ABSTRACT
The objective of these studies was to evaluate the exposures of meropenem and vaborbactam that would produce antibacterial activity and prevent resistance development in carbapenem-resistant Klebsiella pneumoniae carbapenemase (KPC)-producing Enterobacteriaceae strains when tested at an inoculum of 108 CFU/ml. Thirteen K. pneumoniae isolates, three Enterobacter cloacae isolates, and one Escherichia coli isolate were examined in an in vitro hollow-fiber model over 32 h. Simulated dosage regimens of 1 to 2 g of meropenem with 1 to 2 g of vaborbactam, with meropenem administered every 8 h by a 3-h infusion based on phase 1 or phase 3 patient pharmacokinetic data, were studied in the model. A dosage of 2 g of meropenem in combination with 2 g of vaborbactam was bactericidal against K. pneumoniae, E. cloacae, and E. coli strains, with meropenem-vaborbactam MICs of up to 8 mg/liter. When the vaborbactam exposure was adjusted to the levels observed in patients enrolled in phase 3 trials (24-h free AUC, ∼550 mg · h/liter, versus 320 mg · h/liter in the phase 1 studies), 2 g of meropenem with 2 g of vaborbactam was also bactericidal against strains with meropenem-vaborbactam MICs of 16 mg/liter. In addition, this level of vaborbactam also suppressed the development of resistance observed using phase 1 exposures. In this pharmacodynamic model, exposures similar to 2 g of meropenem in combination with 2 g of vaborbactam administered every 8 h by a 3-h infusion in phase 3 trials produced antibacterial activity and suppressed the development of resistance against carbapenem-resistant KPC-producing strains of Enterobacteriaceae.
KEYWORDS: meropenem, vaborbactam, KPC, hollow fiber
INTRODUCTION
Resistance to carbapenem antimicrobial agents has become a major threat to public health worldwide (1, 2). The three major mechanisms for generating carbapenem resistance are hydrolyzing enzymes, loss or modification of porins, and increased efflux (3–5). The most common resistance mechanism involves enzymatic degradation by a variety of enzymes collectively called carbapenemases, which are produced by genes located either on plasmids or chromosomes (6, 7). Class A serine carbapenemases, especially Klebsiella pneumoniae carbapenemase (KPC) carbapenemases, have become the most common in the United States and globally (8). Mortality rates range between 23 and 75% in patients infected with K. pneumoniae strains producing carbapenemases (9).
Vaborbactam is a novel highly potent boronic acid-based serine beta-lactamase inhibitor being developed in combination with meropenem (10). Vaborbactam has a broad spectrum of beta-lactamase activity but has been optimized to specifically inhibit the KPC carbapenemase and thus restore the activity of carbapenem antibiotics against KPC-producing strains of carbapenem-resistant Enterobacteriaceae. Recent in vitro studies have shown that this combination of meropenem and vaborbactam has excellent potency against Gram-negative bacteria, especially KPC-producing strains of Enterobacteriaceae (11).
The goal of these pharmacodynamic studies was to demonstrate the activity of meropenem in combination with vaborbactam against KPC-producing carbapenem-resistant Enterobacteriaceae isolates using simulated human exposures for various dosage regimens in an in vitro hollow-fiber model.
(This work was presented in part at the 54th Interscience Conference on Antimicrobial Agents and Chemotherapy, 5 to 9 September 2014, Washington, DC.)
RESULTS
Susceptibility testing.
The MICs for the strains used in these studies are shown in Table 1. All strains were resistant to meropenem, with MICs of greater than 8 mg/liter. The addition of either 4 or 8 mg/liter vaborbactam restored the activity of meropenem in most strains.
TABLE 1.
MICs for the strains used in these experiments
| Strain | Beta-lactamase(s) | Porin mutation(s)a |
Meropenem MIC (mg/liter) |
|||
|---|---|---|---|---|---|---|
| OmpK35 | OmpK36 | Alone | With vaborbactam at: |
|||
| 4 mg/liter | 8 mg/liter | |||||
| E. coli EC1007 | KPC-3 | ND | ND | 8 | ≤0.06 | ≤0.06 |
| E. cloacae ECL1058 | KPC-3, SHV-11, TEM-1 | FL | FL | 8 | 1 | 0.125 |
| E. cloacae ECL1061 | KPC-3, AmpC hyperexpression | FS aa#287 | FL | 16 | 0.5 | 0.125 |
| E. cloacae ECL1079 | KPC-3 | Stop aa#60 | Stop aa#77 | >64 | 16 | 8 |
| K. pneumoniae KP1004 | KPC-2, TEM-1, SHV-11 | ND | ND | 16 | ≤0.06 | ≤0.06 |
| K. pneumoniae KP1061 | KPC-3, SHV-11, TEM-1 | FS aa#42 | FL | 16 | ≤0.06 | ≤0.06 |
| K. pneumoniae KP1074 | KPC-3, SHV-11, TEM | FS aa#42 | GD | >64 | 1 | 0.5 |
| K. pneumoniae KP1087 | KPC-2, CTX-M-15, SHV-11, TEM-1 | FS aa#208 | GD | 32 | 0.5 | 0.25 |
| K. pneumoniae KP1092 | SHV-11, TEM-1, KPC | FS aa#42 | IS at −45 | >64 | 64 | 32 |
| K. pneumoniae KP1093 | KPC-3, SHV-11, TEM | FS aa#42 | GD | >64 | 4 | 0.5 |
| K. pneumoniae KP1094 | KPC-2, TEM-1, LEN-17 | stop aa#230 | stop aa#92 | >64 | 32 | 4 |
| K. pneumoniae KP1096 | KPC-2, TEM, SHV-11 | L63V, E132K | IS at nt#126 | >64 | 64 | 16 |
| K. pneumoniae KP1099 | KPC-2 SHV-11 SHV-12 CTX-M-14 | FS aa#29 | GD | >64 | 4 | 1 |
| K. pneumoniae KP1100 | KPC-3 TEM SHV | FS aa#42 | GD | >64 | 8 | 4 |
| K. pneumoniae KP1194 | KPC TEM SHV | FS aa#42 | IS at −45 | >64 | 64 | 8 |
| K. pneumoniae KP1223 | KPC2, SHV, TEM | FS aa#29 | GD | >64 | 64 | 8 |
| K. pneumoniae KP1254 | KPC, SHV, TEM | FS aa#42 | IS and ΔopmK36 | >64 | ND | 64 |
ND, not determined; FL, full length (functional); stop aa#, nonsense mutations resulting in a truncated non-functional protein; FS aa#, frameshift mutation resulting in a nonfunctional protein; GD, insertion of two amino acids Gly134Asp135 resulting in a narrow semifunctional channel; IS at −45, promoter insertion of an IS element resulting in downregulation of expression; T333N is loss-of-function mutations in ompK36 of KP1096.
Pharmacokinetic parameters.
The target and observed pharmacokinetic parameters for meropenem and vaborbactam in the hollow-fiber model are shown in Table 2.
TABLE 2.
Simulated pharmacokinetic parameters based on phase 1 studies
| Parametera | Meropenem target | Meropenem actual | Vaborbactam target | Vaborbactam actual |
|---|---|---|---|---|
| 1 g of meropenem with 1 g of vaborbactam administered every 8 h by 3-h infusion | ||||
| Half-life (h) | 1 | 1.13 ± 0.03 | 1 | 1.29 ± 0.13 |
| Cmax (mg/liter) | 19.5 | 16.45 ± 0.30 | 15 | 13.99 ± 0.04 |
| AUC0–8 (h · mg/liter) | 70 | 58.69 ± 0.60 | 53 | 52.38 ± 2.05 |
| AUC0–24 (h · mg/liter) | 210 | 176.1 ± 1.80 | 159 | 157.14 ± 6.15 |
| 1 g of meropenem with 2 g of vaborbactam administered every 8 h by 3-h infusion | ||||
| Half-life (h) | 1 | 1.26 ± 0.00 | 1 | 1.34 ± 0.01 |
| Cmax (mg/liter) | 19.3 | 16.48 ± 0.75 | 30 | 28.54 ± 4.14 |
| AUC0–8 (h · mg/liter) | 70 | 58.15 ± 6.94 | 106 | 108.54 ± 15.97 |
| AUC0–24 (h · mg/liter) | 210 | 174.45 ± 20.83 | 318 | 325.25 ± 48.43 |
| 2 g of meropenem with 2 g of vaborbactam administered every 8 h by 3-h infusion | ||||
| Half-life (h) | 1 | 1.36 ± 0.07 | 1 | 1.47 ± 0.14 |
| Cmax (mg/liter) | 39 | 34.47 ± 3.95 | 30 | 24.1 ± 5.84 |
| AUC0–8 (h · mg/liter) | 140 | 132.63 ± 15.23 | 106 | 105.57 ± 4.42 |
| AUC0–24 (h · mg/liter) | 420 | 397.88 ± 45.70 | 318 | 316.7 ± 13.26 |
Cmax, maximum concentration of drug in serum; AUC0–8, AUC from 0 to 8 h; AUC0–24, AUC from 0 to 24 h.
When a dosage regimen of 1 g of meropenem with 1 g of vaborbactam administered every 8 h by a 3-h infusion was simulated, meropenem levels were above 4 mg/liter for 50% of the dosing interval. The average vaborbactam 24-h area under the concentration-time curve (AUC) was 157 mg · h/liter.
For a simulated regimen of 1 g of meropenem with 2 g of vaborbactam every 8 h by a 3-h infusion, meropenem concentrations were above 4 mg/liter for 50% of the dosing interval. The mean vaborbactam 24-h AUC across studies was 325 mg · h/liter.
For 2 g of meropenem with 2 g of vaborbactam every 8 h by a 3-h infusion using data from normal subjects enrolled in phase 1 studies, meropenem concentrations were above 8 mg/liter for 75% of the dosing interval and were above 16 mg/liter for 40 to 50% of the dosing interval. The mean vaborbactam 24-h AUC across studies was 317 mg · h/liter (Table 2).
The target and measured pharmacokinetic parameters for a regimen of 2 g of meropenem and 2 g of vaborbactam based on phase 3 studies are shown in Table 3. Meropenem exposures were unchanged from phase 1 data and were above 8 mg/liter for 75% of the dosing interval and above 16 mg/liter for 40 to 50% of the dosing interval. The vaborbactam 24-h AUC increased from ∼320 mg · h/liter to ∼550 mg · h/liter.
TABLE 3.
Simulated and measured pharmacokinetic parameters based on phase 3 studies from patients with CLCR >50a
| Parameter | Meropenem target | Meropenem actual | Vaborbactam target | Vaborbactam actual |
|---|---|---|---|---|
| Half-life (h) | 1 | 1.13 | 1 | 1.19 |
| Cmax (mg/liter) | 39 | 35.83 | 52 | 50.22 |
| AUC0–24 (h · mg/liter) | 420 | 383.43 | 550 | 547.29 |
Dosing was 2 g of meropenem with 2 g of vaborbactam administered every 8 h by 3-h infusion. CLCR, creatinine clearance.
Antibacterial activity.
A simulated dosage regimen of 1 g of meropenem with 1 g of vaborbactam (24-h free vaborbactam AUC, 157 mg · h/liter) was bactericidal against K. pneumoniae strains, with meropenem-vaborbactam MICs of less than 1 mg/liter (using 8 mg/liter vaborbactam) producing 5 to 6 logs of bacterial killing (Fig. 1). Of the strains tested, only K. pneumoniae KP1099 with a meropenem-vaborbactam MIC of 1 mg/liter (using 8 mg/liter vaborbactam) regrew at this exposure. The posttreatment MIC for this strain increased from 1 mg/liter to ≥32 mg/liter. The posttreatment MICs of the other strains tested with this dosage regimen did not change from those at pretreatment.
FIG 1.
Activity of simulated exposures similar to 1 g of meropenem with 1 g of vaborbactam based on phase 1 data administered every 8 h by a 3-h infusion against carbapenem-resistant KPC-containing Enterobacteriaceae. (MICs shown are meropenem-vaborbactam MICs with vaborbactam at 8 mg/liter). Tick marks on x axis are the start of each infusion.
When the simulated dosage regimen was modified to 1 g of meropenem with 2 g of vaborbactam (24-h free vaborbactam AUC, 325 mg · h/liter), the increased vaborbactam exposure produced 6 logs of bacterial killing against strain K. pneumoniae KP1099 and prevented the development of resistance (MIC increase) observed with 1 g of vaborbactam. The increased exposure of vaborbactam also produced 6 logs of bacterial killing against K. pneumoniae strains KP1094 and KP1100, both with meropenem-vaborbactam MICs of 4 mg/liter (using 8 mg/liter vaborbactam). No regrowth or change in MICs was detected in any of the strains tested (Fig. 2).
FIG 2.
Activity of simulated exposures similar to 1 g of meropenem with 2 g of vaborbactam based on phase 1 data administered every 8 h by a 3-h infusion against carbapenem-resistant KPC-containing Enterobacteriaceae. (MICs shown are meropenem-vaborbactam MICs with vaborbactam at 8 mg/liter). Tick marks on x axis are the start of each infusion.
A simulated dosage regimen of 2 g of meropenem with 2 g of vaborbactam (24-h free vaborbactam AUC, 317 mg · h/liter) produced 6 logs of bacterial killing against all strains tested, with meropenem-vaborbactam MICs of up to 8 mg/liter (using 8 mg/liter vaborbactam), and suppressed the development of resistance in these isolates based on a comparison of the MICs from pre- and posttreatment isolates (Fig. 3). This dosage regimen also produced over 4 logs of bacterial killing against K. pneumoniae KP1096, with a meropenem-vaborbactam MIC of 16 mg/liter (using 8 mg/liter vaborbactam), but after 10 h of exposure, a resistant population was selected with a meropenem-vaborbactam MIC of 64 mg/liter (using 8 mg/liter vaborbactam), and the strain regrew (Fig. 4). This dosage regimen was also able to produce 2 to 3 logs of bacterial killing against K. pneumoniae KP1092 and KP1254, with meropenem-vaborbactam MICs of 32 and 64 mg/liter (using 8 mg/liter vaborbactam), respectively. Both isolates regrew to the level of the starting inoculum 16 h after the start of treatment; however, changes in MICs were not observed (data not shown).
FIG 3.
Activity of simulated exposures similar to 2 g of meropenem with 2 g of vaborbactam based on phase 1 data administered every 8 h by a 3-h infusion against carbapenem-resistant KPC-containing Enterobacteriaceae. (MICs shown are meropenem-vaborbactam MICs with vaborbactam at 8 mg/liter). Tick marks on x axis are the start of each infusion.
FIG 4.
Activity of simulated exposures similar to 2 g of meropenem with 2 g of vaborbactam based on phase 1 data administered every 8 h by a 3-h infusion against carbapenem-resistant KPC-containing Enterobacteriaceae with meropenem-vaborbactam MICs of ≥8 mg/liter. (MICs shown are meropenem-vaborbactam MICs with vaborbactam at 8 mg/liter). Tick marks on x axis are the start of each infusion.
When the exposure of vaborbactam was increased to levels observed in patients enrolled in phase 3 trials (24-h free vaborbactam AUC, 550 mg · h/liter) (Table 3), 2 g of meropenem with 2 g of vaborbactam produced ∼6 logs of bacterial killing against K. pneumoniae KP1096 and suppressed the resistance development observed with the lower exposure of vaborbactam, with no changes in MICs between pre- and posttreatment isolates (Fig. 5).
FIG 5.
Activity of simulated exposures similar to 2 g of meropenem with 2 g of vaborbactam based on phase 3 data administered every 8 h by a 3-h infusion against carbapenem-resistant KPC-containing Enterobacteriaceae with meropenem-vaborbactam MICs of ≥8 mg/liter. (MICs shown are meropenem-vaborbactam MICs with vaborbactam at 8 mg/liter). Tick marks on x axis are the start of each infusion.
DISCUSSION
While meropenem has excellent stability with many bacterial beta-lactamases, resistance to meropenem can be mediated by class A serine carbapenemases, especially KPC. Vaborbactam is a novel cyclic boronic acid beta-lactamase inhibitor, which restores the activity of meropenem against several clinically important bacterial beta-lactamases, particularly the KPC.
The aim of these studies was to determine the exposures of meropenem and vaborbactam that would maximize efficacy and prevent the development of resistance over a 32-h experiment using a high inoculum (∼108 CFU/ml) in strains with meropenem-vaborbactam MICs of up to 16 mg/liter (with vaborbactam at 8 mg/liter).
The strains tested were selected to represent a broad range of potential resistance mechanisms that could affect the activity of meropenem-vaborbactam and included multiple beta-lactamases in combination with KPC, as well as multiple porin mutations. The loss or alteration in bacterial porin proteins changes the outer membrane permeability and the uptake of antibiotics, resulting a significant increase in bacterial resistance. In the case of K. pneumoniae, two major porins, OmpK35 and OmpK36 (12–15), have been demonstrated to facilitate the uptake of meropenem, and mutations or deletions in these porins are associated with meropenem resistance in clinical settings. In our studies, we used a wide variety of strains with or without a functional OmpK35/OmpK36. The functional OmpK35/OmpK36 strains were highly susceptible to meropenem-vaborbactam and had MICs of ≤0.06 mg/liter. However, meropenem-vaborbactam MICs against the strains with nonfunctional OmpK35/OmpK36 varied from ≤0.06 to 64 mg/liter. The two isolates with meropenem-vaborbactam MICs greater than 8 mg/liter, KP1092 and KP1254, had loss-of-function mutations to their OmpK36 porins.
The results from 3 strains (KP1096, KP1194, and KP1223) should be noted. In these strains, meropenem MIC testing with vaborbactam at 4 mg/liter reduced meropenem MICs to 32 to 64 mg/liter (Table 1). When tested with vaborbactam at 8 mg/liter, meropenem MICs were ≤8 mg/liter. Given the bacterial killing and lack of regrowth in the hollow fiber using a simulated dosage regimen of 2 g of meropenem plus 2 g of vaborbactam every 8 h, these data suggest that meropenem MIC testing with a fixed concentrations of vaborbactam at 8 mg/liter best predicts results with this dosage regimen.
The data obtained from these studies show that the simulated exposures produced from a 2-g dose of vaborbactam in combination with a 2-g dose of meropenem administered every 8 h by a 3-h infusion was sufficient to produce bacterial killing and suppress the development of resistance over the 32-h study in KPC-producing strains of Enterobacteriaceae with multiple mechanisms of carbapenem resistance in strains with a meropenem-vaborbactam MIC of up to 8 mg/liter (with vaborbactam at 8 mg/liter). In addition, when the exposure of vaborbactam was adjusted to that observed in patients enrolled in the phase 3 trials (24-h AUC, ∼500 versus 320 mg · h/liter in the phase 1 studies), a strain with a meropenem-vaborbactam MIC of 16 mg/liter (with vaborbactam at 8 mg/liter) was effectively treated with 6 logs of bacterial killing, and the resistance that developed with a lower exposure of vaborbactam was suppressed. These studies suggest that a dosage regimen of 2 g of meropenem plus 2 g of vaborbactam every 8 h by a 3-h infusion will be effective against Enterobacteriaceae strains with meropenem-vaborbactam MICs of 8 mg/liter (with vaborbactam at 8 mg/liter) and provide support for the study of subjects with infections due to carbapenem-resistant Enterobacteriaceae in phase 3 studies (ClinicalTrials.gov identifier no. NCT02168946).
MATERIALS AND METHODS
Antimicrobial agents.
Meropenem for injection (lot no. DF-3297; Sandoz) was purchased from commercial sources. Vaborbactam (lot no. P-232-159-2) was manufactured by The Medicines Company. Meropenem was dissolved according to the directions in the package insert. Vaborbactam was solubilized in water and the pH adjusted by the addition of NaOH. Both drugs were admixed and further diluted in Mueller-Hinton broth (MHB) for use in the model.
Bacterial isolates and susceptibility testing.
A total of 17 isolates were tested: 13 K. pneumoniae isolates, three E. cloacae isolates, and one E. coli isolate (Table 1). The strains were selected to represent a broad range of potential resistance mechanisms that could affect the activity of meropenem-vaborbactam and included multiple beta-lactamases in combination with KPC and mutations in porins OmpK35 and OmpK36. The MICs of meropenem alone or in combination with vaborbactam were determined using fixed concentrations of vaborbactam at 4 or 8 mg/liter pre- and posttreatment, according to CLSI reference methods (16). Briefly, assays were performed using a final volume of 100 μl. The inocula were adjusted to yield a final cell density of ca. 5 × 105 CFU/ml. Meropenem was diluted directly into 96-well microtiter plates by serial 2-fold dilutions, and then vaborbactam was added at a fixed concentration. Microtiter plates were read using a plate reader (Molecular Devices, Sunnyvale, CA) at 600 nm, as well as by visual observation using a reading mirror. The MIC was defined as the lowest concentration of antibiotic at which the visible growth of the organism was completely inhibited.
In vitro pharmacodynamic model.
The in vitro pharmacodynamic model consists of central and peripheral compartments (17). The peripheral compartments consist of artificial capillary units (cartridge C-3001; FiberCell Systems, Inc., Frederick, MD) arranged in series with the central compartment (18). Each capillary unit has a bundle of small semipermeable fibers with a molecular size retention of ca. 5,000 molecular weight (MW) to allow the passage of nutrients but not bacteria. The entire system was set up in a dry-heat incubator adjusted to 37°C and included six artificial capillary units so that three strains can be tested in duplicate. Drug doses were administered into the central compartment by a 3-h infusion and circulated to peripheral hollow-fiber units by peristaltic pumps (MasterFlex L/S; Cole-Parmer Instrument Co., Vernon Hills, IL).
Bacterial preparation and CFU determination.
Prior to inoculation into the hollow-fiber chambers, all strains were grown in MHB at 37°C under constant aeration. After 20 h, each inoculum was subcultured into fresh MHB and allowed to regrow at 37°C, under constant aeration, for 3 h to yield ∼108 CFU/ml by correlation of absorbance at 600 nm with predetermined plate counts. Three strains in duplicate were used in each experiment. Each bacterial suspension was injected into the peripheral chambers of two hollow-fiber units allowed to grow for 2 h. Samples were collected from the peripheral compartment at designated time points, serially diluted, and plated on Mueller-Hinton agar (MHA) plates. The limit of detection was 100 CFU/ml, with the exception of the 32-h time point, where the limit of detection was 1 CFU/ml.
Dosage regimens.
Dosage regimens studied were 1 g of meropenem with 1 g of vaborbactam, 1 g of meropenem with 2 g of vaborbactam, and 2 g of meropenem with 2 g of vaborbactam administered every 8 h by a 3-h infusion. Pharmacokinetic data for meropenem and vaborbactam were obtained from phase 1 studies and the literature (19, 20). Following the completion of population pharmacokinetic modeling from phase 3 studies, a dosage regimen of 2 g of meropenem with 2 g of vaborbactam using vaborbactam exposure from patients was also studied (21).
Pharmacokinetic evaluation.
Samples were collected at designated time points from the central compartment over a 32-h period and assayed for meropenem and vaborbactam concentrations. An equal volume of 3-(N-morpholino) propanesulfonic acid (MOPS) buffer (pH 7) was added to stabilize meropenem, and the samples were stored at −80°C until bioanalysis.
Bioanalytical assay.
Meropenem and vaborbactam standards for a bioanalytical assay were prepared in MHB at concentrations of 0.25 to 100 μg/ml. Twenty-microliter aliquots of sample were placed in 1.5-ml microcentrifuge tubes containing 20 μl of 10.0 μg/ml doripenem (internal standard for meropenem) in water, 20 μl of 5.0 μg/ml RPX7015 (internal standard for vaborbactam) in water, and 600 μl of water. The samples were mixed using a vortex mixer, and then 75 μl was added to 1,000 μl of water in a 96-well plate. The samples were mixed again using a vortex mixer. Twenty microliters of each sample was injected onto a high-performance liquid chromatography–mass spectrometer (HPLC-MS) for quantification using a 20-μl loop in loop overfill injection mode. Sample concentrations were fitted to a one-compartment pharmacokinetic model (Phoenix WinNonlin version 6.4; Certara USA, Inc., Princeton, NJ).
ACKNOWLEDGMENTS
This work, including the efforts of M. Sabet, Z. Tarazi, D. Rubio-Aparicio, T. G. Nolan, J. Parkinson, O. Lomovskaya, M. N. Dudley, and D. C. Griffith, was funded in part with federal funds from the Department of Health and Human Services, the Office of the Assistant Secretary for Preparedness and Response, and the Biomedical Advanced Research and Development Authority (BARDA), under contract no. HHSO100201400002C with Rempex Pharmaceuticals, a wholly owned subsidiary of The Medicines Company.
All authors meet the International Committee of Medical Journal Editors (ICMJE) criteria for authorship for the manuscript, take responsibility for the integrity of the work as a whole, and have given final approval to the version to be published.
M. Sabet, Z. Tarazi, T. G. Nolan, J. Parkinson, D. Rubio-Aparicio, O. Lomovskaya, M. N. Dudley, and D. C. Griffith are employees of The Medicines Company.
We acknowledge writing assistance provided by Starr Grundy of SD Scientific, Inc. that was funded by The Medicines Company.
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