Development, validation and comparison of surrogate matrix and surrogate analyte approaches with UHPLC-MS/MS to simultaneously quantify dopamine, serotonin and γ-aminobutyric acid in four rat brain regions.

As biomarkers, endogenous neurotransmitters play critical roles in the process of neuropsychiatric diseases, and neurotransmitter levels in different brain regions can contribute to neurological disease diagnosis and treatment. Due to the lack of a blank matrix for endogenous neurotransmitters, surrogate-matrix and surrogate-analyte approaches have been used for the determination of neurotransmitters to solve this problem. In this study, we capitalised on the high accuracy, precision, and throughput of UHPLC-MS/MS and developed new methods based on the two approaches. Both approaches satisfied FDA and EMA validation criterias after an appropriate parallelism assessment, and they were used to further quantify the three endogenous neurotransmitters, including dopamine (DA), serotonin (5-HT) and γ-aminobutyric acid (GABA) in rat brain four regions (cortex, striatum, hypothalamus and hippocampus) which represent the catecholamines, indolamines, and amino acids, respectively. Comparison of the results in the same rats (n = 10) showed there was no significant difference in DA, 5-HT, or GABA levels between the two approaches (P > 0.05). The concentrations of DA and GABA were highest in striatum and hypothalamus, respectively, and the levels of 5-HT were paralleled in striatum and hippocampus almost 2-fold higher than other regions. This is the first study to compare these two approaches in the determination of endogenous neurotransmitter content in the rat brain, and the surrogate-matrix approach proved to be simple, rapid, and reliable, considering cost, matrix similarity, and practicality.

IR-780-loaded polymeric micelles enhance the efficacy of photothermal therapy in treating breast cancer lymphatic metastasis in mice.

Cancer metastasis is responsible for over 90% of breast cancer-related deaths, and inhibiting lymph node metastasis is an option to treat metastatic disease. Herein, we report the use of IR-780-loaded polymeric micelles (IPMs) for effective photothermal therapy (PTT) of breast cancer lymphatic metastasis. The IPMs were nanometer-sized micelles with a mean diameter of 25.6 nm and had good stability in simulated physiological solutions. Under 808-nm laser irradiation, IPMs exhibited high heat-generating capability in both in vitro and in vivo experiments. After intravenous injection, IPMs specifically accumulated in the tumor and metastatic lymph nodes and penetrated into these tissues. Moreover, a single IPMs treatment plus laser irradiation significantly inhibited primary tumor growth and suppressed lymphatic metastasis by 88.2%. Therefore, IPMs are an encouraging platform for PTT applications in treatment of metastatic breast cancer.

Lower irritation microemulsion-based rotigotine gel: formulation optimization and in vitro and in vivo studies.

BACKGROUND: Rotigotine is a potent and selective D1, D2, and D3 dopaminergic receptor agonist. Due to an extensive first-pass effect, it has a very low oral bioavailability (approximately 0.5% in rats). PURPOSE: The present investigation aimed to develop a microemulsion-based hydrogel for transdermal rotigotine delivery with lower application site reactions. METHODS: Pseudoternary phase diagrams were constructed to determine the region of oil in water (o/w)-type microemulsion. Central composite design was used to support the pseudoternary phase diagrams and to select homogeneous and stable microemulsions with an optimal amount of rotigotine permeation within 24 hours. In vitro skin permeation experiments were performed, using Franz diffusion cells, to compare rotigotine-loaded microemulsions with rotigotine solutions in oil. The optimized formulation was used to prepare a microemulsion-based hydrogel, which was subjected to bioavailability and skin irritancy studies. RESULTS: The selected formulations of rotigotine-loaded microemulsions had enhanced flux and permeation coefficients compared with rotigotine in oil. The optimum microemulsion contained 68% water, 6.8% Labrafil(®), 13.44% Cremophor(®) RH40, 6.72% Labrasol(®), and 5.04% Transcutol(®) HP; the drug-loading rate was 2%. To form a microemulsion gel, 1% Carbomer 1342 was added to the microemulsion. The bioavailability of the rotigotine-loaded microemulsion gel was 105.76%±20.52% with respect to the marketed rotigotine patch (Neupro(®)). The microemulsion gel irritated the skin less than Neupro. CONCLUSION: A rotigotine microemulsion-based hydrogel was successfully developed, and an optimal formulation for drug delivery was identified. This product could improve patient compliance and have broad marketability.

[Study on preparation of ligustrazine ocular implant and correlation between in vivo and in vitro drug release].

OBJECTIVE: To prepare ligustrazine (TMPZ) ocular sustained-release implant, and investigate its in vitro drug release, pharmacokinetics in rabbit vitreum and in vitro correlation. METHOD: Ligustrazine ocular sustained-release implants were prepared by micro-twin conical screw mixers with hot-melting extrusion method, with polyactic-co-glycolic acid (PLGA) as the matrix. HPLC was adopted to determine the concentration in vitreum after ligustrazine was implanted in rabbit eyes, in order to examine its in vivo sustained-release behavior, and study the correlation between in vitro and in vivo. RESULT: Ligustrazine implants were prepared with a drug-loading rate between 10% and 30%, which was in conformity to the pharmacopoeia in terms of the content uniformity. Its in vitro release was in conformity to the zero-order release model. With PLGA 5050, 2. 5A as a vector, ligustrazine implants with a drug-loading rate of 30% could slowly release drug for more than 3 weeks, indicating a good correlation between in vitro and in vivo release. CONCLUSION: Ligustrazine ocular implants prepared with hot-melting extrusion method is practicable. Ligustrazine ocular implants release drug smoothly in rabbit vitreous vitreums, suggesting good sustained-release effect.

Determination of puerarin in rabbit aqueous humor by liquid chromatography tandem mass spectrometry using microdialysis sampling after topical administration of puerarin PAMAM dendrimer complex.

To study pharmacokinetic properties of puerarin poly(amido amine) (PAMAM) dendrimer complex, a sensitive liquid chromatography tandem mass spectrometry method (LC-MS/MS) was developed and validated to determine puerarin in rabbit aqueous humor using microdialysis sampling. Astilbin was used as the internal standard. The linear range for puerarin was from 2 to 1000ng/mL (r=0.9986) based on 20µL of aqueous humor. The coefficients of variations for intra-day and inter-day precisions were less than 10.0%, and the relative error of accuracy was within ±6.3%. The mean extraction recovery of puerarin varied from 80.4% to 85.5%. Microdialysis provides a complete concentration versus time profile. A significant difference was observed in main pharmacokinetic parameters of C(max), AUC and t(1/2) between puerarin solution and puerarin PAMAM dendrimer complex. Complex formation resulted in an obvious increase in bioavailability of puerarin after topical administration to rabbit according to the above LC-MS/MS assay method.

Effect of poly(amidoamine) dendrimers on corneal penetration of puerarin.

The aim of this study was to investigate the effect of Poly(amidoamine) (PAMAM) dendrimers on corneal permeation of puerarin (PUE). Permeation studies were performed using excised cornea of rabbits by a Valia-Chien diffusion apparatus. Drug-treatment studies were carried out by measuring the penetration of puerarin on cornea in PAMAM-PUE physical mixture or PAMAM-PUE complex, and cornea-treatment studies were carried out by measuring the penetration of puerarin on PAMAM dendrimer pretreated cornea in puerarin solution. The results showed that the permeability coefficient of puerarin in PAMAM-PUE physical mixture was enhanced by 2.48 (G3), 1.99 (G4) and 1.36 (G5) times on average, respectively compared to control. However, no significant permeability enhancement of puerarin in PAMAM-PUE complex was found compared to control. This may attribute to free drug concentration was lower in PAMAM-PUE complex which served as a depot and exhibited slow-released behavior of drug. Cornea-treatment studies showed that the lag time of puerarin was decreased, while the cumulative amount within 2.5 h (Q(2.5)) and the permeability coefficient of puerarin increased compared to control. The permeability coefficient of puerarin was linear correlated to the molecular weight of PAMAM dendrimer (r(2)=0.99). This indicates that higher generation of PAMAM dendrimer more easily interact with cornea or loosen the epithelium cell junctions than lower generation to increase the flux of puerarin. Overall, the study showed that PAMAM dendrimer increased the corneal permeation of puerarin mainly by altering the corneal barrier.

[Preparation of cyclosporine A loaded mPEG-PLGA copolymer micelles and study its pharmacokinetics in rats].

To prepare cyclosporine A (CyA) loaded block copolymer micelles and observe its release behaviors in vitro and pharmacokinetics in rats, methoxylpoly (ethylene glycol)-poly (D, L-lactide-co-glycolide) (mPEG-PLGA) was synthesized by ring-opening copolymerization of lactide and glycolide in the presence of methoxylpoly (ethylene glycol) (mPEG) as initiator. The structure of the mPEG-PLGA copolymer was confirmed with 1H NMR and FT-IR. The cyclosporine A loaded micelles (CyA-PM) were prepared by solvent evaporation method and their morphology was observed by the transmission electron microscope (TEM). The mean size and size distribution were determined by dynamic light scattering (DLS). The release behaviors in vitro and pharmacokinetics in rats were investigated by HPLC method using cyclosporine A injection commercial agent, sandimmune, as the reference. The obtained CyA-PM showed spherical shape with the core-shell structure, the mean particle sizes are in the range of 136.1-141.9 nm. The drug loading amount and entrapment efficiency were increased and the particle size became smaller with decreasing the ratio of acetone to water. With the increasing of the amount of cyclosporine A fed the drug loading increased, entrapment efficiency decreased and the particle size had no change. CyA-PM showed significant sustained release behave in vitro compared with sandimmune and only 9.7% of encapsulated cyclosporine A was released after 12 hours, the release characteristics was well fitted with Higuchi equation (r = 0.999). The Pharmacokinetics study at equal administration dosage (5 mg x kg(-1)) in rats showed the half-life (t1/2) of CyA-PM extended and the area under concentration-time curve (AUC) increased compared to sandimmune. The results also showed that cyclosporine A concentration-time data were all in accord with two compartment model. Cyclosporine A loaded mPEG-PLGA micelles showed obviously solubility enhancement, sustained release and overcome the side effect and toxicity of sandimmune resulted from solubiling agent-polyoxyethylene castor oil (Cremophor EL) and might be developed as a novel dosage form of cyclosporine A.

[Preparation of sustained release multivesicular liposome for thymopentin and preliminary study on its pharmacokinetics in rats].

To optimize the formulation and preparation method of multivesicular liposome of thymopentin and to investigate its pharmacokinetics in rats, the multivesicular liposome of thymopentin was prepared by double emulsification method and the formulation was optimized by orthogonal design. The release characteristics of thymopentin from multivesicular liposome in PBS (pH 7.4) and in plasma were investigated. The multivesicular liposome of thymopentin labeled with fluorescein isothiocyanate was prepared by double emulsification method. Its pharmacokinetics was evaluated following intramuscular injection in rats. The optimal formulation of multivesicular liposome of thymopentin were formulated with 7.5% glucose in aqueous phase and 2.25 mol x L(-1) triolein, 2.68 mol x L(-1) DPPG and 16.96 mol x L(-1) DOPC in organic phase. The entrapment efficiency of the multivesicular liposome of thymopentin was above 85% and the mean particle size was about 22 microm. The in vitro release of thymopentin from multivesicular liposome in PBS (pH 7.4) and in plasma was found to be in a sustained manner. The release curves were fitted to Higuchi equation. The pharmacokinetics following intramuscular injection of the multivesicular liposome of thymopentin labeled with fluorescein isothiocyanate in rats showed that the peak concentration of thymopentin was lower and elimination of it was slower significantly than that of thymopentin labeled with fluorescein isothiocyanate solution in the same dose. The plasma concentration of thymopentin maintained above quantitative limitation at 120 h after administration of multivesicular liposome of thymopentin. The optimized formulation and preparation technology of multivesicular liposome of thymopentin with higher entrapment efficiency are feasible with good reproducibility. Multivesicular liposome of thymopentin showed significant sustained-release property following intramuscular injection in rats.

[Preparation of diclofenac sodium liposomes and its ocular pharmacokinetics].

AIM: To prepare diclofenac sodium liposomes and observe its ocular pharmacokinetics in rabbits. METHODS: The diclofenac sodium cationic liposomes were prepared by reverse-phase evaporation methods and the formula of liposome was optimized with uniform design. HPLC method was established and validated for the determination of diclofenac sodium in precornea, cornea and aqueous humor of rabbit eye. Liposome and eyedrop solution 50 microL with total 50 microg diclofenac sodium were instilled to eyes of rabbits, separately. Samples of tear, cornea and aqueous humor were collected at different time intervals after rabbits were sacrificed. The ocular pharmacokinetics was investigated by the concentration-time data of tear, cornea and aqueous humor. RESULTS: The mean particle size of the diclofenac sodium liposomes was 226.5 nm with zeta potential of + 18. 1 mV. The entrapment efficiency reached 63%. Compared with solution, liposome was characterized by slower clearance in precornea. The concentration of diclotenac in cornea and aqueous humor instilled with liposome were higher than that with eye-drop solution. Cmax of diclofenac sodium in aqueous humor instilled with liposome and eye-drop solution were (0.69 +/- 0.25) and (0.48 +/- 0.19) microg x mL(-1) and (36.68 +/- 11.7) and (21.82 +/- 8.6) microg x g(-1) in cornea, respectively. But no significant difference were found to Tmax in aqueous humor and cornea between liposome and eyedrop, T(1/2) of diclofenac in aqueous humor and cornea with liposoine were longer than that with eye-drop solution. The ocular bioavailability of liposome in aqueous humor was 211% compared with that of eyedrop. CONCLUSION: Diclofenac sodium cationic liposomes can increase the corneal contact time, enhance the corneal permeability of diclofenac sodium and improve its ocular bioavailability.