In Vitro and In Vivo Activities, Absorption, Tissue Distribution, and Excretion of OBP-4, a Potential Anti-Clostridioides difficile Agent

ABSTRACT

Clostridioides difficile infection (CDI) is considered a major concern of the health care system globally, with an increasing need for alternative therapies. OBP-4, a new oxazolidinone-fluoroquinolone hybrid with excellent in vitro activities and good safety, shows promising features as an antibacterial agent. Here, we further evaluated the in vitro and in vivo activities of OBP-4 against C. difficile and its absorption (A), distribution (D), and excretion (E) profiles in rats. In vitro assays indicated that OBP-4 was active against all tested C. difficile strains, with MICs ranging from 0.25 to 1 mg/liter. In addition, OBP-4 showed complete inhibition of spore formation at 0.5× MIC. In the mouse model of CDI, 5-day oral treatment with OBP-4 provided complete protection from death and CDI recurrence in infected mice. However, cadazolid (CZD) and vancomycin (VAN) showed less protection of infected mice than did OBP-4 in terms of diarrhea and weight loss, especially VAN. Subsequently, ADE investigations of OBP-4 with a reliable liquid chromatography-tandem mass spectrometry (LC-MS/MS) method showed extremely low systemic exposure and predominantly fecal excretion, resulting in a high local concentration of OBP-4 in the intestinal tract—the site of CDI. These results demonstrated that OBP-4 possesses good activity against C. difficile and favorable ADE characteristics for oral treatment of CDI, which support further development of OBP-4 as a potential anti-CDI agent.

TEXT

Clostridioides difficile, a Gram-positive, anaerobic, spore-forming bacterial pathogen, is a component of the normal gut microbiota in healthy individuals (1). However, it is known to cause a spectrum of intestinal diseases that range from severe diarrhea to life-threatening colitis and toxic megacolon in infected patients (2). It is estimated that C. difficile infection (CDI) causes approximately 223,900 cases and 12,800 deaths annually in the United States alone, with an annual financial burden of more than 1 billion dollars (3). Worse still, the mortality and economic burden caused by CDI have increased dramatically in recent years, partly due to the emergence and spread of hypervirulent C. difficile strains (4–6). Another major challenge for treating CDI is the high recurrence rate, making CDI a major public health problem worldwide (7).

Although antibiotics including metronidazole and vancomycin (VAN) remain the mainstay of therapy for CDI (8), their poor efficacy, high rate of recurrences (9), and resistance problems (10) frequently lead to treatment failures. Oral fidaxomicin is also a treatment option for CDI, but there is still a high recurrence rate (24%) for BI/NAP1/027 hypervirulent strains (11). Many new antibacterial molecules to fight CDI are now being studied, but most of them have been shown to have some undesired characteristics (12). Therefore, the lack of available therapeutic options for CDI emphasizes the urgent need for exploring new and effective anti-CDI agents.

Cadazolid (CZD), an oxazolidinone-fluoroquinolone hybrid, was sponsored by Actelion Pharmaceuticals and is in development for the treatment of CDI (13). It has been shown that protein synthesis inhibition is the main antibacterial action exerted by CZD (14), and thus, its antibacterial spectrum is largely limited to Gram-positive bacteria (15). In addition, CZD has extremely poor aqueous solubility, which prevents its gastrointestinal absorption, leading to high drug concentrations at the site of action (16). Therefore, CZD presents many promising key features for CDI treatment, such as potent efficacy, favorable pharmacokinetic profile, low risk of resistance development, and lower impact on the gut flora (12, 13). However, the rate of CDI recurrence with CZD treatment is still high (18.2% to 25%), while it was significantly lower than that seen with VAN (50%) in a clinical trial (17). Recently, we reported a series of new oxazolidinone-fluoroquinolone hybrids and tested their antibacterial activities against antibiotic-susceptible and antibiotic-resistant Gram-positive bacteria. Among them, OBP-4 (Fig. 1) displays potent in vitro antibacterial activities against these Gram-positive bacteria, with MICs ranging from 0.016 to 0.5 µg/ml, which are generally lower than those of CZD (18). It was also found that OBP-4 displayed potent protein synthesis inhibition compared with DNA synthesis inhibition in the in vitro assays. Furthermore, the acute oral 50% lethal dose (LD₅₀) of OBP-4 in mice was greater than 2,000 mg/kg of body weight, indicating a good safety margin. Collectively, OBP-4 has shown some promising results for the treatment of bacterial infections. Here, we further investigated the in vitro and in vivo activities of OBP-4 against C. difficile. Subsequently, absorption, distribution, and excretion (ADE) studies of OBP-4 were performed in rats to provide better insights into its activity and safety.

FIG 1.

Chemical structure of OBP-4: 9-fluoro-8-(4-((2-fluoro-4-((R)-5-(hydroxymethyl)-2-oxooxazolidin-3-yl)phenoxy)methyl)-4-hydroxypiperidin-1-yl)-5-methyl-1-oxo-6,7-dihydro-1,5-dihydro-benzo(3,2,1-ij)quinoline-2-carboxylic acid.

RESULTS

In vitro antibacterial activity against C. difficile.

The in vitro antibacterial activities of OBP-4 and comparators against a group of C. difficile strains with different toxin types (Table 1) were tested, and the results are presented in Table 2. OBP-4 showed excellent activities against all C. difficile strains tested, with a MIC range of 0.25 to 1 mg/liter (Table 2). Moreover, OBP-4 inhibited spore formation markedly at sub-growth-inhibitory concentrations, and no new spore formation was observed in the presence of OBP-4 at 0.5× MIC (Table 3). In contrast, CZD showed activities similar to those of OBP-4 against C. difficile strains and C. difficile spores (ATCC BAA-1803) (Tables 2 and 3). However, VAN showed significantly less activity than OBP-4 and CZD against C. difficile strains and had no inhibitory effect on spore formation of C. difficile at concentrations of 0.25× and 0.5× MIC (Tables 2 and 3). Meanwhile, OBP-4 was also active against C. perfringens at a concentration of 0.5 mg/liter, but activity against B. fragilis was not detectable even at the highest concentration tested (16 mg/liter) (Table 2).

TABLE 1.

Information for strains in the in vitro susceptibility test

Species	Strain	Ribotype or sequence type	Toxinotypea
C. difficile	SH 0201-018	017	A− B+, CDTb−
	TAFY 36	ST3	A+ B+, CDTb−
	ZR22	ST35	A+ B+, CDTb−
	ATCC 700057	038	A− B−, CDTb−
	XA 0204-005	ST54	A+ B+, CDTb−
	GZ15	ST119	A− B+, CDTb−
	HZ 0206-001	ST1	A+ B+, CDTb−
	ATCC 9689	001	A+ B+, CDTb−
	ATCC BAA-1803	027	A+ B+, CDTb+
	ATCC 43598	017	A− B+, CDTb−
	ATCC 43255	087	A+ B+, CDTb−
C. perfringens	ATCC 13124
B. fragilis	ATCC 25285

^aCDTb, C. difficile binary toxin b.

TABLE 2.

In vitro antibacterial activities of OBP-4 against C. difficile

Organism	MIC (mg/liter)
	OBP-4	CZD	VAN
C. difficile SH 0201-018	0.5	0.25	1
C. difficile TAFY 36	0.25	0.125	0.5
C. difficile ZR22	0.5	0.25	1
C. difficile ATCC 700057	0.5	0.25	1
C. difficile XA 0204-005	0.5	0.125	1
C. difficile GZ15	0.25	0.125	1
C. difficile HZ 0206-001	1.0	0.25	4
C. difficile ATCC 9689	0.5	0.25	1
C. difficile ATCC BAA-1803	0.25	0.25	2
C. difficile ATCC 43598	0.5	0.25	1
C. difficile ATCC 43255	0.5	0.25	1
C. perfringens ATCC 13124	0.5	0.125	1
B. fragilis ATCC 25285	>16	>16	>16

TABLE 3.

Effect of OBP-4 on spore formation in C. difficile (ATCC BAA-1803)

Dose	Sporulation rate (96 h)a
	OBP-4	CZD	VAN
0.25× MIC	3.9 × 10−6	3.6 × 10−6	2.2 × 10−4
0.5× MIC	0	0	2.0 × 10−4

^aThe value for the untreated control was 2.6 × 10⁻⁴.

Activity in the mouse model of CDI.

The mouse model was used to evaluate the in vivo activity of OBP-4 and the comparators with 5-day oral treatment against lethal challenge with the hypertoxigenic C. difficile strain VPI 10463. Uninfected mice with 100% survival (Fig. 2) showed no diarrhea (Fig. 3), weight loss (Fig. 4), or other abnormal signs, which were incorporated into the clinical scores (Fig. 5). For the infection control group, the death of mice began to occur at day 2 after infection and peaked at 75% mortality at day 4 after infection (Fig. 2). At the end of observation, surviving mice in the infection control group returned to normal activity levels (Fig. 3 to 5). Animals treated with OBP-4 at a 10-mg/kg dose fared well, with 100% survival (Fig. 2) during the 22-day monitoring period. We also note that the OBP-4-treated mice showed little weight loss and other abnormal signs (Fig. 3 to 5) during the posttreatment phase (day 5 to 22). Although CZD at a dose of 10 mg/kg also showed a good protective effect in the infected mice, with 100% survival (Fig. 2), mice started exhibiting diarrhea (Fig. 3) and weight loss (Fig. 4) after the stoppage of treatment. Compared with OBP-4-treated mice, the survival rate of mice treated with VAN at a dose of 20 mg/kg was 87.5% (Fig. 2), and the surviving mice fared poorly, with significant diarrhea (Fig. 3) and weight loss (Fig. 4) 8 to 14 days after infection (Table 4). Furthermore, clinical scores of animals treated with VAN and CZD were increased significantly after withdrawal of administration compared with OBP-4 (Fig. 5; Table 4). Therefore, the activity of OBP-4 was superior to that of the comparators VAN and CZD in terms of diarrhea, body weight change, and clinical scores in the mouse model of CDI (Table 4).

FIG 2.

Survival curves of mice with OBP-4 and comparator treatment in a mouse model of CDI. Mice were pretreated with cefoperazone and clindamycin and challenged with C. difficile (VPI 10463) on day 0. Oral treatment began on day 0 with either blank vehicle, vancomycin (VAN), cadazolid (CZD), or OBP-4, administered once daily for 5 consecutive days. *, P < 0.05, and **, P < 0.01, versus infection control.

FIG 3.

Mean diarrhea scores of surviving mice in each group during the 22-day monitoring period. Diarrhea scores of each mouse in the mouse CDI model were evaluated as indicated in Table S1 in the supplemental material.

FIG 4.

Mean body weight change (%) of surviving mice in each group during the 22-day monitoring period. Body weight change of each mouse in the mouse CDI model was calculated based on the body weight on day 0, and euthanasia was performed when weight loss exceeded 20%.

FIG 5.

Mean clinical scores of surviving mice in each group during the 22-day monitoring period. Clinical scores of each mouse in the mouse CDI model were evaluated as indicated in Table S1 in the supplemental material.

TABLE 4.

Statistical analysis of diarrhea scores, body weight change, and clinical scores^a

Comparison	Significance of difference at time interval (days) for:
	Diarrhea score		BW change (%)		Clinical score
	8–14	0–22	8–14	0–22	8–14	0–22
HC vs IC	**	**	**	**	**	**
VAN vs IC	NS	*	NS	**	NS	*
CZD vs IC	*	**	**	**	**	**
OBP-4 vs IC	**	**	**	**	**	**
OBP-4 vs VAN	##	##	##	##	##	##
OBP-4 vs CZD	##	##	##	#	#	##

^a** and ##, P < 0.01; * and #, P < 0.05; NS, not significant (P > 0.05). Abbreviations: HC, healthy control; IC, infection control; VAN, vancomycin; CZD, cadazolid; BW, body weight.

Pharmacokinetics.

Mean plasma concentration-time profiles of OBP-4 and the major pharmacokinetic parameters (mean ± SD) for oral (5, 10, and 20 mg/kg) and intravenous (1 mg/kg) administration are displayed in Fig. 6 and Table 5, respectively.

FIG 6.

Mean concentration-time profiles of OBP-4 in rat plasma after oral (5, 10, and 20 mg/kg) and intravenous (1 mg/kg) administrations. Data are means ± SD (n = 6).

TABLE 5.

Mean pharmacokinetic parameters of OBP-4 in rats after oral and intravenous administration^a

Dose (mg/kg)	AUC0–t (ng·h/ml)	AUC0–∞ (ng·h/ml)	Cmax (ng/ml)	Tmax (h)	t1/2 (h)	CL (liters/h/kg)	F (%)
Oral
5	26.45 ± 11.02	29.23 ± 11.22	3.74 ± 0.70	1.75 ± 0.19	3.81 ± 1.74	198.52 ± 87.73	0.81
10	28.11 ± 8.75	31.36 ± 9.57	5.07 ± 1.56	1.49 ± 0.32	3.05 ± 1.06	318.91 ± 128.15	0.44
20	33.92 ± 8.88	36.45 ± 9.26	6.42 ± 0.94	1.49 ± 0.11	2.41 ± 0.99	609.29 ± 167.34	0.25
Intravenous
1	616.73 ± 234.57	719.71 ± 257.94	2,216.09 ± 773.37		0.23 ± 0.06	1.53 ± 0.49

^aData are means ± SD (n = 6).

Plasma concentrations of OBP-4 peaked rapidly, with time to reach peak concentration (T_max) ranging from 1.49 h to 1.75 h after oral administrations, while plasma peak concentration (C_max) was not more than 6.42 ng/ml at three dosages (Table 5). Subsequently, the plasma concentrations of OBP-4 decreased, with elimination half-life (t_1/2) values of 2.41 to 3.81 h. The C_max and the area under the concentration-time curve from time zero to the time of the last quantifiable concentration (AUC_0–t) and from time zero to infinity (AUC_0–∞) obtained from three oral administration groups showed a lack of linearity, along with the augmentation of total body clearance (CL) with increased dosage (Table 5). Moreover, the mean plasma concentrations of OBP-4 dropped below the lower limit of quantitation (LLOQ) at 12 h after oral administration (Fig. 6). For intravenous administration, C_max was found to be 2,216.09 ± 773.37 ng/ml, which was much higher than that of oral administration; then, OBP-4 was eliminated rapidly, with a t_1/2 of 0.23 h (Table 5). The calculated oral bioavailability (F) of OBP-4 at the three dosages was 0.81%, 0.44%, and 0.25%, respectively.

Tissue distribution.

Concentration-time profiles of OBP-4 in various tissues at 0.5, 1, 2, 4, and 8 h following a single oral administration at 10 mg/kg are presented in Fig. 7. Results demonstrated that the maximum tissue concentration of OBP-4 following oral administration was detected in the intestinal tract (4,428.1 ± 756.1 ng/g), a site of drug absorption for oral administration. Next is the stomach, with a concentration of 1,337.6 ± 182.5 ng/g (Fig. 7). When OBP-4 was absorbed into systemic circulation, the highest concentration of OBP-4 was observed in liver (29.2 ± 5.7 ng/g), followed by lung (15.2 ± 4.9 ng/g), kidney (11.0 ± 1.7 ng/g), and heart (8.5 ± 1.7 ng/g) (Fig. 7), which were significantly lower than those in the stomach and small intestine. The concentrations of OBP-4 in rat tissues first increased and then decreased with a peak time of 2 h, but they continually decreased in the stomach (Fig. 7). Additionally, OBP-4 was not detected in the spleen and brain (data not shown).

FIG 7.

Mean concentration-time profiles of OBP-4 in various rat tissues with a single oral dose of 10 mg/kg. Data are means ± SD (n = 6).

Excretion.

Cumulative excretions of OBP-4 in rat urine and feces after a single oral administration at 10 mg/kg are shown in Fig. 8. Following complete urine and feces collection, the fecal cumulative excretion of OBP-4 was up to 92.8% of the administered dose within 144 h, but the urinary cumulative excretion of OBP-4 was only 0.015% of the administered dose within 144 h (Fig. 8). Moreover, the excretion of OBP-4 was concentrated in the first 24 h and then rapidly decreased until the end of the study (Fig. 8). In addition, approximately 0.7% of OBP-4 was detected in cage wash samples collected after administration. In total, the amount of OBP-4 excreted in the urine and feces was 93.5% of the administered dose.

FIG 8.

Urinary and fecal cumulative excretion profile of OBP-4 in rats following a single oral administration at 10 mg/kg. Data are means ± SD (n = 6).

DISCUSSION

Currently, the recommended first-line therapeutic agents for CDI, including metronidazole and VAN, are suboptimal, with high rates of treatment failure and recurrence (19). In recent years, a new class of antibiotics termed quinoxolidinones, incorporating oxazolidinone and fluoroquinolone pharmacophores, has been found to have potent activity against C. difficile, such as CZD and MCB-3681 (13, 20). CZD is the first member of the quinoxolidinone antibiotics, and its in vitro and in vivo activity against C. difficile has been well documented (21–23). In this study, we evaluated the in vitro activity of OBP-4, a new quinoxolidinone antibiotic, against C. difficile strains and its effect on spore formation at 0.25× and 0.5× MIC. The data demonstrated that OBP-4 is as potent as CZD and more potent than VAN. Consistent with previous studies, we also found that VAN did not inhibit the spore formation of C. difficile at sub-growth-inhibitory concentrations (21), which may be associated with CDI recurrence during VAN treatment. Furthermore, OBP-4 exhibited enhanced potency against other Gram-positive bacteria compared with CZD and linezolid (LZD), while the activity against Gram-negative bacteria is still weak (18). However, it is believed that the narrow antibacterial spectrum has a limited effect on the normal bowel flora, which plays a central role in fighting the development of CDI (24). In fact, CZD with a narrow antibacterial spectrum against Gram-positive bacteria has been shown to have a very limited impact on the normal gut microflora in an in vitro human gut model (22), even though CZD showed improved potency against Gram-positive bacteria compared to LZD. Although OBP-4, as an analogue of CZD, may have a comparable impact on the normal gut microflora, further investigations are necessary to clarify the impact of OBP-4 on the gut microbiota.

In the in vivo phase, a mouse model of CDI was used to assess the activity of OBP-4 as a potential therapeutic for CDI. In the mouse model, OBP-4 outperformed CZD and VAN in providing complete protection from CDI recurrence in infected mice during the whole monitoring period. Despite the fact that VAN was effective in controlling CDI in the treatment phase, the surviving animals treated with VAN suffered severe signs of recurrent infection after withdrawal of treatment. Similar to our findings, relapse of CDI involving diarrhea, weight loss, and death following treatment with VAN has been widely reported (9, 25, 26). Furthermore, clinical recurrence associated with VAN treatment has been attributed to antibiotic-induced disorder of the gut microbiota (27). Although the mice treated with CZD did not die during the treatment stage, they succumbed to diarrhea and weight loss thereafter. Consistent with our findings, patients receiving CZD in a phase 2 study also experienced clinical recurrence, while the recurrence rate was significantly lower than that of VAN (17). Previous studies have demonstrated that CDI recurrence may result from the incomplete eradication of existing C. difficile (28), reinfection with C. difficile from the environment (29), or failure to reestablish the normal gut microflora (30, 31). In order to minimize the opportunity for C. difficile dissemination, the sterile cages and daily diet of all mice were changed over time in our study. Therefore, the reasons for the CDI recurrence in animals receiving VAN or CZD are not clear from these experiments. Meanwhile, an in-depth evaluation on the risk of CDI recurrence with OBP-4 treatment should be considered in subsequent studies.

Given the excellent activity of OBP-4 against C. difficile, the ADE profiles of OBP-4 were further investigated with a reliable liquid chromatography-tandem mass spectrometry (LC-MS/MS) method to obtain a more comprehensive understanding of the dynamic process of OBP-4 in vivo.

After oral administration of OBP-4 to rats, C_max and AUC were found to be generally low. However, C_max and AUC for intravenous administration (1 mg/kg) were significantly greater than the corresponding values determined after oral administration, indicating an extremely low oral bioavailability. Considering the poor aqueous solubility of OBP-4, we speculated that the poor intestinal absorption played a vital role in the low bioavailability of OBP-4 (32, 33). A similar pharmacokinetic profile was also reported previously in clinical trials of CZD (16, 34), which is probably due to their similar structures. Meanwhile, the impact of different doses within the range of 5 to 20 mg/kg on the pharmacokinetic behavior of OBP-4 was also investigated. The C_max and AUC of OBP-4 increased slightly in a dose-disproportional manner, resulting in a nonlinear pharmacokinetic characteristic across the tested dosage range.

It is well recognized that drug response is closely related to its target concentration (35). In the present study, the concentrations of OBP-4 in the rat gastrointestinal tract are much higher than MICs of OBP-4 against C. difficile, which provides an appealing advantage for the treatment of CDI. Nevertheless, only a minor amount of OBP-4 was found in the internal tissues, such as heart, liver, lung, and kidney, in line with that detected in plasma. A low-level distribution of OBP-4 in internal tissues indicated that it has no tendency to accumulate in tissue compartments. In addition, OBP-4 was not detected in brain, suggesting that it could not transport through the blood-brain barrier (BBB). The physiochemical properties, such as high molecular mass (36, 37), should be the foremost relevant factors in preventing OBP-4 from crossing the BBB. These findings demonstrated that OBP-4 has an extremely low systemic exposure, which would provide helpful information for toxicity studies of OBP-4.

Consistent with excretion profile of CZD in healthy subjects (16), the majority of OBP-4 was found to be excreted in rat feces. The predominant fecal excretion of OBP-4, along with the low systemic bioavailability, supports the speculation that OBP-4 has poor intestinal absorption, resulting in high intestinal exposure. Meanwhile, the low systemic bioavailability also explains the extremely low urinary excretion of OBP-4. Overall, the elimination of OBP-4 was relatively complete, with a total recovery of 93.5%. Furthermore, the high exposure of OBP-4 prototype drug in feces indicated that the major elimination route of OBP-4 is fecal excretion rather than metabolism.

Collectively, the ADE characteristics of OBP-4 presented here will be helpful for gaining a better understanding of its in vivo activity.

Conclusion.

In summary, we evaluated the activity of OBP-4, a new oxazolidinone-fluoroquinolone hybrid, against C. difficile and its ADE profiles in rats for the first time. OBP-4 exhibited desirable features for a potential anti-CDI agent, including good potency, reduction of the risk of recurrence, and favorable ADE characteristics. Extensive susceptibility tests of OBP-4 against various C. difficile strains together with determination of the impact on resident flora are required to support further development of OBP-4 as a promising therapeutic option for CDI.

MATERIALS AND METHODS

Chemicals and reagents.

OBP-4 and CZD with the purity ≥98% were provided by Orbiepharm Co., Ltd. (Beijing, China). Cefoperazone, clindamycin, VAN, and linezolid (LZD) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Acetonitrile, methanol, formic acid, and ammonium formate (high-performance liquid chromatography [HPLC] grade) were obtained from Thermo Fisher Scientific Inc. (Waltham, MA, USA). A Milli-Q system to produce ultrapure water (Millipore, MA, USA) was used.

Bacterial strains.

A panel of well-characterized C. difficile strains (Table 1), including five standard strains from ATCC and six clinical isolates from hospitals, was collected by Orbiepharm Co., Ltd. (Beijing, China), and used for the in vitro activity evaluation of OBP-4. In addition, a strain of Clostridium perfringens and a strain of Bacteroides fragilis were also tested to evaluate the activity of OBP-4. C. difficile VPI 10463 (ATCC 43255) was used to establish the mouse model of CDI and investigate the in vivo activity of OBP-4.

Animals.

Specific-pathogen-free (SPF) female C57BL/6 mice (6 to 8 weeks) were obtained from the experimental center of Shanghai Lingchang Biotechnology Co., Ltd. (animal license number: SCXK2018-003, Shanghai, China). Sprague-Dawley male rats (6 to 8 weeks) were obtained from the laboratory animal center of Lanzhou Veterinary Research Institute (animal license number SCXK2015-001; Lanzhou, China). During the 7-day acclimatization period, all animals were reared under standard conditions (temperature of 22 ± 2°C, relative humidity of 60% ± 10% and a 12 h light/dark cycle) with an unlimited supply of food and water. For ADE studies, all rats fasted overnight prior to experiments. Finally, the surviving and moribund animals were humanely killed by CO₂ inhalation. All animal experiments followed the guidelines for the care and use of laboratory animals issued by the U.S. National Institutes of Health (38) and were approved by the Animal Ethics Committee of Lanzhou Institute of Husbandry and Pharmaceutical Sciences.

In vitro antibacterial activity.

The susceptibility testing (MICs) of OBP-4 against C. difficile strains was conducted by the agar dilution method as outlined by the Clinical and Laboratory Standards Institute (CLSI) (39). Antibacterial stock solutions at a concentration of 3.84 mg/ml were prepared in dimethyl sulfoxide (DMSO) (OBP-4 and CZD) or double-distilled water (ddH₂O) (VAN) and then serially diluted 2-fold to provide the required concentrations. The tested microorganisms were cultured in brucella agar plates supplemented with 5% sheep blood, 1‰ hemin, and 1‰ vitamin K. The active cultures (final concentration of 3 × 10⁵ CFU/ml) were inoculated onto antibacterial-containing or control agar plates. The plates were incubated at 37°C under anaerobic conditions and observed after 48 h of incubation. The MIC was defined as the lowest concentration of the antibacterial at which visible growth was completely inhibited. The effects of OBP-4, CZD, and VAN on spore formation with C. difficile strain ATCC BAA-1803 were investigated following the steps described by Ochsner et al. (40). Briefly, antibacterials at 0.25× and 0.5× MIC or control solvent were added to growing cells at late exponential growth phase. Up to 96 h, the total viable cells and ethanol-resistant spores were enumerated to calculate the sporulation rate. Each determination was repeated in triplicate.

Mouse model of CDI.

C57BL/6 mice were randomly assigned into five groups (n = 8 per group, 4 animals per cage): the healthy control group, infection control group, VAN group, CZD group, and OBP-4 group. The mouse CDI model was established according to the protocol published previously (41). Briefly, all mice were first pretreated with cefoperazone (0.5 mg/ml) in drinking water for a total of 10 days (day −13 to day −4). Subsequently, mice were allowed access to regular feeding (from day −3) and administered a single intraperitoneal injection of clindamycin at 10 mg/kg before the C. difficile infection (day −1). After an interval of 24 h, all mice except those in the healthy control group were challenged with 5 × 10⁶ CFU of C. difficile VPI 10463 spores by oral gavage (day 0). The treatment was initiated 4 h postchallenge. Based on previous studies, the treatment doses of VAN and CZD were set at 20 mg/kg (25, 42) and 10 mg/kg (21), respectively. As OBP-4 is an analogue of CZD, the dose of OBP-4 was also set at 10 mg/kg for comparative analysis. All test compounds, including OBP-4, CZD, and VAN were orally administered once daily for 5 consecutive days (day 0 to day 4). Animals in the healthy control group and infection control group were given the equal volume of blank vehicle (0.5% methylcellulose with 0.05% Tween 80). Clinical scores of each mouse based on weight loss, activity levels, posture, eyes/nose, skin/fur, and diarrhea, as well as deaths, were recorded daily (see Table S1 in the supplemental material for the scoring system) (42) from day 0 to day 22 postinfection. Surviving and moribund mice were humanely killed by CO₂ inhalation.

In vivo ADE studies. (i) Sample collection.

To determine the concentration of OBP-4, plasma, tissue, urine, and feces samples were collected and frozen at −80°C prior to assays. In the pharmacokinetic study, rats were randomly divided into four groups (n = 6 per group). One group (1 mg/kg) was intravenously injected with OBP-4 dissolved in a mixed solvent (N,N-dimethylacetamide, polyethylene glycol 400 [PEG 400], physiological saline; 1:9:10 [vol/vol/vol]), and the other three groups were orally administered OBP-4 (5, 10, and 20 mg/kg) suspended in 0.5% (wt/vol) sodium carboxymethyl cellulose (CMC-Na). Blood samples were collected predose, at 0.25, 0.5, 1, 2, 4, 6, 8, 12, and 24 h after oral administration, and at 0.083, 0.25, 0.5, 1, 2, 4, 6, 8, and 12 h after an intravenous injection. To separate plasma, blood samples were centrifuged at 3,000 rpm for 10 min.

Rats were anesthetized with 2% sodium pentobarbital intraperitoneally, and tissue samples, including heart, liver, spleen, lung, kidney, stomach, small intestine, and brain, were promptly collected predose and at 0.5, 1, 2, 4, and 8 h after a single oral dose of 10 mg/kg OBP-4 (n = 6 per time point). After residual blood or gastrointestinal contents were washed off, the weights of tissue samples were recorded.

Rats (n = 6) were housed in individual stainless-steel metabolic cages and received a single oral dose of 10 mg/kg OBP-4. Urine and feces samples were collected before dosing and at intervals of 0 to 6, 6 to 12, 12 to 24, 24 to 36, 36 to 48, 48 to 72, 72 to 96, 96 to 120, and 120 to 144 h after dosing. The volumes of urine samples and weights of dried feces samples in each time interval were recorded.

(ii) Sample preparation.

For plasma samples, 10 µl of IS (internal standard, LZD) solution was added to 100 µl plasma sample and mixed in a 2-ml centrifuge tube. To precipitate proteins, 300 µl acetonitrile was added to the mixture and then manually vortexed for 2 min. The clear supernatant was transferred into another new centrifuge tube after centrifugation (12,000 rpm, 10 min) and evaporated to dryness with a vacuum concentration system (RapidVap Vertex evaporator, Labconco, USA) at 45°C. The residue was redissolved with 200 µl acetonitrile and further filtered with a 0.22-µm filter.

Tissue samples were homogenized with normal saline (1:3 [wt/vol]) and then processed like plasma samples. The processing steps for urine samples were identical to those of plasma except that extract solvent was replaced by ethyl acetate-chloroform (3:7 [vol/vol], 600 µl). Fecal samples were homogenized in normal saline (1:10 [wt/vol]) and subsequently treated by the same method as plasma with acetonitrile-ethyl acetate (3:7 [vol/vol], 1 ml) as extract solvent.

Finally, an aliquot of filtrate (5 µl for plasma and tissues, 10 µl for urine and feces) was injected into the LC-MS/MS system.

(iii) Sample assay.

All samples were analyzed with an Agilent 1200 LC system coupled to an Agilent G6410A triple-quadruple tandem mass spectrometer (Agilent Technologies, USA) in the positive ionization mode. Acetonitrile spiked with 0.1% formic acid (B) and water spiked with 0.1% formic acid and 10 mM ammonium formate (A) were used as the mobile phase. Analyte separation was performed on a Poroshell EC-C₁₈ column (3.0 by 100 mm, 2.7 µm; Agilent Technologies, USA) with the following gradient program: 0 to 1 min, 90% A; 1 to 2 min, 90 to 50% A; 2 to 5 min, 50 to 5% A; 5 to 6 min, 5 to 90% A. Quantification was carried out by multiple-reaction monitoring (MRM) mode at m/z 600.3→582.3 (OBP-4) and m/z 338.2→296.2 (IS). MassHunter workstation software (version B 01.03) was used for collection and analysis of data.

The method was validated in accordance with FDA guidance for bioanalytical method validation (43). No significant endogenous interference was determined in rat biological samples (see Fig. S1 in the supplemental material). The lower limit of quantification (LLOQ) of OBP-4 in rat plasma, various tissues, and urine was 1 ng/ml, and that in feces was 2 ng/ml. The method has good linearity across the concentration range of 1 or 2 to 1,000 ng/ml (R² ≥ 0.99). For all quality control (QC) samples, the intra- and interday precisions (relative standard deviation, %) were both less than 9.0%, and the intra- and interday accuracies (relative error, %) were within −9.5% to 9.0% (see Table S2 in the supplemental material). Furthermore, OBP-4 was stable under different storage conditions (see Table S3 in the supplemental material).

Data analysis.

Data obtained from the mouse model of CDI were analyzed using the statistical software SPSS 19.0 (SPSS Inc., Chicago, IL, USA). For statistical analyses of body weight change, diarrhea scores, and clinical scores, the endpoint values from dead or euthanized mice were included at each time point after death but not shown in the graphs. Differences between three or more groups across time periods were analyzed by one-way analysis of variance (ANOVA), and differences between two groups were analyzed using Student's t test. Survival rates were analyzed using the Cox regression and log rank tests by the GraphPad Prism 8.3.0 software (San Diego, CA, USA). A P value below 0.05 was considered statistically significant.

Pharmacokinetic parameters, including plasma peak concentration (C_max), time to reach peak concentration (T_max), area under the plasma concentration-time curve from time zero to infinity (AUC_0–∞) and from time zero to the time of the last quantifiable concentration (AUC_0–t), elimination half-life (t_1/2), and total body clearance (CL) were obtained from WinNonlin professional software (version 5.2; Pharsight Co., Mountain View, CA, USA) based on a one-compartment model approach. AUC_0–∞ values were used to calculate the oral bioavailability (F) of OBP-4 with the following equation: F (%) = (AUC_oral × dose_i.v.)/(AUC_i.v. × dose_oral) × 100. The concentrations of OBP-4 in rat tissues were calculated according to a previous report (44) and expressed in nanograms per gram. All results are presented as means with standard deviations (SD).