VASCULOPROTECTOR

Abstract: The invention relates to a method of inducing a vasculoprotective effect in a subject, the method comprising treating the subject with an ER beta agonist.

Description: VASCULOPROTECTOR This invention relates to the use of ligands of the second estrogen receptor, ERβ, or compounds which affect ERβ for vasculoprotection, that is to say in inducing protective effects m the vascular wall particularly in fibroproliferative disorders (such as atherosclerosis, ateπosclerosis, diabetic and autoimmune angiopathies), after injury (such as restenosis after angioplasty and bypass surgery) and m chronic allograft rejection. Recently, a second estrogen receptor, ERβ, has been revealed (WO97/09348) Vascular mtimal dysplasia and remodelling are characteπstic features of remjury following balloon angioplasty, coronary bypass surgery (Holmes, D. et al. Am J Cardiol (1984); 53: 77C-81C, Holmes, D et al J Am Coll Cardiol (1988); 22 1 149-55), and in chronic allograft rejection (Lemstrom KB, Koskinen PK Circulation (1997); 96' 1240-1249, Hayry, P et al. Immunol Rev (1993) Aug; 134: 33-81, Hayry P et al. Faseb J (1993); 7: (11) 1055-60) The initial response to vascular injury is inflammatory and involves the attraction of lymphocytes, macrophages and thrombocytes to the site of injury and the secretion of cytokines, eikosanoids and growth factors (Ross. R. Nature (1993); 362(6423)' 801-9). Under the influence of growth factors and cytokines, smooth muscle cells (SMC) proliferate and migrate from the media into the intima and contπbute to mtimal hyperplasia and stenosis Estrogen has several protective effects on the vascular wall (Farhat MY et al. J Pharmacol Exp Ther (1996), 276- 652-7). Some of these are rapid, presumably direct membrane effects, whereas others require transcπptional activation of genes (Farhat MY. et al. Biochem Pharmacol (1996); 51(5)- 571-6). The inhibitory effect of estrogen on the replication, migration and extracellular matrix deposition by vascular smooth muscle cells, the key event m vascular fibroproliferative dysplasias, is presumably a genomic effect mediated through a vaπety of mechanisms including regulation of several growth factors and/or their receptors and possibly by a direct antiprohferative effect of estrogen on smooth muscle cells (Farhat MY, et al. Faseb J (1996); 10(5): 615-24) The vasculoprotective effect of estrogen was first demonstrated in population studies in humans, where estrogen replacement therapy demonstrated a protective effect on atherosclerotic vascular disease in post menopausal women (Stampfer M J et al (1991) N Engl J Med 325 756-762, Grady D et al (1992) Ann Intern Med 111 1016-1037), as later confirmed in monkeys (Wagner JD et al Metabolism (1997), 46(6) 698-705) Later, the vasculoprotective effect has been documented m more detail in animal models and in vitro Estrogen has been found to inhibit the intimal thickening after mechanical carotid balloon injury in rabbits (Foegh ML et al J Vase Surg (1994), 19(4) 722-6), rats (Chen SJ et al Circulation (1996), 93(3) 577-84) and in mice (Sullivan TJ et al J Clin Invest (1995), 96 2482-8), as well as the immunologically-mduced vascular fibroproliferative dysplasia in rabbit aorta (Cheng LP, et al Transplantation (1991), 52(6) 967-72) and heart (Foegh ML et al Transplant Proc (1987) 90-5) allogratts In vitro, it has been demonstrated that estrogen inhibits migration and replication of vascular smooth muscle cells (Akishita M et al Atherosclerosis (1997), 130(1-2) 1-10, Kolodgie FD, et al Am J Pathol (1996), 148(3) 969-76, Morev AK, et al Endocπnology (1997), 138(8) 3330-9, Suzuki A, et al Cardiovasc Res (1996), 32(3) 516-23) These observations are consistant with findings in reporter gene assays that functional estrogen receptors are expressed in vascular smooth muscle cells of bovine (Balica M et al Circulation (1997), 95(7) 1954-60, rat ((Bayard F et al Endocrinology (1995), 136(4) 1523-9 Bayard F et al Ciba Found Symp (1995), 191(122) 122-32. Bei M et al J Steπod Biochem Mol Biol (1996), 58(1) 83-8), guinea pig (Bhalla RC, et al Am J Physiol (1997) H1996-2003), and human (Karas RH. et al Febs Lett (1995), 377(2) 103-8) origin In addition to being anti-prohferative and anti-migratory to smooth muscle cells, estrogen may also display vasculoprotective effects via the vascular wall endothehum Functional estrogen receptors have been demonstrated in endothehal cells (Venkov CD et al Circulation (1996), 94 727-33) Estrogen downregulates cytokine-induced adhesion molecule expression in human endothehum in vitro (Cauhn GT et al J Chn Invest (1996), 98(1) 36-42), it is anti-apoptotic to endothehal cells (Spyπdopoulos, I et al Circulation (1997), 95(6) 1505-14) and it enhances functional endothehal recovery after denudation assay in vivo (Krasmski K et al Circulation (1997), 95(7) 1768-72) There are also additional, indirect pathways whereby estrogen may mediate vasculoprotective effects In addition to being directly anti-prohferative to smooth muscle cells and protective to the vascular endothehal cells, the estrogen effect may be mediated indirectly via hpoprotem metabolism and via promoting vasodilation by stimulating prostacyclm and nitric oxide synthesis and via regulation of the cell membrane voltage-dependent calcium channels resulting in inhibition of extracellular calcium mobilization and flux (see Farhat MY et al. J Pharmacol Exp Ther (1996); 276(2): 652-7, Farhat MY. et al Biochem Pharmacol (1996); 51(5). 571-6) None of these mechanisms alone explain the beneficial effect of estrogen. For example, although the endothehal nitπc oxide synthetase (e-NOS) mRNA and protein are upregulated in vascular endothehum by estrogen in vitro (MacRitchie AN, et al. Circ Res ( 1997), 81(3) 355-62), the effect is only partially inhibited by the e-NOS inhibitor NAME (Holm P et al. J C n Invest (1997); 100(4) 821-8) The development of vasculoprotective drug therapies based on the protective effect of estrogen has been difficult, as it has not been possible to differentiate the desired vasculoprotective effect of estrogen from its undesirable effects on the reproductive system - e.g its utero trophic effect Recent work on the vasculoprotective effect of estrogen m ERα-deficient mice by Iafrati and coworkers (Iafrati MD et al Nat Med (1997); 3(5) 545-8). suggests that ERα may not be responsible for the vasculoprotective estrogemc response The inventors have unexpectedly managed to differentiate the vasculoprotective effect of estrogen from its uterotrophic effect using ligands with different binding affinity to ERα and ERβ. The inventors have discovered that the genistem, an lsoflavonic phytoestrogen, which shows approximately 20 x higher binding affinity to ERβ compared its binding affinity for ERα displays a full vasculoprotective effect but is devoid of any uterotrophic effect. In addition, the inventors have demonstrated that ERβ is strongly upregulated in the vascular wall as a consequence of injury, whereas ERα remains expressed at a constitutive background level only The discovery of a second estrogen receptor ERβ, and the recent finding that disruption of the "classical" ERα gene in mice preserves the vasculoprotective effect of estrogen, offer better targeting of estrogen in vasculoprotective drug therapies The inventors have unexpectedly demonstrated that, after endothehal denudation injury of rat carotid artery, ERα mRNA (and protein) are constitutively expressed at a low level in the vascular wall, whereas the expression of ERβ mRNA increases >30-fold after injury In in situ hybridization, the ERβ mRNA co-localizes with the replicating and migrating SMC m the media and in the neointima Treatment of ovariectomized female rats on a soybean deficient diet with the lsoflavonic phytoestrogen genistein, which shows approximately 20x higher binding affinity to ERβ than to ERα, and with 17β -estradiol, which does not differentiate between the two receptor subtypes, at a dose range of 0 00250 through 2 50 mg/kg/d, provides on both occasions a dose-dependent vasculoprotective effect However, treatment with 17β-estradιol only, but not with genistein, is accompanied by a dose-dependent uterotrophic effect Thus the vaculoprotective effect of estrogen has been for the first time differentiated from the uterotrophic effect using ligands with different binding affinity to ERβ vs ERα The in vivo experiments used genistein doses of less than 2 mg/kg/d. which is well known below the dose level (50 - 100 mM) where the protein tyrosine kinase inhibitory effect of genistein and lsoflavones have been demonstrated As genistein at this dose range is likely to function via the ERβ, and is devoid of inhibitory effect on protein tyrosine kmases, the vasculoprotective effect of genistein is therefore mediated by ERβ The colocahzation of ERβ mRNA in in situ hybridization on the replicating/migratory smooth muscle cells dunng the rephcative and migratory bursts after endothehal trauma, is of relevance in explaining the results obtained First, on day 7 after denudation injury the endothehal regrowth from both ends of the vessel is only at its very beginning (Clowes AW, et al Lab Invest (1983); 49(3): 327-33) and the dose dependent inhibitory effect with both ligands may be discussed in terms of inhibition of smooth muscle cell replication, only Although the binding affinity of genistein to ER-β is only 20-fold better than 17β-estradιol, (the best pair of ligands at the moment to differentiate between the two receptors -Kuiper. G et al Endocrinology (1997), 138(3) 863-70), the anti-pro ferative effect and the effect on mtimal thickening vs. the uterotrophic effect were clearly different. In regard to mtimal thickening and in vivo replication after endothehal denudation, the linear plots show a slight advantage for genistein vs. 17-β-estradιol This advantage is also seen m the in vitro vascular smooth muscle cell inhibition studies However, at the tested dose range genistein displayed no uterogenic effect in vivo, whereas the uterogenic effect of 17-β-estradιol was clearly visible Thus, it can be calculated that the vasculoprotective effect of the estrogen is most likely mediated via ERβ This is clearly supported by the observation of Iafrati et al Nat Med (1997); 3(5): 545-8 where selective disruption of the ERα gene m mice did not have an effect on the vasculoprotective effect of estrogen Therefore, in summary, the inventors have demonstrated that ERβ is strongly upregulated in the vascular wall as a consequence of injury, whereas ERα remains expressed at constitutive low background level, only In situ hybridization demonstrated that ERβ mRNA co-localizes on the replicating and migrating vascular smooth muscle cells in the media and neointima suggesting that both genes are transcnbed and expressed in functional form This was tested by using two different ligands with approximately 20-fold affinity difference for ERα and ERβ, 17-β-estradιol and genistein These two ligands clearly differentiated the vasculoprotective vs uterogenic effect Withm the dose range tested both of the ligands demonstrated a dose-dependent vasculoprotective effect, whereas only 17-β-estradιol but not genistein demonstrated a dose-dependent uterotrophic effect Finally, the anti-prohferative effect of these two ligands on vascular smooth muscle cell cultures deriving from rat aorta was confirmed in vitro, a dose-dependent inhibition of smooth muscle cell proliferation was again observed. Taken together, the results presented in this study and previous observations strongly suggest that the vascular protective effect of estrogen is mediated predominantly or exclusively by ERβ. These results will enable the generation of vasculoprotective estrogen mimetics without classical uterotrophic side-effects. A first aspect of the invention provides a vasculoprotective composition comprising an ERβ hgand, preferably an agonist, although the compound may be Erβ antagonist In particular, post menopausal women suffer from an increased risk of vascular disease and heart disease and are therefore a target population for treatment with agonists of ERβ This was unexpected because it was possible that the vasculoprotective effect operates via the recently-discovered ERβ or by another hitherto unknown ER subtype Another possibility was that the desired vasculoprotective effect is obtained via modification of the signalling to the response elements of "vasculoprotective" genes via intermediary transcription factors, such as SRC-1, TIF-2, A1B1, or via ER-interactmg proteins, such as RIP 140, RIP 160, TIF 1, etc According to another aspect of the invention, there is provided a pharmaceutical composition useful for the treatment of vasculopathies compπsing an ERβ agonist According to another aspect of the invention, there is provided the use of an ERβ agonist in the treament of vasculopathies According to further aspects of the invention there are provided methods of treating, or preventing, a vasculopathy comprising treating a subject with an ERβ agonist, preferably an ERβ-selective agonist The vasculopathy may be a fibroproliferative condition For example, it may be a fibroproliferative condition such as restenosis, angioplasty, chronic allograft rejection, diabetic angiopathy, autoimmune angiopathy, atreπosclerosis, and atherosclerosis According to another aspect of the invention there is provided a method of inducing a vasculoprotective effect in a subject, the method compπsing contacting the subject with an ERβ agonist Alternatively, the vasculoprotective effect may be induced in cells, tissues such as blood vessels or organs. The cells tissues on which may have been produced in vitro, may than be placed in a subject. For example in a vasculoprotective effect may be induced and in vitro generated or seeded vascular grafts pπor to implantation in humans. The gene may be inserted by a virus. The vasculoprotective effect may be, for example, reducing intimal thickness. Preferably, the ERβ agonist is selective for ERβ. For example, it may have a binding affinity for ERβ which is at least 10 times, preferably at least 20 times, greater than for ERα According to a further aspect of the invention there is provided a method of producing artificial tissues or organs the method including the step of treating the tissue or organ with an ERβ agonist For example it is known to produce blood vessels by seeding cells, usually from the patient to receive the blood vessel about a former tube and growing those cells The invention embraces articificial tissues or organs obtainable by such a method. Methods and compositions m accordance with the invention will now be described, by way of example only, with reference to the accompanying drawings Figs 1 to 5. in which; Fig. 1 shows the results of in situ expression of ERα and ERβ at 15 mm and 7 days after injury Sense controls (not shown) were negative; Fig. 2 shows the results of ERα and ERβ expression at different time points, as quantitated as number of grams/400 μm. separately for media and neointima; Fig. 3 shows dose response plots of 17α estradiol and genistein on a 7 day denudation injury, as quantiated as number of intima nuclei, and on the uterotrophic effect, as quantitated as the 7 day weight of rat uteruses; Fig 4 shows a dose response plot of the effect of estradiol and genistein on the prohferative response of rat vascular smooth muscle cells after serum starvation and PDGF-stimulation in vitro, and Fig 5 gives details of antibodies used in Examples 1. Expression and localization of ERα and ERβ after carotid denudation trauma in male rats Expression and localization of ERα and ERβ was investigated by in situ hybπdisation from paraformaldehyde-fixed parrafm embedded specimens with sense controls Carotid denudations were made to Wistar rats purchased from the Laboratory Animal Center, University of Helsinki, Finland The rats were anaesthetized with 240 mg/kg chloral hydrate I p Buprenorphine (Temgesic, Reckitt Coleman, Hull, England) was given for pen- and postoperative pain relief Male and ovaπectomized female rats weighing 300-400g were used for all experiments A transverse incision of the neck was performed A full exposure of the carotid system was made by cleaving of the ventral edge of the left stemomastoid muscle and omohyoid muscles The proximal and distal control of the carotid artery was obtained with a l l mm micro vascular clip A 2-I , rench Fogarty balloon catheter (Baxter Healthcare Corp, Santa Ana, CA) was introduced into the common carotid artery through the left external carotid artery and inflated with 0 2 ml air To produce adequate vessel injury the catheter was passed 3 times, balloon inflated, through the common carotid artery The external carotid artery was hgated after removal of the catheter and the wound was closed Evaluation of histological changes was made from midcarotid sections at 0, 15 mm, 3 days, 7 days, 14 days and 30 days after denudation injury The carotids were removed "en block" and fixed in paraformaldehyde Histological specimens were fixed in 3% paraphormaldehyde solution for 4 hours, transferred to saline and embedded in paraffin The number of cell nuclei in the adventia, media and intima was quantitated from paraffin sections stained with Meyer's hematoxylm-eosin using 400x magnification For in situ hybridization, the left carotid of male rats was denuded of endothehum and the rats were sacπfied at 15 mm, 3 days, 7 days, 14 days and 28 days after injury, with a minimum 3 of rats per time point. The specimens of different time points and the non-denuded control specimen were placed on a single organosilane-treated microscopy slide and in situ hybridization was performed as descπbed below. 2. In situ hybridization After deparaffinization and rehydration. sections were denatured in 0 2 M HC1, heat-denatured in 2x Sahne-Socmm Citrate (SSC) at 70°C and treated with proteinase K (1 μg/ml) Sections were then post-fixed with 4% paraformaldehyde, acetylated with 0 25% acetic anhydride in 0 1 M tnethanolamine, dehydrated and air-dπed. Slides were hybπdized with antisense or sense RNA probes (described below) overnight at 60°C, washed in 4xSSC, treated with RNAse A (20 μg/ml) and washed sequentially in SSC solutions (2xSSC, lxSSC, 0.5xSSC, all at room temperature; O. lxSCC at 50°C; O. lxSCC at RT) with ImM DTT Finally, the slides were πnsed in O. lxSSC (with 1 mM DTT), dehydrated in graded ethanols and air-dried Slides were dipped into autography emulsion (NBT 3, Kodak), exposed for 7-14 days, developed, counterstained, dehydrated and mounted with Permount 3. Probe preparation The complementary RNA probes were synthesized according to manufacturer's directions (Promega, Madison, WI) in the presence of 35 S-UTP (Amersham, International. Willshire, UK) using following cDNA fragments as templates. For ERβ ; a 400 bp EcoRI-AccI fragment (from the 5'UTR region) of the rat ERβ cDNA subcloned in a pBluescπpt KS vector was linearized with EcoRI or Accl enzymes for the production of antisense and sense transcripts, respectively. For ERα, a 200 bp BstxI-EcoRI fragment (from the 3' UTR, F-domain region) of the rat ERα cDNA subcloned to a Bluescπpt vector was hneaπzed with Sad or EcoRI enzymes pπor to synthesis of antisense and sense probes, respectively. RNA probes transcπpted from opposite strands of the same plasmid template, yielding antisense and sense probes, were adjusted to the same specific radioactivity (minimum 10 000 cpm μl). Control non-denuded carotids were compared to denuded carotids removed at 15 mm, and 3, 7, 14, and 30 days post denudation. To ensure that the expression levels between different specimens were comparable, all tissue specimens were placed on one slide and hybπdized in identical conditions either for ERα or β. ERα and β mRNAs were expressed constitutively at low level in the vascular tunica media in normal non-denuded cartotids The level of expression of ERα mRNA remained unaltered throughout the experiment as can be seen from Figs 1 and 2 Figure 1 shows the in situ expression of ERα and ERβ m rat carotid seven days past denudation. Antisense RNA probes were used with the Lumen (L) facing up. Compared to specimens obtained 15 minutes post denudation, ERβ expression is strongly enhanced in the media (MED) and particularly in the vascular mtima (INT) whereas the level of ERα expression was not elevated Figure 2 shows the time course of the events. Male rats were denuded as previously descπbed, and the animals were sacrificed at the same points, I e 15 minutes, 3, 7, and 14 days post injury Three-fold up-regulation of ERβ mRNA was observed three days after denudation in the media and the level of expression in the media increased to 8-fold on day 7, whereafter it declined Even more prominent changes in the expression levels were observed in the hyperplastic mtima/neomtima. Whereas the ERα expression in the intima remained at the level observed in the control vessel media, or at most doubled, the level of ERβ expression in the mtima increased nearly 40-fold on day 7 (Fig. 2), whereafter it declined but remained elevated even after 14 days post injury 4. Dose responses to 17β-E2 and genistein on post denudation carotid trauma and on uterine weight in female rats. Female adult rats were ovariectomized on day -7 and carotid denudation was performed on day 0 and the animals were killed on day 7 (at the end of the experiment also the uterus was removed, weighed and histology was performed). Female rats were ovariectomized, placed on a soy-bean free diet (Special Diet Services, UK) (to eliminate effects of phytoestrogens from the diet) for 7 days and both carotids were denuded. One group of animals received 17β-estradiol (17β-E2) (Sigma, St Louis, MO) and the other group genistein (kindly donated by Dr. William Helferich, Michigan State University, or purchased from Plantech, UK) at reducing doses from 2.5 mg/kg/d s.c. downwards, whilst the third group received vehicle only and served as control. 17β-E2 and genistein were dissolved in dimethylsulphoxide (Sigma). Animals were weighed daily, and both drugs were administered subcutaneously (s.c.) using the following doses: 2.5, 0.25, 0.025 and 0.0025 mg/kg in one s.c. injection per day. The animals were killed at 7 days post injury, the uterus and both carotids were removed, the uterus was weighed and both organs were processed for histology as previously described. Ten denuded carotids of rats receiving only vehicle (DMSO 200 ml/kg/day) were compared to denuded carotids of rats receiving 17-β estradiol and to carotids of rats receiving genistein at escalating doses of 0.0025, 0.025, 0.25 and 2.5 mg/kg/d, three to five carotids at each dose level. Both 17β-E2 and genistein had a dose-dependent effect on nuclear number in mtima, but no measurable effect on the number of nuclei in the media (not shown). (Fig. 3). The line plots indicate that genistein might have been slightly more efficacious (r 2 0.838 vs 0.746) in its vasculoprotective effect. On the other hand, in the dose range employed, only 17-β estradiol displayed a dose-dependent stimulatory effect on uterine weight (r 2 0.954) while genistein had no effect (r 2 0.96) (Figure 3). 5. Effect on estradiol vs genistein on SMC replication in vivo An aqueous solution of 5-bromo-2'-deoxyuridine and 5-fluoro-2'deoxyuridine (Zymed Laboratories, Inc, San Francisco,CA) was used for labelling of proliferating cells after denudation. For "pulse labelling" a dose of 400 μl of labelling suspension was injected i.v. according to manufacturer's instructions, and the rats were killed exactly 3 h after the pulse. The carotids were fixed as described above and processed for paraffin embedding. BrdU stainings of cross sections were performed using a mouse primary antibody (Bu20a, Dako, A/S, Denmark) and Vectastain Elite ABC kit (Vector Laboratories, Burlingame, CA). Sections were deparaffinized and microwave-treated at 500 W for 2 x 5 min in 0.1 M citrate buffer, pH 6, followed by treatment in 95% formamide in 0.15 M tri-sodium citrate at 70°C for 45 min. Antibody dilutions were made according to manufacturer's instructions. Sections were counterstained with Mayers' haematoxylin and eosin, and the number of positive cells was counted separately from the intimal, medial and adventitial layers. Both of these ligands also reduced, dose dependently, the replication rate in the intima, as quantitated by the number of BrdU incorporating cells after pulse labelling of the rat. Table 1 shows the effect of E2 and genistein on the number of proliferating (BrdU-incorporating) cells in the vascular intima seven days after denudation injury.# No of BrdU incorporating cells Drug dose (mg/kg/d) 17β estradiol genistein Nil 36.7 + 7.5 0.0025 27.3 + 12.5 32.3 + 12. 0.025 6.0 + 3.8 6.0 + 3.8 2.5 10.5 + 2.9 12.3 + 3.7 # Animals received BrdU pulse 3 hours before sacrifice. 6. Dose-responses to 17β-E2 and genistein on vascular smooth muscle cell proliferation in vitro. As the results regarding the in vivo responses of genistem vs 17β-E2 to vascular trauma suggested a marginally better efficacy of genistem, this possibility was investigated further in vitro in the proliferation assays of vascular smooth muscle cells Rat thoracic aorta smooth muscle cells at 10-12 passage were plated in 96 well tissue culture plates on day -2, left to attach phenyl-red free RPMI 1640 and depπved from serum for additional two days On day 0, the cells were stimulated by 20 ng/ml Platelet-Deπved Growth Factor-B (PDGF-B) (Sigma), or left non-stimulated Genistein and 17-β estradiol were added to the cultures at the indicated concentrations on day -1, and all cultures were harvested on day 1 after a 24 hour 3 H-thymιdme (Η-TdR) pulse on day 1 As seen in Figure 4, both E2 and genistem displayed a dose-dependent anti-prohferative effect on baby rat smooth muscle cells in culture 7. Expression and localization of ERα AND ERβ in primates after carotid denudation trauma Because species differences may exist m gene expression, we wanted to confirm the rat results descπbed above in subhuman pπmates and also to extend the observations to other types of vasculopathies, particularly to allograft fibroproliferative vascular disease in a rat cardiac transplant model and to human cardiac transplantation Baboons are a particularly useful animal model as they have 98% sequence identity with the human genome Carotid/iliac denudations were performed to baboons Balloon catheter denudation of carotid artenes was performed in 8 male baboons (Papio ursmus) weighing 16-18 kg Additional 4 baboons served as non-operated controls.The animals were purchased from the Animal Laboratory of the Medical Faculty, University of Stellenbosch, South Africa. The baboons were sedated with ketamine hydrochloπde (5mg/kg EM) and anesthesia was induced with thiopental sodιum(5mg/kg IV) and maintained with inhaled halothane. Prophylactic cefazohn sodium (25mg/kg IM,Elι Lilly) was administered and the left common carotid artery was explored by vertical neck incision following the anteπor border of sternocleidomastoid muscle The carotid system was exposed in the carotid tπangle and proximal and distal control of the common carotid artery was obtained with small vascular clamps, just proximal to its bifurcation No hepaπn was administered. Through a small artenotomy, a 4-Fr Fogarty balloon catheter (Baxter Healthcare Corp, Santa Ana , CA) was introduced into the distal common carotid artery It was passed retrograde into the aortic arch and inflated with 1 7 ml of air resulting m a 1.5 lbs pull force and a balloon size of 9 mm when inflated The inflated balloon was then retπeved under tension while rotating the shaft of the catheter to produce uniform injury This was repeated three times to ensure sufficient arteπal denudation All procedures were done by the same individual The catheter was then removed; the artenotomy closed with interrupted 7-0 monofilament polypropylene sutures, and flow restored The wound was then closed in layers and buprenorphine (Temgesic, Reckitt, Coleman, Hull, England) 0.25mg/kg IV was given as required, for postoperative pam relief One baboon was sacπficed at each time point by administering a overdose of pentobarbitol (100 mg kg IV) and TV potassium chloπde. Three minutes before extermination, standard hepaπn (300U/kg IV) was given Both carotid arteries were then removed and evaluation of histological changes was madefrom midcarotid sections at 0,15 mm and at 2,3,4,7,14 and 28 days post injury Pieces of rat uterus were obtained for ER-α specificity controls All animals received humane care in compliance with guidelines set forth by the National Institutes of Health, publication No.86-23,Guιde for the Care and Use of Laboratory Animals, and the project was approved by the Ethical Committee of the Faculty of Medicine of the University of Stellenbosch, South Africa Immunohistochemistry was made from paraffin cross sections using a mouse or rabbit pπmary antibody and Vectastain Elite ABC kit (Vector Laboratoπes, Burhngame, CA). Sections were deparaffinized and microwave-treated at 500 W for 2 x 5 mm in 0 1 M citrate buffer, pH 6, followed by treatment in 95%> formamide in 0.15 M tπ-sodium citrate at +70°C for 45 min. Antibody dilutions were made according to manufacturers instructions. Sections were counterstained with Mayers haematoxylin and eosin, and the total number of positive cells was counted separately from the intimal, medial and adventitial layers using 400x magnification. At least 5 sections were investigated separately of each carotid and the specimen with median intensity of intimal changes was counted. In the baboon carotid/iliac denudation model 4 baboons were sacrificed w/o injury and at least 1 baboon was sacrificed at 15 min, 2, 3, 4, 7, 14 and 28 d time points post injury. ERβ was found as exclusive receptor in baboon arteries and ERα in baboon uterus. After injury, the intensity of immunohistochemical staining with all three commercial antibodies for ERβ increased considerably and staining colocalized with the vascular SMC. There was no staining with ERα antibodies though the control uterus stained strongly. ERβ was the only ER located in the baboon ateries found by immunohistochemistry. Specifically tissue samples were contacted with commercially-available ERβ or ERα-selective antibodies. The staining colocalized with vascular SMC. 8. Analysis of ERα and ERβ expression in rat and human allograft vessels. In rat and human heart allograft vessels ERβ is the exclusive receptor and ERα in uterus. During acute rejection, the ERβ was shown to be strongly upregulated (same panel of antibodies) and ERα remain non-existent. Human specimens were obtained from routine enbdomyocardial biopsies performed for diagnostic purposes to U of Helsinki Hospital heart transplant receipients. The biopsies represented no rejection, acute rejections of different histological intensities and of chronic rejection, least 10 specimens per group. Rat heart transplants were made to abdominal vessels as descnbed (Lemstrom, K., Sihvola, R., Bruggeman C, Hayry, P., and Koskmen, P. abolished by DHPG prophylaxis m the rat. Circulation 1997:95:2614-2616). Specimens for immunohistochemistry were obtained as descnbed above in relation to baboons. and following antibodies shown in Fig. 5 were employed, with consistent results: In rat and human heart allograft vessels ERβ was also found to be the exclusive receptor and ERα dominated in uterus. Duπng acute rejection, the staining for ERβ was strongly upregulated (same panel of antibodies) and the ERα reactivity remain non-existent Taken together, the expression patterns of ERβ vs ERα in subhuman pnmates and in human and rat allograft models entirely agree with the rat carotid results descπbed in the application and demonstrate ERβ as the exclusive estrogen receptor in arteπal tissue and vascular SMC making this a ideal target for drug therapies

Claims: Claims 1. A vasculoprotective composition comprising an ERβ ligand. 2. A vasculoprotective composition according to claim 1 wherein the ERβ ligand is an ERβ agonist. 3. A vasculoprotective composition according to claim 1 wherein the ERβ ligand is an ERβ antagonist 4. A vasculoprotective composition according to claim 1 or claim 2 comprising an ERβ-selective agonist. 5. A pharmaceutical composition useful for the treatment of vasculopathies comprising an ERβ agonist. 6. A pharmaceutical composition according to claim 5 comprising an ERβ-selective agonist. 7. A composition according to claim 4 or 6 in which the binding affinity of the ERβ agonist to ERβ is at least 10 times greater than the binding affinity to ERα. 8. A composition according to claim 7 in which the binding affinity of the agonist to ERβ is at least 20 times greater than to ERα. 9. The use of an ERβ agonist in the treatment of vasculopathies. 10. The use of an ERβ-selective agonist in the treatment of vasculopathies. 11. The use according to claim 10 in which the vasculopathy is a fibroproliferative condition. 12. The use according to claim 11 in which the fibroproliferative vasculopathy is selected from restenosis, angioplasty, chronic allograft rejection, diabetic angiopathy, autoimmune angiopathy, arteriosclerosis, and atherosclerosis. 13. A method of inducing a vasculoprotective effect in a subject, the method comprising treating the subject with an ERβ agonist. 14. A method of inducing a vasculoprotective effect according to claim 13 in which the ERβ agonist has a higher affinity for ERβ than ERα. 15. A method of inducing a vasculoprotective effect in a subject according to claim 14 in which the binding affinity of the agonist to ERβ is at least 10 times greater than to ERα. 16. A method of inducing a vasculoprotective effect in a subject according to claim 15 in which the binding affinity of the agonist to ERβ is at least 20 times greater than to ERα. 17. A method of inducing a vasculoprotective effect in which the effect is decrease of intimal thickness. 18. A method according to any one of claims 13 to 17 in which the vasculoprotective effect is induced to treat a fibroproliferative vasculopathy. 19. A method according to claim 18 in which the fibroproliferative vasculopathy is selected from restenosis, angioplasty, chronic allograft rejection, diabetic angiopathy, autoimmune angiopathy, ateriosclerosis and atherosclerosis. 20. A composition, use or method according to any preceding claim in which the ERβ selective agonist is genistein or a chemical derivative or structural analogue thereof. 21. A use or method according to any one of claims 9 to 20 in which uterotrophic effects are minimised or do not result. 22. A method according to any one of claims 13 to 21 in which the subject is a mammal. 23. A method according to claim 22 in which the mammal is a primate. 24. A method according to claim 23 in which the mammal is human. 25. A method according to claim 22, 23 or 24 in which the mammal is female. 26. A method according to claim 25 in which the female is post-menopausal. 27. A method of producing artificial tissues or organs the method including the step of treating the tissue or organ with an ERβ agonist. 28. A method according to claim 27 in which the tissue or organ is a blood vessel. 29. Articificial tissues or organs obtainable by a method according to claim 27 or 28.

Inventors: GUSTAFSSON JAN-AKE, HAYRY PEKKA, MAKELA SARI, GUSTAFSSON, JAN-AKE, HAYRY, PEKKA, MAKELA, SARI
Applicants: DEAN JOHN PAUL, GUSTAFSSON JAN AKE, HAYRY PEKKA, KAROBIO AB, MAKELA SARI, DEAN, JOHN, PAUL, GUSTAFSSON, JAN-AKE, HAYRY, PEKKA, KARO BIO AB, MAKELA, SARI
Application year: 1999
Main classification: C07K14
IPC codes: A61K31, A61K45, A61P9, A61P37, C07D471, C12N5