The preclinical pharmacological profile of WAY-132983, a potent M1 preferring agonist.
(9/274)Muscarinic M1 preferring agonists may improve cognitive deficits associated with Alzheimer's disease. Side effect assessment of the M1 preferring agonist WAY-132983 showed significant salivation (10 mg/kg i.p. or p.o.) and produced dose-dependent hypothermia after i. p. or p.o. administration. WAY-132983 significantly reduced scopolamine (0.3 mg/kg i.p.)-induced hyperswimming in mice. Cognitive assessment in rats used pretrained animals in a forced choice, 1-h delayed nonmatch-to-sample radial arm maze task. WAY-132983 (0.3 mg/kg i.p) significantly reduced scopolamine (0.3 mg/kg s.c.)-induced errors. Oral WAY-132983 attenuated scopolamine-induced errors; that is, errors produced after combining scopolamine and WAY-132983 (to 3 mg/kg p.o.) were not significantly increased compared with those of vehicle-treated control animals, whereas errors after scopolamine were significantly higher than those of control animals. With the use of miniosmotic pumps, 0.03 mg/kg/day (s.c.) WAY-132983 significantly reduced AF64A (3 nmol/3 microliter/lateral ventricle)-induced errors. Verification of AF64A cholinotoxicity showed significantly lower choline acetyltransferase activity in the hippocampi of AF64A-treated animals, with no significant changes in the striatal or frontal cortex. Cognitive assessment in primates involved the use of pretrained aged animals in a visual delayed match-to-sample procedure. Oral WAY-132983 significantly increased the number of correct responses during short and long delay interval testing. These effects were also apparent 24 h after administration. WAY-132983 exhibited cognitive benefit at doses lower than those producing undesirable effects; therefore, WAY-132983 is a potential candidate for improving the cognitive status of patients with Alzheimer's disease. (+info)
Prostate-specific antigen promoter/enhancer driven gene therapy for prostate cancer: construction and testing of a tissue-specific adenovirus vector.
(10/274)A range of luciferase reporter vectors was constructed, incorporating 5'-flanking sequences from the prostate-specific antigen (PSA), human glandular kallikrein 2 (hKLK2), and cytomegalovirus (CMV) promoters for expression control. Tissue specificity was evaluated in the PSA-positive line LNCaP and PSA-negative cells from different tissues of origin (CoLo320, DG75, EJ, A2780, and Jurkat). The minimal 628-bp PSA and hKLK2 promoters showed only low-level expression in either PSA-positive or PSA-negative cells and showed no increase with the addition of androgen. Tandem duplication of the PSA promoter slightly increased expression in PSA-positive LNCaP cells. The addition of CMV enhancer sequences upstream of a single PSA or hKLK2 promoter substantially but nonspecifically increased luciferase expression in all cell lines tested. However, placing a 1455-bp PSA enhancer sequence upstream of either the PSA or hKLK2 promoters increased expression 20-fold in the PSA-positive cell line LNCaP but not in the PSA-negative lines. Tandem duplication of the PSA enhancer increased expression to approximately 50-fold higher than either promoter alone while retaining tissue-specific control. The level of expression was reduced by the addition of a third copy of the PSA enhancer. Expression from all enhancer constructs was increased 100-fold above basal levels when induced with the androgen dihydrotestosterone, with the PSA-based constructs consistently exhibiting roughly twice the level of expression of the hKLK2-based constructs at all androgen concentrations. Adenovirus vectors were produced in which either enhanced green fluorescent protein or nitroreductase could be expressed from the optimized PSA double enhancer-promoter construct and evaluated in LNCaP cells and the bladder-derived line EJ. Control vectors with the CMV promoter gave good levels of expression in both cell lines, whereas the PSA constructs only produced detectable levels of protein in the LNCaP cells as assessed by fluorescence of enhanced green fluorescent protein or by Western blotting of nitroreductase. LNCaP but not EJ cells were selectively sensitized to the prodrug CB1954 following infection with Ad-PSA(EEP)-NR. The PSA-based nitroreductase virus produced comparable amounts of nitroreductase and sensitization to CB1954 approaching that of the CMV-driven virus. Plasmid and adenovirus constructs combining PSA enhancer and promoter sequences demonstrate selective expression of linked genes in PSA-positive cells. The expression is induced by androgen and gives therapeutically relevant levels of effector proteins. (+info)
The nitroreductase/CB1954 combination in Epstein-Barr virus-positive B-cell lines: induction of bystander killing in vitro and in vivo.
(11/274)Epstein-Barr virus (EBV)-based gene delivery vectors that preferentially express toxic genes in EBV-infected cells could be used to target EBV-positive tumors for destruction. We have shown previously that the cytosine deaminase (CD) enzyme, which converts the prodrug 5-fluorocytosine (5-FC) into the toxic compound 5-fluorouracil efficiently kills EBV-positive cells in the presence of 5-FC, with a substantial bystander killing effect in vitro and in vivo. To identify the optimal enzyme/prodrug combination for treating EBV-positive lymphomas, we have compared the effectiveness of the CD/5-FC combination with the nitroreductase (NTR)/CB1954 combination for killing EBV-positive B-cell lines. NTR metabolizes CB1954 into an alkylating agent that cross-links DNA. When the CD gene or the NTR gene were transfected into two different EBV-positive B-cell lines in vitro, approximately 90% of cells were killed in a prodrug-dependent manner, although the transfection efficiency was <5%. However, severe combined immunodeficient mouse tumors containing either 30% or 100% of NTR-expressing Burkitt lymphoma (Jijoye) cells were growth inhibited, but not cured, by treatment with intraperitoneal CB1954 (20 mg/kg/day) for 10 days. These results suggest that the NTR/CB1954 combination induces efficient bystander killing of EBV-positive B-cell lines in vitro but may not be as effective as the CD/5-FC combination for treating B-cell lymphomas in vivo. (+info)
Expression of Escherichia coli B nitroreductase in established human tumor xenografts in mice results in potent antitumoral and bystander effects upon systemic administration of the prodrug CB1954.
(12/274)Expression of the Escherichia coli enzyme nitroreductase (NTR) in mammalian cells enables them to activate the prodrug 5-(aziridin-1-yl)-2,4-dinitrobenzamide (CB1954), leading to interstrand DNA cross-linking and apoptosis in both proliferating and quiescent cells. In the work reported here, we used human hepatocellular carcinoma and squamous carcinoma cell lines constitutively expressing NTR to demonstrate that the ntr/CB1954 system results in potent, long-lasting antitumoral effects in mice. We also demonstrate that this enzyme/prodrug combination results in antitumoral effects in vivo when only a minority of tumor cells express the enzyme, using either cells constitutively expressing NTR or ntr gene delivery in situ. (+info)
Activity-dependent neurotrophic factor: intranasal administration of femtomolar-acting peptides improve performance in a water maze.
(13/274)Activity-dependent neurotrophic factor (ADNF) is a glia-derived protein that is neuroprotective at femtomolar concentrations. A nine-amino acid peptide derived from ADNF (Ser-Ala-Leu-Leu-Arg-Ser-Ile-Pro-Ala; ADNF-9) captured the activity of the parent protein and has been reported to protect cultured neurons from multiple neurotoxins. Antibodies recognizing ADNF-9 produced neuronal apoptosis, and identified an additional, structurally related, glia-derived peptide, Asn-Ala-Pro-Val-Ser-Ile-Pro-Gln (NAP). Previous comparative studies have characterized s.c.-injected NAP as most efficacious in protecting against developmental retardation and learning impairments in apolipoprotein E-deficient mice. This study was designed to assess 1) neuroprotection after intranasal administration of ADNF-9 and NAP to rats treated with the cholinotoxin ethylcholine aziridium; and 2) bioavailability and pharmacokinetics after intranasal administration. Results showed significant improvements in short-term spatial memory, as assessed in a water maze, after daily intranasal administration of 1 microg of peptide (ADNF-9 or NAP) per animal. However, a 5-day pretreatment with ADNF-9 did not improve performance measured after cessation of treatment. Compared with rats treated with ADNF-9, NAP-pretreated animals exhibited a significantly better performance. Furthermore, NAP (and not ADNF-9) protected against loss of choline acetyl transferase activity. Significant amounts of (3)H-labeled NAP reached the brain, remained intact 30 min after administration, and dissipated 60 min after administration. This study revealed efficacy for ADNF-related peptides in rodent models for neurodegeneration. The small size of the molecules, the low dosage required, the noninvasive administration route, and the demonstrated activity in a relevant paradigm suggest NAP as a lead compound for future drug design. (+info)
Cellular origin of regenerating parenchyma in a mouse model of severe hepatic injury.
(14/274)Several treatments in rodents, including administration of the alkylating agent dipin, followed by two-thirds partial hepatectomy in mice combine destruction of liver parenchyma with hepatocyte mitoinhibition. These treatments induce proliferation of bile epithelial-like cells (termed oval cells), development of foci composed of small hepatocytes, and eventual replacement of damaged parenchyma by healthy hepatocytes. It has been proposed that these oval cells represent transitional cells in a nonhepatocytic liver facultative stem cell lineage that can give rise to the small hepatocyte foci, and that these foci eventually become confluent and replace liver parenchyma. In this study, we used in vivo cell lineage marking in genetically chimeric livers to test the hypothesis that hepatocytes can serve as the precursor cell type to the small hepatocyte foci that develop in mouse liver after treatment with dipin plus partial hepatectomy. Although we do not exclude the possibility that some small hepatocyte foci may be stem cell-derived, we demonstrate that hepatocyte-derived foci are present after dipin-induced liver damage in mice. (+info)
Bioactivation of 5-(aziridin-1-yl)-2,4-dinitrobenzamide (CB 1954) by human NAD(P)H quinone oxidoreductase 2: a novel co-substrate-mediated antitumor prodrug therapy.
(15/274)A novel prodrug activation system, endogenous in human tumor cells, is described. A latent enzyme-prodrug system is switched on by a simple synthetic, small molecule co-substrate. This ternary system is inactive if any one of the components is absent. CB 1954 [5-(aziridin-1-yl)-2,4-dinitrobenzamide] is an antitumor prodrug that is activated in certain rat tumors via its 4-hydroxylamine derivative to a potent bifunctional alkylating agent. However, human tumor cells are resistant to CB 1954 because they are unable to catalyze this bioactivation efficiently. A human enzyme has been discovered that can activate CB 1954, and it has been shown to be commonly present in human tumor cells. The enzyme is NQO2 [NAD(P)H quinone oxidoreductase 2], but its activity is normally latent, and a nonbiogenic co-substrate such as NRH [nicotinamide riboside (reduced)] is required for enzymatic activity. There is a very large (100-3000-fold) increase in CB 1954 cytotoxicity toward either NQO2-transfected rodent or nontransfected human tumor cell lines in the presence of NRH. Other reduced pyridinium compounds can also act as co-substrates for NQO2. Thus, the simplest quaternary salt of nicotinamide, 1-methyl-3-carboxamidopyridinium iodide, was a co-substrate for NQO2 when reduced to the corresponding 1,4-dihydropyridine derivative. Increased chain length and/or alkyl load at the 1-position of the dihydropyridine ring improved specific activity, and compounds more active than NRH were found. However, little activity was seen with either the 1-benzyl or 1-(2-phenylethyl) derivatives. A negatively charged substituent at the 3-position of the reduced pyridine ring also negated the ability of these compounds to act as cosubstrates for NQO2. In particular, 1-carbamoylmethyl-3-carbamoyl-1,4dihydropyridine was shown to be a co-substrate for NQO2 with greater stability than NRH, with the ability to enter cells and potentiate the cytotoxicity of CB 1954. Furthermore, this agent is synthetically accessible and suitable for further pharmaceutical development. NQO2 activity appears to be related to expression of NQO1 (DT-diaphorase), an enzyme that is known to have a favorable distribution toward certain human cancers. NQO2 is a novel target for prodrug therapy and has a unique activation mechanism that relies on a synthetic co-substrate to activate an apparently latent enzyme. Our findings may reopen the use of CB 1954 for the direct therapy of human malignant disease. (+info)
Melatonin is protective in necrotic but not in caspase-dependent, free radical-independent apoptotic neuronal cell death in primary neuronal cultures.
(16/274)To assess the neuroprotective potential of melatonin in apoptotic neuronal cell death, we investigated the efficacy of melatonin in serum-free primary neuronal cultures of rat cortex by using three different models of caspase-dependent apoptotic, excitotoxin-independent neurodegeneration and compared it to that in necrotic neuronal damage. Neuronal apoptosis was induced by either staurosporine or the neurotoxin ethylcholine aziridinium (AF64A) with a delayed occurrence of apoptotic cell death (within 72 h). The apoptotic component of oxygen-glucose deprivation (OGD) unmasked by glutamate antagonists served as a third model. As a model for necrotic cell death, OGD was applied. Neuronal injury was quantified by LDH release and loss of metabolic activity. Although melatonin (0.5 mM) partly protected cortical neurons from OGD-induced necrosis, as measured by a significant reduction in LDH release, it was not effective in all three models of apoptotic cell death. In contrast, exaggeration of neuronal damage by melatonin was observed in native cultures as well as after induction of apoptosis. The present data suggest that the neuroprotectiveness of melatonin strongly depends on the model of neuronal cell death applied. As demonstrated in three different models of neuronal apoptosis, the progression of the apoptotic type of neuronal cell death cannot be withhold or is even exaggerated by melatonin, in contrast to its beneficial effect in the necrotic type of cell death. (+info)