PK analysis of hsBCL9CT-24 and hsBCL9CT-35 was conducted in 35 male ICR outbred mice (Shanghai SLAC Laboratory Animal Co. Ltd.) in accordance with the standard protocol (Viva Biotech) and ethical regulations (IACUC). hsBCL9CT-24 and hsBCL9CT-35 were administered in the tail vein of murine models through i.v. bolus and i.p. injections. Positive control vehicles were prepared with 10% DMSO and 90% deionized H2O to generate a mixed solvent, with peptides tested at nominal concentrations of 0.5 mg/ml (incubated at room temperature before administration). Blood samples (300 μl) were collected periodically at the retro-orbital vein during 0.25, 1, 2, 4, 6, 7, 12, and 24 hours after dose, followed by plasma separation for pending bioanalysis. Additional details are described in Supplementary Materials and Methods.

Analytic concentrations were determined through LC-MS/MS (Agilent 1100 HPLC, AB Sciex API 4000). The internal standard osalmid (Sigma), acetonitrile (MeCN), and all solvents and chemicals met or exceeded analytical grade standards. MS was completed through electrospray ionization and positive MRM (multi reaction monitor) scans, while chromatographic conditions were set as follows: column [Thermo Betasil C18 (2.1 mm × 50 mm, 5 μm)], mobile phase A (0.1% HCOOH in H2O), mobile phase B (0.1% HCOOH in MeCN), elution rate (500 μl/min), temperature (40°C), and injection volume (15 μl). Standard stock solution was prepared with an analyte (1 mg/ml) in DMSO (70% MeCN in UP H2O), with calibration samples serially diluted to 10, 50, 100, 500, 1000, 2000, and 3000 ng/ml, while quality control final concentrations were spread over 40, 800, 1600, and 2400 ng/ml. The investigator was blinded to the group allocation during the experiment. Standard PK parameters were calculated by noncompartmental analysis modules in WinNonlin Professional v5.2 (Pharsight, U.S. Food and Drug Administration–certified) and included area under curve (AUClast and AUCINF), terminal half-life [terminal t1/2 = ln(2)/λz], maximum plasma concentration (Cmax), time to reach maximum plasma concentration (Tmax), clearance (CL), volume of distribution (Vz), apparent volume of distribution at steady state (Vss), and mean residence time from time of dosing to infinity (MRTINF). Bioavailability (F) was calculated as follows: F = [(AUCint(i.p.) × dosei.v.)/(AUCint(i.v.) × dosei.p.)] × 100%. Each data point was performed in triplicate, and each experiment was repeated twice.

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