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A Phase I and Pharmacokinetic Study of Short Infusions of UCN-01 in Patients with Refractory Solid Tumors

Divisions of ¹ Medical Oncology and ² Experimental Therapeutics, Sidney Kimmel Comprehensive Cancer Center at Johns Hopkins, Baltimore, Maryland and ³ Walter Reed Army Medical Center, Washington, District of Columbia

Requests for reprints: Ross C. Donehower, Sidney Kimmel Comprehensive Cancer Center at Johns Hopkins, 1650 Orleans Street, Room 187, Baltimore, MD 21231. Phone: 410-955-8838; Fax: 410-955-0125; E-mail: rdonehow{at}jhmi.edu.

	ABSTRACT

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Purpose: To define the maximum tolerated dose and the dose-limiting toxicity of the kinase modulator UCN-01 administered as a short (1-3 hours) infusion to patients with refractory solid tumors and to evaluate the pharmacokinetics of this novel agent.

Experimental Design: Twenty-four patients (15 men, 9 women; median age, 59 years; Eastern Cooperative Oncology Group Performance Status, 0-2) were treated with UCN-01 in this phase I study. Using an accelerated titration design, six dose levels were evaluated ranging from 3 mg/m² over 3 hours to 95 mg/m² over 1 to 3 hours administered every 28 days. Plasma, urine, and saliva samples were collected for pharmacokinetic analysis.

Results: Seventy courses were evaluable for toxicity. The most frequent adverse events were grade 1 to 2 nausea, vomiting, hyperglycemia, and hypotension. Hypotension was dose limiting at 95 mg/m² when UCN-01 was administered over 1 hour. The recommended dose of UCN-01 as a short infusion is 95 mg/m² over 3 hours for the first course and 47.5 mg/m² over 3 hours for second and subsequent courses. No objective responses were observed. Mean (SD) pharmacokinetic variable values in nine patients treated at 95 mg/m² over 3 hours were volume of distribution at steady state, 14 (5.4) L; ß half-life, 406 (151) hours; systemic clearance, 0.028 (0.017) L/h; C_max, 51 (16) µmol/L; and area under the curve, 19,732 (12,195) µmol/L h.

Conclusions: UCN-01 is well tolerated when given at doses of 95 mg/m² over 3 hours every 28 days with second and subsequent courses given at 50 % of the first course dose.

Key Words: UCN-01 • antineoplastic agent • toxicity • pharmacokinetics • kinase modulator

	INTRODUCTION

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Over 50 % of patients who develop cancer will die of this disease despite numerous advances in the development of cytotoxic chemotherapy over the past 50 years. Recently, a better understanding of the mechanisms of cell cycle control, apoptosis, and drug resistance has identified new targets for cancer chemotherapy and led to the development of new classes of agents. UCN-01 (Fig. 1) is a staurosporine analogue isolated from the culture broth of Streptomyces sp. (1). UCN-01 was selected for drug development partly because its pattern of activity against a National Cancer Institute (Bethesda, MD) screen of 60 human cancer cell lines was unlike that of other classes of drugs already in use (2). Unique patterns of differential activity in this cell line panel have been used to select compounds with novel mechanisms of antineoplastic effect (3).

View larger version (17K): [in this window] [in a new window]   Fig. 1 UCN-01 (7-hydroxystaurosporine).

Work from several groups supports the hypothesis that UCN-01 promotes its antitumor activity through induction of apoptosis by either modulation of cyclin-dependent kinases or inhibition of cell cycle checkpoints. UCN-01 may act by abrogating the G₂ block often induced by cellular damage, thus causing inappropriate progression to G₂ and subsequently apoptosis (7, 8). It has been shown to inhibit chk1 and, at much higher concentrations, chk2 (9). In human leukemic cell lines, UCN-01-induced apoptosis is associated with a decrease in tyrosine phosphorylation of cdk1 and cdk2 and premature or inappropriate activation of this family (10). This mode of cytotoxicity in UCN-01 seems directly related to cdc2 activation, as the effect can be reversed with inactivation of this enzyme by heat in cdc2 temperature-sensitive FT210 cell lines (11). UCN-01 has also been shown to abrogate the S-phase arrest induced by DNA-damaging agents. It may abrogate the S-phase arrest by translocating proliferating cell nuclear antigen, a sliding clamp for DNA polymerase. In damaged cells, sequestration of this proliferating cell nuclear antigen (via p21) is required for p53-dependent G₁ arrest (12). Finally, it has been shown that caspases and serine proteases are activated during (and necessary for) UCN-01-induced apoptosis (13).

Interestingly, UCN-01 may also act by creating a block at the G₁-S transition of the cell cycle and thereby arresting cell growth. UCN-01 has been shown to act by developing a G₁-S block in MDA-MB-468 cells (14). This G₁ arrest may be dependent on Rb (retinoblastoma gene), which plays a role in the G₁-S transition (15), although this remains controversial. For example, in lung cancer cell lines, UCN-01 decreased Rb phosphorylation is associated with inhibition of G₁-S transition (16). In epidermoid cancer cell lines, G₁ accumulation induced by UCN-01 was associated with dephosphorylation of Rb and cdk2 and induction of p21 (17). However, others have shown UCN-01-mediated G₁ arrest and antiproliferative effect independent of Rb function (18).

Although it is not yet clear where in the cell cycle UCN-01 acts to inhibit tumor growth or whether its effect is Rb dependent, it has been shown that UCN-01 activity may be p53 independent (19). Data from three experiments with p53-null and genetically matched p53 wild-type mice show equal cytotoxicity and induction of cell cycle–independent apoptosis following 24-hour exposure to UCN-01 (19). In a breast cancer cell line with defective p53 function, UCN-01 markedly enhanced the cytotoxicity of cisplatin (7). UCN-01 has also been shown to potentiate camptothecin-induced cytotoxicity in three different p53-deficient cell lines more than in three comparable wild-type cell lines (8). p53 tumor suppressor gene mutations confer both a poor response to most chemotherapeutics and an inferior survival. Early identification and use of therapies that abrogate in vivo resistance associated with p53 mutations would potentially allow targeted molecular therapy toward these poor risk patients.

There is extensive preclinical evidence supporting the antitumor activity of UCN-01 alone and in combination with other chemotherapeutics. UCN-01 has been shown to have antitumor activity in a variety of preclinical models, including breast, ovarian, lung, and kidney cancer cell lines and breast, lung, fibrosarcoma, and epidermoid carcinoma xenografts (3, 14, 20–26). UCN-01 is synergistic with thiotepa, mitomycin, cisplatin, melphalan, topotecan, gemcitabine, fludarabine, 5-fluorouracil, and radiation therapy in preclinical models.

A phase I study of UCN-01 administered as a 72-hour continuous infusion has been reported previously (27). Pharmacokinetic data showed that UCN-01 has a prolonged half-life (>400 hours). This is thought to be due to species-specific high-affinity binding to plasma proteins, such as ₁-acid glycoprotein (AAG). Therefore, shorter durations of infusion administered every 28 days with subsequent doses given at 50 % the first course dose may be optimal.

The present trial was designed to evaluate UCN-01 administered over 1 to 3 hours every 28 days. The study objectives were to define the maximum tolerated dose of UCN-01 on this schedule and to recommend a dose for further clinical evaluation. Additionally, this study was designed to describe the toxicities and pharmacokinetics of UCN-01 on this schedule and look for preliminary evidence of antineoplastic activity.

	PATIENTS AND METHODS

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Patient Eligibility. Only patients with a solid tumor malignancy refractory to conventional therapy or for whom no standard therapy existed were candidates for this study. Eligibility criteria included the following: (a) age, >18 years; (b) Eastern Cooperative Oncology Group performance status, <2 (ambulatory and capable of self-care); (c) a life expectancy of at least 12 weeks; (d) no major surgery, radiotherapy, or chemotherapy within 28 days of study entry (or 6 weeks if treatment included a nitrosurea or mitomycin C); (e) adequate hematopoietic (hemoglobin, >8.0 g/dL; absolute neutrophil count, >1,500/µL; platelets, >100,000/µL), hepatic (total bilirubin,<1.2 mg/dL; transaminases, <2.5 times the upper limits of normal), and renal (creatinine, <1.5 mg/dL) functions; (f) not pregnant or nursing; and (g) no other coexisting medical problems of sufficient severity to limit full compliance with the study or which would expose them to undue risk. As UCN-01 is a vesicant, all patients were required to have a central venous access catheter. Patients were ineligible if they had active infections, autoimmune hemolytic anemia, or history of brain tumor or brain metastases or if they required anticonvulsant or antiretroviral medications because of potential drug interactions and difficulty assessing toxicity. Low-dose warfarin therapy for prophylactic management of central lines was allowed, but therapeutic anticoagulation for venous thrombosis rendered patients ineligible. All patients gave written, informed consent according to federal and institutional guidelines before treatment.

Dose Escalation and Trial Design. In this phase I study, an accelerated titration design modeled after that of Simon et al. (28) was employed. In simulations, these designs significantly reduced the number of patients who would be anticipated to have grade 0 to 1 toxicity but only modestly increased the average number of patients experiencing grade 3 or 4 toxicity, thereby decreasing the number of patients treated at ineffective doses and more quickly identifying the dose for further study. The plan therefore was that one patient would be enrolled per cohort, first shortening the infusion from 3 to 2 hours to 1 hour and then escalating the dose in 100 % increments between cohorts until grade 2 toxicity felt to be drug related was observed. The goal of the decrease in infusion time was to evaluate the safety of a short 1-hour infusion, which was thought to be more convenient for patients than 3-hour infusions. At the point that grade 2 toxicity was observed in this accelerated titration design, the plan was to expand each cohort to three patients and escalate by 40 % increments. The starting dose was 3 mg/m² based on recommendations from a phase I study of a longer infusion, which was ongoing at the time (27). The dose escalation and infusion duration schemes are displayed in Table 1. After two patients were enrolled, the dose was increased to 12 mg/m². At 48 mg/m², the number of patients per cohort was expanded to 3 and escalation slowed to 40 % increments, because toxicity in the form of dose-limiting nausea, vomiting, and pancreatitis had been observed at 68 mg/m² in the ongoing trial in Japan. At the dose level of 95 mg/m² over 1 hour, dose-limiting hypotension was observed, which was thought to be related to the rapid infusion. Therefore, the infusion duration was extended to 3 hours in a subsequent cohort of patients. Because of the long plasma half-life of UCN-01, the second and later doses for all dose levels were 50 % of the first course dose listed. No intrapatient dose escalation was permitted; however, dose reductions to the prior dose level were required in patients who experienced dose-limiting toxicity provided they had recovered from the toxicity in time for the second course.

Drug Administration. UCN-01 was supplied by the DCTD, National Cancer Institute as sterile, single-use vials each of which contained 10 mg UCN-01 lyophilized powder, 11 mg citric acid USP, 22.2 mg anhydrous dibasic sodium phosphate USP, and 200 mg lactose NF. Each 10 mg vial was reconstituted with 10 mL of water for injection USP to produce a 1 mg/mL solution of UCN-01. The intact vials were stored in the refrigerator (2-8°C). UCN-01 solutions were further diluted with either normal saline or D5W. UCN-01 was administered as a 1- to 3-hour i.v. infusion every 28 days. An inline filter with a pore size of 1.2 µm was used. Because this agent is an irritant, central venous access was required. No hematopoietic growth factors were used.

Pretreatment and Follow-up Studies. History, physical examination, performance status assessment, and routine laboratory studies were done before treatment and weekly. Routine laboratory studies included a complete blood cell count, differential WBC, electrolyte and chemistry panel, and liver function variables. Samples for determination of AAG concentrations were obtained at baseline before beginning treatment. A toxicity assessment was done weekly during course 1 and every 2 weeks thereafter. Tumor response was evaluated after every two courses using standard criteria. Patients were given the option of continuing on protocol as long as progressive disease or intolerable toxicity had not occurred. Progressive disease was defined as the appearance of new lesions or a 25 % increase in the sum of the products of the bidimensional diameters of any measurable lesions. A complete response was defined as disappearance of all disease on two measurements separated by a minimum period of 4 weeks. A partial response required a 50 % reduction in the sum of the products of the bidimensional diameters of any measurable lesions. Stable disease was defined as no significant change (including decreases <50 % or increases <25 %) in measurable or evaluable disease for at least 4 weeks and no new lesions.

Pharmacokinetic Sampling and Analytic Assay. Pharmacokinetic studies were done in all patients during cycle 1. Blood samples was collected at the following times: pretreatment; at 0.5, 1, and 2 hours during the infusion; immediately before the end of infusion; and postinfusion at 0.17, 0.5, 1, 2, 3, 4, 6, 24, 48, and 72 or 96 hours after the end of the infusion. Samples were also obtained at 1, 2, and 3 weeks after the end of the infusion and immediately before the second infusion. For patients receiving a 1- or 2-hour infusion, blood samples were collected preinfusion, at three time points during the infusion (0.25, 0.5, and 0.75 hour during the 1-hour infusion; 0.5, 1, and 1.5 hours during the 2-hour infusion), and immediately before the end of the infusion. Blood samples were collected in heparinized tubes and centrifuged at 1,000 x g for 10 minutes, and plasma was frozen at –20°C until the time of analysis. Urine samples were collected during cycle 1 pretreatment and then continuously during the time intervals 0 to 12 and 12 to 24 hours after the start of the infusion. At the time of processing, the container was mixed, the volume was recorded, and a 12 mL sample was frozen at –20°C until analysis. Starting at the 68 mg/m² dose level, 1 mL saliva was collected when possible before the end of infusion, between 5 and 7 hours and at 24 hours after the start of the infusion, and once during weeks 1, 2, and 3. The sample was frozen at –20°C until analysis.

Plasma samples were analyzed using a modification of a high-performance liquid chromatography method using an UV spectrophotometer (29). Briefly, 0.25 mL plasma was deproteinized with 0.5 mL acetonitrile. Separation was accomplished on a Luna phenyl-hexyl column, 5 µmol/L, 150 x 4.6 mm (Phenomenex) with a Phenyl Guard Pak pre-column (Phenomenex, Torrance, CA), UCN-01 eluted using a gradient profile consisting of ammonium acetate (50 mmol/L) and acetonitrile with a flow rate of 1.0 mL/min. The total run time was 20 minutes. The UV detection was set at 295 nm. UCN-01 plasma concentrations were quantitated over the range of 0.1 to 100 µmol/L. For measurement of urine and saliva concentrations, a 200 µL sample of urine and a 500 µL sample of saliva were processed according to the method described for plasma. UCN-01 was quantitated in urine and saliva over the concentration range of 0.1 to 50 µmol/L and 4 to 1000 nmol/L, respectively. Quality assurance samples were assayed with each analytic run and were within 20 % of the nominal concentration.

Pharmacokinetic Data Analysis. For 11 patients, one or more blood samples obtained at 24 hours and/or later were obtained improperly and were not usable for analysis. All samples from one patient were incorrectly collected and were not usable. After excluding these samples, an iterative two-stage approach was implemented to estimate individual pharmacokinetic variables for all patients. First, a two-compartmental linear model was fit to UCN-01 plasma concentrations from 12 patients with complete concentration data using weighed least-squares regression. Values for the mean and SD for each variable estimated by weighed least-squares regression were then used to establish Bayesian priors for the structural model variables, which included V_c, K₁₂, K₂₁, and K_e. An iterative two-stage approach was used, in each iteration updating the Bayesian priors, until the mean estimates of all variables differed by <5 % from the previous mean estimate, which was our arbitrarily predefined stopping point. In the final model fit, data from 23 patients, including 11 with incomplete observations (11-15 observations), were analyzed using a Bayesian algorithm to estimate individual pharmacokinetic variables. Calculated secondary pharmacokinetic variables included V_ss, t, t;ß, and Cl_s. Area under the curve (AUC) was calculated as dose divided by Cl_s. C_max values were obtained from the model-estimated plasma concentration at the end of the drug infusion.

Statistical Analysis. Univariate linear correlation analysis was used to assess the relationship between body surface area (BSA) and AAG concentrations and UCN-01 exposure variables (C_max and AUC). ANOVA was used to determine the association among patient demographics, baseline AAG serum concentrations before cycle 1, and UCN-01 exposure and worst grade of hypotension and hyperglycemia during course 1. The method of Tukey-Kramer was used to adjust for multiple comparisons of mean values. The association between categorical variables was assessed using Fisher's exact test. Statistical analysis was done using JMP Statistical Discovery Software version 3.2.6 (SAS Institute, Cary, NC). The a priori level of significance was P < 0.05.

	RESULTS

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Patient Characteristics. Twenty-four patients received 70 courses of therapy through six dose levels as shown in Table 1. The median number of courses per patient was 2 (range, 1-24). Patient characteristics are listed in Table 2.

In an ongoing study in Japan, dose-limiting hypotension was observed in a patient who received 120 mg/m² over 3 hours. This patient required pressor support for 3 days but then recovered. He also experienced grade 4 hyperglycemia. Given this information and our own experience, we enrolled an additional six patients at the final dose level of 95 mg/m² over 3 hours. Four of six had grade 1 to 2 hypotension, but no further dose-limiting hypotension was observed. Therefore, this level was considered the maximum tolerated dose and the recommended dose for further testing. Patient demographics (age, BSA, and sex), AAG concentrations, and UCN-01 plasma exposure (C_max and AUC) were not related to worst grade of hypotension during course 1 (P 0.20); saliva concentrations were not obtained in all patients to assess the relationship with hypotension.

Mild hyperglycemia was the most frequent adverse event in this study. Fourteen patients treated at dose levels of 12 mg/m² experienced mild hyperglycemia. However, eight of these had baseline blood glucose levels of 100 mg/dL. Typically, the hyperglycemia was mild and resolved without intervention before the next cycle. Two patients treated at 95 mg/m² over 3 hours had grade 3 hyperglycemia. These patients had baseline blood glucose levels of 200 secondary to poorly controlled diabetes. One of these patients presented with headache, dizziness, some confusion, polyuria, polydipsia, and blood glucose of 642 mg/dL 7 days after his fourth course. He was admitted and treated with fluids and insulin, and his symptoms resolved. No other patients required insulin. Exploratory analysis was done to identify potential factors that were associated with the occurrence of hyperglycemia during course 1. Age was associated with grade 2 to 3 hyperglycemia, where the mean age was 64 versus 53 years in those that experienced grade 0 to 1 hyperglycemia (P = 0.026). Other patient demographics (BSA and sex), AAG concentrations, and UCN-01 plasma exposure (C_max and AUC) were not related to worst grade of hyperglycemia during course 1 (P 0.61). C-reactive peptide was not measured in the present study.

Mild nausea was the second most frequent adverse event, affecting 14 patients and 18 (26 %) courses. Thirteen patients had grade 1 to 2 vomiting, and seven of these were treated at the 95 mg/m² dose level. When nausea was consistently observed at the 48 and 68 mg/m² dose levels, ondansetron was added as standard pretreatment for patients receiving UCN-01 95 mg/m². Despite this, 7 of 12 patients treated at 95 mg/m² had some degree of nausea. Three of the patients who experienced nausea and vomiting had these symptoms acutely during or shortly after drug administration.

Three patients in this study developed neurologic symptoms after receiving multiple courses of therapy. One patient treated with 12 mg/m², who had stable disease and remained on study for 24 courses, developed seizures following the 24th course and was found to have progressive disease in the central nervous system. Therefore, her seizures were thought not to be drug related. One patient treated with 95 mg/m² over 1 hour, who developed grade 3 hyperglycemia, seized while admitted for hyperglycemia after his fourth course. His blood glucose was >300 mg/dL at the time. Head computed tomography, magnetic resonance imaging of brain, and lumbar puncture were unrevealing. Electroencephalography showed slow asynchronous activity in central temporal regions bilaterally. He was placed on Dilantin and taken off study. The investigators felt it was possible the study drug contributed to his symptoms. Another patient treated with 95 mg/m² over 3 hours developed grade 3 behavioral changes accompanied by grade 2 confusion and memory loss during the fourth and fifth cycles of therapy. This event was not associated with hypotension; however, the patient did have concomitant use of narcotics and a history of heavy alcohol use. He was taken off study. His symptoms were not thought to be drug related.

Tumor Response. No responses were observed in this heavily pretreated population receiving single agent UCN-01. One patient with cervical cancer had stable disease and remained on study for 24 courses (1 year). Two patients were removed from study for toxicity and thereafter found to have progressive disease. Twenty-one patients had tumor progression after one to five courses (median, 2).

Pharmacokinetic Studies. Plasma samples were obtained from all 24 patients, and pharmacokinetic data were evaluable for 20 patients. UCN-01 plasma pharmacokinetic variables are listed in Table 5. At the recommended dose for further testing, 95 mg/m² administered over 3 hours, average values for V_ss, t_1/2ß, and Cl_s were 14 L, 406 hours, and 0.028 L/h, respectively. At this dose level, mean values for C_max and AUC were 51 µmol/L and 19,732 µmol/L h, and values for these exposure variables varied 2.9- and 5.9-fold, respectively.

Urine concentration data were evaluable for 21 patients. The mean percentage of UCN-01 excreted unchanged in urine from time 0 to 24 hours was 0.79 % (range, 0.049-2.9). Saliva concentrations were obtained in three and eight patients at the 65 and 95 mg/m² dose levels, respectively. Saliva concentrations were measurable (above the assay limit of quantitation) in one of three patients treated at 68 mg/m² and seven of eight patients treated at 95 mg/m². Maximum UCN-01 saliva concentrations were achieved after the end of the infusion at the 24-hour time point on day 2 in post-patients; at the 95 mg/m² dose level, the average C_max value was 889 nmol/L (range, 434-1,425). In one patient at the 95 mg/m² dose level, a saliva sample was obtained at 3.5 weeks after the infusion, with a measurable concentration of 183 nmol/L; samples were not obtained after day 8 in the other seven patients at this dose level. The average saliva-plasma concentration ratio, expressed as a percentage, was 0.82 % (range, 0.62-1.3) and 1.57 % (range, 0.14-4.09) at the 68 and 96 mg/m² dose levels, respectively.

	DISCUSSION

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UCN-01 (Fig. 1), a potent inhibitor of protein kinase C, was selected for development because of its broad and unique pattern of activity against a National Cancer Institute drug discovery screen of 60 human cancer cell lines and because of its mechanism of action, which may include modulation of cyclin-dependent kinases, induction of G₁ accumulation and apoptosis, and abrogation of G₂ arrest induced by DNA-damaging agents leading to apoptosis. Early clinical testing of prolonged infusions revealed that UCN-01 is highly protein bound in humans, with a half-life over 2 weeks. Therefore, this phase I trial was conducted to evaluate UCN-01 given as a short infusion (1-3 hours) every 28 days. As described, dose-limiting hypotension was seen in one of three patients treated with 95 mg/m² over 1 hour. Mild hypotension was seen in seven of nine patients treated with 95 mg/m² over 3 hours, but no dose-limiting toxicity was seen at this dose level. The recommended dose for further study is 95 mg/m² administered over 3 hours for the first course and half that dose for subsequent courses given at 28-day intervals.

A phase I trial of UCN-01 given as a prolonged 72-hour infusion has been published (27). This study defined 42.5 mg/m²/d for 3 days to be the maximum tolerated dose and the dose recommended for further study. It was recommended that subsequent doses be given every 28 days at half the first course dose. The dose-limiting toxicities were nausea, vomiting, hyperglycemia, and hypoxia. It is interesting to note that the side effect profile is somewhat different in pattern and severity between the two trials. In the present study, hyperglycemia for the most part was mild and reversible and in many cases thought not to be drug related because of baseline blood glucose elevations. However, in the study of 72-hour infusions, dose-limiting hyperglycemia was seen at 53 mg/m². In that study, the UCN-01-associated hyperglycemia seen at 53 mg/m² was due to peripheral insulin resistance. C-peptide was increased in 23 of 29 patients studied after exposure to UCN-01. C-peptide was not measured in our study of short infusions. In the present study, nausea and vomiting were common but not dose limiting and only partially treated with ondansetron. However, in the study of 72-hour infusions, dose-limiting nausea and vomiting were only observed at the highest dose level. In our study of short infusions, hypotension was the dose-limiting toxicity. In the study of 72-hour infusion, mild hypotension was seen at 24 mg/m²/d doses and grade 3 hypotension was also observed at 53 mg/m². In contrast to the study of 72-hour infusion, pulmonary toxicity and myalgias were not observed in the present study. Although pulse oximetry was not routinely monitored in this trial, there were no reports of dyspnea as seen in the previous study. There is not a clear pharmacokinetic explanation for the differences in toxicity profiles between the two studies. Differences in efficacy cannot be evaluated in these phase I trials. Shorter infusions would likely be more convenient for patients; however, further study and comparison is needed.

Similar to results from the phase I study of 72-hour infusions (27), when administered over shorter infusion times of 1 to 3 hours, UCN-01 exhibited a long half-life (406 hours), low clearance (0.028 L/h), and marked variability in AUC values (6-fold). However, assessment of the relationship between UCN-01 dose and exposure (C_max and AUC) was not possible due to the utilization of an accelerated dose escalation scheme in the present study, where several dose levels have only one patient. The recommended phase II dose was thus expanded to nine patients to obtain more thorough pharmacokinetic data. At this dose level (95 mg/m² over 3 hours), a higher mean C_max of 51 µmol/L was achieved compared with another phase I study where the recommended phase II dose was 42.5 mg/m² over 72 hours (36 µmol/L); a similar mean AUC value of 19,732 µmol/L h was achieved with the 3-hour infusion compared with the 72-hour infusion (21,212 µmol/L h).

The long half-life for UCN-01 has been attributed partly to specific high-affinity binding to plasma proteins, specifically AAG. UCN-01 has a half-life that is far longer that for AAG (3-5 days), suggesting that other proteins may play a role as well (30). In the prior study, a correlation between AAG and UCN-01 plasma concentration was observed (23). In our study, no correlation between UCN-01 volume of distribution or clearance and AAG was observed. This is not unexpected for a highly protein bound drug where measurement of total drug concentrations in plasma is often a poor surrogate for unbound drug. Accurate assessment of relationships between pharmacokinetic variables and plasma proteins would best be accomplished by measuring unbound drug concentrations. Kurata et al. (31) have shown recently that the binding site of UCN-01 on AAG overlaps with the sites for propranolol, warfarin, and progesterone. This raises the possibility of important drug interactions in cancer patients receiving UCN-01 along with other highly protein-bound medications.

Despite the extensive protein binding, maximum UCN-01 saliva concentrations at values ranging from 434 to 1,425 nmol/L were achieved at the 95 mg/m² dose level, a concentration of 183 nmol/L was measured at 3.5 weeks in one patient with sampling out this far after the drug infusion. Concentrations of 30 to 300 nmol/L have been sufficient for growth inhibition, abrogation of G₂ arrest and induction of apoptosis in in vitro systems (4, 13, 16). This suggests that potentially biologically active free drug concentrations are achieved at the dose recommended here, although the optimal UCN-01 concentrations and duration of exposure to accomplish therapeutic effects have not been defined. Of interest, neither patient demographics (age, BSA, and sex), AAG concentrations, nor UCN-01 plasma exposure (C_max and AUC) were related to worst grade of hypotension during course 1 or to hyperglycemia during course 1.

No tumor responses and only one prolonged stabilization of disease were observed in the present study of single agent UCN-01 administered as a short infusion. Perhaps its best application will be in combination with conventional cytotoxic agents. Invitro, UCN-01 has been shown to enhance the cytotoxicity of a variety of conventional chemotherapeutics. For example, UCN-01 enhanced the cytotoxicity of cisplatin by a factor of 3 in ovary cancer cell lines (20) and breast cancer cell lines (22). UCN-01 has shown synergy when given with mitomycin C in human epidermoid cancer cell lines and xenografts (23) and breast and gastric cancer cell lines (24) and increased the cytotoxicity of paclitaxel in gastric and breast cancer cell lines (22, 24, 25). Finally, UCN-01 exhibited synergistic cytotoxicity with camptothecin and topotecan in breast cancer cell lines (8, 22, 26) and in colon cancer cell lines where UCN-01 potentiated camptothecin cytotoxicity up to 50-fold (8). The mechanism for synergy is thought to be that UCN-01 abrogates cell cycle arrest induced by the cytotoxic agents. For example, UCN-01 suppresses S-phase accumulation induced by camptothecin in colon and breast carcinoma cell lines (8, 26). UCN-01 is thought to enhance the cytotoxicity of cisplatin by abrogating the G₂ arrest necessary for DNA repair (20). Clinical trials combining UCN-01 with fludarabine, cisplatin, 5-fluorouracil, and cytarabine are ongoing.

This study used an accelerated titration design (28). This method was developed because studies using existing designs often take a long time to complete and provide little information about interpatient variability or cumulative toxicity. Furthermore, using the standard design, many patients in phase I studies are treated at subtherapeutic doses (28). Using data from 20 phase I trials of anticancer agents, which used standard dose escalation schemes, mathematical modeling was used to analyze these data and to develop alternative dose escalation schemes that could overcome the shortcomings of existing studies. In the present study, using the accelerated titration design and modifications for dose escalation based on events experienced in concurrent trials, we used only six dose levels and 15 patients to reach the final dose level. The additional six patients were all treated at the recommended dose for further study. By contrast, using a modified Fibonacci design with three patients per level and escalation steps of 33 % to 50 %, it would have required nine dose levels and 27 patients to reach 100 mg/m², even if the information from the other trials had been used to modify the doses. Thus, the accelerated titration design, even modified as it was, saved three dose levels and 12 patients at lower doses. However, one drawback to this design is the relative paucity of pharmacokinetic information at lower dose levels. Therefore, the linearity of the relationship between dose and concentration is harder to assess than in more traditional designs.

In summary, the results of this phase I trial show that UCN-01 is well tolerated when given at doses of 95 mg/m² over 3 hours every 28 days with second and subsequent courses given at 50 % the first course dose. In the absence of notable antitumor activity with single agent therapy, ongoing clinical trials will define its application in combination with conventional cytotoxic agents.

	FOOTNOTES

 
Grant support: NIH grants U01CA70095 and 5P30CA06973.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Note: E. Dees is currently at the Division of Hematology and Oncology, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599. S. O'Reilly is urrently in Cork Regional Hospital, Cork, Ireland.

Received 8/26/04; accepted 10/27/04.

	REFERENCES

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