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1999;285:1276C1279. useful β-Secretase Inhibitor IV in the treatment of patients with atherosclerotic cardiovascular disease. experiments were done following protocols approved by the Animal Use and Care Committee of University of California-Davis. Homozygous ApoE-deficient (C57BL/6 background) male mice were purchased from the Jackson β-Secretase Inhibitor IV Laboratory (Bar Harbor, ME). Atherosclerosis was induced in 5 month old mice by infusion of angiotensin II and an atherogenic diet (Research Diets, Inc) as previously described 14, 15. β-Secretase Inhibitor IV Angiotensin II was delivered via subcutaneously implanted osmotic minipumps (Alzet, model 1002) for 4 weeks. Osmotic minipumps were inserted into a subcutaneous pocket under light anesthesia with a mixture of 80 mg/kg ketamine and 12 mg/kg xylazine (Sigma-Aldrich, St.Louis). The minipumps contained angiotensin II in sterile ddH2O at a concentration calculated to deliver 1000 ng ? min?1 ? kg?1 This dose of angiotensin II was selected on the basis of previous studies 9 which demonstrated that this dose of angiotensin II increases MAP by approximately 30 mmHg which is relevant to clinical hypertension. Mice were fed an atherogenic diet for 4 weeks concurrent with angiotensin II infusion that consists of purified components designed to match the original Paigens Atherogenic Rodent Diet. The components list can be obtained from the manufacturer (Research Diets, Inc). Drug Delivery The highly potent soluble epoxide hydrolase inhibitor AEPU was chosen for this study 16 to overcome the limitations of previous high melting (mp 142C) and lipophilic inhibitors of sEH such as the earlier generation sEH inhibitor AUDA (12-(3-adamantan-1-yl-ureido)dodecanoic acid) (Figure S1A). In addition, AUDA is thought to not only be a potent transition state inhibitor of the sEH but also to be a weak mimic of 14,15-EET 17. In contrast, AEPU has a low melting temp (mp 79C), is definitely more water soluble such that can it be delivered orally through the drinking water without the need for hydroxypropyl–cyclodextrin to solubilize the compound. AEPU also has little structural resemblance to the EETs (Number S1B). Separate studies have shown AEPU to penetrate into cells much faster than AUDA and to have high oral availability. AEPU is also β-Secretase Inhibitor IV more potent within the murine sEH than AUDA (Table 1). This improvement in physical properties was vital for this study, since the -cyclodextrin used to solubilize AUDA sequesters cholesterol18 and is itself capable of inhibiting atherosclerotic plaque formation. The sEH inhibitor AEPU was delivered at a dose of 90 g/ml in drinking water for 8 weeks, starting 4 weeks prior to the initiation of the 4 week treatment with the atherogenic diet and angiotensin II infusion. This dose was selected based on water solubility data and earlier pharmacokinetic studies in mice. Mice were observed to drink approximately 3 ml of Ngfr water per day consistent with additional published studies19, 20. Parallel studies on additional biological end points that have been carried out in our laboratory, show this procedure gives a dose of approximately 10 mg AEPU per kg per day. TABLE 1 Properties of the sEH inhibitors AEPU and AUDA. prior to experimentation. AEPU (4.5 mg) was dissolved in 3 ml of oleic ester rich triglyceride to a final concentration of 1 1.5 mg/ml. Each mouse was treated with 10 mg/kg of AEPU in 100 uL of triglyceride and 10 L blood samples were collected from your tail vein using a heparinized pipet tip at 0, 0.5, 1, 2, 3, 4, 6, 24 hr after drug administration. After sample collection, each blood sample was diluted with 50 L distilled water, extracted with ethyl acetate twice with 10 L of surrogate remedy (250 ng/ml of 1-(5-butoxypentyl)-3-adamantylurea in methanol) and reconstituted with 50 L of internal standard remedy (100 ng/ml of 1-adamantanyl-3-decylurea in methanol) following a drying step under nitrogen. The extracted samples were analyzed by liquid chromatography coupled with β-Secretase Inhibitor IV mass spectrometry (LC/MS-MS). Specifically, chromatographic separation was performed on an ACQUITY ultra overall performance liquid chromatography (UPLC) instrument equipped with a 2.1 50.