ABSTRACT
Tebipenem pivoxil is the first orally available carbapenem antibiotic and has been approved in Japan for treating ear, nose, and throat and respiratory infections in pediatric patients. Its active moiety, tebipenem, has shown potent antimicrobial activity in vitro against clinical isolates of Enterobacterales species from patients with urinary tract infections (UTIs), including those producing extended-spectrum β-lactamases (ESBLs) and/or AmpC β-lactamase. In the present study, tebipenem was tested for stability to hydrolysis by a set of clinically relevant β-lactamases, including TEM-1, AmpC, CTX-M, OXA-48, KPC, and NDM-1 enzymes. In addition, hydrolysis rates of other carbapenems, including imipenem, meropenem, and ertapenem, were determined for comparison. It was found that, similar to other carbapenems, tebipenem was resistant to hydrolysis by TEM-1, CTX-M, and AmpC β-lactamases but was susceptible to hydrolysis by KPC, OXA-48, and NDM-1 enzymes with catalytic efficiency values (kcat/Km) ranging from 0.1 to 2 × 106 M−1s−1. This supports the reported results of antimicrobial activity of tebipenem against ESBL- and AmpC-producing but not carbapenemase-producing Enterobacterales isolates. Considering that CTX-M and AmpC β-lactamases represent the primary determinants of multidrug-resistant complicated UTIs (cUTIs), the stability of tebipenem to hydrolysis by these enzymes supports the utility of its prodrug tebipenem, tebipenem pivoxil hydrobromide (TBP-PI-HBr), as an oral therapy for adult cUTIs.
KEYWORDS: ESBL, β-lactamases, AmpC, carbapenems, cUTI, tebipenem, SPR994, tebipenem pivoxil hydrobromide
INTRODUCTION
Urinary tract infections (UTIs) are among the most frequent infectious diseases affecting humans and represent an important public health problem with a substantial economic burden (1). They are caused primarily by Gram-negative bacteria, among which Enterobacterales species such as Escherichia coli, Klebsiella pneumoniae, and Proteus mirabilis are prominent (1). Complicated UTIs (cUTIs) occur in patients with a structural or functional abnormality of the genitourinary tract (2).
β-Lactam antibiotics, including penicillins, cephalosporins, carbapenems, and monobactams, have been the mainstay in combating bacterial infections, including cUTIs (1, 2). However, their efficacy has been challenged by the occurrence of resistant Enterobacterales species in patients with cUTIs. In these bacteria, resistance occurs primarily through the expression of β-lactamases, the enzymes that inactivate β-lactam antibiotics via hydrolysis of the β-lactam ring. Among various β-lactamases associated with cUTIs, extended-spectrum β-lactamases (ESBLs) are of particular concern as they are prevalent in cUTIs and capable of hydrolyzing penicillins, cephalosporins, and monobactams (1, 2). Therefore, ESBL-producing organisms are resistant to virtually all β-lactam agents except carbapenems. In addition, 55% to 100% of Gram-negative pathogens producing ESBLs have been reported to be coresistant to fluroquinolones and other antimicrobials that are often used to treat cUTIs (3, 4).
Carbapenems remain the “antibiotics of last resort” for the treatment of multidrug-resistant (MDR) cUTIs due to their broad-spectrum antimicrobial activity and stability to hydrolysis by ESBLs (5). However, they are available only as intravenous therapeutics for such indications. This has led to a growing number of hospitalizations and extended lengths of stay for patients with cUTIs. Therefore, orally available carbapenems are warranted to facilitate the management of cUTIs in ambulatory care settings.
Tebipenem is the first oral carbapenem with tebipenem pivoxil as the prodrug and has been marketed in Japan since 2009 to treat pediatric otitis media, sinusitis, and pneumonia (Fig. 1) (6). Tebipenem was recently shown to be potent in inhibiting clinical isolates of Enterobacterales species that cause adult UTIs, including those producing ESBLs and/or AmpC enzymes, another β-lactamase that is prevalent in cUTIs and capable of hydrolyzing penicillins and cephalosporins (7). A new oral formulation of tebipenem, tebipenem pivoxil hydrobromide (TBP-PI-HBr, formerly SPR994), is under development for treatment of adult cUTIs/acute pyelonephritis (AP) (8, 9).
FIG 1.
Structure of tebipenem.
In order to understand the molecular mechanism for its antimicrobial activity, tebipenem was tested for stability to hydrolysis by a set of β-lactamases that are often found in organisms causing cUTIs, including ESBLs and carbapenemases. In addition, other clinically available carbapenems were used as comparators. It was found that, similar to other carbapenems, tebipenem was stable to hydrolysis by TEM-1, AmpC, and CTX-M enzymes but was susceptible to hydrolysis by OXA-48, KPC, and NDM-1 enzymes with catalytic efficiency (kcat/Km) values ranging from 0.1 × 106 to 2 × 106 M−1s−1. Therefore, the β-lactamase stability data of tebipenem together with in vitro antimicrobial activity of tebipenem and pharmacokinetics/pharmacodynamics (PK-PD) of TBP-PI-HBr support the development of TBP-PI-HBr as an oral drug to treat adult cUTI/AP.
RESULTS
Hydrolysis of tebipenem by TEM-1, AmpC, and CTX-M enzymes.
As the prototype serine β-lactamase, the TEM-1 enzyme is known to be very efficient in hydrolyzing penicillins but deficient in hydrolyzing oxyimino-cephalosporins and carbapenems (10). Consistent with this spectrum, we found that TEM-1 exhibited no or very low enzyme activity in hydrolyzing ceftazidime, ceftibuten, or any carbapenems. The low catalytic efficiency (kcat/Km) was attributed primarily to a low turnover rate (kcat), although hydrolysis of ceftazidime also showed a high Km value.
AmpC, together with ESBLs such as CTX-M-14 and CTX-M-15, represents clinically important cephalosporinases (11, 12). We found that these enzymes exhibit significantly higher catalytic efficiency than the TEM-1 enzyme in hydrolyzing both ceftazidime and ceftibuten (Table 1). However, similar to the TEM-1 enzyme, these enzymes displayed negligible hydrolysis activity against carbapenems (Table 1). This accounts for the in vitro antimicrobial activity of tebipenem and other carbapenems in inhibiting Enterobacterales species that produce AmpC and/or ESBLs. The CTX-M-15 enzyme hydrolyzes ceftazidime with a kcat/Km approximately 10-fold higher than that observed for CTX-M-14 (Table 1). CTX-M-15 contains a D240G substitution compared to CTX-M-14, and it has been shown that the addition of this substitution to the CTX-M-14 enzyme increases kcat/Km by 10-fold (13). Therefore, the D240G substitution is likely responsible for the observed higher kcat/Km values for ceftazidime hydrolysis by CTX-M-15. In addition, the P167S substitution is found in CTX-M variant enzymes that display higher kcat/Km values for ceftazidime hydrolysis (14). However, the addition of the P167S substitution to CTX-M-15 reduces ceftazidime hydrolysis levels (Table 1). This result is consistent with the previous finding that the P167S and D240G substitutions are antagonistic. Each substitution alone increases ceftazidime hydrolysis, but the combination results in lower levels of hydrolysis than observed for either of the single mutants (13).
TABLE 1.
β-Lactam hydrolysis by TEM-1, AmpC, and CTX-M enzymes
| Substrate | Parameter | Value for enzyme: |
||||
|---|---|---|---|---|---|---|
| TEM-1 | AmpC | CTX-M-14 | CTX-M-15 | CTX-M-15 P167S | ||
| Nitrocefin | kcat (s−1) | 710 ± 80 | 940 ± 180 | 370 ± 27 | 210 ± 8 | 77 ± 3 |
| Km (μM) | 30 ± 10 | 760 ± 100 | 21 ± 3 | 6.3 ± 0.9 | 7.6 ± 1.2 | |
| kcat/Km (μM−1 s−1) | 24 ± 8.4 | 0.8 ± 0.19 | 18 ± 2.9 | 33 ± 4.9 | 10 ± 1.6 | |
| Ceftazidime | kcat (s−1) | NDa | 0.11 ± 0.0094 | ND | ND | 3.09 ± 0.61 |
| Km (μM) | >1,000 | 45 ± 13 | ND | ND | 600 ± 170 | |
| kcat/Km (μM−1 s−1) | 0.0002 | 0.0025 ± 0.00075 | 0.0011 ± 0.000070 | 0.014 ± 0.0010 | 0.0051 ± 0.0018 | |
| Ceftibuten | kcat (s−1) | HNDb | 0.15 ± 0.011 | 26 ± 3 | 34 ± 1 | 33 ± 2 |
| Km (μM) | HND | 1.8 ± 0.77 | 250 ± 49 | 98 ± 11 | 190 ± 23 | |
| kcat/Km (μM−1 s−1) | HND | 0.085 ± 0.037 | 0.10 ± 0.023 | 0.35 ± 0.041 | 0.17 ± 0.023 | |
| Imipenem | kcat (s−1) | 0.02 ± 0.0011 | 0.058 ± 0.0026 | 0.0048 ± 0.00039 | 0.0046 ± 0.00027 | 0.0022 |
| Km (μM) | 16 ± 2.8 | 30 ± 3 | 2.2 ± 0.92 | 3.1 ± 0.75 | <2.5 | |
| kcat/Km (μM−1 s−1) | 0.0013 ± 0.00024 | 0.0019 ± 0.00021 | 0.0022 ± 0.00094 | 0.0016 ± 0.00040 | >0.009 | |
| Meropenem | kcat (s−1) | 0.00053 ± 0.000022 | 0.013 ± 0.0019 | 0.003 ± 0.00020 | 0.0072 ± 0.00052 | 0.0045 ± 0.0017 |
| Km (μM) | <5 | 83 ± 22 | 4.2 ± 1.1 | 2.2 ± 0.16 | 3.6 ± 2.0 | |
| kcat/Km (μM−1 s−1) | >0.00011 | 0.00016 ± 0.000048 | 0.00072 ± 0.00020 | 0.0032 ± 0.00033 | 0.0013 ± 0.00087 | |
| Ertapenem | kcat (s−1) | 0.0010 ± 0.000036 | 0.012 ± 0.0004 | 0.0024 ± 0.00044 | 0.0038 | 0.0058 ± 0.00074 |
| Km (μM) | <5 | 4.6 ± 0.54 | 8.5 ± 4.7 | <5 | 4.5 ± 2.0 | |
| kcat/Km (μM−1 s−1) | >0.00021 | 0.0026 ± 0.00032 | 0.00028 ± 0.00016 | >0.00076 | 0.0013 ± 0.00060 | |
| Tebipenem | kcat (s−1) | 0.00072 ± 0.000034 | 0.0022 ± 0.00028 | 0.0031 ± 0.00032 | 0.008 | 0.0036 ± 0.00052 |
| Km (μM) | 5.1 ± 1.2 | 7.2 ± 2.7 | 11 ± 2.8 | <5 | 6.1 ± 2.7 | |
| kcat/Km (μM−1 s−1) | 0.00014 ± 0.000034 | 0.00031 ± 0.00012 | 0.00028 ± 0.000077 | >0.0016 | 0.00059 ± 0.00028 | |
ND, not determined.
HND, hydrolysis not detected.
Hydrolysis of tebipenem and comparator antibiotics by carbapenemases.
OXA-, KPC-, and NDM-type β-lactamases represent the most prevalent carbapenemases in the clinic that contribute to carbapenem-resistant Enterobacterales (CRE) (5). Therefore, tebipenem and comparator antibiotics were tested for stability to hydrolysis by representative members for these types of enzymes, i.e., OXA-48, KPC-2 and KPC-3, and NDM-1. As shown in Table 2, although OXA-48 and KPC-2 were not highly active against tested cephalosporins, they displayed comparable high efficiency in hydrolyzing all tested carbapenems. KPC-3 differs from KPC-2 by one amino acid residue (Tyr274 in KPC-3 versus His274 in KPC-2) but displayed 10-fold higher efficiency in hydrolyzing ceftazidime and 20-fold higher efficiency in hydrolyzing ceftibuten, similar to what was found by Mehta et al. (15). In addition, KPC-3 is efficient in hydrolyzing all tested carbapenems, although its meropenem hydrolysis activity is 5-fold lower than that of KPC-2 (Table 2). Further, the KPC-2 D179Y mutant, which has been reported to provide resistance to the inhibitor avibactam, displays low catalytic activity toward carbapenems, including tebipenem. NDM-1, however, displays significantly higher kcat and kcat/Km values than OXA-48 and KPC enzymes in hydrolyzing all tested cephalosporins and carbapenems (Table 2). This is consistent with previous reports on the high catalytic activity of NDM-1 (16–18).
TABLE 2.
β-lactam hydrolysis by carbapenemases (NDM-1, OXA-48, KPC-2, KPC-3, and KPC-2 D179Y)
| Substrate | Parameter | Value for enzyme: |
||||
|---|---|---|---|---|---|---|
| NDM-1 | OXA-48 | KPC-2 | KPC-3 | KPC-2 D179Y | ||
| Nitrocefin | kcat (s−1) | 1.3 ± 0.2 | 140 ± 24 | 120 ± 5 | 84 ± 1 | 0.0052 ± 0.00028 |
| Km (μM) | <2.5 | 36 ± 7 | 43 ± 2 | 33 ± 1 | 7.8 ± 1.7 | |
| kcat/Km (μM−1 s−1) | >0.5 | 4.0 ± 1.0 | 2.8 ± 0.17 | 2.5 ± 0.081 | 0.00066 ± 0.00015 | |
| Ceftazidime | kcat (s−1) | 70 ± 3 | HNDa | NDb | ND | 0.027 ± 0.0057 |
| Km (μM) | 30 ± 0.4 | HND | ND | ND | 7.1 ± 3.9 | |
| kcat/Km (μM−1 s−1) | 2.3 ± 0.10 | HND | 0.0012 ± 0.00012 | 0.016 ± 0.00012 | 0.0038 ± 0.0022 | |
| Ceftibuten | kcat (s−1) | 31 ± 0.6 | 0.0090 ± 0.00054 | ND | ND | 0.013 ± 0.00094 |
| Km (μM) | 5 ± 0.02 | 69 ± 11 | ND | ND | 9.6 ± 3.0 | |
| kcat/Km (μM−1 s−1) | 6.2 ± 0.12 | 0.00013 ± 0.000022 | 0.0048 | 0.011 | 0.0013 ± 0.00042 | |
| Imipenem | kcat (s−1) | 240 ± 16 | 2.7 ± 0.2 | 88 ± 21 | 34 ± 4 | HND |
| Km (μM) | 65 ± 10 | 3.7 ± 0.7 | 200 ± 6 | 110 ± 25 | HND | |
| kcat/Km (μM−1 s−1) | 3.7 ± 0.25 | 0.73 ± 0.054 | 0.45 ± 0.11 | 0.32 ± 0.038 | HND | |
| Meropenem | kcat (s−1) | 180 ± 0.6 | 0.11 ± 0.01 | 4.0 ± 0.02 | 1.6 ± 0.1 | HND |
| Km (μM) | 37 ± 2 | 6.0 ± 1.2 | 16 ± 0.2 | 31 ± 8 | HND | |
| kcat/Km (μM−1 s−1) | 4.8 ± 0.26 | 0.017 ± 0.0032 | 0.26 ± 0.0035 | 0.050 ± 0.013 | HND | |
| Ertapenem | kcat (s−1) | 140 ± 1 | 0.19 ± 0.003 | 5.8 ± 0.39 | 8.0 ± 0.05 | HND |
| Km (μM) | 17 ± 2 | <1.25 | 23 ± 3 | 12 ± 1 | HND | |
| kcat/Km (μM−1 s−1) | 8.5 ± 1.0 | >0.15 | 0.26 ± 0.038 | 0.65 ± 0.054 | HND | |
| Tebipenem | kcat (s−1) | 63 ± 1 | 0.18 ± 0.0034 | 46 ± 11 | 32 ± 1 | 0.0050 ± 0.00061 |
| Km (μM) | 31 ± 8 | 1.59 ± 0.19 | 48 ± 7 | 35 ± 3 | 9.8 ± 3.8 | |
| kcat/Km (μM−1 s−1) | 2.0 ± 0.52 | 0.11 ± 0.013 | 0.96 ± 0.27 | 0.92 ± 0.084 | 0.00051 ± 0.00021 | |
HND, hydrolysis not detected.
ND, not determined.
DISCUSSION
Antibiotics such as trimethoprim sulfamethoxazole, ciprofloxacin, and ampicillin represent the most commonly recommended therapeutics for both uncomplicated and complicated UTIs (2). However, UTIs, especially complicated UTIs (cUTIs), are becoming increasingly difficult to treat because of the widespread emergence of antibiotic resistance mechanisms in UTI-causing Enterobacterales species, including E. coli, K. pneumoniae, and P. mirabilis (2). Of particular concern are extended-spectrum β-lactamases (ESBLs), as they confer resistance to Enterobacterales for most β-lactam antibiotics except carbapenems. In addition, ESBL-producing organisms are often resistant to ciprofloxacin and trimethoprim sulfamethoxazole (3, 4). Therefore, carbapenems remain the last-line antibiotic to treat multidrug-resistant cUTIs (5).
Despite their important clinical utility over the past 30 years, the currently marketed carbapenems (i.e., imipenem, meropenem, ertapenem, and doripenem) are administered almost exclusively intravenously (5). This situation was changed by tebipenem, which was introduced in Japan in 2009 as an orally administered prodrug, tebipenem pivoxil, to treat pediatric otitis media, sinusitis, and pneumonia (6). Recently, tebipenem was tested in vitro for antimicrobial activity against clinical isolates of Enterobacterales species that caused UTIs, including E. coli, K. pneumoniae, and P. mirabilis (7). It was found that tebipenem displayed efficacy very comparable to that of other carbapenems in inhibiting growth of clinical isolates, including those expressing ESBLs and/or the AmpC enzyme (7). Here, we show that tebipenem and its carbapenem comparators were stable to hydrolysis by CTX-M type ESBLs and the AmpC enzyme (Table 1), consistent with the in vitro antimicrobial activity of tebipenem. In contrast, tebipenem, similar to its carbapenem comparators, is susceptible to hydrolysis by carbapenemases such as the OXA-48, KPC, and NDM-1 enzymes (Table 2). This explains the failure of tebipenem and other carbapenems in inhibiting carbapenemase-producing Enterobacterales species that cause UTIs.
In order to develop an orally available prodrug of tebipenem with improved availability and stability, tebipenem pivoxil was modified to tebipenem pivoxil hydrobromide (TBP-PI-HBr, formerly SPR994). We also showed that tebipenem, which lacks the pivoxil group, is stable to ESBL and AmpC enzymes but is hydrolyzed by carbapenemases. Pharmacokinetic studies of TBP-PI-HBr with healthy human subjects have shown that orally administered TBP-PI-HBr is readily converted to tebipenem, most of which can be recovered in the urine of human subjects (9). In addition, TBP-PI-HBr was shown to be well tolerated by human subjects (9). These studies, together with in vitro β-lactamase stability analysis in the present study and in vitro antimicrobial activity results, support use of TBP-PI-HBr for treatment of adult cUTI/AP.
MATERIALS AND METHODS
Antibiotics.
Ceftazidime, clavulanate lithium, imipenem, and meropenem were purchased from U.S. Pharmacopeial Convention. Nitrocefin, ceftibuten, and ertapenem were purchased from Millipore Sigma. Avibactam was purchased from Advanced ChemBlocks Inc. Tebipenem (TBP) was provided by Spero Therapeutics, Inc. as powder.
Expression and purification of β-lactamases.
The TEM-1, CTX-M-14, OXA-48, and NDM-1 β-lactamases were overexpressed in E. coli BL21(DE3) and purified as described previously (10, 17, 19). For the expression of KPC-2 and KPC-3 enzymes, the DNA sequences encoding the mature form of the enzymes were amplified from the pTP123-KPC-2 and pTP123-KPC-3 plasmids (15), respectively, and cloned into the pET28a-TEV vector to obtain the recombinant vector for KPC-2 and KPC-3, respectively. The expression vector for KPC-2 D179Y was constructed by site-directed mutagenesis using pET28a-TEV-KPC-2 as the template DNA. The DNA sequence encoding the mature form of AmpC was amplified from the chromosomal DNA of E. coli MG1655 and cloned into pET28a-TEV vector. The pET28a-TEV-CTX-M-15 and pET28a-TEV-CTX-M-15 P167S were from our laboratory collections. With all of these recombinant vectors, β-lactamases were produced in E. coli BL21(DE3) with a 6×His tag at their N termini and a TEV protease recognition sequence between the His tag and the enzyme. The His tag β-lactamase enzymes were purified with a HisTrap FF column (GE Healthcare), and the His tag was cleaved by incubation with TEV protease. After removal of the TEV protease by incubation with a Ni Sepharose resin (GE Healthcare), β-lactamases were further purified by gel-filtration chromatography using a Superdex 75 GL 16/600 sizing column (GE Healthcare) with 20 mM HEPES (pH 7.4) and 50 mM NaCl as running buffer. Fractions containing β-lactamase were pooled and concentrated with 10 kDa cutoff Amicon concentrator units (EMD Millipore). Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) analysis of the purified enzymes showed that their purity was >90%. The final protein concentrations were determined by measuring absorbance at 280 nm with a DU800 spectrophotometer (Beckman Coulter) and using their corresponding extinction coefficient of ε280, which was calculated using the ExPASy ProtParam tool.
Determination of kinetic parameters.
Enzyme kinetic parameters for the hydrolysis of nitrocefin, ceftazidime, ceftibuten, imipenem, meropenem, ertapenem, tebipenem, and tebipenem pivoxil hydrobromide by various β-lactamases were determined under different conditions for different enzymes. For TEM-1, they were determined at 30°C in 50 mM sodium phosphate buffer (pH 7.2). For AmpC, they were determined at 28°C in 50 mM sodium phosphate buffer (pH 7.0). For CTX-M-14, CTX-M-15, and CTX-M-15 P167S, they were determined at 30°C in sodium phosphate buffer (pH 6.0). For KPC-2, KPC-2 D179Y, and KPC-3, they were determined at 28°C in sodium phosphate buffer (pH 7.0). For OXA-48, they were determined at 30°C in sodium phosphate buffer (pH 7.0). For NDM-1, they were determined at 25°C in 50 mM HEPES (pH 7.4) supplemented with 10 μM ZnSO4. Bovine serum albumin (BSA) was included in the buffers at the final concentration of 1 μg/mL to stabilize the enzymes.
Antibiotic hydrolysis was monitored with a DU800 spectrophotometer (Beckman Coulter) equipped with a thermostatically controlled cell by following the absorbance change of nitrocefin at 482 nm (Δε482 nm = 15,000 M−1 cm−1), ceftazidime at 260 nm (Δε260 nm = −10,500 M−1 cm−1), ceftibuten at 260 nm (Δε260 nm = −3,510 M−1 cm−1), tebipenem at 300 nm (Δε300 nm = −10,400 M−1 cm−1), imipenem at 300 nm (Δε300 nm = −9,000 M−1 cm−1), meropenem at 300 nm (Δε300 nm = −8,416 M−1 cm−1), and ertapenem at 300 nm (Δε300 nm = −7,100 M−1 cm−1). kcat and Km parameters were determined under initial-rate conditions by fitting the initial velocity (vo) at various substrate concentrations ([S]) to the Michaelis–Menten equation, v = Vmax [S]/(Km + [S]), using GraphPad Prism5. When Vmax could not be determined because Km was too high, kcat/Km was determined by analyzing the complete hydrolysis time courses at low antibiotic concentration and fitting the data to the equation v = kcat/Km [E][S], where E represents enzyme concentration. Kinetic parameters were averaged from at least two independent determinations.
ACKNOWLEDGMENTS
This research was supported by a contract between Baylor College of Medicine and Spero Therapeutics, Inc. and funded in whole or in part with federal funds from the Department of Health and Human Services; Office of the Assistant Secretary for Preparedness and Response, Biomedical Advanced Research and Development Authority, under contract no. HHSO100201800015C.
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