CHO-produced RBD-Fc subunit vaccines with alternative adjuvants generate immune responses against SARS-CoV-2.

Subunit vaccines feature critical advantages over other vaccine platforms such as stability, price, and minimal adverse effects. To maximize immunological protection of subunit vaccines, adjuvants are considered as main components that are formulated within the subunit vaccine. They can modulate adverse effects and enhance immune outcomes. However, the most suitable formulation providing the best immunological outcomes and safety are still under investigation. In this report, we combined recombinant RBD with human IgG1 Fc to create an RBD dimer. This fusion protein was expressed in CHO and formulated with alternative adjuvants with different immune activation including Montanide ISA51, Poly (I:C), and MPLA/Quil-A® as potential vaccine candidate formulations. Using the murine model, a potent induction of anti-RBD IgG antibodies in immunized mice sera were observed. IgG subclass analyses (IgG1/IgG2a) illustrated that all adjuvanted formulations could stimulate both Th1 and Th2-type immune responses in particular Poly (I:C) and MPLA/Quil-A®, eliciting greater balance. In addition, Montanide ISA51-formulated RBD-Fc vaccination provided a promising level of neutralizing antibodies against live wild-type SARS-CoV-2 in vitro followed by Poly (I:C) and MPLA/Quil-A®, respectively. Also, mice sera from adjuvanted formulations could strongly inhibit RBD:ACE2 interaction. This study offers immunogenicity profiles, forecasted safety based on Vaccine-associated enhanced disease (VAED) caused by Th1-skewed immunity, and neutralizing antibody analysis of candidates of RBD-Fc-based subunit vaccine formulations to obtain an alternative subunit vaccine formulation against SARS-CoV-2.

Quantification of CYP3A and Drug Transporters Activity in Healthy Young, Healthy Elderly and Chronic Kidney Disease Elderly Patients by a Microdose Cocktail Approach.

Background: Ageing and chronic kidney disease (CKD) affect pharmacokinetic (PK) parameters. Since mechanisms are related and remain unclear, cytochrome P450 (CYP) 3A and drug transporter activities were investigated in the elderly with or without CKD and compared to healthy adults using a microdose cocktail. Methods: Healthy young participants (n = 20), healthy elderly participants (n = 16) and elderly patients with CKD (n = 17) received, in study period 1, a single dose of microdose cocktail probe containing 30 µg midazolam, 750 µg dabigatran etexilate, 100 µg atorvastatin, 10 µg pitavastatin, and 50 µg rosuvastatin. After a 14-day wash-out period, healthy young participants continued to study period 2 with the microdose cocktail plus rifampicin. PK parameters including area under the plasma concentration-time curve (AUC), maximum plasma drug concentration (Cmax), and half-life were estimated before making pairwise comparisons of geometric mean ratios (GMR) between groups. Results: AUC and Cmax GMR (95% confidence interval; CI) of midazolam, a CYP3A probe substrate, were increased 2.30 (1.70-3.09) and 2.90 (2.16-3.88) fold in healthy elderly and elderly patients with CKD, respectively, together with a prolonged half-life. AUC and Cmax GMR (95%CI) of atorvastatin, another CYP3A substrate, was increased 2.14 (1.52-3.02) fold in healthy elderly and 4.15 (2.98-5.79) fold in elderly patients with CKD, indicating decreased CYP3A activity related to ageing. Associated AUC changes in the probe drug whose activity could be modified by intestinal P-glycoprotein (P-gp) activity, dabigatran etexilate, were observed in patients with CKD. However, whether the activity of pitavastatin and rosuvastatin is modified by organic anion transporting polypeptide 1B (OATP1B) and of breast cancer resistance protein (BCRP), respectively, in elderly participants with or without CKD was inconclusive. Conclusions: CYP3A activity is reduced in ageing. Intestinal P-gp function might be affected by CKD, but further confirmation appears warranted. Clinical Trial Registration:http://www.thaiclinicaltrials.org/ (TCTR 20180312002 registered on March 07, 2018).

Genome analysis and optimization of γ-aminobutyric acid (GABA) production by lactic acid bacteria from plant materials.

Gamma-aminobutyric acid (GABA) plays a key role as an inhibitory neurotransmitter in the mammalian sympathetic nervous system and has other health benefits. Molecular characterization, genome analysis, and optimization were investigated to improve GABA production of a selected strain of lactic acid bacteria. Eleven isolates from plant materials were screened for GABA productivity and were identified based on phenotypic and genotypic characteristics. The most potent strain was chosen for genome analysis and GABA production optimization using the response surface methodology (RSM). Each of the two strains was closely related to Lactobacillus plantarum, Lactobacillus brevis, Weissella cibaria, Leuconostoc pseudomesenteroides while each strain was similar to Lactobacillus pentosus, Enterococcus lactis, and Leuconostoc mesenteroides. They produced GABA ranging from 0.036 ± 0.000 to 17.315 ± 0.171 g/L at 72 h-cultivation. Among them, the most potent strain, SL9-6, showed the highest GABA production (17.315 g/L) when cultivated with 10% (v/v) inoculum for 48 h. The draft genome sequence of strain SL9-6 exhibited 96.90% average nucleotide identity value and 74.50% digital DNA-DNA hybridization to Lactobacillus brevis NCTC 13768T. This strain contained a glutamate decarboxylase gene system (gadA, gadB, and gadC). Optimal culture conditions were determined as 40.00 g/L glucose, 49.90 g/L monosodium glutamate, pH 5.94, and 31.10°C by RSM, giving maximum GABA production of 32.48 g/L. Results from RSM also indicated that monosodium glutamate concentration, pH, and temperature were significant variables. GABA production significantly improved here could promise further application of strain SL9-6.

Development and Qualification of a Physiologically Based Pharmacokinetic Model of Finasteride and Minoxidil Following Scalp Application.

In this study, we aimed to develop and qualify a PBPK model for scalp application using two drugs with marked differences in physicochemical properties and PK profiles. The parameters related to scalp physiology, drug PK, and formulations were incorporated into a Multi-Phase and Multi-Layer (MPML) Mechanistic Dermal Absorption (MechDermA) model within the Simcyp® Simulator V17. The finasteride PBPK model was linked to its effect on dihydrotestosterone (DHT) levels in plasma and scalp using an indirect response model. Predicted PK (and PD for finasteride) profiles and parameters were compared against the clinically reported data and justified by visual predictive checks and two-fold error criteria for model verification. The PBPK/PD model for finasteride reasonably demonstrated an ability to predict its respective PK and PD profiles, and parameters following scalp application under various clinical scenarios. Using the same scalp physiological input parameters, the minoxidil PBPK model was then developed and satisfactorily qualified with independent clinical datasets. Collectively, these results suggested that the established PBPK model may have broader utility for other topical formulations intended for scalp application.

Asiaticoside but not its aglycone exhibits neuritogenicity through TrkA receptor signaling: a bridge between ERK1/2-CREB and Akt-GSK3ß/RhoA.

The neuritogenicity and the neuroregenerative potential of asiaticoside (AS) and its aglycone, asiatic acid (AA), has been generally reported. We recently identified the participation of extracellular signal-regulated protein kinases 1/2 (ERK1/2) and protein kinase B (Akt) in the neuritogenic mechanism of AS and AA. In this study, we further investigated the possible upstream target molecule and the associated downstream signaling of both triterpenoids in mouse neuroblastoma Neuro-2a cells. Our immunoblotting and immunofluorescence assays revealed that either AS or AA exerted neurite extension activity through inhibitory effect on glycogen synthase kinase 3ß (GSK3ß) and Ras homolog gene family member A (RhoA). AS appeared significantly more potent in promoting neurite elongation than AA, and concurrently expressed a higher degree of inhibition on GSK3ß and RhoA activations. The mediation of GSK3ß and RhoA activities in AS-treated cells involved Akt signaling. Moreover, when using GW441756, a specific tropomyosin receptor kinase A (TrkA) receptor signaling inhibitor, the ERK1/2 and Akt phosphorylation, the inhibitory effects on GSK3ß and RhoA and the neurite outgrowth induced by AS, but not AA, were totally suppressed. In conclusion, our findings supported the different upstream regulators of AS and AA in promoting neuritogenicity in Neuro-2a cells. Although both AS and AA could enhance neurite elongation through the suppression of GSK3ß and RhoA activities, only AS could modulate the effect through TrkA receptor signaling.

Interspecies differences in stability kinetics and plasma esterases involved in hydrolytic activation of curcumin diethyl disuccinate, a prodrug of curcumin.

The investigation of in vitro plasma metabolism of ester prodrugs is an important part of in vitro ADME assays during preclinical drug development. Here, we show that the in vitro metabolism including plasma stability and metabolizing enzymes of curcumin diethyl disuccinate (CDD), an ester prodrug of curcumin, in dog and human plasma are similar but markedly different from those in rat plasma. HPLC and nonlinear regression analyses indicated that the hydrolysis of CDD in plasma followed a consecutive pseudo-first order reaction. The rapid hydrolytic cleavage of CDD in rat, dog, and human plasma was accelerated by plasma esterases in the following order: rat ≫ human > dog. LC-Q-TOF/MS analysis showed that the cleavage of ester bonds of CDD is preferential at the phenolic ester. Monoethylsuccinyl curcumin is the only intermediate metabolite found in plasma metabolism of CDD in all tested species. Further investigation using different esterase inhibitors revealed that carboxylesterase is the major enzyme involved in the hydrolysis of CDD in rats while multiple plasma esterases play a role in dogs and humans. Thus, the difference in the hydrolysis rates and the metabolizing enzymes of CDD metabolism in rat, dog and human plasma observed here is of benefit to further in vivo studies and provides a rationale for designing ester prodrugs of CUR with esterase-specific bioactivation.

Differences in Neuritogenic Activity and Signaling Activation of Madecassoside, Asiaticoside, and Their Aglycones in Neuro-2a cells.

Madecassoside (MS) and asiaticoside (AS) along with their aglycones, madecassic acid (MA) and asiatic acid (AA), are considered the major neuroactive triterpenoid constituents of Centella asiatica. In this study, we aimed to compare MS, AS, MA, and AA for their neurite outgrowth activities and mechanisms in Neuro-2a cells. Immunofluorescent cell staining showed MS and AS significantly increased the percentage of neurite-bearing cells (%NBC) and the neurite length with higher potency than MA and AA. The triterpenoid glycosides induced sustained extracellular signal-regulated protein kinases 1 and 2 (ERK1/2) phosphorylation, while their aglycones activated only transient signaling of ERK1/2. Suppression of ERK1/2 activation significantly abolished not only cAMP response element-binding protein (CREB) phosphorylation but also the increment of %NBC and neurite length in MS- and AS-treated cells. Inhibition of ERK phosphorylation did not produce similar blockage of CREB activation and neurite outgrowth in MA- and AA-treated cells. On the other hand, inactivation of protein kinase B (Akt) resulted in a suppression of neurite lengthening in all studied triterpenoids. This is the first study discerning the different signaling pathways of neurite outgrowth activity induced by C. asiatica triterpenoid glycosides and aglycones. Neurite outgrowth activity of the glycosides MS and AS was found to involve the activation of sustained ERK phosphorylation leading to CREB activation, while ERK activation was not associated with MA- and AA-induced neurite outgrowth. In addition, Akt activation was evident to be more involved in neurite elongation process.

Simultaneous determination of curcumin diethyl disuccinate and its active metabolite curcumin in rat plasma by LC-MS/MS: Application of esterase inhibitors in the stabilization of an ester-containing prodrug.

Four esterase inhibitors, ethylenediamine tetraacetic acid disodium (Na2EDTA), sodium fluoride (NaF), bis(4-nitrophenyl) phosphate (BNPP) and phenylmethanesulfonyl fluoride (PMSF), were evaluated for their inhibitory effects on enzymatic hydrolysis of labile phenolate esters in curcumin diethyl disuccinate (CDD), a prodrug of curcumin (CUR), in rat plasma. BNPP and PMSF at 10mM exhibited stabilization by preventing degradation of CDD. BNPP at a final concentration of 10mM was subsequently selected to prevent ex vivo metabolism of CDD throughout LC-MS/MS analysis of CDD and CUR in rat plasma. A simple protein precipitation technique using acetonitrile as a precipitating agent was used to extract CDD, CUR and dimethylcurcumin (DMC), an internal standard, from rat plasma. Chromatographic separation was performed on a Halo C8 column (4.6×50mm, 2.7µm) using an isocratic mobile phase containing acetonitrile-0.2% formic acid in water (73:27v/v) with a flow rate of 0.4mLmin(-1). An AB SCIEX QTRAP(®) 6500 mass spectrometer was operated using a positive ion electrospray mode for ionization and detection of analytes and internal standard. Calibration curves for CDD and CUR were established using 50µL of rat plasma over the concentration range of 1-500ngmL(-1). The developed method was fully validated according to US Food and Drug Administration (FDA) guidelines for selectivity, sensitivity, linearity, accuracy, precision, dilution integrity, recovery, matrix effect, and stability. The validated method was applied to evaluate the pharmacokinetics of CDD and CUR in rats after a single intravenous dose of 40mgkg(-1). The method using BNPP as an esterase inhibitor was successful in determining the remaining CDD in rat plasma. The pharmacokinetic results indicate that CDD in rats is converted instantaneously to CUR after intravenous administration and a higher CUR plasma concentration at 5min is achieved in comparison with direct intravenous injection of CUR.

Pharmacokinetics of Compound D, the Major Bioactive Component of Zingiber cassumunar, in Rats.

Rhizomes of Zingiber cassumunar have been used for many years in traditional Thai medicine as an anti-inflammatory agent. The major bioactive component of this plant is Compound D [E-4-(3', 4'-dimethoxyphenyl)but-3-en-1-ol], which is a strong smooth muscle relaxant, and has antihistamine and anti-inflammatory actions. There is, however, incomplete information available for the pharmacokinetics of Compound D in mammals. In this study, we examined the pharmacokinetic profiles of Compound D in male Wistar rats. A standardized extract of Z. cassumunar containing 4 % w/w Compound D was administered intravenously at 25 mg/kg or by oral gavage at 25, 75, or 250 mg/kg to Wistar rats. Blood, tissues, urine, and feces were collected from 0 to 48 h after dosing and the level of Compound D was determined by liquid chromatography-tandem mass spectrometry. The concentration of Compound D ranged from 10-100 µg/L, reached a maximum approximately 0.15 h after oral dosing. Compound D exhibited an excellent tissue to plasma ratio, ranging from 1- to 1000 in several organs at 1-4 h after oral dosing. Less than 1 % of unchanged Compound D was excreted in the urine and feces. Further studies on tissue uptake and metabolite identification are required to obtain complete pharmacokinetic information and to develop appropriate dosing strategies of Compound D and the standardized extract of Z. cassumunar.

Canarosine: a new guanidine alkaloid from Canavalia rosea with inhibitory activity on dopamine D1 receptors.

A new acyclic guanidine alkaloid, canarosine (1), together with five known compounds, beta-sitosterol (2), stigmasterol (3), daucosterol (4), epi-inositol 6-O-methyl ether (5), and rutin (6), were isolated from the aerial parts of Canavalia rosea. Their structures were established on the basis of their spectroscopic data. In the radioligand receptor binding assay, canarosine (1), at a concentration of 100 microg/ml, caused 91% inhibition of the dopamine D1 receptor binding with an IC50 value of 39.4 +/- 5.8 microM.

Influence of incorporation methods on partitioning behavior of lipophilic drugs into various phases of a parenteral lipid emulsion.

The purpose of this study was to investigate the effect of drug incorporation methods on the partitioning behavior of lipophilic drugs in parenteral lipid emulsions. Four lipophilic benzodiazepines, alprazolam, clonazepam, diazepam, and lorazepam, were used as model drugs. Two methods were used to incorporate drugs into an emulsion: dissolving the compound in the oil phase prior to emulsification (de novo emulsification), and directly adding a concentrated solution of drug in a solubilizer to the emulsion base (extemporaneous addition). Based on the molecular structures and determination of the oil and aqueous solubilities and the partition coefficients of the drugs, the lipophilicity was ranked as diazepam > clonazepam > lorazepam > alprazolam. Ultracentrifugation was used to separate the emulsion into four phases, the oil phase, the phospholipid-rich phase, the aqueous phase and the mesophase, and the drug content in each phase was determined. Partitioning of diazepam, which has the highest lipophilicity and oil solubility among the four drugs, was unaffected by the drug incorporation method, with both methods giving a high proportion of drug in the inner oil phase and the phospholipid-rich phase, compared to the aqueous phase and mesophase. Partitioning of the less lipophilic drugs (alprazolam, clonazepam, and lorazepam) in the phases of the emulsion system was dependent on the method of incorporation and the drug solubility properties. Emulsions of the three drugs prepared by de novo emulsification exhibited higher drug localization in the phospholipid-rich phase compared to those made by extemporaneous addition. With the latter method, the drugs tended to localize in the outer aqueous phase and mesophase, with less deposition in the phospholipid-rich phase and no partitioning into the inner oil phase.

Preparation and characterization of propylthiouracil niosomes.

Propylthiouracil, a lyophobic drug with an antiproliferative activity, was formulated into niosomes using various classes of nonionic surfactants. Feasibility of vesicle formation by the sonication method was evaluated. Size and size distribution was measured by laser diffraction. Entrapment and drug release over 24 h were monitored by UV spectrophotometric method at 275 nm. The results revealed that niosomes readily formed from various compositions of nonionic surfactant and cholesterol, with or without a stabilizer. Entrapment of PTU in niosomes depended on bilayer composition. The release of PTU from all niosomal formulations studied was retarded and followed the first-order kinetics. Degree of slow release had a negative correlation with drug entrapment. The release rate also depended on the physical state of the bilayer. The results of this study indicate that PTU niosomes were able to control the release of PTU and might be of value to develop further into topical formulations.

The influence of physicochemical properties of preservative compounds on their distribution into various phases of oil in water submicron emulsion.

Phospholipids--stabilized submicron emulsions require the addition of preservatives to destroy or inhibit the growth of microorganisms when these preparations are non-sterile products or when packed in multi-dose containers. This study examined the distribution of four paraben esters--methylparaben, ethylparaben, propylparaben, and butylparaben--that were added into submicron emulsions by de novo emulsification. The distribution of preservative compounds among different phases was determined after separation of submicron emulsions by ultracentrifugation. The compounds with higher lipophilicity were concentrated in the oil phase and phospholipids-rich phase, leading to an increase in oil droplet size. However, the effect of the paraben distribution on zeta potential and pH of emulsion fluctuated depending on the type of phospholipid used. The lower lipophilic compounds were mostly found in the aqueous phase and mesophase. These results signify the possibility that the chemical structures and lipophilicity of preservative compounds affected their distribution in phospholipid-stabilized submicron emulsions. We conclude that the higher concentration of preservatives or their combination may be required for efficient preservation of submicron emulsion products.