Current Pharmaceutical Biotechnology - Volume 14, Issue 12, 2013

Objective: The goals of this study were to observe how luteolin protects endothelial cells (ECs) from injury stimulated by Angiotensin II (Ang II), investigate the role of vascular endothelial dysfunction in vascular smooth muscle cell proliferation and migration in vitro and investigate its primary mechanism of action. Methods: A non-contact coculture system was established using a transwell system; ECs were cultured in the lower wells, while the smooth muscle cells (SMCs) were cultured in the upper wells. Cell proliferation was assessed by the MTT assay. The number of SMCs that migrated through the membrane of transwell system were observed and counted. The expression levels of various proteins (VEGF, p-Akt, Nox4) expressed in ECs were determined by Western blotting. VEGF mRNA expression was detected by reverse transcription-polymerase chain reaction (RT-PCR). The supernatants of ECs were measured by enzyme linked immunosorbent assay (ELISA) to assay VEGF concentration. Results: Ang II-stimulated ECs significantly increased the proliferation and migration of SMCs, and these effects were inhibited by luteolin pretreatment. Luteolin suppressed the Ang II-induced upregulation of Nox4, p-AKT and VEGF expression in ECs. Conclusion: These results demonstrate that luteolin is capable of inhibiting endothelial dysfunction induced by Ang II by suppressing the upregulation of Nox4, p-Akt and VEGF, thereby restraining the proliferation and migration of SMCs induced by injured ECs.

Graphene-based nanosheets (GNS) are atomic-thickness monolayers of hexagonally arranged, graphite-derived carbon atoms that may be composed of graphene, graphene oxide, or reduced graphene oxide. They have attracted tremendous interest for their potential in pharmaceutical applications, due to their unique physical, chemical, and mechanical properties GNS exhibit highly uniform surface areas and may have hydroxyl (-OH), epoxide (-O-), and carboxyl functional groups at their basal surfaces and plane edges, depending on their oxidized and reduced surface properties. GNS show high-level optical absorption of near infrared (NIR) light and elevate the temperature of nearby environments. Furthermore, they can be loaded with anticancer drugs via hydrophobic interactions, π−π stacking, or electrostatic binding. Given these properties, GNS can be used in chemotherapy, photodynamic therapy, photothermal therapy, and theranostics. However, although GNS appear to have far-reaching potential in the field of biomedical research, their widespread pharmaceutical application has been limited by issues such as poor stability in physiological buffers, undefined mechanisms of cellular uptake, toxicity problems, and a lack of standard preparation methods. Here, we review the current pharmaceutical applications of GNS, focusing on chemotherapy, phototherapy, combo therapy and theranostic applications with challenging issues.

Pterostilbene is a naturally occurring dimethyl ether analog of resveratrol identified in several plant species. Telomerase is important in tumor initiation and cellular immortalization. Given the striking correlations between telomerase activity and proliferation capacity in tumor cells, telomerase had been considered as a potentially important molecular target in cancer therapeutics. Molecular docking studies were performed on pterostilbene with the crystal structure of telomerase (3DU6). Pterostilbene was evaluated for its in vitro cytotoxicity in breast (MCF7) and lung cancer (NCI H-460) cell lines, antimitotic activity in green grams and telomerase activity. Curcumin was used as a standard. Docking results indicated good interaction between pterostilbene and the active site of telomerase and the docked energy of pterostilbene was -7.10 kcal/mol. Pterostilbene showed strong inhibitory effect on in vitro telomerase activity and cell growth in both the cell lines tested in a dose dependent manner. Cancer cells treated with 80 μM pterostilbene exhibited significant telomerase inhibition, after 72 hours (MCF-7 and NCI H-460; 81.52% and 74.69% reduction, respectively, compared to control). The IC50 of pterostilbene for anti-proliferative activity in MCF7 and NCI H-460 cell lines were found to be 30.0 and 47.2 μM, respectively. The best antimitotic activity was obtained with 80 μM of pterostilbene (100% reduction in water imbibition). All the above results were comparable to that of curcumin. The drug-related properties of pterostilbene were calculated using Molinspiration, Osiris Property Explorer and ACD/Chemsketch softwares. Pterostilbene obeyed Lipinski’s Rule of Five indicating its therapeutic potential in humans. It was found that the telomerase inhibitory activity exhibited by pterostilbene was dependent of the cell viability and has the potential to be a new drug candidate against breast and lung cancers.

Nitrofurazone (NTZ) is usually employed in the topical treatment of infected wounds and lesions of both skin and mucosa. Microencapsulation is a process utilized in the incorporation of active ingredients within polymers aiming at, among other objectives, the prolonged release of pharmaceutical compounds and protection from atmospheric agents (viz. moisture, light, heat and/or oxidation). With the goal of utilizing the microparticles containing encapsulated NTZ in pharmaceutical formulations, one prepared microparticles containing NTZ via ionotropic gelation of sodium alginate. The microparticles were characterized via scanning electron microscopy analyses, Fourier transform infrared spectroscopy (FTIR) analyses, via determination of encapsulation efficiency, and via thermal analyses (both TGA and DSC). The final gel formulation was also characterized rheologically. The extrusion/solidification technique employed to obtain the calcium alginate microparticles with encapsulated NTZ was found to be adequate, and produced an NTZ encapsulation efficiency of ca. 97.8% ± 1.1%. The calcium alginate microparticles thus obtained, with encapsulated NTZ, exhibited an oval shape and hydrodynamic diameters between 500 μm and 800 μm. From the thermal analyses performed, together with information from the infrared spectra, one may conclude that NTZ did not strongly bind to the polymer, which may be favorable for the release of the active ingredient. From the results obtained in the present research effort, one may conclude that the microparticles produced possess the potential to be utilized as carriers for NTZ in pharmaceutical formulations such as gels, ointments, and solutions.

IL-1β and TNF-α play key roles in the inflammatory response. Their abnormal expression may cause the occurrence of various diseases, such as RA. Recently, medicines of target TNF-α and IL-1β have become popular in the clinical practice. Although these biological agents can get mostly good results, they are not effective in all patients. The reason for this result may be that these biological agents could not fully inhibit a variety of inflammatory cytokines in the inflammatory response. In the present study, a fusion protein gene which encoded human interleukin-1β scfv and soluble TNF receptor I (sTNFRI) was cloned. A number of in vitro assays demonstrated that anti-IL-1β scfv/TNFRI simultaneously bound to both targets. The bioactivity assay showed that the fusion protein could inhibit both the cytotoxicity of hTNF-α on L929 cells and hIL-1β-induced proliferation of L929 cells, indicating that the fusion protein has the ability to neutralize both hTNF-α and hIL-1β. In this study, we established the chicken type II collagen-induced rheumatoid arthritis model in Kunming mice, and evaluated the pharmacological effect of the fusion protein in vivo. Model mice were randomly divided into 8 groups (n=8): CIA model control group, DEX treatment group (1 mg/kg), intraperitoneal treatment group (highdose: 5 mg/kg; medium-dose: 2 mg/kg; low-dose: 0.8 mg/kg), subcutaneous treatment group (high-dose: 5 mg/kg; medium- dose: 2 mg/kg; low-dose: 0.8 mg/kg), and healthy mice as control. The control group received the same volume of saline. The mice were administrated once every 2 days. Arthritis index, anti-CII antibody titers, cytokine levels, histopathological changes were examined. The results showed that anti-IL-1β scfv/TNFRI fusion protein could reduce the degree of joint swelling, inflammatory cell infiltration, synovial cell proliferation and the level of CII antibody in the sera. The Real-time PCR analysis showed that anti-IL-1β scfv/TNFRI had the ability to reduce the expression of IL-1β, TNF-α, IL-17A, MMP-3, IL-6 and improve the expression of IL-10 in a dose-dependent manner, suggesting that the fusion protein is the mediator for IL-1β and TNF-α involved in the RA process. Compared with DEX positive medicine control, anti-IL-1β scfv/TNFRI appeared more beneficial in treatment of CIA mice. The therapeutic effect of the anti-IL-1β scfv/TNFRI at 5mg/kg was significantly better than that of DEX treatment. So the anti-IL-1β scfv/TNFRI can become a candidate for treatment of RA.

To improve the solubility, bioavailability and anti-tumor effect of lapatinib, lapatinib-incorporated lipid nanoparticles (LTNPs) were prepared and characterized. The particle size of LTNPs was 88.6 nm with a zeta potential of 20 mV. Laptinib was loaded into LTNPs with a non-crystal structure as determined by FT-IR. In vitro, LTNPs could be effectively uptaken into C6 glioma cells at a concentration-dependent manner. In vivo, LTNPs showed a relative higher AUC, which was 5.27- and 3.21-fold as that of Tykerb and lapatinib suspension (LTS) group. LTNPs also showed highest glioma concentration, which may benefit from the enhanced permeability and retention effect and active targeting ability. In toxicity studies, LTNPs displayed a half lethal dose over 250 mg/kg. Repeated administering 30 mg/kg of LTNPs could led to toxicity to hematology which might owe to the bovine serum albumin, a foreign protein to mice. However, there was no organic change observed through HE staining. In conclusion, LTNPs could target to glioma with high concentration and low side effect.

Gene therapy has become a clinical reality as demonstrated by remarkable benefits seen in Phase I/II clinical trials for hemophilia B, lipoprotein lipase deficiency and Leber’s congenital amarousis. The choice of, and the improved understanding in vector characteristics have contributed significantly to this success. The adeno-associated virus (AAV) vectors used in these trials have been long known to be relatively safe and efficacious. However, certain factors, most notably host immunity to the vector, prevent their widespread use. In patients who have pre-existing antibodies to AAV, these vectors will be rapidly cleared. Administration of a relatively high initial dose of vector to achieve and sustain a higher margin of therapeutic benefit is limited by concerns of vector dose-dependent T cell response. Frequent vector administration necessitated by the non-integrating nature of the virus is difficult due to the variable, yet significant host immunological memory. Thus generation of AAV vectors that are immunologically inert is pivotal for the long-term success with this promising vector system. Several strategies, that aim targeted disruption of antigenic sites or those that chemically modify the vectors have been proposed for host immune evasion. While these approaches have been successful in the pre-clinical model systems, this continues to be a field of intense experimentation and constant improvisation due to limited information available on vector immunology or data from human studies. This review forms a comprehensive report on current strategies available to generate immunologically inert AAV vectors and their potential in mediating longterm gene transfer.