ABSTRACT
BILN 2061 is a potent, reversible inhibitor of hepatitis C virus NS3/NS4A serine protease. Early clinical proof of principle with the drug was offset by the results of subsequent safety studies in Rhesus monkeys revealing cardiotoxicity that featured myocardial vacuolation corresponding to mitochondrial swelling. Here we describe an investigation into the nature, onset, and reversibility of the lesion, and an assessment of potentially predictive biomarkers for the change. Rhesus monkeys were orally administered 1,000 mg/kg/day BILN 2061 and either necropsied after one, three, fourteen, or twenty-eight doses or afforded a ten-week recovery period. The results of electrocardiographic and plasma troponin I and T measurements were unaffected by BILN 2061, but cardiac myocytic vacuolation, correlated with mitochondrial swelling, was observed after three or more doses. Echocardiographic traces obtained after twenty-eight consecutive days of dosing revealed two animals with diminished left ventricular cardiac ejection fraction. One animal was immediately necropsied and exhibited marked cardiotoxicity. The other was afforded a ten-week treatment-free period during which the left ventricular ejection fraction returned to normal. All recovery animal hearts were microscopically and ultrastructurally normal. High-dose BILN 2061 cardiotoxicity in Rhesus monkeys appeared early in the treatment regimen and exhibited reversibility. A reliable biomarker has yet to be identified.
Subject(s)
Carbamates/toxicity , Hepacivirus/drug effects , Macrocyclic Compounds/toxicity , Protease Inhibitors/toxicity , Quinolines/toxicity , Thiazoles/toxicity , Administration, Oral , Animals , Antiviral Agents/pharmacology , Antiviral Agents/toxicity , Biomarkers , Carbamates/pharmacology , Cardiotoxins/pharmacology , Cardiotoxins/toxicity , Drug Evaluation, Preclinical , Female , Macaca mulatta , Macrocyclic Compounds/pharmacology , Male , Mitochondrial Swelling/drug effects , Myocytes, Cardiac/drug effects , Protease Inhibitors/pharmacology , Quinolines/pharmacology , Serine Proteases/metabolism , Thiazoles/pharmacology , Viral Nonstructural ProteinsABSTRACT
Monocyte chemoattractant protein-1 (MCP-1) is a stimulator of collateral artery growth and has been shown to increase collateral artery conductance in rabbits and pigs. The minimal infusion duration and the minimally effective dose of MCP-1 are currently unknown, as is the sustainability of the therapeutic effect over a longer observation period than tested before. MCP-1 was infused intra-arterially in pigs after unilateral femoral artery occlusion in different doses and infusion durations between 2 hours and 2 weeks. Two weeks after ligation, arterial conductance under maximal vasodilatation was measured. The long-term efficacy was investigated in 2 additional groups of animals after 6 weeks. Infusion with 2 microg/min of MCP-1 for 6 hours was sufficient to double arterial conductance, and arterial conductance after 6 weeks was still significantly increased.
Subject(s)
Arteries/drug effects , Chemokine CCL2/pharmacology , Peripheral Vascular Diseases/drug therapy , Animals , Arteries/growth & development , Arteries/physiopathology , CHO Cells , Chemokine CCL2/administration & dosage , Chemokine CCL2/genetics , Collateral Circulation/drug effects , Cricetinae , Cricetulus , Disease Models, Animal , Dose-Response Relationship, Drug , Female , Femoral Artery/surgery , Humans , Infusions, Intra-Arterial , Ligation/adverse effects , Male , Peripheral Vascular Diseases/etiology , Peripheral Vascular Diseases/physiopathology , Receptors, CCR2 , Receptors, Chemokine/genetics , Receptors, Chemokine/physiology , Recombinant Proteins/administration & dosage , Recombinant Proteins/pharmacology , Swine , Swine, Miniature , Time Factors , Treatment OutcomeABSTRACT
PURPOSE: To test whether blocking the Na+/H+ antiport reduces intraocular pressure (IOP) in the mouse. METHODS: The electrophysiologic approach (the servo-null micropipette system, SNMS) that had been adapted for continuously monitoring IOP in the mouse was used in a study of the effects of a series of transport inhibitors. RESULTS: Topical application of three direct blockers of Na+/H+ exchangers produced comparable reductions in mouse IOP: dimethylamiloride (DMA, -5.0 +/- 0.7 mm Hg), ethylisopropylamiloride (EIPA, -4.1 +/- 1.0), and BIIB723 (-4.9 +/- 1.7 mm Hg). These effects were mediated locally, not systemically, because adding DMA to one eye had no effect on IOP in the contralateral eye. In contrast to the actions of selective inhibitors of Na+/H+ exchange, neither the low-potency inhibitor amiloride nor the inhibitor of Na+-K+-2Cl- cotransport bumetanide by itself was effective. Dorzolamide, which slows delivery of H+ and HCO3- to Na+/H+ and Cl-/HCO3- antiports, also reduced IOP by 2.9 +/- 0.6 mm Hg. After first blocking Na+/H+ exchange with DMA, EIPA, BIIB723, or dorzolamide, application of bumetanide produced an additional reduction in IOP of 3.8 to 4.0 mm Hg. CONCLUSIONS: The first step in formation of aqueous humor is uptake of NaCl by the ciliary epithelial cells from the stroma, possibly by both paired Na+/H+ and Cl-/HCO3- antiports and a bumetanide-sensitive Na+-K+-2Cl- symport. The present data are consistent with electron probe x-ray microanalyses of rabbit ciliary epithelium indicating that the antiports are the dominant mechanism. That bumetanide can produce a previously unobserved lowering of IOP when the Na+/H+ antiport is also inhibited substantiates a dominant antiport mechanism.
Subject(s)
Amiloride/analogs & derivatives , Amiloride/pharmacology , Intraocular Pressure/drug effects , Sodium-Hydrogen Exchangers/antagonists & inhibitors , Sulfonamides/pharmacology , Thiophenes/pharmacology , Administration, Topical , Amiloride/administration & dosage , Animals , Antihypertensive Agents/administration & dosage , Antihypertensive Agents/pharmacology , Aqueous Humor/metabolism , Bumetanide/pharmacology , Diuretics/administration & dosage , Diuretics/pharmacology , Electrophysiology , Female , Male , MiceABSTRACT
PURPOSE: The purpose of this study was to develop a nonviral gene transfer method for therapeutic delivery of the human monocyte chemoattractant protein-1 (MCP-1) in patients with peripheral artery disease, using local catheter-mediated electrotransfer of naked plasmid DNA into arteries. METHODS: Arterial walls of the A. profunda femoris of pigs were transfected either with a human MCP-1 or with a firefly luciferase-encoding DNA construct. The efficacy of electrotransfer of DNA was analyzed after 2 days by quantitative polymerase chain reaction (PCR) or luciferase activity measurements. To optimize MCP-1 gene transfer conditions, a voltage range of 60-150 V was applied as a train of six square pulses of 20 ms each at 1 Hz and was combined with a dose of 150 microg DNA. Subsequently, the optimized voltage was used to test a dose range of 80-300 microg DNA. RESULTS: The voltage optimum for arterial transfection was observed at 80 volts. Using this setting, the dose application of 300 microg MCP-1 plasmid DNA (the maximal dose tested) demonstrated the highest MCP-1 expression signal. The electric pulses and the transfer and expression of human MCP-1 per se did not induce endogenous porcine MCP-1 expression in treated arteries. Interestingly, angioplastic predilation of the artery before gene transfer, which had originally been postulated to enhance transfection by improving access of the plasmid to subendothelial cell layers, resulted in an attenuated transfection efficacy. CONCLUSIONS: The present study demonstrates that transluminal catheter-based electroporation provides an efficient technology for nonviral intravascular gene transfer by just applying unformulated DNA.
Subject(s)
Carotid Arteries/drug effects , Catheterization/methods , Chemokine CCL2/genetics , Electroporation/methods , Transfection/methods , Animals , Catheterization/instrumentation , Chemokine CCL2/metabolism , Humans , Luciferases/genetics , Luciferases/metabolism , Swine , Transfection/instrumentationABSTRACT
Cardiac hypertrophy is often associated with an increased sympathetic drive, and both in vitro and in vivo studies have demonstrated the development of cardiomyocyte hypertrophy in response to either alpha- or beta-adrenergic stimulation. Because an association between the Na+/H+ exchanger and cellular growth has been proposed, this study aimed to analyze the possible role of the antiporter in isoproterenol-induced cardiac hypertrophy. Isoproterenol alone (5 mg/kg IP once daily) or combined with a selective inhibitor of the Na+/H+ exchanger activity (3 mg x kg(-1) x d(-1) BIIB723) was given to male Wistar rats for 30 days. Sex- and age-matched rats that received 0.9% saline IP daily served as controls. Echocardiographic follow-up showed a 33% increase in left ventricular mass in the isoproterenol-treated group, whereas it did not increase in the isoproterenol+BIIB723-treated group. Heart weight-to-body weight ratio at necropsy was 2.44+/-0.11 in controls and increased to 3.35+/-0.10 (P<0.05) with isoproterenol, an effect that was markedly attenuated by BIIB723 (2.82+/-0.07). Intense cardiomyocyte enlargement and severe subendocardial fibrosis were found in isoproterenol-treated rats, and both effects were attenuated by BIIB723. Myocardial Na+/H+ exchanger activity and protein expression significantly increased in isoproterenol-treated rats compared with the control group (1.45+/-0.11 vs 0.91+/-0.05 arbitrary units, P<0.05). This effect was significantly reduced by BIIB723 (1.17+/-0.02, P<0.05). In conclusion, our results show that Na+/H+ exchanger inhibition prevented the development of isoproterenol-induced hypertrophy and fibrosis, providing strong evidence in favor of a key role played by the antiporter in this model of cardiac hypertrophy.