Clinical Cancer Research Vol. 10, 7127-7135, November 1, 2004
© 2004 American Association for Cancer Research
A Phase I Open Label Study of the Farnesyltransferase Inhibitor CP-609,754 in Patients with Advanced Malignant Tumors
Stacy L. Moulder1,
John J. Mahany1,
Richard Lush1,
Caio Rocha-Lima2,
Michael Langevin1,
Karen J. Ferrante3,
Lisa Michele Bartkowski3,
Shama M. Kajiji3,
Dennis A. Noe3,
Simone Paillet3 and
Daniel M. Sullivan1
1 Experimental Therapeutics and Phase I Programs, Department of Interdisciplinary Oncology, H. Lee Moffitt Cancer Center and Research Institute, University of South Florida, Tampa, Florida; 2
Department of Medicine, Sylvester Cancer Center, University of Miami, Miami, Florida; and 3
Pfizer Global Research and Development, Groton, Connecticut
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ABSTRACT
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Purpose: The purpose of this phase I clinical trial was to determine the maximum-tolerated dose and toxicity of CP-609,754 in patients with solid tumors refractory to standard therapies, to determine the cellular effects of CP-609,754 on its molecular target (farnesyltransferase), and to determine the recommended phase II dose (RP2D) of this agent.
Experimental Design: Consenting patients with adequate bone marrow, liver, and renal function were enrolled with an accelerated dose strategy with single-patient parallel cohorts in whom the drug was given orally either once or twice daily. Once a dose-limiting toxicity was encountered or two patients developed Common Toxicity Criteria 
grade 2 toxicities, a modified Fibonacci sequence was initiated. Blood samples were collected during cycle 1 for pharmacokinetic and pharmacodynamic analyses.
Results: A total of 68 cycles of CP-609,754 was administered to 21 patients enrolled in this study. The dose escalation was from 20 mg once daily to 640 mg twice per day, and at the highest dose level, one of six patients developed a dose-limiting toxicity of grade 3 neuropathy. The drug was otherwise well tolerated, and the maximum-tolerated dose was not reached because of the large number of tablets that would have been required for additional dose escalation. Pharmacokinetic analyses showed a proportional increase in exposure with dose, rapid oral absorption, and a half-life of 
3 hours. Pharmacodynamic results predict a 95% maximal inhibition of peripheral blood mononuclear cell farnesyltransferase activity 2 hours postdose, on average, with a dose of 400 mg twice per day of CP-609,754.
Conclusions: On the basis of the safety findings and the pharmacokinetic and pharmacodynamic analyses, the RP2D of CP-609,754 is 640 mg twice per day.
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INTRODUCTION
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The Ras oncogene has an activating mutation in 30% of all human tumors and plays a key role in transducing extracellular signals involved in cancer cell proliferation and survival (1)
. Blocking mutated Ras has been shown to reverse malignant transformation in preclinical models (2)
, additionally supporting the hypothesis that inhibition of Ras can be used as antineoplastic therapy. Ras proteins transduce growth and differentiation signals from receptor tyrosine kinases to the cell nucleus where gene transcription is initiated (3)
. Protein prenylation (attachment of a thiol-ester) is required for membrane localization and subsequent signal transduction by the Ras oncoprotein (4
, 5)
. The thiol-ester attachment of a farnesyl moiety is catalyzed by farnesyltransferase, an enzyme that can be specifically targeted to inhibit posttranslational processing of proteins such as Ras (6)
.
Although ras-driven cell proliferation is a valid target for biological therapy, farnesylated proteins other than Ras may also contribute to the mechanism of action of these drugs (7)
. RhoB is a protein that can undergo farnesylation or geranylgeranylation and is involved with proliferation, adhesion, cytoskeleton organization, and induction of apoptosis (8
, 9)
. Treatment with farnesyl transferase inhibitors increases levels of geranylgeranylated RhoB, which can induce apoptosis (10
, 11)
. Additional research is needed to determine the exact mechanism of action of farnesyl transferase inhibitors; however, because of the largely cytostatic effects of these compounds in preclinical experiments, it is likely that these drugs will require prolonged and continuous exposure to maximize efficacy (12)
.
CP-609,754 [chemical name: 6-[(4-chlorophenyl) hydroxyl (1-methyl-1-H-imidazol-5-yl) methyl]-4-(3-ethynylphenyl)-1-methyl-2-(1H)-quinolinone, (2R,3R)-2,3-dihydroxybutanediote (1:1)] is a D(-) tartrate salt (Fig. 1)
that shows selective inhibition of farnesyltransferase with both in vitro and in vivo models (Table 1)
. The IC50 for inhibiting farnesylation of recombinant human H-Ras is 0.57 ng/mL and recombinant K-Ras is 46 ng/mL. Kinetic studies with recombinant human farnesyltransferase indicate that CP-609,754 is competitive for the prenyl acceptor (H-Ras protein) and noncompetitive for the prenyl donor farnesyl PPI, suggesting that the compound interacts with the farnesyltransferase-farnesyl PPI complex and competes for the binding of the Ras protein. Additional studies indicate that CP-609,754 is a reversible inhibitor of farnesyltransferase with a slow on/off rate.
CP-609,754 inhibits farnesylation (IC50 = 1.72 ng/mL) of mutant H-Ras in 3T3 H-ras (61L)-transfected cell lines with SDS-PAGE analysis of [35S]methionine-labeled material. This effect is not seen in K-Ras transfected cell lines even at concentrations up to 4790 ng/mL. Because K-Ras protein is alternatively prenylated by GGTase-1 in the presence of farnesyltransferase inhibitors, it cannot be separated from farnesylated K-Ras during SDS-PAGE analysis of [35S]methionine-labeled proteins. Similar analysis with tritiated prenyl precursors instead of [35S]methionine confirms that CP-609,754 selectively inhibits farnesylation of both H- and K-Ras proteins in 3T3 transfectants.
CP-609,754 has in vivo antitumor activity against 3T3 H-ras (61L) tumors. With twice daily oral dosing of CP-609,754, tumor regression is achieved with a dose of 100 mg/kg; the ED50 for tumor growth inhibition is 28 mg/kg. With continuous i.p. infusion of CP-609,754, tumor growth is inhibited by >50%, and tumor farnesyltransferase activity inhibited by >30% in mice in which the plasma concentration of CP-609,754 is maintained above 118 ng/mL. On the basis of these findings, it was projected that CP-609,754 will inhibit farnesyltransferase activity and be clinically efficacious against ras-expressing tumors if plasma concentrations of CP-609,754 are maintained above 118 ng/mL.
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PATIENTS AND METHODS
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Patient Selection.
Twenty-one patients were enrolled onto this phase I trial. The University of South Florida Institutional Review Board approved this trial, and verbal and written informed consent was obtained from all patients. Eligible patients had a diagnosis of an advanced solid tumor that was refractory to standard therapies or for which no standard therapy existed. Enrollment criteria included measurable disease, adequate bone marrow function (absolute neutrophil count 1500 cells/mm3, platelets 100,000 cells/mm3), bilirubin 
1.5 mg/dL, aspartate aminotransferase and alanine aminotransferase 2.5x the upper limits of normal or 5x the upper limits of normal with documented liver metastasis, serum creatinine 1.5x the upper limits of normal or an estimated creatinine clearance 60 mL/min, Eastern Cooperative Oncology Group performance status of 0 to 2, life expectancy > 12 weeks, and age 18 years. Exclusion criteria included a history of chemotherapy, radiation therapy, immunotherapy, and/or other investigational agents within 4 weeks of study entry (6 weeks for previous treatments with carboplatin, nitrosoureas, or mitomycin), radiotherapy to >30% of bone marrow containing areas, history of bone marrow transplantation, history of central nervous system malignancy, serious concomitant medical disorders incompatible with evaluating the study drug, clinically significant gastrointestinal abnormalities, including requirement for i.v. alimentation, malabsorption syndromes, and active peptic ulcer disease. Patients with a history of cerebrovascular events, uncontrolled seizure disorder, uncontrolled infection, history of sensitivity to imidazole containing drugs, chronic steroid therapy, significant laboratory abnormalities requiring medical intervention, and pregnant and breastfeeding women were also excluded from study entry.
Drug Supply and Treatment Schema.
CP-609,754 was manufactured by Pfizer, Inc., and supplied as 10- or 50-mg tablets administered on an empty stomach (at least 1 hour before or 2 hours after food intake). This was an open-label, dual cohort, phase I dose escalation trial to establish the maximum-tolerated dose, dose-limiting toxicity (DLT) and a recommended phase II dose (RP2D) of CP-609,754. Initially, the drug was administered on a once daily oral dosing schedule with a cycle of therapy defined as a 28-day period of study drug administration; however, a second parallel cohort was added to explore a twice daily dosing schedule after initial pharmacokinetic analysis revealed the drug’s half life to be <12 hours. The starting dose for this study was 20 mg/day based on preclinical pharmacology and toxicity data. In cycle 1, day 1 and day 15 doses were administered during a 24-hour hospitalization to monitor for toxicity and to collect blood for pharmacokinetic and pharmacodynamic study end points. All other doses were given on an outpatient basis with weekly monitoring for toxicity. After the first three patients completed 15 days of therapy at a starting dose of 20 mg/day, two new cohorts were initiated with one patient per cohort. The first cohort received the same total daily dose, but CP-609,754 was administered on a twice daily dosing schedule. The second cohort received an escalated dose administered once daily. Dose escalation initially followed an accelerated dose strategy with one subject per cohort and a 100% dose escalation once the lower dose cohort had safely completed 15 days of therapy. A modified Fibonacci dose escalation design was initiated at the dose level in which one subject experienced a DLT or two subjects at the same or different dose level developed Common Toxicity Criteria grade 2 toxicities. This design increased all subsequent cohorts to three to six patients with subsequent dose escalations of 67, 50, 40, and 33% of the prior dose level. The maximum-tolerated dose was defined as the dose of CP-609,574 that produced DLT in <33% of subjects treated at a given dose level. At the maximum-tolerated dose, each of the two dosing regimens, once daily and twice per day, was to be evaluated by expanding the cohort to six subjects.
DLT was defined by adverse events according to the National Cancer Institute, Common Toxicity Criteria (version 2.0) and included grade 4 neutropenia persisting for >7 consecutive days or associated with fever > 38°C; grade 4 thrombocytopenia or grade 3 thrombocytopenia associated with bleeding requiring platelet transfusion; grade 4 nonhematologic toxicity related to the study drug; grade 3 nonhematologic toxicity that failed to resolve to <grade 2 within 7 days (except nausea, vomiting, fatigue, or asthenia); or nausea and/or vomiting (>grade 2) that persisted with maximum treatment and/or prophylaxis.
Chemistry and hematologic measurements were completed weekly while patients were receiving therapy with additional clinic visits scheduled at the discretion of the investigator. In the absence of disease progression or unacceptable toxicity, treatment was to continue for a maximum of 12 cycles (48 weeks), with provisions for additional treatment in responding patients.
Response Evaluation.
Tumor measurements were repeated every two cycles. Standard Response Evaluation Criteria in Solid Tumors were used to evaluate response. The best overall response was recorded from the start of treatment until disease progression or recurrence.
Pharmacokinetics.
Blood specimens for CP-609,754 analysis were collected in sodium heparinized tubes on days 1 and 15 of cycle 1. For once daily dosing, the sampling schedule was predose and 1, 2, 3, 4, 6, 8, 12, 16, and 24 hours after dosing. For twice daily dosing, the sampling schedule was predose and 1, 2, 3, 4, 6, 8, and 12 hours after each dose. Specimens were centrifuged at 1500 relative centrifugal force for 10 to 15 minutes at 5°C, and the separated plasma was stored in labeled, screw-capped polypropylene tubes at temperatures –20°C within 1 hour of collection.
Plasma CP-609,754 concentrations were assayed at Pfizer Global Research and Development (Groton, CT) with a validated assay method. Plasma aliquots had internal standard added (Pfizer compound CP-595,730) and were acidified with 1% acetic acid. Drug and internal standard were extracted with a Waters OASIS MCX (10 mg) SPE 96-well extraction plate and were eluted with 5% ammonium hydroxide/95% methanol. The eluates were dried down and reconstituted in 50:50 acetonitrile:10 mmol/L ammonium acetate. Analytical separation was done with reverse phase liquid chromatography with a LUNA 5-µm C8(2) 2.00 x 50-mm column (Phenomenex, Torrance, CA) preceded by a 2.0-µm stainless steel precolumn filter. The detection method was turbospray tandem mass spectrometry done on a Sciex API 3000 mass spectrometer (PE Biosystems, Foster City CA). The lower limit of quantification for the assay was 1 ng/mL, and the upper limit of quantification was 250 ng/mL. The intra-assay accuracy and precision (as coefficient of variation) were, respectively, 94 and 4.4% at the lower limit of quantification, 93 and 2.3%, at the upper limit of quantification, and 93 to 110% and 2.3 to 5.5% over the quantifiable range.
Individual patient pharmacokinetic parameter values were estimated from each patient’s concentration-time data with a noncompartmental approach. The parameters estimated were observed maximum plasma concentration (Cmax), the time of occurrence of Cmax (Tmax), the observed end-of-dosing-interval plasma concentration (Cmin), and the terminal phase half-life (T1/2). For day 1 data, the area under the plasma concentration-time curve extrapolated to infinity (AUC0-inf), and for day 15 data, the AUC over the dosing interval (AUC0-
) and the accumulation ratio were calculated. Cmax, Tmax, and Cmin were determined empirically. T1/2 was calculated as ln(2) divided by the terminal phase rate constant, which was estimated with ordinary least-squares regression of time on log-transformed plasma concentration data from the terminal phase; visual examination of the aggregate plasma concentration data indicated that the terminal phase was present beginning 4 hours after dosing, so the 4-hour time point was used as the start of the terminal phase for all of the patients. AUC was calculated with the linear trapezoidal rule over the range of observed data; the extrapolated portion of AUC0-inf was calculated with the estimate of the terminal rate constant found in calculating T1/2. The accumulation ratio was calculated as day 15 AUC0- divided by day 1 AUC0-inf.
Pharmacodynamics.
The pharmacodynamic marker evaluated in this study was farnesyl transferase activity in peripheral blood mononuclear cells. Blood was collected into sodium citrate-containing mononuclear cell preparation tubes (Vacutainer CPT#362753, 8 mL; Becton Dickinson; Franklin Lakes, NJ) on day 1 and day 15 of cycle 1. For both once daily and twice daily dosing, the sampling schedule was predose and 2, 12, and 24 hours after the morning dose of CP-609,754.
Blood samples were stored upright at room temperature (18°C to 25°C) for no longer than 2 hours until processed at the study site for the collection of peripheral blood mononuclear cells. Peripheral blood mononuclear cell pellets were prepared at the study site with the following procedure: Vacutainer tubes were inverted 8 to 12 times to mix the anticoagulant with blood. The samples were centrifuged at 1500 to 1800 relative centrifugal force at room temperature in a horizontal (swing-out head) rotor for 30 minutes (with no brake). The upper half of the plasma layer was discarded without disturbing the cell layer containing the peripheral blood mononuclear cells. The peripheral blood mononuclear cell layer was collected and transferred to a conical polypropylene centrifuge tube. The pooled peripheral blood mononuclear cells were resuspended in an excess of room temperature sterile PBS and then centrifuged for 10 to 15 minutes at 300 relative centrifugal force. The supernatant was aspirated and discarded and the wash procedure repeated two more times (total of three PBS washes). The pellet was resuspended in room temperature sterile PBS and a cell count done with an aliquot from the resuspended pellet. The sample was then recentrifuged (15 minutes at 300 relative centrifugal force) and the supernatant completely removed. The cell pellets were immediately flash-frozen in liquid nitrogen and the sample stored at –80°C until shipped.
Peripheral blood mononuclear cell homogenates were assayed for farnesyltransferase activity at Pfizer Global Research and Development by measuring the transfer of the farnesyl group from [3H]farnesyl PPI (FPP) to a prenyl acceptor (biotinylated-KTKCVIS peptide) with the Scintillation Proximity Assay (Amersham Life Science Products, Arlington Heights, IL).
Pharmacokinetic/Pharmacodynamic Analysis.
Peripheral blood mononuclear cell farnesyltransferase activity (expressed as fraction of pretreatment activity) was modeled as being related directly to the plasma CP-609,754 concentration measured at the corresponding time with the Emax model of drug action,
where Imax is the maximum extent of inhibition of farnesyltransferase activity, IC50 is the Michaelis constant of the inhibition process, and Cp is the plasma concentration of CP-609,754 measured at the same time as the peripheral blood mononuclear cell farnesyltransferase activity. The model was applied to all of the 2-hour postdose CP-609,754 concentrations and peripheral blood mononuclear cell farnesyltransferase activity data simultaneously to derive population estimates of Imax and IC50. The 2-hour postdose data were selected because they are the observed data closest to the time of maximal plasma concentration of CP-609,754 and, therefore, of maximal inhibition of peripheral blood mononuclear cell farnesyltransferase activity. Parameter estimation was done by nonlinear regression analysis (WinNonlin, Pharsight Corporation, Mountain View CA).
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RESULTS
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Demographics.
A total of 21 patients were enrolled in the study. One patient with non–small-cell lung cancer withdrew from the study after developing a coagulopathy with an increased prothrombin time after 14 days of therapy at the 20 mg once daily dose. The patient was receiving warfarin for a history of deep venous thrombosis, so the possibility of drug interaction could not be excluded. Patient characteristics are as outlined in Table 2
.
Dose Escalation.
A total of 68 cycles was administered, and the median number of cycles per patient was 6.3 (range, <1 to 13). The mean duration of therapy for all patients treated was 89 days. Dose escalation proceeded per protocol guidelines until the 1280 mg once daily dose level was reached. As a result of the large number of tablets required for dose administration, the 1280 mg once daily dose level was abandoned, and the 640 mg twice daily dosing cohort was expanded to include three patients. Although no DLTs were reported during the first 14 days of treatment for each of the three patients enrolled at this dose level, additional accrual to a higher dose level was interrupted for 46 days due to the lack of available study drug. During this interval, a single patient within the 640 mg twice daily dosing cohort experienced grade 3 febrile neutropenia after two cycles of therapy (symptoms resolved within 7 days) and grade 3 peripheral neuropathy after three cycles of therapy. The peripheral neuropathy lasted >7 days and resulted in discontinuation of the study drug; however, this was not considered a DLT because the patient had completed more than one cycle of therapy. Although this patient was not considered to have a DLT per protocol specifications, this dose level was expanded to include a total of six patients. One additional patient developed grade 3 neuropathy consisting of dizziness and unsteady gait after 11 days of therapy. The drug was held for 5 days and then restarted after the neuropathy decreased to < grade 2. After taking the drug for an additional 3 days, the neuropathy returned, and the patient discontinued study protocol, which resulted in the only DLT reported during the study. No additional DLTs occurred; however, the study was halted at the 640 mg twice daily dose level due to lack of available study drug and concerns about patient compliance with the large number of tablets required for additional dose escalation.
Hematologic Toxicity.
When considering both treatment- and nontreatment-related toxicities, lymphopenia and anemia were the most common hematologic toxicities encountered during the study. Grade 3 lymphopenia occurred throughout most of the dose levels studied. Anemia also occurred throughout the dose levels studied but was usually mild (grade 1 or 2). One patient treated at the 640 once daily dose level did, however, develop grade 3 anemia. Four patients developed leukopenia (one patient, grade 1; one patient, grade 2; and two patients, grade 3) within the 640 mg twice daily dosing cohort. One patient in the 640 mg twice daily dosing cohort developed grade 3 febrile neutropenia after 2 months of therapy. Although no source was identified, the fever resolved with broad spectrum antibiotics, and the patient restarted the study drug after resolution of symptoms. An additional cycle of therapy was administered without recurrence of neutropenia or fever. Thrombocytopenia was not seen in the lower dose levels, but two patients in the 640 mg twice daily dosing cohort developed grade 2 and 3 thrombocytopenia, respectively. All treatment-related grade 3 and grade 4 adverse events are outlined in Table 3
.
Nonhematologic Toxicity.
When considering both treatment-related and nontreatment-related toxicities, grade 1 to 2 elevations or reductions in serum electrolytes were seen at all dose levels studied. Grade 3 hypokalemia was reported in patients at the 20, 160, 320 mg once daily dose levels and at the 320 mg twice daily dose level (one patient at each dose level); the events at the 20 and 160 mg once daily dose levels were not attributed to study drug. Grade 3 hypocalcemia occurred in one patient at the 20 mg once daily dose level but was not considered to be due to study treatment. One patient in the 10 mg twice daily dose level developed grade 2 hyperbilirubinemia, and grade 4 elevations in alkaline phosphatase and gamma glutamyltransferase that were attributed to progression of disease. Two additional patients developed grade 3 elevations of gamma glutamyltransferase at the 80 mg once daily and 40 mg twice daily dose levels, respectively. Grade 4 hyponatremia and grade 3 elevation in serum creatinine also occurred in one patient with colorectal carcinoma treated at the 160 mg once daily dose level. Additional evaluation revealed that the patient had progression of disease within the pelvis causing ureteral obstruction.
The most frequent adverse events encountered at the 640 mg twice daily dose level are listed in Table 4
. Grade 3 peripheral neuropathy occurred in one patient after three cycles of therapy, and the study drug was discontinued after the neuropathy did not resolve to grade 1 within 7 days. A second patient also developed grade 3 neuropathy after 11 days of therapy at the 640 mg twice daily dose level. Other grade 1 nonhematologic toxicities (not listed in Table 4
) included (one each), myasthenia, pharyngitis, taste perversion, dyspepsia, abdominal pain, and fever.
Tumor Response.
Patients were evaluated for response after the first two cycles and every two cycles thereafter. There were no objective responses observed, but 10 patients had prolonged stabilization of disease for >2 months (range, 68 to 367 days, see Table 5
). The majority of patients with stable disease had colorectal cancer (four patients), bronchoalveolar carcinoma (two patients), or renal cell carcinoma (two patients). An additional patient with hepatocellular carcinoma had stable disease for 3 months before discontinuing the study drug secondary to neuropathy. The longest duration of stable disease (367 days) was observed in a patient with thyroid carcinoma who later discontinued the drug due to mild renal insufficiency. The renal insufficiency did not improve after discontinuation of the drug and was attributed to the patient’s diabetes mellitus.
Pharmacokinetics.
Pharmacokinetic data were obtained for eight patients receiving CP-609,754 by once daily dosing and for 11 patients receiving CP-609,754 by twice daily dosing. The pharmacokinetic parameter values for patients receiving CP-609,754 in individual subject twice daily dosing cohorts are listed in Table 6
. The mean pharmacokinetic parameter values for patients in the 640 mg twice daily dosing cohort, the only twice daily cohort with multiple patients, are listed in Table 7
, and the mean day 15 plasma CP-609,754 concentration-time curve for patients in this cohort is shown in Fig. 2
. Within the 640 mg twice daily dosing cohort, the absorption of CP-609,754 is fairly rapid, with Tmax between 1 and 2 hours after morning dosing and 4 hours after evening dosing. The half-life is short, 3 hours. The accumulation of CP-609,754 with multiple dosing is less than expected, with a mean accumulation ratio of 0.75 for the morning dose and 0.54 for the evening dose. Cmin for the evening dose is close to the target concentration of 118 ng/mL.
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Table 6 Pharmacokinetic parameters for patients receiving CP-609,754 in individual subject twice daily dosing cohorts
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Table 7 Mean (SD) of pharmacokinetic parameters for patients receiving CP-609,754 in 640 mg twice daily dosing cohort
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Fig. 2. Mean (±SE) day 15 plasma CP-609,754 concentration-time curve for patients receiving 640 mg CP-609,754 twice daily (n = 5).
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Pharmacodynamics.
Peripheral blood mononuclear cell farnesyltransferase activity data were obtained for three patients receiving CP-609,754 by once daily dosing and for nine patients receiving CP-609,754 by twice daily dosing. Plasma concentration data were obtained for all of these patients. Table 8
lists the day 1 and day 15 2-hour postmorning dose CP-609,754 concentrations and peripheral blood mononuclear cell farnesyltransferase activity values according to dose. The 2-hour postdose peripheral blood mononuclear cell farnesyltransferase activity represents the lowest measured peripheral blood mononuclear cell farnesyltransferase activity for most patients. Greater than 70% inhibition of peripheral blood mononuclear cell farnesyltransferase activity was achieved at that time point by all patients receiving 160 mg CP-609,754 once daily or twice daily dosing. The 2-hour postdose peripheral blood mononuclear cell farnesyltransferase activity is inversely correlated with the daily dose (r = –0.81). Table 9
lists the day 15 morning and evening predose CP-609,754 concentrations and peripheral blood mononuclear cell farnesyltransferase activity values. For twice daily dosing, the morning predose peripheral blood mononuclear cell farnesyltransferase activities are mostly lower than the evening predose activities. This is so because the end of dosing interval concentrations after the evening dose are, for the most part, higher than for the morning dose, likely due to a food effect from the evening meal (see Fig. 2
). For the patients dosed at 640 mg twice daily dosing, the peripheral blood mononuclear cell farnesyltransferase activity is, on average, 60% inhibited at the evening predose time point, which is the time of nadir plasma concentration for twice daily dosing of CP-609,754.
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Table 8 Day 1 and day 15 2-hour postmorning dose plasma CP-606,754 concentrations (C2 h) and fractional FTase activity values
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Table 9 Day 15 morning and evening predose plasma CP-606,754 concentrations (C0 h) and fractional FTase activity values
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The Emax model yielded a good fit of the combined day 1 and day 15 2-hour postmorning dose data, as shown in Fig. 3
(r2, 0.51). The population estimate of Imax is 0.92 (SE, 0.03) and of IC50 is 31.6 (SE, 8.5) ng/mL. The relationship between the 2-hour postdose peripheral blood mononuclear cell farnesyltransferase activity and twice daily dose was calculated with plasma CP-609,754 concentrations predicted from a linear relationship between morning twice daily dose and 2-hour postdose concentration. Using this relationship, a 200 (95% confidence interval, 5–375) mg twice daily dose is predicted to provide, on average, 90% maximal inhibition of peripheral blood mononuclear cell farnesyltransferase activity, and a dose of 400 (95% confidence interval, 225–580) mg is predicted to result in, on average, 95% maximal inhibition.
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Fig. 3. The Emax pharmacokinetic/pharmacodynamic model fit of the combined day 1 and day 15 2-hour postmorning dose fractional peripheral blood mononuclear cell (PBMN) Farnesyltransferase (FTase) activity data. The observed data are shown as symbols, whereas the model fit of the data are shown as a solid line.
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There is no predictive relationship between the degree of inhibition of farnesyltransferase and the clinical response of the patients (stable disease versus progressive disease) in the small number of patients in whom farnesyltransferase data were available.
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DISCUSSION
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In the phase I setting, where patients have advanced refractory disease and most are enrolled at dose levels below those thought to be maximally efficacious, it is only infrequently possible to show a biological effect of therapy by monitoring tumor response. In contrast, cellular effects can often be showed convincingly, even in the phase I setting, with pharmacodynamic measures. Such a demonstration increases the confidence-in-mechanism for the investigational agent, thereby providing impetus for its continued development. Pharmacodynamic monitoring was undertaken in this study to show the desired cellular effect of treatment with CP-609,754 and the inhibition of its molecular target, farnesyl transferase. Farnesyltransferase activity was measured in peripheral blood mononuclear cell cells because of the ease of obtaining serial specimens. The data, although constituting a small data set, show that a clinically significant degree of inhibition of farnesyltransferase activity was likely achieved with the higher doses of CP-609,754 evaluated in this study.
On the basis of mouse tumor experiments, it is predicted that CP-609,754 will be clinically efficacious against ras-expressing tumors if concentrations are maintained above 118 ng/mL. Cmin values after the evening dose of 640 mg twice daily approached this target value (Table 7)
. After the morning dose, concentrations of CP-609,754 fell below the target value after 8 hours (Fig. 2)
. This indicates that a dose of 640 mg twice daily is close to but slightly less than the dose needed to achieve the preclinical target concentration values.
The toxicity profile for 640 mg twice daily dosing, the highest dose evaluated in this study, was favorable, although two patients developed significant neurotoxicity, which resulted in discontinuation of study drug. One patient developed peripheral neuropathy after 3 months of therapy; however, this patient was not considered a DLT because more than one cycle of therapy was administered before developing neuropathy that resulted in study drug discontinuation. Although the event was not considered a DLT, this dose cohort was expanded to six patients to additionally assess delayed toxicity at the 640 mg twice daily dose level. A second patient developed dizziness and unsteady gait after 11 days of therapy. The second patient’s symptoms resolved with discontinuation of CP-609,754 but recurred shortly after the drug was restarted, resulting in the study’s only reported DLT. Although the etiology of the neuropathy is unknown, similar reports of neurotoxicity have been seen in phase I testing of other drugs designed to inhibit farnesyltransferase (12
, 13)
.
Elevations in creatinine have also been seen in patients treated with other farnesyltransferase inhibitors in phase I or II clinical trials (14, 15, 16)
. Two patients treated on this study developed elevations in serum creatinine; however, neither was considered to be secondary to drug therapy. One patient with an elevated creatinine developed progression of disease, resulting in urinary obstruction, and the second patient developed mild renal insufficiency after completing 13 cycles of therapy. The renal insufficiency did not resolve with discontinuation of study drug and was attributed to an underlying diagnosis of diabetic nephropathy. Although gastrointestinal side effects have been dose limiting for other farnesyltransferase inhibitors in clinical development (16
, 17) , only mild to moderate gastrointestinal side effects were reported with CP-609,754. All patients responded to supportive care and these events were not dose limiting.
Hematologic toxicity was mild in the lower dose levels with anemia being the most common laboratory abnormality noted. One patient developed febrile neutropenia at the 640 mg twice daily dose level during the second cycle of therapy. After treatment with antibiotics and study drug discontinuation for 5 days, the neutropenic fever resolved, and the patient restarted CP-609,754 without dose reduction. The patient received 30 additional days of therapy without recurrence of neutropenia or fever. However, the study drug was discontinued after three cycles when the patient developed grade 3 peripheral neuropathy that did not resolve with discontinuation of study drug. Mild to moderate leukopenia and lymphopenia were also seen. This is not unexpected because Ras plays an important role in the activation of T cells and natural killer cells (18)
, and lymphopenia has been seen as a side effect during preclinical, as well as phase I development of other farnesyltransferase inhibitors (12
, 19)
. The significance of this finding is also difficult to determine in patients who have received extensive therapy for metastatic cancer; however, the absence of opportunistic infections is clinically reassuring. One patient with non–small-cell lung cancer developed coagulopathy with an increased prothrombin time after receiving 14 days of therapy at the 20 mg once daily dose and was withdrawn from study. The patient was receiving warfarin for a history of deep venous thrombosis, so the possibility of drug interaction could not be excluded. Grade 3 hypokalemia was also frequently seen on weekly lab evaluation. The etiology of the hypokalemia is unknown and has not been reported for other farnesyltransferase inhibitors in clinical development.
Although no measurable responses occurred during the study, 10 patients had prolonged stabilization of disease > 2 months (see Table 5
). Patients with stable disease had variety of tumor types, including colorectal, bronchoalveolar, renal cell, hepatocellular, and thyroid carcinoma. All patients treated at the 640 mg twice daily dose had stable disease for >2 months, except for one patient who developed neurotoxicity and stopped therapy after 11 days of treatment. The longest duration of stable disease (367 days) was observed in the patient with thyroid carcinoma treated at the 640 mg twice daily dose level. The mean duration of response for patients who tolerated therapy in this cohort was 160 days (range, 71 to 367 days), which shows that chronic oral dosing of CP-609,754 is feasible and could possibly result in prolonged disease stabilization.
In conclusion, this study has showed that chronic oral dosing with CP-609,754 can inhibit farnesylation in peripheral blood mononuclear cell cells at doses that were easily tolerable. The maximum-tolerated dose was not reached in this study due to the large number of tablets that would have been required for additional dose escalation. On the basis of the safety findings and the pharmacokinetic and pharmacodynamic analyses, the recommended dose for phase II testing is 640 mg twice daily. Treatment with CP-609,754 resulted in stabilization of disease for >2 months in all of the patients who could tolerate the 640 mg twice daily dose; therefore, future study should be considered.
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FOOTNOTES
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Grant support: Pfizer Global Research & Development.
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.
Requests for reprints: Daniel M. Sullivan, 12902 Magnolia Drive, Tampa, FL 33612-9497. Phone: (813) 979-3878; Fax: (813) 979-7265; E-mail: sullivad{at}moffitt.usf.edu
Received 5/ 6/04;
revised 7/19/04;
accepted 7/21/04.
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ABSTRACT
INTRODUCTION
