Studies of human pancreatic elastase treatment of rabbit and human vein rings to predict human therapeutic doses.

Vascular tissue contains abundant elastic fibers that contribute to vessel elasticity. Vonapanitase (formerly PRT-201) is a recombinant human chymotrypsin-like elastase family member 1 (CELA1) shown to cleave the elastin component of elastic fibers, resulting in increased vessel diameter. The purpose of these current studies was to determine vein diameter, wall thickness, elastin content, and vonapanitase potency in veins used in a model of arteriovenous fistula (AVF) and in patients undergoing AVF creation for hemodialysis access to guide dose selection for human trials. Rabbit linguofacial, maxillary, and external jugular veins, and human basilic and upper and lower arm cephalic veins were dissected postmortem and sectioned into 2 mm length rings. Rings were incubated in vonapanitase at 37°C at varying concentrations and times. Elastin content was estimated histologically and by quantifying desmosine, a protein cross-link unique to elastin. Rabbit veins were substantially thinner and contained less elastin than human veins. In human veins, elastin content was greatest in basilic and least in lower arm cephalic. Vonapanitase removed elastin in a time- and concentration-dependent manner in all vein types. A lower concentration of vonapanitase was required to remove elastin from rabbit relative to human veins. In summary, vonapanitase reduced the elastin content of rabbit and human veins but did so at a lower concentration in the rabbit veins. Rabbit models may overestimate the potency of vonapanitase in humans. These results indicate that human dose selection should be guided by human vein ring experiments.

Recombinant Human Elastase Treatment of Cephalic Veins.

BACKGROUND: Vessel injury at the time of Arteriovenous Fistula (AVF) creation may lead to neointimal hyperplasia that impairs AVF maturation. Vonapanitase, a recombinant human chymotrypsin-like elastase family member 1, is an investigational drug under development to improve AVF maturation and patency. The current studies were designed to document vonapanitase effects in human cephalic veins that are used in AVF creation. METHODS: Human cephalic veins were mounted on a perfusion myograph. Vonapanitase 1.2, 4, 13.2, and 40 µg/ml or saline was applied drop wise on the vein followed by saline rinse. Vein segments were cut into rings for elastin content determination by desmosine radioimmunoassay and histology. Fluorescently-labelled vonapanitase was applied to veins and adventitial imaging was performed using laser scanning confocal microscopy. In vivo time course experiments were performed by treating rabbit jugular veins and harvesting 1 h and 4 h after vonapanitase treatment. RESULTS / CONCLUSION: Vonapanitase reduced desmosine content in a dose-related manner. Histology also confirmed a dose-related reduction in elastic fiber staining. Fluorescently-labelled vonapanitase persistently localized to elastic fibers in the vein adventitia. In vivo experiments showed a reduction in desmosine content in jugular veins from 1 h to 4 h following treatment. These data suggest that vonapanitase targets elastin in elastic fibers in a dose related manner and that elastase remains in the vessel wall and has catalytic activity for at least 1 h.

Recombinant Human Elastase Alters the Compliance of Atherosclerotic Tibial Arteries After Ex Vivo Angioplasty.

PURPOSE: This study was designed to determine whether vonapanitase (formerly PRT-201), a recombinant human elastase, treatment can fragment the protein elastin in elastic fibers and cause dilation of atherosclerotic human peripheral arteries subjected to ex vivo balloon angioplasty. MATERIALS AND METHODS: Seven patients undergoing lower limb amputation for peripheral artery disease or who died and donated their bodies to science donated 11 tibial arteries (5 anterior, 6 posterior) for this study. All arteries were atherosclerotic by visual inspection. The arteries underwent ex vivo balloon angioplasty and thereafter were cut into rings and studied on wire myographs where the rings were stretched and tension was recorded. After treatment with vonapanitase 2 mg/mL or vehicle control, myography was repeated and the rings were then subject to elastin content measurement using a desmosine radioimmunoassay and elastic fiber visualization by histology. The wire myography data were used to derive compliance, stress-strain, and incremental elastic modulus curves. RESULTS: Vonapanitase treatment reduced elastin (desmosine) content by 60% and decreased elastic fiber histologic staining. Vonapanitase-treated rings experienced less tension at any level of stretch and as a result had shifts in the compliance and stress-strain curves relative to vehicle-treated rings. Vonapanitase treatment did not alter the incremental elastic modulus curve. CONCLUSIONS: Vonapanitase treatment of atherosclerotic human peripheral arteries after ex vivo balloon angioplasty fragmented elastin in elastic fibers, decreased tension in the rings at any level of stretch, and altered the compliance and stress-strain curves in a manner predicting arterial dilation in vivo. Based on this result, local treatment of balloon angioplasty sites may increase blood vessel diameter and thereby improve the success of balloon angioplasty in peripheral artery disease.

Effects of recombinant human type I pancreatic elastase on human atherosclerotic arteries.

RATIONALE: At physiologic pressures, elastic fibers constrain artery diameter. Local treatment of atherosclerotic arteries with PRT-201, a recombinant type I elastase, could result in fragmentation and removal of elastin fibers and increased vessel diameter. OBJECTIVE: To investigate the use of PRT-201 as a treatment for human atherosclerotic arteries. METHODS AND RESULTS: Arteries were harvested from donor legs amputated due to severe peripheral artery disease or from recently deceased persons who donated their bodies to science. Three- to four-centimeter artery segments were studied on a perfusion myograph to obtain baseline diameter data. After treatment with PRT-201 3.6 mg/mL or saline for 30 minutes myography was repeated. PRT-201 treatment resulted in an increase in vessel diameter across a range of transmural pressures. Average anterior tibial artery diameter increased by 0.78 ± 0.21 mm (27% ± 12%), whereas average posterior tibial artery diameter increased by 0.58 ± 0.30 mm (21% ± 11%), both P < 0.001. Elastin content as measured by desmosine radioimmunoassay was reduced by approximately 50%, P < 0.001. CONCLUSIONS: The results suggest that PRT-201 treatment of atherosclerotic peripheral arteries in patients could increase artery diameter, and thus luminal area, possibly alleviating some of the symptoms of peripheral artery disease.

A novel system for the investigation of microvascular dysfunction including vascular permeability and flow-mediated dilatation in pressurised human arteries.

INTRODUCTION: Adverse drug reactions may be manifested through changes in microvascular function (e.g. angioedema) or by subtle modification of the mechanisms controlling vascular tone, such as flow-mediated dilatation. Until now the early detection of such adverse drug reactions has been hampered by the lack of a predictive in vitro model. This in vitro model can be utilised to test potential effect of drugs on the normal responses of the vascular system. METHODS: The PM-1, a new automated perfusion myograph, allows detection of the external and internal dimensions of tubular biological structures and regulates both the intraluminal pressure and flow independently. Drugs can be infused intraluminally or extraluminally (by adding to the bathing solution) to determine effects on constriction, relaxation or modulation of vascular tone. The novel imaging system also facilitates the measurement of vascular permeability using dyes introduced intraluminally into the vessel. RESULTS: To assess effects on flow-mediated dilatation we increased flow rate in pressurised human subcutaneous arteries (<500mum diameter) in the absence and presence of various drugs. Increasing flow from 0.04ml/min to 0.3ml/min resulted in a 39+/-3% relaxation of a U46619 pre-constriction (10(-6)M). This was enhanced in the presence of Ivermectin and inhibited in the presence of 100microM L-NAME (316+/-169% and 16+/-1% respectively).To assess effects on vascular permeability we infused albumin-bound Evans blue dye through the lumen of human subcutaneous arteries as a marker, in the absence and presence of a modulatory drug. Infusion of thrombin (0.5units/ml) through the vessel lumen caused an 11.8% increase in vessel permeability compared to vehicle infusion. CONCLUSION: The development of the PM-1 allows new drugs to be tested in relevant human or animal tissues at an early stage allowing crucial go/no-go decisions to be made early in development and giving a more complete picture of the overall effects of test compounds on vascular function.