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BISPHOSPHONATE QUINOLONE CONJUGATES AND USES THEREOF
CROSS-REFERENCE TO RELATED APPLICATIONS
This application claims the benefit of and priority to co-pending US
Provisional Patent
Application No. 62/695,583, filed on July 9, 2018, entitled "BISPHOSPHONATE
QUINOLONE
CONJUGATES AND USES THEREOF", which is incorporated by reference herein in its
entirety.
STATEMENT REGARDING FEDERALY SPONSORED RESEARCH OR DEVELOPMENT
This invention was made with government support under grant number R41DE025789-
01
and under grant number R42DE025789-02 awarded by the NIH/NIDCR as well as
under grant
number R43AR073727 awarded by the NIH/NIAMS. The US government has certain
rights in
the invention.
BACKGROUND
Bone and joint infections affect millions of adults and children worldwide.
The overall
incidence in the United States is 3-6 million persons, with specific
populations having different
risks. For diabetics, the annual incidence of foot ulcers is about 1 in 30,
with underlying
osteomyelitis in up to two-thirds of the cases. In children, recently reported
annual incidence
ranges from 1/4000 to 1/15000. However, in the Pediatric Health Information
System (PHIS)
database of administrative data from U.S. pediatric hospitals, we found 10,245
(0.5%) discharges
with a diagnosis of osteomyelitis among 2,247,889 in a 5-year period from 2009-
2013, fora rough
annual incidence of approximately 1/1100 hospitalizations.
A number of Gram-positive and Gram-negative bacteria, as well as fungi and
mycobacteria can cause bone and joint infections. By far the most common
organism implicated
in bone and joint infections is Staphylococcus aureus (S.aureus), both
methicillin-susceptible
.. (MSSA) and methicillin-resistant (MRSA).
The standard of care for bone and joint infections usually requires systemic
administration
of antibiotics. For acute infections, intravenous antibiotics are generally
prescribed for 2 ¨ 6
weeks. Prolonged courses of oral antibiotics may follow for chronic
infections, or infections
associated with retained implanted hardware. Both for acute and chronic
infections, these
extended courses of therapy can lead to drug-related adverse events in a
significant percentage
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of patients ¨ 15% in one estimate for a cohort treated for infections with
MSSA. Moreover, it is
known that nephrotoxicity with vancomycin, the most common therapy for MRSA
infections,
occurs in as much as 43% of patients, and increases with duration of therapy.
Persistent bone infections such as jaw osteomyelitis, osteomyelitis at other
skeletal sites
and osteonecrosis can culminate in significant bone resorption and destruction
of bone and
hydroxyapatite (HA) mineral. Bone and HA resorption is thought to be induced
and mediated not
only by bone cells, i.e. osteoclasts, but also microbial biofilm pathogens in
combination with host
inflammatory responses and osteoclastogenic activity. Biofilms are a complex
microbial
community composed of one or more bacterial species attached to a substrate
and surrounded
by a self- produced extracellular matrix. Many different types of microbial
infections are known to
be caused by organisms growing in a biofilm state. Bacterial biofilms of
Staphylococcus aureus
(S. aureus) are the dominant cause of biofilm- associated infections in health
care systems and
are associated with serious infections such as osteomyelitis.
Osteomyelitis is associated with significant morbidity and mortality.
Surgery and
antimicrobial therapy, often intravenous and longer-term antibiotics, are
mainstays of
osteomyelitis management. Surgery can involve conservative removal of infected
bone or more
aggressive modalities such as resection. Thus, treatment of infectious bone
disease is mainly
antimicrobial therapy with or without surgical intervention depending on
clinicopathologic factors.
Antibiotics, however, have poor bone absorption and pharmacokinetics in vivo.
Therefore, any
improvement in bone bioavailability of therapeutic antibiotics would be a
significant advancement
in treating osteomyelitis.
To overcome the many challenges associated with treating bone infections, it
has become
common practice by clinicians to use local delivery systems for achieving
higher therapeutic
antibacterial concentrations in bone. For example, polymethylmethacrylate
beads represent the
majority of non-biodegradable carrier systems used to deliver antibiotics to
orthopedic infections,
but they require surgical removal upon completion of drug release. They also
tend to release
antibiotics in an initial burst pattern that quickly depletes the bulk of the
drug from the carrier
beads, followed by a slow release at lower concentrations that may not be
adequate to control
infection and may foster development of resistance. These concerns limit the
usefulness of this
approach in the majority of bone and joint infections.
Dentistry has used local delivery of antimicrobials to treat infected jawbone
associated
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with conditions like periodontal bone loss, jaw osteomyelitis and
osteonecrosis in order to reach
high local concentrations of drug, but these modalities are often ineffective
without surgical
intervention and bone bioavailability of antibiotic is poor. Antibiotic-
impregnated cement, used
primarily at the time of first debridement of an infected implant to improve
control of the infection,
.. is not generally used in the treatment of bone and joint infections of
native bone without implanted
hardware. Concerns about prolonged sub-therapeutic antibiotic concentrations
and selection of
resistant organisms also apply to cement.
Localized delivery of antimicrobial agents to bone could be a significant step
forward in
treating infectious bone disease, but still has penetration limitations and
potential eukaryotic cell
cytotoxicity; thus, research and the development of more effective and
physiologically targeted
delivery systems are in high demand. An ideal antibiotic delivery system is
one that targets bone
tissue without the need for surgical implantation or removal. Such targeting
also minimizes
systemic doses and exposure of tissues other than bone to antibiotics,
therefore reducing the risk
of adverse effects or selective pressure facilitating the emergence of
resistant organisms.
Reduced dosing frequency made possible by achieving prolonged concentrations
of the antibiotic
at the site of infection is another potential major benefit.
The inadequate efficacy of current antimicrobial treatments for osteomyelitis
has been
ascribed to the limited access of systemically administered antibiotics to
sites where causative
bacteria can reside, including as biofilms on bone surfaces, even surfaces
within the osteocytic
.. canalicular network, where a class of drugs known as bisphosphonates (BPs)
readily gain access.
SUMMARY
Provided herein, in various aspects, are BP quinolone antibiotic compounds,
conjugates
and formulations, and various methods of use thereof, to address the
aforementioned needs.
In any one or more aspects herein, to exploit BP affinity for bone, a "target
and release"
chemistry approach involving delivery of a quinolone antibiotic to bone or
hydroxyapatite (HA)
surfaces via BP conjugates is provided, in particular to sites where bone
infections have initiated
and elevated bone metabolism has taken place. Relatively serum-stable drug- BP
linkers can be
utilized that metabolize and release the parent quinolone antibiotic most
preferentially at the bone
surface.
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The BP quinolone compounds, conjugates and formulations can contain a
bisphosphonate (BP) that can be releasably conjugated to a quinolone compound
or analog. In
any one or more embodiments herein, the BP has an alpha substituent and the
alpha subsitutent
is a hydroxy, amino or thiol group. The quinolone can be conjugated directly
or indirectly to the
BP at the geminal carbon alpha substituent of the BP, a described in any one
or more aspects
herein. In any one or more embodiments, the quinolone is reversibly coupled or
conjugated to a
geminal hydroxy, amino or thiol group on the carbon between the two
phosphonate groups of the
BP. When conjugated in this manner the two phosphonate groups operate to
weaken the linkage
of the quinolone to the BP, and the BP is activating the linker that
reversibly couples or conjugates
.. the quinolone to the BP upon release of the quinolone from the BP. In
particular embodiments,
the BP can be etidronate, methylene hydroxy bisphosphonate (MHBP) or
pamidronate, preferably
etidronate or MHBP. In particular embodiments, the BP can be an inactive or a
low active BP, as
described herein.
In any one or more embodiments, the BP quinolone compound or conjugate can be
administered systemically to selectively deliver a quinolone to the skeleton
and, in particular, to
infected bone sites, or locally when combined with bone grafts or bone graft
substitutes (i.e., can
target bone, bone infections, or other high bone metabolism sites) in a
subject. In any one or more
embodiments, the BP quinolone compound or conjugate can release the quinolone,
in particular
a quinolone compound, substituent or derivative thereof. Also provided herein
are methods of
synthesizing BP quinolone compounds, conjugates and methods of treating or
preventing
osteomyelitis or other bone infections with one or more of the BP quinolone
compounds,
conjugates and/or formulations provided herein.
Provided herein, in various aspects, are BP quinolone compounds and conjugates
that
can contain a bisphosphonate (BP) that can be releasably conjugated to a
quinolone. In
embodiments, the BP quinolone compound or conjugate can selectively deliver a
quinolone to
bone, bone grafts, and or bone graft substitutes in particular to sites of
higher bone metabolism
where bone infections have initiated in a subject. In any one or more
embodiments, the BP
quinolone compound or conjugate can release the quinolone. Also provided
herein are methods
of synthesizing BP quinolone compounds and conjugates and methods of treating
or preventing
osteomyelitis or other bone infections with one or more of the BP quinolone
compounds or
conjugates provided herein.
In particular embodiments herein, the BP is etidronate conjugated to a
fluoroquinolone
antibiotic such as ciprofloxacin, moxifloxacin or sitafloxacin. In
embodiments, the BP is etidronate
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conjugated to a non-fluoroquinolone such as nemonoxacin. For example, the
conjugate can be
a compound according to Formula (41), Formula (43), Formula (44) or Formula
(45).
0 OH
, 0
rN
HO pH 0
ThrN)
0,p/CH3 0
l OH
HO
Formula (41)
0 OH
0
() A
(H0)20P
)70
(H0)20P CH3
Formula (43)
OH
0
0 I .0H
.õF HN 0 p:OH
ci O' OH
Nii<1
HO
0 0
Formula (44)
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HO
0
-=P -OH
HNT N`o OH
7 0-
HO
0 0
Formula (45)
Also provided herein are pharmaceutical compositions or formulations
containing a
compound according to Formula (41), Formula (43), Formula (44) and/or Formula
(45), and a
pharmaceutically acceptable carrier.
Also provided herein are methods of treating a bone infection in a subject in
need thereof
that can include the step of administering an amount of the compound according
to Formula (41),
Formula (43), Formula (44) and/or Formula (45), or a pharmaceutical
formulation containing a
compound according to Formula (41), Formula (43), Formula (44) and/or Formula
(45), to a
subject in need thereof.
Also provided herein are compounds, conjugates and antimicrobial and
antibiotic agents
containing a bisphosphonate (BP) and a quinolone compound, wherein the
quinolone compound
is releasably or reversibly coupled to the bisphosphonate via a linker, as
described herein.
Preferred releasable linkers, as described herein, are more or less stable in
the bloodstream
shortly after administration and more or less slowly cleaved in the bone /
skeletal compartments
of the body to slowly release quinolone antibiotic compounds, substituents or
derivatives locally.
In any one or more aspects, the BP quinolone compound can be comprised of a
quinolone
antibiotic analog or substituent according to the following structure or
Formula (A),
R40 0
R5
OH
R1
Ro R2
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Formula (A)
wherein R1 can be either
90H
rN \ HO
1_
HN 0 " \
OH
or NH or
L = linker or
cp \ cp \
0
HO -OHP N \ HO \
\ L HO \
HO NL) / =
13¨K OH
1_ p
"' L linker HO Ho/ ,-,
OH L = linker or HO ,-1 = ner or "i L =
linker
>1--
and wherein R2 can be
and wherein R3 can be either H or OCH3,
and where R4 can be H,
and wherein R5 can be H or F.
As shown, the quinolone of Formula (A) can be linked to a bisphosphonate (BP).
In any
one or more aspects or embodiments herein, a compound or conjugate comprising
a
bisphosphonate (BP) and a quinolone compound or analog is provided wherein the
BP can have
an alpha substituent and the alpha substituent can be a hydroxy, amino or
thiol group. The
quinolone can be directly or indirectly conjugated to the BP at the germinal
carbon alpha
substituent (X) of the BP, as illustrated in the formula below.
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quinolone
HO X
OH
HO ¨P --n P ¨OH
0 R "co conjugates between alpha-X containing BP and
quinolone
X= 0, NH, NRI, S
R1 can be alkyl or substituted alkyl, aryl or subsituted aryl groups
wherein R can be H, substituted and unsubstituted alkyl, alkyl amino, alkyl-
aryl, aryl,
alkylheteroaryl, or heteroaryl.
Preferred BPs are those that have a geminal hydroxy group on the carbon
between the
two phosphonate groups. A generic analog of such a BP is illustrated in Fig.
25. The BP be an
alpha-OH containing BP and wherein the quinolone is directly or indirectly
conjugated to the BP
at the geminal OH of the BP.
In any one or more aspects, the bisphosphonate can be ethylidenebisphosphonate
moiety
(etidronate) that can be substituted by hydroxy (an alpha-hydroxy), amino or
thiol. In some
aspects, the bisphosphonate can include a para-hydroxyphenylethylidene group
or derivative
thereof. In embodiments, the BP can be a clinically known BP, such as
pamidronate, alendronate,
risedronate, zoledronate, minodronate, neridronate, and etidronate, which can
be unmodified or
modified as described herein.
In any one or more preferred embodiments, the BP can be etidronate. Etidronate
can be
linked to a quinolone to form a quinolone antibiotic etidronate-ciprofloxacin
(FCC) conjugate, such
as in Formula (41) or to form an etidronate moxifloxacin (ECX) conjugate such
as in Formula (43)
herein.
The linker, L, can be a compound that is cleavable, meaning that it reversibly
couples the
quinolone analog or compound, in particular a quinolone antimicrobial or
antibiotic analog or
substituent thereof, to the BP. As used herein, the term "cleavable" can mean
a group that is
chemically or biochemically unstable under physiological conditions. In any
one or more aspects,
the linker can be a carbamate, having a structure or Formula (B) below
11
81õ, õõC, R2
0 N
R3
Formula (B)
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for coupling a quinolone, R2, to a BP, R1, as described herein, and R3 can be
substituted and
unsubstituted alkyl, acetyl, benzoyl or other amides, phenyl and substituted
phenyl, preferably H.
In any one or more aspects, the linker can be a carbonate, having a structure
or Formula
(C) below
ii
¨ 0 ¨FP
Formula (C)
for coupling a quinolone, R2, to a BP, R1, as described herein.
In any one or more aspects of any one or more embodiments herein, the linker
can be an
alkyl or an aryl carbamate linker. The linker can be an 0-thioaryl or
thioalkyl carbamate linker.
The linker can be an S-thioaryl or thioalkyl carbamate linker. The linker can
be a phenyl carbamate
linker. The linker can be a thiocarbamate linker. The linker can be an 0-
thiocarbamate linker. The
linker can be an S-thiocarbamate linker. The linker can be an ester linker.
The linker can be a
dithiocarbamate. The linker can be a urea linker. The linker can be part of
the R1 group of Formula
(A) along with the BP and couple the BP to the quinolone, as described herein.
In any one or
more aspects, the linker can be exemplified by any one of Formula (D) ¨
Formula (H) below,
wherein: R2 can be a quinolone or a quinolone substituent or derivative and R1
can be a BP, both
as described herein; and R3 can be substituted and unsubstituted alkyl,
acetyl, benzoyl or other
amides, phenyl and substituted phenyl, preferably H.
0
R' II_132 111 A _132
N N 0
111
R3 R3
Formula (D) Formula (E) Formula (F)
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0
Ill II R2 R1 ,,R2
N =N N
R3 R4 R3
Formula (G) Formula (H)
In some aspects, the BP is etidronate. In some aspects, the quinolone is
ciprofloxacin or
moxifloxacin. In some aspects, the BP is etidronate, the quinolone is
ciprofloxacin and the linker
is an alkyl or an aryl carbamate or a linker of Formula (F) providing the
compound of Formula
(41). In some aspects, the BP is etidronate, the quinolone is moxifloxacin and
the linker is an
alkyl or an aryl carbamate or a linker of Formula (F) providing the compound
of Formula (43). In
some aspects, the BP is etidronate, the quinolone is sitafloxacin or
nemonoxacin and the linker
is an alkyl or an aryl carbamate or a linker of Formula (F) providing the
compound of Formula (44)
or Formula (45) below.
OH
I 0 ..OH
.õ
HN F p -OH
yOH
HO
0 0
Formula (44)
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HO
zipt -OH
0
OP-OH
HN
\ 0 OH
7 rc
N
HO
0 0
Formula (45)
In other aspects, the BP can be another BP described herein, such as
pamidronate,
neridronate, olpadronate, alendronate, ibandronate, minodronate, risedronate,
zoledronate,
hydroxymethylenebisphosphonate, and combinations thereof.
Also provided herein are pharmaceutical formulations that can contain a
bisphosphonate
(BP) and a quinolone compound of Formula (A), wherein the quinolone compound
is releasably
coupled to the bisphosphonate via a linker, L; and a pharmaceutically
acceptable carrier. The
bisphosphonate (BP) and the linker, L, can be as described herein in any one
or more aspects.
Also provided, in any one or more aspects of any one or more embodiments
herein, are
compounds and conjugates containing a bisphosphonate (BP) and a quinolone
compound,
wherein the quinolone compound is releasably coupled to the bisphosphonate via
a linker. The
BP can be selected from the group of: hydroxyl phenyl alkyl or aryl
bisphosphonates, hydroxyl
phenyl (or aryl) alkyl hydroxyl bisphosphonates, amino phenyl(or aryl) alkyl
bisphosphonates,
amino phenyl(or aryl) alkyl hydroxyl bisphosphonates, hydroxyl alkyl
bisphosphonates, hydroxyl
alkyl hydroxyl bisphosphonates, hydroxyl alkyl phenyl(or aryl) alkyl
bisphosphonates, hydroxyl
phenyl(or aryl) alkyl hydroxyl bisphosphonates, amino phenyl(or aryl) alkyl
bisphosphonates,
amino phenyl(or aryl) alkyl hydroxyl bisphosphonates, hydroxyl alkyl
bisphosphonates, hydroxyl
alkyl hydroxyl bisphosphonates, hydroxypyridyl alkyl bisphosphonates, pyridyl
alkyl
bisphosphonates, hydroxyl imadazoyl alkyl bisphosphonates, imidazoyl alkyl
bisphosphonates,
etidronate, pamidronate, neridronate, olpadronate, alendronate, ibandronate,
risedronate,
zoledronate, minodronate, hydroxymethylenebisphosphonate, and combinations
thereof, wherein
all the compounds can be optionally further substituted or are unsubsituted.
In any one or more aspects of any one or more embodiments herein, the
quinolone
compound can be a fluoroquinolone or a non-fluoroquinolone. The quinolone
compound can be
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selected from the group of: alatrofloxacin, amifloxacin, balofloxacin,
besifloxacin, cadazolid,
ciprofloxacin, clinafloxacin, danofloxacin, delafloxacin, difloxacin,
enoxacin, enrofloxacin,
finafloxacin, flerofloxacin, flumequine, gatifloxacin, gem ifloxacin,
grepafloxacin, ibafloxacin, JNJ-
Q2, levofloxacin, lomefloxacin, marbofloxacin, moxifloxacin, nadifloxacin,
norfloxacin, ofloxacin,
orbifloxacin, pazufloxacin, pefloxacin, pradofloxacin, prulifloxacin,
rufloxacin, sarafloxacin,
sitafloxacin, sparfloxacin, temafloxacin, tosufloxacin, trvafloxacin,
zabofloxacin, nemonoxacin and
combinations thereof.
In some aspects, the BP is etidronate. In some aspects, the quinolone is
ciprofloxacin or
moxifloxacin. In other aspects, the BP can be another BP described herein,
such as pamidronate,
neridronate, olpadronate, alendronate, ibandronate, minodronate, risedronate,
zoledronate,
hydroxymethylenebisphosphonate, and combinations thereof.
The quinolone compound can have a structure according to Formula (A),
R40 0
R5
OH
R1
IR,) R2
Formula (A),
where R1 can be substituents including alkyl, substituted alkyl, alkenyl,
substituted alkenyl,
alkynyl, substituted alkynyl, phenyl, substituted phenyl, aryl, substituted
aryl, heteroaryl,
substituted heteroaryl, halo, hydroxyl, alkoxy, substituted alkoxy, phenoxy,
substituted phenoxy,
aroxy, substituted aroxy, alkylthio, substituted alkylthio, phenylthio,
substituted phenylthio,
arylthio, substituted arylthio, cyano, isocyano, substituted isocyano,
carbonyl, substituted
carbonyl, carboxyl, substituted carboxyl, amino, substituted amino, amido,
substituted amido,
sulfonyl, substituted sulfonyl, sulfonic acid, phosphoryl, substituted
phosphoryl, phosphonyl,
substituted phosphonyl, polyaryl, substituted polyaryl, C3-C20 cyclic,
substituted C3-C20 cyclic,
heterocyclic, substituted heterocyclic, amino acid, peptide, and polypeptide
groups,
where R2 can be substituents including alkyl, substituted alkyl, alkenyl,
substituted alkenyl,
alkynyl, substituted alkynyl, phenyl, substituted phenyl, aryl, substituted
aryl, heteroaryl,
substituted heteroaryl, halo, hydroxyl, alkoxy, substituted alkoxy, phenoxy,
substituted phenoxy,
aroxy, substituted aroxy, alkylthio, substituted alkylthio, phenylthio,
substituted phenylthio,
arylthio, substituted arylthio, cyano, isocyano, substituted isocyano,
carbonyl, substituted
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carbonyl, carboxyl, substituted carboxyl, amino, substituted amino, amido,
substituted amido,
sulfonyl, substituted sulfonyl, sulfonic acid, phosphoryl, substituted
phosphoryl, phosphonyl,
substituted phosphonyl, polyaryl, substituted polyaryl, C3-C20 cyclic,
substituted C3-C20 cyclic,
heterocyclic, substituted heterocyclic, amino acid, peptide, and polypeptide
groups,
where R3 can be substituents including alkyl, substituted alkyl, alkenyl,
substituted alkenyl,
alkynyl, substituted alkynyl, phenyl, substituted phenyl, aryl, substituted
aryl, heteroaryl,
substituted heteroaryl, halo, hydroxyl, alkoxy, substituted alkoxy, phenoxy,
substituted phenoxy,
aroxy, substituted aroxy, alkylthio, substituted alkylthio, phenylthio,
substituted phenylthio,
arylthio, substituted arylthio, cyano, isocyano, substituted isocyano,
carbonyl, substituted
carbonyl, carboxyl, substituted carboxyl, amino, substituted amino, amido,
substituted amido,
sulfonyl, substituted sulfonyl, sulfonic acid, phosphoryl, substituted
phosphoryl, phosphonyl,
substituted phosphonyl, polyaryl, substituted polyaryl, C3-C20 cyclic,
substituted C3-C20 cyclic,
heterocyclic, substituted heterocyclic, amino acid, peptide, and polypeptide
groups,
where R4 can be substituents including alkyl, substituted alkyl, alkenyl,
substituted
alkenyl, alkynyl, substituted alkynyl, phenyl, substituted phenyl, aryl,
substituted aryl, heteroaryl,
substituted heteroaryl, halo, hydroxyl, alkoxy, substituted alkoxy, phenoxy,
substituted phenoxy,
aroxy, substituted aroxy, alkylthio, substituted alkylthio, phenylthio,
substituted phenylthio,
arylthio, substituted arylthio, cyano, isocyano, substituted isocyano,
carbonyl, substituted
carbonyl, carboxyl, substituted carboxyl, amino, substituted amino, amido,
substituted amido,
sulfonyl, substituted sulfonyl, sulfonic acid, phosphoryl, substituted
phosphoryl, phosphonyl,
substituted phosphonyl, polyaryl, substituted polyaryl, C3-C20 cyclic,
substituted C3-C20 cyclic,
heterocyclic, substituted heterocyclic, amino acid, peptide, and polypeptide
groups, and
and wherein R5 can be H or F.
In any one or more aspects, the linker can be as described in any one or more
aspects
elsewhere herein. The linker can be attached to the R1 group of Formula (A).
In any one or more aspects or embodiments herein, the BP can have an alpha
substituent
and the alpha substituent is a hydroxy, amino or thiol group. The quinolone
can be directly or
indirectly conjugated to the BP at the germinal carbon alpha substituent (X)
of the BP, as
illustrated in the formula below.
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quinolone
X
HO pH
HO 2P +P¨OH
R \\=0 conjugates between alpha-X containing BP and
quinolone
0
X= 0, NH, NR1, S
R1 can be alkyl or substituted alkyl, aryl or subsituted aryl groups
wherein R can be H, substituted and unsubstituted alkyl, alkyl amino, alkyl-
aryl, aryl,
alkylheteroaryl, or heteroaryl. Preferred BPs are those that have a geminal
hydroxy group on the
carbon between the two phosphonate groups.
In any one or more aspects or embodiments herein, the bisphosphonate can be an
ethylidenebisphosphonate moiety (etidronate) that can be substituted by
hydroxy (an alpha-
hydroxy), amino or thiol.
In some aspects, the bisphosphonate can include a para-
hydroxyphenylethylidene group or derivative thereof. In embodiments, the BP
can be a clinically
known BP, such as pamidronate, alendronate, risedronate, zoledronate,
minodronate,
neridronate, and etidronate, which can be unmodified or modified as described
herein.
In some aspects, the compound has a formula according to Formula (41), Formula
(43),
Formula (44) or Formula (45).
0 OH
I
rN
HO OH
')<cH Thr N
n=0. _. .3 0
I OH
HO
Formula (41)
0 OH
ccI
iN 0 N
A
(H0)20P\
ko
(H0)20p cH3
Formula (43)
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OH
0 I
0 -OH
HN ---\p -OH
V CI Ni-----3c1 / =
OH
HO
00
Formula (44)
HO
0
0P-OH
HN OH
7 0- rc
N
HOyyILJ
I
00
Formula (45)
Also provided herein are pharmaceutical formulations that can contain a
bisphosphonate
and a quinolone compound, wherein the quinolone compound is releasably coupled
to the
bisphosphonate via a linker; and a pharmaceutically acceptable carrier. The
bisphosphonate can
be selected from the group of: hydroxyl phenyl alkyl or aryl bisphosphonates,
hydroxyl phenyl (or
aryl) alkyl hydroxyl bisphosphonates, amino phenyl(or aryl) alkyl
bisphosphonates, amino
phenyl(or aryl) alkyl hydroxyl bisphosphonates, hydroxyl alkyl
bisphosphonates, hydroxyl alkyl
hydroxyl bisphosphonates, hydroxyl alkyl phenyl(or aryl) alkyl
bisphosphonates, hydroxyl
phenyl(or aryl) alkyl hydroxyl bisphosphonates, amino phenyl(or aryl) alkyl
bisphosphonates,
amino phenyl(or aryl) alkyl hydroxyl bisphosphonates, hydroxyl alkyl
bisphosphonates, hydroxyl
alkyl hydroxyl bisphosphonates, hydroxypyridyl alkyl bisphosphonates, pyridyl
alkyl
bisphosphonates, hydroxyl imadazoyl alkyl bisphosphonates, imidazoyl alkyl
bisphosphonates,
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etidronate, pamidronate, neridronate, olpadronate, alendronate, ibandronate,
risedronate,
zoledronate, minodronate, hydroxymethylenebisphosphonate, and combinations
thereof, wherein
all the compounds can be optionally futher substituted or are unsubsituted.
The quinolone compound can be a fluoroquinolone or a non-fluoroquinolone. The
quinolone compound can be selected from the group of: alatrofloxacin,
amifloxacin, balofloxacin,
besifloxacin, cadazolid, ciprofloxacin, clinafloxacin, danofloxacin,
delafloxacin, difloxacin,
enoxacin, enrofloxacin, finafloxacin, flerofloxacin, flumequine, gatifloxacin,
gemifloxacin,
grepafloxacin, ibafloxacin, JNJ-Q2, levofloxacin, lomefloxacin, marbofloxacin,
moxifloxacin,
nadifloxacin, norfloxacin, ofloxacin, orbifloxacin, pazufloxacin, pefloxacin,
pradofloxacin,
prulifloxacin, rufloxacin, sarafloxacin, sitafloxacin, sparfloxacin,
temafloxacin, tosufloxacin,
trvafloxacin, zabofloxacin, nemonoxacin and combinations thereof.
The quinolone compound can have a structure according to Formula (A),
R40 0
R5
OH
R1
IR,) R2
Formula (A),
where R1 can be substituents including alkyl, substituted alkyl, alkenyl,
substituted alkenyl,
alkynyl, substituted alkynyl, phenyl, substituted phenyl, aryl, substituted
aryl, heteroaryl,
substituted heteroaryl, halo, hydroxyl, alkoxy, substituted alkoxy, phenoxy,
substituted phenoxy,
aroxy, substituted aroxy, alkylthio, substituted alkylthio, phenylthio,
substituted phenylthio,
arylthio, substituted arylthio, cyano, isocyano, substituted isocyano,
carbonyl, substituted
carbonyl, carboxyl, substituted carboxyl, amino, substituted amino, amido,
substituted amido,
sulfonyl, substituted sulfonyl, sulfonic acid, phosphoryl, substituted
phosphoryl, phosphonyl,
substituted phosphonyl, polyaryl, substituted polyaryl, C3-C20 cyclic,
substituted C3-C20 cyclic,
heterocyclic, substituted heterocyclic, amino acid, peptide, and polypeptide
groups,
where R2 can be substituents including alkyl, substituted alkyl, alkenyl,
substituted alkenyl,
alkynyl, substituted alkynyl, phenyl, substituted phenyl, aryl, substituted
aryl, heteroaryl,
substituted heteroaryl, halo, hydroxyl, alkoxy, substituted alkoxy, phenoxy,
substituted phenoxy,
aroxy, substituted aroxy, alkylthio, substituted alkylthio, phenylthio,
substituted phenylthio,
arylthio, substituted arylthio, cyano, isocyano, substituted isocyano,
carbonyl, substituted
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carbonyl, carboxyl, substituted carboxyl, amino, substituted amino, amido,
substituted amido,
sulfonyl, substituted sulfonyl, sulfonic acid, phosphoryl, substituted
phosphoryl, phosphonyl,
substituted phosphonyl, polyaryl, substituted polyaryl, C3-C20 cyclic,
substituted C3-C20 cyclic,
heterocyclic, substituted heterocyclic, amino acid, peptide, and polypeptide
groups,
where R3 can be substituents including alkyl, substituted alkyl, alkenyl,
substituted alkenyl,
alkynyl, substituted alkynyl, phenyl, substituted phenyl, aryl, substituted
aryl, heteroaryl,
substituted heteroaryl, halo, hydroxyl, alkoxy, substituted alkoxy, phenoxy,
substituted phenoxy,
aroxy, substituted aroxy, alkylthio, substituted alkylthio, phenylthio,
substituted phenylthio,
arylthio, substituted arylthio, cyano, isocyano, substituted isocyano,
carbonyl, substituted
carbonyl, carboxyl, substituted carboxyl, amino, substituted amino, amido,
substituted amido,
sulfonyl, substituted sulfonyl, sulfonic acid, phosphoryl, substituted
phosphoryl, phosphonyl,
substituted phosphonyl, polyaryl, substituted polyaryl, C3-C20 cyclic,
substituted C3-C20 cyclic,
heterocyclic, substituted heterocyclic, amino acid, peptide, and polypeptide
groups,
where R4 can be substituents including alkyl, substituted alkyl, alkenyl,
substituted
alkenyl, alkynyl, substituted alkynyl, phenyl, substituted phenyl, aryl,
substituted aryl, heteroaryl,
substituted heteroaryl, halo, hydroxyl, alkoxy, substituted alkoxy, phenoxy,
substituted phenoxy,
aroxy, substituted aroxy, alkylthio, substituted alkylthio, phenylthio,
substituted phenylthio,
arylthio, substituted arylthio, cyano, isocyano, substituted isocyano,
carbonyl, substituted
carbonyl, carboxyl, substituted carboxyl, amino, substituted amino, amido,
substituted amido,
sulfonyl, substituted sulfonyl, sulfonic acid, phosphoryl, substituted
phosphoryl, phosphonyl,
substituted phosphonyl, polyaryl, substituted polyaryl, C3-C20 cyclic,
substituted C3-C20 cyclic,
heterocyclic, substituted heterocyclic, amino acid, peptide, and polypeptide
groups, and
and wherein R5 can be H or F.
In any one or more aspects, the linker can be as described in any one or more
aspects
elsewhere herein. The linker can be attached to the R1 group of Formula (A).
In any one or more aspects, the BP can have an alpha substituent and the alpha
substituent is a hydroxy, amino or thiol group. The quinolone can be directly
or indirectly
conjugated to the BP at the germinal carbon alpha substituent (X) of the BP,
as illustrated in the
formula below.
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quinolone
X).<
HO, pH
HO ¨P ¨OH
R o conjugates between alpha-X containing BP and
quinolone
0
X= 0, NH, NRI, S
121 can be alkyl or substituted alkyl, aryl or subsituted aryl groups
wherein R can be H, substituted and unsubstituted alkyl, alkyl amino, alkyl-
aryl, aryl,
alkylheteroaryl, or heteroaryl. Preferred BPs are those that have a geminal
hydroxy group on the
carbon between the two phosphonate groups.
In any one or more aspects, the bisphosphonate can be an
ethylidenebisphosphonate
moiety (etidronate) that can be substituted by hydroxy (an alpha-hydroxy),
amino or thiol. In some
aspects, the bisphosphonate can include a para-hydroxyphenylethylidene group
or derivative
thereof. In embodiments, the BP can be a clinically known BP, such as
pamidronate, alendronate,
risedronate, zoledronate, minodronate, neridronate, and etidronate, which can
be unmodified or
modified as described herein.
In some aspects, the formulation includes a compound that has a formula
according to
Formula (41), Formula (43), Formula (44) and/or Formula (45) herein.
The amount of the compound or conjugate in the pharmaceutical formulation can
be an
amount effective to kill or inhibit bacteria. The amount of the compound or
conjugate in the
pharmaceutical formulation can be an amount effective to treat, inhibit, or
prevent a bone disease.
The amount of the compound or conjugate in the pharmaceutical formulation can
be an amount
effective to treat, inhibit, or prevent osteomyelitis, osteonecrosis, peri-
implantitis, and/or
periodontitis. The amount of the compound or conjugate in the pharmaceutical
formulation can
be an amount effective for prophylaxis treatment of any of the foregoing.
Further, in one or more embodiments, methods are provided of preparing a
bisphosphonate-quinolone compound, conjugate or formulation thereof,
comprising linking a
bisphosphonate with a quinolone compound or substituent as described in any
one or more
aspects herein. Methods are also provided for use of any one or more
bisphosphonate-quinolone
compounds or conjugates in the preparation of a pharmaceutical or medicament
for the treatment
of any one or more of the diseases mentioned herein.
Also provided herein are methods of treating a bone disease or infection, such
as
osteomyelitis, in a subject in need thereof that can include the step of
administering an amount,
in particular an effective amount, of a compound as provided herein or
pharmaceutical formulation
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thereof to the subject in need thereof. Also provided herein are methods of
treating a bone
disease, such as a hematogenous or local osteomyelitis including juvenile
osteomyelitis and
infections related to prosthetic joint replacements or osteonecrosis, in a
subject in need thereof
that can include the step of administering an amount, in particular an
effective amount of a
compound as provided herein or pharmaceutical formulation thereof to the
subject in need
thereof.
Also provided herein are methods of treating peri-implantitis or periodontitis
in a subject in
need thereof, the method comprising administering an amount of administering
an amount, in
particular an effective amount, of a compound as provided herein or
pharmaceutical formulation
thereof to the subject in need thereof.
Also provided herein are methods of treating diabetic foot disease in a
subject in need
thereof, the method comprising administering an amount, in particular an
effective amount, of a
compound as provided herein or pharmaceutical formulation thereof to the
subject in need
thereof. Also provided herein are methods of treating bone infections in
diabetic patients including
diabetic foot diseases in a subject in need thereof, the method comprising
administering an
amount, in particular an effective amount, of a compound as provided herein or
pharmaceutical
formulation thereof to the subject in need thereof. A reduction in related
amputations,
debridement, of limbs and infected skeletal sites can result from these more
powerful localized
modes of antibiotic therapies.
Also provided herein are bone graft compositions that can include a bone graft
material
and a compound as described herein or a pharmaceutical formulation thereof,
wherein the
compound or pharmaceutical formulation thereof is attached to, integrated
with, chemisorbed to,
or mixed with the bone graft material. The bone graft material can be
autograft bone material,
allograft bone material, xenograft bone material, a synthetic bone graft
material, or any
combination thereof.
Also provided herein are methods that can include the step of implanting the
bone graft
composition as described herein in a subject in need thereof.
Also provided herein are methods of preventing or prophylaxis treatment of,
biofilm
infection at an osseous or implant surgical site, or at a surgical site where
bone grafting is
performed, where the methods can include the step of administering a compound
as described
herein to a subject in need thereof.
Also provided herein are methods of preventing, or prophylaxis treatment, of
biofilm
infection at an osseous or implant surgical site, or at a surgical site where
bone grafting is
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performed, where the method can include the step of implanting a bone graft
composition as
described herein to a subject in need thereof.
Other compounds, compositions, formulations, methods, features, and advantages
of the
present disclosure of a fabrication system for nanowire template synthesis,
will be or become
apparent to one with skill in the art upon examination of the following
drawings and detailed
description. It is intended that all such additional systems, methods,
features, and advantages be
included within this description, be within the scope of the present
disclosure, and be protected
by the accompanying claims.
BRIEF DESCRIPTION OF THE DRAWINGS
Further aspects of the present disclosure will be readily appreciated upon
review of the
detailed description of its various embodiments, described below, when taken
in conjunction with
the accompanying drawings.
Fig. 1 shows a scanning electron micrograph (SEM; 100x magnification) of a
surgical
specimen from a patient with chronic osteomyelitis showing characteristic
multi-layered and
matrix-enclosed biofilms colonizing bone surfaces internally and externally;
inset top right shows
high-power view (5000x magnification) of the causative staphylococcal biofilm
pathogens. [The
sample was processed for SEM, sputter coated with platinum and imaged with an
XL 30S SEM
(FEG, FEI Co., Hillsboro, OR) operating at 5 kV in the secondary electron
mode].
Fig. 2 demonstrates the general BP quinolone conjugate targeting strategy.
Fig. 3 shows an embodiment of a BP-FQ conjugate.
Fig. 4 shows additional BP-Ab conjugate design.
Fig. 5 shows an embodiment of a synthesis scheme for synthesis of BP-Ab
conjugates
with an 0-thiocarbamate linker.
Fig. 6 shows an embodiment of a scheme for synthesis of alpha-OH protected BP
esters.
Fig. 7 shows an embodiment of a scheme for synthesis of BP 3-linker 3-
ciprofloxacin.
Fig. 8 shows a BP-carbamate-moxifloxacin BP conjugate and synthesis scheme.
Fig. 9 shows a BP-carbamate-gatifloxacin BP conjugate and synthesis scheme.
Fig. 10 shows a BP-p-Hydroxyphenyl Acetic Acid-ciprofloxacin BP conjugate and
synthesis scheme.
Fig. 11 shows a BP-OH-ciprofloxacin BP conjugate and synthesis scheme.
Fig. 12 shows a BP-O-Thiocarbamate-ciprofloxacin BP conjugate and synthesis
scheme.
Fig. 13 shows a BP-S-Thiocarbamate-ciprofloxacin BP conjugate and synthesis
scheme.
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Fig. 14 shows a BP-Resorcinol-ciprofloxacin BP conjugate and synthesis scheme.
Fig. 15 shows a BP-Hydroquinone-ciprofloxacin BP conjugate and synthesis
scheme.
Fig. 16 shows one embodiment of a genus structure for a genus of BP-
Fluoroquinolones.
Fig. 17 shows various BP-fluoroquinolone conjugates.
Fig. 18 shows one embodiment of a genus structure for a genus of a phosphonate
containing an aryl group.
Fig. 19 shows various BPs, where X can be F, Cl, Br, or I.
Fig. 20 shows various BP's with terminal primary amines.
Fig. 21 shows various BPs coupled to a linker containing a terminal hydroxyl
and amine
.. functional groups where R can be Risedronate, Zoledronate, Minodronate,
Pamidronate, or
Alendronate.
Fig. 22 shows various BP-pamidronate-ciprofloxacin conjugates.
Fig. 23 shows various BP-Alendronate-ciprofloxacin conjugates.
FIG. 24 depicts examples of pharmacologically inert BPs used in the present
conjugation:
medium (A/E), high (B/F), and low (C/G) affinity BPs and longer phenylalkyl
chain BP (D/H).
FIG. 25 depicts examples of pharmacologically low active BPs that can be used
in the
present conjugation.
FIG. 26 depicts the results of dynamic monitoring of biofilm growth in the
presence of
different concentrations of conjugates. Culture ( ------------------- ), S.
aureus with ECC 2 pg/ml I ( ),
------------ ECC 5 pg/ml I ( -- ), ECC 10 pg/ml ( -------------------- ), ECX
2 pg/ml I ( ), ECX 5 pg/ml I ( ), ECX
10 pg/ml ( ---- ).
FIG. 27 depicts S. aureus MICK analysis for ECC & ECX. ECC ( -------- ), ECX (
).
FIG. 28 depicts the results of dynamic monitoring of biofilm growth in the
presence of
different concentrations of parent antibiotics. Culture ( -------------------
), S. aureus with Cipro 0.1 pg/ml I (---
------------------ ----), Cipro 0.25 pg/ml ( --- ), Cipro 1 pg/ml ( -- ),
Cipro 2 pg/ml ( ), Moxi 0.05 pg/ml (
--), Moxi 0.1 pg/ml ( -- ), Moxi 0.2 pg/ml ( ---------- ), Moxi 0.5 pg/ml (
).
FIG. 29 depicts S. aureus MICK analysis for Cipro & Moxi. Moxi ( -- ), Cipro (
).
FIG. 30 depicts the results of dynamic monitoring of biofilm growth in the
presence of
Cipro/Moxi+HA(10 pg/ml). Culture ( -- ), S. aureus with Cipro 0.01 pg/ml ( --
), Cipro 0.075
----- pg/ml ( ---------- ), Cipro 0.1 pg/ml ( -- ), Cipro 0.25 pg/ml ( ),
Cipro 0.50 pg/ml ( ), Moxi
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0.02 pg/ml ( ---- ), Moxi 0.04 pg/ml ( ------------ ), Moxi 0.08 pg/ml ( -- ),
Moxi 0.1 pg/ml ( ),
Moxi 0.2 pg/ml ( --- ).
FIG. 31 depicts S. aureus M IC50 analysis for Cipro/Moxi+HA. Moxi ( ---------
), Cipro ( ).
FIG. 32 depicts the results of dynamic monitoring of biofilm growth in the
presence of
---------------------------- ECC/ECX+HA(10 pg/ml). Culture ( ---------- ), S.
aureus with ECC 1 pg/ml ( ), ECC 5 pg/ml (----
---), ECC 7.5 pg/ml ( -- ), ECC 10 pg/ml ( -- ), ECX 1 pg/ml ( -------------
), ECX 2.5 pg/ml ( ),
ECX 7.5 pg/ml ( ---- ), ECX 10 pg/ml ( -- ).
FIG. 33 depicts S. aureus MICK analysis for ECC/ECX+HA. ECX ( -- ), ECC ( --
).
DETAILED DESCRIPTION
Before the present disclosure is described in greater detail, it is to be
understood that this
disclosure is not limited to particular embodiments described, and as such
may, of course, vary.
It is also to be understood that the terminology used herein is for the
purpose of describing
particular embodiments only, and is not intended to be limiting.
Where a range of values is provided, it is understood that each intervening
value, to the
tenth of the unit of the lower limit unless the context clearly dictates
otherwise, between the upper
and lower limit of that range and any other stated or intervening value in
that stated range, is
encompassed within the disclosure. The upper and lower limits of these smaller
ranges may
independently be included in the smaller ranges and are also encompassed
within the disclosure,
subject to any specifically excluded limit in the stated range. VVhere the
stated range includes one
or both of the limits, ranges excluding either or both of those included
limits are also included in
the disclosure.
Unless defined otherwise, all technical and scientific terms used herein have
the same
meaning as commonly understood by one of ordinary skill in the art to which
this disclosure
belongs. Although any methods and materials similar or equivalent to those
described herein can
also be used in the practice or testing of the present disclosure, the
preferred methods and
materials are now described.
All publications and patents cited in this specification are herein
incorporated by reference
as if each individual publication or patent were specifically and individually
indicated to be
incorporated by reference and are incorporated herein by reference to disclose
and describe the
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methods and/or materials in connection with which the publications are cited.
The citation of any
publication is for its disclosure prior to the filing date and should not be
construed as an admission
that the present disclosure is not entitled to antedate such publication by
virtue of prior disclosure.
Further, the dates of publication provided could be different from the actual
publication dates that
may need to be independently confirmed.
As will be apparent to those of skill in the art upon reading this disclosure,
each of the
individual embodiments described and illustrated herein has discrete
components and features
which may be readily separated from or combined with the features of any of
the other several
embodiments without departing from the scope or spirit of the present
disclosure. Any recited
method can be carried out in the order of events recited or in any other order
that is logically
possible.
Embodiments of the present disclosure will employ, unless otherwise indicated,
techniques of molecular biology, microbiology, nanotechnology, pharmacology,
organic
chemistry, biochemistry, botany and the like, which are within the skill of
the art. Such techniques
are explained fully in the literature.
Definitions
Unless otherwise specified herein, the following definitions are provided.
As used herein, "about," "approximately," and the like, when used in
connection with a
numerical variable, generally refers to the value of the variable and to all
values of the variable
that are within the experimental error (e.g., within the 95% confidence
interval for the mean) or
within 10% of the indicated value, whichever is greater.
As used interchangeably herein, "subject," "individual," or "patient," refers
to a
vertebrate, preferably a mammal, more preferably a human. Mammals include, but
are not limited
to, murines, simians, humans, farm animals, sport animals, and pets. The term
"pet" includes a
dog, cat, guinea pig, mouse, rat, rabbit, ferret, and the like. The term "farm
animal" includes a
horse, sheep, goat, chicken, pig, cow, donkey, llama, alpaca, turkey, and the
like.
As used herein, "control" can refer to an alternative subject or sample used
in an
experiment for comparison purposes and included to minimize or distinguish the
effect of
variables other than an independent variable.
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As used herein, "analog" or "analogue," such as an analogue of a
bisphosphonate
described herein, can refer to a structurally close member of the parent
molecule or an appended
parent molecule such as a bisphosphonate.
As used herein, "conjugated" can refer to direct attachment of two or more
compounds
to one another via one or more covalent or non-covalent bonds. The term
"conjugated" as used
herein can also refer to indirect attachment of two or more compounds to one
another through an
intermediate compound, such as a linker.
As used herein, "pharmaceutical formulation" refers to the combination of an
active
agent, compound, or ingredient with a pharmaceutically acceptable carrier or
excipient, making
the composition suitable for diagnostic, therapeutic, or preventive use in
vitro, in vivo, or ex vivo.
As used herein, "pharmaceutically acceptable carrier or excipient" refers to a
carrier
or excipient that is useful in preparing a pharmaceutical formulation that is
generally safe, non-
toxic, and is neither biologically or otherwise undesirable, and includes a
carrier or excipient that
is acceptable for veterinary use as well as human pharmaceutical use. A
"pharmaceutically
acceptable carrier or excipient" as used in the specification and claims
includes both one and
more than one such carrier or excipient.
As used herein, "pharmaceutically acceptable salt" refers to any acid or base
addition
salt whose counter-ions are non-toxic to the subject to which they are
administered in
pharmaceutical doses of the salts.
As used herein, "active agent" or "active ingredient" refers to a component or
components of a composition to which the whole or part of the effect of the
composition is
attributed.
As used herein, "dose," "unit dose," or "dosage" refers to physically discrete
units
suitable for use in a subject, each unit containing a predetermined quantity
of a BP conjugate,
such as a BP quinolone conjugate, composition or formulation described herein
calculated to
produce the desired response or responses in association with its
administration.
As used herein, "derivative" refers to any compound having the same or a
similar core
structure to the compound but having at least one structural difference,
including substituting,
deleting, and/or adding one or more atoms or functional groups. The term
"derivative" does not
mean that the derivative is synthesized from the parent compound either as a
starting material or
intermediate, although this may be the case. The term "derivative" can include
prodrugs, or
metabolites of the parent compound. Derivatives include compounds in which
free amino groups
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in the parent compound have been derivatized to form amine hydrochlorides, p-
toluene
sulfoamides, benzoxycarboamides, t-butyloxycarboamides, thiourethane-type
derivatives,
trifluoroacetylamides, chloroacetylamides, or formamides. Derivatives include
compounds in
which carboxyl groups in the parent compound have been derivatized to form
methyl and ethyl
esters, or other types of esters, amides, hydroxamic acids, or hydrazides.
Derivatives include
compounds in which hydroxyl groups in the parent compound have been
derivatized to form 0-
acyl, 0-carbamoyl, or 0-alkyl derivatives. Derivatives include compounds in
which a hydrogen
bond donating group in the parent compound is replaced with another hydrogen
bond donating
group such as OH, NH, or SH. Derivatives include replacing a hydrogen bond
acceptor group in
.. the parent compound with another hydrogen bond acceptor group such as
esters, ethers,
ketones, carbonates, tertiary amines, innine, thiones, sulfones, tertiary
amides, and sulfides.
"Derivatives" also includes extensions of the replacement of the cyclopentane
ring, as an
example, with saturated or unsaturated cyclohexane or other more complex,
e.g., nitrogen-
containing rings, and extensions of these rings with various groups.
As used herein, "administering" refers to an administration that is oral,
topical,
intravenous, subcutaneous, transcutaneous, transdermal, intramuscular, intra-
joint, parenteral,
intra-arteriole, intradermal, intraventricular, intracranial, intraperitoneal,
intralesional, intranasal,
rectal, vaginal, by inhalation, or via an implanted reservoir. The term
"parenteral" includes
subcutaneous, intravenous, intramuscular, intra-articular, intra-synovial,
intrasternal, intrathecal,
intrahepatic, intralesional, and intracranial injections or infusion
techniques.
The term "substituted" as used herein, refers to all permissible substituents
of the
compounds described herein. In the broadest sense, the permissible
substituents include acyclic
and cyclic, branched and unbranched, carbocyclic and heterocyclic, aromatic
and nonaromatic
substituents of organic compounds. Illustrative substituents include, but are
not limited to,
.. halogens, hydroxyl groups, or any other organic groupings containing any
number of carbon
atoms, e.g. 1-14 carbon atoms, and optionally include one or more heteroatoms
such as oxygen,
sulfur, or nitrogen grouping in linear, branched, or cyclic structural
formats. Representative
substituents include alkyl, substituted alkyl, alkenyl, substituted alkenyl,
alkynyl, substituted
alkynyl, phenyl, substituted phenyl, aryl, substituted aryl, heteroaryl,
substituted heteroaryl, halo,
hydroxyl, alkoxy, substituted alkoxy, phenoxy, substituted phenoxy, aroxy,
substituted aroxy,
alkylthio, substituted alkylthio, phenylthio, substituted phenylthio,
arylthio, substituted arylthio,
cyano, isocyano, substituted isocyano, carbonyl, substituted carbonyl,
carboxyl, substituted
carboxyl, amino, substituted amino, amido, substituted amido, sulfonyl,
substituted sulfonyl,
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sulfonic acid, phosphoryl, substituted phosphoryl, phosphonyl, substituted
phosphonyl, polyaryl,
substituted polyaryl, C3-C20 cyclic, substituted 03-020 cyclic, heterocyclic,
substituted heterocyclic,
amino acid, peptide, and polypeptide groups.
As used herein, "substituent" or "suitable substituent" means a chemically and
pharmaceutically acceptable group, i.e., a moiety that does not significantly
interfere with the
preparation of or negate the efficacy of the inventive compounds. Such
suitable substituents may
be routinely chosen by those skilled in the art. Suitable substituents include
but are not limited to
the following: a halo, 01-06 alkyl, 02-06 alkenyl, C1-C6 haloalkyl, 01-06
alkoxy, 01-06 haloalkoxy,
02-06 alkynyl, 03-08 cycloalkenyl, (03-08 cycloalkyl)Ci-06 alkyl, (03-08
cycloalkyl)02-C6 alkenyl,
(03-08 cycloalkyl)Ci-06 alkoxy, 03-C7 heterocycloalkyl, (03-C7
heterocycloalkyl)Ci-06 alkyl, (03-
07 heterocycloalkyl) 02-06 alkenyl, (03-C7 heterocycloalkyl)Ci-06 alkoxyl,
hydroxy, carboxy, oxo,
sulfanyl, 01-06 alkylsulfanyl, aryl, heteroaryl, aryloxy, heteroaryloxy,
aralkyl, heteroaralkyl,
aralkoxy, heteroaralkoxy, nitro, cyano, amino, 01-06 alkylamino, di-(Ci-
C6alkyl)amino, carbamoyl,
(C1-06 alkyl)carbonyl, (C1-06 alkoxy)carbonyl, (C1-C6 alkyl)aminocarbonyl, di-
(C1-06
alkyl)aminocarbonyl, arylcarbonyl, aryloxycarbonyl, (C1-C6 alkyl)sulfonyl, and
arylsulfonyl. The
groups listed above as suitable substituents are as defined hereinafter except
that a suitable
substituent may not be further optionally substituted.
The term "alkyl" refers to the radical of saturated aliphatic groups (i.e., an
alkane with one
hydrogen atom removed), including straight-chain alkyl groups, branched-chain
alkyl groups,
cycloalkyl (alicyclic) groups, alkyl-substituted cycloalkyl groups, and
cycloalkyl-substituted alkyl
groups.
In some embodiments, a straight chain or branched chain alkyl can have 30 or
fewer
carbon atoms in its backbone (e.g., 01-030 for straight chains, and C3-C30 for
branched chains).
In other embodiments, a straight chain or branched chain alkyl can contain 20
or fewer, 15 or
fewer, or 10 or fewer carbon atoms in its backbone. Likewise, in some
embodiments cycloalkyls
can have 3-10 carbon atoms in their ring structure. In some of these
embodiments, the cycloalkyl
can have 5, 6, or 7 carbons in the ring structure.
The term "alkyl" (or "lower alkyl") as used herein is intended to include both
"unsubstituted alkyls" and "substituted alkyls," the latter of which refers to
alkyl moieties having
one or more substituents replacing a hydrogen on one or more carbons of the
hydrocarbon
backbone. Such substituents include, but are not limited to, halogen,
hydroxyl, carbonyl (such as
a carboxyl, alkoxycarbonyl, formyl, or an acyl), thiocarbonyl (such as a
thioester, a thioacetate, or
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a thioformate), alkoxyl, phosphoryl, phosphate, phosphonate, phosphinate,
amino, amido,
amidine, imine, cyano, nitro, azido, sulfhydryl, alkylthio, sulfate,
sulfonate, sulfamoyl, sulfonamido,
sulfonyl, heterocyclyl, aralkyl, or an aromatic or heteroaromatic moiety.
Unless the number of carbons is otherwise specified, "lower alkyl" as used
herein means
an alkyl group, as defined above, but having from one to ten carbons in its
backbone structure.
Likewise, "lower alkenyl" and "lower alkynyl" have similar chain lengths.
It will be understood by those skilled in the art that the moieties
substituted on the
hydrocarbon chain can themselves be substituted, if appropriate. For instance,
the substituents
of a substituted alkyl may include halogen, hydroxy, nitro, thiols, amino,
azido, imino, amido,
phosphoryl (including phosphonate and phosphinate), sulfonyl (including
sulfate, sulfonamido,
sulfamoyl and sulfonate), and silyl groups, as well as ethers, alkylthios,
carbonyls (including
ketones, aldehydes, carboxylates, and esters), -CF3, -CN and the like.
Cycloalkyls can be
substituted in the same manner.
The term "heteroalkyl," as used herein, refers to straight or branched chain,
or cyclic
carbon-containing radicals, or combinations thereof, containing at least one
heteroatom. Suitable
heteroatoms include, but are not limited to, 0, N, Si, P, Se, B, and S,
wherein the phosphorous
and sulfur atoms are optionally oxidized, and the nitrogen heteroatom is
optionally quaternized.
Heteroalkyls can be substituted as defined above for alkyl groups.
The term "alkylthio" refers to an alkyl group, as defined above, having a
sulfur radical
attached thereto. In preferred embodiments, the "alkylthio" moiety is
represented by one of -S-
alkyl, -S-alkenyl, and -S-alkynyl. Representative alkylthio groups include
methylthio, ethylthio, and
the like. The term "alkylthio" also encompasses cycloalkyl groups, alkene and
cycloalkene
groups, and alkyne groups. "Arylthio" refers to aryl or heteroaryl groups.
Alkylthio groups can be
substituted as defined above for alkyl groups.
The terms "alkenyl" and "alkynyl", refer to unsaturated aliphatic groups
analogous in
length and possible substitution to the alkyls described above, but that
contain at least one double
or triple bond respectively.
The terms "alkoxyl" or "alkoxy," as used herein, refers to an alkyl group, as
defined
above, having an oxygen radical attached thereto. Representative alkoxyl
groups include
methoxy, ethoxy, propyloxy, tert-butoxy and the like. An "ether is two
hydrocarbons covalently
linked by an oxygen. Accordingly, the substituent of an alkyl that renders
that alkyl is an ether or
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resembles an alkoxyl, such as can be represented by one of -0-alkyl, -0-
alkenyl, and -0-alkynyl.
The terms "aroxy" and "aryloxy", as used interchangeably herein, can be
represented by ¨0-aryl
or 0-heteroaryl, wherein aryl and heteroaryl are as defined below. The alkoxy
and aroxy groups
can be substituted as described above for alkyl.
The terms "amine" and "amino" (and its protonated form) are art-recognized and
refer to
both unsubstituted and substituted amines, e.g., a moiety that can be
represented by the general
formula:
R' R"
¨N or ¨N ¨R'
wherein R, R', and R" each independently represent a hydrogen, an alkyl, an
alkenyl, -
(CH2)m-Rc or R and R' taken together with the N atom to which they are
attached complete a
heterocycle having from 4 to 8 atoms in the ring structure; Rc represents an
aryl, a cycloalkyl, a
cycloalkenyl, a heterocycle or a polycycle; and m is zero or an integer in the
range of 1 to 8. In
some embodiments, only one of R or R' can be a carbonyl, e.g., R, R' and the
nitrogen together
do not form an imide. In other embodiments, the term "amine" does not
encompass amides, e.g.,
wherein one of R and R' represents a carbonyl. In further embodiments, R and
R' (and optionally
R") each independently represent a hydrogen, an alkyl or cycloakly, an alkenyl
or cycloalkenyl, or
alkynyl. Thus, the term "alkylamine" as used herein means an amine group, as
defined above,
having a substituted (as described above for alkyl) or unsubstituted alkyl
attached thereto, i.e., at
least one of R and R' is an alkyl group.
The term "amido" is art-recognized as an amino-substituted carbonyl and
includes a
moiety that can be represented by the general formula:
0
R'
wherein R and R' are as defined above.
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As used herein, "aryl" refers to 05-C10-membered aromatic, heterocyclic, fused
aromatic,
fused heterocyclic, biaromatic, or bihetereocyclic ring systems. Broadly
defined, "aryl", as used
herein, includes 5-, 6-, 7-, 8-, 9-, and 10-membered single-ring aromatic
groups that may include
from zero to four heteroatoms, for example, benzene, pyrrole, furan,
thiophene, imidazole,
oxazole, thiazole, triazole, pyrazole, pyridine, pyrazine, pyridazine,
pyrimidine, and the like. Those
aryl groups having heteroatoms in the ring structure may also be referred to
as "aryl heterocycles"
or "heteroaromatics." The aromatic ring can be substituted at one or more ring
positions with one
or more substituents including, but not limited to, halogen, azide, alkyl,
aralkyl, alkenyl, alkynyl,
cycloalkyl, hydroxyl, alkoxyl, amino (or quaternized amino), nitro,
sulfhydryl, imino, amido,
phosphonate, phosphinate, carbonyl, carboxyl, silyl, ether, alkylthio,
sulfonyl, sulfonamido,
ketone, aldehyde, ester, heterocyclyl, aromatic or heteroaromatic moieties, -
CF3, -CN, and
combinations thereof. The term "aryl" includes phenyl.
The term "aryl" also includes polycyclic ring systems having two or more
cyclic rings in
which two or more carbons are common to two adjoining rings (i.e., "fused
rings") wherein at least
one of the rings is aromatic, e.g., the other cyclic ring or rings can be
cycloalkyls, cycloalkenyls,
cycloalkynyls, aryls and/or heterocycles. Examples of heterocyclic rings
include, but are not
limited to, benzimidazolyl, benzofuranyl, benzothiofuranyl, benzothiophenyl,
benzoxazolyl,
benzoxazolinyl, benzthiazolyl, benztriazolyl, benztetrazolyl, benzisoxazolyl,
benzisothiazolyl,
benzimidazolinyl, carbazolyl, 4aH carbazolyl, carbolinyl, chromanyl,
chromenyl, cinnolinyl,
decahydroquinolinyl, 2H, 6H-1,5,2-dithiazinyl, dihydrofuro[2,3
b]tetrahydrofuran, furanyl,
furazanyl, imidazolidinyl, imidazolinyl, imidazolyl, /H-indazolyl, indolenyl,
indolinyl, indolizinyl,
indolyl, 3H-indolyl, isatinoyl, isobenzofuranyl, isochromanyl, isoindazolyl,
isoindolinyl, isoindolyl,
isoquinolinyl, isothiazolyl, isoxazolyl, methylenedioxyphenyl, morpholinyl,
naphthyridinyl,
octahydroisoquinolinyl, oxadiazolyl, 1,2,3-oxadiazolyl, 1,2,4-oxadiazolyl,
1,2,5-oxadiazolyl, 1,3,4-
oxadiazolyl, oxazolidinyl, oxazolyl, oxindolyl, pyrimidinyl, phenanthridinyl,
phenanthrolinyl,
phenazinyl, phenothiazinyl, phenoxathinyl, phenoxazinyl, phthalazinyl,
piperazinyl, piperidinyl,
piperidonyl, 4-piperidonyl, piperonyl, pteridinyl, purinyl, pyranyl,
pyrazinyl, pyrazolidinyl,
pyrazolinyl, pyrazolyl, pyridazinyl, pyridooxazole, pyridoimidazole,
pyridothiazole, pyridinyl,
pyridyl, pyrimidinyl, pyrrolidinyl, pyrrolinyl, 2H-pyrrolyl, pyrrolyl,
quinazolinyl, quinolinyl, 4H-
quinolizinyl, quinoxalinyl, quinuclidinyl,
tetrahydrofuranyl, tetrahydroisoquinolinyl,
tetrahydroquinolinyl, tetrazolyl, 6H-1,2,5-thiadiazinyl, 1,2,3-thiadiazolyl,
1,2,4-thiadiazolyl, 1,2,5-
thiadiazolyl, 1,3,4-thiadiazolyl, thianthrenyl, thiazolyl, thienyl,
thienothiazolyl, thienooxazolyl,
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thienoimidazolyl, thiophenyl, and xanthenyl. One or more of the rings can be
substituted as
defined above for "aryl."
The term "aralkyl," as used herein, refers to an alkyl group substituted with
an aryl group
(e.g., an aromatic or heteroaromatic group).
The term "aralkyloxy" can be represented by ¨0-aralkyl, wherein aralkyl is as
defined
above.
The term "carbocycle," as used herein, refers to an aromatic or non-aromatic
ring(s) in
which each atom of the ring(s) is carbon.
"Heterocycle" or "heterocyclic," as used herein, refers to a monocyclic or
bicyclic
structure containing 3-10 ring atoms, and in some embodiments, containing from
5-6 ring atoms,
wherein the ring atoms are carbon and one to four heteroatoms each selected
from the following
group of non-peroxide oxygen, sulfur, and N(Y) wherein Y is absent or is H, 0,
(C1-C1o) alkyl,
phenyl or benzyl, and optionally containing 1-3 double bonds and optionally
substituted with one
or more substituents. Examples of heterocyclic rings include, but are not
limited to,
benzimidazolyl, benzofuranyl, benzothiofuranyl, benzothiophenyl, benzoxazolyl,
benzoxazolinyl,
benzthiazolyl, benztriazolyl, benztetrazolyl, benzisoxazolyl,
benzisothiazolyl, benzimidazolinyl,
carbazolyl, 4aH carbazolyl, carbolinyl, chromanyl, chromenyl, cinnolinyl,
decahydroquinolinyl,
2H,6H-1,5,2-dithiazinyl, dihydrofuro[2,3 b]tetrahydrofuran, furanyl,
furazanyl, imidazolidinyl,
imidazolinyl, imidazolyl, 1H-indazolyl, indolenyl, indolinyl, indolizinyl,
indolyl, 3H-indolyl, isatinoyl,
isobenzofuranyl, isochromanyl, isoindazolyl, isoindolinyl, isoindolyl,
isoquinolinyl, isothiazolyl,
isoxazolyl, methylenedioxyphenyl, morpholinyl, naphthyridinyl,
octahydroisoquinolinyl,
oxadiazolyl, 1,2,3-oxadiazolyl, 1,2,4-oxadiazolyl,
1,2,5-oxadiazolyl, 1,3,4-oxadiazolyl,
oxazolidinyl, oxazolyl, oxepanyl, oxetanyl, oxindolyl, pyrimidinyl,
phenanthridinyl, phenanthrolinyl,
phenazinyl, phenothiazinyl, phenoxathinyl, phenoxazinyl, phthalazinyl,
piperazinyl, piperidinyl,
piperidonyl, 4-piperidonyl, piperonyl, pteridinyl, purinyl, pyranyl,
pyrazinyl, pyrazolidinyl,
pyrazolinyl, pyrazolyl, pyridazinyl, pyridooxazole, pyridoimidazole,
pyridothiazole, pyridinyl,
pyridyl, pyrimidinyl, pyrrolidinyl, pyrrolinyl, 2H-pyrrolyl, pyrrolyl,
quinazolinyl, quinolinyl, 4H-
quinolizi nyl, quinoxalinyl, quinuclidinyl,
tetrahydrofuranyl, tetrahydroisoquinolinyl,
tetrahydropyranyl, tetrahydroquinolinyl, tetrazolyl, 6H-1,2,5-thiadiazinyl,
1,2,3-thiadiazolyl, 1,2,4-
thiadiazolyl, 1,2,5-thiadiazolyl, 1,3,4-thiadiazolyl, thianthrenyl, thiazolyl,
thienyl, thienothiazolyl,
thienooxazolyl, thienoimidazolyl, thiophenyl, and xanthenyl. Heterocyclic
groups can optionally be
substituted with one or more substituents at one or more positions as defined
above for alkyl and
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aryl, for example, halogen, alkyl, aralkyl, alkenyl, alkynyl, cycloalkyl,
hydroxyl, amino, nitro,
sulfhydryl, imino, amido, phosphate, phosphonate, phosphinate, carbonyl,
carboxyl, silyl, ether,
alkylthio, sulfonyl, ketone, aldehyde, ester, a heterocyclyl, an aromatic or
heteroaromatic moiety,
-CF3, -CN, or the like. The terms "heterocycle" or "heterocyclic" can be used
to describe a
compound that can include a heterocycle or heterocyclic ring.
The term "carbonyl" is art-recognized and includes such moieties as can be
represented
by the general formula:
0 0
____________________________ X R or X ______ R'
wherein X is a bond or represents an oxygen or a sulfur, and R and R' are as
defined
above. Where X is an oxygen and R or R' is not hydrogen, the formula
represents an "ester".
Where X is an oxygen and R is as defined above, the moiety is referred to
herein as a carboxyl
group, and particularly when R is a hydrogen, the formula represents a
"carboxylic acid." Where
X is an oxygen and R' is hydrogen, the formula represents a "formate." In
general, where the
oxygen atom of the above formula is replaced by sulfur, the formula represents
a "thiocarbonyl"
group. Where X is a sulfur and R or R' is not hydrogen, the formula represents
a "thioester."
Where X is a sulfur and R is hydrogen, the formula represents a
"thiocarboxylic acid." Where X is
a sulfur and R' is hydrogen, the formula represents a "thioformate." On the
other hand, where X
is a bond, and R is not hydrogen, the above formula represents a "ketone"
group. Where X is a
bond, and R is hydrogen, the above formula represents an "aldehyde" group.
The term "heteroatom" as used herein means an atom of any element other than
carbon
or hydrogen. Exemplary heteroatoms include, but are not limited to, boron,
nitrogen, oxygen,
phosphorus, sulfur, silicon, arsenic, and selenium. Heteroatoms, such as
nitrogen, can have
hydrogen substituents and/or any permissible substituents of organic compounds
described
herein which satisfy the valences of the heteroatoms. It is understood that
"substitution" or
"substituted" includes the implicit proviso that such substitution is in
accordance with permitted
valence of the substituted atom and the substituent, and that the substitution
results in a stable
compound, i.e., a compound that does not spontaneously undergo transformation
such as by
rearrangement, cyclization, elimination, etc.
The term "hydroxy" refers to a ¨OH radical.
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As used herein, the term "nitro" refers to -NO2; the term "halogen" designates
-F, -Cl, -Br,
or -I; the term "sulfhydryl" refers to -SH; the term "hydroxyl" refers to -OH;
and the term "sulfonyl"
refers to -SO2-
As used herein, "carbamate" can be used to refer to a compound derived from
carbamic
acid (NH2COOH) and can include carbamate esters. "Carbamates" can have the
general structure
of:
0
R1 N/ R2
0
R3
where R1, R2, and R3 can be any permissible substituent.
As used herein, "carbonate" can be used to refer to a compound derived from
carbonic
acid (H2CO3) and can include carbonate esters. "Carbonates" can have the
general structure of:
¨0 As used herein, "effective amount" can refer to the amount of a composition
described
herein or pharmaceutical formulation described herein that will elicit a
desired biological or
medical response of a tissue, system, animal, plant, protozoan, bacteria,
yeast or human that is
being sought by the researcher, veterinarian, medical doctor or other
clinician. The desired
biological response can be modulation of bone formation and/or remodeling,
including but not
limited to modulation of bone resorption and/or uptake of the BP conjugates,
such as the BP
quinolone conjugates, described herein. The effective amount will vary
depending on the exact
chemical structure of the composition or pharmaceutical formulation, the
causative agent and/or
severity of the infection, disease, disorder, syndrome, or symptom thereof
being treated or
prevented, the route of administration, the time of administration, the rate
of excretion, the drug
combination, the judgment of the treating physician, the dosage form, and the
age, weight,
general health, sex and/or diet of the subject to be treated. "Effective
amount" can refer to the
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amount of a compositions described herein that is effective to inhibit the
growth of or reproduction
of a microorganism, including but not limited to a bacterium or population
thereof. "Effective
amount" can refer to the amount of a compositions described herein that is
kill a microorganism,
including but not limited to a bacterium or population thereof. "Effective
amount" can refer to the
.. amount of a compositions described herein that is effective to treat and/or
prevent osteomyelitis
in a subject in need thereof.
The terms "quinolone," "quinolone antimicrobial molecule," and "oxazolidinone
antimicrobial agent," or "substituents" or "derivatives thereof and related
terms, have the same
meaning and refer to antimicrobial agents which are part of the well-known
class of "quinolones,"
.. as described in more detail herein.
As used herein, "therapeutic" generally can refer to treating, healing, and/or
ameliorating
a disease, disorder, condition, or side effect, or to decreasing in the rate
of advancement of a
disease, disorder, condition, or side effect. The term also includes within
its scope enhancing
normal physiological function, palliative treatment, and partial remediation
of a disease, disorder,
condition, side effect, or symptom thereof.
The term "antibacterial" includes those compounds that inhibit, halt or
reverse growth of
bacteria, those compounds that inhibit, halt, or reverse the activity of
bacterial enzymes or
biochemical pathways, those compounds that kill or injure bacteria, and those
compounds that
block or slow the development of a bacterial infection.
As used herein, the terms "treating" and "treatment" can refer generally to
obtaining a
desired pharmacological and/or physiological effect. The effect may be
prophylactic in terms of
preventing or partially preventing a disease, symptom or condition thereof.
And as used herein
are intended to mean, at least, the mitigation of a disease condition
associated with a bacterial
infection in a subject, including mammals, such as a human, that is alleviated
by a reduction of
growth, replication, and/or propagation of any bacterium such as Gram-positive
organisms, and
includes curing, healing, inhibiting, relieving from, improving and/or
alleviating, in whole or in part,
the disease condition.
The term "prophylaxis" is intended to mean at least a reduction in the
likelihood that a
disease condition associated with a bacterial infection will develop in a
mammal, preferably a
human. The terms "prevent" and "prevention" are intended to mean blocking or
stopping a
disease condition associated with a bacterial infection from developing in a
mammal, preferably
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a human. In particular, the terms are related to the treatment of a mammal to
reduce the likelihood
("prophylaxis") or prevent the occurrence of a bacterial infection, such as
bacterial infection that
may occur during or following a surgery involving bone reparation or
replacement. The terms also
include reducing the likelihood ("prophylaxis") of or preventing a bacterial
infection when the
mammal is found to be predisposed to having a disease condition but not yet
diagnosed as having
it. For example, one can reduce the likelihood or prevent a bacterial
infection in a mammal by
administering a compound of Formula (1) and/or Formula (2), or a
pharmaceutically acceptable
prodrug, salt, active metabolite, or solvate thereof, before occurrence of
such infection.
As used herein, "synergistic effect," "synergism," or "synergy" refers to an
effect arising
between two or more molecules, compounds, substances, factors, or compositions
that is greater
than or different from the sum of their individual effects.
As used herein, "additive effect" refers to an effect arising between two or
more
molecules, compounds, substances, factors, or compositions that is equal to or
the same as the
sum of their individual effects.
The term "biocompatible", as used herein, refers to a material that along with
any
metabolites or degradation products thereof that are generally non-toxic to
the recipient and do
not cause any significant adverse effects to the recipient. Generally
speaking, biocompatible
materials are materials which do not elicit a significant inflammatory or
immune response when
administered to a patient.
As used herein, the term "osteomyelitis" can refer to acute or chronic
osteomyelitis,
and/or diabetic foot osteomyelitis, diabetic chronic osteomyelitis, prosthetic
joint infections,
periodontitis, peri-implantitis, osteonecrosis, and/or hematogenous
osteomyelitis and/or other
bone infections.
Discussion
Infectious bone disease, or osteomyelitis, is a major problem worldwide in
human and
veterinary medicine and can be devastating due to the potential for limb-
threatening sequelae and
mortality. The treatment approach to osteomyelitis is mainly antimicrobial,
and often long-term,
with surgical intervention in many cases to control infection. The causative
pathogens in most
cases of long bone osteomyelitis are infections of Staphylococcus aureus, and
corresponding
biofilms, which are bound to bone in contrast to their planktonic (free-
floating) counterparts. Other
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bone infections and corresponding biofilms are known to arise from a broad
spectrum of both
gram positive and gram-negative bacteria.
The biofilm-mediated nature of osteomyelitis is important in clinical and
experimental
settings because many biofilm pathogens are uncultivable and exhibit an
altered phenotype with
respect to growth rate and antimicrobial resistance (as compared to their
planktonic counterparts).
The difficulty in eradicating biofilms with conventional antibiotics partly
explains why the higher
success rates of antimicrobial therapy in general have not yet been realized
for orthopedic
infections, along with the development of resistant biofilm pathogens, the
poor penetration of
antimicrobial agents in bone, and adverse events related to systemic toxicity.
To overcome the many challenges associated with osteomyelitis treatment, there
is
increasing interest in drug delivery approaches using bone-targeting
conjugates to achieve higher
or more sustained local therapeutic concentrations of antibiotic in bone while
minimizing systemic
exposure.
Methylenebisphosphonates or substituted methylidine bisphosphonate moieties,
commonly referred to as "bisphosphonates" (BPs) are therapeutic agents for the
treatment of
many bone disorders. The bisphosphonate P-C-P group mimics the P-O-P bond of
the naturally-
occurring mediator of bone metabolism, inorganic pyrophosphate. The structural
relationship of
pyrophosphate and methylene bisphosphonates in acid form is shown below.
Individual BPs can
be defined by the covalently attached substituents R1, and R2.
0 0 0 0
HO.." Ø" , .0H HO .OH
HO -13 OH HO -13r 2 P OH
Pyrophosphate Bisphosphonate
The bridging carbon of the bisphosphonate can be substituted with modifying
groups (Ri,
R2) to confer specific biological properties on the derivative. BPs exhibit
strong binding affinity to
HA, the major inorganic material found in bone, particularly at sites of high
bone turnover, and
they are exceptionally stable to both chemical and biological degradation. It
is often
underappreciated that BPs also traverse through soft and hard tissues of the
body (e.g.
endothelium, periosteum, HA) to target bone and the canalicular network and
vascular canals
within bones. These highly specific bone-targeting properties of BPs make them
ideal carriers to
deliver drugs or macromolecules to bone surfaces.
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Quinolone, and in particular fluoroquinolone, antibiotics conjugated to
bisphosphonates
(BPs), for example osteoadsorptive BPs, represents a promising approach
because of the long
clinical track-record of safety of each constituent, and their advantageous
biochemical properties.
In early investigations of the fluoroquinolone family in this context,
ciprofloxacin demonstrated the
best binding and microbiological properties when bound to a BP. Ciprofloxacin
has several
advantages for repurposing in this context: it can be administered orally or
intravenously with
relative bioequivalence, it has broad spectrum antimicrobial activity that
includes the most
commonly encountered osteomyelitis pathogens, it demonstrates bactericidal
activity in clinically
achievable doses, and it is the least expensive drug in the fluoroquinolone
family.
The specific bone-targeting properties of the BP family makes them ideal
carriers for
introducing antibiotics to bone in osteomyelitis pharmacotherapy. BPs form
strong bidentate and
tridentate bonds with calcium and as a result concentrate in hydroxyapatite
(HA), particularly at
sites of active metabolism or infection and inflammation. BPs also exhibit
exceptional stability
against both chemical and biological degradation. The concept of targeting
ciprofloxacin to bone
via conjugation with a BP has been discussed in a number of reports over the
years.
Despite these positive attributes of BPs and quinolones, such as
ciprofloxacin, current
attempts at generating prodrugs containing BPs and quinolones have been
unsuccessful. Most
attempts resulted in either systemically unstable prodrugs or non-cleavable
conjugates that were
found to mostly inactivate either component of the conjugate by interfering
with the
pharmacophoric requirements. For example, Delorme et al. (WO 2007/138381)
describe use of
acyloxy chemical neighbors to activate an alkyl carbamate linker for cleavage
of an oxazolidinone
from a bisphosphonate, but this appears to be too actively cleaved in the
bloodstream. Similar
findings have been described by Houghton et al 2008 J. Medicinal Chemistry,
51:6955-6969. And
it has been shown by Morioka et. al ("Design, synthesis, and biological
evaluation of novel
estradiol¨bisphosphonate conjugates as bone-specific estrogens," Bioorganic &
Medicinal
Chemistry 18 (2010) 1143-1148) and by Arns et. al ("Design and synthesis of
novel bone-
targeting dual-action pro-drugs for the treatment and reversal of
osteoporosis," Bioorganic &
Medicinal Chemistry 20 (2012) 2131-2140) that alkyl carbamates are too stable
to be useful as
a "target and release" linkage for bone targeted, active conjugates.
VVith the deficiencies of current BP quinolone conjugates in mind, described
herein are BP
quinolone conjugates that can contain a BP that can be releasably conjugated
to a quinolone,
such as ciprofloxacin, moxifloxacin, sitafloxacin or nemonoxacin. In
embodiments, the BP
quinolone conjugate can selectively deliver a quinolone to bone, bone grafts,
and or bone graft
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substitutes (i.e. can target bone, bone grafts, or bone graft substitutes) in
a subject. In some
embodiments, the BP quinolone conjugate can release the quinolone. Also
provided herein are
methods of synthesizing BP quinolone conjugates and methods of treating or
preventing
osteomyelitis or other bone infections with one or more of the BP quinolone
conjugates provided
herein.
The compositions provided herein can employ a "target and release linker"
strategy, where
a releasable and bone-specific targeting bisphosphonate-antibiotic (BP-Ab)
conjugate can be
made by attaching the antibiotic or antimicrobial agent, e.g., a quinolone, to
a BP. In any one or
more aspects, the BP can be a pharmacologically inactive BP or
pharmacologically low active BP
using a cleavable or reversible linker, such as carbamate, thiocarbamate,
hydrazone, hydrazone,
et al., or carbonate, so that the antimicrobial agent can be released upon
binding to bone surfaces
by the decreased pH and/or enzymatic environment which is typically found at
active sites of bone
resorption or infection.
In any one or more aspects, the quinolone can be attached to a hydroxy BP,
directly or
indirectly, to the geminal hydroxyl group on the carbon between the two
phosphonate groups of
the BP. This is in contrast to use of an aryl carbamate to otherwise attach or
link a quinolone to
the BP at a site other than an alpha hydroxyl, alpha thiol, or alpha amino
site. To activate the
attachment or linker enough for cleavage (the "target and release" concept)
the present disclosure
utilizes carbamates (and relatives) that are uniquely activated by the alpha
carbon or substituent
of bisphosphonates for adequate release. In any one or more aspects herein,
all analogs of
known clinically used BPs are preferred. Etidronate and MH DP or methylene
hydroxy BP may
be most preferred.
The present chemistry and conjugate design also allows the cleavage to release
two
known (clinically used) drugs, the quinolone and the BP (in particular
clinically used BPs). Carbon
dioxide is the only other released component from the linker. Thus, no new
safety questions exist
for the released components. Previously unknown was whether a linkage to the
geminal hydroxy
group of a bisphosphonate would cleave appropriately for this use to
accomplish this ("target and
release" based efficacy) goal in vivo. Previously, unknown BPs, that have not
been used clinically,
were used to create aryl carbamate linkages at sites other than the alpha
carbon of the BP. These
BPs, that had not previously been studied in a human subject, allowed creation
of an aryl
carbamate-based conjugate to allow a release rate/cleavage rate that was
useful for bioactivity.
Here it has been found in vitro that release still occurs at a useful rate
because a carbamate, via
linkage to the geminal hydroxy group is sufficiently activated by the adjacent
phosphonate groups
to cleave adequately. Since this has now been found to occur, the present
compounds,
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conjugates and formulations offer the opportunity to use many clinically known
bisphosphonates
within the conjugate because most have a geminal hydroxy group.
An exemplary BP-quinolone release mechanism is depicted in FIG. 2 using
ciprofloxacin
as an exemplary quinolone. However, a non-fluoroquinolone can also be
conjugated to the BP as
described herein. This BP-Ab conjugate can have the ability to deliver and
release the
antimicrobial agent specifically to the infectious osteolytic site where
higher bone metabolism is
occurring. Use of an inactive or low active BP can also offer a unique
treatment option by
providing a higher concentration of antimicrobial agent at the disease site
and relatively lower
systemic levels than with higher active BPs. Other BP-quinolone compounds and
conjugates, as
described herein, can have the same or similar activity.
Also provided herein are formulations that can include an amount of a
compound,
conjugate or composition described herein and an additional compound (such as,
but not limited
to, a carrier, diluent, or other active agent or ingredient). The formulation
can be a pharmaceutical
formulation that can contain a pharmaceutically acceptable carrier. The
compositions and/or
formulations can be administered to a subject. The subject can have a bone
infection. The
compositions and formulations provided herein can be used to treat and/or
prevent bone infection.
The compositions and formulations provide herein can provide, in some
embodiments, bone
specific delivery of an antimicrobial agent.
The general concept of targeting active drug species to the bone compartment
with BP
has been discussed in a number of reports. However, no drugs have yet been
developed, as early
attempts led to either systemically unstable prodrugs or non-cleavable
conjugates that were found
mostly to inactivate either component of the conjugate by interfering with the
pharmacophoric
requirements. This suggests that target and release strategies are likely
chemical class-
dependent (taking into consideration compatibilities of the functional groups
of each component)
as well as biochemical target-dependent, and the design for any particular
chemical class must
be customized for its use. Therefore, provided herein are embodiments of a
novel approach to
develop a bone targeted antibiotic or antimicrobial agent with a linkage that
can be metabolically
stable in the bloodstream and metabolically labile on bone to facilitate
appropriate release.
In particular, in any one or more aspects herein, the linkages utilized herein
are designed
to allow maximum local antibacterial efficacy at the site of an infection
where higher bone turnover
exists, while also limiting exposure of lower turnover skeletal sites, non-
skeletal sites, and distant
compartments throughout the body from any adverse effect due to antibiotic or
bisphosphonate
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components or conjugate. Thus, for example, the alpha hydoxy carbamate linkers
and other
related alpha carbon oriented linkers herein are specifically selected to have
maximum stability in
the bloodstream, while still having sensitivity to chemically cleave and
release quinolone
antibiotics at skeletal sites of bacterial infection, due to their sensitivity
to the enzymatic processes
and pH characteristics of that environment. Furthermore, select, but not all,
embodiments in this
disclosure, include the use of bisphosphonates that do not have significant
pharmacological
activity for the targeting component of these drug conjugates. These
"nonantiresorptive" or weak
antiresorptive bisphosphonates have the characteristics of only targeting the
antibiotic to the bone
compartments described and do not have properties to otherwise affect bone
metabolism directly.
Examples include aryl carbamates and aryl thiocarbamates derived from
substituted and
unsubstituted 2-[4-aminophenyl]ethane 1,1 bisphosphonate and 2-[4-
hydroxyphenyl]ethane 1,1
bisphosphonate. Also, included are carbamates derived from substituted and
unsubstituted 2-[3-
aminophenyl]ethane 1,1 bisphosphonate and 2[3-hydroxyphenyl]ethane 1,1
bisphosphonate
substituted and unsubstituted 2-[2-aminophenyl]ethane 1,1 bisphosphonate and 2-
[2-
hydroxyphenyl]ethane 1,1 bisphosphonate. Furthermore, aryl dithiocarbamates,
derived from
substituted and unsubstituted 2-[4-thiophenyflethane 1,1 bisphosphonate, 2[3-
thiophenyl]ethane
1,1 bisphosphonate, and 2[2-thiophenyl]ethane 1,1 bisphosphonate.
In any one or more aspects, the BP of the conjugate can be a pharmacologically
inert or
inactive BP. Examples of pharmacologically inert or inactive BPs that can be
conjugated with a
quinolone as described herein are shown in FIG. 24.
As an example, the inert or inactive BP series for the conjugation can be 4-
hydroxyphenylethylidene BP (FIG. 24A) or 4-aminophenylethylidene BP (FIG. 24E)
having a
medium mineral affinity. Further analogs such as hydroxy BP (FIGS. 24B and
24F) (higher
mineral affinity) and methyl BP (FIGS. 24C and 24G) (lower mineral affinity)
can be used to adjust
the concentration of the BP-Ab conjugates at bone. Phenyl alkyl BPs with
different chain lengths
such as in FIGS. 24D and 24H (propyl or butyl vs. ethyl phenyl) can also be
utilized to optimize
the conjugation chemistry yields and conjugate stability.
In any one or more aspects, the BP of the conjugate can be a pharmacologically
low active
BP. Examples of pharmacologically low active BPs that can be conjugated with a
quinolone as
described herein are shown in FIG. 25. By "low active BP" we mean either the
bisphosphonate
or the dosage level of the bisphosphonate is not so high as to effect bone
metabolism. However,
in some aspects, a higher active BP may be desired where it is desired to both
effect bone
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metabolism and to deliver a quinolone antibiotic to inhibit and/or kill/effect
bacteria on bone.
Other compositions, compounds, methods, features, and advantages of the
present
disclosure will be or become apparent to one having ordinary skill in the art
upon examination of
the following drawings, detailed description, and examples. It is intended
that all such additional
compositions, compounds, methods, features, and advantages be included within
this
description, and be within the scope of the present disclosure.
Bisphosphonate (BP) Quinolone Conjugates and Formulations Thereof
BP Quinolone Conjugates
Provided herein are BP quinolone compounds, conjugates and formulations
thereof. A BP
can be conjugated to a quinolone via a linker. In embodiments, the linker is a
releasable linker.
The quinolone can be releasably attached via a linker to the BP. Thus, in some
embodiments, the
BP quinolone conjugate can selectively deliver and release the quinolone at or
near bone, bone
grafts, or bone graft substitutes (Fig. 2). In other words, for example, a BP
fluoroquinolone
conjugate can provide targeted delivery of a fluoroquinolone to bone and/or
the areas proximate
to bone.
The BP of the BP quinolone conjugates provided herein can be conjugated to any
BP
including but not limited to, hydroxyl phenyl alkyl or aryl bisphosphonates,
hydroxyl phenyl (or
aryl) alkyl hydroxyl bisphosphonates, amino phenyl(or aryl) alkyl
bisphosphonates, amino
phenyl(or aryl) alkyl hydroxyl bisphosphonates, hydroxyl alkyl
bisphosphonates, hydroxyl alkyl
hydroxyl bisphosphonates hydroxyl alkyl phenyl(or aryl) alkyl bisphosphonates,
hydroxyl
phenyl(or aryl) alkyl hydroxyl bisphosphonates, amino phenyl(or aryl) alkyl
bisphosphonates,
amino phenyl(or aryl) alkyl hydroxyl bisphosphonates, hydroxyl alkyl
bisphosphonates, hydroxyl
alkyl hydroxyl bisphosphonates, all of the former being further unsubstituted
or substituted. In
particular, the BP can be etidronate, pamidronate, neridronate, olpadronate,
alendronate,
ibandronate, minodronate, risedronate, zoledronate,
hydroxymethylenebisphosphonate, and
combinations thereof. The bisphosphonate may also be substituted for phosphono
phosphinic
acid or phosphono carboxylic acid. In embodiments, the BP can be pamidronate,
alendronate,
risedronate, zoledronate, minodronate, neridronate, etidronate, which can be
unmodified or
modified as described herein. In preferred embodiments, the BP is etidronate,
MHBP, or
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pamidronate, unmodified or modified.
The BP can contain, or be modified to contain, linkages to an alpha
subsitutent and the
alpha subsitutent can be a hydroxy, amino or thiol group. A antibiotic
quinolone compound or
analog can be conjugated directly or indirectly to the BP at a geminal carbon
substituent of the
BP. The quinolone and/or a linker can also be coupled to a BP having an anti-
resorptive effect
that is significantly reduced or eliminated.
In BPs containing an aryl or phenyl, the aryl or phenyl can be substituted
with a suitable
substitutent at any position on the ring. In some embodiments, the aryl or
phenyl ring of the BP is
substituted with one or more electron donating species (e.g. F, N, and Cl).
Non-pharmacologically active BP variants may also be used for the purpose of
quinolone
delivery absent BP action.
The quinolone can be any quinolone, a fluoroquinolone or a non-fluoroquinolone
including
but not limited to alatrofloxacin, amifloxacin, balofloxacin, besifloxacin,
cadazolid, ciprofloxacin,
clinafloxacin, danofloxacin, delafloxacin, difloxacin, enoxacin, enrofloxacin,
finafloxacin,
flerofloxacin, flumequine, gatifloxacin, gemifloxacin, grepafloxacin,
ibafloxacin, JNJ-Q2,
levofloxacin, lomefloxacin, marbofloxacin, moxifloxacin, nadifloxacin,
norfloxacin, ofloxacin,
orbifloxacin, pazufloxacin, pefloxacin, pradofloxacin, prulifloxacin,
rufloxacin, sarafloxacin,
sitafloxacin, sparfloxacin, temafloxacin, tosufloxacin, trvafloxacin,
zabofloxacin, nemonoxacin and
any combination thereof. In particular, the quinolone can be a
fluoroquinolone, preferably
ciprofloxacin or moxifloxacin.
In any one or more aspects or embodiments herein, the BP quinolone compound
can be
comprised of a quinolone analog or substituent according to the following
structure or Formula
(A),
R40 0
R5
OH
R1
IR,' R2
Formula (A)
wherein R1 can be either
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\ (LOH
HO -P
rN HO N
HN 0
OH
or (¨NH or
L = linker or
ccif
Cr\PN
0 oil
HO -P 3%. L
HO \,
\ HO4 \
0 '
HO )( IsL) / =
-< OH
p p .0H
OH L = linker or HO 0 L = linker or HO 0 L
= linker
>l¨
and wherein R2 can be
and wherein R3 can be either H or OCH3,
and where R4 can be H,
and wherein R5 can be H or F.
As shown, the quinolone of Formula (A) can be linked to a bisphosphonate (BP).
In any
one or more aspects or embodiments herein, the BP can have an alpha
substituent and the alpha
substituent is a hydroxy, amino or thiol group. The quinolone can be directly
or indirectly
conjugated to the BP at the germinal carbon alpha substituent (X) of the BP,
as illustrated in the
formula below.
/quinolone
x;\
HO, I pH
HO -P -OH
o= R Nµco conjugates between alpha-X containing BP and quinolone
X= 0, NH, NR1, S
R1 can be alkyl or substituted alkyl, aryl or subsituted aryl groups
wherein R can be H, substituted and unsubstituted alkyl, alkyl amino, alkyl-
aryl, aryl,
alkylheteroaryl, or heteroaryl.
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Preferred BPs are those that have a geminal hydroxy group on the carbon
between the
two phosphonate groups. A generic analog of such a BP is illustrated in Fig.
25._In any one or
more aspects, the bisphosphonate can be ethylidenebisphosphonate moiety
(etidronate) that can
be substituted by hydroxy (an alpha-hydroxy), amino or thiol.
In some aspects, the
bisphosphonate can include a para-hydroxyphenylethylidene group or derivative
thereof. In
embodiments, the BP can be a clinically known BP, such as pamidronate,
alendronate,
risedronate, zoledronate, minodronate, neridronate, and etidronate, which can
be unmodified or
modified as described herein.
In any one or more embodiments, the BP can be etidronate. Etidronate can be
linked to
a quinolone to form a quinolone antibiotic etidronate-ciprofloxacin (ECC)
conjugate, such as in
Formula (41) or to form an etidronate moxifloxacin (ECX) conjugate such as in
Formula (43)
herein.
The linker, L, can be a compound that is cleavable, meaning that it reversibly
couples the
quinolone analog or compound, in particular a quinolone antimicrobial or
antibiotic analog or
substituent thereof, to the BP. As used herein, the term "cleavable" can mean
a group that is
chemically or biochemically unstable under physiological conditions. In any
one or more aspects,
the linker can be a carbamate, having a structure or Formula (B) below
0
I
R2
N
R3
Formula (B)
for coupling a quinolone, R2, to a BP, R1, as described herein, and R3 can be
substituted and
unsubstituted alkyl, acetyl, benzoyl or other amides, phenyl and substituted
phenyl, preferably H.
In any one or more aspects, the linker can be a carbonate, having a structure
or Formula
(C) below
0
It
RI ¨0
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Formula (C)
for coupling a quinolone, R2, to a BP, R1, as described herein.
In any one or more aspects, the linker can be an alkyl or an aryl carbamate
linker. The
linker can be an 0-thioaryl or thioalklyl carbamate linker. The linker can be
an S-thioaryl or
thioalkyl carbamate linker. The linker can be a phenyl carbamate linker. The
linker can be a
thiocarbamate linker. The linker can be an 0-thiocarbamate linker. The linker
can be an S-
thiocarbamate linker. The linker can be an ester linker. The linker can be a
dithiocarbamate. The
linker can be a urea linker. The linker can be part of the R1 group of Formula
(A) along with the
BP and couple the BP to the quinolone, as described herein. In any one or more
aspects, the
linker can be exemplified by any one of Formula (D) ¨ Formula (H) below,
wherein: R2 can be a
quinolone or a quinolone substituent or derivative and R1 can be a BP, both as
described herein;
and R3 can be substituted and unsubstituted alkyl, acetyl, benzoyl or other
amides, phenyl and
substituted phenyl, preferably H.
0
131 II 131 A
N N 0
111 k
R3 R3 R2
Formula (D) Formula (E) Formula (F)
0
R2 R1 II
, R2
N N
R3 R4 R3
Formula (G) Formula (H)
In some aspects, the BP is etidronate. In some aspects, the quinolone is
ciprofloxacin or
moxifloxacin. In some aspects, the BP is etidronate, the quinolone is
ciprofloxacin and the linker
is an aryl or alkyl carbamate or a linker of Formula (F) providing the
compound of Formula (41).
In some aspects, the BP is etidronate, the quinolone is moxifloxacin and the
linker is an aryl or
alkyl carbamate or a linker of Formula (F) providing the compound of Formula
(43). In some
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aspects, the BP is etidronate, the quinolone is sitafloxacin or nemonoxacin
and the linker is an
alkyl or aryl carbamate or a linker of Formula (F) providing the compound of
Formula (44) or
Formula (45) herein.
In other aspects, the BP can be another BP described herein, such as
pamidronate,
.. neridronate, olpadronate, alendronate, ibandronate, minodronate,
risedronate, zoledronate,
hydroxymethylenebisphosphonate (HM BP), and combinations thereof.
In any one or more aspects, the bisphosphonate can have an alpha substituent
that is
substituted by hydroxy (an alpha-hydroxy), amino, or thiol. In any one or more
aspects, the
bisphosphonate can be an ethylidenebisphosphonate moiety (etidronate) that can
be substituted
by hydroxy (an alpha-hydroxy), amino or thiol. In some aspects, the
bisphosphonate can include
a para-hydroxyphenylethylidene group or derivative thereof. In embodiments,
the BP can be a
clinically known BP, such as pannidronate, alendronate, risedronate,
zoledronate, minodronate,
neridronate, and etidronate, which can be unmodified or modified as described
herein.
Also provided herein are pharmaceutical formulations that can contain a
bisphosphonate
(BP) and a quinolone compound of Formula (A), wherein the quinolone compound
is releasably
coupled to the bisphosphonate via a linker, L; and a pharmaceutically
acceptable carrier. The
bisphosphonate (BP) and the linker, L, can be as described herein in any one
or more aspects.
In any one or more embodiments and aspects herein, the quinolone can have a
generic
structure according to Formula (A), where R1 can be substituents including
alkyl, substituted alkyl,
alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, phenyl,
substituted phenyl, aryl,
substituted aryl, heteroaryl, substituted heteroaryl, halo, hydroxyl, alkoxy,
substituted alkoxy,
phenoxy, substituted phenoxy, aroxy, substituted aroxy, alkylthio, substituted
alkylthio,
phenylthio, substituted phenylthio, arylthio, substituted arylthio, cyano,
isocyano, substituted
isocyano, carbonyl, substituted carbonyl, carboxyl, substituted carboxyl,
amino, substituted
amino, amido, substituted amido, sulfonyl, substituted sulfonyl, sulfonic
acid, phosphoryl,
substituted phosphoryl, phosphonyl, substituted phosphonyl, polyaryl,
substituted polyaryl, C3-020
cyclic, substituted C3-C20 cyclic, heterocyclic, substituted heterocyclic,
amino acid, peptide, and
polypeptide groups, where R2 can be substituents including alkyl, substituted
alkyl, alkenyl,
substituted alkenyl, alkynyl, substituted alkynyl, phenyl, substituted phenyl,
aryl, substituted aryl,
heteroaryl, substituted heteroaryl, halo, hydroxyl, alkoxy, substituted
alkoxy, phenoxy, substituted
phenoxy, aroxy, substituted aroxy, alkylthio, substituted alkylthio,
phenylthio, substituted
phenylthio, arylthio, substituted arylthio, cyano, isocyano, substituted
isocyano, carbonyl,
substituted carbonyl, carboxyl, substituted carboxyl, amino, substituted
amino, amido, substituted
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amido, sulfonyl, substituted sulfonyl, sulfonic acid, phosphoryl, substituted
phosphoryl,
phosphonyl, substituted phosphonyl, polyaryl, substituted polyaryl, 03-C20
cyclic, substituted 03-
C20 cyclic, heterocyclic, substituted heterocyclic, amino acid, peptide, and
polypeptide groups,
where R3 can be substituents including alkyl, substituted alkyl, alkenyl,
substituted alkenyl,
alkynyl, substituted alkynyl, phenyl, substituted phenyl, aryl, substituted
aryl, heteroaryl,
substituted heteroaryl, halo, hydroxyl, alkoxy, substituted alkoxy, phenoxy,
substituted phenoxy,
aroxy, substituted aroxy, alkylthio, substituted alkylthio, phenylthio,
substituted phenylthio,
arylthio, substituted arylthio, cyano, isocyano, substituted isocyano,
carbonyl, substituted
carbonyl, carboxyl, substituted carboxyl, amino, substituted amino, amido,
substituted amido,
sulfonyl, substituted sulfonyl, sulfonic acid, phosphoryl, substituted
phosphoryl, phosphonyl,
substituted phosphonyl, polyaryl, substituted polyaryl, C3-C20 cyclic,
substituted C3-C20 cyclic,
heterocyclic, substituted heterocyclic, amino acid, peptide, and polypeptide
groups, and where
R4 can be substituents including alkyl, substituted alkyl, alkenyl,
substituted alkenyl, alkynyl,
substituted alkynyl, phenyl, substituted phenyl, aryl, substituted aryl,
heteroaryl, substituted
heteroaryl, halo, hydroxyl, alkoxy, substituted alkoxy, phenoxy, substituted
phenoxy, aroxy,
substituted aroxy, alkylthio, substituted alkylthio, phenylthio, substituted
phenylthio, arylthio,
substituted arylthio, cyano, isocyano, substituted isocyano, carbonyl,
substituted carbonyl,
carboxyl, substituted carboxyl, amino, substituted amino, amido, substituted
amido, sulfonyl,
substituted sulfonyl, sulfonic acid, phosphoryl, substituted phosphoryl,
phosphonyl, substituted
phosphonyl, polyaryl, substituted polyaryl, C3-C20 cyclic, substituted 03-C20
cyclic, heterocyclic,
substituted heterocyclic, amino acid, peptide, and polypeptide groups, and
wherein R5 can be H
or F.
R2 R3
(NyLR1
HO I
0 0 R4
Formula (A)
The BP can be conjugated to the quinolone, either a fluoroquinolone or a non-
fluoroquinolone, preferably a fluoroquinolone, via a releasable linker. In any
one or more aspects,
the linker can be an alkyl or an aryl carbamate linker. The linker can be an 0-
thioaryl or thioalkyl
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carbamate linker. The linker can be an S-thioaryl or thio alkyl carbamate
linker. The linker can be
a phenyl carbamate linker. The linker can be a thiocarbamate linker. The
linker can be an 0-
thiocarbamate linker. The linker can be an S-thiocarbamate linker. The linker
can be an ester
linker. The linker can be a dithiocarbamate. The linker can be a urea linker.
The linker can be
part of the R1 group of Formula (A) along with the BP and couple the BP to the
quinolone, as
described herein. In any one or more aspects, the linker can be exemplified by
any one of
Formula (D) ¨ Formula (H) below, wherein: R2 can be a quinolone or a quinolone
substituent or
derivative and R1 can be a BP, both as described herein; and R3 can be
substituted and
unsubstituted alkyl, acetyl, benzoyl or other amides, phenyl and substituted
phenyl, preferably H.
0
111 II R2 111 II R2
N N 0
R1
R3 R3 'o R2
Formula (D) Formula (E) Formula (F)
0
111 A R2 R1 jt, R2
S N N
R3 R4 R3
Formula (G) Formula (H)
In some aspects, the BP is etidronate. In some aspects, the quinolone is
ciprofloxacin or
moxifloxacin. In some aspects, the BP is etidronate, the quinolone is
ciprofloxacin and the linker
is an alkyl or an aryl carbamate or a linker of Formula (F) providing the
compound of Formula (41)
herein. In some aspects, the BP is etidronate, the quinolone is moxifloxacin
and the linker is an
alkyl or an aryl carbamate or a linker of Formula (F) providing the compound
of Formula (43)
herein. In some aspects, the BP is etidronate, the quinolone is sitafloxacin
or nemonoxacin and
the linker is an alkyl or an aryl carbamate or a linker of Formula (F)
providing the compound of
Formula (44) or Formula (45) herein.
In any one or more aspects or embodiments herein, the BP has an alpha
substituent and
the alpha substituent is a hydroxy, amino or thiol group and the quinolone is
directly or indirectly
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conjugated to the BP at the germinal carbon alpha substituent (X) of the BP,
as illustrated in the
formula below.
quinolone
X)<-
+ pH
HO ¨P P ¨OH
R
0 0 conjugates between alpha-X containing BP and
quinolone
X= 0, NH, NRI, S
RI can be alkyl or substituted alkyl, aryl or subsituted aryl groups
wherein R can be H, substituted and unsubstituted alkyl, alkyl amino, alkyl-
aryl, aryl,
alkylheteroaryl, or heteroaryl.
In any one or more aspects, the BP is an alpha-OH containing BP that can be
conjugated
to the quinolone, such as a fluoroquinolone, at a geminal OH group on the BP
as shown below.
In various aspects the quinolone, such as a fluoroquinolone, can be directly
conjugated (e.g.,
without use of a linker other than C=0) to a geminal OH group of the BP. In
various aspects, the
quinolone can be indirectly conjugated via a linker at the geminal OH group of
the BP.
In some aspects, the compound can have a formula according to Formula (41),
Formula
(43), Formula (44) or Formula (45) herein.
BP Quinolone Conjugate Pharmaceutical Formulations
Also described herein are formulations, including pharmaceutical formulations,
which can
contain an amount of a BP quinolone compound or conjugate described elsewhere
herein in any
one or more aspects or embodiments. The amount can be an effective amount. The
amount can
be effective to inhibit the growth and/or reproduction of a bacterium. The
amount can be effective
to kill a bacterium. Formulations, including pharmaceutical formulations can
be formulated for
delivery via a variety of routes and can contain a pharmaceutically acceptable
carrier. Techniques
and formulations generally can be found in Remmington's Pharmaceutical
Sciences, Meade
Publishing Co., Easton, Pa. (20th Ed., 2000), the entire disclosure of which
is herein incorporated
by reference. For systemic administration, an injection is useful, including
intramuscular,
intravenous, intraperitoneal, and subcutaneous. For injection, the therapeutic
compositions of the
invention can be formulated in liquid solutions, for example in
physiologically compatible buffers
such as Hank's solution or Ringer's solution. In addition, the BP quinolone
conjugates and/or
components thereof can be formulated in solid form and redissolved or
suspended immediately
prior to use. Lyophilized forms are also included. Formulations, including
pharmaceutical
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formulations, of the BP quinolone conjugates can be characterized as being at
least sterile and
pyrogen-free. These formulations include formulations for human and veterinary
use.
Suitable pharmaceutically acceptable carriers include, but are not limited to
water, salt
solutions, alcohols, gum arabic, vegetable oils, benzyl alcohols, polyethylene
glycols, gelatin,
carbohydrates such as lactose, amylose or starch, magnesium stearate, talc,
silicic acid, viscous
paraffin, perfume oil, fatty acid esters, hydroxyl methylcellulose, and
polyvinyl pyrrolidone, which
do not deleteriously react with the BP quinolone conjugate.
The pharmaceutical formulations can be sterilized, and if desired, mixed with
auxiliary
agents, such as lubricants, preservatives, stabilizers, wetting agents,
emulsifiers, salts for
influencing osmotic pressure, buffers, coloring, flavoring and/or aromatic
substances, and the like
which do not deleteriously react with the BP quinolone conjugate.
Another formulation includes the addition of the BP quinolone conjugates to
bone graft
material or bone void fillers for the prevention or treatment of
osteomyelitis, peri-implantitis or perk
prosthetic infections, and for socket preservation after dental extractions.
A pharmaceutical formulation can be formulated to be compatible with its
intended route
of administration. Examples of routes of administration include parenteral,
e.g., intravenous,
intradermal, subcutaneous, oral (e.g., inhalation), transdermal (topical),
transmucosal, and rectal
administration. Solutions or suspensions used for parenteral, intradermal, or
subcutaneous
application can include the following components: a sterile diluent such as
water for injection,
saline solution, fixed oils, polyethylene glycols, glycerin, propylene glycol
or other synthetic
solvents; antibacterial agents such as benzyl alcohol or methyl parabens;
antioxidants such as
ascorbic acid or sodium bisulfite; chelating agents such as
ethylenediaminetetraacetic acid;
buffers such as acetates, citrates or phosphates and agents for the adjustment
of tonicity such as
sodium chloride or dextrose. pH can be adjusted with acids or bases, such as
hydrochloric acid
or sodium hydroxide. The parenteral preparation can be enclosed in ampoules,
disposable
syringes or multiple dose vials made of glass or plastic.
Formulations, including pharmaceutical formulations, suitable for injectable
use can
include sterile aqueous solutions (where water soluble) or dispersions and
sterile powders for the
extemporaneous preparation of sterile injectable solutions or dispersions. For
intravenous
administration, suitable carriers can include physiological saline,
bacteriostatic water, Cremophor
EM TM (BASF, Parsippany, N.J.) or phosphate buffered saline (PBS). Injectable
pharmaceutical
formulations can be sterile and can be fluid to the extent that easy
syringability exists. Injectable
pharmaceutical formulations can be stable under the conditions of manufacture
and storage and
must be preserved against the contaminating action of microorganisms such as
bacteria and
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fungi. The carrier can be a solvent or dispersion medium containing, for
example, water, ethanol,
a pharmaceutically acceptable polyol like glycerol, propylene glycol, liquid
polyetheylene glycol,
and suitable mixtures thereof. The proper fluidity can be maintained, for
example, by the use of a
coating such as lecithin, by the maintenance of the required particle size in
the case of dispersion
and by the use of surfactants. Prevention of the action of microorganisms can
be achieved by
various antibacterial and antifungal agents, for example, parabens,
chlorobutanol, phenol,
ascorbic acid, thimerosal, and the like. In some embodiments, it can be useful
to include isotonic
agents, for example, sugars, polyalcohols such as mannitol, sorbitol, and
sodium chloride in the
composition.
Sterile injectable solutions can be prepared by incorporating any of the BP
quinolone
conjugates described herein in an amount in an appropriate solvent with one or
a combination of
ingredients enumerated herein, as required, followed by filtered
sterilization. Generally,
dispersions can be prepared by incorporating the BP quinolone conjugate into a
sterile vehicle
which contains a basic dispersion medium and the required other ingredients
from those
enumerated herein. In the case of sterile powders for the preparation of
sterile injectable solutions,
examples of useful preparation methods are vacuum drying and freeze-drying
which yields a
powder of the active ingredient plus any additional desired ingredient from a
previously sterile-
filtered solution thereof.
Systemic administration can also be by transmucosal or transdermal means. For
transmucosal or transdermal administration, penetrants appropriate to the
barrier to be permeated
can be used in the formulation. Such penetrants are generally known in the
art, and include, for
example, for transmucosal administration, detergents, bile salts, and fluidic
acid derivatives.
Transmucosal administration can be accomplished through the use of nasal
sprays or
suppositories. For transdermal administration, the BP quinolone conjugates can
be formulated
into ointments, salves, gels, or creams as generally known in the art. In some
embodiments, the
BP quinolone conjugates can be applied via transdermal delivery systems, which
can slowly
release the BP quinolone conjugates for percutaneous absorption. Permeation
enhancers can be
used to facilitate transdermal penetration of the active factors in the
conditioned media.
Transdermal patches are described in for example, U.S. Pat. No. 5,407,713;
U.S. Pat. No.
5,352,456; U.S. Pat. No. 5,332,213; U.S. Pat. No. 5,336,168; U.S. Pat. No.
5,290,561; U.S. Pat.
No. 5,254,346; U.S. Pat. No. 5,164,189; U.S. Pat. No. 5,163,899; U.S. Pat. No.
5,088,977; U.S.
Pat. No. 5,087,240; U.S. Pat. No. 5,008,110; and U.S. Pat. No. 4,921,475.
For oral administration, a formulation as described herein can be presented as
capsules,
tablets, powders, granules, or as a suspension or solution. The formulation
can contain
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conventional additives, such as lactose, mannitol, cornstarch or potato
starch, binders, crystalline
cellulose, cellulose derivatives, acacia, cornstarch, gelatins,
disintegrators, potato starch, sodium
carboxymethylcellulose, dibasic calcium phosphate, anhydrous or sodium starch
glycolate,
lubricants, and/or or magnesium stearate.
For parenteral administration (i.e., administration through a route other than
the alimentary
canal), the formulations described herein can be combined with a sterile
aqueous solution that is
isotonic with the blood of the subject. Such a formulation can be prepared by
dissolving the active
ingredient (e.g. the BP quinolone conjugate) in water containing
physiologically-compatible
substances, such as sodium chloride, glycine and the like, and having a
buffered pH compatible
with physiological conditions, so as to produce an aqueous solution, then
rendering the solution
sterile. The formulation can be presented in unit or multi-dose containers,
such as sealed
ampoules or vials. The formulation can be delivered by injection, infusion, or
other means known
in the art.
For transdermal administration, the formulations described herein can be
combined with
skin penetration enhancers, such as propylene glycol, polyethylene glycol,
isopropanol, ethanol,
oleic acid, N-methylpyrrolidone and the like, which increase the permeability
of the skin to the
nucleic acid vectors of the invention and permit the nucleic acid vectors to
penetrate through the
skin and into the bloodstream. The formulations and/or compositions described
herein can be
further combined with a polymeric substance, such as ethylcellulose,
hydroxypropyl cellulose,
ethylene/vinyl acetate, polyvinyl pyrrolidone, and the like, to provide the
composition in gel form,
which can be dissolved in a solvent, such as methylene chloride, evaporated to
the desired
viscosity and then applied to backing material to provide a patch.
For inclusion in bone graft substitutes or bone void fillers to prevent local
post-operative
infection or graft failure after surgery, and to provide sustained local
release of antibiotic at the
graft site, the formulations described herein can be combined with any
xenograft (bovine),
autograft (self) or allograft (cadaver) material or synthetic bone substitute.
For example, a powder
formulation can be premixed by the treating surgeon or clinician
bedside/chairside with any
existing bone graft substitute on the market or with an autologous graft. This
formulation can be
further combined with any previously described formulation, and can be
combined with products
containing hydroxyapatites, tricalcium phosphates, collagen, aliphatic
polyesters (poly(lactic)
acids (PLA), poly(glycolic)acids (PGA), and polycaprolactone (PC L),
polyhydroxybutyrate (PH B),
methacrylates, polymethylmethacrylates, resins, monomers, polymers, cancellous
bone
allografts, human fibrin, platelet rich plasma, platelet rich fibrin, plaster
of Paris, apatite, synthetic
hydroxyapaptite, coralline hydroxyapatite, wollastonite (calcium silicate),
calcium sulfate,
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bioactive glasses, ceramics, titanium, devitalized bone matrix, non-
collagenous proteins,
collagen, and autolyzed antigen extracted allogenic bone. In this embodiment
the bone graft
material combined with BP quinolone conjugate can be in the formulation of a
paste, powder,
putty, gel, hydrogel, matrix, granules, particles, freeze-dried powder, freeze-
dried bone,
demineralized freeze-dried bone, fresh or fresh-frozen bone, corticocancellous
mix, pellets, strips,
plugs, membranes, lyophilized powder reconstituted to form wet paste,
spherules, sponges,
blocks, morsels, sticks, wedges, cements, or amorphous particles; many of
these may also be in
injectable formulations or as a combination of two or more aforementioned
formulations (e.g.
injectable paste with sponge).
In another embodiment, the BP-quinolone conjugate can be combined with factor-
based
bone grafts containing natural or recombinant growth factors, such as
transforming growth factor-
beta (TGF-beta), platelet-derived growth factor (PDGF), fibroblast growth
factors (FGF), and/or
bone morphogenic protein (BMP). In another embodiment, the BP quinolone
conjugate can be
combined with cell-based bone grafts used in regenerative medicine and
dentistry including
embryonic stem cells and/or adults stem cells, tissue-specific stem cells,
hematopoietic stem
cells, epidermal stem cells, epithelial stem cells, gingival stem cells,
periodontal ligament stem
cells, adipose stem cells, bone marrow stem cells, and blood stem cells.
Therefore, a bone graft
with the property of osteoconduction, osteoinduction, osteopromotion,
osteogenesis, or any
combination thereof can be combined with the BP quinolone conjugate for
clinical or therapeutic
use.
Dosage forms
The BP quinolone compounds, conjugates and formulations thereof described
herein in
any one or more aspects or embodiments can be provided in unit dose form such
as a tablet,
capsule, single-dose injection or infusion vial, or as a predetermined dose
for mixing with bone
graft material as in formulations described above. Where appropriate, the
dosage forms described
herein can be microencapsulated. The dosage form can also be prepared to
prolong or sustain
the release of any ingredient. In some embodiments, the complexed active agent
can be the
ingredient whose release is delayed. In other embodiments, the release of an
auxiliary ingredient
is delayed. Suitable methods for delaying the release of an ingredient
include, but are not limited
to, coating or embedding the ingredients in material in polymers, wax, gels,
and the like. Delayed
release dosage formulations can be prepared as described in standard
references such as
"Pharmaceutical dosage form tablets," eds. Liberman et. al. (New York, Marcel
Dekker, Inc.,
1989), "Remington ¨ The science and practice of pharmacy", 20th ed.,
Lippincott Williams &
Wilkins, Baltimore, MD, 2000, and "Pharmaceutical dosage forms and drug
delivery systems", 6th
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Edition, Ansel et al., (Media, PA: Williams and Wilkins, 1995). These
references provide
information on excipients, materials, equipment, and processes for preparing
tablets and capsules
and delayed release dosage forms of tablets and pellets, capsules, and
granules. The delayed
release can be anywhere from about an hour to about 3 months or more.
Coatings may be formed with a different ratio of water soluble polymer, water
insoluble
polymers, and/or pH dependent polymers, with or without water insoluble/water
soluble non
polymeric excipient, to produce the desired release profile. The coatings can
be either performed
on the dosage form (matrix or simple) which includes, but is not limited to,
tablets (compressed
with or without coated beads), capsules (with or without coated beads), beads,
particle
compositions, "ingredient as is" formulated as, but not limited to, suspension
form or as a sprinkle
dosage form.
Examples of suitable coating materials include, but are not limited to,
cellulose polymers
such as cellulose acetate phthalate, hydroxypropyl cellulose, hydroxypropyl
methylcellulose,
hydroxypropyl methylcellulose phthalate, and hydroxypropyl methylcellulose
acetate succinate;
polyvinyl acetate phthalate, acrylic acid polymers and copolymers, and
methacrylic resins that are
commercially available under the trade name EUDRAGITO (Roth Pharma,
Westerstadt,
Germany), zein, shellac, and polysaccharides.
Effective Amounts
The formulations can contain an effective amount of a BP quinolone compound or
conjugate (effective for inhibiting and/or killing a bacterium) described
herein in any one or more
aspects or embodiments. In some embodiments, the effective amount ranges from
about 0.001
pg to about 1,000 g or more of the BP quinolone conjugate described herein. In
some
embodiments, the effective amount of the BP quinolone conjugate described
herein can range
from about 0.001 mg/kg body weight to about 1,000 mg/kg body weight. In yet
other embodiments,
the effective amount of the BP quinolone conjugate can range from about 1% w/w
to about 99%
or more w/w, w/v, or v/v of the total formulation. In some embodiments, the
effective amount of
the BP quinolone conjugate is effective at killing a bacterium that is the
causative agent of
osteomyelitis and all its subtypes (e.g. diabetic foot osteomyelitis), jaw
osteonecrosis, and
periodontitis including, but not limited to any strain or species of
Staphylococcus, Pseudomonas,
Aggregatibacter, Actinomyces, Streptococcus, Haemophilus, Salmonella,
Serratia, Enterobacter,
Fusobacterium, Bacteroides, Porphyromonas, Prevotella, Veillonella,
Campylobacter,
Peptostreptococcus, Eikenella, Treponema, Dialister, Micromonas, Yersinia,
Tannerella, and
Escherichia.
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Methods of Using the BP Quinolone Coniuqates
An amount, including an effective amount, of the BP quinolone compounds,
conjugates
and formulations thereof described herein in any one or more aspects or
embodiments can be
administered to a subject in need thereof. In some embodiments the subject in
need thereof can
have a bone infection, disease, disorder, or a symptom thereof. In some
embodiments, the subject
in need thereof can be suspected of having or is otherwise predisposed to
having a bone infection,
disease, disorder, or a symptom thereof. In some embodiments, the subject in
need thereof may
be at risk for developing an osteomyelitis, osteonecrosis, pen-prosthetic
infection, and/or pen-
implantitis. In embodiments, the disease or disorder can be osteomyelitis and
all its subtypes,
osteonecrosis, peri-implantitis or periodontitis. In some embodiments the
subject in need thereof
has a bone that is infected with a microorganism, such as a bacteria. In some
embodiments, the
bacteria can be any strain or species of Staphylococcus, Pseudomonas,
Aggregatibacter,
Actinomyces, Streptococcus, Haemophilus, Salmonella, Serratia, Enterobacter,
Fusobacterium,
Bacteroides, Porphyromonas, Prevotella, Veillonella, Campylobacter,
Peptostreptococcus,
Eikenella, Treponema, Dialister, Micromonas, Yersinia, Tannerella, and
Escherichia. In some
embodiments, the bacteria can form biofilms. In some embodiments,
osteomyelitis can be treated
in a subject in need thereof by administering an amount, such as an effective
amount, of BP
quinolone conjugate or formulation thereof described herein to the subject in
need thereof. In
some embodimnets, the compositions and compounds provided herein can be used
in
osteonecrosis treatment and/or prevention, distraction osteogenesis, cleft
repair, repair of critical
supra-alveolar defects, jawbone reconstruction, and any other reconstructions
or repair of a bone
and/or joint.
Administration of the BP quinolone compounds or conjugates is not restricted
to a single
route, but can encompass administration by multiple routes. For instance,
exemplary
administrations by multiple routes include, among others, a combination of
intradermal and
intramuscular administration, or intradermal and subcutaneous administration.
Multiple
administrations can be sequential or concurrent. Other modes of application by
multiple routes
will be apparent to the skilled artisan.
The pharmaceutical formulations can be administered to a subject by any
suitable method
that allows the agent to exert its effect on the subject in vivo. For example,
the formulations and
other compositions described herein can be administered to the subject by
known procedures
including, but not limited to, by oral administration, sublingual or buccal
administration, parenteral
administration, transdermal administration, via inhalation, via nasal
delivery, vaginally, rectally,
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and intramuscularly. The formulations or other compositions described herein
can be
administered parenterally, by epifascial, intracapsular, intracutaneous,
subcutaneous,
intradermal, intrathecal, intramuscular, intraperitoneal, intrasternal,
intravascular, intravenous,
parenchymatous, and/or sublingual delivery. Delivery can be by injection,
infusion, catheter
delivery, or some other means, such as by tablet or spray. Delivery can also
be by a carrier such
as hydroxyapatite or bone in the case of anti-infective bone graft material at
a surgical site.
Delivery can be via attachment or other association with a bone graft
material.
EXAMPLES
Now having described the embodiments of the present disclosure, in general,
the following
Examples describe some additional embodiments of the present disclosure. While
embodiments
of the present disclosure are described in connection with the following
examples and the
corresponding text and figures, there is no intent to limit embodiments of the
present disclosure
to this description. On the contrary, the intent is to cover all alternatives,
modifications, and
equivalents included within the spirit and scope of embodiments of the present
disclosure.
Introduction
Infectious bone disease, or osteomyelitis, is a major problem worldwide in
human and
veterinary medicine and can be devastating due to the potential for limb-
threatening sequelae and
mortality (Lew, et al., Osteomyelitis. Lancet 2004;364:369-79; Desrochers, et
al, Limb amputation
and prosthesis. Vet Clin North Am Food Anim Pract 2014;30:143-55; Stoodley, et
al., Orthopaedic
biofilm infections. Curr Orthop Pract 2011;22:558-63; Huang, et al., Chronic
osteomyelitis
increases long-term mortality risk in the elderly: a nationwide population-
based cohort study. BMC
Geriatr 2016;16:72). The treatment approach to osteomyelitis is mainly
antimicrobial, and often
long-term, with surgical intervention in many cases to control infection. The
causative pathogens
in most cases of long bone osteomyelitis are biofilms of Staphylococcus
aureus; by definition
these microbes are bound to bone (Fig. 1) in contrast to their planktonic
(free-floating)
counterparts (Wolcott, et al., Biofilms and chronic infections. J Am Med Assoc
2008;299:2682-
2684).
The biofilm-mediated nature of osteomyelitis is important in clinical and
experimental
settings because many biofilm pathogens are uncultivable and exhibit an
altered phenotype with
respect to growth rate and antimicrobial resistance (as compared to their
planktonic counterparts)
(Junka, et al., Microbial biofilms are able to destroy hydroxyapatite in the
absence of host
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immunity in vitro. J Oral Maxillofac Surg 2015;73:451-64; Herczegh, et al.,
Osteoadsorptive
bisphosphonate derivatives of fluoroquinolone antibacterials. J Med Chem 2002;
45:2338-41).
The difficulty in eradicating biofilms with conventional antibiotics partly
explains why the higher
success rates of antimicrobial therapy in general have not yet been realized
for orthopedic
.. infections, along with the development of resistant biofilm pathogens, the
poor penetration of
antimicrobial agents in bone, and adverse events related to systemic toxicity
(Buxton, et al.,
Bisphosphonate-ciprofloxacin bound to Skelite is a prototype for enhancing
experimental local
antibiotic delivery to injured bone. Br J Surg 2004;91:1192-6).
To overcome the many challenges associated with osteomyelitis treatment, there
is
increasing interest in drug delivery approaches using bone-targeting
conjugates to achieve higher
or more sustained local therapeutic concentrations of antibiotic in bone while
minimizing systemic
exposure (Panagopoulos, et al., Local Antibiotic Delivery Systems in Diabetic
Foot Osteomyelitis:
Time for One Step Beyond? Int J Low Extern Wounds 2015;14:87-91; Puga, et al.,
Hot melt poly-
epsilon-caprolactone/poloxam me implantable matrices for sustained delivery of
ciprofloxacin.
Acta biomaterialia 2012;8:1507-18). Fluoroquinolone antibiotics conjugated to
osteoadsorptive
bisphosphonates (BPs) represents a promising approach because of the long
clinical track-record
of safety of each constituent, and their advantageous biochemical properties
(Buxton, et al.,
Bisphosphonate-ciprofloxacin bound to Skelite is a prototype for enhancing
experimental local
antibiotic delivery to injured bone. Br J Surg 2004;91:1192-6). In early
investigations of the
fluoroquinolone family in this context, ciprofloxacin demonstrated the best
binding and
microbiological properties when bound to BP (Herczegh, et al., Osteoadsorptive
bisphosphonate
derivatives of fluoroquinolone antibacterials. J Med Chem 2002;45:2338-41).
Ciprofloxacin has
several advantages for repurposing in this context: it can be administered
orally or intravenously
with relative bioequivalence, it has broad spectrum antimicrobial activity
that includes the most
commonly encountered osteomyelitis pathogens, it demonstrates bactericidal
activity in clinically
achievable doses, and it is the least expensive drug in the fluoroquinolone
family (Houghton, et
al., Linking bisphosphonates to the free amino groups in fluoroquinolones:
preparation of
osteotropic prodrugs for the prevention of osteomyelitis. J Med Chem
2008;51:6955-69).
Example 1:
Non-limiting examples of quinolones that can be included in the BP conjugates
herein include the
following quinolones.
Fluorinated Quinolones
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F
00
/
HN
H H (,,-
N.õANNH2 I
F 1.1 N 1\1)
N ,N NIFFi H
HO,1 F HO 1
F
0 0
O 0 Alatrofloxacin
Amifloxacin
00
y0- H 1
OH
N N....N.
J N
1 H
O QFCI A
F
O 0 Li ,ci- H
F-121,4
Balofloxacin Besifloxacin
o--0 OH
7 OH
,0 Y
N NH
N
N N,,- F
I
1 HO
HO F
F 00
00
Ciprofloxacin
Cadazolid
NH2
7 a 0 0
F
HO 1
1 I
HO N
F Nh.
O 0 NH2
Clinafloxacin Danofloxacin
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F 00
NH2 F
1 1 OH
F kif'. CI r,,,, OH (-N N
HO N)
1 40
F
0 0 F
Delafloxacin Difloxacin
(NH 0 0
,.N 1\( N) F
1 I HO 1
HOI-r-rF N N1
0 0
1
Enoxacin Enrofloxacin
00
0 0
F
HO 1 F
H HO 1
I
A N N
1 i
N Fr 0) i) F
Finafloxacin F Flerofloxacin
F
y 0- NH
N N..
0
HO 1
HO N / F
0 0 NH2
0
Flumequine Gatifloxacin
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H2N
7 p_ 7
N NH
.)
N I\L N ----N N
I I HO I
HO /
F F
0 0
O 0
Gem ifloxacin Grepafloxacin
O0 7 r"
F N NF
HO 1
HO
N F 1\11-1
2
00 F
lbafloxacin JNJ-Q2
rNH
N N N 1\1)
HO I HO I
F F
O 0 00
Levofloxacin Lomefloxacin
0 0 7
F N
1 OH
rN N HO I
F
1\kr) 0N,, 0 0
Marbofloxacin Moxifloxacin
r-OH
'1 r NH
N N N 1\1)
I I
HO HO
iiIIIF F
00 00
Nadifloxacin Norfloxacin
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-L-,-rc, rN- 0 0 F
N I\1) HO 1 F
I s,
HO NN' '
F
A, F L,NH
0 0
Ofloxacin Orbifloxacin
'I,,.
rN-
N
I NH2 N I\1)
HO I
F HO
F
00 00
Pazufloxacin Pefloxacin
0 0
F r No
OH S N N) 0--i
H
1-116iN _______________________________ N HO
I I A _________________________________ F 0
H N _________________________________ 0 0
Pradofloxacin Prulifloxacin
0 50
rs rN- HO F
1
N N.) N N1
HO 1 F NH
0
0 0
Rufloxacin FSarafloxacin
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______________ soF NH2
y F r-NH
N Ni---K1
I H HO I
O F
F 0 0 NH2
0 0
Sitafloxacin Sparfloxacin
F
F
NH2
F' NH
F'
N N
N N
TMS I I I
F HOI.,.-wF
0 0
00
Temafloxacin Tosufloxacin
F
00
H
HO./1-...}. .õ----....õ- F
F NH2 * I
N I\L NI---. :
NNI\nCN
I H
HOYYIF A .--\ -
N-0
0 0 \
Trovafloxacin Zabofloxacin
The following is an example of a non-flouronated quinolone.
NH2
HO 1
00
Nemonoxacin
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Example 2:
Dental implants are a critical part of modern dental practice and it is
estimated that up to
35 million Americans are missing all of their teeth in one or both jaws. The
overall market for these
implants to replace and reconstruct teeth is expected to reach $4.2 billion by
2022. While the
majority of implants are successful, some of these prosthetics fail due to
peri-implantitis, leading
to supporting bone destruction. Peri-implantitis has a bimodal incidence,
incluiding early stage
(<12 months) and late stage (>5 years) failures; both of these critical
failure points are largely the
result of bacterial biofilm infections on and around the implant. Peri-
implantitis is a common
reason for implant failure. Dental implants failures are generally caused by
biomechanical or
biological/microbiological reasons. The prevalence of peri-implantitis, the
most severe form of
microbiological-related implant disease leading to the destruction of
supporting bone is difficult to
ascertain from the current literature. However, recent studies indicate that
peri-implantites is a
growing problem with increasing prevalence4. A recent study of 150 patients
followed 5 to 10
years showed a rate of peri-implantitis of approximately 17% and 30%
respectively, indicating
that it is a significant issue5. Early implant failure or lack of
osseointegration is a separate problem
and occurs in roughly 9% of implanted jaws . This is more prevalent in the
maxilla and is
associated with bacterial infection during surgery or from a nearby site (e.g.
periodontitis) as well
as other well-recognized and modifiable risk factors such as smoking,
diabetes, excess cement,
and poor oral hygiene2.
Biofilm infection can be involved in the etiophathogeneiss of peri-
implantitis. Biofilm
infections represent a unique problem for treatment and are often difficult to
diagnose, resistant
to standard antibiotic therapy, resistant to host immune responses, and lead
to persistent
intractable infections7.The biofilm hypothesis of infection has been steadily
expanded since the
early elucidation that bacteria live in matrix supported communities5s. It is
now established that
over 65% of chronic infections are caused by bacteria living in biofilms7.
This implies that
approximately 12 million people in the US are affected by, and almost half a
million people die in
the US each year, from these infections. Peri-implantitis and periodontitis
are among the most
common biofilm infections encountered. Peri-implantitis has been found to be a
comparatively
simpler infection with less diverse communities (and keystone pathogens) than
periodontitis
infections10. Typically, gram negative species predominate. Other orthopedic
or osseous
infections including those of the jaw, are also caused by bacterial biofilm
communities12 making
the technology developed here amenable for use in these diseases as well.
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Currently treatment approaches to peri-implantits have their limitations.
While peri-
implantitis has several causes, the predominant etiology is bacterial biofilm.
There are no
universally accepted guidelines or protocols for peri-implantitis therapy,
many of the clinical
regimens for bacterial peri-implantitis treatment comprise local and systemic
antibiotic delivery13
and surgical debridement of the lesion, including restorative grafting with
bone graft
substitutes14,16. Clinical experience has shown, however, that it is difficult
to advance even a local
antibiotic delivery device to the bottom of a deep pen-implant pocket and to
infected jawbone, or
to get systemic antibiotics to penetrate adequately into infected jawbone to
kill biofilm
pathogens16, which is largely due to the intrinsic poor bone (and pen-implant)
biodistribution or
pharmacokinetics of the antibiotics17. In previous long-term studies, even
when infected implants
were cleaned locally with an antiseptic agent and systemic antibiotics were
administered, there
was additional loss of supporting bone in more than 40% of the advanced peri-
implantitis
lesions16.
In addition, longer-term systemic antibiotic therapy could result in systemic
toxicity or
adverse effects, and also resistance. Therefore, it has become common practice
by clinicians to
use local delivery systems for achieving higher therapeutic antibacterial
concentrations in bone.
For example, dentists use chairside mixing of minocycline or doxycycline
powder (e.g. Arestine),
or chlorhexidine solution (e.g. PerioChip9, with bone graft material for local
delivery18. Such
approaches are merely a slurry and do not represent a strong binding between
the antibiotic and
the bone substitute as in the BioVinc approach, and thus suffer from
comparatively earlier
washout and less efficient pharmacokinetics as previously discussed. In
addition, investigators
have also used several biodegradable and non-biodegradable local antibiotic
delivery systems16.
However, these approaches have several limitations, e.g., non-biodegradable
approaches (e.g.
polymethylmethacrylate cements) require a second surgery to remove the
antibiotic loaded
device, are incompatible with certain antibiotics, and suffer from inefficient
release kinetics; in
some cases, <10% of the total delivered antibiotic is released17.
Biodegradable materials including
fibers, gels, and beads are receiving increasing interest, however, their
clinical efficacy for the
treatment of peri-implantitis is not well-documented3. Even when effective
antimicrobials/antiseptics are used to treat peri-implantitis in the jaw, such
as local chlorhexidine
delivery, there is minor influence on treatment outcomes as demonstrated in
prospective animal
and human studies15,17. These data taken together further support the poor
pharmacokinetics of
antibiotics in bone as previously mentioned, and highlight the need for bone-
binding/bone-
targeted and sustained antibiotic release strategies.
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BP-Conjugates
Considering the limitations of current treatment approaches, it is a
significant advance in
the field to develop a bone/biofilm-targeting antimicrobial agent. The BP-
antibiotic (BP-Ab)
conjugates provided herein can overcome many challenges associated with poor
antibiotic
pharmacokinetics or bioavailability in bone and within bone-bound biofilms.
These componds can
reduce infection via a "targeting and release approach," which can reduce
concern with systemic
toxicity and/or drug exposure in other (e.g. non-infected) tissues. The BP-Ab
conjugates can be
integrated into a bone graft substitute. The BP-Ab can be a BP-fluoroquinolone
conjugate. In
some instances, the BP-Ab can be a bisphosphonate-carbamate-ciprofloxacin
(BCC, compound
6), as shown in Fig. 3. The exemplary structure of Fig. 3 is also referred to
herein as BCC
(compound 6). When integrated into a bone graft the BP-Ab bone graft material
can also be
referred to as a BP-Ab-bone graft. For example, when the antiboiotic is a
fluoroquinolone, it can
be referred to as a BP-FQ-bone graft. These compound(s) can effectively adsorb
to
hydroxyapatite (HA)/bone, and can achieve a sustained release and
antimicrobial efficacy against
biofilm pathogens over time. The compounds and graft material integrating the
compound(s)
provided herein can be used as an anti-infective bone graft substitute for
adjunct treatment or
prevention of peri-implantitis. The conjugate will be released locally from
the graft material with
sustained release kinetics and cleaved in the presence of bacterial or
osteoclastic activity as we
have previously demonstrated, in vitro and in vivo, in other results provided
elsewhere herein. In
this way the grafts can provide greater local concentrations of the FQ, such
as ciprofloxacin, as
compared to current delivery routes. In sum the compounds and bone-graft
materials provided
herein can contain an antibiotic that is conjugated to a safe or
pharmacologically inactive (non-
antiresorptive) BP moiety bound to calcium/HA in the graft material via strong
polydentate
electrostatic interactions, and the antibiotic releases over time; it does not
simply represent a
topical antibiotic that is merely mixed in as a slurry with existing bone
graft material as some
current clinical approaches in this context. This chemisorbed drug attached to
calcium phosphate
mineral (HA) is therefore a major advance in the field and overcomes many of
the limitations in
antibiotic delivery to pen-implant bone for effective bactericidal activity
against biofilm pathogens.
The general concept of targeting bone by linking active drug molecules to BPs
has been
.. discussed in a review30. However, as of this time no FDA approved drugs
have been developed,
as early attempts led to either systemically unstable prodrugs or non-
cleavable conjugates that
were found mostly to inactivate either component of the conjugate by
interfering with the
pharmacophoric requirements. In the quinolone field a prominent example was
described by
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Herczegh where antibacterial properties of the fluoroquinolone were diminished
upon conjugation
with a stable BP-linked congener31-32. Therefore, a target and release linker
strategy is needed.
Recently, medicinal chemistry strategies exploiting less stable linking
techniques start to
emerge. Others have linked fluoroquinolones via the carboxylic acid group to
several different BP
moieties. They found that glycolamide ester prodrugs of the antibiotics
moxifloxacin and
gatifloxacin reduced infection when used prophylactically in a rat
osteomyelitis mode133. This
same group has used acyloxycarbamate and phenylpropanone based linkers to
tether the same
antibiotics via amine functionality to simple BP systems34. They show using
the same prophylactic
rat model that these conjugates are also better than the parent antibiotic at
inhibiting the
establishment of infection. The Targanta team33 has carried several of these
prodrug strategies
on into use with the glycopeptide antibiotic oritavancin35. This dual function
drug seems to be
somewhat effective in preventing infection. However, to date they have not
published studies
showing that they can treat an established infection and they also have not
published
pharmacokinetics of the prodrug. It is believed that these analogs are too
labile in the bloodstream
to fully realize success with this therapeutic approach as their drug
candidate selection was based
in part on plasma instability. Thus, it is believed that these compounds
developed by these groups
fail to achieve effective local concentrations of the antibiotic.
The BCC compound(s) (Fig. 3) can incorporate the phenyl moity of the phenyl
carbamate
linker directly into the BP portion of the molecule. Release kinetics can be
modified or tuned via
modification of the phenyl ring with electron withdrawing or donating groups,
which can alter the
liability of the linker. Additionally, the BP core lacks effectiveness as an
antiresporptive agent, and
thus, does not carry the risk of medication-related osteonecrosis of the jaw
like the more potent
nitrogen-containing BP drugs (e.g., zoledronate39,40. It is demonstrate herein
and in other
Examples herein that this target and release strategy using the phenyl
carbamate linker very likely
releases the active drug directly into the bacterial biofilm in the bone
milieu. The bone targeting
is so effective that it works better than ciprofloxacin against biofilms grown
on HA bone matrix
surrogate than on planktonic cultures grown in plastic vessels. An analog
conjugate made with a
non-cleavable amide linkage (bisphosphonate-amide-ciprofloxacin, leaving out
the phenolic
oxygen of the carbamate, was found to have very little effect on bacterial
growth under any
circumstances, demonstrating that active cleavage of the conjugate is required
for antimicrobial
activity.
Example 3
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Design and synthesis of additional BP-Ab conjugates (Fig. 4). Additional BP-Ab
conjugates can be designed using, for example, ciprofloxacin and moxifloxacin
conjugated to BPs
(e.g. 4-hydroxyphenylethylidene BP (BP 1, Fig. 4), its hydroxy-containing
analog (BP 2, Fig. 4,
with higher bone affinity) and pamidronate (BP 3, Fig. 4), via carbamate based
linkers (e.g.
carbamate, S-thiocarbamate, and 0-thiocarbamate). Fig. 5 shows an exemplary
synthesis
scheme for synthesis of BP-Ab conjugates with an 0-thiocarbamate linker.
Conjugates with S-
thiocarbamate linkage (slightly more labile) can be obtained by isomerization
of conjugates with
0-thiocarbamate linkage via the Newman-Kwart rearrangement (ref. 47, 48).
Preliminary
chemistry has already been conducted to demonstrate the feasibility of the
quick synthesis of
these targets. Adding bone affinity is therefore well demonstrated using the a-
OH containing BPs
(49). Added bone affinity will enhance concentrations of the conjugate at the
bone surface and
facilitate higher local concentrations of drug short term and long term. For
the synthesis of
conjugates with a-OH containing BPs (BP 2 and pamidronate, Fig. 4), since the
a-OH
bisphosphonate ester is prone to rearrangement to a phosphonophosphate, the a-
OH can be
protected with the tert-butyldimethylsilyl (TBS) group (Scheme 2, Fig. 6)
(50). Then the a-O-TBS
BP 2 ester are activated by 4-nitrophenyl chloroformate and reacted with
ciprofloxacin or
moxifloxacin similarly as in Fig. 5. For a-O-TBS BP 3 ester, a linker with
phenol group (e.g., linker
1 (resorcinol), linker 2 (hydroquinone), linker 3 (4-hydroxyphenylacetic
acid), Figure 20) are used
to tether BP and antimicrobial agents, and the synthesis route using linker 3
is illustrated as an
example here (Scheme 3, Fig. 7). All BP-Ab conjugates are characterized by 1H,
31P, 13C NM R,
MS, HPLC, and elemental analysis to assure identity.
The mineral binding affinity of the BP-Ab conjugates can be determined.
Briefly, Anorganic
bovine bone large particle size (uniformly 1-2mm) can be accurately weighed
(1.4-1.6 mg) and
suspended in a 4 mL clear vial containing the appropriate volume of assay
buffer [0.05% (wt/vol)
Tween20, 10pM EDTA and 100mM HEPES pH=7.4] for 3hr. This bone material can
then be
incubated with increasing amounts of BP-Ab (0, 25, 50, 100, 200 and 300pM).
Samples can be
gently shaken for 3h at 37 C in the assay buffer. Subsequent to the
equilibrium period, the vials
can be centrifuged at 10,000 rpm for 5 min to separate solids and supernatant.
The supernatant
(0.3 mL) can be collected and the concentration of the equilibrium solution
are measured using a
Shimadzu UV-VIS spectrometer (275nm wavelength). Fluorescent emission can also
be used to
calculate binding parameters. Nonspecific binding can be measured with a
similar procedure in
the absence of HA as control. The amount of parent drug/BP-Ab conjugates bound
to HA is
deduced from the difference between the input amount and the amount recovered
in the
supernatants after binding. Binding parameters (Kd and Bmax represent the
equilibrium
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dissociation constant and maximum number of binding sites, respectively) can
be calculated using
the PRISM program (Graphpad, USA) and measured in 5 independent experiments.
Compounds
with an equilibrium dissociation constant (Kd) lower than 20 pM (- 2x Kd of
parent BPs) can be
preferred. A two-sample t-test can be used to evaluate the binding parameters
of the BP-Abs.
The sample size (n=5) in each group can be used to detect the effect size 1.72
for this hypothesis
at a power of 80% and a one-side Type I error of 0.05.
The linkage-stability of the BP-Ab conjugates can be determined. Briefly, the
linker stability
of each BP-Ab conjugate can be tested in PBS buffers with different pH (pH =
1, 4, 7.4, 10) and
human or canine serum. BP-Ab can be suspended in 400 pL of above-mentioned PBS
or in 400
pL of 50% (v/v in PBS) human or canine serum. The suspension/solution can be
incubated for 24
h at 37 C and centrifuged at 13000 rpm for 2 min, and the supernatant can be
recovered.
Methanol (5X volume relative to supernatant) can be added to each supernatant,
and the mixture
can be vortexed for 15 min to extract released fluoroquinolone. The mixture
can be then
centrifuged at 10000 rpm for 15 min to pellet the insoluble material. The
supernatant containing
the extracted fluoroquinolone can be recovered and evaporated to dryness. The
dried pellets can
be resuspended in PBS, and the amount of released fluoroquinolone can be
determined by UV-
VIS measurements as described previously. The percentage of fluoroquinolone
drug released
can then be calculated based on the input amount and the measured amount of
released drug.
The identity of released drug can be confirmed by LC-MS analysis and/or NMR if
the
concentrations are sufficient.
The in vitro inhibition of biofilm growth on HA discs can be determined.
Briefly, for custom
disc manufacturing, commercially available HA powder can be used. Powder
pellets of 9.6mm in
diameter can be pressed without a binder. Sintering can be performed at 900
C. The tablets can
be compressed using the Universal Testing System for static tensile,
compression, and bending
tests (Instron model 3384; Instron, Norwood, MA). The quality of the
manufactured HA discs can
be checked by means of confocal microscopy and microcomputed tomography (micro-
CT) using
an LEXT OLS4000 microscope (Olympus, Center Valley, PA) and Metrotom 1500
microtomograph (Carl Zeiss, Oberkochen, Germany), respectively. HA discs can
then be
introduced to the following concentrations [mg/mL] of each BP-Ab conjugate and
.. ciprofloxacin/moxifloxacin: 800, 400, 200, 100, 50, 25, 10, 5, 1 and left
for 24h/37 C. After
incubation, HA discs can be removed and introduced to 1 mL of PBS and left for
5 min in gentle
rocker shaker; 3 subsequent rinsings are performed this way. After rinsing,
1mL of Aa suspension
can be introduced to discs and left for 24h/37 C. Discs can then be rinsed to
remove non-bound
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bacteria and subjected to vortex shaking. The serial dilutions of suspension
obtained can then be
culture plated on modified TSB agar plates and colony growth is counted after
24h.
The oseeointegration effect of the BP-FQ-bone grafts on critical size can be
evaluated in
supra-alveolar pen-implant defect model for bone grafting. Briefly, in this
split mouth design,
mandibular PM2-PM4 are bilaterally extracted in 6 beagle dogs (3 males, 3
females) and are
allowed to heal for 12 weeks. Crestal incision are made followed by
mucoperiosteal flap reflection.
Ostectomy are performed to create a 6mm supra-alveolar defect. Implant site
osteotomy
preparations are made in each of the premolar regions by sequential cutting
with internally
irrigated drills in graduated diameters under copious irrigation. Implants
(Astra Tech Osseospeed
Tx 3 x 11 mm) are placed in the position of PM2-PM4 on each side in such
manner that the
implants are positioned 4mm supracrestally in relation to the created defect
and at the same
distance from the buccal cortical bone plate. Dogs are divided randomly into 3
different groups (2
dogs per group):
1. Anorganic bovine bone (1g large particle size 1-2mm) chemisorbed with BP-
fluoroquinolone are used on the right side and collagen plugs (negative
control) are used on the
left side.
2. Anorganic bovine bone (1g large particle size 1-2mm, positive control)
are used
on the right side and collagen plugs (negative control) are used on the left
side.
3. Bio-OssO (1g large particle size 1-2mm) chemisorbed with BP-
fluoroquinolone are
used on the right side and Bio-Oss (1g large particle size 1-2mm, positive
control) are used on
the left side.
Chemistry and antimicrobial assay results from experiments described above can
inform
calculations of the ideal standardized quantity of the conjugate for
adsorption to graft material for
use in all in vivo experiments described here. Early calculations predicated
based on the
preliminary results indicate that 5mg or less of conjugate adsorbed to 1g of
graft material will
provide 2-3 orders of magnitude bactericidal activity above the MIC of tested
pathogens. Our BP-
fluoroquinolone conjugate can be applied in a range of bone graft materials
including
commercially available ones, e.g., Bio-Osse; thus we choose house-made
anorganic bovine
bone and BioOss as a positive control in the study for a demonstration of wide
applications of the
conjugate. All defects are filled (depending on the groups above) with a
standardized amount of
biomaterial up to the platform of each implant on both sides, and Bio-Gidee
membranes are used
to cover the graft and the implants for improved stability. The flaps are
closed in a tension free
manner with the use of periosteal releasing incisions, internal mattress and
finally marginal single
interrupted sutures (PTFE 4,0, Cytoplast, USA). MicroCT are acquired at this
point and animals
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are monitored clinically for inflammation and adverse events. Additionally, as
described in the
experiments to follow, these animals undergo PK studies to assess for any
systemic exposure to
the components within the graft material (e.g. intact conjugate, BP,
antibiotic, or linker). Animals
are sacrificed after 12 weeks and the mandibles are resected and examined by
micro-CT followed
by histologic preparation. Baseline micro-CT scans of the jaws are taken for
comparison to post-
experimental scans. Quantitative 3D volumetric micro-CT and histomorphometric
analyses are
performed to examine the volume of new bone present in pen-implant sites, as
well as first bone-
to-implant contact, total defect area, regenerated area, regenerated area
within total defect area,
regenerated bone, residual bone substitute material, percentage of mineralized
tissue, soft tissue,
and void. Finally, necropsy are performed for post-mortem evaluation of organs
and systems for
gross and microscopic signs of tolerability issues from local oral therapy.
Antimicrobial efficacy of the BP-FQ-bone grafts can be evaluated in a canine
peri-
implantitis model. Briefly, in this split mouth design, mandibular PM2-PM4 are
extracted bilaterally
in 8 beagle dogs (4 males, 4 females; 48 teeth total) using minimally
traumatic technique. After 3
months of healing mucoperiosteal flaps are elevated on both sides of the jaw
and osteotomy
preparations are made in each of the premolar regions by sequential cutting
with internally
irrigated drills in graduated diameters under copious external irrigation.
Using a non-submerged
technique, implants (Astra Tech Osseospeed Tx 3 x 11 mm) are installed at
each site. The
sequence of implant placement are identical in both sides but randomized with
a computer
generated randomization scheme between dogs. Healing abutments are connected
to the
implants and flaps approximated with resorbable sutures. A plaque control
regimen comprising
brushing with dentifrice is then initiated four times a week. Twelve weeks
after implant placement
just prior to initiation of experimental peri-implantitis, microbiological
samples are obtained from
all pen-implant sites with sterile paper points (Dentsply, Maillefer, size 35,
Ballaigues,
Switzerland) and placed immediately in Eppendorf tubes (Starlab, Ahrensburg,
Germany) for
microbiological analysis. Microbiologic analysis are performed as we have
previously detailed via
DNA extraction and 16S rRNA PCR amplification.(55) PCR amplicons are sequenced
using the
Roche 454 GS FLX platform and data analyzed with the Quantitative Insights
into Microbial
Ecology (QIIME) software package (56). Colony forming unit counts (CFU/mL) are
determined
from samples as in our Phase I study as described earlier. At this point
experimental peri-
implantitis are initiated as follows. Aggregatibacter actinomycetemcomitans
(Aa) biofilm, a
keystone periodontal pathogen, which is not endogenous to canine flora, are
initiated on the
healing abutments in vitro as performed in our previous experiment in a rat
animal model and also
in our previous animal peri-implantitis study. The biofilm inoculated healing
abutments are placed
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on the implants and cotton ligatures are placed in a submarginal position
around the neck of
implants. After 10 weeks of bacterial infection, microbial sampling and
analysis are done again
as before and micro-CT scans are taken as the baseline for the peri-
implantitis defect. Treatment
of this experimental peri-implantitis model are initiated by surgical
debridement of all implant sites
by raising full-thickness buccolingual flaps, removing any existing calculus
from implant surfaces
using an air-powder abrasion device, and wiping of the implant surfaces with
gauze soaked in
chlorhexidine gluconate 0.12%. The animals are divided into 4 groups as
follows (2 dogs per
group):
1. Anorganic bovine bone (1g large particle size 1-2mm) with chemisorbed BP-
fluoroquinolone are used on the right side and collagen plugs (negative
control) are used on the
left side.
2. Anorganic bovine bone (1g large particle size 1-2mm, positive control)
are used
on the right side and collagen plugs (negative control) are used on the left
side.
3. Anorganic bovine bone (1g large particle size 1-2mm) with chemisorbed BP-
fluoroquinolone are used on the right side and an antimicrobial releasing
device (100 mg topical
minocycline, positive control) are used on the left side.
4. Bio-Osse (1g large particle size 1-2 mm) with chemisorbed BP-
fluoroquinolone
(positive control) are used on the right side and an antimicrobial releasing
device (100 mg topical
minocycline, positive control) are used on the left side.
Treatment group assignments are blinded to future investigators for data
analysis.
Standardized and comparable amounts of antimicrobials are used in treatment
groups. After
treatment, flaps are repositioned and sutured (PTFE 4,0, Cytoplast, USA) and
oral hygiene
measures reinstituted after 1 week following suture removal. Clinical and
micro-CT scan
examinations are performed again at 3 months after surgery and also
microbiological samples
are acquired at this time point for analysis as described above. Six months
after peri-implantitis
surgery animals are euthanized and micro-CT scans are performed, and the jaws
are resected
for assessment of histopathologic parameters as detailed in the section
"critical size supra-
alveolar pen-implant defect model." An inflammatory score are determined from
histologic
sections as previously detailed (ref. 57) for correlation with clinical and
radiologic findings.
Statistical analysis: Statistical calculations are performed with SPSS 22.0
(IBM, Armonk,
NY) and Excel 2016 (Microsoft Corporation, Redmond, WA). Power analyses were
performed to
determine sample size estimations for all animal studies using G Power 3
software58. Following
data collection from these animal studies, quantitative outcomes are analyzed
first with descriptive
statistics to understand the distribution of the data (parametric or non-
parametric) and to generate
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the mean, standard error, standard deviation, kurtosis and skewness, and 95%
confidence levels.
The data are analyzed using the Kruskall-Wallis test, ANOVA, or mixed linear
models as
applicable and statistical significance are carried out at a=0.05 level when
comparing groups.
Post-hoc testing using unpaired t-tests and Dunnett's test for multiple
comparisons are also
performed to further validate findings. All animal experiments are described
using the ARRIVE
guidelines for reporting on animal research to ensure the quality,
reliability, validity, and
reproducibility of results59.
The drug compound and component stability and in vitro ADME of BCC (6) can be
evaluated. This data can help establish if there is likely to be any large
differences in human
metabolism vs. experimental animals. Incubation of 6 with human, rat, and dog
liver microsomes
and hepatocytes followed by LC/MS analysis of the metabolite mixture are
performed. The
metabolic profile of ciprofloxacin is known62,63, and so our focus are on any
metabolites of the BP
portion of the molecule and of the parent (e.g. piperazine ring cleavage as is
known for
ciprofloxacin). Once metabolites have been determined in vitro, plasma samples
from other in
vivo experiments described abvoe are used to determine these compounds at
steady state in
vivo.
The toxicology of the BCC (6) can be evaluated in rat and dog to determine
NOAEL. In
order to determine the NOAEL and maximum tolerated dosage (MTD) in rat and dog
we first carry
out dose ranging studies. Groups of 6 rats (3 males, 3 females), are given a
single intravenous
dose of 10 mg/kg for 6, or based on our best assessment at the time. The dose
are escalated by
doubling until acute toxicity is noted (MTD) then this dose are reduced by 20%
sequentially until
no effects are seen, this will be the NOAEL for the compound. Toxicity are
assessed as mild,
moderate or substantial, and moderate toxicity in
or substantial toxicity in animal define the
MTD64. Animals are followed for body weight and clinical observations for 5
days. After 5 days,
animals are euthanized and necropsy performed to assess for organ weight and
histology (15
sections to include liver and kidney based on clinical BP toxicology). A
similar dose range study
are carried out in dogs (1/sex, starting at the equivalent dose as determined
from allometric
scaling 4 mg/kg assuming 250 g rats and 10 kg dogs) and include hematology and
clinical
chemistry in addition to identical terminal studies as in rat. This can use a
total of 4-6 cohorts.
An expanded acute toxicity testing in groups of animals including
toxicokinetics and
recovery testing at the NOAEL and the MTD can be performed. Gropus of 48 rats
including 10/sex
can be used for each dose for assessment of toxicity and 9/sex for
toxicokinetics and 5/sex for
recovery. Toxicokinetics are determined at 6 time points (3 rats/time point
chosen randomly from
male or female) following administration of each dose. Time points are 5, 30,
60, 120 mins, 12
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hrs, and 24 hrs post dosing. Recovery animals are observed for 14 days
followed by assessment
of organ weight and histology as in the above study. From the toxicokinetic
study, PK parameters
are determined by non-compartmental analysis (NCA) including Cmax, AUC and
half-life. An
identical experiment was carried out in canines but including 10 total animals
(3/sex for dosing
and 2/sex for recovery) with multiple blood draws from each animal at the same
time points as for
the rats. The AUC at the NOAEL for canines are used to calculate the maximum
allowable
exposure from the bone graft/BP-fluoroquinolone conjugate as described in aim
2 and PK
experiments in canines are used to determine if there is systemic exposure
above 1/100 of this
level.
For population modeling, a unique 3-compartment (blood/urine/bone)
mathematical model
of BP pharmacokinetics which has been validated clinically and are applied to
the current
project65. From the canine study, in each animal at the time of euthanasia, we
sample bone (jaw
and femur), tendon (gastrocnemius) for determination of BP and fluoroquinolone
concentrations.
We combine these data and our model to describe the time course in dogs. From
this model we
can simulate the expected exposure of bone and cartilage to both BP and
fluoroquinolone with
alternative dosing or repeated dosing. This can inform subsequent human
dosing. The
nonparametric adaptive grid (NPAG) algorithm with adaptive gamma implemented
within the
Pmetrics package for R (Laboratory of Applied Pharmacokinetics and
Bioinformatics, Los
Angeles, CA) are used for all PK model-fitting procedures as previously
described66-68. Assay
error (SD) is accounted for using an error polynomial as a function of the
measured concentration,
and comparative performance evaluation are completed using Akaike's
information criterion, a
regression of observed versus predicted concentrations, visual plots of PK
parameter-covariate
regressions, and the rule of parsimony.
Example 4
The BP-Ab conjugates can be integrated into grafts and grafting devices. In
embodiments,
one or more of the BP-Ab conjugates can be integrated into an already approved
bone graft
product, such as the bovine bone materials from BioOss (Geistlich Pharma AG,
Switzerland) or
MinerOss (BioHorizons, Birmingham, AL) to name a few. The BP-Ab conjugate(s)
can be
admixed with a support material for use as a dental bone graft substitute. The
product will
comprise the conjugate adsorbed to anorganic bovine bone material. This
material will allow the
local delivery of antibiotic to the region of bone graft implantation to
reduce bacterial infection
rates and associated dental pathology such as peri-implantitis and other
infections. The dental
applications for our product could include not only peri-implantitis
treatment, but also socket
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preservation after tooth extraction, ridge or sinus augmentation,
periodontitis prevention or
treatment, osteomyelitis or osteonecrosis treatment or prevention, or other
oral and periodontal
surgery applications where such a bone graft could be beneficial. The BP-
fluoroquinolone
conjugate material will be intimately adsorbed on the bone graft substitute
and our preliminary
data show sustained release into the area of bone destruction in the case of
infections, which
allows our product to more effectively deliver antibiotic to the site of
infection with negligible to no
systemic exposure to either component of the conjugate compound.
The grafting material can also be beneficial for non-dental grafting
procedures, such as
sinus grafting procedures.
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Example 5:
This Example demonstrates various BP conjugate compounds and synthesis
schemes.
BP-carbamate-moxifloxacin BP conjugate and synthesis scheme is demonstrated in
Fig. 8. Fig.
9 shows a BP-carbamate-gatifloxacin BP conjugate and synthesis scheme. Fig. 10
shows a BP-
p-Hydroxyphenyl Acetic Acid-ciprofloxacin BP conjugate and synthesis scheme.
Fig. 11 shows a
BP-OH-ciprofloxacin BP conjugate and synthesis scheme. Fig. 12 shows a BP-O-
Thiocarbamate-
ciprofloxacin BP conjugate and synthesis scheme. Fig. 13 shows a BP-S-
Thiocarbamate-
ciprofloxacin BP conjugate and synthesis scheme. Fig. 14 shows a BP-Resorcinol-
ciprofloxacin
BP conjugate and synthesis scheme. Fig. 15 shows a BP-Hydroquinone-
ciprofloxacin BP
conjugate and synthesis scheme.
Fig. 16 shows one embodiment of a genus structure for a BP-fluoroquinolone
conjugate,
where W can be 0 or S or N, X can be 0, S, N, CH20, CH2N, or CH2S, Y can be H,
CH3, NO2, F,
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CI, Br, I, or CO2H, Z can be H, CH3, OH, NH2, SH, F, CI, Br, or I, and n can
be 1-5. Fig. 17 shows
various BP-fluoroquinolone conjugates.
Fig. 18 shows one embodiment of a genus structure for a genus of a phosphonate
containing an aryl group, where X can be H, CH3, OH, NH2, SH, F, CI, Br, or 1,
Y can be P03H2,
or CO2H. Z can be OH, NH2, SH, or N3, and n can be 1 or 2. Fig. 19 shows
various BPs, where
X can be F, Cl, Br, or I and n can be 1 or 2.
Fig. 20 shows various BP's with terminal primary amines. Fig. 21 shows various
BPs
coupled to a linker containing a terminal hydroxyl and amine functional groups
where R can be
Risedronate, Zoledronate, Minodronate, Pamidronate, or Alendronate. Fig. 22
shows various BP-
pamidronate-ciprofloxacin conjuagtes. Fig. 23 shows various BP-Alendronate-
ciprofloxacin
conjuagtes.
Example 6.
1. Dimethyl acetylphosphonate (37)
0 Me0 Neat 0
sP¨OMe if ?Me
-F-OMe
Me0 DC-it
0
C21-13C103H903P C4H904P
78.49 g/mol 124.07 g/mol 152.08 g/mol
(37)
Trimethyl phosphite (2.36 mL, 20 mmol) was added to ice-cold acetyl chloride
(1.44 mL,
20.2 mmol) under N2 over a period of 20 mins. The colorless solution was
warmed to room
temperature, stirred for 30 mins, and concentrated under vacuum to afford 2.89
g (94%) product
as colorless oil which was used in next reaction as is. iHNMR (300 MHz,
0D0I3): O 3.84 (d, J =
12 Hz, 6H), 2.46 (d, J = 5.4 Hz, 3H). 31PNMR (121 MHz, 0D013): 6 -1.10.
2. Tetramethyl(1-hydroxyethylidene)-bisphosphonate (38)
OMe 0 Me0 OMe
X \
H-P=0 it ?Me `-0
OMe / j_ -OMe Et20, N2
NH' 0 0 C - rt, 3h HO p=0
Me0 OMe
C2H703P C6l-119N1 041-1904P C6H1607P2
110.04 g/mol 129.24 g/mol 152.08 g/mol 262.13 g/mol
81
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(37) (38)
Dimethyl acetylphosphate (37) (2.2 g, 14. 44 mmol) was added dropwisely to an
ice-cold
solution of dimethyl phosphite (1.63 mL, 15.91 mmol) and dibutylamine (0.767
mL, 1.44 mmol) in
dry ether (30 mL) under N2. The ice bath was removed, and the mixture was
stirred at room
temperature for 3h. The resulting precipitate was filtered, washed with ether,
and dried under
vacuum overnight to afford 3.24 g (85%) of product as white solid. 11-INMR
(300 MHz, CDCI3): 6
3.94 - 3.82 (m, 12H), 3.44 (t, J = 8.4 Hz, 1H), 1.68 (t, J = 16.2 Hz, 3H).
31PNMR (121 MHz, CDCI3):
O22.21. MS-ESI: 263.1 [M+H]+.
3. Tetramethyl (1-{[(4-nitrophenoxy)carbonyl]oxylethane-1,1-
diyl)bis(phosphonate) (39)
Me0,
HO P0(0Me)2 ome
=OyCI DCM, N2
I 02N 4)<P0(0Me) 2 0 oc _ rt, 3h X 0
0 0
02N
Me0' bMe
061-11607P2 C7H10N2 C71-14CINO4
013H19N011P2
262.13 g/mol 122.16 g/mol 201.56 g/mol
427.23 g/mol
(39)
p-nitrophenyl chloroformate (768 mg, 3.81 mmol) was added to an ice-cold
solution of
DMAP (466 mg, 3.81 mmol) in DCM (20 mL) under N2. After stirring for 10 mins,
the tetramethyl(1-
hydroxyethylidene)-bisphosphonate (1 g, 3.81 mmol) was added in one portion.
The ice-bath was
removed, and the mixture was stirred at room temperature for 3h. Next, the
reaction mixture was
extracted with 20 mL each of cold aqueous 0.1 N HCI (2x), water, brine, dried
over MgSO4, and
concentrated. The crude mixture was separated by column chromatography using
Et0Ac/Me0H
(1-3%) to afford 1.16g (71%) light-yellow oil. 1HNMR (300 MHz, CDCI3): 6 8.27
(d, J = 9 Hz, 2H),
.. 7.40 (d, J = 9 Hz, 2H), 3.97 - 3.87 (m, 12H), 2.02 (t, J = 15.6 Hz, 3H).
31PNMR (121 MHz, CDCI3):
6 17.98. MS-ESI: 445.3 [M+NH4]+.
4: Ciprofloxacin carbamoyl etidronate tetramethyl ester (40)
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0 OH 0 OH
Me0, ome NaHCO3,
02N 0 ji..)
0
H20/THF 0
Me0 OMe
Mee OMe N
HN 0 - õ,,..) A N
A
me6 OMe
Ci3Hi9N0n P2 CO,FN303 C241-132FN30,P2
427.23 g/mol 331.34 g/mol 619.47 g/mol
(40)
To a solution of NaHCO3 (239.5 mg, 2.85 mmol) in H20 (20 mL) was added
ciprofloxacin
(899.6 mg, 2.71 mmol) and the suspension was cooled in ice-bath. Next, the
tetramethyl (1-{[(4-
nitrophenoxy)carbonyl]oxy}ethane-1,1-diy1)bis(phosphonate) dissolved in THF
(20 mL) was
added dropwisely over a period of 20 mins. The yellow suspension was stirred
overnight (14 h)
at room temperature. The reaction mixture was concentrated, and the crude
mixture was
separated by column chromatography using DCM/Me0H (1-5%) to provide 832 mg
(49%) of light-
yellow solid. 11-INMR (300 MHz, CDCI3): 6 8.77 (s, 1H), 8.03 (d, J = 12.6 Hz,
1H), 7.36 (d, J = 6.9
Hz, 1H) 3.98 - 3.80 (m, 12H), 3.79 -3.68 (br s, 4H), 3.58 - 3.50 (m, 1H), 3.30
(t, J = 9.6 Hz, 4H),
1.95(t, J = 15.6 Hz, 3H), 1.40(q, J = 6.8 Hz, 2H), 1.23- 1.16(m, 2H). 31PNMR
(121 MHz, 0DCI3):
6 20.19. MS-ESI: 620.3 [M+H]+.
5. Etidronate-carbamate-Ciprofloxacin (41)
0 OH
0 OH
0
1] CH3CN
0
Me0 OMe
Si-Br 2] Me0H
HO 0H
,
0--.1=fy 0 Nj
y
0=P 0
0=P, rj I , -OH
Me0 -OMe HO
C241-132FN3 11P2 C31-19BrSi C20 H24FN3 11P2
619.47 g/mol 153.09 g/mol 563.36 g/mol
(41)
A mixture of tetramethyl etidronate-carbamate-ciprofloxacin (775 mg, 1.25
mmol) and
bromotrimethylsilane (1.53 g, 10 mmol) in ACN (28 mL) was stirred for 2 h. The
volatiles were
evaporated under vacuum and Me0H (28 mL) was added to the residue. After
stirring for 30mins
the resulting suspension was filtered, washed with Me0H (10 mL x 2), and dried
under vacuum
overnight to afford 662 mg (93%) off-white solid. 1H NMR (300 MHz, 20% CD3CN
in DMSO-d6) 6
8.66 (s, 1H), 7.92 (d, J = 13.2 Hz, 1H), 7.57 (d, J = 7.4 Hz, 1H), 3.78 (p, J
= 3.1 Hz, 1H), 3.64 (br
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d, J = 32.1 Hz, 4H), 3.32 (br s, 4H), 1.82 (t, J = 15.1 Hz, 3H), 1.32 (d, J =
6.5 Hz, 2H), 1.16 (s,
2H). 31PNMR (121 MHz, 20% CD3CN in DMSO-d6): b 15.57. MS-ESI: 564.2 [M+H]+.
6. Moxifloxacin carbamoyl etidronate tetramethyl ester (42)
Me0 oMe 0 OH 0 OH
0 Ne2CO, 0
,
02N At 0 I H20/THF
114P
Me0' OMe
\-14C1
(Me0) 20P
(Me0) 20P
k0
3F1 gN011 P2 C211-125C1FN304 C28H38FN3012P2
427.23 g/mol 437.89 g/mol 689.56 g/mol
(42)
Moxifloxacin HCI was added to a solution of Na2003 in H20 (20 mL) and the
solution
was cooled in ice bath. Next, the tetramethyl (1-{[(4-
nitrophenoxy)carbonyl]oxy}ethane-1,1-
diy1)bis(phosphonate) dissolved in THF (20 mL ) was added dropwisely over 30
min. The ice bath
was removed, the flask was covered with aluminum foil, and the reaction was
stirred for 20 h at
room temperature. Next, the reaction mixture was concentrated, and the crude
purified by column
chromatography using DCM/Me0H (1-5%) to afford 624 mg (29%) of product as off-
white foam.
1H NMR (300 MHz, Chloroform-d) 6 8.78 (s, 1H), 7.81 (d, J = 13.8 Hz, 1H), 4.82
(br s, 1H), 4.16
-4.04 (m, 2H), 4.02 - 3.92 (m, 2H), 3.92 - 3.80 (m, 12H), 3.56 (s, 3H), 3.48
(t, J = 10.5 Hz, 1H),
3.24 (d, J = 10.5 Hz, 1H), 3.00 ( br s, 1H), 2.40 - 2.24 (m, 1H), 1.94 (t, J =
15.9 Hz, 3H), 1.87 -
1.74 (m, 2H), 1.60- 1.44 (m, 2H), 1.35- 1.21 (m, 1H), 1.17 ¨ 1.01 (m, 2H),
0.88 - 0.75 (m, 1H).
31PNMR (121 MHz, CDCI3): 6 20.36. MS-ESI: 690.4 [M+N+
7. Etidronate-carbamate-Moxifloxacin (43)
o OH
0 OH
0
0
1] CH3CN
CcSi-Br ] 0, + , 2] Me0H c-ri 0, A
0.F,F)V
(Me0)20P
(Me0)20Pko wa0
õ OH
b
C28 H38FN3 12P2 C3H3BrSi C24H30FN3012P2
689.56 g/mol 153.09 g/mol 633.45
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(43)
A mixture of tetramethyl etidronate-carbamate-moxifloxacin tetramethyl ester
(764 mg,
1.10 mmol) and bromotrimethylsilane (1.35 g, 8.86 mmol) in ACN (25 mL) was
stirred for 2h. The
volatiles were evaporated under vacuum and Me0H (25 mL) was added to the
residue. After
stirring for 30 mins, the solvent was evaporated, and the residue was
triturated with minimum
volume of DCM for 30min5. The solid was filtered, and dried under high vacuum
to afford 757 mg
of product (quantitative yield). 1H NMR (300 MHz, Methanol-d4) 6 8.98 (s, 1H),
7.79 (d, J = 14.5
Hz, 1H), 4.39 - 4.25 (m, 1H), 4.24 - 4.07 (m, 2H), 4.01 (t, J = 10.3 Hz, 1H),
3.65 (s, 3H), 3.61 -
3.51 (m, 2H), 3.41 (d, J = 10.7 Hz, 1H), 3.04 (br s, 1H), 2.43 - 2.27 (m, 1H),
1.90 (t, J = 15.2 Hz,
3H), 1.83- 1.71 (m, 2H), 1.55 (q, J = 10.8 Hz, 2H), 1.43- 1.32 (m, 1H), 1.30-
1.18 (m, 1H), 1.17
- 1.03 (m, 1H), 1.01 - 0.83 (m, 1H). 31PNMR (121 MHz, Methanol-d4): 6 16.60.
MS-ESI: 634.2
[M+1-1]+.
Example 7.
The following is a general structure of BP-quinolone as can be described in
one or more
aspects herein.
/quinolone
X211
HO, /OH
HO -P -OH
R \\(:) conjugates between alpha-X containing BP and
quinolone
0
X= 0, NH, NRI, S
R1 can be alkyl or substituted alkyl, aryl or subsituted aryl groups
wherein R can be H, substituted and unsubstituted alkyl, alkyl amino, alkyl-
aryl, aryl,
alkylheteroaryl, or heteroaryl.
Example 8.
The following are non-limiting examples of BP-quinolone conjugates as
described in one
or more aspects herein.
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OH
F 'I OH
' P - HO OH
'
HO P =0
:L.,,,FNi . ?[ IC,i NH 4
O* OH ---,;): 0 ;p( 0
HO II 0 --IL Nr
F
0 OH 0 N
' - 0 I
H N N
N ,1\1 4 ,Ai
I
HO
HO lry-- F F
00XI
00
Eti-Alatrofloxacin-1 Eti-Amifloxacin
HO OH
HO \ 11=0 HO
F \ ,OH
HO -,µI) ---X 0 0 :-.-_p
0 0 -4
H 1 0 0 ,k/
OH
0
N.TAN )-LNH2 OH
y 0- P
0 il '
F H N aN ,4
0
NI- - --': I I
HO
HO Irmr- F F:
00
0 0 Eti-Alatrofloxacin-2 Eti-Balofloxacin
OH
HO, /, 0
F HO\ \ ,p - o---0 OH
0
/-----\ 0 HO -P II 0 N,--'/
HO , 0 N 0 0 " 0
HO-
HO ID = ?
CI N /
OH 7 0
0
-p 0 i_i H N F
N
/ sb I
:
HO HO
F
Eti-Besifloxacin Eti-Cadazolid-2
0 0
OH
0 0, /
7 OH is P -OH
-0
0
N 0 ID -OH
HO / NO- -- \ 0 0 *OH
F
0 0 F Eti-Cadazolid-1
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OH
0 - 1121 -OH OH
0 , 1
=p -OH
HO
, N CI H p,<___
7 , ,,,, s_
OH
0.:' OH
N rNA0 '
N 0 N
\---- 0 0 OH
I HO F 0
H2N F
0 0 Eti-Ciprofloxacin Eti-Clinafloxacin-1
F
NH2 ).y. NH 2 OH
7 ci
1 N 0 - I -OH
- P
N 6 F CI
1----r01r
HO p Co
1
Ire J_Is -OH HO F
0 0 HN
T OH II II
0 0
0 ,..,,P 'OH
ti bH
Eti-Clinafloxacin-2 Eti-Delafloxacin-1
0, PH
-OH OH
0 ----\, .0H I n 0- - H
''13
-OH
CI OH
P-4:0H
F
1 IV r___õ N N N
, '1 rNo c; :OH
HO '--
N N -../ .-- -...-- ...-;---
1 I
I HO .1.r.i.r.E
F 0 0
00
Eti-Enoxacin
Eti-Delafloxacin-2 OH
HO 0 s /
\ ,OH
µP -OH
0 0 Oz-.p 0 (
F,P -OH
7 0- r N 0 0 ' bH
HO AU.J1
I
N N
N N
HRLN 0 o
çiti A I I HO
F
_____________________ N I-I'. 0 i
0 0 NH2 Eti-Gatifloxacin-1
Eti-Finafloxacin
87
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y 0- (NH
HO
/ N,1' 0 s /OH
'P -OH
N N
I F o o------(¨
HO HN --\< ,P-OH
/ N
0 1 N 0 0 OH
0 0 HN ..fr-0j;)
-P -OH N -0
0 - OH \
Eti-Gatifloxacin-2 Eti-Gemifloxacin
OH
0.1
13-0H OH
0 -OH
--k'
- ='p -OH
p
7 r õ. --,N-0 0 OH
I N HO N
N'CF---OH
N N
HO
F N --k d' 'OH
I 0 0 F H 0
F
0 0 Eti-Grepafloxacin Eti-JNJ-Q2
, os pH 7 0- E-r'_-
OOH
-- N
IXIIX
0 'OH
N ,N ¨ -\----- HO 0
0 \---/ 0 --P -OH
OH F -P
0 0 0 OH
F
Eti-Lomefloxacin Eti-Moxifloxacin
HO P 0, 0/ H
0,, pH
/ N '13 -OH
¨ H P -OH
0
0 N 0-4--
NaCl/O p
0 N\ 5 0 0,, 'OH 0 - -OH
0 ' OH
0
F
Eti-Nemonoxacin - Eti-Nadifloxacin
OH 0 0 F
OH
g./ F
HO 1 0,1
/----\ 0 P - 1 = p
N N --< OH -OH ------- N y 0--('-
0 \/ 0 ,P-OH N OH
0 0 ' ' X P N __ µ ,f:
F OH 0 0 OH
Eti-Norfloxacin Eti-Orbifloxacin -
88
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OH
0 , I 0H H0,20H 0 0
-13 -
HO, \ ro F
.'r0 0 ----
p -OH .õp 0 1 OH
1
N f\A o' OH
, . HO
HO H 0 __ t. H
I o 6 y N
F H H NA
O0
Eti-Pazufloxacin Eti-Pradofloxacin
OH
0 0 o . IF0 _
- H
F OH
HO 1
I 0 ,1 0
- p - H p> = . i F 0 ----
p OH
N
NO -*Inw HO N CI
HN --4 d' :OH
N .../ ,p, ..... -- / 0
1.1 \\O 0 OH
0 Nv,
0
F Eti-Sarafloxacin F Eti-Sitafloxacin
--,..
(-7P> 0 OH <:( F N
HO , N F OH
/ 0
/----- 0..(....._H -OH N , /
O N N- \' F .13 -
OH
\ 0 (
0 \----! OH 0 0--P, -OH
H2N
HN -.,µ
HO ,P-OH
,F 0
0 0 0 'OH
Eti-Sparfloxacin-1 Eti-Sparfloxacin-2
OH
F 0 ,1
p - -OH
F .
OH F 0 ---(
.0H
TMS N 0,1 HN "0 0
0
/ = p -OH
F
o /---\ 0¨k--
N N 6
N N ---( ,p : OH
0 I .......,
\----'c 0 0' OH HO
F
Eti-Temafloxacin 0 0 Eti-Tosufloxacin
OH
. 010H a a OH
F sp
F -
H ri_e--- OH
- 0k-rp-OH HO 0,1
K.).1.,,,_.õ..,.....õ, F -p-OH
,' 1 1 o ¨(----
OH 0 N N NQN --\K /, =
N N NI- --"Ei
A 0 0 OH
\
r I
HO õ N-0
\
0 0 Eti-Trovafloxacin Eti-Zabofloxacin
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OH
J OH OH HO
'
F P - HO 0
0 0 ----( -OH --µ1D---\ _A /
:Lookil . it it kil 4 õ ,OH P HO II 0 N
0 0 ri\J
F '- -N - - '. 0 I
H N N)
N ,,N1 NI A
I
I HO
F
HO Ir-y-F
00XI
00
MHDP-Alatrofloxacin-1 MHDP-Amifloxacin
HO pH
HO I, =0 HO
F HO ----µ,1 ¨ 0 0 ....,\p
,OH
0 H 0-1 ,
0 0
),..,, .0H
F r---
N.,rANNH2 y 0- 0 r-
OH
N a 4
H N 0
HO 0
IF:
HO lry-F
00
0 0 MHDP-Alatrofloxacin-2 MHDP-Balofloxacin
OH
HO, /, 0
F HO p - o--- 0 OH
0
Z-----\ 0 HO ---( A 0
HO , 0 N__4>0
1,) 0 0
HO-
HO 0 P = o
)' KNCj CI N /
OH 7 r- 0
-p H H N F
N/
/ -0 I
HO HO
F:
MHDP-Besifloxacin 0 0 MHDP-Cadazolid-2
OH
0 0, /
7 OH is 1\17\.....iv
_1(Ø....7sP-OH
¨0
0 \
- OH
HO / 0---NO 0 *OH
F
0 0 F MHDP-Cadazolid-1
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OH
' P OH
0 OH
--( 0
-p -OH
P -
7
.---- i' OH HO / N CI H c)
, p,-- OH
N N 0 N
\--- 0 0 OH
HO 1 0
F: H2N F
0 0 MHDP-Ciprofloxacin MHDP-Clinafloxacin-1
F
NH ).y.N1-12 OH
y cl 1 N c== I OH
'P -
N Nr3 F CI i_____r_0
r:OH
0
HO ' OH
0 1 o
a ig - OH HO
F
0 0 HN ..ir- -1õ-- ,0H
0 0
0 .13 'OH
0. OH
MHDP-Clinafloxacin-2 MHDP-Delafloxacin-1
0, pH
'IP 'OH OH
OH
F a r,- , - OH ' P -
0 0 ---( p OH
i
I r/si -=o d' :OH
,= i__,OH
F N CI N N N
1 1
1 IiThr-i-. F.
HO HO
F-
0 0
00
MHDP-Enoxacin
MHDP-Delafloxacin-2 OH
HO 0, /
\ ,OH ' P -OH
0 0 Ozp 0 ---(
F.- ). ,OH
y 0- Nrsx,--µ0 0'P 'OOH
HO 1 0 P -
I H
0
0 HO N
N N it OH
QLN
A II 1
F
N Fr. 0 i
0 0 NH2 MHDP-Gatifloxacin-1
MHDP-Finafloxacin
91
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0
y 0- rN,_,
HO
/ N,1' 0 , / H
5P-OH
o--(
I o
HN --\< ,P-OH
F.
0 0 01-1
0 0 HN 0
/1 )---I": 0 H \
0 OH F \
-P -OH N -0
\
MHDP-Gatifloxacin-2 N-" ' MHDP-
Gemifloxacin
OH
:131 -OH OH
0 --(ID , OH Y (-- 0 1
- ,-p -OH
7 r.,,,_40 61 'OH
HO I N N NcF
0 ----(p .0H
F N ...-k d' 'OH
I 0 0 F H
HO 0
F
0 0 MHDP-Grepafloxacin MHDP-JNJ-Q2
0
F , pH 7 0- Frp OOH
HO / N i______ ¨\ 0 .....y -OH
N N <HO
,P-OH F -P
0 OH 0 'OH
0 0 0 OH
F
MHDP-Lomefloxacin MHDP-Moxifloxacin
HO P 0, 0/ I-1
0,, pH
i ' p -OH
N
HO /
----rr ,,P -OH Nasq---(
o N\.5 o 0 OH 0 -P -
OH
0 '
OH
0
F
-._
MHDP-Nemonoxacin MHDP-Nadifloxacin
OH 0 0 F
OH
HO / N / HO 1 '
I -OH
----\ 0 P -OH I ' P
N N --< -( N N )=.'µµ 0---( p-OH
0 \_¨/-OH X o o F LN µ
' '
F OH 0''
'OH
0
MHDP-Norfloxacin MHDP-Orbifloxaan
92
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OH
= I OH H0,2F1 0 0
-P "
,õ. HO I" z0 F
.ro o--( -OH
P HO ;11:: --( 0
HO I H
I OH
N , =
N -4 ' OH 0 04 H N N
0 IslJ
H NI I A 0 0
MHDP-Pazufloxacin MHDP-Pradofloxacin
OH
0 0 040H-
F OH
HO 1 I )> = = IF 0 ---(p -OH - I -OH
= P
N N --'i 0 _____( N CI HN -4 s 'OH
0
0 ,p.- OH HO
/
\`0 0OH
0 N5v,
0
F mcH. DJP-
S.i.t.aufloxacin
F MHDP-Sarafloxacin
NH
I F 00H OH
<1 F N
HO /
I\. OOH ..õ...{ -OH N 0, /
0 N N
\--- 0 = 13 -OH
0 0 ' OH
HO HN -i ,P-OH
H2N F 0
0 0 0 bH
MHDP-Sparfloxacin-1 MHDP-Sparfloxacin-2
OH
F 0 , I
p
= -OH
F 41
OH F
O---(p -OH
-4
TMS i N 0 , I
= p -OH HN0 S 'OH
F
0 /-----\ 0-4
p:OH ,,N N Ni
0 \---- 16 s OH I I
HO Irm.r, r
F.
MHDP-Temafloxacin 0 0 MHDP-Tosufloxacin
OH
= I OH OH
F 'P ' 0 0
=I OH
H E N- ....0 ---(),:),-OH
0 OH HO -j*Ljt= F = P -=
0
F 161 NN -..*1',=nCN -C( F:o i
N,N Nr-d'H
H
I I N -0
HO .1.r.1,F
\
0 0 MHDP-Trovafloxacin MHDP-Zabofloxacin
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HO 0
\ HO¨P" OH
/-- 0 OH
0 P --
\ OH y 0- f-Hp HO /
7 r---N----40 N N N)r 0
/OH
I OH
N N)., / \ N
HO I 0 P ,
I -43
Fs
HO 0 0 / 1
F N
O 0 --
...
RIS-Ciprofloxacin RIS-Moxifloxacin
HO
\,.O
0
HO -P " OH
Pi- (:) y 0, 'rip Ho\ ?Fi
0
1
Y OH
r'N ---(3 N ¨\\ N
o OH
N N ,.) c/N HO F I 0
I 1
7N OH
HO 0 0
F VI
O 0 N
ZOL-Ciprofloxacin ZOL-Moxifloxacin
HO 9H
\ /
0
... p OH
OH
- p/_ 0
0 y 0, E-,_'_ Ho\ ro
V OH
,,./
(---N---40 _ N
Y ii )r_04 /OH
N N N / N HO I 0 P
I F ___________________________________________ N I ::
OH
HO 0 0 -.
G
z N
O0 1 ¨
MIN-Ciprofloxacin MIN-Moxifloxacin 2----
OH
HO I , 0
'1=' - PH
/ OH
N co
0t P =0 y 0, p /OH HO \ /
Y
r-.' I
OH
-H >T-0--
N N ,) NH2 HO I 0
P =0
I Fs \
OH
HO 0 0
F H2N
O 0 PAM-Ciprofloxacin PAM-
Moxifloxacin
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OH
HO I , 0
--. p OH
/ H OH
0 P =0 y 0- ,---5,), HO \ /
OH
7 r-N 4 \
OH N N *=:Ei )r 0 /
0 N N ) HO I P
---
I NH2 F 1 --
HO 0 0 OH
F
NH2
0 0 ALN-Ciprofloxacin ALN-Moxifloxacin
0 Y r--- NH Yo
HON 8 N N . n -) N N ,y, NH
HO HO H
\ .,,,
HO -P - cJçXIiX
F ----)--- F
HO ----P ---
I 0 0 0 P =-C) 0 0
HO HO / \
Eti-Ciprofloxacin-V2 OH Eti-Moxifloxacin-V2
V
0 (NH 7 0- rpaiH
HO 8 N N N p HO\
HO --- )____ 0 I
- P - 0 1
F HO )----- F.:
HO ----P --
HO 0 _ID 7:0 0 0
H0\
OH
MHDP-Ciprofloxacin-V2 MHDP-Moxifloxacin-V2
N i H
OH!
0 I 7 r- NH OHNI
HOiP N N 0 I N NH
HOp H
0 =-_-_p 0 F
0 0
HOOH /\ 00
HO OH
RIS-Ciprofloxacin-V2 RIS-Moxifloxacin-V2
OH N ______________________________________________ N _______ Hr_3_
OH /, /
0 / i y
HO --___\\ N -;-' r. NH 01 Ei -n o . N p .. N
N ,..) ,p N N N
HO
I
0 "...p
- P/ \,-, F
/ µ 0 F / i ...,
HO OH 00 HO '
OH 00
ZOL-Ciprofloxacin-V2 ZOL-
Moxifloxacin-V2
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)
n
N / N
Y r---NH N / N 7 0-- r
. NH
N N .
HO
N
OI___
I -H
HO\¨j4__ I 0
F
0:-------P 0 0 ----/
I P -- 0 0 i P , 0 0
HO 0,, \ OH
HO I NOH
HO OH
MIN-Ciprofloxacin-V2 MIN-
Moxifloxacin-V2
H __
=\ ;OH NH2 y
..õ\j- ____________________ (,-NH HO NH2 7
o" 1---3_
HO ----1- N N ,) 4
I
I-1
I I
P 0 '.
F F:
HO" \ HO /
0 ---=P
OH 0 0
HO' OH 0 0
PAM-Ciprofloxacin-V2 PAM-
Moxifloxacin-V2
NH2 H2N
% c\H.,...rj 7
r---NH HO YO rr
HO ---P N N
=-= ::-....7 p N N
.õ, NH
H
0p 0 I
HO/ -4 I
/ \ F
T F
HO OH 0 0 HOOH 0 00 =
ALN-Ciprofloxacin-V2 ALN-
Moxifloxacin-V2
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HO 0
OH
HO P
HO I , 0 HO F'// OH
/
p --,
,,F 0 0
"OH
......08 / OH
0
HO / NJ/ CI HN -4
0
0 1\1\5 0 \--- u NI
V OH 0
I
-.., N 0
F.
RIS-Nemonoxacin RIS-Sitafloxacin
HO\ , 0
HO -P ' OH
HO P HO
\ , 0
HO -P ' OH p....F /
0 =0
I
/ N 0 __ kii 0 p/L,
/ i-P
N CI HN -4 N OH
0 OH u HO
/ -\\..
0 N\.5 0
P
ZOL-Nemonoxacin ZOL-
Sitafloxacin
HO OH
\ /
OH
HO P HO \ / H
_ p OH j2)....F 0 --tl
-t._: I
0 - H P =0 CI HN ---4 OH
N
0 N .,õ..,, 0
II I HO /
OH 0 ¨
0 ¨ Nv N / N
0
N / NJ 0
F (,)/
MIN-Nemonoxacin 0: MIN-
Sitafloxacin
OH
HO I 0
i' OH
OH P ,
/
HO P HO 1 0
\ i= N 0 OH
P / );>. ,,F 0 17=0
/ ..._ kil 0 p,_0
HO / N CI HN --4 OH
0
-1r 1 0
0 N\5 0 OH Nq7, NH2
0
NH2 0
F
PAM-Nemonoxacin PAM-
Sitafloxacin
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OH
HO 1 0
-: OH
OH P ,
/
HO P HO 1 0
-. OH
P /
/ N 0 ¨ 0 ___ pi¨ 0 HO / N CI HN --4 Ps::
0
"tr I 0
0 N\5 0 OH N
0 NH2
0
NH2 F
":.
ALN-Nemonoxacin ALN-Sitafloxacin
NH2 .õF NH2
OH 7 0- rc OH y CI r-5
1 101
>3 N N ..,,,
.0 1
HO I
0 =-P 0 O=p 0 F
/
0 0
HO'OH 00 HO \ H
Eti-Nemonoxacin-V2 Eti-Sitafloxacin-V2
NH2 õF NH2
0 0 OH y
\\, CI
HO 1` /OHNi--5<1
".....p
N ../=.,,,
F
0 -7--P 0
/ = / 0 0
OH 00
HO HO \C)E1
MHDP-Nemonoxacin-V2 MHDP-Sitafloxacin-V2
NH2 õF NH2
N\
Ni
0- OH
0,1 -
.)p N
HO I HO I
0 =---P 0 0 =p 0 F
/ \ / 0 0
OH 00
HO HO \()F1
RIS-Nemonoxacin-V2 RIS-
Sitafloxacin-V2
NH2 õF NH2
OH ,N OH N "
CI
HO -0.4/ cN3 HO -..._õ%p/ N 3 YN NI-
3
N N
0 P 0 F
HO OH 0 0 HO OH 0 0
ZOL-Nemonoxacin-V2 ZOL-Sitafloxacin-V2
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NH2
NH2 N --CD¨ / ____ .,,F
N ----:-- )/ __
HO HO
H i P 1 HO
O
F:
0 / 0
P P -
HO 0 0 HOT 0 0
OH OH
MIN-Nemonoxacin-V2 MIN-
Sitafloxacin-V2
NH2 .õF
NH2
0 7 0- .
(--; 7NI-12 y
õ wOH NH2
HO ¨P N N .., HO
I
0 I 0--
HO V 1 0 0 HO/ \ 0 0
OH OH
PAM-Nemonoxacin-V2 PAM-
Sitafloxacin-V2
NH2 õF
NH2
NH2
\/
NH( 0 rc
0 ;.\..7 y ---1 CI
\\ ,OH
HO ¨P
P 0
HO V 1 0 0 HO / \ 00
OH OH
ALN-Nemonoxacin-V2 ALN-
Sitafloxacin-V2
Example 9:
To exploit BP affinity for bone, a "target and release" chemistry approach was
investigated
involving delivery of antibiotics to bone or hydroxyapatite (HA) via BP
conjugates. Serum-stable
drug-BP linkers were utilized that metabolize and release the parent
antibiotic at the bone surface.
Designed, synthesized, and tested were novel quinolone antibiotic etidronate-
ciprofloxacin (FCC)
conjugate, BV81022, and etidronate-moxifloxacin (ECX) conjugate, BV81051, for
activity against
S. aureus biofilms which are causative in the majority of osteomyelitis cases.
Problems for public health like osteomyelitis caused by bacterial biofilms
have
emphasized the lack of information about biofilm development. Several methods
are available to
study biofilms in vitro or ex vivo, including quantification of sessile
bacteria after detachment from
the surface by scraping, vortexing or sonication, and observations by
microscopy techniques to
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monitor biofilm progression. However, these applications are limited by high
labor intensity,
intrusive sampling and/or long time lags from sampling to obtaining a result.1
Hence, improved
biofilm-monitoring assays are essential for biofilm behavior research and
biomedical applications.
Therefore, sensitive, accurate, reproducible and fast methods are desirable
for real-time
monitoring of biofilms. In this regard, promising advances in impedance
technology based on the
ability of cells to impede an electrical current when adhered to MTP with gold
electrodes have
been developed in recent years.
In the present study, electrode impedance measurements were applied to test
the
therapeutic efficacy and effect on biofilm formation and growth of our novel
ECC and ECX
conjugates in comparison to ciprofloxacin and moxifloxacin against S. aureus.
Real-time
measurements are able to detect whether these biofilms are unaffected,
inhibited, or induced
during antimicrobial therapy. 2
Microbiolgy: For experimental purposes, a robust biofilm forming and well-
studied S.
aureus strain ATCC 6538 was used. The following parent antibiotics were
tested: ciprofloxacin
(C), moxifloxacin (X); the following experimental conjugates were tested:
etidronate-ciprofloxacin
(FCC) and etidronate-moxifloxacin (ECX). Real-time biofilm assays were
performed with an
xCELLigence RTCA SP instrument according to the manufacturer's instructions.4
For monitoring
biofilm formation and RTCA sensitivity assays, 80 pl of TSBYE was added to
each well of non-
reusable 16X microtiter E-plates (ACEA Biosciences) for the impedance
background
measurement using the standard protocol provided by the software. 1 pl of
bacteria suspension
in a total of 120 pl of TSBYE was then added to the 16 E-plate wells. Each
sample was run in
duplicate. E-plates were positioned in the xCELLigence Real-Time Cell Analyzer
MP, incubated
at 37 C and monitored on the RTCA system at 15-min time intervals for 24 h.
Cell-sensor
impedance was expressed as a unit called cell index (Cl) according to the
manufacturer's
instructions. The Cl at each time point is defined as (ZnZb)/15, where Zn is
the cell-electrode
impedance of the well when it contains cells and Zb is the background
impedance with growth
media alone. Standard deviations of duplicates or triplicates of wells were
analyzed with the RTCA
Software
Affinity of antibiotics to HA. 1 pg/mL of each compound was added to a
solution containing
10 pg/mL of HA powder and incubated for 4h/37 C under magnetic stirring. Next,
HA powder was
allowed to sediment for 1h/4 C. After this time, the content of antibiotic in
the supernatant was
assessed using HPLC (Shimadzu Prominence). To evaluate quantity of conjugates
bound to HA,
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we used methodology described in more detail in our previous work.3Affinity of
compounds to HA
powder was estimated as follows: 100% - peak area of tested compound detected
/ peak area of
control sample *100%.
Infection preventative experiment. 80-pl of TSBYE was added to each well to
measure
background impedance. 1 pl of bacteria suspension in a total of 120 pl of
TSBYE containing a
variety of concentrations toward antibiotics was then added to the 16 E-plate
wells. Two replicates
of each antibiotic concentration and negative controls without antibiotic were
also included. Cells
were monitored for 24 h and analysis was done to calculate MICH) values.
Infection preventative experiment with HA. Different concentrations of each
compound
were added to a solution containing 10 pg/mL of HA powder and incubated for
2h/37 C under
magnetic stirring. Then 80-pl of TSBYE was added to each well to measure
background
impedance. 1 pl of bacteria suspension in a total of 120 pl of TSBYE
containing a gradient of
concentrations toward antibiotics plus HA was then added to the 16 E-plate
wells. Two replicates
of each antibiotic concentration and negative controls without antibiotic were
also included. Cells
were monitored for 24 hrs and analysis was performed to calculate M IC50
values.
Results: HPLC results evaluating the binding affinity of tested compounds to
HA indicate
that the conjugates had high binding affinity and retention to HA in
comparison to the
unconjugated antibiotics. Electrode impedance measurements were applied to
test the
therapeutic efficacy and effect on biofilm formation and growth of the novel
ECC and
ECX conjugates in comparison to ciprofloxacin and moxifloxacin against S.
aureus. Real-time
measurements were able to detect whether these biofilms are unaffected,
inhibited, or induced
during antimicrobial therapy (see, Figs. 26, 28, 30, 32).
In the infection preventative experiment using real time monitoring MIC50 has
been
calculated for each conjugate and parent antibiotic as shown in FIGs. 27 and
29. The S. aureus
.. MIC50 for ciprofloxacin, moxifloxacin, ECC, and ECX are: 0.09, 0.11, 4.88,
and 5.10 pg/ml
respectively.
In the infection preventative experiment in the presence of HA using real time
monitoring
MIC50 has been calculated for each conjugate and parent antibiotic as shown in
FIGs 31 and 33.
The S. aureus MIC50 for ciprofloxacin, moxifloxacin, ECC, and ECX are: 0.24,
0.09, 9.60, and 28
pg/ml respectively.
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Conclusion: Thus, by using impedance measurements in microtiter plates with
gold electrodes the antibiotic effect on S. aureus bacterial biofilm growth
were assessed in real
time. Real-time biofilm analysis allowed detection of decreases or increases
in microbial
mass over time during antimicrobial therapy, which can be used to evaluate
antibiotic
susceptibility and efficacy in biofilm-mediated infections clinically. The
novel etidronate-
fluoroquinolone conjugates designed and tested in this study (ECC, ECX)
retained the bone
binding properties of the parent BP drug, and also the antimicrobial activity
of the parent antibiotic
in the presence or absence of HA albeit at lower levels due to the nature of
the
chemical modification and possible partial cleavage at the tested conditions.
This class of
conjugates using BP drugs as biochemical vectors for the delivery of
antibiotic agents to bone
(where osteomyelitis biofilm pathogens reside) represents an advantageous
approach to the
treatment of osteomyelitis by providing improved bone pharmacokinetics while
minimizing
systemic exposure (toxicity) of these drugs.
References for Example 9:
1) Coenye T, Nelis H.J. In vitro and in vivo model systems to study microbial
biofilm
formation. J Microbiol Methods 2010; 83, 89-105.
2) Saginur R, Stdenis M, Ferris W, Aaron, S.D, Chan F, Lee C, Ramotar K.
Multiple
combination bactericidal testing of staphylococcal biofilms from implant-
associated
infections. Antimicrob Agents Chemother 2006; 50, 55-61.
3) Sedghizadeh PP and Ebetino FH et al. Design, synthesis, and antimicrobial
evaluation of
a novel bone-targeting bisphosphonate-ciprofloxacin conjugate for the
treatment of
osteomyelitis biofilms. J Med Chem 2017; 60, 2326-43.
4) Atienza J.M, Zhu J, Wang X, Xu X, Abassi Y. Dynamic monitoring of cell
adhesion and
spreading on microelectronic sensor arrays. J Biomol Screen 2005; 10, 795-805.
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