Gene expression profiling identifies responsive patients with cancer of unknown primary treated with carboplatin, paclitaxel, and everolimus: NCCTG N0871 (alliance).

BACKGROUND: Carboplatin (C) and paclitaxel (P) are standard treatments for carcinoma of unknown primary (CUP). Everolimus, an mTOR inhibitor, exhibits activity in diverse cancer types. We did a phase II trial combining everolimus with CP for CUP. We also evaluated whether a gene expression profiling (GEP) test that predicts tissue of origin (TOO) could identify responsive patients. PATIENTS AND METHODS: A tumor biopsy was required for central confirmation of CUP and GEP. Patients with metastatic, untreated CUP received everolimus (30 mg weekly) with P (200 mg/m(2)) and C (area under the curve 6) every 3 weeks. The primary end point was response rate (RR), with 22% needed for success. The GEP test categorized patients into two groups: those having a TOO where CP is versus is not considered standard therapy. RESULTS: Of 45 assessable patients, the RR was 36% (95% confidence interval 22% to 51%), which met criteria for success. Grade ≥3 toxicities were predominantly hematologic (80%). Adequate tissue for GEP was available in 38 patients and predicted 10 different TOOs. Patients with a TOO where platinum/taxane is a standard (n = 19) tended to have higher RR (53% versus 26%) and significantly longer PFS (6.4 versus 3.5 months) and OS (17.8 versus 8.3 months, P = 0.005), compared with patients (n = 19) with a TOO where platinum/taxane is not standard. CONCLUSIONS: Everolimus combined with CP demonstrated promising antitumor activity and an acceptable side-effect profile. A tumor biomarker identifying TOO may be useful to select CUP patients for specific antitumor regimens. CLINICALTRIALSGOV: NCT00936702.

Multicenter phase II study of the AKT inhibitor MK-2206 in recurrent or metastatic nasopharyngeal carcinoma from patients in the mayo phase II consortium and the cancer therapeutics research group (MC1079).

BACKGROUND: This study investigated the activity of MK-2206, an AKT inhibitor, in metastatic or recurrent nasopharyngeal carcinoma (NPC). METHOD: Oral MK-2206 at a dose of 200 mg was administered on days 1, 8, 15 and 22 of a 28-day cycle until progression. Plasma EBV DNA clearance during the first month of treatment was measured, and archived tumors were analyzed for the expression of AKT and PIK3CA mutation and PIK3CA amplification. The dual primary endpoint was objective response rate and 6-month progression-free survival (PFS) rate. RESULTS: 21 patients were enrolled and one patient achieved a partial response (5 %) and 11 had stable disease (52 %), with a median PFS of 3.5 months (95 % confidence interval, CI: 0.9-7.3). The 6-month PFS rate was 43 % (95 % CI: 22-66 %) and the median OS was 10 months (95 % CI: 5.9 months-not reached). Seven patients (33 %) experienced grade 3 toxicities which could be related to MK-2206. Macular-papular rash was the most common (n = 6), followed by hyperglycemia (n = 2) and fatigue (n = 1). In the 12 tumor samples analyzed, PIK3CA amplification was detected in one patient's primary NPC, who had SD lasting over 12 months. Patients with decreasing EBV DNA values over time were more likely to be alive and progression-free for at least 6 months than those without a decrease (p = 0.001). CONCLUSION: The study was terminated due to the limited activity observed in this heavily pre-treated group of patients. Further studies are needed to elucidate the optimal way of selecting patients for AKT inhibitors.

Heat shock protein inhibitors increase the efficacy of measles virotherapy.

Oncolytic measles virus strains have activity against multiple tumor types and are currently in phase I clinical testing. Induction of the heat shock protein 70 (HSP70) constitutes one of the earliest changes in cellular gene expression following infection with RNA viruses including measles virus, and HSP70 upregulation induced by heat shock has been shown to result in increased measles virus cytotoxicity. HSP90 inhibitors such as geldanamycin (GA) or 17-allylaminogeldanamycin result in pharmacologic upregulation of HSP70 and they are currently in clinical testing as cancer therapeutics. We therefore investigated the hypothesis that heat shock protein inhibitors could augment the measles virus-induced cytopathic effect. We tested the combination of a measles virus derivative expressing soluble human carcinoembryonic antigen (MV-CEA) and GA in MDA-MB-231 (breast), SKOV3.IP (ovarian) and TE671 (rhabdomyosarcoma) cancer cell lines. Optimal synergy was accomplished when GA treatment was initiated 6-24 h following MV infection. Western immunoblotting confirmed HSP70 upregulation in combination-treated cells. Combination treatment resulted in statistically significant increase in syncytia formation as compared to MV-CEA infection alone. Clonogenic assays demonstrated significant decrease in tumor colony formation in MV-CEA/GA combination-treated cells. In addition there was increase in apoptosis by 4,6-diamidino-2-phenylindole staining. Western immunoblotting for caspase-9, caspase-8, caspase-3 and poly(ADP-ribose) polymerase (PARP) demonstrated increase in cleaved caspase-8 and PARP. The pan-caspase inhibitor Z-VAD-FMK and caspase-8 inhibitor Z-IETD-FMK, but not the caspase-9 inhibitor Z-IEHD-FMK, protected tumor cells from MV-CEA/GA-induced PARP activation, indicating that apoptosis in combination-treated cells occurs mainly via the extrinsic caspase pathway. Treatment of normal cells, such as normal human fibroblasts, however, with the MV-CEA/GA combination, did not result in cytopathic effect, indicating that GA did not alter the MV-CEA specificity for tumor cells. One-step viral growth curves, western immunoblotting for MV-N protein expression, QRT-PCR quantitation of MV-genome copy number and CEA levels showed comparable proliferation of MV-CEA in GA-treated vs -untreated tumor cells. Rho activation assays and western blot for total RhoA, a GTPase associated with the actin cytoskeleton, demonstrated decrease in RhoA activation in combination-treated cells, a change previously shown to be associated with increase in paramyxovirus-induced cell-cell fusion. The enhanced cytopathic effect resulting from measles virus/GA combination supports the translational potential of this approach in the treatment of cancer.

A phase II trial of tipifarnib in myelofibrosis: primary, post-polycythemia vera and post-essential thrombocythemia.

Patients with primary myelofibrosis (PMF) or post-polycythemia vera or post-essential thrombocythemia myelofibrosis (post-PV/ET MF) have limited therapeutic options. The farnesyltransferase-inhibitor tipifarnib inhibits in vitro proliferation of myeloid progenitors from such patients. In the current phase II clinical trial, single-agent oral tipifarnib (300 mg twice daily x 21 of 28 days) was given to 34 symptomatic patients with either PMF (n=28) or post-PV/ET MF (n=6). Median time to discontinuation of protocol therapy was 4.6 months; reasons for early termination (n=19; 56%) included disease progression (21%) and adverse drug effects (18%). Toxicities (>/=grade 3) included myelosuppression (n=16), neuropathy (n=2), fatigue (n=1), rash (n=1) and hyponatremia (n=1). Response rate was 33% for hepatosplenomegaly and 38% for transfusion-requiring anemia. No favorable changes occurred in bone marrow fibrosis, angiogenesis or cytogenetic status. Pre- and post-treatment patient sample analysis for in vitro myeloid colony growth revealed substantial reduction in the latter. Clinical response did not correlate with either degree of colony growth, measurable decrease in quantitative JAK2(V617F) levels or tipifarnib IC(50) values (median 11.8 nM) seen in pretreatment samples. The current study indicates both in vitro and in vivo tipifarnib activity in PMF and post-PV/ET MF.

A phase I study of 153Sm-EDTMP with fixed high-dose melphalan as a peripheral blood stem cell conditioning regimen in patients with multiple myeloma.

Despite response rates of 30% after high-dose chemotherapy with autologous hematopoietic stem cell transplant, patients with multiple myeloma are not cured. 153Samarium ethylenediaminetetramethylenephosphonate (153Sm-EDTMP; Quadramet) is a short-range, beta-emitting therapeutic radiopharmaceutical with avid skeletal uptake. In total, 12 patients were treated with escalating doses of 153Sm-EDTMP (N=3/group; 6, 12, 19.8, and 30 mCi/kg) and a fixed dose of melphalan (200 mg/m(2)). No dose limiting toxicity was seen. To better standardize the marrow compartment radiation dose, the study was modified such that an additional six patients were treated at a targeted absorbed radiation dose to the red marrow of 40 Gy based on a trace labeled infusion 1 week prior to the therapy. Despite rapid elimination of unbound radiopharmaceutical via kidneys and bladder, no episodes of nephrotoxicity, hemorrhagic cystitis, or delayed radiation nephritis were observed with a median follow-up of 31 months (range 8.5-44). Median times to ANC>0.5 and platelet >20 x 10(6)/l were 12 and 11 days, respectively, with no graft failures. Overall response rate was 94% including seven very good partial responses and five complete responses. Addition of 153Sm EDTMP to melphalan conditioning appears to be safe, well-tolerated and worthy of further study.

Cytotoxicity of cisplatin and cisdiammine-1,1-cyclobutane dicarboxylate in MGH-U1 cells grown as monolayers, spheroids, and xenografts.

The cytotoxicity of cisplatin and cisdiammine-1,1-cyclobutane dicarboxylate (CBDCA) was examined using the MGH-U1 human bladder carcinoma cell line, grown as monolayer cultures, multicellular tumor spheroid(s) (MTS), and xenografts in immune-deprived CBA/CaJ mice. The cell survival of exponentially growing monolayers and MTS treated with cisplatin declined in a monoexponential fashion with a concentration of drug resulting in 10% colony survival (D10) of 7.75 micrograms/ml and 9.5 micrograms/ml, respectively. MTS growth delay determination demonstrated a drug concentration-dependent increase in growth delay and a correlation between decreasing surviving fraction and increasing growth delay. In vivo treatment of MGH-U1 xenografts with cisplatin caused a modest decrease in surviving fraction although the xenografted cells treated in vitro demonstrated the same sensitivity to cisplatin as those cells maintained continuously in vitro. The D10 for CBDCA treatment was 246 micrograms/ml for exponentially growing monolayer cells and 196 micrograms/ml for MTS. Growth delay studies with CBDCA showed a concentration-dependent increase in spheroid growth delay and a correlation between decreasing surviving fraction and growth delay similar to cisplatin. The conclusions were that: 1) cisplatin and CBDCA did not have any difficulty penetrating into spheroids, 2) both agents appeared to be active against the noncycling poorly nourished cells found near the necrotic center of spheroids, 3) both cisplatin and CBDCA were cytotoxic toward MGH-U1 cells but cisplatin was 20-30 times more effective, and 4) the limited cytotoxic effect of cisplatin in vivo may be due to the low area under the concentration times the time curve achieved in vivo and not due to intrinsic cell resistance.

Phase I-phase II trial of N-phosphonacetyl-L-aspartic acid given by intravenous infusion and 5-fluorouracil given by bolus injection.

A phase I clinical trial of N-phosphonacetyl-L-aspartic acid (PALA) and 5-fluorouracil (FUra) was performed on 30 patients. PALA was given as a 15-minute iv infusion once daily for 5 days, and FUra was given as a bolus injection on days 2, 3, 4, and 5. Cycles of treatment were repeated every 3 weeks. Dose-limiting toxicity was manifested by stomatitis and diarrhea. Skin rash was observed also but was not dose limiting. No consistent hematopoietic or renal toxicity was observed. Seventeen patients with disseminated metastatic melanoma and measurable disease were evaluated for response. One partial response was seen; however, the response was associated with significant toxicity, and the treatment could not be repeated. Stable disease was observed in 3 patients with melanoma, 1 patient with colon carcinoma, and 1 patient with ovarian carcinoma. Our findings suggest that the clinical activity of PALA and FUra given according to the above schedule for melanoma is less than 25% (P less than 0.05). Pharmacokinetic studies of FUra revealed no consistent effect of PALA pretreatment on FUra disappearance in plasma. The mean FUra elimination half-line in plasma was 7.11 +/- 0.84 minutes (SEM), which is no different from that reported for FUra alone. The recommended doses on this schedule for phase II studies are 1,000 mg PALA/m2/day iv daily for 5 days and 200 mg FUra/m2/day iv on days 2, 3, 4, and 5.

Superiority of granisetron to dexamethasone plus prochlorperazine in the prevention of chemotherapy-induced emesis.

Trials of selective 5-hydroxytryptamine3 receptor antagonists have shown excellent antiemetic activity for chemotherapy containing cisplatin when compared with high-dose metoclopramide. There is little information about the efficacy of these new agents for chemotherapy other than for high-dose cisplatin. We performed a double-blind, randomized trial comparing a single dose of the 5-hydroxytryptamine3 receptor antagonist granisetron (BRL 43694A) as a single intravenous dose with dexamethasone plus prochlorperazine in 152 patients receiving their first course of moderately emetogenic chemotherapy (mainly doxorubicin- and cyclophosphamide-containing combinations). During the first 24 hours, there was a statistically significant advantage for the granisetron group in terms of the prevention of both nausea and emesis. There was no difference in the frequency of reported adverse events. We conclude that granisetron is more effective than dexamethasone plus prochlorperazine in patients who are receiving moderately emetogenic cytotoxic agents.

Cytotoxicity of adriamycin in MGH-U1 cells grown as monolayer cultures, spheroids, and xenografts in immune-deprived mice.

The cytotoxic activity of Adriamycin was examined in the MGH-U1 human bladder carcinoma line, grown as monolayer culture, as spheroids, and as xenografts in immune-deprived mice. The MGH-U1 cells grown as spheroids were much more resistant to Adriamycin (concentration of drug resulting in 37% cell survival, 4.5 micrograms/ml) than when treated as monolayer cultures (concentration of drug resulting in 37% cell survival, 0.9 microgram/ml). Adriamycin fluorescence was demonstrated only in the outer two layers of cells forming the spheroids, suggesting that limited drug penetration is an important factor in the resistance of spheroids to Adriamycin. Sequential trypsinization of spheroids 750 micron in diameter allowed us to determine the cytotoxic effects of Adriamycin in MGH-U1 cells derived from different depths of the spheroid. We found that cells near the surface of the spheroid had a survival similar to those of exponentially growing monolayer cells treated with Adriamycin. Cells located in the middle of the viable rim were more resistant to Adriamycin, and those found near the necrotic center were most resistant to Adriamycin. The effects of Adriamycin treatment on spheroid growth delay were determined also. In spite of a small cytotoxic effect on the clonogenic fraction of cells in MGH-U1 spheroids, the growth delay effect of Adriamycin in intact spheroids was marked. This observation is consistent with Adriamycin killing primarily the cells in the outer layers of the spheroid, where most of the proliferation in the spheroid occurs. In vivo treatment of MGH-U1 xenografts with Adriamycin followed by assessment of cell survival in vitro showed minimal evidence of cytotoxicity, consistent with the poor drug penetration observed in the spheroid model. These studies suggest that: (a) Adriamycin penetrates poorly into solid tissues; (b) in vitro clonogenic survival following Adriamycin exposure of a cell suspension may predict falsely for drug sensitivity to chemotherapy; (c) a small decrease in clonogenic survival can be translated into a long growth delay but, ultimately, the tumor regrows because some clonogenic cells are spared; and (d) for Adriamycin, the spheroid model more closely parallels the in vivo effects than does monolayer culture. The use of the spheroid model for the study of Adriamycin cytotoxicity gives further insight into the action of this drug in solid tumors.

Effects of thymidylate synthase inhibition on thymidine kinase activity and nucleoside transporter expression.

The effects of de novo dTMP inhibition by N-(5-[N-(3,4-dihydro-2-methyl-4-oxoquinazolin-6-ylmethyl)-N- methylamino]-2- thenoyl)-L-glutamic acid (D1694) or N6-[4-(morpholinosulfonyl)benz]-N6-diaminobenz[cd]indole glucuronate (AG-331) on clonogenic survival, thymidylate synthase (TS) and thymidine kinase (TK) activity, and expression of S-(p-nitrobenzyl)-6-thioinosine-sensitive nucleoside transporter (NT) sites were addressed in the human bladder cancer cell line, MGH-U1. These two TS inhibitors are structurally diverse. D1694 is a folate-based TS inhibitor, whereas AG-331 is a novel agent that inhibits the cofactor binding site of the enzyme. They also differ with respect to their cytotoxic effects in this cell line; D1694 cytotoxic 50% inhibitory concentration (IC50) and IC90 were 6.0 and 9.0 nM, respectively and IC50 and IC90 for TS inhibition were 2.5 and 4.8 nM, respectively. In contrast, AG-331 cytotoxic IC50 could not be achieved even at concentrations of up to 20 microM for 24-h exposures, and IC50 and IC90 for TS inhibition were 0.7 and 3.0 microM, respectively. Similar effects for D1694 and AG-331 were observed in their modulation of TK activity and NT expression. 5-(SAENTA-x8)-Fluorescein, a highly modified form of adenosine incorporating a fluorescein molecule which binds with a 1:1 stoichiometry to S-(p-nitrobenzyl)-6-thioinosine-sensitive NT sites, was used to investigate the expression of NT following exposure of cells to D1694 and AG-331. TK activity was addressed by the metabolism of [3H]thymidine to [3H]TMP by cellular extracted protein and by an alternative flow cytometric method using a modified form of thymidine incorporating a fluorescent molecule, dansyl-5-amino-2-deoxyuridine. Results obtained by both methods were comparable. At concentrations of 5 and 10 nM, D1694 increased TK activity 2.3-4.5-fold and NT expression 34-39-fold. AG-331, at concentrations of 5 and 10 microM, increased TK activity 1.8-2.5-fold and NT expression 22-31-fold, respectively. These data suggest that TK activity and NT expression have a common regulatory mechanism which is sensitive to endogenous dTTP pools and that the salvage pathway is a complex system of kinases coordinated with transport of nucleosides.

ICI D1694 and idoxuridine: a synergistic antitumor combination.

The cytotoxicity of idoxuridine (IdUrd), a thymidine analogue, and ICI D1694 (D1694), a folate-based thymidylate synthase (TS) inhibitor, were examined individually and in combination in two human tumor cell lines. MGH-U1 bladder cancer and HCT-8 colon cancer cells were grown as monolayer cultures with and without thymidine. The cytotoxicity of these agents alone and in combination were determined using normal human bone marrow colony-forming unit, granulocyte-macrophage (CFU-GM) as a surrogate for myelosuppression in vivo. Thymidylate synthase inhibition, IdUrd incorporation into DNA, and DNA single-strand breaks were measured in each cell line and related to cytotoxicity. The cytotoxicity of a 24-h exposure to IdUrd or D1694 increased with drug concentration in each cell line. The drug concentrations producing 50% and 10% clonogenic survival in MGH-U1 cells, respectively, were 0.006 and 0.009 microM for D1694 and 13.0 and 81.0 microM for IdUrd. Those for HCT-8 cells, respectively, were 0.009 and 0.018 microM for D1694 and 7.5 and 20.5 microM for IdUrd. The cytotoxicity of IdUrd combined with D1694 was synergistic in both MGH-U1 and HCT-8 cells as determined by median-effect analysis. The addition of thymidine at concentrations of 0.1, 0.3, and 1.0 microM to the culture medium did not decrease the cytotoxicity of D1694 in either tumor cell line. TS inhibition using the whole cell assay was observed with only D1694, producing 50% inhibition of TS activity at 0.002 microM for MGH-U1 and 0.007 microM for HCT-8 cells. IdUrd did not inhibit TS activity, nor did it enhance the TS inhibitory effects of D1694. The incorporation of IdUrd into DNA increased with increasing concentrations of D1694. This increased DNA incorporation correlated with the increase in DNA single-strand breaks. DNA single-strand breaks paralleled cytotoxicity. CFU-GM survival, exposed to the same drug concentrations as those used in the tumor cell lines, revealed that the therapeutic index was greater for the combination than for either agent alone. These findings suggest that IdUrd plus D1694 is a promising new drug combination, which may have a favorable therapeutic index in vivo.

Application of a simple competitive protein-binding assay technique to the pharmacokinetics of N-(phosphonacetyl)-L-aspartate in humans.

A competitive protein-binding assay for N-(phosphonacetyl)-L-aspartate (PALA) using aspartate transcarbamylase as the receptor protein and [14C]PALA as the radioactive ligand is described here and has been applied to study the pharmacokinetics of PALA in humans. A protein-free ultrafiltrate of plasma, prepared by centrifugation of 1-ml samples through Amicon Centriflo membrane cones, was used in the assay, which had a maximun sensitivity of 0.7 microM PALA in plasma. At this level, the coefficient of variation was less than 10%. Comparison of the competitive binding assay to a gas chromatographic-mass spectrometric technique shows that the two methods yield equivalent results in the concentration range of 1 microM to 1 mM. However, the competitive binding assay possesses practical advantages because of its simplicity and the ease with which multiple samples may be assayed. PALA disappearance from plasma was studied in seven patients and was found to be consistent with a two-compartment open model. The t1/2 alpha (elimination half-life for initial phase) and t1/2 beta (elimination half-life for terminal phase) were 0.93 +/- 0.73 (S.D.) hr and 4.82 +/- 1.48 hr, respectively. The cumulative urinary excretion of PALA int two patients was 70 and 90% of the administered dose 16 hr after the infusion was completed.

A bioassay to measure cytotoxicity of plasma from patients treated with mitomycin C.

The unpredictable clinical toxicity observed in patients treated with mitomycin C and the observation that this agent must be reduced to an active form before alkylating target molecules have led to the development of a bioassay which is capable of detecting biologically active forms of mitomycin C in the plasma of drug-treated patients. The bioassay makes use of a repair-deficient mutant of Chinese hamster ovary cells, UV-20, which is 40 to 60 times more sensitive to mitomycin C than its wild-type parent. A standard curve relating in vitro cell colony-forming ability of UV-20 versus drug concentration in the plating medium has been determined. Mitomycin C levels in patient plasma as low as 1 ng/ml can be detected, compared to the 5-ng/ml limit of detection obtained with a high-pressure liquid chromatography assay for the parent compound. This assay has been utilized to detect active drug in plasma obtained from patients with colorectal cancer treated with mitomycin C as a single agent. At the completion of drug injection, serial blood samples were collected in heparinized tubes, and aliquots of plasma were extracted and assayed for mitomycin C levels by high-pressure liquid chromatography, diluted and assayed directly for their toxicity for UV-20 cells, or frozen at -20 degrees C to be assayed at a later time. The activity detected by immediate bioassay was stable up to 2 mo in frozen samples. Plasma pharmacokinetics determined by the bioassay in seven patients were no different than those determined by high-pressure liquid chromatography. No stable, cytotoxic species other than the parent compound were detected by the bioassay in the plasma of patients treated with mitomycin C.

Serum uridine levels in patients receiving N-(phosphonacetyl)-L-aspartate.

Serum uridine levels of N-(phosphonacetyl)-L-aspartate-treated patients from Phase 1 and continuing trials receiving 1000 to 2000 mg/sq m/day were measured by reverse-phase high-pressure liquid chromatography. For the five patients studied, the predose serum levels ranged from 2.6 to 64. microM, well within the normal range of 1.9 to 8.9 microM. All serum uridine levels decreased in the first 24 hr, but the drop was quite variable (7 to 65%). Uridine levels in all five patients remained being 37 to 85% at differing time points. The only patient to show a daily decrease was the only responder and the patient with the largest decrease. N-(Phosphonacetyl)-L-aspartate appears to have a limited reductive effect on human serum uridine levels.

Phase I pharmacokinetic and pharmacodynamic study of a new anthrapyrazole, CI-937 (DUP937).

We performed a phase I trial of CI-937 (DUP937), an anthrapyrazole, with the following objectives: (a) to determine the maximally tolerated dose in humans; (b) to define the toxicity spectrum of this agent; (c) to describe the pharmacokinetics of the drug; (d) to test a pharmacokinetics based hypothesis of dose escalation; and (e) to relate drug pharmacokinetics to pharmacodynamics. CI-937 was administered as a single bolus injection every 3-4 weeks at doses ranging from 3.6 to 25.2 mg/m2. Thirty-two patients and 57 courses were evaluable for toxicity. Pharmacokinetic analysis was performed in 30 patients on the first course using a sensitive and selective radioimmunoassay. The maximally tolerated dose in patients with no prior therapy was 25.2 mg/m2 and dose-limiting toxicity was neutropenia. Thrombocytopenia, nausea, vomiting, stomatitis, and alopecia were mild. A partial response was recorded in a patient with mesothelioma. The area under the curve increased linearly with dose, and total body clearance of CI-937 was independent of dose. The mean total body clearance was 107 +/- 55.8 ml/min/m2, mean steady state volume of distribution was 492 +/- 469 liters/m2, and terminal half-life was 3.78 +/- 2.86 days. The extended factors of 2 methods of pharmacologically guided dose escalation were intended for use but ultimately were equivalent to that of the modified Fibonacci dose escalation method. Dose and the area under the curve were significant predictors of a percentage change in WBC and neutrophil count in a univariate analysis. Only dose and baseline neutrophil count predicted a percentage change in WBCs in a multifactor analysis. Dose and prior chemotherapy predicted percentage change in neutrophil count in a multifactor analysis. We conclude that the dose-limiting toxicity of CI-937 is neutropenia and that the recommended phase II starting dose is 22 mg/m2.

Phase I pharmacokinetic study of cyclosporin A combined with doxorubicin.

We performed a phase I trial of cyclosporin A (CsA) in combination with doxorubicin (dox) to determine the maximally tolerated dose (MTD) of the combination in man, to define the quantitative and qualitative toxicities of the combination, and to determine the pharmacokinetics of the two drugs when used together. CsA was administered as a continuous infusion for 6 days, and dox was administered as a single 10-min infusion 24 h after the initiation of CsA. The starting CsA infusion rate was 5 micrograms/kg/min, and the dox starting dose was 30 mg/m2. Courses were administered every 4 weeks with first CsA and then dox being escalated in consecutive cohorts of patients until the MTD was determined. Twenty-three patients and 40 courses were evaluable for toxicity. Pharmacokinetic analysis was performed in 23 patients on the first course for whole blood CsA and plasma dox and doxorubicinol. The MTD of CsA was 6 micrograms/kg/min, and for dox it was 45 mg/m2. Dose-limiting toxicity was neutropenia. Serum creatinine and creatinine clearance did not change over the infusion period. Bilirubin increased from a median of 10 mumol/liter at the initiation of the infusion to a median of 40.4 mumol/liter at the end of the infusion but returned to normal before the next cycle of therapy. Nausea and vomiting were common and marked, whereas thrombocytopenia was mild. Two patients, one with small cell lung cancer and one with breast cancer, had stable disease while receiving treatment for 5 and 6 months, respectively. Mean whole blood steady state concentrations of CsA were 2210 ng/ml during the infusion with total body clearance of 0.177 liter/h/kg. The area under the concentration x time curve (AUC) increased linearly with dose of dox, and total body clearance was independent of dose. The mean total body clearance was 2.46 liters/h/m2, and terminal half-life was 49.6 h. The AUC for dox was greater and clearance was less than has been previously reported at the doses administered in this study. The ratio of AUC for doxorubicinol to AUC for dox was less than expected, suggesting that the metabolism and/or excretion of dox was decreased when administered with CsA. We conclude that dox can be combined with infusioned CsA but at a lower dose than when given alone. This may be due to altered metabolism and/or excretion of dox or increased bone marrow stem cell sensitivity to dox.

Phase I trial of N-(phosphonacetyl)-L-aspartate.

A Phase I clinical trial of N-(phosphonacetyl)-L-aspartate, an antimetabolite which inhibits a key enzyme in the de novo pathway of pyrimidine biosynthesis, was conducted. N-(Phosphonacetyl)-L-aspartate was given as an i.v. 15-min infusion once daily for five days; cycles of treatment were repeated every three weeks. Thirty-four patients received treatment. Dose-limiting toxicity was observed at 1500 to 2000 mg/sq m/day and was manifested by skin rash, diarrhea, and stomatitis. Rash and diarrhea usually began during the first week of treatment and persisted up to Day 17 of a cycle of therapy. No consistent hematopoietic, hepatic, or renal toxicity was observed. One partial response in a patient with colon carcinoma was seen and continues at more than eight months. Stable disease was observed in three patients with colon carcinoma, two patients with hypernephroma, one patient with pancreatic carcinoma, and one patient with melanoma. The predictability and reversibility of toxicity and the suggestion of antitumor activity in humans are observations which support the further evaluation of N-(phosphonacetyl)-L-aspartate in Phase II studies.

The HER tyrosine kinase inhibitor CI1033 enhances cytotoxicity of 7-ethyl-10-hydroxycamptothecin and topotecan by inhibiting breast cancer resistance protein-mediated drug efflux.

Because the activities of HER family members are elevated and/or aberrant in a variety of human neoplasms, these cell surface receptors are receiving increasing attention as potential therapeutic targets. In the present study, we examined the effect of combining the HER family tyrosine kinase inhibitor CI1033 (PD 183805) with the topoisomerase (topo) I poison 7-ethyl-10-hydroxycamptothecin (SN-38), the active metabolite of irinotecan, in a number of different cell lines. Colony-forming assays revealed that the antiproliferative effects of simultaneous treatment with CI1033 and SN-38 were synergistic in T98G glioblastoma cells and HCT8 colorectal carcinoma cells, whereas sequential treatments were additive at best. In additional studies examining the mechanistic basis for these findings in T98G cells, immunoblotting revealed that the inhibitory effects of CI1033 on epidermal growth factor receptor autophosphorylation were unaffected by SN-38. Likewise, CI1033 had no effect on topo I polypeptide levels, localization, or activity. Nonetheless, CI1033 markedly enhanced the number of covalent topo I-DNA complexes stabilized by SN-38 or the related agent topotecan (TPT). Analysis of intracellular SN-38 levels by high-performance liquid chromatography and intracellular TPT levels by flow microfluorometry revealed that CI1033 increased the steady-state accumulation of SN-38 and TPT by 9.4 +/- 1.9- and 1.8 +/- 0.2-fold, respectively. Further evaluation revealed that the initial rate of TPT uptake was unaffected by CI1033, whereas the rate of efflux was markedly diminished. Additional studies demonstrated that T98G and HCT8 cells express the breast cancer resistance protein (BCRP), a recently cloned ATP binding cassette transporter. Moreover, CI1033 enhanced the uptake and cytotoxicity of SN-38 and TPT in cells transfected with BCRP but not empty vector. Conversely, CI1033 accumulation was diminished in cells expressing BCRP, suggesting that CI1033 is a substrate for this efflux pump. These results indicate that CI1033 can modulate the accumulation and subsequent cytotoxicity of two widely used topo I poisons in cells that have no history of previous exposure to these agents.

A Phase I trial of the farnesyl transferase inhibitor SCH66336: evidence for biological and clinical activity.

Farnesyl protein transferase (FT), an enzyme that catalyzes the first step in the posttranslational modification of ras and a number of other polypeptides, has emerged as an important target for the development of anticancer agents. SCH66336 is one of the first FT inhibitors to undergo clinical testing. We report a Phase I trial to assess the maximum tolerated dose, toxicities, and biological effectiveness of SCH66336 in inhibiting FT in vivo. Twenty patients with solid tumors received 92 courses of escalating SCH66336 doses given orally twice a day (b.i.d.) for 7 days out of every 3 weeks. Gastrointestinal toxicity (nausea, vomiting, and diarrhea) and fatigue were dose-limiting at 400 mg of SCH66336 b.i.d. Moderate reversible renal insufficiency, secondary to dehydration from gastrointestinal toxicity, was also seen. Inhibition of prelamin A farnesylation in buccal mucosa cells of patients treated with SCH66336 was demonstrated, confirming that SCH66336 inhibits protein farnesylation in vivo. One partial response was observed in a patient with previously treated metastatic non-small cell lung cancer, who remained on study for 14 months. This study not only establishes the dose for future testing on this schedule (350 mg b.i.d.) but also provides the first evidence of successful inhibition of FT in the clinical setting and the first hint of clinical activity for this class of agents.

A randomized trial of fluorouracil and folinic acid in patients with metastatic colorectal carcinoma.

We determined the therapeutic effect of fluorouracil (5-FU) in combination with folinic acid (FA) in patients with measurable recurrent or metastatic carcinoma of the colon or rectum by comparing it to standard 5-FU therapy in a prospective randomized controlled trial. Patients were randomized to receive either FA, 200 mg/m2/d for five consecutive days, or nothing. All patients received 5-FU, 370 mg/m2/d for five days on the first course, with subsequent dose modifications to maintain equal toxicity in the two arms. One hundred thirty patients were entered on trial and only five were excluded from the analysis because they did not meet the eligibility criteria or they refused therapy after randomization. The two treatment arms were balanced for 11 clinical characteristics. Patients were evaluated for response at the end of every two treatment courses and toxicity after every course of therapy. Median follow-up was 1.45 years. Dose-limiting toxicity was mucositis and diarrhea on this treatment schedule, although neutropenia was apparent. The response rate was 33% (21 of 63 patients) in the 5-FU and FA arm and was 7% (four of 61 patients) in the 5-FU arm (P less than .0005). Time to disease progression was significantly different in the combination arm as compared with the single-agent arm (P = .023). Overall survival was significantly longer for patients treated with 5-FU and FA as compared with those receiving 5-FU alone (P = .05). The median survival was 12.6 months for patients receiving the combination, and 9.6 months for those receiving 5-FU alone. Our results indicate that the combination of 5-FU and FA is effective treatment for patients with metastatic or recurrent carcinoma of the rectum and colon who have not received prior chemotherapy.