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In Vivo Pharmacodynamics of β-Lactams/Nacubactam against Carbapenem-Resistant and/or Carbapenemase-Producing Enterobacter cloacae and Klebsiella pneumoniae in Murine Pneumonia Model

In Vivo Pharmacodynamics of β-Lactams/Nacubactam against Carbapenem-Resistant and/or... antibiotics Article In Vivo Pharmacodynamics of -Lactams/Nacubactam against Carbapenem-Resistant and/or Carbapenemase-Producing Enterobacter cloacae and Klebsiella pneumoniae in Murine Pneumonia Model 1 , 2 2 3 3 3 2 Mao Hagihara , Hideo Kato , Toshie Sugano , Hayato Okade , Nobuo Sato , Yuichi Shibata , 2 2 2 2 2 Daisuke Sakanashi , Jun Hirai , Nobuhiro Asai , Hiroyuki Suematsu , Yuka Yamagishi 2 , and Hiroshige Mikamo * Department of Molecular Epidemiology and Biomedical Sciences, Aichi Medical University, Nagakute 480-1195, Japan; hagimao@aichi-med-u.ac.jp Department of Clinical Infectious Diseases, Aichi Medical University, Nagakute 480-1195, Japan; katou.hideo.233@mail.aichi-med-u.ac.jp (H.K.); shibata.yuuichi.414@mail.aichi-med-u.ac.jp (Y.S.); saka74d@aichi-med-u.ac.jp (D.S.); hiraichimed@gmail.com (J.H.); nobuhiro0204@gmail.com (N.A.); hsuemat@aichi-med-u.ac.jp (H.S.); y.yamagishi@mac.com (Y.Y.) Meiji Seika Pharma Co., Ltd., Tokyo 104-8002, Japan; toshie.sugano@meiji.com (T.S.); hayato.okade@meiji.com (H.O.); nobuo.satou@meiji.com (N.S.) * Correspondence: mikamo@aichi-med-u.ac.jp; Tel./Fax: +81-561-61-1842 Citation: Hagihara, M.; Kato, H.; Sugano, T.; Okade, H.; Sato, N.; Abstract: Carbapenem-resistant Enterobacterales (CRE) and carbapenemase-producing Enterobac- Shibata, Y.; Sakanashi, D.; Hirai, J.; terales (CPE) have become global threats. CRE and CPE derived infections have been associ- Asai, N.; Suematsu, H.; et al. In Vivo ated with high mortality due to limited treatment options. Nacubactam is a -lactamase inhibitor Pharmacodynamics of and belongs to the new class of diazabicyclooctane. The agent has an in vitro antimicrobial ac- -Lactams/Nacubactam against Carbapenem-Resistant and/or tivity against several classes of -lactamase-producing Enterobacterales. This study evaluated Carbapenemase-Producing antimicrobial activity of combination therapies including -lactams (aztreonam, cefepime, and Enterobacter cloacae and Klebsiella meropenem) and nacubactam against four Enterobacter cloacae and six Klebsiella pneumoniae isolates pneumoniae in Murine Pneumonia with murine pneumonia model. Based on changes in bacterial quantity, antimicrobial activities of Model. Antibiotics 2021, 10, 1179. some regimens were assessed. Combination therapies including -lactams (aztreonam, cefepime, https://doi.org/10.3390/ and meropenem) with nacubactam showed enhanced antimicrobial activity against CRE E. cloacae antibiotics10101179 (3.70 to 2.08 Dlog CFU/lungs) and K. pneumoniae (4.24 to 1.47 Dlog CFU/lungs) with IMP-1, 10 10 IMP-6, or KPC genes, compared with aztreonam, cefepime, meropenem, and nacubactam monothera- Academic Editor: Nicholas Dixon pies. Most combination therapies showed bacteriostatic (3.0 to 0 Dlog CFU/lungs) to bactericidal (<3.0 Dlog CFU/lungs) activities against CRE isolates. This study revealed that combination Received: 26 August 2021 regimens with -lactams (aztreonam, cefepime, and meropenem) and nacubactam are preferable Accepted: 23 September 2021 Published: 28 September 2021 candidates to treat pneumonia due to CRE and CPE. Keywords: aztreonam; cefepime; meropenem; nacubactam; carbapenemase-producing Enterobac- Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in terales; carbapenem-resistant Enterobacterales; Enterobacter cloacae; Klebsiella pneumoniae; pneumonia published maps and institutional affil- iations. 1. Introduction Carbapenem-resistant Enterobacterales (CRE)-caused infections have become a global Copyright: © 2021 by the authors. issue. As therapeutic options are limited, the infections showed high mortality [1,2]. Licensee MDPI, Basel, Switzerland. Carbapenemase-producing Enterobacterales (CPE) have spread globally at an alarming This article is an open access article rate [3,4]. These bacterial strains have potentials to be highly resistant to not only for distributed under the terms and carbapenems, but also other commonly used antimicrobials, through the propagation of conditions of the Creative Commons plasmids encoding carbapenem-hydrolyzing enzymes [5,6]. Attribution (CC BY) license (https:// Escherichia coli and Klebsiella pneumoniae occupy the majority among detected CPE creativecommons.org/licenses/by/ strains from infected patients [7–9]. However, Enterobacter cloacae was the second-most 4.0/). Antibiotics 2021, 10, 1179. https://doi.org/10.3390/antibiotics10101179 https://www.mdpi.com/journal/antibiotics Antibiotics 2021, 10, x FOR PEER REVIEW  2 of 11  for carbapenems, but also other commonly used antimicrobials, through the propagation  of plasmids encoding carbapenem‐hydrolyzing enzymes [5,6].  Antibiotics 2021, 10, 1179 2 of 11 Escherichia coli and Klebsiella pneumoniae occupy the majority among detected CPE  strains from infected patients [7–9]. However, Enterobacter cloacae was the second‐most  detected pathogen with carbapenemase genes in sputum specimens, followed by K. pneu‐ moniae [10]. Among Enterobacterales strains, the production of β‐lactamases was the main  detected pathogen with carbapenemase genes in sputum specimens, followed by K. pneu- reason for resistance to β‐lactams. Then, owing to the hydrolysis of almost all β‐lactams,  moniae [10]. Among Enterobacterales strains, the production of -lactamases was the main infections due to carbapenemase‐producing Enterobacterales strains are the one of the  reason for resistance to -lactams. Then, owing to the hydrolysis of almost all -lactams, most serious issues to manage in treatment.  infections due to carbapenemase-producing Enterobacterales strains are the one of the Nacubactam (NAC: OP0595) is a novel β‐lactamase inhibitor that belongs to non‐β‐ most serious issues to manage in treatment. lactam diazabicyclooctane [11]. The agent can inhibit bacterial growth of Enterobacterales  Nacubactam (NAC: OP0595) is a novel -lactamase inhibitor that belongs to non- - expressing  some  classes  of β‐lactamases  (classes  A,  C,  and  D).  Comparing  protective  lactam diazabicyclooctane [11]. The agent can inhibit bacterial growth of Enterobacterales expr mecha essing nisms, some  NAC classes  has siof mila -lactamases r effects to other (classes  β‐laA, ctama C, and se inhib D). iComparing tors [11–13].pr Howe otective ver,  mechanisms, the agent has an NAC  inhib has itory similar  effect ef of fects  penic toill other in‐bi nd -lactamase ing protein inhibitors  2 (PBP2) of [11 Entero –13]. However bacterales ,  the too agent [11]. Ther has efore, an inhibitory  NAC no etf fect only of ha penici s direct llin-binding  antimicropr bial otein  effe2ct(PBP2) s, but al of soEnter  enhan obacterales cer effects  too of  co [11‐admin ]. Theriefor stered e, NAC  β‐lact not am only s  agai hasnst dir  Enterobacterales ect antimicrobial efha fects, ve  sev buteral also  cenhancer lasses  of β effects ‐lac‐ of co-administered -lactams against Enterobacterales have several classes of -lactamases tamases producing genes in an in vitro study [11–13].  producing genes in an in vitro study [11–13]. In our previous in vivo study with murine thigh infection model, combination ther‐ In our previous in vivo study with murine thigh infection model, combination ther- apies including β‐lactams (aztreonam (ATM), cefepime (FEP), and meropenem (MEM))  apies including -lactams (aztreonam (ATM), cefepime (FEP), and meropenem (MEM)) and NAC showed enhanced antibacterial activities against CRE pathogens [14]. However,  and NAC showed enhanced antibacterial activities against CRE pathogens [14]. However, these combination therapies have not been evaluated the antimicrobial activities against  these combination therapies have not been evaluated the antimicrobial activities against CRE− and/or CPE− derived pneumonia. Therefore, the antimicrobial efficacy of these com‐ CRE and/or CPE derived pneumonia. Therefore, the antimicrobial efficacy of these bination therapies against E. cloacae and K. pneumoniae were assessed with a murine pneu‐ combination therapies against E. cloacae and K. pneumoniae were assessed with a murine monia model (Figure 1).  pneumonia model (Figure 1). Figure 1. Study flow. MIC: minimum inhibitory concentration; CRE: carbapenem-resistant Enter- Figure 1. Study flow. MIC: minimum inhibitory concentration; CRE: carbapenem‐resistant Entero‐ obacterales; ATM: aztreonam; FEP: cefepime; MEM: meropenem, NAC: nacubactam, PK: pharma- bacterales; ATM: aztreonam; FEP: cefepime; MEM: meropenem, NAC: nacubactam, PK: pharma‐ cokinetics, PD: pharmacodynamics, ELF: epithelial lining fluid, %f T > MIC: the percentage of free cokinetics, PD: pharmacodynamics, ELF: epithelial lining fluid, %fT > MIC: the percentage of free  drug time above MIC. drug time above MIC.  2. Results 2. Results  2.1. The Minimum Inhibitory Concentrations (MICs) of ATM, FEP, MEM, and NAC 2.1. The Minimum Inhibitory Concentrations (MICs) of ATM, FEP, MEM, and NAC  Table 1 shows MICs of ATM, FEP, MEM, and NAC against the 10 study isolates. Table 1 shows MICs of ATM, FEP, MEM, and NAC against the 10 study isolates.  Among them, seven isolates are resistance to MEM according to the Clinical and Labo- Among them, seven isolates are resistance to MEM according to the Clinical and Labora‐ ratory Standards Institute (CLSI) breakpoints [15]. Combination regimens of ATM, FEP, tory Standards Institute (CLSI) breakpoints [15]. Combination regimens of ATM, FEP, and  and MEM with NAC showed 2- to >128-fold lower MICs, compared with those of - MEM with NAC showed 2‐ to >128‐fold lower MICs, compared with those of β‐lactam  lactam monotherapies against the CRE+ isolates. Additionally, these combination regimens monotherapies  against  the  CRE+  isolates.  Additionally,  these  combination  regimens  showed the same to >128-fold lower MICs than those of -lactam monotherapies against showed the same to >128‐fold lower MICs than those of β‐lactam monotherapies against  the CRE/CPE+ isolates. Same MICs were observed between combinations and monother- apies against CRE/CPE E. cloacae (20-5694). Antibiotics 2021, 10, 1179 3 of 11 Table 1. Characteristics of E. cloacae and K. pneumoniae isolates utilized in this study. MIC (g/mL) Types Species Strains Genotypes MEM MEM/NAC * FEP FEP/NAC * ATM ATM/NAC * NAC 13-4983 IMP-1, 4 1 * 8 2 * 0.12 0.03 * 2 E. cloacae 990235 IMP-1 64 8 * 64 4 * 64 1 * 8 CRE+/CPE+ ATCC KPC 8 0.25 * 32 1 * >128 1 * 2 BAA-1705 K. pneumoniae 13-3445 IMP-1 64 4 * 128 2 * 0.5 0.25 * 2 594 IMP-6 32 2 * 4 2 * 8 0.25 * >64 E. cloacae 16-0483 4 2 * 4 1 * 128 2 * 4 CRE+/CPE K. pneumoniae 16-2183 CTX-M-9 8 2 * >128 4 * >128 4 * >128 DHA-1, 990645 0.25 0.25 2 1 64 1 2 CRE/CPE+ K. pneumoniae IMP-1 K. pneumoniae ATCC700603 SHV-18 0.03 0.03 1 0.25 * >128 1 * 4 CRE/CPE E. cloacae 20-5694 0.03 0.03 0.03 0.03 0.12 0.12 4 MIC: minimum inhibitory concentration; CPE: carbapenemase-producing Enterobacterales; CRE: carbapenem-resistant Enterobac- terales; ATM: aztreonam; FEP: cefepime; MEM: meropenem, NAC: nacubactam. *: p < 0.05, comparing MIC values of corresponding -lactam monotherapy. 2.2. ATM, FEP, MEM, and NAC Pharmacokinetics (PK) in the Epithelial Lining Fluid (ELF) and Plasma ATM, FEP, MEM, and NAC PK profiles in ELF and plasma are depicted in Figure S1. In this study, single doses of NAC and each -lactam monotherapies were used at 100 mg/kg (subcutaneous doses). Table 2 shows the calculated PK parameters of the antimicrobials in plasma and ELF. Table 2. PK parameters in ELF and plasma in murine pneumonia model. Dose Cmax AUC0–¥ Vd/F CL/F Antimicrobials Tmax (h) T1/2 (h) (mg/kg) (g/mL) (g h/mL) (L/kg) (L/h/kg) In ELF Nacubactam 100 0.5 31 39 1.63 NA NA Meropenem 100 0.5 3 6 0.69 NA NA Cefepime 100 1 18 32 0.84 NA NA Aztreonam 100 0.5 16 27 0.94 NA NA In plasma Nacubactam 100 0.25 116 131 0.61 0.67 0.76 Meropenem 100 0.25 53 36 0.4 1.6 2.79 Cefepime 100 0.25 80 84 0.41 0.71 1.19 Aztreonam 100 0.5 96 120 0.36 0.43 0.83 PK: pharmacokinetic; ELF: epithelial lining fluid; C : maximum drug concentration; T : time to reach maximum plasma concentration; max max AUC : AUC in ELF or plasma from time 0 to ¥; T : half-life; CL/F: apparent clearance; Vd/F: apparent distribution volume; NA: 0–¥ 1/2 not applicable. 2.3. Pharmacodynamic (PD) Study with Murine Pneumonia Model 2.3.1. Antimicrobial Efficacies of NAC Monotherapy At the start of antimicrobial therapies (0 h), bacterial counts in lungs of control mice were between 6.79 to 7.00 log colony forming units (cfu)/lungs for E. cloacae and 6.59 to 7.21 log cfu/lungs for K. pneumoniae. Bacterial number differences of growth control were between 0.06 to 2.58 Dlog CFU/lungs for E. cloacae and 0.77 to 2.74 Dlog CFU/lungs 10 10 for K. pneumoniae after 24 h from the start of antimicrobial therapies. Bacterial number differences of NAC monotherapy (320, 160, and 80 mg/kg q8h) were between 2.27 and 1.19 Dlog CFU/lungs for four E. cloacae isolates and 1.34 to 2.66 Dlog CFU/lungs for 10 10 six K. pneumoniae isolates (Figure S2). Among total 10 study isolates, six isolates showed higher bacterial numbers in lungs at 24 h than at 0 h, while maximum dosage of NAC was adopted (320 mg/kg q8h). Antibiotics 2021, 10, 1179 4 of 11 2.3.2. Antimicrobial Efficacies against CRE+ Isolates Antibiotics 2021, 10, x FOR PEER REVIEW  4 of 11  Bacterial number differences in K. pneumoniae-infected mice received combination therapies were between 4.24 and 1.47 Dlog CFU/lungs (Figure 2) and 3.70 and 2.08 Dlog CFU/lungs for E. cloacae-infected mice (Figure 3). Compared with the corre- bacterial  numbers  in  lungs  at  24  h  than  at  0  h,  while  maximum  dosage  of  NAC  was  sponding -lactam monotherapies, combination therapies showed higher antimicrobial adopted (320 mg/kg q8h).  activities against CRE+ isolates (p < 0.05), except when combined with MEM + NAC against CRE+/CPE isolate (E. cloacae 16-0483) and combined with ATM + NAC against 2.3.2. Antimicrobial Efficacies against CRE+ Isolates  CRE+/CPE+ isolate (13-4983). Additionally, the NAC dose-dependent antimicrobial activ- ity of combination therapy with NAC and ATM against CRE+/CPE isolate (K. pneumoniae Bacterial number differences in  K. pneumoniae‐infected mice received combination  16-2183) was observed. When ATM, FEP, and MEM were combined with NAC, similar therapies were between −4.24 and 1.47 Δlog10 CFU/lungs (Figure 2) and −3.70 and −2.08  trends of antimicrobial activity were also observed against CRE+/CPE+ isolate (K. pneumo- Δlog10 CFU/lungs for E. cloacae‐infected mice (Figure 3). Compared with the correspond‐ niae ATCC BAA-1705). Moreover, while most combination therapies showed bacteriostatic ing β‐lactam monotherapies, combination therapies showed higher antimicrobial activi‐ (3.0 to 0 Dlog CFU/lungs) to bactericidal (<3.0 Dlog CFU/lungs) activities against 10 10 ties against CRE+ isolates (p < 0.05), except when combined with MEM + NAC against  CRE isolates, bacterial counts in lungs of MEM + NAC combination therapy group against CRE+/CPE−  isolate  (E.  cloacae  16‐0483)  and  combined  with  ATM  +  NAC  against  K. pneumoniae (594) were higher at 24 h than at 0 h in the control (>0 Dlog CFU/lungs). CRE+/CPE+ isolate (13‐4983). Additionally, the NAC dose‐dependent antimicrobial activ‐ ity of combination therapy with NAC and ATM against CRE+/CPE− isolate (K. pneumoniae  2.3.3. Antimicrobial Efficacies against CRE Isolates 16‐2183) was observed. When ATM, FEP, and MEM were combined with NAC, similar  Bacterial number differences in combination therapies were between 3.56 and trends of antimicrobial activity were also observed against CRE+/CPE+ isolate (K. pneu‐ 2.81 Dlog CFU/lungs for E. cloacae and 4.46 and 1.70 Dlog CFU/lungs for 10 10 moniae ATCC BAA‐1705). Moreover, while most combination therapies showed bacterio‐ K. pneumoniae (Figure 4). Comparing with the corresponding -lactam monotherapies, combination therapies showed enhanced antimicrobial activities, when combined with FEP static  (−3.0  to  0 Δlog10  CFU/lungs)  to  bactericidal  (<−3.0 Δlog10  CFU/lungs)  activities  and AZT with NAC against CRE/CPE (K. pneumoniae ATCC700603) and CRE/CPE+ against CRE isolates, bacterial counts in lungs of MEM + NAC combination therapy group  (K. pneumoniae 990645). However, NAC did not affect antimicrobial activity of MEM ther- against  K.  pneumoniae  (594)  were  higher  at  24  h  than  at  0  h  in  the  control  (>0 Δlog10  apy against these isolates. Moreover, no enhanced antimicrobial activity was found in CFU/lungs).  combination therapies against E. cloacae (20-5694). Figure 2. Antimicrobial efficacy of study regimens against CRE+ Klebsiella pneumoniae isolates. Figure 2. Antimicrobial efficacy of study regimens against CRE+ Klebsiella pneumoniae isolates.  Control (), -lactam (aztreonam, cefepime, meropenem) monotherapy (), add nacubactam Control (□), β‐lactam (aztreonam, cefepime, meropenem) monotherapy (■), add nacubactam 80  80 mg/kg q8h (), add nacubactam 160 mg/kg q8h (), add nacubactam 320 mg/kg q8h (). mg/kg q8h (■), add nacubactam 160 mg/kg q8h (■), add nacubactam 320 mg/kg q8h (■). White bar  White bar control applies to all the drug combinations shown. *: vs. control p < 0.05, : vs. -lactam control applies to all the drug combinations shown. *: vs. control p < 0.05,  : vs. β‐lactam monother‐ monotherapy p < 0.05, [: vs. add nacubactam 80 mg/kg q8h p < 0.05. CRE: carbapenem-resistant apy p < 0.05, ♭: vs. add nacubactam 80 mg/kg q8h p < 0.05. CRE: carbapenem‐resistant Enterobac‐ Enterobacterales, CPE: carbapenemase-producing Enterobacterales, Aztreonam: ATM, cefepime: FEP, terales, CPE: carbapenemase‐producing Enterobacterales, Aztreonam: ATM, cefepime: FEP, mero‐ meropenem: MEM. All data are shown as average  SD. penem: MEM. All data are shown as average ± SD.  Antibiotics 2021, 10, x FOR PEER REVIEW  5 of 11  Antibiotics 2021, 10, x FOR PEER REVIEW  5 of 11  Antibiotics 2021, 10, 1179 5 of 11 Figure 3. Antimicrobial efficacy of study regimens against CRE+ Enterobacter cloacae isolates. Con‐ trol (□), β‐lactam (aztreonam, cefepime, meropenem) monotherapy (■), add nacubactam 80  mg/kg q8h (■), add nacubactam 160 mg/kg q8h (■), add nacubactam 320 mg/kg q8h (■). White  bar control applies to all the drug combinations shown. *: vs. control p < 0.05,  : vs. β‐lactam mono‐ therapy p < 0.05. CRE: carbapenem‐resistant Enterobacterales, CPE: carbapenemase‐producing  Enterobacterales, Aztreonam: ATM, cefepime: FEP, meropenem: MEM. All data are shown as av‐ erage ± SD.  2.3.3. Antimicrobial Efficacies against CRE− Isolates  Bacterial number differences in combination therapies were between −3.56 and −2.81  Δlog10 CFU/lungs for E. cloacae and −4.46 and −1.70 Δlog10 CFU/lungs for K. pneumoniae  (Figure  4).  Comparing  with  the  corresponding β‐lactam  monotherapies,  combination  Figure 3. Antimicrobial efficacy of study regimens against CRE+ Enterobacter cloacae isolates. Control Figure 3. Antimicrobial efficacy of study regimens against CRE+ Enterobacter cloacae isolates. Con‐ therapies showed enhanced antimicrobial activities, when combined with FEP and AZT  (), -lactam (aztreonam, cefepime, meropenem) monotherapy (), add nacubactam 80 mg/kg q8h trol (□), β‐lactam (aztreonam, cefepime, meropenem) monotherapy (■), add nacubactam 80  with NAC against CRE−/CPE− (K. pneumoniae ATCC700603) and CRE−/CPE+ (K. pneu‐ (), add nacubactam 160 mg/kg q8h (), add nacubactam 320 mg/kg q8h (). White bar control mg/kg q8h (■), add nacubactam 160 mg/kg q8h (■), add nacubactam 320 mg/kg q8h (■). White  moniae  990645).  However,  NAC  did  not  affect  antimicrobial  activity  of  MEM  therapy  applies to all the drug combinations shown. *: vs. control p < 0.05, : vs. -lactam monotherapy bar control applies to all the drug combinations shown. *: vs. control p < 0.05,  : vs. β‐lactam mono‐ against these isolates. Moreover, no enhanced antimicrobial activity was found in combi‐ p < 0.05. CRE: carbapenem-resistant Enterobacterales, CPE: carbapenemase-producing Enterobacterales, therapy p < 0.05. CRE: carbapenem‐resistant Enterobacterales, CPE: carbapenemase‐producing  nation therapies against E. cloacae (20‐5694).  Aztreonam: ATM, cefepime: FEP, meropenem: MEM. All data are shown as average  SD. Enterobacterales, Aztreonam: ATM, cefepime: FEP, meropenem: MEM. All data are shown as av‐ erage ± SD.  2.3.3. Antimicrobial Efficacies against CRE− Isolates  Bacterial number differences in combination therapies were between −3.56 and −2.81  Δlog10 CFU/lungs for E. cloacae and −4.46 and −1.70 Δlog10 CFU/lungs for K. pneumoniae  (Figure  4).  Comparing  with  the  corresponding β‐lactam  monotherapies,  combination  therapies showed enhanced antimicrobial activities, when combined with FEP and AZT  with NAC against CRE−/CPE− (K. pneumoniae ATCC700603) and CRE−/CPE+ (K. pneu‐ moniae  990645).  However,  NAC  did  not  affect  antimicrobial  activity  of  MEM  therapy  against these isolates. Moreover, no enhanced antimicrobial activity was found in combi‐ nation therapies against E. cloacae (20‐5694).  Figure 4. Antimicrobial efficacy of study regimens against CRE isolates. Control (), -lactam (aztreonam, cefepime, meropenem) monotherapy (), add nacubactam 80 mg/kg q8h (), add Figure 4. Antimicrobial efficacy of study regimens against CRE− isolates. Control (□), β‐lactam  nacubactam 160 mg/kg q8h (), add nacubactam 320 mg/kg q8h (). White bar control ap- (aztreonam, cefepime, meropenem) monotherapy (■), add nacubactam 80 mg/kg q8h (■), add  plies to all the drug combinations shown. *: vs. control p < 0.05, : vs. -lactam monotherapy nacubactam 160 mg/kg q8h (■), add nacubactam 320 mg/kg q8h (■). White bar control applies to  p < 0.05. CRE: carbapenem-resistant Enterobacterales, CPE: # carbapenemase-producing Enterobac- all the drug combinations shown. *: vs. control p < 0.05,  : vs. β‐lactam monotherapy p < 0.05. CRE:  terales, Aztreonam: ATM, cefepime: FEP, meropenem: MEM. All data shown as average  SD. carbapenem‐resistant Enterobacterales, CPE: carbapenemase‐producing Enterobacterales, Aztre‐ onam: ATM, cefepime: FEP, meropenem: MEM. All data shown as average ± SD.  2.4. Relationships between Antimicrobial Activities and the Percentage of Free Drug Time above MIC (%fT > MIC) 2.4. Relationships between Antimicrobial Activities and the Percentage of Free Drug Time above  Figure 5 shows the relationships between Dlog CFU/lungs and %fT > MIC against MIC (%fT > MIC)  study isolates. The %fT > MIC in ELF showed higher correlations with Dlog CFU/lungs (R = 0.88 for ATM, 0.75 for FEP, 0.45 for MEM). Similarly, the %fT > MIC in plasma was also correlated with Dlog CFU/lungs well (R = 0.89 for ATM, 0.74 for FEP, 0.55 for MEM). Along with the elevation of %fT > MIC value, the bacterial density was reduced after 24 h Figure 4. Antimicrobial efficacy of study regimens against CRE− isolates. Control (□), β‐lactam  of antimicrobial treatments. (aztreonam, cefepime, meropenem) monotherapy (■), add nacubactam 80 mg/kg q8h (■), add  nacubactam 160 mg/kg q8h (■), add nacubactam 320 mg/kg q8h (■). White bar control applies to  all the drug combinations shown. *: vs. control p < 0.05,  : vs. β‐lactam monotherapy p < 0.05. CRE:  carbapenem‐resistant Enterobacterales, CPE: carbapenemase‐producing Enterobacterales, Aztre‐ onam: ATM, cefepime: FEP, meropenem: MEM. All data shown as average ± SD.  2.4. Relationships between Antimicrobial Activities and the Percentage of Free Drug Time above  MIC (%fT > MIC)  Antibiotics 2021, 10, x FOR PEER REVIEW  6 of 11  Figure 5 shows the relationships between Δlog10 CFU/lungs and %fT > MIC against  study isolates. The %fT > MIC in ELF showed higher correlations with Δlog10 CFU/lungs  (R  = 0.88 for ATM, 0.75 for FEP, 0.45 for MEM). Similarly, the %fT > MIC in plasma was  also correlated with Δlog10 CFU/lungs well (R  = 0.89 for ATM, 0.74 for FEP, 0.55 for MEM).  Antibiotics 2021, 10, 1179 6 of 11 Along with the elevation of %fT > MIC value, the bacterial density was reduced after 24 h  of antimicrobial treatments.  Figure 5. Relationships between %fT > MIC of -lactams and Dlog CFU/lungs at 24 h. An inhibitory effect sigmoid Imax Figure 5. Relationships between %fT > MIC of β‐lactams and Δlog10 CFU/lungs at 24 h. An inhibitory effect sigmoid Imax  model was adapted to reveal the correlations between %fT > MIC of each drug and Dlog CFU/lungs of each antimicrobial model was adapted to reveal the correlations between %fT > MIC of each drug and Δlog10 CFU/lungs of each antimicrobial  regimen with Phoenix WinNonlin software (ver. 8.1; Certara, L.P.). The 2 R represents the coefficient of determination. regimen with Phoenix WinNonlin software (ver. 8.1; Certara, L.P.). The R  represents the coefficient of determination. %fT  %fT > MIC: the percentage of free drug time above MIC, ATM: aztreonam, FEP: cefepime, MEM: meropenem, > MIC: the percentage of free drug time above MIC, ATM: aztreonam, FEP: cefepime, MEM: meropenem, NAC: nacubac‐ tam. NAC:  nacubactam. 3. Discussion 3. Discussion  NAC has a dual mechanism of action. The agent inhibits some classes of serine NAC has a dual mechanism of action. The agent inhibits some classes of serine β‐ -lactamases and PBP2 in Enterobacterales [11,16,17]. In our previous study, the same lactamases and PBP2 in Enterobacterales [11,16,17]. In our previous study, the same com‐ combination therapies including NAC and -lactams showed enhanced antimicrobial ac- bination therapies including NAC and β‐lactams showed enhanced antimicrobial activi‐ tivities against Enterobacterales have some carbapenemase-producing genes with a murine ties against Enterobacterales have some carbapenemase‐producing genes with a murine  thigh infection model [14]. However, these combination therapies have not been evalu- thigh infection model [14]. However, these combination therapies have not been evalu‐ ated against CRE and CPEcaused pneumonia. In this in vitro study, the combination ated against CRE− and CPE−caused pneumonia. In this in vitro study, the combination  regimens of -lactams with NAC showed 1- to >128-fold lower MICs against CPE and regimens of β‐lactams with NAC showed 1‐ to >128‐fold lower MICs against CPE and  CRE isolates, comparing with the corresponding -lactam monotherapies (Table 1). As CRE isolates, comparing with the corresponding β‐lactam monotherapies (Table 1). As  previous in vitro study suggested, our results also suggested that NAC enhanced in vitro previous in vitro study suggested, our results also suggested that NAC enhanced in vitro  antimicrobial activities of -lactams against several classes of -lactamase-producing antimicrobial activities of β‐lactams against several classes of β‐lactamase‐producing E.  E. cloacae and K. pneumoniae isolates [11–13]. cloacae and K. pneumoniae isolates [11–13].  Moreover, in this pharmacodynamics evaluation, NAC monotherapy could not show Moreover, in this pharmacodynamics evaluation, NAC monotherapy could not show  a bactericidal effect against all study isolates (Figure S2). In contrast, combination therapies a bactericidal effect against all study isolates (Figure S2). In contrast, combination thera‐ showed enhanced antimicrobial activities against CRE+ E. cloacae and K. pneumoniae isolates pies showed enhanced antimicrobial activities against CRE+ E. cloacae and K. pneumoniae  (Figures 2 and 3). At least one regimen among combination therapies of ATM, FEP, and isolates (Figures 2 and 3). At least one regimen among combination therapies of ATM,  MEM with NAC showed bactericidal effects against all CRE+ E. cloacae and K. pneumoniae isolates, excluding the K. pneumoniae isolate (594). Similar to our previous study using a murine thigh infection model [14], combina- tion therapy of MEM and NAC was not effective (>0Dlog CFU/lungs) against CRE+ K. pneumoniae (594) (Figure 2). The isolate has the IMP-6 gene and showed low susceptibil- ity to MEM and NAC, whereas MEM + NAC resulted in relatively lower MIC (2 g/mL) (Table 1). In vitro studies of the other group conducted showed that the efficacy of NAC is Antibiotics 2021, 10, 1179 7 of 11 restricted to bacteria with IMP-type carbapenemase-producing genes [11–13]. NAC only binds to PBP2. On the other hand, ATM and FEP bind to PBP3 rather than PBP2 [11]. Thus, the NAC-derived enhancer effect was expected when the agent was co-administrated with ATM and FEP than with MEM. Furthermore, we could speculate that NAC was not effective to work as an PBP2 inhibitor to CRE+ K. pneumoniae (594) as the isolate has low susceptibility to both MEM and NAC. Although further study to clarify the mechanism is needed, MEM + NAC combination therapy against IMP-type carbapenemase-producing Enterobacterales could be unsuitable, especially when causative bacteria have high MICs (low susceptibilities) of MEM and NAC. In general, optimizing the antimicrobial dosage according to the PK/PD theory is one of the important components to improve infected patient outcomes. As the PK/PD breakpoint of NAC has not been fully evaluated, it was difficult to interpret our PD study results. However, the antimicrobial activities of -lactams correlate with %f T > MIC well [18,19]. NAC co-administration had no impact on PK profiles of -lactams (ATM, FEP, and MEM) in plasma [14]. Additionally, we did not observe significant differ- ences in the PK data of plasma between the thigh infection model and pneumonia model [14, Figure S1]. Due to the fact that the reappearance of human PK profiles of antimicro- bials was difficult for us, as mice have higher clearance ability than that of humans, we used the same dosage regimens as previous in vivo studies [14] to obtain similar areas under the free drug concentration-time curves (f AUC) when -lactams (ATM, FEP, and MEM) were dosed at 1 g q8h, and NAC (2, 1, and 0.5 g q8h) [20–26]. Using calculated PK parameters, we conducted PK/PD analysis and found the good relationships between %f T > MIC of -lactams (ATM, FEP, and MEM) in ELF and plasma and antimicrobial activities (Figure 5). Our data will give important insights to explore the optimal dosage regimen for pneumonia patients. The combination therapy of -lactams (ATM, FEP, and MEM) and NAC was highly effective against CPE and CRE caused pneumonia. However, we evaluated limited classes of carbapenemases producing Enterobacteriales in this study. In Japan, IMP-1 and -6 type CPE isolates are the most frequently detected [7,8]. However, there are regional differences of the -lactamases prevalence in Enterobacterales [7,9,27]. Therefore, further studies are needed. As the other limitation, we did not evaluate inoculum effects, while we evaluated antimicrobial activities with similar inoculum size to previous report [28–30]. Although our study has a few limitations, this is the first in vivo study to evaluate antimicrobial activities of combination therapies including -lactams and NAC with pneu- monia model. Our PD data suggested combination therapied including NAC is a potent candidate for CRE caused pneumonia. This fact can give new therapeutic choice to antimi- crobial treatment for CRE caused pneumonia, while the prevalence and burden of CRE infection are rising. Additionally, we revealed the relationship between these antimicrobial activities and antimicrobial PK data (in plasma and ELF). Therefore, our data would be a preferable reference to explore optimal antimicrobial dosages in the future. 4. Materials and Methods 4.1. Antimicrobials To determine the antimicrobial concentrations in ELF and plasma, we used analytical- grade ATM (lot LFSZK: Tokyo Chemical Industry Co., Ltd., Tokyo, Japan), FEP (lot I0L200USP: USP) and MEM hydrate (lot YCY8L: Tokyo Chemical Industry Co., Ltd.). Meiji Seika Pharma Co., Ltd. (Tokyo, Japan) provided us analytical-grade NAC (lot 510- 015-4097-01). For all in vivo study, ATM (Eisai Co., Ltd., Tokyo, Japan), FEP (Bristol-Myers Squibb K.K., Tokyo, Japan), MEM (Meiji Seika Pharma Co., Ltd.), and NAC (lot BS1807SB03) were used. Stock solutions of ATM, FEP, and MEM were stored at 4 C. The stock solutions were reconstituted with normal saline (NS) at desired concentrations. Antibiotics 2021, 10, 1179 8 of 11 4.2. Microorganisms Four E. cloacae and six K. pneumoniae isolates were used in this study (Table 1). Clinical isolates of E. cloacae and K. pneumoniae were provided from department of Microbial laboratory in Aichi Medical University Hospital. CRE isolates have MEM MIC  4 g/mL. CPE isolates have any detectable carbapenemase genes. According to previous our study methods [14], direct sequencing was conducted to screen carbapenemase and other - lactamase-encoding genes. 4.3. Susceptibility Test The antimicrobial susceptibilities of ATM, FEP, MEM, and NAC were determined with the broth microdilution method, following recommended methods by CLSI [15]. For the combination regimens, the broth microdilution method was conducted in fixed concentra- tion ratios of each -lactam and NAC (1:1 (w/w)), according to CLSI method (M100-ED31: Table 5A-2). The -lactam concentration was used to show MICs in combination thera- pies. The MIC studies were conducted a minimum of three times, and the geometric MIC was reported. 4.4. Animals Pathogen-free, ICR mice (4-week-old) were acquired from Charles River Laboratories Japan, Inc. (Yokohama, Japan). They were provided food and water ad libitum. They were housed and used following the National Research Council recommendations. 4.5. Pneumonia Model Neutropenic murine pneumonia model was used to evaluate antimicrobial activities of various antimicrobial regimens against E. cloacae and K. pneumoniae in the lungs of mice [30]. Mice were rendered transiently neutropenic with cyclophosphamide of intraperitoneal doses at Day-4 (150 mg/kg) and Day-1 (100 mg/kg) before inoculation. Study isolates for inoculation were stored at 80 C. A bacterial suspension was prepared at approxi- mately 10 cfu/mL with study isolates after 24 h incubation of the second transfer. Under anesthesia, mice were orally instilled the bacterial suspension (0.075 mL), and the nares were blocked while being held vertically for 60 s to aspirate the suspension into the lungs. After inoculation, mice were randomly divided into control or each antimicrobial treatment group (n = 6, respectively). 4.6. PK Studies For the PK study of ATM, FEP, MEM, and NAC in ELF and plasma, each antimi- crobial was dosed subcutaneously (single dose) as monotherapies to the infected mice. The neutropenic pneumonia mice were infected with K. pneumoniae ATCC43816. The mice were injected with ATM, FEP, MEM, or NAC (at 100 mg/kg, respectively). Mice were sacrificed at each time point (0.25, 0.5, 1, 2, 3, 4, and 5 h). Then, blood samples from the axillary artery and bronchoalveolar lavage fluid (BALF) samples were col- lected (n = 3 in each group). After the detection of urea concentration in each BALF and plasma sample with a urea assay kit (Urea Assay Kit, BioChain Institute, Inc., Newark, CA, USA), the antimicrobial concentrations in ELF were calculated: ELF antimicrobial concentration = BALF antimicrobial concentration  (the ratio of urea concentrations in plasma/BALF). AUC in ELF and plasma from time 0 to ¥ (AUC0–¥) were calculated according to the trapezoidal method. The PK parameters in the ELF and plasma were calculated with Phoenix WinNonlin software (ver. 8.1; Certara, L.P.). 4.7. Instrumentation and Chromatographic Conditions Liquid chromatography-tandem mass spectrometry (LC-MS/MS) was used to analyze ATM, FEP, MEM, and NAC concentrations at Meiji Seika Pharma Co., Ltd. Detailed conditions and procedures are in the Supplementary document. Antibiotics 2021, 10, 1179 9 of 11 4.8. PD Study One hour after inoculation of study isolates, mice received antimicrobials (0.2 mL) subcutaneously (n = 6 in each group). We used same dosage of -lactams and NAC with our previous in vivo study [14] to be similar f AUC values of NAC (2.0, 1.0, and 0.5 g), and -lactams (1.0 g) in blood with clinical PK study data. The following eight-hourly (q8h) doses were evaluated: 700 mg/kg for ATM, 260 mg/kg for FEP, and 140 mg/kg for MEM, 320, 160, and 80 mg/kg for NAC. Control mice were administered NS (0.2 mL). Twenty-four hours after antimicrobial treatments started, lungs were harvested from mice. Then, mice were sacrificed by CO exposure. Removed lungs from each mouse were homogenized individually, and the homogenates were plated on trypticase soy agar with 5% sheep blood after serial dilutions with NS to determine the bacterial counts in lungs (cfu/lungs). In addition, six infected mice were harvested just before the start of antimicrobial therapies to know bacterial number in lungs at 0 h. The antimicrobial efficacies were defined as follows: detected bacterial counts in the treated group at 24 h and bacterial counts in the control group at 0 h (Dlog CFU/lungs). 4.9. PK/PD Analysis The %T > MIC in ELF and plasma for unbound ATM, FEP, MEM, and NAC was calculated with PK parameters (Table S1 and Table S2) in pneumonia mice, serum protein binding ratio [14,20–22], and MIC values, with the R software (ver. 3.6.1). For combination therapies, MICs of combinations were used. An inhibitory effect sigmoid I model was max adapted to reveal the correlations between %f T > MIC of each drug and Dlog CFU/lungs of each antimicrobial regimen with Phoenix WinNonlin software (ver. 8.1; Certara, L.P.). To estimate the percentage of variance in efficacy, the coefficient of determination (R ) was used. 4.10. Statistical Analysis Mann–Whitney U test was used to compare in vitro MIC values of each -lactam monotherapy and corresponding combination therapies. To compare the in vivo antimicro- bial efficacy between the regimens, one-way ANOVA with Bonferroni correction was used. Statistical analysis was performed with JMP, version 10.0 (SAS, Tokyo, Japan). Differences were considered statistically significant at p < 0.05. 5. Conclusions Combination therapies including ATM, FEP, and MEM with NAC showed potent in vivo antimicrobial activities against pneumonia caused by CRE and CPE E. cloacae and K. pneumoniae. These translational data support the potential role of NAC in combination with ATM, FEP, and MEM as a therapy for CRE and CPE K. pneumoniae and E. cloacae caused human pneumonia. Supplementary Materials: The following are available online at https://www.mdpi.com/article/ 10.3390/antibiotics10101179/s1, Figure S1: In vivo pharmacokinetic data of nacubactam, meropenem, cefepime, and aztreonam in ELF and plasma, Figure S2: In vivo efficacy of nacubactam monotherapy, Table S1: Apparent pharmacokinetic parameters in ELF in murine pneumonia model for calculation of f T > MIC, Table S2: Pharmacokinetic parameters in plasma in murine pneumonia model for calculation of %f T > MIC. Author Contributions: Conceptualization, H.M., H.O., T.S. and M.H.; data curation, T.S., H.O. and M.H.; formal analysis, N.S., T.S. and M.H.; investigation, M.H., H.K., Y.S., D.S., J.H., N.A., H.S., Y.Y. and N.A.; project administration, M.H. and H.M.; resources, H.M.; supervision, H.M.; validation, T.S.; writing—original draft, M.H.; writing—review and editing, T.S. and H.M. All authors have read and agreed to the published version of the manuscript. Funding: This work was supported by AMED (grant number JP18pc0101028). Institutional Review Board Statement: The ethics committee of Aichi medical university reviewed this study protocol and approved (2020-30). To conduct PK studies of -lactams and NAC, the animal Antibiotics 2021, 10, 1179 10 of 11 experiment management committee and pharmaceutical research laboratories at Meiji Seika Pharma Co., Ltd. reviewed this study protocol and approved (C2020-010 and C2020-013). Informed Consent Statement: Not applicable. Data Availability Statement: The datasets analyzed during this study are available and can be obtained, at request, on reasonable enquiry. Conflicts of Interest: T.S., H.O. and N.S. are employees of Meiji Seika Pharma Co., Ltd., Japan. H.M. received partial research funding from Meiji Seika Pharma Co., Ltd., Japan. The authors declare no conflict of interest. References 1. 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In Vivo Pharmacodynamics of β-Lactams/Nacubactam against Carbapenem-Resistant and/or Carbapenemase-Producing Enterobacter cloacae and Klebsiella pneumoniae in Murine Pneumonia Model

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2079-6382
DOI
10.3390/antibiotics10101179
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antibiotics Article In Vivo Pharmacodynamics of -Lactams/Nacubactam against Carbapenem-Resistant and/or Carbapenemase-Producing Enterobacter cloacae and Klebsiella pneumoniae in Murine Pneumonia Model 1 , 2 2 3 3 3 2 Mao Hagihara , Hideo Kato , Toshie Sugano , Hayato Okade , Nobuo Sato , Yuichi Shibata , 2 2 2 2 2 Daisuke Sakanashi , Jun Hirai , Nobuhiro Asai , Hiroyuki Suematsu , Yuka Yamagishi 2 , and Hiroshige Mikamo * Department of Molecular Epidemiology and Biomedical Sciences, Aichi Medical University, Nagakute 480-1195, Japan; hagimao@aichi-med-u.ac.jp Department of Clinical Infectious Diseases, Aichi Medical University, Nagakute 480-1195, Japan; katou.hideo.233@mail.aichi-med-u.ac.jp (H.K.); shibata.yuuichi.414@mail.aichi-med-u.ac.jp (Y.S.); saka74d@aichi-med-u.ac.jp (D.S.); hiraichimed@gmail.com (J.H.); nobuhiro0204@gmail.com (N.A.); hsuemat@aichi-med-u.ac.jp (H.S.); y.yamagishi@mac.com (Y.Y.) Meiji Seika Pharma Co., Ltd., Tokyo 104-8002, Japan; toshie.sugano@meiji.com (T.S.); hayato.okade@meiji.com (H.O.); nobuo.satou@meiji.com (N.S.) * Correspondence: mikamo@aichi-med-u.ac.jp; Tel./Fax: +81-561-61-1842 Citation: Hagihara, M.; Kato, H.; Sugano, T.; Okade, H.; Sato, N.; Abstract: Carbapenem-resistant Enterobacterales (CRE) and carbapenemase-producing Enterobac- Shibata, Y.; Sakanashi, D.; Hirai, J.; terales (CPE) have become global threats. CRE and CPE derived infections have been associ- Asai, N.; Suematsu, H.; et al. In Vivo ated with high mortality due to limited treatment options. Nacubactam is a -lactamase inhibitor Pharmacodynamics of and belongs to the new class of diazabicyclooctane. The agent has an in vitro antimicrobial ac- -Lactams/Nacubactam against Carbapenem-Resistant and/or tivity against several classes of -lactamase-producing Enterobacterales. This study evaluated Carbapenemase-Producing antimicrobial activity of combination therapies including -lactams (aztreonam, cefepime, and Enterobacter cloacae and Klebsiella meropenem) and nacubactam against four Enterobacter cloacae and six Klebsiella pneumoniae isolates pneumoniae in Murine Pneumonia with murine pneumonia model. Based on changes in bacterial quantity, antimicrobial activities of Model. Antibiotics 2021, 10, 1179. some regimens were assessed. Combination therapies including -lactams (aztreonam, cefepime, https://doi.org/10.3390/ and meropenem) with nacubactam showed enhanced antimicrobial activity against CRE E. cloacae antibiotics10101179 (3.70 to 2.08 Dlog CFU/lungs) and K. pneumoniae (4.24 to 1.47 Dlog CFU/lungs) with IMP-1, 10 10 IMP-6, or KPC genes, compared with aztreonam, cefepime, meropenem, and nacubactam monothera- Academic Editor: Nicholas Dixon pies. Most combination therapies showed bacteriostatic (3.0 to 0 Dlog CFU/lungs) to bactericidal (<3.0 Dlog CFU/lungs) activities against CRE isolates. This study revealed that combination Received: 26 August 2021 regimens with -lactams (aztreonam, cefepime, and meropenem) and nacubactam are preferable Accepted: 23 September 2021 Published: 28 September 2021 candidates to treat pneumonia due to CRE and CPE. Keywords: aztreonam; cefepime; meropenem; nacubactam; carbapenemase-producing Enterobac- Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in terales; carbapenem-resistant Enterobacterales; Enterobacter cloacae; Klebsiella pneumoniae; pneumonia published maps and institutional affil- iations. 1. Introduction Carbapenem-resistant Enterobacterales (CRE)-caused infections have become a global Copyright: © 2021 by the authors. issue. As therapeutic options are limited, the infections showed high mortality [1,2]. Licensee MDPI, Basel, Switzerland. Carbapenemase-producing Enterobacterales (CPE) have spread globally at an alarming This article is an open access article rate [3,4]. These bacterial strains have potentials to be highly resistant to not only for distributed under the terms and carbapenems, but also other commonly used antimicrobials, through the propagation of conditions of the Creative Commons plasmids encoding carbapenem-hydrolyzing enzymes [5,6]. Attribution (CC BY) license (https:// Escherichia coli and Klebsiella pneumoniae occupy the majority among detected CPE creativecommons.org/licenses/by/ strains from infected patients [7–9]. However, Enterobacter cloacae was the second-most 4.0/). Antibiotics 2021, 10, 1179. https://doi.org/10.3390/antibiotics10101179 https://www.mdpi.com/journal/antibiotics Antibiotics 2021, 10, x FOR PEER REVIEW  2 of 11  for carbapenems, but also other commonly used antimicrobials, through the propagation  of plasmids encoding carbapenem‐hydrolyzing enzymes [5,6].  Antibiotics 2021, 10, 1179 2 of 11 Escherichia coli and Klebsiella pneumoniae occupy the majority among detected CPE  strains from infected patients [7–9]. However, Enterobacter cloacae was the second‐most  detected pathogen with carbapenemase genes in sputum specimens, followed by K. pneu‐ moniae [10]. Among Enterobacterales strains, the production of β‐lactamases was the main  detected pathogen with carbapenemase genes in sputum specimens, followed by K. pneu- reason for resistance to β‐lactams. Then, owing to the hydrolysis of almost all β‐lactams,  moniae [10]. Among Enterobacterales strains, the production of -lactamases was the main infections due to carbapenemase‐producing Enterobacterales strains are the one of the  reason for resistance to -lactams. Then, owing to the hydrolysis of almost all -lactams, most serious issues to manage in treatment.  infections due to carbapenemase-producing Enterobacterales strains are the one of the Nacubactam (NAC: OP0595) is a novel β‐lactamase inhibitor that belongs to non‐β‐ most serious issues to manage in treatment. lactam diazabicyclooctane [11]. The agent can inhibit bacterial growth of Enterobacterales  Nacubactam (NAC: OP0595) is a novel -lactamase inhibitor that belongs to non- - expressing  some  classes  of β‐lactamases  (classes  A,  C,  and  D).  Comparing  protective  lactam diazabicyclooctane [11]. The agent can inhibit bacterial growth of Enterobacterales expr mecha essing nisms, some  NAC classes  has siof mila -lactamases r effects to other (classes  β‐laA, ctama C, and se inhib D). iComparing tors [11–13].pr Howe otective ver,  mechanisms, the agent has an NAC  inhib has itory similar  effect ef of fects  penic toill other in‐bi nd -lactamase ing protein inhibitors  2 (PBP2) of [11 Entero –13]. However bacterales ,  the too agent [11]. Ther has efore, an inhibitory  NAC no etf fect only of ha penici s direct llin-binding  antimicropr bial otein  effe2ct(PBP2) s, but al of soEnter  enhan obacterales cer effects  too of  co [11‐admin ]. Theriefor stered e, NAC  β‐lact not am only s  agai hasnst dir  Enterobacterales ect antimicrobial efha fects, ve  sev buteral also  cenhancer lasses  of β effects ‐lac‐ of co-administered -lactams against Enterobacterales have several classes of -lactamases tamases producing genes in an in vitro study [11–13].  producing genes in an in vitro study [11–13]. In our previous in vivo study with murine thigh infection model, combination ther‐ In our previous in vivo study with murine thigh infection model, combination ther- apies including β‐lactams (aztreonam (ATM), cefepime (FEP), and meropenem (MEM))  apies including -lactams (aztreonam (ATM), cefepime (FEP), and meropenem (MEM)) and NAC showed enhanced antibacterial activities against CRE pathogens [14]. However,  and NAC showed enhanced antibacterial activities against CRE pathogens [14]. However, these combination therapies have not been evaluated the antimicrobial activities against  these combination therapies have not been evaluated the antimicrobial activities against CRE− and/or CPE− derived pneumonia. Therefore, the antimicrobial efficacy of these com‐ CRE and/or CPE derived pneumonia. Therefore, the antimicrobial efficacy of these bination therapies against E. cloacae and K. pneumoniae were assessed with a murine pneu‐ combination therapies against E. cloacae and K. pneumoniae were assessed with a murine monia model (Figure 1).  pneumonia model (Figure 1). Figure 1. Study flow. MIC: minimum inhibitory concentration; CRE: carbapenem-resistant Enter- Figure 1. Study flow. MIC: minimum inhibitory concentration; CRE: carbapenem‐resistant Entero‐ obacterales; ATM: aztreonam; FEP: cefepime; MEM: meropenem, NAC: nacubactam, PK: pharma- bacterales; ATM: aztreonam; FEP: cefepime; MEM: meropenem, NAC: nacubactam, PK: pharma‐ cokinetics, PD: pharmacodynamics, ELF: epithelial lining fluid, %f T > MIC: the percentage of free cokinetics, PD: pharmacodynamics, ELF: epithelial lining fluid, %fT > MIC: the percentage of free  drug time above MIC. drug time above MIC.  2. Results 2. Results  2.1. The Minimum Inhibitory Concentrations (MICs) of ATM, FEP, MEM, and NAC 2.1. The Minimum Inhibitory Concentrations (MICs) of ATM, FEP, MEM, and NAC  Table 1 shows MICs of ATM, FEP, MEM, and NAC against the 10 study isolates. Table 1 shows MICs of ATM, FEP, MEM, and NAC against the 10 study isolates.  Among them, seven isolates are resistance to MEM according to the Clinical and Labo- Among them, seven isolates are resistance to MEM according to the Clinical and Labora‐ ratory Standards Institute (CLSI) breakpoints [15]. Combination regimens of ATM, FEP, tory Standards Institute (CLSI) breakpoints [15]. Combination regimens of ATM, FEP, and  and MEM with NAC showed 2- to >128-fold lower MICs, compared with those of - MEM with NAC showed 2‐ to >128‐fold lower MICs, compared with those of β‐lactam  lactam monotherapies against the CRE+ isolates. Additionally, these combination regimens monotherapies  against  the  CRE+  isolates.  Additionally,  these  combination  regimens  showed the same to >128-fold lower MICs than those of -lactam monotherapies against showed the same to >128‐fold lower MICs than those of β‐lactam monotherapies against  the CRE/CPE+ isolates. Same MICs were observed between combinations and monother- apies against CRE/CPE E. cloacae (20-5694). Antibiotics 2021, 10, 1179 3 of 11 Table 1. Characteristics of E. cloacae and K. pneumoniae isolates utilized in this study. MIC (g/mL) Types Species Strains Genotypes MEM MEM/NAC * FEP FEP/NAC * ATM ATM/NAC * NAC 13-4983 IMP-1, 4 1 * 8 2 * 0.12 0.03 * 2 E. cloacae 990235 IMP-1 64 8 * 64 4 * 64 1 * 8 CRE+/CPE+ ATCC KPC 8 0.25 * 32 1 * >128 1 * 2 BAA-1705 K. pneumoniae 13-3445 IMP-1 64 4 * 128 2 * 0.5 0.25 * 2 594 IMP-6 32 2 * 4 2 * 8 0.25 * >64 E. cloacae 16-0483 4 2 * 4 1 * 128 2 * 4 CRE+/CPE K. pneumoniae 16-2183 CTX-M-9 8 2 * >128 4 * >128 4 * >128 DHA-1, 990645 0.25 0.25 2 1 64 1 2 CRE/CPE+ K. pneumoniae IMP-1 K. pneumoniae ATCC700603 SHV-18 0.03 0.03 1 0.25 * >128 1 * 4 CRE/CPE E. cloacae 20-5694 0.03 0.03 0.03 0.03 0.12 0.12 4 MIC: minimum inhibitory concentration; CPE: carbapenemase-producing Enterobacterales; CRE: carbapenem-resistant Enterobac- terales; ATM: aztreonam; FEP: cefepime; MEM: meropenem, NAC: nacubactam. *: p < 0.05, comparing MIC values of corresponding -lactam monotherapy. 2.2. ATM, FEP, MEM, and NAC Pharmacokinetics (PK) in the Epithelial Lining Fluid (ELF) and Plasma ATM, FEP, MEM, and NAC PK profiles in ELF and plasma are depicted in Figure S1. In this study, single doses of NAC and each -lactam monotherapies were used at 100 mg/kg (subcutaneous doses). Table 2 shows the calculated PK parameters of the antimicrobials in plasma and ELF. Table 2. PK parameters in ELF and plasma in murine pneumonia model. Dose Cmax AUC0–¥ Vd/F CL/F Antimicrobials Tmax (h) T1/2 (h) (mg/kg) (g/mL) (g h/mL) (L/kg) (L/h/kg) In ELF Nacubactam 100 0.5 31 39 1.63 NA NA Meropenem 100 0.5 3 6 0.69 NA NA Cefepime 100 1 18 32 0.84 NA NA Aztreonam 100 0.5 16 27 0.94 NA NA In plasma Nacubactam 100 0.25 116 131 0.61 0.67 0.76 Meropenem 100 0.25 53 36 0.4 1.6 2.79 Cefepime 100 0.25 80 84 0.41 0.71 1.19 Aztreonam 100 0.5 96 120 0.36 0.43 0.83 PK: pharmacokinetic; ELF: epithelial lining fluid; C : maximum drug concentration; T : time to reach maximum plasma concentration; max max AUC : AUC in ELF or plasma from time 0 to ¥; T : half-life; CL/F: apparent clearance; Vd/F: apparent distribution volume; NA: 0–¥ 1/2 not applicable. 2.3. Pharmacodynamic (PD) Study with Murine Pneumonia Model 2.3.1. Antimicrobial Efficacies of NAC Monotherapy At the start of antimicrobial therapies (0 h), bacterial counts in lungs of control mice were between 6.79 to 7.00 log colony forming units (cfu)/lungs for E. cloacae and 6.59 to 7.21 log cfu/lungs for K. pneumoniae. Bacterial number differences of growth control were between 0.06 to 2.58 Dlog CFU/lungs for E. cloacae and 0.77 to 2.74 Dlog CFU/lungs 10 10 for K. pneumoniae after 24 h from the start of antimicrobial therapies. Bacterial number differences of NAC monotherapy (320, 160, and 80 mg/kg q8h) were between 2.27 and 1.19 Dlog CFU/lungs for four E. cloacae isolates and 1.34 to 2.66 Dlog CFU/lungs for 10 10 six K. pneumoniae isolates (Figure S2). Among total 10 study isolates, six isolates showed higher bacterial numbers in lungs at 24 h than at 0 h, while maximum dosage of NAC was adopted (320 mg/kg q8h). Antibiotics 2021, 10, 1179 4 of 11 2.3.2. Antimicrobial Efficacies against CRE+ Isolates Antibiotics 2021, 10, x FOR PEER REVIEW  4 of 11  Bacterial number differences in K. pneumoniae-infected mice received combination therapies were between 4.24 and 1.47 Dlog CFU/lungs (Figure 2) and 3.70 and 2.08 Dlog CFU/lungs for E. cloacae-infected mice (Figure 3). Compared with the corre- bacterial  numbers  in  lungs  at  24  h  than  at  0  h,  while  maximum  dosage  of  NAC  was  sponding -lactam monotherapies, combination therapies showed higher antimicrobial adopted (320 mg/kg q8h).  activities against CRE+ isolates (p < 0.05), except when combined with MEM + NAC against CRE+/CPE isolate (E. cloacae 16-0483) and combined with ATM + NAC against 2.3.2. Antimicrobial Efficacies against CRE+ Isolates  CRE+/CPE+ isolate (13-4983). Additionally, the NAC dose-dependent antimicrobial activ- ity of combination therapy with NAC and ATM against CRE+/CPE isolate (K. pneumoniae Bacterial number differences in  K. pneumoniae‐infected mice received combination  16-2183) was observed. When ATM, FEP, and MEM were combined with NAC, similar therapies were between −4.24 and 1.47 Δlog10 CFU/lungs (Figure 2) and −3.70 and −2.08  trends of antimicrobial activity were also observed against CRE+/CPE+ isolate (K. pneumo- Δlog10 CFU/lungs for E. cloacae‐infected mice (Figure 3). Compared with the correspond‐ niae ATCC BAA-1705). Moreover, while most combination therapies showed bacteriostatic ing β‐lactam monotherapies, combination therapies showed higher antimicrobial activi‐ (3.0 to 0 Dlog CFU/lungs) to bactericidal (<3.0 Dlog CFU/lungs) activities against 10 10 ties against CRE+ isolates (p < 0.05), except when combined with MEM + NAC against  CRE isolates, bacterial counts in lungs of MEM + NAC combination therapy group against CRE+/CPE−  isolate  (E.  cloacae  16‐0483)  and  combined  with  ATM  +  NAC  against  K. pneumoniae (594) were higher at 24 h than at 0 h in the control (>0 Dlog CFU/lungs). CRE+/CPE+ isolate (13‐4983). Additionally, the NAC dose‐dependent antimicrobial activ‐ ity of combination therapy with NAC and ATM against CRE+/CPE− isolate (K. pneumoniae  2.3.3. Antimicrobial Efficacies against CRE Isolates 16‐2183) was observed. When ATM, FEP, and MEM were combined with NAC, similar  Bacterial number differences in combination therapies were between 3.56 and trends of antimicrobial activity were also observed against CRE+/CPE+ isolate (K. pneu‐ 2.81 Dlog CFU/lungs for E. cloacae and 4.46 and 1.70 Dlog CFU/lungs for 10 10 moniae ATCC BAA‐1705). Moreover, while most combination therapies showed bacterio‐ K. pneumoniae (Figure 4). Comparing with the corresponding -lactam monotherapies, combination therapies showed enhanced antimicrobial activities, when combined with FEP static  (−3.0  to  0 Δlog10  CFU/lungs)  to  bactericidal  (<−3.0 Δlog10  CFU/lungs)  activities  and AZT with NAC against CRE/CPE (K. pneumoniae ATCC700603) and CRE/CPE+ against CRE isolates, bacterial counts in lungs of MEM + NAC combination therapy group  (K. pneumoniae 990645). However, NAC did not affect antimicrobial activity of MEM ther- against  K.  pneumoniae  (594)  were  higher  at  24  h  than  at  0  h  in  the  control  (>0 Δlog10  apy against these isolates. Moreover, no enhanced antimicrobial activity was found in CFU/lungs).  combination therapies against E. cloacae (20-5694). Figure 2. Antimicrobial efficacy of study regimens against CRE+ Klebsiella pneumoniae isolates. Figure 2. Antimicrobial efficacy of study regimens against CRE+ Klebsiella pneumoniae isolates.  Control (), -lactam (aztreonam, cefepime, meropenem) monotherapy (), add nacubactam Control (□), β‐lactam (aztreonam, cefepime, meropenem) monotherapy (■), add nacubactam 80  80 mg/kg q8h (), add nacubactam 160 mg/kg q8h (), add nacubactam 320 mg/kg q8h (). mg/kg q8h (■), add nacubactam 160 mg/kg q8h (■), add nacubactam 320 mg/kg q8h (■). White bar  White bar control applies to all the drug combinations shown. *: vs. control p < 0.05, : vs. -lactam control applies to all the drug combinations shown. *: vs. control p < 0.05,  : vs. β‐lactam monother‐ monotherapy p < 0.05, [: vs. add nacubactam 80 mg/kg q8h p < 0.05. CRE: carbapenem-resistant apy p < 0.05, ♭: vs. add nacubactam 80 mg/kg q8h p < 0.05. CRE: carbapenem‐resistant Enterobac‐ Enterobacterales, CPE: carbapenemase-producing Enterobacterales, Aztreonam: ATM, cefepime: FEP, terales, CPE: carbapenemase‐producing Enterobacterales, Aztreonam: ATM, cefepime: FEP, mero‐ meropenem: MEM. All data are shown as average  SD. penem: MEM. All data are shown as average ± SD.  Antibiotics 2021, 10, x FOR PEER REVIEW  5 of 11  Antibiotics 2021, 10, x FOR PEER REVIEW  5 of 11  Antibiotics 2021, 10, 1179 5 of 11 Figure 3. Antimicrobial efficacy of study regimens against CRE+ Enterobacter cloacae isolates. Con‐ trol (□), β‐lactam (aztreonam, cefepime, meropenem) monotherapy (■), add nacubactam 80  mg/kg q8h (■), add nacubactam 160 mg/kg q8h (■), add nacubactam 320 mg/kg q8h (■). White  bar control applies to all the drug combinations shown. *: vs. control p < 0.05,  : vs. β‐lactam mono‐ therapy p < 0.05. CRE: carbapenem‐resistant Enterobacterales, CPE: carbapenemase‐producing  Enterobacterales, Aztreonam: ATM, cefepime: FEP, meropenem: MEM. All data are shown as av‐ erage ± SD.  2.3.3. Antimicrobial Efficacies against CRE− Isolates  Bacterial number differences in combination therapies were between −3.56 and −2.81  Δlog10 CFU/lungs for E. cloacae and −4.46 and −1.70 Δlog10 CFU/lungs for K. pneumoniae  (Figure  4).  Comparing  with  the  corresponding β‐lactam  monotherapies,  combination  Figure 3. Antimicrobial efficacy of study regimens against CRE+ Enterobacter cloacae isolates. Control Figure 3. Antimicrobial efficacy of study regimens against CRE+ Enterobacter cloacae isolates. Con‐ therapies showed enhanced antimicrobial activities, when combined with FEP and AZT  (), -lactam (aztreonam, cefepime, meropenem) monotherapy (), add nacubactam 80 mg/kg q8h trol (□), β‐lactam (aztreonam, cefepime, meropenem) monotherapy (■), add nacubactam 80  with NAC against CRE−/CPE− (K. pneumoniae ATCC700603) and CRE−/CPE+ (K. pneu‐ (), add nacubactam 160 mg/kg q8h (), add nacubactam 320 mg/kg q8h (). White bar control mg/kg q8h (■), add nacubactam 160 mg/kg q8h (■), add nacubactam 320 mg/kg q8h (■). White  moniae  990645).  However,  NAC  did  not  affect  antimicrobial  activity  of  MEM  therapy  applies to all the drug combinations shown. *: vs. control p < 0.05, : vs. -lactam monotherapy bar control applies to all the drug combinations shown. *: vs. control p < 0.05,  : vs. β‐lactam mono‐ against these isolates. Moreover, no enhanced antimicrobial activity was found in combi‐ p < 0.05. CRE: carbapenem-resistant Enterobacterales, CPE: carbapenemase-producing Enterobacterales, therapy p < 0.05. CRE: carbapenem‐resistant Enterobacterales, CPE: carbapenemase‐producing  nation therapies against E. cloacae (20‐5694).  Aztreonam: ATM, cefepime: FEP, meropenem: MEM. All data are shown as average  SD. Enterobacterales, Aztreonam: ATM, cefepime: FEP, meropenem: MEM. All data are shown as av‐ erage ± SD.  2.3.3. Antimicrobial Efficacies against CRE− Isolates  Bacterial number differences in combination therapies were between −3.56 and −2.81  Δlog10 CFU/lungs for E. cloacae and −4.46 and −1.70 Δlog10 CFU/lungs for K. pneumoniae  (Figure  4).  Comparing  with  the  corresponding β‐lactam  monotherapies,  combination  therapies showed enhanced antimicrobial activities, when combined with FEP and AZT  with NAC against CRE−/CPE− (K. pneumoniae ATCC700603) and CRE−/CPE+ (K. pneu‐ moniae  990645).  However,  NAC  did  not  affect  antimicrobial  activity  of  MEM  therapy  against these isolates. Moreover, no enhanced antimicrobial activity was found in combi‐ nation therapies against E. cloacae (20‐5694).  Figure 4. Antimicrobial efficacy of study regimens against CRE isolates. Control (), -lactam (aztreonam, cefepime, meropenem) monotherapy (), add nacubactam 80 mg/kg q8h (), add Figure 4. Antimicrobial efficacy of study regimens against CRE− isolates. Control (□), β‐lactam  nacubactam 160 mg/kg q8h (), add nacubactam 320 mg/kg q8h (). White bar control ap- (aztreonam, cefepime, meropenem) monotherapy (■), add nacubactam 80 mg/kg q8h (■), add  plies to all the drug combinations shown. *: vs. control p < 0.05, : vs. -lactam monotherapy nacubactam 160 mg/kg q8h (■), add nacubactam 320 mg/kg q8h (■). White bar control applies to  p < 0.05. CRE: carbapenem-resistant Enterobacterales, CPE: # carbapenemase-producing Enterobac- all the drug combinations shown. *: vs. control p < 0.05,  : vs. β‐lactam monotherapy p < 0.05. CRE:  terales, Aztreonam: ATM, cefepime: FEP, meropenem: MEM. All data shown as average  SD. carbapenem‐resistant Enterobacterales, CPE: carbapenemase‐producing Enterobacterales, Aztre‐ onam: ATM, cefepime: FEP, meropenem: MEM. All data shown as average ± SD.  2.4. Relationships between Antimicrobial Activities and the Percentage of Free Drug Time above MIC (%fT > MIC) 2.4. Relationships between Antimicrobial Activities and the Percentage of Free Drug Time above  Figure 5 shows the relationships between Dlog CFU/lungs and %fT > MIC against MIC (%fT > MIC)  study isolates. The %fT > MIC in ELF showed higher correlations with Dlog CFU/lungs (R = 0.88 for ATM, 0.75 for FEP, 0.45 for MEM). Similarly, the %fT > MIC in plasma was also correlated with Dlog CFU/lungs well (R = 0.89 for ATM, 0.74 for FEP, 0.55 for MEM). Along with the elevation of %fT > MIC value, the bacterial density was reduced after 24 h Figure 4. Antimicrobial efficacy of study regimens against CRE− isolates. Control (□), β‐lactam  of antimicrobial treatments. (aztreonam, cefepime, meropenem) monotherapy (■), add nacubactam 80 mg/kg q8h (■), add  nacubactam 160 mg/kg q8h (■), add nacubactam 320 mg/kg q8h (■). White bar control applies to  all the drug combinations shown. *: vs. control p < 0.05,  : vs. β‐lactam monotherapy p < 0.05. CRE:  carbapenem‐resistant Enterobacterales, CPE: carbapenemase‐producing Enterobacterales, Aztre‐ onam: ATM, cefepime: FEP, meropenem: MEM. All data shown as average ± SD.  2.4. Relationships between Antimicrobial Activities and the Percentage of Free Drug Time above  MIC (%fT > MIC)  Antibiotics 2021, 10, x FOR PEER REVIEW  6 of 11  Figure 5 shows the relationships between Δlog10 CFU/lungs and %fT > MIC against  study isolates. The %fT > MIC in ELF showed higher correlations with Δlog10 CFU/lungs  (R  = 0.88 for ATM, 0.75 for FEP, 0.45 for MEM). Similarly, the %fT > MIC in plasma was  also correlated with Δlog10 CFU/lungs well (R  = 0.89 for ATM, 0.74 for FEP, 0.55 for MEM).  Antibiotics 2021, 10, 1179 6 of 11 Along with the elevation of %fT > MIC value, the bacterial density was reduced after 24 h  of antimicrobial treatments.  Figure 5. Relationships between %fT > MIC of -lactams and Dlog CFU/lungs at 24 h. An inhibitory effect sigmoid Imax Figure 5. Relationships between %fT > MIC of β‐lactams and Δlog10 CFU/lungs at 24 h. An inhibitory effect sigmoid Imax  model was adapted to reveal the correlations between %fT > MIC of each drug and Dlog CFU/lungs of each antimicrobial model was adapted to reveal the correlations between %fT > MIC of each drug and Δlog10 CFU/lungs of each antimicrobial  regimen with Phoenix WinNonlin software (ver. 8.1; Certara, L.P.). The 2 R represents the coefficient of determination. regimen with Phoenix WinNonlin software (ver. 8.1; Certara, L.P.). The R  represents the coefficient of determination. %fT  %fT > MIC: the percentage of free drug time above MIC, ATM: aztreonam, FEP: cefepime, MEM: meropenem, > MIC: the percentage of free drug time above MIC, ATM: aztreonam, FEP: cefepime, MEM: meropenem, NAC: nacubac‐ tam. NAC:  nacubactam. 3. Discussion 3. Discussion  NAC has a dual mechanism of action. The agent inhibits some classes of serine NAC has a dual mechanism of action. The agent inhibits some classes of serine β‐ -lactamases and PBP2 in Enterobacterales [11,16,17]. In our previous study, the same lactamases and PBP2 in Enterobacterales [11,16,17]. In our previous study, the same com‐ combination therapies including NAC and -lactams showed enhanced antimicrobial ac- bination therapies including NAC and β‐lactams showed enhanced antimicrobial activi‐ tivities against Enterobacterales have some carbapenemase-producing genes with a murine ties against Enterobacterales have some carbapenemase‐producing genes with a murine  thigh infection model [14]. However, these combination therapies have not been evalu- thigh infection model [14]. However, these combination therapies have not been evalu‐ ated against CRE and CPEcaused pneumonia. In this in vitro study, the combination ated against CRE− and CPE−caused pneumonia. In this in vitro study, the combination  regimens of -lactams with NAC showed 1- to >128-fold lower MICs against CPE and regimens of β‐lactams with NAC showed 1‐ to >128‐fold lower MICs against CPE and  CRE isolates, comparing with the corresponding -lactam monotherapies (Table 1). As CRE isolates, comparing with the corresponding β‐lactam monotherapies (Table 1). As  previous in vitro study suggested, our results also suggested that NAC enhanced in vitro previous in vitro study suggested, our results also suggested that NAC enhanced in vitro  antimicrobial activities of -lactams against several classes of -lactamase-producing antimicrobial activities of β‐lactams against several classes of β‐lactamase‐producing E.  E. cloacae and K. pneumoniae isolates [11–13]. cloacae and K. pneumoniae isolates [11–13].  Moreover, in this pharmacodynamics evaluation, NAC monotherapy could not show Moreover, in this pharmacodynamics evaluation, NAC monotherapy could not show  a bactericidal effect against all study isolates (Figure S2). In contrast, combination therapies a bactericidal effect against all study isolates (Figure S2). In contrast, combination thera‐ showed enhanced antimicrobial activities against CRE+ E. cloacae and K. pneumoniae isolates pies showed enhanced antimicrobial activities against CRE+ E. cloacae and K. pneumoniae  (Figures 2 and 3). At least one regimen among combination therapies of ATM, FEP, and isolates (Figures 2 and 3). At least one regimen among combination therapies of ATM,  MEM with NAC showed bactericidal effects against all CRE+ E. cloacae and K. pneumoniae isolates, excluding the K. pneumoniae isolate (594). Similar to our previous study using a murine thigh infection model [14], combina- tion therapy of MEM and NAC was not effective (>0Dlog CFU/lungs) against CRE+ K. pneumoniae (594) (Figure 2). The isolate has the IMP-6 gene and showed low susceptibil- ity to MEM and NAC, whereas MEM + NAC resulted in relatively lower MIC (2 g/mL) (Table 1). In vitro studies of the other group conducted showed that the efficacy of NAC is Antibiotics 2021, 10, 1179 7 of 11 restricted to bacteria with IMP-type carbapenemase-producing genes [11–13]. NAC only binds to PBP2. On the other hand, ATM and FEP bind to PBP3 rather than PBP2 [11]. Thus, the NAC-derived enhancer effect was expected when the agent was co-administrated with ATM and FEP than with MEM. Furthermore, we could speculate that NAC was not effective to work as an PBP2 inhibitor to CRE+ K. pneumoniae (594) as the isolate has low susceptibility to both MEM and NAC. Although further study to clarify the mechanism is needed, MEM + NAC combination therapy against IMP-type carbapenemase-producing Enterobacterales could be unsuitable, especially when causative bacteria have high MICs (low susceptibilities) of MEM and NAC. In general, optimizing the antimicrobial dosage according to the PK/PD theory is one of the important components to improve infected patient outcomes. As the PK/PD breakpoint of NAC has not been fully evaluated, it was difficult to interpret our PD study results. However, the antimicrobial activities of -lactams correlate with %f T > MIC well [18,19]. NAC co-administration had no impact on PK profiles of -lactams (ATM, FEP, and MEM) in plasma [14]. Additionally, we did not observe significant differ- ences in the PK data of plasma between the thigh infection model and pneumonia model [14, Figure S1]. Due to the fact that the reappearance of human PK profiles of antimicro- bials was difficult for us, as mice have higher clearance ability than that of humans, we used the same dosage regimens as previous in vivo studies [14] to obtain similar areas under the free drug concentration-time curves (f AUC) when -lactams (ATM, FEP, and MEM) were dosed at 1 g q8h, and NAC (2, 1, and 0.5 g q8h) [20–26]. Using calculated PK parameters, we conducted PK/PD analysis and found the good relationships between %f T > MIC of -lactams (ATM, FEP, and MEM) in ELF and plasma and antimicrobial activities (Figure 5). Our data will give important insights to explore the optimal dosage regimen for pneumonia patients. The combination therapy of -lactams (ATM, FEP, and MEM) and NAC was highly effective against CPE and CRE caused pneumonia. However, we evaluated limited classes of carbapenemases producing Enterobacteriales in this study. In Japan, IMP-1 and -6 type CPE isolates are the most frequently detected [7,8]. However, there are regional differences of the -lactamases prevalence in Enterobacterales [7,9,27]. Therefore, further studies are needed. As the other limitation, we did not evaluate inoculum effects, while we evaluated antimicrobial activities with similar inoculum size to previous report [28–30]. Although our study has a few limitations, this is the first in vivo study to evaluate antimicrobial activities of combination therapies including -lactams and NAC with pneu- monia model. Our PD data suggested combination therapied including NAC is a potent candidate for CRE caused pneumonia. This fact can give new therapeutic choice to antimi- crobial treatment for CRE caused pneumonia, while the prevalence and burden of CRE infection are rising. Additionally, we revealed the relationship between these antimicrobial activities and antimicrobial PK data (in plasma and ELF). Therefore, our data would be a preferable reference to explore optimal antimicrobial dosages in the future. 4. Materials and Methods 4.1. Antimicrobials To determine the antimicrobial concentrations in ELF and plasma, we used analytical- grade ATM (lot LFSZK: Tokyo Chemical Industry Co., Ltd., Tokyo, Japan), FEP (lot I0L200USP: USP) and MEM hydrate (lot YCY8L: Tokyo Chemical Industry Co., Ltd.). Meiji Seika Pharma Co., Ltd. (Tokyo, Japan) provided us analytical-grade NAC (lot 510- 015-4097-01). For all in vivo study, ATM (Eisai Co., Ltd., Tokyo, Japan), FEP (Bristol-Myers Squibb K.K., Tokyo, Japan), MEM (Meiji Seika Pharma Co., Ltd.), and NAC (lot BS1807SB03) were used. Stock solutions of ATM, FEP, and MEM were stored at 4 C. The stock solutions were reconstituted with normal saline (NS) at desired concentrations. Antibiotics 2021, 10, 1179 8 of 11 4.2. Microorganisms Four E. cloacae and six K. pneumoniae isolates were used in this study (Table 1). Clinical isolates of E. cloacae and K. pneumoniae were provided from department of Microbial laboratory in Aichi Medical University Hospital. CRE isolates have MEM MIC  4 g/mL. CPE isolates have any detectable carbapenemase genes. According to previous our study methods [14], direct sequencing was conducted to screen carbapenemase and other - lactamase-encoding genes. 4.3. Susceptibility Test The antimicrobial susceptibilities of ATM, FEP, MEM, and NAC were determined with the broth microdilution method, following recommended methods by CLSI [15]. For the combination regimens, the broth microdilution method was conducted in fixed concentra- tion ratios of each -lactam and NAC (1:1 (w/w)), according to CLSI method (M100-ED31: Table 5A-2). The -lactam concentration was used to show MICs in combination thera- pies. The MIC studies were conducted a minimum of three times, and the geometric MIC was reported. 4.4. Animals Pathogen-free, ICR mice (4-week-old) were acquired from Charles River Laboratories Japan, Inc. (Yokohama, Japan). They were provided food and water ad libitum. They were housed and used following the National Research Council recommendations. 4.5. Pneumonia Model Neutropenic murine pneumonia model was used to evaluate antimicrobial activities of various antimicrobial regimens against E. cloacae and K. pneumoniae in the lungs of mice [30]. Mice were rendered transiently neutropenic with cyclophosphamide of intraperitoneal doses at Day-4 (150 mg/kg) and Day-1 (100 mg/kg) before inoculation. Study isolates for inoculation were stored at 80 C. A bacterial suspension was prepared at approxi- mately 10 cfu/mL with study isolates after 24 h incubation of the second transfer. Under anesthesia, mice were orally instilled the bacterial suspension (0.075 mL), and the nares were blocked while being held vertically for 60 s to aspirate the suspension into the lungs. After inoculation, mice were randomly divided into control or each antimicrobial treatment group (n = 6, respectively). 4.6. PK Studies For the PK study of ATM, FEP, MEM, and NAC in ELF and plasma, each antimi- crobial was dosed subcutaneously (single dose) as monotherapies to the infected mice. The neutropenic pneumonia mice were infected with K. pneumoniae ATCC43816. The mice were injected with ATM, FEP, MEM, or NAC (at 100 mg/kg, respectively). Mice were sacrificed at each time point (0.25, 0.5, 1, 2, 3, 4, and 5 h). Then, blood samples from the axillary artery and bronchoalveolar lavage fluid (BALF) samples were col- lected (n = 3 in each group). After the detection of urea concentration in each BALF and plasma sample with a urea assay kit (Urea Assay Kit, BioChain Institute, Inc., Newark, CA, USA), the antimicrobial concentrations in ELF were calculated: ELF antimicrobial concentration = BALF antimicrobial concentration  (the ratio of urea concentrations in plasma/BALF). AUC in ELF and plasma from time 0 to ¥ (AUC0–¥) were calculated according to the trapezoidal method. The PK parameters in the ELF and plasma were calculated with Phoenix WinNonlin software (ver. 8.1; Certara, L.P.). 4.7. Instrumentation and Chromatographic Conditions Liquid chromatography-tandem mass spectrometry (LC-MS/MS) was used to analyze ATM, FEP, MEM, and NAC concentrations at Meiji Seika Pharma Co., Ltd. Detailed conditions and procedures are in the Supplementary document. Antibiotics 2021, 10, 1179 9 of 11 4.8. PD Study One hour after inoculation of study isolates, mice received antimicrobials (0.2 mL) subcutaneously (n = 6 in each group). We used same dosage of -lactams and NAC with our previous in vivo study [14] to be similar f AUC values of NAC (2.0, 1.0, and 0.5 g), and -lactams (1.0 g) in blood with clinical PK study data. The following eight-hourly (q8h) doses were evaluated: 700 mg/kg for ATM, 260 mg/kg for FEP, and 140 mg/kg for MEM, 320, 160, and 80 mg/kg for NAC. Control mice were administered NS (0.2 mL). Twenty-four hours after antimicrobial treatments started, lungs were harvested from mice. Then, mice were sacrificed by CO exposure. Removed lungs from each mouse were homogenized individually, and the homogenates were plated on trypticase soy agar with 5% sheep blood after serial dilutions with NS to determine the bacterial counts in lungs (cfu/lungs). In addition, six infected mice were harvested just before the start of antimicrobial therapies to know bacterial number in lungs at 0 h. The antimicrobial efficacies were defined as follows: detected bacterial counts in the treated group at 24 h and bacterial counts in the control group at 0 h (Dlog CFU/lungs). 4.9. PK/PD Analysis The %T > MIC in ELF and plasma for unbound ATM, FEP, MEM, and NAC was calculated with PK parameters (Table S1 and Table S2) in pneumonia mice, serum protein binding ratio [14,20–22], and MIC values, with the R software (ver. 3.6.1). For combination therapies, MICs of combinations were used. An inhibitory effect sigmoid I model was max adapted to reveal the correlations between %f T > MIC of each drug and Dlog CFU/lungs of each antimicrobial regimen with Phoenix WinNonlin software (ver. 8.1; Certara, L.P.). To estimate the percentage of variance in efficacy, the coefficient of determination (R ) was used. 4.10. Statistical Analysis Mann–Whitney U test was used to compare in vitro MIC values of each -lactam monotherapy and corresponding combination therapies. To compare the in vivo antimicro- bial efficacy between the regimens, one-way ANOVA with Bonferroni correction was used. Statistical analysis was performed with JMP, version 10.0 (SAS, Tokyo, Japan). Differences were considered statistically significant at p < 0.05. 5. Conclusions Combination therapies including ATM, FEP, and MEM with NAC showed potent in vivo antimicrobial activities against pneumonia caused by CRE and CPE E. cloacae and K. pneumoniae. These translational data support the potential role of NAC in combination with ATM, FEP, and MEM as a therapy for CRE and CPE K. pneumoniae and E. cloacae caused human pneumonia. Supplementary Materials: The following are available online at https://www.mdpi.com/article/ 10.3390/antibiotics10101179/s1, Figure S1: In vivo pharmacokinetic data of nacubactam, meropenem, cefepime, and aztreonam in ELF and plasma, Figure S2: In vivo efficacy of nacubactam monotherapy, Table S1: Apparent pharmacokinetic parameters in ELF in murine pneumonia model for calculation of f T > MIC, Table S2: Pharmacokinetic parameters in plasma in murine pneumonia model for calculation of %f T > MIC. Author Contributions: Conceptualization, H.M., H.O., T.S. and M.H.; data curation, T.S., H.O. and M.H.; formal analysis, N.S., T.S. and M.H.; investigation, M.H., H.K., Y.S., D.S., J.H., N.A., H.S., Y.Y. and N.A.; project administration, M.H. and H.M.; resources, H.M.; supervision, H.M.; validation, T.S.; writing—original draft, M.H.; writing—review and editing, T.S. and H.M. All authors have read and agreed to the published version of the manuscript. Funding: This work was supported by AMED (grant number JP18pc0101028). Institutional Review Board Statement: The ethics committee of Aichi medical university reviewed this study protocol and approved (2020-30). To conduct PK studies of -lactams and NAC, the animal Antibiotics 2021, 10, 1179 10 of 11 experiment management committee and pharmaceutical research laboratories at Meiji Seika Pharma Co., Ltd. reviewed this study protocol and approved (C2020-010 and C2020-013). Informed Consent Statement: Not applicable. Data Availability Statement: The datasets analyzed during this study are available and can be obtained, at request, on reasonable enquiry. Conflicts of Interest: T.S., H.O. and N.S. are employees of Meiji Seika Pharma Co., Ltd., Japan. H.M. received partial research funding from Meiji Seika Pharma Co., Ltd., Japan. The authors declare no conflict of interest. References 1. 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Journal

Antibiotics – Multidisciplinary Digital Publishing Institute

Published: Sep 28, 2021

Keywords: aztreonam; cefepime; meropenem; nacubactam; carbapenemase-producing Enterobacterales; carbapenem-resistant Enterobacterales; Enterobacter cloacae; Klebsiella pneumoniae; pneumonia

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