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Papers by Steven Duddy

Journal of Pharmacology and Experimental Therapeutics, Apr 17, 2017

Alzheimer's disease (AD) is characterized neuropathologically by an abundance of 1) neuritic plaq... more Alzheimer's disease (AD) is characterized neuropathologically by an abundance of 1) neuritic plaques, which are primarily composed of a fibrillar 42-amino-acid amyloid-b peptide (Ab), as well as 2) neurofibrillary tangles composed of aggregates of hyperphosporylated tau. Elevations in the concentrations of the Ab42 peptide in the brain, as a result of either increased production or decreased clearance, are postulated to initiate and drive the AD pathologic process. We initially introduced a novel class of bridged aromatics referred tg-secretase modulatoro as g-secretase modulators that inhibited the production of the Ab42 peptide and to a lesser degree the Ab40 peptide while concomitantly increasing the production of the carboxyltruncated Ab38 and Ab37 peptides. These modulators potently lower Ab42 levels without inhibiting the g-secretase-mediated proteolysis of Notch or causing accumulation of carboxyl-terminal fragments of APP. In this study, we report a large number of pharmacological studies and early assessment of toxicology characterizing a highly potent g-secretase modulator (GSM), (S)-N-(1-(4-fluorophenyl)ethyl)-6-(6-methoxy-5-(4-methyl-1H-imidazol-1-yl)pyridin-2-yl)-4-methylpyridazin-3-amine (BPN-15606). BPN-15606 displayed the ability to significantly lower Ab42 levels in the central nervous system of rats and mice at doses as low as 5-10 mg/kg, significantly reduce Ab neuritic plaque load in an AD transgenic mouse model, and significantly reduce levels of insoluble Ab42 and pThr181 tau in a threedimensional human neural cell culture model. Results from repeatdose toxicity studies in rats and dose escalation/repeat-dose toxicity studies in nonhuman primates have designated this GSM for 28day Investigational New Drug-enabling good laboratory practice studies and positioned it as a candidate for human clinical trials.

Preclinical development of an EP2 antagonist for post-seizure cognitive deficits

Neuropharmacology

Macrophage Activation, and Increased Angiogenic Growth Factors in Mice but Not in Rats Pregabalin-Treated Mice: Evidence of Increased Bicarbonate, Dysregulated Erythropoiesis, Key Components of the Mode of Action for Hemangiosarcoma Induction in

2,5-Di-(tert-butyl)-1,4-benzohydroquinone rapidly elevates cytosolic Ca2+ concentration by mobilizing the inositol 1,4,5-trisphosphate-sensitive Ca2+ pool

Journal of Biological Chemistry, 1989

2,5-Di-(tert-butyl)-1,4-benzohydroquinone (tBuBHQ), a potent inhibitor of liver microsomal ATP-de... more 2,5-Di-(tert-butyl)-1,4-benzohydroquinone (tBuBHQ), a potent inhibitor of liver microsomal ATP-dependent Ca2+ sequestration (Moore, G. A., McConkey, D. J., Kass, G. E. N., O'Brien, P. J., and Orrenius, S. (1987) FEBS Lett. 224, 331-336), produced a concentration-dependent, rapid increase in cytosolic free Ca2+ concentration ([Ca2+]i) in isolated rat hepatocytes (EC50 = 1-2 microM). The amplitude of the [Ca2+]i increase was essentially identical with that produced by vasopressin, but the tBuBHQ-stimulated [Ca2+]i increase remained sustained for 15-20 min. Vasopressin added 2-3 min after tBuBHQ caused [Ca2+]i to rapidly return to basal levels; however, tBuBHQ added after vasopressin resulted in a Ca2+ transient rather than a sustained [Ca2+]i elevation. Ca2+ influx was not stimulated in tBuBHQ-treated hepatocytes, but was markedly enhanced upon addition of vasopressin. Depletion of the endoplasmic reticular Ca2+ pool by the addition of vasopressin to hepatocytes incubated in low Ca2+ medium virtually abolished the tBuBHQ-mediated [Ca2+]i rise and vice versa. In saponin-permeabilized hepatocytes, tBuBHQ released Ca2+ from the same nonmitochondrial, ATP-dependent Ca2+ pool which was released by inositol 1,4,5-trisphosphate. Furthermore, tBuBHQ-induced Ca2+ release in saponin-permeabilized cells was not inhibited by neomycin, and tBuBHQ did not produce any apparent accumulation of inositol phosphates in intact hepatocytes. The rate of passive efflux of Ca2+ from Ca2+-loaded hepatic microsomes was unaltered by tBuBHQ. Thus, tBuBHQ inhibits ATP-dependent Ca2+ sequestration via a direct effect on the endoplasmic reticulum Ca2+ pump, resulting in net Ca2+ release and elevation of [Ca2+]i. Taken together, our results show that in the absence of hormonal stimuli, excess Ca2+ is only slowly cleared from the hepatocyte cytosol, indicating that the basal rate of Ca2+ removal by the plasma membrane Ca2+ pump and mitochondria is slow. Furthermore, Ca2+-mobilizing hormones appear to stimulate an active process of Ca2+ removal from hepatocyte cytosol which does not depend on re-uptake into the endoplasmic reticulum.

The Journal of pharmacology and experimental therapeutics, Jan 17, 2017

Alzheimer's disease is characterized neuropathologically by an abundance of 1) neuritic plaqu... more Alzheimer's disease is characterized neuropathologically by an abundance of 1) neuritic plaques, which are primarily composed of a fibrillar 42 amino acid amyloid β peptide, as well as 2) neurofibrillary tangles composed of aggregates of hyperphosporylated tau. Elevations in the concentrations of the Aβ42 peptide in the brain, as a result of either increased production or decreased clearance are postulated to initiate and drive the AD pathological process. We initially introduced a novel class of bridged aromatics referred to as γ-secretase modulators that inhibited the production of the Aβ42 peptide and to a lesser degree the Aβ40 peptide while concomitantly increasing the production of the carboxyl-truncated Aβ38 and Aβ37 peptides. These modulators potently lower Aβ42 levels without inhibiting the γ-secretase-mediated proteolysis of Notch or causing accumulation of carboxyl-terminal fragments of APP. Herein we report a large number of pharmacological studies and early assessme...

BHQ/rapidly elevates cytosolic Ca2+ concentration by mobilizing the inositol 1, 4,5-trisphosphate sensitive Ca2+ pool

Journal of Biological Chemistry

ABSTRACT

Oncogene, 1999

Relatively little is known about the molecular mechanisms of tumor promotion/progression in mamma... more Relatively little is known about the molecular mechanisms of tumor promotion/progression in mammary carcinogenesis. Increased protein kinase C (PKC) activity is known to promote tumor formation in several tissues; however, its role in mammary carcinogenesis is not yet known. To determine if individual PKCs may selectively regulate properties of mammary tumor cells, we compared PKC isozyme levels in mammary tumor cell lines with low, moderate and high metastatic potential. All three cell lines expressed a, d, e and z PKCs; however, PKCd levels were relatively increased in the highly metastatic cells. To determine if increased PKCd could contribute to promotion/progression, we overexpressed PKCd in the low and moderately metastatic cell lines. PKCd overexpression had no signi®cant eect on growth of adherent cells, but signi®cantly increased anchorage-independent growth. Conversely, expressing the regulatory domain of PKCd (RDd), a putative PKCd inhibitory fragment, inhibited anchorage-independent growth. The ecacy of RDd as a PKCd inhibitor was demonstrated by showing that RDd selectively interfered with PKCd subcellular location and signi®cantly interfered with phosphorylation of the PKC cytoskeletal substrate, adducin. PKC-dependent phosphorylation of cytoskeletal substrate proteins, such as adducin, provides a mechanistic link between increased PKCd activity and phenotypic changes in cytoskeletaldependent processes such as migration and attachment, two processes that are relevant to metastatic potential. The reciprocal growth eects of expressing PKCd and RDd as gain and loss of function constructs, respectively, provide strong evidence that PKCd regulates processes important for anchorage-independent growth in these mammary tumor cells.

2,5-Di(TERT-BUTYL)-1,4-BENZOHYDROQUINONE - TRISPHOSPHATE-SENSITIVE Ca2+ POOL A NOVEL MOBILIZER OF THE INOSITOL 1,4,5

Isolated hepatocytes and the isolated perfused rat liver have been used to study the alterations ... more Isolated hepatocytes and the isolated perfused rat liver have been used to study the alterations of cytosolic free Ca’+ concentration ([Ca”],) produced by 2.5-di(tert-butyl)-l.4-benzohydroquinone (tBuBHQ), a potent inhibitor of hepatic microsomal Ca’+ sequestration (Moore. G.A., McConkey. D.J., Kass, G.E.N., OBrien, P.J. and Orrenius, S. FEBS Lett.. 224, 331-336). (1987). Addition of tBuBHQ to isolated hepatocytes caused a rapid increase in [Ca’+], which was similar in magnitude to the [CaL+], elevation induced by the Ca’+ mobilizing hormone, vasopressin. In contrast with vasopressin which caused a Ca” transient, tBuBHQ elevated [Ca”], to a new steady state that was maintained for up to 15-20min. When vasopressin was administered during the t BuBHQ-induced period of elevated [Ca” I,. [Ca” 1, rapidly returned to basal levels. Similarly, if vasopressin was administered just prior to tBuBHQ, the resultant tBuBHQ-dependent change in [Ca’+ 1, was transient. and not sustained. The hydroqu...

FUNDAMENTAL AND APPLIED TOXICOLOGY 9, 304-3 13(1987) Cytoprotective Effects of Modulators of Oxidative Xenobiotic Metabolism in

304-313. This investigation was conducted to evaluate the effects of modulation of several phase ... more 304-313. This investigation was conducted to evaluate the effects of modulation of several phase I xenobiotic-metabolizing enzyme activities on the expression of precocene H-induced hepato-toxicity. Precocene II (175-200 mg/kg) was given intraperitoneally to male Sprague-Dawley rats that had been exposed previously to inducers (phenobarbital and 3-methylcholanthrene) or inhibitors (SICF 525-A and cimetidine) of oxidative xenobiotic metabolism. Hepatic damage was measured both biochemically (leakage of aspartate aminotransferase and alanine amino-transferase into the serum) and histologically. Significant protection from precocene Il-induced hepatotoxicity was observed in all treated animals regardless of whether the modulator em-ployed was an inducer or an inhibitor of microsomal oxidative enzymes. These results indicate that the level of activity of various forms of cytochrome P-450 significantly influences the sever-ity of hepatic necrosis induced by precocene II. Furthermore, the...

Stabilisierte multifunktionale antioxidative verbindungen und verfahren zur verwendung

Cell growth & differentiation : the molecular biology journal of the American Association for Cancer Research, 1995

The tumorigenic phenotype in rat liver epithelial cells overexpressing c-myc may depend on a tran... more The tumorigenic phenotype in rat liver epithelial cells overexpressing c-myc may depend on a transforming growth factor (TGF)-alpha/epidermal growth factor receptor autocrine loop (L. W. Lee et al., Cancer Res., 51: 5238-5244, 1991). In the present study, we have used constitutive sense and antisense TGF-alpha expression vectors to modify TGF-alpha production in carcinogen-transformed clonal derivatives of a rat liver epithelial cell line, WB-F344, that variably express c-myc, endogenous TGF-alpha, and tumorigenicity. Transgene-mediated TGF-alpha protein production was elevated 2- to 9-fold in derivatives of a low c-myc-expressing transformed cell line, GN4, and 35-fold in a derivative of a high c-myc-expressing cell line, GN6. Although the GN4- and GN6-derived cell lines expressed functional EGF receptor and steady-state c-myc mRNA levels that were comparable to their respective parental cell lines, increased TGF-alpha expression did not increase the tumorigenicity of the derivativ...

Spontaneous and Thiazolidinedione-Induced B6C3F1 Mouse Hemangiosarcomas Exhibit Low ras Oncogene Mutation Frequencies

Toxicology and Applied Pharmacology, 1999

Hemangiosarcomasare uncommon malignant endothelial cell tumors in humans and experimental animal ... more Hemangiosarcomasare uncommon malignant endothelial cell tumors in humans and experimental animal species. The mechanisms giving rise to these tumors are poorly understood even though the histotypes are comparable between humans and rodents. Activating mutations in cellular ras protooncogenes have been detected in sporadic and chemically induced human and rodent hemangiosarcomas. Ras activation significantly modulates tumor angiogenesis, suggesting that mutations in ras genes might be causally related to vascular tumorigenesis. To more clearly define the role of ras in experimental vascular tumorigenesis, mutations in the Ki- and Ha-ras genes were characterized in 63 hemangiosarcomas that arose unexpectedly in control and treated B6C3F1 mice during a two-year carcinogenicity study of the thiazolidinedione troglitazone. DNA was extracted from paraffin sections of mouse hemangiosarcomas, control liver, or positive control hepatocellular carcinomas with defined mutations in the Ki- or Ha-ras genes. Exons 1 and 2 of the Ki- and Ha-ras genes were independently amplified using primer extension preamplification/locus-specific heminested PCR, and PCR amplicons were directly sequenced to identify mutations in codons 12, 13, or 61. Activating mutations were detected in 3 of 63 hemangiosarcomas: a single G-->A transition in the second position of Ki-ras codon 13 in a tumor from a treated animal and two G-->T transversions in the second position of Ha-ras codon 13, one in a single tumor from a control animal and one in a tumor from a treated animal. These mutations are consistent with endogenous mutagenesis arising from oxidative DNA damage. The low frequency of mutation (<5%) indicates that ras mutations did not contribute significantly to hemangiosarcoma development and suggests that mutational ras activation may not be a necessary step in vascular tumorigenesis in mice.

Journal of Biological Chemistry, 1996

We have used an interaction cloning strategy to isolate cDNAs for sequences that interact with pr... more We have used an interaction cloning strategy to isolate cDNAs for sequences that interact with protein ki

Mutation Research/Environmental Mutagenesis and Related Subjects, 1990

Calcium and signal transduction in oxidative cell damage

Calcium, Oxygen Radicals and Cellular Damage, 1991

... In Calcium and Cell Function vol. IV, ed. WY Cheung, pp. 376-410. Orlando, Fl: Academic Press... more ... In Calcium and Cell Function vol. IV, ed. WY Cheung, pp. 376-410. Orlando, Fl: Academic Press. ... Role of thiols in protection against biological reactive intermediates. In Biological Reactive Intermediates III, ed. JJ Kocsis, DJ Jollow, CM Witmer, JO Nelson & R. Snyder, pp. 41-51. ...

Eicosanoids released following inhibition of the endoplasmic reticulum Ca2+ pump stimulate Ca2+ efflux in the perfused rat liver

Biochemical Pharmacology, 1993

In the isolated perfused rat liver 2,5-di(tert-butyl)hydroquinone (tBuHQ), a selective inhibitor ... more In the isolated perfused rat liver 2,5-di(tert-butyl)hydroquinone (tBuHQ), a selective inhibitor of the endoplasmic reticulum Ca2+ pump, induces a prolonged glucose output and stimulates Ca2+ efflux. The present study shows that tBuHQ depleted the hormone-sensitive Ca2+ pool in the perfused liver, abolishing the vasopressin- or phenylephrine-induced Ca2+ efflux. The effects of tBuHQ were reversible, since the response to these agonists gradually returned within 1 hr of perfusion, and protein synthesis was not required for this recovery. Since tBuHQ does not cause Ca2+ efflux from isolated hepatocytes, we examined the mechanism responsible for the tBuHQ-induced Ca2+ efflux observed in the intact liver. The cyclooxygenase inhibitor indomethacin prevented the Ca2+ extrusion stimulated by tBuHQ, but not that induced by vasopressin. During infusion of tBuHQ there was a 9-fold increase in the concentration of thromboxane B2 in the perfusate. The Ca2+ efflux response to tBuHQ was inhibited by the thromboxane/prostaglandin endoperoxide receptor antagonist, L-655,240 (3-[1-(4-chlorobenzyl)-5-fluoro-3-methyl-indol-2-yl]2,2-dimethylpropa noic acid) in the absence of any effect on thromboxane B2 release. Thus, the inhibition of the endoplasmic reticulum Ca2+ pump by tBuHQ results in a rise in the cytosolic Ca2+ concentration in non-parenchymal cells, leading to the formation of cyclooxygenase products. The released eicosanoids, in turn, stimulate Ca2+ efflux from hepatocytes.

Biochemical Pharmacology, 1989

Ebselen (PZ51, 2-phenyl-1,2-benzoisoselenazol-3-(2H)-one) was shown to be an inhibitor of human g... more Ebselen (PZ51, 2-phenyl-1,2-benzoisoselenazol-3-(2H)-one) was shown to be an inhibitor of human granulocyte oxidative burst stimulated by phorbol myristate acetate (ICY 25 PM). Estimation of the primary oxygen metabolites of the burst was complicated by the redox chemistry of Ebselen. Ebselen inhibited NADPH-stimulated superoxide generation by a partially purified NADPH oxidase preparation with an ICY,, of OS-l.0 PM. Ebselen was also shown to inhibit the activity of partially purified Ca*+-and phospholipid-dependent protein kinase C (ICY cn. 0.5 PM). Phorbol ester-stimulated phosphorylation of protein in intact cells was inhibited by Ebselen (IC,, cu. 50 ,uM). These pharmacodynamic properties of Ebselen are discussed in terms of its anti-inflammatory activity in uiuo. The findings are also discussed in terms of Ebselen's known abilitv to interact with sulfhydryl components of cells, particularly critical

Metabolism of Fluvalinate by Laying Hens

Journal of Agricultural and Food Chemistry, 1982

... SOC. 1960,82, 2027. Fieser, LF; Fieser, M. Steroids; Reinhold: New York, 1959; pp 53,424.Fi... more ... SOC. 1960,82, 2027. Fieser, LF; Fieser, M. Steroids; Reinhold: New York, 1959; pp 53,424.Fieser, M.; Fieser, L. 'Reagents for Organic Synthesis; Wiley-Interscience: New York, 1969; Vol. 2, p 363. ... Luana E. Staiger, Gary B. Quietad,* Steven K. Duddy, and David A. Schooley ...

Preclinical validation of a potent γ-secretase modulator for Alzheimer’s disease prevention

Journal of Experimental Medicine

A potent γ-secretase modulator (GSM) has been developed to circumvent problems associated with γ-... more A potent γ-secretase modulator (GSM) has been developed to circumvent problems associated with γ-secretase inhibitors (GSIs) and to potentially enable use in primary prevention of early-onset familial Alzheimer’s disease (EOFAD). Unlike GSIs, GSMs do not inhibit γ-secretase activity but rather allosterically modulate γ-secretase, reducing the net production of Aβ42 and to a lesser extent Aβ40, while concomitantly augmenting production of Aβ38 and Aβ37. This GSM demonstrated robust time- and dose-dependent efficacy in acute, subchronic, and chronic studies across multiple species, including primary and secondary prevention studies in a transgenic mouse model. The GSM displayed a >40-fold safety margin in rats based on a comparison of the systemic exposure (AUC) at the no observed adverse effect level (NOAEL) to the 50% effective AUC or AUCeffective, the systemic exposure required for reducing levels of Aβ42 in rat brain by 50%.