The Engineered Lysin, CF-370, is Active Against Antibiotic-Resistant Gram-Negative Pathogens In vitro and Synergizes with Meropenem in Experimental Pseudomonas aeruginosa Pneumonia.

BACKGROUND: Lysins (cell wall hydrolases) targeting Gram-negative organisms require engineering to permeabilize the outer membrane and access subjacent peptidoglycan to facilitate killing. In the current study, the potential clinical utility for engineered lysin, CF-370, was examined in vitro and in vivo against Gram-negative pathogens important in human infections. METHODS: MICs and bactericidal activity were determined using standard methods. An in vivo proof-of-concept efficacy study was conducted using a rabbit acute pneumonia model caused by Pseudomonas aeruginosa. RESULTS: CF-370 exhibited potent antimicrobial activity, with MIC50/90 values (in µg/mL) for: P. aeruginosa, 1/2; Acinetobacter baumannii, 1/1; Escherichia coli, 0.25/1; Klebsiella pneumoniae, 2/4; Enterobacter cloacae 1/4; and Stenotrophomonas maltophilia 2/8. CF-370 furthermore demonstrated: i) bactericidal activity; (ii) activity in serum; iii) a low propensity for resistance; iv) anti-biofilm activity; and v) synergy with antibiotics. In the pneumonia model, CF-370 alone decreased bacterial densities in lungs, kidneys and spleen vs. vehicle control, and demonstrated significantly increased efficacy when combined with meropenem (vs either agent alone). CONCLUSIONS: CF-370 is the first engineered lysin described with potent broad spectrum in vitro activity against multiple clinically-relevant Gram-negative pathogens, as well as potent in vivo efficacy in an animal model of severe invasive multi-system infection.

In vitro activity of exebacase against methicillin-resistant Staphylococcus aureus biofilms on orthopedic Kirschner wires.

Orthopedic foreign body-associated infection can be difficult to treat due to the formation of biofilms protecting microorganisms from both antimicrobials and the immune system. Exebacase is an antistaphylococcal lysin (cell wall hydrolase) under consideration for local treatment for biofilm-based infections caused by methicillin-resistant Staphylococcus aureus (MRSA). To determine the activity of exebacase, we formed MRSA biofilms on orthopedic Kirschner wires and exposed them to varying concentrations (0.098, 0.98, 9.8 mg/ml) of exebacase and/or daptomycin over 24 h. The biofilm consisted of 5.49 log10 colony forming units (cfu)/K-wire prior to treatment and remained steady throughout the experiment. Exebacase showed significant biofilm reduction at all timepoints (up to 5.78 log10 cfu/K-wire; P < 0.0495) compared to the controls at all concentrations and all time points with bactericidal activity (> 3 log10 cfu/K-wire reduction) observed for up to 12 h for the 0.098 and 0.98 mg/ml concentrations and at 24 h for 9.8 mg/ml. Daptomycin showed significant biofilm reduction, although non-bactericidal, at all time points for 0.98 and 9.8 mg/ml and at 4 and 8 h with 0.098 mg/ml (P < 0.0495). This study supports further evaluation of local administration of exebacase as a potential treatment for orthopedic implant infections.

The N-Acetylgalactosamine-conjugated small interfering RNA inclisiran can be coadministered safely with atorvastatin in cynomolgus monkeys resulting in additive low-density lipoprotein cholesterol reductions.

Inclisiran, a small interfering RNA, selectively inhibits proprotein convertase subtilisin/kexin type 9 (PCSK9) synthesis in the liver and has been shown to reduce low-density lipoprotein cholesterol (LDL-C) by ≥50% in patients with hypercholesterolemia receiving maximally tolerated statins. The toxicokinetic, pharmacodynamic, and safety profiles of inclisiran when coadministered with a statin were characterized in cynomolgus monkeys. Six cohorts of monkeys were administered either atorvastatin (40 mg/kg, reduced to 25 mg/kg during the study, daily, oral gavage), inclisiran (300 mg/kg every 28 days, subcutaneous administration), atorvastatin (40/25 mg/kg) and inclisiran combinations (30, 100, or 300 mg/kg), or control vehicles over 85 days followed by 90 days' recovery. Inclisiran and atorvastatin toxicokinetic parameters were similar in cohorts administered either agent alone or in combination. Inclisiran exposure increased in a dose-proportional manner. At Day 86, atorvastatin increased plasma PCSK9 levels four-fold from pretreatment levels but did not significantly lower serum LDL-C levels. Inclisiran (alone or in combination) reduced PCSK9 (mean decrease 66-85%) and LDL-C (mean decrease 65-92%) from pretreatment levels at Day 86; levels were significantly lower than the control group (p ≤ .05) and remained decreased during the 90-day recovery. Coadministration of inclisiran with atorvastatin resulted in greater reductions in LDL-C and total cholesterol compared with either drug alone. No toxicities or adverse effects were observed in any cohort receiving inclisiran, either alone or in combination. In summary, inclisiran significantly inhibited PCSK9 synthesis and decreased LDL-C in cynomolgus monkeys without increasing the risk of adverse effects when coadministered with atorvastatin.

Locally delivered antistaphylococcal lysin exebacase or CF-296 is active in methicillin-resistant Staphylococcus aureus implant-associated osteomyelitis.

Introduction: Staphylococcus aureus is the most common cause of orthopedic infections and can be challenging to treat, especially in the presence of a foreign body. The antistaphylococcal lysins exebacase and CF-296 have rapid bactericidal activity, a low propensity for resistance development, and synergize with some antibiotics. Methods: Rabbit implant-associated osteomyelitis was induced by drilling into the medial tibia followed by locally delivering exebacase, CF-296, or lysin carrier. A titanium screw colonized with methicillin-resistant S. aureus (MRSA) IDRL-6169 was inserted. Intravenous daptomycin or saline was administered and continued daily for 4 d. On day 5, rabbits were euthanized, and the tibiae and implants were collected for culture. Results were reported as log 10 colony forming units (cfu) per gram of bone or log 10  cfu per implant, and comparisons among the six groups were performed using the Wilcoxon rank sum test. Results: Based on implant and bone cultures, all treatments resulted in significantly lower bacterial counts than those of controls ( P ≤ 0.0025 ). Exebacase alone or with daptomycin as well as CF-296 with daptomycin were more active than daptomycin alone ( P ≤ 0.0098 ) or CF-296 alone ( P ≤ 0.0154 ) based on implant cultures. CF-296 with daptomycin was more active than either CF-296 alone ( P = 0.0040 ) or daptomycin alone ( P = 0.0098 ) based on bone cultures. Conclusion: Local delivery of either exebacase or CF-296 offers a promising complement to conventional antibiotics in implant-associated infections.

Evaluation of the distribution and excretion of [14C]-inclisiran following single subcutaneous administration in cynomolgus monkeys.

Inclisiran is a small interfering RNA molecule that was designed to reduce plasma low-density lipoprotein cholesterol (LDL-C) levels by inhibiting proprotein convertase subtilisin/kexin type 9 synthesis in the liver. This study aimed to characterize the tissue distribution and excretion of inclisiran after dosing in monkeys. A single 20 mg/kg subcutaneous injection of [14C]-inclisiran was administered to 12 male cynomolgus monkeys. Plasma concentrations and tissue binding parameters for inclisiran were assessed up to 42 days after injection using liquid scintillation of blood samples and tissue homogenates, as well as quantitative whole-body autoradiography. Radioactivity was highest in the liver at all time points from 24 h onward and remained elevated throughout the entire study period. Radioactivity was also detected in the kidneys and bladder wall, returning to low levels by 24 h. The concentration of radioactivity in the liver (402.97 µg equivalent/g) was 15.7-fold higher than in the kidneys (25.70 µg equivalent/g). Very low amounts of radioactivity were detected in all other tissues examined. The highest radioactivity in tissue homogenates was in the liver and kidney pyramid (327 and 351 µg equivalent/g, respectively). This study confirmed the selective uptake of inclisiran by the liver, indicating that the N-acetylgalactosamine linker allows for selective uptake via the asialoglycoprotein receptors expressed on hepatocytes compared with other tissues that lack asialoglycoprotein receptors. The long tissue retention in the liver supports the infrequent, biannual dosing schedule for inclisiran in the clinic and the temporal disconnect between short-term systemic exposure and sustained lowering of LDL-C.

Exebacase in Addition to Daptomycin against MRSA.

Exebacase is a lysin (cell wall hydrolase) with direct lytic activity against Staphylococcus aureus including methicillin-resistant S. aureus (MRSA). Time-kill analysis experiments illustrated bactericidal activity of exebacase-daptomycin against MRSA strains MW2 and 494. Furthermore, exebacase in addition to daptomycin (10, 6, and 4 mg/kg/day) in a two-compartment ex vivo pharmacokinetic/pharmacodynamic simulated endocardial vegetation model with humanized doses resulted in reductions of 6.01, 4.99, and 2.81 log10 CFU/g (from initial inoculum) against MRSA strain MW2.

Efficacy assessment of lysin CF-296 in addition to daptomycin or vancomycin against Staphylococcus aureus in the murine thigh infection model.

OBJECTIVES: CF-296 is a lysin in pre-clinical development for the treatment of MSSA and MRSA infections, used in addition to standard-of-care (SOC) antibiotics. We evaluated the efficacy of CF-296 alone and in addition to daptomycin or vancomycin against Staphylococcus aureus in the neutropenic mouse thigh infection model. METHODS: Eight isolates (one MSSA and seven MRSA) were studied. Mice were administered five CF-296 monotherapy doses ranging from 0.5 to 50 mg/kg intravenously. To assess adjunctive therapy, mice received sub-therapeutic daptomycin alone, sub-therapeutic vancomycin alone, or the five CF-296 doses in addition to either daptomycin or vancomycin. RESULTS: Relative to starting inoculum (5.80 ± 0.31 log10 cfu/thigh), bacterial density in vehicle controls increased by +2.49 ± 0.98 across all eight strains. Relative to 24 h controls, a dose-response in bacterial killing (range -0.22 ± 0.87 to -2.01 ± 1.71 log10 cfu/thigh) was observed with increasing CF-296 monotherapy against the eight isolates. Daptomycin and vancomycin resulted in -1.36 ± 0.77 and -1.37 ± 1.01 log10 cfu/thigh bacteria reduction, respectively, relative to 24 h controls. Escalating CF-296 exposures (0.5-50 mg/kg) in addition to daptomycin resulted in an enhanced dose-response, ranging from bacterial killing of -0.69 to -2.13 log10 cfu/thigh, relative to daptomycin alone. Similarly, in addition to vancomycin, escalating CF-296 exposures resulted in bacterial reduction ranging from -1.37 to -2.29 log10 cfu/thigh, relative to vancomycin alone. CONCLUSIONS: Relative to SOC antibiotics (daptomycin or vancomycin), addition of CF-296 resulted in robust and enhanced antibacterial dose-response, achieving ≥1 log10 cfu/thigh decrease across most doses, highlighting a potential role for CF-296 adjunctive therapy against MSSA and MRSA isolates.

Effect of the Lysin Exebacase on Cardiac Vegetation Progression in a Rabbit Model of Methicillin-Resistant Staphylococcus aureus Endocarditis as Determined by Echocardiography.

Methicillin-resistant Staphylococcus aureus (MRSA) poses significant therapeutic challenges related to its frequency in clinical infections, innate virulence properties, and propensity for multiantibiotic resistance. MRSA is among the most common causes of endovascular infections, including infective endocarditis (IE). Our objective was to employ transthoracic echocardiography (TTE) to evaluate the effect of exebacase, a novel direct lytic agent, in experimental aortic valve MRSA IE. TTE was utilized to evaluate the in vivo effect of exebacase on MRSA-infected vegetation progression when combined with daptomycin (versus daptomycin alone). Primary intravegetation outcomes were maximum size, weights at sacrifice, and MRSA counts at infection baseline versus after 4 days of daptomycin treatment (alone or in addition to exebacase administered once on treatment day 1). A single dose of exebacase in addition to daptomycin cleared significantly more intravegetation MRSA than daptomycin alone. This was associated with a statistical trend toward reduced maximum vegetation size in the exebacase plus daptomycin versus the daptomycin alone therapy groups (P = 0.07). Also, mean vegetation weights in the exebacase-treated group were significantly lower than those of the daptomycin alone group (P < 0.0001). Maximum vegetation size by TTE correlated with vegetation weight (P = 0.005). In addition, intravegetation MRSA counts in the combination group were significantly lower than those of untreated controls (P < 0.0001) and the daptomycin alone group (P < 0.0001). This study suggests that exebacase has a salutary impact on MRSA-infected vegetation progression when combined with daptomycin, especially in terms of vegetation MRSA burden, size, and weight. Moreover, TTE appears to be an efficient noninvasive tool to assess therapeutic efficacies in experimental MRSA IE.

Exebacase in Addition to Daptomycin Is More Active than Daptomycin or Exebacase Alone in Methicillin-Resistant Staphylococcus aureus Osteomyelitis in Rats.

Bacteriophage-derived lysins are being developed as anti-infective agents. In an acute osteomyelitis methicillin-resistant Staphylococcus aureus (MRSA) model, rats receiving no treatment or treatment with daptomycin, exebacase (CF-301), or daptomycin plus exebacase had means of 5.13, 4.09, 4.65, and 3.57 log10 CFU/gram of bone, respectively. All treated animals had fewer bacteria than did untreated animals (P ≤ 0.0001), with daptomycin plus exebacase being more active than daptomycin (P = 0.0042) or exebacase (P < 0.001) alone.

Toxicologic and Inhibitory Receptor Actions of the Etomidate Analog ABP-700 and Its Metabolite CPM-Acid.

BACKGROUND: The etomidate analog ABP-700 produces involuntary muscle movements that could be manifestations of seizures. To define the relationship (if any) between involuntary muscle movements and seizures, electroencephalographic studies were performed in Beagle dogs receiving supra-therapeutic (~10× clinical) ABP-700 doses. γ-aminobutyric acid type A (GABAA) and glycine receptor studies were undertaken to test receptor inhibition as the potential mechanism for ABP-700 seizures. METHODS: ABP-700 was administered to 14 dogs (6 mg/kg bolus followed by a 2-h infusion at 1 mg · kg(-1) · min(-1), 1.5 mg · kg(-1) · min(-1), or 2.3 mg · kg(-1) · min(-1)). Involuntary muscle movements were documented, electroencephalograph was recorded, and plasma ABP-700 and CPM-acid concentrations were measured during and after ABP-700 administration. The concentration-dependent modulatory actions of ABP-700 and CPM-acid were defined in oocyte-expressed α1ß3γ2L GABAA and α1ß glycine receptors (n = 5 oocytes/concentration) using electrophysiologic techniques. RESULTS: ABP-700 produced both involuntary muscle movements (14 of 14 dogs) and seizures (5 of 14 dogs). However, these phenomena were temporally and electroencephalographically distinct. Mean peak plasma concentrations were (from lowest to highest dosed groups) 35 µM, 45 µM, and 102 µM (ABP-700) and 282 µM, 478 µM, and 1,110 µM (CPM-acid). ABP-700 and CPM-acid concentration-GABAA receptor response curves defined using 6 µM γ-aminobutyric acid exhibited potentiation at low and/or intermediate concentrations and inhibition at high ones. The half-maximal inhibitory concentrations of ABP-700 and CPM-acid defined using 1 mM γ-aminobutyric acid were 770 µM (95% CI, 590 to 1,010 µM) and 1,450 µM (95% CI, 1,340 to 1,560 µM), respectively. CPM-acid similarly inhibited glycine receptors activated by 1 mM glycine with a half-maximal inhibitory concentration of 1,290 µM (95% CI, 1,240 to 1,330 µM). CONCLUSIONS: High dose ABP-700 infusions produce involuntary muscle movements and seizures in Beagle dogs via distinct mechanisms. CPM-acid inhibits both GABAA and glycine receptors at the high (~100× clinical) plasma concentrations achieved during the dog studies, providing a plausible mechanism for the seizures.

Oritavancin Pharmacokinetics and Bone Penetration in Rabbits.

The pharmacokinetics and bone concentrations of oritavancin were investigated after a single intravenous dose was administered to rabbits. The pharmacokinetic profile of oritavancin in rabbits showed that it is rapidly distributed to bone tissues, with concentrations remaining stable for up to 168 h, the last measured time point. Based on these findings, further evaluation of oritavancin for the treatment of infections in bone tissues is warranted.

Oritavancin does not induce Clostridium difficile germination and toxin production in hamsters or a human gut model.

OBJECTIVES: To evaluate the relative propensities of oritavancin and vancomycin to induce Clostridium difficile infection (CDI) in hamster and in vitro human gut models. METHODS: Hamsters received clindamycin (100 mg/kg orally or subcutaneously), oritavancin (50 mg/kg orally) or vancomycin (50 mg/kg orally). C. difficile spores were administered orally the next day. Control hamsters received vehicle only (polyethylene glycol 400) plus spores or clindamycin but no spores. Hamsters were monitored for clinical signs for 20 days. Caecal contents were analysed for C. difficile cells, spores and the presence of (cyto)toxin. Oritavancin and vancomycin were instilled over 7 days into separate in vitro gut models primed with pooled human faeces and inoculated with C. difficile ribotype 027 spores. Gut flora, C. difficile total viable and spore counts, toxin titres and antimicrobial concentrations were determined. RESULTS: All hamsters treated with oritavancin survived up to 20 days, with no evidence of C. difficile spores, vegetative cells or toxin in their caeca. No hamsters treated with clindamycin or vancomycin survived >6 days after spore administration. Death was associated with high C. difficile counts and toxin in caecal contents. In the gut model, oritavancin dosing elicited a rapid, marked decrease in total viable C. difficile and spore counts to below the limit of detection. Vancomycin did not elicit germination or toxin production in the gut model, but C. difficile remained present as spores throughout. CONCLUSIONS: Oritavancin exposure, unlike exposure to vancomycin or clindamycin, did not lead to CDI in hamsters. In both models, oritavancin reduced C. difficile total counts and spores to below detectable limits. The data indicate the potential of oritavancin for CDI treatment, since exposure did not induce C. difficile germination and toxin production, which are known to exacerbate the disease state.

Linking bisphosphonates to the free amino groups in fluoroquinolones: preparation of osteotropic prodrugs for the prevention of osteomyelitis.

Osteomyelitis is an infection located in bone and a notoriously difficult disease to manage, requiring frequent and heavy doses of systemically administered antibiotics. Targeting antibiotics to the bone after systemic administration may provide both greater efficacy of treatment and less frequent administration. By taking advantage of the affinity of the bisphosphonate group for bone mineral, we have prepared a set of 13 bisphosphonated antibacterial prodrugs based on eight different linkers tethered to the free amino functionality on fluoroquinolone antibiotics. While all but one of the prodrugs were shown in vitro to be effective and rapid bone binders (over 90% in 1 h), only eight of them demonstrated the capacity to significantly regenerate the parent drug. In a rat model of the disease, a selected group of agents demonstrated their ability to prevent osteomyelitis when used in circumstances under which the parent drug had already been cleared and is thus inactive.

Bisphosphonated fluoroquinolone esters as osteotropic prodrugs for the prevention of osteomyelitis.

Osteomyelitis is a difficult to treat bacterial infection of the bone. Delivering antibacterial agents to the bone may overcome the difficulties in treating this illness by effectively concentrating the antibiotic at the site of infection and by limiting the toxicity that may result from systemic exposure to the large doses conventionally used. Using bisphosphonates as osteophilic functional groups, different forms of fluoroquinolone esters were synthesized and evaluated for their ability to bind bone and to release the parent antibacterial agent. Bisphosphonated glycolamide fluoroquinolone esters were found to present a profile consistent with effective and rapid bone binding and efficient release of the active drug moiety. They were assessed for their ability to prevent bone infection in vivo and were found to be effective when the free fluoroquinolones were not.

Overexpression of IL-21 promotes massive CD8+ memory T cell accumulation.

The ability of IL-21 to promote in vitro T cell survival led us to investigate its biological activity in vivo. We report that overexpression of IL-21 in transgenic mice drives CD8(+) memory T cell accumulation with a concomitant reduction in naive T cell numbers. These memory T cells are functional, given their ability to rapidly produce IFN-gamma and proliferate following stimulation. Since the homeostasis of naive and memory T cells is controlled by cytokines, we evaluated whether IL-21 influences cytokine receptor expression. We show that IL-21 inhibits IL-7R expression on naive T cells in vitro, suggesting impaired IL-7-mediated naive T cell survival in IL-21-transgenic mice. In contrast, IL-7R expression on CD4(+) memory T cells is not affected, allowing their IL-7-dependent survival in IL-21-transgenic mice. Although IL-21 decreases IL-7R expression on CD8(+) memory T cells, this has no impact on their survival since their maintenance in the T cell pool is IL-7-independent. Rather, we demonstrate that CD8(+) memory T cells are receptive to IL-21 survival signals allowing for their accumulation in IL-21-transgenic mice. This study identifies new roles for IL-21 in T cell homeostasis and in the regulation of T cell responses to cytokines.

A new class of small molecule RNA polymerase inhibitors with activity against rifampicin-resistant Staphylococcus aureus.

The RNA polymerase holoenzyme is a proven target for antibacterial agents. A high-throughput screening program based on this enzyme from Staphylococcus aureus had previously identified a 2-ureidothiophene-3-carboxylate as a low micromolar inhibitor. An investigation of the relationships between the structures of this class of compounds and their inhibitory- and antibacterial activities is described here, leading to a set of potent RNA polymerase inhibitors with antibacterial activity. Characterization of this bioactivity, including studies of the mechanism of action, is provided, highlighting the power of the reverse chemical genetics approach in providing tools to inhibit the bacterial RNA polymerase.

Triaminotriazine DNA helicase inhibitors with antibacterial activity.

Screening of a chemical library in a DNA helicase assay involving the Pseudomonas aeruginosa DnaB helicase provided a triaminotriazine inhibitor with good antibacterial activity but associated cytotoxicity toward mammalian cells. Synthesis of analogs provided a few inhibitors that retained antibacterial activity and demonstrated a significant reduction in cytotoxicity. The impact of serum and initial investigations toward a mode of action highlight several features of this class of compounds as antibacterials.