▶ Development of novel agents for fracture healing and management of osteoporosis through modern drug design, scientific validation of traditional remedies and generation of new knowledge.

The screening program is actively carrying out osteogenic, anti-resorptive and dual action screenings of synthetic and phytochemicals and standardized extract from Indian medicinal plants. Current status of major developments out of this effort is summarized below:

1.1.1 Compound S011-341

Compound S011-341 and its diastereomers S-016-969 and S-016-970 were found to have osteoprotective effects. Further, two enantiomers from each of the diastereomers have been isolated. Further studies are in progress.

1.1.2 Compound S016-1436

Compound S016-1436 has been evaluated for bone forming activity in female osteopenic Sprague Dawley rat model and compound exhibits osteoprotective effect at dose of 5mg/kg body weight. Further studies in combination with PTH are in progress.

1.1.3 S007-1500: Fracture healing agent

GPCR liability studies for S007-1500 were carried out at CEREP Eurofins, France. Compound had no GPCR liability. 30-DAY repeat dose oral toxicity and toxicokinetic study in Beagle dogs with 14-day recovery period have been completed and compound is safe. Future plans include submission of IND application dossier.

1.1.4 Development of a standardized extract from stem of Cassia occidentalis L. for mitigating glucocorticoid-induced bone loss

Cassia occidentalis L. (belongs to Caesalpiniaceae family) is an annual plant. Pursuing a lead from a folk practice prevalent since the late nineteenth century in Andhra Pradesh, we have not only confirmed its fracture healing activity but also demonstrated efficacy in preventing glucocorticoid-induced osteoporosis (GIO), the commonest form of medication-induced bone loss caused chiefly due to impairment of bone formation. Our study found that standardized extract of an ethanolic extract and its butanolic fraction from the stem of Cassia occidentalis has bone anabolic as well as anti-catabolic effects on skeleton, resulting in the protection against glucocorticoid-induced bone loss. As many as six osteogenic compounds were isolated out of which apigenin-6C-glucopyranoside was most effective in vitro. Our results contribute towards validation of the traditional use of Cassia occidentalis in fracture healing and also suggest its beneficial use in GIO. (J EthnopharmacolPMID: 30703497) Presently, the standardized extract of the Cassia occidentalis stem is undergoing IND-enabling studies for filing phase I clinical trial approval to DCG(I) under the recently launched Phyto-pharmaceutical mode. To this effect all regulatory studies in rats have been submitted and IND compilation is in process.

1.1.5 Synthesis of Pentoxifylline derivatives for Postmenopausal Osteoporosis

Pentoxifylline (PTX),a derivative of theobromine, a methylated xanthine has been investigated from last three decades because of its primary pharmacological actions on hemorheology and other anti-inflammatory effects. It was approved in 1984 for the prevention of intermittent claudication in chronic occlusive arterial disease. It has salutary effects in segmental bone defect and fracture healing, as well as stimulation of bone formation (Pal et al. Bone 123 (2019) 28–38). Therefore, in view of the potential fracture healing and osteogenic properties of PTX, derivatives of pentoxifylline were designed and synthesized by treating pentoxifylline with different derivatives of 2H-pyran-2-ones to afford functionalized benzene-cored pentoxifyllines in good yields.

1.1.6 Synthesis of Fluorescent Medicarpin derivatives for deciphering anti-osteoporosis activity

Fluorescent probes have been highly attractive and versatile analytical and imaging tools because of their inherent unique characteristics. Researchers are now routinely tagging the non-fluorescent molecules of interest with a fluorescent moiety for visualization. It can be used to identify the mechanism of action, localization, target identification, and quantification of molecules in living cells and organisms. Medicarpin was found to be active for the treatment of osteoporosis. Now in order to identify its target and mechanism of action, fluorescent derivatives of Medicarpin have been designed and synthesized by tagging fluorescein/phenothiazine moiety as a fluorophore.

1.1.7 Therapeutic repurposing

1.1.7.1 Pentoxifylline restores bone mass and quality in osteopenic rabbits by an osteogenic mechanism: A comparative study with human parathyroid hormone

The non-selective phosphodiesterase inhibitor, pentoxifylline (PTX) belongs to methylxanthine class and is used for the treatment of intermittent claudication due to artery occlusion. Previous studies reported salutary effect of PTX in segmental bone defect and fracture healing in rats and stimulation of bone formation in mice. We studied the effect of orally dosed PTX in skeletally mature OVX rabbits with established osteopenia. EC50 of PTX in rabbit bone marrow stromal cells was 3.07±1.37nM. Plasma PTX level was 2.05±0.522nM after a single oral dosing of 12.5 mg/kg, which was 1/6th adult human dose of PTX. After daily oral dosing for four months at 12.5 mg/kg to osteopenic rabbits, PTX restored BMD, bone mineral content (BMC), micro-architecture and bone strength to the level of sham operated (ovary intact) group. These effects were observed at both axial and appendicular bones. Furthermore, similar to PTH, PTX had no effect on bone resorption. Taken together, PTX completely restored bone mass, bone strength and bone biomaterial properties by an anabolic mechanism, and hold the potential for becoming the first oral osteogenic drug for the treatment of post-menopausal osteoporosis. (Bone: PMID: 30858147).

Team Members:

▶ Design, synthesis and bioevaluation of novel molecules/isolates from natural sources for new lead generation and/or development of agents for the management of osteoporosis, and bone related disorders.
▶ Scientific validation of traditional remedies
▶ Therapeutic repurposing
▶ Molecular mechanism of action of promising agents;
▶ Advancing in knowledge frontiers

1.1.7.2 Daflon has osteogenic and anti-resorptive effects and enhances the skeletal effect of teriparatide: evidence of estrogen receptor  in mediating skeletal effects of the drug

Flavanone glycoside containing drug, daflonR (diosmin/hesperidin, 9:1 combination) is in clinical use for the treatment of chronic venous insufficiency for many years. Here, we showed that at the human equivalent dose daflon promoted bone regeneration at the osteotomy site and peak bone accrual in rat models. Daflon restored trabecular bones and strength with attendant increases in surface referent bone formation parameters and serum osteogenic marker in osteopenic OVX rats. Daflon also suppressed bone resorption in OVX rats and OVX rats treated with teriparatide [human parathyroid hormone (PTH 1-34), TDPD] without inhibiting the osteoanabolic effect of TDPD. Moreover, the combination of daflon and TDPD showed greater skeletal effect than the monotherapies. PK studies revealed that oral administration of daflon resulted in the formation of diosmetin, the aglycone form of diosmin. Subsequent studies showed that diosmetin specifically activated estrogen receptor- (ER) resulting in the stimulation of osteoblast differentiation and suppressed the anti-osteoblastogenic factor, sclerostin. Our findings of osteoanabolic effect of daflon accompanied by its ability to enhance the skeletal effect of TDPD could lead to a new class for anti-osteoporosis therapy through therapeutic repurposing (Biomed PharmacotherPMID: 31306971)

1.1.7.3 The osteogenic effect of liraglutide involves enhanced mitochondrial biogenesis in osteoblasts

Liraglutide (Lira), a long-acting glucagon-like peptide 1 receptor (GLP-1R) agonist reduces glycosylated hemoglobin in type 2 diabetes mellitus patients. Here, we investigated the osteoanabolic effect of Lira and studied the underlying mechanism. In established osteopenic OVX rats, Lira completely restored bone mass and strength comparable to parathyroid hormone (PTH). The serum levels of osteogenic surrogate pro-collagen type 1 N-terminal pro-peptide (P1NP) and surface referent bone formation parameters were comparable between Lira and PTH. GLP-1R, adiponectin receptor 1 (AdipoR1) and peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC1) levels in bones were downregulated in the OVX group but restored in the Lira group whereas PTH had no effect. In cultured osteoblasts, Lira time-dependently increased GLP-1R, AdipoR1 and PGC-1. In osteoblasts, Lira rapidly phosphorylated AMP-dependent protein kinase (AMPK), the cellular energy sensor. Exendin 3, a selective GLP-1R antagonist and PKA inhibitor H89 blocked Lira-induced increases in osteoblast differentiation, and expression levels of AdipoR1 and PGC-1. Lira increased mitochondrial number, respiratory proteins and respiration in osteoblasts in vitro and in vivo, and blocking mitochondrial respiration mitigated Lira-induced osteoblast differentiation. Taken together, our data show that Lira has a strong osteoanabolic effect which involves upregulation of mitochondrial function.(BiochemPharmacolPMID: 30885766)

2.2 Progress on advancing in knowledge frontiers

2.2.1 MicroRNA-672-5p identified during weaning reverses osteopenia and sarcopenia in ovariectomized mice

Post-menopausal condition augments the biological aging process, characterized by multiple metabolic disorders in which bone loss is the most prevalent outcome and usually coupled with sarcopenia. Coexistence of such associated pathogenesis have much worse health outcomes, compared to individuals with osteoporosis only. Pre- and post-natal bone development demands calcium from mother to fetus during pregnancy and lactation leading to a significant maternal skeletal loss. It follows an anabolic phase around weaning during which there is a notable recovery of the maternal skeleton. Here, we have studied the therapeutic effect of microRNA-672-5p identified during weaning when it is predominantly expressed, in ovariectomized mice for both osteopenia and sarcopenia. miR-672-5p induced osteoblast differentiation and mineralization. These actions were mediated through inhibition of Smurf1 with enhanced Runx2 transcriptional activation. In vivo, miR-672-5p significantly increased osteoblastogenesis and mineralization, thus reversing bone loss caused by ovariectomy. It also improved bone-mineral density, load-bearing capacity, and bone quality. Sarcopenia was also alleviated by miR-672-5p, as we observed increased cross-sectional area and Feret’s diameter of muscle fibers. We hypothesize that elevated miR-672-5p expression has therapeutic efficacy in estrogen-deficiency-induced osteopenia along with sarcopenia. (MolTher Nucleic Acids PMID: 30769134)

2.2.2 MiR-487b-3p impairs osteoblastogenesis by targeting Notch-regulated ankyrin-repeat protein

The study reports the role of miR-487b-3p in regulation of osteoblast functions. Over-expression of miR-487b-3p leads to inhibition of osteoblastic differentiation. Using in silico approaches, Nrarp was found to be the direct target of miR-487b-3p which was further validated by luciferase 3′ UTR reporter assay. Nrarp inhibits Notch-1 signaling and promotes Wnt signaling by stabilization of LEF-1. Protein levels of Nrarp, RUNX-2, Lef1 and β catenin were reduced in osteoblasts cells transfected with miR-487b-3p whereas protein levels of Notch1, Hes1 and P- β catenin were up regulated when osteoblast cells were transfected with miR-487b-3p. These outcomes were reversed after treating cells with anti-miR-487b-3p. Further silencing of miR-487b-3p in neonatal and Ovx Balb/c mice attenuated all the inhibitory actions of miR-487b-3p on osteoblast differentiation. Overall, miR-487b-3p negatively regulates osteogenesis by suppressing Nrarp expression, which in turn, suppresses Runx-2 and Wntsignalling, both of which play a pivotal action in osteoblast differentiation. (J Endocrinol. 2019;241(3):249-263.).

2.2.3 Increased bone marrow-specific adipogenesis by clofazimine causes impaired fracture healing, osteopenia and osteonecrosis without extra-skeletal effects in rats

Mycobacterium leprae infection causes bone lesions and osteoporosis, however, the effect of anti-leprosy drugs on the bone is unknown. We, therefore, set out to address it by investigating osteogenic differentiation from bone marrow derived mesenchyme stem cells. Out of seven anti-leprosy drugs, only clofazimine (CFZ) reduced MSCs viability (IC50 ~1M) and their osteogenic differentiation but increased adipogenic differentiation on a par with rosiglitazone, and this effect was blocked by a peroxisome proliferator-activated receptor gamma(PPARγ) antagonist, GW9662. CFZ also decreased osteoblast viability and resulted in impaired bone regeneration in a rat femur osteotomy model at 1/3rd human drug dose owing to increased callus adipogenesis as GW9662 prevented this effect. In adult rats, CFZ caused osteopenia in long bones marked by suppressed osteoblast function due to enhanced adipogenesis and increased osteoclast functions. A robust increase in marrow adipose tissue (MAT) by CFZ did not alter hematologic parameters but likely reduced BM vascular bed leading to osteonecrosis (ON) characterized by empty osteocyte lacunae. From these data, we conclude that CFZ has skeletal toxicity and could be used for creating a rodent ON model devoid of extra-skeletal effects. (ToxicolSciPMID: 31393584)

2.2.4 Estrogen receptor activation in response to Azadirachtin A stimulates osteoblast differentiation and bone formation in mice

In this study, it was seen that a natural pure compound Azadirachtin A (Aza A) isolated from Azadirachtaindica binds selectively to a site in the estrogen receptor, identifying itself to be a selective tissue modifier. Using computational and medicinal chemistry, Aza A was shown to bind electively to estrogen receptor‐α (ERα) as compared with ERβ. This preferential binding of Aza A to ERα with good pharmacokinetic distribution in the body forms metabolites, showing that it is well absorbed. In in vivo estrogen deficiency models for osteoporosis, Aza A at a much lower dose enhances new bone formation at both sites of the trabecular and cortical bone with increased bone strength and presents with no hyperplastic effect in the uterus. (J Cell PhysiolPMID:31225646)