Pharmacology of Novel Heteroaromatic Polycycle Antibacterials

Bacterial strains and susceptibility testing.In vitro antimicrobial susceptibility was determined by the standard NCCLS broth microdilution method and as described previously (17). While these HARP compounds exhibit activities against a broad range of gram-positive organisms, the following subset of strains is represented herein: S. aureus (ATCC 13709, methicillin susceptible; ATCC 27660 and ATCC 33591, methicillin resistant), Streptococcus pneumoniae (ATCC 49619), Enterococcus faecalis (ATCC 29212), and Bacillus cereus (ATCC 11778). An Enterococcus faecium strain, ATCC 51559, resistant to vancomycin (and other drugs) was used for some specific MIC studies. The reference control drugs used in vitro and in vivo were vancomycin (Sigma Chemical Co., St. Louis, Mo.) and linezolid (Pharmacia, Kalamazoo, Mich.).

DNA-binding assay.A previously described (21) DNase I footprinting protocol was used in this study. Plasmid pTrc99a (Amersham Pharmacia Biotech, Inc., Piscataway, N.J.) was used as a DNA-binding probe for all DNase I footprint titration experiments. A 348-bp DNA fragment labeled with ³²P at the 3′ end was prepared by digesting pTrc99a with EcoRI and PvuII with simultaneous fill in with Sequenase (version 2.0) [α-³²P]ATP and [α-³²P]TTP.

DNA binding to the sequence 5′-TTGACAATTAATCAT-3′, an AT-rich sequence that occurs in many prokaryotic regulatory elements and as the Trc promoter, was measured (1, 22).

Protein binding.The HARP compounds were dissolved in dimethyl sulfoxide to obtain a stock solution of 10 mM. The dimethyl sulfoxide stock solution was then diluted 1/1,000, 1/2,000, and 1/5,000 in plasma to result in incubation concentrations of 10, 5, and 2 μM, respectively. The spiked plasma samples were incubated at 37°C for 30 min. Two 1.5-ml aliquots were transferred into 2-ml Quick Seal centrifuge tubes (Beckman Instruments, Fullerton, Calif.) and subjected to ultracentrifugation in a Beckman 110K rotor operated at 85,000 rpm (295,000 × g) for 2 h at 37°C. The remainder of the aliquot was incubated for an additional 2 h at 37°C and served as a control. After ultracentrifugation, a 100-μl aliquot of the clear supernatant and a 50-μl aliquot of the control serum were used for analysis. Proteins were precipitated with acetonitrile (with an internal standard), and the extract was cleared by centrifugation. The clear supernatant was concentrated under a stream of nitrogen (TurboVap; Zymark, Hopkinton, Mass.), and 10 μl was subjected to liquid chromatography-mass spectrometry-mass spectrometry (LC-MS/MS) analysis on an LCQ-Duo instrument (Thermo Finnigan, San Jose, Calif.) equipped with an electrospray ionization source and an Agilent 1100 solvent delivery system. The extract was separated on a Beta-Basic C₄ column (5 μm, 30 by 2 mm), with separation performed at 300 μl/min at 25°C with 0.05% formic acid in water (solution A) and 0.05% formic acid in acetonitrile (solution B) as the mobile phase. Separation was achieved with the following gradient: 5% solution B for 0.5 min, a change to 50% solution B in 1 min, a change to 90% solution B in 2.5 min, and holding at 90% for 1 min, followed by reequilibration to 5% solution B in 9 min.

Pharmacokinetics and tissue disposition. (i) PharmacokineticsMale Sprague-Dawley rats (weight, ∼250 g) with jugular vein cannulae were purchased from Hilltop Labs (Scottdale, Pa.) and fasted overnight prior to dosing. The in-life portion of the rat pharmacokinetic studies were generally performed at Northview Pacific Laboratories in Hercules, Calif., but have been validated in-house as well. Groups of two to three rats were given intravenous (i.v.) bolus injections in the lateral tail vein at a dose volume of 2.5 ml/kg of body weight to achieve doses of 3, 10, and 50 mg/kg. Blood samples were collected from the jugular vein cannulae and placed in sodium heparin-coated tubes at several time points: 30 min before dosing and 5, 15, and 30 min and 1, 2, 4, 8, 12, and 24 h postdosing. Samples were stored on ice and centrifuged, and the plasma was collected and frozen on dry ice. The samples were stored at −80°C prior to analysis.

A single pharmacokinetic study with 10-kg beagle dogs was performed with GSQ-2287 at LAB Preclinical (Laval, Quebec, Canada). GSQ-2287 was administered via a 10-min i.v. infusion to three dogs at a dose of 3 mg/kg. Plasma samples were obtained at the same time points used for the rats and as described above for the rats.

Limited pharmacokinetic studies of GSQ-2287 and GSQ-10547 were performed with mice. Groups of three ICR mice (females) per time point were treated i.v. or subcutaneously at 7.5 ml/kg. At serial time points (i.e., 5, 20, 60, 120, and 240 min), the animals were killed, and blood was collected at termination by cardiac puncture. Subcutaneous dosing was done in the context of preliminary pharmacokinetic-pharmacodynamic determinations.

(ii) Tissue distribution.The tissue dispositions of efficacious doses of GSQ-2287 (50 mg/kg) and the reference drug, vancomycin (10 mg/kg), were determined after the administration of a single i.v. dose to female ICR mice over a time period of up to 5 h. Mice were exsanguinated, and the organs (liver, lung, kidneys, spleen, brain, thigh muscle) were rapidly isolated and flash frozen.

(iii) Data analysis.The pharmacokinetic analyses were based on the average concentration in plasma for three animals per treatment group at each nominal sample time. Noncompartmental modeling was performed with WinNonlin software (Pharsight, Corp., Mountainview, Calif.).

Bioanalytical analysis of plasma and tissue.Samples containing GSQ and linezolid were analyzed at GeneSoft Pharmaceuticals (South San Francisco, Calif.) and samples containing vancomycin were analyzed at Bay Bioanalytics Laboratory (Hercules, Calif.) by the methods described below.

(i) Analysis of GSQ and linezolid in plasma.Protein precipitation was performed by adding 0.5 ml of acetonitrile and 10 ng of internal standard (diphenhydramine) to 0.1 ml of plasma and blank plasma samples. The mixture was vortexed for 2 min and centrifuged at 3,500 rpm in an Allegra 6R centrifuge equipped with an S2096 microtiter rotor (Beckman Coulter) for 5 min at 4°C. The clear supernatant was transferred to a clean 96-well plate and concentrated under a stream of nitrogen in a TurboVap 96 concentrator (Zymark). The sample extracts were reconstituted in 250 μl of methanol-water (1:1), and 10 μl was used for LC-MS/MS analysis. LC-MS/MS analysis was carried out on an LCQ-Deca mass spectrometer (Thermo Finnigan) equipped with an electrospray ionization source operated in positive ion mode, a Surveyor solvent delivery system (Thermo Finnigan), and a Surveyor 96-well plate autoinjector (Thermo Finnigan). The temperature of the sample compartment was set to 10°C. The LCQ-Deca mass spectrometer was operated with a spray voltage of 4.5 kV and a capillary temperature of 250°C. The sheath gas flow rate, auxiliary gas flow rate, capillary voltage, tube lens offset, and collision energy were optimized for each analyte. Data were acquired via single-reaction monitoring. The ions representing the (M + H)⁺ species for the analyte and internal standard were selected with an isolation width of 4 nm (atomic mass units) and were fragmented by collision with nitrogen (nitrogen quotient, 0.250; time, 30 ms) to form specific product ions, which were subsequently monitored. The extract was chromatographed on a Beta-Basic C₄ column (5 μm, 30 by 2 mm; Thermo Keystone, Bellefonte, Pa.) run at 300 μl/min at 25°C with 0.05% formic acid in water (solution A) and 0.05% formic acid in acetonitrile (solution B) as the mobile phase. Separation was achieved with the following gradient: 20% solution B, which was changed to 50% B in 1 min, held at 50% solution B for 1 min, changed to 90% solution B in 1 min, and held at 90% for 1.5 min, followed by reequilibration to 20% solution B within 1.5 min. The column effluent was diverted to the mass spectrometer column from 2 to 5.4 min; otherwise, it was diverted to waste.

(ii) Analysis of GSQ and linezolid in tissue.Aliquots of 50 mg of tissue were used. Water (100 μl), ice-cold acetonitrile (200 μl), and internal standard (10 ng; diphenhydramine) were added to each tissue aliquot. The mixture was put on ice and homogenized with a PowerGen 125 homogenizer for 20 s. After homogenization, 700 μl of acetonitrile was added, the samples were mixed, and the precipitate was pelleted by centrifugation in an Allegra 6R centrifuge equipped with an S2096 microtiter rotor (Beckman Coulter) for 10 min at 4°C. The clear supernatant was transferred to a clean 96-well plate and concentrated under a stream of nitrogen in a TurboVap 96 concentrator (Zymark). The tissue extracts were reconstituted in 250 μl of methanol-water (1:1), and 10 μl was used for LC-MS/MS analysis. Tissue aliquots were quantitated by use of a plasma standard curve. The LC-MS/MS conditions were identical to those described above.

(iii) Analysis of vancomycin in plasma.Plasma (50 μl) was transferred to a 96-well plate and treated with 150 μl of acetonitrile containing 0.1 μg of internal standard (diphenhydramine) per ml. The plates were sealed, and the contents of the wells were mixed thoroughly before the precipitate was cleared by centrifugation (5,800 × g for 3 min). After centrifugation the plates were cooled in a freezer (−20°C) for 5 to 10 min before 40 μl of clear supernatant was transferred to an autosampler microplate. The samples were dried under nitrogen and reconstituted in 100 μl of 0.2% formic acid. A total of 50 μl was used for LC-MS/MS analysis. LC-MS/MS analysis was carried out on a PE Sciex API 3000 mass spectrometer equipped with a turbo ion spray source and a Shimadzu high-pressure liquid chromatography system consisting of two LC-10 ADvp pumps, a SIL-HT autosampler, and a SPD-10 A UV detector. A multiple-reaction monitoring method was performed in the positive ion mode for quantitation of the analyte and the internal standard by using Q1-Q3 transitions in unit resolution. The dwell times were 450 ms on the analyte and 50 ms on the internal standard. The nebulizing and curtain gases were both set to 10 liters/min, the collision-activated dissociation gas was set to 4 liters/min, and the source temperature was 400°C. An optimization procedure was performed for each analyte to select the Q1-Q3 transition used for quantitation and to optimize the declustering potential, focusing potential, collision energy, and collision cell exiting potential for that particular transition.

The extracts were chromatographed on a Higgins Analytical Targ cartridge guard column (2 by 20 mm) with 10 mM ammonium formate and 0.5% formic acid in water (solution A) and 10 mM ammonium formate and 0.5% formic acid in 90% acetonitrile (solution B) as the mobile phase, which was run at 400 μl/min. Separation was achieved with the following gradient: 5% solution B, which was changed to 50% solution B in 1 min, held at 50% solution B, changed to 95% solution B in 3 min, and held at 95% solution B for 0.25 min, followed by reequilibration to 5% solution B within 0.75 min. The unbound wash peak and the wash peak with high organic contents were diverted to waste.

(iv) Analysis of vancomycin in tissue.A total of 50 μl of water, followed by 150 μl of acetonitrile (containing internal standard), was added to each 50-mg aliquot of tissue. The mixture was homogenized with a Polytron homogenizer equipped with a serrated microtip. Homogenized samples were centrifuged at 14,000 × g for 5 min. A total of 40 μl of the clear supernatant was transferred to a clean 96-well microplate and concentrated under a stream of nitrogen. All subsequent procedures were identical to those described above for the analysis of plasma.

Calibration curves.The calibration curves (y = mx + b) were generated by linear least-squares regression of the peak area ratios (y) of the analytes to their internal standards versus the concentrations (x) of the calibration standards. The concentrations of the analytes in plasma and tissue samples were calculated by using the resulting peak area ratios and the regression equations of the calibration curves. Calibration standards were prepared by adding 20 μg of analyte to 1 ml of blank plasma. This 20-μg/ml standard was subsequently serially diluted (threefold) with blank plasma to cover a concentration range of 1 to 20,000 ng/ml. The calibration standards were extracted and analyzed by the methods described above. The backcalculated concentrations of the standards and quality control samples did not deviate by more than 40% from the nominal concentrations.

Murine peritonitis model.Two strains of S. aureus, ATCC 13709 (methicillin susceptible) and ATCC 27660 (methicillin resistant, β-lactamase positive), were used for the murine peritonitis model. Fresh colonies were grown in brain heart infusion broth (International BioProducts, Bothell, Wash.) at 37°C with gentle agitation and adjusted to a concentration of 2 × 10⁷ CFU/ml. A solution of 10% gastric mucin porcine (ICN Biomedicals Inc., Aurora, Ohio) was added at an equal volume to achieve the final inoculum of 1.0 × 10⁷ CFU/ml in 5% mucin. Both ATCC 27660 and ATCC 13709 were prepared in this manner. This represented an inoculum that was 5 to 10 times the 50% lethal dose, which caused a 100% mortality rate by 48 h in vehicle-treated animals infected with each of the strains (M. Gross, R. Tanaka, M. Garcia, W. Aparicio, B. Batiste, M. Powers, T. Annamalai, G. Patou, H. Moser, R. Bürli, P. Jones, J. Kaizerman, M. Iwamoto, V. Jiang, L. Lin, J. Ge, S. Difuntorum, R. Lyons, and K. Johnson, Abstr. 42nd Intersci. Conf. Antimicrob. Agents Chemother., abstr., F-2036, p. 233, 2002).

Groups of five ICR female mice (weight, ∼25 g; Taconic Farms) were inoculated intraperitoneally with 0.5 ml of the inoculum described above. The groups were treated i.v. with the vehicle, a positive control (i.e., vancomycin and linezolid), or the test compounds at 1 h postinfection. The mice were clinically monitored for 5 days, with survival being the primary end point. All animal studies were implemented with strict adherence to federal guidelines and the regulations of the institutional animal care and use committee.

For all in vivo studies described herein, test antibacterials were formulated in 30% hydroxypropyl-β-cyclodextrin-10% d-mannitol-50 mM sodium acetate buffer (pH 4.5 ± 0.1) (denoted G06) to achieve concentrations necessary for in vivo profiling. Notably, the pharmacokinetic properties of these HARP antibiotics did not differ significantly when they were formulated in other solution vehicles (e.g., polyethylene glycol or propylene glycol) (data not shown). Data analysis for determination of the 50% effective dose (ED₅₀), confidence intervals, and coefficient of variance was performed with the WinNonLin pharmacodynamic analysis package.

Murine neutropenic thigh infection model.Two strains of S. aureus, ATCC 13709 (methicillin susceptible) and ATCC 33591 (methicillin resistant, MecA⁺), were used for the murine neutropenic thigh infection model. Overnight cultures in brain heart infusion broth were adjusted by dilution in phosphate-buffered saline (PBS; HyClone, Logan, Utah) to 10^5.9 ± 10^0.4 CFU of methicillin-resistant S. aureus (MRSA) strain ATCC 33591 per ml and 10^5.6 ± 10^0.1 CFU of methicillin-susceptible S. aureus (MSSA) strain ATCC 13709 per ml.

The protocol was essentially that described by Andes et al. (2). ICR mice were rendered neutropenic with 200-mg/kg intraperitoneal injections of cyclophosphamide (Sigma) 4 days and 1 day prior to infection. Groups of three to five neutropenic mice under anesthesia with isoflurane (Baxter, Deerfield, Ill.) were inoculated intramuscularly (0.05 ml). At time −2 h, each anterior thigh muscle was inoculated with 10^4.6 ± 10^0.4 CFU of MRSA strain ATCC 33591, and the opposite thigh was inoculated with 10^4.3 ± 10^0.1 CFU of MSSA strain ATCC 13709. At time zero, the mice were treated i.v. with one of the test compounds or a negative or positive control compound. At predetermined time points posttreatment (e.g., 2 to 24 h), the mice were killed and the thighs were collected aseptically and placed in 10 ml of 1× sterile PBS.

Quantitation of the bacterial load was accomplished by homogenizing the thighs with a Polytron homogenizer (PT 10/35; Brinkman, Westbury, N.Y.). Serial dilutions were prepared with PBS as the diluent. Three dilutions of each homogenate (100 μl each) were plated onto Trypticase soy agar plates (Becton Dickinson Biosciences, Sparks, Md.). Efficacy was determined by comparing the bacterial counts for the negative control (vehicle-treated) groups to those for the groups treated with the test compounds.

S. pneumoniae lung infection. S. pneumoniae ATCC 6303 was grown overnight in Mueller-Hinton broth (Becton Dickinson Biosciences) containing 5% lysed horse blood to a concentration of ∼5 × 10⁶ CFU/ml. Mice were anesthetized with isoflurane (Baxter), and 50 μl of the inoculum described above was instilled into each mouse via the intranasal route. A group of animals was killed at 30 min postinoculation, and the lungs were collected aseptically and placed in 1 ml of sterile 1× PBS to assess the bacterial the loads in the lungs. Treatments were administered i.v. at 1 and 5 h postinoculation. Survival was monitored over 7 days.

Quantitation of the bacterial load was accomplished by homogenizing the lungs with a polytron homogenizer in 1 ml of sterile PBS. Serial dilutions were performed with PBS as the diluent. Three dilutions of each homogenate (100 μl each) were plated onto Trypticase soy agar plates containing 5% sheep blood.

Safety pharmacology and genotoxicity screens. (i) Target selectivity.The activities of the HARP compounds against a wide array of different receptors were evaluated by extensive binding of the compounds at concentrations of 10 μM to ∼60 different targets (LeadProfilingScreen; MDS Pharma Services, Bothell, Wash.).

(ii) HERG channel activity.As a means of assessing the potential for interruption of cardiac I_Kr channel activity with a possible influence on the QT interval, whole-cell patch clamp studies were performed at 0.1 Hz with channel-transfected human embryonic kidney (HEK-293) cells across a range of GSQ-2287 concentrations from 0.1 to 30 μM (0.068 to 20.4 μg/ml). The positive control was Eisai-4031, tested at 1, 10, 50, and 100 nM and with pacing at 0.1 Hz (HERG current assay; Porsolt Pharmacology, Boulogne-Billancourt Cedex, France, with Zenas Technologies, New Orleans, La.).

(iii) Study of physiological and behavioral effects.Evaluation of the effect of a HARP compound on the physiological and central nervous system (CNS) status of intact animals was performed by a standard Irwin screen (Porsolt Pharmacology) with mice (n = 3 mice per group) administered a i.v. vehicle control or GSQ-2287 (10, 30, 100, 300 mg/kg) and observed for up to 24 h.

(iv) Genotoxicity assays.The potential genotoxicities of the HARP compounds were tested with prototype compound GSQ-2287 over a broad range of concentrations in several different systems (Sitek Research, Rockville, Md.). The compound was tested in vitro by Ames bacterial mutagenesis assays with Salmonella strains TA-98, TA-100, TA-1535, and TA-1537 and Escherichia coli SP2 uvrA at concentrations up to 75 μg/plate and by chromosomal aberration and hypoxanthine guanine phosphoribosyl transferase (HGPRT) mutation assays with CHO cells at concentrations up to 160 μg/ml. Finally, potential genotoxicity in vivo was assessed by mouse bone marrow micronucleus testing after i.v. administration of the compounds at concentrations ranging from 10 to 120 mg/kg. All assays were performed as described elsewhere (Sitek Research) with negative and positive control groups.

Rodent tolerability testing. (i) Acute toxicity studies.ICR female mice (weight, ∼25 g) received a single i.v. bolus in the lateral tail vein. The mice were monitored for clinical signs for 48 to 72 h. The mice were then killed by CO₂ inhalation; and a gross necropsy, including determination of body and organ weights, was performed. Blood was collected at the time of euthanasia; and the alanine aminotransferase, aspartate aminotransferase, blood urea nitrogen, creatinine, alkaline phosphotase, glucose, calcium, phosphorus, triglyceride, and total bilirubin levels were analyzed.

Hematology analysis included the following: white blood cell counts, red blood cell counts, hemoglobin concentrations, hematocrit levels, mean corpuscular volume, mean corpuscular hemoglobin concentration, mean corpuscular hemoglobin concentration, and platelet counts.

(ii) Multiday studies.ICR female mice (weight, ∼25 g) received an i.v. bolus in the lateral tail vein once daily for 4 days. The mice were clinically monitored throughout the study. On day 5, 24 h following the last administration of a test compound, the mice were killed by CO₂ inhalation and a gross necropsy was performed. Blood samples were collected for clinical chemistry and hematology analyses at the time of euthanasia (see above for the specific blood analyses performed). Body weights and the weights of selected organs were determined and compared to those for the vehicle-treated control animals.

In vitro activity and protein or DNA binding.The in vitro MICs and the DNA and plasma protein binding results are depicted in Table 1. The HARP compounds were active (MICs ≤ 1 μg/ml) against resistant strains of S. aureus (vancomycin-insensitive, MRSA) and S. pneumoniae (penicillin resistant). The in vitro MIC profile was consistent with those for other drugs with activities against gram-positive bacteria (6, 12; Ge et al., 41st ICAAC). GSQ-10547, in particular, was more potent than linezolid and vancomycin (Table 1). Notably, all three compounds exhibited good activities against a multidrug-resistant (including vancomycin-resistant) E. faecium strain, ATCC 51559: the MICs of GSQ-2287, GSQ-11203, and GSQ-10547 were 1, 0.125, and 1 μg/ml, respectively. In addition, these three HARP antibiotics may potentially exhibit cytostatic and cytocidal activities in mammalian cell cultures containing 5% fetal calf serum, but at concentrations ≥20-fold the MICs (data not shown). The binding to an AT-rich DNA sequence to which the HARP compounds bind (6) was measured. While the K_ds of all compounds were ≤20 nM, GSQ-11203 showed a particularly high binding affinity (K_d, ≤0.1 nM). Potential binding to plasma proteins was also measured to facilitate evaluation of structure-activity and pharmacokinetic-pharmacodynamic relationships. All three HARP compounds exhibited high levels of protein binding, ranging from 90 to 96% for mouse, rat, and dog plasma proteins, as noted in Table 1. The potential effects of plasma proteins on the in vitro antibacterial activities were investigated by measuring the MICs in the presence of 40 mg of human plasma albumin per ml. Eight- to 16-fold shifts in the MICs of all three compounds were observed, consistent with high levels of protein binding.

Pharmacokinetics and tissue disposition.The time courses of the concentrations of the HARP compounds and the reference drugs with activities against gram-positive organisms in plasma following the administration of a 3-mg/kg i.v. bolus to rats are shown in Fig. 2, with the derived pharmacokinetic parameters depicted in Table 2. The pharmacokinetics were characterized by biphasic dispositions with relatively high maximum concentrations of the drugs in plasma (C_maxs), including rapid distribution phases relative to those for the reference drugs linezolid and vancomycin. Clearance approximated liver blood flow (for GSQ-2287 and GSQ-11203), and the terminal elimination phase was relatively extended (2 to 4 h). The volume of distribution was moderate to high in rats (9 to 34 liters/kg).

• Open in new tab
• Download powerpoint

FIG. 2.

Pharmacokinetics of HARP compounds in rat plasma after i.v. administration. The concentrations of parent compound GSQ-2287, GSQ-10547, GSQ-11203, and vancomycin in plasma were determined after the administration a single 3-mg/kg bolus i.v. Each point represents mean ± standard deviation concentrations in plasma for three rats. Analysis of the concentrations in plasma was performed by LC-MS/MS.

The pharmacokinetics of GSQ-2287 were further evaluated in ICR mice and beagle dogs. As shown in Table 2, the pharmacokinetics were similar in all three species, with a trend for the relative area under the concentration-time curve (AUC) and half-life to be greater in dogs than in rodents. The substantial distribution-phase decay is likely related to an extensive and rapid distribution instead of an association with vascular surfaces or cellular components (see below and data not shown).

Analysis of the concentrations in selected tissues following i.v. dosing of the test compounds was used to evaluate the nature of the distribution of the HARP antibiotics to peripheral organs. Such an analysis has never been performed with DNA minor-groove binders of this type and was deemed helpful for both pharmacodynamic and toxicodynamic interpretations. Mice were administered GSQ-2287 or vancomycin as a reference control (Fig. 3A and B, respectively). Notably, the test compounds were administered at comparable efficacious dose levels: 50 mg/kg for GSQ-2287 and 10 mg/kg for vancomycin. Vancomycin showed proportionally greater concentrations in plasma and tissue than GSQ-2287 and, correspondingly, showed greater efficacy. Both compounds were rapidly distributed to peripheral organs, with the liver and kidney exhibiting higher concentrations than plasma or the other organs assessed. The distributions of GSQ-2287 and vancomycin to thigh muscle were intermediate compared to those to liver and plasma, and the distributions of vancomycin to liver and plasma were still higher, despite the use of a fivefold lower dosage. The distribution to the spleen was high and between those to the liver and kidney. The concentrations in the lung (data not shown) were intermediate compared to those in liver and muscle. GSQ-2287 did not exhibit significant concentration in the brain at any time point (Fig. 3A). Whereas the kinetics of GSQ-2287 decay in most organs was similar to that in plasma, the spleen had a greater retention as a function of time. Similar studies were performed with GSQ-10547 and GSQ-11203 (data not shown), and they had profiles generally similar to that for GSQ-2287.

• Open in new tab
• Download powerpoint

FIG. 3.

Tissue disposition of GSQ-2287 (A) and vancomycin (B) in mice. (A) Concentrations of GSQ-2287 in plasma and selected tissues following administration of a 50-mg/kg i.v. bolus. Each point represents mean ± standard deviation concentrations in plasma or tissue for three mice. Tissues samples (50 mg each) were collected immediately after the mice were killed and flash frozen in dry ice. Analysis of the concentrations in plasma and tissue was performed by LC-MS/MS. (B) Mean ± standard deviation concentrations of vancomycin in plasma and selected tissues following administration of a 10-mg/kg i.v. bolus. Each point represents the mean ± standard deviation concentrations in plasma or tissue for three mice, as described for panel A.

Efficacy against mouse peritonitis.As a first examination, the HARP compounds were evaluated in the classic mouse lethal peritonitis model of in vivo antibacterial efficacy: Table 3 presents a summary of the ED₅₀s from a number of studies with S. aureus. The compounds were dosed once i.v. at 1 h postinfection, and the results were compared directly to those for vancomycin. All three representative HARP antibiotics protected the mice against a lethal MSSA infection, with the rank order of potency correlating with the MICs obtained in vitro. In addition, the compounds were tested for efficacy in a more challenging setting: infection with MRSA. With a shift in effective dose levels relative to those of the reference drugs, GSQ-10547 and GSQ-11203 provided protection against MRSA. The ED₅₀ of the most potent HARP compound, GSQ-10547, was 13 mg/kg for MRSA, whereas the ED₅₀ was 7 mg/kg for MSSA. A detailed representation of the full dose-response profile for GSQ-10547 is shown in Fig. 4. The calculated ED₅₀ (13 mg/kg) of GSQ-10547 was a few fold higher than that of vancomycin. Notably, distamycin A was examined in the peritonitis models and exhibited limited efficacy against MSSA when it was administered i.v. (20% survival with two i.v. doses of 20 mg/kg). The acute toxicity of distamycin A at higher doses (e.g., 50 mg/kg twice a day) precluded meaningful efficacy studies with MRSA-infected animals.

• Open in new tab
• Download powerpoint

FIG. 4.

Dose-response curve for GSQ-10547 for determination of its efficacy against MRSA sepsis. The results reflect survival at day 5 after an intraperitoneal injection of a lethal inoculum of MRSA and administration of the test compound at 1 h postinfection. Percent survival data (from 19 individual studies) were fit to a sigmoid maximum effectiveness model by using the WinNonlin (version 4.0) program. The ED₅₀ (12 mg/kg) and gamma (shape parameter) were determined along with the coefficient of variance (11%).

Neutropenic thigh infection model. (i) S. aureus ATCC 13709 (MSSA).The results from studies with the murine neutropenic thigh infection model of MSSA infection and subsequent exposure to the test compounds are depicted in Fig. 5A. Treatment with GSQ-10547 and GSQ-2287 at 50 to 60 mg/kg resulted in substantial reductions in the bacterial burdens in the thighs at 5 h. For the more potent compound, GSQ-10547, this reduction was retained for 24 h, and the results were comparable to those for vancomycin (dose, 20 mg/kg) at that time point.

• Open in new tab
• Download powerpoint

FIG. 5.

In vivo efficacies of the HARP compounds in neutropenic mice with MSSA (A) or MRSA (B) thigh infection. (A) Time course of mean ± standard deviation log CFU per thigh of MSSA strain ATCC 13709 following i.v. dosing with GSQ-2287 (▵; 60 mg/kg), GSQ-10547 (×; 50 mg/kg), and vancomycin (□; 20 mg/kg). ○, vehicle-treated control. Each point represents the mean ± standard deviation log CFU per thigh for more than three mice combined from multiple studies. (B) Time course of mean ± standard deviation log CFU per thigh of MRSA strain ATCC 33591 following i.v. dosing of GSQ-2287 (○; 60 mg/kg), GSQ-10547 (•; 50 mg/kg), and vancomycin (⋄; 20 mg/kg). ×, vehicle-treated control. Each point represents the mean ± standard deviation log CFU per thigh for three or more mice combined from at least two studies with each compound.

(ii) S. aureus ATCC 33591 (MRSA). The efficacies of the test compounds against MRSA strain ATCC 33591 in the thighs are presented in Fig. 5B. GSQ-11203 and GSQ-10547 showed potent activities (1 log reduction in the bacterial load compared with that for the vehicle-treated group) against MRSA at doses of ≤25 and 30 mg/kg, respectively. In comparison, the doses of vancomycin and linezolid that yielded a 1 log reduction in the bacterial load compared with that for the vehicle-treated group were both approximately 10 mg/kg. These values were derived from individual studies with at least two doses for three mice per group in at least three studies. Thigh burden was determined 5 to 6 h after drug administration. All of the test compounds including vancomycin and linezolid yielded minimal reductions in thigh burden 24 h after a single i.v. administration. It is noteworthy that GSQ-11203 reproducibly showed potency similar to or better than that of GSQ-10547 (at 50 mg/kg, a 1.7 log reduction at 5 h) in this particular model, even though the MIC for ATCC 33591 was higher (Table 1) and the pharmacokinetic profile (Table 2; Fig. 2) would not imply significantly superior exposure.

Lung infection.The most potent HARP antibiotic, GSQ-10547, was further evaluated for its in vivo efficacy in a stringent lung pneumonia model. GSQ-10547 (MIC = 0.25 μg/ml) dosed at 50 mg/kg i.v. at 1 and 5 h postinfection protected 100% of mice from a lethal lung infection with S. pneumoniae ATCC 6303 (penicillin susceptible). All vehicle-treated control animals died within 48 to 72 h. Amoxicillin-clavulanate (Augmentin; MIC = 0.031 μg/ml) administered orally via the same regimen at 10 mg/kg provided 100% efficacy.

Safety pharmacology and genotoxicity screens.A broad in vitro receptor interaction screen can unveil off-target activities which may influence both safety and efficacy. Of more than 60 receptors screened with 10 μM GSQ-2287, only 3 targets, all G-protein-coupled receptors, were inhibited >50% (but <80%): adenosine A_2a, 55%; dopamine D₃, 76%; and serotonin 5-HT_2a, 62%. The other receptors for which <50% inhibition was detected were as follows: adenosine (A₁, A_2b), adrenergic receptor (α_1a, α_1b, α_1d, α_2a, α_2b, β₁, β₂, NE transporter), bradykinin (B₁, B₂), calcium channel L (BZT, DHP), calcium channel N, dopamine (D₁, D_2L, D_4.1, Transporter), endothelin (ET_a, ET_b), epidermal growth factor, estrogen α, γ-aminobutyric acid (Transporter, A, BZD, B), glucocorticoid, glutamate (kainate, N-methyl-d-aspartates), histamine (H₁, H₂, H₃), imidazoline I₂, interleukin 1α, leukotriene, (B₄, D₄), muscarinin (M₁, M₂, M₃), neuropeptide (Y₁, Y₂), nicotinic (Ach), opiate (δ, κ, μ), platelet-aggregating factor, K⁺ channel, purinergic (2_y, 2_x), serotonin (5HT-3, Transporter), sigma (1, 2), Na⁺ channel, tachykinin NK₁, and testosterone. Interestingly, some structure-activity correlates were detected when the same study was performed with GSQ-10547 and GSQ-11203 (Table 4).

Another important in vitro safety pharmacology test is for potential alteration of HERG K⁺ channel conductance. Blockage of the HERG channel can lead to abnormalities in the QT phase of the cardiac action potential. GSQ-2287 at concentrations ranging from 0.1 to 30 μM caused a ≤5% reduction in the QT phase of the cardiac action potential relative to that for the controls, which is essentially considered negative.

A classic in vivo screen (Irwin test) used to evaluate the potential alteration of physiological and CNS or motor function was performed. The vehicle control (G06) or GSQ-2287 at 10, 30, 100, and 300 mg/kg was administered i.v. to groups of three mice each; and the mice were monitored for up to 24 h. A significant response involved at least two of three animals, and the response was clearly different from that for the vehicle-treated controls. The following was observed: at 10 and 30 mg/kg there were no significant changes; at 100 mg/kg there was mild and transient (at 15 to 30 min postdosing) sedation, abnormal gait, reduced muscle tone, and hypothermia; and at 300 mg/kg there was transient (at 15 to 60 min postdosing) sedation, abnormal gait (some rolling), tremor, reduced muscle tone and traction, and hypothermia.

Last, a comprehensive evaluation of the potential genotoxicity of GSQ-2287 was performed in vitro and in vivo. Considering that the apparent mode of action is reversible DNA binding, such prospective analysis could be critical to ultimate therapeutic applications. The results of Ames mutagenesis assays with five strains of bacteria and the test compounds at concentrations up to 75 μg/plate were uniformly negative. Genotoxicity against mammalian cells was determined with CHO cells in two assays: HGPRT mutation and chromosomal aberration assays. In a manner consistent with the studies with bacteria, GSQ-2287 at concentrations up to 160 μg/ml had no significant effect on CHO cells relative to the findings for the vehicle-treated control cultures. Finally, potential genotoxicity in vivo was assessed by a mouse micronucleus test, in which animals (three mice per group) were administered either the control solution (G06), GSQ-2287, or GSQ-10547 (10 to 120 mg/kg) i.v. Bone marrow was subsequently analyzed from euthanized mice at 24 and 48 h postadministration. Once again, neither compound was observed to cause significant abnormalities.

Acute and multiple-dose tolerability studies.Tolerability studies were performed with mice by monitoring changes in body and organ weights, serum chemistry, and hematology. The acute toxicity screening assay results for GSQ-10547 are summarized in Tables 5 and 6. GSQ-10547 was well tolerated at 50 mg/kg i.v. at all of the end points mentioned above. GSQ-11203 was also well tolerated at 50 mg/kg, while GSQ-2287 was well tolerated, with no significant adverse effects at any of the end points after administration of single doses up to 300 mg/kg (Gross et al., 42nd ICAAC).

As a preliminary means of assessing tolerability to multiple exposures, prototype compound GSQ-2287 and the overall more active lead compound, GSQ-10547, were administered to mice i.v. once daily for 5 days. These multiday toxicology screening studies monitored changes in body and organ weights, clinical parameters, serum chemistries, and hematology. The HARP antibacterials GSQ-2287 and GSQ-10547 were both tested in two separate multiple-exposure studies with the same end points listed in Tables 5 and 6, and neither compound showed any significant adverse effects relative to the findings for the vehicle-treated controls at the 30-mg/kg screening doses (Gross et al., 42nd ICAAC).