WO1999049884A1 - A method for the prevention of a patient's fibroproliferative vasculopathy - Google Patents

Abstract

This invention relates to the use of an agonist specific for at least one somatostatin (SST) receptor showing an increased expression in a patient's vascular wall subsequent to injury or operation, for the manufacture of a pharmaceutical preparation. Said pharmaceutical preparation is intended for use in the treatment or the prevention of said patient's fibroproliferative vasculopathy following vascular injury or a vascular surgical operation. Said pharmaceutical composition is intended for administering to said patient for one to two weeks after the injury or surgical operation.

Description

A METHOD FOR THE PREVENTION OF A PATIENT'S FIBROPROLIFERATIVE VASCULOPATHY

FIELD OF THE INVENTION

This mvention relates to the use of an agonist specific for at least one somatostatin (SST) receptor showing an increased expression in the patient's vascular wall subsequent to vascular trauma, for the treatment or prevention of the patient's fϊbroproliferative vasculopathy such as chronic allograft rejection or vascular restenosis following vascular trauma.

BACKGROUND OF THE INVENTION

The publications and other materials used herein to illuminate the background of the invention, and in particular, cases to provide additional details respecting the practice, are incorporated by reference.

Fibroproliferative vasculopathy includes restenosis following coronary bypass surgery and PTCA (percutaneous transluminal coronary angioplasty). allograft arteriosclerosis in chronic allograft rejection, diabetic angiopathy and all forms of common arteriosclerosis.

Vascular intimal dysplasia and remodelling are characteristic features of rein jury following balloon angioplasty, coronaiy bypass surgery (Holmes et alJ984; Holmes et al. 1988) and in chronic allograft rejection (Lemstrom and Koskinen. 1997; Hayry et al. 1993). The initial response to vascular injury is inflammatory and involves attraction of lymphocytes, macrophages and thromocytes to the site of injury and secretion of cytokines, eicosanoids and growth factors (Ross 1993). Under the influence of growth factors and cytokines, smooth muscle cells (SMC) proliferate and migrate from the media to the inti a and contribute to intimal 2

hyperplasia and stenosis The key mediatoi s of SMC proliferation and migiation ai e IL- l . TNF PDGF IGF 1 bFGF EGF TGFβ and VEGF ( Asahara et al 1 95 Bornfeldt et al 1994. Feins et al 1991 , Libby and Gahs 1995. Gahs et al 1995. Gi onwald et al 1989, Hancock et al 1994, Hayry et al 1995. Lindnei and Reidin 1991, Myllarmerm et al 1997, Nabel et al 1993 , Shi et al 1996. Tanaka et al 1996) and the matrix metalloproteinases in SMC locomotion truough the extracellulai matrix (Bendeck et al 1996, Gahs et al 1995) In view of the central lole of SMC proliferation, therapeutic sϋategies designed to prevent stenosis have attempted to suppress SMC proliferation by blocking the production and action of growth tactoi s and cytokines with receptor antagomsts or antisense ohgonucleotides directed against cell cycle l egulatory molecules (Hayry et al 1995, Myllarniemi et l 199^"7 Sπois et al 1997 \\ πghton et al 1996) One important inhibitor of rmtogenic signalling is somatostatin (SST) (Gi ant et al 1994 Hong et al 1993 Yu i et al 1997)

The ne ohormone SST is produced widely in the body and acts both systemicalK via the circulation and locally to inhibit cell prohfeiation as well as the secretion of various hormones, growth factors and neurotransmitter substances SST and its metabolically moie stable synthetic analogs, such as SMS201 -995 (octreotide) and BIM23014 (lanreotide, angiopeptm), exert a numbei of vascular effects such as vasoconstrict on m the gut and inhibition of angiogenesis The actions of SST aie mediated by a family of five heptahelical G protein coupled receptors termed SSTR 1-5 All five SSTRs are functionally coupled to inhibition of adenylyl cyclase Some of the leceptor isotypes also modulate other effectors such as phosphotyiosine phosphatase, K and voltage-dependent Ca^~ ion channels, a

Na / H exchanger, phosphohpase Λ₂ and MAP kinase (MAPK.) Based on structuial similarity and the ability to ieact with octapeptide and hexapeptide SST analogs, the SST receptor family can be subdivided into two categones the SSTR2.3.5 category with high affinity to these analogs and the SSTR 1.4 category with low affinity to these compounds (see Table) In experiments using arterial, venous, and vascular transplant models in various ariimal species, tire administration of octreotide or lanreotide prevents the formation of dysplastic lesions (Foegh et al. 1994, Foegh and Ramwell 1995. Giant et al 1994, Hong et al. 1993; Yumi et al. 1997, Hayry et al 1993). These results, however, have been inconsistent in different experimental models In randomized placebo controlled clinical trials, lanreotide in some studies has been shown to prevent restenosis after percutaneous transluminal angioplasty as quantitated by angiography or as clinical events (Enksen et al 1995. Emanuelsson et al 1995 ). whereas the same therapeutic response has not been achieved with octreotide (von Essen et al 1997) Differences in the binding specificity of the SST analogs for the five SSTRs as w ell as the dose and duration of administration of SST analogs ma\ contribute m part to the inconsistent lesults obtained in these studies For instance octreotide and lanreotide both bind with high affinity to SSTR2 and SSTRS but display species-dependent variability in binding to SSTR3, octreotide binds well to human SSTR3 but shows only moderate affinity for the rodent receptor, whereas the opposite is the case for lanreotide. To optimize the vasculoprotective effect of SST. the ideal approach would be to characterize the pattern of expression of SSTRs in the vascular wall after trauma, and to target the subtypes involved with appropiate agonists Towards this objective, we have determined the time course of expression of mRNA for SSTR1 -5 in rat aorta after endothelial denudation (balloon injury) by reverse transcription polymerase chain reaction (RT-PCR) and localized the reseptors directly by lrrrmunocytochemistry with rabbit polyclonal antibodies to receptor subtype specific peptides

OBJECT AND SUMMARY OF THE INVENTION

The object of this invention is thus to characterize level of expression of somatostatin receptors (SSTRs) versus time in the intimal layer of the vascular wall after trauma (v ascular injury or vasculai suiger ) . and to target the receptoi subtypes showing increased expiession aftei trauma with appropiate agonists so as to avoid fibroprohfeiative vasculopathy of the patient's blood vessels which have been subjected to trauma

The mvention thus concerns the use of an agonist specific for at least one somatostatin (SST) receptoi showing an inci eased expi ession in a patient s v asculai wall subsequent to mjurv 01 operation, for the manufactuie of a pharmaceutical preparation for use in the treatment or the pi evention of said patient s fibroprohferative v asculopathy following vascular injury or a vascular suigical operation Said pharmaceutical composition is intended for administering to said patient foi one to two weeks after the injury oi suigical operation

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 shows Southern blots of mRNA RT-PCR products for the five SSTR subtypes at different times in control and in denuded aortic samples.

Figure 2 shows the mean expression level versus time for mRNA of five SSTR subtypes following vascular injury, and

Figure 3 shows the immunoreactivity of the five SSTR subtypes at diffeient times in control and in denuded aortic samples

DETAILED DESCRIPTION OF THE INVENTION

The expeπments carried out and discussed below were performed in rat The expression of the individual subtypes of the SST receptors are, however, believed to follow the same pattern in other mammals, including man According to a preferred embodiment, an SST receptor subtype which is present in the intimal layer of the vasculai wall and shows an increased expression din ing the first week following the injury 01 the surgical operation should be activated bv the agonist The process of wound healing is essentially completed after two weeks A prolonged activation of an highly expressed SST receptor after this point would not have any restenosis preventing action

According to a preferred embodiment, the SSTR 1 or SSTR4 receptoi should be activated

10

According to a preferred embodiment, the SS T receptor active agonist is a compound such as somatostatin SR1F- 14. somatostatin SR1F-28. or D AD1-SST 14 (des.AA ^' "[D-Trp \-p-ιsopropyl-4-amιnomethyl-L-phenylalanιne]-S T- 14) The structure and binding affinities of somatostatin and its analogues mentioned abov e

15 have been disclosed m Patel YC ( 1997)

The table below shows a companson of binding affinities K, (nM) somatostatin and its key cyclic analogs lanreotide and octeoπde for five human somatostatin receptors m transfected CHO cell lines The data are provided by YC Patel

20

Ligand hSSTR l hSSTR2 hSSTR3 hSSTR4 hSTR5

Somatostatin 2.2 4 1 6 1 1 1 0 07 (SRIF-28)

Somatostatin 1 1 1 3 1 6 0 53 0 9 (SRIF-14)

DADI- 3 2 >1000 > 1000 4 3 > 1000 SST 14

Lanreotide >1000 1 8 43 66 0 62

Octeoride >1000 2 1 4 4 > 1000 5 6

6

According to a pieferable embodiment, the selected SST-ieceptor is the SS Y- l eceptoi subtype 1 oi subtype 4 and the agonist is somatostatin SRI V- 14

The method according to this mvention is useful in the prevention of the patient^'s fibroproliferative vasculopathy following vascular trauma

According to one embodiment, the method accoiding to this invention is useful to prevent vascular restenosis following balloon angioplasty or a coronary bypass operation However, the invention is not restricted heieto The pattern of expression for the v arious SST receptoi subtypes found in the aorta is believed to exist thioughout the patient^'s vascular system The inv ention is ther efore applicable to the pi e ention of restenosis following anv v ascular tr auma in the patient^'s bod\

According to another embodiment, the method according to this invention is useful for the pievention of a patient's chronic allograft rejection

The term "SST receptor active agonist" shall be understood to include the compound as such as well as any pharmaceutically acceptable deπvative thei eot such as salt, ester etc

For the purpose of the mvention, the SST receptor active agonist can be administered by vaπous routes but pieferable by systemic routes The suitable administration forms include, for example, oial formulations, parenteral injections including inti avenous. intiamusculai . lntradermal and subcutanous injections, oi transdermal administration forms

The required dosage of the compounds of the SST ieceptoi agonist w ill v an, with the particular condition being treated, the seventy of the condition, the duration of the treatment, the administration loute and the specific compound being emplov ed 7

The therapeutically effective dose for a particulai compound can. for example, be in the range 1 - 10 μg pei kg of body weight and day The suitable period of treatment is one to two weeks after the trauma, perfeiably two weeks If the trauma is caused by surgical operation, the administration of the agent is pieferable started one da before the surgical operation

The invention is demonstrated by the following expeπments

MATERIALS AND METHODS

Aortic denudations

Male Wistar lats weighing 200-300 g were anesthetized with chloral hydiate ( 240 mg/kg l p ) The thoracic aorta was denuded of endothehum using a 2F Fogartv arterial embolectomy catheter (Baxter Healthcaie Corpoiation. Santa Ann. C A) The cathetei was introduced into the thoracic aorta via the left iliac artery, inflated with 0 2 ml an. and passed five times to iemove the endothehum The iliac artery as ligated and the animals allowed to lecovei (Clowes et al 1983 Haudenschild and Swartz 1979) Buprenorphine (Temgesic, Reckitt Colman, Hull England) w ^ admmistered for pen- and postopeiative pain relief Gioups of 3-5 rats wei e sacnficed at 15 min, 3 days, 7 days, and 14 days and 60 days and aortic tissue removed for evaluation of SSTR expression All animals received humane caie in compliance with the Pπnciples of Laboiatory Animal Caie and the Guide foi the Care and Use of Laboratory Animals prepaied and foπnulated by the National Institute of Health (NIH Publication No 86-23, revised 1985)

Three separate expeπments were performed In the fust experiment. 12 iats w ere denuded and 15 coded speciments of thoracic vascular tissue (3 conti ol 12 denuded. 3 specimens/time point) were collected foi RNA isolation and RT-PCR In the second expeπment, 20 iats weie denuded and 25 coded speciments weie 8

collected for RNA isolation and RT-PCR (5 specimens/time pojnt) Foui of these were used for RNA isolation and RT-PCR and the fifth specimen foi routine histology, quantitation of cell replication, and SSTR lmmunocytochemistry In the third expeπment, frozen sections of 20 aortas (4 control. 16 denuded) were processed for immunocytochemisti'y for SSTR 1 -5 The results described here derive from experiments 2 and 3

For RN A isolation, aortic tissue specimens were flash frozen in liquid nitr ogen and stored at -80 °C for R A isolation For e aluation of morphological changes, aortic cioss sections from the mid segment of the denuded aiea wei e fixed in 3°o paraformalhyde (pH 7 4), embedded in paraffin foi sectioning and stained with Mayei s haematoxylin and eosin (HE) Foi lmmunocytochemistry. aortic specimens were embedded m Tissue-Tek (Miles lnc . Elkhard, 1ND) and snap frozen in hguid nitrogen Serial frozen sections (4-6 μm) weie air dried on silane coated slides. fixed m acetone at -20 °C for 20 mm and stoied at -20°C until used

Revei se Transcription Polvmerase Chain Reaction (RT-PCR)

Weighed vascular tissue samples were pulverized in liquid nitrogen using a mortal and pestle and total RNA isolated usmg the standard technique of quanidinium lsothiocyanate-phenol-chloroform exti action For reverse transcription. 20 μg total RNA was treated with 10 units/mg RQ 1 Rnase-free Dnase l (Promega) in 40 mM Tns buffered HCl. pH 7.9, 10 mM NaC l . 6 mM MgC l₂. 10 mM CaC L foi 30 min at 37 °C The DNasel was inactivated by phenol chloroform extraction followed by ethanol precipitation Five μg of DNA-free RNA was then incubated in 20 ul reaction containing 20 mM Tπs-HC I , pH 8 4. 50 mM KC l , 5 mM MgC U 1 mM dNTPs. 20 units of RNasin (Promega). 100 pmol of random hexanucleotides (Pharmacia), 200 units of Moloney muπne leukemia virus (M M LV). revei se transcnptase (Gibco BRL) at 42 °C for 30 min Foui μl of the resulting cD\ A samples were denatured at 94 °C in 20 m\l Tris HCl, pH 8 4. 50 mM KC 1 1 5 mM MgCl .200 mM dNTPs, and 20 pmol each of SST1-5 pπmers in 50 μl reaction volume for 10 min. The following primers were used for the PCR amplification

rSSTRl Sense: 5^'ATGTTCCCCAATGGCACC 3^' (nt 1-18) (SEQ ID NO: 1 ) Antisense: 5^' CAGATTCTCAGGCTGGAAGTCCTC 3(nt 1093-1115)

(SEQIDNO:2) rSSTR2 Sense- 5^'AGCAACGCGGTCCTCACGTT 3^' (nt 124-143) (SEQIDNO:3)

Antisense: 5^' GGAGGTCTCCATTGAGGAGG 3 (nt 1077-1196) (SEQIDNO:4) rSSTR3 Sense 5^' ATGAGCACGTGCCACATGCAG 3^'(nt 565-585) (SEQID O:5)

Antisense: 5^' ACAGATGGCTCAGCGTGCTG 3^'(nt 1266-1286) (SEQIDNO:6) rSSTR4 Sense: 5^' ATGGTAACTATCCAGTGCAT 3^'(nt 127-147) (SEQIDNO:7)

Antisense: 5^' GTGAGGCAGAAGACACTCGTGAACAT 3^' ( nt 376- 401)(SEQIDNO:8) rSSTR5 Sense: 5 ^' TGGTCACTGGTGGGCTCAGC 3 (nt 70-89) (SEQIDNO:9)

Antisense: 5^' CCTGCTGGTCTGCATGAGCC 3^'(nt 1067-1086) (SEQ ID NO: 10) β-actin: Sense: 5 ^' ATCATGAAGTGTGACGTGGAC 3 ^'(nt 90- 110) (SEQIDNOJ1) Antisense: 5 ^' AACCGACTGCTGTC ACCTTCA 3 ^'(nt 529-549)

(SEQIDNOJ2)

PCR reaction was initiated by the addition of 25 units of Taq polymerase (Gibco BRL) at 85 °C (hot start). The following conditions were used. SSTR 1.2.4 - denaturation at 94 °C for 1 min annealing at 55 °C for 30 seconds and extension at 10

72 °C for 90 second' SSTR3.5 - denaturation at 94 °C for 1 min. annealing at 64 °C for 30 seconds and extension at 72 °C for 90 seconds The receptors were coamphfied with β-actm for 30 cycles followed by final extension at 72 'T foi 10 min

Southern transfer and hybridization

10 μl PCR products were separated by electrophoresis on 1.2% agarose gels, transferred to Genescreen Plus Membranes (Dupont), and hybπdized with ³²P labelled SSTR1-5. and β-actin-specific cDNA probes labelled to high specific activity by random hexanucleotide primers using a Life Technologies Kit Following hybridization for 20-22 h at 70 ° C, filters wei e washed and exposed to Kodak XAR film at four different times The hybridization signals were quantitated usmg a Java Video Analysis Software Package (Jandel Scientific. Corte Madera. CA) and used as an index of SST and actin mRNA To ensure that the hybridization bands were quantitated in the linear range, each blot was exposed to x-ray film foi several different times Only bands that did not reach saturation density of exposure were subjected to quantitative analysis. The units derived from the Java analysis were arbitraπly assigned a pixel density corrected for background Values of SSTR 1-5 mRNA expression were normalized to those of actm mRNA on the same gels. All expenments were performed at least 3 times and each mRNA quantitation represents the average of six measurements

BrdU Staining of Proliferating Cells

The method used was modified from the radioisotope method of Goldberg et al 1979 Cell proliferation was quantitated by Bromodeuxyuπdine labelling (BidU 1 1

Zymed Laboratoπes, San Francisco CA) according to the manufactur er s instructions Rats wei e injected with 0 3 ml Bidl labelling l eagent 4 h befor e sacrifice and cellular incorporation was visualized by staining of paraffin cross sections usmg a mouse primary antibody (Bu20a. Dako A/S. Denmark) and Vectastam Elite ABC Kit (Vector Laboratones, Burlingarne, CA). Sections were deparaffϊmzed and microwave-treated at 500 W for 2 x 5 mm in O l citrate buffer. pH 6. followed by treatment in 95% formamide in 0 15 M tπ-sodium cm ate at 70 °C for 45 mm Antibody dilutions were made according to the manufacturer s instructions Sections were counterstained with Mayers haematoxyhn and eosm and positive cells in mtimal, medial, and adventitial layers were separately counted and analysed

Antibodies to SSTR 1 -5 and lmmunohlS■Ochem^sπλ^, of SSTR 1 -5 antι-.ens

Antipeptide labbit polyclonal antibodies specific to SSTR 1-5 weie pioduced and characterized as previously described Synthetic ohgopeptides corresponding to deduced sequences in the amino terminal segment or extracellular loop 3 of hSST l-5 were conjugated to keyhole limpet hemocyanm and used to immunize New Zealand white rabbits The sequences selected were identical or nearly identical between the human and rat SSTR isoforms Anti-SSTR activity m rabbit sera was screened by the ability to inhibit [^12:>I-LTT] SST-28 binding to membrane SSTRs. by lmmunocytochemistry of stable CHO-K1 cells individually transfected with hSSTRl-5, and by Western blot analysis (SSTR2) Before lmmunostaining. the slides weie refixed with chlorofoπn and dned in air as described by Lemstiom and Koskinen 1997 Aftei incubation with 1 5 % noπnal goat serum, frozen sections were incubated with the panel of SSTR1 -5 pπmary antibodies (diluted 1 200 to 1.500) at 4° for 12 h With intervening washes in Tris buffered saline, the sections were incubated with goat antrrabbit rat absorbed secondary antibody at i oom temperature for 30 min followed by exposuie to avidm-biotmylated horseiadish peroxidase complex (Vectastatin Elite, ABC Kit) in phosphate buffeied saline at 12 room temperature for 30 min The reaction was revealed by chromogen 3-amιno-9- ethylcarbazole ( AEC Sigma) containing 0 1% hydiogen peroxide, yielding a br own- red reaction product. Specimens were counterstained with hematoxyhn and covershps were mounted (Aquamount BDH Ltd ) The controls used to validate the specificity of the SSTR immunoreactivity mcluded preimmune serum in place of pnmary antibody and pnmary antibody absorbed with excess antigen

Statistical Analysis

Statistical significance at diffeient time points after vessel jurv was deteπnined b\ analysis of variance followed by the Bonferron t-test P values " o 05 w ei e considered statistically significant

RESULTS

Vasculai Cell Proliferation in Response to Denudation Iniur

To validate the model against pievious reports (Clowes et al 1983. Haudenschild and Swartz 1979. Hayiy et al 1995) we tested the piohfeiative and intimal lesponsc of denuded aorta at the time points selected for the deteπnination of SSTR N \ and protein levels Following denudation, the endothelial lining w as completely removed Quiescent cells in the media were induced to proliferation beginning on day 3 followed by migration and further proliferation in the mtima on day 7- 14 Staining with antibody to α-SMC actin and α-leukocyte common antigen (α-LC A) demostrated that virtually all cells in the media and >95% of cells in the mtima expressed α-actin and <5% of the intimal cells weie positive foi -LCA indicating that they were muscle cells (not shown)

Expression of SSTR 1 -5 mRNA in the Vasculai Wall Aftei Trauma Figure 1 depicts Southern blots of RT-PCR products showing the expression of the quadruple specimens of mRNA for the five SSTR subtypes at different times in control and denuded aortic samples The time course of the mean levels of expression of expression of mRNA for the five SSTR subtypes following vasculai injury is summaπzed in Figure 2 Control aorta expressed ieadily detectable levels of SSTR3 mRNA. moderate levels of SSTR 1 and SSTR4 mR\ A. and baielv detectable concentrations of SSTR2 and SSTRS mRN A Follow ma. lnjurv . SSTR 1 mRNA displayed a dramatic 2-fold increase at day 3 (p< 0 01 ) concomitantly with the SMC proliferation in the media and intima The mRNA lev el r emained elevated at day 7 (p < 0 01 ). concomitantly with SMC pi ohfeiation in the intima. but declined theieafter to baseline concentration by day 14 when the piohfeiation was over A parallel increase in SSTR2 mRNA was also observed at day 3 (p < 0 01 ) although the magnitude of the change (~ 20%) was considerably smallei than that of SSTR 1 mRNA Unlike SSTR 1 and SSTR2. SSTR3 mRNA showed a more gradual increase with no change at day 3 followed by a significant inci ease bv 30 % at da 7 (p < 0 01 ) and by 40 % at day 14 (p < 0 001 ) SSTR4 mRNA followed a similar pattern but the magnitude of the increase was smallei (20%) and statistically significant (p < 0 05) at day 7 and (p < 0 001) at day 14 In conti ast to the other 4 subtypes, SSTR5 mRNA iemamed virtually undetectable and its expi ession patteπi did not change following injury

Expression of hSSTR l-5 by Immunohistochemistry

Figure 3 depicts the expression and localization of the five SSTR subtype leceptor proteins in injured vesssel wall after denudation injury, as detected by subtype specific rabbit antisera and lmmunoperoxidase staining In all microphotographs lumen is facing down and adventitia is facing up. as exemplified in the microphotograph in the left uppei comei It should be noted that all 14

immunoreactivity is observed in the media and mtima and none in the adventitia and that the cells in the mtima display mostly SSTR subtypes 1 and 4

The results largely confirm the RNA expression analysis The SSTR 1.2.4 subty pes were expressed at a low levels in the media of the non-denuded (control) aorta, but very little if any of SSTR3 and SSTR5 was seen After denudation, SSTR1 was expressed m the media on day 3, when media proliferation occurred, and strongly in the mtima on day 7 and 14 at the time of proliferation in the intima The expression of SSTR4 subtype was faint m the media on days 3 and 7, but increased in the mtima on day s 14 and 60 and coπelated w ith inci eased intimal thickening SS TR was recorded faintly in the media and mtima until day 60 when it was expressed m the innermost la ei ( luminal side) of the intima SSTR2 and SSTRS w ei e seen m the mtima only faintly if at all

DISCUSSION

The model used in this study for the quantitation of SMC replication and migration after endothlial injury of rat aorta has been well established in the past (Goldberg et al. 1979, Clow es et al 1983, Clowes et al 1983) Needless to say, the injury in this model is performed m healthy vessel and not to a vessel with atheromatous changes as for example coronary balloon dilatation in man is performed Nevertheless the model can be used to investigate smooth mucle cell migratory and proliferative responses The model has previously been extensively chai acteπzed by a number of investigators (Clowes et al 1983, Clowes and Swartz 1985. Hayry et al 1995. Majesky et al 1990; Majesky et al. 1992) demonstrating that aftei injury the medial cells begin to proliferate on day 2. reach the proliferation peak on day 3 and decline to base level on day 5 The migration of the cells into the intima begins on day 4 the mtimal proliferation peaks on day 7 and declines to baseline around day 14 Having this information available, we considered it sufficient to validate our experimental conditions only at the time points of SSTR subset mRNA and protein 15 determinations. 1 e . before denudation and at 15 minutes. 3 day s.7 day s and 14 da\ ^ post denudation Our results on the SMC prohferativ e and mtimal [espouses w er e in complete agreement with the previously published results

Here we find that all five SSTR mRNAs are expressed in rat aorta both as mRNA and protein. Importantly, the SSTR 1 ,4 were clearly present both m the intimal and medial layers of the vascular- wall Aortic denudation induced a time-dependent subtype-selectiv e response in pattern of SSTR expression The earliest change occured in the case of SSTR 1 and SSTR2 whose mRNA mci eased following denudation, reached peak levels between days 3-7 and declined to basal levels by day 14 SSTR3 and SSTR4 displayed a different pattern with a delayed, moi e gradual increase in mRNA beginning at day 3-7 and remaining elevated thei eaftei SSTR5 was costitutively expressed with no change in level of expiession duπng the 2 weeks post injury By lmmunohistochemistry. SSTR antigens wei e localized predominantly in SVIC that weie present in the media oi which had migiated into the mtima In general, the level of expression of SSTR I -5 by mRNA measurement correlated with receptor protein expression by lmmunohistochemistry

Our results provide the first evidence for die expression of all five SSTRs in the aorta, and suggest that like other tissues, e.g., brain, pituitary, and islet cells which also express the five SSTR isoforms, the aorta is an important target of SST action Previously by RT-PCR, only SSTR2 mRNA was detected in rat iliac artery (Chen et al. 1997) By autoradiography, a πch concentration of SST binding sites has been described in pentumoral (but not normal) vessels, suggesting that SSTRs may be induced by a tumor product We found a predominant cellular localization of SS TRs in SMC although lower levels of expression in endothelial or inflammatory cells cannot be ruled out Functional SSTRs have also been identified in glomerulai mesangial cells and in cultuπng intestinal SMC w hich express SSTRs with the pharmacological profile of the subtype 3 receptoi 16

What is the mechanism of SSTR induction by vascular injury⁰ Steady state SSTR mRNA levels are augmented by cAMP, gastrin, EGF. and SST itself. Glucocorticoids acutely induce SSTR1 and SSTR2 mRNA whereas estrogen induces SSTR2 and SSTR3 mRNA and thyroid hormone upregulates SSTR1 and SSTR5 mRNA. The 5 upstream promoter regions of the four receptor genes that have been sequenced (SSTR 1,2,4,5), display a number of consensus sequences that confer responsiveness to cAMP, AP I , AP2. Pit I . and thyroid hormone. The time course of increase in SSTR3 and SSTR4 mRNA in our study approximated the temporal profile of SMC hyperplasia suggesting that induction of these two subtypes may be a consequence of SMC replication. The earlier onset of induction of SSTR 1 and SSTR2 suggests a different mediator, possibly a growth factor such as EGF. Induction of endogenous SSTRs in response to vascular injury may represent a compensatory attempt to modulate the proliferative response by SST produced locally by inflammatory cells.

All five SSTRs are capable of inhibiting cell proliferation. SSTR1-4 act by stimulating PTP which dephosphorylates receptor tyrosine kinases thereby attenuating the mitogenic signal. SSTR5. on the other hand, inhibits guanylate cyclase and cGMP-dependent phosphorylation and activation of MAPK. Since the five SSTRs are expressed in the arterial wall, they could all be potential targets for the direct antiproliferative effects of SST. To date, however, only the effects of myointimale proliferation have been tested. Our findings suggest that SSTR 1 or SSTR4 may be the optimal subtypes to target, given their appropriate localisation and time related induction during the proliferative stage in the vascular wall. In this respect. SSTR1 which is upregulated to a greater extent than SSTR2, and which is insensitive to the current generation of clinically used SST analogs, should be a prime candidate for further testing with SSTR 1 -selective compounds.

It will be appreciated that the methods of the present invention can be incorporated in the form of a vanetv of embodiments, onlv a few of which are disclosed herein. I 17 will be apparent for the specialist in the field that other embodiments exist and do not depart from the spirit of the invention. Thus, the described embodiments are illustrative and should not be construed as restrictive.

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SEQUENCE LISTING

(1) GENERAL INFORMATION: ) APPLICANT: Hayry, Pekka

Patel, Yogesh C

(n) TITLE OF INVENTION: METHOD FOR THE PREVENTION OF A PATIENT'S FIBROPROLIFERATIVE VASCULOPATHY

(in) NUMBER OF SEQUENCES: 12

(lv) CORRESPONDENCE ADDRESS:

(A) ADDRESSEE: ADDUCI, MASTRIANI & S HAUMBERG, LLP

(3) STREET: 1140 Connecticut Avenue, NW

(C) CITY: Washington

(D) STATE: DC

(E) COUNTRY: USA

(F) ZIP: 20036

(v) COMPUTER READABLE FORM:

(A) MEDIUM TYPE: Floppy dιs<

(3) COMPUTER: IBM PC compat ole

< C ) OPERATING SYSTEM: PC-DOS/MS-DOS

(D) SOFTWARE: Patentln Release #1.C, Version #1.30

(vi ) CURRENT APPLICATION DATA:

(A) APPLICATION NUMBER: US C9/049,C2-

(B) FILING DATE: 27-MAR-1998

(C) CLASSIFICATION:

(vm) ATTORNEY/AGENT INFORMATION:

(A) NAME: Lydon, James C

(B) REGISTRATION NUMBER: 30,082

(C) REFERENCE/DOCKET NUMBER: TUR-0Ξ2

(IX) TELECOMMUNICATION INFORMATION: (A) TELEPHONE: 202-467-6300 (3) TELEFAX: 202-466-2006

(2) INFORMATION FOR SEQ ID NO:l:

(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 18 base pairs (3) TYPE: nucleic acid (C) STRANDEDNEΞS: single .0 ) TOPOLOGY: -.-.near

(n) MOLECULE TYPE: o ner nucleic aciα (A) DESCRIPTION: /αesc = "crimer"

(xi) SEQUENCE DESCRIPTION: SEQ I θ:l: ATGTTCCCCA ATGGCACC (2) INFORMATION FOR SEQ ID NO: 2: 24

( ) SEQUENCE CHARACTERISTICS: (A! LENGTH: 24 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS : single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: other nucleic acid (A) DESCRIPTION: /desc = "primer"

(lv) ANTI-SENSE: YES

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:2: CAGATTCTCA GGCTGGAAGT CCTC 24

(2) INFORMATION FOR SEQ ID NO:3:

(l) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 20 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(n) MOLECULE TYPE: other nucleic acid (A) DESCRIPTION: /desc = "primer"

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:3: AGCAACGCGG TCCTCACGTT 20

(2) INFORMATION FOR SEQ ID NO: 4:

(l) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 20 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: other nucleic aciα (A) DESCRIPTION: /desc = "primer"

(lv) ANTI-SENSE: YES

(xi) SEQUENCE DESCRIPTION: SEQ ID O:4:

GGAGGTCTCC A7TGAGGAGG

(2) INFORMATION FOR SEQ ID NO: 5:

(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 21 base pairs 25

(3) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: other nucleic acid (A) DESCRIPTION: /desc = "primer"

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:5: ATGAGCACGT GCCACATGCA G 21

(2) INFORMATION FOR SEQ ID NO: 6:

(l) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 20 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: other nucleic acid (A) DESCRIPTION: /desc = "primer"

(lv) ANTI-SENSE: YES

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 6: ACAGATGGCT CAGCGTGCTG 20

(2) INFORMATION FOR SEQ ID NO: 7:

(l) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 20 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: other nucleic acid (A) DESCRIPTION: /desc = "primer"

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 7: ATGGTAACTA TCCAGTGCAT 20

(2) INFORMATION FOR SEQ ID NO: 8:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 26 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: other nucleic acid 26

₍A) DESCRIPTION: /αesc = "primer" (lv) ANTI-SENSE: YES

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 8: GTGAGGCAGA AGACACTCGT GAACAT 26

(2) INFORMATION FOR SEQ ID NO: 9:

(_) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 20 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: other nucleic acid (A) DESCRIPTION: /desc = "primer"

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 9: TGGTCACTGG TGGGCTCAGC 20

(2) INFORMATION FOR SEQ ID NO: 10:

(l) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 20 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: other nucleic acid (A) DESCRIPTION: /desc = "primer"

(lv) ANTI-SENSE: YES

(xi) SEQUENCE DESCRIPTION: SEQ ID N0:1C: CCTGCTGGTC TGCATGAGCC 20

(2) INFORMATION FOR SEQ ID NO: 11:

(l) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 21 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: other nucleic aciα (A) DESCRIPTION: /desc = "primer" 27

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:ll: ATCATGAAGT GTGACGTGGA C 21

(2) INFORMATION FOR SEQ ID NO: 12:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 21 base pairs

(B) TYPE: nucleic aciα

(C) STRANDEDNESS : single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: other nucleic acid (A) DESCRIPTION: /desc = "primer"

(lv) ANTI-SENSE: YES

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 12: AACCGACTGC TGTCACCTTC A 21

Claims

28

1 The use of an agonist specific for at least one somatostatin (SST) receptoi showing an increased expression in a patient's vasculai wall subsequent to injuiv oi operation, for the manufacture of a pharmaceutical pieparation for use in the treatment or the pievention of said patient^'s fibropioliferative vasculopathv following vasculai injury or a vasculai suigical operation, wheiem said pharmaceutical composition is intended for administering to said patient foi one to two weeks aftei the injury oi surgical operation

2 The use according to claim 1 wheiem the fibroprohferative vasculopathy is selected fi om the gioup consisting of lestenosis follow ing coionary b pass surgei v PTC A (percutaneous tianslumrnal coionarv angioplasty ) allograft aiteπoscleiosis in chionic allograft i ejection doabetic angiopathy and all fonns of common arteriosclerosis

3 The use according to claim 2 wheiem the fibropiolifeiative vasculopathy is vascular restenosis

4 The use accordmg to claim 3 wherein the suigical opeiation is balloon angioplasty or a coronary bypass operation

5 The use accordmg to claim 2 wherein the fibtopiohfeiative vasculopathy is chronic allograft rejection

6 The use accordmg to claim 1 wheiem said SST-ieceptor shows an increased expression during the fust week following the injury oi the surgical opeiation

7 The use according to claim 6 wherein the SST-ieceptoi is the SST-receptor subtype 1 or subtype 4 29

8 The use accoidmg to claim 1 wheiem the administeimg of the teceptoi agonist is stalled a few days before the surgical operation

9 The use accordmg to claim 1 wherein the receptor agonist is a compound selected from the group consisting of somatostatin SRIF-14. somatostatin SRIF-28 and DADI-SST14

10 The use accordmg to claim 1 wherein the agonist is somatostatm SRIF- 14