Abstract
MK-826 (formerly L-749,345), is a potent 1-β-methyl carbapenem with a long half-life and broad spectrum of activity. This compound is presently in phase-II clinical trials. Its activity against a number of gram-positive and gram-negative organisms was compared to those of imipenem (IPM) and eight other β-lactam agents in two in vivo murine infection models. The distribution in tissue and pharmacokinetic properties of MK-826 and ceftriaxone (CTRX) were also evaluated in CD-1 mice following a single intraperitoneal dose (10 mg/kg of body weight). In addition, concentrations in plasma as well as biliary and urinary recovery of MK-826 were compared to that of CTRX in a cannulated rat model. In a localized murine thigh infection model, MK-826 and IPM were superior to a variety of β-lactam antibiotics in reduction of Staphylococcus aureus CFU compared with results from nontreated controls (eliminating ≥4 log10 CFU). Similar activities of IPM and MK-826 were observed in a gram-positive bacterial murine systemic infection model. While IPM demonstrated greater efficacy than MK-826 against Enterobacter cloacae (50% effective doses [ED50s] of 0.062 and 0.227 mg/kg, respectively) and Pseudomonas aeruginosa (ED50s of 0.142 and 3.0 mg/kg, respectively) systemic infections, MK-826 was 8- to 350-fold more efficacious than IPM against all other gram-negative organisms in this infection model. In mice, MK-826 demonstrated a higher peak concentration in serum (62.8 versus 42.6 μg/ml) and a larger area under the curve (AUC) (150.8 versus 90.0 μg · hr/ml) than CTRX. The concentrations of MK-826 and CTRX in serum declined slowly, with levels of 3.6 and 2.0 μg/ml remaining, respectively, at 6 h posttreatment. The rat pharmacokinetic model showed the average AUC of MK-826 to be greater than that of CTRX (284 versus 142 μg · hr/ml) following a single 10-mg/kg dose. Also, a half-life of MK-826 longer than that of CTRX (3.2 versus 2.3 h) was observed in this species. The total amount of drug excreted in the bile in 8 h was greater for CTRX (55 to 64% of the dose) than for MK-826 (6 to 12.5% of the dose). Urinary recovery was similar for both antibiotics, with 16 to 18% of the dose recovered over an 8-h period. This excellent broad-spectrum in vivo efficacy of MK-826, together with advantageous pharmacokinetics, supports the argument for its further clinical development.
An important mechanism of bacterial resistance to β-lactam antibiotics is inactivation by existing and evolving β-lactam-hydrolyzing enzymes (β-lactamases) (2, 16). The evolution of extended-spectrum β-lactamases (ESBLs) is associated with extensive use of β-lactam antibiotics, particularly broad-spectrum cephalosporins, and represents a serious threat to reliable therapy with these antibiotics (6, 8, 15). ESBLs are plasmid-encoded enzymes frequently found in gram-negative organisms such as Escherichia coli and Klebsiella pneumoniae. ESBLs are found throughout the world in hospital, long-term care, and community settings.
MK-826 (formerly L-749,345) is a new broad-spectrum 1-β-methyl carbapenem antibiotic with an extended in vivo half-life (t1/2) (Fig. 1). Its antimicrobial activity demonstrates that this antibiotic is resistant to hydrolysis by bacterial, plasmid, and chromosomally mediated non-metallo-β-lactamases and is able to penetrate the cell wall of clinically significant human pathogens (11). MK-826 exhibits extensive reversible protein binding, which is a basis for its extended duration of in vivo activity. Due to its improved stability to renal dehydropeptidase-I (DHP-I), MK-826 can be administered as a single agent, achieving high enough levels in urine to provide more than adequate coverage of bacteria in the urinary tract (21).
FIG. 1.
Chemical structure of MK-826.
This report compares the in vivo efficacy of this novel compound with those of other β-lactam antibiotics against a number of gram-positive and gram-negative organisms in a localized (thigh) methicillin-susceptible Staphylococcus aureus (MSSA) infection as well as in systemic infection models. Also the distribution in tissue and pharmacokinetic properties of MK-826 were studied in both mouse and rat models.
(Part of this work was presented in abstract form at the 36th Interscience Conference on Antimicrobial Agents and Chemotherapy, 15 to 18 September 1996, New Orleans, La. [3, 4].)
MATERIALS AND METHODS
Bacterial strains and preparation.
A 10-h broth culture of S. aureus MB2985 (Smith) was washed once by centrifugation (5,000 rpm for 20 min at 4°C, Sorvall RC5 rotor). The cell pellet was reconstituted in half the original volume (∼109 CFU/ml) and further diluted (1:5) with Trypticase soy broth for use as the challenge inoculum in the thigh infection model. Except for Streptococcus pneumoniae (MB212, CL4997, CL4983, and CL5631) and Streptococcus pyogenes (MB2874), all bacterial cultures used in the systemic infection models were grown in brain heart broth (BHB) for 10 h on a shaker (250 rpm) at 35°C. The streptococci were grown stationary, overnight (16 to 18 h), under 5% CO2 in BHB plus 10% horse serum. Further dilutions of the cultures to prepare the infectious inocula are listed in Table 1.
TABLE 1.
Bacterial inocula used in the systemic infection model
| Challenge strain | Dilutiona | Inoculum size (CFU/mouse) | No. of LD50sb |
|---|---|---|---|
| S. aureus MB2985 | 1:25 BHB | 2 × 107 | 65 |
| S. pneumoniae MB212 | 1:105 BHB | 8 × 102 | 572 |
| S. pneumoniae MCL4997 | 1:25 BHB | 1.9 × 106 | 510 |
| S. pneumoniae MCL4983 | 1:25 BHB | 1.9 × 106 | 140 |
| S. pneumoniae MCL5631 | 1:25 BHB | 1.4 × 106 | 11 |
| S. pyogenes MB2874 | 1:106 BHB | 1.9 × 102 | 91 |
| E. cloacae MB2646 | 1:5 × 103 HGM | 1.4 × 105 | 9 |
| E. coli MB2884 | 1:105 HGM | 3.5 × 103 | 572 |
| K. pneumoniae MB2883 | 1:5 × 103 HGM | 1.6 × 105 | 516 |
| M. morganii MB2833 | 1:5 × 105 HGM | 5.3 × 102 | 104 |
| P. aeruginosa MB2835 | 1:5 × 104 HGM | 5.2 × 104 | 215 |
| P. aeruginosa MB3286 | 1:5 × 104 HGM | 3.3 × 104 | 19 |
| P. mirabilis MB3125 | 1:104 HGM | 2.6 × 105 | 297 |
| S. marcescens MB3548 | 1:5 × 102 HGM | 1.3 × 106 | 1,400 |
Bacterial cultures diluted in either BHB or 5% hog gastric mucin (HGM).
Challenge inoculum size equals this multiple of the LD50 inoculum.
Antibiotic preparation.
Imipenem (IPM), meropenem (MER), cefepime (FEP), ceftriaxone (CTRX), ceftazidime (CAZ), cefazolin (CEF), cefonicid (CID), cefotaxime (CTX), and penicillin G (PEN) were either commercially available or were synthesized at Merck Research Laboratories. MK-826 (ZD4433) was made by Merck or Zeneca Pharmaceutical Co. Ltd. Antibiotic stock solutions were prepared in 10 mM morpholinopropanesulfonic acid (MOPS) buffer, pH 7.1, and stored at −70°C. For all carbapenems, unless otherwise stated, the MOPS buffer described above also contained cilastatin at a final concentration of 2.0 mg/ml. Injection of 0.5 ml of this solution yields a final concentration of cilastatin of 40 mg/kg of body weight/mouse for each antibiotic treatment regardless of antibiotic concentration. In one experiment, the efficacy of MER was evaluated in the presence and absence of cilastatin. In the thigh model, antibiotic test concentrations generally ranged from 0.5 to 10 mg/kg/dose. In the systemic models, frozen stock solutions were diluted to yield a series of fourfold antibiotic concentrations. In the pharmacokinetic studies, 10-mg/kg solutions of MK-826–cilastatin and CTRX were prepared in 10 mM MOPS buffer.
In vitro MICs of test compounds against the test organisms were determined according to procedures recommended by the National Committee for Clinical Laboratory Standards (14).
Animals.
DBA/2 female mice (Taconic Laboratories, Germantown, N.Y.) weighing 25 ± 1 g were used in the thigh infection model. DBA/2 female mice (19 to 21 g; Taconic) and viral antibody-free CD-1 female mice (Charles River Laboratories, Wilmington, Mass.) weighing 19 to 21 g were used in the systemic infection models. CD-1 mice (19 to 21 g) and female Sprague-Dawley rats (Sasco, St. Louis, Mo.) weighing 235 to 305 g were used in the pharmacokinetic assays.
All animal procedures were performed in accordance with the highest standards for the humane handling, care, and treatment of research animals and were approved by the Merck Institutional Animal Care and Use Committee. The care and use of research animals at Merck meet or exceed all applicable local, state, and federal laws and regulations.
Localized soft tissue infection model.
On day 0, 0.2 ml of the challenge inoculum was injected intramuscularly into the right thigh of each mouse. Antibiotic therapy was initiated 2 h after challenge (0 h), i.e., after the first of eight antibiotic treatments, was administered subcutaneously (s.c.) (0.5 ml) into the upper back. Subsequent antibiotic dosing was performed at 6, 10, 24, 48, 72, 96, and 120 h postinfection for a total of eight doses over a 5-day period. Two days after the final antibiotic dose (day 7) mice were euthanatized, the lower abdominal areas and thighs were flushed with 70% ethanol, and the outer skin was removed. Denuded thighs were then surgically removed aseptically and placed in sterile tubes containing 4 ml of sterile phosphate-buffered saline–10% glycerol for temporary storage at −70°C or for same-day processing. Usually, plating for bacterial counts was done at a later date. On the day of plating, the tubes were thawed in cold water and thighs were ground with a Polytron homogenizer (Brinkmann Instruments) for ∼5 to 10 s each and placed on ice to await further dilution. Tenfold serial dilutions of each sample were made in tubes containing 0.9 ml of cold TSB prior to plating 0.1 ml from selected dilutions onto staphylococcus-selective mannitol salt agar plates. Plates were incubated for 2 days at 35°C to determine the number of CFU remaining per thigh. The geometric mean of the CFU remaining and the log10 change in CFU were also determined for each group.
Systemic infection models.
Mice were challenged intraperitoneally (i.p.) with the inocula described above in 0.5 ml. All antibiotics were prepared from frozen stock cultures to yield a series of fourfold antibiotic concentrations, which were maintained at 4°C for dosing (0.5 ml; s.c.) of five mice per group. Mice were monitored daily for morbidity and mortality over the 7-day test period.
The 50% effective doses (ED50s) of antibiotics and 50% lethal doses (LD50s) of bacteria were determined by the method of Knudsen and Curtis by using a computer program written in Microsoft Quick Basic (9).
Mouse tissue drug distribution and pharmacokinetic studies.
At time zero, groups of 50 CD-1 female mice were injected (0.5 ml; i.p.) with a 10-mg single dose of either MK-826 or CTRX. Cilastatin was coadministered with MK-826 to improve its β-terminal elimination plasma t1/2 (t1/2β) and urinary recovery due to extra renal DHP-I metabolism of carbapenems encountered in rodent species (12). It may be important that in mice, the half-life of IPM in plasma was increased approximately twofold when administered with cilastatin and that of MK-826 was increased nearly threefold (16 to 44 min). Mice were given a 0.5-ml oral dose of water to stimulate urine flow just prior to placement in metabolism cages (five per cage) designed to collect urine free from fecal contamination. At 1, 5, 15, 30, 60, 120, 180, 240, 360, and 1,440 min post-antibiotic treatment, pooled blood samples from each group were allowed to clot, and serum was collected at 4°C, following centrifugation at 10,000 rpm for 10 min (Beckman Microfuge 12 rotor). The volume and pH of urine samples collected during the same time intervals also were recorded. Following blood collection, the left kidney, liver, lung, spleen, brain, heart, and right thigh muscle were removed from individual mice, weighed, and pooled by test groups and tissue types prior to homogenization in four volumes (wt/vol) of buffer to a concentration of ∼200 mg of tissue/ml. CTRX samples were homogenized in Sorensen’s buffer, pH 7.1, while MK-826 samples were prepared in 25 mM 2-(N-morpholino)ethanesulfonic acid (MES) buffer (pH 5.5)–3% MeOH containing cilastatin (50 μg/ml). Urine samples were stored at −70°C until bioassayed, while serum and other tissue samples were placed on ice for immediate assay.
Determination of antibiotic content in fluids and tissues.
A direct-injection, column-switching, reversed-phase high-performance liquid chromatography technique was used to assay serum and other tissue samples. Stabilized samples were injected onto a short pellicular C18 column and washed with 0.15% trifluoroacetic acid (clean-up mobile phase). This clean-up column was then switched in line with an analytical C18 column (4.6 by 250 mm; 40°C; Zorbax SB-C18) and the chromatogram was developed at 1 ml/min with an acetonitrile-methanol gradient in 0.1% trifluoroacetic acid. MK-826 and CTRX were detected at 307 and 254 nm, respectively, with a 165 Variable Wavelength Detector (Beckman Instruments, Palo Alto, Calif.). The limit of detection was ∼0.39 μg/ml of serum for both MK-826 and CTRX and 0.25 to 0.63 and 0.49 to 0.98 μg/g of tissue for MK-826 and CTRX, respectively.
Standard agar disk diffusion procedures were used to determine the bioactivity of each antibiotic recovered in urine. The MK-826 bioassay employs a Bacillus subtilis MB32 spore suspension (Difco, Detroit, Mich.), while the procedure for CTRX uses K. pneumoniae MB1264, both in Mueller-Hinton agar. The total bioactivity of each sample was calculated from the regression line of the appropriate standard curve (MK-826, R2 = 0.9999; CTRX, R2 = 0.9983). The coefficients of variation for the MK-826 and CTRX assays were 1.6 and 1.5%, respectively. Urine assay sensitivities ranged from 0.2 to 0.3 μg/ml for both antibiotics.
Rat plasma, bile, and urine antibiotic concentration studies.
Female Sprague-Dawley rats were anesthetized i.p. with ∼50 mg of pentobarbitol sodium (Nembutol; Abbott, Chicago, Ill.) per kg. The surgical areas of each rat were shaved and swabbed with providone-iodine-alcohol. A small horizontal incision (3 to 4 cm) was made below the xiphoid process. The bile duct was exposed and a cannula of Silastic tubing was threaded into the duct and anchored with ligatures. The abdominal incision was closed with stainless steel wound clips. To facilitate blood collection, an incision was made in the right hind leg of the rat, exposing the femoral artery. A sterile PE 10 catheter was fed into the abdominal aorta and secured in place with silk ligatures. Once normal bile flow was established, each rat was given either an s.c. dose of cilastatin (40 mg/kg) followed by an intravenous (i.v.) injection of MK-826 or an i.v. injection of CTRX in the femoral artery at a dose equivalent to 10 mg/kg. Heparinized blood samples were taken at 5, 30, 60, 120, 240, 360, and 480 min postdose. The plasma was separated by centrifugation and stabilized with an equal volume of ethylene glycol-MES buffer mixture and frozen at −70°C until bioassayed. Bile samples were collected continuously at 0 to 30, 30 to 120, 120 to 360, and 360 to 480 min post-antibiotic treatment in tubes suspended in an ice-water bath. Each sample was stabilized with an equal volume of ethylene glycol-MOPS buffer and frozen at −70°C. At the termination of the study, residual urine was removed from the bladder.
The total bioactivities of the plasma, bile, and urine samples were determined by the standard agar disk diffusion methods described above. The area under the plasma concentration curve (AUC) versus the AUC from 0 to 8 h (AUC0–8) and the AUC from 0 h to infinity (AUC0–∞), the t1/2β, and the plasma clearance (Clp) were calculated.
RESULTS
Localized infection model.
In the S. aureus thigh tissue infection model MK-826, IPM, FEP, and CTRX were all efficacious at 10 mg/kg, with >3 log10 CFU reduction of organism compared to non-antibiotic-treated controls. However, at a fivefold-lower concentration (2 mg/kg), only MK-826 and IPM, with 3.3 and 4.4 log10 CFU eliminated, respectively, maintained this activity (Fig. 2). MER, while not as active as MK-826 or IPM, did exhibit some activity at all levels tested. Interestingly, >4 logs of bacterial clearance was observed with 5 mg of MER per kg in the presence of cilastatin while a <1 log10 CFU decrease was observed with the same dose of MER in the absence of cilastatin (Fig. 3). CAZ and CTX were not efficacious in this MSSA localized infection model.
FIG. 2.
Efficacy of MK-826 and other agents in the mouse thigh model: experiment 1. DBA/2 mice were challenged (0.2 ml; i.p.) with S. aureus MB2985 in the right thigh at 0 h. Antibiotic treatment (0.5 ml; s.c.) was given at 2, 6, 10, 24, 48, 72, 96, and 120 h after challenge. Circles show individual values for mouse 1 to 5; bars show geometric means.
FIG. 3.
Efficacy of MK-826 and other agents in the mouse thigh model: experiment 2. DBA/2 mice were challenged (0.2 ml; i.p.) with S. aureus MB2985 in the right thigh at 0 h. Antibiotic treatment (0.5 ml; s.c.) was given at 2, 6, 10, 24, 48, 72, 96, and 120 h after challenge. Cilastatin was used except where noted. Circles show individual values for mouse 1 to 5; bars show geometric means.
Systemic model.
MK-826 exhibited good activity against all gram-positive organisms tested in the systemic infection model, including highly PEN-resistant pneumococci (Table 2). While not as potent as IPM or CEF against S. aureus (MSSA), MK-826 was two- to fourfold more active than FEP, CID, and CTRX. All four compounds tested against a PEN-susceptible strain of S. pneumoniae were equally efficacious, while MK-826 and CTRX were slightly more active than IPM and PEN against the moderately susceptible strain. IPM was the most active agent against the two PEN-resistant S. pneumoniae strains tested, while MK-826 and CTRX were equipotent against these strains. MK-826 was approximately twofold more active than FEP and CTRX against S. pyogenes. MK-826 was also very active against the gram-negative organisms tested with ED50s of <0.25 mg/kg/dose for Enterobacter cloacae, E. coli, K. pneumoniae, M. morganii, Proteus mirabilis, and Serratia marcescens (Table 3). MK-826 exhibited good activity against two Pseudomonas aeruginosa isolates tested, with ED50s of 1.2 and 3.0 mg/kg/dose, respectively.
TABLE 2.
In vivo efficacy of MK-826 and other agents against gram-positive organisms in a murine systemic infection model
| Challenge strain | Com- pound | MICa (μg/ml) | ED50 (95% CL)b |
|---|---|---|---|
| S. aureus MB2985 (MSSA)c | MK-826 | 0.125 | 1.580 (0.800–3.100) |
| IPM | 0.008 | 0.038 (0.022–0.066) | |
| CEF | 0.250 | 0.460 (0.260–0.800) | |
| FEP | 1.000 | 2.940 (1.490–5.800) | |
| CID | 1.000 | 6.620 (3.360–13.060) | |
| CTRX | 2.000 | 6.250 (ND) | |
| S. pneumoniae MB212d | MK-826 | 0.031 | 0.156 (ND) |
| IPM | 0.004 | 0.052 (0.030–0.090) | |
| FEP | 0.016 | 0.432 (0.140–1.310) | |
| CTRX | 0.016 | 0.094 (0.050–0.160) | |
| S. pneumoniae MCL4997c | MK-826 | 0.125 | 3.190 (1.330–7.660) |
| IPM | 0.062 | 8.010 (3.330–19.240) | |
| CTRX | 0.500 | 3.300 (1.920–5.820) | |
| PEN | 0.500 | 15.110 (8.680–26.300) | |
| S. pneumoniae MCL4983d | MK-826 | 2.000 | 12.500 (ND) |
| IPM | 0.250 | 5.230 (3.010–9.110) | |
| CTRX | 1.000 | 10.340 (5.940–18.010) | |
| PEN | 2.000 | 85.430 (43.320–168.480) | |
| S. pneumoniae MCL5631c | MK-826 | 2.000 | 12.500 (ND) |
| IPM | 0.500 | 2.450 (1.240–4.840) | |
| CTRX | 2.000 | 5.900 (3.000–11.600) | |
| PEN | 4.000 | 41.400 (23.760–72.040) | |
| S. pyogenes MB2874d | MK-826 | 0.008 | 0.059 (0.030–0.116) |
| IPM | 0.004 | 0.012 (0.006–0.023) | |
| FEP | 0.016 | 0.100 (0.042–0.240) | |
| CTRX | 0.016 | 0.120 (0.055–0.263) |
MIC determined by procedures recommended by the National Committee for Clinical Laboratory Standards (14).
The ED50 (calculated on day 7) that protects 50% of infected mice and the 95% confidence limit (95% CL) (both in milligrams per kilogram) are reported. ND, not defined.
Challenge with test organism (0.5 ml; i.p.) and treatment (0.5 ml; s.c.) begun immediately after infection (total of 1 dose).
Challenge with test organism (0.5 ml; i.p.) and treatment (0.5 ml; s.c.) begun immediately and 4 h after infection (total of 2 doses).
TABLE 3.
In vivo efficacy of MK-826 and other agents against gram-negative organisms in a murine systemic infection model
| Challenge strain | Com- pound | MICa (μg/ml) | ED50 (95% CL)b |
|---|---|---|---|
| E. cloacae MB2646c | MK-826 | 0.500 | 0.227 (ND) |
| IPM | 0.250 | 0.062 (0.024–0.161) | |
| FEP | 4.000 | 0.235 (0.135–0.410) | |
| CTRX | ≥256.000 | 30.400 (17.400–52.900) | |
| E. coli MB2884 | MK-826 | 0.008 | 0.022 (ND) |
| IPM | 0.125 | 0.172 (ND) | |
| FEP | 0.031 | 0.013 (0.006–0.028) | |
| CAZ | 0.125 | 0.062 (ND) | |
| CTRX | 0.031 | 0.028 (ND) | |
| K. pneumoniae MB2883 | MK-826 | ≤0.016 | 0.057 (ND) |
| IPM | 0.125 | 6.080 (3.490–10.580) | |
| FEP | ≤0.016 | 1.880 (1.080–3.280) | |
| CTRX | 0.031 | 0.033 (0.015–0.072) | |
| M. morganii MB2833 | MK-826 | 0.008 | 0.034 (0.020–0.060) |
| IPM | 2.000 | 3.400 (1.950–5.920) | |
| FEP | 0.016 | 0.074 (0.034–0.161) | |
| CAZ | 0.125 | 0.318 (0.160–0.630) | |
| CTRX | 0.008 | ≤0.018 (ND) | |
| P. aeruginosa MB2835 | MK-826 | 1.000 | 1.215 (0.698–2.116) |
| IPM | 1.000 | 0.455 (ND) | |
| FEP | 8.000 | ≥9.100 (ND) | |
| CAZ | 1.000 | 8.756 (3.997–19.180) | |
| P. aeruginosa MB3286 | MK-826 | 8.000 | 3.000 (1.060–8.470) |
| IPM | 2.000 | 0.142 (0.059–0.341) | |
| FEP | 2.000 | 0.422 (0.170–1.010) | |
| CAZ | 2.000 | 0.394 (0.151–1.030) | |
| P. mirabilis MB3125 | MK-826 | 0.031 | 0.034 (0.020–0.060) |
| IPM | 4.000 | 12.200 (6.980–21.200) | |
| FEP | 0.031 | 0.071 (ND) | |
| CTRX | 0.031 | ≤0.004 (ND) | |
| S. marcescens MB3548 | MK-826 | 0.500 | 0.121 (0.061–0.238) |
| IPM | 1.000 | 4.720 (2.150–10.340) | |
| FEP | 0.500 | 0.535 (0.271–1.054) | |
| CTRX | 0.500 | 0.469 (0.270–0.817) |
MIC determined by procedures recommended by the National Committee for Clinical Laboratory Standards (14).
The ED50 (calculated on day 7) that protects 50% of infected mice and the 95% confidence limit (95% CL) (both in milligrams per kilogram) are reported. ND, not defined.
Challenge with test organisms (0.5 ml; i.p.) and treatment (0.5 ml; s.c.) begun immediately and 4 h after infection (total of 2 doses).
Mouse pharmacokinetic studies.
The serum and urine parameters of MK-826 (with cilastatin) and CTRX following i.p. administration of a 10-mg/kg dose in CD-1 mice were similar, with peak concentrations in serum of 62.8 μg/ml at 30 min for MK-826 and 42.6 μg/ml at 15 min for CTRX. The AUC0–∞ was 150.8 μg · hr/ml for MK-826, compared to 90.0 μg · hr/ml for CTRX. The t1/2s in serum were also similar for MK-826 (1.27 h) and CTRX (1.22 h). The Clp for MK-826 was 1.11 ml/min/kg, while a slightly higher rate was seen for CTRX (1.85 ml/min/kg). The volume of distribution for CTRX, 170 ml/kg, was slightly higher than that of MK-826 (107 ml/kg). Urinary recoveries over a 24-h period were similar, at 46% for MK-826 and 49% for CTRX.
The distribution of MK-826 and CTRX in selected mouse tissues occurred rapidly from the vascular compartment (Table 4). At 1 h postdose, levels of both antibacterials were detected in kidney, liver, lung, spleen, thigh, and heart tissue. Levels in tissue were low in comparison to levels in serum; however, detectable amounts of MK-826 and CTRX were still present in kidney, liver, lung, and heart tissue 6 h postdose.
TABLE 4.
Distribution of MK-826 and CTRX in tissue in mice following a 10-mg/kg i.p. dosea
| Tissue | AUC0–∞ (μg · hr/ml)
|
|
|---|---|---|
| MK-826 | CTRX | |
| Kidney | 19.98 | 18.24 |
| Liver | 16.75 | 22.23 |
| Lung | 21.82 | 31.89 |
| Spleen | 6.33 | 8.38 |
| Thigh | 6.86 | 7.80 |
| Heart | 17.18 | 16.65 |
| Brain | 1.80 | NDb |
Distribution in tissue determined by high-performance liquid chromatography. Tissue samples were obtained from a group of five mice for each period.
ND, not defined.
Rat pharmacokinetic studies.
Results of the pharmacokinetic distribution of MK-826 (with cilastatin) and CTRX in rats are shown in Table 5. MK-826 was cleared very slowly from the plasma of all three rats, with an average concentration of 15.10 μg/ml remaining 8 h posttreatment. The t1/2β averaged 3.2 h. The Clp averaged 0.47 ml/min/kg. The biliary recoveries in two of three rats were the same, at 12%, compared with only 6% in the third rat. This may have been due to cannulation of the bile duct above the bifurcation, resulting in collection of only half the available bile. Difficulties in establishing normal bile flow in one rat resulted in CTRX analysis in only two rats. Five minutes following i.v. administration of CTRX, an average concentration of 175 μg/ml was observed. At 8 h postdose measurable levels of CTRX in plasma were considerably lower than those seen with MK-826. In addition, the average plasma t1/2 was 2.3 h, compared to 3.2 h for MK-826. As expected, >55% of CTRX was recovered in the bile over the 8-h test period. Urinary recoveries of MK-826 and CTRX were similar, with 18 and 16%, respectively, recovered in 8 h.
TABLE 5.
Pharmacokinetic profile of MK-826 and CTRX in rats following a 10-mg/kg i.v. dosea
| Com- pound | Concn in plasma at time (h)b:
|
AUC0–8 (μg · h/ml) | AUC0–∞ (μg · h/ml) | t1/2β (h) | Clp (ml/min/kg) | % Biliary recovery at time (h):
|
% Urinary recovery (0–8 h) | ||||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| 0.08 | 0.5 | 1 | 2 | 4 | 6 | 8 | 0–0.5 | 0.5–2 | 2–6 | 6–8 | 0–8 | ||||||
| MK-826 | 82.40 | 66.60 | 56.61 | 43.49 | 32.91 | 23.26 | 15.10 | 284.15 | 354.06 | 3.19 | 0.47 | 2.60 | 4.20 | 3.12 | 0.42 | 10.34 | 18.21 |
| CTRX | 174.47 | 68.95 | 36.40 | 19.90 | 5.92 | 2.58 | 1.43 | 142.28 | 147.28 | 2.33 | 1.13 | 22.83 | 22.30 | 13.62 | 1.09 | 59.84 | 16.39 |
MK-826 (10 mg/kg; i.v.) was administered into the right femoral artery with cilastatin (40 mg/kg; s.c.). CTRX was administered in a manner similar to that for MK-826 but minus cilastatin. Values for MK-826 and CTRX are averages for three and two rats, respectively.
Determined by the disk diffusion method using B. subtilis MB32 and K. pneumoniae MB1264 as the assay organisms for MK-826 and CTRX, respectively. 0.08 h = 5 min.
DISCUSSION
Multidrug-resistant bacteria, especially those harboring ESBLs, are of increasing clinical concern (1). These emerging nosocomial pathogens leave clinicians with few treatment options. A once-a-day antibiotic with broad-spectrum gram-positive and gram-negative activity, including activity against organisms with ESBLs, could offer a solution to this problem. MK-826 is a long-acting carbapenem antibiotic which possesses such a broad antibacterial spectrum (5, 7, 10).
In a thigh tissue infection model, MK-826 was highly efficacious, at a level as low as 2 mg/kg, in organism clearance. Neither the cephalosporins nor MER was as active at this level. CTRX and FEP were equally efficacious as MK-826 at 10 mg/kg, but their activities were significantly reduced at lower concentrations. MK-826 also exhibited potent activity in the systemic models of infection against a variety of both gram-positive and gram-negative organisms. Except for one PEN-resistant S. pneumoniae isolate (MCL4997), IPM was the most active agent against all the gram-positive isolates tested. MK-826 in most cases was either equipotent or more efficacious than either CTRX or FEP against these organisms. MK-826 also was highly efficacious against the gram-negative organisms in this infection model. With the exception of P. aeruginosa the ED50s of MK-826 obtained for these organisms were all <0.25 mg/kg/dose. MK-826, while not as efficacious as IPM, did exhibit some activity against two P. aeruginosa isolates tested. These in vivo outcomes accurately confirm the in vitro susceptibility endpoints. In vitro studies have found MK-826 to be very potent against other bacterial species (3, 18, 19). The extensive protein binding of MK-826, while a major factor in its long t1/2, is not deleterious to its in vivo efficacy against organisms for which the MICs are low. As Craig et al. (1) have shown, the most important factor influencing the efficacy of β-lactams, including carbapenems, is the amount of time when drug concentrations are above the MIC. Carbapenems were found to exert maximal cell killing when levels in serum were above the MIC for at least 40% of the dosing interval. Thus, the lower the MIC is, the more negligible the effect of protein binding is, since less free drug would be required to eradicate the organism and more would be available, extending the time when the concentration is above the MIC.
Pharmacokinetically the serum and urine parameters of MK-826 (with cilastatin) and CTRX in mice were similar (4). A slightly higher peak concentration in serum and a larger AUC was observed for MK-826 compared to that of CTRX. Urinary recovery was the same for both compounds, and distribution in tissue was effective and sustained. In rats, the average AUC for MK-826 (with cilastatin) was greater than that of CTRX and the calculated t1/2 of MK-826 was approximately twice as long as that for CTRX. Urinary recovery again was similar for both antibiotics. Like CTRX, MK-826 was eliminated via the hepato-biliary route; however, the total amount of drug excreted over 8 h (55 to 64% versus 6 to 12%) was greater for CTRX than for MK-826. These levels for CTRX were slightly higher than the 40 to 50% obtained in humans by other investigators (17, 20). Similar comparative results of MK-826 and CTRX were obtained in nonhuman primates (22). Presently, results from phase-I clinical trials indicate a mean t1/2β of 4.5 h and an average 48-h urinary excretion of intact MK-826 of 30 to 45% (13).
In summary, MK-826 is a new, long-acting, 1-β-methyl carbapenem presently in phase-II clinical trials. It exhibits potent activity which is comparable or superior to established agents against both gram-positive and gram-negative organisms in systemic and tissue infection models of disease. Importantly, MK-826 exhibits a broad antibacterial spectrum that includes activities against organisms which harbor ESBLs. Advantageous pharmacokinetics, including an extended t1/2β and improved stability to renal DHP-I, support the argument for further development of MK-826, perhaps as a single once-a-day dosing agent.
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