A novel snake venom-derived GPIb antagonist, anfibatide, protects mice from acute experimental ischaemic stroke and reperfusion injury.

BACKGROUND AND PURPOSE: Ischaemic stroke is a serious disease with limited therapy options. Glycoprotein (GP)Ib binding to von Willebrand factor (vWF) exposed at vascular injury initiates platelet adhesion and contributes to platelet aggregation. GPIb has been suggested as an effective target for antithrombotic therapy in stroke. Anfibatide is a GPIb antagonist derived from snake venom and we investigated its protective effect on experimental brain ischaemia in mice. EXPERIMENTAL APPROACH: Focal cerebral ischaemia was induced by 90 min of transient middle cerebral artery occlusion (MCAO). These mice were then treated with anfibatide (4, 2, 1 µg·kg(-1) ), injected i.v., after 90 min of MCAO, followed by 1 h of reperfusion. Tirofiban, a GPIIb/IIIα antagonist, was used as a positive control. KEY RESULTS: Twenty-four hours after MCAO, anfibatide-treated mice showed significantly improved ischaemic lesions in a dose-dependent manner. The mice had smaller infarct volumes, less severe neurological deficits and histopathology of cerebrum tissues compared with the untreated MCAO mice. Moreover, anfibatide decreased the amount of GPIbα, vWF and accumulation of fibrin(ogen) in the vasculature of the ischaemic hemisphere. Tirofiban had similar effects on infarct size and fibrin(ogen) deposition compared with the MCAO group. Importantly, the anfibatide-treated mice showed a lower incidence of intracerebral haemorrhage and shorter tail bleeding time compared with the tirofiban-treated mice. CONCLUSIONS AND IMPLICATIONS: Our data indicate anfibatide is a safe GPIb antagonist that exerts a protective effect on cerebral ischaemia and reperfusion injury. Anfibatide is a promising candidate that could be beneficial for the treatment of ischaemic stroke.

Protective effect of iridoid glycosides from Paederia scandens (LOUR.) MERRILL (Rubiaceae) on uric acid nephropathy rats induced by yeast and potassium oxonate.

Iridoid glycosides of Paederia scandens (IGPS) are an active component isolated from Chinese herb P. scandens (LOUR.) MERRILL (Rubiaceae). Uric acid nephropathy (UAN) is caused by excessive uric acid, which results in damage of kidney tissue via urate crystals deposition in the kidneys. This study aimed to investigate the protective effects of IGPS on UAN in rats induced by yeast and potassium oxonate. Treatment groups received different doses of IGPS and allopurinol (AP) daily for 35 days respectively. The results showed that treatment with IGPS significantly prevented the increases of uric acid in serum and the elevation of systolic blood pressure (SBP), attenuated renal tissue injury, improved renal function and reserved the biological activity of NOS-1. IGPS also inhibited the biological activity of TNF-α and TGF-ß1, and suppressed the mRNA expressions of TNF-α and TGF-ß1 in renal tissue. Taken together, the present and our previous findings suggest that IGPS exerts protective effects against kidney damage in UAN rats through its uric acid-lowering, anti-inflammatory and immunomodulatory properties. Furthermore, decreasing SBP by up regulation of NOS-1 expression and down regulation of TNF-α and TGF-ß1 expression are involved in the effect of IGPS on high uric acid-induced nephropathy.

Trans-resveratrol loaded chitosan nanoparticles modified with biotin and avidin to target hepatic carcinoma.

Conventional liver targeted system focuses on delivering drugs to liver, bringing toxicity on hepatic normal tissues. The purpose of this study is to construct a new system capable of specially targeting to hepatic carcinoma instead of the whole liver. Based on the fact that nanoparticles (NPs) bound with either biotin or avidin tend to accumulate in tumors and avidin-attached reagents were quickly eliminated from blood circulation and assembled in liver, trans-resveratrol loaded chitosan nanoparticles (CS-NPs), CS-NPs with the surface modified either by biotin (B-CS-NPs) or by both biotin and avidin (A-B-CS-NPs) were prepared and their physiochemical properties were investigated. The in vitro release profiles of the three NPs all conformed to bioexponential equation. Pharmacokinetic experiment indicated that A-B-CS-NPs rapidly assembled in liver after injection, with the highest liver targeting index of 2.70, while the modification of biotin attenuated the liver targeting ability of NPs. Inhibitory study on HepG2 cells declared that compared to trans-resveratrol solution and CS-NPs, both B-CS-NPs and A-B-CS-NPs significantly improved the anticancer activity. When incubated with HepG2 cells at high concentration for longer time, A-B-CS-NPs exhibited superior cytotoxicity than B-CS-NPs. This study exclaims that A-B-CS-NPs may be a potent drug delivery vector specially targeting to hepatic carcinoma.

Development of PLGA nanoparticles simultaneously loaded with vincristine and verapamil for treatment of hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) is one of the malignant tumors with poor chemo-sensitivity to vincristine sulfate (VCR) due to multi-drug resistance (MDR). Combinations of encapsulated VCR and verapamil hydrochloride (VRP, a chemo-sensitizer) might be a potential strategy to improve HCC therapeutic efficacy of VCR. PLGA nanoparticles (PLGANPs) simultaneously loaded with VCR and VRP (CVn) were prepared. The entrapment efficiencies of VCR and VRP were 70.92 ± 3.78% and 85.78 ± 3.23%, respectively (n = 3). The HCC therapeutic activity of CVn was assessed using MTT assay. In BEL7402 and BEL7402/5-FU human hepatocarcinoma cell lines, CVn had the same antitumor effect as one free drug/another agent-loaded PLGANPs (C + Vn or Cn + V) combination and coadministration of two single-agent-loaded PLGANPs (Cn + Vn), which was slightly higher than that of the free VCR/VRP combination (C - V). CVn might cause lower normal tissue drug toxicity by the enhanced permeation and retention effect in vivo. Additionally, CVn might cause fewer drug-drug interaction and be the most potential formulation to simultaneously deliver VCR and VRP to the target cell in vivo than the other three nanoparticle formulations (C + Vn, Cn + V, and Cn + Vn). Therefore, we speculate that CVn might be the most effective preparation in the treatment of drug-resistant human HCC in vivo.

Preparation, characterization, and in vivo evaluation of mitoxantrone-loaded, folate-conjugated albumin nanoparticles.

Folic acid was covalently conjugated to bovine serum albumin nanoparticles (BSANP) to target the nanoparticles to SKOV3 cells expressing folate receptors. Mitoxantrone was incorporated into the folate-conjugated albumin nanoparticles, and the final nanoparticle size was 68 nm, as measured by a laser light scattering particle analyzer. The cytotoxic activity of mitoxantrone- loaded, folate-conjugated albumin nanoparticles (MTO-BSANP-folate), which was quantitated by (3)H-thymidine incorporation, was higher than mitoxantrone-loaded BSANP (MTO-BSANP) and MTO solution, and could be inhibited by free folic acid. MTO-BSANPfolate may be endocytosed via the folate receptor on the surface of SKOV3 cells. MTO-BSANPfolate also inhibited tumor growth better than the MTO-BSANP and MTO solution in vivo. These results indicate that folate-conjugated BSANP may have therapeutic potential as a vector for anticancer drugs in cancer chemotherapy.

Reversion of multidrug resistance by co-encapsulation of vincristine and verapamil in PLGA nanoparticles.

Multidrug resistant (MDR) cancer may be treated using combinations of encapsulated cytotoxic drugs and chemosensitizers. To optimize the effectiveness of this combinational approach, poly(d,l-lactide-co-glycolide acid) (PLGA) nanoparticles formulations capable of delivering a cytotoxic drug, vincristine, a chemosensitizer, verapamil, or their combination were prepared via combining O/W emulsion solvent evaporation and salting-out method. Moreover, this work evaluated a number of approaches for the administration of chemosensitizer-cytotoxic drug combinations in a systematic fashion. The results showed that the administration sequence of anticancer drug and chemosensitizer was critical for maximal therapeutic efficacy and the simultaneous administration of vincristine and verapamil could achieve the highest reversal efficacy. In addition, PLGA nanoparticles (PLGANPs) showed moderate MDR reversal activity on MCF-7/ADR cells resistant to vincristine. The dual-agent loaded PLGA nanoparticles system resulted in the similar cytotoxicity to one free drug/another agent loaded PLGANPs combination and co-administration of two single-agent loaded PLGANPs, which was slightly higher than that of the free vincristine/verapamil combination. Co-encapsulation of anticancer drug and chemosensitizer might cause lower normal tissue drug toxicity and fewer drug-drug interactions. Therefore, we speculate that PLGANPs simultaneously loaded with anticancer drug and chemosensitizer might be the most potential formulation in the treatment of drug resistant cancers in vivo.

[Studies on in vivo release of berberine hydrochloride from carboxymethyl konjac glucomannan pellets in rats].

OBJECTIVE: To observe the absorption and concentration of berberine hydrochloride (BH) in gastric, entric, colonic tissue after intragastric administration of BH-containing carboxymethyl konjac glucomannan pellets for evaluating colon-specific drug delivery characteristics of the pellets. METHOD: BH-containing carboxymethyl konjac glucomannan pellets (pellets group) and BH-containing carboxymethyl cellulose suspension (control group) were intragastric administrated to rats at the dose of 50 mg x kg(-1), respectively. A high performance liquid chromatography method determinated BH concentration in rat plasma and tissue. Drug delivery index (DDI) was calculated. RESULT: The range of BH in plasma and tissue in rats were 0.025 2-2.52 mg x L(-1) (r = 0.999 2) and 0.126-25.22 mg x L(-1) (R > 0.99),respectively. The detection of BH in plasma and tissue were 10 microg x L(-1) and 8 microg x L(-1), respectively. The area under the curve (AUC(0 --> infinity)) in the plasma samples of pellets group was 0.477 times that of the control group; in the gastric, entric, colonic tissue, the AUC(0 --> infinity) of pellets group was as much as 0.187, 0.228, 2.00 times that of the control group, respectively. The DDI of the pellets was 0.392 4, 0.478 6, 4.193 in the gastric, entric, colonic tissue of the rat, respectively. CONCLUSION: Carboxymethyl konjac glucomannan pellets may be a useful carrier of BH for colon-specific delivery.

[Effect of several penetration enharcers on transdermal permeation and skin accumulation of artemether].

OBJECTIVE: To investigate penetration characteristics of artemether and the effect of different permeation enhancer on transdermal permeation of artemether through rat skin. METHOD: The permeation experiments were performed using rat skin on modified Franz diffusion cells in vitro. The concentrations of artemether in receptor compartment at specified time points were determined by HPLC. RESULT: The permeating ratio through human skin of artemether solution was Js (2.78 +/- 0.78) microg x cm(-2) x h(-1), the quantity of drug penetrated through and accumulated in the skin by the end of the experiment were (69.07 +/- 3.01) microg x cm(-2), (58.93 +/- 3.56) microg x cm(-2) respectively. Four different permeation enhancers can improve the transdermal permeation of artemether. CONCLUSION: Artemether have the potential to be developed to new transdermal preparation.

[Preparation of self-microemusifying drug delivery system for volatile oil from rhizome of ligusticum Chuanxiong].

OBJECTIVE: To search on the optimum formula for the seif-microemulsifying drug delivery system of volatile oil from rhizome of ligusticum Chuanxiong. METHODS: Through solubility experiment, orthogonal screen and drawing phase diagram, taking the degree of emulsifying, the volume of the rest oil and emulsion particle size as parameters, the appropriate proportion composed of Chuanxiong oil, nonionic surfactant and flux was screened for the optimum formulation of self-microemulsifying drug delivery system. RESULTS: In the formulation of self-microemulsifying drug delivery system for volatile oil from rhizome of ligusticum Chuanxiong, taking S1 as the nonionic surfactant and C1 as the co-surfactant could get the best effect of emulsifying. CONCLUSION: The optimum formula for seif-microemulsification of volatile oil from rhizome of ligusticum Chuanxiong is volatile oil from rhizome of ligusticum Chuanxiong, S1 and C1, with their proportion being 10 : 9 : 1.

[Study on drug release of gastrodin ion-activated nasal in situ gel in vitro].

OBJECTIVE: To study on the drug release characteristics and mechanism of gastrodin ion-activated nasal in situ gel in vitro. METHOD: Regularity and mechanism of the drug release of gastrodin nasal in situ gel were studied by using the diffusion cell model and the membrane-less dissolution model, respectively. A novel kinesis diffusion cell model was designed according to the characteristics of release environment of nasal cavity. It was used to investigate the effect of adhesiveness on the release of the in situ gel. RESULT: Drug release of gastrodin nasal in situ gel followed the one order release model. Erosion rate of the gel was low and not linearly correlated with the release rate. Compared with gastrodin solution, the nasal in situ gel could increase release time and release amount. CONCLUSION: Gastrodin in the nasal in situ gel is released mainly by diffusion rather than erosion. Release amount of the in situ gel in nasal cavity may be obviously increased because of its adhesiveness.

[In vitro dexamethasone release from nanoparticles and its pharmacokinetics in the inner ear after administration of the drug-loaded nanoparticles via the round window].

OBJECTIVE: To investigate the feasibility of local drug delivery into the inner ear using solid lipid nanoparticles (SLN) and evaluate its potential for inner ear disease treatment in terms of the pharmacokinetics of the delivered drug in the inner ear. METHODS: Dexamethasone acetate (DA)-loaded SLN was prepared with Compritol 888 ATO as the matrix by means of hot dispersion-ultrasonic technique. A high-performance liquid chromatography (HPLC) was established for determining DA and dexamethasone (Dex). The pharmaceutical properties of DA-loaded SLN including the particle size, entrapment ratio and in vitro release were estimated. DA-loaded SLN was administered via intratympanic injection or intravenous injection in guinea pigs and Dex concentration in the perilymph was measured with HPLC for estimation of the pharmacokinetic parameters. RESULTS: The mean diameter of the DA-loaded SLN was 106.8 nm with entrapment ratio of 83.8%, and the in vitro DA release from the nanoparticles well conformed to Weibull distribution, with sustained-release of DA from the SLN exceeding 6 days. After intravenous injection of DA-loaded SLN in guinea pigs, Dex failed to be detected in the perilymph. Compared with Dex-loaded in situ gel following intratympanic injection, the relative bioavailability of Dex in the perilymph was 504% following intratympanic injection of DA-loaded SLN, which also resulted in increased t(1/2) and mean residence time (MRT) by 0.5 and 1.9 folds respectively. CONCLUSION: Nanoparticles can be a promising tympanic drug delivery system for topical drug administration in the treatment of inner ear diseases.

[Brain targeting of gastrodin nasal in situ gel].

OBJECTIVE: To investigate the biodistribution of gastrodin ion-activated nasal in situ gel in rat blood and brain tissues and to evaluate its brain targeting. METHODS: Intravenous administration of gastrodin solution or intranasal administration of gastrodin nasal in situ gel were given to 32 rats, respectively. The concentrations of gastrodin in the plasma and gastrodigenin in the brain tissues of the rats were determined by HPLC. RESULTS: The intranasal administration of the in situ gel of gastrodin produced more significant brain targeting effect than the intravencus administration of gstrodin solution (P < 0.01). The area under curve (AUC) of cerebrum, cerebellum and olfactory bulb increased by 1.16, 0.77 and 3.34 times, with 2.66. 2.18 and 5.34 brain targeting indexes (BTI), respectively. The mean residence time (MRT) increased by nearly four-folds. CONCLUSION: Gastrodin nasal in situ gel can improve the brain targeting of gastrodin and slow its release.

[Study on release mechanism of berberine hydrochloride-loaded carboxymethyl konjac glucomannan pellets for colonic delivery].

OBJECTIVE: To study release mechanism of berberine hydrochloride (BH) from carboxymethyl konjac glucomannan pellets for colonic delivery. METHOD: The pellets were prepared by ionotropic gelation technique. The effects of the kinds of enzyme and enzyme concentration of dissolution media on the release of BH and the erosion properties of the pellets were studied. RESULT: Compared with the dissolution media without enzymes, the release of BH and the erosion of the pellets were increased obviously in the media with rat cecal and colonic content or beta-mannase, the degradation of the carrier material of pellets by enzymes was the main factor which result in the erosion of the pellets. With the increased of beta-mannase concentration, the release of BH and the erosion of the pellets increased, the amount relationships of the release of BH and the erosion of the pellets were approximately 1:1. The release of BH exhibit Peppas equation, the n value was more than 1. CONCLUSION: The release mechanism of BH from the pellets was enzymatic erosion-controlled, which indicates the potential of the pellets to serve as a colon-specific drug delivery system.

[Comparison of kinetic behavior in both plasma and tissue after intravenous administration of alpha-asarone in lipid emulsion and aqueous solution in rats and mice].

OBJECTIVE: To compare the pharmacokinetics and tissue distribution of alpha-asarone in lipid emulsion and aqueous solution for injection and study the feasibility of lipid emulsion of alpha-asarone as the parenteral drug delivery system. METHOD: HPLC was used to determine the drug concentration in rat plasma and mice tissues after intravenous (i.v.) administration of lipid emulsion and aqueous solution of alpha-asarone at a single dose (40 mg x kg(-1)), respectively. RESULT: The plasma concentration-time profiles of lipid emulsion and aqueous solution of alpha-asarone after intravenous administration of them are similar and the drug concentration-time data were fitted to a two-compartment open model. The results of tissues distribution showed that distribution contents of alpha-asarone from lipid emulsion and aqueous solution in vivo are similar in lungs but lipid emulsion increased the uptake in livers and spleens, and decreased the uptake in hearts and kidneys for alpha-asarone. CONCLUSION: The plasma concentration-time profiles of alpha-asarone in lipid emulsion and aqueous solution are similar, but lipid emulsion significantly altered the tissue distribution of alpha-asarone, which may be beneficial to decrease its potential toxicity to heart and kidney.

[In vitro evaluation of self-emulsifying drug delivery system of volatile oil from rhizome of Ligusticum chuanxiong].

OBJECTIVE: To investigate the evaluation method for self-emulsifying drug delivery system of volatile oil from rhizome of Ligusticum chuanxiong (VOC SEDDS). METHOD: The self-emulsifying ability, the efficiency of self-emulsification, the properties of emulsion, the dissolution of volatile oil from Rhizome of Ligusticum Chuanxiong and the stability of the emulsion were determined. RESULT: The optimized formulation can fully emulsify in 5 min and the particle sizes were around 102 nm. Zeta potential was about -30 mV. The O/W emulsions were stable through centrifugation with high reproducibility. In vitro dissolution test indicated that over 80% of drug dissolved in 30 min and VOC SEDDS was stable under light and high temperature in 10 d. CONCLUSION: VOC SEDDS has strong self-emulsifying ability, fine stability and high dissolution rate in vitro.

Preparation, characterization and uptake by primary cultured rat hepatocytes of liposomes surface-modified with glycyrrhetinic acid.

3-succinyl-30-stearyl glycyrrhetinic acid (Suc-GLAOSt) was synthesized as a targeting molecule, and incorporated in ordinary to liposomes (LP) to prepare a liposome surface-modified with glycyrrhetinic acid (LP-GLA), which could bind to the hepatocyte through the specific binding site of glycyrrhetinic acid (GLA) on the surface of rat cellular membrane. The maximal molar ratio of Suc-GLAOSt to total lipids in LP-GLA was 1:10. Calcein loaded liposome (Cal-LP) and calcein loaded LP-GLA (Cal-LP-GLA) were prepared by an ethanol injection method. The average diameter of Cal-LP and Cal-LP-GLA was 65 nm +/- 16 nm and 68 nm +/- 21 nm, respectively. The characteristics of cellular uptake of the two types of liposome were investigated through cellular uptake and competitive inhibition experiments. The uptake of Cal-LP-GLA by rat hepatocytes was markedly higher (3.3-fold) than that of Cal-LP (P < 0.01). The uptake of Cal-LP-GLA was inhibited, but the uptake of Cal-LP was not influenced by adding extraneous GLA. LP-GLA may be internalized by hepatocytes via the specific binding site, and can be used as a novel and promising carrier for targeting drug delivery to hepatocytes.

[Preparation and characterization of biotinylated chitosan nanoparticles].

Biotinylated chitosan nanoparticles (Bio-CS-NP) were prepared for the active delivery to cancer cells and its characterization was investigated in this study. The preparation process included two steps. First, biotinylated chitosan ( Bio-CS ) was obtained through a reaction between sulfosuccinimidobiotin and chitosan (CS). Second, Bio-CS-NP were prepared by the precipitation of Bio-CS with sodium chloride solution. With a biotin reagent box, the conjugation densities of biotin on the surface of Bio-CS-NP were determined. The morphology and diameter of the nanoparticles were assayed by transmission electron microscope (TEM) and laser light scattering particle analyzer, respectively. The uptake of nanoparticles by human hepotacarcinoma HepG2 cells, for example, Bio-CS-NP and chitosan nanoparticles (CS-NP) without any modification, was quantitatively examined. The results indicated that the conjugation densities of biotin on the surface of Bio-CS-NP were 2.2 biotin CS. Bio-CS-NP were spherical, smooth on the surface. The average diameter was 296.8 nm. The polydispersion index was 0.155. The uptake of Bio-CS-NP by HepG2 cells was much higher than that of CS-NP (P < 0.05). It demonstrated that Bio-CS-NP can be applied as a new vehicle to actively deliver anticancer drugs to tumor cells. The method for the determination of biotin was simple and practical.

[Study on drug release in vitro and rat intestinal absorption of resveratrol nanoliposomes].

OBJECTIVE: To study the release feature of Res-nanoliposomes in vitro and clarify the difference in absorption of Res-nanoliposomes from varied intestinal segments and the absorptive mechanism in vivo. METHOD: Dialytic method was used to determine resveratrol release rate of Res-nanoliposomes in vitro. An in situ rat perfusion method was used to investigate the intestinal absorption of Res-nanoliposomes. RESULT: Resveratrol release from nanoliposomes in vitro fitted the log-normal distribution equation and had a property of sustained release. Compared with other intestinal segments, significantly high percentage of Res-nanoliposomes was absorbed in ileum (P < 0.001). The absorption rate constants (ka) of Res-nanoliposomes in intestine were not significantly different. CONCLUSION: Res-nanoliposomes could sustain to release drug in vitro. The absorption was a first-order process with the passive diffusion mechanism. The Res-nanoliposomes could promote the absorption of Res in rat small intestine.

[Preparation of liposome entrapped vincristine sulfate and mitoxantrone chlorhydric acid].

OBJECTIVE: To investigate the prepation of the liposome carried with vincristine sulfate (VCR) and mitoxantrone chlorhydric acid (MTO) and to evaluate the quality of the liposome. METHOD: The liposome carried with VCR and MIT was prepared by pH-gradients method and reverse evaporation technique. HPLC was employed to determine VCR and MIT entrapping efficiency of liposomal. Laser particle analyzer was applied to determine the size and zeta potential of the liposomes carried with VCR and MTO. RESULT: The mean diameter of the liposome carried with MIT and VCR was 72.22 nm, with the entrapping rate of 95.77% for VCR and 99.53% for MTO. The liposome had perfect shape. CONCLUSION: The liposomes with high entrapping rate and small particle size had been prepared by pH-gradient method and reverse evaporation technique.