Extracellular Vesicles as Surrogates for the Regulation of the Drug Transporters ABCC2 (MRP2) and ABCG2 (BCRP).

Drug efflux transporters of the ATP-binding-cassette superfamily play a major role in the availability and concentration of drugs at their site of action. ABCC2 (MRP2) and ABCG2 (BCRP) are among the most important drug transporters that determine the pharmacokinetics of many drugs and whose overexpression is associated with cancer chemoresistance. ABCC2 and ABCG2 expression is frequently altered during treatment, thus influencing efficacy and toxicity. Currently, there are no routine approaches available to closely monitor transporter expression. Here, we developed and validated a UPLC-MS/MS method to quantify ABCC2 and ABCG2 in extracellular vesicles (EVs) from cell culture and plasma. In this way, an association between ABCC2 protein levels and transporter activity in HepG2 cells treated with rifampicin and hypericin and their derived EVs was observed. Although ABCG2 was detected in MCF7 cell-derived EVs, the transporter levels in the vesicles did not reflect the expression in the cells. An analysis of plasma EVs from healthy volunteers confirmed, for the first time at the protein level, the presence of both transporters in more than half of the samples. Our findings support the potential of analyzing ABC transporters, and especially ABCC2, in EVs to estimate the transporter expression in HepG2 cells.

Effect of Tacrolimus Formulation (Prolonged-Release vs Immediate-Release) on Its Susceptibility to Drug-Drug Interactions with St. John's Wort.

Tacrolimus is metabolized by cytochrome P450 3A (CYP3A) and is susceptible to interactions with the CYP3A and P-glycoprotein inducer St. John's Wort (SJW). CYP3A isozymes are predominantly expressed in the small intestine and liver. Prolonged-release tacrolimus (PR-Tac) is largely absorbed in distal intestinal segments and is less susceptible to CYP3A inhibition. The effect of induction by SJW is unknown. In this randomized, crossover trial, 18 healthy volunteers received single oral tacrolimus doses (immediate-release [IR]-Tac or PR-Tac, 5 mg each) alone and during induction by SJW. Concentrations were quantified using ultra-high performance liquid chromatography coupled with tandem mass spectrometry and non-compartmental pharmacokinetics were evaluated. SJW decreased IR-Tac exposure (area under the concentration-time curve) to 73% (95% confidence interval 60%-88%) and maximum concentration (Cmax ) to 61% (52%-73%), and PR-Tac exposure to 67% (55%-81%) and Cmax to 69% (58%-82%), with no statistical difference between the 2 formulations. The extent of interaction appeared to be less pronounced in volunteers with higher baseline CYP3A4 activity and in CYP3A5 expressors. In contrast to CYP3A inhibition, CYP3A induction by SJW showed a similar extent of interaction with both tacrolimus formulations. A higher metabolic baseline capacity appeared to attenuate the extent of induction by SJW.

In vitro evaluation of the reductive carbonyl idarubicin metabolism to evaluate inhibitors of the formation of cardiotoxic idarubicinol via carbonyl and aldo-keto reductases.

The most important dose-limiting factor of the anthracycline idarubicin is the high risk of cardiotoxicity, in which the secondary alcohol metabolite idarubicinol plays an important role. It is not yet clear which enzymes are most important for the formation of idarubicinol and which inhibitors might be suitable to suppress this metabolic step and thus would be promising concomitant drugs to reduce idarubicin-associated cardiotoxicity. We, therefore, established and validated a mass spectrometry method for intracellular quantification of idarubicin and idarubicinol and investigated idarubicinol formation in different cell lines and its inhibition by known inhibitors of the aldo-keto reductases AKR1A1, AKR1B1, and AKR1C3 and the carbonyl reductases CBR1/3. The enzyme expression pattern differed among the cell lines with dominant expression of CBR1/3 in HEK293 and MCF-7 and very high expression of AKR1C3 in HepG2 cells. In HEK293 and MCF-7 cells, menadione was the most potent inhibitor (IC50 = 1.6 and 9.8 µM), while in HepG2 cells, ranirestat was most potent (IC50 = 0.4 µM), suggesting that ranirestat is not a selective AKR1B1 inhibitor, but also an AKR1C3 inhibitor. Over-expression of AKR1C3 verified the importance of AKR1C3 for idarubicinol formation and showed that ranirestat is also a potent inhibitor of this enzyme. Taken together, our study underlines the importance of AKR1C3 and CBR1 for the reduction of idarubicin and identifies potent inhibitors of metabolic formation of the cardiotoxic idarubicinol, which should now be tested in vivo to evaluate whether such combinations can increase the cardiac safety of idarubicin therapies while preserving its efficacy.

Bioanalysis of the Ex Vivo Labile PACE4 Inhibitory Peptide Ac-[d-Leu]LLLRVK-Amba in Whole Blood Using Ultra-Performance Liquid Chromatography-Tandem Mass Spectrometry Quantification.

The calcium-dependent serine endoprotease PACE4 is evaluated as a therapeutic target for prostate cancer. The peptide Ac-[d-Leu]LLLRVK-amba inhibits PACE4 with high affinity and has shown efficacy in preclinical mice xenograft models of prostate cancer. To support in vivo examinations of the potential therapeutic peptide Ac-[d-Leu]LLLRVK-amba, we established a highly sensitive assay for its quantification in mouse whole blood microsamples based on UPLC-MS/MS determination. Ac-[d-Leu]LLLRVK-amba was very labile during sample processing, which was particularly pronounced in plasma. High resolution mass spectrometric investigations of the metabolism/degradation in plasma revealed that no peptide bond hydrolysis generated products were formed, leaving the cause of the observed consumption of the peptide elusive. As a consequence, whole-blood quantification was developed relying on the immediate snap-freezing of blood samples after collection and immediate sample processing after serial thawing to ensure accurate and reliable quantification. The assay was validated according to the applicable recommendations of the FDA and EMA in a range of 10-10,000 ng/mL and applied to determine the pharmacokinetics of Ac-[d-Leu]LLLRVK-amba after intravenous and intraperitoneal administration to mice. Individual pharmacokinetic profiles were assessed using four microsamplings per animal. Intraperitoneal absorption was found to be efficient, demonstrating that this well-manageable route of administration is feasible for preclinical efficacy experiments with Ac-[d-Leu]LLLRVK-amba.

Influence of a Short Course of Ritonavir Used as Booster in Antiviral Therapies Against SARS-CoV-2 on the Exposure of Atorvastatin and Rosuvastatin.

PURPOSE: Early antiviral treatment with nirmatrelvir/ritonavir is recommended for SARS-CoV-2-infected patients at high risk for severe courses. Such patients are usually chronically ill and susceptible to adverse drug interactions caused by ritonavir. We investigated the interactions of short-term low-dose ritonavir therapy with atorvastatin and rosuvastatin, two statins commonly used in this population. METHOD: We assessed exposure changes (area under the concentration-time curve (AUC∞) and maximum concentration (Cmax)) of a single dose of 10 mg atorvastatin and 10 mg rosuvastatin before and on the fifth day of ritonavir treatment (2 × 100 mg/day) in healthy volunteers and developed a semi-mechanistic pharmacokinetic model to estimate dose adjustment of atorvastatin during ritonavir treatment. RESULTS: By the fifth day of ritonavir treatment, the AUC∞ of atorvastatin increased 4.76-fold and Cmax 3.78-fold, and concurrently, the concentration of atorvastatin metabolites decreased to values below the lower limit of quantification. Pharmacokinetic modelling indicated that a stepwise reduction in atorvastatin dose during ritonavir treatment with a stepwise increase up to 4 days after ritonavir discontinuation can keep atorvastatin exposure within safe and effective margins. Rosuvastatin pharmacokinetics were only mildly modified; ritonavir significantly increased the Cmax 1.94-fold, while AUC∞ was unchanged. CONCLUSION: Atorvastatin doses should likely be adjusted during nirmatrelvir/ritonavir treatment. For patients on a 20-mg dose, we recommend half of the original dose. In patients taking 40 mg or more, a quarter of the dose should be taken until 2 days after discontinuation of nirmatrelvir/ritonavir. Patients receiving rosuvastatin do not need to change their treatment regimen. TRIAL REGISTRATION: EudraCT number: 2021-006634-39. DRKS00027838.

Persister cell phenotypes contribute to poor patient outcomes after neoadjuvant chemotherapy in PDAC.

Neoadjuvant chemotherapy can improve the survival of individuals with borderline and unresectable pancreatic ductal adenocarcinoma; however, heterogeneous responses to chemotherapy remain a significant clinical challenge. Here, we performed RNA sequencing (n = 97) and multiplexed immunofluorescence (n = 122) on chemo-naive and postchemotherapy (post-CTX) resected patient samples (chemoradiotherapy excluded) to define the impact of neoadjuvant chemotherapy. Transcriptome analysis combined with high-resolution mapping of whole-tissue sections identified GATA6 (classical), KRT17 (basal-like) and cytochrome P450 3A (CYP3A) coexpressing cells that were preferentially enriched in post-CTX resected samples. The persistence of GATA6hi and KRT17hi cells post-CTX was significantly associated with poor survival after mFOLFIRINOX (mFFX), but not gemcitabine (GEM), treatment. Analysis of organoid models derived from chemo-naive and post-CTX samples demonstrated that CYP3A expression is a predictor of chemotherapy response and that CYP3A-expressing drug detoxification pathways can metabolize the prodrug irinotecan, a constituent of mFFX. These findings identify CYP3A-expressing drug-tolerant cell phenotypes in residual disease that may ultimately inform adjuvant treatment selection.

Comprehensive in vitro analysis evaluating the variable drug-drug interaction risk of rifampicin compared to rifabutin.

Compared to rifampicin (600 mg/day), standard doses of rifabutin (300 mg/day) have a lower risk of drug-drug interactions due to induction of cytochrome P450 3A4 (CYP3A4) or P-glycoprotein (Pgp/ABCB1) mediated by the pregnane X receptor (PXR). However, clinical comparisons with equal rifamycin doses or in vitro experiments respecting actual intracellular concentrations are lacking. Thus, the genuine pharmacological differences and the potential molecular mechanisms of the discordant perpetrator effects are unknown. Consequently, the cellular uptake kinetics (mass spectrometry), PXR activation (luciferase reporter gene assays), and impact on CYP3A4 and Pgp/ABCB1 expression and activity (polymerase chain reaction, enzymatic assays, flow cytometry) were evaluated in LS180 cells after treatment with different rifampicin or rifabutin concentrations for variable exposure times and eventually normalized to actual intracellular concentrations. In addition, inhibitory effects on CYP3A4 and Pgp activities were investigated. While rifampicin is poorly taken up by LS180 cells, it strongly activates PXR and leads to enhanced expression and activity of CYP3A4 and Pgp. In contrast, rifabutin is a significantly less potent and less efficient PXR activator and gene inducer, despite sixfold to eightfold higher intracellular accumulation. Finally, rifabutin is a potent inhibitor of Pgp (IC50 = 0.3 µM) compared to rifampicin (IC50 = 12.9 µM). Together, rifampicin and rifabutin significantly differ by their effects on the regulation and function of CYP3A4 and Pgp, even when controlled for intracellular concentrations. Rifabutin's concurrent Pgp inhibitory action might partly compensate the inducing effects, explaining its weaker clinical perpetrator characteristics.

Correction: Bay et al. Functional Characterization of the Solute Carrier LAT-1 (SLC7A5/SLC3A2) in Human Brain Capillary Endothelial Cells with Rapid UPLC-MS/MS Quantification of Intracellular Isotopically Labelled L-Leucine. Int. J. Mol. Sci. 2022, 23, 3637.

The authors would like to make the following corrections to the publication [...].

The first-in-class ERK inhibitor ulixertinib shows promising activity in mitogen-activated protein kinase (MAPK)-driven pediatric low-grade glioma models.

BACKGROUND: Pediatric low-grade gliomas (pLGG) are the most common pediatric central nervous system tumors, with driving alterations typically occurring in the MAPK pathway. The ERK1/2 inhibitor ulixertinib (BVD-523) has shown promising responses in adult patients with mitogen-activated protein kinase (MAPK)-driven solid tumors. METHODS: We investigated the antitumoral activity of ulixertinib monotherapy as well as in combination with MEK inhibitors (MEKi), BH3-mimetics, or chemotherapy in pLGG. Patient-derived pLGG models reflecting the two most common alterations in the disease, KIAA1549:BRAF-fusion and BRAFV600E mutation (DKFZ-BT66 and BT40, respectively) were used for in vitro and in vivo (zebrafish embryos and mice) efficacy testing. RESULTS: Ulixertinib inhibited MAPK pathway activity in both models, and reduced cell viability in BT40 with clinically achievable concentrations in the low nanomolar range. Combination treatment of ulixertinib with MEKi or BH3-mimetics showed strong evidence of antiproliferative synergy in vitro. Ulixertinib showed on-target activity in all tested combinations. In vivo, sufficient penetrance of the drug into brain tumor tissue in concentrations above the in vitro IC50 and reduction of MAPK pathway activity was achieved. In a preclinical mouse trial, ulixertinib mono- and combined therapies slowed tumor growth and increased survival. CONCLUSIONS: These data indicate a high clinical potential of ulixertinib for the treatment of pLGG and strongly support its first clinical evaluation in pLGG as single agent and in combination therapy in a currently planned international phase I/II umbrella trial.

Metabolic Effect of Blocking Sodium-Taurocholate Co-Transporting Polypeptide in Hypercholesterolemic Humans with a Twelve-Week Course of Bulevirtide-An Exploratory Phase I Clinical Trial.

Bile acids (BA) play an important role in cholesterol metabolism and possess further beneficial metabolic effects as signalling molecules. Blocking the hepatocellular uptake of BA via sodium-taurocholate co-transporting polypeptide (NTCP) with the first-in-class drug bulevirtide, we expected to observe a decrease in plasma LDL cholesterol. In this exploratory phase I clinical trial, volunteers with LDL cholesterol > 130 mg/dL but without overt atherosclerotic disease were included. Thirteen participants received bulevirtide 5 mg/d subcutaneously for 12 weeks. The primary aim was to estimate the change in LDL cholesterol after 12 weeks. Secondary endpoints included changes in total cholesterol, HDL cholesterol, lipoprotein(a), inflammatory biomarkers, and glucose after 12 weeks. In addition, cardiac magnetic resonance imaging (CMR) was performed at four time points. BA were measured as biomarkers of the inhibition of hepatocellular uptake. After 12 weeks, LDL cholesterol decreased not statistically significantly by 19.6 mg/dL [−41.8; 2.85] (Hodges−Lehmann estimator with 95% confidence interval). HDL cholesterol showed a significant increase by 5.5 mg/dL [1.00; 10.50]. Lipoprotein(a) decreased by 1.87 mg/dL [−7.65; 0]. Inflammatory biomarkers, glucose, and cardiac function were unchanged. Pre-dose total BA increased nearly five-fold (from 2026 nmol/L ± 2158 (mean ± SD) at baseline to 9922 nmol/L ± 7357 after 12 weeks of treatment). Bulevirtide was generally well tolerated, with most adverse events being administration site reactions. The exploratory nature of the trial with a limited number of participants allows the estimation of potential effects, which are crucial for future pharmacological research on bile acid metabolism in humans.

Evaluation of PepT1 (SLC15A1) Substrate Characteristics of Therapeutic Cyclic Peptides.

The human peptide transporter hPepT1 (SLC15A1), physiologically transporting dipeptides and tripeptides generated during food digestion, also plays a role in the uptake of small bioactive peptides and peptide-like drugs. Moreover, it might be addressed in prodrug strategies of poorly absorbed drugs. We hypothesised that the cyclic drug peptides octreotide and pasireotide could be substrates of this transporter because their diameter can resemble the size of dipeptides or tripeptides due to their strong structural curvature and because they reach the systemic circulation in Beagle dogs. For investigating possible hPepT1 substrate characteristics, we generated and characterised a CHO-K1 cell line overexpressing SLC15A1 by transfection and selection via magnetic beads. Possible inhibition of the uptake of the prototypical substrate Gly-Sar by octreotide and pasireotide was screened, followed by quantifying the uptake of the cyclic peptides in cells overexpressing SLC15A1 compared with the parental cell line. Although inhibition of Gly-Sar uptake was observed, uptake of octreotide and pasireotide was not increased in SLC15A1 overexpressing cells, indicating a lack of transport by hPepT1. Our data clearly indicate that octreotide and pasireotide are nonsubstrate inhibitors of hPepT1 and that their oral bioavailability cannot be explained by absorption via hPepT1.

Functional Characterization of the Solute Carrier LAT-1 (SLC7A5/SLC2A3) in Human Brain Capillary Endothelial Cells with Rapid UPLC-MS/MS Quantification of Intracellular Isotopically Labelled L-Leucine.

The solute carrier L-type amino acid transporter 1 (LAT-1/SLC7A5) is a viable target for drug delivery to the central nervous system (CNS) and tumors due to its high abundance at the blood-brain barrier and in tumor tissue. LAT-1 is only localized on the cell surface as a heterodimer with CD98, which is not required for transporter function. To support future CNS drug-delivery development based on LAT-1 targeting, we established an ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) assay for stable isotopically labeled leucine ([13C6, 15N]-L-leucine), with a dynamic range of 0.1-1000 ng/mL that can be applied for the functional testing of LAT-1 activity when combined with specific inhibitors and, consequently, the LAT-1 inhibition capacity of new compounds. The assay was established in a 96-well format, facilitating high-throughput experiments, and, hence, can support the screening for novel inhibitors. Applicable recommendations of the US Food and Drug Administration and European Medicines Agency for bioanalytical method validation were followed to validate the assay. The assay was applied to investigate the IC50 of two well-known LAT-1 inhibitors on hCMEC/D3 cells: the highly specific LAT-1 inhibitor JPH203, which was also used to demonstrate LAT-1 specific uptake, and the general system L inhibitor BCH. In addition, the [13C6, 15N]-L-leucine uptake was determined on two human brain capillary endothelial cell lines (NKIM-6 and hCMEC/D3), which were characterized for their expressional differences of LAT-1 at the protein and mRNA level and the surface amount of CD98. The IC50 values of the inhibitors were in concordance with previously reported values. Furthermore, the [13C6, 15N]-L-leucine uptake was significantly higher in hCMEC/D3 cells compared to NKIM-6 cells, which correlated with higher expression of LAT-1 and a higher surface amount of CD98. Therefore, the UPLC-MS/MS quantification of ([13C6, 15N]-L-leucine is a feasible strategy for the functional characterization of LAT-1 activity in cells or tissue.

Simultaneous Quantification and Pharmacokinetic Characterization of Doxapram and 2-Ketodoxapram in Porcine Plasma and Brain Tissue.

Atrial fibrillation (AF) is an arrhythmia associated with an increased stroke risk and mortality rate. Current treatment options leave unmet needs in AF therapy. Recently, doxapram has been introduced as a possible new option for AF treatment in a porcine animal model. To better understand its pharmacokinetics, three German Landrace pigs were treated with intravenous doxapram (1 mg/kg). Plasma and brain tissue samples were collected. For the analysis of these samples, an ultra performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS) assay for the simultaneous measurement of doxapram and its active metabolite 2-ketodoxapram was developed and validated. The assay had a lower limit of quantification (LLOQ) of 10 pg/mL for plasma and 1 pg/sample for brain tissue. In pigs, doxapram pharmacokinetics were biphasic with a terminal elimination half-life (t1/2) of 1.38 ± 0.22 h and a maximal plasma concentration (cmax) of 1780 ± 275 ng/mL. Its active metabolite 2-ketodoxapram had a t1/2 of 2.42 ± 0.04 h and cmax of 32.3 ± 5.5 h after administration of doxapram. Protein binding was 95.5 ± 0.9% for doxapram and 98.4 ± 0.3% for 2-ketodoxapram with a brain-to-plasma ratio of 0.58 ± 0.24 for doxapram and 0.12 ± 0.02 for 2-ketodoxapram. In conclusion, the developed assay was successfully applied to the creation of pharmacokinetic data for doxapram, possibly improving the safety of its usage.

Effect of Pantoprazole on the Absorption of Hydroxychloroquinea A Randomized Drug-Drug Interaction Trial in Healthy Adults.

Hydroxychloroquine as a weak basic compound with two amines is strongly enriched in cell compartments with low pH, suggesting that modification of gastric pH by coadministered proton pump inhibitors might reduce its solubility and absorption and thus its efficacy in patients. We addressed this question in a single-center, open-label, randomized, parallel drug-drug interaction trial in healthy adults (EudraCT No. 2020-001470-30). All participants received a single oral dose of 400-mg hydroxychloroquine, and one group additionally received 40 mg of pantoprazole once daily for 9 days dosed to steady state. Whole-blood samples were collected for 72 hours, and hydroxychloroquine was quantified by liquid chromatography-tandem mass spectrometry. Primary endpoints were whole-blood hydroxychloroquine areas under the concentration-time curve from 0 to 72 hours (AUC0-72h ) and peak concentrations (Cmax ). Unpaired 2-sided t-tests of the log transformed pharmacokinetic parameters were performed to compare both groups. Twenty-four participants (12 per group) were included. Hydroxychloroquine AUC0-72h and Cmax did not differ between groups without and with pantoprazole (arithmetic mean; AUC0-72h , 7649 ng/ml • h, and 8429 ng/ml • h, P = .50; Cmax , 448 ng/mL and 451.5 ng/mL, P = .96, respectively). Pantoprazole did not alter hydroxychloroquine absorption, indicating that proton pump inhibitors do not affect its bioavailability.

Investigating the Central Nervous System Disposition of Actinomycin D: Implementation and Evaluation of Cerebral Microdialysis and Brain Tissue Measurements Supported by UPLC-MS/MS Quantification.

Actinomycin D is a potent cytotoxic drug against pediatric (and other) tumors that is thought to barely cross the blood-brain barrier. To evaluate its potential applicability for the treatment of patients with central nervous system (CNS) tumors, we established a cerebral microdialysis model in freely moving mice and investigated its CNS disposition by quantifying actinomycin D in cerebral microdialysate, brain tissue homogenate, and plasma. For this purpose, we developed and validated an ultraperformance liquid chromatography-tandem mass spectrometry assay suitable for ultra-sensitive quantification of actinomycin D in the pertinent biological matrices in micro-samples of only 20 µL, with a lower limit of quantification of 0.05 ng/mL. In parallel, we confirmed actinomycin D as a substrate of P-glycoprotein (P-gp) in in vitro experiments. Two hours after intravenous administration of 0.5 mg/kg, actinomycin D reached total brain tissue concentrations of 4.1 ± 0.7 ng/g corresponding to a brain-to-plasma ratio of 0.18 ± 0.03, while it was not detectable in intracerebral microdialysate. This tissue concentration exceeds the concentrations of actinomycin D that have been shown to be effective in in vitro experiments. Elimination of the drug from brain tissue was substantially slower than from plasma, as shown in a brain-to-plasma ratio of approximately 0.53 after 22 h. Because actinomycin D reached potentially effective concentrations in brain tissue in our experiments, the drug should be further investigated as a therapeutic agent in potentially susceptible CNS malignancies, such as ependymoma.

Rapid and Sensitive Quantification of Intracellular Glycyl-Sarcosine for Semi-High-Throughput Screening for Inhibitors of PEPT-1.

The peptide transporter PEPT-1 (SLC15A1) plays a major role in nutritional supply with amino acids by mediating the intestinal influx of dipeptides and tripeptides generated during food digestion. Its role in the uptake of small bioactive peptides and various therapeutics makes it an important target for the investigation of the systemic absorption of small peptide-like active compounds and prodrug strategies of poorly absorbed therapeutics. The dipeptide glycyl-sarcosine (Gly-Sar), which comprises an N-methylated peptide bond that increases stability against enzymatic degradation, is widely utilized for studying PEPT-1-mediated transport. To support experiments on PEPT-1 inhibitor screening to identify potential substrates, we developed a highly sensitive Gly-Sar quantification assay for Caco-2 cell lysates with a dynamic range of 0.1 to 1000 ng/mL (lower limit of quantification 0.68 nM) in 50 µL of cell lysate. The assay was validated following the applicable recommendations for bioanalytic method validation of the FDA and EMA. Sample preparation and quantification were established in 96-well cell culture plates that were also used for the cellular uptake studies, resulting in a rapid and robust screening assay for PEPT-1 inhibitors. This sample preparation principle, combined with the high sensitivity of the UPLC-MS/MS quantification, is suitable for screening assays for PEPT-1 inhibitors and substrates in high-throughput formats and holds the potential for automation. Applicability was demonstrated by IC50 determinations of the known PEPT-1 inhibitor losartan, the known substrates glycyl-proline (Gly-Pro), and valaciclovir, the prodrug of aciclovir, which itself is no substrate of PEPT-1 and consequently showed no inhibition in our assay.

Bioanalysis of selinexor in mouse plasma micro-samples utilizing UPLC-MS/MS.

Selinexor, a first-in-class inhibitor of the nuclear export protein Exportin-1 (XPO1), was recently approved for the treatment of multiple myeloma in combination with dexamethasone, and as monotherapy for diffuse large B-cell lymphoma. To enable investigations of selinexor in mice, we established and validated an ultrahigh-performance liquid chromatography - tandem mass spectrometry (UPLC-MS/MS) assay in the plasma concentration range of 1-1000 ng/mL using plasma microsamples of 5 µL. Protein depletion with acetonitrile was used for efficient isolation of selinexor which was followed by a dilution step, resulting in a scalable sample processing. Quantification was performed with positive electrospray ionization tandem mass spectrometry in the selected reaction monitoring mode. Due to the high sensitivity of the quantification and the scalable sample processing procedure, the assay can be used for different concentration ranges to either further decrease the achievable lower limit of quantification or to reduce the amount of plasma used. The assay showed interday and intraday accuracy of 89.0-109.0% with a corresponding precision ≤ 14.1%. Suitability for investigations of selinexor in small animal experiments was demonstrated by determination of plasma selinexor in mice after oral administration.

Intact plasma quantification of the large therapeutic lipopeptide bulevirtide.

Bulevirtide is a first-in-class entry inhibitor of the hepatitis B and hepatitis delta virus blocking the sodium/bile acid co-transporter NTCP, and was recently approved for the treatment of hepatitis D as a priority medicine (prime) in an accelerated assessment by the European Medicines Agency. It is a very large lipopeptide comprising 47 amino acids in its sequence and a myristoylation at the N-terminus. For support of clinical development, we established highly sensitive plasma quantification assays using 100 µL of plasma, spanning concentrations of 0.1 to 100 ng/mL and 1 to 1000 ng/mL with the option to measure ten-fold diluted samples up to 10,000 ng/mL. Quantification was performed with UPLC-MS/MS measurements after extraction with protein precipitation. Both assays were fully validated according to the pertinent guidelines of the FDA and EMA, including incurred sample reanalyses and cross-validation using clinical study samples. Graphical abstract.

Application of triple quadrupole tandem mass spectrometry to the bioanalysis of collision-induced dissociation-resistant cyclic peptides - Ultra-sensitive quantification of the somatostatin-analog pasireotide utilizing UHPLC-MS/MS.

Cyclic peptides are considered collision-induced dissociation (CID)-resistant due to immobile protons, and the necessity of at least two consecutive dissociation reactions to produce fragments with deviating m/z values. Therefore, the bioanalysis of cyclic peptides by tandem mass spectrometry (MS/MS) poses a major challenge, especially on triple quadrupole (TQ) instruments. One of these peptides is the somatostatin analog pasireotide, a cyclic hexapeptide administered to treat Cushing's disease and acromegaly. To support oral formulation development, sub-therapeutic quantification of pasireotide is highly beneficial. Regardless of the considered CID-resistance, we investigated the CID-characteristics of pasireotide and subsequently developed an ultra-sensitive UHPLC-MS/MS assay with a lower limit of quantification (LLOQ) of 5 pg/mL (4.9 pM) when using 100 µL of plasma and validated it according to the guidelines of the FDA and EMA. The achieved sensitivity, which is the highest thus far reported, demonstrates that TQ-MS/MS is a feasible approach to sensitive quantification of cyclic peptides despite their CID-resistance. Pasireotide was fast and efficiently extracted by protein depletion via precipitation using acetonitrile. Correlation coefficients > 0.99 were achieved for all calibration curves with linear regression. Inter-run and intra-run accuracy ranged from 89.4 to 99.3 % with corresponding precision of ≤ 7.5 % in the calibrated range, and from 94.6 to 105.6 % with corresponding precision of ≤ 14.5 % at the LLOQ. Quantification of 10-fold diluted samples showed an accuracy of 90.8 % and corresponding precision of 4.0 %. The assay was applied to the quantification of pasireotide plasma concentrations after intravenous administration to beagle dogs.

Development and Validation of an LC-MS-Based Quantification Assay for New Therapeutic Antibodies: Application to a Novel Therapy against Herpes Simplex Virus.

Multiple therapeutic monoclonal antibodies (mAbs) are currently under development or in (pre)clinical study phases to reach regulatory approval. Among these, a new mAb against herpes simplex virus, HDIT101, was recently tested in healthy volunteers during a phase I clinical trial (first-in-human, dose escalation). In the frame of the pharmacokinetic evaluation of this new therapy, a mass spectrometric (MS)-based method was developed for the quantification of HDIT101 in human plasma using liquid chromatography coupled to tandem mass spectrometry. In this work, we describe the development of this bioanalytical assay using the quantification of a HDIT101 surrogate peptide, the assay validation procedure according to the FDA guidelines within the calibration range from 20 to 5000 µg/mL, and its application to plasma samples from the first-in-human clinical trial. This work presents a generic workflow for the development of MS-based quantification assays of new therapeutic antibodies that allows reaching high immunopurification recovery (>98% for HDIT101 over the full calibration range with a precision of 6.9% CV). Surrogate peptide and stable isotopically labeled internal standard were stable, and batch-to-batch accuracies and precisions at the four quality standard levels ranged between -2 and 5% bias and 8 and 11% CV, respectively.