Fostriecin: chemistry and biology

A review of the current status of the chemistry and biology of fostriecin (CI-920) is provided. Fostriecin is a structurally unique, naturally-occurring phosphate monoester that exhibits potent and efficacious antitumor activity. Initially it was suggested that its activity could be attributed to a direct, albeit weak, inhibition of the enzyme topoisomerase II. However, recent studies have shown that fostriecin inhibits the mitotic entry checkpoint through the much more potent and selective inhibition of protein phosphatase 2A (PP2A) and protein phosphatase 4 (PP4). In fact, it is the most selective small molecule inhibitor of a protein phosphatase disclosed to date. The contribution, if any, that topoisomerase II versus PP2A/PP4 inhibition makes to fostriecin's antitumor activity has not yet been fully defined. Initial phase I clinical trials with fostriecin never reached dose-limiting toxicity or therapeutic dose levels and were halted due to its storage instability and unpredictable chemical purity. Hence, the total synthesis of fostriecin has been pursued in order to confirm its structure and stereochemistry, to provide access to quantities of the pure natural product, and to access key partial structures or simplified/stable analogs. Several additional natural products have been isolated which contain similar structural features (phospholine, phoslactomycins, phosphazomycin, leustroducsins, sultriecin, and cytostatin), and some exhibit comparable biological properties.     

Differential effects of tautomycetin and its derivatives on protein phosphatase inhibition, immunosuppressive function and antitumor activity

In the present work, we studied the structure-activity relationship (SAR) of tautomycetin (TMC) and its derivatives. Further, we demonstrated the correlation between the immunosuppressive fuction, anticancer activity and protein phosphatase type 1 (PP1) inhibition of TMC and its derivatives. We have prepared some TMC derivatives via combinatorial biosynthesis, isolation from fermentation broth or chemical degradation of TMC. We found that the immunosuppressive activity was correlated with anticancer activity for TMC and its analog compounds, indicating that TMC may home at the same targets for its immunosuppressive and anticancer activities. Interestingly, TMC-F1, TMC-D1 and TMC-D2 all retained significant, albeit reduced PP1 inhibitory activity compared to TMC. However, only TMC-D2 showed immunosuppressive and anticancer activities in studies carried out in cell lines. Moreover, TMC-Chain did not show any significant inhibitory activity towards PP1 but showed strong growth inhibitory effect. This observation implicates that the maleic anhydride moiety of TMC is critical for its phosphatase inhibitory activity whereas the C1-C18 moiety of TMC is essential for the inhibition of tumor cell proliferation. Furthermore, we measured in vivo phosphatase activities of PP1 in MCF-7 cell extracts treated with TMC and its related compounds, and the results indicate that the cytotoxicity of TMC doesn't correlate with its in vivo PP1 inhibition activity. Taken together, our study suggests that the immunosuppressive and anticancer activities of TMC are not due to the inhibition of PP1. Our results provide a novel insight for the elucidation of the underlying molecular mechanisms of TMC's important biological functions.     

Regulation of immunoglobulin E-mediated secretion by protein phosphatases in human basophils and mast cells of skin and lung.

A wide range of serine/threonine protein phosphatase (PP) inhibitors were studied for effects on the immunoglobulin E (IgE)-mediated release of histamine from human lung mast cells, human skin mast cells and basophils. Okadaic acid (OA) inhibited the release of histamine from all three cell types in a concentration-dependent manner. Two structural analogues of okadaic acid, okadaol and okadaone, known to be less active than the parent molecule as inhibitors of PP, were less active than okadaic acid as inhibitors of histamine release in these three cell types. A number of PP inhibitors, showing differences in selectivity for PP1 and PP2A, were also evaluated. Calyculin, which is roughly equipotent as a PP1 and PP2A inhibitor, attenuated the release of histamine from all three cell types. Similarly, tautomycin (TAU), which shows greater selectivity for PP1 over PP2A, was also effective at inhibiting histamine release in all three cell types. In contrast, fostriecin, which is very much more potent as an inhibitor of PP2A over PP1, was ineffective as an inhibitor in all three cell types. These data indicate that the regulation of mediator release by PPs is similar in lung mast cells, skin mast cells and basophils. Moreover, the data suggest that PP1 is important in the control of cellular activity.     

Phase I study of temsirolimus in combination with EKB-569 in patients with advanced solid tumors

Purpose Activation of EGFR can stimulate proliferative and survival signaling through mTOR. Preclinical data demonstrates synergistic activity of combined EGFR and mTOR inhibition. We undertook a phase I trial of temsirolimus (T, an mTOR inhibitor) and EKB-569 (E, an EGFR inhibitor) to determine the safety and tolerability. Methods The primary aim was to determine the maximally tolerated dose (MTD) of this combination in adults with solid tumors. Following the dose-escalation phase, (Cohort A), two subsequent cohorts were used to assess any pharmacokinetic (PK) interaction between the agents. Results Forty eight patients were enrolled. The MTD of this combination was E, 35 mg daily and T, 30 mg on days 1-3 and 15-17 using a 28-day cycle. The most common toxicities were nausea, diarrhea, fatigue, anorexia, stomatitis, rash, anemia, neutropenia, thrombocytopenia, and hypertriglyceridemia. Sixteen patients (36%) had at least one grade 3 toxicity. The most frequent grade 3/4 toxicities were diarrhea, dehydration, and nausea and vomiting (19% each). No grade 5 events were seen. Four patients had a partial response and 15 had stable disease. Clinical benefit was seen across a range of tumor types and in all cohorts. PK analysis revealed no significant interaction between E and T. Conclusions This combination of agents is associated with tolerable toxicities at doses that induced responses. PK studies revealed no interaction between the drugs. Further investigations of this targeting strategy may be attractive in renal cell carcinoma, non-small cell lung cancer, alveolar sarcoma, and carcinoid tumor.     

A phase 1 trial of XK469: Toxicity profile of a selective topoisomerase IIβ inhibitor

Summary Purpose: XK469, a member of the quinoxaline family of antitumor agents, is believed to be unique in its ability to selectively target topoisomerase IIβ. Based on encouraging pre-clinical data, a phase I trial was conducted to determine the dose limiting toxicity (DLT) and the maximum tolerated dose (MTD). Methods: A 2B accelerated titration schema was employed. XK469 was administered as a 5 or 20 min IV infusion on days 1–5 every 21 days. The starting dose was 9 mg/m². Pharmacokinetics (PK) were conducted in cycles 1–3. Results: 22 patients (21 evaluable, mean age: 56 years, median performance status: 1) were enrolled. At dose level 11 (260 mg/m²/daily X 5), 1/6 patients experienced a DLT of grade 4 neutropenia. At 346 mg/m²/daily X 5, 2/2 patients experienced DLT's with one episode of febrile neutropenia and one grade 3 infection. The MTD was identified as 260 mg/m²/day. XK469 peak plasma levels and systemic exposure were proportional to dose indicating linear pharmacokinetics. However, secondary peaks in the PK profiles and a rapid decline in drug level from 23 to 24 h occurred in some patients. Drug infusion in the afternoon followed by dense sampling of levels during the elimination phase supported the hypothesis that the drug was being sequestered. No anti-tumor activity was identified. Conclusions: Traditional PK sampling designs were inadequate to describe XK469 disposition. XK469 and related structures work through a unique mechanism of action. A further understanding of the specific mechanism of these compounds might uncover a unique avenue for future drug development.     

Pharmacokinetic study of oxaliplatin iv chronomodulated infusion combined with 5-fluorouracil iv continuous infusion in the treatment of advanced colorectal cancer

We investigated the pharmacokinetics (PK), preliminary clinical results and toxicity of chronomodulated oxaliplatin (OHP) plus 5-fluorouracil (5-FU) without folinic acid (FA) in 13 patients with metastatic colorectal cancer. 5-FU (200 mg/m2/day as 14-day continuous iv infusion for six cycles) plus OHP at increasing doses (25-30-35 mg/m2/day, as 12 h chronomodulated iv infusion on days 1-2-3-4, every 14 days for six cycles) were administered to reach maximum tolerated dose (MTD). At MTD (30 mg/m2/day), a PK study of 5-FU and OHP (in total and ultrafiltered-UF plasma) was performed. 5-FU plasma levels were fairly stable, below that reported in similar studies and closely related to the lack of the most typical 5-FU toxicities. OHP Cmax occurred 7 h after infusion start; a progressive accumulation of free Pt and ultrafiltered Pt (UF-OHP) through cycles 1-6 was noted. A marked difference between total plasma and UF Pt was seen in the elimination phase. OHP plasma clearance decrease was related to Vz (volume of distribution of late elimination phase), whereas in UF-OHP was due to a change in Ke or t1/2. In conclusion, the association of 5-FU with chronomodulated OHP do not seem to influence PK parameters of either drugs. Toxicity was modest/acceptable and clinical efficacy good: preliminary data showed a threshold neurotoxicity at total plasma Pt concentrations >1500 ng/ml and UF plasma Pt concentrations >150 ng/ml.     

A phase I study of oral ZD 1839 given daily in patients with solid tumors: IND.122, a study of the Investigational New Drug Program of the National Cancer Institute of Canada Clinical Trials Group

Summary Purpose: To define the maximum tolerated dose (MTD), the dose limiting toxicity (DLT), the biological active (BA) dose and the pharmacokinetics (PK) of the epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor ZD1839 (Iressa) when administered continuously as a once daily dose in patients with advanced, incurable solid tumours. Patients and methods: Twenty-eight patients were enrolled in cohorts of three from three National Cancer Institute of Canada Clinical Trials Group (NCIC CTG) centers. ZD1839 was given at doses from 150 to 800 mg daily orally and patients underwent a pretreatment and a 28 day post treatment tumor biopsy, while PK sampling was performed on days 8, 15, 22, 29, and a toxicity assessment every 28 days. Results: All twenty-eight patients were evaluable for non-hematological and hematological toxicity. Twenty-seven were evaluable for response. The MTD was not reached but DLT included reversible rash and diarrhea. One patient with urachal cancer had a transient 55% decrease in tumor size and two other patients (breast and non-small cell lung cancer) had minor responses; three additional patients had pharmacodynamic evidence of target effect. PK demonstrated steady state within the first 2 weeks of dosing and dose dependent exposure. Conclusion: It appears that ZD 1839 at a dose of 800 m/day was tolerable, although some patients required dose modification for diarrhea. Doses above 250 m/day demonatrate biologic activity and could be consider for future study in a variety of EGFR positive tumor types.     

Cyanobacterial nodularin is a potent inhibitor of type 1 and type 2A protein phosphatases.

The present study characterizes the inhibitory effects of nodularin, a recently isolated hepatotoxic compound from the cyanobacterium Nodularia spumigena, on type 1 (PP1), type 2A, (PP2A), type 2B (PP2B), and type 2C (PP2C) protein phosphatases. Both PP2A and PP1 were potently inhibited (IC50 = 0.026 and 1.8 nM, respectively) by nodularin, whereas PP2B was inhibited to a lesser extent (IC50 = 8.7 microM). Nodularin had no apparent effect on PP2C, alkaline phosphatase, acid phosphatase, insulin receptor tyrosine kinase, protein kinase A, phosphorylase kinase, or protein kinase C. In a whole-cell extract of T51B liver cells, nodularin inhibited PP1 and PP2A activity with a potency similar to that seen with their purified catalytic subunits. Thus, due to the high specificity of nodularin for PP2A and PP1, this hepatotoxin may prove to be useful as a probe for distinguishing the activity of these protein phosphatases in cell extracts.     

Hepatic toxicity and persistence of ser/thr protein phosphatase inhibition by microcystin in the little skate Raja erinacea

Microcystin-induced ser/thr protein phosphatase (PP) inhibition and toxicity were examined in the little skate (Raja erinacea), an evolutionarily primitive marine vertebrate. As in mammals, PP inhibition and toxicity were exclusively hepatocellular, but were much more persistent in the skate. A dose of 63 microg/kg given iv to adult male skates resulted in the near complete inhibition of hepatic PP activity at 24 h. PP activity was still 95% inhibited 7 days after dosing in skates given 125 microg/kg microcystin. Mortality occurred at doses of 500 microg/kg or more. Hepatic lesions were only seen in animals with fully inhibited PP activity in liver. The histological changes seen at 125 microg/kg were mild periportal inflammatory changes increasing in severity together with hepatocyte necrosis at higher doses of microcystin. Microcystin persisted and could be detected in plasma up to 7 days after dosing. This finding shows that, in the skate, as in mammals, the liver is the only organ capable of uptake of microcystin, since there was no significant inhibition of PP activity in the rectal gland and small decreases in PP activity of the kidney that were not time or dose dependent. In vitro microcystin caused dose-dependent inhibition of PP activity in isolated skate hepatocytes, while it was without effect in cultured rectal glands. Uptake of microcystin and the accompanying inhibition of PP activity in skate hepatocytes was prevented by the addition of a series of organic dyes and bile acids. The spectrum of inhibitors of microcystin uptake in skate is similar to that seen in the rat, indicating common features of the carrier(s) in these diverse species.     

A phase I and pharmacokinetic study of docetaxel combined with Doxil (pegylated liposomal doxorubicin) without and with granulocyte colony stimulating factor

The purpose of this study was (i) to determine the maximum tolerated dose (MTD) of docetaxel that can be administered in combination with Doxil, given without and with granulocyte colony stimulating factor (G-CSF), (ii) to define the pharmacokinetics (PK) of docetaxel when used in combination with Doxil, and (iii) to make preliminary observations on the anti-tumor activity of this combination in patients with metastatic solid tumors. Thirty-seven patients with metastatic cancer were enrolled. Courses were repeated every 3 weeks. Patients received a fixed dose of Doxil 30 mg/m(2) in combination with escalating doses of docetaxel ranging from 40 to 100 mg/m(2). After encountering dose-limiting febrile neutropenia, subsequent escalation was accomplished with G-CSF support. Selected patients at the recommended phase II dose underwent PK evaluation. The most common toxicity observed was neutropenia. Dose-limiting toxicity (30 mg/m(2) Doxil + 80 mg/m(2) docetaxel) was febrile neutropenia in three of six patients treated without G-CSF. Major non-hematological toxicities included alopecia, mucositis and hand-foot syndrome, and were observed after cumulative doses of chemotherapy. Objective responses (complete/partial) were documented in eight of 37 patients (four with breast cancer) and stable disease was seen in 17 patients. PK studies showed an increased tissue retention (decreased clearance) of docetaxel when given with Doxil. The recommended phase II dose of Doxil/docetaxel is 30/60 mg/m(2), q3 weeks, without G-CSF. Further dose escalation to 30/80 mg/m(2) is safe with G-CSF support. Anti-tumor activity, particularly against breast cancer, was observed at various dose levels. Our observations should provide evidence for phase II studies of this combination in patients with breast cancer and other anthracycline/taxane-sensitive cancers.     

Clinical pharmacology of the novel marine-derived anticancer agent Ecteinascidin 743 administered as a 1- and 3-h infusion in a phase I study

Ecteinascidin 743 (ET-743) is an anticancer agent derived from the Caribbean tunicate Ecteinascidia turbinata. In the present article, the pharmacokinetics and pharmacodynamics of ET-743 are described within a phase I study. Forty patients with solid tumors initially received ET-743 as a 1-h i.v. infusion every 21 days at nine dose levels (50-1100 microg/m(2)). The maximal tolerated dose (MTD) was 1100 microg/m(2), with thrombocytopenia and fatigue as dose-limiting toxicities (DLTs). As this MTD was substantially lower than in parallel phase I studies, dose escalation continued using a prolonged, 3-h infusion. Thirty-two patients were entered at five dose levels (1000-1800 microg/m(2)). The MTD was 1800 microg/m(2) with pancytopenia and fatigue as DLTs. The recommended phase II dose was 1650 microg/m(2) given over 3 h at which 12 patients were treated. Pharmacokinetic monitoring was performed for both treatment schedules. Non-compartmental pharmacokinetic parameters at the recommended dose with the 3-h infusion were (mean value+/-SD): clearance 87+/-30 l/h and mean elimination half-life 26+/-7 h. Pharmacokinetics were linear at the dose range tested with this schedule. The percentage decrease in platelets, white blood cells and neutrophils correlated with the area under the plasma concentration versus time curve (AUC), dose and maximal plasma concentration (C(max)). Hepatic toxicity increased with dose, AUC and C(max). Administration of 1650 microg/m(2) ET-743 over 3 h seemed clinically feasible; pharmacokinetics were linear with this schedule. Hepatic and hematological toxicities correlated with exposure to ET-743.     

Potential anti-tumor effects of FTY720 associated with PP2A activation: a brief review

FTY720 (Fingolimod, Gilenya^?) is an FDA-approved immunosuppressant currently used in the treatment of multiple sclerosis. However, a large number of studies over the last few years have shown that FTY720 shows potent antitumor properties that suggest its potential usefulness as a novel anticancer agent. Interestingly, the restoration of protein phosphatase 2A (PP2A) activity mediated by FTY720 could play a key role in its antitumor effects. Taking into account that PP2A inactivation is a common event that determines poor outcome in several tumor types, FTY720 could serve as an alternative therapeutic strategy for cancer patients with such alterations.     

Dose Escalation in Phase I Clinical Studies of Anticancer Agents in Japan and Comparison of Pharmacokinetic Parameters Between Japanese and Non-Japanese

Abstract

The published reports of phase I clinical studies of anticancer agents conducted in Japan from 1981 to 1991 were reviewed. Data of 30 drugs administered by 50 different schedules were available. the median ratio of MTD to starting dose was 7.8 for domestic agents as compared with 3.4 for imported drugs. the median number of dose escalation steps required to reach the MTD was 7 for domestic agents as compared with 5 for imported drugs (not significant). No differences were found in AUCs at the MTD of 7 anticancer agents between Japanese reports and the trials conducted in the United States and/or Europe. Based on these observations, a simple and efficient strategy of phase I clinical trials for imported agents was proposed.     

Phase I study of 17-allylamino-17 demethoxygeldanamycin,gemcitabine and/or cisplatin in patients with refractory solid tumors

Purpose: To determine the maximum tolerated dose (MTD) and characterize the dose-limiting toxicities (DLT) of 17-AAG, gemcitabine and/or cisplatin. Levels of the proteins Hsp90, Hsp70 and ILK were measured in peripheral blood mononuclear cell (PMBC) lysates to assess the effects of 17-AAG. Experimental design: Phase I dose-escalating trial using a “3 + 3” design performed in patients with advanced solid tumors. Once the MTD of gemcitabine + 17-AAG + cisplatin was determined, dose escalation of 17-AAG with constant doses of gemcitabine and cisplatin was attempted. After significant hematologic toxicity occurred, the protocol was amended to evaluate three cohorts: gemcitabine and 17-AAG; 17-AAG and cisplatin; and gemcitabine, 17-AAG and cisplatin with modified dosing. Results: The 39 patients enrolled were evaluable for toxicity and response. The MTD for cohort A was 154 mg/m² of 17-AAG, 750 mg/m² of gemcitabine, and 40 mg/m² of cisplatin. In cohort A, DLTs were observed at the higher dose level and included neutropenia, hyperbilirubinemia, dehydration, GGT elevation, hyponatremia, nausea, vomiting, and thrombocytopenia. The MTD for cohort C was 154 mg/m² of 17-AAG and 750 mg/m² of gemcitabine, with one DLT observed (alkaline phosphatase elevation) observed. In cohort C, DLTs of thrombocytopenia, fever and dyspnea were seen at the higher dose level. The remaining cohorts were closed to accrual due to toxicity. Six patients experienced partial responses. Mean Hsp90 levels were decreased and levels of Hsp70 were increased compared to baseline. Conclusions: 17-AAG in combination with gemcitabine and cisplatin demonstrated antitumor activity, but significant hematologic toxicities were encountered. 17-AAG combined with gemcitabine is tolerable and has demonstrated evidence of activity at the MTD. The recommended phase II dose is defined as 154 mg/m² of 17-AAG and 750 mg/m² of gemcitabine, and is currently being investigated in phase II studies in ovarian and pancreatic cancers. There is no recommended phase II dose for the cisplatin-containing combinations.     

Prolonged Sublethal Exposure to the Protein Phosphatase Inhibitor Microcystin-LR Results in Multiple Dose-Dependent Hepatotoxic Effects

The purpose of this study was to relate dose-dependent hepatotoxicitystemming from prolonged exposure to sublethal concentrationsof the cyclic heptapeptide microcystin-LR (Mcyst) to hepaticMcyst concentrations and protein phosphatase activity. Mcystis a potent inhibitor of protein phosphatase types 1 and 2A(PP1 and PP2A). Twenty male Sprague-Dawley rats were infusedcontinuously with 0, 3, 6, or 9 µg Mcyst/days for 28 daysusing intraperitoneal mini-osmotic pumps containing highly purifiedtoxin or saline. At the end of 28 days, dose-dependent increasesin several serum biochemical tests including sorbitol dehydrogenase,aspartate amhotransferase, -glutamyl transferase, alkaline phosphatase,and bile acids had occurred. Serum albumin decreased in a dose-dependentfashion. Liver activity of both PP1 and PP2A decreased in adose-dependent manner, but with a relatively greater effecton PP2A than PP1. Liver cytosol Mcyst concentrations, measuredby direct competitive ELISA, also increased in a dose-dependentmanner, although at a higher rate than would be predicted fromthe incremental increase in dose given. This disproportionalincrease is suggestive of the bioaccumulation of Mcyst withincreasing dose. Histopathological abnormalities included hepatocellularapoptosis and cytosolic vacuolation of principally zone 3 hepatocytes.Immunohistochemical stains revealed Mcyst predominantly withinpericanalicular regions of zone 3 hepatocytes. It was concludedthat prolonged exposure to sublethal concentrations of Mcystresults in multiple dose-depen-dent hepatotoxic effects thatcorrespond to decreased hepatic serine/threonine protein phosphataseactivity and increasing cytosolic Mcyst concentrations. Thedisproportional increase of hepatic Mcyst concentrations observedmay suggest the bioaccumulation of toxin and an increasing relativerisk of hepatotoxicity with increasing dose.     

Knockdown of Clusterin Expression Increases the In Vitro Sensitivity of Human Prostate Cancer Cells to Paclitaxel

Clusterin/apolipoprotein J is a secreted heterodimeric glycoprotein that is implicated in several pathophysiological processes, including tissue remodeling, reproduction, lipid transport, and apoptosis. Although previous studies demonstrated that clusterin is able to protect against apoptosis, the role of the clusterin in cellular proliferation remains elusive. To determine whether clusterin plays an important role in cellular proliferation, the function of clusterin was examined using a small interfering RNA (siRNA) in PC3 human prostate cancer cells. Transient transfection with clusterin siRNA resulted in significant suppression of clusterin mRNA and protein expression. Clusterin knockdown resulted in a decrease in protein expression of phospho-Akt and an increase in expression of proteins phosphatase type 2AC (PP2AC) and phosphorylation of p38. However, treatment with PP2AC siRNA exerted minimal effects on clusterin expression. Interestingly, clusterin mRNA expression was reduced in paclitaxel-treated cells, and the cytotoxic effect of paclitaxel was more potent when cells were incubated with clusterin siRNA. In addition, co-treatment with paclitaxel and clusterin siRNA significantly enhanced PP2AC levels. Taken together, these results indicate that clusterin plays a crucial role in PC3 cell proliferation and that clusterin depletion may contribute to enhanced sensitivity of PC3 cells to anticancer agents such as paclitaxel.     

Phase I Trial of the Polyamine Analog N1,N14-Diethylhomospermine (DEHSPM) in Patients with Advanced Solid Tumors

BACKGROUND: This phase I study was conducted to determine maximal tolerated dose (MTD) and dose-limiting toxicities (DLT) in patients with advanced solid tumors treated with the polyamine analog N1, N14-diethylhomospermine (DEHSPM). METHODS: Patients were treated with DEHSPM administered as a subcutaneous (SC) injection daily for five consecutive days repeated every 4 weeks. Three dose levels were examined starting at 12.5 mg/m2/day, escalating to 37.5 mg/m2/day. RESULTS: A total of 15 patients were enrolled. Dose limiting toxicities (grade 3 or 4) included nausea, vomiting, constipation, ileus, elevations of aspartate aminotransferase (AST) and alkaline phosphatase, hyperbilirubinemia, and ventricular bigeminy. CONCLUSION: DEHSPM given as a SC injection is overall well tolerated at lower doses, but significant toxicities were observed at the 37.5mg/m2/day dose level. MTD was established at 25 mg/m2/day but further investigation with this study drug is not recommended secondary to the potential for neurotoxicities and hepatic damage as a result of cumulative doses.     

A Phase I Trial of Daily Oral 4′-N-Benzoyl-Staurosporine in Combination with Protracted Continuous Infusion 5-Fluorouracil in Patients with Advanced Solid Malignancies

PURPOSE: 4'- N -Benzoyl-staurosporine (PKC412) is an orally available staurosporine derivative that inhibits protein kinase C. The objectives of this phase I trial were to determine the maximum tolerated dose (MTD), the dose limiting toxicities (DLTs), and the pharmacokinetics of PKC412 when co-administered with 5-Fluorouracil (5-FU). EXPERIMENTAL DESIGN: PKC412 was given daily with a 21-day continuous i.v. infusion of 5-FU 200 mg/m2/day, repeated every 4 weeks. The PKC412 dose was escalated by a modified continual reassessment method. The steady-state plasma pharmacokinetics of 5-FU, PKC412, and two of its circulating metabolites were determined during the first cycle of therapy. RESULTS: A total of 33 patients were treated with 70 cycles (median: 2, range: 1-4) of PKC412 at doses ranging from 25 to 225 mg/day. No significant toxicities were encountered with doses up to 150 mg/day. Among nine patients treated with 225 mg/day of PKC412, one experienced grade 3 fatigue and nausea, another developed grade 3 hyperglycemia, and three had grade 2 emesis and stomatitis, leading to early treatment discontinuation. Minor responses consisting of a 40-45% tumor reduction were observed in two patients, one with gall bladder carcinoma and one with breast cancer. Mean values of steady-state pharmacokinetic variables for both PKC412 and 5-FU were comparable to single agent studies. CONCLUSIONS: The recommended phase II dose of PKC412 is 150 mg/day when combined with a continuous infusion of 200 mg/m2/day 5-FU. The dose limiting toxicity was grade 2 emesis and stomatitis and the regimen showed indications of activity. There was no evidence of a pharmacokinetic interaction between the two drugs.