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Rationale: A common strategy for antibody-drug conjugate (ADC) quantitation from in vivo study samples involves measurement of total antibody, conjugated ADC, and free payload concentrations using multiple reaction monitoring (MRM) mass spectrometry. This not only provides a limited picture of biotransformation but can also involve lengthy method development. Quantitation of ADCs directly at the intact protein level in native conditions using high-resolution mass spectrometers presents the advantage of measuring exposure readout as well as monitoring the change in average drug-to-antibody ratio (DAR) and in vivo stability of new linker payloads with minimal method development. Furthermore, site-specific cysteineconjugated ADCs often rely on non-covalent association to retain their quaternary structure, which highlights the unique capabilities of native mass spectrometry (nMS) for intact ADC quantitation. Methods: We developed an intact quantitation workflow involving three stages: automated affinity purification, nMS analysis, and data processing in batch fashion. The sample preparation method was modified to include only volatile ion-pairing reagents in the buffer systems. A capillary size-exclusion chromatography (SEC) column was coupled to a quadrupole time-of-flight high-resolution mass spectrometer for highthroughput nMS analysis. Samples from two mouse pharmacokinetic (PK) studies were analyzed using both intact quantitation workflow and the conventional MRM-based approach. Results: A linear dynamic range of 5-100 µg/mL was achieved using 20 µL of serum sample volume. The results of mouse in vivo PK measurement using the intact quantitation workflow and the MRM-based approach were compared, revealing excellent method agreement. Conclusions: We demonstrated the feasibility of utilizing nMS for the quantitation of ADCs at the intact protein level in preclinical PK studies. Our results indicate that this intact quantitation workflow can serve as an alternative generic method for highthroughput analysis, enabling an in-depth understanding of ADC stability and safety in vivo. [ABSTRACT FROM AUTHOR]

On behalf of the team I am pleased to present the second annual 'novel insights into drug transporter sciences review' focused on peer-reviewed articles that were published in the year 2021. In compiling the articles for inclusion, preprints available in 2021 but officially published in 2022 were considered to be in scope. To support this review the contributing authors independently selected one or two articles that were thought to be impactful and of interest to the broader research community. A similar approach as published last year was adopted whereby key observations, methods and analysis of each paper is concisely summarized in the synopsis followed by a commentary highlighting the impact of the paper in understanding drug transporters' role in drug disposition. As the goal of this review is not to provide a comprehensive overview of each paper but rather highlight important findings that are well supported by the data, the reader is encouraged to consult the original articles for additional information. Further, and keeping in line with the goals of this review, it should be noted that all authors actively contributed by writing synopsis and commentary for individual papers and no attempt was made to standardize language or writing styles. In this way, the review article is reflective of not only the diversity of the articles but also that of the contributors. I extend my thanks to the authors for their continued support and also welcome Diane Ramsden and Pallabi Mitra as contributing authors for this issue (Table 1). Table 1. Selected articles for this review. Title Area Authors Source 1. Atorvastatin-associated rhabdomyolysis in a patient with a novel variant of the SLCO1B1 gene: a case report Polymorphism Kiage et al. J Clin Lipidol. 2022;16(1):23–27 2. Quantitation of plasma membrane drug transporters in kidney tissue and cell lines using a novel proteomic approach enabled a prospective prediction of metformin disposition Proteomics Kikuchi et al. Drug Metab Dispos. 2021;49(10):938–946 3. Proteomics-informed prediction of rosuvastatin plasma profiles in patients with a wide range of body weight Proteomics Wegler et al. Clin Pharmacol Ther. 2021;109(3):762–771 4. Performance of plasma coproporphyrin I and III as OATP1B1 biomarkers in humans Biomarker Neuvonen et al. Clin Pharmacol Ther. 2021 Dec;110(6):1622–1632 5. Identification of appropriate endogenous biomarker for risk assessment of multidrug and toxin extrusion protein-mediated drug-drug interactions in healthy volunteers Biomarker Miyake et al. Clin Pharmacol Ther. 2021;109(2):507–516 6. Population pharmacokinetic modeling and simulation to support qualification of pyridoxic acid as endogenous biomarker of OAT1/3 renal transporters Biomarker Ahmad et al. CPT Pharmacometrics Syst Pharmacol. 2021 May;10(5):467–477 7. Acute and chronic effects of rifampin on letermovir suggest transporter inhibition and induction contribute to letermovir Pharmacokinetics Induction/Biomarkers Robbins et al. Clin Pharmacol Ther. 2022;111(3):664–675 8. Exploring the use of serum-derived small extracellular vesicles as liquid biopsy to study the induction of hepatic cytochromes P450 and organic anion transporting polypeptides Induction/Biomarkers Rodrigues et al. Clin Pharmacol Ther. 2021;110(1):248–258 9. Unraveling pleiotropic effects of rifampicin by using physiologically based pharmacokinetic modeling: assessing the induction magnitude of P-glycoprotein-cytochrome P450 3A4 dual substrates Induction Pan et al. CPT Pharmacometrics Syst Pharmacol. 2021;10:1485–1496. 10. Quantification of CYP3A and Drug transporters activity in healthy young, healthy elderly and chronic kidney disease elderly patients by a microdose cocktail approach Clinical pharmacology Rattanacheeworn et al. Front Pharmacol. 2021;12:726669. 11. Quantitative prediction of P-glycoprotein-mediated drug–drug interactions and intestinal absorption using humanized mice DDI Miyake et al. Br J Pharmacol. 2021;178(21):4335–4351. 12. Regulation of OATP1B1 function by tyrosine kinase-mediated phosphorylation Regulation Hayden et al. Clin Cancer Res. 2021;27(15):4301–4310. [ABSTRACT FROM AUTHOR]

Reported the stress and coping experience of community volunteers who counseled dying clients and their families. Volunteers most frequently reported turning to themselves or to the client when distressed, also to peers, friends, and family. A common role-defined purpose was significant in stress response and coping. (Author)

It is established that neuronal nitric-oxide synthase (nNOS) is ubiquitylated and proteasomally degraded. The proteasomal degradation of nNOS is enhanced by suicide inactivation of nNOS or by the inhibition of hsp90, which is a chaperone found in a native complex with nNOS. In the current study, we have examined whether CHIP, a chaperone-dependent E3 ubiquitin-protein isopeptide ligase that is known to ubiquitylate other hsp90-chaperoned proteins, could act as an ubiquitin ligase for nNOS. We found with the use of HEK293T or COS-7 cells and transient transfection methods that CHIP overexpression causes a decrease in immunodetectable levels of nNOS. The extent of the loss of nNOS is dependent on the amount of CHIP cDNA used for transfection. Lactacystin (10 µM), a selective proteasome inhibitor, attenuates the loss of nNOS in part by causing the nNOS to be found in a detergent-insoluble form. Immunoprecipitation of the nNOS and subsequent Western blotting with an anti-ubiquitin IgG shows an increase in nNOS-ubiquitin conjugates because of CHIP. Moreover, incubation of nNOS with a purified system containing an E1 ubiquitin-activating enzyme, an E2 ubiquitin carrier protein conjugating enzyme (UbcH5a), CHIP, glutathione S-transferase-tagged ubiquitin, and an ATP-generating system leads to the ubiquitylation of nNOS. The addition of purified hsp70 and hsp40 to this in vitro system greatly enhances the amount of nNOS-ubiquitin conjugates, suggesting that CHIP is an E3 ligase for nNOS whose action is facilitated by (and possibly requires) its interaction with nNOS-bound hsp70. [ABSTRACT FROM AUTHOR]

A variety of signaling proteins form heterocomplexes with and are regulated by the heat shock protein chaperone hsp90. These complexes are formed by a multiprotein machinery, including hsp90 and hsp70 as essential and abundant components and Hop, hsp40, and p23 as nonessential cochaperones that are present in much lower abundance in cells. Overexpression of signaling proteins can overwhelm the capacity of this machinery to properly assemble heterocomplexes with hsp90. Here, we show that the limiting component of this assembly machinery in vitro in reticulocyte lysate and in vivo in Sf9 cells is p23. Only a fraction of glucocorticoid receptors (GR) overexpressed in Sf9 cells are in heterocomplex with hsp90 and have steroid binding activity, with the majority of the receptors present as both insoluble and cytosolic GR aggregates. Coexpression of p23 with the GR increases the proportion of cytosolic receptors that are in stable GR-hsp90 heterocomplexes with steroid binding activity, a strictly hsp90-dependent activity for the GR. Coexpression of p23 eliminates the insoluble GR aggregates and shifts the cytosolic receptor from very large aggregates without steroid binding activity to ∼600-kDa heterocomplexes with steroid binding activity. These data lead us to conclude that p23 acts in vivo to stabilize hsp90 binding to client protein. [ABSTRACT FROM AUTHOR]

Rationale: A common strategy for antibody-drug conjugate (ADC) quantitation from in vivo study samples involves measurement of total antibody, conjugated ADC, and free payload concentrations using multiple reaction monitoring (MRM) mass spectrometry. This not only provides a limited picture of biotransformation but can also involve lengthy method development. Quantitation of ADCs directly at the intact protein level in native conditions using high-resolution mass spectrometers presents the advantage of measuring exposure readout as well as monitoring the change in average drug-to-antibody ratio (DAR) and in vivo stability of new linker payloads with minimal method development. Furthermore, site-specific cysteine-conjugated ADCs often rely on non-covalent association to retain their quaternary structure, which highlights the unique capabilities of native mass spectrometry (nMS) for intact ADC quantitation., Methods: We developed an intact quantitation workflow involving three stages: automated affinity purification, nMS analysis, and data processing in batch fashion. The sample preparation method was modified to include only volatile ion-pairing reagents in the buffer systems. A capillary size-exclusion chromatography (SEC) column was coupled to a quadrupole time-of-flight high-resolution mass spectrometer for high-throughput nMS analysis. Samples from two mouse pharmacokinetic (PK) studies were analyzed using both intact quantitation workflow and the conventional MRM-based approach., Results: A linear dynamic range of 5-100 μg/mL was achieved using 20 μL of serum sample volume. The results of mouse in vivo PK measurement using the intact quantitation workflow and the MRM-based approach were compared, revealing excellent method agreement., Conclusions: We demonstrated the feasibility of utilizing nMS for the quantitation of ADCs at the intact protein level in preclinical PK studies. Our results indicate that this intact quantitation workflow can serve as an alternative generic method for high-throughput analysis, enabling an in-depth understanding of ADC stability and safety in vivo., (© 2024 John Wiley & Sons Ltd.)

A high throughput, semi-automated clearance screening assay in hepatocytes was developed allowing a scientist to generate data for 96 compounds in one week. The 384-well format assay utilizes a Thermo Multidrop Combi and an optimized LC-MS/MS method. The previously reported LCMS/ MS method reduced the analytical run time by 3-fold, down to 1.2 min injection-to-injection. The Multidrop was able to deliver hepatocytes to 384-well plates with minimal viability loss. Comparison of results from the new 384-well and historical 24-well assays yielded a correlation of 0.95. In addition, results obtained for 25 marketed drugs with various metabolism pathways had a correlation of 0.75 when compared with literature values. Precision was maintained in the new format as 8 compounds tested in ≥39 independent experiments had coefficients of variation ≤21%. The ability to predict in vivo clearances using the new stability assay format was also investigated using 22 marketed drugs and 26 AbbVie compounds. Correction of intrinsic clearance values with binding to hepatocytes (in vitro data) and plasma (in vivo data) resulted in a higher in vitro to in vivo correlation when comparing 22 marketed compounds in human (0.80 vs 0.35) and 26 AbbVie Discovery compounds in rat (0.56 vs 0.17), demonstrating the importance of correcting for binding in clearance studies. This newly developed high throughput, semi-automated clearance assay allows for rapid screening of Discovery compounds to enable Structure Activity Relationship (SAR) analysis based on high quality hepatocyte stability data in sufficient quantity and quality to drive the next round of compound synthesis.

The opioid buprenorphine has been shown to provide adequate postoperative analgesia in both companion and laboratory animals. However, its use is still hindered by the need for multiple parenteral injections to achieve continuous analgesia. The purpose of the current study was to conduct a pharmacokinetic analysis of 2 new long-acting formulations of buprenorphine-an injectable sustained-release buprenorphine (SRB) and a transdermal buprenorphine (TDB) patch-in healthy Göttingen minipigs by using liquid chromatography-electrospray ionization-tandem mass spectrometry. Administration of 0.18 mg/kg SC SRB and 30 μ g/h TDB achieved AUC(0-Tlast) of 221.6 ± 26.8 and 25.2 ± 3.9 ng × h/mL, respectively, compared with 9.7 ± 1.4 ng*h/mL for 0.02 mg/kg IV buprenorphine. By using a hypothesized therapeutic plasma buprenorphine concentration threshold of 0.1 ng/mL, therapeutic concentrations were achieved at the first study time point (5 to 30 min) and lasted an average of 8.0 ± 1.3 h for intravenous buprenorphine and 264.0 ± 32.2 h for SRB. TDB achieved therapeutic concentrations in 12 to 24 h after patch application, which lasted until the patch was removed at 72 h. The results of this study suggest that SRB and TDB are long-acting alternatives for pain management, and their use could decrease animal handling and stress, thereby simplifying pain management and improving welfare in laboratory swine.

The goal of the current study was to compare the efficacy, adverse effects, and plasma buprenorphine concentrations of sustained-release buprenorphine (SRB) and buprenorphine after subcutaneous administration in dogs undergoing ovariohysterectomy. In a prospective, randomized, blinded design, 20 healthy adult female Beagle dogs underwent routine ovariohysterectomy and received multimodal analgesia consisting of meloxicam and one of two buprenorphine formulations. Dogs were randomly assigned to receive either SRB (0.2 mg/kg SC, once) or buprenorphine (0.02 mg/kg SC every 12 h for 3 d). Blinded observers assessed all dogs by using sedation scores, pain scores, temperature, HR, RR, and general wellbeing. Dogs were provided rescue analgesia with 0.02 mg/kg buprenorphine SC if the postoperative pain score exceeded a prede- termined threshold. Blood samples were collected, and mass spectrometry was used to determine plasma buprenorphine concentrations. Data were analyzed with a linear mixed model and Tukey-Kramer multiple comparison. Age, body weight, anesthetic duration, surgical duration, sevoflurane concentration, and cardiorespiratory variables did not differ significantly between groups. Dogs in both formulation groups had comparable postoperative sedation and pain scores. One dog from each formulation group had breakthrough pain requiring rescue analgesia. Plasma buprenorphine concentrations remained above a hypothesized therapeutic concentration of 0.6 ng/mL for 136.0 ± 11.3 and 10.67 ± 0.84 h for SRB and buprenorphine, respectively. Based on the results of this study, multimodal analgesic regimens consisting of meloxicam and either buprenorphine or SRB are equally efficacious in managing pain associated with an ovariohysterectomy and show comparable side effects.

Tetrahydrobiopterin is a necessary cofactor for the synthesis of nitric oxide by the hemeprotein enzyme, NO-synthase (NOS). It is widely thought that inadequate levels of tetrahydrobiopterin lead to tissue injury and organ dysfunction due, in part, to formation of superoxide from pterin-deficient NOS. In the course of studies on the ubiquitylation of neuronal NOS (nNOS), we have found that certain substrate analogs, such as N(G)-nitro-L-arginine, stabilize the dimeric form of nNOS and protect the enzyme from ubiquitylation. Since tetrahydrobiopterin is known to bind near heme and confers stability to the active dimeric structure of nNOS, we wondered if the loss of tetrahydrobiopterin could be an endogenous signal for nNOS ubiquitylation and degradation. We show here in HEK293 cells stably transfected with nNOS that depletion of tetrahydrobiopterin by treatment with 2,4-diamino-6-hydroxypyrimidine leads to destabilization of the dimeric form and enhances ubiquitylation of nNOS. Sepiapterin, a precursor to tetrahydrobiopterin in the salvage pathway, completely reverses the effect of 2,4-diamino-6-hydroxypyrimidine on nNOS ubiquitylation. Consistent with that found in cells, the in vitro ubiquitylation of nNOS by reticulocyte proteins decreases when tetrahydrobiopterin is present. Thus, inadequate amounts of tetrahydrobiopterin may lead to a sustained decrease in the steady state level of nNOS that is not readily reversed.