Pharmaceutical compositions comprising lignans and their derivatives for treating hyperplastic diseases (2025)

U.S. patent number 9,669,000 [Application Number 14/282,941] was granted by the patent office on 2017-06-06 for pharmaceutical compositions comprising lignans and their derivatives for treating hyperplastic diseases. This patent grant is currently assigned to Universitat Innsbruck. The grantee listed for this patent is Universitat Innsbruck. Invention is credited to David Bernhard, Gunther Laufer, Stefan Schwaiger, Hermann Stuppner.

Pharmaceutical compositions comprising lignans and theirderivatives for treating hyperplastic diseases

Abstract

The present invention relates to a pharmaceutical compositioncomprising specific compounds which may be obtained fromLeontopodium alpinum Cass. (Edelweiss). A preferred compound isleoligin(=(2S,3R,4R)-4-(3,4-dimethoxybenzyl)-2-(3,4-dimethoxyphenyl)tetrahydrofur-an-3-yl]methyl(2Z)-2-methylbut-2-enoat]). Corresponding means andmethods in respect of medical uses of the compounds are described.The present invention also provides a medical device comprising,containing or having been contacted with the compound. Thecompounds provided herein may particularly be used in the treatmentof hyperplastic diseases, in particular intimal hyperplasia, e.g.stenosis, restenosis, atherosclerosis and the like. Also envisagedherein is the use of these compounds in the treatment ofproliferative diseases, such as leukemia, prostate cancer and lungcancer.

Prior Publication Data

Related U.S. Patent Documents

References Cited [Referenced By]

U.S. Patent Documents

Foreign Patent Documents

Other References

Primary Examiner: Carter; Kendra D
Attorney, Agent or Firm: Parker Highlander PLLC

Parent Case Text

The present application is a continuation of U.S. application Ser.No. 13/054,385, filed Apr. 19, 2011, which is a national phaseapplication under 35 U.S.C. .sctn.371 of International ApplicationNo. PCT/EP2009/059256, filed Jul. 17, 2009, which claims benefit ofpriority to U.S. Provisional Application No. 61/135,287, filed Jul.18, 2008. The entire text of each of the above referenceddisclosures is specifically incorporated herein by reference.

Claims

The invention claimed is:

1. A method for treating or ameliorating intimal hyperplasiacomprising the administration of a compound of formula (I) to asubject in an amount effective to treat or ameliorate said intimalhyperplasia: ##STR00007## wherein: R.sup.1, R.sup.2 and R.sup.3 areindependently selected from H, OH, halogen, alkyl, or alkoxy; andR.sup.4, R.sup.5 and R.sup.6 are independently selected from H, OH,halogen, alkyl, or alkoxy; R.sup.7 is --OC(O)R.sup.9 or--C(O)OR.sup.9; wherein R.sup.9 is alkyl or alkenyl; and whereinany alkyl or alkenyl group comprised in R.sup.7 may beunsubstituted or substituted by one or more substituents, selectedfrom OH, halogen or alkoxy; and X is O or S; and the dashed linesin the ring structure containing the group X indicate that therespective bond is a single bond; or any pharmaceuticallyacceptable salt or solvate thereof.

2. The method of claim 1, wherein the compound of formula (I) hasthe stereochemistry indicated in formula (Ia): ##STR00008## whereinR.sup.1 to R.sup.7 and X are defined as in claim 1.

3. The method of claim 1, wherein at least one of R.sup.1, R.sup.2and R.sup.3 and at least one of R.sup.4, R.sup.5 and R.sup.6 is analkoxy group.

4. The method of claim 1, wherein at least two of R.sup.1, R.sup.2and R.sup.3 and at least two of R.sup.4, R.sup.5 and R.sup.6 arealkoxy groups.

5. The method of claim 1, wherein the compound of formula (I) hasthe following structure: ##STR00009##

6. The method of claim 1, wherein five or all six of R.sup.1 toR.sup.6 are alkoxy, and the remaining group of R.sup.1 to R.sup.6,if applicable, is hydrogen.

7. The method of claim 1, wherein said intimal hyperplasia isstenosis or restenosis.

8. The method of claim 1, wherein said intimal hyperplasia isatherosclerosis.

9. The method of claim 1, whereby said compound is administered byany one of a parenteral route, oral route, intravenous route,subcutaneous route, intranasal route or transdermal route.

10. The method of claim 1, wherein said subject is a human.

Description

BACKGROUND OF THE INVENTION

The present invention relates to a pharmaceutical compositioncomprising specific compounds which may be obtained fromLeontopodium alpinum Cass. (Edelweiss), or structurally relatedcompounds. A preferred compound is leoligin(=(2S,3R,4R)-4-(3,4-dimethoxybenzyl)-2-(3,4-dimethoxyphenyl)tetr-ahydrofuran-3-yl]methyl (2Z)-2-methylbut-2-enoat]). Correspondingmeans and methods in respect of medical uses of the compounds aredescribed. The present invention also provides a medical devicecomprising, containing or having been contacted with the compound.The compounds provided herein may particularly be used in thetreatment of hyperplastic diseases, in particular intimalhyperplasia, e.g. stenosis, restenosis, atherosclerosis and thelike. Also envisaged herein is the use of these compounds in thetreatment of proliferative diseases, such as leukaemia, prostatecancer and lung cancer.

Coronary artery bypass grafting (CABG) and percutaneous coronaryintervention (PCI) are the two invasive options to treat coronaryartery disease (CAD), being one of the leading causes of morbidityand mortality worldwide; see WHO, Cardiovascular diseases, InternetCommunication (2007); see alsowww.who.int/cardiovascular_diseases/en/. The success of boththerapeutic approaches is however often limited by restenosis andgraft failure which are considered as hyperplasticdiseases/disorders. With respect to graft patency rates after CABGthe vessels of choice are clearly the internal mammary arteries;see Tatoulis, Ann Thorac Surg, 77(1), 93-101 (2004). However due tolimitations in availability saphenous vein grafts are morefrequently used in CABG than arterial grafts (e.g. in 2004 at theInnsbruck Medical University 51% of bypass grafts were saphenousveins; see Schachner, European Surgery 39(2), 72-5 (2007). In pastyears clinical optimization, like graft handling (e.g. "no touchtechniques") and lipid lowering therapy has impressively increasedthe patency rates of saphenous vein conduits and is currentlyapproximately at 60% 10 years after CABG (Schachner (2007) loc.cit; Lau, Semin Vasc Med 4(2), 153-9 (2004); Tsui, Eur J VascEndovasc Surg 23(3), 202-8 (2002). Still, the major reasons for aloss of patency at earlier time points are thromboses, neointimaformation, and intimal hyperplasia, (10-20% loss of patency afterthe first year), and graft atherosclerosis later after CABG (Lau(2004), loc. cit.; Hozumi, Heart 76(4), 317-20 (1996); Marin, JVasc Surg 18(3):407-14 (1993). Thus, graft disease stillsignificantly limits the durability of venous bypasses.

Generally, the causative factors and the pathophysiologicalprocesses that underlie vein graft disease are not well understood.It is thought that vein graft disease is a result of a variety ofevents initiated by vascular damage that does occur due to surgicalhandling, ischemia, and arterialisation (blood pressure, bloodflow) of grafts. This initial damage is though to provoke adaptiverepair processes in the vessel wall, like tissue remodelling(positive and negative) and intimal hyperplasia; see Lau (2004),loc. cit., Hozumi (1996), loc. cit., Marin (1993), loc. cit; Lau,Circulation 4, 114(1 Suppl):1435-1440 (2006). On one hand thisresponse is vital for the adaptation of the graft to the arterialenvironment, but an excessive response is thought to give raise tograft disease that ultimately results in graft failure.

Despite a complex array of intra and inter cellular signallingevents in the development of graft disease after CABG and/or PCIthe core elements on the histological level are endothelial damage(denudation) and smooth muscle cell (SMC) proliferation andinfiltration of the intima. Pro-inflammatory signalling due totissue damage and cellular necrosis but also as an element ofadaptive tissue remodelling is another highly relevant factor; seeMitra, Immunol Cell Biol 84(2), 115-24 (2006). Although, theexcellent concept of using drug eluting stents/matrices instead ofpurely mechanical devices will most likely prevail in PCI- andCABG-based prevention of restenosis and graft failure, at themoment there is a significant lack of functional drugs, screened ordesigned precisely for these applications. Currently used drugs aremainly chemotherapeutic agents developed for cancer orimmunosuppressive therapy, which may be too aggressive orunspecific for the treatment of restenosis and graft disease, sincealso endothelial healing--important for the prevention ofthromboses--is impaired by these drugs.

Lignans are considered as potential candidate molecules which maybe used in the treatment of diseases/disorders associated with thecardiovascular system and will be discussed herein below in moredetail. However, only a limited number of publications havereported on the impact of lignans on the cardiovascular system ingeneral, and only a few different lignans have been tested so far.It is of note that a treatment of hyperplastic diseases/disorderswith lignans has not been described in the art. The existing datarather suggest that lignans are cardiovascular protective agentswith lipid lowering, anti-oxidative, anti-hypertensive,anti-thrombotic, and anti-inflammatory activities.

A large number of lignan-based cancer therapy studies (in vitro andin vivo) showed profound cytotoxicity and cell death induction bythese compounds, see Kim Planta Med, 68(3), 271-4 (2002) and Lin JCell Biochem 84(3), 532-44 (2002). The use of cytotoxic compoundsin the treatment of a hyperplastic disease/disorder, and inparticular vein graft disease, is generally considered asdetrimental since also healthy cells, such as EC cells can bedamaged. Hence, the use of cytotoxic lignans known in the artshould be avoided in the treatment of these diseases. Therefore,there is still a demand for compounds which may be used in thetreatment of hyperplastic diseases/disorders and which avoid thedisadvantages of compounds known in the art.

SUMMARY OF THE INVENTION

Thus, the technical problem underlying the present invention is theprovision means and methods for the medical interference inhyperplastic diseases or hyperplastic disorders.

The technical problem is solved by provision of the embodimentscharacterized in the claims.

Accordingly, the present invention relates to a pharmaceuticalcomposition comprising a compound of formula (I)

##STR00001##

wherein R.sup.1, R.sup.2 and R.sup.3 are independently selectedfrom H, OH, halogen, alkyl, or alkoxy; and R.sup.4, R.sup.5 andR.sup.6 are independently selected from H, OH, halogen, alkyl, oralkoxy; R.sup.7 is selected from --OR.sup.8, --N(R.sup.8')R.sup.8,--SR.sup.8, --C(O)R.sup.8, --OC(O)R.sup.9, --C(O)OR.sup.9,--N(R.sup.9')C(O)R.sup.9, --C(O)N(R.sup.9')R.sup.9 or--S(O)R.sup.9; wherein R.sup.8 and R.sup.9 are independentlyselected from alkyl or alkenyl and R.sup.8' and R.sup.9' areindependently selected from H, alkyl or alkenyl; and wherein anyalkyl or alkenyl group comprised in R.sup.7 may be unsubstituted orsubstituted by one or more substituents, selected from OH, halogenor alkoxy; X is selected from O, S, C(R.sup.10)R.sup.10 andNR.sup.10 wherein R.sup.10 is H, alkyl or alkenyl; and the dashedlines in the ring structure containing group X indicate that therespective bond may be a single or a double bond; or anypharmaceutically acceptable salt or solvate thereof.

In a preferred embodiment, the compound of formula (I) comprised inthe pharmaceutical composition has the stereochemistry indicated informula (Ia):

##STR00002##

wherein R.sup.1 to R.sup.7 and X are defined as described hereinabove.

For the above formulae (I) and (Ia), the following embodiments arepreferred in the context of the invention.

Alkyl substituents, as they may be present as R.sup.1 to R.sup.6,are preferably C1 to C6 alkyl groups, more strongly preferred areC1 to C3 alkyl groups, and further preferred is methyl.

Halogen substituents include fluoro-, chloro-, bromo- andiodo-atoms, with preference given to chloro and bromo.

As set out above, X is selected from O, S, C(R.sup.10)R.sup.10 andNR.sup.10; wherein R.sup.10, independently for each occurrence, isH, alkyl or alkenyl. Preferred as alkyl group is a C1 to C6 alkylgroup, particularly preferred are methyl and ethyl. Preferred as analkenyl group is a C2 to C6 alkenyl group.

Preferably, X is O or NR.sup.10, and particularly preferred is O.Preferred groups R.sup.10 are H and C1 to C6 alkyl, particularlypreferred are H and methyl.

As further explained above, the dashed lines in the ring structurecontaining group X indicates that the respective bond may be asingle or a double bond. The ring structure may contain no doublebond, one double bond or two double bonds at the respectiveposition. Preferred are cases where no double bond is present, i.e.the ring structure containing group X is a saturated ring.

It is generally preferred that at least one of R.sup.1 to R.sup.3represents an alkoxy group, and it is more preferred that two orall three of them represent an alkoxy group. Among suitable alkoxygroups, general preference is given to C1 to C6 alkoxy groups, morestrongly preferred are C1 to C3 alkoxy groups and particularpreference is given to methoxy groups. If two of R.sup.1 to R.sup.3represent an alkoxy group, it is preferred that one of them isR.sup.2.

In the preferred compounds referred to above, wherein one or two ofR.sup.1 to R.sup.3 represent an alkoxy group, it is furtherpreferred that the remaining groups of R.sup.1 to R.sup.3 representH or an alkyl group, preferably H. Preferred alkyl groups are C1 toC6 alkyl groups, more strongly preferred are C1 to C3 alkyl groups,and further preferred is methyl.

It is generally preferred that at least one of R.sup.4 to R.sup.6represents an alkoxy group, and it is more preferred that two orall three of them represent an alkoxy group. Among suitable alkoxygroups, general preference is given to C1 to C6 alkoxy groups, morestrongly preferred are C1 to C3 alkoxy groups and particularpreference is given to methoxy groups. If two of R.sup.4 to R.sup.6represent an alkoxy group, it is preferred that one of them isR.sup.5.

In the preferred compounds referred to above, wherein one or two ofR.sup.4 to R.sup.6 represent an alkoxy group, it is furtherpreferred that the remaining groups of R.sup.4 to R.sup.6 representH or an alkyl group, preferably H. Preferred alkyl groups are C1 toC6 alkyl groups, more strongly preferred are C1 to C3 alkyl groups,and further preferred is methyl.

Thus, particularly preferred are compounds wherein four, five orall six of R.sup.1 to R.sup.6 are alkoxy, and the remaining groupsof R.sup.1 to R.sup.6, if any, are hydrogen. Mention may be made inthis respect specifically of compounds wherein R.sup.1 is H andR.sup.2 and R.sup.3 are alkoxy, or all of R.sup.1 to R.sup.3 arealkoxy; and wherein R.sup.4 is H and R.sup.5 and R.sup.6 arealkoxy, or all of R.sup.4 to R.sup.6 are alkoxy. Among suitablealkoxy groups, general preference is given to C1 to C6 alkoxygroups, more strongly preferred are C1 to C3 alkoxy groups andparticular preference is given to methoxy groups.

R.sup.7 is preferably --OC(O)R.sup.9, --C(O)OR.sup.9,--N(R.sup.9')C(O)R.sup.9, --C(O)N(R.sup.9')R.sup.9 or--S(O)R.sup.9, i.e. an ester, amide or sulfoxy group, with aparticular preference for the ester groups --OC(O)R.sup.9or

--C(O)OR.sup.9. Most preferred as R.sup.7 is a group--OC(O)R.sup.9.

R.sup.8 is preferably an alkyl or alkenyl group which isunsubstituted. Preferred alkyl groups have 2 or more, particularly3 or more carbon atoms. It is further preferred that they have 14or less, such as 10 or less, particularly 8 or less or 6 or lesscarbon atoms. Preferred alkenyl groups have 3 or more carbon atoms.It is further preferred that they have 14 or less, such as 10 orless, particularly 8 or less or 6 or less carbon atoms. Independentof the number of carbon atoms, it is preferred that the alkenylgroups have one C--C double bond.

R.sup.8' is preferably H or any alkyl group having 10 or less, suchas 8 or less, preferably 6 or less carbon atoms, such as methyl,ethyl, or propyl.

R.sup.9 is preferably an alkyl or alkenyl group which isunsubstituted. Preferred alkyl groups have 2 or more, particularly3 or more carbon atoms. It is further preferred that they have 14or less, such as 10 or less, particularly 8 or less or 6 or lesscarbon atoms. Preferred alkenyl groups have 3 or more carbon atoms.It is further preferred that they have 14 or less, such as 10 orless, particularly 8 or less or 6 or less carbon atoms.Independently of the number of carbon atoms, it is preferable thatthe alkenyl groups have one C--C double bond. Particularlypreferred as R.sup.9 is a branched alkenyl group as it occurs inleoligin of the formula --C(CH.sub.3)CH--CH.sub.3. In this group,the methyl substituents at the double bond may be in E- orZ-configuration with respect to each other, with preference for theZ-configuration.

R.sup.9' is preferably H or any alkyl group having 10 or less, suchas 8 or less, preferably 6 or less carbon atoms, such as methyl,ethyl, or propyl.

In a strongly preferred embodiment, the present invention concernspharmaceutical compositions comprising compounds of formula (1) or(1a) wherein X is O; wherein, in the case of formula (1), the ringstructure containing X has no double bonds; wherein four, five orall six of R.sup.1 to R.sup.6 are alkoxy, and the remaining groupsof R.sup.1 to R.sup.6, if any, are hydrogen; R.sup.7 is--OC(O)R.sup.9 or --C(O)OR.sup.9, particularly --OC(O)R.sup.9; andR.sup.9 is an unsubstituted alkenyl group having one double bondand 8 or less carbon atoms or an unsubstituted alkyl group havingtwo or more and 8 or less carbon atoms.

While the invention has been described as relating topharmaceutical compositions, it should be understood that thecompounds contained in these pharmaceutical compositions as activeagents play an important role in the context of the invention.Thus, the claims also encompass preferred compounds per se, such asthe compounds of formula (1) or (1a) defined above, wherein five orall six of R.sup.1 to R.sup.6 are alkoxy; the remaining group ofR.sup.1 to R.sup.6, if applicable, is hydrogen; and the ringstructure containing X, the group X and R.sup.7 are as definedabove, including preferred embodiments thereof.

A further strongly preferred embodiment relates to a pharmaceuticalcomposition, wherein the compound of formula (I) has the followingstructure:

##STR00003##

The chemical structure given herein above is(2S,3R,4R)-4-(3,4-dimethoxybenzyl)-2-(3,4-dimethoxyphenyl)tetrahydrofuran--3-yl]methyl (2Z)-2-methylbut-2-enoat] also commonly known underthe trivial name "leoligin". Leoligin has been shown in theappended examples as a particularly strong inhibitor of cellproliferation, in particular proliferation of SMCs. It is knownthat proliferation of SMCs is a central mechanism specificallyinvolved in a hyperplastic disease/disorder, in particular intimalhyperplasia. Proliferation of SMCs is also involved in vein graftdisease, which will be described herein below in more detail.

The present invention solves the above identified technical problemsince, as documented herein below and in the appended examples, itwas surprisingly found that a lignan derived from the roots ofEdelweiss (Leontopodium alpinum Cass.), namely leoligin[(2S,3R,4R)-4-(3,4-dimethoxybenzyl)-2-(3,4-dimethoxyphenyl)tetrahydrofura-n-3-yl]methyl (2Z)-2-methylbut-2-enoat] and derivatives thereofexhibit a highly beneficial effect in a medical setting. Leoliginhas been shown herein to be a stronger inhibitor of vascular smoothmuscle cell (SMC) proliferation compared to other compounds knownin the art, such as lariciresinol (see in particular Example 2 andFIG. 6; formula given herein below).

##STR00004##

Isolated primary human vascular smooth muscle cells represent thecentral cell type in intimal thickening and intimal hyperplasia.Thus, SMC proliferation and migration is a central mechanismunderlying hyperplastic diseases/disorders and inhibitors of thismechanism may interfere with the development and/or progression oftheses diseases.

Another, even more surprising finding was that not only leoliginbut compounds of formula (I) in general and leoligin derivatives asdescribed herein inhibit vascular smooth muscle cell (SMC)proliferation. Some derivatives of leoligin, such as the 5-methoxy-and 5,5'-dimethoxy-derivative, inhibit SMC proliferation at acomparable or at even lower concentration than leoligin, seeExample 3 and FIG. 7. In contrast thereto, lariciresinol(IC.sub.50>100 .mu.M) induced a weak inhibition of SMC whencompared to leoligin (IC.sub.50=54.5 .mu.M), and its5-methoxy-derivatives, e.g. 5-methoxy-leoligin (IC.sub.50=45.9.mu.M) and 5,5'-dimethoxy-derivatives, e.g. 5,5'-dimethoxy-leoligin(IC.sub.50=48.6 .mu.M) after 72 h. Without being bound by theory,it is believed that the methoxy-groups may contribute to anincrease in lipophilicity of the compounds to be used in accordancewith the present invention, thus possibly enhancing and/orfacilitating their cellular uptake. This may be one reason why e.g.5-methoxy-derivatives of leoligin can be used at comparable orlower concentrations than leoligin.

Structural formulas of exemplary methoxy-derivatives anddimethoxy-derivatives, which also represent preferred compounds inthe context of the present invention, are given herein below:

##STR00005##

A further advantage of the compound comprised in the pharmaceuticalcomposition of the present invention, in particular leoligin andderivatives thereof, is its property not to induce cell death inSMCs, and most importantly also not in endothelial cells (ECs).Endothelial cells form a thin layer of cells, the so calledendothelium, that line the interior surface of blood vessels. Theendothelium forms an interface between the circulating blood andthe rest of the vessel wall. Proper endothelial function isessential for blood vessel integrity and loss of its function is ahallmark for vascular diseases.

The property of the compounds to be used in accordance with thepresent invention not to induce cell death is in strong contrast tocompounds known in the art, where cytotoxic effects were observed,e.g. the lignans honokiol and magnolol described herein below. Incontrast, the compounds provided herein represent lignans whichdiffer from known lignans by the lack of toxicity and celldeath-inducing activity and also by their cell cycle inhibitoryactivity. The compounds of the present invention are therefore ofparticular advantage in the treatment of (a) hyperplasticdisease(s)/disorder(s).

In the appended experimental section herein below it is shown thatthe compounds of the present invention comprised in apharmaceutical composition can successfully be used as inhibitor ofintimal hyperplasia in a human saphenous vein organ culture modelfor graft disease. The compounds and in particular leoligin,potently inhibited intimal hyperplasia, and even reversed graftdisease in pre-damaged vessels. In a mouse model for venous bypassgraft disease leoligin potently inhibited intimal hyperplasia invivo, and had no negative effect on the integrity of the vascularendothelium. Such an assessment can also be carried out in largeranimals/animal models. An exemplary protocol is provided in theexperimental section herein below. In particular, an exemplaryprotocol using a porcine animal model (i.e. "Landschwein") toassess the efficacy of the particular compound known under thetrivial name "Leoligin" is given in the appended examples. Pigs(such as the well-known "Landschwein", a particular pig race) are apreferred animal model of (bypass-) intimal hyperplasia andstenosis to be used in context of the present invention, since thecirculatory system of pigs is very similar to that of humans. Aperson skilled in the art is readily in the position to adapt thisprotocol (e.g. to compounds of formula (I), in particular to (a)(di)methoxy-derivative(s) of leoligin (in various concentrations)or to other large animal models) and assess that compounds offormula (I) as described herein inhibit intimal hyperplasia also inlarge animals in vivo. It is apparent from the above that resultsobtained in pigs can, to a large extent, be extrapolated tohumans.

The following parameters/effects define independently of each othera treatment success evaluated for example by the above animalmodels (e.g. porcine model) of compounds of formula (I), inparticular Leoligin (and/or its ((di)methoxy-)derivative(s): 1) Anintima thickness and/or intima-media thickness of the treatmentgroup below the control group. 2) A smaller number of smooth musclecells in the intima of the treatment group compared to the control.3) The presence of a higher number of p27 and or p21 positive cellsin the treatment group compared to the control. 4) A smaller degreeof neointima formation in the treatment group compared to thecontrol. 5) A reduced presence of tissue remodelling processes inthe treatment group compared to the control. 6) A lower number ofpro-inflammatory cells in the vessel wall in the treatment groupcompared to the control. 7) An intact endothelium. 8) Aphysiological degree of contractility of the vessels. 9) A lowdegree of adhesion molecule expression on the endothelial surface.10) patency of grafts and no signs of thrombus formation. 10)Conserved elasticity of grafts in the treatment group compared tothe control. And 11) Conserved contractility of grafts in thetreatment group compared to the control.

In the prevention of vein graft failure after CABG the majortherapeutic targets are neointima formation and intimal hyperplasia(early to intermediate complications) as well as graftatherosclerosis (long term complication). Although some progresshas been made in past years a major limit in current strategies isthe lack of appropriate compounds, as mentioned above. Most of theapplied agents are too aggressive, and often cause not only SMCapoptosis and inhibition of proliferation, but also significantlyreduce endothelial viability. Since the closure of the vascularendothelium after PCI or CABG is very important in vascular healingand anti-thrombosis, compounds that are not toxic for ECs areinteresting agents for intra- and extravascular drug eluting stentsand matrices. The compounds provided herein and in particularleoligin have exactly this active profile. Although leoligininhibits EC proliferation (see FIG. 4), which may reduce localendothelial healing, a wound repair via the circulation (ECprecursors and circulating ECs) is possible (see FIG. 5). Thereason for this in vivo observation may be due to the fact thatleoligin is not toxic for ECs, which could facilitate are-colonisation of denuded vessel areas, and consequently reducethromboses. Compounds of formula (I) and, in particular leoliginand ((di)methoxy)derivatives thereof may be advantageously used inthe treatment, prevention and amelioration of (a) hyperplysticdisease(s)/disorder(s), in particular intimal hyperplasia andthromboses in CABG and PCI.

In sum, it has been surprisingly found in the context of thepresent invention that compounds of formula (I) as described hereinabove, such as leoligin and ((di)methoxy)derivatives thereof, cansuccessfully be used in a medical setting for the inhibition ofcellular proliferation, in particular the proliferation of SMCs. Incontrast to compounds known in the art, the compounds of thepresent invention are non-toxic and can be used at lowconcentrations. Thus, the pharmaceutical composition of the presentinvention comprising these compounds is particularly useful in thetreatment of hyperplastic diseases/disorders, in particular intimalhyperplasia, such as vein graft disease. The compounds may--due totheir EC preserving character--also be applied in drug elutingstents.

Such a beneficial use of these compounds in a medical setting hasnot been described in the art, even though anti-angiogenic activityof some lignans was reported; see Bai, J Biol Chem 278(37), 35501-7(2003) and Bergman Clin Cancer Res 13(3), 1061-7 (2007). However,these lignans are structurally different from the compoundsprovided in the present invention.

For example, lignans to be used in the prior art are honokiol andmagnolol which are derived from Magnolia species. The respectiveformulas are given herein below:

##STR00006##

It is evident from these formulas that neither honokiol normagnolol have a structural similarity to the compound to be used incontext of the present invention. Honokiol, a lignan constituent ofthe plant Magnolia officinalis, was shown to inhibit cell death inECs but also to potentiate cell death in vivo and in vitro; seeZhang, Eur J Pharmacol, 554(1), 1-7 (2007) and Ahn, Mol Cancer Res4(9), 621-33 (2006). Honokiol was further shown to caused an arrestin the G1 phase of the cell cycle in SMCs which was associated byan upregulation of p21/WAF1; see Lee (2006), loc. cit. Magnolol,another lignan isolated from Magnolia officinalis was shown toinduce cell death in SMCs in a capase-dependent manner and also toinhibit TNFalpha-mediated VCAM-1 expression as well as to preventIL-6-induced STAT3 expression in ECs; see Chen, NaunynSchmiedebergs Arch Pharmacol 368(2), 127-33 (2003); Chen, Br JPharmacol 135(1), 37-47 (2002); Chen Br J Pharmacol 148(2), 226-32(2006). A study by Razuvaev (J Vasc Surg 46(1), 108-15 (2007))reported that the cyclolignan picropodophyllin inhibits intimalhyperplasia after balloon injury in vivo via an interaction withIGF-receptor and ERK signalling. However, a potential toxicity ofpicropodophyllin on endothelial cells has not been tested. A fewother lignan type compounds, like flax seed lignans have beentested in different model systems of inflammation, cancer, andcardiovascular diseases. Due to a lack of knowledge concerningspecificities and characteristics of different lignans however themechanisms underlying the interaction of lignans with thecardiovascular system are not well defined.

Without being bound by theory, one mechanism by which the compoundof the present invention may exert its effect in a medical settingis its induction of a cell cycle arrest in the G1-phase, which isassociated by an accumulation of the cell cycle inhibitor p27/KIP.The underlying mechanism of the effect conferred by the compoundused and provided in accordance with the present invention may beits induction of an arrest in the cell cycle. In contrast tohonokiol which causes a cell cycle arrest in the G1 phase by anupregulation of p21 (see Lee, FEBS Lett 580(22), 5177-84 (2006)),leoligin leads to an increase in p27/KIP protein levels. Althoughthe result i.e. a G1 phase arrest is similar for both compounds,the underlying signalling processes involved, and the mechanism ofaction may differ. P27/KIP is well known to bind and therebyinactivate the cyclinE/cdk2 complex which phosphorylates pRB.Phosphorylated pRB looses its ability to inhibit transcriptionfactors like E2F, which upon release serve as transcription factorsfor proliferation-promoting genes. Although p27/KIP was alsoreported to cause an arrest at the G2-S transition in a limitednumber of cell types via an interference with cyclinA/cdk2 andcyclinB/cdk2 complexes (see Pagano, Mol Cell 14(4), 414-6 (2004)),in the case of SMCs the usually observed interaction of p27/KIPwith cyclinE/cdk2 complexes which leads to an arrest in the G1phase seems to be at play. The shift in the molecular weight ofp27/KIP-1 at 50 .mu.M leoligin from 27 kD to 58 and 85 kDrespectively, may indicate the binding of p27/KIP-1 to thecyclinE/cdk2 complex, or an oligomerisation of molecules(dimers/trimers). The 23 kD band may represent a cleavage productof p27/KIP. The signalling pathways via which leoligin leads to theaccumulation and change in molecular weight of p27/KIP-1 proteinremain to be elucidated.

Only recently, Edelweiss root extracts have been chemicallycharacterized; see Dobner et al. (2003), loc. cit. Edelweiss rootextracts show a complex pattern of secondary plant metabolites, ofseveral compound classes like coumarins, lignans, sesquiterpenes,polyacetylens, diterpenes, and others; see Schwaiger, Planta Med,70(10), 978-85 (2004). In general, lignans are polyphenolic plantmetabolites derived from phenylalanine, which are synthesized bythe coupling of two phenylpropanoid units by a bond between the.beta.-positions in the propane side chains. One of these lignanswhich has been isolated from the roots of Edelweiss isleoligin-IUPAC name[(2S,3R,4R)-4-(3,4-dimethoxybenzyl)-2-(3,4-dimethoxyphenyl)tetrahydrofura-n-3-yl]methyl (2Z)-2-methylbut-2-enoat]. Although it is thus knownthat roots of Edelweiss (Leontopodium alpinum Cass.), one of themost popular alpine plants, which has been used in folk medicinefor the treatment of diarrhea, fever, and "abdominal aches" containlignans, a medical use of any of these lignans, and in particularleoligin and its ((di)methoxy)derivatives, as an inhibitor ofcellular proliferation, in particular the proliferation of SMCs,has neither been described nor proposed in the art. Also a medicaluse of the isolated compounds of the general formula (I) as givenherein above, has not been described in the prior art.

As mentioned above, edelweiss and extracts thereof have been usedin folk medicine. However, it is of note that only the upper parts(i.e. flowers, leaves and stems) of the Edelweiss plant have beenused because these contain the bulk of the biomass and have thusbeen easier available. Historical references from the year 1582mention that Edelweiss and its relatives are mainly used for thetreatment of diarrhea and dysentery; see Tabernaemontanus, J. T.(1582): Das Ander Buch von Kreutern. In: Bauhin, H. (ed.) (1731):D. Jacobi Theodori Tabernaemontani neu vollkommen Kraeuter-Buch.Reprint Basel, Konig, 1731. Verlag Kolbl, Grunwald (Munchen) 1993).Further information on the traditional use of Edelweiss wascollected by several diploma theses on the usage of Alpine plantsin folk medicine, performed at the Institute of Pharmacognosy ofthe University of Vienna. Interviews of elder inhabitants of alpineregions in Austria and Northern Italy revealed a variety of localknowledge. In Vorarlberg Edelweiss flower heads were boiled inmilk, preparations of which were used for the therapy of abdominalaches and diarrhea in humans, and particularly also in domesticstock; see Kiene, Volksmedizin in verschiedenen GebietenVorarlberg, Master Thesis at the University of Vienna (1992);Bitschnau, Arzneidrogen der Volksmedizin im Montafon, Master Thesisat the University of Vienna (1991).

Similar information was also obtained for North-Tyrol, East-Tyroland South-Tyrol, where Edelweiss was, furthermore, used to curetonsillitis, angina and bronchitis, and as an antipyretic to lowerfever; see Knechtl, Volksmedizinisch verwendete Heilpflanzen undHausmittel im Inntal und umgebenden Seitentalern (Tirol), MasterThesis at the University of Vienna (1992); "Wieser,Volksmedizinische Verwendung von Heilpflanzen und Hausmitteln imOsttiroler Pustertal mit Seitentilem und im Lesachtal", MasterThesis at the University of Vienna (1995); Pickl-Herck,Volksmedizinische Anwendung im Norden Sudtirols. Master Thesis atthe University of Vienna (1995). In Polish traditional medicine, L.alpinum was used for the therapy of breast cancer by localapplication of a poultice of the aerial plant parts; see Hartwell,J. Nat. Prod. (Lloydia) 31, 71-170 (1968). Knechtl (1992; loc.cit.) also describes that infusions of edelweiss flowers can beused to ameliorate stomach-ache. In particular diarrhea in childrenis to be treated with milk in which flowers from edelweiss plantshas been boiled; see Knechtl (1992; loc. cit.). Wieser (1995; loc.cit.) points out that upper parts of edelweiss plants are used infolk medicine, since edelweiss plants are selected out of the cutgrass of alpine meadows (i.e. the upper parts of edelweiss plantsare collected) and dried. This has been particularly described forthe Villgratental in Kalkstein (1650 m above sea level). Theedelweiss plant is described in Wieser (1995; loc. cit.) as the"chamomile" of the Alps, since it is used in medicine similar tochamomile. According to Wieser (1995; loc. cit.) an edelweissinfusion is used to ameliorate stomach ache, while edelweiss boiledin milk is helpful in abdominal cramping. Pickl-Herk (1995; loc.cit.) describes the following medical use of edelweiss flowers:infusion of flowers is beneficial in ameliorating stomach ache (inparticular caused by foul drinking water), stomach flatulencies,and diarrhea with vomiting. Again, infusions of edelweiss flowersare intended to be administered in particular to children."Edelweissmilch" (i.e. 4-5 flowers boiled in 0.5 l milk) is usedfor the following disorders: diarrhea, vertigo, poisoning (leads tovomiting), snake-bites, blood poisoning, indigestion, abdominalcramping, stomach ache, stomach flatulencies, or hangover; seePickl-Herk (1995; loc. cit.). Also the use of "Edelweissmilch" inveterinary medicine is disclosed in this document, i.e. thetreatment of calves suffering from diarrhea and of calves/cowssuffering from stomach flatulencies is described.

Again, the use of Edelweiss in folk medicine as described in theprior art documents above involves only extracts from the upperparts of the plant, but not of the roots. Also a specific compoundcontained in this extract to be used in folk medicine has not beendescribed in this context.

As mentioned above and shown in detail in the experimental sectionherein below, the compound to be used in accordance with thepresent invention or the compound as comprised in thepharmaceutical composition of the present invention may be obtainedfrom plants belonging to the genus Leontopodium, optionallyfollowed by standard derivatization reactions. It is particularlypreferred that the compounds provided herein may be obtained fromLeontopodium alpinum, in particular Leontopodium alpinum Cass.,which is commonly known under the trivial name "edelweiss".According to another nomenclature "edelweiss" may also be knownunder the scientific term "Leontopodium nivale subsp. alpinum(Cass.) Greuter". However, the terms "Leontopodium alpinum Cass"and "Leontopodium nivale subsp. alpinum (Cass.) Greuter" refer tothe same plant species and merely reflect a regrouping of thespecies in botanical nomenclature. Accordingly, the these terms canbe used interchangeably in context of the present invention and anydefinitions and explanations given herein in respect ofLeontopodium alpinum Cass. also applies to Leontopodium nivalesubsp. alpinum (Cass.) Greuter, mutatis mutandis, and viceversa.

Of course, it is envisaged herein that the compounds to be usedaccording to the present invention may be obtained from otherLeontopodium species, including but not limited to commercialcultivars, such as Leontopodium hybrids. Accordingly the compoundsmay be obtained from the following, exemplary Leontopodium speciesand cultivars: L. catipes (DC.) F. Muell., L. gnaphalioidesHieron., L. japonicum var. sandwicense H. Le., L. linearifoliumBritton, L. meredithae (F. Muell.) F. Muell., L. albogriseumHand.-Mazz., L. aloysiodorum Hort. ex Hand.-Mazz., L. alpinumCass., L. alpinum Colm. ex Willk. & Lange, L. alpinum Cass.subsp. nivale (Ten.) Tutin, L. amrheinii Hort. ex Mollers, L.andersonii C. B. Clarke, L. antennarioides Socz., L. arbusculaBeauverd, L. artemisiifolium Beauverd, L. aurantiacum Hand.-Mazz.,L. beerianum Beauverd ex Murr, L. blagoveshczenskyi Vorosch., L.bonatii Beauverd, L. brachyactis Gand., L. caespitosum Beauverd, L.caespitosum Diels, L. calocephalum Beauverd, L. campestreHand.-Mazz., L. catipes F. Muell., L. chamaejasme Beauverd, L.charkeviczii V. Yu. Barkalov, L. chuii Hand.-Mazz., L. conglobatumHand.-Mazz., L. coreanum Nakai, L. dedekensi Beauverd, L.delavayanum Hand.-Mazz., L. discolor Beauverd, L. dubium Beauverd,L. evax Beauverd, L. fangingense Ling, L. fauriei Hand.-Mazz., L.fedtschenkoanum Beauverd, L. fimbrilligerum J. R. Drumm., L.fischerianum Beauverd, L. foliosum Beauverd, L. forrestianumHand.-Mazz., L. francheti Beauverd, L. futtereri Diels, L. giraldiiDiels, L. gnaphalioides Hieron. ex Sod., L. gracile Hand.-Mazz., L.haastioides Hand.-Mazz., L. hallaisanense Hand.-Mazz., L.haplophylloides Hand.-Mazz., L. hastatum Beavera, L. hayachinense(Takeda) Hara & Kitam., L. helveticum D. Don ex G. Don, L.himalayanum DC., Leontopodium.times.intermedium Sunderm., L.jacotianum Beauverd, L. jacotianum Beauverd var. haastioides(Hand.-Mazz.) R. C. Srivastava, L. jamesonii Beauverd, L. japonicumMiq., L. japonicum Miq. f. happoense Hid. Takah. ex T. Shimizu, L.javanicum Zoll. & Mor., L. junpeianum Kitam., L. kamtschaticumKomarov, L. krasense Derganc, L. kurilense Takeda, L. leiolepisNakai, L. leiolepis Nakai var. crinulosum H. S. Pak, L. leiolepisNakai var. curvicollum H. S. Pak, L. leontopodinum Hand.-Mazz., L.leontopodioides Beauverd, L. leontopodium Karst.,Leontopodium.times.lindavicum Sunderm., L. linearifolium Britton,L. linearifolium Benth. & Hook. f., L. linearifoliumHand.-Mazz., L. longifolium Ling, Leontopodium.times.macranthumSunderm., L. maireanum Beauverd ex Hand.-Mazz., L. makianum Kitam.,L. mariae Muell., L. melanolepis Ling, L. meredithae F. Muell., L.micranthum Ling, L. microcephalum (Hand.-Mazz.) Ling, L.microphyllum Hayata, L. monocephalum Edgew., L. monoicum Benth.& Hook. f., L. montisganeshii S. Akiyama, L. muscoidesHand.-Mazz., L. nanum Hand.-Mazz., L. nivale (Ten.) Huet exHand.-Mazz., L. nivale (Ten.) Huet ex Hand.-Mazz. subsp. alpinum(Cass.) Greuter, L. niveum Hand.-Mazz., L. nobile Beauverd, L.ochroleucum Beauverd, L. ochroleucum Beauverd subsp. campestre(Ledeb.) V. M. Khanminchun, L. ochroleucum Beauverd subsp.campestre (Hand.-Mazz.) Khanm., L. ochroleucum Beauverd subsp.conglobatum (Turcz.) V. M. Khanminchun, L. ochroleucum Beauverdsubsp. conglobatum (Hand.-Mazz.) Khanm., L. omeiense Ling, L.palibinianum Beauverd, L. paradoxum J. R. Drumm., L. perniveumHonda, L. pirinicum Hand.-Mazz., L. pulchellum Beauverd, L.pusillum Hand.-Mazz., L. roseum Hand.-Mazz., L. rosmarinoidesHand.-Mazz., L. sachalinense Miyabe & Kudo, L. sandwicenseRock, L. shinanense Kitam., L. sibiricum Cass., L. sinense Hemsl.ex Forb. & Hemsl., L. smithianum Hand.-Mazz., L. soulieiBeauverd, L. spathulatum Kitam., L. stellatum A. P. Khokhr., L.stoechas Hand.-Mazz., L. stoloniferum Hand.-Mazz., L. stracheyi C.B. Clarke ex Hemsl., L. subulatum Beauverd, L. suffruticosum Y. L.Chen, L. tataricum Kom., L. thomsonianum Beauverd, L. umbellatumBluff. & Fingerh., L. villosulum A. P. Khokhr., L. villosumHand.-Mazz., and L. wilsonii Beauverd.

Of course, the compounds provided herein may also be obtained fromcorresponding cell culture, cell suspension culture or a comparablein vitro cultivation technique, such as callus culture and thelike. A person skilled in the art will be aware of correspondingmeans and methods for establishing and maintaining correspondingcultures. In a preferred embodiment of the invention, the cellculture is derived from roots of Leontopodium species describedherein above, in particular Leontopodium alpinum (edelweiss). Mostpreferably, the cell culture is derived from hairy roots.

Based on his general knowledge and the teaching provided herein askilled person is readily in the position to obtain the compoundsto be used herein, in particular leoligin, from Leontopodiumspecies. Generally, the person skilled in the art is capable ofpreparing an extract from plants belonging to the genusLeontopodium by standard techniques. A preferred method forextracting these compounds from the roots of Leontopodium alpinumis provided in Example 1 herein below. An artisan will be aware howto adapt this protocol for extracting the compounds from furtherLeontopodium species and in particular from roots of these plants.A skilled person will also be aware of alternative protocols to beused in this context. The term extract is well known in the art andused accordingly herein. For example, this term may refer topreparations of fluid consistence (fluid extracts and tinctures),semisolid consistence (viscous extracts, syrup concentrate) orsolid consistence (dried extracts), which are usually preparedusing fresh or dried plant material.

The extract obtained from Leontopodium species is an extract thatis received by the use of an organic or non-organic solvent.Suitable solvents are hexane, heptane, petroleum benzene, acetone,chloroform, dichloromethane, ethyl acetate, diethylether, liquidcarbon dioxide, ethanol, ternary butyl methyl ether (tBMe) andmixtures of water and alcohol. The extract may be obtained byextracting the plant material, in particular roots, with any of thesolvents separately. It is further possible to subsequently extractthe obtained extract with a second solvent or mixtures of differentsolvents. An exemplary, non-limiting solvent to be used in a firstextraction step is hexane. However, any of the above solvents canbe used in such a first extraction step. This first extraction stepmay be followed by (a) subsequent second (or further) extractionstep with at least one of the above exemplary solvents, e.g.dichloromethane, chloroform or ternary butyl methyl ether (tBMe).Extraction of the compounds disclosed herein (in particularcompounds of formula (I), such as leoligin and/or its(di)methoxy-derivative(s)) in accordance with the present inventionis also illustrated in the appended examples. Preferably,dichloromethane and methanol are used as extraction solvents. Insubsequent extraction, it is preferred that the compounds are firstextracted with n-hexane, followed by a subsequent extraction withdichloromethane, chloroform or tBMe. As shown herein, the lignancontent (i.e. content of compounds of formula (I), such as leoliginand/or its (di)methoxy-derivative(s)) can be increased by a secondor further extracting steps using the herein described methods, andin particular the above solvents. Also the use of chromatographicmethods, such as Sephadex-LH20-column chromatography and inparticular silica gel column chromatograph is advantageous in thiscontext. As also demonstrated in the appended examples, an increasein the leoligin content from about 0.7% to about 2.2% can beachieved using Sephadex-LH20-column chromatography. It is shownherein that a pronounced increase in the leoligin content fromabout 1.4% to about 10% can be achieved using silica gelchromatograph.

It is envisaged herein that further chromatographic methods toincrease the content of the herein disclosed compounds (inparticular compounds of formula (I), such as leoligin and/or its(di)methoxy-derivative(s)) can used in addition or in thealternative to the above described methods. Exemplary, non-limitingchromatographic methods to be used in this context are reversedphase column chromatography or (semi)-preparative HPLC usingwater/acetonitrile mixtures or comparable solvent mixtures known inthe art. Alternatively, techniques of liquid-liquid extractions(discontinuous or continuous methods) can be used to increase thecontent of the herein disclosed compounds (in particular compoundsof formula (I), such as leoligin and its(di)methoxy-derivative(s)). An exemplary liquid-liquid extractionis high speed counter current chromatography using a solvent systemof two not mixable solvents.

The preparation of the basic extract of Leontopodium species, inparticular Leontopodium alpinum, may comprise mechanical pulping,sonication, use of mortars and pestles, freeze-thawing cycles, useof blenders (like Waring-Blenders, Polytron), liquid homogenizationand maceration (see also appended examples), or e.g. Douncehomogenization, Potter-Elvehjem, French Press etc. In the appendedexamples, a mechanical maceration is used. However, the extractsmay be obtained by disrupting the cells and cells from theLeontopodium species by any mechanical/physical or chemical means,like by use of detergents.

Mechanical methods rely on the use of rotating blades to grind anddisperse large amounts of complex tissue, such as plant leaves,flowers, seeds and in particular roots. The Waring blender and thePolytron are commonly used for this purpose. Unlike the Waringblender, which is similar to a standard household blender, thePolytron draws tissue into a long shaft containing rotatingblades.

Liquid-based homogenization is the most widely used cell disruptiontechnique for cultured cells. Cells are lyzed by forcing the cellor tissue suspension through a narrow space, thereby shearing thecell membranes. Three different types of homogenizers are in commonuse. A Dounce homogenizer consists of a round glass pestle that ismanually driven into a glass tube. A Potter-Elvehjem homogenizerconsists of a manually or mechanically driven Teflon pestle shapedto fit a rounded or conical vessel. The number of strokes and thespeed at which the strokes are administered influences theeffectiveness of Dounce and Potter-Elvehjem homogenization methods.Both homogenizers can be obtained in a variety of sizes toaccommodate a range of volumes. A French press consists of a pistonthat is used to apply high pressure to a sample volume of 40 to 250ml, forcing it through a tiny hole in the press. Only two passesare required for efficient lysis due to the high pressures usedwith this process. It is of note that in more industrialapplications also other, larger devices may be employed to preparethe extracts from Leontopodium species.

Sonication is also a physical disruption commonly used to breakopen cells. The method uses pulsed, high frequency sound waves toagitate and lyse cells and finely diced tissue. To preventexcessive heating, ultrasonic treatment may be applied in multipleshort bursts to a sample immersed in an ice bath. Sonication isbest suited for volumes <100 ml.

The freeze/thaw method is commonly used to lyse bacterial and cellsfrom higher organism. The technique involves freezing a cellsuspension in a dry ice/ethanol bath or freezer and then thawingthe material at room temperature or 37.degree. C. This method oflysis causes cells to swell and ultimately break as ice crystalsform during the freezing process and then contract during thawing.Multiple cycles are necessary for efficient lysis, and the processcan be quite lengthy.

Cells, organisms as well as tissue might be treated with variousagents to aid the disruption process. Chemical substances, such ashexane, petroleum benzene, chloroform, dichloromethane, acetone,ethyl acetate, diethyl ether, ethanol and mixtures of water andalcohol or mixtures of different solvents may be added during orbefore mechanical disruption. Lysis can also be promoted bysuspending cells in a hypotonic buffer, which cause them to swelland burst more readily under physical shearing. Processing can beexpedited by treating cells with glass beads in order to facilitatethe crushing of cell walls. Viscosity of a sample typicallyincreases during lysis due to the release of nucleic acid material.DNase may be added to samples along with to reduce thisproblem.

Less preferred, however envisaged, is the use of detergents in thepreparation of the extracts to be treated in accordance with thepresent invention. Detergents are a class of molecules whose uniqueproperties enable manipulation (disruption or formation) ofhydrophobic-hydrophilic interactions among molecules in biologicalsamples. Such detergents may be used to lyse cells, solubilizemembrane proteins and lipids. Generally, moderate concentrations ofmild (i.e., nonionic) detergents compromise the integrity of cellmembranes, thereby facilitating lysis of cells and extraction ofsoluble protein, often in native form. Using other conditions,detergents effectively penetrate between the membrane bilayers atconcentrations sufficient to form mixed micelles with isolatedphospholipids. Detergents may be, e.g. Triton X-100.RTM.,Triton-X-114.RTM., NP-40.RTM.; CHAPS, Tween-20.RTM., Tween-40.RTM.,Tween-80.RTM., Octyl Glucoside, Octylthio Glucoside, Brij-35,Brij-58, SDS and the like. However, it may be useful to stabilizethe extract by certain chemical means. Illustrative stabilizers arediscussed herein below in context of pharmaceutical or cosmeticcompositions.

The cells and plants to be employed in order to obtain the basicextract may be cells of natural origin as well as cultured cells orplants. It is preferred herein that the cells or plants and inparticular roots of the plants are dried before mechanicaldisruption/maceration as described herein above. The cells orplants may be air dried, lyophilized (freeze-dried) or, though lesspreferred, dried in an oven. It is preferred herein that the"cell(s)" and "plant(s)" to be used as a basic material are fresh,i.e. harvested shortly before the extract is prepared. Nonetheless,it is possible to store the basic material before its use in thepreparation of the extract. For example, the basic material may belyophilized (freeze-dried) or simply frozen and stored at lowtemperatures, e.g. at about -20 to -30.degree. C. or as low as-80.degree. C.

In context of the present invention, the term "cell" and "plant" tobe used as basic material for preparing the extract to be treatedby the method of the present invention also comprises the use of"tissues". Such tissues may be leaves, sprouts, or reproductiveorgans e.g. flowers. Preferably, the tissues are roots, inparticular hairy roots. In addition, callus or cell cultures may beused which may be derived from tissues described above, inparticular roots, and which are grown in liquid culture or onsolidified culture medium. The appropriate culturing methods ofcalli or cell cultures are known to a person skilled in the art. Aculture medium may be for example a MS (Murashige and Skoog) mediumwhile a solidifying agent may be agarose, plant agar or bacto agar.A basic culture medium such as a MS medium may be modified inrespect to pH range, carbon or nitrogen source, amino acids orvitamins amongst others. The use of plants regenerated from suchcallus or cell culture is also envisaged, as well as plants ororganisms generally grown or propagated in vitro.

Methods for preparing the extract are known in the art and alsodescribed herein. Preferably, the extract is further processedshortly after its preparation (e.g. the extract is used in thepreparation of a herein disclosed pharmaceutical composition);however, it is also possible to store the extract for some timebefore they are used in accordance with the present invention. Theextracts may, for example, be stored in lyophilized form or in formof dried extracts. However, each storage form known in the art isbe employed, as long as the storage has the effect that the extract(and its components) remain efficacious over a long time period,i.e. the stored extract has, preferably, substantially the sameefficacy as the fresh extract.

Dried extracts can be routinely prepared by methods known in theart. For example, following mechanical disruption of the basic(plant) material by e.g. maceration or percolation, the materialcan be extracted using (a) solvent(s) or mixtures thereof asdescribed herein. After separation of the fluid phase and theextract residue (which contains e.g. cellulose, pectin and the likeand which does, preferably, not contain the active substance(s) asdisclosed herein, i.e. predominantly leoligin and its((di)methoxy)-derivative(s). The fluid extract (i.e. the fluidphase of the obtained extract) may be concentrated taking advantageof routine techniques, some of which are exemplarily describedherein below. Such concentration techniques include, but are notlimited to fluidised-bed drying, concentration to a syrup orconcentrated fluid extract, spray drying, freeze drying or the useof a vacuum dryer, a drying tunnel, vacuum band dryer or a dryinghurdle. Often organic-hydrous fluid extracts (such as the fluidextract obtained herein using an organic solvent) are concentratedby nucleate boiling or surface evaporation.

Routine drying techniques employed in the pharmaceutical fieldcomprise distillation and drying under normal conditions (i.e. roomtemperature) also methods which take advantage of variations inpressure and temperature in order to obtain the dried extracts. Onewell known method for preparing a dried extract is as follows:First, a fluid extract or tincture is prepared; after subsequentdistillation of the solvent a viscous extract is obtained, to whichoften adjuvants and/or excipients (e.g. lactose,polyvinylpyrrolidone, sucrose, silicon dioxide and the like areadded. This moist mass is then dried in suitable driers. Alsoemployed in this context is the use of a vacuum band dryer(Mitchell Dryers Ltd), wherein a dried extract is obtained from theviscous extract after a pre-drying step using downdraftvaporizers.

Also envisaged herein is the use of commercially availableextracts, in particular dried extracts, obtained from (a) plant(s)belonging to the genus Leontopodium.

After mechanical disruption of the cell(s), tissue(s) or wholeplant(s) the plant material may be further macerated and/ordissolved/suspended in an organic solvent, such as hexane,petroleum benzene, chloroform, dichloromethane, acetone, ethylacetate, diethyl ether, liquid carbon dioxide, ethanol and mixturesof water and alcohol with any of the solvents separately orsubsequently with a second solvent or mixtures of differentsolvents. Preferably, dichloromethane and methanol are used asextraction solvents.

As shown in the appended examples, a hexane extract comprising0.67% leoligin and 1.47% leoligin and its methoxy-derivative(s) caneasily be prepared by routine techniques. However, it is preferredherein that the extract is enriched in the compounds described andprovided herein, in particular compounds of formula (I), such asleoligin and/or its ((di)methoxy) derivative(s). As also shown inthe appended examples, higher yields (relative to the leoligincontent [w/w %] in the extract) typically in a range between 0.7%to 1.5% can easily be obtained using standard extraction methodsand solvents (such as dichloromethane). Using these standardextraction methods, yields of up to about 2.2% of leoligin and its5-methoxy-derivative can be obtained. As described herein, thecontent of compounds of formula (I), in particular leoligin (and/orits (di)methoxy-derivative(s)), can be further increased bymultiple extraction rounds, e.g. a first extraction step usinghexane followed by (a) subsequent extraction step(s) using e.g.dichloromethane, chloroform or ternary butyl methyl ether (=tBMe).A total lignan content (predominantly compounds of formula (I), inparticular leoligin and/or its (di)methoxy-derivative(s)) of atleast 2.4% can be achieved if subsequent extraction steps areapplied The concentration of lignans (predominantly compounds offormula (I), in particular leoligin and/or its(di)methoxy-derivative(s)) can also be increased by the use ofSephadex-LH20-column chromatography (increase in the leoligincontent from about 0.7% to about 2.2%).

Preferably, the extract is an enriched extract, i.e. containsleoligin and its ((di)methoxy)-derivative(s) in a high amount. Suchan enriched extract can, for example, be obtained by takingadvantage of silica gel chromatography as demonstrated in theappended examples. Silica gel column chromatography is well knownin the art and described in detail in standard textbooks, such as"Preparative Chromatography Techniques" by Hostettmann, K. Marston,Andrew Hostettmann, Maryse, Springer-Verlag GmbH, 2007, 260 p. Inthe experimental section, it was shown that a pronounced increasein the leoligin content from 1.36% to 9.76% [w/w] can be achievedusing silica gel chromatograph (mobile phase: petroleumether-acetone).

Accordingly, it is preferred herein that the solid components ofthe extract (e.g. after evaporating the solvent by any of thedrying methods described herein) comprise at least 0.05%, 0.1%,0.5%, 0.7%, 1%, 1.5%, 2.0%, 2.5% or 3.0% of the compounds offormula (I), in particular leoligin and/or((di)methoxy)-derivative(s) thereof, wherein an extract the solidcomponents of which comprise at least 0.7% of these compounds canbe considered an "enriched" extract in context of the presentinvention. More preferably, the solid components of the extractcomprise at least 5%, 6%, 7%, 8%, and most preferably at least 9%or 10% of the compounds of formula (I), in particular leoliginand/or ((di)methoxy)-derivative(s) thereof. An extract, the solidcomponents of which comprise at least 9% of these compounds can beconsidered a "highly enriched" extract. An "enriched extract", and,in particular a "highly enriched" extract as defined herein,represents therefore a preferred embodiment of the present."Enriched" or "highly enriched" extracts are particularly useful inthe herein disclosed medical context, in particular the treatment,prevention, or amelioration of (a) hyperplastic disorder(s) asdefined herein. In accordance with the present invention, it isalso preferred herein that the solid components of the (highlyenriched) extract comprise at least 15%, 20%, 25%, 30%, 40%, 50%,60%, 80% or 90% of the compounds of formula (I), in particularleoligin and/or (a) derivative(s) thereof (preferably (a)(di)methoxy-derivative(s), more preferably the herein disclosedderivatives 5-Methoxy-Leoligin and/or 5,5'-Dimethoxy-Leoligin).Based on the teaching provided herein a skilled person is readilyin the position to determine whether an extract prepared inaccordance with the present invention is enriched/highly enrichedin compounds of formula (I), in particular leoligin and/or (a)derivative(s) (preferably (a) (di)methoxy-derivative(s), morepreferably the herein disclosed derivatives 5-Methoxy-Leoliginand/or 5,5'-Dimethoxy-Leoligin). Most preferably, pure compounds offormula (I) are obtained, i.e. solid components of the extractcomprise at least 95% of the compounds described and providedherein. In order to obtain a higher yield of the compounds offormula (I), the basic extracted material may be subjected to atleast one further and up to eight further cycles of extraction. Itis preferred that the (enriched/highly enriched) extract isobtained from (a) plant(s) belonging to the genus Leontopodium, inparticular from the roots of such (a) plant(s). Exemplary speciesor cultivars of the above genus and to be used in accordance withthe present invention are known in the art and also disclosedherein.

It is envisaged herein, that the "enriched/highly enriched" extractcomprises predominantly leoligin as active substance, in particularin combination with its methoxy-derivatives. Based on the teachinggiven herein, a skilled person is readily in the position todetermine which amount of the (enriched/highly enriched) extract isto be employed in particular in the preparation of thepharmaceutical compositions comprising/consisting of the extractdepending on the concentration/content of the herein disclosedactive substance (preferably of leoligin and/or its(di)methoxy-derivative(s) and mixtures thereof). Preferably, theextract employed/contained in the pharmaceutical composition exertssubstantially the same medical effect as a pharmaceuticalcomposition comprising (a) compound(s) of formula (I), inparticular leoligin and/or its (di)methoxy-derivative(s) (ormixtures thereof), more particularly leoligin or the(di)methoxy-derivative alone (as shown in the appended examples)."Substantially the same effect" means in context of the presentinvention that the "effect" varies by less than 10%, preferablyless than 5%, most preferably less than 1%. An exemplary "effect"to be measured is inhibition of vascular smooth muscle cell (SMC)proliferation which is a central mechanism underlying hyperplasticdiseases/disorders as described herein above and also demonstratedin the appended examples.

As mentioned above, the herein provided and disclosed extractsobtained from (a) plant(s) belonging to the genus Leontopodium can,in accordance with the present invention, be used in a medicalcontext. Accordingly, the present invention relates in oneembodiment to a pharmaceutical composition comprising a rootextract obtained from a plant belonging to the genus Leontopodium.A further embodiment relates to a pharmaceutical compositioncomprising an extract obtained from a plant belonging to the genusLeontopodium, whereby the extract is highly in enriched in theherein disclosed compounds, in particular compounds of formula (I),such as leoligin and/or its (di)methoxy-derivative(s) (or mixturesthereof). It is preferred herein that the highly enriched extractpredominantly comprises leoligin and/or its(di)methoxy-derivative(s) (or mixtures thereof). Formulas ofleoligin and preferred (di)methoxy-derivatives thereof are alsoprovided herein. The herein disclosed pharmaceutical compositioncomprising a (root) extract obtained from a plant belonging to thegenus Leontopodium, wherein the extract is preferably enriched(most preferably highly enriched) in the compounds of formula (I),in particular leoligin and/or ((di)(methoxy-))derivatives thereof,is used in the treatment, prevention or amelioration of ahyperplastic disorder as defined herein. The term "root extract"used herein means an extract obtained from roots, i.e. plantmaterial from the lower parts of the plants are used, preferablyonly roots are used as raw plant material in the preparation of theextract. It is preferred in this context that the pharmaceuticalcomposition consists of the (preferably enriched, more preferablyhighly enriched) extract. However, furtherexcipients/adjuvants/carriers and the like as described herein andknown in the art may be contained in the pharmaceutical compositionin addition to the extract. In accordance with the above, acomposition comprising (consisting of) a(n) (root) extract obtainedfrom a plant belonging to the genus Leontopodium, whereby theextract is (highly) in enriched in compounds of formula (I), inparticular leoligin and/or its (di)methoxy-derivative(s) (ormixtures thereof), is provided herein for use in medicine or foruse as a medicament. Also a (root) extract obtained from a plantbelonging to the genus Leontopodium, whereby the extract is(highly) in enriched in compounds of formula (I), in particularleoligin and/or its (di)methoxy-derivative(s) (or mixturesthereof), for use in medicine or as a medicament is provided. It isenvisaged that the above (pharmaceutical) compositions/extracts areto be used in accordance with the present invention in thetreatment, prevention or amelioration of a hyperplastic disorder asdisclosed and defined herein.

In order to obtain the single compounds, the extracts may beprepared and evaporated as described above and, submitted tofurther purification by column chromatography using silica gel,silica gel modified by means of AgNO.sub.3, reversed phase material(RP18) or Sephadex LH 20.RTM. as stationary phases. Additionally,other separation techniques e.g. high speed counter currentchromatography or (semi)-preparative HPLC might be used as well.Fractions obtained by the above mentioned chromatographictechniques may be further purified, e.g. by another cycle ofchromatographic purification. For example, a cross-linked dextrangel may be used for such further purification, like e.g. SephadexLH-20.RTM.. This kind of chromatography is usually performed in thepresence of an organic solvent such as methanol, acetonedichloromethane and the like. It is envisaged herein that theherein described pharmaceutical compositions comprising the extractdisclosed herein may also (in addition) comprise the pure (and/or(substantially) purified, e.g. purified from the extract) activesubstances (i.e. compound of formula (I), in particular Leoliginand/or its (di)methoxy derivative(s)). In accordance with theabove, it is preferred herein that the pharmaceutical compositioncomprises essentially the plant extracts disclosed herein andobtained by the herein described methods. Also envisaged herein isa pharmaceutical composition, which does not comprise the hereindescribed extract, but comprises the pure (and/or (substantially)purified, e.g. purified from the extract) active substances (i.e.compound(s) of formula (I), in particular Leoligin and/or its(di)methoxy-derivative(s)). The extract can also be obtained byalternative extraction methods known in the art e.g. supercriticalcarbon dioxide extraction, percolation or Soxhlet-extraction andadaptable for the means and methods of the present invention by oneskilled in the art.

Also envisaged herein, though less preferred, the compounds mayalso be obtained from upper parts of the plants, e.g. flowers,stems, leaves, seeds and the like. Leontopodium alpinum (Edelweiss)plants to be extracted are easily available e.g. from the Stationfederale de recherches en production vegetale de Changins (see alsohttp://www.admin.ch/sar/rac; Revue Suisse Vitic. Arboric. Hortic.31(2), 889-96 (1999)).

In an alternative embodiment, the compounds to be used herein mayalso be synthesized. An exemplary synthetic pathway of leoligin isshown in FIG. 8. The shown synthetic pathway might be adapted by achange of the corresponding educts to obtain other compounds of thepresent invention A skilled person will be aware of methods ofsynthesizing the compounds of the present invention, in particularleoligin, or may deduce corresponding methods e.g. from Li Hong Hu,J. Nat. Prod. 68, 342-8. (2005); Babasaheb P. Bandgar, Monatsheftefur Chemie 135, 1251-5 (2004); J Pijus Kumar Mandal, Org. Chem. 63,2829-34 (1998); Subhas Chandra Roy, J. Org. Chem. 67, 3242-8(2002).

As mentioned above, the active compounds referred to herein mayalso be provided via semi-synthetic methods, e.g. by derivatizing anatural product such as leoligin. Suitable derivatization reactionsknown in the art comprise methods wherein the ester bond present inleoligin is saponified to produce an alcohol. The alcohol may beoxidized to provide a carbonyl/carboxylic acid functionality to bereacted with an alcohol, thiol or amine, or it may be esterifiedwith a different organic acid, it may be converted into an amineetc.

The pharmaceutical composition may comprise the compounds providedin the present invention. The compounds to be used in accordancewith the present invention may be obtained from Leontopodium plantsas described herein above and/or chemically synthesized.

The pharmaceutical composition of the present invention comprisingcompounds of formula (I) and, in particular, leoligin, will beformulated and dosed in a fashion consistent with good medicalpractice, taking into account the clinical condition of theindividual patient, the site of delivery of the pharmaceuticalcomposition, the method of administration, the scheduling ofadministration, and other factors known to practitioners. The"effective amount" of the pharmaceutical composition for purposesherein is thus determined by such considerations.

The skilled person knows that the effective amount ofpharmaceutical composition administered to an individual will,inter alia, depend on the nature of the compound. For example, ifsaid compound is a lignan the total pharmaceutically effectiveamount of pharmaceutical composition administered parenterally perdose will be in the range of about 1 .mu.g/kg/day to 10 mg/kg/dayof patient body weight, although, as noted above, this will besubject to therapeutic discretion. More preferably, this dose is atleast 0.01 mg/kg/day, and most preferably for humans between about0.01 and 1 mg/kg/day. If given continuously, the pharmaceuticalcomposition is typically administered at a dose rate of about 1.mu.g/kg/hour to about 50 .mu.g/kg/hour, either by 1-4 injectionsper day or by continuous subcutaneous infusions, for example, usinga mini-pump. An intravenous bag solution may also be employed. Thelength of treatment needed to observe changes and the intervalfollowing treatment for responses to occur appears to varydepending on the desired effect. The particular amounts may bedetermined by conventional tests which are well known to the personskilled in the art.

Pharmaceutical compositions of the invention may be administeredorally, rectally, parenterally, intracistemally, intravaginally,intraperitoneally, topically (as by powders, ointments, drops ortransdermal patch), bucally, or as an oral or nasal spray.Accordingly, also the compound provided herein may be administeredby any one of a parenteral route, oral route, intravenous route,intraarterial route, intramuscular route, intracardial route,intrapulmonal route, intravesical route, intravitreal route,subcutaneous route, intranasal route or transdermal route.

Pharmaceutical compositions of the invention preferably comprise apharmaceutically acceptable carrier. By "pharmaceuticallyacceptable carrier" is meant a non-toxic solid, semisolid or liquidfiller, diluent, encapsulating material or formulation auxiliary ofany type. The term "parenteral" as used herein refers to modes ofadministration which include intravenous, intramuscular,intraperitoneal, intrasternal, subcutaneous and intraarticularinjection and infusion.

The pharmaceutical composition is also suitably administered bysustained release systems. Suitable examples of sustained-releasecompositions include semi-permeable polymer matrices in the form ofshaped articles, e.g., films, or microcapsules. Sustained-releasematrices include polylactides (U.S. Pat. No. 3,773,919, EP 58,481),copolymers of L-glutamic acid and gamma-ethyl-L-glutamate (Sidman,U. et al., Biopolymers 22:547-556 (1983)), poly(2-hydroxyethylmethacrylate) (R. Langer et al., J. Biomed. Mater. Res. 15:167-277(1981), and R. Langer, Chem. Tech. 12:98-105 (1982)), ethylenevinyl acetate (R. Langer et al., Id.) orpoly-D-(-)-3-hydroxybutyric acid (EP 133,988). Sustained releasepharmaceutical composition also include liposomally entrappedcompound. Liposomes containing the pharmaceutical composition areprepared by methods known per se: DE 3,218,121; Epstein et al.,Proc. Natl. Acad. Sci. (USA) 82:3688-3692 (1985); Hwang et al.,Proc. Natl. Acad. Sci. (USA) 77:4030-4034 (1980); EP 52,322; EP36,676; EP 88,046; EP 143,949; EP 142,641; Japanese Pat. Appl.83-118008; U.S. Pat. Nos. 4,485,045 and 4,544,545; and EP 102,324.Ordinarily, the liposomes are of the small (about 200-800Angstroms) unilamellar type in which the lipid content is greaterthan about 30 mol. percent cholesterol, the selected proportionbeing adjusted for the optimal therapy.

For parenteral administration, the pharmaceutical composition isformulated generally by mixing it at the desired degree of purity,in a unit dosage injectable form (solution, suspension, oremulsion), with a pharmaceutically acceptable carrier, i.e., onethat is non-toxic to recipients at the dosages and concentrationsemployed and is compatible with other ingredients of theformulation.

Generally, the formulations are prepared by contacting thecomponents of the pharmaceutical composition uniformly andintimately with liquid carriers or finely divided solid carriers orboth. Then, if necessary, the product is shaped into the desiredformulation. Preferably the carrier is a parenteral carrier, morepreferably a solution that is isotonic with the blood of therecipient. Examples of such carrier vehicles include water, saline,Ringer's solution, and dextrose solution. Non aqueous vehicles suchas fixed oils and ethyl oleate are also useful herein, as well asliposomes. The carrier suitably contains minor amounts of additivessuch as substances that enhance isotonicity and chemical stability.Such materials are non-toxic to recipients at the dosages andconcentrations employed, and include buffers such as phosphate,citrate, succinate, acetic acid, and other organic acids or theirsalts; antioxidants such as ascorbic acid; low molecular weight(less than about ten residues) (poly)peptides, e.g., polyarginineor tripeptides; proteins, such as serum albumin, gelatin, orimmunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone;amino acids, such as glycine, glutamic acid, aspartic acid, orarginine; monosaccharides, disaccharides, and other carbohydratesincluding cellulose or its derivatives, glucose, manose, ordextrins; chelating agents such as EDTA; sugar alcohols such asmannitol or sorbitol; counterions such as sodium; and/or nonionicsurfactants such as polysorbates, poloxamers, or PEG.

The components of the pharmaceutical composition to be used fortherapeutic administration must be sterile. Sterility is readilyaccomplished by filtration through sterile filtration membranes(e.g., 0.2 micron membranes). Therapeutic components of thepharmaceutical composition generally are placed into a containerhaving a sterile access port, for example, an intravenous solutionbag or vial having a stopper pierceable by a hypodermic injectionneedle.

The components of the pharmaceutical composition ordinarily will bestored in unit or multi-dose containers, for example, sealedampoules or vials, as an aqueous solution or as a lyophilizedformulation for reconstitution. As an example of a lyophilizedformulation, 10-ml vials are filled with 5 ml of sterile-filtered1% (w/v) aqueous solution, and the resulting mixture islyophilized. The infusion solution is prepared by reconstitutingthe lyophilized compound(s) using bacteriostaticWater-for-Injection.

In one embodiment, a compound as defined herein above is for use intreating, preventing or ameliorating of a hyperplasticdisease/disorder. The present invention also relates to the use ofa compound as defined herein for the preparation of apharmaceutical composition for the treatment, prevention oramelioration of a hyperplastic disease/disorder.Hyperplasia/Hyperplastic diseases/disorders (malign and benign) arecharacterised by abnormal (i.e. hyperphysiological) high numbers ofcell divisions in an organ or tissue. Hyperplasia may be apathological or a physiological process which is however alwaysclearly distinguishable from hypertrophy. Hypertrophy is theenlargement of tissue or an organ by enlargement of single cells.Hyperplasia is the physiologic or pathological enlargement oftissues or organs due to an increase in the number of cells due tocell division. An important part of vein graft disease is a processcalled intimal hyperplasia, being a hyperplastic process, whichleads to narrowing of the vessel lumen and graft failure. Anotherprocess that contributes to vein graft disease is atherosclerosis,which is characterised by deposition of lipids in the vessel wall,infiltration of macrophages, foam cell formation and fatty streakformation, tissue remodelling which is associated with theproliferation and infiltration of the intima by smooth musclecells, deposition of extracellular matrix, and plaque formation. Inaddition, inflammatory processes crucially contribute toatherosclerosis, vein graft disease and hyperplasia. Theseprocesses may lead to plaque rupture which may lead to thethrombosis and or occlusion of the vein graft. In the process ofatherosclerosis proliferation of smooth muscle cells plays animportant role, also this proliferation is a hyperplasticprocess.

A preferred hyperplastic disease to be treated, prevented orameliorated in accordance with the present invention is intimalhyperplasia and/or vein graft disease. Vein graft disease ischaracterised by intimal hyperplasia i.e. the abnormally increasedproliferation of smooth muscle cells in the intima and media of thevessel wall. Preferably, the hyperplastic disease/disorder ishyperplasia. The hyperplasia may be intimal hyperplasia. In apreferred embodiment the intimal hyperplasia is stenosis orrestenosis. The intimal hyperplasia may also be atherosclerosis.The meaning of the terms "hyperplasia", "intimal hyperplasia","stenosis", "restenosis" and "atherosclerosis" is well known in theart and may be deduced from standard textbooks such as "Handbook ofCoronary Stents" edited by P. W. Serruys and B. Rensing, 4thedition, published by Taylor & Francis, or from the "Handbookof Drug-eluting stents, edited by P. W. Serruys and A. H.Gershlick, published by Informa Healthcare. It is particularlyenvisaged herein that vein graft diseases may be treated inaccordance with the present invention.

The hyperplastic disease may also be a proliferative or neoplasticdisease. A proliferative disease is generally considered as adisease associated with uncontrolled/increased proliferation ofcells. Neoplasia or neoplastic diseases are charaterised by the newformation of tissues either as a physiological process or as apathological process. Typical pathological neoplastic diseases aretumors/cancers. Non-limiting examples of proliferative diseases arebenign proliferative diseases, such as benign proliferative breastdisease, cancerous disorders, like blood tumors, leukemia, as wellas solid tumors like B-cell lymphomas, myelotic cancer, prostatecancer, breast cancer, colon cancer, lung cancer and skincancer.

In one embodiment, the present invention relates to a method fortreating, preventing or ameliorating a hyperplasticdisease/disorder comprising the administration of a compound asdefined herein to a subject in need of such a treatment, preventionor amelioration. It is preferred that the subject is a human.

A preferred application form is a drug eluting stent systemdescribed herein may be a polymer based drug delivering system or apolymer coated drug delivering system. It is to be understood thatthe compound of formula (I) described and provided herein, and inparticular leoligin and/or (a) ((di)methoxy)-derivative(s) thereof,is applied to the drug delivering system in combination with (a)polymer(s). Therefore the drug component (the active ingredient) isembedded in a non-erodible polymer carrier (base coat formulation)which is surrounded by a suitable topcoat layer to control therelease of the embedded drug. A possible application form would bea system containing parylene C and the following two non-erodiblepolymers: polyethylene-co-vinyl acetate (PEVA) and poly n-butylmethacrylate (PBMA). A combination of the two polymers (67%/33%)mixed with the compound of formula (I) described and providedherein, and in particular leoligin and/or (a)((di)methoxy)-derivative(s) thereof, makes up the basecoatformulation which is applied to a parylene C treated stent. Adrug-free topcoat of PBMA polymer is applied to the stent surfaceto control the release kinetics of the compound of formula (I)described and provided herein, and in particular leoligin and/or(a) ((di)methoxy)-derivative(s) thereof. Alternatively a singlelayer polymer e.g. a Translute.RTM. polymer carrier, might be usedas drug delivering matrix. The drug/polymer coating is adhered tothe entire surface (i.e., luminal and abluminal) of the stent.

In one embodiment, the present invention relates to a medicaldevice comprising, containing or having been contacted with acompound as described herein, i.e. a compound with formula (I), inparticular leoligin.

Preferably, the medical device is a drug delivering system. Such adrug delivering system may, for example, be a balloon catheter. Aballoon catheter is a kind of a catheter having an inflatableballoon at its tip. The balloon catheter can be used to widen anarrow opening or passage within the human or animal body, and maybe of particular advantage in the treatment of a hyperplasticdisease like e.g. intimal hyperplasia, restenosis, stenosis or veingraft disease. In a first step, the deflated balloon catheter ispositioned at the side to be widened and is then in a second stepinflated. After widening the opening or passage the balloon isdeflated and can then easily be removed. It is envisaged that aballoon used in this context may be coated or may contain thecompound of formula (I), in particular leoligin and/or its((di)methoxy)-derivative(s). These compounds may be delivered tothe cells or tissue surrounding the balloon during and/or uponinsertion, inflation and deflation of the balloon. A balloon asdescribed herein above may be particularly beneficial in thiscontext since it can be completely removed after the wideningprocedure, reducing the possible occurrence of inflammatoryresponses.

The drug delivering system described herein may be a polymer baseddrug delivering system or a polymer coated drug delivering system.It is to be understood that the compound of formula (I) describedand provided herein, and in particular leoligin and/or((di)methoxy)-derivative(s) thereof, is applied to the drugdelivering system in combination with (a) polymer(s). Preferably,the drug delivering system provides for a sustained release of thecompounds. Non-limiting examples of polymer to be used for coatingthe drug delivering systems are polyethylene glycol; polystyrene;polyurethane; poly(hydroxyvalerate); poly(L-lactic acid);polycaprolactone; poly(lactide-co-glycolide);poly(hydroxybutyrate); poly(hydroxybutyrate-co-valerate);polydioxanone; polyorthoester; polyanhydride; poly(glycolic acid);poly(D,L-lactic acid); poly(glycolic acid-co-trimethylenecarbonate); polyphosphoester; polyphosphoester urethane; poly(aminoacids); cyanoacrylates; poly(trimethylene carbonate);poly(iminocarbonate); copoly(ether-esters) (e.g., PEO/PLA);polyalkylene oxalates; polyphosphazenes; biomolecules, such asfibrin, fibrinogen, cellulose, starch, collagen and hyaluronicacid; polyurethanes; silicones; polyesters; polyolefins;polyisobutylene and ethylene-alphaolefin copolymers; acrylicpolymers and copolymers; vinyl halide polymers and copolymers, suchas polyvinyl chloride; polyvinyl ethers, such as polyvinyl methylether; polyvinylidene halides, such as polyvinylidene fluoride andpolyvinylidene chloride; polyacrylonitrile; polyvinyl ketones;polyvinyl aromatics, such as polystyrene; polyvinyl esters, such aspolyvinyl acetate; copolymers of vinyl monomers with each other andolefins, such as ethylene-methyl methacrylate copolymers,acrylonitrile-styrene copolymers, ABS resins, and ethylene-vinylacetate copolymers; polyamides, such as Nylon 66 andpolycaprolactam; alkyd resins; polycarbonates; polyoxymethylenes;polyimides; polyethers; epoxy resins; polyurethanes; rayon;rayon-triacetate; cellulose; cellulose acetate; cellulose butyrate;cellulose acetate butyrate; cellophane; cellulose nitrate;cellulose propionate; cellulose ethers; and carboxymethylcellulose.

Preferably, the medical device provided herein is a stent. Incontext of the present invention the term "stent" means a medicaldevice that is inserted into a natural conduit of the body toprevent or counteract a disease-induced localized flowconstriction. Stents provided herein may preferably be used in thetreatment of (a) hyperplastic disease(s)/disorder(s), in particular(a) vein graft disease(s). The meaning of the term "drug elutingstent" is well known in the art and may, for example, be deducedfrom the "Handbook of Coronary Stents" edited by P. W. Serruys andB. Rensing, 4.sup.th edition, published by Taylor & Francis, orfrom the "Handbook of Drug-eluting stents, edited by P. W. Serruysand A. H. Gershlick, published by Informa Healthcare. Based on hisgeneral knowledge and the teaching provided herein and in standardtext books, like the above-mentioned Handbook of Coronary Stents, aperson skilled in the art is readily in the position to develop andprepare a stent and in particular a drug eluting stent comprising,containing or having been contacted with a compound of formula (I),in particular leoligin and/or its ((di)methoxy)-derivative(s). Askilled person will be aware of methods for coating the stent withthe herein described and provided compound, whereby the coatingallows delivery of the compound to cells and/or tissue, e.gendothelial cells/endothelium. The stent, in particular the drugeluting stent, to be used herein may also be a biodegradable stent,i.e. the stent is degraded/dissolved some time after insertion ofthe stent in a passage to be widened as described herein above.

In a preferred embodiment of the present invention, the medicaldevice is used for the delivery of a drug, wherein the drug is thecompound provided herein, i.e. the compound of formula (I) and inparticular leoligin and/or ((di)methoxy)-derivative(s) thereof.Therapeutic systems which are used in the art as "drug deliveringsystem", in particular as "drug eluting stent" contain the agentsPaclitaxel (Taxol.RTM.) or Sirolimus which are structurally notrelated to the compounds to be used in context of the presentinvention. Further, these agents exhibit their effect in acompletely different way compared to the present compounds offormula (I), and in particular, leoligin and/or its((di)methoxy)-derivative(s).

Paclitaxel promotes the assembly of microtubules from tubulindimers and stabilizes microtubules by preventing depolymerization.This stability results in the inhibition of the normal dynamicreorganization of the microtubule network that is essential forvital interphase and mitotic cellular functions Premarket ApprovalApplications (PMA) of the FDA; P030025: TAXUS.TM. Express 2.TM.Paclitaxel-Eluting Coronary Stent System (Monorail andOver-the-Wire). Issued Mar. 4, 2004; Part 2--Summary of Safety andEffectiveness Data. http://www.fda.gov/cdrh/pdf3/P030025.html. Themechanism (or mechanisms) by which a CYPHER.TM. Stent (Sirolimus isthe active agent contained therein) exerts its effect on neointimaproduction as seen in clinical studies has not been established.Sirolimus inhibits T-lymphocyte activation and smooth muscle andendothelial cell proliferation in response to cytokine and growthfactor stimulation. In cells, sirolimus binds to the immunophilin,FK Binding Protein-12 (FKBP-12). The sirolimus-FKBP-12 complexbinds to and inhibits the activation of the mammalian Target ofRapamycin (mTOR), leading to inhibition of cell cycle progressionfrom the GI to the S phase Premarket Approval Applications (PMA) ofthe FDA; P020026: Cypher sirolimus-eluting coronary stent on theraptor over-the-wire delivery system or raptorrail rapid exchangedeliver. Issued Apr. 24, 2003; Part 2--Summary of Safety andEffectiveness Data. http://www.fda.gov/cdrh/pdf2/P0020026.html.

The compound provided herein, such as compounds of formula (I) andin particular leoligin, may also be used in liquids intended forrising and/or storing (a) venous bypass(es), particularly beforeand/or during bypass operations. The rinsing and/or storage of thevenous bypass(es) in such a liquid is particularly advantageoussince such a kind of "pretreatment" of the bypass(es) may preventdamage of endothelial cells and/or inhibit the pathologicalproliferation of smooth muscle cells, and consequently reduce theprobability of hyperplastic disease/disorder, in particular intimalhyperplasia, stenosis, restenosis or vein-graft disease.

Accordingly, a rinsing and/or storage solution for a venous bypassis provided herein which comprises the compound of formula (I) andin particular leoligin and/or its ((di)methoxy)-derivative(s). Thestorage solution may, in addition to the compound described herein,comprise further components, such as stabilizers, preservingagents, buffering agents, salts (like NaCl), osmotically activecompounds, proteins (like albumin). It is to be understood that therinsing and/or storage solution may comprise only one compound offormula (I) or, optionally, different compounds of formula (I),like leoligin and/or different (a) ((di)methoxy)-derivative(s) ofleoligin. The rinsing/storage solution may comprise the same molarconcentration of these different compounds, or, alternatively,different concentrations. For example a first compound may bepresent at a double concentration compared to a second compound.Preferably, the molar concentration of the compound in therinsing/storage solution is between 1 and 500 .mu.M, preferablybetween 10 and 200 .mu.M preferably 50 .mu.M. It is envisagedherein that the leak tightness of the venous bypass(es) rinsed withor stored in the solution as described herein above is to be testedwith a device suitable for such a test. Leak tightness tests ofvenous bypass(es) are well known in the art and a person skilled inthe art is therefore aware of corresponding means and methods toperform such a test.

The compound(s) of formula (I) may also be applied periadventiallyto (a) venous bypass(es) before, during and/or after a bypassoperation, whereby the compound(s) may, optionally, be applied witha gel (e.g. Pluronic gel) or without a gel.

The present invention also relates to a method for rinsing and/orstoring a venous bypass comprising contacting the venous bypasswith the compound of formula (I), in particular leoligin and/or (a)((di)methoxy)-derivative(s) thereof.

The present invention is further described by reference to thefollowing non-limiting figures and examples.

BRIEF DESCRIPTION OF THE DRAWINGS

The Figures show:

FIG. 1. Leoligin is a Constituent of Edelweiss (Leontopodiumalpinum Cass.) Roots

Edelweiss is one of the most popular alpine plants and is also usedin folk medicine for the treatment of indigestion, fever, and"abdominal aches". FIG. 1A shows the flower of Edelweiss(Leontopodium alpinum Cass.). FIG. 1B shows the chemical structureof leoligin-IUPAC name:[(2S,3R,4R)-4-(3,4-dimethoxybenzyl)-2-(3,4-dimethoxyphenyl)tetrahydrofura-n-3-yl]methyl (2Z)-2-methylbut-2-enoat. Leoligin is a lignan, whichwas isolated from the roots of Edelweiss (Leontopodium alpinumCass.).

FIG. 2. Leoligin Inhibits Intimal Hyperplasia of Human SaphenousVeins In Vitro

The diagram in FIG. 2A summarises the data from experiments wherehuman saphenous veins were induced to develop intimal hyperplasiain organ culture. Tissue samples were incubated with DMSO (solventcontrol) or various concentrations of leoligin for 2 weeks. Afterthe incubation tissues were fixed, dehydrated, and embedded inparaffin. After preparation of sections, samples were stained(Elastica van Giesson stain), and the intimal thickness wasmeasured using Image J software. Approximately 30 measurements persample and a total of 5 samples (different donors) perconcentration were analysed. Shown are mean values (inpixel)+/-S.D. Baseline samples were fixed directly afterpreparation of fresh tissue and represent the status of the vesselprior to organ culture.

** . . . p<0.01; *** . . . p<0.001.

FIG. 2B shows the effect of the presence or absence of leoligin onorgan culture-caused intimal thickening of a representative sampleof a saphenous vein with mild pre-existing intimal hyperplasia(upper row, left: baseline; upper row, right, control; lower row,left: leoligin 5 .mu.M; lower row, right: leoligin, 50 .mu.M). Thearea between the arrows indicates the intimal thickness.

FIG. 3. Leoligin Inhibits SMC Proliferation, Induces a Cell CycleArrest in the G1 Phase and Leads to an Accumulation of p27/KIP

In order to investigate the effect of leoligin on the cellularlevel, isolated primary human vascular smooth muscle cells (SMCs)were incubated with the indicated concentrations of leoligin forthe indicated times. The diagram in FIG. 3A shows an analysis ofcell viability determined by the annexin V/propidium iodide methodand FACS analyses. Values shown are mean values from arepresentative experiment performed in triplicates. The diagram inFIG. 3B shows an analysis of cell proliferation by the XTT assay.Values shown are mean values from three independentexperiments+/-S.D. The histogram blots in FIG. 3C show the resultsfrom a DNA content analysis of control-treated or leoligin-treatedSMCs after 24 hours of incubation (upper row, left histogram . . .solvent control; upper row, right histogram . . . leoligin). TheWestern blot (lower row) shows an analysis of p27/KIP of SMCsincubated with leoligin for 24 hours with the indicatedconcentrations of leoligin.

FIG. 4. Leoligin is not Toxic for ECs and InhibitsTNFalpha-Mediated VCAM Expression.

The effects of leoligin on endothelial cells is shown in FIG. 4.Primary human vascular endothelial cells (ECs) were incubated withthe indicated concentrations of leoligin for the indicated times.The diagram in FIG. 4A shows a representative analysis of cellviability determined by the annexin V/propidium iodide method andFACS analyses. Values shown are mean values from a representativeexperiment performed in triplicates. The diagram in FIG. 4B showsan analysis of EC proliferation by the XTT assay. Values shown aremean values from three independent experiments+/-S.D. The impact ofleoligin on TNFalpha-induced surface expression of VCAM-1, ICAM-1,and E-selectin (E-Sel) is shown in the three diagrams in FIG. 4C.Data shown are mean fluorescence intensities (MFI) ofrepresentative experiments. The lower right image in FIG. 4C showsa metabolic protein labelling analysis of EC in the presence of theindicated concentrations of leoligin. After protein preparation,total cellular proteins were separated on polyacrylamide gels, gelwere dried and analysed by exposure to x-ray films.

FIG. 5. Leoligin Inhibits Neointima Formation In Vivo, withoutCausing Endothelial Damage.

The effects of leoligin on intimal hyperplasia of venous bypassconduits in vivo are shown in FIG. 5. The diagram in FIG. 5Adisplays a morphometric analysis of intimal thickness of vena cavainterposed into the carotid artery of control-treated animals andleoligin-treated animals. Before wound closure after thetransplantation, a peradventitial depot of 100 .mu.l of 0.9% NaCl(control) or 100 .mu.l of 100 .mu.M leoligin in 0.9% NaCl wereapplied. After 4 weeks conduits were removed and subjected tomorphometric analyses and to immunohistochemistry. FIG. 5B: theupper two images show a Elastica van Giesson stain of sections ofthe venous conduits. White arrows indicate neointimal thicknesswhich was analysed by morphometry (see FIG. 5A). Central imagesdisplay a staining for the endothelial cell CD31/PECAM-1 antigen(brown, black arrows) combined with a hematoxilin stain. Lowerimages display a immunohistochemical staining of the sections forthe cell cycle inhibitor p27/KIP-1 (brown) combined with ahematoxilin stain. The abbreviations "Lu" and "Li" stand for thefull terms "lumen" and "liver", respectively.

FIG. 6. SMC Comparison

The diagram in FIG. 6 shows a comparison of the smooth muscle cell(SMC) proliferation-inhibitory activity of lariciresinol andleoligin by the XTT assay. Values shown are mean values from threeindependent experiments+/-S.D.

FIG. 7. SMC Comparison

The diagram in FIG. 7 shows a comparison of the smooth muscle cell(SMC) proliferation-inhibitory activity of leoligin and the twoindicated derivatives by the XTT assay. Values shown are meanvalues from three independent experiments+/-S.D.

FIG. 8. Pathway

FIG. 8 shows a possible synthetic pathway of leoligin.

The following Examples illustrate the invention.

EXAMPLE 1: LEOLIGIN INHIBITS INTIMAL HYPERPLASIA OF VENOUS BYPASSGRAFTS

Material and Methods

General

All reagents used were of purissimum or analytical grade qualityand were purchased from Sigma Aldrich (Sigma-Aldrich, Vienna,Austria) if not specified otherwise. Water was produced by reverseosmosis followed by distillation.

Plant Material, Isolation, and Purification of Leoligin

Ground roots (1907.84 g) from L. alpinum Cass. were exhaustivelymacerated with dichloromethane (12.5 l DCM, at RT, eight times).Voucher specimens are deposited at the herbarium of the Institutfer Pharmazie/Pharmakognosie, Leopold-Franzens-UniversitatInnsbruck. Extracts were evaporated to dryness yielding 43.0 gcrude dichloromethane extract. 40.0 g of the obtained crude extractwere redissolved in 100 ml MeOH and separated in a MeOH soluble andinsoluble part. The soluble part was separated by Sephadex.RTM. LH20 (Pharmacia Biotech, Sweden) CC (90.times.3.5 cm) with MeOH asmobile phase yielding 8 fractions. Fraction 5 (15.13 g; 320-410 mlelution volume) was rechromatographed by silica CC (180 g,41.times.3.5 cm) using a PE-acetone gradient with an increasingamount of acetone yielding 40 fractions (A-1 to A-40). A smallamount (28.2 mg) of Fraction A-21 (PE/acetone, 85:15; 441.2 mg) wasseparated by semi preparative HPLC (Phenomenex Synergy Max-RPcolumn (10 .mu.m, 10.times.250 mm); 55% acetonitrile/45% water,isocratic; flow: 3.50 ml/min; 25.degree. C.) yielding 16.0 mg pureleoligin and i4.0 mg of its 5-methoxy derivative. A small amount(26.5 mg) of Fraction A-22 (PE/acetone, 85:15; 77.8 mg) wasseparated by semi preparative HPLC (Waters X-Terra Prep MS C18, 5.mu.m, 7.8.times.100 mm column; 70% MeOH/30% water, isocratic;flow: 1.50 ml/min; 25.degree. C.) yielding 6.3 mg of the5,5'-dimethoxyderivative of leoligin.

Preparation of Extracts Enriched in Leoligin and itsMethoxy-Derivative

In order to quantify the content of leoligin and itsmethoxy-derivative in different extract preparations severalextraction procedures were used. Therefore ground roots (20.00 g)from L. alpinum Cass. were exhaustively macerated withdichloromethane (100 ml DCM, at RT, eight times). After filtrationthe obtained extracts were combined, evaporated to dryness to yielda semi solid DCM-extract. Other extracts were prepared byultrasonic extraction using 20.00 g ground roots which weresonicated for 15 min using 1.times.200 ml and 1.times.100 ml ofsolvent or a second ultrasonic extraction cycle after air drying ofthe plant material (5.00 g; 2.times.15 min; 2.times.100 mlsolvent). The leoligin content was determined by means ofHPLC-quantification using the method of external standard. Eachextract was prepared in duplicate and quantified in triplicate. Thecontent of 5-Methoxy-leoligin was calculated using the calibrationcurve of leoligin.

Cell Culture

Human umbilical vein endothelial cells (HUVECs) were isolated fromumbilical cords (kindly donated by the Gynecology and ObstetricsDepartment, Innsbruck Medical University) by enzymatic detachmentusing collagenase, as previously described; see Bernhard, FASEB J17(15), 2302-4 (2003). Human umbilical vein smooth muscle cells(SMCs) were isolated from the same umbilical cords according toChamley-Campbell, Physiol Rev 59(1), 1-61 (1979). SMCs wereroutinely passaged in 0.2% gelatine-coated (Sigma, Steinheim,Germany) polystyrene culture flasks (Becton Dickinson, MeylanCedex, France) in Medium 231 (Cascade Biologics, Paisley, UK). Theisolation and analysis of human umbilical cord ECs and SMCs hasbeen approved by the Ethics Committee of the Innsbruck MedicalUniversity.

Quantification of Cell Death and Cellular DNA Content

For detection and/or quantification of cell death, forward/sidewardlight scattering analysis, the AnnexinV-propidium iodide method,and staining of nuclear DNA content (cell cycle analyses) were usedas described; see Bernhard FASEB J 13(14), 1991-2001 (1999).

Analysis of Cellular Proliferation

Cell proliferation was measured by the XTT cell proliferation assay(Biomol, Hamburg, Germany) as described by the manufacturer. TheXTT assay is based on the ability of metabolic active cells toreduce the tetrazolium salt XTT to orange coloured compounds offormazan. The dye formed is water soluble and the dye intensity canbe read at a given wavelength with a spectrophotometer. Theintensity of the dye is proportional to the number of metabolicactive cells. In addition to the XTT assay allproliferation-experiments were also evaluated by counting cells ina coulter counter.

Surface Expression Analyses ICAM, VCAM, and E-Selectin.

FACS-based analyses of surface adhesion molecule expression wasperformed according to a protocol by Grabner et al. (see Grabner,Cytometry 40(3), 238-44 (2000). Antibodies used were anti-VCAM-1antibody (clone 1.4 C3, Neomarkers), anti-ICAM-1 antibody (clone28, DAKO Cytomation), and anti-E-selctine antibody (clone 16G4;Novo castra).

Western Blotting

Western blotting was performed as previously described; seeBernhard, Cell Death Differ 8(10), 1014-21 (2001). Primaryantibodies used were anti-p27/KIP-1 antibody (clone 57; BDTransduction Laboratories).

Metabolic Labelling of Proteins

Metabolic labelling with .sup.35S-methionine/cysteine was performedas previously described (Bernhard (2001), loc. cit.).

Human Saphenous Vein Organ Culture

For organ culture experiments, remnants (surgical waste) ofsaphenous veins from patients undergoing CABG were collected. Thesaphenous veins were opened longitudinally and attached to siliconepatches (with the endothelium facing upwards). Tissue pieces wereincubated with culture medium (RPMI1640, 30% serum, 8 heparinmU/ml, antibiotics) for 2 weeks in the presence or absence ofleoligin. to induce intimal hyperplasia. For details see Schachner,Eur J Cardiothorac Surg 32, 906-911 (2007). Leoligin was addedfreshly every second day to organ cultures over a time period of 2weeks. 5 .mu.M leoligin completely inhibited intimal hyperplasia(p=0.003), and 50 .mu.M even reversed pre-existing intimalhyperplasia of saphenous veins (p<0.001). The use of humansaphenous veins has been approved by the Ethics Committee of theInnsbruck Medical University.

Mouse Model--Vein Graft Disease

In the applied mouse model the vena cava of a donor mouse wasinterposed into the carotid artery of a recipient mouse. Aftertransplantation and prior to wound closure a 100 .mu.l depot of0.9% NaCl (control group) or 100 .mu.M leoligin in 0.9% NaCl(leoligin group) was placed around the adventitia of the vein graftin the recipient mouse. 4 weeks after the intervention mice weresacrificed and the interposed venae cavae were harvested foranalyses. Of the 7 animals per group 3 control and 2leoligin-treated animals showed no blood flow due to thrombosesafter 4 weeks and were consequently excluded from further analyses.For details on the surgical procedure see Schachner, Eur JCardiothorac Surg 30(3), 451-63 (2006); Schachner, Heart Surg Forum9(1), E515-E517 (2006). Animal experiments were approved by theCommission for Animal Testing Affairs of the Austrian Ministry forScience and Research.

IHC and Morphometric Analyses

Following fixation in 4% paraformaldehyde and dehydration oftissues from organ culture and mouse experimentation, tissues wereembedded in paraffin (venae cavae from the in vivo experiments wereembedded into mouse liver prior to fixation) and sections wereprepared. Immunohistochemistry was performed with the AccustainElastica Stain (HT25) kit (Sigma-Aldrich, USA) or the EnVision+System-HRP (DAB) (DakoCytomation, Denmark) according to themanufacturers instructions. Primary antibodies used wereanti-p27/KIP-1 antibody (clone 57; BD Transduction Laboratories),and anti-CD31/PECAM-1 antibody (clone JC70A, Dako Cytomation).Image analyses were performed using the Image J software of theNational Institute of Health (USA).

Results

Preparation of Extracts Enriched in Leoligin and IstMethoxy-Derivates

Root extracts comprising leoligin and its derivates5-Methoxy-leoligin und 5,5'-dimethoxy-leoligin have been preparedas described herein above (i.e. roots macerated at room temperatureand extracted using dichlormethane), whereby the yield of theextract lies typically in the range of between 1.03 bis 2.26% andwhereby the maximum level of the leoligin and methoxy-leoligincontent (quantified as a mixture thereof) is 2.14%.

In order to obtain extracts enriched in leoligin and its derivates,the plant material is in a first step extracted with hexane orheptane followed by a subsequent extraction with organic solventsdichloromethane, chloroform or ternary butyl methyl ether. Theresults are summarized in the table below.

TABLE-US-00001 Total lignan Pre- Leoligin content in treatmentYield of content the extract solvent Extraction extract (w %) in(calculated as (defatting) solvent (w %) the extract leoligin; w %)-- dichloro- 1.03-2.26% 0.77-1.36% 1.69-2.14% methane* --n-hexane** 0.07-0.15% 0.67% 1.47% -- n-heptane** 0.12-0.27% 0.74%1.55% n-hexane dichloro- 0.50% 1.32% 2.67% methane*** n-hexanechloroform*** 0.65% 1.31% 2.71% n-hexane tBMe*** 0.39% 1.19% 2.43%*exhaustive mazeration (20.00 g; 8 .times. 100 ml); **ultrasonicextraction (20.00 g; 15 min; 1 .times. 200 ml; 1 .times. 100 ml);***ultrasonic extraction (5.00 g; 2 .times. 15 min; 2 .times. 100ml).

"Yield of extract" refers to the weight of the extract vs. thebasic material used, where a leoligin content refers to thepercentage by weight of Leoligin and lignan, respectively, in theextract.

The concentration of lignans (and derivatives) was also increasedby the use of Sephadex-LH20-column chromatography (increase in theleoligin content from 0.77% to 2.21%). The most pronounced increase(increase in the leoligin content from 1.36% auf 9.76%) wasachieved using silica gel column chromatograph (mobile phase:Petroleum ether-aceton).

Leoligin a Compound from Edelweiss Potently Inhibits IntimalHyperplasia in a Human Saphenous Vein In Vitro Model.

Leoligin[(2S,3R,4R)-4-(3,4-dimethoxybenzyl)-2-(3,4-dimethoxyphenyl)tetra-hydrofuran-3-yl]methyl (2Z)-2-methylbut-2-enoat (see FIG. 1) acompound that was previously isolated from the roots of Edelweiss(Leontopodium alpinum Cass.) is a lignan type secondary plantmetabolite. In our organ culture-based screen for compounds capableof inhibiting human saphenous vein intimal hyperplasia, leoliginshowed a profound inhibitory activity (see FIG. 2). Leoligin whenadded to organ cultures over a time period of 2 weeks (addedfreshly every second day) inhibited intimal hyperplasia in adose-dependent fashion. 5 .mu.M leoligin completely inhibitedintimal hyperplasia (p=0.003), and 50 .mu.M even reversedpre-existing intimal hyperplasia of saphenous veins (p<0.001)(see FIG. 2).

Leoligin Inhibits SMC Proliferation by Inducing a Cell Cycle Arrestin the G1 Phase which is Associated by a Shift in Molecular Weightand an Accumulation of p27/KIP Protein

In order to reveal the mechanism underlying leoligin-mediatedinhibition and reversal of saphenous vein intimal hyperplasia weanalysed the effects of leoligin on isolated primary human vascularsmooth muscle cells, which represent the central cell type inintimal thickening and intimal hyperplasia (SMC proliferation, andmigration). Our analyses clearly showed that leoligin causes only amarginal increase in cell death (apoptosis and necrosis) in SMCsafter 48 hour (FIG. 3, upper panel, left side). Analyses at latertime points were consistent with the findings after 48 hours (datanot shown). In contrast, analyses of cell numbers by the XTTproliferation assay (FIG. 3, upper panel, right side) as well as byCasy-based cell counting (data not shown) revealed a significantSMC proliferation inhibitory effect of leoligin. Consequentmeasurements of nuclear DNA content (FIG. 3, two lower panels, leftside) revealed that leoligin causes a massive accumulation of cellsin the G1-phase of the cell cycle. Since it is well known that anumber of cell cycle regulators can cause a G1 arrest, we performeda Western blot based analyses for changes in G1 arrest-relevantcell cycle regulators. The lower right panel of FIG. 3 shows aWestern blot demonstrating that leoligin induces a complete changein the appearance of p27/KIP protein i.e. from one signal at 27 kDto an intensive signal at 58 kD, and three weak signals at 24, 27(original signal) and 85 kD (the 85 kD signal was hardly visibleand is not shown). The accumulation of p27/KIP is well known tomediate a G1-arrest by binding to and thereby inactivating thecyclin D1/E-cdk2 complex. The observed shift in molecular weightmay indicate the binding of p27/KIP to this complex or anoligomerisation of p27/KIP molecules.

Leoligin is not Toxic for ECs and Inhibits TNFalpha-Mediated VCAM-1Expression.

Since the integrity of the vascular endothelium plays a centralrole in vascular repair and healing, anti-thrombosis, and the graftatherosclerosis-relevant control of cell (macrophages) and compound(cholesterol) exchange between the blood and the vessel wall wealso analysed the effects of leoligin on the vascular endothelium.Although leoligin also inhibited the proliferation of ECs no toxicor cell death-inducing effect of leoligin on ECs could be observed(see FIG. 4, left panels). Analyses at later time points wereconsistent with the findings after 48 hours (data not shown).Interestingly, leoligin inhibited TNFalpha-mediated surfaceexpression of VCAM-1 (upper right panel). To exclude that generalphenomena, like an inhibition of the endothelial translationalmachinery by leoligin accounts for this observation, we performedmetabolic protein labelling experiments (FIG. 4, lower rightpanel), and analysed the effect of leoligin on other adhesionmolecules (FIG. 4, right side, two central panels). These datademonstrate that leoligin does not block protein synthesis, anddoes not interfere with the translocation of proteins to thecellular surface in general, but specifically inhibits stimulatedVCAM-1 expression on the EC surface.

Leoligin Inhibits Neointima Formation In Vivo, without CausingEndothelial Damage.

To test the applicability of leoligin in vivo we analysed leoligineffects in a mouse model for vein graft disease (see Methodssection). Like in human saphenous vein bypass conduits thetransplant develops a severe intimal hyperplasia within a couple ofweeks. Leoligin was applied directly after the transplantation as aperiadvential reservoir (100 .mu.l of 100 .mu.M leoligin in 0.9%NaCl) before surgical closure of the wound. Four weeks after thetransplantation mice were sacrificed and the venous conduits wereremoved for further analyses. Thrombosed veins were excluded formfurther analyses (see Methods section) and the remaining sampleswere analysed by means of immunohistochemistry. FIG. 5 (left side)shows that leoligin treatment, in contrast to control-treatment(0.9% NaCl), potently inhibited intimal hyperplasia in vivo. Veingrafts of mice treated with leoligin showed significantly reducedneointimal thickness (upper central and upper right image). A CD31(endothelial marker) staining of the sections revealed that theendothelial layer of leoligin and control treated conduits wasintact. Finally, a p27/KIP staining of the sections revealed that alarge number of cells/nuclei in the leoligin-treated grafts stainedpositive for p27/KIP even four weeks after the application ofleoligin, indicating a long duration of drug effect.

In the in vivo experiments described herein above leoligin wasadded directly after the surgical procedure. Thereafter, mice werekept for additional 4 week, only then samples were analysed. Sincea large number of SMCs in the leoligin group stained positive forp27/KIP the mechanism via which leoligin inhibits the proliferationof SMCs may be the induction of differentiation. In contrast tocell culture and in vivo experiments where leoligin was added onlyonce and at the beginning of the experiments, leoligin showedprofound inhibition of intimal hyperplasia in the in vitro organculture experiments (leoligin was added every second day) alreadyat a concentration of 5 .mu.M. These data suggest that a constantaddition of leoligin may improve the results but also that apositive therapeutic effect can be achieved by a single applicationof leoligin, the consequence of which being detectable even 4 weeksafter this application in vivo. With respect to the used ofsaphenous veins with pre-existent intimal hyperplasia for CABG,leoligin may help to increase patency rates simply by applyingleoligin to the grafts prior to re-implantation. In addition,differences in the potential to inhibit or reverse intimalhyperplasia by varying the dose and duration of treatment, may helpto reduce complications e.g. graft aneurysms which may occur as aresult of reduced adaptive tissue remodelling in the vesselwall.

The above provided assessment using an in vivo mouse model for veingraft disease can also be carried out in larger animals/animalmodels. An exemplary protocol (experimental set up and evaluationof data) using a porcine animal model (i.e. "Landschwein") toassess the efficacy of the particular compound known under thetrivial name "Leoligin" is given herein below.

Experimental Setup

Leoligin is used in a concentration of 100 .mu.M. Animals areseparated into two treatment groups (control, treatment).Furthermore, animals are separated into groups for a harvestingtime point after 4 weeks and 12 weeks, respectively. The treatmentschedule is illustrated in the following table:

TABLE-US-00002 Time of analysis Number Compound tested(postoperativ) of animals Treatment group 1 Control NaCl 4 weeks 6Leoligin NaCl + Leoligin 4 weeks 6 Total 12 Treatment group 2Control NaCl 12 weeks 6 Leoligin NaCl + Leoligin 12 weeks 6 Total12

Anaesthesia and Surgical Procedure--Vena Saphena as Carotis Graftin the "Landschwein" (Pig Race)

For transportation and to prevent narcosis complications animalsreceive in the cot an intramuscular injection of 4 mg/kg Azaperonand 0.1 mg/kg Atropin one hour prior to narcosis. Sleeping animalsare transported in a transport box with straw filler to theoperating theatre. Animals receive an infusion of 2-3 mg/kgPropofol and 15 mg Piritramid via the ear vein, followed byintubation and ventilation with 30% O.sub.2. Animals receive aninitial muscle relaxation 8 mg Pancuronium and repeatedly 0.2mg/kg/h Pancuronium. For maintenance of narcosis animals receive apermanent infusion of 8-12 mg/kg/h Propofol und 15 mg Piritramid.In order to prevent pain in the animals the dose may be increased.During the preparative phase animals receive in addition to theabove a permanent infusion with 6 ml/kg/h of Ringer-Lactate.

Surgical Procedure: Carotis-Graft

After the initiation of anaesthesia according to the above protocolanimals are kept in dorsal position, shaved, washed with Octinseptfor disinfection, and covered with sterile cloth.

For preparation of the saphenous vein a ca. 10 cm longitudinalincision is made on the hind leg of animals. Following theidentification of the vein, the vein is cleaned from surroundingtissue by "no-touch"-technique. Side arms are clipped and cutthrough. After the preparation of the vein by the above technique,the vein is removed and cannulated via the distal end. To test fortightness and to expand the vessel diameter to the proper size, thevein is expanded by flushing with a pressure of 80-100 mmHg.Flushing and expansion of the sephenous vein in control animals isdone using 0.9% NaCl-solution including 1.Salinity. DMSO, in thetreatment group a 100 .mu.M leoligin (or derivative thereof)solution in 0.9% NaCl-solution including 1.Salinity. DMSO is used(leoligin/derivative is first solubilised in DMSO (100 mM solution)and is then diluted 1:1000 in 0.9% NaCl-solution.). Afterexpansion, veins are incubated for 30 minutes to one hour at roomtemperature in the above solutions. After hind leg wound closure,the surgeon starts with the preparation of the neck region. Firstan incision is placed on the right jugular side medial of themusculus stemocleidomastoideus. After a blunt preparation and cutthrough of the platysma the trionum caroticum is identified. Underprotection of the vena jugularis interna and nervus vagus, thearteria carotis communis is prepared, and a 3 cm segment isisolated. Then 100 units of heparin/kg are injected intravenously.After placing of a vessel clamp at the proximal and distal side ofthe segment, the arteria carotis communis is cut through and thesurgical margin is trimmed to an angle of 45.degree.. A part of theprepared isolated vena saphena magna is shortened to thecorresponding length and is anastomosed on both sides by an end-endtechnique. The anastomose is sutured with 7/0 Prolene in continuostechnique. To increase the exposure time of the vessel to leoligin(or derivative), resorbable haemostypticum (Dabostemp clothes) aresoaked with 0.9% NaCl-solution including 1.Salinity. DMSO(control), or a 100 .mu.M leoligin (or derivative) solution in 0.9%NaCl-solution including 1.Salinity. DMSO (treatment group) arewarped around the interposed vein. The wound is closed in layers,then the skin is closed.

To keep the number of animals low the same procedure is repeated onthe left side according to the above protocol. After surgery,animals are transported to the animal keeping facility in a speciesappropriate manner and receive a pain therapy for additional 7 dayswith Dipidolor and Novalgin.

Harvesting-Surgery

Harvesting is performed 4 and 12 weeks after surgery. Anaesthesiaand transport, washing and covering with sterile clothes isaccomplished as described above. First the surgical scar on theright side is located and the site is re-opened. After thepreparation of the interposed vein vessel claps are places on theproximal and distal site, the vein is removed. The same procedureis accomplished on the left side. Animals are euthanized with abolus of Dormicum and Fenta, followed by an injection of 20 mValKCl.

Analyses

The following analyses are performed on the harvested vein graft:Histology: VanGiessen, Oil red, HE-staining; Immunohistochemistry:.beta.-Aktin, p27, p21, CD31, ICAM-1, VCAM-1, CD3, CD4, CD8, CD56,etc. Analysis is performed using the Image J Software. Evaluationand data analyses will be performed by blinded researchers; seeReisinger (2009) Cardiovasc Res. 82; 542-549, and Messner (2009),Arterioscler. Thromb. Vasc. Biol. Electron microscopical analyses(SEM/TEM) of the endothelial surface and the composition of thevessel wall; see Messner (2009), Arterioscler. Thromb. Vasc. Biol.,and Bernhard (2003), FASEB J. 17:2302-2304. Contractility andfunctionality of grafts are analysed in an organ bath; seeHammerer-Lercher A, Clin Sci (Lond). 2006; 111:225-231. Prior toharvesting the intima media thickness of grafts is analysed bysonography; see Knoflach (2003) Circulation; 108:1064-1069, andKnoflach (2009) Stroke; 40:1063-1069. Collection of blood and serumsamples for the detection of the test compound (i.e. leoligin).

Based on the above analyses, the following parameters/effectsdefine independently of each other a treatment success by Leoligin(or derivative): 1) An intima thickness and/or intima-mediathickness of the treatment group below the control group. 2) Asmaller number of smooth muscle cells in the intima of thetreatment group compared to the control. 3) The presence of ahigher number of p27 and or p21 positive cells in the treatmentgroup compared to the control. 4) A smaller degree of neointimaformation in the treatment group compared to the control. 5) Areduced presence of tissue remodelling processes in the treatmentgroup compared to the control. 6) A lower number ofpro-inflammatory cells in the vessel wall in the treatment groupcompared to the control. 7) An intact endothelium. 8) Aphysiological degree of contractility of the vessels. 9) A lowdegree of adhesion molecule expression on the endothelial surface.10) patency of grafts and no signs of thrombus formation. 10)Conserved elasticity of grafts in the treatment group compared tothe control. And 11) Conserved contractility of grafts in thetreatment group compared to the control.

EXAMPLE 2: LEOLIGIN IS A STRONGER INHIBITOR OF SMC (SMOOTH MUSCLECELL PROLIFERATION) THAN Lariciresinol

The diagram in FIG. 6 shows a comparison of the smooth muscle cell(SMC) proliferation-inhibitory activity of lariciresinol andleoligin by the XTT assay after 72 h. Values shown are mean valuesfrom three independent experiments+/-S.D. The obtained IC.sub.50value of leoligin (54.5 .mu.M; CI.sub.95: 49.4-59.4 .mu.M) wasfound to be more effective than the lariciresinol (IC.sub.50>100.mu.M).

Methods FIG. 6

Analysis of Cellular Proliferation

As described above (Example 1) cell proliferation was measured bythe XTT cell proliferation assay (Biomol, Hamburg, Germany) asdescribed by the manufacturer. The XTT assay is based on theability of metabolic active cells to reduce the tetrazolium saltXTT to orange coloured compounds of formazan. The dye formed iswater soluble and the dye intensity can be read at a givenwavelength with a spectrophotometer. The intensity of the dye isproportional to the number of metabolic active cells.

A use of lariciresinol in the context of stent implantation hasalso been proposed in DE 10 2004 046 244.

EXAMPLE 3: LEOLIGIN AND ITS METHOXY-DERIVATIVES INHIBIT SMCPROLIFERATION

The diagram in FIG. 7 shows a comparison of the smooth muscle cell(SMC) proliferation-inhibitory activity of leoligin and its naturalderivatives by the XTT assay after 72 h. Values shown are meanvalues from three independent experiments+/-S.D. The obtainedIC.sub.50 value of leoligin (54.5 .mu.M; CI.sub.95: 49.4-59.4.mu.M) was found to be not significant different from those of5'methoxyleoligin (45.9 .mu.M; CI.sub.95: 37.3-53.9 .mu.M) or5,5-dimethoxyleoligin (IC.sub.50 48.6 .mu.M; CI.sub.95: 39.9-56.6.mu.M).

Methods FIG. 7

Analysis of Cellular Proliferation

As described above (Example 1) cell proliferation was measured bythe XTT cell proliferation assay (Biomol, Hamburg, Germany) asdescribed by the manufacturer. The XTT assay is based on theability of metabolic active cells to reduce the tetrazolium saltXTT to orange coloured compounds of formazan. The dye formed iswater soluble and the dye intensity can be read at a givenwavelength with a spectrophotometer. The intensity of the dye isproportional to the number of metabolic active cells.

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References

• who.int/cardiovascular_diseases/en
• admin.ch/sar/rac
• fda.gov/cdrh/pdf3/P030025.html
• fda.gov/cdrh/pdf2/P0020026.html