these devices is pressure-driven delivery that causes addi- tional vessel damage and low efficacy. Viral vectors or different lipid carriers may increase the efficacy of delivery. Fibrin meshwork is an alternative vehicle for sustained release of antisense, a factor that may be important in the case of stent implantation. Polymer-coated stents have been used successfully to deliver micromolar concentrations of c-myc antisense PMO into the vessel wall (74) (Fig. 2). Zhang et al. (75) reported effective local delivery of c-myc antisense ODN by gelatin- coated platinum–ipidium stents in rabbits. These experiences showed that ultimate success will require polymers that are capable of rapid elution of the oligonucleotide with minimal capacity to inflame or otherwise cause additional injury to the vessel wall. Perfluorobutane gas microbubbles with a coating of dextrose and albumin efficiently bind antisense oligomers (76). These 0.3- to 10-␮m particles bind to sites of vascular injury. Furthermore, perfluorobutane gas is an effective cell membrane fluidizer. The potential advantages of microbubble carrier delivery include minimal additional vessel injury from delivery; no resident polymer to degrade, leading to eventual inflammation; rapid bolus delivery; and the high likelihood of repeated delivery. In addition, the potential for perfluorocar- bon gas microbubble carriers (PGMC) to deliver to vessel regions both proximal and distal to stents in vessels suggests this mode of delivery will serve as an excellent adjuvant to a variety of catheter and coated-stent delivery techniques. First clinical experience of antisense therapy in the treatment of restenosis The clinical applicability of antisense technology remains limited by a relative lack of specificity, slow uptake across the cell membrane, and rapid degradation of oligonucleotides. Promising results emerged from the PREVENT trial (77), which showed efficacy of ex vivo gene therapy of human vascular bypass grafts with an antisense oligonucleotide to E2F transcription factor, which is essential for VSMC proliferation in lowering the incidence of venous bypass graft failure. Recently reported results of another clinical trial (ITALICS) in Rotterdam (78) that examined the effectiveness of antisense compound directed against c-myc, however, were disap- pointing. The authors considered several reasons for the observed lack of effect of the antisense compound. Among them, the local concentration of antisense compound achieved may not have been high enough to show a signifi- cant effect. Also, the single administration of the antisense compound might not be effective in suppressive c-myc, which showed biphasic response to the vessel injury. The authors also used a self-expanding stent, which can cause chronic injury of stented arteries. Under these circumstances, a single injection of antisense may not be adequate to reduce myointimal response. Optimistic results have been obtained with the newly introduced AVI-4126, which belongs to a family of mole- cules known as the PMOs (28). These oligomers are comprised of (dimethylamino)phosphinylideneoxy-linked morpholino subunits, which contain a heterocyclic base recognition moiety of DNA attached to a substituted morpholine ring system. In general, PMOs are capable of binding to RNA in a sequence-specific fashion with sufficient avidity to be useful for the inhibition of the translation of mRNA into protein in vivo. Although PMOs share many similarities with other substances that are capable of producing antisense effects [e.g., DNA, RNA, and their analogous oligonucleotide analogs such as the phosphorothioates (PSOs)], there are several critical differences. Most importantly, PMOs are uncharged and resistant to degradation under biological conditions, exceptionally stable at temperature extremes, and resistant to degradation in plasma and to the nucleases found in serum and liver extracts (79). They also exhibit a high degree of specificity and efficacy, both in vitro and in cell culture (80), which averts a variety of potentially significant limitations observed in PSO chemistry. The antisense mechanism of action appears to be through the PMO hybrid duplex with mRNA to inhibit translation. Finally, PMOs have 376 Antisense approach Figure 2 ( See color plate .) Polymer-coated stent delivery of c-myc antisense phosphorodiamidate morpholino oligomers into swine vessels. 1180 Chap32 3/14/07 11:39 AM Page 376 demonstrated antisense activity against c-myc pre-mRNA in living human cells (81). The combined efficacy, potency, and lack of nonspecific activities of PMO chemistry have compelled us to re-examine the approach to antisense c-myc in the prevention of restenosis following balloon angioplasty. PMOs have been evaluated for adverse effects after intra- venous bolus injections in both primates (GLP studies by Sierra Biomedical) and man (GCP studies at MDS Harris). No alterations in heart rate, blood pressure, or cardiac output were observed. In summary, bolus injections of PMO by local catheter-based delivery devices are feasible. Our studies with endoluminal delivery of advanced c-myc antisense PMO into the area of PTCA (Transport Catheter™; rabbit iliac artery model) (82) and into coronary arteries following stent implantation (Infiltrator™ delivery system; pig model) (83) demonstrated complete inhibition of c-myc expression and a significant reduction of the neointimal formation in the treated vessels in a dose-dependent fashion while allowing for complete vascular healing. Similar results were obtained after implantation of advanced c-myc anti- sense PMO-eluting phosphorylcholine-coated stents in the porcine coronary restenosis model (74). We also observed less inflammation after implantation of the antisense-loaded stent. This favorable influence on hyperplasia (a 40% reduc- tion of intima) in the absence of endothelial toxicity may represent an advantage of antisense PMO over more destructive methods such as brachytherapy (84) or cytotoxic inhibitors (85). We also tested novel perfluorocarbon gas microbubble carriers (PGMS) for site-specific delivery of AVI- 4126 to the injured vessel wall and obtained encouraging results (86). The most robust of observations to date by multiple inves- tigators is the finding that AVI-4126 is safe and effective in vascular application in a number of species. Different meth- ods for local delivery have also been tested, but these observations fall short of proof that AVI-4126 will be effective in the treatment of human restenosis. Efficacy in animal models has also been encouraging. Furthermore, all these studies with AVI-4126 indicated that the agent is safe. The last remaining question is if AVI-4126 will find a place in future therapeutic regimens for the prevention of resteno- sis; this answer might be found in the results of phase II clinical studies currently being conducted, such as AVAIL. Our recent data on six-month follow-up on the patients enrolled in the AVAIL study (87) showed that AVI-4126 is effective in reduc- ing neointimal formation, particularly when locally delivered in high dose. We also concluded that local delivery of antisense is safe and feasible. The results indicate that antisense (AVI- 4126) can be as effective in prevention of the restenosis as most of the well-known antiproliferative agents do, but in contrast to other chemotherapeutics (paclitaxel, actinomycin D) c-myc antisense inhibits cell cycle in the G-1 phase, which make its effect less toxic and comparable with that of rapamycin. Conclusion Proof of principle has been established that inhibition of several cellular proto-oncogenes including DNA-binding protein c-myb, nonmuscle myosin heavy chain, proliferat- ing-cell nuclear antigen, PDGF, bFGF, and c-myc inhibit SMC proliferation in vitro and in several animal models. 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Local paclitaxel delivery for the prevention of restenosis: biological effects and efficacy in vivo. J Am Coll Cardiol 2000; 35(7): 1969–1976. 86 Kipshidze NN, Porter TR, Dangas G, et al. Systemic targeted delivery of antisense with perflourobutane gas microbubble carrier reduced neointimal formation in the porcine coronary restenosis model. Cardiovasc Radiat Med 2003; 4(3): 152–159. 87 Kipshidze N, lversen P, Overlie P, et al. First human experience with local delivery of novel antisense AVI-4126 with infiltrator catheter in de novo native and restenotic coronary arteries: six-month clinical and angiographic follow-up from AVAIL study. Cardiovasc Revasc Med 2007 (in press). 380 Antisense approach 1180 Chap32 3/14/07 11:39 AM Page 380 Introduction Phototherapy, the therapeutic application of light in the treatment of diseases has evolved over thousands of years from its origins in Asia. The earliest understanding that photonic energy in visible light could be harnessed through the presence of a photoreactive substance to promote a biological effect in an oxygenated tissue is attributed to the work of Professor von Tappeiner on xanthene derivatives, first published in 1900 (1). This work led to the realization that the destructive skin lesions observed in porphyrias could be attributable to a photodynamic effect. Early photodynamic agents were naturally derived porphyrins such as hematoporphyrin and typically were mixtures of many porphyrins leading to inconsistent biological results. A purified form, hematoporphyrin derivative (HpD), was shown through red fluorescence under ultraviolet light to localize in tumors (2). Thus, the synthesis of first-generation photoreactive agents was directed toward their use in disease diagnosis. It was not until the observation by Diamond et al. in 1972 that the photodynamic effect caused selective necro- sis of a glioma implant in a rat (3), that the term photodynamic treatment (PDT) was coined. The 1980s saw the advent of second-generation photore- active agents characterized by greater purity, favorable pharmacokinetics, and stronger absorption of light in the far red part of the spectrum that is least attenuated on transmis- sion through tissue. PDT development programs have resulted in marketing approval of several photoreactive agents by the Food and Drug Administration (FDA) and other regula- tory agencies including Photofrin ® (esophageal/bronchial cancer), Levulan ® (actinic keratosis), and Visudyne ® (age- related macular degeneration). The use of PDT in interventional cardiovascular therapy is experimental. However, the unique combination of site-specific, endovascu- lar activity and potential application for focal or regional inter- vention makes PDT an attractive concept for primary treatment of atherosclerosis or as an adjunct to inhibit restenosis. The following sections provide an overview of the principles of photodynamic effect and highlight potential appli- cation of PDT to structural targets underlying certain cardiovascular diseases. Mechanisms of photodynamic effect and modes of cell death in photodynamic treatment At the core of the photodynamic effect is a photoreactive agent with a stable electronic configuration that exists as a singlet in the ground state—(Fig. 1). Upon excitation by the absorption of photonic energy from light of a specific wave- length (h␯ exc ) the photoreactive molecule is elevated to a higher though short-lived first excited energy state, which is also a singlet. The molecule either relaxes to its ground singlet state releasing energy as a photon through fluorescence (h␯ F ), or may convert to a triplet state by intersystem crossing (ISX). The photoreactive triplet has greater longevity than its parent singlet, in the order of milliseconds, increasing the probability of interaction with the surrounding oxygen molecules. Higher intersystem crossing probabilities and higher triplet quantum yields are inherent in those photoreactive agents selected for clinical development, as these parameters indicate the quantity of cytotoxic species produced. Energy in the photoreactive triplet state molecule provides the basis for biomol- ecular interactions in photodynamic treatment. The predominant mechanism involves generation of singlet oxygen ( 1 O 2 ). 33 Principles of photodynamic treatment Thomas L. Wenger and Nicholas H. G. Yeo 1180 Chap33 3/14/07 11:40 AM Page 381 The diffusion distance of 1 O 2 is around 0.01–0.02 ␮ before being quenched (4) and so the photoreactive drug must be associated intimately with the target substrate for maximal impact. Biomolecules present in cellular membranes react rapidly with 1 O 2 and are prime targets for PDT. Membranous intracellular organelles such as mitochondria, lysosomes, and nuclei are also potential targets for attack by 1 O 2 . Photodynamic cytotoxicity is initiated through various signaling pathways. Both apoptotic and necrotic modes of cell death have been described (5). Modulating the components of PDT dosimetry (e.g., administered doses of photoreactive agent and light, and the time interval between these) together with the specific binding characteristics of the photoreactive agent, can alter the balance between apoptosis and necrosis (6–9). Endovascular PDT of injury-induced hyperplastic arteries has been shown to induce neointimal and medial apoptosis in vivo (10). PDT-mediated translocation of a pro-apoptotic mitochon- drial protein (apoptosis-inducing factor) from the mitochondria to the nucleus appears to play a role in smooth muscle cell (SMC) apoptosis (11). Cytotoxic free radicals formed during PDT also inactivate cell-associated basic fibroblast growth factor and inhibit the stimulation of SMC mitogenesis after tissue injury (12). Managing photodynamic treatment at the threshold The principles of photodynamic effect require that each of the elements (e.g., photoreactive molecules, photons of the appropriate wavelength, and molecular oxygen) is present at the site of the intended treatment effect coincidently and in such numbers that the yield of 1 O 2 is sufficient to overcome the target’s ability to sustain itself against the oxidative stress being inflicted. The corollary is also important, namely that where any one or more of the elements is present below the threshold the target may tolerate the resultant oxidative stress. It is self-evident then that dosimetry is critical. The challenge is thus to establish dosimetry parameters that provide a working surface of safety and efficacy that accom- modates the biologic and pathologic variability present in patients undergoing treatment. Figure 2 highlights the required intersection of the three elements of PDT necessary to generate 1 O 2 . The principal criteria influencing each element’s contribution to the photo- dynamic effect are also listed. 382 Principles of photodynamic treatment Necrosis Apoptosis Oxidation Cellular / subcellular substrates ISX hv exc hv F Photoreactive agent singlet ground state (S 0 ) First excited triplet state (T 1 ) T 1 excess energy transfers to 3 O 2 , and returns to ground state for repeat cycles of excitation First excited singlet state (S 1 ) 1 O 2 First excited singlet state 3 O 2 Triplet oxygen ground state Excess energy Figure 1 Summary of the photodynamic effect. 1 O 2 • Wavelength • Light source • Delivery device • Irradiance (mW/cm 2 ) • Dose (J/cm 2 ) • Photodynamic efficiency • Pharmacokinetics • Route of administration • Dose • Systemic tolerability • Localization time course • O 2 tension in target • Replenishment capacity during PDT Activating Light Molecular Oxygen Photoreactive Agent Figure 2 ( See color plate .) Summary of the interaction of the three elements required for photodynamic effect. Abbreviation : PDT, photodynamic treatment. 1180 Chap33 3/14/07 11:40 AM Page 382 Configuring photodynamic therapy for endovascular intervention Photoreactive agents Table 1 provides a summary of the principal photoreactive agents that have been investigated clinically or are currently undergoing industry-sponsored development for endovas- cular use. A small number of other photoreactive agents (including various porphyrin and phthalocyanine derivatives) have been investigated in basic cardiovascular research stud- ies or in in vivo models of cardiovascular disease. Certain photophysical and pharmacokinetic characteristics are of particular importance in determining the potential util- ity of photoreactive agents in endovascular treatment of cardiovascular disease. Agents having a high triplet state quan- tum yield are more efficient generators of 1 O 2 and this productivity advantage can translate to less photosensitivity burden on the patient and a lower energy requirement for effective activation. In the cath lab, more efficient photoreac- tive agents requiring shorter activation times may minimize procedure times for endovascular PDT. Selection of photoreactive agents has been largely directed toward those having strong absorption in the far red part of the visible spectrum, offering the deeper tissue effect that goes with longer wavelength activation (see section on Light Activation). This characteristic has been a long-held holy grail of PDT researchers seeking to enlarge the volume of tissue ablation to treat advanced cancers. Most of the photoreactive agents under investigation today have evolved from this selection process. Another important characteristic of photoreactive agents is their apparent affinity for certain targets that are of special interest for interventional vascular therapists. As most photo- sensitizers fluoresce, the kinetics of their distribution in vascular tissue can be investigated both at macroscopic and microscopic levels using fluorescence imaging techniques (Fig. 3). Numerous studies on porphyrin, chlorin, texaphryin, pheophorbide and phthalocyanine photosensitizers in vari- ous animal models have documented selective localization in Configuring photodynamic therapy for endovascular intervention 383 Drug ID (cardiovascular Code/generic name Cardiovascular Sponsor-defined clinical Other information sponsor) development development target status (from company publication) Preclinical Clinical phase (P) Antrin ® Motexafin lutetium ✔ Coronary P1 Vulnerable plaque ( Pharmacyclics ) Peripheral P2 Photofrin ® Porfimer sodium ✔ Coronary P1 — Marketed (pilot study) internationally as Photofrin for PDT of cancer ALA Aminolevulinic ✔ Peripheral P1 — Can be acid/ALA-induced (pilot study) administered protoporphrin-IX orally LS11 Talaporfin, NPe6 ✔ — SFA restenosis and Marketed in ( Vascular Mono-L-aspartyl vulnerable plaque Japan as Reconditioning ™) chlorin e6, MACE Laserphyrin ® for PDT of cancer PhotoPoint ® ( Miravant ) MV0633 ✔ — Vulnerable plaque and coronary restenosis MV2101 ✔ — Vascular access failure in hemodialysis patients Abbreviations : ALA, 5-aminolevulinic acid; PDT, photodynamic treatment; SFA, superficial femoral artery. Table 1 Principal photoreactive agents with cardiovascular development experience 1180 Chap33 3/14/07 11:40 AM Page 383 atheromatous plaque and sites of endothelial injury (13–26). Despite differences in the molecular configurations and physicochemical properties of these photoreactive agents their affinities follow a remarkably consistent pattern of uptake. In normal uninjured and nonatheromatous control arteries there is little accumulation except in the endothe- lium. In atheromatous lesions there is typically strong accumulation in the intima, weak accumulation in the media, and rare presence in the adventitia. In balloon-injured, but nonatheromatous arteries, there is strong uptake into the media, less in the intima, and no uptake in the adventitia. Balloon-injured, atheromatous lesions show both intimal and medial accumulation. Uptake into diffuse atherosclerotic lesions in a model of vein graft disease has also been demon- strated (27). Factors such as the structure, charge, and lipophilicity of a photoreactive agent will determine serum protein binding, cellular uptake, subcellular localization and ultimately the biological effect at the time of light activation. The mechanism of photoreactive agent accumulation in plaque has not been fully elucidated but may relate to a tendency to bind to low- density lipoproteins (LDL). During the development of atherosclerosis, scavenger receptors present on the surface of accumulating macrophages mediate the uptake of modified (oxidized) lipoproteins transforming the cells into foam cells (28). The level of expression of scavenger receptors on macrophage-derived foam cells increases dramatically as the disease progresses (29). This may increase the cellular uptake of photoreactive agents that are carried on LDL particles. For example, electron microscopy has revealed the presence of the gold salt of talaporfin (LS11/NPe6) in macrophages within an atherosclerotic plaque (30). Furthermore, the uptake of LDL by another key interventional cardiovascular target— arterial smooth muscle cells (SMC)—is reported to be significantly increased by hypoxia exclusive of LDL receptor activity (31). LDL transport may thus provide receptor-mediated and direct modes of entry of photoreac- tive agents into macrophages and SMCs within a thickening intima as atherosclerosis progresses. Perhaps these processes also explain the uptake of photoreactive agents in the media of vessels injured by angioplasty. Time-dependent accumula- tion of motexafin lutetium within murine macrophages and human SMCs has been shown by real-time monitoring of the agent’s fluorescence emission at 750 nm (32). Some photoreactive agents, especially those that are hydrophobic or amphiphilic, may also be transported in complexes loosely or tightly formed with serum albumin. It is believed that albumin-binding proteins on the surface of endothelial cells create a specific pathway for gp60-mediated transcytosis of the albumin-photoreactive agent complex across the endothelial cell monolayer (33). Drug to light activation interval Although there may be a number of similarities in the process of uptake of photoreactive agents into sites of atherosclerosis and vascular injury, there may be substantial differences in the time during which this occurs. The ideal time to undertake light activation is when the photoreactive agent is present in the pathologic target and absent elsewhere. Thus, careful selection of the drug to light activation interval (DLI) is an important parameter in maximizing the benefit versus the risk in this treatment. The real attraction of endovascular PDT as a regional intervention for diffuse atherosclerotic disease is based on the opportunity to combine an agent that self-local- izes in pathologic foci, coupled with regionally distributed light energy that itself has no affect on the tissue in absence of the photoreactive agent. This also provides a basis to mitigate 384 Principles of photodynamic treatment Figure 3 ( See color plate .) Microscopy with 405 nm excitation reveals red fluorescence from talaporfin (LS11/NPe6) in macrophages within atheromatous plaque on abdominal aorta in hyper- cholesterolemic rabbit, 24 hours after 5 mg/kg intra- venous administration. Note green autofluorescence from elastic fibers in adventitia with no detected LS11. Source : Courtesy of Prof. K Aizawa, Tokyo Medical University, Tokyo, Japan. 1180 Chap33 3/14/07 11:40 AM Page 384 geographic miss during adjunctive use through extending light activation beyond the edge of the lesion. The presence of photoreactive agent in blood within the light activation field may mask the activation site by absorbing the activating light’s energy before it reaches the intended target. However, delaying activation while the photoreactive agent clears from the blood may require many hours. Preadministering a photoreactive agent hours or days in antic- ipation of an intended intervention, so as to achieve an accumulation threshold in a cellular target but not in blood, may be inconvenient. The ideal is a photoreactive agent that can be administered during an interventional procedure, which rapidly accumulates within the target and can be effi- ciently activated by light with only a marginal increment in the overall procedure time. Light activation Longer wavelengths of light at the far red end of the visible light spectrum penetrate tissues more deeply than shorter wavelengths near the blue part of the spectrum. When light passes into tissue, the optical properties of the tissue deter- mine the extent to which it is reflected, transmitted, scattered, or absorbed. The optical properties of tissue are defined by the presence of chromophores that absorb energy in the light, and structures within the tissue (e.g., cells and subcellular organelles) that scatter light. Scattering becomes more significant as wavelength decreases toward the blue- violet (i.e., 390–420 nm) and ultraviolet (i.e., Ͻ380 nm) parts of the spectrum limiting the depth that light penetrates. As wavelength increases toward the infrared (i.e., beyond 1000 nm) the depth of light penetration is reduced by water absorption. Between these regions in the visible part of the spectrum, and with specific reference to the photodynamic treatment of arterial disease, the major light-absorbing chro- mophores are oxyhemoglobin, which absorbs strongly in the blue-green regions (420 and 540–580 nm), and yellow chro- mophores in carotenoids contained in the atheroma that strongly absorb blue-green light at 420–530 nm (34) with a peak absorption around 470 nm. Thus, blue light will not penetrate deeply into tissue and yellow light will be variably attenuated. While blue light may be a viable choice for a subendothelial treatment field, red light can activate photoreactive drugs more deeply into the tissue and is perhaps a better choice for targeting SMCs in the media, for example, after angioplasty. Atherosclerotic plaque evolves to be an optically complex lesion ranging from diffuse intimal thickening through lipid-rich regions and the presence of calcification, neovascularization, and intraplaque hemorrhage. In this setting, red light above 650 nm wavelength may be the most effective activation strategy. Alternatively, as photoreactive agents typically have several wavelengths at which they activate strongly within the blue to red color range (although the 1 O 2 yields may be very different) contemporaneous light activation with multiple acti- vating wavelengths may potentially enable “through the lesion” treatment. Light transmission through blood to the arterial wall must contend with scattering by blood elements, absorption by oxyhemoglobin, and absorption by the photoreactive drug present in the blood. It is claimed that motexafin lutetium which absorbs around 730 nm does not require blood exclu- sion from the vascular treatment field during light activation. Other photoreactive agents under cardiovascular develop- ment with activation wavelengths in the region 630 to 670 nm are believed to require blood exclusion. It is unclear whether these perceived distinctions are real. With oxyhemoglobin, the absorption nadir is between 660 and 710 nm, whereas with de-oxyhemoglobin the absorption graph declines across the range 580–800 nm with two inflections around 750 nm. However, hemoglobin in arterial blood is greater than 90% saturated with oxygen; thus, the absorption of light by oxyhe- moglobin carries greater weight in considering appropriate wavelengths for efficient light transmission through arterial blood. In this regard, there appears to be little to differentiate between photoreactive agents that are activated across the range 650 to 730 nm (Fig. 4). Light transmission may depend on hematocrit, hemoglobin concentration, light catheter diameter to vessel diameter distance relationships, drug phar- macokinetics and DLI, and other factors. Various strategies have been used to eliminate blood from the lumen including balloon occlusion of blood flow at the light delivery site and saline flush [hemodilution technique (35)]. Ultimately, whether complete blood exclusion is needed is uncertain. The duration of light delivery can be defined by the total optical energy required (i.e., light dose or fluence measured in J/cm 2 across the endovascular surface being treated) and the optical power (i.e., irradiance, measured in mW/cm 2 ) applied to the endovascular surface, according to the formula: Time (sec) = Joules ( J)/ Watts (W) Long durations of light exposure, where occlusion is required, may require light delivery to be fractionated with one or more reperfusion intervals, especially in coronary applications. Light activation protocols based on intense energy delivery may appear attractive in terms of shortened light exposure but may lead to photobleaching (destruction of the photoreactive agent), and, in the presence of hypoxia or restricted re-oxygenation capacity, may be ineffective. Light for endovascular PDT has typically been generated by pumped-dye or solid-state diode lasers and delivered to the site of treatment through a fiberoptic with a diffusing segment at the distal end of the device that provides radial distribution of the light. Where blood flow occlusion is required, the fiberoptic may be delivered to the treatment site through the guidewire channel of an angioplasty catheter with the diffusing segment positioned within the translucent balloon (36,37). 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