Evaluation of the In Vitro Activity of Eravacycline against a Broad Spectrum of Recent Clinical Anaerobic Isolates.

The novel fluorocycline antibiotic eravacycline is in development for use in the treatment of serious infections caused by susceptible and multidrug-resistant (MDR) aerobic and anaerobic Gram-negative and Gram-positive pathogens. Eravacycline and 11 comparator antibiotics were tested against recent anaerobic clinical isolates, including MDR Bacteroides spp. and Clostridium difficile Eravacycline was potent in vitro against all the isolates tested, including strains with tetracycline-specific resistance determinants and MDR anaerobic pathogens resistant to carbapenems and/or ß-lactam-ß-lactamase inhibitor combinations.

Heterocyclyl tetracyclines. 2. 7-Methoxy-8-pyrrolidinyltetracyclines: discovery of TP-2758, a potent, orally efficacious antimicrobial against Gram-negative pathogens.

A convergent total synthesis platform led to the discovery of TP-2758 from a series of novel 7-methoxy-8-heterocyclyl tetracycline analogs. TP-2758 demonstrated high in vitro potency against key Gram-negative pathogens including extended spectrum ß-lactamases- and carbapenemase-producing Enterobacteriaceae and Acinetobacter spp. strains. This compound was efficacious when administered either intravenously or orally in multiple murine infection models and displayed a favorable preclinical pharmacological profile supporting its advancement into clinical development.

In-vitro activity of the novel fluorocycline eravacycline against carbapenem non-susceptible Acinetobacter baumannii.

The activity of eravacycline was compared with that of anti-Acinetobacter reference antimicrobials against carbapenem non-susceptible Acinetobacter baumannii isolates associated with an acquired OXA or up-regulation of the intrinsic OXA-51-like enzyme. Antimicrobial susceptibility testing was performed by broth microdilution of 286 non-duplicate, carbapenem non-susceptible A. baumannii isolates to eravacycline, amikacin colistin, doxycycline, imipenem, levofloxacin, meropenem, minocycline, sulbactam, tigecycline and tobramycin. Eravacycline showed greater activity than the comparators of the tetracycline class, levofloxacin, amikacin, tobramycin and colistin. The eravacycline MIC50/90 values were 0.5/1 mg/L and those for tigecycline, minocycline and doxycycline were 1/2, 4/8 and 32/ ≥ 64 mg/L, respectively. In conclusion, eravacycline was the most potent antibiotic of those tested against A. baumannii, including isolates that were resistant to sulbactam, imipenem/meropenem, levofloxacin and amikacin/tobramycin. Eravacycline has the potential to become a useful addition to the limited armamentarium of drugs that can be used to treat this problem pathogen.

The Fluorocycline TP-271 Is Efficacious in Models of Aerosolized Bacillus anthracis Infection in BALB/c Mice and Cynomolgus Macaques.

The fluorocycline TP-271 was evaluated in mouse and nonhuman primate (NHP) models of inhalational anthrax. BALB/c mice were exposed by nose-only aerosol to Bacillus anthracis Ames spores at a level of 18 to 88 lethal doses sufficient to kill 50% of exposed individuals (LD50). When 21 days of once-daily dosing was initiated at 24 h postchallenge (the postexposure prophylaxis [PEP] study), the rates of survival for the groups treated with TP-271 at 3, 6, 12, and 18 mg/kg of body weight were 90%, 95%, 95%, and 84%, respectively. When 21 days of dosing was initiated at 48 h postchallenge (the treatment [Tx] study), the rates of survival for the groups treated with TP-271 at 6, 12, and 18 mg/kg TP-271 were 100%, 91%, and 81%, respectively. No deaths of TP-271-treated mice occurred during the 39-day posttreatment observation period. In the NHP model, cynomolgus macaques received an average dose of 197 LD50 of B. anthracis Ames spore equivalents using a head-only inhalation exposure chamber, and once-daily treatment of 1 mg/kg TP-271 lasting for 14 or 21 days was initiated within 3 h of detection of protective antigen (PA) in the blood. No (0/8) animals in the vehicle control-treated group survived, whereas all 8 infected macaques treated for 21 days and 4 of 6 macaques in the 14-day treatment group survived to the end of the study (56 days postchallenge). All survivors developed toxin-neutralizing and anti-PA IgG antibodies, indicating an immunologic response. On the basis of the results obtained with the mouse and NHP models, TP-271 shows promise as a countermeasure for the treatment of inhalational anthrax.

The Fluorocycline TP-271 Is Efficacious in Models of Aerosolized Francisella tularensis SCHU S4 Infection in BALB/c Mice and Cynomolgus Macaques.

TP-271 is a novel, fully synthetic fluorocycline in development for complicated bacterial respiratory infections. TP-271 was active in vitro against a panel of 29 Francisella tularensis isolates, showing MICs against 50% and 90% of isolates of 0.25 and 0.5 µg/ml, respectively. In a mouse model of inhalational tularemia, animals were exposed by aerosol to 91 to 283 50% lethal doses (LD50)/mouse of F. tularensis SCHU S4. Following 21 days of once-daily intraperitoneal dosing with TP-271 at 3, 6, 12, and 18 mg/kg of body weight/day, initiating at 24 h postchallenge, survival was 80%, 100%, 100%, and 100%, respectively. When treatment was initiated at 72 h postchallenge, survival was 89%, 100%, 100%, and 100% in the 3-, 6-, 12-, and 18-mg/kg/day TP-271 groups, respectively. No mice treated with the vehicle control survived. Surviving mice treated with TP-271 showed little to no relapse during 14 days posttreatment. In a nonhuman primate model of inhalational tularemia, cynomolgus macaques received an average aerosol exposure of 1,144 CFU of F. tularensis SCHU S4. Once-daily intravenous infusion with 1 or 3 mg/kg TP-271, or vehicle control, for 21 days was initiated within 6 h of confirmed fever. All animals treated with TP-271 survived to the end of the study, with no relapse during 14 days after the last treatment, whereas no vehicle control-treated animals survived. The protection and low relapse afforded by TP-271 treatment in these studies support continued investigation of TP-271 for use in the event of aerosolized exposure to F. tularensis.

Fluorocycline TP-271 Is Potent against Complicated Community-Acquired Bacterial Pneumonia Pathogens.

TP-271 is a novel, fully synthetic fluorocycline antibiotic in clinical development for the treatment of respiratory infections caused by susceptible and multidrug-resistant pathogens. TP-271 was active in MIC assays against key community respiratory Gram-positive and Gram-negative pathogens, including Streptococcus pneumoniae (MIC90 = 0.03 µg/ml), methicillin-sensitive Staphylococcus aureus (MSSA; MIC90 = 0.25 µg/ml), methicillin-resistant S. aureus (MRSA; MIC90 = 0.12 µg/ml), Streptococcus pyogenes (MIC90 = 0.03 µg/ml), Haemophilus influenzae (MIC90 = 0.12 µg/ml), and Moraxella catarrhalis (MIC90 ≤0.016 µg/ml). TP-271 showed activity (MIC90 = 0.12 µg/ml) against community-acquired MRSA expressing Panton-Valentine leukocidin (PVL). MIC90 values against Mycoplasma pneumoniae, Legionella pneumophila, and Chlamydia pneumoniae were 0.004, 1, and 4 µg/ml, respectively. TP-271 was efficacious in neutropenic and immunocompetent animal pneumonia models, generally showing, compared to the burden at the start of dosing, ~2 to 5 log10 CFU reductions against MRSA, S. pneumoniae, and H. influenzae infections when given intravenously (i.v.) and ~1 to 4 log10 CFU reductions when given orally (p.o.). TP-271 was potent against key community-acquired bacterial pneumonia (CABP) pathogens and was minimally affected, or unaffected, by tetracycline-specific resistance mechanisms and fluoroquinolone or macrolide drug resistance phenotypes. IMPORTANCE Rising resistance rates for macrolides, fluoroquinolones, and ß-lactams in the most common pathogens associated with community-acquired bacterial pneumonia (CABP) are of concern, especially for cases of moderate to severe infections in vulnerable populations such as the very young and the elderly. New antibiotics that are active against multidrug-resistant Streptococcus pneumoniae and Staphylococcus aureus are needed for use in the empirical treatment of the most severe forms of this disease. TP-271 is a promising new fluorocycline antibiotic demonstrating in vitro potency and nonclinical efficacy by intravenous and oral administration against the major pathogens associated with moderate to severe CABP.

Heterocyclyl Tetracyclines. 1. 7-Trifluoromethyl-8-Pyrrolidinyltetracyclines: Potent, Broad Spectrum Antibacterial Agents with Enhanced Activity against Pseudomonas aeruginosa.

Utilizing a total synthesis approach, the first 8-heterocyclyltetracyclines were designed, synthesized, and evaluated against panels of tetracycline- and multidrug-resistant Gram-positive and Gram-negative pathogens. Several compounds with balanced, highly potent in vitro activity against a broad range of bacterial isolates were identified through structure-activity relationships (SAR) studies. One compound demonstrated the best antibacterial activity against Pseudomonas aeruginosa both in vitro and in vivo for tetracyclines reported to date.

Assessing the Efficacy and Safety of Eravacycline vs Ertapenem in Complicated Intra-abdominal Infections in the Investigating Gram-Negative Infections Treated With Eravacycline (IGNITE 1) Trial: A Randomized Clinical Trial.

Importance: Eravacycline is a novel, fully synthetic fluorocycline antibiotic of the tetracycline class with in vitro activity against clinically important gram-negative, gram-positive aerobic, and facultative bacteria including most of those resistant to cephalosporins, fluoroquinolones, ß-lactam/ß-lactamase inhibitors, multidrug resistant strains and carbapenem-resistant Enterobacteriaceae, and most anaerobic pathogens. Objective: To evaluate the efficacy and safety of eravacycline compared with ertapenem in adult hospitalized patients with complicated intra-abdominal infections (cIAIs). Design, Setting, and Participants: This was a phase III, randomized, double-blind, multicenter study that evaluated the efficacy and safety of eravacycline in comparison with ertapenem in patients with cIAI requiring surgical or percutaneous intervention. The test-of-cure evaluation was conducted 25 to 31 days after the first dose of the study drug and the follow-up visit was conducted 38 to 50 days after the first dose of the study drug. All patients recruited into this study were hospitalized. Five hundred forty-one patients were recruited for this study; 270 patients were randomized to receive eravacycline, and 271 patients were randomized to receive ertapenem. Patients had to meet all of the following criteria: hospitalized for cIAI requiring intervention; 18 years or older; evidence of systemic inflammatory response; pain caused by cIAI; able to provide informed consent; and diagnosis of cIAI with sonogram or radiographic imaging or visual confirmation. Analyses were done in intent-to-treat and evaluable populations. Interventions: Patients received eravacycline, 1.0 mg/kg every 12 hours, or ertapenem, 1.0 g every 24 hours, for a minimum of four 24-hour dosing cycles. Main Outcomes and Measures: Clinical outcome assessments were made at the end of treatment, test of cure, and follow-up visits and were classified as clinical cure, clinical failure, or indeterminate/missing. Results: In total, 541 patients were randomly assigned to treatment: 270 in the eravacycline group and 271 in the ertapenem group. The mean ages were 54.9 years and 55.4 years for the eravacycline and ertapenem groups, respectively. Most patients were white (263 of 270 patients [97.4%] in the eravacycline group and 260 of 271 patients [95.9%] in the ertapenem group). For the microbiological intent-to-treat population, the rates of clinical cure at the test-of-cure visit were 86.8% in the eravacycline group and 87.6% in the ertapenem group. The difference in clinical cure rates between the groups was -0.80% (95% CI, -7.1% to 5.5%), meeting the prespecified noninferiority margin and allowing for statistical noninferiority of eravacycline to ertapenem to be declared for this study. Both study drugs were well tolerated. Conclusions and Relevance: Overall, eravacycline demonstrated noninferiority to ertapenem for the treatment of patients with cIAI. Trial Registration: Clinicaltrials.gov Identifier: NCT01844856.

Design, Synthesis, and Biological Evaluation of Hexacyclic Tetracyclines as Potent, Broad Spectrum Antibacterial Agents.

A series of novel hexacyclic tetracycline analogues ("hexacyclines") was designed, synthesized, and evaluated for antibacterial activity against a wide range of clinically important bacteria isolates, including multidrug-resistant, Gram-negative pathogens. Valuable structure-activity relationships were identified, and several hexacyclines displayed potent, broad spectrum antibacterial activity, including promising anti-Pseudomonas aeruginosa activity in vitro and in vivo.

Eravacycline (TP-434) is efficacious in animal models of infection.

Eravacycline is a novel broad-spectrum fluorocycline antibiotic being developed for a wide range of serious infections. Eravacycline was efficacious in mouse septicemia models, demonstrating 50% protective dose (PD50) values of ≤ 1 mg/kg of body weight once a day (q.d.) against Staphylococcus aureus, including tetracycline-resistant isolates of methicillin-resistant S. aureus (MRSA), and Streptococcus pyogenes. The PD50 values against Escherichia coli isolates were 1.2 to 4.4 mg/kg q.d. In neutropenic mouse thigh infection models with methicillin-sensitive S. aureus (MSSA) and S. pyogenes, eravacycline produced 2 log10 reductions in CFU at single intravenous (i.v.) doses ranging from 0.2 to 9.5 mg/kg. In a neutropenic mouse lung infection model, eravacycline administered i.v. at 10 mg/kg twice a day (b.i.d.) reduced the level of tetracycline-resistant MRSA in the lung equivalent to that of linezolid given orally (p.o.) at 30 mg/kg b.i.d. At i.v. doses of 3 to 12 mg/kg b.i.d., eravacycline was more efficacious against tetracycline-resistant Streptococcus pneumoniae in a neutropenic lung infection model than linezolid p.o. at 30 mg/kg b.i.d. Eravacycline showed good efficacy at 2 to 10 mg/kg i.v. b.i.d., producing up to a 4.6 log10 CFU reduction in kidney bacterial burden in a model challenged with a uropathogenic E. coli isolate. Eravacycline was active in multiple murine models of infection against clinically important Gram-positive and Gram-negative pathogens.

In vitro susceptibility testing of eravacycline is unaffected by medium age and nonstandard assay parameters.

Eravacycline is a fluorocycline antibiotic in phase 3 clinical development for complicated intra-abdominal and urinary tract infections. To support its clinical development, a study was conducted to evaluate the effects of various susceptibility test parameters on the MIC values for aerobic bacteria. The results showed that eravacycline appears to be largely unaffected by medium age, medium additives, and other nonstandard assay conditions.

Eravacycline (TP-434) is active in vitro against biofilms formed by uropathogenic Escherichia coli.

Eravacycline (formerly TP-434) was evaluated in vitro against pre-established biofilms formed by a uropathogenic Escherichia coli strain. Biofilms were eradicated by 0.5 µg/ml eravacycline, which was within 2-fold of the MIC for planktonic cells. In contrast, colistin and meropenem disrupted biofilms at 32 and 2 µg/ml, respectively, concentrations well above their respective MICs of 0.5 and 0.03 µg/ml. Gentamicin and levofloxacin eradicated biofilms at concentrations within 2-fold of their MICs.

Phase 2, randomized, double-blind study of the efficacy and safety of two dose regimens of eravacycline versus ertapenem for adult community-acquired complicated intra-abdominal infections.

Eravacycline is a novel fluorocycline, highly active against Gram-positive and Gram-negative pathogens in vitro, including those with tetracycline and multidrug resistance. This phase 2, randomized, double-blind study was conducted to evaluate the efficacy and safety of two dose regimens of eravacycline compared with ertapenem in adult hospitalized patients with complicated intra-abdominal infections (cIAIs). Patients with confirmed cIAI requiring surgical or percutaneous intervention and antibacterial therapy were randomized (2:2:1) to receive eravacycline at 1.5 mg/kg of body weight every 24 h (q24h), eravacycline at 1.0 mg/kg every 12 h (q12h), or ertapenem at 1 g (q24h) for a minimum of 4 days and a maximum of 14 days. The primary efficacy endpoint was the clinical response in microbiologically evaluable (ME) patients at the test-of-cure (TOC) visit 10 to 14 days after the last dose of study drug therapy. Overall, 53 patients received eravacycline at 1.5 mg/kg q24h, 56 received eravacycline at 1.0 mg/kg q12h, and 30 received ertapenem. For the ME population, the clinical success rate at the TOC visit was 92.9% (39/42) in the group receiving eravacycline at 1.5 mg/kg q24h, 100% (41/41) in the group receiving eravacycline at 1.0 mg/kg q12h, and 92.3% (24/26) in the ertapenem group. The incidences of treatment-emergent adverse events were 35.8%, 28.6%, and 26.7%, respectively. Incidence rates of nausea and vomiting were low in both eravacycline groups. Both dose regimens of eravacycline were as efficacious as the comparator, ertapenem, in patients with cIAI and were well tolerated. These results support the continued development of eravacycline for the treatment of serious infections, including those caused by drug-resistant Gram-negative pathogens. (This study has been registered at ClinicalTrials.gov under registration no. NCT01265784.).

Synthesis and biological evaluation of 8-aminomethyltetracycline derivatives as novel antibacterial agents.

The C-8 position of the tetracyclines has been largely underexplored because of limitations in traditional semisynthetic techniques. Employing a total synthetic approach allowed for modifications at the C-7 and C-8 positions, enabling the generation of structure-activity relationships for overcoming the two most common tetracycline bacterial-resistance mechanisms: ribosomal protection (tet(M)) and efflux (tet(A)). Ultimately, several compounds were identified with balanced activity against both Gram-positive and Gram-negative bacteria, including pathogens bearing both types of tetracycline-resistance mechanisms. Compounds were screened in a murine systemic infection model to rapidly identify compounds with oral bioavailability, leading to the discovery of several compounds that exhibited efficacy when administered orally in murine pyelonephritis and pneumonia models.

Development and characterization of a Pseudomonas aeruginosa in vitro coupled transcription-translation assay system for evaluation of translation inhibitors.

Bacterial transcription and translation have proven to be effective targets for broad-spectrum antimicrobial therapies owing to the critical role they play in bacterial propagation and the overall conservation of the associated machinery involved. Escherichia coli is the most common source of S30 extract used in bacterial in vitro coupled transcription-translation assays, however, transcription-translation assays in other important pathogens including Staphylococcus aureus and Streptococcus pneumoniae have been described (Murray et al., 2001; Dandliker et al., 2003). Pseudomonas aeruginosa is an important and difficult-to-treat Gram-negative pathogen. In a drug discovery program, to de-risk any potential species specificity of novel inhibitors, we developed and optimized a robust method for the preparation of S30 extract from P. aeruginosa strain PAO1. Further, a P. aeruginosa transcription-translation assay using a firefly luciferase reporter plasmid was validated and compared to an E. coli S30-based system using a wide range of antibiotics encompassing multiple classes of translation inhibitors. Results showed a similar ranking of the activities of known inhibitors, illustrative of the high degree of conservation between the transcription-translation pathways in both organisms.

Target- and resistance-based mechanistic studies with TP-434, a novel fluorocycline antibiotic.

TP-434 is a novel, broad-spectrum fluorocycline antibiotic with activity against bacteria expressing major antibiotic resistance mechanisms, including tetracycline-specific efflux and ribosomal protection. The mechanism of action of TP-434 was assessed using both cell-based and in vitro assays. In Escherichia coli cells expressing recombinant tetracycline resistance genes, the MIC of TP-434 (0.063 µg/ml) was unaffected by tet(M), tet(K), and tet(B) and increased to 0.25 and 4 µg/ml in the presence of tet(A) and tet(X), respectively. Tetracycline, in contrast, was significantly less potent (MIC ≥ 128 µg/ml) against E. coli cells when any of these resistance mechanisms were present. TP-434 showed potent inhibition in E. coli in vitro transcription/translation (50% inhibitory concentration [IC(50)] = 0.29 ± 0.09 µg/ml) and [(3)H]tetracycline ribosome-binding competition (IC(50) = 0.22 ± 0.07 µM) assays. The antibacterial potencies of TP-434 and all other tetracycline class antibiotics tested were reduced by 4- to 16-fold, compared to that of the wild-type control strain, against Propionibacterium acnes strains carrying a 16S rRNA mutation, G1058C, a modification that changes the conformation of the primary binding site of tetracycline in the ribosome. Taken together, the findings support the idea that TP-434, like other tetracyclines, binds the ribosome and inhibits protein synthesis and that this activity is largely unaffected by the common tetracycline resistance mechanisms.

Fluorocyclines. 1. 7-fluoro-9-pyrrolidinoacetamido-6-demethyl-6-deoxytetracycline: a potent, broad spectrum antibacterial agent.

This and the accompanying report (DOI: 10.1021/jm201467r ) describe the design, synthesis, and evaluation of a new generation of tetracycline antibacterial agents, 7-fluoro-9-substituted-6-demethyl-6-deoxytetracyclines ("fluorocyclines"), accessible through a recently developed total synthesis approach. These fluorocyclines possess potent antibacterial activities against multidrug resistant (MDR) Gram-positive and Gram-negative pathogens. One of the fluorocyclines, 7-fluoro-9-pyrrolidinoacetamido-6-demethyl-6-deoxytetracycline (17j, also known as TP-434, 50th Interscience Conference on Antimicrobial Agents and Chemotherapy Conference , Boston, MA , September 12-15, 2010 , poster F1 - 2157 ), is currently undergoing phase 2 clinical trials in patients with complicated intra-abdominal infections (cIAI).

Fluorocyclines. 2. Optimization of the C-9 side-chain for antibacterial activity and oral efficacy.

Utilizing a fully synthetic route to tetracycline analogues, the C-9 side-chain of the fluorocyclines was optimized for both antibacterial activity and oral efficacy. Compounds were identified that overcome both efflux (tet(K), tet(A)) and ribosomal protection (tet(M)) tetracycline-resistance mechanisms and are active against Gram-positive and Gram-negative organisms. A murine systemic infection model was used as an oral efficacy screen to rapidly identify compounds with oral bioavailability. Two compounds were identified that exhibit both oral bioavailability in rat and clinically relevant bacterial susceptibility profiles against major respiratory pathogens. One compound demonstrated oral efficacy in rodent lung infection models that was comparable to marketed antibacterial agents.