Pharmacokinetic and Preclinical Safety Studies of Endolysin-Based Therapeutic for Intravenous Administration.

Pharmacokinetics and safety studies of innovative drugs is an essential part of drug development process. Previously we have developed a novel drug for intravenous administration (lyophilizate) containing modified endolysin LysECD7-SMAP that showed notable antibacterial effect in different animal models of systemic infections. Here we present data on pharmacokinetics of endolysin in mice after single and multiple injections. Time-concentration curves were obtained, and pharmacokinetic parameters for preparation (C0, kel t1/2, AUC0-∞, MRT, ClT, Vss) were calculated. It was shown that although endolysin is rather short-lived in blood serum (t1/2 = 12.5 min), the therapeutic concentrations of LysECD7-SMAP (in degraded and non-degraded form) were detected for 60 minutes after injection that is sufficient for antibacterial effect. Based on the obtained data, it was proposed that endolysin distributes presumably in murine blood, degrades in blood and liver, and is eliminated via glomerular filtration. Safety profile of the preparation relating to general toxicity, immunotoxicity and allergenicity was assessed in rodents. It was demonstrated that LysECD7-SMAP in potential therapeutic (12.5 mg/kg), 10-fold (125 mg/kg) and 40-fold (500 mg/kg) doses showed no signs of intoxication and significant abnormalities after single and repeated i.v. administrations, preparation was non-immunogenic and induced minor and reversible allergic reaction in animals.

Development of novel antimicrobials with engineered endolysin LysECD7-SMAP to combat Gram-negative bacterial infections.

BACKGROUND: Among the non-traditional antibacterial agents in development, only a few targets critical Gram-negative bacteria such as carbapenem-resistant Pseudomonas aeruginosa, Acinetobacter baumannii or cephalosporin-resistant Enterobacteriaceae. Endolysins and their genetically modified versions meet the World Health Organization criteria for innovation, have a novel mode of antibacterial action, no known bacterial cross-resistance, and are being intensively studied for application against Gram-negative pathogens. METHODS: The study presents a multidisciplinary approach, including genetic engineering of LysECD7-SMAP and production of recombinant endolysin, its analysis by crystal structure solution following molecular dynamics simulations and evaluation of antibacterial properties. Two types of antimicrobial dosage forms were formulated, resulting in lyophilized powder for injection and hydroxyethylcellulose gel for topical administration. Their efficacy was estimated in the treatment of sepsis, and pneumonia models in BALB/c mice, diabetes-associated wound infection in the leptin receptor-deficient db/db mice and infected burn wounds in rats. RESULTS: In this work, we investigate the application strategies of the engineered endolysin LysECD7-SMAP and its dosage forms evaluated in preclinical studies. The catalytic domain of the enzyme shares the conserved structure of endopeptidases containing a putative antimicrobial peptide at the C-terminus of polypeptide chain. The activity of endolysins has been demonstrated against a range of pathogens, such as Klebsiella pneumoniae, A. baumannii, P. aeruginosa, Staphylococcus haemolyticus, Achromobacter spp, Burkholderia cepacia complex and Haemophylus influenzae, including those with multidrug resistance. The efficacy of candidate dosage forms has been confirmed in in vivo studies. Some aspects of the interaction of LysECD7-SMAP with cell wall molecular targets are also discussed. CONCLUSIONS: Our studies demonstrate the potential of LysECD7-SMAP therapeutics for the systemic or topical treatment of infectious diseases caused by susceptible Gram-negative bacterial species and are critical to proceed LysECD7-SMAP-based antimicrobials trials to advanced stages.

Pharmacokinetics and Preclinical Safety Studies of Modified Endolysin-based Gel for Topical Application.

Antibacterial therapy with phage-encoded endolysins or their modified derivatives with improved antibacterial, biochemical and pharmacokinetic properties is one of the most promising strategies that can supply existing antibacterial drugs array. Gram-negative bacteria-induced infections treatment is especially challenging because of rapidly spreading bacterial resistance. We have developed modified endolysin LysECD7-SMAP with a significant antibacterial activity and broad spectra of action against gram-negative bacteria. Endolysin was formulated in a bactericidal gel for topical application with pronounced effectivity in local animal infectious models. Here we present preclinical safety studies and pharmacokinetics of LysECD7-SMAP-based gel. We have detected LysECD7-SMAP in the skin and underlying muscle at therapeutic concentrations when the gel is applied topically to intact or injured skin. Moreover, the protein does not enter the bloodstream, and has no systemic bioavailability, assuming no systemic adverse effects. In studies of general toxicology, local tolerance, and immunotoxicology it was approved that LysECD7-SMAP gel local application results in the absence of toxic effects after single and multiple administration. Thus, LysECD7-SMAP-containing gel has appropriate pharmacokinetics and can be considered as safe that supports the initiation of the phase I clinical trials of novel antibacterial drug intending to treat acute wound infections caused by resistant gram-negative bacteria.

Discovering the Potentials of Four Phage Endolysins to Combat Gram-Negative Infections.

Endolysin-based therapeutics are promising antibacterial agents and can successfully supplement the existing antibacterial drugs array. It is specifically important in the case of Gram-negative pathogens, e.g., ESKAPE group bacteria, which includes Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter species, and are highly inclined to gain multiple antibiotic resistance. Despite numerous works devoted to the screening of new lytic enzymes and investigations of their biochemical properties, there are significant breaches in some aspects of their operating characteristics, including safety issues of endolysin use. Here, we provide a comprehensive study of the antimicrobial efficacy aspects of four Gram-negative bacteria-targeting endolysins LysAm24, LysAp22, LysECD7, and LysSi3, their in vitro and in vivo activity, and their biological safety. These endolysins possess a wide spectrum of action, are active against planktonic bacteria and bacterial biofilms, and are effective in wound and burn skin infection animal models. In terms of safety, these enzymes do not contribute to the development of short-term resistance, are not cytotoxic, and do not significantly affect the normal intestinal microflora in vivo. Our results provide a confident base for the development of effective and safe candidate dosage forms for the treatment of local and systemic infections caused by Gram-negative bacterial species.

Efficacy of the Endolysin-Based Antibacterial Gel for Treatment of Anaerobic Infection Caused by Fusobacterium necrophorum.

Abscess formation is a common complication of severe life-threatening infections caused by obligate anaerobes. Fusobacterium necrophorum is among the frequently detected anaerobic pathogens from clinical specimens associated with liver abscesses, skin and soft tissue infections, or oral abscesses. The antimicrobial therapy for this kind of infection needs to be optimized. Here, we examined the possibility of treating F. necrophorum-induced abscess wound infections with candidate therapeutics based on three endolysins with activity against a broad spectrum of aerobe Gram-negative pathogens. Antibacterial gel containing three Gram-negative bacteria-targeting endolysins, LysAm24, LysAp22, and LysECD7, was formulated for topical use. Abscess formation was induced in rabbits with F. necrophorum and caused systemic infection. The survival and lifespan of the animals, general parameters, and biochemical and hematological blood tests were analyzed to assess the effectiveness of the gel treatment for the wound infection. The administration of the investigated gel twice per day for 5 days resulted in less acute inflammation, with decreased leukocytes and segmented neutrophils in the blood, retardation of infection progression, and an almost two-fold increase in the lifespan of the animals compared to the placebo group. The results indicate that endolysin-based therapy is an effective approach to treat anaerobic bacterial infections. The use of endolysins as independent pharmaceuticals, or their combination with antibiotics, could significantly reduce the development of complications in infectious diseases caused by sensitive bacterial species.