The study protocol was approved by the Institutional Animal Care and Use Committee of Shanghai Medicilon Inc (ethics approval numbers MED1405_P001, MED1405_P003, 10012-14007, and 10012-14008).

The data are reported as the mean ± standard deviation. The dose–response curves and the half-maximal inhibitory concentration (IC₅₀) were determined by non-linear regression using GraphPad Prism 8.0. All statistical analyses were performed using SPSS Statistics 21.0 software (SPSS Korea, Seoul, Republic of Korea). P < 0.05 was considered statistically significant.

X842 was designed as a linaprazan prodrug by side chain esterification with glutaric acid (Figure 2). We investigated the pharmacokinetic profile of X842 after IV and oral administration. To determine the biotransformation of X842 (prodrug) to linaprazan (parent drug), the pharmacokinetic characteristics of both compounds were evaluated. Sixty Sprague-Dawley rats (equal distribution of males and females) were randomly assigned to five groups. Group 1 underwent IV administration of the test drugs (Figure 3; Table 1). Groups 2, 3, and 4 received increasing concentrations of orally administered drugs (Figure 4; Table 2). Group 5 received repeated administrations of orally administered drugs for seven consecutive days (Figure 5; Table 3).

The plasma concentration of X842 was barely detectable within 0.5 h after IV administration in both male and female rats (Figure 3). This rapid decline in the X842 concentration indicated that X842 was quickly converted into its active metabolite, linaprazan, in vivo. For single-dose oral administration, t_1/2 ranged from 2.0 to 2.7 h in male rats, and from 2.1 to 4.1 h in female rats (Table 2). Notably, the blood concentration of linaprazan was significantly higher than that of its prodrug X842 after 4 h when administered at doses of 2.4 mg/kg and 9.6 mg/kg (P < 0.05; Figure 4). This significant difference in the blood concentrations between the prodrug and its active metabolite is critical for understanding the pharmacokinetics of X842.

The plasma exposure calculation showed that the AUC_(0–24h) of the parent drug was 5.1–9.8 times that of the prodrug, indicating significantly greater overall exposure. Notably, the pharmacokinetic profile was consistent with the reported data for similar compounds, such as vonoprazan, which also demonstrates rapid absorption and significant conversion to active metabolites in vivo (Kong et al., 2020). Rapid conversion to the active metabolite ensures that the drug rapidly reaches its therapeutic concentration, which is essential for the effective treatment of acid-related diseases, such as GERD (Ashida et al., 2015).

Following single oral dosing, female rats consistently exhibited higher systemic exposure than males (Table 2). C_max and AUC_0–24h were significantly greater at every dose tested (P < 0.05), whereas no sex difference was seen after IV administration, indicating that absorption rather than clearance underlies the disparity. When the same doses were administered orally for seven consecutive days (Table 3), modest accumulation occurred in males (AUC _Day7 ÷ AUC _Day1 = 1.8 for the prodrug and 1.3 for the parent drug), but not in females (ratio = 0.9). This suggests that saturation or auto-inhibition of first-pass metabolism develops only in males upon repeated dosing. Consequently, the absolute oral bioavailability at 0.6 mg/kg (calculated from the combined exposure of parent drug plus active metabolites) was 11% in males versus 27% in females (Table 4), confirming both the sex-dependent absorption and the mild accumulation observed in males.

In dogs, X842 was evaluated after IV, single oral, and repeated oral dosing. Following IV administration (Supplementary Table S1), t_1/2 was short and was similar between the sexes (∼0.6 h), indicating rapid clearance. After a single oral dose (Supplementary Table S2), t_1/2 increased to 2.0–3.4 h in males and to 2.1–3.2 h in females, consistent with rapid first-pass conversion of X842 to linaprazan. Unlike in rats, where females displayed significantly higher C_max and AUC_0–24h than males, oral exposure in dogs showed no sex-related differences (Supplementary Tables S2, S3). Across species, AUC_0–24h increased with the dose in both rats and dogs (Figure 6). In dogs, the increase from 1.2 to 4.8 mg/kg reached statistical significance (P < 0.05), confirming dose proportionality. Where female rats exhibited markedly higher parent drug exposure (P < 0.05), female dogs showed only a non-significant trend toward lower AUC_0–24h at the highest dose. Collectively, these data indicate a clear dose–response relationship in dogs and an absence of sex differences in oral absorption.

After evaluating the pharmacokinetic profile of X842, its pharmacodynamic profile was investigated. The effect of X842 on H⁺/K⁺-ATPase activity was analyzed using an MLG-based assay, an indirect colorimetric method in which the readout has been cross-validated against radiometric ATP hydrolysis assays (Bandhuvula et al., 2007; Baykov et al., 2021) with correlation coefficients of >0.98, confirming its reliability for quantifying H⁺/K⁺-ATPase inhibition. The ATPase activity was not directly measured in this study because the inhibitory effects were quantified by calculating the percentage inhibition relative to the control wells. In the presence of K⁺, linaprazan and X842 inhibited H⁺/K⁺-ATPase activity in a concentration-dependent manner. The inhibitory potency of these three compounds ranked as follows: vonoprazan > linaprazan > X842. Their IC₅₀ values were 17.15 nM (95% confidence interval [CI] 10.81–26.87 nM), 40.21 nM (95% CI 24.02–66.49 nM), and 436.20 nM (95% CI 227.3–806.6 nM), respectively (Figure 7). No measurable inhibitory effect was observed in the absence of K⁺. Compared with linaprazan, X842 exhibited nearly 10-fold lower inhibitory activity against H⁺/K⁺-ATPase in the presence of K⁺, which may be partially due to structural modifications.

In the pylorus-ligated rat model, we systematically compared the antisecretory potency of X842 with that of the positive control vonoprazan under conditions of basal acid secretion following simple pyloric ligation. Vonoprazan (2 mg/kg) suppressed total gastric acid by 47% relative to vehicle-treated controls (P < 0.05). Under identical conditions, X842 produced clear dose-dependent inhibition; 1.5, 1.0, and 0.5 mg/kg reduced acid by 85%, 61%, and 44%, respectively (all P < 0.05), whereas 0.15 mg/kg X842 had no significant effect (6% inhibition, P > 0.05) (Table 5). The dose required for 50% inhibition (ID₅₀) was 0.55 mg/kg for X842, substantially lower than the dose of 2 mg/kg vonoprazan. X842 administered at ≥1 mg/kg consistently outperformed vonoprazan at 2 mg/kg in terms of both absolute acid reduction and fractional inhibition. Collectively, this demonstrated that X842 was a more potent acid suppressant than vonoprazan in this validated preclinical model, as well as evidencing its therapeutic potential in the histamine-stimulated pylorus-ligation model (Supplementary Figure S3; Supplementary Table S4).

Linaprazan and vonoprazan are well characterized P-CABs (Gedda et al., 2007; Hori et al., 2011). In the present study, we compared the gastric acid inhibitory properties of linaprazan, vonoprazan, and X842. X842 showed significant inhibitory effects on H⁺/K⁺-ATPase in the presence of K⁺ compared with in the absence of K⁺ (Figure 7). The potency of X842 in vitro was 20-times lower than that of linaprazan and vonoprazan when cellular esterase was not present (i.e., when X842 had not been metabolized). However, in vivo experiments using a pylorus-ligated rat model (Figure 8) showed that X842 for inhibiting acid secretion was more potent and had a longer duration of action than vonoprazan. The prodrug nature of X842 was confirmed by two additional in vivo experiments. The acid inhibitory effects were exerted by the metabolites of X842. After administration to rats, the percentage of X842 that was transformed into linaprazan ranged from 48.69% to 59.48% (Table 2). After administration to dogs, the transformed percentage ranged from 34.23% to 57.02% (Supplementary Table S2). Thus, the desired pharmacological properties of X842 were achieved by its designation as a prodrug of linaprazan.

Vonoprazan was a first-in-class P-CAB that entered the market with higher potency and longer-lasting acid suppression than conventional PPIs (Otake et al., 2016; Garnock-Jones, 2015). X842 surpassed vonoprazan in vivo. For instance, at doses of ≥1 mg/kg, it achieved a higher rate of acid suppression than 2 mg/kg vonoprazan (Figure 8). Moreover, even under non-metabolic conditions in vitro, X842 retained measurable activity against H⁺/K⁺-ATPase in the presence of K⁺, but it was 20-fold weaker than linaprazan or vonoprazan (Figure 7). This indicates that both the prodrug and its active metabolite contribute to the effect, a profile already hinted at in preclinical and phase I studies (data to be published) and characterized by rapid onset and prolonged action. Although these observations suggest a dual pharmacodynamic mechanism, we cannot rule out esterase-mediated conversion within the enzyme preparation. Experiments with purified enzyme systems or esterase inhibitors will be required for definitive confirmation. Collectively, the data show that X842 is a potent, fast-acting, and long-lasting H⁺/K⁺-ATPase inhibitor. Such properties position it as a promising therapeutic option for acid-related disorders, particularly in clinical settings where current PPI therapy remains inadequate.

As described previously (Rawla et al., 2018), linaprazan was quickly absorbed with a relatively short t_1/2, but it failed to achieve 24-h inhibition of gastric acid secretion. In the pharmacokinetic analysis, X842 reduced C_max and extended the t_1/2 of linaprazan (Table 1; Table 2). As mentioned, X842 may have a dual pharmacodynamic action in which the prodrug acts on the H⁺/K⁺-ATPase in the stomach before being metabolized. This could be an advantage of X842, conferring both fast-onset and prolonged-action properties on the inhibition of gastric acid secretion after oral administration. Compared with linaprazan, another advantage of X842 was the reduced toxicity (data to be published). No significant adverse effects, including acute, reproductive, teratogenic, or carcinogenic toxicity, were observed in either rats or dogs. The prodrug X842 minimizes hepatic exposure to linaprazan owing to its two-compartment pharmacokinetics, which also effectively minimizes toxicity.

As a novel P-CAB, the structural features of X842 in terms of the ester component are designed to influence its pharmacokinetic properties. In vivo, the prodrug is rapidly hydrolyzed by carboxylesterase 2 to yield the active metabolite linaprazan (for detailed experimental evidence see our companion study), which subsequently undergoes oxidation, dehydrogenation, and glucuronidation (Zhang et al., 2024). We noted that the plasma concentrations of both X842 and its metabolite linaprazan in female rats were higher than in male rats (Figures 4, 5; Table 2). This sex difference was not observed after IV administration, suggesting differences in the absorption and metabolism of X842 in rodents after oral administration. In fact, imidazopyridine derivatives are major inhibitors of cytochrome P450 (CYP) 3A4 enzymes (Martignoni et al., 2006). The CYP3A subfamily of isoforms differ between the sexes in rats (Song et al., 2012). However, in contrast to the results seen in rats, no sex differences in the absorption or plasma concentrations of X842 or linaprazan were observed in dogs (Supplementary Table S2; Figure 6). Although differences in drug absorption and metabolism among animal species are not uncommon, these observed species differences should be considered in further studies in humans. However, no sex differences have been observed in phase I trials involving more than 80 participants with equal numbers of both men and women (data to be published) after oral administration of X842. The consistency between the pharmacokinetic characteristics of X842 and linaprazan in humans and dogs is understandable, as CYP polymorphisms in humans and canines are much more closely related to each other than to rodents (Song et al., 2012).