Nature-inspired synthesis of antibacterial glucovanillin derivatives.

The ongoing threat of Antimicrobial Resistance (AMR) complicated by the rise of Multidrug-Resistant (MDR) pathogens calls for increased efforts in the search for novel treatment options. While deriving inspiration from antibacterial natural compounds, this study aimed at using synthetic approaches to generate a series of glucovanillin derivatives and explore their antibacterial potentials. Among the synthesized derivatives, optimum antibacterial activities were exhibited by those containing 2,4- and 3,5-dichlorophenylamino group coupled to a glucovanillin moiety (compounds 6h and 8d respectively). In those compounds, the Minimum Inhibitory Concentrations (MIC) of 128-256 µg/mL were observed against reference and MDR strains of Klebsiella pneumoniae, Methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin-resistant Enterococcus faecium (VRE). Moreover, these findings emphasize the claims from previous reports on the essence of smaller molecular size, the presence of protonatable amino groups and halogens in potential antibacterial agents. The observed moderate and broad-spectrum activities of the stated derivatives point to their suitability as potential leads towards further efforts to improve their antibacterial activities.

Isolation and Characterization of Galloylglucoses Effective against Multidrug-Resistant Strains of Escherichia coli and Klebsiella pneumoniae.

The search for new antibiotics against multidrug-resistant (MDR), Gram-negative bacteria is crucial with respect to filling the antibiotics development pipeline, which is subject to a critical shortage of novel molecules. Screening of natural products is a promising approach for identifying antimicrobial compounds hosting a higher degree of novelty. Here, we report the isolation and characterization of four galloylglucoses active against different MDR strains of Escherichia coli and Klebsiella pneumoniae. A crude acetone extract was prepared from Paeonia officinalis Linnaeus leaves, and bioautography-guided isolation of active compounds from the extract was performed by liquid-liquid extraction, as well as open column, flash, and preparative chromatographic methods. Isolated active compounds were characterized and elucidated by a combination of spectroscopic and spectrometric techniques. In vitro antimicrobial susceptibility testing was carried out on E. coli and K. pneumoniae using 2 reference strains and 13 strains hosting a wide range of MDR phenotypes. Furthermore, in vivo antibacterial activities were assessed using Galleria mellonella larvae, and compounds 1,2,3,4,6-penta-O-galloyl-ß-d-glucose, 3-O-digalloyl-1,2,4,6-tetra-O-galloyl-ß-d-glucose, 6-O-digalloyl-1,2,3,4-tetra-O-galloyl-ß-d-glucose, and 3,6-bis-O-digalloyl-1,2,4-tri-O-galloyl-ß-d-glucose were isolated and characterized. They showed minimum inhibitory concentration (MIC) values in the range of 2-256 µg/mL across tested bacterial strains. These findings have added to the number of known galloylglucoses from P. officinalis and highlight their potential against MDR Gram-negative bacteria.

Targeting interleukin-4 to the arthritic joint.

Anti-inflammatory cytokines are a promising class of therapeutics for treatment of rheumatoid arthritis (RA), but their use is currently limited by a rapid clearance and systemic toxicity. Interleukin-4 is a small cytokine with potential for RA therapy. To increase its pharmacokinetic features, we engineered a murine IL4 conjugate by incorporating an unnatural amino acid through genetic code expansion to which PEG-folate, as a targeting moiety and PEG alone as control, were site-specifically bound. Both IL4 conjugates retained bioactivity and induced primary murine macrophage polarization into an alternatively activated (M2) related phenotype. The PEGylated conjugates had a terminal half-life of about four hours in healthy mice compared to unPEGylated IL4 (0.76 h). We showed that both conjugates successfully accumulated into arthritic joints in an antigen-induced arthritis (AIA) mouse model, as assessed by non-invasive fluorescence imaging. The modular nature of the IL4 conjugate chemistry presented herein facilitates easy adaption of PEG chain length and targeting moieties for further improvement of half-life and targeting function for future efficacy studies.

Geometrical and Structural Dynamics of Imatinib within Biorelevant Colloids.

Solubilization of lipophilic drugs is essential for efficient uptake. We detail the solubilization of imatinib in simulated gastrointestinal fluids containing taurocholate (TC) and lecithin (L) and reflecting fasted versus fed states using NMR spectroscopy, X-ray diffractometry, transmission electron microscopy, and dynamic light scattering analysis. Imatinib concentration impacted colloidal geometries and molecular dynamics in a fasted state. At drug substance concentrations up to 250 µM, imatinib was mainly engulfed within the core of >110 nm in diameter vesicles. At higher drug concentrations, the colloids collapsed to <40 nm, and imatinib migrated into the shell of the micelles, mainly being associated with the lipophilic face of TC but not with L. Simulating the fed state resulted in the formation of small micelles independent of the drug concentration. Furthermore, a hydrogel was formed, effectively keeping the drug substance in an amorphous state even when stressed by drying. In conclusion, this study detailed the fascinating dynamics of colloidal structures and molecular assembly as a function of imatinib concentration in biorelevant conditions. This approach may provide a blueprint for the rational development of future pharmaceutical formulations, taking the molecular interactions with bile salts/phospholipids into account.

Influence of salt type and ionic strength on self-assembly of dextran sulfate-ciprofloxacin nanoplexes.

We evaluated an analytical setup to identify optimal preparation conditions for nanoplex formation of small molecule drugs and polyelectrolytes using ciprofloxacin (CIP) and dextran sulfate (DS) as model compounds. The suitability of isothermal titration calorimetry (ITC) as a screening tool for rational formulation optimization was assessed. Besides ITC, static and dynamic light scattering, zeta potential measurements and scanning electron microscopy were applied to analyze the influence of different salt types and ionic strengths on CIP/DS nanoplex formation. The addition of low amounts of salt, especially 0.1M NaCl, improved the formation of CIP/DS nanoplexes. The presence of low amounts of salt led to smaller and more numerous particles of higher uniformity but had no influence on the release of CIP from nanoplexes. Furthermore, the molar range, within which efficient complexation was achieved, was broader in the presence of 0.1M NaCl than in the absence of salt with overall comparable complexation efficiency. Importantly, binding affinity correlated with particle shape and morphology, potentially enabling optimization of critical quality attributes based on ITC data. Altogether, ITC along with supplemental methods is a versatile screening tool for the evaluation of nanoplex formulation conditions regarding mixing ratio, salt type and ionic strength.

Effects of chondrogenic and osteogenic regulatory factors on composite constructs grown using human mesenchymal stem cells, silk scaffolds and bioreactors.

Human mesenchymal stem cells (hMSCs) isolated from bone marrow aspirates were cultured on silk scaffolds in rotating bioreactors for three weeks with either chondrogenic or osteogenic medium supplements to engineer cartilage- or bone-like tissue constructs. Osteochondral composites formed from these cartilage and bone constructs were cultured for an additional three weeks in culture medium that was supplemented with chondrogenic factors, supplemented with osteogenic factors or unsupplemented. Progression of cartilage and bone formation and the integration between the two regions were assessed by medical imaging (magnetic resonance imaging and micro-computerized tomography imaging), and by biochemical, histological and mechanical assays. During composite culture (three to six weeks), bone-like tissue formation progressed in all three media to a markedly larger extent than cartilage-like tissue formation. The integration of the constructs was most enhanced in composites cultured in chondrogenic medium. The results suggest that tissue composites with well-mineralized regions and substantially less developed cartilage regions can be generated in vitro by culturing hMSCs on silk scaffolds in bioreactors, that hMSCs have markedly higher capacity for producing engineered bone than engineered cartilage, and that chondrogenic factors play major roles at early stages of bone formation by hMSCs and in the integration of the two tissue constructs into a tissue composite.

Porous silk fibroin 3-D scaffolds for delivery of bone morphogenetic protein-2 in vitro and in vivo.

Bone morphogenetic protein-2 (BMP-2) plays a key role in osteogenesis. Biomaterials used for the sustained delivery of BMP-2 in vivo have shown therapeutic benefits. In the present study, BMP-2 was loaded in porous silk fibroin scaffolds derived from silkworm cocoons (2.4 +/- 0.14 microg per scaffold). The release profile of BMP-2 under dynamic culture conditions (spinner flasks) showed that after 1 week in culture 25% of the initial BMP-2 was retained adsorbed to the scaffold; up to 4 weeks no additional BMP-2 was released. BMP-2 induced human bone marrow stromal cells (hMSCs) to undergo osteogenic differentiation when the seeded scaffolds were cultured in medium supplemented with osteogenic stimulants for 4 weeks, based on elevated alkaline phosphatase activity, calcium deposition, and transcript levels for bone sialoprotein, osteopontin, osteocalcin, BMP-2, and cbfa-1. Micro-computed tomography revealed densely deposited mineral at the center of the scaffolds. In contrast, hMSCs cultured in control scaffolds (no BMP-2) exhibited limited osteogenesis. When implanted in critical sized cranial defects in mice, scaffolds loaded with BMP-2 and seeded with hMSCs resulted in significant bone ingrowth. These results were qualitatively similar to scaffolds loaded with BMP-2 but no hMSCs or with BMP-2 and hMSCs but not pregrown into bone-like tissue. Bone-related outcomes were improved when compared with the scaffold controls implanted without BMP-2. These studies illustrate the potential use of slow degrading silk fibroin 3-D scaffolds loaded with BMP-2, in combination with hMSCs, in osteogenesis studies in vitro and in vivo, and provide a new range of material properties for these applications.

Bone morphogenetic protein-2 decorated silk fibroin films induce osteogenic differentiation of human bone marrow stromal cells.

Bone morphogenetic protein (BMP)-2 has a critical role in bone formation and regeneration. Therefore, the ability to immobilize this molecule in certain matrices may be crucial in bone tissue engineering. Using carbodiimide chemistry, BMP-2 was directly immobilized on silk fibroin films. Whereas human bone marrow stromal cells cultured on unmodified silk fibroin films in the presence of osteogenic stimulants exhibited little if any osteogenesis, the same cells cultured on BMP-2 decorated films in the presence of osteogenic stimulants differentiated into an osteoblastic lineage as assessed by their significantly elevated alkaline phosphatase activity, calcium deposition, and higher transcript levels of collagen type I, bone sialoprotein, osteopontin, osteocalcin, BMP-2, and cbfa1. Using cell culture inserts, it was demonstrated that differentiation was induced by the immobilized protein and not by protein released into the culture medium. Comparison with a similar amount of medium-supplemented BMP-2, where no additional protein was added with medium changes, showed that delivery of BMP-2 immobilized on the biomaterial surface was more efficient than soluble delivery. The results illustrate that BMP-2 covalently coupled on silk biomaterial matrices retains biological function in vitro based on the induction of osteogenic markers in seeded bone marrow stromal cells.

IGF-I and GH stimulate Phex mRNA expression in lungs and bones and 1,25-dihydroxyvitamin D(3) production in hypophysectomized rats.

OBJECTIVE: X-linked hypophosphatemia, a renal phosphate (Pi)-wasting disorder with defective bone mineralization, is caused by mutations in the PHEX gene (a Pi-regulating gene with homology to endopeptidases on the X chromosome). We wondered whether changes in Phex and neprilysin (NEP) (another member of the family of zinc endopeptidases) mRNA expression could be observed in relation to vitamin D and Pi metabolism during GH- and IGF-I-stimulated growth of hypophysectomized rats. DESIGN: Animals were infused s.c. for 2 days with vehicle, 200 mU (67 microg) GH or 300 microg IGF-I/rat per 24 h. We determined serum osteocalcin and osteocalcin mRNA in bone, Phex mRNA in bone and lungs, serum 1,25-dihydroxyvitamin D(3) (1,25-(OH)(2)D(3)) and serum Pi levels, and renal expression of 25-hydroxyvitamin D(3)-1alpha-hydroxylase (1alpha-hydroxylase), of 25-hydroxyvitamin D(3)-24-hydroxylase (24-hydroxylase) and of the Na-dependent Pi-cotransporter type I and II (Na(d)Pi-I and -II). RESULTS: As compared with vehicle-treated controls, body weight and tibial epiphyseal width significantly increased in GH- and IGF-I-treated animals. Serum osteocalcin and osteocalcin mRNA levels in bone, Phex mRNA in bone and lungs, serum 1,25-(OH)(2)D(3) and renal 1alpha-hydroxylase mRNA rose concomitantly, whereas expression of NEP in lungs was barely affected and renal 24-hydroxylase mRNA decreased. Na(d)Pi-I and -II gene expression in the kidney and serum Pi levels remained unchanged. CONCLUSIONS: Our findings suggest a coordinate regulation of Phex mRNA expression in lungs and bone and vitamin D metabolism during GH- and IGF-I-stimulated growth.