U.S. patent application number 15/951804 was filed with the patent office on 2018-08-16 for pharmaceutical composition, substrate comprising a pharmaceutical composition, and use of a pharmaceutical composition.
The applicant listed for this patent is Biomet Deutschland GmbH. Invention is credited to Nicole Duewelhenke.
Application Number | 20180228941 15/951804 |
Document ID | / |
Family ID | 38680223 |
Filed Date | 2018-08-16 |
United States Patent
Application |
20180228941 |
Kind Code |
A1 |
Duewelhenke; Nicole |
August 16, 2018 |
PHARMACEUTICAL COMPOSITION, SUBSTRATE COMPRISING A PHARMACEUTICAL
COMPOSITION, AND USE OF A PHARMACEUTICAL COMPOSITION
Abstract
Use of a pharmaceutical composition for the local treatment or
prevention of a tissue infection at an infection site, the
pharmaceutical composition comprising at least two different
antibiotics of group A or pharmaceutically acceptable derivatives
thereof, or an antibiotic of group A and at least one antibiotic of
group B or pharmaceutically acceptable derivatives thereof. Group A
comprises primarily intracellular active antibiotics working as
inhibitor of bacterial RNA polymerase; as inhibitor of gyrase; or
as inhibitor of bacterial protein synthesis. Group B comprises
primarily extracellular active antibiotics working as inhibitor of
bacterial cell wall synthesis; or inhibitor of bacterial protein
synthesis; or by direct destabilisation or rupture of the bacterial
cell wall.
Inventors: |
Duewelhenke; Nicole;
(Berlin, DE) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Biomet Deutschland GmbH |
Berlin |
|
DE |
|
|
Family ID: |
38680223 |
Appl. No.: |
15/951804 |
Filed: |
April 12, 2018 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
14566465 |
Dec 10, 2014 |
9968710 |
|
|
15951804 |
|
|
|
|
12670354 |
Jan 22, 2010 |
8921365 |
|
|
PCT/EP2008/006046 |
Jul 23, 2008 |
|
|
|
14566465 |
|
|
|
|
Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61P 31/00 20180101;
A61F 2/40 20130101; A61L 27/06 20130101; A61K 31/496 20130101; A61F
2/38 20130101; A61L 27/54 20130101; A61L 2300/45 20130101; A61L
2300/406 20130101; A61L 27/28 20130101; A61K 45/06 20130101; A61K
31/7048 20130101; A61F 2/32 20130101; A61F 2/3804 20130101; A61K
31/395 20130101; A61P 31/04 20180101; A61L 2420/06 20130101; A61P
43/00 20180101; A61K 31/7036 20130101; A61K 31/4709 20130101; A61K
38/12 20130101; A61K 31/65 20130101; A61K 38/08 20130101; A61K
31/7088 20130101; A61L 31/16 20130101; A61F 2/44 20130101; A61K
31/665 20130101; A61K 31/4709 20130101; A61K 2300/00 20130101; A61K
31/65 20130101; A61K 2300/00 20130101; A61K 31/665 20130101; A61K
2300/00 20130101; A61K 31/7048 20130101; A61K 2300/00 20130101;
A61K 31/7088 20130101; A61K 2300/00 20130101; A61K 38/12 20130101;
A61K 2300/00 20130101 |
International
Class: |
A61L 27/54 20060101
A61L027/54; A61K 31/7048 20060101 A61K031/7048; A61F 2/38 20060101
A61F002/38; A61F 2/40 20060101 A61F002/40; A61L 31/16 20060101
A61L031/16; A61L 27/28 20060101 A61L027/28; A61L 27/06 20060101
A61L027/06; A61K 45/06 20060101 A61K045/06; A61K 38/12 20060101
A61K038/12; A61K 38/08 20060101 A61K038/08; A61K 31/7088 20060101
A61K031/7088; A61F 2/32 20060101 A61F002/32; A61K 31/7036 20060101
A61K031/7036; A61K 31/665 20060101 A61K031/665; A61K 31/65 20060101
A61K031/65; A61K 31/496 20060101 A61K031/496; A61K 31/4709 20060101
A61K031/4709; A61K 31/395 20060101 A61K031/395; A61F 2/44 20060101
A61F002/44 |
Foreign Application Data
Date |
Code |
Application Number |
Jul 23, 2007 |
EP |
07 075 639.0 |
Claims
1. A pharmaceutical composition for use in the local treatment and
prevention of a tissue infection, the pharmaceutical composition
comprising a rifamycin and fosfomycin or a rifamycin and daptomycin
in a coating, wherein the coating comprises 10 to 1000
.mu.g/cm.sup.2 antibiotics.
2. The composition according to claim 1, characterized in that the
rifamycin is rifampin, rifabutin, rifapentine or rifamixin.
3. The composition according to claim 1, characterized in that,
said composition comprises rifampin and fosfomycin.
4. The composition according to claim 1, characterized in that said
composition further comprises a biofilm formation inhibitor.
5. The composition according to claim 4, characterized in that the
biofilm formation inhibitor comprises salicylic acid or a
pharmaceutically acceptable derivative or salt thereof.
6. The composition according to claim 1 characterized in that the
infected tissue to be treated is acutely or chronically
infected.
7. The composition according to claim 1, characterized in that said
composition is used in the treatment of extracellular and/or
intracellular microbial infected tissue cells and/or for the
prevention of extracellular and/or intracellular microbial
infections of tissue cells.
8. The composition according to claim 7, characterized in that the
infected tissue cells are soft tissue cells and/or bone tissue
cells.
9. The composition according to claim 8, characterized in that the
infected tissue soft tissue cells and/or bone tissue cells are
osteoblasts, leucocytes, erythrocytes, keratinocytes, fibroblasts,
fat cells, muscle cells and/or endothelial cells.
10. Composition according to claim 7, characterized in that the
tissue infection is caused by gram-negative and/or gram-positive
bacteria, preferably by the Staphyloccoci type, most preferably by
Staphylococcus aureus.
11. The composition according to claim 1, characterized in that
said composition comprises rifamycin and fosfomycin in such a
concentration that the rifamycin reaches a concentration of 0.005
to 100 .mu.g/ml, preferably 0.006 to 80 .mu.g/ml, most preferably
0.0075 to 10 .mu.g/ml at the site to be treated, and fosfomycin
reaches a concentration of 1 to 1000 .mu.g/ml, preferably 5 to 800
.mu.g/ml, most preferably 10 to 200 .mu.g/ml fosfomycin at the site
to be treated.
12. The composition according to claim 1, characterized in that
said composition comprises rifamycin and daptomycin in such a
concentration that the rifamycin reaches a concentration of 0.005
to 100 .mu.g/ml, preferably 0.006 to 80 .mu.g/ml, most preferably
0.0075 to 20 .mu.g/ml at the site to be treated, and daptomycin
reaches a concentration of 0.1 to 100 .mu.g/ml, preferably 0.5 to
80 .mu.g/ml, most preferably 1 to 20 .mu.g/ml at the site to be
treated.
13. The composition according to claim 1, characterized in that the
coating comprises a substrate arranged and provided for being used
as carrier of said composition, in particular for local antibiotic
therapy of an acute or chronic infection of a tissue of a
subject.
14. Composition according to claim 13, characterized in that said
substrate comprises a fleece, a fabric, a polymethyl methacrylate,
a copolymer of methylmethacrylate and methylacrylate, a
biodegradable polymer, polyethylene, a metal, a ceramic, a bone
cement, a bone substitute, or a combination of any of the
foregoing.
15. The composition according to claim 14, characterized in that
the fleece or the fabric comprises a natural or synthetic fibre,
particularly polylactide, and/or collagen.
16. The composition according to claim 13, characterized in that
said substrate comprises an implantable prosthesis, in particular a
hip prosthesis, a shoulder prosthesis, an elbow prosthesis, a knee
prosthesis, a vertebral implant, or an implant for trauma surgery.
Description
CROSS-REFERENCE TO A RELATED APPLICATION
[0001] This application is a National Phase Patent Application of
International Patent Application Number PCT/EP2008/006046, filed on
Jul. 23, 2008, which claims priority of European Patent Application
Number 07075639.0, filed on Jul. 23, 2007.
INTRODUCTION AND SUMMARY
[0002] The invention relates to the use of a pharmaceutical
composition, a pharmaceutical composition, a pharmaceutical
composition for treatment of extracellular and/or intracellular
microbial infected cells, and a substrate comprising a
pharmaceutical composition. The invention relates further to the
use of antibiotics as anti-adhesives against microorganisms on
surfaces.
[0003] Infections of bone and tissue are the severest problem of
orthopaedics and surgery, in particular due to increasing operation
frequency. 30% of all bone infections become chronic despite of
treatment. Further, many cases are known in which an infection
reoccurred after alleged successful earlier treatment. In 3% of all
cases, amputation is the only remaining option. Systemic treatment
with antibiotics is difficult since antibiotics penetrate through
bone generally only very poorly and thus concentrations being
sufficiently high to eliminate an infection are hardly
achievable.
[0004] Local application of antibiotics is better suited for
therapy of infections of bone and other tissue than systemic
antibiotic therapy since by local application higher concentrations
of antibiotics can be achieved at the treatment site than by
systemic application. A prerequisite for a successful local
antibiotics therapy is a preceding radical surgical therapy,
including debridement of all bone or tissue necroses and excision
of all foreign material. Local antibiotics carrier known from prior
art are bone cements made from polymethylmethacrylate (PMMA), beads
made from PMMA, collagen fleeces and bone substituents. These
carriers are commercially available with a limited number of
antibiotics applied onto them: gentamicin, tobramycin, clindamycin,
vancomycin and teicoplanin.
[0005] Though local antibiotics therapy employing the
above-mentioned antibiotics have already improved treatment of bone
and joint infections, such therapy fails in a significant number of
cases (up to 16%). Therapy failure, however, often finally leads to
the necessity of an amputation.
[0006] The major reasons for therapy failure are a) resistances
against certain antibiotics, b) ineffectiveness of antibiotics
against sessile bacteria, c) intracellular localised bacteria and
d) induction of Small Colony Variants. In this context,
ineffectiveness according to item b) is due to biofilm formation
and cessation of proliferation of the bacteria to be eliminated.
Further, in this context, intracellular is to be understood with
respect to a cell of the host, i.e. of the subject to be treated.
Thus, if bacteria are inside a cell of the host, antibiotics being
unable to penetrate to the inner of the cell cannot act on the
bacteria to be eliminated.
[0007] It has been known for some time that Staphylococci can
survive inside leucocytes. Further, it is known that strains of
Staphylococcus aureus showing the so-called Small Colony Variant
phenotype can be internalised by keratinocytes and endothelial
cells and can persist intracellularly. It was demonstrated that
they remain intracellularly inside lysosomes. S. aurerus of normal
phenotype can also be internalised by endothelial cells,
fibroblasts and keratinocytes and can remain intracellularly inside
lysosomes.
[0008] It was demonstrated that S. aurerus isolates display a
dichotomy: Whereas cytotoxic strains survive in keratinocytes and
fibroblasts and induce significant cytotoxicity to their host cells
due to intracellular division, non-cytotoxic strains are killed
inside of keratinocytes and fibroblasts, indicating that uptake of
S. aurerus represents an important mechanism of cell-autonomous
host defence (Krut, O. et al., Infection and Immunity, 2003, 71:
2716-2723).
[0009] S. aurerus can also be internalized by osteoblasts, but
osteoblasts cannot kill internalized non-cytotoxic staphylococci,
instead they can persist over days and weeks inside osteoblasts
without proliferation. After lysis of the osteoblasts, the
Staphylococci can proliferate again. Intracellular persistence of
Staphylococci and possibly other bacteria in osteoblasts and their
potential to persist intracellularly inside lysosomes may play a
particular role when looking at bone infections. This could be
causative for chronic progression of bone infections.
[0010] Though it is still not known exactly whether pseudomonades,
streptococci and enterococci can persist in osteoblasts,
intracellular persistence of these bacteria could be shown in
general. This intracellular persistence was hitherto only thought
to be possibly related with chronic progression of other diseases,
but since pseudomonades, streptococci and enterococci are frequent
pathogens with respect to bone infections, their intracellular
persistence might be causative for chronic progression of bone
infections.
[0011] Based on this assumption, it is explainable why allegedly
successfully treated bone infections can re-outbreak even after
years. Inside a host cell the bacteria are protected against
numerous antibiotics which cannot penetrate the cell membrane (e.
g. penicillins, glycopeptides). Though an acute infection being
induced by planktonic (floating) bacteria might be treated with
these antibiotics, bacteria can remain intracellularly and cause a
re-infection after release from the host cell.
[0012] In prior art, bone and soft tissue infections are treated
locally mainly with aminoglycosides (gentamicin, tobramycin) which
usually cannot penetrate the cell membrane of host cells. On the
other hand, it was reported that aminoglycosides can accumulate in
lysosomes of fibroblasts but are inactive due to the low pH of
lysosomes.
[0013] Consequently, antibiotics used in prior art for local
therapy of infections of bone and other tissue are not suited to
treat all these infections successfully. In particular, local
antibiotics carrier containing only gentamicin are ineffective
against infections of bacteria showing a Small Colony Variant
phenotype and can even induce formation of Small Colony Variant
phenotypes. None of the antibiotics used at present for local
therapy of infection can eliminate intracellular localized
bacteria.
[0014] WO 2006/064517 discloses an antibiotic composition
comprising a first antibiotic inhibiting the bacterial protein
synthesis and a second antibiotic not inhibiting the bacterial
protein synthesis.
[0015] U.S. Pat. No. 5,217,493 discloses an implantable medical
device which is coated against biofilm colonization with rifampin
and novobiocin, or rifampin and minocycline.
[0016] Given an embodiment it is possible to provide a
pharmaceutical composition for treating and preventing extra- and
intracellular infections of cells, especially tissue cells, a
substrate carrying such composition, and a method of applying such
a composition and substrate.
[0017] It is further possible to decrease the adhesion rate of
microorganisms on different substrate surfaces.
[0018] In an embodiment of the invention, the local treatment and
prevention is done at an infection site. The tissue to be treated
can e.g. be soft tissue and/or bone tissue including what is
generally denoted as "bone". The pharmaceutical composition
comprises at least two different antibiotics of group A or
pharmaceutically acceptable derivatives thereof, or an antibiotic
of group A and at least one antibiotic of group B or respective
pharmaceutically acceptable derivatives thereof. Group A comprises
primarily intracellular active antibiotics working as inhibitor of
bacterial RNA polymerase, as inhibitor of gyrase or as inhibitor of
bacterial protein synthesis. Group B comprises primarily
extracellular active antibiotics working as inhibitor of bacterial
cell wall synthesis or as inhibitor of bacterial protein synthesis
or destabilise or rupture the bacterial cell wall directly.
[0019] In the context of the present description a tissue infection
is understood as an extracellular and intracellular infection of
tissue cells caused by microorganisms.
[0020] In order to circumvent resistances against the antibiotics
used, particularly in long-term treatments, a combination of at
least two antibiotics can be chosen. Such a combination results
also in higher efficacy. Though it is generally considerable to use
only intracellular active antibiotics, a combination of an
intracellular active antibiotic (group A) with an extracellular
active antibiotic (group B) may also be chosen. Though antibiotics
of group B are not intracellular active, they can inhibit formation
of resistances since they act on extracellular bacteria in a
bactericidal manner and resistances are only formed in planktonic,
proliferating populations of bacteria. Since the extracellular
active antibiotics of group B show a different mechanism of action
than the antibiotics of group A, parallel resistances can hardly
occur.
[0021] The pharmaceutical composition to be used can comprise
further additives, dispersants, solvents or carrier substances etc.
known per se.
[0022] In order to achieve good results in treating infections of
bone and other tissue, at least one of the antibiotics chosen
should, in an embodiment, fulfil at least one of the following
criteria: [0023] a) It should penetrate the cell membrane of the
host cell (i. e. the cell of the subject to be treated inside which
the bacteria to be eliminated are located). [0024] b) It should be
able to reach the inside of the lysosomes of the host cell. [0025]
c) It should be active at low pH (particularly at that pH being
present in lysosomes, i. e. ca. pH 4 to pH 5). [0026] d) It should
have a bactericidal activity. [0027] e) It should show its
bactericidal activity also against non-proliferating bacteria.
[0028] In an embodiment at least one of the antibiotics chosen
fulfils a plurality of the criteria mentioned above. In another
embodiment, fulfilment of all of these criteria is achieved. In
still another embodiment, a fulfilment of all criteria by all
antibiotics chosen is achieved.
[0029] In an embodiment said antibiotics of group A working as
inhibitor of bacterial RNA polymerase comprise ansamycins,
particularly rifamycins. Particularly, rifampin, rifabutin,
rifapentine or rifamixin may be chosen. A pharmaceutical
composition containing rifampin is particularly suited in
eliminating intracellular Staphylococci, which were shown to be
eliminated within 3 days after local administration of an according
pharmaceutical composition.
[0030] In a further embodiment said antibiotics of group A working
as inhibitor of gyrase comprise fluoroquinolones. The
fluoroquinolone moxifloxacin is particularly chosen.
[0031] In an embodiment said antibiotics of group A working as
inhibitor of bacterial protein synthesis comprise streptogramins
like, e. g., quinupristin or dalfopristin. In an embodiment, a
combination of quinupristin and dalfopristin is used. It is to be
noted that the pharmaceutical composition to be used may contain
more than a single antibiotic of each group (and more than two
antibiotics of group A if no antibiotic of group B is used) and
thus more than two antibiotics in total.
[0032] In an embodiment said antibiotics of group B working as
inhibitor of bacterial cell wall synthesis or destabilising and
rupturing the cell wall directly comprise glycopeptides, fosfomycin
and polypeptides. In an embodiment the glycopeptides chosen are
vancomycin and teicoplanin. In the same or another embodiment the
polypeptides chosen are bacitracin, polymyxin B as well as other
polymyxins and daptomycin.
[0033] In an embodiment said antibiotics of group B working as
inhibitor of bacterial protein synthesis comprise aminoglycosides.
In this context, particularly arbekacin may be chosen.
[0034] An exemplary pharmaceutical composition to be used comprises
a rifamycin and an aminoglycoside. Another exemplary pharmaceutical
composition comprises rifampin and arbekacin; such a composition
essentially covers the entire germ spectrum to be eliminated and is
effective against problematic bacteria like methicillin-resistant
S. aurerus (MRSA) or methicillin-resistant S.epidermidis (MRSE).
Both antibiotics are effective also against non-proliferating
(resting) bacteria and are temperature resistant (heat stable) so
that they can be added to a bone cement made of
poly(methylmethacrylate) (PMMA), to PMMA bead chains, and to
spacers for revision operations.
[0035] Another pharmaceutical composition to be used comprises a
rifamycin and fosfomycin. Still another pharmaceutical composition
comprises rifampin and fosfomycin; such a composition also
essentially covers the entire germ spectrum to be eliminated and is
also effective against problematic bacteria like MRSA and MRSE.
Fosfomycin has the further property that it binds reversibly to
hydroxyl apatite and thus remains, even after release from a
carrier, longer in a bone than other antibiotics. Further,
fosfomycin is the smallest antibiotic known and diffuses or
penetrates very well through or into bone tissue.
[0036] A further pharmaceutical composition to be used comprises a
rifamycin and a fluoroquinolone. Another pharmaceutical composition
comprises rifampin and moxifloxacin.
[0037] One object is also addressed by providing a pharmaceutical
composition. Such a pharmaceutical composition can be used for the
local treatment and prevention of a tissue infection at an
infection site, whereby further embodiments of such a use are
analogous to those explained above and to which in entirety
reference is made hereby.
[0038] Such a pharmaceutical composition comprises at least two
different antibiotics of group A' or pharmaceutically acceptable
derivatives thereof, or an antibiotic of group A' and an antibiotic
of group B' or pharmaceutically acceptable derivatives thereof. In
this case, group A' comprises the primarily intracellular active
antibiotics ansamycins, particularly rifamycins such as rifampin,
rifabutin, rifapentine or rifamixin; fluoroquinolones, particularly
moxifloxacin; streptogramins, particularly quinupristin and/or
dalfopristin. Group B' comprises the primarily extracellular active
antibiotics glycopeptides, particularly vancomycin or teicoplanin;
fosfomycin; polypeptides, particularly bacitracin, daptomycin, or
polymyxin B; and aminoglycosides, particularly arbekacin. It is to
be noted that glycopeptides cannot be the second antibiotic of a
pharmaceutical composition comprising only two antibiotics and
comprising an ansamycin as first antibiotic.
[0039] In an embodiment the pharmaceutical composition comprises
only a glycopeptide, a polypeptide or fosfomycin as possible
antibiotic of group B', but no aminoglycosides are used as
antibiotic of group B'. In another embodiment no streptogramins are
used as antibiotic of group A'.
[0040] In an embodiment the antibiotics are chosen in such a way
that either none or all antibiotics in the pharmaceutical
composition work as inhibitors of protein synthesis, i.e. either a)
only different streptogramins, or a streptogramin and an
aminoglycoside may be used or b) no streptogramins and no
aminoglycosides may be used at all.
[0041] In an alternative embodiment the pharmaceutical composition
comprises a rifamycin and an aminoglycoside, particularly rifampin
and arbekacin.
[0042] In another embodiment the pharmaceutical composition
comprises a rifamycin and fosfomycin, particularly rifampin and
fosfomycin.
[0043] Such a composition comprises rifamycin and fosmycin in such
a concentration that the rifamycin reaches a concentration of 0.005
to 100 .mu.g/ml, preferably 0.006 to 80 .mu.g/ml, most preferably
0.0075 to 20 .mu.g/ml at the site to be treated. Fosfomycin reaches
a concentration of 1 to 1000 .mu.g/ml, preferably 5 to 800
.mu.g/ml, most preferably 10 to 200 .mu.g/ml at the site to be
treated.
[0044] In yet another embodiment the pharmaceutical composition
comprises a rifamycin and polypeptide, particularly rifampin and
daptomycin.
[0045] Such a composition comprises rifamycin and daptomycin in
such a concentration that the rifamycin reaches a concentration of
0.005 to 100 .mu.g/ml, preferably 0.006 to 80 .mu.g/ml, most
preferably 0.0075 to 20 .mu.g/ml at the site to be treated.
Daptomycin reaches a concentration of 01 to 100 .mu.g/ml,
preferably 0.5 to 80 .mu.g/ml, most preferably 1 to 20 .mu.g/ml at
the site to be treated.
[0046] In still another embodiment the pharmaceutical composition
comprises a rifamycin and a fluoroquinolone, particularly rifampin
and moxifloxacin.
[0047] One object is also achieved by a pharmaceutical composition
for the treatment of extracellular and/or intracellular microbial
infected cells and/or for the prevention of microbial infections of
cells comprising at least one antibiotic acting as an inhibitor of
bacterial RNA polymerase, at least one antibiotic affecting the
bacterial cell wall or its synthesis, and/or at least one
antibiotic acting as a gyrase inhibitor.
[0048] The treatment preferably occurs locally or systemically.
[0049] Advantageously, ansamycins, particulary rifamycins such as
rifampin, rifabutin, rifapentine or rifamixin are used as
inhibitors of bacterial RNA polymerase. As antibiotics affecting
the bacterial cell wall or its synthesis glycopeptides,
particularly vancomycin or teicoplanin, fosfomycin and
polypeptides, particularly bacitracin and daptomycin are chosen. As
a gyrase-inhibitors fluoroquinolones, particularly moxifloxacin, is
applied.
[0050] Rifamycin is used in concentrations between 0.005 to 100
.mu.g/ml, preferably 0.006 to 80 .mu.g/ml, most preferably 0.0075
to 20 .mu.g/ml. Fosfomycin is used in concentrations of 1 to 1000
.mu.g/ml, preferably 5 to 800 .mu.g/ml, most preferably 10 to 200
.mu.g/ml. Moxifloxacin is applied in a concentration between 0.1 to
500 .mu.g/ml, preferably 0.5 to 200 .mu.g/ml, most preferably 1 to
100 .mu.g/ml. Daptomycin is used in concentrations of 0.1 to 100
.mu.g/ml, preferably 0.5 to 80 .mu.g/ml, most preferably 1 to 20
.mu.g/ml. The same concentrations are preferably used in a
combination of rifamycin, fosfomycin, daptomycin and/or
moxifloxacin.
[0051] The pharmaceutical composition is especially effective in
case of infected cells such as osteoblasts, leucocytes,
erythrocytes, keratinocytes, fibroblasts, fat cells, muscle cells
and/or endothelial cells.
[0052] Furthermore, the pharmaceutical composition is effective
against microbial infection caused by gram-negative and/or
gram-positive bacteria, preferably by the Staphyloccoci type, most
preferably by Staphylococcus aureus.
[0053] In an embodiment the pharmaceutical compositions to be used
further comprise a biofilm formation inhibitor. Every substance
reducing or inhibiting at least partially the attachment of germs,
especially bacteria on a surface or the ability of germs to
accumulate on a surface to form a biofilm on that surface is
considered as biofilm formation inhibitor.
[0054] In an embodiment salicylic acid or a pharmaceutical active
derivative or salt thereof is used as biofilm formation inhibitor.
Particularly, a combination of salicylic acid and an aminoglycoside
may be used. Salicylic acid enhances the microbial activity of
aminoglycosides against bacteria, especially against E. coli and
Klebsiella pneumoniae: Salicylates enter a cell in a protonated
form, thereby increasing the membrane potential of the cell. This,
in turn, simplifies the uptake of aminoglycosides into the interior
of the cell.
[0055] Even salicylic acid itself shows an effect on bacteria.
Growth of encapsulated Klebsiella pneumoniae in the presence of
salicylate results in reduced synthesis of capsular
polysaccharides. The loss of capsular material exposes the cell
surface of K. pneumoniae to the host defence mechanisms, thus
shortening the time required for infection clearance. Salicylic
acid reduces the ability of bacteria to adhere onto surfaces and to
form biofilms. Though salicylic acid does not provide 100%
protection against biofilm formation, it supports the effect of
antibiotics.
[0056] Acetylsalicylic acid and/or its predominant metabolite
salicylic acid exhibit definable impacts both in vitro and in vivo
on microbial virulence phenotypes. Bacterial virulence factors help
mediate infection by bacteria in a host organism. The following
effects have been noted: reduction of adhesion to relevant
biomatrices, reduction of capsule production, mitigation of biofilm
formation, and diminution of vegetation growth, intravegetation
bacterial proliferation, and hematogenous dissemination in
experimental infective endocarditis. Salicylic acid also regulates
positively the translation of specific gene loci including multiple
antibiotic-resistance loci. Further, it induces cytoplasmic
proteins; and increases quinolone resistance.
[0057] The synthesis of some types of fimbriae in E. coli e.g.
colonization factor antigen, P fimbriae and type 1 fimbriae are
reduced following growth in the presence of salicylate. Because
fimbriae play a critical role in the attachment of E. coli to
epithelial surfaces, salicylate treatment might prevent infection
caused by some strains of fimbriated E. coli. Salicylate also
limits adherence of E. coli to silastic catheters.
[0058] Chemotaxis in bacteria is modulated through regulation of
flagella rotation. This rotation, when counterclockwise, leads to
swimming along a linear trajectory and, when clockwise, leads to
tumbling. Salicylate is recognized as a chemorepellant by the E.
coli tsr gene product. This recognition leads to prolonged tumbling
of motile E. coli and ultimately causes cells to migrate away from
salicylate. Swarming behaviour of E. coli is also inhibited by
salicylate in a concentration-dependent manner. Production of the
flagellum itself in E. coli is inhibited by growth in the presence
of salicylate. This is mediated by inhibiting the production of
flagellin, the protein monomer constituting the flagella. It has
also been speculated that inhibition of flagella synthesis and
motility in E. coli by salicylate is due to reduced synthesis in
OmpF synthesis, which may be required for flagella assembly.
[0059] Biofilms consist of microorganisms and other matter encased
in a polysaccharide matrix of microbial origin. Growth of
Pseudomonas aeruginosa and Staphylococcus epidermidis in the
presence of salicylate reduces the production of extracellular
polysaccharide required for biofilm formation. The reduction in
biofilm formation decreases the ability of these organisms to
adhere to contact lenses and medical polymers. A component of
biofilm production in S. epidermidis is extracellular slime which
is composed of a complex mixture of polysaccharides, teichoic acids
and proteins. Production of slime-associated proteins and teichoic
acids is inhibited in S. epidermidis by salicylate.
[0060] In case of S. aurerus, salicylic acid mitigates two distinct
virulence phenotypes that are of key relevance for matrix binding,
i.e. to fibrinogen and fibronectin, and .alpha.-hemolysin activity.
These effects are specifically associated with salicylic
acid-mediated reduction in the expression of the respective
structural genes, i.e., fnbA, fnbB, and hla. In addition to the
suppression of matrix protein binding and cytolytic profiles,
enhanced exoenzyme and protein A production occurs in the presence
of salicylic acid. These findings raise the likelihood that
salicylic acid executed its antimicrobial effects through one or
more global regulatory networks rather than a decrease in general
gene transcription. Global regulon sarA and the global regulon agr
are mitigated by salicylic acid, corresponding to the reduced
expression in of the hla and fnbA genes in vitro. It should be
noted that S. aurerus virulence parameters were not completely
suppressed by salicylic acid but were reduced, in a drug
concentration-dependent manner, by a maximum of approximately
50%.
[0061] In an embodiment the infected tissue to be treated is
acutely or chronically infected. A combination of an acute and a
chronic infection, i.e. the acute infection overlying the chronic
infection, might also be treated.
[0062] The object is also achieved by providing a substrate for
medical purposes according to claim 21. The substrate is preferably
used as carrier of the pharmaceutical composition when locally
treating and preventing the tissue infection. In a further
embodiment the substrate is also used locally after removal of the
infected tissue as a supplement in surgical debridement.
[0063] In one embodiment the substrate can be soaked with the
pharmaceutical composition to be used. In another embodiment the
pharmaceutical composition can be dispersed in a base material of
the substrate. In still another embodiment, the pharmaceutical
composition can be polymerised with the base material. Thus, it is
possible to coat the substrate with the pharmaceutical composition
and/or to incorporate the pharmaceutical composition into the
substrate.
[0064] In a preferred embodiment the substrate underwent special
treatment e.g. sand blasting or hydroxyl apatite coating before the
pharmaceutical composition is applied.
[0065] Within the scope of the present description is also a
coating made of a support material in which the pharmaceutical
composition is present e.g. in a dispersed form. Such support
material can include polylactides. The support material with the
dispersed pharmaceutical composition is then applied as a coating
onto the substrate--either directly onto the surface of the latter
or onto a layer being already present on that surface or on another
layer.
[0066] In an embodiment the substrate comprises a fleece, a fabric,
a poly ethyl methacrylate, a copolymer of methylmethacrylate and
methylacrylate, a resorbable polymer, polyethylene, a metal or a
metal alloy e.g. a Ti6Al4V alloy or another titaniumium alloy, a
ceramic, a bone cement, particularly made from a polymeric material
or from calcium phosphate and/or a bone substitute. Thus, PMMA bead
chains consisting mainly of a copolymer of methylmethacrylate and
methylacrylate as well as glycine and a specific pharmaceutical
composition to be administered as local antibiotics carrier are a
possible substrate. Further, the bone cement may be intended to be
used for spacer and for revision operations.
[0067] In case of PMMA bead chains, the following mode of use is
possible: firstly, the pharmaceutical composition is dispersed
within the PMMA base material. The powder is heated to 180.degree.
C. and filled into forms by injection moulding. The pharmaceutical
composition is being distributed all over the base material and can
diffuse from the inner parts of a PMMA bead towards the surface,
where it may interact with bacteria being present around the PMMA
bead chain. The PMMA bead chains may comprise 0.1-10 wt %,
preferably 0.5-8 wt %, most preferably 1-5 wt % antibiotic(s).
[0068] In another embodiment, particularly in case of revision
operations, the substrate is an implantable prosthesis, wherein
joint prostheses and particularly knee, hip, shoulder, elbow
prostheses as well as vertebral implants are respective examples.
Furthermore, all implants for trauma surgery like screws, plate,
etc. may be used as substrate. The substrate coating may comprise
10-1000 .mu.g/cm.sup.2, preferably 20-500 .mu.g/cm.sup.2, most
preferably 50-300 .mu.g/cm.sup.2 antibiotic(s) per cm.sup.2
substrate surface area.
[0069] In an embodiment the fleece or fabric comprises a natural or
synthetic fibre, which can be biodegradable, wherein polylactide
(polylactic acid) is an exemplary material. In another embodiment
the fleece or fabric comprises collagen, wherein the fleece may
consist essentially of collagen. In the latter case, the collagen
fleece is also completely biodegradable. The fleece may comprise
0.01-10 mg/cm.sup.2, preferably 0.1-8 mg/cm.sup.2, most preferably
0.5-5 mg/cm.sup.2 antibiotic(s) per cm.sup.2 fleece.
[0070] Further a method for locally treating a subject with a
pharmaceutical composition is described, the pharmaceutical
composition comprising: [0071] at least two different antibiotics
of group A or pharmaceutically acceptable derivatives thereof or
[0072] an antibiotic of group A and at least one antibiotic of
group B or pharmaceutically acceptable derivatives thereof, wherein
[0073] group A comprises intracellular active antibiotics working
as [0074] inhibitor of bacterial RNA polymerase, [0075] inhibitor
of gyrase or [0076] inhibitor of bacterial protein synthesis and
group B comprises extracellular active antibiotics working [0077]
as inhibitor of bacterial cell wall synthesis, [0078] as inhibitor
of bacterial protein synthesis or [0079] by direct destabilisation
or rupture of the bacterial cell wall.
[0080] This method may be particularly used for treating a tissue
infection of said subject, wherein the tissue may be, e.g., soft
tissue and/or bone tissue and/or bone. These infections might occur
due to a surgical operation, particularly due to an operation
related to implanting an implant into a human or non-human body.
Thus, the treatment might be applied to a human or non-human
body.
[0081] With respect to further embodiments of this aspect reference
is made the explanations given above which are analogously
applicable for said method, particularly regarding the substrate to
be used and the antibiotics to be chosen.
[0082] A second object is achieved by using a combination of at
least antibiotic acting as an inhibitor of bacterial RNA polymerase
and at least one antibiotic affecting the bacterial cell wall or
its synthesis as anti-adhesives against microorganisms on
surfaces.
[0083] The inhibitor of bacterial RNA polymerase is preferably
selected from the group comprising ansamycins, particulary
rifamycins such as rifampin, rifabutin, rifapentine or
rifamixin.
[0084] The antibiotic affecting the bacterial cell wall or its
synthesis is preferably selected from the group comprising
glycopeptides, particularly vancomycin or teicoplanin, fosfomycin
and polypeptides, particularly bacitracin or daptomycin. A
preferred combination comprises rifamycin and fosfomycin.
[0085] In a further embodiment the microorganisms are gram-negative
and/or gram-positive bacteria, preferably of the Staphyloccoci
type, most preferably Staphylococcus aureus.
[0086] The combination of the at least one inhibitor of bacterial
RNA polymerase and the at least one antibiotic affecting the
bacterial cell wall or its synthesis is preferably attached or
coated onto surfaces made of metal, preferably titanium, steel or
metal alloy, ceramics, and bone cement or hydroxyl apatite.
[0087] When coated on a substrate the combination may comprise
rifamycin and fosfomycin in a concentration between 10 and 1000
.mu.g/cm.sup.2, preferably 20 to 500 .mu.g/cm.sup.2, most
preferably 50-200 .mu.g/cm.sup.2, respectively.
[0088] Advantageously, the antiadhesive effect is accompanied by a
bactericidal effect on the tissue surrounding the coated
surfaces.
BRIEF DESCRIPTION OF THE DRAWINGS
[0089] Examples of embodiments are explained in further detail by
means of the following figures and examples.
[0090] FIG. 1 shows CFU of S. aurerus ATTC 6538P in cell culture
supernatant of osteoblastic MG63 cells.
[0091] FIG. 2 shows CFU of S. aurerus BAA44 in cell culture
supernatant of osteoblastic G63 cells.
[0092] FIG. 3a shows metabolic activity of osteoblastic MG63 cells
after infection with S. aureus ATTC 6538P followed by addition of
rifampin to the cell culture supernatant after treatment with
lysostaphin to remove extracellular bacteria.
[0093] FIG. 3b shows metabolic activity of osteoblastic MG63 cells
after infection with S. aureus ATTC 6538P followed by addition of
fosfomycin to the cell culture supernatant after treatment with
lysostaphin to remove extracellular bacteria.
[0094] FIG. 3c shows metabolic activity of osteoblastic MG63 cells
after infection with S. aureus ATTC 6538P followed by addition of
fosfomycin, rifampin and their combination to the cell culture
supernatant after treatment with lysostaphin to remove
extracellular bacteria.
[0095] FIG. 3d shows metabolic activity of osteoblastic MG63 cells
alter infection with S. aureus ATTC 6538P followed by addition of
after adding a mixture containing 10 .mu.g/ml fosfomycin and
0.006-0.0075 .mu.g/ml rifampin to the cell culture supernatant
after treatment with lysostaphin to remove extracellular
bacteria.
[0096] FIG. 3e shows metabolic activity of osteoblastic MG63 cells
after infection with S. aureus ATTC 6538P followed by addition of
moxifloxacin to the cell culture supernatant after treatment with
lysostaphin to remove extracellular bacteria.
[0097] FIG. 4a shows CFU of S. aurerus BAA44 located in
osteoblastic MG63 cells after adding fosfomycin to the cell culture
supernatant of osteoblastic MG63 cells after treatment with
lysostaphin to remove extracellular bacteria.
[0098] FIG. 4b shows CFU of S. aurerus BAA44 located in
osteoblastic MG63 cells after adding rifampin to the cell culture
supernatant of osteoblastic MG63 cells after treatment with
lysostaphin to remove extracellular bacteria.
[0099] FIG. 4c shows CFU of S. aurerus BAA44 located in
osteoblastic MG63 cells after adding a mixture containing 50
.mu.g/ml fosfomycin and 2.5-40 .mu.g/ml rifampin to the cell
culture supernatant of osteoblastic MG63 cells after treatment with
lysostaphin to remove extracellular bacteria.
[0100] FIG. 4d: shows CFU of S. aurerus BAA44 located in
osteoblastic MG63 cells after adding a mixture containing 25-400
.mu.g/ml fosfomycin and 2.5 .mu.g/ml rifampin to the cell culture
supernatant of osteoblastic MG63 cells after treatment with
lysostaphin to remove extracelluar bacteria.
[0101] FIG. 4e shows CFU of S. aurerus BAA44 located in
osteoblastic MG63 cells after adding fosfomycin, rifampin and a
mixture containing 50 .mu.g/ml fosfomycin and 10 .mu.g/ml rifampin
to the cell culture supernatant of osteoblastic MG63 cells after
treatment with lysostaphin to remove extracellular bacteria.
[0102] FIG. 4f shows CFU of S. aurerus BAA44 located in
osteoblastic MG63 cells after adding fosfomycin, rifampin and a
mixture containing 25 .mu.g/ml fosfomycin and 2.5 .mu.g ml rifampin
to the cell culture supernatant of osteoblastic MG63 cells after
treatment with lysostaphin to remove extracellular bacteria.
[0103] FIG. 4g: shows CFU of S. aurerus BAA44 located in
osteoblastic MG63 cells after adding fosfomycin, rifampin and a
mixture containing 50 .mu.g/ml fosfomycin and 20 .mu.g/ml rifampin
to the cell culture supernatant of osteoblastic MG63 cells after
treatment with lysostaphin to remove extracellular bacteria.
[0104] FIG. 5a: shows CFU of S. aurerus ATTC 6538P per titanium
disc after 1.5 h incubation of S. aurerus on vancomycin or
rifampin/fosfomycin coated titanium disc, which were either washed
once or thrice before incubation.
[0105] FIG. 5b: shows CFU of S.epidermidis ATTC 35984 per titanium
disc after 2 h incubation with S.epidermis on rifampin/fosfomycin
coated titanium discs, which were washed twice before
incubation.
[0106] FIG. 5c: shows CFU of S. aurerus BAA44 per titanium disc
after 1.5 h incubation of with S. aurerus on vancomycin or
rifampin/fosfomycin coated titanium discs, which were washed either
once or twice before incubation.
[0107] FIG. 5d: shows CFU of S. aurerus BAA44 on titanium discs or
in the supernatant after overnight incubation of S. aurerus with
vancomycin or rifampin/fosfomycin coated titanium discs in Minimal
Medium.
[0108] FIG. 6: shows CFU of in osteoblastic MG63 cells
intracellular located or to osteoblastic MG63 cells adhered S.
aurerus BAA44 after incubation overnight with S. aurerus BAA44 and
antibiotics.
DETAILED DESCRIPTION
[0109] 1. Use of Rifampin and Fosfomycin or their Combination for
the Treatment of Extracellular Infections
[0110] 1.1. Use of Rifampin and Fosfomycin or their Combination for
the Treatment of Extracellular Infections of Osteoblastic MG63
Cells Infected with Staphylococcus aureus Subsp. aureus Rosenbach
(ATTC 6538P)
[0111] Osteoblastic MG63 cells were detached with the cell
detachment medium Accutase 24 hours before infection. The cell
number was determined using the Neubauer counting chamber. Cells
were seeded onto uncoated 24 well plates with a cell density of
1.5.times.10.sup.4 cells/cm.sup.2 in 1 ml DMEM (Dulbecco's Modified
Eagle's Medium) with 10% FOS (fetal calf serum), 1% Glutamax-1 and
1% Natrium Pyruvat and incubated at 37.degree. C. and 5%
CO.sub.2.
[0112] An overnight culture of S. aurerus ATTC 6538P was prepared
by infecting 5 ml Caso-Bouillon medium with S. aurerus ATTC 6538P.
The cultures were incubated with shaking (450 U/min) over night at
37.degree. C. 100 .mu.l of the overnight cultures were transferred
into 5 ml Caso Boulillon medium and incubated for 2 h at 37.degree.
C. with shaking (450 U/min) prior to infection.
[0113] The cell culture supernatant of the osteoblastic MG63 cells
was removed with a pipette from the wells. 1 ml containing
1.times.10.sup.6 S. aurerus ATTC 6538P cells was added to each
well. Two 24 well plates were incubated with S. aurerus ATTC 6538P.
The combined osteoblastic cells and bacteria were incubated for 1.5
h at 37.degree. C. under 5% CO.sub.2 atmosphere. The presence of
bacteria was determined using a microscope.
[0114] After 1.5 h the supernatant was removed and the wells were
carefully washed twice with 37.degree. C. warm DMEM without
additives. It was microscopically checked, if not too many cells
were detached during the washing procedure. During the washing only
the planktonic cells were removed, bacteria adhered to cells and
the cell culture plastics were visible in great numbers. Afterwards
1 ml of cell culture complete medium was added to each well
containing following antibiotics: [0115] 100 .mu.g/ml gentamicin
[0116] 1 .mu.g/ml rifampin [0117] 100 .mu.g/ml fosfomycin disodium
[0118] 1 .mu.g/ml rifampin+100 .mu.g/ml fosfomycin disodium [0119]
1 .mu.g/ml rifampin+100 .mu.g/ml gentamicin No negative control
without antibiotics was used since the strong bacterial growth in
absence of antibiotics would damage the osteoblastic cells.
[0120] After incubation for 24 h, 100 .mu.l of the cell culture
supernatant was streaked out on Caso agar plates (Casein-peptone
soymeal-peptone broth) directly, e.g. in case of rifampin,
fosfomycin, rifampin/fosfomycin and rifmpicin/gentamicin, or after
appropriate dilution, e.g. 1:100 in case of gentamicin, and
incubated overnight at 37.degree. C. The supernatant of two wells
per group was streaked out.
[0121] The person skilled in the art will recognize that the above
given description is just one possibility out of many
alternatives.
[0122] The CFU (colony forming unit) of the supernatant was
determined and is shown in FIG. 1.
[0123] FIG. 1 shows clearly a sensitivity of S. aurerus ATTC 6538P
located in the culture supernatant of osteoblastic MG63 cells
towards the different antibiotics with the exception of gentamicin
Although the used concentrations were high compared to the MIC
values (minimal inhibitory concentration) determined for this S.
aurerus strain, the bacteria could not be removed completely with
the antibiotic treatment. This is due to the fact that the bacteria
settled on the surface of the cells or the cell culture plastic,
which results in reduced sensitivity to antibiotics. This simulates
the in vivo situation where staphylococci readily bind to the
extracellular matrix and foreign bodies. The effect of rifampin,
fosfomycin and the combination of rifampin/gentamicin is moderate,
whereas the combination rifampin/fosfomycin shows a strong,
synergistic effect.
[0124] 1.2. Use of Rifampin and Fosfomycin or their Combination for
the Treatment of Extracellular Infections of Osteoblastic MG63
Cells Infected with Staphylococcus aureus Subsp. aureus
(BAA44).
[0125] The experimental set up for the infection of osteoblastic
MG63 cells infected with S. aurerus BAA44, a MRSA strain with
additional resistance against multiple antibiotics, was basically
the same as above.
[0126] Following antibiotics were used: [0127] 100 .mu.g/ml
vancomycin [0128] 10 .mu.g/ml rifampin [0129] 100 .mu.g/ml
fosfomycin [0130] 10 .mu.g/ml rifampin+100 .mu.g/ml fosfomycin
[0131] 10 .mu.g/ml rifampin+100 .mu.g/ml vancomycin.
[0132] After incubation for 24 h 100 .mu.l of the cell culture were
streaked out on Caso agar plates (Casein-peptone soymeal-peptone
broth) directly, e.g. in case of vancomycin, rifampin/fosfomycin
and rifampin/vancomycin, or after appropriate dilution, e.g. 1:100
in case of rifampin and fosfomycin, and incubated overnight at
37.degree. C. The supernatant of two wells per group was streaked
out.
[0133] The person skilled in the art will recognize that the above
given description is just one possibility out of many
alternatives.
[0134] The CFU (colony forming unit) of the supernatant was
determined and is shown in FIG. 2.
[0135] FIG. 2 shows clearly a sensitivity of S. aurerus BAA44
located in the culture supernatant of osteoblastic MG63 cells
towards the different antibiotics. It is pointed out that it was
necessary to adapt the CFU values logarithmical.
[0136] Rifampin in the used concentration shows as expected hardly
any efficacy, since S. aurerus BAA44 is a rifampin resistant
strain. Also the antibiotic effect of fosfomycin is relatively
small. However, the combination rifampin/fosfomycin shows a strong,
synergistic effect on extracellular S. aurerus BAA44, which was
surprising and not expected due to the weak effect of the single
compounds.
[0137] The effect of the combined rifampin/fosfomycin is even
slightly better than the effect of vancomycin, which is one of the
most important antibiotics for the treatment of MRSA infections.
The combination of vancomycin and rifampin also shows a slight
synergistic effect. It is noteworthy that the concentration of
vancomycin used in this experiment was very high to increase the
otherwise weak bactericidal effect of vancomycin. A concentration
of 100 .mu.g/ml vancomycin cannot be achieved with intravenous
application.
[0138] 2. Use of Different Antibiotics, i.e. Rifampin and
Fosfomycin or their Combination for the Treatment of Intracellular
Infections
[0139] 2.1. Use of Different Antibiotics, i.e. Rifampin and
Fosfomycin or their Combination for the Treatment of Intracellular
Infections of Osteoblasts MG63 Infected with Staphylococcus aureus
Subsp. aureus Rosenbach (ATTC 6538P)
[0140] The experimental set up for determination of intracellular
infection of osteoblastic MG63 cells infected with S. aurerus ATTC
6538P was essentially the same as above.
[0141] However, in order to eliminate extracellular S. aurerus ATTC
6538P each cell culture was treated with lysostaphin after
infection before adding the antibiotics.
[0142] For this purpose the bacterial suspension was removed from
each well and the cells were washed once with warm DMEM containing
10% FCS. 250 .mu.g/ml 25 .mu.g/ml lysostaphin solution was added to
each well. The cells were incubated for 10 min at 37.degree. C.
Afterwards no extracellular bacteria could be observed
microscopically. The lysostaphin solution was removed completely
and the cells were washed once with 1 ml warm DMEM. Afterwards the
antibiotic solutions having the following compositions were added:
[0143] 100 .mu.g/ml vancomycin [0144] 100 .mu.g/ml gentamicin
[0145] 0.01-100 .mu.g/ml rifampin [0146] 10-1000 .mu.g/ml
fosfomycin [0147] 1 .mu.g/ml rifampin+100 .mu.g/ml fosfomycin
[0148] 1 .mu.g/ml rifampin+100 .mu.g/ml gentamicin [0149] 1-100
.mu.g/ml moxifloxacin The infected cells were incubated for 24 h at
37.degree. C. under CO.sub.2 atmosphere.
[0150] In order to determine the metabolic activity of osteoblastic
MG63 cells after infection, the cell supernatant was removed and 1
ml of warm fresh cell culture medium was added to each well.
Afterwards 200 .mu.l of MTT solution
(3[4,5-Dimethylthiazol-2-yl]-2,5-diphenaltetrazoliumbromide) was
added to each well. The cultures were incubated for 2 h at
37.degree. C. under 5% 00.sub.2 atmosphere. The cell culture
supernatant was removed and the formazan, which was formed due to
metabolic activity, was solubilised with 1 ml isopropanol. 200
.mu.l of each suspension were transferred to a 96 well microtiter
plate and the absorbance at 540 nm was measured with an ELISA
reader (Tecan).
[0151] The absorbance at 540 nm is an indicator for the metabolic
activity of the osteoblastic MG63 cells. The intracellular
propagation of the cytotoxic S. aurerus strain ATTC 6538P in
osteoblastic MG63 cells leads to the death of the infected cell.
The lower the extinction is the lower is the metabolic activity of
the cells and thus the stronger is the infection of the cells with
S. aurerus ATTC 6538P.
[0152] The person skilled in the art will recognize that the above
given description is just one possibility out of many
alternatives.
[0153] FIGS. 3 a-d show he influence of the different antibiotics
on the metabolic activities of osteoblastic MG63 cells
[0154] Vancomycin is unable to penetrate into the cell and thus
does not influence the intracellular propagation of S. aurerus
inside the osteoblastic cells. Therefore, the metabolic activity of
the osteoblastic cells is strongly reduced due to the infection
with S. aurerus ATTC 6538P (FIGS. 3a-d). The same applies to
gentamicin. Nevertheless, in combination of gentamicin with
rifampin the metabolic activity was higher than for rifampin alone
(data not shwon).
[0155] Rifampin on the other hand is able to reduce the cell death
caused by S. aurerus ATTC 6538P drastically (FIG. 3a). Already
small concentrations (0.006 .mu.g/ml) are sufficient in increasing
the metabolic activity.
[0156] Fosfomycin also influences the intracellular propagation of
S. aurerus ATTC 6538P and thus the metabolic activity of the
infected osteoblastic cells (FIG. 3b). 10 .mu.g/ml fosfomycin
increases the metabolic activity only slightly, whereby 100
.mu.g/ml had the maximal effect and almost doubled the metabolic
activity. This result is surprising since so far it has not been
known that fosfomycin is be able to penetrate into cells. It is
only known that fosfomycin can penetrate into neutrophils.
[0157] The combination of rifampin and fosfomycin also leads to an
increase of metabolic activity (FIG. 3d), even showing a
synergistic effect (FIG. 3c).
[0158] Also the application of 1 to 100 .mu.g/ml moxifloxacin can
inhibit intracellular growth of S. aurerus ATTC 6538P and thus
increase the metabolic activity up to more than two fold (FIG.
3e).
[0159] 2.2. Use of Rifampin, Fosfomycin or their Combination for
the Treatment of Intracellular Infections of Osteoblasts MG63
Infected with Staphylococcus aureus Subsp aureus (BAA44)
[0160] The experimental set up for determination of intracellular
infection of osteoblastic MG63 cells infected with S. aurerus BAA44
was essentially the same as above described for S. aurerus ATTC
6538P,
[0161] Because the non-cytotoxic S. aurerus BAA44 persists in
osteoblasts and does not divide intracellularly like the cytotoxic
strain S. aurerus ATTC6538P, the intracellular localisation of S.
aurerus BAA44 does not result in cell death of the osteoblastic
cells. The intracellular infection of the osteoblastic MG63 cells
with S. aurerus BAA44 could therefore not be determined on basis of
the metabolic activity of the cells and was determinded via cell
lysis and counting of the intracellular CFU instead.
[0162] Antibiotic solutions having the following compositions were
added: [0163] 100 .mu.ml vancomycin [0164] 2.5-40 .mu.g/ml rifampin
[0165] 25-400 .mu.g/ml fosfomycin [0166] and their mixtures in
different ratios as given below. The infected cells were incubated
with the antiobioics for 24 h at 37.degree. C. under 5% CO.sub.2
atmosphere.
[0167] Afterwards the cells are washed once with PBS pH 7.4
(phosphate buffer solution) followed by lysis with 1 ml 0.1% Triton
X100 in ringer's solution. The lysates were treated for 5 min with
ultrasound. The lysates are thoroughly resuspended with a pipette.
Only one 24 well plate was handled and the other plates were stored
at 4.degree. C. in order to minimize bacterial growth in the
lysate. 100 .mu.l lysate were undiluted streaked out on Caso agar
plates, incubated over night at 37.degree. C. and the colonies were
counted.
[0168] FIGS. 4a-4g show the CFU value per well as an indicator for
the degree of intracellular S. aurerus BAA44 infections of
osteoblastic MG63 cells. The lower the CFU value is the lower is
the infection rate of the osteoblastic cells with S. aurerus BAA44.
This correlates to the efficacy of the added antibiotic. Due to the
weak intracellular growth of S. aurerus BAA44, a decrease in CFU is
caused by the bactericidial effect of the antibiotics.
[0169] Fosfomycin in concentration between 50-400 .mu.g/ml shows a
good efficacy on the infection rate with intracellular located S.
aurerus BAA44 (FIG. 4a). Surprisingly the effect of fosfomycin can
be achieved at concentrations allowing for intravenous application
(100-400 .mu.g/ml, preferably 132-297 .mu.g/ml in serum). Because
of its excellent tissue penetration high fosfomycin concentrations
are also achieved in bone. Therefore, fosfomycin is successfully
applied in the treatment of osteomyelitis
[0170] The person skilled in the art will recognize that the above
given description is just one possibility out of many
alternatives.
[0171] Athough S. aurerus BAA44 is a rifampin resistant strain
rifampin shows a good intracellular potency (FIG. 4b).
[0172] Rifampin and fosfomycin show clearly a synergistic effect in
varying concentration ratios (FIG. 4c-g).
[0173] Even in case of the rifampin resistant S. aurerus BAA44 the
applied concentrations were sufficient enough in order to allow a
systemic treatment of bone infections. Therefore, the combination
of rifamycin and fosfomycin is suitable for treating osteomyelitis
and can also be applied systemically.
[0174] 3. Use of a Combination of Rifampin and Fosfornvcin as
Anti-Adhesives on Surfaces of Medical Substrates
[0175] 3.1. Adhesion of Staphylococcus aureus Subsp. aureus
Rosenbach (ATTC 6538P) on a Titanium Substrate Coated with Rifampin
and Fosfomycin
[0176] An overnight culture of S. aurerus ATTC 6538P was prepared
by infecting 5 ml Caso-Bouillon medium with S. aurerus ATTC 6538P.
The cultures were incubated with shaking (450 U/min) over night at
37.degree. C. 100 .mu.l of the overnight cultures were transferred
into 5 ml Caso Boulillon medium and incubated for 2 h at 37.degree.
C. with shaking (450 U/min). The bacterial density was determined
photometrically. The bacterial suspension was diluted 1:2 in Caso
Bouillon prior measurement. A bacterial suspension with a density
of 1.times.10.sup.5 CFU/ml in Caso Boullion with 10% FCS was used
for the adhesion experiments.
[0177] Differently coated 2 cm titanium discs were used as samples:
[0178] titanium discs sand blasted as negative control, [0179]
titanium discs sand blasted and coated with 200 .mu.g/cm.sup.2
vancomycin, [0180] titanium discs simultaneously coated with 50
.mu.g/cm.sup.2 rifampin and 200 .mu.g/cm.sup.2 fosfomycin calcium,
[0181] titanium discs coated in a first step with 50 .mu.g/cm.sup.2
rifampin and in a second step with 200 .mu.g/cm.sup.2fosfomycin
calcium, and [0182] titanium discs coated in a first step with 200
.mu.g/cm.sup.2fosfomycin calcium and in a second step with 50
.mu.g/cm.sup.2 rifampin.
[0183] After coating the titanium discs were washed either one time
or three times with PBS. The coated and uncoated titanium discs
were incubated for 5 min at room temperature with 5 ml PBS. This
was repeated two more times. In the third circle the titanium discs
were incubated for 1 h at room temperature. Before removing the PBS
solution the titanium discs were turned or swivelled in order to
increase the detachment of the antibiotics. Afterwards the titanium
discs were transferred into sterile 12 well plates.
The different titanium samples were incubated with 2 ml bacterial
suspension for 1.5 h at 37.degree. C. without shaking.
[0184] Afterwards the bacterial suspension was removed and the
discs were washed three times with 2.5 ml PBS. After the last
washing cycle each discs was placed in 10 ml sterile ringer's
solution. Only one disc of each group was simultaneously examined
while the other discs were stored at 4.degree. C. The titanium
discs in the ringer's solution were exposed to ultra sound for 10
min in order to detach the adhered bacteria. The suspensions
comprising the detached bacteria were diluted (1:10, 1:100) and
streaked out on a Caso agar plate. The agar plates ere incubated
over night at 37.degree. C. and the next day the colonies were
counted.
[0185] The person skilled in the art will recognize that the above
given description is just one possibility out of many
alternatives.
[0186] FIG. 5a shows the CFU of S. aurerus ATTC 6538P per titanium
disc afterincubation for 1.5 h with the bacteria. Vancomycin had
only an anti-adhesive effect after one washing step, but did not
reveal any anti-adhesive effect after three washing steps. In fact,
the number of S. aurerus cells adhered to the vancomycin coated
discs was identicalto the uncoated discs. However, the combination
rifampin and fosfomycin showed a strong anti-adhesive effect. The
effect depended only slightly on the number of washing steps.
Obviously, the rifampin/fosfomycin coating was less likely to be
removed completely from the titanium surface by several washing
steps than vancomycin.
[0187] The order of coating the discs with rifampin and
fosfomycin--together, first rifampin then fosfomycin; first
fosfomycin then rifampin--does not seem to influence the effect
(FIG. 5a).
[0188] 3.2. Adhesion of Staphylococcus epidermis ATTC 35984 on a
Titanium Substrate Coated with a Combination of Rifampin and
Fosfomycin
[0189] The experimental set up was essentially the same as
described above for S. aurerus ATTC 6538P.
[0190] Differently coated 2 cm titanium discs were used as samples:
[0191] titanium discs (sand blasted) as negative control, [0192]
titanium discs coated in a first step with 50 .mu.g/cm.sup.2
rifampin and in a second step with 200 .mu.g/cm.sup.2fosiomycin
calcium, and [0193] titanium discs coated in a first step with 200
.mu.g/cm.sup.2fosfomycin calcium and in a second step with 50
.mu.g/cm.sup.2 rifampin.
[0194] The coated titanium discs were washed three times with 5 ml
PBS before incubation with S.epidermis.
[0195] The person skilled in the art will recognize that the above
given description is just one possibility out of many
alternatives.
[0196] The experimental results for S.epidermis (FIG. 5b) support
the results found in case of S. aurerus ATTC6538P. The adhesion of
S.epidermis ATTC 35984 on uncoated titanium was lower than the
adhesion of S. aurerus ATTC6538P. This can relate to the fact that
S.epidermis preferably attaches to plastics or hydroxyapatite but
less to titanium. Although the titanium discs were washed three
times before incubation with the bacteria, the combination rifampin
and fosfomycin shows a strong anti-adhesive effect.
[0197] 3.3. Adhesion of Staphylococcus aureus BAA44 on a Titanium
Substrate Coated with a Combination of Rifampin and Fosfomycin
[0198] The experimental set up was essentially the same as
described above for S. aurerus ATTC 6538P.
[0199] The following coated 2 cm titanium discs were used as
samples: [0200] titanium discs sand blasted as negative control,
[0201] titanium discs coated with 200 .mu.g/cm.sup.2 vancomycin
[0202] titanium discs sand blasted and coated in a first step with
50 .mu.g/cm.sup.2 rifampin and in a second step with 200
.mu.g/cm.sup.2 fosfomycin calcium.
[0203] The coated titanium discs were washed either once or twice
with 5 ml PBS before incubation with S. aurerus BAA44 for 1.5
h.
[0204] The person skilled in the art will recognize that the above
given description is just one possibility out of many
alternatives.
[0205] FIG. 5c shows the CFU on the discs after incubation with S.
aurerus BAA44 . Vancomycin reduced the adhesion of S. aurerus BAA44
only if the disc were washed once: After two washing steps all
vancomycin seems to be removed and no reduction of bacterial
adhesion could be observed. Despite the rifampin-resistance of S.
aurerus BAA44 the combination rifampin/fosfomycin had a strong
anti-adhesive effect, which was only slightly diminished if the
discs were washed twice instead of once before incubation with the
bacteria.
[0206] 3.4. Bactericidal Activity of Titanium Substrate Coated with
Rifampin and Fosfomycin Against S. aurerus BAA44
[0207] An overnight culture of S. aurerus BAA44 was prepared by
infecting 5 ml Caso-Bouillon medium with S. aurerus BAA44. The
cultures were incubated with shaking (450 U/min) over night at
37.degree. C. 100 .mu.l of the overnight cultures were transferred
into 5 ml Casio Boulillon medium and incubated for 2 h at
37.degree. C. with shaking (450 U/min) prior to the incubation with
the titanium discs. The bacterial density was determined
photometrically.
[0208] A bacterial suspension with a density of 1.times.10.sup.4
CFU/ml in Minimal Medium (PBS, 0.2% ammonium chloride, 0.2% sodium
sulphate, 0.25% glucose, 1% Caso Bouillon, 50 .mu.g/ml
glucose-6-phosphate) was used in the adhesion assay. The Minimal
Medium was used instead of Casa Bouillon to minimize the bacterial
growth.
[0209] Differently coated 2 cm titanium discs were used as samples:
[0210] titanium discs (sand blasted) as negative control, [0211]
titanium discs (sand blasted) coated with 200 .mu.g/cm.sup.2
vancomycin, [0212] titanium discs coated in a first step with 300
.mu.g/cm.sup.2fosiomycin calcium and in a second step with 70
.mu.g/cm.sup.2 rifampin.
[0213] After coating, the titanium discs were washed three times
with 2.5 ml PBS at room temperature.
[0214] The different titanium samples were incubated with 2 ml
bacterial suspension for 15.5 h at 37.degree. C. without
shaking.
[0215] Afterwards the CFU in the supernatant as well as the adhered
bacteria on the titanium discs were analysed. The supernatant was
diluted 1:10 in PBS, 100 .mu.l of the dilution were streaked out on
Caso agar plates. The discs were washed four times with 2.5 ml PBS
to remove not adherent bacteria. After the last washing cycle each
discs was placed in 10 ml sterile ringer's solution. Only one disc
of each group was simultaneously examined while the other discs
were stored at 4.degree. C. The titanium discs in the ringer's
solution were exposed to ultra sound for 10 min in order to detach
the adhered bacteria. The suspensions comprising the detached
bacteria were diluted (1:10, 1:100, 1:1000) and streaked out on a
Caso agar plate. The agar plates were incubated over night at
37.degree. C. and the next day the colonies were counted.
[0216] The person skilled in the art will recognize that the above
given description is just one possibility out of many
alternatives.
[0217] The results are shown in FIG. 5d. The bacterial growth in
the negative controls was reduced by using Minimal Medium, but
nevertheless the bacterial CFU in the supernatant increased ten
times during the incubation period. Surprisingly, more CFU could be
found adhered to the uncoated disc than in the supernatant.
[0218] Vancomycin reduced the bacterial growth in the cell culture
supernatant compared to the uncoated control slightly, but the
adherence of the bacteria was even more reduced. However,
vancomycin could not exhibit any bactericidal effect and more than
50,000 CFU could be found on the vancomycin coated titanium
samples.
[0219] Although the titanium discs were washed three times before
the adhesion assay, the fosfomycin/rifampin combination displayed a
dear bactericidal activity against the rifampin-resistant strain
BAA44. No CFU could be detected in the supernatant and less than
100 CFU adhered to the titanium surface. This corresponds to a
86,000-fold reduction in bacterial adherence compared to uncoated
titanium and a 470-fold reduction compared to the vancomycin
coating.
[0220] It was expected that a soluble antibiotic coating without
carrier matrix e.g. polymer matrix unfolds its efficacy by
dissolving into the tissue fluid after implantation. The
colonization of the implant or prosthesis is then hampered by
killing the planktonic bacteria before colonization and reduction
of bacterial propagation due to the efficacy of the dissolved
antibiotics. After several washing steps the amount of rifampin and
fosfomycin left on the discs, and thus available in the
supernatant, was still high enough for showing antibacterial
efficacy in the supernatant. Therefore, the rifampin/fosfomycin
coating is stable enough to get in contact with tissue fluids and
blood during implantation and is still effective in preventing
bacterial adherence to the implant surface and the surrounding
tissue. This property is especially important for staphylococci
infections, because staphylococci do not adhere exclusively to
implants but to the extracellular matrix of tissue as well.
[0221] 4. Use of Rifampin and Daptomycin or their Combination for
Treatment of Acute Infection of Osteoblasts MG63 Cells with
Staphylococcus aureus Subsp. aureus (BAA44)
[0222] Osteoblastic MG63 cells were detached with the cell
detachment medium Accutase 24 hours before infection. The cell
number was determined using the Neubauer counting chamber. Cells
were seeded onto uncoated 24well plates with a cell density of
1.5.times.10.sup.4 cells/cm.sup.2 in 1 ml DMEM (Dulbecco's Modified
Eagle's Medium) with 10% FCS (fetal calf serum), 1% Glutamax-I and
1% Natrium Pyruvat and incubated at 37.degree. C. and 5%
CO.sub.2.
[0223] An overnight culture of S. aurerus BAA44 was prepared by
infecting 5 ml Caso-Bouillon medium with S. aurerus BAA44. The
cultures were incubated with shaking (450 U/min) over night at
37.degree. C. 100 .mu.l of the overnight cultures were transferred
into 5 ml Caso Boulillon medium and incubated for 2 h at 37.degree.
C. with shaking (450 U/min) prior to infection.
[0224] The cell culture supernatant of the osteoblastic MG63 cells
was removed with a pipette from the wells. 1 ml containing
1.times.10.sup.6 S. aurerus BAA44 CFU was added to each well
containing also antibiotics having the following compositions:
[0225] 50 .mu.g/ml vancomycin [0226] 2.5 .mu.g/ml rifampin [0227]
1.25-10 .mu.g/ml daptomycin [0228] and their mixtures in different
ratios as given below.
[0229] The combined osteoblastic cells, bacteria, and antibiotic
compositions were incubated for 18 h at 37.degree. C. under 5%
CO.sub.2 atmosphere.
[0230] Afterwards the cells were washed once with PBS pH 7.4
(phosphate buffer solution) followed by lysis with 1 ml 0.1% Triton
X100 in ringer's solution. The lysates were thoroughly resuspended
with a pipette. Only one 24 well plate was handled and the other
plates were stored at 4.degree. C. in order to minimize bacterial
growth in the lysate. The lysates were diluted 1:10 in PBS, 100
.mu.l of diluted lysate were streaked out on Caso agar plates,
incubated over night at 37.degree. C. and the colonies were
counted.
[0231] The person skilled in the art will recognize that the above
given description is just one possibility out of many
alternatives.
[0232] Because the cells were not treated with lysostaphin after
infection, the CFU value per well (FIG. 6) is an indicator for the
degree of intracellular infection of osteoblastic MG63 cells with
S. aurerus BAA44 as well as for S. aurerus BAA44 adhered
extracellularly to osteoblastic MG63 cells. The lower the CFU value
is the lower is the infection rate of the osteoblastic cells with
S. aurerus BAA44. This correlates to the efficacy of the added
antibiotic.
[0233] Because the strain is rifampin-resistant the effect of 2.5
.mu.g rifampin was less than for vancomycin and daptomycin, but
overgrowth of the MG63 cells with planktonic S. aurerus BAA44 was
prevented efficacious (data not shown)
[0234] Daptomycin alone showed good efficacy already in
concentrations of 1.25 .mu.g/ml and 2.5 .mu.g/ml, whereas 5
.mu.g/ml and 10 .mu.g/ml could eradicate the infection
completely.
[0235] Despite the ineffectiveness of rifampin alone, the
combination 2.5 .mu.g/ml rifampin and 1.25 .mu.g/ml or 2.5 .mu.g/ml
daptomycin respectively was synergistic in eliminating all
intracellular and extracellular adhered bacteria.
[0236] Because vancomycin is only weak bactericidal a very high
concentration of vancomycin was used in this experiment to increase
its efficacy. This concentration can never be achieved by
intravenous application of vancomycin. However, several hundred S.
aurerus could escape vancoymicin by invading the osteoblastic
cells, a phenomenon that has relevance in vivo especially in the
treatment of bone infections.
[0237] Daptomycin is in contrast to glycopeptides like vancomycin
rapidly bactericidal and the bactericidal activity is concentration
dependent. Therefore the higher concentrations of 5 and 10 .mu.g/ml
could eliminate all bacteria before they were able to invade the
osteoblastic cells.
[0238] Local application of rifampin and daptomycin could be an
efficient treatment for acute bone infections. Daptomycin
eliminates in high concentrations very efficiently all
extracellular bacteria and thus prevents infection of new
osteoblasts, while rifampin is able to eradicate intracellular
infected osteoblasts.
[0239] 5. Coated or Impregnated Substrates for Medical Purposes
[0240] Rifampin was diluted in methanol in a concentration of 30-40
mg/ml. Fosfomycin calcium was suspended in ultrapure water in a
concentration of 100-140 .mu.g/ml. No further additives were used.
The titanium endoprosthesis with different surface modifications
(sand-blasted, porous coated, or hydroxyapatite coated) was coated
directly with the antibiotic solutions using the ink-jet or the
spray coating process. The surface can be coated with rifampin
first, followed by fosfomycin calcium, the other way around, or
both antibiotics simultaneously. The resulting covering density was
50-70 .mu.g/cm.sup.2 rifampin and 300-350 .mu.g/cm.sup.2
fosfomycin.
[0241] Rifampin, fosfomycin disodium, and fosfomycin calcium were
incorporated into collagen fleeces during the production process of
the fleeces. Rifampin and fosfomycin disodium were added dissolved
in acidified buffer, while fosfomycin calcium was added in watery
suspension. The final concentrations were 0.1-0.2 mg rifampin per
cm.sup.2 collagen fleece and 0.5 mg-2 mg fosfomycin per cm.sup.2
collagen fleece, whereas fosfomycin disodium and fosfomycin calcium
could contribute in varying proportions to the final concentration
of fosfomycin.
[0242] Rifampin and fosfomycin disodium were mixed with two
different polymers on PMMA basis, zirconium dioxid, and glycine.
Rifampin was added in an amount of 0.5-1.5% of the total weight,
while fosfomycin disodium was added in an amount of 2.5-7.5% of the
total weight. The polymer/antibiotic mixture was heated to
160-180.degree. C. and PMMA beads were manufactured directly on
metal wires by injection moulding.
[0243] The person skilled in the art will recognize that the above
given description is just one possibility out of many
alternatives.
[0244] Numerous modifications and variations of practicing the
present invention are possible in light of the above teachings and
therefore will fall within the scope of the following claims.
* * * * *