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 regarding the practice, are incorporated by reference.

Somatostatin, also known as somatotropin release-inhibiting factor (SRIF), is a widely distributed peptide occurring in two forms, SRIF-14 and SRIF-28. SRIF has been connected to multiple physiological functions, such as the regulation of neurotransmission in the brain, the modulation of cell proliferation and the secretion of growth hormone, insulin, glucagon, pancreatic enzymes and gastric acid.

The biological responses of SRIF are mediated via high affinity G protein-coupled receptors (GPCR), of which five different subtypes (SSTR1- 5) have been cloned and characterized in humans (1,2). The affinities of the two endogenous ligands, SRIF-14 and SRIF-28, on the five subtypes are relatively similar, even though SRIF-28 has been reported to have a moderate preference for the SSTR5. However, to the extent that this has been addressed, it has already become clear that the expression profile of the five subtypes in different tissues can be distinctly different. In addition, the five subtypes also show some degree of difference in their interactions with a number of signaling pathways. Thus, the pleiotropic physiological responses linked to SRIF are a reflection of its widespread distribution, the existence of multiple receptor subtypes with unequal expression patterns, and the at least partly differential coupling of these subtypes to specific signalling pathways.

In concordance with the above highlighted complex physiology of somatostatin receptors, the acute administration of SRIF or one of its analogs induces a large number of biological effects (1,3). However, these initially potent responses often diminish with continuing or repeated exposure (1,4). The processes connected to the fading of receptor- mediated responses upon continuous or repeated exposure to SRIF or SRIF-analogs are most likely based on adaptive mechanisms such as desensitization and down-regulation. Homologous receptor desensitization is one of the major regulatory principles of GPCR-mediated signalling. It represents an agonist-dependent phenomenon, which occurs rapidly after the binding of the agonist to the receptor and is characterized by a reduced cellular responsiveness following continuous or repeated stimulation.

Desensitization appears to be a means for cells to adapt rapidly to the overall level of stimulatory input, presumably in order to avoid an excessive degree of stimulation. In line with this notion is the commonly observed phenomenon that the rate and magnitude in the desensitization of biological responses caused by different agonists on a given receptor tend to correlate with the efficacy of these compounds, i. e. strong agonists usually give rise to more rapid and more pronounced desensitizations of responses than weak agonists (5). Concerning somatostatin receptor, Hukovic et al. (1996) have shown in CHO cells stably transfected with individual receptor subtypes that SSTR2, 3, 4 and 5, but not SSTR1, display rapid internalisation upon prolonged agonist exposure (6). Smalley et al. (1998) have demonstrated that the response of SSTR4 to SRIF, but not to its synthetic analog L-362,855, is susceptible to agonist-induced desensitization (7,8).

As stated above, SRIF appears to control cell proliferation in normal and tumorous tissues (9,10). Somatostatin and its stable analogs have been shown to suppress the growth of various cancer cells and tumors, including neuroendocrine tumors that express somatostatin receptors. It is therefore believed that somatostatin does not only produce indirect anti-proliferative effects, based on the inhibition of growth hormone

and growth factor secretion, but does also possess direct anti-proliferative efficacy (11,12).

While a casual survey of the scientific literature on SRIF and somatostatin receptor might suggest unanimous acceptance for the notion of SRIF exerting, if anything then inhibitory effects on cellular proliferation, there have been a small number of reports describing SSTR-related growth- promoting effects (13-18). However, in all of these cases the observation of a proliferative response has been limited to in vitro cell culture models. Due to the absence of any supporting in vivo evidence for such a proliferative response upon the activation of somatostatin receptor, the relevance of these in vitro observations has so far remained obscure.

The rather complex physiology of SRIF makes the design and synthesis of subtype-selective ligands highly desirable, not only for the purpose of dissecting the physiological functions of individual subtypes, but also because subtype-selective drug candidates hold the potential to develop novel clinical applications based on the specific targeting of particular subtypes. Because of the scarcity of truly subtype selective ligands and the only rather recent appearance of such tools, relatively little is known about the identity of the somatostatin receptor subtypes mediating anti-proliferative or, for that matter, proliferative responses. The occasionally contrasting nature of cell growth responses observed upon application of somatostatin receptor agonists to cells expressing multiple SSTRs may well be caused by the simultaneous activation of several subtypes with mutually opposing effects. A recent review on literature evidence implicating different SSTR subtypes in anti-proliferative or proliferative responses and the possibly underlying molecular mechanisms has been published in the year 2001 by Lu et al. (19).

Concerning the SSTR4 it seems that the question of whether activation of the receptor by SRIF causes an anti-proliferative or a proliferative response is completely unclear since the available literature data appears to be in direct conflict. Florio et al. (1996) have reported on an anti-proliferative effect in PC C13 cells endogenously expressing the

SSTR4, while Sellers et al. (1999) have described a proliferative response in CHO cells heterologously expressing the SSTR4 at a relatively high receptor density of about 2 pmol/mg membrane protein (18,20). While the findings on cell proliferation are diametrically opposed, there appears to be evidence in both cellular systems that prolonged stimulation of the SSTR4 by SRIF leads to desensitization. In the case of SSTR4-transfected CHO cells the evidence is of a direct nature because Smalley et al. (1998) have demonstrated that repeated application of SRIF-14 to SSTR4-transfected CHO cells causes a loss of responsiveness as measured by extracellular acidification rates (7). For PC C13 cells Florio et al. (1996) noted a loss of the antiproliferative effect after 32 hours and speculated that this may be due to either receptor down-regulation or the degradation of the peptide (20).

When looking at protein kinase pathways in the CHO-SSTR4 cells Sellers et al. (1999) observed that SRIF-14 gave rise to a rapid initial peak of MAP/ERK kinase (MEK) activity which then returned to a considerably lower but elevated level of activity that was sustained over a period of at least four hours (18). Intriguingly, using some additional. experimental tools Sellers et al. (1999) arrived at the conclusion that it is not the initial peak in MEK activity that is responsible for the proliferative outcome in the CHO-SSTR4 cells but rather the more moderate yet sustained activity seen after four hours (18,21). In connection with the documented propensity of the SSTR4 receptor to undergo desensitisation, this conclusion by Sellers et al. (1999) raises the interesting question, to what extent the observation of a proliferative response in the CHO-SSTR4 cells may have been dependent on the artificially large number of SSTR4 receptor present in these cells. In other words, is it conceivable that it was the large number of receptor that allowed for the generation of a sufficiently large and sustained level of MEK activity despite the inclination of the receptor to undergo desensitization. If that were the case, the anti- proliferative response observed by Florio et al. (1996) in the PC C13 cells may then well be the consequence of different temporal activation pattern in

protein kinase pathways, in particular the absence of sustained MEK activity several hours after setting the SRIF stimulation. As suggested by Lu et al.

(2001), the MEK pathway may play a positive or a negative role in the regulation of cell proliferation depending on the intensity and duration of receptor activation (19).

A scenario such as the one outlined above in an attempt to reconcile the otherwise diametrically opposed observations by Florio et al.

(20) and Sellers et al. (21) in terms of SSTR4-mediated proliferative or antiproliferative responses suggests that the default mode of action for the SSTR4 upon activation by SRIF-14 is more likely to be an anti-proliferative one. However, we would now like to offer the hypothesis that the outcome of an agonist-mediated SSTR4 activation can be turned towards a proliferative response, provided a sufficiently large and sustained stimulatory input via the SSTR4 can be achieved. The hypothesis is based on our surprising finding that the endogenous somatostatin receptor agonists, SRIF-14 and SRIF-28, are clearly not defining the maximal agonist response possible at the SSTR4. As a matter of fact, we have identified a new class of agonists that possess up to 2.5-3 times higher efficacies than the two aforementioned endogenous agonists. When the classical convention of considering endogenous ligands as full agonists is maintained and a term recently introduced by Perret et al. 2002 is adopted, these highly efficacious compounds can be designated SSTR4 superagonists (22).

Equally surprisingly we have also found that there are compounds in this group of SSTR4 superagonists, which, despite their high efficacies, show a much lower propensity to cause desensitization of SSTR4-mediated responses than SRIF-14 or SRIF-28. These two properties of superagonism and low propensity for causing desensitization allow, each individually but especially in their combination, for much more pronounced and sustained stimulations of SSTR4 receptor than has previously been possible. Using such a superagonist with reduced propensity for causing desensitization, and in agreement with the hypothesis developed above, we have also for the first time been able to demonstrate a pronounced

proliferative effect in vivo upon activation of a somatostatin receptor subtype. Besides the possibility to achieve a proliferative response, a superior agonist efficacy in combination with reduced receptor desensitization also offers other therapeutic benefits. This makes it for example possible to avoid escalating drug doses in order to compensate for the waning responsiveness of the receptor. Since higher doses of drugs are very often associated with an increased liability towards unwanted side effects, superior efficacies and the absence or a much reduced degree of receptor desensitisation hold the clear promise of better side effect profiles.

SUMMARY OF THE INVENTION The present invention relates to a method for the treatment or prevention of a disease or condition in a mammal, said method requiring or benefiting from the strong activation of the somatostatin receptor subtype 4 (SSTR4), wherein said method comprises of administering to said mammal a therapeutical effective amount of at least one highly efficacious SSTR4 agonist.

BRIEF DESCRIPTION OF THE DRAWINGS Fig. 1A. shows the stimulation of [35S] guanosine-5'-O- (3- thio) tri phosphate ( [35 S] GTP7S) binding in membranes of Chinese hamster ovary (CHO) cells expressing the human SSTR4 receptor as a function of agonist concentrations. The agonists are SRIF-14, SRIF-28 and compound A (an SSTR4 agonist). The stimulatory response at each concentration is expressed in % of the maximal effect produced by SRIF-14, which was set as 100 %.

Fig. 1 B. shows the stimulation of [35S] guanosine-5'-O- (3- thio) triphosphate ([35S] GTPyS) binding in membranes of CHO cells expressing the human alpha-2A adrenoceptor as a function of agonist concentrations. The agonists are SRIF-14, compound A and the alpha-2 reference agonist epinephrine. The stimulatory response at each

concentration is expressed in % of the maximal effect produced by epinephrine, which was set as 100 %.

Fig. 2A-C. shows the extracellular acidification rate (luV/s) of CHO cells expressing the human SSTR4 after the applications of the SRIF-14 (Fig. 2A.), SRIF-28 (Fig. 2B. ) and compound A (Fig. 2C). The addition of test substances is indicated by arrows.

Fig. 3A-B. shows the extracellular acidification rate (V/s) of CHO cells expressing the human alpha-2 adrenoceptor after applications of SRIF- 14 (Fig. 3A. ) or compound A (Fig. 3B. ) The addition of test substances is indicated by arrows.

Fig. 4A. shows the extracellular acidification rate (uV/s) of CHO cells expressing the human SSTR4 after consecutive applications of progressively increasing concentrations of SRIF-14 or compound A.

Fig. 4B. shows dose-response curves for SRIF-14 and compound A. The extracellular acidification rate of CHO cells expressing the human SSTR4 is given as a function of the concentrations of SRIF-14 and compound A. The source of the data is the same as in Fig. 4A with the responses at individual concentrations normalized relative to the maximum response obtained for 1 uM compound A, which was set as 100%.

Fig. 5A. shows the extracellular acidification rate (in luts) of CHO cells expressing the human SSTR4. The addition of 3 pM SRIF-14 or 1 uM compound A is indicated by arrows.

Fig. 5B. shows the extracellular acidification rate (in luts) of CHO cells expressing the human SSTR4. The addition of 3 uM SRIF-14 or 1 uM compound A is indicated by arrows.

Fig. 6. shows the extracellular acidification rate (in pV/s) of pertussis toxin (PTX) -pretreated or non-pretreated CHO cells expressing the human SSTR4 after application of 1 uM compound A. The addition of SRIF-14 or compound A is indicated by arrows.

Fig. 7. shows the effects of compound A on the re- endothelialization of rat carotid artery after denudation. Presented is the morphometrically determined thickness of the intimal and the medial area in

rat carotid artery after once daily applications (s. c. ) of the vehicle (sterile phosphate-buffered saline) or 200 llg/kg compound A for fourteen days. The data shown are the mean SD of n = 5 determinations in each case. The star above the column of compound A in the intima indicates a statistically significant difference (p < 0.05) between compound A and the corresponding vehicle group.

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to the treatment of conditions or diseases, wherein strong somatostatin receptor subtype 4 (SSTR4) activation is beneficial or required. It was surprisingly found that a new class of agonists possess up to 2.5-3 times higher efficacies at the SSTR4 than the endogenous agonists SRIF-14 and SRIF-28 ; these agonists are therefore termed superagonists. Equally surprisingly, it was found that in this group of superagonists there are compounds that show a much lower propensity than SRIF-14 or SRIF-28 to cause desensitization of SSTR4- mediated responses. Moreover, a pronounced proliferative effect in vivo was demonstrated for the first time upon strong and prolonged SSTR4- activation. The two properties of superagonism and a low propensity to cause desensitization of SSTR4-mediated responses, either individually, but especially in their combination, allow it to achieve a much more pronounced and sustained stimulation of SSTR4 receptor than has previously been possible.

As is demonstrated in the examples of the current invention, the superior agonist efficacy of the SSTR4 selective compound relative to the endogenous somatostatin peptides is mediated by the SSTR4 and involves, at least in part, the participation of pertussis toxin-sensitive members of the Gjxo family of G proteins.

Definitions The term"treatment"shall be understood to include the complete curing of a disease condition, as well as the amelioration or alleviation of said disease or condition.

The term"prevention"shall be understood to include the complete prevention, prophylaxis, as well as the lowering of the risk of individuals to fall ill with said disease or condition.

The term"mammal"refers to a human or an animal subject.

The term"SSTR4 agonist"shall be understood as a substance or drug that has affinity for and that activates the somatostatin receptor subtype SSTR4.

The wording"selective SSTR4 agonist"shall be understood to mean an agonist having a binding affinity (expressed for example in the form of Ki values) for the SSTR4 that is at least 10 times better than those for the other somatostatin receptor subtypes.

The wording"highly efficacious SSTR4 agonist"shall mean an agonist which gives rise to a significantly higher maximal response at the SSTR4 than the responses produced by the endogenous ligands SRIF-14 or SRIF-28.

The term"desensitization"shall mean a decreasing responsiveness to the further application of SSTR4 agonists after repeated or prolonged stimulation of SSTR4 receptor by such SSTR4 agonists.

The wording"reduced propensity to cause agonist-mediated desensitization of SSTR4 receptor"shall be understood as the ability of an SSTR4 agonist to produce an SSTR4-mediated response, which, upon repeated or prolonged application of the corresponding agonist, is better maintained than the SSTR4-mediated response obtainable with SRIF-14 or SRIF-28 under otherwise identical application regiments.

Preferred embodiments According to a preferred embodiment, the SSTR4 agonist is a selective SSTR4 agonist. Preferably, its affinity (e. g. in form of its Ki value) for the somatostatin subtype 4 receptor should be at least 10 times better than for any of the somatostatin subtype receptors 1,2, 3, or 5. More preferably, its affinity for the somatostatin receptor subtype 4 should be at least 100 times better than for any of the receptor subtypes 1,2, 3, or 5.

According to another preferred embodiment, the SSTR4 agonist is highly efficacious. The efficacy should preferably be 30 % higher compared to SRIF-14, more preferably at least 100 % higher compared to SRIF-14 when tested in the assay described in Example 2.

In yet another preferred embodiment the highly efficacious SSTR4 agonist possesses a significantly decreased propensity compared to SRIF-14 or SRIF-28 to cause the desensitisation of SSTR4-mediated responses. This is to say that the highly efficacious SSTR4 agonist is expected to maintain a significantly larger response than SRIF-14 or SRIF- 28 upon repeated application in an indicative assay such as the microphysiometry assay described in Example 3.

The SSTR4 agonist should preferably have one or more of the following biological activities: stimulation of cell proliferation; stimulation of vascularization of a damaged and/or ischaemic organ, transplants or grafts; normalization of myocardial perfusion as a consequence of chronic cardiac ischaemia or myocardial infarction; augmentation of collateral vessel development after vascular occlusion or to ischaemic tissues or organs; promotion of endothelial growth in vascular grafts and transplants ; promotion of proliferation or differentiation of a stem cell (e. g. embryonic stem cell, neural stem cell), neural progenitor cell and/or neurocyte for transplantation; proliferation of fibroblasts, neurons, glia, oligodendrocytes, Schwann cells, ganglion cells or progenitors thereof; proliferation of bone marrow cells ; proliferation of hematopoietic stem cells and/or hematopoietic precursor cells ; proliferating the large intestine; proliferation-mediated generation of new hair cells ; modulation of the cell differentiation; induction of embryonic development; neurite outgrowth; enhancement of recovery from nerve or neuronal damage; myelination ; angiogenesis or arteriogenesis.

An especially preferred SSTR4 agonist is the compound (1'S, <BR> <BR> 2S)-N- (l'-carbamoyl-2'-phenylethyl)-5-guanidino-2- (4"-methyl-l"-naphtha- lenesulfonylamino) pentanamide, which has been disclosed in Finnish patent

applications F120031454 and F120031455. This compound is in the following called"compound A".

Typical groups of diseases or conditions that can be treated or prevented by the method according to this invention are, for example, any hypoproliferation disorder; wound healing ; tissue healing or tissue repair; respiratory diseases such asthma; hair loss ; psoriasis ; atopic dermatitis; mastocytosis; diabetic complications, such as diabetic angiopathy, retinopathy, nephropathy or ischaemic disease; ocular diseases, such as age-related macular degeneration, maculopathy, glaucoma, or retinitis pigmentosa; brain aneurysms; leg ulcers or other forms of skin ulcerations ; variceal bleeding in cirrhosis; conditions related to portal hypertension; muscle deterioration, for example muscular dystrophy, or damage caused after a heart attack; low cognitive function; impaired learning ; impaired memory; age-related cognitive decline ; sleep disorders; neurodegenerative disease such as Alzheimer's disease, Parkinson's disease, amyotropic lateral sclerosis, Huntington's disease, spinocerebellar degeneration and the like ; demyelinating diseases such as multiple sclerosis and acute transverse myelitis; extrapyramidal and cerebella disorders such as lesions of the corticospinal system; spinocerebellar degenerations such as spinal ataxia, Friedreich's ataxia, cerebella cortical degenerations; multiple systems degenerations such as Mencel, Dejerine-Thomas sporadic or recessive disorder, Shi-Drager and Machado-Joseph disease; disorders of the motor unit such as neurogenic muscular atrophies (anterior horn cell degeneration, such as amyotrophi lateral sclerosis (ALS), infantile spinal muscular atrophy and juvenile spinal muscular atrophy); neurological developmental diseases such as those due to premature delivery or cocaine addiction; psychoneural diseases such as schizophrenia; epilepsy; autism; anxiety; aggression; social withdrawal; motion sickness; cranial trauma; spinal damage; cerebrovascular disorder; cerebrovascular dementia and the like.

Doses and administration routes The method includes a step of administering a therapeutical effective amount of a highly efficacious SSTR4 agonist (e. g. , an SSTR4

selective agonist) to a mammal in need of the treatment. The therapeutical effective amount depends on the condition being treated, the route of administration chosen, the specific activity of the compound being used and ultimately will be decided by the attending physician or veterinarian.

Depending on the disease or condition to be treated, the duration of administration of the somatostatin agonist to the patient may last until the disease or condition has subsided or the somatostatin agonist may be administered for the lifetime of the patient.

The SSTR4 agonists within the scope of the invention may be formulated in a conventional manner using one or more pharmaceutical acceptable carrier or excipient to produce pharmaceutical compositions for the treatment of diseases or disorders disclosed. Formulations can for instance enable oral, buccal, topical, intranasal, parenteral (e. g. intravenous, intramuscular or subcutaneous) or rectal administration respectively administration by inhalation or insufflation. Furthermore, the SSTR4 agonists belonging to the scope of the invention may also be formulated for sustained delivery.

For oral administration forms of suitable compositions include, but are not limited to, tablets, chewable tablets and capsules. These may be prepared by conventional means with pharmaceutical acceptable excipients, such as binding agents (e. g. pregelatinized maize starch), disintegrants (e. g. potato starch), fillers (e. g. lactose) or lubricants (e. g. magnesium stearate). Tablets may be coated by methods well known in the art. For oral administration, possible liquid preparations include, but are not limited to, solutions, syrups or suspensions, or they may exist as dry powder for constitution with water or other suitable vehicle prior use. These liquid preparations may be prepared by conventional means with pharmaceutical acceptable agents, such as suspending agents, non-aqueous vehicles, preservatives and emulsifiers.

A possible dose of the therapeutics within the scope of the invention for oral, parenteral, buccal or topical dose to the adult human is

between 0.1 and 500 mg of the active compound per unit dose, which may administered, for instance, 1 to 4 times in a day.

It is well recognized that the precise dose, the route of administration and the dosing interval can be determined by those skilled in the art. It is also well recognized that these variables depend on multiple factors including, but not restricted to, the activity of the therapeutic compound, the formulation thereof, pharmacokinetic properties (such as absorption, distribution, metabolism and excretion) of the therapeutic compound, the nature and location of the target tissue or organ and the issues connected to the state of a disease or disorder in a patient in need of treatment. Additionally, when the compounds within the scope of the invention are administered with additional pharmaceutically active ingredients, one or more pharmaceutical compositions may be used for the delivery of all the agents, which may be administered together, or at different times, as determined by those skilled in the art.

The invention will be illuminated by the following non-restrictive experimental section.

EXPERIMENTAL SECTION List of abbreviations aMEM alpha Minimum Essential Medium BSA bovine serum albumin CHO chinese hamster ovary EDTA ethylenediaminetetraacetic acid Hepes 4- (2-hydroxyethyl) piperazine-1-ethanesulfonic acid PBS phosphate-buffered saline PTX pertussis toxin [35S] GTPyS [35S] guanosine-5'-O- (3-thio) triphosphate Tris tris (hydroxymethyl) aminomethane Cell culture and transfections Chinese hamster ovary (CHO) cells (K1 strain) were cultured in Ham's F12 medium (Nutrient Mixture Ham's F12; Invitrogen, Glasgow, UK)

containing 10 % fetal calf serum. The cDNA encoding the human SSTR1 (hSSTR1) or human SSTR4 (hSSTR4) was subcloned from a construct in pBluescript (Stratagene, San Diego, CA), into a bicistronic mammalian expression construct (Clontech, Palo Alto, CA) with an amino-terminal hemagglutinin tag (Clontech, Palo Alto, CA) (and EF1 promoter (Invitrogen, Groningen, Netherlands) in hSSTR4 cloning) using FUGEN TU 6 Transfection Reagent (Roche Diagnostics Corporation, Indianapolis, IN).

Stable transfectants were selected in Ham's F12 containing G418 at 500 pg/ml. After selection, transfected CHO-hSSTR4 cells were sorted and the highest-expressing cells collected by fluorescence-activated cell sorting (Flow Cytometry Core Facility, Turku, Finland). Hereafter, both the sorted CHO-hSSTR4 and the unsorted CHO-hSSTR1 cells were cloned by dilution- plating. The highest expressing clone was identified through SRIF binding assays using ('251-Tyr)- [Leu8, DTrp22]-somatostatin-28 (1251-LTT-SRIF-28) (Anawa, Zurich, Switzerland ; specific activity: 2000 Ci/mmol) as tracer. CHO cells expressing the human SSTR2 (hSSTR2), human SSTR3 (hSSTR3) and human SSTR5 (hSSTR5) were obtained from Prof. Kjell Oberg (Uppsala University Hospital, Sweden). The recombinant CHO cells expressing the SSTR subtypes were grown in Ham's F12 medium supplemented with 5 % foetal bovine calf serum and 200-250 ug/ml G418.

Membrane preparation Recombinant CHO cells expressing the human SSTRs were harvested in phosphate-buffered saline (PBS), and frozen at-70°C. To prepare the membranes, thawed cell pellets were homogenized on ice in a Potter Elvehjem homogeniser in 10 mM Tris-HCI, 0.1 mM EDTA, pH 7.5 at RT supplemented with 320 mM sucrose. The nuclear pellet obtained by centrifugation of the homogenate at 1 000 g for 15 min at 4°C was discarded. From the supernatant, a membrane fraction was collected by centrifugation at 48 000 x gmaxfor 30 min at 4°C. The 48 000 x g pellet was re-suspended in 10 mM Tris-HCI, 0.1 mM EDTA, pH 7.5 at RT without sucrose and centrifuged again at 48 000 x 9max for 30 min. The supernatant

was discarded and the washed membranes were resuspended in 10 mM Tris-HCI, 0.1 mM EDTA, pH 7.5 at RT and thereafter stored at-70°C.

Binding affinity at the human somatostatin receptor subtypes The affinity of the compound A for the five human somatostatin receptor subtypes (hSSTR1-5) was determined in competition binding assays with (1251-Tyr)-[Leu8, DTrp22]-somatostatin-28 ('251-LTT-SRIF-28). The biological material for these experiments consisted of membranes from Chinese hamster ovary (CHO) cells stably transfected with one of the five human somatostatin receptor subtypes. Membranes (3-20 lug of total protein per sample) and trace amount of 1251-LTT-SRIF-28 were incubated in 10 mM Hepes, 1 mM EDTA, 5 mM MgC12, 5 mg/ml of BSA and 30 ug/ml bacitracin, pH 7.6 with six concentrations of the compounds. Each concentration was run in duplicate. Nonspecific binding was defined by 1 uM somatostatin-14 (SRIF-14) and corresponded to 5-25% of total binding. After 60 min at room temperature, incubations were terminated by rapid vacuum filtration through GF/B glass fiber filter mats (presoaked at 4°C in 200 mi of 10 mM Hepes, 1 mM EDTA, 5 mM MgCI2, pH 7.6) and three 5 ml washes with ice-cold wash buffer (20 mM TRIS, 1 mM EDTA, 5 mM MgCiz, pH 7.4). The filters were then dried, impregnated with scintillate and their radioactivity was measured by scintillation counting. The analysis of the experiments was carried out by nonlinear least square curve fitting. Affinity constants (Ki) were calculated from the IC50-values according to the Cheng-Prusoff's equation (25).

Experiments were repeated a minimum of three times.

[35S] Guanosine-5'-O- (3-thio) triphosphate binding assay The ability of the ligands to stimulate receptor-mediated binding of [35S] guanosine-5-0-(3-thio) triphosphate ([35S] GTPyS) (PerkinElmer Life Sciences, Boston, MA; specific activity: 2000 Ci/mmol) to G proteins in membranes of CHO cells expressing hSSTR4 was measured. Membranes (about 10 lug of protein per sample), prepared as described above, were incubated in 20 mM Hepes, pH 7.4, 10 mM MgCI2, 10 uM GDP, 100 mM NaCI, and 10 ug/ml saponin with different concentrations of test ligand.

Each concentration was run in duplicate. After a 45-min incubation at 30°C

(15-min pre-incubation without label followed by a 30-min stimulation after addition of label), the reaction was terminated by rapid vacuum filtration GF/B glass fiber filter mats. Filters were then washed four times with 5 ml of ice-cold wash buffer (20 mM TRIS, 1 mM EDTA, 5 mM MgCI2, pH 7.4 at room temperature), dried, and counted for radioactivity in a scintillation counter. In control experiments, membranes of CHO-C10 cells, which express the adrenergic alpha-2A receptors, were used and the assay was run basically as described above.

Measurement of the extracellular acidification rate A four-channel CytosensorR Microphysiometer instrument (Molecular Devices, Menlo Park, CA, USA) was used to evaluate receptor desensitization. For this purpose the extracellular acidification rate of CHO cells expressing the hSSTR4 was determined after application of SSTR4 agonists (SRIF-14, compound A or SRIF-28). Cultured CHO-hSSTR4 cells were seeded into 12-mm capsule cups at 3 x 105 cells/cup and incubated in 5% C02 at 37°C for 18 h. The capsule cups were loaded into the sensor chambers of the instrument and the chambers were perfused with running medium (bicarbonate-free aMEM supplemented with 2 mM glutamin, 26 mM NaCI, 50 U/ml penicillin and 50 pg/ml streptomycin) at a flow rate of 100 lul/min. Agonists were diluted into running medium and perfused through a second fluid path. Valves directed the flow from either fluid path to the sensor chamber. During each 2 min pump cycle, the cells were exposed to the agonists for 10 seconds or 50 seconds, and the metabolic rate measurement (acidification rate) was accomplished during a pause in cell perfusion by recording the pH of the running medium for the time segment 1: 25 to 1: 55 min. The pump was restarted at 2 min to initiate the next cycle.

Carotid denudation Carotid denudations were performed in male Wistar rats 300-400 g in weight as described by Aavik et al. (23). This model was chosen because it has previously been shown by Khare et al. (24) that the two somatostatin receptor subtypes SSTR4 and SSTR1 are prominently expressed in carotid artery recovering from denudation. Briefly, the rats

were anaesthetized with chloral hydrate (240 mg/kg, ip), and buprenorphine (0.25 mg/kg, sc) was given for peri-and postoperative pain relief. A 2F Fogarty balloon catheter (Baxter Healthcare Corp, Santa Ana, CA) was introduced into the common carotid artery through the left external carotid artery and inflated with 0.2 ml of air resulting in a 0. 5-lb pull force and a balloon size of 4 mm when inflated. To produce adequate vessel injury, the catheter was retrieved three times with the balloon inflated through the common carotid artery. The external carotid artery was ligated after removal of the catheter, and the wound was closed. To determine the effects of compound A on the re-endothelialization of the denuded carotid artery, animals were injected subcutaneously with 200 ug/kg of the compound or vehicle (sterile phosphate-buffered saline) during the operation and thereafter once daily for another 13 days. On day 14 the rats were anaesthesized as above before they were sacrificed. The animals were venesected and in situ fixed with 3% neutral paraformaldehyde intracardially for 1 h, post-fixed thereafter in the same solution for additional 4 hours and transferred to PBS for embedding. Histological evaluations were made from midcarotid sections of five animals each in the vehicle group and the group treated with compound A. The histological pictures were digitised with an Olympus video microscope and quantitated using the version 1.62 NIH- Image software package. The length of the internal and external elastic laminae (perimeter) and areas inside the internal elastic lamina, external elastic lamina and the carotid lumen were measured. Intimal and medial areas and intima-media area ratios were calculated from these values.

Example 1 Compound A is selective for hSSTR4 The affinity of SSTR ligands was tested in competition binding assays with 1251-LTT-SRIF-28 as the radioligand and membranes from CHO cells expressing one of the five human somatostatin receptor subtypes (hSSTR1-5).

Using the protocol described above the following test results were obtained.

Compound Kj (hSSTR1) Ki (hSSTR2) Ki (hSSTR3) Ki (hSSTR4) Kj (hSSTR5)<BR> /nM/nM/nM/nM/nM Compound A 73 t 19 >10000 >10000 3. 6 0. 7 >10000 SRIF-14 1. 4 t 0. 2 0. 62 t 0. 1 5 1. 6 0. 2 4. 1 t 0. 6 4. 1 t 0. 4 The table above shows that compound A binds to hSSTR1 and hSSTR4, but has a 20 times higher affinity for the hSSTR4 compared to the hSSTR1. Compound A does not, at concentrations up to 10 uM, bind to the other hSSTRs. SRIF-14 was included for reference purposes in the competition binding assays.

Example 2 Compound A has a superior efficacy compared to SRIF-14 and SRIF-28 at hSSTR4 The functional activity of SSTR ligands was tested as their ability to stimulate [35S] GTPyS binding to G proteins in membranes (10 ug/sample) of CHO cells expressing the hSSTR4 or the human alpha-2 adrenoceptor (about 5 pg/sample).

Fig. 1A. shows that compound A elicits a significantly higher agonist response compared to SRIF-14 and SRIF-28 at the hSSTR4 receptor subtype.

Fig. 1B. shows that the dose-dependent increase in [35S] GTPyS binding caused by SRIF-14 and compound A is mediated through the hSSTR4 receptor, since membranes of CHO cells lacking the hSSTR4, but instead expressing the human alpha-2A adrenoceptor, failed to give rise to any significant increase in [35S] GTPyS binding when challenged with micromolar concentrations of either SRIF-14 or compound A. In contrast, increasing concentrations of the alpha-2 adrenoceptor agonist epinephrine resulted in a dose-dependent increase in [35S] GTPyS binding.

Example 3 Differential desensitization of hSSTR4 by compound A compared to SRIF- 14 and SRIF-28 Receptor desensitization was assessed through CytosensorR Microphysiometry by examining the extracellular acidification rate of CHO cells expressing the hSSTR4 after application of SSTR4 agonist (SRIF-14, SRIF-28 and compound A). <BR> <BR> <P>Fig. 2. demonstrates that responses to SRIF-14 (Fig. 2A. ) and<BR> SRIF-28 (Fig. 2B. ), but not compound A (Fig. 2C. ), are susceptible to agonist-induced hSSTR4 desensitization. The acidification rates of CHO cells transfected with hSSTR4 to 10-second pulses of 3 uM SRIF-14/SRIF- 28 or 1 pM compound A, followed by a 30-minute wash-out period before the repeat exposure, are shown. The acidification rates in response to the first exposure increased by 20-25 %. Repeat exposures to 3 uM SRIF-14 or SRIF-28 resulted in much smaller increases in acidification rates, showing that SRIF-14 and SRIF-28 cause desensitization of the hSSTR4-mediated response. In contrast, repeat exposures to 1 uM compound A continued to produce similar responses as in the first exposure, thus showing that compound A does not, or only very weakly, desensitize the hSSTR4- mediated response.

The fact that the responses in acidification rates towards SRIF-14 and compound A were specific for the hSSTR4 receptor subtype is demonstrated in Fig. 3. This figure shows the acidification rate responses of CHO cells devoid of hSSTR4 receptors but instead transfected with human alpha-2A adrenoceptors. Repeated 10-second pulses of 3 uM SRIF-14 (Fig.

3A) or 1 uM compound A (Fig. 3B), followed by a 30 minute wash-out period did not lead to any discernible response to the SSTR4 agonists (i. e. SRIF- 14 or compound A). However, a clear increase in the acidification rate (30%- 40 %) was observed in response to epinephrine, an alpha-2 agonist.

Fig. 4. shows the acidification rate responses of CHO cells transfected with hSSTR4 upon exposure to progressively increasing concentrations of SRIF-14 or compound A. The cells were challenged with

SSTR4 agonists during 50-second pulses separated by washout periods of 30 minutes duration. Dose-dependent increases in acidification rates were observed for both SRIF-14 and compound A. However, while the dose- response-curve for compound A was sigmoid (EC50 = 0.09 0.04 nM), that for SRIF-14 was bell-shaped with the peak effect corresponding only to about 60 % of the maximum effect elicited by compound A. The reduced amplitude and the bell-shaped nature of the SRIF-14 dose-response-curve conform to the expectation of an initially blunted and then increasingly suppressed response towards an agonist, which causes desensitization in a cumulative dose-response experiment. The dose-response curves in Fig.

4B. were plotted from the pooled data of three separate experiments, one of them exemplified in Fig. 4A.

Fig. 5. demonstrates further that the SRIF-14 induced desensitization of hSSTR4 is agonist-specific. Fig. 5A. shows the acidification rate of CHO cells transfected with hSSTR4 upon a 10-second pulse of 3 uM SRIF-14 followed 30 minutes later by a 10-second pulse of 1 , uM compound A. Fig. 5B. shows the acidification rate of cells upon a 10- second pulse of 1 uM compound A followed 30 minutes later by a 10- second pulse of 3 uM SRIF-14. The increases in acidification rates during the first exposure amounted to about 20-25% in both cases, independent of the identity of the agonist used. The subsequent exposure of the cells to 1 uM compound A (after they had experienced 3 uM SRIF-14 in the initial exposure) resulted in a much smaller response (an about 7-9 % increase in the acidification rate) towards compound A, strongly suggesting that the hSSTR4 had undergone desensitization by SRIF-14 prior to the exposure to compound A. In contrast, the subsequent exposure of the cells to 3 uM SRIF-14 (after they had experienced 1 uM compound A in the initial exposure) produced a very similar second response, confirming that the hSSTR4 had not been desensitized to a discernable degree by the initial exposure to compound A.

Fig. 6. demonstrates that the observed increases in acidification rate are due to the coupling of hSSTR4 to pertussis toxin (PTX) -sensitive G-

proteins of the Gi/Go family. The figure shows the acidification rate of CHO cells transfected with hSSTR4 receptor, with and without PTX pretreatment of the cells, in response to a 10-second pulse of 1 pM compound A. While compound A produced the usual increase in acidifcation rate in non-PTX- treated cells, following PTX-pretreatment, the response to 1 pM compound A was abolished.

Example 4 Compound A gives rise to a profound proliferative effect in vivo Fig. 7 shows that compared to vehicle, compound A (200 pg/kg s. c. daily) gives rise to a statistically significant increase in the thickness of the intimal area after carotid denudation. In contrast, no differences between compound A and vehicle were observed in the thickness of the medial area.

It will be appreciated that the methods of the present invention can be incorporated in the form of a variety of embodiments, only a few of which are disclosed herein. It will be apparent for the expert skilled 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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