Validation of the Transferrin Receptor for Drug Targeting to Brain Capillary Endothelial Cells In Vitro

Recently, we have shown that transferrin (Tf) is actively endocytosed by the Tf R on primary cultured bovine brain capillary endothelial cells (BCEC). The objective of this investigation is to determine whether the Tf R can facilitate endocytosis of a (protein) model drug, using Tf as a targeting vector. Secondly, the mechanism of endocytosis was investigated. Horseradish peroxidase (HRP, 40 kDa) was chosen as a model drug, since it normally does not cross the blood–brain barrier (BBB) and its concentration in biological media can be easily quantified.

Tf-HRP conjugates (1:1) are actively and specifically endocytosed by BCEC in vitro in a concentration and time-dependent manner. At an applied concentration of 3 μg/ml, association (a combination of binding and endocytosis) of Tf-HRP reached equilibrium at a concentration of 2 ng/mg cell protein after 1 h of incubation at 37°C. This was approximately 3-fold higher compared to binding at 4°C (0.6 ng/mg cell protein). Association of Tf-HRP was compared to BSA-HRP. After 2 h of incubation at 37°C association levels were 5.2 and 2.5 ng/mg cell protein, for Tf-HRP and BSA-HRP, respectively. Under those conditions, association of Tf-HRP could be inhibited to approximately 30% of total association by an excess of non-conjugated Tf, but not with BSA, while association of BSA-HRP could be inhibited by both proteins. Furthermore, by using specific inhibitors of endocytotic processes, it was shown that association of Tf-HRP is via clathrin-coated vesicles. Association of Tf-HRP is inhibited by phenylarsine oxide (an inhibitor of clathrin-mediated endocytosis) to 0.4 ng/mg cell protein, but not by indomethacin, which inhibits formation of caveolae. Finally, following iron scavenging by deferoxamine mesylate (DFO, resulting in a higher Tf R expression) a 5-fold increase in association of Tf-HRP to 15.8 ng/mg cell protein was observed.

In conclusion, the Tf R is potentially suitable for targeting of a (protein) cargo to the BBB and to facilitate its endocytosis by the BCEC.     

The percutaneous permeability and absorption of dexamethasone esters in diabetic rats: a preliminary study

Abstract

To evaluate the influence of diabetes on the permeation of dexamethasone acetate (DA) and dexamethansone sodium phosphate (DSP), the two major dexamethansone esters in clinical practice, when applied percutaneously, histochemical staining was used to determine the skin morphology; improved Franz diffusion cells and microdialysis were used to assess the percutaneous permeation of DA and DSP in normal and diabetic rats. Histopathological examination showed that the epidermal tissue of diabetic rat was much thinner, the epidermal cell layer was less clear and the stratified arrangement of epidemic cell had almost disappeared and progressive atrophy were developed on the subcutaneous fat. In vitro studies showed that the cumulative and the penetrated DSP amount in Group DM were higher. The mean flux value and the mean depositional amount of Group DM were increased significantly compared to those of Group CTL, whereas the amount of DA penetrating was of no difference. Microdialysis indicated that there was no significant difference between Group CTL and Group DM for all the pharmacokinetic parameters of DA. In contrast, the subcutaneous AUC_all values and the C_max of DSP were significantly increased compared to the control. In conclusion, diabetic rat skin significantly increased the percutaneous permeation of DSP but had no effect on that of DA. It suggests that patients with diabetes should consider the dose of administration when using DA, DSP or other glucocorticoids topically, as different liposolubilities may play some role in the permeability of these compounds via diabetic skin.     

Synthesis of a novel phosphate analog of 20-hydroxylecdysone with potent hypoglycemic activity

A novel, water-soluble 20-hydroxylecdysono-20,22-phosphoric acid 2 and its sodium salt 3 were designed and synthesized from 20-hydroxylecdysone 1 in six steps and with 67% overall yield. The synthesized phosphoric acid 2 exhibited hypoglycemic activity >40-fold more potent than that of 20-hydroxylecdysone 1 at concentrations between 2 × 10^? 7 and 2 × 10^? 8 mol/l in a glucose consumption test in HepG2 cells. At a concentration of 2 × 10^? 9 mol/l, phosphoric acid 2 was still active, causing a maximum increase in glucose consumption of more than 500%, while 20-hydroxylecdysone 1 was inactive.     

α-干扰素和磷酸肌酸钠联合治疗病毒性心肌炎的临床观察

目的探讨α-干扰素和磷酸肌酸钠联合应用治疗病毒性心肌炎的效果。方法选取病毒性心肌炎患者92例,随机分为观察组和对照组各46例,两组均采用基础治疗和对症处理。观察组另加用α-干扰素和磷酸肌酸钠治疗,对照组另加用利巴韦林和泛癸利酮治疗。治疗4周后对比两组的疗效。结果观察组的总有效率为95.6%,高于对照组的80.4%,两组比较,差异有统计学意义（P〈0.05）。观察组治疗后肌酸磷酸激酶、天冬氨酸转氨酶和乳酸脱氢酶较治疗前明显改善（P〈0.05）,且改善的情况优于对照组（P〈0.05）。结论α-干扰素和磷酸肌酸钠联合治疗病毒性心肌炎效果显著,值得推广应用。     

Influence of Y2O3 Content on Structural,Optical, Spectroscopic,and Laser Properties of Er3+, Yb3+ Co-Doped Phosphate Glasses

The influence of the addition of Y₂O₃ on the structural, spectroscopic, and laser properties of newly prepared Er, Yb-doped strontium-sodium phosphate glass was investigated. While the addition of Y₂O₃ has a small influence on the absorption spectra and fluorescence lifetime, it has a strong impact on the emission cross-section and on OH⁻ content. The glasses were used as the active medium for diode-pumped laser emitting at 1556 nm. The increase in Y₂O₃ content leads to a significant 35% increase in laser slope efficiency up to 10.4%, but at the expense of the substantial reduction of the wavelength tunability from 82 to 54 nm.     

Performance of Calcium Phosphate Cements in the Augmentation of Sheep Vertebrae—An Ex Vivo Study

Oil-based calcium phosphate cement (Paste-CPC) shows not only prolonged shelf life and injection times, but also improved cohesion and reproducibility during application, while retaining the advantages of fast setting, mechanical strength, and biocompatibility. In addition, poly(L-lactide-co-glycolide) (PLGA) fiber reinforcement may decrease the risk for local extrusion. Bone defects (diameter 5 mm; depth 15 mm) generated ex vivo in lumbar (L) spines of female Merino sheep (2–4 years) were augmented using: (i) water-based CPC with 10% PLGA fiber reinforcement (L3); (ii) Paste-CPC (L4); or (iii) clinically established polymethylmethacrylate (PMMA) bone cement (L5). Untouched (L1) and empty vertebrae (L2) served as controls. Cement performance was analyzed using micro-computed tomography, histology, and biomechanical testing. Extrusion was comparable for Paste-CPC(-PLGA) and PMMA, but significantly lower for CPC + PLGA. Compressive strength and Young’s modulus were similar for Paste-CPC and PMMA, but significantly higher compared to those for empty defects and/or CPC + PLGA. Expectedly, all experimental groups showed significantly or numerically lower compressive strength and Young’s modulus than those of untouched controls. Ready-to-use Paste-CPC demonstrates a performance similar to that of PMMA, but improved biomechanics compared to those of water-based CPC + PLGA, expanding the therapeutic arsenal for bone defects. O, significantly lower extrusion of CPC + PLGA fibers into adjacent lumbar spongiosa may help to reduce the risk of local extrusion in spinal surgery.     

Necrosis targeted combinational theragnostic approach to treat cancer

Residual cancer cells and subsequent tumor relapse is an obstacle for curative cancer treatment. Tumor necrosis therapy (TNT) has recently been developed to cause residual tumor regression or destruction. Here, we exploited the avidity of the sennidin A (SA) tracer and radioiodinated SA (¹³¹I-SA) to necrotic tumors in order to further empower TNT. We showed high uptake and prolonged retention of SA in necrotic tumors and a quick clearance in other non-targeted tissues including the liver. On SPECT-CT images, tumor mass appeared persistently as a hotspot. Based on the prominent targetability of ¹³¹I-SA to the tumor necrosis, we designed a combinational theragnostic modality. The vascular disrupting agent (VDA) combretastatin A4 phosphate (CA4P) was used to cause massive tumor necrosis, which formed the target of ¹³¹I-SA that subsequently killed the residual tumor cells by cross-fire irradiation of beta particles. Consequently, ¹³¹I-SA combined with CA4P significantly inhibited tumor growth, extended tumor doubling time and prolonged mean animal survival. In conclusion, ¹³¹I-SA in combination with necrosis inducing drugs/therapies may generate synergetic tumoricidal effects on solid malignancies by means of primary debulking and secondary cleansing process.     

Self-hardening calcium phosphate composite scaffold for bone tissue engineering.

Calcium phosphate cement (CPC) sets in situ to form solid hydroxyapatite, can conform to complex cavity shapes without machining, has excellent osteoconductivity, and is able to be resorbed and replaced by new bone. Therefore, CPC is promising for craniofacial and orthopaedic repairs. However, its low strength and lack of macroporosity limit its use. This study investigated CPC reinforcement with absorbable fibers, the effects of fiber volume fraction on mechanical properties and macroporosity, and the cytotoxicity of CPC-fiber composite. The rationale was that large-diameter absorbable fibers would initially strengthen the CPC graft, then dissolve to form long cylindrical macropores for colonization by osteoblasts. Flexural strength, work-of-fracture (toughness), and elastic modulus were measured vs. fiber volume fraction from 0% (CPC Control without fibers) to 60%. Cell culture was performed with osteoblast-like cells, and cell viability was quantified using an enzymatic assay. Flexural strength (mean+/-SD; n=6) of CPC with 60% fibers was 13.5+/-4.4 MPa, three times higher than 3.9+/-0.5 MPa of CPC Control. Work-of-fracture was increased by 182 times. Long cylindrical macropores 293+/-46 microm in diameter were created in CPC after fiber dissolution, and the CPC-fiber scaffold reached a macroporosity of 55% and a total porosity of 81%. The new CPC-fiber formulation supported cell adhesion, proliferation and viability. The method of using large-diameter absorbable fibers in bone graft for mechanical properties and formation of long cylindrical macropores for bone ingrowth may be applicable to other tissue engineering materials.     

Mechanical augmentation of the vertebral body by calcium phosphate cement injection

The effectiveness of transpedicular calcium phosphate cement (CPC) injection as a new treatment for osteoporotic compression fracture of vertebrae was evaluated by measuring the compressive strength and the mode of failure in vertebrae experimentally injected with CPC. Forty-five human cadaver vertebrae were divided into three groups: a control group; group A, in which CPC was injected into the upper half of the vertebral body; and group B, in which CPC was injected into the whole vertebra. The load-displacement curve characteristically had two peaks in group A, and decreased rapidly after failure in group B. The failure site was the cancellous bone immediately below the cranial endplate in the control group, cancellous bone immediately below the CPC injection area in group A, and in the CPC injection area in group B. Although mechanical strength was greatest in those vertebrae in which the entire cancellous bone was replaced with CPC, the compressive strength of the vertebrae was also increased by partial replacement of cancellous bone with CPC injection. In terms of mode of failure and mechanical gradient with adjacent vertebrae, there were several advantages for those vertebrae in which the cranial half of the cancellous bone was replaced with CPC. Received: May 29, 2000 / Accepted: September 20, 2000     

清心解毒汤治疗邪毒侵心型病毒性心肌炎

目的探讨清心解毒汤治疗邪毒侵心型病毒性心肌炎的疗效。方法采用前瞻性研究方法,选取我院2013年1月-6月收治的病毒性心肌炎患者160例,随机分为治疗组与对照组,各80例,治疗组给予清心解毒汤(麻黄、杏仁、生石膏、黄芩等,2次/d,200 mL/次),对照组给予常规西医对症治疗(卧床休息、抗病毒及调整免疫等常规对症治疗)。结果治疗组总有效率为86.25%,高于对照组的75.00%,差异有统计学意义(P<0.05);治疗组显效率为40.00%,高于对照组的30.00%(P<0.05);2组治疗后SV、CO和EF均高于治疗前(P<0.05);治疗组治疗后SV、CO和EF分别为(61.33±7.28)mL、(4.50±0.61)L/min和(55.01±6.64)%,均高于对照组(P<0.05);2组治疗后CK、CKMB、AST和LDH均低于治疗前(P<0.05);治疗组治疗后CK、CKMB、AST和LDH分别为(80.30±10.62)u/L、(18.61±5.69)u/L、(31.47±4.92)u/L和(92.42±10.73)u/L,均低于对照组(P<0.05)。结论清心解毒汤可以减轻心脏损害程度,治疗热毒侵心型病毒性心肌炎。