U.S. patent application number 10/787627 was filed with the patent office on 2004-08-26 for combination of antimicrobial agents and bacterial interference to coat medical devices.
Invention is credited to Darouiche, Rabih O., Hull, Richard.
Application Number | 20040166102 10/787627 |
Document ID | / |
Family ID | 22783988 |
Filed Date | 2004-08-26 |
United States Patent
Application |
20040166102 |
Kind Code |
A1 |
Darouiche, Rabih O. ; et
al. |
August 26, 2004 |
Combination of antimicrobial agents and bacterial interference to
coat medical devices
Abstract
This invention relates to a method for coating a medical device
comprising the steps of applying to at least a portion of the
surface of said medical device, an antimicrobial coating layer and
a non-pathogenic bacterial coating layer, wherein the antimicrobial
and non-pathogenic bacterial coating layers inhibit the growth of
pathogenic bacterial and fungal organisms. The non-pathogenic
bacterium used in the bacterial coating layer is resistant to the
antimicrobial agent. Furthermore, the non-pathogenic bacterium
layer includes at least one of the following: viable whole cells,
non-viable whole cells, or cellular structures or extracts. The
antimicrobial agent and non-pathogenic bacterium are used to
develop a kit comprising these compositions in one container or in
separate containers. The kit is used to coat a catheter prior to
implantation in a mammal.
Inventors: |
Darouiche, Rabih O.;
(Houston, TX) ; Hull, Richard; (Houston,
TX) |
Correspondence
Address: |
FULBRIGHT & JAWORSKI, LLP
1301 MCKINNEY
SUITE 5100
HOUSTON
TX
77010-3095
US
|
Family ID: |
22783988 |
Appl. No.: |
10/787627 |
Filed: |
February 26, 2004 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
10787627 |
Feb 26, 2004 |
|
|
|
09877898 |
Jun 8, 2001 |
|
|
|
6719991 |
|
|
|
|
60210715 |
Jun 9, 2000 |
|
|
|
Current U.S.
Class: |
424/93.45 ;
427/2.24 |
Current CPC
Class: |
A61L 2300/30 20130101;
A61L 2300/45 20130101; A61L 29/005 20130101; A61L 2420/02 20130101;
A61L 29/08 20130101; A61L 29/16 20130101; A61L 2300/61 20130101;
A61L 2300/404 20130101; A61L 2300/406 20130101 |
Class at
Publication: |
424/093.45 ;
427/002.24 |
International
Class: |
A61K 045/00; B05D
003/00 |
Goverment Interests
[0002] The work herein was supported by grants from the United
States Government. The United States government may have certain
rights in the invention.
Claims
We claim:
1. A kit comprising compositions to coat the surfaces of medical
devices prior to implantation into a mammal comprising an
antimicrobial agent and a culture from a non-pathogenic bacterium,
wherein said non-pathogenic bacterium has been genetically modified
to enhance the adherence of the bacterium to the implant
surface.
2. The kit of claim 1, wherein the compositions are in the same
container.
3. The kit of claim 1, wherein the compositions are in different
containers.
4. A kit comprising compositions to coat the surfaces of medical
devices prior to implantation into a mammal comprising an
antimicrobial agent and a culture from a non-pathogenic bacterium,
wherein said non-pathogenic bacterium has been genetically modified
to decrease the sensitivity of the bacterium to antimicrobial
agents.
5. A kit comprising compositions to coat the surfaces of medical
devices prior to implantation into a mammal comprising an
antimicrobial agent and a culture from a non-pathogenic bacterium,
wherein said non-pathogenic bacterium has been genetically modified
to increase the stability of the bacterium at room temperature.
6. A kit comprising compositions to coat the surfaces of medical
devices prior to implantation into a mammal comprising an
antimicrobial agent and a culture from a non-pathogenic bacterium,
wherein said non-pathogenic bacterium has been lyophilized and
reconstituted prior to application to the surface of the
implant.
7. A kit comprising a medical device pre-coated with an
antimicrobial agent and compositions to coat said medical device
prior to implantation into a mammal comprising a culture from a
non-pathogenic bacterium.
8. The kit of claim 7, wherein said antimicrobial agent is selected
from the group consisting of an antibiotic, an antiseptic, a
disinfectant and a combination thereof.
9. The kit of claim 8, wherein said antimicrobial agent is selected
from the group of antibiotics consisting of penicillins,
cephalosporins, carbepenems, other beta-lactams antibiotics,
aminoglycosides, macrolides, lincosamides, glycopeptides,
tetracylines, chloramphenicol, quinolones, fucidins, sulfonamides,
trimethoprims, rifamycins, oxalines, streptogramins, lipopeptides,
ketolides, polyenes, azoles, and echinocandins.
10. The kit of claim 8, wherein said antimicrobial agent is
selected from the group of antiseptics consisting of
.alpha.-terpineol, methylisothiazolone, cetylpyridinium chloride,
chloroxyleneol, hexachlorophene, chlorhexidine and other cationic
biguanides, methylene chloride, iodine and iodophores, triclosan,
taurinamides, nitrofurantoin, methenamine, aldehydes, azylic acid,
silver, benzyl peroxide, alcohols, and carboxylic acids and
salts.
11. The kit of claim 7, wherein said non-pathogenic bacterium is a
gram-negative bacterium.
12. The kit of claim 11, wherein said gram-negative bacterium is
selected from the group consisting of Enterobacteriacea,
Pseudomonas aeruginosa, Stenotrophomonas maltophilia, Burkholderia
cepacia, Gardnerella vaginalis, and Acinetobacter species.
13. The kit of claim 11, wherein said non-pathogenic gram-negative
bacterium is selected from the group of Enterobacteriacea
consisting of Escherichia, Shigella, Edwardsiella, Salmonella,
Citrobacter, Klebsiella, Enterobacter, Hafnia, Serratia, Proteus,
Morganella, Providencia, Yersinia, Erwinia, Buttlauxella, Cedecea,
Ewingella, Kluyvera, Tatumella and Rahnella.
Description
[0001] This application is a divisional application of application
Ser. No. 09/877,898 filed Jun. 8, 2001 which claims priority to
Provisional Application No. 60/210,715, which was filed on Jun. 9,
2000.
FIELD OF INVENTION
[0003] The present invention relates to a method of coating a
medical device with an antimicrobial agent and a non-pathogenic
bacterium which is resistant to the antimicrobial coating.
Additionally, the invention relates to a kit that contains
compositions of the antimicrobial agent and the non-pathogenic
bacterium that are applied to the medical device before
implantation in the mammal. Furthermore, this invention relates to
a method for preventing a urinary tract infection comprising the
use of an antimicrobial agent and a non-pathogenic bacterium.
BACKGROUND OF INVENTION
[0004] Indwelling vascular and urinary catheters are becoming
essential in the management of hospitalized patients. Implanted
orthopedic devices are also becoming more prevalent, partly to meet
the needs of a growing elderly population. The benefit derived from
these catheters and orthopedic devices, as well as other types of
medical devices is often offset by infectious complications.
[0005] The most common hospital-acquired infection is urinary tract
infection (UTI). The majority of cases of UTI are associated with
the use of urinary catheters, including transurethral foley,
suprapubic and nephrostomy catheters. These urinary catheters are
inserted in a variety of populations, including the elderly, stroke
victims, spinal cord-injured patients, post-operative patients, and
those with obstructive uropathy. Despite adherence to sterile
guidelines for the insertion and maintenance of urinary catheters,
catheter-associated UTI continues to pose a major problem. For
instance, it is estimated that almost one-quarter of hospitalized
spinal cord-injured patients develop symptomatic UTI during their
hospital course. Gram-negative bacilli account for almost 60-70%,
enterococci for about 25% and Candida species for about 10% of
cases of UTI.
[0006] Colonization of bacteria on the surfaces of the implant or
other part of the device can produce serious patient problems,
including the need to remove and/or replace the implanted device
and to vigorously treat secondary infective conditions. A
considerable amount of attention and study has been directed toward
preventing such colonization by the use of antimicrobial agents,
such as antibiotics, bound to the surface of the materials employed
in such devices.
[0007] Various methods have previously been employed to contact or
coat the surfaces of medical devices with an antimicrobial agent.
For example, one method would be to flush the surfaces of the
device with an antimicrobial containing solution. Generally, the
flushing technique would require convenient access to the
implantable device. For example, catheters are generally amenable
to flushing with a solution of rifampin and minocycline or rifampin
and novobiocin. For use in flushing solutions, the effective
concentration of the antibiotic would range from about 1 to 10
mg/ml for minocycline, preferably about 2 mg/ml; 1 to 10 mg/ml for
rifampin, preferably about 2 mg/ml; and 1 to 10 mg/ml for
novobiocin, preferably about 2 mg/ml. The flushing solution would
normally be composed of sterile water or sterile normal saline
solutions.
[0008] A known method of coating the devices is to first apply or
absorb to the surface of the medical device a layer of
tridodecylmethyl ammonium chloride (TDMAC) surfacant followed by an
antiobiotic coating layer. For example, a medical device having a
polymeric surface, such as polyethylene, silastic esaltomers,
polytetrafluoroethylene or Dacron, can be soaked in a 5% by weight
solution of TDMAC for 30 minutes at room temperature, air dried,
and rinsed in water to remove excess TDMAC. Alternatively, TDMAC
precoated vascular catheters are commercially available. The device
carrying the absorbed TDMAC surfactant coating can then be
incubated in an antibiotic solution for up to one hour or so,
allowed to dry, then washed in sterile water to remove unbound
antibiotic and stored in a sterile package until ready for
implantation. In general, the antiobiotic solution is composed of a
concentration of 0.01 mg/ml to 60 mg/ml of each antiobiotic in an
aqueous pH 7.4-7.6 buffered solution, sterile water, or methanol.
According to one method, an antibiotic solution of 60 mg of
minocycline and 30 mg of rifampin per ml of solution is applied to
the TDMAC coated catheter.
[0009] Another successful coating method is impregnation of an
antimicrobial agent. The antimicrobial agent penetrates and is
incorporated in the exposed surfaces. The antimicrobial composition
is formed by dissolving an antimicrobial agent in an organic
solvent, adding a penetrating agent, and adding an alkalinizing
agent to the composition. The composition is heated to a
temperature between 30.degree. C. and 70.degree. C. prior to
applying to the medical device. See, e.g., U.S. Pat. No. 5,902,283
and U.S. Pat. No. 5,624,704.
[0010] A further method known to coat the surface of medical
devices with antiobiotics involves first coating the selected
surfaces with benzalkonium chloride followed by ionic bonding of
the antiobiotic composition. See, e.g., Solomon, D. D. and
Sherertz, R. J., J. Controlled Release, 6:343-352 (1987) and U.S.
Pat. No. 4,442,133.
[0011] These and many other methods of coating medical devices with
antibiotics appear in numerous patents and medical journal
articles. Practice of the prior art coating methods results in a
catheter or medical device wherein only the surface of the device
is coated with an antibiotic. While the surface coated catheter
does provide effective protection against bacteria initially, the
effectiveness of the coating diminishes over time. During use of
the medical device or catheter, the antimicrobials leach from the
surface of the device into the surrounding environment. Over a
period of time, the amount of antibiotics present on the surface
decreases to a point where the protection against bacteria is no
longer effective.
[0012] Previously there have been several approaches to prophylaxis
of urinary tract infection in chronically catheter dependent
patients. Antibacterial compounds applied at the urethral meatus,
silver impregnated catheters, intravesical instillation of various
chemicals and antimicrobial agents, such as methenamine, cranberry
juice and ascorbic acid, have been used with mixed success at best.
Prophylactic oral antibiotics may reduce the incidence of
asymptomatic bacteruria in patients on clean intermittent
catheterization but do not reduce that of symptomatic infection. A
prospective study found a higher incidence of symptomatic infection
among patients who received prophylactic antibiotics. Furthermore,
prolonged treatment with antimicrobial agents, creates drug
resistant pathogens, breakthrough infections and disruption of the
normal flora.
[0013] With the world wide emergence of increased antibiotic
resistant agents, an interest has developed in the use of bacterial
interference as a means to cope with this problem. In nature,
bacteria interact with each other as they attempt to establish
themselves and dominate their environment. Some of the interactions
are synergistic, whereas others are antagonistic. It has been
suggested that these antagonistic interactions, so-called bacterial
interference, may act in the prevention of certain infectious
diseases. Bacterial interference operates through several
mechanisms, i.e., production of antagonistic substances, changes in
the bacterial microenvironment, and reduction of needed nutritional
substances. Typically, the therapeutic approach of using bacterial
interference involves the implantation of low-virulence bacterial
strains that are potentially capable of interfering with the
colonization and infection of more virulent microorganisms.
[0014] In recent years, the use of Lactobacillus has been
investigated as a possible treatment for UTI. It is well known that
indigenous, non-pathogenic bacteria predominate on intestinal,
vaginal and uro-epithelial cells and associated mucus in the health
state, and that pathogenic organisms (such as bacteria, yeast and
viruses) predominate in the stages leading to and during
infections. Organisms such as Escherichia coli, enterococci,
Candida, Gardnerella and Klebsiella originate from the bowel,
colonize the perineum, vagina, urethra and can infect the bladder
and vagina. See e.g., U.S. Pat. No. 5,645,830 and U.S. Pat. No.
6,004,551.
[0015] In addition to the increased risk of infection associated
with the use of urinary catheters, these patients are subjected to
an increase in medical expenses. Typically, urinary catheters are
replaced every 2-4 weeks. This time frame was established by the
medical community based upon the safety concern of a biofilm of
pathogenic bacteria developing on the catheter surface. Thus,
patients may need to schedule an appointment every 2-4 weeks to
have the catheter replaced resulting in the expense of office
visits and the cost of approximately 24 catheters per year.
[0016] There is a general appreciation in the medical community
that better methods to prevent the development of urinary
catheter-associated UTI are needed. This invention describes for
the first time the use of a non-pathogenic bacterium in combination
with an antimicrobial agent to prevent UTI. It is noteworthy that
the non-pathogenic bacterium used in this invention had been
previously considered a pathogenic bacterium that results in UTI,
thus suggesting, that this invention is indeed non-obvious.
[0017] Furthermore, this invention addresses the long-felt need of
reducing the medical expenses incurred by patients that require a
urinary catheter. Coating a urinary catheter with both an
antimicrobial agent and a non-pathogenic bacterium will prolong the
time frame between replacements of catheters. This invention could
potentially increase the time from 2-4 weeks up to several months,
thus, the amount of catheters and incurred medical expenses are
reduced.
SUMMARY OF THE INVENTION
[0018] An embodiment of the present invention is a method for
coating a medical device comprising the steps of applying to at
least a portion of the surface of said medical device, an
antimicrobial coating layer, wherein said antimicrobial coating
layer comprises an antimicrobial agent in an effective
concentration to inhibit the growth of bacterial and fungal
organisms relative to uncoated medical devices; and applying to at
least a portion of the surface of said medical device, a
non-pathogenic bacterial coating layer, wherein said non-pathogenic
bacterial coating layer comprises a non-pathogenic gram-negative
bacterium in an effective concentration to inhibit the growth of
pathogenic bacterial and fungal organisms, wherein said
non-pathogenic gram-negative bacterium is resistant to said
antimicrobial agent.
[0019] In specific embodiments, the antimicrobial agent is selected
from the group consisting of an antibiotic, an antiseptic, a
disinfectant and a combination thereof. The present invention also
encompasses lipid and other complex formulations of antimicrobial
agents or derivatives thereof.
[0020] Another specific embodiment is that the antimicrobial agent
is selected from the group of antibiotics consisting of
penicillins, cephalosporins, carbepenems, other beta-lactams
antibiotics, aminoglycosides, macrolides, lincosamides,
glycopeptides, tetracylines, chloramphenicol, quinolones, fucidins,
sulfonamides, trimethoprims, rifamycins, oxalines, streptogramins,
lipopeptides, ketolides, polyenes, azoles, and echinocandins.
[0021] A further embodiment of the present invention is that the
antimicrobial agent is selected from the group of antiseptics
consisting of .alpha.-terpineol, methylisothiazolone,
cetylpyridinium chloride, chloroxyleneol, hexachlorophene,
chlorhexidine and other cationic biguanides, methylene chloride,
iodine and iodophores, triclosan, taurinamides, nitrofurantoin,
methenamine, aldehydes, azylic acid, silver, benzyl peroxide,
alcohols, and carboxylic acids and salts.
[0022] In specific embodiments of the present invention, the
non-pathogenic gram-negative bacterium is selected from the group
consisting of Enterobacteriacea, Pseudomonas aeruginosa,
Stenotrophomonas maltophilia, Burkholderia cepacia, Gardnerella
vaginalis, and Acinetobacter species.
[0023] A specific embodiment is that the non-pathogenic
gram-negative bacterium is Pseudomonas aeruginosa.
[0024] Another embodiment of the present invention is that the
non-pathogenic gram-negative bacterium is selected from the group
of Enterobacteriacea consisting of Escherichia, Shigella,
Edwardsiella, Salmonella, Citrobacter, Klebsiella, Enterobacter,
Hafnia, Serratia, Proteus, Morganella, Providencia, Yersinia,
Erwinia, Buttlauxella, Cedecea, Ewingella, Kluyvera, Tatumella and
Rahnella.
[0025] In a specific embodiment, the Enterobacteriacea is
Escherichia coli 83972 (E. coli 83972) or mutants thereof.
[0026] In a further embodiment of the present invention, the
non-pathogenic gram-negative bacterium is a bacterium which adheres
to urinary catheters selected from the group consisting of
Providencia, Proteus, Pseudomonas aeruginosa, Escherichia coli, and
other urinary organisms.
[0027] A further embodiment is that the non-pathogenic bacterial
coating layer further comprises viable whole cells of the
non-pathogenic gram-negative bacterium.
[0028] Another embodiment is that the non-pathogenic bacterial
coating layer further comprises non-viable whole cells or cellular
structures or extracts of the non-pathogenic gram-negative
bacterium.
[0029] In a further embodiment, the non-pathogenic bacterial
coating layer further comprises at least one or more viable whole
cells, non-viable whole cells or cellular structures or extracts of
the non-pathogenic gram-negative bacterium.
[0030] Another embodiment of the present invention is that the
non-pathogenic bacterial coating layer further comprises at least
two non-pathogenic gram-negative bacteria.
[0031] An embodiment of the present invention is a method for
coating a medical device comprising the steps of applying to at
least a portion of the surface of said medical device, an
antimicrobial coating layer, wherein said antimicrobial coating
layer comprises an antimicrobial agent in an effective
concentration to inhibit the growth of bacterial and fungal
organisms relative to uncoated medical devices; and applying to at
least a portion of the surface of said medical device, a
non-pathogenic bacterial coating layer, wherein said non-pathogenic
bacterial coating layer comprises non-pathogenic gram-positive
bacterium in an effective concentration to inhibit the growth of
pathogenic bacterial and fungal organisms, wherein said
non-pathogenic gram-positive bacterium is resistant to said
antimicrobial agent.
[0032] In specific embodiments, the non-pathogenic gram-positive
bacterium is selected from the group consisting of Staphylococcus
aureus, coagulase-negative staphylococci, streptococci,
enterococci, corynebacteria, and Bacillus species.
[0033] In another specific embodiment, the antimicrobial agent is
selected from the group of antibiotics consisting of penicillins,
cephalosporins, carbepenems, other beta-lactams antibiotics,
aminoglycosides, macrolides, lincosamides, glycopeptides,
tetracylines, chloramphenicol, quinolones, fucidins, sulfonamides,
trimethoprims, rifamycins, oxalines, streptogramins, lipopeptides,
ketolides, polyenes, azoles, and echinocandins.
[0034] A further embodiment of the present invention is that the
non-pathogenic bacterial coating layer further comprises viable
whole cell of the non-pathogenic gram-positive bacterium.
[0035] Another specific embodiment is that the non-pathogenic
bacterial coating layer further comprises non-viable whole cells or
cellular structures or extracts of the non-pathogenic gram-positive
bacterium.
[0036] In specific embodiments, the non-pathogenic bacterial
coating layer further comprises at least one or more viable whole
cells, non-viable whole cells or cellular structures or extracts of
the non-pathogenic gram-positive bacterium.
[0037] In a further embodiment, the non-pathogenic bacterial
coating layer further comprises at least two non-pathogenic
gram-positive bacteria.
[0038] Another specific embodiment is that the non-pathogenic
bacterial coating layer further comprises at least one
non-pathogenic gram-positive bacterium and at least one
non-pathogenic gram-negative bacterium.
[0039] Another embodiment of the present invention is a method for
coating a medical device comprising the steps of applying to at
least a portion of the surface of said medical device, an
antimicrobial coating layer, wherein said antimicrobial coating
layer comprises an antimicrobial agent in an effective
concentration to inhibit the growth of bacterial and fungal
organisms relative to uncoated medical devices; and applying to at
least a portion of the surface of said medical device, a fungal
coating layer, wherein said fungal coating layer comprises a fungus
in an effective concentration to inhibit the growth of pathogenic
bacterial and fungal organisms, wherein said fungus is resistant to
said antimicrobial agent. A specific embodiment is that the fungus
is Candida.
[0040] A further embodiment of the present invention is a method
for preventing a urinary tract infection comprising the steps of
pre-treating a patient with antibiotics for five to seven days;
inoculating said patient with a culture of non-pathogenic
bacterium; and applying to at least a portion of the surface of a
urinary catheter, an antimicrobial coating layer having an
antimicrobial agent in an effective concentration to inhibit the
growth of bacterial and fungal organisms relative to uncoated
medical devices.
[0041] A specific embodiment of the present invention is a kit
comprising compositions to coat the surfaces of medical devices
prior to implantation into a mammal comprising an antimicrobial
agent and a culture from a non-pathogenic bacterium, wherein said
non-pathogenic bacterium has been genetically modified to enhance
the adherence of the bacterium to the implant surface. In a further
embodiment of the kit, the compositions are in the same container.
In another embodiment of the kit, the compositions are in different
containers.
[0042] Another specific embodiment of the present invention is a
kit comprising compositions to coat the surfaces of medical devices
prior to implantation into a mammal comprising an antimicrobial
agent and a culture from a non-pathogenic bacterium, wherein said
non-pathogenic bacterium has been genetically modified to the
decrease the sensitivity of the bacterium to antimicrobial
agents.
[0043] A further embodiment is a kit comprising compositions to
coat the surfaces of medical devices prior to implantation into a
mammal comprising an antimicrobial agent and a culture from a
non-pathogenic bacterium, wherein said non-pathogenic bacterium has
been genetically modified to increase the stability of the
bacterium at room temperature.
[0044] Another embodiment of the present invention is a kit
comprising compositions to coat the surfaces of medical devices
prior to implantation into a mammal comprising an antimicrobial
agent and a culture from a non-pathogenic bacterium, wherein said
non-pathogenic bacterium has been lyophilized and reconstituted
prior to application to the surface of the implant.
[0045] Yet further, another embodiment of the present invention is
a kit comprising a medical device pre-coated with an antimicrobial
agent and compositions to coat said medical device prior to
implantation into a mammal comprising a culture from a
non-pathogenic bacterium.
[0046] Other and further objects, features and advantages would be
apparent and eventually more readily understood by reading the
following specification and or any examples of the present
preferred embodiments of the invention are given for the purpose of
the disclosure.
DETAILED DESCRIPTION OF THE INVENTION
[0047] As used herein in the specification, "a" or "an" may mean
one or more. As used herein in the claim(s), when used in
conjunction with the word "comprising", the words "a" or "an" may
mean one or more than one. As used herein "another" may mean at
least a second or more.
[0048] The term "antiseptics" as used herein is defined as an
antimicrobial substance that inhibits the action of microorganisms,
including but not limited to .alpha.-terpineol,
methylisothiazolone, cetylpyridinium chloride, chloroxyleneol,
hexachlorophene, chlorhexidine and other cationic biguanides,
methylene chloride, iodine and iodophores, triclosan, taurinamides,
nitrofurantoin, methenamine, aldehydes, azylic acid, silver, benzyl
peroxide, alcohols, and carboxylic acids and salts.
[0049] One skilled in the art is cognizant that these antiseptics
can be used in combinations of two or more to obtain a synergistic
effect. Furthermore, the antiseptics are dispersed along the
surface of the medical device.
[0050] Some examples of combinations of antiseptics include a
mixture of chlorhexidine, chlorhexidine and chloroxylenol,
chlorhexidine and methylisothiazolone, chlorhexidine and
.alpha.-terpineol, methylisothiazolone and .alpha.-terpineol;
thymol and chloroxylenol; chlorhexidine and cetylpyridinium
chloride; or chlorhexidine, methylisothiazolone and thymol. These
combinations provide a broad spectrum of activity against a wide
variety of organisms.
[0051] The term "antibiotics" as used herein is defined as a
substance that inhibits the growth of microorganisms without damage
to the host. For example, the antibiotic may inhibit cell wall
synthesis, protein synthesis, nucleic acid synthesis, or alter cell
membrane function.
[0052] Classes of antibiotics that can possibly be used include,
but are not limited to, macrolides (i.e., erythromycin),
penicillins (i.e., nafcillin), cephalosporins (i.e., cefazolin),
carbepenems (i.e., imipenem, aztreonam), other beta-lactam
antibiotics, beta-lactam inhibitors (i.e., sulbactam), oxalines
(i.e. linezolid), aminoglycosides (i.e., gentamicin),
chloramphenicol, sufonamides (i.e., sulfamethoxazole),
glycopeptides (i.e., vancomycin), quinolones (i.e., ciprofloxacin),
tetracyclines (i.e., minocycline), fusidic acid, trimethoprim,
metronidazole, clindamycin, mupirocin, rifamycins (i.e., rifampin),
streptogramins (i.e., quinupristin and dalfopristin) lipoprotein
(i.e., daptomycin), polyenes (i.e., amphotericin B), azoles (i.e.,
fluconazole), and echinocandins (i.e., caspofungin acetate).
[0053] Examples of specific antibiotics that can be used include,
but are not limited to, erythromycin, nafcillin, cefazolin,
imipenem, aztreonam, gentamicin, sulfamethoxazole, vancomycin,
ciprofloxacin, trimethoprim, rifampin, metronidazole, clindamycin,
teicoplanin, mupirocin, azithromycin, clarithromycin, ofloxacin,
lomefloxacin, norfloxacin, nalidixic acid, sparfloxacin,
pefloxacin, amifloxacin, gatifloxacin, moxifloxacin, gemifloxacin,
enoxacin, fleroxacin, minocycline, linezolid, temafloxacin,
tosufloxacin, clinafloxacin, sulbactam, clavulanic acid,
amphotericin B, fluconazole, itraconazole, ketoconazole, and
nystatin. Other examples of antibiotics, such as those listed in
Sakamoto et al, U.S. Pat. No. 4,642,104 herein incorporated by
reference will readily suggest themselves to those of ordinary
skill in the art.
[0054] The term "bacterial interference" as used herein is defined
as an antagonistic interactions among bacteria to establish
themselves and dominate their environment. Bacterial interference
operates through several mechanisms, i.e., production of
antagonistic substances, changes in the bacterial microenvironment,
and reduction of needed nutritional substances.
[0055] The term "coating" as used herein is defined as a layer of
material covering a medical device. The coating can be applied to
the surface or impregnated within the material of the implant.
[0056] The term "effective concentration" means that a sufficient
amount of the antimicrobial agent is added to decrease, prevent or
inhibit the growth of bacterial and/or fungal organisms. The amount
will vary for each compound and upon known factors such as
pharmaceutical characteristics; the type of medical device; age,
sex, health and weight of the recipient; and the use and length of
use. It is within the skilled artisan's ability to relatively
easily determine an effective concentration for each compound.
[0057] The term "gram-negative bacteria" or "gram-negative
bacterium" as used herein is defined as bacteria which have been
classified by the Gram stain as having a red stain. Gram-negative
bacteria have thin walled cell membranes consisting of a single
layer of peptidoglycan and an outer layer of lipopolysacchacide,
lipoprotein, and phospholipid. Exemplary organisms include, but are
not limited to, Enterobacteriacea consisting of Escherichia,
Shigella, Edwardsiella, Salmonella, Citrobacter, Klebsiella,
Enterobacter, Hafnia, Serratia, Proteus, Morganella, Providencia,
Yersinia, Erwinia, Buttlauxella, Cedecea, Ewingella, Kluyvera,
Tatumella and Rahnella. Other exemplary gram-negative organisms not
in the family Enterobacteriacea include, but are not limited to,
Pseudomonas aeruginosa, Stenotrophomonas maltophilia, Burkholderia,
Cepacia, Gardenerella, Vaginalis, and Acinetobacter species.
[0058] The term "gram-positive bacteria" or "gram-positive
bacterium" as used herein refers to bacteria, which have been
classified using the Gram stain as having a blue stain.
Gram-positive bacteria have a thick cell membrane consisting of
multiple layers of peptidoglycan and an outside layer of teichoic
acid. Exemplary organisms include, but are not limited to,
Staphylococcus aureus, coagulase-negative staphylococci,
streptococci, enterococci, corynebacteria, and Bacillus
species.
[0059] The term "medical device" as used herein refers to any
material, natural or artificial that is inserted into a mammal.
Particular medical devices especially suited for application of the
antimicrobial combinations of this invention include, but are not
limited to, peripherally insertable central venous catheters,
dialysis catheters, long term tunneled central venous catheters,
long term non-tunneled central venous catheters, peripheral venous
catheters, short-term central venous catheters, arterial catheters,
pulmonary artery Swan-Ganz catheters, urinary catheters, artificial
urinary sphincters, long term urinary devices, urinary dilators,
urinary stents, other urinary devices, tissue bonding urinary
devices, penile prostheses, vascular grafts, vascular catheter
ports, vascular dilators, extravascular dilators, vascular stents,
extravascular stents, wound drain tubes, hydrocephalus shunts,
ventricular catheters, peritoneal catheters, pacemaker systems,
small or temporary joint replacements, heart valves, cardiac assist
devices and the like and bone prosthesis, joint prosthesis and
dental prosthesis.
[0060] The term "mutant" as defined herein refers to a bacterium
that has been mutated using standard mutagenesis techniques such as
site-directed mutagenesis. One skilled in the art recognizes that
the term mutant includes, but is not limited to base changes,
truncations, deletions or insertions of the wild-type bacterium.
Thus, the size of the mutant bacterium may be larger or smaller
than the wild-type or native bacterium. Yet further, one skilled in
the art realizes that the term mutant also includes different
strains of bacteria or bacteria that has been chemically or
physically modified as used herein.
[0061] The term "non-pathogenic bacteria" or "non-pathogenic
bacterium" includes all known and unknown non-pathogenic bacterium
(gram positive or gram negative) and any pathogenic bacteria that
has been mutated or converted to a non-pathogenic bacterium.
Furthermore, a skilled artisan recognizes that some bacteria may be
pathogenic to specific species and non-pathogenic to other species;
thus, these bacteria can be utilized in the species in which it is
non-pathogenic or mutated so that it is non-pathogenic.
[0062] One specific embodiment of the present invention is a method
for coating a medical device comprising the steps of applying to at
least a portion of the surface of said medical device, an
antimicrobial coating layer, wherein said antimicrobial coating
layer comprises an antimicrobial agent in an effective
concentration to inhibit the growth of bacterial and fungal
organisms relative to uncoated medical devices; and applying to at
least a portion of the surface of said medical device, a
non-pathogenic bacterial coating layer, wherein said non-pathogenic
bacterial coating layer comprises a non-pathogenic gram-negative
bacterium in an effective concentration to inhibit the growth of
pathogenic bacterial and fungal organisms, wherein said
non-pathogenic gram-negative bacterium is resistant to said
antimicrobial agent.
[0063] The medical devices that are amenable to impregnation by the
antimicrobial combinations are generally comprised of a
non-metallic material such as thermoplastic or polymeric materials.
Examples of such materials are rubber, plastic, polyethylene,
polyurethane, silicone, Gortex (polytetrafluoroethylene), Dacron
(polyethylene tetraphthalate), polyvinyl chloride, Teflon
(polytetrafluoroethylene), latex, elastomers, nylon and Dacron
sealed with gelatin, collagen or albumin.
[0064] The amount of each antimicrobial agent used to coat the
medical device varies to some extent, but is at least a sufficient
amount to form an effective concentration to inhibit the growth of
bacterial and fungal organisms.
[0065] The antimicrobial agents can be used alone or in combination
of two or more of them. The antimicrobial agents are dispersed
throughout the surface of the medical device. The amount of each
antimicrobial agent used to impregnate the medical device varies to
some extent, but is at least of an effective concentration to
inhibit the growth of bacterial and fungal organisms.
[0066] The antimicrobial agent and the non-pathogenic bacteria can
be applied to the medical device in a variety of methods. Exemplary
application methods include, but are not limited to, spraying,
painting, dipping, sponging, atomizing, smearing, impregnating and
spreading.
[0067] A skilled artisan is cognizant that the development of
microorganisms in culture media is dependent upon a number of very
important factors, e.g., the proper nutrients must be available;
oxygen or other gases must be available as required; a certain
degree of moisture is necessary; the media must be of the proper
reaction; proper temperature relations must prevail; the media must
be sterile; and contamination must be prevented.
[0068] A satisfactory microbiological culture contains available
sources of hydrogen donors and acceptors, carbon, nitrogen, sulfur,
phosphorus, inorganic salts, and, in certain cases, vitamins or
other growth promoting substances. The addition of peptone provides
a readily available source of nitrogen and carbon. Furthermore,
different media results in different growth rates and different
stationary phase densities. A rich media results in a short
doubling time and higher cell density at a stationary phase.
Minimal media results in slow growth and low final cell densities.
Efficient agitation and aeration increases final cell densities. A
skilled artisan will be able to determine which type of media is
best suited to culture a specific type of microorganism. For
example, since 1927, the DIFCO manual has been used in the art as a
guide for culture media and nutritive agents for microbiology.
[0069] The method of the present invention preferably comprises a
single step of applying an antimicrobial composition to the
surfaces of a medical device and a single step of applying a
non-pathogenic bacterium to the surfaces of a medical device.
However, it is expected that several applications of the
antimicrobial agent and/or non-pathogenic bacterium, or other
substances, can be applied to the surface of the implant without
affecting the adherence of the antimicrobial agent or the
non-pathogenic bacterium. Furthermore, one skilled in the art is
cognizant that the antimicrobial agent and the non-pathogenic
bacterium can be applied together in a single step. Thus, the
method of the application of the antimicrobial agent and the
non-pathogenic bacterium can vary and should not be limited to the
described methods. Furthermore, a skilled artisan recognizes that
the order of the application of the compositions (i.e.,
antimicrobial agent and non-pathogenic bacterium) is not relevant
and can vary for any given application to a medical device.
[0070] In specific embodiments, the antimicrobial agent is selected
from the group consisting of an antibiotic, an antiseptic, a
disinfectant and a combination thereof. More specifically, the
antimicrobial agent is selected from the group of antibiotics
consisting of penicillins, penicillins, cephalosporins,
carbepenems, other beta-lactams antibiotics, aminoglycosides,
macrolides, lincosamides, glycopeptides, tetracylines,
chloramphenicol, quinolones, fucidins, sulfonamides, trimethoprims,
rifamycins, oxalines, streptogramins, lipopeptides, ketolides,
polyenes, azoles, and echinocandins.
[0071] In further specific embodiments, the antimicrobial agent is
selected from the group of antiseptics consisting of
.alpha.-terpineol, methylisothiazolone, cetylpyridinium chloride,
chloroxyleneol, hexachlorophene, cationic biguanides, methylene
chloride, iodine and iodophores, triclosan, nitrofurantoin,
methenamine, aldehydes, azylic acid, silver, and benzyl
peroxide.
[0072] Another embodiment of the present invention is that the
non-pathogenic gram-negative bacterium is selected from the group
consisting of Enterobacteriacea, Pseudomonas aeruginosa,
Stenotrophomonas maltophilia, Burkholderia cepacia, Gardnerella
vaginalis, and Acinetobacter species. In a specific embodiment, the
non-pathogenic gram-negative bacterium is Pseudomonas
aeruginosa.
[0073] In specific embodiments, the non-pathogenic gram-negative
bacterium is selected from the group of Enterobacteriacea
consisting of Escherichia, Shigella, Edwardsiella, Salmonella,
Citrobacter, Klebsiella, Enterobacter, Hafnia, Serratia, Proteus,
Morganella, Providencia, Yersinia, Erwinia, Buttlauxella, Cedecea,
Ewingella, Kluyvera, Tatumella and Rahnella.
[0074] More specifically, the Enterobacteriacea is Escherichia coli
83972 or mutants thereof. E. coli 83972 (or, Knt, H) was originally
isolated from a young woman as an asymptomatic bacteruria
associated isolate. It expressed none of the adherence phenotype
associated with uropathogenic E. coli. Preliminary studies
suggested that E. coli 83972 possessed genes associated with type 1
(fim) but not P (pap) pili. However, a more recent analysis
revealed that it possessed genes for type I and P pili synthesis
(although it does not appear to express the P pili in vivo) as well
as gene sequences homologous with foc (type 1C pili) and uca (G
pili) genes.
[0075] Another specific embodiment of the present invention, is
that the non-pathogenic gram-negative bacterium is a bacterium
which adheres to urinary catheters selected from the group
consisting of Providencia, Proteus, Pseudomonas aeruginosa and
Escherichia coli.
[0076] In further embodiments of the present invention, the
non-pathogenic bacterial coating layer further comprises viable
whole cells of the non-pathogenic gram-negative bacterium. In
addition to the use of viable whole cells, the non-pathogenic
bacterial coating layer further comprises non-viable whole cells or
cellular structures or extracts of the non-pathogenic gram-negative
bacterium. In a specific embodiment, the non-pathogenic bacterial
coating layer further comprises at least one or more viable whole
cells, non-viable whole cells or cellular structures or extracts of
the non-pathogenic gram-negative bacterium. Furthermore, the
non-pathogenic bacterial coating layer further comprises at least
two non-pathogenic gram-negative bacteria.
[0077] Furthermore, one skilled in the art is cognizant that the
factor or factors which are responsible for the inhibition of the
pathogens may be isolated and utilized, thus eliminating the
necessity of using viable whole cells, non-viable whole cells or
cellular structures or extracts. These inhibitory substances may be
readily separated from cultured bacterial cells by techniques such
as filtration, precipitation and centrifugation, which are readily
known in the art.
[0078] A specific embodiment of the present invention is a method
for coating a medical device comprising the steps of applying to at
least a portion of the surface of said medical device, an
antimicrobial coating layer, wherein said animicrobial coating
layer comprises an antimicrobial agent in an effective
concentration to inhibit the growth of bacterial and fungal
organisms relative to uncoated medical devices; and applying to at
least a portion of the surface of said medical device, a
non-pathogenic bacterial coating layer, wherein said non-pathogenic
bacterial coating layer comprises a non-pathogenic gram-positive
bacterium in an effective concentration to inhibit the growth of
pathogenic bacterial and fungal organisms, wherein said
non-pathogenic gram-positive bacterium is resistant to said
antimicrobial agent.
[0079] In specific embodiments of the present invention, the
non-pathogenic gram-positive bacterium is selected from the group
consisting of Staphylococcus aureus, coagulase-negative
staphylococci, streptococci, enterococci, corynebacteria, and
Bacillus species.
[0080] Another specific embodiment of the present inventions is
that the antimicrobial agent is selected from the group of
antibiotics consisting of penicillins, cephalosporins, carbepenems,
other beta-lactams antibiotics, aminoglycosides, macrolides,
lincosamides, glycopeptides, tetracylines, chloramphenicol,
quinolones, fucidins, sulfonamides, trimethoprims, rifamycins,
oxalines, streptogramins, lipopeptides, ketolides, polyenes,
azoles, and echinocandins.
[0081] In specific embodiments of the present invention, the
non-pathogenic bacterial coating layer further comprises viable
whole cells of the non-pathogenic gram-positive bacterium. In
addition, the non-pathogenic bacterial coating layer further
comprises non-viable whole cells or cellular structures or extracts
of the non-pathogenic gram-positive bacterium. In further
embodiments, the non-pathogenic bacterial coating layer further
comprises at least one or more viable whole cells, non-viable whole
cells or cellular structures or extracts of the non-pathogenic
gram-positive bacterium.
[0082] In specific embodiments, the non-pathogenic bacterial
coating layer further comprises at least two non-pathogenic
gram-positive bacteria. Another specific embodiment includes that
the non-pathogenic bacterial coating layer further comprises at
least one non-pathogenic gram-positive bacterium and at least one
non-pathogenic gram-negative bacterium.
[0083] Another specific embodiment is a method for coating a
medical device comprising the steps of applying to at least a
portion of the surface of said medical device, an antimicrobial
coating layer, wherein said antimicrobial coating layer comprises
an antimicrobial agent in an effective concentration to inhibit the
growth of bacterial and fungal organisms relative to uncoated
medical devices; and applying to at least a portion of the surface
of said medical device, a fungal coating layer, wherein said fungal
coating layer comprises a fungus in an effective concentration to
inhibit the growth of pathogenic bacterial and fungal organisms,
wherein said fungus is resistant to said antimicrobial agent. More
specifically, the fungus is Candida.
[0084] One specific embodiment of the present invention is a method
for preventing a urinary tract infection comprising the steps of
pretreating a patient with antibiotics for five to seven days;
inoculating the patient with a culture of a non-pathogenic
bacterium; and applying to at least a portion of the surface of a
urinary catheter, an antimicrobial coating layer having an
antimicrobial agent in an effective concentration to inhibit the
growth of bacterial and fungal organisms relative to uncoated
medical devices.
[0085] Another specific embodiment of the present invention is a
kit comprising compositions to coat the surfaces of medical devices
prior to implantation into a mammal comprising an antimicrobial
agent and a culture from a non-pathogenic bacterium, wherein said
non-pathogenic bacterium has been genetically modified to enhance
the adherence of the bacterium to the implant surface. More
specifically, the compositions are in the same container. Another
embodiment includes the kit with the compositions in different
containers.
[0086] The preferable mammal in the present invention is humans.
However, other mammals may be used. Exemplary mammals include, but
are not limited to, dogs, cats, cows, horses, rats, mice, monkeys,
and rabbits.
[0087] One skilled in the art readily recognizes the significance
of the development of a kit comprising the compositions to coat
catheters prior to use in mammals. These kits may be readily
prepared by utilizing standard bacterial culturing and storing
techniques and standard preparations of antimicrobial solutions,
which are readily known and applied in the art. The compositions
used in the kit may be in the following forms, but are not limited
to these forms, creams, capsules, gels, pastes, powders, liquids
and particles.
[0088] It is also contemplated that a kit may comprise a medical
device that has been pre-coated with an antimicrobial agent and
compositions to coat the medical device prior to implantation into
a mammal comprising a culture from a non-pathogenic bacterium.
Thus, the medical staff only needs to apply the non-pathogenic
bacterium composition to the medical device prior to implantation.
One skilled in the art realizes that a kit containing a pre-coated
medical device will reduce the amount of time that is needed for
the implantation.
[0089] A further embodiment is a kit comprising compositions to
coat the surfaces of medical devices prior to implantation into a
mammal comprising an antimicrobial agent and a culture from a
non-pathogenic bacterium, wherein said non-pathogenic bacterium has
been genetically modified to decrease the sensitivity of the
bacterium to antimicrobial agents. One skilled in the art is
cognizant that mutations can be made to any given bacteria to alter
the sensitivity to antimicrobial agents. Furthermore, a skilled
artisan is well versed in the various methods to modify bacteria.
For example, a standard modification is the insertion of an
antibiotic resistant gene using transposons.
[0090] Where employed, mutagenesis will be accomplished by a
variety of standard, mutagenic procedures. Mutation is the process
whereby changes occur in the quantity or structure of an organism.
Mutation can involve modification of the nucleotide sequence of a
single gene, blocks of genes or whole chromosome. Changes in single
genes may be the consequence of point mutations which involve the
removal, addition or substitution of a single nucleotide base
within a DNA sequence, or they may be the consequence of changes
involving the insertion or deletion of large numbers of
nucleotides.
[0091] Mutations can arise spontaneously as a result of events such
as errors in the fidelity of DNA replication or the movement of
transposable genetic elements (transposons) within the genome. They
also are induced following exposure to chemical or physical
mutagens. Such mutation-inducing agents include ionizing
radiations, ultraviolet light and a diverse array of chemical such
as alkylating agents and polycyclic aromatic hydrocarbons all of
which are capable of interacting either directly or indirectly
(generally following some metabolic biotransformations) with
nucleic acids. The DNA lesions induced by such environmental agents
may lead to modifications of base sequence when the affected DNA is
replicated or repaired and thus to a mutation. Mutation also can be
site-directed through the use of particular targeting methods.
[0092] Chemical mutagenesis. Chemical mutagenesis offers certain
advantages, such as the ability to find a full range of mutant
alleles with degrees of phenotypic severity, and is facile and
inexpensive to perform. The majority of chemical carcinogens
produce mutations in DNA. Benzo[a]pyrene, N-acetoxy-2-acetyl
aminofluorene and aflotoxin B1 cause GC to TA transversions in
bacteria and mammalian cells. Benzo[a]pyrene also can produce base
substitutions such as AT to TA. N-nitroso compounds produce GC to
AT transitions. Alkylation of the 04 position of thymine induced by
exposure to n-nitrosoureas results in TA to CG transitions.
[0093] Radiation Mutagenesis. The integrity of biological molecules
is degraded by the ionizing radiation. Adsorption of the incident
energy leads to the formation of ions and free radicals, and
breakage of some covalent bonds. Susceptibility to radiation damage
appears quite variable between molecules, and between different
crystalline forms of the same molecule. It depends on the total
accumulated dose, and also on the dose rate (as once free radicals
are present, the molecular damage they cause depends on their
natural diffusion rate and thus upon real time). Damage is reduced
and controlled by making the sample as cold as possible.
[0094] Transposon mutagenesis. The genes in microorganisms are not
static, but are capable under certain conditions to move around the
genome. The process by which a gene moves from one place to another
is transposition. If the transposon becomes inserted in a gene,
then it usually results in the inactivation of the gene. Two
transposons widely used for mutagenesis are Tn5, which confers
neomycin and kanamycin resistance, and Tn10, which contains a
marker for tetracycline resistance. Because the presence of the
transposon itself can be followed by its antibiotic resistance
properties, selection of antibiotic resistant cells after
transposition is used to isolate a wide variety of mutants. Thus,
transposon mutagenesis provides a useful tool for creating mutants
throughout the chromosome.
[0095] One skilled in the art is cognizant that this simple
bacterial mutagenesis can be utilized to alter the antibiotic
resistance of specific bacteria to decrease the sensitivity of the
bacteria to the antimicrobial agent used in the present invention.
Furthermore, a skilled artisan is cognizant that the present
invention does not propose the addition of an exorbitant amount of
antimicrobial resistance genes. The present invention proposes the
use of one or a maximum of a few anitmicrobial resistance genes,
which are typically present in the bacteria which constitute the
normal flora. The use of a non-pathogenic bacterium that is
resistant to one or a maximum of few antimicrobial agents does not
pose additional risks to the patient because 1) non-pathogenic
bacterium is intended to prevent infection by the typically more
resistant pathogenic bacterium; 2) the non-pathogenic bacterium
should not cause symptomatic infections which require antibiotic
therapy; and 3) the non pathogenic bacterium is typically made
resistant to antimicrobial agents that are not usually used to
treat established infection.
[0096] One specific embodiment is a kit comprising compositions to
coat the surfaces of medical devices prior to implantation into a
mammal comprising an antimicrobial agent and a culture from a
non-pathogenic bacterium, wherein said non-pathogenic bacterium has
been genetically modified to increase the stability of the
bacterium at room temperature. Standard methods that are
well-established in the art can be utilized to modify the bacteria,
i.e., bacterial mutagenesis.
[0097] Another specific embodiment is a kit comprising compositions
to coat the surfaces of medical devices prior to implantation into
a mammal comprising an antimicrobial agent and a culture from a
non-pathogenic bacterium, wherein said non-pathogenic bacterium has
been lyophilized and reconstituted prior to application to the
surface of the implant. A skilled artisan is cognizant that
lyophilization of bacteria are standard techniques used in
microbiology to increase the stability and preserve the
microorganism indefinitely in a dried state.
[0098] Bacteria are lyophilized to increase the stability of the
bacteria for long-term storage. Lyophilization stabilizes the
formulation by removing the solvent component or components to
levels that no longer support chemical reactions. This removal is
accomplished by first freezing the formulation, thus separating the
solutes from the solvent. Then the solvent is removed by primary
drying or sublimation followed by a secondary drying or desorption.
The formulation consists of three basic components--active
ingredient, excipient, and solvent system. In general, the active
ingredient in the pharmaceutical industry is defined by its potency
and, in the diagnostic industry, by its reactivity. Depending on
means of production, there may be variations in the composition of
the active component from batch to batch.
[0099] Excipients serve several functions. They primarily provide a
stable liquid environment for the active ingredient for some finite
time. The excipient cryoprotects the active ingredient during the
freezing process. In the freezing of formulations containing
biological organisms, the formation of ice within leads to the
organism's destruction by cell membrane rupture. Sucrose, glucose,
and dextran are excipients used to cryoprotect organisms.
[0100] The excipient serves as a bulking agent. When solid
concentrations of a formulation reach <2%, the resulting cake
has poor structural qualities and leaves the container during the
drying process. The addition of bulking agents such as mannitol and
dextran strengthen cake structure. The role of the solvent system
is often overlooked. Most formulations are totally aqueous
solutions, although others contain solvents such as tertiary butyl
alcohol to increase the solubility of some compounds. The solvent
system is removed during drying, but its thermal properties have a
major impact on the cosmetic properties of the final product.
[0101] Freezing and Drying the Formulation. Formation of ice during
freezing results in dramatic changes in concentrations of the
active ingredient and the excipient or excipients of the
formulation.
[0102] In most formulations containing an active ingredient and an
excipient, freezing greatly increases the concentration of the
active ingredient and the excipient or excipients, but does not
produce a well defined eutectic mixture. Instead, freezing produces
a complex, glassy system that may be homogeneous or heterogeneous.
This complex system, at this time, is produced in the interstitial
region of ice crystals as a result of the freezing process.
[0103] Drying. For lyophilization to occur, the solvent is first
removed by sublimation while the temperature of the frozen matrix
is maintained below the eutectic (eutectic temperature is a point
on a phase diagram where the temperature of the system or the
concentration of the solution at the point cannot be altered
without changing the number of phases present) or collapse
temperature of the formulation. This is the primary drying process.
The chamber pressure and product and shelf temperatures, during
primary drying, are based on the formulation's eutectic or collapse
temperature.
[0104] After primary drying, the residual moisture on the resulting
cake surface is reduced to levels that no longer support biological
growth and chemical reaction. This process is secondary drying. The
reduction of moisture in the cake during secondary drying is
accomplished by increasing the shelf temperature and reducing the
partial pressure of water vapor in the container. The required
partial pressure of water vapor and shelf temperature are
ascertained from stability studies of lyophilized or vacuum-dried
products having varied amounts of residual moisture.
[0105] The following examples are offered by way of example, and
are not intended to limit the scope of the invention in any
manner.
EXAMPLE 1
Bacterial Interference
[0106] Patient selection. Patients were excluded with remediable
etiologies for recurrent urinary tract infection, other than change
in bladder management. Other exclusion criteria were existing
urolithiasis, nephrostomy tube, ureteral stent, vesicoureteral
reflux, immunosuppression, vascular or genitourinary prostheses,
cardiovascular disease requiring antibiotic prophylaxis, on-going
or anticipated need for antibiotic therapy for nonurological
infection, pregnancy, fertility of female subjects not using some
form of accepted birth control, age younger than 18 years or
inability to give informed consent.
[0107] Symptomatic urinary tract infection was defined as the
presence of bacteriuria (greater than 10.sup.5 CFU/ml) with fever,
dysuria, increased urinary urgency, frequency or incontinence,
flank, suprapublic or scrotal pain, exacerbation of baseline
spasticity or autonomic dysreflexis, nausea and/or vomiting gross
hematuria, coatovertebral or suprapubic tenderness, acrotal mass
consistent with epididymitis or periurethral abscess, and/or a
fluctant or exquisitely tender prostate. Significant laboratory
values included white blood count greater than 10,500 cells per mm,
and/or a left shift in the count and positive blood cultures, All
patients discontinued all measures to suppress urinary tract
infection for at least 2 weeks prior to the study, and all
demonstrated a positive urine culture. No patient altered the form
of bladder management during the study.
[0108] Inoculation protocol. Bacteria were stored frozen in 1%
peptone per 30% glycerol solution at -80.degree. C. Before patient
inoculation, bacteria were grown overnight on agar plates and a
single colony was inoculated into 30 ml nutrient extract broth. The
culture was then incubated overnight at 37.degree. C. with
aeration. Bacteria were harvested by centrifugation and washed with
25 ml sterile irrigation saline and resuspended in irrigation
saline at a final concentration of 10.sup.5 to 10.sup.6 CFU/ml.
[0109] At study entry, a urine culture was obtained. Each patient
was treated as indicated with appropriate antibiotics for 5 to 7
days and any existing urinary catheter was changed 3 days after
beginning therapy. The urine was recultured and the lower urinary
tract was inoculated with E. coli 83972, 48 to 72 hours after
completion of the antibiotic course. Inoculation consisted of
instillation via a sterile bladder catheter of 30 ml. normal saline
containing 10.sup.5 to 10.sup.6 colony-forming units per ml. Each
inoculation cycle consisted of 2-3 inoculations on 3 consecutive
days. Patients that were incontinent at low bladder volumes were
treated with a Foley catheter balloon occluding the bladder neck or
diversion stoma for 30 minutes after inoculation. If colonization
was unsuccessful after 1 cycle of inoculations, the protocol was
repeated up to 3 cycles.
[0110] For colonized patients, post-inoculation urine cultures and
antibiotic susceptibilities were obtained weekly for 1 month,
monthly for 1 year and quarterly thereafter. Successful stable
colonization was defined as the presence of E. coli 83972 in the
urine at detectable levels (greater than 10.sup.3 CFU/ml) for
greater than 1 month after inoculation. E. coli isolated from urine
were identified as E. coli 83972 using whole cell DNA fingerprint
analysis. At each follow-up visit patients were queried about
symptoms and signs of urinary tract infection, symptoms of
extragenitourinary infection and the institution of antibiotic
therapy since the preceding follow-up visit. Routine annual (more
frequent if indicated) urological surveillance consisted of
physical examination, serum creatinine determination, urinary tract
imaging and urodynamic evaluation.
[0111] Long-term colonization. Persistent colonization (greater
than 1 month) was achieved in 13 cases (Table 1). Mean duration of
colonization was 12.3 months (range 2 to 40). The urine
concentration of E. coli 83972 was maintained at greater than
10.sup.5 CFU/ml. E. coli 83972 existed in pure culture (mean
duration 4.6 months, range 1 to 13) or in the presence of
contaminating organisms. Other bacterial genera were present as
transient co-colonizers or persisted for longer intervals (mean
10.2 months, range 7 to 30) together with E. coli 83972.
Co-colonizing bacteria, except for enterococcus, were present at
reduced concentration of 0.1% of the concentration of E. coli
83972. When present, entereococci existed at the same concentration
as E. coli 83972. In seven patients, E. coli 83972 was
spontaneously eliminated from the bladder and replaced by other
organisms. In five patients, E. coli 83972 was eliminated following
antibiotic treatment for non-gentiourinary infections.
1TABLE 1 Duration of colonization and outcome in colonized subjects
Colonization Colonization Duration Co-Colonizing Pt. Attempts
(months) Organisms* Outcome DLD 1 2 K. pneumoniae, Spontaneous
group B elimination Streptococcus JDG 1 5 None Treated for
respiratory tract infection 2 37 K. pneumoniae, Lost to followup
Enterococcus, P. aeruginosa JHK 1 2 Enterococcus, Treated for group
B respiratory tract Streptococcus infection 2 40 K. crytoca,
Treated for toe Enterococcus.dagger. infection RA 1 80 K.
pneumoniae, Spontaneous Enterococcus, S. elimination aureus.dagger.
2 16 Enterococcus Ongoing RWR 1 7 Group B Treated for toe
Streptococcus infection 2 26 Group B Ongoing Streptococcus,
Marganella.dagger. HK 1 2 None Spontaneous elimination EKM 1 2 K.
pneumoniae, Treated for finger P. aeruginosa.dagger. infection TAK
1 2 Enterococcus Spontaneous elimination 2 14 K. pneumoniae,
Ongoing group B Streptococcus AM 1 9 None Ongoing RGR 1 3 Group B
Spontaneous Streptococcus, elimination Enterococcus.dagger. HK 1 16
P. aeruginosa, Ongoing Entercoccus.dagger. RB 1 6 S. aureus
Spontaneous elimination BR 1 4 P. aeruginosa, K. Spontaneous
pneumoniae, elimination Enterococcus.dagger. *Organism coexisted
with E. coli 83972 greater than 3 months duration.
.dagger.Transient colonizations less than 3 months
[0112] Patients had no symptomatic urinary tract infections while
colonized with E. coli 83972 (0 infections per 18.4 patient-years).
Successfully colonized patients had a mean of 3.1 urinary tract
infections per year (range 2 to 7) before colonization. During the
study 11 subjects had 1 or more infections. Symptomatic infection
occurred in 4 subjects who were not successfully colonized with E.
coli 83972 (Table 2). No correlation was found between other
genitourinary or nongenitournary related adverse events and bladder
colonization with E. coli 83972.
2TABLE 2 Adverse events Pt. Genitourinary related Nongenitourinary
related EAD Urinary tract infection,* urinary Tooth abscess, upper
tract infection* respiratory tract infection BJV None None DLD Mild
incontinence Impacted wisdom tooth JDG None Upper respiratory tract
infection, bronchitis, superficial cellulitis of scrotum, sinusitis
JHK None Upper respiratory tract infection, coccygeal decubitus,
ingrown toe nail RA Suprapubic catheter occlusion Sacral decubitus,
topical antibiotic treatment RWR Urinary tract infection*
Toothache, muscle/chest pain, toe infection, growth of birthmark BR
Urinary tract infection* Diagnosis of diabetes mallitus, chronic
back pain EKM Urinary tract infection* Finger burn TAK Urinary
tract infection*, urinary Flu, sore throat, toe infection, tract
infection* pneumonia SS Dysreflexia at inoculation None AM None
Right arm cellulitis RGR Urethral discharge + urinary None tract
infection* RK None Gastrointestinal evaluation, phantom pains BB
Urosepsis* Atrial fibrillation, warfarin toxicity, hyperglycemia BR
Suprapubic catheter occlusion + Constipation, blood in stool,
hematuria, kidney cyst, gastrointestinal evaluation, cellulitis +
pus at suprapubic hemorrhoids, dizziness, panic catheter site,
urinary tract attack on quinolone antibiotic infection* DM Urinary
tract infection* Fall from gurney, hypokalemia DNM Urinary tract
infection,* urinary Bilat. Hip pain tract infection* AWR None None
TJP None None CB Urinary tract infection* None *Not while colonized
with E. coli 83972.
[0113] Other methods of coating surfaces of medical devices with
antibiotics are taught in U.S. Pat. No. 4,895,566 (a medical device
substrate carrying a negatively charged group having a pKa of less
than 6 and a cationic antibiotic bound to the negatively charged
group); U.S. Pat. No. 4,917,686 (antibiotics are dissolved in a
swelling agent which is absorbed into the matrix of the surface
material of the medical device); U.S. Pat. No. 4,107,121
(constructing the medical device with ionogenic hydrogels, which
thereafter absorb or ionically bind antibiotics); U.S. Pat. No.
5,013,306 (laminating an antibiotic to a polymeric surface layer of
a medical device); U.S. Pat. No. 5,902,283 (antimicrobial agents
are impregnated in catheters) and U.S. Pat. No. 4,952,419 (applying
a film of silicone oil to the surface of an implant and then
contacting the silicone film bearing surface with antibiotic
powders). One skilled in the art realizes that the above procedure
can be modified, i.e., the length of time the implant is in the
antimicrobial solution, the concentration of the antimicrobial
agent and the drying time.
EXAMPLE 2
Culturing of Microorgansims
[0114] Before inoculation or application of the bacteria to the
catheter, the bacteria are grown utilizing the appropriate
conditions as defined in the art. Typically, the bacteria are grown
on an agar plate overnight at 37.degree. C. A single colony is
chosen from the overnight culture plate and is used to inoculate 30
ml of nutrient extract broth. The culture is incubated overnight at
37.degree. C. with aeration. The bacteria are stored at room
temperature or lyophilized for future use. If the bacteria are used
immediately, then the bacteria are harvested by centrifugation and
washed with sterile saline and resuspended in sterile saline at a
final concentration of 10.sup.5 to 10.sup.6 CFU/ml.
EXAMPLE 3
Bacterial Interference and Antimicrobial Coating
[0115] Three types of 1.times.1 cm square-shaped catheter material
were tested: (1) uncoated latex catheter material; (2) latex
catheter material coated with a "low concentration" of
sulfamethoxazole (100 mg of sulfamethoxazole per ml of coating
solution); and (3) latex catheter material coated with a "high
concentration" of sulfamethoxazole (200 mg of sulfamethoxazole per
ml of coating solution).
[0116] Two strains of pap-negative E. coli 83972 were tested:
sulfamethoxazole-susceptible E. coli (strain HU2117), and
sulfamethoxazole-resistant E. coli strain (HU2209).
[0117] Tested E. coli strains were grown overnight on L agar
(HU2117) or L agar containing 100 micrograms per ml of
sulfathiazole (HU2209) at 37.degree. C., then inoculated into
minimal media in screw cap tubes and incubated at 37.degree. C.
until the culture reached the early-log phase of growth. The
culture was then diluted 1:100 into minimal media in a screw-cap
tube which also contained a square of the catheter material. The
catheter square was wedged into the tube approximately two
centimeters below the surface of the liquid in a vertical position
so that bacteria could not merely settle onto it. After 48 hours at
37.degree. C., an aliquot of the culture was removed, diluted and
plated onto L agar to determine the viable counts of the planktonic
bacteria. The catheter squares were removed from the culture tubes
aseptically and placed into 10 mls of buffered saline containing
0.01% SDS in snap-cap tubes. The tubes were vortexed briefly to
wash the catheter square of remaining planktonic bacteria. Squares
were removed aseptically and placed individually into standard
glass scintillation vials containing 10 mls of buffered saline/SDS.
The vials containing the squares were then subjected to 10 minutes
of treatment in a sonic water bath. Following the treatment, which
removes the attached bacteria from the membrane material, an
aliquot was removed from each vial, diluted and plated onto L agar
to determine the number of bacteria attached per square centimeter.
A ratio of adherent bacteria to bacteria in the supernatant
solution was determined (Table 3).
3TABLE 3 Ratio of adherent bacteria to bacteria in the supernatant
solution (.times.10.sup.-7) Latex coated with Latex coated with
Uncoated low conc. of high conc. of E. coli Strain Latex
Sulfamethoxazole Sulfamethoxazole HU2117 10.3 1.1 4.3
(sulfa-susceptible) 0.6 1.1 10 4.0 7.2 Mean of observations: 5.0
3.1 7.2 HU2209 22 0.5 56 (sulfa-resistant) 3.8 5.0 36 Mean of
observations: 13 2.8 46
[0118] The sulfamethoxazole-resistant HU2209 E. coli strain tended
to adhere better than the sulfamethoxazole-susceptible HU2117 E.
coli strain to both uncoated latex catheter material (mean ratio of
adherent bacteria to bacteria in the supernatant
solution.times.10.sup.-7 of 13 vs. 5.0) and latex material coated
with high concentration of sulfamethoxazole (mean ratio of adherent
bacteria to bacteria in the supernatant solution.times.10.sup.-7 of
46 vs. 7.2). Thus, one skilled in the art realizes that the
introduction of sulfamethoxazole resistance into E. coli does not
reduce bacterial adherence to latex catheter material. This finding
ensures that antimicrobial-resistant non-pathogenic strains of E.
coli can avidly adhere to the surface of catheters.
[0119] The HU2117 sulfamethoxazole-susceptible E. coli strain
tended to adhere less to latex coated with low concentration of
sulfamethoxazole vs. uncoated latex material (mean ratio of
adherent bacteria to bacteria in the supernatant
solution.times.10.sup.-7 of 3.1 vs. 5.0). However, this trend for
reduction in bacterial adherence to sulfamethoxazole-coated vs.
uncoated latex could not be established when comparing adherence of
the HU2117 sulfamethoxazole-susceptible E. coli strain to latex
coated with high concentration of sulfamethoxazole vs. uncoated
latex material (mean ratio of adherent bacteria to bacteria in the
supernatant solution.times.10.sup.-7 of 7.2 vs. 5.0). Thus, a
skilled artisan realizes that coating of latex catheter material
with sulfamethoxazole may not consistently reduce bacterial
adherence. This finding underscores the importance of assessing the
potential impact of combining antimicrobial coating with another
potentially protective measure, such as bacterial interference.
[0120] The HU2209 sulfamethoxazole-resistant E. coli strain adhered
more to latex material coated with high concentration of
sulfamethoxazole than to uncoated latex (mean ratio of adherent
bacteria to bacteria in the supernatant solution.times.10.sup.-7 of
46 vs. 13). One skilled in the art realizes that the use of
sulfamethoxazole-resistant strain of E. coli in combination with
high concentration sulfamethoxazole-coated latex enhances bacterial
adherence of this non-pathogenic strain. As a result,
antimicrobial-resistant non-pathogenic strains persist in larger
concentrations and for longer periods of time than
antimicrobial-susceptible non-pathogenic strains on the surface of
catheters. Thus, these results indicate that the use of a
combination of bacterial interference plus antimicrobial coating
has a higher efficacy than antimicrobial coating alone in
preventing bacterial pathogens from colonizing the catheters
resulting in an even lower likelihood of developing clinical
catheter-related infections.
EXAMPLE 4
Application of Antimicrobial Agent and Non-Pathogenic Bacterium and
Use In Vivo
[0121] The urinary catheter is coated with an antimicrobial agent
and a non-pathogenic bacterium. The non-pathogenic bacterium can be
in a liquid composition or in a powdered composition. The powdered
composition is derived from lyophilization. Both the antimicrobial
agent and non-pathogenic bacterium are applied to the catheter and
allowed to dry. After the antimicrobial agent and the
non-pathogenic bacterium have been applied to the catheter, the
coated catheter is then implanted into a patient similar to
standard procedures. After implantation, the patients are followed
and queried about symptoms and signs of urinary tract infections.
The catheters are replaced at given times. The replaced catheters
are also coated with the antimicrobial agent and the non-pathogenic
bacterium.
[0122] One skilled in the art is cognizant that the urinary
catheter treated with an antimicrobial agent and a non-pathogenic
bacterium is potentially capable of withstanding the growth of a
pathogenic biofilm longer than a non-treated catheter, thus
increasing the time between catheter removal and exchange. The
length of time necessary between removal of the catheters is
determined by the strength of the antimicrobial agent and the
non-pathogenic bacterium to inhibit the growth of pathogenic
bacteria.
REFERENCES CITED
[0123] All patents and publications mentioned in the specification
are indicative of the level of those skilled in the art to which
the invention pertains. All patents and publications are herein
incorporated by reference to the same extent as if each individual
publication was specifically and individually indicated to be
incorporated by reference.
[0124] Ditunno, J. F., Jr. and Farmal, C. S., N Engl J Med, 330:
550, 1994
[0125] Hackler, R. H., J Urol, 117: 486, 1977
[0126] Maynard, F. M. and Diakno, A. C. J Urol, 132: 943, 1984
[0127] Whiteneck, G. G. et al., Paraplegia, 30: 817, 1992
[0128] Cardenas, D. D. and Hooton, T. M. Arch Phys Med Rehabil, 76:
272, 1995
[0129] Waites, K. B., et al., J. Arch Phys Med Rehabil, 704: 691,
1993
[0130] Stover, S. L., et al., Arch Phys Med Rehabil, 70: 47,
1989
[0131] Stark, R. P. and Maki, D. G. N Engl J Med, 911: 560,
1984
[0132] Sotolange, J. R., Jr. and Knleilat, N. J Urol, 143: 979,
1990
[0133] Warren, J. W., Med Clin North Am, 75: 481, 1991
[0134] Hansson, S., et al:, BMJ, 298: 853, 1989
[0135] Hansson, S., et a.l, BMJ, 298: 856, 1989
[0136] Nicolle, L. E. Infect Dia Clin North Am, 11: 647, 1997
[0137] Reid, G., et al., Clin Microbiol Rev. 3: 335, 1990
[0138] Lindberg, U.: Acts Paediatr Scand, 64: 718, 1975
[0139] Anderson, P., et al., Infect Immun, 58: 2915, 1991
[0140] Agace, W. W., et al., J Clin Invest, 92: 780, 1999
[0141] Hull, R. A., et al., Infect Immun, 67: 429, 1999
[0142] Gouby, A., et al., J Clin Microbiol, 30:1588, 1992
[0143] Castello, T., et al., Spinal Cord, 34: 592, 1996
[0144] Avorn, J., et al., JAMA, 271: 761, 1994
[0145] Banover, K., et al., J Am Paraplegia Soc. 14: 52, 1991
[0146] Johnson, J. R., et al., J Infect Dis. 182: 1145, 1990
[0147] Riley, D. K., et al., Am J Med, 88: 349, 1995
[0148] Mohler, J. L., et al., J Urol, 138: 336, 1987
[0149] Bakke, A. and Vollset, S. E.: J Urol, 148: 527, 1999
[0150] Wulit, B., et al., J Urol, 150: 2057, 1998
[0151] One skilled the art readily appreciates that the present
invention is well adapted to carry out the objectives and obtain
the ends and advantages mentioned as well as those inherent
therein. Methods, procedures and techniques described herein are
presently representative of the preferred embodiments and are
intended to be exemplary and are not intended as limitations of the
scope. Changes therein and other uses will occur to those skilled
in the art which are encompassed within the spirit of the invention
or defined by the scope of the pending claims.
* * * * *