WO1996030005A1

WO1996030005A1 - Anti-stroke effects of calcium antagonists

Info

Publication number: WO1996030005A1
Application number: PCT/US1996/004374
Authority: WO
Inventors: Frank M. Yatsu; Nargis A. Alam; Satoshi Kataoka
Original assignee: Research Development Foundation
Priority date: 1995-03-30
Filing date: 1996-03-29
Publication date: 1996-10-03
Also published as: AU5436896A; IL117560A0; ZA962472B

Abstract

The present invention provides a method of treating a cerebrovascular disease in an animal comprising the step of administering to an animal in need of such treatment a therapeutically effective dose of a calcium channel antagonist. Also provided is a method of inhibiting the proliferation of vascular smooth muscle cells in an animal comprising the step of administering to an animal in need of such treatment a pharmacologically effective dose of a calcium channel antagonist. Further provided is a method of inhibiting the expression of transcription factor AP-1 in an animal comprising the step of administering to an animal in need of such treatment a pharmacologically effective dose of a calcium channel antagonist.

Description

ANTI-STROKE EFFECTS OF CALCIUM ANTAGONISTS

BACKGROUND OF THE INVENTION

Fiøiq of tbφ Ipvptjon The present invention relates generally to the fields of neurology and biochemical pharmacology. More specifically, the present invention relates to novel anti-atherosclerogenic effects of calcium antagonists in the cerebrovasculature. Description of the Related Art The proliferation of vascular smooth muscle cells

(VSMCs) is a key event in the pathogenesis of atherogenesis. Platelet-derived growth factor (PDGF) is thought to play an important role in the formation of such lesion formation in vivo. Platelet-derived growth factor, which is thought to be a major mitogen promotes the proliferation of vascular smooth muscle cells in vivo. The effects of platelet-derived growth factor are believed to be transmitted via a platelet-derived growth factor receptor by modulating mitogenesis, phosphoinositide turnover and expression of proto-oncogenes. Previous reports illustrated various actions of calcium antagonists in vitro. The mechanisms underlying the anti-atherogenic effect of calcium antagonists include: an antioxidant activity in cell membranes, a reduction of intracellular lipid accumulation by stimulation of cholesterol ester hydrolase activity and the promotion of cholesterol efflux. , the prevention of the deposition of cholesterol esters in the acrophages and the inhibition of the proliferation of smooth muscle cells. Calcium antagonists impair the development and progression of the atherosclerotic plaque by reducing the proliferation of vascular smooth muscle cells. Moreover, such agents may also affect the transformation of contractile to the synthetic phenotype of vascular smooth muscle cells.

It has been shown that calcium antagonists have an inhibitory effect on platelet-derived growth factor-induced signal transduction pathways, particularly that of phosphatidyl inositol turnover. However, the mechanism by which calcium antagonists induce anti-mitogenic effect on vascular smooth muscle cells is less clear. Elucidation of anti-mitogenic mechanisms of calcium antagonists at the cellular levels is of major interest.

The atherosclerotic lesion or atheroma has been the focus of study for many years, and the histological features have been well delineated in showing four major characteristics. These features are: (1) cellular proliferation, particularly smooth muscle cells; (2) increase in cholesterol deposition, especially cholesterol esters; (3) prominence of macrophages, particularly those which are lipid-laden, so-called "foamy cells" because of their appearance of being fat-filled, plus associated cytokines, produced by macrophages among other cellular elements including those in the blood; and (4) enhanced synthesis of connective tissue elements, such as elastin and glycosaminoglycans. Each of these four areas of histological prominence has been the focus of intense research, and while the definitive sequence of atherogenesis is still debated and uncertain, it clearly is multifactorial and includes the conspiracy of impaired cholesterol metabolism in conjunction with increased proliferation of smooth muscle cells and with heightened activity of cytokines. Certain cytokines are known to be important in regulating leukocyte adhesion, cellular growth, vasomotor functions, remodeling of the vascular matrix and regulating blood compatibility in order to minimize or influence thrombosis on arterial endothelium.

No single element provokes atherosclerosis in isolation but likely control of one of these interactive elements should assist in substantially reducing the process of atherogenesis. For example, cholesterol reduction has already made an impact in decreasing the occurrence of coronary heart disease in subjects at risk who have reduced their serum cholesterol levels below 170 mg/dl. Vascular injury, as occurs with hypertension, smoking, oral contraceptives and other insults unrelated to plasma cholesterol levels, causes the adhesion of platelets to the site of injury. This adhesion provokes a "release reaction" in platelets with the secretion of a variety of compounds, but particularly thromboxane A2 and adenosine diphosphate and mobilizes the recruitment of platelets to increase aggregation. In addition, the platelets release platelet-derived growth factor which provokes the proliferation of smooth muscle cells and their migration to the endothelium where the smooth muscle cells form the initial elements of an atheroma.

The athero atous process is limited primarily to the large vessels such as the aorta and conducting arteries, such as the carotid, renal, coronary or middle cerebral arteries. However, smaller arterioles can be affected with a proliferative process as in lacunar strokes wherein fat laden cells and proliferation is termed "lipohyalinosis". Furthermore, in Binswanger'ε Disease or subcortical arteriopathic encephalopathy, the small penetrating arterioles of the white matter are "end vessels" in processing of unknown nature. The proliferative process of these smaller vessels may represent uncontrolled proliferation under a normally present growth factor. The normal production of nerve growth factor (NGF) by smooth muscle cells of arteries and the response of the NGF protein, the trk-oncogene, suggests a paracrine function. Aberrant or complete loss of regulation of this paracrine control could lead to increased proliferation of arteriolar smooth muscle cells and narrowed luminal area and subsequent reduced blood flow and ischemia. Thus, the normal proliferation of smooth muscle cells probably participates in atherosclerosis and the same uncontrolled process in cerebral arterioles can lead to luminal narrowing, brain ischemia of conducting fibers and stroke-like symptoms and dementia.

The prior art is deficient in the lack of effective means of preventing or therapeutically treating a wide variety of cerebrovascular diseases. The present invention fulfills this longstanding need and desire in the art. SUMMARY OF THE INVENTION

In one embodiment of the present invention, there is provided a method of treating a cerebrovascular disease in an ani al comprising the step of administering to an animal in need of such treatment a therapeutically effective dose of a calcium channel antagonist.

In another embodiment of the present invention, there is provided a method of inhibiting the proliferation of vascular smooth muscle cells in an animal comprising the step of administering to an animal in need of such treatment a pharmacologically effective dose of a calcium channel antagonist. In yet another embodiment of the present invention, there is provided a method of inhibiting the expression of transcription factor AP-1 in an animal comprising the step of administering to an animal in need of such treatment a pharmacologically effective dose of a calcium channel antagonist.

Other and further aspects, features, and advantages of the present invention will be apparent from the following description of the presently preferred embodiments of the invention given for the purpose of disclosure. BRIEF DESCRIPTION OF THE DRAWINGS

So that the matter in which the above-recited features, advantages and objects of the invention, as well as others which will become clear, are attained and can be understood in detail, more particular descriptions of the invention briefly summarized above may be had by reference to certain embodiments thereof which are illustrated in the appended drawings. These drawings form a part of the specification. It is to be noted, however, that the appended drawings illustrate preferred embodiments of the invention and therefore are not to be considered limiting in their scope.

Figure 1 shows the effect of calcium antagonists on [³H]thymidine incorporation in vascular smooth muscle cells. The final concentration of calcium antagonists were tested under unsti ulated condition. The results are expressed as the mean±SD of three independent experiments. Concentration of each calcium antagonists was 10^*5 M, 10^'6 M, and 10^"7 M. *p<0.05, **p<0.01 control vs each calcium antagonist. Figure 2 shows the effect of calcium antagonists on mitogenic response of vascular smooth muscle cells (vascular smooth muscle cells) to recombinant platelet derived growth factor-AA, platelet derived growth factor-BB, and platelet derived growth factor-AB in vascular smooth muscle cells. The final concentration of the recombinant platelet derived growth factor-AA, platelet derived growth factor-BB, and platelet derived growth factor-AB was 40 ng/ml. Final concentration of clentiazem was 10^"5 M and those of verapamil, diltiazem, and nifedipine were 10* M. Data are the mean±SD of three independent experiments. * p<0.05, **p<0.01, control vs each calcium antagonist.

Figure 3 shows the effect of clentiazem on the production of inositol phosphates induced by recombinant platelet derived growth factor-AA, -BB in vascular smooth muscle cells. The final concentration of recombinant platelet derived growth factor-AA and recombinant platelet derived growth factor-BB was 10 ng/ml. The final concentration of clentiazem was 10⁵ M. Data are ean+SD of three independent experiments. *p<0.05, **P<0.01.

Figure 4 shows the phosphorylation of 80 kD protein mediated by PDBu and platelet derived growth factor-BB in vascular smooth muscle cells. Figure 4A shows the phosphorylation of an 80 kD protein mediated by PDBu. The final concentration of PDBu was 200 nM. The concentration of calcium antagonist was 10^*5 M. Figure 4B shows the phosphorylation of 80 kD protein mediated by platelet derived growth factor-BB. The final concentration of platelet derived growth factor-BB was 40 μg/ml. The final concentration of each calcium antagonist was 10^"5 M. The final concentration of staurosporine was 20 mM.

Figure 5 shows the effect of clentiazem on expression of transcription factor AP-1 induced by recombinant platelet derived growth factor in vascular smooth muscle cell. The final concentration of platelet derived growth factor-AA, platelet derived growth factor-BB, and platelet derived growth factor-AB was 10 ng/ml respectively. The final concentration of clentiazem was 10-5 M. The cold AP-1 contained 100 fold of unlabelled AP-1 oligonucleotide.

Figure 6 shows the effect of calcium antagonists on expression of transcription factor AP- 1 in vascular smooth muscle cell. Figure 6A shows the effect of calcium antagonists on expression of AP-1 induced by recombinant platelet derived growth factor-BB. The final concentration of platelet derived growth factor-BB was 10 ng/ml. The final concentration of clentiazem, verapamil, diltiazem, and nifedipine was 10^"5 M. Figure 6B shows the effect of clentiazem and diltiazem on expression of transcription factor AP-1 induced by phorbol 12- myristate 13-acetate. The final concentration of phorbol 12- myristate 13-acetate was 200 nM. The final concentration of clentiazem and diltiazem was 10^'5 M. Figure 7 shows the effect of clentiazem on expression of transcription factor AP-1 in vascular smooth muscle cell. Figure 7A shows the effect of clentiazem on expression of AP- 1 induced by platelet derived growth factor-BB. The final concentration of recombinant platelet derived growth factor-BB was 10 ng/ml. The final concentration of clentiazem was 10^"5 M, 10-6 M, 10-7 M, respectively. Figure 7B shows the effect of clentiazem on expression of AP-1 induced by basic FGF. The final concentration of b-FGF was 10 ng/ml.

DETAILED DESCRIPTION OF THE INVENTION The present invention is directed to a method of treating a cerebrovascular disease in an animal comprising the step of administering to an animal in need of such treatment a therapeutically effective dose of a calcium channel antagonist. Representative examples of cerebrovascular disease include intracerebral hemorrhage, subarachnoid hemorrhage due to aneurysms, migraine, intracerebral atherosclerosis, lipohyalinosis, Binswanger's disease or subcortical arteriopathic encephalopathy, Moyamoya disease and impairment of the blood brain barrier with brain edema formation. Generally, representative calcium channel antagonists useful in the methods of the present invention include clentiazem, verapamil, diltiazem, and nifedipine. The pharmaceutical compositions comprising calcium channel blockers are suitable for use in a variety of drug delivery systems. For a brief review of present methods for drug delivery, see Langer, Science , 249:1527-1533 (1990). Methods for preparing administrable compounds are known or apparent to those skilled in the art and are described in more detail, for example, in Remington's Pharmaceutical Science , 17th ed. , Mack Publishing Company, Easton, PA (1988) . Generally, the calcium channel antagonist is administered in a dose of from about 0.3 mg/kg to about 10 mg/kg.

The present invention is also directed to a method of inhibiting the proliferation of vascular smooth muscle cells in an animal comprising the step of administering to an animal in need of such treatment a pharmacologically effective dose of a calcium channel antagonist.

In yet another embodiment, the present invention is also directed to a method of inhibiting the expression of transcription factor AP-1 in an animal comprising the step of administering to an animal in need of such treatment a pharmacologically effective dose of a calcium channel antagonist.

The following examples are given for the purpose of illustrating various embodiments of the invention and are not meant to limit the present invention in any fashion. EXAMPLE 1

Tissue culture plastics were obtained from Falcon Labware. Growth media and fetal bovine serum were obtained from Sigma. Natural platelet derived growth factor (nplatelet derived growth factor) , and recombinant platelet derived growth factor-AA, platelet derived growth factor-BB, platelet derived growth factor-AB were purchased from Upstate Biotechnology Inc. (Lake Placid NY) . Basic fibroblast growth factor (FGF) was purchased from Gibco BRL (Grand Island NY) . Methyl [³H]thymidine, [³H]myo-inositol, [7-³²P]ATP, and [7~³²P]dCT were obtained from Amersham International. Phorbol 12, 13 dibutylate (PDBu) , phorbol 12-myristate 13-acetate, and staurosporine were purchased from Sigma. Calcium antagonists (veramapil, diltiaze , and nifedipine) were also purchased from Sigma. Poly (dl-dc) was purchased from Pharmacia. Reverse transcriptase (M- MLV) was purchased from Amersham Life Science. Clentiazem (TA- 3090) was obtained from Marion Laboratories Inc. (Kansas City) . EXAMPLE 2

Cell culture

Vascular smooth muscle cells from the rat thoracic aorta (A-10) was obtained from American Type Culture Collection (Rockville, Maryland) . Vascular smooth muscle cells were cultured in Dulbecco's modified Eagles medium (DMEM) supplemented with 10% fetal bovine serum (FBS) , penicillin and streptomycin (each 100 μg/ml) . The cells were used between the fifteenth and eighteenth passage in these experiments.

EXAMPLE 3 Measurement of DNA synthesis

Mitogenesis was assayed by measuring [³H]thymidine incorporation into the DNA. Subconfluent (70-80%) vascular smooth muscle cells were starved with FBS free DMEM, which contains 0.1% bovine serum albumin (BSA) and 0.1% glucose at 37°C for 24 hours. Control dishes received no addition of agents in FBS free DMEM which contains 0.1% BSA and glucose. While test dishes received the addition of recombinant platelet derived growth factor-AA (40 ng/ml) , platelet derived growth factor-BB (40 ng/ml) , platelet derived growth factor-AB (40 ng/ml) and/or clentiazem (10^"5 M) , verapamil (10^"6M), diltiazem (10 ^"6 M) and nifedipine (10^"6 M) . Quiescent vascular smooth muscle cells were supplemented with 1 μCi/ml of [³H]thymidine, incubated for 24 hours at 37°C, washed with phosphate buffered saline (PBS) and 5% trichloro acetic acid (TCA) , then dissolved in 0.25% N NaOH containing 0.1% SDS. The radioactivity associated with the cell lysates was then determined by LS 3133 Beckman scintillation counter.

EXAMPLE 4 Phosphatidyl inositol turnover Confluent vascular smooth muscle cells were cultured for 48 hours in a medium free of myo-inositol that contained 10% dialysed serum and 1 μCi/ml of [³H]myo-inositol. The medium was removed, cells were washed with PBS, and incubated with PBS containing 10 mM LiCl for 10 minutes. Cells were treated with rplatelet derived growth factor-AA (10 ng/ml) , rplatelet derived growth factor-BB (10 ng/ml) , and/or clentiazem (10^*5 M) for 20 minutes. They were then rinsed twice with PBS, and treated with 5% TCA solution. Cells were then scraped into Eppendorf tubes and the cellular debris was pelleted by centrifugation. Cells were then dissolved in 0.1 M NaOH, and cellular radioactivity and protein were determined. The supernatant was extracted with ether and then the aqueous phase was applied to an ion exchange column (Bio-Rad AG 1X8) to separate the inositol phosphates. The column was washed with 5 mM myo-inositol until no radioactivity was detected in the eluate. This was followed by elution of the bound inositol phosphates with 0.2 M, 0.5 M, 1 M and 1.5 M ammonium formate in 0.1 M formic acid. Radioactivity of each inositol phosphate (IP1, IP2, IP3, and IP4) fraction was determined from the eluted solutions.

EXAMPLE S Phosphorylation of protein kinase C substrate Confluent and quiescent cultures of vascular smooth muscle cells were starved with FBS free DMEM for 48 hours. Subsequently, vascular smooth muscle cells were treated with a calcium antagonist (10^*5 M) for 30 minutes before the addition of PDBu or rplatelet derived growth factor-BB. The cells were then washed twice with FBS free DMEM and twice with an isotonic KC1 salt solution (120 mM KC1, 30 mM NaCl, 1 mM MgCl₂, 1 mM K₂HP0₄, 10 mM sodium PIPES pH 7.0, 1 mM EGTA, and 0.037 mM CaCl₂) at 37°C immediately before the experiment. Phosphorylation was initiated by replacing the salt solution from the last wash with 1 ml of permeabilization medium containing 40 μM digitonin and 10 μM [7³²P]ATP (0.2-1 Ci/mmol) in isotonic KC1 solution. The cells were then incubated at 37°C for 5 minutes with 200 nM phorbol 12, 13-dibutylate (PDBu), rplatelet derived growth factor-BB and/or calcium antagonists. The cells were immediately scraped off the dishes into hot SD ample buffer. Samples were resolved by one-dimensional SDS-PAGE. After electrophoresis the gels were dried, exposed to Kodak x-ray film at -70^βC for autoradiography. The intensity of the 80kD protein band was quantified by Bio-Rad imaging densitometer (Model GS- 670) .

EXAMPLE 6 Expression of transcriptional factor AP-1

To demonstrate the effect of several different calcium antagonists on the induction of AP-1 complex in vascular smooth muscle cells induced by recombinant platelet derived growth factor-AA, BB, or AB, basic-fibroblast growth factor (b-FGF) , and phorbol 12-myristate 13-acetate (phorbol 12-myristate 13- acetate) , 90% confluent vascular smooth muscle cells were first deprived of serum for 24 hours. The cells were next treated with a calcium antagonists for 15 minutes before the addition of growth factors or phorbol 12-myristate 13-acetate. Recombinant platelet derived growth factor-AA, rplatelet derived growth factor-BB, or rplatelet derived growth factor-AB (10 ng/ml) , b-fibroblast growth factor (b-FGF) (20 ng/ml) and phorbol 12-myristate 13-acetate (200 nM) were added, followed by incubation at 37°C for 45 minutes. The reaction was terminated by removing the incubation medium by aspiration followed by washing with 5 ml ice cold PBS.

EXAMPLE 7 Nuclear extract

Nuclear extracts were prepared by a modification of the procedure described by Dignam et al. Subconfluent vascular smooth muscle cells were washed with ice cold PBS and scraped into 5 ml PBS. The cells were sedimented by centrifugation (500 xg for 5 minutes) , then re-suspended in 5 ml hypotonic solution (10 mM Tris-HCl [pH 7.9], 1.5 mM MgCl₂, 10 mM KC1, 0.5 mM DTT) and allowed to swell on the ice for 10 minutes. The cells were then homogenized by 20 strokes of a Dounce glass homogenizer and nuclei were sedimented by centrifugation at 1000 xg for 5 minutes. The nuclei were then re-suspended in the nucleic re- suspension buffer (20 mM Tris-HCl [pH 7.9-, 20% glycerol, 1.5 mM MgC12, 1 mM PMSF and 0.5 mM DTT) followed by the addition of 4 M KC1 to a final concentration of 0.3 M KC1. The suspension was rocked gently at 4°C for 30 minutes, then centrifuged at 13000 xg at 40°C for 15 minutes. The supernatant containing the nuclear extract was stored at -70°C until assayed.

EXAMPLE 8 Gel mobility shift assay The AP-1 binding site was prepared by hybridizing two oligonucleotides, consensus sequences:

5'-GATCTGTGACTCAGCGCGA-3' and 5'-GATCTCGCGCTGAGTCACA-3' . The probes containing the AP-1 sequence were radiolabeled using α³²P-dCTP and MMLV. Nuclear extract (2-5 mg protein) was incubated in 20 ml binding buffer (10 mM Tris-HCI [pH 7.9], 5 mM MgCl₂, 0.5 mM DTT, 0.5% glycerol) containing 2 μg poly(dl-dc) poly(dl-dc) . The ³²P labeled AP-1 probe (0.5-1.0 μg) was added and incubated for 30 minutes at room temperature. The reaction mixture was loaded directly onto a 5% polyacrylamide (30:0.8/acrylamide:bisacrylamide) gel in 0.25 TBE (25 mM Trizma base, 25 mM boric acid, 1 mM EDTA) and electrophoresed at 150 V for 1.5 hours. The gel was dried and analyzed by autoradiography. The intensity of AP-1 bands was quantified as the integrated area of optical density value by Bio-Rad imaging densito eter (Model GS-650) .

EXAMPLE 9 Statistical analysis

Data were expressed as mean+SD. Comparisons between groups were done by the use of student's paired t test. For all analysis, significance was defined as a P value of less than 0.05.

EXAMPLE 10 Anti-mitoσenic effect of calcium antagonists on vascular smooth muscle cells Under unstimulated condition, clentiazem (10^"5 M) , verapamil (10^"5 M) , diltiazem (10^"5 M) , and nifedipine (10^"5 M) each significantly inhibited the incorporation of [³H]thymidine by 86.1%, 61.7%, 57.0%, 67.3%, on vascular smooth muscle cells, respectively (p<0.05, p<0.01, p<0.01, p<0.01). These inhibitory effect of calcium antagonists were seen in a dose dependent manner (Figure 1) . rplatelet derived growth factor-BB increased thymidine incorporation, with a dose of 40 ng/ml producing a stimulation of approximately 130%. In contrast, rplatelet derived growth factor-AA (40 ng/ml) and rplatelet derived growth factor-AB (40 ng/ml) failed to stimulate thymidine incorporation. All calcium antagonists did not inhibit the thymidine incorporation induced by rplatelet derived growth factor-AA(40 ng/ml). However, clentiazem (10'⁵ M) , verapamil, diltiazem, and nifedipine (10^'6M) each produced a significant inhibition of the thymidine incorporation induced by rplatelet derived growth factor-BB (40 ng/ml) (p<0.0l, p<0.01, p<0.05, p<0.01 respectively). As shown in Figure 2, diltiazem, verapamil and nifedipine (10^*5-10^*7M) significantly decreased the incorporation of thymidine induced by rplatelet derived growth factor-AB at a concentration of 40 ng/ml (p<0.05).

EXAMPLE 11 Effect of clentiazem on production of inositol phosphates

Natural platelet derived growth factor (40 ng/ml) significantly stimulated the production of total inositol phosphates (IP1, IP2, IP3, IP4) by 227% over the control level. Natural platelet derived growth factor also significantly stimulated the production of inositol phosphate-3 in vascular smooth muscle cells by 149% (p<0.01, p<0.05, p<0.05, p<0.05). Clentiazem (10^"5M) had no significant effect on the production of inositol phosphates under unstimulated condition, and also failed to inhibit the platelet derived growth factor-induced production of inositol phosphate-3 in vascular smooth muscle cells (data not shown) . Recombinant platelet derived growth factor-BB (10 ng/ml) also significantly stimulated the production of total inositol phosphate by 176% over control (p<0.01) . Recombinant platelet derived growth factor-BB significantly stimulated the production of inositol phosphate-3 by 133% (p<0.05, p<0.05). Clentiazem (10^*5M) failed to inhibit the production of inositol phosphate-3 induced by rplatelet derived growth factor-BB in vascular smooth muscle cells, rplatelet derived growth factor-AA (10 ng/ml) failed to stimulate the production of inositol phosphate. Figure 3 shows that clentiazem did not decrease the production of inositol phosphate-3 induced by rplatelet derived growth factor-AA. EXAMPLE »

Phosphorylation of protein kinase C substrate

PDBu (200 nM) markedly stimulated the phosphorylation of the 80 kD protein yristoylated, alanine-rich C kinase substrate, a substrate of protein kinase C, on digitonin- permeabilized vascular smooth muscle cells by 240%. Each calcium antagonist (10^~5 M) reduced the PDBu mediated phosphorylation of the myristoylated, alanine-rich C kinase substrate by 163% in clentiazem, 133% in diltiazem, 140% in verapamil, and 116% in nifedipine respectively (Figure 4A) . In addition, rplatelet derived growth factor-BB(40 ng/ml) phosphorylated the 80 kD protein myristoylated, alanine-rich C kinase substrate by 230%. Calcium antagonists (10^'s M) each reduced the rplatelet derived growth factor-BB mediated phosphorylation of the myristoylated, alanine-rich C kinase substrate by 205% in clentiazem, 180% in diltiazem, 167% in verapamil, and 186% in nifedipine, respectively compared to controls (Figure 4B) . Staurosporine (20 μM) completely inhibited the phosphorylation of 80 kD protein myristoylated, alanine-rich C kinase substrate mediated by PDBu and rplatelet derived growth factor-BB.

EXAMPLE 13 Expression of AP-1 induced by growth factor

The expression of AP-1 complex induced by rplatelet derived growth factor-AA (10 ng/ml) was increased over control level by 183%, rplatelet derived growth factor-BB (10 ng/ml) by 193%, and rplatelet derived growth factor-AB (10 ng/ml) by 156%. The expression of AP-1 induced by rplatelet derived growth factor-BB (lOng/ml) and platelet derived growth factor-AB (10 ng/ml) was reduced by pretreatment with clentiazem (10^*5 M) , by rplatelet derived growth factorBB+clentiazem (94%) , and by rplatelet derived growth factor-AB+clentiazem (127%) (Figure 5) . The expression of AP-1 induced by r platelet derived growth factor-BB (10 ng/ml) (144%) was also reduced by pretreatment with verapamil (10^"6 M) by 102%. Diltiazem (10^"6 M) and nifedipine (10^"6M) each had no significant inhibitory effect on the platelet derived growth factor-BB induced expression of AP-1 (Figure 6A) . The AP-1 induced by phorbol 12-myristate 13- acetate (200 nM) by 251% was reduced by 160% by clentiazem (10'⁵ M) and by 198% by verapamil (10^"5 M) (Figure 6B) . Clentiazem reduced the expression of AP-1 induced by rplatelet derived growth factor-BB (175%) in a dose-dependent manner, at 10^"5 M in 138%, 10^'6 M in 151%, 10^*7 M in 195% (Figure 7A) . The AP-1 expression complex induced by basic FGF (20 ng/ml) (170%) was inhibited by clentiazem (10^*5 M) by 71% (Figure 7B) . The unlabelled AP-1 oligo er (100 fold excess) abolished the binding activity in the extract from vascular smooth muscle cells, indicating the binding is specific for the AP-1 sequence.

The present invention illustrates the inhibitory effect of calcium antagonists on DNA synthesis, including rplatelet derived growth factor-BB mediated signal transduction pathway of transcription factor AP-1 expression and protein kinase C phosphorylation on vascular smooth muscle cells. The present invention demonstrated that calcium antagonists have inhibitory effect on DNA synthesis reducing the AP-1 expression and phosphorylation of protein kinase C substrate induced by rplatelet derived growth factor-BB via the signal transduction pathway of vascular smooth muscle cells. Clentiazem and verapamil inhibited the expression of AP-1 complex induced by rplatelet derived growth factor-BB.

The transcription factor AP-l is a heterodimer of c- fos and c-jun with the binding site can act as a cellular enhancer capable of stimulating transcription in vitro and is inducible by 12-o-tetradecanoylphorbol-13 acetate (TPA) . The expression of transcription factor AP-l is associated with cell differentiation, development, and proliferation. The inhibitory effect of calcium antagonists on the transcription factor AP-l expression induced by rplatelet derived growth factor-BB suggests a specific inhibition of the expression of the B chain gene of platelet derived growth factor. It has been known that platelet derived growth factor-B chain gene transcript level is elevated in the human atherosclerotic plaque as compared with the normal artery in vivo as well as proliferation of vascular smooth muscle cells in vitro. Therefore, rplatelet derived growth factor-BB is considered to be one of the most potent mitogenε to vascular smooth muscle cells, which can induce myointimal hyperplasia of the vascular wall. Basic-FGF is also thought to be an important mitogen for vascular smooth muscle cells replication in injured arteries for growth of intimal lesion. Since calcium antagonists reduced the expression of transcription factor AP-l induced by rplatelet derived growth factor-BB and b-FGF, those would have some inhibitory effect on initial transcription regulation in proliferation of vascular smooth muscle cells.

Transcriptional factor AP-l is an enhancing protein inducible by phorbol esters, a TPA-responsive element (TRE) , and an activator of early transcription that represses late transcription. The activation of protein kinase C mediated by TPA may initiate the phosphorylation and dephosphorylation of various cellular components. Protein kinase C also appears to be the receptor protein for tumor-promoting phorbol ester, and may be located at important point of various pathway in cell proliferation and play a major role in transmembrane signal transduction of hormones and growth factors leading to cellular responses. The activation of protein kinase C induced by phorbol ester or platelet derived growth factor, also mediates the phosphorylation of the L-type voltage dependent calcium channel on the cell membrane of vascular smooth muscle cells, such phosphorylation can then stimulate the influx of Ca2+ to cytosol. Calcium antagonists are thought to bind the a-subunits of the voltage operated calcium channel, and inhibit the phosphorylation of dyhydropyridine receptor or L-type calcium channel and the transmembrane flux of Ca2+. The present invention has shown that calcium antagonists reduce the phosphorylation of protein kinase C substrate MARCKS, which is an indicator or marker for protein kinase C activation, mediated by rplatelet derived growth factor-BB and PDBu. The inhibitory effect of different calcium antagonists on the phosphorylation of MARCKS mediated by rplatelet derived growth factor-BB appeared to be varied. But the ability of calcium antagonists to affect the changes induced gamma-platelet derived growth factor indicates that protein kinase C plays a role in nuclear events and transcription of the early genes c-fos and c-jun which entail components of the transcription factor AP-l.

Platelet derived growth factor-BB involves a variety of second messengers including the phosphorylation on the tyrosine of phospholipase C-7I, the hydrolysis of phosphatidylinositol, the formation of IP3, diacylglycerol, the phosphorylation of protein kinase C, and an increase in the intracellular mobilization of Ca2+. The present invention also showed that rplatelet derived growth factor-BB exhibits a greater mitogenic effect than either rplatelet derived growth factor-AA in vascular smooth muscle cells, because of the induction of the AP-l complex via an increase in the production of IP3. The mechanism of the increase in intracellular Ca2+ by rplatelet derived growth factor-BB involves the mobilization of intracellular calcium from the endoplasmic reticulum as the result of an increased production of IP3 and the influx of calcium ions through the calcium channel. Platelet derived growth factor-AA induced the expression of transcription factor AP-l without the increase of total inositol phosphates indicates an action via other signal transduction pathways. The mitogenicity of rplatelet derived growth factor-BB and mediated signal transduction pathway therefore were considered to differ from those of rplatelet derived growth factor-AA in vascular smooth muscle cells. Although some calcium antagonists have been shown to inhibit two isomers of the platelet derived growth factor-induced signal transduction pathway, increase the formation of IP3, DAG, cellular Ca2+, clentiazem failed to show a specific inhibitory effect on the production of IP3 in vascular smooth muscle cells.

Since the expression of transcription factor AP-l is likely to play a central role in the regulation of biological process like cell proliferation and differentiation, the specific efficacy of calcium antagonists on inhibition of vascular smooth muscle cell proliferation which is due to reducing rplatelet derived growth factor-BB induced transcription factor AP-l expression was strongly indicated. In this study, calcium antagonists were shown have some inhibitory effect on phosphorylation of protein kinase C substrate induced by rplatelet derived growth factor-BB. The action of calcium antagonists in preventing vascular smooth muscle cells proliferation may be at the fundamental genomic level in inhibiting the activation of the transcription factor AP-l, via rplatelet derived growth factor-BB mediated signal transduction pathway. Calcium antagonists would therefore be beneficial in preventing the formation of the atherosclerotic lesion, especially with respect to myointimal hyperplasia of vascular smooth muscle cells. In conclusion, the anti- mitogenic effect of calcium channel blockers may be due to their ability to reduce the DNA synthesis and the inhibition of AP-l expression induced by rplatelet derived growth factor-BB and phorbol ester via the reduction on of protein kinase C phosphorylation. The definite inhibitory effects on the rplatelet derived growth factor-BB-induced signal transduction pathway including the expression of transcription factor AP-l would help in preventing the formation of atheromatous lesions. Any patents or publications mentioned in this specification are indicative of the levels of those skilled in the art to which the invention pertains. These patents and publications are herein incorporated by reference to the same extent as if each individual publication was specifically and individually indicated to be incorporated by reference.

One skilled in the art will readily appreciate that the present invention is well adapted to carry out the objects and obtain the ends and advantages mentioned, as well as those inherent therein. The present examples along with the methods, procedures, treatments, molecules, and specific compounds described herein are presently representative of preferred embodiments, are exemplary, and are not intended as limitations on the scope of the invention. Changes therein and other uses will occur to those skilled in the art which are encompassed within the spirit of the invention as defined by the scope of the claims. SEQUENCE LISTING

(1) GENERAL INFORMATION:

(i) APPLICANTS: Yatsu, Frank M. ; Alam, Nargis A. ; and Satoshi Kataoka (ϋ) TITLE OF INVENTION: ANTI-STROKE EFFECTS OF CALCIUM ANTAGONISTS

(iii) NUMBER OF SEQUENCES: 2 (iv) CORRESPONDENCE ADDRESS:

(A) ADDRESSEE: James F. Weiler (B) STREET: One Riverway, Suite 1560

(C) CITY: Houston

(D) STATE: Texas

(E) COUNTRY: USA

(F) ZIP: 77056 (v) COMPUTER READABLE FORM:

(A) MEDIUM TYPE: Floppy disk

(B) COMPUTER: IBM PC compatible

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

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(B) FILING DATE:

(C) CLASSIFICATION:

(viii) ATTORNEY/AGENT INFORMATION: (A) NAME: Weiler, James F.

(B) REGISTRATION NUMBER: 16,040

(C) REFERENCE/DOCKET NUMBER: D-5739 (ix) TELECOMMUNICATION INFORMATION:

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(B) TYPE: nucleic acid (C) STRANDEDNESS: double

(D) TOPOLOGY: linear (ii) MOLECULE TYPE:

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(A) LENGTH: 19

(B) TYPE: nucleic acid

(C) STRANDEDNESS: double

(D) TOPOLOGY: linear (ii) MOLECULE TYPE:

(A) Description: other nucleic acid (iii) HYPOTHETICAL: No (iv) ANTISENSE: No (Vi) ORIGINAL SOURCE: (B) STRAIN:

(C) INDIVIDUAL ISOLATE:

(D) DEVELOPMENTAL STAGE:

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Claims

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1. A method of treating a cerebrovascular disease in an animal comprising the step of administering to an animal in need of such treatment a therapeutically effective dose of a calcium channel antagonist.

2. The method of claim 1, wherein said cerebrovascular disease is selected from the group consisting of intracerebral hemorrhage, subarachnoid hemorrhage due to aneurysms, migraine, intracerebral atherosclerosis, lipohyalinosis, Binswanger's disease or subcortical arteriopathic encephalopathy, Moyamoya disease and impairment of the blood brain barrier with brain edema formation.

3. The method of claim 1, wherein said calcium channel antagonist is selected from the group consisting of clentiazem, verapamil, diltiazem, and nifedipine.

4. The method of claim 1, wherein said calcium channel antagonist is administered in a dose of from about 0.3 mg/kg to about 10 mg/kg.

5. A method of inhibiting the proliferation of vascular smooth muscle cells in an animal comprising the step of administering to an animal in need of such treatment a pharmacologically effective dose of a calcium channel antagonist.

6. The method of claim 5, wherein said calcium channel antagonist is selected from the group consisting of clentiazem, verapamil, diltiazem, and nifedipine.

7. The method of claim 5, wherein said calcium channel antagonist is administered in a dose of from about 0.3 mg/kg to about 10 mg/kg.

8. A method of inhibiting the expression of transcription factor AP-l in an animal comprising the step of administering to an animal in need of such treatment a pharmacologically effective dose of a calcium channel antagonist.

9. The method of claim 8, wherein said calcium channel antagonist is selected from the group consisting of clentiazem, verapamil, diltiazem, and nifedipine.

10. The method of claim 8, wherein said calcium channel antagonist is administered in a dose of from about 0.3 mg/kg to about 10 mg/kg.