CN112672787A

CN112672787A - Bisphosphonic acid quinolone conjugates and uses thereof

Info

Publication number: CN112672787A
Application number: CN201980058610.8A
Authority: CN
Inventors: 弗兰克·H·埃贝蒂诺; 舒廷·孙; 菲利普·T·谢里安
Original assignee: Biovinc LLC
Current assignee: Biovinc LLC
Priority date: 2018-07-09
Filing date: 2019-07-09
Publication date: 2021-04-16
Also published as: JP2021532087A; CA3105829A1; EP3820565A4; WO2020014269A9; WO2020014269A1; EP3820565A1

Abstract

Bisphosphonic quinolone compounds, conjugates, and pharmaceutical formulations thereof are described that may include a Bisphosphonate (BP) and a quinolone, wherein the BP has an alpha substituent and the alpha substituent is a hydroxyl, amino, or thiol group, and wherein the quinolone is directly or indirectly conjugated to the BP at a geminal carbon alpha substituent (X) of the BP. In one or more embodiments, the BP can be an α -OH-containing BP and the quinolone can be reversibly conjugated to the BP directly or indirectly at a geminal OH of the BP. Also provided herein are methods of making and using the bisphosphonate quinolone conjugates and pharmaceutical formulations thereof.

Description

Bisphosphonic acid quinolone conjugates and uses thereof

Cross Reference to Related Applications

The benefit AND priority of co-pending U.S. provisional patent application No. 62/695,583 entitled "bisphosphonic QUINOLONE CONJUGATES AND USES THEREOF," filed on 2018, 7, 9, AND incorporated herein by reference in its entirety.

Statement regarding federally sponsored research or development

The invention is made with government support under grant numbers R41DE025789-01 and grant number R42DE025789-02 issued by NIH/NIDCR and grant number R43AR073727 issued by NIH/NIAMS. The U.S. government has certain rights in this invention.

Background

Bone and joint infections affect millions of adults and children worldwide. The overall incidence in the united states is 3-6 million people, with a particular population having different risks. For diabetic patients, the annual incidence of foot ulcers is about 1 in 30, up to two-thirds of cases with underlying osteomyelitis. In children, the annual incidence has recently been reported in the range of 1/4000 to 1/15000. However, in the Pediatric Health Information System (PHIS) database of the management data of the american pediatric hospital, we found that during the 5 years 2009-2013, 10,245 (0.5%) of 2,247,889 was diagnosed as osteomyelitis at the time of discharge and the gross annual incidence was about 1/1100 hospitalization.

Many gram-positive and gram-negative bacteria as well as fungi and mycobacteria can cause bone and joint infections. To date, the most common organism implicated in bone and joint infections is Staphylococcus aureus (s.aureus), both methicillin-sensitive (MSSA) and methicillin-resistant (MRSA).

The standard of care for bone and joint infections generally requires systemic administration of antibiotics. For acute infections, the prescription is usually: the antibiotic is injected intravenously for 2-6 weeks. For chronic infections or infections associated with retained implanted hardware, a prolonged course of oral antibiotics may be followed. For both acute and chronic infections, these extended courses of treatment may lead to drug-related adverse events in a significant percentage of patients — 15% in one estimate of the cohort for treatment of infected MSSA. Furthermore, it is known that nephrotoxicity of vancomycin, the most common treatment of MRSA infections, occurs in up to 43% of patients and increases with the duration of treatment.

Persistent bone infections such as osteomyelitis of the jaw bone, osteomyelitis and osteonecrosis in other skeletal sites eventually lead to significant bone resorption and destruction of bone and Hydroxyapatite (HA) minerals. Bone and HA resorption is thought to be induced and mediated not only by osteocytes (i.e., osteoclasts), but also by microbial biofilm pathogens in combination with host inflammatory responses and osteoclastogenesis activity. Biofilms are complex microbial communities composed of one or more bacterial species attached to a matrix and surrounded by a self-produced extracellular matrix. Many different types of microbial infections are known to be caused by organisms that grow in a biofilm state. Bacterial biofilms of Staphylococcus aureus (s. aureus) are the leading cause of biofilm-related 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, usually intravenous and long-term antibiotic, are the mainstays of osteomyelitis management. Surgery may include conservative removal of the affected bone or a more aggressive approach, such as resection. Thus, the treatment of infectious bone diseases is primarily based on clinical pathology with or without surgical intervention for antimicrobial therapy. However, antibiotics have poor bone resorption and pharmacokinetics in vivo. Thus, any improvement in the bone bioavailability of a therapeutic antibiotic would be a significant advance in the treatment of osteomyelitis.

To overcome many of the challenges associated with treating bone infections, it has become routine for clinicians to use local delivery systems to achieve higher therapeutic antibacterial concentrations in bone. For example, polymethylmethacrylate beads represent the most non-biodegradable carrier systems for delivering antibiotics to orthopedic infections, but they require surgical removal after drug release is complete. They also tend to release the antibiotic in an initial burst mode that quickly consumes the majority of the drug from the carrier beads, followed by a slow release at a lower concentration that may not be sufficient to control the infection and may promote the development of drug resistance. These concerns limit the usefulness of this approach in most bone and joint infections.

Dentistry has used local delivery of antimicrobial agents to treat infected jaw bone associated with conditions such as periodontal bone loss, jaw osteomyelitis and osteonecrosis to achieve high local concentrations of the drug, but without surgical intervention, these approaches are generally ineffective and the bone bioavailability of antibiotics is poor. Antibiotic impregnated cementum, which is used primarily when debriding infected implants for the first time to improve control of infection, is not generally used to treat bone and joint infections of natural bone without implanted hardware. Concerns regarding prolonged sub-therapeutic antibiotic concentrations and selection of drug-resistant bacteria also apply to cementum.

Local delivery of antimicrobial agents to bone can be an important advance in the treatment of infectious bone diseases, but still has osmotic limitations and potential eukaryotic cellular cytotoxicity; therefore, there is a high need to research and develop more efficient delivery systems with physiological targeting. The ideal antibiotic delivery system is one that targets bone tissue without the need for surgical implantation or removal. This targeting also minimizes systemic dose and exposure of tissues other than bone to antibiotics, thus reducing the risk of selective stress that adversely affects or promotes the emergence of resistant bacteria. Another potential major benefit is that by achieving prolonged antibiotic concentrations at the site of infection, it is possible to reduce the frequency of administration.

The lack of efficacy of current antimicrobial treatments for osteomyelitis has been attributed to the restricted access of antibiotics to sites where pathogenic bacteria (including biofilms such as on the surface of bone, even within the network of bone tubules) can reside systemically, a class of drugs known as bisphosphonic acids (BPs) are readily available.

Disclosure of Invention

BP quinolone antibiotic compounds, conjugates, and formulations thereof and various methods of use are provided herein in various aspects to address the above stated needs.

In any one or more aspects herein, to exploit the affinity of BP for bone, a "targeting and release" chemistry approach is provided that involves delivery of quinolone antibiotics to the bone or Hydroxyapatite (HA) surface by BP conjugates, particularly to sites where bone infection HAs been initiated and elevated bone metabolism HAs occurred. A relatively serum-stable drug-BP linker that is most preferentially metabolized at the bone surface and releases the parent quinolone antibiotic can be utilized.

The BP quinolone compounds, conjugates, and formulations may comprise a Bisphosphonate (BP) that may 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 substituent is a hydroxyl group, an amino group, or a thiol group. The quinolone may be conjugated directly or indirectly to the BP at the geminal carbon alpha substituent of the BP, as described in any one or more aspects herein. In any one or more embodiments, the quinolone is reversibly coupled or conjugated to a geminal hydroxyl, amino, or thiol group on a carbon between two phosphonic acid groups of the BP. When conjugated in this manner, two phosphonic acid groups act to weaken the attachment of the quinolone to the BP, and when the quinolone is released from the BP, the BP activates a linker that reversibly couples or conjugates the quinolone to the BP. In a particular embodiment, the BP may be etidronic acid, methylenehydroxy bisphosphonic acid (MHBP) or pamidronic acid, preferably etidronic acid or MHBP. In particular embodiments, the BP may be an inactive or low active BP, as described herein.

In any one or more embodiments, the BP quinolone compound or conjugate may be administered systemically to a subject to selectively deliver the quinolone to bone, and particularly to an infected bone site, or locally to a subject when combined with a bone graft or bone graft substitute (i.e., may target bone, bone infection, or other high bone metabolic site). In any one or more embodiments, the BP quinolone compound or conjugate may release the quinolone, particularly a quinolone compound, a substituent, or a 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 include a Bisphosphonate (BP) that can be releasably conjugated to a quinolone. In embodiments, the BP quinolone compound or conjugate may selectively deliver the quinolone to bone, bone graft, and or bone graft substitute of a subject, particularly to higher bone metabolic sites where bone infection has been triggered. In any one or more embodiments, the BP quinolone compound or conjugate may 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 certain embodiments herein, the BP is etidronic acid conjugated to a fluoroquinolone antibiotic, such as ciprofloxacin, moxifloxacin, or sitafloxacin. In embodiments, the BP is etidronic acid conjugated to a non-fluoroquinolone, such as neruoxacin. For example, the conjugate may be a compound according to formula (41), formula (43), formula (44) or formula (45).

Also provided herein are pharmaceutical compositions or formulations comprising 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, which may include the steps of: administering to a subject in need thereof an amount of a compound according to formula (41), formula (43), formula (44), and/or formula (45), or a pharmaceutical formulation comprising a compound according to formula (41), formula (43), formula (44), and/or formula (45).

Also provided herein are compounds, conjugates, and antimicrobial and antibiotic agents comprising a Bisphosphonate (BP) and a quinolone compound, wherein the quinolone compound is releasably or reversibly coupled to the bisphosphonate through a linker, as described herein. As described herein, preferred releasable linkers are more or less stable in the bloodstream shortly after administration and more or less slowly lyse in the bone/skeletal zones of the body to slowly release locally the quinolone antibiotic compound, substituent or derivative.

In any one or more aspects, the BP quinolone compound may consist of a quinolone antibiotic analog or substituent according to structure or formula (a),

wherein R is¹Can be

And wherein R²Can be

And wherein R³Can be H or OCH3 and can be,

and wherein R⁴It may be a compound of formula (I) and (II),

and wherein R⁵May be H or F.

As shown, the quinolone of formula (a) may be linked to a Bisphosphonate (BP). In any one or more aspects or embodiments herein, there is provided a compound or conjugate comprising a Bisphosphonate (BP) and a quinolone compound or analog, wherein the BP may have an alpha substituent, and the alpha substituent may be a hydroxyl, amino, or thiol group. The quinolone may be conjugated directly or indirectly to the BP at the geminal carbon alpha substituent (X) of the BP, as illustrated by the following formula.

Conjugates between BP containing a-X and quinolones

X＝0、NH、NR¹、S

R¹May be an alkyl or substituted alkyl, aryl or substituted aryl group

Wherein R can be H, substituted and unsubstituted alkyl, alkylamino, alkyl-aryl, alkylheteroaryl, or heteroaryl.

Preferred BPs are those having a geminal hydroxyl group on the carbon between the two phosphonic acid groups. A generic analog of such BP is illustrated in fig. 25. The BP is 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 may be an ethylidene bisphosphonate (etidronic acid) moiety that may be substituted with a hydroxyl group (α -hydroxyl), an amino group, or a thiol. In some aspects, the bisphosphonic acid may include a p-hydroxyphenyl ethylidene group or a derivative thereof. In embodiments, the BP can be a clinically known BP, such as pamidronic acid, alendronic acid, risedronic acid, zoledronic acid, minodronic acid, neridronic acid, and etidronic acid, which can be unmodified or modified as described herein.

In any one or more preferred embodiments, the BP can be etidronic acid. Etidronic acid can be linked to a quinolone to form a quinolone antibiotic etidronic acid-ciprofloxacin (ECC) conjugate, such as in formula (41), or to form an etidronic acid moxifloxacin (ECX) conjugate, such as in formula (43) herein.

Said linker (L) may be a cleavable compound, meaning that it reversibly couples a quinolone analogue or compound, in particular a quinolone antimicrobial or antibiotic analogue or a substituent thereof, to said BP. As used herein, the term "cleavable" may mean a group that is chemically or biochemically labile under physiological conditions. In any one or more aspects, the linker can be a carbamate having the structure or formula (B)

For the preparation of quinolones, R²With BP, R¹Is coupled, as described herein, and R³Can 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 the structure or formula (C)

For the preparation of quinolones, R²With BP, R¹Coupling, as described herein.

In any one or more aspects of any one or more embodiments herein, the linker can be an alkyl carbamate or aryl carbamate linker. The linker may be a thiocarbamic-O-aryl ester or a thiocarbamic alkyl ester linker. The linker may be a thiourethane-S-aryl ester or a thiourethane alkyl ester linker. The linker may be a phenyl carbamate linker. The linker may be a thiocarbamate linker. The linker may be an O-thiocarbamate linker. The linker may be an S-thiocarbamate linker. The linker may be an ester linker. The linker may be a dithiocarbamate. The linker may be a urea linker. The linker may be R of formula (A) together with BP¹A moiety, and coupling said BP to said quinolone, as described herein. At any rateIn one or more aspects, the linker can be exemplified by any one of the following formulas (D) to (H), wherein: r²May be a quinolone or quinolone substituent or derivative and R¹May be BP, both as described herein; and R is³Can be substituted and unsubstituted alkyl, acetyl, benzoyl or other amides, phenyl and substituted phenyl, preferably H.

In some aspects, the BP is etidronic acid. In some aspects, the quinolone is ciprofloxacin or moxifloxacin. In some aspects, the BP is etidronic acid, the quinolone is ciprofloxacin, and the linker is an alkyl or aryl carbamate or a linker of formula (F), providing a compound of formula (41). In some aspects, the BP is etidronic acid, the quinolone is moxifloxacin, and the linker is an alkyl or aryl carbamate or a linker of formula (F), providing a compound of formula (43). In some aspects, the BP is etidronic acid, the quinolone is sitafloxacin or nerofloxacin, and the linker is an alkyl or aryl carbamate or a linker of formula (F), providing a compound of formula (44) or formula (45) below.

In other aspects, the BP can be other BPs described herein, such as pamidronic acid, neridronic acid, olpadronic acid, alendronic acid, ibandronic acid, minodronic acid, risedronic acid, zoledronic acid, hydroxymethylbisphosphonic acid, and combinations thereof.

Also provided herein are pharmaceutical formulations that can comprise a Bisphosphonate (BP) and a quinolone compound of formula (a) releasably coupled to a bisphosphonate through a linker (L), and a pharmaceutically acceptable carrier. In any one or more aspects, the Bisphosphonate (BP) and linker (L) may be as described herein.

In any one or more aspects of any one or more embodiments herein, there are also provided compounds and conjugates comprising a Bisphosphonate (BP) and a quinolone compound, wherein the quinolone compound is releasably coupled to the bisphosphonate through a linker. The BP may be selected from the group of: hydroxyphenylalkyl or aryl bisphosphonic acids, hydroxyphenyl (or aryl) alkylhydroxy bisphosphonic acids, aminophenyl (or aryl) alkylbisphosphonic acids, aminophenyl (or aryl) alkylhydroxy bisphosphonic acids, hydroxyalkyl hydroxybisphosphonic acids, hydroxyalkylphenyl (or aryl) alkylphosphonic acids, hydroxyphenyl (or aryl) alkylhydroxy bisphosphonic acids, aminophenyl (or aryl) alkylphosphonic acids, aminophenyl (or aryl) alkylhydroxy bisphosphonic acids, hydroxyalkyl hydroxybisphosphonic acids, hydroxypyridinylalkyl bisphosphonic acids, pyridylalkyl bisphosphonic acids, hydroxyimidazolylalkyl bisphosphonic acids, imidazolylalkyl bisphosphonic acids, etidronic acid, pamidronic acid, neridronic acid, olpadronic acid, alendronic acid, ibandronic acid, risedronic acid, zoledronic acid, minodronic acid, hydroxymethylenediphosphonic acid, and combinations thereof, wherein all compounds may be optionally further substituted or unsubstituted.

In any one or more aspects of any one or more embodiments herein, the quinolone compound may be a fluoroquinolone or a non-fluoroquinolone. The quinolone compound may be selected from the group consisting of: alafloxacin, amifloxacin, balofloxacin, besifloxacin (besifloxacin), cadazolide, ciprofloxacin, clinafloxacin, danofloxacin, delafloxacin (delafloxacin), difloxacin, enoxacin, enrofloxacin, finafloxacin, fleroxacin (flerofloxacin), flumequine, gatifloxacin, gemifloxacin, ebafloxacin, JNJ-Q2, levofloxacin, lomefloxacin, marbofloxacin, moxifloxacin, nadifloxacin, norfloxacin, ofloxacin, orbifloxacin, pazufloxacin, pefloxacin, prarofloxacin, fleroxacin, sarafloxacin, sitafloxacin, sparfloxacin, temafloxacin, tosufloxacin, trovafloxacin (travafloxacin), zabofloxacin, norfloxacin and combinations thereof.

In some aspects, the BP is etidronic acid. In some aspects, the quinolone is ciprofloxacin or moxifloxacin. In other aspects, the BP can be other BPs described herein, such as pamidronic acid, neridronic acid, olpadronic acid, alendronic acid, ibandronic acid, minodronic acid, risedronic acid, zoledronic acid, hydroxymethylbisphosphonic acid, and combinations thereof.

The quinolone compound may have a structure according to formula (a),

wherein R is¹May be a substituent, which includes: alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, phenyl, substituted phenyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, halogen, hydroxy, alkoxy, substituted alkoxy, phenoxy, substituted phenoxy, aryloxy, substituted aryloxy, 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-C₂₀Cyclic, substituted C₃-C₂₀Rings, heterocycles, substituted heterocycles, amino acids, peptides and polypeptide groups,

wherein R is²May be a substituent, which includes: alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, phenyl, substituted phenyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, halogen, hydroxyA group, alkoxy, substituted alkoxy, phenoxy, substituted phenoxy, aryloxy, substituted aryloxy, 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-C₂₀Cyclic, substituted C₃-C₂₀Rings, heterocycles, substituted heterocycles, amino acids, peptides and polypeptide groups,

wherein R is³May be a substituent, which includes: alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, phenyl, substituted phenyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, halogen, hydroxy, alkoxy, substituted alkoxy, phenoxy, substituted phenoxy, aryloxy, substituted aryloxy, 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-C₂₀Cyclic, substituted C₃-C₂₀Rings, heterocycles, substituted heterocycles, amino acids, peptides and polypeptide groups,

wherein R is⁴May be a substituent, which includes: alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, phenyl, substituted phenyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, halogen, hydroxy, alkoxy, substituted alkoxy, phenoxy, substituted phenoxy, aryloxy, substituted aryloxy, alkylthio, substituted alkylthio, phenylthio, substituted phenylthio, arylthio, substituted arylthio, cyano, isocyano, substituted isocyanoCyano, 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-C₂₀Cyclic, substituted C₃-C₂₀A ring, a heterocycle, a substituted heterocycle, an amino acid, a peptide and a polypeptide group, and

and wherein R⁵May 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 may be attached to R of formula (A)¹The groups are attached.

In any one or more aspects or embodiments herein, the BP can have an alpha substituent, and the alpha substituent is a hydroxyl, amino, or thiol group. The quinolone may be conjugated directly or indirectly to the BP at the geminal carbon alpha substituent (X) of the BP, as illustrated by the following formula.

Conjugates between BP containing alpha-X and quinolones

X＝0、NH、NR¹、S

R¹May be an alkyl or substituted alkyl, aryl or substituted aryl group

Wherein R can be H, substituted and unsubstituted alkyl, alkylamino, alkyl-aryl, alkylheteroaryl, or heteroaryl. Preferred BPs are those having a geminal hydroxyl group on the carbon between the two phosphonic acid groups.

In any one or more aspects or embodiments herein, the bisphosphonate may be an ethylidene bisphosphonate moiety (etidronic acid) that may be substituted with a hydroxyl (α -hydroxyl), amino, or thiol. In some aspects, the bisphosphonic acid may include a p-hydroxyphenyl ethylidene group or a derivative thereof. In embodiments, the BP can be a clinically known BP, such as pamidronic acid, alendronic acid, risedronic acid, zoledronic acid, minodronic acid, neridronic acid, and etidronic acid, 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).

Also provided herein are pharmaceutical formulations that can include a bisphosphonate and a quinolone compound releasably coupled to the bisphosphonate through a linker, and a pharmaceutically acceptable carrier. The bisphosphonic acid may be selected from the group of: hydroxyphenylalkyl or aryl bisphosphonic acids, hydroxyphenyl (or aryl) alkylhydroxy bisphosphonic acids, aminophenyl (or aryl) alkylbisphosphonic acids, aminophenyl (or aryl) alkylhydroxy bisphosphonic acids, hydroxyalkyl hydroxybisphosphonic acids, hydroxyalkylphenyl (or aryl) alkylphosphonic acids, hydroxyphenyl (or aryl) alkylhydroxy bisphosphonic acids, aminophenyl (or aryl) alkylphosphonic acids, aminophenyl (or aryl) alkylhydroxy bisphosphonic acids, hydroxyalkyl hydroxybisphosphonic acids, hydroxypyridinylalkyl bisphosphonic acids, pyridylalkyl bisphosphonic acids, hydroxyimidazolylalkyl bisphosphonic acids, imidazolylalkyl bisphosphonic acids, etidronic acid, pamidronic acid, neridronic acid, olpadronic acid, alendronic acid, ibandronic acid, risedronic acid, zoledronic acid, minodronic acid, hydroxymethylenediphosphonic acid, and combinations thereof, wherein all compounds may be optionally further substituted or unsubstituted.

The quinolone compound may be a fluoroquinolone or a non-fluoroquinolone. The quinolone compound may be selected from the group consisting of: alafloxacin, aminofloxacin, balofloxacin, besifloxacin, cadazolide, ciprofloxacin, clinafloxacin, danofloxacin, delafloxacin, difloxacin, enoxacin, enrofloxacin, finafloxacin, fleroxacin, flumequine, gatifloxacin, gemifloxacin, grepafloxacin, ebafloxacin, JNJ-Q2, levofloxacin, lomefloxacin, marbofloxacin, moxifloxacin, nadifloxacin, norfloxacin, ofloxacin, orbifloxacin, pazufloxacin, pefloxacin, prafloxacin, prarofloxacin, rufloxacin, sarofloxacin, sitafloxacin, sparfloxacin, temafloxacin, tosufloxacin, trovafloxacin, zabofloxacin, nemonoxacin and combinations thereof.

The quinolone compound may have a structure according to formula (a),

wherein R is²May be a substituent, which includes: alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, phenyl, substituted phenyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, halogen, hydroxy, alkoxy, substituted alkoxy, phenoxy, substituted phenoxy, aryloxy, substituted aryloxy, alkylthio, substituted alkylthioPhenylthio, 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-C₂₀Cyclic, substituted C₃-C₂₀Rings, heterocycles, substituted heterocycles, amino acids, peptides and polypeptide groups,

wherein R is⁴May be a substituent, which includes: alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, phenyl, substituted phenyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, halogen, hydroxy, alkoxy, substituted alkoxy, phenoxy, substituted phenoxy, aryloxy, substituted aryloxy, 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 aryl, substituted heteroaryl, halogen, hydroxy, alkoxy, substituted alkoxy, phenoxy, substituted phenoxy, aryloxy, substituted aryloxy, alkylthio, substituted alkylthio, phenylthio, substituted phenylthio, arylthio, substituted arylthio, cyano, isocyano, substituted isocyano, carbonyl, substituted carbonyl, carboxyl, substituted carboxyl, amino,Substituted sulfonyl, sulfonic acid, phosphoryl, substituted phosphoryl, phosphonyl, substituted phosphonyl, polyaryl, substituted polyaryl, C₃-C₂₀Cyclic, substituted C₃-C₂₀A ring, a heterocycle, a substituted heterocycle, an amino acid, a peptide and a polypeptide group, and

and wherein R⁵May be H or F.

In any one or more aspects, the BP can have an alpha substituent, and the alpha substituent is a hydroxyl, amino, or thiol group. The quinolone may be conjugated directly or indirectly to the BP at the geminal carbon alpha substituent (X) of the BP, as illustrated by the following formula.

Conjugates between BP containing a-X and quinolones

X＝0、NH、NR¹、S

R¹May be an alkyl or substituted alkyl, aryl or substituted aryl group

In any one or more aspects, the bisphosphonate may be an ethylidene bisphosphonate moiety (etidronic acid) that may be substituted with a hydroxyl group (α -hydroxyl), an amino group, or a thiol. In some aspects, the bisphosphonic acid may include a p-hydroxyphenyl ethylidene group or a derivative thereof. In embodiments, the BP can be a clinically known BP, such as pamidronic acid, alendronic acid, risedronic acid, zoledronic acid, minodronic acid, neridronic acid, and etidronic acid, which can be unmodified or modified as described herein.

In some aspects, the formulation comprises a compound having 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 may be an amount effective to kill or inhibit bacteria. The amount of the compound or conjugate in the pharmaceutical formulation may be an amount effective to treat, inhibit or prevent bone disease. The amount of the compound or conjugate in the pharmaceutical formulation may 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 may be an effective amount for prophylactic treatment of any of the foregoing.

Further, in one or more embodiments, there is provided a method of preparing a bisphosphonate-quinolone compound, conjugate, or formulation thereof, the method comprising linking a bisphosphonate with a quinolone compound or substituent, as described in any one or more aspects herein. Also provided are methods of using any one or more bisphosphonate-quinolone compounds or conjugates in the manufacture of a medicament or medicament for treating 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, which may comprise the steps of: administering to said subject in need thereof an amount, in particular an effective amount, of a compound as provided herein or a pharmaceutical formulation thereof. Also provided herein are methods of treating bone diseases, such as hematogenous or local osteomyelitis including juvenile osteomyelitis and infections associated with prosthetic joint replacement or osteonecrosis, in a subject in need thereof, which may comprise the steps of: administering to said subject in need thereof an amount, in particular an effective amount, of a compound as provided herein or a pharmaceutical formulation thereof.

Also provided herein are methods of treating peri-implantitis or periodontitis in a subject in need thereof, comprising administering to the subject in need thereof an amount, particularly an effective amount, of a compound as provided herein or a pharmaceutical formulation thereof.

Also provided herein are methods of treating diabetic foot disease in a subject in need thereof, comprising administering to the subject in need thereof an amount, particularly an effective amount, of a compound as provided herein or a pharmaceutical formulation thereof. Also provided herein are methods of treating bone infections (including diabetic foot disease) in a diabetic patient in a subject in need thereof, comprising administering to the subject in need thereof an amount, particularly an effective amount, of a compound as provided herein or a pharmaceutical formulation thereof. The associated reduction in amputation, debridement of limbs and infected bone sites can be caused by these more powerful local antibiotic treatment modalities.

Also provided herein are bone graft compositions that can include a bone graft material and a compound or pharmaceutical formulation thereof as described herein, wherein the compound or pharmaceutical formulation thereof is attached, bound, chemisorbed, or mixed with the bone graft material. The bone graft material may be an autograft bone material, an allograft bone material, a xenograft bone material, a synthetic bone graft material, or any combination thereof.

Also provided herein are methods that can include the step of implanting a bone graft composition as described herein into a subject in need thereof.

Also provided herein are methods of prophylactic or preventative treatment of a biofilm infection at a bone or an implant surgical site or at a surgical site where a bone graft is performed, wherein the methods may comprise the step of administering to a subject in need thereof a compound as described herein.

Also provided herein are methods of prophylactic or preventative treatment of biofilm infection at a bone or implant surgical site or at a surgical site where a bone graft is performed, wherein the methods may comprise the step of implanting a bone graft composition as described herein into a subject in need thereof.

Other compounds, compositions, formulations, methods, features and advantages of the presently disclosed fabrication systems for nanowire template synthesis will be or become apparent to one with skill in the art upon examination of the following figures 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.

Drawings

Further aspects of the present disclosure will be more readily understood after a review of the following detailed description of various embodiments of the disclosure taken in conjunction with the accompanying drawings.

FIG. 1 shows a scanning electron micrograph (SEM; magnification 100X) of a surgical specimen from a patient with chronic osteomyelitis showing characteristic multi-layered and matrix-enclosed biofilms for colonization of the internal and external surfaces of bones; the top right panel shows a high magnification view (5000 x magnification) of a pathogenic staphylococcal biofilm pathogen. [ samples were treated for SEM, sputter coated with platinum and imaged with XL 30S SEM (FEG, FEI Co., Hillsboro, OR) operating at 5kv in secondary electron mode ].

Figure 2 demonstrates a general BP quinolone conjugate targeting strategy.

Fig. 3 shows an embodiment of a BP-FQ conjugate.

Figure 4 shows an additional BP-Ab conjugate design.

Fig. 5 shows an embodiment of a synthesis scheme for synthesizing BP-Ab conjugates with O-thiocarbamate linkers.

Fig. 6 shows an embodiment of a scheme for the synthesis of an α -OH protected BP ester.

FIG. 7 shows an embodiment of a protocol for the synthesis of BP 3-linker 3-ciprofloxacin.

Figure 8 shows BP-carbamate-moxifloxacin BP conjugates and the synthesis scheme.

Fig. 9 shows BP-carbamate-gatifloxacin BP conjugates and the synthetic scheme.

Figure 10 shows BP-p-hydroxyphenylacetic acid-ciprofloxacin BP conjugates and the synthetic scheme.

FIG. 11 shows BP-OH-ciprofloxacin BP conjugate and the synthetic scheme.

Fig. 12 shows BP-O-thiocarbamate-ciprofloxacin BP conjugates and the synthetic scheme.

Fig. 13 shows BP-S-thiocarbamate-ciprofloxacin BP conjugates and the synthetic scheme.

Figure 14 shows BP-resorcinol-ciprofloxacin BP conjugates and the synthetic scheme.

Figure 15 shows BP-hydroquinone-ciprofloxacin BP conjugates and the synthetic scheme.

FIG. 16 shows one embodiment of the general structure of BP-fluoroquinolones.

Figure 17 shows various BP-fluoroquinolone conjugates.

FIG. 18 illustrates one embodiment of the general structure of phosphonic acids containing an aryl group.

FIG. 19 shows various BPs, where X can be F, Cl, Br or I.

Fig. 20 shows various BPs with terminal primary amines.

Fig. 21 shows the coupling of various BPs to a linker comprising a terminal hydroxyl and amine functional group, where R can be risedronic acid, zoledronic acid, minodronic acid, pamidronic acid, or alendronic acid.

Figure 22 shows various BP-pamidronic acid-ciprofloxacin conjugates.

Figure 23 shows various BP-alendronate-ciprofloxacin conjugates.

Fig. 24 depicts an example of a pharmacologically inert BP for conjugation in the present invention: medium (A/E), high (B/F) and low (C/G) affinity BP and longer phenyl alkyl chain BP (D/H).

Figure 25 depicts an example of a pharmacologically low activity BP that can be used in the conjugation of the present invention.

Figure 26 depicts the results of dynamically monitoring biofilm growth in the presence of different concentrations of conjugate. Culture (- - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -.

FIG. 27 depicts the MIC of Staphylococcus aureus for ECC and ECX₅₀And (6) analyzing. ECC (- - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -.

Figure 28 depicts the results of dynamically monitoring biofilm growth in the presence of different concentrations of parent antibiotics. The culture (- - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -, 0.05 mu g/ml of moxifloxacin (- - - - - -), 0.1 mu g/ml of moxifloxacin (- - - - - - -), 0.2 mu g/ml of moxifloxacin (- - - - - - - - -), and 0.5 mu g/ml of moxifloxacin (- - - - - - - - - -).

FIG. 29 depicts the MIC of Staphylococcus aureus for ciprofloxacin and moxifloxacin₅₀And (6) analyzing. Moxifloxacin (- - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -.

Figure 30 depicts the results of dynamically monitoring biofilm growth in the presence of ciprofloxacin/moxifloxacin + HA (10 μ g/ml). The culture comprises a culture (- - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -, 0.1 mu g/ml (- - - - - - -) of moxifloxacin and 0.2 mu g/ml (- - - - - - -) of moxifloxacin.

FIG. 31 depicts Staphylococcus aureus MICs for ciprofloxacin/moxifloxacin + HA₅₀And (6) analyzing. Moxifloxacin (- - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -.

FIG. 32 depicts the results of dynamically monitoring biofilm growth in the presence of ECC/ECX + HA (10 μ g/ml). Culture (- - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -.

FIG. 33 depicts Staphylococcus aureus MICs for ECC/ECX + HA₅₀And (6) analyzing. ECX (- - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -.

Detailed Description

Before the present disclosure is described in more 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. Where the 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 were incorporated by reference to disclose and describe the methods and/or materials in connection with which the publications were cited. The citation of any publication is a publication prior to the filing date thereof and should not be construed as an admission that the present disclosure is not entitled to antedate such publication by virtue of prior publication. Further, the dates of publication provided may be different from the actual publication dates which may need to be independently confirmed.

It will be apparent to those of skill in the art upon reading this disclosure that 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 listed methods may be performed in the order of the events listed or in any other order that is logically possible.

Unless otherwise indicated, embodiments of the present disclosure will employ techniques in the art of molecular biology, microbiology, nanotechnology, pharmacology, organic chemistry, biochemistry, botany, and the like. These techniques are fully described in the literature.

Definition of

Unless otherwise stated herein, the following definitions are provided.

As used herein, "about", "approximately", and the like, when used in connection with a numerical variable, generally refer to the value of the variable as well as all values of the variable that are within experimental error (e.g., within 95% confidence interval of the mean) or within ± 10% of the specified value (whichever is larger).

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, domestic animals, sports animals, and pets. The term "pet" includes dogs, cats, guinea pigs, mice, rats, rabbits, ferrets, and the like. The term "livestock" includes horses, sheep, goats, chickens, pigs, cattle, donkeys, llamas, alpacas, turkeys, and the like.

As used herein, "control" may refer to an alternative subject or sample used in an experiment for comparison purposes and is included to minimize or distinguish the effects of variables other than independent variables.

As used herein, an "analog" or "analog" such as an analog of a bisphosphonate, as described herein, may refer to a member that is structurally similar to the parent molecule or an additional parent molecule, such as a bisphosphonate.

As used herein, "conjugated" may refer to two or more compounds directly attached to each other by one or more covalent or non-covalent bonds. The term "conjugated" as used herein may also refer to two or more compounds indirectly attached to each other 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 prophylactic use in vitro, in vivo or ex vivo.

As used herein, "pharmaceutically acceptable carrier or excipient" refers to a carrier or excipient that is generally safe, non-toxic, and not biologically or otherwise undesirable for use in the preparation of pharmaceutical formulations, and includes carriers or excipients that are acceptable for veterinary as well as human pharmaceutical use. As used in the specification and claims, "pharmaceutically acceptable carrier or excipient" 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, the counter ion of which is non-toxic to a subject administered a pharmaceutical dose of the salt.

As used herein, "active agent" or "active ingredient" refers to one or more components of a composition to which all or part of the effect of the composition is attributed.

As used herein, "dose," "unit dose," or "amount" refers to discrete units suitable for use on the body of a subject, each unit comprising a predetermined amount of a BP conjugate, such as a BP quinolone conjugate, composition, or formulation described herein, calculated to produce a desired response or a response associated with administration thereof.

As used herein, "derivative" refers to any compound having the same or similar core structure as the compound, but with at least one structural difference, including substitution, deletion, and/or addition of one or more atoms or functional groups. The term "derivative" does not imply that the derivative is synthesized from the parent compound as a starting material or intermediate, although this may be the case. The term "derivative" can include prodrugs or metabolites of the parent compound. The derivatives include compounds in which the free amino groups in the parent compound have been derivatized to form amine salts, p-toluenesulfonamides, benzyloxycarboxamides, t-butoxycarboxamides, ethyl thiocarbamate derivatives, trifluoroacetamides, chloroacetamides or carboxamides. Derivatives include compounds in which the carboxyl group of the parent compound has been derivatized to form methyl and ethyl esters or other types of esters, amides, hydroxamic acids or hydrazides. Derivatives include those in which the hydroxy group in the parent compound has been derivatized to form an O-acyl, O-carbamoyl or O-alkyl derivative. Derivatives include compounds in which a hydrogen bond donor group in the parent compound is replaced by another hydrogen bond donor group (such as OH, NH or SH). Derivatives include the replacement of a hydrogen bond accepting group in the parent compound with another hydrogen bond accepting group such as esters, ethers, ketones, carbonates, tertiary amines, imines, thiones, sulfones, tertiary amides, and sulfides. "derivatives" also include extensions such as the replacement of the cyclopentane ring with saturated or unsaturated cyclohexane or other more complex, e.g., nitrogen-containing, rings, as well as extensions of these rings with various groups.

As used herein, "administering" means orally, topically, intravenously, subcutaneously, transdermally, intramuscularly, intra-articularly, parenterally, intra-arteriolar, intradermally, intraventricularly, intracranially, intraperitoneally, intralesionally, intranasally, rectally, vaginally, by inhalation, or by implantable drug depot. The term "parenteral" includes subcutaneous, intravenous, intramuscular, intraarticular, intrasynovial, intrasternal, intrathecal, intrahepatic, intralesional and intracranial injection or infusion techniques.

The term "substituted" as used herein refers to all permissible substituents of the compounds described herein. In a broad sense, the permissible substituents include acyclic and cyclic, branched and unbranched, carbocyclic and heterocyclic, aromatic and nonaromatic substituents of organic compounds. Exemplary substituents include, but are not limited to, halogen, hydroxyl group, or any other organic group containing any number of carbon atoms (e.g., 1-14 carbon atoms), and also optionally include one or more heteroatoms, such as oxygen, sulfur, or nitrogen, grouped 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, hydroxy, alkoxy, substituted alkoxy, phenoxy, substituted phenoxy, aryloxy, substituted aryloxy, alkylthio, substituted alkenyl, substituted alkynyl, substituted aryl, heteroarylPhenyl, substituted phenyl, aryl, substituted aryl, 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-C₂₀Cyclic, substituted C₃-C₂₀Rings, heterocycles, substituted heterocycles, amino acids, peptides 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 compounds of the present invention. Such suitable substituents may be routinely selected by those skilled in the art. Suitable substituents include, but are not limited to, the following: halogen, C₁-C₆Alkyl radical, C₂-C₆Alkenyl radical, C₁-C₆Haloalkyl, C₁-C₆Alkoxy radical, C₁-C₆Haloalkoxy, C₂-C₆Alkynyl, C₃-C₈Cycloalkenyl group, (C)₃-C₈Cycloalkyl) C₁-C₆Alkyl, (C)₃-C₈Cycloalkyl) C₂-C₆Alkenyl, (C)₃-C₈Cycloalkyl) C₁-C₆Alkoxy radical, C₃-C₇Heterocycloalkyl group, (C)₃-C₇Heterocycloalkyl) C₁-C₆Alkyl, (C)₃-C₇Heterocycloalkyl) C₂-C₆Alkenyl, (C)₃-C₇Heterocycloalkyl) C₁-C₆Alkoxy, hydroxy, carboxy, oxo, sulfonyl, C₁-C₆Alkylsulfonyl, aryl, heteroaryl, aryloxy, heteroaryloxy, aralkyl, heteroaralkyl, aralkoxy, heteroaralkoxy, nitro, cyano, amino, C₁-C₆Alkylamino, di- (C)₁-C₆Alkyl) amino, carbamoyl, (C)₁-C₆Alkyl) carbonyl (C)₁-C₆Alkoxy) carbonyl、(C₁-C₆Alkyl) aminocarbonyl, di- (C)₁-C₆Alkyl) aminocarbonyl, arylcarbonyl, aryloxycarbonyl, (C)₁-C₆Alkyl) sulfonyl and arylsulfonyl. The groups listed above as suitable substituents are as defined below, except that suitable substituents may not be further optionally substituted.

The term "alkyl" refers to the radical of a saturated aliphatic group (i.e., an alkane from which one hydrogen atom has been 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, the linear or branched alkyl group may have 30 or fewer carbon atoms in its backbone (e.g., C for linear chain)₁-C₃₀And for the side chain is C₃-C₃₀). In other embodiments, the linear or branched alkyl group may contain 20 or fewer, 15 or fewer, or 10 or fewer carbon atoms in its backbone. Likewise, in some embodiments, cycloalkyl groups may have 3 to 10 carbon atoms in their ring structure. In some of these embodiments, the cycloalkyl group 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 carboxyl, alkoxycarbonyl, formyl, or acyl), thiocarbonyl (such as thioester, thioacetate, or thioformate), alkoxy, phosphoryl, phosphate, phosphonate, phosphinate, amino, amide, amidine, imine, cyano, nitro, azido, sulfhydryl, alkylthio, sulfate, sulfonate, sulfamoyl, sulfonamide, sulfonyl, heterocyclyl, aralkyl, or an aromatic or heteroaromatic moiety.

As used herein, "lower alkyl" means an alkyl group as defined above, but having one to ten carbons in its backbone structure, unless the number of carbons is otherwise specified. Likewise, "lower alkenyl" and "lower alkynyl" have similar chain lengths.

It will be appreciated by those skilled in the art that the moiety substituted on the hydrocarbon chain may itself be substituted, if appropriate. For example, substituents of substituted alkyl groups may include halogen, hydroxy, nitro, thiol, amino, azido, imino, amido, phosphoryl (including phosphonato and phosphonato), sulfonyl (including sulfate, sulfonamido, sulfamoyl and sulfonate), and silyl groups, as well as ether, alkylthio, carbonyl (including ketones, aldehydes, carboxylic acid groups and esters), -CF₃CN, -CN, etc. Cycloalkyl groups may be substituted in the same manner.

The term "heteroalkyl," as used herein, refers to a straight or branched chain or cyclic carbon-containing group, or a combination thereof, that contains at least one heteroatom. Suitable heteroatoms include, but are not limited to, O, N, Si, P, Se, B, and S, wherein the phosphorus and sulfur atoms are optionally oxidized, and the nitrogen heteroatom is optionally quaternized. The heteroalkyl group may be substituted as defined above for the alkyl group.

The term "alkylthio" refers to an alkyl group as defined above having a thio group attached thereto. In a preferred embodiment, an "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 includes cycloalkyl groups, alkene and cycloalkene groups, and alkyne groups. "arylthio" refers to an aryl or heteroaryl group. The arylthio group may be substituted as defined above for the alkyl group.

The terms "alkenyl" and "alkynyl" refer to unsaturated aliphatic groups that are similar in length and possible substitution as alkyl groups described above, but each contain at least one double or triple bond.

The term "alkoxy" or "alkoxy" as used herein refers to an alkyl group as defined above having an oxy group attached thereto. Representative alkoxy groups include methoxy, ethoxy, propoxy, tert-butoxy and the like. An "ether" is two hydrocarbons covalently linked by oxygen. Thus, substituents that render an alkyl group as an ether or an alkyl group similar to an alkoxy group, such as may be represented by one of-O-alkyl, -O-alkenyl, and-O-alkynyl. The terms "aryloxy" and "aryloxy", as used interchangeably herein, may be represented by-O-aryl or O-heteroaryl, wherein aryl and heteroaryl are defined as follows. Alkoxy and aryloxy groups may be substituted as described above for alkyl groups.

The terms "amine" and "amino" (and protonated forms thereof) are well known in the art and refer to both unsubstituted and substituted amines, e.g., moieties that can be represented by the general formula:

wherein R, R 'and R' each independently represent hydrogen, alkyl, alkenyl, (CH2)_m-R_COr R and R' together with the N atom to which they are attached form a heterocyclic ring having from 4 to 8 atoms in the ring structure; r_CRepresents aryl, cycloalkyl, cycloalkenyl, heterocycle or polycycle; and m is zero or an integer in the range of 1 to 8. In some embodiments, only one of R or R 'may be a carbonyl group, e.g., R, R' and the nitrogen together do not form an imide. In other embodiments, the term "amine" does not include amides, e.g., where one of R and R' represents a carbonyl group. In a further embodiment, R and R' (and optionally R ") each independently represent hydrogen, alkyl or cycloalkyl, 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 group attached thereto, i.e. at least one of R and R' is an alkyl group.

The term "amido" is known in the art as an amino-substituted carbonyl and includes moieties that can be represented by the general formula:

wherein R and R' are as defined above.

As used herein, "aryl" refers to C₅-C₁₀-aromatic, heterocyclic, fused aromatic, fused heterocyclic, biaromatic or diheterocyclic systems. As broadly defined, "aryl" as used herein includes 5-, 6-, 7-, 8-, 9-, and 10-membered monocyclic aromatic groups that may include from zero to four heteroatoms, such as 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 may 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, hydroxy, alkoxy, amino (or quaternized amino), nitro, mercapto, imino, amido, phosphonato, carbonyl, carboxy, silyl, ether, alkylthio, sulfonyl, sulfonamido, ketone, aldehyde, ester, heterocyclyl, aromatic or heteroaromatic moieties, -CF₃CN, -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 cycloalkyl, cycloalkenyl, cycloalkynyl, aryl, and/or heterocyclic. Examples of heterocycles include, but are not limited to, benzimidazolyl, benzofuranyl, benzothiofuranyl, benzothiophenyl, benzoxazolyl, benzoxazolinyl, benzothiazolyl, benzotriazolyl, benzotetrazolyl, benzisoxazolyl, benzisothiazolyl, benzimidazolinyl, carbazolyl, 4aH carbazolyl, carbolinyl, chromanyl, benzopyranyl, cinnolinyl, decahydroquinolinyl, 2H,6H-1,5, 2-dithiazinyl, dihydrofuro [2,3b ] tetrahydrofuran, furanyl, furazanyl, imidazolidinyl, imidazolinyl, imidazolyl, 1H-indazolyl, pseudoindolyl (indolynyl), indolinyl, indolizinyl, indolyl, 3H-indolyl, isatinoyl (isatinoyl), isobenzofuranyl, isobenzodihydropyranyl, isoindolinyl, isoindolyl, isoquinolyl, isothiazolyl, isoxazolyl, methylenedioxyphenyl, morpholinyl, naphthyridinyl, octahydroisoquinolyl, 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, phenoxathiin, phenoxazinyl, phthalazinyl, piperazinyl, piperidinyl, piperidonyl, 4-piperidonyl, piperonyl, pteridinyl, purinyl, pyranyl, pyrazinyl, pyrazolidinyl, pyrazolinyl, pyrazolyl, pyridazinyl, pyridooxazole, pyridoimidazole, pyridothiazole, pyridyl (pyridinyl), pyridyl (pyridylal), pyrimidinyl, pyrrolidinyl, pyrrolinyl, pyrazolinyl, pyrazolidinyl, pyridylal, and the like, 2H-pyrrolyl, quinazolinyl, quinolinyl, 4H-quinolizinyl, quinoxalinyl, quinuclidinyl, tetrahydrofuryl, tetrahydroisoquinolinyl, tetrahydroquinolinyl, tetrazolyl, 6H-1,2, 5-thiadiazolyl, 1,2, 3-thiadiazolyl, 1,2, 4-thiadiazolyl, 1,2, 5-thiadiazolyl, 1,3, 4-thiadiazolyl, thianthrenyl, thiazolyl, thienyl, thienothiazolyl, thienooxazolyl, thienoimidazolyl, thienyl, and xanthenyl. One or more of the rings may 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 "aralkoxy" may be represented by-O-aralkyl, wherein aralkyl is as defined above.

The term "carbocycle" as used herein refers to one or more aromatic or non-aromatic rings in which each atom of the ring or rings is carbon.

As used herein, "heterocycle" or "heterocyclic" refers to a monocyclic or bicyclic structure comprising 3 to 10 ring atoms (and in some embodiments 5 to 6 ring atoms) wherein the ring atoms are carbon and one to four heteroatoms each selected from the following group: non-peroxidized oxygen, sulfur and N (Y), wherein YIs absent or is H, O, (C)₁-C₁₀) Alkyl, phenyl or benzyl, and optionally contains 1-3 double bonds, and is optionally substituted with one or more substituents. Examples of heterocycles include, but are not limited to, benzimidazolyl, benzofuranyl, benzothiofuranyl, benzothiophenyl, benzoxazolyl, benzoxazolinyl, benzothiazolyl, benzotriazolyl, benzotetrazolyl, benzisoxazolyl, benzisothiazolyl, benzimidazolinyl, carbazolyl, 4aH carbazolyl, carbolinyl, chromanyl, benzopyranyl, cinnolinyl, decahydroquinolinyl, 2H,6H-1,5, 2-dithiazinyl, dihydrofuro [2,3b ] group]Tetrahydrofuran, furyl, furazanyl, imidazolidinyl, imidazolinyl, imidazolyl, 1H-indazolyl, isoindolyl, indolinyl, indolizinyl, indolyl, 3H-indolyl, isatoiyl, isobenzofuryl, isobenzodihydropyranyl, isoindolyl, isoindolinyl, isoindolyl, isothiazolyl, isoxazolyl, methylenedioxyphenyl, morpholinyl, naphthyridinyl, octahydroisoquinolyl, 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, phenoxazinyl, phenazinyl, phthalazinyl, isoquinolyl, isothiazolyl, isoxazolyl, methylindolyl, morpholinyl, isoquinolyl, isothiazolyl, isoxazolyl, 1, 4-oxadiazolyl, morpholinyl, naphthyridinyl, octahydroisoquinolyl, oxadiazolyl, and phenanthridinyl, Piperazinyl, piperidinyl, piperidonyl, 4-piperidonyl, piperonyl, pteridinyl, purinyl, pyranyl, pyrazinyl, pyrazolidinyl, pyrazolinyl, pyrazolyl, pyridazinyl, pyridooxazole, pyridoimidazole, pyridothiazole, pyridyl (pyridinyl), pyridyl (pyridil), pyrimidinyl, pyrrolidinyl, pyrrolinyl, 2H-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, pyranyl, pyrazinyl, pyrazolidinyl, quinazolinyl, quinolizyl, quinoliz, Thienothiazolyl, thienooxazolyl, thienoimidazolyl, thienyl and xanthyl groups. The heterocyclic group may optionally be para-alkane as hereinbefore describedDefined as radicals and aryl radicals, substituted at one or more positions by one or more substituents, e.g. halogen, alkyl, aralkyl, alkenyl, alkynyl, cycloalkyl, hydroxy, amino, nitro, mercapto, imino, amido, phospho, phosphonato, carbonyl, carboxy, silyl, ether, alkylthio, sulfonyl, ketone, aldehyde, ester, heterocyclyl, aromatic or heteroaromatic moieties, -CF₃CN, -CN, etc. The term "heterocycle" or "heterocyclic" may be used to describe a compound that may include a heterocycle or heterocyclic ring.

The term "carbonyl" is art-recognized and includes such moieties as may be represented by the general formula:

wherein X is a bond or represents oxygen or sulphur and R' are as defined above. When X is oxygen and R or R' is not hydrogen, the formula represents an "ester". When X is oxygen and R is as defined above, the moiety is referred to herein as a carboxyl group, and in particular when R is hydrogen, the formula represents a "carboxylic acid". When X is oxygen and R' is hydrogen, the formula represents "formate". Typically, when the oxygen atom in the above formula is replaced by sulfur, the formula represents a "thiocarbonyl" group. When X is sulfur and R or R' is not hydrogen, the formula represents a "thioester". When X is sulfur and R is hydrogen, the formula represents a "thiocarboxylic acid". When X is sulfur and R' is hydrogen, the formula represents "thiocarboxylate". On the other hand, when X is a bond and R is not hydrogen, the above formula represents a "ketone" group. When 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, may have hydrogen substituents and/or any permissible substituents of organic compounds described herein that satisfy the valencies of the heteroatoms. It is understood that the implication of "substituted" or "substituted" is that such substitution is in accordance with the allowed valences of the atoms and substituents being replaced, and that the substitution results in a stable compound, i.e. a compound that does not spontaneously undergo a transformation such as rearrangement, cyclization, elimination, etc.

The term "hydroxy" refers to the "-OH" group.

As used herein, the term "nitro" refers to-NO₂(ii) a The term "halogen" means-F, -Cl, -Br, or-I; the term "mercapto" refers to-SH; the term "hydroxy" refers to-OH; and the term "sulfonyl" refers to-SO₂-。

As used herein, "carbamate" may be used to refer to a compound derived from carbamic acid (NH)₂COOH) and may include carbamates. "carbamates" may have the following general structure:

wherein R is₁、R₂And R₃May be any permissible substituents.

As used herein, "carbonate" may be used to refer to a compound derived from carbonic acid (H)₂CO₃) Derivatized compounds, and may include carbonates. "carbonates" can have the following general structure:

as used herein, an "effective amount" may refer to an amount of a composition described herein or a pharmaceutical formulation described herein that will elicit the desired biological or medical response of a tissue, system, animal, plant, protozoan, bacteria, yeast or human that is being sought by a researcher, veterinarian, medical doctor or other clinician. The desired biological response may be modulation of bone formation and/or remodeling, including but not limited to modulation of bone resorption and/or uptake of a BP conjugate (such as a BP quinolone conjugate described herein). The effective amount will vary depending on the exact chemical structure of the composition or pharmaceutical preparation, the pathogen being treated or prevented and/or the severity of its infection, disease, disorder, syndrome or symptom, the route of administration, time of administration, rate of excretion, 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. An "effective amount" can refer to an amount of a composition described herein that is effective to inhibit the growth or reproduction of a microorganism (including, but not limited to, a bacterium or population thereof). An "effective amount" can refer to an amount of a composition described herein that kills a microorganism (including, but not limited to, a bacterium or population thereof). An "effective amount" can refer to an amount of a composition 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 "substituent" or "derivative" thereof and related terms have the same meaning and refer to antimicrobial agents that are part of the well-known class of "quinolone" as described in more detail herein.

As used herein, "therapeutic" may generally refer to treating, curing and/or ameliorating a disease, condition, disorder or side effect, or reducing the rate of progression of a disease, condition, disorder or side effect. The term also includes within its scope enhancement of normal physiological function, palliative treatment, and partial remediation of its disease, disorder, condition, side effect, or symptom.

The term "antibacterial" includes those compounds that inhibit, prevent or reverse bacterial growth, those compounds that inhibit, prevent or reverse the activity of bacterial enzymes or biochemical pathways, those compounds that kill or damage bacteria, and those compounds that block or slow the development of bacterial infections.

As used herein, the terms "treating" and "treatment" may generally refer 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 is intended to mean alleviation of a disease condition associated with at least a bacterial infection in a subject (including a mammal, such as a human being), which disease condition is alleviated by reducing the growth, replication and/or reproduction of any bacteria, such as gram-positive organisms, and which alleviation includes completely or partially curing, inhibiting, alleviating, ameliorating and/or alleviating the disease condition.

The term "preventing" is intended to mean at least reducing 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 halting the progression of a disease condition associated with a bacterial infection in a mammal, preferably a human. In particular, these terms are relevant to the treatment of mammals to reduce the likelihood ("prevention") or prevent the occurrence of bacterial infections, such as may occur during or after surgery involving bone repair or replacement. These terms also include reducing the likelihood of a bacterial infection ("prophylaxis") or preventing a bacterial infection when a mammal is found to be predisposed to a disease condition but has not yet been diagnosed as having a disease condition. For example, by administering a compound of formula (1) and/or formula (2), or a pharmaceutically acceptable prodrug, salt, active metabolite, or solvate thereof, before such infection occurs, the likelihood of a bacterial infection in a mammal can be reduced or prevented.

As used herein, "synergistic effect," "synergistic effect," or "synergy" refers to an effect produced between two or more molecules, compounds, substances, factors, or compositions that is greater than or different from the sum of their respective effects.

As used herein, "additive effect" refers to an effect produced between two or more molecules, compounds, substances, factors or compositions that is equal or equivalent to the sum of their respective effects.

As used herein, the term "biocompatible" means that the material, along with any metabolites or degradation products thereof, is generally non-toxic to a recipient and does not cause any significant adverse effects to the recipient. Generally, a biocompatible material is one that does not cause a significant inflammatory or immune response when administered to a patient.

As used herein, the term "osteomyelitis" may refer to acute or chronic osteomyelitis, and/or diabetic foot osteomyelitis, diabetic chronic osteomyelitis, prosthetic joint infection, periodontitis, peri-implantitis, osteonecrosis, and/or hematogenous osteomyelitis and/or other bone infections.

Discussion of the related Art

Infectious bone disease, or osteomyelitis, is a major problem in human and veterinary medicine worldwide and can be catastrophic due to potentially limb-threatening sequelae and death. The treatment of osteomyelitis is mainly antimicrobial and is usually long-term and in many cases controls the infection by surgical intervention. In most cases of long bone osteomyelitis, the causative pathogen is an infection with staphylococcus aureus and the corresponding biofilm, which, unlike their planktonic (free-floating) counterparts, is osseointegrated. Other bone infections and corresponding biofilms are known to be caused by a wide range of gram-positive and gram-negative bacteria.

The biofilm-mediated nature of osteomyelitis is important in clinical and experimental settings, as many biofilm pathogens are unculturable and exhibit altered phenotypes in terms of growth rate and antimicrobial resistance (compared to their planktonic counterparts). The difficulty in eliminating biofilms with conventional antibiotics explains why antimicrobial therapy, which is often successful, is not successful in orthopedic infections with the development of resistant biofilm pathogens, poor penetration of the antimicrobial agent into the bone, and adverse events related to systemic toxicity.

To overcome many of the challenges associated with osteomyelitis treatment, there is increasing interest in drug delivery methods using bone-targeting conjugates to achieve higher or longer-lasting local antibiotic therapeutic concentrations in bone while minimizing systemic exposure.

Methylenebisphosphonic acid or substituted methylenebisphosphonic acid moieties, commonly referred to as "bisphosphonic acids" (BPs) which are therapeutic agents, are used to treat a number of bone conditions. The bisphosphonate P-C-P group mimics the P-O-P bond of the naturally occurring mediator inorganic pyrophosphate of bone metabolism. The following shows the structural relationship between acid-form pyrophosphoric acid and methylenebisphosphonic acid. Each BP may be formed by covalently attached substituents R₁And R₂And (4) limiting.

The bridging carbon of the bisphosphonic acids may be modified (R)₁、R₂) Substitution to confer specific biological properties to the derivative. BP exhibits strong binding affinity for HA (the major inorganic material found in bone, especially at sites of high bone turnover), and they are exceptionally stable against both chemical and biological degradation. It is generally not known that BP also traverses the soft and hard tissues of the body (e.g., endothelium, periosteum, HA) to target bone and small tubular networks and vascular pathways in bone. These highly specific bone targeting properties of BP make it an ideal vehicle for drug or macromolecule delivery to the bone surface.

Quinolone conjugated to bisphosphonic acids (BPs) (e.g., bone adsorbed BPs) and particularly fluoroquinolone antibiotics represent a promising approach because the clinical follow-up record of the safety of each component is long and their biochemical properties are also superior. In early studies of the fluoroquinolone family in this context, ciprofloxacin showed the best binding and microbiological properties when bound to BP. Reuse of ciprofloxacin in this context has several advantages: it can be administered orally or intravenously, is relatively bioequivalent, has broad spectrum antimicrobial activity against pathogens including the most common osteomyelitis, exhibits bactericidal activity at clinically achievable doses, and is the least expensive drug of the fluoroquinolone family.

The specific bone targeting properties of the BP family make them ideal carriers for the introduction of antibiotics into bone in the pharmacotherapy of osteomyelitis. BP forms strong bidentate and tridentate bonds with calcium and therefore accumulates in Hydroxyapatite (HA), particularly at sites of active metabolism or infection and inflammation. BP also exhibits excellent stability to both chemical and biological degradation. The concept of targeting ciprofloxacin to bone by conjugation to BP has been discussed for many years in a number of reports.

Despite these positive properties of BP and quinolone drugs (such as ciprofloxacin), no current attempts to generate prodrugs comprising BP and quinolone have been successful. Most attempts have resulted in systemically unstable prodrugs or non-cleavable conjugates, which have been found to be largely inactive by interfering with pharmacodynamic requirements. For example, Delorme et al (WO 2007/138381) describes the use of acyloxy chemical vicinal elements (chemical linkers) to activate alkyl carbamate linkers to cleave oxazolidinones from bisphosphonic acids, but this appears to be too actively cleaved in the bloodstream. Similar findings have been described by Houghton et al 2008J. medicinal Chemistry,51: 6955-. And alkyl carbamates have been shown by Morioka et al ("Design, synthesis, and biological evaluation of novel and biological conjugates as a bone-specific evaluation," biological & Medicinal Chemistry 18(2010) 1143) and by Arns et al ("Design and synthesis of novel bone-targeting dual-action pro-drugs for the same and reverse of bone, and" biological & Medicinal Chemistry 20(2012)2131 "2140) to be too stable to be used as a" targeted and released "linkage for an active conjugate for bone targeting.

Recognizing the deficiencies of current BP quinolone conjugates, described herein are BP quinolone conjugates that may comprise BP that may be releasably conjugated to a quinolone (such as ciprofloxacin, moxifloxacin, sitafloxacin, or nerofloxacin). In embodiments, the BP quinolone conjugate may selectively deliver the quinolone to a bone, a bone graft, and or a bone graft substitute (i.e., may target the bone, bone graft, or bone graft substitute) of a subject. In some embodiments, the BP quinolone conjugate may 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 use a "targeting and releasing linker" strategy, in which releasable and bone-specifically targeted bisphosphonate-antibiotic (BP-Ab) conjugates can be made by attaching antibiotics or antimicrobial agents (e.g., quinolones) to BP. In any one or more aspects, the BP can be a pharmacologically inactive BP or a pharmacologically low active BP using a cleavable or reversible linker, such as a carbamate, thiocarbamate, hydrazone, or carbonate, such that the antimicrobial agent can be released upon binding to the bone surface through a reduced pH and/or enzymatic environment, which is typically found at the active site of bone resorption or infection.

In any one or more aspects, the quinolone can be attached to the hydroxy BP, a geminal hydroxy group attached directly or indirectly to a carbon between two phosphonic acid groups of BP. This is in contrast to using aryl carbamates to attach or attach a quinolone to BP in other ways at sites other than the alpha hydroxyl, alpha thiol, or alpha amino site. To activate the attachment or linker sufficiently for cleavage ("targeting and release" concept), the present disclosure utilizes carbamates (and related) that are uniquely activated by the alpha carbon or substituent of the bisphosphonic acid for sufficient release. In any one or more aspects herein, all analogs of BP known to be clinically useful are preferred. Etidronic acid and MHDP or methylene hydroxy BP may be most preferred.

The chemistry and conjugate design of the present invention also allows cleavage to release two known (clinically used) drugs, the quinolone and BP (especially the clinically used BP). Carbon dioxide is the only other releasing component from the linker. Thus, there are no new safety issues with the release composition. It was previously unknown whether the linkage to the geminal hydroxyl group of bisphosphonic acids would be properly cleaved for use in vivo to achieve this (based on "targeting and release" efficacy) goal. Previously, unknown BPs (not yet used clinically) were used to generate aryl carbamate linkages at sites other than the alpha carbon of BP. These BPs, which have not been previously studied in human subjects, allow creation of aryl carbamate-based conjugates to allow release/cleavage rates useful for bioactivity. It has been found herein in vitro that release still occurs at a useful rate, since the carbamate, via the linkage to the geminal hydroxyl group, is sufficiently activated by the adjacent phosphonic acid group for sufficient cleavage. As this has now been found to occur, the compounds, conjugates and formulations of the present invention provide an opportunity to use many clinically known bisphosphonic acids within conjugates, as most have geminal hydroxyl groups.

An exemplary BP-quinolone release mechanism is depicted in fig. 2, using ciprofloxacin as an exemplary quinolone. However, non-fluoroquinolones can also be conjugated to BP as described herein. Such BP-Ab conjugates may have the ability to specifically deliver and release antimicrobial agents to infectious osteolytic sites where higher bone metabolism occurs. The use of inactive or low active BP can also provide unique treatment options by providing higher concentrations of antimicrobial agents and relatively lower systemic levels at the disease site compared to higher active BP. Other BP-quinolone compounds and conjugates as described herein may 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 may be a pharmaceutical formulation which may comprise a pharmaceutically acceptable carrier. The compositions and/or formulations can be administered to a subject. The subject may have a bone infection. The compositions and formulations provided herein can be used to treat and/or prevent bone infections. In some embodiments, the compositions and formulations provided herein can provide bone-specific delivery of antimicrobial agents.

The general concept of using BP to target active drug substances to bone regions has been discussed in a number of reports. However, no drug has been developed, as early attempts resulted in systemically unstable prodrugs or non-cleavable conjugates, which were found to be largely inactive by interfering with pharmacodynamic requirements. This suggests that the target and release strategies may be chemical class dependent (taking into account the compatibility of the functional groups of each component) as well as biochemical target dependent, and that the design of any particular chemical class must be tailored for its use. Accordingly, provided herein are embodiments of novel methods of developing bone-targeted antibiotics or antimicrobial agents with linkages that are metabolically stable in the bloodstream and metabolically unstable on bone to facilitate proper release.

In particular, in any one or more aspects herein, the linkages used herein are designed to allow maximum local antibacterial efficacy at the site of infection where there is a higher bone turnover, while also limiting exposure to lower turnover bone sites, non-bone sites, and distal compartments throughout the body, from any adverse effects due to antibiotics or bisphosphonate components or conjugates. Thus, for example, the α hydroxy carbamate linker and other related α carbon-directed linkers herein are specifically selected to have maximum stability in blood, while still being sensitive to chemical cleavage and release of quinolone antibiotics at the bacterial infected skeletal sites, due to their sensitivity to the enzymatic processes and pH characteristics of that environment. In addition, selected, but not all embodiments in this disclosure include the use of bisphosphonic acids that have no significant pharmacological activity with respect to the targeting component of these drug conjugates. These "non-or weakly-resorptive bisphosphonates have the feature of targeting antibiotics only to the described bone compartment and do not have the properties that otherwise directly affect bone metabolism. Examples include aryl carbamates and aryl thiocarbamates derived from substituted and unsubstituted 2- [ 4-aminophenyl ]

ethane

1,1 bisphosphonic acids and 2- [ 4-hydroxyphenyl ]

ethane

1,1 bisphosphonic acids. Also included are carbamates derived from substituted and unsubstituted 2- [ 3-aminophenyl ]

ethane

1,1 bisphosphonic acids and 2- [ 3-hydroxyphenyl ]

ethane

1,1 bisphosphonic acids, substituted and unsubstituted 2- [ 2-aminophenyl ]

ethane

1,1 bisphosphonic acids and 2- [ 2-hydroxyphenyl ]

ethane

1,1 bisphosphonic acids. In addition, aryl dithiocarbamates derived from substituted and unsubstituted 2- [ 4-thiophenyl ]

ethane

1,1 bisphosphonic acids, 2- [ 3-thiophenyl ]

ethane

1,1 bisphosphonic acids, and 2- [ 2-thiophenyl ]

ethane

1,1 bisphosphonic acids.

In any one or more aspects, the BP of the conjugate can be a pharmacologically inactive or inactive BP. An example of a pharmacologically inert or inactive BP that can be conjugated to a quinolone as described herein is shown in figure 24.

As an example, the inert or inactive BP set used for conjugation can be 4-hydroxyphenylethylidene BP (fig. 24A) or 4-aminophenylethylidene BP (fig. 24E) with moderate mineral affinity. Additional analogs, such as hydroxy BP (fig. 24B and 24F) (higher mineral affinity) and methyl BP (fig. 24C and 24G) (lower mineral affinity) can be used to modulate the concentration of BP-Ab conjugate at bone. Phenylalkyl BPs with different chain lengths, such as in fig. 24D and 24H (propyl or butyl versus ethylphenyl), can also be used to optimize conjugation chemistry yield and conjugate stability.

In any one or more aspects, the BP of the conjugate can be a pharmacologically low activity BP. An example of a pharmacologically low active BP that can be conjugated to a quinolone as described herein is shown in fig. 25. By "low activity BP" is meant a bisphosphonate or a dosage level of bisphosphonate that is not so high as to affect bone metabolism. However, in some aspects, a higher activity BP may be required in cases where it is desirable to both affect bone metabolism and deliver a quinolone antibiotic to inhibit and/or kill/affect bacteria on bone.

Other compositions, compounds, methods, features and advantages of the disclosure will be or become apparent to one with skill in the art upon examination of the following figures, detailed description and examples. It is intended that all such additional compositions, compounds, methods, features and advantages be included within this description, be within the scope of the present disclosure.

Bisphosphonic acid (BP) quinolone conjugates and formulations thereof

BP quinolone conjugates

Provided herein are BP quinolone compounds, conjugates, and formulations thereof. BP can be conjugated to a quinolone through a linker. In an embodiment, the linker is a releasable linker. The quinolone may be releasably attached to the BP through a linker. Thus, in some embodiments, BP quinolone conjugates can selectively deliver and release the quinolone at or near a bone, bone graft, or bone graft substitute (fig. 2). In other words, for example, BP fluoroquinolone conjugates can provide targeted delivery of fluoroquinolones to bone and/or regions proximate to bone.

The BP of the BP quinolone conjugates provided herein may be conjugated to any BP, including, but not limited to, hydroxyphenylalkyl or aryl bisphosphonic acids, hydroxyphenyl (or aryl) alkylhydroxy bisphosphonic acids, aminophenyl (or aryl) alkyl bisphosphonic acids, aminophenyl (or aryl) alkylhydroxy bisphosphonic acids, hydroxyalkyl hydroxy bisphosphonic acids, hydroxyalkyl phenyl (or aryl) alkyl bisphosphonic acids, hydroxyphenyl (or aryl) alkylhydroxy bisphosphonic acids, aminophenyl (or aryl) alkyl bisphosphonic acids, aminophenyl (or aryl) alkylhydroxy bisphosphonic acids, hydroxyalkyl hydroxy bisphosphonic acids, all of which are further substituted or unsubstituted. In particular, BP can be etidronic acid, pamidronic acid, neridronic acid, olpadronic acid, alendronic acid, ibandronic acid, minodronic acid, risedronic acid, zoledronic acid, hydroxymethylenediphosphonic acid, and combinations thereof. Bisphosphonic acids may also be substituted with phosphonophosphinic acids or phosphonocarboxylic acids. In embodiments, BP can be pamidronic acid, alendronic acid, risedronic acid, zoledronic acid, minodronic acid, neridronic acid, etidronic acid, which can be unmodified or modified as described herein. In a preferred embodiment, BP is unmodified or modified etidronic acid, MHBP or pamidronic acid.

BP may comprise or be modified to comprise a linkage to an alpha substituent, and the alpha substituent may be a hydroxyl, amino or thiol group. The antibiotic quinolone compound or analog may be conjugated directly or indirectly to BP at the geminal carbon substituent of BP. The quinolone and/or the linker may also be coupled to BP with significantly reduced or eliminated anti-absorption.

In BP comprising an aryl or phenyl group, the aryl or phenyl group may be substituted at any position on the ring with a suitable substituent. 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).

The non-pharmacologically active BP variants can also be used for the purpose of quinolone delivery without BP effect.

The quinolone may be any quinolone, fluoroquinolone, or non-fluoroquinolone, including but not limited to: alafloxacin, amifloxacin, balofloxacin, besifloxacin, cadozolamide, ciprofloxacin, clinafloxacin, danofloxacin, delafloxacin, difloxacin, enoxacin, enrofloxacin, finafloxacin, fleroxacin, flumequine, gatifloxacin, gemifloxacin, grepafloxacin, ebafloxacin, JNJ-Q2, levofloxacin, lomefloxacin, marbofloxacin, moxifloxacin, nadifloxacin, norfloxacin, ofloxacin, orbifloxacin, pazufloxacin, pefloxacin, prafloxacin, prarofloxacin, rufloxacin, sarofloxacin, sitafloxacin, sparfloxacin, temafloxacin, tosufloxacin, trovafloxacin, zabofloxacin, nemonoxacin and any combination thereof. In particular, the quinolone may be a fluoroquinolone, preferably ciprofloxacin or moxifloxacin.

In any one or more aspects or embodiments herein, the BP carbostyril compound can be composed of a carbostyril analog or substituent according to the following structure or formula (a),

wherein R is¹Can be

And wherein R²Can be

And wherein R³Can be H or OCH3 and can be,

and wherein R⁴It may be a compound of formula (I) and (II),

and wherein R⁵May be H or F.

As shown, the quinolone of formula (a) may 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 hydroxyl, amino, or thiol group. The quinolone may be conjugated directly or indirectly to the BP at the geminal carbon alpha substituent (X) of the BP, as illustrated by the following formula.

Conjugates between BP containing a-X and quinolones

X＝0、NH、NR¹、S

R¹May be an alkyl or substituted alkyl, aryl or substituted aryl group

Preferred BPs are those having a geminal hydroxyl group on the carbon between the two phosphonic acid groups. A generic analog of such BP is illustrated in fig. 25. In any one or more aspects, the bisphosphonate can be an ethylidene bisphosphonate moiety (etidronic acid) that can be substituted with a hydroxyl group (α -hydroxyl), an amino group, or a thiol. In some aspects, the bisphosphonic acid may include a p-hydroxyphenyl ethylidene group or a derivative thereof. In embodiments, the BP can be a clinically known BP, such as pamidronic acid, alendronic acid, risedronic acid, zoledronic acid, minodronic acid, neridronic acid, and etidronic acid, which can be unmodified or modified as described herein.

In any one or more embodiments, BP can be etidronic acid. Etidronic acid can be linked to a quinolone to form a quinolone antibiotic etidronic acid-ciprofloxacin (ECC) conjugate, such as in formula (41), or to form an etidronic acid moxifloxacin (ECX) conjugate, such as in formula (43) herein.

In any one or more aspects, the linker can be an alkyl carbamate or aryl carbamate linker. The linker may be a thiocarbamic-O-aryl ester or a thiocarbamic alkyl ester linker. The linker may be a thiourethane-S-aryl ester or a thiourethane alkyl ester linker. The linker may be a phenyl carbamate linker. The linker may be a thiocarbamate linker. The linker may be an O-thiocarbamate linker. The linker may be an S-thiocarbamate linker. The linker may be an ester linker. The linker may be a dithiocarbamate. The linker may be a urea linker. The linker may be R of formula (A) together with BP¹A moiety, and coupling said BP to said quinolone, as described herein. In any one or more aspects, the linker can be exemplified by any one of the following formulas (D) -formula (H), wherein: r²May be a quinolone or quinolone substituent or derivative and R¹May be BP, both as described herein; and R is³Can be substituted and unsubstituted alkyl, acetyl, benzoyl or other amides, phenyl and substituted phenyl, preferably H.

In some aspects, the BP is etidronic acid. In some aspects, the quinolone is ciprofloxacin or moxifloxacin. In some aspects, the BP is etidronic acid, the quinolone is ciprofloxacin, and the linker is an aryl or alkyl carbamate or a linker of formula (F), providing a compound of formula (41). In some aspects, the BP is etidronic acid, the quinolone is moxifloxacin, and the linker is an aryl or alkyl carbamate or a linker of formula (F), providing a compound of formula (43). In some aspects, the BP is etidronic acid, the quinolone is sitafloxacin or nerofloxacin, and the linker is an alkyl or aryl carbamate or a linker of formula (F), providing compounds of formula (44) or formula (45) herein.

In other aspects, the BP can be other BPs described herein, such as pamidronic acid, neridronic acid, olpadronic acid, alendronic acid, ibandronic acid, minodronic acid, risedronic acid, zoledronic acid, hydroxymethylbisphosphonic acid (HMBP), and combinations thereof.

In any one or more aspects, the bisphosphonic acids may have an α substituent substituted with a hydroxyl (α -hydroxyl), amino, or thiol. In any one or more aspects, the bisphosphonate may be an ethylidene bisphosphonate moiety (etidronic acid) that may be substituted with a hydroxyl group (α -hydroxyl), an amino group, or a thiol. In some aspects, the bisphosphonic acid may include a p-hydroxyphenyl ethylidene group or a derivative thereof. In embodiments, the BP can be a clinically known BP, such as pamidronic acid, alendronic acid, risedronic acid, zoledronic acid, minodronic acid, neridronic acid, and etidronic acid, which can be unmodified or modified as described herein.

In any one or more of the embodiments and aspects herein, the quinolone is a quinoloneThe ketones may have the general structure according to formula (A), wherein R¹May be a substituent, which includes: alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, phenyl, substituted phenyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, halogen, hydroxy, alkoxy, substituted alkoxy, phenoxy, substituted phenoxy, aryloxy, substituted aryloxy, 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-C₂₀Cyclic, substituted C₃-C₂₀A ring, heterocycle, substituted heterocycle, amino acid, peptide and polypeptide group, wherein R is²May be a substituent, which includes: alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, phenyl, substituted phenyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, halogen, hydroxy, alkoxy, substituted alkoxy, phenoxy, substituted phenoxy, aryloxy, substituted aryloxy, 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-C₂₀Cyclic, substituted C₃-C₂₀A ring, heterocycle, substituted heterocycle, amino acid, peptide and polypeptide group, wherein R is³May be a substituent, which includes: alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, phenyl, substituted phenyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, halogen, hydroxy, alkoxy, substituted alkoxyA group, phenoxy group, substituted phenoxy group, aryloxy group, substituted aryloxy group, alkylthio group, substituted alkylthio group, phenylthio group, substituted phenylthio group, arylthio group, substituted arylthio group, cyano group, isocyano group, substituted isocyano group, carbonyl group, substituted carbonyl group, carboxyl group, substituted carboxyl group, amino group, substituted amino group, amide group, substituted amide group, sulfonyl group, substituted sulfonyl group, sulfonic acid, phosphoryl group, substituted phosphoryl group, phosphono group, substituted phosphono group, polyaryl group, substituted polyaryl group, C₃-C₂₀Cyclic, substituted C₃-C₂₀Rings, heterocycles, substituted heterocycles, amino acids, peptides and polypeptide groups, and wherein R4 can be a substituent, including: alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, phenyl, substituted phenyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, halogen, hydroxy, alkoxy, substituted alkoxy, phenoxy, substituted phenoxy, aryloxy, substituted aryloxy, 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-C₂₀Cyclic, substituted C₃-C₂₀A ring, heterocycle, substituted heterocycle, amino acid, peptide and polypeptide group, and wherein R is⁵May be H or F.

BP can be conjugated to a quinolone (fluoroquinolone or non-fluoroquinolone, preferably fluoroquinolone) through a releasable linker. In any one or more aspects, the linker can be an alkyl carbamate or aryl carbamate linker. The linker may be a thiocarbamic-O-aryl ester or a thiocarbamic alkyl ester linker. The jointThe head may be a thiourethane-S-aryl ester or a thiourethane alkyl ester linker. The linker may be a phenyl carbamate linker. The linker may be a thiocarbamate linker. The linker may be an O-thiocarbamate linker. The linker may be an S-thiocarbamate linker. The linker may be an ester linker. The linker may be a dithiocarbamate. The linker may be a urea linker. The linker may be R of formula (A) together with BP¹A moiety, and coupling said BP to said quinolone, as described herein. In any one or more aspects, the linker can be exemplified by any one of the following formulas (D) -formula (H), wherein: r²May be a quinolone or quinolone substituent or derivative and R¹May be BP, both as described herein; and R is³Can be substituted and unsubstituted alkyl, acetyl, benzoyl or other amides, phenyl and substituted phenyl, preferably H.

In some aspects, the BP is etidronic acid. In some aspects, the quinolone is ciprofloxacin or moxifloxacin. In some aspects, the BP is etidronic acid, the quinolone is ciprofloxacin, and the linker is an alkyl or aryl carbamate or a linker of formula (F), providing a compound of formula (41) herein. In some aspects, the BP is etidronic acid, the quinolone is moxifloxacin, and the linker is an alkyl or aryl carbamate or a linker of formula (F), providing compounds of formula (43) herein. In some aspects, the BP is etidronic acid, the quinolone is sitafloxacin or nerofloxacin, and the linker is an alkyl or aryl carbamate or a linker of formula (F), providing compounds 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 hydroxyl, amino, or thiol group, and the quinolone is conjugated directly or indirectly to the BP at the geminal carbon alpha substituent (X) of the BP, as illustrated in the formula below.

Conjugates between BP containing a-X and quinolones

X＝0、NH、NR¹、S

R¹May be an alkyl or substituted alkyl, aryl or substituted aryl group

In any one or more aspects, BP is an α -OH containing BP that can be conjugated to a quinolone, such as a fluoroquinolone, at a geminal OH group on the BP, as shown below. In various aspects, a quinolone such as a fluoroquinolone may be conjugated directly (e.g., without the use of a linker other than C ═ O) to the geminal OH group of BP. In various aspects, the quinolone can be indirectly conjugated at the geminal OH group of the BP through a linker.

In some aspects, the compound may 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, that can include an amount of a BP quinolone compound or conjugate as described herein, elsewhere in any one or more aspects or embodiments. The amount may be an effective amount. The amount is effective to inhibit the growth and/or reproduction of bacteria. This amount is effective to kill the bacteria. Formulations, including pharmaceutical formulations, can be formulated for delivery by a variety of routes and can include a pharmaceutically acceptable carrier. Techniques and formulations are generally available in Remmington's Pharmaceutical Sciences, Meade Publishing Co., Easton, Pa. (20)^thEd.,2000) toThe entire disclosure of which is incorporated herein by reference. For systemic administration, injections are available, including intramuscular, intravenous, intraperitoneal, and subcutaneous injections. For injection, the therapeutic compositions of the present invention may 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 may be formulated in solid form and redissolved or suspended immediately prior to use. Lyophilized forms are also included. Formulations of BP quinolone conjugates, including pharmaceutical formulations, may be characterized as being at least sterile and pyrogen-free. These formulations include both for human use and veterinary formulations.

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, hydroxymethylcellulose, and polyvinylpyrrolidone, which do not deleteriously react with BP quinolone conjugates.

The pharmaceutical formulations can be sterilized and, if desired, can be mixed with adjuvants such as lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts for influencing osmotic pressure, buffers, colorants, flavors, and/or aromatic substances, and the like, which do not deleteriously react with the BP quinolone conjugate.

Another formulation includes the addition of BP quinolone conjugates to bone graft materials or interstitial fillers for the prevention or treatment of osteomyelitis, peri-implantitis or peri-prosthetic infection, and for alveolar protection after tooth extraction.

The pharmaceutical formulation may 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 for parenteral, intradermal, or subcutaneous administration may include the following components: sterile diluents 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 paraben; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents, such as ethylenediaminetetraacetic acid; buffers such as acetate, citrate or phosphate, and agents for adjusting tonicity such as sodium chloride or glucose. The adjustment of the pH can be with an acid or base, 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 suitable for injectable use, including pharmaceutical formulations, may 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 may include physiological saline, bacteriostatic water, Cremophor EMTM (BASF, Parsippany, n.j.) or Phosphate Buffered Saline (PBS). To the extent that easy injection is achieved, the injectable pharmaceutical formulation may be sterile and may be fluid. 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 fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, and pharmaceutically acceptable polyols such as glycerol, propylene glycol, liquid polyethylene glycol, and suitable mixtures thereof. 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 may be useful to include isotonic agents (e.g., sugars, polyalcohols such as mannitol, sorbitol, and sodium chloride) in the composition.

Sterile injectable solutions can be prepared by incorporating an amount of any of the BP quinolone conjugates described herein in a suitable solvent with one or a combination of ingredients enumerated herein, as required, followed by filter sterilization. In general, the preparation of the dispersion can be carried out by incorporating the BP quinolone conjugate into a sterile vehicle comprising the base 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 methods of preparation are vacuum drying and freeze-drying which yields a powder of the active ingredient plus any additional desired ingredient in 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 are used in the formulation. Such penetrants are generally known in the art, and include, for example, for transmucosal administration, surfactants (detergens), bile salts, and fusidic acid derivatives. Transmucosal administration can be accomplished through the use of nasal sprays or suppositories. For transdermal administration, the BP quinolone conjugate may be formulated into ointments, salves, gels, or creams as generally known in the art. In some embodiments, the BP quinolone conjugate may be administered through a transdermal delivery system, which may slowly release the BP quinolone conjugate for transdermal absorption. The penetration enhancer may be used to enhance transdermal penetration of the active factor in the conditioned medium. Transdermal patches are described, for example, in U.S. Pat. Nos. 5,407,713; U.S. patent nos. 5,352,456; U.S. patent nos. 5,332,213; U.S. Pat. Nos. 5,336,168; U.S. patent nos. 5,290,561; U.S. patent nos. 5,254,346; U.S. patent nos. 5,164,189; U.S. patent nos. 5,163,899; U.S. patent nos. 5,088,977; U.S. patent nos. 5,087,240; U.S. patent nos. 5,008,110; and U.S. patent No.4,921,475.

For oral administration, the formulations as described herein may be presented as capsules, tablets, powders, granules, or as a suspension or solution. The formulations may contain conventional additives such as lactose, mannitol, corn or potato starch, binders, crystalline cellulose, cellulose derivatives, acacia, corn starch, gelatin, disintegrants, potato starch, sodium carboxymethylcellulose, dibasic calcium phosphate, anhydrous or sodium starch glycolate, lubricants and/or magnesium stearate.

For parenteral administration (i.e., administration by a route other than through the digestive tract), the formulations described herein may be combined with a sterile aqueous solution that is isotonic with the blood of the subject. Such formulations can be prepared by dissolving the active ingredient (e.g., 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 to produce an aqueous solution, and then rendering the solution sterile. The formulations may be presented in unit-or multi-dose containers, such as sealed ampoules or vials. The formulation may be delivered by injection, infusion or other means known in the art.

For transdermal administration, the formulations described herein may be combined with skin penetration enhancers, such as propylene glycol, polyethylene glycol, isopropyl alcohol, ethanol, oleic acid, N-methyl pyrrolidone, and the like, which increase the permeability of the skin to the nucleic acid vectors (vectors) of the present invention and allow the nucleic acid vectors to permeate into the bloodstream through the skin. The formulations and/or compositions described herein may be further combined with a polymeric substance, such as ethyl cellulose, hydroxypropyl cellulose, ethylene/vinyl acetate, polyvinylpyrrolidone, and the like, to provide a composition in the form of a gel, which may be dissolved in a solvent such as methylene chloride, evaporated to a desired viscosity and then applied to a base material to provide a patch.

The formulations described herein may be combined with any xenograft (bovine), autograft (autologous) or allograft (cadaveric) material or synthetic bone substitute in order to be included in a bone graft substitute or bone gap filler to prevent local post-operative infection or post-operative graft failure and to provide sustained local release of antibiotics at the site of the graft. For example, the treating surgeon or clinician may premix the powder formulation with any commercially available bone graft substitute or with autograft at the bedside/couch side. This formulation may be further combined with any of the previously described formulations, and may be combined with products comprising hydroxyapatite, tricalciumphosphate, collagen, aliphatic polyesters, polylactic acids (PLA), polyglycolic acids (PGA), and Polycaprolactone (PCL), Polyhydroxybutyrate (PHB), methacrylates, polymethylmethacrylate, resins, monomers, polymers, cancellous bone allograft, human fibrin, platelet rich plasma, platelet rich fibrin, plaster of paris, apatite, synthetic hydroxyapatite, coral hydroxyapatite, wollastonite (calcium silicate), calcium sulfate, bioactive glass, ceramics, titanium, devitalized bone matrix, non-collagenous protein, collagen, and autolytic de-antigenic allogeneic bone. In this embodiment, the bone graft material combined with the BP quinolone conjugate may be in the formulation of a paste, powder, cement, gel, hydrogel, matrix, granule, freeze-dried powder, freeze-dried bone, demineralized freeze-dried bone, fresh or fresh frozen bone, pith-skin mixture, pellet, strip, embolic agent (plugs), membrane, lyophilized powder reconstituted to form a wet paste, pellet, sponge, block, slug (morsels), rod, wedge, cement (cement), or amorphous granule; of these, many can also be in an injectable formulation or as a combination of two or more of the above (e.g., an injectable paste of a sponge).

In another embodiment, the BP-quinolone conjugate may be combined with a factor-based bone graft comprising a natural or recombinant growth factor, such as transforming growth factor-beta (TGF- β), Platelet Derived Growth Factor (PDGF), Fibroblast Growth Factor (FGF), and/or Bone Morphogenic Protein (BMP). In another embodiment, the BP quinolone conjugate may be combined with a cell-based bone graft for regenerative medicine and dentistry including embryonic stem cells and/or adult 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. Thus, bone grafts having properties of osteoconduction, osteoinduction, osteopromotion, osteogenesis, or any combination thereof, may be used in combination with BP quinolone conjugates 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 may be provided in unit dosage form, such as tablets, capsules, single dose injections or infusion vials, or as a predetermined dose in a formulation as described above for mixing with bone graft material. Where appropriate, the dosage forms described herein may be microencapsulated. The dosage form may also be formulated to prolong or sustain the release of any of the ingredients. In some embodiments, the complex active agent may be a delayed release component. In other embodiments, the release of the secondary ingredient is delayed. Suitable methods for delaying release of the ingredients include, but are not limited to, coating or embedding the ingredients in a polymer, wax, gel, or like material. Delayed release dosage formulations may be prepared as described in standard references, such as "Pharmaceutical dosage forms" or "eds. Liberman et al. (New York, Marcel Dekker, Inc.,1989)," Remington-The science and practice of medicine ", 20th ed., Lippincott Williams & Wilkins, Baltimore, MD,2000, and" Pharmaceutical dosage forms and drug delivery systems ", 6th Edition, Ansel et al. (Media, PA: Williams and Wilkins, 1995). These references provide 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 may be any time from about one hour to about 3 months or more.

Coatings may be formed from water-soluble polymers, water-insoluble polymers and/or pH-dependent polymers, with or without water-insoluble/water-soluble non-polymeric excipients, in varying ratios to produce the desired release profile. Coatings may be applied to the dosage form (matrix or simply) including, but not limited to, tablets (compressed with or without coated beads), capsules (with or without coated beads), beads, granular compositions, the ingredients themselves formulated as, but not limited to, a suspension or sprinkled dosage form.

Examples of suitable coating materials include, but are not limited to, cellulosic polymers such as cellulose acetate phthalate, hydroxypropyl cellulose, hydroxypropyl methylcellulose phthalate, and hydroxypropyl methylcellulose acetate succinate; polyvinyl acetate phthalate, acrylic polymers and copolymers, and commercially available under the trade name

(Roth Pharma, Westerstadt, Germany), zein, shellac, and polysaccharides.

An effective amount

The formulation can include an effective amount of a BP quinolone compound or conjugate (effective to inhibit and/or kill bacteria) described herein in any one or more aspects or embodiments. In some embodiments, an effective amount of a BP quinolone conjugate described herein ranges from about 0.001pg to about 1,000g or more. In some embodiments, an effective amount of a BP quinolone conjugate described herein may range from about 0.001mg/kg body weight to about 1,000mg/kg body weight. In yet other embodiments, an effective amount of the BP quinolone conjugate may 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 to kill bacteria that are causative agents of osteomyelitis and all subtypes thereof (e.g., diabetic foot osteomyelitis), jaw necrosis, and periodontitis, including, but not limited to, Staphylococcus (Staphylococcus), Pseudomonas (Pseudomonas), agglomerans (agregabacter), Actinomyces (Actinomyces), Streptococcus (Streptococcus), Haemophilus (haemaphilus), Salmonella (Salmonella), Serratia (Serratia), Enterobacter (Enterobacter), clostridium (Fusobacterium), Bacteroides (Bacteroides), Porphyromonas (Porphyromonas), Prevotella (Prevotella), vemura (Veillonella), Campylobacter (Campylobacter), Peptostreptococcus (peptococcus), echococcus (eekenella), trellifera (Yersinia), Yersinia (Yersinia), yersinica), streptomyces (peptococcus), Streptococcus (peptococcus), Pseudomonas (yersinica), Yersinia (yersinica), corynebacterium (bacillus (Pseudomonas), corynebacterium (bacillus (Pseudomonas), Pseudomonas (Pseudomonas), Pseudomonas (yersinica), Pseudomonas (Yersinia), Pseudomonas (yersinica), Pseudomonas (p (Pseudomonas), any strain or species of the genera tanneria (tannorella) and Escherichia (Escherichia).

Methods of using BP quinolone conjugates

An amount (including an effective amount) of the BP quinolone compound, the conjugate, and the formulation thereof described herein in any one or more aspects or embodiments can be administered to a subject in need thereof. In some embodiments, a subject in need thereof can have a bone infection, disease, disorder, or symptom thereof. In some embodiments, a subject in need thereof may be suspected of having, or otherwise susceptible to having, a bone infection, disease, disorder, or symptom thereof. In some embodiments, a subject in need thereof may be at risk of developing osteomyelitis, osteonecrosis, periprosthetic infection, and/or peri-implantitis. In embodiments, the disease or condition may be osteomyelitis and all subtypes thereof, osteonecrosis, peri-implantitis, or periodontitis. In some embodiments, a subject in need thereof has bone infected with a microorganism (such as a bacterium). In some embodiments, the bacterium can be any strain or species of staphylococcus, pseudomonas, coacervate bacillus, actinomyces, streptococcus, haemophilus, salmonella, serratia, enterobacter, clostridium, bacteroides, porphyromonas, prevotella, veyonococcus, campylobacter, peptostreptococcus, akerma, treponema, alisteribacter, micromonospora, yersinia, tannophila, and escherichia. In some embodiments, the bacteria may form a biofilm. In some embodiments, osteomyelitis in a subject in need thereof can be treated by administering to the subject in need thereof an amount (such as an effective amount) of a BP quinolone conjugate described herein or a formulation thereof. In some embodiments, the compositions and compounds provided herein may be used for osteonecrosis treatment and/or prevention, stretch osteogenesis, fracture repair, repair of critical upper socket defects, jaw bone reconstruction, and any other reconstruction or repair of bone and/or joint.

Administration of the BP quinolone compound or conjugate is not limited to a single route, but may include administration by a variety of routes. For example, exemplary administration by various routes includes a combination of intradermal and intramuscular administration, or a combination of intradermal and subcutaneous administration, among others. The multiple administrations may be sequential or simultaneous. Other modes of administration by various routes will be apparent to those skilled in the art.

The pharmaceutical formulation may 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 a subject by known procedures, including, but not limited to, by oral administration, sublingual or buccal administration, parenteral administration, transdermal administration, by inhalation, by nasal delivery, vaginally, rectally, and intramuscularly. The formulations or other compositions described herein may be administered parenterally, by extrafascial, intracapsular, intradermal (intracutaneous), subcutaneous, intradermal, intrathecal, intramuscular, intraperitoneal, intrasternal, intravascular, intravenous, parenchymal, and/or sublingual delivery. Delivery may be by injection, infusion, catheter delivery, or some other means, such as by tablet or spray. In the case of infection-resistant bone graft materials at the surgical site, delivery may also be by a carrier such as hydroxyapatite or bone. Delivery may be by attachment or other bonding with bone graft material.

Examples

Having now described embodiments of the present disclosure, the following examples, in general, describe some additional embodiments of the present disclosure. While embodiments of the present disclosure have been described in connection with the following examples and the corresponding text and drawings, there is no intent to limit embodiments of the present disclosure to such illustrations. On the contrary, the intent is to cover all alternatives, modifications and equivalents as included within the spirit and scope of the embodiments of the disclosure.

Introduction to the design reside in

Infectious bone disease, or osteomyelitis, is a major problem in human and veterinary medicine worldwide and can be catastrophic due to potentially Limb threatening sequelae and death (Lew, et al, Osteomyelitis. Lancet 2004; 364: 369-79; Desrochers, et al, Limb amplification and prosthetisis. vet Clin North Am Food animal practice 2014; 30: 143-55; Stodley, et al, orthopaedics biological in-fection. Current Orthoverse practice 2011; 22: 558-63; Huang, et al, Chonic genetic engineering in great-term mortalities, bone marrow in the same bone marrow, BMC 2016: 16). The treatment of osteomyelitis is mainly antimicrobial and is usually long-term and in many cases controls the infection by surgical intervention. In most cases of long bone osteomyelitis, the causative pathogen is the biofilm of staphylococcus aureus; unlike their planktonic (free-floating) counterparts, these microorganisms are, by definition, synostotic (FIG. 1) (Wolcott, et al, Biofilms and chrononic infections. J. Am Med. Assoc 2008; 299: 2682-.

The biofilm-mediated nature of osteomyelitis is important in clinical and experimental settings because many biofilm pathogens are non-culturable and exhibit altered phenotypes in terms of growth rate and antimicrobial resistance (compared to their planktonic counterparts) (Junka, et al, Microbial biolofilms area available to depression in the ability of host immunity in vitro. J. Oral Maxillac of Surg 2015; 73: 451-64; Herczegh, et al, Ostepsorptive biofilm differentiation derivatives of fluoroquinolone antibiotics. J. Med. Chem 2002; 45: 2338-41). The difficulty in eliminating biofilms with conventional antibiotics explains why antimicrobial therapy, which is generally successful with higher success rates, has been unsuccessful in orthopedic infections with the development of drug-resistant biofilm pathogens, poor penetration of the antimicrobial agent into the bone, and adverse events associated with systemic toxicity (Buxton, et al, Bisphostate-ciprofloxacin bone bound to Skelite is a protocol for enhancing experimental local antibacterial delivery to injected bone, Br J Surg 2004; 91: 1192-6).

To overcome many of the challenges associated with Osteomyelitis treatment, there is an increasing interest in drug Delivery methods using bone-targeting conjugates to achieve higher or more sustained Local Antibiotic treatment concentrations in bone while minimizing systemic exposure (Panagopoulos, et al, Local Antibiotic Delivery Systems in diabetes Foot tissue: Time for One Step Beyond int J Low Extrem Wounds 2015; 14: 87-91; Puga, et al, Hot melt poly-epsilon-capto/poloxamine imaging materials for sustained Delivery of ciprofloxacin. Fluoroquinolone antibiotics conjugated to bone-adsorbed bisphosphonic acids (BP) represent a promising approach because the safety clinical follow-up record for each component is long and their biochemical properties are superior (Buxton, et al, Bispho-cifloxacin bound to skin is a protocol for enhancing experimental local antibiotic bound. Br J Surg 2004; 91: 1192-6). In early studies of the fluoroquinolone family in this context, ciprofloxacin showed the best binding and microbiological properties when bound to BP (Herczegh, et al, Osteoadesorptive bisphosphate derivatives of fluoroquinolone antibiotics. J Med Chem 2002; 45: 2338-41). Reuse of ciprofloxacin in this context has several advantages: it can be administered orally or intravenously, is relatively bioequivalent, has broad spectrum antimicrobial activity against pathogens including the most common osteomyelitis, exhibits bactericidal activity at clinically achievable doses, and is the least expensive drug of the fluoroquinolone family (Houghton, et al, Linking bisphonates to the free amino groups in fluoroquinolones: preparation of optoelectronic drugs for the prevention of osteoporosis. 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

The following are examples of non-fluorinated quinolones.

Example 2:

dental implants are an important part of modern dental practice, and it is estimated that up to 3500 tens of thousands of americans have lost all teeth in either one or both jaws. By 2022, the total market for these implants for replacement and reconstruction of teeth is expected to reach $ 42 billion. While most implants have been successful, some of these prostheses fail due to peri-implantitis, resulting in failure of the supporting bone. Periimplantitis has a bimodal incidence, including early (< 12 months) and late (> 5 years) failure; both of these critical failure points are largely the result of bacterial biofilm infection on and around the implant. Periimplantitis is a common cause of implant failure. Dental implant failure is often due to biomechanical or biological/microbiological reasons. From the current literature, it is difficult to determine the prevalence of peri-implantitis, the most severe form of microbial-related implant disease that causes destruction of supporting bone. However, recent studies have shown that peri-implantitis is an increasingly serious problem with increasing prevalence⁴. A recent study of 150 patients followed for 5 to 10 years showed that peri-implantitis occurred at approximately 17% and 30%, respectively, suggesting that this is a significant problem⁵. Early implant failure or lack of bony union is a separate problem and occurs in about 9% of implanted jawbones⁶. This is more common in the maxilla⁶And are associated with bacterial infections (e.g., periodontitis) during or near surgery and other recognized and modifiable risk factors such as smoking, diabetes, excessive flutemastin, and oral hygiene²。

Biofilm infection may be withThe pathogenesis of peri-implantitis is related. Biofilm infection represents a unique therapeutic problem and is often difficult to diagnose, resistant to standard antibiotic therapy, resistant to host immune response, and results in persistent refractory infection⁷. The biofilm hypothesis of infection has been expanding since the early elucidation of bacterial growth in matrix-supported communities^8,9. It has been demonstrated that over 65% of chronic infections are caused by bacteria growing in biofilms⁷. This means that approximately 1200 million people in the united states suffer from these infections each year, and nearly 50 million people die from these infections. Periimplantitis and periodontitis are the most commonly encountered biofilm infections. Periimplantitis has been found to be a relatively simple infection with less community diversity (and key pathogens) than periodontitis infections¹⁰. Typically, gram-negative species predominate¹¹. Other bone or bone infections, including jaw infections, are also caused by bacterial biofilm communities¹²Making the techniques developed herein equally applicable to these diseases.

Current methods of treating peri-implantitis have their limitations. Although peri-implantitis has multiple causes, the main cause is bacterial biofilm. There is currently no generally accepted guideline or protocol for peri-implantitis treatment, and many clinical protocols for bacterial peri-implantitis treatment include local and systemic antibiotic delivery¹³And surgical debridement of lesions, including restorative bone grafts using bone graft substitutes^14,15. However, clinical experience has shown that it is difficult to advance even a local antibiotic delivery device to the bottom of the deep peri-implant pocket and the infected jaw bone, or to allow sufficient penetration of systemic antibiotics into the infected jaw bone to kill biofilm pathogens¹⁶This is mainly due to the intrinsic poor bone (and peri-implant) biodistribution or pharmacokinetics of antibiotics¹⁷. In previous long-term studies, even when infected implants were cleaned locally with antiseptic and systemic antibiotics were administered, there was additional loss of supporting bone in more than 40% of the advanced peri-implant inflammatory lesions¹⁵。

In addition, long-term systemic antibiotic treatment may lead to systemic toxicity or adverse reactions as well as drug resistance. Therefore, it has become common practice for clinicians to use local delivery systems to achieve higher therapeutic antibacterial concentrations in bone. For example, a dentist uses minocycline (minocycline) or doxycycline powder (e.g., as

) Or chlorhexidine solutions (e.g. of the type

) Chair-side mixing with bone graft material for local delivery¹⁸. Such methods are mere slurries and do not form strong bonds between antibiotics and bone substitutes as in the BioVinc method, and therefore suffer from relatively early clearance and less efficient pharmacokinetics as previously described. In addition, researchers have used several biodegradable and non-biodegradable topical antibiotic delivery systems¹⁹. However, these methods have some limitations, for example, non-biodegradable methods (e.g., polymethylmethacrylate fluanten) 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 attention, however, their clinical effectiveness in treating peri-implantitis has not been well documented³. Even when effective antimicrobial/antiseptic agents are used to treat peri-implantitis in the jaw, such as topical chlorhexidine delivery, the effect on the therapeutic effect is small, as demonstrated in prospective animal and human studies^15,17. Taken together, these data further support the poor pharmacokinetics of the aforementioned antibiotics in bone and highlight the need for osseointegration/bone targeting and sustained antibiotic release strategies.

BP-conjugates

Given the limitations of current therapeutic approaches, the development of antimicrobial agents targeting bone/biofilm is a significant advance in the art. The BP-antibiotic (BP-Ab) conjugates provided herein can overcome many of the challenges associated with poor antibiotic pharmacokinetics or bioavailability in bone and within synoviously bonded biofilms. These compounds can reduce infection by "targeting and release methods," which can reduce concerns about systemic toxicity and/or drug exposure in other (e.g., non-infected) tissues. The BP-Ab conjugate can be incorporated into a bone graft substitute. The BP-Ab may be a BP-fluoroquinolone conjugate. In some cases, BP-Ab may be bisphosphonate-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 incorporated into a bone graft, the BP-Ab bone graft material may also be referred to as a BP-Ab-bone graft. For example, when the antibiotic is fluoroquinolone, it may be referred to as a BP-FQ-bone graft. These compounds are effectively adsorbed on Hydroxyapatite (HA)/bone and enable sustained release over time and antimicrobial effectiveness against biofilm pathogens. The compounds provided herein, as well as graft materials incorporating the compounds, can be used as anti-infective bone graft substitutes for the adjuvant treatment or prevention of peri-implantitis. The conjugate will be released locally from the graft material with sustained release kinetics and will lyse in the presence of bacterial activity or osteoclast activity, as we have previously demonstrated in vitro and in vivo in other results provided elsewhere herein. In this way the graft can provide greater local concentrations of FQ (such as ciprofloxacin) than current delivery routes. In summary, the compounds and bone graft materials provided herein can comprise an antibiotic conjugated to a safe or pharmacologically inactive (non-absorption inhibiting) BP moiety that binds to calcium/HA in the graft material through strong multidentate electrostatic interactions, and the antibiotic is released over time; it does not simply represent a topical antibiotic that is mixed with existing bone graft material only in the form of a slurry, as some current clinical methods do in this context. Thus, this chemisorbed drug attached to calcium phosphate minerals (HA) is an important advance in the field and overcomes many limitations in delivering antibiotics to peri-implant bone to achieve effective bactericidal activity against biofilm pathogens.

The general concept of targeting bone by attaching an active drug molecule to BP has been reviewed in one section³⁰Discussed in (1). However, since the results of early attempts were systemically unstable prodrugs or non-cleavable conjugates, which were found to be largely inactive by interfering with pharmacophore requirements, no FDA-approved drugs have been developed at this time. In the field of quinolones, Herczegh describes a prominent example in which the antibacterial properties of fluoroquinolones are reduced by conjugation in the case of stable BP-linked homologues^31-32. Thus, targeting and releasing linker strategies are needed.

In recent years, pharmacochemical strategies using less stable ligation techniques have begun to emerge. Others have fluoroquinolones attached to several different BP moieties through carboxylic acid groups. They found that the antibiotics moxifloxacin and the hydroxyacetamide ester prodrug of gatifloxacin reduced infection when used prophylactically in the rat osteomyelitis model³³. This same group used acyloxycarbamate and propiophenone based linkers to tether the same antibiotics to a simple BP system via amine functionality³⁴. They showed that these conjugates also outperform the parent antibiotics in inhibiting the establishment of infection using the same prophylactic rat model. Targana team³³Has been associated with the glycopeptide antibiotic oxomycin³⁵Several of these prodrug strategies have been performed for use together. This dual function drug appears to have some degree of efficacy in preventing infection. However, to date, they have not published studies showing their ability to treat established infections, and they have not published the pharmacokinetics of the prodrug. It is believed that because their drug candidate selection is based in part on plasma instability, these analogs are too unstable in the blood to be fully successful with this therapeutic approach. Thus, it is believed that these compounds developed by these groups do not achieve effective local concentrations of antibiotics.

BCC compounds (FIG. 3) can convert ammoniaThe phenyl moiety of the phenyl formate linker is directly incorporated into the BP portion of the molecule. The release kinetics can be modified or tuned by modifying the benzene ring with electron withdrawing or electron donating groups, which can alter the reliability of the linker. Furthermore, the BP core lacks efficacy as an anti-resorptive agent and therefore does not carry the risk of drug-related osteonecrosis of the jaw as do more potent nitrogen-containing BP drugs (e.g., zoledronic acid)^39,40. This targeting and release strategy using a phenyl carbamate linker is demonstrated here and in other examples herein to be likely to release the active drug directly into bacterial biofilms in the bone environment. Bone targeting is so effective that it is better than ciprofloxacin against biofilms growing on HA bone matrix substitutes, and also better against planktonic cultures grown in plastic containers. It was found that similar conjugates made with non-cleavable amide linkages (bisphosphonic acid-amide-ciprofloxacin), which discard the phenolic oxygen of the carbamate, had little effect on bacterial growth in any case, indicating that active cleavage of the conjugate is required for antimicrobial activity.

Example 3

Design and synthesis of additional BP-Ab conjugates (fig. 4). Additional BP-Ab conjugates can be designed by conjugating a carbamate-based linker (e.g., carbamate, S-thiocarbamate, and O-thiocarbamate) with BP (e.g., 4-hydroxyphenylethylidene BP (BP 1, fig. 4), its hydroxyl-containing analog (BP 2, fig. 4, with higher bone affinity), and pamidronic acid (BP 3, fig. 4) using, for example, ciprofloxacin and moxifloxacin fig. 5 shows an exemplary synthetic scheme for synthesizing a BP-Ab conjugate with an O-thiocarbamate linker fig. a conjugate with an S-thiocarbamate linkage (slightly more labile) can be obtained by isomerizing a conjugate with an O-thiocarbamate linkage by a Newman-Kwart rearrangement (reference 47, 48). Preliminary chemical experiments have been performed to demonstrate the feasibility of rapidly synthesizing these targets. Thus the additive bone affinity was well demonstrated using a-OH containing BP (49). The addition of bone affinity will enhance the concentration of bone surface conjugates and promote higher short and long term local drug concentrations. For the synthesis of conjugates with α -OH containing BP (BP 2 and pamidronic acid, fig. 4), α -OH can be protected with a tert-butyldimethylsilyl (TBS) group (scheme 2, fig. 6) (50) since α -OH bisphosphonates rearrange easily into phosphono-phosphates. The α -O-TBS BP 2 ester was then activated by 4-nitrophenyl chloroformate and reacted similarly with ciprofloxacin or moxifloxacin as shown in fig. 5. For α -O-TBS BP 3 ester, BP and the antimicrobial agent are linked using a linker with a phenolic group (e.g., linker 1 (resorcinol), linker 2 (hydroquinone), linker 3 (4-hydroxyphenylacetic acid), fig. 20), and the synthetic route using linker 3 is illustrated here as an example (scheme 3, fig. 7). All BP-Ab conjugates were characterized by 1H, 31P, 13C NMR, MS, HPLC and elemental analysis to ensure identity (identity).

The mineral binding affinity of the BP-Ab conjugate can be determined. Briefly, large particle size (1.4-1.6mg) inorganic bovine bone (1-2 mM uniformly) can be accurately weighed (1.4-1.6mg) and suspended in assay buffer containing appropriate volumes [ 0.05% (wt/vol) Tween20, 10. mu.M EDTA and 100mM HEPES pH 7.4]4mL clear vial for 3 hr. This bone material can then be incubated with increasing amounts of BP-Ab (0, 25, 50, 100, 200 and 300. mu.M). The sample in assay buffer can be gently shaken at 37 ℃ for 3 h. After the equilibration period, the vial may be centrifuged at 10,000rpm for 5min to separate the solids and supernatant. The supernatant (0.3mL) was collected and the concentration of the equilibration solution was measured using a Shimadzu UV-VIS spectrometer (275nm wavelength). Fluorescence emission can also be used to calculate binding parameters. In the absence of HA, as a control, nonspecific binding can be measured using a similar procedure. The amount of parent drug/BP-Ab conjugate bound to HA was deduced from the difference between the input and the amount recovered in the supernatant after binding. Binding parameter (K)_dAnd Bmax, representing the equilibrium dissociation constant and the maximum number of binding sites, respectively) can be calculated using the PRISM program (Graphpad, usa) and measured in 5 independent experiments. Equilibrium dissociation constant (K)_d) Less than 20. mu.M (. about.2X K of parent BP)_d) The compounds of (4) may be preferred. The binding parameters of BP-Ab can be assessed using a two-sample t-assay. The amount of sample in each set (n-5) can be used for detectionThe amount of effect to this hypothesis was 1.72, 80% efficacy, and 0.05 for unilateral type I error.

The linkage stability of the BP-Ab conjugate can be determined. Briefly, the ligation stability of each BP-Ab conjugate can be tested in PBS buffer with different pH (pH 1,4, 7.4, 10) and human or canine serum. BP-Ab can be suspended in 400. mu.L of the above PBS or 400. mu.L of 50% (v/v in PBS) human or canine serum. The suspension/solution can be incubated at 37 ℃ for 24h and centrifuged at 13000rpm for 2min, and the supernatant recovered. Methanol (5 x volume relative to the supernatant) can be added to each supernatant and the mixture can be vortexed for 15min to extract the released fluoroquinolones. Then, the mixture may be centrifuged at 10000rpm for 15min to precipitate insoluble materials. 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 fluoroquinolone released determined by UV-VIS measurement as previously described. The percentage of fluoroquinolone drug released may then be calculated based on the input amount and the measured amount of drug released. If the concentration is sufficient, the identity of the released drug can be confirmed by LC-MS analysis and/or NMR.

Inhibition of biofilm growth on HA discs in vitro can be determined. Briefly, commercially available HA powder can be used for the manufacture of custom discs. Powder pellets with a diameter of 9.6mm can be pressed without binder. Sintering may be carried out at 900 ℃. Tablets may be compressed using a general purpose test system for static tensile, compression and flexural testing (Instron model 3384; Instron, Norwood, Mass.). The quality of the manufactured HA discs can be checked by confocal microscopy and micro computer tomography (micro CT) using an LEXT OLS4000 microscope (Olympus, Center Valley, PA) and a Metrotom 1500 micro tomograph (Carl Zeiss, Oberkochen, germany), respectively. The HA discs can then be introduced into each BP-Ab conjugate and ciprofloxacin/moxifloxacin at the following concentrations [ mg/mL ]: 800. 400, 200, 100, 50, 25, 10, 5,1 and standing for 24h/37 ℃. After incubation, the HA discs can be removed and introduced into 1mL PBS and placed in a gentle shaking shaker for 5 min; subsequently 3 rinses were performed in this manner. After washing, 1mL of Aa suspension can be introduced onto the dish and left at 24h/37 ℃. The disc may then be rinsed to remove unbound bacteria and vortexed. Serial dilutions of the obtained suspension can then be plated on modified TSB agar plates for culture and colony growth counted after 24 h.

The bone fusion effect of the BP-FQ-bone graft on critical dimensions can be evaluated in an upper dental socket periimplant defect model for bone grafting. Briefly, in this split oral design, mandible PM2-PM4 was bilaterally extracted from 6 beagle dogs (3 males, 3 females) and allowed to heal for 12 weeks. Cristae dissection was performed after the flap was everted. An osteotomy was performed to create a 6mm superior alveolar defect. Implant site osteotomy preparations were performed at each premolar region by successively cutting with an internal irrigation drill (intercalary irrigated drills) of progressively changing diameter under sufficient irrigation. The implant (AstraTech Osseoped) was implanted in this manner

3 x 11mm) was placed at each side of the PM2-PM4 so that the implant was positioned 4mm above the crest relative to the defect created and at the same distance from the buccal cortex lamina. Dogs were randomized into 3 different groups (2 dogs each):

1. inorganic bovine bone (1g large particle size 1-2mm) chemisorbed with BP-fluoroquinolone was used on the right side and collagen plugs (negative control) were used on the left side.

2. Inorganic bovine bone (1g large particle size 1-2mm, positive control) was used on the right side, and collagen plugs (negative control) were used on the left side.

3. With BP-fluoroquinolone chemisorbed on the right side

(1g large particle size 1-2mm), and used on the left side

(1g large particle size 1-2mm, positive control).

The chemistry derived from the above experiments andthe antimicrobial assay results can inform the calculation of the ideal standardized amount of conjugate for adsorption to the graft material for use in all in vivo experiments described herein. Early calculations based on preliminary results predictions indicate that adsorption of 5mg or less of the conjugate to 1g of graft material will provide 2-3 orders of magnitude higher bactericidal activity than the MIC of the pathogen tested. Our BP-fluoroquinolone conjugates are applicable to a range of bone graft materials, including commercially available bone graft materials, for example

Therefore, we selected homemade inorganic bovine bones and BioOss in the study as positive controls to demonstrate the broad application of the conjugates. All defects (according to the above group) were filled with standardized amounts of biomaterial up to the platform of each implant on both sides and used

The membrane covers the graft and implant for improved stability. The flap is closed in a tensionless manner by means of a periosteal release incision, an internal cushion and finally a marginal single interrupted suture (PTFE 4.0, Cytoplast, usa). At this point, microscopic CT is taken and animals are monitored clinically for inflammation and adverse events. In addition, as described in the experiments that follow, PK studies were performed on these animals to assess any systemic exposure to components within the graft material (e.g., intact conjugate, BP, antibiotic or linker). Animals were sacrificed and the mandible was excised after 12 weeks and examined by micro CT followed by histological preparation. Baseline micro-CT scans of the jaw bone were compared to post-experimental scans. Quantitative 3D volume micro CT and histomorphometric analysis were performed to examine the volume of new bone present at the peri-implant site, as well as first bone-to-implant contact rate/area, total defect area, regeneration area within total defect area, regenerated bone, residual bone substitute material, percentage of mineralized tissue, soft tissue and voids. Finally, autopsy was performed to obtain necropsy assessments of organs and systems, gross and microscopic to obtain tolerance problems caused by topical oral treatmentThe following signs.

The antimicrobial effectiveness of the BP-FQ-bone graft can be evaluated in a canine periphyto-arthritis model. Briefly, in this split mouth design, the mandibles PM2-PM4 were extracted bilaterally using minimally invasive techniques on 8 beagle dogs (4 males, 4 females; 48 teeth total). After 3 months of healing, the mucoperiosteal flap was lifted on both sides of the jaw and prepared for implant osteotomy by successive cuts with sufficient external rinsing at each premolar region with an internal rinsing burr of gradually changing diameter. Using non-embedding technology, the implant (AstraTech Osseospeed)

3 x 11mm) was installed at each site. The order of implant placement was the same on both sides, but the randomization schedule between the computer-generated dogs was randomized. The healing abutment is attached to the implant and the flap, approximated with absorbable sutures. The plaque control regimen included brushing the teeth four times a week with toothpaste. Twelve weeks after implant placement, just before the onset of experimental peri-implantitis, microbiological samples were taken from all peri-implant sites with sterile paper twist (dentspray, Maillefer, size 35, balaigues, switzerland) and immediately placed in Eppendorf tubes (Starlab, Ahrensburg, germany) for microbiological analysis. As we detailed earlier, microbiological analysis was performed by DNA extraction and 16S rRNA PCR amplification (55). PCR amplicons were sequenced using the Roche 454GS FLX platform and the data were analyzed with quantitative insight microbial ecology (QIIME) software package (56). Colony forming unit counts (CFU/mL) in the samples were determined as described earlier in our phase I study. At this time, experimental peri-implantitis started as follows. As we performed in our previous experiments on rat animal models and our previous studies of peri-implantitis of animals, the formation of a biofilm of the key periodontal pathogen actinobacillus conglobata (Aa) is initiated on the in vitro healing abutment, which is not endogenous to the canine flora. Placing the healed abutment incubated with biofilm on the implant and placing a cotton ligature around the neck of the implant near the edgeThe rim position. After 10 weeks of bacterial infection, the samples were again taken and analyzed as before, and a micro-CT scan was taken as a baseline for peri-implant inflammatory defects. Treatment of this experimental peri-implantitis model was initiated by surgical debridement of all implant sites by lifting the full thickness bucco-lingual flap, removal of any existing stones from the implant surface using a gas powder abrasion device, and rubbing the implant surface with gauze soaked in chlorhexidine gluconate 0.12%. Animals were divided into 4 groups (2 dogs each) as follows:

1. inorganic bovine bone with chemisorbed BP-fluoroquinolone was used on the right (1g large particle size 1-2mm) and collagen plugs were used on the left (negative control).

3. Inorganic bovine bone with chemisorbed BP-fluoroquinolone was used on the right (1g large particle size 1-2mm) and an antimicrobial release device (100mg topical minocycline, positive control) was used on the left.

4. With chemisorbed BP-fluoroquinolone on the right

(1g Large particle size 1-2mm) (positive control), and the left side using antimicrobial release device (100mg local minocycline, positive control).

Future researchers who do data analysis are unaware of treatment group assignments. Standardized and equivalent amounts of antimicrobial agents were used in the treatment groups. After treatment, the flap was repositioned and sutured (PTFE 4,0, Cytoplast, usa), and oral hygiene measures were reestablished after 1 week after the stitches were removed. At 3 months post-surgery, clinical and micro-CT scan examinations were again performed and a microbial sample was taken at this point for analysis as also described above. Six months after peri-implantitis surgery, animals were euthanized and subjected to micro-CT scanning, and the jaw bone was excised for assessment of histopathological parameters as detailed in the partial "superior alveolar peri-implant defect model on critical dimension". Inflammation scores were determined from histological sections as detailed previously (reference 57) for correlation with clinical and radiological outcomes.

Statistical analysis: statistical calculations were performed with SPSS 22.0(IBM, Armonk, NY) and Excel2016(Microsoft Corporation, Redmond, WA). Efficacy analysis was performed to determine sample size estimates for all animal studies using G Power 3 software⁵⁸. After data is collected from these animal studies, the quantitative results are first analyzed using descriptive statistics to understand the distribution of the data (parametric or non-parametric) and to generate mean, standard error, standard deviation, kurtosis and skewness and 95% confidence levels. Data were analyzed using Kruskall-Wallis test, ANOVA or mixed linear model where applicable, and statistical significance was performed at a 0.05 level when multiple groups were compared. Post hoc analysis was also performed using unpaired t-test and Dunnett's test for multiple comparisons to further confirm the results. All animal experiments were described using ARRIVE guidelines for reporting animal studies to ensure the quality, confidence, efficacy and reproducibility of results⁵⁹。

BCC (6) can be evaluated for drug compound and ingredient stability as well as in vitro ADME. These data can help determine if there is likely to be any large difference between human metabolism and experimental animals. Incubate with human, rat and dog liver microsomes and hepatocytes 6, and then perform LC/MS analysis on the metabolite mixture. The metabolic profile of ciprofloxacin is known^62,63And therefore our focus is on any metabolite of the BP portion of the molecule and any metabolite of the parent (e.g. piperazine ring cleavage, known for ciprofloxacin). Once the metabolites were determined in vitro, these compounds were determined in vivo at steady state using plasma samples from the other in vivo experiments described above.

The toxicology of BCC (6) can be assessed in rats and dogs to determine NOAEL. To determine NOAEL and Maximum Tolerant Dose (MTD) in rats and dogs, we first performed a dose range study. Groups of 6 rats (3 males, 3 females) were given a single intravenous dose of 10mg/kg of 6, or based on our best assessment at this time. By increasing the dose incrementally by doubling until acute toxicity (MTD) is found,this dose was then reduced by 20% in order until no effect, which would be the NOAEL of the compound. Toxicity was evaluated as mild, moderate or large and MTD was defined as moderate toxicity in 2 animals or large toxicity in 1 animal⁶⁴. Animals were subjected to 5 day duration body weight and clinical observations. After 5 days, animals were euthanized and necropsied to assess organ weight and histology (15 sections including liver and kidney based on clinical BP toxicology). Similar dose range studies were also performed on dogs (1/sex, starting with an equivalent dose, as determined by the allometric scaling method (4 mg/kg), assuming 250g in rats and 10kg in dogs) and including hematology and clinical chemistry in addition to the same endpoint studies as in rats. This may use a total of 4-6 queues.

Extended acute toxicity tests, including pharmacokinetic and recovery tests, can be performed in groups of animals with NOAEL and MTD. A group comprising 48 rats of 10/sex can be used for each dose to assess toxicity, with 9/sex for toxicity kinetics, and 5/sex for recovery. After administration of each dose, pharmacokinetics were determined at 6 time points (randomly selected 3 rats from male or female per time point). Time points were 5, 30, 60, 120min, 12hr and 24hr post-administration. The recovered animals were observed for 14 days and then assessed for organ weight and histology as in the above study. From the pharmacokinetic studies, PK parameters were determined by non-compartmental analysis (NCA) including Cmax, AUC and half-life. The same experiment was performed in canines, but included a total of 10 animals (3/sex for dosing and 2/sex for recovery) and blood was drawn from each animal multiple times at the same time points as for rats. The maximum allowable exposure from the bone graft/BP-fluoroquinolone conjugate was calculated using the AUC for NOAEL in canines, as described in target 2, and PK experiments in canines were used to determine if there was systemic exposure of 1/100 above this level.

For the population model, a unique 3-compartmental (blood/urine/bone) mathematical model of BP pharmacokinetics has been clinically validated and applied to the current project⁶⁵. In the canine study, we sampled the bone in each animal (jaw) at euthanasiaBone and femur), tendon (gastrocnemius) were used to determine BP and fluoroquinolone concentrations. We combined these data with our model to describe the time course in dogs. According to this model, we can simulate the expected exposure of bone and cartilage to both BP and fluoroquinolone with alternative or repeated administrations. This may provide information for subsequent human administration. The non-parametric adaptive mesh (NPAG) algorithm of the adaptive gamma, performed in the Pmetrics package of R (Laboratory of Applied pharmaceuticals and Bioinformatics, Los Angeles, Calif.), is used for all PK model-fitting procedures as previously described^66-68. Analytical error (SD) was considered using an error polynomial as a function of measured concentration, and comparative performance evaluation was done using Akaike's information criteria, regression of observed versus predicted concentrations, visualization of PK parameters-covariate regression, and rules of brevity.

Example 4

The BP-Ab conjugate can be incorporated into grafts and graft devices. In embodiments, one or more BP-Ab conjugates can be incorporated into an approved bone graft product, such as

(Geistlich Pharma AG, Switzerland) or

Bovine bone material (BioHorizons, Birmingham, AL), and the like. One or more BP-Ab conjugates can be used as a dental bone graft substitute in admixture with a support material. The product will include the conjugate adsorbed on inorganic bovine bone material. Such a material would allow for the local delivery of antibiotics to the area where the bone graft is implanted to reduce the rate of bacterial infection and associated dental pathologies, such as peri-implantitis and other infections. Dental applications of our product may include not only peri-implantitis treatment, but also post-extraction alveolar protection, crest or sinus lifting, periodontitis prevention or treatment, osteomyelitis or osteonecrosis treatment or prevention, or other oral and periodontal surgical applications where such bone grafts are beneficial. The BP-fluoroquinolone conjugate material is tightly boundClosely adsorbed on the bone graft substitute and our preliminary data show that in case of infection it is released continuously to the area of bone destruction, which allows our product to deliver antibiotics more efficiently to the site of infection with negligible to no systemic exposure to either component of the conjugate compound.

The graft material may also be beneficial for non-dental implants, such as sinus implants.

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Example 5:

this example demonstrates various BP conjugate compounds and synthetic schemes. The BP-carbamate-moxifloxacin BP conjugate and the synthesis scheme are shown in fig. 8. Fig. 9 shows BP-carbamate-gatifloxacin BP conjugates and the synthetic scheme. Figure 10 shows BP-p-hydroxyphenylacetic acid-ciprofloxacin BP conjugates and the synthetic scheme. FIG. 11 shows BP-OH-ciprofloxacin BP conjugate and the synthetic scheme. Fig. 12 shows BP-O-thiocarbamate-ciprofloxacin BP conjugates and the synthetic scheme. Fig. 13 shows BP-S-thiocarbamate-ciprofloxacin BP conjugates and the synthetic scheme. Figure 14 shows BP-resorcinol-ciprofloxacin BP conjugates and the synthetic scheme. Figure 15 shows BP-hydroquinone-ciprofloxacin BP conjugates and the synthetic scheme.

FIG. 16 shows one embodiment of the general structure of a BP-fluoroquinolone conjugate, wherein W may be O or S or N, and X may be O, S, N, CH₂O、CH₂N or CH₂S, Y may be H, CH₃、NO₂F, Cl, Br, I or CO₂H, Z may be H, CH₃、OH、NH₂SH, F, Cl, Br or I, and n may be 1-5. Figure 17 shows various BP-fluoroquinolone conjugates.

FIG. 18 shows one embodiment of the general structure of phosphonic acids containing an aryl group, where X can be H, CH₃、OH、NH₂SH, F, Cl, Br or I, Y may be PO₃H₂Or CO₂H. Z can be OH or NH₂SH or N₃And n may 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 BPs with terminal primary amines. Fig. 21 shows the coupling of various BPs to a linker comprising a terminal hydroxyl and amine functional group, where R can be risedronic acid, zoledronic acid, minodronic acid, pamidronic acid, or alendronic acid. Figure 22 shows various BP-pamidronic acid-ciprofloxacin conjugates. Figure 23 shows various BP-alendronate-ciprofloxacin conjugates.

Example 6.

1. Dimethyl acetylphosphonate (37)

In N₂Trimethyl phosphite (2.36mL,20mmol) was added to ice cold acetyl chloride (1.44mL,20.2mmol) over a period of 20 min. Warming the colorless solutionTo room temperature, stirred for 30min and concentrated in vacuo to give 2.89g (94%) of the product as a colourless oil, which was used as such in the next reaction.¹HNMR(300MHz,CDCl3):δ3.84(d,J＝12Hz,6H),2.46(d,J＝5.4Hz,3H)。³¹PNMR(121MHz,CDCl₃):δ-1.10。

2. Tetramethyl (1-hydroxyethylidene) bisphosphonic acid (38)

In N₂Next, acetyl dimethyl phosphate (37) (2.2g,14.44mmol) was added dropwise to an ice-cold solution of dimethyl phosphite (1.63mL,15.91mmol) and dibutylamine (0.767mL,1.44mmol) in dry ether (30 mL). The ice bath was removed and the mixture was stirred at room temperature for 3 h. The resulting precipitate was filtered, washed with ether and dried under vacuum overnight to give 3.24g (85%) of the product as a white solid.¹HNMR(300MHz,CDCl₃):δ3.94–3.82(m,12H),3.44(t,J＝8.4Hz,1H),1.68(t,J＝16.2Hz,3H)。³¹PNMR(121MHz,CDCl₃):δ22.21。MS-ESI:263.1[M+H]+。

3. Tetramethyl (1- { [ (4-nitrophenoxy) carbonyl ] oxy } ethan-1, 1-diyl) bis (phosphonic acid) (39)

In N₂Next, p-nitrophenyl chloroformate (768mg,3.81mmol) was added to an ice-cold solution of DMAP (466mg,3.81mmol) in DCM (20 mL). After stirring for 10min, tetramethyl (1-hydroxyethylidene) -bisphosphonic acid (1g,3.81mmol) was added in one portion. The ice bath was removed and the mixture was stirred at room temperature for 3 h. Next, the reaction mixtures were each extracted with 20mL of cold 0.1N aqueous HCl (2X), water, brine, over MgSO₄Drying and concentrating. The crude mixture was separated by column chromatography using EtOAc/MeOH (1-3%) to give 1.16g (71%) of a light yellow oil.¹HNMR(300MHz,CDCl3):δ8.27(d,J＝9Hz,2H),7.40(d,J＝9Hz,2H),3.97–3.87(m,12H),2.02(t,J＝15.6Hz,3H)。³¹PNMR(121MHz,CDCl₃):δ17.98。MS-ESI:445.3[M+NH4]+。

4: ciprofloxacin carbamoyl etidronate tetramethyl ester (40)

To NaHCO₃(239.5mg, 2.85mmol) in H₂To a solution in O (20mL) was added ciprofloxacin (899.6mg,2.71mmol), and the suspension was cooled in an ice bath. Next, tetramethyl (1- { [ (4-nitrophenoxy) carbonyl) dissolved in THF (20mL) was added dropwise over a period of 20min]Oxy } ethan-1, 1-diyl) bis (phosphonic acid). The yellow suspension was stirred at room temperature overnight (14 h). The reaction mixture was concentrated and the crude mixture was isolated by column chromatography using DCM/MeOH (1-5%) to give 832mg (49%) of a light yellow solid.¹HNMR(300MHz,CDCl₃):δ8.77(s,1H),8.03(d,J＝12.6Hz,1H),7.36(d,J＝6.9Hz,1H)3.98–3.80(m,12H),3.79–3.68(br s,4H),3.58–3.50(m,1H),3.30(t,J＝9.6Hz,4H),1.95(t,J＝15.6Hz,3H),1.40(q,J＝6.8Hz,2H),1.23–1.16(m,2H)。³¹PNMR(121MHz,CDCl3):δ20.19。MS-ESI:620.3[M+H]+。

5. Etidronic acid-carbamate-ciprofloxacin (41)

A mixture of tetramethyletidronate-carbamate-ciprofloxacin (775mg, 1.25mmol) and bromotrimethylsilane (1.53g,10mmol) in ACN (28mL) was stirred for 2 h. Volatiles were evaporated under vacuum and MeOH (28mL) was added to the residue. After stirring for 30min, the resulting suspension was filtered, washed with MeOH (10mL × 2), and dried under vacuum overnight to give 662 mg: (b: (r))93%) as an off-white solid.¹H NMR(300MHz,20％CD₃CN, DMSO-d 6) δ 8.66(s,1H),7.92(d, J ═ 13.2Hz,1H),7.57(d, J ═ 7.4Hz,1H),3.78(p, J ═ 3.1Hz,1H),3.64(br d, J ═ 32.1Hz,4H),3.32(br s,4H),1.82(t, J ═ 15.1Hz,3H),1.32(d, J ═ 6.5Hz,2H),1.16(s, 2H).³¹PNMR (121MHz, 20% CD3CN in DMSO-d 6): delta 15.57. MS-ESI 564.2[ M + H ]]+。

6. Moxifloxacin carbamoyl etidronate tetramethylester (42)

Moxifloxacin HCl was added to a solution of Na2CO3 in H2O (20mL) and the solution was cooled in an ice bath. Next, tetramethyl (1- { [ (4-nitrophenoxy) carbonyl dissolved in THF (20mL) was added dropwise over 30min]Oxy } ethan-1, 1-diyl) bis (phosphonic acid). The ice bath was removed, the flask was covered with aluminum foil, and the reaction was stirred at room temperature for 20 h. Next, the reaction mixture was concentrated and the crude product was purified by column chromatography using DCM/MeOH (1-5%) to give 624mg (29%) of the product as an off-white foam.¹H NMR (300MHz, chloroform-d) δ 8.78(s,1H),7.81(d, J ═ 13.8Hz,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 ═ 10.5Hz,1H),3.24(d, J ═ 10.5Hz,1H),3.00(br s,1H),2.40-2.24(m,1H),1.94(t, J ═ 15.9Hz,3H),1.87-1.74(m,2H),1.60-1.44(m,2H),1.35-1.21(m,1H), 1.17-1.01 (m,1H), 0.75(m, 1H).³¹PNMR(121MHz,CDCl3):δ20.36。MS-ESI:690.4[M+H]+

7. Etidronic acid-carbamate-moxifloxacin (43)

Tetramethylisathosphine in ACN (25mL)A mixture of acid-carbamate-moxifloxacin tetramethylester (764mg, 1.10mmol) and bromotrimethylsilane (1.35g,8.86mmol) was stirred for 2 h. Volatiles were evaporated under vacuum and MeOH (25mL) was added to the residue. After stirring for 30min, the solvent was evaporated and the residue was triturated with the minimum volume of DCM for 30 min. The solid was filtered and dried under high vacuum to give 757mg of product (quantitative yield).¹H NMR (300MHz, methanol-d 4) δ 8.98(s,1H),7.79(d, J ═ 14.5Hz,1H),4.39-4.25(m,1H),4.24-4.07(m,2H),4.01(t, J ═ 10.3Hz,1H),3.65(s,3H),3.61-3.51(m,2H),3.41(d, J ═ 10.7Hz,1H),3.04(br s,1H), 2.43-2.27 (m,1H),1.90(t, J ═ 15.2Hz,3H),1.83-1.71(m,2H),1.55(q, J ═ 10.8Hz,2H),1.43-1.32(m,1H), 1.30-1.18H), 1.01-1.83 (m,1H), 1.83-1H), 1.83 (m, 1H).³¹PNMR (121MHz, methanol-d 4): delta 16.60. MS-ESI 634.2[ M + H ]]+。

Example 7.

The following is a general structure of BP-quinolones as may be described in one or more aspects herein.

Conjugates between BP containing alpha-X and quinolones

X＝O、NH、NR¹、S

R¹May be an alkyl or substituted alkyl, aryl or substituted aryl group

Example 8.

The following are non-limiting examples of BP-quinolone conjugates as described in one or more aspects herein.

Example 9:

to exploit the affinity of BP for bone, a "targeting and release" chemistry approach was investigated, which involves the delivery of antibiotics to bone or Hydroxyapatite (HA) via a BP conjugate. Serum-stabilized drug-BP linkers are utilized that metabolize and release the parent antibiotic at the bone surface. The activity of the novel quinolone antibiotics etidronate-ciprofloxacin (ECC) conjugate BV81022 and etidronate-moxifloxacin (ECX) conjugate BV81051 on staphylococcus aureus biofilms, which are pathogenic in most cases of osteomyelitis, were designed, synthesized and tested.

Public health problems caused by bacterial biofilms, such as osteomyelitis, have been highlighted to lack information about biofilm development. Several methods are available for studying biofilms in vitro or ex vivo, including quantification of sessile bacteria after separation from surfaces by scraping, vortexing or sonication, and observation by microscopy techniques to monitor biofilm progression. However, these applications are limited by high labor intensity, invasive sampling, and/or long lag time from sampling to obtaining results¹. Thus, improved biofilm monitoring assays are of crucial importance for biofilm behavior studies and biomedical applications. Thus, sensitive, accurate, reproducible, and fast methods are desirable for real-time monitoring of biofilms. In this regard, promising advances have been developed in recent years in impedance technology based on the ability of cells to block current when attached to an MTP with gold electrodes.

In this study, electrode impedance measurements were applied to test the therapeutic efficacy of our novel ECC and ECX conjugates versus ciprofloxacin and moxifloxacin against staphylococcus aureus as well as the effect on biofilm formation and growth. Real-time measurements can detect whether these biofilms are unaffected, inhibited or induced during antimicrobial therapy²。

Microbiology: for experimental purposes, robust biofilm formation 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: etidronic acid-ciprofloxacin (ECC) and etidronic acid-moxifloxacin (ECX). Real-time biofilm assays were performed using an xCELLigence RTCA SP instrument according to the manufacturer's instructions⁴. To monitor biofilm formation and RTCA sensitivity assays, 80 μ Ι of TSBYE was added to each well of a non-reusable 16X microtiter E plate (ACEA Biosciences) for impedance background measurements using standard protocols provided by the software. Then 1. mu.l of bacterial suspension in a total of 120. mu.l of TSBYE was added to 16E-plate wells. Each sample was run in duplicate. The E plate was positioned in an xCELLigence real-time cell analyzer MP, incubated at 37 ℃ and monitored on an RTCA system for 24h at 15min intervals. According to the manufacturer's instructions, cell sensor impedance is expressed in units called the Cell Index (CI). The CI at each time point is defined as (ZnZb)/15, where Zn is the cell electrode impedance of the well when the well contains cells, and Zb is the background impedance with only the growth medium. Duplicate or triplicate standard deviations of wells were analyzed with RTCA software

1 μ g/mL of each compound was added to a solution containing 10 μ g/mL of HA powder and incubated for 4h/37 ℃ with magnetic stirring. Next, the HA powder was allowed to settle for 1h/4 ℃. Thereafter, the antibiotic content in the supernatant was evaluated using hplc (shimadzu promience). To assess the amount of conjugate bound to HA, we used the method described in more detail in our previous work³. The affinity of the compound for HA powder was estimated as follows: 100% -peak area of detected compound/peak area of control sample 100%.

Add 80- μ l of TSBYE to each well to measure background impedance. Then 1 μ l of bacterial suspension in a total of 120 μ l TSBYE containing various concentrations of antibiotics was added to 16E-plate wells. Two replicates per antibiotic concentration and antibiotic-free anionsSexual controls are also included. Cells were monitored for 24h and analyzed to calculate MIC₅₀The value is obtained.

Different concentrations of each compound were added to a solution containing 10 μ g/mL HA powder and incubated for 2h/37 ℃ with magnetic stirring. Then 80- μ l of TSBYE was added to each well to measure background impedance. Then 1 μ l of bacterial suspension in a total of 120 μ l TSBYE containing a gradient concentration of antibiotics plus HA was added to 16E-plate wells. Two replicates of each antibiotic concentration and a negative control without antibiotic were also included. Cells were monitored for 24hr and analyzed to calculate MIC₅₀The value is obtained.

As a result: HPLC results evaluating the binding affinity of the tested compounds to HA indicated that the conjugates have high binding affinity and retention of HA compared to the unconjugated antibiotic. Electrode impedance measurements were applied to test the therapeutic efficacy of the novel ECC and ECX conjugates compared to ciprofloxacin and moxifloxacin against staphylococcus aureus and the effect on biofilm formation and growth. Real-time measurements can detect whether these biofilms are unaffected, inhibited, or induced during antimicrobial therapy (see, fig. 26, 28, 30, 32).

In infection preventative experiments using real-time monitoring, MICs have been calculated for each conjugate and the parent antibiotic₅₀As shown in fig. 27 and 29. Staphylococcus aureus MIC for ciprofloxacin, moxifloxacin, ECC, and ECX₅₀Respectively as follows: 0.09, 0.11, 4.88 and 5.10. mu.g/ml.

In infection preventative experiments using real-time monitoring in the presence of HA, MICs have been calculated for each conjugate and the parent antibiotic₅₀As shown in fig. 31 and 33. Staphylococcus aureus MIC for ciprofloxacin, moxifloxacin, ECC, and ECX₅₀Respectively as follows: 0.24, 0.09, 9.60 and 28. mu.g/ml.

And (4) conclusion: thus, the antibiotic effect on the growth of staphylococcus aureus bacterial biofilms was assessed in real time by impedance measurements using microtiter plates with gold electrodes. Real-time biofilm analysis allows detection of a decrease or increase in microbial mass over time during antimicrobial therapy, which can be used clinically to assess the sensitivity and efficacy of antibiotics in biofilm-mediated infections. The novel etidronic acid-fluoroquinolone conjugates (ECC, ECX) designed and tested in this study retained the osseointegrative properties of the parent BP drug, and also the antimicrobial activity of the parent antibiotic in the presence or absence of HA, albeit at a lower level due to the nature of the chemical modification and possible partial cleavage under the conditions tested. Such conjugates using BP drugs as biochemical carriers (vectors) to deliver antibiotic agents to bone where the osteomyelitis biofilm pathogens reside represent an advantageous approach to the treatment of osteomyelitis by providing improved bone pharmacokinetics while minimizing systemic exposure (toxicity) of these drugs.

Reference to 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 Screen2005；10,795–805.

Claims

1. a compound comprising a Bisphosphonate (BP) and a quinolone, wherein the BP has an alpha substituent and the alpha substituent is a hydroxyl, amino, or thiol group, and wherein the quinolone is conjugated directly or indirectly to the BP at a geminal carbon alpha substituent (X) of the BP in the formula

2. The compound of claim 1, wherein said BP is an alpha-OH-containing BP and wherein said quinolone is conjugated directly or indirectly to said BP at a geminal OH of said BP.

3. A compound according to claim 1 or 2, wherein the quinolone is a fluoroquinolone.

4. The compound of any of claims 1 or 2, wherein the quinolone is selected from the group consisting of: alafloxacin, aminofloxacin, balofloxacin, besifloxacin, cadazolide, ciprofloxacin, clinafloxacin, danofloxacin, delafloxacin, difloxacin, enoxacin, enrofloxacin, finafloxacin, fleroxacin, flumequine, gatifloxacin, gemifloxacin, grepafloxacin, ebafloxacin, JNJ-Q2, levofloxacin, lomefloxacin, marbofloxacin, moxifloxacin, nadifloxacin, norfloxacin, ofloxacin, orbifloxacin, pazufloxacin, pefloxacin, prafloxacin, prarofloxacin, rufloxacin, sarofloxacin, sitafloxacin, sparfloxacin, temafloxacin, tosufloxacin, trovafloxacin, zabofloxacin, nemonoxacin and combinations thereof.

5. A compound according to claim 1 or 2, according to formula (41), formula (43), formula (44) or formula (45)

6. The compound of claim 1 according to the formula

7. The compound of claim 1, wherein said quinolone or said BP quinolone compound consists of a quinolone antibiotic analog or substituent according to formula (A),

wherein R is¹Can be

And wherein R²Can be

And wherein R³Can be H or OCH3 and can be,

and wherein R⁴It may be a compound of formula (I) and (II),

and wherein R⁵May be H or F.

8. The compound of claim 1, wherein said bisphosphonate is selected from the group consisting of: modified or unmodified etidronic acid, methylenehydroxy bisphosphonic acid (MHBP), risedronic acid, zoledronic acid, minodronic acid, neridronic acid, pamidronic acid, alendronic acid, and combinations thereof.

9. The compound of claim 7 or claim 8, wherein the quinolone compound is a fluoroquinolone.

10. The compound of claim 7 or claim 8, wherein the quinolone compound is selected from the group consisting of: alafloxacin, aminofloxacin, balofloxacin, besifloxacin, cadazolide, ciprofloxacin, clinafloxacin, danofloxacin, delafloxacin, difloxacin, enoxacin, enrofloxacin, finafloxacin, fleroxacin, flumequine, gatifloxacin, gemifloxacin, grepafloxacin, ebafloxacin, JNJ-Q2, levofloxacin, lomefloxacin, marbofloxacin, moxifloxacin, nadifloxacin, norfloxacin, ofloxacin, orbifloxacin, pazufloxacin, pefloxacin, prafloxacin, prarofloxacin, rufloxacin, sarofloxacin, sitafloxacin, sparfloxacin, temafloxacin, tosufloxacin, trovafloxacin, zabofloxacin, nemonoxacin and combinations thereof.

11. The compound of claim 7 or claim 8, wherein the quinolone compound is ciprofloxacin, moxifloxacin, sitafloxacin, or nemonoxacin.

12. The compound of any one of claims 7-11, wherein the linker is a carbamate, thiocarbamate, hydrazine, or carbonate or ester, or urea.

13. The compound of any one of claims 7-11, wherein the linker is a carbamate linker or an ester linker.

14. The compound of any of claims 7-11, wherein the linker is an alkyl carbamate or aryl carbamate linker.

15. The compound of any of claims 7-11, wherein the linker is a thiocarbamic-O-aryl ester or thiocarbamic alkyl ester linker.

16. The compound of any of claims 7-11, wherein the linker is a thiocarbamic-S-aryl ester or thiocarbamic alkyl ester linker.

17. The compound of any one of claims 7-11, wherein the linker is a phenyl carbamate linker.

18. The compound of any one of claims 7-11, wherein the linker is a thiocarbamate linker.

19. The compound of any one of claims 7-11, wherein the linker is an O-thiocarbamate linker.

20. The compound of any one of claims 7-11, wherein the linker is an S-thiocarbamate linker.

21. A pharmaceutical formulation comprising:

an amount of a compound according to any one of claims 1-20; and

a pharmaceutically acceptable carrier.

22. The pharmaceutical formulation of claim 21, wherein the amount of the compound is an amount effective to kill or inhibit bacteria.

23. The pharmaceutical formulation of claim 21, wherein the amount of the compound is an amount effective to treat or prevent a bone disease having: abnormal bone resorption, osteoporosis, bone infection, osteomyelitis, osteonecrosis, peri-implantitis, and periodontitis.

24. A method of treating a bone infection in a subject in need thereof, the method comprising:

administering to the subject in need thereof an amount of a compound according to any one of claims 1-20 or a pharmaceutical formulation thereof.

25. A method of treating osteomyelitis in a subject in need thereof, said method comprising:

26. A method of treating peri-implantitis or periodontitis in a subject in need thereof, the method comprising administering to the subject in need thereof an amount of a compound of any one of claims 1-20, or a pharmaceutical formulation thereof.

27. A method of treating diabetic foot in a subject in need thereof, the method comprising administering to the subject in need thereof an amount of a compound according to any one of claims 1-20 or a pharmaceutical formulation thereof.

28. A method, comprising:

administering to a subject an amount of a compound according to any one of claims 1-20 or a pharmaceutical formulation thereof.

29. A bone graft composition comprising:

a bone graft material and a compound or pharmaceutical formulation thereof according to any one of claims 1 to 20, wherein the compound or pharmaceutical formulation is attached to, bound to, chemisorbed or mixed with the bone graft material.

30. The bone graft composition of claim 29, wherein said bone graft material is an autograft bone material, an allograft bone material, a xenograft bone material, a synthetic bone graft material, or any combination thereof.

31. A method, comprising:

implanting the bone graft composition of any one of claims 29-30 into a subject in need thereof.

32. A method of preventing biofilm infection at a bone or an implant surgical site or at a surgical site where a bone graft is performed, wherein the method comprises:

administering to a subject in need thereof a compound according to any one of claims 1-20 or a pharmaceutical formulation thereof.

33. A method of preventing biofilm infection at a bone or an implant surgical site or at a surgical site where a bone graft is performed, wherein the method comprises:

34. Use of a composition according to any one of claims 1-20 for the manufacture of a pharmaceutical formulation for treating a bone infection in a subject in need thereof.