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Preface for special issue on new analytical techniques and methods in drug metabolism and pharmacokinetics

Guangbo Ge, Mingshe Zhu

Guangbo Ge, Mingshe Zhu Analytical technologies and approaches for drug metabolism and pharmacokinetics (DMPK) research in the pharmaceutical industry and academic research institutes have evolved rapidly over the past decade. On one hand. the discovery and development of small molecule drug candidates requires earlier and better understanding of their absorption, distribution,metabolism and excretion (ADME) in human as well as their interactions with metabolizing enzymes, transporters and drug targets, such as receptors and DNA. On the other hand. several new drug modalities and deliver systems. such as peptide and protein therapeutics. antibody-drug conjugates (ADC) and nano drug delivery systems, have been introduced into the drug pipelines. Journal of Pharmaceutical Analysis. 2020 10(3): 前插1-前插2.

Current status and future directions of high-throughput ADME screening in drug discovery

Wilson Z. Shou

Wilson Z. Shou During the last decade high-throughput in vitro absorption, distribution, metabolism and excretion (HT-ADME) screening has become an essential part of any drug discovery effort of synthetic molecules. The conduct of HT-ADME screening has been"industrialized"due to the extensive development of software and automation tools in cell culture, assay incubation, sample analysis and data analysis. The HT-ADME assay portfolio continues to expand in emerging areas such as drug-transporter interactions, early soft spot identification, and ADME screening of peptide drug candidates. Additionally, thanks to the very large and high-quality HT-ADME data sets available in many biopharma companies, in silico prediction of ADME properties using machine learning has also gained much momentum in recent years. In this re-view, we discuss the current state-of-the-art practices in HT-ADME screening including assay portfolio, assay automation, sample analysis, data processing, and prediction model building. In addition, we also offer perspectives in future development of this exciting field. Journal of Pharmaceutical Analysis. 2020 10(3): 201-208.

Current LC-MS-based strategies for characterization and quantification of antibody-drug conjugates

Xiaoyu Zhu, Shihan Huo, Chao Xue, Bo An, Jun Qu

Xiaoyu Zhu, Shihan Huo, Chao Xue, Bo An, Jun Qu The past few years have witnessed enormous progresses in the development of antibody-drug conju-gates (ADCs). Consequently, comprehensive analysis of ADCs in biological systems is critical in sup-porting discovery, development and evaluation of these agents. Liquid chromatography-mass spectrometry (LC-MS) has emerged as a promising and versatile tool for ADC analysis across a wide range of scenarios, owing to its multiplexing ability, rapid method development, as well as the capability of analyzing a variety of targets ranging from small-molecule payloads to the intact protein with a high, molecular resolution. However, despite this tremendous potential, challenges persist due to the high complexity in both the ADC molecules and the related biological systems. This review summarizes the up-to-date LC-MS-based strategies in ADC analysis and discusses the challenges and opportunities in this rapidly-evolving field. Journal of Pharmaceutical Analysis. 2020 10(3): 209-220.

Current status of in vivo bioanalysis of nano drug delivery systems

Tingting Wang, Di Zhang, Dong Sun, Jingkai Gu

Tingting Wang, Di Zhang, Dong Sun, Jingkai Gu The development of nano drug delivery systems (NDDSs) provides new approaches to fighting against diseases. The NDDSs are specially designed to serve as carriers for the delivery of active pharmaceutical ingredients (APIs) to their target sites, which would certainly extend the benefit of their unique physi-cochemical characteristics, such as prolonged circulation time, improved targeting and avoiding of drug-resistance. Despite the remarkable progress achieved over the last three decades, the understanding of the relationships between the in vivo pharmacokinetics of NDDSs and their safety profiles is insufficient. Analysis of NDDSs is far more complicated than the monitoring of small molecular drugs in terms of structure, composition and aggregation state, whereby almost all of the conventional techniques are inadequate for accurate profiling their pharmacokinetic behavior in vivo. Herein, the advanced bio-analysis for tracing the in vivo fate of NDDSs is summarized, including liquid chromatography tandem-mass spectrometry (LC-MS/MS), F(o)rster resonance energy transfer (FRET), aggregation-caused quench-ing (ACQ) fluorophore, aggregation-induced emission (AIE) fluorophores, enzyme-linked immunosor-bent assay (ELISA), magnetic resonance imaging (MRI), radiolabeling, fluorescence spectroscopy, laser ablation inductively coupled plasma MS (LA-ICP-MS), and size-exclusion chromatography (SEC). Based on these technologies, a comprehensive survey of monitoring the dynamic changes of NDDSs in struc-ture, composition and existing form in system (i.e. carrier polymers, released and encapsulated drug) with recent progress is provided. We hope that this review will be helpful in appropriate application methodology for investigating the pharmacokinetics and evaluating the efficacy and safety profiles of NDDSs. Journal of Pharmaceutical Analysis. 2020 10(3): 221-232.

Ultra-sensitive bioanalysis of the therapeutic peptide exenatide for accurate pharmacokinetic analyses at effective plasma concentrations utilizing UPLC-MS/MS

Max Sauter, Philipp Uhl, Jürgen Burhenne, Walter E. Haefeli

Max Sauter, Philipp Uhl, Jürgen Burhenne, Walter E. Haefeli Exenatide is the first approved glucagon-like peptide 1 receptor agonist subcutaneously or intramus-cularly injected for the treatment of type 2 diabetes mellitus. Typical therapeutic plasma concentrations are in the low pg/mL range, therefore requiring ultra-sensitive quantification. To enable the accurate evaluation of pharmacokinetic studies, we established a UPLC-MS/MS assay with a lower limit of quantification (LLOQ) of 5 pg/mL (1.2 pM) using 200μL of plasma, validated according to FDA''s and EMA''s pertinent guidelines. Exenatide was isolated from plasma with solid phase extraction utilizing anion-exchange sorbent. Quantification was performed with positive electrospray ionization tandem mass spectrometry in the selected reaction monitoring mode. The calibrated concentration range of 5-10,000 pg/mL was linear showing correlation coefficients>0.99. Interday and intraday accuracy ranged from 97.5%to 105.4%with corresponding precision of<10.9%. Accuracy at the LLOQ ranged from 93.0%to 102.5%with corresponding precision of<15.9%. Because of the validity of a 10-fold dilution QC (accuracy 111.2%), the assay is suitable for exenatide quantification up to 100,000 pg/mL. The ultra-sensitive assay''s applicability was demonstrated by the quantification of exenatide plasma concentrations and pharma-cokinetics after intravenous and nasal administration to beagle dogs. Journal of Pharmaceutical Analysis. 2020 10(3): 233-239.

Software-aided detection and structural characterization of cyclic peptide metabolites in biological matrix by high-resolution mass spectrometry

Ming Yao, Tingting Cai, Eva Duchoslav, Li Ma, Xu Guo, Mingshe Zhu

Ming Yao, Tingting Cai, Eva Duchoslav, Li Ma, Xu Guo, Mingshe Zhu Compared to their linear counterparts, cyclic peptides show better biological activities, such as anti-bacterial, immunosuppressive, and anti-tumor activities, and pharmaceutical properties due to their conformational rigidity. However, cyclic peptides could form numerous putative metabolites from po-tential hydrolytic cleavages and their fragments are very difficult to interpret. These characteristics pose a great challenge when analyzing metabolites of cyclic peptides by mass spectrometry. This study was to assess and apply a software-aided analytical workflow for the detection and structural characterization of cyclic peptide metabolites. Insulin and atrial natriuretic peptide (ANP) as model cyclic peptides were incubated with trypsin/chymotrypsin and/or rat liver S9, followed by data acquisition using TripleTOF? 5600. Resultant full-scan MS and MS/MS datasets were automatically processed through a combination of targeted and untargeted peak finding strategies. MS/MS spectra of predicted metabolites were interrogated against putative metabolite sequences, in light of a, b, y and internal fragment series. The resulting fragment assignments led to the confirmation and ranking of the metabolite sequences and identification of metabolic modification. As a result, 29 metabolites with linear or cyclic structures were detected in the insulin incubation with the hydrolytic enzymes. Sequences of twenty insulin metabolites were further determined, which were consistent with the hydrolytic sites of these enzymes. In the same manner, multiple metabolites of insulin and ANP formed in rat liver S9 incubation were detected and structurally characterized, some of which have not been previously reported. The results demonstrated the utility of software-aided data processing tool in detection and identification of cyclic peptide metabolites. Journal of Pharmaceutical Analysis. 2020 10(3): 240-246.

Quantitation of DNA by nuclease P1 digestion and UPLC-MS/MS to assess binding efficiency of pyrrolobenzodiazepine

Yong Ma, Buyun Chen, Donglu Zhang

Yong Ma, Buyun Chen, Donglu Zhang Accurate DNA quantitation is a prerequisite in many biomedical and pharmaceutical studies. Here we established a new DNA quantitation method by nuclease P1 digestion and UPLC-MS/MS analysis. DNA fragments can be efficiently hydrolyzed to single deoxyribonucleotides by nuclease P1 in a short time. The decent stabilities of all the four deoxyribonucleotides were confirmed under different conditions. Deoxyadenosine monophosphate (dAMP) was selected as the surrogate for DNA quantitation because dAMP showed the highest sensitivity among the four deoxyribonucleotides in the UPLC-MS/MS analysis. The linear range in DNA quantitation by this method is 1.2-5000 ng/mL. In the validation, the inter-day and intra-day accuracies were within 90%-110%, and the inter-day and intra-day precision were acceptable (RSD<10%). The validated method was successfully applied to quantitate DNA isolated from tumors and organs of a mouse xenograft model. Compared to the quantitation methods using UV absorbance, the reported method provides an enhanced sensitivity, and it allows for the accurate quantitation of isolated DNA with contamination of RNA and ribonucleotide. Journal of Pharmaceutical Analysis. 2020 10(3): 247-252.

Rapid bioluminescence assay for monitoring rat CES1 activity and its alteration by traditional Chinese medicines

Jun Zhang, Dandan Wang, Liwei Zou, Min Xiao, Yufeng Zhang, Ziwei Li, Ling Yang, Guangbo Ge, Zhong Zuo In traditional Chinese medicine herbs (TCM), including Radix Salviae Miltiorrhizae (Danshen), Radix Puerariae Lobatae (Gegen), Radix Angelicae Sinensis (Danggui), and Rhizoma Chuanxiong (Chuanxiong) are widely used for the prevention and treatment of cardiovascular diseases and also often co-administered with Western drugs as a part of integrative medicine practice. Carboxylesterase 1 (CES1) plays a pivotal role in the metabolisms of pro-drugs. Since (S)-2-(2-(6-dimethylamino)-benzothiazole)-4,5-dihydro-thiazole-4-carboxylate (NLMe) has recently been identified by us as a selective CES1 bioluminescent sensor, we developed a rapid method using this substrate for the direct measurement of CES1 activity in rats. This bioluminescence assay was applied to determine CES1 activity in rat tissues after a two-week oral administration of each of the four herbs noted above. The results demonstrated the presence of CES1 enzyme in rat blood and all tested tissues with much higher enzyme activity in the blood, liver, kidney and heart than that in the small intestine, spleen, lung, pancreas, brain and stomach. In addition, the four herbs showed tissue-specific effects on rat CES1 expression. Based on the CES1 biodistribution and its changes after treatment in rats, the possibility that Danshen, Gegen and Danggui might alter CES1 ac-tivities in human blood and kidney should be considered. In summary, a selective and sensitive biolu-minescence assay was developed to rapidly evaluate CES1 activity and the effects of orally administered TCMs in rats. Journal of Pharmaceutical Analysis. 2020 10(3): 253-262.

An ultra-sensitive and easy-to-use assay for sensing human UGT1A1 activities in biological systems

Ya-Di Zhu, Hui-Lin Pang, Qi-Hang Zhou, Zi-Fei Qin, Qiang Jin, Moshe Finel, Yi-Nan Wang, Wei-Wei Qin, Yin Lu, Dan-Dan Wang, Guang-Bo Ge The human UDP-glucuronosyltransferase 1A1 (UGT1A1), one of the most essential conjugative enzymes, is responsible for the metabolism and detoxification of bilirubin and other endogenous substances, as well as many different xenobiotic compounds. Deciphering UGT1A1 relevance to human diseases and characterizing the effects of small molecules on the activities of UGT1A1 requires reliable tools for probing the function of this key enzyme in complex biological matrices. Herein, an easy-to-use assay for highly-selective and sensitive monitoring of UGT1A1 activities in various biological matrices, using liquid chromatography with fluorescence detection (LC-FD), has been developed and validated. The newly developed LC-FD based assay has been confirmed in terms of sensitivity, specificity, precision, quanti-tative linear range and stability. One of its main advantages is lowering the limits of detection and quantification by about 100-fold in comparison to the previous assay that used the same probe substrate, enabling reliable quantification of lower amounts of active enzyme than any other method. The precision test demonstrated that both intra- and inter-day variations for this assay were less than 5.5%. Further-more, the newly developed assay has also been successfully used to screen and characterize the regu-latory effects of small molecules on the expression level of UGT1A1 in living cells. Overall, an easy-to-use LC-FD based assay has been developed for ultra-sensitive UGT1A1 activities measurements in various biological systems, providing an inexpensive and practical approach for exploring the role of UGT1A1 in human diseases, interactions with xenobiotics, and characterization modulatory effects of small mole-cules on this conjugative enzyme. Journal of Pharmaceutical Analysis. 2020 10(3): 263-270.

Screening of the whole human cytochrome P450 complement (CYPome) with enzyme bag cocktails

Sangeeta Shrestha Sharma, Shishir Sharma, Matthias Bureik

Sangeeta Shrestha Sharma, Shishir Sharma, Matthias Bureik We have previously introduced the use of permeabilized fission yeast cells (enzyme bags) that recom-binantly express full-length CYPs for drug metabolism studies. Such enzyme bags are cells with pores that function as enzymes in situ. They can easily be prepared without a need for ultracentrifugation and may be used in similar protocols as microsomes. In this study we report the preparation of enzyme bag cocktails that permit the testing of multiple CYPs in a single enzyme bag reaction. Moreover, we established a convenient testing scheme that permits a rapid screen of all human CYPs for activity to-wards any given candidate substrate. An important aspect of this approach is the reduction of individual CYP test assays. If a cocktail containing many CYPs tests negative, it follows that all CYPs included in that cocktail need not be tested individually, thus saving time and resources. The new protocol was validated using two probe substrates. Journal of Pharmaceutical Analysis. 2020 10(3): 271-276.