Pyrazinamide-resistant Tuberculosis Obscured From Common Targeted Molecular Diagnostics.

Here, we describe a clinical case of pyrazinamide-resistant (PZA-R) tuberculosis (TB) reported as PZA-susceptible (PZA-S) by common molecular diagnostics. Phenotypic susceptibility testing (pDST) indicated PZA-R TB. Targeted Sanger sequencing reported wild-type PncA, indicating PZA-S TB. Whole Genome Sequencing (WGS) by PacBio and IonTorrent both detected deletion of a large portion of pncA, indicating PZA-R. Importantly, both WGS methods showed deletion of part of the primer region targeted by Sanger sequencing. Repeating Sanger sequencing from a culture in presence of PZA returned no result, revealing that 1) two minority susceptible subpopulations had vanished, 2) the PZA-R majority subpopulation harboring the pncA deletion could not be amplified by Sanger primers, and was thus obscured by amplification process. This case demonstrates how a small susceptible subpopulation can entirely obscure majority resistant populations from targeted molecular diagnostics and falsely imply homogenous susceptibility, leading to incorrect diagnosis. To our knowledge, this is the first report of a minority susceptible subpopulation masking a majority resistant population, causing targeted molecular diagnostics to call false susceptibility. The consequence of such genomic events is not limited to PZA. This phenomenon can impact molecular diagnostics' sensitivity whenever the resistance-conferring mutation is not fully within primer-targeted regions. This can be caused by structural changes of genomic context with phenotypic consequence as we report here, or by uncommon mechanisms of resistance. Such false susceptibility calls promote suboptimal treatment and spread of strains that challenge targeted molecular diagnostics. This motivates development of molecular diagnostics unreliant on primer conservation, and impels frequent WGS surveillance for variants that evade prevailing molecular diagnostics.

Potent dual MAGL/FAAH inhibitor AKU-005 engages endocannabinoids to diminish meningeal nociception implicated in migraine pain.

BACKGROUND: Engaging the endocannabinoid system through inhibition of monoacylglycerol lipase (MAGL) and fatty acid amide hydrolase (FAAH), degrading endocannabinoids (endoCBs) 2-arachidonoylglycerol (2-AG) and anandamide (AEA), was proposed as a promising approach to ameliorate migraine pain. However, the activity of MAGL and FAAH and action of endoCB on spiking activity of meningeal afferents, from which migraine pain originates, has not been explored thus far. Therefore, we here explored the analgesic effects of endoCB enhancement in rat and human meningeal tissues. METHODS: Both MAGL and FAAH activity and local 2-AG and AEA levels were measured by activity-based protein profiling (ABPP) and LC-MS/MS, respectively, in rat meninges obtained from hemiskulls of P38-P40 Wistar rats and human meninges from elderly patients undergoing non-migraine related neurosurgery. The action on endoCBs upon administration of novel dual MAGL/FAAH inhibitor AKU-005 on meningeal afferents excitability was tested by investigating paired KCl-induced spiking and validation with local (co-)application of either AEA or 2-AG. Finally, the specific TRPV1 agonist capsaicin and blocker capsazepine were tested. RESULTS: The basal level of 2-AG exceeded that of AEA in rat and human meninges. KCl-induced depolarization doubled the level of AEA. AKU-005 slightly increased spontaneous spiking activity whereas the dual MAGL/FAAH inhibitor significantly decreased excitation of nerve fibres induced by KCl. Similar inhibitory effects on meningeal afferents were observed with local applications of 2-AG or AEA. The action of AKU-005 was reversed by CB1 antagonist AM-251, implying CB1 receptor involvement in the anti-nociceptive effect. The inhibitory action of AEA was also reversed by AM-251, but not with the TRPV1 antagonist capsazepine. Data cluster analysis revealed that both AKU-005 and AEA largely increased long-term depression-like meningeal spiking activity upon paired KCl-induced spiking. CONCLUSIONS: In the meninges, high anti-nociceptive 2-AG levels can tonically counteract meningeal signalling, whereas AEA can be engaged on demand by local depolarization. AEA-mediated anti-nociceptive effects through CB1 receptors have therapeutic potential. Together with previously detected MAGL activity in trigeminal ganglia, dual MAGL/FAAH inhibitor AKU-005 appears promising as migraine treatment.

Potentiation of endocannabinoids and other lipid amides prevents hyperalgesia and inflammation in a pre-clinical model of migraine.

Targeting fatty acid amide hydrolase (FAAH) is a promising therapeutic strategy to combat certain forms of pain, including migraine headache. FAAH inhibitors, such as the O-biphenyl-3-yl carbamate URB597, have been shown to produce anti-hyperalgesic effects in animal models of migraine. The objective of this study was to investigate the behavioral and biochemical effects of compounds ARN14633 and ARN14280, two URB597 analogs with improved solubility and bioavailability, in a migraine-specific rat model in which trigeminal hyperalgesia is induced by nitroglycerin (NTG) administration. ARN14633 (1 mg/kg, i.p.) and ARN14280 (3 mg/kg, i.p.) were administered to adult male Sprague-Dawley rats 3 hours after NTG injection. One hour after the administration of either compound, rats were subjected to the orofacial formalin test. ARN14633 and ARN14280 attenuated NTG-induced nocifensive behavior and reduced transcription of genes encoding neuronal nitric oxide synthase, pain mediators peptides (calcitonin gene-related peptide, substance P) and pro-inflammatory cytokines (tumor necrosis factor-alpha, interleukin-1beta and 6) in the trigeminal ganglion, cervical spinal cord and medulla. Finally, both compounds strongly elevated levels of endocannabinoids and/or other FAAH substrates in cervical spinal cord and medulla, and, to a lesser extent, in the trigeminal ganglia. The results indicate that the novel global FAAH inhibitors ARN14633 and ARN14280 elicit significant anti-hyperalgesic effects in a migraine-specific animal model and inhibit the associated peptidergic-inflammatory response. Although the precise mechanism underlying these effects remains to be elucidated, our results support further investigational studies of FAAH blockade as a potential therapeutic strategy to treat migraine conditions.

[Prevalence and transmission of pyrazinamide-resistant Mycobacterium tuberculosis in Hunan Province,China].

Objective: To provide a scientific reference for the prevention and treatment of pyrazinamide-resistant tuberculosis (PZA-R TB), we analyzed the prevalence and risk factors of pyrazinamide-resistant tuberculosis in Hunan province and described the genotyping and clustering characteristics of the pyrazinamide-resistant Mycobacterium tuberculosis (PZA-R MTB) isolates. Methods: The drug susceptibility test results of first-line anti-tuberculosis drugs including isoniazid (INH), rifampicin (RFP), streptomycin (SM), ethambutol (EMB) and pyrazinamide (PZA), and the characteristics of patients were collected from 3 862 tuberculosis patients in Hunan Chest Hospital (Institute of Tuberculosis Control and Prevention) from January 2016 to December 2018. The prevalence of PZA-R TB was calculated and risk factors were analyzed by univariate and multivariable logistic regression analysis. Two hundred and twelve Mycobacterium tuberculosis isolates selected from June 2017 to June 2018 were genotyped using the 24-loci MIRU-VNTR system. The genetic difference value (h), and the Hunter-Gaston index (HGI) were used to evaluate the resolution and variation for the 24 loci. MIRU-VNTR results were analyzed using BioNumerics 5.0 software to conduct cluster analysis. Clustered isolates were further analyzed by pncA gene sequencing. Results: The rate of PZA-R TB among tuberculosis patients and MDR patients was 14.7%(566/3 862) and 60.5%(511/844), respectively. Multivariable logistic regression analysis showed that patients who were INH mono-resistance and MDR had a higher risk of developing PZA resistance, compared with TB patients who were pan-sensitive to anti-TB drugs (INH, RFP, SM, and EMB). The adjusted OR value (95%CI) was 13.08(5.67-30.18), 298.41(164.88-540.08), respectively, and P values were all less than 0.01. Clustering analysis showed that 65 strains formed 19 clusters, the clustering rate was 30.7%(65/212). Of 19 clusters, eight clusters had at least two isolates with identical pncA mutation types within each cluster. In eight clusters, cluster 4, 6, 16 had four, three, and two patients who lived in the same county, respectively, thus providing probable epidemiological links for the recent transmission of PZA-R Mycobacterium tuberculosis. At least 47.6%(101/212) of PZA drug-resistant TB patients were suggestive of primary drug resistance caused by transmission. Conclusions: The prevalence of PZA-R TB was severe in Hunan province. PZA susceptibility testing should be performed for isolates resistant to any first-line anti-tuberculosis drugs, especially for MDR-MTB isolates. Nearly half of tuberculosis patients were suggestive of primary drug resistance caused by transmission. The prevention and treatment strategy of PZA-R TB should focus on the standardized treatment and management of patients as well as control of the source of infection.

Specific Lowering of Asymmetric Dimethylarginine by Pharmacological Dimethylarginine Dimethylaminohydrolase Improves Endothelial Function, Reduces Blood Pressure and Ischemia-Reperfusion Injury.

Multiple clinical and preclinical studies have demonstrated that plasma levels of asymmetric dimethylarginine (ADMA) are strongly associated with hypertension, diabetes, and cardiovascular and renal disease. Genetic studies in rodents have provided evidence that ADMA metabolizing dimethylarginine dimethylaminohydrolase (DDAH)-1 plays a role in hypertension and cardiovascular disease. However, it remains to be established whether ADMA is a bystander, biomarker, or sufficient contributor to the pathogenesis of hypertension and cardiovascular and renal disease. The goal of the present investigation was to develop a pharmacological molecule to specifically lower ADMA and determine the physiologic consequences of ADMA lowering in animal models. Further, we sought to determine whether ADMA lowering will produce therapeutic benefits in vascular disease in which high ADMA levels are produced. A novel long-acting recombinant DDAH (M-DDAH) was produced by post-translational modification, which effectively lowered ADMA in vitro and in vivo. Treatment with M-DDAH improved endothelial function as measured by increase in cGMP and in vitro angiogenesis. In a rat model of hypertension, M-DDAH significantly reduced blood pressure (vehicle: 187 ± 19 mm Hg vs. M-DDAH: 157 ± 23 mm Hg; P < 0.05). Similarly, in a rat model of ischemia-reperfusion injury, M-DDAH significantly improved renal function as measured by reduction in serum creatinine (vehicle: 3.14 ± 0.74 mg/dl vs. M-DDAH: 1.1 ± 0.75 mg/dl; P < 0.01), inflammation, and injured tubules (vehicle: 73.1 ± 11.1% vs. M-DDAH: 22.1 ± 18.4%; P < 0.001). These pharmacological studies have provided direct evidence for a pathologic role of ADMA and the therapeutic benefits of ADMA lowering in preclinical models of endothelial dysfunction, hypertension, and ischemia-reperfusion injury. SIGNIFICANCE STATEMENT: High levels of ADMA occur in patients with cardiovascular and renal disease. A novel modified dimethylarginine dimethylaminohydrolase by PEGylation effectively lowers ADMA, improves endothelial function, reduces blood pressure and protects from ischemia-reperfusion renal injury.

The fatty acid amide hydrolase and cyclooxygenase-inhibitory properties of novel amide derivatives of carprofen.

In experimental animals, inhibition of fatty acid amide hydrolase (FAAH) reduces the gastrointestinal damage produced by non-steroidal anti-inflammatory agents that act by inhibition of cyclooxygenase (COX). This suggests that compounds able to inhibit both enzymes may be potentially useful therapeutic agents. In the present study, we have investigated eight novel amide analogues of carprofen, ketoprofen and fenoprofen as potential FAAH/COX dual action inhibitors. Carpro-AM1 (2-(6-Chloro-9H-carbazol-2-yl)-N-(3-methylpyridin-2-yl)propenamide) and Carpro-AM6 (2-(6-Chloro-9H-carbazol-2-yl)-N-(3-chloropyridin-2-yl)propenamide) were found to be fully reversible inhibitors of the hydrolysis of 0.5 µM [3H]anandamide in rat brain homogenates with IC50 values of 94 and 23 nM, respectively, i.e. 2-3 orders of magnitude more potent than carprofen in this respect. Both compounds inhibited the cyclooxygenation of arachidonic acid by ovine COX-1, and were more potent inhibitors of human recombinant COX-2 when 2-arachidonoylglycerol was used as substrate than when arachidonic acid was used. It is concluded that Carpro-AM1 and Carpro-AM6 are dual-acting FAAH/substrate-selective COX inhibitors.

Aspartoacylase suppresses prostate cancer progression by blocking LYN activation.

BACKGROUND: Globally, despite prostate cancer (PCa) representing second most prevalent malignancy in male, the precise molecular mechanisms implicated in its pathogenesis remain unclear. Consequently, elucidating the key molecular regulators that govern disease progression could substantially contribute to the establishment of novel therapeutic strategies, ultimately advancing the management of PCa. METHODS: A total of 49 PCa tissues and 43 adjacent normal tissues were collected from January 2017 to December 2021 at Zhongnan Hospital of Wuhan University. The advanced transcriptomic methodologies were employed to identify differentially expressed mRNAs in PCa. The expression of aspartoacylase (ASPA) in PCa was thoroughly evaluated using quantitative real-time PCR and Western blotting techniques. To elucidate the inhibitory role of ASPA in PCa cell proliferation and metastasis, a comprehensive set of in vitro and in vivo assays were conducted, including orthotopic and tumor-bearing mouse models (n = 8 for each group). A combination of experimental approaches, such as Western blotting, luciferase assays, immunoprecipitation assays, mass spectrometry, glutathione S-transferase pull-down experiments, and rescue studies, were employed to investigate the underlying molecular mechanisms of ASPA's action in PCa. The Student's t-test was employed to assess the statistical significance between two distinct groups, while one-way analysis of variance was utilized for comparisons involving more than two groups. A two-sided P value of less than 0.05 was deemed to indicate statistical significance. RESULTS: ASPA was identified as a novel inhibitor of PCa progression. The expression of ASPA was found to be significantly down-regulated in PCa tissue samples, and its decreased expression was independently associated with patients' prognosis (HR = 0.60, 95% CI 0.40-0.92, P = 0.018). Our experiments demonstrated that modulation of ASPA activity, either through gain- or loss-of-function, led to the suppression or enhancement of PCa cell proliferation, migration, and invasion, respectively. The inhibitory role of ASPA in PCa was further confirmed using orthotopic and tumor-bearing mouse models. Mechanistically, ASPA was shown to directly interact with the LYN and inhibit the phosphorylation of LYN as well as its downstream targets, JNK1/2 and C-Jun, in both PCa cells and mouse models, in an enzyme-independent manner. Importantly, the inhibition of LYN activation by bafetinib abrogated the promoting effect of ASPA knockdown on PCa progression in both in vitro and in vivo models. Moreover, we observed an inverse relationship between ASPA expression and LYN activity in clinical PCa samples, suggesting a potential regulatory role of ASPA in modulating LYN signaling. CONCLUSION: Our findings provide novel insights into the tumor-suppressive function of ASPA in PCa and highlight its potential as a prognostic biomarker and therapeutic target for the management of this malignancy.

Modification of aspartoacylase for potential use in enzyme replacement therapy for the treatment of Canavan disease.

Canavan disease is a fatal neurological disease without any effective treatments to slow the relentless progress of this disorder. Enzyme replacement therapy has been used effectively to treat a number of metabolic disorders, but the presence of the blood-brain-barrier presents an additional challenge in the treatment of neurological disorders. Studies have begun with the aim of establishing a treatment protocol that can effectively replace the defective enzyme in Canavan disease patients. The human enzyme, aspartoacylase, has been cloned, expressed and purified, and the surface lysyl groups modified through PEGylation. Fully active modified enzymes were administered to mice that are defective in this enzyme and that show many of the symptoms of Canavan disease. Statistically significant increases in brain enzyme activity levels have been achieved in this animal model, as well as decreases in the elevated substrate levels that mimic those found in Canavan disease patients. These results demonstrate that the modified enzyme is gaining access to the brain and functions to correct this metabolic defect. The stage is now set for a long term study to optimize this enzyme replacement approach for the development of a treatment protocol.

N-Acylethanolamine Acid Amidase Inhibition Potentiates Morphine Analgesia and Delays the Development of Tolerance.

Opioids are essential drugs for pain management, although long-term use is accompanied by tolerance, necessitating dose escalation, and dependence. Pharmacological treatments that enhance opioid analgesic effects and/or attenuate the development of tolerance (with a desirable opioid-sparing effect in treating pain) are actively sought. Among them, N-palmitoylethanolamide (PEA), an endogenous lipid neuromodulator with anti-inflammatory and neuroprotective properties, was shown to exert anti-hyperalgesic effects and to delay the emergence of morphine tolerance. A selective augmentation in endogenous PEA levels can be achieved by inhibiting N-acylethanolamine acid amidase (NAAA), one of its primary hydrolyzing enzymes. This study aimed to test the hypothesis that NAAA inhibition, with the novel brain permeable NAAA inhibitor AM11095, modulates morphine's antinociceptive effects and attenuates the development of morphine tolerance in rats. We tested this hypothesis by measuring the pain threshold to noxious mechanical stimuli and, as a neural correlate, we conducted in vivo electrophysiological recordings from pain-sensitive locus coeruleus (LC) noradrenergic neurons in anesthetized rats. AM11095 dose-dependently (3-30 mg/kg) enhanced the antinociceptive effects of morphine and delayed the development of tolerance to chronic morphine in behaving rats. Consistently, AM11095 enhanced morphine-induced attenuation of the response of LC neurons to foot-shocks and prevented the attenuation of morphine effects following chronic treatment. Behavioral and electrophysiological effects of AM11095 on chronic morphine were paralleled by a decrease in glial activation in the spinal cord, an index of opioid-induced neuroinflammation. NAAA inhibition might represent a potential novel therapeutic approach to increase the analgesic effects of opioids and delay the development of tolerance.

Suppression of murine leukemias by L-asparaginase. Incidence of sensitivity among leukemias of various types: comparative inhibitory activities of guinea pig serum L-asparaginase and Escherichia coli L-asparaginase.

A survey of 109 recently derived leukemias of the mouse revealed that sensitivity to suppression by guinea pig serum is a common property of transplanted leukemias of certain classes. The sensitive leukemias included five that arose spontaneously in mice of strains with a low incidence of leukemia and 21 that were induced by X-radiation. Two GPS-sensitive leukemias were not more sensitive than a GPS-resistant leukemia to a range of standard chemotherapeutic agents. The effectiveness of L-asparaginase EC-2 from Escherichia coli in suppression of the GPS-sensitive leukemia EARAD1 depends upon the conditions of assay. Whereas it is not inhibitory when administered as a single dose at the time of inoculation of the leukemia it is considerably more effective than GPS when used in the treatment of established leukemia. Permanent cures of 7-day generalized transplants of EARAD1 can be effected by the administration of 2000 or more units of EC-2. Immunological factors apparently do not contribute to cure as treated survivors are fully susceptible to rechallenge with minimal numbers of cells from the same leukemia. Reinoculated survivors with progressively growing transplants have been successfully retreated with EC-2. The blood clearance of EC-2 L-asparaginase injected into mice is much more rapid than that of GPS L-asparaginase. After intraperitoneal inoculation of the EC-1 L-asparaginase, which does not have leukemia-inhibitory activity, only very low levels of enzyme activity could be detected in the serum. The effectiveness of EC-2 from E. coli and its availability from a virtually limitless source will make it possible to extend the study of inhibition of leukemias and other tumors by L-asparaginase to species other than small rodents.

The hematopoietic stem cell marker VNN2 is associated with chemoresistance in pediatric B-cell precursor ALL.

Most relapses of acute lymphoblastic leukemia (ALL) occur in patients with a medium risk (MR) for relapse on the Associazione Italiana di Ematologia e Oncologia Pediatrica and Berlin-Frankfurt-Münster (AIEOP-BFM) ALL protocol, based on persistence of minimal residual disease (MRD). New insights into biological features that are associated with MRD are needed. Here, we identify the glycosylphosphatidylinositol-anchored cell surface protein vanin-2 (VNN2; GPI-80) by charting the cell surface proteome of MRD very high-risk (HR) B-cell precursor (BCP) ALL using a chemoproteomics strategy. The correlation between VNN2 transcript and surface protein expression enabled a retrospective analysis (ALL-BFM 2000; N = 770 cases) using quantitative polymerase chain reaction to confirm the association of VNN2 with MRD and independent prediction of worse outcome. Using flow cytometry, we detected VNN2 expression in 2 waves, in human adult bone marrow stem and progenitor cells and in the mature myeloid compartment, in line with proposed roles for fetal hematopoietic stem cells and inflammation. Prospective validation by flow cytometry in the ongoing clinical trial (AIEOP-BFM 2009) identified 10% (103/1069) of VNN2+ BCP ALL patients at first diagnosis, primarily in the MRD MR (48/103, 47%) and HR (37/103, 36%) groups, across various cytogenetic subtypes. We also detected frequent mutations in epigenetic regulators in VNN2+ ALLs, including histone H3 methyltransferases MLL2, SETD2, and EZH2 and demethylase KDM6A. Inactivation of the VNN2 gene did not impair leukemia repopulation capacity in xenografts. Taken together, VNN2 marks a cellular state of increased resistance to chemotherapy that warrants further investigations. Therefore, this marker should be included in diagnostic flow cytometry panels.

Investigation of Safety and Tolerability of ASP3652 Based on Clinical Studies of Cerebrospinal Fluid Transfer After Multiple Doses and Exposure After Single Doses at High Dose Levels.

INTRODUCTION: The studies described here were conducted to investigate the central nervous system (CNS) transfer of ASP3652, a peripherally acting inhibitor of fatty acid amide hydrolase, after multiple doses at around the anticipated therapeutic dose and the safety, tolerability, and pharmacokinetics after single doses at corresponding supratherapeutic doses in healthy subjects. METHODS: Study 1 was an open-label multiple dose study in which ASP3652 (300 mg bid) or matching placebo was administered in multiple doses to healthy subjects. Study 2 was a placebo-controlled, randomized 4 × 4 crossover study in which ASP3652 was given as three single ascending doses of ASP3652 (600-1800 mg) or matching placebo to healthy subjects. Levels of ASP3652 and endocannabinoids (eCBs) in plasma, cerebrospinal fluid (CSF) (study 1 only), and safety were evaluated. RESULTS: In study 1, ASP3652 was readily absorbed to reach Cmax at 1 h after dosing. AUCtau and Cmax of ASP3652 in CSF were approximately 0.2% and 0.06% of the AUCtau and Cmax in plasma after multiple doses of ASP3652 300 mg bid. At steady state the area under the response-time curve (AURC) from 0 to 12 h and the maximum response for anandamide in plasma were approximately 550-fold and 230-fold higher than those in CSF. In study 2, the Cmax and AUC of ASP3652 increased higher than dose proportionally in subjects receiving 600-1800 mg ASP3652. For eCBs, although the AURC increased less than dose proportionally, maximum plasma levels were comparable across all treatment groups. The incidence of adverse events (AEs) was similar across all treatment groups including the placebo group. There was no evidence of CNS-related side effects. CONCLUSIONS: ASP3652 showed low CNS penetration at the anticipated therapeutic dose and was well tolerable without any CNS-related AEs at supratherapeutic doses, supporting that the drug can be safely tested at the anticipated therapeutic dose. TRIAL REGISTRATION: ClinicalTrials.gov identifier, NCT02034734 for study 1, NCT01815684 for study 2.

The Safety, Tolerability, Pharmacokinetics, and Pharmacodynamics of ASP3652 in First-in-Human and Ascending Multiple Oral Dose Studies in Healthy Subjects.

INTRODUCTION: Inhibitors of fatty acid amide hydrolase (FAAH) increase the levels of endocannabinoids and have shown analgesic and anti-inflammatory activity in animal models. ASP3652 is a peripherally acting FAAH inhibitor in development for the treatment of chronic bladder and pelvic pain disorders. Here we describe the safety, pharmacokinetics, and pharmacodynamics of single and multiple oral doses of ASP3652 administered in healthy non-elderly and elderly male and female volunteers. METHODS: Study 1 was a combined single-ascending dose and food-effect study in which ASP3652 was given as single doses (1-600 mg) or matching placebo in healthy subjects. Study 2 was a multiple ascending dose study in which ASP3652 or matching placebo was administered in multiple oral doses (10-300 mg bid and 600 mg qd for 14 days) to healthy subjects. In both studies, the levels of ASP3652, FAAH, endocannabinoids (eCBs) and safety were evaluated. RESULTS: ASP3652 was readily absorbed to reach Cmax at 1 h after a single dose. Steady state was reached within 3 days after the start of multiple dosing. The Cmax and AUC of ASP3652 increased in a slightly more than dose-proportional manner after a single dose of ASP3652 at 30-600 mg. There was some accumulation (15-38%) based on Cmax and AUC12h upon multiple doses. Cmax was 47% lower in combination with food. There was no significant effect of gender or age on the pharmacokinetics of ASP3652. FAAH activity was inhibited in a dose-dependent manner in all dose groups after single and multiple doses of ASP3652, paralleled by an increase in plasma levels of anandamide (AEA). The incidence of adverse events following multiple doses was similar across all treatment groups including the placebo group. CONCLUSIONS: Single and multiple doses of ASP3652 were safe and well tolerated and increased endogenous cannabinoid plasma levels.

Antagonism of purified asparaginase from guinea pig serum toward lymphoma.

The tumor inhibitory activity of highly purified asparaginase from guinea pig serum toward the Gardner lymphosarcoma in C3H mice was compared with that of the serum itself. The purified enzyme, homogeneous by ultracentrifugation and immunoelectrophoresis, had activity comparable to that of the serum. The serum was also effective in mice which were made immunologically incompetent by radiation with coba't-60.

PvdQ Quorum Quenching Acylase Attenuates Pseudomonas aeruginosa Virulence in a Mouse Model of Pulmonary Infection.

Pseudomonas aeruginosa is the predominant pathogen in pulmonary infections associated with cystic fibrosis. Quorum sensing (QS) systems regulate the production of virulence factors and play an important role in the establishment of successful P. aeruginosa infections. Inhibition of the QS system (termed quorum quenching) renders the bacteria avirulent thus serving as an alternative approach in the development of novel antibiotics. Quorum quenching in Gram negative bacteria can be achieved by preventing the accumulation of N-acyl homoserine lactone (AHL) signaling molecule via enzymatic degradation. Previous work by us has shown that PvdQ acylase hydrolyzes AHL signaling molecules irreversibly, thereby inhibiting QS in P. aeruginosa in vitro and in a Caenorhabditis elegans model of P. aeruginosa infection. The aim of the present study is to assess the therapeutic efficacy of intranasally instilled PvdQ acylase in a mouse model of pulmonary P. aeruginosa infection. First, we evaluated the deposition pattern of intranasally administered fluorochrome-tagged PvdQ (PvdQ-VT) in mice at different stages of pulmonary infection by in vivo imaging studies. Following intranasal instillation, PvdQ-VT could be traced in all lung lobes with 42 ± 7.5% of the delivered dose being deposited at 0 h post-bacterial-infection, and 34 ± 5.2% at 72 h post bacterial-infection. We then treated mice with PvdQ during lethal P. aeruginosa pulmonary infection and that resulted in a 5-fold reduction of lung bacterial load and a prolonged survival of the infected animals with the median survival time of 57 hin comparison to 42 h for the PBS-treated group. In a sublethal P. aeruginosa pulmonary infection, PvdQ treatment resulted in less lung inflammation as well as decrease of CXCL2 and TNF-α levels at 24 h post-bacterial-infection by 15 and 20%, respectively. In conclusion, our study has shown therapeutic efficacy of PvdQ acylase as a quorum quenching agent during P. aeruginosa infection.

Therapeutic Potential of Fatty Acid Amide Hydrolase, Monoacylglycerol Lipase, and N-Acylethanolamine Acid Amidase Inhibitors.

Fatty acid ethanolamides (FAEs) and endocannabinoids (ECs) have been shown to alleviate pain and inflammation, regulate motility and appetite, and produce anticancer, anxiolytic, and neuroprotective efficacies via cannabinoid receptor type 1 (CB1) or type 2 (CB2) or via peroxisome proliferator-activated receptor α (PPAR-α) stimulation. FAEs and ECs are synthesized by a series of endogenous enzymes, including N-acylphosphatidylethanolaminephospholipase D (NAPE-PLD), diacylglycerol lipase (DAGL), or phospholipase C (PLC), and their metabolism is mediated by several metabolic enzymes, including fatty acid amide hydrolase (FAAH), monoacylglycerol lipase (MAGL), N-acylethanolamine acid amidase (NAAA), or cyclooxygenase 2 (COX-2). Over the past decades, increasing the concentration of FAEs and ECs through the inhibition of degrading enzymes has been considered to be a viable therapeutic approach to enhance their antinociceptive and anti-inflammatory effects, as well as to protect the nervous system.

Effects of AAV-2-mediated aspartoacylase gene transfer in the tremor rat model of Canavan disease.

The tremor rat is a spontaneous epilepsy model with a seizure phenotype caused by a deletion in the aspartoacylase (ASPA) gene. The absence of ASPA expression in these animals results in undetectable levels of enzyme activity and the accumulation of the substrate N-acetyl-aspartate (NAA) in brain, leading to generalized myelin vacuolation and severe motor and cognitive impairment. In support of human gene therapy for CD, recombinant adeno-associated viral vector (AAV-2) expressing ASPA was stereotactically delivered to the tremor rat brain and effects on the mutant phenotype were measured. AAV-ASPA gene transfer resulted in elevated aspartoacylase bioactivity compared to untreated mutant animals and elicited a significant decrease in the pathologically elevated whole-brain NAA levels. Assessment of motor function via quantitative rotorod testing demonstrated that rats injected with AAV-ASPA significantly improved on tests of balance and coordinated locomotion compared to animals receiving control vectors. This study provides evidence that AAV-2-mediated aspartoacylase gene transfer to the brain improves biochemical and behavioral deficits in tremor rat mutants (tm/tm) and supports the rationale of human gene transfer for Canavan disease.

High-yield production of a functional bacteriophage lysin with antipneumococcal activity using a plant virus-based expression system.

Streptococcus pneumoniae is the causative agent of several serious infectious diseases. It is becoming increasingly antibiotic resistant worldwide, and thus new antimicrobials are needed. One alternative to antibiotics may be the use of peptidoglycan hydrolases, the bacteriophage lytic enzymes. In this study, we demonstrated high level expression of the S. pneumoniae bacteriophage lysin Pal in Nicotiana benthamiana - TMV (Tobacco Mosaic Virus) transient expression system. The protein was purified to homogeneity and tested for streptococci killing activity in vitro and in vivo. In vitro, Pal was able to lyse three tested S. pneumoniae strains: NCTC12695, NCTC12977 and NCTC11888. The treatment of BALB/c mice with 100 µg, 200 µg and 400 µg of Pal 1h post-challenge with double lethal dose of S. pneumoniae NCTC12695 strain showed a clear dose response and protected from lethal sepsis 30%, 40% and 50% of mice, respectively. The improved mice survival correlated with decreased blood bacterial titers. In conclusion, these results suggest that plant-expressed bacteriophage lysins may have potential use as antimicrobial agents.