Anthony F. Kreft

Acute γ-Secretase Inhibition Improves Contextual Fear Conditioning in the Tg2576 Mouse Model of Alzheimer's Disease

Transgenic mice (Tg2576) overexpressing the Swedish mutation of the human amyloid precursor protein display biochemical, pathological, and behavioral markers consistent with many aspects of Alzheimers disease, including impaired hippocampal function. Impaired, hippocampal-dependent, contextual fear conditioning (CFC) is observed in mice as young as 20 weeks of age. This impairment can be attenuated after treatment before training with the phosphodiesterase-4 inhibitor rolipram (0.1 mg/kg, i.p.). A rolipram-associated improvement is also observed in the littermate controls, suggesting that the effect of rolipram is independent of β-amyloid. Acute treatment before training (but not after training or before testing) with the γ-secretase inhibitor (GSI) N-[N-(3,5-difluorophenacetyl)-l-alanyl]-S-phenylglycine-t-butylester (DAPT), at a dose that reduces brain concentrations of β-amyloid (100 mg/kg), attenuates the impairment in 20- to 65-week-old Tg2576 mice. Importantly, DAPT had no effect on performance of control littermates. These data are supportive of a role of β-amyloid in the impairment of CFC in Tg2576 mice. Furthermore, they suggest that acute treatment with GSI may provide improved cognitive functioning as well as disease-modifying effects in Alzheimers disease.

Etodolac selectively inhibits human prostaglandin G/H synthase 2 (PGHS-2) versus human PGHS-1

The isozymes of prostaglandin G/H synthase (PGHS) are shown to be differentially inhibited in vitro by currently marketed nonsteroidal anti-inflammatory drugs (NSAIDs) using microsomal rhPGHS-1 and rhPGHS-2. Comparison of selectivity ratios (IC50 rhPGHS-1/IC50 rhPGHS-2) demonstrated a 10-fold selectivity of etodolac (Lodine) for rhPGHS-2, whereas the other NSAIDs evaluated demonstrated no preference or a slight preference for inhibition of rhPGHS-1. In vitro enzyme results were supported by a human whole blood assay where etodolac also demonstrated a 10-fold selectivity for inhibition of PGHS-2 mediated TxB2 production. Taken together, these data may be key to explaining the clinically observed gastrointestinal safety of etodolac versus other marketed NSAIDs.

Begacestat (GSI-953): A Novel, Selective Thiophene Sulfonamide Inhibitor of Amyloid Precursor Protein γ-Secretase for the Treatment of Alzheimer's Disease

The presenilin containing γ-secretase complex is responsible for the regulated intramembraneous proteolysis of the amyloid precursor protein (APP), the Notch receptor, and a multitude of other substrates. γ-Secretase catalyzes the final step in the generation of Aβ40 and Aβ42 peptides from APP. Amyloid β-peptides (Aβ peptides) aggregate to form neurotoxic oligomers, senile plaques, and congophilic angiopathy, some of the cardinal pathologies associated with Alzheimers disease. Although inhibition of this protease acting on APP may result in potentially therapeutic reductions of neurotoxic Aβ peptides, nonselective inhibition of the enzyme may cause severe adverse events as a result of impaired Notch receptor processing. Here, we report the preclinical pharmacological profile of GSI-953 (begacestat), a novel thiophene sulfonamide γ-secretase inhibitor (GSI) that selectively inhibits cleavage of APP over Notch. This GSI inhibits Aβ production with low nanomolar potency in cellular and cell-free assays of γ-secretase function, and displaces a tritiated analog of GSI-953 from enriched γ-secretase enzyme complexes with similar potency. Cellular assays of Notch cleavage reveal that this compound is approximately 16-fold selective for the inhibition of APP cleavage. In the human APP-overexpressing Tg2576 transgenic mouse, treatment with this orally active compound results in a robust reduction in brain, plasma, and cerebral spinal fluid Aβ levels, and a reversal of contextual fear-conditioning deficits that are correlated with Aβ load. In healthy human volunteers, oral administration of a single dose of GSI-953 produces dose-dependent changes in plasma Aβ levels, confirming pharmacodynamic activity of GSI-953 in humans.

Discovery of Begacestat, a Notch-1-Sparing γ-Secretase Inhibitor for the Treatment of Alzheimer's Disease

SAR on HTS hits 1 and 2 led to the potent, Notch-1-sparing GSI 9, which lowered brain Abeta in Tg2576 mice at 100 mg/kg po. Converting the metabolically labile methyl groups in 9 to trifluoromethyl groups afforded the more stable analogue 10, which had improved in vivo potency. Further side chain modification afforded the potent Notch-1-sparing GSI begacestat (5), which was selected for development for the treatment of Alzheimers disease.

Inhibition of platelet and neutrophil phospholipase A2 by hydroxyeicosatetraenoic acids (HETES): a novel pharmacological mechanism for regulating free fatty acid release

The present study demonstrated that acid-extracted platelet phospholipase A2 (PLA2) exhibited marked hydrolytic activity against both [1-14C]oleic acid- and [1-14C]arachidonic acid-labeled Escherichia coli. The rate of hydrolysis was linear up to 30 min and was directly proportional to the amount of enzyme added to the reaction mixture. The data further indicated that 5-hydroxy-6,8,11,15-eicosatetraenoic acid (5-HETE) inhibited platelet PLA2 in a dose-dependent manner (IC50 = 42 microM), whereas 5-lactone HETE had no inhibitory effect up to 100 microM. The degree of inhibition of PLA2 activity was unaffected by Ca2+ concentrations but was reduced in the presence of increasing amounts of E. coli substrate. Both 12-hydroxy-5,8,10,14-eicosatetraenoic acid (12-HETE) and 15-hydroxy-5,8,11,13-eicosatetraenoic acid (15-HETE) also inhibited platelet PLA2 activity (IC50 = 26 and 72 microM respectively). Furthermore, the inhibitory effects of these monoHETEs were confirmed with a PLA2 preparation derived from rat neutrophils. Thus, these data suggest a novel pharmacological action of HETEs on PLA2 which may have potential ramifications in the regulation of arachidonic acid metabolism.

Modulation by hydroxyeicosatetraenoic acids (HETEs) of arachidonic acid metabolism in mouse resident peritoneal macrophages

The effects of 5-, 5-lactone, 12- and 15-hydroxyeicosatetraenoic acids (HETEs) on the synthesis of leukotriene C4 (LTC4), thromboxane B2 (TXB2) and prostaglandin E2 (PGE2) by mouse resident peritoneal macrophages incubated with zymosan particles (100 micrograms/ml) were investigated. Zymosan phagocytosis stimulated a 110-, 16-, and 16-fold increase in LTC4, TXB2 and PGE2 synthesis, respectively. 15-HETE inhibited zymosan-induced LTC4 (IC50 = 1.1 microM) and TXB2 (IC50 = 38.9 microM) synthesis; in contrast, 15-HETE induced a consistent but variable enhancement of PGE2 synthesis. 5-HETE (IC50 = 15 microM), 5-lactone HETE (IC50 = 10.4 microM) and 12-HETE (IC50 = 13 microM) also inhibited LTC4 synthesis but they were approximately an order of magnitude less potent than 15-HETE. Furthermore, 5-HETE, 5-lactone HETE and 12-HETE inhibited TXB2 (IC50 = 20.4, 16.9 and 11.8 microM, respectively) and PGE2 (IC50 = 38.6, 2.3 and 11.6 microM, respectively) synthesis. Thus, monoHETEs exert modulatory actions on arachidonic acid metabolism and the different isomers of HETE differ quantitatively and qualitatively in their actions.

Effect of a 5-lipoxygenase (5-LO)/cyclooxygenase (CO) inhibitor, WY-47, 288, on cutaneous models of inflammation.

WY-47,288 (2-[(1-naphthalenyloxy)methyl]quinoline) demonstrated topical antiinflammatory activity in several animal models of skin inflammation. Application of WY-47,288 to mouse ear surfaces inhibited arachidonic acid (ED50=0.3 mg/ear) and tetradecanoylphorbol acetate (TPA)-induced inflammation (40% at 1 mg/ear). Administration of WY-47,288 (1 mg/ear) at 30 min and 5 h after TPA reduced ear edema and epidermal proliferation by 50%. WY-47,288 also inhibited oxazolone-induced contact hypersensitivity in mouse ears (ED50=0.4 mg/ear) and UVB-induced guinea pig skin erythema (ED50≈0.25 mg/spot). These antiinflammatory effects may be due to inhibition of 5-lipoxygenase (5-LO) and cyclooxygenase (CO) since the synthesis of 5-LO and CO products by rat neutrophils and mouse macrophages was dose-dependently reduced by WY-47,288. By contrast, WY-47,288 demonstrated no appreciable inhibition of 12-LO (rabbit platelet), 15-LO (soybean) or phospholipase A2 (human platelet). Furthermore, no systemic adverse effects were observed after topical, parenteral or oral administration of WY-47,288, suggesting that WY-47,288 is a safe topical 5-LO/CO inhibitor for treating skin inflammation.

Discovery of a novel series of Notch-sparing γ-secretase inhibitors

Using a cell-based assay, we have identified a new series of Notch-sparing gamma-secretase inhibitors from HTS screening leads 2a and 2e. Lead optimization studies led to the discovery of analog 8e with improved gamma-secretase inhibitory potency and Notch-sparing selectivity.

Wy-48,252 (1,1,1-trifluoro-N-[3-(2-quinolinylmethoxy)phenyl]methane sulfonamide) an orally active leukotriene D4 antagonist: Pharmacological characterization in vitro and in vivo in the guinea pig

The following communicates the pharmacology of Wy-48,252 (1,1,1-trifluoro-N-[3-(2-quinolinylmethoxy)phenyl]methanesulfonamide) a chemically novel and orally potent leukotriene (LT) D4 receptor antagonist. In the isolated guinea-pig trachea pretreated with indomethacin (5 microM) and L-cysteine (10 mM), Wy-48,252 antagonized TD4-induced contraction with a pKB = 7.6. Against LTC4 on tissues pretreated with IND and glutathione (10 mM), Wy-48,252 had a pKB greater than 5. Wy-48,252 (10 microM) did not antagonize pilocarpine-, histamine- or PGF2 alpha-induced tracheal contraction. Further, in the presence of indomethacin and chlorpheniramine (1 microM), Wy-48,252 dose-dependently inhibited the antigen-induced contraction of guinea-pig trachea in a manner consistent with antagonism at the LTD4 receptor and inhibition of LT synthesis. In the Konzett-Rossler model of i.v. LTD4-induced bronchoconstriction in indomethacin treated guinea pigs, intragastric Wy-48,252 (2 hr) had an ID50 of 100 micrograms/kg and a functional half-life of 5 hr. Against i.v. antigen-induced bronchoconstriction in guinea pigs treated with indomethacin and chlorpheniramine, intragastric Wy-48,252 (2 hr) had an ID50 of 0.6 mg/kg and a 5 hr half life. Intragastric Wy-48,252 also selectively blocked the cutaneous wheal reaction to intradermal LTD4 but not histamine. We conclude that Wy-48,252 is distinguished from other selective LTD4 receptor antagonists by its oral potency and should be useful in ascertaining the role of LTD4 mediated processes in asthma, allergy and animal models.

(S)-N-(5-Chlorothiophene-2-sulfonyl)-β,β-diethylalaninol a Notch-1-sparing γ-secretase inhibitor

Accumulation of beta-amyloid (Abeta), produced by the proteolytic cleavage of amyloid precursor protein (APP) by beta- and gamma-secretase, is widely believed to be associated with Alzheimers disease (AD). Research around the high-throughput screening hit (S)-4-chlorophenylsulfonyl isoleucinol led to the identification of the Notch-1-sparing (9.5-fold) gamma-secretase inhibitor (S)-N-(5-chlorothiophene-2-sulfonyl)-beta,beta-diethylalaninol 7.b.2 (Abeta(40/42) EC(50)=28 nM), which is efficacious in reduction of Abeta production in vivo.