Extended supercooled storage of red blood cells.

Red blood cell (RBC) transfusions facilitate many life-saving acute and chronic interventions. Transfusions are enabled through the gold-standard hypothermic storage of RBCs. Today, the demand for RBC units is unfulfilled, partially due to the limited storage time, 6 weeks, in hypothermic storage. This time limit stems from high metabolism-driven storage lesions at +1-6 °C. A recent and promising alternative to hypothermic storage is the supercooled storage of RBCs at subzero temperatures, pioneered by our group. Here, we report on long-term supercooled storage of human RBCs at physiological hematocrit levels for up to 23 weeks. Specifically, we assess hypothermic RBC additive solutions for their ability to sustain supercooled storage. We find that a commercially formulated next-generation solution (Erythro-Sol 5) enables the best storage performance and can form the basis for further improvements to supercooled storage. Our analyses indicate that oxidative stress is a prominent time- and temperature-dependent injury during supercooled storage. Thus, we report on improved supercooled storage of RBCs at -5 °C by supplementing Erythro-Sol 5 with the exogenous antioxidants, resveratrol, serotonin, melatonin, and Trolox. Overall, this study shows the long-term preservation potential of supercooled storage of RBCs and establishes a foundation for further improvement toward clinical translation.

P-Glycoprotein (ABCB1/MDR1) and BCRP (ABCG2) Limit Brain Accumulation and Cytochrome P450-3A (CYP3A) Restricts Oral Exposure of the RET Inhibitor Selpercatinib (RETEVMO).

Selpercatinib is a targeted, FDA-approved, oral, small-molecule inhibitor for the treatment of rearranged during transfection (RET) proto-oncogene mutation-positive cancer. Using genetically modified mouse models, we investigated the roles of the multidrug efflux transporters ABCB1 and ABCG2, the OATP1A/1B uptake transporters, and the drug-metabolizing CYP3A complex in selpercatinib pharmacokinetics. Selpercatinib was efficiently transported by hABCB1 and mAbcg2, but not hABCG2, and was not a substrate of human OATP1A2, -1B1 or -1B3 in vitro. In vivo, brain and testis penetration were increased by 3.0- and 2.7-fold in Abcb1a/1b-/- mice and by 6.2- and 6.4-fold in Abcb1a/1b;Abcg2-/- mice, respectively. Oatp1a/1b deficiency did not alter selpercatinib pharmacokinetics. The ABCB1/ABCG2 inhibitor elacridar boosted selpercatinib brain penetration in wild-type mice to the levels seen in Abcb1a/1b;Abcg2-/- mice. Cyp3a-/- mice showed a 1.4-fold higher plasma AUC0-4h than wild-type mice, which was then 1.6-fold decreased upon transgenic overexpression of human CYP3A4 in liver and intestine. In summary, ABCG2, and especially ABCB1, limit brain and testis penetration of selpercatinib. Elacridar coadministration could mostly reverse these effects, without causing acute toxicity. CYP3A-mediated metabolism can limit selpercatinib oral exposure and hence its tissue concentrations. These insights may be useful in the further clinical development of selpercatinib.

Quantitative bioanalytical assay for the selective RET inhibitors selpercatinib and pralsetinib in mouse plasma and tissue homogenates using liquid chromatography-tandem mass spectrometry.

Selpercatinib and pralsetinib are potent and selective tyrosine kinase inhibitors targeting the rearranged during transfection (RET) receptor in various types of cancer. In this study, a bioanalytical assay was developed and fully validated for selpercatinib and pralsetinib in mouse plasma and partially in eight mouse tissue homogenates using liquid chromatograph-tandem mass spectrometry. Samples were pre-treated by protein precipitation with acetonitrile using erlotinib as internal standard. Separation of the analytes was performed on an ethylene bridged octadecyl silica C18 column by gradient elution using ammonium hydroxide (in water) and methanol. Analytes were detected by positive electrospray ionization in selected reaction monitoring mode. A linear concentration range of 2-2000 ng/ml was used for the validation of the assay for both inhibitors. The precision values (within-day and between-day) ranged between 3.4 and 10.2% for selpercatinib and 3.1-14.6% for pralsetinib in all matrices. Furthermore, data obtained for accuracy were between 91.7 and 109.3% and 85.1-114.1% for selpercatinib and pralsetinib, respectively. No significant matrix effects or extraction losses were observed and both analytes were stable under all investigated conditions. Finally, a pilot study for selpercatinib in mice was conducted employing this method, followed by a successful incurred sample reanalysis.