CZ-48

9-Nitrocamptothecin-20-O-propionate (CZ112) and 9-Nitrocamptothecin (9NC) are the bioactive derivatives of camptothecin (CPT), an alkaloid isolated from Camptotheca acuminata, and have been confirmed to possess high anti-cancer properties. In the present study, 9NC was identified as the major metabolite of CZ112 in rat plasma through HPLC/photodiode array detection (PDA) and liquid chromatography tandem mass spectrometry (LC-MS/MS) analysis. A highly sensitive LC-MS/MS method was developed and validated for the simultaneous analysis of CZ112 and 9NC in rat plasma, and camptothecin-20-O-acetate (CZ44) was used as an internal standard (IS). The calibration curves were linear (r² > 0.999) over concentrations from 2.5 to 320 ng/mL for both CZ112 and 9NC. The method had an accuracy of 96.7-109.6%, and the intra- and inter-day precision (RSD%) were 10.9% or less for CZ112 and 9NC. The stability data showed no significant degradation occurred under the experimental conditions. This method was successfully applied to the pharmacokinetic study of CZ112 and its metabolite 9NC in rat plasma after intravenous and intragastric administration. The oral bioavailability of CZ112 was 6.2 ± 3.3% (n = 6).

SARM1, an NAD-utilizing enzyme, regulates axonal degeneration. We show that CZ-48, a cell-permeant mimetic of NMN, activated SARM1 in vitro and in cellulo to cyclize NAD and produce a Ca²⁺ messenger, cADPR, with similar efficiency as NMN. Knockout of NMN-adenylyltransferase elevated cellular NMN and activated SARM1 to produce cADPR, confirming NMN was its endogenous activator. Determinants for the activating effects and cell permeability of CZ-48 were identified. CZ-48 activated SARM1 via a conformational change of the auto-inhibitory domain and dimerization of its catalytic domain. SARM1 catalysis was similar to CD38, despite having no sequence similarity. Both catalyzed similar set of reactions, but SARM1 had much higher NAD-cyclizing activity, making it more efficient in elevating cADPR. CZ-48 acted selectively, activating SARM1 but inhibiting CD38. In SARM1-overexpressing cells, CZ-48 elevated cADPR, depleted NAD and ATP, and induced non-apoptotic death. CZ-48 is a specific modulator of SARM1 functions in cells.