The angiogenic effect of ONO-1301, a novel long-acting prostacyclin agonist loaded in PLGA microspheres prepared using different molecular weights of PLGA,in a murine sponge model

The purpose of this study was to evaluate the angiogenic effect of topical application of three types of ONO-1301-loaded poly (lactide-co-glycolide) microspheres (ONO-1301 PLGA MS). ONO-1301 PLGA MS were prepared with PLGA 5010, 5020 and 5050 (with molecular weights of 10?K, 20?K and 50?K, respectively), using the solvent evaporation method. The lactide:glycolide ratio was fixed at 50:50; only the molecular weight was varied. The microspheres had an average diameter of almost 25?µm, and a loading efficiency of at least 70%. The sustained-release effect and its dependence on the molecular weight of the polymer used was confirmed in an in vitro drug-release test and by measuring subcutaneous plasma levels after administration of the three types of ONO-1301 PLGA MS to rats for 28 days. In the murine sponge model, the three types of ONO-1301 PLGA MS were administered to mice in a subcutaneously placed sponge and hemoglobin and hepatocyte growth factor (HGF) levels in the sponge were measured at predefined intervals up to 28 days. The hemoglobin and HGF levels obtained were significantly higher than those obtained after daily administration of ONO-1301 powder. Additional in vivo fluorescence imaging showed that PLGA MS remained in the sponge for 28 days. In conclusion, the three types of ONO-1301 PLGA MS prepared with PLGA three different molecular weight suppress the burst release, stimulate angiogenesis on topical application in a murine sponge model. This formulation may therefore be capable of improving the clinical picture in some types of vascular disease.     

Synthetic collagen fibers coated with a synthetic peptide containing the YIGSR sequence of laminin to promote peripheral nerve regeneration in vivo

The usefulness of collagen fibers and the YIGSR sequence (Tyr-lle-Gly-Ser-Arg) of laminin for nerve regeneration were examined in vivo. Type I collagen gel (G-group), Type I collagen fibers (F-group), Type I collagen fibers coated with laminin (L-group) or the YIGSR sequence (Y-group) were packed into silicone tubes, 15 mm long, and transplanted to the sciatic nerves of Wistar rats. Empty silicone tubes were used as the control. The animals were sacrificed 8 weeks after transplantation. Bridging of the nerve was confirmed in the F-(7/12), Y-(7/10) and L-group (6/10), but no bridging was observed in any of the animals of the G- and control group. Nerve regeneration among the space of collagen fibers was observed, and it was suggested that fibroblasts infiltrated the gap in the substance of the degenerated collagen fibers were followed by Schwann cells on the basis of immunocytochemistry. The number of myelinated axons per regenerated tissue in the tube (density), and total area of myelinated axons per measured regenerated tissue in the tube (% axon area) in each the L- and Y-group were significantly higher than that in the F-group (P < 0.05). These results suggest the possibility of obtaining adequate nerve regeneration with new artificial materials only. © 1999 Kluwer Academic Publishers     

An in vivo murine model for screening cranial bone regenerative materials: testing of a novel synthetic collagen gel

Rapid and efficient animal models are needed for evaluating the effectiveness of many new candidate bone regenerative materials. We developed an in vivo model screening for calvarial bone regeneration in lipopolysaccharide (LPS)-treated mice, in which materials were overlaid on the periosteum of the calvaria in a 20 min surgery and results were detectable in 1 week. Intraperitoneal LPS injection reduced spontaneous bone formation, and local application of basic fibroblast growth factor (bFGF) increased the bone-forming activities of osteoblasts. A novel synthetic collagen gel, alkali-treated collagen (AlCol) cross-linked with trisuccinimidyl citrate (TSC), acted as a reservoir for basic substances such as bFGF. The AlCol–TSC gel in conjunction with bFGF activated osteoblast activity without the delay in osteoid maturation caused by bFGF administration alone. The AlCol–TSC gel may slow the release of bFGF to improve the imbalance between osteoid formation and bone mineralization. These findings suggest that our model is suitable for screening bone regenerative materials and that the AlCOl–TSC gel functions as a candidate reservoir for the slow release of bFGF.     

In vitro and in vivo anti-tumor effects of gemcitabine loaded with a new drug delivery system

In recent years, much attention has been given to liposomal formulation as an efficient drug loading system (DDS) in chemotherapy of cancer. In this study, the advantages of magnetic nanoparticles and Polyethylene Glyco (PEG) materials were considered to synthesize magnetic gemcitabine long-circulating liposomes (MGLL) and the potential of MGLL as a brand new delivery system was evaluated. MGLL was prepared using the reverse-phase evaporation method. In the optimized preparation, MGLL had an average diameter of 201 nm with a narrow size distribution measured by dynamic light scattering (DLS), which could be easily dispersed in ultrapure water under a stable state for 90 days. The encapsulation efficiency of gemcitabine in MGLL reached 87.2% as determined by HPLC. In vitro MTT assay showed that MGLL had significant cytotoxicity to MCF-7 cells compared with the conventional modalities. In vivo, the inhibition of tumor growth in MGLL group was more remarkable than that of other groups (P < 0.05). In conclusion, MGLL under optimized condition could be used as an effective carrier for tumor-targeted therapy.     

Preparation of novel cationic copolymer microspheres and evaluation of their function by in vitro and in vivo tests as pH-sensitive drug carrier systems

Novel pH-sensitive copolymer microspheres containing methylacrylic acid and styrene cross-linking with divinylbenzene were synthesized by free radical polymerization. The microspheres that were formed were then characterized by Fourier-Transform infrared (FT-IR) spectroscopy, differential scanning calorimetry (DSC), size analysis, and X-ray analysis. The copolymer microspheres showed pulsatile swelling behavior whenthe pH of the media changed. The pH-sensitive microspheres were loaded with diltiazem hydrochloride (DH). The release characteristics of the free drug and the drug-loaded microspheres were studied under both simulated gastric conditions and intestinal pH conditions. The in vivo evaluation of the pulsatile preparation was subsequently carried out using beagle dogs as experimental subjects. The results demonstrated that the drug release exhibited a pulsatile character both in vitro and in vivo.     

Bone repair of defects filled with a phosphocalcic hydraulic cement: an in vivo study

A quickly setting calcium phosphate-based hydraulic cement mixed with particles of tricalcium phosphate (TCP) ceramic was implanted in 56 metaphysial defects made in the long bones of ten adult mongrel dogs. Microradiography, histology and scanning electron microscopy (SEM) demonstrated the slow resorption of the cement and the bony incorporation of the calcium phosphate ceramic particles which were consistently embedded in bone. The original structural pattern of the bone tended to be restored 7 months after implantation. The cement did not hinder the incorporation of the calcium phosphate ceramic particles, neither did it elicit any inflammatory or foreign-body response. The cement was easily shaped and allowed a perfect filling of any defect, resulting in close contact of the whole implant surface with the host bone at the time of surgery, associated with appreciable mechanical strength. Most of the practical problems associated with the use of calcium phosphate ceramics in the repair of bone defects could be overcome with the cement.     

Transverse electric scattering by a dielectric biconvex cylinder loaded with a shallow circular trough in a ground plane

The scattering problem of transverse electric wave from a dielectric biconvex cylinder buried in a shallow circular trough of a ground plane is investigated and a rigorous series solution is also derived. Based on the region-matching method, the analysed region is decomposed into two subregions by introducing a semi-circular auxiliary boundary. The magnetic field of each subregion is expressed in terms of cylindrical wave functions with unknown expansion coefficients. After imposing the matching conditions and the boundary condition on the trough surface with the aid of Graf's addition theorem, the unknown coefficients are determined. Comparisons with published data for a dielectric circular cylinder case show very good agreement. Visible effects of depth-to-half-width ratios of a dielectric biconvex cylinder on echo width, far- field pattern and near-field distribution are illustrated in graphical form.     

Estimating the stresses in cantilever beam loaded by a parabolically distributed load with Airy stress functions

This study presents three mathematical methods namely the polynomial stress function approach, the Fourier series form approach and the approximated equations form approach for finding the stress distribution in a cantilever beam with rectangular cross section loaded by a parabolically distributed load. By taking the stress function as a polynomial of the seventh degree, it is attempted to find the coefficients either in complete or in full shape of the polynomial. In the Fourier series approach, the load is discreted to unlimited series of harmonic loads and superposing resultant stresses is the affect of parabolically distributed load on the beam. The resultant stresses are compared with some approximated stress formulas which have been provided before. Finite element analysis are done for square, short, medium and long cantilever beams and the mathematical results of stress distribution in five different height of the beam was compared with FEM results. It was found good results for τ _yy and τ _xy in all cross section of the beams and acceptable results for τ _xx only in y = 0. It is found that discreting loads to even a limit number of harmonic loads and superposing the resultant stresses can give the distribution of τ _yy and τ _xy with the acceptable precision in medium and long cantilever beams with rectangular cross section.     

In vitro and in vivo evaluation of a novel push-pull osmotic pump with orifices on both side surfaces

A novel push-pull osmotic pump (PPOP) was developed for delivery of water-insoluble drug gliclazide. Compared to conventional PPOP, which only had orifice(s) on the side of the drug layer, the novel PPOP had orifices of the same diameter on both side surfaces. The in vitro drug-release behavior of both novel PPOP and conventional PPOP were studied and compared; it was found that the drug-release rate of both kinds of PPOP could be influenced by coating level and core hardness whereas orifice size did not have much influence on it, and the study also showed that none of the former factors could influence the similarity of the drug-release profiles of the two kinds of PPOP. Mechanism of drug release from novel PPOP was illustrated using Poiseuille's law of lamina flow, and it was found that under regular formulation, the dissolution profiles of the two kinds of PPOP were similar. In vivo study also showed that the concentration-time profiles of gliclazide in plasma of the two PPOP were comparable and both of them had good in vitro-in vivo correlation. By simply drilled on both side surfaces, the novel PPOP did not need side identification when drilled, so it was more suitable for industrial manufacture than the conventional ones.     

Accuracy in the determination of isoelectric points of some proteins and a peptide by capillary isoelectric focusing: utility of synthetic peptides as isoelectric point markers

To evaluate the accuracy ofisoelectric point determination by capillary isoelectric focusing, the pI values of nine proteins and a peptide, the pI values of which had been determined by other methods and ranging pI 3.55-9.60, were determined by capillary isoelectric focusing by cofocusing of recently developed peptide pI markers ranging 3.38-10.17, and the consistency of the pI values was examined. Isoelectric focusing was carried out in neutral polymer-coated capillaries, and the pH gradient was mobilized by pressure toward the cathode, to detect samples with absorption at 280 nm at a fixed detection point. Carrier ampholytes from two different suppliers and in different pH ranges were used. The sharp peaks of the highly pure peptide pI markers greatly facilitated the unambiguous identification of the peaks. When a carrier ampholyte ranging over the acidic side was used, the detection of acidic pI samples was anomalously delayed. This could be partly mitigated by reducing the viscosity of the anode solution in comparison with the pH gradient formed in the capillary. Since the detection times vs the pH relationships were not linear in most cases, the use of a linear calibration line over an entire pH gradient would be erroneous. Instead, the pI values of samples were calculated by assuming a linear relation for pH against detection time between two flanking marker peptides. Close agreement between the pI values, determined by capillary isoelectric focusing, and the reference values of the samples was observed within an average difference range of 0.04-0.08 pH unit with a sample consumption of 10-100 ng within 30-60 min. Some carrier ampholytes were preferentially more effective at either the acidic or the basic side of the pH gradient. For confirmation of the completion of focusing, the use of two different focusing times is recommended.     

Free form fabricated features on CoCr implants with and without hydroxyapatite coating in vivo: a comparative study of bone contact and bone growth induction

The current study evaluates the in vivo response to free form fabricated cobalt chromium (CoCr) implants with and without hydroxyapatite (HA) plasma sprayed coatings. The free form fabrication method allowed for integration of complicated pyramidal surface structures on the cylindrical implant. Implants were press fit into the tibial metaphysis of nine New Zealand white rabbits. Animals were sacrificed and implants were removed and embedded. Histological analysis, histomorphometry and electron microscopy studies were performed. Focused ion beam was used to prepare thin sections for high-resolution transmission electron microscopy examination. The fabricated features allowed for effective bone in-growth and firm fixation after 6 weeks. Transmission electron microscopy investigations revealed intimate bone-implant integration at the nanometre scale for the HA coated samples. In addition, histomorphometry revealed a significantly higher bone contact on HA coated implants compared to native CoCr implants. It is concluded that free form fabrication in combination with HA coating improves the early fixation in bone under experimental conditions.     

Immobilization of a human epidermal growth factor receptor 2 mimotope-derived synthetic peptide on Au and its potential application for detection of herceptin in human serum by quartz crystal microbalance

Therapeutic antibodies are antigenically similar to human antibodies and are difficult to detect in assays of human serum samples without the use of the therapeutic antibody's complementary antigen. Herein for the first time, we established a platform to detect Herceptin in solutions by using a small (<2.2 kDa), inexpensive, highly stable human epidermal growth factor receptor (HER2) mimotope-derived synthetic peptide immobilized on the surface of a Au quartz electrode. We used the HER2 mimotope as a substitute for the HER2 receptor protein in piezoimmunosensor or quartz crystal microbalance (QCM) assays to detect Herceptin in human serum. We demonstrated that assay sensitivity was dependent upon the amino acids used to tether and link the peptide to the sensor surface and the buffers used to carry out the assays. The detection limit of the piezoimmunosensor assay was 0.038 nM with a linear operating range of 0.038-0.859 nM. Little nonspecific binding to other therapeutic antibodies (Avastin and Rituxan) was observed. Levels of Herceptin in serum samples obtained from treated patients, as ascertained using the synthetic peptide-based QCM assay, were typical for those treated with Herceptin. The findings of this study are significant in that low-cost synthetic peptides could be used in a QCM assay, in lieu of native or recombinant antigens or capture antibodies, to rapidly detect a therapeutic antibody in human serum. The results suggested that a synthetic peptide bearing a particular functional sequence could be applied for developing a new generation of affinity-based immunosensors to detect a broad range of clinical biomarkers.     

Feasibility, safety and pharmacokinetic study of hepatic administration of drug-eluting beads loaded with irinotecan (DEBIRI) followed by intravenous administration of irinotecan in a porcine model

Irinotecan eluting embolization beads (DEBIRI) are currently being evaluated in the clinic for the treatment of colorectal cancer metastases to the liver. The aim of this study was to determine the safety and pharmacokinetics associated with two cycles of hepatic embolization using DEBIRI followed by intravenous administration of irinotecan. Pigs were embolized with DEBIRI (100–300 μm, 100 mg dose, n = 6) and blood samples taken over 24 h to determine plasma levels of irinotecan and SN-38 metabolite and for haematology and biochemistry. At 24 h an IV infusion of 250 mg/m² of irinotecan was administered and the plasma levels taken again. This cycle was repeated 3 weeks later. A single animal was subjected to a more aggressive regimen of embolization with 200 mg bead dose and IV of 350 mg/m² for two cycles. Three animals were sacrificed at 6 weeks and the remaining four (n = 3 standard dose, n = 1 high dose) animals at 12 weeks and detailed histopathology performed. All animals tolerated the treatments well, with only minor changes in haematological and biochemical parameters. There was no overlap in drug plasma levels observed from the bead and IV treatments when given 24 h apart and no difference between the pharmacokinetic profiles of the two cycles separated by 3 weeks. Irinotecan plasma AUC values were similar in both the embolization and IV arms of the study. C_max values obtained during the IV arms of the study are approximately double that of the embolization arms whilst T_max times are shorter in the IV arms, supporting extended release of drug from the beads. Bioavailability for bead-based delivery was double that for IV administration, which was attributed to reduced clearance of the drug when delivered by this route. No additive toxicity was observed as a consequence of the combined treatments. The combination of irinotecan delivery via drug eluting bead and IV was well-tolerated with no significant clinical effects. Pharmacokinetic analyses suggest the bioavailability from bead-based delivery of drug is double that of IV infusion, attributable to reduced drug clearance for the former.     

Physiological responses induced in tomato plants by a two-component nanostructural system composed of carbon nanotubes conjugated with quantum dots and its in vivo multimodal detection

Plant seedlings were exposed to single-walled carbon nanotube-quantum dot conjugates (SWCNT-QD) mixed in the growth medium in order to understand the interactions between these multicomponent nanosystems and plants. A combination of fluorescent and Raman-scattering 2D mapping analysis was used to clearly monitor the presence of the SWCNT-QD conjugates in various parts of the tomato seedlings. We found that the addition of QDs to SWCNTs dramatically changed the biological viability of the tomato plants by significantly accelerating leaf senescence and inhibiting root formation. Although the exposure of SWCNTs only to the plants induced positive effects, the chlorophyll content decreased by 1.5-fold in leaves, and the total weight of the root system decreased four times for the tomato plants exposed to SWCNT-QDs (50 μg ml(-1)) compared to plants grown on regular medium as controls. Our results clearly indicate that the exposure of plants to multicomponent nanomaterials is highly influenced by the presence and bioactivity of each component, individually. Such studies could be the foundation for understanding how complex nanosized systems affect the activity of various biological systems with a major impact on ecotoxicology.     

The in vitro and in vivo investigation of inhaled migraine therapies using a novel aerosol delivery system consisting of an air pressurized capsule device (APCD) in combination with a pMDI spacer for endotracheal dosing into beagle dogs

Abstract

Context: Aerosol delivery to animals in preclinical settings has historically been very challenging, requiring the use of techniques, such as intratracheal instillation and dry powder insufflation, that are somewhat invasive, inefficient and not representative of clinical inhalation.

Objective: The objective of this work is to develop a system to deliver dry powder to dogs in an efficient and effective manner for the study of new anti-migraine compounds in development.

Materials and methods: The new device uses a metered aliquot of a dry gas to force dry powder drug from a pre-filled HPMC capsule into an AeroChamber® spacer for subsequent inhalation by the animal.

Results: The delivery of two invesigational migraine drugs via the new device was assessed in vitro using abbreviated Andersen cascade impaction and showed the device is capable of generating a reproducible delivered dose of up to ～68% with more than 50% of the dose in the respirable range. In vivo studies have also been performed showing that this device effectively delivered the migraine drugs to spontaneously breathing dogs using a proprietary validated dog inhalation model.

Discussion: Results confirmed that the air pressurized capsule device (APCD) was effective in delivering the APIs to lungs of the animals. The in vivo data verified the advantages of inhaled delivery over oral delivery for this class of drugs and were used to establish the cardiopulmonary and respiratory side effect liability profile for these compounds.

Conclusions: This work has demonstrated the utility of this device for quick and accurate screening of prospective drug candidates, representing a significant improvement in ease of use and reprodicibility over current delivery methods.     

Finite-difference time-domain and steady-state analysis with novel boundary conditions of optical transport in a three-dimensional scattering medium illuminated by an isotropic point source

We evaluate the numerical accuracy of finite-difference time-domain (FDTD) analysis of optical transport in a three-dimensional scattering medium illuminated by an isotropic point source. This analysis employs novel boundary conditions for the diffusion equation. The power radiated from an isotropic point source located at a depth equal to the reciprocal of the reduced scattering coefficient (1/μ'(s)) below the surface at the irradiated position is introduced to the integral form of the diffusion equation. Finite-difference approximations of the diffusion equation for a surface cell are derived by utilizing new boundary conditions that include an isotropic source even in a surface cell. Steady-state and time-resolved reflectances are calculated by FDTD analysis for a semi-infinite uniform scattering medium illuminated by an isotropic point source. The numerical results agree reasonably with the analytical solutions for μ'(s)=1-3 mm(-1) without resizing the mesh elements.