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Antimicrobial Agents and Chemotherapy, August 2005, p. 3302-3310, Vol. 49, No. 8
0066-4804/05/$08.00+0     doi:10.1128/AAC.49.8.3302-3310.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

Systemic Antibacterial Activity of Novel Synthetic Cyclic Peptides

Véronique Dartois,^1,* Jorge Sanchez-Quesada,^1,, Edelmira Cabezas,¹ Ellen Chi,¹ Chad Dubbelde,¹ Carrie Dunn,¹ Juan Granja,^1, Colleen Gritzen,¹ Dana Weinberger,¹ M. Reza Ghadiri,² and Thomas R. Parr Jr.^1,¶

Adaptive Therapeutics, Inc., 5820 Nancy Ridge Drive, San Diego, California 92121,¹ The Scripps Research Institute, La Jolla, California 92037²

Received 8 February 2005/ Returned for modification 18 March 2005/ Accepted 16 May 2005

Top Abstract Introduction Materials and Methods Results Discussion References

 
Cyclic peptides with an even number of alternating D,L--amino acid residues are known to self-assemble into organic nanotubes. Such peptides previously have been shown to be stable upon protease treatment, membrane active, and bactericidal and to exert antimicrobial activity against Staphylococcus aureus and other gram-positive bacteria. The present report describes the in vitro and in vivo pharmacology of selected members of this cyclic peptide family. The intravenous (i.v.) efficacy of six compounds with MICs of less than 12 µg/ml was tested in peritonitis and neutropenic-mouse thigh infection models. Four of the six peptides were efficacious in vivo, with 50% effective doses in the peritonitis model ranging between 4.0 and 6.7 mg/kg against methicillin-sensitive S. aureus (MSSA). In the thigh infection model, the four peptides reduced the bacterial load 2.1 to 3.0 log units following administration of an 8-mg/kg i.v. dose. Activity against methicillin-resistant S. aureus was similar to MSSA. The murine pharmacokinetic profile of each compound was determined following i.v. bolus injection. Interestingly, those compounds with poor efficacy in vivo displayed a significantly lower maximum concentration of the drug in serum and a higher volume of distribution at steady state than compounds with good therapeutic properties. S. aureus was unable to easily develop spontaneous resistance upon prolonged exposure to the peptides at sublethal concentrations, in agreement with the proposed interaction with multiple components of the bacterial membrane canopy. Although additional structure-activity relationship studies are required to improve the therapeutic window of this class of antimicrobial peptides, our results suggest that these amphipathic cyclic D,L--peptides have potential for systemic administration and treatment of otherwise antibiotic-resistant infections.

Top Abstract Introduction Materials and Methods Results Discussion References

 
The incidence of community-acquired and nosocomially acquired infections due to the bacterium Staphylococcus aureus is rising (25). From 1990 to 1992, this microorganism was the most common cause of nosocomial pneumonias and surgical wound infections (14). The overall growing crisis in antibiotic resistance and the rise in the incidence of methicillin-resistant S. aureus (MRSA) strains (32, 33) have emphasized the need for therapeutic alternatives to currently available antibiotics. Vancomycin remains the mainstay of therapy against several resistant gram-positive pathogens. However, vancomycin is slowly bactericidal, and with the recent increase in nosocomial infections caused by vancomycin-resistant enterococci and S. aureus (4, 13, 16), there is a growing need for antimicrobial agents with novel mechanisms of action to attack these resistant pathogens. The Food and Drug Administration recently approved the use of daptomycin, a cyclic lipodepsipeptide antibiotic, for the treatment of complicated skin and skin structure infections caused by several gram-positive bacteria. Its mode of action seems to be related to the disruption of the membrane potential of the bacterium, which is caused by the favored oligomerization of daptomycin upon extracellular calcium binding (21).

Cyclic peptides composed of an even number of alternating D- and L--amino acids adopt a planar ring conformation. This conformation orients the amino acid side chains to the outside of the ring structure and the amide groups perpendicular to the plane of the ring. The amide groups are responsible for the hydrogen bond-directed stacking of cyclic peptide subunits in environments that favor hydrogen bond formation, such as lipid bilayers (18, 19). The self-assembling properties of individual cyclic D,L--peptides result in the formation of multimeric, hollow tubular structures also called peptide nanotubes (15). The interaction of these supramolecular structures with biological membranes is highly dependent upon the amino acid composition of the D,L--peptides and the chemical properties of the residues that are in contact with the components of the cell membrane. Peptide nanotubes formed from amphipathic cyclic peptides adopt an orientation parallel to the membrane plane, where the hydrophobic side chains are inserted into the lipidic components of the membrane and the hydrophilic residues remain exposed to the hydrophilic components of the cell membrane. In this format, peptide nanotubes are believed to permeate membranes through a carpet-like mechanism, collapse transmembrane potential and/or gradient, and cause rapid cell death (15, 30).

In this report, we describe the discovery of novel antibacterial compounds resulting from the combinatorial synthesis of short cyclic peptides made of alternating D- and L--amino acids. Six representative compounds were chosen based on their in vitro potency and their in vivo tolerability and efficacy in murine models of infection. The present study was conducted to evaluate the in vivo pharmacodynamics and initial pharmacokinetics of these six cyclic peptides in comparison to vancomycin and oxacillin.

Top Abstract Introduction Materials and Methods Results Discussion References

 
Animals. Four-week-old, specific-pathogen-free, ICR female mice (Harlan Sprague-Dawley, Indianapolis, IN) weighing 21 to 24 g were used for all studies. The Adaptive Therapeutics Animal Care and Use Committee approved all the experimental protocols.

Bacterial strains and media. Methicillin-sensitive S. aureus (MSSA) strains ATCC 25923, 29213, and 13709 (Smith) and MRSA strains ATCC 43300 and 33591 were used for in vitro and in vivo studies. Organisms were grown, subcultured, and quantified in Mueller-Hinton broth (MHB), cation-adjusted MHB (CAMHB), or tryptic soy broth (TSB) and on tryptic soy agar (TSA) (Difco Laboratories, Detroit, MI) or blood agar plates (Hardy Diagnostics, Santa Maria, CA).

Compounds. Vancomycin, oxacillin, and rifampin were obtained from Sigma-Aldrich (St. Louis, MO) and solubilized in 5% dimethyl sulfoxide (DMSO)-water for in vitro use or phosphate-buffered saline (PBS) for in vivo use. Peptide test compounds were purified by high-performance liquid chromatography (HPLC) using mixtures of trifluoroacetic acid (TFA), water, and acetonitrile as the solvent system. Purity values of compounds used for in vivo experiments were between 95% and 98%.

Primary screening of peptide libraries. Primary cyclic peptide libraries were routinely screened at either 8 µg/ml or 16 µg/ml for antibacterial activity against S. aureus ATCC 29213. Nominal 200-µg/ml stock solutions of the peptides in 5% DMSO in water were aliquoted into sterile 96-well flat-bottom polystyrene plates using a Tomtec Quadra 96 liquid handler. For screening compounds at 8 µg/ml, 4 µl of 200-µg/ml stocks were distributed into plates prefilled with 4 µl of 5% DMSO in water per well. For screening compounds at 16 µg/ml, 8 µl of the 200-µg/ml stocks was distributed into empty plates.

S. aureus was grown in a shaking incubator at 35°C and 200 rpm in 20 ml TSB using individual colonies retrieved from a fresh overnight TSA-sheep blood plate. The culture was grown to a density of approximately 1 x 10⁸ cells/ml and then diluted to 5 x 10⁵ cells/ml in CAMHB. This 5 x 10⁵-cell/ml dilution was dispensed into the compound-containing plates (90 µl/well) using a Titan Multidrop 384. Controls on each plate included a blank medium control, a 100% inhibition control (vancomycin at a final concentration of 4 µg/ml), and a 100% growth control (5% DMSO in water only). After addition of the cell suspension, plates were sealed and incubated at 35°C for 18 h. The optical density at 595 nm (OD₅₉₅) of each well was measured after 18 h using a Tecan Genios spectrophotometer. OD data were uploaded and processed by an internal database and expressed as percent inhibition relative to that of the 100% inhibition controls.

MICs and minimum bactericidal concentrations (MBCs). MICs of the various compounds against a panel of gram-positive pathogens were determined by standard National Committee for Clinical Laboratory Standards microdilution methods (26a) with twofold serial dilutions in CAMHB. Each well contained 100 µl (50 µl inoculum plus 50 µl of drug-containing MHB). The final cell density was 1 x 10⁵ to 5 x 10⁵/ml. MIC endpoints were determined by visual inspection after incubation at 35°C for 18 to 24 h. Final results were expressed as means of two to four determinations. MBCs were defined as the minimum concentration of agent that brought about >99.9% killing of the organism.

Antimicrobial killing kinetics. The kinetics of bactericidal activity was determined by measuring changes in the viable counts of bacteria exposed to various test compounds. A 20-ml liquid culture of S. aureus ATCC 25923 was grown in TSB at 35°C for 1 h or until the OD₆₂₅ reached 0.08 to 0.1 (10⁸ CFU/ml). The culture was diluted 1:100 in 4 ml (10⁶ CFU/ml) of prewarmed medium containing the test compound, control antibiotic, or vehicle-only control at a concentration of four times the MIC and incubated at 35°C. Samples were drawn at 0, 2, 5, 10, 30, 60, and 90 min. Serial dilutions of these samples were plated on CAMHB and incubated overnight at 35°C. The number of CFU on plates with an appropriate density was scored the next day.

Resistance development studies. MICs of six peptide compounds (6752, 7251, 1316, 1150, 6756, and 6853), vancomycin, and rifampin were newly determined as described above using a single culture of S. aureus ATCC 29213 inoculated with colonies from a fresh streak plate of this strain. The final concentrations of the serial twofold dilutions of the compounds ranged from 32 µg/ml to 0.25 µg/ml, and those of vancomycin and rifampin ranged from 16 µg/ml to 0.125 µg/ml and 0.25 µg/ml to 0.00195 µg/ml, respectively, for these MIC experiments.

After this initial MIC experiment, MICs were then determined daily for 15 days for each of the eight compounds using cells from the well in which the compound concentration was one-half the MIC (1/2 MIC well). For each compound, the 1/2 MIC well from the previous day's MIC assay plate was resuspended and the OD₅₉₅ of the resuspension was determined. The resuspension was then diluted to 5 x 10⁵ cells/ml in CAMHB and used to again determine the MIC of the same compound to which those cells had previously been exposed. Compound concentration ranges were adjusted during the course of the 15-day experiment when necessary to keep them useful for MIC determination. All MIC determinations were done in duplicate.

Aliquots of the 5 x 10⁵-cell/ml dilutions for each 1/2 MIC well were periodically plated on TSA-sheep blood plates and incubated at 35°C for 18 h to verify the cell densities by colony counts and to visually inspect the phenotype of the resultant S. aureus colonies.

Hemolysis assay. Serial twofold dilutions of the compounds were prepared in 20% DMSO in water, and 20 µl-aliquots were distributed in 96-well plates. Mouse red blood cells (RBC) were obtained by centrifuging whole blood at 1,000 x g, washed in PBS, and resuspended in PBS containing 10% fetal bovine serum (FBS) at a final RBC concentration of 5%. The RBC suspension (80 µl) was added to each well, and the plate was incubated at 35°C for 30 min. Following centrifugation at 1,000 x g, hemolysis was assessed by measuring the OD₅₉₅ of the serum layer. Hemolysis is calculated as the percentage of total hemolysis as defined by hemolysis achieved by 100 µg/ml melittin (Sigma Chemical Company, St. Louis, MO) as the control. Fifty percent hemolysis (HD₅₀) values were calculated as the compound concentrations required to lyse 50% of the cells.

Cytotoxicity assay. Cellular cytotoxicity was measured by a colorimetric assay that makes use of the tetrazolium salt MTS [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium]. A subconfluent monolayer culture of rat hepatoma cells (H-4-II-E) (ATCC CRL1548) was trypsinized in 0.25% trypsin-1 mM EDTA in Hanks balanced salt solution without Ca²⁺ or Mg²⁺. Cells were collected in complete Eagle's minimum essential medium containing 10% FBS. Complete growth medium was Eagle's minimal essential medium combined with FBS to a final concentration of 10% and antibiotic-antimycotic liquid so as to contain 100 U/ml penicillin, 100 µg/ml streptomycin, and 0.25 µg/ml amphotericin B in 0.85% saline. Dilutions of cells were made in complete medium at 5 x 10⁴ cells/ml, and 90 µl of the resultant mixture was aliquoted onto 96-well plates.

Cells were allowed to incubate at 37°C in the presence of 5% CO₂, and 10 µl of serial twofold dilutions of compound in 5% DMSO in water was then added to the 90 µl of medium and cells. Thioridazine was included as a positive control. Post 24-h incubation in the presence of compound, cells were tested with MTS (25 µl each of MTS at 40 mg/ml in 100% DMSO and phenazine methosulfate at 3 mM in PBS per well). Initial readings of the plates were performed at 490 nm immediately following addition of the reagents. Plates were then allowed to incubate 3 h more, and an endpoint reading was taken at 490 nm with 5 s of linear shaking immediately before reading. The net increase between the initial and final readings was determined for each well. Percent cytotoxicity was calculated as follows: (control value – test value) x 100/control value. Fifty percent inhibitory concentrations were obtained by using the GraphPad Prizm software (www.graphpad.com; San Diego, CA).

Tolerance testing. To determine the maximum tolerated dose in the mouse, compounds were prepared in 5% (wt/vol) dextrose in water and injected intravenously (i.v.) starting at 10 mg/kg; an up-down protocol, based on the outcome obtained with the initial dose of 10 mg/kg, was followed. The following signs were recorded: reduced motor activity, piloerection, redness in the ear lobe, cyanosis, protruding eyeballs, slow or labored breathing, loss of response in the rear legs, convulsions, and death. A score was given based on the intensity and number of the observed signs listed above as follows: 5, no signs observed; 4, light redness in the ear lobe; 3, reduced motor activity and redness in the ear lobe; 2, reduced motor activity, piloerection, and pronounced redness in the ear lobe; 1, protruding eyeballs, temporary loss of motor activity, piloerection, redness in the earlobes and legs, and labored breathing; 0.5, protruding eyeballs, temporary loss of motor activity, piloerection, redness in the earlobes and legs, convulsions with subsequent loss of response in the rear legs, and labored breathing; 0, death.

Neutropenic-mouse thigh model of infection. Mice were rendered neutropenic by injecting cyclophosphamide (Henry Schein, Melville, NY) intraperitoneally 4 days (150 mg/kg of body weight) and 1 day (100 mg/kg) before experimental infection. Previous studies have shown that this regimen produces neutropenia in this model for 5 days (12). Broth cultures of freshly plated bacteria were grown overnight in MHB. After a 1:4,000 dilution into PBS, bacterial counts of the inoculum ranged between 10⁶ and 2 x 10⁶ CFU/ml. Mice were anesthetized briefly with approximately 4% isoflurane just prior to inoculation. The bacterial suspension (0.1 ml) was injected intramuscularly into each thigh (approximately 10⁵ CFU/thigh). In all studies, treatment was initiated 2 h following bacterial inoculation through an i.v. route. At various time points following treatment, groups of three mice were humanely sacrificed by CO₂ asphyxiation. The thigh muscle mass, including the bone, was homogenized using a tissue homogenizer (Polytron; Kinematica AG) and decimally diluted in iced PBS, and 5-µl aliquots of five serial dilutions were plated on TSA. Following overnight incubation at 35°C, CFU were enumerated for each thigh and expressed as the log₁₀ CFU/thigh. The limit of detection was 3.3 log CFU/thigh. Efficacy (as presented in Table 2) was calculated by subtracting the mean log₁₀ CFU/thigh of each group of mice at a given time point following therapy from the mean log₁₀ CFU/thigh of untreated control mice taken at the corresponding time point. The postantibiotic effect (PAE) was calculated by subtracting the time it took for organisms to increase 1 log in the thighs of control animals from the time it took organisms to grow the same amount in treated animals after serum drug levels fell below the MIC (11): PAE = T – C, where C is the time for 1 log₁₀ control growth and T is the time for 1 log₁₀ treatment growth after levels have fallen below the MIC.

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TABLE 2. In vivo efficacy of cyclic peptides in the thigh and peritonitis infection models

Mouse peritonitis model. Type II mucin (Sigma-Aldrich, St. Louis, MO), an enzyme extract of porcine stomach, was used as an adjuvant for inoculation of the mice and was prepared as a 7% stock solution in PBS supplemented with 0.2 mM FeNH₄-citrate. The bacterial suspensions were diluted 1:10 with ice-cold mucin solution to give a final mucin concentration of 6.3% and kept on ice prior to inoculation. Inoculation was performed by intraperitoneal injection of 0.2 ml of a mucin-bacterial suspension via a 25-gauge syringe. The inoculum contained 3 x 10⁶ CFU/mouse of S. aureus ATCC 13709 (Smith), which causes death of 100% of untreated animals within approximately 18 h following inoculation. Groups of 10 to 12 mice were treated i.v. with decreasing doses of test and control compounds at 0 h and 2 h following inoculation. For each experiment, a group of mice treated with mucin was included as a control for the lethality of the infection. The endpoint was determined at 48 h. The ED₅₀, the single dose providing protection to 50% of the mice, was calculated using the Reed and Muench equation (29).

Pharmacokinetic study. Mice received an 8-mg/kg dose of test compounds prepared in 5% dextrose via tail vein injection. Groups of three mice were euthanized by CO₂ asphyxiation at different intervals from 2 min to 8 h after dosing, and blood samples were collected by cardiac puncture. After 15 min at room temperature, coagulated blood was centrifuged and serum was recovered and stored frozen at –20°C. The serum samples were analyzed and quantified for peptide content by reverse-phase HPLC coupled with mass spectrometry. All plasma samples were deproteinized with 2 volumes acetonitrile, diluted 1:1 in running buffer, and injected onto a Waters Atlantics C₁₈ column (2.1 by 150 mm) maintained at room temperature. The peptide was eluted using a flow rate of 200 µl/min with a 1:1 mixture of 0.1% formic acid in water and 50% acetonitrile using an isocratic gradient. An ABI API 2000 liquid chromatography-tandem mass spectrometry system was used to quantify the peptide in each serum sample. For quantitative calibration, standard curves were established using spiked compounds in serum. The limit of detection was between 0.1 and 1.0 µg/ml.

Data analysis. One- and two-compartment pharmacokinetic models with bolus input and first-order elimination from the central compartment were fitted to the serum concentration-time data using WinNonlin (Pharsight, Mountain View, CA). The optimal number of exponentials was selected using the Akaike information criterion (37). The pharmacokinetic parameters total drug clearance, volume of the central compartment, volume of distribution at steady state (V_ss), and half-lives at the alpha and beta phases were obtained directly from the modeling output.

Peptide synthesis: solvents and reagents. Acetonitrile (HPLC grade; Aldrich, St. Louis, MO), dichloromethane (DCM, HPLC grade; Aldrich), diethyl ether (American Chemical Society grade; Fisher), isopropanol (American Chemical Society grade; Fisher), N,N-dimethylformamide (DMF, sequencing grade; Aldrich), N-methylpyrrolidinone (NMP, peptide synthesis grade; Aldrich), and N,N-diisopropylethylamine (DIEA, peptide synthesis grade; Aldrich) were obtained from the noted providers and used without further purification. TFA (reagent grade; Aldrich), diisopropylcarbodiimide (DIC; Aldrich), N-hydroxybenzotriazole (anhydrous; Fisher), 1-hydroxy-7azabenzotriazole (Applied Biosystems, Foster City, CA), 2-(1-H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate (HBTU; Novabiochem EMD Biosciences, San Diego, CA), phenylsilane (Aldrich), tetrakistriphenylphosphine palladium (0) [Pd(PPh₃)₄; Aldrich], and benzotriazole-1-yl-oxy-tris-pyrrolidino-phosphonium hexafluorophosphate (NovaBiochem) were used as obtained. Trityl chloride polystyrene macrobeads (500 to 560 µm in diameter) were obtained from Peptides International (Louisville, KY). Commercially available amino acids and resins were used as obtained from Novabiochem, Advanced Chemtech (Louisville, KY), or NeoMPS (San Diego, CA). The side chain protection groups were as follows for 9-fluorenylmethoxy carbonyl (Fmoc) synthesis: Arg (Pbf), His (Trt), Lys (Boc), Ser (t-Bu), Thr (t-Bu), Tyr (t-Bu), Asp (Ot-Bu), Glu (Ot-Bu), Asn (Trt), and Gln (Trt). All other chemicals were used as obtained from Aldrich, Sigma (St. Louis, MO), or Fisher (Fluka, Acros).

Single-compound-per-bead combinatorial cyclic peptide libraries. The peptides were prepared on 500- to 560-µm-diameter macrobeads of trityl chloride polystyrene-based resin using the split-and-pool approach (17, 24). Coupling of the first allyl amino acid to the resin was conducted using 1.5 equivalents (eq) of N--deprotected N--Fmoc-L-Lys-allyl ester and 4 eq DIEA in DCM overnight (0.2 M Lys). After coupling, the resin was washed with DCM (3 x 15 min), 10% DIEA, 10% methanol (MeOH), 80% DCM (3 x 15 min), and DCM (3 x 15 min). After drying the beads in vacuo, the resin loading level was calculated by weight or based on Fmoc release upon treatment with piperidine and monitoring UV absorbance at 290 nm. Beads were swollen in DMF for at least 30 min prior to initial Fmoc deprotection. Sequential growing of the peptide chain was accomplished via repeated cycles of deprotecting with 25% piperidine in DMF (2 x 15 min); washing with DMF (4 x 15 min); coupling with 4 eq amino acid, 10 eq DIEA, and 4 eq HBTU in DMF (1 x 4 h); and washing with DMF (4 x 15 min). Following the synthesis of the linear peptide, the beads were washed with DCM. To the resin was added a degassed solution of 0.5 eq Pd(PPh₃)₄ in 90% DCM-10% phenylsilane. After shaking under nitrogen for 2 h, the resin was washed sequentially with DMF, DCM, and DMF. Resin was treated again for 2 h with 0.5 eq Pd(PPh₃)₄ in 90% DCM-10% phenylsilane under nitrogen and washed with a solution of 1% sodium dimethylthiocarbamate in DMF (3 x 15 min) and 1% DIEA in DMF (3 x 15 min). Cyclization was accomplished by deprotection of the terminal Fmoc using 25% piperidine-DMF (2 x 15 min); washing with DMF (3 x 15 min), 10% DIEA-DMF (3 x 15 min), and NMP (1 x 15 min); and cyclizing overnight with 3 eq each of HATU, HOAT, and DIEA in dry NMP (0.2 M HATU). After cyclization, the resin was washed with NMP (4 x 15 min), DCM (2 x 15 min), and MeOH (1 x 15 min). The beads were dried in vacuo and arrayed (one bead per well) in 96-well plates (1-ml capacity per well) analogously to the arraying reported by Clemons et al. (7). To each well, 95 µl of a cleavage cocktail (2.5% triisopropylsilane, 2.5% H₂O, 95% TFA) was added and the hydrolysis was allowed to continue for 2 h. After cleavage, diethyl ether was added to precipitate the peptide. The plates were centrifuged, the supernatant was removed, and the volatiles were evaporated. The residue was dissolved in 5% DMSO in water to prepare nominal 200-µg/ml stock solutions (concentration estimate based on average theoretical yields and resin loading) for biological assays and mass spectrometry sequence determination (28). The final yield per bead was approximately 100 µg.

Individual peptide synthesis. Peptides were synthesized using standard solid-phase Fmoc protocols (36) on the Fmoc-Lys-O-allyl ester-loaded trityl resin with coupling reaction times ranging from 4 to 12 h either manually (using HBTU as the coupling reagent, reaction times ranging from 0.5 to 1 h) or in a peptide synthesizer (Advanced Chemtech APEX 396; DIC-N-hydroxybenzotriazole as the coupling reagent and employing a double-coupling protocol, 2 x 1 h). Following synthesis of the linear peptide, the resin was swollen in dry DCM for 20 min. To the resin was added a degassed solution of 0.5 eq Pd(PPh₃)₄ in 90% DCM-10% phenylsilane. After shaking under nitrogen for 2 h, the resin was washed sequentially with DMF, DCM, and DMF. Resin was treated again for 2 h with 0.5 eq Pd(PPh₃)₄ in 90% DCM-10% phenylsilane under nitrogen and washed with a solution of 1% sodium dimethylthiocarbamate in DMF (3 x 15 min) and 1% DIEA in DMF (3 x 15 min). After the final Fmoc deprotection (25% piperidine in DMF, 2 x 15 min), the resin was washed thoroughly with DMF (3 x 15 min), 10% DIEA-DMF (3 x 15 min), and 0.8 M LiCl-DMF (3 x 15 min). The resin was treated with 5 eq benzotriazole-1-yl-oxy-tris-pyrrolidino-phosphonium hexafluorophosphate-5 eq 1-hydroxy-7azabenzotriazole-20 eq DIEA in 0.8 M LiCl-DMF for at least 12 h. After washing with DMF (3 x 15 min) and DCM (2 x 15 min), followed by MeOH, the peptide was cleaved from the resin and deprotected by treatment with 95% TFA-2.5% water-2.5% triisopropylsilane for 2 h. Peptides were recovered by precipitation with diethyl ether and purified by HPLC using mixtures of TFA, water, and acetonitrile as eluents.

Mass spectrometric sequence determination of cyclic peptide library members. Mass spectrometry was used as previously reported (28) to determine the likely sequence of cyclic peptide library members. Fidelity for the identification routinely was >90%.

Top Abstract Introduction Materials and Methods Results Discussion References

 
Library design and screening. Combinatorial libraries of novel cyclic D,L--peptides were synthesized and screened for antimicrobial activity. Similar molecules have previously been shown to form self-assembling supramolecular structures with bioactivity (15). We generated libraries of both cyclo-D,L--hexa- and octapeptides designed to determine the effect of the global amino acid composition of the peptides on their antimicrobial activity and the optimal ratio of hydrophobic to hydrophilic residues for potency and selectivity toward bacterial cells. Studies were initiated with a hexamer library that was designed to provide members possessing between two and four consecutive hydrophilic residues chosen from among six amino acids having either charged or noncharged side chains (Lys, Arg, His, Glu, Asn, and Ser) but always having a lysine residue at position 1 for resin attachment purposes. Positions within the hydrophobic region were filled with Trp and Leu. The design of the compounds in the octamer library is analogous, but the number of consecutive hydrophilic residues ranged from three to five. A schematic depiction of these original libraries is given in Fig. 1. In peptide sequences described throughout this work, the conventional one-letter code for amino acids is used, except for the unnatural amino acids ornithine (O) and O-benzyltyrosine (BY); residues having D stereochemistry are indicated by lowercase letters, and a trailing dash at the end of the peptide sequence indicates that the peptide is cyclic.

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FIG. 1. Schematic description of hexamer (left) and octamer (right) libraries. Hydrophilic residue positions are shown in black circles and hydrophobic positions in gray circles. Positions indicated by white circles are those that include either hydrophilic or hydrophobic amino acids.

The above-mentioned libraries were screened for antibacterial activity, and the resulting data were used to design subsequent libraries in an effort to optimize the biological data and physical properties (i.e., solubility) of the analyzed members. Over the full course of our study, approximately 140,000 representative compounds from a collection of >1 x 10⁶ synthesized hexapeptides and octapeptides were assayed for antibacterial activity against S. aureus 29213 in primary screens at a final peptide concentration of either 8 µg/ml or 16 µg/ml. Approximately 8,000 of the tested peptides (0.57%) were found during this screening to inhibit S. aureus growth by 90% or greater relative to the 100% growth controls under the specific screening conditions used. These compounds were recovered and tested again to confirm antibiotic activity using the same protocol followed during primary screening. A success rate of 80 to 90% was routinely obtained upon secondary screening. Compounds with confirmed activity were then analyzed by mass spectrometry to determine the sequence of the cyclic peptide. Compounds for which amino acid sequences were generated with high confidence were then synthesized on a larger scale (10 to 100 mg) for further testing.

Identification of lead compounds. After scaled-up synthesis, compounds were again tested for in vitro antimicrobial activity against S. aureus. Preliminary in vivo characterization of compounds with confirmed activity led to the identification of several compounds which were further investigated (Table 1). The hexamer peptide l-L-w-H-s-K- (not shown) and the octamer peptide k-K-h-K-w-L-w-K- (1150) were isolated directly from the starting combinatorial libraries (Fig. 1). The hexamer peptide properties were optimized by performing point substitutions, resulting in the cyclo-D,L--hexapeptide l-L-w-H-o-K- (1316). Similarly, octamers with confirmed activity were optimized by amino acid substitution at specific peptide residues to yield S-w-F-k-T-k-S-k- (6752), S-w-F-k-H-k-S-k- (6756), and S-w-BY-k-N-k-S-k- (6853). These second-generation compounds have certain improved in vitro or in vivo traits over those of their parent molecules. Additionally, the sequence s-W-f-K-t-K-s-K- (7251), the enantiomer of 6752, also was synthesized to investigate whether stereochemistry plays a critical role in biological activity.

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TABLE 1. Summary of in vitro potency and toxicity and in vivo tolerability of peptide compounds

In vitro inhibitory activity. Table 1 lists the MICs of the six lead compounds against MSSA ATCC 29213 in CAMHB. MICs were also obtained in CAMHB supplemented with 50% mouse serum to assess potential protein binding issues. With the exception of 6853, serum MICs remained within a twofold dilution of the standard MICs, suggesting only minimum serum protein binding or inactivation. Interestingly, 6752 and its enantiomer 7251 exhibited similar potency in vitro. This observation is consistent with the thought that small antimicrobial peptides exert their effect on bacteria via nonchiral interactions with membrane lipids (35), even though enzymatic inhibitory activities of most small bioactive molecules are enantioselective with a highly selective fit for one of the two enantiomeric forms (9, 10, 34). The MIC₉₀ (the concentration at which 90% of the isolates tested were inhibited) was determined for 6752 and 6756 using a panel of 10 strains that included MRSA strains ATCC 43300 and 33591. The MIC₉₀s of 6752 and 6756 were 8 and 12 µg/ml, respectively, identical to their corresponding MICs against S. aureus 29213. Bactericidal activity was assessed by measuring MBCs as shown in Table 1. Time-kill curves were obtained to study the kinetics of bactericidal activity. Compounds 6752 and 1150 and vancomycin were tested against MSSA strain ATCC 25923 at four times the compound's MIC. Consistent with their mechanism of action, both peptides displayed much more rapid bacterial killing than did vancomycin. Compound 1150 decreased the viable counts by 100% within 5 min, while 6752 reduced the viable counts by 3 log units after the same incubation period. On the other hand, vancomycin caused no change in CFU in this period and a 1.6-log CFU/ml decrease over an incubation period of 6 h at four times the MIC.

Attempts to generate spontaneous resistance in vitro. The ability of S. aureus 29213 to develop resistance to 6752, 7251, 1316, 1150, 6756, and 6853, and to vancomycin and rifampin as controls, was evaluated by repeated passaging and MIC determination. S. aureus cells growing in the presence of a compound at half the MIC on a particular day were used the next day in an MIC assay of that same compound. In this manner, cells were continually exposed to a single compound at one-half the MIC while being passaged over 15 days. The MIC of rifampin increased as much as 512-fold over 15 days—rifampin is an agent to which resistance is known to arise quite easily by spontaneous chromosomal point mutations (31). In contrast, the MICs of 7251, 6853, and 6756 increased only twofold, while the MIC of 6752 increased fourfold and that of 1150 remained unchanged. Again, both 6752 and its enantiomer 7251 were very similar with respect to the capacity of S. aureus to acquire resistance to each compound. Similarly to the tested peptides, the MIC of vancomycin under these conditions only increased twofold; vancomycin is generally regarded as an antibiotic to which spontaneous resistance development is unlikely to occur under conditions where horizontal genetic transfer between species is excluded (22).

To confirm that the observed increases in MICs were due to adaptive changes in the S. aureus cells and not due to instability of the anti-infective compounds, MICs of the peptides for the passaged bacteria were determined at the end of the 15 days in parallel with S. aureus cells that had not previously been exposed to antibiotics. MICs obtained with fresh S. aureus cells for all compounds tested were the same as those at the start of the experiment. This indicated that the compounds were stable during the period of the experiment and that the cells that had been continuously exposed to test compounds had acquired mechanisms allowing for increased resistance (data not shown). Due to the poor diffusion properties of the peptides in solid growth medium, we were unable to experimentally perform the conventional assay using multiples of the MICs and detecting spontaneous mutants arising in conventional growth agar medium. Similar behavior has been observed for oritavancin, a new semisynthetic derivative of vancomycin in clinical development (Thomas R. Parr, unpublished observation).

In vitro toxicity and in vivo tolerability studies. Hemolytic activity of the peptides was tested using a mouse RBC lysis assay. As shown in Table 1, 6752, 6756, and 7251 exhibited no significant hemolytic activity at clinically relevant concentrations, while peptides 6853, 1316, and 1150 had HD₅₀s ranging between 100 and 200 µg/ml, with 1150 being the most hemolytic compound. Melittin was used as a positive control and showed an HD₅₀ of 2 µg/ml under those conditions. Compound cytotoxicity was measured using a dye exclusion method. Trends in cytotoxicity measured in a rat hepatoma cell line mirror those obtained in the hemolysis assay, with 6752, 6756, and 7251 having no significant cytotoxicity below 100 µg/ml, while 6853, 1150, and 1316 display increasing toxicity in vitro. Consistent with these results, 6752 and its enantiomer 7251 were well tolerated in vivo in the mouse with no visible signs of discomfort when administered as a bolus i.v. injection at 8 mg/kg. The maximum tolerated dose was above 20 mg/kg. Both 1316 and 1150 were extremely poorly tolerated, with 1150 being tolerated only at doses of 5 mg/kg or less (Table 1).

Thigh model of infection. Mice were made profoundly neutropenic during the course of the thigh model experiments by administration of cyclophosphamide. Initial inocula were, per thigh, 5.1 ± 0.1 log₁₀ CFU of S. aureus ATCC 25923. Control and test compounds were administered i.v. 2 h following thigh infection, and thigh bacterial counts were determined at regular intervals following treatment. The organisms expanded 3.1 ± 0.3 log₁₀ CFU/thigh after 10 h in untreated control mice. The viable recovery of S. aureus from infected thighs following a single injection of vancomycin, oxacillin, and 6752 is shown in Fig. 2A. Single 16-mg/kg doses of 6752 and vancomycin produced a prolonged antibacterial effect against S. aureus, while oxacillin at 16 mg/kg had a weak bactericidal effect which lasted for 1 h posttreatment. Escalating doses of 6752 resulted in dose-dependent killing of S. aureus, with a maximum effect at single doses exceeding 8 mg/kg (Fig. 2B). In vivo PAEs also increased with dose and were 3.75 h and >7 h for 6752 at 8 mg/kg and 16 mg/kg, respectively (see Materials and Methods for the PAE calculation method used). The in vivo efficacy of 6752 against MRSA 33591 was very similar, with a maximum bactericidal effect of 2.2 log₁₀ CFU/thigh at 8 h following a single dose of 8 mg/kg (data not shown).

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FIG. 2. (A) Efficacies of vancomycin, oxacillin, and 6752 against S. aureus ATCC 25923 in the neutropenic-mouse thigh model. Each curve represents the bacterial titer observed in the thighs versus time. Treated animals received a 16-mg/kg dose injected i.v. at 2 h postinfection. Symbols: , untreated control animals; , 6752; , oxacillin; , vancomycin. The time above the MIC or the time during which the serum concentration of each compound was above its corresponding MIC is indicated in the upper left corner as follows: black rectangle, oxacillin; hatched rectangle, vancomycin; gray rectangle, 6752. (B) Dose-response study of 6752 against S. aureus ATCC 25923 in the neutropenic-mouse thigh model. Each curve represents the bacterial titer observed in the thighs versus time. Symbols: , untreated animals; , 6752 at 2 mg/kg (i.v.); , 6752 at 4 mg/kg (i.v.); , 6752 at 8 mg/kg (i.v.); , 6752 at 16 mg/kg (i.v.).

The efficacy of 7251, 6756, 6853, 1316, and 1150 against MSSA strain ATCC 25923 in the neutropenic-mouse thigh model is shown in Table 2. The enantiomer of 6752, 7251 (Table 1), as well as 6756 and 6853, produced a bactericidal effect similar to that of 6752 at a dose of 8 mg/kg. Overall, the reduction in bacterial counts produced by the four compounds was similar to that obtained with vancomycin at the same dose. In contrast, 1316 and 1150 caused no significant reduction in the thigh bacterial counts, despite significant potency in vitro. The latter two compounds were also very poorly tolerated in the mouse.

Peritonitis model of infection. The in vivo efficacy of 6752, 7251, 6756, 6853, and vancomycin in sepsis studies with MSSA ATCC 13709 (Smith) is shown in Table 2. All four compounds exhibited similar 50% protective doses. Following the 24-h experiment, the spleens and kidneys were recovered from survivors treated with 6752 (8 mg/kg at 0 h and 2 h following intraperitoneal infection). Bacterial counts were obtained and compared to those of untreated animals (Table 3). On average, mice treated with 6752 showed decreases of 2.9 log₁₀ CFU/spleen and 3.6 log₁₀ CFU/kidney pair, while vancomycin at the same dose produced CFU decreases of 3.2 log₁₀ and 3.9 log₁₀ in the spleen and kidneys, respectively. The two peptides which had no activity against MSSA in the neutropenic-mouse thigh model, 1316 and 1150, were not tested in the peritonitis model due to poor systemic tolerance.

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TABLE 3. Bacterial counts in the spleens and kidneys of survivors in the S. aureus peritonitis infection modela

In vivo pharmacokinetic studies. The serum concentrations of 6752 following administration of single i.v. injections of 2, 4, 8, and 16 mg/kg to naive mice are shown in Fig. 3. The decline in concentration was best described by a two-compartment model. The values for the pharmacokinetic parameters are shown in Table 4. In these preliminary experiments, the serum concentrations showed dose proportionality for doses of up to 8 mg/kg, as indicated by the steady increase in the maximum concentration of the drug in serum (C_max) and the area under the concentration-time curve (AUC). However, the increase in C_max and AUC was much less pronounced between 8 and 16 mg/kg. Accordingly, the elimination half-life and V_ss remained within a comparable range at 2, 4, and 8 mg/kg but significantly increased at 16 mg/kg (Table 4). Figure 3 also shows a more drastic decrease in the initial serum concentration of 6752 at 16 mg/kg than at lower doses.

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FIG. 3. Mean serum concentration-time profiles of 6752 following i.v. injection of the compound at 2 mg/kg (), 4 mg/kg (), 8 mg/kg (), and 16 mg/kg ().

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TABLE 4. Pharmacokinetic parameters of cyclic peptides in serum following administration of a single-bolus i.v. injection to micea

Pharmacokinetic profiles were also established for three other peptides, one showing in vivo efficacy in the thigh model of infection, namely, 6853, and the two peptides with no apparent efficacy in vivo, 1316 and 1150. Interestingly, the C_max of 6853 was similar to that of 6752, while both 1316 and 1150 showed significantly lower peak concentrations and AUC values. Also, 1316 and 1150 displayed increased volumes of distribution, with 1150 having a 30-fold increased V_ss value compared to that of 6752 injected at the same dose. Thus, some level of correlation appears to exist among C_max, V_ss, and efficacy in the mouse for all four compounds examined. Specifically, a high C_max may correlate with good efficacy in vivo whereas large volumes of distribution may be indicators of a lower C_max and therefore decreased efficacy in infection models.

Top Abstract Introduction Materials and Methods Results Discussion References

 
Until now, application of antimicrobial peptides to the clinic has been hampered by various barriers, and most therapeutic peptides are being developed for topical uses only (5, 8, 38), with the exception of the anionic lipodepsipeptide daptomycin (21). Daptomycin has been recently introduced into clinical medicine for the treatment of complicated skin and skin structure infections caused by susceptible strains of several gram-positive microorganisms. Though the molecules reported here and daptomycin both have cell membrane-mediated activity, there are several differences to be noted. Daptomycin is an anionic molecule. It has been reported to have cell wall activity and does not exhibit activity against stationary-phase cells (1). Furthermore, daptomycin is not recognized to require self-assembly for its activity. We have obtained the first conclusive examples of a new class of cationic peptide antimicrobial agents showing prolonged systemic antimicrobial activity in infection models, where the molecules are administered i.v. to treat an infection localized beyond the i.v. compartment.

Positive in vivo efficacy effects in the neutropenic-mouse thigh model of infection show a more rapid and drastic initial drop in the bacterial counts for 6752 than for vancomycin and oxacillin (Fig. 2A), in agreement with the putative bactericidal mechanism of action of the cyclic peptides. In contrast to the nearly instantaneous membrane disruption by the cyclic peptides, both vancomycin and oxacillin exert their activity by inhibiting enzymes involved in cell wall synthesis, and their effect are therefore seen more slowly. In the absence of human pharmacokinetic and efficacy data for the cyclic peptides, a 16-mg/kg single dose was chosen based on the tolerance data for 6752 and on the clinical dose recommended for vancomycin (1 g i.v. once daily for a 70-kg individual, which corresponds to 14.3 mg/kg). The same dose was used for 6752, vancomycin, and oxacillin in order to compare the intensity and duration of the bactericidal effect of each compound at similar initial serum concentrations. At 16 mg/kg, 6752 clearly exhibits a more prolonged antibacterial activity in the thigh model than oxacillin but is similar to vancomycin. In our experiments, the calculated PAEs were >6 h for vancomycin, in agreement with values found in the literature (20), and >7 h for 6752. We observed a minimal PAE for oxacillin, in contrast with what has been reported with other, similar, ß-lactams using the same model of infection, where the PAE could reach up to 4 h against S. aureus strains. In the neutropenic-mouse thigh model, 6752 also demonstrated efficacy against MRSA strain 33591 identical to the activity seen against MSSA strain ATCC 25923.

In vitro, these new peptide molecules displayed fast bactericidal activity in an enantiomer-independent manner, with failure of S. aureus to easily develop spontaneous resistance upon prolonged exposure to the peptides at sublethal concentrations. Taken together, our in vitro and in vivo results are consistent with a previously proposed mechanism of action for these cyclo-D,L--peptides: insertion into the bacterial membrane with a subsequent increase in membrane permeability, potential collapse, and fast cell death (3, 6, 15). When tested against MRSA either in vitro or in vivo, the cyclic peptides had similar potencies against MSSA and MRSA, in agreement with the putative mechanism of action of various classes of antimicrobial peptides thought to interact with negatively charged components of the bacterial membrane as a whole, rather than with individual enzymatic processes (23, 27). This complex set of interactions between the amino acid side chains and components of the bacterial membrane canopy could explain the failure of S. aureus to easily develop spontaneous resistance upon prolonged exposure to the peptides at sublethal concentrations. The proposed mechanism of action is not likely to be influenced by individual target alterations, which would not grossly alter the membrane structure to the extent required for the appearance of resistant mutants. Furthermore, the unique abiotic structure of the D,L-cyclic peptides may contribute to a reduced risk of drug-resistant bacterial emergence. The putative mode of action is further supported by the MBCs and MICs being quite similar.

The hypothesis that primary antimicrobial activity occurs at the membrane level is strengthened by the observation that enantiomeric cyclopeptides have the same activity in vitro and show comparable behavior in more complex environments, as is proven with the results in the murine model experiments with compounds 6752 and 7251. It is unlikely that the critical mode of action involves interaction with a specific receptor where changes in stereochemistry would be expected to have drastic consequences with regard to potency and activity. Other antimicrobial peptides thought to disrupt bacterial membranes by a physical mechanism have been shown to be active regardless of their enantiomeric form (2, 26, 35).

For the peptides described in Table 1, we observed a good correlation between the toxicity of the cyclic peptides in vitro, as indicated by the ED₅₀s and HD₅₀s, and their respective tolerability in vivo. Among the six compounds examined in detail and presented here, those with low ED₅₀s and/or HD₅₀s showed very poor acute tolerability in vivo. We have observed that prototypic cyclic peptides from this structural class, when prone to amorphous aggregation as measured by in vitro methods, lead to a terminal blood pressure drop within seconds to minutes when injected i.v. into instrument-monitored rats (Neil Granger, Louisiana State University, personal communication). It must be noted, however, that a correlation between in vitro cytotoxicity and tolerance in vivo was not always observed for the other cyclic peptides tested. Overall, structure-activity relationship studies are required to correlate specificity (or lack thereof) with the primary sequence of the peptides in order to increase the tolerability levels and thereby the therapeutic window of these cyclic-D,L--peptides.

Analysis of our pharmacokinetic data and correlation with pharmacodynamic properties of the cyclic peptides revealed the following. Compounds with poor efficacy in vivo, namely, 1150 and 1316, had lower initial C_maxs than those peptides which showed good therapeutic activity in vivo. Also, both 1150 and 1316 exhibited a clear increase in their V_sss compared to the peptides showing in vivo efficacy (Table 4). Interestingly, those same compounds with high V_sss and lower C_maxs were significantly more hemolytic and cytotoxic in vitro and were also very poorly tolerated systemically. We therefore hypothesize that poor tolerability upon i.v. injection into the mouse may be due to vascular leakage and partial blood vessel collapse, with a subsequent severe drop in blood pressure and diffusion of the peptides in deep tissue compartments. Such a phenomenon could explain the drastic initial decrease in serum concentrations, the resulting poor efficacy of 1150 and 1316 in the various infection models, and the lack of correlation between in vitro potency and positive therapeutic activity for these compounds. Also, our preliminary pharmacokinetic and pharmacodynamic results suggest that C_max could be the pharmacokinetic index which best correlates with efficacy in vivo, though further work needs to be done to verify this assumption. Other components of the innate immune system, such as neutrophil activation to produce superoxide, may also be involved in the positive biological effect of these compounds (26).

Preliminary studies showed poor bioavailability of these cyclic peptides following oral or subcutaneous administration. Intravenous administration was required for therapeutic efficacy. This is most likely due to the size of these compounds, with a molecular mass of around 1,000 Da for octamers, and to their polycationic nature. On the other hand, these alternating D,L-cyclopeptides are highly resistant to proteolysis and stable in serum and other biological fluids. This constitutes an important advantage which distinguishes them from other peptides with therapeutic potential (38). Finally, pharmacokinetic and pharmacodynamic studies performed with 6752 and 6756 in renally impaired mice indicated that a significant portion of the injected compounds is cleared renally based on the dramatic decrease in the clearance rate and more prolonged in vivo efficacy seen in the presence of uranyl nitrate (data not shown).

In this paper, we detail the first conclusive examples of a new class of cationic peptide antimicrobial agents showing prolonged systemic antimicrobial activity in infection models, where the molecules are administered i.v. to treat an infection localized beyond the blood compartment. Although additional structure-activity relationship studies are required to improve the therapeutic window of this class of antimicrobial peptides, our data suggest that the amphipathic cyclic D,L-peptides, with their self-assembling and specific membrane disruption mechanism of action, may have potential for systemic administration and treatment of otherwise antibiotic-resistant infections.

 
The assistance of Emmett Bond, David Griffith, Shirley Carigo-Cairel, Dana Johnson, Morag Day, Christian Solem, and Tim Hoffman is gratefully acknowledged.

 
* Corresponding author. Mailing address: Novartis Institute for Tropical Diseases, 10 Biopolis Rd., #05-01 Chromos, Singapore 138670. Phone: 65 6722 2930. Fax: 65 6722 2910. E-mail: veronique.dartois{at}novartis.com.

V.D. and J.S.-Q. contributed equally to the present study.

Present address: Departamento de Química Orgánica, Universidad Autónoma de Madrid, Cantoblanco, 1049 Madrid, Spain.

Present address: Departamento de Química Orgánica, Facultad de Química, Universidad de Santiago, E-15782 Santiago de Compostela, Spain.

^¶ Present address: Targanta Therapeutics Inc., 7170 Frederick Banting, St. Laurent H4S 2A1, Quebec, Canada.

Top Abstract Introduction Materials and Methods Results Discussion References

 

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Antimicrobial Agents and Chemotherapy, August 2005, p. 3302-3310, Vol. 49, No. 8
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