Pharmacokinetic Screening Research Articles

POTENTIAL ANTIVIRAL OF CATECHINS AND THEIR DERIVATIVES TO INHIBIT SARS-COV-2 RECEPTORS OF M pro PROTEIN AND SPIKE GLYCOPROTEIN IN COVID-19 THROUGH THE IN SILICO APPROACH

Catechin and their derivatives have been studied to have antiviral potential against COVID-19 through in silico method “pharmacokinetics screening and molecular docking”. Pharmacokinetics and toxicity profiles were obtained through the ADMETSAR server and SwissADME server. Then proceed with the prediction of affinity through the method molecular docking using the software application MOE 2007.09. The testmaterial is in the form of a 3D catechin structure and its derivatives as well as several control ligands downloaded via Pubmed. While templatethe Receptor M pro protein and the Spike glycoprotein are downloaded from pdb.org (PDB ID: 6LU7 and 6LXT). The pharmacokinetic profile of catechins is relatively better than all control ligands with the lowest toxicity. Molecular docking results also show that catechins and theirderivatives have a stronger affinity than control ligands. This research proves that catechin has antiviral potential through inhibition of Mpro protein and Spike glycoprotein COVID-19 virus.

In vitro antioxidant activity of defatted seed extracts of Ocimum sanctum on rat PC-12 cells and its inhibitory efficacy with receptors of oral squamous cell carcinoma

Oil cakes, also known as defatted seeds used as by-product and has not studied against oral squamous cell carcinoma (OSCC). In present study, polyphenolic flavonoids of defatted seed extracts of Ocimum sanctum were investigated. Prepared extracts were analysed for thin layer chromatography & high pressure liquid chromatography to identify the polyphenolic flavonoid in extracts. In vitro antioxidant activity and in silico studies were observed by molecular docking with receptors of OSCC using Swiss docking and its pharmacokinetic screening. Present study revealed that quercetin, rutin and tannic acid were found in the defatted seed extracts. Antioxidant capacity was found to be high in ethanolic and aqueous extract of defatted seeds extract, whereas ethanolic extract has shown enzymatic activity and maximum level of non enzyme content in treated rat PC-12 cells that were exposed to neurotoxic shock. Molecular docking of quercetin, rutin and tannin acid has shown best binding energy results with epidermal growth factor receptor (EGFR), β2 adrenergic receptor (β2-AR) and keap1/nrf2 receptors of OSCC. Authors would to conclude that polyphenolic flavonoids has proven its antioxidant activity with rat PC-12 cells as well has revealed its binding efficacy against receptors that can be utilize against OSCC as herbal remedy.

LC-MS/MS analysis of puerarin and 18β-glycyrrhetinic acid in human plasma after oral administration of Samso-eum and its application to pharmacokinetic study.

The aim of this study was to confirm pharmacokinetic screening of multiple components in healthy Korean subjects after oral administration of Samso-eum and perform quantitation of active components in the human plasma. Thirteen potential bioactive components [puerarin (PRR), daidzin, nodakenin, ginsenoside Rb1, 18β-glycyrrhetinic acid (18β-GTA), 6-shogaol, naringin, glycyrrhizin, hesperidin, platycodin D, naringenin, hesperetin, and 6-gingerol] were screened based on literature. The results showed that three analytes (daidzin, naringenin, and hesperetin) were detected in trace amounts. In addition, PRR and 18β-GTA were detected in human plasma after the oral administration of Samso-eum. In this study, a liquid chromatography-electrospray ionization-tandem mass spectrometry method was validated for the simultaneous determination of PRR and 18β-GTA in human plasma. This was the first study to evaluate pharmacokinetics of PRR and 18β-GTA after the usual oral dose of Samso-eum (30 g containing 102.48 mg PRR, 48.18 mg glycyrrhizin) in human subjects.

Screening of promising molecules against MurG as drug target in multi-drug-resistant-Acinetobacter baumannii - insights from comparative protein modeling, molecular docking and molecular dynamics simulation

The UDP-N-acetylglucosamine-N-acetylmuramyl-(pentapeptide) pyrophosphoryl-undecaprenol N-acetylglucosamine transferase (MurG) is located in plasma membrane which plays a crucial role for peptidoglycan biosynthesis in Gram-negative bacteria. Recently, this protein is considered as an important and unique drug target in Acinetobacter baumannii since it plays a key role during the synthesis of peptidoglycan as well as which is not found in Homo sapiens. In this study, initially we performed comparative protein modeling approach to predict the three-dimensional model of MurG based on crystal structure of UDP-N-acetylglucosamine-N-acetylmuramyl-(pentapeptide) pyrophosphoryl-undecaprenol N-acetylglucosamine transferase (PDB ID: 1F0K) from E.coli K12. MurG model has two important functional domains located in N and C- terminus which are separated by a deep cleft. Active site residues are located between two domains and they are Gly20, Arg170, Gly200, Ser201, Gln227, Phe254, Leu275, Thr276, and Glu279 which play essential role for the function of MurG. In order to inhibit the function of MurG, we employed the High Throughput Virtual Screening (HTVS) and docking techniques to identify the promising molecules which will further subjected into screening for computing their drug like and pharmacokinetic properties. From the HTVS, we identified 5279 molecules, among these, 12 were passed the drug-like and pharmacokinetic screening analysis. Based on the interaction analysis in terms of binding affinity, inhibition constant and intermolecular interactions, we selected four molecules for further MD simulation to understand the structural stability of protein-ligand complexes. All the analysis of MD simulation suggested that ZINC09186673 and ZINC09956120 are identified as most promising putative inhibitors for MurG protein in A. baumannii.Communicated by Ramaswamy H. Sarma

O-Hydroxycinnamic derivatives as prospective anti-platelet candidates: in silico pharmacokinetic screening and evaluation of their binding sites on COX-1 and P2Y12 receptors.

Background The high prevalence of thrombotic abnormalities has become a major concern in the health sector. This is triggered by uncontrolled platelet aggregation, which causes complications and death. The problem becomes more complicated because of the undesirable side effects of the drugs currently in use, some of which have reportedly become resistant. This study aims to evaluate the potency of o-hydroxycinnamic acid derivatives (OCA1a-22a) and their pharmacokinetic properties and toxicity for them to be developed as new antiplatelet candidates. Methods In silico analysis of pharmacokinetics was carried out using pKCSM. Molecular docking of the compounds OCA 1a-22a was performed using the Molegro Virtual Docker. In silico evaluation of the potency of biological activity was done by measuring the bonding energy of each tested compound to the target receptor i.e. COX-1 and P2Y12, as the Moldock score (MDS). Results pKCSM analyses showed that more than 90% of OCA 1a-22a are absorbed through the intestine and distributed in plasma. Most tested compounds are not hepatotoxic, and none is mutagenic. An evaluation of the COX-1 receptor showed that OCA 2a-22a have lower binding energy compared to aspirin, which is the COX-1 inhibitor used today. So, it can be predicted that OCA 2-22a have stronger activity. Interactions with P2Y12 show lower MDS than aspirin, but slightly higher than ibuprofen, which is the standard ligand. Conclusions ADMET (absorption, distribution, metabolism, excretion, and toxicity) profile prediction shows that OCA 1a-22a have the potential to be developed as oral preparations. OCA 1a-22a have strong potential to interact with COX-1 and P2Y12 receptors, so they are prospective anti-platelet candidates.

Dissection of Pharmacological Mechanism of Chinese Herbal Medicine Yihuo Huatan Formula on Chronic Obstructive Pulmonary Disease: A Systems Pharmacology-Based Study

Chronic obstructive pulmonary disease (COPD) is one of the most common respiratory diseases. Yihuo Huatan Formula (YHF), as a proven Chinese Herbal Medicine (CHM), has been verified to be effective in the treatment of stable COPD through years’ of practice. Nevertheless, its working mechanism is still unclear. We sought to systematically decipher the mechanism of YHF for treating stable COPD using systems pharmacology-based method that integrates pharmacokinetic screening, target prediction, network analyses, GO and KEGG enrichment analyses. Firstly, a total of 1267 chemicals out of 15 herbal components were included in YHF chemical database. Among them, 180 potential active molecules were screened out through pharmacokinetic evaluation. Then 258 targets of the active molecules were predicted, of which 84 were chosen for further analyses. Finally, the network analyses and GO and KEGG enrichment methods suggested a therapeutic effect of YHF on the alleviation of airway inflammation, decrease of mucus secretion, maintenance of immune homeostasis and benefit of COPD comorbidities, by regulating multiple targets and pathways. The systems pharmacology-based approach helps to understand the underlying working mechanism of YHF in stable COPD from a holistic perspective, and offers an exemplification for systematically uncovering the action mechanisms of CHM.

A Systems Pharmacology-Based Study of the Molecular Mechanisms of San Cao Decoction for Treating Hypertension.

Traditional Chinese medicine (TCM) has a longstanding history and has gained widespread clinical applications. San Cao Decoction (SCD) is an experience prescription first formulated by Prof. Duzhou Liu. We previously demonstrated its antihypertensive effects; however, to systematically explain the underlying mechanisms of action, we employed a systems pharmacology approach for pharmacokinetic screening and target prediction by constructing protein-protein interaction networks of hypertension-related and putative SCD-related targets, and Database for Annotation, Visualization, and Integrated Discovery enrichment analysis. We identified 123 active compounds in SCD and 116 hypertension-related targets. Furthermore, the enrichment analysis of the drug-target network showed that SCD acts in a multidimensional manner to regulate PI3K-Akt-endothelial nitric oxide synthase signaling to maintain blood pressure. Our results highlighted the molecular mechanisms of antihypertensive actions of medicinal herbs at a systematic level.

Analysis of Biological Samples in a Contemporary Laboratory Practice (Review)

Introduction. In the present publication highlights the key points of the main stages of development of methods for determining trace amounts of drugs and metabolites in biological samples using chromatographic and chromatography-mass spectrometry methods. The main sources of errors are specified. The main attention is paid to chromatography-mass spectrometry, which is the basic method of analysis of small molecules in biological samples. Examples from literary sources and authors' own practice are given.Text. The review highlights some of the practical issues of preparation of calibration samples, method of increasing the stability of the sample at the stage of sampling and plasma preparation. In particular, the influence of various anticoagulants on the accuracy of the analysis is reflected. Specify the method of reducing back conversion of some metabolites of carboxyl-containing drugs to parent compound to prevent overestimation of the results of quantitative determination. Some methods of sample preparation, which have become widespread recently, are noted. For example, solid supported liquid-liquid extraction, based on the extraction of the component of interest from the water sample into the liquid layer distributed on a solid high-polar carrier, followed by eluting by a system of non-polar solvents that do not mix with this layer. Recommendations on the use of internal standards, the preparation of the mobile phase for HPLC, on chromatographic separation, validation techniques are given. In the section «Mass spectrometric detection» features of preparation of a mobile phase for chromatography-mass spectrometry experiments are given. The questions of carry-over reduction, ion suppression, matrix effect are covered. The phenomenon of cross-talk in the study of drug metabolism by chromatography-mass spectrometry is discussed. It consists in the mutual distortion of the mass spectrometric response, when the same mass fragments are formed from different ions-precursors. Features of development of techniques for high-performance pharmacokinetic screening are given.Conclusion. The authors hope that the presented material will be useful for scientists and specialists in the field of pharmacokinetics, biomarker discovery and clinical analyses.

Identification of novel small molecule non-peptidomimetic inhibitor for prolyl oligopeptidase through in silico and in vitro approaches

Prolyl oligopeptidase (POP) enzyme has been studied for various disorders, viz. Schizophrenia, Alzheimer’s, Parkinson’s, Depression, Inflammation, etc., for three decades, but no drug has passed through the clinical trials, possibly because of indigent pharmacokinetics. This might have been a result of similar structures of drug candidates. This study aimed at identifying novel small non-peptidomimetic inhibitors for POP enzyme that could serve as a lead for developing newer drugs. Structure-based virtual screening of molecules of MolMall database was conducted on the POP enzyme (PDB ID 3DDU) to identify potential hits. The hits identified were subjected to computational pharmacokinetic screening followed by molecular mechanics/generalized Born and surface area studies to estimate the binding free energy of the docked complexes. After that, nine hits were selected and tested for POP inhibitory activity, among which one compound MM 4 was found to be most potent with EC50 of 100 µM. Compound MM 4 was further subjected to molecular dynamics simulations to study the overall stability of the ligand–protein complex. The compound interacted strongly with catalytic amino acid Arg643 by forming salt and water bridges; it also interacted well with amino acids Phe173, Arg252 and Met235. This study provides a lead molecule for further development of POP inhibitors.Communicated by Ramaswamy H. Sarma

Pharmacogenomic Profiling of ADME Gene Variants: Current Challenges and Validation Perspectives.

In the past decades, many efforts have been made to individualize medical treatments, taking into account molecular profiles and the individual genetic background. The development of molecularly targeted drugs and immunotherapy have revolutionized medical treatments but the inter-patient variability in the anti-tumor drug pharmacokinetics (PK) and pharmacodynamics can be explained, at least in part, by genetic variations in genes encoding drug metabolizing enzymes and transporters (ADME) or in genes encoding drug receptors. Here, we focus on high-throughput technologies applied for PK screening for the identification of predictive biomarkers of efficacy or toxicity in cancer treatment, whose application in clinical practice could promote personalized treatments tailored on individual’s genetic make-up. Pharmacogenomic tools have been implemented and the clinical utility of pharmacogenetic screening could increase safety in patients for the identification of drug metabolism-related biomarkers for a personalized medicine. Although pharmacogenomic studies were performed in adult cohorts, pharmacogenetic pediatric research has yielded promising results. Additionally, we discuss the current challenges and theoretical bases for the implementation of pharmacogenetic tests for translation in the clinical practice taking into account that pharmacogenomics platforms are discovery oriented and must open the way for the setting of robust tests suitable for daily practice.

Abstract 4905: Utility of human FcRn transgenic mice for preclinical screening of immunotherapeutics

Abstract Preclinical assessment is required for the growing number of immunotherapeutics in development. Clinically relevant pharmacokinetic analysis can be achieved by using transgenic mice that uniquely express the human Fc receptor neonatal (hFcRn). To demonstrate the utility of the human FcRn Tg mouse model platform, three immunotherapeutics (pembrolizumab, ipilimumab, and belatacept) were administered IV to Tg32, Tg276, FcRn null, and B6 wild type mice. The mice were blood sampled (25 µL) at 1, 3, 5, 7, 9, 12, 16, 19, 22, 26, and 30 days. Immunotherapeutic plasma concentations, assessed by human IgG ELISA, were pharmacokinetically analyzed. Tg32 mice yielded half-life values for these immunotherapeutics with ranges that mimicked patient data. Though reduced in scale, Tg276 mice also produced half-life data that correlated with the established human half-life data for pembrolizumab, ipilimumab, and belatacept. These results confirm that the human FcRn Tg model platform can be broadly applied to preclinical pharmacokinetic screening of mAb and Fc-fusion based immunotherapeutics. Citation Format: Gregory J. Christianson, Emily Lowell, Cat Lutz. Utility of human FcRn transgenic mice for preclinical screening of immunotherapeutics [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4905.

Systematic investigation of the Erigeron breviscapus mechanism for treating cerebrovascular disease

Ethnopharmacological relevanceCerebrovascular diseases (CBVDs), characterized by striking morbidity and mortality, have become the most common life-threatening diseases. The existing drugs of CBVDs target one or a few of pathogenic factors, the efficacy of which is limited because of the complexity of CBVDs. Traditional Chinese medicine (TCM), featured by multi-component and multi-target endows the great effectiveness in CBVDs treatment. For instance, Erigeron breviscapus (vant.) Hand. Mazz. （Erigeron breviscapus) has been used to treat CBVDs for a long time and the efficacy has been verified through years’ of practice. Nevertheless, the mechanisms of Erigeron breviscapus for treating CBVDs are still unclear. The aim of the studySystematically decipher the mechanisms of Erigeron breviscapus for treating CBVDs. Materials and methodsThe systems pharmacology approach is utilized by integrating ADME pharmacokinetic screening, target fishing, protein-protein interaction (PPI), network analysis and in vitro experiments verification. ResultsFirst, 14 potentially active molecules were screened out through in silico ADME pharmacokinetic evaluation, most of which have been reported with excellent biological activities. Then 169 targets of active molecules were read out using our in-house softwares, systems drug targeting (sysDT) and Weighted Ensemble Similarity(WES). We found that the targets of the active compounds were significantly enriched to the CBVDs therapeutic targets by analyzing their biological processes and protein-protein interactions (PPIs). A multi-layer network analysis including compound-target network, target-pathway network and “CBVDs pathway” indicated that the Erigeron breviscapus exerts a protective effect on CBVDs via regulating multiple pathways and hitting on multiple targets. Meanwhile in vitro experiments confirmed that the stigmasterol, scutellarein, and daucosterol from Erigeron breviscapus increased the MEK and PLCγ proteins levels, and decreased the expression of Bax, PI3K, and eNOS, which led to the cell survival, proliferation and contraction. ConclusionThe approach used in this work offers a new exemplification for systematically understanding the mechanisms of herbal medicines, which will give an impulse to the CBVDs drug development.

Rats and rabbits as pharmacokinetic screening tools for long acting intramuscular depots: case study with paliperidone palmitate suspension

Development of prodrug of 9-hydroxyrisperidone (paliperidone) long-acting intramuscular injection has enabled delivery over four-week time period with improved compliance.The key aim of this work was to establish a reliable preclinical model which may potentially serve as a screening tool for judging the pharmacokinetics of paliperidone formulation(s) prior to human clinical work.Sparse sampling composite study was used in rats, (Wistar/Sprague–Dawley (SD; n = 10)) and a serial blood sampling study design was used in rabbits (n = 4). Animals received intramuscular injection of paliperidone palmitate in the thigh muscle at dose of 16 (rats) and 4.5 mg/kg (rabbits). Samples were drawn in rats (retro-orbital sinus) and rabbits (central ear artery) and were analysed for paliperidone using liquid chromatography–mass spectrometry/ mass spectrometry (LC-MS/MS) assay. The plasma data was subjected to pharmacokinetic analysis.Following intramuscular injection of depot formulation in Wistar/SD rats and rabbits, absorption of paliperidone was slow and gradual with median value of time to reach maximum concentration (Tmax) occurring on day 7. The exposures (i.e. area under the curve (AUC; 0–28) days) were 18,597, 21,865 and 18,120 ng.h/mL, in Wistar, SD and rabbits, respectively. The clearance was slow and supported long half-life (8–10 days).Either one of the two models can serve as a research tool for establishing pharmacokinetics of paliperidone formulation(s).

Single jugular vein cannulated rats may not be suitable for intravenous pharmacokinetic screening of high logP compounds

Rat is commonly used for pharmacokinetic screening during pharmaceutical lead optimization. To handle the large number of compounds, rats with a single jugular vein cannulation are commonly utilized for intravenous pharmacokinetic studies, where the same cannula is used both for dose administration and blood sampling. We demonstrate that the single cannula method is not suitable for all compounds, especially for high logP compounds. We propose an alternative dual cannulation technique in which two cannulas are placed in the same jugular vein, thus avoiding an additional surgery. Compounds were administered orally or via intravenous infusion to compare PK parameters, including bioavailability, using both procedures. For itraconazole and amiodarone, known to bind to the cannula, the measured plasma exposures were substantially higher in the single cannulated rats than those from dual cannulated rats. Area under the plasma concentration time curve differed by 79% and 74% for itraconazole and amiodarone, respectively. When compared to the single cannulation approach, clearance, volume of distribution and bioavailability determined by dual cannulation were 39%, 60% and 38% higher for itraconazole, and 46%, 34% and 42% higher for amiodarone, respectively. In contrast, all pharmacokinetic parameters were similar between single and dual-cannulated rats for the hydrophilic compound atenolol. Based on these results, we recommend the use of dual cannulated rats for intravenous pharmacokinetic studies when testing a series of hydrophobic compounds that may be prone to non-specific binding to the cannula. If single cannulated model is selected for pharmacokinetic screening, we recommend a bridging study with dual cannulated rats with representative compounds of a given chemical series.

The pharmacokinetic screening of multiple components of the Nao Mai Tong formula in rat plasma by liquid chromatography tandem mass spectrometry combined with pattern recognition method and its application to comparative pharmacokinetics

The Nao Mai Tong formula (NMT) is composed of Rhubarb, Ginseng, Ligusticum wallichii and Pueraria in a ratio of 3:3:2:2 (w/w) and is a well-known traditional Chinese prescription that has been clinically employed for treating ischemia cerebrovascular disease. The goal of this study was to investigate the pharmacokinetics of multiple components (chryohol-8-O-β-D-glucoyroide, physcion-8-O-β-D-glucopyranoside, aloe-emodin, rhein, emodin, chrysophanol, ginsenoside Rg1, ginsenoside Rb1, ginsenoside Rb3, ginsenoside Rc, senkyunolide I, ligustilide puerarin, daidzein, 3′-methoxy puerarin) after the oral administration of the NMT formula in rats. A rapid and sensitive UHPLC-Quadrupole-Orbitrap-MS with a sequential positive and negative ionization mode was developed to determine the 15 absorbed ingredients. After extraction from blood, the analytes and internal standards were subjected to ultra-high performance liquid chromatography with Agela Venusil MPC18 (2.1mm×100mm, 3μm, Agela, USA). The mobile phase consisted of methanol and ammonium acetate (3mmolL−1) under gradient elution conditions. This validated method was successfully applied to a comparative pharmacokinetic study of fifteen components in rat plasma after oral administration of the NMT formula or single herb extracts to normal and stroke-afflicted rats. A principal component analysis (PCA) was utilized to evaluate the differences in the pharmacokinetic behavior (time-course) of the absorbed components of NMT, and the absorbed components were assigned to 3 separate clusters. A comparison of the body dynamics of each group indicated that cluster B (ginsenoside Rg1, ginsenoside Rb1, ginsenoside Rb3, ginsenoside Rc) might be the most important constituents controlling the pharmacological effects of NMT. The comparative pharmacokinetic study showed that the different groups had different pharmacokinetic characteristics. The pharmacokinetics-based UHPLC Quadrupole-Orbitrap-MS using a full-scan mode combined with a pattern recognition approach can provide a reliable and suitable means of screening and identifying potentially bioactive components that contribute to the pharmacological effects of Traditional Chinese Medicine (TCM).

A novel prodrug strategy to improve the oral absorption of O-desmethylvenlafaxine.

O-Desmethylvenlafaxine (desvenlafaxine, ODV) is the active metabolite of venlafaxine, with similar activity and less risk for pharmacokinetic drug interactions compared to its parent compound venlafaxine. The purpose of this study was to design a series of esters of ODV and assess their potential as ODV prodrugs with improved bioavailability and brain uptake. Seven esters were synthesized and pharmacokinetic screening was performed in rats. The monoester formed on the phenolic hydroxyl of ODV (ODVP-1, ODVP-2, ODVP-3 and ODVP-5) could be degraded to ODV in rat plasma. These four compounds confirmed as possible prodrugs were then studied to evaluated the relative bioavailability of ODV they produced in beagle dogs. ODVP-1, ODVP-2 and ODVP-3 demonstrated higher relative bioavailability of ODV. Finally, ODVP-1, ODVP-2 and ODVP-3 were studied to evaluate their brain uptake in rats. The concentration of ODV in the rat plasma, brain and hypothalamus after administration of ODVP-1, ODVP-2 or ODVP-3 was higher compared with that of ODV. The higher bioavailability, improved pharmacokineics properties and more rapid penetration and translation of ODV suggest that ODVP-1, ODVP-2 or ODVP-3 may warrant further development and application as ODV prodrugs.

Utility of capillary microsampling for rat pharmacokinetic studies: Comparison of tail-vein bleed to jugular vein cannula sampling

IntroductionSerial sampling methods have been used for rat pharmacokinetic (PK) studies for over 20years. Currently, it is still common to take 200–250μL of blood at each timepoint when performing a PK study in rats and using serial sampling. While several techniques have been employed for collecting blood samples from rats, there is only limited published data to compare these methods. Recently, microsampling (≤50μL) techniques have been reported as an alternative process for collecting blood samples from rats. MethodsIn this report, five compounds were dosed orally into rats. For three proprietary compounds, jugular vein cannula (JVC) sampling was used to collect whole blood and plasma samples and capillary microsampling (CMS) was used to collect blood samples from the tail vein of the same animal. For the two other compounds, marketed drugs fluoxetine and glipizide, JVC sampling was used to collect both whole blood and blood CMS samples while tail-vein sampling from the same rats was also used to collect both whole blood and blood CMS samples. ResultsFor the three proprietary compounds, the blood AUC as well as the blood concentration-time profile that were obtained from the tail vein were different from those obtained via JVC sampling. For fluoxetine, the blood total exposure (AUC) was not statistically different when comparing tail-vein sampling to JVC sampling, however the blood concentration-time profile that was obtained from the tail vein was different than the one obtained from JVC sampling. For glipizide, the blood AUC and concentration-time profile were not statistically different when comparing the tail-vein sampling to the JVC sampling. For both fluoxetine and glipizide, the blood concentration profiles obtained from CMS were equivalent to the blood concentration profiles obtained from the standard whole blood sampling, collected at the same sampling site. DiscussionThe data in this report provide strong evidence that blood CMS is a valuable small volume blood sampling approach for rats and that it provides results for test compound concentrations that are equivalent to those obtained from traditional whole blood sampling. The data also suggest that for some compounds, the concentration-time profile that is obtained for a test compound based on sampling from a rat tail vein may be different from that obtained from rat JVC sampling. In some cases, this shift in the concentration-time profile will result in different PK parameters for the test compound.Based on these observations, it is recommended that a consistent blood sampling method should be used for serial microsampling in discovery rat PK studies when testing multiple new chemical entities. If the rat tail vein sampling method is selected for PK screening, then conducting a bridging study on the lead compound is recommended to confirm that the rat PK obtained from JVC sampling is comparable to the tail-vein sampling.

Noscapine recirculates enterohepatically and induces self-clearance

Noscapine (Nos), an antitussive benzylisoquinoline opium alkaloid, is a non-toxic tubulin-binding agent currently in Phase II clinical trials for cancer chemotherapy. While preclinical studies have established its tumor-inhibitory properties in various cancers, poor absorptivity and rapid first-pass metabolism producing several uncharacterized metabolites for efficacy, present an impediment in translating its efficacy in humans. Here we report novel formulations of Nos in combination with dietary agents like capsaicin (Cap), piperine (Pip), eugenol (Eu) and curcumin (Cur) known for modulating Phase I and II drug metabolizing enzymes. In vivo pharmacokinetic (PK), organ toxicity evaluation of combinations, microsomal stability and in vitro cytochrome P450 (CYP) inhibition effects of Nos, Cap and Pip using human liver microsomes were performed. Single-dose PK screening of combinations revealed that the relative exposure of Nos (2μgh/mL) was enhanced by 2-fold (4μgh/mL) by Cap and Pip and their plasma concentration–time profiles showed multiple peaking phenomena for Nos indicating enterohepatic recirculation or differential absorption from intestine. CYP inhibition studies confirmed that Nos, Cap and Pip are not potent CYP inhibitors (IC50>1μM). Repeated oral dosing of Nos, Nos+Cap and Nos+Pip showed lower exposure (Cmax and AUClast) of Nos on day 7 compared to day 1. Nos Cmax decreased from 3087ng/mL to 684ng/mL and AUClast from 1024ngh/mL to 508ngh/mL. In presence of Cap and Pip, the decrease in Cmax and AUClast of Nos was similar. This may be due to potential enzyme induction leading to rapid clearance of Nos as the trend was observed in Nos alone group also. The lack of effect on intrinsic clearance of Nos suggests that the potential drug biotransformation modulators employed in this study did not contribute toward increased exposure of Nos on repeated dosing. We envision that Nos-induced enzyme induction could alter the therapeutic efficacy of co-administered drugs, hence emphasizing the need for strategic evaluation of the metabolism of Nos to reap its maximum efficacy.

Comparison of Pharmacokinetics of Dapsone in Male Sprague Dawley Rats Following Retro Orbital, Jugular Vein and Saphenous Vein Blood Sampling

Pharmacokinetic (PK) studies play an important role in identifying lead compounds for further development. Typically rats are used for PK screening of New Chemical Entities (NCEs) as the compound requirements are minimal (<10 mg), multiple blood sampling (up to 10 samples) can be performed from the same animal and in small volumes (5-25μL) for sample analysis. Blood sampling site is critical in obtaining multiple blood samples of good quality and in small volumes with minimal stress to animals. However, it is not known whether PK parameters can be influenced by sampling site. Thus, in this study, we evaluated the effect of different blood sampling sites like retro-orbital plexus, jugular vein and saphenous vein on PK parameters of Dapsone. Dapsone was administered both orally and intravenously at a dose of 12 mg/kg to a group of 4 male Sprague Dawley rats and blood samples were collected up to 24 h. Samples were analyzed by LC/MS/MS and PK parameters were calculated. With all the sampling techniques, PK parameters like clearance, volume of distribution, half-life and bioavailability were similar. Due to the control on the blood volume withdrawn at each time point, quick sampling with minimal hemolysis and minimal animal handling stress during sampling, Jugular Vein (JV) or Saphenous Vein (SV) sampled rats can be used for PK studies. Further for saphenous sampling no pre-study preparation like cannulation is required before dosing the animals therefore sampling of rats through saphenous vein is recommended for pharmacokinetic and toxicokinetic studies. To summarize, SV sampling reduce the number of animals in different Pharmacokinetic (PK) (mouse) and Toxicokinetic (TK) (mouse and rat) studies by using serial draws, offers reduction and refinement over the othersampling techniques with minimal preparation upfront and with a potential to replace them.

Application of human FcRn transgenic mice as a pharmacokinetic screening tool of monoclonal antibody

1. For drug discovery, useful screening tools are essential to select superior candidates. Here, we evaluated the applicability of transgenic mice expressing human neonatal Fc receptor (FcRn) (hFcRn Tgm) as a pharmacokinetic screening tool of therapeutic monoclonal antibodies (mAbs) and Fc-fusion proteins that overcomes the species difference in FcRn binding.2. Marketed 11 mAbs and 2 Fc-fusion proteins were intravenously administered to hFcRn Tgm and WT mice. The half-lives in hFcRn Tgm and WT mice were compared with those in human obtained from literature. The linear half-lives in human and monkey were also calculated by nonlinear pharmacokinetic analysis. For comparison, correlations of half-lives between monkey and human were also evaluated.3. The half-lives of mAbs and Fc-fusion proteins after intravenous administration ranged from 1.1 to 13.2 days in hFcRn Tgm and from 1.2 to 30.3 days in WT mice. The half-lives in human correlated more closely with those in hFcRn Tgm than in WT mice and monkey.4. Our results suggest that hFcRn Tgm are a valuable and useful tool for pharmacokinetic screening of mAbs and Fc-fusion proteins in the preclinical stage. Furthermore, we believe that hFcRn Tgm are broadly applicable to preclinical pharmacokinetic screening of mAbs-based therapeutics.