U.S. patent application number 09/140888 was filed with the patent office on 2002-05-30 for compositions including glycosaminoglycans degrading enzymes and use of same against surface protected bacteria.
Invention is credited to YACOBY-ZEEVI, ORON.
Application Number | 20020064858 09/140888 |
Document ID | / |
Family ID | 21943675 |
Filed Date | 2002-05-30 |
United States Patent
Application |
20020064858 |
Kind Code |
A1 |
YACOBY-ZEEVI, ORON |
May 30, 2002 |
COMPOSITIONS INCLUDING GLYCOSAMINOGLYCANS DEGRADING ENZYMES AND USE
OF SAME AGAINST SURFACE PROTECTED BACTERIA
Abstract
A method of rendering a surface protected bacteria more
susceptible to an anti-bacterial agent effected by subjecting the
bacteria to a glycosaminoglycans degrading enzyme.
Inventors: |
YACOBY-ZEEVI, ORON; (MEITAR,
IL) |
Correspondence
Address: |
Sol Steinbein
G. E. Ehrlich Ltd
c/o Anthony Castorina
2001 Jefferson Davis Highway Ste. 207
Arlington
VA
22202
US
|
Family ID: |
21943675 |
Appl. No.: |
09/140888 |
Filed: |
August 27, 1998 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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09140888 |
Aug 27, 1998 |
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09046475 |
Mar 25, 1998 |
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09140888 |
Aug 27, 1998 |
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08922170 |
Sep 2, 1997 |
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Current U.S.
Class: |
435/232 |
Current CPC
Class: |
C12Y 302/01166 20130101;
A61M 15/009 20130101; A61K 38/00 20130101; A61K 2300/00 20130101;
A61K 9/0073 20130101; C07K 16/40 20130101; A61K 38/51 20130101;
A61K 45/06 20130101; A61K 38/51 20130101; C12N 9/2402 20130101;
G01N 33/573 20130101; G01N 2400/40 20130101; C12Y 402/02008
20130101; G01N 2333/988 20130101 |
Class at
Publication: |
435/232 |
International
Class: |
C12N 009/88 |
Claims
What is claimed is:
1. A method of rendering a surface protected bacteria more
susceptible to an anti-bacterial agent comprising the step of
subjecting said bacteria to a glycosaminoglycans degrading
enzyme.
2. The method of claim 1, wherein said surface protected bacteria
is a mucoid bacteria.
3. The method of claim 1, wherein said surface protected bacteria
is an alginate-producing bacteria.
4. The method of claim 1, wherein said surface protected bacteria
is a biofilm-producing bacteria.
5. The method of claim 1, wherein said anti-bacterial agent is a
bactericide.
6. The method of claim 1, wherein said anti-bacterial agent is an
antibiotic.
7. The method of claim 1, wherein said anti-bacterial agent is an
immune moiety.
8. The method of claim 1, wherein said glycosaminoglycans degrading
enzyme is selected from the group consisting of a lysosomal
hydrolase and a bacterial lyase.
9. The method of claim 1, wherein said glycosaminoglycans degrading
enzyme is selected from the group consisting of an endoglycosidase,
an exoglycosidase and a sulfatase.
10. The method of claim 1, wherein said glycosaminoglycans
degrading enzyme is selected from the group consisting of
heparanse, connective tissue activating peptide III, hyaluronidase,
glucoronidase, iduronate sulfatase, heparinase I, heparinase II
heparinase III, chondroitinase ABC, chondroitinase AC,
chondroitinase B and chondroitinase C.
11. The method of claim 1, wherein said bacteria is of a genus
selected from the group consisting of Pseudomonas, Azotobacter,
Azomonas, Serpens, Fusobacterium, Klebsiella, Streptococcus,
Staphylococcus and Treponema.
12. The method of claim 1, wherein said bacteria is of a genus
Pseudomonas.
13. The method of claim 1, wherein said bacteria is Pseudomonas
aeruginosa.
14. The method of claim 1, wherein said bacteria is in a lung of a
patient suffering chronic pulmonary infection, the method being for
relieving symptoms associated with said chronic pulmonary
infection.
15. The method of claim 1, wherein said bacteria is in a lung of a
cystic fibrosis patient suffering chronic pulmonary infection, the
method being for relieving symptoms associated with said chronic
pulmonary infection.
16. The method of claim 1, wherein said bacteria is growing on a
non-living substratum.
17. The method of claim 16, wherein said non-living substratum
forms a part of a medical device.
18. The method of claim 17, wherein said medical device is selected
from the group consisting of an infusion device, a catheter device,
a contact lens device, a dialysis device and a draining device.
19. A method of rendering a surface protected bacteria less capable
of adhering to a substratum comprising the step of subjecting said
bacteria to a glycosaminoglycans degrading enzyme.
20. The method of claim 19, wherein said surface protected bacteria
is a mucoid bacteria.
21. The method of claim 19, wherein said surface protected bacteria
is an alginate-producing bacteria.
22. The method of claim 19, wherein said surface protected bacteria
is a biofilm-producing bacteria.
23. The method of claim 19, wherein said glycosaminoglycans
degrading enzyme is selected from the group consisting of a
lysosomal hydrolase and a bacterial lyase.
24. The method of claim 19, wherein said glycosaminoglycans
degrading enzyme is selected from the group consisting of an
endoglycosidase, an exoglycosidase and a sulfatase.
25. The method of claim 19, wherein said glycosaminoglycans
degrading enzyme is selected from the group consisting of
heparanse, connective tissue activating peptide III, hyaluronidase,
glucoronidase, iduronate sulfatase, heparinase I, heparinase II
heparinase III, chondroitinase ABC, chondroitinase AC,
chondroitinase B and chondroitinase C.
26. The method of claim 19, wherein said bacteria is of a genus
selected from the group consisting of Pseudomonas, Azotobacter,
Azomonas, Serpens, Fusobacterium, Klebsiella, Streptococcus,
Staphylococcus and Treponema.
27. The method of claim 19, wherein said bacteria is of a genus
Pseudomonas.
28. The method of claim 19, wherein said bacteria is Pseudomonas
aeruginosa.
29. The method of claim 19, wherein said substratum is a living
tissue.
30. The method of claim 19, wherein said substratum is a lung
tissue of a patient suffering chronic pulmonary infection, the
method being for relieving symptoms associated with said chronic
pulmonary infection.
31. The method of claim 19, wherein said substratum is a lung
tissue of a cystic fibrosis patient suffering chronic pulmonary
infection, the method being for relieving symptoms associated with
said chronic pulmonary infection.
32. The method of claim 19, wherein said substratum is a non-living
substratum.
33. The method of claim 32, wherein said non-living substratum
forms a part of a medical device.
34. The method of claim 33, wherein said medical device is selected
from the group consisting of an infusion device, a catheter device,
a contact lens device, a dialysis device and a draining device.
35. A method of treating a disease for relieving disease associated
symptoms comprising the step of administering a therapeutical
composition including a glycosaminoglycans degrading enzyme.
36. The method of claim 35, wherein said disease is cystic
fibrosis, whereas administering said composition is effected by
inhaling aerosols and further wherein the disease associated
symptoms include chronic pulmonary infection by a surface protected
bacteria.
37. A therapeutic composition for treating a surface protected
bacteria associated disease or symptoms comprising a
glycosaminoglycans degrading enzyme and an antibiotic.
38. The therapeutic composition of claim 37, wherein said disease
is cystic fibrosis and wherein said symptoms are chronic pulmonary
infection.
39. The therapeutic composition of claim 37, wherein said
glycosaminoglycans degrading enzyme is selected from the group
consisting of heparanse, connective tissue activating peptide III,
hyaluronidase, glucoronidase, iduronate sulfatase, heparinase I,
heparinase II heparinase III, chondroitinase ABC, chondroitinase
AC, chondroitinase B and chondroitinase C.
40. A bactericide composition effective in eliminating a surface
protected bacteria comprising a glycosaminoglycans degrading enzyme
and a bactericide.
41. The bactericide composition of claim 40, wherein said
glycosaminoglycans degrading enzyme is selected from the group
consisting of heparanse, connective tissue activating peptide III,
hyaluronidase, glucoronidase, iduronate sulfatase, heparinase I,
heparinase II heparinase III, chondroitinase ABC, chondroitinase
AC, chondroitinase B and chondroitinase C.
Description
[0001] This is a continuation in part of U.S. patent application
Ser. No. 09/046,475, filed Mar. 25, 1998, which is a
continuation-in-part of U.S. patent application Ser. No.
08/922,170, filed Sep. 2, 1997.
FIELD AND BACKGROUND OF THE INVENTION
[0002] 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.
[0003] 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
(.beta.-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.
[0004] 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).
[0005] 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. aeruginosa 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.
[0006] 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 .beta.-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.
[0007] 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 March 1995; 163(3):
217-222).
[0008] 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.sup.+, Cl.sup.- and K.sup.+
ions.
[0009] 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.
[0010] Collectively, the regulation of the alginate biosynthetic
pathway in P. aeruginosa is multignenic and appears to be
relatively complex, which suggests that this system has a long
evolutionary history. Alginate is secreted in copious amounts
(e.g., 2 mg/ml in culture supernatants), thus channeling much of
the available carbon and energy sources toward its production. It
is not surprising that alginate biosynthesis would be tightly
regulated. However, when production of alginate is advantageous to
the organism (such as in the CF respiratory tract) the rare mucoid
cells in the population become predominant because of their
selective advantage. When alginate production is no longer
advantageous, the instability of the Alg+ phenotype (controlled by
the genetic switch algS) allows the nonmucoid population to quickly
become predominant as a result of the ability to conserve carbon
and energy resources. Environmental factors may also play a role in
the frequency of alginate conversion. Because virtually all CF
isolates of P. aeruginosa are mucoid, it was postulated that the
lung environment of CF patients provides the trigger required to
turn on the production of the alginate adhesin. To identify
virulence genes of P. aeruginosa that are important in infection of
CF patients, an in vivo selection system (IVST) was used to
identify promoters that are specifically inducible by respiratory
mucus derived from CF patients. Three genetic loci that are highly
inducible by the mucus were identified (Wang J et al.; Mol
Microbiol December 1996; 22(5): 1005-12).
[0011] It is unlikely that this complex regulatory scheme to
activate alginate production evolved solely as a pathogenic
mechanism specific for the infection of CF patients. P. aeruginosa
normally dwells in the soil environment, and alginate conversion
may have evolved to protect the bacterial population from
destruction due to attack by bacteriophages or bactriocins or from
desiccate during periods of dryness. However, P. aeruginosa is a
remarkable opportunistic pathogen and has adapted the alginate
conversion system to promote debilitating and life threatening
pulmonary infections of CF patients. Understanding alginate gene
regulation in P. aeruginosa may lead to treatments that could turn
off alginate production by the organisms resident in the CF lung,
thus improving the longevity and quality of life for these patients
(Pseudomonas: biotransformations, pathogenesis, and evolving
biotechnology. Edited by Simon Silver et al., 1990 Am. Soc. For
Microbiol. Ch. 2 and 3, pp. 15-36)
[0012] Adhesion mechanisms of P. aeruginosa: Adherence through
carbohydrate-binding adhesins is an early step in colonization of
the lung by gram-negative organisms (Azghani AO et al.;
Glycobiology February 1995; 5(1): 39-44). Pseusomonas aeruginosa is
an opportunistic pathogen capable of causing serious localized
infections of the cerebrospinal fluid (CSF), urinary tract, eye,
ear, lung, skin, and other parts of the body. The organism is often
isolated from peritoneal dialysis membranes. Generalized systemic
infections tend to occur only in injured, immunodeficient, or
otherwise compromised patients. It is not surprising, therefore,
that this genus has developed a number of adhesion mechanisms, each
being specific for a particular type of substratum. There are
numerous articles reporting various mechanisms of adhesion for P.
aeruginosa. These include the hydrophobic effect, adhesion to and
by alginates, and lectin-dependent adhesion. Some reports maintain
that the adhesion of P. aeruginosa is fimbriae-dependent. The
exopolysaccharide alginate binds to buccal epithelial and tracheal
cells, as well as to bronchotracheal mucin. Antipolysaccharide
inhibits binding of the organisms to the tracheal cells. Also,
mucoid strains of Pseudomonas adhere much better to tracheal cells
compared to nonmucoid strains or compared to alginate-producing
bacteria grown in an antibiotic medium to reduce alginate
production. Alginate appears to play a role in the adhesion of
Pseudomonas to contact lenses. Microscopic evidence suggests that
mucoid strains may adhere to ciliated tracheal cells and to inert
surfaces, including contact lenses, by polysaccharide-like
materials. The presence of alginate in the reaction mixture causes
an increase in the number of bacteria adherent to tracheal cells or
immobilized mucin. It has been suggested that the alginate may act
to trap and tether the organisms to the substrata, thereby allowing
other adhesins, such as the fimbrial adhesin complex, to bind the
specific receptors. If the target animal cell or substrata lacks
the ability to bind alginate, then the presence of an
exopolysaccharide coat on the bacterial surface may actually impede
the ability of the organisms to attach to such animal cells. This
may explain the rescued ability of the mucoid strains to bind to
phagocytic cells or to primary cultures of cilliated epithelial
cells. The phagocytic cells lack the ability to bind alginate.
[0013] There are several reports showing that adhesion of P.
aeruginosa to various substrata may involve lectins specific for
carbohydrates other than sialic acid. For example, adhesion of the
bacteria to the tracheal epithelium is inhibited by D-galactose and
N-acetyl-D-glucosamine. Moreover, putative nonfimbrial adhesins
produced by both mucoid and nonmucoid strains of Pseudomonas,
specific for Gal.beta.1, 4GlcNAc or Gal.beta.1, 3GlcNAc sequences,
may be involved in mediating binding of the organisms to human
bronchial mucins (Bacterial Adhesion to Cells and Tissues/Ofek I
and Doyle R J, 1994 Chapman & Hall, Inc. pp. 114-116,
418-421).
[0014] P. aeruginosa biofilms: Bacteria in nature often exist as
sessile communities called biofilms. These communities develop
structures that are morphologically and physiologically
differentiated from free living bacteria. A cell-to-cell signal is
involved in the development of P. aeruginosa biofilms. The
involvement of an intercellular signal molecule in the development
of P. aeruginosa biofilms suggests possible targets to control
biofilm growth on catheters, in CF and in other environments where
P. aerugiosa biofilms are a persistent problem. Biofilms of P.
aeruginosa develop on solid surfaces exposed to a continuous flow
of nutrients. The biofilm structures consist primarily of an
exopolysaccharide matrix or glycocalyx in which the bacteria are
embedded. Cell to cell signal is required for the differentiation
of individual cells of the common bacterium P. aeruginosa into
complex multicellular structures. P. aeruginosa cells in biofilms
secrete a particular homoserine lactone, 3-oxododecanoylhomoserine
(OdDHL), that helps to control biofilm differentiation. OdDHL binds
an R-protein inside the target cell, activating RNA polymerases
that are involved in forming the biofilm. A mutation that blocks
generation of the signal molecule hinders differentiation, and the
resulting abnormal biofilm appears to be sensitive to the detergent
biocide SDS. The control of biofilm differentiation and integrity
by quorum sensing has important implications in medicine. Because
of their innate resistance to antibiotics and other biocides,
biofilms in these environments are difficult, if not impossible, to
eradicate. Bacterial biofilms also present other problems of
significant economic importance in both industry and medicine. The
finding of a connection between biofilm differentiation into
clusters of bacteria resistant to the detergent biocide SDS and a
quorum-sensing signal suggests that inhibition of these
cell-to-cell signals could aid in the treatment of biofilms (Davies
D G et al.; Science vol 280 Apr. 10, 1998).
[0015] It was shown that in continuous flow biofilm cultures in
medium resembling CF bronchial secretions, P. aeruginosa was not
eradicated form biofilms by 1 week of treatment with high
concentrations of ceftazidime and gentamicin, to which they are
sensitive on conventional testing. The addition of rifampicin,
which has little activity against the strains as measured by the
minimum inhibitory concentration, led to the apparent elimination
of the bacteria from the biofilms. The effect was not strain
specific. Ghani M and Soothill J S; Can J microbial November 1997;
43(11): 999-1004.
[0016] Scanning electron microscopy (SEM) showed that the internal
surfaces of catheters and drainage systems are commonly colonized
by thick layers or biofilms of organisms embedded in a
polysaccharide matrix. There is evidence from laboratory and
clinical studies that the progressive spread of the biofilm along
the luminal surfaces of the bag, tube and catheter leads to bladder
infection (Stickler D J et al.; Br J Urol. 1996, 78; 579-88).
[0017] Biofilms are also an integral part of dental plaque, thereby
contributing to tooth decay and periodontal disease. One target is
Fusobacterium nucleatum, an oral bacteria found in biofilms coating
the area between teeth and gums (Potera C; ASM News, 64(6),
1998).
[0018] The incidence of gram-negative bacteria in reviews of
postoperative acute endophthalmitis ranges from 3-22% with P.
aeruginosa being one of the more common etiological agents of
fulminant infections. These infections usually develop within days
of the surgery and have a poor prognosis. P. aeruginosa has been
reported to adhere to damaged corneal cells as well as to contact
lenses. Its adherence to silicone and latex materials is
significantly greater that of several isolates of Staph.
Epidermidis. The in vitro adherence of P. aeruginosa to a variety
of hydrogel and rigid gas-permeable contact lenses correlates with
clinical data. P. aeruginosa adheres to the optic material of
intraoccular lenses: AcrySof-acrylic<PMMA<silicone
1<silicon 2, and form biofilms (Manal M et al.; J Cataract
Refract Surg vol. 24, January 1998).
[0019] CF lung predilection for excessive inflammation and
infection with P. aeruginosa: In CF, defective function of the CFTR
in airway epithelial cells and submucosal glands, results in
chronic pulmonary infection with P. aeruginosa. The pulmonary
infection incites an intense host inflammatory response, causing
progressive suppurative pulmonary disease. Several hypotheses have
been proposed to explain CF lung predilection for excessive
inflammation and infection with P. aeruginosa.
[0020] The role of alginate in pathogenesis is complex and appears
to confer antiphagocytic properties and an adherence mechanism upon
the organism. Autopsies show that mucoid P. aeruginosa forms
adherent microcolonies in the lung. Alginate does not firmly adhere
to the organisms but is released in large quantities into the
respiratory environment. Because alginate is very viscous in
aqueous solution, it probably contributes to the high viscosity of
the bronchial secretions in the CF lung, resulting in obstruction
of small airways, interference with mucociliary airway clearance,
and impaired movement to phagocytes. The mucoid organisms may be
more adapted to a chronic infection because they secrete lower
levels of proteases, which would otherwise cause extensive lung
damage and acute infection. Also P. aeuruginosa can utilize the
respiratory secretions of the CF lung to support rapid growth and
alginate biosynthesis; thus, the mucus-congested CF respiratory
tract provides P. aeruginosa with a nutritionally rich environment
favorable to clonization. The initial colonization of the CF upper
respiratory tract appears to be with a nonmucoid strain and is
often asympomatic. This usually precedes the emergence of mucoid
variants of the original strain and is followed by chronic
infection and a poor prognosis for the patient.
[0021] CF patients do produce opsonic antibodies to mucoid P.
aeruginosa that are in a planktonic or suspended state, but these
antibodies fail to kill P. aeruginosa growing in a biofilm (Pier G
B; Behring Inst Mitt February 1997; 98: 350-60). Results showed
that alginate and neutral polysaccharides are involved in
phagocytic impairment of P. aeruginosa (Pasquier C et al.; FEMS
Microbiol Lett Feb. 15, 1997; 147(2): 195-202). Alginate production
inhibits opsonic and nonopsonic phagocytosis, protects cells form
reactive oxygen intermediates and plays additional roles associated
with biofilm phenomena (Hatano K et al.; Infect Immun. January
1995; 63(1): 21-26; Meluleni G J et al.; J. Immunol. Aug. 15, 1995;
155(4): 2029-38). The results support the findings of the previous
extensive work carried out in vitro, suggesting that the phagocytic
and other bactericidal systems in the lung are impaired: Some
investigators suggest that the elevated salt content in the surface
fluid of the CF airway renders human .beta.-defensin-1
nonfunctional, eliminating the bactericidal activity of the
respiratory epithelium. Another hypothesis suggests that failure of
the respiratory epithelial cells in the CF lung to ingest bacteria
and be sloughed allows for P. aeruginosa retention at the
endobronchial surface. The airway epithelial cell ingestion of
bacteria followed by cellular desquamation may protect the lung
form infection, and epithelial cells expressing mutant forms of the
CFTR may be defective in this function. It was found that
transformed human airway epithelial cells homozygous for the delta
F508 allele of CFTR were significantly defective in uptake of P.
aeruginosa compared with the same cell line complemented with the
wild-type allele of CFTR. Defective epithelial cell internalization
of P. aeruginosa may be a critical factor in hyper susceptibility
of CF patients to chronic lung infections (Pier G B et al.; Am J
Respir Crit Care Med October 1996; 154(4 Pt 2): S175-82; Proc Natl
Acad Sci U S A Oct. 28, 1997; 94(22): 12088-93). The majority of CF
mucoid isolates carry mucA mutations which allow transcription of
alginate biosynthetic genes, resulting in a mucoid phenotype.
Mucoidy is caused by muc mutations that depress the alternative
sigma factor encoded by algU, which in turn activates alginate
biosynthetic and ancillary regulatory genes (Boucher J C et al.;
Infec Immun September 1997; 65: 3838-46; Boucher J C et al.; J
Bacteriol January 1996; 178(2): 511-23). Mucoid cells are cleared
less efficiently and appears to linger in the lung longer that
nonmucoid organisms. This finding suggests that mucoidy may confer
an ability to resist innate clearance mechanisms in the lung and,
along with other potentially contributing factors, could be the
basis for selection of mucA mutants in CF (Yu H et al.; Infec and
Immun. 1998, 66(1): 280-88). Another possibility is that
Pseudomonas adheres to epithelial cells in the CF airway in greater
numbers because of the abnormal surface properties of the cells,
thus leading to infection.
[0022] Although details of the mechanism differ, all of these
hypotheses predict that the basic defect in CF permits retention of
bacteria at an otherwise sterile site, providing the stimulus for
inflammation. Furthermore, it is suggested that the CF genotype is
associated with excessive inflammatory response compared with the
normal response, even if the initiating stimulus is similar. In
order to understand the pathogenesis of pulmonary disease
characteristic of CF, it is required to examine not only the
impaired clearance of the bacteria, but also the excessive host
response to P. aeruginosa (Van Heeckeren A et al.; J. Clin Inves.
December 1997; 100(11): 2810-5).
[0023] Thus, P. aeruginosa is a remarkable opportunistic pathogen
and has adapted the alginate conversion system to promote
debilitating and life threatening pulmonary infections of CF
patients. The environment of the CF lung is unique in its capacity
to induce alginate production by P. aeruginosa. However, the
factors which contribute to this unusual host-pathogen interaction
have not yet been determined.
[0024] Understanding alginate gene regulation in P. aeruginosa may
lead to treatments that could turn off alginate production by the
organisms resident in the CF lung, thus improving the longevity and
quality of life for these patients.
[0025] CF is the most common fatal genetic disease among the
Caucasian population, affecting approximately 1:2500 newborns. The
median age of survival of patients with CF has dramatically
increased over the past 2 decades from less than 10 to more than 30
years. This progress has occurred primarily through improved
nutritional support and aggressive management with antibiotic
therapy of acute pulmonary infections. Currently there is no
effective combination of therapies which completely eradicates
alginate-producing P. aeruginosa from the CF lung environment. The
development of new compounds effective in preventing alginate
synthesis represents a major step towards reaching this goal. Such
inhibitors of alginate synthesis have potential clinical
applications in that elimination of the alginate capsule might
render P. aeruginosa more susceptible to both antibiotic therapy
and the host's immune system. Therefore, many laboratories are
involved in an extensive study of the genetics and regulation of
the alginate biosynthetic pathway in P. aeruginosa in an effort to
identify factors unique to the CF lung environment that trigger
expression of the genes involved in alginate biosynthesis and in an
attempt to find nontoxic compounds that inhibit alginate synthesis
by inhibiting the enzymes directly involved in the pathway.
[0026] Mrnsy R J et al. have investigated the use of an alginate
lyase obtained from a bacterial source to disrupt P. aeruginosa
alginate's polymeric nature and effect a change in the Theological
properties of CF sputum in vitro. Their results suggested that
bacterial alginate present within purulent CF sputum may be quite
stable, that endogenous alginate lyase activities appear to be
limited and that the in vitro addition of exogenous alginate lyase
can lead to the disruption of alginate and a change in the
viscoelastic properties of some purulent CF sputum samples (Mrsny R
J et al.; Pulm Pharmacol December 1994; 7(6): 357-66).
[0027] A suspension of 2% P. aeruginosa alginate completely blocked
the diffusion of gentamycin and tobramycin, but not that of
carbenicillin, illustrating how alginate production can help
protect P. aeruginosa growing within alginate microcolonies in
patients with CF from the effects of aminoglycosides. This
aminoglycoside diffusion barrier was degraded with a semipurified
preparation of P. aeruginosa alginate lyase (Hatch R A, Schiller N
L; Antimicrob Agents Chemother. April 1998; 42(4): 974-7).
[0028] A 41 kDa alginate lyase capable of degrading alginic acid of
P. aeruginosa was prepared from the culture of Bacillus strain
ATB-1015. The enzyme was found useful for the treatment of
respiratory diseases caused by infection by P. aeruginosa (JP
95-181047, JP 09009962 A2 to Akira Nakagawa). Alginic acid lyase(s)
which decompose alginic acid into sugar and the 4-deoxy-5-keto
uronic acid is used for treatment of pulmonary cystic fibrosis (JP
06197760 A to Yakuhin Otsuka). However, these lyases fail to
degrade glycosaminoglycans.
[0029] More therapeutic approaches: Phagocytosis of P. aeruginosa
by macrophages is a unique two-step process; binding is
glucose-independent but ingestion occurs only in the presence of
D-glucose or D-mannose. Since glucose is present in only negligible
quantities in the endobroncheal space, P. aeruginosa may be
pathogenic by virtue of its capacity to exploit the opportunity
presented in the lower airway to resist normal nonspecific
phagocytic defenses. Because delivery of simple glucose by aerosol
would not be effective, various approaches for targeting glucose to
alvelolar macrophages by receptor-mediated endocytosis are under
investigation (Speert D P et al.; Behring Inst Mitt February 1997;
98: 274-82).
[0030] The ongoing lung tissue damage in chronically P. aeruginosa
infected CF patients has been shown to be caused by elastase
liberated from polymorphonuclear leukocytes (PMN). Alginate alone
appeared to be a weak inhibitor of the hydrolysis of long synthetic
peptide substrates and [.sup.14C]elastin by elastase. Alginate also
had effects on the antielastase function of naturally occurring
protease inhibitors in the lung: It reduces the association rate of
elastase and alpha 1-proteinase inhibitor, whereas it increases the
association rate of elastase and secretory leukoprotease
inhibitor.
[0031] Based on these finding, alginate may be an important factor
in determining the local distribution of leukocyte elastase and
perturbing the overall protease-antiprotease balance in the
infected lungs of CF patients (Ying Q L et al.; Am j Respir Cell
Mol Biol. August 1996; 15(2): 283-91).
[0032] Thus, prevention of the onset of the chronic infection or
prevention of the dominance of the inflammation by PMNs would be
important goals for a vaccine strategy against P. aeruginosa.
Findings suggested that change from the Th2 like response seen in
CF patients towards a Th1 response might improve their prognosis
(Johansen H K et al.; Behring Inst Mitt February 1997; 98:
269-73).
[0033] Care should be taken when treating nonmucoid P. aeruginosa
with gyrase inhibitors such as ciprofloxacin, norfloxacin and
ofloxacin, which target the A subunit of topoisomerase II, since it
resulted in 100% conversion to the mucoid phenotype. An increase in
resistance was observed in populations that expressed the mucoid
phenotype. Both mucoid conversion and antibiotic resistance were
completely reversible when ciprofloxacin pressure was withdrawn,
but only partially reversible by the removal of norfloxacin and
ofloxacin. Thus, these experiments indicate that in the presence of
some fluoroquinolones, a conditional response resulting in mucoid
conversion and antibiotic resistance may occur (Pina S E, Mattingly
S J; Curr Microbiol August 1997; 35(2): 103-8).
[0034] Other mucoid bacteria: Klebsiella Pneumoniae K1 synthesizes
capsular polysaccharide. Non mucoid variants thereof are more
susceptible to some bacteriophages, possibly due to the reduction
or absence of capsular polysaccharide (Mengistu Y et al.; J Appl
Bacteriol May 1994, 76(5): 424-30). There are two virulence factors
of K. pneumoniae: aerobactin and the mucoid phenotype. Aerobactin
is always associated with the mucoid phenotype (FEMS Microbiol
Lett. Jul. 15, 1995; 130(1): 51-57).
[0035] Mucoid or highly encapsulated strains of group A
Streptococci have been associated both with unusually sever
infections and with acute rheumatic fever. The mucoid M-type 18
strain of a group A Streptococcus has a hyaluronic acid capsule
which plays an important role in virulence. The region of the
chromosome essential for capsular polysaccharide expression is
conserved among diverse group A streptococcal strains. Wessels M R
et al.; Infec Immun February 1994; 62(2): 433-41. In communities,
where increases in cases of rheumatic fever had been reported, the
serotypes M-1, 3, 5, and 18 were isolated which, on culture,
produced characteristic mucoid colonies (Spencer R C; Eur J Clin
Microbiol Infect Dis. 1995, 14 Suppl 1: S26-32). The mucoid
serotype 3 of S. pneumoniae cause rapid fatal infections, despite
adequate antibiotic therapy (Hsueh P R et al.; J Formaos Med Assoc
May 1996; 95(5): 364-71).
[0036] The antiphagocytic effect of M protein has been considered a
critical element in virulence of the group A Streptococcus. The
hyaluronic acid capsule also appeared to play an important role:
studies of an acapsular mutant derived form the mucoid or highly
encapsulated M protein type 18 group A strepococcal strain 282
indicated that loss of capsule expression was associated with
decreased resistance to phagocytic killing and with reduced
virulence in mice. The results provide further evidence that the
hyaluronic acid capsule confers resistance to phagocytosis and
enhances group A streptococcal virulence. Moses A E et al.; Infect
Immun. January 1997; 65(1): 64-71.
[0037] Staphylococcus aureus arthritis is a rapidly progressive and
highly erosive disease of the joints in which both host and
bacterial factors are of pathogenic importance. One potential
bacterial virulence factor is the ability to express a
polysaccharide capsule (CP). Among 11 reported capsular serotypes,
CP type 5 (CP5) and CP8 comprise 80-85% of all clinical blood
isolates. The results clearly indicated that the expression of CP5
is a determinant of the virulence of S. aureus in arthritis and
septicemia (Nilsson I M et al.; Infec Immun 1 October; 65(10):
4216-21).
[0038] Treponema denticola, which has been associated with
periodontitis, synthesizes or acquires and extracellular
polysaccharide layer (Scott D et al; Oral Microbiol Immunol April
1997, 12(2): 121-5).
[0039] The above described data implies that there is a widely
recognized need for, and it would be highly advantageous to have
agents effective in reducing mucus production by bacteria, by, for
example, degradation or prevention of synthesis of the bacterial
exopolysaccharide alginate. The benefits from employing such agents
for the degradation of bacterial exopolysaccharide alginate include
(i) viscosity reduction of alginate related hyperviscous bronchial
secretions; (ii) disruption of bacterial biofilms which may render
the bacteria more susceptible to host immune defense systems and
antibiotic therapy; (iii) inhibition of alginate associated
adhesion to host cells and enhancement of bacterial clearance,
resulting in reduction of infection rate; (vi) reduction of host's
inflammatory response to infection.
SUMMARY OF THE INVENTION
[0040] According to one aspect of the present invention there is
provided a method of rendering a surface protected bacteria more
susceptible to an anti-bacterial agent comprising the step of
subjecting the bacteria to a glycosaminoglycans degrading
enzyme.
[0041] According to another aspect of the present invention there
is provided a method of rendering a surface protected bacteria less
capable of adhering to a substratum comprising the step of
subjecting the bacteria to a glycosaminoglycans degrading
enzyme.
[0042] According to yet another aspect of the present invention
there is provided a method of treating a disease for relieving
disease associated symptoms comprising the step of administering a
therapeutical composition including a glycosaminoglycans degrading
enzyme.
[0043] According to still another aspect of the present invention
there is provided a therapeutic composition for treating a surface
protected bacteria associated disease or symptoms comprising a
glycosaminoglycans degrading enzyme and an antibiotic.
[0044] According to yet another aspect of the present invention
there is provided a bactericide composition effective in
eliminating a surface protected bacteria comprising a
glycosaminoglycans degrading enzyme and a bactericide.
[0045] According to further features in preferred embodiments of
the invention described below, the surface protected bacteria is a
mucoid bacteria.
[0046] According to still further features in the described
preferred embodiments the surface protected bacteria is an
alginate-producing bacteria.
[0047] According to still further features in the described
preferred embodiments the surface protected bacteria is a
biofilm-producing bacteria.
[0048] According to still further features in the described
preferred embodiments the anti-bacterial agent is a
bactericide.
[0049] According to still further features in the described
preferred embodiments the anti-bacterial agent is an
antibiotic.
[0050] According to still further features in the described
preferred embodiments the anti-bacterial agent is an immune
moiety.
[0051] According to still further features in the described
preferred embodiments the glycosaminoglycans degrading enzyme is
selected from the group consisting of a lysosomal hydrolase and a
bacterial lyase.
[0052] According to still further features in the described
preferred embodiments the glycosaminoglycans degrading enzyme is
selected from the group consisting of an endoglycosidase, an
exoglycosidase and a sulfatase.
[0053] According to still further features in the described
preferred embodiments the glycosaminoglycans degrading enzyme is
selected from the group consisting of heparanse, connective tissue
activating peptide III (CTAP), hyaluronidase, glucoronidase,
iduronate sulfatase, heparinase I, heparinase II heparinase III,
chondroitinase ABC, chondroitinase AC, chondroitinase B and
chondroitinase C.
[0054] According to still further features in the described
preferred embodiments the bacteria is of a genus selected from the
group consisting of Pseudomonas, Azotobacter, Azomonas, Serpens,
Fusobacterium, Klebsiella, Streptococcus, Staphylococcus and
Treponema
[0055] According to still further features in the described
preferred embodiments the bacteria is of a genus Pseudomonas
[0056] According to still further features in the described
preferred embodiments the bacteria is Pseudomonas aeruginosa
[0057] According to still further features in the described
preferred embodiments the bacteria is in a lung of a patient
suffering chronic pulmonary infection, the method being for
relieving symptoms associated with the chronic pulmonary
infection.
[0058] According to still further features in the described
preferred embodiments the bacteria is in a lung of a cystic
fibrosis patient suffering chronic pulmonary infection, the method
being for relieving symptoms associated with the chronic pulmonary
infection.
[0059] According to still further features in the described
preferred embodiments the bacteria is growing on a non-living
substratum.
[0060] According to still further features in the described
preferred embodiments the non-living substratum forms a part of a
medical device.
[0061] According to still further features in the described
preferred embodiments the medical device is selected from the group
consisting of an infusion device, a catheter device, a contact lens
device, a dialysis device and a draining device.
[0062] The present invention successfully addresses the
shortcomings of the presently known configurations by providing
methods and compositions effective in combating surface protected
bacteria (e.g., mucoid-, alginate-, biofilm-producing bacteria), by
subjecting such bacteria to glycosaminoglycans degrading enzyme,
rendering such bacteria surface non-protected and therefore more
susceptible to anti-bacterial agents and less capable of adhering
to various living and non-living substrata.
BRIEF DESCRIPTION OF THE DRAWINGS
[0063] The invention herein described, by way of example only, with
reference to the accompanying drawings, wherein:
[0064] FIG. 1 demonstrates the monosaccharide composition of mucoid
P. aeruginosa isolate No. 1 (samples 11, 12) and of three sputum
samples from CF patients (samples 1, 6, 24), as well as of heparan
sulphate standard (HS1), chondroitin sulfate A (G9), and of
monosaccharide standard ladder (G8).
[0065] FIGS. 2a-e demonstrate the effect of heparanase on the
amount of mucus produced by mucoid P. aeruginosa isolate No. 1.
Bacteria were plated on tryptic soy agar (2a-d) or McConkey agar
(2e). Discs soaked with the examined compound were placed on top.
E=heparanase, I=boiled heparanase, B=heparanase buffer,
C=chondroitinase ABC, C-I=boiled chondroitinase, D=DNase,
D-I=boiled Dnase, Hy=hyaluronidase, H-II=heparinase II.
[0066] FIGS. 3a-b demonstrate the effect of heparanase on
sensitivity to antibiotics. (2a) Bacteria were plated on tryptic
soy agar following treatment with (from left to right, top to
bottom) heparanase buffer, heparanase, boiled heparanase,
chondroitinase ABC, hyaluronidase or heparinase II. Antibiotic
discs were placed according to the scheme in FIG. 3b.
[0067] FIGS. 4a-e demonstrate the effect of heparanase on mucoid P.
aeruginosa isolate No. 40 proliferation rate and sensitivity to
antibiotics. (4a) The proliferation rate following treatment with
I=boiled heparanase, E=heparanase, H-II=heparinase II,
Hy=hyaluronidase. (4b-e) The proliferation rate as in FIG. 4a, in
the presence of antibiotics, (4b) amikacin at MIC-2 (0.78
.mu.g/ml); (4c) amikacin at MIC-1 (1.56 .mu.g/ml); (4d) gentamycin
at MIC-2 (0.39 .mu.g/ml); (4e) gentamycin at MIC-1 (0.78
.mu.g/ml).
DESCRIPTION OF THE PREFERRED EMBODIMENTS
[0068] The present invention is of methods and compositions
effective in combating mucoid, alginate-producing and/or
biofilm-producing bacteria, by subjecting such bacteria to
glycosaminoglycans degrading enzymes, which can be used, for
example, to treat infections caused by such virulent bacteria.
Specifically, the present invention can be used for the reduction
of mucus production by mucoid P. aeruginosa and other mucus
producing bacteria, disruption of bacterial biofilms, and
increasing the susceptibility to antibacterial treatment, for the
management of respiratory diseases including, but not limited to,
cystic fibrosis, chronic bronchitis, pulmonary emphysema,
infectious pneumonia, chronic obstructive lung/pulmonary disease
(COLD/COPD), tuberculosis, and fungal infection, to improve
clearance of lung secretions by reducing their viscoelastic
properties, reduce the frequency of respiratory infections and/or
inflammation requiring parenteral antibiotics and/or
anti-inflammatory drugs, respectively, and to improve pulmonary
function, due to degradation of mucus produced by mucus producing
bacteria in general and P. aeruginosa in particular. It is also
recommended for the treatment of infected wounds and bums, ear and
eye infections, urinary tract and other catheter associated
infections.
[0069] The principles and operation of the present invention may be
better understood with reference to the drawings and accompanying
descriptions.
[0070] Before explaining at least one embodiment of the invention
in detail, it is to be understood that the invention is not limited
in its application to the details of construction and the
arrangement of the components set forth in the following
description or illustrated in the drawings. The invention is
capable of other embodiments or of being practiced or carried out
in various ways. Also, it is to be understood that the phraseology
and terminology employed herein is for the purpose of description
and should not be regarded as limiting.
[0071] U.S. patent application Ser. Nos. 08/922,170 and 09/109,386,
which are incorporated by reference as if fully set forth herein
teach the cloning of the heparanase gene. U.S. patent application
Ser. No. 09/071,618, filed May 1, 1998, which is incorporated by
reference as if fully set forth herein, teaches the production of
recombinant heparanase by a variety of heterologous expression
systems and the purification of recombinant heparanase. U.S. patent
application Ser. No. 09/046,475, which is incorporated by reference
as if fully set forth herein, teaches the use of glycosaminoglycans
degrading enzymes for the management, treatment and relieve of
symptoms of respiratory diseases associate with accumulation of
mucoid, mucopurulent or purulent material containing
glycosaminoglycans. The present invention, on the other hand,
relates to the use of glycosaminoglycans degrading enzymes to
combat mucoid-, alginate- and/or biofilm-producing bacteria.
[0072] Thus, according to one aspect of the present invention there
is provided a method of rendering a surface protected bacteria more
susceptible to an anti-bacterial agent. The method is effected by
subjecting the surface protected bacteria to a glycosaminoglycans
degrading enzyme.
[0073] According to another aspect of the present invention there
is provided a method of rendering a surface protected bacteria less
capable of adhering to a substratum. Again, the method is effected
by subjecting the surface protected bacteria to a
glycosaminoglycans degrading enzyme.
[0074] Further in accordance with the teachings of the present
invention there is provided a method of treating a disease for
relieving disease associated symptoms effected by the
administration a therapeutical composition including a
glycosaminoglycans degrading enzyme.
[0075] According to still another aspect of the present invention
there is provided a therapeutic composition for treating a surface
protected bacteria associated disease or symptoms. The comprising a
glycosaminoglycans degrading enzyme and an antibiotic. Examples of
antibiotics are given in the Examples section hereinunder.
[0076] According to yet another aspect of the present invention
there is provided a bactericide composition effective in
eliminating a surface protected bacteria. The bactericide
composition includes a glycosaminoglycans degrading enzyme and a
bactericide.
[0077] As used herein, the term "glycosaminoglycans" refers to
polysaccharide-protein conjugates, such as, but not limited to,
heparan sulfate, hyaluronic acid, chondroitin sulfate, keratan
sulfate I, II, dermatan sulfate and heparin.
[0078] As used herein in the specification and in the claims
section below, the term "surface protected bacteria" refers to
mucoid bacteria, alginate-producing bacteria and/or
biofilm-producing bacteria.
[0079] As used herein in the specification and in the claims
section below, the term "anti-bacterial agent" refers to natural
and man-made agents effective in killing and/or inhibiting the
growth rate of bacteria. Anti-bacterial agents according to the
present invention include bactericides, such as, but not limited
to, biocide SDS, antibiotics and immune moieties, such as
antibodies and immune cells, e.g., macrophages, T cells and the
like.
[0080] The glycosaminoglycans degrading enzyme according to the
present invention can be of any type. It can be a lysosomal
hydrolase or a bacterial lyase. It can be an endoglycosidase, an
exoglycosidase or a sulfatase. It can, for example, be any one or
combination of the following enzymes: heparanse, connective tissue
activating peptide III (CTAP), hyaluronidase, glucoronidase,
iduronate sulfatase, heparinase I, heparinase II heparinase III,
chondroitinase ABC, chondroitinase AC, chondroitinase B and/or
chondroitinase C.
[0081] The surface-protected bacteria according to the present
invention can be of any genus capable of surface protection,
including, but not limited to, Pseudomonas, Azotobacter, Azomonas,
Serpens, Fusobacterium, Klebsiella, Streptococcus, Staphylococcus
or Treponema. Pseudomonas aeruginosa which is known to cause
pulmonary infection in CF patients is of particular interest.
[0082] Thus, the method and composition according to the present
invention is effective in treating lungs of patients suffering
chronic pulmonary infection, associated, for example, with cystic
fibrosis, for relieving symptoms associated therewith. Treating
infected lungs according to the present invention is preferably
effected by an inhaler device designed to generate an aerosol of
the therapeutic composition according to the present invention.
Yet, the present invention is also effective in treating other
organs of the body and/or other conditions, such as, but not
limited to, ears, eyes, teeth, gums, wounds and bums which can be
infected by surface protected bacteria.
[0083] As used herein in the specification and in the claims
section below, the term "treating" when used in conjunction with a
disease refers to substantially inhibiting, slowing or reversing
the progression of a disease, substantially ameliorating clinical
symptoms of a disease or substantially preventing the appearance of
clinical symptoms of a disease.
[0084] For therapeutic and/or prophylactic treatment, the
glycosaminoglycans degrading enzyme according to the present
invention can be formulated in a composition, which may include
thickeners, carriers, buffers, diluents, surface active agents,
preservatives, and the like, all as well known in the art.
Pharmaceutical compositions may also include one or more active
ingredients such as but not limited to anti inflammatory agents,
anti microbial agents, anesthetics, bactericides, antibiotics and
the like in addition to the glycosaminoglycans degrading enzyme.
Other enzymes, such as DNase can also be included for some
applications (e.g., treatment of CF lung).
[0085] The pharmaceutical composition can be administered in either
one or more of ways depending on whether local or systemic
treatment is of choice, and on the area to be treated.
Administration may be done topically (including ophtalmically,
vaginally, rectally, intranasally), orally, by inhalation or
parenterally.
[0086] Formulations for topical administration may include but are
not limited to lotions, ointments, gels, creams, suppositories,
drops, liquids, sprays and powders. Conventional pharmaceutical
carriers, aqueous, powder or oily bases, thickeners and the like
may be necessary or desirable.
[0087] Formulations for oral administration include powders or
granules, suspensions or solutions in water or non-aqueous media,
sachets, capsules or tablets. Thickeners, diluents, flavorings,
dispersing aids, emulsifiers or binders may be desirable.
[0088] Formulations for parenteral administration may include but
are not limited to sterile aqueous solutions which may also contain
buffers, diluents and other suitable additives.
[0089] Dosing is dependent on severity and responsiveness of the
condition to be treated, but will normally be one or more doses per
day, with course of treatment lasting from several days to several
months or until a cure is effected or a diminution of disease state
is achieved. Persons ordinarily skilled in the art can easily
determine optimum dosages, dosing methodologies and repetition
rates.
[0090] The method and bactericide composition according to the
present invention are effective in inhibiting or killing surface
protected bacteria growing on a non-living substratum, which forms,
for example, a part of a medical device, such as, but not limited
to, infusion device, a catheter device, a contact lens device, a
dialysis device and a draining device.
[0091] The present invention successfully addresses the
shortcomings of the presently known configurations by providing new
means to combat surface protected bacteria (e.g., mucoid-,
alginate-, biofilm-producing bacteria), which is effected by
subjecting such bacteria to GAG degrading enzyme, rendering such
bacteria surface non-protected and susceptible to anti-bacterial
agents and less capable of adhering to various substrata.
[0092] Thus, according to the present invention glycosaminoglycans
degrading enzymes, such as, but not limited to, heparanases,
connective tissue activating peptide, heparinases, glucoronidases,
heparitinases, hyaluronidases, sulfatases and chondroitinases are
used, alone, in combination, or in combination with conventional
substances, preferably as aerosol, for the management of diseases
associated with infection by surface protected bacteria.
[0093] The effects of glycosaminoglycans degrading enzymes on the
amounts of bacterial alginate production and the associated
payoffs, in addition to their effects on reducing the viscosity of
sinuses and airway secretions with associated implications derived
from cellular heparan sulfate degradation on curtailing the rate of
infection and inflammatory responses (see U.S. patent application
Ser. No. 09/046,475), will result in reduction and/or prevention of
chronic infection, reduction of tissue damage, thus improving the
patients quality of life and prolong life span.
[0094] Each of the various embodiments and aspects of the present
invention as delineated hereinabove and as claimed in the claims
section below finds experimental support in the Examples section
that follows.
EXAMPLES
[0095] Reference is now made to the following examples, which
together with the above descriptions, illustrate the invention in a
non limiting fashion.
Materials and Experimental Methods
[0096] Bacterial strain: Clinical isolates of a mucoid Pseudomonas
aeruginosa strains from CF patients were kindly provided by Dr. H
Berkovier from the Hadassa hospital, Ein Kerem, and Prof. E. Kerem
from Shaarei Tsedek hospital, Jerusalem. Bacteria were grown on
either McConkey agar plates (Hi Labs, Israel), or tryptic soy broth
(TSB)+1.5% agar (Difco) or 1.5% agarose (FMC BioProducts, USA)
plates, or in tryptic soy broth (Difco Laboratories, USA).
[0097] Monosaccharide composition of P. aeruginosa alginate:
Monosaccharide composition of P. aeruginosa alginate was tested
using FACE Glycosaminoglycan Identification Kit (Glyco, Inc.,
Novato, Calif., USA), according to manufacture's instructions.
[0098] Reduction of alginate:
[0099] 1. TSB+1.5% agar or 1.5% agarose plates were prepared
according to manufactures instructions. Plates were covered with
mucoid P. aeruginosa isolate using a cotton swab. Whatman's 25 mm 3
MM discs were then placed on the seeded bacteria. The discs were
loaded with either 50 .mu.l 0.1 .mu.g/.mu.l recombinant human
heparanase, or 50 .mu.l 0.1 .mu.g/.mu.l heat inactivated (10 min at
100.degree. C.) recombinant human heparanase, or 50 .mu.l
heparanase buffer (10 mM buffer phosphate pH 6.8, 0.4 M NaCl, with
or without 1 mM DTT), or 25 .mu.l (3 units/.mu.l) hyaluronidase
(Sigma), or 25 .mu.l heparinase II (0.01 unit/.mu.l) (kindly
provided by Prof. I. Vlodavsky of the Hebrew University of
Jerusalem)+25 .mu.l saline, or 25 .mu.l (1 unit /.mu.l) DNase
(Promega)+25 .mu.l saline, or 25 .mu.l (0.01 u/.mu.l)
chondroitinase ABC (Sigma)+25 .mu.l saline. The plates were
incubated at 37.degree. C. for 18 hours. The disc's surfaces were
examined for the presence of mucus. Then, each disc was tapped with
a bacterial needle and the bacteria were cultured 18 hours at
37.degree. C. on TS agar plates. The phenotype of the colonies was
observed and further tested for sensitivity to antibiotics.
[0100] 2. A bacterial colony was collected with a sterile tip and
suspended in 500 .mu.l saline. Fifty .mu.l were added to a culture
medium containing 50% TSB, 50% saline, 20 mM citrate-phosphate
buffer pH 5.6 with either heparanase 2.5 .mu.g/ml (50 .mu.l), or 50
.mu.l boiled heparanase, or 50 .mu.l heparanase buffer. Culture was
shaken 18 hours at 37.degree. C. A sample from each tube was
recultured on a TS agar plate and incubated at 37.degree. C., 18
hours, and the cultures phenotype was recorded. 0.5 ml from each
tube was sampled for viscosity testing, using microviscosometer
(Haake, W. Germany).
[0101] Resistance of mucoid vs. nonmucoid bacteria:
[0102] Antibiotic susceptibility test discs: The enzyme-treated
discs were tapped with a coffon swab that was then used to seed the
"treated" bacteria onto a new TS plate. Susceptibility discs were
then placed on the seeded plates and incubated for 18 hours at
37.degree. C. The zone of inhibition of bacterial growth was
measured with a ruler.
[0103] MIC. The minimal inhibitory concentration (MIC) was
determined by serial dilutions of various types of antibiotics
(Enrofloxacin, sulfa/trimethoprim, gentamycin and amikacin), for
various P. aeruginosa isolates obtained.
[0104] The effect of a combined treatment (enzyme and antibiotics)
at either MIC or MIC-1, or the treatment with the enzyme followed
by the antibacterial treatment at MIC, MIC-1 or MIC-2, on the
number of bacteria grown was assessed by O.D. readings at 630
nm.
Experimental Results
[0105] In an attempt to elucidate the composition of the alginate,
and find whether it has the characteristics of glycosaminoglycans
making it a substrate for GAG degrading enzymes, mucoid P.
aeruginosa isolate No. 1 were collected, and analyzed using the GAG
identification kit. The results, which are shown in FIG. 1,
indicate that the alginate has amino sugars and uronic acid and
galactose, which compose the disaccharides units of the GAG.
[0106] In order to test the possibility that GAG degrading enzymes
may reduce the amount of mucoid bacteria or reduce the amount of
alginate, Watmann's 3 MM disc were used following the concept of
susceptibility discs. The discs were placed on a TS agar plate that
was previously seeded with P. aeruginosa isolate No. 1. The
results, which are shown in FIGS. 2a-e, demonstrate that discs
soaked with heparanase, heparinase II and chondroitinase ABC had a
dry appearance, while discs soaked with hyaluronidase, DNase,
buffer, boiled heparanase and DNase were covered with mucus and had
a shiny appearance. Tapping the center of each disc, and replating,
revealed that the discs were contaminated by live mucoid P.
aeruginosa, (not shown). After the addition of 50 .mu.l of
gentamycin at MIC-2 (3.15 .mu.g/ml, MIC=12.5 .mu.g/ml) to the
center of each disc and incubating for 5 hours, the center of each
disc was tapped with a cotton swab and the bacteria were seeded on
TS plates. A variety of susceptibility discs were then placed on
the seeded plates and incubated for 18 hours at 37.degree. C. The
results, which are presented in FIGS. 3a-b and Table 1 below,
reveal that the bacteria that were treated by heparanase,
heparinase II and chondroitinase ABC followed by gentamycin at
MIC-1, were more susceptible to 5 of 7 antibiotics tested and
produced less alginate, as is compared to the bacteria treated by
hyaluronidase, buffer and boiled heparanase.
1TABLE 1 Diameter (mm) of bacterial growth inhibition E I B Hy H-II
C Amikacin 26 23 22 21 24 25 Doxylin 29 24 19 22 27 27 Ciproflox 18
15 13 12 18 17 Enroflox 21 15 13 14 18 21 Gentam 23 19 17 17 21 23
SxT ns ns ns ns ns ns Amox/Cla ns ns ns ns ns ns E = heparanase, I
= boiled heparanase, B = heparanase buffer, Hy = hyaluronidase,
H-II = heparinase II, C = chondroitinase ABC, ns = not
sensitive.
[0107] In order to further establish the supposition that
heparanase may induce changes in alginate producing bacterial
count, changes in the total amount of alginate, and alterations in
sensitivity to antibiotics, P. aeruginosa (isolate No. 40) was
seeded on a TS agar plate. 3 MM discs soaked with either
heparanase, boiled heparanase, heparanase-buffer, hyaluronidase or
heparinase II were placed on top. After 18 hours of incubation at
37.degree. C., the discs that were treated with heparanase, boiled
heparanase, heparanase buffer and heparinase-II had a dry
appearance, and the disc that was treated with hyaluronidase had a
shiny appearance. This bacterial isolate produced less alginate in
general and was more sensitive to antibiotics (Table 2). The disks
that were soaked with either heparanase, boiled heparanase,
heparinase II or hyaloronidase were then washed in 50 ml tubes
containing TSB. The bacteria containing broth was further diluted
and divided into 96 well plates with various antibiotics at
different concentrations (MIC-1, MIC-2). The results, which are
summarized in FIGS. 4a-e, show that the proliferation rate of
bacteria treated previously with heparanase or heparinase II is
slower compared to bacteria treated with boiled heparanase or
hyaluronidase.
2TABLE 2 Minimal inhibitory concentration Isolate No. Gentamycin
Enrofloxacin Amikacin No. 1 12.5 6.25 25 No. 38 12.5 6.25 nd No. 39
3.12 0.78 nd No. 40 1.56 3.12 3.12 No. 41 0.78 1.56 nd nd = not
determined
[0108] Thus, heparanase, heparinase II and chondroitinase ABC, all
of which are GAG degrading enzymes, induce phenotypic changes of
mucoid P. aeruginosa, exhibiting a reduced amount of alginate,
slower proliferation rate and higher susceptibility to a wide range
of antibiotics. The mechanism by which these enzymes induce these
changes is still under investigation, yet several possible
mechanisms are postulated herein in a non-limiting fashion (i) the
enzymes degrade the alginate; (ii) the enzymes interfere with
alginate production; (iii) the enzymes interfere with intercellular
signaling for alginate production or alginate degradation by
lyases; (iv) the enzymes inhibit bacterial proliferation.
[0109] Although the invention has been described in conjunction
with specific embodiments thereof, it is evident that many
alternatives, modifications and variations will be apparent to
those skilled in the art. modifications and variations will be
apparent to those skilled in the art. Accordingly, it is intended
to embrace all such alternatives, modifications and variations that
fall within the spirit and broad scope of the appended claims.
* * * * *