Antimicrobial Agents and Chemotherapy, May 2004, p . 1708-1712, Vol . 48, No . 5

In Vivo Efficacy and Pharmacokinetics of AC98-6446, a Novel Cyclic Glycopeptide, in Experimental Infection Models

William J . Weiss,^1* Timothy Murphy,¹ Eileen Lenoy,¹ and Mairead Young²

Infectious Disease Section,¹ Discovery Analytical Chemistry, Wyeth Research, Pearl River, New York 10965²

Received 8 October 2003/ Returned for modification 19 December 2003/ Accepted 29 January 2004

The methicillin-resistant staphylococci (MRSA) are prevalent in most major medical centers, compromising a large percentage of staphylococcal infections encountered . The incidence of these isolated pathogens has continued to increase . Their prevalence varies by geographic regions, but they remain a clinical problem due to multidrug resistance (22) . Decreased susceptibility to erythromycin, gentamicin, tetracycline, rifampin, and the fluoroquinolones severely limits the therapeutic options for treatment of patients with severe MRSA infections (6) . Glycopeptide-intermediate Staphylococcus aureus strains were first identified in the late 1990s, with the first reported vancomycin-resistant strain recently isolated from a patient in a Michigan medical center (14) . Staphylococcal strains with reduced susceptibility to vancomycin have now been identified in several countries (3, 13) .

Glycopeptide resistance in enterococci is recognized as a clinical problem of increasing importance (7) . In particular, vancomycin-resistant Enterococcus (VRE) represents an important nosocomial pathogen capable of spread from colonized infected patients to uncolonized ones (30) . Since it was first isolated in Europe in 1986, VRE has reached upwards of 26% in isolates tested from intensive care units in the United States (2) . The intrinsic resistance of the enterococci precludes the use of conventional antibiotic therapy (1, 21) .

AC98-6446 (Fig . 1), the ketal derivative of a core-modified mannopeptimycin, is a semisynthetic glycopeptide derived from Streptomyces hygroscopicus (8, 28) . Esterified mannopeptimycins produced from this organism have demonstrated excellent in vitro activity . MICs of 0.03 to 0.06 µg/ml have been exhibited by AC98-6446 against methicillin-susceptible and -resistant staphylococci . In vitro activity against vancomycin-susceptible and -resistant enterococci has been demonstrated at concentrations of 0.12 to 0.25 µg/ml and 0.008 µg/ml for Streptococcus pneumoniae (P . Petersen, P . Labthavikul, T . Wang, R . Dushin, and P . Bradford, Abstr . 42nd Intersci . Conf . Antimicrob . Agents Chemother., abstr . F-353, 2002) . Additionally, AC98-6446 has been shown to be bactericidal, exhibits a long (2 to 4 h) gram-positive postantibiotic effect (P . Petersen, H . Hartman . T . Wang, R . Dushin, and P . Bradford, Abstr . 42nd Intersci . Conf . Antimicrob . Agents Chemother., abstr . F-354, 2002), and exhibits limited inoculum effect and activity against adherent cells in a biofilm (P . Labthavikul, P . Petersen, T . Wang, R . Dushin, and P . Bradford, Abstr . 42nd Intersci . Conf . Antimicrob . Agents Chemother., abstr . F-355, 2002) . These properties make it a potential candidate for treatment of serious infection caused by resistant staphylococci and enterococci .

 
The present study was undertaken to evaluate the in vivo efficacy of AC98-6446 in several experimental infection models with these organisms as well as to assess the pharmacokinetics of the compound in several species .

(Data from this study were presented in part at the 42nd Interscience Conference on Antimicrobial Agents and Chemotherapy, San Diego, Calif., 27 to 30 September, 2002 [T . Murphy, E . Lenoy, M . Young, and W . Weiss, Abstr . 42nd Intersci . Conf . Antimicrob . Agents Chemother., abstr . F-356, 2002].)

Compounds. AC98-6446 was prepared by Wyeth Research (Pearl River, N.Y.), and vancomycin was obtained from Sigma Chemical Corp . (St . Louis, Mo.) . AC98-6446 was prepared in 5% dextrose water, and vancomycin was prepared in sterile saline for all the models performed .

Acute lethal infection model. Female mice, strain CD-1 (Charles River Laboratories, Kingston, N.Y.), 20 ± 2 g of body weight each, were challenged by intraperitoneal injection of bacterial cells from 5-h broth cultures suspended in either trypticase soy broth or 5% hog gastric mucin . Five animals were infected at each of five i.v.-administered dose levels, covering a twofold dilution series for AC98-6446 and vancomycin for each organism . The bacterial inoculum level for infection was sufficient to result in death of untreated controls within 24 to 48 h . The 7-day survival ratios from three separate tests were pooled for estimation of the 50% effective dose (ED₅₀) by a computerized program for probit analysis (5) . All procedures were carried out by using protocols approved by the Wyeth Research Animal Care and Use Committee .

Murine thigh infection model. Female CD-1 mice (Charles River Laboratories) weighing 18 to 22 g were rendered neutropenic by intraperitoneal injection of Cytoxan (cyclophosphamide) on days –4 (150 mg/kg of body weight) and –1 (100 mg/kg) before infection . On the day of infection, random colonies of Staphylococcus aureus Smith were selected off a trypticase soy agar plate that was incubated for 24 h at 37°C by using the Prompt inoculation system (BBL Microbiology Systems, Sparks, Md.) . This was then diluted in trypticase soy broth to yield a final bacterial concentration of 10⁷ CFU/ml . The thigh infection was induced by intramuscular injection of 0.1 ml of the inoculum into the left thigh of each mouse 1.5 h prior to initiation of treatment . AC98-6446 was administered by intravenous injection of 0.2 ml in twofold dilutions ranging from 0.06 to 16 mg/kg with three mice per dose level . Mice were euthanized 24 h after dose administration and thighs were removed, homogenized, and plated for determination of viable bacteria . The limit of detection of bacterial counts was 10² CFU/thigh . Counts were analyzed by using a sigmoid E_max model (WinNonlin Pro, version 3.0; Pharsight Corporation, Mountain View, Calif.) .

Rat endocarditis. Endocarditis was produced in male Wistar rats (250 g; Charles River Laboratories) by insertion of a sealed polyethylene cannula (PE10) through the right carotid artery into the left ventricle; the cannula was sutured in place as a point of adherence for bacterial infection (5, 6, 15) . At 48 h after implantation of the cannula, a 5-h bacterial culture was diluted to 10⁵ to 10⁶ CFU/ml in sterile saline, and 1 ml was injected i.v . (5) . Isogenic strains of Enterococcus faecalis were used: GC 6181, a vancomycin-susceptible isolate, and GC 6191, its isogenic vancomycin-resistant (VanA) derivative . Inoculum infection concentration was verified by plate counts . Antibacterial treatment was initiated 24 h after bacterial challenge . Treatments were delivered by intravenous administration every 12 h for 3 days . The dose ranges tested were from 1 to 10 mg/kg/day . Untreated control rats received injections of phosphate-buffered saline . Both treated and control rats were euthanized by CO₂ inhalation 24 h after the last treatment . Hearts were aseptically removed, weighed, homogenized, and serially diluted in saline for determination of viable bacteria (the limit of detection was 10² CFU/heart), expressed as log₁₀ CFU per heart .

Pharmacokinetics. The pharmacokinetics of AC98-6446 were investigated in the mouse, rat, dog, and monkey . Female CD-1 mice (Charles River Laboratories) weighing 18 to 22 g were administered a single dose of AC98-6446 i.v . at 20 mg/kg . Blood samples were taken at selected time points by cardiac puncture, and the blood from three mice was pooled for serum collection at each time point . Male Wistar rats (250 g; Charles River Laboratories) received 20 mg of AC98-6446/kg by i.v . injection . Three rats were used for each dose tested . Blood samples were obtained via a surgically implanted jugular cannula at selected time points . Female beagle dogs (9 to 12 kg) and female cynomolgus monkeys (4 to 6 kg) were administered AC98-6446 by i.v . injection of 20 mg/kg . Three animals were used for each species at each dose . Blood samples were collected from the femoral artery for analysis at selected time points .

Sample analysis. Analysis of the pharmacokinetic samples was performed by LC-MS (HP1100) via direct injection of the plasma sample (10 µl) . A gradient of 10 to 60% (0.02% trifluoracetic acid) acetonitrile over 15 min was used in a Prodigy ODS3 (dimensions, 4.6 by 150 mm, 5-µm column; flow rate, 1 ml/min) . Detection was performed by using an MSD detector (single ion monitoring at 717.5; fragmentor, 100; gain, 1) (Agilent Technologies, Palo Alto, Calif.) . Limit of detection of AC98-6446 was 50 ng/ml . Pharmacokinetic parameters were determined by using the WinNonlin program (WinNonlin Pro, version 3.0; Pharsight Corporation) .

 
Murine thigh infection model. Figure 2 shows the effect of i.v . administration of AC98-6446 to neutropenic CD-1 mice infected intramuscularly with S . aureus Smith . AC98-6446 was administered 1.5 h after infection as a single dose over a range from 0.06 to 16 mg/kg . The bacterial density taken from control animals prior to initiation of dosing was determined to be 7.2 ± 0.35 log₁₀ CFU/thigh, which increased to 11.2 ± 0.78 log₁₀ CFU/thigh after 24 h in the untreated controls . The maximal effect represented by a 2 log₁₀ reduction in counts was observed for doses exceeding 0.5 mg/kg . The dose that resulted in no change of bacterial growth from the initial titer (static dose) was determined to be 0.39 mg/kg . The EC₅₀, or dose resulting in 50% of maximal bacterial killing, was 0.37 mg/kg, with an E_max value of 5.2 CFU/thigh . AC98-6446 exhibited an extreme difference in bactericidal activity between the 0.25 and 0.5 mg/kg doses . The twofold dose level change resulted in a 4.5 log₁₀ difference in bacterial thigh density . The steep dose response between these two doses represents the difference from no effect to maximal effect observed with all doses administered .

 
Rat endocarditis. Bacterial vegetation densities were determined in untreated controls at the end of therapy for E . faecalis GC 6181 and at both the initiation and end of therapy for GC 6191 . Results are shown in Table 2 . The mean bacterial density for untreated control animals (mean log₁₀ numbers of CFU per heart ± the standard deviation) at 4 days after bacterial challenge for GC 6181 was 9.52 ± 0.9 . Mean bacterial densities for GC 6191 untreated controls at initiation of therapy and after 4 days were 7.39 ± 0.7 and 9.96 ± 1.2, respectively . Both E . faecalis cardiac infections were treated i.v . with AC98-6446 administered twice a day for total daily doses of 1, 2, 6, and 10 mg/kg . Against E . faecalis GC 6181, a vancomycin-susceptible strain, AC98-6446 demonstrated a dose response reduction in viable bacterial counts compared to counts observed in infected control animals . A dose of 2 mg/kg/day resulted in vegetative counts that were 1.96 log₁₀ lower than those of untreated animals at the end of therapy . Increasing the dose to 6 and 10 mg/kg/day resulted in bacterial reductions of 3.88 and 3.84 log₁₀ CFU, respectively . Total doses of 1, 2, 6, and 10 mg/kg/day of AC98-6446 administered to animals infected with E . faecalis GC 6191 (vancomycin-resistant) resulted in bacterial count reductions of 2.53, 2.29, 4.58, and 5.92 log₁₀ CFU from untreated control titers at the end of therapy, respectively . Compared to the bacterial counts at the initiation of therapy, doses of 6 and 10 mg/kg/day resulted in titer reductions of 2.01 log₁₀ and 3.35 log₁₀, respectively . AC98-6446 therefore demonstrated a bactericidal effect (>3 log₁₀) for the vancomycin-resistant E . faecalis infection when administered at 10 mg/kg/day .

 
Pharmacokinetics. The pharmacokinetics of i.v.-administered AC98-6446 in four species are shown in Table 3 . The maximum observed concentration of AC98-6446 after 20-mg/kg i.v . administration to mice, rats, monkeys, and dogs was 31.6, 231, 188, and 201 µg/ml, respectively . The compound demonstrates increased exposure as measured by the area under the concentration curve with higher species . The area under the concentration curve increases from 164 µg · h/ml in mice to 241 µg · h/ml in rats, 860 µg · h/ml in monkeys, and 1,247 µg · h/ml in dogs . The observed increased exposure corresponds to a decreased clearance of the compound from the plasma in the higher species . Clearance values (in milliliters per hour per kilogram) decrease from 121 and 83 in the mouse and rat to 23 and 16 in the monkey and dog . The plasma t_1/2 also reflects this with values of 3.3, 1.5, 11.5, and 11 h observed for the mouse, rat, monkey, and dog, respectively .

 
The increase in the number of gram-positive bacteria with multidrug resistance occurring in the last several years has resulted in infections that are difficult to treat . This is of particular concern regarding staphylococci and enterococci . The incidence of MRSA has risen to be the cause of up to 21% of skin infections and 59% of nosocomial pneumonia in certain areas (20) . Infections with MRSA have become a concern in long-term care facilities from patients colonized with MRSA in hospitals prior to transfer to the facility (15) . Until recently, vancomycin has been the drug of choice for these infections (24) . However, the emergence of S . aureus with reduced susceptibility to vancomycin has been reported from the United States, France, Spain, United Kingdom, Japan, Korea, and China (29) . These isolates, with vancomycin MICs ranging from 4 to 8 µg/ml, have been referred to as VISA, for vancomycin-intermediate S . aureus (13) . Many of these isolates exhibiting reduced susceptibility to the glycopeptides have been associated with therapeutic failures with vancomycin (29) . Resistance has been reported to be associated with increased cell wall synthesis and a thickened or aggregated cell wall (23) . Infections with these isolates represents a therapeutic challenge .

The enterococci have emerged as an increasingly important pathogen because of acquired resistance to the glycopeptides (VRE) and other agents . Glycopeptide resistance rates vary from extremely low for E . faecalis in the Asia Pacific and Latin America to over 50% for E . faecium in North America (17) . VRE colonization appears to be more frequent than actual infection (11) and predominates in the intensive care unit, with nosocomial transmission a particular concern (19, 21) . Resistance rates for vancomycin and some of the newer agents underscore the need for alternative therapy (10, 18, 27) .

AC98-6446 is a semisynthetic derivative belonging to the mannopeptimycin family of glycopeptide antibiotics that selectively targets bacterial cell wall synthesis (25) . It has been shown to be a potent bactericidal inhibitor of resistant gram-positive bacteria, including methicillin-resistant and glycopeptide-intermediate S . aureus as well as vancomycin-resistant E . faecalis and E . faecium (Petersen et al., 42nd ICAAC) . The in vitro activity observed in these studies was also exhibited by in vivo efficacy .

Our results indicate that AC98-6446 was more effective than vancomycin against infections with MSSA and MRSA, vancomycin-resistant E . faecalis, and penicillin-susceptible and -resistant S . pneumoniae . Efficacy of AC98-6446 was not dependent on the resistance phenotype of the organism . We observed equivalent efficacy regardless of methicillin resistance in S . aureus or penicillin resistance in S . pneumoniae . Of particular note is that AC98-6446 was over 10 and 50 times more efficacious than vancomycin against infections with the MRSA and VRE strains, respectively .

As previously reported, AC98-6446 exhibited bactericidal activity against staphylococci, streptococci, and enterococci as well as a long postantibiotic effect (Petersen et al., 42nd ICAAC) . The results of the thigh infection model from this study show a sharp decline in bacterial counts in infected thighs within a twofold dose range . Increasing the dose administered from 0.25 to 0.5 mg/kg resulted in the difference between a 3 log₁₀ increase and a 1.5 log₁₀ decrease from the initial infection level . Increasing doses up to 16 mg/kg did not affect the observed bactericidal activity of the compound . Further pharmacodynamic studies are required to assess the implications of this and the parameters required for efficacy of the compound .

Infective endocarditis due to complications from intravenous drug use, prosthetic valves, and nosocomial bacteremia results in extended hospital stays and high mortality rates (26) . This is especially true when infection is due to VRE or MRSA (4) . AC98-6446 was clearly effective in preventing growth of both vancomycin-susceptible and vancomycin-resistant E . faecalis isolates in an infective endocarditis model . It exhibited greater efficacy at a lower dose than was previously reported for vancomycin against these same strains (1.5 and 0.14 log₁₀ reduction against GC 6181 and GC 6191, respectively, at a dose of 40 mg/kg/day) (16) . In addition, bactericidal activity (>3 log₁₀ reduction in viable counts) was observed against the VRE strain from counts at the initiation of therapy . These results underscore the excellent activity of AC98-6446 against VRE in addition to its ability to treat this difficult deep-seated infection .

The infection models described in this study involved the use of rodent species (mouse and rat) only . Pharmacokinetic exposure of AC98-6446 was moderate in the mouse and rat, with t_1/2 of 3.3 and 1.5 h and plasma clearance of 121 and 83 ml/h/kg . Higher species (monkey and dog) exhibited longer plasma exposure and slower clearance values . Efficacy, if related to overall exposure, could therefore require a lower administered dose in higher species .

Overall, AC98-6446, representative of a novel class of glycopeptide antibiotics, exhibits excellent in vitro activity against resistant gram-positive pathogens, which was also demonstrated in vivo in three different animal models of infection . Efficacy against infections involving MRSA and vancomycin-resistant E . faecalis and an acceptable pharmacokinetic profile coupled with a unique mechanism of action (A . Ruzin, G . Singh, A . Severin, Y . Yang, R . Dushin, A . Sutherland, A . Minnick, M . Greenstein, M . May, D . Shlaes, and P . Bradford, unpublished results) make this compound an excellent candidate for further study .

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