Brief overview of cell cycle regulation

The regulation of cell cycle and proliferation has been extensively studied in the last few years and a consensus paradigm of the regulation of the cell cycle regulation has been developed (Sherr, 1996; Morgan, 1997). According to this paradigm, the master switch of the cell cycle is the retinoblastoma (Rb) family of proteins. Proliferation occurs by the phosphorylation of these family of proteins by the cyclin-dependent kinases (cdk's) (Figure 1) (Sherr, 1996). There are at least nine cdk's (Sherr, 1996; Morgan, 1997). These kinases are activated by D-type cyclins (D1, D2, and D3) and cyclin E, and inhibited by two families of cdk inhibitors (CKI's), the Ink (p16^Ink4a, p15^Ink4b, p18^Ink4c, and p19^Ink4d) and Cip/Kip families (p21^Cip1, p27^Kipl, and p57^Kip2) (Sherr, 1996; Morgan, 1997).

Figure 1.

Schematic representation of cdk's and cell cycle control

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Rb proteins are pocket proteins that sequester E2F transcription factors preventing them from activating critical genes in cell proliferation (Wang et al., 2001; Yu et al., 2001). After Rb phosphorylation by cdk4 and/or cdk6 complexes during the G₁ phase and cdk2 at the G₁/S interphase, E2F proteins are released and promote the transcription of genes essential for transition to the S phase of the cell cycle (Lees et al., 1993; DeGregori et al., 1995). cdk4,6/D-type cyclins therefore execute their critical functions during the mid-to-late G1 phase, as cells cross a G₁ restriction point and become independent of mitogens for completion of the division cycle. These features suggest that the fundamental role of these complexes is to integrate extracellular signals with the cell cycle machinery (Sherr, 1996; Morgan, 1997).

Other important points of regulation have been described in G₂ and mitosis. In these phases also, the specific expression of certain regulators is essential to control the correct sequence of events that lead to cell division. Basically, the cyclins B1, B2, and its partner cdc2 (cdk1) together with other kinases and phosphatases (wee1, cdc25) regulated the final phases of the cell cycle (Figure 1)

In the last decade, several proteins that participate in the tight control of cell division have been found to be mutated, deleted, amplified, or overexpressed in human tumors. In the first part of this review, we summarize the principal points of deregulation found in human tumors with particular emphasis on lung cancer.

Cell cycle alterations in human neoplasms

In the last few years, it became clear that cyclins, cdk complexes, and other cell cycle regulators are mechanistically involved in the development of human tumors (Motokura and Arnold, 1993; Weinberg, 1996; Jacks and Weinberg, 1998; Robles et al., 1998; Rodriguez-Puebla et al., 1999). This is consistent with a large body of literature showing the importance of inactivation of the Rb pathway in tumor development (Hatakeyama and Weinberg, 1995; Weinberg, 1996; Senderowicz, 2002b). The inactivation of Rb can be produced by direct mutation of the Rb protein, but this is a relatively rare event occurring only in Rb's, osteosarcomas, and a minority of breast and some other tumors (Hunter and Pines, 1994; Sherr, 1996; Weinberg, 1996). More frequent alterations of this pathway occur by functional inactivation of Rb by hyperphosphorylation. This is normally the result of elevated cdk activities caused by overexpression of cyclins, cdk's.

For example, several laboratories have reported that some tumors show loss of Rb or, alternatively, overexpression of cyclin D1 (Bartek et al., 1993; Bartkova et al., 1995, 1996). Similarly, in other tumors, loss of p16^Ink4a and Rb are mutually exclusive (Aagaard et al., 1995; Lukas et al., 1995a, 1995b). This observation led to the hypothesis that inactivation of the cyclin D/CDK/p16/pRb pathway can promote tumor development and that either loss of the suppressor activity of Rb or p16^Ink4a, or overexpression of cyclin D1 can over-ride this checkpoint (Sherr, 1996; Weinberg, 1996). Several reports have implicated D-type cyclins in neoplastic development, although limited information is available on the participation of its partner, cdk4, in these events. The involvement of cdk4 in the neoplastic process was suggested by the fact that cdk4 amplification and/or overexpression were detected in human glioblastomas, but in these tumors overexpression and/or amplification of D-type cyclins were not detected (Sonoda et al., 1995; Ichimura et al., 1996). In addition, cdk4 mutations were identified in patients with familial melanoma (Wolfel et al., 1995), and, recently, amplification and overexpression of cdk4 were also detected in sporadic breast carcinomas (An et al., 1999), ovarian carcinomas (Masciullo et al., 1997), and sarcomas (Kanoe et al., 1998). Taken together, proteins that govern cell cycle control are reasonable targets for cancer therapy (Meijer, 2000; Senderowicz, 2002b).

Manipulation of cdk activity for therapeutic purposes

Several strategies could be considered to modulate cdk activity (see Figure 2). These strategies are divided into direct effects on the catalytic cdk subunit or indirect modulation of regulatory pathways that govern cdk activity(Senderowicz, 2000; Senderowicz and Sausville, 2000). Small molecular endogenous cdk inhibitors (SCDKI) are compounds that directly target the catalytic cdk subunit. Most of these compounds modulate cdk activity by interacting specifically with the ATP-binding site of cdk's (De Azevedo et al., 1997; Meijer and Kim, 1997; Zaharevitz et al., 1999; Senderowicz, 2000; Senderowicz and Sausville, 2000; Senderowicz, 2002b). Examples of this class include flavopiridol, roscovitine, aminothiazole, UCN-01 (7-hydroxystaurosporine), and alsterpaullone (See Figure 2). The second class are compounds that modulate cdk activity by targeting the regulatory upstream pathways that govern cdk activity: by altering the expression and synthesis of the cdk/cyclin subunits or the cdk inhibitory proteins; by modulating the phosphorylation of cdk's; by targeting cdk-activating kinase (CAK), cdc25, and wee1/myt1; or by manipulating the proteolytic machinery that regulates the catabolism of cdk/cyclin complexes or their regulators (see Figure 2, panel b) (Senderowicz, 2000; Senderowicz and Sausville, 2000; Senderowicz, 2002a, 2002b). Examples for this class of compounds include perifosine and UCN-01, among others.

Figure 2.

Modes of action for cell cycle modulators. There are two families of small-molecule cdk modulators, the direct and the indirect cdk modulators. Indirect modulators interact with upstream pathways, leading to loss in cdk activity. Loss in cdk function may occur due to loss in mass of catalytic subunit and/or cofactors, increased endogenous inhibitors, by increased wee1/myt1, or by loss in cdk7 or cdc25c activity. However, the most successful way of modulating cdk is by competing with ATP binding for cdks

Full figure and legend (90K)

Small-molecule cdk modulators

(Tables 1 and 2)

Table 1 - Direct cdk modulators.

Full table

Table 2 - Indirect cdk modulators.

Full table

As mentioned previously, cdk's can be modulated by direct effects on the catalytic subunit and/or by disruption of upstream regulatory pathways. Several examples and mechanisms are described in Tables 1 and 2 and elsewhere (Meijer, 2000; Senderowicz, 2000; Senderowicz, 2001a, 2001b; Ortega et al., 2002; Schoepfer et al., 2002; Senderowicz, 2002a, 2002b; Mettey et al., 2003).

Cdk modulators in clinical trials

Flavopiridol

Mechanism of antiproliferative effects

Flavopiridol (L86-8275 or HMR 1275, see Figure 3) is a semisynthetic flavonoid derived from rohitukine, an indigenous plant from India. Initial studies with this flavonoid revealed clear evidence of G₁/S or G₂/M arrest, due to loss in cdk1 and cdk2 (Kaur et al., 1992; Worland et al., 1993; Losiewicz et al., 1994). Studies using purified cdks showed that the inhibition observed is reversible and competitively blocked by ATP, with a K_i of 41 nM (Kaur et al., 1992; Worland et al., 1993; Losiewicz et al., 1994; Carlson et al., 1996a, 1996b). Furthermore, 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 (De Azevedo et al., 1996), confirming the earlier biochemical studies with flavopiridol (Losiewicz et al., 1994). Flavopiridol inhibits all cdks thus far examined (IC₅₀100 nM), but it inhibits cdk7 (CAK) less potently (IC₅₀300 nM) (Losiewicz et al., 1994; Carlson et al., 1996a, 1996b).

Figure 3.

Structure of small-molecule direct cdk modulators in clinical trials

Full figure and legend (32K)

In addition to directly inhibiting cdks, flavopiridol promotes a decrease in the level of cyclin D1, an oncogene that is overexpressed in many human neoplasias. Of note, neoplasms that overexpress cyclin D1 have a poor prognosis (Michalides et al., 1995; Fredersdorf et al., 1997; Gansauge et al., 1997). Depletion of cyclin D1 appears to lead to the loss of cdk activity (Carlson et al., 1999). Cyclin D1 decrease is caused by depletion of cyclin D1 mRNA and was associated with a specific decline in cyclin D1 promoter, measured by a luciferase reporter assay (Carlson et al., 1999). The transcriptional repression of cyclin D1 observed after treatment with flavopiridol is consistent with the effects of flavopiridol on yeast cells (see above) and underscores the conserved effect of flavopiridol on eucaryotic cyclin transcription (Gray et al., 1998). In summary, flavopiridol can induce cell cycle arrest by at least three mechanisms: (1) direct inhibition of cdk activities by binding to the ATP-binding site; (2) prevention of the phosphorylation of cdks at threonine-160/161 by inhibition of cdk7/cyclin H (Worland et al., 1993; Carlson et al., 1996a); and (3) decrease in the amount of cyclin D1, an important cofactor for cdk4 and cdk6 activation (G₁/S arrest only).

In part, flavopiridol regulates transcription due to potent inhibition of P-TEFb (also known as cdk9/cyclin T), with a K_i of 3 nM, leading to inhibition of transcription by RNA polymerase II by blocking the transition into productive elongation. Interestingly, in contrast with all cdk's tested so far, flavopiridol was not competitive with ATP in this reaction. P-TEFb is a required cellular cofactor for the human immunodeficiency virus (HIV-1) transactivator, Tat. Consistent with its ability to inhibit P-TEFb, flavopiridol blocked Tat transactivation of the viral promoter in vitro. Furthermore, flavopiridol blocked HIV-1 replication in both single-round and viral spread assays with an IC₅₀ of less than 10 nM (Chao et al., 2000). These actions of the drug led to the testing of flavopiridol through clinical trials for patients with HIV-related malignancies (Wright et al., 1998).

An important biochemical effect involved in the antiproliferative activity of flavopiridol is the induction of apoptotic cell death. Hematopoietic cell lines are often quite sensitive to flavopiridol-induced apoptotic cell death, (Konig et al., 1997; Arguello et al., 1998; Byrd et al., 1998; Parker et al., 1998), but the mechanism(s) by which flavopiridol induces apoptosis have not yet been elucidated. Flavopiridol does not modulate topoisomerase I/II activity (Parker et al., 1998). In certain hematopoietic cell lines, neither BCL-2/BAX nor p53 appeared to be affected (Parker et al., 1998; Shapiro et al., 1999b), whereas, in other systems, BCL-2 may be inhibited (Konig et al., 1997). Preliminary evidence from one laboratory demonstrated that flavopiridol-induced apoptosis in leukemia cells is associated with early activation of the MAPK protein kinase family of proteins (MEK, p38 and JNK) (Lahusen et al., 2000). This activation may lead to the activation of caspases (Lahusen et al., 2000). As seen in this and other models, caspase inhibitors prevent flavopiridol-induced apoptosis (Byrd et al., 1998; Lahusen et al., 2000). It is unclear whether the putative flavopiridol-induced inhibition of cdk activity is required for induction of apoptosis.

Clear evidence of cell cycle arrest along with apoptosis was observed in a panel of squamous head and neck cancer cell lines, including a cell line (HN30) that is refractory to several DNA-damaging agents, such as -irradiation and bleomycin (Patel et al., 1998). Again, the apoptotic effect was independent of p53 status and was associated with the depletion of cyclin D1 (Patel et al., 1998). These findings have been corroborated in other preclinical models (Bible and Kaufmann, 1996; Schrump et al., 1998; Chien et al., 1999; Shapiro et al., 1999b). Efforts to understand flavopiridol-induced apoptosis are under intense investigation.

Flavopiridol targets not only tumor cells but also angiogenesis pathways. Brusselbach et al. (1998) incubated primary human umbilical vein endothelial cells (HUVECs) with flavopiridol and observed apoptotic cell death even in cells that were not cycling, leading to the notion that flavopiridol may have antiangiogenic properties due to endothelial cytotoxicity. In other model systems, Kerr et al. (1999) tested flavopiridol in an in vivo Matrigel model of angiogenesis and found that flavopiridol decreased blood vessel formation, a surrogate marker for the antiangiogenic effect of this compound. Furthermore, as mentioned earlier, Melillo et al. (1999) demonstrated that, at low nanomolar concentrations, flavopiridol prevented the induction of vascular endothelial growth factor (VEGF) by hypoxic conditions 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 observed may be in part due to antiangiogenic effects. Whether the various antiangiogenic actions of flavopiridol result from its interaction with a cdk target or other targets requires further study.

The antitumor effect observed with flavopiridol can also be explained by activation of differentiation pathways. It became clear recently that cells become differentiated when exit of the cell cycle (G₀) and loss of cdk2 activity occur. Based on this information, Lee et al. (1999) tested flavopiridol and roscovitine, both known cdk2 inhibitors, to determine if they induce a differentiated phenotype. For this purpose, NCI-H358 lung carcinoma cell lines were exposed to cdk2 antisense construct, flavopiridol, or roscovitine. Clear evidence of mucinous differentiation along with loss in cdk2 activity was observed in this lung carcinoma model. Thus, it is plausible that the antitumor effect of flavopiridol in lung carcinoma models may be due to induction of differentiation, among others (Lee et al., 1999).

Several investigators have attempted to determine if flavopiridol has synergistic effects with standard chemotherapeutic agents. For example, synergistic effects in A549 lung carcinoma cells were demonstrated when treatment with flavopiridol followed treatment with paclitaxel, cytarabine, topotecan, doxorubicin, or etoposide (Bible and Kaufmann, 1997; Schwartz et al., 1997). In contrast, a synergistic effect was observed with 5-fluorouracil only when cells were treated with flavopiridol for 24 h before the addition of 5-fluorouracil. Furthermore, synergistic effects with cisplatin were not schedule dependent (Bible and Kaufmann, 1997). However, Chien et al. (1999) failed to demonstrate a synergistic effect between flavopiridol and cisplatin and/or -irradiation in bladder carcinoma models. One important issue to mention is that most of these studies were performed in in vitro models. Thus, confirmatory studies in in vivo animal models are needed.

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 (Sedlacek et al., 1996; Drees et al., 1997). These demonstrations led to human clinical trials of flavopiridol administered as a 72-h continuous infusion every 2 weeks (Senderowicz et al., 1998b) (see below). Subsequent studies in 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 (Arguello et al., 1998). Moreover, in HN-12 head and neck cancer xenografts flavopiridol administered intraperitoneally for 5 days demonstrated a substantial growth delay (Patel et al., 1998). Again, apoptotic cell death and cyclin D1 depletion were observed in tissues from xenografts treated with flavopiridol (Arguello et al., 1998). Based on these results, a phase I trial of 1 h daily infusional flavopiridol every 3 weeks has been conducted at the NCI (Tan et al., 2002).

Clinical experience with flavopiridol

Two phase I clinical trials of flavopiridol administered as a 72-h continuous infusion every 2 weeks have been completed (Senderowicz et al., 1998b; Thomas et al., 2002). In the NCI phase I trial (N=76) of infusional flavopiridol, dose-limiting toxicity (DLT) was secretory diarrhea with a maximal-tolerated dose (MTD) of 50 mg/m²/day for 3 days. In the presence of antidiarrheal prophylaxis (a combination of cholestyramine and loperamide), patients tolerated higher doses, defining a second MTD, 78 mg/m²/day for 3 days. The DLT observed at the higher dose level was a substantial proinflammatory syndrome (fever, fatigue, local tumor pain, and modulation of acute-phase reactants) and reversible hypotension (Senderowicz et al., 1998b). Minor responses were observed in patients with non-Hodgkin's lymphoma, colon, and kidney cancer for more than 6 months. Moreover, one patient with refractory renal cancer achieved a partial response for more than 8 months (Senderowicz et al., 1998b). 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 (Senderowicz et al., 1998b). Plasma concentrations of 300–500 nM flavopiridol, which inhibit cdk activity in vitro, were safely achieved during this trial (Senderowicz et al., 1998b).

In a complementary phase I trial also exploring the same schedule (72-h continuous infusion every 2 weeks), Thomas et al (2002) found that the DLT was diarrhea, corroborating the NCI experience. Moreover, plasma concentrations of 300–500 nM flavopiridol were also observed. Interestingly, there was one patient in this trial with refractory metastatic gastric cancer that progressed after a treatment regimen containing 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.

The first phase I trial of a daily 1-h infusion of flavopiridol for 5 consecutive days every 3 weeks was recently completed (Senderowicz et al., 1998b; Tan et al., 2002). This schedule was based on antitumor results observed in leukemia/lymphoma and head and neck cancer xenografts treated with flavopiridol (Arguello et al., 1998; Patel et al., 1998). A total of 55 patients were treated in this trial. The recommended phase II dose is 37.5 mg/m²/day for 5 consecutive days. DLTs observed at 52.5 mg/m²/day are nausea/vomiting, neutropenia, fatigue, and diarrhea (Senderowicz et al., 1998b; Tan et al., 2002). Other (nondose-limiting) side effects are local tumor pain and anorexia. To reach higher flavopiridol concentrations, the protocol was amended to administer flavopiridol for 3 days and then for 1 day only. With these protocol modifications, we were able to achieve concentrations (4 M) necessary to induce apoptosis in xenograft models (Arguello et al., 1998; Patel et al., 1998; Senderowicz et al., 2000; Tan et al., 2002) Of note, the half-life observed in this trial is much shorter (3 h) than the infusional trial (10 h). Thus, the high micromolar concentrations achieved in the 1-h infusional trial could be maintained only for short periods of time (Senderowicz et al., 2000; Tan et al., 2002). Several phase II trials in patients with refractory head and neck cancer, chronic lymphocytic leukemia (CLL), and mantle cell lymphoma (MCL) are currently being tested using this schedule (see below). A phase I trial testing the combination of paclitaxel and infusional (24 h) flavopiridol demonstrated good tolerability with a dose-limiting pulmonary toxicity (Schwartz et al., 2002).

Phase II trials of flavopiridol given as a 72-h continuous infusion with the MTD in the absence of antidiarrheal prophylaxis (50 mg/m²/day) to patients with CLL, non-small-cell lung cancer (see below), non-Hodgkin's lymphoma, and colon, prostate, gastric, head and neck, and kidney cancer, and phase I trials of flavopiridol administered on novel schedules and in combination with standard chemotherapeutic agents are being performed (Werner et al., 1998; Wright et al., 1998; Bennett et al., 1999; Shapiro et al., 1999a; Stadler et al., 2000).

In a recently published phase II trial of flavopiridol in metastatic renal cancer, two objective responses (response rate=6%, 95% confidence interval, 1–20%) were observed. Most patients developed grade 1–2 diarrhea and asthenia (Stadler et al., 2000). In this trial, patients who demonstrated glucuronide flavopiridol metabolites in plasma have less pronounced diarrhea in comparison to nonmetabolizers (Innocenti et al., 2000). Thus, it may be possible that patients with higher metabolic rates may tolerate higher doses of flavopiridol. Phase II trials of shorter (1-h) infusional flavopiridol are being conducted in MCL, CLL, and HNSCC. Of interest, several refractory CLL and MCL patients demonstrated evidence of clinical responses (partial responses) in these trials (Jose Ramon Suarez, personal communications).

Based on the interesting results obtained in several preclinical lung carcinoma models, a phase II trial of patients with metastatic lung carcinoma was performed (Shapiro et al., 2001). A total of 20 patients were treated with a 72-h continuous infusion of flavopiridol every 14 days at a dose of 50 mg/m²/day. The most common toxicities included grade 2 diarrhea, asthenia, and venous thromboses. The median overall survival for the 20 patients who received treatment was 7.5 months. Of note, the median survival of 7.5 months achieved with flavopiridol is similar to the range of 7.4–8.2 months reported recently in a randomized trial of four chemotherapy regimens containing platinum analogues in combination with taxanes or gemcitabine (Schiller et al., 2002). Moreover, a similar overall survival was obtained with gefitinib (Iressa), a recently approved EGFR inhibitor for the treatment of advanced lung cancer (Kris et al., 2002). Based on these encouraging results, a phase III trial comparing standard combination chemotherapy versus combination chemotherapy plus flavopiridol is currently under investigation.

UCN-01

Mechanism of antiproliferative activity

Staurosporine is a potent nonspecific protein and tyrosine kinase inhibitor, with a very low therapeutic index in animals (Tamaoki, 1991). Thus, efforts to find staurosporine analogues of staurosporine have identified compounds specific for protein kinases. One staurosporine analogue, UCN-01 (see Figure 3), has potent activity against several protein kinase C isoenzymes, particularly the Ca²⁺-dependent protein kinase C with an IC₅₀ 30 nM (Seynaeve et al., 1994; Takahashi et al., 1987, 1989). In addition to its effects on protein kinase C, UCN-01 has antiproliferative activity in several human tumor cell lines (Akinaga et al., 1991, 1994; Seynaeve et al., 1993; Wang et al., 1995; Akiyama et al., 1997). These effects appear not to be related to the effects of UCN-01 in PKC signaling (Wang et al., 1995).

Another interesting feature, again unrelated to PKC, is 'inappropriate activation' of cdk kinases in intact cells (Wang et al., 1995). This phenomenon correlates with the G2 abrogation checkpoint observed with this agent. Experimental evidence suggests that DNA damage leads to cell cycle arrest to allow DNA repair. In the presence of UCN-01, irradiated cells are unable to accumulate in the G₂ phase with subsequent early mitosis with the onset of apoptotic cell death (Wang et al., 1996). The accelerated mitosis is due to activation of cdc2 kinase. These activations could be partially explained by the inactivation of Wee1, the kinase that negatively regulates the G₂/M phase transition (Yu et al., 1998). Moreover, UCN-01 can have a direct effect on chk1, the protein kinase that regulates the G2 checkpoint (Sarkaria et al., 1999; Busby et al., 2000; Graves et al., 2000). 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. Studies from other groups suggest that not only is UCN-01 able to abrogate the G₂ checkpoint induced by DNA-damaging agents but also, in some circumstances, UCN-01 is able to abrogate the DNA damage-induced S phase checkpoint (Bunch and Eastman, 1997; Shao et al., 1997).

Another interesting property of UCN-01 is its ability to arrest cells in the G₁ phase of the cell cycle (Seynaeve et al., 1993; Akinaga et al., 1994; Kawakami et al., 1996; Shimizu et al., 1996; Akiyama et al., 1997; Akiyama et al., 1999; Chen et al., 1999a; Usuda et al., 2000). When human epidermoid carcinoma A431 cells (mutated p53) or HN12 head and neck carcinoma cell lines are incubated with UCN-01, these cells were arrested in the G₁ phase with Rb hypophosphorylation and p21^waf1/p27^kip1 accumulation (Akiyama et al., 1997; Patel et al., 2002a). Chen et al. (1999a) suggest that Rb, but not p53, function is essential for UCN-01-mediated G1 arrest. However, Shimizu et al. (1996) demonstrated that lung carcinoma cell lines with either absent, mutant, or wild-type Rb exposed to UCN-01 displayed G₁ arrest and antiproliferative effects irrespective of Rb function. Thus, the exact role of Rb or p53 in the G₁arrest induced by UCN-01 is still unknown. Further studies on the putative target(s) for UCN-01 in the G₁ phase arrest of cells are warranted. UCN-01 demonstrated enhanced cytotoxicity in cells with mutant p53 (Wang et al., 1996).

In CA-46 and HT-29 tumor cell lines carrying mutated p53 genes, potent cytotoxicity results following exposure to UCN-01. To extend these observations further, the MCF-7 cell line with no endogenous p53 because of the ectopic expression of E6, a human papillomavirus type-16 protein, showed enhanced cytotoxicity when treated with a DNA-damaging agent, such as cisplatin, and UCN-01, compared with the isogenic wild-type MCF-7 cell line. Thus, a common feature observed in more than 50% of human neoplasias, associated with poor outcome and refractoriness to standard chemotherapies (Marchetti et al., 1993; Lowe et al., 1994), may render tumor cells more sensitive to UCN-01.

A very exciting recent development is the discovery that UCN-01 can modulate the PI3 kinase/AKT survival pathway (Testa and Bellacosa, 2001; Sato et al., 2002). UCN-01 displays a potent inhibition in vitro of the pdk1 serine/threonine kinase, leading to dephosphorylation and inactivation of akt (Sato et al., 2002). Although this is an exciting novel feature of UCN-01, it is of utmost importance to demonstrate whether the antitumor effects of UCN-01 are mediated by this action. Moreover, demonstration that these effects also occur in in vivo settings is crucial.

As previously mentioned, synergistic effects of UCN-01 have been observed with many chemotherapeutic agents, including mitomycin C, 5-fluorouracil, carmustine, and camptothecin, among others (Akinaga et al., 1993; Bunch and Eastman, 1996; Pollack et al., 1996; Husain et al., 1997; Shao et al., 1997; Tsuchida and Urano, 1997; Hsueh et al., 1998; Jones et al., 2000; Sugiyama et al., 2000). Therefore, it is possible that combining UCN-01 with these or other agents could improve its therapeutic index. Clinical trials exploring these possibilities are currently being developed.

UCN-01 administered by an intravenous or intraperitoneal route displayed antitumor activity in xenograft model systems with breast carcinoma (MCF-7 cells), renal carcinoma (A498 cells), and leukemia (MOLT-4 and HL-60) cells (A Senderowicz, 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 the greatest antitumor activity observed when UCN-01 was present for 72 h (Seynaeve et al., 1993). Thus, a clinical trial using a 72-h continuous infusion every 2 weeks was conducted.

Clinical trials of UCN-01

The first phase I trial of UCN-01 was recently completed (Senderowicz et al., 1998a; Sausville et al., 2001). UCN-01 was initially administered as a 72-h continuous infusion every 2 weeks based on data from in vitro and xenograft preclinical models. However, it became apparent in the first few patients that the drug had an unexpectedly long half-life (30 days). This half-life was 100 times longer than the half-life observed in preclinical models, most likely due to the avid binding of UCN-01 to ₁-acid glycoprotein (Fuse et al., 1998; Sausville et al., 1998). Thus, the protocol was modified to administer UCN-01 every 4 weeks (one half-life) and in subsequent courses, the duration of infusion was decreased by half (total 36 h). Thus, it was possible to reach similar peak plasma concentrations in subsequent courses with no evidence of drug accumulation. There was no evidence of myelotoxicity or gastrointestinal toxicity (prominent side effects observed in animal models), despite very high plasma concentrations achieved (35–50 M) (Fuse et al., 1998; Sausville et al., 1998, 2001; Senderowicz et al., 1998a). DLTs were nausea/vomiting (amenable to standard antiemetic treatments), symptomatic hyperglycemia associated with an insulin-resistance state (increase in insulin and c-peptide levels while receiving UCN-01), and pulmonary toxicity characterized by substantial hypoxemia without obvious radiologic changes. The recommended phase II dose of UCN-01 given on a 72-h continuous infusion schedule was 42.5 mg/m²/day (Sausville et al., 2001). One patient with refractory metastatic melanoma developed a partial response that lasted 8 months. Another patient with refractory anaplastic large-cell lymphoma that had failed multiple chemotherapeutic regimens including high-dose chemotherapy has no evidence of disease more than 4 years after the initiation of UCN-01. Moreover, a few patients with leiomyosarcoma, non-Hodgkin's lymphoma, and lung cancer demonstrated stable disease for 6 months (Senderowicz et al., 1999; Sausville et al., 2001).

In order to estimate 'free UCN-01 concentrations' in body fluids, several efforts were considered. Plasma ultracentrifugation and salivary determination of UCN-01 revealed similar results. At the recommended phase II dose (37.5 mg/m²/day over 72 h), concentrations of 'free-salivary' UCN-01 (100 nM) that may cause G₂ checkpoint abrogation can be achieved. As mentioned earlier, UCN-01 is a potent PKC inhibitor. In order to determine the putative signaling effects of UCN-01 in tissues, bone marrow aspirates and tumor cells were obtained from patients before and during the first cycle of UCN-01 administration. Western blot studies were performed in those samples against phosphorylated adducin, a cytoskeletal membrane protein, a specific substrate phosphorylated by PKC (Fowler et al., 1998). A clear loss in phosphoadducin content in the post-treatment samples was observed in all tumor and bone marrow samples tested, concluding that UCN-01 can modulate PKC activity in tissues from patients in this trial (Senderowicz et al., 1999; Sausville et al., 2001).

Several groups are conducting shorter duration (3-h) infusional trials of UCN-01. Interestingly, the toxicity profile of shorter infusions is similar to the toxicities observed with the 72-h infusion trial (Tamura et al., 1999; Dees et al., 2000). However, with shorter infusions, more pronounced hypotension was observed (Tamura et al., 1999; Dees et al., 2000). The determination of free UCN-01 in these trials is of utmost importance as higher free concentrations for shorter periods may be more or less beneficial compared with the free concentrations observed in the 72-h infusion trial.

Based on the unique pharmacological features and anecdotal clinical evidence of synergistic effects in one patient with refractory disease (Wilson et al., 2000), several combination trials with standard chemotherapeutic agents recently commenced. A phase I/II trial of gemcitabine followed by 72-h infusional UCN-01 in CLL started at the NCI. Other studies of UCN-01 in combination with cisplatin, 5-fluorouracil, among other agents, also commenced recently.

Summary

Most human malignancies have an aberration in the Rb pathway due to 'cdk hyperactivation'. Several small-molecule cdk modulators are being discovered and tested in the clinic. The first ATP competitive cdk inhibitors tested in clinical trials, flavopiridol and UCN-01, showed promising results with evidence of antitumor activity and plasma concentrations sufficient to inhibit cdk-related functions. The best schedule to be administered, combination with standard chemotherapeutic agents, best tumor types to be targeted, and demonstration of cdk modulation from tumor samples from patients in these trials are important issues that need to be answered in order to advance these agents to the clinical arena.

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