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Lea Scheppke,
Edith Aguilar,
Ray F Gariano,
Ruth Jacobson,
John Hood,
John Doukas,
Jon Cao,
Glenn Noronha, Shiyin Yee,
Sara Weis,
Michael B Martin,
Richard Soll,
David A Cheresh,
Martin Friedlander
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ABSTRACT: Retinal and choroidal vascular diseases, with their associated abnormalities in vascular permeability, account for the majority of patients with vision loss in industrialized nations. VEGF is upregulated in ischemic retinopathies such as diabetes and is known to dramatically alter vascular permeability in a number of nonocular tissues via Src kinase-regulated signaling pathways. VEGF antagonists are currently in clinical use for treating the new blood vessels and retinal edema associated with neovascular eye diseases, but such therapies require repeated intraocular injections. We have found that vascular leakage following intravitreal administration of VEGF in mice was abolished by systemic or topical delivery of what we believe is a novel VEGFR2/Src kinase inhibitor; this was confirmed in rabbits. The relevance of Src inhibition to VEGF-associated alterations in vascular permeability was further substantiated by genetic studies in which VEGF injection or laser-induced vascular permeability failed to augment retinal vascular permeability in Src-/- and Yes-/- mice (Src and Yes are ubiquitously expressed Src kinase family members; Src-/- and Yes-/- mice lacking expression of these kinases show no vascular leak in response to VEGF). These findings establish a role for Src kinase in VEGF-mediated retinal vascular permeability and establish a potentially safe and painless topically applied therapeutic option for treating vision loss due to neovascular-associated retinal edema.
Journal of Clinical Investigation 07/2008; 118(6):2337-46. · 15.39 Impact Factor
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Moorthy S S Palanki,
Hideo Akiyama,
Peter Campochiaro,
Jianguo Cao,
Chun P Chow,
Luis Dellamary,
John Doukas,
Richard Fine,
Colleen Gritzen,
John D Hood, [......],
Andrew McPherson,
Ved P Pathak,
Joel Renick,
Richard Soll,
Naoyasu Umeda, Shiyin Yee,
Katsutoshi Yokoi,
Binqi Zeng,
Hong Zhu,
Glenn Noronha
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ABSTRACT: Age-related macular degeneration (AMD) is one of the leading causes of loss of vision in the industrialized world. Attenuating the VEGF signal in the eye to treat AMD has been validated clinically. A large body of evidence suggests that inhibitors targeting the VEGFr pathway may be effective for the treatment of AMD. Recent studies using Src/YES knockout mice suggest that along with VEGF, Src and YES play a crucial role in vascular leak and might be useful in treating edema associated with AMD. Therefore, we have developed several potent benzotriazine inhibitors designed to target VEGFr2, Src, and YES. One of the most potent compounds is 4-chloro-3-{5-methyl-3-[4-(2-pyrrolidin-1-yl-ethoxy)phenylamino]benzo[1,2,4]triazin-7-yl}phenol ( 5), a dual inhibitor of both VEGFr2 and the Src family (Src and YES) kinases. Several ester analogues of 5 were prepared as prodrugs to improve the concentration of 5 at the back of the eye after topical administration. The thermal stability of these esters was studied, and it was found that benzoyl and substituted benzoyl esters of 5 showed good thermal stability. The hydrolysis rates of these prodrugs were studied to analyze their ability to undergo conversion to 5 in vivo so that appropriate concentrations of 5 are available in the back-of-the-eye tissues. From these studies, we identified 4-chloro-3-(5-methyl-3-{[4-(2-pyrrolidin-1-ylethoxy)phenyl]amino}-1,2,4-benzotriazin-7-yl)phenyl benzoate ( 12), a topically administered prodrug delivered as an eye drop that is readily converted to the active compound 5 in the eye. This topically delivered compound exhibited excellent ocular pharmacokinetics and poor systemic circulation and showed good efficacy in the laser induced choroidal neovascularization model. On the basis of its superior profile, compound 12 was advanced. It is currently in a clinical trial as a first in class, VEGFr2 targeting, topically applied compound for the treatment of AMD.
Journal of Medicinal Chemistry 04/2008; 51(6):1546-59. · 5.25 Impact Factor
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ABSTRACT: [7-(2,6-Dichloro-phenyl)-5-methyl-benzo[1,2,4]triazin-3-yl]-[4-(2-pyrrolidin-1-yl-ethoxy)-phenyl]-amine (TG100435) is a novel multi-targeted Src family kinase inhibitor with demonstrated anticancer activity in preclinical species. Potent kinase inhibition is associated with TG100435 and its major N-oxide metabolite [7-(2,6-dichlorophenyl)-5-methyl-benzo[1,2,4]triazin-3-yl]-{4-[2-(1-oxy-pyrrolidin-1-yl)-ethoxy]-phenyl}-amine (TG100855). The objectives of the current study were to identify the hepatic enzyme(s) responsible for 1) the total metabolic flux of TG100435, 2) the formation of TG100855, and 3) the subsequent metabolism of TG100855. Flavin-containing monooxygenases (FMO) and cytochrome P450 monooxygenases (P450s) contribute to TG100435 total metabolic flux. TG100435 metabolic flux was completely inhibited by methimazole and ketoconazole, suggesting only FMO- and CYP3A4-mediated metabolism. TG100855 formation was markedly inhibited (~90%) by methimazole or heat inactivation (>99%). FMO3 was the primary enzyme responsible for TG100855 formation. In addition, an enzyme mediated retroreduction of TG100855 back to TG100435 was observed. The N-oxidation reaction was approximately 15 times faster than the retroreduction reaction. Interestingly, the retroreduction of TG100855 to TG100435 in recombinant P450 or liver microsomes lacked inhibition by the P450 inhibitors. TG100435 formation in the human liver microsomes or recombinant P450 increased as a function of cytochrome P450 reductase activity, suggesting potential involvement of cytochrome P450 reductase. The results of this in vitro study demonstrate the potential of TG100435 and TG100855 to be interconverted metabolically. FMO seem to be the major N-oxidizing enzymes, whereas cytochrome P450 reductase seems to be responsible for the retroreduction reaction.
Drug Metabolism and Disposition 01/2008; 35(12):2242-51. · 3.73 Impact Factor
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Moorthy S S Palanki,
Elena Dneprovskaia,
John Doukas,
Richard M Fine,
John Hood,
Xinshan Kang,
Dan Lohse,
Michael Martin,
Glenn Noronha,
Richard M Soll,
Wolfgang Wrasidlo, Shiyin Yee,
Hong Zhu
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ABSTRACT: In studies aimed toward identifying effective and safe inhibitors of kinase signaling cascades that underlie ischemia/reperfusion (I/R) injury, we synthesized a series of pteridines and pyridopyrazines. The design strategy was inspired by the examination of naturally occurring PI3K inhibitors such as wortmannin and quercetin, and building a pharmacophore-based model used for optimization. Structural modifications led to hybrid molecules which incorporated aminopyrimidine and aminopyridine moieties with ATP mimetic characteristics into the pharmacophore motifs to modulate kinase affinity and selectivity. Elaborations involving substitutions of the 2 and 4 positions of the pyrimidine or pyridine ring and the 6 and 7 positions of the central pyrazine ring resulted in in vivo activity profiles which identified potent inhibitors of vascular endothelial growth factor (VEGF) induced vascular leakage. Pathway analysis identified a diaminopteridine-diphenol as a potent and selective phosphatidylinositol-3-kinase (PI3K) inhibitor. The structure-activity relationship studies of various analogues of diaminopteridine-diphenol-based on biochemical assays resulted in potent inhibitors of PI3K.
Journal of Medicinal Chemistry 10/2007; 50(18):4279-94. · 5.25 Impact Factor
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Steven X Hu,
Richard Soll, Shiyin Yee,
Daniel L Lohse,
Ahmed Kousba,
Binqi Zeng,
Xiyun Yu,
Andrew McPherson,
Joel Renick,
Jianguo Cao,
Arek Tabak,
John Hood,
John Doukas,
Glenn Noronha,
Michael Martin
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ABSTRACT: TG100435 ([7-(2,6-dichloro-phenyl)-5-methyl-benzo[1,2,4]triazin-3-yl]-[4-(2-pyrrolidin-1-yl-ethoxy)-phenyl]-amine) is a novel multitargeted, orally active protein tyrosine kinase inhibitor. The inhibition constants (K(i)) of TG100435 against Src, Lyn, Abl, Yes, Lck, and EphB4 range from 13 to 64 nM. TG100435 has systemic clearance values of 20.1, 12.7, and 14.5 ml/min/kg and oral bioavailability of 74%, 23%, and 11% in mouse, rat, and dog, respectively. Four oxidation metabolites of TG100435 have been found in human, dog, and rat in vitro and in vivo. The ethylpyrrolidine N-oxide of TG100435 is the predominant metabolite (TG100855; [7-(2,6-dichloro-phenyl)-5-methyl-benzo[1,2,4]triazin-3-yl]-{4-[2-(1-oxy-pyrrolidin-1-yl)-ethoxy]-phenyl}-amine) in human, dog, and rat. TG100855 is 2 to 9 times more potent than the parent compound. Flavin-containing monooxygenases are the primary enzymes mediating the biotransformation. Significant conversion of TG100435 to TG100855 has been observed in rat and dog after oral administration. Systemic exposure of TG100855 is 1.1- and 2.1-fold greater than that of TG100435 in rat and dog after oral dosing of TG100435. Since TG100435 is predominantly converted to the more potent N-oxide metabolite across species in vivo and in vitro, the overall tyrosine kinase inhibition in animal models may be substantially increased after oral administration of TG100435.
Drug Metabolism and Disposition 07/2007; 35(6):929-36. · 3.73 Impact Factor
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Glenn Noronha,
Kathy Barrett,
Antonio Boccia,
Tessa Brodhag,
Jianguo Cao,
Chun P Chow,
Elena Dneprovskaia,
John Doukas,
Richard Fine,
Xianchang Gong, [......],
Joel Renick,
Feng Shi,
Richard Soll,
Ute Splittgerber,
Silva Stoughton,
Suhan Tang, Shiyin Yee,
Binqi Zeng,
Ningning Zhao,
Hong Zhu
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ABSTRACT: We describe the identification of [7-(2,6-dichlorophenyl)-5-methylbenzo [1,2,4]triazin-3-yl]-[4-(2-pyrrolidin-1-ylethoxy)phenyl]amine (3), a potent, orally active Src inhibitor with desirable PK properties, demonstrated activity in human tumor cell lines and in animal models of tumor growth.
Bioorganic & Medicinal Chemistry Letters 03/2007; 17(3):602-8. · 2.55 Impact Factor
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ABSTRACT: The absorption, metabolism, and excretion of N-[3-fluoro-4-[2-(propylamino)ethoxy]phenyl]-4,5,6,7-tetrahydro-4-oxo-1H-indole-3-carboxamide monomethanesulfonate (1), a GABAA receptor partial agonist potentially useful in treating generalized anxiety disorder, have been evaluated in both Sprague-Dawley rats and cynomolgus monkeys using [14C]1. In both species, mass balance was achieved within 48 h postdose, with the majority of drug-related material excreted within the feces; the clearance of 1 in each species had both metabolic and renal components. In addition to the metabolites produced by aliphatic hydroxylation and/or N-dealkylation of 1, two unique metabolites were detected: a putative carbamic acid (M7) in rat plasma and monkey bile, and an N-carbamoyl glucuronide (M8) in both rat and monkey bile. Metabolite M8 was structurally deciphered by liquid chromatographytandem mass spectrometry and NMR, and was readily generated in vitro upon incubation of [14C]1 with rat liver microsomes fortified with uridine 5'-diphosphoglucuronic acid trisodium salt and alamethicin under a CO2 atmosphere. Treatment of M8 with beta-glucuronidase afforded 1 directly. The presence of M8 in bile and its notable absence from other matrices suggests the enterohepatic cycling of 1 via M8. Although the structure of M7 was not elucidated unequivocally due to its inability to be formed in vitro and its minimal absolute quantities in limited biological matrices, data herein clearly support its structural rationalization. Furthermore, since M7 is the precursor of M8, detection of M8 is indirect evidence of its existence. It is proposed that M7 arises from an equilibrium between 1 and dissolved CO2-equivalents both in vivo and in vitro, similar to carbamino bonds observed in hemoglobin and certain amino acids, respectively.
Drug Metabolism and Disposition 12/2005; 33(11):1688-99. · 3.73 Impact Factor
