In-situ forming chitosan implant-loaded with raloxifene hydrochloride and bioactive glass nanoparticles for treatment of bone injuries: Formulation and biological evaluation in animal model.

In-situ forming implants receive great attention for repairing serious bone injuries. The aim of the present study was to prepare novel chitosan in-situ forming implants (CIFI) loaded with bioactive glass nanoparticles and/or raloxifene hydrochloride (RLX). Incorporating raloxifene hydrochloride (RLX) as a selective estrogen receptor modulator was essential to make use of its anti-resorptive properties. The prepared formulae were tested for their in-vitro gelation time, drug release, injectability, rheological properties, erosion rate and morphological properties. Results revealed that the formulation composed of 1% (w/v) chitosan with 2% (w/v) NaHCO3 and 1% (w/v) bioactive glass nanoparticles (CIFI-BG) possessed the most sustained drug release profile which extended over four months with low burst release effect compared to the same formulation lacking bioactive glass nanoparticles (CIFI). Selected formulations were tested for their ability to enhance bone regeneration in induced puncture in rate tibia. Results declared that these formulations were able to enhance bone regeneration after 12 weeks in comparison to the untreated tibial punctures and that containing bioactive glass could be considered as novel approach for treatment of serious bone injuries which require long term treatment and internal mechanical bone support during healing.

A Novel H2S-releasing Amino-Bisphosphonate which combines bone anti-catabolic and anabolic functions.

Bisphosphonates (BPs) are the first-line treatment of bone loss resulting from various pathological conditions. Due to their high affinity to bone they have been used to develop conjugates with pro-anabolic or anti-catabolic drugs. We recently demontrated that hydrogen sulfide (H2S), promotes osteogenesis and inhibits osteoclast differentiation. Here we developed an innovative molecule, named DM-22, obtained from the combination of alendronate (AL) and the H2S-releasing moiety aryl-isothiocyanate. DM-22 and AL were assayed in vitro in the concentration range 1-33 µM for effects on viability and function of human osteoclasts (h-OCs) and mesenchymal stromal cells (h-MSCs) undergoing osteogenic differentiation. Amperometric measures revealed that DM-22 releases H2S at a slow rate with a thiol-dependent mechanism. DM-22 significantly inhibited h-OCs differentiation and function, maintaining a residual h-OCs viability even at the high dose of 33 µM. Contrary to AL, in h-MSCs DM-22 did not induce cytotoxicity as revealed by LDH assay, significantly stimulated mineralization as measured by Alizarin Red staining and increased mRNA expression of Collagen I as compared to control cultures. In conclusion, DM-22 is a new BP which inhibits h-OCs function and stimulate osteogenic differentiation of h-MSCs, without cytotoxicity. DM-22 is an ideal candidate for a novel family of osteoanabolic drugs.

Preparation and anti-osteoporotic activities in vivo of phosphorylated peptides from Antarctic krill (Euphausia superba).

Antarctic krill (Euphausia superba) protein serves as a novel sustainable protein source for human. Krill protein isolate was phosphorylated by the dry-heating method with sodium pyrophosphate. Phosphorylated peptides from Antarctic krill (PP-AKP) were obtained from phosphorylated protein through tryptic hydrolysis. Two types of phosphate bonds were introduced by phosphorylation, i.e. PO and PO bonds. The anti-osteoporotic activities of PP-AKP at two doses (400 and 800mg/kg body weight) were investigated with an osteoporotic rat model, which was established with bilateral ovariectomy surgery. Different doses of PP-AKP were given intraperitoneal injections to rats once a day with alendronate as a positive control. Phosphorylated peptides from Antarctic krill dose-dependently preserved bone mineral density in osteoporotic rats by increasing the degree of bone mineralization. Both trabecular and cortical bone strength in osteoporotic rats was significantly improved with PP-AKP treatment. The mechanism by which PP-AKP augmented bone mineral density and bone strength was relation to the reduction in osteoclast-mediated bone remodeling, as was supported by the decrease in bone resorption markers. Phosphorylated peptides from Antarctic krill could be developed as functional food or nutritional supplements.

Novel nonsecosteroidal vitamin D3 carboxylic acid analogs for osteoporosis, and SAR analysis.

Novel vitamin D(3) analogs with carboxylic acid were explored, focusing on a nonsecosteroidal analog, LG190178, with a bisphenyl skeleton. From X-ray analysis of these analogs with vitamin D receptor (VDR), the carboxyl groups had very unique hydrogen bonding interactions in VDR and mimicked 1α-hydroxy group and/or 3ß-hydroxy group of 1α,25-dihydroxyvitamin D(3). A highly potent analog, 6a, with good in vitro activity and pharmacokinetic profiles was identified from an SAR study. Compound 6a showed significant prevention of bone loss in a rat osteoporosis model by oral administration.

Synthesis and in vitro evaluation of bisphosphonated glycopeptide prodrugs for the treatment of osteomyelitis.

As therapeutic agents of choice in the treatment of complicated infections, glycopeptide antibiotics are often preferentially used in cases of osteomyelitis, an infection located in bone and notoriously difficult to successfully manage. Yet frequent and heavy doses of these systemically administered antibiotics are conventionally prescribed to obtain higher antibiotic levels in the bone and reduce the high recurrence rates. Targeting antibiotics to the bone after systemic administration would present at least three potential advantages: (i) greater efficacy, by concentrating the therapeutic agent in bone; (ii) greater convenience, through a reduction in the frequency of administration; and (iii) greater safety, by reducing the levels of systemic drug exposure. We present here the design, synthesis and in vitro evaluation of eight prodrugs of the glycopeptide antibacterial agents vancomycin and oritavancin taking advantage of the affinity of the bisphosphonate group for bone for delivery to osseous tissues.

Effect of chitooligosaccharides on calcium bioavailability and bone strength in ovariectomized rats.

Chitosan polymer with deacetylation degree of 93% was hydrolyzed with an endo-type chitosanase (35,000 U/g protein) with substrate to enzyme ratio of 1 to 1.5 for 18 h in a batch reactor, and then the resultant hydrolysates were fractionated into four different molecular weights using an ultrafiltration (UF) membrane reactor system. An in vitro study elucidated that four kinds of chitooligosaccharides (COSs) could efficiently inhibit the formation of insoluble calcium salts in the neutral pH. In vivo effects of COSs on Ca bioavailability were further studied in the osteoporosis modeling rats induced by ovariectomy and concurrent low calcium intake. During the experimental period corresponding to the menopause with the osteoporosis disease, calcium retention was increased and bone turnover was decreased by COS IV supplementation in the ovariectomized (OVX) rats. After the low Ca diet, COS IV diet including both normal level of calcium and vitamin D significantly decreased calcium loss in feces and increased calcium retention compared to the control diet. The levels of femoral total calcium, bone mineral density (BMD), and femoral strength were also significantly increased by the COS IV diet in a similar level to those of CPP diet group. In the present study, the results proved the beneficial effects of low molecular chitooligosaccharide (COS IV) in preventing negative mineral balance.