Low- versus high-dose recombinant urokinase for the treatment of chronic saphenous vein graft occlusion.

Recanalization of a totally occluded saphenous vein graft (SVG) using commercially available urokinase from human kidney cells has been shown to be effective, but the duration of infusion and complications such as allergic reactions, bleeding events, and non-Q-wave myocardial infarction have limited its acceptance. Recently, genetic engineering has allowed the synthesis of recombinant urokinase (r-UK). Patients with an occluded SVG from 37 centers were randomized to receive a 6-hour infusion of either low-dose (125,000 IU/hour) or high-dose (350,000 IU/hour) r-UK followed by up to a maximum of 18 hours of r-UK (125,000 IU/hour) via a subselective catheter directly into the occluded vein graft. The primary study end point was final preintervention achievement of Thrombolysis In Myocardial Infarction (TIMI) flow > or = 2 using core angiographic analysis. One hundred seven patients were randomized and 98 received the study drug (low dose 52 patients, high dose 46 patients). TIMI flow > or = 2 after completion of the study drug was higher in the high-dose group (51% vs 24%, p = 0.019). This difference narrowed, but a trend was still evident on the final angiogram after adjunctive mechanical intervention (72% vs 58%, p = 0.254). Bleeding complications were frequent; severe or life-threatening bleeding occurred in 12% of patients on the low dose and 11% of patients on the high dose (p = NS), including 2 intracerebral bleeds, both of which were fatal with 1 in each group. Thus, in patients with an occluded SVG, a randomized trial of direct low-dose versus high-dose r-UK infusion demonstrated increased recanalization rates (TIMI flow > or = 2) in the high-dose arm. Percutaneous revascularization of SVG with r-UK can be accomplished with acceptable success rates, but complications are frequent.

Efficacy of intra-arterial and intravenous prourokinase in an embolic stroke model evaluated by diffusion-perfusion magnetic resonance imaging.

OBJECTIVE: To explore the utility of intravenous (i.v.) prourokinase treatment, we compared intra-arterial (i.a.) and i.v. delivery in a rat embolic stroke model, using diffusion (DWI) and perfusion (PI) magnetic resonance imaging to assess in vivo effects on ischemic lesion evolution and reperfusion. BACKGROUND: Thrombolytic therapy with recombinant tissue-type plasminogen activator (rt-PA) for acute ischemic stroke is useful during the initial hours after onset. Prourokinase is a novel thrombolytic agent with potential safety advantages in comparison to rt-PA. METHODS: Twenty-four male Sprague-Dawley rats were embolized with autologous blood clots into the middle cerebral artery territory and then randomly assigned at 30 minutes after embolization to a 2-hour bolus infusion with i.a. prourokinase, i.v. prourokinase, or vehicle. DWI and PI were performed before treatment and repeated during and at the end of the treatment. RESULTS: PI demonstrated that both i.a. and i.v. significantly improved the percentage of the ischemic hemisphere that was normally perfused when the 20-minute, pretreatment, and 145-minute after embolization time points were compared; in the control group, the hypoperfused volume increased over time. DWI disclosed that the ischemic lesion evolution slightly decreased in the i.a. group, remained stable in the i.v. group, and increased over time in the control group. Infarct volume by triphenyltetrazolium chloride (TTC) staining was significantly smaller in both treatment groups than controls. CONCLUSIONS: These results demonstrate that i.a. and i.v. therapy with prourokinase are equally effective in promoting reperfusion and inhibiting the development of focal ischemic injury in this rat embolic stroke model.

Ex vivo inhibition of beta-thromboglobulin release following administration to man of ABT-299, a novel prodrug of a potent platelet activating factor antagonist.

OBJECTIVE AND DESIGN: ABT-299 is a prodrug that is converted by serum esterase to a potent platelet activating factor (PAF) antagonist (A-85783). In order to evaluate the pharmacological activity of this antagonist in man the effect of ABT-299 given to healthy volunteers on ex vivo PAF-induced beta-thromboglobulin (beta-TG) release in blood was assessed. SUBJECTS: 37 healthy male volunteers, age 18 to 40 (mean age of 23.6 years) and free of medication, participated in the study. TREATMENT: Subjects were administered intravenously 0.8 mg, 2 mg, or 70 mg doses of ABT-299 (6-7 subjects per group) or placebo (9 subjects, pooled). METHODS: Peripheral blood taken over 12 h after dosing was used for ex vivo beta-TG release and, in the case of the 70 mg dose, measurement of plasma drug concentration. Data were compared by Student's t-test. RESULTS: All three doses produced highly significant inhibition (p < 0.005 compared to predose values) of PAF-induced beta-TG release (units/ml plasma +/- SEM) 12 h after drug administration (54 +/- 14 vs. 405 +/- 51, n = 8; 79 +/- 23 vs. 480 +/- 127, n = 7; 21 +/- 10 vs. 327 +/- 72, n = 6, respectively) whereas there was no significant difference in beta-TG release in the placebo group (449 +/- 90 vs. 307 +/- 49, n = 9). Inhibition was associated with the rapid appearance in plasma of A-85783 and the pyridine N-oxide metabolite of A-85783. Within 2 h, the plasma concentration of the metabolite exceeded that of the parent drug. Both the parent drug and the metabolite exhibited potent in vitro inhibition of PAF-induced beta-TG release (A2 values of 4 and 1 nM respectively). CONCLUSIONS: These studies are the first to illustrate the utility of the beta-TG release assay for assessing ex vivo activity of PAF antagonists. These studies also demonstrate that the administration of ABT-299 to man results in potent, long lasting inhibition of PAF-mediated platelet activation, due in part to the pyridine-N-oxide metabolite, and support the potential therapeutic utility of this prodrug in treating PAF-mediated diseases.

Hemodynamic effects of renin inhibitors.

Renin inhibitors may offer an exciting new therapeutic means of blocking the action of the renin-angiotensin-aldosterone system. These compounds interfere with the first, rate-limiting step in the synthesis of angiotensin II by binding directly to the highly specific enzyme, renin. This approach may represent a more focused alternative to angiotensin-converting enzyme inhibitor therapy with an improved side-effect profile. Renin inhibitors given parenterally safely lower blood pressure in patients with essential heart failure. Under conditions of salt limitation, normal subjects show increases in renal plasma flow during infusion of renin inhibitors. The systemic and renal hemodynamic responses to renin inhibition are accompanied by suppression of plasma renin activity, plasma angiotensin II and plasma aldosterone levels. Recent scientific advances in discovering how to address the limitations of the low oral bioavailability of peptide-based compounds have made it possible to invent orally active renin inhibitors. Orally active renin inhibitors are presently being evaluated in clinical trials. The availability of intravenous and oral delivery of renin inhibitors broadens the potential of these agents to treat a wide variety of acute and chronic cardiorenal disorders.

Renin inhibition.

Modification of the renin-angiotensin-aldosterone system by renin inhibitors may be an alternative to angiotensin-converting enzyme inhibitors in the treatment of cardiovascular disease. The development of clinically useful renin inhibitors has been hampered by a variety of pharmacologic problems, most notably the poor oral bioavailability of these peptide-related compounds. Peptidomimetic renin inhibitors that have been stabilized to enzymatic degradation in conjunction with optimizing physical characteristics amenable to intestinal absorption offer the greatest promise to date. Studies in animal models demonstrate that renin inhibitors are capable of reducing both systolic and diastolic blood pressures without causing reflex tachycardia. The response appears to be sustained with chronic administration. The beneficial cardiovascular effects of these compounds have been confirmed in the few studies conducted in patients with hypertension and in those with congestive heart failure. Further development of renin inhibitors is warranted.

Dose-dependent effects of the renin inhibitor zankiren HCl after a single oral dose in mildly sodium-depleted normotensive subjects.

BACKGROUND: Zankiren HCl (A-72517) is a potent renin inhibitor shown to have substantial bioavailability in several animal species and to produce dose-related reductions in blood pressure, plasma renin activity, and angiotensin II (Ang II) in salt-depleted dogs. The present study was designed to evaluate the hemodynamic effects of oral zankiren HCl administration in healthy volunteers and to characterize the response of the renin-angiotensin system (RAS) to specific blockade by this new renin inhibitor. METHODS AND RESULTS: Twenty-four male volunteers participated in a double-blind randomized, placebo-controlled in-hospital study to evaluate the effects of zankiren HCl (10 to 250 mg). All subjects were pretreated with 40 mg furosemide 12 hours before study drug administration. Blood pressure and heart rate were monitored by an automated oscillometric device, and blood samples were obtained for active renin, total renin, plasma renin activity, angiotensin I (Ang I), Ang II, aldosterone, and plasma zankiren concentration. Satisfactory absorption of zankiren HCl was demonstrated by the results of plasma drug concentration determinations, and renin inhibitory activity was confirmed by dose-related suppression of plasma renin activity, Ang I, Ang II, and aldosterone and increases in plasma active renin concentration. Furthermore, hypotensive activity was readily observed in these normotensive subjects, as evidenced by statistically significant dose-related blood pressure reductions (P < .01). CONCLUSIONS: Results from this study demonstrate for the first time that oral administration of a renin inhibitor can dose-dependently decrease blood pressure and circulating components of the RAS in normotensive volunteers as a result of documented absorption.

Renin inhibitors: C-terminal oxetanes as potent transition-state mimics.

A novel transition-state mimic containing a C-terminal oxetane has been developed. Renin inhibitors incorporating this fragment exhibit enhanced potency against human plasma renin at physiological pH. The binding affinity of this new species has allowed size reductions at other sites.

Cardiovascular effects and hemodynamic mechanism of action of the novel, nonpeptidic renin inhibitor A-74273 in dogs.

A-74273 is a nonpeptidic, potent inhibitor of human and canine renin (IC50 = 3.1 and 43 nM, respectively, in plasma at pH 7.4) and has been shown to be orally active in dogs. To determine the hemodynamic mechanism underlying this renin inhibitor's hypotensive activity, the cardiac and hemodynamic effects of A-74273 were studied in sodium-depleted and sodium-replete pentobarbital-anesthetized dogs. Vehicle [5% dextrose in water (V, D5W), n = 8] or a single dose of A-74273 was administered intravenously (i.v.) as a bolus followed by a 30-min infusion (one tenth the bolus dose per minute). Baseline mean arterial pressure (MAP) was similar among all treatment groups, but baseline plasma renin activity (PRA) was increased in the sodium-depleted dogs as compared with the sodium-replete dogs. In sodium-depleted dogs (n = 7-8/dose), MAP decreased maximally as compared with baseline by 4 +/- 1, 19 +/- 3, and 23 +/- 3% during infusion of A-74273 at doses of 0.001, 0.01, and 0.1 mg/kg/min, respectively (p < 0.05 vs. baseline or V). The two highest infusion doses also produced significant reductions (p < 0.05 vs. baseline and V) in systemic vascular resistance (SVR, 21 +/- 2 and 25 +/- 2%) and left ventricular end-diastolic pressure (LVEDP, 40 +/- 8 and 47 +/- 12%). In sodium-replete dogs (n = 4/dose), an infusion dose of 0.01 mg/kg/min elicited no hemodynamic response, whereas 0.1 mg/kg/min reduced MAP by 13 +/- 2% (p < 0.05 vs. baseline) and SVR by 7 +/- 6%.(ABSTRACT TRUNCATED AT 250 WORDS)

Effects of renin inhibitor A-72517 on hemodynamics and cardiac function in sodium-depleted dogs.

A-72517 is a potent inhibitor of human renin (IC50 = 1.0 nmol/L, pH 7.4 in plasma) and, aside from displaying modest activity against canine plasma renin (IC50 = 110 nmol/L), has been shown to be orally active in the dog and other animals. Renin inhibitors, in general, are presumed to exert their hypotensive effect through a reduction in total peripheral resistance. To elucidate the hemodynamic mechanism of action of this new dipeptidic renin inhibitor, the cardiac and systemic hemodynamic effects of A-72517 were studied in sodium-depleted, pentobarbital-anesthetized dogs. Each dog received either vehicle (n = 8) or a single dose (n = 8/dose) of A-72517 administered intravenously as a priming bolus followed by a 30 min constant infusion; infusion doses were 0.01, 0.05, and 0.1 mg/kg/min. A-72517 elicited significant (P < .05) dose-related reductions in mean arterial pressure (MAP) and systemic vascular resistance (SVR) compared to baseline values and the vehicle-treated group, and the recoveries of MAP and SVR were also dose-related. Plasma renin activity, measured by radioimmunoassay, was nearly completely suppressed during drug infusion at all doses. The hypotensive responses did not alter cardiac output nor did they induce reflex tachycardia at any dose. Left ventricular dP/dtmax did not change during infusion of A-72517, but, when corrected for changes in afterload, showed dose-related increases with drug treatment. Moreover, left ventricular end-diastolic pressure and pulmonary arterial wedge pressure were significantly reduced at the high dose.(ABSTRACT TRUNCATED AT 250 WORDS)

Discovery of a well-absorbed, efficacious renin inhibitor, A-74273.

The development of orally active renin inhibitors has been plagued by limited bioavailability in animals and humans. A-74273 is a novel, potent nonpeptide inhibitor of human renin (IC50 = 3.1 nM). This compound was absorbed into the portal and systemic circulations of anesthetized rats, ferrets, monkeys, and dogs after intraduodenal dosing. This favorable pattern also was observed after oral dosing in conscious animals, except in monkeys. Hepatic extraction of A-74273 was more efficient in rats and monkeys than in dogs or ferrets. A-74273 modestly inhibits dog renin, and when given orally as the base (0, 0.3, 1, 3, 10, and 30 mg/kg; n = 8 per dose) to conscious, salt-depleted dogs it induced dose-related reductions in mean arterial pressure and plasma renin activity. Peak falls in mean arterial pressure from normotensive baselines were -14 +/- 1, -26 +/- 3, and -44 +/- 3 mm Hg for the 3, 10, and 30 mg/kg groups, respectively (p < 0.05). Baseline plasma renin activity values (10.9 +/- 1.1-12.7 +/- 1.1 ng angiotensin I/ml/hr) were maximally inhibited, ranging from 43 +/- 8% at 0.3 mg/kg to 98 +/- 1% at 30 mg/kg. Bioavailability in this model was estimated to be 54 +/- 13% when plasma drug levels were determined by a renin inhibitory activity assay, but bioavailability was lower when compared with high-performance liquid chromatographic analysis of A-74273. This discrepancy was accounted for by the identification of structurally similar metabolites that are as active as the parent drug against human renin but much less potent against dog renin.(ABSTRACT TRUNCATED AT 250 WORDS)

Discovery of a peptide-based renin inhibitor with oral bioavailability and efficacy.

Peptidic renin inhibitors have been poorly absorbed across the intestine or rapidly eliminated by the liver and have been reported to have oral bioavailabilities of less than 2%. A peptide-based renin inhibitor, A-72517 (molecular mass of 706 daltons), was devised that has oral bioavailabilities of 8, 24, 32, and 53% in the monkey, rat, ferret, and dog, respectively. Dose-related reductions in blood pressure, plasma renin activity, and plasma angiotensin II in parallel with increased plasma drug concentrations were observed after oral administration of A-72517 to conscious, salt-depleted dogs. Thus, peptide-based molecules of sizable molecular mass can be absorbed intact into the systemic circulation of animals. These findings support the potential of peptide-based drugs for oral administration.

Nonpeptide renin inhibitors employing a novel 3-aza(or oxa)-2,4-dialkyl glutaric acid moiety as a P2/P3 amide bond replacement.

A new series of renin inhibitors has been developed. The inhibitors feature a novel replacement for the P2/P3 dipeptide moiety normally associated with renin inhibitors. The dipeptide replacement was a (2S,4S)-3-aza(or oxa)-2,4-dialkylglutaric acid amide. Extensive structure-activity relationship studies determined that optimum potency was achieved when inhibitors employed a benzyl and butyl group at the C(4) and C(2) carbon position, respectively. In addition, maximum in vitro potency was obtained when the N-terminus was functionalized by incorporating a 4-(1,3-dioxabutyl)piperidine amide. SAR data suggested that the 1,3-dioxabutyl group (methoxymethyl ether) interacted by hydrogen bonding to groups in the S4 domain of renin. This hypothesis was strengthened when a 4-butylpiperidine amide was substituted and inhibitor potency decreased dramatically. Inhibitors employing this novel dipeptide mimic were prepared by coupling the glutaric acid amides with either the transition-state mimic (2S,3R,4S)-2-amino-1-cyclohexyl-3,4-dihydroxy-6- methylheptane (18) or the hydroxyethylene dipeptide isostere. The glutaric acid amides were prepared by two general procedures. The first procedure involved the reductive amination of alpha-amino acid esters with alpha-keto esters. The second procedure involved the displacement reaction of alpha-bromo esters or acids with alpha-amino acid amides.

Water-soluble renin inhibitors: design of a subnanomolar inhibitor with a prolonged duration of action.

Incorporation of nonreactive polar functionalities at the C- and N-termini of renin inhibitors led to the development of a subnanomolar compound (21) with millimolar solubility. This inhibitor demonstrated excellent efficacy and a long duration of action upon intravenous administration to monkeys. While activity was also observed intraduodenally, a comparison of the blood pressure responses indicated low bioavailability. Subsequent experiments in rats showed that, although the compound was absorbed from the gastrointestinal tract, extensive liver extraction severely limited bioavailability.

Effect of intrarenal renin inhibition on renal hemodynamics and excretory function.

This study was designed to investigate in sodium-depleted monkeys the renal hemodynamic and excretory effects resulting from blockade of the renin-angiotensin system induced by intrarenal infusion of the primate-selective renin inhibitor A-65317. Intrarenal infusion of A-65317 (n = 6) at a dose of 0.01 micrograms.kg-1.min-1 elicited an increase (P less than 0.05) in renal blood flow (RBF) from 43.5 +/- 2.7 to 49.4 +/- 4.4 ml/min and glomerular filtration rate (GFR) from 6.3 +/- 0.3 to 6.9 +/- 0.4 ml/min, with no significant changes in mean arterial pressure (MAP) or plasma renin activity (PRA). Increases (P less than 0.05) in the urine flow rate (0.18 +/- 0.04 to 0.28 +/- 0.04 ml/min) and the fractional excretion of sodium (0.18 +/- 0.06 to 0.35 +/- 0.13%) were also observed. After a recovery period, the intrarenal infusion dose of A-65317 was increased to 0.1 microgram.kg-1.min-1 and RBF increased (P less than 0.05) from 42.9 +/- 3.9 to 53.0 +/- 3.7 ml/min in conjunction with a significant 85 +/- 4% inhibition of PRA and a 14 +/- 4 mmHg reduction in MAP. GFR and electrolyte excretion remained at control levels. Intrarenal infusion of vehicle (n = 6) had no significant effect on any of the variables studied. In a separate group of monkeys, intravenous (iv) infusion of A-65317 at 0.01 microgram.kg-1.min-1 (n = 5) did not result in significant changes from control.(ABSTRACT TRUNCATED AT 250 WORDS)

Prolonged duration of blood pressure response to enalkiren, the novel dipeptide renin inhibitor, in essential hypertension.

The effects of sustained renin inhibition by repeated administration of enalkiren (A-64662), the novel dipeptide renin inhibitor, were evaluated in a randomized, double-blind, placebo-controlled, parallel-group study of 32 inpatients (eight per group) with essential hypertension who were maintained on a diet containing 60 meq/day sodium. Three different dosage regimens of enalkiren were studied: 1) 1.2 mg/kg quotid., 2) 0.3 mg/kg q.i.d., and 3) 0.1 mg/kg q.i.d. Each patient received an intravenous infusion every 6 hours for 1 week. Placebo infusions were used to mimic the 4 times/day dosing schedule. Blood pressure was measured periodically via 24-hour automated monitoring equipment. Mean plasma renin activity in the patient groups ranged from 1.58 to 2.68 ng angiotensin I/ml/hr. Plasma renin activity was promptly suppressed in all groups receiving enalkiren. Prolonged duration of plasma renin activity suppression (greater than or equal to 24 hours) was demonstrated after the administration of 1.2 mg/kg enalkiren. The 0.3 mg/kg q.i.d. and 1.2 mg/kg quotid. regimens produced statistically significant reductions (p less than or equal to 0.05) in systolic and diastolic blood pressures with clear evidence of persistent antihypertensive activity for 12 hours or more when compared with the placebo group. Despite relatively large reductions in mean systolic and diastolic blood pressure, mean pulse rates were essentially unchanged. The prolonged reduction in blood pressure with enalkiren without evidence of tachyphylaxis after 1 week of treatment suggests that renin inhibitors may emerge as useful therapeutic agents for the treatment of hypertension.

Renal hemodynamic and excretory responses to renin inhibition induced by A-64662.

Experiments were conducted in sodium-depleted anesthetized monkeys to determine the effects of the primate-selective renin inhibitor A-64662 on renal function. Five groups of monkeys were examined with each group receiving an i.v. infusion of vehicle or A-64662 at doses (bolus plus continuous infusion) of 0.1 + 0.01, 1.0 + 0.1, 10 + 1.0 or 100 micrograms/kg + 10 micrograms/kg/min (n = 6/dose). Plasma renin activity was inhibited (P less than .05) at all infusion doses ranging from 33 +/- 8% at the lowest dose to 95 +/- 3% at the highest dose. Inhibition of plasma renin activity was accompanied by renal vasodilation as renal blood flow (RBF) increased (P less than .05) in a dose-dependent manner beginning at the dose of 1.0 microgram/kg + 0.1 micrograms/kg/min. RBF increased 36 +/- 7% at the highest dose of A-64662 examined. Associated with the increments in RBF, renal vascular resistance progressively decreased (P less than .05) by 12 +/- 3, 31 +/- 3 and 40 +/- 6%, respectively, with increasing doses of A-64662. Glomerular filtration rate was unchanged at all doses of A-64662. As a result, a significant (P less than .05) fall in the filtration fraction was observed as the dose of A-64662 increased. Mean arterial pressure was unaffected by the two lowest doses of A-64662, but decreased (P less than 0.05) by 13 +/- 1 and 18 +/- 4 mm Hg, respectively, at the two highest doses of A-64662 infusion.(ABSTRACT TRUNCATED AT 250 WORDS)