Rat Pharmacokinetics and In Vitro Metabolite Identification of KM-819, a Parkinson's Disease Candidate, Using LC-MS/MS and LC-HRMS.

FAF1 (FAS-associated factor 1) is involved in the activation of Fas cell surface death receptors and plays a role in apoptosis and necrosis. In patients with Parkinson's disease, FAF1 is overexpressed in dopaminergic neurons in the substantia nigra. KM-819, an FAF1 inhibitor, has shown potential for preventing dopaminergic neuronal cell death, promoting the degradation of α-synuclein and preventing its accumulation. This study aimed to develop and validate a quantitative analytical method for determining KM-819 levels in rat plasma using liquid chromatography-tandem mass spectrometry. This method was then applied to pharmacokinetic (PK) studies in rats. The metabolic stability of KM-819 was assessed in rat, dog, and human hepatocytes. In vitro metabolite identification and metabolic pathways were investigated in rat, dog, and human hepatocytes. The structural analog of KM-819, namely N-[1-(4-bromobenzyl)-3,5-dimethyl-1H-pyrazol-4-yl]-2-(phenylsulfanyl) acetamide, served as the internal standard (IS). Proteins were precipitated from plasma samples using acetonitrile. Analysis was carried out using a reverse-phase C18 column with a mobile phase consisting of 0.1% formic acid in distilled water and 0.1% formic acid in acetonitrile. The analytical method developed for KM-819 exhibited linearity within the concentration range of 0.002-10 µg/mL in rat plasma. The precision and accuracy of the intra- and inter-day measurements were <15% for the lower limit of quantification (LLOQ) and all quality control samples. KM-819 demonstrated stability under various sample storage conditions (6 h at room temperature (25 °C), four weeks at -20 °C, three freeze-thaw cycles, and pretreated samples in the autosampler). The matrix effect and dilution integrity met the criteria set by the Food and Drug Administration and the European Medicines Agency. This sensitive, rapid, and reliable analytical method was successfully applied in pharmacokinetic studies in rats. Pharmacokinetic analysis revealed the dose-independent kinetics of KM-819 at 0.5-5 mg/kg, with a moderate oral bioavailability of ~20% in rats. The metabolic stability of KM-819 was also found to be moderate in rat, dog, and human hepatocytes. Metabolite identification in rat, dog, and human hepatocytes resulted in the discovery of six, six, and eight metabolites, respectively. Glucuronidation and mono-oxidation have been proposed as the major metabolic pathways. Overall, these findings contribute to a better understanding of the pharmacokinetic characteristics of KM-819, thereby aiding future clinical studies.

Synthesis and biological evaluation of quinolone derivatives as transthyretin amyloidogenesis inhibitors and fluorescence sensors.

Under certain conditions, numerous soluble proteins possess an inherent tendency to convert into insoluble amyloid aggregates, which are associated with several sporadic and genetic human diseases. Transthyretin (TTR) is one of the more than 30 human amyloidogenic proteins involved in conditions such as senile systemic amyloidosis, familial amyloid polyneuropathy, and familial amyloid cardiomyopathy. Considerable effort has been focused on identifying the native tetrameric TTR stabilizers to inhibit rate-limiting tetramer dissociation and, consequently, ameliorate TTR amyloidogenesis. Here, we describe the design and synthesis of quinolin-2(1H)-one derivatives that could be structurally complementary to the thyroxine-binding site within tetrameric TTR. Among these quinolin-2(1H)-one derivatives, compound 7a allowed 16.7% of V30M-TTR (3.6 µM) fibril formation at the same concentration and 49.6% at a concentration of 1.8 µM. Compound 7a exhibited much greater potency in complex biological samples like human plasma than that observed with tafamidis, the drug approved for the treatment of TTR amyloid cardiomyopathy for wild-type or hereditary TTR-mediated amyloidosis. Furthermore, the unique spectral properties of compound 7a demonstrated its high potential for TTR quantification, imaging sensors, and fluorescent tools to study the mechanism of TTR amyloidogenesis.

Development of an LC-MS/MS Method for ARV-110, a PROTAC Molecule, and Applications to Pharmacokinetic Studies.

ARV-110, a novel proteolysis-targeting chimera (PROTAC), has been reported to show satisfactory safety and tolerability for prostate cancer therapy in phase I clinical trials. However, there is a lack of bioanalytical assays for ARV-110 determination in biological samples. In this study, we developed and validated an LC-MS/MS method for the quantitation of ARV-110 in rat and mouse plasma and applied it to pharmacokinetic studies. ARV-110 and pomalidomide (internal standard) were extracted from the plasma samples using the protein precipitation method. Sample separation was performed using a C18 column and a mobile phase of 0.1% formic acid in distilled water-0.1% formic acid in acetonitrile (30:70, v/v). Multiple reaction monitoring was used to quantify ARV-110 and pomalidomide with ion transitions at m/z 813.4 → 452.2 and 273.8 → 201.0, respectively. The developed method showed good linearity in the concentration range of 2-3000 ng/mL with acceptable accuracy, precision, matrix effect, process efficiency, and recovery. ARV-110 was stable in rat and mouse plasma under long-term storage, three freeze-thaw cycles, and in an autosampler, but unstable at room temperature and 37 °C. Furthermore, the elimination of ARV-110 via phase 1 metabolism in rat, mouse, and human hepatic microsomes was shown to be unlikely. Application of the developed method to pharmacokinetic studies revealed that the oral bioavailability of ARV-110 in rats and mice was moderate (23.83% and 37.89%, respectively). These pharmacokinetic findings are beneficial for future preclinical and clinical studies of ARV-110 and/or other PROTACs.

ERRγ ligand HPB2 upregulates BDNF-TrkB and enhances dopaminergic neuronal phenotype.

Brain derived neurotrophic factor (BDNF) promotes maturation of dopaminergic (DAergic) neurons in the midbrain and positively regulates their maintenance and outgrowth. Therefore, understanding the mechanisms regulating the BDNF signaling pathway in DAergic neurons may help discover potential therapeutic strategies for neuropsychological disorders associated with dysregulation of DAergic neurotransmission. Because estrogen-related receptor gamma (ERRγ) is highly expressed in both the fetal nervous system and adult brains during DAergic neuronal differentiation, and it is involved in regulating the DAergic neuronal phenotype, we asked in this study whether ERRγ ligand regulates BDNF signaling and subsequent DAergic neuronal phenotype. Based on the X-ray crystal structures of the ligand binding domain of ERRγ, we designed and synthesized the ERRγ agonist, (E)-4-hydroxy-N'-(4-(phenylethynyl)benzylidene)benzohydrazide (HPB2) (Kd value, 8.35 µmol/L). HPB2 increased BDNF mRNA and protein levels, and enhanced the expression of the BDNF receptor tropomyosin receptor kinase B (TrkB) in human neuroblastoma SH-SY5Y, differentiated Lund human mesencephalic (LUHMES) cells, and primary ventral mesencephalic (VM) neurons. HPB2-induced upregulation of BDNF was attenuated by GSK5182, an antagonist of ERRγ, and siRNA-mediated ERRγ silencing. HPB2-induced activation of extracellular-signal-regulated kinase (ERK) and phosphorylation of cAMP-response element binding protein (CREB) was responsible for BDNF upregulation in SH-SY5Y cells. HPB2 enhanced the DAergic neuronal phenotype, namely upregulation of tyrosine hydroxylase (TH) and DA transporter (DAT) with neurite outgrowth, both in SH-SY5Y and primary VM neurons, which was interfered by the inhibition of BDNF-TrkB signaling, ERRγ knockdown, or blockade of ERK activation. HPB2 also upregulated BDNF and TH in the striatum and induced neurite elongation in the substantia nigra of mice brain. In conclusion, ERRγ activation regulated BDNF expression and the subsequent DAergic neuronal phenotype in neuronal cells. Our results might provide new insights into the mechanism underlying the regulation of BDNF expression, leading to novel therapeutic strategies for neuropsychological disorders associated with DAergic dysregulation.

Pharmacokinetics and diuretic effect of furosemide after single intravenous, oral tablet, and newly developed oral disintegrating film administration in healthy beagle dogs.

BACKGROUND: Furosemide, a diuretic that acts on the loop of Henle, is commonly used to treat congestive heart failure in veterinary medicine. Some owners have difficulty in administering oral tablet medication to animal patients, which leads to noncompliance, especially during long-term administration. Oral disintegrating film (ODF) has the advantages of easy administration via a non-invasive route, rapid dissolution, and low suffocating risk. The objective of this study was to research the pharmacokinetic (PK) profiles and diuretic effect of furosemide after intravenous (IV), orally uncoated tablet (OUT), and newly developed ODF administration in healthy beagle dogs. In this study, a furosemide-loaded ODF (FS-ODF) formulation was developed and five beagle dogs were administered a single dose (2 mg/kg) of furosemide via each route using a cross-over design. RESULTS: The most suitable film-forming agent was sodium alginate; thus, this was used to develop an ODF for easy drug administration. No significant differences were detected in the PK profiles between OUT and FS-ODF. In the blood profiles, the concentration of total protein was significantly increased compared to the baseline (0 h), whereas no significant difference was detected in the concentration of creatinine and hematocrit compared to the baseline. FS-ODF resulted in a similar hourly urinary output to OUT during the initial 2 h after administration. The urine specific gravity was significantly decreased compared to the baseline in each group. The peak times of urine electrolyte (sodium and chloride) excretion per hour were 1 h (IV), 2 h (OUT), and 2 h (FS-ODF). CONCLUSIONS: These results suggest that the PK/PD of furosemide after administration of newly developed FS-ODF are similar to those of OUT in healthy dogs. Therefore, the ODF formulation has the benefits of ease and convenience, which would be helpful to owners of companion animals, such as small dogs (< 10 kg), for the management of congestive heart failure.

Protective effect of RIPK1-inhibitory compound in in vivo models for retinal degenerative disease.

Receptor interacting protein kinase 1 (RIPK1) plays a key role in necroptosis, which is a type of programmed necrosis that is involved in ocular diseases, including glaucoma and dry age-related macular degeneration (AMD). We previously introduced RIPK1-inhibitory compound (RIC), which has biochemical characteristics and a mode of action that are distinct from those of the prototype RIPK1 inhibitor necrostatin-1. The intraperitoneal administration of RIC exerts a protective effect on retinal ganglion cells against a glaucomatous insult. In this study, we examined the protective effect of RIC on retinal pigment epithelium (RPE) against sodium iodate (SI) insult, which is associated with dry AMD pathogenesis. The eye drop administration of RIC that reached on the retina prevented RPE loss in SI-induced retinal degeneration. RIC consistently demonstrated retinal protection in the funduscopy and electroretinogram analyses in SI-injected rabbits and iodoacetic acid-treated mini-pigs. Moreover, the in vivo protective effects of RIC were superior to those of ACU-4429 and doxycycline, which are other medications investigated in clinical trials for the treatment of dry AMD, and RIC did not induce retinal toxicity following topical administration in rats. Collectively, RIC displayed excellent retinal penetration and prevented retinal degeneration in the pathogenesis of dry AMD with a high in vivo efficacy.

Enhanced Solubility, In-Vitro Dissolution and Lipase Inhibition of a Self-Nanoemulsifying Drug Delivery System Containing Orlistat.

The aim of this work was to prepare and assess a self-nanoemulsifying drug delivery system (SNEDDS) containing water-insoluble orlistat that was used for the inhibition of lipase activity in gastrointestinal lumen. The pseudo-ternary phase diagram, composed of orlistat and medium chain triglycerides (MCT) as oil phase and Cremophor EL as surfactant, was made for the confirmation of giving SNEDDS preconcentrate. The physical state, particle size dispersed in water, dissolution and lipase inhibition of SNEDDS preconcentrates were investigated. The appointed SNEDDS preconcentrates in the pseudo-ternary phase diagram showed no endothermic peak of orlistat and a liquid state. The particle sizes of SNEDDS dispersed with water were uniform and in the range of <200 nm. In the dissolution test, the liquid SNEDDS preconcentrate and solid state mixture exhibited 90.89±2.03% versus 22.42±3.71% at 60 min., respectively, whereas the raw orlistat showed no significant dissolution rate. The SNEDDS preconcentrate showed a lipase inhibition of 92.42±1.58% until 60 min., with no significant inhibition activity of orlistat. Therefore, the SNEDDS preconcentrate presented in this work solubilized orlistat and increased its dissolution rate, and resulted in sufficient inhibitory action on lipase.

Comparative single-dose pharmacokinetics of sildenafil after oral and rectal administration in healthy beagle dogs.

BACKGROUND: Sildenafil citrate, a highly selective phosphodiesterase type 5 inhibitor, is used to treat pulmonary hypertension (PH) in veterinary medicine. The objective of this study was to investigate pharmacokinetic profiles by oral administration of orally disintegrating film (ODF) and film coated tablet (FCT) formulations and rectal administration of ODF formulation in healthy dogs. Twelve healthy beagle dogs were administered four separate doses of sildenafil: FCT formulation 2 mg/kg orally, ODF formulation 2 mg/kg orally, ODF formulation 2 mg/kg rectally, and ODF formulation 10 mg/kg rectally. For 24 hours following administration, blood samples were collected and the plasma concentrations of sildenafil were assayed by liquid chromatography-tandem mass spectrometry. RESULTS: There were no significant differences in all the pharmacokinetic parameters between FCT and ODF formulations when administrated orally. Cmax at the time of rectal administration was lower when the same dose was given as that orally administered. No serious systemic adverse events (AEs) were observed. CONCLUSIONS: These findings suggest that sildenafil ODF formulation can be used as an alternative to FCT formulation in the treatment of canine PH patients; additionally, rectal administration of sildenafil ODF may be a beneficial treatment option for canine patients who are unable to receive medication orally.

Pharmacokinetics of tafamidis, a transthyretin amyloidosis drug, in rats.

1. We characterized the pharmacokinetics of tafamidis, a novel drug to treat transthyretin-related amyloidosis, in rats after intravenous and oral administration at doses of 0.3-3 mg/kg. In vitro Caco-2 cell permeability and liver microsomal stability, as well as in vivo tissue distribution and plasma protein binding were also examined. 2. After intravenous injection, systemic clearance (CL), volumes of distribution at steady state (Vss) and half-life (T½) remained unaltered as a function of dose, with values in the ranges of 6.41-7.03 mL/h/kg, 270-354 mL/kg and 39.5-46.9 h, respectively. Following oral administration, absolute bioavailability was 99.7-104% and was independent of doses from 0.3 to 3 mg/kg. In the urine and faeces, 4.36% and 48.9% of tafamidis, respectively, were recovered. 3. Tafamidis was distributed primarily in the liver and not in the brain, kidney, testis, heart, spleen, lung, gut, muscle, or adipose tissue. Further, tafamidis was very stable in rat liver microsomes, and its plasma protein binding was 99.9%. 4. In conclusion, tafamidis showed dose-independent pharmacokinetics with intravenous and oral doses of 0.3-3 mg/kg. Tafamidis undergoes minimal first-pass metabolism, distributes mostly in the liver and plasma, and appears to be eliminated primarily via biliary excretion.

Subacute toxicity evaluation of KR-33493, FAF1 inhibitor for a new anti-parkinson's disease agent, after oral administration in rats and dogs.

KR33493, a newly developed FAS-associated factor 1 (FAF1) inhibitor for Parkinson's disease, is being evaluated in a Phase I clinical trial. In the present study, the subchronic toxicity of KR33493 in Sprague-Dawley (SD) rats and beagle dogs was investigated at various oral doses for 28 and 14 days, respectively. During the study, food consumption, body weights, organ weights, gross findings, and mortality were examined; and ophthalmoscopy, electrocardiography, hematology, serum biochemistry, urinalysis, histopathology, and toxicokinetics were performed. In rats, weight gain decreased in both sexes at 500 mg/kg/day, with no significant differences. In dogs, some significant differences compared with the control were found during the trial; however, at the end of recovery periods, these were no longer observed and there was no dose correlation. Some histopathological findings were observed, but these were considered as incidental changes. Since no other significant changes were observed, doses above 500 and 1000 mg/kg KR33493 in rat and dogs, respectively, caused no observed adverse effects. Therefore, based on these results, the Phase 1 clinical trial for KR33493 was approved by the Korean Food & Drug Administration.

Discovery of Tyk2 inhibitors via the virtual site-directed fragment-based drug design.

In this study, we synthesized compound 12 with potent Tyk2 inhibitory activity from FBDD study and carried out a cell-based assay for Tyk2/STAT3 signaling activation upon IFNα5 stimulation. Compound 12 completely suppressed the IFNα5-mediated Tyk2/STAT3 signaling pathway as well as the basal levels of pSTAT3. Stimulation with IFNα/ß leads to the tyrosine phosphorylation of the JAK1 and Tyk2 receptor-associated kinases with subsequent STATs activation, transmitting signals from the cell surface receptor to the nucleus. In conclusion, the potency of compound 12 to interrupt the signal transmission of Tyk2/STAT3 appeared to be equivalent or superior to that of the reference compound.

Hair-growth-promoting effect of conditioned medium of high integrin α6 and low CD 71 (α6bri/CD71dim) positive keratinocyte cells.

Keratinocyte stem/progenitor cells (KSCs) reside in the bulge region of the hair follicles and may be involved in hair growth. Hair follicle dermal papilla cells (HFDPCs) and outer root sheath (ORS) cells were treated with conditioned medium (CM) of KSCs. Moreover, the effects of KSC-CM on hair growth were examined ex vivo and in vivo. A human growth factor chip array and RT-PCR were employed to identify enriched proteins in KSC-CM as compared with CM from keratinocytes. KSC-CM significantly increased the proliferation of HFDPCs and ORS cells, and increased the S-phase of the cell cycle in HFDPCs. KSC-CM led to the phosphorylation of ATK and ERK1/2 in both cell types. After subcutaneous injection of KSC-CM in C3H/HeN mice, a significant increase in hair growth and increased proliferation of hair matrix keratinocytes ex vivo was observed. We identified six proteins enriched in KSC-CM (amphiregulin, insulin-like growth factor binding protein-2, insulin-like growth factor binding protein-5, granulocyte macrophage-colony stimulating factor, Platelet-derived growth factor-AA, and vascular endothelial growth factor). A growth-factor cocktail that contains these six recombinant growth factors significantly increased the proliferation of HFDPCs and ORS cells and enhanced the hair growth of mouse models. These results collectively indicate that KSC-CM has the potential to increase hair growth via the proliferative capacity of HFDPCs and ORS cells.

Simultaneous quantification of methylene blue and its major metabolite, azure B, in plasma by LC-MS/MS and its application for a pharmacokinetic study.

A simple and sensitive liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was developed for the quantification of methylene blue (MB) and its major metabolite, azure B (AZB), in rat plasma. A simple protein precipitation using acetonitrile was followed by injection of the supernatant on to a Zorbax HILIC Plus column (3.5 µm, 2.1 × 100 mm) with isocratic mobile phase consisting of 5 mM ammonium acetate in 10:90 (v/v) water:methanol at a flow rate of 0.3 mL/min and detection in positive ionization mode. The standard curve was linear over the concentration range from 1 to 1000 ng/mL for MB and AZB with coefficient of determination above 0.9930. The lower limit of quantification was 1 ng/mL using 20 µL of rat plasma sample. The intra- and inter-assay precision and accuracy were <12%. The developed analytical method was successfully applied to the pharmacokinetic study of MB and AZB in rats.

Quantitative determination of enzalutamide, an anti-prostate cancer drug, in rat plasma using liquid chromatography-tandem mass spectrometry, and its application to a pharmacokinetic study.

This report details a method using liquid chromatography-tandem mass spectrometry (LC-MS/MS) that allows one to determine the concentration of an atypical anticancer drug, enzalutamide, in rat plasma. Specifically, this method involves the addition of an acetonitrile and bicalutamide (internal standard) solution to plasma samples. Following centrifugation of this mixture, an aliquot of the supernatant was directly injected into the LC-MS/MS system. Separation was achieved using a column packed with octadecylsilica (5 µm, 2.1 × 50 mm) with 10 mM ammonium acetate in acetonitrile as the mobile phase; detection was accomplished using MS/MS by multiple-reaction monitoring via an electrospray ionization source. This method demonstrated a linear standard curve (r = 0.997) over a concentration range of 0.001-1 µg/mL, as well as an intra- and inter-assay precision of 2.7 and 5.1%, respectively, and an accuracy range from 100.8 to 105.6%. The lower limit of quantification was 1.0 ng/mL in 50 µL of rat plasma sample. We also demonstrated that this analytical method could be successfully applied to the pharmacokinetic study of enzalutamide in rats.

Pharmacokinetic interactions of niclosamide in rats: Involvement of organic anion transporters 1 and 3 and organic cation transporter 2.

Niclosamide is an anthelmintic drug with a long history of use and is generally safe and well tolerated in humans. As the conventional dose of niclosamide results in a low but certain level in systemic circulation, drug interactions with concomitant drugs should be considered. We aimed to investigate the interaction between niclosamide and drug transporters, as such information is currently limited. Niclosamide inhibited the transport activity of OATP1B1, OATP1B3, OAT1, OAT3, and OCT2 in vitro. Among them, the inhibitory effects on OAT1, OAT3, and OCT2 were strong, with IC50 values of less than 1 µM. When 3 mg/kg of niclosamide was co-administered to rats, systemic exposure to furosemide (a substrate of OAT1/3) and metformin (a substrate of OCT2) increased, and the renal clearance (CLr) of the drugs significantly decreased. These results suggest that niclosamide inhibits renal transporters, OAT1/3 and OCT2, not only in vitro but also in vivo, resulting in increased systemic exposure to the substrates of the transporters by strongly blocking the urinary elimination pathway in rats. The findings of this study will support a meticulous understanding of the transporter-mediated drug interactions of niclosamide and consequently aid in effective and safe use of niclosamide.

Discovery of non-LBD inhibitor for androgen receptor by structure-guide design.

In this study, we synthesized the BF-3 binding small molecules, a series of pyridazinone-based compounds, as a novel class of non-LBP antiandrogens for treating prostate cancer by inhibiting androgen receptor. The new class compound was discovered to inhibitor the viability of AR-dependent human prostate LNCap cells and AR activity combining with the computational method. It showed a good physicochemical and PK property.

The Development of Super-Saturated Rebamipide Eye Drops for Enhanced Solubility, Stability, Patient Compliance, and Bioavailability.

The present study aimed to develop clear aqueous rebamipide (REB) eye drops to enhance solubility, stability, patient compliance, and bioavailability. For the preparation of a super-saturated 1.5% REB solution, the pH-modification method using NaOH and a hydrophilic polymer was employed. Low-viscosity hydroxypropyl methylcellulose (HPMC 4.5cp) was selected and worked efficiently to suppress REB precipitation at 40 °C for 16 days. The additionally optimized eye drops formulation (F18 and F19) using aminocaproic acid and D-sorbitol as a buffering agent and an osmotic agent, respectively, demonstrated long-term physicochemical stability at 25 °C and 40 °C for 6 months. The hypotonicity (<230 mOsm) for F18 and F19 noticeably extended the stable period, since the pressure causing the REB precipitation was relieved compared to the isotonic. In the rat study, the optimized REB eye drops showed significantly long-lasting pharmacokinetic results, suggesting the possibility of reducing daily administration times and increasing patient compliance (0.50- and 0.83-times lower Cmax and 2.60- and 3.64-times higher exposure in the cornea and aqueous humor). In conclusion, the formulations suggested in the present study are promising candidates and offer enhanced solubility, stability, patient compliance, and bioavailability.

Novel Strategy To Inhibit Transthyretin Amyloidosis via the Synergetic Effect of Chemoselective Acylation and Noncovalent Inhibitor Release.

Strategies for developing targeted covalent inhibitors (TCIs), which have the advantages of a prolonged duration of action and selectivity toward a drug target, have attracted great interest in drug discovery. Herein, we report chemoselective covalent inhibitors that specifically target lysine ε-amine groups that conjugate with an endogenous protein to prevent disease-causing protein misfolding and aggregation. These TCIs are unique because the benzoyl group is preferentially conjugated to Lys15 at the top of the T4 binding site within transthyretin (TTR) while simultaneously releasing a potent noncovalent TTR kinetic stabilizer. The potency of these covalent inhibitors is superior to tafamidis, the only FDA-approved drug for the treatment of hereditary TTR amyloidosis. In addition to investigations into the covalent modification of TTR via reverse-phase high-performance liquid chromatography, direct methods are performed to confirm and visualize the presumed covalent interaction via mass spectrometry and X-ray crystallography.

Liquid chromatography-tandem mass spectrometry for quantification of lacosamide, an antiepileptic drug, in rat plasma and its application to pharmacokinetic study.

A rapid, simple and sensitive liquid chromatography-tandem mass spectrometry (LC/MS/MS) was developed for the determination of an antiepileptic drug, lacosamide, in rat plasma. The method involves the addition of acetonitrile and internal standard solution to plasma samples, followed by centrifugation. An aliquot of the supernatant was diluted with water and directly injected into the LC/MS/MS system. The separations were performed on column packed with octadecylsilica (5 µm, 2.0 × 50 mm) with 0.1% formic acid and acetonitrile as mobile phase, and the detection was performed on tandem mass spectrometry by the multiple-reaction monitoring via an electrospray ionization source. The standard curve was linear over the concentration range from 0.3 to 1000 ng/mL. The lower limit of quantification was 0.3 ng/mL using 50 µL of rat plasma sample. The intra- and inter-assay precision and accuracy were found to be less than 11.7 and 8.8%, respectively. The developed analytical method was successfully applied to the pharmacokinetic study of lacosamide in rats.

Clinical Anti-aging Efficacy of Propolis Polymeric Nanoparticles Prepared by a Temperature-induced Phase Transition Method.

BACKGROUND: Collagen forms a dermal matrix in the skin. Biosynthesis and decomposition of collagen are the major processes in skin aging. Propolis is rich in flavonoids and phenolic compounds, which are known to be effective in preventing skin aging, including the enhancement of fibroblast proliferation, activation, and growth capacity. OBJECTIVES: The aim of this study was to develop a poorly soluble propolis extract as an active ingredient in cosmetic products for anti-aging efficacy. METHODS & RESULTS: Polymeric nanoparticles containing propolis extract, polyethylene glycol 400, and poloxamer 407 were prepared via a temperature-induced phase transition method. The particle size of the polymeric nanoparticles was approximately 20.75 nm. The results of an in vitro procollagen type I carboxy-terminal peptide assay and a matrix metalloproteinase-1 inhibition assay showed that the polymeric nanoparticles increased collagen production by 19.81%-24.59% compared to blank (p < 0.05), and significantly reduced intracellular collagenase activity by 7.46%-31.52% compared to blank (p < 0.05). In a clinical trial, polymeric nanoparticles in a cosmetic formulation were applied around the eyes of 24 female subjects for 8 weeks. Five skin parameters were significantly improved after the application of the test ampoule. Visual evaluation using the Global Photo Damage Score showed a significant reduction in wrinkles after the application of the test ampoules (p < 0.001). CONCLUSIONS: This study outlines the development of stable polymeric nanoparticles containing poorly soluble propolis in a cosmetic formulation, and its efficacy in wrinkle improvement. The developed polymeric nanoparticles were effective for alleviating wrinkles and can be used for pharmaceutical applications that utilize propolis as antiseptic, anti-inflammatory, antimycotic, antifungal, antibacterial, antiulcer, anticancer, and immunomodulatory agents.