ENACT: a protocol for a randomised placebo-controlled trial investigating the efficacy and mechanisms of action of adjunctive N-acetylcysteine for first-episode psychosis.

BACKGROUND: First-episode psychosis (FEP) may lead to a progressive, potentially disabling and lifelong chronic illness; however, evidence suggests that the illness course can be improved if appropriate treatments are given at the early stages. Nonetheless, the efficacy of antipsychotic medications is suboptimal, particularly for negative and cognitive symptoms, and more efficacious and benign treatments are needed. Previous studies have shown that the antioxidant amino acid N-acetylcysteine (NAC) reduces negative symptoms and improves functioning in chronic schizophrenia and bipolar disorder. Research is scarce as to whether NAC is beneficial earlier in the course of illness. The primary aim of this study is to determine the efficacy of treatment with adjunctive NAC (2 g/day for 26 weeks) compared with placebo to improve psychiatric symptoms in young people experiencing FEP. Secondary aims are to explore the neurobiological mechanisms underpinning NAC and how they relate to various clinical and functional outcomes at 26- and 52-week follow-ups. METHODS/DESIGN: ENACT is a 26-week, randomised controlled trial of adjunctive NAC versus placebo, with a 26-week non-treatment follow-up period, for FEP. We will be recruiting 162 young people aged 15-25 years who have recently presented to, and are being treated at, the Early Psychosis Prevention and Intervention Centre, Melbourne, Australia. The primary outcome is the Total Score on the Positive and Negative Syndrome Scale which will be administered at baseline, and weeks 4, 8, 12, 26 (primary endpoint), and 52 (end of study). Secondary outcomes include: symptomatology, functioning, quality of life, neurocognition, blood-derived measures of: inflammation, oxidative and nitrosative stress, and magnetic resonance spectroscopy measures of glutathione concentration. DISCUSSION: Targeted drug development for FEP to date has generally not involved the exploration of neuroprotective agents. This study has the potential to offer a new, safe, and efficacious treatment for people with FEP, leading to better treatment outcomes. Additionally, the neuroprotective dimension of this study may lead to a better long-term prognosis for people with FEP. It has the potential to uncover a novel treatment that targets the neurobiological mechanisms of FEP and, if successful, will be a major advance for psychiatry. TRIAL REGISTRATION: Australian New Zealand Clinical Trials Registry, ID: ACTRN12618000413224. Registered on 21 March 2018.

GABA(A) receptor cell surface number and subunit stability are regulated by the ubiquitin-like protein Plic-1.

Controlling the number of functional gamma-aminobutyric acid A (GABA(A)) receptors in neuronal membranes is a crucial factor for the efficacy of inhibitory neurotransmission. Here we describe the direct interaction of GABA(A) receptors with the ubiquitin-like protein Plic-1. Furthermore, Plic-1 is enriched at inhibitory synapses and is associated with subsynaptic membranes. Functionally, Plic-1 facilitates GABA(A) receptor cell surface expression without affecting the rate of receptor internalization. Plic-1 also enhances the stability of intracellular GABA(A) receptor subunits, increasing the number of receptors available for insertion into the plasma membrane. Our study identifies a previously unknown role for Plic-1, a modulation of GABA(A) receptor cell surface number, which suggests that Plic-1 facilitates accumulation of these receptors in dendritic membranes.

Improvement in the therapeutic, immunological, and clearance properties of Escherichia coli and Erwinia carotovora L-asparaginases by attachment of poly-DL-alanyl peptides.

The chemical modification of both Escherichia coli and Erwinia carotovora asparaginases by a DL-alanine-N-carboxyanhydride polymerization technique produced modified enzymes which had greater protease stability, retained most of their catalytic activity, and demonstrated a 7- to 10-fold prolongation in plasma clearance properties in normal mice and rats. Concomitantly, plasma substrate depletion was also extended 5 to 13 days longer for the modified as compared with the native enzymes. For preparations of modified enzymes with plasma half-lives longer than 24 hr, the therapeutic activity was superior to that of the native enzymes. In addition, the modified E. coli preparations were less immunogenic in mice than was the native enzyme, and they cross-reacted with antibodies developed to the native enzyme to a 300-fold lesser degree, such that the modified enzyme still showed prolonged clearance in an animal which had been immunized previously to the native enzyme. The native enzyme was immediately cleared from the plasma of such immune animals, although hyperimmune animals would rapidly clear both the native and modified enzymes. Similarly, the modified E. carotovora enzyme would cross-react to a 500-fold lesser degree with antibodies developed against the native E. carotovora enzyme.

Immunological and pharmacological characterization of poly-DL-alanyl-modified Erwinia carotovora L-asparaginase.

The covalent attachment of poly-DL-alanine peptides to lysyl residues on the surface of Erwinia carotovora L-asparaginase has produced a modified enzyme which is much less immunogenic in mice and demonstrates 100-fold longer plasma half-life in the rhesus monkey. Immunogenic responses towards both the immunoglobulin G (IgG) and immunoglobulin E (IgE) antibody subclasses were evaluated in C57BL x DBA/2 F1 mice exposed to 250 rads of whole-body irradiation 4 hr prior to immunization with 5-diazo-4-oxynorvaline-inactivated native and modified L-asparaginase in complete Freund's adjuvant. Under these immunologically stressful conditions, the native enzyme evoked an IgE and IgG response which could be further amplified by a secondary immunization, whereas the modified enzyme evoked no IgE or IgG response even after a tertiary immunization. In experiments mimicking an intensive therapeutic schedule, whereby two groups of mice were given weekly injections of 5 to 10 units of either native or modified asparaginase for up to 14 weeks, neither enzyme form evoked a significant IgE response, and only the mice given injections of the native enzyme produced an IgG response. In a preliminary patient study, skin testing of a child who had shown an allergic reaction to the native enzyme resulted in a negative response after an intradermal injection of the modified enzyme, whereas a wheal and flare reaction was observed to both the native enzyme and a histamine control. All of these results suggest that the modified enzyme should show a definite reduction in immunological reactions associated with L-asparaginase treatment of childhood leukemia.

Subunit-specific association of protein kinase C and the receptor for activated C kinase with GABA type A receptors.

GABA receptors (GABA(A)) are the major sites of fast synaptic inhibition in the brain and can be assembled from five subunit classes: alpha, beta, gamma, delta, and epsilon. Receptor function can be regulated by direct phosphorylation of beta and gamma2 subunits, but how kinases are targeted to GABA(A) receptors is unknown. Here we show that protein kinase C-betaII (PKC-betaII) is capable of directly binding to the intracellular domain of the receptor beta1 and beta3 subunits, but not to those of the alpha1 or gamma2 subunits. Moreover, associating PKC-betaII is capable of specifically phosphorylating serine 409 in beta1 subunit and serines 408/409 within the beta3 subunit, key residues for modulating GABA(A) receptor function. The receptor for activated C kinase (RACK-1) was found also to bind to the beta1 subunit intracellular domain, but PKC binding appeared to be independent of this protein. Using immunoprecipitation, the association of PKC isoforms and RACK-1 with neuronal GABA(A) receptors was seen. Furthermore, PKC isoforms associating with neuronal receptors were capable of phosphorylating the receptor beta3 subunit. Together, these observations suggest GABA(A) receptors are intimately associated with PKC isoforms via a direct interaction with receptor beta subunits. This interaction may serve to localize PKC activity to GABA(A) receptors in neurons allowing the rapid regulation of receptor activity by cell-signaling pathways that modify PKC activity.

Methotrexate analogues. 14. Synthesis of new gamma-substituted derivatives as dihydrofolate reductase inhibitors and potential anticancer agents.

The gamma-tert-butyl ester (1), gamma-hydrazide (2), gamma-n-butylamide (3), and gamma-benzylamide (4) derivatives of methotrexate (MTX) were synthesized from 4-amino-4-deoxy-N10-methylpteroic acid (APA) and the appropriate blocked L-glutamic acid precursors with the aid of the peptide bond forming reagent diethyl phosphorocyanidate. The affinity of these side chain modified products for dihydrofolate reductase (DHFR) from Lactobacillus casei and L1210 mouse leukemic cells was determined spectrophotometrically or by competitive radioligand binding assay, and their cytotoxicity was evaluated against L1210 leukemic cells in culture. The results provide continuing support for the view that the "gamma-terminal region" of the MTX side chain is an attractive site for molecular modification of this anticancer agent.

Methotrexate analogues. 13. Chemical and pharmacological studies on amide, hydrazide, and hydroxamic acid derivatives of the glutamate side chain.

Carbodiimide-mediated condensation of 4-amino-4-deoxy-N10-methylpteroic acid (APA) with several alkyl, aralkyl, and aryl amines, in the presence or absence of N-hydroxysuccinimide, was employed in order to prepare new lipid-soluble bis(amide) derivatives of methotrexate (MTX) as potential prodrugs. MTX dianilide was likewise prepared, in comparable yield, from APA and L-glutamic acid dianilide via the mixed carboxylic--carbonic anhydride method. Dihydrazide and bis(N-methylhydrazide) derivatives of MTX were formed readily from MTX diethyl ester. However, reaction with hydroxylamine led to MTX gamma-monohydroxamic acid as the sole isolated product. The bis adduct appears to form, but is unstable during workup. The identity of the product was confirmed by independent mixed anhydride synthesis from APA and the gamma-monohydroxamate of L-glutamic acid. Treatment of MTX dimethyl ester with N,N-dimethylhydrazine unexpectedly yielded MTX gamma-monomethyl ester. MTX dianilide was active against L1210 leukemia in mice, with a +155% increase in life span at a dose of 160 mg/kg given ip in 10% Tween 80 on a q3d X 3 schedule. The bis(p-chlorobenzylamide), bis(p-methoxybenzylamide), and dihydrazide were also active against L1210 leukemia in vivo, but to a lesser extent than the dianilide. The gamma-monohydroxamic acid derivative showed activity (+111% ILS at 40 mg/kg) similar to that of MTX and was found to bind to a partially purified dihydrofolate reductase preparation from L1210 cells with an ID50 of 0.005 microM as compared to 0.007 microM for MTX. In vivo experiments in mice indicated that the pharmacokinetic properties of this compound and of MTX are similar but failed to demonstrate any advantage over MTX in terms of selective uptake into tumor (sc implanted P388 leukemia) or improved penetration of the central nervous system. The activities of the dianilide, bis(benzylamide), and dihydrazide derivatives in vivo are of interest in view of their low toxicity relative to MTX against cells in culture, which suggests that these derivatives are probably acting as prodrugs in the intact animal.

Methotrexate analogues. 15. A methotrexate analogue designed for active-site-directed irreversible inactivation of dihydrofolate reductase.

N alpha-(4-Amino-4-deoxy-N10-methylpteroyl)-N epsilon-(iodoacetyl)-L-lysine (1) was synthesized as a potential active-site-directed irreversible inhibitor of dihydrofolate reductase (DHFR). In an ultraviolet spectrophotometric assay of dihydrofolate reduction of Lactobacillus casei DHFR, 1 and methotrexate (MTX, 4-amino-4-deoxy-N10-methylpteroyl-L-glutamic acid) had ID50 values of 4.5 and 6.2 nM. The corresponding ID50 values in a competitive radioligand binding assay against [3H]MTX were 31 and 16 nM. Thus, as reversible inhibitors of this enzyme over a short exposure time, 1 and MTX had comparable activity. On the other hand, when L. casei DHFR was incubated for up to 6 h with 0.1 or 1.0 microM 1, a progressive decrease in the ability of [3H]MTX to subsequently displace the drug was observed. When MTX itself was used at the same concentrations, the extent of displacement of [3H]MTX did not decrease with time. These results were consistent with rapid reversible binding of 1 to the enzyme, followed more slowly by covalent bond formation near the active site. The pH profile for this effect followed a curve with a sigmoidal shape. The apparent inflection point near pH 7.2 was consistent with alkylation of a histidine residue.

Methotrexate analogues. 20. Replacement of glutamate by longer-chain amino diacids: effects on dihydrofolate reductase inhibition, cytotoxicity, and in vivo antitumor activity.

Chain-extended analogues of methotrexate were synthesized by condensation of 4-amino-4-deoxy-N10-methylpteroic acid with esters of L-alpha-aminoadipic, L-alpha-aminopimelic, and L-alpha-aminosuberic acids, followed by ester hydrolysis with acid or base. Coupling was accomplished in up to 85% yield by the use of the peptide bond forming reagent diethyl phosphorocyanidate at room temperature. The products were found to bind bacterial (Lactobacillus casei) and mammalian (L1210 mouse leukemia) dihydrofolate reductase with an affinity comparable to methotrexate and were also equitoxic to L1210 cells in culture. Cytotoxicity increased up to 3-fold as the number of CH2 groups in the amino acid side chain was extended from two to five. The alpha-aminoadipate and alpha-aminopimelate analogues were poor substrates for carboxypeptidase G1, confirming that this enzyme has a strict requirement for a C-terminal L-glutamic acid residue. The in vivo antitumor activity of the chain-extended analogues against L1210 leukemia in mice was comparable to that of the parent drug on the qd X 9 schedule, but higher doses were required to achieve the same increase in survival. The results were consistent with findings, reported separately, that these compounds are poor substrates for folate polyglutamate synthetase and therefore would not be expected to form gamma-polyglutamates once they enter a cell. This distinctive property has potential therapeutic implications for the treatment of certain MTX-resistant tumors whose resistance may be associated with a lower than normal capacity to form gamma-polyglutamates in comparison with proliferative tissues such as intestinal mucosa or marrow.

Methotrexate analogues--XVII. Antitumor activity of 4-amino-4-deoxy-N10-methylpteroyl-D,L-homocysteic acid and its dual inhibition of dihydrofolate reductase and folyl polyglutamate synthetase.

A new analogue of methotrexate was synthesized from 4-amino-4-deoxy-N10-methylpteroic acid and D,L-homocysteic acid. The product (mAPA-HCysA) was bound tightly to L1210 mouse leukemia dihydrofolate reductase (IC50 = 1 nM), inhibited L1210 cell proliferation in culture (IC50 = 0.3 microM), and prolonged the survival of L1210 leukemic mice (98% increase in lifespan at 120 mg/kg, qdx9). Studies on the interaction of mAPA-HCysA with partially purified mouse liver folyl polyglutamate synthetase revealed that mAPA-HCysA was not a substrate. Hence, the increased dose of mAPA-HCysA required to inhibit tumor growth in vitro and in vivo relative to methotrexate may reflect, in part, the inability of this compound to form non-effluxing polyglutamates. Folyl polyglutamate synthetase was competitively inhibited by mAPA-HCysA (K1 = 190 +/- 70 microM) when folate was the variable substrate. Thus, mAPA-HCysA is the first known compound to inhibit both mammalian dihydrofolate reductase and mammalian folyl polyglutamate synthetase.