Patent Application
20090124539
The present invention relates to methods of inhibiting the proliferation of bacteria in a patient by administering to the patient an antibiotic compound. The invention also presents ex vivo methods of use for the same antibiotic compound such as methods of sanitizing surfaces and/or objects, and methods of assaying Gram positive bacteria.
Bacteria are unicellular microorganisms. They are typically a few micrometers long and have many shapes including spheres, rods, and spirals. Bacteria are ubiquitous in every habitat on Earth, growing in soil, acidic hot springs, radioactive waste [Fredrickson J, Zachara J, Balkwill D, et al (2004). “Geomicrobiology of high-level nuclear waste-contaminated vadose sediments at the hanford site, Washington state”. Appl Environ Microbiol 70 (7): 4230-41], seawater, and deep in the earth's crust. Some bacteria can even survive in the extreme cold and vacuum of outer space. There are typically 40 million bacterial cells in a gram of soil and a million bacterial cells in a milliliter of fresh water; in all, there are approximately five nonillion (5×1030) bacteria in the world. Whitman W, Coleman D, Wiebe W (1998). “Prokaryotes: the unseen majority”. Proc Natl Acad Sci USA 95 (12): 6578-83. Bacteria are vital in recycling nutrients, and many important steps in nutrient cycles depend on bacteria, such as the fixation of nitrogen from the atmosphere. However, most of these bacteria have not been characterized, and only about half of the phyla of bacteria have species that can be cultured in the laboratory. Rappé M, Giovannoni S. “The uncultured microbial majority”. Annu Rev Microbiol 57: 369-94.
Although the vast majority of these bacteria are rendered harmless or beneficial by the protective effects of the mammalian immune system, a few pathogenic bacteria cause infectious diseases, including cholera, syphilis, anthrax, leprosy and bubonic plague. The most common fatal bacterial diseases are respiratory infections, with tuberculosis alone killing about 2 million people a year, mostly in sub-Saharan Africa. See http://www.who.int/healthinfo/bodgbd2002revised/en/index.html.
Although there are numerous antibiotics that are effective in treating patients suffering from bacterial infections, several recent generations of disease causing bacteria possess multiple drug resistance and have become serious clinical problems.
The number of patients treated for antibiotics-resistant infections has increased drastically in recent years. What started in the 1980s as problem primarily associated with hospital-acquired Enterococcus infections in long-term care patients has become a problem that has moved into the general community and has grown to include a number of common and very serious human pathogens. Drug-resistant Streptococci, Staphylococci and Pseudomonas strains are quite common. In fact, currently as many as 70% of hospital-acquired infections in the US are resistant to at least one antibiotic, and about 40% of S. aureus infections are multidrug-resistant. Coates, A., Hu, Y., Bax, R., and Page, C. (2002) “The Future Challenges Facing the Development of New Antimicrobial Drugs. Nat. Rev. Drug Discov. 1:895-910.
Even very powerful drugs like vancomycin and teicoplanin, which for years represented the “agents of last resort” for treatment of antibiotics-resistant infections, are no longer efficacious against certain strains of bacteria (see e.g., Smith, T. L., and Jarvis, W. R. (1999) Antimicrobial resistance in Staphylococcus aureus. Microb. Infect. 1:795-805; Ge, M., Chen, Z., Onishi, H. R., Kohler, J., Silver, L. L., Kerns, R., Fuzukawa, S., Thompson, C., and Kahne, D. (1999) Vancomycin derivatives that inhibit peptidoglycan biosynthesis without binding D-Ala-D-Ala. Science 284:507-511; and Goldman, R. C., and Gange, D. (2000) Inhibition of transglycosylation involved in bacterial peptidoglycan synthesis. Curr. Med. Chem. 7:801-820). Hence, these compounds are predicted to be of little use for the treatment of future infections. In this context, it is important to realize that the loss of efficacy of vancomycin and related compounds leaves very few treatment options for patients with multi-drug resistant infections. The seriousness of the situation is clearly illustrated by the fact that as many as 90,000, of the two million people who acquired a bacterial infection in US hospitals in 2004, died as a result of it (Leeb, M. (2004) A shot in the arm. Nature 431:892-893). There is clearly an immediate need for new antibiotics with novel modes of action. Thus, there is a strong demand for a compound having excellent antibacterial activity against antibiotic resistant strains of disease causing bacteria.
The present invention provides methods of inhibiting bacterial proliferation including providing a pharmaceutical composition comprising Empedopeptin or a pharmaceutically acceptable salt thereof, wherein the bacteria comprises at least one Gram positive strain.
In several embodiments, the Gram positive strain is resistant to glycopeptides, aminoglycosides, oxazolidinones, penicillins, macrolides, rifamycins, polypeptides, lipopeptides, chloramphenicol, or any combination thereof. For example, the Gram positive strain further comprises Enterococcus faecalis, Staphylococcus aureus, Staphylococcus epidermidis, Streptococcus pneumoniae, Streptococcus pyogenes, or any combination thereof.
In other embodiments, the Gram positive strain is resistant to at least one of linezolid, oxacillin, vancomycin, daptomycin, erythromycin, methicillin, gentamicin, chloramphenicol, fusidic acid, rifampin, or combinations thereof. For instance, the Gram positive strain is resistant to methicillin.
In several embodiments, the Gram positive strain consists essentially of Enterococcus faecalis, the Gram positive strain consists essentially of Staphylococcus aureus, the Gram positive strain consists essentially of Staphylococcus epidermidis, the Gram positive strain consists essentially of Streptococcus pneumoniae, or the Gram positive strain consists essentially of Streptococcus pyogenes.
In some embodiments, the method further includes providing a second antibiotic agent. For instance, some methods further include providing a second pharmaceutical composition, wherein the second pharmaceutical composition comprises a second antibiotic agent, or providing a single pharmaceutical composition comprising Empedopeptin and a second antibiotic agent.
Another aspect of the present invention provides methods of treating a patient infected with bacteria including providing a pharmaceutical composition comprising Empedopeptin or a pharmaceutically acceptable salt thereof, wherein the bacteria comprises at least one Gram positive strain.
In several embodiments, the Gram positive strain is resistant to one or more of glycopeptides, oxazolidinones, penicillins, macrolides, rifamycins, polypeptides, lipopeptides, chloramphenicol, or combinations thereof. For example, the Gram positive strain further comprises Enterococcus faecalis, Staphylococcus aureus, Staphylococcus epidermidis, Streptococcus pneumoniae, Streptococcus pyogenes, or any combination thereof. For example, the Gram positive strain is resistant to linezolid, oxacillin, vancomycin, daptomycin, methicillin, gentamicin, erythromycin, chloramphenicol, fusidic acid, rifampin, or any combination thereof. In other examples, the Gram positive strain is resistant to methicillin.
In several embodiments, the Gram positive strain consists essentially of Enterococcus faecalis, the Gram positive strain consists essentially of Staphylococcus aureus, the Gram positive strain consists essentially of Staphylococcus epidermidis, the Gram positive strain consists essentially of Streptococcus pneumoniae, or the Gram positive strain consists essentially of Streptococcus pyogenes.
In some embodiments, the method further includes providing a second antibiotic agent. For instance, some methods further include providing a second pharmaceutical composition, wherein the second pharmaceutical composition comprises a second antibiotic agent, or providing a single pharmaceutical composition comprising Empedopeptin and a second antibiotic agent.
Another aspect of the present invention provides methods of treating a patient infected with Staphylococcus aureus or Staphylococcus epidermidis, either of which is resistant to glycopeptides, oxazolidinones, penicillins, macrolides, rifamycins, polypeptides, lipopeptides, chloramphenicol, or any combination thereof, comprising administering to the patient an effective amount of a pharmaceutical composition comprising Empedopeptin or a pharmaceutically acceptable salt thereof.
In several embodiments, the pharmaceutical composition is administered to the patient parenterally or intravenously. In other embodiments, the pharmaceutical composition is intravenously administered to the patient, or the pharmaceutical composition is topically administered to the patient.
Another aspect of the present invention provides methods of sanitizing a surface or object comprising contacting the surface or object with a cleaning composition comprising Empedopeptin and a carrier.
In several embodiments, the carrier comprises water or alcohol.
In other embodiments, the surface is skin, or the object is an agricultural product, a medical instrument, a kitchen utensil, or an article of clothing.
In some embodiments, the cleaning composition further comprises a second antibiotic agent, e.g., one that does not substantially affect the antibiotic activity of Empedobactin.
Another aspect of the present invention provides methods of assaying bacteria for Empedopeptin resistance comprising colonizing bacteria in a medium; and incubating the medium, wherein the medium comprises Empedopeptin.
Another aspect of the present invention provides an isolated nucleotide sequence comprising SEQ. ID. NO. 1.
Another aspect of the present invention provides an isolated protein sequence comprising SEQ. ID. NO. 2.
Another aspect of the present invention provides an isolated nucleotide sequence comprising SEQ. ID. NO. 3.
Another aspect of the present invention provides an isolated protein sequence comprising SEQ. ID. NO. 4.
Another aspect of the present invention provides an isolated nucleotide sequence comprising SEQ. ID. NO. 5.
Another aspect of the present invention provides an isolated protein sequence comprising SEQ. ID. NO. 6.
Another aspect of the present invention provides an isolated nucleotide sequence comprising SEQ. ID. NO. 7
Another aspect of the present invention provides an isolated protein sequence comprising SEQ. ID. NO. 8.
The present invention provides methods of restricting bacterial proliferation by providing a pharmaceutical composition comprising Empedopeptin, wherein the bacteria comprises at least one Gram positive strain that is resistant to one or more of aminoglycosides, carbacephems, carbapenems, cephalosporins (e.g., first generation, second generation, third generation, or fourth generation), glycopeptides, lipopeptides, macrolides, monobactams, penicillins, polypeptides, quinolones, sulfonamides, tetracyclines, oxazolidinones, rifamycins, other unclassified antibiotics (e.g., chloramphenicol), or combinations thereof. This method is useful for ex vivo or in vivo purposes.
As used herein, “Empedopeptin”, refers to a cyclic peptide having the structure:
As used herein, “antibiotic” or “antibiotic agent” refers to a compound, such as penicillin, streptomycin, methicillin, vancomycin, erythromycin, daptomycin, and/or bacitracin produced by or derived from certain fungi, bacteria, and other organisms, or are synthetically produced, that can destroy or inhibit the growth of other microorganisms. Antibiotics are widely used in the prevention and treatment of infectious diseases such as bacterial infection. Common antibiotics are discussed below.
As used herein, “antibiotic resistant” or “antibiotic resistance” refers to a characteristic of some bacteria, wherein at least some portion of a population of bacteria can survive and proliferate despite being treated with large amounts of antibiotic. For example, antibiotic resistance is used to mean that the bacteria does not lyse or is not otherwise destroyed by the antibiotic. Antibiotic resistance can also mean that the bacteria actively grows and proliferates in the presence of the antibiotic. In several examples, antibiotic resistant bacteria are those that when treated with one or more antibiotics yield a minimal inhibitory concentration from between about 2-fold to more than about 100-fold higher (e.g., from about 3 fold to about more than 100 fold, from about 4 fold to about more than 100 fold, or the like) than that observed for bacteria sensitive to the one or more antibiotic(s), or bacteria having intermediate resistance to the one or more antibiotic(s).
As used herein, “alcohol” refers to an organic compound in any physical state (e.g., solid, gas, or liquid) that includes a carbon atom that is bonded to a hydroxy (—OH) functional group. Without limitation, exemplary alcohols include methanol, ethanol, propanol, isopropanol, or the like.
As used herein, “bacteria” means ubiquitous one-celled organisms, spherical, spiral, or rod-shaped and appearing singly or in chains, comprising the Schizomycota, a phylum of the kingdom Monera (in some classification systems the plant class Schizomycetes), various species of which are involved in fermentation, putrefaction, infectious diseases, or nitrogen fixation.
As used herein, “bacterial proliferation” means growth or reproduction of bacteria.
As used herein, “an effective amount” is defined as the amount required to confer a therapeutic effect on the treated patient, and is typically determined based on age, surface area, weight, and condition of the patient. The interrelationship of dosages for animals and humans (based on milliGrams per meter squared of body surface) is described by Freireich et al., Cancer Chemother. Rep., 50: 219 (1966). Body surface area may be approximately determined from height and weight of the patient. See, e.g., Scientific Tables, Geigy Pharmaceuticals, Ardsley, New York, 537 (1970).
As used herein, “agricultural product” means fruits, vegetables, nuts, flowers, honey, and animal products such as beef, pork, chicken, fish, lamb, or the like.
As used herein, “medical instrument” means instruments associated with medical uses such as a scalpels, hemostats, saws, retractors, forceps, surgical needles, catheters, drills, bandages, rib spreaders, tongue depressors, and any other instrument that is commonly inserted into a living organism.
As used herein, “kitchen utensils” means instruments commonly used in food preparation such as knives, forks, spoons, tongs, spatulas, any other instruments that are commonly used in food preparation.
As used herein, “Gram positive” refers to bacteria that retain a crystal violet color during the Gram stain process. Gram positive bacteria will appear blue or violet under a microscope.
As used herein, “Gram negative” refers to bacteria that retain a red or pink color during the Gram stain process. Gram negative bacteria will appear red or pink under a microscope. The difference in classification between Gram positive and Gram negative bacteria is largely based on a difference in the bacteria's cell wall structure.
As used herein, “patient” refers to a mammal, including a human.
Unless otherwise stated, structures depicted herein are also meant to include all isomeric (e.g., enantiomeric, diastereomeric, and geometric (or conformational)) forms of the structure; for example, the R and S configurations for each asymmetric center, (Z) and (E) double bond isomers, and (Z) and (E) conformational isomers. Therefore, single stereochemical isomers as well as enantiomeric, diastereomeric, and geometric (or conformational) mixtures of the present compounds are within the scope of the invention.
Unless otherwise stated, all tautomeric forms of the compounds of the invention are within the scope of the invention. Additionally, unless otherwise stated, structures depicted herein are also meant to include compounds that differ only in the presence of one or more isotopically enriched atoms. For example, compounds having the present structures except for the replacement of hydrogen by deuterium or tritium, or the replacement of a carbon by a 13C- or 14C-enriched carbon are within the scope of this invention. Such compounds are useful, for example, as analytical tools or probes in biological assays, or as therapeutic agents.
Abbreviations used herein have the following meanings:
L-Arg: L-Arginine
D-Ser: D-Serine
L-Pro: L-Proline
D-Pro: D-Proline
L-Ala: L-Alanine
L-Thr: L-Threonine
D-aThr: D-allo-Threonine
L-hyPro: L-trans-3-hydroxyproline
D-hyAsp: D-threo-β-hydroxyaspartic acid
L-hyAsp: L-threo-β-hydroxyaspartic acid
The present invention provides methods of inhibiting bacterial proliferation comprising providing a pharmaceutical composition comprising Empedopeptin or a pharmaceutically acceptable salt thereof, wherein the bacteria comprises at least one Gram positive strain, and the Gram positive strain is resistant to one or more of glycopeptides, lipopeptides, aminoglycosides, oxazolidinones, penicillins, macrolides, rifamycins, polypeptides, other unclassified antibiotics (e.g., chloramphenicol), or combinations thereof. In several methods, the Gram positive strain further comprises Enterococcus faecalis, Staphylococcus aureus, Staphylococcus epidermidis, Streptococcus pneumoniae, Streptococcus pyogenes, or any combination thereof.
For example, in one group of methods, the Gram positive strain comprises Enterococcus faecalis, Staphylococcus aureus, Staphylococcus epidermidis, Streptococcus pneumoniae, Streptococcus pyogenes, or any combination thereof; and the Gram positive strain is further resistant to one or more glycopeptides including amikacin, gentamicin, kanamycin, neomycin, netilmicin, paromomycin, streptomycin, tobramycin, vancomcin, teicoplanin, and apramycin. In other methods, the Gram positive strain comprises Enterococcus faecalis, Staphylococcus aureus, Staphylococcus epidermidis, Streptococcus pneumoniae, Streptococcus pyogenes, or any combination thereof; and the Gram positive strain is further resistant to one or more penicillins including methicillin, dicloxacillin, flucloxacillin, oxacillin, nafcillin, amoxicillin, ampicillin, azlocillin, carbenicillin, cloxacillin, mezlocillin, penicillin, piperacillin, ticarcillin, or any combination thereof. In another method, the Gram positive strain comprises Enterococcus faecalis, Staphylococcus aureus, Staphylococcus epidermidis, Streptococcus pneumoniae, Streptococcus pyogenes, or any combination thereof; and the Gram positive strain is further resistant to one or more aminoglycosides including amikacin, gentamicin, kanamycin, neomycin, netilmicin, paromomycin, streptomycin, tobramycin, apramycin, or combinations thereof. In another method, the Gram positive strain comprises Enterococcus faecalis, Staphylococcus aureus, Staphylococcus epidermidis, Streptococcus pneumoniae, Streptococcus pyogenes, or combinations thereof; and the Gram positive strain is further resistant to one or more macrolides including erythromycin, azithromycin, troleandomycin, clarithromycin, dirithromycin, roxithromycin, or any combination thereof. In another method, the Gram positive strain comprises Enterococcus faecalis, Staphylococcus aureus, Staphylococcus epidermidis, Streptococcus pneumoniae, Streptococcus pyogenes, or any combination thereof; and the Gram positive strain is further resistant to one or more rifamycins including rifampin, rifabutin, rifapentine, or any combination thereof. In another method, the Gram positive strain comprises Enterococcus faecalis, Staphylococcus aureus, Staphylococcus epidermidis, Streptococcus pneumoniae, Streptococcus pyogenes, or any combination thereof; and the Gram positive strain is further resistant to one or more polypeptides or lipopeptides including daptomycin, bacitracin, colistin, polymyxin B, or any combination thereof. In other methods, the Gram positive strain comprises Enterococcus faecalis, Staphylococcus aureus, Staphylococcus epidermidis, Streptococcus pneumoniae, Streptococcus pyogenes, or any combination thereof; and the Gram positive strain is further resistant to one or more of linezolid, oxacillin, vancomycin, daptomycin, methicillin, gentamicin, chloramphenicol, fusidic acid, rifampin, or any combination thereof.
In several alternative methods, the Gram positive strain consists essentially of Enterococcus faecalis that is resistant to one or more of linezolid, oxacillin, vancomycin, daptomycin, methicillin, gentamicin, chloramphenicol, fusidic acid, rifampin, or combinations thereof. In other methods, the Gram positive strain consists essentially of Staphylococcus aureus that is resistant to one or more of linezolid, oxacillin, vancomycin, daptomycin, methicillin, gentamicin, chloramphenicol, fusidic acid, rifampin, or combinations thereof. In several methods, the Gram positive strain consists essentially of Staphylococcus epidermidis that is resistant to one or more of linezolid, oxacillin, vancomycin, daptomycin, methicillin, gentamicin, chloramphenicol, fusidic acid, rifampin, or combinations thereof. In other methods, the Gram positive strain consists essentially of Streptococcus pneumoniae that is resistant to one or more of linezolid, oxacillin, vancomycin, daptomycin, methicillin, gentamicin, chloramphenicol, fusidic acid, rifampin, or combinations thereof. In other methods, the Gram positive strain consists essentially of Streptococcus pyogenes that is resistant to one or more of linezolid, oxacillin, vancomycin, daptomycin, methicillin, gentamicin, chloramphenicol, fusidic acid, rifampin, or combinations thereof.
The methods of inhibiting bacterial proliferation are also useful for treating a patient infected with bacteria, wherein the bacteria is a Gram positive strain that is resistant to glycopeptides, lipopeptides, aminoglycosides, oxazolidinones, penicillins, macrolides, rifamycins, polypeptides, or other unclassified antibiotics (e.g., chloramphenicol), or any combination thereof.
Such methods comprise providing a pharmaceutical composition comprising Empedopeptin or a pharmaceutically acceptable salt thereof to treat an infection of Gram positive bacteria that are resistant to glycopeptides, lipopeptides, aminoglycosides, oxazolidinones, penicillins, macrolides, rifamycins, polypeptides, unclassified antibiotics (e.g., chloramphenicol), or combinations thereof.
In several methods, a patient infected with bacteria is treated with a pharmaceutical composition comprising Empedopeptin or a pharmaceutically acceptable salt thereof, wherein the bacteria comprises at least one Gram positive strain, and the Gram positive strain is resistant to one or more glycopeptides, lipopeptides, aminoglycosides, oxazolidinones, penicillins, macrolides, rifamycins, polypeptides, unclassified antibiotics (e.g., chloramphenicol), or combinations thereof. In other methods, patient is infected with Enterococcus faecalis that is resistant to glycopeptides, aminoglycosides, oxazolidinones, lipopeptides, penicillins, macrolides, rifamycins, polypeptides, unclassified antibiotics (e.g., chloramphenicol), or combinations thereof. In several methods, the patient is infected with Staphylococcus aureus that is resistant to one or more glycopeptides, aminoglycosides, lipopeptides, oxazolidinones, penicillins, macrolides, rifamycins, polypeptides, unclassified antibiotics (e.g., chloramphenicol), or combinations thereof. In several methods, the patient is infected with Staphylococcus epidermidis that is resistant to one or more glycopeptides, lipopeptides, aminoglycosides, oxazolidinones, penicillins, macrolides, rifamycins, polypeptides, unclassified antibiotics (e.g., chloramphenicol), or combinations thereof. In other methods, the patient is infected with Streptococcus pneumoniae that is resistant to one or more glycopeptides, lipopeptides, aminoglycosides, oxazolidinones, penicillins, macrolides, rifamycins, polypeptides, or unclassified antibiotics (e.g., chloramphenicol), or combinations thereof. In some methods, the patient is infected with Streptococcus pyogenes that is resistant to one or more of linezolid, oxacillin, vancomycin, daptomycin, methicillin, gentamicin, chloramphenicol, fusidic acid, rifampin, or combinations thereof.
Other methods provide for treating a patient infected with bacteria comprising providing Empedopeptin, or a pharmaceutically acceptable salt thereof, wherein the bacteria comprises Enterococcus faecalis, Staphylococcus aureus, Staphylococcus epidermidis, Streptococcus pneumoniae, Streptococcus pyogenes, or combinations thereof. More specifically, the bacteria comprises methicillin resistant Staphylococcus aureus, methicillin resistant Streptococcus pneumoniae, methicillin resistant Streptococcus pyogenes, or combinations thereof. In several methods, the population of bacteria is resistant to linezolid, oxacillin, vancomycin, daptomycin, methicillin, gentamicin, chloramphenicol, fusidic acid, rifampin, or combinations thereof. In other embodiments, the population of bacteria consists essentially of Enterococcus faecalis. In still more embodiments, the population of bacteria consists essentially of Staphylococcus aureus. Alternatively, the population of bacteria consists essentially of Staphylococcus epidermidis. Or, the population of bacteria consists essentially of Streptococcus pneumoniae. In some embodiments, the population of bacteria consists essentially of Streptococcus pyogenes.
Other embodiments of the present invention provide methods of treating a patient infected with Staphylococcus aureus or Staphylococcus epidermidis, either of which is resistant to linezolid, oxacillin, vancomycin, daptomycin, methicillin, gentamicin, chloramphenicol, fusidic acid, rifampin, or combinations thereof, comprising administering to the patient an effective amount of Empedopeptin or a pharmaceutically acceptable salt thereof.
Still more embodiments provide methods of sanitizing a surface or object comprising contacting the surface or object with a cleaning composition comprising Empedopeptin and an effective carrier. Several cleaning compositions of the present invention include a carrier comprising water, alcohol, or mixtures thereof. In other examples, the solvent comprises ethanol, methanol, isopropanol, water, or combinations thereof. This method is well-suited for sanitizing surfaces such as skin, countertops, tabletops, and other surfaces that can host infectious bacteria. Moreover, this method is well-suited for sanitizing objects such as surgical instruments (e.g., scalpel, oral thermometer, retractor, saw blades, forceps, hemostat, scissors, or the like), kitchen utensils, or the like.
In several embodiments, the pharmaceutical composition useful for treating infection or restricting the proliferation of bacteria can optionally include a second antibiotic agent. For instance the pharmaceutical composition can comprise Empedopeptin and one or more antibiotic agents independently selected from glycopeptides, lipopeptides, aminoglycosides, oxazolidinones, penicillins, macrolides, rifamycins, polypeptides, or unclassified antibiotics (e.g., chloramphenicol).
Another aspect of the present invention provides methods of assaying bacteria for Empedopeptin resistance comprising colonizing bacteria in a medium comprising Empedopetin, and incubating the bacteria. Any bacteria can be assayed using this method.
Antibiotics are often classified by the scope of their respective bioactivities. An antibiotic's scope of bioactivity is qualitatively assessed as being narrow spectrum, moderate spectrum, or broad spectrum.
Narrow spectrum antibiotics have activity in only a few strains of bacteria or small family of bacteria, while antibiotics having activities in multiple strains or families of bacteria are classified as moderate spectrum antibiotics, and those antibiotics having activities in a large number of strains or families of bacteria (e.g., Gram negative bacteria and/or Gram positive bacteria) are classifies as broad spectrum antibiotics.
Antibiotics can also be classified by the organisms against which they are effective, and by the type of infection in which they are useful, which depends on the sensitivities of the organisms that most commonly cause the infection and the concentration of antibiotic obtainable in the affected tissue.
At the most generic level, antibiotics can be classified as either bactericidal or bacteriostatic. Bactericidals kill bacteria directly where bacteriostatics prevent them from dividing. However, these classifications are based on laboratory behavior; in practice, both of these can end a bacterial infection.
Common commercial antibiotics include aminoglycosides, carbacephems, carbapenems, cephalosporins (e.g., first generation, second generation, third generation, or fourth generation), glycopeptides, lipopeptides, macrolides, monobactams, penicillins, polypeptides, quinolones, sulfonamides, tetracyclines, oxazolidinones, rifamycins, and unclassified antibiotics (e.g., chloramphenicol). Each class of antibiotic is briefly discussed below.
Penicillins include those antibiotic drugs obtained from penicillium molds or produced synthetically, which are most active against Gram-positive bacteria and used in the treatment of various infections and diseases. Penicillin is one of the beta-lactam antibiotics, all of which possess a four-ring beta-lactam structure fused with a five-membered thiazolidine ring. These antibiotics are nontoxic and kill sensitive bacteria during their growth stage by the inhibition of biosynthesis of their cell wall mucopeptide. Penicillin antibiotics provide narrow spectrum bioactivity, moderate or intermediate spectrum bioactivity, and broad spectrum bioactivity. Without limitation, narrow spectrum penicillins include methicillin, dicloxacillin, flucloxacillin, oxacillin, nafcillin, or the like. Without limitation, moderate or intermediate spectrum penicillins include amoxicillin, ampicillin, or the like. Penicillins include, without limitation, ampicillin, azlocillin, carbenicillin, cloxacillin, dicloxacillin, flucloxacillin, mezlocillin, nafcillin, penicillin, piperacillin, and ticarcillin.
Aminoglycosides are a group of antibiotics that are effective against certain types of bacteria. They include amikacin, gentamicin, kanamycin, neomycin, netilmicin, paromomycin, streptomycin, tobramycin and apramycin. Those which are derived from Streptomyces genus are named with the suffix -mycin, while those which are derived from micromonospora are named with the suffix -micin. Aminoglycosides are useful primarily in infections involving aerobic, Gram-negative bacteria, such as Pseudomonas, Acinetobacter, and Enterobacter. In addition, some mycobacteria, including the bacteria that cause tuberculosis, are susceptible to aminoglycosides. The most frequent use of aminoglycosides is empiric therapy for serious infections such as septicemia, complicated intraabdominal infections, complicated urinary tract infections, and nosocomial respiratory tract infections. Usually, once cultures of the causal organism are grown and their susceptibilities tested, aminoglycosides are discontinued in favor of less toxic antibiotics.
Carbacephem is a class of antibiotic medication, specifically modified forms of cephalosporin. It prevents bacterial cell division by inhibiting cell wall synthesis. Without limitation, carbacephems include loracarbef, or the like.
Carbapenems are a class of beta-lactam antibiotics, the structure of which renders them highly resistant to beta-lactamases. Carbapenems include, without limitation, imipenem (often given as part of imipenem/cilastatin), meropenem, ertapenem, faropenem, doripenem, panipenem/betamipron, or the like.
Cephalosporins are a class of beta-lactam antibiotics. Together with cephamycins they belong to a sub-group called cephems. First-generation cephalosporins are predominantly active against Gram positive bacteria. First generation cephalosporins are moderate spectrum agents, with a spectrum of activity that includes penicillinase-producing, methicillin-susceptible staphylococci and streptococci, though they are not the drugs of choice for such infections. They also have activity against some Escherichia coli, Klebsiella pneumoniae and Proteus mirabilis, but have no activity against Bacteroides fragilis, enterococci, methicillin-resistant staphylococci, Pseudomonas, Acinetobacter, Enterobacter, indole-positive Proteus or Serratia. First generation cephalosporins include, without limitation, cefadroxil, cefazolin, and cephalexin.
The second generation cephalosporins have a greater Gram negative spectrum while retaining some activity against Gram positive cocci. They are also more resistant to beta-lactamase. Second generation cephalosporins include, for example, cefonicid, cefprozil, cefproxil, cefuroxime, cefuzonam, cefaclor, cefamandole, ceforanide, and cefotiam.
Third generation cephalosporins have a broad spectrum of activity and further increased activity against Gram negative organisms. Some members of this group (particularly those available in an oral formulation, and those with anti-pseudomonal activity) have decreased activity against Gram positive organisms. They may be particularly useful in treating hospital-acquired infections, although increasing levels of extended-spectrum beta-lactamases are reducing the clinical utility of this class of antibiotics. Without limitation, third generation cephalosporins include cefcapene, cefdaloxime, cefdinir, cefditoren, cefetamet, cefixime, cefmenoxime, cefodizime, cefoperazone, cefotaxime, cefpimizole, cefpodoxime, cefteram, ceftibuten, ceftiofur, ceftiolene, ceftizoxime, and ceftriaxone. Third generation cephalosporins with antipseudomonal activity include ceftazidime, cefpiramide, and cefsulodin.
Oxacephems are also sometimes grouped with third-generation cephalosporins and include latamoxef and flomoxef.
Fourth generation cephalosporins are extended-spectrum agents with similar activity against Gram positive organisms as first-generation cephalosporins. They also have a greater resistance to beta-lactamases than the third generation cephalosporins. Many can cross blood brain barrier and are effective in meningitis. Exemplary fourth generation cephalosporins include cefclidine, cefepime, cefluprenam, cefoselis, cefozopran, cefpirome, and cefquinome.
These cephems have progressed far enough to be named, but have not been assigned to a particular generation: ceftobiprole, cefaclomezine, cefaloram, cefaparole, cefcanel, cefedrolor, cefempidone, cefetrizole, cefivitril, cefmatilen, cefmepidium, cefovecin, cefoxazole, cefrotil, cefsumide, ceftioxide, ceftobiprole, ceftobiprole, and cefuracetime.
Glycopeptide antibiotics are another class of antibiotic drugs. They consist of a glycosylated cyclic or polycyclic nonribosomal peptide. Exemplary glycopeptide antibiotics include vancomycin, teicoplanin, ramoplanin, and decaplanin.
Macrolides are a group of drugs (typically antibiotics) whose activity stems from the presence of a macrolide ring, a large lactone ring to which one or more deoxy sugars, usually cladinose and desosamine, are attached. The lactone ring can be either 14-, 15- or 16-membered. Macrolides belong to the polyketide class of natural products. Common antibiotic macrolides include erythromycin, azithromycin, troleandomycin, clarithromycin, dirithromycin, and roxithromycin.
Monobactams are beta-lactam antibiotics wherein the beta-lactam ring is alone, and not fused to another ring (in contrast to most other beta-lactams, which have at least two rings). An example is aztreonam.
Polypeptide antibiotics include bacitracin, colistin, and polymyxin B.
Quinolones are another family of broad spectrum antibiotics. The parent of the group is nalidixic acid. The majority of quinolones in clinical use belong to the subset of fluoroquinolones, which have a fluoro group attached the central ring system. Exemplary quinolone antibiotics include cinoxacin, flumequine, nalidixic acid, oxolinic acid, piromidic acid, pipemidic acid, ciprofloxacin, enoxacin, fleroxacin, lomefloxacin, nadifloxacin, norfloxacin, ofloxacin, pefloxacin, rufloxacin, balofloxacin, grepafloxacin, levofloxacin, pazufloxacin mesilate, sparfloxacin, temafloxacin, tosufloxacin, clinafloxacin, gemifloxacin, moxifloxacin, gatifloxacin, sitafloxacin, and trovafloxacin.
Antibacterial sulfonamides (sometimes called simply sulfa drugs) are synthetic antimicrobial agents that contain the sulfonamide group. In bacteria, antibacterial sulfonamides act as competitive inhibitors of the enzyme dihydropteroate synthetase, DHPS. Several antibacterial sulfonamides include mafenide prontosil, sulfacetamide, sulfamethizole, sulfanilamide, sulfasalazine, sulfisoxazole, trimethoprim, and trimethoprim-sulfamethoxazole.
Tetracyclines are a group of broad-spectrum antibiotics named for their four (“tetra-”) hydrocarbon rings (“-cycl-”) derivation (“-ine”). Exemplary tetracyclines include tetracycline, chlortetracycline, oxytetracycline, demeclocycline, doxycycline, lymecycline, meclocycline, methacycline, minocycline, rolitetracycline, and tigecycline.
Oxazolidinones are a class of compounds containing 2-oxazolidone in their structures. Oxazolidinones are useful antibiotics. Some of the most important oxazolidinones are the last generation of antibiotics used against Gram positive bacterial strains. One example of an oxazolidinone is linezolid.
Rifamycins are a group antibiotics that are synthesized either naturally by the bacterium Amycolatopsis mediterranei, or artificially. Rifamycins are particularly effective against mycobacteria, and are therefore used to treat tuberculosis, leprosy, and mycobacterium avium complex (MAC) infections. The rifamycin antibiotic group includes, without limitation, rifampin, rifL.
Lipopeptide antibiotics includes peptides with attached lipids or a mixture of lipids and peptides such as the cyclic lipopeptide, daptomycin.
Other unclassified antibiotics include chloramphenicol, clindamycin, ethambutol, fosfomycin, furazolidone, isoniazid, metronidazole, mupirocin, nitrofurantoin, platensimycin, pyrazinamide, quinupristin/dalfopristin, spectinomycin, and telithromycin.
Pharmaceutical compositions comprising the abovementioned antibiotics can comprise a combination of antibiotics.
Furthermore, the abovementioned antibiotics can be administered via any suitable method (e.g., orally, topically, intravenously, ip injection, muscular injection (IM), or by any combination thereof). These antibiotics can further be administered concurrently, i.e., at approximately the same time, or sequentially, i.e., at different times.
Recent generations of bacteria have developed resistance to one or more of the abovementioned antibiotic agents. Such bacteria include Enterococcus faecalis, Staphylococcus aureus, Staphylococcus epidermidis, Streptococcus pneumoniae, Streptococcus pyogenes, each of which can cause illness in mammals; especially humans.
Cyclic peptides are composed of several biosynthetic units, typically amino acids, linked in sequence to form a closed ring structure. The producing organisms contain large enzyme complexes referred to as non-ribosomal peptide synthetase (NRPS) complexes, which are responsible for the synthesis of these molecules. NRPS complexes have an assembly line-like organization comprising a number of biosynthetic modules, each of which is responsible for the addition of one, specific amino acid (biosynthetic unit) to the sequence of the cyclic peptide.
Because each biosynthetic module in the NRPS complex is specific for a certain amino acid, the sequential arrangement of the modules in the complex does, in itself, determine the sequence and structure of the cyclic peptide produced. From this follows that if the sequence, or order, of the modules is changed, the amino acid sequence of the peptide will also change. That is, if a biosynthetic module specific for a particular amino acid is substituted for a module specific for another amino acid, the net effect will be a different amino acid, at that position, in the peptide produced by the modified NRPS complex. Moreover, since the arrangement of modules in an NRPS complex is a direct reflection of the arrangement of the module-encoding gene sequences in the corresponding NRPS gene, deletion, insertion and/or substitution of biosynthetic modules in an NRPS complex can be accomplished by deletion, insertion and/or substitution of the relevant sequence segments in the corresponding NRPS gene. Consequently, genetic engineering (of the relevant cyclic peptide-producing organism) can now be used to generate molecules with features that previously could only be introduced using the complicated and expensive synthetic chemistry methods discussed above.
Nonetheless, utilization of the genetic engineering approach outlined above, for introduction of modifications to the structure of Empedopeptin, requires knowledge of the sequence and structure of the NRPS gene encoding the Empedopeptin synthetase. This gene has to date not been identified or cloned. Consequently, with the aim of cloning this gene, a set of degenerate PCR primers, targeted at the coding regions of the highly conserved core adenylation domain sequence motifs A3 (AUG1470: GGWTCYACWGGWACWCCWTTRCC; forward) and A8 (AUG1473: CCWARYTCWATACGRAAWCCACG; reverse; with R=A or G; W=A or T; Y═C or T), were prepared. The design of the primers was optimized with regard to the codon usage of the Empedopeptin-producer organism Empedobacter spp. ATCC 31962. A PCR amplification was subsequently carried out using these primers, standard reaction conditions and the Expand High-Fidelity PCR system (Roche), according to the manufacturer's protocol. The reaction yielded an 806 bp DNA fragment, which was cloned and subjected to sequence analysis. This revealed that the fragment encodes a portion of an NRPS adenylation domain. The amplified fragment shares highest amino acid sequence homology (55% identity, 66% similarity) with the proline-activating adenylation domain of module 2 in the syringopeptin synthetase from Pseudomonas syringae pv. syringae. Determination and analysis of the presumed substrate-binding constituents, in the fragment sequence, revealed that the adenylation domain amplified from Empedobacter spp. likely recognizes and activates proline. Together these observations suggest that the cloned PCR fragment represents a fragment of the Empedopeptin synthetase NRPS gene.
The sequence of the putative Empedopeptin synthase fragment is SEQ ID NO 1, and is provided in the sequence listing below.
The corresponding protein sequence is SEQ. ID. NO. 2, and is also provided in the sequence listing below.
The first step in the cloning of the remaining portion(s) the Empedopeptin synthetase NRPS gene (epp) cluster involved construction of an Empedobacter haloabium fosmid library. This was done using the CopyControl Cloning System (Epicentre) which combines the clone stability afforded by single copy cloning with the advantages of high yields of DNA obtained by on-demand induction of clones to a high copy number (usually 10-200 copies per cell). First, high-molecular-weight E. haloabium genomic DNA (>80 kb) was prepared, using standard procedures. The genomic DNA was then sheared to approximately 40 kb fragments which, subsequently, were end-repaired to generate the appropriate blunt and 5′-phosphorylated ends. The end-repaired DNA was then size-fractioned on a low-melting-point agarose gel, using field-inversion gel electrophoresis (FIGE). DNA fragments of the appropriate size (approx. 40 kb) were excised, extracted from the gel, and, subsequently, ligated into the CopyControl pCC1FOS cloning vector. Following packaging of the ligated DNA into Lambda phage particles, the packaging reaction mix was used for transfection of Escherichia coli EPI300-T1, to determine the library's titer. And, once the titer was determined the library was plated and screened.
Individual clones derived from plating of the fosmid library were screened by PCR, using primers designed to amplify the NRPS gene fragment, previously amplified from E. haloabium genomic DNA (see above). E. haloabium belongs to the family of Flavobacteriaceae (e.g. Flavobacterium johnsoniae, Flavobacterium pschrophilum, and Flavobacterium sp. MED217), which has an average genome size of approximately 4.4 Mb. Consequently, about 500 clones were screened to ensure a 99% probability of finding at least one clone that contained the (entire) sequence information of the (putative) empedopeptin biosynthetic gene cluster (predicted size: approx. 30 kb).
Twelve 48-well-microtiter plates were prepared by adding 0.8 ml of Luria-Broth (LB) medium, supplemented with 12.5 μg/ml chloramphenicol, and inoculating the medium in each well with a single clone from the plated fosmid library (see above). Following overnight incubation at 30° C./250 rpm, 20 μl of each culture was used as inoculum for the copy number amplification procedure outlined below. The remainder of the cultures were supplemented with 0.4 ml glycerol and stored, as a master plate, at −80° C. The aliquots induced for copy number amplification produced the (high) yields of fosmid DNA required for PCR analysis and fingerprinting. Fresh 48-well-microtiter plates were prepared by adding 0.8 ml LB medium, supplemented with 12.5 μg/ml chloramphenicol and 0.1% arabinose, and inoculating the medium in each well with 20 μl of the pre-culture prepared earlier. The cultures were incubated overnight at 30° C./250 rpm. To reduce the time and effort involved in the screening of the fosmid clones, small aliquots of the individual cultures were combined into defined pools (of 24 clones each), and the (fosmid) DNA present in each pool was isolated using standard procedures. The pooled fosmid DNAs was used as template in PCR amplifications with primers designed to amplify the NRPS gene fragment isolated previously by degenerate primer PCR (see above). Genomic E. haloabium DNA and/or the previously cloned putative empedopeptin NRPS gene fragment was used as positive controls for these experiments. Fosmid DNA from the individual clones in the clone pools that produced an amplicon of the expected size (in the first round of PCR) were subsequently prepared and analyzed individually in the same manner. This second round of PCR identified two individual fosmid clone(s) that, upon sequencing, were found to both contain the entire NRPS portion of the (putative) empedopeptin biosynthetic gene cluster.
An illustration of the gene cluster sequence identified in two fosmid clones prepared from E. haloabium genomic DNA is provided as
In
The isolated nucleotide and protein sequences of the three NRPS genes comprising the Empedopeptin biosynthetic gene cluster are also provided as follows:
Following assembly of the sequence derived from the two clones, a comparative analysis with published NRPS gene clusters was carried out to determine the module and domain organization of the deduced (putative) Empedopeptin biosynthetic NRPS complex, and any associated gene sequences. Associated sequences could encode enzymes involved in “tailoring” reactions, such as hydroxylation of the proline and aspartic acid residues in the peptide, or in the regulation of expression or export of the peptide.
The observed module and domain organization of the identified gene is illustrated in
As illustrated in
Also as illustrated in
In
The present invention includes within its scope pharmaceutically acceptable prodrugs of the compounds of the present invention. A “pharmaceutically acceptable prodrug” means any pharmaceutically acceptable salt, ester, salt of an ester, or other derivative of a compound of the present invention which, upon administration to a recipient, is capable of providing (directly or indirectly) a compound of this invention or an active metabolite or residue thereof. Preferred prodrugs are those that increase the bioavailability of the compounds of this invention when such compounds are administered to a mammal or which enhance delivery of the parent compound to a biological compartment relative to the parent species.
The term “pharmaceutically acceptable carrier, adjuvant, or vehicle” refers to a non-toxic carrier, adjuvant, or vehicle that does not destroy the pharmacological activity of the Empedopeptin with which it is formulated. Pharmaceutically acceptable carriers, adjuvants or vehicles that may be used in the compositions of this invention include, but are not limited to, ion exchangers, alumina, aluminum stearate, lecithin, serum proteins, such as human serum albumin, buffer substances such as phosphates, glycine, sorbic acid, potassium sorbate, partial glyceride mixtures of saturated vegetable fatty acids, water, salts or electrolytes, such as protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zinc salts, colloidal silica, magnesium trisilicate, polyvinyl pyrrolidone, cellulose-based substances, polyethylene glycol, sodium carboxymethylcellulose, polyacrylates, waxes, polyethylene-polyoxypropylene-block polymers, polyethylene glycol and wool fat.
Pharmaceutically acceptable salts of the compounds of this invention include those derived from pharmaceutically acceptable inorganic and organic acids and bases. Examples of suitable acid salts include acetate, adipate, alginate, aspartate, benzoate, benzenesulfonate, bisulfate, butyrate, citrate, camphorate, camphorsulfonate, cyclopentanepropionate, digluconate, dodecylsulfate, ethanesulfonate, formate, fumarate, glucoheptanoate, glycerophosphate, glycolate, hemisulfate, heptanoate, hexanoate, hydrochloride, hydrobromide, hydroiodide, 2-hydroxyethanesulfonate, lactate, maleate, malonate, methanesulfonate, 2-naphthalenesulfonate, nicotinate, nitrate, oxalate, palmoate, pectinate, persulfate, 3-phenylpropionate, phosphate, picrate, pivalate, propionate, salicylate, succinate, sulfate, tartrate, thiocyanate, tosylate and undecanoate. Other acids, such as oxalic, while not in themselves pharmaceutically acceptable, may be employed in the preparation of salts useful as intermediates in obtaining the compounds of the invention and their pharmaceutically acceptable acid addition salts.
Salts derived from appropriate bases include alkali metal (e.g., sodium and potassium), alkaline earth metal (e.g., magnesium), ammonium and N+(C1-4 alkyl)4 salts. This invention also envisions the quaternization of any basic nitrogen-containing groups of the compounds disclosed herein. Water or oil-soluble or dispersible products may be obtained by such quaternization.
The compositions of the present invention may be administered orally, parenterally, by inhalation spray, topically, rectally, nasally, buccally, vaginally or via an implanted reservoir. The term “parenteral” as used herein includes subcutaneous, intravenous, intramuscular, intra-articular, intra-synovial, intrasternal, intrathecal, intrahepatic, intralesional and intracranial injection or infusion techniques. Preferably, the compositions are administered orally, intraperitoneally or intravenously. Sterile injectable forms of the compositions of this invention may be aqueous or oleaginous suspension. These suspensions may be formulated according to techniques known in the art using suitable dispersing or wetting agents and suspending agents. The sterile injectable preparation may also be a sterile injectable solution or suspension in a non-toxic parenterally-acceptable diluent or solvent, for example as a solution in 1,3-butanediol. Among the acceptable vehicles and solvents that may be employed are water, Ringer's solution and isotonic sodium chloride solution. In addition, sterile, fixed oils are conventionally employed as a solvent or suspending medium.
For this purpose, any bland fixed oil may be employed including synthetic mono- or di-glycerides. Fatty acids, such as oleic acid and its glyceride derivatives are useful in the preparation of injectables, as are natural pharmaceutically-acceptable oils, such as olive oil or castor oil, especially in their polyoxyethylated versions. These oil solutions or suspensions may also contain a long-chain alcohol diluent or dispersant, such as carboxymethyl cellulose or similar dispersing agents that are commonly used in the formulation of pharmaceutically acceptable dosage forms including emulsions and suspensions. Other commonly used surfactants, such as Tweens, Spans and other emulsifying agents or bioavailability enhancers which are commonly used in the manufacture of pharmaceutically acceptable solid, liquid, or other dosage forms may also be used for the purposes of formulation.
The pharmaceutically acceptable compositions of this invention may be orally administered in any orally acceptable dosage form including, but not limited to, capsules, tablets, aqueous suspensions or solutions. In the case of tablets for oral use, carriers commonly used include lactose and corn starch. Lubricating agents, such as magnesium stearate, are also typically added. For oral administration in a capsule form, useful diluents include lactose and dried cornstarch. When aqueous suspensions are required for oral use, the active ingredient is combined with emulsifying and suspending agents. If desired, certain sweetening, flavoring or coloring agents may also be added.
Alternatively, the pharmaceutically acceptable compositions of this invention may be administered in the form of suppositories for rectal administration. These can be prepared by mixing the agent with a suitable non-irritating excipient that is solid at room temperature but liquid at rectal temperature and therefore will melt in the rectum to release the drug. Such materials include cocoa butter, beeswax and polyethylene glycols.
The pharmaceutically acceptable compositions of this invention may also be administered topically, especially when the target of treatment includes areas or organs readily accessible by topical application, including diseases of the eye, the skin, or the lower intestinal tract. Suitable topical formulations are readily prepared for each of these areas or organs.
Topical application for the lower intestinal tract can be effected in a rectal suppository formulation (see above) or in a suitable enema formulation. Topically-transdermal patches may also be used.
For topical applications, the pharmaceutically acceptable compositions may be formulated in a suitable ointment containing the active component suspended or dissolved in one or more carriers. Carriers for topical administration of the compounds of this invention include, but are not limited to, mineral oil, liquid petrolatum, white petrolatum, propylene glycol, polyoxyethylene, polyoxypropylene compound, emulsifying wax and water. Alternatively, the pharmaceutically acceptable compositions can be formulated in a suitable lotion or cream containing the active components suspended or dissolved in one or more pharmaceutically acceptable carriers. Suitable carriers include, but are not limited to, mineral oil, sorbitan monostearate, polysorbate 60, cetyl esters wax, cetearyl alcohol, 2-octyldodecanol, benzyl alcohol and water.
For ophthalmic use, the pharmaceutically acceptable compositions may be formulated as micronized suspensions in isotonic, pH adjusted sterile saline, or, preferably, as solutions in isotonic, pH adjusted sterile saline, either with or without a preservative such as benzylalkonium chloride. Alternatively, for ophthalmic uses, the pharmaceutically acceptable compositions may be formulated in an ointment such as petrolatum.
The pharmaceutically acceptable compositions of this invention may also be administered by nasal aerosol or inhalation. Such compositions are prepared according to techniques well-known in the art of pharmaceutical formulation and may be prepared as solutions in saline, employing benzyl alcohol or other suitable preservatives, absorption promoters to enhance bioavailability, fluorocarbons, and/or other conventional solubilizing or dispersing agents.
Most preferably, the pharmaceutically acceptable compositions of this invention are formulated for parenteral administration or specifically intramuscular injection.
The amount of the compounds of the present invention that may be combined with the carrier materials to produce a composition in a single dosage form will vary depending upon the host treated, the particular mode of administration. Preferably, the compositions should be formulated so that a dosage of between 0.01-100 mg/kg body weight/day of the modulator can be administered to a patient receiving these compositions.
It should also be understood that a specific dosage and treatment regimen for any particular patient will depend upon a variety of factors, including the activity of the specific compound employed, the age, body weight, general health, sex, diet, time of administration, rate of excretion, drug combination, and the judgment of the treating physician and the severity of the particular disease being treated. The amount of a compound of the present invention in the composition will also depend upon the particular compound in the composition.
Depending upon the particular condition, or disease, to be treated or prevented, additional therapeutic agents, which are normally administered to treat or prevent that condition, may also be present in the compositions of this invention. As used herein, additional therapeutic agents that are normally administered to treat or prevent a particular disease, or condition, are known as “appropriate for the disease, or condition, being treated.”
In several pharmaceutical compositions comprising Empedopeptin, the carrier is water or saline.
Compounds:
The investigational agent, Empedopeptin, was purified from the culture broth of Empedobacter haloabium strain No. G393-B445 (ATCC 31962) as provided in Konishi, M., Sugawara, K., Hanada, M., Tomita, K., Tomatsu, K., Miyaki, T., and Kawaguchi, H. (1984) Empedopeptin (BMY-28117), a new depsipeptideantibiotic. 1. Production, isolation and properties. J. Antibiot. 37:949-957. The Empedopeptin was stored at −20° C. until the day of the MIC assay. Daptomycin (Lot# CDCX01) was obtained from Cubist, linezolid (Lot# LZD05003) from Pfizer, vancomycin (Lot# 016K1102) from Sigma-Aldrich, and oxacillin (Lot# 1101952) from BioChemika.
The solvent for all of the compounds was deionized water (DIW), and all of the compounds dissolved in the solvent. The stock solutions were allowed to stand in DIW for one hour at room temperature prior to testing to allow time for auto-sterilization. The stock concentration of the test compounds was 5120 μg/mL, resulting in the final test concentration range of 128-0.12 μg/mL.
The test organisms were originally received from clinical sources, or from the American Type Culture Collection. When received, the organisms were sub-cultured onto an appropriate agar medium. Following incubation, colonies were harvested from these plates and cell suspensions prepared and frozen at −80° C. On the day prior to assay, a frozen vial of each culture was thawed and the contents were streaked for isolation onto either Tryptic Soy Agar (Becton Dickinson, Sparks, Md.) or Tryptic Soy Agar (Enhanced Hemolysis; Becton Dickinson) supplemented with 5% sheep blood for streptococci. The agar plates were incubated overnight at 35° C. Staphylococcus aureus ATCC 29213 and Enterococcus faecalis ATCC 29212 were included as quality control isolates in the assay.
Test Medium:
The test medium for the broth microdilution testing was Mueller Hinton II broth (MHB II; BBL# 212322, Lot # 6024003, Becton Dickinson). The broth was prepared at 1.05× normal weight/volume to offset the 5% volume of the drug solution in the final test plates.
For streptococci, lysed horse blood (Lot # H88621; Cleveland Scientific, Bath, Ohio) was added to the MHB II at a final concentration of 2%.
CLSI guidelines recommend that Mueller-Hinton II broth be adjusted to contain 50 mg/L of Ca++ ions for proper daptomycin MIC results. Since Mueller-Hinton II broth has already been adjusted by the manufacturer to contain approximately 25 mg/L of Ca++ ions, an additional 25 mg/L of Ca++ ions was adjusted with 10 mg/mL of CaCl2.2H2O (Lot# 084K0215; Sigma-Aldrich) added at a rate of 0.1 mL/L of broth, for each desired increment of 1 mg/L. This supplemented Mueller-Hinton II broth was used only in wells containing daptomycin.
MIC Methodology:
MIC values were determined using a broth microdilution method as recommended by the Clinical and Laboratory Standards Institute (Clinical and Laboratory Standards Institutea. Methods for Dilution Antimicrobial Susceptibility Tests for Bacteria That Grow Aerobically; Approved Standard-Seventh Edition. Clinical and Laboratory Standards Institute document M7-A7 [ISBN 1-56238-587-9]. Clinical and Laboratory Standards Institute, 940 West Valley Road, Suite 1400, Wayne, Pa. 19087-1898 USA, 2006). Automated liquid handlers (Multidrop 384, Labsystems, Helsinki, Finland; Biomek 2000 and Multimek 96, Beckman Coulter, Fullerton Calif.) were used to conduct serial dilutions and make liquid transfers.
Wells of two standard 96-well microdilution plates (Falcon 3918; Becton Dickinson) were filled with 150 μL of DMSO using the Multidrop 384. These plates were used to prepare the drug “mother plates” that provided the serial drug dilutions for replicate “daughter plates”. The Biomek 2000 was used to transfer 150 μl of each stock solution from the wells of column 1 of a deep well plate to the corresponding wells in column 1 of the mother plate and to make eleven 2-fold serial dilutions in the mother plates. The wells of column 12 contained no drug and were the organism growth control wells. Each mother plate has the capacity to create a total of 12 daughter plates.
The daughter plates were loaded with 180 μL of one of the media described above using the Multidrop 384. The wells of the daughter plates ultimately contained 180 μL of MHB II, 10 μL of drug solution, and 10 μL of bacterial inoculum prepared in broth appropriate to the test organism (1.05×). The daughter plates were prepared on the Multimek 96 instrument, which transferred 10 μL of drug solution from each well of the mother plate to each corresponding well of each daughter plate in a single step.
Standardized inoculum of each organism was prepared following Clinical and Laboratory Standards Institute (Clinical and Laboratory Standards Instituteb. Performance Standards for Antimicrobial Susceptibility Testing; Sixteenth Informational Supplement. CLSI document M100-S16 [ISBN 1-56238-588-7]. Clinical and Laboratory Standards Institute, 940 West Valley Road, Suite 1400, Wayne, Pa. 19087-1898 USA, 2006) methods. The inoculum for each organism was dispensed into sterile reservoirs divided by length (Beckman Coulter), and the Biomek 2000 was used to inoculate the plates. Daughter plates were placed on the Biomek 2000 work surface in a reversed position so that inoculation occurred from low to high drug concentration. The Biomek 2000 delivered 10 μL of standardized inoculum into each well. This yielded a final cell concentration in the daughter plates of approximately 5×105 colony-forming-units/mL.
Plates were stacked 3 high, covered with a lid on the top plate, placed in plastic bags, and incubated at 35° C. for approximately 20 h. Following incubation, the microplates were removed from the incubator and viewed from the bottom using a plate viewer. An un-inoculated solubility control plate was observed for evidence of drug precipitation. The MIC was read and recorded as the lowest concentration of drug that inhibited visible growth of the organism.
Results:
All of the compounds were soluble in the stock solutions and in the microbiological test media (data not shown). Table 1 details the test organisms and phenotypes and the MIC data for the test agents.
Enterococcus
faecalis
Enterococcus
4LZDR
faecalis
Enterococcus
faecalis
Enterococcus
faecalis
Enterococcus
faecium
Enterococcus
6DAPR
faecium
Enterococcus
faecium
Enterococcus
faecium
Staphylococcus
aureus
Staphylococcus
aureus
Staphylococcus
aureus
9FAR
Staphylococcus
aureus
Staphylococcus
aureus
Staphylococcus
aureus
Staphylococcus
11GMR
aureus
Staphylococcus
aureus
Staphylococcus
12CHLR
aureus
Staphylococcus
13RAR
aureus
Staphylococcus
epidermidis
Staphylococcus
epidermidis
Streptococcus
pneumoniae
Streptococcus
pneumoniae
Streptococcus
pneumoniae
Streptococcus
pneumoniae
Streptococcus
16mef(A)
pneumoniae
Streptococcus
17erm(B)
pneumoniae
Streptococcus
pyogenes
Streptococcus
pyogenes
1VSE—vancomycin-sensitive Enterococcus
2CLSI—Clinical and Laboratory Standards Institute
3QC—Quality Control
4LZD—linezolid
5Van—vancomycin
6DAP—daptomycin
7MSSA—methicillin-sensitive Staphylococcus aureus
8MRSA—methicillin-resistant Staphylococcus aureus
9FA—fusidic acid
10VISA—vancomycin-intermediate Staphylococcus aureus
11GM—gentamicin
12CHL—chloramphenicol
13RA—rifampin
14MSSE—methicillin-sensitive Staphylococcus epidermidis
15MRSE—methicillin-resistant Staphylococcus epidermidis
16mefA—macrolide resistance via efflux
17ermB—ribosomal erythromycin resistance
The quality control strain MIC data (Table 2) demonstrated that daptomycin, oxacillin, and vancomycin had MIC results within the CLSI quality control ranges for each, thereby validating the assay results for these agents. However, linezolid demonstrated MIC values one dilution higher than the specified CLSI range for both quality control organisms, therefore, the data for linezolid are not acceptable. Overall, linezolid yielded MIC values higher than typically seen for these organisms, consistent with the out-of-range quality control values. The linezolid data are included in Table 1; however, the values should be viewed with caution.
Staphylococcus
aureus
Enterococcus
faecalis
a Clinical and Laboratory Standards Institute (2)
b
Staphylococcus aureus ATCC 29213
c
Enterococcus faecalis ATCC 29212
The phenotypic characteristics were confirmed for all strains where the subject drug was included in the assay (for example, vancomycin-resistance evident for VRE, etc.). Empedopeptin demonstrated broad activity against Gram-positive bacteria, including strains resistant to other antibacterial agents. Against Enterococci, the range of MIC values was 4-32 μg/mL with most strains inhibited at 8-16 μg/mL. The most sensitive Enterococcal strain was E. faecalis 101 (MIC≈4 μg/mL) and the least sensitive was the daptomycin-resistant strain E. faecium 1721. Empedopeptin demonstrated activity against Van A and Van B Enterococci, as well as the linezolid-resistant strain.
Against staphylococci, Empedopeptin demonstrated MIC values in the range of 0.5-8 μg/mL, with the majority of strains inhibited in the range of 4-8 μg/mL. This included isolates resistant to oxacillin, linezolid, fusidic acid, gentamicin, chloramphenicol, and rifampin as well as intermediate-resistance to vancomycin.
Empedopeptin demonstrated greater potency against Streptococci than Enterococci or Staphylococci, inhibiting all strains of S. pneumoniae in the range of ≦0.12-2 μg/mL. This included strains carrying common quinolone resistance mutations, ermB (ribosomal erythromycin resistance), and mefA (macrolide resistance via efflux). Interestingly, the mefA strain was highly susceptible to Empedopeptin. Empedopeptin was also highly active against S. pyogenes inhibiting both test strains at ≦0.12 μg/mL (including the macrolide-resistant strain).
From these results, Empedopeptin has demonstrated activity against several Gram-positive bacteria; and, more importantly, Empedopeptin also demonstrated broad activity against several different antibiotic-resistant strains of bacteria.
It is to be understood that while the invention has been described in conjunction with the detailed description thereof, the foregoing description is intended to illustrate and not limit the scope of the invention, which is defined by the scope of the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims.
This application is a continuation-in-part of PCT patent application serial no PCT/US2008/002435, filed on Feb. 25, 2008, under 35 U.S.C. § 363, which claims priority to U.S. provisional application Ser. No. 60/903,487, filed Feb. 26, 2007; each of which is hereby incorporated by reference entirely.
| Number | Date | Country | |
|---|---|---|---|
| 60903487 | Feb 2007 | US |
| Number | Date | Country | |
|---|---|---|---|
| Parent | PCT/US2008/002435 | Feb 2008 | US |
| Child | 12284954 | US |
