Maximal Relative Efficacy Research Articles

The Polyaminoisoprenyl Potentiator NV716 Revives Old Disused Antibiotics against Intracellular Forms of Infection by Pseudomonas aeruginosa.

Active efflux confers intrinsic resistance to multiple antibiotics in Pseudomonas aeruginosa, including old disused molecules. Beside resistance, intracellular survival is another reason for failure to eradicate bacteria with antibiotics. We evaluated the capacity of polyaminoisoprenyl potentiators (designed as efflux pump inhibitors [EPIs]) NV716 and NV731 compared to PAβN to restore the activity of disused antibiotics (doxycycline, chloramphenicol [substrates for efflux], and rifampin [nonsubstrate]) in comparison with ciprofloxacin against intracellular P. aeruginosa (strains with variable efflux levels) in THP-1 monocytes exposed over 24 h to antibiotics alone (0.003 to 100× MIC) or combined with EPIs. Pharmacodynamic parameters (apparent static concentrations [Cs] and maximal relative efficacy [Emax]) were calculated using the Hill equation of concentration-response curves. PAβN and NV731 moderately reduced (0 to 4 doubling dilutions) antibiotic MICs but did not affect their intracellular activity. NV716 markedly reduced (1 to 16 doubling dilutions) the MIC of all antibiotics (substrates or not for efflux; strains expressing efflux or not); it also improved their relative potency and maximal efficacy (i.e., lower Cs; more negative Emax) intracellularly. In parallel, NV716 reduced the persister fraction in stationary cultures when combined with ciprofloxacin. In contrast to PAβN and NV731, which act only as EPIs against extracellular bacteria, NV716 can resensitize P. aeruginosa to antibiotics whether they are substrates or not for efflux, both extracellularly and intracellularly. This suggests a complex mode of action that goes beyond a simple inhibition of efflux to reduce bacterial persistence. NV716 appears to be a useful adjuvant, including to disused antibiotics with low antipseudomonal activity, to improve their activity, including against intracellular P. aeruginosa.

Influence of pH on the activity of finafloxacin against extracellular and intracellular Burkholderia thailandensis, Yersinia pseudotuberculosis and Francisella philomiragia and on its cellular pharmacokinetics in THP-1 monocytes

ObjectivesBurkholderia pseudomallei, Yersinia pestis and Francisella tularensis are facultative intracellular bacteria causing life-threatening infections. We have (a) compared the activity of finafloxacin (a fluoroquinolone in development showing improved activity at acidic pH) with that of ciprofloxacin, levofloxacin and imipenem against the extracellular and intracellular (THP-1 monocytes) forms of infection by attenuated surrogates of these species (B. thailandensis, Y. pseudotuberculosis, F. philomiragia) and (b) assessed finafloxacin cellular pharmacokinetics (accumulation, distribution, efflux). MethodsBacteria in broth or in infected monocytes were exposed to antibiotics at pH 7.4 or 5.5 for 24 hr. Maximal relative efficacies (Emax) and static concentrations (Cs) were calculated using the Hill equation (concentration–response curves). Finafloxacin pharmacokinetics in cells at pH 7.4 or 5.5 was investigated using 14C-labelled drug. ResultsExtracellularly, all drugs sterilized the cultures, with finafloxacin being two to six times more potent at acidic pH. Intracellularly, Emax reached the limit of detection (4–5 log10 cfu decrease) for finafloxacin against all species, but only against B. thailandensis and F. philomiragia for ciprofloxacin and levofloxacin, while imipenem caused less than 2 log10 cfu decrease for all species. At acid pH, Cs shifted to two to five times lower values for finafloxacin and to one to four times higher values for the other drugs. Finafloxacin accumulated in THP-1 cells by approximately fivefold at pH 7.4 but up to 20-fold at pH 5.5, and distributed in the cytosol. ConclusionsFluoroquinolones have proven to be effective in reducing the intracellular reservoirs of B. thailandensis, Y. pseudotuberculosis and F. philomiragia, with finafloxacin demonstrating an additional advantage in acidic environments.

Increased intracellular activity of MP1102 and NZ2114 against Staphylococcus aureus in vitro and in vivo

Treatment of Staphylococcus aureus infections remains very difficult due to its capacity to survive intracellularly and its multidrug resistance. In this study, the extracellular/intracellular activities of plectasin derivatives-MP1102/NZ2114 were investigated against three methicillin-susceptible/-resistant S. aureus (MSSA/MRSA) strains in RAW 264.7 macrophages and mice to overcome poor intracellular activity. Antibacterial activities decreased 4–16-fold under a mimic phagolysosomal environment. MP1102/NZ2114 were internalized into the cells via clathrin-mediated endocytosis and macropinocytosis and distributed in the cytoplasm; they regulated tumor necrosis factor-α, interleukin-1β and interleukin-10 levels. The extracellular maximal relative efficacy (Emax) values of MP1102/NZ2114 towards the three S. aureus strains were >5-log decrease in colony forming units (CFU). In the methicillin-resistant and virulent strains, MP1102/NZ2114 exhibited intracellular bacteriostatic efficacy with an Emax of 0.42–1.07-log CFU reduction. In the MSSA ATCC25923 mouse peritonitis model, 5 mg/kg MP1102/NZ2114 significantly reduced the bacterial load at 24 h, which was superior to vancomycin. In MRSA ATCC43300, their activity was similar to that of vancomycin. The high virulent CVCC546 strain displayed a relatively lower efficiency, with log CFU decreases of 2.88–2.91 (total), 3.41–3.50 (extracellular) and 2.11–2.51 (intracellular) compared with vancomycin (3.70). This suggests that MP1102/NZ2114 can be used as candidates for treating intracellular S. aureus.

Pharmacodynamics of ceftazidime/avibactam against extracellular and intracellular forms of Pseudomonas aeruginosa.

When tested in broth, avibactam reverses ceftazidime resistance in many Pseudomonas aeruginosa that express ESBLs. We examined whether similar reversal is observed against intracellular forms of P. aeruginosa . Strains: reference strains; two engineered strains with basal non-inducible expression of AmpC and their isogenic mutants with stably derepressed AmpC; and clinical isolates with complete, partial or no resistance to reversion with avibactam. Pharmacodynamic model: 24 h concentration-response to ceftazidime [0.01-200 mg/L alone or with avibactam (4 mg/L)] of bacteria in broth or bacteria phagocytosed by THP-1 monocytes, with calculation of ceftazidime relative potency ( C s : concentration yielding a static effect) and maximal relative effect [ E max : cfu decrease at infinitely large antibiotic concentrations (efficacy in the model)] using the Hill equation. Cellular content of avibactam: quantification by LC-MS/MS. For both extracellular and intracellular bacteria, ceftazidime C s was always close to its MIC. For ceftazidime-resistant strains, avibactam addition shifted ceftazidime C s to values close to the MIC of the combination in broth. E max was systematically below the detection limit (-5 log 10 ) for extracellular bacteria, but limited to -1.3 log 10 for intracellular bacteria (except for two isolates) with no effect of avibactam. The cellular concentration of avibactam reflected extracellular concentration and was not influenced by ceftazidime (0-160 mg/L). The potential for avibactam to inhibit β-lactamases does not differ for extracellular and intracellular forms of P. aeruginosa , denoting an unhindered access to its target in both situations. The loss of maximal relative efficacy of ceftazidime against intracellular P. aeruginosa was unrelated to resistance via avibactam-inhibitable β-lactamases.

Cellular pharmacokinetics and intracellular activity of the novel peptide deformylase inhibitor GSK1322322 against Staphylococcus aureus laboratory and clinical strains with various resistance phenotypes: studies with human THP-1 monocytes and J774 murine macrophages.

GSK1322322 is a peptide deformylase inhibitor active against Staphylococcus aureus strains resistant to currently marketed antibiotics. Our aim was to assess the activity of GSK1322322 against intracellular S. aureus using an in vitro pharmacodynamic model and, in parallel, to examine its cellular pharmacokinetics and intracellular disposition. For intracellular activity analysis, we used an established model of human THP-1 monocytes and tested one fully susceptible S. aureus strain (ATCC 25923) and 8 clinical strains with resistance to oxacillin, vancomycin, daptomycin, macrolides, clindamycin, linezolid, or moxifloxacin. Uptake, accumulation, release, and subcellular distribution (cell fractionation) of [(14)C]GSK1322322 were examined in uninfected murine J774 macrophages and uninfected and infected THP-1 monocytes. GSK1322322 demonstrated a uniform activity against the intracellular forms of all S. aureus strains tested, disregarding their resistance phenotypes, with a maximal relative efficacy (E max) of a 0.5 to 1 log10 CFU decrease compared to the original inoculum within 24 h and a static concentration (C s) close to its MIC in broth. Influx and efflux were very fast (<5 min to equilibrium), and accumulation was about 4-fold, with no or a minimal effect of the broad-spectrum eukaryotic efflux transporter inhibitors gemfibrozil and verapamil. GSK1322322 was recovered in the cell-soluble fraction and was dissociated from the main subcellular organelles and from bacteria (in infected cells). The results of this study show that GSK1322322, as a typical novel deformylase inhibitor, may act against intracellular forms of S. aureus. They also suggest that GSK1322322 has the ability to freely diffuse into and out of eukaryotic cells as well as within subcellular compartments.

RX-P873, a Novel Protein Synthesis Inhibitor, Accumulates in Human THP-1 Monocytes and Is Active against Intracellular Infections by Gram-Positive (Staphylococcus aureus) and Gram-Negative (Pseudomonas aeruginosa) Bacteria.

The pyrrolocytosine RX-P873, a new broad-spectrum antibiotic in preclinical development, inhibits protein synthesis at the translation step. The aims of this work were to study RX-P873's ability to accumulate in eukaryotic cells, together with its activity against extracellular and intracellular forms of infection by Staphylococcus aureus and Pseudomonas aeruginosa, using a pharmacodynamic approach allowing the determination of maximal relative efficacies (Emax values) and bacteriostatic concentrations (Cs values) on the basis of Hill equations of the concentration-response curves. RX-P873's apparent concentration in human THP-1 monocytes was about 6-fold higher than the extracellular one. In broth, MICs ranged from 0.125 to 0.5 mg/liter (S. aureus) and 2 to 8 mg/liter (P. aeruginosa), with no significant shift in these values against strains resistant to currently used antibiotics being noted. In concentration-dependent experiments, the pharmacodynamic profile of RX-P873 was not influenced by the resistance phenotype of the strains. Emax values (expressed as the decrease in the number of CFU from that in the initial inoculum) against S. aureus and P. aeruginosa reached more than 4 log units and 5 log units in broth, respectively, and 0.7 log unit and 2.7 log units in infected THP-1 cells, respectively, after 24 h. Cs values remained close to the MIC in all cases, making RX-P873 more potent than antibiotics to which the strains were resistant (moxifloxacin, vancomycin, and daptomycin for S. aureus; ciprofloxacin and ceftazidime for P. aeruginosa). Kill curves in broth showed that RX-P873 was more rapidly bactericidal against P. aeruginosa than against S. aureus. Taken together, these data suggest that RX-P873 may constitute a useful alternative for infections involving intracellular bacteria, especially Gram-negative species.

Pharmacodynamic Evaluation of the Intracellular Activity of Antibiotics towards Pseudomonas aeruginosa PAO1 in a Model of THP-1 Human Monocytes

Pseudomonas aeruginosa invades epithelial and phagocytic cells, which may play an important role in the persistence of infection. We have developed a 24-h model of THP-1 monocyte infection with P. aeruginosa PAO1 in which bacteria are seen multiplying in vacuoles by electron microscopy. The model has been used to quantitatively assess antibiotic activity against intracellular and extracellular bacteria by using a pharmacodynamic approach (concentration-dependent experiments over a wide range of extracellular concentrations to calculate bacteriostatic concentrations [Cs] and maximal relative efficacies [Emax]; Hill-Langmuir equation). Using 16 antipseudomonal antibiotics (three aminoglycosides, nine β-lactams, three fluoroquinolones, and colistin), dose-response curves were found to be undistinguishable for antibiotics of the same pharmacological class if data were expressed as a function of the corresponding MICs. Extracellularly, all of the antibiotics reached a bacteriostatic effect at their MIC, and their Emax exceeded the limit of detection (-4.5 log(10) CFU compared to the initial inoculum). Intracellularly, Cs values remained unchanged for β-lactams, fluoroquinolones, and colistin but were approximately 10 times higher for aminoglycosides, whereas Emax values were markedly reduced (less negative), reaching -3 log(10) CFU for fluoroquinolones and only -1 to -1.5 log(10) CFU for all other antibiotics. The decrease in intracellular aminoglycoside potency (higher Cs) can be ascribed to the acid pH to which bacteria are exposed in vacuoles. The decrease in the Emax may reflect a reversible alteration of bacterial responsiveness to antibiotics in the intracellular milieu. The model may prove useful for comparison of antipseudomonal antibiotics to reduce the risk of persistence or relapse of pseudomonal infections.

Activity of ceftaroline against extracellular (broth) and intracellular (THP-1 monocytes) forms of methicillin-resistant Staphylococcus aureus: comparison with vancomycin, linezolid and daptomycin

Ceftaroline fosamil is approved for treatment of acute bacterial skin and skin structure infections caused by methicillin-resistant Staphylococcus aureus (MRSA). We examined the activity of its active metabolite (ceftaroline) against intracellular forms of S. aureus in comparison with vancomycin, daptomycin and linezolid. Two methicillin-susceptible S. aureus (MSSA) and 11 MRSA strains with ceftaroline MICs from 0.125 to 2 mg/L [two strains vancomycin- and one strain linezolid-resistant (EUCAST interpretative criteria); VISA and cfr+] were investigated. The activity was measured in broth and after phagocytosis by THP-1 monocytes in concentration-dependent experiments (24 h of incubation) to determine: (i) relative potencies (EC(50)) and static concentrations (C(s)) (mg/L and × MIC); and (ii) relative activities at human C(max) (E(C)(max)) and maximal relative efficacies (E(max)) (change in log(10) cfu compared with initial inoculum). Ceftaroline stability and cellular accumulation (at 24 h) were measured by mass spectrometry. Ceftaroline showed similar activities in broth and in monocytes compared with vancomycin, daptomycin and linezolid, with no impact of resistance mechanisms to vancomycin or linezolid. For all four antibiotics, intracellular E(C)(max) and E(max) were considerably lower than in broth (∼0.5 log(10) versus 4-5 log(10) cfu decrease), but the EC(50) and C(s) showed comparatively little change (all values between ∼0.3 and ∼6× MIC). The mean cellular to extracellular ceftaroline concentration ratios (20 mg/L; 24 h) were 0.66 ± 0.05 and 0.90 ± 0.36 in uninfected and infected cells, respectively. In vitro, ceftaroline controls the growth of intracellular MRSA to an extent similar to that of vancomycin, linezolid and daptomycin for strains with a ceftaroline MIC ≤ 2 mg/L.

Activity of finafloxacin, a novel fluoroquinolone with increased activity at acid pH, towards extracellular and intracellular Staphylococcus aureus, Listeria monocytogenes and Legionella pneumophila

Finafloxacin, an 8-cyano-substituted fluoroquinolone, expresses enhanced activity at acidic pH and is less susceptible to several fluoroquinolone resistance determinants. In this study, we compared finafloxacin and ciprofloxacin for (i) activity against ciprofloxacin-susceptible and -resistant Staphylococcus aureus as well as wild-type and Lde efflux-positive (Lde+) Listeria monocytogenes, (ii) accumulation in THP-1 macrophages and (iii) intracellular activity towards phagocytised S. aureus, L. monocytogenes and Legionella pneumophila (developing in acidic, neutral and mildly acidic environments, respectively), using a pharmacological approach assessing drug potencies and maximal relative efficacies (Emax). Finafloxacin minimum inhibitory concentrations (MICs) were two-fold lower than those of ciprofloxacin against meticillin-susceptible S. aureus ATCC 25923, were only modestly increased in an isogenic strain overexpressing NorA and were ≤0.25mg/L for community-acquired meticillin-resistant S. aureus. No loss of activity was seen in Lde+ L. monocytogenes. An acidic pH decreased the MIC of finafloxacin and increased that of ciprofloxacin both for S. aureus and L. monocytogenes, in parallel with corresponding changes in drug accumulation (tested with S. aureus ATCC 25923 only). Finafloxacin accumulated less than ciprofloxacin in THP-1 cells, but the situation was reversed by exposure of cells to acid pH. In S. aureus-infected cells, acid pH increased the potency of finafloxacin without change of Emax, whilst decreasing the potency and the maximal relative efficacy of ciprofloxacin (less negative Emax). Finafloxacin was more potent and showed larger Emax than ciprofloxacin against phagocytised L. pneumophila, but was less potent against phagocytised L. monocytogenes. Finafloxacin appears to be an acid-pH-favoured antibiotic that may find useful applications in infections where the local pH is low.

Activity of moxifloxacin against intracellular community-acquired methicillin-resistant Staphylococcus aureus: comparison with clindamycin, linezolid and co-trimoxazole and attempt at defining an intracellular susceptibility breakpoint

Co-trimoxazole, clindamycin and linezolid are used to treat community-acquired methicillin-resistant Staphylococcus aureus (CA-MRSA) infections, but little is known about intracellular activity. Moxifloxacin is active against intracellular methicillin-susceptible S. aureus (MSSA), but CA-MRSA has not been studied. We used 12 clinical CA-MRSA, 1 MSSA overexpressing norA and 2 hospital-acquired MRSA (moxifloxacin MICs: 0.03 to 4 mg/L). Activity was assessed in broth and after phagocytosis by THP-1 macrophages or keratinocytes {concentration-dependent experiments [24 h of incubation] to determine relative potencies [EC(50)], static concentrations [C(s)] and maximal relative efficacies [E(max) (change in log(10) cfu compared with initial inoculum)] and time-dependent experiments [0-72 h] at human C(max)}. Concentration-dependent experiments: in broth, EC(50) and C(s) were correlated with the MIC for all antibiotics, but moxifloxacin achieved significantly (P < 0.01) greater killing (more negative E(max)) than the comparators; and in THP-1 cells and keratinocytes, moxifloxacin acted more slowly but still reached a near bactericidal effect (2 to 3 log(10) cfu decrease) at 24 h with unchanged EC(50) and C(s) as long as its MIC was ≤0.125 mg/L (recursive partitioning analysis). Clindamycin and linezolid were static, and co-trimoxazole was unable to suppress the intracellular growth of CA-MRSA. At human C(max) in broth, moxifloxacin killed more rapidly and more extensively (≥5 log(10) cfu decrease at 10 h) than clindamycin (4 log(10) cfu at 48 h) or co-trimoxazole and linezolid (1-2 log(10) cfu at 72 h). Moxifloxacin is active against both extracellular and intracellular CA-MRSA if the MIC is low, and is more effective than clindamycin, co-trimoxazole and linezolid.

Contrasting effects of acidic pH on the extracellular and intracellular activities of the anti-gram-positive fluoroquinolones moxifloxacin and delafloxacin against Staphylococcus aureus.

In contrast to currently marketed fluoroquinolones, which are zwitterionic, delafloxacin is an investigational fluoroquinolone with an anionic character that is highly active against Gram-positive bacteria. We have examined the effect of acidic pH on its accumulation in Staphylococcus aureus and in human THP-1 cells, in parallel with its activity against extracellular and intracellular S. aureus. Moxifloxacin was used as a comparator. Delafloxacin showed MICs 3 to 5 log(2) dilutions lower than those of moxifloxacin for a collection of 35 strains with relevant resistance mechanisms and also proved to be 10-fold more potent against intracellular S. aureus ATCC 25923. In medium at pH 5.5, this difference was further enhanced, with the MIC decreasing by 5 log(2) dilutions. In infected cells incubated in acidic medium, the relative potency was 10-fold higher than that at neutral pH and the maximal relative efficacy reached a bactericidal effect at 24 h. These results can be explained by a 10-fold increase in delafloxacin accumulation in both bacteria and cells at acidic pH, making delafloxacin one of the most efficient drugs tested in this model. Opposite effects were seen for moxifloxacin with respect to both activity and accumulation. As reported for zwitterionic fluoroquinolones, delafloxacin was found associated with the soluble fraction in homogenates of eukaryotic cells. Taken together, these properties may confer to delafloxacin an advantage for the eradication of S. aureus in acidic environments, including intracellular infections.

Activity of quinupristin/dalfopristin against extracellular and intracellular Staphylococcus aureus with various resistance phenotypes

Treatment of chronic or recurrent Staphylococcus aureus infections may require using antibiotics with activity against intracellular multiresistant organisms. Quinupristin/dalfopristin (3:7) has been examined in this context. Quinupristin and dalfopristin were used separately or mixed. Strains used were: (i) methicillin-susceptible and -resistant S. aureus (MSSA and MRSA); (ii) one vat(B) MSSA and msr(A/B) MRSA; (iii) erm(A)+ [MSSA, MRSA, vancomycin-intermediate S. aureus (VISA) and vancomycin-resistant S. aureus (VRSA)]; and (iv) one erm(A/B)+ cfr+ MRSA resistant to quinupristin, dalfopristin and their combination. Assessment of activity was determined by: (i) MICs (CLSI method); and (ii) concentration-response curves in broth and after phagocytosis by THP-1 macrophages, with descriptors of the model (Emin) and the pharmacodynamic response [maximal relative efficacy (Emax), relative potency (EC50) and apparent static concentration (Cstatic)]. erm(A)-positive strains were all susceptible to quinupristin/dalfopristin (except strain CM05), with MICs not adversely influenced by acid pH or by the MRSA, VISA or VRSA character of the strain. In concentration-response experiments, quinupristin/dalfopristin showed similar patterns for all strains (except strain CM05), with a >3 log10 cfu decrease in broth and a 1.3 [erm(A) strain] to 2.6 [fully susceptible, vat(B) and msr(A/B) strains] log10 cfu decrease for intracellular bacteria at the maximal extracellular concentration tested (25 mg/L). Maximal extracellular and intracellular activity was obtained for a quinupristin/dalfopristin ratio of 3:7. For strain CM05, quinupristin/dalfopristin was static in all conditions. Based on historical comparisons with rifampicin, fluoroquinolones, lipoglycopeptides and other antistaphylococcal drugs with a large accumulation in eukaryotic cells, quinupristin/dalfopristin appears to be one of the most active antibiotics against intracellular S. aureus studied in this model so far, largely irrespective of its resistance phenotype.

Intra- and extracellular activity of linezolid against Staphylococcus aureus in vivo and in vitro

Treatment of Staphylococcus aureus infections remains problematic (slow responses and frequent recurrences). Intracellular persistence of the S. aureus could explain those difficulties because of impaired intracellular efficacy of antibiotics. Our aim was to study linezolid for its intracellular activity. (i) Pharmacodynamic (PD) analysis of intracellular activity using in vitro (THP-1 macrophages) and in vivo (mouse peritonitis) models with determination of key dose-response parameters [maximal relative efficacy (E(max)), relative potency (EC(50)) and static concentration (C(static))] towards methicillin-susceptible S. aureus (ATCC 25923; clinical isolate) with linezolid MICs of 4 mg/L; (ii) pharmacokinetic (PK) analysis in uninfected mice for determination of C(max), AUC and half-life for total and free drug; and (iii) determination of the predictive PK/PD parameter (fT > MIC, fAUC(24)/MIC or fC(max)/MIC) for therapeutic outcome. In vitro, linezolid showed an E(max) of approximately 1 log(10) cfu reduction compared with initial inoculum both intra- and extracellularly and an approximately 3-fold increased relative potency (lower EC(50) and C(static)) intracellularly. In vivo, the efficacy of linezolid was impaired (<0.5 log(10) reduction extracellularly; failure to reduce the cfu to less than the initial load intracellularly) with, however, an increased intracellular potency (lower EC(50)). Infection outcome correlated better with the fAUC(24)/MIC (R(2) = 55%) than with the fT > MIC parameter (R(2) = 51%) for the extracellular compartment, but no parameter emerged as significant for the intracellular compartment. Linezolid exerts only a weak intracellular activity against the strains of S. aureus tested, even though, in contrast to most other antibiotics, its potency does not appear impaired in comparison with the extracellular activity.

Plectasin Shows Intracellular Activity against Staphylococcus aureus in Human THP-1 Monocytes and in a Mouse Peritonitis Model

Antimicrobial therapy of infections with Staphylococcus aureus can pose a challenge due to slow response to therapy and recurrence of infection. These treatment difficulties can partly be explained by intracellular survival of staphylococci, which is why the intracellular activity of antistaphylococcal compounds has received increased attention within recent years. The intracellular activity of plectasin, an antimicrobial peptide, against S. aureus was determined both in vitro and in vivo. In vitro studies using THP-1 monocytes showed that some intracellular antibacterial activity of plectasin was maintained (maximal relative efficacy [E(max)], 1.0- to 1.3-log reduction in CFU) even though efficacy was inferior to that of extracellular killing (E(max), >4.5-log CFU reduction). Animal studies included a novel use of the mouse peritonitis model, exploiting extra- and intracellular differentiation assays, and assessment of the correlations between activity and pharmacokinetic (PK) parameters. The intracellular activity of plectasin was in accordance with the in vitro studies, with an E(max) of a 1.1-log CFU reduction. The parameter most important for activity was fC(peak)/MIC, where fC(peak) is the free peak concentration. These findings stress the importance of performing studies of extra- and intracellular activity since these features cannot be predicted from traditional MIC and killing kinetic studies. Application of both the THP-1 and the mouse peritonitis models showed that the in vitro results were similar to findings in the in vivo model with respect to demonstration of intracellular activity. Therefore the in vitro model was a good screening model for intracellular activity. However, animal models should be applied if further information on activity, PK/pharmacodynamic parameters, and optimal dosing regimens is required.