Drug – bio-affecting and body treating compositions – Designated organic active ingredient containing – Peptide containing doai
Reexamination Certificate
1999-03-23
2003-05-13
Low, Christopher S. F. (Department: 1654)
Drug, bio-affecting and body treating compositions
Designated organic active ingredient containing
Peptide containing doai
C530S317000, C530S320000, C435S032000, C435S810000
Reexamination Certificate
active
06562785
ABSTRACT:
BACKGROUND OF THE INVENTION
Bacterial, fungal, and plant cells have a cell wall outside the cell membrane, while animal cells in general, and human cells in particular, do not. Compounds which interfere with synthesis of the cell wall, such as antibiotics in the penicillin and cephalosporin families, can kill bacteria without significant toxicity to an infected host or patient. It is known that rupture of the bacterial cell membrane occurs when bacteria are killed by antibiotics such as penicillin that are administered at least minimum inhibitory concentration levels; this membrane damage allows normally impermeant compounds such as propidium iodide to enter the bacteria.
There has been an alarming increase in recent years in the number of infections due to bacteria resistant to nearly all, and in some cases all available, antibiotics. Such bacteria are, by definition, not killed by the drugs. While much emphasis has been placed on the discovery of new antibiotics, it would be useful to develop methods and reagents to potentiate already existing antibiotics. In addition, such potentiating mechanisms would also be useful in combination with agents that injure bacteria but are not considered traditional antibiotic compounds. Furthermore, the treatment of other pathogens, such as fungi, is often deleterious to the host or patient due to toxicity of the therapy to the host. The potentiation of such therapy, allowing the use of lower doses of the therapeutic agent is also desirable.
SUMMARY OF THE INVENTION
The present invention relates to the discovery by the Applicant that bacteria treated with a concentration of antibiotic less than that needed to kill 100% of the organisms in culture take up propidium iodide or TO-PRO-3™ (an asymmetrical cyanine dye), although their membranes appear intact, i.e., unruptured, by virtue of their maintenance of a normal membrane potential. The subsequent growth pattern of cultures exposed to this sublethal dose of antibiotic indicates that the induced permeability was transient and reversible and that the sublethally injured bacteria eventually resumed growth. The coexistence of propidium iodide permeability and an unruptured membrane in antibiotic treated cells is a novel discovery, and raises the possibility of treating bacteria with sublethal doses of antibiotic to induce permeability to toxic agents which are impermeant, and thus harmless, to host cells.
The toxicity of most organic compounds to cells is dependent upon the compounds entering the cells; the facility with which compounds enter the cells can, in many cases, be manipulated. Both bacterial and mammalian cells are susceptible to the toxicity of the nucleic acid binding dye ethidium bromide, which shares the heterocyclic ring structure of propidium iodide but carries only a single positive charge, bearing an N-ethyl group instead of the N-propyltrimethyl (quaternary) ammonium group found in propidium iodide. Both ethidium and propidium form complexes with DNA and RNA, and are toxic to cells once taken up. However, ethidium normally enters cells, albeit slowly, while propidium is normally excluded by its additional charge.
There are many chemical modifications capable of modifying permeant organic molecules or agents to render them impermeant in addition to those described by Yue et al (U.S. Pat. No. 5,321,130 (1994) and International Application (WO96/13552)), the teachings of which are incorporated herein in their entirety. It is thus possible to prepare many derivatives of generally toxic organic molecules or agents, such as nucleic acid binding compounds, inhibitors of nucleic acid synthesis, inhibitors of protein synthesis, and inhibitors of energy metabolism, which will exhibit markedly reduced toxicity to intact mammalian and other eukaryotic cells and bacteria, but which will enter and kill bacteria or other cells such as fungi, which have been transiently permeabilized. The combination of a permeabilizing compound, such as a beta-lactam antibiotic, and a modified organic toxic agent with reduced permeability provides a novel and general therapeutic approach to treating infections by microorganisms, including those otherwise resistant to antibiotic treatment.
The phenomenon of transient membrane permeability induced by sublethal injury may be quite general. Some bacteria become dormant in the absence of nutrients and can survive for months at low or no membrane potential. Organisms in this condition have also been reported to take up dyes such as propidium and TO-PRO-3™ (Davey, H. M., Weichart, D. H., Kell D. B., Kaprelyants, A. S.:
Current Protocols in Cytometry,
Wiley-Liss, New York, 1999, 11.3.1-11.3.20.). It is probable that many antibiotics of other membrane permeabilizing compounds can induce the state of membrane permeability even in resistant organisms. Furthermore, in such dormant bacteria, it is likely that treatment with membrane impermeant toxic agents alone will result in their death.
Transient permeabilization of cell membranes with retention of viability may be induced by chemical agents such as lysolecithin or by physical agents such as electric fields (electroporation); this may be done in vitro, as for the purpose of introducing gene constructs into the cells, but is difficult or impossible to accomplish in vivo. Thus, until the present invention, there has been no incentive to develop impermeant derivatives of generally toxic agents, such as toxic nucleic acid binding compounds, inhibitors of nucleic acid synthesis, inhibitors of protein synthesis, inhibitors of energy metabolism, etc., as therapeutic agents against bacteria including gram negative bacteria such as
Pseudomonas aeruginosa,
and gram positive bacteria, such as
S. aureus
or
M. luteus,
or other pathogens, such as fungi.
The present invention provides a method of killing bacteria or other pathogens by contacting the bacteria with a membrane permeabilizing compound or combination of compounds in conjunction with an otherwise membrane impermeant toxic agent or agents. The present invention potentiates the killing effect of compounds that permeabilize bacteria or other pathogens such as fungi, including known antibiotics; this potentiation of the killing effect of antibiotics can occur even in antibiotic resistant bacteria. The present invention also provides a method to detect the presence of permeability in bacteria with intact cell membranes as evidenced by maintenance of membrane potential, allowing the detailed study of antibiotic-induced changes in bacterial physiology. The ability to differentiate among the effects of different antibiotics could lead to improvement in the treatment regimen for bacterial infection, including infection by antibiotic resistant organisms.
The present invention relates to a method of rendering pathogens such as bacteria and fungi permeable with sublethal doses of permeabilizing compounds, and thus lethally susceptible to (i.e., susceptible to killing by) a toxic agent or agents normally impermeant to, and thus inactive against, bacterial and eukaryotic cells with intact membranes.
The present invention is also drawn to a method of rendering antibiotic resistant bacteria lethally susceptible to said antibiotic or antibiotics, comprising contacting the bacteria with said antibiotic in combination with a lethal dose of an agent or agents, wherein the agent is impermeant to bacterial and eukaryotic cells with intact membranes in the absence of the antibiotic, resulting in the death of the bacteria. The present invention is further drawn to a method of preventing survival or generation of antibiotic resistant bacteria. This method comprises contacting the bacteria with a combination of said antibiotic or antibiotics at a concentration sufficient to permeabilize said bacteria and a lethal amount of an agent or agents, wherein the agent is impermeant to bacterial and eukaryotic cells with intact membranes in the absence of the antibiotic, resulting in the death of the bacteria. The present invention also relates to a method of killing bacteria comprising contacti
Hamilton Brook Smith & Reynolds P.C.
Low Christopher S. F.
Mohamed Abdel A.
Shapiro Howard M.
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