Drug – bio-affecting and body treating compositions – Designated organic active ingredient containing – Heterocyclic carbon compounds containing a hetero ring...
Reexamination Certificate
2001-09-26
2003-06-03
Raymond, Richard L. (Department: 1624)
Drug, bio-affecting and body treating compositions
Designated organic active ingredient containing
Heterocyclic carbon compounds containing a hetero ring...
C514S333000, C514S338000, C514S410000, C540S145000
Reexamination Certificate
active
06573258
ABSTRACT:
BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention relates to antimicrobial agents. More specifically, the present invention relates to a series of novel positively-charged porphyrins having at least two and up to four positive charges situated in peripheral substituents of the tetrapyrrolic macrocycle and at least one hydrophobic tail originating from one of such charged sites or two adjacent charged sites which are active as photodynamic agents in killing both Gram-positive and Gram-negative bacteria.
2. Description of Related Art
The resistance to antibiotics developed by an increasing number of microorganisms is recognized to be a worldwide health problem. Tunger et al.,
Int. J. Antimicrob. Agents,
15:131-135 (2000); Jorgensen & Ferraro,
Clin. Infect. Dis.
30:799-808 (2000). Historically, the discovery and development of new antibiotics has been a slow process plagued with uncertainty, disappointment, and only relatively rare successes. Even after discovery, prospective drug candidates must undergo rigorous testing to assure their safety and efficacy. Because of this, the highly reported advent of drug-resistant microorganisms has caused alarm among medical professionals and the public, with many left wondering whether novel antibiotics can be developed quickly enough to forestall possible problems.
As a result of this, researchers have begun to explore the possibilities of developing nontraditional antibiotic approaches for killing microorganisms. The goals of such development efforts include not only controlling antibiotic-untreatable infections, but in addition, limiting the development of additional antibiotic-resistant microbe strains by selecting a killing mechanism which does not involve the target's genetic material, or which is not otherwise mutagenic. This acts to prevent, at least in part, selection for or creation of strains potentially resistant to the action of the killing agent.
One such method being evaluated is the treatment of microbial infections by photodynamic therapy (“PDT”). This appears to be a valuable alternative method of eradicating bacteria, in part, because it appears to utilize a mechanism that is different from that typical of most antibiotics. Generally, PDT is based on the use of a photosensitizing molecule that, once activated by light, generates reactive oxygen species (“ROS”) that are toxic to a large variety of prokaryotic and eukaryotic cells including bacteria, mycoplasma, and yeasts. Malik et al.,
J. Photochem. Photobiol. B: Biol.,
5:281-293 (1990); Bertolini et al.,
Microbios,
71:33-46 (1992).
One important feature of this approach is that the photosensitizing activity of many photodynamic agents is not impaired by bacterial resistance to antibiotics. Instead, it largely depends on the chemical structure of the photosensitizing agents themselves. Malik et al,
J. Photochem. Photobiol. B: Biol.,
14:262-266 (1992). Various types of known neutral and anionic photosensitizers, for example, exhibit a pronounced phototoxic activity against Gram-positive bacteria while exhibiting no appreciable cytotoxic activity against Gram-negative bacteria unless the permeability of the outer membrane of the Gram-negative bacteria is altered by treatment with EDTA or polycations. Bertolini et al.,
FEMS Microbiol. Lett.,
71:149-156 (1990); Nitzan et al.,
Photochem. Photobiol.,
55:89-97 (1992). Without being limited to any one theory, it appears, in light of current research, that the more complex and thicker cellular envelope of Gram-negative bacteria (as compared to that of Gram-positive bacteria) may prevent the efficient binding of these photosensitizer molecules. In addition, the envelope may simply intercept and deactivate the cytotoxic reactive oxygen species generated by the photosensitizer molecules before fatal damage can be inflicted. Ehrenberg et al.,
Photochem. Photobiol.,
41:429-435 (1985); and Valduga et al.,
Photochem. Photobiol. B: Biol.,
21:81-86 (1993).
In contrast, positively charged photosensitizers, including porphyrins and phthalocyanines, promote the efficient inactivation of Gram-negative bacteria without the need of modifying the natural structure of the cellular envelope. Merchat et al.,
J. Photochem. Photobiol. B: Biol.,
32:158-163 (1996); and Minnock et al.,
J. Photochem. Photobiol. B: Biol.,
32:159-164 (1996). Again, without being limited to any one theory, it appears that the positive charge favors the binding of the photosensitizer molecule at critical cellular sites which, once damaged by exposure to light, cause the loss of cell viability. Merchat et al.,
J. Photochem. Photobiol. B: Biol.,
35:149-157 (1996).
One of the families of positively-charged photosensitizers currently being investigated is based on the porphyrin molecule. Porphyrins are macrocyclic molecular compounds with a ring-shaped tetrapyrrolic core. As such, porphyrins are commonly found in their dianionic form coordinated to a metal ion. The unique properties of the tetrapyrrolic core have made porphyrins central in many biological systems that play a vital role in many life processes. Several compounds which are critically important for essential biological processes, such as chlorophyll and heme, are derived from the coordination of a metal ion with a porphyrin nucleus. H. R. Mahler and E. H. Cordes,
Biological Chemistry,
2d ed. 418, 1966.
Porphyrins are generally derived from the parent tetrapyrrole porphin by replacing hydrogens at one or more of the positions 1 and 8 as well as at one or more of the meso-(pyrrole bridging) carbon atoms with side chains such as, for example, methyls, ethyls, vinyls, propionic acids, or aromatic groups. Id. Porphyrins are often classified based on the side chains they contain. Hydrogenation of one or two pyrrole moieties generates the corresponding chlorophyll, and, respectively, bacteriochlorophyll derivatives.
As briefly noted above, porphyrins are able to form metal chelates with a large variety of metal ions, including: cobalt, copper, iron, magnesium, nickel, silver, and zinc. Id. at 419. Heme is an iron chelate of a porphyrin, while chlorophyll and bacteriochlorophyll are magnesium chelates. Porphyrins such as these are generally synthesized from the precursors glycine and succinyl CoA. See L. Stryer,
Biochemistry,
2d ed. 504-507 (1981).
Present porphyrins and techniques for their use require that, in order to act as antimicrobial agents in the treatment of bacterial and other microbial infections, or even for use in the photosterilization of water, they must be employed in concentrations of at least 10 micromolar. They must then be irradiated for as long as about 30 minutes. See id.
One porphyrin-based cationic photosensitizer shown to be effective in killing both Gram-positive and Gram-negative bacteria, including the ability to efficiently inactivate
Escherichia coli
, is cationic meso-tetra(N-methyl-4-pyridyl)porphine, or “T
4
MPyP”. See Merchat, et al.,
J. Photochem.
&
Photobiol. B: Biol.
32:153-157, (1996); Merchat, et al.,
J. Photochem.
&
Photobiol B: Biol.,
35:149-157, (1996); Okuno, Synthesis, July 1980, 537, and Valduga et al.,
Biochem. Biophys. Res. Comm.,
256:84-88 (1999). Without being limited to any one theory, it appears that the phototoxic activity of this porphyrin is mediated by the impairment of enzymatic and transport functions of both the outer and cytoplasmic membrane. DNA was found not to be a primary target of T
4
MPyP photosensitization.
It has further been well established that the hydro- or lipo-philicity of a photosensitizer strongly affects the binding of the photosensitizer to a target cell, and as a consequence, its cytotoxic activity. Merchat et al.,
J. Photochem. Photobiol. B: Biol.,
35:149-157 (1996).
Currently known porphyrin photosensitizers, current methods of their synthesis, and known techniques for their use are inadequate for many intended applications. This is true in part due to the need for high concentrations of reagent and the requirement of extended irradiation periods. These factors render the m
Bommer Jerry C.
Jori Giulio
Frontier Scientific, Inc.
Madson & Metcalf
Raymond Richard L.
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