TP0586532, a Novel Non-Hydroxamate LpxC Inhibitor: Potentiating Effect on In Vitro Activity of Meropenem against Carbapenem-Resistant Enterobacteriaceae.

Carbapenem-resistant Enterobacteriaceae (CRE) are an urgent threat to public health requiring the development of novel therapies. TP0586532 is a novel non-hydroxamate LpxC inhibitor that inhibits the synthesis of lipopolysaccharides, which are components of the outer membranes of Gram-negative bacteria. Based on the mechanism of action of TP0586532, we hypothesized that it might enhance the antibacterial activity of other antibiotics by increasing the permeability of the outer bacterial membrane. The combination of TP0586532 with meropenem, amikacin, cefepime, piperacillin, and tigecycline showed synergistic and additive effects against carbapenem-susceptible Klebsiella pneumoniae and Escherichia coli. Checkerboard experiments against 21 carbapenem-resistant K. pneumoniae and E. coli strains (13 blaKPC+, 5 blaNDM-1+, 2 blaVIM+, and 1 blaIMP+) showed that the combination of TP0586532 with meropenem yielded synergistic and additive effects against 9 and 12 strains, respectively. In a time-kill assay examining 12 CRE strains, synergistic effects were observed when TP0586532 was combined with meropenem against many of the strains. A membrane permeability assay using ethidium bromide (EtBr) was performed to investigate the mechanism of the potentiating effect. TP0586532 increased the influx of EtBr into a CRE strain, suggesting that TP0586532 increased membrane permeability and facilitated intracellular access for the antibiotics. Our study demonstrates that TP0586532 potentiates the in vitro antibacterial activity of meropenem against CRE. Combination therapy consisting of TP0586532 and meropenem has potential as a treatment for CRE infections. IMPORTANCE Carbapenem-resistant Enterobacteriaceae (CRE) are an urgent public health threat, as therapeutic options are limited. TP0586532 is a novel LpxC inhibitor that inhibits the synthesis of lipopolysaccharides in the outer membranes of Gram-negative bacteria. Here, we demonstrated the potentiating effects of TP0586532 on the antibacterial activity of meropenem against CRE harboring various types of carbapenemase genes (blaKPC+, blaNDM-1+ blaVIM+, and blaIMP+). TP0586532 also augmented the bactericidal effects of meropenem against CRE strains, even against those with a high level of resistance to meropenem. The potentiating effects were suggested to be mediated by an increase in bacterial membrane permeability. Our study revealed that a combination therapy consisting of TP0586532 and meropenem has the potential to be a novel therapeutic option for CRE infections.

Pharmacodynamic target assessment and prediction of clinically effective dosing regimen of TP0586532, a novel non-hydroxamate LpxC inhibitor, using a murine lung infection model.

INTRODUCTION: TP0586532 is a novel non-hydroxamate UDP-3-O-acyl-N-acetylglucosamine deacetylase (LpxC) inhibitor. Pharmacokinetic/pharmacodynamic (PK/PD) indices and magnitude of index that correlated with the efficacy of TP0586532 were determined and used to estimate the clinically effective doses of TP0586532. METHODS: Dose-fractionation studies were conducted using a murine neutropenic lung infection model caused by carbapenem-resistant Enterobacteriaceae. The relationships between the efficacy and the PK/PD index (the maximum unbound plasma concentration divided by the MIC [fCmax/MIC], the area under the unbound plasma concentration-time curve from 0 to 24 h divided by the MIC, and the cumulative percentage of a 24-h period that the unbound plasma concentration exceeds the MIC) were determined using an inhibitory sigmoid maximum-effect model. In addition, the magnitudes of fCmax/MIC were evaluated using the dose-response relationships for each of the seven carbapenem-resistant strains of Enterobacteriaceae. Furthermore, the clinically effective doses of TP0586532 were estimated using the predicted human PK parameters, the geometric mean of fCmax/MIC, and the MIC90 for carbapenem-resistant Klebsiella pneumoniae. RESULTS: The PK/PD index that best correlated with the efficacy was the fCmax/MIC. The geometric means of the fCmax/MIC associated with the net stasis and 1-log reduction endpoints were 2.30 and 3.28, respectively. The clinically effective doses of TP0586532 were estimated to be 1.24-2.74 g/day. CONCLUSION: These results indicate the potential for TP0586532 to have clinical efficacy at reasonable doses against infections caused by carbapenem-resistant Enterobacteriaceae. This study provided helpful information for a clinically effective dosing regimen of TP0586532.

TP0586532, a non-hydroxamate LpxC inhibitor, reduces LPS release and IL-6 production both in vitro and in vivo.

UDP-3-O-acyl-N-acetylglucosamine deacetylase (LpxC) is an essential enzyme in the biosynthesis of Lipid A, an active component of lipopolysaccharide (LPS), from UDP-3-O-acyl-N-acetylglicosamine. LPS is a major component of the cell surface of Gram-negative bacteria. LPS is known to be one of causative factors of sepsis and has been associated with high mortality in septic shock. TP0586532 is a novel non-hydroxamate LpxC enzyme inhibitor. In this study, we examined the inhibitory effect of TP0586532 on the LPS release from Klebsiella pneumoniae both in vitro and in vivo. Our results confirmed the inhibitory effect of TP0586532 on LPS release from the pathogenic bacterial species. On the other hand, meropenem and ciprofloxacin increase the level of LPS release. Furthermore, the effects of TP0586532 on LPS release and interleukin (IL)-6 production in the lung were determined using a murine model of pneumonia caused by K. pneumoniae. As observed in the in vitro study, TP0586532 showed the marked inhibitory effect on LPS release in the lungs, whereas meropenem- and ciprofloxacin-treated mice showed higher levels of LPS release and IL-6 production in the lungs as compared to those in the lungs of vehicle-treated mice. Moreover, TP0586532 used in combination with meropenem and ciprofloxacin attenuated the LPS release and IL-6 production induced by meropenem and ciprofloxacin in the lung. These results indicate that the inhibitory effect of TP0586532 on LPS release from pathogenic bacteria might be of benefit in patients with sepsis.

A spreading roots sign: Characteristic sign of the preliminary stage of medial meniscus posterior root tear on magnetic resonance imaging.

BACKGROUND: Medial meniscus posterior root tears (MMPRTs) can result in the development of osteoarthritis or osteonecrosis. Clinical experience suggests that symptoms such as dull pain or discomfort in the popliteal area or the calf area, which are sometimes misdiagnosed as sciatic nerve pain, may precede impending rupture. We found that bone marrow edema emanating from the meniscal root on magnetic resonance imaging (MRI) scans-spreading roots sign-may indicate the preliminary stage of an MMPRT. The purpose of this study was to evaluate the efficacy of the spreading roots sign as an MMPRT-predictor. METHODS: In a retrospective study, we reviewed the chart data and MRI results of patients who had required surgery for an acute MMPRT. We grouped patients by whether or not they had reported the above-mentioned precursory symptoms prior to acute rupture (precursory symptom group/non-precursor group), and when possible, we examined MRI scans to identify with which events the appearance/disappearance of the spreading roots sign coincided. Sex, age, body mass index, bone mineral density, radiological parameters, and MRI parameters were compared between groups. RESULTS: Data from 24 patients (precursory symptom group, n = 17 [70.8 %]; non-precursor group, n = 7 [29.2 %]) were included; data from 5 patients included MRI scans prior to acute rupture. There were no significant differences between precursory symptom and non-precursor groups, except for the ratio of the presence of the spreading roots sign (p = 0.005). The appearance of the spreading roots sign on MRI scans coincided with the onset of precursory symptoms, and its disappearance coincided with acute rupture and the appearance of other MRI signs typical of MMPRT (white meniscus/truncation/meniscal extrusion/giraffe neck signs). CONCLUSIONS: Our findings suggest that the spreading roots sign can be used as a unique precursory sign for MMPRT.

TP0586532, a non-hydroxamate LpxC inhibitor, has in vitro and in vivo antibacterial activities against Enterobacteriaceae.

The emergence of multi-drug resistant pathogenic bacteria, especially Gram-negative bacteria, is a worldwide health problem. New antibiotics directed at previously unexplored targets are urgently needed to overcome resistance to existing antibiotic classes. UDP-3-O-acyl-N-acetylglucosamine deacetylase (LpxC) is an attractive target for a new antibacterial agent. Although a number of LpxC inhibitors have been identified, none have been approved as antibacterial agents. These LpxC inhibitors contain a hydroxamate moiety, which is a robust zinc ion chelator. The nonspecific inhibition of metalloenzymes through zinc ion chelation is one of possibilities leading to unwanted side effects. Herein, we report that TP0586532, a non-hydroxamate LpxC inhibitor, has a broad spectrum of antibacterial activity against carbapenem-resistant Enterobacteriaceae. The MIC90 of TP0586532 against clinical isolates of carbapenem-resistant Klebsiella pneumoniae was 4 µg ml-1. TP0586532 also showed an in vivo efficacy against murine systemic, urinary tract and lung infection models caused by meropenem- or ciprofloxacin-resistant strains. The estimated maximum unbound plasma concentration value at the effective dose of TP0586532 in murine infection models was around 13 µg ml-1. TP0586532 is predicted to exhibit a in vivo efficacy without cardiovascular toxicity and showed the potential of non-hydroxamate LpxC inhibitors as antibacterial agents against carbapenem-resistant Enterobacteriaceae.

Discovery of a potent dual inhibitor of wild-type and mutant respiratory syncytial virus fusion proteins through the modulation of atropisomer interconversion properties.

The development of effective respiratory syncytial virus (RSV) fusion glycoprotein (F protein) inhibitors against both wild-type and the D486N-mutant F protein is urgently required. We recently reported a 15-membered macrocyclic pyrazolo[1,5-a]pyrimidine derivative 4 that exhibited potent anti-RSV activities against not only wild-type, but also D486N-mutant F protein. However, NMR studies revealed that the 15-membered derivative 4 existed as a mixture of atropisomers. An optimization study of the linker moiety between the 2-position of the benzoyl moiety and the 7-position of the pyrazolo[1,5-a]pyrimidine scaffold identified a 16-membered derivative 42c with an amide linker that showed a rapid interconversion of atropisomers. Subsequent optimization of the 5-position of the pyrazolo[1,5-a]pyrimidine scaffold and the 5-position of the benzoyl moiety resulted in the discovery of a potent clinical candidate 60b for the treatment of RSV infections.

Pharmacological Characterization of TP0591816, a Novel Macrocyclic Respiratory Syncytial Virus Fusion Inhibitor with Antiviral Activity against F Protein Mutants.

Human respiratory syncytial virus (RSV) is a major cause of lower respiratory tract infections in early childhood. However, no vaccines have yet been approved for prevention of RSV infection, and the treatment options are limited. Therefore, development of effective and safe anti-RSV drugs is needed. In this study, we evaluated the antiviral activity and mechanism of action of a novel macrocyclic anti-RSV compound, TP0591816. TP0591816 showed significant antiviral activities against both subgroup A and subgroup B RSV, while exerting no cytotoxicity. Notably, the antiviral activity of TP0591816 was maintained against a known fusion inhibitor-resistant RSV strain with a mutation in the cysteine-rich region or in heptad repeat B. Results of a time-of-addition assay and a temperature shift assay indicated that TP0591816 inhibited fusion of RSV with the cell membrane during viral entry. In addition, TP0591816 added after cell infection also inhibited cell-cell fusion. A TP0591816-resistant virus strain selected by serial passage had an L141F mutation, but no mutation in the cysteine-rich region or in heptad repeat B in the fusion (F) protein. Treatment with TP0591816 reduced lung virus titers in a dose-dependent manner in a mouse model of RSV infection. Furthermore, the estimated effective dose of TP0591816 for use against F protein mutants was thought to be clinically realistic and potentially tolerable. Taken together, these findings suggest that TP0591816 is a promising novel candidate for the treatment of resistant RSV infection.

Discovery of a Potent Dual Inhibitor of Wild-Type and Mutant Respiratory Syncytial Virus Fusion Proteins.

A novel series of macrocyclic pyrazolo[1,5-a]pyrimidine derivatives as respiratory syncytial virus (RSV) fusion glycoprotein (F protein) inhibitors were designed and synthesized based on docking studies of acyclic inhibitors. This effort resulted in the discovery of several macrocyclic compounds, such as 12b, 12f, and 12h, with low nanomolar to subnanomolar activities against the wild-type RSV F protein A2. In addition, 12h showed a single-digit nanomolar potency against the previously reported drug-resistant mutant D486N. Molecular modeling and computational analyses suggested that 12h binds to the D486N mutant while maintaining a rigid bioactive conformation via macrocyclization and that it interacts with a hydrophobic cavity of the mutant using a new interaction surface of 12h. This report describes the rational design of macrocyclic compounds with dual inhibitory activities against wild-type and mutant RSV F proteins.

The effect on subjective quality of life of occupational therapy based on adjusting the challenge-skill balance: a randomized controlled trial.

OBJECTIVE: To verify the effect of adjusting the challenge-skill balance with respect to rehabilitation process. DESIGN: A single-blind, two-arm, parallel-group, randomized controlled trial. SETTING: Recovery rehabilitation unit of Harue Hospital, Japan. SUBJECTS: The trial included 72 clients (mean (SD): age, 74.64 (9.51) years; Functional Independence Measure score, 98.26 (15.27)) with cerebral or spinal disease or musculoskeletal disease. INTERVENTIONS: Clients were randomly divided into two groups: the experimental group, who received occupational therapy with adjustment of the challenge-skill balance, and the control group who received conventional occupational therapy. Time from admission to discharge was considered the implementation period; the final evaluation was conducted at three months after discharge. MAIN MEASURES: The primary outcome was subjective quality of life (Ikigai-9). Secondary outcomes were the health-related quality of life (EuroQol-5 Dimensions, Five Levels (EQ-5D-5L)), the Flow State Scale for Occupational Tasks, and the Functional Independence Measure. A cost-effectiveness analysis was conducted using total cost and quality-adjusted life-year based on the EQ-5D-5L. RESULTS: Significant differences were observed between the experimental and control groups with respect to the Ikigai-9 score (P = 0.008) and EQ-5D-5L (P = 0.038), and the effect sizes were 0.76 (95% confidence interval [CI]: 0.27-1.24) and 0.62 (95% CI: 0.14-1.10), respectively. No significant between-group differences in other outcomes were observed, for example, the Functional Independence Measure score improved in both experimental and control groups (119.80 (5.50) and 118.84 (6.97), respectively. The incremental cost-effectiveness ratio was US$5518.38. CONCLUSIONS: Adjusting the challenge-skill balance may be a useful approach to improve the participant's subjective quality of life in the rehabilitation process.

Effect of adjusting the challenge-skill balance for occupational therapy: study protocol for a randomised controlled trial.

INTRODUCTION: Occupational therapy (OT) is defined as the promotion of client health and well-being through a client-centred practice. However, there is a tendency to rely on the therapist's experiences and values, and there is a difference between the client's and therapist's perceptions regarding the current activity that the client is engaged in. In previous studies that have applied 'flow', activities supported by OT in elderly people were analysed, indicating a difference in recognition. Therefore, we thought that more effective OT could be implemented by adjusting the challenge-skill (ACS) balance, and we invented a novel process termed as ACS balance for OT. The purpose of this study is to verify the effect of ACS-OT on clients in the recovery rehabilitation unit and to prepare a protocol for randomised controlled trial (RCT) implementation. METHOD AND ANALYSIS: This single-blind RCT will recruit 80 clients aged 50-99 years admitted to the recovery rehabilitation unit who meet eligibility criteria. Clients will be randomly allocated to receive ACS-OT or standard OT. Both interventions will be performed during the clients' residence at the unit. The primary outcome measure will be subjective quality of life and will be measured at entry into (pre) and at discharge from (post) the unit and at 3 months afterwards (follow-up). Outcomes will be analysed using a linear mixed model fitted with a maximum likelihood estimation. ETHICS AND DISSEMINATION: This protocol has been approved by the ethics review committee of the Tokyo Metropolitan University (No.17020). Results of this trial will be submitted for publication in a peer-reviewed journal. TRIAL REGISTRATION NUMBER: UMIN-CTR number, UMIN000029505; Pre-results.

Adjusting Challenge-Skill Balance to Improve Quality of Life in Older Adults: A Randomized Controlled Trial.

OBJECTIVE: We sought to investigate whether occupational therapy that includes adjusting the challenge-skill balance improves health-related quality of life (HRQOL) for older adults in comparison with standard occupational therapy. METHOD: In this single-blind, randomized controlled trial, 56 older adults were assigned to two groups that received 10 sessions of occupational therapy with and without adjustment of challenge-skill balance. The primary outcome was change in HRQOL after 10 sessions of occupational therapy. RESULTS: Significant differences were observed in HRQOL using the EuroQol-5 Dimension score (p = .022, d = 0.76) and the eight-item Short-Form Health Survey scores for general health (p = .001, d = 0.99) and in flow experience using the Flow State Scale for Occupational Tasks (p = .008, d = 0.82). CONCLUSION: Assessment and adjustment of the challenge-skill balance of activities may effectively improve older adults' HRQOL.

In vitro antibacterial activity of α-methoxyimino acylide derivatives against macrolide-resistant pathogens and mutation analysis in 23S rRNA.

We characterized in vitro activities of α-methoxyimino acylides against macrolide-resistant clinical isolates of Streptococcus pneumoniae, Streptococcus pyogenes and Mycoplasma pneumoniae with ribosome modification or substitution and selected acylide-resistant mutants to clarify the binding point of the acylides. The acylides had low MICs against erm(B) gene-containing S. pneumoniae and S. pyogenes (MIC90s, 1-4 µg ml-1). For M. pneumoniae, although they had poor potencies against macrolide-resistant strains with the A2058G (Escherichia coli numbering) mutation in 23S rRNA (MICs, >32 µg ml-1), one of them showed in vitro activities against macrolide-resistant strains with the A2058U or A2059G mutations (MICs, 0.5-1 µg ml-1). These A2058U and A2059G mutant strains were used for the selection of acylide-resistant mutants. A genetic analysis showed that new point mutations in acylide-resistant mutants were found at G2576 in domain V of 23S rRNA and at Lys90 in L22 ribosomal protein. Furthermore, a molecular modeling study revealed that G2505/C2610, which enables stacking with G2576, might interact with a pyridyl moiety or an α-methoxyimino group at the 3-position of acylides. The α-methoxyimino acylides were shown to possess a tertiary binding point at G2505/C2610 in 23S rRNA. Our results suggest that α-methoxyimino acylides represent significant progress in macrolide antimicrobials.