Fig. 4: Estimating effective concentration and dose to infer iPK/PD parameters for S-XL6.
S-XL6’s concentration-%TE relationship was established by titration into target tissue homogenate, a minimum effective dose was estimated and tested in vivo. a Titration of covalent drug in whole blood and target tissue homogenate followed by intact protein LC-MS analysis. S-XL6 was spiked at increasing concentrations in whole blood and brain homogenate (target tissue) prepared from fast-line B6SJL-Tg (SOD1*G93A)1Gur/J transgenic mouse model, incubated at 37°C for 1 h, extracted using chloroform-ethanol precipitation, and %TE was analyzed by LC-MS. Dose estimation of S-XL6 was based upon 37% METE. b In vivo administration of covalent binder S-XL6 to achieve effective target engagement in systemic (whole blood) and target organ (brain). Dosed ALS SOD1G93A mice achieved METE at all predicted doses (10 mg/kg intravenously, 12.5 mg/kg and 30 mg/kg subcutaneously). In vitro and in vivo experiments were performed in singlet and duplicate, respectively. c Intravenous (IV) administration of covalent drug S-XL6 at 10 mg/kg and (d) oral (PO) administration of S-XL6 at 150 mg/kg in transgenic SOD1G93A mice (n = 2 per RoA). Whole blood was collected at 1, 4, 8 24-, 48-, 72-, and 168-h post-dose (for IV), and up to 72-h for PO, and extracted and analyzed as described above. Area under the curves (AUCs) were estimated for IV and PO curves. Additionally, brain (target) tissue was collected 1-hr post IV dose and processed as described above. Kp[brain] was calculated using %TE values (%TE brain divided by %TE blood) was c.a. ~1. For c and d raw %TE data are shown as discrete data points, and the lines illustrate fits obtained to the two term iPK/PD model [\(C\left(t\right)=A{e}^{-\alpha t}+B{e}^{-\beta t}\)] derived in Supplementary Fig. 2 and described in Supplementary File. Data (c, d) are presented as Mean ± SD. RoA = route of administration, METE = Minimally effective target engagement. Source data are provided as a Source Data file.
