(M1030-06-33) The Application of 19F-NMR in the Analysis of Drug-Related Substances of Leritrelvir (RAY1216) in Human Urine and Fecal Samples to Support a Phase I Clinical Study

Purpose: In this study, sensitive and specific 19F-NMR bioanalytical methods in urine and fecal matrices have been developed and qualified to support a Phase I clinical trial of Leritrelvir (RAY1216) to obtain preliminary human mass balance data. The established 19F-NMR methods can be used as fast and reliable alternatives to traditional radiolabeled human mass balance for fluorinated compounds. Human mass balance studies can provide critical data for evaluating the absorption, distribution, metabolism, and excretion (ADME) properties of a new chemical entity (NCE). Traditionally, this has been performed by administering radiolabeled drug substances, typically 14C or occasionally 3H, as detection methods for these isotopes, which allows for absolute quantification of drug-related substances (DRMs) in blood, plasma, and excreta. However, radiolabeling studies are usually costly and time-consuming. As an alternative, Nuclear Magnetic Resonance (NMR) spectroscopy has recently emerged as a powerful analytical technique in mass balance study. Its non-destructive and non-invasive nature and its ability to provide detailed structural information have made it an invaluable tool for quantitative analysis. Furthermore, the advent of high field magnets and cryoprobes significantly improves the sensitivity of NMR for its application in drug in vivo ADME research. Among the NMR-based bioanalytical methods, 19F-NMR can provide qualitative insights into human ADME endpoints of fluorinated compounds, including mass balance, total DRM determination and metabolite profiling and identification in plasma and excreta. In this study, we developed 19F-NMR methods in urine and fecal samples in early clinical studies of Leritrelvir (RAY1216) to obtain preliminary data for human mass balance.

Methods: For the urine matrix, 2 mL of urine were weighed into 4 mL polypropylene tubes. After adding 50 μL of PF-07321332 internal standard solution and drying under a stream of nitrogen at room temperature, the samples were reconstituted in 0.65 mL DMSO-D6 for NMR analysis. For the fecal sample, 0.4 g of fecal matter was homogenized with 3.6 mL of methanol and then centrifuged at 3,000g for 15 min. Three mL of the supernatant were taken out and spiked with 50 μL of PF-07321332 internal standard solution. After being dried under a stream of nitrogen at room temperature, the samples were reconstituted in 1 mL DMSO-D6 for NMR analysis. 1D ¹⁹F spectra were acquired at a temperature of 300 K using a Bruker AVANCE NEO 400MHz NMR instrument equipped with a 5 mm BBO probe with a z-gradient system. A total of 1,024 scans were accumulated for each sample using a 1D inverse gated decoupling pulse sequence with a relaxation delay of five seconds. A total of 32,768 complex points covering 7462.7Hz were recorded at a transmitter frequency offset of -73.2 ppm. The data was zero-filled to 65,536 complex points prior to Fourier transformation, and an exponential window function was applied with a line-broadening factor of 1.0 Hz. The spectra were manually phased, and the baseline was corrected and referenced to the internal standard PF-07321332 (= –72.9 ppm). Based on these parameters, the total acquisition time was approximately 123 minutes per sample.

Results: During the method development, different options of internal standards were carefully evaluated. Firstly, the internal standard should have a distinct and well-separated chemical shift from Leritrelvir (RAY1216) in the NMR spectrum to ensure the absence of signal overlap and interference with peak integration. Secondly, to avoid off-resonance effects, the fluorine shift should not differ too much from Leritrelvir (RAY1216). Thirdly, the signal intensity of the internal standard should not differ more than 50-fold from the expected lowest and highest concentrations of Leritrelvir (RAY1216). The standard curve and quality control sample for the urine and fecal matrix were prepared by adding the working solution of Leritrelvir (RAY1216) into the responding blank matrix. The standard curve ranges for urine and fecal are 3.50-175 μg/mL and 35.0-3500 μg/g, respectively. The response ratio of Leritrelvir (RAY1216) to the internal standard is plotted against the concentration of Leritrelvir (RAY1216) in the standard curve. No matrix effect was observed, and the standard curve regression fit well. The standard curve for urine and fecal is y=0.03711x-0.008574 (r²=0.9980) (shown in Figure 1) and y=0.003321x-0.02395 (r²=0.9998) (shown in Figure 2), respectively. The accuracy of quality control samples for urine and fecal method are between -9.7 to 9.7% and -4.0 to 5.1%, respectively.

Conclusion: This qualified method has been applied to support urine and fecal sample analysis in a clinical trial titled: A randomized, double-blind, placebo-controlled Phase I trial evaluating the tolerability, pharmacokinetics, and food effects of Leritrelvir (RAY1216) tablets in adult subjects.

Figure 1. Standard curve for Leritrelvir (RAY1216) in a urine matrix

Figure 2. Standard curve for Leritrelvir (RAY1216) in a fecal matrix