Drug – bio-affecting and body treating compositions – Enzyme or coenzyme containing – Multienzyme complexes or mixtures of enzymes
Reexamination Certificate
2000-08-10
2004-07-06
Witz, Jean C. (Department: 1651)
Drug, bio-affecting and body treating compositions
Enzyme or coenzyme containing
Multienzyme complexes or mixtures of enzymes
C424S094600, C435S264000
Reexamination Certificate
active
06759040
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates to a method for preparing biofilm degrading, multiple specificity, hydrolytic enzyme mixtures which are specifically tailored to remove targeted biofilms. The present invention also is directed to methods for using hydrolytic enzyme mixtures in both industrial and therapeutic applications. The industrial applications include but are not limited to the use of biofilm-degrading, multiple specificity, hydrolytic enzyme mixtures for removing or preventing the formation of biofilms in water cooling towers, industrial process piping, heat exchangers, in food processing or food preparation, in potable water systems, reservoirs, swimming pools, or related sanitary water systems, and on membranes such as those used for desalinization, industrial processes, or related applications.
The therapeutic applications include but are not limited to the use of therapeutically-useful, multiple-specificity, hydrolytic enzyme mixtures or components thereof for the prevention or treatment of dental caries and periodontal disease, improving the treatment of cystic fibrosis or the complications or symptoms of cystic fibrosis, and diseases or complications associated with biofilm formation on implantable medical devices such as cardiovascular devices. The route of administration can be by any means including delivering the hydrolytic enzyme mixture by aerosol to the lungs and applying the hydrolytic enzyme mixture topically.
BACKGROUND OF THE INVENTION
Naturally occurring biofilms are continuously produced and often accumulate on numerous industrial surfaces and on biological surfaces. In an industrial setting, the presence of these biofilms causes a decrease in the efficiency of industrial machinery, requires increased maintenance, and presents potential health hazards. For example, the surfaces of water cooling towers become increasingly coated with microbially produced biofilm slime which both constricts water flow and reduces heat exchange capacity. Water cooling tower biofilms may also harbor pathogenic microorganisms such as
Legionella pneumophila
. Food preparation lines are routinely plagued by biofilm build-up both on the machinery and on the food product where biofilms often include potential pathogens. Industrial biofilms are complex assemblages of insoluble polysaccharide-rich biopolymers which are produced and elaborated by surface dwelling microorganisms. The chemical composition of industrial biofilms are diverse and are specific to each species of surface dwelling microorganism. Because of this complexity and diversity, non-specific hydrolytic enzymes are ineffective in degrading these biofilms and consequently ineffective in reducing or eliminating the undesirable biofilm.
On a biological surface, the presence of these biofilms results in the growth of, and subsequent colonization by, pathogenic microorganisms on an internal or external surface of a host animal or on the surface of objects introduced into the animal (e.g surgical implants). Animal pathogens which colonize surfaces are often maintained and protected by unique polysaccharide rich biofilms produced by the pathogen. Such biofilms coat the infected or colonized surface of the animal or implanted object and continue to be produced during the disease process. For many diseases, biofilms are required for the disease process to become established and to progress. The chemical compositions of pathogen-associated surface biofilms, which consist of complex mixtures of biopolymers, are specific to each species of pathogen. Because of this complexity, non-specific hydrolytic enzymes or hydrolytic enzymes with a single specificity are ineffective in degrading these biofilms and consequently ineffective in reducing or eliminating the disease condition. At the present time, there are no therapeutic products which are commercially employed to degrade and remove these disease related, pathogen-produced biofilms.
Currently, biofilms are most commonly removed using physical abrasion a process which is both inefficient and incomplete. Antimicrobials (biocides and antibiotics) are employed to slow biofilm build-up by killing the microbes that produce biofilms; however, once established, the biofilms protect the embedded, biofilm-producing bacteria from the action of these agents. Furthermore, many antimicrobial agents are toxic and damaging to the environment. Consequently, there is a need for a method to readily remove and control biofilms that does not depend solely on physical abrasion or on the action of antimicrobial agents. This need could be met by a mixture of multiple specificity, hydrolytic enzymes which have been tailored to degrade the specific complex biopolymer composition of a target biofilm. A tailored mixture of multiple hydrolytic enzymes could be employed to degrade biofilms resulting in their more complete removal and in enhanced antimicrobial activity.
It has recently become apparent that insoluble complex polysaccharides (ICP) in the environment are most efficiently degraded by a cascade of enzymes acting in concert. The degradation of these insoluble complex polysaccharides require more than “simple” exoenzymes. Normally, an array of enzymes, part of a complex system, is required to fully hydrolyze the polysaccharide into its final monosaccharide end product (Belas et al., 1988; Bassler et al., 1991b; Bayer & Lamed 1992; Salyers et al., 1996; Svitil et al., 1997). Most of the carbohydrate-degrading enzymes are highly specific for glycosidic sugar and the anomeric configuration of the glycosidic bond. They can act endolytically, hydrolyzing internal carbohydrate bonds, generating oligosaccharide intermediates resulting in relatively rapid viscosity decreases of the polymer; others act exolytically, degrading the polymer from the non-reducing termini generating a single monosaccharide end product. These enzymes tend to show a higher specificity with high molecular weight substrates than lower molecular weight substrates.
For the degradation of the insoluble complex polysaccharides, enzyme localization relative to other enzymes and biomolecules is often important for enzyme efficiency, as is the chemistry of its active site. Many reports have been published describing the properties of numerous isolated polysaccharide-degrading bacteria; however, relatively little is understood concerning how intact bacteria degrade insoluble complex polysaccharides or how the multiple enzymes produced by the organism interact (Salyers et al., 1996). It should be noted that degradation of the insoluble complex polysaccharides into its monosaccharide requires multiple enzymes and possibly other proteins (e.g. substrate-binding).
The present invention teaches general methods for preparing biofilm-degrading, multiple-specificity hydrolytic enzyme mixtures which are specifically tailored to remove targeted industrial and/or disease-related biofilms. These biofilm degrading hydrolytic enzyme mixtures can be employed to remove or degrade biofilms from the target surface causing a reduction of the biofilm and resulting in increased efficiency and improved hygiene in industrial settings and in improved treatment in therapeutic settings. The present invention will find application in numerous settings where biofilms currently present efficiency and health problems.
Hydrolytic enzyme mixtures can be employed, via direct application to the biofilm, to remove or degrade disease-associated and/or industrial biofilms from the surfaces colonized by the pathogen. The present invention will find application in industrial settings, such as water cooling towers, waste water piping, heat exchangers, and food preparation lines. The present invention will also find application as a therapeutic agent for the treatment of numerous currently uncontrolled animal, and particularly human, diseases. For example: i) Oral plaque-forming bacterial species, the causal agents of dental caries, are maintained by complex biofilms required for their continued colonization of the tooth surface and their disease causing ac
Carlson Peter S.
Manyak David M.
Quintero Ernesto J.
Weiner Ronald M.
Arent Fox Kintner & Plotkin & Kahn, PLLC
University of Maryland College Park
Witz Jean C.
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