WO2001012831A2 - Anti-bacterial and anti-viral compositions and methods - Google Patents

Abstract

Provided are preparations obtained from a bacterial isolate from a bacterial-amoebae consortium that have anti-reverse transcriptase or anti-microbial activity. One of the preparations is present in the supernatant from a culture of the bacteria, while the other preparation comprises a soluble green pigment having blue pigment and yellow pigment components. The blue pigment component comprises an N-methyl quinoline compound. Uses of the preparations, for example in inhibiting reverse transcriptase activity or inhibiting the growth of certain microorganisms, as well as anti-reverse transcriptase and anti-bacterial kits, are also provided.

Description

ANTI-BACTERIAL AND ANTI-VIRAL COMPOSITIONS AND METHODS

BACKGROUND OF THE INVENTION

The present application is a continuation-in-part of co-pending U.S. patent application

Serial No. 08/929,648, filed on September 15, 1997, the entire text of which is specifically incorporated herein by reference without disclaimer. The invention was made with government support under contract DE-AC05-96OR22464 awarded by the United States Department of Energy to Lockheed Martin Energy Research Corporation, and the United States government has certain rights in the present invention.

1. Field of the Invention

The present invention relates generally to the field of microbiology. In particular, the present invention provides preparations that have anti-reverse transcriptase and/or anti- bacterial activities, which are obtained from a bacteria isolated from a bacterial-amoebae consortium. One of the preparations is present in the supernatant from a culture of the bacteria, while the other preparation comprises a soluble green pigment having blue pigment and yellow pigment components. The blue pigment component comprises an N-methyl quinoline compound. Uses of the preparations, for example in inhibiting reverse transcriptase activity and inhibiting the growth of certain microorganisms, as well as anti- reverse trancriptase and anti-bacterial kits, are also provided.

2. Description of Related Art

The emerging antibiotic resistance in many bacterial species including Staphylococcus aureus and various other microbial species is seen as a potential threat to mankind (Begley, 1994). It is clear that while a variety of approaches to the treatment of bacterial diseases have experienced some success, the growing problems of antibiotic resistance, variability of antigens between species and in the same species through mutation of antigens, and the inefficient immune systems in young children, the elderly and other immunocompromised patients, all present difficulties that need to be overcome. Thus, there exists today an immediate need for an effective treatment for streptococcal and staphylococcal pathogens that can be used for a variety of infections in both man and animals. Particularly worrisome is the multidrug resistance of Staphylococcus aureus. Today, almost half of the staphylococcal strains causing nosocomial infections are resistant to all antibiotics except vancomycin, and it appears to be only a question of time before vancomycin will become ineffective (Begley, 1994). It is in this scenario that one must consider new strategies in attempts to prevent and treat infection. In addition to these problems with bacterial infections, viral infections caused by

RNA viruses are also on the rise. A worldwide epidemic of acquired immune deficiency syndrome (AIDS), a disease caused by the retrovirus human immunodeficiency virus (HIV), is currently being experienced. As of December, 1994, more than one million cases of AIDS had been reported to the World Health Organization (WHO). The WHO has estimated that actual AIDS cases as of 1994 totaled more than 4.5 million worldwide. An estimate by the WHO predicts that there will be 10 million AIDS cases and 30-40 million people infected with HIV worldwide by the year 2000.

The human immunodeficiency virus, a lentivirus, is a member of the Retroviridae family of retroviruses. This family includes other members that are causal agents of devastating diseases of humans. For example, certain cancers are caused by oncornaviruses, oncogenic viruses that cause transformation of cells. Retroviruses are particularly refractory to treatment, largely because of their unique replication strategy.

Retroviruses are single-stranded RNA viruses. The virions of retroviruses include reverse transcriptase, an enzyme that catalyzes the synthesis of a single-stranded DNA molecule, using the single-stranded viral RNA as a template. Second strand synthesis, also catalyzed by reverse transcriptase, yields a double-stranded DNA provirus that integrates into the host's genome. In the case of lentiviruses such as HIV, the virus may lie dormant for a period of several years before becoming activated again. The human immunodeficiency virus infects CD4+ T lymphocytes, cells that play an important role in combating infections. Proliferation of HIV within the T cells results in destruction of the cells and interruption of the cells' normal function. As a consequence, people with AIDS are susceptible to opportunistic infections and the development of neoplasms.

Although millions of dollars have been directed toward the development of drugs for the treatment of persons infected with the human immunodeficiency virus (HIV), none of these drugs has been shown to be therapeutically effective against AIDS. One promising class of chemotherapeutics includes inhibitors of reverse transcriptase, an enzyme that is required for the replication of HIV and other members of the Retroviridae family of viruses. However, to date, these compounds have shown only limited effectiveness in slowing the progression of AIDS. Problems of cellular toxicity together with development of drug resistant variants of the virus have compromised the effective utility of these drugs. Therefore, current AIDS treatment protocols call for the use of a combination of available drugs.

Thus, new drugs useful in the treatment and/or prevention of bacterial and viral infections would represent a significant advance in the art.

SUMMARY OF THE INVENTION

The present invention overcomes these and other shortcomings in the art by providing bacterial preparations and compositions that have anti-bacterial/anti-microbial or anti-viral activities. The anti-bacterial or anti-microbial preparations and compositions have a variety of uses, including, but not limited to, inhibiting the growth of one or more selected microorganisms and treating or preventing a variety of bacterial or microbial infections. The anti-viral preparations and compositions have a variety of uses, including, but not limited to, inhibiting the activity of reverse transcriptase, either in vitro or in vivo, and treating or preventing a variety of RNA virus infections. The present invention provides compositions comprising an active ingredient having anti-reverse transcriptase activity, prepared by growing a biologically pure culture of a bacterial isolate having all of the identifying characteristics of Pseudomonas aeruginosa isolate #15 (ATCC #55638) in a liquid media containing organic carbon under conditions effective to produce the active ingredient, and isolating a supernatant fraction of the culture, the supernatant fraction comprising the active ingredient. In all aspects of the present invention, a preferred bacterial isolate is Pseudomonas aeruginosa isolate #15 (ATCC # 55638).

A wide variety of liquid media containing organic carbon can be used to generate the active ingredient having anti-reverse transcriptase activity, including, but not limited to, tryptic soy broth. Other suitable media are disclosed herein, and will be well known to those of ordinary skill in the art in light of the present disclosure. Likewise, a variety of growth conditions are "effective to produce the active ingredient" having anti-reverse transcriptase activity, for example, growth in tryptic soy broth for about four days at about room temperature (about 25°C) or about 30°C. However, in certain aspects of the invention, the culture can be grown for about 2 days, about 3 days, about 5 days, about 6 days or about 7 days or more, at between about 20°C to about 37°C. In further aspects of the present invention, particularly in vivo aspects, the anti-reverse transcriptase composition is dispersed in a pharmaceutically acceptable excipient. The present invention also provides compositions comprising an active ingredient prepared by growing a biologically pure culture of a bacterial isolate having all of the identifying characteristics of Pseudomonas aeruginosa isolate #15 (ATCC #55638) in a media containing organic carbon under conditions effective to produce the active ingredient, and obtaining the active ingredient from the media, the active ingredient having absorption peaks at about 694 nm and about 845 nm and anti-bacterial activity.

In certain embodiments of the invention, the active ingredient having anti-bacterial or anti-microbial activity comprises a quinoline compound N-substituted with a lower alkyl group. As used herein, "a lower alkyl group" will be understood to include alkyl groups with 1 to 6 carbon atoms, including, but not limited to methyl, ethyl, propyl, isopropyl, butyl, pentyl and hexyl. In preferred aspects of the invention, the active ingredient having antibacterial activity is an N-methyl quinoline compound.

As discussed above, a variety of growth conditions are "effective to produce the active ingredient" having anti-bacterial activity, for example, growth for about 2 days, about 3 days, about 4 days, about 5 days, about 6 days or about 7 days or more, at a temperature of from between about 20°C to about 37°C. As discussed above, a number of different media containing organic carbon can be used to grow the bacterial isolate, and the media can be either liquid or solid. Solid media is typically prepared by adding agar or agarose to the desired liquid media. The active ingredient having anti-bacterial activity is soluble in aqueous solvents, and thus is typically isolated from the broth or a culture of the bacterial isolate, or extracted from the solid media used to grow the bacterial isolate. A number of different aqueous solvents or solutions can be used to extract the active ingredient having anti-bacterial properties from the solid media, such as water, liquid media, buffered solutions and the like. In certain aspects of the invention, the water used to extract the active ingredient having anti-bacterial activity is distilled, filtered, sterile filtered or autoclaved. In a preferred embodiment of the present invention, the bacterial isolate is grown on trypticase soy agar plates, and the active ingredient is extracted from the agar with water. In certain aspects of the invention, the active ingredient having anti-bacterial activity is further purified by filtration, such as sterile filtration.

In preferred embodiments of the present invention, the active ingredient having anti-bacterial or anti-microbial activity inhibits the growth of at least a first microorganism from the genus Staphylococcus, Aeromonas, Legionella, Bacillus or Micrococcus, including, but not limited to, Staphylococcus aureus, Aeromonas hydrophila, Legionella pneumophila or Bacillus laterosporus. In certain aspects of the invention, the microorganism is located within an animal. In those embodiments in which the active ingredient having anti-bacterial activity is administered to a mammal, the active ingredient is preferably dispersed in a pharmaceutically acceptable excipient. The invention further provides compositions comprising an active ingredient prepared by growing a biologically pure culture of a bacterial isolate having all of the identifying characteristics of Pseudomonas aeruginosa isolate #15 (ATCC #55638) in a media containing organic carbon under conditions effective to produce the active ingredient, extracting a green pigment from the media using an aqueous solvent, and extracting the active ingredient from the green pigment using an organic solvent, such as chloroform, the active ingredient comprising a blue pigment comprising an N-methyl quinoline compound and having anti-bacterial activity.

The present invention also provides an N-substituted quinoline compound having anti-microbial activity. The quinoline compound is N-substituted with a lower alkyl group. As discussed above, "a lower alkyl group" will be understood to include alkyl groups with 1 to 6 carbon atoms, including, but not limited to methyl, ethyl, propyl, isopropyl, butyl, pentyl and hexyl. In preferred embodiments of the invention, the compound is an N-methyl- quinoline. In aspects of the invention in which the N-substituted quinoline compound is administered to an animal, the compound is preferably dispersed in a pharmaceutically acceptable excipient.

The invention additionally provides a composition that is isolatable from a biologically pure culture of a bacterial isolate having all of the identifying characteristics of Pseudomonas aeruginosa isolate #15 (ATCC #55638) grown in a media containing organic carbon, is soluble in water, has green pigmentation, has absorption peaks at about 694 nm and about 845 nm, and inhibits growth of at least a first selected microorganism.

The present invention provides kits for inhibiting the activity of a reverse transcriptase, comprising, in a suitable container a composition comprising an active ingredient having anti-reverse transcriptase activity prepared by growing a biologically pure culture of a bacterial isolate having all of the identifying characteristics of Pseudomonas aeruginosa isolate #15 (ATCC #55638) in a liquid media containing organic carbon under conditions effective to produce the active ingredient having anti-reverse transcriptase activity and isolating a supernatant fraction of the culture, the supernatant fraction comprising the active ingredient having anti-reverse transcriptase activity, and at least a second, distinct anti- reverse transcriptase composition.

The present invention also provides kits for inhibiting the growth of at least a first selected microorganism, comprising, in a suitable container a composition comprising an active ingredient having anti-bacterial activity prepared by growing a biologically pure culture of a bacterial isolate having all of the identifying characteristics of Pseudomonas aeruginosa isolate #15 (ATCC #55638) in a media containing organic carbon under conditions effective to produce the active ingredient and obtaining the active ingredient from the media, the active ingredient having absorption peaks at about 694 nm and about 845 nm and anti-bacterial activity, and at least a second, distinct anti-microbial composition.

The present invention further provides kits for inhibiting the growth of at least a first selected microorganism, comprising, in a suitable container, a quinoline compound N- substituted with a lower alkyl group, and at least a second, distinct anti-microbial composition. In preferred embodiments of the invention, the quinoline compound is an N- methyl-quinoline compound.

The present invention thus also provides methods of inhibiting the activity of a reverse transcriptase, comprising contacting the reverse transcriptase with an effective amount of a composition comprising an active ingredient having anti-reverse transcriptase activity prepared by growing a biologically pure culture of a bacterial isolate having all of the identifying characteristics of Pseudomonas aeruginosa isolate #15 (ATCC #55638) in a liquid media containing organic carbon under conditions effective to produce the active ingredient having anti-reverse transcriptase activity and isolating a supernatant fraction of the culture, the supernatant fraction comprising the active ingredient having anti-reverse transcriptase activity. In certain aspects, the active ingredient having anti-reverse transcriptase activity is further purified by filtration, or alternatively by sterile filtration.

In certain methods of the present invention, the reverse transcriptase is comprised within a cell. In some methods, the cell is a prokaryotic cell, while in others, the cell is a eukaryotic cell. In further methods of the invention, the cell is comprised within an animal, such as a human subject. In the methods where the cell is comprised within an animal, the active ingredient having anti-reverse transcriptase activity is preferably dispersed in a pharmaceutically acceptable excipient. In still further aspects of the invention, the reverse transcriptase is contacted with at least a second, distinct anti-reverse transcriptase compound. The present invention further provides a method of inhibiting the growth of a selected microorganism, comprising contacting the microorganism with an effective amount of a composition comprising an active ingredient having anti-bacterial activity, prepared by growing a biologically pure culture of a bacterial isolate having all of the identifying characteristics of Pseudomonas aeruginosa isolate #15 (ATCC #55638) in a media containing organic carbon under conditions effective to produce the active ingredient having anti -bacterial activity, and obtaining the active ingredient having anti-bacterial activity from the media, the active ingredient having absorption peaks at about 694 nm and about 845 nm.

In preferred methods, the active ingredient having anti-bacterial activity comprises a quinoline compound N-substituted by a lower alkyl group, such as an N-methyl quinoline compound. In preferred anti-microbial methods of the invention, the selected microorganism is from the genus Staphylococcus, Aeromonas, Legionella, Bacillus or Micrococcus, including, but not limited to, Staphylococcus aureus, Aeromonas hydrophila, Legionella pneumophila or Bacillus laterosporus. In particular methods, the selected microorganism is comprised within an animal, including, but not limited to, a human subject. In these methods, the composition comprising the active ingredient having anti-bacterial activity is preferably dispersed in a pharmaceutically acceptable excipient.

In further methods of the invention, the anti-microbial composition comprises at least a second, distinct compound that inhibits the growth of the selected microorganism. The present invention also provides a method of inhibiting the growth of a selected microorganism, comprising contacting the microorganism with an effective amount of a quinoline compound N-substituted with a lower alkyl group.

Further provided are methods of treating or preventing a disease caused by an RNA virus in an animal having or at risk of developing the disease, which comprise administering to the animal a therapeutically or prophylactically effective amount of a pharmaceutical composition comprising an active ingredient having anti-reverse transcriptase activity prepared by growing a biologically pure culture of a bacterial isolate having all of the identifying characteristics of Pseudomonas aeruginosa isolate #15 (ATCC #55638) in a media containing organic carbon under conditions effective to produce the active ingredient having anti-reverse transcriptase activity, isolating a supernatant fraction of the culture, the supernatant fraction comprising the active ingredient having anti-reverse transcriptase activity, and further purifying the active ingredient having anti-reverse transcriptase activity by sterile filtration. In certain treatment and prevention methods, the animal is a human subject. In particular methods the viral disease is AIDS or hepatitis.

In further treatment and prevention methods of the present invention, at least a second, distinct anti-viral compound is administered to the animal in combination with the pharmaceutical composition comprising an active ingredient having anti-reverse transcriptase activity. The at least a second anti-viral compound can be administered prior to, in conjunction with, or after administration of the pharmaceutical composition comprising an active ingredient having anti-reverse transcriptase activity. Additionally provided by the present invention are methods of treating or preventing a bacterial infection in an animal having or at risk of developing the bacterial infection, comprising administering to the animal a therapeutically or prophylactically effective amount of a pharmaceutical composition comprising an active ingredient having anti-bacterial activity prepared by growing a biologically pure culture of a bacterial isolate having all of the identifying characteristics of Pseudomonas aeruginosa isolate #15 (ATCC #55638) in a media containing organic carbon under conditions effective to produce the active ingredient having anti-bacterial activity, obtaining the active ingredient having anti-bacterial activity from the media, the active ingredient having absorption peaks at about 694 nm and about 845 nm, and further purifying the active ingredient having anti-bacterial activity by sterile filtration.

In certain treatment and prevention methods, the bacterial infection is caused by a microorganism from the genus Staphylococcus, Aeromonas, Legionella, Bacillus or Micrococcus, including, but not limited to, Staphylococcus aureus, Aeromonas hydrophila, Legionella pneumophila or Bacillus laterosporus. In further treatment and prevention methods of the invention, at least a second, distinct anti-bacterial compound is administered to the animal. The at least a second anti-bacterial compound can be administered prior to, in conjunction with, or after administration of the pharmaceutical composition comprising an active ingredient having anti-bacterial activity. The present invention also provides a method of treating or preventing a bacterial infection in an animal having or at risk of developing the bacterial infection, comprising administering to the animal a therapeutically or prophylactically effective amount of a quinoline compound N-substituted with a lower alkyl group.

BRIEF DESCRIPTION OF THE DRAWINGS

The following drawings form part of the present specification and are included to further demonstrate certain aspects of the present invention. The invention may be better understood by reference to one or more of these drawings in combination with the detailed description of specific embodiments presented herein.

FIG. 1A and FIG. IB. Spectroscopic profiles of green (FIG. 1A) and red

(FIG. IB) pigments.

DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS

A. Bacterial Isolate #15

Bacterial isolate #15 (ATCC 55638) was found to be effective in the practice of the present invention. The isolation and characterization of isolate #15 is disclosed in U.S. Patent No. 5,518,919, which is incorporated herein in its entirety by reference. Bacterial isolate #15 (ATCC 55638) has been classified as Pseudomonas aeruginosa, based on a comparison of the 16S RNA sequences. The isolate will be referred to herein as bacterial isolate #15 (ATCC 55638) or Pseudomonas aeruginosa isolate #15 (ATCC 55638). It is anticipated that other isolates having the characteristics of Pseudomonas aeruginosa isolate #15 (ATCC 55638) will be useful in this invention. Pseudomonas aeruginosa isolate #15 (ATCC 55638) is characterized as an amoeba-associated bacterial species that produces products having anti-reverse transcriptase or anti-bacterial activity when grown to stationary phase. An "amoeba-associated bacterial species" is defined herein as a bacterial species that is found to coexist with a free-living amoeba in an ecto- or endosymbiotic relationship.

By "aged culture" it is meant that the culture is allowed to grow until stationary phase is reached. In the examples below, the bacterial cultures were allowed to grow on Trypticase Soy Agar (TSA) or for about 4 days in Trypticase Soy Broth (TSB) at room temperature. The number of days that it takes a bacterial culture to reach a particular phase of growth is dependent on a number of factors, including the medium in which the bacteria are cultured, the temperature, and aeration. Varying these parameters may affect the time it takes for the culture to reach stationary phase. Aged cultures obtained using such variations in culture conditions are within the scope and spirit of the present invention.

Other liquid and solid culture media containing organic carbon can also be used to grow the bacterial isolate of the present invention, including, but not limited to, medium 3, medium 44, medium 122, medium 129, medium 147, medium 225, medium 260, medium 265, medium 368, medium 1287 or medium 1356, either with or without agar, as disclosed in the ATCC Catalogue of Bacteria and Bacteriophages (1992). Additionally, in different embodiments of the present invention, the cultures are grown at temperatures of from between about 20°C to about 37°C, but in preferred aspects, the cultures are grown at room temperature (about 25°C), about 30°C or about 37°C.

The inventors have discovered that bacterial isolate #15 produces a novel soluble green pigment when grown in air on an agar medium rich in organic carbon. Pigment formation was inhibited when the culture was grown in a 100% carbon dioxide atmosphere. The inventors have also discovered that bacterial isolate #15 also produces a soluble red pigment when grown on Pseudosel agar (Atlas, 1993), or in organic medium in the presence of Acanthamoebae royreba and preferentially in a carbon dioxide atmosphere. Spectroscopic analysis of the green and red pigments indicated that they differed significantly from common plant and bacterial pigments. For example, pseudomonads are known to produce a number of pigments. Pseudomonas aeruginosa is known to produce at least three pigments, pyocyanin (N-methyl- 1-hydroxyphenazine; Cox, 1986), pyoverdine (6,7-dihydroxy quinoline; Stintzi et al, 1996) and pyochelin (Ankenbauer et al, 1988). However, the inventors have determined that the present pigments are distinct from these previously characterized pseudomonad pigments.

B. Inhibition of Reverse Transcriptase Certain of the preparations described herein have anti-reverse transcriptase activity.

The Retroviridae family of RNA-containing tumor viruses is characterized by the presence of the enzyme, reverse transcriptase, in the virions. These viruses are able to infect and replicate only in their natural eucaryotic hosts. The genome of these viruses is composed of two single-stranded RNA molecules that replicate through a double-stranded DNA intermediate. In the early stages of the retroviral life cycle, viral RNA is copied to form a double-stranded DNA, which is integrated into host DNA to generate the provirus (Varmus and Swanstrom, 1982). The provirus induces virus multiplication and transformation.

The synthesis of the proviral DNA is catalyzed by the enzyme reverse transcriptase, which may efficiently utilize either RNA or DNA templates for DNA synthesis by the elongation of a primer bearing a paired 3' hydroxyl terminus. Reverse transcriptase is an RNA-dependent DNA polymerase that elongates an oligonucleotide primer (such as a tRNA) paired to a template strand (either RNA or DNA) and thus synthesizes a DNA molecule that is complementary to the template strand (complementary DNA, cDNA). Inherent in the same protein is a second activity, RNAse H, which degrades RNA present as a duplex RNA:DNA hybrid. Discovery of this enzyme was a decisive step in understanding the mechanism by which a virus with an RNA genome could replicate its genetic information in infected cells (Baltimore, 1970; Temin and Mizutani, 1970).

Reverse transcriptase is also widely used as a means of producing complementary DNA (cDNA) copies of messenger RNA (mRNA) molecules. These cDNAs may be inserted into expression vectors that are used to transform cells so that the resulting cells produce a desired polypeptide encoded by the original mRNA.

1. Sources of Reverse Transcriptase Reverse transcriptases useful in the methods of the invention include, but are not limited to those derived from retroviruses, such as of the genus Cisternavirus A; Oncovirus B, including mouse mammary tumor viruses (MMTV-S (Bitmer's virus), MMTV-P (GR virus), MMTV-L); Oncovirus C, such as Rous sarcoma virus, Rous-associated virus, avian sarcoma viruses, chicken sarcoma viruses, leukosis viruses, reticuloendotheliosis viruses, pheasant viruses, murine sarcoma viruses, murine leukosis virus G (Gross or AKR virus), murine leukosis viruses (MLV-F, MLV-M, MLV-R (Friend, Maloney, Rauscher viruses)), murine radiation leukemia virus, murine endogenous viruses, rat leukosis virus, feline leukosis viruses, feline sarcoma virus, feline endogenous virus (RD114), hamster leukosis virus (HLV), porcine leukosis virus, bovine leukosis virus, primate sarcoma viruses (woolly monkey, gibbon, ape), primate sarcoma-associated virus, primate endogenous viruses (baboon endogenous virus, stumptail monkey virus, MAC-1, owl monkey virus (OMC-1)); Oncovirus D, including reptilian viruses, such as the viper virus, and non-reptilian viruses such as Mason-Pfizer monkey virus (MPMV), langur virus, and squirrel monkey virus; Lentivirus E, including Visna virus of sheep and Maedi virus; and Spumavirus F, including foamy viruses of primates, cats, humans, and bovids.

Reverse transcriptases of the invention can also be derived from any of the human retroviruses, particularly the human T cell leukemia viruses and human immunodeficiency viruses, as well as from the hepadnairuses, including hepatitis viruses A, B, C, non-A/non-B and delta agent, Caulimoviruses, avian myoblastosis virus, simian immunodeficiency viruses, feline immunodeficiency viruses, and equine infectious anemia viruses.

2. Inhibition Assays

In addition to the assays described herein below, a number of other assays known to those of ordinary skill in the art can be used to determine the extent of inhibition of reverse transcriptase activity by the compositions disclosed herein. Examples of in vitro reverse transcription assays include, but are not limited to, those described by Lacey et al. (1992), Ma et al. (1992), Reardon (1992), Izuta et al. (1991), Parker et al. (1991); Vrang et al. (1987); Olsen et al. (1992), Gronowitz et al. (1991), White et al. (1990 and 1991), Furman et al. (1988), Vogt et a/. (1989) and U.S. Patent No. 5,124,327.

3. Combination with Other Reverse Transcriptase Inhibitors

The instant anti-reverse transcriptase preparations can be used in combination with any number of other well-known reverse transcriptase inhibitors. Two pharmacological classes of inhibitor molecules, nucleoside and nonnucleoside, have been found to be effective in halting the enzymatic function of the reverse transcriptase (Larder, 1993). Nucleoside inhibitors such as AZT (zidovudine, azidothymidine; Boucher et al, 1993; Fischl et al, 1987, 1990; Lambert et al, 1990; Meng et al, 1990; Skowron et al, 1993; Yarchoan et al, 1986), ddC (Zalcitabine, 2', 3'-dideoxycytidine, Hivid), ddl (didanosine, 2',3'-dideoxyinosine, Videx), and d4T (Stavudine, 2', 3'-didehydro-2', 3'-dideoxythymine) are chemically similar to the normal nucleosides and therefore can be converted to their triphosphate form and then used in the synthesis of DNA during reverse transcription. However, elongation of the DNA chain is blocked since these compounds lack a 3'-OH group that is essential for incorporation of additional nucleotides. Problems of cellular toxicity together with development of drug resistant variants of the virus have compromised the effective utility of these drugs.

A number of pharmacologically active nonnucleoside inhibitors (NNI) have also been identified. Many of these inhibitors appear highly potent, relatively nontoxic, and specifically inhibit HIV reverse transcriptase. Examples of such compounds include, but are not limited to, nevirapine (BI-RG-587, l l-cyclopropyl-5, 11 -dihydro-4-methyl-6H- dipyrido[3,2-b:2',3']-e(l,4)diazepin-6-one), TIBO (Tetrahydroimidazo[4,5,l- jk][l ,4]benzodiazepin-2(lH)-one), HEPT (1 -[(2-hydroxyethoxymethyl)]-6-

(phenylthio)thymine), BHAP (bis(heteroaryl)piperazine), and alpha-APA (alpha- anilinophenylacetamide). However, the rapid emergence of HIV strains resistant to these compounds in vitro has become a major concern that may affect further development of these types of drugs (Larder, 1993). Rapid mutations, in some cases within weeks or months, in the HIV-1 RT have been reported upon exposure of HIV-infected cells to these compounds.

Therapeutic compounds and reverse transcriptase inhibitors and metabolites thereof useful in any of the methods of the invention also include, but are not limited to dideoxynucleotide triphosphate analogs, including 2',3'-dideoxynucleoside 5'-triphosphates (Izuta et al, 1991); including, for example, dideoxyinosine and dideoxycytidine (Shirasaka et al, 1990); anti-reverse transcriptase antibodies and sFvs; Carbovir (carbocyclic analog of 2',3'-didehydro-2',3'-dideoxyguanosine; White et al, 1990); 3'-azido-3'-deoxythymidine triphosphate, (Furman et al, 1986); 3'-azido-3'-deoxythymidine (Mitsuya et al, 1985; Tavares et al, 1987); , thymidine 5'-[ ,β-imido]-triphosphate, 3'-azido-3'-deoxythymidine 5'-[α,β-imido]-triphosphate, dideoxythymidine 5'-[α,β-imido]-triphosphate, 3'- azidothymidine 5 '-[β,γ-imido] -triphosphate, thymidine 5'-[α,β:β,γ-diimido]-triphosphate (Ma et al, 1992); R82913 ((+)-S-4,5,6,7-tetrahydro-9-chloro-5-methyl-6-(3-methyl-2-butenyl)- imidazo[4,5,l-jk][l,4]-benzodiazepin-2(lH)-thione (a TIBO derivative); (White et al, 1991); 3'-deoxy-2',3'-didehydrothymidine 5'-triphosphate, 2',3 '-dideoxycytidine 5'-triphosphate; 2',3'-dideoxyadenosine 5'-triphosphate; 2',3'-dideoxyguanosine 5'-triphosphate; 2',3'- dideoxythymidine 5'-triphosphate; (Reardon, 1992); 5 '-triphosphate of carbovir (the carbocyclic analog of 2'-3'-didehydro-2'-3'-dideoxyguanosine; Parker et al, 1991, White et al, 1991); threo- and erythro- isomers of 3'-azido-3'-deoxythimidine triphosphate (Vrang et al, 1987); 2'.3'-didehydro-2',3'-dideoxythimidine (D4T) (Wainberg et al, 1990); purines comprising a 2',3'-dideoxyribose moiety, nucleosides comprising a 2',3'-didehydro-2',3'- deoxyribose moiety, 2',3'-dideoxythymidinene (ddE Thd) (Masood et al, 1989); galolyl derivatives of quinic acid, particularly 3',4',5-tri-O-galoylquinic acid (Tri GQA), and 3,4-di-O-galloyl-5-digalloylquinic acid, Tetra GQA plus 3'-azido-3-deoxy thymidine triphosphate or phosphonoformic acid (Parker et al, 1989); Merck compound L-697,661 (Olsen et al, 1992); 3'-azido-2',3'-dideoxyadenosine AZA (Shirasaka et al, 1990); 3'-azido- 2'-3'-dideoxyguanosine (AZG), carbovir monophosphate; (-Et, -nPr, -nPre, -iPre, -Ce) 5'- triphosphates of 5 '-substituted 2'-deoxy-uridine; phosphonoacidic acid and phosphonoformic acid (Pei-Zhen, 1989); 3-amino-thymidine 5 '-triphosphate (Lacey et al, 1992); zidovudine monophosphate and diphosphate; 2',3'-dideoxynucleosides; R 12913; Ribavirin poly(A)*poly(U), (Hovanessian et al, 1991); AZT plus interferon; anhydro-AZT; phosphoformate ("Foscarnet"); deoxy-thiacytidine (Wainberg et al, 1990); anhydro-N3, - UdR and the nonnucleoside inhibitors shown in U.S. Patent No. 5,917,033 (incorporated herein in its entirety by reference).

Any combination of the above reverse transcriptase inhibitors can be used in combination with the agents disclosed herein.

4. Diseases Caused by RNA Viruses The present preparations and compositions are contemplated to have broad application in the treatment of retrovirus infection, particularly HIV infection and AIDS. The compositions are also contemplated to be useful for individuals infected by a hepadnavirus, especially Hepatitis B virus. Other conditions contemplated for treatment with the compositions disclosed herein include, but are not limited to, HIV 1 or HIV 2 infection, Kaposi's sarcoma, pneumocytis pneumonia, mycobacterium infection, AIDS Related Complex, AIDS dementia and systemic candidiasis.

C. Inhibition of Microbial Growth

1. Microorganisms Inhibited by Green and/or Blue Pigment The filter sterilized green pigment containing material and the blue pigment containing material were shown to be effective in inhibiting the growth of a variety of microorganisms, including species of the Staphylococcus, Aeromonas, Legionella, Bacillus and Micrococcus genus. The zones of inhibition were comparable to those seen with commercially available preparations of gentamycin and ampicillin. However, the material appeared to have no effect on the growth of Streptococcus faecalis, Salmonella typhimurium, Citrobacter freundeii, Klebsiella oxytoca, Listeria grayeii and Eschenchia coli under the conditions employed herein. 2. Assays For Inhibition of Microbial Growth

There are a number of methods for measuring antimicrobial sensitivity that are well known to those of skill in the art (Nester et al, 1978). The three main tests are disk diffusion susceptibility tests, broth microdilution minimal inhibitory concentration (MIC) tests and β-lactamase tests. The disk diffusion test was used herein to test the green and blue pigment containing materials for their ability to inhibit microbial growth, and are described in greater detail in Example 3.

3. Combination with Other Anti-Microbial Compounds The instant active agents can be used in combination with any number of other well- known antibiotics. Classes of antibiotics contemplated for use in combination with the antimicrobial agents disclosed herein include, but are not limited to, tetracyclines, rifamycins, macrolides, penicillins, cephalosporins, other beta-lactam antibiotics, aminoglycosides, chloramphenicol, sufonamides, glycopeptides, quinolones, fusidic acid, polyenes, azoles and beta-lactam inhibitors.

Examples of specific antibiotics that can be used include, but are not limited to, minocycline, rifampin, erythromycin, azithromycin, clarithromycin, roxithromycin, oleandomycin, spiramycin, josamycin, miocamycin, midecamycin, rosaramycin, troleandomycin, flurithromycin, rokitamycin, dirithromycin, nafcillin, cefazolin, imipenem, aztreonam, gentamicin, sulfamethoxazole, vancomycin, ciprofloxacin, trimethoprim, metronidazole, clindamycin, lincomycin, celesticetin, teicoplanin, mupirocin, azithromycin, clarithromycin, ofloxacin, lomefloxacin, norfloxacin, nalidixic acid, sparfloxacin, pefloxacin, amifloxacin, enoxacin, fleroxacin, temafloxacin, tosufloxacin, clinafloxacin, sulbactam, clavulanic acid, amphotericin B, fluconazole, itraconazole, ketoconazole, and nystatin. Other examples of antibiotics, such as those listed in Sakamoto et al., U.S. Pat. No. 4,642,104, herein incorporated by reference, are also contemplated for use in the combination aspects of the present invention. Multiple antimicrobial agents can be used in combination with the agents disclosed herein. Tables 1 and 2 below list a number of common antibiotics and their standard oral and parenteral doses.

Table 1 Common Antibiotics And Usual Oral Doses

ANTIBIOTIC DOSAGE

Penicillin V (Rugby (generic), V-cillin K) 250 mg qid

Dicloxacillin (Glenlawn (generic), Dynapen) 250 mg qid

Cloxacillin (Tegopen) 250 mg qid

Amoxicillin (Rugby (generic), Polymox) 250 mg tid

Ampicillin (Moore (generic), Polycillin) 250 mg qid

Augmentin (250 mg (tablets or chewables) or 125 mg tid (suspension or chewables))

Carbenicillin (Geocillin) 1 tab (382 mg) or 2 tab qid

Cephalexin (Rugby (generic), Keflex) 250 or 500 mg qid

Cefadroxil (Rugby (generic), Duricef) 1 gm bid

Cephradine (Rugby (generic), Velosef) 250 or 500 mg qid

Cefaclor (Ceclor) 250 mg tid

Cefuroxime axetil (Ceftin) 125, 250 or 500 mg bid

Cefixime (Suprax) 400 mg q24h

Cefprozil (Cefzil) 250 mg ql2h

Loracarbef (Lorabid) 200 mg bid

Cefpodoxime proxetil (Vantin) 200 mg bid

Clindamycin (Cleocin) 300 mg q8h

TMP/SMZ (Bactrim, Septra (generic)) 1 double-strength bid

Trimethoprim (Rugby (generic), Proloprim) 100 mg bid

Erythromycin (base, Abbott, or E-mycin (delayed 250 mg qid release)

Erythromycin stearate (Rugby (generic)) 250 mg qid

Azithromycin (Zithromax) 1 g once only 500 mg, day 1, plus ANTIBIOTIC DOSAGE

250 mg, day 2-5

Clarithromycin (Biaxin) 250 or 500 mg bid

Tetracycline hydrochloride (Mylan, Sumycin 250) 250 mg qid

Doxycycline (Lederle (generic), Vibramycin) 100 mg qd (with 200-mg initial load)

Vancomycin (Vancocin HCl (oral sol'n/powder)) Capsules 125 mg q6h PO

Metronidazole (Rugby (generic), Flagyl) 250 mg qid

Norfloxacin (Noroxin) 400 mg bid

Ciprofloxacin (Cipro) 250, 500 or 750 mg bid

Ofloxacin (Floxin) 200, 300 or 400 mg bid

Lomefloxacin Maxaquin 400 mg once qd

Table 2 Common Antibiotics And Usual Parenteral Doses

ANTIBIOTIC DOSAGE

Penicillin G (Pfizerpen G (Pfizer)) 2.4 or 12 million units

Oxacillin (Prostaphlin (Bristol)) 1 g

Nafcillin (Nafcil (Bristol)) 12 g

Ampicillin (Omnipen (Wyeth)) 6 g

Ticarcillin (Ticar (Beecham)) 18 g

Piperacillin (Pipracil (Lederle)) 16 or 18 g

Mezlocillin (Mezlin (Miles)) 16 or 18 g

Ticarcillin-clavulanate (Timentin (Beecham)) 18 g/0.6 g or 12 g/0.4 g

Ampicillin-sulbactam (Unasyn (Roerig)) 6 or 12 g

Cephalothin (Keflin (Lilly)) 9 g (1.5 g q4h)

Cefazolin (Ancef (SKF)) 4 g (1 g q6h) or 3 g (1 g q8h)

Cefuroxime (Zinacef (Glaxo)) 6 g (750 mg q8h) or 4.5 g (1.5 g q8h)

Cefamandole (Mandol (Lilly)) 9 g (1.5 g q4h)

Cefoxitin (Mefoxin (MSD)) 8 g (2 g q6h) or 6 g (2 g q8h)

Cefonicid (Monicid (SKF)) l g ql2h ANTIBIOTIC DOSAGE

Cefotetan (Cefotan (Stuart)) 2 g ql2h

Cefmetazole (Zefazone (Upjohn)) 2 g q8h

Ceftriaxone (Rocephin (Roche)) 2 g (2.0 g q24h) or l g (1.0 g q24h)

Ceftazidime (Fortax (Glaxo), Taxicef (SKF), 6 g (2 g q8h) Tozidime (Lilly))

Cefotaxime (Claforan (Hoechst)) 2 g q6h or 2 g q8h

Cefoperazone (Cefobid (Pfizer)) 8 g (2 g q6h) or 6 g (2 g q8h)

Ceftizoxime (Ceftizox (SKF)) 2 g q8h

Aztreonam (Azactam (Squibb)) 2 g q8h or 1 g q8h

Imipenem (Primaxin (MSD)) 2000 mg (500 mg 16h)

Gentamicin (Garamycin (Schering, generic) (Elkins- 360 mg (1.5 mg/kg q8h for an 80-kg Sinn) patient)

Tobramycin (Nebcin (Dista)) 360 mg (120 mg q8h)

Amikacin (Amikin (Bristol)) 1200 mg (600 mg ql2h)

Clindamycin (Cleocin (Upjohn)) 2400 mg (600 mg q6h), 2700 mg (900 mg q8h) or 1800 mg (600 mg q8h)

Chloramphenicol (Chloromycetin (P/D)) 4 g (1 g q6h)

TMP/SMZ (Septra (Burroughs Wellcome)) 1400 mg TMP (350 mg TMP q6h) or 700 mg TMP (350 mg TMP ql2h)

Erythromycin (Elkins-Sinn) 2000 mg (500 mg q6h)

Doxycycline (Vibramycin (Pfizer)) 200 mg (100 mg ql2h)

Vancomycin (Vancocin (Lilly)) 2000 mg (500 mg q6h)

Metronidazole ((generic) (Elkins-Sinn)) 2000 mg (500 mg q6h)

Ciprofloxacin (Cipro) 200 mg ql2h or 400 mg ql2h

Pentamidine (Pentam (LyphoMed)) 280 mg q24h 4. Diseases Caused by Microbial Infections

The present compositions are contemplated for use in treating a number of bacterial infections, including, but not limited to, Staphylococcus, Aeromonas, Legionella, Bacillus and Micrococcus infections, as well as for treating anthrax.

D. Pharmaceutical Compositions and Routes of Administration

Pharmaceutical compositions of the present invention will generally comprise an effective amount of composition comprising one of the active ingredients disclosed herein, such as a green pigment or blue pigment preparation, or component thereof, dissolved or dispersed in a pharmaceutically acceptable carrier or aqueous medium.

The phrases "pharmaceutically or pharmacologically acceptable" refer to molecular entities and compositions that do not produce an adverse, allergic or other untoward reaction when administered to an animal, or a human, as appropriate. As used herein, "pharmaceutically acceptable excipient or carrier" includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents and the like. The use of such media and agents for pharmaceutical active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active ingredient, its use in the therapeutic compositions is contemplated. Supplementary active ingredients can also be incorporated into the compositions. The agents of the present invention will often be formulated for parenteral administration, e.g., formulated for injection via the intravenous, intramuscular, subcutaneous or other such routes. The preparation of an aqueous composition that contains one or more of the instant active ingredients, such as a green pigment or blue pigment preparation, or component thereof, will be known to those of skill in the art in light of the present disclosure. Typically, such compositions can be prepared as injectables, either as liquid solutions or suspensions; solid forms suitable for using to prepare solutions or suspensions upon the addition of a liquid prior to injection can also be prepared; and the preparations can also be emulsified. Solutions of the active compounds as free base or pharmacologically acceptable salts can be prepared in water suitably mixed with a surfactant, such as hydroxypropylcellulose.

Dispersions can also be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof and in oils. Under ordinary conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms.

The pharmaceutical forms suitable for injectable use include sterile aqueous solutions or dispersions; formulations including sesame oil, peanut oil or aqueous propylene glycol; and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. In all cases the form must be sterile and must be fluid to the extent that easy syringability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms, such as bacteria and fungi.

Compositions comprising the agents of the present invention can be formulated into a composition in a neutral or salt form. Pharmaceutically acceptable salts, include the acid addition salts (formed with the free amino groups of the protein) and which are formed with inorganic acids such as, for example, hydrochloric or phosphoric acids, or such organic acids as acetic, oxalic, tartaric, mandelic, and the like. Salts formed with the free carboxyl groups can also be derived from inorganic bases such as, for example, sodium, potassium, ammonium, calcium, or ferric hydroxides, and such organic bases as isopropylamine, trimethylamine, histidine, procaine and the like.

The carrier can also be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), dimethylsulfoxide (DMSO), suitable mixtures thereof, and vegetable oils. The proper fluidity can be maintained, for example, by the use of a coating, such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. The prevention of the action of microorganisms can be brought about by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars or sodium chloride. Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminum monostearate and gelatin.

Sterile injectable solutions are prepared by incorporating the active compounds in the required amount in the appropriate solvent followed by filtered sterilization. Generally, dispersions are prepared by incorporating the various sterilized active ingredients into a sterile vehicle which contains the basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum-drying and freeze- drying techniques which yield a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.

Upon formulation, solutions will be administered in a manner compatible with the dosage formulation and in such amount as is therapeutically effective. Formulations are easily administered in a variety of dosage forms, such as the type of injectable solutions described above, but drug release capsules and the like can also be employed. Suitable pharmaceutical compositions in accordance with the invention will generally include an amount of one or more of the active agents of the present invention admixed with an acceptable pharmaceutical diluent or excipient, such as a sterile aqueous solution, to give a range of final concentrations, depending on the intended use. The techniques of preparation are generally well known in the art as exemplified by Remington's Pharmaceutical Sciences, 16th Ed. Mack Publishing Company, 1980, incorporated herein by reference. It should be appreciated that endotoxin contamination should be kept minimally at a safe level, for example, less that 0.5 ng/mg protein. Moreover, for human administration, preparations should meet sterility, pyrogenicity, and general safety and purity standards as required by FDA Office of Biological Standards. The therapeutically effective doses are readily determinable using an animal model.

Experimental animals susceptible to or having a viral or microbial infection are frequently used to optimize appropriate therapeutic doses prior to translating to a clinical environment. Such models are known to be very reliable in predicting effective clinical strategies. In addition to the compounds formulated for parenteral administration, such as intravenous or intramuscular injection, other pharmaceutically acceptable forms are also contemplated, e.g., tablets or other solids for oral administration, time release capsules, liposomal forms and the like. Other pharmaceutical formulations may also be used, dependent on the condition to be treated. Of course, methods for the determination of optimal dosages for conditions such as these would be evident to those of skill in the art in light of the instant specification, and the knowledge of the skilled artisan.

It is also contemplated that certain benefits will result from the manipulation of the agents of the present invention to provide them with a longer in vivo half-life. Slow release formulations are generally designed to give a constant drug level over an extended period. Increasing the half-life of a drug, such as agents of the present invention, is intended to result in high intravenous levels upon administration, which levels are maintained for a longer time, but which levels generally decay depending on the pharmacokinetics of the construct.

E. Kits

The present invention also provides therapeutic kits comprising the active agents of the present invention described herein. Such kits will generally contain, in suitable container, a pharmaceutically acceptable formulation of a green pigment or blue pigment preparation, or component thereof, in accordance with the invention. The kits may also contain other pharmaceutically acceptable formulations, such as any one or more of a range of therapeutically beneficial drugs.

The kits may have a single container that contains the agent, with or without any additional components, or they may have distinct container means for each desired agent. Certain preferred kits of the present invention include a green pigment or blue pigment preparation, or component thereof, packaged in a kit for use in combination with the co-administration of a second agent, such as a distinct anti-reverse transcriptase, anti-viral or anti-microbial compound. In such kits, the components may be pre-complexed, either in a molar equivalent combination, or with one component in excess of the other; or each of the components of the kit may be maintained separately within distinct containers prior to administration to a patient.

When the components of the kit are provided in one or more liquid solutions, the liquid solution is an aqueous solution, with a sterile aqueous solution being particularly preferred. However, the components of the kit may be provided as dried powder(s). When reagents or components are provided as a dry powder, the powder can be reconstituted by the addition of a suitable solvent. It is envisioned that the solvent may also be provided in another container means. One of the components of the kit may be provided in capsules for oral administration. The container means of the kit will generally include at least one vial, test tube, flask, bottle, syringe or other container means, into which the green pigment or blue pigment preparation, or component thereof, and any other desired agent, may be placed and, preferably, suitably aliquoted. Where additional components are included, the kit will also generally contain a second vial or other container into which these are placed, enabling the administration of separated designed doses. The kits may also comprise a second/third container means for containing a sterile, pharmaceutically acceptable buffer or other diluent.

The kits may also contain a means by which to administer a green pigment or blue pigment preparation, or component thereof, to an animal or patient, e.g., one or more needles or syringes, or even an eye dropper, pipette, or other such like apparatus, from which the formulation may be injected into the animal or applied to a diseased area of the body. The kits of the present invention will also typically include a means for containing the vials, or such like, and other component, in close confinement for commercial sale, such as, e.g., injection or blow-molded plastic containers into which the desired vials and other apparatus are placed and retained.

The following examples are included to demonstrate preferred embodiments of the invention. It should be appreciated by those of skill in the art that the techniques disclosed in the examples which follow represent techniques discovered by the inventors to function well in the practice of the invention, and thus can be considered to constitute preferred modes for its practice. However, those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments which are disclosed and still obtain a like or similar result without departing from the spirit and scope of the invention.

EXAMPLE 1 Production of Green and Red Pigments From Bacterial Isolate #15

Free-living amoebae and other protozoa interact with a variety of bacteria. In the case of amoebae, this interaction ranges from bacteria that serve as food sources or are sequestered as endosymbionts within amoebae to bacterial species that are amplified by amoebae.

In previous studies, the inventors have shown that many bacteria associated with free- living amoebae isolated from environmental sources have unusual characteristics such as pigmentation, crystal formation and biodispersant production (Tyndall et al, 1991; Dietz et al, 1994). Also, in recent studies, the inventors have shown that infection of Acanthamoebae royreba with Legionella or exposure of A. royreba to megarad doses of gamma radiation can result in the appearance of bacterial derivars not detectable after extensive testing of unirradiated or uninfected amoebae control cultures (Tyndall and Domingue, 1982; Vass and Tyndall, 1994; Vass et al, 1995).

The initial observations in the present study were part of the aforementioned radiation studies where selected amoebic and bacterial combinations were plated for viability after radiation exposure. In control unirradiated cultures, a mixture of bacterial isolate #15, originally cultured from amoebae isolated from environmental sources, and A. royreba produced a soluble green pigment. Subsequently, a series of experiments was undertaken to expand this initial observation and define the conditions necessary for pigment production and for spectroscopic analysis of the pigments. A. Materials and Methods

Amoebae/Bacterial Growth. Intra-amoebic bacteria from environmental free-living amoebae, Oak Ridge consortium 46C (Tyndall et al, 1991), were originally isolated and subsequently cultured on Trypticase Soy Agar (TSA) and modified peptone medium (MPM). All other bacteria were grown on TSA. Bacterial isolate #15, one of the pseudomonads obtained from the consortium of amoebae and bacteria, is a Gram-negative rod shaped organism. It is motile, oxidase positive, catalase positive, and does not ferment glucose, mannose, inositol, sorbitol, rhamnose, saccharose, melibiose, amyglandin, or arabinose. Like many such amoebae-associated isolates, this pseudomonad produces extracellular crystals and a biodispersant. The amoeba used in this study, Acanthamoebae royreba, was grown in 712 medium at 37°C (ATCC Media Handbook, 1984).

Pisment Production on Asar Rich in Orεanic Carbon. Culture #15 was grown on TSA plates for two days at 35°C. A drop of A. royreba suspension was added to the center of some plates and the plates were then reincubated in the presence or absence of CO₂, with and without light and at 35°C or room temperature. Test plates were exposed to a CO₂ atmosphere by placing them in a desiccator jar flushed with CO₂. After 48 hours the plates were checked for the production of pigments and reincubated. Similarly, after 96 and 216 hours the plates were checked for subsequent changes. Because Pseudomonas chlororaphis can also produce small amounts of green pigment, this bacterium was grown on TSA and compared to isolate #15.

UV-visible Spectroscopy. Green and red pigments were extracted from TSA plates with water. After pigment production was established, the agar plates were cut into sections and placed in sterile water in culture flasks for 5 to 7 days at 4°C. The water was then removed and centrifuged at 2000 x g for 30 minutes, and then filter sterilized using a 0.2 μm filter.

Spectroscopy in the visible range from 300-1000 nm was performed using an SLM Amino DW-2000 UV-VIS spectrophotometer interfaced to an IBM PS/2 model 60 computer. Base lines were collected, stored in the computer, and used to correct the absoφtion spectra of the pigments.

B. Results

Amoebae-associated bacterial isolate #15 alone produced a soluble green pigment when grown on TSA plates in air, in either light or dark, and at either room temperature or 35°C (Table 3). Pigment formation did not occur under any experimental conditions in a 100% CO₂ atmosphere.

Table 3 Inhibitory or Non-Inhibitory Conditions for Green or Red Pigment Production by

Bacterial Isolate #15

Only formed in the presence of Acanthamoebae royreba

Red pigment was produced on TSA plates only by combining isolate #15 and A. royreba, preferentially in the CO₂ atmosphere in light or dark and at 35°C (Table 3). Neither red nor green pigments were produced when Pseudomonas chlororaphis was plated under any of the aforementioned test conditions.

UV-visible spectroscopic analysis of the soluble green pigment produced by isolate #15 on TSA showed two major peaks at 694 and 845 nm. Analysis of the red pigment showed two major peaks at 376 and 858 (FIG. 1). The green pigment is composed of a blue pigment and a yellow pigment. The blue pigment and the yellow pigment were isolated from the green pigment by layering the green pigment onto a silicic acid column made from a Pasteur pipette and plugged with glass wool. The column was first eluted with 5 ml of methanol, which eluted the yellow pigment, and then with 5 ml of chloroform, which eluted the blue pigment. Preliminary structural analysis of the blue pigment indicates that it comprises an N-methyl quinoline having the following basic structure:

Bacterial pseudomonad isolate #15, which was originally cultured from environmental amoebae, produces both pigments and crystals. In the presence of organic carbon, green and red diffusible pigments are produced either alone or in combination with A. royreba, respectively. Occasionally, under certain conditions, even the copious biogenic crystals produced by isolate #15 are also green. To the inventors' knowledge, no other bacterium exhibits such properties. Because the green pigment produced by this isolate might have been related to the pigment produced by R. chlororaphis, this bacterium was grown under the same conditions that trigger pigment production by isolate #15. Unlike isolate #15, however, pigment production did not occur with P. chlororaphis. Moreover, the peak absoφtion wavelengths of the green and red pigments analyzed in this study do not correlate with those of common bacterial or plant photosynthetic pigments. EXAMPLE 2 Anti-Reverse Transcriptase Activity of Supernatant From Isolate #15 Cultures

The present Example illustrates the ability of the supernatant from a culture of bacterial isolate #15 to inhibit reverse transcriptase activity.

A. Materials and Methods

Production of Anti-Reverse Transcriptase Activity

A bacterial preparation containing anti-reverse transcriptase activity (anti-RT) was obtained as follows. One well-isolated single colony of Pseudomonas aeruginosa isolate #15 (ATCC No. 55638) was used to inoculate tryptic soy broth. The bacteria were allowed to grow four days stationary phase at 30°C with gyrorotatory shaking at approximately 40 φm. The bacteria were then harvested and stored at -20°C until tested for the presence of a reverse transcriptase inhibitor. Prior to testing, the cultures were sterilized by autoclaving. For each culture to be tested, a 10 ml aliquot was centrifuged for 10 min at 11 x g. The pellets were resuspended in 1.5 ml sterile PBS and centrifuged for 10 min in a sterile Eppendorf tube. The supernatant was then examined for the presence of anti-reverse transcriptase activity.

Genprobe Assay for Inhibition of Reverse Transcriptase Activity The presence of an inhibitor of reverse transcriptase was assayed using the Gen-Probe assay (Gibco/BRL, Grand Island, NY). The Gen-Probe assay employs a reverse transcription reaction to effect first strand synthesis, followed by amplification of the product by polymerase chain reaction (PCR). Reaction products are detected by agarose gel electrophoresis and staining with ethidium bromide. The presence of a band of approximately 600 base pairs indicates reverse transcriptase activity, whereas the absence of the band indicates inhibition of reverse transcriptase activity.

To test the bacterial preparations for the presence of a reverse transcriptase inhibitor, 5 μl aliquots of each preparation, made as described in the preceding section, were used in a 25 μl reverse transcription reaction volume. The preparation was undiluted, or diluted 1 :10 or 1 :100, using distilled water as the diluent. Following the reverse transcription reaction, a 2 μl aliquot of the reaction mixture was used for the PCR reaction, which was conducted by standard methods in a 20 μl reaction volume. The samples were loaded onto a TBE-1% agarose gel, electrophoretically separated, and stained with ethidium bromide (EtBr), according to standard methods. Included as a molecular size standard was Hαelll-digested φX174.

The bacterial preparations tested for inhibition of reverse transcriptase included fresh or aged cultures of Pseudomonas aeruginosa isolate #15, which were either gamma- irradiated (1.5 mR gamma irradiation total; 0.3 mR h^"1 for 5 hours) or unirradiated. Also included was a preparation from a culture of Pseudomonas chlororaphis isolate (ATCC 9446).

RNA Preparation

Total RNA was isolated using a cloned M-MLV RT kit (GIBCO BRL, Rockville, MD), and the manufacturers' instructions provided with the kit. The total RNA (lOμg) was incubated with 3 units of RNase-free DNase in lχ DNase buffer (50 mM Tris-Cl (pΗ 8.3), 75 mM KC1, 3 mM MgCl₂), in a total volume of 203 μl at 37°C for 1 hour. The reaction was terminated by the addition of 10 μl of 0.25 M EDTA, 10 μl of 10% SDS and 2 μl of proteinase K (lOmg/ml; Sigma- Aldrich, St. Louis, MO), and incubating the mixture at 56°C for 30 minutes. The reaction was extracted with phenol/chloroform, and the RNA was precipitated with 1/10 volume of NaOAc (pΗ 5.2) and 2 volumes of 100% ethanol at -20°C overnight. The RNA was then spun down, washed with 75% EtOΗ, dried, and resuspended in 50 μl of DEPC-treated water.

Reverse Transcription Reaction

After the DNase treatment, reverse transcriptase reactions were conducted as outlined above with an equal amount of DNA-free total RNA. The DNA-free total RNA (approximately 1 μg) was incubated at 70°C for 5 minutes, chilled on ice, and then 1 mM of dATP, dGTP, dCTP and dTTP, 5 mM MgCl₂, lx PCR Buffer, 1 unit of RNase inhibitor, 2.5 units of reverse transcriptase and 2.5 μM random hexamers was added. The reaction was incubated at room temperature for 10 minutes, at 42°C for 20 minutes, and then at 99°C for 5 minutes. After chilling the reaction on ice, 1/20 to 1/10 of the reaction was used for PCR™.

PCR ™ Reaction

The samples from the reverse transcriptase reactions above were added to 2 mM MgCl₂, lχ PCR Buffer, 0.2 mM dGTP, dATP, dCTP and dTTP, 0.5 μM of primer I (5'-CACAGGACTAGAACACCTGC-3'; SEQ ID NO:l) and primer II (5'-GCTGGTGAAAAGGACCTCT-3'; SEQ ID NO:2) and 1 unit of Ampli-Taq™ DNA polymerase, and PCR was conducted as follows. The samples were denatured at 95°C for 7 minutes, and then 40 cycles of amplification (denaturation at 94°C for 30 seconds, annealing at 50°C for 30 seconds, and extension at 72°C for 1 minute) were conducted, followed by a 72°C incubation for 10 minutes. The PCR reactions were analyzed by agarose gel electrophoresis.

B. Results

Bacterial preparations made from aged cultures of ATCC No. 55638 were found to inhibit reverse transcriptase. The inhibitory effect appears to be independent of whether the preparation was irradiated, because preparations of both irradiated and unirradiated aged cultures inhibit reverse transcriptase.

At all concentrations tested (undiluted, 10-fold diluted and 100-fold diluted), the irradiated and non-irradiated tryptic soy broth (TSB) from the bacterial isolate #15 culture inhibited reverse transcriptase. Undiluted and 10-fold diluted TSB from bacterial isolate #15 culture grown with an N₂ headspace also inhibited reverse transcriptase. Undiluted TSB from the Pseudomonas chlororaphis isolate also appeared to inhibit reverse transcriptase. The TSB alone did not inhibit reverse transcriptase activity. EXAMPLE 3 Anti-Bacterial Activity of Green and Blue Pigments

The present Example illustrates the anti-microbial effects of the green and blue pigments produced by isolate #15.

A. Materials and Methods

As described above, isolate #15 was grown on trypticase soy agar plates at 35°C in a 5% CO₂ atmosphere for 48 hours. Once green pigment production was established, the agar plates were cut into sections and placed in sterile water in culture flasks for 5 to 7 days at 4°C. The water was then removed and centrifuged at 2000 x g for 30 minutes, and then filter sterilized using a 0.2 μm filter.

Ten ml of the green water soluble pigment was layered onto a silicic acid column made from a Pasteur pipette and plugged with glass wool. The column was first eluted with 5 ml of methanol, which eluted the yellow pigment, and then with 5 ml of chloroform, which eluted the blue pigment. The eluted pigments were concentrated with a stream of nitrogen.

Twenty μl of the filter sterilized soluble green pigment containing material and 10 μl of the blue pigment containing material were then soaked onto sterile, blank antibiotic sensitivity disks and allowed to dry at room temperature. These disks were then used to test microbial inhibition by placing them onto plates spread with the microorganisms of interest and measuring the zones of inhibition of growth (Clinical Microbiology Procedures Handbook, 1992). Briefly, the organisms is log phase were plated on Mueller-Hinton agar plates at a concentration of 0.5 McFarland turbidity, and the pigment soaked disks were positioned on the plates, which were then incubated at 34-35°C for 16-18 hours.

B. Results

The filter sterilized green pigment containing material and the blue pigment containing material were effective in inhibiting the growth of a variety of microorganisms, including species of the Staphylococcus, Aeromonas, Legionella, Bacillus and Micrococcus genus. For example, the zones of inhibition for Staphylococcus aureus was > 20 mm, for Aeromonas hydrophila was > 16 mm, for Legionella pneumophila was > 17 mm, and for Bacillus laterosporus was >17 mm. These zones of inhibition were comparable to those seen with commercially available preparations of gentamycin and ampicillin. However, the material appeared to have no effect on the growth of Streptococcus faecalis, Salmonella typhimurium, Citrobacter freundeii, Klebsiella oxytoca, Listena grayed and Eschenchia coli under the conditions employed.

All of the compositions and/or methods disclosed and claimed herein can be made and executed without undue experimentation in light of the present disclosure. While the compositions and methods of this invention have been described in terms of preferred embodiments, it will be apparent to those of skill in the art that variations may be applied to the compositions and/or methods, and in the steps or in the sequence of steps of the methods described herein, without departing from the concept, spirit and scope of the invention. More specifically, it will be apparent that certain agents that are both chemically and physiologically related may be substituted for the agents described herein while the same or similar results would be achieved. All such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the spirit, scope and concept of the invention as defined by the appended claims.

REFERENCES

The following references, to the extent that they provide exemplary procedural or other details supplementary to those set forth herein, are specifically incoφorated herein by reference.

U.S. Patent No. 5,124,327, issued June 23, 1992. U.S. Patent No. 5,917,033, Issued June 29, 1999.

Ankenbauer et al, "Synthesis and biological activity of pyochelin, a siderophore of

Pseudomonas aeruginosa," J. Bacteriol 170:5344-5351, 1988. ATCC Catalogue of Bacteria and Bacteriophages, 18^th Edition (Gherna, R., Pienta, P. and

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Claims

WHAT IS CLAIMED IS:

1. A composition having the following properties:

produced by Pseudomonas aeruginosa having ATCC accession number 55638 grown in the presence of organic carbon;

comprising a red pigment and a green pigment;

exhibiting absoφtion peaks at 376nm and 858 nm;

having antibacterial activity; and

exhibiting anti-reverse transcriptase activity.

2. The composition of claim 1 wherein the green pigment comprises a yellow pigment and a blue pigment.

3. The composition of claim 2 wherein the green pigment has absoφtion peaks at 694 nm and 845 nm.

4. The composition of claim 3 wherein the green pigment is characterized by antimicrobial activity against Staphylococcus, Aeromonas, Legionella, Bacillus and Micrococcus.

5. The composition of claim 4 wherein the Staphylococcus, Aeromonas, Legionella,

Bacillus and Micrococcus are Staphylococcus aureus, Aeromonas hydrophila, Legionella pneumophila and Bacillus laterosporus

6. The composition of claim 2 wherein the blue pigment inhibits the growth of microorganisms selected from the group consisting of Staphylococcus, Aeromonas, Legionella and Bacillus.

1. The composition of claim 6 wherein the Staphylococcus, Aeromonas, Legionella,

Bacillus and Micrococcus are Staphylococcus aureus, Aeromonas hydrophila, Legionella pneumophila and Bacillus laterosporus

8. The composition of claim 2 wherein the blue pigment comprises an N-methyl quinoline compound.

9. The composition of claim 1 wherein the red pigment has absoφtion peaks at 376 nm and 858 nm.

10. The composition of any of claims 1 -9 disposed in a pharmaceutically acceptable excipient.

11. An antibacterial compound produced by culturing an amoeba-associated bacterial isolate in media including an organic carbon source in a normal atmosphere wherein said compound comprises a green pigment that inhibits microbial growth of bacteria selected from the group consisting of Staphylococcus aureus, Aeromonas hydrophila, Legionella pneumophila and Bacillus laterosporus.

12. The antibacterial compound of claim 11 wherein the green pigment comprises a blue pigment.

13. The antibacterial compound of claim 11 wherein the amoeba-associated bacterial isolate is Pseudomonas aeruginosa having ATCC accession number 55638.

14. A red pigment produced by culturing Pseudomonas aeruginosa isolate # 15 having

ATCC accession number 55638 in the presence of Acanthamoebae royreba in a carbon dioxide atmosphere at a temperature of about 35 C.

15. Use of the composition or compound of any of claims 1-13 to treat a condition requiring inhibition of a viral reverse transcriptase or inhibition of the growth of a microorganism such as Staphylococcus, Aeromonas, Legionella or Bacillus, comprising administering to a mammal an amount of the composition effective to inhibit viral reverse transcriptase or to inhibit microbial growth.

16. The use of claim 15 wherein the microorganism is Staphylococcus aureus,

Aeromonas hydrophila, Legionella pneumophila ox Bacillus laterosporus.

17. A kit comprising in suitable containers any of the compositions or compounds of claims 1-13, optionally a second antimicrobial agent or reverse transcriptase inhibitor, and instructions for use in inhibiting microbial or retroviral infections.