In Vivo Efficacy of Apramycin in Murine Infection Models

Apramycin is a unique aminoglycoside with a dissociation of antibacterial activity and ototoxicity. We assessed the antibacterial efficacy of apramycin in two murine models of infection, Mycobacterium tuberculosis aerosol infection and Staphylococcus aureus septicemia. In both infection models, the efficacy of apramycin was comparable to that of amikacin. These results suggest that apramycin has the potential to become a clinically useful agent against drug-resistant pathogens and support further development of this promising unique aminoglycoside.     

In Vivo Bioluminescence Imaging of Murine Xenograft Cancer Models with a Red-shifted Thermostable Luciferase

Purpose

Conventional in vivo bioluminescence imaging using wild-type green-emitting luciferase is limited by absorption and scattering of the bioluminescent signal through tissues. Imaging methods using a red-shifted thermostable luciferase from Photinus pyralis were optimized to improve the sensitivity and image resolution. In vivo bioluminescence imaging performance of red- and green-emitting luciferases were compared in two different xenograft mouse models for cancer.

Methods

Human hepatoblastoma cell line (HepG2) and human acute monocytic leukemia cell line (Thp1) cells were genetically engineered using retroviral vector technology to stably express the red-shifted or the wild-type green luciferase. A xenograft model of liver cancer was established by subcutaneous injection of the HepG2-engineered cells in the flank regions of mice, and a leukemia model was generated by intravenous injection of the engineered Thp1 cells. The cancer progression was monitored with an ultrasensitive charge-coupled device camera. The relative intensities of the green- and red-emitting luciferases were measured, and the resulting spatial resolutions of the images were compared. Imaging was performed with both intact and scarified live animals to quantify the absorption effects of the skin and deep tissue.

Results

The red-emitting luciferase was found to emit a bioluminescence signal with improved transmission properties compared to the green-emitting luciferase. By imaging the HepG2 models, which contained tumors just beneath the skin, before and after scarification, the percentage of light absorbed by the skin was calculated. The green bioluminescent signal was 75?±?8% absorbed by the skin, whereas the red signal was only 20?±?6% absorbed. The Thp1 model, which contains cancer cells within the bones, was likewise imaged before and after scarification to calculate the percentage of light absorbed by all tissue under the skin. This tissue was responsible for 90?±?5% absorption of the green signal, but only 65?±?6% absorption of the red signal.

Conclusion

Two different bioluminescent mouse cancer models demonstrate the utility of a new red-shifted thermostable luciferase, Ppy RE-TS, that improved the in vivo imaging performance when compared with wild-type P. Pyralis luciferase. While wild-type luciferase is currently a popular reporter for in vivo imaging methods, this study demonstrates the potential of red-emitting firefly luciferase mutants to enhance the performance of bioluminescence imaging experiments.     

In Vivo Activity of Ceftobiprole in Murine Skin Infections Due to Staphylococcus aureus and Pseudomonas aeruginosa

Ceftobiprole, a broad-spectrum cephalosporin with activity against methicillin-resistant Staphylococcus aureus (MRSA) (P. Hebeisen et al., Antimicrob. Agents Chemother. 45:825-836, 2001), was evaluated in a subcutaneous skin infection model with Staphylococcus aureus Smith OC 4172 (methicillin-susceptible S. aureus [MSSA]), S. aureus OC 8525 (MRSA), Pseudomonas aeruginosa OC 4351 (having an inducible AmpC β-lactamase), and P. aeruginosa OC 4354 (overproducing AmpC β-lactamase). In the MSSA and MRSA infection models, ceftobiprole, administered as the prodrug ceftobiprole medocaril, was more effective in reducing CFU/g skin (P < 0.001) than were cefazolin, vancomycin, or linezolid based on the dose-response profiles. Skin lesion volumes in MSSA-infected animals treated with ceftobiprole were 19 to 29% lower than those for cefazolin-, vancomycin-, or linezolid-treated animals (P < 0.001). In MRSA infections, lesion size in ceftobiprole-treated mice was 34% less than that with cefazolin or linezolid treatment (P < 0.001). Against P. aeruginosa, ceftobiprole at similar doses was as effective as meropenem-cilastatin in reductions of CFU/g skin, despite 8- and 32-fold-lower MICs for meropenem; both treatments were more effective than was cefepime (P < 0.001) against the inducible and overproducing AmpC β-lactamase strains of P. aeruginosa. Ceftobiprole was similar to meropenem-cilastatin and 47 to 54% more effective than cefepime (P < 0.01) in reducing the size of the lesion caused by either strain of P. aeruginosa in this study. These studies indicate that ceftobiprole is effective in reducing both bacterial load and lesion volume associated with infections due to MSSA, MRSA, and P. aeruginosa in this murine model of skin and soft tissue infection.Antimicrobial resistance is becoming more problematic in the health care setting, with infections mediated by drug-resistant pathogens being associated with increased morbidity and mortality and corresponding increased health care costs and longer hospital stays (12). β-Lactams, including cefazolin, have been used for many years to treat methicillin-susceptible Staphylococcus aureus (MSSA) skin and soft tissue infections (9, 52). The rising proportion of methicillin resistance in staphylococcal infections in hospitals and in the community has compromised the effectiveness of this antibiotic class for staphylococci and resulted in the increased use of vancomycin to treat patients with these infections (21, 41). As a consequence, vancomycin-intermediate S. aureus (VISA) strains continue to increase in prevalence, and vancomycin-resistant S. aureus (VRSA) strains have begun to appear (24, 33, 34, 49, 51). Linezolid, an oxazolidinone antibiotic with a gram-positive antibacterial spectrum of activity including methicillin-resistant S. aureus (MRSA) (27), was introduced in 2000 to address the growing problem of MRSA, but safety concerns have limited its utility (4, 29).Prior to the advent of methicillin resistance (and therein resistance to all approved β-lactams), cephalosporins were considered the drugs of choice to treat staphylococcal infections (45). Ceftobiprole is a new cephalosporin that, unlike currently marketed β-lactams, has high binding affinity for penicillin-binding protein 2a (PBP2a) (19), resulting in potent in vitro and in vivo activity against MRSA. The prodrug form of this agent, ceftobiprole medocaril, has completed two phase III complicated skin and skin structure infection (cSSSI) clinical trials in which efficacy was demonstrated against a broad spectrum of bacteria, including MRSA (1, 44).Antimicrobial resistance in gram-negative organisms is also a serious problem that has limited the number of effective antimicrobial agents available to physicians to treat infection in the hospital setting. While skin infections with Pseudomonas aeruginosa are found with less frequency (6.2%) than are staphylococcal skin infections (19.1%), they remain a common cause of morbidity in surgical site and burn infections (2) and with immunocompromised patients. (26, 52). Antipseudomonal cephalosporins are frequently used for the treatment of P. aeruginosa; however, the overproduction of AmpC β-lactamases reduces the antibiotic choices available for treatment (40).Ceftobiprole binds tightly to PBP3 in P. aeruginosa, as do other cephalosporins (20), and consequently has also been shown to have in vitro and in vivo activity against this bacterium. MICs for ceftobiprole against recent clinical isolates are similar to those of cefepime and meropenem (10, 25). The in vivo antipseudomonal activity of ceftobiprole has previously been characterized in the mouse septicemia and the neutropenic thigh infection models (23, 30, 32), but the effect of AmpC β-lactamases on in vivo activity has not been reported.In this study, ceftobiprole medocaril was evaluated in a murine skin and soft tissue infection model utilizing methicillin-susceptible and -resistant strains of S. aureus and strains of P. aeruginosa that express various levels of AmpC. In these models, the broad-spectrum (gram-positive and gram-negative) activity of ceftobiprole was demonstrated against these important skin pathogens.     

In Vivo Characterization of the Peptide Deformylase Inhibitor LBM415 in Murine Infection Models

LBM415 is an antibacterial agent belonging to the peptide deformylase inhibitor class of compounds. It has previously been shown to demonstrate good activity in vitro against a range of pathogens. In this study, the in vivo efficacy of LBM415 was evaluated in various mouse infection models. We investigated activity against a systemic infection model caused by intraperitoneal inoculation of Staphylococcus aureus (methicillin [meticillin] susceptible [MSSA] and methicillin resistant [MRSA]) and Streptococcus pneumoniae (penicillin susceptible [PSSP] and multidrug resistant [MDRSP]), a thigh infection model caused by intramuscular injection of MRSA, and a lung infection produced by intranasal inoculation of PSSP. In the systemic MSSA and MRSA infections, LBM415 was equivalent to linezolid and vancomycin. In the systemic PSSP infection, LBM415 was equivalent to linezolid, whereas against systemic MDRSP infection, the LBM415 50% effective dose (ED₅₀) was 4.8 mg/kg (dosed subcutaneously) and 36.6 mg/kg (dosed orally), compared to 13.2 mg/kg for telithromycin and >60 mg/kg for penicillin V and clarithromycin. In the MRSA thigh infection, LBM415 significantly reduced thigh bacterial levels compared to those of untreated mice, with levels similar to those after treatment with linezolid at the same dose levels. In the pneumonia model, the ED₅₀ to reduce the bacterial lung burden by >4 log₁₀ in 50% of treated animals was 23.3 mg/kg for LBM415, whereas moxifloxacin showed an ED₅₀ of 14.3 mg/kg. In summary, LBM415 showed in vivo efficacy in sepsis and specific organ infection models irrespective of resistance to other antibiotics. Results suggest the potential of peptide deformylase inhibitors as a novel class of therapeutic agents against antibiotic-resistant pathogens.With the emergence of pathogens resistant to current clinically used antibiotics, the need for new therapies has become of paramount importance. A novel class of antibacterial agents to emerge from research in this field is the peptide deformylase (PDF) inhibitors. PDF is a highly conserved metalloenzyme which deformylates the initial N-formyl methionine of newly synthesized bacterial polypeptides. This is an important step in bacterial protein synthesis, thus making it an attractive antibacterial target. The role of PDF and its attractiveness as an antibacterial target have previously been reviewed (10, 11, 15, 16).LBM415 is one of the first compounds of the PDF inhibitor class to advance to clinical trials for the oral (p.o.) and parenteral treatment of respiratory tract and skin and skin structure infections caused by susceptible gram-positive and -negative organisms. LBM415 has been evaluated previously in vitro in comparison with other antibiotics and demonstrated potent activity against clinical strains of staphylococci, streptococci, enterococci, Moraxella catarrhalis, Legionella pneumophila, and Haemophilus influenzae (2, 5, 6, 9, 13, 14). There was no difference in activity against strains classified as being susceptible or resistant to other classes of antibiotics. LBM415 also displayed activity against a collection of other gram-positive species, including Aerococcus spp., Bacillus spp., Corynebacterium spp., Gemella spp., Lactobacillus spp., Lactococcus spp., Leuconostoc spp., Listeria spp., Micrococcus spp., Nocardia spp., and Stomatococcus spp. (9). In the present study, the in vivo efficacy of LBM415 was evaluated in mice against systemic infections caused by Staphylococcus aureus (methicillin [meticillin] susceptible [MSSA] and methicillin resistant [MRSA]) and Streptococcus pneumoniae (penicillin susceptible [PSSP] and penicillin resistant [PRSP]). Furthermore, the therapeutic efficacy of LBM415 was evaluated using a mouse thigh infection and a mouse pneumonia lung infection model.     

In Vitro and In Vivo Antibacterial Evaluation of Cadazolid,a New Antibiotic for Treatment of Clostridium difficile Infections

Clostridium difficile is a leading cause of health care-associated diarrhea with significant morbidity and mortality, and new options for the treatment of C. difficile-associated diarrhea (CDAD) are needed. Cadazolid is a new oxazolidinone-type antibiotic that is currently in clinical development for treatment of CDAD. Here, we report the in vitro and in vivo antibacterial evaluation of cadazolid against C. difficile. Cadazolid showed potent in vitro activity against C. difficile with a MIC range of 0.125 to 0.5 μg/ml, including strains resistant to linezolid and fluoroquinolones. In time-kill kinetics experiments, cadazolid showed a bactericidal effect against C. difficile isolates, with >99.9% killing in 24 h, and was more bactericidal than vancomycin. In contrast to metronidazole and vancomycin, cadazolid strongly inhibited de novo toxin A and B formation in stationary-phase cultures of toxigenic C. difficile. Cadazolid also inhibited C. difficile spore formation substantially at growth-inhibitory concentrations. In the hamster and mouse models for CDAD, cadazolid was active, conferring full protection from diarrhea and death with a potency similar to that of vancomycin. These findings support further investigations of cadazolid for the treatment of CDAD.     

In Vivo Effects of Immunomodulators in a Murine Model of Fluorouracil-Induced Mucositis

Background

Fluorouracil (5-FU) is a pyrimidine analogue used as a cancer treatment. Its toxic side effects, including mucositis, are reported to occur in 40% of the treated patients. Because of the inflammatory component of mucositis, we explored the possibility of modulating this condition with an immunomodulatory agent and a tumor necrosis factor-α inhibitor.

Objective

The aim of this study was to evaluate the effect of 2 immunosuppressive agents, etanercept and cyclosporine, in a murine model of 5-FU–induced mucositis.

Methods

To study the short-term effects of 5-FU on mucositis, cyclosporine and etanercept were administered to mice after an injection of 5-FU. The animals (n = 8) were euthanized at 6 hours post-challenge. Hematoxylin and eosin–stained histologic sections of the small intestine were examined for signs of apoptosis. To further examine the potential of cyclosporine in the treatment of 5-FU–induced mucositis in a longer duration, the animals (N = 15) were given 2 challenges of 5-FU within 6 hours. All mice were dosed daily until day 9 with either cyclosporine (100 mg/kg) or phosphate-buffered saline (PBS).

Results

Six hours after 5-FU challenge, 25 mg/kg etanercept and 50 mg/kg cyclosporine had no effect on 5-FU–induced apoptosis (P > 0.05). However, 100 mg/kg cyclosporine significantly reduced the cumulative level of apoptosis >41.6% of the intestinal crypt surface (P < 0.05). During long-term observation, all mice began to lose weight at a rate of approximately 0.8 g/day after 5-FU exposure. The rates of weight loss and survival were not affected by cyclosporine treatment. The diarrhea onset began on day 4 with 46.7% of the PBS-treated mice showing signs of diarrhea compared with 53.3% in the cyclosporine group. The diarrhea score for both groups plateaued on day 7, with a cumulative score of 41 for the PBS group and 50 for the cyclosporine group. Cyclosporine treatment did not affect the diarrhea onset day or severity compared with the PBS-treated group (P > 0.05).

Conclusions

Our data indicated that etanercept is not a suitable treatment for 5-FU–induced mucositis. Despite decreased apoptosis in the gut, cyclosporine did not affect the severity of the diarrhea or survival. Therefore, we concluded that cyclosporine treatment was only effective in mediating the short-term apoptotic events in the intestines but has no long-term effect on the animals' survival and diarrhea.     

Correlation of In Vitro Activity,Serum Levels,and In Vivo Efficacy of Posaconazole against Rhizopus microsporus in a Murine Disseminated Infection

A broth microdilution method was used to evaluate the in vitro activities of seven antifungal agents against 15 clinical strains of Rhizopus microsporus. Amphotericin B (AMB) and posaconazole (POS) were the most active drugs. In a model of disseminated R. microsporus infection in immunosuppressed mice, we studied the efficacy of POS administered once or twice daily against four of the strains previously tested in vitro and compared it with that of liposomal AMB (LAMB). LAMB was the most effective treatment for the two strains with intermediate susceptibility to POS. For the two POS-susceptible strains, LAMB and POS at 20 mg/kg of body weight twice a day orally showed similar efficacies. The in vivo efficacy of POS administered twice a day orally correlated with the in vitro susceptibility data and the serum drug concentrations.Zygomycosis is a frequently lethal invasive infection that occurs predominantly in immunocompromised patients (4), a population with a very poor prognosis and a high mortality rate (8). The clinical manifestations include rhino-orbito-cerebral, cutaneous, pulmonary, gastrointestinal, and disseminated infections (4). In a recent study in which a large number of clinical isolates of zygomycetes from different regions of the United States were molecularly identified, it was demonstrated that Rhizopus oryzae and Rhizopus microsporus were the most common species (3). Traditionally, amphotericin B (AMB) and, more recently, its lipid formulations are the front-line agents for the treatment of zygomycosis (4). Specifically, liposomal amphotericin B (LAMB) is less nephrotoxic than AMB and has better central nervous system penetration than AMB and the other lipid formulations (21). Posaconazole (POS) is a broad-spectrum triazole antifungal with a large volume of distribution into tissues (12). This drug has shown good in vitro activity against zygomycetes (1, 2) and has been used successfully as salvage therapy in some case reports and clinical trials of disseminated zygomycosis (8, 22, 23). However, its effectiveness remains controversial, since in experimental studies it has shown poor activity against R. oryzae, the main species causing zygomycosis (6, 9, 17). Several in vitro studies have shown that POS also exhibits significant activity against R. microsporus, another relevant clinical species (1, 2, 11), and a few clinical (14) and experimental (6) studies seem to demonstrate in vivo efficacy as well.In this study, after confirming the significant in vitro activity of POS and AMB, we evaluated the efficacy of POS against four strains of R. microsporus in a murine model of disseminated infection. Considering that antifungal susceptibility can differ substantially among different strains of a given species, which could explain the variable percentages of success demonstrated by POS and AMB in clinical trials (8, 18, 23), we tested multiple strains exhibiting various in vitro responses to obtain more-robust results.     

Characterizing In Vivo Pharmacodynamics of Carbapenems against Acinetobacter baumannii in a Murine Thigh Infection Model To Support Breakpoint Determinations

Pharmacodynamic profiling data of carbapenems for Acinetobacter spp. are sparse. This study aimed to determine the pharmacodynamic targets of carbapenems for Acinetobacter baumannii based on a range of percentages of the dosing interval in which free drug concentrations remained above the MIC (fT>MIC) in the neutropenic murine thigh infection model. fT>MIC values of 23.7%, 32.8%, and 47.5% resulted in stasis, 1-log reductions, and 2-log reductions in bacterial density after 24 h, respectively. The pharmacodynamic targets of carbapenems for A. baumannii demonstrated in vivo are similar to those of other Gram-negative bacteria.     

In Vitro and In Vivo Activity of a Synthetic Halogenated Quinoline Against Cryptococcus neoformans

The minimum inhibitory concentrations of a halogenated quinoline, 3-amino-7-chloro-3,4-dihydro-1-hydroxycarbostyril (CBS), against nine clinical strains of Cryptococcus neoformans were determined by in vitro testing. The CBS was fungistatic at a minimum concentration of 0.2 μg/ml at 48 h for several strains. In vivo toxicity studies were carried out in mice. Mice were also infected with C. neoformans strain Price and injected with various concentrations of CBS. Mean life expectancy of treated groups of animals was increased over infected untreated controls.     

In Vitro and In Vivo Inhibition of Murine Gamma Herpesvirus 68 Replication by Selected Antiviral Agents

We have evaluated the susceptibility of the murine gamma herpesvirus 68 (MHV-68) to a variety of antiviral agents. The acyclic nucleoside phosphonate analogs cidofovir [(S)-1-(3-hydroxy-2-phosphonylmethoxypropyl)cytosine], (S)-1-(3-hydroxy-2-phosphonylmethoxypropyl)adenine (HPMPA), and adefovir [9-(2-phosphonylmethoxyethyl)adenine] efficiently inhibited the replication of the virus in Vero cells (50% effective concentrations [EC₅₀s], 0.008, 0.06, and 2.2 μg/ml, respectively). Acyclovir, ganciclovir, and brivudin [(E)-5-(2-bromovinyl)-2′-deoxyuridine] had equipotent activities (EC₅₀s, 1.5 to 8 μg/ml), whereas foscarnet and penciclovir were less effective (EC₅₀s, 23 and ≥30 μg/ml, respectively). The novel N-7-substituted nucleoside analog S2242 [7-(1,3-dihydroxy-2-propoxymethyl)purine] inhibited MHV-68 replication by 50% at 0.2 μg/ml. The susceptibilities of MHV-68 and Epstein-Barr virus (EBV) to cidofovir, HPMPA, adefovir, and acyclovir were found to be comparable. However, for penciclovir, ganciclovir, brivudin, and S2242, major differences in the sensitivity of MHV-68 and EBV were observed, suggesting that MHV-68 is not always an optimal surrogate for the study of antiviral strategies for EBV. When evaluated with a model for lethal MHV-68 infections in mice with severe combined immunodeficiency, cidofovir proved to be very efficient in protecting against virus-induced mortality (100% survival at 50 days postinfection), whereas acyclovir, brivudin, and adefovir had little or no effect.     

Assessment of aquo-ethanolic extract of Camellia sinensis against Carbapenem Resistant Escherichia coli: In Vivo Trials in a Murine Model

The prevalence of Carbapenem Resistant Escherichia coli (CRE) has increased considerably during the last decade, which can be ascribed to relative scarcity of effective non toxic antimicrobial agents. The present study was conducted to evaluate the antimicrobial activity of aquo-ethanolic (1:1) extract of leaves of Camellia sinensis (PTRC-31911-A) against Carbapenem Resistant Escherichia coli at preclinical level using peritonitis infection model in Sprague Dawley rats. Efficacy analysis of PTRC-31911-A involved enumeration of CRE colonies in blood and urine samples of test animals for a period of 5 days from infection. A reduction in microbial count of biological fluids was considered as the primary endpoint of the selected murine model. Physical, biochemical, hematological and histological indices of toxicity were employed as secondary relative indicators of the induced disease. Physical manifestations of infected rats included significantly high body temperature (Temp_Infected = 103.18 °F, ∼5% increase) and noteworthy reduction in weight (Weight_Infected = 126.83 g, ∼15% decrease) as compared to control. Significant (P < 0.05) increase in total white blood cells, eosinophil and monocyte counts as well as a significant decrease (P < 0.05) in erythrocytes count, hematocrit volume, red blood cell distribution width and hemoglobin concentration were observed in the infected group as compared to the control group. Furthermore, noteworthy increase in liver and kidney function test parameters were observed in case of infected groups. All the hematological and biochemical parameters were found to be within optimum range in case of treatment group, indicating restoration of homeostasis. Histopathological studies also presented symptoms of hemorrhage and glomerular damage with structural distortion in glomerular capillary loops of infected groups, which were later recovered in treated groups, indicating the nephro-protective potential of PTRC-31911-A. The study clearly points out that Camellia sinensis extract (PTRC-31911-A; single dose of 5 mg/Kg bwt; oral, + 24 h) is highly effective against Carbapenem Resistant Escherichia coli owing mainly to the presence of flavonoids and polyphenolic compounds, identified by LCMS. Ongoing studies are expected to further unravel the mechanism of action and bioactivity determinants of this broad spectrum plant extract.     

In Vivo Activity of HSR-903, a New Fluoroquinolone, against Respiratory Pathogens

The in vivo activity of HSR-903, a new fluoroquinolone, against major bacteria which cause respiratory tract infections was evaluated. HSR-903 was active against experimental respiratory tract infections in mice challenged with penicillin-susceptible and penicillin-resistant Streptococcus pneumoniae and Haemophilus influenzae strains. Treatment with HSR-903 reduced the bacterial numbers in infected murine lungs. In accord with the pulmonary clearance results, the rates of survival for mice treated with HSR-903, sparfloxacin, levofloxacin, ciprofloxacin, and benzylpenicillin were 50, 30, 10, 0, and 0%, respectively, 14 days after being infected with penicillin-resistant S. pneumoniae. A pharmacokinetic study with pneumonic mice showed that the levels of HSR-903 in the lungs were seven to eight times higher than those in the plasma. These results indicate that clinical studies of HSR-903 against respiratory tract infections may be warranted.     

NBTI 5463 Is a Novel Bacterial Type II Topoisomerase Inhibitor with Activity against Gram-Negative Bacteria and In Vivo Efficacy

The need for new antibiotics that address serious Gram-negative infections is well recognized. Our efforts with a series of novel bacterial type II topoisomerase inhibitors (NBTIs) led to the discovery of NBTI 5463, an agent with improved activity over other NBTIs against Gram-negative bacteria, in particular against Pseudomonas aeruginosa (F. Reck, D. E. Ehmann, T. J. Dougherty, J. V. Newman, S. Hopkins, G. Stone, N. Agrawal, P. Ciaccio, J. McNulty, H. Barthlow, J. O''Donnell, K. Goteti, J. Breen, J. Comita-Prevoir, M. Cornebise, M. Cronin, C. J. Eyermann, B. Geng, G. R. Carr, L. Pandarinathan, X. Tang, A. Cottone, L. Zhao, N. Bezdenejnih-Snyder, submitted for publication). In the present work, NBTI 5463 demonstrated promising activity against a broad range of Gram-negative pathogens. In contrast to fluoroquinolones, the compound did not form a double-strand DNA cleavable complex with Escherichia coli DNA gyrase and DNA, but it was a potent inhibitor of both DNA gyrase and E. coli topoisomerase IV catalytic activities. In studies with P. aeruginosa, NBTI 5463 was bactericidal. Resistant mutants arose at a low rate, and the mutations were found exclusively in the nfxB gene, a regulator of the MexCD-OprJ efflux system. Levofloxacin-selected resistance mutations in GyrA did not result in decreased susceptibility to NBTI 5463. Animal infection studies demonstrated that NBTI 5463 was efficacious in mouse models of lung, thigh, and ascending urinary tract infections.     

In Vivo Pharmacodynamic Target Investigation of Two Bacterial Topoisomerase Inhibitors,ACT-387042 and ACT-292706, in the Neutropenic Murine Thigh Model against Streptococcus pneumoniae and Staphylococcus aureus

ACT-387042 and ACT-292706 are two novel bacterial topoisomerase inhibitors with broad-spectrum activity against Gram-positive and -negative bacteria, including methicillin-resistant Staphylococcus aureus and penicillin- and fluoroquinolone-resistant Streptococcus pneumoniae. We used the neutropenic murine thigh infection model to characterize the pharmacokinetics (PK)/pharmacodynamics (PD) of these investigational compounds against a group of 10 S. aureus and S. pneumoniae isolates with phenotypic resistance to beta-lactams and fluoroquinolones. The in vitro activities of the two compounds were very similar (MIC range, 0.03 to 0.125 mg/liter). Plasma pharmacokinetics were determined for each compound by using four escalating doses administered by the subcutaneous route. In treatment studies, mice had 10^7.4 to 10⁸ CFU/thigh at the start of therapy with ACT-387042 and 10^6.7 to 10^8.3 CFU/thigh at the start of therapy with ACT-292706. A dose-response relationship was observed with all isolates over the dose range. Maximal kill approached 3 to 4 log₁₀ CFU/thigh compared to the burden at the start of therapy for the highest doses examined. There was a strong relationship between the PK/PD index AUC/MIC ratio (area under the concentration-time curve over 24 h in the steady state divided by the MIC) and therapeutic efficacy in the model (R², 0.63 to 0.82). The 24-h free-drug AUC/MIC ratios associated with net stasis for ACT-387042 against S. aureus and S. pneumoniae were 43 and 10, respectively. The 24-h free-drug AUC/MIC ratios associated with net stasis for ACT-292706 against S. aureus and S. pneumoniae were 69 and 25, respectively. The stasis PD targets were significantly lower for S. pneumoniae (P < 0.05) for both compounds. The 1-log-kill AUC/MIC ratio targets were ∼2- to 4-fold higher than stasis targets. Methicillin, penicillin, or ciprofloxacin resistance did not alter the magnitude of the AUC/MIC ratio required for efficacy. These results should be helpful in the design of clinical trials for topoisomerase inhibitors.