Discovery and evaluation of novel FAAH inhibitors in neuropathic pain model

Conceptual design and modification of urea moiety in chemotype PF-3845/04457845, the bench marking irreversible inhibitor of fatty acid amide hydrolase (FAAH), led to discovery of a novel nicotinamide-based lead 12a having reversible mechanism of action. Focused SAR around the pyridine heterocycle (Ar) in 12a (Tables 1 and 2) resulted into four shortlisted compounds, (?)-12a, (?)-12i, (?)-12l–m. The required (?)-enantiomers were obtained via diastereomeric resolution of a novel chiral dissymmetric intermediate 15. Based on comparative profile of FAAH potency, metabolic stability in liver microsome, liability of inhibiting major hCYP450 isoforms, rat PK, and brain penetration ability, two SAR optimized compounds, (?)-12l and (?)-12m, were selected for efficacy study in rat model of chemotherapy-induced peripheral neuropathy (CIPN). Both the compounds exhibited dose related antihyperalgesic effects, when treated with 3–30?mg/kg po for 7?days. The effects at 30?mg/kg are comparable to that of PF-04457845 (10?mg/kg) and Tramadol (40?mg/kg).     

Patellofemoral cartilage stresses are most sensitive to variations in vastus medialis muscle forces

The purpose of this study was to evaluate the effects of variations in quadriceps muscle forces on patellofemoral stress. We created subject-specific finite element models for 21 individuals with chronic patellofemoral pain and 16 pain-free control subjects. We extracted three-dimensional geometries from high resolution magnetic resonance images and registered the geometries to magnetic resonance images from an upright weight bearing squat with the knees flexed at 60°. We estimated quadriceps muscle forces corresponding to 60° knee flexion during a stair climb task from motion analysis and electromyography-driven musculoskeletal modelling. We applied the quadriceps muscle forces to our finite element models and evaluated patellofemoral cartilage stress. We quantified cartilage stress using an energy-based effective stress, a scalar quantity representing the local stress intensity in the tissue. We used probabilistic methods to evaluate the effects of variations in quadriceps muscle forces from five trials of the stair climb task for each subject. Patellofemoral effective stress was most sensitive to variations in forces in the two branches of the vastus medialis muscle. Femur cartilage effective stress was most sensitive to variations in vastus medialis forces in 29/37 (78%) subjects, and patella cartilage effective stress was most sensitive to variations in vastus medialis forces in 21/37 (57%) subjects. Femur cartilage effective stress was more sensitive to variations in vastus medialis longus forces in subjects classified as maltrackers compared to normal tracking subjects (p?=?0.006). This study provides new evidence of the importance of the vastus medialis muscle in the treatment of patellofemoral pain.     

A Protein Disulfide Isomerase Controls Neuronal Migration through Regulation of Wnt Secretion

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Toxoplasma gondii targets a protein phosphatase 2C to the nuclei of infected host cells

Intracellular pathogens have evolved a wide array of mechanisms to invade and co-opt their host cells for intracellular survival. Apicomplexan parasites such as Toxoplasma gondii employ the action of unique secretory organelles named rhoptries for internalization of the parasite and formation of a specialized niche within the host cell. We demonstrate that Toxoplasma gondii also uses secretion from the rhoptries during invasion to deliver a parasite-derived protein phosphatase 2C (PP2C-hn) into the host cell and direct it to the host nucleus. Delivery to the host nucleus does not require completion of invasion, as evidenced by the fact that parasites blocked in the initial stages of invasion with cytochalasin D are able to target PP2C-hn to the host nucleus. We have disrupted the gene encoding PP2C-hn and shown that PP2C-hn-knockout parasites exhibit a mild growth defect that can be rescued by complementation with the wild-type gene. The delivery of parasite effector proteins via the rhoptries provides a novel mechanism for Toxoplasma to directly access the command center of its host cell during infection by the parasite.     

Daily dosing of rifapentine cures tuberculosis in three months or less in the murine model

Background

Availability of an ultra-short-course drug regimen capable of curing patients with tuberculosis in 2 to 3 mo would significantly improve global control efforts. Because immediate prospects for novel treatment-shortening drugs remain uncertain, we examined whether better use of existing drugs could shorten the duration of treatment. Rifapentine is a long-lived rifamycin derivative currently recommended only in once-weekly continuation-phase regimens. Moxifloxacin is an 8-methoxyfluoroquinolone currently used in second-line regimens.

Methods and Findings

Using a well-established mouse model with a high bacterial burden and human-equivalent drug dosing, we compared the efficacy of rifapentine- and moxifloxacin-containing regimens with that of the standard daily short-course regimen based on rifampin, isoniazid, and pyrazinamide. Bactericidal activity was assessed by lung colony-forming unit counts, and sterilizing activity was assessed by the proportion of mice with culture-positive relapse after 2, 3, 4, and 6 mo of treatment. Here, we demonstrate that replacing rifampin with rifapentine and isoniazid with moxifloxacin dramatically increased the activity of the standard daily regimen. After just 2 mo of treatment, mice receiving rifapentine- and moxifloxacin-containing regimens were found to have negative lung cultures, while those given the standard regimen still harbored 3.17 log₁₀ colony-forming units in the lungs (p < 0.01). No relapse was observed after just 3 mo of treatment with daily and thrice-weekly administered rifapentine- and moxifloxacin-containing regimens, whereas the standard daily regimen required 6 mo to prevent relapse in all mice.

Conclusions

Rifapentine should no longer be viewed solely as a rifamycin for once-weekly administration. Our results suggest that treatment regimens based on daily and thrice-weekly administration of rifapentine and moxifloxacin may permit shortening the current 6 mo duration of treatment to 3 mo or less. Such regimens warrant urgent clinical investigation.     

Liver mtDNA content increases during development: a comparison of methods and the importance of age- and tissue-specific controls for the diagnosis of mtDNA depletion

BACKGROUND: The quantitative loss of mitochondrial DNA (mtDNA) known as mtDNA depletion, often gives rise to liver disease. The diagnosis of mtDNA depletion syndrome is frequently imprecise, both for technical reasons and because of the lack of established age-adjusted normal ranges. We aimed to refine quantitative methods for diagnosing the hepatic type of mtDNA depletion syndrome, firstly by establishing an age-matched reference range for mitochondrial to nuclear DNA ratio (henceforth "mtDNA content") and secondly by investigating mtDNA in fibroblasts. METHODS: By comparing realtime PCR with an established method for quantifying mtDNA content we established a reference range for young children using biopsy and post-mortem material from patients <15 years. In addition, we investigated the arrangement of mtDNA in nucleoids from fibroblasts using fluorescence microscopy. RESULTS: Both methods showed that the mtDNA content of liver increases rapidly over the perinatal period. In a patient whose liver mtDNA content fell, but remained within the reference range, early investigation and age-matched controls were essential, as we found a progressive increase in muscle mtDNA copy number, respiratory chain activity and muscle power with age. In three further patients, fluorescence microscopy of the fibroblasts proved diagnostic. In one case a movement disorder was an important pointer. CONCLUSIONS: These cases highlight the (i) need for comparing mtDNA copy number data generated from patients to DNA isolated from an age-matched normal range from the tissue of interest and (ii) the utility of mtDNA staining with PicoGreen as a method to detect aberrant nucleoid morphology in mtDNA depletion patient fibroblast lines when affected tissues are not available for measuring mtDNA copy number.