Rescue bisphosphonate treatment of alveolar bone improves extraction socket healing and reduces osteonecrosis in zoledronate-treated mice.

Bisphosphonate (BP)-related osteonecrosis of the jaw, previously known as BRONJ, now referred to more broadly as medication-related osteonecrosis of the jaw (MRONJ), is a morbid condition that represents a significant risk for oncology patients who have received high dose intravenous (IV) infusion of a potent nitrogen containing BP (N-BP) drug. At present, no clinical procedure is available to prevent or effectively treat MRONJ. Although the pathophysiological basis is not yet fully understood, legacy adsorbed N-BP in jawbone has been proposed to be associated with BRONJ by one or more mechanisms. We hypothesized that removal of the pre-adsorbed N-BP drug common to these pathological mechanisms from alveolar bone could be an effective preventative/therapeutic strategy. This study demonstrates that fluorescently labeled BP pre-adsorbed on the surface of murine maxillo-cranial bone in vivo can be displaced by subsequent application of other BPs. We previously described rodent BRONJ models involving the combination of N-BP treatment such as zoledronate (ZOL) and dental initiating factors such as tooth extraction. We further refined our mouse model by using gel food during the first 7 days of the tooth extraction wound healing period, which decreased confounding food pellet impaction problems in the open boney socket. This refined mouse model does not manifest BRONJ-like severe jawbone exposure, but development of osteonecrosis around the extraction socket and chronic gingival inflammation are clearly exhibited. In this study, we examined the effect of benign BP displacement of legacy N-BP on tooth extraction wound healing in the in vivo model. Systemic IV administration of a low potency BP (lpBP: defined as inactive at 100 µM in a standard protein anti-prenylation assay) did not significantly attenuate jawbone osteonecrosis. We then developed an intra-oral formulation of lpBP, which when injected into the gingiva adjacent to the tooth prior to extraction, dramatically reduced the osteocyte necrosis area. Furthermore, the tooth extraction wound healing pattern was normalized, as evidenced by timely closure of oral soft tissue without epithelial hyperplasia, significantly reduced gingival inflammation and increased new bone filling in the extraction socket. Our results are consistent with the hypothesis that local application of a rescue BP prior to dental surgery can decrease the amount of a legacy N-BP drug in proximate jawbone surfaces below the threshold that promotes osteocyte necrosis. This observation should provide a conceptual basis for a novel strategy to improve socket healing in patients treated with BPs while preserving therapeutic benefit from anti-resorptive N-BP drug in vertebral and appendicular bones.

Clickable prodrugs bearing potent and hydrolytically cleavable nicotinamide phosphoribosyltransferase inhibitors.

PURPOSE: Our previous study indicated that carborane containing small-molecule 1-(hydroxymethyl)-7-(4'-(trans-3″-(3'″-pyridyl)acrylamido)butyl)-1,7-dicarbadodecaborane (hm-MC4-PPEA), was a potent inhibitor of nicotinamide phosphoribosyltransferase (Nampt). Nampt has been shown to be upregulated in most cancers and is a promising target for the treatment of many different types of cancers, including breast cancers. PATIENTS AND METHODS: To increase the selectivity of hm-MC4-PPEA toward cancer cells, three prodrugs were synthesized with different hydrolyzable linkers: ester, carbonate, and carbamate. Using click chemistry a fluorophore was attached to these prodrugs to act as a model for our conjugation strategy and to serve as an aid for prodrug stability studies. The stabilities of these drug conjugates were tested in phosphate-buffered saline (PBS) at normothermia (37°C) using three different pH levels, 5.5, 7.5, and 9.5, as well as in horse serum at physiological pH. The stability of each was monitored using reversed-phase HPLC equipped with both diode array and fluorescence detection. The inhibitory activity of hm-MC4-PPEA was also measured using a commercially available colorimetric assay. The biological activities of the drug conjugates as well as those of the free drug (hm-MC4-PPEA), were evaluated using the MTT assay against the human breast cancer cell lines T47D and MCF7, as well as the noncancerous, transformed, Nampt-dependent human breast epithelium cell line 184A1. RESULTS: hm-MC4-PPEA showed to be a potent inhibitor of recombinant Nampt activity, exhibiting an IC50 concentration of 6.8 nM. The prodrugs showed great stability towards hydrolytic degradation under neutral, mildly acidic and mildly basic conditions. The carbamate prodrug also showed to be stable in rat serum. However, the carbonate and the ester prodrug release at various rates in serum presumably owing to the presence of several different classes of esterase. The biological activities of the drug conjugates correlate with the stability of their cleavable linkers observed in serum. CONCLUSION: The targeted and selective delivery of potent Nampt inhibitors to cancer cells is a potentially new route for the treatment of many cancers. These prodrugs linked to small cancer-associated peptides may be optimum for their use as targetable Nampt inhibitors.

MC-PPEA as a new and more potent inhibitor of CLP-induced sepsis and pulmonary inflammation than FK866.

Our previous study indicated that overexpression of nicotinamide phosphoribosyltransferase (NAMPT) aggravated acute lung injury, while knockdown of NAMPT expression attenuated ventilator-induced lung injury. Recently, we found that meta-carborane-butyl-3-(3-pyridinyl)-2E-propenamide (MC-PPEA, MC4), in which the benzoylpiperidine moiety of FK866 has been replaced by a carborane, displayed a 100-fold increase in NAMPT inhibition over FK866. Here, we determined the effects of MC4 and FK866 on cecal ligation and puncture (CLP) surgery-induced sepsis in C57BL/6J mice. MC4 showed stronger inhibitory effects than FK866 on CLP-induced mortality, serum tumor necrosis factor α (TNFα) levels, pulmonary myeloperoxidase activity, alveolar injury, and interleukin 6 and interleukin1ß messenger RNA levels. In vitro cell permeability and electric cell-substrate impedance sensing assays demonstrated that MC4 inhibited TNFα- and thrombin-mediated pulmonary endothelial cell permeability better than FK866. MC4 also exerted more potent effects than FK866, at concentrations as low as 0.3 nM, to attenuate TNFα-mediated intracellular cytokine expression, nicotinamide adenine dinucleotide (NAD+) and its reduced form NADH levels, and nuclear factor kappa B p65 phosphorylation and nuclear translocation in A549 cells. Our results strongly suggest that the newly developed MC4 is a more potent suppressor of CLP-induced pulmonary inflammation and sepsis than FK866, with potential clinical application as a new treatment agent for sepsis and inflammation.

Design, Synthesis, and Antimicrobial Evaluation of a Novel Bone-Targeting Bisphosphonate-Ciprofloxacin Conjugate for the Treatment of Osteomyelitis Biofilms.

Osteomyelitis is a major problem worldwide and is devastating due to the potential for limb-threatening sequelae and mortality. Osteomyelitis pathogens are bone-attached biofilms, making antibiotic delivery challenging. Here we describe a novel osteoadsorptive bisphosphonate-ciprofloxacin conjugate (BV600022), utilizing a "target and release" chemical strategy, which demonstrated a significantly enhanced therapeutic index versus ciprofloxacin for the treatment of osteomyelitis in vivo. In vitro antimicrobial susceptibility testing of the conjugate against common osteomyelitis pathogens revealed an effective bactericidal profile and sustained release of the parent antibiotic over time. Efficacy and safety were demonstrated in an animal model of periprosthetic osteomyelitis, where a single dose of 10 mg/kg (15.6 µmol/kg) conjugate reduced the bacterial load by 99% and demonstrated nearly an order of magnitude greater activity than the parent antibiotic ciprofloxacin (30 mg/kg, 90.6 µmol/kg) given in multiple doses. Conjugates incorporating a bisphosphonate and an antibiotic for bone-targeted delivery to treat osteomyelitis biofilm pathogens constitute a promising approach to providing high bone-antimicrobial potency while minimizing systemic exposure.

Improved synthesis of MC4-PPEA and the biological evaluation of its hydroxymethyl derivative.

Nicotinamide phosphoribosyltransferase (Nampt) is an intriguing target for the treatment of many diseases, including cancer. Previously, our group demonstrated that carborane clusters may be used to increase the potency of small molecule inhibitors of Nampt over other, similarly sized organic moieties. Herein, we report a greatly improved, gram-scale synthesis of our most potent agent: 1-(4'-(trans-3″-(3‴-pyridyl) acrylamide)butyl)-1,7-dicarba-closo-dodecaborane (MC4-PPEA). Additionally, the carborane moiety of the molecule has been modified with a hydroxymethyl functional group to allow for its covalent attachment to targeted prodrugs, the synthesis of which are underway. Using cell viability assays, we demonstrate that this new derivative exhibits low, to mid-nanomolar potencies against human breast cancer cell lines in vitro.