Indian Patents. 224306:A KIT COMPRISING 4-H-1-BENZOPYRAN-4-ONE DERIVATIVES

Title of Invention	A KIT COMPRISING 4-H-1-BENZOPYRAN-4-ONE DERIVATIVES
Abstract	ABSTRACT "A KIT COMPRISING 4-H-1-BENZOPYRAN-4-ONE DERIVATIVES" IN/PCT/2001/01010/CHE A kit comprising (-)-cis,5-7-dihydroxy-2-X2-chlorophenyI)-8-[4-(3-hydroxy-l-methyl)-piperidiniy]]-4H-benzopyran-4-one(flaropiridol) together with excipients or additives for the treatment of lesions after balloon injury.

Full Text	Field of the Invention The instant invention relates to the use of 4-H-l -benzopyran-4-one derivatives as inhibitors of smooth muscle cell (SMC) proliferation. Statement Regarding Federally Sponsored Research or Development The present invention was made with support from the National Institutes of Health under Grant Nos. HL03658 and AG 15234. Background of the Invention The cellular responses to vascular injury-cellular dysfunction, activation, dedifferentiation proliferation and migration - culminate in clinical events - such as restenosis, which occurs following balloon angioplasty and stent placement for the treatment of human atherosclerotic disease1. Smooth muscle cell proliferation is a common, and perhaps unifying feature of vascular injury models, and SMC are the major cellular component of neointimal lesions3,3. Renewed interest in inhibiting SMC proliferation has accompanied the increased use of stents for the treatment of coronary disease, since in-stent restenosis is almost entirely dependent on neointimal formation and SMC hyperplasia4. It is estimated that as many as 100,000 patients with in-stent restenosis required treatment in 1997 alone1; therefore, an easily administered, effective inhibitor of SMC hyperplasia would have profound clinical and economic ramfications6. Efforts to inhibit SMC proliferation in vascular injury models, either by modulating cellular mediators of the proliferative response or by directly interfering with the cell cycle machinery, have provided important insights into neointimal formation. Cell cycle progression is a tightly controlled event regulated positively by cyelin-dependent kinases (Cdks) and their cyclin regulatory subunits7, and negatively by Cdk~ inhibitors and tumor suppressor genes such as retinoblastoma protein (Rb) and p53s. Adenoviral-mediated overexpression of endogenous Cdk inhibitors p21 and p27kipi or of a constitutively active form of Rb blocks neointimal formation in the rat carotid injury model9"11; similarly, inhibition of the activity of transcription factor E2F by competitively overexpressing cognate DNA binding sites also inhibits SMC proliferation and neointimal formation12. Such studies support that cell cycle inhibition is an attractive target for intervention in vascular lesion formation. Whereas genetic interventions have aided in the dissection of the mechanisms regulating neointimal formation, they suffer from the shortcoming of not being, at present, clinically suitable for the treatment of vascular disease in humans. A water-soluble, low molecular weight compound with specific cell cycle-regulatory effects, particularly one with oral activity, would have broad applicability both experimentally and, potentially, clinically. The recently identified flavone, flavopiridol, is a Cdk inhibitor that potently blocks the activity of Cdk2, Cdc2, and Cdk4 13-16, in contrast with other pharmacological inhibitors of Cdks, flavopiridol is remarkable for its kinase specificity, its oral availability, and its potency, being effective in nanomolar concentrations 16. These unique features result in a favorable side effect profile that has led to the testing of flavopiridol in Phase I clinical trials for the treatment of refractory neoplasms ' ?. Given these properties, we have examined the ability of flavopiridol to inhibit SMC proliferation in vitro and after balloon injury to the rat carotid artery. We demonstrate that flavopiridol is a potent and selective inhibitor of cell cycle progression and that it arrests SMC proliferation both in vivo and in vitro; moreover, neointimal formation is effectively blocked by oral doses of flavopiridol lower than those known to have toxic effects in humans. It has now surprisingly been found, that 4-H-l -benzopyran-4-one derivatives are suitable SMC proliferation inhibitor. It is know that 4-H-l -benzopyran-4-one derivatives are suitable for controlling tumors. However it is surprising that the 4-H-l-benzopyran-4-one derivatives according to the instant invention effectively act as an SMC proliferation inhibitor at dosage levels which have no effect on cellular viability and thus at much lower dosage level than the dosage levels which have to be used in the control of tumor growth. Accordingly, a subject of the instant invention is the use of 4-H-l-benzopyran-4-one derivatives as inhibitors of smooth muscle cell proliferation. Figure 1. Effect of flavopiridol on HASMC DNA synthesis. A. Quiescent HASMC were treated in the absence (-) or presence (+) of bFGF (10 ng/ml) and with the indicated concentrations of flavopiridol (nmol/L) for 24 h. BrdU incorporation as a measure of proliferation was determined by an EUSA-based assay and expressed as percentage of incorporation in the absence of bFGF treatment. p treated with bFGF (10 ng/ml), thrombin (2 U/ml), or vehicle in the presence of absence of flavopiridol (75 nmol/L) and BrdU incorporation was measured. p untreated cells. »p treatment with thrombin alone. Figure 2. Effect of flavopiridol on HASMC proliferation. Quiescent HASMC were treated with bFGF (10 ng/ml) alone (a), bFGF and flavopiridol (75 nmol/L) (6), or vehicle (8) for the times indicated and cell numbers after treatment were determined. Results are expressed as the cell counts per well (X 103), Figure 3. Effect offlavopiridol on cyclin-dependent kinase activity in HASMC. Quiescent HASMC were treated with bFGF (10 ng/ml), thrombin (2 U/ml), or vehicle in the presence or absence offlavopiridol (75 nmol/L), and phosphorylation of histone HI was quantified as a measure of Cdk activity and expressed as a percentage of Cdk activity in the absence of bFGF treatment. p bFGF alone, fp Figure 4. Regulation of cell cycle-related proteins by flavopiridol. Quiescent HASMC were treated in the presence (+) or absence (-) of bFGF (10 ng/ml), thrombin (2 U/ml), and/or flavopiridol (75 nmol/L) for 24 h. Immunoblotting of cellular lysates was performed with specific antibodies recognizing cycltn Dl (top panel), PCNA (middle panel), and phosphorylated (pRb) and hyperphosphorylated (ppRb) Rb (tower panel). Figure 5. Effects offlavopiridol on MAP kinase activity in HASMC, Quiescent HASMC were treated in the presence (+) or absence (-) of bFGF (10 ng/ml), thrombin (2 U/ml), PD98059 (30 umot/L) and/or flavopiridol (75 nmol/L) for 30 min. Levels of phosphorylated Erkl (pErkl) and Erk2 (pErk2) were measured by immunoblotting with a phosphorylation-specific antibody recognizing both proteins (upper panel). MAP kinase activity was measured with an in-gel kinase assay, using myelin basic protein as a substrate (lower panel). Figure 6. HASMC viability after treatment with flavopiridol. Quiescent HASMC were treated with flavopiridol (75 nmol/L), TNF-a (50 ng/ml), or vehicle for the times indicated. Cellular viability was assessed by trypan blue exclusion. Results are expressed as the percentage of viable cells to total cells counted. Figure 7. Inhibition of rot carotid artery neointimal formation by flavopiridol after balloon injury. Neointima/media ratios were measured in histologic sections or rat carotid arteries treated with or without flavopiridol (5 mg/kg) for 5 days after injury. Arteries were examined 7 (n = 12) and 14 (n = 12) days after injury. The percentage of PCNA-positive nuclei (± SEM, expressed as a percentage of counted nuclei) in the neointima of arteries from each time point and treatment group is also given. p Suitable 4-H-I-benzopyran-4-ojie derivatives are compounds of the formula I wherein R1 is hydrogen, alky! having 1 to 6 carbon atoms, aryl-Ci-C4-aIkyl; Ci-Ce-alky! substituted by halogen, hydroxy, or carboxy; C3-C6-cycloalkyl, pyridyl, thienyl, C3-C6-cycloalkyl-C\|-C4-alkyl, C2-C6-alkenyI, C2-C6-alkynyI, phenyl; phenyl, mono- or polysubstituted by halogen, Cr Ct-alkyl, C\|-C4-alkoxy, hydroxyl, carboxyl, COO-alkyl, CONH2, CONH-alkyt, CON(alxyi)2, nitro, trifluoromethyl, amino, Ci-C4-alkylamino, di-Ci-Q-alkylamino, or phenyl; naphthyl, carboxyl, -CHO, COO-Ci-CU-alkyI, a primary amino, alkylamino, aralkylamino, dialkylamino, amido, arylamino, diarylamino, or -CHiO-Ci-Q-alkyl; R2 is hydrogen, alkyl having 1 to 6 carbon atoms, aryl, nitro, amino, di-Ci-C4-alkylamino, a halogen, hydroxy!, alkoxy, -COOH, -COO-Cl-C4-alkyl, -CHO, -CH2OH or -CH20-Cr C4alkyl; R3 is hydrogen, C\|-C4-alkyI; C\|-C4-alkyl substituted by halogen, hydroxy or carboxy, hydroxy!, carboxyl, nitro, amino, Ci-CValkylammo, di- Ci-C4-alkylamino, halogen, -O-alkyl-C(0)-alkyI, -CHO, -CH2OH, -CH20- C,-C4-alkyl or R2N-C(0)-0-, wherein R is H, C,-C6-alkyl, cycloalkyl; or -O-alky!-C(0)-alkyl or aryl; R4 is hydrogen, hydroxy!, C\|-C4-a(koxy, C\|-C4-alkanoyIoxy, C[-C4-alkoxycarbonyI, aryloxy, amino, Ci-C4-alkylamino, di- Ci-C4-alkylamino, or RVN-C(0)-0- wherein R' is H, Cj-Ce-alkyl, cycloalkyl or aryl; Rs is hydrogen, Cj-Q-atkyl, aryl- C1-C4 alkyl, Ca-Cs-cycloalkyl, C3-C6-cycloalkyl- C1-C4- alkyl, alky! amino, C]-C4-aIkanoyI, -C(0)-0- C\|-C4-alky! or aroyl, where the aryl group in Ri, Ri, Rs, R4, and R5 is unsubstituted phenyl or phenyl that is mono or pofysubstituted by halogen, CrC4-alkyl, C-Ci-alkoxy, hydroxy!, carboxyl, COO-atkyl, CONH2, CONH-alkyl, CON(alkyl)2, nitro, trifluoromethyl, amino, Ct-C4-alkyl amino, di- C,-C4-aikyIamino or phenyl; m is an integer between 0 and 3 and n is 1, or a pharmacologically acceptable acid addition salt thereof. The compounds according to the invention have two centers of asymmetry, one where the heterocyclic ring containing nitrogen is fused to the benzopyran moiety (C-4'), the other at the rVsubstituted carbon atom (C-31), which means that two pairs of optical isomers are possible. The definition of the compounds according to the invention embraces all possible stereoisomers and their mixtures. It very particularly embraces the racemic forms and the isolated optical isomers having the specified activity. The two racemates can be resolved by physical methods, such as, for example, fractional crystallization. The individual optical isomers can be obtained from the racemates by conventional methods, such as, for example, salt formation with an optically active acid followed by crystallization. Examples of alkyl groups which are suitable for R\| to Rs are straight-chain or branched radicals having up to 6, preferably up to 5, carbon atoms, for example methyl, ethyl, propyl, isopropyl, t-butyl, pentyl or isopentyl groups. Examples of substituted alkyl groups which are suitable for Rj to Rs are haloalkyl, such as trifluoromethyl, hydroxyalkyl, such as hydroxyethyl, or carboxyalkyl, such as carboxyethyl. Suitable examples of a cycloalkyl group which has 3 to 6 carbon atoms and is represented by Ri to Rs are cyclopropyl, cyclobutyl, cyclopentyl or cyclohexyl, Cyclopropylmethyl is an example of cycloalkylalkyl. An example of an aralkyl group which is represented by R, to R5 is a phenylalkyl group in which the phenyl group is unsubstituted or monosubstituted or polysubstituted by substituents such as halogen, Ci-O-alkyl, C\|-C4-alkoxy or nitro or by a trifluoromethyl group, amino group and substituted amino group. An example of an aryl group which is represented by Ri to Rs is a phenyl group which is unsubstituted or monosubstituted or polysubstituted by substituents such as halogen, C\~C2, nitro or trifluoromethyl, amino, C\|-C4-alkylamino, di- C\|-C4-alkylamino, aromatic heterocyclic groups such as pyridyl groups, and polycyclic aromatic radicals, such as naphthyl groups. A suitable example of an alkylamino group which is represented by R] to R5 is (CHj),,-NRfiR?, where n is 1 to 3 and Re and R7 are alkyl and are as defined as above in the case of alkyl Ri to R5; moreover, R and R? together with the nitrogen atom to which they are bonded can be a heterocyclic ring having one or more hetero atoms. Suitable examples of heterocyclic rings which are formed by R^ and R? together with the nitrogen to which they are bonded are piperidine. pyrrolidine, morpholine, piperazine or imidazole, all of which can be unsubstituted or substituted in one or more positions by CrC4-alkyl, Ci-Gpalkoxy or aryl or by a hydroxyl or amino group. Suitable examples of salts of the compounds according to the invention with inorganic or organic acids are hydrochloride, hydrobromide, sulfate, phosphate, acetate, oxalate, tartrate, citrate, maleate or fumarate. in which Ri is hydrogen, Ci-Cj-alkyl, naphthyl, phenyl; phenyl mono- or polysubstituted by halogen, C,-C4-alky], d-C4-alkoxy, hydroxyl, carboxyl, COO-alkyl. CONH2, CONH-alkyl, CON(alkyI)2, nitro, trifluoromethyl, amino, Ci-Gi-alkylammo, di- C]-C4-alkyJamino, or phenyl; pyridyl, or thienyl; R2 is hydrogen or Cj-C3-alkyI; Rs is C,-C3-alkyl, Cs-Cs-cycloalkyl. or C3-Cj-cycloaIkyl- C\|-C4-alkyI. Particularly preferred are compounds of formula la, in which R\| is phenyl, thienyl, pyridyl, chlorophenyl, dichlorophenyl, methylphenyl, aminophenyl, bromophenyl, hydroxyphenyl or naphthyl; Ri is hydrogen and Rj is methyl. A compound of special importance is (-)-cis,-5,7-dihydroxy-2-)(2-chlorophenyl)-8-[4-(3-hydroxy-l-methyl)-piperidinyl]-4H-benzopyran-4-one (Flavopiridol), particularly in fee form of ist hydrochloride. The compounds according to the instant invention can be prepared according to the disclosure of U.S. Pat. No. 4,900,727 and U.S. Pat. No. 5,284,856 which are incorporated herein by reference. The examples of said U.S. patents are also of relevance for the instant application. The compounds according to the instant invention inhibit smooth muscle cell proliferation. Further subject-matter of the invention are therefore also pharmaceuticals for the inhibition of smooth muscle cell proliferation, which contain at least one compound of the formula I as defined above or at least one of its pharmacologically acceptable acid addition salts, and the use of a compound of the formula I as defined above for the preparation of a pharmaceutical having an smooth muscie cell proliferation inhibitory action. Typical application areas for the compounds according to the instant invention are diseases/disorders/injuries which are accompanied by smooth muscle cell-rich vascular lesions. A very important example therefor are lesions after balloon injury. Another important application area is the prevention of restenosis after stent implantation. The 4H-1 -benzopyran-4-one derivatives are used according to the invention in the generally known fashion which is known to the expert. For pharmaceuticals, an effective amount of the active substance mentioned is employed either per se or preferably in combination with suitable pharmaceutical auxiliaries in the form of tablets, coated tablets, capsules, suppositories, emulsions, suspensions or solutions, the active compound content being up to about 95%, preferably between 10 and 75%. The expert will know which auxiliaries are suitable for the desired formulation of the pharmaceutical because of his expert knowledge. Besides auxiliaries for tablets, or solvents, gel formers, bases for suppositories and other excipients for the active substance, it is possible to use, for example, antioxidants, dispersants, emulsifiers, defbamers, flavor corrigants, preservatives, solubilizers or colorants. The active substance can be administered orally, parenterally, intravenously or rectally, oral administration being preferred. For a form of oral administration, the active substance may be mixed with other compounds together with the additives which are suitable for this purpose, such as excipients, stabilizers or inert diluents, and customary methods can be used for bringing it into suitable administration forms, such as tablets, coated tablets, hard-gelatin capsules, and aqueous alcoholic or oily suspensions or solutions. Examples of inert excipients which can be used are gum arabic, magnesia, lactose, glucose or starch, in particular corn starch. In this context, the formulation can be prepared as dry granules or moist granules. Examples of suitable oily excipients or solvents are vegetable or animal oils, such as sunflower oil or cod-liver oil. For subcutaneous or intravenous administration, a solution, suspension or emulsion of the active substance is formed, if appropriate using substances which are conventional for this purpose, such as solubilizers, emulsifiers or other auxiliaries. Examples of suitable solvents are water, physiological sodium chloride solution or alcohols, for example, ethanol, propanol or glycerol, and also sugar solutions, such as glucose solutions or mannitol solutions, or a mixture of the various solvents which have been mentioned. The dose of 4H-l-benzopyran-4-one derivatives which is to be administered daily is to be selected to suit the desired effect. The 4H-I-benzopyran-4-one derivatives can be administered in a dose which is less than 70%, preferably less that 60%, in particular less than 50% of the dosage, which is used to control tumor growth in the respective mammal. An example would be - in a nude mouse xenograft model - a dose of about Smg/kg bodyweight administered orally once daily. This is half of the dosage which inhibits tumor growth in the same animal model (Drees et al. Clin. Cancer Res. 1997; 3:273-279). The compound according to the instant invention can also be comprised in a kit along with other excipients or additives for treatment of lesions of balloon injury or stent implant. The pharmacokinetic properties of the 4H-l-benzopyran-4-one derivatives could make it necessary to administer the said compound several times a day or to chose slow release formulations. Examples 1. Flavopiridol inhibits smooth muscle cell proliferation and neointimal formation in vivo in a rat carotid injury model of vascular injury. The well-established rat carotid injury model, in which neointimal lesion formation after catheter-induced injury is critically dependent on SMC proliferation (Clowes et al. Lab. Invest. 1983; 49:327-333, Clowes et al. Circ. Res. 1985; 56:139-145) to examine whether Flavopiridol induces growth arrest of SMC in vivo, as it does in vitro. Flavopiridol was administered orally at a dose of 5 mgAcg once daily, beginning on the day of injury and for four days after, since this time period covers the initial induction of Cdk2 and the first wave of SMC proliferation in this model (Circ. Res.1995; 77:445-465, Circ. Res.1997; 80:418-426). Mean tntimal and medial areas were quantified 7 and 14 days after injury, and neointimal lesion size was expressed as the ratio of the neointimal to the medial area. Twelve animals each were in the treated and untreated groups. The ratio at 7 days was 1.00+/-0.05 in arteries of vehicle-treated rats and 0.65+/-0.04 in Flavopiridol-treated rat arteries, a reduction of 35%. At 14 days, the neointimal/media ratio was 1.08 +/-0.Q4 in vehicle-treated rats and 0.66+/-0.03 In Flavopiridol-treated rats, a reduction of 38.9%. These effects were statistically significant at both time points (P Methods Materials- Flavopiridol (L86-8275, (ycis, -5,7-dihydroxy-2- (bFGF) was purchased from Collaborative Biochemical and thrombin from Sigma. The MEK1 inhibitor PD98059 was obtained from New England Biolabs. Cell culture- Human aortic smooth muscle cells (HASMC) were obtained from Clonetics and were cultured as previously described 18. Cells were used at passages 5-9. Prior to performing experiments, cells were growth-arrested at 80% confluence for 48 h with medium containing 0.2% fetal bovine serum. Cell proliferation EUSA- Cell proliferation was measured by ELISA (Amersham Life Science). HASMC were grown in gelatin-coated 96-well plates and made quiescent. Cells were treated with 10 ng/ml bFGF, 2 U/ml thrombin, or vehicle for 24 h. Flavopiridol (75 nmoi/L) was administered I h prior to growth factor treatment. 5-Bromo-2'-deoxyuridine (BrdU) was added to a final concentration of 10 umoI/L during the last 2 h of treatment. BrdU incorporation was measured as described 19, Results are expressed as mean ± SEM for 12 samples per condition from two independent experiments. Cell counts- Growth-arrested HASMC grown to 50% confluence in 6-weII plates were treated with or without flavopiridol (75 nmol/L) or bFGF (10 ng/ml). At intervals after treatment, cells were trypsinized and cell numbers determined using a hemocytometer. Western blot analysis- Quiescent HASMC were treated in the presence or absence of growth factors and/or flavopiridol as indicated. Western blot analysis was performed as previously described '8. The primary antibodies were: a polyclonal anti-human cyclin Dl antibody (M-20, Santa Cruz), a monoclonal anti-human proliferating cell nuclear antigen (PCNA) antibody (PC10, Sigma), a phosphorylation-specific p44/42 (Erkl/Erk2) MAP kinase monoclonal antibody (New England Biolabs), and a monoclonal anti-Rb antibody (G3-245, Pharmigen), which recognizes the phosphorylated (pRb) and highly phosphorylated (ppRb) Rb species. For immunoblotting studies, experiments were repeated at least three times. Cdk activity- Quiescent HASMC were treated with agonists and inhibitors for 24 h and total cell lysates were prepared as described for Western blotting. The kinase assay was performed with a histone HI kinase assay kit (Upstate Biotechnology) following the manufacturer's instructions, fn brief, 10 \|il of peptide inhibitors for protein kinase C (2 umol/L) and protein kinase A (2 fjmol/L), 100 jig cell lysate, 10 ul assay buffer and 10 uJofa mixture containing 75 umol/L magnesium chloride, 500 umol/L ATP and I uCi/ml [v-J2P]ATP were mixed in a microcentrifuge tube. After incubation at 30°C for 10 min, aliquots were pipetted onto phosphocellulose papers. The papers were washed in 0.75% phosphoric acid, followed by measurement of cpm in a scintillation counter (Beckman). Results are expressed as the mean ± SEM for three samples and are representative of three independent experiments. In-gel kinase assay- Quiescent HASMC were treated with growth factors for 30 min and total cell lysates were prepared as described for Western blotting. In some experiments, HASMC were pretreated for 60 min with 30 umol/L PD98059, flavopiridol, or vehicle. Equal amounts of proteins (50 jig/lane) were resolved on a polyacrylamide gel that was copolymerized with 350 ug/ml myelin basic protein. The gel was treated with [y-MP]ATP and autoradiography was performed as described '^. Trypan-blue exclusion- HASMC were grown in 5-cm dishes at low confluence and growth-arrested as described. Cells were treated with flavopiridol (75 nmol/L) or tumor necrosis factor-D (TNF-D; 50 ng/ml) for the indicated times. Following the removal of the medium, 0.4% trypan blue in phosphate-buffered saline was added to the dishes. After 5 min, the cells in the dishes were counted under the microscope. Blue celts were counted as nonviable cells. Rat carotid injury model- Injury to the rat carotid artery was performed essentially as described 2. Adult male Sprague-Dawley rats (400-500 g, Zivic-Miiler) were anesthetized with an intraperitoneal injection of ketamine (2 mg/kg) and xylazine (4 mg/Wg). The left internal carotid was then cannulated with a 2F embolectomy catheter. The balloon was inflated with saline and withdrawn across the artery three times to produce a distending and denuding injury. The right carotid artery was uninjured and served as a control of injury for each animal. Immediately following recovery from anesthesia and for four additional days thereafter, rats were administered flavopiridol (5 mg/kg in water) or water by gavage in a blinded manner. All rats survived the surgery and mere were no overt signs of toxicity related to drug administration in the doses used. At specified time points after carotid injury, rats were anesthetized as above and perfiision-flxed systemtcally with 4% paraformaldehyde in phosphate-buffered saline. Right and left carotid arteries were removed and distended by injection of 4% paraformaldehyde through the lumen, after which they were dehydrated and stored in 70% ethanol at 4°C. Immunohistochemistry was performed as previously described l8, using the monoclonal PCNA antibody and a polyclonal anti-human Cdk2 antibody {M2-G, Santa Cruz). Image analysis- The extreme distal and proximal regions of each artery (approx. 500 um) were removed. Ten intermediate cross-sections (8 urn each) taken 500 um apart were analyzed from each artery. Slides were fixed and stained with hematoxylin and eostn as previously described '8. Using a Nikon Diaphot 300 microscope and a 4X objective, each cross-section was captured as a digital image using a Hamamatsu C5985 video camera and TCPro 2.41 (Coreco, Inc.). Medial and neointimal areas were determined using NIH Image software. Medial and neointimal boundaries were determined by one slide reviewer (A.M.) and verified in a blinded fashion by a second reviewer (C.P.). Lesion size was expressed as the neointima/media ratio. Results for each group were expressed as the mean ± SEM. 92% or more of the images were interpretable in each group; the remainder suffered from fixation artifacts and were not analyzed. Statistical analysis- When appropriate, data from quantitative studies were expressed as the mean ± SEM. For multiple treatment groups, a factorial ANOVA followed by Fisher's least significant difference test was applied. Statistical significance was accepted at p Results Flavopiridol inhibits HASMC proliferation- On the basis of flavopiridol's ability to inhibit the proliferation of a variety of tumor cell lines, we tested the hypothesis that its use would block the growth of primary culture human SMC. Growth-arrested HASMC were treated with the SMC mitogen bFGF (10 ng/ml) for 24 h in the presence of increasing concentrations of flavopiridol and proliferation was measured by an ELISA-based assay. In comparison with untreated cells, proliferation of bFGF-treated cells was increased 5.4-fold (Figure I A). Pretreatment for 1 h with as little as 50 nmol/L flavopiridol significantly decreased HASMC proliferation (to 3.9-fold,p To test the generality of flavopiridol's effects on SMC proliferation, we examined its effect on mttogenesis elicited by thrombin (2 U(m\), which acts through a G protein-coupled receptor, in comparison with bFGF, which stimulates a member of the receptor tyrosine kinase family. Flavopiridol (75 nmol/L) significantly and potently inhibited both bFGF- and thrombin-induced HASMC proliferation (5.4-fold vs. 1.8-fold and 2.4-fold vs. 0.7-fold, respectively,p Flavopiridol inhibils Cdk activity and cell cycle-related gene expression In HASMC- To assess the specific effect of flavopiridol on the cell cycle machinery, we measured histone HI kinase activity in cellular lysates from growth factor-stimulated HASMC. Phosphorylation of histone HI reflects the activities of Cdc2 and Cdk2 20. Treatment of HASMC with bFGF and thrombin resulted in 4.4-fold and 3.6-fold mcreases, respectively, in histone HI kinase activity (Figure 3). These increases in cycl in-dependent kinase activity were totally blocked by pretreatment with flavopiridol (75 nmol/L). By Western blot analysis, we also addressed whether flavopiridol influenced growth factor-induced regulation of cell cycle-related proteins in HASMC. Cycl in Dl is a Gl phase cycl in that is upregulated by growth factor stimulation and is rapidly degraded during withdrawal from the cell cycle 21, Cyclin Dl protein levels were upregulated 6.3-fold and 3.2-fold, respectively, in response to bFGF and thrombin treatment for 24 h (Figure 4), an effect which could be completely blocked by pretreafment with flavopiridol, Similarly, increased expression of PCNA, which is synthesized predominantly during S phase in conjunction with DNA replication 22, was also blocked by flavopiridol pretreatment. As a final measure of cell cycle-related proteins, we examined Rb phosphorylation in response to growth factor expression using an antibody that recognizes phosphorylated Rb. Rb is a cell cycle regulator that binds to and inactivates transcription factor E2F when Rb is in the unphosphorylated state 23 and induces SMC growth arrest in vivo '. Phosphorylation inactivates Rb and allows progression through S phase to proceed. Analysis of Rb phosphorylation is particularly relevant because Rb is a target of Cdk2 and Cdk4 in vivo. Both thrombin and bFGF induced hyperphosphorylation of Rb, an effect that was inhibited by flavopiridol. Taken together, these results indicate that flavopiridol influences the expression and activity of Gl and S phase-related cell cycle control elements in HASMC in association with its growth-inhibitory effects. Flavopiridol has no effect on MAP kinase phosphorylation or activity- To ensure that flavopiridol was acting specifically at the level of the cell cycle, rather than nonspecifically on upstream kinase pathways, we measured phosphorylation and activity of ErkI (p44 MA? kinase) and Erk2 (p42 MAP kinase), two members of the MAP kinase family. We chose these kinases because they are immediately upstream of transcriptional events occurring in response to growth stimuli and downstream of a number of critical mitogenic signaling pathways 2 activation 25, increased amounts of phosphorylated ErkI and Erk2 were detected after treatment of HASMC with thrombin and bFGF for 30 min, compared with untreated cells (Figure 5, upper panel). Phosphorylation of ErkI and Erk2 by both thrombin and bFGF was blocked by pretreatment with PD98059, but not with flavopiridol. To confirm these findings, we measured Erk 1 and Erk2 activity by an in-gel kinase assay (Figure 5, lower panel). Again, we found that ErkI and Erk2 activities were increased in response to thrombin and bFGF, an effect that was inhibitable by PD98059 but not by flavopiridol. These experiments, in conjunction with those presented in Figures 3 and 4, provide evidence that the effects of flavopiridol on HASMC proliferation are due to a specific arrest of the cell cycle machinery by blocking Cdk activity without affecting upstream signaling events. Flavopiridol does not decrease HASMC viability- Previous reports of flavopiridol activity in other cell types have demonstrated that, depending on the cell line, flavopiridol may either induce growth arrest without affecting viability, or it may cause apoptosis ' 6\26-29, we therefore assessed whether flavopiridol decreased the viability of HASMC by measuring trypan blue exclusion at various time points after treatment. Quiescent HASMC were treated with flavopiridol (75 nmol/L)» vehicle, or TNF-0 (50 ng/ml), a cytokine known to induce apoptosis in this cell type 30. Whereas TNF-D potently decreased the viability of HASMC, resulting in the death of essentially all cells treated for 24 h, flavopiridol had no such effect (Figure 6). We have noted that with higher concentrations and longer incubations, some decreases in viability in the presence of flavopiridol may occur (not shown). However, under the conditions tested, flavopiridol primarily induces growth arrest, without affecting SMC viability. Flavopiridol inhibits smooth muscle cell proliferation and neointimal formation in vivo in a rat carotid injury model of vascular Injury- We utilized the well-established rat carotid injury model, in which neointimal lesion formation after catheter-induced injury is critically dependent on SMC proliferation 2,3_ (0 examine whether flavopiridol induces growth arrest of SMC in vivo, as it does in vitro. We administered flavopiridol orally at a dose of 5 mg/kg once daily, beginning on the day of injury and for four days after, since this time period covers the initial induction of Cdk2 and the first wave of SMC proliferation in this model 31,32. Mean intimal and medial areas were quantified 7 and 14 days after injury, and neointimal lesion size was expressed as the ratio of the neointimal to the medial area. Twelve animals each were in the treated and untreated groups. The neointima/media ratio at 7 days was 1.00 ± 0.05 in arteries of vehicle-treated rats and 0.65 ± 0.04 in flavopiridol-treated rat arteries, a reduction of 35.0% (Figure 7). At 14 days, the neointima/media ratio was 1.08 ± 0.04 in vehicle-treated rats and 0.66 ± 0.03 in flavopiridol-treated rats, a reduction of 38.9%. These effects were statistically significant at both time points ip In order to demonstrate directly that flavopiridol inhibited smooth muscle cell proliferation, we stained sections for PCNA expression in representative fields from each artery and determined the percentage of PCNA-positive nuclei in the neointima. At 7 days, 31.1 ± 7.2% of nuclei in injured arteries from untreated rats were PCNA-positive, whereas only 11.8 ± 1.5% of injured arteries in flavopiridol-treated rats were PCNA-positive (Figure 7; p Discussion In the present study, we have examined whether the novel Cdk inhibitor, flavopiridol, the most potent and specific inhibitor of Cdks known, is a suitable candidate for inhibiting SMC proliferation in vivo, particularly in the setting of vascular injury. Previous attempts to target the cell cycle machinery therapeutically for the treatment of vascular disease have provided a rationale for the present studies 9'12; however, methods employed to this end have relied on gene transfer technologies to inhibit cell cycle progression. At present, formidable obstacles prevent the clinical application of these techniques 33, During the course of our studies, it was reported that CVT-313, a recently identified compound that also has Cdk inhibitory properties but at micromolar concentrations, can also inhibit neointimal formation; however, it was required that CVT-313 be instilled into the carotid artery at the time of injury to produce this effect 34. in contrast, we show that flavopiridol, when administered orally, can potently inhibit neointimal formation, to a degree comparable with other clinically relevant agents ' U 35,36.7he ora\| activity of flavopiridol makes it virtually unique among agents shown to be active in animal models of vascular injury. Its selectivity, potency, and ease of administration make flavopiridol an excellent candidate for examining the therapeutic benefits of cell cycle inhibition in vivo in human vascular lesions. We chose to administer flavopiridol orally in a concentration (5 mg/kg) half that which inhibits tumor growth in a nude mouse xenograft model 27. It is notable that flavopiridol concentrations of 75 nmol/L result in near complete inhibition of SMC proliferation in our studies, whereas median serum concentrations of 425 nmol/L were attained at doses below the toxic threshold in Phase I human studies of refractory carcinoma 17. Our results suggest that much lower doses of cell cycle inhibitors than those used for neoplasia may be effective in the setting of vascular diseases such as restenosis, with the concomitant benefit of increased toletability. Whereas we have demonstrated that flavopiridol induces growth arrest without affecting viability of HASMC in culture, and have shown decreased neointimal formation after flavopiridol treatment in vivo, we cannot be sure that cell cycle arrest is the only factor reducing neointimal formation in carotid lesions. Flavopiridol can induce growth arrest with or without inducing apoptosis, depending on the cell type observed 29. Interestingly, flavopiridol inhibits apoptosis in PC 12 cells that have been terminally differentiated, yet it induces apoptosis in undifferentiated PC12 cells that are proliferating 28. Although our in vitro experiments were performed under conditions that would mimic the phenotype of SMC prior to injury, it is possible that SMC may respond differently to flavopiridol after injury and may even undergo apoptosis. While the role of apoptosis in vascular lesions is unclear, expression of the Fas ligand in SMC induces apoptosis and blocks neointimal formation in rabbits after balloon injury 37, suggesting that if flavopindol does indeed induce apoptosis of SMC in vivo, as it does in proliferating PC 12 cells, this may be a salutary phenomenon in the context of neointimal formation. Other mechanisms may also contribute to the effects of flavopiridol on lesion formation. For example, antisense oligodeoxynucleotide-mediated cell cycle inhibition improves endothelial function in rabbit vein grafts 38. Further studies will be needed to address mechanisms of flavopiridol's effects, other than growth arrest, on SMC in vivo. Given the demonstrated role of SMC proliferation in lesion formation after rat carotid injury 2 3, it is interesting to note that, despite the profound effects of flavopiridol in vitro, its ability to inhibit neointimal formation (though significant) was more modest under the conditions of our in vivo experiments. We have considered several explanations for this observation. It is unlikely that accelerated SMC proliferation occurs after cessation of flavopiridol, since differences in proliferative indices are sustained as long as 14 days after injury (Figures 7 and 9), It is more plausible either that other components of lesion formation, such as SMC migration and extracellular matrix production, still contribute to lesion formation, even in the absence of significant SMC proliferation, or that delivery of flavopiridol once daily is insufficient to arrest proliferation entirely in this model. Recent data indicating that the biological half-life of flavopiridol is as short as 2.5 hrs suggests that the latter hypothesis may be correct; further studies may be able to identify an even more effective dosing regimen 39, Our results indicate that flavopiridol can inhibit SMC proliferation and, hence, neointimal formation in a well-accepted small animal model of vascular disease. It must be pointed out that the relevance of inhibition of SMC proliferation is controversial in human vascular lesions, and may differ depending on the nature of the lesion and the time at which observations of proliferation are made. The proliferative index of SMC in human atherectomy specimens is remarkably low 40, although these specimens may not reflect proliferative changes at earlier, more critical stages in lesion development. In addition, arterial remodeling independent of neointimal growth may account for a significant proportion of luminal obstruction after angioplasty in humans 41. In contrast, indices of mitotic activity in SMC are much higher (25% PCNA-positive) in atherectomy specimens from human lesions with in-stent restenosis, consistent with the established role of SMC hyperplasia, but not remodeling, / in this process "*. As stent placement and the clinical problem of in-stent restenosis increases, so will the need for an effective means to arrest SMC hyperplasia and neointimal formation. Since flavopiridol is a potent, orally available drug with specific Cdk-inhibitory activity, and since safe doses of fiavopiridol are known in humans, flavopiridol can be considered as a pharmacologic candidate for prevention of in-stent restenosis in humans. Methods and Results: Using cultured human aortic smooth muscle cells, we found that flavopiridol in concentrations as low as 75 nmol/L resulted in near complete inhibition of basic fibroblast growth factor- and thrombin-induced proliferation. At this dose, flavopiridol inhibited cyclin-dependent kinase activity, as measured by histone HI phosphorylation, but had no effect on MAP kinase activation. Induction of cell cycle-related proteins cyclin Dl, proliferating cell nuclear antigen, and phosphorylated retinoblastoma protein was also blocked by flavopiridol. Flavopiridol had no effect on cellular viability. To test whether flavopiridol had a similar activity in vivo when administered orally, we examined neoindmal formadon in rat carotid arteries after balloon injury. Fiavopiridol (5 mg/kg) administered by gavage reduced neointimal area by 35% and 39% at 7 and 14 days, respectively, after injury. Conclusions: Flavopiridol inhibits SMC growth in vitro and in vivo. It's oral availability and selectivity for cyclin-dependent kinases make it a potential therapeutic tool in the treatment of SMC-rich vascular lesions. REFERENCES \. Ross R. The pathogenesis of atherosclerosis: a perspective for the 1990s. Nature 1993; 362:801-9. 2. Clowes AW, Reidy MA, Clowes MM. Kinetics of cellular proliferation after arterial injury. Lab. Invest. 1983;49:327-333. 3. Clowes A, Schwartz S. Significance of quiescent smooth muscle migration in the injured rat carotid artery. Circ. Res. 1985; 56:139-145. 4. Kearney M, Pieczek A, Haley L, Losordo DW, Andres V, Schainfeld R, Rosenfield K, Isner JM. Histopathology of in-stent restenosis in patients with peripheral artery disease. Circulation 1997; 95:1998-2002. 5. Mintz GS, Hoffmann R, Mehran R, Pichard AD, Kent KM, Satler LF, Popma JJ, Leon MB. In-stent restenosis: the Washington Hospital Center experience. Am. J. Cardiol. 1998; 81-.7E-13E. 6- CaliffRM. Restenosis: the cost to society. Am. Heart J. 1995;130:680-684. 7. Sherr CJ. Mammalian Gj cyclins. Cell 1993; 73:1059-1065. 8. Hunter T. Braking the cycle. Ceil 1993; 75:839-841. 9. Chen D, Krasinski D, Chen D, Sylvester A, Chen J, Nisen PD, Andres V. Downregulation of cyclin-dependent kinase 2 activity and cyclin A promoter activity in vascular smooth muscle cells by p27^n>l, an inhibitor of neointima formation in the rat carotid artery. J. Clin. Invest. 1997; 99:2334-2341. 10. Chang MW, Barr E, Lu MM, Barton K, Leiden JM. Adenovirus-mediated over-expression of the cyclin/cyclin-dependent kinase inhibitor, p21 inhibits vascular smooth muscle cell proliferation and neointima formation in the rat carotid artery model of balloon angioplasty. J. Clin. Invest. 1995; 96:2260-2268. 11. Chang MW, Barr E, Seltzer J, Jiang Y-Q, Nabel EG, Parmacek MS, Leiden JM. Cytostatic gene therapy for vascular proliferative disorders with a constitutively active fonn of the retinoblastoma gene product. Science 1995; 267:518-522. 12. Morishita R, Gibbons GH, Horiuchi M, Ellison KE, Nakajima M, Zhang L, Kaneda Y, Ogihara T, Dzau VJ. A gene therapy strategy using a transcription factor decoy of the E2F binding site inhibits smooth muscle proliferation in vivo. Proc. Natl. Acad. Sci. USA 1995; 92:5855-5859. 13. Losiewitz MD, Carlson BA, Kaur G, Sausville EA, Worland PJ. Potent inhibition of Cdc2 kinase activity by the flavanoid, L86-8275. Biochem. Biophys. Res. Commun 1994; 201:589-595. 14. Carlson BA, Dubay MM, Sausville EA, Brizuela L, Worland PJ. Flavopiridol induces G] arrest with inhibition of cyclin-dependent kinase (CDK) 2 and CDK4 in human breast carcinoma cells. Cane. Res. 1996; 56:2973-2978. 15. de Azevedo WF, Mueller-Dieckmann H-J, Schuze-Gahmen U, Worland PJ, Sausville E, Kim S-H. Structural basts for specificity and potency of a flavanoid inhibitor of human Cdk2, a cell cycle kinase. Proc. Natl. Acad. Sci USA 1996; 93:2735-2740. 16. Kaur G, Stetlcr-Stevenson M, Sebers S, Worland P, Sedlacek H, Myers C, Czech J, Naik R, Sausville E. Growth inhibition with reversible cell cycle arrest of carcinoma cells by flavone L86-8275. J. Natl. Cane. Inst. 1992; 84:1736-1740. 17. Senderowicz AM, Headlee D, Stinson S, Lush RM, Figg WD, Pluda J, Sausville EA. A Phase 1 trial of flavopiridol (FLA), a novel cyclin-dependent kinase inhibitor, in patients with refractory neoplasm. Proc. Am. Soc. Clin. Oncol. 1997; 16:226a. Abstract. 18. Ruef i, Hu ZY, Yin L-Y, Wu Y, Hanson SR, Kelly AB, Harker LA, Rao GN, Runge MS, Patterson C. Induction of vascular endothelial growth factor in balloon-injured baboon arteries. Circ. Res. 1997; 81:24-33. 19. Ruef J, Rao GN, Li F, Bode Cv Patterson C, Bhatnagar A, Runge MS. Induction of rat aortic smooth muscle cell growth by the lipid peroxidation product 4-hydroxy-2-nonenal. Circulation 1998; 97:1071-1078. 20. Swank RA, Th'ng JP, Guo XW, Valdez J, Bradbury EM, Gurley LR. Four distinct cyclin-dependent kinases phosphorylate histone HI at all of its growth related phosphorylation sites. Biochemistry 1997; 36:13761-13768. 21. Matsushime H, Roussel MF, Ashmun RA, Sherr CJ. Colony-stimulating factor 1 regulates novel cyclins during the Gl phase of the cell cycle. Cell 1991; 65:701-713. 22. Bravo R. Synthesis of the nuclear protein (PCNA) and its relationships with DNA replication. Exp. Cell Res. 1986; 163:287-293. 23. Ewen ME, Sluss HK, Sherr CJ, Matsushime H, Kato JY, Livingston DM. Functional interaction of the retinoblastoma protein with mammalian D-type cyclins. Cell 1993; 7:487-497. 24. Hunter T. Protein kinases and phosphatases: The yin and yang of protein phosphorylation and signaling. Cell 1995; 80:225-236. 25. Payne DM, Rossomando AJ, Martino P, Erickson AK, Her J-H, Shabanowitz J, Hunt DF, Weber MJ, Sturgill TW. Identification of the regulatory phosphorylation sites in pp42/mitogen-activated protein kinase (MAP kinase). EMBOJ. 1991; 10:885-892. 26. Konig A, Schwartz GK, Mohammad RM, Al-Katib A, Gabnlove JL. The novel cyclin-dependent kinase inhibitor flavopiridol downregulates Bcl-2 and induces growth arrest and apoptosis in chronic B-cell leukemia lines. Blood 1997; 90:4307-4312. 27. Drees M, Dengler WA, Roth T, Labonte H, Mayo J, Malspeis L, Grever M, Sausville EA, Fieberg HH. Flavopiridol (L86-8275): Selective antitumor activity in vitro and activity in vivo for prostate carcinoma cells. C/in. CancerRes. 1997; 3-.273-279. 28. Park DS, Rarinelli SE, Greene LA. Inhibitors of cyclin-dependent kinases promote survival of post-mitotic neuronally differentiated PC12 cells and sympathetic neurons. J. Biol. Chem. 1996;271:8161-8169. 29. Parker BW, Kaur G, Nieves-Niera W, Taimi M, Kohlhagcn G, Shimizu T, Losiewicz MD, Pommier Y, Sausville EA, Senderowicz AM. Early induction of apoptosis in hematopoietic cell lines after exposure to flavopiridol. Blood 1998; 91:458-465. 30. Geng YJ, Wu Q, Muszynski M, Hansson GK, Libby P. Apoptosis of vascular smooth muscle cells induced by in vitro stimulation with interferon-gamma, tumor necrosis factor-alpha, and interleukin-1 beta. Arterioscler. Thromb.Biol. 1996; 16:19-27. 31. Schwartz SM, deBIois D, O'Brien ERM. The intima: soil for atherosclerosis and restenosis. Circ. Res. 1995; 77:445-465. 32. Wei GL, Krasinkski K, Kearney M, Isner JM, Walsh K, Andres V. Temporally and spatially coordinated expression of cell cycle regulatory factors after angioplasty. Circ. Res. 1997; 80:418-426. 33. de Young MB, Dichek DA. Gene therapy for restenosis. Circ. Res. 1997; 82:306-313. 34. Brooks EE, Gray NS, Joly A, Kerwar SS, Lum R, Mackman RL, Norman TC, Rosete J, Rowe M, Schow SR, Schultz TG, Wang X, Wick MM, Shiftman D. CVT-313, a specific and potent inhibitor ofCDK2 that prevents neointimal formation.,/ Biol Chem. 1997; 272:299207-29911. 35. Sirois MG, Simons M, Edelman ER. Antisense oligonucleotide inhibition of PDGFR-b receptor subunit expression directs suppression of intimal thickening. Circulation 1997; 95:669-676. 36- Rade JJ, Schulick AH, Virmani R, Dichek DA. Local adenoviral-mediated expression of recombinant hirudin reduces neointima formation after arterial injury. Nature Med. 1996; 2:293-298. 37- Sata M, Perlman H, Muruve DA, Silver M, Ikebe M, Libermann TA, Oettgen P, Walsh K. Fas Hgand gene transfer to the vessel wall inhibits neointima formation and overrides the adenovirus-mediated Tcell response. Proc. Natl. Acad. Sci. USA 1998; 95:1213-1217. 38- Mann MJ, Gibbons GH, Tsao PS, von der Leyen HE, Cooke JP, Buitrago R, Kernoff R, Dzau VJ. Cell cycle inhibition preserves endothelial function in genetically engineered rabbit vein grafts. J. Clin. Invest. 1997; 99:1295-1301. 39. Arguello F, Alexander M, Sterry JA, Tudor G, Smith EM, Kalavar NT, Greene JF, Koss W, Morgan CD, Stinson SF, Siford TJ, Alvord WG, Klabansky RL, Sausville EA. Flavopiridol induces apoptosis of normal lymphoid cells, causes immunosuppression, and has potent antitumor activity in vivo against human leukemia and lymphoma xenografts. Blood 1998;91:2482-2490. 40. O'Brien ER, Aipers CE, Stewart DK, Ferguson M, Tran N, Gordon D, Benditt EP, HinoharaT, Simpson JB, Schwartz SM. Proliferation in primary and restenotic coronary atherectomy tissue: implications for anti-proliferative therapy. Circ. Res. 1993; 73:223-231. 41. Mintz GS, Popma JJ, Pichard AD, Kent KM, Satler LR, Wong SC, Hong MD, Kovach JA, Leon MB. Arterial remodeling after coronary angioplasty: a serial intravascular ultrasound study. Circulation 1996; 94:35-43. We Claim: 1. A kit comprising (-)-cis,5-7-dihydroxy-2-)(2-chIorophenyl)-8-[4-(3- hydroxy-l-methyl)-piperidiniyl]-4H-benzopyran-4-one(flaropiridol) together with excipients or additives for the treatment of lesions after balloon injury. 2. A kit comprising (-)-cis,5-7-dihydroxy-2-X2-chIorophenyI)-8-l4-(3- hydroxy-l-methyl)-piperidiniyl]-4H-benzopyran-4-one(flaropiridol) together with excipients or additivies for the treatment after stent implantation.

Full Text

Field of the Invention
The instant invention relates to the use of 4-H-l -benzopyran-4-one derivatives as inhibitors of smooth muscle cell (SMC) proliferation.
Statement Regarding Federally Sponsored Research or Development
The present invention was made with support from the National Institutes of Health under Grant Nos. HL03658 and AG 15234.
Background of the Invention
The cellular responses to vascular injury-cellular dysfunction, activation, dedifferentiation proliferation and migration - culminate in clinical events - such as restenosis, which occurs following balloon angioplasty and stent placement for the treatment of human atherosclerotic disease1. Smooth muscle cell proliferation is a common, and perhaps unifying feature of vascular injury models, and SMC are the major cellular component of neointimal lesions3,3. Renewed interest in inhibiting SMC proliferation has accompanied the increased use of stents for the treatment of coronary disease, since in-stent restenosis is almost entirely dependent on neointimal formation and SMC hyperplasia4. It is estimated that as many as 100,000 patients with in-stent restenosis required treatment in 1997 alone1; therefore, an easily administered, effective inhibitor of SMC hyperplasia would have profound clinical and economic ramfications6.
Efforts to inhibit SMC proliferation in vascular injury models, either by modulating cellular mediators of the proliferative response or by directly interfering with the cell cycle machinery, have provided important insights into neointimal formation. Cell cycle progression is a tightly controlled event regulated positively by cyelin-dependent kinases (Cdks) and their cyclin regulatory subunits7, and negatively by Cdk~ inhibitors and tumor suppressor genes such as retinoblastoma protein (Rb) and p53s. Adenoviral-mediated overexpression of endogenous Cdk inhibitors p21 and p27kipi or of a constitutively active form of Rb blocks

neointimal formation in the rat carotid injury model9"11; similarly, inhibition of the activity of transcription factor E2F by competitively overexpressing cognate DNA binding sites also inhibits SMC proliferation and neointimal formation12. Such studies support that cell cycle inhibition is an attractive target for intervention in vascular lesion formation.
Whereas genetic interventions have aided in the dissection of the mechanisms regulating neointimal formation, they suffer from the shortcoming of not being, at present, clinically suitable for the treatment of vascular disease in humans. A water-soluble, low molecular weight compound with specific cell cycle-regulatory effects, particularly one with oral activity, would have broad applicability both experimentally and, potentially, clinically. The recently identified flavone, flavopiridol, is a Cdk inhibitor that potently blocks the activity
of Cdk2, Cdc2, and Cdk4 13-16, in contrast with other pharmacological inhibitors of Cdks, flavopiridol is remarkable for its kinase specificity, its oral availability, and its potency, being
effective in nanomolar concentrations 16. These unique features result in a favorable side effect profile that has led to the testing of flavopiridol in Phase I clinical trials for the
treatment of refractory neoplasms ' ?. Given these properties, we have examined the ability of flavopiridol to inhibit SMC proliferation in vitro and after balloon injury to the rat carotid artery. We demonstrate that flavopiridol is a potent and selective inhibitor of cell cycle progression and that it arrests SMC proliferation both in vivo and in vitro; moreover, neointimal formation is effectively blocked by oral doses of flavopiridol lower than those known to have toxic effects in humans.
It has now surprisingly been found, that 4-H-l -benzopyran-4-one derivatives are suitable SMC proliferation inhibitor. It is know that 4-H-l -benzopyran-4-one derivatives are suitable for controlling tumors. However it is surprising that the 4-H-l-benzopyran-4-one derivatives according to the instant invention effectively act as an SMC proliferation inhibitor at dosage levels which have no effect on cellular viability and thus at much lower dosage level than the dosage levels which have to be used in the control of tumor growth.
Accordingly, a subject of the instant invention is the use of 4-H-l-benzopyran-4-one derivatives as inhibitors of smooth muscle cell proliferation.
Figure 1. Effect of flavopiridol on HASMC DNA synthesis. A. Quiescent HASMC were treated in the absence (-) or presence (+) of bFGF (10 ng/ml) and with the indicated concentrations of flavopiridol (nmol/L) for 24 h. BrdU incorporation as a measure of proliferation was determined by an EUSA-based assay and expressed as percentage of incorporation in the absence of bFGF treatment. *p
treated with bFGF (10 ng/ml), thrombin (2 U/ml), or vehicle in the presence of absence of
flavopiridol (75 nmol/L) and BrdU incorporation was measured. *p untreated cells. *»p treatment with thrombin alone.
Figure 2. Effect of flavopiridol on HASMC proliferation. Quiescent HASMC were treated
with bFGF (10 ng/ml) alone (a), bFGF and flavopiridol (75 nmol/L) (6), or vehicle (8) for the
times indicated and cell numbers after treatment were determined. Results are expressed as the
cell counts per well (X 103),
Figure 3. Effect offlavopiridol on cyclin-dependent kinase activity in HASMC. Quiescent
HASMC were treated with bFGF (10 ng/ml), thrombin (2 U/ml), or vehicle in the presence or
absence offlavopiridol (75 nmol/L), and phosphorylation of histone HI was quantified as a
measure of Cdk activity and expressed as a percentage of Cdk activity in the absence of bFGF
treatment. *p bFGF alone, fp Figure 4. Regulation of cell cycle-related proteins by flavopiridol. Quiescent HASMC were
treated in the presence (+) or absence (-) of bFGF (10 ng/ml), thrombin (2 U/ml), and/or
flavopiridol (75 nmol/L) for 24 h. Immunoblotting of cellular lysates was performed with
specific antibodies recognizing cycltn Dl (top panel), PCNA (middle panel), and
phosphorylated (pRb) and hyperphosphorylated (ppRb) Rb (tower panel).
Figure 5. Effects offlavopiridol on MAP kinase activity in HASMC, Quiescent HASMC were
treated in the presence (+) or absence (-) of bFGF (10 ng/ml), thrombin (2 U/ml), PD98059
(30 umot/L) and/or flavopiridol (75 nmol/L) for 30 min. Levels of phosphorylated Erkl
(pErkl) and Erk2 (pErk2) were measured by immunoblotting with a phosphorylation-specific
antibody recognizing both proteins (upper panel). MAP kinase activity was measured with an
in-gel kinase assay, using myelin basic protein as a substrate (lower panel).
Figure 6. HASMC viability after treatment with flavopiridol. Quiescent HASMC were treated
with flavopiridol (75 nmol/L), TNF-a (50 ng/ml), or vehicle for the times indicated. Cellular
viability was assessed by trypan blue exclusion. Results are expressed as the percentage of
viable cells to total cells counted.
Figure 7. Inhibition of rot carotid artery neointimal formation by flavopiridol after balloon
injury. Neointima/media ratios were measured in histologic sections or rat carotid arteries
treated with or without flavopiridol (5 mg/kg) for 5 days after injury. Arteries were examined
7 (n = 12) and 14 (n = 12) days after injury. The percentage of PCNA-positive nuclei (± SEM,

expressed as a percentage of counted nuclei) in the neointima of arteries from each time point and treatment group is also given. *p Suitable 4-H-I-benzopyran-4-ojie derivatives are compounds of the formula I

wherein
R1 is hydrogen, alky! having 1 to 6 carbon atoms, aryl-Ci-C4-aIkyl; Ci-Ce-alky! substituted by halogen, hydroxy, or carboxy; C3-C6-cycloalkyl, pyridyl, thienyl, C3-C6-cycloalkyl-C|-C4-alkyl, C2-C6-alkenyI, C2-C6-alkynyI, phenyl; phenyl, mono- or polysubstituted by halogen, Cr Ct-alkyl, C|-C4-alkoxy, hydroxyl, carboxyl, COO-alkyl, CONH2, CONH-alkyt, CON(alxyi)2, nitro, trifluoromethyl, amino, Ci-C4-alkylamino, di-Ci-Q-alkylamino, or phenyl; naphthyl, carboxyl, -CHO, COO-Ci-CU-alkyI, a primary amino, alkylamino, aralkylamino, dialkylamino, amido, arylamino, diarylamino, or -CHiO-Ci-Q-alkyl;

R2 is hydrogen, alkyl having 1 to 6 carbon atoms, aryl, nitro, amino, di-Ci-C4-alkylamino, a halogen, hydroxy!, alkoxy, -COOH, -COO-Cl-C4-alkyl, -CHO, -CH2OH or -CH20-Cr C4alkyl;
R3 is hydrogen, C|-C4-alkyI; C|-C4-alkyl substituted by halogen, hydroxy or carboxy, hydroxy!, carboxyl, nitro, amino, Ci-CValkylammo, di- Ci-C4-alkylamino, halogen, -O-alkyl-C(0)-alkyI, -CHO, -CH2OH, -CH20- C,-C4-alkyl or R2N-C(0)-0-, wherein R is H, C,-C6-alkyl, cycloalkyl; or -O-alky!-C(0)-alkyl or aryl;
R4 is hydrogen, hydroxy!, C|-C4-a(koxy, C|-C4-alkanoyIoxy, C[-C4-alkoxycarbonyI, aryloxy,
amino, Ci-C4-alkylamino, di- Ci-C4-alkylamino, or
RVN-C(0)-0-
wherein R' is H, Cj-Ce-alkyl, cycloalkyl or aryl;
Rs is hydrogen, Cj-Q-atkyl, aryl- C1-C4 alkyl, Ca-Cs-cycloalkyl, C3-C6-cycloalkyl- C1-C4-
alkyl, alky! amino, C]-C4-aIkanoyI, -C(0)-0- C|-C4-alky! or aroyl, where the aryl group in Ri,
Ri, Rs, R4, and R5 is unsubstituted phenyl or phenyl that is mono or pofysubstituted by
halogen, CrC4-alkyl, C-Ci-alkoxy, hydroxy!, carboxyl, COO-atkyl, CONH2, CONH-alkyl,
CON(alkyl)2, nitro, trifluoromethyl, amino, Ct-C4-alkyl amino, di- C,-C4-aikyIamino or
phenyl;
m is an integer between 0 and 3 and n is 1,
or a pharmacologically acceptable acid addition salt thereof.
The compounds according to the invention have two centers of asymmetry, one where the heterocyclic ring containing nitrogen is fused to the benzopyran moiety (C-4'), the other at the rVsubstituted carbon atom (C-31), which means that two pairs of optical isomers are possible. The definition of the compounds according to the invention embraces all possible stereoisomers and their mixtures. It very particularly embraces the racemic forms and the isolated optical isomers having the specified activity. The two racemates can be resolved by physical methods, such as, for example, fractional crystallization. The individual optical isomers can be obtained from the racemates by conventional methods, such as, for example, salt formation with an optically active acid followed by crystallization.

Examples of alkyl groups which are suitable for R| to Rs are straight-chain or branched radicals having up to 6, preferably up to 5, carbon atoms, for example methyl, ethyl, propyl, isopropyl, t-butyl, pentyl or isopentyl groups.
Examples of substituted alkyl groups which are suitable for Rj to Rs are haloalkyl, such as trifluoromethyl, hydroxyalkyl, such as hydroxyethyl, or carboxyalkyl, such as carboxyethyl.
Suitable examples of a cycloalkyl group which has 3 to 6 carbon atoms and is represented by Ri to Rs are cyclopropyl, cyclobutyl, cyclopentyl or cyclohexyl, Cyclopropylmethyl is an example of cycloalkylalkyl.
An example of an aralkyl group which is represented by R, to R5 is a phenylalkyl group in which the phenyl group is unsubstituted or monosubstituted or polysubstituted by substituents such as halogen, Ci-O-alkyl, C|-C4-alkoxy or nitro or by a trifluoromethyl group, amino group and substituted amino group.
An example of an aryl group which is represented by Ri to Rs is a phenyl group which is unsubstituted or monosubstituted or polysubstituted by substituents such as halogen, C\~C2, nitro or trifluoromethyl, amino, C|-C4-alkylamino, di- C|-C4-alkylamino, aromatic heterocyclic groups such as pyridyl groups, and polycyclic aromatic radicals, such as naphthyl groups.
A suitable example of an alkylamino group which is represented by R] to R5 is (CHj),,-NRfiR?, where n is 1 to 3 and Re and R7 are alkyl and are as defined as above in the case of alkyl Ri to R5; moreover, R* and R? together with the nitrogen atom to which they are bonded can be a heterocyclic ring having one or more hetero atoms. Suitable examples of heterocyclic rings which are formed by R^ and R? together with the nitrogen to which they are bonded are piperidine. pyrrolidine, morpholine, piperazine or imidazole, all of which can be unsubstituted or substituted in one or more positions by CrC4-alkyl, Ci-Gpalkoxy or aryl or by a hydroxyl or amino group.
Suitable examples of salts of the compounds according to the invention with inorganic or organic acids are hydrochloride, hydrobromide, sulfate, phosphate, acetate, oxalate, tartrate, citrate, maleate or fumarate.

in which Ri is hydrogen, Ci-Cj-alkyl, naphthyl, phenyl; phenyl mono- or polysubstituted by
halogen, C,-C4-alky], d-C4-alkoxy, hydroxyl, carboxyl, COO-alkyl. CONH2, CONH-alkyl,
CON(alkyI)2, nitro, trifluoromethyl, amino, Ci-Gi-alkylammo, di- C]-C4-alkyJamino, or
phenyl; pyridyl, or thienyl;
R2 is hydrogen or Cj-C3-alkyI;
Rs is C,-C3-alkyl, Cs-Cs-cycloalkyl. or C3-Cj-cycloaIkyl- C|-C4-alkyI.
Particularly preferred are compounds of formula la, in which R| is phenyl, thienyl, pyridyl, chlorophenyl, dichlorophenyl, methylphenyl, aminophenyl, bromophenyl, hydroxyphenyl or naphthyl; Ri is hydrogen and Rj is methyl.
A compound of special importance is (-)-cis,-5,7-dihydroxy-2-)(2-chlorophenyl)-8-[4-(3-hydroxy-l-methyl)-piperidinyl]-4H-benzopyran-4-one (Flavopiridol), particularly in fee form of ist hydrochloride.
The compounds according to the instant invention can be prepared according to the disclosure of U.S. Pat. No. 4,900,727 and U.S. Pat. No. 5,284,856 which are incorporated herein by reference. The examples of said U.S. patents are also of relevance for the instant application.
The compounds according to the instant invention inhibit smooth muscle cell proliferation. Further subject-matter of the invention are therefore also pharmaceuticals for the inhibition of smooth muscle cell proliferation, which contain at least one compound of the formula I as defined above or at least one of its pharmacologically acceptable acid addition

salts, and the use of a compound of the formula I as defined above for the preparation of a pharmaceutical having an smooth muscie cell proliferation inhibitory action. Typical application areas for the compounds according to the instant invention are diseases/disorders/injuries which are accompanied by smooth muscle cell-rich vascular lesions. A very important example therefor are lesions after balloon injury. Another important application area is the prevention of restenosis after stent implantation.
The 4H-1 -benzopyran-4-one derivatives are used according to the invention in the generally known fashion which is known to the expert. For pharmaceuticals, an effective amount of the active substance mentioned is employed either per se or preferably in combination with suitable pharmaceutical auxiliaries in the form of tablets, coated tablets, capsules, suppositories, emulsions, suspensions or solutions, the active compound content being up to about 95%, preferably between 10 and 75%.
The expert will know which auxiliaries are suitable for the desired formulation of the pharmaceutical because of his expert knowledge. Besides auxiliaries for tablets, or solvents, gel formers, bases for suppositories and other excipients for the active substance, it is possible to use, for example, antioxidants, dispersants, emulsifiers, defbamers, flavor corrigants, preservatives, solubilizers or colorants.
The active substance can be administered orally, parenterally, intravenously or rectally, oral administration being preferred. For a form of oral administration, the active substance may be mixed with other compounds together with the additives which are suitable for this purpose, such as excipients, stabilizers or inert diluents, and customary methods can be used for bringing it into suitable administration forms, such as tablets, coated tablets, hard-gelatin capsules, and aqueous alcoholic or oily suspensions or solutions. Examples of inert excipients which can be used are gum arabic, magnesia, lactose, glucose or starch, in particular corn starch. In this context, the formulation can be prepared as dry granules or moist granules. Examples of suitable oily excipients or solvents are vegetable or animal oils, such as sunflower oil or cod-liver oil.
For subcutaneous or intravenous administration, a solution, suspension or emulsion of the active substance is formed, if appropriate using substances which are conventional for this purpose, such as solubilizers, emulsifiers or other auxiliaries. Examples of suitable solvents are water, physiological sodium chloride solution or alcohols, for example, ethanol, propanol or glycerol, and also sugar solutions, such as glucose solutions or mannitol solutions, or a mixture of the various solvents which have been mentioned.

The dose of 4H-l-benzopyran-4-one derivatives which is to be administered daily is to be selected to suit the desired effect. The 4H-I-benzopyran-4-one derivatives can be administered in a dose which is less than 70%, preferably less that 60%, in particular less than 50% of the dosage, which is used to control tumor growth in the respective mammal. An example would be - in a nude mouse xenograft model - a dose of about Smg/kg bodyweight administered orally once daily. This is half of the dosage which inhibits tumor growth in the same animal model (Drees et al. Clin. Cancer Res. 1997; 3:273-279).
The compound according to the instant invention can also be comprised in a kit along with other excipients or additives for treatment of lesions of balloon injury or stent implant.
The pharmacokinetic properties of the 4H-l-benzopyran-4-one derivatives could make it necessary to administer the said compound several times a day or to chose slow release formulations.
Examples 1. Flavopiridol inhibits smooth muscle cell proliferation and neointimal formation in vivo in a rat carotid injury model of vascular injury.
The well-established rat carotid injury model, in which neointimal lesion formation after catheter-induced injury is critically dependent on SMC proliferation (Clowes et al. Lab. Invest. 1983; 49:327-333, Clowes et al. Circ. Res. 1985; 56:139-145) to examine whether Flavopiridol induces growth arrest of SMC in vivo, as it does in vitro. Flavopiridol was administered orally at a dose of 5 mgAcg once daily, beginning on the day of injury and for four days after, since this time period covers the initial induction of Cdk2 and the first wave of SMC proliferation in this model (Circ. Res.1995; 77:445-465, Circ. Res.1997; 80:418-426). Mean tntimal and medial areas were quantified 7 and 14 days after injury, and neointimal lesion size was expressed as the ratio of the neointimal to the medial area. Twelve animals each were in the treated and untreated groups. The ratio at 7 days was 1.00+/-0.05 in arteries of vehicle-treated rats and 0.65+/-0.04 in Flavopiridol-treated rat arteries, a reduction of 35%. At 14 days, the neointimal/media ratio was 1.08 +/-0.Q4 in vehicle-treated rats and 0.66+/-0.03 In Flavopiridol-treated rats, a reduction of 38.9%. These effects were statistically significant at both time points (P Methods Materials- Flavopiridol (L86-8275, (*ycis, -5,7-dihydroxy-2-
(bFGF) was purchased from Collaborative Biochemical and thrombin from Sigma. The MEK1 inhibitor PD98059 was obtained from New England Biolabs.
Cell culture- Human aortic smooth muscle cells (HASMC) were obtained from Clonetics and were cultured as previously described 18. Cells were used at passages 5-9. Prior to performing experiments, cells were growth-arrested at 80% confluence for 48 h with medium containing 0.2% fetal bovine serum.
Cell proliferation EUSA- Cell proliferation was measured by ELISA (Amersham Life Science). HASMC were grown in gelatin-coated 96-well plates and made quiescent. Cells were treated with 10 ng/ml bFGF, 2 U/ml thrombin, or vehicle for 24 h. Flavopiridol (75 nmoi/L) was administered I h prior to growth factor treatment. 5-Bromo-2'-deoxyuridine (BrdU) was added to a final concentration of 10 umoI/L during the last 2 h of treatment. BrdU
incorporation was measured as described 19, Results are expressed as mean ± SEM for 12 samples per condition from two independent experiments.
Cell counts- Growth-arrested HASMC grown to 50% confluence in 6-weII plates were treated with or without flavopiridol (75 nmol/L) or bFGF (10 ng/ml). At intervals after treatment, cells were trypsinized and cell numbers determined using a hemocytometer.
Western blot analysis- Quiescent HASMC were treated in the presence or absence of growth factors and/or flavopiridol as indicated. Western blot analysis was performed as previously
described '8. The primary antibodies were: a polyclonal anti-human cyclin Dl antibody (M-20, Santa Cruz), a monoclonal anti-human proliferating cell nuclear antigen (PCNA) antibody (PC10, Sigma), a phosphorylation-specific p44/42 (Erkl/Erk2) MAP kinase monoclonal antibody (New England Biolabs), and a monoclonal anti-Rb antibody (G3-245, Pharmigen), which recognizes the phosphorylated (pRb) and highly phosphorylated (ppRb) Rb species. For immunoblotting studies, experiments were repeated at least three times.
Cdk activity- Quiescent HASMC were treated with agonists and inhibitors for 24 h and total cell lysates were prepared as described for Western blotting. The kinase assay was performed with a histone HI kinase assay kit (Upstate Biotechnology) following the manufacturer's instructions, fn brief, 10 |il of peptide inhibitors for protein kinase C (2 umol/L) and protein kinase A (2 fjmol/L), 100 jig cell lysate, 10 ul assay buffer and 10 uJofa mixture containing

75 umol/L magnesium chloride, 500 umol/L ATP and I uCi/ml [v-J2P]ATP were mixed in a microcentrifuge tube. After incubation at 30°C for 10 min, aliquots were pipetted onto phosphocellulose papers. The papers were washed in 0.75% phosphoric acid, followed by measurement of cpm in a scintillation counter (Beckman). Results are expressed as the mean ± SEM for three samples and are representative of three independent experiments.
In-gel kinase assay- Quiescent HASMC were treated with growth factors for 30 min and total cell lysates were prepared as described for Western blotting. In some experiments, HASMC were pretreated for 60 min with 30 umol/L PD98059, flavopiridol, or vehicle. Equal amounts of proteins (50 jig/lane) were resolved on a polyacrylamide gel that was copolymerized with 350 ug/ml myelin basic protein. The gel was treated with [y-MP]ATP and autoradiography was performed as described '^.
Trypan-blue exclusion- HASMC were grown in 5-cm dishes at low confluence and growth-arrested as described. Cells were treated with flavopiridol (75 nmol/L) or tumor necrosis factor-D (TNF-D; 50 ng/ml) for the indicated times. Following the removal of the medium, 0.4% trypan blue in phosphate-buffered saline was added to the dishes. After 5 min, the cells in the dishes were counted under the microscope. Blue celts were counted as nonviable cells.
Rat carotid injury model- Injury to the rat carotid artery was performed essentially as
described 2. Adult male Sprague-Dawley rats (400-500 g, Zivic-Miiler) were anesthetized with an intraperitoneal injection of ketamine (2 mg/kg) and xylazine (4 mg/Wg). The left internal carotid was then cannulated with a 2F embolectomy catheter. The balloon was inflated with saline and withdrawn across the artery three times to produce a distending and denuding injury. The right carotid artery was uninjured and served as a control of injury for each animal. Immediately following recovery from anesthesia and for four additional days thereafter, rats were administered flavopiridol (5 mg/kg in water) or water by gavage in a blinded manner. All rats survived the surgery and mere were no overt signs of toxicity related to drug administration in the doses used. At specified time points after carotid injury, rats were anesthetized as above and perfiision-flxed systemtcally with 4% paraformaldehyde in phosphate-buffered saline. Right and left carotid arteries were removed and distended by injection of 4% paraformaldehyde through the lumen, after which they were dehydrated and stored in 70% ethanol at 4°C. Immunohistochemistry was performed as previously described

l8, using the monoclonal PCNA antibody and a polyclonal anti-human Cdk2 antibody {M2-G, Santa Cruz).
Image analysis- The extreme distal and proximal regions of each artery (approx. 500 um) were removed. Ten intermediate cross-sections (8 urn each) taken 500 um apart were analyzed from each artery. Slides were fixed and stained with hematoxylin and eostn as previously described '8. Using a Nikon Diaphot 300 microscope and a 4X objective, each cross-section was captured as a digital image using a Hamamatsu C5985 video camera and TCPro 2.41 (Coreco, Inc.). Medial and neointimal areas were determined using NIH Image software. Medial and neointimal boundaries were determined by one slide reviewer (A.M.) and verified in a blinded fashion by a second reviewer (C.P.). Lesion size was expressed as the neointima/media ratio. Results for each group were expressed as the mean ± SEM. 92% or more of the images were interpretable in each group; the remainder suffered from fixation artifacts and were not analyzed.
Statistical analysis- When appropriate, data from quantitative studies were expressed as the mean ± SEM. For multiple treatment groups, a factorial ANOVA followed by Fisher's least significant difference test was applied. Statistical significance was accepted at p Results
Flavopiridol inhibits HASMC proliferation- On the basis of flavopiridol's ability to inhibit the proliferation of a variety of tumor cell lines, we tested the hypothesis that its use would block the growth of primary culture human SMC. Growth-arrested HASMC were treated with the SMC mitogen bFGF (10 ng/ml) for 24 h in the presence of increasing concentrations of flavopiridol and proliferation was measured by an ELISA-based assay. In comparison with untreated cells, proliferation of bFGF-treated cells was increased 5.4-fold (Figure I A). Pretreatment for 1 h with as little as 50 nmol/L flavopiridol significantly decreased HASMC proliferation (to 3.9-fold,p To test the generality of flavopiridol's effects on SMC proliferation, we examined its effect on mttogenesis elicited by thrombin (2 U(m\), which acts through a G protein-coupled receptor, in comparison with bFGF, which stimulates a member of the receptor tyrosine kinase family.

Flavopiridol (75 nmol/L) significantly and potently inhibited both bFGF- and thrombin-induced HASMC proliferation (5.4-fold vs. 1.8-fold and 2.4-fold vs. 0.7-fold, respectively,p Flavopiridol inhibils Cdk activity and cell cycle-related gene expression In HASMC- To assess the specific effect of flavopiridol on the cell cycle machinery, we measured histone HI kinase activity in cellular lysates from growth factor-stimulated HASMC. Phosphorylation of
histone HI reflects the activities of Cdc2 and Cdk2 20. Treatment of HASMC with bFGF and thrombin resulted in 4.4-fold and 3.6-fold mcreases, respectively, in histone HI kinase activity (Figure 3). These increases in cycl in-dependent kinase activity were totally blocked by pretreatment with flavopiridol (75 nmol/L).
By Western blot analysis, we also addressed whether flavopiridol influenced growth factor-induced regulation of cell cycle-related proteins in HASMC. Cycl in Dl is a Gl phase cycl in that is upregulated by growth factor stimulation and is rapidly degraded during
withdrawal from the cell cycle 21, Cyclin Dl protein levels were upregulated 6.3-fold and 3.2-fold, respectively, in response to bFGF and thrombin treatment for 24 h (Figure 4), an effect which could be completely blocked by pretreafment with flavopiridol, Similarly, increased expression of PCNA, which is synthesized predominantly during S phase in conjunction with DNA replication 22, was also blocked by flavopiridol pretreatment. As a final measure of cell cycle-related proteins, we examined Rb phosphorylation in response to growth factor expression using an antibody that recognizes phosphorylated Rb. Rb is a cell cycle regulator that binds to and inactivates transcription factor E2F when Rb is in the unphosphorylated state 23 and induces SMC growth arrest in vivo '. Phosphorylation inactivates Rb and allows progression through S phase to proceed. Analysis of Rb phosphorylation is particularly relevant because Rb is a target of Cdk2 and Cdk4 in vivo. Both thrombin and bFGF induced hyperphosphorylation of Rb, an effect that was inhibited by flavopiridol. Taken together, these results indicate that flavopiridol influences the expression and activity of Gl and S phase-related cell cycle control elements in HASMC in association with its growth-inhibitory effects.

Flavopiridol has no effect on MAP kinase phosphorylation or activity- To ensure that flavopiridol was acting specifically at the level of the cell cycle, rather than nonspecifically on upstream kinase pathways, we measured phosphorylation and activity of ErkI (p44 MA? kinase) and Erk2 (p42 MAP kinase), two members of the MAP kinase family. We chose these kinases because they are immediately upstream of transcriptional events occurring in response to growth stimuli and downstream of a number of critical mitogenic signaling pathways 2 activation 25, increased amounts of phosphorylated ErkI and Erk2 were detected after treatment of HASMC with thrombin and bFGF for 30 min, compared with untreated cells (Figure 5, upper panel). Phosphorylation of ErkI and Erk2 by both thrombin and bFGF was blocked by pretreatment with PD98059, but not with flavopiridol. To confirm these findings, we measured Erk 1 and Erk2 activity by an in-gel kinase assay (Figure 5, lower panel). Again, we found that ErkI and Erk2 activities were increased in response to thrombin and bFGF, an effect that was inhibitable by PD98059 but not by flavopiridol. These experiments, in conjunction with those presented in Figures 3 and 4, provide evidence that the effects of flavopiridol on HASMC proliferation are due to a specific arrest of the cell cycle machinery by blocking Cdk activity without affecting upstream signaling events.
Flavopiridol does not decrease HASMC viability- Previous reports of flavopiridol activity in other cell types have demonstrated that, depending on the cell line, flavopiridol may either induce growth arrest without affecting viability, or it may cause apoptosis ' 6\26-29, we therefore assessed whether flavopiridol decreased the viability of HASMC by measuring trypan blue exclusion at various time points after treatment. Quiescent HASMC were treated with flavopiridol (75 nmol/L)» vehicle, or TNF-0 (50 ng/ml), a cytokine known to induce apoptosis in this cell type 30. Whereas TNF-D potently decreased the viability of HASMC, resulting in the death of essentially all cells treated for 24 h, flavopiridol had no such effect (Figure 6). We have noted that with higher concentrations and longer incubations, some decreases in viability in the presence of flavopiridol may occur (not shown). However, under

the conditions tested, flavopiridol primarily induces growth arrest, without affecting SMC viability.
Flavopiridol inhibits smooth muscle cell proliferation and neointimal formation in vivo in a rat carotid injury model of vascular Injury- We utilized the well-established rat carotid injury model, in which neointimal lesion formation after catheter-induced injury is critically dependent on SMC proliferation 2,3_ (0 examine whether flavopiridol induces growth arrest of SMC in vivo, as it does in vitro. We administered flavopiridol orally at a dose of 5 mg/kg once daily, beginning on the day of injury and for four days after, since this time period covers the initial induction of Cdk2 and the first wave of SMC proliferation in this model 31,32. Mean intimal and medial areas were quantified 7 and 14 days after injury, and neointimal lesion size was expressed as the ratio of the neointimal to the medial area. Twelve animals each were in the treated and untreated groups. The neointima/media ratio at 7 days was 1.00 ± 0.05 in arteries of vehicle-treated rats and 0.65 ± 0.04 in flavopiridol-treated rat arteries, a reduction of 35.0% (Figure 7). At 14 days, the neointima/media ratio was 1.08 ± 0.04 in vehicle-treated rats and 0.66 ± 0.03 in flavopiridol-treated rats, a reduction of 38.9%. These effects were statistically significant at both time points ip In order to demonstrate directly that flavopiridol inhibited smooth muscle cell proliferation, we stained sections for PCNA expression in representative fields from each artery and determined the percentage of PCNA-positive nuclei in the neointima. At 7 days, 31.1 ± 7.2% of nuclei in injured arteries from untreated rats were PCNA-positive, whereas only 11.8 ± 1.5% of injured arteries in flavopiridol-treated rats were PCNA-positive (Figure 7; p Discussion In the present study, we have examined whether the novel Cdk inhibitor, flavopiridol, the most potent and specific inhibitor of Cdks known, is a suitable candidate for inhibiting SMC proliferation in vivo, particularly in the setting of vascular injury. Previous attempts to target

the cell cycle machinery therapeutically for the treatment of vascular disease have provided a rationale for the present studies 9'12; however, methods employed to this end have relied on gene transfer technologies to inhibit cell cycle progression. At present, formidable obstacles prevent the clinical application of these techniques 33, During the course of our studies, it was reported that CVT-313, a recently identified compound that also has Cdk inhibitory properties but at micromolar concentrations, can also inhibit neointimal formation; however, it was required that CVT-313 be instilled into the carotid artery at the time of injury to produce this effect 34. in contrast, we show that flavopiridol, when administered orally, can potently inhibit neointimal formation, to a degree comparable with other clinically relevant agents ' U 35,36.7he ora| activity of flavopiridol makes it virtually unique among agents shown to be active in animal models of vascular injury. Its selectivity, potency, and ease of administration make flavopiridol an excellent candidate for examining the therapeutic benefits of cell cycle inhibition in vivo in human vascular lesions.
We chose to administer flavopiridol orally in a concentration (5 mg/kg) half that which
inhibits tumor growth in a nude mouse xenograft model 27. It is notable that flavopiridol concentrations of 75 nmol/L result in near complete inhibition of SMC proliferation in our studies, whereas median serum concentrations of 425 nmol/L were attained at doses below the
toxic threshold in Phase I human studies of refractory carcinoma 17. Our results suggest that much lower doses of cell cycle inhibitors than those used for neoplasia may be effective in the setting of vascular diseases such as restenosis, with the concomitant benefit of increased toletability.
Whereas we have demonstrated that flavopiridol induces growth arrest without affecting viability of HASMC in culture, and have shown decreased neointimal formation after flavopiridol treatment in vivo, we cannot be sure that cell cycle arrest is the only factor reducing neointimal formation in carotid lesions. Flavopiridol can induce growth arrest with or without inducing apoptosis, depending on the cell type observed 29. Interestingly, flavopiridol inhibits apoptosis in PC 12 cells that have been terminally differentiated, yet it
induces apoptosis in undifferentiated PC12 cells that are proliferating 28. Although our in vitro experiments were performed under conditions that would mimic the phenotype of SMC prior to injury, it is possible that SMC may respond differently to flavopiridol after injury and may even undergo apoptosis. While the role of apoptosis in vascular lesions is unclear,

expression of the Fas ligand in SMC induces apoptosis and blocks neointimal formation in rabbits after balloon injury 37, suggesting that if flavopindol does indeed induce apoptosis of SMC in vivo, as it does in proliferating PC 12 cells, this may be a salutary phenomenon in the context of neointimal formation. Other mechanisms may also contribute to the effects of flavopiridol on lesion formation. For example, antisense oligodeoxynucleotide-mediated cell cycle inhibition improves endothelial function in rabbit vein grafts 38. Further studies will be needed to address mechanisms of flavopiridol's effects, other than growth arrest, on SMC in vivo.
Given the demonstrated role of SMC proliferation in lesion formation after rat carotid injury
2* 3, it is interesting to note that, despite the profound effects of flavopiridol in vitro, its ability to inhibit neointimal formation (though significant) was more modest under the conditions of our in vivo experiments. We have considered several explanations for this observation. It is unlikely that accelerated SMC proliferation occurs after cessation of flavopiridol, since differences in proliferative indices are sustained as long as 14 days after injury (Figures 7 and 9), It is more plausible either that other components of lesion formation, such as SMC migration and extracellular matrix production, still contribute to lesion formation, even in the absence of significant SMC proliferation, or that delivery of flavopiridol once daily is insufficient to arrest proliferation entirely in this model. Recent data indicating that the biological half-life of flavopiridol is as short as 2.5 hrs suggests that the latter hypothesis may be correct; further studies may be able to identify an even more effective dosing regimen 39,
Our results indicate that flavopiridol can inhibit SMC proliferation and, hence, neointimal formation in a well-accepted small animal model of vascular disease. It must be pointed out that the relevance of inhibition of SMC proliferation is controversial in human vascular lesions, and may differ depending on the nature of the lesion and the time at which observations of proliferation are made. The proliferative index of SMC in human atherectomy specimens is remarkably low 40, although these specimens may not reflect proliferative changes at earlier, more critical stages in lesion development. In addition, arterial remodeling independent of neointimal growth may account for a significant proportion of luminal
obstruction after angioplasty in humans 41. In contrast, indices of mitotic activity in SMC are much higher (25% PCNA-positive) in atherectomy specimens from human lesions with in-stent restenosis, consistent with the established role of SMC hyperplasia, but not remodeling,

/
in this process "*. As stent placement and the clinical problem of in-stent restenosis increases, so will the need for an effective means to arrest SMC hyperplasia and neointimal formation. Since flavopiridol is a potent, orally available drug with specific Cdk-inhibitory activity, and since safe doses of fiavopiridol are known in humans, flavopiridol can be considered as a pharmacologic candidate for prevention of in-stent restenosis in humans. Methods and Results: Using cultured human aortic smooth muscle cells, we found that flavopiridol in concentrations as low as 75 nmol/L resulted in near complete inhibition of basic fibroblast growth factor- and thrombin-induced proliferation. At this dose, flavopiridol inhibited cyclin-dependent kinase activity, as measured by histone HI phosphorylation, but had no effect on MAP kinase activation. Induction of cell cycle-related proteins cyclin Dl, proliferating cell nuclear antigen, and phosphorylated retinoblastoma protein was also blocked by flavopiridol. Flavopiridol had no effect on cellular viability. To test whether flavopiridol had a similar activity in vivo when administered orally, we examined neoindmal formadon in rat carotid arteries after balloon injury. Fiavopiridol (5 mg/kg) administered by gavage reduced neointimal area by 35% and 39% at 7 and 14 days, respectively, after injury. Conclusions: Flavopiridol inhibits SMC growth in vitro and in vivo. It's oral availability and selectivity for cyclin-dependent kinases make it a potential therapeutic tool in the treatment of SMC-rich vascular lesions.
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We Claim:
1. A kit comprising (-)-cis,5-7-dihydroxy-2-)(2-chIorophenyl)-8-[4-(3-
hydroxy-l-methyl)-piperidiniyl]-4H-benzopyran-4-one(flaropiridol)
together with excipients or additives for the treatment of lesions after
balloon injury.
2. A kit comprising (-)-cis,5-7-dihydroxy-2-X2-chIorophenyI)-8-l4-(3-
hydroxy-l-methyl)-piperidiniyl]-4H-benzopyran-4-one(flaropiridol)
together with excipients or additivies for the treatment after stent
implantation.

Documents:

in-pct-2001-1010-che abstract-duplicate.pdf

in-pct-2001-1010-che abstract.pdf

in-pct-2001-1010-che claims-duplicate.pdf

in-pct-2001-1010-che claims.pdf

in-pct-2001-1010-che correspondence-others.pdf

in-pct-2001-1010-che correspondence-po.pdf

in-pct-2001-1010-che description (complete)-duplicate.pdf

in-pct-2001-1010-che description (complete).pdf

in-pct-2001-1010-che drawings.pdf

in-pct-2001-1010-che form-1.pdf

in-pct-2001-1010-che form-19.pdf

in-pct-2001-1010-che form-26.pdf

in-pct-2001-1010-che form-3.pdf

in-pct-2001-1010-che form-5.pdf

in-pct-2001-1010-che pct.pdf

in-pct-2001-1010-che petition.pdf

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Patent Number

224306

Indian Patent Application Number

IN/PCT/2001/1010/CHE

PG Journal Number

47/2008

Publication Date

21-Nov-2008

Grant Date

10-Oct-2008

Date of Filing

18-Jul-2001

Name of Patentee

AVENTIS PHARMACEUTICALS INC

Applicant Address

ROUTE 202-206, P.O. BOX 6800 BRIDGEWATER NEW JERSEY 08807-0800

Inventors:

#	Inventor's Name	Inventor's Address
1	PATTERSON, WINSTON, CAMPBELL	1915 AVENUE M, GALVESTON, TX 77550
2	DUMONT, JENNIFER, A	37 FAWN TERRACE LANE, GROTON, MA 01450,

PCT International Classification Number

A61K31/00

PCT International Application Number

PCT/US00/01104

PCT International Filing date

2000-01-18

PCT Conventions:

#	PCT Application Number	Date of Convention	Priority Country
1	09/243,380	1999-02-01	U.S.A.
2	09/468,665	1999-12-21	U.S.A.