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© 2001 American Society for Clinical Oncology Phase I Trial of 72-Hour Continuous Infusion UCN-01 in Patients With Refractory NeoplasmsFrom the Developmental Therapeutics Program Clinical Trials Unit, Medicine Branch, and Investigational Drug Branch, Cancer Therapy Evaluation Program, National Cancer Institute, Bethesda, MD; Department of Pathology, College of Medicine, University of Vermont, Burlington, VT; Department of Biochemistry and Molecular Biology, University of British Columbia, Vancouver, British Columbia, Canada; and Pharmaceutical Research Institute, Kyowa Hakko Kogyo Co, Ltd, Shizuoka, Japan. Address reprint requests to Edward A. Sausville, MD, PhD, Developmental Therapeutics Program, Division of Cancer Treatment and Diagnosis, National Cancer Institute, Executive Plaza North Building, Ste 8000, 6130 Executive Blvd, Rockville, MD 20852; email: Sausville{at}nih.gov
PURPOSE: To define the maximum tolerated dose (MTD) and dose-limiting toxicity (DLT) of the novel protein kinase inhibitor, UCN-01 (7-hydroxystaurosporine), administered as a 72-hour continuous intravenous infusion (CIV). PATIENTS AND METHODS: Forty-seven patients with refractory neoplasms received UCN-01 during this phase I trial. Total, free plasma, and salivary concentrations were determined; the latter were used to address the influence of plasma protein binding on peripheral tissue distribution. The phosphorylation state of the protein kinase C (PKC) substrate alpha-adducin and the abrogation of DNA damage checkpoint also were assessed. RESULTS: The recommended phase II dose of UCN-01 as a 72-hour CIV is 42.5 mg/m2/d for 3 days. Avid plasma protein binding of UCN-01, as measured during the trial, dictated a change in dose escalation and administration schedules. Therefore, nine patients received drug on the initial 2-week schedule, and 38 received drug on the recommended 4-week schedule. DLTs at 53 mg/m2/d for 3 days included hyperglycemia with resultant metabolic acidosis, pulmonary dysfunction, nausea, vomiting, and hypotension. Pharmacokinetic determinations at the recommended dose of 42.5 mg/m2/d for 3 days included mean total plasma concentration of 36.4 µM (terminal elimination half-life range, 447 to 1176 hours), steady-state volume of distribution of 9.3 to 14.2 L, and clearances of 0.005 to 0.033 L/h. The mean total salivary concentration was 111 nmol/L of UCN-01. One partial response was observed in a patient with melanoma, and one protracted period ( > 2.5 years) of disease stability was observed in a patient with alk-positive anaplastic large-cell lymphoma. Preliminary evidence suggests UCN-01 modulation of both PKC substrate phosphorylation and the DNA damage-related G2 checkpoint. CONCLUSION: UCN-01 can be administered safely as an initial 72-hour CIV with subsequent monthly doses administered as 36-hour infusions.
NOVEL CANCER treatment strategies are in development to target the molecular abnormalities that drive cancer cell proliferation. Signal transduction refers to the processes responsible for orchestration of the cellular response to environmental cues or stresses. Protein kinases participate in growth factors and oncogene product-dependent signal transduction pathways, and have emerged as key regulators of cell proliferation.1 Protein kinases transfer the gamma phosphate of adenosine 5c-triphosphate to protein substrates, whose function or localization is then altered. Protein kinase inhibitors conceivably could act to interrupt these signals and convey a therapeutic effect in cancers driven by protein kinase-mediated signal transduction. Staurosporine is a natural product derived from fermentation extracts of a number of bacterial species.2 Staurosporine initially was recognized as a potent inhibitor of protein kinase C (PKC), a Ca2+ and phospholipid-activated kinase. Different isoforms of PKC are activated in response to growth factors that act on receptor tyrosine kinases as well as 7-transmembrane domain receptors. Subsequent studies have revealed that staurosporine is a broad-acting kinase inhibitor with little specificity or selectivity for PKC. UCN-01 (7-hydroxystaurosporine) was then defined as a derivative of staurosporine that occurs naturally and has greater selectivity for PKC3 as well as the ability to inhibit numerous other kinases.4 PKC inhibition in UCN-01-treated cells is not critically related to growth inhibition by various criteria.5-7 These results suggest an additional target or targets as the basis for the antiproliferative action of UCN-01. UCN-01 can mediate three distinct cellular effects in vitro: cell cycle arrest, induction of apoptosis, and potentiation of DNA damage-related toxicity. Cell cycle arrest occurs in the G1 or S phase, dependent on drug concentration and synchronization of the protocol used.8-12
Studies in the A498 renal carcinoma cell xenograft with in vivo and various other models suggest that relatively frequent administration of drug would convey the best antineoplastic effects.13,14 Because certain cell types, eg, the MDA-MB-468 breast carcinoma cells, required 72 hours of drug exposure before irreversible growth inhibition occurred,8 the initial schedule for this protocol was a 72-hour infusion administered every 2 weeks. The first nine patients treated on that schedule, however, demonstrated unexpectedly high concentrations of drug, with a long terminal elimination half-life (t1/2). Because of the potential implications of that finding for the development of staurosporine analogs, the pharmacology of the initial four patients was reported in detail previously15 with demonstration of species-specific binding of UCN-01 to the alpha1-acidic glycoprotein (
Patients were eligible for entry onto the study if they were 18 years of age or older and had a histologic diagnosis of solid tumor or lymphoma, effective standard therapy was not available to them, and their Eastern Cooperative Oncology Group performance status was 0 to 2. Laboratory requirements included ALT and AST  2.5 times normal, total bilirubin less than 1.5 mg/dL, creatinine clearance > 55 mL/min, hemoglobin  9 g/dL, platelets 100,000/mm3, absolute granulocyte count 1,500/mm3, normal prothrombin time, and normal partial thromboplastin time. Patients were excluded if they had evidence of active infection or other significant medical problems including unstable or newly diagnosed angina, myocardial infarction within 6 months, class II to IV congestive heart failure, severe chronic obstructive lung disease with forced vital capacity less than 1,000 mL, uncontrolled seizures, or grade 2 or worse neuropathy. Patients who had received radiation previously to 30% or more of their bone marrow, those with greater than 50% of the liver replaced by tumor, and those who were administered corticosteroids for reasons other than physiologic replacement also were excluded. Patients had received no chemotherapy or radiation therapy for at least 4 weeks, and had recovered from toxicities associated with prior therapy. The protocol was approved by the National Cancer Institute institutional review board. All patients provided signed informed consent.
Baseline Evaluation
Drug Preparation and Administration
Study Design Once the prolonged plasma t1/2 was documented, the consent form was revised to warn patients that although limited studies had been performed with commonly used drugs in vitro and had not identified drug interactions (unpublished data), interactions with other drugs were possible. Patients were precluded from receipt of other investigational drugs for at least 2 months after administration of the last dose of UCN-01 on this protocol (approximately two half-lives), and they were warned of possible interactions with standard chemotherapy. Dose-limiting toxicity (DLT) was defined as reversible grade 3 or 4 nonhematologic toxicity (excluding alopecia, fever, nausea, and vomiting), irreversible nonhematologic toxicity of at least grade 2, grade 4 granulocytopenia, or thrombocytopenia of at least 4 days’ duration. After documentation of hyperglycemia as a conventional DLT, the protocol was modified to allow dose escalation by redefinition of evidence of ketoacidosis or nonketotic acidosis as grade 3 or 4 hyperglycemia, despite treatment of hyperglycemia with insulin. Three to six patients were entered at each dose level. If one of the first three patients had DLT, up to three additional patients were entered. If any of these patients experienced DLT, the MTD was exceeded and additional patients were entered at one dose level below. If only one of six patients had DLT, patients were entered onto the next highest dose level. The dose recommended for phase II evaluation was defined as a dose associated with no more than one of six patients who had DLT. For a patient to receive subsequent courses, it was necessary for drug-related toxicities to resolve and any laboratory abnormalities to recover to meet study entry criteria. Patients who had DLT could be retreated after recovery at the next lowest dose level. Patients could continue therapy in the absence of progressive disease or unacceptable toxicity. Intrapatient dose escalation was not allowed.
Response and Progression of Disease
Pharmacokinetic and Pharmacodynamic Studies
Selected patients at higher dose levels (first course The precise cellular target(s) responsible for the antiproliferative or proapoptotic effect of UCN-01 is a matter of current investigation, as discussed below. UCN-01 is a potent inhibitor of "classic" PKC isoforms with an IC50 against the Ca2+-dependent PKCs of approximately 30 nmol/L.18 Therefore, PKC activity represents a potential means to assess the molecular pharmacodynamics of UCN-01 action, although effects on PKC activity may not be the basis for the drug’s antiproliferative effect. The cytoskeletal protein adducin is a prominent PKC substrate. The antibody to alpha-adducin was raised against the carboxy terminus of recombinant expressed protein, and the antiphosphoadducin antibody recognizes both phospho-alpha and phospho-gamma adducins.19 At or near the MTD, selected patients with tumor specimens that could be sampled easily, as well as bone marrow aspirates (N = 4) and peripheral mononuclear cells (N = 1), were obtained for evaluation of PKC activity via assay of alpha-adducin phosphorylation state19 by Western blot. For these studies, mononuclear cells from bone marrow or effusion were isolated by Ficoll-Paque separation (Amersham Pharmacia Biotech, Piscataway, NJ). Samples were evaluated before treatment and at the end of the infusion, under separate informed consent. Briefly, 10 mL of bone marrow aspirate was mixed with 10 mL of phosphate-buffered saline, layered slowly over 10 mL of Ficoll, and centrifuged at 2,000 rpm for 30 minutes in 50-mL centrifuge tubes. The resulting mononuclear cell layer was washed with phosphate-buffered saline and stored at -70°C. Bone marrow or tumor cells were lysed in sodium phosphate buffer that contained the protease inhibitor diisopropyl fluorophosphate at a final concentration of 1 mg/mL (Sigma Chemical Co, St Louis, MO). Next, 30 µg of protein were electrophoresed and transferred to polyvinylidene fluoride membranes.20 The PKC-dependent phosphorylated epitope of alpha-adducin, a cytoskeletal protein and total alpha-adducin, was determined by the phosphorylation state-specific and nonphosphorylation state-specific antibodies against alpha-adducin.19 Assessment of plasma from treated patients to abrogate the G2 checkpoint of irradiated MCF-7 cells was conducted as described by Roberge et al21 and as detailed in the figure legends in this article.
Patient Demographics Forty-seven patients were entered onto the trial between April 1996 and March 1999. Patient characteristics are summarized in Table 1. The majority of patients were male, and the median Eastern Cooperative Oncology Group performance status was 1. Nine patients received drug on an every-other-week administration schedule, and 38 received an amended schedule, with second and subsequent courses administered at 4-week intervals at one half the initial administered first course dose (as discussed below). Two of the patients had not received previous systemic therapy.
Dose Escalation and Toxicity As listed in Table 2, the first course of the first three dose levels, including 1.8, 3.6, and 6 mg/m2/d for 3 days administered every 2 weeks, was tolerated with no DLT that was clearly and initially identified as drug-related. Table 3 demonstrates that the non-DLTs encountered at those dose levels consisted largely of nonspecific constitutional symptoms (eg, fever); the exception was reversible asymptomatic grade 3 hyperglycemia (initially considered unrelated to drug because the patients had episodic prebaseline glucose elevations) observed in one patient at the first dose level and in an additional patient who received 6 mg/m2/d for 3 days. As listed in Table 4, patients who received these first three dose levels had plasma drug concentrations above 2 µmol/L and a markedly long UCN-01 half-life (ie, hundreds of hours). This was not expected from preclinical studies in animal models. Because of the immediate implications of that finding for the study design of other staurosporine-derived molecules, accrual to the trial was halted temporarily, and the pharmacology of the initial three dose levels was studied in detail. Results from the first four patients have been reported elsewhere,13,15 with the elucidation that a species-specific high affinity binding of UCN-01 to  AG, a normal plasma component, probably accounts for the unexpected human pharmacology.
Concern regarding possible accumulation of UCN-01, if administered on an every-other-week schedule, led to modification of the protocol for initial doses 12 mg/m2/d for 3 days. Second and subsequent courses of UCN-01 at these doses were administered at 4-week rather than 2-week intervals, and the second and subsequent doses were one half of the patient’s initial dose. The rationale for this approach was based on modeling studies that predicted approximately one half of a given dose probably would be cleared during a 4-week period. Studies to be reported elsewhere confirm approximate maintenance of peak and trough levels of UCN-01 through a patient’s course of treatment (unpublished data). Using this approach, Table 2 demonstrates that through doses of 12, 17, and 24 mg/m2/d for 3 days, only one DLT was recorded, a transient episode of asymptomatic grade 3 hyperglycemia that was initially considered unrelated to the drug. Table 3 demonstrates that these levels were well tolerated with the exception of mainly grade 1 or 2 constitutional symptoms. The first serious toxicity occurred at 34 mg/m2/d for 3 days in a patient who experienced a grade 4 pulmonary adverse experience on day 3 of cycle 1. The patient described a sudden onset of nausea, fatigue, and mild shortness of breath, which was accompanied by a transient decline in hemoglobin oxygen saturation, from 98% at baseline to 89% at room air oxygen concentrations. The hypoxia was corrected within 15 minutes of administration of supplemental oxygen via nasal cannula, diphenhydramine, and single-dose corticosteroids, and within 48 hours the patient experienced grade 2 myalgia described as "aching muscles" and "cramping." During this time, serial electrocardiography revealed tachycardia without other abnormalities, and a ventilation/perfusion scan, chest x-ray, and echocardiogram did not suggest a structural cause of the episode. An additional five conventionally assessable patients were accrued to this dose level without DLT. The first three patients at 42.5 mg/m2/d for 3 days tolerated the therapy without DLT, except for one transient episode of grade 3 hyperglycemia with an uncertain relation to drug at that time. At 53 mg/m2/d for 3 days, the second patient treated experienced grade 4 hyperglycemia and grade 4 vomiting. A subsequent patient at 53 mg/m2/d for 3 days experienced only moderate nausea but grade 4 hyperglycemia; thus 53 mg/m2/d for 3 days was defined as surpassing the MTD. Additional toxicities experienced at 53 mg/m2/d for 3 days included prominent (grade 2 or 3) fever, headache, fatigue, nausea, hypotension, myalgia, anorexia, diarrhea, dyspnea, and stomatitis. Five additional patients were studied at 42.5 mg/m2/d for 3 days, and none encountered major toxicity ( Table 5). Thus, of eight patients initially treated at 42.5 mg/m2/d for 3 days, one experienced DLT, and 42.5 mg/m2/d for 3 days was defined as the recommended phase II dose for UCN-01 administered as a first course 72-hour continuous infusion.
Mechanism of Hyperglycemia and Attempt to Dose Escalate Although isolated episodes of grade 3 asymptomatic, reversible hyperglycemia were noted in three of 19 patients treated at 24 mg/m2/d for 3 days (Table 3), transient hyperglycemia of grade 2 or less was present at all dose levels. The incidence and severity increased at doses 42.5 mg/m2/d for 3 days ( Fig 1A). Hyperglycemia can reflect either insulin resistance in the peripheral tissues or primary loss of insulin output. We hypothesized that UCN-01-induced hyperglycemia might be reversed by coadministration with insulin. After modification of the informed consent and consultation with the endocrinology service, patients were treated at 53 mg/m2/d for 3 days; at the first documentation of hyperglycemia > 200 mg/dL, an insulin drip was initiated in a monitored setting, with an algorithm to administer 10% dextrose for glucose < 200 mg/dL. Figure 1B illustrates the course of a patient who received 53 mg/m2/d for 3 days and was treated expectantly with insulin at the first documentation of significant hyperglycemia. By day 2 of the 72-hour infusion, UCN-01-induced hyperglycemia was accompanied by increases in the immunoreactive C peptide derived from proinsulin. Elaboration of C peptide is a normal islet response to a glucose stress, and it is evidence that UCN-01-induced hyperglycemia arises from peripheral tissue insulin resistance. Immunoreactive C peptide was increased in 23 of 29 patients evaluated after exposure to UCN-01. Figure 1C demonstrates that UCN-01 treatment also caused increased free fatty acids and lactate in the same patient described in Fig 1B. Two additional patients were treated at 53 mg/m2/d for 3 days with insulin infusion for UCN-01-induced hyperglycemia. Despite successful management of hyperglycemia in one patient, both patients experienced grade 3 nausea or vomiting that did not respond to antiemetics. Furthermore, a subsequent patient experienced grade 3 pulmonary toxicity, grade 2 ileus, and hypotensive toxicities that reversed during a 1- to 2-day period off drug. Thus, even with careful efforts to control glucose and support nausea or vomiting, the emergence of serious pulmonary toxicity argued against the practical value of continued efforts to administer UCN-01 at or above 53 mg/m2/d for 3 days.
To confirm that 42.5 mg/m2/d for 3 days was a tolerable dose, three additional patients were accrued to that level. Two patients tolerated this initial dose with, at worst, transient grade 2 fatigue or nausea (Table 5). In the third patient, grade 3 hypoxia was experienced in the setting of bronchospasm that was attributed to an exacerbation of underlying COPD. One dose of methylprednisolone was administered, and within less than 12 hours the patient experienced grade 4 hyperglycemia as well as grade 2 nausea and vomiting. The patient’s hyperglycemia was reversed with insulin administration and hydration. Thus, of 11 patients treated at 42.5 mg/m2/d for 3 days, three UCN-01-related first course DLTs occurred, including two episodes of transient hyperglycemia of no clinical significance and one episode of transient hypoxia. In contrast, of nine patients treated at 53 mg/m2/d for 3 days, three grade 4 toxicities that involved hyperglycemia and vomiting occurred in two patients. No patient deaths were attributed to UCN-01. Overall, during 47 total courses at the dose level of 42.5 mg/m2/d for 3 days (Table 5), 18 grade 2 episodes, one grade 3 episode, and one grade 4 episode of hyperglycemia occurred (the latter instance was associated with corticosteroid administration). In addition, lactic acid was increased in six of 17 assessable patients at this dose level. Because of the reversible nature of toxicities at 42.5 mg/m2/d for 3 days, we propose this as the MTD and recommended phase II dose as a 72-hour infusion, with prophylactic treatment of nausea and vomiting.
Other Clinical Toxicities Headache without photophobia or focal neurologic signs was a frequent adverse experience from day 2 of the infusion, and it was prominent at doses > 34 mg/m2/d for 3 days. Headaches resolved 1 or 2 days after drug infusion and tended to recur with rechallenge. Asymptomatic hypotension of grade 2 or less occurred at doses of 24, 34, and 42.5 mg/m2/d for 3 days, and it was a DLT in one patient who received 53 mg/m2/d for 3 days. In general, these occurrences were characterized by a moderate decrease in baseline systolic blood pressure that resolved promptly with gentle normal saline hydration. Myalgia was experienced in 16 of 47 initial courses, and it was described characteristically as muscle aches with occasional cramps in the lower extremities that were not altered by exercise and not associated with muscle weakness. Symptoms usually subsided during the week after drug administration, although occasionally patients described persistence of these symptoms for as long as 2 weeks. Myalgia also tended to recur with retreatment and was notable after 10 of 25 courses administered on the every 2-week regimen for the first three dose levels. Grade 1 elevation of creatine phosphokinase occurred in a total of four patients during the first cycle of drug administration: one at the dose of 34 mg/m2/d for 3 days, two at 42.5 mg/m2/d for 3 days, and one at 53 mg/m2/d for 3 days.
Additional Laboratory Abnormalities
Pharmacology and Pharmacokinetics
The increase in Cpmax appears linear until approximately 24 mg/m2/d for 3 days ( Fig 2A), or total UCN-01 concentration of approximately 20 µmol/L. Thereafter, notable heterogeneity in Cpmax is observed compared with the administered dose, and the curve appears to trend to a plateau. Data to be presented in detail elsewhere suggest a positive correlation between
Unexpected tight binding to human plasma proteins raised considerable doubt as to whether UCN-01 would distribute successfully to tissues; therefore, measurement of salivary UCN-01 was undertaken to serve as a "surrogate" measure of effective free drug in the plasma. Table 4 and Fig 2B demonstrate a median salivary concentration of 111 nmol/L at the MTD of 42.5 mg/m2/d for 3 days. The mean plasma and saliva concentrations versus time curves are presented in Fig 3. As observed in these figures, the initial postinfusion half-life in saliva seems to decline rapidly and immediately after cessation of the UCN-01 infusion, and this decline is more rapid than that in plasma. After this rapid decrease, however, salivary concentrations are maintained for several weeks. The saliva concentrations measured at 4 weeks postinfusion are maintained above the assay detection limit of 10 nmol/L for patients at the UCN-01 dose of 53 mg/m2/d for 3 days; patients at the dose of 42.5 mg/m2/d for 3 days demonstrate levels only at or below the detection threshold of the assay for several weeks.
As an additional assessment of potentially "free" unbound UCN-01, plasma was ultracentrifuged according to the method outlined by Fuse et al,15 and the UCN-01 present in the supernatant was defined as "free" drug. Figure 2C demonstrates that "free" UCN-01 of 200 to 600 nmol/L was detected at the end of infusion of doses 24 mg/m2/d for 3 days. Considerable variability in the data is apparent, however, in patients treated at the same dose level.
Tumor Responses and Pharmacodynamic Effects
To determine whether normal tissue and tumor tissue from patients demonstrate an effect on cellular PKC function, we assayed the expression of phosphoadducin and unphosphorylated alpha-adducin in samples retrieved from patients treated at or above the MTD. Model experiments in vitro with Jurkat T lymphoblasts revealed that cells exposed to 300 nmol/L of UCN-01 display a rapid (approximately 1 hour) decrease in phospho-alpha-adducin content, which is sustained for up to 9 hours ( Fig 5A). Nonphosphorylated alpha-adducin expression is minimally altered for up to 15 hours, at which time decreased mass of unphosphorylated alpha-adducin also is apparent. At the same time, the expression of beta-actin is unaltered. Figure 5Bshows that before treatment, bone marrow cells from two patients and tumor cells from an effusion had detectable phosphorylated and unphosphorylated adducin. When bone marrow or tumor tissues from the same patients were obtained on day 3 of treatment, however, a decrease in phosphoadducin and unphosphorylated adducin was evident; this is similar to observations in Jurkat cells exposed to UCN-01 for up to 15 hours. Thus, effective "free" plasma concentrations may be present in patients treated with these doses of UCN-01, which may modulate intracellular signaling events.
We tested whether plasma from patients who receive UCN-01 may modulate G2 checkpoint function ex vivo. In a model experiment, irradiated MCF-7 cells were exposed to UCN-01 in medium only or in medium with a 1:10 volume of added human plasma, with the indicated final concentration of UCN-01 ( Fig 6A). When plasma samples obtained at the end of initial UCN-01 infusion in these trials were tested in this assay, clear evidence of G2 checkpoint modulation in the reporter cell line was observed (Fig 6B). Plasma from four of six patients at doses of 34 mg/m2/d for 3 days, three of three patients at 42.5 mg/m2/d for 3 days, and three of four patients at 53 mg/m2/d for 3 days demonstrated 20% or greater abrogation of the G2 checkpoint. The greatest degree of G2 checkpoint abrogation was observed at the dose of 42.5 mg/m2/d, although the differences among the 34, 42.5, and 53 mg/m2/d doses were not statistically significant. Despite the presence of human plasma protein and evidence of PKC modulation in vivo, the observations of G2 checkpoint abrogation from patient samples reinforce the notion that despite plasma protein binding, sufficient free UCN-01 to promote biologically relevant end points can potentially be achieved at the recommended phase II dose of 42.5 mg/m2/d for 3 days.
The recommended phase II dose of UCN-01 administered as a 72-hour continuous infusion is 42.5 mg/m2/d for 3 days. Subsequent courses are administered at 4-week intervals during a 36-hour period (the second dose is one half by mass the first dose). DLTs experienced at the next highest dose level (53 mg/m2/d for 3 days) included hyperglycemia, nausea and vomiting, and pulmonary dysfunction characterized as hypoxia without infiltrate but with transient small pleural effusions. End infusion drug concentrations at the recommended phase II dose were approximately 35 µmol/L (total) and 400 nmol/L (free measured by ultracentrifugation), with a prolonged t1/2 of 618 hours. An average salivary drug concentration of 111 nmol/L at 42.5 mg/m2/d for 3 days suggested that despite plasma protein binding, drug was distributed to peripheral tissues. Initial evidence was obtained that sufficient drug concentrations could be achieved in surrogate or tumor tissues to modulate the phosphorylation state of adducin, a PKC substrate, and that plasma from treated patients could modulate the G2 checkpoint in an irradiated reporter cell line.
Preclinical studies in rats and dogs indicated that expected toxicities might include local injection site irritation, reversible bone marrow depression, and gastrointestinal toxicity. Because of the radically different human pharmacology that results from
The basis for pulmonary toxicity is less clear. No consistent anatomic findings (infiltrate, atelectasis, or altered ventilation/perfusion [V/Q] scans) correlated with this finding, with the exception of small transient pleural effusions with volume that could not account for the degree of hypoxia observed. Altered cardiac motility or pulmonary thrombosis was not demonstrated. Perhaps UCN-01 alters V/Q ratios and allows a functional right-to-left shunt to emerge. It is noteworthy that staurosporine has been reported to cause induction of nitric oxide synthase in vascular smooth muscle cells. This reflects an actual increase in inducible nitric oxide synthase gene expression, with evidence of increased nitric oxide accumulation in the medium.24 This effect has been proposed to result from staurosporine-induced activation of the C/EBP family of transcription factors. Endogenous, locally generated nitrate in the pulmonary circulation may have an increased propensity to arise because of the administration of the drug through a central venous catheter, so the hypoxia might then extend from altered pulmonary vascular or microvascular tone and cause altered V/Q balance. It is remarkable in this regard that a nondose-limiting but notable side effect of UCN-01 is headache, which was virtually universal at doses These adverse effects probably reflect interference of UCN-01 with cellular signaling systems not directly relatable to cell proliferation, and they emphasize a limitation of UCN-01. Although UCN-01 is highly active against PKC isoforms alpha, beta, and gamma, with less activity against PKCs delta and epsilon,18 its antiproliferative activity seems dissociated from its effects on PKC.5,6 Akinaga et al25 have emphasized its capacity to cause G1 arrest in cells that express the Rb tumor suppressor protein. This cell cycle arrest has occurred with decreased pRb-kinase activity, which suggests an effect of the drug on the activity of the cyclin-dependent kinase (CDK) family of cell cycle regulatory enzymes. In vitro studies with adenosine 5'triphosphate concentrations sufficiently high to begin to mimic the cellular environment, however, do not suggest that CDK1 or 2 is potently and directly inhibited by UCN-015, although it is apparent that the proper activating phosphorylations on threonine 160 (in CDK2) seem to be lost. This implies a regulation of kinases "upstream" of the CDKs to explain UCN-01’s capacity to block cell cycle progression, or its effects that result in increased endogenous inhibitors, eg, p21WAF1/CIP1. It is apparent that the "free" UCN-01 plasma concentrations attained at well-tolerated doses (estimated after ultracentrifugation of plasma) (Fig 2C) approach, but do not always reach the concentrations necessary for persistent inhibition of cell cycle progression in different cell types.25 Certain purine-like molecules or their derivatives are known to abrogate the G2 cell cycle checkpoint that is initiated in response to DNA damage or other stresses. This checkpoint may also operate in non-DNA-damaged cells to ensure that all nuclear DNA is replicated before commencement of the G2 to M transition. A striking early observation was that UCN-01 seemed to share this property, but at concentrations 1/10,000 of those at which purines or methylxanthines are effective.26,27 Recent studies in yeast and in mammalian cells have demonstrated that the human homologs of the cell cycle checkpoint kinases, chk128,29 and chk2,30 may be responsible for DNA damage-dependent cell cycle arrest and perhaps participate in "normal" cell cycle regulation by inhibiting the state of activation of the cdc25C phosphatase. This would maintain CDKs in their inactive tyrosine-phosphorylated state. An early effect of UCN-01 in Jurkat cells is decreased tyrosine phosphorylation of CDK1 and 2.5 Thus, there are two attractive potential targets for UCN-01 action: CDK regulatory kinases that act upstream of CDKs, or components of the chk1 pathway that coordinate cellular response to DNA-damaging agent action and whose participation in the "normal" cellular economy is currently an intense focus of investigation. Concordant with this view, recent studies have demonstrated that UCN-01 action causes the inactivation of wee1Hu kinase, with activation of cdc25C phosphatase,31 and it potently inhibits chk1 but not chk2.32-34 The median salivary concentration achieved at the completion of the recommended phase II dose of UCN-01 (42 mg/m2/d for 3 days) is 111 nmol/L (range, 60 to 250 nmol/L). It is apparent that these concentrations are well within a range expected to effect G2 checkpoint abrogation. This data might be construed to argue in favor of UCN-01 development as a potential modulator of chemotherapy or radiation therapy.
UCN-01 is unique in the tightness of its binding to
The optimal durations of UCN-01 exposure to accomplish its three potentially therapeutic effects, namely cell cycle arrest, induction of apoptosis, or sensitization to DNA-damaging agents, have not been defined with precision. They could range from as short as 10 to 12 hours (induction of apoptosis in T lymphoblasts)5 to approximately 72 hours (irreversible inhibition of cell growth in the MDA-MB 468).8 The desired schedule must achieve concentrations that might lead to one or all of the above effects. That salivary UCN-01 concentration decreases during a 24-hour period after cessation of infusion, in association with resolution of toxicities such as headache, nausea, and hyperglycemia (Fig 3) is noteworthy. This supports the distribution of at least some level of free drug to peripheral tissues for the period of the continuous infusion and shortly thereafter, and it might further imply that distribution from
PKC412 (N-benzoyl-staurosporine) is the only other staurosporine inhibitor of protein kinases that has entered a clinical trial. PKC412 was studied on an oral, once daily administration schedule, and it did not have a conventional MTD established because of the number of capsules considered acceptable for dosing. By 225 mg/d, nausea, vomiting, and fatigue were prominent toxicities.36,37 Of interest, that agent also displayed complex pharmacology because of binding to UCN-01 has demonstrated preliminary evidence of ability to affect biologic endpoints. Specifically, in three patients whose bone marrow or tumor was studied (Fig 5), evidence of decrease in the phosphorylation of alpha-adducin, a PKC substrate, was demonstrated. Plasma from UCN-01-treated patients was able to abrogate the G2 checkpoint in an irradiated reporter cell line (Fig 6). Although these results are preliminary and require more widespread confirmation from patients treated on this and other regimens, they suggest that at well-tolerated dose levels, some modulation of PKC signaling and DNA damage-directed checkpoint function may be possible with this regimen of UCN-01.
An alternative view is that analogs of UCN-01, and indeed of PKC412, which lack or have greatly attenuated
1. Hunter T: 1001 protein kinases redux: Towards 2000. Semin Cell Biol 5: 367-376, 1994[Medline] 2. Furusaki A, Hashiba N, Matsumoto T, et al: The crystal and molecular structure of staurosporine, a new alkaloid from a streptomyces strain. Bull Chem Soc Japan 55: 3681-3685, 1982 3. Takahashi I, Saitoh Y, Yoshia M, et al. UCN-01 and UCN-02, new selective inhibitors of protein kinase C: II. Purification, physico-chemical properties, structural determination and biological activities. J Antibiot (Tokyo) 42:571-576, 1989 4. Tamaoki T, Nakano M: Potent and specific inhibitors of protein kinase C of microbial origin. Biotechnology (NY) 8: 732-735, 1990[Medline] 5. Wang Q, Worland PJ, Clark JL, et al: Apoptosis in 7-hydroxystaurosporine treated T-lymphoblasts correlates with activation of cyclin-dependent kinases 1 and 2. Cell Growth Differ 6: 927-936, 1995[Abstract] 6. Courage C, Snowden R, Gescher A: Differential effects of staurosporine analogues on cell cycle, growth and viability in A549 cells. Br J Cancer 74: 1199-1205, 1996[Medline]
7.
Shao RG, Cao CX, Pommier Y: Activation of PKC
8.
Seynaeve CM, Stetler-Stevenson MA, Sebers S, et al: Cell cycle arrest and growth inhibition by the protein kinase antagonist UCN-01 in human breast carcinoma cells. Cancer Res 53: 2081-2086, 1993 9. Akinaga S, Nomura K, Gomi K, et al. Effect of UCN-01, a selective inhibitor of protein kinase C, on the cell cycle distribution of human epidermoid carcinoma, A431 cells. Cancer Chemother Pharmacol 33:273-280, 1994
10.
Akiyama T, Yoshida T, Tsujita T, et al: G1 phase accumulation induced by UCN-01 is associated with dephosphorylation of Rb and CDK2 proteins as well as induction of CDK inhibitor p21/Cipl/WAF1/Sdi1 in p53-mutated human epidermoid carcinoma A431 cells. Cancer Res 57: 1495-1501, 1997 11. Akiyama T, Shimizu M, Okabe M, et al: Differential effects of UCN-01, staurosporine and CGP 41251 on cell cycle progression and CDC2/cyclin B1 regulation in A431 cells synchronized at M phase by nocodazole. Anticancer Drugs 10: 67-78, 1999[Medline] 12. Chen X, Lowe M, Keyomarsi K: UCN-01-mediated G1 arrest in normal but not tumor breast cells is pRb-dependent and p53 independent. Oncogene 18: 5691-5702, 1999[Medline] 13. Sausville EA, Lush RD, Headlee D, et al: Clinical pharmacology of UCN-01: Initial observation and comparison to preclinical models. Cancer Chemother Pharmacol 42: S54-S59, 1998 (suppl)
14.
Akinaga S, Gomi K, Morimoto M, et al: Antitumor activity of UCN-01, a selective inhibition of protein kinase C, in murine and human tumor models. Cancer Res 51: 4888-4892, 1991
15.
Fuse E, Tanii H, Kurata N, et al: Unpredicted clinical pharmacology of UCN-01 caused by specific binding to human alpha1-acid glycoprotein. Cancer Res 58: 3248-3253, 1998 16. Wittes RE: Manual of Oncologic Therapeutics. Philadelphia, PA, Lippincott, 1991 17. Bauer KS, Lush RM, Rudek MA, et al: A high-performance liquid chromatography method using ultraviolet and fluorescence detection for the quantitation of UCN-01, 7-hydroxystaurosporine, from human plasma and saliva. Biomed Chromatogr 14: 338-343, 2000[Medline] 18. Seynaeve CM, Kazanietz M, Blumberg P, et al: Differential inhibition of protein kinase C isozymes by UCN-01, a staurosporine analogue. Mol Pharmacol 45: 1207-1214, 1994[Abstract] 19. Fowler L, Everitt J, Stevens JL, et al: Redistribution and enhanced protein kinase C-mediated phosphorylation of alpha and gamma-adducin during renal tumor progression. Cell Growth Differ 9: 405-413, 1998[Abstract]
20.
Carlson B, Lahusen T, Singh S, et al: Downregulation of cyclin D1 by transcriptional regression in MCF-7 human breast carcinoma cells induced by flavopiridol. Cancer Res 59: 4634-4641, 1999
21.
Roberge M, Berlinck RGS, Xu L, et al: High-throughput assay for G2 checkpoint inhibitors and identification of the structurally novel compound isogranulatimide. Cancer Res 58: 5701-5706, 1998 22. Rondinone CM, Zarnowski MJ, Londos C, et al. The inhibitory effect of staurosporine on insulin action is prevented by okadaic acid: Evidence for an important role of serial threonine phosphorylation in eliciting insulin like effects. Biochim Biophys Acta 1314:49-56, 1996 23. Nishimura H, Simpson IA: Staurosporine inhibits phorbol 12-myristate 13-acetate and insulin-stimulated translocation of GLUT1 and GLUT4 glucose transporters in rat adipose cells. Biochem J 302: 271-277, 1994 24. Hecker M, Preiss B, Schini-Kerth VB: Induction by staurosporine of nitric oxide synthase expression in vascular smooth muscle cells: Role of NEKB, CRED, and C/EPBß. Br J Pharm 120: 1067-1074, 1997[Medline] 25. Akinaga S, Sugiyama K, Akiyama T: UCN-01 (7-hydroxystaurosporine) and other indolocarbazole compounds: A new generation of anticancer agents for the new century? Anticancer Drug Des 15: 43-52, 2000[Medline]
26.
Wang Q, Fan S, Eastman A, et al: UCN-01: a potent abrogator of G2 checkpoint function in cancer cells with disrupted p53. J Natl Cancer Inst 88: 956-965, 1996 27. Bunch RT, Eastman A: Enhancement of cisplatin-induced cytotoxicity by 7-hydroxy- staurosporine (UCN-01), a new G2-checkpoint inhibitor. Clin Cancer Res 2: 791-797, 1996[Abstract]
28.
Sanchez Y, Wong C, Thoma RS: Conservation of the Chk1 checkpoint pathway in mammals: Linkage of DNA damage to Cdk regulation through Cdc25. Science 277: 1497-1531, 1997 29. Kaneko Y, Watanabe N, Morisaki H, et al: Cell-cycle-dependent and ATM-independent expression of human Chk1 kinase. Oncogene 18: 3673-3681, 1999[Medline]
30.
Tominaga K, Morisaki H, Kaneko Y, et al: Role of human Cds1 (Chk2) kinase in DNA damage checkpoint and its regulation by p53. J Biol Chem 274: 31463-31467, 1999
31.
Yu L, Orlandi L, Wang P, et al: UCN-01 abrogates G2 arrest though a Cdc2-dependent pathway that is associated with inactivation of the Wee1Hu kinase and activation of the Cdc25C phosphatase. J Biol Chem 273: 33455-33464, 1998
32.
Sarkaria JN, Busby EC, Tibbetts RS, et al: Inhibition of ATM and ATR kinase activities by the radiosensitizing agent, caffeine. Cancer Res 59: 4375-4382, 1999
33.
Graves PR, Yu L, Schwartz JK, et al: The Chk1 protein kinase and the Cdc25C regulatory pathway are targets of the anticancer agent UCN-01. J Biol Chem 275: 5600-5605, 2000
34.
Busby EC, Leistritz DF, Abraham RT, et al: The radiosensitizing agent 7-hydroxystaurosporine (UCN-01) inhibits the DNA damage checkpoint kinase hChk1. Cancer Res 60: 2108-2112, 2000 35. Kremer JA, Wilting J, Janssen LHM: Drug binding to human alpha-1-acid glycoprotein in health and disease. Pharmacol Rev 40: 1-47, 1998[Medline] 36. MacDonald AC, Propper D, King D, et al: Phase I and pharmacokinetic study of CGP 41251, an inhibitor of PKC. Proc Am Soc Clin Oncol 16: 212a, 1997 (abstr 742) 37. Fabbro D, Ruetz S, Bodis S, et al: PKC412: A protein kinase inhibitor with a broad therapeutic potential. Anticancer Drug Des 15: 17-28, 2000[Medline] 38. Virchis AE, Ganeshagura K, Fletcher L, et al: Updated results of an exploratory phase II study on a protein kinase C inhibition in low grade lymphoproliferative disorders. Blood 92: 284b, 1998 (abstr 4226)
39.
Thavasu P, Propper D, McDonald A, et al: The protein kinase C inhibitor CGP412151 suppresses cytokine release and extracellular signal-related kinase 2 expression in cancer patients. Cancer Res 59: 3980-3984, 1999 40. Liang C, Sun L, Tran N, et al: Discovery and design of angiogenesis inhibitors that inhibit tyrosine kinase activities associated with VEGF, FGF, and PDGF receptors. Proc Am Assoc Cancer Res 40: 452, 1999 (abstr) Submitted July 27, 2000; accepted January 16, 2001. This article has been cited by other articles:
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ABSTRACT
INTRODUCTION
, and the fitted average (—) is contrasted with the expected increase with linear pharmacology (---). (B) Salivary UCN-01 concentration at the indicated dose. The median value is the center line; the 25th and 75th percentile ranges are within the box and 10th and 90th percentiles are within the error bars. (C) "Free" UCN-01 concentrations at end infusion were estimated by ultracentrifugation, as described in Patients and Methods.
). The percentage of mitotic cells was assessed by use of the antibody described by Roberge et al
109. In comparison, although many drugs associate with 
