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.

Immobilization of antimicrobial and anti-quorum sensing enzymes onto GMA-grafted poly(vinyl chloride) catheters.

Catheter-associated infections still represent a challenging thread because of the likelihood of biofilm formation. The aim of this work was the surface modification of catheters to immobilize lysozyme and acylase under mild conditions while preserving antimicrobial and anti-quorum sensing performances. Glycidyl methacrylate (GMA) was grafted onto poly(vinyl chloride) (PVC) catheters by a pre-irradiation method. The effects of monomer concentration, pre-irradiation dose, reaction time, monomer concentration and reaction temperature were investigated. The grafting process was monitored using FTIR-ATR spectroscopy, differential scanning calorimetry, thermogravimetric analysis, and swelling data. Lysozyme was directly immobilized onto PVC-g-GMA maintaining the hydrolytic activity, which hindered Staphylococcus aureus adhesion. For acylase immobilization, the PVC-g-GMA catheters were reacted with ethylenediamine and glutaraldehyde in order to facilitate acylase covalent binding. Free acylase in solution demonstrated notably capability to act as quorum sensing inhibitor, as observed using Chromobacterium violaceum as biosensor, by degrading a wide variety of acylated homoserine lactones (AHLs), including those produced by Pseudomonas aeruginosa and Acinetobacter baumannii. Acylase-immobilized PVC-g-GMA catheters were challenged against degradation of AHLs and the activity monitored using both the biosensor and HPLC-MS. Relevantly, the functionalized catheters completely degraded all tested AHL signals, opening new ways of preventing biofilm formation on medical devices.

Uncoupling N-acetylaspartate from brain pathology: implications for Canavan disease gene therapy.

N-Acetylaspartate (NAA) is the second most abundant organic metabolite in the brain, but its physiological significance remains enigmatic. Toxic NAA accumulation appears to be the key factor for neurological decline in Canavan disease-a fatal neurometabolic disorder caused by deficiency in the NAA-degrading enzyme aspartoacylase. To date clinical outcome of gene replacement therapy for this spongiform leukodystrophy has not met expectations. To identify the target tissue and cells for maximum anticipated treatment benefit, we employed comprehensive phenotyping of novel mouse models to assess cell type-specific consequences of NAA depletion or elevation. We show that NAA-deficiency causes neurological deficits affecting unconscious defensive reactions aimed at protecting the body from external threat. This finding suggests, while NAA reduction is pivotal to treat Canavan disease, abrogating NAA synthesis should be avoided. At the other end of the spectrum, while predicting pathological severity in Canavan disease mice, increased brain NAA levels are not neurotoxic per se. In fact, in transgenic mice overexpressing the NAA synthesising enzyme Nat8l in neurons, supra-physiological NAA levels were uncoupled from neurological deficits. In contrast, elimination of aspartoacylase expression exclusively in oligodendrocytes elicited Canavan disease like pathology. Although conditional aspartoacylase deletion in oligodendrocytes abolished expression in the entire CNS, the remaining aspartoacylase in peripheral organs was sufficient to lower NAA levels, delay disease onset and ameliorate histopathology. However, comparable endpoints of the conditional and complete aspartoacylase knockout indicate that optimal Canavan disease gene replacement therapies should restore aspartoacylase expression in oligodendrocytes. On the basis of these findings we executed an ASPA gene replacement therapy targeting oligodendrocytes in Canavan disease mice resulting in reversal of pre-existing CNS pathology and lasting neurological benefits. This finding signifies the first successful post-symptomatic treatment of a white matter disorder using an adeno-associated virus vector tailored towards oligodendroglial-restricted transgene expression.

Intrathecal Delivery of ssAAV9-DAO Extends Survival in SOD1G93A ALS Mice.

Amyotrophic lateral sclerosis (ALS) is an adult-onset, irreversible neurodegenerative disease that leads to progressive paralysis and inevitable death 3-5 years after diagnosis. The mechanisms underlying this process remain unknown, but new evidence indicates that accumulating levels of D-serine result from the downregulation of D-amino acid oxidase (DAO) and that this is a novel mechanism that leads to motoneuronal death in ALS via N-methyl-D-aspartate receptor-mediated cell toxicity. Here, we explored a new therapeutic approach to ALS by overexpressing DAO in the lumbar region of the mouse spinal cord using a single stranded adeno-associated virus serotype 9 (ssAAV9) vector. A single intrathecal injection of ssAAV9-DAO was made in SOD1G93A mice, a well-established mouse model of ALS. Treatment resulted in moderate expression of exogenous DAO in motorneurons in the lumbar spinal cord, reduced immunoreactivity of D-serine, alleviated motoneuronal loss and glial activation, and extended survival. The potential mechanisms underlying these effects were associated with the down-regulation of NF-κB and the restoration of the phosphorylation of Akt. In conclusion, administering ssAAV9-DAO may be an effective complementary approach to gene therapy to extend lifespans in symptomatic ALS.

Acylase-containing polyurethane coatings with anti-biofilm activity.

Due to the prevalence of biofilm-related infections, which are mediated by bacterial quorum sensing, there is a critical need for materials and coatings that resist biofilm formation. We have developed novel anti-biofilm coatings that disrupt quorum sensing in surface-associated bacteria via the immobilization of acylase in polyurethane films. Specifically, acylase from Aspergillus melleus was covalently immobilized in biomedical grade polyurethane coatings via multipoint covalent immobilization. Coatings containing acylase were enzymatically active and catalyzed the hydrolysis of the quorum sensing (QS) molecules N-butyryl-L-homoserine lactone (C4-LHL), N-hexanoyl-L-homoserine lactone (C6-LHL), and N-(3-oxododecanoyl)-L-homoserine lactone (3-oxo-C12-LHL). In biofilm inhibition assays, immobilization of acylase led to an approximately 60% reduction in biofilm formation by Pseudomonas aeruginosa ATCC 10145 and PAO1. Inhibition of biofilm formation was consistent with a reduction in the secretion of pyocyanin, indicating the disruption of quorum sensing as the mechanism of the coating activity. Scanning electron microscopy further showed that acylase-containing coatings contained far fewer bacterial cells than control coatings that lacked acylase. Moreover, acylase-containing coatings retained 90% activity when stored dry at 37°C for 7 days and were more stable than the free enzyme in physiological conditions, including artificial urine. Ultimately, such coatings hold considerable promise for the clinical management of catheter-related infections as well as the prevention of infections in orthopedic applications (i.e., on hip and knee prostheses) and on contact lenses. Biotechnol. Bioeng. 2016;113: 2535-2543. © 2016 Wiley Periodicals, Inc.

Development of a dry, stable and inhalable acyl-homoserine-lactone-acylase powder formulation for the treatment of pulmonary Pseudomonas aeruginosa infections.

In the lungs of cystic fibrosis (CF) patients, Pseudomonas aeruginosa commonly causes chronic infections. It has been shown that the P. aeruginosa quorum sensing (QS) system controls the expression of virulence factors during invasion and infection to host cells. PvdQ is an acyl-homoserine lactone (AHL) acylase able to degrade the signal molecule of P. aeruginosa QS. The role of PvdQ in inhibiting the QS and its successive virulence determinants has been established in in vitro as well as in in vivo, the latter in a Caenorabdhitis elegans model. For the treatment of pulmonary P. aeruginosa infections, we propose that PvdQ can be best administered directly to the lungs of the patients as a dry powder because this is expected to give specific advantages in delivery as compared to nebulizing. Therefore in this study we investigated the production of a PvdQ powder by spray-freeze drying using mannitol, trehalose and inulin as excipient. The activity of PvdQ in the powder was determined immediately after production and after subsequent storage during 4 weeks at 20°C and 55°C. We found that the enzymatic activity of PvdQ is fully maintained during spray-freeze drying using mannitol, trehalose or inulin as excipient. However, mannitol was not able to stabilize the protein during storage, while PvdQ incorporated in trehalose or inulin was fully stabilized even during storage at 55°C for at least 4 weeks. The poor stabilizing capacities of mannitol during storage could be related to its crystalline nature while the excellent stabilizing capacities of trehalose and inulin during storage could be related to their amorphous nature. The trehalose and inulin-based particles consisted of porous spheres with a volume average aerodynamical diameter of ∼1.8 µm implying that they are suitable for pulmonary delivery. This is the first study in which an AHL-degrading enzyme is processed into spray-freeze-dried powder suitable for inhalation.

In vitro analysis of protection of the enzyme bile salt hydrolase against enteric conditions by whey protein-gum arabic microencapsulation.

The interest in efficient intestinal delivery of health-promoting substances is increasing. However, the delivery of vulnerable substances such as enzymes requires specific attention. The transit through the stomach, where the pH is very low, can be detrimental to the enzymatic activity of the protein to be delivered. Here, we describe the microencapsulation of the model enzyme bile salt hydrolase (Bsh) using whey protein-gum arabic microencapsulates for food-grade and targeted enzyme delivery in the proximal region of the small intestine. Furthermore, the efficacy of enteric coating microencapsulates for site-specific enzyme delivery was compared in vitro with living Lactobacillus plantarum WCFS1 bacteria that endogenously produce the Bsh enzyme. Microencapsulates allowed highly effective protection of the enzyme under gastric conditions. Moreover, Bsh release under intestinal conditions appeared to be very efficient, although in the presence of pancreatin, the Bsh activity decreased in time due to proteolytic degradation. In comparison, L. plantarum appeared to be capable to withstand gastric conditions as well as pancreatin challenge. Delivery using encapsulates and live bacteria each have different (dis)advantages that are discussed. In conclusion, live bacteria and food-grade microencapsulates provide alternatives for dedicated enteric delivery of specific enzymes, and the choice of enzyme to be delivered may determine which mode of delivery is most suitable.

Toxicity due to 2- and 13-wk inhalation exposures of rats and mice to N, N-dimethylformamide.

In order to better characterize the toxicity of N,N-dimethylformamide (DMF) and to provide its basic toxicity data for risk assessment of workers exposed to DMF, F344 rats and BDF1 mice of both sexes were exposed by inhalation (6 h/d x 5 d/wk) to 100, 200, 400, 800 or 1,600 ppm DMF for 2 wk, and 50, 100, 200, 400 or 800 ppm DMF for 13 wk. Three male and 7 female rats died during the 2-wk exposure to 1,600 ppm DMF, but no death of the exposed rats or mice occurred under any other exposure conditions. Massive, focal and single cell necroses were observed in the liver of DMF-exposed rats and mice. The massive necrosis associated with the centrilobular fibrosis occurred at the highest exposure concentration. The single cell necrosis was associated with fragmentation of the nucleoli as well as an increased mitotic figure. The 13-wk exposures of rats and mice to DMF were characterized by increases in the relative liver weight and the incidence of the centrilobular hepatocellular hypertrophy as well as increased serum levels of AST, ALT, LDH, total cholesterol and phospholipid. Lower confidence limits of the benchmark dose yielding the response with a 10% extra risk (BMDL10) were determined for the relative liver weight and the incidence of hepatocellular hypertrophy of the 13-wk exposed animals. The BMDL10 resulted in 1 ppm for the increased relative liver weight of male rats and mice and 17 ppm for the hepatocellular hypertrophy of male mice.

[Effect of L-lysine-alpha-oxidase gel on development of ophthalmic herpes and herpetic skin lesions in rabbit]. / Izuchenie vliianiia gelevoi formy L-lizin-alpha-oksidazy na razvitie oftal'mogerpesa i gerpeticheskikh porazhenii kozhi krolika.

The effect of various doses of L-lysine-alpha-oxidase gel (1.4-3.5 and 70 mcg/ml) on development of eye and skin herpetic lesions due to type 1 herpes simplex virus was studied on rabbits. The doses of 1.4 to 3.5 mcg/ml were not sufficient for the therapeutic effect. The dose of 70 mcg/ml provided complete healing of the lesions in 3 to 4 days. The results were confirmed by the immunological tests.

Antibody-enzyme conjugates for cancer therapy.

The use of antibody-enzyme conjugates directed at tumor-associated antigens to achieve site-specific activation of prodrugs to potent cytotoxic species, termed "antibody-directed enzyme prodrug therapy" (ADEPT), has attracted considerable interest since the concept was first described in 1987. Prodrug forms of both clinically used anticancer agents and novel cytotoxic compounds have been developed to take advantage of potential prodrug-generating technology employing a variety of enzymes with widely differing substrate specificities. A particular advantage of the ADEPT approach is that it may allow the use of extremely potent agents such as nitrogen mustards and palytoxin, which are too toxic to be readily used in conventional chemotherapy. Preliminary studies using an antibody-enzyme conjugate constructed with a bacterial enzyme and a murine monoclonal antibody not only have established the value of the ADEPT technique, but also have highlighted the potential problem of immunogenicity of proteins of nonhuman origin. This problem has been tackled in the first instance by the use of immunosuppressive agents, but long-term solutions are being investigated in the development of second-generation ADEPT systems, including the development of human antibody-human enzyme fusion proteins and catalytic antibodies. Such improvements, coupled with further refinement of the prodrug-drug element of the system and the wide variety of antibody-enzyme-drug combinations available, should mean that ADEPT-based approaches will form an important element of the search for the anticancer drugs of the future.

Treatment of L5178Y tumor-bearing BDF1 mice with a nonimmunogenic L-glutaminase-L-asparaginase.

An L-glutaminase-L-asparaginase from Achromobacter has been rendered nonimmunogenic by the covalent attachment of polyethylene glycol (PEG) to nonessential amine groups of the enzyme. PEG-L-glutaminase-L-asparaginase exhibits a greatly enhanced half-life in the bloodstream compared to the unmodified enzyme in normal mice, and is effective in prolonging the survival of BDF1 mice inoculated ip with L5178Y cells. PEG-L-glutaminase-L-asparaginase appears rapidly in the blood following ip injection.