Author + information
- Received September 10, 1996
- Revision received February 10, 1997
- Accepted March 4, 1997
- Published online June 1, 1997.
- ↵*Mark A. Lovich, Division of Health Sciences and Technology, Massachusetts Institute of Technology, 56-322, Cambridge, Massachusetts 02139.
Objectives. We attempted to characterize how drug released into the perivascular space enters the arterial wall and how it is cleared from the local environment.
Background. Drug released into the perivascular space can enter the artery either from the adventitial aspect or from the lumen after absorption by the extraarterial capillaries and mixing within the systemic circulation. Some investigators suggest that this latter mechanism dominates, and they question whether local drug release is synonymous with local deposition.
Methods. We investigated both the pathways by which adventitially released drug is cleared from the perivascular space and those by which drug enters the blood vessel wall. Inulin was used to follow drug release from implanted devices and subsequent entry to the circulation, because of its first-pass urinary excretion. Heparin was used to follow arterial deposition because of its vasoactivity and tissue-binding properties. The different potential pathways of drug entry and egress were systematically removed and the effects on metabolism and deposition determined.
Results. Ligature occlusion of the artery did not decrease inulin excretion or heparin deposition. Extravascular wraps designed to shield the device from extramural capillaries reduced inulin excretion rates 10-fold but did not alter heparin deposition into the vessel wall. The deposition of drug after perivascular delivery was 500 times higher than after intraperitoneal administration.
Conclusions. Although almost all the drug released into the perivascular space is cleared through the extravascular capillaries, virtually all the deposited drug diffuses directly from the perivascular space, and little arrives from the endovascular aspect. These data support the view that local drug release leads directly to increased local drug concentration.
(J Am Coll Cardiol 1997;29:1645–50)
It has been hoped for some time that the systemic toxicity of novel vasoactive compounds could be circumvented with local vascular drug delivery, directing potent drugs to injured blood vessels without the potential adverse complications of systemic administration ([1, 2]). Polymeric and cell-based delivery systems have been implanted perivascularly to deliver growth-modulating compounds to arterial cells and tissues ([3–5]). Yet the precise mechanisms by which the compounds travel from perivascular devices and enter vascular tissues are still elusive, making their pharmacokinetics difficult to assess and frustrating appreciation of their full clinical utility. For example, it might be argued that drug released into the perivascular space principally enters the adjacent artery from the lumen after absorption by extramural capillaries and mixture within the systemic circulation, rendering periarterial release functionally identical to any other form of intravascular delivery. Moreover, one must then reconsider the perceived advantages of local perivascular implantation and release. On the other hand, the success of perivascular release strategies in comparison with modes of systemic administration suggests a physiologic advantage to both local sustained release and direct delivery of drug to the adventitial aspect of the artery (). Elucidation of the transport and metabolic pathways for drugs placed outside the artery is therefore critical to optimizing release device formulations and implantation regimens for maximal biologic effect.
We therefore sought to characterize the pathways by which drug is cleared from the perivascular space and subsequently deposited within the blood vessel wall. Drug can be cleared from the perivascular space by transmural diffusion (pathway a, Fig. 1a), or absorption by extraarterial microvessels (pathway b). Extraarterial capillaries are defined as any source of clearance outside the arterial wall proper and includes the capillaries of skeletal muscle or other tissues and lymphatic vessels. The relative importance of these pathways was determined by measuring the urinary excretion rate of perivascularly released inulin as each route was systematically eliminated. As we have previously documented (), urinary inulin excretion can be used as a proxy for drug entry into the general circulation as this polysaccharide is first-pass excreted and eliminated from plasma through the renal glomerulus without metabolism, secretion or tubular reabsorption. Drug absorbed by these capillaries could join the systemic circulation and enter the vessel wall from its endovascular aspect. This possibility, as well as the potential for drug to diffuse indirectly from the perivascular aspect, was tested by following the deposition of heparin released from outside the blood vessel. Heparin was chosen as a model vasotherapeutic drug because it binds reversibly to elements of the blood vessel wall ([7, 8]) and is a potent inhibitor of vascular smooth muscle cell proliferation ([9–11]). Heparin deposition was measured in the native unmanipulated state and after elimination of pathways that could lead to endovascular administration from the systemic circulation (pathway c, Fig. 1b), extraarterial capillary uptake (pathway d, Fig. 1b) or direct diffusion from the perivascular space (pathway e, Fig. 1b). These studies may allow for greater understanding of local vascular drug metabolism and more rational design of regional vascular drug therapy.
We measured the individual in vitro release kinetics of 14C-inulin or 3H-heparin from 28% (wt/vol) gels of Poloxamer copolymer (poly(oxyethylene)-poly(oxypropylene) [Pluronic 407, BASF Wyandotte Corp. and Anti-adhesion 28, MDV Technologies]). Poloxamer 407 solutions undergo reverse phase gelation, remaining as free-flowing liquids until the ambient temperature is raised above the critical threshold of 15°C, well below room and body temperature. Above this temperature, viscosity increases markedly and the gel solidifies into a solid mass. Erosion of, and subsequent release from, such a gel is then prolonged, enabling use as drug depots. Poloxamer solution containing either 14C-inulin (3.5 μCi, 1.4 mg, NEN-Dupont) or 3H-heparin (10.0 μCi, 14 μg, NEN-Dupont) was kept in liquid form on ice (3° to 5°C), and then 100-μl aliquots were injected into a Silastic tube (inner diameter [ID] 3.18 mm, Dow Corning) maintained at 23°C. The rise in temperature from 4°C to 23°C promoted reverse phase gelation. The resultant cylindric plug of Poloxamer was removed from the tubing, placed in 50 ml of phosphate-buffered saline solution (pH 7.4 and 37°C) under agitation, and 0.5-ml samples were removed every 15 min for hour 1 and every 30 min for the next 4 h. All in vitro release experiments were performed in triplicate.
1.1 Routes of Clearance From the Perivascular Space.
The protocol conformed to the “Position of the American Heart Association on Research Animal Use” adopted by the Association in November 1984, and it was approved by the Massachusetts Institute of Technology Committee on Animal Care. Sprague-Dawley female rats (275 to 325 g) were anesthetized with an intraperitoneal injection of ketamine (50 mg/kg body weight) and xylazine (10 mg/kg). Urinary inulin excretion was monitored continuously through the placement of a catheter into the bladder. The bladder, exposed after a midline abdominal incision, was cannulated with a 20-cm polyethylene tube (ID 0.58 mm, Clay Adams) and secured with a purse-string suture. Incisions were closed, and supplementary anesthesia with ketamine (12.5 mg/kg) and xylazine (2.5 mg/kg) was administered as necessary.
The left common carotid artery was exposed and cleaned of excess fat and fascia. Histologic and immunohistochemical examination of arteries that had undergone similar manipulation showed complete absence of vasa vasorum and no overt damage. In one experimental group, a 100-μl dose of 14C-inulin in 28% (wt/vol) Poloxamer solution (0.35 μCi, 0.14 mg) kept on ice (3° to 5°C) was injected into the perivascular space. The gel, which had remained fluid while cool, gelled immediately on contact with the artery at 37°C, conforming to the irregular surface of the vessel (). Urinary flow through the bladder cannula was directed into a clean scintillation vial every 15 min for 1 h and every 30 min for the next 3 h. In a second experimental group, the potential for transarterial inulin clearance was eliminated by ligating arteries at proximal and distal sites spaced 1 cm apart (blocking pathway a, Fig. 1a). Any inulin that traversed the wall was then trapped within the occluded segment and could not mix with systemic circulation for excretion. In a third group, the Poloxamer gel and extraarterial capillary beds were separated by a circumferential Silastic sheath (ID 3.18 mm) whose ends and seam were plugged with a silicon glue (Type A Medical Adhesive, Dow Corning). The Poloxamer solution was injected only into the space within the sheath, retaining arterial contact with the gel but eliminating possible capillary bed absorption of inulin (blocking pathway b). After all of these experiments the 14C-inulin content within each urine sample was determined by liquid scintillation spectroscopy (1214 RackBeta, LKB-Wallac). In a fourth groupthe integrity of the Silastic wrap was determined. Each of the manipulations described for the previous two groups was employed and both transmural and extraarterial capillary clearance were eliminated (blocking pathways a and b, Fig. 1a). Four rats were examined in each of the four experimental groups. To visualize potential leaks from the seam or plugs at the ends of this barrier, Evans blue dye (Sigma) was mixed into the Poloxamer solution (25 mg/ml), and then injected into a wrapped artery of an additional animal.
1.2 Routes of Entry to the Blood Vessel Wall.
In a similar fashion, drug entry and deposition into the arterial wall were quantified with heparin-releasing Poloxamer gels fabricated as described above. Female Sprague-Dawley rats (275 to 325 g) were anesthetized and their left carotid arteries isolated as before. Four animals were examined in each experimental group. In one group, 100 μl of 3H-heparin (1.0 μCi, 1.4 μg) in 28% Poloxamer solution kept on ice (3° to 5°C) was injected into the perivascular space of the left carotid artery. The Poloxamer solution gelled immediately on contact with the artery. One hour after administration of the heparin-gel, the left carotid artery was excised, blotted to remove excess fluid, and dipped into 100% ethanol to dissolve adhering Poloxamer gel. The artery was dehydrated, weighed, solubilized with 0.5 ml of Soluene-350 (Packard) and counted by liquid scintillation spectrometry for 3H-heparin deposition. In addition, 10- to 60-mg tissue samples from the abdominal aorta, iliac and femoral arteries and liver were harvested, dehydrated, weighed, solubilized and counted. The liver was assayed in particular because its high density of endothelial cells created a large potential sink for heparin binding ([13, 14]). Liver samples were bleached to reduce color quenching of tritium by adding 0.5 ml of hydrogen peroxide before scintillation counting.
In a second experimental group, the possibility that heparin could be deposited from blood flowing in the lumen was eliminated by occluding the artery with proximal and distal ligatures. Any heparin detected in the wall could have only arrived directly from the perivascular space (blocking pathway c, Fig. 1b). In a third group, the possibility that heparin might enter the systemic circulation from extraarterial capillary drug absorption was prevented with a Silastic wrap. The heparin-Poloxamer solution was injected around the artery but inside a silicon glue–sealed Silastic sheath (blocking pathway d). In a fourth group, heparin deposition within the carotid artery was quantified after the Poloxamer solution was injected into the highly vascularized peritoneal cavity (blocking pathway e). The contribution of direct diffusion from the perivascular space could then be determined by comparison with data obtained after local arterial delivery. In this group, care was taken to remove liver samples that were not in contact with any injected Poloxamer gel.
All data are presented as the mean value ± SEM. Linear regression correlation coefficients were determined for in vitro release data and for urinary inulin excretion data. The former were compared using two-tailed ttests of equal variance and the latter through analysis of variance (ANOVA). Heparin deposition measurements for the various manipulations to the carotid artery were compared through ANOVA. All data were deemed statistically different when p < 0.05.
In vitro release kinetic profiles for inulin and heparin from Poloxamer 407 gels cast into cylindric geometries were linear with time and statistically indistinguishable (Fig. 2). Inulin was administered from these gels into the perivascular space of unmanipulated native arteries or ligature-occluded arteries whose transarterial pathways to the systemic circulation were removed (Fig. 1a). Rates of inulin clearance from the perivascular space of these two vessels, native and occluded, were statistically indistinguishable (Fig. 3a), implying that drug released into the perivascular space was cleared through the extraarterial capillaries and not through the wall of the carotid artery into the lumen. These observations were further supported by tracking inulin excretion rates when the polysaccharide was released from the perivascular space of wrapped arteries. The wraps served as an impermeable barrier preventing drug from clearing through the extraarterial capillaries. Inulin excretion rates from wrapped arteries were ∼10-fold lower than for native or occluded arteries, but indistinguishable from rates observed in arteries that were both occluded and wrapped (Fig. 3b). Thus, the minimal amount of inulin excretion detected with wrapped arteries reflects leakage through imperfections in the Silastic barrier rather than transmural clearance. The presence of barrier leaks was visually detected when Evans blue dye delivered from the Poloxamer gel extravasated from the ends of the wrap and migrated along the artery, ultimately spreading to the capillaries in the perivascular space.
Deposition of heparin released into the perivascular space was followed as each potential pathway from release device to the tissue—extraarterial capillary absorption, delivery from the general circulation and direct diffusion—was systematically eliminated (Fig. 1b). After 1 h of release, the amount of heparin in the carotid artery was <1.2% of the administered dose, 500-fold higher than in distant arteries or the liver and not statistically altered by ligature occlusion of the blood vessel (Fig. 4). After intraperitoneal application of the heparin-Poloxamer gel, drug deposition in the carotid artery was similar to that in other tissues. Drug delivery from inside a wrap surrounding the carotid artery did not result in statistically increased local arterial concentrations. In addition, concentrations in distant structures were not diminished, because the wrap failed to eliminate extraarterial capillary absorption.
Although local perivascular drug delivery systems have demonstrated successful release of a range of compounds and achieved diverse biologic effects in injured blood vessels ([3, 4, 12, 15–21]), limitations remain both in understanding the mechanisms of action and in optimal design of these systems. The pharmacokinetics that result from such systems are complex, as they impart large time-dependent drug concentration gradients across vascular tissues (). Precise elucidation of the pharmacokinetics requires not only quantitative evaluation of the forces that govern transport and binding within arterial tissues but also a description of how drug arrives at the boundary of the blood vessel wall. Transport and binding constants have been evaluated for a model vasotherapeutic compound, heparin, in vascular tissues ([7, 23]); however, the boundary conditions remain obscure. Controversy exists as to whether drug released into the perivascular space diffuses directly to the artery or whether drug is absorbed by capillaries outside the artery, mixed with the systemic circulation and only then returned to the endovascular aspect. Countless studies have exhaustively characterized both the mechanisms and kinetics of drug release from polymeric systems, and devices can now be fabricated to deliver virtually any pattern of release. However, there is a dearth of information regarding the fate of the drug once freed. It has been further noted () with other compounds that only a small fraction of the drug released into the perivascular space can be found within the blood vessel, suggesting that steps might be taken to increase the efficiency of local delivery. Additional debate exists as to whether wrapping the artery and release device within an impermeable barrier that shields extraarterial microvessels from the drug will improve vascular localization and further minimize systemic levels.
3.1 Model Pathways From the Perivascular Space to the Blood Vessel Wall.
The present study was designed to show how perivascularly released drug arrives at the arterial wall by independently determining the relative importance of each of the pathways by which drug leaves the perivascular space and by which pathways it enters the blood vessel wall. We delivered model drugs, inulin and heparin, perivascularly and observed how the urinary excretion and arterial deposition were altered as the pathways of vessel wall clearance and entry were systematically eliminated, respectively. The potential routes of drug clearance from the perivascular space are absorption by extraarterial microvessels or transarterial diffusion directly into the circulation (Fig. 1a), whereas the pathways to the arterial wall are endovascular application from the circulation or direct diffusion through the adventitial aspect (Fig. 1b). Ligature occlusion of the blood vessel did not decrease inulin excretion (Fig. 3a) or decrease heparin deposition (Fig. 4). Extravascular wraps that were designed to prevent exposure of the extravascular capillaries to the drug reduced inulin excretion but left heparin deposition unchanged. However, the deposition of heparin after perivascular delivery was much higher than for intraperitoneal administration. The combination of these analyses reveals that the overwhelming amount of perivascularly released drug is absorbed by the extraarterial capillaries, yet virtually all the drug found in the artery diffuses directly from the device through the perivascular space and into the arterial wall.
These seemingly contrary processes are neither paradoxic nor mutually exclusive. The total surface area for absorption of the extraarterial capillaries is much larger than that of the carotid artery. Moreover, the transmural diffusive resistance of the artery is much greater than that of an individual capillary, simply because it is much thicker (). Both of these effects cause the vast majority of drug to be cleared by the extraarterial capillaries. That all of the drug deposited in the arterial wall comes from the perivascular space, and not from the circulation, reflects systemic dilution far below the drug concentration in the perivascular interstitial fluids. Therefore, there is a large transmural concentration gradient forcing drug into the vessel wall from the perivascular space. These arguments reflect the anatomy of the blood vessel wall and the surrounding extraarterial capillaries, and are valid for any compound. Therefore, the balance between transarterial transport and absorption by these capillaries, to a first-order approximation, is insensitive to differences in diffusion, partitioning and binding exhibited by heparin, inulin or other compounds. We have, therefore, combined these two results to provide a unified qualitative model of how drug is handled by vascular tissues and their local environments.
The rat carotid artery is thin and has few if any vasa vasorum. In thicker arteries the transvascular resistance is greater and transmural clearance will be even further overwhelmed by absorption from extraarterial capillaries. However, larger arteries are vascularized by vasa vasorum (), which potentially absorb drug and carry it deeper into the media, thus enhancing transmural clearance. Both increased transvascular resistance and the contributions of vasa vasorum are important considerations for extrapolating the results of these studies to larger arteries.
3.2 Is There a Need to Wrap the Device With the Artery?
The potential loss of drug to extraarterial capillaries has prompted some investigators ([3, 26]) to advocate that perivascular drug delivery might be more efficient if the artery and release device were wrap-isolated with impermeable barriers. Our data show that inulin still leaked out of wrapped spaces, even after the ends were plugged (Fig. 3b). It is possible that the continuity of the artery through the plug and beyond support a continuous aqueous layer on the adventitial surface that provides a low resistance pathway for drug to escape from within the wrap. This leak was noted visually by administering Evans blue dye in Poloxamer gel from within a wrap around the carotid artery and observing the diffusion along the arterial surface. The arterial wrap did not increase local vascular heparin concentrations or decrease deposition in distant structures (Fig. 4), in part because it failed to completely eliminate extraarterial capillary absorption. However, the wrap did slow the release of drug from Poloxamer 407 gels, presumably by limiting the exposure to interstitial fluids (Fig. 3). The rush to wrap arteries and release devices should be tempered in light of evidence that wrapping arteries can impose deleterious ischemic and proliferative injury ([27, 28]).
Drug was released into the perivascular space in vivo in an attempt to settle a long-running debate on how drug moves from local release devices to the blood vessel wall. These studies have shown that the overwhelming majority of drug leaves the perivascular space through the extraarterial capillaries, and yet little enters the vessel wall from the endovascular aspect. All of the small fraction of drug that enters the blood vessel wall arrives by direct diffusion from the perivascular space. In addition, placing barriers around the device and artery does not eliminate extraarterial capillary uptake completely, significantly diminish concentrations in distant organs or increase the amount of drug found in the blood vessel wall.
We thank Renata Yang and Lily Huang for their assistance in the laboratory.
☆ This study was supported in part by Grant GM/HL 49039 from the National Institutes of Health, Bethesda, Maryland; the Burroughs-Wellcome Fund in Experimental Therapeutics, Durham, North Carolina, and the Whitaker Foundation for Biomedical Engineering, Rosslyn, Virginia.
- analysis of variance
- inner diameter
- Received September 10, 1996.
- Revision received February 10, 1997.
- Accepted March 4, 1997.
- The American College of Cardiology
- Wolinsky H
- Riessen R,
- Isner JM
- Edelman ER,
- Adams DA,
- Karnovsky MJ
- Nathan AN MA,
- Edelman ER
- Simons M,
- Edelman ER,
- Langer R,
- DeKeyser JL,
- Rosenberg RD
- Rogers C,
- Edelman ER
- Lambert T,
- Dev V,
- Rechavia E,
- Forrester JS,
- Litvack F,
- Eigler NL
- Gimple LW, Gertz DG, Haber HL, et al. Effect of chronic subcutaneous or intramural administration of heparin on femoral artery restenosis after balloon angioplasty in hypercholesterolemic rabbits: a quantitative angiographic and histopathological study 86 1992;86:1536–46.
- Edelman ER,
- Simons M,
- Sirois MG,
- Rosenberg RD
- March KL,
- Mohanraj S,
- Ho PPK,
- Wilensky RL,
- Hathaway DR
- Lovich MA,
- Edelman ER
- Lovich MA,
- Edelman ER
- Edelman ER,
- Nugent MA,
- Karnovsky MJ
- Wolinsky H,
- Glagov S
- Mayberg MR