Rat Plasma Proteins Research Articles

Development and validation of LC-MS/MS method for estimating the pharmacokinetics, protein binding, and metabolic stability of soluble epoxide hydrolase inhibitor EC5026

EC5026 is a soluble epoxide hydrolase (SEH) inhibitor which is being developed clinically (Phase-1) as a first-in-class analgesic for the treatment of pain. In the present study, we report the development and validation of the LC-MS/MS method for the estimation of EC5026 from rat plasma. The developed method is simple, specific, and sensitive for the quantification of EC5026 from rat plasma. The method was applied to investigate in-vivo pharmacokinetics after single oral administration (P.O.) of EC5026 (5.0 mg/kg) in Wistar rats. Further, the in-vitro metabolic stability and rat plasma protein binding of EC5026 were evaluated using the developed method. The in-vitro results demonstrated a moderate clearance with a value of 10.8 mL/min/kg and half-life (t1/2) of 57.75 min. The results show moderate clearance by the liver manifesting in satisfactory oral bioavailability. The rat plasma protein binding was estimated to be 96.24% ± 0.97% and 96.38% ± 0.56% at 1 µM and 10 µM concentrations, respectively. The developed analytical method is expected to facilitate future pre-clinical, clinical investigations of EC5026.

Evidence of nerve agent VX exposure in rat plasma by detection of albumin-adducts in vitro and in vivo

VX is a highly toxic organophosphorus nerve agent that reacts with a variety of endogenous proteins such as serum albumin under formation of adducts that can be targeted by analytical methods for biomedical verification of exposure. Albumin is phosphonylated by the ethyl methylphosphonic acid moiety (EMP) of VX at various tyrosine residues. Additionally, the released leaving group of VX, 2-(diisopropylamino)ethanethiol (DPAET), may react with cysteine residues in diverse proteins. We developed and validated a microbore liquid chromatography-electrospray ionization high-resolution tandem mass spectrometry (µLC-ESI MS/HR MS) method enabling simultaneous detection of three albumin-derived biomarkers for the analysis of rat plasma. After pronase-catalyzed cleavage of rat plasma proteins single phosphonylated tyrosine residues (Tyr-EMP), the Cys34(-DPAET)Pro dipeptide as well as the rat-specific LeuProCys448(-DPAET) tripeptide were obtained. The time-dependent adduct formation in rat plasma was investigated in vitro and biomarker formation during proteolysis was optimized. Biomarkers were shown to be stable for a minimum of four freeze-and-thaw cycles and for at least 24 h in the autosampler at 15 °C thus making the adducts highly suited for bioanalysis. Cys34(-DPAET)Pro was superior compared to the other serum biomarkers considering the limit of identification and stability in plasma at 37 °C. For the first time, Cys34(-DPAET)Pro was detected in in vivo specimens showing a time-dependent concentration increase after subcutaneous exposure of rats underlining the benefit of the dipeptide disulfide biomarker for sensitive analysis.

Prospective Validation of Machine Learning Algorithms for Absorption, Distribution, Metabolism, and Excretion Prediction: An Industrial Perspective.

Absorption, distribution, metabolism, and excretion (ADME), which collectively define the concentration profile of a drug at the site of action, are of critical importance to the success of a drug candidate. Recent advances in machine learning algorithms and the availability of larger proprietary as well as public ADME data sets have generated renewed interest within the academic and pharmaceutical science communities in predicting pharmacokinetic and physicochemical endpoints in early drug discovery. In this study, we collected 120 internal prospective data sets over 20 months across six ADME in vitro endpoints: human and rat liver microsomal stability, MDR1-MDCK efflux ratio, solubility, and human and rat plasma protein binding. A variety of machine learning algorithms in combination with different molecular representations were evaluated. Our results suggest that gradient boosting decision tree and deep learning models consistently outperformed random forest over time. We also observed better performance when models were retrained on a fixed schedule, and the more frequent retraining generally resulted in increased accuracy, while hyperparameters tuning only improved the prospective predictions marginally.

Utilization of Methionine by the Adult Rat: II. Absorption and Tissue Uptake of L- and DL-Methionine

The absorption of l- and dl-methionine-C14H3 from the alimentary tract and the distribution of radiocarbon from these sources and dl-methionine-2-C14 in blood components and 18 tissues of rats were investigated. Small, but statistically significant differences in the absorption and tissue uptake of l- and dl-methionine were observed in these experiments. The absorption of l-methionine is calculated to be 1.1 times that of its d- isomer.After 30 minutes, the plasma and plasma proteins of rats fed l-methionine-methyl-C14 contained greater activity than the corresponding components of rats fed the dl-form of this compound. More radiocarbon was observed in plasma, protein-free plasma, and plasma protein of rats fed dl-methionine-2-C14 than in those components of rats fed the methyl-labeled dl-compound.The variation in tissue response of individual animals similarly treated to uniform doses of l- and dl-methionine-methyl-C14 and dl-methionine-2-C14 has been measured. During a 30-minute interval, the greatest amount of variability in uptake of l-methionine occurred in pancreas, liver, bone marrow and thyroid.Significantly greater concentrations of radiocarbon were present in brain, lungs, liver, and heart of rats fed l-methionine-methyl-C14 than in those tissues of rats fed the dl- form of this amino acid. The concentration of the methyl carbon of methionine in liver was significantly greater than that of the alpha-carbon.

Development of Combretastatin A-4 Analogues as Potential Anticancer Agents with Improved Aqueous Solubility.

Combretastatin A-4 (CA-4) is a potent tubulin polymerisation inhibitor. However, the clinical application of CA-4 is limited owing to its low aqueous solubility and the easy conversion of the olefin double bond from the more active cis- to the less active trans-configuration. Several structural modifications were investigated to improve the solubility of CA-4 derivatives. Among the compounds we synthesized, the kinetic solubility assay revealed that the solubility of compounds containing a piperazine ring increased the most, and the solubility of compounds 12a1, 12a2, 15 and 18 was increased 230-2494 times compared with that of the control compound (Z)-3-(4-aminophenyl)-2-(3,4,5-trimethoxyphenyl)acrylonitrile (9a). In addition, these synthesised stilbene nitriles had high anticancer cell (AGS, BEL-7402, MCF-7, and HCT-116) selectivity over L-02 and MCF-10A normal cells while maintaining micromolar activity against cancer cells. The most cytotoxic compound is 9a, and the IC50 value is 20 nM against HCT-116 cancer cells. Preliminary studies indicated that compound 12a1 had excellent plasma stability and moderate binding to rat plasma proteins, suggesting it is a promising lead compound for the development of an anticancer agent.

Developmental changes in the extent of drug binding to rat plasma proteins

Binding of therapeutics to proteins in blood plasma is important in influencing their distribution as it is their free (unbound) form that is able to cross cellular membranes to enter tissues and exert their actions. The concentration and composition of plasma proteins vary during pregnancy and development, resulting in potential changes to drug protein binding. Here, we describe an ultrafiltration method to investigate the extent of protein binding of six drugs (digoxin, paracetamol, olanzapine, ivacaftor, valproate and lamotrigine) and two water soluble inert markers (sucrose and glycerol) to plasma proteins from pregnant and developing rats. Results showed that the free fraction of most drugs was lower in the non-pregnant adult plasma where protein concentration is the highest. However, plasma of equivalent protein concentration to younger pups obtained by diluting adult plasma did not always exhibit the same extent of drug binding, reinforcing the likelihood that both concentration and composition of proteins in plasma influence drug binding. Comparison between protein binding and brain drug accumulation in vivo revealed a correlation for some drugs, but not others. Results suggests that plasma protein concentration should be considered when using medications in pregnant and paediatric patients to minimise potential for fetal and neonatal drug exposure.

Pharmacokinetics, bioavailability, and plasma protein binding study of glytrexate, a novel multitarget antifolate.

Glytrexate, developed by our team, as a novel multitarget folate antagonist, has inhibitory effects on a variety of cancer cell types, especially KB tumor cells (IC50 0.078 nM), and thus has antitumor drug development prospects. However, its pharmacokinetics and plasma protein binding properties remain unknown. In this study a selective and sensitive liquid chromatography-tandem mass spectrometry (LC‒MS/MS) method was developed and verified to facilitate biological analysis. The bioanalysis method was applied to evaluate the stability, plasma protein binding, and pharmacokinetics of glytrexate. Glytrexate is more stable in human plasma than in rat plasma and in human liver microsomes. The binding of glytrexate to human plasma proteins was higher than that to rat plasma proteins, both of which were less than 30%, suggesting that glytrexate may be at a higher concentration at the pharmacologic target receptor(s) in tissues. Pharmacokinetic characteristics were determined by noncompartmental analysis after administration of single oral (12.5, 25 and 50 mg/kg) and intravenous (2 mg/kg) doses in rats. According to the rat oral pharmacokinetic characteristics, glytrexate had linear dynamics in a dose range of 12.5–50 mg/kg and a poor oral bioavailability of 0.57–1.15%. The investigation revealed that the intravenous half-life, AUC, and Cmax of glytrexate were higher than those of pemetrexed. Pemetrexed is generally produced as an injection preparation. This provides ideas for the development of glytrexate formulations. Therefore, glytrexate injection has clinical application prospects compared to oral administration. This study provides a basis for further investigations into the pharmacological effects and clinical uses of glytrexate.

Plasma proteomic analysis of diabetes improvement by vitamin D supplementation

To analyze the effect of vitamin D supplementation on the improvement of diabetes mellitus based on plasma proteomics. Five-week-old SPF spontaneously obese rats with type 2 diabetes were randomly divided into a diabetic group and a diabetic vitamin D intervention group, and the control group was Zucker lean rats. The fasting blood glucose of the rats in each group was compared with that of the diabetic vitamin D group, and the plasma proteins of the rats in each group were compared by quantitative analysis of the high-resolution mass spectrometry system iTRAQ, and KEGG signaling pathway analysis was performed. The fasting blood glucose of rats in the diabetic vitamin D intervention group was significantly lower than that of the diabetic group, and the proteins that were differentially expressed in the diabetic vitamin D intervention group were significantly improved. KEGG signaling pathway analysis revealed that the differential proteins in the diabetic group were mainly distributed among enzymes, exosomal proteins, and peptidases and inhibitors, and that the number of differences in these three classes of proteins was significantly reduced in the diabetic intervention group. Vitamin D supplementation can improve the differential expression of fasting glucose and plasma proteins in the diabetic rats.

Rat plasma protein binding of kaempferol-3-O-rutinoside from Lu'an GuaPian tea and its anti-inflammatory mechanism for cardiovascular protection.

Previous study found a high content of kaempferol-3-O-rutinoside (KR) in Lu'an GuaPian tea, however, the rat plasma protein binding and mechanism of KR for cardiovascular protection are unclear. Thus, we studied plasma protein binding using ultrafiltration followed by UPLC, and screened its inhibition against LPS-induced inflammation injury in vitro as well as the underlying mechanism by molecular docking and western blot. KR showed over 74% plasma protein binding ratio. Furthermore, KR may act on the toll-like receptor 4 (TLR4) and myeloid differentiation factor 88 (MyD88). In vitro experiments showed that KR decreases the overexpression of TLR4, MyD88, and nuclear factor-κB (NF-κB), which further validates the molecular docking results, suggesting that KR could block TLR4/MyD88/NF-κB signaling. These results indicate that KR could be a potential active agent in the protection of myocardial injury. PRACTICAL APPLICATIONS: Health benefits of tea are largely dependent on the intake of flavonoids. Flavonoids are a group of compounds beneficial to cardiovascular disease and an important part of "functional foods." Lu'an GuaPian tea is mainly produced in Lu'an City, Anhui Province and is one of the top 10 famous teas in China. Kaempferol-3-O-rutinoside in Lu'an GuaPian has good hypoglycemic effect, mainly manifested in a strong inhibition of α-glucosidase and α-amylase activities. Present study showed that kaempferol-3-O-rutinoside could block TLR4/MyD88/NF-κB signaling, suggesting that it could be a potential active agent in the protection of myocardial injury.

Tailor-made deep eutectic solvents extraction combined with UPLC-MS/MS determination of icarrin and icarisid II in rat plasma and its comparative pharmacokinetic application

Using green and high efficient solvents to extract and trace active ingredients of traditional Chinese medicine (TCM) in the complex biological samples was still challenging. In this paper, a co-friendly, fast pretreatment method with high extraction efficiency, based on the tailor-made deep eutectic solvent (DES) system, combined with ultra performance liquid chromatography-triple quadrupole tandem mass spectrometry (UPLC-MS/MS) was developed and validated for the determination of icarrin and icarisid II in rat plasma samples, which can be further applied to comparative pharmacokinetic studies after oral administration of Herba Epimedii and icarrin monomer in rats, respectively. PrE (l-proline: ethylene glycol = 1:4 mol/mol) and acetonitrile were optimized and combined as the tailor-made DES at the volumetric ratio of 3:7 to extract icarrin and icarisid II, and to precipitate the protein in rat plasma in one step simultaneously. The extraction efficiency of the tailor-made DES was about 1.7 times of DES (PrE). The extraction recovery of icarrin and icarisid II in rat plasma samples by this method were within the range of 90–110 %, and the lower limits of quantification (LLOQ) were 0.32 ng mL−1 (icarrin) and 0.43 ng mL−1 (icarisid II). There was a linear relationship between 0.32−80.16 ng mL−1 (icarrin) and 0.43−107.4 ng mL−1 (icarisid II), which effectively reduced the detection limit. In this comparative pharmacokinetic study, the maximum plasma concentration (Cmax) and the area under plasma concentration-time curve (AUC0-∞) of two analytes in rat plasma of Herba Epimedii group were both much higher than those in the icarrin monomer group, which suggested that other ingredients in Herba Epimedii may contribute to the in vivo absorption of icarrin and icarisid II. This simple, rapid, relatively green and high effeicient method would provide a new approach for the extraction of active ingredients from complex biological samples.

THE EFFECT OF INTRAGASTRICALLY ADMINISTERED SESAME (SESAMUM INDICUM L) OIL ON GROWTH RATE AND SOME BIOCHEMICAL PROFILE OF RATS FED A HIGH-FAT DIET.

The effect(s) of sesame (Sesamum Indicum L) seed oil on the growth rate, performance and some biochemical parameters in adult albino (Wister) rats maintained on a high fat diet was examined. Adult rats maintained on a high fat diet were intragastrically fed 10mg/ml of sesame seed oil suspended in normal saline, daily for 60 days. Two other groups maintained on the high fat diet only (High fat diet control) and the normal diet (Normal diet control) were included. Water and diet were provided ad libitum. The rats were weighed weekly, while feed supply and left-overs were recorded daily. At 60 days, all animals were killed by decapitation. Blood and liver samples were taken for biochemical analyses. Feeding high fat diet significantly (P<0.05) increased the final body weight, liver and kidney weights of the rats compared to those on normal diet. Administration of sesame seed oil along with high fat diet further increased (P <0.05) the final rat body weight while decrease (P<0.05) was observed in the liver weight and a significant (P 0.05) decrease in the kidney weight was observed compared to the high fat diet fed rats. Biochemical profile shows significantly (P<0.01) higher levels of plasma proteins and globulins in the normal diet-fed rats compared to the high fat-diet-fed rats. Administration of sesame seed oil along with high fat diet significantly (P<0.01) increased the plasma glucose, total proteins, globulins and uric acid concentrations compared to the high fat diet fed rats. Significantly (P<0.01) higher glucose and uric acid levels and a significantly (P<0.01) lower globulin levels were observed in the sesame seed oil group compared to the normal diet control group. The plasma albumin concentration did not differ significantly among the groups.

Протеїнограма крові тварин в стані штучного гіпобіозу за ешерихіозу

The features of fractional distribution of plasma proteins in rats with Escherichia coli at different stages of infection are established. It was studied the effect of hypobiosis on the proteinogramma under and after artificial sleep too. Artificial hypobiosis is important for therapeutic use at bacterial infection and proteinogramma is informatively significant as a diagnostic element of the functional changes provoked by a hypobiosis. We used the nephelometric method. Experiments were performed on young rats with Escherichia coli, in a state of hypobiosis and one day after leaving it. It was established the infection provoked changes in plasma proteins such as increasing globulins and decreasing albumin at the beginning of the disease and the opposite trend during the period of total depletion. The introduction of sick animals into a state of hypobiosis causes an increase in nonspecific resistance of the organism level due to an increase in the content of γ-globulin fraction and a decrease in the level of albumin, which increases the survival of animals after removal from hypobiosis. Indicators of the proteinogram of blood of animals that were introduced into the state of hypobiosis at the initial stage of the disease and the stage of progressive symptoms return to control values on the first day after the elimination of hypobiosis factors. Further research should be aimed at a comprehensive study of functional changes in the body according to the proposed model.

Abstract 578: Pharmacokinetic proprieties of a locked nucleic acid oligonucleotide targeting miR-221

Abstract MiR-221 is overexpressed in several malignancies where it promotes tumor growth and survival by interfering with gene transcripts, including p27Kip1, PUMA, PTEN, and p57Kip2. We previously demonstrated that a novel 13-mer miR-221 inhibitor (LNA-i-miR-221) exerts antitumor activity against human cancer with a pilot favorable pharmacokinetics and safety profile in mice and non-naive monkeys. Next we performed and here report a non-GLP/GLP dose-finding investigation of LNA-i-miR-221 in Sprague Dawley rats. The safety of the intravenous dose (125 mg/kg/day) for four consecutive days, two treatment cycles, was investigated by a first non-GLP study. Toxicokinetics profile of LNA-i-miR-221 was next explored in a GLP study at three different doses (5, 12.5 and 125 mg/kg/day). Slight changes in blood parameters and histological findings in kidney were observed at the highest dose. These effects were reversible and consistent with in vivo ASO class effect. Based on these findings the NOAEL was established at 5 mg/kg/day. The plasma exposure of LNA-i-miR-221, based on C0 and AUC, suggested no differential sex effect. Slight accumulation occurred between cycles one and two but was not observed after four consecutive administrations. These findings demonstrate a safety profile of LNA-i-miR-221 in Sprague Dawley rats and provide a reference translational framework and path for the development of other LNA miR inhibitors in Phase I clinical study. In addition to understand the pharmacokinetics properties of this new agent, including unbound/total clearance, we investigated the LNA-i-miR-221 protein binding in three different species including rat (Sprague Dawley), monkey (Macaca fascicularis) and human. To this aim, we generated a suitable ultrafiltration method to study the binding of LNA-i-miR-221 to plasma proteins. We identified that the fraction of LNA-i-miR-221 (at concentration of 1 and 10 µM) bound to rat, monkey and human plasma proteins is high and ranges from 98.2 to 99.05%. This high protein binding of LNA-i-miR-221 to plasma proteins in all the species tested translates into a pharmacokinetic advantage by preventing rapid renal clearance. The integration of these results into multiple allometric interspecies scaling methods have been then used to draw inferences about LNA-i-miR-221 pharmacokinetics in humans, therefore providing a framework for definition of safe starting and escalation doses toward a first-in-human clinical trial of LNA-i-miR-221. This work was supported by the Italian Association for Cancer Research (AIRC), PI: PT. “Special Program Molecular Clinical Oncology - 5 per mille" n. 9980, 2010/15 and its Extension Program 2016/17 n.9980. Citation Format: Maria Teresa Di Martino. Pharmacokinetic proprieties of a locked nucleic acid oligonucleotide targeting miR-221 [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 578.

Plasma protein binding, metabolism, reaction phenotyping and toxicokinetic studies of fenarimol after oral and intravenous administration in rats

Fenarimol (FNL), an organic chlorinated fungicide, is widely used in agriculture for protection from fungal spores and fungi. Despite being an endocrine disruptor, no toxicokinetic data is reported for this fungicide. In the present work, we determined the plasma protein binding, metabolic pathways and toxicokinetics of FNL in rats. In vitro binding of FNL to rat and human plasma proteins was ∼90%, suggesting that FNL is a highly protein bound fungicide. The predicted in vivo hepatic clearance of FNL in rats and humans was estimated to be 36.71 and 14.39 mL/min/kg, respectively, indicating it to be an intermediate clearance compound. Reaction phenotyping assay showed that CYP3A4 mainly contributed to the overall metabolism of FNL. The oral toxicokinetic study of FNL in rats at no observed adverse effect level dose (1 mg/kg) showed maximum plasma concentration (C max) of 33.97 ± 4.45 ng/mL at 1 h (T max). The AUC0–∞ obtained was 180.18 ± 17.76 h*ng/mL, whereas, the t 1/2 was ∼4.74 h. Following intravenous administration, FNL displayed a clearance of 42.48 mL/min/kg which was close to the predicted in vivo hepatic clearance. The absolute oral bioavailability of FNL at 1 mg/kg dose in rats was 45.25%. FNL at 10 mg/kg oral dose exhibited non-linear toxicokinetics with greater than dose-proportional increase in the systemic exposure (AUC0–∞ 8270.53 ± 1798.59 h*ng/mL).

Avocado Pear Pulp (Persea americana)-Supplemented Cake Improved Some Serum Lipid Profile and Plasma Protein in Rats.

Avocado pear pulp (Persea americana) is indigenous to Mexico. It is widely cultivated and consumed all over the world. Its oil is rich in monounsaturated fatty acids (SFAs) and can be used to replace SFAs in a diet to lower low-density lipoprotein (LDL) and raise high-density lipoprotein (HDL). Consumers seek healthy foods that aid in reducing metabolic syndrome. The aim of this study is to evaluate the effects of the best acceptable cakes supplemented with 0%, 10%, 30%, and 50% avocado on the serum lipid profile and plasma protein in rats. Twenty-five rats separated into five groups of four test groups and one control group were fed individually for 14 days, after which blood samples were withdrawn and subjected to biochemical analysis. Intake of cake supplemented with avocado pear pulp resulted in significant (P < .05) increment in the body weight, total cholesterol, triglycerides and HDL, and serum protein and decrement in LDL of the rats. The group fed 50% supplemented cake showed highest increase in HDL and least decrease in LDL. Cake supplemented with 50% avocado pear pulp proved to be a better supplementation in reducing serum LDL and increasing serum HDL and serum protein in rats, indicating to be a promising nutraceutical for the management of cardiovascular diseases and its associated complications.

Absorption, Distribution and Excretion of [14C] Zamicastat after Single Oral and Intravenous Administration in Rats and Dogs

BackgroundZamicastat is a dopamine‐β‐hydroxylase inhibitor (1), previously showed to increase the survival rate in the monocrotaline rat model of pulmonary arterial hypertension.ObjectivesThe purpose of this study was to characterize the absorption, distribution and elimination of 14C‐zamicastat in rat and dog, after single oral (p.o.) or intravenous (i.v.) administration.MethodsWistar rats and Beagle dogs were administered with 14C‐zamicastat at 30 mg/kg orally or intravenously with 2.5 mg/kg (rat) and 0.1 mg/kg (dog). Plasma, urine and faeces samples were collected at different time‐points to measure the plasma exposure and the recovery of total radioactivity up to 168 h post‐dose. Whole body phosphor imaging (WBPI) was evaluated in rat after p.o. administration of 30 mg/kg of 14C‐zamicastat. The extent of plasma protein binding was determined by equilibrium dialysis and blood cell binding was determined, using centrifugation, on pooled male samples from each species.ResultsAfter single oral dosing of 14C‐zamicastat, more than 95% of total radioactivity was recovered at five and seven days from rats and dogs, respectively. The radioactivity peaked in plasma between 6 to 8 h and 2 to 4 h post‐dosing in rats and dogs, respectively, followed by a slow elimination over time. Zamicastat related radioactivity was mainly recovered in faeces with very low levels (less than 20% of total radioactivity) excreted in urine in both species. Following i.v. administration more than 80% of radioactivity was recovered in both species mainly via feaces excretion. The WBPI study indicates that radioactivity was distributed throughout the body with the majority of tissues achieving a tissue:blood ratio greater than 2. Elimination of radioactivity progressed over time with most tissues reaching the low limit of quantification at 168h. The highest tissue:blood ratios were noted in thyroid gland, lung and harderian gland at, respectively, 168h, 8h and 8h post‐dosing. Protein binding data indicates that zamicastat is preferentially bound to plasma proteins in a concentration independent manner, with 98% and 96% bound to rat and dog plasma proteins, respectively.ConclusionZamicastat is a highly plasma protein bound compound after oral administration and it is extensively distributed to tissues. After oral administration to rats and dogs, radioactivity was almost completely recovered with most of zamicastat‐related radioactivity excreted through faeces.

Mathematical Modeling of In Vitro‐In Vivo Atypical Kinetics of Diazepam

Cytochrome P450 (CYP) and uridine 5'‐diphospho‐glucuronosyltransferase (UGT) enzymes are responsible for metabolism of most marketed drugs. Due to the large binding sites of certain CYPs and UGTs, CYP and UGT substrates tend to show non ‐ Michaelis‐Menten kinetics (atypical kinetics). Although several in vitro data provide evidence for the existence of atypical kinetics, this characteristic is seldom observed with in vivo data. The purpose of this project is to characterize the atypical kinetics of diazepam and its sequential metabolism in in vitro and in vivo systems. Numerical methods will be used to obtain kinetic parameters, which will be used for in vitro‐in vivo correlations (IVIVC).Diazepam, which exhibits atypical kinetics in vitro, and its primary metabolites (temazepam, nordiazepam), and secondary metabolite (oxazepam) were characterized with in vitro studies with pooled male Sprague Dawley rat liver microsome (MRLM), pooled female Sprague Dawley (FRLM), and purified rat cyp isoforms (cyp3a1, cyp3a2, and cyp2c11) separately. Rat microsomal partition and rat plasma protein binding assays were conducted via equilibrium dialysis to determine the free fraction of the drug. Two substrate binding (ESS) models provided better fits than one substrate (ES) models, as determined from the resulting AICc values. Preliminary in vitro data from MRLM and cyp3a1 showed obvious sigmoidal saturation curves for temazepam formation. Only cyp3a1 show significant sigmoidal curves for nordiazepam formation. For cyp3a1, Km and Vmax values for temazepam and nordiazepam calculated with the EES model are significantly different from those with the ES model. The intrinsic clearance (CLint) values calculated from the MRLM and cyp3a1 ESS models is not constant within the observed range of diazepam plasma levels of rats. No obvious sigmoidicity of diazepam saturation curves was observed for cyp3a2 (male‐specific) and cyp2c11 (male‐specific) isoforms, which are other cyp isoforms responsible for diazepam metabolism in male rats. In order to characterize in vitro‐in vivo correlations for diazepam, in vivo PK studies with female and male rats are currently underway.Support or Funding InformationThis research was supported by NIGMS grants: R01GM114369 and R01GM104178.

Identification of the differentially expressed protein biomarkers in rat blood plasma in response to gamma irradiation

Purpose: Simple, rapid and high-throughput dose assessment is critical for clinical diagnosis, treatment and emergency intervention in a large-scale radiological accident. The goal of this study is to screen and identify new ionizing radiation-responsive protein biomarkers in rat plasma.Materials and methods: Sprague-Dawley rats were exposed to single doses of 0, 1, 3, 5 Gy of Cobalt-60 γ-rays total body irradiation at a dose rate of 1 Gy/min. The tandem mass tag labeling (TMT) combined with liquid chromatography mass spectrometry (LC-MS/MS) approach was used to screen the differentially expressed proteins in rat plasma collected at 1, 3, 5 and 7 days post-irradiation. Bioinformatics analysis was conducted to explore the biological functions of these proteins. The expression levels of candidate radiation-sensitive protein biomarkers were confirmed using enzyme-linked immune-sorbent assay (ELISA).Results: A total of 503 differentially expressed proteins were identified. Most of these proteins were implicated in immune response, phagocytosis and signal transduction following ionizing radiation. Five up-regulated proteins including alpha-2-macroglobulin (A2m), chromogranin-A (CHGA), glutathione pertidase 3 (GPX3), clusterin (Clu) and ceruloplasmin (Cp) were selected for ELISA analysis. It was found that the expression levels of A2m, CHGA and GPX3 protein were increased in a dose-dependent manner at 1, 3 and 5 days after irradiation.Conclusion: Proteomics analysis revealed radiation-induced differentially expressed proteins in rat plasma. Our results suggested that A2m, CHGA, GPX3 protein expressions alterations in rat plasma may have potential as biomarkers to evaluate radiation exposure.

Lablab purpureus (L) bean flour ameliorates plasma proteins and accretion of docosahexaenoic acid (DHA, 22:6, ω‐3) in the plasma, liver, and brain of malnourished rats

AbstractLablab purpureus (L) bean is an undervalued or underutilized orphan crop in many tropical countries, where different forms of malnutrition are associated with stunting growth and cognitive deficiencies. We previously reported that L. purpureus contains ω‐3 α‐linolenic acid (ALA, C18:3, ω‐3), which can act as the precursor of ω‐3 docosahexaenoic acid (DHA, C22:6, ω‐3). Inadequate level of DHA impairs growth, development, and cognitive performance. Therefore, we evaluated if supplementation of L. purpureus seed flour (LPS) affects the nutritional status, in terms of body weight gain, plasma proteins, and DHA levels of malnourished model rats. Three groups of rats, namely, controls, malnourished alone (MN), and 15% LPS‐supplemented malnourished (MN + LPS) rats were fed with LPS for 12 weeks. Afterward, body weight, liver weight, brain weight, plasma proteins, micronutrients, lipid profile, and fatty acid profile of plasma, liver, and brains were determined by standard methods. The levels of liver lipid peroxide (LPO) and proinflammatory TNFα were also measured. The body weight, liver weight, serum total proteins and micronutrients (iron/potassium), and the levels of docosahexaenoic acid significantly increased in the plasma, liver, and brain of MN + LPS rats. Moreover, LPO and TNFα levels reduced significantly in MN + LPS rats. in vitro analysis revealed a significant free radical scavenging and antioxidative potential of L. purpureus seed extract. Thus, L. purpureus not only replenishes protein‐energy malnutrition, but also increases the levels of DHA, an indispensable polyunsaturated fatty acid for brain cognition. Finally, our results suggest that L. purpureus might benefit human malnutrition and related cognitive deficits.

Adrenergic alpha-2 receptor antagonists cease augmented oxidation of plasma proteins and anxiety of rats caused by chronic noise exposure.

Background:Noise is one of the environmental factors, which is considered as a powerful stressor for the organism. Generally, the acoustic stress affects the behavior and physiological state of humans and animals.Aims:The goal of this study is to investigate the relationship between chronic noise exposure and the effects of adrenergic alpha-2 receptor antagonists, beditin and mesedin, on the anxiety and oxidation of plasma proteins and fibrinogen in rats.Methods:The experiments were carried out on non-linear albino male rats, divided into four groups (six animals in each): 1. Healthy controls 2. Exposed to noise of a level 91 dB(A), eight hours daily, during 7, 30 and 60 days; 3. Injected with 2 mg/kg of beditin (2-(2-amino-4-thiazolyl)-1,4-benzodioxane hydrochloride)); 4. Injected with 10 mg/kg mesedin (2-(2-methyl-amino-thiozolyl)-1,4-benzodioxane hydrochloride). For evaluating the cognitive impairment, the Any-maze test was applied. The level of carbonylation of proteins was assessed by reaction with 2,4-dinitrophenylhydrazine, spectrophotometrically.Results:Chronic noise decreased locomotor activity and increased anxiety and oxidation of plasma protein and fibrinogen. Intensity of these changes were dependent on the duration of noise exposure.Conclusion:The Alpha 2 adrenoblockers alleviate oxidative modification of plasma proteins and reduce the cognitive impairment caused by chronic exposure to noise.