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Pharmacology

Pharmacology of Novel Heteroaromatic Polycycle Antibacterials

M. Gross, R. Bürli, P. Jones, M. Garcia, B. Batiste, J. Kaizerman, H. Moser, V. Jiang, U. Hoch, J.-X. Duan, R. Tanaka, K. W. Johnson
M. Gross
1Pharmacology
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R. Bürli
2Chemistry Departments, GeneSoft Pharmaceuticals, South San Francisco, California 94080
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P. Jones
2Chemistry Departments, GeneSoft Pharmaceuticals, South San Francisco, California 94080
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M. Garcia
1Pharmacology
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B. Batiste
1Pharmacology
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J. Kaizerman
2Chemistry Departments, GeneSoft Pharmaceuticals, South San Francisco, California 94080
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H. Moser
2Chemistry Departments, GeneSoft Pharmaceuticals, South San Francisco, California 94080
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V. Jiang
2Chemistry Departments, GeneSoft Pharmaceuticals, South San Francisco, California 94080
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U. Hoch
1Pharmacology
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J.-X. Duan
2Chemistry Departments, GeneSoft Pharmaceuticals, South San Francisco, California 94080
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R. Tanaka
1Pharmacology
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K. W. Johnson
1Pharmacology
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  • For correspondence: kjohnson@genesoft.com
DOI: 10.1128/AAC.47.11.3448-3457.2003
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ABSTRACT

Heteroaromatic polycycle (HARP) compounds are a novel class of small (Mw, 600 to 650) DNA-binding antibacterials. HARP compounds exhibit a novel mechanism of action by preferentially binding to AT-rich sites commonly found in bacterial promoters and replication origins. Noncovalent binding in the minor groove of DNA results in inhibition of DNA replication and DNA-dependent RNA transcription and subsequent bacterial growth. HARP compounds have previously been shown to have potent in vitro activities against a broad spectrum of gram-positive organisms. The present report describes the extensive profiling of the in vitro and in vivo pharmacology of HARP antibacterials. The efficacies of representative compounds (GSQ-2287, GSQ-10547, and GSQ-11203), which exhibited good MIC activity, were tested in murine lethal peritonitis and neutropenic thigh infection models following intravenous (i.v.) administration. All compounds were efficacious in vivo, with potencies generally correlating with MICs. GSQ-10547 was the most potent compound in vitro and in vivo, with a 50% effective dose in the murine lethal peritonitis model of 7 mg/kg of body weight against methicillin-sensitive Staphylococcus aureus (MSSA) and 13 mg/kg against methicillin-resistant S. aureus (MRSA). In the neutropenic mouse thigh infection model, GSQ-11203 reduced the bacterial load (MRSA and MSSA) 2 log units following administration of a 25-mg/kg i.v. dose. In a murine lung infection model, treatment with GSQ-10547 at a dose of 50 mg/kg resulted in 100% survival. In addition to determination of efficacy in animals, the pharmacokinetic and tissue disposition profiles in animals following administration of an i.v. dose were determined. The compounds were advanced into broad safety screening studies, including screening for safety pharmacology, genotoxicity, and rodent toxicity. The results support further development of this novel class of antibiotics.

The increase in the number of infections caused by drug-resistant gram-positive organisms in the clinic represents a medical need and a commercial opportunity for the development of bactericidal agents with new modes of action (10, 14, 16). Such candidates are expected to lack cross-resistance to existing classes and, provided they exhibit efficacy in vivo, might be of great therapeutic utility. The present report provides a proof of concept for novel chemical entities denoted heteroaromatic polycycle (HARP) antibacterials which are thought to act by a unique DNA-binding mode of action.

HARP antibacterials are small (Mw, ∼600) compounds whose design was initially based on the natural product distamycin A, which was recognized to possess weak antibiotic activity in the 1960s (1, 3, 4, 5). The mechanism of action of distamycin A was unclear, although it was postulated to involve interactions with minor-groove, AT-rich sites of DNA (1, 3, 4, 5, 7); and related compounds with in vitro antibacterial activities were shown to bind to an AT-rich dodecamer (9). Despite some intriguing properties, distamycin A was never developed as an antibacterial due to insufficient potency. Beginning 3 years ago, we applied medicinal chemistry approaches to generate novel polyamide-based compounds with improved antibacterial potency in vitro (6, 13). Moreover, we devoted additional efforts at better defining the bactericidal and bacteriostatic modes of action (12; Y. Ge, J. Wu, and S. White, Abstr. 41st Intersci. Conf. Antimicrob. Agents Chemother., abstr. F-1686, p. 241, 2001). We have optimized this class of compounds for reversible nanomolar binding to the minor groove of AT-rich DNA (1; Ge et al., 41st ICAAC) and potent broad-spectrum activity against gram-positive organisms (6, 12, 13; Ge et al., 41st ICAAC). Consistent with the notion that novel chemical entities with novel modes of action might have activities against drug-resistant organisms, it is clear that the optimized HARP compounds are potent against methicillin-resistant Staphylococcus aureus (MRSA), vancomycin-insensitive S. aureus, vancomycin-resistant enterococci, and drug-resistant pneumococci (6, 12; Ge et al., 41st ICAAC; data not shown).

The present studies were designed to profile the pharmacology of lead compounds. We selected an early prototype, GSQ-2287, and two more potent derivatives, GSQ-10547 and GSQ-11203, to advance into preclinical animal studies and certain in vitro assays related to selectivity and safety. Herein we present for the first time the pharmacological characterization of HARP antibacterials. In addition to pharmacokinetic and safety profiling, we provide a proof of concept of the broad and potent antibacterial efficacies of these agents in vivo. These studies support the further development of HARP antibacterials as novel therapeutic agents.

The structures of distamycin A and the GSQ compounds are depicted in Fig. 1. GSQ-2287 represents a prototype from which the more potent derivatives GSQ-10547 and GSQ-11203 were derived. Optimization of GSQ-2287 led to replacement of the N-terminal chlorothiophene unit by an isoquinoline moiety and the replacement of the central N-methyl-pyrrole by an NH-pyrrole (GSQ-11203) and a benzene moiety in GSQ-10547.

FIG. 1.
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FIG. 1.

Chemical structures of distamycin A and the HARP compounds.

(This work was presented in part at the 42nd Interscience Conference on Antimicrobial Agents and Chemotherapy, 27 to 30 September 2002, San Diego, Calif.)

MATERIALS AND METHODS

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 105.9 ± 100.4 CFU of methicillin-resistant S. aureus (MRSA) strain ATCC 33591 per ml and 105.6 ± 100.1 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 104.6 ± 100.4 CFU of MRSA strain ATCC 33591, and the opposite thigh was inoculated with 104.3 ± 100.1 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 IKr 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.

RESULTS

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 Kds of all compounds were ≤20 nM, GSQ-11203 showed a particularly high binding affinity (Kd, ≤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.

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TABLE 1.

In vitro activities of HARP antibiotics

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 (Cmaxs), 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).

FIG. 2.
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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.

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TABLE 2.

Pharmacokinetic parameters of HARP antibiotics in rats and comparative pharmacokinetics of GSQ-2287 in mice rats and dogsa

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.

FIG. 3.
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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.

FIG. 4.
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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%).

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TABLE 3.

Efficacies of HARP antibiotics in mouse peritonitis model

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.

FIG. 5.
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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 A2a, 55%; dopamine D3, 76%; and serotonin 5-HT2a, 62%. The other receptors for which <50% inhibition was detected were as follows: adenosine (A1, A2b), adrenergic receptor (α1a, α1b, α1d, α2a, α2b, β1, β2, NE transporter), bradykinin (B1, B2), calcium channel L (BZT, DHP), calcium channel N, dopamine (D1, D2L, D4.1, Transporter), endothelin (ETa, ETb), 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 (2y, 2x), 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).

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TABLE 4.

Structure-activity relationship for off-target binding

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).

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TABLE 5.

Clinical chemistries from acute toxicity screeninga

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TABLE 6.

Hematology from acute toxicity screeninga

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).

DISCUSSION

The need for antibacterials with novel modes of action to combat drug-resistant bacteria is well documented (10, 14, 16). HARP compounds, which are derived from natural polyamides, represent antibacterials with novel mechanisms of action, in that they strongly but reversibly and preferentially bind to DNA at AT-rich regions, such as those found in bacterial promoter regions (1, 15, 19, 20, 21). Bacterial transcription and coupled translation are subsequently inhibited, leading to potent and broad-spectrum antibacterial activity (Table 1) (6, 12; Ge et al., 41st ICAAC). The present studies were undertaken to characterize the pharmacological properties of this class of compounds to establish initial in vitro versus in vivo relationships and to provide a stringent means of assessing drug development potential. The in vivo activities displayed by the HARPs in three bacterial infection models in which activity generally correlates well with clinical efficacy provide therapeutic proof of concept. Acceptable in vivo pharmacokinetics and efficacies in combination with additional in vitro and in vivo safety profiles, including genotoxicity, safety pharmacology, and preliminary acute and multiple-dose toxicity screens, provide a composite view which supports further development of this novel class of antibacterials.

The field of molecular recognition of DNA by small-molecule polyamides has evolved extensively over the last 40 years, as recently reviewed by Dervan (11), Sharma et al. (20), and Cozzi and Mongelli (8). While many applications have been investigated and are evolving toward sequence-specific gene regulation for potential anticancer and antiviral applications (8, 11, 20), we have focused on empirical antibacterial optimization. As indicated in a comparison of the structures GSQ-2287 and distamycin A (8), modification of the end cap resulted in significantly improved antibacterial activity in vitro (13), and alteration of the terminal amino group improved antibacterial efficacy and tolerability in vivo (13; Gross et al., 42nd ICAAC). A medicinal chemistry optimization path was established and was driven by in vitro and in vivo evaluations. In the evolution of the evaluation, GSQ-2287 served as an i.v. antibacterial prototype compound and GSQ-10547 represented a more advanced lead compound.

The structural modifications of GSQ-2287 resulted in compounds with improved potencies of DNA binding to the sequences tested. The replacement of the central N-methyl-pyrrole with a benzene group improved both DNA binding and potency in terms of the MIC. A strict correlation between DNA-binding affinity and MICs has not been observed. Bacterial uptake and/or efflux appears to be an important variable: some of the more potent compounds tested in vitro are not the most potent AT-rich DNA binders (e.g., GSQ-11203 versus GSQ-10547), thus adding to the complexity of lead compound optimization. Notably, within this class of molecules, we have not identified compounds which do not bind to DNA (i.e., compounds with Kds >10 μM) and yet still exhibit antibacterial activity (i.e., MICs < 16 μg/ml). An interesting structure-function relationship was apparent from evaluation of off-target (i.e., protein receptor) interactions (Table 4). In summary, of the receptors inhibited by at least one compound by ≥50%, it appears that reactivity with 5-HT2A (range, 30 to 62%) is the only one that is shared activity. Adenosine A2A binding of GSQ-2287 was related to the presence of an N-terminal chlorothiophene moiety, as it was not observed with the isoquinolines (GSQ-11203 and GSQ-10547). The core pyrrole composition influenced the adrenergic α2B and dopamine D3 receptors, whereas replacement of the central pyrrole with a benzene (GSQ-10547) appeared to enhance neuropeptide Y1 binding activity.

The pharmacokinetics and tissue disposition of the HARP antibacterials shared some common traits. First, the levels of plasma protein binding ranged from ∼90 to 95%, depending on the molecule, and were in part influenced by the nature of the end groups, as, for instance, distamycin A is ∼60% plasma protein bound (data not shown). Second, the distribution of the HARP compounds after i.v. administration was rapid and extensive, with sustained terminal elimination half-lives. Third, the compounds had similar tissue exposure profiles: the levels in the brain were low, likely due to failure to cross the blood-brain barrier on the basis of size and polarity. Highly perfused organs which may also be involved in elimination, namely, the liver and kidney, tended to exhibit the highest steady-state concentrations. In addition, the trend for concentration in the lymphatics may be related to reticuloendothelial system uptake and transport. In terms of comparative kinetics in plasma between different species (mouse, rat, dog), the pharmacokinetic profiles of the compounds were generally conserved, with a potential trend for greater exposures (Cmax and AUC) per given dose in dogs relative to those in rodents. While oral formulation optimization was not undertaken, none of the HARP compounds described in this report and tested in G06 or a polyethylene glycol solution vehicle exhibited >5% oral bioavailability.

Assessment of the efficacies of the HARP compounds in murine models of infection indicated that the in vivo antibacterial activity of GSQ-10547 was potent, durable, and broad in scope. Specifically, GSQ-10547 displayed efficacy against multiple S. aureus strains, including MSSA and MRSA strains, in classic animal models of sepsis and thigh infection. Further in vivo efficacy was evident in a model of lethal S. pneumoniae lung infection, in which GSQ-10547 at 50 mg/kg provided a complete survival benefit, while the efficacy of ofloxacin (MIC = 2.0 μg/ml for penicillin-susceptible S. pneumoniae strains) for sustained survival in the same lung infection model required 100 to 200 mg/kg/day.

We have performed preliminary pharmacokinetic-pharmacodynamic evaluations with GSQ-2287 and GSQ-10547 in the mouse peritonitis model. The variables Cmax, AUC, and the time at which the concentration remains above the MIC were controlled by bolus administration by the i.v. or subcutaneous route and by comparison of those results with the results obtained with controlled i.v. infusion. The results pointed toward efficacy being concentration driven, particularly Cmax (data not shown).

In general, the rank order of potency in vivo of GSQ-2287, GSQ-11203, and GSQ-10547 correlated well with the in vitro MICs, with GSQ-10547 being the most potent analog overall (GSQ-2287 < GSQ-11203 < GSQ-10547). In terms of relative in vivo potency, vancomycin as a reference drug exhibited better potency than GSQ-10547, which is likely due to two key traits: the level of plasma protein binding is significantly less for vancomycin (∼30%), and the levels of tissue exposure for vancomycin relative to those for the polyamides are particularly high in mice. The attractiveness of these new antibacterial agents lies at least partly in the realization of meaningful efficacy in vivo by a novel class which retains activity against drug-resistant organisms, including vancomycin-resistant staphylococci and enterococci (see Results) (12; Ge et al., 41st ICAAC).

As HARP compounds represent a novel therapeutic class of antibacterials with a unique, DNA-targeting mechanism, careful evaluation of selectivity and safety were mandated. Accordingly, a broad approach toward early safety assessment was undertaken. In terms of selectivity, GSQ-2287 (and the other HARP compounds; data not shown) exhibited minimal off-target activity (Table 4) at concentrations that exceeded the MICs and that correlated with maximal efficacious levels in plasma. A potentially undesirable effect of capitalizing on a novel DNA-binding mechanism might be the observation of genetic toxicity. The results of extensive genotoxicity testing were negative with GSQ-2287 and GSQ-10547. There were no significant increases in mutation frequencies of either bacterial or mammalian cells exposed to the HARP compounds. Furthermore, no significant indication of bone marrow micronucleus formation relative to the findings for the vehicle-treated controls was observed following i.v. administration of the compounds to mice, and yet the results obtained with the positive control agents in all assays validated the integrity of the study. Such uniformly negative genotoxicity outcomes further attest to the therapeutic potential of this novel class of antibacterials.

Finally, general systemic tolerability was evaluated in preliminary Irwin (functional observation battery) tests and acute and multidose toxicity screens with rodents. In the Irwin test, the observation of some deficits in CNS and motor activity at the highest doses was not discouraging, considering the route (i.v.), the relatively high dose levels (100 to 300 mg/kg), and the transient nature. In toxicity screens with dose levels exceeding those producing antibacterial efficacy in vivo, there were no observations of overt changes in any end point. Such claims are not intended to discount the potential toxicity of HARP compounds in general, as some of the optimization effort has addressed tolerability. Further dose-escalating, longer-term studies are necessary to better define margins of safety.

In summary, the pharmacological evaluation of HARP DNA binders described here supports the continued research optimization and subsequent development of this class as novel antibacterial therapeutic agents. The magnitude and spectrum of antibacterial efficacy in vitro and in vivo support their potential utility as i.v. administered agents for the treatment of infections caused by gram-positive bacteria. Treatment of infections caused by drug-resistant organisms, including emerging vancomycin- and linezolid-resistant organisms, may represent special opportunities for the HARP compounds. Their activities against drug-resistant isolates is evident from the MICs, and the potential translation to in vivo efficacy is demonstrated by the present work.

ACKNOWLEDGMENTS

This work was in part funded by a grant (grant N65236-99-1-5427) from DARPA.

We are very appreciative of the excellent technical support provided by Willie Aparicio, Stacey Difuntorum, Lin Lin, Mike Powers, Thamil Annamalai, Mari Iwamoto, Hsiu Chen, James Ge, and Zhijun Ye. Eric Taylor at Bay Bioanalytics is appreciated for scientific contributions. Yuan Chao is acknowledged for excellent patent support and advice. Sandhya Girish and Jackie Gibbons are appreciated for their assistance with data analysis. Finally, we recognize the editorial, scientific, and management contributions of Gary Patou, Ken Drazan, Sarah White, Nick Marini, Denene Lofland, Sofia Touami, and Eldon Baird.

FOOTNOTES

    • Received 22 January 2003.
    • Returned for modification 9 June 2003.
    • Accepted 17 July 2003.
  • Copyright © 2003 American Society for Microbiology

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Pharmacology of Novel Heteroaromatic Polycycle Antibacterials
M. Gross, R. Bürli, P. Jones, M. Garcia, B. Batiste, J. Kaizerman, H. Moser, V. Jiang, U. Hoch, J.-X. Duan, R. Tanaka, K. W. Johnson
Antimicrobial Agents and Chemotherapy Oct 2003, 47 (11) 3448-3457; DOI: 10.1128/AAC.47.11.3448-3457.2003

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Pharmacology of Novel Heteroaromatic Polycycle Antibacterials
M. Gross, R. Bürli, P. Jones, M. Garcia, B. Batiste, J. Kaizerman, H. Moser, V. Jiang, U. Hoch, J.-X. Duan, R. Tanaka, K. W. Johnson
Antimicrobial Agents and Chemotherapy Oct 2003, 47 (11) 3448-3457; DOI: 10.1128/AAC.47.11.3448-3457.2003
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KEYWORDS

Anti-Bacterial Agents
Heterocyclic Compounds
Potassium Channels, Voltage-Gated

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