Affiliation of authors: DTP Clinical Trials Unit, Developmental Therapeutics Program, Division of Cancer Treatment and Diagnosis, National Cancer Institute, Bethesda, MD.
Correspondence to: Adrian M. Senderowicz, M.D., National Institutes of Health, Bldg. 10, Rm. 6N113, Bethesda, MD 20892 (e-mail: sendero{at}helix. nih.gov).
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ABSTRACT |
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INTRODUCTION TO CELL CYCLE REGULATION |
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Specific cdks operate in distinct phases of the cell cycle. Complexed with their respective D-type cyclin partners, cdk4 and cdk6 are responsible for the cell's progression through G1 phase (Fig. 1). A complex of cdk2 and cyclin E is responsible for the cell's progression from G1 phase to S phase. A complex of cdk2 and cyclin A is required for the cell's progression through S phase, and a complex of cdk1 (also known as cdc2) and cyclin B is required for mitosis (1). These complexes are in turn regulated by a stoichiometric combination with small inhibitory proteins called endogenous cdk inhibitors. The INK4 (inhibitor of cdk4) family includes p16ink4a, p15ink4b, p18ink4c, and p19ink4d, and its members specifically inhibit cyclin D-associated kinases. Members of the kinase inhibitor protein family p21waf1, p27Kip1, and p57kip2 bind and inhibit the activity of complexes of cyclin E and cdk2 and complexes of cyclin A and cdk2 (13-15). Although members of the kinase inhibitor protein family were initially thought to exclusively regulate G1 and S phases, several reports (16-18) demonstrated that these proteins can also regulate the G2/M-phase transition.
DNA synthesis (S phase) begins with the cdk4- and/or cdk6-mediated phosphorylation of Rb protein (which is complexed with the transcriptional factor E2F). Phosphorylated Rb is released from its complex with E2F. The released E2F then promotes the transcription of numerous genes required for the cell to progress through S phase, including thymidylate synthase and dihydrofolate reductase, among others (2,19,20). Additional information about cell cycle regulation can be found in several reviews (21-24).
The vast majority of human cancers have abnormalities in some component of the Rb pathway (Fig. 2) because of hyperactivation of cdks resulting from the overexpression of positive cofactors (cyclins/cdks) or a decrease in negative factors (endogenous cdk inhibitors) or Rb gene mutations (Fig. 2). Therefore, a pharmacologic cdk inhibitor that could be used in "mechanism-based therapy" would be of great theoretical interest as a treatment for many neoplasms (25). This possibility is intriguing because, for cancer patients, the loss of endogenous cdk inhibitors confers poor prognosis. For example, loss of p27kip1 protein predicts a poor outcome in patients with breast, prostate, lung, colon, or gastric carcinoma [reviewed in (26)]. Loss of p16ink4a is clearly associated with poor prognosis in patients with non-small-cell lung cancer or melanoma (26). However, the results with p21waf1 are inconclusive; loss of p21 may be prognostic in certain cancers, but inconsistent results were obtained for breast cancer (26). If validated in larger clinical studies, these markers could be incorporated in the routine pathologic examination of many tumors to determine prognosis.
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TARGETS FOR INTERVENTION IN THE CELL CYCLE |
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INDIRECT CDK MODULATORS |
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When the cdk inhibitor p16ink4A was introduced into a lung cancer cell line with defective endogenous p16ink4A, the cells were arrested in G1 phase (30). This effect occurs only in cells with functional Rb (31). When both p53 and p16ink4a were introduced into cells, apoptotic cell death followed (32). When p21waf1 or p27kip1 were introduced, in vitro and in vivo antitumor effects and G1/S arrest were observed (33,34). When p27 was introduced in several preclinical tumor models, apoptotic cell death was induced, but the relationship between the putative cdk inhibition of p27kip1 and its induction of apoptotic cell death is unclear (35,36).
Several small molecule chemical inhibitors of cdks appear to modulate the expression of cdk inhibitors. For example, lovastatin blocks cells in G1 phase by induction of p21waf1and p27kip1, which leads to the loss of cdk activity (37,38). Rapamycin blocks lymphocytes in G1 phase by preventing the interleukin 2-stimulated degradation of p27kip1 (39). However, it is unclear whether the cell cycle inhibitory effects of these small molecule chemical inhibitors can be solely explained by the induction of p27kip1.
cdk Inhibition by Peptidomimetic-Based Approaches
Another strategy to block cell cycle progression by loss of cdk activity is to use small peptides that mimic the effects of endogenous cdk inhibitors. Several carriers have been tested that introduce peptides into cells, including a 16-residue segment derived from the Drosophila antenappedia protein. When this 16-residue transmembrane carrier was linked to the third ankyrin repeat of the p16ink4A protein and inserted into cells, Rb-dependent G1 arrest and cell senescence were observed (40). Hybrids containing the antenappedia peptide and different p21waf1 peptides were constructed. In a breast-derived cell line, the chimera containing the carboxyl-terminal peptide of p21, amino acids 141-160, had a higher specificity for cdk4/cyclin D than for cdk2/cyclin E and arrested the cells in G1 phase (41). In contrast, in vitro the chimera containing amino-terminal peptides of p21, amino acids 17-33 and 63-77, inhibited both cdk1 and cdk2, and cells transduced with this chimera were arrested in all phases of the cell cycle (42).
Another approach to inhibiting cdk activity is to develop peptides that bind to cdks and inhibit cdk kinase activity (43,44). Colas et al. (43) demonstrated that a 20-amino acid peptide, identified by use of a combinatorial library, specifically binds cdk2 and inhibits its activity at low nanomolar concentrations in vitro. This peptide could act by blocking the interaction of the catalytic subunit with substrates or cyclin cofactors (43). Chen et al. (44) have shown that 8-amino acid peptides derived from the putative cyclin-cdk2-binding region of p21waf1 (PVKRRLFG) and E2F1 (PVKRRLDL) linked to N-terminal residues derived from human immunodeficiency virus Tat protein or antennapedia protein can block cells in S phase. This effect was associated with a loss of cdk2 activity. Although all of the cells tested with these chimeras showed clear evidence of G1/S-phase arrest, immortalized/transformed cells were more prone to apoptotic cell death.
cdk Inhibition by Depletion in cdk/Cyclin Subunits
Antisense technology has been used to deplete messenger RNAs (mRNAs) for cdk and/or cyclins (45-47). When cyclin D1 was depleted from tumor cell lines, a substantial antiproliferative effect was observed that was synergistic with different standard chemotherapeutic agents (45).
Several compounds can inhibit tumor progression by the modulation of cdk/cyclin subunits. In breast carcinoma cell lines, antiestrogens, such as tamoxifen, inhibit the expression of cyclin D and other cell cycle-related proteins and inhibit cdk activity (48). In breast carcinoma cell lines, retinoids, such as all-trans-retinoic acid and 9-cis-retinoic acid, inhibit the expression of multiple cell cycle regulators, including cyclin D1, cyclin D3, cdk2, and cdk4 (49). In some model systems, rapamycin, an inhibitor of FKBP (FK-506-binding protein)/mTOR (mammalian target of rapamycin), was also associated with a decline in cyclin D1 protein (39). Although treatment of cells with any of these compounds may lead to the decline of cyclin proteins and the perturbation of other cell cycle-related proteins, it is unclear how these compounds act. Perhaps the changes result from a direct interaction between the drug and the pathways that regulate the production of cyclin/cdk or result from the G1-phase arrest and/or Rb dephosphorylation, which are observed with these compounds.
cdk Inhibition by Modulation of Proteasomal Machinery
Sequential turnover of certain cell cycle regulators, including cyclins and p27kip1, is mediated by the 20S proteasome, which promotes proteolytic degradation through the ubiquitin/proteasome pathway. Increased turnover of cyclins with the associated loss of cdk activity may lead to cell cycle arrest with or without apoptotic cell death. Inhibiting 20S proteasome-mediated degradation could lead to accumulation of cdk inhibitors, such as p27ink4a, and to cell cycle arrest with or without apoptotic cell death (50). An important unresolved issue is the net effect and/or specificity of modulating proteasomal pathways. Nonspecific proteasome modulation could alter many signaling pathways (by the accumulation of proteins that activate or inhibit cdks) and thus could have a final effect on cells that is difficult to predict.
cdk Activation by Modulation of Upstream Phosphatases/Kinases
The abrogation (overriding) of intact cell cycle checkpoints by
upstream phosphatases and/or kinases could induce the "inappropriate
acceleration" of certain phases of the cell cycle. For example, when
the G2-phase checkpoint is activated by genotoxic stress
(i.e., -irradiation), the G2 period is extended to allow
DNA repair. In the presence of agents that abrogate (override) this
checkpoint, premature mitosis occurs, resulting in apoptotic cell death
(51). Thus, abolishing the G2 checkpoint might
sensitize cells to agents that would normally cause cells to pause or
arrest in G2 phase (51,52).
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DIRECT CDK MODULATORS (SMALL MOLECULAR CDK INHIBITORS) |
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The first cdk inhibitor discovered was dimethylaminopurine (56). This compound was initially shown to inhibit mitosis in sea urchin embryos without inhibiting protein synthesis. Later, dimethylaminopurine was shown to inhibit cdk1 activity (IC50 [concentration that inhibits activity by 50%] = 120 µM) but to be relatively nonspecific (27). Isopentenyladenine, a derivative of dimethylaminopurine, was somewhat more potent and selective for the cdks (IC50 = 55 µM) (57). Other active purine derivatives have been identified in screening campaigns for more specific and potent cdk inhibitors. Olomoucine potently inhibited cdk1 and cdk2 activities (IC50 = 7 µM) (27,57). Roscovitine, a derivative of olomoucine, is a more potent cdk inhibitor (IC50 values for cdk1/cdk2 = 0.7 µM) (27).
The crystal structures of cdk2 complexed with isopentenyladenine, olomoucine, or roscovitine showed that all three inhibitors bind to the ATP site (29,58). CVT-313, another purine analogue, was identified by use of a combinatorial library strategy and the crystal structure of cdk2. Similar to previous analogues, CVT-313 was specific for cdk1 and cdk2 with IC50 values of 4.2 and 1.5 µM, respectively (59).
A combinatorial approach was then used to modify the purine scaffold of 2-fluoro-6-chloropurine, and several compounds that potently and specifically inhibited cdc2 and cdk2 were identified. Four novel compounds (purvalanol-A, purvalanol-B, compound 52, and compound 52E) were characterized through a battery of in vitro kinases experiments (60). The crystal structure of purvalanol-B complexed with cdk2 showed that purvalanol-B bound to the ATP-binding site resembles the binding of olomoucine to cdk2. The more membrane-permeable purvalanol-A was tested on the National Cancer Institute's (NCI's) 60-cell-line anticancer drug screen panel. The average IC50 value of purvalanol-A was 2 µM, demonstrating that it was a more active antiproliferative agent than purvalanol-B (60). Cell cycle studies of purvalanol-A on human fibroblasts showed that it arrested cells in G1/S phase and G2/M phase, compatible with the putative inhibitory properties in cdk1 and cdk2, respectively (60).
Paullones
With the use of the antiproliferative in vitro profile of flavopiridol in NCI's anticancer drug screen panel and the computational algorithm COMPARE, several members of the paullone family were identified (61). Kenpaullone (NSC 664704) potently inhibited cdk1/cyclin B (IC50 = 0.4 µM), cdk2/cyclin A (IC50 = 0.68 µM), cdk2/cyclin E (IC50 = 7.5 µM), and cdk5/p35 (IC50 = 0.85 µM) but had much lower activity toward other kinases (28). Kenpaullone competitively inhibits the binding of ATP, with an apparent Ki (i.e., inhibitory constant) for cdk1/cyclin B of about 2.5 µM. Molecular modeling studies demonstrated that kenpaullone may bind to the ATP-binding site with residue contacts similar to other cdk2 inhibitors (28). Cell cycle effects of kenpaullone were characterized with the MCF10A breast epithelial cell line. Cells were synchronized in G0/G1 phase by serum starvation and then stimulated to re-enter the cell cycle in the presence of vehicle or kenpaullone at its approximate IC50 concentration (30 µM). Twenty hours later, vehicle-treated cells entered S phase. However, cells exposed to kenpaullone were arrested at the G1/S boundary. A similar effect was obtained with another paullone analogue, 10-bromo-paullone (NSC 672234) (28).
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CONSEQUENCES OF CDK INHIBITION |
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Initial cell cycle studies by van den Heuvel and Harlow (62) demonstrated that ectopic expression of cdk1-dominant negative alleles was able to block U2OS osteosarcoma cell lines at the G2/M-phase boundary. In contrast, expression of dominant negative alleles of cdk2 or cdk3 blocked cells in S phase (62). Thus, roles for each cdk in human cell cycle began to be assigned.
Ectopic expression of endogenous cdk inhibitors (such as p16, p21, or p27) or peptidomimetics derived from p21, p16, or E2F1, as described above in detail, demonstrates the feasibility of using this method to arrest cells in the cell cycle.
As described above, several small molecular cdk inhibitors, including roscovitine, olomoucine, purvalanol, and flavopiridol, arrest cells at either the G1/S- or the G2/M-phase boundaries (53,59,60,63,64). It is unclear why these agents arrest some cells in G1/S phase and other cells in G2/M phase or both.
Apoptotic Cell Death
The antiproliferative effects of olomoucine, flavopiridol, and roscovitine were accompanied by the induction of apoptotic cell death in certain cell types (64-66). In one study (66), the ability of flavopiridol and olomoucine to induce apoptotic cell death varied, depending on the growth status of the cells. That is, flavopiridol or olomoucine protected postmitotic nondividing PC12 neuronal cells from apoptotic cell death after the withdrawal of nerve growth factor. However, flavopiridol did not protect cycling PC12 cells from apoptotic cell death after the withdrawal of nerve growth factor (66). Similarly, cdk4 and cdk6 proteins from dominant negative alleles, but not cdk2 or cdk3 proteins from dominant negative alleles, protected neurons from apoptotic cell death after the withdrawal of nerve growth factor (67). Thus, susceptibility of PC12 cells to flavopiridol- and olomucine-induced apoptotic cell death may vary, depending on the growth state of the cells.
HeLa cervical carcinoma cells treated with staurosporine and tumor necrosis factor-
were protected from apoptotic cell death by cdc2, cdk2, or cdk3 encoded by dominant negative
alleles. However, only cdk2 encoded by a dominant negative allele protected cells from apoptotic
cell death induced by ectopic expression of topoisomerase-II
(68).
Finally, certain apoptotic stimuli induce the caspase-mediated cleavage in endogenous cdk
inhibitors (p21/p27) or cdk-inhibitory proteins (wee1 and cdc27) leading to activation of cdks (69-71). Thus, cell cycle arrest and/or apoptosis induced by the inhibition
of cdks depends on several factors, including the mechanism of inhibition, the type of cells, and
the proliferation status of the cells.
Differentiation
During differentiation, cells exit the cell cycle and lose cdk2 activity. Lee et al. (72) tested whether the chemical cdk2 inhibitors flavopiridol and roscovitine could induce a differentiated phenotype by exposing NCI-H358 lung carcinoma cells to a cdk2 antisense construct, flavopiridol, or roscovitine. They observed that all three cdk2 inhibitors could induce mucinous differentiation with the loss of cdk2 activity.
When U937 myelomonocytic leukemia cells were treated with aminopurvalanol, the cells acquired a phenotype characteristic of differentiated macrophages. Moreover, aminopurvalanol, a potent inhibitor of cdk1 and cdk2, appeared to arrest cells at the G2/M boundary and then to induce apoptotic cell death (73). Other investigators (74) observed a similar phenomenon; ectopic expression of p21waf1 or p27kip1 resulted in a "differentiated phenotype" with cells arrested in G1 or G2 phases and a 4N amount of DNA.
Transcriptional Effects
To compare the effects of several chemical cdk inhibitors on the expression of complementary DNA in yeast cells, Gray et al. (60) incubated Saccharomyces cerevisiae with compound 52 and flavopiridol (each at 25 µM ) for 2 hours and measured mRNA by oligonucleotide array methods. Two percent to 3% of the 6200 yeast genes examined showed a greater than twofold change in transcript levels in the presence of these agents. Moreover, almost 50% of affected transcripts were affected by both compound 52 and flavopiridol. These genes fell into distinct groups, including genes that regulate progression of cell cycle, genes that regulate phosphate and cellular energy metabolism, and genes that regulate guanosine 5'-triphosphate (GTP)- or ATP-binding proteins. However, more than 40% of the changes in mRNA were not concordant between flavopiridol and compound 52. These discrepancies might be explained 1) by the broad cdk-inhibitory activity of flavopiridol compared with the selective cdk2/cdk1-inhibitory activities of compound 52, 2) by the different intracellular concentrations achieved by these inhibitors, 3) by the distinctive molecular structures of these inhibitors, or 4) from their putative effects on other cellular targets.
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PRECLINICAL PHARMACOLOGY OF FLAVOPIRIDOL |
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Mechanism of Action
Flavopiridol, also known as L86-8275 or HMR 1275, is a semisynthetic
flavonoid derived from rohitukine, an alkaloid isolated from a plant
indigenous to India (Fig. 4). Table 1 contains a
summary of the most important preclinical in vitro effects of
flavopiridol. Initially, flavopiridol displayed modest activity in
vitro as an inhibitor of tyrosine kinase of the epidermal growth
factor receptor and an inhibitor of protein kinase A (IC50 =
21 and 122 µM, respectively) (75). However, when
flavopiridol was tested in the NCI's 60-cell-line anticancer drug
screen panel, its IC50 was 66 nM. This concentration
is about 1000 times lower than the concentration required to inhibit
protein kinase A and the tyrosine kinase of the epidermal growth factor
receptor (75). The antiproliferative effect was not associated
with the presence of the epidermal growth factor receptor
(75,76). Flavopiridol was shown to arrest cells in
G1 phase or at the G2/M boundary, raising the
possibility that flavopiridol may inhibit cdk2 and cdk1 (76).
Studies using purified cdks showed that flavopiridol inhibits the
activities of cdk1, cdk2, and cdk4; this inhibition is competitively
blocked by ATP, with a Ki of 41 nM
(53,54,76-78). The crystal structure of the complex of
deschloroflavopiridol and cdk2 showed that flavopiridol binds to
the ATP-binding pocket, with the benzopyran occupying the same region
as the purine ring of ATP (79). This observation was
concordant with our earlier biochemical studies with flavopiridol
(54). Flavopiridol inhibits all cdks thus far examined
(IC50 = approximately 100 nM), but it inhibits cdk7
(cdk-activating kinase) less potently (IC50 = approximately
300 nM) (53,54,77).
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Another important action of flavopiridol is the selective induction of apoptotic cell death. Hematopoietic cell lines are often quite sensitive to flavopiridol-induced apoptotic cell death (65,84-86), but the mechanism(s) by which flavopiridol induces apoptosis have not yet been elucidated. Flavopiridol does not modulate topoisomerase I/II activity (65). In certain hematopoietic cell lines, neither BCL-2/BAX nor p53 appeared to be affected (65,85), whereas, in other systems, BCL-2 may be inhibited (86). It is unclear whether the putative flavopiridol-induced inhibition of cdk activity is required for induction of apoptosis.
Cell cycle arrest, but again with clear evidence of apoptotic cell death, was observed with a
panel of head and neck squamous cell carcinoma cell lines, including a cell line (HN30) that is
refractory to several DNA-damaging agents, such as -irradiation and bleomycin (87). The apoptotic effect was independent of p53 status and was
associated with the depletion of cyclin D1 (87). These findings have been
corroborated in other preclinical models (88-90). Efforts to understand
flavopiridol-induced apoptosis are under way.
To determine whether flavopiridol has antiangiogenic properties, Brusselbach et al. (91) incubated human umbilical vein endothelial cells (HUVECs) with flavopiridol and observed apoptotic cell death even in cells that were not cycling. In another report, Kerr et al. (92) tested flavopiridol in an in vivo angiogenesis model and found that flavopiridol decreased blood vessel formation in the mouse Matrigel model of angiogenesis. Melillo et al. (93) demonstrated that, at low nanomolar concentrations, flavopiridol blunted the induction of vascular endothelial growth factor (VEGF) by hypoxia in human monocytes. This effect was caused by a decreased stability of VEGF mRNA, which paralleled the decline in VEGF protein. Thus, the antitumor activity of flavopiridol may be supplemented by antiangiogenic effects. Whether the various antiangiogenic actions of flavopiridol result from its interaction with a cdk target or other targets requires further study.
To test for synergistic effects with other compounds, cytotoxic assays of flavopiridol in
combination with standard chemotherapeutic agents were performed (94,95). Synergistic effects in A549 lung carcinoma cells were demonstrated when
treatment with flavopiridol followed treatment with paclitaxel, cytarabine, topotecan,
doxorubicin, or etoposide. In contrast, a synergistic effect was observed with 5-fluorouracil only
when cells were treated with flavopiridol for 24 hours before addition of 5-fluorouracil.
Synergistic effects with cisplatin were not schedule dependent (95).
However, Chien et al. (88) failed to demonstrate a synergistic effect
between flavopiridol and cisplatin and/or -irradiation in bladder carcinoma models.
Several questions about the antiproliferative activity of flavopiridol remain unanswered. Why does treatment with flavopiridol cause some cells to arrest at the G1/S-phase boundary and other cells to arrest at the G2/M-phase boundary? What role does the depletion of D-type cyclins play in flavopiridol-induced G1/S arrest? What is the relationship between cdk inhibition and apoptotic cell death, and what are the targets for flavopiridol-induced apoptotic cell death?
Antitumor Effect in Preclinical Models
Experiments using colorectal (Colo205) and prostate (LnCap/DU-145) carcinoma xenograft models in which flavopiridol was administered frequently over a protracted period demonstrated that flavopiridol is cytostatic (75). This demonstration led to clinical trials in humans of flavopiridol administered as a 72-hour continuous infusion every 2 weeks (96) (see below).
Subsequent studies in some models of human leukemia/lymphoma xenografts demonstrated that flavopiridol administered intravenously as a bolus rendered animals tumor free, whereas flavopiridol administered as an infusion only delayed tumor growth (84). In head and neck (HN-12) xenografts, when flavopiridol was administered as an intraperitoneal bolus daily at 5 mg/kg for 5 days, a substantial growth delay was observed (87). Again, apoptotic cell death and cyclin D1 depletion were observed in tissues from xenografts treated with flavopiridol at peak plasma concentrations of 5-8 µM (84). Based on these results, the feasibility of a phase I trial with the administration of flavopiridol as a 1-hour infusion is currently being explored at the NCI (see below).
Preclinical Pharmacokinetics and Toxicology
Murine plasma concentration-time profiles for flavopiridol
exhibited biexponential behavior with mean and ß half-lives of
16.4 and 201.0 minutes, respectively. The mean total-body plasma
clearance was 22.6 mL/minute per kg, and the mean oral bioavailability
after bolus intragavage was approximately 20%. Pharmacokinetic studies
in dogs (75) had very similar results.
The metabolism of flavopiridol was investigated in isolated liver perfusion models. Flavopiridol was glucuronidated in the liver, and then this flavopiridol metabolite excreted in the biliary tract. This property underlies flavopiridol's propensity to undergo enterohepatic circulation (97,98). Preclinical pharmacologic and toxicologic evaluations have identified dose-limiting toxic effects as reversible hematopoietic and gastrointestinal effects.
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HUMAN CLINICAL TRIALS OF FLAVOPIRIDOL |
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In the presence of antidiarrheal prophylaxis (a combination of cholestyramine and loperamide), patients tolerated higher doses, defining a second maximal tolerated dose, 78 mg/m2 per day for 3 days. The dose-limiting toxicity observed at the higher dose level was reversible hypotension and a substantial proinflammatory syndrome (fever, fatigue, local tumor pain, and modulation of acute-phase reactants) (96).
Tumors in one patient with non-Hodgkin's lymphoma, one patient with colon cancer, and one patient with kidney cancer decreased in size (minor responses = shrinkage of <50%) for more than 6 months. Moreover, one patient with refractory renal cancer achieved a partial response (shrinkage of >50% of masses) (96). Of 14 patients who received flavopiridol for more than 6 months, five patients received flavopiridol for more than 1 year and one patient received flavopiridol for more than 2 years (96). This potential "disease stabilization," which may have been noted in this trial, is consistent with preclinical models, where tumor stasis is observed. Appropriate measurements of cytostatic effects are necessary to confirm that cdk inhibition might be related to this clinical outcome. Plasma concentrations of 300-500 nM flavopiridol, which inhibit cdk activity in vitro, were safely achieved during our trial (96).
In a complementary phase I trial also exploring the use of a 72-hour continuous infusion of flavopiridol every 2 weeks, Thomas et al. (99) found that the dose-limiting toxicity is diarrhea, corroborating the experience of the NCI. Moreover, plasma concentrations of 300-500 nM flavopiridol were also observed. It is interesting that there was one patient in this trial with refractory gastric cancer that had metastasized to the liver who was initially treated surgically and subsequently failed to respond to one treatment regimen of 5-fluorouracil. When treated with flavopiridol, this patient achieved a sustained complete response without any evidence of disease for more than 2 years after treatment was completed.
In September 1998, we began the first phase I trial of a daily 1-hour infusion of flavopiridol for 5 consecutive days every 3 weeks. This dose schedule was based on our antitumor results observed in leukemia/lymphoma and head and neck xenografts treated with flavopiridol (see above). At this time, 27 patients have been treated in this phase I trial. The recommended phase II dose is 37.5 mg/m2 per day for 5 consecutive days. Dose-limiting toxic effects observed at 52.5 mg/m2 per day are nausea/vomiting, neutropenia, fatigue, and diarrhea. Other (non-dose-limiting) side effects are "local tumor pain" and anorexia (Senderowicz AM: unpublished results). To reach higher flavopiridol concentrations, the protocol was amended to administer flavopiridol for 3 days only. Higher peak plasma flavopiridol concentrations (approximately 4 µM) may be obtained with this schedule (Senderowicz AM: unpublished results).
A phase I trial testing the combination of paclitaxel and flavopiridol demonstrated good tolerability with a dose-limiting pulmonary toxicity (100).
Phase II trials of flavopiridol given as a 72-hour continuous infusion to patients with chronic lymphocytic leukemia, non-small-cell lung cancer, non-Hodgkin's lymphoma, or colon, prostate, gastric, head and neck, or kidney cancer, etc., and phase I trials of flavopiridol administered on novel schedules and in combination with standard chemotherapeutic agents are being explored (101-104).
Several important clinical questions remain to be answered in these trials. Is flavopiridol an "effective" anticancer agent? Which is the best schedule for flavopiridol monotherapy? What is the best method to combine flavopiridol and other agents? Which is the most reliable pharmacodynamic parameter to follow in patients? How should "stable disease" be defined in phase II trials?
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PRECLINICAL PHARMACOLOGY OF UCN-01 |
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Mechanism of Action
One staurosporine analogue, UCN-01 (7-hydroxystaurosporine; Fig. 4), has potent activity against several protein kinase C isoenzymes, particularly the Ca2+-dependent protein kinase C with an IC50 of about 30 nM. UCN-01 has lower potency against the novel Ca2+-independent protein kinases C (IC50 = approximately 500 nM) and no effect against the atypical protein kinases C (106-108), similar to the activity of staurosporine. In addition to its effects on protein kinase C, UCN-01 has antiproliferative activity in several human tumor cell lines (109-113). In contrast, another highly selective potent protein kinase C inhibitor, GF 109203X, has a modest antiproliferative activity, despite a similar capacity to inhibit protein kinase C in vitro (110). Thus, these results suggest that the antiproliferative activity of UCN-01 is probably not explained solely by inhibition of protein kinase C. UCN-01 moderately inhibited the activity of immunoprecipitated cdk1 (cdc2) and cdk2 (IC50 = 300-600 nM). However, when intact cells were exposed to UCN-01, "inappropriate activation" of the same kinases occurred (110).
Experimental evidence suggests that DNA damage leads to cell cycle arrest to allow DNA repair. In cells where the G1-phase checkpoint is not active because of p53 inactivation, irradiated cells accumulate in G2 phase because the G2 checkpoint is mediated by the inactivation of cyclin B/cdc2 by wee1 kinase (Fig. 5, A). In contrast, UCN-01 (Fig. 5, B) induces the activation of cdc2/cyclin B and thus promotes cells to enter early mitosis with the onset of apoptotic cell death. These effects could be partially explained by the inactivation of wee1, the kinase that negatively regulates the G2/M-phase transition or activation of cdc25 phosphatase (114). Thus, although UCN-01 at high concentrations can directly inhibit cdks in vitro, UCN-01 can modulate cellular upstream regulators at much lower concentration, leading to inappropriate cdc2 activation by acting on targets that remain to be defined. DNA-damaging agents not only can activate the G2-phase checkpoint but also can activate the S-phase checkpoint (115). Studies from other groups suggest that UCN-01 not only is able to abrogate the G2 checkpoint induced by DNA-damaging agents but also, in some circumstances, is able to abrogate the DNA damage-induced S-phase checkpoint (115).
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Another interesting aspect of UCN-01 is its ability to arrest cells in G1 phase of the cell cycle (109,112,113,123-125). Human epidermoid carcinoma A431 cells contain mutated p53. When incubated with UCN-01, these cells were arrested in G1 phase, Rb was hypophosphorylated, and p21waf1 and p27kip1 accumulated (113). However, in another report (125), the antiproliferative effect of UCN-01 was not dependent on the functional status of Rb. Thus, the G1 arrest observed with UCN-01 is apparently independent of the status of p53 and Rb. Further studies on the putative target(s) for UCN-01 in the G1-phase arrest of cells are under way.
Courage et al. (126) found that UCN-01-resistant A549 lung
carcinoma cells were sensitive to two other protein kinase C inhibitors, CGP 41251 and Ro
31-8220, and were marginally resistant (about twofold) to etoposide. These UCN-01-resistant
cells had lost several protein kinase C isoenzymes, protein kinase C ,
, and |gv.
However, the levels of these isoenzymes returned to baseline when these resistant cells were
cultured in UCN-01-free medium. Thus, as described above, it is unlikely that the protein kinase
C family of signaling proteins is the only target for UCN-01 cytotoxicity (110,126).
Although many important questions have been answered, several questions remain. What is the real target for UCN-01 in G1/S phase-arrested cells? Does protein kinase C play any role in UCN-01-induced cell cycle arrest and/or apoptotic cell death? What is the real target for the G2 checkpoint abrogation?
Spectrum of In Vivo Antitumor Activity
UCN-01 administered by an intravenous or intraperitoneal route displayed antitumor activity in xenograft model systems with breast carcinoma (MCF-7), renal carcinoma (A498), and leukemia (MOLT-4 and HL-60) cell lines (Senderowicz AM: unpublished results). The antitumor effect was greater when UCN-01 was given over a longer period. This requirement for a longer period of treatment was also observed in in vitro models, with highest antitumor activity observed when UCN-01 was present for 72 hours (109). Thus, a clinical trial using a 72-hour continuous infusion every 2 weeks was conducted (described below).
Preclinical Pharmacokinetics and Toxicology
Pharmacokinetics and toxicologic studies using several schedules were done in rats and dogs. When beagle dogs were given a 72-hour continuous infusion of UCN-01, local (site of injection) and gastrointestinal toxic effects were dose-limiting and a steady-state plasma concentration of 330 nM UCN-01 was achieved. Pharmacokinetic parameters were a volume of distribution (6.09 L/kg), a total clearance of 0.6 L/kg per hour with a ß half-life of about 12 hours (127).
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PHASE I CLINICAL TRIALS OF UCN-01 |
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SUMMARY |
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NOTES |
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We are indebted to colleagues from the Laboratory of Biological Chemistry and Developmental Therapeutics Program Clinical Trials Unit, National Cancer Institute, for all the encouragement and heavy work provided for these projects. We also want to acknowledge the National Cancer Institute and the National Institutes of Health staff for their contributions in these clinical trials. Finally, we want to acknowledge the patients and their relatives for their willingness to participate in our clinical trials and contribute to the advancement of medicine.
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Manuscript received June 15, 1999; revised December 7, 1999; accepted December 15, 1999.
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