- Email: [email protected]
Comparison of local intravascular drug-delivery catheter systems
Comparison of local intravascular drug-delivery catheter systems P e t e r Gonschior, MD, a C l e m e n s Pail], T a n y a Y. H u e h n s , MRCP, a Florian G e r h e u s e r , MD, a Aysel Erdemci, MD, a K a t h a r i n a Larisch, MD, a M a r c Dellian, MD, b S t e f a n Deil, a Alwin-E. Goetz, MD, c H a n s A. Lehr, MD, PhD, d a n d B e r t h o l d H6fling, MD a Munich, Germany, and Seattle, Wash. Systemic and local delivery of the photosensitive drug Photofrin polyporphyrin was investigated in normal porcine arteries (n = 192). A macroporous balloon and a novel needle injection catheter were used for local drug delivery and compared with systemic delrvery. Fluorescence microscopy combined with digital image analysis was used to quantify the drug-related fluorescence. Systemic delivery showed a maximum in the intima at 4 hours. Application with the porous balloon revealed maximum indicator-related fluorescence intensity in the intima after 5 minutes; delivery with the needle injection catheter resulted in a several-fold enhanced maximum in adventitia after 30 minutes compared with the maximum achieved with either systemic injection or porous balloon application. After 21 days fluorescence was detectable in arteries treated with the new needle injection catheter. Local drug delivery is feasible with either system, but prolonged delivery was achieved only with the needle injection catheter. (AM HEARTJ 1995; 130:1174-81 .)
T h e l o n g - t e r m success o f p e r c u t a n e o u s t r a n s l u m i n a l c o r o n a r y a n g i o p l a s t y r e m a i n s limited b y the 20% to 50% r a t e of restenosis, 1, 2 w h i c h is t h o u g h t to r e s u l t f r o m tissue h y p e r p l a s i a . 3-5 Antiproliferative or even cytotoxic a g e n t s m a y be of t h e r a p e u t i c v a l u e in prev e n t i n g tissue h y p e r p l a s i a a n d t h u s m a y reduce restenosis. To date, systemic a d m i n i s t r a t i o n of such s u b s t a n c e s is limited e i t h e r b y t h e i r a d v e r s e effects or b y a lack of local t h e r a p e u t i c effect at t h e site of restenosis.5, 6 Local delivery of a n y antiproliferative or cytotoxic a g e n t m i g h t limit the t h e r a p e u t i c effect to specific a r e a s a n d avoid systemic a d v e r s e effects. Such application s y s t e m s would s u b s t a n t i a l l y de-
From the aMedical Department I, bInstitute of Surgical Research, and CInstitute of Anesthesiology, Klinikum Grosshadern; University of Munich; and the dDepartment of Pathology, University of Washington. Supported by the Deutsche Forschungsgemeinschai~ (grant 1076/1-2) and Friedrich Baur Foundation, Germany (grant 58/94); Dr. Huehns is supported by the Peel Medical Research Trust, London, U.K. Received for publication Dec. 29, 1994; accepted May 30, 1995. Reprint requests: P. Genschior, MD, University of Munich, Medical Dept I, Klinikum Grosshadern, Marchioninistr. 15, D-81377 Munich, Germany. Copyright © 1995 by Mosby-Year Book, Inc. 0002-8703/95/$5.00 + 0 4/1167121
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crease the overall a m o u n t of t h e d r u g n e e d e d and, at the s a m e time, p e r m i t a h i g h e r concentration of t h e active c o m p o n e n t to be o b t a i n e d in the region of interest. M a n y of the p r e v i o u s l y developed s y s t e m s h a v e only a limited efficiency in deploying s u b s t a n c e s locally. 7-9 I n this study, two local c a t h e t e r - b a s e d drugdelivery s y s t e m s w e r e designed to overcome t h e s e shortcomings. One s y s t e m consisted of a modified porous balloon design; the second s y s t e m w a s a cathe t e r w i t h six c i r c u m f e r e n t i a l needles t h a t could be e x t e n d e d a n d u s e d to inject the d r u g into the vessel wall. Local application of a p h o t o s e n s i t i z i n g a n d fluorescing s u b s t a n c e w a s u s e d to t e s t the feasibility a n d efficacy of t h e s e two n e w s y s t e m s c o m p a r e d w i t h systemic application in porcine a r t e r i a l vessels. METHODS Catheter systems for selective drug application
System 1. The development of the porous balloon-catheter system was implemented in cooperation with Carl Baasel-Lasertech (Munich, Germany). A conventional balloon catheter with an inflated balloon diameter of 3 m m and a length of 25 mm was perforated by using a focused carbon dioxide laser beam creating 12 pores, each with an average diameter of 75 !am. Three rows with 4 pores per row were positioned centrally in the balloon to guarantee circumferential distribution of the drug; the distance between pores was approximately 3 mm. Preliminary testing showed that the balloon tolerated an inflation pressure of up to 10 atm for >60 seconds. The balloon was designed to adhere to the vessel wall when inflated, allowing the drug within to leak out. Prior in vitro experiments determined that the amount of drug delivered correlated linearly with the perforated area of the balloon and the inflation pressure (Table I). System 2. The needle catheter device was a 5F flexible polyethylene catheter with a central lumen for a guide wire and six outer needles of 250 ~m (31G) positioned symmetrically. The design of the device was developed in close cooperation with a catheter manufacturer (BMT Inc., Oberpfaffenhofen, Germany). With the catheter placed in the vessel lumen, the needles are advanced by a mechanism at the external end of the catheter, thus extending the nee-
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Fig. 1. Distal p a r t of new needle injection catheter. Metal capsule a t tip (length 10 to 15 mm) houses six p r e s h a p e d needles t h a t can be advanced by mechanism (not shown) at distal e x t r a v a s c u l a r end of catheter.
dles proximally so t h a t t h e y fan out to encompass a diameter of 5.5 mm. The needles are preshaped, with a curve at the end, so t h a t t h e y p e n e t r a t e laterally into the media or perivascular a r e a (Fig. 1). In this study, local drug deposition in the perivascular tissue was achieved by injection of Photofrin through the extended needles. Fluorescent drug application. The fluorescing hematoporphyrin-derivative solution Photofrin (QLT, Vancouver, Canada, 2.5 mg/ml) was injected systemically (2.5 mg/kg) and with system 1 and system 2 (5 mg in both cases). Experimental procedures. Investigations were performed in 48 pigs of both sexes with an average weight of 31 kg (range 20 to 36 kg). All a n i m a l experiments were approved by the F e d e r a l Animal Care Committee and were performed according to the rules and principles of the American Physiological Society. The pigs received a normal chow diet during the schedule of experiments. In each pig, four vessels were excised (two carotid and two femoral arteries). A n e s t h e s i a was induced with i n t r a m u s c u l a r k e t a m i n e (250 to 500 mg; Ketanest, P a r k e Davies, Berlin, Germany), f l u n i t r a z e p a m (4 to 8 mg; Rohypnol, Hoffman-La Roche, Grenzach-Whylen, Germany) and atropine (1 mg, BraunMelsungen, Melsungen, Germany). The a n i m a l s were orotracheally i n t u b a t e d and mechanically ventilated (Servo 900B ventilator, Siemens, Erlangen, G e r m a n y ) with a mixture of 30% oxygen, 70% nitrous oxide, and either isoflurane or enflurane (1.0 to 2.0 vol%; Abbott, Wiesbaden, Germany). P i r i t r a m i d (0.5 to 1.0 mg; Dipidolor, Janssen, Neuss, Germany) was used for analgesia, and relaxation was obtained by i n t e r m i t t e n t application of pancuroniumbromide (4 mg; Organon, Eppelheim, Germany). Continuous monitoring of electrocardiogram, body t e m p e r a t u r e , and end-expiratory carbon dioxide was performed in all experiments. A catheter was inserted into a j u g u l a r vein for intravenous infusion. In chronic experiments 5000 IU of h e p a r i n were intravenously administered. No guide wires were used to position the local drug systems; after
Table ]. A m o u n t of isotonic saline solution t h a t expels in vitro depending on perforated a r e a of porous balloon and inflation p r e s s u r e
Perforated area (Itm2) 2500 Inflation pressure (atm) 1 0.2 2 0.5 3 0.7
5000
7500
0.5 1 1.5
0.7 1.5 2.2
local d r u g application, arteriotomies were closed with single polypropylene sutures (Prolene 6-0, Ethicon, Hamburg, Germany). Pigs were r a n d o m l y assigned to one of three groups, receiving either systemic, macroporous balloon, or needle c a t h e t e r application of drug. Systemic drug application in untreated vessels. There were 44 u n t r e a t e d vessels, 22 carotid and 22 femoral arteries. Photofrin was injected intravenously at a dose of 2.5 mg/kg. Hemodynamic p a r a m e t e r s were k e p t constant. In each pig, vessel segments were excised from femoral and carotid arteries 30 m i n u t e s or 1, 4, 12, 24, or 48 hours after Photofrin injection. Local drug application, porous balloon catheter. System 1 consisted of 84 vessels, 42 carotid and 42 femoral arteries. F e m o r a l and carotid arteries on both sides were exposed by surgical cutdown. Care was t a k e n not to mobilize the vessel so t h a t the wall was not externally injured at preparation. After a r t e r y incision the porous balloon catheter was inserted and Photofrin (5 m g in 2 ml) was injected with a pressure of 2 a t m over a 30-second period. This inflation pressure was chosen because it allows reliable injection of the dye in a time t h a t would be well tolerated even in coronary perfusion systems and minimizes potential vessel wall alteration t h a t could occur when porous
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Fig. 2. Histologic section of arterial vessel 7 days after local drug application with needle catheter. Arrow indicates needle-induced perforation t h a t was filled with organized thrombus after 7 days (elastic van Gieson stain, original magnification x40).
balloons are used a t higher inflation pressures. 1° At the pressure selected for these studies, the inflated balloon measures 2.9 m m in diameter, with a m e a n ratio of arterial to inflated balloon size of 0.8 (range 0.6 to 1.0). Vessels were excised 5, 15, 30, 60, or 120 minutes or 7, 14, or 21 days after the application of Photofrin. Local drug application, needle injection catheter. System 2 consisted of 64 vessels, 32 carotid and 32 femoral arteries. W i t h system 2 the arteries were similarly exposed and the catheter introduced by incision. Within the lumen, the needles were advanced into the perivascular tissue and, when fully deployed, Photofrin (5 mg in 2 ml) was injected over a 30-second period. The relation between the width of the needle injection catheter with needles extended and the vessel size was always > 1.0. Vessels were excised after 15, 30, 60, or 120 m i n u t e s or 7, 14, or 21 days. Microscopic analysis. All vascular tissue was processed in a d a r k room to avoid bleaching of Photofrin. Vessel segments were fixed i m m e d i a t e l y after excision by being embedded in Tissue-Tek (OCT compound, Miles Inc., Indiana) and frozen in liquid nitrogen. Serial sections (5 pm) were designated for either histologic or fluorescence analysis. Sections for histologic analysis were routinely processed with hematoxylin-eosin or elastic van Gieson's stains. Fluorescence was stimulated by the light of a xenon highpressure bulb (0.1 mW/cm2). A filter limited the emitted light to wavelengths between 355 and 425 nm. A digital image processing system m e a s u r e d the intensity of the emitted light, and fluorescence detection was used to quantify Photofrin content as previously described. 11,12 In brief, nonhomogenous light intensities of the irradiation unit were digitally subtracted; as reference, silicon particles with constant fluorescence were used (Impregum, Seefeld, Germany). Three silicon particles were used for
every m e a s u r e m e n t , and these were compared on a weekly basis with s t a n d a r d s stored in a d a r k place. Results indicated no m e a s u r a b l e bleaching effect after r e p e a t e d use and t h a t the fluorescence between particles was similar. Fluorescence intensity was m e a s u r e d within intima, media, and adventitia by investigators blinded to the randomization of each animal. A computer program allowed definition of six r a n d o m l y selected regions of interest, each with a size of 60 x 60 p m in every vessel layer. Results from the six areas were averaged. Ratios of vessel fluorescence versus s t a n d a r d fluorescence (1.0) were calculated. The autofluorescence was determined before m e a s u r e m e n t in control vessels and automatically subtracted. Gain was set equally for all m e a s u r e m e n t s . With the sensitivity used in this study, no significant autofluorescence could be m e a s u r e d in femoral or carotid arteries. Previous studies have revealed autofluorescence in elastic arteries (including carotid vessels) when the gain was set twofold to threefold higher, mostly localized where the elastic fibers are in the tissue (unpublished data); other vessels had no detectable autofluorescence level. Total vessel a r e a and fluorescing a r e a were d e t e r m i n e d according to a s t a n d a r d protocol. Statistics. Mean and SD were calculated for each set of fluorescence values. Comparisons of the different groups were m a d e with variance analysis followed by Wilcoxon and Wilcox multiple comparisons. Within groups, differences were tested with the F r i e d m a n test. A p value of <0.01 was considered to be statistically significant if not indicated otherwise.
RESULTS Feasibility of intravascular local drug delivery. With
system 1, adequate drug delivery was achieved in all
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x
~,o,5
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30 min.
•
60 rain.
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12h
•
24h
•
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x p< 0.001 0
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Fig. 3. Photofrin-related fluorescence after systemic drug administration (mean -+ SD).
vessel segments as verified by fluorescence analysis at low power. At histologic examination, no necrosis, local dissection, bleeding, or o t h e r signs of vessel t r a u m a were found. At l a t e r time points, one or two e x t r a cell layers in the i n t i m a were seen only in certain regions, with no overall resulting change in int i m a / m e d i a ratio at a n y time. With system 2, catheter applications were successful in 63 of 64 arterial segments in t e r m s of drug delivery as shown by the presence of fluorescence in all vessel wall layers at sequential analysis. In one case the needles could not be r e t r a c t e d into the c a t h e t e r after d r u g delivery w i t h o u t mechanical force, which induced a unilateral vessel t e a r and local periadventitial bleeding. All other e x p e r i m e n t a l applications were performed w i t h o u t complications. Cross-sections were macroscopically and microscopically intact with circumscript catheter-induced perforations seen in the vascular wall b u t no e x t r a v a s c u l a r bleeding or perivascular h e m a t o m a . In chronic experiments, no tissue h y p e r p l a s i a was found at the sites of needle insertion after 7, 14, or 21 days (Fig. 2) with no change in the i n t i m a / m e d i a ratio. All arteries were p a t e n t at the time of excision. T h e r e were no significant differences b e t w e e n results obtained in the femoral or carotid arteries, e i t h e r at histologic exa m i n a t i o n or fluorescence microscopy. Autofluorescence. The autofluorescence of femoral vessels in the p r e s e n t s t u d y was 3 _+ 3; in carotid vessels it was 4 + 3. Fluorescence after systemic application. The fluorescence of photosensitizing substance increased over early time points up to a m a x i m u m at 4 hours (0.64 _+ 0.01), t h e n decreased after 24 hours (Fig. 3). Fluorescence was always highest in the i n t i m a (Fig. 4); the relation of fluorescence between i n t i m a and media at the 4-hour time point was 1.5:1.
Fig. 4. Fluorescence in artery after systemic administration of Photofrin. Intense fluorescence is seen as yellow localized in intima, with lower fluorescence (red) in adjacent tissue and in adventitia. Fluorescence after local application System 1. W h e n Photofrin was applied locally with the porous balloon catheter, m a x i m u m Photofrin-related fluorescence was detected 5 m i n u t e s after t r e a t m e n t (Fig. 5). This value (ratio to s t a n d a r d 0.66 _+ 0.08) was comparable with the value obtained after 4 hours with systemic application. Fluorescence tailed offafter the 5-minute time point and was only 50% of the initial value at 1 hour. Again, i n t i m a showed the highest fluorescence, decreasing t o w a r d the outer region of the vessel wall with a relation to the i n t i m a of 6:1 (Fig. 6). No fluorescence was detected at time points l a t e r t h a n 2 hours.
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~0,5
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autofluorescence
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5 rain.
•
15 min.
[]
30 min.
•
60 min.
•
120 min.
x p< 0.001 Intima
Media
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Fig. 5. Photofrin-related fluorescence after local drug delivery with porous balloon (mean _+SD).
(media 0.55 +_ 0.05) compared with reference. At 14 days, a decrease in detectable fluorescence was found, with 0.80 _+ 0.20 in adventitia compared with reference. At 21 days, drug-related fluorescence was further reduced but still measurable in adventitia at 0.40 _+ 0.10 (media 0.20). DISCUSSION
Fig. 6. Fluorescence in artery after administration of Photofrin with porous balloon. Intense fluorescence is seen as white~yellow localized in intima, with lower fluorescence (red) in adjacent tissue and in adventitia.
System 2. Photofrin-related fluorescence was sixfold enhanced after needle catheter application (p < 0.001) compared with systemic delivery and porous balloon application. Fluorescence measurement with the needle injection catheter indicated that there was maximum fluorescence in the adventitia 30 minutes after drug application (Figs. 7 and 8); at this time point, the relation of fluorescence between adventitia and intima was 1.33:1. Prolonged drugrelated fluorescence was obtained in chronic experiments. After 7 days, a ratio of 1.15 _+ 0.77 drug-related fluorescence was determined in adventitia
The present study describes local drug delivery with two catheter systems compared with systemic administration. Normal porcine vessels were selected for the experiments because of certain similarities between h u m a n and porcine vascular systems. 5 The investigation was a feasibility study designed to test the ability of two devices to deliver drug into arteries; hence normal uninjured porcine vessels were used. A local strategy to prevent restenosis is particularly attractive because the site of delivery is accessible immediately after angioplasty, and the pathophysiologic process is limited to the lesion treated. To date, several devices designed to achieve local drug delivery in arteries have been investigated; they appear to have varying efficiency and long-term effeats.S, 9 The major problem seems to be achieving sufficient concentrations of drug locally in the arterial wall without leakage or toxicity. Two new drugdelivery devices were designed to overcome some of these shortcomings. The original porous balloon was developed to administer drugs with high pressure, forcing delivery into the arterial wall through the pores. 1° Experimental data indicate that a significant jet effect can occur with high-pressure inflation of this device. 13To minimize this pressure-related effect, a device with pores of 75 pm size was designed in conjunction with a laser manufacturer. Earlier studies (unpublished data) indicated significant leakage with perforations
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Fig. 7. Fluorescence in artery after local drug delivery of Photofrin with needle injection catheter. Intense fluorescence is localized in intima (white signal at center) and adventitia.
xlX 4-4
x
32-
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30 min
[]
60 min
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120 min
[]
7d
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14d
[]
21d
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Fig. 8. Photofrin-related fluorescence after local drug delivery with needle injection catheter (mean _+SD).
>100 l~m; pores of <75 pm require extremely high pressures to expel drug and could become blocked with blood products in vivo. The needle injection catheter was designed in collaboration with a catheter manufacturer specifically to deliver large amounts of drug into the adventitia without leakage. It has been suggested t h a t the adventitia plays a pivotal role in the vessel response to catheter-induced injury and that drug delivery into the adven-" titia creates a depot, which may provide a method of prolonged therapeutic action through the vasa vasorum. 14, 15
It is important that local drug-delivery devices do not in themselves cause injury and tissue hyperplasia. In this study, neither local drug-delivery method resulted in significant tissue hyperplasia at later time points. With regard to the porous balloon, this finding could be attributed to the low pressures used for drug injection, although the low pressures used may have resulted in a certain amount of leakage because the balloon was not in close apposition with vessel wall. Injection with the needle catheter was not associated with local bleeding or excessive vascular t r a u m a at early time points; in addition,
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chronic experiments indicated no significant tissue hyperplasia at laceration sites. Puncture with the six needles led to circumscript wound healing with a little organized thrombus. Such minimal bleeding at the time of needle puncture can probably be attributed to the elasticity of the vessels. Furthermore, the needles that extend from this catheter are much finer than the smallest needles used in vessel cannulation. Results obtained with 7F devices (unpublished data) demonstrate that with larger needles there was also no indication at chronic time points of significant perivascular bleeding at the puncture sites. Both systemic administration and the two local delivery systems resulted in fluorescence in arterial vessel segments. In vivo studies with amelanotic melanoma cell lines implanted into hamster skin by means of various porphyrinoids and fluorescence microscopy and chemical tissue extraction indicate that measured fluorescence correlates linearly with Photofrin concentration. 11, 12 However, assessment of the amount achieved in target tissue is compared with a standard and is not a direct measurement and therefore can only indirectly be used to compare efficiencies between the delivery systems, in contrast with the injection of radiolabeled molecules. Measurement of fluorescence as a way of assessing tissue content was selected in this study because this will establish any relation with histologic features in the various tissue layers. Photofrin accumulates in a perinuclear fashion in cells 16, 17; in this study, no local cytotoxicity was observed relating to Photofrin deposition. The dose chosen for systemic delivery of Photofrin (2.5 mg/kg) reflects the dose used as cancer therapy in clinical studies of photodynamic therapy17; approximately 10% of this dose was used for local drug delivery. Photofrin was our drug choice in part because it may have potential in conjunction with local photodynamic therapy in the prevention of restenosis.16, 17 In comparison with systemic administration, which resulted in a maximum fluorescence at 4 hours after injection, local application of Photofrin resulted in a maximum concentration in the vessel wall at an earlier time point. After delivery with the porous balloon, there was maximum fluorescence after 5 minutes in all intimal areas. Fluorescence in the vessel wall subsequently decreased, probably as the drug was washed out by the circulating blood in the lumen or through the vasa vasorum; additionally, side branches at the site of drug delivery might have contributed to the washout effect of drug with the balloon inflated. This early maximal drug delivery into the artery wall, especially into the intima, sug-
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gests that the system might be best suited to therapeutic approaches that make use of drugs with an immediate effect, for example, photodynamic therapy with local photosensitizer application and subsequent intravascular illumination. After local Photofrin delivery with the needle injection catheter, maximum fluorescence in adventitial layers was reached after 30 minutes. Compared with both the porous balloon and systemic administration, a significantly higher maximum fluorescence was achieved with the needle injection catheter, and the total fluorescing cross-sectional area was enhanced. After 21 days, indicator-related fluorescence was detectable only in arteries treated with the needle catheter. The new needle catheter system might therefore be best suited to therapeutic approaches that make use of drugs that need to be present in the wall for several weeks or more to take full effect or for gene therapy. Further studies are needed to evaluate both longer-term effects and the applicability of this device to an injury model. From this feasibility study it is concluded that the selective application of photosensitive drug by these two catheter devices results in measurable fluorescence in the arterial wall, indicating drug content. Although the porous balloon had a lower efficiency of delivery compared with the needle catheter, it revealed comparable fluorescence in arteries to that after systemic administration. This particular porous balloon might be adequate for immediate treatment strategies after angioplasty that incorporate direct drug activation. The highest fluorescence was achieved with the new needle injection system, which allowed prolonged drug delivery that was detectable for up to 21 days after delivery, with no apparent local adverse effects.
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1. Serruys PW, Luijten HE, Beatt KJ, Geuskens R, de Feyter F, van den Brand M, Reiber JHC, ten Katen HJ, van Es GA, Hugenholtz PG. Incidence of restenosis after successful coronary angioplasty: a time-related phenomenon: A quantitative angiographic study in 342 consecutive patients at 1, 2, 3 and 4 months. Circulation 1988;77:361-71. 2. Landau C, Lange RA, Hillis D. Percutaneous transluminal coronary angioplasty. N Engl J Med 1994;330:981-93. 3. Ross R. The pathogenesis ofatherosclerosis: a perspective for the 1990s. Nature 1993;362:801-9. 4. H(ifling B, Welsch U, Heimerl J, Gonschior P, Bauriedel G. Analysis of atherectomy specimens. Am J Cardiol 1993;72:96-107E. 5. Muller DWM, Ellis SG, Topol EJ. Experimental models of coronary artery restenosis. J Am Coll Cardiol 1992;19:418-32. 6. Herrmann JPR, Hermans WRM, Vos J, Serruys PW. Pharmacologic approaches to the prevention ofrestenosis following angioplasty. Drugs 1993;46:18-52. 7. Wilensky RL, March KL, Gradus-Pizlo I, Schauwecker D, Miehaels MB, Robinson J, Carlson K, Hathaway DR. Regional and arterial localization of radioactive microparticles after local delivery by unsupported or supported balloon catheters. AM HEARTJ 1995;129:852-9.
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8. Riessen R, Isner JM. Prospects for site-specific delivery of pharmacologic and molecular therapies. J Am Coll Cardiol 1994;23:1234-44. 9. H6fling B, Huehns TY. Intravascular local drug delivery after angioplasty. Eur Heart J 1995;15:437-40. 10. Wolinsky H, Thung SN. Use of a perforated balloon catheter to deliver concentrated heparin into the wall of the normal canine artery. J Am Coll Cardiol 1990;15:475-81. 11. Leunig M, Richert C, Gamarra F, Lumper W, Vogel E, Jocham D, Goetz AE. Tumour localisation kinetics of photofrin and three synthetic porphyrinoids in an amelanotic melanoma of the hamster. Br J Cancer 1993;68:225-34. 12. Abels C, Heil P, Dellian M, Kuhnle GEH, Baumgartner R, Goetz AE. In vivo kinetics and spectra of 5-aminolaevulinic acid-induced fluorescence in an amelanotic melanoma of the hamster. Br J Cancer 1994; 70:826-33.
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13. Plante S, Dupuis G, Mongeau CJ, Durand P. Porous balloon catheters for local delivery: assessment of vascular damage in a rabbit iliac angioplasty model. J Am Coll Cardiol 1994;24:820-4. 14. Barker SGE, Tilling LC, Miller GC, Beesley JE, Fleetwood G, Stavri GT, Baskerville PA, Martin JF. The adventitia and atherogenesis: removal initiates intimal proliferation in the rabbit which regresses on generation of neoadventitia. Atherosclerosis 1994;105:131-44. 15. Barger CA, Beeuwkes R, Lainey LL, Silverman KJ. Hypothesis: vasa vasorum and neovascularization of human coronary arteries. N Engl J Med 1984;310:175-7. 16. Gonschior P, Fleuchaus M, Gerheuser F, Mack B, H6fling B. Local drug delivery and photodynamic therapy in an experimental restenosis model. J Am Coll Cardiol 1994;23:473A. 17. Pass HI. Photodynamic therapy in oncology: mechanisms and clinical use. J Natl Cancer Inst 1993;85:443-56.
T h e l o n g - t e r m success o f p e r c u t a n e o u s t r a n s l u m i n a l c o r o n a r y a n g i o p l a s t y r e m a i n s limited b y the 20% to 50% r a t e of restenosis, 1, 2 w h i c h is t h o u g h t to r e s u l t f r o m tissue h y p e r p l a s i a . 3-5 Antiproliferative or even cytotoxic a g e n t s m a y be of t h e r a p e u t i c v a l u e in prev e n t i n g tissue h y p e r p l a s i a a n d t h u s m a y reduce restenosis. To date, systemic a d m i n i s t r a t i o n of such s u b s t a n c e s is limited e i t h e r b y t h e i r a d v e r s e effects or b y a lack of local t h e r a p e u t i c effect at t h e site of restenosis.5, 6 Local delivery of a n y antiproliferative or cytotoxic a g e n t m i g h t limit the t h e r a p e u t i c effect to specific a r e a s a n d avoid systemic a d v e r s e effects. Such application s y s t e m s would s u b s t a n t i a l l y de-
From the aMedical Department I, bInstitute of Surgical Research, and CInstitute of Anesthesiology, Klinikum Grosshadern; University of Munich; and the dDepartment of Pathology, University of Washington. Supported by the Deutsche Forschungsgemeinschai~ (grant 1076/1-2) and Friedrich Baur Foundation, Germany (grant 58/94); Dr. Huehns is supported by the Peel Medical Research Trust, London, U.K. Received for publication Dec. 29, 1994; accepted May 30, 1995. Reprint requests: P. Genschior, MD, University of Munich, Medical Dept I, Klinikum Grosshadern, Marchioninistr. 15, D-81377 Munich, Germany. Copyright © 1995 by Mosby-Year Book, Inc. 0002-8703/95/$5.00 + 0 4/1167121
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crease the overall a m o u n t of t h e d r u g n e e d e d and, at the s a m e time, p e r m i t a h i g h e r concentration of t h e active c o m p o n e n t to be o b t a i n e d in the region of interest. M a n y of the p r e v i o u s l y developed s y s t e m s h a v e only a limited efficiency in deploying s u b s t a n c e s locally. 7-9 I n this study, two local c a t h e t e r - b a s e d drugdelivery s y s t e m s w e r e designed to overcome t h e s e shortcomings. One s y s t e m consisted of a modified porous balloon design; the second s y s t e m w a s a cathe t e r w i t h six c i r c u m f e r e n t i a l needles t h a t could be e x t e n d e d a n d u s e d to inject the d r u g into the vessel wall. Local application of a p h o t o s e n s i t i z i n g a n d fluorescing s u b s t a n c e w a s u s e d to t e s t the feasibility a n d efficacy of t h e s e two n e w s y s t e m s c o m p a r e d w i t h systemic application in porcine a r t e r i a l vessels. METHODS Catheter systems for selective drug application
System 1. The development of the porous balloon-catheter system was implemented in cooperation with Carl Baasel-Lasertech (Munich, Germany). A conventional balloon catheter with an inflated balloon diameter of 3 m m and a length of 25 mm was perforated by using a focused carbon dioxide laser beam creating 12 pores, each with an average diameter of 75 !am. Three rows with 4 pores per row were positioned centrally in the balloon to guarantee circumferential distribution of the drug; the distance between pores was approximately 3 mm. Preliminary testing showed that the balloon tolerated an inflation pressure of up to 10 atm for >60 seconds. The balloon was designed to adhere to the vessel wall when inflated, allowing the drug within to leak out. Prior in vitro experiments determined that the amount of drug delivered correlated linearly with the perforated area of the balloon and the inflation pressure (Table I). System 2. The needle catheter device was a 5F flexible polyethylene catheter with a central lumen for a guide wire and six outer needles of 250 ~m (31G) positioned symmetrically. The design of the device was developed in close cooperation with a catheter manufacturer (BMT Inc., Oberpfaffenhofen, Germany). With the catheter placed in the vessel lumen, the needles are advanced by a mechanism at the external end of the catheter, thus extending the nee-
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Fig. 1. Distal p a r t of new needle injection catheter. Metal capsule a t tip (length 10 to 15 mm) houses six p r e s h a p e d needles t h a t can be advanced by mechanism (not shown) at distal e x t r a v a s c u l a r end of catheter.
dles proximally so t h a t t h e y fan out to encompass a diameter of 5.5 mm. The needles are preshaped, with a curve at the end, so t h a t t h e y p e n e t r a t e laterally into the media or perivascular a r e a (Fig. 1). In this study, local drug deposition in the perivascular tissue was achieved by injection of Photofrin through the extended needles. Fluorescent drug application. The fluorescing hematoporphyrin-derivative solution Photofrin (QLT, Vancouver, Canada, 2.5 mg/ml) was injected systemically (2.5 mg/kg) and with system 1 and system 2 (5 mg in both cases). Experimental procedures. Investigations were performed in 48 pigs of both sexes with an average weight of 31 kg (range 20 to 36 kg). All a n i m a l experiments were approved by the F e d e r a l Animal Care Committee and were performed according to the rules and principles of the American Physiological Society. The pigs received a normal chow diet during the schedule of experiments. In each pig, four vessels were excised (two carotid and two femoral arteries). A n e s t h e s i a was induced with i n t r a m u s c u l a r k e t a m i n e (250 to 500 mg; Ketanest, P a r k e Davies, Berlin, Germany), f l u n i t r a z e p a m (4 to 8 mg; Rohypnol, Hoffman-La Roche, Grenzach-Whylen, Germany) and atropine (1 mg, BraunMelsungen, Melsungen, Germany). The a n i m a l s were orotracheally i n t u b a t e d and mechanically ventilated (Servo 900B ventilator, Siemens, Erlangen, G e r m a n y ) with a mixture of 30% oxygen, 70% nitrous oxide, and either isoflurane or enflurane (1.0 to 2.0 vol%; Abbott, Wiesbaden, Germany). P i r i t r a m i d (0.5 to 1.0 mg; Dipidolor, Janssen, Neuss, Germany) was used for analgesia, and relaxation was obtained by i n t e r m i t t e n t application of pancuroniumbromide (4 mg; Organon, Eppelheim, Germany). Continuous monitoring of electrocardiogram, body t e m p e r a t u r e , and end-expiratory carbon dioxide was performed in all experiments. A catheter was inserted into a j u g u l a r vein for intravenous infusion. In chronic experiments 5000 IU of h e p a r i n were intravenously administered. No guide wires were used to position the local drug systems; after
Table ]. A m o u n t of isotonic saline solution t h a t expels in vitro depending on perforated a r e a of porous balloon and inflation p r e s s u r e
Perforated area (Itm2) 2500 Inflation pressure (atm) 1 0.2 2 0.5 3 0.7
5000
7500
0.5 1 1.5
0.7 1.5 2.2
local d r u g application, arteriotomies were closed with single polypropylene sutures (Prolene 6-0, Ethicon, Hamburg, Germany). Pigs were r a n d o m l y assigned to one of three groups, receiving either systemic, macroporous balloon, or needle c a t h e t e r application of drug. Systemic drug application in untreated vessels. There were 44 u n t r e a t e d vessels, 22 carotid and 22 femoral arteries. Photofrin was injected intravenously at a dose of 2.5 mg/kg. Hemodynamic p a r a m e t e r s were k e p t constant. In each pig, vessel segments were excised from femoral and carotid arteries 30 m i n u t e s or 1, 4, 12, 24, or 48 hours after Photofrin injection. Local drug application, porous balloon catheter. System 1 consisted of 84 vessels, 42 carotid and 42 femoral arteries. F e m o r a l and carotid arteries on both sides were exposed by surgical cutdown. Care was t a k e n not to mobilize the vessel so t h a t the wall was not externally injured at preparation. After a r t e r y incision the porous balloon catheter was inserted and Photofrin (5 m g in 2 ml) was injected with a pressure of 2 a t m over a 30-second period. This inflation pressure was chosen because it allows reliable injection of the dye in a time t h a t would be well tolerated even in coronary perfusion systems and minimizes potential vessel wall alteration t h a t could occur when porous
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Fig. 2. Histologic section of arterial vessel 7 days after local drug application with needle catheter. Arrow indicates needle-induced perforation t h a t was filled with organized thrombus after 7 days (elastic van Gieson stain, original magnification x40).
balloons are used a t higher inflation pressures. 1° At the pressure selected for these studies, the inflated balloon measures 2.9 m m in diameter, with a m e a n ratio of arterial to inflated balloon size of 0.8 (range 0.6 to 1.0). Vessels were excised 5, 15, 30, 60, or 120 minutes or 7, 14, or 21 days after the application of Photofrin. Local drug application, needle injection catheter. System 2 consisted of 64 vessels, 32 carotid and 32 femoral arteries. W i t h system 2 the arteries were similarly exposed and the catheter introduced by incision. Within the lumen, the needles were advanced into the perivascular tissue and, when fully deployed, Photofrin (5 mg in 2 ml) was injected over a 30-second period. The relation between the width of the needle injection catheter with needles extended and the vessel size was always > 1.0. Vessels were excised after 15, 30, 60, or 120 m i n u t e s or 7, 14, or 21 days. Microscopic analysis. All vascular tissue was processed in a d a r k room to avoid bleaching of Photofrin. Vessel segments were fixed i m m e d i a t e l y after excision by being embedded in Tissue-Tek (OCT compound, Miles Inc., Indiana) and frozen in liquid nitrogen. Serial sections (5 pm) were designated for either histologic or fluorescence analysis. Sections for histologic analysis were routinely processed with hematoxylin-eosin or elastic van Gieson's stains. Fluorescence was stimulated by the light of a xenon highpressure bulb (0.1 mW/cm2). A filter limited the emitted light to wavelengths between 355 and 425 nm. A digital image processing system m e a s u r e d the intensity of the emitted light, and fluorescence detection was used to quantify Photofrin content as previously described. 11,12 In brief, nonhomogenous light intensities of the irradiation unit were digitally subtracted; as reference, silicon particles with constant fluorescence were used (Impregum, Seefeld, Germany). Three silicon particles were used for
every m e a s u r e m e n t , and these were compared on a weekly basis with s t a n d a r d s stored in a d a r k place. Results indicated no m e a s u r a b l e bleaching effect after r e p e a t e d use and t h a t the fluorescence between particles was similar. Fluorescence intensity was m e a s u r e d within intima, media, and adventitia by investigators blinded to the randomization of each animal. A computer program allowed definition of six r a n d o m l y selected regions of interest, each with a size of 60 x 60 p m in every vessel layer. Results from the six areas were averaged. Ratios of vessel fluorescence versus s t a n d a r d fluorescence (1.0) were calculated. The autofluorescence was determined before m e a s u r e m e n t in control vessels and automatically subtracted. Gain was set equally for all m e a s u r e m e n t s . With the sensitivity used in this study, no significant autofluorescence could be m e a s u r e d in femoral or carotid arteries. Previous studies have revealed autofluorescence in elastic arteries (including carotid vessels) when the gain was set twofold to threefold higher, mostly localized where the elastic fibers are in the tissue (unpublished data); other vessels had no detectable autofluorescence level. Total vessel a r e a and fluorescing a r e a were d e t e r m i n e d according to a s t a n d a r d protocol. Statistics. Mean and SD were calculated for each set of fluorescence values. Comparisons of the different groups were m a d e with variance analysis followed by Wilcoxon and Wilcox multiple comparisons. Within groups, differences were tested with the F r i e d m a n test. A p value of <0.01 was considered to be statistically significant if not indicated otherwise.
RESULTS Feasibility of intravascular local drug delivery. With
system 1, adequate drug delivery was achieved in all
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30 min.
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12h
•
24h
•
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Fig. 3. Photofrin-related fluorescence after systemic drug administration (mean -+ SD).
vessel segments as verified by fluorescence analysis at low power. At histologic examination, no necrosis, local dissection, bleeding, or o t h e r signs of vessel t r a u m a were found. At l a t e r time points, one or two e x t r a cell layers in the i n t i m a were seen only in certain regions, with no overall resulting change in int i m a / m e d i a ratio at a n y time. With system 2, catheter applications were successful in 63 of 64 arterial segments in t e r m s of drug delivery as shown by the presence of fluorescence in all vessel wall layers at sequential analysis. In one case the needles could not be r e t r a c t e d into the c a t h e t e r after d r u g delivery w i t h o u t mechanical force, which induced a unilateral vessel t e a r and local periadventitial bleeding. All other e x p e r i m e n t a l applications were performed w i t h o u t complications. Cross-sections were macroscopically and microscopically intact with circumscript catheter-induced perforations seen in the vascular wall b u t no e x t r a v a s c u l a r bleeding or perivascular h e m a t o m a . In chronic experiments, no tissue h y p e r p l a s i a was found at the sites of needle insertion after 7, 14, or 21 days (Fig. 2) with no change in the i n t i m a / m e d i a ratio. All arteries were p a t e n t at the time of excision. T h e r e were no significant differences b e t w e e n results obtained in the femoral or carotid arteries, e i t h e r at histologic exa m i n a t i o n or fluorescence microscopy. Autofluorescence. The autofluorescence of femoral vessels in the p r e s e n t s t u d y was 3 _+ 3; in carotid vessels it was 4 + 3. Fluorescence after systemic application. The fluorescence of photosensitizing substance increased over early time points up to a m a x i m u m at 4 hours (0.64 _+ 0.01), t h e n decreased after 24 hours (Fig. 3). Fluorescence was always highest in the i n t i m a (Fig. 4); the relation of fluorescence between i n t i m a and media at the 4-hour time point was 1.5:1.
Fig. 4. Fluorescence in artery after systemic administration of Photofrin. Intense fluorescence is seen as yellow localized in intima, with lower fluorescence (red) in adjacent tissue and in adventitia. Fluorescence after local application System 1. W h e n Photofrin was applied locally with the porous balloon catheter, m a x i m u m Photofrin-related fluorescence was detected 5 m i n u t e s after t r e a t m e n t (Fig. 5). This value (ratio to s t a n d a r d 0.66 _+ 0.08) was comparable with the value obtained after 4 hours with systemic application. Fluorescence tailed offafter the 5-minute time point and was only 50% of the initial value at 1 hour. Again, i n t i m a showed the highest fluorescence, decreasing t o w a r d the outer region of the vessel wall with a relation to the i n t i m a of 6:1 (Fig. 6). No fluorescence was detected at time points l a t e r t h a n 2 hours.
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~0,5
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5 rain.
•
15 min.
[]
30 min.
•
60 min.
•
120 min.
x p< 0.001 Intima
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Fig. 5. Photofrin-related fluorescence after local drug delivery with porous balloon (mean _+SD).
(media 0.55 +_ 0.05) compared with reference. At 14 days, a decrease in detectable fluorescence was found, with 0.80 _+ 0.20 in adventitia compared with reference. At 21 days, drug-related fluorescence was further reduced but still measurable in adventitia at 0.40 _+ 0.10 (media 0.20). DISCUSSION
Fig. 6. Fluorescence in artery after administration of Photofrin with porous balloon. Intense fluorescence is seen as white~yellow localized in intima, with lower fluorescence (red) in adjacent tissue and in adventitia.
System 2. Photofrin-related fluorescence was sixfold enhanced after needle catheter application (p < 0.001) compared with systemic delivery and porous balloon application. Fluorescence measurement with the needle injection catheter indicated that there was maximum fluorescence in the adventitia 30 minutes after drug application (Figs. 7 and 8); at this time point, the relation of fluorescence between adventitia and intima was 1.33:1. Prolonged drugrelated fluorescence was obtained in chronic experiments. After 7 days, a ratio of 1.15 _+ 0.77 drug-related fluorescence was determined in adventitia
The present study describes local drug delivery with two catheter systems compared with systemic administration. Normal porcine vessels were selected for the experiments because of certain similarities between h u m a n and porcine vascular systems. 5 The investigation was a feasibility study designed to test the ability of two devices to deliver drug into arteries; hence normal uninjured porcine vessels were used. A local strategy to prevent restenosis is particularly attractive because the site of delivery is accessible immediately after angioplasty, and the pathophysiologic process is limited to the lesion treated. To date, several devices designed to achieve local drug delivery in arteries have been investigated; they appear to have varying efficiency and long-term effeats.S, 9 The major problem seems to be achieving sufficient concentrations of drug locally in the arterial wall without leakage or toxicity. Two new drugdelivery devices were designed to overcome some of these shortcomings. The original porous balloon was developed to administer drugs with high pressure, forcing delivery into the arterial wall through the pores. 1° Experimental data indicate that a significant jet effect can occur with high-pressure inflation of this device. 13To minimize this pressure-related effect, a device with pores of 75 pm size was designed in conjunction with a laser manufacturer. Earlier studies (unpublished data) indicated significant leakage with perforations
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Fig. 7. Fluorescence in artery after local drug delivery of Photofrin with needle injection catheter. Intense fluorescence is localized in intima (white signal at center) and adventitia.
xlX 4-4
x
32-
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30 min
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60 min
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120 min
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7d
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14d
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21d
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Fig. 8. Photofrin-related fluorescence after local drug delivery with needle injection catheter (mean _+SD).
>100 l~m; pores of <75 pm require extremely high pressures to expel drug and could become blocked with blood products in vivo. The needle injection catheter was designed in collaboration with a catheter manufacturer specifically to deliver large amounts of drug into the adventitia without leakage. It has been suggested t h a t the adventitia plays a pivotal role in the vessel response to catheter-induced injury and that drug delivery into the adven-" titia creates a depot, which may provide a method of prolonged therapeutic action through the vasa vasorum. 14, 15
It is important that local drug-delivery devices do not in themselves cause injury and tissue hyperplasia. In this study, neither local drug-delivery method resulted in significant tissue hyperplasia at later time points. With regard to the porous balloon, this finding could be attributed to the low pressures used for drug injection, although the low pressures used may have resulted in a certain amount of leakage because the balloon was not in close apposition with vessel wall. Injection with the needle catheter was not associated with local bleeding or excessive vascular t r a u m a at early time points; in addition,
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chronic experiments indicated no significant tissue hyperplasia at laceration sites. Puncture with the six needles led to circumscript wound healing with a little organized thrombus. Such minimal bleeding at the time of needle puncture can probably be attributed to the elasticity of the vessels. Furthermore, the needles that extend from this catheter are much finer than the smallest needles used in vessel cannulation. Results obtained with 7F devices (unpublished data) demonstrate that with larger needles there was also no indication at chronic time points of significant perivascular bleeding at the puncture sites. Both systemic administration and the two local delivery systems resulted in fluorescence in arterial vessel segments. In vivo studies with amelanotic melanoma cell lines implanted into hamster skin by means of various porphyrinoids and fluorescence microscopy and chemical tissue extraction indicate that measured fluorescence correlates linearly with Photofrin concentration. 11, 12 However, assessment of the amount achieved in target tissue is compared with a standard and is not a direct measurement and therefore can only indirectly be used to compare efficiencies between the delivery systems, in contrast with the injection of radiolabeled molecules. Measurement of fluorescence as a way of assessing tissue content was selected in this study because this will establish any relation with histologic features in the various tissue layers. Photofrin accumulates in a perinuclear fashion in cells 16, 17; in this study, no local cytotoxicity was observed relating to Photofrin deposition. The dose chosen for systemic delivery of Photofrin (2.5 mg/kg) reflects the dose used as cancer therapy in clinical studies of photodynamic therapy17; approximately 10% of this dose was used for local drug delivery. Photofrin was our drug choice in part because it may have potential in conjunction with local photodynamic therapy in the prevention of restenosis.16, 17 In comparison with systemic administration, which resulted in a maximum fluorescence at 4 hours after injection, local application of Photofrin resulted in a maximum concentration in the vessel wall at an earlier time point. After delivery with the porous balloon, there was maximum fluorescence after 5 minutes in all intimal areas. Fluorescence in the vessel wall subsequently decreased, probably as the drug was washed out by the circulating blood in the lumen or through the vasa vasorum; additionally, side branches at the site of drug delivery might have contributed to the washout effect of drug with the balloon inflated. This early maximal drug delivery into the artery wall, especially into the intima, sug-
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gests that the system might be best suited to therapeutic approaches that make use of drugs with an immediate effect, for example, photodynamic therapy with local photosensitizer application and subsequent intravascular illumination. After local Photofrin delivery with the needle injection catheter, maximum fluorescence in adventitial layers was reached after 30 minutes. Compared with both the porous balloon and systemic administration, a significantly higher maximum fluorescence was achieved with the needle injection catheter, and the total fluorescing cross-sectional area was enhanced. After 21 days, indicator-related fluorescence was detectable only in arteries treated with the needle catheter. The new needle catheter system might therefore be best suited to therapeutic approaches that make use of drugs that need to be present in the wall for several weeks or more to take full effect or for gene therapy. Further studies are needed to evaluate both longer-term effects and the applicability of this device to an injury model. From this feasibility study it is concluded that the selective application of photosensitive drug by these two catheter devices results in measurable fluorescence in the arterial wall, indicating drug content. Although the porous balloon had a lower efficiency of delivery compared with the needle catheter, it revealed comparable fluorescence in arteries to that after systemic administration. This particular porous balloon might be adequate for immediate treatment strategies after angioplasty that incorporate direct drug activation. The highest fluorescence was achieved with the new needle injection system, which allowed prolonged drug delivery that was detectable for up to 21 days after delivery, with no apparent local adverse effects.
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8. Riessen R, Isner JM. Prospects for site-specific delivery of pharmacologic and molecular therapies. J Am Coll Cardiol 1994;23:1234-44. 9. H6fling B, Huehns TY. Intravascular local drug delivery after angioplasty. Eur Heart J 1995;15:437-40. 10. Wolinsky H, Thung SN. Use of a perforated balloon catheter to deliver concentrated heparin into the wall of the normal canine artery. J Am Coll Cardiol 1990;15:475-81. 11. Leunig M, Richert C, Gamarra F, Lumper W, Vogel E, Jocham D, Goetz AE. Tumour localisation kinetics of photofrin and three synthetic porphyrinoids in an amelanotic melanoma of the hamster. Br J Cancer 1993;68:225-34. 12. Abels C, Heil P, Dellian M, Kuhnle GEH, Baumgartner R, Goetz AE. In vivo kinetics and spectra of 5-aminolaevulinic acid-induced fluorescence in an amelanotic melanoma of the hamster. Br J Cancer 1994; 70:826-33.
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13. Plante S, Dupuis G, Mongeau CJ, Durand P. Porous balloon catheters for local delivery: assessment of vascular damage in a rabbit iliac angioplasty model. J Am Coll Cardiol 1994;24:820-4. 14. Barker SGE, Tilling LC, Miller GC, Beesley JE, Fleetwood G, Stavri GT, Baskerville PA, Martin JF. The adventitia and atherogenesis: removal initiates intimal proliferation in the rabbit which regresses on generation of neoadventitia. Atherosclerosis 1994;105:131-44. 15. Barger CA, Beeuwkes R, Lainey LL, Silverman KJ. Hypothesis: vasa vasorum and neovascularization of human coronary arteries. N Engl J Med 1984;310:175-7. 16. Gonschior P, Fleuchaus M, Gerheuser F, Mack B, H6fling B. Local drug delivery and photodynamic therapy in an experimental restenosis model. J Am Coll Cardiol 1994;23:473A. 17. Pass HI. Photodynamic therapy in oncology: mechanisms and clinical use. J Natl Cancer Inst 1993;85:443-56.