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.

Cisplatin not only targets DNA but also RNA. However, it is largely unknown whether platinated RNA (Pt-RNA) causes apoptosis and thus contributes to the cytotoxic effects of cisplatin. Consequently, cellular RNA was isolated from HepG2 and LS180 cells, exposed to cisplatin, and the resulting Pt-RNA (20 ng Pt/µg RNA) was transfected into these cancer cell lines or used to treat an apoptosis reporter Caenorhabditis elegans (C. elegans) strain (MD701, expressing CED-1::GFP). Cellular and molecular effects of Pt-RNA were evaluated by luminogenic caspase 3/7 assays, PCR array analysis, and fluorescence microscopy-based quantification of apoptosis in C. elegans gonads. Assuming RNA cross-linking (pseudo double-stranded RNA), the contribution of the Toll-like receptor 3 (TLR3, a sensor of double-stranded RNA) to apoptosis induction in cancer cell lines was investigated by pharmacological TLR3 inhibition and overexpression. In contrast to controls, Pt-RNA significantly enhanced apoptosis in C. elegans (2-fold) and in the cancer cell lines (2-fold to 4-fold). TLR3 overexpression significantly enhanced the pro-apoptotic effects of Pt-RNA in HepG2 cells. TLR3 inhibition reduced the pro-apoptotic effects of Pt-RNA and cisplatin, but not of paclitaxel (off-target control). Gene expression analysis showed that Pt-RNA (but not RNA) significantly enhanced the mRNA levels of nuclear factor kappa B subunit 2 and interleukin-8 in HepG2 cells, suggesting that Pt-RNA is a damage-associated molecular pattern that additionally causes pro-inflammatory responses. Together, this data suggests that not only DNA but also cellular RNA is a functionally relevant target of cisplatin, leading to pro-apoptotic and immunogenic effects.

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 authors would like to make the following corrections to the publication