Boundary layer infusion of nitric oxide reduces early smooth muscle cell proliferation in the endarterectomized canine artery.

To evaluate the direct effect of nitric oxide (NO) on vascular smooth muscle cell (SMC) proliferation in vivo, we used an expanded polytetrafluoroethylene (ePTFE)-based local infusion device to deliver an NO donor, proline/NO (PROLI/NO), to the luminal boundary layer of endarterectomized artery and the distal anastomosis of the graft in a canine model. Once delivered to the blood, PROLI/NO releases NO by a mechanism involving pH-dependent decomposition. Six dogs underwent bilateral femoral artery endarterectomies. ePTFE infusion devices, blindly primed with PROLI/NO to one artery or proline to the contralateral vessel, were anastomosed proximal to the injured segments so that each animal served as its own control. PROLI/NO or proline was continuously delivered for 7 days from an osmotic reservoir, through the wall of the graft infusion device. Euthanasia was carried out at 7 days, and the processed specimens were blindly analyzed for SMC proliferation at both graft anastomoses and endarterectomized segments by a bromodeoxyuridine index assay. All dogs survived with no clinical side effects. In comparing the treated and control vessels, NO released from PROLI/NO significantly reduced SMC proliferation by 43% (13.24 +/- 1.24% versus 23.24 +/- 1.01%, P = 0.004) at the distal anastomoses and by 68% (10.58 +/- 1.63% versus 25.17 +/- 3.39%, P = 0.007) at endarterectomized segments. However, there was no significant difference in blood flow measurements between treated and control arteries (56.25 +/- 6.50 ml/min versus 46.50 +/- 3.20 ml/min, P = 0.094). These data demonstrate that local boundary layer infusion of NO released from PROLI/NO significantly reduces SMC proliferation in injured arteries with no effect on regional blood flow. This study suggests a new strategy to inhibit early SMC proliferation in injured arteries and probably to control intimal hyperplastic lesion formation in the manipulated vessels.

Inhibition of acute stent thrombosis under high-shear flow conditions by a nitric oxide donor, DMHD/NO. An ex vivo porcine arteriovenous shunt study.

BACKGROUND: Coronary stenting is limited by subacute thrombosis, especially in smaller-diameter vessels, in which shear rates are high. The objective of the present study was to determine whether local delivery of a new type of NO donor, the NO adduct of N,N'-dimethylhexanediamine (DMHD/NO), inhibits acute stent thrombosis (ST) at high-shear flow. METHODS AND RESULTS: Effects of local infusion of DMHD/NO; intravenous aspirin, and heparin on ST were evaluated in an ex vivo porcine AV shunt model. Nitinol stents (2 mum in diameter, n = 120) were placed in a tubular chamber and perfused with blood from pigs (n = 13) at a shear rate of 2100s-1 for 20 minutes. ST was quantified by measurement of dry thrombus weight(TW). Effects on platelet aggregation (PA), blood pressure, bleeding time, and activated clotting time (ACT) were also examined. There was a dose-dependent inhibition of ST and PA by DMHD/NO. TW was reduced by 95% (1 +/- 2 versus 16 +/- 4 mg control, mean +/- SD, P < .001), and PA was reduced by 75% (4 +/- 3 versus 14 +/- 9 omega/min control, P < .05) at the highest dose of 10 mumol/L. DMHD/NO had no effects on bleeding time, ACT, or blood pressure. In contrast, aspirin (10 mg/kg), despite inhibiting PA, had no effects on TW (12 +/- 5 versus 16 +/- 8 mg control, P = .3). Heparin (200 U/kg) reduced TW by 33% (14 +/- 4 versus 21 +/- 3 mg control, P < .05) and prolonged ACT. CONCLUSIONS: Local delivery of DMHD/NO produced a 15-fold inhibition of acute ST at high-shear flow without producing adverse systemic hemostatic or hemodynamic effects. Thus, treatment with DMHD/NO may be an effective strategy for prevention of stent thrombosis.

Nitric oxide-releasing polymers containing the [N(O)NO]- group.

Ions of structure X[N(O)NO]- display broad-spectrum pharmacological activity that correlates with the rate and extent of their spontaneous, first-order decomposition to nitric oxide when dissolved. We report incorporation of this functional group into polymeric matrices that can be used for altering the time course of nitric oxide release and/or targeting it to tissues with which the polymers are in physical contact. Structural types prepared include those in which the [N(O)NO]- group is attached to heteroatoms in low molecular weight species that are noncovalently distributed throughout the polymeric matrix, in groupings pendant to the polymer backbone, and in the polymer backbone itself. They range in physical form from films that can be coated onto other surfaces to microspheres, gels, powders, and moldable resins. Chemiluminescence measurements confirm that polymers to which the [N(O)NO]- group is attached can serve as localized sources of nitric oxide, with one prototype providing sustained NO release for 5 weeks in pH 7.4 buffer at 37 degrees C. The latter composition, a cross-linked poly-(ethylenimine) that had been exposed to NO, inhibited the in vitro proliferation of rat aorta smooth muscle cells when added as a powder to the culture medium and showed potent antiplatelet activity when coated on a normally thrombogenic vascular graft situated in an arteriovenous shunt in a baboon's circulatory system. The results suggest that polymers containing the [N(O)NO]- functional group may hold considerable promise for a variety of biomedical applications in which local delivery of NO is desired.

Nebulized nitric oxide/nucleophile adduct reduces chronic pulmonary hypertension.

OBJECTIVE: Inhaled nitric oxide (NO) is a selective pulmonary vasodilator, but its use has been restricted almost exclusively to the intensive care setting due to the complexity of its delivery. NO/nucleophile adducts, such as diethylenetriamine/NO (DETA/NO), spontaneously release NO in aqueous solutions. We hypothesized that a nebulized DETA/NO (half-time of NO release > 20 h) would stay in the lower airways and continuously supply sufficient NO to achieve sustained vasodilation in chronic pulmonary hypertension. METHODS: Chronic pulmonary hypertension was induced in rats by a monocrotaline injection. Nineteen days later, nebulizations of DETA/NO were given on 4 consecutive days (5 and 50 mu mol; 10 min/day). One day after the last nebulization, pulmonary and systemic arterial pressure and cardiac output were measured after thoracotomy. The lungs were isolated and perfused to study the pressure-flow relationship. The effect of DETA/NO nebulization on acute vasoconstrictor reactivity was studied in additional isolated lungs. RESULTS: Total pulmonary, but not systemic, vascular resistance was significantly reduced by both DETA/NO doses, suggesting that DETA/NO, like NO, causes preferential dilation of the pulmonary circulation. The pulmonary perfusion pressure-flow curves were shifted downwards by DETA/NO treatment, indicating improved resistive properties of the pulmonary vasculature. DETA/NO nebulization into isolated lungs increased exhaled NO levels and progressively reduced vasoconstrictor responses to angiotensin II and acute hypoxia. These effects were not reversed by perfusate exchange. In intact rats, carotid artery pressure and plasma NO2- + NO3- levels did not change during and after DETA/NO nebulization. CONCLUSION: DETA/NO nebulization offers a possibility of once a day, ambulatory delivery of NO and is a potential treatment for chronic pulmonary hypertension, although further studies are needed to establish safety and selectivity.

Nitric oxide (NO) donor molecules: effect of NO release rate on vascular smooth muscle cell proliferation in vitro.

Nitric oxide (NO) inhibits vascular smooth muscle cell (SMC) growth in vitro. To determine the effects of release rate and exposure time on SMC growth inhibition by NO, we compared the activities of five NO donors that generate NO with half-lives of 2 min (DEA/NO, Et2N[N2O2]Na), 15 min (PAPA/NO, CH3(CH2)2N[N2O2]-(CH2)3NH3+), 39 min, (SPER/NO, H2N(CH2)3NH2+(CH2)4N[N2O2]-(CH2)3NH2), 3 h (DPTA/NO, H2N(CH2)3N[N2O2]-(CH2)3NH3+), and 20 h (DETA/NO, H2N(CH2)2N[N2O2]-(CH2)2NH3+). After 22-h treatment, rat aorta SMC (RA-SMC) DNA synthesis was inhibited with IC50 values of 180, 60, and 40 microM for SPER/NO, DPTA/NO, and DETA/NO, respectively. DEA/NO and PAPA/NO did not inhibit DNA synthesis significantly at any concentration tested (20-500 microM). The inhibitory effect of NO on RA-SMC DNA synthesis was thus greatest when a given molar dose of NO was delivered slowly throughout the 22-h period. The antiproliferative effect of DETA/NO was confirmed by measurement of cell numbers for 7 days. When RA-SMC were treated with 500 microM DETA/NO on days 1, 3, and 5, growth was completely suppressed. Cell viability was > 95%, confirming that DETA/NO was not cytotoxic. The results suggest that NO donors may be useful inhibitors of intimal hyperplasia and restenosis after vascular injury such as balloon angioplasty.

Spironolactone. II. Bioavailability.

The bioavailability of commercial 25-mg spironolactone tablets and a new tablet preparation containing 100 mg of the drug has been determined in 12 healthy male subjects. After a 200-mg oral dose of the drug given in a solution of polyethylene glycol-400, the peak plasma level of the dethioacetylated metabolite canrenone was 633 +/- 154 ng/ml (mean +/- SD) and was reached at 1.4 +/- 0.43 hr. This peak was higher and was achieved earlier than after either eight 25-mg tablets (480 +/- 155 ng/ml at 2.9 +/- 1.03 hr) or two 100-mg tablets (474 +/- 182 ng/ml at 3.0 +/- 1.37 hr). From the ratio of the 24-hr area under the plasma concentration-time curves, the bioavailabilities of the two tablet preparations relative to the solution were 99.6 +/- 18.2% and 92.1 +/- 22.9%, respectively. The amount of canrenone excreted in the urine by 48 hr was 4.48 +/- 1.26 mg (solution), 6.36 +/- 2.02 mg (eight 25-mg tablets), and 7.81 +/- 1.87 mg (two 100-mg tablets), representing 2% to 4% of the administered dose. It is concluded that urinary excretion of canrenone alone is not a reliable method for determining the bioavailability of spironolactone.

Spironolactone. III. Canrenone--maximum and minimum steady-state plasma levels.

Steady-state plasma levels of canrenone have been examined in 23 healthy men who received repeated doses of spironolactone for 15 days. The dose regimens were 200 mg once a day and 50 mg 4 times a day. With both treatments the steady-state levels were reached after 3 to 4 days. With once-a-day treatment, the maximum and minimum steady-state levels were about 500 and 100 ng/ml, respectively. The minimum levels with treatment 4 times a day were about 200 ng/ml. The post steady-state half-life of canrenone with once-a-day treatment (19.2 +/- 6.57 hr) was longer (p less than 0.01) than the half-life obtained in the 4 times-a-day treatment (12.5 +/- 3.39 hr). The former half-life was similar to the log-linear phase half-life of canrenone in the single-dose studies.

Determination of disopyramide and its mono-N-dealkylated metabolite in blood serum and urine.

A rapid, precise and accurate assay for disopyramide and mono-N-dealkylated disopyramide concentrations in blood serum and urine is described. The method involves extraction of the drugs from a basic aqueous medium into chloroform, derivatization of the metabolite, purification of the extract and gas chromatographic analysis using an OV-17 liquid phase and flame ionization detection. Unique characteristics of the procedure, direct derivatization in the organic phase and the use of Florisil to separate the drugs from interfering materials, should be applicable to the analysis of other basic drugs in biological specimens.

Reduction of atherogenicity of natural fats by small additions of ethyl linoleate in the diet of the rat.

Ethyl linoleate was substituted in part for the 20% of butterfat, hydrogenated coconut oil, lard, or tallow in an atherogenic diet fed to rats throughout a 40-week experimental period. Aortic degeneration, evidenced by lipid infiltration of the intima, was observed in the control groups but not in the linoleate-fed groups. Groups that received butterfat or hydrogenated coconut oil showed reduced plasma and hepatic cholesterol levels when fed 2% of ethyl linoleate; groups that received lard or tallow showed no significant change in plasma and hepatic cholesterol levels when fed 2% of ethyl linoleate; and groups that received a fat-free diet with 2% of ethyl linoleate showed lower plasma and hepatic cholesterol levels and more complete aortic protection than groups that were fed 20% of corn oil or cottonseed oil. The data suggest that, in the cholesterol-fed rat, the kind and amount of dietary fatty esters may influence aortic condition via some route(s) other than control of plasma and hepatic cholesterol levels.