Abstract
This annex study to a phase 1 study aimed to correlate urinary concentrations and bactericidal titers (UBTs) of BAL30072, a novel siderophore monosulfactam, in healthy subjects in order to evaluate which dosage of BAL30072 should be investigated in a clinical study on complicated urinary tract infection (UTI). Three cohorts of a total of 19 healthy male subjects were included in the add-on study and received the following BAL30072 dosages. The 1st cohort received 1 g once a day (q.d.) intravenously (i.v.) (1 h) on day 1 and 1 g thrice daily (t.i.d.) on day 2, the 2nd cohort received 2 g q.d. i.v. (1 h) on day 1 and 2 g t.i.d. on day 2, and the 3rd cohort received 1 g q.d. i.v. (4-h infusion) on day 8. Urine was collected up to 24 h after drug administration. UBTs were determined for seven Escherichia coli isolates (three wild type [WT], CTX-M-15, TEM-3, TEM-5, NDM-1), two Klebsiella pneumoniae isolates (WT, KPC), one Proteus mirabilis isolate (WT), and two Pseudomonas aeruginosa isolates (WT, VIM-1 plus AmpC). Urine drug concentrations were measured by liquid chromatography-tandem mass spectrometry (LC-MS/MS). The median urinary excretions of BAL30072 ranged between 38% and 46% (3 cohorts). The median UBTs after i.v. administration of 1 or 2 g q.d. and after 1 or 2 g t.i.d. showed positive UBTs for 24 h after the lowest dosage (1 g q.d.) for 5 of 7 of the Enterobacteriaceae strains and after the higher dosage of 2 g administered i.v. t.i.d. for all strains tested. After i.v. infusion of 1 g over 4 h, positive UBTs were demonstrated for three E. coli strains for up to 12 h, for the K. pneumoniae (KPC) strain for up to 8 h, and for the P. aeruginosa (VIM-1 plus AmpC) strain for up to only 4 h. The minimal bactericidal concentrations (MBCs) of the E. coli (NDM-1) strain and the K. pneumoniae (WT) strain correlated well between broth and urine but did not correlate well for the two P. aeruginosa strains. BAL30072 exhibits positive UBTs for 24 h even after a dosage of 1 g administered i.v. q.d. for 5 of 7 Enterobacteriaceae strains and after 2 g administered i.v. t.i.d. for all strains except one P. aeruginosa strain (50% of the time). In general, the UBTs correlated well with the MICs of the Enterobacteriaceae but were lower for P. aeruginosa. The clinical efficacy with a dosage regimen of BAL30072 of 2 g administered i.v. t.i.d. should be evaluated in the treatment of complicated UTI.
INTRODUCTION
For initial empirical therapy of severe urinary tract infections (UTIs), including acute pyelonephritis, parenteral antibiotics are recommended (1). Due to increasing rates of β-lactam-resistant and multidrug-resistant (MDR) Gram-negative uropathogens, especially in hospital-acquired but also in community-acquired UTIs (2), new antibiotics need to be developed.
BAL30072 is a recently developed siderophore–β-lactam conjugate. This monocyclic β-lactam contains an iron-chelating dihydroxypyridone substituent. Using a transcriptome approach, the FecIRA Fe3+-dicitrate transporter was identified as a regulatory cascade, and the PiuA iron receptor was identified as a putative uptake system affecting susceptibility to BAL30072 (3). The molecule has potent in vitro activity against many species of MDR isolates of the Enterobacteriaceae family, including isolates harboring a class A carbapenemase or a metallo-β-lactamase (4, 5). BAL30072 also exhibits potent activity against MDR Pseudomonas aeruginosa and Acinetobacter sp. isolates, including many carbapenem-resistant strains (5, 6). Unlike other monocyclic β-lactams, BAL30072 was found to trigger the spheroplasting and lysis of Escherichia coli cells rather than the formation of extensive filaments. The basis for this unusual property is its inhibition of the penicillin-binding proteins PBP 1a and PBP 1b in addition to its high affinity for PBP 3 (7). PBP 3 is the highest-affinity target of the monobactam aztreonam (4).
Whereas pharmacokinetics/pharmacodynamics (PK/PD) parameters derived from plasma concentrations are predictive for many systemic infections, urinary bactericidal activity may be a better predictive parameter for the treatment of severe UTIs (8). Therefore, in an ex vivo annex study to a phase 1 study published in part elsewhere (9), urinary concentrations and urinary bactericidal titers (UBT) after escalating intravenous (i.v.) dosages of BAL30072 were determined against various Gram-negative pathogens, including ATCC test strains, wild types (WTs), and specific extended-spectrum β-lactamase (ESBL) producers, to evaluate which dosage of BAL30072 should be investigated in a clinical study on complicated UTIs caused by such pathogens.
(This work was presented in part as posters at the 52nd Interscience Conference on Antimicrobial Agents and Chemotherapy, 2012 [10], and at the 54th Interscience Conference on Antimicrobial Agents and Chemotherapy, 2014 [11].)
MATERIALS AND METHODS
Study design and subject population.
This study was performed as an ex vivo annex study to a phase I clinical study to assess the pharmacokinetics and safety of BAL30072 after multiple ascending dose infusions in healthy male subjects. The clinical study was performed at Momentum Pharma Services GmbH (Hamburg, Germany) under the supervision of Basilea Pharmaceutica International Ltd. The clinical study was conducted under protocol numbers SFM-CP-002 and SFM-CP-004 (Basilea Pharmaceutica International Ltd.) and EudraCT numbers 2011-002374-23 and 2012-003118-05, respectively, and was approved by the corresponding ethical committee (Ethik-Kommission der Ärztekammer Hamburg, Hamburg, Germany).
The clinical phase 1 study was performed with healthy adult subjects (25 males, including 6 on placebo, aged 28 to 45 years). A total of 19 male subjects, determined to be healthy by a general physical examination, medical history, vital signs, 12-lead electrocardiogram (ECG), and laboratory safety tests, received BAL30072 i.v. All subjects were Caucasian, with a median (range) age of 33 (28 to 45) years, a weight of 80.4 (59.8 to 103.7) kg, a height of 180 (170 to 197) cm, and a body mass index of 24.9 (18.8 to 30.1) kg/m2.
The annex studies in cohorts 1 and 2 (SFM-CP-002) were performed only on days 1 and 2 of the phase 1 study. Cohorts 1 and 2 (n = 7 and 6, respectively) received 1 g and 2 g i.v. (1-h infusion) once a day (q.d.) (day 1) followed by 1 g and 2 g i.v. three times a day (t.i.d.) (day 2), respectively. In cohort 3 (SFM-CP-004) (n = 6), 1,000 mg of BAL30072 was infused i.v. t.i.d. over 4 h, except on pharmacokinetics days 1, 8, and 14, when BAL30072 was infused i.v. q.d. over 4 h. The annex study, including determinations of UBTs, was performed only on day 8.
Urine samples from all subjects were shipped (+4°C) to the Prokaryon Institute (Nuremberg, Germany) for the ex vivo study. Laboratory personnel at the Prokaryon Institute were provided with unblinded subject information to exclude urine samples from the six subjects who had received placebo because the phase 1 study was designed to determine not only pharmacokinetics but also safety.
Drug administration.
Seven subjects (cohort 1) received 1 g i.v. BAL30072 (1-h infusion) q.d. (day 1) followed by 1 g i.v. BAL30072 (1-h infusion) t.i.d (day 2) at 0, 8, and 16 h, and 6 subjects (cohort 2) received 2 g i.v. BAL30072 q.d. (day 1) followed by 2 g i.v. BAL30072 t.i.d (day 2) at 0, 8, and 16 h. Six subjects (cohort 3) received 1 g i.v. BAL30072 (4-h infusion) q.d. (day 8).
The study drug was administered on study day 1 after an overnight (8-h) fast. After drug administration on day 1, subjects fasted for 3 h. Water was given ad libitum overnight. At predose and during the first 6 h after the start of the infusion, subjects were asked to drink 200 ml/h; thereafter, they were asked to drink 100 ml/h until 16 h after the start of infusion. Afterward, water was served ad libitum. The same rules for water intake applied on day 2 and on day 8 because of intensive urine collection for PK and UBT.
Alcohol and grapefruit juice were not allowed 72 h before drug administration until the end of the study (day 21 ± 1). Caffeine-containing beverages were not allowed 24 h before drug administration until study day 17.
Sample collection.
On day −1, approximately 250 ml of urine was collected from each subject from admission until the next morning and at the following time intervals thereafter: 0 to 2 h, 2 to 4 h, 4 to 8 h, 8 to 12 h, and 12 to 24 h (day 1 and day 8) and 0 to 2 h, 2 to 4 h, 4 to 8 h, 8 to 12 h, 12 to 16 h, and 16 to 24 h (day 2) after drug administration. All samples were stored at −80°C.
Drug concentrations in urine.
The determination of BAL30072 in human urine samples from the clinical studies SFM-CP-002 and SFM-CP-004 were performed by liquid chromatography-tandem mass spectrometry (LC-MS/MS) according to a fully validated method. In-study validation was performed according to FDA guidelines (12) and according to European Medicines Agency (EMA) guidelines on bioanalytical method validation (13).
The urine was diluted with methanol containing the internal standards BAL30072-D6 and BAL104936-D6. After dilution and centrifugation, the diluted sample was further diluted with water containing 19 μM citric acid. After mixing, an aliquot of 10 μl of the diluted sample was injected onto the high-pressure liquid chromatography (HPLC) system.
BAL30072 and BAL104936 were quantified by LC-MS/MS in the selected reaction monitoring mode using heated electrospray ionization in positive ion mode. The lower limit of quantification (LLOQ) for the two analytes was set to 0.200 μg/ml, and the upper limit of quantification (ULOQ) for the two analytes was set to 200 μg/ml.
In the SFM-CP-002 study (cohorts 1 and 2), a total of 284 human urine samples, an additional 70 calibration samples, and 42 quality control samples (QC1 to QC3) were analyzed. The descriptive statistics of the QC for BAL30072 showed that the interbatch precision was between 1.9% and 5.8%, whereas the interbatch accuracy was in the range of 97.5% to 103.5% of nominal concentration.
In the SFM-CP-004 study (cohort 3), a total of 426 human urine samples, an additional 100 calibration samples, and 60 quality control samples (QC1 to QC3) were analyzed. The descriptive statistics of the QC for BAL30072 showed that the interbatch precision was between 2.3% and 8.3%, whereas the interbatch accuracy was in the range of 102.7% to 104.7% of nominal concentration.
Test organisms.
There were six wild-type uropathogens or ATCC reference strains without acquired resistance mechanisms: E. coli (3 strains), Klebsiella pneumoniae (1 strain), Proteus mirabilis (1 strain), and P. aeruginosa (1 strain). In addition, there were six strains with a specifically identified β-lactamase enzyme(s): four E. coli strains (CTX-M15, TEM-3, TEM-5, NDM-1), one Klebsiella pneumoniae strain (KPC), and one Pseudomonas aeruginosa (VIM-1 plus AmpC) strain.
Determination of MICs and MBCs.
MICs of BAL30072 and 7 additional antibiotics (ampicillin, cefepime, ceftazidime, cefotaxime, cefoxitin, meropenem, ciprofloxacin) were determined in triplicate by a broth dilution method using Iso-Sensitest broth (CM0473; Oxoid, Wesel, Germany) with an inoculum of 5 × 106 CFU/ml determined by optical density. The MIC was defined as the lowest concentration inhibiting visible growth after incubation at 37°C for 18 h in ambient air. BAL30072 was provided by Basilea (Basel, Switzerland), and for the other antibiotics, a prepared test set (Merlin Diagnostika GmbH, Bornheim-Hersel, Germany) was used.
Minimal bactericidal concentrations (MBCs) were determined in duplicate by dilution of BAL30072 in Iso-Sensitest broth (pH 7.5, CM0473; Oxoid) and in pooled urine (pH 5.3) from 6 subjects of cohort 3. The final inoculum, which was confirmed by actual counting, ranged from 1.5 × 106 to 9.4 × 106 CFU/ml. The plates were then incubated at 37°C for 18 h in ambient air. In a second step, 10 μl of the subcultured urine was transferred onto Iso-Sensitest agar supplemented with 5% blood (Oxoid, Wesel, Germany). The plates were again incubated for 24 h at 37°C. The number of colonies subsequently grown was used to determine the bactericidal endpoint. The MBC was defined as the concentration that produced a >99.9% (>3-log) reduction of the initially inoculated colony counts.
Determination of UBTs.
For determination of urinary bactericidal titers (UBTs), a 2-fold serial dilution (dilution range, 1:0, 1:1 to 1:1,024) of the urine samples was prepared. The individual subject's antimicrobial agent-free urine or pooled urine from 4 different healthy study subjects collected prior to drug administration was used as a diluent. A microdilution test was used to determine UBTs. Each well of the microplates contained 100 μl of the prepared urinary dilution. Bacterial strains were then added to the wells of the microplates using a multipoint inoculator (10 μl). The final inoculum, which was confirmed by actual counting, ranged from 3.8 × 106 to 9.4 × 106 CFU/ml. The plates were then incubated at 37°C for 18 h in ambient air. After this first incubation, 10 μl of the subcultured urine was transferred onto Iso-Sensitest agar supplemented with 5% blood (Oxoid, Wesel, Germany). The plates were again incubated for 24 h at 37°C. The number of colonies subsequently grown was used to determine the bactericidal endpoint. Urinary bactericidal activity was defined as a >99.9% (>3-log) reduction of the initially inoculated colony counts. A UBT of 0 was defined as no bactericidal activity, and a UBT of 1 was used when only undiluted urine displayed bactericidal activity.
The laboratory reproducibility of UBTs was determined for one strain (P. mirabilis 414) for 4 time intervals, each in triplicate (12 results), in one subject after i.v. administration of 2 g BAL30072 to get a broad range of results. Pooled urine from 4 healthy study subjects was used as a diluent. The 2-fold dilution steps for each of the 4 time intervals ranged up to 3 dilution steps with a standard deviation between 0.49 and 0.80 dilution step.
The area under the UBT-versus-time curve (AUBT) was calculated for 24 h of the 1st and 2nd days for cohorts 1 and 2 and of the 8th day for cohort 3 as the sum of the products of the reciprocal UBT values and the respective time intervals in hours for each test organism and for each drug.
Iron content.
The iron content in broth was determined by a photometric method and in the pooled urine of cohort 3 by an ionic chromatographic method (Synlab Medical Laboratory, Weiden, Germany) (14, 15).
Statistical analyses.
Statistical analyses were descriptive; calculations were performed using the Microsoft Excel 2010 program (Microsoft Co., Redmond, WA, USA).
RESULTS
Urine volumes, concentrations, and excretions.
The median (range) urine volumes (ml) and BAL30072 urinary concentrations (mg/liter) and excretions (mg) are given in Table 1 for subjects in cohort 1 who received 1 g BAL30072 i.v., in Table 2 for subjects in cohort 2 who received 2 g BAL30072 i.v., and in Table 3 for subjects in cohort 3 who received a 1-g i.v. infusion of BAL30072 over 4 h.
TABLE 1.
Median urine volume and BAL30072 concentration (mg/liter) in urine samples of 7 male subjects of cohort 1a
| Collection period (h) | Median (range) volume (ml) | Median (range) concentration (mg/liter) | Median (range) urinary excretion (mg) |
|---|---|---|---|
| 0–1b | |||
| 0–2 | 399 (165–589) | 708 (509–2,070) | 300 (258–342) |
| 2–4 | 395 (91–643) | 243 (156–444) | 119 (40.4–130) |
| 4–8 | 1,037 (782–1215) | 39.1 (35.4–75.6) | 43.3 (38.3–65.6) |
| 8–12 | 653 (193–1,086) | 11.2 (5.31–21.5) | 5.77 (4.15–7.81) |
| 12–24 | 1,155 (961–2,481) | 1.08 (0.72–2.21) | 1.79 (1.03–2.33) |
| 24–25b | |||
| 24–26 | 415 (53–829) | 667 (354–6,030) | 278 (60.8–320) |
| 26–28 | 416 (63–516) | 372 (210–1,840) | 130 (106–175) |
| 28–32 | 880 (429–1,291) | 57.8 (39.1–195) | 47.6 (34.6–205) |
| 32–33b | |||
| 32–36 | 783 (616–1,368) | 451 (307–730) | 418 (353–473) |
| 36–40 | 528 (441–795) | 80.6 (41.4–114) | 43.8 (32.9–59.1) |
| 40–41b | |||
| 40–48 | 1,016 (423–1,639) | 412 (298–640) | 419 (253–503) |
TABLE 2.
| Collection period (h) | Median (range) volume (ml) | Median (range) concentration (mg/liter) | Median (range) urinary excretion (mg) |
|---|---|---|---|
| 0–1b | |||
| 0–2 | 327 (87–1,121) | 1,659 (387–6,800) | 485 (218–762) |
| 2–4 | 473 (83–1,012) | 445 (269–3,130) | 241 (206–304) |
| 4–8 | 1,256 (677–1,554) | 97.4 (47.8–179) | 122 (59.9–233) |
| 8–12 | 952 (500–2,398) | 12.0 (6.09–48.9) | 12.0 (5.58–30.9) |
| 12–24 | 1,518 (1,059–3,456) | 2.13 (1.43–6.08) | 3.03 (2.24–9.42) |
| 24–25b | |||
| 24–26 | 244 (125–387) | 2,195 (1,510–5,600) | 550 (433–700) |
| 26–28 | 191 (133–693) | 1,525 (430–2,280) | 306 (249–363) |
| 28–32 | 1,048 (775–1,136) | 86.0 (58.8–170) | 96.0 (45.6–168) |
| 32–33b | |||
| 32–36 | 1,015 (500–1,256) | 702 (582–1,090) | 772 (324–1,072) |
| 36–40 | 622 (412–978) | 157 (92.3–358) | 89.0 (72.0–148) |
| 40–41b | |||
| 40–48 | 1,177 (995–1,415) | 679 (523–984) | 849 (573–1,025) |
TABLE 3.
Median urine volume and BAL30072 concentration (mg/liter) in urine samples of 6 male subjects of cohort 3a
| Collection period (h) | Median (range) volume (ml) | Median (range) concentration (mg/liter) | Median (range) urinary excretion (mg) |
|---|---|---|---|
| 0–2 | 790 (469–1,025) | 128 (69.6–232) | 99.6 (66.9–112) |
| 2–4 | 593 (462–822) | 309 (138–359) | 168 (113–197) |
| 4–8 | 669 (443–1,023) | 127 (96.9–208) | 106 (42.9–125) |
| 8–12 | 736 (335–1,140) | 11.0 (3.60–22.5) | 8.51 (3.01–11.2) |
| 12–24 | 1,406 (1,085–2,326) | 1.90 (0.80–4.40) | 2.85 (1.21–4.77) |
Subjects were administered a 1-g i.v. BAL30072 infusion over 4 h on the 8th day.
The highest median BAL30072 urinary concentration in cohort 1 was found in the 0- to 2-h period on the first day (708 mg/liter), and the corresponding peak on the 2nd day reached 667 mg/liter. The highest median BAL30072 urinary concentration in cohort 2 on the first day was found in the 0- to 2-h period (1,659 mg/liter), and the corresponding peak on the 2nd day reached 2,195 mg/liter. The highest median BAL30072 urinary concentration in cohort 3 was found in the 2- to 4-h period (309 mg/liter). The accumulated median (range) urinary excretion of BAL30072 in cohort 1 was 449 mg (410 to 544 mg) on the first day, corresponding to 44.9% (40.9% to 54.4%) of the administered dose and was 1,283 mg (977 to 1,526 mg) on the second day corresponding to 42.8% (32.6% to 50.9%) of the administered dose. The accumulated median (range) urinary excretion of BAL30072 in cohort 2 was 966 mg (532 to 1,119 mg) on the first day, corresponding to 48.3% (26.6% to 56.0%) of the administered dose, and was 2,512 mg (2,116 to 3,230 mg) on the second day, corresponding to 41.9% (35.3% to 53.8%) of the administered dose. The accumulated median (range) urinary excretion of BAL30072 in cohort 3 was 380 mg (269 to 426 mg) on study day 8, corresponding to 38.0% (26.9% to 42.6%) of the administered dose.
MICs.
The MICs of BAL30072 for the test strains are shown in Table 4, and the MICs of the 7 additional antibiotics are shown in Table S1 in the supplemental material. The MICs of BAL30072 for the 12 test strains ranged between ≤0.06 and 32 mg/liter. Concerning the 7 additional antibiotics tested for the 5 wild-type Enterobacteriaceae strains, the MICs were ≤1 mg/liter of the third-generation cephalosporins (cefotaxime, ceftazidime, cefepime) and ≤4 mg/liter of cefoxitin. For the wild-type P. aeruginosa strain, the MIC was ≤1 mg/liter of ceftazidime and cefepime. The E. coli strain positive for NDM-1 was resistant to all 7 additional antibiotics tested, including meropenem. In contrast, the MIC of BAL30072 was 1 mg/liter against this E. coli strain.
TABLE 4.
MICs of BAL30072 determined in triplicate (median shown) by a broth dilution method using Iso-Sensitest broth for the test strains with an identified β-lactamase enzyme(s)
| Strain | Enzyme(s) presentd | MIC (mg/liter) of BAL30072 |
|---|---|---|
| E. coli ATCC 25922 | — | ≤0.06 |
| E. coli BAL-3 WT | — | ≤0.06 |
| E. coli BAL-5 WT | — | ≤0.06 |
| E. coli NCTC 13451 | CTX-M15 | 0.125 |
| E. coli CF102a | TEM-3 | 4 |
| E. coli HH | TEM-5 | 32 |
| E. coli IR3b | NDM-1 | 1 |
| P. mirabilis 414 WT | — | 0.25 |
| K. pneumoniae 595 WT | — | 2 |
| K. pneumoniae ATCC BAA 1705 | KPC | 4 |
| P. aeruginosa ATCC 27853 | — | 2 |
| P. aeruginosa HPA10586a,c | VIM-1, AmpC | 1 |
UBTs.
All predose urine samples were used as positive controls and showed bacterial growth of all strains tested after incubation for 24 h at 37°C. The median (range) UBTs after i.v. administration of 1 g or 2 g q.d. on day 1 and of 1 g or 2 g t.i.d. on day 2 (administered at 0, 8, and 16 h) and after 1 g i.v. infusion over 4 h on day 8 are shown in Fig. 1 to 5 and in Tables S2 to S6 in the supplemental material.
FIG 1.
Median reciprocal urinary bactericidal titers (UBTs) of BAL30072 for E. coli strains tested in urine samples of 7 male subjects of cohort 1 (1 g i.v. BAL30072 over 1 h q.d. on the 1st day followed by 1 g i.v. BAL30072 over 1 h t.i.d. on the 2nd day).
FIG 5.
Median reciprocal urinary bactericidal titers (UBTs) of BAL30072 for five bacterial strains tested in urine samples of 6 male subjects of cohort 3 (1-g i.v. BAL30072 infusion over 4 h q.d. on the 8th day).
FIG 2.
Median reciprocal urinary bactericidal titers (UBTs) of BAL30072 for K. pneumoniae, P. mirabilis, and P. aeruginosa strains tested in urine samples of 7 male subjects of cohort 1 (1 g i.v. BAL30072 over 1 h q.d. on the 1st day followed by 1 g i.v. BAL30072 over 1 h t.i.d. on the 2nd day).
Median positive UBTs, i.e., at least undiluted urine exhibits bactericidal activity, could already be demonstrated for 24 h after once daily dosage of 1 or 2 g i.v. BAL30072 for all wild-type Enterobacteriaceae tested and for the CTX-M-15-positive E. coli strain but not for the E. coli TEM-1- and TEM-3-positive strains or the two P. aeruginosa strains tested (Fig. 1 to 4; see also Tables S2 to S5 in the supplemental material). Only after a dosage of 2 g i.v. BAL30072 t.i.d. were median UBTs positive throughout the 24-h period for all strains tested, including for the P. aeruginosa strain that was positive for VIM-1 and AmpC (Fig. 3 and 4; see also Tables S4 and S5).
FIG 4.
Median reciprocal urinary bactericidal titers (UBTs) of BAL30072 for K. pneumoniae, P. mirabilis, and P. aeruginosa strains tested in urine samples of 6 male subjects of cohort 2 (2 g i.v. BAL30072 over 1 h q.d. on the 1st day followed by 2 g i.v. BAL30072 over 1 h t.i.d. on the 2nd day).
FIG 3.
Median reciprocal urinary bactericidal titers (UBTs) of BAL30072 for E. coli strains tested in urine samples of 6 male subjects of cohort 2 (2 g i.v. BAL30072 over 1 h q.d. on the 1st day followed by 2 g i.v. BAL30072 over 1 h t.i.d. on the 2nd day).
Of the five strains tested after the 1-g i.v. infusion over 4 h, the three E. coli strains (ATCC 25922, CTX-M15, NDM-1) showed positive UBTs for up to 12 h, the one K. pneumoniae (WT) strain showed positive UBTs for up to 8 h, and the P. aeruginosa (VIM-1 plus AmpC) strain showed positive UBTs for only up to 4 h (Fig. 5; see also Table S6 in the supplemental material).
In general, the UBTs correlated well with the MICs of the Enterobacteriaceae but were proportionally lower for P. aeruginosa.
MBCs and iron concentrations in broth and urine.
Table 5 shows the MBCs of BAL30072 for four test strains with similar MICs performed in duplicate in Iso-Sensitest broth (pH 7.5) and pooled urine (pH 5.3) from cohort 3. Whereas the MBCs of the two strains of the Enterobacteriaceae family correlated well between broth and urine, the MBCs of the two strains of P. aeruginosa were 16 and more times higher in urine than they were in broth.
TABLE 5.
MICs and MBCs of BAL30072 determined in duplicate in Iso-Sensitest broth (pH 7.5) and pooled urine (pH 5.3) from cohort 3
| Strain | MIC in broth (mg/liter) | MBC in broth (mg/liter) | MBC in urine (mg/liter) |
|---|---|---|---|
| E. coli (NDM-1) | 1 | 2 | 4 |
| K. pneumoniae (WT) | 2 | 2–4 | 2–4 |
| P. aeruginosa (ATCC 27953) | 2 | 4 | 64 |
| P. aeruginosa (VIM-1, AmpC) | 1 | 1–2 | >64 |
The total iron content in broth was 420 μg/liter, and in the pooled urine, it was 27.6 μg/liter.
DISCUSSION
The present study is an ex vivo annex study of a double-blind, randomized, single-ascending dose phase 1 study of healthy male subjects that has been published in part elsewhere (9). In summary, the mean maximum concentrations of drug in serum (Cmax) of BAL30072 were 28.5, 76.0, 103, 226, and 286 mg/liter, with corresponding area under the concentration-time curve (AUC) values of 43.8, 140, 183, 549, and 1,040 mg · h/liter, following infusions of 500, 1,000, 2,000, 4,000, and 8,000 mg, respectively. The elimination half-life of BAL30072 ranged from 1.6 to 2.3 h, the volume of distribution at steady state was between 11.1 and 15.3 liters, and the total systemic clearance was 7.4 to 12 liters/h. Approximately 50% of the BAL30072 dose administered was recovered unchanged in urine. The main metabolite, the ring-open product (BAL104936), contributed on average to 25% of the systemic exposure, and approximately 12% was recovered in urine.
In this ex vivo annex study from parts of the phase 1 study, UBT was determined as an additional PK/PD parameter because it results from the actual urinary drug concentration and the susceptibility of a pathogen in a specific individual urinary milieu, which might be quite different from the condition standard against which MIC is usually measured. In contrast to plasma, various ion contents and the pH of the urine may considerably influence the results (16). MICs of BAL30072 for E. coli ATCC 25922 and P. aeruginosa ATCC 27853 that were determined in broth with pH 6.0 were generally higher than those determined at pH 7.2 to 7.4 (standard) or at pH 8.0 (data on file; Basilea Pharmaceutica International Ltd., Basel, Switzerland). Furthermore, iron, which is essential for enterobacterial growth, is present at a much lower concentration in urine, a fact that acts as a defensive mechanism aiming to inhibit bacterial growth in the urinary tract.
BAL30072, a novel siderophore monosulfactam, is a beta-lactam antibiotic protected against some of the ESBLs frequently encountered in Gram-negative uropathogens. Since renal excretion is close to about 50%, it was justified to determine UBTs for a variety of uropathogens with different ESBL-related beta-lactam resistances.
The results of the UBT study have shown that a single 1-h infusion of 1 g i.v. results in subsequent bactericidal activity in the urine for a 24-h period only for non-ESBL and CTX-M-15 β-lactamase-producing Enterobacteriaceae. Higher doses, such as 2 g t.i.d., were required to produce positive UBTs for all strains tested. Since BAL30072 belongs to the β-lactam antibiotics and the time above MIC may be the crucial PK parameter, a more continuous i.v. infusion over 4 h was also investigated. This was done to show whether a 1-g dose administered t.i.d. as a prolonged i.v. infusion of 4 h could possibly result in positive UBTs over 24 h for all strains tested. From the single-dose study, it could already be concluded that this would not be the case. Therefore, a dosage regimen of 2 g i.v. BAL30072 t.i.d. may be required if in a clinical study complicated UTI needs to be evaluated. In such a study, urinary bactericidal activity, determined as UBT, should also be correlated with microbiological success because the underlying conditions of a complicated UTI are very heterogeneous. It may become evident that, especially for biofilm infections such as catheter-associated UTIs, higher degrees of UBTs are needed than those for other conditions that are less likely to involve biofilm infections.
The different activities of the Enterobacteriaceae and the Pseudomonas strains in relation to their MICs as determined in Iso-Sensitest broth are not only shown by the lower UBTs but also by the MBCs measured in broth and pooled urine. Whereas the MBCs of the E. coli and K. pneumoniae strains correlated well in the two media, the MBCs for the two Pseudomonas strains were 16 and ≥32 times higher in urine than in broth. One explanation for the lower antipseudomonal activity of BAL30072 in urine than in broth may be that the iron content in urine is about 15 times lower than that in broth, which has an iron content that is close to the normal range in serum. The normal range of iron in serum for females is 370 to 1,450 μg/liter (6.6 to 26 μmol/liter), and for males, it is 590 to 1,580 μg/liter (11 to 28 μmol/liter); iron excretion in urine is <98 μg/24 h (<1.8 μmol/day) (17). It can be assumed that the total iron contents in broth and urine correspond to free iron because no iron-binding structures, e.g., proteins, are present in these two matrices. There also seem to be characteristic genus-specific differences in the utilization of hydroxamate and catecholate siderophores (18), which also may contribute to the different activity in urine found for Enterobacteriaceae strains compared to that of the Pseudomonas strains tested.
BAL30072, a siderophore monosulfactam, follows the concept of siderophore conjugation, which facilitates compound uptake across the outer membrane by hijacking bacterial iron acquisition systems. Tomaras et al. (19) demonstrated for MB-1, a novel siderophore-conjugated monobactam, excellent in vitro activity against P. aeruginosa when tested using standard assay conditions. Unfortunately, the in vitro findings did not correlate with the in vivo results, as multiple strains were not effectively treated by MB-1 despite having low MICs. The authors provided evidence that competition with native siderophores may contribute to the recalcitrance of some P. aeruginosa isolates in vivo. Van Delden et al. (3) selected mutants with decreased susceptibilities to BAL30072 in P. aeruginosa PAO1 under a variety of conditions. Under iron-deficient conditions, mutants with overexpression of AmpC β-lactamase predominated. Similar mutants were obtained after selection at >16 times the MIC under iron-sufficient conditions. These results show that the activity of BAL30072 against Pseudomonas strains may be unfavorably be affected under iron-deficient conditions, which is usually the case in urine. The exact mechanisms, however, need to be elucidated.
BAL30072, a novel siderophore monosulfactam, may represent a promising new antibacterial agent for the treatment of complicated UTIs caused by multidrug-resistant Enterobacteriaceae. Further clinical studies supported by population PK/PD modeling and Monte Carlo simulations are required to identify the optimal dosing regimen of BAL30072 considering the urinary antibiotic activity, which may be different as expected from the activity in broth, especially for Pseudomonas strains.
Conclusion.
Bactericidal activity in the urine of healthy subjects was already detected for 24 h after the lower dose of 1 g i.v. q.d. for all wild-type Enterobacteriaceae tested and CTX-M-15-positive E. coli. After the higher dosage (2 g i.v. t.i.d.), urinary bactericidal activity was also detected for TEM-3-positive and TEM-5-positive E. coli and for P. aeruginosa (including the VIM-1- and AmpC-positive strain). After a 1-g i.v. infusion over 4 h, positive urinary bactericidal activity was detected for up to 12 h for the E. coli strains, for up to 8 h for the K. pneumoniae strain, but for up to only 4 h for the P. aeruginosa strain tested. The lower activity of BAL30072 in urine, as expected from the MICs in broth for the two strains of P. aeruginosa, may be due to the about 15 times lower iron content in urine than in broth. Further studies to investigate UBTs of different dosing regimens for other ESBL-producing uropathogens and the clinical efficacy of BAL30072 in the treatment of complicated UTIs are warranted.
Supplementary Material
ACKNOWLEDGMENT
We thank Momentum Pharma Services GmbH (Hamburg, Germany) and Basilea Pharmaceutica International Ltd. (Basel, Switzerland) for giving us the data of the phase I study to include in this publication and Synlab Medical Laboratory (Weiden, Germany) for their determination of the iron contents in broth and urine.
This study was supported by an unrestricted grant from Basilea Pharmaceutica International Ltd., Basel, Switzerland.
Funding Statement
The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.
Footnotes
Supplemental material for this article may be found at http://dx.doi.org/10.1128/AAC.02425-15.
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