Pharmacokinetic Evidence of Steady and Sustained Drug Release from Long-Acting Implantable Corticosteroid Matrices for Chronic Rhinosinusitis.

BACKGROUND: The efficacy of topical corticosteroids is limited in chronic rhinosinusitis (CRS) due to rapid clearance from the nasal cavity and insufficient drug delivery to inflamed sinonasal passages. LYR-210 is an implantable corticosteroid matrix designed to provide up to 24 weeks of treatment to patients with CRS by locally delivering mometasone furoate (MF) to the sinonasal mucosa. In a randomized, controlled, dose-ranging LANTERN study, LYR-210 (7500 µg) achieved clinically relevant improvement in CRS cardinal symptom composite scores, the 22-item Sinonasal Outcome Test (SNOT-22), ethmoid opacification, and the need for rescue treatment at 24 weeks. OBJECTIVE: As the plasma MF concentrations of LYR-210 (2500 µg) and LYR-210 (7500 µg) were evaluated at weeks 4, 12, and 24 in the LANTERN study (data on file at Lyra Therapeutics, Inc.), this study aims to characterize the pharmacokinetic profiles of both doses of LYR-210 at earlier timepoints post-placement in patients with CRS. METHODS: Twenty-four surgically naïve adult patients with CRS were enrolled in an open-label, multicenter study and underwent in-office bilateral administration of LYR-210 (2500 µg) (n = 12 patients) or LYR-210 (7500 µg) (n = 12 patients) into the middle meatus. Plasma MF concentrations were determined pre-placement and 1-h post-placement (day 1), and on days 2, 3, 7, 14, 21, 28, 42, and 56 by liquid chromatography-tandem mass spectrometry. RESULTS: Both LYR-210 doses were well-tolerated with no serious adverse events. Systemic MF levels were dose-dependent and lower than reported values of other respiratory MF products. Plasma MF concentrations showed steady drug release from LYR-210 (2500 µg) and LYR-210 (7500 µg) that persisted through day 56. CONCLUSION: LYR-210 achieved dose-dependent, continuous local MF delivery at a steady rate with low systemic exposure for months.

Drug Release and Pharmacokinetic Evaluation of Novel Implantable Mometasone Furoate Matrices in Rabbit Maxillary Sinuses.

BACKGROUND: Intranasal corticosteroid sprays (INCSs) used to treat chronic rhinosinusitis are suboptimal due to limited penetration into the middle meatus, rapid clearance, and poor patient compliance. A bioresorbable drug matrix, developed with the XTreoTM drug delivery platform, may overcome the limitations of INCS by providing continuous dosing over several months. OBJECTIVE: To evaluate the in vitro drug release and in vivo pharmacokinetics of novel mometasone furoate (MF) matrices in a rabbit dorsal maxillary osteotomy model. METHODS: XTreoTM matrices were formulated to consistently elute MF for up to 6 months. Matrices were surgically placed bilaterally into the maxillary sinuses of New Zealand White (NZW) rabbits. Tissue and plasma MF concentrations were measured to assess the in vivo drug delivery. The in vivo and in vitro drug release kinetics of the matrices were quantified and compared to those of rabbits receiving daily Nasonex® MF nasal sprays. RESULTS: XTreoTM matrices self-expanded upon deployment to conform to the irregular geometry of the maxillary sinus cavities in the NZW rabbits. Sustained release of MF was demonstrated in vitro and in vivo for 2 MF matrices of distinct release durations and an in vitro-in vivo correlation was established. Therapeutic levels of MF in local tissues were measured throughout the intended dosing durations. In contrast to the variable peaks and troughs of daily nasal sprays, sustained dosing via a single administration of MF matrices was confirmed by quantifiable plasma MF concentrations over the intended dosing duration. CONCLUSION: The XTreoTM MF matrices provided targeted and efficient dosing to local sinus tissues that was superior to INCS. Sustained drug release was confirmed both in vitro and in vivo. The novel XTreoTM technology may provide precisely tuned, long-lasting drug delivery to sinus tissues with a single treatment.

Calcium-sensing receptor activating ERK1/2 and PI3K-Akt pathways to induce the proliferation of osteosarcoma cells.

Our results showed that the expression of calcium-sensing receptor (CaSR) and the concentration of intracellular calcium were enhanced in the osteosarcoma cells MG-63 compared with in the normal osteoblasts hFOB1.19. GdCl3 (CaSR agonist) significantly increased the proliferation rate of MG-63 cells, the expression of PCNA and Cyclin D1 and decreased the expression of p21Cip/WAF-1 . The effect of NPS2390 (CaSR antagonist) on the above indicators was opposite to GdCl3 . In addition, GdCl3 up-regulated the phosphorylation of ERK1/2, PI3K and Akt and the proliferation rate of MG-63 cells. However, these effects of GdCl3 were cancelled by LY294002 (a PI3K inhibitor) or PD98059 (an ERK1/2 inhibitor). Our results demonstrate that activation of CaSR promotes osteosarcoma cell multiplication by up-regulating ERK1/2 and PI3K-Akt pathways.

Synovial fibroblast-targeting liposomes encapsulating an NF-κB-blocking peptide ameliorates zymosan-induced synovial inflammation.

Synovial fibroblasts (SFs) play a crucial role in the inflammatory process of rheumatoid arthritis (RA). The highly activated NF-κB signal in SFs is responsible for most of the synovial inflammation associated with this disease. In this study, we have developed an SF-targeting liposomal system that encapsulates the NF-κB-blocking peptide (NBD peptide) HAP-lipo/NBD. HAP-lipo/NBDs demonstrated efficient SF-specific targeting in vitro and in vivo. Our study also showed a significant inhibitory effect of HAP-lipo/NBD on NF-κB activation, inflammatory cytokine release and SF migration capability after zymosan stimulation. Furthermore, the systemic administration of HAP-lipo/NBDs significantly inhibited synovial inflammation and improved the pathological scores of arthritis induced by zymosan. Thus, these results suggest that an SF-targeting NF-κB-blocking strategy is a potential approach for the development of alternative, targeted anti-RA therapies.

The development of bioresorbable composite polymeric implants with high mechanical strength.

Implants for the treatment of tissue defects should mimic the mechanical properties of the native tissue of interest and should be resorbable as well as biocompatible. In this work, we developed a scaffold from variants of poly(glycolic) acid which were braided and coated with an elastomer of poly(glycolide-co-caprolactone) and crosslinked. The coating of the scaffold with the elastomer led to higher mechanical strength in terms of compression, expansion and elasticity compared to braids without the elastomer coating. These composite scaffolds were found to have expansion properties similar to metallic stents, utilizing materials which are typically much weaker than metal. We optimized the mechanical properties of the implant by tuning the elastomer branching structure, crosslink density, and molecular weight. The scaffolds were shown to be highly resorbable following implantation in a porcine femoral artery. Biocompatibility was studied in vivo in an ovine model by implanting the scaffolds into femoral arteries. The scaffolds were able to support an expanded open lumen over 12 months in vivo and also fully resorbed by 18 months in the ovine model.

Modification of Human Umbilical Cord Blood Stem Cells Using Polyethylenimine Combined with Modified TAT Peptide to Enhance BMP-2 Production.

With the emerging role of umbilical cord blood-derived mesenchymal stem cells (hUCB-MSC) for bone regeneration and delivery of therapeutic proteins, there is an increasing need for effective gene delivery systems to modify such cells. mTAT, a TAT peptide sequence bearing histidine and cysteine residues, has been successfully used for intracellular gene delivery. Using a gWiz-GFP plasmid, we demonstrated that polyethylenimine combined with mTAT (mTAT/PEI) displayed good transfection efficacy in hUCB-MSC. hUCB-MSC transfected with mTAT/PEI were shown to express more BMP-2 protein and mRNA, indicating the feasibility of using the cells as a BMP-2 delivery system. Importantly, compared to PEI25, a "gold standard" nonviral transfection polymer, mTAT/PEI had limited toxicity to the cells. Furthermore, we demonstrated enhanced osteogenic activity in vitro for BMP-2 expressing hUCB-MSC. These results provide encouraging evidence for the potential use of mTAT/PEI to genetically modify hUCB-MSC as an approach to enhance tissue regeneration.

Hypoxia-induced resistance to cisplatin-mediated apoptosis in osteosarcoma cells is reversed by gambogic acid independently of HIF-1α.

In vitro evidence of hypoxia-induced resistance to cisplatin (CDDP)-mediated apoptosis exists in human osteosarcoma (OS). Gambogic acid (GA) is a promising chemotherapeutic compound that could increase the chemotherapeutic effectiveness of CDDP in human OS cells by inducing cell cycle arrest and promoting apoptosis. This study examined whether GA could overcome OS cell resistance to CDDP. Hypoxia significantly reduced levels of CDDP-induced apoptosis in the OS cell lines MG63 and HOS. However, combined treatment with GA and CDDP revealed a strong synergistic action between these drugs, and higher protein levels of the apoptosis-related factor Fas, cleaved caspase-8 and cleaved caspase-3 and lower expression of hypoxia-inducible factor (HIF)-1α are detected in both cell lines. Meanwhile, drug resistance was not reversed by exposure to the HIF-1α inhibitor 2-methoxyestradiol. These findings strongly suggest that hypoxia-induced resistance to CDDP is reversed by GA in OS cells independently of HIF-1α. Furthermore, in vivo studies using xenograft mouse models revealed that combination therapy with CDDP and GA exerted increased antitumor effects by inducing apoptosis. Taken together, our results demonstrate that GA may be a new potent therapeutic agent useful for targeting human OS cells.

Aquaporin 3 protects against lumbar intervertebral disc degeneration via the Wnt/ß-catenin pathway.

Previous studies have demonstrated that the expression of aquaporin 3 (AQP3), a water channel which promotes glycerol permeability and water transport across cell membranes, is reduced in degenerative lumbar intervertebral disc (IVD) tissues. However, the role of AQP3 in the pathogenesis of IVD degeneration has not recieved much scholarly attention. The objective of the present study was to investigate the effect of AQP3 on cell proliferation and extracellular matrix (ECM) degradation in human nucleus pulposus cells (hNPCs) using gain-of-function and loss-of-function experiments, and to determine whether Wnt/ß-catenin signaling is involved in the effect of AQP3 on IVD degeneration. hNPCs were transfected with the AQP3-pcDNA3.1 plasmid or AQP3 siRNA to overexpress or suppress AQP3. An MTT assay was performed to determine cell proliferation, and we found that AQP3 promoted hNPC proliferation. The expression of aggrecan, a disintegrin and metalloproteinase with thrombospondin motifs (ADAMTS)4 and ADAMTS5 was detected using western blot analysis, to examine the effect of AQP3 on ECM degradation in hNPCs. The results revealed that AQP3 inhibited ECM degradation in hNPCs. In addition, we found that Wnt/ß-catenin signaling was suppressed by AQP3. However, the effect of AQP3 on hNPC proliferation and ECM degradation was reversed by treatment with lithium chloride, a known activator of Wnt/ß­catenin signaling. In conclusion, using in vitro and in vivo tests, we have reported for the first time, to the best of our knowledge, that AQP3 exerts protective effects against IVD degeneration, and these are effected, at least partially, through the inhibition of Wnt/ß-catenin signaling.

Tumor necrosis factor-α-dependent infiltration of macrophages into the dorsal root ganglion in a rat disc herniation model.

STUDY DESIGN: A prospective molecular mechanism of macrophages infiltration in experimental disc herniation. OBJECTIVE: To investigate the mechanisms of macrophages infiltration into the dorsal root ganglion (DRG) in a rat model of disc herniation. SUMMARY OF BACKGROUND DATA: Macrophages infiltrate the DRG after application of nucleus pulposus (NP) on the DRG, and may play an important role in radiculopathy. However, the mechanisms of macrophages infiltration after NP application remain poorly understood. METHODS: After experimental disc herniation in this study, we investigated changes in the expression of ED1 (a marker of macrophages) and vascular cell adhesion molecule-1 (VCAM-1) in DRG using immunofluorescence. We also investigated the expression of ED1 and VCAM-1 in DRG by treatment with tumor necrosis factor-α (TNF-α) inhibitor at the time of surgery. RESULTS: We found a massive ED1-positive macrophages infiltrated the DRG, and VCAM-1-like immunoreactivity vessels became evident after NP application. Furthermore, both macrophage infiltration and VCAM-1 expression were prevented by treatment with TNF-α inhibitor at the time of surgery. CONCLUSION: These findings indicated that macrophages infiltration into the DRG was TNF-α-dependent, and might be partly mediated by VCAM-1 in the early stage of experimental lumbar disc herination. Taken together, this study provides important preliminary data suggesting that TNF-α plays an important role in the macrophage infiltration. LEVEL OF EVIDENCE: N/A.

Viability inhibition effect of gambogic acid combined with cisplatin on osteosarcoma cells via mitochondria-independent apoptotic pathway.

We previously demonstrated that gambogic acid (GA) is a promising chemotherapeutic compound for human osteosarcoma treatment. The aim of this study was to detect whether the combination of lower-dose GA (0.3 mg/L) and cisplatin (CDDP) (1 mg/L) could perform a synergistic effect on inhibiting tumor in four osteosarcoma cell lines. Our results showed that the combination between GA at lower dose and CDDP significantly exerts a synergistic effect on inhibiting the cellular viability in MG63, HOS, and U2OS cells. In contrast, an antagonistic character was detected in SAOS2 cells exposed to the combined use of lower-dose GA (0.3 mg/L) and CDDP (1 mg/L). Then, analysis of cell cycle showed the combination of both drugs significantly induced the G2/M phase arrest, without any difference relative to GA treatment alone, in MG63 cells. Flow-cytometric analysis of cell apoptosis displayed that the apoptotic rate in the combination group is higher than that in GA treatment alone in MG63, HOS, and U2OS cells. The combined use of both drugs had no effect on mitochondrial membrane potential, but promoted the apoptosis-inducing function through triggering of CDDP in the three cell lines. By measurement of mitochondrial membrane potential, the activity of caspase-3 and the expressions of caspase-8 and caspase-9, it was showed that the apoptosis-promoting effect of the combined use of both drugs could be dependent on the death receptor apoptosis pathway, not dependent on the mitochondria apoptosis mechanism. This research, for the first time, demonstrates that GA could increase the chemotherapeutic effect of CDDP in human osteosarcoma treatment through inducing the cell cycle arrest and promoting cell apoptosis.

Enzyme-amplified array sensing of proteins in solution and in biofluids.

We have developed an enzyme-nanoparticle sensor array where the sensitivity is amplified through enzymatic catalysis. In this approach cationic gold nanoparticles are electrostatically bound to an enzyme (beta-galactosidase, beta-Gal), inhibiting enzyme activity. Analyte proteins release the beta-Gal, restoring activity and providing an amplified readout of the binding event. Using this strategy we have been able to identify proteins in buffer at a concentration of 1 nM, substantially lower than current strategies for array-based protein sensing. Moreover, we have obtained identical sensitivity in studies where the proteins are spiked into the complex protein matrix provided by desalted human urine ( approximately 1.5 muM total protein; spiked protein concentrations were 0.067% of the overall protein concentration), demonstrating the potential of the method for diagnostic applications.

Nanoparticles for detection and diagnosis.

Nanoparticle based platforms for identification of chemical and biological agents offer substantial benefits to biomedical and environmental science. These platforms benefit from the availability of a wide variety of core materials as well as the unique physical and chemical properties of these nanoscale materials. This review surveys some of the emerging approaches in the field of nanoparticle based detection systems, highlighting the nanoparticle based screening methods for metal ions, proteins, nucleic acids, and biologically relevant small molecules.

Photoregulated release of caged anticancer drugs from gold nanoparticles.

An anticancer drug (5-fluorouracil) was conjugated to the surface of gold nanoparticles through a photocleavable o-nitrobenzyl linkage. In this system, the particle serves as both cage and carrier for the therapeutic, providing a nontoxic conjugate that effectively releases the payload upon long wavelength UV irradiation.

Photoinduced energy and electron-transfer processes in porphyrin-perylene bisimide symmetric triads.

The photophysics of two symmetric triads, (ZnP)2PBI and (H2P)2PBI, made of two zinc or free-base porphyrins covalently attached to a central perylene bisimide unit has been investigated in dichloromethane and in toluene. The solvent has been shown to affect not only quantitatively but also qualitatively the photophysical behavior. A variety of intercomponent processes (singlet energy transfer, triplet energy transfer, photoinduced charge separation, and recombination) have been time-resolved using a combination of emission spectroscopy and femtosecond and nanosecond time-resolved absorption techniques yielding a very detailed picture of the photophysics of these systems. The singlet excited state of the lowest energy chromophore (perylene bisimide in the case of (ZnP)2PBI, porphyrin in the case of (H2P)2PBI) is always quantitatively populated, besides by direct light absorption, by ultrafast singlet energy transfer (few picosecond time constant) from the higher energy chromophore. In dichloromethane, the lowest excited singlet state is efficiently quenched by electron transfer leading to a charge-separated state where the porphyrin is oxidized and the perylene bisimide is reduced. The systems then go back to the ground state by charge recombination. The four charge separation and recombination processes observed for (ZnP)2PBI and (H2P)2PBI in dichloromethane take place in the sub-nanosecond time scale. They obey standard free-energy correlations with charge separation lying in the normal regime and charge recombination in the Marcus inverted region. In less polar solvents, such as toluene, the energy of the charge-separated states is substantially lifted leading to sharp changes in photophysical mechanism. With (ZnP)2PBI, the electron-transfer quenching is still fast, but charge recombination takes place now in the nanosecond time scale and to triplet state products rather than to the ground state. Triplet-triplet energy transfer from the porphyrin to the perylene bisimide is also involved in the subsequent deactivation of the triplet manifold to the ground state. With (H2P)2PBI, on the other hand, the driving force for charge separation is too small for electron-transfer quenching, and the deactivation of the porphyrin excited singlet takes place via intersystem crossing to the triplet followed by triplet energy transfer to the perylene bisimide and final decay to the ground state.

Controlled nanoparticleassembly through protein conformational changes.

Selective surface recognition by proteins provides programmed bottom-up assembly of synthetic nanomaterials. We have investigated the controlled self-assembly of functionalized gold nanoparticles (Au-TAsp) with cytochrome c (Cyt c) and apoCyt c through complementary electrostatic interactions. Au-TAsp formed discrete, water-soluble adducts with native Cyt c, whereas unfolded apoCyt c induced nanocomposite formation at high Cyt c : Au-TAsp ratios. The binding of random-coil apoCyt c to Au-TAsp at low ratios induced α-helix formation in soluble nanocomposites, but at elevated ratios insoluble micron-scale aggregates were formed. The local structure of the assemblies was critically dependent on the Cyt c : Au-TAsp ratio. The dispersibility of apoCyt c-Au-TAsp was pH dependent, providing rapid and reversible control over nanocomposite assembly. The apoCyt c-Au-TAsp aggregates could likewise be disassembled through proteolytic cleavage of apoCyt c, demonstrating the ability to selectively remodel these hybrid materials.

Isomeric control of protein recognition with amino acid- and dipeptide-functionalized gold nanoparticles.

Amino acid and dipeptide-functionalized gold nanoparticles (NPs) possessing L/D-leucine and/or L/D-phenylalanine residues have been constructed in order to target the surfaces of alpha-chymotrypsin (ChT) and cytochrome c (CytC). Isothermal titration calorimetry (ITC) was conducted to evaluate the binding thermodynamics and selectivity of these NP-protein interactions. The chirality of the NP end-groups substantially affects the resultant complex stability, with up to 20-fold differences seen between particles of identical hydrophobicity, demonstrating that structural information from the ligands can be used to control protein recognition.

Biomimetic interactions of proteins with functionalized nanoparticles: a thermodynamic study.

Gold nanoparticles (NPs) functionalized with L-amino acid-terminated monolayers provide an effective platform for the recognition of protein surfaces. Isothermal titration calorimetry (ITC) was used to quantify the binding thermodynamics of these functional NPs with alpha-chymotrypsin (ChT), histone, and cytochrome c (CytC). The enthalpy and entropy changes for the complex formation depend upon the nanoparticle structure and the surface characteristics of the proteins, e.g., distributions of charged and hydrophobic residues on the surface. Enthalpy-entropy compensation studies on these NP-protein systems indicate an excellent linear correlation between DeltaH and TDeltaS with a slope (alpha) of 1.07 and an intercept (TDeltaS0) of 35.2 kJ mol(-1). This behavior is closer to those of native protein-protein systems (alpha = 0.92 and TDeltaS0 = 41.1 kJ mol(-1)) than other protein-ligand and synthetic host-guest systems.