A Preliminary Study of Pharmacokinetics and Pharmacodynamics of Oral Fingolimod in Dogs.

BACKGROUND/AIM: Fingolimod is a sphingosine-1-phosphate receptor modulator that prevents lymphocytes egress from lymphoid organs. It has been used as a disease-modifying drug for human multiple sclerosis and has shown better therapeutic effects than other conventional therapies. Therefore, this study was performed to obtain preclinical data of fingolimod in dogs. MATERIALS AND METHODS: Nine laboratory Beagle dogs were used and randomized into three groups for pharmacokinetics (PK) and pharmacodynamics (PD). The dogs were administered once with a low-dose (0.01 mg/kg, n=3), medium-dose (0.05 mg/kg, n=3), and high-dose (0.1 mg/kg, n=3) of fingolimod, orally. Samples were collected serially at predetermined time points, and whole blood fingolimod concentrations were measured using high-performance liquid chromatography-mass spectrometry. Differential counts of leukocytes over time were determined to identify immune cells' response to fingolimod. RESULTS: Regarding PK, the concentration of fingolimod in the blood increased in a dose-dependent manner, but it was not proportional. Regarding PD, the number of lymphocytes significantly decreased compared to baseline in all dose groups (low-dose, p=0.0002; medium-dose, p<0.0001; high-dose, p=0.0012). Eosinophils were significantly reduced in low- (p=0.0006) and medium- (p=0.0006) doses, and neutrophils were also significantly reduced in medium-(p=0.0345) and high- (p=0.0016) doses. CONCLUSION: This study provides the basis for future clinical applications of fingolimod in dogs with immune-mediated diseases, such as meningoencephalitis of unknown etiology.

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.

Pharmacokinetics of fluconazole after oral administration to healthy beagle dogs.

BACKGROUND: Fluconazole can be effective in the treatment of superficial mycoses in dogs. However, the pharmacokinetics of fluconazole have not yet been evaluated to determine its optimal dosing regimen. OBJECTIVES: This study aimed to determine the plasma concentration of fluconazole after single and multiple administrations at two different dosages in dogs. METHODS AND MATERIALS: Eight healthy beagle dogs were divided into two groups, and each group received either 5 or 10 mg/kg of fluconazole per os. The pharmacokinetics of fluconazole was determined following single and multiple administrations p.o. Single- and multiple-dose treatment periods were separated by a washout period of seven days. Plasma concentrations of fluconazole were determined by established high-performance liquid chromatography coupled with tandem mass spectrometry system. RESULTS: In the 5 mg/kg group, the mean maximum concentrations (Cmax ) and the area under the plasma concentrations (AUC0-24h ) were 4.84 µg/mL and 85.56 µg*h/mL, respectively, after single administration and 6.58 µg/mL and 119.52 µg*h/mL, respectively, after multiple administrations. In the 10 mg/kg group, the Cmax and AUC0-24h were 5.67 µg/mL and 109.19 µg*h/mL, respectively, after single administration and 15.10 µg/mL and 291.51 µg*h/mL, respectively, after multiple administrations. The Cmax (p < 0.001) and AUC0-24h (p < 0.001) were significantly lower in the 5 mg/kg group than those in the 10 mg/kg group at multiple administrations. CONCLUSIONS AND CLINICAL RELEVANCE: Fluconazole accumulates in plasma and exhibits dose-proportional pharmacokinetics after multiple doses, and was safe and well tolerated at these doses for short-term administration.

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.

The Development and Optimization of Hot-Melt Extruded Amorphous Solid Dispersions Containing Rivaroxaban in Combination with Polymers.

Rivaroxaban (RXB), a novel oral anticoagulant that directly inhibits factor Xa, is a poorly soluble drug belonging to Biopharmaceutics Classification System (BCS) class II. In this study, a hot-melt extruded amorphous solid dispersion (HME-ASD) containing RXB is prepared by changing the drug:polymer ratio (Polyvinylpyrrolidione-vinyl acetate 64, 1:1-1:4) and barrel temperature (200-240 °C), fixed at 20% of Cremophor® RH 40 and 15 rpm of the screw speed, using the hot-melt extruding technique. This study evaluates the solubility, dissolution behavior, and bioavailability for application to oral drug delivery and optimizes the formulation of rivaroxaban amorphous solid dispersion (RXB-ASD). Based on a central composite design, optimized RXB-ASD (PVP VA 64 ratio 1:4.1, barrel temperature 216.1 °C, Cremophor® RH 40 20%, screw speed 15 rpm) showed satisfactory results for dependent variables. An in vitro drug dissolution study exhibited relatively high dissolution in four media and achieved around an 80% cumulative drug release in 120 min. Optimized RXB-ASD was stable under the accelerated condition for three months without a change in crystallinity and the dissolution rate. A pharmacokinetic study of RXB-ASD in rats showed that the absorption was markedly increased in terms of rate and amount, i.e., the systemic exposure values, compared to raw RXB powder. These results showed the application of quality by design (QbD) in the formulation development of hot-melt extruded RXB-ASD, which can be used as an oral drug delivery system by increasing the dissolution rate and bioavailability.

Pharmacokinetic Characterization of Supinoxin and Its Physiologically Based Pharmacokinetic Modeling in Rats.

Supinoxin is a novel anticancer drug candidate targeting the Y593 phospho-p68 RNA helicase, by exhibiting antiproliferative activity and/or suppression of tumor growth. This study aimed to characterize the in vitro and in vivo pharmacokinetics of supinoxin and attempt physiologically based pharmacokinetic (PBPK) modeling in rats. Supinoxin has good permeability, comparable to that of metoprolol (high permeability compound) in Caco-2 cells, with negligible net absorptive or secretory transport observed. After an intravenous injection at a dose range of 0.5-5 mg/kg, the terminal half-life (i.e., 2.54-2.80 h), systemic clearance (i.e., 691-865 mL/h/kg), and steady state volume of distribution (i.e., 2040-3500 mL/kg) of supinoxin remained unchanged, suggesting dose-independent (i.e., dose-proportional) pharmacokinetics for the dose ranges studied. After oral administration, supinoxin showed modest absorption with an absolute oral bioavailability of 56.9-57.4%. The fecal recovery following intravenous and oral administration was 16.5% and 46.8%, respectively, whereas the urinary recoveries in both administration routes were negligible. Supinoxin was mainly eliminated via NADPH-dependent phase I metabolism (i.e., 58.5% of total clearance), while UDPGA-dependent phase II metabolism appeared negligible in the rat liver microsome. Supinoxin was most abundantly distributed in the adipose tissue, gut, and liver among the nine major tissues studied (i.e., the brain, liver, kidneys, heart, lungs, spleen, gut, muscles, and adipose tissue), and the tissue exposure profiles of supinoxin were well predicted with physiologically based pharmacokinetics.

Determination of motolimod concentration in rat plasma by liquid chromatography-tandem mass spectrometry and its application in a pharmacokinetic study.

Motolimod (VTX-2337) is an agonist of toll-like receptor 8. In this study, a novel and sensitive liquid chromatography-tandem mass spectrometry method was developed for quantifying motolimod in rat plasma and subsequently used in a pharmacokinetic study. Proteins were precipitated from plasma samples using acetonitrile prior to the analysis. GS-9620 was used as an internal standard. High-performance liquid chromatography was performed using a Spursil C18-EP column (3 µm, 50 × 2.1 mm). Aqueous ammonium formate and acetonitrile were used as the mobile phase. Motolimod was detected using an electrospray ionization interface under multiple reaction monitoring conditions in the positive ion mode. The developed method produced a linear correlation over a concentration range of 1-1000 ng/mL (r = 0.9944). Intra- and inter-day precision values ranged from 4.8%-10.7% (the lower limit of quantification precision value was 16.3 %), whereas intra- and inter-day accuracy values ranged from 0.3%-9.1 %. The mean recovery of motolimod from rat plasma was 109.4 %. Additionally, motolimod was found to be stable under various conditions (three freeze-thaw cycles, 6-h storage at room temperature, short- and long-term stability tests, and processing). The developed method was successfully used in a pharmacokinetic study in rats. Motolimod showed non-linear pharmacokinetics following its intravenous administration to rats at 0.6-6 mg/kg. Additionally, very low exposure (<1 %) was obtained following oral administration of the drug to rats. The results also showed that motolimod has a low metabolic stability in the liver microsomes and exhibits extensive binding to the plasma proteins.

Pharmacokinetics of intravenous administered two different high doses of ascorbic acid in healthy beagle dogs.

OBJECTIVE: We performed a randomized two-way crossover study to evaluate the pharmacokinetic profiles of two high-dose ascorbic acid (AA) after IV infusion in healthy beagle dogs. MATERIALS AND METHODS: The dogs were administered IV AA at two doses of 1.5 and 3 gm/kg for 4 h, and the AA concentration in plasma and urine pH was measured before and after administration. RESULTS: The plasma concentrations of AA in both groups peaked 3 h after administration. Among the two groups, the urine pH was not significantly different (p = 0.1299-0.7944). High-dose IV AA did not induce serious adverse events in dogs. CONCLUSION: The results of this study suggest that the high dose of AA which reaches the therapeutic dose for cancer and supports the safety of high-dose IV AA in dogs.

Pharmacophoric Site Identification and Inhibitor Design for Autotaxin.

Autotaxin (ATX) is a potential drug target that is associated with inflammatory diseases and various cancers. In our previous studies, we have designed several inhibitors targeting ATX using computational and experimental approaches. Here, we have analyzed topological water networks (TWNs) in the binding pocket of ATX. TWN analysis revealed a pharmacophoric site inside the pocket. We designed and synthesized compounds considering the identified pharmacophoric site. Furthermore, we performed biological experiments to determine their ATX inhibitory activities. High potency of the designed compounds supports the predictions of the TWN analysis.

Effects of formulation types on pharmacodynamics of warfarin in patients with cerebral infarction and dysphagia.

PURPOSE: The purpose of this study was to investigate the effects of the type of formulation on the efficacy of warfarin. MATERIALS AND METHODS: The electronic medical records of patients with cerebral infarction, who were administered tablet or powder formulations of warfarin from 2013-2015, were retrospectively analyzed. Clinical data, changes in the international normalized ratio (INR), the warfarin dose, and the time to reach the plasma warfarin concentration that could induce an adverse effect, such as bleeding, were evaluated. Coefficients of variation of INR and of the warfarin dose, as well as the warfarin sensitivity index (WSI), were used to evaluate the INR stability. Statistical analysis of the data was performed using a independent t-test. Additionally, survival analysis was performed. RESULTS: The data showed that 57 and 137 patients were administered warfarin as powder and tablet formulations, respectively. We noted that INR, WSI, and INR/dose × body weight differed significantly between the two groups of patients. The median survival times to reach the plasma warfarin concentration that could induce adverse effects were 3.6 and 4.2 days of treatment with the powder and tablet formulations, respectively. The efficacy of warfarin was higher when the drug was administered as a powder than when it was administered as a tablet. CONCLUSION: The findings of this study indicate that INR should be carefully monitored in the first 4 days of warfarin administration as a powder formulation.

Liquid chromatography-tandem mass spectrometry of recombinant human extracellular superoxide dismutase (rhSOD3) in mouse plasma and its application to pharmacokinetic study.

The antioxidant enzyme human extracellular superoxide dismutase (SOD3) is a promising biopharmaceutical candidate for the treatment of various diseases. To support the early development of SOD3 as a biopharmaceutical, a simple, sensitive, and rapid liquid chromatography tandem mass spectrometry procedure was developed and validated for the determination of SOD3 levels in the plasma of ICR mice. After purification with Ni-NTA magnetic beads and digestion with trypsin, SOD3 signature peptides and internal standard signature peptide (ISP) were separated via high performance liquid chromatography using a Zorbax C18 column (2.1 × 50 mm, 3.5 µm) and a mobile phase consisting of 10 mM ammonium formate, 0.1% formic acid, and acetonitrile. The analyte and ISP were detected via a tandem mass spectrometer in electrospray ionization and multiple reaction monitoring modes to select both the signature peptide for SOD3 at m/z 669 to 969 and the ISP at m/z 655 to 941 in the positive ion mode. The calibration curves were linear (r > 0.99) between 5 and 1000 µg/mL with a lower limit of quantification of 5 µg/mL. The relative standard deviation ranged from 3.08 to 8.84% while the relative error ranged from -0.13 to -9.56%. This method was successfully applied to a preclinical pharmacokinetic study of SOD3 in male ICR mice.

Pharmacokinetics and pharmacodynamics of intravenous esomeprazole at 2 different dosages in dogs.

BACKGROUND: Although the demand for esomeprazole is increasing in veterinary medicine, the pharmacokinetics (PK) and pharmacodynamics of esomeprazole have been described in only a few studies. OBJECTIVE: To determine the PK of 0.5 and 1 mg/kg esomeprazole administered IV q12h and to investigate its effects on intragastric pH in healthy dogs. ANIMALS: Six adult Beagles. METHODS: Open-label, randomized, and crossover design. The dogs received 0.5 or 1 mg/kg esomeprazole IV q12h for 48 hours. Plasma concentrations of esomeprazole were measured by high-performance liquid chromatography-tandem mass spectrometry. Intragastric pH was determined using the Bravo pH monitoring system and recorded as mean percentage time (MPT) for which pH was ≥3 and ≥4 for 24 hours in each group. RESULTS: The peak plasma concentration and area under the curve from the time of dosing to the last measurable concentration in the 1 mg/kg group were higher than those in the 0.5 mg/kg group. However, when the dosage normalized, intergroup differences were not significant. The MPTs for which intragastric pH was ≥3 and ≥4 for 48 hours were 88% ± 7% and 81% ± 9% for the 0.5 mg/kg group and 90% ± 9% and 85% ± 11% for the 1 mg/kg group, respectively, with no significant intergroup differences. CONCLUSIONS AND CLINICAL IMPORTANCE: The pharmacokinetic parameters and acid suppressant effect for 0.5 and 1 mg/kg esomeprazole were not significantly different. Furthermore, the efficacy of esomeprazole 0.5 mg/kg IV q12h was sufficient to increase intragastric pH in Beagles.

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.

Discovery and optimization of ATX inhibitors via modeling, synthesis and biological evaluation.

Autotaxin (ATX) is a potential target for the treatment of various cancers. A new series of ATX inhibitors was rationally designed and synthesized based on our previous study. Biological evaluation and structure-activity relationship (SAR) of this series are discussed. Among fourteen synthesized derivatives, six compounds (2, 3, 4, 12, 13 and 14) exhibited enhanced ATX inhibitory activities with IC50 values in the low nanomolar range. Molecular interactions of all the synthesized compounds within the active site of ATX were studied through molecular docking studies. Herein, we describe our lead optimization efforts that resulted in the identification of a potent ATX inhibitor (compound 4 with IC50 = 1.23 nM, FS-3 and 2.18 nM, bis-pNPP). Furthermore, pharmacokinetic properties of this most promising compound are profiled.

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.

Development and validation of a liquid chromatography-tandem mass spectrometry method for the assay of tafamidis in rat plasma: Application to a pharmacokinetic study in rats.

Tafamidis is a first-in-class inhibitor of transthyretin amyloid fibril formation. It has been available in Argentina, Japan, and Mexico for the treatment of transthyretin amyloidosis in adult patients with early-stage symptomatic polyneuropathy. In this study, a rapid and sensitive liquid chromatography-tandem mass spectrometry method was developed and validated for the assay of tafamidis in rat plasma. The method was also assessed for its applicability to pharmacokinetic studies in rats. Tafamidis was extracted from rat plasma by the liquid-liquid extraction method using hydrochloric acid and ethyl acetate. A reversed-phase C18 column and a mobile phase consisting of 10mM ammonium formate and acetonitrile were used to achieve chromatographic separation. The flow rate for the mobile phase was set at 0.3mL/min. Tafamidis and 2-CBC, which was used as the internal standard (IS), were analyzed by multiple reaction monitoring in negative ESI mode at m/z transitions of 305.4→261.4 for tafamidis and 271.7→227.8 for the IS. The lower limit of quantification of tafamidis was obtained as 3ng/mL, and the calibration curve was linear over a concentration range of 3-3000ng/mL (R2>0.99). The validation parameters investigated, which were specificity, precision, accuracy, matrix effect, recovery, and stability, were well within acceptable limits. The method was successfully used for the evaluation of the pharmacokinetics of tafamidis in rats.

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.

Pharmacokinetics of enzalutamide, an anti-prostate cancer drug, in rats.

We characterized the pharmacokinetics of enzalutamide, a novel anti-prostate cancer drug, in rats after intravenous and oral administration in the dose range 0.5-5 mg/kg. Tissue distribution, liver microsomal stability, and plasma protein binding were also examined. After intravenous injection, systemic clearance, volumes of distribution at steady state (Vss), and half-life (T½) remained unaltered as a function of dose, with values in the ranges of 80.4-86.3 mL/h/kg, 1020-1250 mL/kg, and 9.13-10.6 h, respectively. Following oral administration, absolute oral bioavailability was 89.7 % and not dose-dependent. The recoveries of enzalutamide in urine and feces were 0.0620 and 2.04 %, respectively. Enzalutamide was distributed primarily in 10 tissues (brain, liver, kidneys, testis, heart, spleen, lungs, gut, muscle, and adipose) and tissue-to-plasma ratios of enzalutamide ranged from 0.406 (brain) to 10.2 (adipose tissue). Further, enzalutamide was stable in rat liver microsomes, and its plasma protein binding was 94.7 %. In conclusion, enzalutamide showed dose-independent pharmacokinetics at intravenous and oral doses of 0.5-5 mg/kg. Enzalutamide distributed primarily to 10 tissues and appeared to be eliminated primarily by metabolism.