 5-azacytidine, or vidarabine.
In a preferred embodiment, the additional second antiviral agent is AZT, VX-478, acyclovir or famciclovir.
Any conventional pharmaceutically acceptable carrier or adjuvant may be utilized in the combinations of
this invention. These carriers and adjuvants include, for example, ion exchangers, alumina, aluminum stearate, lecithin, serum proteins, such as human serum albumin, buffer substances, such as phosphates, glycine, sorbic acid, potassium sorbate, partial glyceride mixtures of saturated vegetable fatty acids, water, salts or electrolytes such as protamine sulfate, disodium hydrogen phosphate, sodium chloride, zinc salts, colloidal silica, magnesium, trisilicate, polyvinyl pyrrolidone, cellulose-based substances and polyethylene glycol.
Adjuvants for topical or gel base forms may be selected from the group consisting of sodium carboxymethylcellulose, polyacrylates, polyoxyethylene-polyoxpropylene-block polymers, polyethylene glycol, natural and synthetic gum bases, and wood wax alcohols.
Formulations may include any excipient or carrier which may be added to sucrose octasulfate or pharmaceutical salts thereof, without affecting its biological effect. Pharmaceutical combinations of the present invention may be administered to epithelial surfaces or the body, or may be adminstered by parenteral techniques or by an implanted resevoir. The term "parenteral" as used herein includes subcutaneous, intravenous, intramuscular, intra- articular, intra-synovial, intrasternal, intrathecal, intrahepatic, intralesional and intracranial injection or infusion techniques.
In a preferred embodiment, the combinations of the present invention may be topically administered to any epithelial surface. An "epithelial surface" according to this invention is defined as an area of tissue that covers external surfaces of a body, or which and lines hollow structures including, but not limited to, cutaneous and mucosal surfaces. Such epithelial surfaces include oral, pharyngeal, esophogeal, pulmonary, ocular, aural, nasal,
buccal, lingual, vaginal, cervical, genitourinary, alimentary, and anorectal surfaces.
Pharmaceutical combinations of the present invention, may be formulated in a variety of conventional forms employed for topical administration. These include, for example, semi-solid and liquid dosage forms, such as liquid solutions or suspensions, suppositories, douches, enemas, gels, creams, emulsions, lotions, slurries, powders, sprays, lipsticks, foams, pastes, toothpastes, ointments, salves, balms, douches, drops, troches, chewing gums, lozenges, mouthwashes, rinses. Standard and well-understood formulation strategies for topical agents can be applied to sucrose octasulfate or a pharmaceutically acceptable salt thereof in order to enhance the persistence and residence time of the drug, and to improve the prophylactic efficacy achieved.
According to one preferred embodiment of this invention, the sucrose octasulfate combination is formulated into a topical cream or gel. Such formulations are particularly useful, for example, for treatment or prevention of vaginal enveloped viral infections and treatment of viral infections of the oral cavity, including cold sores caused by herpes labialis .
Furthermore, the pharmaceutical combinations of this invention may also be administered topically. Such topical formulations may be used to treat or prevent enveloped viral infections of eye, the skin, or the lower intestinal tract. Suitable topical formulations are may be readily prepared for each of these areas or organs. Topical application for the lower intestinal tract can be effected in a rectal suppository formulation or in a suitable enema formulation. Topically-transdermal patches may also be used.
The pharmaceutical combinations of this invention
may also be administered by nasal aerosol or inhalation. Such combinations are prepared according to techniques well- known in the art of pharmaceutical formulation and may be prepared as solutions in saline, employing benzyl alcohol or other suitable preservatives, absorption promoters to enhance bioavailability, fluorocarbons, and/or other conventional solubilizing or dispersing agents.
For ophthalmic use, the pharmaceutical combinations may be formulated as micronized suspensions in isotonic, pH adjusted sterile saline, or, preferably, as solutions in isotonic, pH adjusted sterile saline, either with our without a preservative such as benzylalkonium chloride. Alternatively, for ophthalmic uses, the pharmaceutical combinations may be formulated in an ointment such as petrolatum.
Alternatively, the pharmaceutical combinations of this invention may be administered in the form of suppositories for rectal administration. These can be prepared by mixing the agent with a suitable non-irritating excipient which is solid at room temperature but liquid at rectal temperature and therefore will melt in the rectum to release the drug. Such materials include cocoa butter, beeswax and polyethylene glycols.
Sucrose octasulfate is a particularly preferred pharmaceutical salt of sucrose octasulfate in the combinations of this invention due to its formation of an anionic gel in dilute acid and its ability to bind to and protect ulcerated areas of the gastrointestinal tract. Orally administered sucrose octasulfate, which is presumably in a gel form in the gastrointestinal tract, may have benefits as a slow acting virucide as a result of slow release from its gel-like state.
The pharmaceutical combinations of this invention may be orally administered in any orally acceptable dosage
form including, but not limited to, capsules, tablets, gums, aqueous suspensions or solutions. In the case of tablets for oral use, carriers which are commonly used include lactose and corn starch. Lubricating agents, such as magnesium stearate, are also typically added. For oral administration in a capsule form, useful diluents include lactose and dried corn starch. When aqueous suspensions are required for oral use, the active ingredient is combined with emulsifying and suspending agents. If desired, certain sweetening, flavoring or coloring agents may also be added. Sterile injectable forms of the combinations of this invention may be aqueous or oleaginous suspension. These suspensions may be formulated according to techniques known in the art using suitable dispersing or wetting agents and suspending agents. The sterile injectable preparation may also be a sterile injectable solution or suspension in a non-toxic parenterally-acceptable diluent or solvent, for example as a solution in 1, 3-butanediol . Among the acceptable vehicles and solvents that may be employed are water, Ringer's solution and isotonic sodium chloride solution. In addition, sterile, fixed oils are conventionally employed as a solvent or suspending medium. For this purpose, any bland fixed oil may be employed including synthetic mono- or di- glycerides. Fatty acids, such as oleic acid and its glyceride derivatives are useful in the preparation of injectables, as are natural pharmaceutically-acceptable oils, such as olive oil or castor oil, especially in their polyoxyethylated versions. These oil solutions or suspensions may also contain a long-chain alcohol diluent or dispersant, such as Ph. Helv or similar alcohol.
In the combinations of this invention, the antiviral agent is present in amounts ranging from 50% to 100% of the amounts normally administered for that agent
when it is used in a monotherapy. The remaining part of the combination will be made up of carrier and, if appropriate, water.
Sucrose octasulfate or a pharmaceutically acceptable salt thereof will represent some percentage of the total dose in other dosage forms, including liquid solutions or suspensions, suppositories, douches, enemas, gels, creams, emulsions, lotions, slurries, powders, sprays, lipsticks, foams, pastes, toothpastes, ointments, salves, balms, douches, drops, troches, lozenges, mouthwashes, rinses and others. Creams and gels, for example, are typically limited by the physical chemical properties of the delivery medium to concentrations less than 20% (e.g., 200mg/gm) . For special uses, far less concentrated preparations can be prepared, (e.g., lower percent formulations for pediatric applications) .
Given that no toxicity has been observed for sucrose octasulfate or a pharmaceutically acceptable salt thereof over many decades of clinical use as an anti-ulcerant [W. R. Garnett, Clin. Pharm. , 1, pp. 307-14
(1982); R. N. Brogden et al., Drugs, 27, pp. 194-209 (1984); D. M. McCarthy, New Enσ. J. Med., 325, pp. 1017-25 (1991)], one cannot establish an upper limit for the therapeutically effective dose short of mechanical compaction at the site of application. For most forms of sucrose octasulfate the minimum amount present in the combinations of this invention that is effective in treating or preventing viral disease due to direct interaction with the virus should produce be 1 x 10 mg/ml. If sucralfate is employed the minimum effective concentration should be 1 x 10 mg/ml.
According to another embodiment, the invention provides a pharmaceutical combination for preventing conception and for the prophylaxis or treatment of an
enveloped viral infection in a patient comprising: an amount of a sucrose octasulfate or a pharmaceutically acceptable salt thereof, sufficient to reduce or prevent viral replication in said patient via direct interaction with said virus; an amount of a contraceptive effective to prevent conception; a pharmaceutically acceptable carrier.
In this embodiment, the sucrose octasulfate component can be in any salt form mentioned above. Preferably, it is the aluminum salt sucralfate. Preferably, the contraceptive agent is selected from nonoxynol-9, nonoxynol-11, octoxynol, sodium docusate, HPA-23, gossypol, menfegol, arildone, gramicidin, magainins, defensins, melittin, or amphotercin B. Most preferably, the contraceptive agent is nonoxynol-9. The carrier employed in the sucrose octasulfate/contraceptice agent combinations of this invention should be compatible with vaginal administration and/or coating of contraceptive devices. The choice of particular carrier for these uses is well known in the art. These combinations may be utilized in solid, semi-solid and liquid dosage forms, such as diaphragm jelly, douches, foams, ointments, creams, balms, gels, salves, pastes, slurries, vaginal suppositories, sexual lubricants, and coatings for devices such as condoms, contraceptive sponges, cervical caps and diaphragms.
According to another embodiment, the invention provides a method for treating or preventing an enveloped virus infection in a patient comprising the step of administering to said patient a composition comprising: an amount of a sucrose octasulfate or a pharmaceutically acceptable salt thereof, sufficient to reduce or prevent viral replication in said patient via direct interaction with said virus; and a pharmaceutically acceptable carrier. The choice of sucrose octasulfate form and of
carrier are the same as described above for sucrose octasulfate/antiviral agent combinations.
According to one preferred embodiment, this method comprises the additional step of administering to said patient, either as part of the same dosage form or as a separate dosage form, an antiviral agent selected from a reverse transcription inhibitor, a viral protease inhibitor, a viral polymerase inhibitor and an anionic sulfated polysaccharide polymer comprising at least three repeating units.
Choices and preferences for the various antiviral agents are the same as disclosed above for sucrose octasulfate/antiviral agent pharmaceutical combinations. According to an alternate preferred embodiment, the methods comprises the additional step of administering to said patient, either as part of the same dosage form or as a separate dosage form, an amount of a contraceptive effective to prevent conception.
Choices and preferences for the form of sucrose octasulfate and for the contaceptive agent are the same as those described above for the sucrose octasulfate/ contraceptive agent pharmaceutical combinations.
Combinational therapies utilized in the methods of this invention may also exert an additive or synergistic effect, particularly when the viricidal activity of each component operates via a different mechanism. The reduction in effective therapeutic dose achieved with such combinations may be additive or synergistic. In the latter case, dramatically lower doses of both the conventional antiviral agent and of sucrose octasulfate or a pharmaceutically acceptable salt thereof will lead to effective therapy nonetheless. Accordingly, the occurrence of any side effects associated with the added conventional antiviral agent may be reduced or avoided — where, as
stated above, the toxicity of sucrose octasulfate or its basic aluminum salt, sucralfate is known to be minimal from prior clinical experience. Another advantage of such combination therpy regimens is that they may delay the emergence of viral resistance.
A patient according to this invention is an animal, including a human. Animal patients include, but are not limited to, other mammals, birds, and fish. Preferably, the patient is a human. The methods of this invention are carried out for a period of time sufficient to prevent viral replication in a patient or to prevent transmission of enveloped virus from an infected patient to another patient through the direct interaction of sucrose octasulfate or its salts with the virus.
Such enveloped viruses include, for example, virus from the genera Retroviridae , Herpesviridae, Orthomyxoviridae, Paramyxoviridae, Hepadnaviridae , Flaviviridae , Togaviridae, Rhabdoviridae, Poxviridae, Arenaviridae, Coronoviridae, Bunyaviridae and Filoviridae. Retroviridae that may be treated or prevented using the methods of this invention include, for example, human immunodeficiency virus type 1 and the human immunodeficiency virus type 2 lentiviruses, foamy viruses and human T-cell leukemia viruses.
Herpesviridae that may be treated or prevented using the methods of this invention include, for example, herpes simplex type 1 and herpes simplex type 2 viruses, varicella-zoster viruses, cytomegaloviruses, lymphoproliferative herpesviruses and Epstein-Barr virus.
Orthomyxoviridae that may be treated or prevented using the methods of this invention include, for example, influenza A, influenza B and influenza C viruses.
Paramyxoviridae that may be treated or prevented using the methods of this invention include, for example, respiratory syncytial virus, mumps virus, parainfluenza viruses and measles-like viruses. Hepadnaviridae that may be treated or prevented using the methods of this invention include, for example, hepatitis B virus.
Flaviviridae that may be treated or prevented using the methods of this invention include, for example, hepatitis C virus, yellow fever virus, dengue virus and tick-borne encephalitis viruses.
Togaviridae that may be treated or prevented using the methods of this invention include, for example, rubella virus . Rhabdoviridae that may be treated or prevented using the methods of this invention include, for example, rabies virus and vesicular stomatitis virus.
Poxviridae that may be treated or prevented using the methods of this invention include, for example, vertebrate and avian poxviruses and vaccinia viruses.
Arenaviridae that may be treated or prevented using the methods of this invention include, for example, the arenaviruses.
Coronaviridae that may be treated or prevented using the methods of this invention include, for example, the coronaviruses.
Bunyaviridae that may be treated or prevented using the methods of this invention include, for example, the hantaviruses. Filoviridae that may be treated or prevented using the methods of this invention include, for example, Marburg, Reston and Ebola viruses.
Preferably, the enveloped viral infection to be
treated is one wherein the virus if from the genera Retroviridae, Herpesviridae, Orthomyxoviridae, Paramyxoviridae, Hepadnaviridae, Flaviviridae, or Rhabdoviridae. More preferably, the virus is type 1 or type 2 human immunodeficiency virus, type 1 or type 2 herpes simplex virus, varicella zoster virus,
Epstein-Barr virus, cytomegalovirus, influenza type A, B, or C virus, respiratory scincytial virus, mumps virus, hepatitis B virus, hepatitis C virus, encephalitis virus, rabies virus, or dengue fever-inducing virus. Most preferably, the virus is type 1 or type 2 human immunodeficiency virus or type 1 or type 2 herpes simplex virus.
Pharmaceutical compositions of this invention are also useful in the treatment or prevention of respiratory complications in neonates resulting from paramyxovirus infection, measles, German measles, conjunctivitis, other non-sexually transmitted enveloped virus-induced diseases, herpes-associated Karposi's sarcoma, other viral-assocated cancers, encephalitis and yellow fever.
Preferably, the methods of this invention are used to treat an epithelial surface of a patient. Most preferably, the epithelial surface is selected from oral, pharyngeal, esophogeal, pulmonary, ocular, aural, nasal, buccal, lingual, vaginal, cervical, genitourinary, alimentary, or anorectal surfaces.
In one preferred embodiment, the methods of this invention are useful to prevent and treat infections that cause oral and esophageal sores. Oral sores may arise by infection from a number of enveloped viruses including
Herpesviridae, including herpes simplex type I and type II viruses, and Retroviridae such as the human immunodeficiency viruses. Sucrose octasulfate or a pharmaceutically acceptable form thereof, used in this embodiment may be in
several forms including solid, semi-solid and liquid dosage forms, including liquid solutions or suspensions, troches, lozenges, mouthwashes, rinses, pastes toothpastes, ointments, salves, balms, lipsticks, gels, creams, emulsions, lotions, slurries, powders, sprays, foams, and drops .
In another preferred embodiment, the methods of this invention are useful for treating and preventing infections that cause genital sores. Genital sores may arise from infection by a number of enveloped viruses including Herpesviridae such as the herpes simplex type I and type II viruses, retroviridae such as the human immunodeficiency viruses. Sucrose octasulfate or a pharmaceutically acceptable salt thereof, used in this embodiment may be incorporated into several forms including solid, semi-solid and liquid dosage forms, diaphragm jelly, douches, sprays, foams, lotions, ointments, creams, balms, powders, gels, salves, pastes, slurries, suppositories, sexual lubricants, and including coatings or for devices such as condoms, diaphragms, feminine napkins, and tampons. In another preferred embodiment, the methods of this invention are useful for treating and preventing respiratory infections. Respiratory infections be caused by a number of enveloped viruses including Orthomyxoviridae such as the influenza virus, Paramyxoviridae such as the respiratory syncytial virus, parainfluenza virus, hantavirus, and cytomegalovirus. Sucrose octasulfate or pharmaceutically acceptable salt thereof, used in this embodiment may be in several forms including solid, semi- solid and liquid dosage forms, including liquid solutions or suspensions, troches, lozenges, mouthwashes, rinses, pastes toothpastes, ointments, salves, balms, lipsticks, gels, creams, emulsions, lotions, slurries, powders, sprays, foams, and drops.
According to another embodiment, the invention provides a method for preventing vertical transmission of an enveloped virus from a mother to a child during childbirth, which comprises the step of treating said mother prior to or during labor with a composition comprising: an amount of a sucrose octasulfate or a pharmaceutically acceptable salt thereof, sufficient to reduce or prevent viral replication in said child via direct interaction with said virus; and a pharmaceutically acceptable carrier. This method is carried out by topically administering to the mother's birth canal during or prior to labor a therapeutically effective concentration of sucrose octasulfate or a pharmaceutically acceptable salt thereof. Recent data suggests a correlation between maternal viral load and extended contact with vaginal and cervical secretions by the neonate, with the incidence of vertical transmission of HIV to newborns [T. Taha et al., XI Intl. Conf. on AIDS, abstract Th.C.410 (1996); R.J. Simonds et al., ibid, abstract Tu.C.440 (1996); R.W. Steketee et al., ibid, abstract Tu.C.441 (1996); R. Ravinathan et al., ibid, abstract We.C.3580 (1996)].
According to this embodiment, the sucrose octasulfate composition or combination utilized should be compatible with the treatment of neonates. The choice of carrier and antiviral agent if utilized for use in childbirth is well known in the art.
According to another embodiment, the invention provides a method for disinfecting a health care device which is designed to come into contact with a patient, said method comprising the step of contacting the health care device with a composition comprising an amount of a sucrose octasulfate or a pharmaceutically acceptable salt thereof, sufficient to reduce the viral titer on said health care device prior to said device coming into contact with said
patient. Preferably, the device is selected from a contraceptive device, a plastering material, a bandage, a sponge, a napkin, a strip, a tissue, a protective external covering used in medicine, a surgical device, a dental device, a laboratory device, surgical gloves, a surgical gown, and a surgical mask. For example, sucrose octasulfate or a pharmaceutically acceptable salt thereof, may be mixed with a talcum lubricant powder used to line surgical gloves. The amounts of sucrose octasulfate salts, as well as the antiviral agent (if present), carriers and adjuvants that are necessary for the coating or treatment of said devices that are necessary to treat the aforementioned devices and biological materials for the purpose of disinfecting said devices prior to coming into contact with animals will be dependent on the type, size, surface area, consistency, intended use of this device. Preferably, the devices will be prepared in such a way as to be treated with a therapeutically or prophylactically effective amount and for a period of time sufficient to prevent the transmission of an enveloped virus from said device. One skilled in the art will be able to determine the most appropriate formulations for treatment of said devices.
According to another embodiment, the invention provides a method for disinfecting a biological fluid comprising the step of contacting said fluid with an amount of sucrose octasulfate or a pharmaceutically acceptable salt thereof, sufficient to reduce the viral titer in said biological fluid. Preferably, the biological fluid is selected from blood, plasma, ova, or semen. Because of its benign nature, the sucrose octasulfate or salt thereof may be added directly to the biological fluid. Alternatively, it may be coupled to a solid support comprising, for example, plastic or glass beads, or a filter, which is placed in contact with those samples. Sucrose octasulfate
and its salts are particularly advantageous for purifying blood because of its lack of anticoagulant activity.
According to yet another embodiment, the invention provides a method for preventing the transmission of an enveloped viral infection between animals, birds or fish, comprising the step of contacting a food, water, or living environment of said animals, birds or fish with an amount of a sucrose octasulfate or a pharmaceutically acceptable salt thereof, sufficient to reduce or prevent viral replication in said animals, birds or fish via direct interaction with said virus.
The most effective mode of administration and dosage regimen of sucrose octasulfate or pharmaceutically acceptable salts thereof for living things depends upon the target virus, previous therapy, the extent of desired treatment, the target area of treatment, the body weight of patient, drug combinations, severity of the disease, the patient's health status and response to the sucrose octasulfate and the judgment of the treating physician. The sucrose octasulfate may be administered to the patient in any pharmaceutically acceptable topical dosage form, at one time or over a series of treatments.
Upon improvement of a patient's condition, a maintenance dose of the sucrose octasulfate or a pharmaceutically acceptable salt thereof alone or in combination with a second antiviral agent may be administered, if necessary. Subsequently, the dosage or the frequency of administration, or both, may be reduced, as a function of the symptoms, to a level at which the improved condition is retained. When the symptoms have been alleviated to the desired level, treatment should cease. Patients may, however, require intermittent treatment on a long-term basis upon any recurrence of disease symptoms.
In order that the invention described herein may
be more fully understood, the following examples are set forth. It should be understood that these examples are set forth for illustrative purposes only and are not to be construed as limiting this invention in any manner.
Example l — cell lines and Materials Sucralfate was purchased as a powder from Spectrum Chemical Manufacturing. Corp. (Gardena, CA) or as a tablet from Marion Merril Dow (Kansas City, Mo) . Sucrose octasulfate powder was purchased from U.S. Pharmacopeial Convention, Inc., Rockville, MD.
Sucralfate stock suspensions were prepared by dissolving lg of sucralfate powder in 4mL of RPMI-1640 medium with 0.01 N HC1 added. The sucralfate stock suspension (at 250 mg/ml) was stored at 4°C prior to use. Sucrose octasulfate solutions were prepared by dissolving 100 mg sucrose octasulfate in 4mL of RPMI-1640 medium; the 25 mg/mL sucrose octasulfate solutions were also stored as above . Human monocytes and cell lines (C8166, H9, U-937) were prepared in RPMI-1640 media, supplemented with 20% fetal calf serum. Peripheral blood mononuclear cells (PBMCs) were grown in the same medium to which IL-2 (5%, Cellular Products) and phytohemagglutinin (5μg/mL) had been added.
HTLV-IIIB was provided by R.C. Gallo, NCI, Bethesda, MD. HIV-1 18a and 18c was provided by Dr. V.A. Johnson, University of Alabama, Birmingham, AL. HIV-1-9400232 (HIV-194) , is a low passage clinical isolate obtained from a participant in an AIDS clinical trial.
Example 2 — Cvtotoxicity Assays Given that sucralfate suspensions interfere visually with the determination of cell viability by trypan blue staining, cytotoxic effects of the drug were determined
by monitoring cellular proliferation, as assayed by 3H- thymidine incorporation. One μCi thymidine (DuPont-NEN, Boston, MA) was added to 105 cells in 200 μL of medium in 96-well plates which had been cultured for several days with various concentrations of sucralfate or sucrose octasulfate, or in the absence of drug. Following the addition of 3H- thymidine, cultures were incubated for 4-6 hours at 37°C and 3H-TdR incorporation was determined.
Example 3 — Inhibition of Enveloped Virus
Activity in Infected Cell Types Cell-free HIV stocks were obtained from supernatant fluids of HTLV-III infected H9 cultures or C8166 cells (a T4+ transformed human umbilical cord blood lymphocyte line) [J. Sodroski et. al., Nature, 322, pp. 470- 74 (1986)] (culture conditions: 1 x 106 cells/ml with 75% viable cells) . Stock solutions of sucralfate suspensions (both powder and tablet) were prepared in RPMI medium containing 0.01N HC1. Stock solutions of sucrose octasulfate were prepared in RPMI alone. Serial dilutions of sucralfate and sucrose octasulfate ranging from 0.0064 mg/ml to 100 mg/ml were prepared from the stock solutions so that equivalent aliquots of diluted sucralfate and sucrose octasulfate suspensions could be added to cells for a series of final concentrations ranging from 0 to 4 mg/ml.
2 x 105 PBMCs, or 1 x 105 monocytes, C8166, H9, or U-937 cells in 100 μL were exposed to 100 TCID50 of HIV-1 inoculum in 50 μL without a subsequent wash (where TCID50 is defined as the viral titer that will infect 50% of the cells in a tissue culture experiment) . Simultaneously, 100 μL aliquots of the sucralfate or sucrose octasulfate dilutions (described above) were added to each well. Four days after infection, supernatants from the test cultures were assayed for viral replication as described below.
HIV virus replication assays were performed essentially as according to D.D. Ho et al., J. Virol.. 61, pp. 2024-28 (1987) and Sodroski et al . , supra. In this assay, the ability of sucralfate to block HIV replication was measured by HIV p24 antigen production. An assay kit [HTLV-III p24 Radioimmunoassay System, Catalogue No. NEK-040, NEK-040A, Biotechnology Systems, New Research Products, Dupont] which contained affinity purified 125i labelled HIV p24 antigen, a rabbit anti-p24 antibody and a second goat anti-rabbit antibody was used to precipitate p24 antigen-antibody complexes. The assay was carried out according to the protocol included with the kit as further described below.
Specifically, a sample containing unlabelled p24 antigen was mixed with a fixed amount of 125ι labelled p24 and a fixed limiting amount of rabbit anti-p24 antibody. The samples were incubated overnight at room temperature. A goat anti-rabbit immunoglobulin preparation was then added to the test mixture, to precipitate the anti-p24 antibody. The samples were centrifuged and the supernatant was aspirated. Pelletted 125I labelled p24 was quantitated for each sample by gamma counting and comparison to a standard curve .
Based on the measurements of p24 production over the dosage range of sucrose octasulfate and sucralfate tested, the concentration of sucralfate and sucrose octasulfate required to inhibit 50% of viral replication (IC50 values) were determined. Sucrose octasulfate and sucralfate reproducibly caused concentration-dependent inhibition of HIV-1 replication. TC50 values indicate the concentration of sucralfate or sucrose octasulfate that inhibited 50% of cellular proliferation as measured by tritiated thymidine incorporation (see protocol Example 2, results Table 1) .
The inhibitory activity of sucralfate tablet and sucralfate powder can be compared within cell lines on Table 1. For instance, sucralfate tablet is approximately 4.5 fold more effective against an acutely infected H9 cell line than a chronically infected cell line. Sucralfate powder and sucrose octasulfate both were more effective against acutely infected H9 cells than chronically infected H9 cells .
Sucralfate activity varied depending on the viral strain and cell line tested. For example, in PMBC cells, sucralfate tablet suspension inhibited HIV replication more effectively in HIV 18C and HIV 194 isolates than in HIV 18a isolate. However, sucralfate tablet suspensions did not have different effects on the level of HIV infection in monocytes challenged with HIV 18a or HIV 18c (see IC50 values, Table 1) . Therefore, the treatment or prophylaxis of HIV infection using sucralfate and sucrose octasulfate will need to be adjusted depending on the targeted cell and viral strain.
TABLE 1
Example 4 — Svnerσistic Effect of AZT and Sucrose Octasulfate Aσainst HTV 2 x 105 PBMCs in 100 μL were exposed to 100 TCID50 of HIV-1 inoculum (isolates 18c and 18c) in 50 μL without subsequent washing of cells. Simultaneously, 100 μl aliquots of pre-diluted sucrose octasulfate or AZT suspensions were added to each well for a final concentration of 4 nM, InM, 0.25nM, 0.064nM, or 0.016nM of AZT or a final concentration 4 mg/ml, 1 mg/ml, 0.25 mg/ml, 0.064 mg/ml, or 0.016 mg/ml of sucrose octasulfate. In another set of experiments, AZT was tested in combination with sucrose octasulfate such that 4 nM AZT was tested with 4 mg/ml sucrose octasulfate, 1 nM AZT was tested with 1 mg/ml of sucrose octasulfate, and so on. Viral replication was assayed by quantitating HIV p24 antigen production four days after infection (see Example 3) .
In tests involving HIV isolate 18a, sucrose octasulfate alone inhibited the infection of 50% of the cells (IC50) at 0.99 mg/ml. AZT inhibited the infection of 50% of the cells (IC50) at 3.35 nM. Sucrose octasulfate and AZT combined was able to inhibit 50% of the tested cells at a concentration of 0.65 nM AZT and 0.35-0.45 mg/ml sucrose octasulfate. Therefore, the results indicate that five fold less AZT and approximately 2.5 fold less sucrose octasulfate are required to achieve 50% viral inhibition than by either AZT or sucrose octasulfate alone. In a similar study using HIV isolate 18c, ten fold less AZT and five fold less
sucrose octasulfate was needed to achieve 50% viral inhibition than when either one was used alone.
Example 5 Effect of pH on the
Enveloped Virus Killing Activity of Sucralfate
Sucralfate was dissolved in solutions with varying pH prior to addition to cells. Inhibition of viral replication was measured four days after infection by p24 antigen production (see Example 3) . Sucralfate dissolved in a solution of pH 6.0 was more inhibitory than sucralfate dissolved in pH 3.0 (compare IC50 values, Table 2). Sucralfate was also found to be more soluble at pH 5.61-6.40 than pH 3.0. Therefore, the activity of sucralfate appears related to its solubility. TABLE 2 p24 Antigen ng/mL (Day 4 after infection)
Example 6 — Sucrose Octasulfate and Salts Have
Anti-Herpes Simplex Virus Activity HSV-1 (strain HSV-690) and HSV-2 (strain SKB-1) and the AS49/20S cell line were obtained from Dr. Adriana Weinberg, University of Colorado Health Sciences. The aniviral activity of sucralfate tablets and sucralfate octasulfate was tested at final concentrations (ranging from
0 to 80 mg/ml) against herpes simplex virus, type 1 (HSV-1) or against herpes simples virus, type 2 (HSV-2) . Trisodium phosphonoformate (PFA) , a viral polymerase inhibitor, was used as a positive control. In these studies, HSV-1 or HSV- 2 (MOI 0.005 pfu/cell) and sucralfate or sucrose octasulfate was added to AS49/20S cells, incubated at 37°C in 5% C02, and observed for evidence of cytopathic effect (CPE) over a period of 0 to 48 hours after viral innoculation. Observation of CPE is commonly used by those of skill in the art as a measurement of viral activity (Table 3) .
Alternatively, plaque formation was observed and quantitated (Table 4) .
Based on CPE observations, sucralfate and sucrose octasulfate inhibited 50% of CPE in HSV-1 infected cells at approximately 0.125 to 2 mg/ml. Severity of CPE depended upon the point of addition of drug or viral challenge of HSV-1. For example, in Tables 3a-c, sucralfate and sucrose octasulfate were more effective against HSV-1 when added simultaneously with virus than when added after the virus was allowed to contact the cell (compare IC50 values of preinfected cells and cells not preinfected) .
TABLE 3
Anti-HSV-1 Activity of Sucralfate and Sucrose Octasulfate
CPE (Cytopathic Effect)
The inhibitory activity of sucrose octasulfate (over a final concentration range of 0 to 8 mg/ml) was tested essentially as described above. However, viral activity was quantitated by counting the number of plaques formed for each infection (see results in Table 4, below) .
HSV-2, like HSV-1, was more effectively inhibited by sucrose ocatasulfate or sucralfate when the cells were not pre-treated with virus (See Table 4, compare IC50 values of preinfected cells and cells not preinfected) . At concentrations of 0.03 to 0.5 mg/ml, sucrose octasulfate inhibited 50% of plaque formation by HSV-2 when no drug was added. No significant toxicity was observed at the concentrations and formulations of sucralfate or sucrose octasulfate as recited in Table 3 or Table 4.
TABLE 4
Anti-HSV-2 Activity of Sucrose Octasulfate in Human AS49/20S Cells
PFU (Plaque Forming Units)/0. lmL
While we have hereinbefore presented a number of embodiments of this invention, it is appartent that our basic construction can be altered to provide other embodiments which utilize the methods of this invention. Therefore, it will be appreciated that the scope of this
invention is to be defined by the claims appended hereto rather than the specific embodiments which have been presented hereinbefore by way of example.