Catalpol attenuates osteoporosis in ovariectomized rats through promoting osteoclast apoptosis via the Sirt6-ERα-FasL axis.

BACKGROUND: Catalpol, a major active component of the Chinese herb Rehmannia glutinosa, possesses various pharmacological benefits, including anti-inflammatory, antidiabetic, and antitumor properties. Recent studies have reported that catalpol can attenuate bone loss and enhance bone formation. Nevertheless, the molecular mechanisms underlying its effects on osteoporosis pathogenesis remain unclear. PURPOSE: We investigated whether catalpol had a protective effect against postmenopausal osteoporosis (PMOP) and explored its exact mechanism of action. METHODS: Seventy-two rats were randomly divided into six groups: sham, model, low-dose catalpol (5 mg/kg/day), medium-dose catalpol (10 mg/kg/day), high-dose catalpol (20 mg/kg/day), and positive control (alendronate, 2.5 mg/kg). In this experiment, a ovariectomy was performed to establish a female rat model of PMOP. After 12 weeks of gavage, micro-computed tomography (micro-CT) and histochemical staining were performed to evaluate bone mass, bone microstructure and histological parameters. Furthermore, RAW 264.7 cells were induced by RANKL to form mature osteoclasts to investigate the effect of catalpol on osteoclast differentiation and apoptosis in vitro. Additionally, the osteoclast apoptosis-related proteins of Sirt6, ERα, FasL, NFATc1, cleaved-caspase 8, cleaved-caspase 3, and Bax were assessed using western blotting. The expressions of NFATc1, Ctsk, Oscar, and Trap were quantified using RT-qPCR. The apoptotic rate of the osteoclasts was determined using flow cytometry. Sirt6 knockdown was performed using siRNA gene silencing in experiments to investigate its role in catalpol-mediated osteoclast apoptosis. The deacetylation of ERα in osteoclasts was tested via co-immunoprecipitation. RESULTS: Catalpol (10 and 20 mg/kg) and alendronate (2.5 mg/kg) could significantly improve bone mineral density (BMD) and microstructure and decrease osteoclast density in ovariectomized (OVX) rats. In addition, catalpol (10 and 20 mg/kg) upregulated the expression of Sirt6, ERα, FasL, cleaved-caspase 8, cleaved-caspase 3, Bax, and downregulated the expression of NFATc1, Ctsk, Oscar, Trap both in vivo and in vitro. Catalpol also promoted ERα deacetylation and stabilized ERα protein to enhance the expression of FasL. In addition, Sirt6 knockdown by siRNA prevented ERα deacetylation and eliminated catalpol-mediated osteoclast apoptosis. CONCLUSIONS: The present study demonstrated that catalpol prevents estrogen deficiency-induced osteoporosis by promoting osteoclast apoptosis via the Sirt6-ERα-FasL axis. These findings revealed a novel molecular mechanism underpinning the impact of catalpol in the progression of osteoporosis and provided novel insights into the treatment of osteoporosis.

Novel insights into the pharmacological modulation of human periodontal ligament stem cells by the amino-bisphosphonate Alendronate.

Alendronate (ALN) is a second-generation bisphosphonate widely used for osteoporosis and cancer-induced bone lesions. Many studies have confirmed a strong relationship between osteonecrosis of the jaws (ONJ) development and oral bisphosphonates, especially ALN, although the molecular mechanisms underlying this pathology have not yet been elucidated. The reduction in bone turnover and vascularization usually observed in ONJ are the result of ALN action on different cell types harboured in oral microenvironment, such as osteoclasts, endothelial cells, and periodontal ligament stem cells (PDLSCs). In this perspective, the present study aims to investigate the effects of different ALN concentrations (2 µM, 5 µM, 10 µM, 25 µM, 50 µM) on the phenotype and functional properties of human PDLSCs (hPDLSCs). hPDLSCs showed a decrease in cell viability (MTT assay) only when treated with ALN concentration of 10 µM or larger for 48 h and 72 h. Cell cycle analysis revealed a moderate increase in proportion of S-phase cells after exposure to low ALN concentration (2-5 µM), an effect that was reverted after exposure to 10-50 µM ALN. Conversely, cell death was evidenced via Annexin V/PI assay at very high concentration of ALN (50 µM) after 4 days of treatment. In addition, we explored whether the effects of ALN on hPDLSCs growth and survival can be mediated by its ability to modulate oxidative stress. To this, we quantified the intracellular ROS amount and lipid peroxidation by using DCF probe and Bodipy staining, respectively. Flow cytometry analysis showed that ALN induced a dose-dependent reduction of intracellular oxidative stress and lipid peroxidation upon treatment with low concentrations at both 48 h and 72 h. Increased levels of oxidative stress was reported at 50 µM ALN and was also confirmed via TEM analysis. Despite the stability of the cellular immunophenotype, hPDLSCs showed impaired mobility after ALN exposure. Chronic exposure (7-14 days) to ALN in the range of 2-10 µM significantly decreased the expression of the differentiation-related factors ALP, RUNX2, COLI, and OPN as well as the osteogenic ability of hPDLSCs compared with untreated cells. Conversely, higher doses were found to be neutral. Our findings indicated that the effects of ALN on hPDLSCs behavior are dose-dependent and suggest a role for oxidative stress in ALN-induced cell death that may lead to novel therapeutic approaches for ONJ.

Enhancement of isoprene production in engineered Synechococcus elongatus UTEX 2973 by metabolic pathway inhibition and machine learning-based optimization strategy.

An engineered Synechococcus elongatus UTEX 2973-IspS.IDI is used to enhance isoprene production through geranyl diphosphate synthase (CrtE) inhibition and process parameters (light intensity, NaHCO3 and growth temperature) optimization approach. A cumulative isoprene production of 1.21 mg/gDCW was achieved with productivity of 12.6 µg/gDCW/h in culture supplemented with 20 µg/mL alendronate. This inhibition strategy improvises the cumulative isoprene production 5.76-fold in presence of alendronate. The maximum cumulative production of isoprene is observed to be 5.22 and 6.20 mg/gDCW (54.4 and 64.6 µg/gDCW/h) at statistical and artificial neural network genetic algorithm (ANN-GA) optimized conditions, respectively. The overall increase of isoprene production is found to be 29.52-fold using an integrated approach of inhibition and ANN-GA optimization in comparison to unoptimized cultures without alendronate. This study reveals that alendronate use as a potential inhibitor and machine learning based optimization is a better approach in comparison to statistical optimization to enhance the isoprene production.

Engineering Bifunctional Calcium Alendronate Gene-Delivery Nanoneedle for Synergistic Chemo/Immuno-Therapy Against HER2 Positive Ovarian Cancer.

Ovarian cancer is the most lethal gynecological malignancy. Most patients are diagnosed at an advanced stage with widespread peritoneal dissemination and ascites. Bispecific T-cell engagers (BiTEs) have demonstrated impressive antitumor efficacy in hematological malignancies, but the clinical potency is limited by their short half-life, inconvenient continuous intravenous infusion, and severe toxicity at relevant therapeutic levels in solid tumors. To address these critical issues, the design and engineering of alendronate calcium (CaALN) based gene-delivery system is reported to express therapeutic level of BiTE (HER2×CD3) for efficient ovarian cancer immunotherapy. Controllable construction of CaALN nanosphere and nanoneedle is achieved by the simple and green coordination reactions that the distinct nanoneedle-like alendronate calcium (CaALN-N) with a high aspect ratio enabled efficient gene delivery to the peritoneum without system in vivo toxicity. Especially, CaALN-N induced apoptosis of SKOV3-luc cell via down-regulation of HER2 signaling pathway and synergized with HER2×CD3 to generate high antitumor response. In vivo administration of CaALN-N/minicircle DNA encoding HER2×CD3 (MC-HER2×CD3) produces sustained therapeutic levels of BiTE and suppresses tumor growth in a human ovarian cancer xenograft model. Collectively, the engineered alendronate calcium nanoneedle represents a bifunctional gene delivery platform for the efficient and synergistic treatment of ovarian cancer.

Anabolic Response of Intermittent Parathyroid Hormone and Alendronate on the Osteochondral Tissue of TMJ.

OBJECTIVE: To characterize the effects of parathyroid hormone (PTH) and alendronate (Alend) on the osteochondral tissue of temporomandibular joint (TMJ). MATERIALS AND METHODS: Ninety-six male and female transgenic reporter mice, 4 to 5 weeks old were divided into 6 groups: (1) Control group: Saline was injected daily for 14 days; (2) PTH: PTH was injected daily for 14 days; (3) Alend: Alend was injected every alternate days for 14 days; (4) Combined PTH and Alend: PTH was injected daily and Alend injected every alternate days for 14 days; (5) PTH then Alend: PTH was injected daily for 14 days followed by Alend injections in alternate days for 14 days; and (6) PTH wait Alend: PTH was injected daily for 14 days. There was a waiting period of 1 week before administration of Alend in alternate days for 14 days. Mice were injected with 5-ethnyl-2'-deoxyuridine (EdU), 48 and 24 hours prior to euthanization. RESULTS: There was significant increase in bone volume and decrease in osteoclastic activity in groups in which Alend was administered after PTH in both gender. There was significant increase in cartilage thickness with PTH or Alend alone in females, whereas in males, PTH alone led to increase in cartilage thickness. Chondrocyte apoptosis was significantly decreased with PTH or Alend alone in both male and female. Matrix metallopeptidase 13, and aggreganase-2 (ADAMTS5) expression were significantly decreased with PTH and Alend alone in both gender. CONCLUSION: PTH and Alend administration causes anabolic effects in the osteochondral tissue of TMJ.

Anti-osteoporosis Effect of Fisetin against Ovariectomy Induced Osteoporosis in Rats: In silico, in vitro and in vivo Activity.

Osteoporosis is a bone related disease that is characterised by bone loss that further increases the susceptibility to bone fractures and bone frailty due to disturbances in the micro-architecture of bone tissue. Fisetin (flavonoids) exhibited anti-inflammatory and antioxidative stress effects against various diseases. In this protocol, we make an effort to comfort the anti-osteoporosis effect of fisetin against ovariectomy (OVX) induced osteoporosis. A docking study of fisetin and alendronate on the estrogen (α and ß) and vitamin D receptors was carried out. SaOS-2 (osteoblast like human) cells were used for the estimation of cell proliferation. The OVX induced OVX model was used and three doses of fisetin and alendronate was given to rats till 16 weeks. The hormone levels, bone turnover markers and biochemical parameters were estimated. Fisetin was docked into estrogen (α and ß) and vitamin D receptors, resulting in stable complexes with lower binding scores. Fisetin significantly (p < 0.001) exhibited the induction of cell proliferation against the SaOS-2 cells. OVX induced osteoporosis rats exhibited a suppression of body weight and uterus index, after the Fisetin treatment. Fisetin treatment significantly (p < 0.001) improved the level of bone mineral content (BMC), bone mineral density (BMD) and biochemical parameters such as energy, maximum load, stiffness, young modules, maximum stress and reduced the level of 1,25(OH) 2 D3 and E 2 . Fisetin treatment significantly (p < 0.001) declined the level of phosphorus (P), calcium (Ca) and boosted the level of VitD. Fisetin treatment significantly (p < 0.001) reduced the malonaldehyde (MDA) level and enhanced the glutathione (GSH), catalase (CAT), superoxide dismutase (SOD) level in the bone, intestine and hepatic tissue. Fisetin treatment suppressed the cytokines, RANKL/OPG ratio, receptor activator of nuclear factor-κB ligand (RANKL) and improved the level of osteoprotegerin (OPG). The findings suggest that fisetin could be a beneficial phytoconstituent for the treatment and prevention of postmenopausal osteoporotic complications.

Enhancement of Cancer Chemotherapeutic Efficacy via Bone-Targeted Drug Delivery Carrier in Bone Metastases.

PURPOSE: Bone metastases are common in malignant tumors, especially for the advanced cancers. Chemotherapy is an important treatment in clinic, but the application is limited due to the severe adverse reactions. We try to design bone-targeted drug delivery systems (DDS) for the delivery of chemotherapeutic drugs in bone metastatic carcinoma. MATERIAL AND METHODS: We added alendronate (Aln) to metal organic framework (MOF) to synthesize a new bone-targeted DDS named Aln-MOF. Doxorubicin (DOX) as a classic anti-cancer drug was encapsulated. The material characterization, drug release and bone affinity were detected. In vitro experiment, the cell toxicity was detected by cck-8 test and cellular uptake were detected by laser scanning confocal microscope and flow cytometry. In vivo experiment, the pharmacokinetics of DDS in the blood was analyzed by fluorescence spectrophotometer and the biodistribution was detected by a multi-mode optical in vivo imaging system. The anti-tumor effects of MOFDOX and Aln-MOFDOX were evaluated by monitoring the tumor volume and weight during the animal experiment. In addition, the toxicity of DDS to different organs was determined by HE staining. RESULTS: Aln-MOF showed good stability, no cytotoxicity and better bone affinity than MOF. Both MOFDOX and Aln-MOFDOX could release DOX, and the release rate at pH = 5.5 was faster than the rate at pH = 7.4. The cellular uptake of Aln-MOF and MOF showed no difference. Aln-MOF had a long retention time in blood, which is beneficial for the enrichment of Aln-MOF in tumor sites. Aln-MOF mainly concentrated at bone metastases in mice. MOFDOX and Aln-MOFDOX could effectively delay tumor progression, and the effect of Aln-MOFDOX was more obvious (P < 0.05). CONCLUSION: Our study confirmed that Aln-MOF has good stability, bone targeting and biosafety. Aln-MOFDOX could release DOX and effectively kill tumor cells of bone metastases. Aln-MOFDOX has a promising prospect in the treatment of bone metastasis.

Alendronate inhibits triglyceride accumulation and oxidative stress in adipocytes and the inflammatory response of macrophages which are associated with adipose tissue dysfunction.

Alendronate, a bisphosphonate used to prevent osteoporosis, stimulates osteogenesis but impairs adipogenesis. Different clinical trials suggest that the incidence of diabetes may be lower in patients treated with alendronate. Taking into account the importance of adipocytes and macrophages of adipose tissue in insulin resistance and type 2 diabetes, it is necessary to evaluate the effect of alendronate in both cell types. In this paper, we investigated the effect of alendronate on the differentiation to adipocytes of 3T3-L1 fibroblasts, the cell line most used to study adipogenesis, and also its effect on lipid content and oxidative stress in mature adipocytes as well as on the inflammatory response of macrophages. We found that alendronate inhibits differentiation of 3T3-L1 fibroblasts to adipocytes in keeping with reports in other cell lines. On the other hand, treatment of 3T3-L1 adipocytes with alendronate was able to decrease triglyceride content and to prevent H2O2-induced lipid peroxidation which was evaluated as an indicator of oxidative stress. In addition, it was found that activation of RAW 264.7 macrophages to a pro-inflammatory M1 type is inhibited by this bisphosphonate. These results suggest that alendronate may contribute to prevent adipocyte excessive enlargement and the induction of oxidative stress in 3T3-L1 adipocytes as well as the activation of macrophages to a pro-inflammatory M1 type, which are events associated with adipose tissue dysfunction and insulin resistance. In this study, we unraveled the underlying mechanisms of events that were previously observed in clinical trials.

Alendronate promotes the gene expression of extracellular matrix mediated by SP-1/SOX-9.

BACKGROUND AND PURPOSE: Osteoarthritis (OA) is a disease with significant degenerative changes of articular cartilage, which is reported to be closely related to the integrity of chondrocytes extracellular matrix (ECM). Alendronate belongs to the family of bisphosphonates with promising cartilage repair function. In the present study, the effects of Alendronate on the gene expression of chondrocytes ECM and the potential mechanism will be investigated to explore the potential therapeutic property of Alendronate on OA. METHODS: Human SW1353 chondrocytes were stimulated with 1 and 2 µM Alendronate for 12 h. The gene expression of Col2α1, COL9α2, and Acan in the treated chondrocytes was determined by qRT-PCR. QRT-PCR and western blot analysis were used to evaluate the expression level of SOX-9 in the treated chondrocytes. The expression level of SP-1 was checked by qRT-PCR and immunostaining. SiRNA against SP-1 was transfected into chondrocytes to knockdown the expression of SP-1. The levels of p-ERK1/2 and total ERK1/2 were examined using western blot analysis. TNF-α was used to induce an OA-like in vitro model in the chondrocytes for therapeutic evaluations. RESULTS: Treatment with Alendronate increased the levels of ECM related genes (Col2α1, COL9α2, and Acan) in a dose-dependent manner through increasing the expression of SOX-9, a central regulator of ECM genes. Additionally, our findings demonstrate that the effects of Alendronate in the expression of SOX-9 are mediated by SP-1 as silencing of SP-1 abolished these effects. Notably, Alendronate increased the phosphorylation of ERK1/2 and inhibition of ERK1/2 using its specific inhibitor U0126 blocked the expression of SP-1. Finally, we found that treatment with Alendronate could rescue TNF-α-induced reduction of Col2α1, COL9α2, Acan and SOX-9. CONCLUSION: Our data indicated that Alendronate might promote the gene expression of extracellular matrix through SOX-9 mediated by the ERK1/2/SP1 signaling pathway.

Absorption-Enhancing Mechanisms of Capryol 90, a Novel Absorption Enhancer, for Improving the Intestinal Absorption of Poorly Absorbed Drugs: Contributions to Trans- or Para-Cellular Pathways.

PURPOSE: We have previously reported that Capryol 90 improves the intestinal absorption of insulin, a peptide drug, without causing serious damage to the intestinal epithelium. However, the effects of Capryol 90 and its related formulations on the intestinal absorption of other drugs, and their absorption-enhancing mechanisms are still unclear. The aim of this study is to evaluate the effects of Capryol 90 and its related formulations on the intestinal absorption of drugs and elucidate their absorption-enhancing mechanisms. METHODS: The intestinal absorption of 5(6)-carboxyfluorescein, fluorescein isothiocyanate-dextrans, and alendronate was evaluated using an in situ closed loop method. Brush border membrane vesicles (BBMVs) were labeled with fluorescent probes, and the fluidity of membrane was evaluated by a fluorescence depolarization method. The expression levels of tight junction (TJ) proteins were measured using a Western blot method and immunofluorescence staining. RESULTS: Among the tested excipients, Capryol 90 significantly improved the small and large intestinal absorption of drugs. In mechanistic studies, Capryol 90 increased the membrane fluidity of lipid bilayers in BBMVs. Additionally, Capryol 90 decreased the expression levels of TJ-associated proteins, namely claudin-4, occludin, and ZO-1. CONCLUSIONS: Capryol 90 is an effective absorption enhancer for improving the intestinal absorption of poorly absorbed drugs via both transcellular and paracellular pathways.

Microfluidic fabrication of alendronate-loaded chitosan nanoparticles for enhanced osteogenic differentiation of stem cells.

AIMS: In this study, we used a cross-junction microfluidic device for preparation of alendronate-loaded chitosan nanoparticles with desired characteristics to introduce a suitable element for bone tissue engineering scaffolds. MAIN METHODS: By controlling the reaction condition in microfluidic device, six types of alendronate-loaded chitosan nanoparticles were fabricated which had different physical properties. Hydrodynamic diameter of synthetized particles was evaluated by dynamic light scattering (102 to 215 nm). Nanoparticle morphology was determined by SEM and AFM images. The osteogenic effects of prepared selected nanoparticles on human adipose stem cells (hA-MSCs) were evaluated by assessment of alkaline phosphatase (ALP) activity, calcium deposition, ALP and osteopontin gene expression. KEY FINDINGS: The highest loading efficiency percentage (%LE) was %32.42 ± 2.02. Based on MTT assessment, two samples which had no significant cytotoxicity were chosen for further studies (particle sizes and %LE were 142 ± 6.1 nm, 198 ± 16.56 nm, %16.76 ± 3.91 and %32.42 ± 2.02, respectively). In vitro release behavior of nanoparticles displayed pH responsive characteristics. Significant faster release was seen in acidic pH = 5.8 than neutral pH = 7.4. The selected nanoparticles demonstrated higher ALP activity at 14 days in comparison to selected blank sample and osteogenic differentiation media (ODM) and a downregulation at 21 days in comparison to 14 days. Calcium content assay at 21 days displayed significant differences between alendronate-loaded nanoparticles and ODM. ALP and osteopontin mRNA expression was significantly higher than the cells cultured in ODM at 14 and 21 days. SIGNIFICANCE: We concluded that our prepared nanoparticles significantly enhanced osteogenic differentiation of hA-MSCs and can be a suitable compartment of bone tissue engineering scaffolds.

Evaluating two nanocarrier systems for the transdermal delivery of sodium alendronate.

Sodium alendronate is a nitrogen-containing bisphosphonate, widely used for osteoporosis treatment. However, due to its several oral administration drawbacks, the transdermal route represents an interesting option. The aim of this study was to formulate sodium alendronate in two submicron delivery systems, microemulsions, and solid-in-oil nanosuspensions, both systems possessing permeation enhancing properties. The composition of microemulsions was determined through the construction of pseudo-ternary phase diagrams. Solid-in-oil nanosuspensions were prepared by an emulsification-freeze-drying method, evaluating the effect of sonication time and the type of surfactant. According to the results of drug loading capacity, droplet/particle size, and polydispersity index, two microemulsions and two nanosuspensions were selected to perform the subsequent evaluations. The results showed that microemulsions allowed a faster release of alendronate than nanosuspensions. The permeation capacity of alendronate formulations was assessed through the synthetic membrane Strat M®, as well as through pigskin, finding higher fluxes with microemulsions than with nanosuspensions. In order to elucidate the effect of the formulations on the permeability barrier of the stratum corneum, techniques such as ATR-FTIR and TEWL were used. Finally, measurements of erythema intensity showed that neither of the two nanosystems caused skin irritation after 2 h of contact. The results suggest that alendronate formulated in a microemulsion can be a viable transdermal nanocarrier for osteoporosis treatment.

Alendronate-Functionalized Poly(2-oxazoline)s with Tunable Affinity for Calcium Cations.

A library of poly(2-oxazoline)s functionalized with controllable amounts of alendronate, hydroxyl, and carboxylic acid side groups was successfully synthesized to create novel polymers with tunable affinity for calcium cations. The affinity of alendronate-containing polymers for calcium cations was quantified using isothermal titration calorimetry. Thermodynamic measurements revealed that the Ca2+-binding affinity of these polymers increased linearly with the amount of alendronate functionalization, up to values (KCa2+ = 2.4 × 105 M-1) that were about 120-fold higher than those for previously reported polymers. The calcium-binding capacity of alendronate-functionalized poly(2-oxazoline)s was exploited to form robust hydrogel networks cross-linked using reversible physical bonds. Oscillatory rheology showed that these hydrogels recovered more than 100% of their initial storage modulus after severe network destruction. The versatile synthesis of alendronate-functionalized polymers and their strong and tunable affinity for calcium cations render these polymers promising candidates for various biomedical applications.

Elimination of intravenous alendronate by hemodialysis: A kinetic study.

INTRODUCTION: The potential utility of intravenous alendronate for the treatment of osteoporosis in hemodialysis patients was recently reported. However, the pharmacokinetics of intravenous alendronate in patients on hemodialysis is not clear. METHODS: Six hemodialysis patients (mean age, 80.5 years) with osteoporosis who had received intravenous alendronate prior to the study were enrolled. The participants received a 30-min infusion of 900-µg alendronate intravenously at the beginning of the dialysis session. The blood flow rate (Qb) and dialysate flow rate (Qd) were set at 200 mL/min and 500 mL/min, respectively. All patients used the same dialyzer (1.5-m2 polysulfone membrane). At the completion of administration, plasma and dialysate samples were collected, and alendronate concentrations were determined using metal-free high-performance liquid chromatography (HPLC)-tandem mass spectrometry (MS/MS). RESULTS: The plasma arterial alendronate concentration was 150.9 ± 46.09 ng/mL. It decreased through the dialyzer to 76.1 ± 34.1 ng/mL (venous alendronate concentration). Mean alendronate clearance was 113.9 ± 25.6 mL/min. Mean alendronate removal by hemodialysis, measured by the difference in arterial-venous concentrations, was 51.8%. CONCLUSIONS: Fifty percent of intravenous alendronate was removed by hemodialysis, which is nearly equal to elimination of alendronate in patients with normal renal function. The elimination by hemodialysis would decrease the risk of excessive accumulation in bone. UMIN 000027182.

Synthesis and evaluation of a novel 99m Tc nitrido radiopharmaceutical with alendronate dithiocarbamate as a potential bone-imaging agent.

Currently, a popular strategy for designing novel radioprobes as bone-imaging agents is based on the concept of bifunctional radiopharmaceuticals. Considering the dithiocarbamate ligand can act as a suitable bifunctional linking agent to attach technetium-99m (99m Tc) to corresponding target molecules, in this study, alendronate dithiocarbamate (ALNDTC) was synthesized and radiolabeled with [99m Tc≡N]2+ core by ligand exchange reaction to produce 99m TcN-ALNDTC complex, for the potential use as a novel probe for bone imaging. The radiochemical purity of the complex was over 90%. The complex was stable in vitro and could bind to hydroxyapatite. The partition coefficient result indicated it was hydrophilic, and an evaluation of biodistribution in mice indicated that the complex exhibited a higher bone uptake than did 99m Tc-labeled methylenediphosphonate (99m Tc-MDP). Further, single photon emission computed tomography imaging study indicated clear accumulation in bone, suggesting that 99m TcN-ALNDTC would be a promising candidate for bone imaging.

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.

Design, Synthesis, and Pharmacokinetics of a Bone-Targeting Dual-Action Prodrug for the Treatment of Osteoporosis.

A dual-action bone-targeting prodrug has been designed, synthesized, and evaluated for in vitro and in vivo metabolic stability, in vivo tissue distribution, and rates of release of the active constituents after binding to bones through the use of differentially double-labeled derivatives. The conjugate (general structure 7) embodies the merger of a very potent and proven anabolic selective agonist of the prostaglandin EP4 receptor, compound 5, and alendronic acid, a potent inhibitor of bone resorption, optimally linked through a differentially hydrolyzable linker unit, N-4-carboxymethylphenyl-methyloxycarbonyl-leucinyl-argininyl-para-aminophenylmethylalcohol (Leu-Arg-PABA). Optimized conjugate 16 was designed so that esterase activity will liberate 5 and cathepsin K cleavage of the Leu-Arg-PABA element will liberate alendronic acid. Studies with doubly radiolabeled 16 provide a proof-of-concept for the use of a cathepsin K cleavable peptide-linked conjugate for targeting of bisphosphonate prodrugs to bone and slow release liberation of the active constituents in vivo. Such conjugates are potential therapies for the treatment of bone disorders such as osteoporosis.

Calcium phosphate nanoparticles functionalized with alendronate-conjugated polyethylene glycol (PEG) for the treatment of bone metastasis.

Because of the peculiarity of the bone microstructure, the uptake of chemotherapeutics often happens at non-targeted sites, which induces side effects. In order to solve this problem, we designed a bone-targeting drug delivery system that can release drug exclusively in the nidus of the bone. Alendronate (ALN), which has a high ability to target to hydroxyapatite, was used to fabricate double ALN-conjugated poly (ethylene glycol) 2000 material (ALN-PEG2k-ALN). The ALN-PEG2k-ALN was characterized using 1H NMR and 31P NMR and FTIR. ALN-PEG2k-ALN-modified calcium phosphate nanoparticles (APA-CPNPs) with an ALN targeting moiety and hydrophilic poly (ethylene glycol) arms tiled on the surface was prepared for bone-targeted drug delivery. The distribution of ALN-PEG2k-ALN was tested by X-ray photoelectron spectroscopy. Isothermal titration calorimetry data indicated that similar to free ALN, both ALN-PEG2k-ALN and APA-CPNPs can bind to calcium ions. The bone-binding ability of APA-CPNPs was verified via ex vivo imaging of bone fragments. An in vitro release experiment demonstrated that APA-CPNPs can release drug faster in an acid environment than a neutral environment. Cell viability experiments indicated that blank APA-CPNPs possessed excellent biocompatibility with normal cells. Methotrexate (MTX) loaded APA-CPNPs have the same ability to inhibit cancer cells as free drug at high concentrations, while they are slightly weaker at low concentrations. All of these experiments verified the prospective application of APA-CPNPs as a bone-targeting drug delivery system.

Enhanced oral delivery of alendronate by sucrose fatty acids esters in rats and their absorption-enhancing mechanisms.

Oral delivery is the most fascinating route for interminable drug remedy. However, the intestinal absorption of alendronate (ALN), a bisphosphonate drug after oral administration is very poor. Absorption enhancers, which help to achieve the efficiency-safety balance, are considered one of the most promising agents for the improvement the intestinal absorption of drugs. In the current study, we focused on using sucrose fatty acid esters (SEs) as promising absorption enhancers to enhance the intestinal absorption of alendronate using an in situ closed-loop method in rats. The intestinal absorption of alendronate was significantly enhanced in the presence of SEs, especially L-1695. In addition, no considerable increase was observed in the activity of lactate dehydrogenase (LDH) or in protein release from the intestinal epithelium in the presence of sugar esters at concentrations equivalent to or lower than 1.0% (w/v), suggesting that these compounds are safe. Furthermore, mechanistic studies revealed increased membrane fluidity and loosening of the tight junctions (TJs) might be the underlying mechanism by which SEs improve the intestinal intake of alendronate, via transcellular and paracellular routes, respectively. These findings suggest that SEs are effective absorption enhancers for improving the intestinal absorption of alendronate, without causing serious damage to the enteric epithelium.

Enhanced Oral Delivery of Bisphosphonate by Novel Absorption Enhancers: Improvement of Intestinal Absorption of Alendronate by N-Acyl Amino Acids and N-Acyl Taurates and Their Absorption-Enhancing Mechanisms.

Bisphosphonates (BPs) are carbon-substituted pyrophosphate analogs that exhibit a high affinity to hydroxyapatite and specifically inhibit bone resorption. Alendronate sodium (sodium 4-amino-1-hydroxybutylidene-1,1-bisphosphonate trihydrate) is a typical BP compound in clinical use. BPs have very low bioavailability, typically <1% after their oral administration, and their intestinal absorption is further reduced by co-administered drugs or food. In this study, we examined the effects of N-acyl amino acids and N-acyl taurates on the small intestinal absorption of alendronate. All N-acyl amino acids and N-acyl taurates increased the small intestinal absorption of alendronate, especially 1% (wt/vol) sodium palmitoyl sarcosinate (PN), which elicited a 14-fold increase. In addition, the absorption-enhancing effects of these enhancers were reversible and they may not cause continuous and irreversible membrane toxicity in the rat small intestine. Furthermore, we examined the absorption-promoting mechanisms of PN and found that it increased the membrane fluidity of the lipid bilayers. In addition, it was found that PN may open the tight junctions by reducing the expression level of claudin-4, which is a major tight junction protein. These findings indicate that these enhancers are useful for promoting the intestinal absorption of alendronate.