Recombinant Tissue Plasminogen Activator-conjugated Nanoparticles Effectively Targets Thrombolysis in a Rat Model of Middle Cerebral Artery Occlusion.

The efficacy and safety of recombinant tissue plasminogen activator (rtPA) need to be improved due to its low bioavailability and requirement of large dose administration. The purpose of this study was to develop a fibrin-targeted nanoparticle (NP) drug delivery system for thrombosis combination therapy. We conjugated rtPA to poly(ethylene glycol)- poly(e-caprolactone) (PEG-PCL) nanoparticles (rtPA-NP) and investigated its physicochemical characteristics such as particle size, zeta potential, enzyme activity of conjugated rtPA and its storage stability at 4°C. The thrombolytic activity of rtPA-NP was evaluated in vitro and in vivo as well as the half-life of rtPA-NP, the properties to fibrin targeting and its influences on systemic hemostasis in vivo. The results showed that rtPA-NP equivalent to 10% of a typical dose of rtPA could dissolve fibrin clots and were demonstrated to have a neuroprotective effect after focal cerebral ischemia as evidenced by decreased infarct volume and improved neurological deficit (P<0.001). RtPA-NP did not influence the in vivo hemostasis or coagulation system. The half-life of conjugated rtPA was shown to be approximately 18 times longer than that of free rtPA. These experiments suggested that rtPA-conjugated PEG-PCL nanoparticles might be a promising fibrin-targeted delivery system for a combination treatment of thrombosis.

Determination of sulphasalazine and its main metabolite sulphapyridine and 5-aminosalicylic acid in human plasma by liquid chromatography/tandem mass spectrometry and its application to a pharmacokinetic study.

A simple and sensitive liquid chromatography/positive-ion electrospray ionization mass spectrometry (LC-ESI-MS/MS) method has been developed for the simultaneous determination of sulphasalazine (SASP) and its main metabolite sulphapyridine (SP) and 5-aminosalicylic acid (5-ASA) with 100 µL of human plasma using dimenhydrinate as the internal standard (I.S.). The API-3000 LC-MS/MS was operated under the multiple reaction-monitoring mode (MRM) using the electrospray ionization technique. Protein precipitation process was used to extract SASP, SP, 5-ASA and I.S. from human plasma. The total run time was 9.0 min and the elution of SASP, SP and 5-ASA was at 4.8 min, 2.5 min and 2.0 min, respectively. The separation was achieved with a mobile phase consisting of 0.2% formic acid, 2 mM ammonium acetate in water (mobile phase A) and 0.2% formic acid, 2 mM ammonium acetate in methanol (mobile phase B) by using gradient elution on a XBP Phenyl column (100 mm × 2.1 mm, 5 µm). The developed method was validated in human plasma with a lower limit of quantitation of 10 ng/mL for SASP, SP and 5-ASA, respectively. A linear response function was established for the range of concentrations 10-10,000 ng/mL (r>0.99) for SASP and 10-1000 ng/mL (r>0.99) for SP and 5-ASA. The intra and inter-day precision values for SASP, SP and 5-ASA met the acceptance as per FDA guidelines. SASP, SP and 5-ASA were stable during stability studies, i.e., long term, auto-sampler and freeze/thaw cycles. The method was successfully applied for the evaluation of pharmacokinetics of SASP, SP and 5-ASA after single oral doses of 250 mg SASP to 10 healthy volunteers.

Determination of vinpocetine and its primary metabolite, apovincaminic acid, in rat plasma by liquid chromatography-tandem mass spectrometry.

A precise and sensitive liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for simultaneous determination of vinpocetine (VP) and its primary metabolite, apovincaminic acid (AVA), in rat plasma was developed and validated. The analytes and the internal standard-dimenhydrinate were extracted from 50 microL aliquots of rat plasma via solid-liquid extraction. Chromatographic separation was achieved in a run time of 3.5 min on a C(18) column under isocratic conditions. Detection of analytes and IS was done by tandem mass spectrometry, operating in positive ion and multiple reaction monitoring (MRM) acquisition mode. The protonated precursor to product ion transitions monitored for VP, AVA and IS were m/z 351.4-->280.2, 323.2-->280.2 and 256.2-->167.3 respectively. The method was fully validated for its sensitivity, selectivity, accuracy and precision, matrix effect, stability study and dilution integrity. A linear dynamic range of 0.5-500 ng/mL for both VP and AVA was evaluated with mean correlation coefficient (r) of 0.9970 and 0.9984 respectively. The precision of the assay (RSD%) was less than 8.55% at all concentrations levels for both VP and AVA. This method was successfully applied to a pharmacokinetic study of VP in rats after intravenous (1 mg/kg) and oral (1 mg/kg) administration.

Effect of lactoferrin- and transferrin-conjugated polymersomes in brain targeting: in vitro and in vivo evaluations.

AIM: To evaluate the effect of lactoferrin (Lf) and transferrin (Tf) in brain targeting. METHODS: Polymersomes (PSs), employed as vectors, were conjugated with Lf or Tf and were characterized by morphology, particle size, zeta potential, and surface densities of the Lf or Tf molecules. In vitro uptake of Lf-PS and Tf-PS by bEnd.3 cells was investigated using coumarin-6 as a fluorescent probe. In vivo tissue distribution and pharmacokinetics of (125)I-Lf-PS and (125)I-Tf-PS were also examined. RESULTS: The mean particle size of PS, Lf-PS, and Tf-PS was around 150 nm and the zeta potential of the PSs was about -20 mV. Less than 0.12% of the coumarin was released from coumarin-6-loaded PS in 84 h indicating that coumarin-6 was an accurate probe for the PSs' behavior in vitro. It was shown that the uptake of Lf-PS and Tf-PS by bEnd.3 cells was time-, temperature-, and concentration-dependent. Both Lf and Tf could increase the cell uptake of PSs at 37 degrees C, but the uptake of Tf-PS was significantly greater than that of Lf-PS. In vivo tissue distribution and pharmacokinetics in mice revealed higher brain uptake and distribution of Tf-PS than Lf-PS, which was in accordance with in vitro uptake results. The drug targeting index (DTI) of Tf-PS with regard to Lf-PS was 1.51. CONCLUSION: Using a PS as the delivery vector and bEnd.3 cells as the model of the blood-brain barrier (BBB), Tf was more effective than Lf in brain targeting.

[The pharmacokinetics of FK506 and its nanoparticles in aqueous humor of rabbits].

OBJECTIVE: To investigate the pharmacokinetics of FK506 and its nanoparticles in aqueous humor of rabbits applied with eye drops or subconjunctiva injections. METHODS: 42 New Zealand albino rabbits were divided into 2 groups. (1) Nanoparticle solution containing 10 microg FK506 was injected into subconjunctiva or dropped on conjunctival sac of rabbits (68 eyes in 34 cases). (2) Eye drops containing 20 or 40 microg FK506 without nanoparticles were dropped on conjunctival sac of rabbits (16 eyes in 8 cases). Aqueous humor was collected at different times after local administration and FK506 concentrations were measured by ELISA. Ocular pharmacokinetic parameters of FK506 were calculated by 3p87 software. RESULTS: In the solution containing nanoparticle, the effective FK506 concentration in aqueous humor could be kept up to 16 h in eye drops group. The range of FK506 concentration was between (15.50 +/- 3.39) - (2.59 +/- 0.83) ng/ml. FK506 in aqueous humor can be maintained for 96 h by injecting into subconjunctiva and FK506 concentration were between (9.62 +/- 2.19) - (2.60 +/- 0.21) ng/ml from 6 - 96 h. T(max) and C(max) in eye drops were (1.25 +/- 0.50) h and (15.52 +/- 2.37) ng/ml respectively; while T(max) and C(max) in subconjunctiva injection were (64.00 +/- 13.86) h and (10.16 +/- 1.37) ng/ml respectively. Each AUC(0-->t) was (152.44 +/- 16.74) ng.ml(-1).h(-1) and (612.48 +/- 54.39) ng.ml(-1).h(-1) respectively; each Ka was 3.790 +/- 0.730 and 0.040 +/- 0.004 respectively; and each MRT was (8.20 +/- 1.28) h and (58.53 +/- 5.42) h respectively. In the eye drops containing 20 microg or 40 microg FK506 but without nanoparticles applied in conjunctival sac of rabbits, all effective FK506 concentrations in aqueous humor could not be kept over 4 h. T(max) was 1 h and C(max) was (18.93 +/- 6.95) ng/ml in 20 microg FK506, while in 40 microg FK506 T(max) was 2h and C(max) was (28.33 +/- 1.31) ng/ml. CONCLUSION: Solution with FK506 nanoparticle dropping onto the eye or injecting into subconjunctiva could be sustained in rabbit aqueous humor for a longer time than non-nanoparticle FK506 solution, while with injecting of nanoparticle FK506 solution it could be detected with a relative low but longer effective concentration.