WO1995015182A1 - Compositions anti-infectieuses et procedes d'utilisation - Google Patents

Compositions anti-infectieuses et procedes d'utilisation Download PDF

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Publication number
WO1995015182A1
WO1995015182A1 PCT/US1994/013834 US9413834W WO9515182A1 WO 1995015182 A1 WO1995015182 A1 WO 1995015182A1 US 9413834 W US9413834 W US 9413834W WO 9515182 A1 WO9515182 A1 WO 9515182A1
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approximately
copolymer
molecular weight
weight represented
crl
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PCT/US1994/013834
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English (en)
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Robert L. Hunter
R. Martin Emanuele
Hameedsulthan S. Allaudeen
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Emory University
Cytrx Corporation
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Priority to AU12990/95A priority Critical patent/AU1299095A/en
Publication of WO1995015182A1 publication Critical patent/WO1995015182A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08G—MACROMOLECULAR COMPOUNDS OBTAINED OTHERWISE THAN BY REACTIONS ONLY INVOLVING UNSATURATED CARBON-TO-CARBON BONDS
    • C08G65/00—Macromolecular compounds obtained by reactions forming an ether link in the main chain of the macromolecule
    • C08G65/02—Macromolecular compounds obtained by reactions forming an ether link in the main chain of the macromolecule from cyclic ethers by opening of the heterocyclic ring
    • C08G65/30—Post-polymerisation treatment, e.g. recovery, purification, drying
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/74—Synthetic polymeric materials
    • A61K31/765—Polymers containing oxygen
    • A61K31/77—Polymers containing oxygen of oxiranes

Definitions

  • the present invention relates to antiinfective compounds and more particularly to compounds and methods that kill or suppress the growth of bacteria, viruses, fungi and protozoa.
  • the compounds and methods are effective in controlling intracellular organisms.
  • Tuberculosis has been a major killing disease of civilization for most of recorded history. The incidence of the disease declined in parallel with advancing standards of living since at least the mid-nineteenth century. However, in spite of the efforts of numerous health organizations worldwide, the eradication of tuberculosis (TB) has never been achieved, nor is it imminent. Nearly half of the world's population is infected with M. tuberculosis, with approximately 8 million new cases and there million deaths attributable to TB yearly.
  • TB is on the rise, even in the United States where up to 10 million individuals are believed to be infected. Almost 28,000 new cases were reported in 1990, a 9.4 percent increase over 1989. A sixteen percent increase was observed from 1985 to 1990.
  • TB is acquired by the respiratory route; actively infected individuals spread this infection efficiently by coughing or sneezing "droplet nuclei" which contain viable bacilli. Overcrowded living conditions and shared air spaces are especially conducive to the spread of TB, underlying the increase in instances that have been observed in the U.S. in prison inmates and among the homeless in larger cities.
  • mycobacteria other than M. tuberculosis are also becoming increasingly problematic as elements in the list of opportunistic infections that plague the AIDS patient.
  • MAC Avium-intracellulare complex
  • Mycobacteria including Mycobacterium avium, are intracellular parasites that are capable of growth within cells in the host such as macrophages.
  • the mycobacteria grow slowly, produce no endotoxin and are not motile. They multiply within the macrophages, kill the macrophage and are taken up by new macrophages to start the process over.
  • Host resistance depends upon activation of the macrophages. Activated macrophages are able to kill the bacteria that reside within the cell. This activation depends upon specific T-cells which are produced as the result of a cell mediated immune reaction against proteins of the mycobacteria.
  • Mycobacterial infections have been likened to a war of attrition in which there is a delicate balance between the ability of the mycobacteria to survive within the macrophages and the ability of the host to activate macrophages sufficiently to kill them. In the absence of rapidly acting antiinfective compounds, the goal of therapy is to tip the balance in favor of the host. There is still no clear understanding of the factors which contribute to the virulence of mycobacteria. Many investigators have implicated lipids of the cell wall and bacterial surface as contributors to colony morphology and virulence. Evidence suggests that C-mycosides, on the surface of certain mycobacterial cells, are important in facilitating survival of the organism within macrophages. Trehalose 6,6' dimycolate, a cord factor, has been implicated for other mycobacteria.
  • Mycobacterium avium bacilli occur in several distinct colony forms. Bacilli which grow as transparent or rough colonies on conventional laboratory media are able to multiply within macrophages in tissue culture, are virulent when injected into susceptible mice, and are resistant to antibiotics. Rough or transparent bacilli which are maintained on laboratory culture media often spontaneously assume an opaque colony morphology at which time they fail to grow in macrophages, are avirulent in mice, and are highly susceptible to antibiotics. The differences in colony morphology between the transparent, rough and opaque strains of Mycobacterium avium are almost certainly due to the presence of a glycolipid coating on the surface of transparent and rough organisms which acts as a protective capsule.
  • This capsule, or coating is composed primarily of C-mycosides which apparently shield the virulent Mycobacterium avium organisms from lysosomal enzymes and antibiotics.
  • the non-virulent opaque forms of Mycobacterium avium have very little C-mycoside on their surface.
  • Both resistance to antibiotics and resistance to killing by macrophages have been attributed to the glycolipid barrier on the surface of Mycobacterium avium.
  • Fatty acids have been shown to exert either an inhibitory or enhancing effect on mycobacterial growth depending on the length of the carbon chain, the degree of saturation, and the presence and type of hydrophilic groups. The mechanism of the growth inhibition effect of fatty acids is not understood, but they are known to be surface-active agents which can interact with glycolipids on the surface of the mycobacteria.
  • Triton WR1339 and Triton A20 are mixtures of numerous similar compounds and are generally toxic. Cornforth and his colleagues synthesized a number of similar materials having greater purity, and found that the antituberculosis effect increased with the molecular weight of the hydrophobic portion of the molecules. The most effective pure preparation had four alkylphenol groups attached in a ring configuration. The size of the hydrophilic moiety was also important. Preparations with an average of 15 to 20 polyoxyethylene (POE) moieties per phenolic group were most effective in treating tuberculosis. However, increasing the number of POE moieties to 60 produced a compound which caused infection to progress more rapidly than untreated controls.
  • POE polyoxyethylene
  • tuberculosis and antituberculosis effects were found to impede the growth of virulent tuberculosis bacteria in intact animals and in macrophages in tissue culture. However, they had no effect on the growth of the organisms in bacterial culture. Consequently, the agents affected either the host or the host-parasite interactions, but had no direct effect on mycobacteria. The bulk of evidence suggested that the tuberculosis and antituberculosis effects were due to modification of surface lipids of the mycobacteria.
  • the purified Cornforth compounds were not developed commercially as antimycobacterial agents. The reasons for this decision are not known, but several factors may have contributed. First, none of the preparations were pure. Second, the compounds suppressed the growth of mycobacteria in animals but did not produce cures. Finally, the compounds were found to be significantly toxic, producing disorders such as necrosis of the liver.
  • nonionic surfactants in general, are much less toxic than either anionic or cationic surfactants.
  • the nonionic block copolymers are among the least toxic of known surfactants.
  • Nonionic block copolymer surfactants can be synthesized in forms which span virtually the entire range of physical chemical activities of known nonionic surface active agents. Wetting agents with properties reminiscent of Cornforth's antituberculosis agents can be produced in many molecular configurations. They can be produced in a higher molecular weight and in a purer form than is practically feasible with most other surfactants. Problems with toxicity of inactive contaminants can be minimized.
  • the known effects of the block copolymers on serum lipids suggest that the block copolymers have biologic activities similar to those of the agents studied by Cornforth with less toxic effects.
  • AIDS Acquired Immune Deficiency Syndrome, or AIDS, is a disease thought to be caused by a human retrovirus, the Human T Lymphotropic Virus El (HTLV- ⁇ l) which is also called human immunodeficiency virus or HIV.
  • HIV has ribonucleic acid, or RNA, as its genetic material.
  • RNA ribonucleic acid
  • reverse transcriptase exploits the viral RNA as a template to assemble a corresponding molecule of DNA. The DNA travels through the cell nucleus and inserts itself among the host chromosomes, where it provides the basis for viral replication.
  • the host cell is often a T4 lymphocyte, a white blood cell that has a central and regulatory role in the immune system.
  • T4 lymphocyte a white blood cell that has a central and regulatory role in the immune system.
  • the virus may remain latent until the lymphocyte is immunologically stimulated by a secondary infection. Then the virus reproducing itself rapidly killing or rendering ineffective the host cell.
  • the resulting depletion of the T4 cells, and loss of activity leaves the patient vulnerable to "opportunistic" infections by an agent that would not normally harm a healthy person.
  • the virus damages the host by many other mechanisms as well.
  • the chain is terminated.
  • the truncated DNA cannot integrate itself into the host chromosomes or provide the basis for viral replication, and so the spread of the infection is halted.
  • One of the compounds that is thought to act by mimicking a nucleotide is azidothymid ⁇ ne, or AZT.
  • AZT is known to have serious side effects and its efficacy in mitigating the AIDS disease has been questioned.
  • the macrophage is now known to be an additional reservoir of the AIDS virus in the body. Consequently, there is an immediate need for a compound that will suppress or halt the replication and infection of cells by the viruses such as the HTV virus.
  • a composition and method is provided that is effective in treating infections caused by microorganisms such as bacteria, fungi, viruses, and protozoa.
  • the present invention is effective in inhibiting the growth of microorganisms such as
  • the antiinfective composition of the present invention comprises a surface active copolymer.
  • the surface active copolymer can be an ethylene oxide-propylene oxide condensation product with the following general formula:
  • a is an integer such that the hydrophobe represented by (C3H6O) has a molecular weight of approximately 1,200 to approximately 15,000, preferably between approximately 1200 and approximately 5300, more preferably between approximately 1750 and approximately 4500, still more preferably approximately 2250 to approximately 4000, and b is an integer such that the hydrophile portion represented by (C2H4O) constitutes approximately 1% to approximately 50% by weight of the compound, preferably approximately 10% to approximately 50% by weight of the compound, more preferably approximately 5% to approximately 30%, and still more preferably approximately 5% to approximately 20%.
  • the present invention also comprises an antiinfective composition effective against infectious diseases comprising an injectable, topical, transdermal, oral, mucosal or inhalation dosage form of an effective amount of a drug, such as an antibiotic or other therapeutic agent, and an effective amount of a nonionic block copolymer having the following general formula:
  • a is an integer such that the hydrophobe represented by (C3H6O) has a molecular weight of approximately 1,200 to approximately 15,000, preferably between approximately 1200 and approximately 5300, more preferably between approximately 1750 and approximately 4500, still more preferably approximately 2250 to approximately 4000, and b is an integer such that the hydrophile portion represented by (C2H4O) constitutes approximately 1% to approximately 50% by weight of the compound, preferably approximately 10% to approximately 50% by weight of the compound, more preferably approximately 5% to approximately 30%, and still more preferably approximately 5% to approximately 20%.
  • composition of the present invention can be administered by a number of routes including, but not limited to injection, topical, transdermal, inhalation, trans-mucosal, oral ingestion, and a combination of a plurality of modes of administration. Additionally, the therapeutic drug may be administered separately from the block copolymers of the present invention, by the same or different route of administration, either simultaneously or at different times.
  • the present invention provides a composition that can be administered to patients who are infected with Mycobacterium species.
  • the surface active copolymer is effective in inhibiting the growth of Mycobacterium species and also causes the bacterium to be more susceptible to conventional antibiotics.
  • Yet another object of the present invention is to provide a method of treating viral infections in humans or animals.
  • Another object of the present invention is a compound and method that is effective in inhibiting the replication of viruses in both animals and humans.
  • Another object of the present invention is to provide a surfactant compound that can be used to treat mycobacterial infections in persons with AIDS.
  • Another object of the present invention is to provide a compound effective against protozoa and protozoal infections.
  • Another object of the present invention is to provide a compound and method that is effective in inhibiting fungal infections.
  • Another object of the present invention is to provide an antibiotic surfactant compound that is non-toxic for humans.
  • Yet another object of the present invention is to provide a surfactant compound that causes the Mycobacterium species to be more susceptible to conventional antibiotics.
  • Another object of the present invention is to provide a composition that is effective against protozoa such as toxoplasma.
  • Fig. 1 is a grid illustrating block copolymers by molecular weight of hydrophobe and percent hydrophile.
  • Fig. 2 is a grid illustrating preferred antiinfective block copolymers by molecular weight of hydrophobe and percent hydrophile.
  • Fig. 3 is a grid illustrating other preferred antiinfective block copolymers by molecular weight of hydrophobe and percent hydrophile.
  • Fig. 4 is a grid illustrating yet other preferred antiinfective block copolymers by molecular weight of hydrophobe and percent hydrophile.
  • Fig. 5 is a grid illustrating still other preferred antiinfective block copolymers by molecular weight of hydrophobe and percent hydrophile.
  • Fig. 6 is a graphical representation of the effect of CRL-8131 on the growth of Mycobacterium avium.
  • Fig. 7 is a graphical representation of the effect of CRL-9038, CRL-8131, CRL-8133 and CRL-8135 on the growth of Mycobacterium avium.
  • Fig. 8 is a graphical representation of the effect of CRL-8131, CRL-8133, and CRL-8135 on the growth of
  • Fig. 9 is a graphical representation of the effect of CRL-9038 on the growth of Mycobacterium avium.
  • Fig. 10 is a graphical representation of the effect of 11 nonionic copolymers on the growth of Mycobacterium avium.
  • Fig. 11 is a graphical representation of the effect of several non-ionic copolymer on Mycobacterium avium growth.
  • Fig. 12 is a graphical representation of the effect of the CRL-8133 copolymer on HTV infection in H9 cells.
  • Fig. 13 is a graphical representation of the effect of CRL-8143 copolymer on the viability of intracellular Mycobacterium avium.
  • Fig. 14 is a graphical representation of the activity of CRL-8131 plus pyrimethamine in mice infected intraperitoneally with tachyzoites of strain RH of T. gondii.
  • Fig. 15 is a graphical representation of the activity of CRL-8131 plus sulfadiazine in mice infected intraperitoneally with tachyzoites of strain RH of T. gondii.
  • Fig. 16 is a graphical representation of the activity of CRL-8131 plus clindamycin in mice infected intraperitoneally with tachyzoites of strain RH of T. gondii.
  • Fig. 17 is a graphical representation of the activity of CRL-8131 plus pyrimethamine in mice infected orally with cysts of the C56 strain of T. gondii.
  • Fig. 18 is a graphical representation of the activity of CRL-8131 plus sulfadiazine in mice infected orally with cysts of the C56 strain of T. gondii.
  • Fig. 19 is a graphical representation of the activity of CRL-8142 plus pyrimethamine in mice infected intraperitoneally with tachyzoites of strain RH of T. gondii.
  • Fig. 20 is a graphical representation of the activity of CRL-8142 plus sulfadiazine in mice infected intraperitoneally with tachyzoites of strain RH of T. gondii.
  • Fig. 21 is a graphical representation of the activity of CRL-8142 plus clindamycin in mice infected intraperitoneally with tachyzoites of strain RH of T. gondii.
  • Fig. 22 is a graphical representation of the activity of CRL-8142 plus pyrimethamine in mice infected orally with cysts of the C56 strain of T. gondii.
  • Fig. 23 is a graphical representation of the activity of CRL-8142 plus sulfadiazine in mice infected orally with cysts of the C56 strain of T. gondii.
  • Fig. 24 is a graphic illustration of the synergistic effect of poloxamer formulation CRL-8131 in combination with rifampin.
  • the present invention comprises therapeutic compositions and methods which kill or inhibit the growth of microorganisms.
  • An example of the bacteria that the present invention is effective against is mycobacteria species, such as Mycobacterium tuberculosis, Mycobacterium avium, and
  • Mycobacterium leprae Other microorganisms that the invention is effective against include, but are not limited to, Chlamydia trachomatis, Chlamydia pneumoniae, Chlamydia psittaei Listeria monocytogenes, Candida albicans, Cryptococcus neoformans, Toxoplasma gondii, Pneumocystis carinii, Herpes simplex virus type 1, Cytomegalovirus, influenza virus type A and B, and respiratory syncytial virus
  • the present invention also includes therapeutic compositions and methods for treating DNA viruses and RNA viruses, and infections and infectious diseases caused by such viruses in a human or animal, including infections caused by HTV or herpes (such as HSV-1) or antigenically-related strains thereof.
  • Antigenically-related strains are strains that cross react with antibodies specific for HTV.
  • One skilled in the art can readily determine viral strains that are antigenically- related to HIV by conducting standard immunoassay tests using anti-HIV antibodies and the viral strain to be analyzed, and looking for positive cross-reactivity.
  • the surface active copolymers disclosed herein are effective in inhibiting or suppressing the replication of such viruses in cells.
  • the present invention also includes a therapeutic composition useful for delivering antimicrobial drugs and treating disease states comprising an admixture of a nonionic block copolymer and an antibiotic or therapeutic drug.
  • Drugs that can be used with the nonionic copolymers of the present invention include, but are not limited to, rifampin, isoniazid, ethambutol, gentamicin, tetracycline, erythromycin, pyrazinamide, streptomycin, clofazimine, rifabutin, fluoroquinolones such as ofloxacin and sparfloxacin, azithromycin, clarithromycin, dapsone, doxycyline, ciprofloxacin, ampicillin, amphotericin B, fluconazole, ketoconazole, fluconazole, pyrimethamine, sulfadiazine, clindamycin, azithromycin, paromycin, diclazaril, clarithromycin, atovaquone,
  • Mycobacterium tuberculosis Isoniazid, rifampin, ethambutol, pyrazinamide, streptomycin, clofazimine, rifabutin, fluoroquinolones such as ofloxacin and sparfloxacin
  • Cryptococcus neofo ⁇ nans Amphotericin B, ketoconazole, fluconazole
  • HIV AZT HIV AZT, DDI, DDC, foscamat, viral protease inhibitors, peptides, antisense oligonucleotides, triplex and other nucleic acid sequences
  • surfactants and low molecular weight alcohols are added to the therapeutic admixture of antimicrobial drug and nonionic block copolymer.
  • surfactants useful in the present invention include Tween 80 and emulsions with fatty acids such as phospholipids, cholate and amino acids.
  • the preferred surfactant is Tween 80.
  • Surfactants are added to the admixture at a concentration ranging from approximately 0.1% to approximately 5% v/v.
  • the preferred surfactant concentration is approximately 2%.
  • the term “approximately” as it applies to concentrations expressed herein means the stated concentration plus or minus ten percent.
  • the term "low molecular weight alcohols” means alcohols having two to eight carbons.
  • An example of a low molecular weight alcohols useful in the present invention is ethanol, which is the preferred low molecular weight alcohol.
  • Low molecular weight alcohols are added to the admixture at a concentration ranging from approximately 0.5% to approximately 5% v/v.
  • the preferred low molecular weight alcohol concentration is between approximately 1% and approximately 3%.
  • the present invention comprises a surface active copolymer that is preferably an ethylene oxide-propylene oxide condensation product with the following general formula:
  • a is an integer such that the hydrophobe represented by (C3H6O) has a molecular weight of between approximately 1,200 and approximately 15,000
  • b is an integer such that the hydrophile portion represented by (C2H 4 O) constitutes approximately 1% to approximately 50% by weight of the compound.
  • Preferred embodiments of the copolymers of the present invention have the following general characteristics.
  • the antiinfective composition of the present invention comprises a surface active copolymer.
  • the surface active copolymer can be an ethylene oxide-propylene oxide condensation product with the following general formula:
  • a is an integer such that the hydrophobe represented by (C3H6O) has a molecular weight of approximately 1,200 to approximately 15,000, preferably between approximately 1200 and approximately 5300, more preferably between approximately 1750 and approximately 4500, still more preferably approximately 2250 to approximately 4000, and b is an integer such that the hydrophile portion represented by (C2H4O) constitutes approximately 1% to approximately 50% by weight of the compound, preferably approximately 10% to approximately 50% by weight of the compound, more preferably approximately 5% to approximately 30%, and still more preferably approximately 5% to approximately 20%.
  • the present invention also comprises an antiinfective composition effective against infectious diseases comprising an injectable, topical, transdermal, trans- mucosal, oral, mucosal or inhalation dosage form of an effective amount of a drug, such as an antibiotic or other therapeutic antimicrobial agent, admixed with an effective amount of a nonionic block copolymer having the following general formula:
  • a is an integer such that the hydrophobe represented by (C3H6O) has a molecular weight of approximately 1,200 to approximately 15,000, preferably between approximately 1200 and approximately 5300, more preferably between approximately 1750 and approximately 4500, still more preferably approximately 2250 to approximately 4000, and b is an integer such that the hydrophile portion represented by (C2H4O) constitutes approximately 1% to approximately 50% by weight of the compound, preferably approximately 10% to approximately 50% by weight of the compound, more preferably approximately 5% to approximately 30%, and still more preferably approximately 5% to approximately 20%.
  • An effective amount is an amount sufficient to treat an infected human or animal by reducing the number of infectious microbes, and/or inhibiting the ability of the infectious microbes to produce toxins and/or reproduce and multiply.
  • compositions of the present invention include, but are not limited to aqueous solutions, suspensions or emulsions, such as oil-in-water emulsions.
  • the polymer blocks are formed by condensation of ethylene oxide and propylene oxide, at elevated temperature and pressure, in the presence of a catalyst. There is some statistical variation in the number of monomer units which combine to form a polymer chain in each copolymer.
  • a preferred ethylene oxide-propylene oxide copolymer for use in the antiinfective composition of the present invention is a copolymer having the following formula:
  • a is an integer such that the hydrophobe represented by (C3H6O) has a molecular weight of about 1,750 to 4,500 and b is an integer such that the hydrophile portion represented by (C2H4O) constitutes approximately 10% to 40% by weight of the compound.
  • An especially preferred embodiment of the antiinfective compound of the present invention is the compound designated CRL-8133 with , the following general formula:
  • POP polyoxypropylene
  • POE polyoxyethylene
  • the antiinfective compound of the present invention is effective in suppressing the growth of Mycobacterium avium, Mycobacterium tuberculosis, Herpes simplex virus type 1 and type 2, Toxoplasmi gondii and other microorganisms affecting humans and animals causing a variety of health disorders.
  • the disorders in which the antiinfective compound of the present invention are effective include, but are not limited to bacterial infections, fungal infections, protozoal infection and viral infections. Examples of specific diseases include tuberculosis, toxoplasmosis, and AIDS. Other diseases caused by microorganisms will be obvious to one of skill in the art.
  • the antiinfective compositions of the present invention have been shown to be effective with only one administration to the patient.
  • the mode of administration can be topical, transdermal, trans-mucosal, oral, inhalation, subcutaneous, intramuscular or intravenous.
  • the preferred mode of injection is intravenous.
  • the optimum amount of the antiinfective compound in an injection varies with the weight of the patient being treated, but appears to be in the range of 1 x 10-3 M to 1 x 10 _ 4 M.
  • the effective dose range generally includes dosages of 0.1 mg/Kg/day to 50 mg/Kg/day.
  • a preferred dosage range is 0.5 mg/Kg/day to 25 mg/Kg/day.
  • a more preferred dosage range is 1 mg/Kg/day to 10 mg/Kg/day. It has been surprisingly found that effective therapy is provided even when the block copolymer and the therapeutic drug are administered separately, either by the same or different routes of administration, and either simultaneously or at different times.
  • Non-ionic block copolymers form micelles above their critical micelle concentration.
  • the non-ionic copolymers have negative thermal coefficients of solubility. In the cold, the kinetic energy of water molecules is reduced and they form weak hydrogen bonds with the oxygen of the POP blocks. This hydration of the hydrophobe promotes solubility at low temperatures. As the temperature rises, the "cloud point" is reached; the increased kinetic energy of the water breaks the hydrogen bonds, the polymer becomes insoluble and micelles form.
  • the copolymers can form physical structures that can be combined or loaded with an additional, distinct therapeutic agent. Consequently, the nonionic block copolymers of the present invention can be used as therapeutic drug delivery vehicles. Admixtures of therapeutic drugs with non-ionic block copolymers have the advantage of synergistic activity of two therapeutic agents. Further, copolymers having specific characteristics can be selected for use with particular therapeutic drugs. For example, CRL-8131, which is hydrophobic, is an excellent carrier for hydrophobic antibiotics such as rifampin. However, other agents which are not distinctly hydrophobic can be used according to the present invention.
  • a therapeutic delivery composition is prepared using any of the antiinfective block copolymers of the present invention in combination with any of a variety of antimicrobial agents.
  • CRL-8131 is used in a concentration of approximately 3% to approximately 5% to construct a therapeutic delivery vehicle.
  • Therapeutic delivery vehicles made using copolymers that are more hydrophilic than CRL-8131 normally require a higher concentration (approximately 5% to approximately 10%) of the copolymer.
  • copolymer-based micelles as a therapeutic drug delivery vehicles is particularly desirable because the micelles are accumulated readily and are present for an extended period of time, in macrophages, the site of HTV and other viral infections and a major target for viral therapy.
  • therapeutic copolymer-based therapeutic compositions include CRL-8131 combined with 2% Tween 80 and 1% ethanol, and CRL-8142 combined with 1% Tween 80 and 5% ethanol.
  • nonionic block copolymers and therapeutic drugs may be administered to a human or animal separately, either simultaneously or at different times.
  • copolymers such as CRL-8131 or CRL-8142 are administered by injection, followed by administration of the therapeutic drug.
  • Administration of the drug may be by any normal route such as, injection, topical or transdermal application, trans-mucosal absorption, inhalation or oral ingestion.
  • the following specific examples illustrate various aspects of the invention, such as in vitro suppression of the growth of colonies of Mycobacterium avium and HTV virus in vitro isolated from humans.
  • Several of the examples also illustrate the invention as it applies to the suppression of growth of Mycobacterium avium and Toxoplasma gondii in macrophages.
  • compositions and methods of the invention useful for gene therapy and compositions and methods of the invention useful for gene- mediated immunization
  • compositions and methods of the invention useful for gene- mediated immunization It will be appreciated that other embodiments and uses will be apparent to those skilled in the art and that the invention is not limited to these specific illustrative examples.
  • the mycobacteria grow in the medium containing l ⁇ C-labeled fatty acid, they utilize the fatty acid and CO2 is produced.
  • the production of CO2 can be detected quantitatively, reflecting the rate and amount of growth occurring in the vial, and is expressed in terms of the "growth index". If an anti ⁇ tuberculosis drug is added to the medium, suppression of growth occurs in the case of susceptible organisms which can be detected by either a decline or a very small increase of the growth index as compared to the control. However, if the organisms are resistant, no suppression occurs in the rate of increase of the growth index on daily testing.
  • the inoculum in the control vial is one hundred fold less than the inoculum used for drug containing vials.
  • Growth index readings are taken each day after inoculation and the increase in growth index over that of the previo ⁇ s day, is compared for the control vial and the vials containing drugs. If the daily increase in growth index, called delta growth index, in the drug vial is equal to or greater than that in the control vial, the test organisms are considered resistant to the drug. For a susceptible organism, the daily increase in the growth index for the control would be much higher than for the drug vial.
  • each of the candidate anti-mycobacterial copolymers ranges in size from 3,600 to 14,000 daltons and the molecular weight attributable to the polyoxypropylene portion of each molecule is approximately 3,250.
  • the hydrophilic portion of each molecule is 10% for CRL-8131, 30% for CRL-8133, 50% for CRL-8135, and 80% for CRL-9038.
  • Each of the candidate anti-mycobacterial copolymers were mixed with the Middlebrook tuberculosis medium at concentration of 1 x 10"3 M and 1 x 10-4 M.
  • each copolymer has a polyoxyethylene portion ranging from 5% to 50% of the total molecule.
  • the molecular weight of the polyoxypropylene portion of each molecule ranges from approximately 1,200 to approximately 4,000.
  • the physical characteristics of each copolymer are summarized in Table H
  • FIG. 11 A correlation between increasing hydrophobicity of the copolymer arid inhibition of M. avium growth is shown in Fig. 11.
  • the copolymers having a polyoxypropylene molecular weight of 1750 were tested for inhibition of M. avium.
  • the copolymers tested are CRL-85171, CRL-85172,
  • inhibition of mycobacterial growth specifically correlates with the molecular structure of the copolymers.
  • the low molecular weight copolymers which have only a small percentage of hydrophil appear to inhibit M . avium growth more than copolymers which are more hydrophilic.
  • the effectiveness of P103 copolymer is examined in preventing HIV replication in H9 cells using a high multiplicity of viral infection.
  • the infections are conducted in the following manner: 10-5 H9 cells in 1 ml medium containing 1.89 x 10" 2 picogram T24 viral antigen per cell, and either 0,
  • CRL-8133 10, 25, or 50 ⁇ g of CRL-8133, are incubated at 37°C in 5% CO 2 for one hour. After this time, the cells are pelleted at 3,000 rpm and washed to remove access non-adsorbed virus. The cells are plated in medium in dishes containing wells of 1 cm diameter. The cultures and the controls, all done in triplicate, are incubated at 37°C, 5% C02, and the volume in each well is maintained at a constant volume of 1 ml. The media contains either 10, 25, or 50 ⁇ g per ml of CRL-8133. Virus yields in terms of total picogram of T24 HIV antigen and cell counts are determined on day seven of the incubation period. Cell viability is determined by trypan blue exclusion.
  • the CRL-8133 copolymer is considered an effective inhibitor of HIV application in H9 cells.
  • PBMC peripheral blood mononuclear cells
  • HTV-1 seronegative and hepatitis B virus sero- negative donors were isolated by Ficoll-Hypaque discontinuous gradient centrifugation at 1,000 x g for 30 minutes, washed twice in phosphate-buffered saline (pH 7.2; PBS), and pelleted at 300 x g for 10 minutes.
  • PHA phytohemagglutinin
  • HIV-1 (strain LAV-1) was obtained from Dr. P. Feorino (Centers for Disease Control, Atlanta, GA). The virus was propagated in human PBMC using RPMI 1640 medium, as described previously (McDougal, J. S., S. P. Cort, M. S. Kennedy, C. D. Cabridilla, P. M. Feorino, D. P. Francis, D. Hicks, V. S. Kalyanaramen, and L. S. Martin. 1985. Immunoassay for the detection and quantitation of infectious human retrovirus, lymphadenopathy-associated virus (LAV). J. Immun. Meth.
  • Virus obtained from cell-free culture supernatant was titrated and stored in aliquots at -70°C until use.
  • Virus particles were pelleted from 5 ml samples at 40,000 rpm for 30 minutes using a Beckman 70.1 Ti rotor and suspended in 200 ⁇ l of virus disrupting buffer (50 mM Tris-HCl, pH 7.8, 800 mM NaCl, 20% glycerol, 0.5 mM phenylmethyl sulfonyl fluoride, and 0.5% Triton X-100).
  • virus disrupting buffer 50 mM Tris-HCl, pH 7.8, 800 mM NaCl, 20% glycerol, 0.5 mM phenylmethyl sulfonyl fluoride, and 0.5% Triton X-100.
  • the RT assay was performed in 96-well microtiter plates, as described by Spira et al. (Spira, T. J., L. H. Bozeman, R. C. Holman, D. T. Warfield, S. K. Phillips, and P. M. Feorino. 1987. Micromethod for assaying the reverse transcriptase of LAV-HTLV-III ⁇ ymphadenopathy- associated virus. J. Clin. Microbiol. 25:97-99).
  • the reaction mixture which contained 50 mM Tris-HCl pH 7.8, 9 mM MgC_2, 5 mM dithiothreitol, 4.7 ⁇ g/ml (rA)n-(dT)i2-l8> 140 ⁇ M dATP, and 0.22 ⁇ M [3H]TTP (specific activity 78.0 Ci/mmol, equivalent to 17,300 cpm/pmol; NEN Research Products, Boston, MA.), was added to* each well. The sample (20 ⁇ l) was added to the reaction mixture which was then incubated at 37°C for 2 hours. The reaction was terminated by the addition of 100 ⁇ l 10% trichloroacetic acid (TCA) containing 0.45 mM sodium pyrophosphate.
  • TCA trichloroacetic acid
  • the acid-insoluble nucleic acids which precipitated were collected on glass filters using a Skatron semi-automatic harvester (setting 9).
  • the filters were washed with 5% TCA and 70% ethanol, dried, and placed in scintillation vials.
  • Four ml of scintillation fluid (Econofluor, NEN Research Products, Boston, MA.) were added and the amount of radioactivity in each sample was determined using a Packard Tri-Carb liquid scintillation analyzer (Model 2,000CA). The results were expressed in dpm/ml of original clarified supernatant.
  • the procedures for the anti-HIV-1 assays in PBMC described above have been published recently (see Schinazi, R.F. et al in Antimicrob. Agents Chemother. 32:1784-1789, December 1988).
  • Vero cells in growth medium 2.5 ml were added to 25 c ⁇ _2 flasks (Falcon) in duplicate at a concentration equivalent to one tenth of cell confluency for each compound under test. After incubation at 25 c ⁇ _2 flasks (Falcon) in duplicate at a concentration equivalent to one tenth of cell confluency for each compound under test. After incubation at 25 c ⁇ _2 flasks (Falcon) in duplicate at a concentration equivalent to one tenth of cell confluency for each compound under test. After incubation at
  • the test compound (2 x final concentration), dissolved in 2.5 ml of the growth medium was added, and two flasks were harvested immediately by decanting the medium, washing once with 3 ml of PBS, and then incubating at 37°C for 5 minutes with 3 ml of trypsin/EDTA (0.125%/0.02%).
  • the cells dislodged from the flask by the latter procedure are generally in clumps and are dispersed by repeated forceful pipetting of the suspension against the surface of the flask.
  • Nahmias, A.J. Effect of combination of acyclovir, and vidarabine or its 5'-monophosphate on herpes simplex viruses in cell culture and in mice. Antimicrob. Agents Chemother. 22:499-507, 1982).
  • the drugs were evaluated for their potential toxic effects on uninfected PHA-stimulated human PBM cells and also in CEM cells.
  • the cells were cultured with and without drug for 6 days at which time aliquots were counted for cell viability as described above. Results are shown in Table HI.
  • PBMC peripheral blood mononuclear cells
  • Copolymers CRL-8131, CRL-8141, LI 03 and LI 23 were solubilized at 400 ⁇ g/ml in ice cold phosphate buffered saline. The cold solutions were filter sterilized on 0.22 ⁇ m filters and stored at 4°C. Each of the four compounds became soluble under these conditions.
  • Macrophage monolayers were pretreated with the indicated copolymers for 18 hours.
  • the macrophages were then challenged with Toxoplasma gondii at a concentration of two T. gondii organisms per macrophage. After one hour, nonphagocytized organisms were removed by washing and the medium plus the copolymer was replenished. Monolayers were fixed and enumerated at 24 hours after challenge.
  • interferon-g, IFN-g (murine recombinant at 200 U/ml) was added to macrophage monolayers 18 hours before challenge with Toxoplasma. These macrophages are activated by the IFN-g and readily kill Toxoplasma.
  • the experimental protocol is as follows: Cultured human macrophages were infected with M. avium and incubated for 7 days with and without the indicated compound. Samples of the macrophages cultures were taken at 0, 4, and 7 days after infection. The macrophages of the samples were lysed, the lysates diluted, and diluted samples of the lysates cultured on 7H10 agar plates to count viable bacteria (CFU).
  • CFU viable bacteria
  • Serovar 4 strain 7497 was purposely used in mixture of phenotypes smooth-transparent (ST) and round-domed (RD).
  • ST is the virulent phenotype and multiplies progressively in macrophages; RD usually do not multiply and may be killed by the macrophages and thus are the avirulent phenotype.
  • Serovar 8 strain T-138 was a highly virulent, pure, ST phenotype.
  • Example IX Experiments were conducted to examine the combination of CRL-8131 and CRL-8142 with pyrimethamine, sulfadiazine or clindamycin for in vivo activity against Toxoplasma gondii.
  • CRL-8131 was combined with two percent Tween 80 and 1 percent ethanol
  • CRL- 8142 was combined with one percent Tween 80 and 5 percent ethanol.
  • IP intraperitoneal
  • mice were Swiss-Webster females weighing 20 grams at the beginning of the experiment. Infection was IP with lO 3 tachyzoites. Treatment with CRL-8131 or CRL- 8142 alone was administered intraperitoneally. Doses of 25 (CRL-8131) or 25 or 50 (CRL-8142) mg/kg/day were used.
  • Treatment was initiated 24 hours after infection and continued for 10 days. Mice dying during treatment and after its discontinuation were examined for presence of T. gondii tachyzoites in intraperitoneal fluid.
  • mice were as above and an infection was with 10 cysts of the C56 strain of T. gondii administered orally by gavage. Treatment with the copolymers alone was administered IP at the doses described above. When combinations were used the copolymers was administered IP and the other drugs, at the concentrations described above, orally by gavage or in the drinking water. Treatment was initiated three days after infection and continued for ten days. Mice were examined for presence of T. gondii as above.
  • Example X Growth studies as described above in Example 1 were conducted to study the effect of rifampin alone, CRL- 8131 alone, and a combination of rifampin and CRL-8131.
  • Rifampin at a concentration of 0.04 ug/ml was effective in inhibiting growth of M. avium as was CRL-8131 at a concentration of 0.01 mg/ml.
  • the combination of rifampin and CRL-8131 at these concentrations however, unexpectedly and dramatically inhibited growth, and provided almost 100% protection. Results are shown in Figure 24.
  • a therapeutic delivery vehicle is prepared by combining any of the antiinfective copolymers, such as CRL- 8131 with any of a variety of antimicrobial agents, such as streptomycin.
  • CRL-8131 a concentration of three to five percent weight per volume is desirable to construct the therapeutic vehicle.
  • hydrophilic copolymer a five to ten percent weight per volume. 300 milligrams of CRL-8131 was added to 10 ml of 0.9% NaCl and the mixture is solubilized by storage at temperatures of 2-4°C until a clear solution is formed. 3.0 grams of streptomycin is added to the clear poloxamer solution and mixed thoroughly until the streptomycin is in solution. The final concentration of streptomycin and copolymer in the mixture is 30% weight per volume and 3% weight per volume, respectively.
  • Micelles associating the copolymer and streptomycin are formed by raising the temperature above 5°C and allowing the suspension of micelles to equilibrate. The equilibrated suspension is suitable for administration.

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Abstract

Cette invention a pour objet une composition et un procédé qui sont efficaces pour traiter les infections provoquées par des microorganismes comprenant les bactéries, les virus et les champignons mais sans pour autant s'y limiter. Cette invention est efficace pour inhiber la croissance de bactéries telles que les Mycobacterium species comprenant, mais sans s'y limiter, le complexe Mycobacterium avium-intracellulare et le M. tuberculosis. Cette invention concerne un copolymère tensioactif, de préférence un produit de condensation oxyde d'éthylène-oxyde de propylène qui correspond à la formule générale suivante: HO(C2H4O)b(C3H6O)a(C2H4O)bH dans laquelle a représente un entier tel que la partie hydrophobe représentée par (C3H6O) présente une masse molaire variant entre approximativement 1200 et 15000, et b représente un entier tel que la partie hydrophile représentée par (C2H4O) constitue approximativement entre 1 % et 50 % en poids du composé.
PCT/US1994/013834 1993-12-02 1994-12-02 Compositions anti-infectieuses et procedes d'utilisation WO1995015182A1 (fr)

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Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1996004924A1 (fr) * 1994-08-09 1996-02-22 Cytrx Corporation Copolymeres ameliores de polyoxypropylene/polyoxyethylene contre les infections et procedes d'utilisation
WO1996013254A1 (fr) * 1994-10-26 1996-05-09 Glaxo Wellcome House Composition pharmaceutique contenant l'atovaquone
WO2002003998A2 (fr) * 2000-07-10 2002-01-17 Chiron Corporation Formulations a base de macrolides destines a l'inhalation et procede de traitement des infections endobronchiales
EP1181937A2 (fr) * 1994-08-09 2002-02-27 Cytrx Corporation Vaccin contenant des acides nucléiquées et adjuvant de vaccin
WO2005056741A1 (fr) 2003-12-13 2005-06-23 Henkel Kommanditgesellschaft Auf Aktien Inhibition de l'adherence de micro-organismes au moyen de tensioactifs non-ioniques
WO2009002652A2 (fr) * 2007-05-29 2008-12-31 Board Of Regents Of The University Of Texas System Compositions et procédés pour le traitement d'infections mycobactériennes

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0003999A2 (fr) * 1978-03-02 1979-09-19 Hoechst Aktiengesellschaft Compositions microbiocides contenant des sels d'alcoyl-diguanidines

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0003999A2 (fr) * 1978-03-02 1979-09-19 Hoechst Aktiengesellschaft Compositions microbiocides contenant des sels d'alcoyl-diguanidines

Cited By (10)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1996004924A1 (fr) * 1994-08-09 1996-02-22 Cytrx Corporation Copolymeres ameliores de polyoxypropylene/polyoxyethylene contre les infections et procedes d'utilisation
EP1181937A2 (fr) * 1994-08-09 2002-02-27 Cytrx Corporation Vaccin contenant des acides nucléiquées et adjuvant de vaccin
EP1181937A3 (fr) * 1994-08-09 2004-02-04 Cytrx Corporation Vaccin contenant des acides nucléiquées et adjuvant de vaccin
WO1996013254A1 (fr) * 1994-10-26 1996-05-09 Glaxo Wellcome House Composition pharmaceutique contenant l'atovaquone
WO2002003998A2 (fr) * 2000-07-10 2002-01-17 Chiron Corporation Formulations a base de macrolides destines a l'inhalation et procede de traitement des infections endobronchiales
WO2002003998A3 (fr) * 2000-07-10 2002-06-13 Chiron Corp Formulations a base de macrolides destines a l'inhalation et procede de traitement des infections endobronchiales
WO2005056741A1 (fr) 2003-12-13 2005-06-23 Henkel Kommanditgesellschaft Auf Aktien Inhibition de l'adherence de micro-organismes au moyen de tensioactifs non-ioniques
US7910647B2 (en) 2003-12-13 2011-03-22 Henkel Ag & Co. Kgaa Adhesion inhibition of microorganisms by non-ionic surfactants
WO2009002652A2 (fr) * 2007-05-29 2008-12-31 Board Of Regents Of The University Of Texas System Compositions et procédés pour le traitement d'infections mycobactériennes
WO2009002652A3 (fr) * 2007-05-29 2009-04-16 Univ Texas Compositions et procédés pour le traitement d'infections mycobactériennes

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