Precise control of liposome size using characteristic time depends on solvent type and membrane properties.

Controlling the sizes of liposomes is critical in drug delivery systems because it directly influences their cellular uptake, transportation, and accumulation behavior. Although hydrodynamic focusing has frequently been employed when synthesizing nano-sized liposomes, little is known regarding how flow characteristics determine liposome formation. Here, various sizes of homogeneous liposomes (50-400 nm) were prepared according to flow rate ratios in two solvents, ethanol, and isopropyl alcohol (IPA). Relatively small liposomes formed in ethanol due to its low viscosity and high diffusivity, whereas larger, more poly-dispersed liposomes formed when using IPA as a solvent. This difference was investigated via numerical simulations using the characteristic time factor to predict the liposome size; this approach was also used to examine the flow characteristics inside the microfluidic channel. In case of the liposomes, the membrane rigidity also has a critical role in determining their size. The increased viscosity and packing density of the membrane by addition of cholesterol confirmed by fluorescence anisotropy and polarity lead to increase in liposome size (40-530 nm). However, the interposition of short-chain lipids de-aligned the bilayer membrane, leading to its degradation; this decreased the liposome size. Adding short-chain lipids linearly decreased the liposome size (130-230 nm), but at a shallower gradient than that of cholesterol. This analytical study expands the understanding of microfluidic environment in the liposome synthesis by offering design parameters and their relation to the size of liposomes.

Controlled rate slow freezing with lyoprotective agent to retain the integrity of lipid nanovesicles during lyophilization.

We designed a novel lyophilization method using controlled rate slow freezing (CSF) with lyoprotective agent (LPA) to achieve intact lipid nanovesicles after lyophilization. During the freezing step, LPA prevented water supercooling, and the freezing rate was controlled by CSF. Regulating the freezing rate by various liquid media was a crucial determinant of membrane disruption, and isopropanol (freezing rate of 0.933 °C/min) was the optimal medium for the CSF system. Lyophilized lipid nanovesicle using both CSF and LPA retained 92.9% of the core material and had uniform size distributions (Z-average diameter = 133.4 nm, polydispersity index = 0.144), similar to intact vesicles (120.7 nm and 0.159, respectively), after rehydration. Only lyophilized lipid nanovesicle using both CSF and LPA showed no changes in membrane fluidity and polarity. This lyophilization method can be applied to improve storage stability of lipid nanocarriers encapsulating drugs while retaining their original activity.

A Simple Method for Continuous Synthesis of Bicelles in Microfluidic Systems.

Bicelle has great potential for drug delivery systems due to its small size and biocompatibility. The conventional method of bicelle preparation contains a long process and harsh conditions, which limit its feasibility and damage the biological substances. For these reasons, a continuous manufacturing method in mild conditions has been demanded. Here, we propose a novel method for DMPC/DHPC bicelle synthesis based on a microfluidic device without heating and freezing processes. Bicelles were successfully prepared using this continuous method, which was identified by the physicochemical properties and morphologies of the synthesized assemblies. Experimental and analytical studies confirm that there is critical lipid concentration and critical mixing time for bicelle synthesis in this microfluidic system. Furthermore, a linear relation between the actual composition of bicelle and initial lipid ratio is deduced, and this enables the size of bicelles to be controlled.

Precise Microfluidic Luminescent Sensor Platform with Controlled Injection System.

Efforts have been devoted to screening various prevalent diseases, such as severe acute respiratory syndrome (SARS) and coronavirus disease 2019 (COVID-19). Real-time polymerase chain reaction (RT-PCR), which is currently the most widely used, has high accuracy, but it requires several facilities and takes a relatively long time to check; so, new testing technology is necessary for a higher test efficiency. A chemiluminescence (CL) sensor is a relatively simple device and suitable as an alternative because it can detect very precise specimens. However, in measurements via CL, the quantitative formulation of reagents that cause color development is important. In the case of mixing using micropipettes, precise analysis is possible, but this technique is limited by uncontrollable errors or deviations in detection amounts. In addition, in using a microfluidic chip to increase field applicability, a syringe pump or other quantification injection tools are required, so problems must be overcome for practical use. Therefore, in this study, a microchip was designed and manufactured to supply a sample of a certain volume by simply blowing air and injecting a sample into the chamber. By utilizing the luminescence reaction of luminol, CuSO4 and H2O2 the performance of the prepared chip was confirmed, and the desired amount of the sample could be injected with a simple device with an error rate of 2% or less. For feasible applications, an experiment was performed to quantitatively analyze thrombin, a biomarker of heart disease. Results demonstrated that biomarkers could be more precisely detected using the proposed microchips than using micropipettes.

Analysis of the Binding of Analyte-Receptor in a Micro-Fluidic Channel for a Biosensor based on Brownian Motion.

This study experimentally analyses the binding characteristics of analytes mixed in liquid samples flowing along a micro-channel to the receptor fixed on the wall of the micro-channel to provide design tools and data for a microfluidic-based biosensor. The binding or detection characteristics are analyzed experimentally by counting the number of analytes bound to the receptor, with sample analyte concentration, sample flow rate, and the position of the receptor along the micro-channel length as the main variables. A mathematical model is also proposed to predict the number of analytes transported and bound to the receptor based on a probability density function for Brownian motion. The coefficient in the mathematical model is obtained by using a dimensionless mathematical model and the experimental results. The coefficient remains valid for all different conditions of the sample analyte concentration, flow rate, and the position of the receptor, which implies the possibility of deriving a generalized model. Based on the mathematical model derived from mathematical and experimental analysis on the detection characteristics of the microfluidic-based biosensor depending on previously mentioned variables and the height of the micro-channel, this study suggests a design for a microfluidic-based biosensor by predicting the binding efficiency according to the channel height. The results show the binding efficiency increases as the flow rate decreases and as the receptor is placed closer to the sample-injecting inlet, but is unaffected by sample concentration.

Pharmacokinetics, Safety, and Tolerability of Tedizolid Phosphate After Single-dose Administration in Healthy Korean Male Subjects.

PURPOSE: Tedizolid phosphate is a next-generation oxazolidinone prodrug that is transformed into the active moiety tedizolid. Its indication is acute bacterial skin and skin structure infections caused by gram-positive species, including methicillin-resistant Staphylococcus aureus. Although tedizolid phosphate has been marketed in Korea, no data on the pharmacokinetic (PK) properties or tolerability of tedizolid phosphate in Korean subjects are available. This study was designed to evaluate the PK properties, oral bioavailability, and tolerability with a single-dose oral and intravenous administration of tedizolid phosphate in healthy Korean male subjects. METHODS: A block-randomized, double-blind, placebo-controlled, single-dose study was conducted in 3 groups (200, 400, and 600 mg; 10 subjects in each group). In the second part of the study, subjects from the 200-mg group received administration orally and intravenously (1-hour infusion) via 2-way crossover for the evaluation of absolute bioavailability. There was a 7-day washout period between treatments in the absolute bioavailability part of the study. Serial blood samples for PK analysis were collected for up to 72 hours. Tolerability was assessed by analysis of adverse events. FINDINGS: Thirty healthy Korean subjects completed the study and were included in the PK and tolerability analyses. Tedizolid phosphate was rapidly converted into tedizolid. After a single oral dose, the Tmax of tedizolid was observed to be 1.5 to 2.5 hours, and the plasma concentration-time curve of tedizolid showed a 2-phase elimination pattern, with a half-life of ~11 hours. Dose-dependent increases were observed in the AUClast value (29,441-78,062 µg · h/L) and in the Cmax value (2679-6980 µg/L) with the administration of tedizolid phosphate 200 to 600 mg PO. The absolute bioavailability of tedizolid was 95.2% (90% CI, 92.7%-97.8%) in the 200-mg administration group. There were no serious adverse events or clinically significant changes in the tolerability assessment. IMPLICATIONS: Tedizolid phosphate at doses of up to 600 mg was well-tolerated in these healthy Korean male subjects. Tedizolid shows dose linearity with oral administration, and no dose adjustment of tedizolid phosphate 200 mg would be needed when switching administration routes. ClinicalTrials.gov identifier: NCT02097043.

Discovery and SAR of N-(1-((substituted piperidin-4-yl)methyl)-3-methoxypiperidin-4-yl)-2-methoxybenzamide derivatives: 5-Hydroxytryptamine receptor 4 agonist as a potent prokinetic agent.

A series of novel benzamide derivatives, altering the 4-fluorophenylalkyl moiety in cisapride, were synthesized as 5-HT4 receptor agonists, and SAR of these analogs was examined on in vitro and in vivo prokinetic activities. These compounds were synthesized for high 5-HT4 receptor binding affinities and low hERG affinities. Several types of analogs were obtained and screened for 5-HT4 binding, hERG blocking, agonism, and gastric emptying assessment. Among the analogues, compound 23g showed promising results compared with the other analogs with respect to gastric emptying rates in rats. Therefore, we suggest that it may be a clinical candidate for the development of a potent prokinetic agent to treat GI disorders.

In vitro activity of tedizolid against gram-positive bacteria in patients with skin and skin structure infections and hospital-acquired pneumonia: a Korean multicenter study.

We compared the activities of tedizolid to those of linezolid and other commonly used antimicrobial agents against gram-positive cocci recovered from patients with skin and skin structure infections (SSSIs) and hospital-acquired pneumonia (HAP) in Korean hospitals. Gram-positive isolates were collected from 356 patients with SSSIs and 144 patients with HAP at eight hospitals in Korea from 2011 to 2014. SSSIs included impetigo, cellulitis, erysipelas, furuncles, abscesses, and infected burns. Antimicrobial susceptibility was tested by using the CLSI agar dilution method. All of the gram-positive isolates were inhibited by ≤1 µg/mL tedizolid. The minimum inhibitory concentration [MIC]90 of tedizolid was 0.5 µg/mL for methicillin-resistant Staphylococcus aureus, which was 4-fold lower than that of linezolid. Tedizolid may become a useful option for the treatment of SSSIs and HAP caused by gram-positive bacteria.

Pharmacological profile of DA-6886, a novel 5-HT4 receptor agonist to accelerate colonic motor activity in mice.

DA-6886, the gastrointestinal prokinetic benzamide derivative is a novel 5-HT4 receptor agonist being developed for the treatment of constipation-predominant irritable bowel syndrome (IBS-C). The purpose of this study was to characterize in vitro and in vivo pharmacological profile of DA-6886. We used various receptor binding assay, cAMP accumulation assay, organ bath experiment and colonic transit assay in normal and chemically constipated mice. DA-6886 exhibited high affinity and selectivity to human 5-HT4 receptor splice variants, with mean pKi of 7.1, 7.5, 7.9 for the human 5-HT4a, 5-HT4b and 5-HT4d, respectively. By contrast, DA-6886 did not show significant affinity for several receptors including dopamine D2 receptor, other 5-HT receptors except for 5-HT2B receptor (pKi value of 6.2). The affinity for 5-HT4 receptor was translated into functional agonist activity in Cos-7 cells expressing 5-HT4 receptor splice variants. Furthermore, DA-6886 induced relaxation of the rat oesophagus preparation (pEC50 value of 7.4) in a 5-HT4 receptor antagonist-sensitive manner. The evaluation of DA-6886 in CHO cells expressing hERG channels revealed that it inhibited hERG channel current with an pIC50 value of 4.3, indicating that the compound was 1000-fold more selective for the 5-HT4 receptor over hERG channels. In the normal ICR mice, oral administration of DA-6886 (0.4 and 2mg/kg) resulted in marked stimulation of colonic transit. Furthermore, in the loperamide-induced constipation mouse model, 2mg/kg of DA-6886 significantly improved the delay of colonic transit, similar to 10mg/kg of tegaserod. Taken together, DA-6886 is a highly potent and selective 5-HT4 receptor agonist to accelerate colonic transit in mice, which might be therapeutic agent having a favorable safety profile in the treatment of gastrointestinal motor disorders such as IBS-C and chronic constipation.

Activity of tedizolid phosphate (TR-701) in murine models of infection with penicillin-resistant and penicillin-sensitive Streptococcus pneumoniae.

The in vitro activity of tedizolid (previously known as torezolid, TR-700) against penicillin-resistant Streptococcus pneumoniae (PRSP) clinical isolates and the in vivo efficacy of tedizolid phosphate (torezolid phosphate, TR-701) in murine models of PRSP systemic infection and penicillin-susceptible S. pneumoniae (PSSP) pneumonia were examined using linezolid as a comparator. The MIC(90) against 28 PRSP isolates was 0.25 µg/ml for tedizolid, whereas it was 1 µg/ml for linezolid. In mice infected systemically with a lethal inoculum of PRSP 1 h prior to a single administration of either antimicrobial, oral tedizolid phosphate was equipotent to linezolid (1 isolate) to 2-fold more potent than linezolid (3 isolates) for survival at day 7, with tedizolid phosphate 50% effective dose (ED(50)) values ranging from 3.19 to 11.53 mg/kg of body weight/day. In the PSSP pneumonia model, the ED(50) for survival at day 15 was 2.80 mg/kg/day for oral tedizolid phosphate, whereas it was 8.09 mg/kg/day for oral linezolid following 48 h of treatment with either agent. At equivalent doses (10 mg/kg once daily tedizolid phosphate or 5 mg/kg twice daily linezolid), pneumococcal titers in the lungs at 52 h postinfection were approximately 3 orders of magnitude lower with tedizolid phosphate treatment than with linezolid treatment or no treatment. Lung histopathology showed less inflammatory cell invasion into alveolar spaces in mice treated with tedizolid phosphate than in untreated or linezolid-treated mice. These results demonstrate that tedizolid phosphate is effective in murine models of PRSP systemic infection and PSSP pneumonia.

Therapeutic effect of a novel oxazolidinone, DA-7867, in BALB/c mice infected with Nocardia brasiliensis.

BACKGROUND: Mycetoma is a chronic infectious disease of tropical and subtropical countries. It is produced by true fungi and actinobacteria. In México, Nocardia brasiliensis is the main causative agent of mycetoma, producing about 86% of the cases; the gold standard for the therapy of mycetoma by N. brasiliensis is the use of sulfonamides which give a 70% cure rate. The addition of amikacin to this regime increases to 95% the cure rate; however, the patients have to be monitored for creatinine clearance and audiometry studies because of the potential development of side effects. Because of that it is important to search for new active compounds. In the present work, we evaluated the in vivo effect of DA-7867, an experimental oxazolidinone, on the development of experimental mycetomas by N. brasiliensis in BALB/c mice. METHODOLOGY/PRINCIPAL FINDINGS: In order to determine the optimal dose utilized to apply to the animals, we first determined by HPLC the plasma levels using several concentrations of the compounds. Based on these results, we used 10 and 25 mg/kg subcutaneously every 24 hr; DA-7867 was also supplied in the drinking water at a calculated dose of 25 mg/kg. As a control we utilized linezolid at 25 mg/kg, a compound active in murine and human infections, three times a day. The mice were infected in the right footpad with a young culture of N. brasiliensis HUJEG-1, and one week later we started the application of the antimicrobials for six more weeks. After that we compared the development of lesions in the groups injected with saline solution or with the antimicrobials; the results were analyzed by the variance ANOVA test. DA-7867 was able to reduce the production of lesions at 25 mg/kg, when given either subcutaneously or in the drinking water. CONCLUSIONS/SIGNIFICANCE: The experimental oxazolidinone DA-7867 is active in vivo against N. brasiliensis, which opens the possibility of using this drug once it is accepted for human application. Since oxazolidinones seem to be active against a wide spectrum of actinobacteria, it is possible they could be used in human cases of mycetoma by other actinomycetales, such as Streptomyces somaliensis, highly prevalent in Sudan, or Actinomadura madurae and A. pelletieri, which are commonly observed in Africa and India.

Efficacy of DA-7218, a new oxazolidinone prodrug, in the treatment of experimental actinomycetoma produced by Nocardia brasiliensis.

Two recently synthesized oxazolidinones: (R)-3-(4-(2-(2-methyltetrazol-5-yl)-pyridin-5-yl)-3-fluorophenyl)-5-hydroxymethyloxazolidin-2-one (DA-7157) and its corresponding pro-drug (R)-3-(4-(2-(2-methyltetrazol-5-yl)-pyridin-5-yl)-3-fluorophenyl)-2-oxo-5-oxazolidinyl) methyl disodium phosphate (DA-7218), have shown very good activity against several Gram positive bacteria, including Nocardia and Mycobacterium. In the present work we evaluated the therapeutic in vivo effects of DA-7218 on Nocardia brasiliensis. We first determined the plasma concentration of the prodrug in BALB/c mice using several doses and then tested its activity in an in vivo experimental actinomycetoma murine model. At the end of treatment, there was a statistically significant difference between the three drug receiving groups (25, 12.5 and 5 mg/kg) and the control group(saline solution) (p=0.001), proving that DA-7218 is effective for the treatment of experimental murine actinomycetoma. This compound could be a potential option for patients affected with mycetoma by Nocardia brasiliensis.