Treatment and prevention of urinary tract infection with orally active FimH inhibitors

Abstract

Chronic and recurrent urinary tract infections pose a serious medical problem because there are few effective treatment options. Patients with chronic urinary tract infections are commonly treated with long-term prophylactic antibiotics that promote the development of antibiotic-resistant forms of uropathogenic Escherichia coli (UPEC), further complicating treatment. We developed small-molecular weight compounds termed mannosides that specifically inhibit the FimH type 1 pilus lectin of UPEC, which mediates bacterial colonization, invasion, and formation of recalcitrant intracellular bacterial communities in the bladder epithelium. Here, we optimized these compounds for oral bioavailability and demonstrated their fast-acting efficacy in treating chronic urinary tract infections in a preclinical murine model. These compounds also prevented infection in vivo when given prophylactically and strongly potentiated the activity of the current standard of care therapy, trimethoprim-sulfamethoxazole, against clinically resistant PBC-1 UPEC bacteria. These compounds have therapeutic efficacy after oral administration for the treatment of established urinary tract infections in vivo. Their unique mechanism of action-targeting the pilus tip adhesin FimH-circumvents the conventional requirement for drug penetration of the outer membrane, minimizing the potential for the development of resistance. The small-molecular weight compounds described herein promise to provide substantial benefit to women suffering from chronic and recurrent urinary tract infections.

Fig. 1

Inhibition, prevention, and disruption of UTI89 biofilm by mannoside. (A) Biphenyl compounds 1 to 6 as described in (38). Cellular HAI titers (EC_>90) from (38) are shown in parentheses. (B) IC₅₀ for compounds 1 to 3 and 6 effects on UTI89 biofilm formation (n = 3). The test mannoside was added at the initiation of biofilm formation. (C) IC₅₀ of 1 to 3 and 6 on UTI89 biofilm inhibition of established UTI89 biofilm. Mannoside was added 24 hours after biofilm growth was initiated, and percent biofilm was calculated 16 hours after addition of mannoside. Bars show the median value of the experiments (n = 3). (D and E) Effect of 6 on biofilm dispersal as measured by confocal microscopy of UTI89 biofilms grown for 24 hours (D), then incubated for an additional 16 hours in the presence of 0.3 µM compound 6 (E). Images along top and left are orthogonal views that show biofilm structures that protrude from the surface (arrow). Scale bars, 26 µm (D) and 28 µm (E).

Fig. 2

Compound 6 pharmacokinetic distribution and effect on chronic infection. (A) Pharmacokinetic analysis of 6 (n ≥ 3 mice) showing concentration in urine over time for each dose indicated. Horizontal dashed line is at IC₅₀ (0.74 µM) determined by the biofilm inhibition assay. IP, intraperitoneally; PO, orally. (B) Compound 6 effect on UTI. Mice chronically infected with UTI89 were treated with PBS, 6 (orally, 100 and 50 mg/kg), or TMP-SMZ (54 and 270 µg/ml, respectively). Six hours after treatment, bacteria in the bladder were counted. In the mannoside- and TMP-SMZ–treated groups, there was a significant drop in bacterial load relative to that in PBS-treated mice. Horizontal lines indicate geometric mean. *P < 0.05; **P < 0.01; ***P < 0.0001, Mann-Whitney U test.

Fig. 3

Compound 6 reduces UTI89 colonization by preventing invasion. (A) Total bacterial CFU at 6 hours after infection from mice treated with PBS or 6, either intraperitoneally (5 mg/kg) or orally (100 mg/kg) 30 min before inoculation with UTI89, revealing significantly less colonization in mannoside-treated mice. ns, not significant; LOD, limit of detection. (B) IBC quantification at 6 hours after infection from mice treated with PBS or 6, either intraperitoneally (5 mg/kg) or orally (100 mg/kg) 30 min before inoculation with UTI89, revealing significantly fewer IBCs in mannoside-treated mice. (C) Bacterial CFU at 6 hours after infection in the ex vivo gentamicin protection assay revealed that both luminal and intracellular bacteria were significantly reduced upon intraperitoneal (5 mg/kg) pretreatment of mice with 6. Horizontal lines indicate geometric mean. *P < 0.05; **P < 0.01; ***P < 0.0001, Mann-Whitney U test. (D and E) Confocal microscopy of bladders from PBS-treated (D) and 6-treated (E) mice. Bacteria were stained with SYTO9 (green; 1:1000 in PBS), and the bladder luminal surface was stained with WGA-594 (red; 1:1000 in PBS). The image in (D) shows a robust IBC; the arrows in (E) indicate luminal bacteria. Scale bars, 10 µm.

Fig. 4

Compound 6 potentiates TMP-SMZ treatment. Total bacterial CFU were quantified 6 hours after infection. UTI89 colonization was reduced in mice treated with 6 (100 mg/kg), TMP-SMZ (54 and 270 µg/ml, respectively), and TMP-SMZ + 6. Horizontal lines indicate geometric mean. *P < 0.05; **P < 0.01; ***P < 0.0001, Mann-Whitney U test.

Fig. 5

Compounds 7 to 10 show enhanced pharmacokinetics and potency at treating infection. (A) Optimized ortho-substituted biphenyl compounds 7 to 10. Cellular HAI titers (EC_>90) are shown in parentheses. (B) Compounds 7 to 10 show improved pharmacokinetics. Compounds 8 and 10 at 50 mg/kg yielded concentrations in the urine 6 hours after treatment equivalent to compound 6 at 100 mg/kg. (C) Chronically infected mice were treated with PBS or compound 6, 8, or 10 (orally, 50 mg/kg). Six hours after treatment, there was a significant drop in bacterial load in mannoside-treated mice relative to PBS-treated mice. The optimized compound 8 showed increased efficacy over 6. (D) Chronically infected mice were treated with PBS or compound 8 at one or three doses every 8 hours. Twenty-four hours after the initial treatment, both compound 8–treated groups showed a significant drop in bacterial counts over the PBS-treated animals. (C and D) Horizontal bars indicate geometric mean. *P < 0.05; **P < 0.01; ***P < 0.0001, Mann-Whitney U test.