GSK3 and lamellipodin balance lamellipodial protrusions and focal adhesion maturation in mouse neural crest migration.

Neural crest cells are multipotent cells that delaminate from the neuroepithelium, migrating throughout the embryo. Aberrant migration causes developmental defects. Animal models are improving our understanding of neural crest anomalies, but in vivo migration behaviors are poorly understood. Here, we demonstrate that murine neural crest cells display actin-based lamellipodia and filopodia in vivo. Using neural crest-specific knockouts or inhibitors, we show that the serine-threonine kinase glycogen synthase kinase-3 (GSK3) and the cytoskeletal regulator lamellipodin (Lpd) are required for lamellipodia formation while preventing focal adhesion maturation. Lpd is a substrate of GSK3, and phosphorylation of Lpd favors interactions with the Scar/WAVE complex (lamellipodia formation) at the expense of VASP and Mena interactions (adhesion maturation and filopodia formation). This improved understanding of cytoskeletal regulation in mammalian neural crest migration has general implications for neural crest anomalies and cancer.

Evaluation of the efficacy and safety of a new formulation-lipid emulsion-based PTX injection: Pharmacokinetics, tissue distributions and anticancer effect on human gastric cancer cells in vitro.

Paclitaxel (PTX) is one of the most widely used chemotherapeutic agents. The commercial PTX formulation was based on Cremophor EL and ethanol owing to its poor aqueous solubility. However, Cremophor EL has been shown to cause toxic effects such as life-threatening anaphylaxis. In our study, we diluted PTX in a commercially available 20% (w/v) lipid emulsion (Lip-PTX) in order to avoid Cremophor EL. The purpose of this study was to evaluate the pharmacokinetics and tissue distributions between Lip-PTX and PTX injection. We also investigated the effects of Lip-PTX and PTX injection on human gastric cancer cells HGC-27 by MTT assay. The apoptosis was detected by flow cytometry with Annexin V/propidium iodide (PI) double staining. Furthermore, the safety such as acute toxicity was also assessed. The results showed that PTX in Sprague-Dawley rats administered Lip-PTX exhibited extended half-life, increased clearance (P < 0.05) and smaller area under the concentration-time curve compared with PTX injection and there was little significant difference in the distribution of PTX in Sprague-Dawley rats or tumor-bearing mice between Lip-PTX and PTX injection. The cells treated with Lip-PTX had a higher percentage of apoptosis and a higher G2 /M phase ratio, which indicated that the anticancer effect of Lip-PTX was significantly better than that of PTX injection. Moreover, our study highlighted the safety of Lip-PTX. This study demonstrated the feasibility and potential advantages of Lip-PTX for clinical therapy.

A fast and high-sensitivity liquid chromatography-tandem mass spectrometry method combined with in vivo microdialysis for quantification of meropenem in rabbits with sepsis under the simultaneous infusion of total parenteral nutrition: Application to a pharmacokinetic study.

A fast and high-sensitivity liquid chromatography-tandem mass spectrometry (LC-MS/MS) method assisted by microdialysis was established for the determination of meropenem in total parenteral nutrition (TPN) infused plasma. A 10-times dilution was arranged for sample preparation to overcome the severe matrix effect caused by the high salt content in dialysate and complex composition of TPN. This quantification method was proved to be satisfied in selectivity, accuracy, precision, linearity (R2 > 0.998), recovery, matrix effect and stability. In the optimized conditions, the calibration curve range was set from 2 to 2000 ng/ml. This validated method was applied to pharmacokinetics study of meropenem in rabbits with sepsis (induced by cecal ligation and punctures) under simultaneous infusion of TPN to simulate the clinical practice. The results demonstrated that the LC-MS/MS method assisted by microdialysis can be used successfully for the determination of meropenem in TPN-infused plasma. Moreover, the area under the curve and the maximum concentrations in the plasma of meropenem in control rabbits were significantly smaller (P < 0.05), while clearance and distribution volumes were significantly greater (P < 0.05) than in those with sepsis. It could be speculated that drug monitoring in patients with sepsis may be necessary.

A simple liquid chromatography tandem mass spectrometric method for fast detection of hydrogen sulfide based on thiolysis of 7-nitro-2, 1, 3-benzoxadiazole ether.

The long identified toxic gas, hydrogen sulfide (H2S), which has also been confirmed as the third gaseous signaling molecule following NO and CO, plays important roles in various physiological and pathological process. The current most established quantification method for H2S is HPLC method coupled with fluorescence detection after derivatization with a costly fluorescent reagent, Monobromobimane (MBB). However, The MBB method is characterized by strict reaction condition, long reaction time, tedious operation, and inconsistent reported results. In this study, based on the thiolysis reaction of 7-nitro-2, 1, 3-benzoxadiazole (NBD) ether, the commonly used chromatographic modifier 4-chloro-7-nitro-2,1,3- benzoxadiazole (NBDCl) and four probes (NBDOMe, NBDOEt, NBDOTFE and NBDOCMR) synthesized from NBDCl were tested as alternatives for fast quantification of H2S by LC-MS/MS. The reaction product between NBD ethers/NBDCl and H2S showed special pink color visible to the naked eye and was easy to synthesize and separate in lab; it also showed good retention on common chromatographic columns and high instrument response; therefore it is a good determinand. After establishment of LC-MS/MS methods for all the related compounds, the reaction conditions were optimized for all the probes with H2S. Then the stability, selectivity, reaction rate, sensitivity and quantitative linear relationship between the reaction product and H2S concentration were studied for each probe. Finally, NBDOEt was selected for LC-MS/MS detection of H2S. In comparision with the MBB method, the established NBDOEt method showed matched sensitivity and linearity, better selectivity, and higher repeatability; and had the advantages of easy operation, simple reaction condition, and cheap raw materials. The method was successfully validated and applied to determination of Na2S content in Na2S∙9H2O bulk drug and injection. In conclusion, NBDOEt is a promising option for quantification of H2S in abiotic matrix.

Field-amplified sample injection in capillary zone electrophoresis for the pharmacokinetic research of trace risperidone and its major metabolite 9-hydroxyrisperidone in beagle dogs.

This article describes a method for the simultaneous quantitation of risperidone and its major metabolite, 9-hydroxyrisperidone, in beagle dog plasma by field-amplified sample injection in capillary zone electrophoresis. The separation was carried out at 25°C in a 48 cm × 75 µm fused-silica capillary with an applied voltage of 20 kV using 60 mM NaH2 PO4 buffer (pH 3.6). The detection wavelength was 280 nm. Clean-up and preconcentration of plasma samples were conducted by 96-well formatted liquid-liquid extraction. In this study, this stacking technique provided a sensitivity enhancement of approximately 158 to 188 fold compared with the same sample without stacking. The method was suitably validated with respect to stability, specificity, linearity, lower limit of quantitation, accuracy, precision, and extraction recovery. Calibration curves exhibited good linearity (r2  > 0.995) over a wide concentration range of 2.5 to 200 ng/mL for both risperidone and 9-hydroxyrisperidone. The intra- and interday precisions at the three quality control levels were less than 11.40%. The intra- and interday accuracies ranged from 87.90 to 107.17% for risperidone and from 88.43 to 105.92% for 9-hydroxyrisperidone. All validation data were within the required limits. In conclusion, the method developed was successfully applied to pharmacokinetic studies of risperidone and 9-hydroxyrisperidone in beagle dogs.

Validation of a universal and highly sensitive two-dimensional liquid chromatography-tandem mass spectrometry methodology for the quantification of pyrazinamide, ethambutol, protionamide, and clofazimine in different biological matrices.

A novel and potent anti-tuberculosis drug combination pyrazinamide (PZA), ethambutol (EMB), protionamide (PTO), and clofazimine (CFZ) that rapidly kills Mycobacterium tuberculosis (Mtb) in the lungs has been identified using the artificial-intelligence-enabled parabolic response surface approach. A universal and highly sensitive two-dimensional liquid chromatography-tandem mass spectrometry (2D-LC-MS/MS) method for the simultaneous determination of PZA, EMB, PTO, and CFZ in various biological samples in different states (liquid samples: plasma, bile, and urine; solid samples: tissue and feces) using simple pretreatment was established and validated. For the first dimension of this column-switching arrangement, the automated purification and enrichment of the drugs were achieved on a Polar-RP column. The subsequent analytical separation was performed on an Agilent Zorbax SB-Aq column, and the total loop time was 7.5 min. The positive-ionization mode with multiple reaction monitoring was used for detection. The sensitivity was good with no carry-over detected, and the lower limit of quantification ranged from 100 to 500 pg/mL. This quantification method was fully validated and proved to be robust in accordance with US Food and Drug Administration guidelines. High recoveries (85.3-111.4%) and accuracies (92.1-109.3%), together with high precision values (0.5-13.8%), were verified in all matrices. All standard curves showed favorable linearities with r2 > 0.995. This validated method was applied to study plasma pharmacokinetics, tissue distribution, and excretion in Sprague-Dawley rats after oral administration of the drug combination.

Preclinical Absorption, Distribution, Metabolism, and Excretion of Sodium Danshensu, One of the Main Water-Soluble Ingredients in Salvia miltiorrhiza, in Rats.

In this study, the absorption, distribution, metabolism and excretion (ADME) of sodium danshensu (Sodium DL-ß-(3, 4-dihydroxyphenyl)lactate), one of the main water-soluble active constituents in Salvia miltiorrhiza, were evaluated in rats. Pharmacokinetic study was evaluated in doses of 15, 30, and 60 mg/kg after intravenous administration of sodium danshensu. Bioavailability study was evaluated by comparing between 30 mg/kg (I.V.) and 180 mg/kg (P.O.) of sodium danshensu. Tissue distribution, metabolism, and excretion were evaluated at 30 mg/kg (I.V.) of sodium danshensu. Following intravenous administration, sodium danshensu exhibited linear pharmacokinetics in the dose range of 15-60 mg/kg. Sodium danshensu appeared to be poorly absorbed after oral administration, with an absolute bioavailability of 13.72%. The primary distribution tissue was kidney, but it was also distributed to lung, stomach, muscle, uterus, heart, etc. Within 96 h after intravenous administration, 46.99% was excreted via urine and 1.16% was excreted via feces as the parent drug. Biliary excretion of sodium danshensu was about 0.83% for 24 h. Metabolites in urine were identified as methylation, sulfation, both methylation and sulfation, and acetylation of danshensu. Sodium danshensu can be developed as an injection because of its poor oral bioavailability. In conclusion, sodium danshensu is widely distributed, mainly phase II metabolized and excreted primarily in urine as an unchanged drug in rats.

Fast and direct determination of catechol-3, 6-bis(methyleiminodiacetic acid) prototype in beagle dog plasma using liquid chromatography tandem mass spectrometry: A simplified and high throughput in-vivo method for the metal chelator.

It is usually somewhat difficult to analyze the metal chelators, especially in complex biological matrix, because of the interference of metal ions in both the matrix and analyzing system. In this study, an innovative and simple bioanalytical method was established and validated for the quantification of a newly developed uranium chelator catechol-3, 6-bis (methyleiminodiacetic acid) (CBMIDA) in beagle dog plasma. Different analytical columns and mobile phase were tested for effective chromatography resolution and sensitive and reproducible response of CBMIDA and the internal standard. An Agilent Zorbax SB AQ column was chosen. Excessive peak tailing, peak asymmetry, low recovery, and poor reproducibility, which are generally observed in chromatographic analysis of metal chelators, were overcome by the use of a pulse gradient method and addition of ethylene diamine tetraacetic acid (EDTA) to the mobile phase at 8 µg mL-1, enabling good peak shape, low matrix interference, high precision and good linearity for CBMIDA quantification in beagle dog plasma. Plasma sample pretreatment was performed by a simple, high throughput protein precipitation step with 2.5 mM EDTA methanol solution in a 96-well protein precipitation plate without complexing with the metal ions, and the sample was directly analyzed by electrospray ionization mass spectrometry. By shifting the analysis target from the metal complex to metal chelator itself, the method has an advantage over the existing method for determination of EDTA and diethylenetriaminepentaacetic acid owing to increased sample throughput and apparent simplicity. The assay was validated in accordance with the United States Food and Drug Administration guidelines and successfully applied to the pharmacokinetic study of CBMIDA in beagles after intramuscular injection of CBMIDA at different doses. The method was sensitive enough for the detection of CBMIDA concentration at 4 elimination half-times. The experimental strategies presented herein may be helpful for the measurement of other radionuclide chelators in biological matrices.

Simultaneous determination of the potent anti-tuberculosis regimen-Pyrazinamide, ethambutol, protionamide, clofazimine in beagle dog plasma using LC-MS/MS method coupled with 96-well format plate.

Tuberculosis is one of the top concerns in the world and acutely threatens human health. A new potent candidate regimen containing pyrazinamide (PZA), ethambutol (EMB), protionamide (PTO) and clofazimine (CFZ) was proposed by Parabolic Response Surface/Feedback System Control (FSC/PRS) system and showed excellent outcomes in vitro and vivo studies. Here, a convenient liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) method was developed for the simultaneously determination of four compounds in beagle dog plasma. The plasma samples, 50 µL for each, were pretreated by methanol on 96-well format plates and a further dilution step was designed to reduce predictable matrix effect and lessen the burden of subsequent analysis. The chromatographic separation was achieved on an Agilent SB-Aq column (4.6 mm × 150 mm, 5 µm) at 30 °C by a gradient elution within 6 min. The mobile phase was a mixture of 0.2% formic acid-5 mM ammonium acetate aqueous solution (phase A) and 0.2% formic acid methanol (phase B) with a total flow rate of 1 mL/min. The 30% of post-column eluant was injected into mass spectrometer, equipped with electrospray ionization (ESI) source under positive mode and multiple-reaction monitoring (MRM). This quantification method was proved to be satisfied in selectivity, accuracy, precision, linearity (r2 > 0.998), recovery, matrix effect and stability. Under the specialized conditions, the calibration curves ranged from 20 to 5000 ng/mL for PZA, 1 to 500 ng/mL for EMB, 1 to 500 ng/mL for PTO, and 1 to 200 ng/mL for CFZ. The quantitative accuracy was further assessed under different degrees of hemolyses in detail. This method was proved to be robust and efficient, and successfully applied to the pharmacokinetic study of the new regimen in Beagle dogs.

High-Throughput Determination of Sodium Danshensu in Beagle Dogs by the LCMS/MS Method, Employing Liquid-Liquid Extraction Based on 96-Well Format Plates.

Sodium Danshensu (sodium d-(+)-ß-(3,4-dihydroxyphenyl) lactate), one of the water-soluble ingredients in Salvia miltiorrhiza, exhibits potent relaxation of the coronary artery and anticoagulation effection. A high-throughput, rapid, and sensitive method combining liquid chromatography with electrospray ionization tandem mass spectrometry to determine the sodium danshensu in beagle dog plasma was developed and validated, using gallic acid as an internal standard (IS). Acidified plasma samples were extracted using 96-well liquid-liquid extraction, and were eluted on a CNW Athena C18 column (3 µm, 2.1 × 100 mm) by using a gradient mobile phase system of methanol and water (containing 0.2% formic acid). The mass spectrometric detection was achieved using negative ion electrospray ionization mode and monitoring the precursor→production combinations of m/z 197→135 for sodium danshensu and 169→125 for IS, in multiple reaction monitoring modes. Good linearity was achieved, and the linear range was 10-1000 ng/mL (R² > 0.996) with a quantification limit of 10 ng/mL for sodium danshensu in beagle dog plasma. The intra- and inter-day precision (RSD) ranged from 2.1% to 9.0%. The accuracy (RE) was between -8.6% and 5.7% at all quality control levels. The validated method was successfully applied to the pharmacokinetics study of sodium danshensu in beagle dog plasma after intravenous injection and oral administration of sodium danshensu.

Determination of Vancomycin in Human Serum by Cyclodextrin-Micellar Electrokinetic Capillary Chromatography (CD-MEKC) and Application for PDAP Patients.

A simple and sensitive cyclodextrin-micellar electrokinetic capillary chromatography (CD-MEKC) method with UV detection was developed and validated for the determination of vancomycin (VCM) in serum. The separation was achieved in 14 min at 25 °C with a fused-silica capillary column of 40.2 cm × 50 mm i.d. (effective length 30.2 cm) and a run buffer containing 25 mM borate buffer with 50 mM sodium dodecylsulfonate (SDS) (pH 9.5) and 2% sulfobutyl-ß-cyclodextrin (sulfobutyl-ß-CD). Under optimal conditions for biological samples, good separations with high efficiency and short analysis time were achieved. Several parameters affecting the drug separation from biological matrices were studied, including buffer types, concentrations, and pHs. The methods were validated over the range of 0.9998-99.98 µg/mL. Calibration curves of VCM also showed good linearity (r² > 0.999). Intra- and interday precisions (relative standard deviation, RSD) were less than 5.80% and 7.38%, and lower limit of quantification (LLOQ) were lower than 1.0 µg/mL. The mean recoveries ranged between 84.03% and 91.69%. The method was successfully applied for monitoring VCM concentrations in serum of patients with peritoneal dialysis-associated peritonitis (PDAP). The assay should be applicable to pharmacokinetic studies and routine therapeutic drug monitoring of this drug in serum.

Hydrodynamic characteristics of the helical flow pump.

The helical flow pump (HFP) was invented to be an ideal pump for developing the TAH and the helical flow TAH (HFTAH) using two HFPs has been developed. However, since the HFP is quite a new pump, hydrodynamic characteristics inside the pump are not clarified. To analyze hydrodynamic characteristics of the HFP, flow visualization study using the particle image velocimetry and computational fluid dynamics analysis were performed. The experimental and computational models were developed to simulate the left HFP of the HFTAH and distributions of flow velocity vectors, shear stress and pressure inside the pump were examined. In distribution of flow velocity vectors, the vortexes in the vane were observed, which indicated that the HFP has a novel and quite unique working principle in which centrifugal force rotates the fluid in the helical volutes and the fluid is transferred from the inlet to the outlet helical volutes according to the helical structure. In distribution of shear stress, the highest shear stress that was considered to be occurred by the shunt flow across the impeller was found around the entrance of the inlet helical volute. However, it was not so high to cause hemolysis. This shunt flow is thought to be improved by redesigning the inlet and outlet helical volutes. In distribution of pressure, negative pressure was found near the entrance of the inlet helical volute. However, it was not high. Negative pressure is thought to be reduced with an improvement in the design of the impeller or the vane shape.

Concept of left atrial pressure estimation using its pulsatile amplitude in the helical flow total artificial heart.

The total artificial heart (TAH) requires physiological control to respond to the metabolic demand of the body. To date, 1/R control is a single physiological control method that can control venous pressure. To realize an implantable 1/R control system, we are developing a new pressure measuring method using absolute pressure sensor. To find a method for absolute pressure sensor, which went well without calibration, concept of left atrial pressure (LAP) estimation using its pulsatile amplitude was proposed. Its possibility was investigated with two long-term survived goats whose hearts were replaced with the helical flow TAHs. In manual control condition, there existed a positive relation between mean LAP (mLAP) and normalized pulsatile amplitude (NPA). Percent systole revealed not to affect the relationship between mLAP and NPA. Dispersion was observed between different pulse rates. As for cardiac output difference (QLD) that is the difference of flow rate between systolic and diastolic phases, similar results were obtained except in low QLDs. In the 1/R control condition, relatively high correlation between mLAP and NPA could be obtained. In estimation of mLAP using the correlating function of individual goat, fairly good correlation was obtained between measured mLAP and estimated mLAP. Despite that further studies are necessary, it was demonstrated that the concept of the LAP estimation could be possible.

Relation between left atrial pressure and the corresponding pulse pressure in the helical flow total artificial heart.

The present control method used in our helical flow total artificial heart (HFTAH) would only need four parameters. Nowadays, gauge pressure sensors are being used to obtain the pressure needed for control parameters. Nevertheless, there are also many following problems such as calibration, maintenance, offset drift and infection due to the skin-penetrative lines for the usage of gauge pressure sensor. Therefore, it is preferable to find another substitutional way instead of the gauge sensor to measure the pressure. In addition, with an eye to completing an implantable HFTAH, we would like to do without any lines through the experiment animal. Therefore, it was confirmed in this study that whether there is a relation between the left atrial pressure (LAP) and its pulse pressure (amplitude). Subsequently the mean value of LAP and its amplitude were quantified. There are two methods used in this study to process the data. Method one, frequency spectrum analysis, is to quantify the signals by getting the absolute value of amplitude for a fixed heartbeat analysis. Method two, by using the synchronous detection method, it is postulated to be more applicable to variant heartbeat data with 1/R control. By the relation of LAP and the pulse pressure acquired in the above two methods, as long as the amplitude of LAP is known by the absolute pressure sensor, it's able to obtain the mean value of LAP (for it suggests a linear relation). Therefore the characteristic could substitute one of the control parameter (that is the LAP), and the other three parameters will be acquired by estimation thus it doesn't need to measure them additionally. Consequently, it is expected that acquiring LAP by absolute pressure sensor for one of the control parameters could attain to an implantable HFTAH.

Pulsatile driving of the helical flow pump.

The helical flow pump (HFP) is newly developed blood pomp for total artificial heart (TAH). HFP can work with lower rotational speed than axial and centrifugal blood pump. It can be seen reasonable feature to generate pulsatile flow because high response performance can be realized. In this article, pulsatility of HFP was evaluated using mock circulation loop. Pulsatile flow was generated by modulating the rotational speed in various amplitude and heart rate. In the experiment, relationship between Pump flow, pump head, rotational speed amplitude, heart rate and power consumption is evaluated. As the result, complete pulsatile flow with mean flow rate of 5 L/min and mean pressure head of 100 mmHg can be obtained at ± 500 rpm with mean rotational speed of 1378 to 1398 rpm in hart rate from 60 to 120. Flow profiles which are non-pulsatile, quasi-pulsatile or complete flow can be adjusted arbitrarily. Therefore, HFP has excellent pulsatility and control flexibility of flow profile.