Efficacy of a Novel Tricyclic Topoisomerase Inhibitor in a Murine Model of Neisseria

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1 AAC Accepted Manuscript Posted Online 20 June 2016 Antimicrob. Agents Chemother. doi: /aac Copyright 2016, American Society for Microbiology. All Rights Reserved. 1 2 Efficacy of a Novel Tricyclic Topoisomerase Inhibitor in a Murine Model of Neisseria gonorrhoeae Infection Victoria J. Savage, Cédric Charrier, Anne-Marie Salisbury, Helen Box, Nathan Chaffer- Malam, Anthony Huxley, Ralph Kirk, Gary M. Noonan, Sarfraz Mohmed, Mark W. Craighead, Andrew J. Ratcliffe, Stuart A. Best, Neil R. Stokes Redx Pharma, Alderley Park, Cheshire, SK10 4TG, United Kingdom Address correspondence to Neil Stokes, n.stokes@redxpharma.com V.J.S. and C.C. contributed equally to this article Running title: Tricyclic Topoisomerase Inhibitors for Gonorrhoea Keywords: gonorrhoea; Neisseria gonorrhoeae; topoisomerase; DNA gyrase; NTTI There is an urgent need for new antibiotics to treat multidrug-resistant Neisseria gonorrhoeae. In this report the microbiology, in vivo pharmacokinetics and efficacy of REDX05931, a representative novel tricyclic topoisomerase inhibitor, were evaluated. REDX05931 demonstrated high oral bioavailability in mice and reduced a N. gonorrhoeae infection following a single dose in a mouse model of gonorrhoea. These data support the potential of this series of small-molecules as a new treatment for drug-resistant gonorrhoeal infections. 1

2 Gonorrhoea is a human sexually-transmitted infection caused by the Gram-negative bacterium Neisseria gonorrhoeae and is a significant global public health problem. In 2008 there were an estimated 106 million new cases of N. gonorrhoeae infection (1). It is the second most commonly-reported infectious disease in the United States. According to the Centers for Disease Control and Prevention (CDC) there are an estimated 820,000 gonococcal infections per year in the United States (2). Untreated infection can lead to clinical complications including urethritis, dysuria, epididymitis, cervicitis and infertility. In rare cases, gonorrhoea may cause a disseminated infection that can manifest as arthritis, endocarditis or meningitis. Historically, gonorrhoea has been treated with a range of antibiotics (3, 4). The sulphonamides were first used, followed by penicillin, tetracycline and spectinomycin. For each of these drugs the development of resistance led to their use being discontinued. Ciprofloxacin and ofloxacin were also previously recommended for the treatment of gonorrhoea. By the middle of the last decade, however, fluoroquinolone resistance rates exceeded 10% of gonococcal isolates and their use was abandoned (5). Current treatment recommendations comprise ceftriaxone in combination with azithromycin (6), although resistance to both agents is emerging (7, 8, 9). CDC estimates that approximately 246,000 of the 820,000 gonococcal infections per year in the United States are drug-resistant (2). Therefore, there is an unmet need for new antibiotics to treat drug-resistant gonorrhoea. We recently disclosed (10, 11) the novel tricyclic topoisomerase inhibitor (NTTI) compounds with a binding mode distinct from the fluoroquinolones and topoisomerase inhibitors in development, such as gepotidacin and zoliflodacin (12, 13). In our prior publication the in vitro potency of compounds against susceptible and antibiotic-resistant strains of N. gonorrhoeae, was described (11). In this study we have extended these investigations by evaluating one of the compounds, REDX05931, in an animal model of gonorrhoea infection. The chemical structure of compound REDX05931 is displayed in Figure 1. 2

3 MICs were determined by the agar dilution method as per the guidelines of the Clinical Laboratory and Standards Institute (14). Bacteriological media were purchased from Oxoid Ltd (Basingstoke, United Kingdom). Strain ATCC was obtained from the American Type Culture Collection via LGC Standards (Teddington, United Kingdom). The MIC of REDX05931 was determined to be 0.03 µg/ml against N. gonorrhoeae ATCC MICs of comparator antibiotics are also displayed in Table 1. The in vivo pharmacokinetic (PK) properties of REDX05931 were evaluated in CD-1 (5 and 30 mg/kg) and BALB/c (60 mg/kg) mice with three animals per time point, following per os (PO) or intravenous (IV) administration. Experiments were conducted by Pharmidex Ltd (London, United Kingdom) under licence from the UK Animals (Scientific Procedures) Act 1986 and cared for under national guidance. For the CD-1 mice studies, the vehicle was 5% DMSO:95% hydroxypropyl-β-cyclodextrin (25% w/v). For the BALB/c mice studies the vehicle compositions were 5% DMSO:95% hydroxypropyl-β-cyclodextrin (25% w/v) (IV) or 0.5% hydroxypropyl methylcellulose in water (PO). Blood was collected at 0.25, 0.5, 1, 2, 3, 6, 8 and 24 h (PO) and 0.08, 0.33, 0.67, 1.5, 3, 6, 8 and 24 h (IV) from CD-1 mice. Blood was collected at 0.5, 1, 3, 8 and 24 h (PO) and 0.08, 0.67, 1.5, 3, 6 and 24 h (IV) from BALB/c mice. Terminal blood was delivered into tubes containing anti-coagulant (EDTA) and centrifuged at 4 C. Plasma samples (50 μl), calibration standard, quality control sample or blank plasma was extracted by the addition of acetonitrile (500 μl) containing generic internal standard (propranolol). Samples were centrifuged at 4000 RPM for 30 minutes and the resulting supernatants (100 μl) were combined with an equal volume of distilled water. Samples were centrifuged for a further 5 min at 4000 RPM prior to analysis by LC-MS/MS. Compound concentrations were quantified from the calibration curve. PK parameters were generated using non-compartmental analysis using Phoenix software (Certara USA Inc, NJ, USA) and are shown in Table 2. REDX05931 showed excellent bioavailability at all three 3

4 doses indicating low first pass effects in mouse. Peak plasma concentration (C max ) was achieved within one hour following PO dosing with values of and ng/ml at 5 and 30 mg/kg, respectively, thereby confirming the approximate dose linearity observed with other compounds from this series (11). At 60 mg/kg, C max was obtained at 0.5 h with a value of ng/ml. Based on 15.7 % fraction unbound in mouse plasma, the free AUC 0-24/MIC ratio for REDX05931 was 20.2, and at 5, 30 and 60 mg/kg PO, respectively. Hence, REDX05931 demonstrated an appropriate PK profile for in vivo efficacy testing. Efficacy was investigated in the mouse model of vaginal N. gonorrhoeae infection (15). Studies were performed by Eurofins Panlabs Taiwan Ltd (Taipei, Taiwan) in general accordance with standard guidelines on animal welfare (16) in an AAALAC-accredited facility. The protocol was reviewed and approved by the Institutional Animal Care and Use Committee. Groups of five ovariectomized BALB/c mice five weeks of age were used. Animals were subcutaneously injected with a water soluble form of estradiol at 0.23 mg/mouse on day -2, 0, 2, 4 and 6. Starting from day -2 and following inoculation to the study end, mice were dosed twice daily with streptomycin (1.2 mg/mouse) and vancomycin (0.6 mg/mouse) by intraperitoneal (IP) injection, plus trimethoprim sulfate (0.04 g/100 ml) in drinking water, to minimize vaginal flora. At day 0, mice were anesthetized with 80 mg/kg pentobarbital through IP injection, and then inoculated intravaginally with CFU of N. gonorrhoeae ATCC Before inoculation, the mouse vagina was first rinsed with 30 µl of 50 mm Hepes (ph 7.4) followed by inoculation with 20 µl gonococci suspension in PBS containing 0.5 mm CaCl 2 and 1 mm MgCl 2. REDX05931 and/or vehicle (1% Tween 80 or 0.9% NaCl, 10 ml/kg) was administered PO two hours after inoculation at 3, 10, 30 or 60 mg/kg. Ciprofloxacin was dosed at 12.5 mg/kg PO as a positive control. Mice were euthanized with CO 2 asphyxiation 24 hours or seven days post-treatment. Lavage was performed with 200 µl GC broth containing 0.05% saponin to recover vaginal bacteria. The bacterial suspensions were plated onto chocolate agar to determine the N. gonorrhoeae 4

5 counts. The results demonstrated a dose-proportional decrease in the number of N. gonorrhoeae CFUs recovered 24 hours after treatment with a single dose of REDX05931 (Figure 2A). At doses of 10, 30 or 60 mg/kg of REDX05931 there was a reduction in mean CFUs of > 2-log relative to the vehicle control, with rapid clearance of the infection observed in all animals at 60 mg/kg. There was a statistically-comparable response between the groups treated with 12.5 mg/kg ciprofloxacin and 10 mg/kg REDX At seven days, a > 3-log mean decrease in bacterial burden was measured for the single dose of 60 mg/kg REDX05931 (Figure 2B), with a low number of CFUs recovered in three of the five animals. In summary, the microbiological, pharmacokinetic and efficacy data presented in this study, as well as our previous report (11), support the potential of the NTTI compounds as a onceonly oral therapy for the treatment of drug-resistant gonorrhoea. ACKNOWLEDGEMENTS This work was funded in part through a collaboration with The Royal Liverpool and Broadgreen University Hospitals NHS Trust. We thank Peter Vince, Gareth Hampton, Kristian Edwards and Jordan Turner for assistance with bioanalysis. 5

6 140 REFERENCES World Health Organization Global incidence and prevalence of selected curable sexually transmitted infections WHO, Geneva, Switzerland. 2. Centers for Disease Control and Prevention Antibiotic resistance threats in the United States. CDC, Atlanta, United States. 3. Unemo, M and Shafer WM Antimicrobial resistance in Neisseria gonorrhoeae in the 21 st century: past, evolution, and future. Clin. Microbiol. Rev. 27: Unemo M Current and future antimicrobial treatment of gonorrhoea the rapidly evolving Neisseria gonorrhoeae continues to challenge. BMC Infectious Diseases 15: Centers for Disease Control and Prevention (CDC) Update to CDC s sexually transmitted diseases treatment guidelines, 2006: fluoroquinolone no longer recommended for treatment of gonococcal infections. MMWR Morb. Mortal. Wkly. Rep. 56: Centers for Disease Control and Prevention (CDC) Sexually transmitted diseases treatment guidelines, MMWR Morb. Mortal. Wkly. Rep. 64(RR-03): Ohnishi M, Golparian D, Shimuta K, Saika T, Hoshina S, Iwasaku K, Nakayam S, Kitawaki J, Unemo M Is Neisseria gonorrhoeae initiating a future era of untreatable gonorrhoea? Detailed characterization of the first strain with high-level resistance to ceftriaxone. Antimicrob. Agents Chemother. 55: Unemo M, Golparian D, Nicholas R, Ohnishi M, Gallay A, Sednaoui P Highlevel cefixime- and ceftriaxone-resistant Neisseria gonorrhoeae in France: novel pena mosaic allele in a successful international clone causes treatment failure. Antimicrob. Agents Chemother. 56: Morita-Ishihara T, Unemo M, Furubayashi K-i, Kawahata T, Shimuta K, Nakayama S-i, Onishi M Treatment failure with 2 g of azithromycin (extended-release 6

7 formulation) in gonorrhoea in Japan caused by the international multidrug-resistant ST1407 strain of Neisseria gonorrhoeae. J. Antimicrob. Chemother. 69: Ratcliffe A, Huxley A, Lyth D, Noonan G, Kirk R, Uosis-Martin M, Stokes N Antibacterial compounds. International Patent Application WO 2015/ Savage VJ, Charrier C, Salisbury A-M, Moyo E, Forward H, Chaffer-Malam N, Metzger R, Huxley A, Kirk R, Uosis-Martin M, Noonan G, Mohmed S, Best SA, Ratcliffe AJ, Stokes NR Biological profiling of novel tricyclic inhibitors of bacterial DNA gyrase and topoisomerase IV. J. Antimicrob. Chemother. 71: Biedenbach DJ, Bouchillon SK, Hackel M, Miller LA, Scangarella-Oman E, Jakielaszek C, Sahm DF In vitro activity of gepotidacin, a novel triazaacenaphthylene bacterial topoisomerase inhibitor, against a broad spectrum of bacterial pathogens. Antimicrob. Agents Chemother. 60: Basarab GS, Kern GH, McNulty J, Mueller JP, Lawrence K, Vishwanathan K, Alm RA, Barvian K, Doig P, Galullo V, Gardner H, Gowravaram M, Huband M, Kimzey A, Morningstar M, Kutschke A, Lahiri SD, Perros M, Singh R, Schuck VJ, Tommasi R, Walkup G, Newman JV Responding to the challenge of untreatable gonorrhea: ETX0914, a first-in-class agent with a distinct mechanism-of-action against bacterial type II topoisomerases. Sci. Rep. 5: Clinical and Laboratory Standards Institute Methods for dilution antimicrobial susceptibility tests for bacteria that grow aerobically; approved standard ninth edition. M07-A Jerse AE Experimental gonococcal genital tract infection and opacity protien expression in estradiol-treated mice. Infect. Immun. 67: National Research Council Guide for the care and use of laboratory animals: eighth edition. National Academy Press, Washington, DC. 7

8 TABLE 1 MICs of REDX05931 and comparator antibiotics against Neisseria gonorrhoeae strain ATCC Compound MIC (µg/ml) REDX Azithromycin 0.25 Cefixime 0.06 Ceftriaxone Ciprofloxacin Doxycycline 0.12 Gepotidacin 0.12 Penicillin 0.06 Spectinomycin 16 Streptomycin >64 Tetracycline 0.06 Zoliflodacin

9 TABLE 2 In vivo pharmacokinetic parameters of REDX05931 in mice following intravenous (IV) and oral (PO) dosing Parameter IV PO Strain CD-1 BALB/c CD-1 CD-1 BALB/c Dose (mg/kg) C max or C 0 (ng/ml) T max (hr) T 1/2 (hr) V dss (L/kg) CL (ml/min/kg) AUC 0-24 (ng.hr/ml) Bioavailability (%)

10 226 FIGURE LEGENDS FIG 1 Chemical structure of compound REDX FIG 2 Efficacy of REDX05931 in a mouse model of N. gonorrhoeae ATCC infection 24 hours (A) or seven days (B) after treatment. Statistical analyses were performed using the Student's t-test where * = a p value < 0.05 and ** = a p value < The dashed horizontal line shows the lower limit of detection of five CFU/mouse. Downloaded from on June 6, 2018 by guest 10

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