ABSTRACT
A series of 2,3,6-pyrazine Rho Kinase inhibitors were optimized for in vivo activity for topical ocular dosing. Modifications of the 2-(piperazin-1-yl)pyrazine derivatives produced compounds with improved solubility and physicochemical properties. Modifications of the 6-pyrazine substituent led to improvements in in vitro potency. Compound 9 had the best in vitro and in vivo potency of EC50=260 nM with a 30% reduction of IOP in a non-human primate model at a dose of 0.33%.
Subject(s)
Glaucoma/drug therapy , Protein Kinase Inhibitors/therapeutic use , Pyrazines/chemical synthesis , Pyrazines/therapeutic use , rho-Associated Kinases/antagonists & inhibitors , Animals , Disease Models, Animal , Dose-Response Relationship, Drug , Guinea Pigs , Humans , Inhibitory Concentration 50 , Models, Molecular , Molecular Structure , Protein Kinase Inhibitors/chemical synthesis , Protein Kinase Inhibitors/chemistry , Protein Kinase Inhibitors/pharmacology , Pyrazines/chemistry , Pyrazines/pharmacology , Pyridines/chemistry , Pyridines/pharmacology , Pyridines/therapeutic useABSTRACT
PURPOSE: To localize mRNA and protein of bradykinin (BK) receptors, BK precursor polypeptide (kininogen) mRNA, and to study functional biochemical pharmacology of the signal transduction processes mediated by B2-receptors in isolated human trabecular meshwork (h-TM) cells. Intraocular pressure (IOP) lowering effects of 2 kinins were also investigated. METHODS: Previously documented procedures were utilized throughout these studies. RESULTS: Kinninogen mRNA was most abundant in TM, ciliary body (CB), and optic nerve head and appeared elevated in glaucomatous h-TM tissue. High levels of B2-receptor mRNA were found in the sclera, iris, TM, and CB. B2-receptor subtype protein was localized in cells of the monkey and h-TM, and the treatment of isolated h-TM cells with transforming growth factor-ß2 (5 ng/mL) caused significant (P<0.04) downregulation of B2-receptor mRNA. In isolated primary h-TM cells, BK (EC50=0.8±0.2 nM; n=19) and Met-Lys-BK (EC50=6.5±1.5 nM) mobilized intracellular Ca(2+) and induced the release of prostaglandins (PGs) that was blocked by 2 B2-receptor antagonists [HOE-140; (S)-WIN-64338]. The cyclooxygenase inhibitor, bromfenac, abolished BK-induced PGs production. BK concentration dependently increased cell impedance, and it significantly (P<0.05) decreased h-TM cell volume in vitro. Intravitreal (ivt) administration of BK (50 µg), but not a B1-agonist (Sar-[D-Phe(9)]-Des-Arg(9)-BK; also at 50 µg), efficaciously lowered IOP (22.9% to 37% from baseline) of Dutch-Belted rabbits that naturally have high IOPs (27-28 mmHg). CONCLUSIONS: BK activates multiple signal transduction pathways in h-TM cells via B2-receptors that also mediate IOP reduction as observed in rabbits following ivt administration of BK. These ocular hypotensive effects of BK may be physiologically important and suggest a novel therapeutic potential of BK-related B2-agonists.
Subject(s)
Intraocular Pressure/physiology , Kininogens/metabolism , RNA, Messenger/metabolism , Receptors, Bradykinin/metabolism , Aged , Animals , Benzophenones/pharmacology , Bradykinin/administration & dosage , Bradykinin/analogs & derivatives , Bradykinin/metabolism , Bradykinin/pharmacology , Bradykinin B2 Receptor Antagonists , Bromobenzenes/pharmacology , Female , Humans , Immunohistochemistry , Intraocular Pressure/drug effects , Kininogens/genetics , Macaca fascicularis , Male , Naphthalenes/pharmacology , Organophosphorus Compounds/pharmacology , Prostaglandins/metabolism , Rabbits , Receptor, Bradykinin B2/genetics , Receptor, Bradykinin B2/metabolism , Signal Transduction/physiology , Trabecular Meshwork/cytology , Trabecular Meshwork/metabolismABSTRACT
The Red Sea sponge Callyspongia (= Siphonochalina) siphonella is a rich source of sipholane triterpenoids. Biocatalysis of the major sipholanes, sipholenol A (1) and sipholenone A (2), respectively, by Mucor ramannianus ATCC 9628 and Cunninghamella elegans ATCC 7929 afforded four new metabolites 3 - 6 along with sipholenol G and 28-hydroxysipholenol A. Major sipholanes along with their biocatalytic products were investigated for their antiproliferative activity against the highly malignant +SA mouse mammary epithelial cell line. Sipholenone A (2) was the most active sipholane inhibiting +SA cell proliferation with an IC(50) value of 20 - 30 microM. Sipholenone A, also, showed cytotoxicity against MCF-7 at a dose of 0.9 microM and antiangiogenic activity in the CAM (chorio-allantoic membrane) assay. This is the first report on anticancer activity of these triterpenoids.
