Pathogens such as Streptococcus pneumoniae (S. pneumoniae), which are easily transmitted in daily life, can cause community-acquired pneumonia (CAP). The risk of bacteremia and sepsis increases if CAP remains untreated; in severe cases, the long-term mortality risk remains high even after treatment [1]. These causative organisms survive and proliferate through type II topoisomerases (DNA gyrase) and topoisomerase IV, which are critical for bacterial DNA replication, transcription, repair, and recombination, thereby facilitating their easy transmission [2]. Therefore, the inhibition of DNA gyrase and topoisomerase IV is essential for the treatment of CAP and acute exacerbation of chronic bronchitis.

Gemifloxacin, moxifloxacin, and levofloxacin are new fluoroquinolone antibiotics. Gemifloxacin primarily targets and inhibits both DNA gyrase and topoisomerase IV, thereby inhibiting DNA synthesis and inducing cell death. Furthermore, gemifloxacin exhibits lower minimum inhibitory concentrations (MICs) against pathogens causing acute exacerbation of chronic bronchitis (AECB) than other fluoroquinolone antibiotics. Clinically, gemifloxacin reduces hospitalization duration and contributes to healthcare cost savings compared to ceftriaxone and clarithromycin. Gemifloxacin has demonstrated higher clinical success rates than levofloxacin over the long term, indicating that gemifloxacin offers multiple advantages over other fluoroquinolone antibiotics [3]. Pharmacokinetic results from previous studies have demonstrated that an average of 61 $\pm$ 9.5% of the dose was excreted in feces following oral administration to healthy subjects, whereas 36 $\pm$ 9.3% was eliminated in urine as an unchanged drug and metabolite. After repeated dosing of 320 mg, the mean renal clearance was approximately 11.6 $\pm$ 3.9 L/h (range 4.6–16 L/h), suggesting that active secretion is involved in the renal excretion of gemifloxacin [4].

Cyclodextrin (CD) is a cyclic oligosaccharide comprising glucose units linked by $\alpha$-1,4-glycosidic bonds. Cyclodextrin is hydrophilic because of the presence of hydroxyl groups on its exterior, whereas its interior is relatively hydrophobic [5]. The application of $\beta$-CD in formulation development is severely limited by both its low intrinsic aqueous solubility due to strong intramolecular hydrogen bonding and a strict acceptable daily intake (ADI) limit of 0.35 g for humans. In contrast, hydroxypropyl-$\beta$-cyclodextrin (HPCD) exhibits drastically improved aqueous solubility through hydroxypropyl substitution and superior oral tolerability (up to 8 g/day) with minimal gastrointestinal irritation [6]. Given these physicochemical and toxicological advantages, HPCD is considered a safer and more effective oral excipient than unmodified $\beta$-CD. In addition, previous studies have demonstrated that CD exhibits specific tissue distribution and clearance characteristics in rats, suggesting that it not only aids absorption but also influences pharmacokinetic changes [7].

Therefore, we identified solubilizing agents that affect the solubility of gemifloxacin, a respiratory antibiotic with multiple advantages. In addition, we used solubilizing agents to compare and evaluate the bioavailability and pharmacokinetics of a new gemifloxacin formulation developed using a reduced active ingredient content compared to a reference drug.

Gemifloxacin is an amphoteric API possessing both carboxylic acid and amino functional groups, and exhibits pH-dependent limited aqueous solubility, which presents constraints in dissolution and absorption [9, 10]. We compared different surfactants and solubility enhancers to improve the solubility of gemifloxacin. Poloxamers are non-ionic surfactants composed of hydrophilic (ethylene oxide, PEO)–hydrophobic (propylene oxide, PPO)–PEO block copolymers that form micelles in aqueous solutions, thereby reducing the interfacial tension between the drug and solvent and improving the wettability and dispersibility of drug particles [11]. However, as depicted in Fig. 1, the solubility values of poloxamer 407 and poloxamer 188 were 27.81 $\pm$ 0.23 mg/mL and 22.62 $\pm$ 0.73 mg/mL, respectively, which were lower than those of the control group. This limited enhancement was attributed to the fact that effective micellization is induced only above the critical micelle concentration (CMC); micelle formation is restricted at relatively low concentrations, preventing the solubility enhancement effect from being fully manifested [12].

In addition, SLS demonstrated a solubility of 33.71 $\pm$ 0.36 mg/mL, lower than that of the control group. This effect is likely due to the strong electrostatic interactions between the amphoteric drug gemifloxacin and SLS, resulting in the precipitation of a poorly soluble lauryl sulfate salt instead of micelle-mediated solubilization [13]. These results suggest that the application of surfactants to amphoteric drugs requires consideration of physical stability and drug–excipient interactions. Similarly, PEG 4000 exhibited a solubility of 19.19 $\pm$ 0.82 mg/mL. PEG functions simply as a solvent modifier and cannot effectively influence the polar or non-polar regions of the API, resulting in low solubility [14]. L-lysine and L-arginine exhibited very low solubilities of 10.37 $\pm$ 0.25 mg/mL and 8.26 $\pm$ 0.19 mg/mL, respectively. Although amino acids can enhance solubility via ionic and non-ionic interactions, the improvement was limited in this study. This suggests that the interaction between the drug and amino acids was insufficient to overcome the strong self-association of the drug molecules [15]. Soluplus® demonstrated a relatively high solubility increase of 51.82 $\pm$ 0.34 mg/mL, which was attributed to its function as a hydrophilic–hydrophobic graft copolymer that forms polymeric micelles and functions as a solid dispersion matrix, thereby enhancing the solubility of poorly soluble drugs. However, its effect has been reported to be limited by the critical micellization concentration and plateau concentration [16]. Polyox N80 displayed a solubility of 43.91 $\pm$ 0.52 mg/mL, similar to the control group. Polyox N80 is a high-molecular-weight polyethylene oxide primarily used as a viscosity enhancer and release matrix; it contributes to viscosity and release control rather than inclusion complex formation or strong micellar solubilization, resulting in relatively low solubility enhancement effects [17]. A comprehensive consideration of these results confirmed HPCD as the solubilizing agent exhibiting the greatest solubility enhancement effect in this study, and a solubility of 69.37 $\pm$ 0.71 mg/mL at 4.1 g. HPCD possesses high aqueous solubility due to hydroxypropyl substitution and significantly increases equilibrium solubility by forming inclusion complexes with drugs through its hydrophobic cavity. The plateau effect observed at HPCD concentrations $\ge$0.1 g can be explained by the phase solubility behavior of cyclodextrin inclusion complexes (Fig. 2). In general, drug–cyclodextrin systems exhibit a limited complexation capacity, where the increase in drug solubility is proportional to cyclodextrin concentration only up to a certain point [17]. Beyond this concentration, the system reaches a saturation state in which most of the drug molecules are already complexed, and further addition of HPCD does not significantly enhance solubility. Although the phase solubility profile suggests the formation of a drug–HPCD inclusion complex, this interpretation is based on indirect evidence. Advanced physicochemical characterization techniques such as differential scanning calorimetry (DSC), Fourier-transform infrared spectroscopy (FTIR), and nuclear magnetic resonance (NMR) were not employed in this study. Therefore, the molecular-level interactions between gemifloxacin and HPCD could not be directly confirmed. This behavior is consistent with the formation of a 1:1 inclusion complex and the transition from a linear phase to a plateau region in the phase solubility diagram. Additionally, the self-association of cyclodextrin molecules and the formation of aggregates at higher concentrations can lead to a deviation from ideal solubilization behavior, thereby limiting the solubilization efficiency [18]. Based on these results, HPCD was determined to be a highly applicable solubility enhancer in terms of physical stability and formulation design. Considering its applicability to tablet formulations, the HPCD concentration was increased in a stepwise manner. The highest solubility of 66.27 $\pm$ 0.42 mg/mL was observed at 0.1 g, suggesting that the solubility increase did not follow a simple dose-dependent pattern. Although a complete concentration–response curve was not established, the observed plateau in solubility above 0.1 g HPCD suggests that further increases in cyclodextrin concentration do not significantly enhance complexation efficiency under the tested conditions (Fig. 2). Previous studies have reported that cyclodextrin–drug complexes exhibit diverse phase–solubility curves and non-linear characteristics in the concentration–solubility relationship [19]. Therefore, 0.1 g of HPCD was determined to be the optimal solubilization condition considering both the solubility enhancement effect and practical formulation applicability.

The bioanalytical method was validated according to the ICH M10 guidelines before conducting in vivo experiments to ensure the reliability and validity of the analytical method for the quantitative analysis of gemifloxacin in plasma. The linear regression equation of the calibration curve established in rat plasma over the range of 0.03–45 µg/mL was y = 0.1009x – 0.0073 (R² = 0.9998, n = 5), demonstrating appropriate linearity. The lower limit of quantification (LLOQ) of gemifloxacin was set at 0.03 µg/mL; the accuracy was within $\pm$1.14%, and precision was up to 5.71% at this concentration, meeting the criteria ($\le$20%) presented in the ICH M10 guidelines. The three QC sample concentrations excluding LLOQ demonstrated accuracy within $\pm$1.28% and precision up to 5.33%, all meeting the acceptance criteria ($\pm$15%) presented in the ICH M10 guidelines [20]. Thus, the analytical method established in this study has sufficient reliability and reproducibility for quantitative analysis of gemifloxacin in rat plasma. In vitro solubility evaluation results confirmed that HPCD significantly improved gemifloxacin solubility. Pharmacokinetic experiments were conducted in rats to evaluate whether this in vitro solubility enhancement translates into improved in vivo absorption. Although the absolute Cmax values were higher in the control group than in Groups A and B, the dose-corrected Cmax/dose values of Group A (1.175 $\pm$ 0.08 µg/mL) and Group B (1.862 $\pm$ 0.08 µg/mL) increased by 1.75-fold and 2.77-fold, respectively, compared to the control group (0.673 $\pm$ 0.13 µg/mL). To clarify the calculation of dose-normalized exposure, the Cmax values were normalized by the administered dose (Cmax/dose). The calculated values were 0.673, 1.175, and 1.862 for the control, Group A, and Group B, respectively. Accordingly, the dose-normalized Cmax values in Group A and Group B were increased by 1.75-fold and 2.77-fold, respectively, compared to the control group. The AUC_0-∞ values of Group A (7.527 $\pm$ 0.60 µg$\cdot$h/mL) and Group B (5.582 $\pm$ 0.55 µg$\cdot$h/mL) increased by 1.53-fold and 1.13-fold, respectively, compared to the control group (4.928 $\pm$ 0.85 µg$\cdot$h/mL). Notably, Group B received half the dose of gemifloxacin compared to Group A, which was intentionally designed to evaluate whether the HPCD formulation could maintain or enhance drug absorption efficiency at reduced doses. Despite the lower administered dose, Group B exhibited a higher dose-normalized Cmax compared to both the control and Group A. This indicates that the HPCD complex improved the absorption efficiency of gemifloxacin, rather than simply increasing systemic exposure due to higher dosing. These findings suggest that the formulation may enable dose reduction while maintaining effective drug absorption. Despite the low administered dose, the observed improvement in absorption cannot be explained solely by an increase in solubility. Rather, the formation of an inclusion complex with HPCD likely inhibited the crystallization of gemifloxacin, thereby reducing precipitation and maintaining a supersaturated state within the intestinal lumen [21]. In particular, to overcome the unstirred water layer (UWL), which is recognized as a primary absorption barrier for poorly water-soluble drugs, the inclusion complex maintains a steep concentration gradient across the entire UWL. As a result, the inclusion complex efficiently transports the drug across the UWL to the lipophilic epithelial surface. Upon reaching the membrane surface, the complex reversibly dissociates, releasing free drug molecules and thereby enhancing mucosal drug permeation [22]. This suggests that the increased solubility of the gemifloxacin–HPCD complex increased the drug concentration in the intestinal lumen, resulting in enhanced absorption by passive diffusion [23]. The interaction between HPCD and phospholipids increases acyl chain disorder, leading to enhanced membrane fluidity and structural perturbation, which in turn increases membrane permeability [24]. This increase in membrane permeability may have partially contributed to the enhanced gemifloxacin absorption observed in this study. These results were consistent with those of previous studies demonstrating that the bioavailability of poorly soluble drugs can be improved by HPCD inclusion complex [25]. HPCD is a widely used pharmaceutical excipient with a well-established safety profile. Previous studies have reported a no-observed-adverse-effect level (NOAEL) of approximately 600 mg/kg/day in rats and low acute toxicity with oral LD₅₀ values exceeding 2000 mg/kg. In the present study, the administered amount of HPCD was substantially lower than these safety thresholds, suggesting minimal risk of toxicity [26]. The increased bioavailability of gemifloxacin is attributed to its improved solubility and enhanced intestinal absorption induced by complexation with HPCD. The plasma concentration of gemifloxacin was the highest at the first sampling time point (1.199 $\pm$ 0.24 µg/mL) and decreased by approximately 48.04% by the third sampling time point (0.623 $\pm$ 0.09 µg/mL) in the control group. Thereafter, no change was observed in the concentration magnitude from the fourth sampling time point (0.621 $\pm$ 0.11 µg/mL), and a biphasic decline pattern was observed where the rate of decrease gradually became more gradual (Fig. 3). This pattern is consistent with that mentioned in previous reports, suggesting that the experimental design and data derived in this study are highly reliable [27]. Plasma concentrations were also the highest at the first sampling time point, at 1.175 $\pm$ 0.08 µg/mL and 0.931 $\pm$ 0.08 µg/mL, respectively, in Groups A and B. The plasma concentrations of Group A (0.905 $\pm$ 0.06 µg/mL) and Group B (0.727 $\pm$ 0.08 µg/mL) decreased by 23% and 22%, respectively, at the third sampling time point, and thereafter demonstrated a gradual decline pattern from subsequent sampling points (Fig. 3). Unlike the rapid plasma concentration decline observed in the control group, the gradual and sustained decline pattern observed in Groups A and B was consistent with that reported in previous pharmacokinetic studies, demonstrating an increased systemic exposure time to drugs in inclusion complexes using HPCD [28]. Various solubilization strategies have been explored to improve the bioavailability of poorly soluble fluoroquinolone antibiotics. However, previously reported approaches have often faced technical limitations. For example, lipid–polymer hybrid nanoparticles have exhibited formulation failure or physical instability due to uncontrolled electrostatic interactions [29]. In addition, polymeric micelle systems that require complex multistep preparation processes and the use of organic solvents have shown only limited improvements in bioavailability, with increases of approximately 1.6-fold [30]. In contrast, the HPCD inclusion strategy employed in the present study significantly improved the Cmax/dose by up to 2.77-fold compared with the control group using a simple preparation process without organic solvents. These results suggest that HPCD complexation represents a highly effective, practical, and promising strategy for improving the oral delivery of gemifloxacin. The improvement of gemifloxacin bioavailability observed in this study is expected to reduce the required amount of API, ultimately enhancing dosing safety and significantly improving patient compliance through smaller tablet sizes [31, 32]. In addition, the reduced API usage may contribute to lowering the drug cost.

These results suggest that HPCD complexation can improve the absorption rate of gemifloxacin, demonstrating its potential to effectively improve the oral delivery of gemifloxacin.

This study has several strengths. First, a systematic and comprehensive screening of ten structurally diverse solubilizing agents including non-ionic surfactants, an anionic surfactant, amino acids, and cyclodextrins was performed under identical experimental conditions, enabling a direct and objective comparison of solubilization capacity for gemifloxacin. Second, the selected HPCD-based formulation was evaluated through both in vitro solubility studies and in vivo pharmacokinetic studies in rats, providing an integrated assessment that bridges bench-level observations with physiologically relevant outcomes. Third, the bioanalytical HPLC method was rigorously validated in accordance with ICH M10 guidelines, demonstrating excellent linearity (R² = 0.9998), precision (CV $\le$5.71%), and accuracy (within $\pm$ 1.28%), which supports the reliability of the pharmacokinetic data reported herein. However, several limitations should be acknowledged. First, the in vivo pharmacokinetic study was conducted using a small sample size (n = 5 per group), which may limit the statistical power and generalizability of the pharmacokinetic findings; future studies with larger cohorts are warranted to confirm these results. Second, the study was restricted to male Sprague-Dawley rats, and it remains unknown whether the observed solubility and bioavailability improvements would translate to the same extent in female animals or in human subjects; further preclinical and clinical studies are needed to evaluate species- and sex-dependent differences. Third, long-term stability testing of the HPCD formulation under various storage conditions was not conducted, and its feasibility for scale-up manufacturing into a solid dosage form (e.g., tablet) remains to be demonstrated. Another limitation of this study is the lack of direct physicochemical characterization of the gemifloxacin–HPCD inclusion complex. Techniques such as DSC, FTIR, NMR, and determination of stability (binding) constants were not performed, which limits the ability to conclusively confirm inclusion complex formation and quantify the strength of interaction. Therefore, the proposed mechanism of solubility enhancement is based on indirect evidence, including phase solubility analysis and pharmacokinetic outcomes. Future studies are warranted to perform detailed structural and thermodynamic characterization of the complex.

This study successfully demonstrated that HPCD effectively enhanced the solubility and bioavailability of gemifloxacin through a systematic evaluation combining in vitro solubility screening and in vivo pharmacokinetic studies. Among the different solubilizing agents evaluated, including non-ionic surfactants (poloxamers, PEG 4000, Soluplus®, Polyox N80), anionic surfactants (SLS), amino acids (L-arginine, L-lysine), and cyclodextrins ($\beta$-cyclodextrin, HPCD), HPCD exhibited superior solubility enhancement, achieving a maximum solubility of 69.37 $\pm$ 0.71 mg/mL at a high concentration (4.1 g), representing a 1.52-fold increase over the control. The optimized HPCD concentration of 0.1 g achieved 66.27 $\pm$ 0.42 mg/mL solubility, demonstrating its practical applicability for tablet formulations. The validated HPLC method provided reliable quantification with excellent linearity (R² = 0.9998), precision (CV $\le$5.71%), and accuracy (within $\pm$1.28%), meeting the ICH M10 guidelines. In vivo pharmacokinetic studies revealed that HPCD formulations significantly improved bioavailability, with AUC_0-∞ increasing by 1.53-fold (Group A) and 1.13-fold (Group B) compared to gemifloxacin alone. The dose-normalized Cmax/dose values increased by 1.75-fold and 2.77-fold, respectively, indicating enhanced absorption efficiency. The sustained plasma concentration profiles observed for the HPCD formulations, characterized by a gradual rather than rapid decline, suggest prolonged systemic exposure and improved pharmacokinetic profiles. These results established HPCD-based solubilization as a promising and practical strategy for the development of improved oral gemifloxacin formulations, potentially leading to enhanced therapeutic outcomes, improved patient compliance, and reduced dosing requirements for clinical applications. Future investigations are warranted to clinically validate, optimize manufacturing processes for commercial development, and explore the underlying molecular mechanisms of drug-CD interactions.

The datasets used and analyzed during the current study are available from corresponding author on reasonable request.

JWK, GEP, JSK, YHK and KMK conceptualized and designed the study and performed the experiments. YHK, KMK and JSK supervised the study and performed data acquisition, analysis, and interpretation. GEP and JWK wrote the manuscript and prepared the figures and tables. YHK and KMK performed the reference search and confirmed the authenticity of all data. All authors contributed to editorial revisions of the manuscript. All authors read and approved the final manuscript and have participated sufficiently in the work and agreed to be accountable for all aspects of the work.

All animal experiments were conducted in accordance with the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. The study was approved by the Institutional Animal Care and Use Committee of Kyungsung University (approval number: study-2025-001A).

Not applicable.

This research was supported by a grant of the Korea Health Technology R&D Project through the Korea Health Industry Development Institute (KHIDI), funded by the Ministry of Health & Welfare, Republic of Korea [grant number: RS-2020-KH088726 (HR20C0026)].

The authors declare no conflicts of interest. We confirm that YHK’s affiliation with Kolon Pharm did not influence data interpretation, the writing of the manuscript, or the scientific judgments made in this study.