CA2300246A1 - Local delivery of 17-beta estradiol decreases neointimal hyperplasia following coronary angioplasty in porcine model - Google Patents
Local delivery of 17-beta estradiol decreases neointimal hyperplasia following coronary angioplasty in porcine model Download PDFInfo
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- CA2300246A1 CA2300246A1 CA002300246A CA2300246A CA2300246A1 CA 2300246 A1 CA2300246 A1 CA 2300246A1 CA 002300246 A CA002300246 A CA 002300246A CA 2300246 A CA2300246 A CA 2300246A CA 2300246 A1 CA2300246 A1 CA 2300246A1
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- beta estradiol
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/56—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids
- A61K31/565—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids not substituted in position 17 beta by a carbon atom, e.g. estrane, estradiol
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P9/00—Drugs for disorders of the cardiovascular system
Abstract
The cardioprotective effects of estrogen are well recognized. In in vitro experiments, and upon systemic administration, 17 - beta estradiol has shown to inhibit vascular smooth muscle cell proliferation and intimal hyperplasia. We hypothesized that locally delivered 17 - beta estradiol could inhibit neointimal proliferation following balloon angioplasty in porcine coronary arteries. Immunohistochemical, and morphometric analyses revealed that, arterial segments treated with local delivery of 17 - beta estradiol showed significantly less smooth muscle cell proliferation and neointima formation. Compared to PTCA
only, or vehicle alone, 17 - beta estradiol decreased neointima formation by 54.6 % and 64.9 % respectively.
only, or vehicle alone, 17 - beta estradiol decreased neointima formation by 54.6 % and 64.9 % respectively.
Description
TITLE OF THE INVENTION
Local Delivery of 17 - beta Estradiol Decreases Neointimal Hyperplasia Following Coronary Angioplasty in Porcine Model FIELD OF THE INVENTION
The present invention relates to the local use of estradiol for preventing restenosis. More specifically, the present invention is concerned with the local use of estradiol for decreasing neointimal hyperplasia that occurs during restenosis.
BACKGROUND OF THE INVENTION
Restenosis is currently the major limitation of percutaneous transluminal coronary angioplasty (PTCA), and is seen in up to 30-40 % of patients.'The most important mechanisms contributing to restenosis are neointimal proliferation, vascular remodelling, and elastic recoil.2 Elastic recoil and vascular remodelling can be reduced to a large extent by stenting.3 Although radiation therapy has been reported to show beneficial effeets,4~5 no effective therapy exists yet for neointimal proliferation.
Vascular smooth muscle cell (SMC) migration and proliferation have been documented to occur as early as 36 hours following arterial injury.s In cell culture assays, 17 - beta estradiol inhibited migration and proliferation of rat vascular SMC.'~8 Similar effects have also been shown with human vascular SMC from saphenous vein.9 Prolonged systemic administration of estrogen has been shown to inhibit intimal hyperplasia in animal studies.'°'" In the present experiment, we tested the hypothesis that local administration of 17 - beta estradiol during PTCA could effectively inhibit neointimal proliferation.
SUMMARY OF THE INVENTION
An object of the present invention is therefore to provide an efficient method by which 17-~i estradiol is used locally during PTCA to prevent restenosis. Compositions for executing this method are also a further object of this invention.
Other objects, advantages and features of the present invention will become more apparent upon reading of the following non restrictive description of preferred embodiments thereof, given by way of example only with reference to the accompanying drawings.
BRIEF DESCRIPTION OF THE DRAWINGS
In the appended drawings:
Figure 1 Representative light micrographs (x 40 magnification) of arterial segments from the same animal, stained with Verhoeffs stain. 17 - beta estradiol (a) treated segment shows markedly less neointimal hyperplasia compared to PTCA only (b), or vehicle alone (c) groups. The extent of injury is similar in all 3 segments.;
Figure 2 Comparison of (A) neointimal area, (B) neointimallmedia area, (C) restenotic index, and (D) % stenosis between PTCA alone vs vehicle only, and PTCA only vs 17 -beta estradiol groups; * p < 0.05, ** p < 0,01 *** p < 0.002. Values are expressed as mean t SEM.
DESCRIPTION OF THE PREFERRED EMBODIMENT
Methods Animal preparation Eighteen juvenile farm pigs (9 female, and 9 castrated male) weighing 20 - 25 kg were studied. The study was approved by, and conducted in accordance with, the guidelines of the Animal Care and Ethical Research Committee of the Montreal Heart Institute. Before the procedure, animals were given 650 mg of acetylsalicylic acid and 30 mg of nifedipine orally, premedicated with intramuscular injection of 6 mg/kg of a mixture of tiletamine hydrochloride and zolazepam hydrochloride, and given 0.05 mg of atropine. The invasive procedure was performed under general anesthesia with a mixture of isoflurane (1 to 1.5 %) and oxygen enriched air. The right femoral artery was cannulated percutaneously, and an 8 Fr arterial sheath was introduced. After arterial access had been obtained, 100 mg of lidocaine and 250 Ulkg of heparin were administered intra-arterially via the sheath. Activated coagulation time was maintained at > 300 seconds throughout the procedure.
Angioplasty and Local Delivery Standard PTCA equipment was used. An 8 Fr right Amplatz guiding catheter and right Judkins guiding catheter were used for cannulation of the left and right coronary arteries, respectively. PTCA was performed with a balloon size chosen to correspond to a balloonlartery ratio of 1.1-1.3.
Three 30-second inflations at 10 atm pressure were performed with a 30-second interval between each inflation. Inflations were performed adjacent to major side branches to facilitate identification during harvesting, taking precaution not to include any side branch in the intended PTCA site. The left anterior descending, left circumflex, and right coronary arteries of each animal were subjected to PTCA. After PTCA, each coronary artery of an animal was randomized to receive either 600 Ng of 17 - beta estradiol locally, or vehicle alone locally, or PTCA only. The chemicals 17 - beta estradiol and its vehicle 2-hydroxypropyl-beta-cyclodextrin (HPCD) were purchased from Sigma Chemical Co. The InfusaSleeve catheter (Local Med, Inc.) was used for local delivery.'2 Five ml of the designated substance was delivered at a driving pressure of 10 atm and support balloon pressure of 6 atm.
Of the 18 animals, 2 died a few days after PTCA, and were excluded;
thus, 16 animals were analyzed. Twelve animals were euthanised at 28 days, and 4 at 7 days. After premedication and anesthesia, the right internal jugular vein and common carotid artery were cannulated.
Following cross-clamping of the descending thoracic aorta exposed via a left lateral thoracotomy, exsanguination was performed, with simultaneous administration of 1 I of 0.9 % NaCI solution. The heart was perfusion-fixed in vivo with 2 1 of 10 % buffered formalin at 200 mm Hg pressure, removed from the animal, and placed in 10 % buffered formalin solution.
Coronary arteries were then dissected free from surrounding tissues. The site of PTCA was identified in relation to adjacent side branches, which served as landmarks. The injured segment was harvested with a 1 cm normal segment proximal and distal to the injured site. Serial sections 3 to 5 mm long were made from the harvested segment, with a minimum of at least 3 sections (maximum 5) from each PTCA site. Sections were 5 stored in buffered 10 % formalin and subjected to dehydration with increasing concentrations of alcohol, followed by treatment with xylene and paraffin. Each section was then cut to slices of 6 pm thickness with a microtome (Olympus cut 4060 E), and stained with VerhoefPs stain for morphometric analysis.
Morphometric analysis Measurements were made with a video microscope (Leitz Diaplan, equipped with a Sony DXC 970 MD color video camera) linked to a 486 personal computer and customized software. A minimum of 3 sections for each injured segment were analyzed and results averaged. Analyses were made by a single observer unaware of the treatment group to which each segment had bee allocated. Randomly selected sections were viewed by a second observer (also blinded to protocol) independently; inter-observer variability was < 5 %. The areas of external elastic lamina (EEL), internal elastic lamina (IEL), and lumen were measured by digital planimetry;
neointimal (I) area (IEL - lumen area) and media (M) area (EEL - IEL area) were obtained. The % neointima was defined as the % of total vessel area occupied by neointima (% neointima = [I/EEL] x 100). Morphologic stenosis was calculated as 100 (1 - IumenIlEL area).'3 The restenotic index was defined as [II(I + M)]I(FIIEL circumference), where F is the fracture length of internal elastic lamina. '4 Histologic injury score was determined as previously defined.'S
Immunohistochemistry Following slicing with a microtome and blocking of non-specific antibodies, the sections were treated with mouse anti - proliferating cell nuclear antigen (PCNA) antibodies and diluted biotynilated goat anti - mouse antibodies. They were then incubated with avidin -biotin (Elite ABC Kit, Vector Laboratories), and developed with 3, 3'- diaminobenzidine (Vector Laboratories). They were finally counter-stained with hematoxylin. Porcine liver cells were used as a positive control. For each section, a 6 Nm slice counter-stained with hematoxylin without treatment with the primary antibody (mouse anti - PCNA) served as a negative control.
The proliferative response to injury was studied by immunohistochemical analysis of samples from animals euthanised at 7 days. The proliferating SMC was obtained by dividing the number of PCNA - positive SMC by the total number of SMC in each field; separate measurements were made for neointimal and media layers. The proliferating cells were identified as SMC by positive staining of parallel sections with a-smooth muscle actin antibody. To standardize comparison among treatment groups, measurements were obtained at 4 fixed locations separated by 90° sites for each section, and the results averaged. For each segment, two sections demonstrating maximal neointimal response were analyzed, and the results averaged.
Local Delivery of 17 - beta Estradiol Decreases Neointimal Hyperplasia Following Coronary Angioplasty in Porcine Model FIELD OF THE INVENTION
The present invention relates to the local use of estradiol for preventing restenosis. More specifically, the present invention is concerned with the local use of estradiol for decreasing neointimal hyperplasia that occurs during restenosis.
BACKGROUND OF THE INVENTION
Restenosis is currently the major limitation of percutaneous transluminal coronary angioplasty (PTCA), and is seen in up to 30-40 % of patients.'The most important mechanisms contributing to restenosis are neointimal proliferation, vascular remodelling, and elastic recoil.2 Elastic recoil and vascular remodelling can be reduced to a large extent by stenting.3 Although radiation therapy has been reported to show beneficial effeets,4~5 no effective therapy exists yet for neointimal proliferation.
Vascular smooth muscle cell (SMC) migration and proliferation have been documented to occur as early as 36 hours following arterial injury.s In cell culture assays, 17 - beta estradiol inhibited migration and proliferation of rat vascular SMC.'~8 Similar effects have also been shown with human vascular SMC from saphenous vein.9 Prolonged systemic administration of estrogen has been shown to inhibit intimal hyperplasia in animal studies.'°'" In the present experiment, we tested the hypothesis that local administration of 17 - beta estradiol during PTCA could effectively inhibit neointimal proliferation.
SUMMARY OF THE INVENTION
An object of the present invention is therefore to provide an efficient method by which 17-~i estradiol is used locally during PTCA to prevent restenosis. Compositions for executing this method are also a further object of this invention.
Other objects, advantages and features of the present invention will become more apparent upon reading of the following non restrictive description of preferred embodiments thereof, given by way of example only with reference to the accompanying drawings.
BRIEF DESCRIPTION OF THE DRAWINGS
In the appended drawings:
Figure 1 Representative light micrographs (x 40 magnification) of arterial segments from the same animal, stained with Verhoeffs stain. 17 - beta estradiol (a) treated segment shows markedly less neointimal hyperplasia compared to PTCA only (b), or vehicle alone (c) groups. The extent of injury is similar in all 3 segments.;
Figure 2 Comparison of (A) neointimal area, (B) neointimallmedia area, (C) restenotic index, and (D) % stenosis between PTCA alone vs vehicle only, and PTCA only vs 17 -beta estradiol groups; * p < 0.05, ** p < 0,01 *** p < 0.002. Values are expressed as mean t SEM.
DESCRIPTION OF THE PREFERRED EMBODIMENT
Methods Animal preparation Eighteen juvenile farm pigs (9 female, and 9 castrated male) weighing 20 - 25 kg were studied. The study was approved by, and conducted in accordance with, the guidelines of the Animal Care and Ethical Research Committee of the Montreal Heart Institute. Before the procedure, animals were given 650 mg of acetylsalicylic acid and 30 mg of nifedipine orally, premedicated with intramuscular injection of 6 mg/kg of a mixture of tiletamine hydrochloride and zolazepam hydrochloride, and given 0.05 mg of atropine. The invasive procedure was performed under general anesthesia with a mixture of isoflurane (1 to 1.5 %) and oxygen enriched air. The right femoral artery was cannulated percutaneously, and an 8 Fr arterial sheath was introduced. After arterial access had been obtained, 100 mg of lidocaine and 250 Ulkg of heparin were administered intra-arterially via the sheath. Activated coagulation time was maintained at > 300 seconds throughout the procedure.
Angioplasty and Local Delivery Standard PTCA equipment was used. An 8 Fr right Amplatz guiding catheter and right Judkins guiding catheter were used for cannulation of the left and right coronary arteries, respectively. PTCA was performed with a balloon size chosen to correspond to a balloonlartery ratio of 1.1-1.3.
Three 30-second inflations at 10 atm pressure were performed with a 30-second interval between each inflation. Inflations were performed adjacent to major side branches to facilitate identification during harvesting, taking precaution not to include any side branch in the intended PTCA site. The left anterior descending, left circumflex, and right coronary arteries of each animal were subjected to PTCA. After PTCA, each coronary artery of an animal was randomized to receive either 600 Ng of 17 - beta estradiol locally, or vehicle alone locally, or PTCA only. The chemicals 17 - beta estradiol and its vehicle 2-hydroxypropyl-beta-cyclodextrin (HPCD) were purchased from Sigma Chemical Co. The InfusaSleeve catheter (Local Med, Inc.) was used for local delivery.'2 Five ml of the designated substance was delivered at a driving pressure of 10 atm and support balloon pressure of 6 atm.
Of the 18 animals, 2 died a few days after PTCA, and were excluded;
thus, 16 animals were analyzed. Twelve animals were euthanised at 28 days, and 4 at 7 days. After premedication and anesthesia, the right internal jugular vein and common carotid artery were cannulated.
Following cross-clamping of the descending thoracic aorta exposed via a left lateral thoracotomy, exsanguination was performed, with simultaneous administration of 1 I of 0.9 % NaCI solution. The heart was perfusion-fixed in vivo with 2 1 of 10 % buffered formalin at 200 mm Hg pressure, removed from the animal, and placed in 10 % buffered formalin solution.
Coronary arteries were then dissected free from surrounding tissues. The site of PTCA was identified in relation to adjacent side branches, which served as landmarks. The injured segment was harvested with a 1 cm normal segment proximal and distal to the injured site. Serial sections 3 to 5 mm long were made from the harvested segment, with a minimum of at least 3 sections (maximum 5) from each PTCA site. Sections were 5 stored in buffered 10 % formalin and subjected to dehydration with increasing concentrations of alcohol, followed by treatment with xylene and paraffin. Each section was then cut to slices of 6 pm thickness with a microtome (Olympus cut 4060 E), and stained with VerhoefPs stain for morphometric analysis.
Morphometric analysis Measurements were made with a video microscope (Leitz Diaplan, equipped with a Sony DXC 970 MD color video camera) linked to a 486 personal computer and customized software. A minimum of 3 sections for each injured segment were analyzed and results averaged. Analyses were made by a single observer unaware of the treatment group to which each segment had bee allocated. Randomly selected sections were viewed by a second observer (also blinded to protocol) independently; inter-observer variability was < 5 %. The areas of external elastic lamina (EEL), internal elastic lamina (IEL), and lumen were measured by digital planimetry;
neointimal (I) area (IEL - lumen area) and media (M) area (EEL - IEL area) were obtained. The % neointima was defined as the % of total vessel area occupied by neointima (% neointima = [I/EEL] x 100). Morphologic stenosis was calculated as 100 (1 - IumenIlEL area).'3 The restenotic index was defined as [II(I + M)]I(FIIEL circumference), where F is the fracture length of internal elastic lamina. '4 Histologic injury score was determined as previously defined.'S
Immunohistochemistry Following slicing with a microtome and blocking of non-specific antibodies, the sections were treated with mouse anti - proliferating cell nuclear antigen (PCNA) antibodies and diluted biotynilated goat anti - mouse antibodies. They were then incubated with avidin -biotin (Elite ABC Kit, Vector Laboratories), and developed with 3, 3'- diaminobenzidine (Vector Laboratories). They were finally counter-stained with hematoxylin. Porcine liver cells were used as a positive control. For each section, a 6 Nm slice counter-stained with hematoxylin without treatment with the primary antibody (mouse anti - PCNA) served as a negative control.
The proliferative response to injury was studied by immunohistochemical analysis of samples from animals euthanised at 7 days. The proliferating SMC was obtained by dividing the number of PCNA - positive SMC by the total number of SMC in each field; separate measurements were made for neointimal and media layers. The proliferating cells were identified as SMC by positive staining of parallel sections with a-smooth muscle actin antibody. To standardize comparison among treatment groups, measurements were obtained at 4 fixed locations separated by 90° sites for each section, and the results averaged. For each segment, two sections demonstrating maximal neointimal response were analyzed, and the results averaged.
Statistical Analysis Values are expressed as mean t standard deviation, except as otherwise indicated. Kruskal - Wallis analysis was used for comparison of data among the 3 groups; subsequently, 17 - beta estradiol and vehicle alone groups were separately compared with the PTCA only group using the Mann - Whitney rank sum test. Chi - square analysis was used for comparison of proportions. The Mann - Whitney rank sum test was also used for comparison of data between male and female animals within the 17 - beta estradiol treated group. Values were considered statistically significant if p < 0.05.
Res a Its Following PTCA and local delivery, animals were allowed to recover, and gained weight steadily. Two animals died 48 and 72 hours after procedure respectively, and were not included; thus 16 animals were studied.
Autopsy of the 2 animals revealed occlusive thrombus at the site of PTCA
(in the 17 - beta estradiol treated vessel in one pig, and in the vessel treated with PTCA only in the other pig).
Injured segments Balloonlartery ratio and artery diameter were not significantly different among the 3 treatment groups (Table 1). Segments with intact IEL in which discernible injury was absent were excluded from analysis (2 from PTCA only group, and 1 from vehicle alone group). Two segments were lost during harvesting and processing (1 of vehicle alone, and 1 of PTCA
only group).
Morphometric analysis Of the 12 animals that underwent morphometric analysis at 28 days, arterial segments treated with local delivery of 17 - beta estradiol showed significantly less neointimal hyperplasia (Figure 1). This beneficial effect was noted in all parameters of neointimal response to injury that were analyzed (Table 1 ). Of note, the extent of morphologic injury was similar among the 3 groups, suggesting that the use of the InfusaSleeve catheter was not associated with an enhanced risk of injury.
It was important to exclude an inhibitory effect on intimal proliferation due to the vehicle, and, to confirm that the effect noted was in response to treatment with 17 - beta estradiol. Analyses comparing segments treated with vehicle alone and PTCA only showed a similar response in terms of the extent of neointimal proliferation. On the other hand, significantly less intimal hyperplasia was observed in 17 - beta estradiol treated segments as compared to segments treated with PTCA only (Figure 2). Compared to PTCA only, or vehicle alone, 17 - beta estradiol decreased neointima formation by 54.6 % and 64.9 % respectively.
To exclude the possibility of influence of sex on response to estrogen, the 7 segments obtained from male pigs treated with 17 - beta estradiol, and 5 segments obtained from female pigs treated with 17 - beta estradiol were analyzed. No statistically significant differences were evident (Table 2).
Immunohistochemistry The number of PCNA - positive SMC was low overall; sacrifice at an earlier time might have yielded a higher number. However, a statistically significant decrease in the proliferative response was seen in animals treated with 17 - beta estradiol. Among the different groups, the % of PCNA - positive SMC in the neointima were 0.43 t 0.52 % in 17 - beta estradiol, 4.26 t 2.33 % in PTCA only, and 4.27 t 2.73 % in vehicle alone groups respectively (p < 0.05 for 17 - beta estradiol vs other 2 groups).
There were no statistically significant differences in % PCNA - positive SMC in the media among the 3 groups: 0.4 t 0.3 %, 1.38 t 1.74 %, and 1.24 t 1.57 % for 17 - beta estradiol, PTCA only, and vehicle alone groups respectively (p = NS).
Vascular remodeling To determine the effect on vascular remodeling of the agents used, the EEL area of the injured segment and of the normal vessel proximal to site of PTCA were obtained, and their ratio calculated. '3 No significant difference among the groups was noted: 1.01 t 0.16, 1.16 t 0.28, 1.31 t 0.37 respectively for 17 - beta estradiol, PTCA only, and vehicle alone groups respectively (p = NS).
Discussion The present study demonstrates, for the first time, that locally delivered 17 - beta estradiol decreases neointimal proliferation following PTCA in pigs.
5 The study also shows that the InfusaSleeve catheter can be used to deliver effectively 17 - beta estradiol intramurally in coronary arteries.
Several previous experiments in animals have demonstrated that estrogen administered subcutaneously for up to 3 weeks inhibited the myointimal 10 response to arterial injury. ,°." Recently, short-term subcutaneous estrogen therapy (6 to 17 days) was also shown to be effective in reducing the injury response in rat carotid artery. '6 Estrogen administered intramuscularly for at least 3 weeks has also demonstrated the potential to inhibit vascular smooth muscle cell proliferation and neointimal hyperplasia in rabbits. " However, the efficacy of local delivery of 17 - beta estradiol to inhibit intimal hyperplasia has not been previously studied.
The biologic effects of estrogen, like other steroid hormones, involve intracellular receptors. The first estrogen receptor (ER) to be discovered was ERa,'e~'9 which was thought to mediate the beneficial effects of estrogen following vascular injury. ERa was also present in coronary arteries obtained from autopsy specimens in both pre- and post-menopausal women,z° and in cell cultures of human saphenous vein and internal mammary artery specimens.2' Recently, a second estrogen receptor, ER(3, has been identified in animals and humans.22,2s The role of ER(3 in response to vascular injury was subsequently demonstrated in experiments with ERa deficient mice. 24 Normal and ERa deficient mice treated with estrogen, when subjected to arterial injury, showed the same extent of inhibition of neointimal proliferation compared to control mice;
thereby demonstrating that inhibition of vascular injury response by estrogen is independent of ERa. Although the present experiment was not designed to study the mechanism of action of 17 - beta estradiol, evidence exists for multiple potential mechanisms by which 17 - beta estradiol can inhibit the vascular response to injury. Of importance may be the effect of 17 - beta estradiol on nitric oxide (NO) synthesis. In cell culture studies with human and bovine endothelial cells, treatment with 17 - beta estradiol stimulated NO synthase and increased NO production.25,zs Postmenopausal women treated with transdermal 17 - beta estradiol showed enhanced in vivo NO synthesis.2' NO has demonstrated inhibitory effects on both migration 28 and proliferation 29 of vascular SMC, and decreased neointima formation after PTCA.'3 Preliminary reports have shown that therapy with 17 - beta estradiol decreases intercellular and vascular cell adhesion molecule expression by human coronary SMC.3o Cellular adhesion molecules are expressed by SMC following arterial injury3' and their suppression with the use of monoclonal antibodies inhibited intimal hyperplasia after arterial injury in rats.3z The regulatory effect of 17 - beta estradiol on vascular endothelial growth factor expression may also be partly responsible.33-s5 perhaps the most important mechanism may be a direct inhibitory effect of 17 - beta estradiol on vascular SMC proliferation.36 The binding of 17 - beta estradiol to its intracellular receptor activates DNA containing "estrogen responsive elements", leading to altered gene expression. 17 - beta estradiol also reduces platelet derived growth factor - induced migration and proliferation of vascular SMC.9 The beneficial effects of 17 - beta estradiol, the predominant circulating estrogen in premenopausal women, on vascular injury response may not be replicated by other kinds of estrogens; for example, conjugated equine estrogen was found to have no effect on neointimal proliferation in non-human primate models.3' Simultaneous administration of progesterone may attenuate the vascular injury response to 17 - beta estradiol.38 A sexually dimorphic response to estrogen in intact rats has been reported following arterial injury, with male rats deriving no benefit with estrogen therapy .39 This sexually dimorphic effect was, however, not observed in another experiment with gonadeetomized rats." I In the present study, too, no significant difference in neointimal proliferative response to 17 - beta estradiol was noted between the sexes. Increased expression of ER~3 mRNA (ER~i is directly associated with inhibition of vascular SMC proliferation) following arterial injury has been demonstrated in intact male rats;4° of additional interest in the study is that no increase in ERa was seen following arterial injury.
17 - beta estradiol is a lipophilic compound with poor solubility in aqueous solutions, thereby needing a vehicle for parenteral administration. HPCD
is a starch derivative that has been successfully tested as an effective excipient for protein drugs. 4' The pharmacokinetics of HPCD are similar to that of inulin, and the toxic dose (nephrotoxicity) has been estimated to be 200 mglkg in rats.42 The dose of HPCD used to dissolve 17 - beta estradiol in the present study was 0.63 mglkg, far below the toxic dose.
Furthermore, HPCD has been used for administration of ophthalmic preparations and intravenous anaesthetic agents in humans.4s,44 HPCD
complexed to 17 - beta estradiol has been used to enhance bioavailability of orally, or, sublingually administered 17 - beta estradiol with no untoward effects in humans.45 Retrospective studies in humans have shown no benefit of hormonal replacement therapy on angiographic restenosis following PTCA;4s although one study did show a beneficial effect after directional atherectomy.4' However, it should be noted that conjugated estrogen (and not 17 - beta estradiol) was the predominant form of estrogen used in many of these patients, and, no information about concommittent use of progesterone is available.
In conclusion, we have shown that, a single dose of 17 - beta estradiol delivered locally during PTCA has the potential to inhibit neointimal proliferation effectively. The delivery of 17 - beta estradiol can be performed easily with the InfusaSleeve catheter, without risk of additional injury. With this approach, it may be possible to avoid potential undesirable effects of long term systemic administration of estrogen. ER~i has been identified in humans, and inhibition of proliferation of human vascular SMC by 17 - beta estradiol has been demonstrated in cell culture assays. The local administration of 17 - beta estradiol is therefore a promising new approach, which might be useful in preventing the proliferative response after PTCA in humans. Its usefulness in preventing restenosis after PTCA merits further investigation.
Res a Its Following PTCA and local delivery, animals were allowed to recover, and gained weight steadily. Two animals died 48 and 72 hours after procedure respectively, and were not included; thus 16 animals were studied.
Autopsy of the 2 animals revealed occlusive thrombus at the site of PTCA
(in the 17 - beta estradiol treated vessel in one pig, and in the vessel treated with PTCA only in the other pig).
Injured segments Balloonlartery ratio and artery diameter were not significantly different among the 3 treatment groups (Table 1). Segments with intact IEL in which discernible injury was absent were excluded from analysis (2 from PTCA only group, and 1 from vehicle alone group). Two segments were lost during harvesting and processing (1 of vehicle alone, and 1 of PTCA
only group).
Morphometric analysis Of the 12 animals that underwent morphometric analysis at 28 days, arterial segments treated with local delivery of 17 - beta estradiol showed significantly less neointimal hyperplasia (Figure 1). This beneficial effect was noted in all parameters of neointimal response to injury that were analyzed (Table 1 ). Of note, the extent of morphologic injury was similar among the 3 groups, suggesting that the use of the InfusaSleeve catheter was not associated with an enhanced risk of injury.
It was important to exclude an inhibitory effect on intimal proliferation due to the vehicle, and, to confirm that the effect noted was in response to treatment with 17 - beta estradiol. Analyses comparing segments treated with vehicle alone and PTCA only showed a similar response in terms of the extent of neointimal proliferation. On the other hand, significantly less intimal hyperplasia was observed in 17 - beta estradiol treated segments as compared to segments treated with PTCA only (Figure 2). Compared to PTCA only, or vehicle alone, 17 - beta estradiol decreased neointima formation by 54.6 % and 64.9 % respectively.
To exclude the possibility of influence of sex on response to estrogen, the 7 segments obtained from male pigs treated with 17 - beta estradiol, and 5 segments obtained from female pigs treated with 17 - beta estradiol were analyzed. No statistically significant differences were evident (Table 2).
Immunohistochemistry The number of PCNA - positive SMC was low overall; sacrifice at an earlier time might have yielded a higher number. However, a statistically significant decrease in the proliferative response was seen in animals treated with 17 - beta estradiol. Among the different groups, the % of PCNA - positive SMC in the neointima were 0.43 t 0.52 % in 17 - beta estradiol, 4.26 t 2.33 % in PTCA only, and 4.27 t 2.73 % in vehicle alone groups respectively (p < 0.05 for 17 - beta estradiol vs other 2 groups).
There were no statistically significant differences in % PCNA - positive SMC in the media among the 3 groups: 0.4 t 0.3 %, 1.38 t 1.74 %, and 1.24 t 1.57 % for 17 - beta estradiol, PTCA only, and vehicle alone groups respectively (p = NS).
Vascular remodeling To determine the effect on vascular remodeling of the agents used, the EEL area of the injured segment and of the normal vessel proximal to site of PTCA were obtained, and their ratio calculated. '3 No significant difference among the groups was noted: 1.01 t 0.16, 1.16 t 0.28, 1.31 t 0.37 respectively for 17 - beta estradiol, PTCA only, and vehicle alone groups respectively (p = NS).
Discussion The present study demonstrates, for the first time, that locally delivered 17 - beta estradiol decreases neointimal proliferation following PTCA in pigs.
5 The study also shows that the InfusaSleeve catheter can be used to deliver effectively 17 - beta estradiol intramurally in coronary arteries.
Several previous experiments in animals have demonstrated that estrogen administered subcutaneously for up to 3 weeks inhibited the myointimal 10 response to arterial injury. ,°." Recently, short-term subcutaneous estrogen therapy (6 to 17 days) was also shown to be effective in reducing the injury response in rat carotid artery. '6 Estrogen administered intramuscularly for at least 3 weeks has also demonstrated the potential to inhibit vascular smooth muscle cell proliferation and neointimal hyperplasia in rabbits. " However, the efficacy of local delivery of 17 - beta estradiol to inhibit intimal hyperplasia has not been previously studied.
The biologic effects of estrogen, like other steroid hormones, involve intracellular receptors. The first estrogen receptor (ER) to be discovered was ERa,'e~'9 which was thought to mediate the beneficial effects of estrogen following vascular injury. ERa was also present in coronary arteries obtained from autopsy specimens in both pre- and post-menopausal women,z° and in cell cultures of human saphenous vein and internal mammary artery specimens.2' Recently, a second estrogen receptor, ER(3, has been identified in animals and humans.22,2s The role of ER(3 in response to vascular injury was subsequently demonstrated in experiments with ERa deficient mice. 24 Normal and ERa deficient mice treated with estrogen, when subjected to arterial injury, showed the same extent of inhibition of neointimal proliferation compared to control mice;
thereby demonstrating that inhibition of vascular injury response by estrogen is independent of ERa. Although the present experiment was not designed to study the mechanism of action of 17 - beta estradiol, evidence exists for multiple potential mechanisms by which 17 - beta estradiol can inhibit the vascular response to injury. Of importance may be the effect of 17 - beta estradiol on nitric oxide (NO) synthesis. In cell culture studies with human and bovine endothelial cells, treatment with 17 - beta estradiol stimulated NO synthase and increased NO production.25,zs Postmenopausal women treated with transdermal 17 - beta estradiol showed enhanced in vivo NO synthesis.2' NO has demonstrated inhibitory effects on both migration 28 and proliferation 29 of vascular SMC, and decreased neointima formation after PTCA.'3 Preliminary reports have shown that therapy with 17 - beta estradiol decreases intercellular and vascular cell adhesion molecule expression by human coronary SMC.3o Cellular adhesion molecules are expressed by SMC following arterial injury3' and their suppression with the use of monoclonal antibodies inhibited intimal hyperplasia after arterial injury in rats.3z The regulatory effect of 17 - beta estradiol on vascular endothelial growth factor expression may also be partly responsible.33-s5 perhaps the most important mechanism may be a direct inhibitory effect of 17 - beta estradiol on vascular SMC proliferation.36 The binding of 17 - beta estradiol to its intracellular receptor activates DNA containing "estrogen responsive elements", leading to altered gene expression. 17 - beta estradiol also reduces platelet derived growth factor - induced migration and proliferation of vascular SMC.9 The beneficial effects of 17 - beta estradiol, the predominant circulating estrogen in premenopausal women, on vascular injury response may not be replicated by other kinds of estrogens; for example, conjugated equine estrogen was found to have no effect on neointimal proliferation in non-human primate models.3' Simultaneous administration of progesterone may attenuate the vascular injury response to 17 - beta estradiol.38 A sexually dimorphic response to estrogen in intact rats has been reported following arterial injury, with male rats deriving no benefit with estrogen therapy .39 This sexually dimorphic effect was, however, not observed in another experiment with gonadeetomized rats." I In the present study, too, no significant difference in neointimal proliferative response to 17 - beta estradiol was noted between the sexes. Increased expression of ER~3 mRNA (ER~i is directly associated with inhibition of vascular SMC proliferation) following arterial injury has been demonstrated in intact male rats;4° of additional interest in the study is that no increase in ERa was seen following arterial injury.
17 - beta estradiol is a lipophilic compound with poor solubility in aqueous solutions, thereby needing a vehicle for parenteral administration. HPCD
is a starch derivative that has been successfully tested as an effective excipient for protein drugs. 4' The pharmacokinetics of HPCD are similar to that of inulin, and the toxic dose (nephrotoxicity) has been estimated to be 200 mglkg in rats.42 The dose of HPCD used to dissolve 17 - beta estradiol in the present study was 0.63 mglkg, far below the toxic dose.
Furthermore, HPCD has been used for administration of ophthalmic preparations and intravenous anaesthetic agents in humans.4s,44 HPCD
complexed to 17 - beta estradiol has been used to enhance bioavailability of orally, or, sublingually administered 17 - beta estradiol with no untoward effects in humans.45 Retrospective studies in humans have shown no benefit of hormonal replacement therapy on angiographic restenosis following PTCA;4s although one study did show a beneficial effect after directional atherectomy.4' However, it should be noted that conjugated estrogen (and not 17 - beta estradiol) was the predominant form of estrogen used in many of these patients, and, no information about concommittent use of progesterone is available.
In conclusion, we have shown that, a single dose of 17 - beta estradiol delivered locally during PTCA has the potential to inhibit neointimal proliferation effectively. The delivery of 17 - beta estradiol can be performed easily with the InfusaSleeve catheter, without risk of additional injury. With this approach, it may be possible to avoid potential undesirable effects of long term systemic administration of estrogen. ER~i has been identified in humans, and inhibition of proliferation of human vascular SMC by 17 - beta estradiol has been demonstrated in cell culture assays. The local administration of 17 - beta estradiol is therefore a promising new approach, which might be useful in preventing the proliferative response after PTCA in humans. Its usefulness in preventing restenosis after PTCA merits further investigation.
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I I
I I
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i lil Table 1. Morphometric Analysis Characteristics 17 - beta estradiolPTCA only Vehicle p value*
alone Segments analyzed12 9 10 NS
Artery size (mm) 2.86 t 0.35 2.94 t 0.242.94 t NS
0.41 BalloonlArtery 1.22 t 0.09 1.2 t 0.06 1.17 t NS
ratio 0.11 EEL~e,/EEL;~~ 1.01 t 0.16 1.31 t 0.371.16 t NS
t 0.28 Neointimal area 0.4 t 0.3 0.88 t 0.611.14 t < 0.05 (mmz) 1.03 neointima 12.16 t 8.89 23.02 t 25.46 t < 0.025 11.91 14.96 NeointimalMedia 0.59 t 0.48 1.67 t 1.291.75 t < 0.01 area 1.29 stenosis 15.67 t 11.13 27.51 t 30.34 t < 0.025 13.17 17.05 Restenotic index 1.3 t 0.5 2.4 t 0.68 2.42 t < 0.005 0.71 Injury score 1.64 t 0.34 1.7 t 0.43 1.77 t NS
0.47 * 17 - beta estradiol vs other 2 groups; tEEL~ef = proximal reference segment external elastic lamina area, EEL;~~ = injured segment external elastic lamina area (averaged).
Table 2. Response to 17 - beta estradiol According to Sex of the Animal Characteristics Male Female p value Restenotic index 1.2 t 0.59 1.37 t 0.45 > 0.1 Neointimal area 0.51 t 0.34 0.25 t 0.15 > 0.1 (mm2) NeointimalMedia 0.78 t 0.55 0.32 t 0.16 > 0.1 area neointima 14.93 t 10.688.29 t 3.72 > 0.1 stenosis 18.93 t 13.3911.09 t 5.16> 0. I
Although the present invention has been described hereinabove by way of preferred embodiments thereof, it can be modified, without departing from the spirit and nature of the subject invention as defined in the appended claims.
Circulation 1997; 95: 1301-7.
10 40. Lindner V, Kim SK, Karas RH, Kuiper GGJM, Gustafsson JA, Mendelsohn ME. Increased expression of estrogen receptor-f3 mRNA in male blood vessels after vascular injury. Circ Res 1998; 83: 224-9.
41. Brewster ME, Hora MS, Simpkins JW, Bodor N. Use of 15 2-hydroxypropyl-betacyclodextrin as a solubilizing and stabilizing excipient for protein drugs. Pharm Res 1991; 8(6): 792-5.
42. Frijlink HW, Visser J, Hefting NR, Oosting R, Meijer DKF, Lerk CF. The pharmacokinetics of beta-cyclodextrin and 2-hydroxypropyl-beta-20 cyclodextrin in the rat. Pharm Res 1990; 7(12): 1248-52.
43. Kristinsson JK, Fridriksdottir H, Thorisdottir S, Sigurdardottir AM, Stefansson E, Loftsson T. Dexamethasone-cyclodextrin-polymer co-complexes in aqueous eye drops: aqueous humor pharmacokinetics in humans. Invest Ophthalmol Vis Sci 1996; 37: 1199-1203.
44. Doenicke A, Roizen MF, Nebauer AE, Kugler A, Hoernecke R, Beger-Hintzen H. A comparison of two formulations for etomidate, 2-hydroxypropyl-beta-cyclodextrin (HPCD) and propylene glycol. Anesth Analg 1994; 79: 933-9.
45. Hoon TJ, Dawood Y, Khan-Dawood FS, Ramos J, Batenhorst RL.
Bioequivalence of a 17 - beta estradiol hydroxypropyl-beta-cyclodextrin complex in postmenopausal women. J Clin Pharmacol 1993; 33: 1116-21.
46. O'Keefe JH, Kim SC, Hall RR, Cochran VC, Lawhorn SL, McCallister BD. Estrogen replacement therapy after coronary angioplasty in women.
J Am Coll Cardiol 1997; 29: 1-5.
47. O'Brien JE, Peterson ED, Keeler GP, Berdan LG, Ohman EM, Faxon DP, Jacobs AK, Topol EJ, CaliffRM. Relation between estrogen replacement therapy and restenosis after percutaneous coronary interventions. J Am Coll Cardiol 1996; 28: 1111-8.
i lil Table 1. Morphometric Analysis Characteristics 17 - beta estradiolPTCA only Vehicle p value*
alone Segments analyzed12 9 10 NS
Artery size (mm) 2.86 t 0.35 2.94 t 0.242.94 t NS
0.41 BalloonlArtery 1.22 t 0.09 1.2 t 0.06 1.17 t NS
ratio 0.11 EEL~e,/EEL;~~ 1.01 t 0.16 1.31 t 0.371.16 t NS
t 0.28 Neointimal area 0.4 t 0.3 0.88 t 0.611.14 t < 0.05 (mmz) 1.03 neointima 12.16 t 8.89 23.02 t 25.46 t < 0.025 11.91 14.96 NeointimalMedia 0.59 t 0.48 1.67 t 1.291.75 t < 0.01 area 1.29 stenosis 15.67 t 11.13 27.51 t 30.34 t < 0.025 13.17 17.05 Restenotic index 1.3 t 0.5 2.4 t 0.68 2.42 t < 0.005 0.71 Injury score 1.64 t 0.34 1.7 t 0.43 1.77 t NS
0.47 * 17 - beta estradiol vs other 2 groups; tEEL~ef = proximal reference segment external elastic lamina area, EEL;~~ = injured segment external elastic lamina area (averaged).
Table 2. Response to 17 - beta estradiol According to Sex of the Animal Characteristics Male Female p value Restenotic index 1.2 t 0.59 1.37 t 0.45 > 0.1 Neointimal area 0.51 t 0.34 0.25 t 0.15 > 0.1 (mm2) NeointimalMedia 0.78 t 0.55 0.32 t 0.16 > 0.1 area neointima 14.93 t 10.688.29 t 3.72 > 0.1 stenosis 18.93 t 13.3911.09 t 5.16> 0. I
Although the present invention has been described hereinabove by way of preferred embodiments thereof, it can be modified, without departing from the spirit and nature of the subject invention as defined in the appended claims.
Claims (3)
1. The use of 17-.beta. estradiol in the making of a medication to prevent restenosis, for in situ administration at a site susceptible to restenosis.
2. A composition for preventing restenosis comprising an anti-vascular smooth cell proliferative amount of 17-.beta. estradiol in a pharmaceutically acceptable carrier.
3. A device comprising 17-.beta. estradiol for in situ delivery to a vascular site susceptible to restenosis.
Priority Applications (14)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CA002300246A CA2300246A1 (en) | 2000-03-09 | 2000-03-09 | Local delivery of 17-beta estradiol decreases neointimal hyperplasia following coronary angioplasty in porcine model |
| JP2001524583A JP3820150B2 (en) | 1999-09-21 | 2000-09-21 | Local delivery of 17-β estradiol to prevent intimal thickening and to improve vascular endothelial function after vascular injury |
| DE60008001T DE60008001T2 (en) | 1999-09-21 | 2000-09-21 | LOCAL ADMINISTRATION OF 17-BETA ESTRADIOL TO IMPROVE VASCULAR ENDOTHELIAL FUNCTION AFTER Vascular Injuries |
| DK00963838T DK1214077T3 (en) | 1999-09-21 | 2000-09-21 | Local administration of 17-beta estradiol to improve vascular endothelial function after vascular injury |
| AU75019/00A AU766208B2 (en) | 1999-09-21 | 2000-09-21 | Local delivery of 17-beta estradiol for preventing vascular intima hyperplasia and for improving vascular endothelium function after vascular injury |
| PT00963838T PT1214077E (en) | 1999-09-21 | 2000-09-21 | LOCAL LIBRARY OF 17-BETA ESTRADIOL TO IMPROVE THE VASCULAR FUNCTION OF ENDOTHELIA IN THE SEQUENCE OF VASCULAR WOUNDS |
| MXPA02003009A MXPA02003009A (en) | 1999-09-21 | 2000-09-21 | Local delivery of 17-beta estradiol for preventing vascular intima hyperplasia and for improving vascular endothelium function after vascular injury. |
| PCT/CA2000/001132 WO2001021157A2 (en) | 1999-09-21 | 2000-09-21 | Local delivery of 17-beta estradiol for preventing vascular intima hyperplasia and for improving vascular endothelium function after vascular injury |
| AT00963838T ATE258441T1 (en) | 1999-09-21 | 2000-09-21 | LOCAL ADMINISTRATION OF 17-BETA ESTRADIOL TO IMPROVE VASCULAR ENDOTHELIAL FUNCTION AFTER VASCULAR INJURY |
| ES00963838T ES2215069T3 (en) | 1999-09-21 | 2000-09-21 | LOCAL SUPPLY OF 17-BETA ESTRADIOL TO AVOID INTIMATE VASCULAR HYPERPLASIA AND TO IMPROVE VASCULAR ENDOTELIAL FUNCTION AFTER VASCULAR DAMAGE. |
| EP00963838A EP1214077B1 (en) | 1999-09-21 | 2000-09-21 | Local delivery of 17-beta estradiol for improving vascular endothelium function after vascular injury |
| CA002381031A CA2381031C (en) | 1999-09-21 | 2000-09-21 | Local delivery of 17-beta estradiol for preventing vascular intima hyperplasia and for improving vascular endothelium function after vascular injury |
| US10/602,934 US20040229856A1 (en) | 1999-09-21 | 2003-06-24 | Local delivery of 17-beta estradiol for preventing vascular intimal hyperplasia and for improving vascular endothelium function after vascular injury |
| US11/672,432 US20070141109A1 (en) | 1999-09-21 | 2007-02-07 | Local Deliver of 17-Beta Estradiol for Preventing Vascular Intimal Hyperplasia and for Improving Vascular Endothelium Function after Vascular Injury |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CA002300246A CA2300246A1 (en) | 2000-03-09 | 2000-03-09 | Local delivery of 17-beta estradiol decreases neointimal hyperplasia following coronary angioplasty in porcine model |
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| CA002300246A Abandoned CA2300246A1 (en) | 1999-09-21 | 2000-03-09 | Local delivery of 17-beta estradiol decreases neointimal hyperplasia following coronary angioplasty in porcine model |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US6471979B2 (en) | 1999-12-29 | 2002-10-29 | Estrogen Vascular Technology, Llc | Apparatus and method for delivering compounds to a living organism |
-
2000
- 2000-03-09 CA CA002300246A patent/CA2300246A1/en not_active Abandoned
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US6471979B2 (en) | 1999-12-29 | 2002-10-29 | Estrogen Vascular Technology, Llc | Apparatus and method for delivering compounds to a living organism |
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