Safety, Pharmacokinetics, and Causal Prophylactic Efficacy of KAF156 in a Plasmodium falciparum Human Infection Study.

BACKGROUND: KAF156 is a novel antimalarial drug that is active against both liver- and blood-stage Plasmodium parasites, including drug-resistant strains. Here, we investigated the causal prophylactic efficacy of KAF156 in a controlled human malaria infection (CHMI) model. METHODS: In part 1, healthy, malaria-naive participants received 800 mg KAF156 or placebo 3 hours before CHMI with P. falciparum-infected mosquitoes. In part 2, KAF156 was administered as single doses of 800, 300, 100, 50, or 20 mg 21 hours post-CHMI. All participants received atovaquone/proguanil treatment if blood-stage infection was detected or on day 29. For each cohort, 7-14 subjects were enrolled to KAF156 treatment and up to 4 subjects to placebo. RESULTS: KAF156 at all dose levels was safe and well tolerated. Two serious adverse events were reported-both resolved without sequelae and neither was considered related to KAF156. In part 1, all participants treated with KAF156 and none of those randomized to placebo were protected against malaria infection. In part 2, all participants treated with placebo or 20 mg KAF156 developed malaria infection. In contrast, 50 mg KAF156 protected 3 of 14 participants from infection, and doses of 800, 300, and 100 mg KAF156 protected all subjects against infection. An exposure-response analysis suggested that a 24-hour postdose concentration of KAF156 of 21.5 ng/mL (90% confidence interval, 17.66-25.32 ng/mL) would ensure a 95% chance of protection from malaria parasite infection. CONCLUSIONS: KAF156 was safe and well tolerated and demonstrated high levels of pre- and post-CHMI protective efficacy. CLINICAL TRIALS REGISTRATION: NCT04072302.

Combinatorial Pharmacodynamics of Ceftolozane-Tazobactam against Genotypically Defined ß-Lactamase-Producing Escherichia coli: Insights into the Pharmacokinetics/Pharmacodynamics of ß-Lactam-ß-Lactamase Inhibitor Combinations.

Despite a dearth of new agents currently being developed to combat multidrug-resistant Gram-negative pathogens, the combination of ceftolozane and tazobactam was recently approved by the Food and Drug Administration to treat complicated intra-abdominal and urinary tract infections. To characterize the activity of the combination product, time-kill studies were conducted against 4 strains ofEscherichia colithat differed in the type of ß-lactamase they expressed. The four investigational strains included 2805 (no ß-lactamase), 2890 (AmpC ß-lactamase), 2842 (CMY-10 ß-lactamase), and 2807 (CTX-M-15 ß-lactamase), with MICs to ceftolozane of 0.25, 4, 8, and >128 mg/liter with no tazobactam, and MICs of 0.25, 1, 4, and 8 mg/liter with 4 mg/liter tazobactam, respectively. All four strains were exposed to a 6 by 5 array of ceftolozane (0, 1, 4, 16, 64, and 256 mg/liter) and tazobactam (0, 1, 4, 16, and 64 mg/liter) over 48 h using starting inocula of 10(6)and 10(8)CFU/ml. While ceftolozane-tazobactam achieved bactericidal activity against all 4 strains, the concentrations of ceftolozane and tazobactam required for a ≥3-log reduction varied between the two starting inocula and the 4 strains. At both inocula, the Hill plots (R(2)> 0.882) of ceftolozane revealed significantly higher 50% effective concentrations (EC50s) at tazobactam concentrations of ≤4 mg/liter than those at concentrations of ≥16 mg/liter (P< 0.01). Moreover, the EC50s at 10(8)CFU/ml were 2.81 to 66.5 times greater than the EC50s at 10(6)CFU/ml (median, 10.7-fold increase;P= 0.002). These promising results indicate that ceftolozane-tazobactam achieves bactericidal activity against a wide range of ß-lactamase-producingE. colistrains.

Polymyxin B in combination with doripenem against heteroresistant Acinetobacter baumannii: pharmacodynamics of new dosing strategies.

OBJECTIVES: Polymyxin B is being increasingly utilized as a last resort against resistant Gram-negative bacteria. We examined the pharmacodynamics of novel dosing strategies for polymyxin B combinations to maximize efficacy and minimize the emergence of resistance and drug exposure against Acinetobacter baumannii. METHODS: The pharmacodynamics of polymyxin B together with doripenem were evaluated in time-kill experiments over 48 h against 108 cfu/mL of two polymyxin-heteroresistant A. baumannii isolates (ATCC 19606 and N16870). Pharmacokinetic/pharmacodynamic relationships were mathematically modelled using S-ADAPT. A hollow-fibre infection model (HFIM) was also used to simulate clinically relevant polymyxin B dosing strategies (traditional, augmented 'front-loaded' and 'burst' regimens), together with doripenem, against an initial inoculum of 109 cfu/mL of ATCC 19606. RESULTS: In static time-kill studies, polymyxin B concentrations >4 mg/L in combination with doripenem 25 mg/L resulted in rapid bactericidal activity against both strains with undetectable bacterial counts by 24 h. The mathematical model described the rapid, concentration-dependent killing as subpopulation and mechanistic synergy. In the HFIM, the traditional polymyxin B combination regimen was synergistic, with a >7.5 log10 reduction by 48 h. The polymyxin B 'front-loaded' combination resulted in more rapid and extensive initial killing (>8 log10) within 24 h, which was sustained over 10 days. With only 25% of the cumulative drug exposure, the polymyxin B 'burst' combination demonstrated antibacterial activity similar to traditional and 'front-loaded' combination strategies. The polymyxin B 'front-loaded' and 'burst' combination regimens suppressed the emergence of resistance. CONCLUSIONS: Early aggressive dosing regimens for polymyxin combinations demonstrate promise for treatment of heteroresistant A. baumannii infections.

The DGAT1 inhibitor pradigastat does not induce photosensitivity in healthy human subjects: a randomized controlled trial using three defined sunlight exposure conditions.

The DGAT1 inhibitor, pradigastat, demonstrated a mild phototoxicity signal in preclinical studies. Therefore, this clinical trial was conducted to assess the risk of photosensitivity in humans. 47 healthy adults were randomized to part A (double-blind, placebo-controlled; 3 : 1 pradigastat : placebo) or part B (open-label positive control ciprofloxacin, investigator blind). Three irradiation conditions (1. full range UVB/UVA, 2. UVA only, 3. 1/2 MED from UVB/UVA + 16 J cm(-2) UVA) were applied to simulate different sunlight exposure conditions. Photosensitizing potential was assessed by determining the minimum erythemal dose (MED) and calculating the photosensitivity index (PI) at 1 and 24 h. Local skin reactions were recorded as a secondary endpoint. Following full UVB/UVA irradiation, there were no significant differences in MED or PI between groups. With UVA-only, no changes in MED or PI were observed for the pradigastat or placebo groups. For ciprofloxacin, there was a significant reduction in MED at 24 h (-32%, vs. 24 h baseline), which correlated to a PI of 1.61. The difference in mean PI between ciprofloxacin-pradigastat, and ciprofloxacin-placebo, was significant at 24 h (p < 0.001). Local skin erythema scores were comparable between pradigastat and placebo, but higher with ciprofloxacin. Pradigastat was not shown to induce photosensitivity reactions, while significant responses were seen with the positive control. These results strongly suggest that pradigastat will not induce photosensitivity reactions in individuals administered doses up to 40 mg per day, which is the highest intended clinical dose. Furthermore, the design of this clinical trial may serve as a prototype for future regulatory clinical photosensitivity studies.

Pharmacodynamic variability beyond that explained by MICs.

Monte Carlo simulations (MCS) present a powerful tool to evaluate candidate regimens by determining the probability of target attainment. Although these assessments have traditionally incorporated variability in pharmacokinetic (PK) parameters and MICs, consideration of interstrain pharmacodynamic (PD) variability has been neglected. A population PK/PD model was developed for doripenem using murine thigh infection data based on 20 bacterial strains. PK data were fit to a linear two-compartment model with first-order input and elimination processes and an absorption lag time from a separate site (r(2) > 0.96). PK parameters were utilized to simulate free-drug profiles for various regimens in PD studies, from which the percentage of the dosing interval for which free-drug concentrations exceed the MIC of the targeted strain (%fT>MIC) was calculated. Doripenem PD was excellently described with Hill-type models (r(2) > 0.98); significant differences between mean PD estimates determined using a two-stage approach versus population analyses were not observed (P > 0.05); however, the variance in 50% effective concentration (EC50) and maximum effect (Emax) among strains was much greater using the two-stage approach. Even using the population approach, interstrain variability in EC50 (coefficient of variation expressed as a percentage [CV%] = 29.2%) and H (CV% = 46.1%) parameters was substantive, while the variability in Emax (CV% = 19.7%) was modest. This resulted in extensive variability in the range of %fT>MIC targets associated with stasis to those associated with a 2-log10 reduction in bacterial burden (CV% ∼ 50%). It appears that MCS, based on the assumption that PD variability is due to MIC alone, underestimates variability and may consequently underestimate treatment failures.

The combination of colistin and doripenem is synergistic against Klebsiella pneumoniae at multiple inocula and suppresses colistin resistance in an in vitro pharmacokinetic/pharmacodynamic model.

Multidrug-resistant (MDR) Klebsiella pneumoniae may require combination therapy. We systematically investigated bacterial killing with colistin and doripenem mono- and combination therapy against MDR K. pneumoniae and emergence of colistin resistance. A one-compartment in vitro pharmacokinetic/pharmacodynamic model was employed over a 72-h period with two inocula (∼10(6) and ∼10(8) CFU/ml); a colistin-heteroresistant reference strain (ATCC 13883) and three clinical isolates (colistin-susceptible FADDI-KP032 [doripenem resistant], colistin-heteroresistant FADDI-KP033, and colistin-resistant FADDI-KP035) were included. Four combinations utilizing clinically achievable concentrations were investigated. Microbiological responses were examined by determining log changes and population analysis profiles (for emergence of colistin resistance) over 72 h. Against colistin-susceptible and -heteroresistant isolates, combinations of colistin (constant concentration regimens of 0.5 or 2 mg/liter) plus doripenem (steady-state peak concentration [C(max)] of 2.5 or 25 mg/liter over 8 h; half-life, 1.5 h) generally resulted in substantial improvements in bacterial killing at both inocula. Combinations were additive or synergistic against ATCC 13883, FADDI-KP032, and FADDI-KP033 in 9, 9, and 14 of 16 cases (4 combinations at 6, 24, 48, and 72 h) at the 10(6)-CFU/ml inoculum and 14, 11, and 12 of 16 cases at the 10(8)-CFU/ml inoculum, respectively. Combinations at the highest dosage regimens resulted in undetectable bacterial counts at 72 h in 5 of 8 cases (4 isolates at 2 inocula). Emergence of colistin-resistant subpopulations in colistin-susceptible and -heteroresistant isolates was virtually eliminated with combination therapy. Against the colistin-resistant isolate, colistin at 2 mg/liter plus doripenem (C(max), 25 mg/liter) at the low inoculum improved bacterial killing. This investigation provides important information for optimization of colistin-doripenem combinations.

Different surface charge of colistin-susceptible and -resistant Acinetobacter baumannii cells measured with zeta potential as a function of growth phase and colistin treatment.

OBJECTIVES: electrostatic forces mediate the initial interaction between cationic colistin and Gram-negative bacterial cells. Lipopolysaccharide (LPS) loss mediates colistin resistance in some A. baumannii strains. Our aim was to determine the surface charge of colistin-susceptible and -resistant A. baumannii as a function of growth phase and in response to polymyxin treatment. METHODS: the zeta potential of A. baumannii ATCC 19606 and 10 clinical multidrug-resistant strains (MICs 0.5-2 mg/L) was assessed. Colistin-resistant derivatives (MIC >128 mg/L) of wild-type strains were selected in the presence of 10 mg/L colistin, including the LPS-deficient lpxA mutant, ATCC 19606R. To determine the contribution of LPS to surface charge, two complemented ATCC 19606R derivatives were examined, namely ATCC 19606R + lpxA (containing an intact lpxA gene) and ATCC 19606R + V (containing empty vector). Investigations were conducted as a function of growth phase and polymyxin treatment (1, 4 and 8 mg/L). RESULTS: wild-type cells exhibited a greater negative charge (-60.5  ±â€Š 2.36 to -26.2  ±â€Š 2.56 mV) thancolistin-resistant cells (-49.2  ±â€Š 3.09 to -19.1  ±  2.80 mV) at mid-log phase (ANOVA, P  <  0.05). Opposing growth-phase trends were observed for both phenotypes: wild-type cells displayed reduced negative charge and colistin-resistant cells displayed increased negative charge at stationary compared with mid-logarithmic phase. Polymyxin exposure resulted in a concentration-dependent increase in zeta potential. Examination of ATCC 19606R and complemented strains supported the importance of LPS in determining surface charge, suggesting a potential mechanism of colistin resistance. CONCLUSIONS: zeta potential differences between A. baumannii phenotypes probably reflect compositional outer-membrane variations that impact the electrostatic component of colistin activity.

Design, synthesis, and evaluation of a new fluorescent probe for measuring polymyxin-lipopolysaccharide binding interactions.

Fluorescence assays employing semisynthetic or commercial dansyl-polymyxin B have been widely employed to assess the affinity of polycations, including polymyxins, for bacterial cells and lipopolysaccharide (LPS). The five primary γ-amines on diaminobutyric acid residues of polymyxin B are potentially derivatized with dansyl-chloride. Mass spectrometric analysis of the commercial product revealed a complex mixture of di- or tetra-dansyl-substituted polymyxin B. We synthesized a mono-substituted fluorescent derivative, dansyl[Lys]¹polymyxin B3. The affinity of polymyxin for purified gram-negative LPS and whole bacterial cells was investigated. The affinity of dansyl[Lys]¹polymyxin B3 for LPS was comparable to polymyxin B and colistin, and considerably greater (K(d)<1 µM) than for whole cells (K(d)∼6-12µM). Isothermal titration calorimetric studies demonstrated exothermic enthalpically driven binding between both polymyxin B and dansyl[Lys]¹polymyxin B3 to LPS, attributed to electrostatic interactions. The hydrophobic dansyl moiety imparted a greater entropic contribution to the dansyl[Lys]¹polymyxin B3-LPS reaction. Molecular modeling revealed a loss of electrostatic contact within the dansyl[Lys]¹polymyxin B3-LPS complex due to steric hindrance from the dansyl[Lys]¹ fluorophore; this corresponded with diminished antibacterial activity (MIC≥16µg/mL). Dansyl[Lys]¹polymyxin B3 may prove useful as a screening tool for drug development.

Atomic force microscopy investigation of the morphology and topography of colistin-heteroresistant Acinetobacter baumannii strains as a function of growth phase and in response to colistin treatment.

The prevalence of infections caused by multidrug-resistant gram-negative Acinetobacter baumannii strains and the lack of novel antibiotics under development are posing a global dilemma, forcing a resurgence of the last-line antibiotic colistin. Our aim was to use atomic force microscopy (AFM) to investigate the morphology and topography of paired colistin-susceptible and -resistant cells from colistin-heteroresistant A. baumannii strains as a function of bacterial growth phase and colistin exposure. An optimal AFM bacterial sample preparation protocol was established and applied to examine three paired strains. Images revealed rod-shaped colistin-susceptible cells (1.65 +/- 0.27 microm by 0.98 +/- 0.07 microm) at mid-logarithmic phase, in contrast to spherical colistin-resistant cells (1.03 +/- 0.09 microm); the latter were also more diverse in appearance and exhibited a rougher surface topography (7.05 +/- 1.3 nm versus 11.4 +/- 2.5 nm for susceptible versus resistant, respectively). Cellular elongation up to approximately 18 microm at stationary phase was more commonly observed in susceptible strains, although these "worm-like" cells were also observed occasionally in the resistant population. The effects of colistin exposure on the cell surface of colistin-susceptible and -resistant cells were found to be similar; topographical changes were minor in response to 0.5 microg/ml colistin; however, at 4 microg/ml colistin, a significant degree of surface disruption was detected. At 32 microg/ml colistin, cellular clumping and surface smoothening were evident. Our study has demonstrated for the first time substantial morphological and topographical differences between colistin-susceptible and -resistant cells from heteroresistant A. baumannii strains. These results contribute to an understanding of colistin action and resistance in regard to this problematic pathogen.

In vitro pharmacodynamic evaluation of ceftolozane/tazobactam against ß-lactamase-producing Escherichia coli in a hollow-fibre infection model.

The proliferation of multidrug-resistant Gram-negative pathogens has been exacerbated by a lack of novel agents in current development by pharmaceutical companies. Ceftolozane/tazobactam was recently approved by the FDA for the treatment of complicated intra-abdominal infections and complicated urinary tract infections. In the present study, the activity of ceftolozane/tazobactam against four isogenic Escherichia coli strains was investigated in a hollow-fibre infection model simulating various clinical dosing regimens. The four investigational E. coli strains included #2805 (no ß-lactamase), #2890 (AmpC ß-lactamase), #2842 (CMY-10 ß-lactamase) and #2807 (CTX-M-15 ß-lactamase). Each strain was exposed to regimens simulating 1 g ceftolozane, 2 g ceftolozane, 1 g ceftolozane/0.5 g tazobactam, and 2 g ceftolozane/1 g tazobactam utilising a starting inoculum of ca. 106 CFU/mL. Whereas 1 g of ceftolozane eradicated strains #2805 and #2842 without subsequent regrowth, 1 g ceftolozane/0.5 g tazobactam was required to eradicate strain #2890. For strain #2890, ceftolozane monotherapy led to bacterial growth on plates impregnated with 20 mg/L ceftolozane by 24 h, whilst combination treatment with tazobactam completely suppressed the development of ceftolozane resistance. In contrast, none of the regimens, including 2 g ceftolozane/1 g tazobactam, were able to entirely suppress bacterial growth in strain #2807, with bacterial counts exceeding 108 CFU/mL by 48 h and ceftolozane-resistant populations being amplified after 24 h. Thus, the combination of ceftolozane and tazobactam achieved bactericidal activity followed by sustained killing over 10 days for three of four isogenic E. coli strains. Ceftolozane/tazobactam is a promising new agent to counter multidrug-resistant Gram-negative bacteria.

Pharmacokinetic/pharmacodynamic evaluation of the efficacy and safety of daptomycin against Staphylococcus aureus.

Daptomycin is a novel lipopeptide exhibiting concentration-dependent bactericidal activity against multidrug-resistant Gram-positive pathogens, including MRSA. Approval of daptomycin is granted at 4-6 mg/kg once daily, however off-label use of doses up to 12 mg/kg daily has been utilised without evidence of significant toxicity. Our aim was to optimise daptomycin regimens by assessing the probability of bacteriological efficacy (pTA) and toxicity (pTOX) at various MICs using Monte Carlo simulation (MCS) techniques. Population pharmacokinetic, pharmacodynamic and toxicodynamic models were developed based on current literature. MCS was performed for 10000 patients, who were assigned true weight and creatinine clearances, and were infected with four Staphylococcus aureus strains at each MIC. Bacteriostatic and bactericidal %pTA was calculated following administration of 6, 8, 10 and 12 mg/kg daptomycin; activity was deemed adequate at %pTA ≥ 90%. Considerable pharmacodynamic variability was observed in derived AUC/MIC targets between strains. Bacteriostatic targets were adequately attained against all strains at MIC ≤ 1 mg/L with daptomycin > 6 mg/kg. However, bactericidal target attainment was only achieved against all strains at the lowest MIC of 0.5 mg/L with daptomycin > 8 mg/kg. At MIC = 2 mg/kg, bactericidal target attainment was extremely poor even at the highest dose of 12 mg/kg. pTOX increased from 3.31% to 17.7% following exposure to 6 mg/kg to 12 mg/kg daily, respectively. Formal benefit:risk analyses favoured doses of 10 mg/kg against infections with MIC < 2 mg/L, whilst modest improvements in activity at 12 mg/kg could not justify the marked increase in pTOX.

Molecular basis for the increased polymyxin susceptibility of Klebsiella pneumoniae strains with under-acylated lipid A.

The impact of under-acylation of lipid A on the interaction between Klebsiella pneumoniae LPS and polymyxins B and E was examined with fluorometric and calorimetric methods, and by (1)H NMR, using a paired wild type (WT) and the ΔlpxM mutant strains B5055 and B5055ΔlpxM, which predominantly express LPS with hexa- and penta-acylated lipid A structures respectively. LPS from B5055ΔlpxM displayed a fourfold increased binding affinity for polymyxins B and E compared with the B5055 WT LPS. EC50 values were consistent with polymyxin minimum inhibitory concentration (MIC) values for each strain. Accordingly, polymyxin exposure considerably enhanced the permeability of the B5055ΔlpxM OM. Analysis of the melting profiles of isolated LPS aggregates suggested that bactericidal polymyxin activity may relate to the acyl chains' phase of the outer membrane (OM). The enhanced polymyxin susceptibility of B5055ΔlpxM may be attributable to the favorable insertion of polymyxins into the more fluid OM compared with B5055. Molecular models of the polymyxin B-lipid A complex illuminate the key role of the lipid A acyl chains for complexation of polymyxin. The data provide important insight into the molecular basis for the increased polymyxin susceptibility of K. pneumoniae strains with under-acylated lipid A. Under-acylation appears to facilitate the integration of the N-terminal fatty-acyl chain of polymyxin into the OM resulting in an increased susceptibility to its antimicrobial activity/activities.

Effect of colistin exposure and growth phase on the surface properties of live Acinetobacter baumannii cells examined by atomic force microscopy.

The diminishing antimicrobial development pipeline has forced the revival of colistin as a last line of defence against infections caused by multidrug-resistant Gram-negative 'superbugs' such as Acinetobacter baumannii. The complete loss of lipopolysaccharide (LPS) mediates colistin resistance in some A. baumannii strains. Atomic force microscopy was used to examine the surface properties of colistin-susceptible and -resistant A. baumannii strains at mid-logarithmic and stationary growth phases in liquid and in response to colistin treatment. The contribution of LPS to surface properties was investigated using A. baumannii strains constructed with and without the lpxA gene. Bacterial spring constant measurements revealed that colistin-susceptible cells were significantly stiffer than colistin-resistant cells at both growth phases (P<0.01), whilst colistin treatment at high concentrations (32 mg/L) resulted in more rigid surfaces for both phenotypes. Multiple, large adhesive peaks frequently noted in force curves captured on colistin-susceptible cells were not evident for colistin-resistant cells. Adhesion events were markedly reduced following colistin exposure. The cell membranes of strains of both phenotypes remained intact following colistin treatment, although fine topographical details were illustrated. These studies, conducted for the first time on live A. baumannii cells in liquid, have contributed to our understanding of the action of colistin in this problematic pathogen.