Cyclin-dependent protein kinases as therapeutic targets in cardiovascular disease
María Dolores Edo, Marta Roldán and Vicente Andrés
Laboratory of Vascular Biology, Department of Molecular and Cellular Pathology and Therapy,
Instituto de Biomedicina de Valencia, Spanish Council for Scientific Research (CSIC), 46010
Valencia, Spain.
Corresponding author:
Vicente Andrés
Tel: +34-96-3391752
FAX: +34-96-3690800
E-mail: vandres@ibv.csic.es
1
ABSTRACT
Excessive cell proliferation is thought to contribute to the development of neointimnal
lesions during the pathogenesis of atherosclerosis, restenosis and vessel bypass graft failure.
Because cell cycle progression requires the sequential activation of cyclin-dependent kinases
(CDKs) through their association with regulatory subunits dubbed cyclins, therapeutic strategies
via CDK inhibition may reduce the burden of vascular proliferative disease. Some of these
approaches may rely on the use of pharmacological CDK inhibitors that target the ATP-binding
pocket of the catalytic site of CDK. Others are based on the overexpression of members of the
family of CDK inhibitory proteins, which associate with and inhibit the activity of CDK/cyclin
holoenzymes. In this review, animal studies that document the efficacy of pharmacological
agents and gene therapy approaches targeting CDK/cyclins for the treatment of vascular
proliferative disease are discussed. We also analyze recent patent applications in this field.
KEY
WORDS:
cardiovascular
disease,
cyclin,
cyclin-dependent
kinase
(inhibitor),
pharmacological therapy, gene therapy
2
LIST OF ABBREVIATIONS: bFGF, basic fibroblast growth factor; CDK, cyclin-dependent
kinase; CKI, CDK inhibitory protein; EC, endothelial cell; ODN,
oligodeoxynucleotide; PCNA, proliferating cell nuclear antigen; PDGF,
platelet-derived
growth
factor;
pRb,
retinoblastoma
protein;
transforming growth factor-; VSMC, vascular smooth muscle cell.
INDEX
1. Introduction
2. Pharmacological CDK inhibitors
3. Gene therapy strategies
3.1. Antisense strategies to inactivate CDKs and cyclins
3.2. Ribozymes
3.3. Overexpression of CKIs
4. Expert opinion
5. Acknowledgements
Bibliography
Patents
3
TGF-
1. Introduction
Atherosclerosis and associated cardiovascular disease (e. g. myocardial infarction and
stroke) are the main cause of mortality and morbidity in industrialized countries. Cardiovascular
risk factors (i. e., hypercholesterolemia, hypertension, smoking) initiate and perpetuate an
inflammatory response within the injured arterial wall that contributes to neointimal lesion
growth during atherosclerosis [1, 2]. Both acellular (e. g. deposition of lipids and extracellular
matrix components) and cellular components become involved in the growth of the
aherosclerotic plaque. Cell types found in neointimal lesion growth include circulating
leukocytes that undergo transendothelial migration, and vascular smooth muscle cells (VSMCs)
that migrate from the tunica media. Recent evidence indicates that circulating precursor cells
also contribute to neointimal lesion development [3-5].
Excessive proliferation of vascular cells is an important component of the chronic
inflammatory response associated to atherosclerosis and related vascular occlusive diseases (i. e.,
in-stent restenosis, transplant vasculopathy, and vessel bypass graft failure) [6, 7]. Thus,
understanding the molecular mechanisms that control hyperplastic growth of vascular cells
should help develop novel therapeutic strategies to attenuate neointimal thickening. Arterial cell
proliferation has been well documented in animal models of atherosclerosis [1, 8-10].
Importantly, studies with hyperlipidemic rabbits have shown an inverse correlation between the
size of the atheromatous plaque and cellular proliferation within the lesion [11-13]. Excessive
proliferation of VSMCs after experimental angioplasty is also followed by the reestablishment of
the quiescent phenotype [14-16]. Thus, vascular cell proliferation appears to prevail at the onset
of atherogenesis and restenosis.
Analysis of human primary atheromatous plaques and restenotic lesions have demonstrated
the expression of proliferation markers [17-29]. It is noteworthy in this regard that several
studies have reported a very low index of cell proliferation within the neointimal lesion [18, 19,
4
21, 23, 25, 29], while others have suggested an abundance of dividing cells [20, 27, 28, 30].
Several methodological issues (i. e., differences in the fixatives used for tissue preservation,
antigen accessibility, diversity of proliferation markers analyzed in these studies) may explain in
part these discrepancies. Moreover, disagreement among these studies might relate to differences
in the source of tissue used for the analysis (i. e., peripheral, coronary and carotid arteries) and
variance in the stage of atherogenesis at the time of tissue harvesting [31].
The cell types that undergo cell proliferation within human atherosclerotic tissue include
VSMCs, leukocytes and endothelial cells (ECs) [17-19, 21-23, 25] [25 bis]. Rekhter et al.
examined human carotid artery primary atherosclerotic tissue retrieved by endarterectomy
surgery [23]. They found greater proliferative activity in the intimal lesion versus the underlying
media. Moreover, monocyte/macrophage proliferation predominated in the intima (46% versus
9.7% -actin immunoreactive VSMCs, 14.3% ECs, 13.1% T lymphocytes), whereas VSMC
proliferation prevailed in the media (44.4% versus 20% ECs, 13.0% monocyte/macrophages, and
14.3% T lymphocytes). Histological examination in 20 patients undergoing antemorten coronary
angioplasty revealed that the extent of intimal proliferation was significantly greater in lesions
with evidence of medial or adventitial tears than in lesions with no or only intimal tears [28]. It is
also noteworthy that cell proliferation is greater in restenotic compared to primary lesions in
human peripheral and coronary arteries [21, 29, 30]. Consistent with this finding, cultured
VSMCs obtained from human advanced primary stenosing displayed lower proliferative activity
than cells from fresh restenosing lesions [32]. Thus, similar to the situation in animal models,
proliferation during human atherosclerosis and restenosis may prevail at the onset of these
disorders.
Tight control of cellular proliferation in higher eukaryotes is essential to ensure normal
tissue patterning during embryonic development and to maintain organ homeostasis in the adult
animal. Cell cycle progression is controlled by several cyclin-dependent kinases (CDKs) that
5
associate with regulatory cyclins [33]. Mitogenic stimuli activate CDK/cyclin holoenzymes, thus
causing hyperphosphorylation of the retinoblastoma protein (pRb) and the related pocket
proteins p107 and p130 from mid G1 to mitosis. The complex interaction among E2F
transcription factors and individual pocket proteins determines whether E2F proteins function as
transcriptional activators or repressors [34]. Interaction of CDK/cyclins with CDK inhibitory
proteins (CKIs) attenuates CDK activity and promotes growth arrest [35]. CKIs of the Cip/Kip
family (p21Cip1, p27Kip1 and p57Kip2) bind to and inhibit a wide spectrum of CDK/cyclin
holoenzymes, while members of the Ink4 family (p16Ink4a, p15Ink4b, p18Ink4c, p19Ink4d) are specific
for cyclin D-associated CDKs. Mitogenic and antimitogenic stimuli affect the rates of synthesis
and degradation of CKIs, as well as their redistribution among different CDK/cyclin pairs [35].
Analysis of the promoter region of CKIs may be an useful tool to identify compounds that
control transcriptional regulation of this family of growth suppressors [101].
The proliferative response of VSMC to balloon angioplasty in the rat carotid artery is
associated with a temporally and spatially coordinated expression of CDKs and cyclins [26, 36].
Importantly, augmented expression of these factors was associated with increased CDK2 and
CDC2 activity [26, 37], demonstrating the assembly of functional CDK/cyclin holoenzymes
within the injured arterial wall. Moreover, CDK2 and cyclin E expression has been detected in
human VSMCs within atherosclerotic and restenotic tissue [20, 26, 38], suggesting that increased
expression (and possibly activation) of positive regulators of cell cycle progression is a
characteristic of vascular proliferative disease in humans.
In the following sections, we will discuss preclinical studies and patents related to the use
of pharmacological agents (Table 1 and 2) and gene therapy strategies (Table 3) targeting
CDK/cyclins to inhibit vascular cell proliferation and reduce neointimal thickening in animal
models of cardiovascular disease. Additional antiproliferative strategies have been discussed
elsewhere [7, 39].
6
2. Pharmacological CDK inhibitors
Low molecular weight inhibitors of CDK activity have been identified that target the ATPbinding pocket of the catalytic site of CDK, thus acting as competitive inhibitors. Structural
information on these agents and their application in cancer therapy has been recently reviewed
[40, 41].
Meijer and coworkers have described an approach to search for new inhibitors for specific
protein kinases using chemical libraries [102]. Table 1 summarizes recent patents in which the
use of pharmacological CDK inhibitors has been claimed for the prevention of vascular cell
proliferation and/or cardiovascular disease.
CVT-313 is a potent CDK2 inhibitor (IC50 = 0.5 M in vitro), which was identified from a
purine analog library [42]. CVT-313 was less effective in inhibiting CDK1 and CDK4, and had
no effect on other, nonrelated ATP-dependent serine/threonine kinases. Treatment of cultured
cells with CVT-313 inhibited hyperphosphorylation of pRb and caused cell cycle arrest at the
G1/S boundary (IC50 ranging from 1.25 to 20 M). Using the rat carotid artery model of balloon
angioplasty, intraluminal delivery of CVT-313 at a dose of 1.25 mg/kg for 15 minutes under
pressure attenuated neointimal lesion formation by more than 80% [42].
Flavopiridol
(L86-8275,
cis-5,7-dihydroxy-2-(2-chlorophenyl)-8-[4-(3-hydroxy-1-
methyl)piperidinyl]-4H-benzopyran-4-one) efficiently inhibited basic fibroblast growth factor
(bFGF)-induced and thrombin-induced proliferation of human VSMCs without affecting cell
viability [43]. This inhibitory effect correlated with reduced CDK activity and blockade of pRb
hyperphosphorylation. Flavopiridol at 5 mg/kg administered orally for 5 days beginning at the
day of balloon angioplasty reduced neointima formation by 35% and by 39% at day 7 and 14
after intervention, respectively [43].
Collectively, the above studies suggest that pharmaceutical inhibition of CDK activity is a
7
potential therapeutic tool in the treatment of proliferative disorders, including VSMC-rich
vascular lesions.
3. Gene therapy strategies
Antiproliferative gene therapy strategies based on direct inhibition of CDK/cyclin
holoenzymes include antisense and ribozyme strategies to inactivate CDK/cyclins, and ectopic
overexpression of CKIs.
3.1. Antisense strategies to inactivate CDKs and cyclins
This approach typically utilizes a synthetic antisense oligodeoxynucleotide (ODN) that
hybridizes in a complementary fashion and stoicheometrically with the target RNA, thereby
inactivating the gene of interest.
Effective reduction of neointimal lesion formation by ODN strategies targeting CDKs and
cyclins has been demonstrated in animal models of balloon angioplasty, including ODN against
cdk2 [37, 44], cdc2 [37, 45] and cyclin B1 [45]. Combined inactivation of cdc2 and cyclin B1 by
cotransfection of antisense ODN against these genes caused greater reduction of neointimal
thickening than blockade of either gene target alone [45]. Of note, combined gene inactivation of
cdc2 and proliferating cell nuclear antigen (pcna) by a single intraluminal delivery of antisense
ODNs resulted in sustained inhibition of neointima formation in the rat carotid artery ballooninjury model [46], whereas this approach had no effect in balloon-injured porcine coronary
arteries [47]. Inactivation of cyclin G1 gene expression by retrovirus-mediated antisense gene
transfer inhibited VSMC proliferation and neointima formation after balloon angioplasty [48].
Moreover, ODN against cdk2 [49], and a combination of antisense ODN against pcna and cdc2
[50], attenuated experimental graft atherosclerosis.
The University Leland Stanford Junior has described a method for inhibiting mammalian
8
VSMC proliferation based on the administration of antisense sequences to cyclins or CDKs
[103]. The methods of this invention may be useful in treating a broad spectrum of vascular
lesions, in particular ex-vivo treatment of vascular grafts prior to surgical grafting. The method
involves direct intraluminal, intramural or periadventitious administration of an effective dosage
of at least one antisense sequence to inhibit the expression of at least one cyclin (cyclin A, B1,
B2, C, D1, D2, D3, E or cyclin X) or a CDK (cdc2, cdk2, cdk4 or cdk5). It is preferable to use
two antisense sequences each from a different cyclin or cdk gene. The antisense sequence to the
target cyclin or cdk gene is preferably administered in combination with an antisense against
pcna. The antisense sequences are incorporated into liposomes, particularly liposomes
containing hemagglutinating virus of Japan.
3.2. Ribozymes
Ribozymes represent a unique class of RNA molecules that catalytically cleave the specific
target RNA, thus resulting in targeted gene disruption. Inactivation of the platelet-derived
growth factor (PDGF) A-chain mRNA by hammerhead ribozyme limited human and rat VSMC
growth in culture [51, 52].
Frimerman et al. have provided the first evidence that ribozymes might represent useful
tools in cardiovascular therapy by demonstrating the efficacy of chimeric hammerhead ribozyme
to pcna in reducing neointimal thickening in a porcine coronary model of in-stent restenosis [53].
Moreover, ribozyme strategy against transforming growth factor-1 (TGF-1) inhibited
neointimal formation after balloon injury in the rat carotid artery model [54].
Immusol Inc. has described methods of producing ribozymes especially targeted to cyclin
B1, cdc2, and pcna to inhibit VSMC proliferation in vascular tissue, as well as ribozyme delivery
systems for anti-restenosis gene therapy [104].
9
3.3. Overexpression of CKIs
The efficacy of CKIs in inhibiting CDK activity and cell cycle progression has been widely
documented in a variety of normal and tumour cells in vitro. Studies arguing for a role of the
CKIs p21Cip1 and p27Kip1 in the pathophysiology of the cardiovascular system include the
following: 1) p21Cip1 and p27Kip1 may contribute to the reestablishment of the quiescent
phenotype after the initial proliferative response to balloon angioplasty in rat and porcine arteries
[24, 55]; 2) p27Kip1 may function as a molecular switch that regulates the phenotypic response of
VSMCs to both hyperplastic and hypertrophic stimuli [56, 57]; 3) p27Kip1 is a negative regulator
of EC proliferation and migration in vitro, and adenovirus-mediated overexpression of p27Kip1
inhibited angiogenesis in vivo [58, 59]; 4) p21Cip1 and p27Kip1 may contribute to integrinmediated control of VSMC proliferation [60]; 5) p27Kip1 may limit cardiomyocyte proliferation
during early postnatal development and after injury in adult mice [61, 62]; 6) changes in p21Cip1
and p27Kip1 expression might regulate human vascular cell proliferation within atherosclerotic
lesions [24, 38], and a causal link between reduced p27Kip1 expression and atherosclerosis has
been established in apolipoprotein E-deficient mice [9]. However, neointimal hyperplasia after
mechanical damage of the arterial wall was similar in wild-type and p27Kip1-null mice [63].
Redundant roles between p21Cip1 and p27Kip1, or compensatory increase in p21Cip1 expression (or
other CKIs) might account for the lack of phenotype of p27 Kip1-null mice in the setting of
mechanical arterial injury.
Different segments of the arterial tree display significant variance in their susceptibility to
atherosclerosis, both in animal models and humans. It is notable that VSMCs display regional
phenotypic variance, both when comparing cells obtained from different compartments of the
same vessel or cells isolated from vessels from different vascular beds [64-70]. Several studies
have suggested a role of CKIs in establishing phenotypic variance in VSMCs from different
vascular beds. By comparing human VSMCs isolated from internal mammary artery and
10
saphenous vein, Yang et al. suggested that sustained p27Kip1 expression in spite of growth stimuli
may contribute to the resistance to growth of VSMCs from internal mammary artery and to the
longer patency of arterial versus venous grafts [69]. Likewise, distinct proliferative response of
intimal and medial VSMCs towards bFGF correlated with distinct expression of p15 Ink4b and
p27Kip1 in these cells [70]. We have recently suggested that intrinsic differences in the regulation
of p27Kip1 may contribute to establishing regional variability in atherogenicity via distinct
regulation of VSMC proliferation and migration [71].
Tanner et al. have reported more frequent expression of p27Kip1 and p21Cip1 within regions
of human coronary atheromas not undergoing proliferation [24]. Concordant expression of TGF receptors I and II in virtually all cells positive for p27Kip1 within human atherosclerotic plaques
suggests that TGF-1 present in these lesions may contribute to p27Kip1 upregulation [38].
Moreover, coexpression of p53 and p21Cip1 in human carotid atheromatous plaque cells that
revealed lack of proliferation markers suggests that induction of p21Cip1 may occur via
transcriptional activation by p53 [72].
Reduced neointimal thickening has been demonstrated in rat, porcine and murine models of
angioplasty by means of intraluminal delivery of replication-defective adenoviral vectors
encoding p21Cip1 and p27Kip1 [55, 73-77]. Neointimal lesion formation in a rabbit model of vein
grafting is also attenuated by ectopic overexpression of p21Cip1 [78].
Methods of preparation and use of recombinant adenoviral vectors capable of expressing
human p21Cip1, p27Kip1 , p16Ink4a and other growth suppressors have been described to inhibit the
cell cycle of proliferating cells, as well as methods for the eradication of cancer and diseased
cells [105]. Likewise, methods of inhibiting cell proliferation using purified p18 Ink4c or p19Ink4d
proteins, and methods of gene therapy using nucleic acids that encode these genes have been
described [106, 107]. Reagents and methods for identifying genes whose expression is
modulated by induction of CKI gene expression have been provided [108]. This invention also
11
provides reagents and methods for identifying compounds that inhibit or potentiate the effects of
CKIs, such as p21Cip1 and p16Ink4a, on cellular gene expression, as a first step in rational drug
design for preventing cellular senescence, carcinogenesis and age-related diseases (such as
atherosclerosis and related disease) or for increasing the efficacy of anticancer therapies.
A method for treating vascular proliferative diseases by administering in vivo a gene
encoding p27Kip1 has been patented [109]. Proteasome degradation of p27Kip1 is thought to play a
major role in the regulation of p27Kip1 expression [35]. Kyushu University has patented the
nucleic acid and amino acid sequence of a new p27Kip1 molecular species exhibiting resistivity to
proteasome degradation, as well as expression vectors encoding this derivative of p27Kip1 for
gene therapy applications targeting cell propagating lesions such as tumours and arteriosclerotic
plaques [110].
3.4. Stem cell gene therapy
Embryonic and adult stem cells display a high proliferative activity, yet they maintain their
pluripotentiality both in vivo and when cultured in vitro. These unique properties of stem cells
has stimulated intense research activity to develop therapeutic strategies to promote the
regeneration of damaged tissues and organs. These studies include the elucidation of the
molecular pathways that govern stem cells differentiation into specific cell types, and methods to
facilitate their expansion in vitro. Of note in this regard, the propagation of a population of stem
cells or progenitor cells by disrupting or inhibiting the CKIs p21Cip1 and/or p27Kip1 has been
claimed in a patent that provides methods of using stem cells with a disrupted p21Cip1 and/or
p27Kip1 gene in gene therapy (e.g., stem cell gene therapy) and bone marrow transplantation
[111]. Methods of stimulating cell growth by blocking the CKIs p18Ink4c and p19Ink4d have been
also described [107].
12
13
4. Expert opinion
Although abundant cell proliferation has been well documented in animal models of
vascular obstructive disease, controversy exists regarding the magnitude of cell proliferation
within human primary atheromatous plaques and restenotic lesions. Cell proliferation appears to
be greater in restenotic compared to primary lesions in human peripheral and coronary arteries.
Furthermore, animal models of atherosclerosis have demonstrated an inverse correlation between
neointimal cell proliferation and atheroma size, suggesting that excessive cell growth might be
limited to early stages of atherogenesis. If this finding is applicable to humans, the potential
benefit of antiproliferative strategies for patients with established (and often advanced)
atherosclerotic plaques is uncertain. Indeed, the antiproliferative approaches used so far for the
treatment of vascular obstructive disease have focused on restenosis and graft atherosclerosis,
during which neointimal hyperplasia is rapid and localized. In general, systemic pharmacological
approaches that proved effective in animal models have been unsuccessful in reducing the
incidence of clinical
restenosis, including antiplatelet, anticoagulant, antithrombotic,
antiproliferative, antioxidant, and antiinflammatory agents [79-82]. The lack of correlation
between animal studies and clinical trials is likely the result of distinct vascular responses to
mechanical injury, and/or dissimilar pharmacokinetics in diverse animal species. Owing that
coronary stents represent almost 80% of the contemporary interventional procedures for
revascularization, local delivery of antiproliferative agents by means of drug releasing stents is
the focus of active research. It is notheworthy in this regard that drug eluting stents to locally
deliver pharmacological agents with antiproliferative, anti-inflammatory and antimigratory
actions (e.g. rapamycin, 7-hexanoyltaxol, and paclitaxel) have shown promising results in
reducing human in-stent restenosis after surgical revascularization.
Because CDKs and cyclins are essential for mammalian cell proliferation, substantial
efforts over the last years have led to the development of antiproliferative strategies based on the
14
inhibition of CDK/cyclin holoenzymes that efficiently limited neointimal hyperplasia in animal
models of cardiovascular disease, including the use of low molecular weight pharmacological
CDK inhibitors. The therapeutic application of these drugs to reduce the incidence of human
restenosis after surgical revascularization awaits clinical evaluation.
Owing that vascular interventions, both endovascular and open surgical, allow minimally
invasive, easily monitored gene delivery, gene therapy is emerging as an attractive strategy in the
treatment of vascular proliferative disease. Gene therapy strategies to inhibit CDK activity
include antisense- and ribozyme-mediated inactivation of CDKs and cyclins, and overexpression
of CKIs. Despite some encouraging results in animal models of cardiovascular disease, further
studies are required to override the current practical barriers and limitations placed on most
clinical trials before gene therapy strategies exhibit wide application in clinic. These should
include the clarification of safety issues, development of better gene delivery vectors and
delivery catheters, and improvement of transgene expression. Aside from these technical
improvements, significant effort in basic research is warranted to identify more effective and
safer treatment genes.
15
5. Acknowledgements
Work in the laboratory of V. Andrés is supported by the Spanish Ministry os Science and
Technology and Fondo Europeo de Desarrollo Regional (grants SAF2001-2358 and SAF20021143), and from the regional government of Valencia (grants GV01-488 and CTGCA/2002/04).
M. D. Edo is the recipient of a fellowship from the regional government of Valencia. M. Roldán
is supported by the Spanish Council for Scientific Research (CSIC) and the European Social
Funds to develop International RTD Management tasks under the Programme "I3P".
16
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First study demonstrating reduced experimental neointimal thickening by targeting CDK
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19
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This study supports the notion that p27Kip1 and p21Cip1 play a critical role in integrindependent control of VSMC proliferation in vitro.
20
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First study reporting the efficacy of CKI overexpression in reducing neointimal hyperplasia
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21
75.
76.
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22
Patents
101.
102.
103.
104.
105.
106.
107.
108.
109.
110.
111.
112.
113.
114.
115.
116.
117.
118.
119.
120.
121.
122.
123.
124.
125.
126.
127.
128.
129.
130.
131.
132.
133.
134.
135.
136.
137.
138.
139.
140.
141.
142.
143.
CHUGAI RES INST FOR MOLECULAR: JP11137251 (2001)
MEIJER L et al: WO9934018 (1999)
UNIV LELAND STANFORD JUNIOR: WO9625491 (1996)
IMMUSOL INC: WO9710334 (1997)
COWAN K et al: WO9625507 (1996)
ROUSSEL MF et al: WO0008153 (2000)
ST JUDE CHILDRENS RES HOSPITAL: WO9624603 (2000)
TRUSTEES OF THE UNIVERSITY OF ILLINOIS et al: WO0138532 (2001)
UNIV MICHIGAN: WO9903508 (2001).
KYUSHU UNIV: JP2001258561 (2001).
CHENG T et al: US2002006663 (200).
TROVA MP: US2002091263 (2002)
ALBANY MOLECULAR RES INC: WO0055161 (2000)
VERMEULEN K et al: WO0149688 (2001)
BREAULT GA et al: WO0164654 (2001)
CALVERT AH et al: WO9950251 (1999)
BASF AG et al: WO0119829 (2001)
WARNER LAMBERT CO: US6498163 (2002)
DOBRUSIN EM et al: WO0155147 (2001)
TRUMPP KSA et al: WO9961444 (1999)
BREAULT GA et al: WO0164655 (2001)
THOMAS AP et al: WO0172717 (2001)
DOBRUSIN EM et al: WO0170741 (2001)
NOVARTIS ERFIND VERWALT GMBH et al: WO0030651 (2000)
SCHERING AG: WO02100401 (2003)
SCHERING AG: WO02092079 (2002)
SCHERING AG: WO02074742 (2002)
SCHERING AG: WO0244148 (2002)
UNIV TEXAS et al: WO0044362 (2002)
SQUIBB BRISTOL MYERS CO: WO9742949 (1997)
CENTRE NAT RECH SCIENT: WO0160374 (2001)
CENTRE NAT RECH SCIENT: WO0141768 (2001)
SINGH R et al: WO0138315 (2001)
KIM EEK et al: WO0183469 (2001)
BOEHRINGER INGELHEIM PHARMA: WO02081445 (2002)
SPEVAK W: WO0116130 (2001)
CHEN BC et al: US2002099217 (2002)
DU PONT RHARM CO: WO0234721 (2002)
CARINI DJ: US2002091127 (2002)
SQUIBB BRISTOL MYERS CO et al: WO0246182 (2002)
DIMEO SV et al: US2001027195 (2001)
CHOI SH et al: WO0185726 (2001)
DENNY WA et al: WO0119825 (2001)
23
Table 1. Patents claiming the use of pharmacological CDK inhibitors to limit vascular cell
proliferation and/or cardiovascular disease
FAMILY
PURINES
COMPOUNDS
Biaryl substituted purine derivatives
6-substituted biaryl purine derivatives
Monosubstituted, disubstituted, and
trisubstituted purine derivatives and their deaza- and azaanalogues
Pyrimidine derivatives
Pyrazolo pyrimidines
REF.
[112]
[113]
[114]
Pyrido[2,3-D]pyrimidines
[115, 116]
[117]
[118]
Pyrido[2,3-d]pyrimidine-2,7-diamine
[119]
4-aminopyrimidines
[118]
Bicyclic pyrimidines
Bicyclic 3,4-dihydropyrimidines
2, 4-di(hetero-)arylamino (-oxy)-5-substituted
pyrimidines
[120]
[120]
[121]
4-amino-5-cyano-2-anilino-pyrimidine derivatives
[122]
5-alkylpyrido[2,3-d]pyrimidines
[123]
ALKALOIDS
Staurosporine
[124]
INDIRUBINS
Indirubin derivatives
Aryl-substituted indirubin derivatives
FLAVONOIDS
Flavopiridol 4-H-1-benzopryan-4-one derivatives
[125-127]
[128]
[129]
2-thio or 2-oxo flavopiridol analogs
Paullone derivatives
Hymenialdisine derivatives
Quinazoline derivatives
3-hydroxychromen-4-one structures
Indolinones substituted in position 6
Substituted indolinones
Azacycloalkanoylaminothiazole derivatives
[130]
[131]
[132]
[133]
[134]
[135]
[136]
[137]
Acylsemicarbazides
5-substituted-indeno[1,2-c]pyrazol-4-ones
Indeno[1,2-c]pyrazol-4-ones
Indazoles substituted with 1,1-dioxoisothiazolidine
Pteridinone derivatives
[138]
[139]
[140, 141]
[142]
[143]
PYRIMIDINES
PAULLONES
HYMENIALDISINES
QUINAZOLINES
HYDROXYCHROMENONES
INDOLINONES
AZACYCLOALKANOYLAMINOTHIAZOLES
SEMICARBAZIDES
INDAZOLES
PTERIDINONES
24
Table 2: Attenuation of neointimal thickening by pharmacological CDK inhibitors
in animal models of vascular proliferative disease.
AGENT
CVT-313
ANIMAL MODEL
Balloon angioplasty (rat)
REF
Flavopiridol
Balloon angioplasty (rat)
[43]
[42]
Table 3: Attenuation of neointimal thickening by antiproliferative gene therapy approaches
targeting CDK/cyclins in animal models of vascular proliferative disease.
STRATEGY
Antisense (ODN)
Antisense (retrovirus)
Oveexpression of
growth suppressors
TARGET GENE
CDK2
CDC2
Cyclin B1
CDC2/PCNA
CDC2/PCNA
CDK2
Cyclin G1
ANIMAL MODEL
Balloon angioplasty (rat)
Balloon angioplasty (rat)
Balloon angioplasty (rat)
Graft arteriosclerosis (rabbit)
Balloon angioplasty (rat)
Graft arteriosclerosis (mouse)
REF
Balloon angioplasty (rat)
[48]
[37, 44]
[37, 45]
[45]
[50]
[46]
[49]
p21Cip1
Balloon angioplasty (rat, mouse, pig) [73-76]
p21Cip1
p27Kip1
Graft arteriosclerosis (rabbit)
Balloon angioplasty (rat, pig)
[78]
[55, 77]
25