Structural basis for species-selective targeting of Hsp90 in a pathogenic fungus.

New strategies are needed to counter the escalating threat posed by drug-resistant fungi. The molecular chaperone Hsp90 affords a promising target because it supports survival, virulence and drug-resistance across diverse pathogens. Inhibitors of human Hsp90 under development as anticancer therapeutics, however, exert host toxicities that preclude their use as antifungals. Seeking a route to species-selectivity, we investigate the nucleotide-binding domain (NBD) of Hsp90 from the most common human fungal pathogen, Candida albicans. Here we report structures for this NBD alone, in complex with ADP or in complex with known Hsp90 inhibitors. Encouraged by the conformational flexibility revealed by these structures, we synthesize an inhibitor with >25-fold binding-selectivity for fungal Hsp90 NBD. Comparing co-crystals occupied by this probe vs. anticancer Hsp90 inhibitors revealed major, previously unreported conformational rearrangements. These insights and our probe's species-selectivity in culture support the feasibility of targeting Hsp90 as a promising antifungal strategy.

Antiallodynic activity of leflunomide is partially inhibited by naltrexone and glibenclamide and associated with reduced production of TNF-α and CXCL-1.

Leflunomide, an immunosuppressive drug approved for the treatment of patients with rheumatoid arthritis, exhibits many mechanisms which may affect the nociceptive processing. Therefore, the present study aimed to evaluate the effect induced by leflunomide on the mechanical allodynia in models of inflammatory and neuropathic pain in mice and investigate mechanisms mediating such effects. Per os (p.o.) administration of leflunomide (25, 50 or 100mg/kg) inhibited the inflammatory edema and mechanical allodynia induced by intraplantar carrageenan. Even ongoing inflammatory edema and mechanical allodynia were reduced by leflunomide. Previous administration of naltrexone (10mg/kg, intraperitoneal) or glibenclamide (40mg/kg, p.o.) partially attenuated leflunomide antiallodynic activity. A single administration of leflunomide (50 or 100mg/kg, p.o.) also partially inhibited ongoing mechanical allodynia induced by chronic constriction injury (CCI) or repeated administrations of paclitaxel. The antiallodynic effect induced by leflunomide (50 or 100mg/kg, p.o.) in the model of neuropathic pain induced by CCI was associated with reduced production of tumor necrosis factor-α both at the injury site and ipsilateral paw. Leflunomide also reduced production of the chemokine CXCL-1 at the paw ipsilateral to the injury site. Concluding, leflunomide partially inhibited ongoing mechanical allodynia in models of inflammatory and neuropathic pain. The antiallodynic effect was associated with activation of opioidergic receptors and ATP-sensitive potassium channels and reduced production of inflammatory mediators. These data indicate leflunomide as a drug that should be further investigated aiming to identify a new analgesic pharmacotherapy and reinforces repositioning as an important strategy to identify new uses for approved drugs.

Phase 2 Study of the HSP-90 Inhibitor AUY922 in Previously Treated and Molecularly Defined Patients with Advanced Non-Small Cell Lung Cancer.

INTRODUCTION: In this phase 2 study, we evaluated the activity of AUY922 in pretreated patients with stage IV NSCLC. METHODS: Patients with advanced NSCLC were divided into molecularly defined strata based on mutations in the EGFR gene, the ALK receptor tyrosine kinase gene (ALK), the KRAS gene, or the wild type of all three. All patients must have received more than two prior lines of therapy, except for those in a fifth stratum for a less pretreated EGFR cohort (EGFR<2). In the EGFR-mutant and ALK-rearranged strata, prior platinum therapy was not required. Patients with EGFR mutation must have received an EGFR tyrosine kinase inhibitor unless they had de novo resistance (e.g., T790M or exon 20 insertions). Eligible patients received weekly intravenous AUY922, 70 mg/m2. The primary objective was to estimate efficacy (complete or partial response, or in the absence of complete or partial response, stable disease) at 18 weeks, by the Response Criteria in Solid Tumors. RESULTS: A total of 153 patients from 21 global centers were enrolled from October 2010 to November 2014. The investigator-assessed overall response rate and stable disease rate at 18 weeks were 31.8% and 9.1% in the ALK-rearranged stratum, 17.1% and 8.6% in EGFR-mutant stratum, 9.7% and 22.6% in the EGFR<2 stratum, 0% and 7.1% in KRAS-mutant stratum, and 8.8% and 8.8% in wild-type stratum. Biomarker data showed activity of AUY922 in EGFR-mutant patients with exon 19 deletion, T790M mutation, and exon 20 insertion. The most common (≥40%) all-causality adverse events were diarrhea, nausea, and decreased appetite. Visual-related disorders were reported in 79.7% of patients (most were grade 1/2). Thirty-five patients (22.9%) reported night blindness. CONCLUSION: AUY922 is active in patients with NSCLC, particularly among patients with ALK rearrangements and EGFR mutations.

Identification of aminosulfonylarylisoxazole as microRNA-31 regulators.

The discovery of small-molecule regulators of microRNAs remains challenging, but a few have been reported. Herein, we describe small-molecule inhibitors of miR-31, a tumor-associated microRNA (miRNA), identified by high-throughput screening using a cell-based reporter assay. Aminosulfonylarylisoxazole compounds exhibited higher specificity for miR-31 than for six other miRNAs, i.e., miR-15a, miR-16, miR-21, miR-92a-1, miR-146a, and miR-155, and increased the expression of miR-31 target genes. The down-regulation of mature miR-31 was observed, while its precursor form increased following treatment with the compounds. Thus, the compounds may target the processing of pre-miR-31 into mature miR-31 and thereby inhibit the production of mature miR-31.

Structural Basis for Small Molecule NDB (N-Benzyl-N-(3-(tert-butyl)-4-hydroxyphenyl)-2,6-dichloro-4-(dimethylamino) Benzamide) as a Selective Antagonist of Farnesoid X Receptor α (FXRα) in Stabilizing the Homodimerization of the Receptor.

Farnesoid X receptor α (FXRα) as a bile acid sensor plays potent roles in multiple metabolic processes, and its antagonist has recently revealed special interests in the treatment of metabolic disorders, although the underlying mechanisms still remain unclear. Here, we identified that the small molecule N-benzyl-N-(3-(tert-butyl)-4-hydroxyphenyl)-2,6-dichloro-4-(dimethylamino) benzamide (NDB) functioned as a selective antagonist of human FXRα (hFXRα), and the crystal structure of hFXRα ligand binding domain (hFXRα-LBD) in complex with NDB was analyzed. It was unexpectedly discovered that NDB induced rearrangements of helix 11 (H11) and helix 12 (H12, AF-2) by forming a homodimer of hFXRα-LBD, totally different from the active conformation in monomer state, and the binding details were further supported by the mutation analysis. Moreover, functional studies demonstrated that NDB effectively antagonized the GW4064-stimulated FXR/RXR interaction and FXRα target gene expression in primary mouse hepatocytes, including the small heterodimer partner (SHP) and bile-salt export pump (BSEP); meanwhile, administration of NDB to db/db mice efficiently decreased the gene expressions of phosphoenolpyruvate carboxykinase (PEPCK), glucose 6-phosphatase (G6-pase), small heterodimer partner, and BSEP. It is expected that our first analyzed crystal structure of hFXRα-LBD·NDB will help expound the antagonistic mechanism of the receptor, and NDB may find its potential as a lead compound in anti-diabetes research.

Inhibiting the teratogenicity of the immunosuppressant leflunomide in mice by supplementation of exogenous uridine.

Leflunomide is an immunosuppressant drug displaying teratogenicity in mice, rats, and rabbits. Its immunosuppressive effect occurs via inhibition of dihydroorotate dehydrogenase (DHODH) and tyrosine kinases. In this study, we coadministered Leflunomide and uridine, a precursor substance of pyrimidine nucleotides, to pregnant CD-1 mice, and examined whether or not a decreased level of intracellular pyrimidine nucleotides with inhibition of DHODH is related to the teratogenicity of Leflunomide. Then we examined the alteration of the nucleotide level in fetal tissue by Leflunomide and the effect of coadministered uridine. We administered Leflunomide with or without uridine to pregnant mice on gestation day 10, and used the vehicle of Leflunomide as a control. Leflunomide caused multiple malformations in all fetuses, but coadministration with uridine inhibited most of its teratogenicity. Leflunomide decreased the concentration of pyrimidine nucleotides, not purine nucleotides, whereas uridine coadministered with Leflunomide partially restored the level of pyrimidine nucleotides. These results indicate that the inhibitory effect of DHODH activity is related to the teratogenicity of Leflunomide.

Impact of multiple inhibitors or substrates of cytochrome P450 2D6 on plasma risperidone levels in patients on polypharmacy.

Studies that focus on multidrug interactions in natural settings are sparse. In this investigation, data from therapeutic drug monitoring (TDM) were used to study the impact of multiple cytochrome P450 enzyme (CYP) 2D6 substrates and inhibitors on plasma risperidone levels. CYP2D6 catalyzes the conversion of risperidone to the active metabolite 9-OH-risperidone. The question whether CYP2D6 activity is important for the level of the "active moiety" (ie, the sum of risperidone and 9-OH-risperidone) is controversial. Concentration-to-dose (C:D) ratios of risperidone and 9-OH-risperidone in 218 patients were associated with the number of concomitantly used substrates or inhibitors of CYP2D6. The C:D ratios of risperidone in patients with 0, 1, and >1 numbers of CYP2D6 inhibitors were 2.6, 8.5, and 17 nmol L mg, respectively. Differences between the groups were highly significant (P < 0.001). All patients with >1 CYP2D6 inhibitors were administered at least 1 potent CYP2D6 inhibitor, that is fluoxetine, paroxetine, thioridazine, and/or levomepromazine. The C:D ratios of the active moiety (risperidone + 9-OH-risperidone) in patients with 0, 1, and >1 numbers of concomitant CYP2D6 inhibitors were 17, 24, and 30 nmol L mg, respectively (P = 0.001), which was explained by higher levels of risperidone without any change in the levels of 9-OH-risperidone. Concomitant use of 1 or several drugs recognized as substrates for CYP2D6, without any proven inhibitory effect, had no apparent influence on the levels of risperidone or 9-OH-risperidone, suggesting that the risk of drug-drug interactions between different substrates of CYP2D6 is low when used in therapeutic doses. In conclusion, the results suggest that an increase in the number of concomitant inhibitors may be associated with a lower CYP2D6 activity, although the type of inhibitor is probably more important. Drug-dependent inhibition of CYP2D6 increases the active moiety of risperidone. An indication for risperidone TDM should therefore include concomitant medication with established CYP inhibitors.

Leflunomide inhibits transendothelial migration of peripheral blood mononuclear cells.

OBJECTIVES: To test whether the active metabolite of leflunomide (LEF-M), in addition to blocking the proliferation of activated lymphocytes by inhibiting dihydro-orotate dehydrogenase (DHODH), influences the transendothelial migration (TEM) of peripheral blood mononuclear cells (PBMC). METHODS: In an in vitro model of PBMC transmigration through an endothelial cell (EC) barrier, PBMC were re-collected in three groups: cells not adherent to the EC, cells bound to, and cells which had migrated through, the EC layer. Experiments in which cells were pretreated with LEF-M (in the absence or in the presence of uridine) were compared with parallel experiments in the presence of medium alone. RESULTS: Preincubation of EC with LEF-M led to a 36 (SEM 16)% reduction in PBMC TEM (p<0.05). Likewise, preincubation of PBMC induced a reduction in their TEM of 39 (9)% (p<0.005). Incubation of both PBMC and EC with LEF-M had an additive effect (mean reduction of 48 (6)%, p<0.005). Incubation of PBMC with LEF-M also decreased monocytic CD44 expression (p<0.005) and PBMC-hyaluronan binding (p<0.05). Incubation of cells with LEF-M and uridine in addition to LEF-M reversed the inhibition of migration, suggesting that the observed effects were due to DHODH inhibition. Fluorocytometric analysis of PBMC subsets within the migrated population showed a decrease of monocytes, but not of B or T cells, after LEF-M treatment. CONCLUSIONS: LEF-M reduces monocytic adhesion molecule expression and TEM and may thus interfere with monocyte and EC activities in RA. Thus, the clinical effects of leflunomide may, at least in part, be due to blocking cell traffic into the inflamed synovia.

Restriction of de novo pyrimidine biosynthesis inhibits Th1 cell activation and promotes Th2 cell differentiation.

Leflunomide, an inhibitor of de novo pyrimidine biosynthesis, has recently been introduced as a treatment for rheumatoid arthritis in an attempt to ameliorate inflammation by inhibiting lymphocyte activation. Although the immunosuppressive ability of leflunomide has been well described in several experimental animal models, the precise effects of a limited pyrimidine supply on T cell differentiation and effector functions have not been elucidated. We investigated the impact of restricted pyrimidine biosynthesis on the activation and differentiation of CD4 T cells in vivo and in vitro. Decreased activation of memory CD4 T cells in the presence of leflunomide resulted in impaired generation and outgrowth of Th1 effectors without an alteration of Th2 cell activation. Moreover, priming of naive T cells in the presence of leflunomide promoted Th2 differentiation from uncommitted precursors in vitro and enhanced Th2 effector functions in vivo, as indicated by an increase in Ag-specific Th2 cells and in the Th2-dependent Ag-specific Ig responses (IgG1) in immunized mice. The effects of leflunomide on T cell proliferation and differentiation could be antagonized by exogenous UTP, suggesting that they were related to a profound inhibition of de novo pyrimidine biosynthesis. These results indicate that leflunomide might exert its anti-inflammatory activities in the treatment of autoimmune diseases by preventing the generation of proinflammatory Th1 effectors and promoting Th2 cell differentiation. Moreover, the results further suggest that differentiation of CD4 T cells can be regulated at the level of nucleotide biosynthesis.

Influence of oral adsorbent AST-120 on anticonvulsive effect of zonisamide in rats.

The influence of oral adsorbent AST-120 (Kremezin) on the anticonvulsive effect and pharmacokinetics of zonisamide was investigated. Oral administration of zonisamide (50 mg/kg) blocked the appearance of the tonic extension induced by maximal electroshock seizure. This effect of zonisamide was inhibited by the oral coadministration of AST-120 (5 g/kg). In pharmacokinetics study, the serum zonisamide concentration after coadministration of zonisamide and AST-120 was significantly lower than that of single administration of zonisamide. However, the anticonvulsive effect of zonisamide was not affected by the administration of AST-120 1.5 h after zonisamide administration. In this condition, the serum zonisamide concentration was not changed. In the in vitro study, AST-120 completely adsorbed zonisamide. These findings suggest that when AST-120 is administered concurrently with zonisamide, a significant inhibition of the anticonvulsive effect of zonisamide occurs, and the decrease in serum zonisamide concentration by the adsorption effect of AST-120 is related to this phenomenon.

Leflunomide suppresses TNF-induced cellular responses: effects on NF-kappa B, activator protein-1, c-Jun N-terminal protein kinase, and apoptosis.

Leflunomide is a pyrimidine biosynthesis inhibitor that has recently been approved for treatment of rheumatoid arthritis. However, the mechanism of leflunomide's antiarthritis activity and is not fully understood. The critical role that TNF plays in rheumatoid arthritis led us to postulate that leflunomide blocks TNF signaling. Previously, we have demonstrated that leflunomide inhibits TNF-induced NF-kappaB activation by suppressing I-kappaBalpha (inhibitory subunit of NF-kappaB) degradation. We in this study show that leflunomide also blocks NF-kappaB reporter gene expression induced by TNFR1, TNFR-associated factor 2, and NF-kappaB-inducing kinase (NIK), but not that activated by the p65 subunit of NF-kappaB, suggesting that leflunomide acts downstream of NIK. Leflunomide suppressed TNF-induced phosphorylation of I-kappaBalpha, as well as activation of I-kappaBalpha kinase-beta located downstream to NIK. Leflunomide also inhibited TNF-induced activation of AP-1 and the c-Jun N-terminal protein kinase activation. TNF-mediated cytotoxicity and caspase-induced poly(ADP-ribose) polymerase cleavage were also completely abrogated by treatment of Jurkat T cells with leflunomide. Leflunomide suppressed TNF-induced reactive oxygen intermediate generation and lipid peroxidation, which may explain most of its effects on TNF signaling. The suppressive effects of leflunomide on TNF signaling were completely reversible by uridine, indicating a critical role for pyrimidine biosynthesis in TNF-mediated cellular responses. Overall, our results suggest that suppression of TNF signaling is one of the possible mechanisms for inhibitory activity of leflunomide against rheumatoid arthritis.

Immunosuppressive leflunomide metabolite (A77 1726) blocks TNF-dependent nuclear factor-kappa B activation and gene expression.

Leflunomide is a novel immunosuppressive and antiinflammatory agent currently being tested for treatment of autoimmune diseases and transplant rejection. NF-kappa B is a transcription factor activated in response to a wide variety of inflammatory stimuli, including TNF, but whether leflunomide blocks NF-kappa B activation is not known. In the present report we demonstrate that treatment of a human T cell line (Jurkat) with leflunomide blocks TNF-mediated NF-kappa B activation in a dose- and time-dependent manner, with maximum inhibition at 5-10 microM. Inhibition was not restricted to TNF-induced activation, because leflunomide also inhibited NF-kappa B activation induced by other inflammatory agents, including phorbol ester, LPS, H2O2, okadaic acid, and ceramide. Leflunomide blocked the degradation of I kappa B alpha and subsequent nuclear translocation of the p65 subunit, steps essential for NF-kappa B activation. This correlated with inhibition of dual specificity-mitogen-activated protein kinase kinase as well as an Src protein tyrosine kinase, p56lck, by leflunomide. Reducing agents did not reverse the effect of leflunomide. Leflunomide also suppressed the TNF-activated NF-kappa B-dependent reporter gene expression. Our results thus indicate that leflunomide is a potent inhibitor of NF-kappa B activation induced by a wide variety of inflammatory stimuli, and this provides the molecular basis for its anti-inflammatory and immunosuppressive effects.

Ionic regulation of proton chemical (pH) and electrical gradients in lung lamellar bodies.

This study investigated the pH (chemical) and electrical gradients in lamellar bodies, the acidic surfactant-secreting organelles of lung epithelial type II cells, by following the uptake of a weak fluorescent base, quinacrine, and a membrane potential-sensitive dye, bis-(3-phenyl-5-oxoisoxazol-4-yl)pentamethine oxonol (oxonol V). In isolated lung lamellar bodies, the ATP-dependent uptake of both agents could be inhibited by bafilomycin A1, a reportedly specific inhibitor of vacuolar-type H(+)-ATPase (V-ATPase) and could be dissipated by a protonophore, carbonyl cyanide p-(trifluoromethoxy)phenylhydrazone, suggesting that the V-ATPase generates an electropositive interior. A closely linked uptake of Cl- neutralizes the positive electrical potential and increases the proton pump activity. The uptake of quinacrine, but not oxonol V, was decreased by Na+. This effect of Na+ could be prevented by dimethylamiloride, suggesting the presence of electroneutral Na+/H+ exchanger in lamellar body membranes. The initial rates of quinacrine and oxonol V uptake were increased by bumetanide, but only in the presence of Na+, K+, and Cl-, suggesting that the lamellar bodies also contain an outwardly directed electroneutral Na(+)-K(+)-2Cl- cotransporter. Thus three ion transporters, H(+)-translocating V-ATPase, Na+/H+ exchanger, and Na(+)-K(+)-2Cl- cotransporter, appear to determine the chemical and electrical gradients across the lamellar body membrane.

Inhibition of smooth muscle cell proliferation in vitro by leflunomide, a new immunosuppressant, is antagonized by uridine.

Chronic rejection in the form of graft vascular disease (GVD) continues to plague clinical transplantation of vascularized organs. The histopathology of this lesion is characterized by neointimal hyperplasia, smooth muscle cell proliferation, and obliterative arteriopathy. Due to the lack of effective medical therapy for preventing or reversing these chronic vascular changes, retransplantation remains the final resort in treatment. Some of the newer immunosuppressive agents, including the new isoxazole derivative leflunomide (LFM), have shown efficacy in preventing chronic rejection in animal models of transplantation. Although its mechanism of action remains incompletely elucidated, previous work using lymphocytes in vitro suggests that the drug might act as a tyrosine kinase inhibitor, an inhibitor of de novo pyrimidine biosynthesis, or both. In order to elucidate whether the efficacy of LFM in vivo is attributable not only to anti-proliferative effects on the recipient immune system but also to direct effects on mesenchymal cells in the donor organ, we examined the effects of LFM on a transformed 9E11G murine smooth muscle cell (M-SMC) line in vitro. We demonstrate here that the active metabolite of LFM, A77 1726, dose-dependently inhibits the constitutive and growth-factor stimulated proliferation of M-SMC in vitro. Furthermore, the anti-proliferative effect of the drug can be reversed by the addition of uridine to the culture medium. These results suggest that inhibition of uridine biosynthesis appears to be a mechanism by which LFM exerts anti-proliferative effects on both lymphocytes and smooth muscle cells, and this dual action may be responsible for its efficacy in preventing GVD in vivo.

Mechanism of the antiproliferative action of leflunomide. A77 1726, the active metabolite of leflunomide, does not block T-cell receptor-mediated signal transduction but its antiproliferative effects are antagonized by pyrimidine nucleosides.

BACKGROUND: Leflunomide, a novel immunosuppressive drug, prolongs experimental graft survival effectively and has been well tolerated in patients with rheumatoid arthritis. A77 1726, the active metabolite of leflunomide, inhibits lymphocyte proliferation in vitro. This study was conducted in Jurkat T cells to investigate the effects of A77 1726 on signal transduction pathways initiated by ligands of the T-cell receptor CD3 complex and to evaluate the effects of A77 1726 on nucleotide biosynthesis. METHODS: Tritiated thymidine incorporation and cell counts quantitated cell proliferation. Spectrofluorescence of Indo/AM dye measured intracellular Ca2+ mobilization. A luciferase assay quantitated interleukin-2 gene promoter activity in stimulated cells transfected with an interleukin-2 promoter-luciferase gene construct. Pyrimidine and purine nucleosides were used to assess antagonism of the antiproliferative activity of A77 1726. RESULTS: (1) A77 1726 dose-dependently inhibited the proliferation of Jurkat T cells (inhibitory concentration of 50% = 6 mumol/L); (2) A77 1726 did not decrease mobilization of intracellular Ca2+ stimulated by phytohemagglutinin or anti-CD3 monoclonal antibody; (3) A77 1726 did not inhibit interleukin-2 gene promoter activity in cells stimulated with ionomycin plus phorbol myristate acetate; (4) inhibition of cell proliferation by A77 1726 was antagonized by addition of uridine, cytidine, or 2(+)-deoxycytidine; (5) addition of uridine 24 hours after treatment with A77 1726 antagonized inhibition of proliferation; (6) A77 1726 was not antagonized by 2'-deoxyuridine, thymidine, adenosine, or guanosine. CONCLUSIONS: (1) A77 1726 inhibited Jurkat T-cell proliferation without inhibiting T-cell receptor-mediated signal transduction events, including tyrosine kinase-dependent intracellular Ca2+ mobilization and activation of the interleukin-2 gene promoter; (2) the antiproliferative effects of A77 1726 on Jurkat T cells are primarily due to interruption of de novo pyrimidine nucleotide biosynthesis. These data provide evidence for a novel in vitro mechanism of the antiproliferative action of this immunosuppressant.

Inhibition of smooth muscle cell proliferation in vitro by leflunomide, a new immunosuppressant, is antagonized by uridine.

Chronic rejection in the form of graft vascular disease (GVD) continues to plague clinical transplantation of vascularized organs. The histopathology of this lesion is characterized by neointimal hyperplasia, smooth muscle cell proliferation, and obliterative arteriopathy. Due to the lack of effective medical therapy for preventing or reversing these chronic vascular changes, retransplantation remains the final resort in treatment. Some of the newer immunosuppressive agents, including the new isoxazole derivative leflunomide (LFM), have shown efficacy in preventing chronic rejection in animal models of transplantation. Although its mechanism of action remains incompletely elucidated, previous work using lymphocytes in vitro suggests that the drug might act as a tyrosine kinase inhibitor, an inhibitor of de novo pyrimidine biosynthesis, or both. In order to elucidate whether the efficacy of LFM in vivo is attributable not only to anti-proliferative effects on the recipient immune system but also to direct effects on mesenchymal cells in the donor organ, we examined the effects of LFM on a transformed 9E11G murine smooth muscle cell (M-SMC) line in vitro. We demonstrate here that the active metabolite of LFM, A77 1726, dose-dependently inhibits the constitutive and growth-factor stimulated proliferation of M-SMC in vitro. Furthermore, the anti-proliferative effect of the drug can be reversed by the addition of uridine to the culture medium. These results suggest that inhibition of uridine biosythesis appears to be a mechanism by which LFM exerts anti-proliferative effects on both lymphocytes and smooth muscle cells, and this dual action may be responsible for its efficacy in preventing GVD in vivo.

In vitro toxicological investigations of isoxazolinone amino acids of Lathyrus sativus.

Two non-protein amino acids of Lathyrus sativus, beta-(isoxazoline-5-on-2-yl)-alanine (BIA) and its higher homologue alpha-amino-gamma-(isoxazoline-5-on-2-yl)-alanine (ACI) were tested for excitotoxic potential. BIA (0.5-2.0 mM) but not ACI (2.0 mM) produced a concentration-dependent neurodegeneration in mouse cortical explants. The neuronal damage was prevented by the prior and simultaneous application of 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX), indicating that it was mediated by non-N-methyl-D-aspartate type receptors. BIA (0.5-2.0 mM) activated CNQX-sensitive currents which were significantly smaller than those activated by 3-N-oxalyl-L-2,3-diaminopropanoic acid (beta-ODAP) or alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) in the majority of neurons. In a small number of cells, BIA (2 mM) produced currents which were similar in amplitude to those activated by beta-ODAP (50 microM). These results suggest that Lathyrus sativus plants engineered to block the synthesis of beta-ODAP may accumulate a neurotoxic precursor and therefore must be tested for the presence of both BIA and beta-ODAP.

Extrasynaptic effects of GABA (gamma-aminobutyric acid) agonists on myelinated axons of peripheral nerve.

Effects of the inhibitory neurotransmitter, GABA (gamma-aminobutyric acid) on the excitability of myelinated fibers of isolated amphibian sciatic nerves and their dorsal and ventral spinal roots have been compared with those of a GABAA agonist, THIP (4,5,6,7-tetrahydroisoxazolo[5,4-c]pyridin-3-ol), and the GABAB agonist baclofen. Graded, prolonged increases in the amplitude of A-fiber half-maximal compound action potentials of Rana ballenderi sciatic nerves were evoked by GABA (Rmax = 49%, EC50 = 0.1 mM); responses to THIP were smaller (Rmax = 34%, EC50 = 1.1 mM) and with a different, distinctly biphasic recovery phase. In studies of Rana catesbeiana nerves and their attached spinal roots, excitability increases produced in fibers of the ventral roots by GABA were smaller than those of the dorsal roots. Peak changes evoked by THIP in both roots were similar to the effects of GABA on the ventral root; however, THIP's ventral root response showed much less sensitivity and was followed by a rapid recovery phase, undershoot, and secondary, prolonged enhancement. Bicuculline methiodide antagonized agonist-induced increases, and revealed the presence of significant decreases in excitability of the ventral root fibers at concentrations of GABA or THIP < 3 mM. Baclofen evoked inconsistent changes in the excitability of whole nerve and root fibers; small increases occurred with lower doses and secondary, delayed decreases with higher doses. The high concentration (> or = 0.1 mM) of the active isomer needed to cause a small response suggests a limited contribution and (or) presence of GABAB receptors.(ABSTRACT TRUNCATED AT 250 WORDS)

Modulation of the seizure threshold for excitatory amino acids in mice by antiepileptic drugs and chemoconvulsants.

A novel method for the assessment of the threshold for clonic seizures induced by excitatory amino acids based on continuous infusion of the glutamate agonists [alpha-amino-3-hydroxy-5-terbutyl-4-isoxazolepropionate (ATPA), kainate or N-methyl-D-aspartate (NMDA)] into the lateral brain ventricle of unrestrained mice is reported. Using this novel method of seizure threshold determination, it was found that systemically administered diphenylhydantoin and carbamazepine elevated the threshold for ATPA and had negligible effects on the threshold for kainate and NMDA. Phenobarbital and trimethadione elevated the threshold for all excitatory amino acids tested, whereas valproate elevated the threshold for ATPA and kainate seizures. Ethosuximide elevated the threshold for ATPA and kainate and decreased the threshold for NMDA seizures. The quisqualate antagonists [2,3-dihydroxy-6-nitro-7-sulfamoyl-benzo(F)quinoxaline and 1-(4-aminophenyl)-4-methyl-7,8-methylenedioxy-5H-2,3-benzodiazepine] elevated the threshold for ATPA and less so for kainate seizures, whereas the NMDA antagonist 3-((+-)2-carboxypiperazine-4-yl)-propyl-1-phosphonate elevated the threshold for NMDA seizures. 1-(4-Aminophenyl)4-methyl-7,8-methylenedioxy-5H-2,3-benzodiazepine in higher doses was also active against NMDA seizures, whereas 3-((+-)2-carboxypiperazine-4-yl)-propyl-1-phosphonate did so with kainate seizures. Among seven different convulsants, pentylenetetrazol, picrotoxin and the beta-carboline methyl 6,7-dimethoxy-4-ethyl-beta-carboline-3-carboxylate lowered the threshold for seizures induced by excitatory amino acids. Pentylenetetrazol and picrotoxin did so with kainate seizures, whereas methyl 6,7-dimethoxy-4-ethyl-beta-carboline-3-carboxylate lowered ATPA thresholds. Bicuculline, 3-mercaptopropionate, strychnine and pilocarpine were inactive.(ABSTRACT TRUNCATED AT 250 WORDS)