Cindy Bay

Evaluation of blood-brain barrier (BBB) permeability is a crucial step in drug research and development, particularly for the pharmacotherapy of targets in the central nervous system. Current preclinical permeability predictions are hindered by limited transferability or BBB model inaccessibility for analysis. In order to generate relevant pharmacological data, it is necessary to establish qualified physiological, translational, and quantitative systems with validated methods. 3D BBB organoids composed of human cerebral microvascular endothelial cells (hCMEC/D3), and primary pericytes and astrocytes were generated. The BBB barrier function was evaluated using the paracellular marker ᴅ-Tyr(Me)Orn. Confocal laser scanning microscopy was used to determine the architecture of the cell arrangements, crucial BBB hallmarks and the apoptosis rate (cleaved caspase 3). An experimental setup was established to evaluate drug uptake into the organoids using ultra-performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS). The concentration- and time-dependent intraorganoidal drug uptake was determined for 13 drugs, and used to calculate the ratio of intra- and extraorganoidal concentrations. These organoid uptake ratios were subsequently correlated with reported in vivo brain-to-plasma ratios. Additionally, the performance of four in silico BBB permeability prediction tools was assessed with the in vivo data. Optimal analysis time point was identified as day 3 post self-assembly based on organoid architecture, endothelial coverage and apoptosis rate. Modular barrier properties were shown by a reduction of intraorganoidal ᴅ-Tyr(Me)Orn after cell connection were compromised, and potential wash out cross-validated by fluorescent Cy5 in the wash solution. Deming regression demonstrated a correlation with pseudo R ² of 0.64 between the organoid uptake ratios and reported mammalian in vivo brain-to-plasma ratios. In contrast, the in silico tools evaluated displayed no correlations with these in vivo data. We established and validated a simple and rapid experimental setup for the UPLC-MS/MS drug quantification into single 3D BBB organoids. We demonstrated that the 3D BBB organoids have translational relevance with regard to human and mammalian in vivo BBB permeability. This system therefore qualifies as New Approach Methodology (NAM) and can be used for approximate evaluation of in vivo brain uptake, while outperforming state of the art in silico models.

For the toxicokinetic evaluation of a lipid conjugate of the cell-penetrating peptide cyclic nonaarginine, we aimed to develop a highly sensitive plasma quantification. Due to the amphiphilic properties and high number of basic amino acids, plasma bioanalysis with LC-MS/MS instruments is difficult. Challenges in particular include chromatographic characteristics, minimal extraction recovery, and resistance against collision-induced dissociation. We developed a quantification of a nonextractable CPP-lipid conjugate by eliminating the lipid part by phospholipase D digestion, enabling the efficient extraction of a surrogate peptide for analysis. Chromatographic separation was feasible only with trifluoroacetic acid (TFA)-based mobile phases. Ion suppression caused by TFA was reversed by postcolumn addition of aqueous ammonia. Efficient extraction of the surrogate peptide fragment was achieved by protein precipitation with TFA. This enabled the highly sensitive quantification of the CPP-lipid conjugate in plasma in the low picomolar range (lower limit of quantification of 0.1 ng/mL; 34 pM). The assay was validated according to the pertinent recommendations of the ICH M10 guideline on bioanalytical method validation and applied to the determination of the intravenous pharmacokinetics of the CPP-lipid conjugate in Beagle dogs. Applicability was further demonstrated by validation of the assay for human plasma. The established strategy can be used as a general approach to the bioanalysis of amphiphilic lipid conjugates by using lipase digestion to generate extractable surrogate fragments. Additionally, TFA-based UPLC-MS/MS analysis with desolvation of TFA adducts by postcolumn addition of ammonia is feasible for the regulated, highly sensitive quantification of arginine-rich peptides.

Lipid-based drug delivery systems can be surface-modified by lipid conjugates of the substance in question. The most important modifications include arginine-rich cell-penetrating peptides (CPP). A toxicokinetic evaluation of these lipid conjugates is im-portant during preclinical and clinical development of drug delivery formulations. Due to their amphiphilic properties and high number of basic amino acid residues, lipid conjugates of CPP exhibit difficult characteristics in regard to their plasma bioanalysis with LC-MS/MS instruments. These especially include challenging chromatography with extensive carry-over and minimal extrac-tion recovery, and, due to large numbers of basic amino acids and the resulting immobility of protons, resistance against collision-induced dissociation. We developed a surrogate quantification of a CPP-lipid conjugate relying on elimination of the lipid part by phospholipase D digestion. Chromatographic separation was only feasible with trifluoro acetic acid (TFA)-based mobile phases. Ion suppression caused by TFA was reversed by post-column addition of aqueous ammonia. Efficient extraction of the surrogate peptide fragment was achieved by protein precipitation with TFA. This enabled the highly sensitive quantification of the CPP-lipid conjugate in plasma in the low picomolar range (lower limit of quantification of 0.1 ng/mL; 34 pM). The assay was validated ac-cording to the pertinent recommendations of the ICH M10 guideline on bioanalytical method validation and applied to the deter-mination of the intravenous pharmacokinetics of the CPP-lipid conjugate in Beagle dogs. The established strategy can be used as a general approach to the bioanalysis of amphiphilic lipid conjugates and especially the TFA-based UPLC-MS/MS analysis with post-column desolvation of TFA adducts by ammonia is a feasible approach for the highly sensitive quantification of arginine-rich peptides and other related substances with challenging chromatographic characteristics.

Metformin is the gold standard substrate for evaluating potential inhibitors of the organic cation transporters (OCTs). Here, we established a UPLC-MS/MS assay to quantify metformin in cell pellets with a range of 0.05–50 ng/mL using 6-deuterated metformin as an internal standard. We used an ion-pairing chromatographic approach with heptafluorobutyric acid, making use of a reverse-phase column, and overcame the associated ion-suppression via previously established post-column injection of aqueous ammonia. The assay was validated according to the Food and Drug Administration (FDA) and the European Medicines Agency (EMA) recommendations for bioanalytical methods. The established extraction procedure was simple, very fast and ensured almost 100% recovery of the analyte. The exceptionally sharp peak form and retention of the ion-pairing chromatography are superior to other methods and allow us to measure as sensitively as 0.05 ng/mL. We used the herein established and validated method to develop a cellular OCT inhibition assay by using metformin as a substrate and human embryonic kidney cells (HEK) overexpressing the OCTs 1-3. The method presented may be useful for identifying new OCT inhibitors, but also for drug–drug interactions and other pharmacokinetic studies, where accurate quantification of low metformin amounts in relevant tissues is mandatory.

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.

The authors would like to make the following corrections to the publication

Targeting myeloid cells, especially microglia, for the treatment of neuroinflammatory diseases such as multiple sclerosis (MS), is underappreciated. Our in silico drug screening reveals topoisomerase 1 (TOP1) inhibitors as promising drug candidates for microglial modulation. We show that TOP1 is highly expressed in neuroinflammatory conditions, and TOP1 inhibition using camptothecin (CPT) and its FDA‐approved analog topotecan (TPT) reduces inflammatory responses in microglia/macrophages and ameliorates neuroinflammation in vivo. Transcriptomic analyses of sorted microglia from LPS‐challenged mice reveal an altered transcriptional phenotype following TPT treatment. To target myeloid cells, we design a nanosystem using β‐glucan‐coated DNA origami (MyloGami) loaded with TPT (TopoGami). MyloGami shows enhanced specificity to myeloid cells while preventing the degradation of the DNA origami scaffold. Myeloid‐specific TOP1 inhibition using TopoGami significantly suppresses the inflammatory response in microglia and mitigates MS‐like disease progression. Our findings suggest that TOP1 inhibition in myeloid cells represents a therapeutic strategy for neuroinflammatory diseases and that the myeloid‐specific nanosystems we designed may also benefit the treatment of other diseases with dysfunctional myeloid cells.

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.

Interferon-alpha (IFN-α) is suggested to cause pharmacokinetic drug interactions by lowering expression of drug disposition genes through affecting the activities of nuclear factor kappa B (NF-ĸB) and pregnane X receptor (PXR). The time-resolved impact of IFN-α 2a (1000 U/mL; 5000 U/mL; 2 h to 30 h) on the activities of NF-ĸB and PXR and mRNA expression (5000 U/mL; 24 h, 48 h) of selected drug disposition genes and on cytochrome P450 (CYP3A4) activity in LS180 cells (5000 U/mL; 24 h, 48 h) was evaluated using luciferase-based reporter gene assays, reverse transcription polymerase chain reaction, and luminescence-based CYP3A4 activity assays. The cross-talk between NF-ĸB activation and PXR suppression was evaluated by NF-ĸB blockage (10 µM parthenolide). IFN-α 2a initially (2 h, 6 h) enhanced NF-ĸB activity 2-fold and suppressed PXR activity by 30%. mRNA of CYP3A4 was halved, whereas UGT1A1 was increased (1.35-fold) after 24 h. After 48 h, ABCB1 expression was increased (1.76-fold). CYP3A4 activity remained unchanged after 24 h, but was enhanced after 48 h (1.35-fold). IFN-α 2a demonstrated short-term suppressive effects on PXR activity and CYP3A4 mRNA expression, likely mediated by activated NF-ĸB. Longer exposure enhanced CYP3A4 activity. Clinical trials should evaluate the relevance by investigating the temporal effects of IFN-α on CYP3A4 using a sensitive marker substrate.