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Cardiovascular Research 2002 53(2):481-486; doi:10.1016/S0008-6363(01)00499-0
© 2002 by European Society of Cardiology
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Copyright © 2001, European Society of Cardiology

Local delivery of low-dose docetaxel, a novel microtubule polymerizing agent, reduces neointimal hyperplasia in a balloon-injured rabbit iliac artery model

Satoshi Yasudaa,*, Teruo Noguchia, Masahiro Gohdaa, Takashi Araib, Nobumasa Tsutsuib, Yasuhide Nakayamac, Takehisa Matsudac and Hiroshi Nonogia

aNational Cardiovascular Center, Division of Cardiology, Department of Medicine, 5-7-1 Fujishiro-dai, Suita, Osaka 565-8565, Japan
bTokai Medical Products Inc, Aichi, Japan
cNational Cardiovascular Center Research Institute, Department of Bioengineering, Osaka, Japan

syasuda{at}hsp.ncvc.go.jp

* Corresponding author. Tel.: +81-6-6833-5012; fax: +81-6-6872-7486

Received 21 June 2001; accepted 4 October 2001


   Abstract
Top
 Abstract
1 Introduction
 2 Methods
 3 Results
 4 Discussion
 References
 
Objective: Docetaxel (DOC) is a novel microtubule polymerizing agent, with superior antiproliferative properties as compared to paclitaxel. DOC is therefore a potential therapeutic tool for the prevention of restenosis following angioplasty. However, DOC has systemic toxicity such as leukocytopenia, which occurs in a dose-dependent manner. To minimize such adverse effects, we carried out local delivery of low-dose DOC directly to injured vessel sites. Methods: The rabbit iliac artery was denuded, and then DOC (2 mg) or control vehicle was administered locally 20 min, via a local drug delivery catheter. Results: The levels of DOC in the plasma were within ng/ml range, eliminating hematopoietic side effects. Seven days after the local delivery (DOC: n=4, control: n=4), DOC decreased the number of Ki-67-labeled cells in the intima (DOC: 22±10 vs. control: 66±18 cells/mm2, P<0.01), indicating a decreased proliferative activity. At 28 days (DOC: n=8, control: n=8), computer-assisted morphometric analysis demonstrated that DOC significantly reduced the intimal area (DOC: 0.15±0.13 vs. control: 0.70±0.13 mm2, P<0.01). There was also a decrease in medial area in the DOC-treated vessels (DOC: 0.62±0.17 vs. control: 1.13±0.38 mm2, P<0.01). Conclusions: Local delivery of DOC, even after a single low-dose administration, effectively inhibits neointimal hyperplasia. Such administration is associated with a minimal likelihood of systemic adverse effects (leukocytopenia), but potentially induces local toxicity (a decrease in medial wall thickness) due to extensive cytotoxic effect.

KEYWORDS DOC=Docetaxel; PTCA=Percutaneous Transluminal Coronary Angioplasty; WBC=White Blood Cell; vWF=von Willebrand's factor

This article is referred to in the Editorial by M. Sturek and H.K. Reddy (pages 292–293) in this issue.


   1 Introduction
Top
 Abstract
 1 Introduction
2 Methods
 3 Results
 4 Discussion
 References
 
Despite extensive effort, including the development of adjunctive therapies and mechanical techniques, 30–50% of patients, undergoing percutaneous transluminal coronary angioplasty (PTCA) develop restenosis within 3 to 6 months of the procedure [1]. In addition to vascular remodeling, neointimal hyperplasia is a major cause of restenosis in response to arterial injury [2]. This process predominantly involves vascular smooth muscle cell proliferation. Therefore, one of the therapeutic strategies for the prevention of restenosis is the suppression of such proliferation using cytostatic or cytotoxic agents [3].

Recent studies have revealed that the microtubules within cells are among the most important components of cellular dynamics, including mitosis, cell signaling and motility [4]. Microtubule polymerizing agents of taxoids, such as paclitaxel, inhibit cell proliferation by blocking mitosis, and are therefore currently used as anticancer drugs [5–7]. In vitro studies have shown that paclitaxel also inhibits vascular smooth muscle cell proliferation [8,9], indicating its potential therapeutic value against restenosis following PTCA.

Docetaxel (DOC: Taxotere®, Rhone Poulenc Rorer, Paris, France) is an analog of paclitaxel (N-debenzoyl-N-tert-butoxycarbonyl-10-deacetyl taxel) that more efficiently induces microtubule polymerization, with superior anticancer and antiproliferative properties as compared to paclitaxel [10,11]. The principal toxicity of DOC is leukocytopenia, which occurs in a dose-dependent manner. To minimize the likelihood of such systemic adverse effects, local administration of the drug directly to the target site has been attracting increasing interest [12]. In the previous studies [9,13], paclitaxel did not inhibit neointimal formation in rabbits after balloon injury. Therefore, in the present study, we tested our hypothesis that local delivery of a low dose of DOC, a novel and potent microtubule polymerizing agent, may attenuate the proliferative response to vascular injury, with reduced risk of systemic toxicity.


   2 Methods
Top
 Abstract
 1 Introduction
 2 Methods
3 Results
 4 Discussion
 References
 
All the procedures were in accordance with the institutional guidelines, which conform to the ‘Position of the American Heart Association on Research Animal Use’.

2.1 The model
New Zealand white rabbits weighing 3.0–3.5 kg were used for this study as in our previous one [14]. Anesthesia was induced by intramuscular injection of ketamine (50 mg/kg), following treatment with xylazine (10 mg/kg). The femoral arteries were exposed by an incision below the inguinal ligament. Heparin sulfate (1000 U) was administered intravenously to prevent blood coagulation. After administration of 2 ml of 1% lidocaine, a 3.6 Fr angioplasty catheter (Tokai Medical Products Inc., Aichi, Japan) [15] was introduced under fluoroscopic guidance by an over-the-wire system into the right iliac artery. This catheter [15] possesses the triple functions of balloon inflation, local administration and blood perfusion. This device was found to maintain 34±7 ml/min of distal blood flow during drug administration at a mean aortic pressure of 100 mm Hg in our pilot studies [15]. Thus, this device enables a drug delivery over a relatively long duration (20 min) with maintaining perfusion. As reported previously [14], the 3.0-mm-diameter balloon, 20 mm in length, was inflated with contrast medium at the right iliac artery immediately distal to the aortic bifurcation (a reliable and reproducible landmark for the removal of the vessels at a later date). The balloon inflated at 6 atm was then retracted and deflated. This procedure was repeated three times in the same segment (20 mm in length from the bifurcation) to ensure complete endothelial denudation.

2.2 Local delivery of docetaxel
Following the balloon-induced injury, local drug delivery was effected via a multifunctional angioplasty catheter. A detailed description of this device appears in our previous reports [14,15]. Briefly, the drug delivery port was positioned at the injured site and the balloon was inflated at a low pressure (2 atm) to allow accumulation of the drug. Then the guidewire was removed to a site proximal to the perfusion port to allow distal perfusion. In pilot studies using this device, the drug permeated into the medial and the adventitial tissues immediately after the delivery, and then persists in the medial tissue for at least 24 h. In addition, this device could deliver the drug homogeneously over 80% of the target site. Via an infusion pump (STC-521, Terumo, Japan), the rabbits were locally administered 2 mg of DOC dissolved in 10 ml saline (DOC group, n=12) or vehicle solution without DOC (Control group, n=12) over 20 min, via the local drug delivery catheter. The dose of DOC used in the present study (2 mg) was one order of magnitude lower than the dose of paclitaxel (~35 mg) used in the previous study in rabbits [9,13].

In the DOC group (n=4), blood samples were obtained 30 min after the drug administration and stored at –20°C. The DOC concentrations in the plasma were then measured at Mitsubishi Chemical BCL (Tokyo, Japan) by high-performance liquid chromatography, as described previously [16]. The concentration range was confirmed to be from 20 to 4000 ng/ml in the plasma [16].

2.3 White blood cell counting
DOC has systemic toxicity such as leukocytopenia, which occurs in a dose-dependent manner. To evaluate the hematopoietic effects of low doses of DOC, the white blood cell (WBC) number was counted in the DOC group (n=4). Blood samples were obtained before, 1, 3, 7, 14 and 28 days after the administration of DOC.

2.4 Postmortem procedures
Seven or 28 days after the procedure, the rabbits were sacrificed by injection of a fatal dose of pentobarbital. For pressure perfusion fixation, a mid-abdominal incision was made and the lower abdominal aorta was isolated, flushed with saline, and fixed with 10% buffered formalin at 80 mm Hg for over 15 min. At least 24 h postfixation, the arterial segments were dehydrated and embedded in paraffin [14].

2.5 Immunohistochemical analysis
In addition, to quantify the degree of smooth muscle cell proliferation, Ki-67 was immunohistochemically stained [17] in tissue specimens obtained 7 days after the drug administration. The primary antibody used was MIB-1 monoclonal antibody (DAKO, Denmark) [17]. The fraction of Ki-67-positive cells per mm2 of the intimal layers was subsequently determined in the control (n=4) and DOC-treated vessels (n=4). Monocytes/macrophages were detected with monoclonal antibody against CD68 (DAKO). Arterial cross sections were also immunostained with primary antibody for von Willebrand's factor (vWF) (DAKO) as a marker for endothelial cells.

2.6 Morphometric analysis
For histology, 5-µm sections were cut and stained with elastic van Gieson or hemotoxylin–eosin. Morphometric analysis was performed on arterial cross sections at 28 days, and imaged using a National Institutes of Health image software package. The endoluminal border, the circumference bounded by the internal elastic lamina, and the external elastic lamina were manually traced, and then the luminal, intimal, and medial areas were calculated. Nuclear counts were also made in the media and intimal area. We compared these parameters between the control (n=8) and the DOC-treated vessels (n=8).

2.7 Statistics
All data were expressed as means±S.D. To compare the DOC group with the Control group, data were analyzed by the unpaired Student's t-test to evaluate the two-tailed levels. In the DOC group, the WBC data were compared by analysis of variance. A value of P<0.05 was considered as denoting significance.


   3 Results
Top
 Abstract
 1 Introduction
 2 Methods
 3 Results
4 Discussion
 References
 
Even 0.5 h after the local administration, the plasma levels of DOC did not exceed 203±80 ng/ml (range of 114 to 270 ng/ml). Fig. 1 shows the serial changes in the WBC counts. There were no significant changes, indicating that the local delivery of a low dose of DOC is associated with minimal risk of leukocytopenia.


Figure 1
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  		Fig. 1 Serial changes in means±S.D. data of white blood cell (WBC) count after local delivery of DOC (n=4).

 
The degree of cell proliferation and the cell type were determined based on immunohistochemical studies (Fig. 2). At 7 days, the number of cells positive for vWF (against endothelial cells) and CD-68 (against macrophages) were not significantly different between the two groups. However, the number of Ki-67-positive cells was 22±10 cells/mm2 in the DOC group, significantly lower than the number, 66±18 cells/mm2 (P<0.01), in the Control group. This finding suggests a decreased proliferative activity of vascular smooth muscle cells in the vessels administered with DOC.


Figure 2
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  		Fig. 2 Quantitative immunohistochemical examinations at 7 days in the Control (untreated-) vessels (open bars: n=4) and Docetaxel (DOC)-treated vessels (closed bars: n=4). The means±S.D. data of the number of cells positive for von Willebrand's factor (vWF) (against endothelial cells), CD-68 (against macrophages), and Ki-67, respectively, are shown. * P<0.05 vs. control by unpaired t-test.

 
Fig. 3 shows representative photomicrographs of histologic cross sections from the injured arterial segments at 28 days. Table 1 summarizes the group morphometric data 28 days after the administration. Local delivery of 2 mg DOC induced a significant reduction in the intimal area, and a decrease in the intima-to-media area ratio. Although these findings indicate suppression of neointimal hyperplasia by the microtubule polymerizing agent, there was also a decrease in medial area in the DOC-treated vessels. The cell density in the media was significantly lower in the DOC group than in the Control group (650±102 vs. 819±124 counts/mm2, P<0.01).


Figure 3
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  		Fig. 3 Cross section (elastin van Gieson stain) of rabbit iliac arteries 28 days after balloon-induced injury. Left panel: Control (untreated-) artery (x40). Right panel: DOC-treated artery (x40). Note marked reduction in intimal hyperplasia in DOC-treated artery.

 

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  		Table 1 Comparison of arterial morphomeric data 28 days after the local administration of docetaxel

 

   4 Discussion
Top
 Abstract
 1 Introduction
 2 Methods
 3 Results
 4 Discussion
References
 
The major finding of this in vivo study is that, even a single low dose local delivery of DOC effectively suppresses neointimal hyperplasia.

Balloon arterial injury activates vascular smooth muscle cell migration and proliferation, thereby inducing neointimal hyperplasia [2]. This response is associated with the induction of a cascade of signaling pathways [3]. Microtubules are important in the transmission of proliferative transmembrane signals from the cell surface receptors to the nucleus [18–20]. Because of their critical role in cell signaling at multiple sites [4], microtubules can be a potential therapeutic target for antiproliferative strategies.

Taxoids, such as paclitaxel and DOC, polymerize tubulin and enhances the assembly of microtubules, and inhibit the proliferation of vascular cells [8,9], as well as tumor cells [10,11]. This inhibitory effect was reported to be sustained for a period of 14 days even after brief (20 min) application in a monoculture of vascular smooth muscle cells [9]. This may be related to its strongly lipophilic structure (facilitating rapid cellular uptake and onset of action) and its strong binding activity to tubulin (leading to sustained action) [21–23]. The recent ex vivo study demonstrated that paclitaxel substantially permeated into the vascular wall (especially into the media) even after 15 min application and that the concentration in vascular tissue exceeded the applied concentration [23]. In addition, paclitaxel affects the proliferation of endothelial cells less than that of smooth muscle cells, as shown in a co-culture experimental model [9,24].

Based on these in vitro and ex vivo findings, we examined the efficacy of in vivo application in suppressing neointimal proliferation that occurs in response to arterial injury. Taxoids were found to exert not only antiproliferative effects but also adverse hematopoietic effects (leukocytopenia) in a dose-dependent manner. The latter is the principal toxicity of this drug in vivo and therefore is of great clinical importance. To minimize the systemic adverse effects, local administration of the drug directly to the target site has been attracting increasing interest [12]. Since the local delivery of {approx}35 mg of paclitaxel (using the microporous balloon over {approx}30 s) did not inhibit intimal growth in a previous study in rabbits [9,13], we focused on DOC, an analog of paclitaxel, in the present study. This novel agent may be particularly advantageous at a low dose, because it is relatively more active as a promoter of tubulin polymerization and as an inhibitor of cell proliferation than paclitaxel [10,11].

We found that the local delivery of DOC to the injured arterial site significantly suppressed intimal hyperplasia, as shown in Table 1 and Fig. 3. This was achieved even after a single low dose (2 mg) administration. The levels of DOC in the plasma were within the ng/ml range, thus being associated with a minimal risk of leukocytopenia (Fig. 1). In immunohistochemical studies (Fig. 2), a significant decrease in smooth muscle cell proliferation but no change in endothelial cell number was observed in DOC-treated vessels. These findings indicate that the local administration of a low dose of DOC enables tissue concentrations sufficient for the cytostasis of vascular smooth muscle cells to be obtained while minimizing the likelihood of systemic side effects in an in vivo experimental model.

However, as shown in Table 1, DOC also induced a significant decrease in the medial area. This was accompanied by the reduction in cell density in the media, and documented in the previous studies using paclitaxel-coating stent [25]. These findings may indicate local toxicity, probably due to extensive injury in the vessels administered with DOC. Tissue toxicity was not observed in our previous study employing local delivery of human recombinant hepatocyte growth factor, a potent endothelial-cell mitogen, using the same experimental model [14]. Different from the re-endothelialization action of hepatocyte growth factor [13,26], the antiproliferative action of DOC may additionally disrupt normal vascular function. Moreover, this study was performed in the otherwise non-atherosclerotic arteries. In the atherosclerotic lesions, the present approach of local drug delivery may have a limitation. Therefore, further development of methods would be desirable for clinical use.

In conclusion, considering their important biological role in cell proliferation [4,5], microtubules are a therapeutic target for the prevention of restenosis following PTCA. For better efficacy and fewer adverse effects, local delivery of microtubule polymerizing agents, such as DOC, could be a potential approach.

Time for primary review 32 days.


   Acknowledgements
 
This study was done with support from the Uehara Memorial Foundation, Osaka Heart Club and Japan Cardiovascular Research Foundation (Dr. Yasuda).


   References
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 Abstract
 1 Introduction
 2 Methods
 3 Results
 4 Discussion
 References
 

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