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.

Targeting the isoprenoid pathway to abrogate progression of pulmonary fibrosis.

Fibrotic remodeling in lung injury is a major cause of morbidity. The mechanism that mediates the ongoing fibrosis is unclear, and there is no available treatment to abate the aberrant repair. Reactive oxygen species (ROS) have a critical role in inducing fibrosis by modulating extracellular matrix deposition. Specifically, mitochondrial hydrogen peroxide (H2O2) production by alveolar macrophages is directly linked to pulmonary fibrosis as inhibition of mitochondrial H2O2 attenuates the fibrotic response in mice. Prior studies indicate that the small GTP-binding protein, Rac1, directly mediates H2O2 generation in the mitochondrial intermembrane space. Geranylgeranylation of the C-terminal cysteine residue (Cys(189)) is required for Rac1 activation and mitochondrial import. We hypothesized that impairment of geranylgeranylation would limit mitochondrial oxidative stress and, thus, abrogate progression of pulmonary fibrosis. By targeting the isoprenoid pathway with a novel agent, digeranyl bisphosphonate (DGBP), which impairs geranylgeranylation, we demonstrate that Rac1 mitochondrial import, mitochondrial oxidative stress, and progression of the fibrotic response to lung injury are significantly attenuated. These observations reveal that targeting the isoprenoid pathway to alter Rac1 geranylgeranylation halts the progression of pulmonary fibrosis after lung injury.

Identification of light-independent inhibition of human immunodeficiency virus-1 infection through bioguided fractionation of Hypericum perforatum.

BACKGROUND: Light-dependent activities against enveloped viruses in St. John's Wort (Hypericum perforatum) extracts have been extensively studied. In contrast, light-independent antiviral activity from this species has not been investigated. RESULTS: Here, we identify the light-independent inhibition of human immunodeficiency virus-1 (HIV-1) by highly purified fractions of chloroform extracts of H. perforatum. Both cytotoxicity and antiviral activity were evident in initial chloroform extracts, but bioassay-guided fractionation produced fractions that inhibited HIV-1 with little to no cytotoxicity. Separation of these two biological activities has not been reported for constituents responsible for the light-dependent antiviral activities. Antiviral activity was associated with more polar subfractions. GC/MS analysis of the two most active subfractions identified 3-hydroxy lauric acid as predominant in one fraction and 3-hydroxy myristic acid as predominant in the other. Synthetic 3-hydroxy lauric acid inhibited HIV infectivity without cytotoxicity, suggesting that this modified fatty acid is likely responsible for observed antiviral activity present in that fraction. As production of 3-hydroxy fatty acids by plants remains controversial, H. perforatum seedlings were grown sterilely and evaluated for presence of 3-hydroxy fatty acids by GC/MS. Small quantities of some 3-hydroxy fatty acids were detected in sterile plants, whereas different 3-hydroxy fatty acids were detected in our chloroform extracts or field-grown material. CONCLUSION: Through bioguided fractionation, we have identified that 3-hydroxy lauric acid found in field grown Hypericum perforatum has anti-HIV activity. This novel anti-HIV activity can be potentially developed into inexpensive therapies, expanding the current arsenal of anti-retroviral agents.

BF3 x Et2O-mediated cascade cyclizations: synthesis of schweinfurthins F and G.

The total synthesis of the natural stilbene (+)-schweinfurthin G (8) has been accomplished through a sequence based on an efficient cationic cascade cyclization. This cascade process is initiated by Lewis acid promoted ring opening of an epoxide and terminated through a novel reaction with a phenolic oxygen "protected" as its MOM ether. Several Lewis acids have been examined for their ability to induce this new reaction, and BF3 x Et2O was found to be the most effective. The only major byproduct under these conditions was one where the expected secondary alcohol was found as its MOM ether derivative (e.g., 30). While this byproduct could be converted to the original target compound through hydrolysis, it also could be employed as a protected alcohol to allow preparation of a benzylic phosphonate (43) without dehydration of the secondary alcohol. The resulting phosphonate was employed in a Horner-Wadsworth-Emmons condensation with an aldehyde representing the right half of the target compounds, an approach complementary to previous studies based on condensation of a right-half phosphonate and a left-half aldehyde.

Pseudohypericin is necessary for the light-activated inhibition of prostaglandin E2 pathways by a 4 component system mimicking an Hypericum perforatum fraction.

Hypericum perforatum (Hp) has been used medicinally to treat a variety of conditions including mild-to-moderate depression. Recently, several anti-inflammatory activities of Hp have been reported. An ethanol extract of Hp was fractionated with the guidance of an anti-inflammatory bioassay (lipopolysaccharide (LPS)-induced prostaglandin E2 production (PGE2)), and four constituents were identified. When combined together at concentrations detected in the Hp fraction to make a 4 component system, these constituents (0.1microM chlorogenic acid (compound 1), 0.08microM amentoflavone (compound 2), 0.07microM quercetin (compound 3), and 0.03microM pseudohypericin (compound 4)) explained the majority of the activity of the fraction when activated by light, but only partially explained the activity of this Hp fraction in dark conditions. One of the constituents, light-activated pseudohypericin, was necessary, but not sufficient to explain the reduction in LPS-induced PGE2 of the 4 component system. The Hp fraction and the 4 component system inhibited lipoxygenase and cytosolic phospholipase A2, two enzymes in the PGE2-mediated inflammatory response. The 4 component system inhibited the production of the pro-inflammatory cytokine tumor necrosis factor-alpha (TNF-alpha), and the Hp fraction inhibited the anti-inflammatory cytokine interleukin-10 (IL-10). Thus, the Hp fraction and selected constituents from this fraction showed evidence of blocking pro-inflammatory mediators but not enhancing inflammation-suppressing mediators.

Synthesis and structure-activity studies of schweinfurthin B analogs: Evidence for the importance of a D-ring hydrogen bond donor in expression of differential cytotoxicity.

The synthesis and biological evaluation of several enantioenriched schweinfurthin B analogs were undertaken to develop structure-activity relationships and guide design of probes for their putative molecular target. The desired stilbenes contain a common left-half hexahydroxanthene ring system and an aromatic right-half with varied substituents. The synthesis involves penultimate Horner-Wadsworth-Emmons coupling of one of several right-half phosphonates with the aldehyde comprising the left-half of 3-deoxyschweinfurthin B. Preparation of the requisite phosphonates, and the respective stilbenes, as well as the cytotoxicity profiles of these new compounds in the National Cancer Institute's 60 cell-line anticancer screen is described. Several of these analogs displayed cytotoxicity patterns well-correlated with the natural product and differences in activity of approximately 10(3) across the various cell lines. Together, these assay results indicate the importance of at least one free phenol group on the aromatic D-ring of this system for differential cytotoxicity.