Pharmacokinetic Study In Rats Research Articles

HPLC-MS/MS Method for the Quantitative Determination of Metformin in Rat Plasma and Its Application to Comparative Bioavailability Assessment

Background: Metformin is a biguanide derivative utilized as a first-line treatment for type 2 diabetes for people over 60 years. However, it faces certain limitations due to its incomplete absorption, resulting in a 50-60% bioavailability. In addition to its blood glucose-lowering effect, the antiproliferative effect of metformin has been demonstrated in vitro. Therefore, it is necessary to consider alternative administration routes that can enhance the bioavailability of metformin, expanding its clinical use beyond its role as an antidiabetic agent. Objective: The aim of the study was to develop a reliable bioanalytical method for the quantitation of metformin in male Sprague-Dawley rat plasma and explore the promising alternative administration route for metformin use. Methods: A robust, high-performance liquid chromatography-tandem mass spectrometry method for the quantification of metformin in rat plasma was developed and validated according to the latest regulatory guidance for bioanalysis. Results: Based on the area under the curves obtained from the rat pharmacokinetic study, subcutaneous injection increased the systemic exposure of metformin by 1.79-fold compared to oral administration in rats. Conclusion: Subcutaneous administration of metformin enhances its bioavailability compared to oral administration, leading to increased antidiabetic effects and potential antitumor activity.

Development and Optimization of Proniosomal Formulation of Irbesartan Using a Box-Behnken Design to Enhance Oral Bioavailability: Physicochemical Characterization and In Vivo Assessment.

This research work aimed to develop and evaluate proniosomes for the oral delivery of the lipophilic drug Irbesartan (IRB) to improve its solubility and bioavailability. Proniosomes of Irbesartan were formulated using a lipid, surfactant, and carrier by a slurry method. Based on the prepared preliminary trial batches and their evaluation, the formulation was optimized by employing a Box-Behnken design (BBD) in which concentrations of span 60 (X1), cholesterol (X2), and mannitol (X3) were used as three independent variables and the vesicular size (VS) (Y1), % entrapment efficiency (% EE) (Y2), and % cumulative drug release (% CDR) (Y3) were used as dependent variables. The optimized batch B1 was obtained from the BBD experiment after validation of checkpoint analysis, and their characterization was done for VS, % EE, % CDR, Fourier transform infrared spectroscopy (FTIR), differential scanning calorimetry (DSC), and X-ray diffraction (XRD) analysis. The optimized batch showed a VS of 199 ± 5.4 nm, a % EE of 99.25 ± 2.24%, and a % CDR of 97.36 ± 1.13% at 24 h. Scanning electron microscopy (SEM) study showed a smooth surface of batch B1. DSC and XRD studies indicated the amorphous nature of the proniosomal formulation. The proniosomal formulation showed increased solubility (2.65 ± 0.2 mg/mL) in phosphate buffer, pH 6.8, as compared to water (0.059 ± 0.02 mg/mL). The pharmacokinetic study in rats confirmed the increased bioavailability of the drug in optimized proniosomal formulation compared with its pure drug suspension. Cmax, Tmax, and AUC0-t of the drug also increased by 2-fold compared to those of drug suspension. Thus, in conclusion, the proniosomal formulation proved to be an efficient carrier for improved oral delivery of Irbesartan by improving the solubility and bioavailability of the drug.

Development of a highly sensitive and green first-derivative synchronous fluorescence spectroscopic method for the simultaneous quantification of telmisartan and rosuvastatin: Greenness metric assessment and application to a pharmacokinetic study in rats

Hypertension and hyperlipidemia frequently coexist and are correlated with elevated cardiovascular adverse outcomes. Fixed dose combination tablets containing antihypertensive and antihyperlipidemic drugs have the potential to improve patient compliance. Telmisartan and rosuvastatin fixed dose combination tablet has been recently formulated. This study provided the first fluorescence spectroscopic method for simultaneously quantifying telmisartan and rosuvastatin in tablet dosage form and plasma. The native fluorescence spectra of telmisartan and rosuvastatin completely overlapped, making direct measurement unachievable. However, through the implementation of synchronous fluorescence measurements of telmisartan and rosuvastatin at a Δλ = 60, distinct narrow bands were observed at 358 nm and 375 nm, respectively. Regrettably, the challenge of overlapping remained unresolved. Nevertheless, by converting these synchronous spectra into first-order spectra, the problem of overlapping was completely resolved. This conversion also allowed for the selective quantification of telmisartan and rosuvastatin at 374 nm and 358 nm, respectively. The validity of this method was confirmed in accordance with ICH guidelines, yielding satisfactory results in terms of the validation characteristics. The method demonstrated linear relationships between the response and the studied drugs concentrations in working range of 50–1000 ng/mL for telmisartan and 100–2000 ng/mL for rosuvastatin. The described methodology was applied for the pharmacokinetic study of telmisartan and rosuvastatin in rat plasma after a single oral dose of 4 mg/kg telmisartan and 50 mg/kg rosuvastatin. Pharmacokinetic analyses revealed a moderate drug-drug interaction between the two drugs, which was not considered to be clinically significant. Moreover, the described method was assessed in terms of sensitivity and environmental sustainability against three previously documented methods. The comparison effectively underscores the supremacy of the proposed technique over the documented techniques.

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.

Discovery of the Novel, Orally Active, and Selective LPA1 Receptor Antagonist ACT-1016-0707 as a Preclinical Candidate for the Treatment of Fibrotic Diseases.

Piperidine 3 is a potent and selective lysophosphatidic acid receptor subtype 1 receptor (LPAR1) antagonist that has shown efficacy in a skin vascular leakage target engagement model in mice. However, compound 3 has very high human plasma protein binding and high clearance in rats, which could significantly hamper its clinical development. Continued lead optimization led to the potent, less protein bound, metabolically stable, and orally active azetidine 17. Rat pharmacokinetics (PK) studies revealed that 17 accumulated in the liver. In vitro studies indicated that 17 is an organic anion co-transporting polypeptide 1B1 (OATP1B1) substrate. Although analogue 24 was no longer a substrate of OATP1B1, PK studies suggested that the compound undergoes enterohepatic recirculation. Replacing the carboxylic acidic side chain by a non-acidic sulfamide moiety and further fine-tuning of the scaffold yielded the potent, orally active LPAR1 antagonist 49, which was selected for preclinical development for the treatment of fibrotic diseases.

A validated LC-MS/MS method for determination of six Anti-SARS-CoV-2 drugs in plasma and its application for a pharmacokinetic study in rats

Antiviral treatment for COVID-19 is considered an effective tool in reducing the rate of severe cases and deaths. As of June 2023, a total of six small molecule antiviral drugs have been conditionally approved for marketing by the National Medical Products Administration (NMPA) within China. In this study, a method of HPLC-MS/MS was established and validated for the determination of six small molecule antiviral drugs in plasma using Lamivudine as an internal standard. The chromatographic separation was performed using gradient elution with an ACE 3 C18-PFP column (3.0 mm × 150 mm, 3 μm), and the mobile phase consisted of deionized water and acetonitrile/water (90:10, v/v), both with 10 mmol/L of ammonium acetate and 0.1 % ammonium hydroxide added. Quantitative analysis of the six small molecule drugs was carried out through selective reaction monitoring based on the positive ion spray ionization mode. The method exhibited excellent precision, accuracy, recovery, and linearity, and it was used to determine the pharmacokinetic characteristics in rats. Our work not only established a bioanalytical method for six small molecule antiviral drugs but also provided scientific references for clinical pharmacokinetic studies.

Development of a high sensitivity UHPLC-MS/MS method to determine the twelve compounds of Physochlainae Radix extract and application to a pharmacokinetic study in rats

Physochlainae Radix (PR) is generally applied for treating cough and asthma. In this study, a sensitive ultra-high-performance liquid chromatography coupled with triple quadrupole mass spectrometry (UHPLC-MS/MS) method was established and validated for the simultaneous determination of twelve components (scopolamine, anisodamine, hyoscyamine, protocatechuic acid, fabiatrin, scopolin, 4-hydroxybenzoic acid, caffeic acid, tropic acid, scopoletin, isoquercitrin, and scoparone) of PR extract in rat plasma. ACQUITY UPLC CSH C18 column was employed for the chromatographic separation with the mobile phase system consisting of acetonitrile and 0.1 % (v/v) formic acid aqueous solution. The intra-day and inter-day precisions of twelve analytes was less than 9.33 %, and the accuracy ranged from −11.08 to 11.89 %. The extraction recoveries of the analytes ranged from 76.42 to 93.17 % and the matrix effects varied from 76.02 to 90.56 %. The results of stability tests demonstrated that the analytes were stable under the different conditions and their relative standard deviation were less than 13.06 %. The developed method was firstly successfully applied to the pharmacokinetic study of the twelve analytes of PR extract in rats, and the pharmacokinetic results showed that the maximum concentration (Cmax, 1553.51 ± 736.52 ng/mL) of tropic acid were relatively large, which was related to the fact that scopolamine and anisodamine were metabolized to tropic acid in vivo. The elimination half-life (T1/2) of fabiatrin, scopolin, 4-hydroxybenzoic acid, and tropic acid were 6.45, 6.16, 9.87, and 7.12 h, suggesting that these analytes were kept for a longer duration in vivo. This research would provide meaningful reference for PR in clinical use.

A robust and validated LC–MS/MS method for the quantification of ramucirumab in rat and human serum using direct enzymatic digestion without immunoassay

A new liquid chromatography tandem mass spectrometry (LC–MS/MS) method was established to quantify the anti-gastric cancer fully human monoclonal antibody (ramucirumab) in rat and human serum. The surrogate peptide (GPSVLPLAPSSK) for ramucirumab was generated by trypsin hydrolysis and quantified using the isotopically labeled peptide GPSVLPLAPSSK[13C6, 15N2]ST containing two more amino acids at the carboxyl end as an internal standard to correct for variations introduced during the enzymatic hydrolysis process and any mass spectrometry changes. Additionally, the oxidation and deamidation of unstable peptides (VVSVLTVLHQDWLNGK and NSLYLQMNSLR) were detected. The quantitative range of the proposed method was 1–1000 μg/mL, and complete methodological validation was performed. The precision, accuracy, matrix effect, sensitivity, stability, selectivity, carryover, and interference of the measurements met the required standards. The validated LC-MS/MS method was applied to pharmacokinetic studies in rats administered ramucirumab at 15 mg/kg intravenously. Overall, a robust, efficient, and cost-effective LC–MS/MS method was successfully developed for quantifying ramucirumab in rat and human serum.

Magnitude of Fruit Juice-Drug Interactions Due to Osmolality-Dependent Fluid Secretion: Differences among Apple, Orange, and Grapefruit Juices.

We recently reported that the gastrointestinal (GI) fluid volume is influenced by the solution osmolality, and proposed that this effect may play a role in beverage-drug interactions. Here, we investigated whether osmolality-dependent fluid secretion can explain the difference in the magnitudes of fruit juice-drug interactions depending on the type of fruit juice (grapefruit juice (GFJ), orange juice (OJ), and apple juice (AJ)). The osmolality of GFJ, OJ, and AJ used in this study was found to be 552, 686, and 749 mOsm/kg, respectively. Measurements of intestinal fluid movement following beverage administration by the in situ closed-loop technique revealed the following rank order for fluid volume in rat ileum: AJ > OJ > GFJ > purified water, suggesting that water movement is dependent on the osmolality of these beverages. Such changes in GI fluid volume are expected to alter the luminal drug concentration, potentially contributing to the magnitude of beverage-drug interactions. Indeed, in vivo pharmacokinetic study in rats revealed that the plasma concentration of atenolol, a low-permeability drug, was the highest after oral administration in purified water, followed by GFJ and OJ, and was the lowest after administration in AJ. In contrast, antipyrine, a high-permeability drug, showed no significant difference in plasma concentration after administration in purified water and fruit juices, suggesting that the absorption of high-permeability drugs is less affected by solution osmolality. Our findings indicate that differences in the magnitude of beverage-drug interactions can be at least partly explained by differences in the osmolality of the beverages ingested.

Microemulsion co-delivering curcumin and DHA-rich algal oil alleviates nonalcoholic fatty liver disease

The combined effect of curcumin and docosahexaenoic acid (DHA) on liver injury has not yet been reported. We determined the synergistic effect of curcumin and DHA on the protection of WRL-68 cells from palmitic acid-induced injury, and on the inhibition of LX2 cells. A microemulsion to co-deliver both compounds was prepared by ultrasonic emulsification. Mice were fed a high-fat diet for 10 weeks to induce mild non-alcoholic fatty liver disease (NAFLD). At a dose of 5 mg/kg curcumin and 30 mg/kg DHA, the microemulsion successfully lowered serum triacylglycerol (TG) and low-density lipoprotein cholesterol levels, and significantly attenuated NAFLD. A pharmacokinetic study in rats showed that the microemulsion had a higher Cmax value for curcumin, a greater AUC0–24 value and a longer t1/2 value for both curcumin and DHA. This investigation provides a novel application of a combination of functional food ingredients to treat chronic liver disease.

Determination of Tyrosine Kinase Inhibitor Icotinib in Rat Plasma using UPLC-MS/MS and its Application In vivo Pharmacokinetic

Objective: The purpose of this study was to develop a UPLC-MS/MS method for the determination of icotinib concentrations in blood plasma. Methods: For plasma sample preparation, protein precipitation with acetonitrile was utilized. Analytes were separated on a Kinetex C18 column using 10 mM ammonium acetate containing 0.2% formic acid and methanol (30:70) as the mobile phase, with a gradient flow rate ranging from 0.2 ml·min-1 to 0.4 ml·min-1. The total chromatographic analysis duration was 4.5 minutes. The UPLC system was connected to a mass spectrometer via an electrospray ionization (ESI) interface operated in positive ion mode. Mass monitoring was conducted in multiple reaction monitoring (MRM) modes, with precursor-to-product transitions being m/z 392.06→304.07 for icotinib and m/z 248.00→120.09 for the internal standard, tinidazole. This method has been used for a pharmacokinetic study in rats that were orally administered a single dose of 30 mg/kg icotinib. Results: The assay showed good linearity over concentration ranges of 1-1000 ng/ml for icotinib, with the correlation coefficient exceeding 0.99. The lower limit of quantitation (LLOQ) was established at 1 ng/ml. Both intra- and inter-day precisions (RSD, %) were below 8.23%. The results demonstrated that stability, matrix effect, extraction recovery, carryover effect and dilution stability were all within the acceptable conditions. The primary pharmacokinetic parameters in SD rats after oral administration of icotinib (30 mg·kg-1 ) were as follows: t1/2 = (2.92 ± 0.87)h, Cmax = (2168.65 ± 268.72)ng/ml, Tmax = (0.70 ± 0.27)h, AUC=(9.69 ± 1.95)ug/mL•h, Vd = (14.51 ± 5.60)L, and CL = (3.19 ± 0.59)L/h. Conclusion: A simple and sensitive UPLC-MS/MS method was developed and validated for the determination of icotinib in pharmacokinetic studies.

Enhancing oral bioavailability of Ca-DTPA by self double emulsifying drug delivery system (SDEDDS)

Objective BCS class III drug (highly soluble, poorly permeable) possesses low oral bioavailability. The research work highlights the utility of self-double emulsifying drug delivery system (SDEDDS) which are stable isotropic mixture of w/o primary emulsion and hydrophilic surfactants for improving oral bioavailability of Ca-DTPA (Calcium diethylenetriamine pentaacetate). Upon oral administration, SDEDDS rapidly emulsifies into w/o/w double emulsions in the aqueous gastrointestinal environment, with hydrophilic drugs entrapped inside oil reservoirs. Methods SDEDDS formulation was successfully developed using excipients, that is, medium chain triglycerides, oleic acid, phospholipids, Span 80, Tween 80 using double emulsification technique. Results The optimized formulation F4 (Aq. phase: 11.6%w,w; MCT & oleic acid: 70.9%w/w; Span 80:17.5%w/w; Lecithin:16%w/w and Tween 80 (10%w/w)) appeared bright yellow liquid which upon dilution appeared milky white within 2 min, droplet size (501.7 nm), pdi value (0.044), zeta potential (-52 mV), entrapment efficiency (79.6 ± 1.63), viscosity (72.2 ± 1.8 mpA.s), significant high cumulative in vitro drug permeation (CDP) and 2.17-fold increase in apparent permeability coefficient. Pharmacokinetic studies in rats showed 1.17-fold increases in AUC of F4 and comparatively higher plasma levels (Cmax) compared with pure drug administered orally. The Absolute (OF4, OD) and Relative bioavailability was found to be 14.52%, 12.35%, and 117.47%, respectively. Conclusion The present studies have clearly demonstrated that SDEDDS could readily form w/o/w double emulsions in vivo with enhanced in vitro and in vivo oral bioavailability. Therefore, considerable augmentation in the rate and extent of oral drug absorption ratified the better performance of the SDEDDS in enhancing the bioavailability of Ca-DTPA.

Applicable Pharmacokinetic Study: Development and Validation of Bioanalytical LC-MS/MS Method for Simultaneous Determination of Tipiracil, Trifluridine and Its Two Metabolites 5-Trifluoromethyluracil, 5-Carboxy 2′-Deoxyuridine in Rat Plasma

A novel liquid chromatography-tandem mass spectrometry (LC-MS/MS) method has been developed and validated for the simultaneous determination of tipiracil (TIP), trifluridine (FTD), and their metabolites, 5-trifluoromethyluracil (FTY) and 5-carboxy-2′-deoxyuridine (5CDU), in rat plasma. This method is highly sensitive, specific, and fast. Paracetamol (PAR) is used as an internal standard (IS). Using acetonitrile-induced protein precipitation, the analytes were extracted from a plasma sample and separated on a Waters BEH C18 (1.7 μm particle size, 50 mm × 2.1 mm ID) column protected by a security guard cartridge (C18, 4 × 2.0 mm). The isocratic mobile phase was made up of methanol and water containing 0.1% formic acid (80:20, v/v) at a flow rate of 0.5 mL/min for 4 min. The quantification was performed using a positive electrospray ionization (ESI) interface and a multiple-reaction monitoring (MRM) mode. The MRM transitions employed were m/z 242.96 → 182.88 for TIP, 296.96 → 116.86 for FTD, 180.98 → 139.85 for FTY, 272.96 → 156.86 for 5CDU, and 151.97 → 92.68 for IS. The validated method complied with the guidelines set by the US-FDA over on a linear concentration range of 5–4000 ng/mL for FTD, FTY, and 5CDU, and 5–1000 ng/mL for TIP. The coefficient of determination (r2) was equal to or greater than 0.997. The corresponding lower limits of detection (LLOD) were 1.5 ng/mL for FTD, FTY, and 5CDU and 1.0 ng/mL for TIP. The recoveries of all analytes from rat plasma ranged from 88.67% to 112.18%, and the mean relative standard deviation (RSD) of accuracy and precision result was less than or equal to 6.84%. FTD, FTY, 5CDU, and TIP demonstrated adequate stability throughout the various circumstances examined. Additionally, no matrix effects were identified for any of the analytes. The assay was effectively utilized to conduct a pharmacokinetic study in rats following the oral administration of FTD and TIP at a dosage of 5.6 mg/kg, with a ratio of 1:0.5 for FTD and TIP, respectively. This indicates that the suggested approach is suitable for future clinical research. The pharmacokinetic parameters Cmax (maximum concentration), Tmax (time to reach maximum concentration), t1/2 (half-life), AUC0-24 (area under the concentration–time curve from 0 to 24 h), AUC total (total area under the concentration–time curve), Ke (elimination rate constant), Vd (volume of distribution), and CL (clearance) of all analytes were assessed. The assay developed exhibits significant advancements compared to earlier bioanalytical methods documented in the literature. These improvements include high sensitivity, specificity, and efficacy in high throughput analysis of complex matrices. Additionally, the assay offers a shorter run time and smaller sample volume (50 μL).

Discovery, Optimization, and Evaluation of Potent and Selective DNA-PK Inhibitors in Combination with Chemotherapy or Radiotherapy for the Treatment of Malignancies.

Given the multifaceted biological functions of DNA-PK encompassing DNA repair pathways and beyond, coupled with the susceptibility of DNA-PK-deficient cells to DNA-damaging agents, significant strides have been made in the pursuit of clinical potential for DNA-PK inhibitors as synergistic adjuncts to chemo- or radiotherapy. Nevertheless, although substantial progress has been made with the discovery of potent inhibitors of DNA-PK, the clinical trial landscape requires even more potent and selective molecules. This necessitates further endeavors to expand the repertoire of clinically accessible DNA-PK inhibitors for the ultimate benefit of patients. Described herein are the obstacles that were encountered and the solutions that were found, which eventually led to the identification of compound 31t. This compound exhibited a remarkable combination of robust potency and exceptional selectivity along with favorable in vivo profiles as substantiated by pharmacokinetic studies in rats and pharmacodynamic assessments in H460, BT474, and A549 xenograft models.

Targeted screening of the synergistic components in Artemisia annua L. leading to enhanced antiplasmodial potency of artemisinin based on a "top down" PD-PK approach

Ethnopharmacological relevanceArtemisinin (ART) showed enhanced antimalarial potency in the herb Artemisia annua L. (A. annua), from which ART is isolated. Increased absorption of ART with inhibited metabolism in the plant matrix is an underlying mechanism. Several synergistic components have been reported based on a "bottom-up" approach, i.e., traditional isolation followed by pharmacokinetic and/or pharmacodynamic evaluation. Aim of the studyIn this study, we employed a "top-down" approach based on in vivo antimalarial and pharmacokinetic studies to identify synergistic components in A. annua. Materials and methodsTwo A. annua extracts in different chemical composition were obtained by extraction using ethyl acetate (EA) and petroleum ether (PE). The synergistic antimalarial activity of ART in two extracts was compared both in vitro (Plasmodium falciparum) and in vivo (murine Plasmodium yoelii). For the PD-PK correlation analysis, the pharmacokinetic profiles of ART and its major metabolite (ART-M) were investigated in healthy rats after a single oral administration of pure ART (20 mg/kg) or equivalent ART in each A. annua extract. A liquid chromatography-tandem high-resolution mass spectrometry (LC-HRMS)-based analytical strategy was then applied for efficient component classification and structural characterization of the differential components in the targeted extract with a higher antimalarial potency. Major components isolated from the targeted extract were then evaluated for their synergistic effect in the same proportion. ResultsCompared with pure ART (ED50, 5.6 mg/kg), ART showed enhanced antimalarial potency in two extracts in vivo (ED50 of EA, 2.9 mg/kg; ED50 of PE, 1.6 mg/kg), but not in vitro (IC50, 15.0–20.0 nM). A significant increase (1.7-fold) in ART absorption (AUC0-t) was found in rats after a single oral dose of equivalent ART in PE but not in EA; however, no significant change in the metabolic capability (AUCART-M/AUCART) was found for ART in either extract. The differential component analysis of the two extracts showed a higher composition of sesquiterpene compounds, especially component AB (3.0% in PE vs. 0.9% in EA) and component AA (14.1% in PE vs. 5.1% in EA). Two target sesquiterpenes were isolated and identified as arteannuin B (AB) and artemisinic acid (AA). The synergism between ART and AB/AA in the same proportion with PE extract (20:1.6:7.6, mg/kg) was verified by a pharmacokinetic study in rats. ConclusionsA "top-down" strategy based on PD-PK studies was successfully employed to identify synergistic components for ART in A. annua. Two sesquiterpene compounds (arteannuin B and artemisinic acid) could enhance the antimalarial potency of ART by increasing its absorption.

Development and validation of synchronous spectrofluorimetric method for the simultaneous determination of duvelisib and moxifloxacin: greenness metric assessment and application to a pharmacokinetic study in rats

Duvelisib (DUV) is a potent anticancer drug whereas Moxifloxacin (MOX) is an antimicrobial drug with anti-proliferative potency against cancerous cells, which is empirically administered in cancer treatment. DUV and MOX combination is commonly prescribed to combat infections in patients while they are under chemotherapy treatment. This study describes, for the first time, the development of a simple and green synchronous spectrofluorimetric (SSF) method for the simultaneous estimation of DUV and MOX in plasma. DUV and MOX were quantified at 273 and 362 nm, respectively without interference between each other at Δλ of 120 nm. The experimental variables influencing fluorescence intensities were thoroughly investigated and the optimum conditions were established. At pH 3.5, the optimum synchronous fluorescence intensity (SFI) was achieved in water solvent by using sodium acetate buffer solution. Calibration curves for DUV and MOX, correlating the SFI with the corresponding drug concentration, were linear in the range of 50–1000 ng mL−1 for both drugs, with good correlation coefficients. The method was extremely sensitive, with limits of detection of 24 and 22 ng mL−1, and limits of quantitation of 40 and 45 ngmL−1 for DUV and MOX, respectively. The SSF method was validated according to the Food and Drug Administration (FDA) guidelines for validation of analytical procedures, and the validation parameters were acceptable. The proposed SSF method was applied to the pharmacokinetic and bioavailability studies in rats’ plasma after single concurrent oral administration of both drugs. The results of the study revealed that caution should be taken with DUV dose when concurrently administered with MOX. The greenness of SSF method was assessed by three different metric tools namely Analytical Eco-scale, Green Analytical Procedure Index, and Analytical Greenness Calculator. The results confirmed that SSF method is an eco-friendly and green analytical approach. In conclusion, the proposed SSF method is a valuable tool for pharmacokinetic/bioavailability studies and therapeutic drug monitoring of simultaneously administered DUV and MOX.

Design of a tulobuterol patch with improved mechanical properties: effect of transdermal permeation enhancers on the release process of metal ligand-based acrylic pressure-sensitive adhesives.

The aim of this study was to design a tulobuterol (TUL) patch with good penetration behavior and mechanical properties. Particular attention was paid to the effect of transdermal permeation enhancers on the release process of metal ligand-based acrylic pressure-sensitive adhesive (AA-NAT/Fe3+). The type and dosage of the enhancers were screened by in vitro transdermal penetration in rat skin. The optimized formulation was evaluated in a pharmacokinetic study in rats. Furthermore, the molecular mechanism by which Azone (AZ) improves the release rate of TUL from AA-NAT/Fe3+ was investigated by FT-IR, shear strength test, rheological study, and molecular simulation. As a result, the optimized formula using AA-NAT/Fe3+ showed better mechanical properties compared to commercial products. Meanwhile, the AUC0-t and Cmax of the optimized patch were 1045 ± 89ng/mL·h and 106.8 ± 28.5ng/mL, respectively, which were not significantly different from those of the commercial product. In addition, AZ increased the mobility of the pressure-sensitive adhesive (PSA) rather than decreasing the drug-PSA interaction, which was the main factor in enhancing TUL release from the patch. In conclusion, a TUL transdermal drug delivery patch was successfully developed using metal-coordinated PSA, and a reference was provided for the design of metal-coordinated acrylic PSA for transdermal patch delivery applications.

Effect of Cimetidine on Metformin Pharmacokinetics and Endogenous Metabolite Levels in Rats.

Tubular secretion is a primary mechanism along with glomerular filtration for renal elimination of drugs and toxicants into urine. Organic cation transporters (OCTs) and multidrug and toxic extrusion (MATE) transporters facilitate the active secretion of cationic substrates, including drugs such as metformin and endogenous cations. We hypothesized that administration of cimetidine, an Oct/Mate inhibitor, will result in increased plasma levels and decreased renal clearance of metformin and endogenous Oct/Mate substrates in rats. A paired rat pharmacokinetic study was carried out in which metformin (5 mg/kg, intravenous) was administered as an exogenous substrate of Oct/Mate transporters to six Sprague-Dawley rats with and without cimetidine (100 mg/kg, intraperitoneal). When co-administered with cimetidine, metformin area under the curve increased significantly by 3.2-fold, and its renal clearance reduced significantly by 73%. Untargeted metabolomics was performed to investigate the effect of cimetidine on endogenous metabolome in the blood and urine samples. Over 8,000 features (metabolites) were detected in the blood, which were shortlisted using optimized criteria, i.e., a significant increase (P value < 0.05) in metabolite peak intensity in the cimetidine-treated group, reproducible retention time, and quality of chromatogram peak. The metabolite hits were classified into three groups that can potentially distinguish inhibition of i) extra-renal uptake transport or catabolism, ii) renal Octs, and iii) renal efflux transporters or metabolite formation. The metabolomics approach identified novel putative endogenous substrates of cationic transporters that could be tested as potential biomarkers to predict Oct/Mate transporter mediated drug-drug interactions in the preclinical stages. SIGNIFICANCE STATEMENT: Endogenous substrates of renal transporters in animal models could be used as potential biomarkers to predict renal drug-drug interactions in early drug development. Here we demonstrated that cimetidine, an inhibitor of organic cation transporters (Oct/Mate), could alter the pharmacokinetics of metformin and endogenous cationic substrates in rats. Several putative endogenous metabolites of Oct/Mate transporters were identified using metabolomics approach, which could be tested as potential transporter biomarkers to predict renal drug-drug interaction of Oct/Mate substrates.

A chitosan/alginate coated nano-liposome to improve intestinal absorption of curcumin for oral administration.

Attempts to improve low absorption and rapid metabolic conversion of curcumin were made by developing curcumin-loaded bilayer nanoliposomes coated with chitosan and alginate for intestinal-specific drug delivery. A curcumin-loaded nano-liposome was prepared with optimized formulations with phosphatidylcholine, curcumin, chitosan, and alginate. The particle size of the optimized formulation was approximately 400nm, and the encapsulation efficiency was more than 99%. In the in vitro release study, curcumin release from the curcumin-loaded nanoliposome with double layers of chitosan/alginate (CNL-CH/AL) was suppressed in the simulated gastric fluid (SGF, pH 1.2) and enhanced in the simulated intestinal fluid (SIF, pH 6.8). In the in vivo pharmacokinetic study in rats, the CNL-CH/AL-treated group showed a prolonged absorption pattern of curcumin and the area under the plasma concentration-time curve from 0 to 24h (AUC0-24) was improved 109-fold compared to the control group treated with a curcumin solution without a nanocarrier.

Atheroprotective CD40-TRAF6 inhibitors: translational pipeline towards a clinical application

Abstract Background The co-stimulatory CD40–CD40 ligand axis is central in atherogenesis. Upon activation, CD40 recruits tumor necrosis factor receptor-associated factors (TRAFs) to induce downstream signaling. Murine studies with mutated CD40-TRAF binding sites in macrophages have shown that the CD40-TRAF6, rather than CD40-TRAF2/3/5, axis is crucial for atherosclerosis. We thus synthesized the small molecule inhibitor TRAF-STOP 6877002, that selectively blocks CD40-TRAF6 signaling. 6877002 successfully reduced the onset and development of atherosclerosis by reducing monocyte recruitment and macrophage activation in ApoE-/- mice without having immunosuppressive side effects. Purpose To assess whether 6877002 is a suitable candidate to pass the translational pipeline towards a clinical application. Methods Elaborate in vitro and in vivo assays evaluating the pharmacological properties and potential toxicity of 6877002. Results Together with Cyprotex, we performed in vitro assays examining the ADME profile of 6877002. In an intestinal permeability assay, 6877002 had low to moderate resorption with no significant efflux, suggesting it is not a substrate for the efflux transporters Pgp/BCRP or MRP2. A plasma protein binding assay showed very low unbound 6877002 fractions, indicating that it is highly bound to plasma proteins. The potential to inhibit cytochrome P450 enzymes was assessed using human liver microsomes in the presence of enzyme-specific probe substrates. While 6877002 inhibited CYP1A2 (IC50=4.01 µM), no potential for inhibiting other CYP isoforms was detected, suggesting low drug-drug interaction potential. To screen for putative cardiovascular risks, hERG ion channel inhibition was assessed and no IC50 could be determined. Hepatic clearance was rapid in mice, rats and dogs, while minipig and human hepatocytes had lower intrinsic clearance. Together with Aptuit, we developed suitable formulations for oral and i.v. administration. In vivo pharmacokinetics studies in rats showed low bioavailability (8.43%) and rapid clearance of orally administered 6877002 compared to i.v. injected 6877002, likely – and in line with our in vitro data – due to fast first-past effect in mouse hepatocytes and uptake by macrophages. To explain the high efficacy of 6877002 despite its rapid metabolism, we assessed whether 6877002 has active metabolites that bind the CD40-TRAF6 pocket and performed an LC-MS/MS-based metabolite profiling. We detected 17 metabolites. Further MD simulations confirmed that Metabolite (M)4, the most abundant metabolite, binds the CD40-TRAF6 pocket, reflected by the highest binding mode (-19.6; defined as Binding Free Energy derived from 40-50 ns of MD run as compared to -16.5 for 6877002). Similar to 6877002, M4 reduced atherosclerosis by 54% in Western diet-fed ApoE-/- mice, indicative of M4 being an active metabolite. Conclusion CD40-TRAF6 inhibitors are an attractive strategy to combat atherosclerosis with high translational potential.