Drug – bio-affecting and body treating compositions – Enzyme or coenzyme containing – Transferases
Reexamination Certificate
1998-08-27
2002-07-23
Prout, Rebecca E. (Department: 1652)
Drug, bio-affecting and body treating compositions
Enzyme or coenzyme containing
Transferases
C424S094100, C424S094610, C424S094620, C435S183000, C435S200000, C435S209000, C435S252100
Reexamination Certificate
active
06423312
ABSTRACT:
FIELD AND BACKGROUND OF THE INVENTION
The present invention relates to the use of glycosaminoglycans degrading enzymes, such as, but not limited to, heparanases, connective tissue activating peptide III (CTAP), heparinases, hyaluronidases and chondroitinases, against surface protected bacteria, for reduction of bacterial alginate and for the disruption of bacterial biofilms. More particularly, the present invention relates to the use of glycosaminoglycans degrading enzymes for treating conditions resulting from infection by mucoid, alginate-producing and/or biofilm-producing bacteria.
Glycosaminoglycans degrading enzymes: Glycosaminoglycans (GAG) are unbranched polyanionic polysaccharides made up of repeating disaccharides. One component of which is always an amino sugar. Degradation of GAG is carried out by a battery of lysosomal hydrolases. These include certain endoglycosidases (heparanse and CTAP degrade heparan sulfate and to a lesser extent heparin, and hyaluronidase from sheep or bovine testes degrade hyaluronic acid and chondroitin sulfate), various exoglycosidases (&bgr;-glucoronidase), and sulfatases (iduronate sulfatase), generally acting in sequence to degrade the various GAG. Bacterial lyases such as heparinase I, II and III from
Flavobacterium heparinum
cleave heparin-like molecules, chondroitinase ABC from
Proteus vulgaris
, AC from
Arthrobacter aurescens
or
Flavobacterium heparinum
, B and C from
Flavobacterium heparinum
degrade chondroitin sulfate.
Bergey's Manual of Determinative Bacteriology describes 149 species of the genus Pseudomonas. However as with species designations in other groups of organisms, many are based on minor points of difference, which may vary under different conditions of growth and nutrition. Most species are motile with polar flagella: straight rods or occasionally coccoid in shape. They grow well on conventional culture media, and many strains thereof produce characteristic pigmentation. All species except
P. maltophilia
are recognized as having a cytochrome C oxidase present when tested with tetramethyl-p-phenylenediamine, a characteristic that distinguishes them from the enterobacteroaceae. Although Pseudomonas are not particularly invasive, once they are established as infective agents, they are very difficult to eradicate (Handbook of Microbiology, Vol. 1 1974 pp. 239-242).
Pseudomonas aeruginosa
is an opportunistic pathogen responsible for a wide range of infections, one of the most debilitating being chronic pulmonary infection in cystic fibrosis (CF) patients. The basic alteration in the bronchial/pulmonary environment of the CF lung causing increased secretion of hyperviscous mucus favors bacterial colonization by
Staphylococcus aureus, Haemophilus influenzae
and
P. aeruginosa
. Prolonged antibiotic therapy and the increasing life expectancy of CF patients may influence the prevalence of all of these organisms in the lung flora.
P. aeruginosa
is found in patients with moderate and severe pulmonary disease, being the sole pathogen found in sputum in the most advanced stages of the disease.
P. aeruginsosa
is particularly resistant to even the most aggressive chemotherapy and has been found to colonize the lungs of 50-90% of all CF patients. It has been shown that the severity of lung infection in CF patients is directly correlated to the presence of mucoid strains. The mucoid
P. aeruginosa
isolates revert at a high frequency to a nonmucoid form upon serial transfers in the laboratory.
The pathogenicity of mucoid
P. aeruginosa
in the CF lung is attributed in part to the synthesis of the exopolysaccharide alginate by the bacterium. Nonmucoid strains of
P. aeruginosa
initially colonize the upper respiratory tract of CF patients. However, mucoid alginate-producing variants appear with prolonged infection and eventually predominate in the CF lung. The alginate produced by these mucoid strains of
P. aeuriginosa
compounds the problems related to the hyperviscous bronchial secretions of CF patients. Alginate-producing strains of
P. aeruginosa
are almost exclusively associated with respiratory tract infections that accompany CF. Although 80% of the
P. aeruginosa
isolates form CF patients are mucoid, only about 1% of clinical
P. aeruginosa
isolates from other types of infections are mucoid. Alginate appears to protect
P. aeruginasa
by shielding it from host immune defense and antibiotic therapy, and possibly enables it to adhere more effectively to respiratory tract tissues. Once established in the CF lung, these mucoid strains tend to persist and parallel the progressive clinical deterioration of the patient. Alginate is a linear acetylated copolymer consisting of &bgr;-1,4-linked D-mannuronic acid and variable amounts of its C-5 epimer L-guluronic acid. Alginate is produced by several bacterial species, the most widely known being
Azotobacter vinelandii
and
P. aeruginosa
. Bacterial alginates differ from algal alginate in that the former contain O-acetyl groups. The viscosity level of alginate may play a role in the pathogenesis of mucoid
P. aeruginosa
in the CF respiratory tract. Several enzymes are involved in the alginate biosynthetic pathway: Phosphomannose isomerase (PMI), GDP-mannose dehydrogenase (GMD), and GDP-mannose pyrophosphorylase (GMP) in mucoid, alginate-producing
P. aeruginose
. Activities of the enzymes are either absent or greatly reduced in nonmucoid strains.
Alginate synthesis by the highly mucoid
P. aeruginosa
8821 M is growth-phase-dependent and the alginate produced per unit of biomass reaches maximum values in the deceleration phase of growth. However, the degree of polymerization increases as batch growth proceeds, reaching maximum values at the stationary phase of growth (Leitao J H, Sa-Correia I; Arch Microbiol 1995, March; 163(3): 217-222).
Regulation of alginate synthesis: The regulation of alginate biosynthesis by
P. aeruginosa
appears to involve fine tuning of several factors. A pivotal step in alginate biosynthesis is the activation of the algD gene in mucoid, alginate-producing
P. aeruginosa
. algD is highly activated in response to increased concentrations of either KCl or NaCl. This is an interesting finding since the CF lung is rich in Na
+
, Cl
−
and K
+
ions.
Alginate-producing strains of three other Pseudomonas species (
P. fluorescens, P. putida,
and
P. mendocina
) have been isolated in vitro by growth on subinhibitory concentrations of carbenicillin. Also, certain phytopathogen Pseudomonas species produce alginate both in planta and in vitro. These observations suggest that many species of Pseudomonas harbor genes involved in alginate biosynthesis, but that they are not normally expressed. Since many of the
P. aeruginosa
alginate genes had been cloned, it was possible to examine genomic DNA from various Pseudomonas species and phylogenetically related organisms for sequences homologous to the
P. aeruginosa
alg genes. Southern hybridization studies using algA, pmm, algD, and algR1 as probes showed some degree of homology with several Pseudomonas species belonging to Pseudomonas RNA homology group 1. Some probes also hybridized with Azotobacter, Azomonas, and Serpens species. In the laboratory, the alginate-producing (alg+) phenotype is somewhat unstable, and nonmucoid (alg−) revertants are commonly seen. Genetic mapping experiments have shown that the switching between alg+ and alg− is due to a genetic change in one region of the chromosome located at about 68 min on the 75-min chromosomal linkage map of Pseudomonas. This was originally referred to as the muc locus. Two additional recognized genes are involved in the regulation of alginate production. These are algR at 9 min and algB at 13 min, both of which are required for high-level alginate production. However, most of the alginate biosynthetic genes appear to be located in a larger gene cluster at 34 min.
Collectively, the regulation of the alginate biosynthetic pathway in
P. aeruginosa
is multignenic and appears to be relatively complex, which
G.E. Ehrlich Ltd.
Insight Strategy & Marketing Ltd.
Prout Rebecca E.
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