Agnostics GmbH, Mannheim, Germany), in accordance with the manufacturer’s JNJ-42253432 custom synthesis guidelines. Animals: All animal studies had been performed applying male ICR mice (25 two gr). The Technion Animal Care and Use committee authorized all procedures, care, and handling of animals. Ethics approval codes: IL0800519, IL0640421, IL0550618, IL1811217. The maximum tolerated dose (MTD) was GNF6702 Cancer determined following a single-dose subcutaneous (S.C) administration of C14(5) OOc10 O working with 3 mice per dose. Animals had been monitored for adverse effects for 7 days immediately after injection. For efficacy assessments, 3 infection models were applied such as a single with topical therapy and two with systemic therapy. 1. Excisional skin wound infection model: mice have been anesthetized by intraperitoneal administration of a mixture of ketamine 100 mg/kg and xylazine 5 mg/kg in PBS and their backs shaved with electric clippers. The following day mice had been similarlyPharmaceutics 2021, 13,four of2.3.anesthetized and were administrated (S.C) 0.05 mg/kg buprenorphine (for discomfort relief). A five mm diameter piece of skin was removed in the middle in the shaved back, with sterile biopsy punch resulting within a full-thickness injury. A total of 20 of a mid-logarithmic culture, containing 5 106 CFU P. aeruginosa 27853 were applied around the wound. Then, 15 min right after inoculation, about 50 of hypromellose gel (prepared as described [43]) containing OAC, antibiotic, or their mixture had been applied around the skin and covered using a piece of health-related tape. As a manage, the car (drug-free gel) was similarly applied on the skin. After a remedy period of four h, about 8 mm diameter of skin surrounding the infected region was removed, suspended in PBS, homogenized, serially diluted 10-fold, and plated for CFU enumeration. The amount of viable bacteria was determined soon after overnight incubation at 37 C. The lower limit of detection was 50 CFU/wound. Thigh infection model (TI): mice had been inoculated intramuscularly with 1 106 CFU/mouse of mid-logarithmic E. coli 25922 and treated subcutaneously 1 and three h right after inoculation. Mice had been sacrificed 24 h soon after infection, their thighs excised, homogenized, serially diluted 10-fold, and plated for CFU enumeration. The amount of viable bacteria was determined soon after overnight incubation at 37 C. The decrease limit of detection was 50 CFU/thigh. Urinary tract infection model (UTI): mice had been anesthetized by means of intraperitoneal injection of one hundred mg/kg ketamine and five mg/kg xylazine. Mice penises were lubricated with an analgesic 2 lidocaine gel. Then, mice have been infected with 50 of 1 108 CFU/mouse of E. coli UPEC CFT073, administrated by an intra-urethral injection applying a catheter (24 GA, 0.156 IN, 0.7 14 mm). Mice have been subcutaneously treated with OAC at 1 and 6 h post infection. Mice had been sacrificed 24 h post inoculation, the bladder and kidneys had been excised, homogenized, serially diluted 10-fold, and plated for CFU enumeration. The number of viable bacteria was determined after overnight incubation at 37 C. The reduced limit of detection was 50 CFU/organ.Blood Circulating Concentrations and Organ Bio-Distribution of C14(five) OOc10 O C14(5) OOc10 O was subjected to preliminary pharmacokinetics (PK) analysis to determine its plasma concentrations or organ bio-distribution following S.C administration to non-neutropenic pathogen-free mice. OAC quantification was performed by LC-MS as follows: blood was drawn at several time intervals and plasma was separated by centrifugation (5000 RCF, ten min).
