Vikram Khedgikar

Withaferin A: a proteasomal inhibitor promotes healing after injury and exerts anabolic effect on osteoporotic bone

Withania somnifera or Ashwagandha is a medicinal herb of Ayurveda. Though the extract and purified molecules, withanolides, from this plant have been shown to have different pharmacological activities, their effect on bone formation has not been studied. Here, we show that one of the withanolide, withaferin A (WFA) acts as a proteasomal inhibitor (PI) and binds to specific catalytic β subunit of the 20S proteasome. It exerts positive effect on osteoblast by increasing osteoblast proliferation and differentiation. WFA increased expression of osteoblast-specific transcription factor and mineralizing genes, promoted osteoblast survival and suppressed inflammatory cytokines. In osteoclast, WFA treatment decreased osteoclast number directly by decreasing expression of tartarate-resistant acid phosphatase and receptor activator of nuclear factor kappa-B (RANK) and indirectly by decreasing osteoprotegrin/RANK ligand ratio. Our data show that in vitro treatment of WFA to calvarial osteoblast cells decreased expression of E3 ubiquitin ligase, Smad ubiquitin regulatory factor 2 (Smurf2), preventing degradation of Runt-related transcription factor 2 (RunX2) and relevant Smad proteins, which are phosphorylated by bone morphogenetic protein 2. Increased Smurf2 expression due to exogenous treatment of tumor necrosis factor α (TNFα) to primary osteoblast cells was decreased by WFA treatment. This was corroborated by using small interfering RNA against Smurf2. Further, WFA also blocked nuclear factor kappa-B (NF-kB) signaling as assessed by tumor necrosis factor stimulated nuclear translocation of p65-subunit of NF-kB. Overall data show that in vitro proteasome inhibition by WFA simultaneously promoted osteoblastogenesis by stabilizing RunX2 and suppressed osteoclast differentiation, by inhibiting osteoclastogenesis. Oral administration of WFA to osteopenic ovariectomized mice increased osteoprogenitor cells in the bone marrow and increased expression of osteogenic genes. WFA supplementation improved trabecular micro-architecture of the long bones, increased biomechanical strength parameters of the vertebra and femur, decreased bone turnover markers (osteocalcin and TNFα) and expression of skeletal osteoclastogenic genes. It also increased new bone formation and expression of osteogenic genes in the femur bone as compared with vehicle groups (Sham) and ovariectomy (OVx), Bortezomib (known PI), injectible parathyroid hormone and alendronate (FDA approved drugs). WFA promoted the process of cortical bone regeneration at drill-holes site in the femur mid-diaphysis region and cortical gap was bridged with woven bone within 11 days of both estrogen sufficient and deficient (ovariectomized, Ovx) mice. Together our data suggest that WFA stimulates bone formation by abrogating proteasomal machinery and provides knowledge base for its clinical evaluation as a bone anabolic agent.

Discovery of coumarin-dihydropyridine hybrids as bone anabolic agents.

The concept of molecular hybridization led us to discover a novel series of coumarin-dihydropyridine hybrids that have potent osteoblastic bone formation in vitro and that prevent ovariectomy-induced bone loss in vivo. In this context, among all the compounds screened for alkaline phosphatase activity, four compounds 10, 14, 18, and 22 showed significant activity at picomolar concentrations. A series of other in vitro data strongly suggested compound 18 as the most promising bone anabolic agent, which was further evaluated for in vivo studies. From these studies compound 18 proved to be useful, which at low oral dose of 1 (mg/kg)/day body weight increased bone mass density and volume, expression of osteogenic genes (RUNX2, BMP-2, and ColI), bone formation rate (BFR), and mineral apposition rate (MAR), improved the trabecular microarchitecture, and decreased bone turn over markers in an ovariectomized rodent model for postmenopausal osteoporosis.

Constituents of Dalbergia sissoo Roxb. leaves with osteogenic activity.

One new isoflavone glucoside, caviunin 7-O-[β-d-apiofuranosyl-(1→6)-β-d-glucopyranoside] (10) and a new itaconic derivative, (E)-4-methoxy-2-(3,4-dihydroxybenzylidene)-4-oxobutanoic acid (15) along with series of isoflavones and flavonols with their glucosides (1-9 and 11-14) and a lignan glucoside (16) were isolated from the ethanolic extract of Dalbergia sissoo leaves. The structures of these compounds were established on the basis of IR, UV, (1)H and (13)C NMR, DEPT, COSY, HSQC, HMBC and MS data. All compounds (1-16) were assessed for osteogenic activity in primary calvarial osteoblast cultures. Compounds 1-4 and 10 increased alkaline phosphatase activity and mineralization thus resulting in significant osteogenic activity.

A novel therapeutic approach with Caviunin-based isoflavonoid that en routes bone marrow cells to bone formation via BMP2/Wnt- β -catenin signaling

Recently, we reported that extract of Dalbergia sissoo made from leaves and pods have antiresorptive and bone-forming effects. The positive skeletal effect attributed because of active molecules present in the extract of Dalbergia sissoo. Caviunin 7-O-[β-D-apiofuranosyl-(1-6)-β-D-glucopyranoside] (CAFG), a novel isoflavonoid show higher percentage present in the extract. Here, we show the osteogenic potential of CAFG as an alternative for anabolic therapy for the treatment of osteoporosis by stimulating bone morphogenetic protein 2 (BMP2) and Wnt/β-catenin mechanism. CAFG supplementation improved trabecular micro-architecture of the long bones, increased biomechanical strength parameters of the vertebra and femur and decreased bone turnover markers better than genistein. Oral administration of CAFG to osteopenic ovariectomized mice increased osteoprogenitor cells in the bone marrow and increased the expression of osteogenic genes in femur and show new bone formation without uterine hyperplasia. CAFG increased mRNA expression of osteoprotegerin in bone and inhibited osteoclast activation by inhibiting the expression of skeletal osteoclastogenic genes. CAFG is also an effective accelerant for chondrogenesis and has stimulatory effect on the repair of cortical bone after drill-hole injury at the tissue, cell and gene level in mouse femur. At cellular levels, CAFG stimulated osteoblast proliferation, survival and differentiation. Signal transduction inhibitors in osteoblast demonstrated involvement of p-38 mitogen-activated protein kinase pathway stimulated by BMP2 to initiate Wnt/β-catenin signaling to reduce phosphorylation of GSK3-β and subsequent nuclear accumulation of β-catenin. Osteogenic effects were abrogated by Dkk1, Wnt-receptor blocker and FH535, inhibitor of TCF-complex by reduction in β-catenin levels. CAFG modulated MSC responsiveness to BMP2, which promoted osteoblast differentiation via Wnt/β-catenin mechanism. CAFG at 1 mg/kg/day dose in ovariectomy mice (human dose ∼0.081 mg/kg) led to enhanced bone formation, reduced bone resorption and bone turnover better than well-known phytoestrogen genistein. Owing to CAFG’s inherent properties for bone, it could be positioned as a potential drug, food supplement, for postmenopausal osteoporosis and fracture repair.

Design and synthesis of ERα/ERβ selective coumarin and chromene derivatives as potential anti-breast cancer and anti-osteoporotic agents

Several new coumarin and chromene prototype derivatives have been synthesised and evaluated for their ERα and ERβ selective activity. Coumarin prototype compounds 18 & 19 were found to be ERα selective and the most active, exhibiting potential antiproliferative activity against both ER +ve & ER −ve breast cancer cell lines. The surprise finding of the series, however, are the novel prototype III chromenes 45 & 46, with aroyl substitution at the 6th position. Both the compounds have shown potent antiproliferative activity against both the breast cancer cell lines, promote alkaline phosphatase activity, enhance osteoblast mineralization in vitro, significantly decrease ERE–ERα dependent transactivation and induce ERβ activity. This specific upregulation of ERβ isoform activity of compound 45 may be responsible for the antiosteoporotic activity at picomolar concentration. In addition, both the compounds were also devoid of any estrogenic activity, which correlates to their antiestrogenic behaviour in the two breast cancer cell lines. Assessment of selectivity using specific SiRNAs for ERα and ERβ revealed that most of the compounds showed ERα and ERβ-mediated action, except compound 28, which showed selectivity to ERα only. Computational docking analysis of active compounds 18 and 45 was conducted to correlate the interaction with the two receptors and it was found that the docked conformations of the coumarin prototype, compound 18 at ERα and ERβ active sites were more or less superimposable on each other. However, the unique orientation of the aminoalkoxy side chain of novel chromene (prototype III) compound 45 in the ERβ binding cavity may be responsible for its potential biological response.

In vivo efficacy studies of layer-by-layer nano-matrix bearing kaempferol for the conditions of osteoporosis: A study in ovariectomized rat model

A prototype formulation based on layer-by-layer (LbL) nano-matrix was developed to increase bioavailability of kaempferol with improved retention in bone marrow to achieve enhanced bone formation. The layer-by-layer nano-matrix was prepared by sequential adsorption of biocompatible polyelectrolytes over the preformed kaempferol-loaded CaCO(3) template. The system was pharmaceutically characterized and evaluated for osteogenic activity in ovariectomized (OVx) rats. Data have been compared to the standard osteogenic agent parathyroid hormone (PTH). Single oral dose of kaempferol loaded LbL nano-matrix formulation increased bioavailability significantly compared to unformulated kaempferol. Three months of Formulated kaempferol administration to osteopenic rats increased plasma and bone marrow Kaempferol levels by 2.8- and 1.75-fold, respectively, compared to free Kaempferol. Formulated Kaempferol increased bone marrow osteoprogenitor cells, osteogenic genes in femur, bone formation rate, and improved trabecular micro-architecture. Withdrawal of Formulated kaempferol-in OVx rats resulted in the maintenance of bone micro-architecture up to 30days, whereas micro-architectural deterioration was readily observed in OVx rats treated with unformulated kaempferol-within 15days of withdrawal. The developed novel formulation has enhanced anabolic effect in osteopenic rats through increased stimulatory effect in osteoblasts. Treatment post-withdrawal sustenance of formulated kaempferol could become a strategy to enhance bioavailability of flavanoids.

Synthesis and evaluation of new coumarin–pyridine hybrids with promising anti-osteoporotic activities

Anti-osteoporotic effects of the newly synthesized coumarin-pyridine hybrids were evaluated in primary cultures of rat calvarial osteoblasts in vitro. Compounds 6a, i, j and k were potent in stimulating osteoblast differentiation and mineralization as assessed by the alkaline phosphatase production and alizarin red-S staining assay, respectively. These compounds were also found to be nontoxic in osteoblast cells as compared to the control group in an MTT assay. Furthermore, the effect of these compounds on the transcript levels of osteogenic genes revealed that the compound 6j robustly enhanced mineralization of the osteogenic genes in calvarial osteoblasts. In this context, compound 6j was selected as a potential lead for further structural optimization in the development of new anti-osteoporotic agents.

Neoflavonoids as potential osteogenic agents from Dalbergia sissoo heartwood

The present study was undertaken to investigate and rationalize the in vitro antiosteoporotic activity of neoflavonoids, isolated from Dalbergia sissoo heartwood. Neoflavonoids were isolated using extensive column chromatography and identified as dalsissooal (1) a new compound and cearoin (2), dalbergin (3), 4-methoxy dalbergion (4), dalbergiphenol (5), dalbergichromene (6), methyl dalbergin (7) and latinone (8) as known compounds by comparison their spectroscopic data with those reported in the literature. Among the screened compounds, compounds 1, 3, 5-8 significantly increased proliferation as assessed by alkaline phosphatase activity and mineralization in calvarial osteoblast cells.

EGb 761 promotes osteoblastogenesis, lowers bone marrow adipogenesis and atherosclerotic plaque formation.

AIM OF THE STUDY Our earlier study has demonstrated that EGb 761 (standardized extract of Ginkgo) has the bone sparing effect on the estrogen deficiency induced bone loss model. In the present study, we have addressed the question whether treatment of osteoporosis benefits arterial calcification or vice versa, because both adipocyte and osteoblast originate from the same mesenchymal cell of the bone marrow cell (BMC) population. MATERIALS AND METHODS Bone marrow cells were isolated to study the effect of EGb 761 on osteoblast and adipocytes. For in vivo effect hamsters were fed high fat diet and the effect of EGb 761 studied on atherosclerotic plaque formation and endothelial function. RESULTS BMCs undergoing induced osteogenic or adipogenic differentiations in the presence of EGb 761 show increase and decrease in mineralization and adipogenesis respectively. Osteogenic and adipogenic mRNAs, reveal lineage dependent expression patterns. Runx-2 (osteoblast transcription factor) showed a progressive increase, whereas PPAR-γ (adipogenic regulator) was attenuated, with same pattern of expression being for late osteogenic and adipogenic genes. EGb 761 led to increase in apoptotic cells and ROS, an important upstream signal. In vivo experiments in hamsters after induction with high cholesterol diet (HCD) show improvement in endothelial function by EGb 761 with lowering in total plasma cholesterol levels. EGb 761 led to vascular preservation of the aortic lumen with impairment of the endothelium dependent relaxation which was corroborated by micro-CT and histological sections of the thoracic region of the aorta. CONCLUSION From this data, it can be implied that EGb 761 controls bone loss, adiposity and lowers atherogenic risk factor after HCD induction.

Osteogenic efficacy enhancement of kaempferol through an engineered layer-by-layer matrix: a study in ovariectomized rats

AIM A layer-by-layer matrix (LBL) comprising kaempferol (LBL-KEM) was prepared for improved osteogenic action. MATERIALS & METHODS The LBL-KEM consisted of alternate layers of sodium alginate and protamine sulfate, which were sequentially deposited on the preformed kaempferol (KEM)-loaded CaCO3 core (CaCO3-KEM) by LBL self-assembly. The LBL matrix developed was evaluated for layer growth by ζ-potential and size alterations after self-assembly of each layer. Its physicochemical properties and intestinal absorption pattern were characterized and its pharmacokinetic behavior, mineralization of bone marrow cells, bone mineral density, bone strength, microcrack formation and estrogenicity were evaluated after oral administration. RESULTS The entrapment efficiency of KEM was 94 ± 2% and the cumulative %KEM released from LBL-KEM was 19.2 and 63.5% at pH 1.4 and 7.4, respectively, after 24 h. Stepwise polyelectrolyte assembly onto initially positively charged particles (+21.2 mV) resulted in alterations between -28.5 and +10.9 mV. A final ζ-potential of -8.9 mV was obtained after terminal surface modification with sodium deoxycholate. Fluorescein isothiocyanate-labeled LBL matrix was diffused into the basolateral lacteal region upon oral administration to rats. The area under the KEM serum concentration curve following oral administration of LBL-KEM to rats was 2479 ± 682 ng·h/ml, nearly twofold higher than free KEM. The concentration-time profile in bone marrow indicated improved penetration and retention of KEM on administration of LBL-KEM. Treatment with LBL-KEM restored bone mineralization, bone mineral density, microcrack formation and empty osteocyte lacunae density in ovariectomized (OVx) rats, which was significantly (p < 0.05) improved in femoral diaphysea, tibial head and vertebrae compared with free KEM treatment. Administration of LBL-KEM to growing female rats for 4 weeks resulted in no estrogenicity when compared with OVx rats. CONCLUSION The data suggests that LBL matrix enhanced drug delivery, improved pharmacokinetics and maintained better bone quality under OVx conditions.