Phase I Pharmacokinetic and Pharmacodynamic Study of Weekly 1-Hour and 24-Hour Infusion BMS-214662, a Farnesyltransferase Inhibitor, in Pati
http://www.100md.com
《临床肿瘤学》
the Departments of Medical Oncology, Pathology, and Radiology, Vall d'Hebron University Hospital, Barcelona, Spain
Bristol-Myers Squibb, Wallingford, CT, Waterloo, Belgium, and Madrid, Spain
ABSTRACT
PURPOSE: BMS-214662 is a potent, nonpeptide, small molecule inhibitor of human farnesyltransferase (FT). We have conducted a phase I pharmacokinetic (PK) and pharmacodynamic study of BMS-214662 administered intravenously weekly with 1- and 24-hour infusions. The objectives were to determine the dose-limiting toxicities and the recommended dose (RD), to describe PKs, and to evaluate the relationships between BMS-214662 exposure, FT inhibition, downstream signaling, and induction of apoptosis in tumor samples.
PATIENTS AND METHODS: Patients with advanced solid tumors and adequate organ function were eligible. The dose was escalated according to a modified Fibonacci schedule.
RESULTS: BMS-214662 was escalated from 56 to 278 mg/m2 in 37 patients in the 1-hour schedule, and from 84 to 492 mg/m2 in 31 patients in the 24-hour schedule. Dose-limiting toxicities included gastrointestinal and renal events. The RDs were 209 mg/m2 and 275 mg/m2 in the 1- and 24-hour schedules, respectively. Five patients (three with breast, one with gastric, and one with renal cell cancer) had clinical benefit from treatment. BMS-214662 exhibited linear PKs with area under the concentration-time curves at the RDs of 27 and 32 μM x h in the 1- and 24-hour schedules, respectively. The pattern of FT inhibition in peripheral-blood mononuclear cells at the RDs was different in the two schedules: high (> 80%) but short-lived ( 6 hours) in the 1-hour infusion and moderate (> 40%) but long-lived (24 hours) in the 24-hour infusion. BMS-214662 induced apoptosis in tumors but did not inhibit MAPK signaling.
CONCLUSION: BMS-214662 can be safely delivered in both the 1-hour and 24-hour infusions at biologically active doses, with the preclinical, PK, and pharmacodynamic profiles favoring the 24-hour schedule.
INTRODUCTION
Ras proteins are critical elements in signaling mediated by receptor tyrosine kinases. The position of these proteins in receptor tyrosine kinase signaling explains their key role in a large number of cellular processes including growth, differentiation, apoptosis, cytoskeletal organization, and membrane trafficking.1-3 After the synthesis of the Pro-Ras protein, a series of post-translational biochemical processes convert this protein to a more hydrophobic one that permits its localization to the cytoplasmic membrane. The first modification of this process is catalyzed by the farnesyltransferase (FT) enzyme, causing the covalent addition of a farnesyl group to the cysteine residue of the C-terminal CAAX sequence of the propeptide. As the first step in this process, farnesylation is the most critical one, and FT blockade causes severe impairment in Ras protein function. Moreover, FT activity in human tumor cells is higher than in normal surrounding cells.4 Therefore, FT has become a relevant target in anticancer drug development.5-11 Initially it was thought that FT inhibitors (FTIs) might selectively decrease the growth of Ras-transformed cells and tumors harboring Ras mutations.12-17 More recent studies have demonstrated that there is no clear correlation between Ras mutations and sensitivity to FTIs.6,18-26 Whereas FTIs effectively inhibit H-ras signaling and transformation, they do not block either the processing or the function of K-ras 4B,27 the isoform of Ras most frequently mutated in human tumors. Indeed, in the presence of FTIs, K-ras can become alternatively prenylated by the FT-related enzyme geranylgeranyltransferase I (GGTI).28,29 Nevertheless, some FTIs can significantly inhibit the growth of tumors containing mutated K-ras4B.30-32 This indicates either that farnesylated proteins other than Ras, like RhoB and the centromere-associated CENP-E and CENP-F, must also play a role in the biologic consequences of FTI treatment,33-36 or that farnesylated and geranylgeranylated Ras function differently, or even that alternative prenylation might also be inhibited by some FTIs. Thus, despite the development of FTIs as drugs targeted to one particular oncoprotein, their actions are complex. Some studies have lightened potential mechanisms of FTIs resistance, in particular, related to K-ras: one is the alternative prenylation of K-ras by GGTI and the other results from K-ras having a higher affinity for FT than H-ras or N-ras, making it more difficult for FTIs to compete with K-ras of FT binding.37
BMS-214662 is a nonpeptide small molecule inhibitor of FT that belongs to a family of compounds distinguished by the presence of an imidazole group attached to the tetrahydrobenzodiazepine nucleus (Fig 1).38 BMS-214662 is a potent in vitro inhibitor of human FT of both H-ras and K-ras, with an IC50 of 1.3 nM and 8.4 nM and an IC90 of 18 and 108 nM, respectively.39 BMS-214662, in contrast with other FTIs, has enhanced cytotoxic effects in preclinical models, resulting in the regression of large, well-established human tumor xenografts in nude mice with either continuous or intermittent dosing schedules.38,40 Another potentially important feature of this compound is that induction of apoptosis occurs both in proliferating and nonproliferating cells. This effect may in part explain the synergistic antitumor activity observed in preclinical models when BMS-214662 is combined with a wide range of cytotoxic drugs.39-42
The objectives of our study were to determine the maximum-tolerated dose (MTD), the dose-limiting toxicities (DLTs), and the recommended dose (RD) of BMS-214662 administered weekly; to further define the pharmacokinetic (PK) profile of this agent; and to determine the relationship between drug exposure and FT inhibition, Ras-mediated signaling, and the induction of apoptosis in both normal and tumor cells. In the first part of the study, we explored a weekly 1-hour infusion. Using this schedule, the toxicity profile was related to maximum plasma concentration (Cmax), and FT inhibition and induction of apoptosis were profound but short lasting. Therefore, we proceeded to evaluate a weekly 24-hour infusion to achieve longer target inhibition.
PATIENTS AND METHODS
Patient Population
Main inclusion criteria were histologically/cytologically confirmed advanced tumors unresponsive to standard therapy; presence of disease accessible to repetitive biopsies; measurable or assessable disease; age 18 years; life expectancy 12 weeks; Eastern Cooperative Oncology Group performance status of 0 to 2; and adequate bone marrow, hepatic, and renal function. Conditions resulting in exclusion included active infection; uncontrolled or significant pulmonary or cardiovascular disease; brain metastasis; or receiving drugs with known significant P-450 3A4 inhibitory effects at trial entry. All patients gave informed consent, and approval was obtained from the ethics committee at Vall d'Hebron University Hospital and the regulatory authorities. The study followed the Declaration of Helsinki and good clinical practice guidelines.
Treatment and Dose Escalation Criteria
BMS-214662 was administered as a single weekly intravenous dose for at least 6 consecutive weeks. In the first part of the study, BMS-214662 was administered as a 1-hour infusion, and in the second part it was administered as a 24-hour infusion. In both cases, a modified Fibonacci dose escalation schema was used. For the 1-hour infusion, planned dose levels were 56, 84, 118, 157, 209, 278, and 370 mg/m2/wk. Due to the appearance of moderate toxicity at the 209 mg/m2 level, a dose level of 245 mg/m2 was added before further escalation to the 278 mg/m2 level. For the 24-hour infusion, planned dose levels were 84, 118, 157, 209, 278, 370, and 492 mg/m2/wk. After observing DLTs in the higher dose levels, a new cohort of 275 mg/m2 was added and this dose was decided in order to be consistent with the RD defined in other phase I studies. In the absence of DLT, a minimum of three assessable patients were to be enrolled to each dose level. If one of the first three patients at a given dose level experienced a DLT, three additional patients were enrolled to the same dose level. If two or more patients presented a DLT at a dose level, enrollment of patients to that dose level was discontinued and the immediately preceding dose level was considered the MTD. DLT was defined as any one of the following toxicities during the first 4 weeks (one cycle): grade 4 National Cancer Institute Common Toxicity Criteria version 1.0 neutropenia for 5 or more consecutive days or febrile neutropenia (fever > 38.5° C with an absolute neutrophil count < 1,000 cells/μL); grade 4 thrombocytopenia (< 10,000 cells/μL) or bleeding episode requiring platelet transfusion; grade 3 or greater nausea and/or emesis despite the use of adequate/maximal medical intervention and/or prophylaxis; any other grade 3 or greater nonhematologic toxicity (except grade 3 injection site reaction); and prolongation of the QTc interval 500 milliseconds. In addition, if the administration of BMS-214662 was held for 2 weeks because treatment-related toxicity failed to recover to grade 1 or baseline, this was also considered to be a dose-limiting event. Transient grade 3 AST/ALT elevations lasting 24 to 48 hours after the end of BMS-214662 infusion and without any clinical sequelae were not considered DLTs. Once the MTD was defined, this dose level would be expanded to include a maximum of 15 patients, to better characterize its safety profile and to determine its suitability as the RD for further phase II development.
Tolerability and Safety
Routine clinical and laboratory assessments were conducted on a weekly basis. After transient (24 to 48 hours after the end of the infusion) grade 3 hematologic and liver toxicities were observed, laboratory assessments were performed 24, 48, and 72 hours after the first infusion of BMS-214662, only in the first week of treatment. Serial ECG monitoring was conducted following the first infusion and thereafter once every 4 weeks. Adverse events were recorded, graded using the National Cancer Institute Common Toxicity Criteria, and assessed by the investigator for any relationship with BMS-214662 treatment. Antiemetics were administered when emesis appeared and thereafter on a prophylactic basis. Patients experiencing DLT could be retreated with BMS-214662 at a dose reduced by one level, provided that all toxicities considered related to BMS-214662 had recovered to baseline or grade 1 severity.
Response Assessments
Objective measurement of tumor mass was assessed in accordance with the revised WHO criteria (Response Evaluation Criteria in Solid Tumors). Patients demonstrating benefit from treatment with BMS-214662 continued therapy until progression or withdrawal criteria were met.
Pharmacokinetic Analysis
Sampling to define the plasma PK profile of BMS-214662 was performed during the first dose of therapy at several time points during the infusion and in the 24-hour period after the end of the infusion. Sample tubes were immediately processed, as described previously.43
The concentration of BMS-214662 in plasma was determined by an analytic method involving reversed-phase, high-performance liquid chromatography with ultraviolet detection, as described previously.43 A noncompartmental PK analysis of the plasma samples was calculated using noncompartmental methods by the PKMENU application using the Statistical Analysis System version 6.12 (SAS Institute, Cary, NC). The following PK parameters of BMS-214662 were estimated: empirical time of peak plasma level (tmax), empirical peak plasma level (Cmax), and apparent elimination rate constant (z), which was calculated by linear regression of the terminal phase of the semilogarithmic plasma levels curve, when this was clearly defined. The elimination half-life (t1/2) was defined as ln2/z. Although there might be a gamma phase with BMS-214662, the lack of sufficient number of concentration time points did not allow to describe if this gamma phase exists or not. The area under the plasma concentration-time curve (AUC) was estimated by the logarithmic-linear trapezoidal algorithm to the data point at approximately 8 hours, with extrapolation to time infinity using the slope of the terminal log concentration versus time data. Total plasma clearance (CL) was calculated as the dose divided by the AUC. Mean values of the PK variables were calculated as the geometric mean of the individual patient values.44,45 Standard deviations for the geometric mean values were estimated by the jackknife method.46 PK data were reported as mean and standard deviation.
Pharmacodynamic Assessments
FT activity in peripheral mononuclear cells The ability of BMS-214662 to inhibit FT activity in peripheral-blood mononuclear cells (PBMCs) was determined in all patients following the first dose of BMS-214662. With the 1-hour infusion, venous blood (8 mL) was collected at 0:00, 1:00, 6:00, 12:00, and 24:00 hours. With the 24-hour infusion, the venous samples were obtained at 0:00, 6:00, 23:58 (before the end of infusion), 30:00, and 48:00 hours. Blood samples (7 mL) were collected in Vacutainer CPT mononuclear-cell preparation tubes containing sodium citrate buffer (Becton Dickinson, Plymouth, UK), and processed as described previously.43
FT activity in tumor and surrounding normal tissue samples The inhibition of FT activity in tumor and surrounding normal tissues by BMS-214662 was determined in selected patients. The timing of tumor and normal tissue biopsies was changed as data from previous samples became available, adjusting the timing as required. With the 1-hour infusion, tissue samples were obtained at baseline, 24:00, and 144:00 hours after the first dose of BMS-214662 at the dose levels of 56 to 157 mg/m2; at baseline, 2:00, and 24:00 hours at the dose levels of 209 to 245 mg/m2; and at baseline, 2:00, 6:00, and 24:00 hours at the dose level of 278 mg/m2. In the 24-hour infusion schedule the samples were obtained at baseline, 6:00, 24:00, and 48:00 hours after the first dose at all dose levels. The tissue samples were immediately frozen after the biopsy procedure and were stored at –70 to –80°C.
Determination of the FT activity FT activity in extracts of PBMC and normal or tumor tissues was determined by a radioenzyme assay using [3H] farnesyl pyrophosphate and human recombinant H-ras proteins produced in bacteria, as described previously.4,40,43,47 FT activity in each sample was determined as fmol/μg protein/h and reported as a percentage of the activity in the pretreatment sample for each patient. The arithmetic mean of the normalized FT activity was calculated from the determinations made at each time point for the group of patients in each dose level.
Signal transduction inhibition and apoptosis in tumor samples MAPK signaling and apoptosis in tumor samples by BMS-214662 were determined in selected patients enrolled in the study. Immunohistochemical analysis of total p42/44 MAPK, phosphorylated p42/44 MAPK at Thr202/Tyr204 (p-MAPK), total Akt, p-Akt at Ser473, p27KIP1 expression, and proliferation marker Ki67 were performed in paraffin-embedded sections from tumor samples, as described previously.48,49 Apoptosis was studied by TUNEL assay49 and by determination of caspase signaling measured as levels of cleaved caspase 3 at Asp175 and cleaved caspase 9 at Asp330 by immunohistochemistry as described previously.50,51 Rabbit polyclonal antitotal p44/42 MAPK #9102, anti-phosphorylated p44/42 (Thr202/Tyr204) MAPK #9101, antitotal Akt #9272, and antiphosphorylated Akt #9277 from Cell Signaling Technology (Beverly, MA); mouse monoclonal anti-p27KIP1 clone SX53G8 and anti-Ki67 clone MIB1 (DakoCytomation, Carpinteria, CA); mouse monoclonal anticleaved caspase 3 at Asp175 #9661 and anticleaved caspase 9 at Asp330 #9501 (Cell Signaling Technology) were used as primary antibodies. Two negative control rabbit polyclonal immunoglobulins (Biogenex, San Ramon, CA; and Santa Cruz Biotech, Santa Cruz, CA) and a negative control mouse monoclonal immunoglobulin (Biogenex) were also used. TUNEL assay 16-dUTP-peroxidase (Roche Diagnostics Gmbh, Mannheim, Germany) was used.
Qualitative changes in the expression of markers were assessed in a blinded fashion. For quantitative analysis, the percentage of cells in the tumor tissue that stained with each antibody was scored from representative sections in 10 high-power (x400) microscope fields, and the average percentage of stained cells was calculated in paired samples. Paired pretherapy and on-therapy samples were analyzed using the Wilcoxon rank test by SPSS Data Analysis Program, version 10.0 (SPSS Inc, Chicago, IL). Statistical tests were conducted at the two-sided 0.05 level of significance.
RESULTS
Characteristics of the 37 patients included in the weekly 1-hour infusion schedule and the 31 patients included in the weekly 24-hour infusion schedule are listed in Table 1. The distribution of patients across dose levels is listed in Tables 2 and 3.
Clinical Toxicities
Hematologic toxicities were observed more frequently in the 1-hour infusion than in the 24-hour infusion schedule (Table 2). Transient grade 3 to 4 neutropenia and leukocytopenia lasting 24 to 48 hours after the end of BMS-214662 infusion without any clinical significance were observed in 38% and 25% of the patients treated with the 1-hour infusion at doses 209 mg/m2, respectively. One patient treated at the 278 mg/m2 level developed grade 4 thrombocytopenia and disseminated intravascular coagulation in the context of sepsis of gastrointestinal origin. This patient died of multiorgan failure after 10 days in an intensive care unit. In contrast, no grade 3 to 4 hematologic toxicities were observed with the 24-hour infusion schedule up to the 492 mg/m2 dose level except for grade 3 anemia. One patient in the 492 mg/m2 cohort experienced grade 4 thrombocytopenia, neutropenia, and leukocytopenia; these events were considered DLTs.
The profile of nonhematologic toxicity was completely different in the two infusion schedules (Table 2). The most frequent toxicities in the 1-hour infusion schedule were of gastrointestinal origin, including nausea/vomiting and diarrhea. One patient at 209 mg/m2 developed grade 3 nausea/vomiting without any prophylactic premedication. This patient also experienced grade 3 hypotension—considered a DLT—during the vomiting episodes. After this case, antiemetics were administered to all patients on a prophylactic basis. One patient at 245 mg/m2 and two patients at 278 mg/m2 presented grade 3 nausea/vomiting despite the antiemetic prophylaxis, all considered to be dose-limiting. One patient at 245 mg/m2 and three patients at 278 mg/m2 developed grade 3 diarrhea despite early and vigorous oral loperamide treatment and these episodes were considered DLTs. One patient at the 245 mg/m2 dose level developed a fatal grade 4 acute pancreatitis, considered to be possibly related to BMS-214662. Transient grade 3 AST/ALT elevations lasting 24 to 48 hours after the end of BMS-214662 infusion and without any clinical sequelae were observed in 19% of the patients treated at doses 245 mg/m2. One patient treated at 278 mg/m2 experienced a reversible grade 3 creatinine elevation considered DLT.
In contrast, the most frequent observed severe toxicity with the 24-hour infusion schedule was of renal origin. In the initial dose escalation, toxicity was limited to grade 1 to 2 events up to the 492 mg/m2 dose level, where two of three patients presented DLTs: one patient with reversible grade 3 creatinine elevation and one patient with reversible grade 3 renal failure, grade 3 nausea/vomiting, grade 3 amylase/lipase elevation, grade 3 infection, grade 4 creatinine elevation, grade 4 hepatitis, grade 4 transient neutropenia, and grade 4 hypovolemic shock. Therefore, the 492 mg/m2 dose level was considered a toxic dose and the lower dose level, 370 mg/m2, was expanded to determine the recommended dose. At the 370 mg/m2 dose level, one of four patients presented DLTs with grade 3 vomiting, reversible grade 3 renal failure, and grade 4 creatinine elevation. This renal toxicity was likely due to acute tubular necrosis since the fractional excretion of sodium was higher than 2% and the normalization of the creatinine levels was slow, with two patients requiring transient dialysis. Kidney biopsies were obtained from the two patients treated at 492 and 370 mg/m2, respectively, who experienced reversible renal failure. In both cases, focal acute tubular damage with regeneration signs and thrombotic microangiopathy limited to the interstitial capillaries were observed. Although only one of four patients at the 370 mg/m2 dose level experienced DLTs, recruitment to this dose level was stopped in light of the severe toxicity. Therefore, the number of patients enrolled at the 275 to 278 mg/m2 dose level was expanded. One of a total of 12 patients treated at 275 to 278 mg/m2 developed a reversible grade 2 increase in the serum creatinine and grade 3 nausea/vomiting.
In summary, DLTs in the 1-hour infusion schedule (Table 2) occurred at the 209 mg/m2 dose level and higher. The 278 mg/m2 dose level was clearly intolerable, with three DLTs occurring in a total of six patients. The 245 mg/m2 dose level was initially considered to be the MTD. However, the case of fatal pancreatitis observed at this dose level and considered related to BMS-214662 makes the 245 mg/m2 difficult to support for phase II use. Therefore, the 209 mg/m2 dose level, which produced grade 3 nausea/vomiting and hypotension in one of eight patients, and specifically in a patient who had not received antiemetic therapy, is recommended as the dose for further development of BMS-214662 when given as a weekly 1-hour infusion. In the 24-hour infusion schedule, DLTs occurred at the 275 to 278 mg/m2 dose level and higher. The 492 and 370 mg/m2 dose levels were clearly intolerable, with DLTs occurring in two of three and one of four patients, respectively. At the 275 to 278 mg/m2 dose level, one of 12 patients presented DLT. Thus, the dose of BMS-214662 recommended as a weekly 24-hour infusion schedule is 275 mg/m2.
The cumulative tolerance of BMS-214662 was globally acceptable (Table 3). In the 1-hour infusion schedule, nine doses out of a total of 287 administered doses were delayed due to toxicity, four in the first 4-week period and five after the initial 4-week period. There were four patients that were dose reduced, one in the first 4-week period and three after the initial 4-week period. In the 24-hour infusion schedule, two doses out of a total of 213 administered doses were delayed due to toxicity, both in the first 4-week period. Four patients needed dose reductions, one in the first 4-week period and three after the initial 4-week period.
Antitumor Activity
There were no objective responses. Two patients (with breast cancer) in the 1-hour infusion schedule appeared to have clinical benefit from the treatment, achieving a minor response and a prolonged stabilization of the disease. Three patients in the 24-hour infusion schedule (with breast, gastric, and renal cell cancer) appeared to benefit from treatment with prolonged stabilization of the disease.
Pharmacokinetics
BMS-214662 exhibited a linear plasma PK between all dose levels in the two different schedules. Cmax and AUC increased in a near dose proportional fashion. The percentage of total AUC that was extrapolated to infinity is greater than 98% across all the dose levels. The mean values of the PK parameters estimated by noncompartimental analysis of the individual patient data are summarized in Table 4. The mean plasma PK profile of every cohort is presented in Figure 2A (1-hour infusion) and 2B (24-hour infusion). Interpatient variability was considerable. There was a complete absence of relationship between the administered dose and the CL, Vss, and t1/2. The CL was very high (388 mL/min/m2, with a 38% coefficient of variation [CV]), the value translating to approximately 50% of the hepatic blood flow, assuming that the blood to plasma partition coefficient of BMS-214662 is 1.0. The mean Vss and t1/2 were 32 l/m2 (with a CV of 50%) and 2.91 hours, respectively. These results would indicate that the fraction of BMS-214662 bound to tissue proteins is close to the same as its binding to plasma proteins, which is around 99%.
Pharmacodynamics
Farnesyltransferase inhibition There was a good correlation between dose and degree of FT inhibition in PBMCs in both infusional schedules. In the 1-hour infusion schedule, the maximum inhibition of FT in PBMCs was observed at the end of the infusion. Greater than 80% inhibition of FT was observed at the 1-hour time point with BMS-214662 doses 118 mg/m2. FT inhibition returned to baseline by the 24-hour time point at all dose-levels (Fig 3A). In contrast, in the 24-hour infusion schedule, FT activity was uniformly inhibited throughout the duration of the infusion, achieving a sustained inhibition of 40% at 275 to 278 mg/m2 and a maximal inhibition of approximately 70% at 370 mg/m2 (Fig 3B). As expected, FT inhibition was more short lasting in the 1-hour infusion (Fig 3C) than in the 24-hour infusion schedule (Fig 3D). There was a good correlation between the BMS-214662 concentration profile and the intensity of FT inhibition in PBMCs in both schedules. The pattern of FT inhibition over time after the exposure of the drug was similar in PBMCs, tumor, and surrounding normal tissue, although there was a trend for a more prolonged inhibition in tumor and surrounding normal tissue than in PBMCs (data not shown).
Effects on signal transduction BMS-214662 did not induce changes in total- and p-MAPK, total- and p-Akt, p27KIP1, and Ki67 in tumor samples. In contrast to this, BMS-214662 induced apoptosis in all but one tumor sample. Apoptosis by TUNEL staining was observed in tumor samples both from patients with signs of antitumor activity and in patients without such activity. Figures 4A through D show the induction of apoptosis in tumor samples obtained from patients in the 1-hour and the 24-hour infusion schedules. Induction of apoptosis—measured by both hematoxylin-eosin and TUNEL stainings—appeared to be related to time after exposure being more prominent at 24- than at 2-hour time points and at the 48-hour than at 24- and 6-hour time points in the 1-hour and 24-hour infusion schedules, respectively. Expression of cleaved caspase 3 and 9 in tumor cells significantly increased after treatment with the two schedules. In the 1-hour infusion schedule, expression of cleaved caspases at 24- and 2-hour time points was significantly increased, being more prominent at the 24-hour time point, whereas in the 24-hour infusion schedule, the expression significantly increased at 48- and 24-hour time points, being more prominent at the 48-hour time point (data not shown). The induction of apoptosis appeared related to dose in the 1-hour infusion schedule and it was observed in all patients with tumor samples at doses 209 mg/m2. However, the induction of apoptosis was not found related to dose in the 24-hour infusion schedule.
DISCUSSION
In this study, BMS-214662 was developed in a weekly schedule, a dosing regimen more consistent with the preclinical activity of the compound. This phase I study was aimed at identifying the most optimal weekly schedule of BMS-214662 based on the toxicity profile, PK, and PD evaluation of FT inhibition and apoptotic induction in normal and tumor cells. We found that the toxicity profiles of the 1- and 24-hour infusion regimens were completely different. Additionally, the patterns of FT inhibition and apoptotic induction were markedly different with the two regimens.
With the 1-hour infusion schedule, DLTs consisted of nausea/vomiting, diarrhea, acute pancreatitis, hypotension, thrombocytopenia and increased serum creatinine. Transient and reversible grade 3 AST/ALT elevations and grade 3 to 4 leukocytopenia/neutropenia lasting 24 to 48 hours after the end of BMS-214662 infusion were also seen, but they did not appear to be of clinical significance. In considering PK and PD studies, FT inhibition completely mirrored the concentrations of BMS-214662 in plasma, with a profound but short-lived FT inhibition in PBMCs and tumor cells.
After completing evaluation of weekly 1-hour infusions, we proceeded to evaluate weekly 24-hour infusions for several reasons. First, the AUC reached with the 1-hour infusion at the RD (27 μM x h) was lower than the AUC predicted in preclinical models to achieve antitumor activity (77 μM x h). To the extent that toxicities might be related to Cmax rather than to AUC, it was anticipated that a higher exposure would be reached with a 24-hour infusion. Second, at the RD with the 1-hour infusion, FT inhibition in tumor cells was nearly complete, but brief ( 6 hours), probably related to the short half-life of the drug (around 3 hours). It was again predicted that a longer infusion would result in a more sustained FT inhibition. Third, in preclinical models, the therapeutic index of BMS-214662 was enhanced by prolonging the exposure time. In in vitro models, the concentration x time product (AUC) required to achieve an IC50 was disproportionately reduced as the exposure time increased from 6 to 24 hours: 100-fold for proliferating cells and 10-fold for quiescent cells. Similarly, in in vivo models, the minimal effective AUC was 29 μM x h when BMS-214662 was administered as a 24-hour infusion and 77 μM x h when it was administered as a 1-hour infusion.
The 24-hour infusion schedule at the highest dose levels resulted in unexpected renal impairment, likely due to acute tubular necrosis. However, at the RD, this schedule was better tolerated without any significant nausea, vomiting, diarrhea, leukocytopenia, or liver toxicity. The differences in the safety profile with the two schedules suggest that some toxicities are related to Cmax, whereas others are related to global exposure to the drug. It is likely that the liver and hematologic toxicities are related to Cmax since Cmax with 1-hour infusion at 209 to 278 mg/m2 was 18.3 to 19.1 μM, whereas the 24-hour infusion yielded a Cmax of 1.1 to 2.9 μM at 209 to 492 mg/m2 dose levels. In contrast, the renal toxicity is likely related to more prolonged exposure. AUC was 23.5 to 55.7 μM x h at the dose levels 209 to 492 mg/m2 with the 24-hour infusion and 27.2 to 32.6 μM x h at 209 to 278 mg/m2 in the 1-hour infusion. In addition to the most favorable safety profile, the 24-hour infusion is preferred over the 1-hour infusion for several reasons. First, delivery of BMS-214662 in a 24-hour infusion at the RD of 278 mg/m2 allows for clinical drug exposures 30 μM x h, consistent with those required preclinically for single-agent activity. Second, the 24-hour infusion provides a longer duration of FT inhibition than does the 1-hour infusion, and third, the 24-hour infusion yields higher degrees of apoptosis.
An important end point of our study was to determine which correlations existed between exposure to the study agent and inhibition of FT in the PBMCs and, of great interest, in the tumor. Although other studies with this43 and other FTIs52,53 had demonstrated a correlation between FTI dose and FT inhibition in PBMCs, and bone marrow cells and buccal mucosa cells, the degree of FT inhibition in the tumor cells with these agents was unknown. Therefore, we evaluated the relationship between the degree of FT inhibition and its timing dependency in the tumor and in selected surrogate tissues. We have demonstrated that there was a good correlation between FT inhibition related to BMS-214662 in PBMCs and normal and tumor tissue samples. As a consequence of this, we suggest that PBMCs can replace tumor tissue to determine the inhibition profile of FT activity related to BMS-214662.
In order to characterize target inhibition, we decided to determine the biologic effects of BMS-214662 in the tumor. Based on the preclinical studies suggesting that BMS-214662 had a profound proapoptotic effect, we studied apoptosis induction in tumor samples. We have clinically demonstrated in the serial tumor PD studies that BMS-214662 induced apoptosis but did not inhibit MAPK signaling. Other FTIs have also demonstrated antitumor activity and induction of apoptosis through Ras-independent pathways.20,24,54,55 The degree and duration of apoptosis was higher in the 24-hour infusion than in the 1-hour infusion schedule. Whereas in the 1-hour infusion, there was a clear correlation between dose and degree of apoptosis, we did not find such correlation in the 24-hour infusion schedule. We could not establish any correlation between the degree of observed apoptosis and clinical benefit and/or toxicity.
Normal tissues such as PBMCs and skin may be good surrogates for evaluating the exposure of the drug and kinetics of the target inhibition in a clinical model. However, tumor PD studies may better explore the biologic effects of a selected agent than normal surrogate tissues, as tumor cells often respond in a different way to targeted drugs than normal cells, also demonstrated with other compounds in the clinical setting, such as the EGFR inhibitors EMD72000SUP>56 and gefitinib.57,58 Therefore, the antineoplastic PD effect of a selected compound on the human tumor cells in the human host can only be evaluated when tumor biopsies are obtained before and during treatment. We and others support PD studies with tumor biopsies from patients enrolled in clinical trials with new targeted therapies.59,60 These studies can not only evaluate the biologic effect of the drug in the tumor, but they may also identify the genomic and proteomic profile of the population with highest chances to benefit from treatment.
BMS-214662 has been administered by other routes and schedules. With the intravenous formulation, BMS-214662 has been administered as 1-hour infusion every 3 weeks,43,61 and once weekly for 4 weeks, followed by 2 weeks of rest.62 BMS-214662 was also formulated in an oral capsule, but the severe gastrointestinal toxicities observed in the clinical setting63 prevented further development with this formulation. Different phase I studies are currently evaluating the synergistic effect of BMS-214662 in combination with cisplatin,64 paclitaxel,65 and both carboplatin and paclitaxel66 in the clinical setting.
In summary, this phase I study with weekly 1-hour and 24-hour infusions of BMS-214662 has shown that this agent can be administered with either infusion duration. However, based on the preclinical evaluation, the clinical safety profile and the PK and PD results, with a more sustained FT inhibition and apoptosis induction with a prolonged infusion, we recommend the 24-hour infusion schedule for further development of this agent.
Authors' Disclosures of Potential Conflicts of Interest
The following authors or their immediate family members have indicated a financial interest. No conflict exists for drugs or devices used in a study if they are not being evaluated as part of the investigation. Employment: Maurizio Voi, Bristol-Myers Squibb; Michael Cooper, Bristol-Myers Squibb; Anne Van Vreckem, Bristol-Myers Squibb; Veeraswamy Manne, Bristol-Myers Squibb; James A. Manning, Bristol-Myers Squibb; Carmen Garrido, Bristol-Myers Squibb. Consultant/Advisory Role: Jose Baselga, Bristol-Myers Squibb. Stock Ownership: Maurizio Voi, Bristol-Myers Squibb; Veeraswamy Manne, Bristol-Myers Squibb; James A. Manning, Bristol-Myers Squibb. For a detailed description of these categories, or for more information about ASCO's conflict of interest policy, please refer to the Author Disclosure Declaration and Disclosures of Potential Conflicts of Interest found in Information for Contributors in the front of each issue.
Acknowledgment
We thank Ramon Salazar for critical review of this manuscript.
NOTES
Presented in part at the 11th NCI-EORTC-AACR Symposium on New Drugs in Cancer Therapy, Amsterdam, Holland, November 7-10, 2000; at the 37th Annual Meeting of the American Society of Clinical Oncology, San Francisco, CA, May 12-15, 2001; and AACR-NCI-EORTC International Conference of Molecular Targets and Cancer Therapeutics, Miami, FL, October 29-November 2, 2001.
Terms in blue are defined in the glossary, found at the end of this issue and online at www.jco.org.
Authors' disclosures of potential conflicts of interest are found at the end of this article.
REFERENCES
Bos JL: ras oncogenes in human cancer: A review. Cancer Res 49:4682-4689, 1989
Barbacid M: ras genes. Annu Rev Biochem 56:779-827, 1987
Boguski MS, McCormick F: Proteins regulating Ras and its relatives. Nature 366:643-654, 1993
Albanell J, Rojo F, Sonnicchsen D, et al: Elevated farnesyltransferase activity in chemo-naive breast cancer tissue as compared to normal surrounding tissue. Proc Am Soc Cancer Res 42:489, 2001 (abstr 2634)
Rowinsky EK, Windle JJ, Von Hoff DD: Ras protein farnesyltransferase: A strategic target for anticancer therapeutic development. J Clin Oncol 17:3631-3652, 1999
Gibbs JB, Graham SL, Hartman GD, et al: Farnesyltransferase inhibitors versus Ras inhibitors. Curr Opin Chem Biol 1:197-203, 1997
Gibbs JB: Ras C-terminal processing enzymes–new drug targets Cell 65:1-4, 1991
Gibbs JB, Oliff A: The potential of farnesyltransferase inhibitors as cancer chemotherapeutics. Annu Rev Pharmacol Toxicol 37:143-166, 1997
Cox AD, Der CJ: Farnesyltransferase inhibitors and cancer treatment: Targeting simply Ras Biochim Biophys Acta 1333:F51-F71, 1997
Lerner EC, Hamilton AD, Sebti SM: Inhibition of Ras prenylation: A signaling target for novel anti-cancer drug design. Anticancer Drug Des 12:229-238, 1997
Tamanoi F: Inhibitors of Ras farnesyltransferases. Trends Biochem Sci 18:349-353, 1993
James GL, Goldstein JL, Brown MS, et al: Benzodiazepine peptidomimetics: potent inhibitors of Ras farnesylation in animal cells. Science 260:1937-1942, 1993
Kohl NE, Mosser SD, deSolms SJ, et al: Selective inhibition of ras-dependent transformation by a farnesyltransferase inhibitor. Science 260:1934-1937, 1993
Kohl NE, Omer CA, Conner MW, et al: Inhibition of farnesyltransferase induces regression of mammary and salivary carcinomas in ras transgenic mice. Nat Med 1:792-797, 1995
Nagasu T, Yoshimatsu K, Rowell C, et al: Inhibition of human tumor xenograft growth by treatment with the farnesyl transferase inhibitor B956. Cancer Res 55:5310-5314, 1995
Manne V, Yan N, Carboni JM, et al: Bisubstrate inhibitors of farnesyltransferase: A novel class of specific inhibitors of ras transformed cells. Oncogene 10:1763-1779, 1995
Bishop WR, Bond R, Petrin J, et al: Novel tricyclic inhibitors of farnesyl protein transferase. Biochemical characterization and inhibition of Ras modification in transfected Cos cells. J Biol Chem 270:30611-30618, 1995
Lebowitz PF, Prendergast GC: Non-Ras targets of farnesyltransferase inhibitors: Focus on Rho. Oncogene 17:1439-1445, 1998
Du W, Lebowitz PF, Prendergast GC: Cell growth inhibition by farnesyltransferase inhibitors is mediated by gain of geranylgeranylated RhoB. Mol Cell Biol 19:1831-1840, 1999
Du W, Liu A, Prendergast GC: Activation of the PI3'K-AKT pathway masks the proapoptotic effects of farnesyltransferase inhibitors. Cancer Res 59:4208-4212, 1999
Lebowitz PF, Davide JP, Prendergast GC: Evidence that farnesyltransferase inhibitors suppress Ras transformation by interfering with Rho activity. Mol Cell Biol 15:6613-6622, 1995
Lebowitz PF, Sakamuro D, Prendergast GC: Farnesyl transferase inhibitors induce apoptosis of Ras-transformed cells denied substratum attachment. Cancer Res 57:708-713, 1997
Law BK, Norgaard P, Moses HL: Farnesyltransferase inhibitor induces rapid growth arrest and blocks p70s6k activation by multiple stimuli. J Biol Chem 275:10796-10801, 2000
Jiang K, Coppola D, Crespo NC, et al: The phosphoinositide 3-OH kinase/AKT2 pathway as a critical target for farnesyltransferase inhibitor-induced apoptosis. Mol Cell Biol 20:139-148, 2000
Prendergast GC, Khosravi-Far R, Solski PA, et al: Critical role of Rho in cell transformation by oncogenic Ras. Oncogene 10:2289-2296, 1995
Prendergast GC, Davide JP, deSolms SJ, et al: Farnesyltransferase inhibition causes morphological reversion of ras-transformed cells by a complex mechanism that involves regulation of the actin cytoskeleton. Mol Cell Biol 14:4193-4202, 1994
Lerner EC, Zhang TT, Knowles DB, et al: Inhibition of the prenylation of K-Ras, but not H- or N-Ras, is highly resistant to CAAX peptidomimetics and requires both a farnesyltransferase and a geranylgeranyltransferase I inhibitor in human tumor cell lines. Oncogene 15:1283-1288, 1997
Whyte DB, Kirschmeier P, Hockenberry TN, et al: K- and N-Ras are geranylgeranylated in cells treated with farnesyl protein transferase inhibitors. J Biol Chem 272:14459-14464, 1997
Rowell CA, Kowalczyk JJ, Lewis MD, et al: Direct demonstration of geranylgeranylation and farnesylation of Ki-Ras in vivo. J Biol Chem 272:14093-14097, 1997
Sun J, Qian Y, Hamilton AD, et al: Ras CAAX peptidomimetic FTI 276 selectively blocks tumor growth in nude mice of a human lung carcinoma with K-Ras mutation and p53 deletion. Cancer Res 55:4243-4247, 1995
Suzuki N, Del Villar K, Tamanoi F: Farnesyltransferase inhibitors induce dramatic morphological changes of KNRK cells that are blocked by microtubule interfering agents. Proc Natl Acad Sci U S A 95:10499-10504, 1998
Suzuki N, Urano J, Tamanoi F: Farnesyltransferase inhibitors induce cytochrome c release and caspase 3 activation preferentially in transformed cells. Proc Natl Acad Sci U S A 95:15356-15361, 1998
Prendergast GC: Farnesyltransferase inhibitors: antineoplastic mechanism and clinical prospects. Curr Opin Cell Biol 12:166-173, 2000
Sebti SM, Hamilton AD: Inhibition of Ras prenylation: A novel approach to cancer chemotherapy. Pharmacol Ther 74:103-114, 1997
Lobell RB, Kohl NE: Pre-clinical development of farnesyltransferase inhibitors. Cancer Metastasis Rev 17:203-210, 1998
Ashar HR, Armstrong L, James LJ, et al: Biological effects and mechanism of action of farnesyl transferase inhibitors. Chem Res Toxicol 13:949-952, 2000
Fiordalisi JJ, Johnson RL, 2nd, Weinbaum CA, et al: High affinity for farnesyltransferase and alternative prenylation contribute individually to K-Ras4B resistance to farnesyltransferase inhibitors. J Biol Chem 278:41718-41727, 2003
Hunt JT, Ding CZ, Batorsky R, et al: Discovery of (R)-7-cyano-2,3,4, 5-tetrahydro-1-(1H-imidazol-4-ylmethyl)-3-(phenylmethyl)-4-(2-thienylsulfonyl)-1H-1,4-benzodiazepine (BMS-214662), a farnesyltransferase inhibitor with potent preclinical antitumor activity. J Med Chem 43:3587-3595, 2000
Manne V, Lee FY, Bol DK, et al: Apoptotic and cytostatic farnesyltransferase inhibitors have distinct pharmacology and efficacy profiles in tumor models. Cancer Res 64:3974-3980, 2004
Rose WC, Lee FY, Fairchild CR, et al: Preclinical antitumor activity of BMS-214662, a highly apoptotic and novel farnesyltransferase inhibitor. Cancer Res 61:7507-7517, 2001
Lee FY, Arico-Gray M, Camuso A, et al: The pro-apoptotic FT inhibitor BMS-214662 produced synergistic effects in combination with chemotherapy with antiproliferative cytotoxic agents. Proceedings of the 2001 AACR-NCI-EORTC International Conference:82, 2001 (abstr 401)
Davis RJ: Signal transduction by the JNK group of MAP kinases. Cell 103:239-252, 2000
Ryan DP, Eder JP, Jr., Puchlaski T, et al: Phase I clinical trial of the farnesyltransferase inhibitor BMS-214662 given as a 1-hour intravenous infusion in patients with advanced solid tumors. Clin Cancer Res 10:2222-2230, 2004
Lacey LF, Keene ON, Pritchard JF, et al: Common noncompartmental pharmacokinetic variables: Are they normally or log-normally distributed J Biopharm Stat 7:171-178, 1997
Mizuta E, Tsubotani A: Preparation of mean drug concentration–time curves in plasma. A study on the frequency distribution of pharmacokinetic parameters. Chem Pharm Bull (Tokyo) 33:1620-1632, 1985
Lam FC, Hung CT, Perrier DG: Estimation of variance for harmonic mean half-lives. J Pharm Sci 74:229-231, 1985
Manne V, Roberts D, Tobin A, et al: Identification and preliminary characterization of protein-cysteine farnesyltransferase. Proc Natl Acad Sci USA 87:7541-7545, 1990
Albanell J, Rojo F, Averbuch S, et al: Pharmacodynamic studies of the epidermal growth factor receptor inhibitor ZD1839 in skin from cancer patients: Histopathologic and molecular consequences of receptor inhibition. J Clin Oncol 20:110-124, 2002
Matar P, Rojo F, Cassia R, et al: Combined epidermal growth factor receptor targeting with the tyrosine kinase inhibitor gefitinib (ZD1839) and the monoclonal antibody cetuximab (IMC-C225): Superiority over single-agent receptor targeting. Clin Cancer Res 10:6487-6501, 2004
Rohn TT, Rissman RA, Davis MC, et al: Caspase-9 activation and caspase cleavage of tau in the Alzheimer's disease brain. Neurobiol Dis 11:341-354, 2002
Woenckhaus C, Giebel J, Failing K, et al: Expression of AP-2alpha, c-kit, and cleaved caspase-6 and -3 in naevi and malignant melanomas of the skin. A possible role for caspases in melanoma progression J Pathol 201:278-287, 2003
Adjei AA, Erlichman C, Davis JN, et al: A Phase I trial of the farnesyl transferase inhibitor SCH66336: evidence for biological and clinical activity. Cancer Res 60:1871-1877, 2000
Karp JE, Lancet JE, Kaufmann SH, et al: Clinical and biologic activity of the farnesyltransferase inhibitor R115777 in adults with refractory and relapsed acute leukemias: A phase 1 clinical-laboratory correlative trial. Blood 97:3361-3369, 2001
Chun KH, Lee HY, Hassan K, et al: Implication of protein kinase B/Akt and Bcl-2/Bcl-XL suppression by the farnesyl transferase inhibitor SCH66336 in apoptosis induction in squamous carcinoma cells. Cancer Res 63:4796-4800, 2003
Mizukami Y, Ura H, Obara T, et al: Requirement of c-jun N-terminal kinase for apoptotic cell death induced by farnesyltransferase inhibitor, farnesylamine, in human pancreatic cancer cells. Biochem Biophys Res Commun 288:198-204, 2001
Tabernero J, Rojo F, Jimenez E, et al: A phase I PK and serial tumor and skin pharmacodynamic (PD) study of weekly (q1w), every 2-week (q2w) or every 3-week (q3w) 1-hour infusion EMD72000, a humanized anti-epidermal growth factor receptor (EGFR) antibody, in patients (pt) with advanced tumors. Proc Am Soc Clin Oncol 22:192, 2003 (abstr 770)
Rojo F, Tabernero J, Van Cutsem E, et al: Pharmacodynamic studies of tumor biopsy specimens from patients with advanced gastric carcinoma undergoing treatment with gefitinib (ZD1839). Proc Am Soc Clin Oncol 22:191, 2003 (abstr 764)
Baselga J, Albanell J, Ruiz A, et al: Phase II and tumor pharmacodynamic study of gefitinib (ZD1839) in patients with advanced breast cancer. Proc Am Soc Clin Oncol 22:7, 2003 (abstr 24)
Baselga J: Skin as a surrogate tissue for pharmacodynamic end points: Is it deep enough Clin Cancer Res 9:2389-2390, 2003
Arteaga CL, Baselga J: Tyrosine kinase inhibitors: Why does the current process of clinical development not apply to them Cancer Cell 5:525-531, 2004
Sonnichsen D, Damle B, Manning J, et al: Pharmocokinetics (PK) and pharmacodynamics (PD) of the farnesyltransferase (FT) inhibitor BMS-214662 in patients with advanced solid tumors. Proc Am Soc Clin Oncol 19:178a, 2000 (abstr 691)
Kim KB, Shin DM, Summey CC, et al: Phase I study of farnesyl transferase inhibitor, BMS-214662 in solid tumors. Proc Am Soc Clin Oncol 20:79a, 2001 (abstr 313)
Camacho LH, Soignet SL, Pezzulli S, et al: Dose escalation of oral farnesyl transferase inhibitor (FTI) BMS-214662 in patients with solid tumors. Proc Am Soc Clin Oncol 20:79a, 2001 (abstr 311)
Mackay HJ, Hoekstra R, Eskens FA, et al: A phase I pharmacokinetic and pharmacodynamic study of the farnesyl transferase inhibitor BMS-214662 in combination with cisplatin in patients with advanced solid tumors. Clin Cancer Res 10:2636-2644, 2004
Bailey HH, Marnocha R, Arzoomanian R, et al: Phase I trial of weekly paclitaxel and BMS214662 in patients with advanced solid tumors. Proc Am Soc Clin Oncol 20:79a, 2001 (abstr 314)
Dy GK, Bruzek LM, Croghan GA, et al: A phase I trial of the farnesyltransferase(FT) inhibitor, BMS-214662 (B) in combination with paclitaxel (P) and carboplatin (C) in patients with advanced cancer. Proc Am Soc Clin Oncol 23:211, 2004 (abstr3066)(Josep Tabernero, Fredy Ro)
Bristol-Myers Squibb, Wallingford, CT, Waterloo, Belgium, and Madrid, Spain
ABSTRACT
PURPOSE: BMS-214662 is a potent, nonpeptide, small molecule inhibitor of human farnesyltransferase (FT). We have conducted a phase I pharmacokinetic (PK) and pharmacodynamic study of BMS-214662 administered intravenously weekly with 1- and 24-hour infusions. The objectives were to determine the dose-limiting toxicities and the recommended dose (RD), to describe PKs, and to evaluate the relationships between BMS-214662 exposure, FT inhibition, downstream signaling, and induction of apoptosis in tumor samples.
PATIENTS AND METHODS: Patients with advanced solid tumors and adequate organ function were eligible. The dose was escalated according to a modified Fibonacci schedule.
RESULTS: BMS-214662 was escalated from 56 to 278 mg/m2 in 37 patients in the 1-hour schedule, and from 84 to 492 mg/m2 in 31 patients in the 24-hour schedule. Dose-limiting toxicities included gastrointestinal and renal events. The RDs were 209 mg/m2 and 275 mg/m2 in the 1- and 24-hour schedules, respectively. Five patients (three with breast, one with gastric, and one with renal cell cancer) had clinical benefit from treatment. BMS-214662 exhibited linear PKs with area under the concentration-time curves at the RDs of 27 and 32 μM x h in the 1- and 24-hour schedules, respectively. The pattern of FT inhibition in peripheral-blood mononuclear cells at the RDs was different in the two schedules: high (> 80%) but short-lived ( 6 hours) in the 1-hour infusion and moderate (> 40%) but long-lived (24 hours) in the 24-hour infusion. BMS-214662 induced apoptosis in tumors but did not inhibit MAPK signaling.
CONCLUSION: BMS-214662 can be safely delivered in both the 1-hour and 24-hour infusions at biologically active doses, with the preclinical, PK, and pharmacodynamic profiles favoring the 24-hour schedule.
INTRODUCTION
Ras proteins are critical elements in signaling mediated by receptor tyrosine kinases. The position of these proteins in receptor tyrosine kinase signaling explains their key role in a large number of cellular processes including growth, differentiation, apoptosis, cytoskeletal organization, and membrane trafficking.1-3 After the synthesis of the Pro-Ras protein, a series of post-translational biochemical processes convert this protein to a more hydrophobic one that permits its localization to the cytoplasmic membrane. The first modification of this process is catalyzed by the farnesyltransferase (FT) enzyme, causing the covalent addition of a farnesyl group to the cysteine residue of the C-terminal CAAX sequence of the propeptide. As the first step in this process, farnesylation is the most critical one, and FT blockade causes severe impairment in Ras protein function. Moreover, FT activity in human tumor cells is higher than in normal surrounding cells.4 Therefore, FT has become a relevant target in anticancer drug development.5-11 Initially it was thought that FT inhibitors (FTIs) might selectively decrease the growth of Ras-transformed cells and tumors harboring Ras mutations.12-17 More recent studies have demonstrated that there is no clear correlation between Ras mutations and sensitivity to FTIs.6,18-26 Whereas FTIs effectively inhibit H-ras signaling and transformation, they do not block either the processing or the function of K-ras 4B,27 the isoform of Ras most frequently mutated in human tumors. Indeed, in the presence of FTIs, K-ras can become alternatively prenylated by the FT-related enzyme geranylgeranyltransferase I (GGTI).28,29 Nevertheless, some FTIs can significantly inhibit the growth of tumors containing mutated K-ras4B.30-32 This indicates either that farnesylated proteins other than Ras, like RhoB and the centromere-associated CENP-E and CENP-F, must also play a role in the biologic consequences of FTI treatment,33-36 or that farnesylated and geranylgeranylated Ras function differently, or even that alternative prenylation might also be inhibited by some FTIs. Thus, despite the development of FTIs as drugs targeted to one particular oncoprotein, their actions are complex. Some studies have lightened potential mechanisms of FTIs resistance, in particular, related to K-ras: one is the alternative prenylation of K-ras by GGTI and the other results from K-ras having a higher affinity for FT than H-ras or N-ras, making it more difficult for FTIs to compete with K-ras of FT binding.37
BMS-214662 is a nonpeptide small molecule inhibitor of FT that belongs to a family of compounds distinguished by the presence of an imidazole group attached to the tetrahydrobenzodiazepine nucleus (Fig 1).38 BMS-214662 is a potent in vitro inhibitor of human FT of both H-ras and K-ras, with an IC50 of 1.3 nM and 8.4 nM and an IC90 of 18 and 108 nM, respectively.39 BMS-214662, in contrast with other FTIs, has enhanced cytotoxic effects in preclinical models, resulting in the regression of large, well-established human tumor xenografts in nude mice with either continuous or intermittent dosing schedules.38,40 Another potentially important feature of this compound is that induction of apoptosis occurs both in proliferating and nonproliferating cells. This effect may in part explain the synergistic antitumor activity observed in preclinical models when BMS-214662 is combined with a wide range of cytotoxic drugs.39-42
The objectives of our study were to determine the maximum-tolerated dose (MTD), the dose-limiting toxicities (DLTs), and the recommended dose (RD) of BMS-214662 administered weekly; to further define the pharmacokinetic (PK) profile of this agent; and to determine the relationship between drug exposure and FT inhibition, Ras-mediated signaling, and the induction of apoptosis in both normal and tumor cells. In the first part of the study, we explored a weekly 1-hour infusion. Using this schedule, the toxicity profile was related to maximum plasma concentration (Cmax), and FT inhibition and induction of apoptosis were profound but short lasting. Therefore, we proceeded to evaluate a weekly 24-hour infusion to achieve longer target inhibition.
PATIENTS AND METHODS
Patient Population
Main inclusion criteria were histologically/cytologically confirmed advanced tumors unresponsive to standard therapy; presence of disease accessible to repetitive biopsies; measurable or assessable disease; age 18 years; life expectancy 12 weeks; Eastern Cooperative Oncology Group performance status of 0 to 2; and adequate bone marrow, hepatic, and renal function. Conditions resulting in exclusion included active infection; uncontrolled or significant pulmonary or cardiovascular disease; brain metastasis; or receiving drugs with known significant P-450 3A4 inhibitory effects at trial entry. All patients gave informed consent, and approval was obtained from the ethics committee at Vall d'Hebron University Hospital and the regulatory authorities. The study followed the Declaration of Helsinki and good clinical practice guidelines.
Treatment and Dose Escalation Criteria
BMS-214662 was administered as a single weekly intravenous dose for at least 6 consecutive weeks. In the first part of the study, BMS-214662 was administered as a 1-hour infusion, and in the second part it was administered as a 24-hour infusion. In both cases, a modified Fibonacci dose escalation schema was used. For the 1-hour infusion, planned dose levels were 56, 84, 118, 157, 209, 278, and 370 mg/m2/wk. Due to the appearance of moderate toxicity at the 209 mg/m2 level, a dose level of 245 mg/m2 was added before further escalation to the 278 mg/m2 level. For the 24-hour infusion, planned dose levels were 84, 118, 157, 209, 278, 370, and 492 mg/m2/wk. After observing DLTs in the higher dose levels, a new cohort of 275 mg/m2 was added and this dose was decided in order to be consistent with the RD defined in other phase I studies. In the absence of DLT, a minimum of three assessable patients were to be enrolled to each dose level. If one of the first three patients at a given dose level experienced a DLT, three additional patients were enrolled to the same dose level. If two or more patients presented a DLT at a dose level, enrollment of patients to that dose level was discontinued and the immediately preceding dose level was considered the MTD. DLT was defined as any one of the following toxicities during the first 4 weeks (one cycle): grade 4 National Cancer Institute Common Toxicity Criteria version 1.0 neutropenia for 5 or more consecutive days or febrile neutropenia (fever > 38.5° C with an absolute neutrophil count < 1,000 cells/μL); grade 4 thrombocytopenia (< 10,000 cells/μL) or bleeding episode requiring platelet transfusion; grade 3 or greater nausea and/or emesis despite the use of adequate/maximal medical intervention and/or prophylaxis; any other grade 3 or greater nonhematologic toxicity (except grade 3 injection site reaction); and prolongation of the QTc interval 500 milliseconds. In addition, if the administration of BMS-214662 was held for 2 weeks because treatment-related toxicity failed to recover to grade 1 or baseline, this was also considered to be a dose-limiting event. Transient grade 3 AST/ALT elevations lasting 24 to 48 hours after the end of BMS-214662 infusion and without any clinical sequelae were not considered DLTs. Once the MTD was defined, this dose level would be expanded to include a maximum of 15 patients, to better characterize its safety profile and to determine its suitability as the RD for further phase II development.
Tolerability and Safety
Routine clinical and laboratory assessments were conducted on a weekly basis. After transient (24 to 48 hours after the end of the infusion) grade 3 hematologic and liver toxicities were observed, laboratory assessments were performed 24, 48, and 72 hours after the first infusion of BMS-214662, only in the first week of treatment. Serial ECG monitoring was conducted following the first infusion and thereafter once every 4 weeks. Adverse events were recorded, graded using the National Cancer Institute Common Toxicity Criteria, and assessed by the investigator for any relationship with BMS-214662 treatment. Antiemetics were administered when emesis appeared and thereafter on a prophylactic basis. Patients experiencing DLT could be retreated with BMS-214662 at a dose reduced by one level, provided that all toxicities considered related to BMS-214662 had recovered to baseline or grade 1 severity.
Response Assessments
Objective measurement of tumor mass was assessed in accordance with the revised WHO criteria (Response Evaluation Criteria in Solid Tumors). Patients demonstrating benefit from treatment with BMS-214662 continued therapy until progression or withdrawal criteria were met.
Pharmacokinetic Analysis
Sampling to define the plasma PK profile of BMS-214662 was performed during the first dose of therapy at several time points during the infusion and in the 24-hour period after the end of the infusion. Sample tubes were immediately processed, as described previously.43
The concentration of BMS-214662 in plasma was determined by an analytic method involving reversed-phase, high-performance liquid chromatography with ultraviolet detection, as described previously.43 A noncompartmental PK analysis of the plasma samples was calculated using noncompartmental methods by the PKMENU application using the Statistical Analysis System version 6.12 (SAS Institute, Cary, NC). The following PK parameters of BMS-214662 were estimated: empirical time of peak plasma level (tmax), empirical peak plasma level (Cmax), and apparent elimination rate constant (z), which was calculated by linear regression of the terminal phase of the semilogarithmic plasma levels curve, when this was clearly defined. The elimination half-life (t1/2) was defined as ln2/z. Although there might be a gamma phase with BMS-214662, the lack of sufficient number of concentration time points did not allow to describe if this gamma phase exists or not. The area under the plasma concentration-time curve (AUC) was estimated by the logarithmic-linear trapezoidal algorithm to the data point at approximately 8 hours, with extrapolation to time infinity using the slope of the terminal log concentration versus time data. Total plasma clearance (CL) was calculated as the dose divided by the AUC. Mean values of the PK variables were calculated as the geometric mean of the individual patient values.44,45 Standard deviations for the geometric mean values were estimated by the jackknife method.46 PK data were reported as mean and standard deviation.
Pharmacodynamic Assessments
FT activity in peripheral mononuclear cells The ability of BMS-214662 to inhibit FT activity in peripheral-blood mononuclear cells (PBMCs) was determined in all patients following the first dose of BMS-214662. With the 1-hour infusion, venous blood (8 mL) was collected at 0:00, 1:00, 6:00, 12:00, and 24:00 hours. With the 24-hour infusion, the venous samples were obtained at 0:00, 6:00, 23:58 (before the end of infusion), 30:00, and 48:00 hours. Blood samples (7 mL) were collected in Vacutainer CPT mononuclear-cell preparation tubes containing sodium citrate buffer (Becton Dickinson, Plymouth, UK), and processed as described previously.43
FT activity in tumor and surrounding normal tissue samples The inhibition of FT activity in tumor and surrounding normal tissues by BMS-214662 was determined in selected patients. The timing of tumor and normal tissue biopsies was changed as data from previous samples became available, adjusting the timing as required. With the 1-hour infusion, tissue samples were obtained at baseline, 24:00, and 144:00 hours after the first dose of BMS-214662 at the dose levels of 56 to 157 mg/m2; at baseline, 2:00, and 24:00 hours at the dose levels of 209 to 245 mg/m2; and at baseline, 2:00, 6:00, and 24:00 hours at the dose level of 278 mg/m2. In the 24-hour infusion schedule the samples were obtained at baseline, 6:00, 24:00, and 48:00 hours after the first dose at all dose levels. The tissue samples were immediately frozen after the biopsy procedure and were stored at –70 to –80°C.
Determination of the FT activity FT activity in extracts of PBMC and normal or tumor tissues was determined by a radioenzyme assay using [3H] farnesyl pyrophosphate and human recombinant H-ras proteins produced in bacteria, as described previously.4,40,43,47 FT activity in each sample was determined as fmol/μg protein/h and reported as a percentage of the activity in the pretreatment sample for each patient. The arithmetic mean of the normalized FT activity was calculated from the determinations made at each time point for the group of patients in each dose level.
Signal transduction inhibition and apoptosis in tumor samples MAPK signaling and apoptosis in tumor samples by BMS-214662 were determined in selected patients enrolled in the study. Immunohistochemical analysis of total p42/44 MAPK, phosphorylated p42/44 MAPK at Thr202/Tyr204 (p-MAPK), total Akt, p-Akt at Ser473, p27KIP1 expression, and proliferation marker Ki67 were performed in paraffin-embedded sections from tumor samples, as described previously.48,49 Apoptosis was studied by TUNEL assay49 and by determination of caspase signaling measured as levels of cleaved caspase 3 at Asp175 and cleaved caspase 9 at Asp330 by immunohistochemistry as described previously.50,51 Rabbit polyclonal antitotal p44/42 MAPK #9102, anti-phosphorylated p44/42 (Thr202/Tyr204) MAPK #9101, antitotal Akt #9272, and antiphosphorylated Akt #9277 from Cell Signaling Technology (Beverly, MA); mouse monoclonal anti-p27KIP1 clone SX53G8 and anti-Ki67 clone MIB1 (DakoCytomation, Carpinteria, CA); mouse monoclonal anticleaved caspase 3 at Asp175 #9661 and anticleaved caspase 9 at Asp330 #9501 (Cell Signaling Technology) were used as primary antibodies. Two negative control rabbit polyclonal immunoglobulins (Biogenex, San Ramon, CA; and Santa Cruz Biotech, Santa Cruz, CA) and a negative control mouse monoclonal immunoglobulin (Biogenex) were also used. TUNEL assay 16-dUTP-peroxidase (Roche Diagnostics Gmbh, Mannheim, Germany) was used.
Qualitative changes in the expression of markers were assessed in a blinded fashion. For quantitative analysis, the percentage of cells in the tumor tissue that stained with each antibody was scored from representative sections in 10 high-power (x400) microscope fields, and the average percentage of stained cells was calculated in paired samples. Paired pretherapy and on-therapy samples were analyzed using the Wilcoxon rank test by SPSS Data Analysis Program, version 10.0 (SPSS Inc, Chicago, IL). Statistical tests were conducted at the two-sided 0.05 level of significance.
RESULTS
Characteristics of the 37 patients included in the weekly 1-hour infusion schedule and the 31 patients included in the weekly 24-hour infusion schedule are listed in Table 1. The distribution of patients across dose levels is listed in Tables 2 and 3.
Clinical Toxicities
Hematologic toxicities were observed more frequently in the 1-hour infusion than in the 24-hour infusion schedule (Table 2). Transient grade 3 to 4 neutropenia and leukocytopenia lasting 24 to 48 hours after the end of BMS-214662 infusion without any clinical significance were observed in 38% and 25% of the patients treated with the 1-hour infusion at doses 209 mg/m2, respectively. One patient treated at the 278 mg/m2 level developed grade 4 thrombocytopenia and disseminated intravascular coagulation in the context of sepsis of gastrointestinal origin. This patient died of multiorgan failure after 10 days in an intensive care unit. In contrast, no grade 3 to 4 hematologic toxicities were observed with the 24-hour infusion schedule up to the 492 mg/m2 dose level except for grade 3 anemia. One patient in the 492 mg/m2 cohort experienced grade 4 thrombocytopenia, neutropenia, and leukocytopenia; these events were considered DLTs.
The profile of nonhematologic toxicity was completely different in the two infusion schedules (Table 2). The most frequent toxicities in the 1-hour infusion schedule were of gastrointestinal origin, including nausea/vomiting and diarrhea. One patient at 209 mg/m2 developed grade 3 nausea/vomiting without any prophylactic premedication. This patient also experienced grade 3 hypotension—considered a DLT—during the vomiting episodes. After this case, antiemetics were administered to all patients on a prophylactic basis. One patient at 245 mg/m2 and two patients at 278 mg/m2 presented grade 3 nausea/vomiting despite the antiemetic prophylaxis, all considered to be dose-limiting. One patient at 245 mg/m2 and three patients at 278 mg/m2 developed grade 3 diarrhea despite early and vigorous oral loperamide treatment and these episodes were considered DLTs. One patient at the 245 mg/m2 dose level developed a fatal grade 4 acute pancreatitis, considered to be possibly related to BMS-214662. Transient grade 3 AST/ALT elevations lasting 24 to 48 hours after the end of BMS-214662 infusion and without any clinical sequelae were observed in 19% of the patients treated at doses 245 mg/m2. One patient treated at 278 mg/m2 experienced a reversible grade 3 creatinine elevation considered DLT.
In contrast, the most frequent observed severe toxicity with the 24-hour infusion schedule was of renal origin. In the initial dose escalation, toxicity was limited to grade 1 to 2 events up to the 492 mg/m2 dose level, where two of three patients presented DLTs: one patient with reversible grade 3 creatinine elevation and one patient with reversible grade 3 renal failure, grade 3 nausea/vomiting, grade 3 amylase/lipase elevation, grade 3 infection, grade 4 creatinine elevation, grade 4 hepatitis, grade 4 transient neutropenia, and grade 4 hypovolemic shock. Therefore, the 492 mg/m2 dose level was considered a toxic dose and the lower dose level, 370 mg/m2, was expanded to determine the recommended dose. At the 370 mg/m2 dose level, one of four patients presented DLTs with grade 3 vomiting, reversible grade 3 renal failure, and grade 4 creatinine elevation. This renal toxicity was likely due to acute tubular necrosis since the fractional excretion of sodium was higher than 2% and the normalization of the creatinine levels was slow, with two patients requiring transient dialysis. Kidney biopsies were obtained from the two patients treated at 492 and 370 mg/m2, respectively, who experienced reversible renal failure. In both cases, focal acute tubular damage with regeneration signs and thrombotic microangiopathy limited to the interstitial capillaries were observed. Although only one of four patients at the 370 mg/m2 dose level experienced DLTs, recruitment to this dose level was stopped in light of the severe toxicity. Therefore, the number of patients enrolled at the 275 to 278 mg/m2 dose level was expanded. One of a total of 12 patients treated at 275 to 278 mg/m2 developed a reversible grade 2 increase in the serum creatinine and grade 3 nausea/vomiting.
In summary, DLTs in the 1-hour infusion schedule (Table 2) occurred at the 209 mg/m2 dose level and higher. The 278 mg/m2 dose level was clearly intolerable, with three DLTs occurring in a total of six patients. The 245 mg/m2 dose level was initially considered to be the MTD. However, the case of fatal pancreatitis observed at this dose level and considered related to BMS-214662 makes the 245 mg/m2 difficult to support for phase II use. Therefore, the 209 mg/m2 dose level, which produced grade 3 nausea/vomiting and hypotension in one of eight patients, and specifically in a patient who had not received antiemetic therapy, is recommended as the dose for further development of BMS-214662 when given as a weekly 1-hour infusion. In the 24-hour infusion schedule, DLTs occurred at the 275 to 278 mg/m2 dose level and higher. The 492 and 370 mg/m2 dose levels were clearly intolerable, with DLTs occurring in two of three and one of four patients, respectively. At the 275 to 278 mg/m2 dose level, one of 12 patients presented DLT. Thus, the dose of BMS-214662 recommended as a weekly 24-hour infusion schedule is 275 mg/m2.
The cumulative tolerance of BMS-214662 was globally acceptable (Table 3). In the 1-hour infusion schedule, nine doses out of a total of 287 administered doses were delayed due to toxicity, four in the first 4-week period and five after the initial 4-week period. There were four patients that were dose reduced, one in the first 4-week period and three after the initial 4-week period. In the 24-hour infusion schedule, two doses out of a total of 213 administered doses were delayed due to toxicity, both in the first 4-week period. Four patients needed dose reductions, one in the first 4-week period and three after the initial 4-week period.
Antitumor Activity
There were no objective responses. Two patients (with breast cancer) in the 1-hour infusion schedule appeared to have clinical benefit from the treatment, achieving a minor response and a prolonged stabilization of the disease. Three patients in the 24-hour infusion schedule (with breast, gastric, and renal cell cancer) appeared to benefit from treatment with prolonged stabilization of the disease.
Pharmacokinetics
BMS-214662 exhibited a linear plasma PK between all dose levels in the two different schedules. Cmax and AUC increased in a near dose proportional fashion. The percentage of total AUC that was extrapolated to infinity is greater than 98% across all the dose levels. The mean values of the PK parameters estimated by noncompartimental analysis of the individual patient data are summarized in Table 4. The mean plasma PK profile of every cohort is presented in Figure 2A (1-hour infusion) and 2B (24-hour infusion). Interpatient variability was considerable. There was a complete absence of relationship between the administered dose and the CL, Vss, and t1/2. The CL was very high (388 mL/min/m2, with a 38% coefficient of variation [CV]), the value translating to approximately 50% of the hepatic blood flow, assuming that the blood to plasma partition coefficient of BMS-214662 is 1.0. The mean Vss and t1/2 were 32 l/m2 (with a CV of 50%) and 2.91 hours, respectively. These results would indicate that the fraction of BMS-214662 bound to tissue proteins is close to the same as its binding to plasma proteins, which is around 99%.
Pharmacodynamics
Farnesyltransferase inhibition There was a good correlation between dose and degree of FT inhibition in PBMCs in both infusional schedules. In the 1-hour infusion schedule, the maximum inhibition of FT in PBMCs was observed at the end of the infusion. Greater than 80% inhibition of FT was observed at the 1-hour time point with BMS-214662 doses 118 mg/m2. FT inhibition returned to baseline by the 24-hour time point at all dose-levels (Fig 3A). In contrast, in the 24-hour infusion schedule, FT activity was uniformly inhibited throughout the duration of the infusion, achieving a sustained inhibition of 40% at 275 to 278 mg/m2 and a maximal inhibition of approximately 70% at 370 mg/m2 (Fig 3B). As expected, FT inhibition was more short lasting in the 1-hour infusion (Fig 3C) than in the 24-hour infusion schedule (Fig 3D). There was a good correlation between the BMS-214662 concentration profile and the intensity of FT inhibition in PBMCs in both schedules. The pattern of FT inhibition over time after the exposure of the drug was similar in PBMCs, tumor, and surrounding normal tissue, although there was a trend for a more prolonged inhibition in tumor and surrounding normal tissue than in PBMCs (data not shown).
Effects on signal transduction BMS-214662 did not induce changes in total- and p-MAPK, total- and p-Akt, p27KIP1, and Ki67 in tumor samples. In contrast to this, BMS-214662 induced apoptosis in all but one tumor sample. Apoptosis by TUNEL staining was observed in tumor samples both from patients with signs of antitumor activity and in patients without such activity. Figures 4A through D show the induction of apoptosis in tumor samples obtained from patients in the 1-hour and the 24-hour infusion schedules. Induction of apoptosis—measured by both hematoxylin-eosin and TUNEL stainings—appeared to be related to time after exposure being more prominent at 24- than at 2-hour time points and at the 48-hour than at 24- and 6-hour time points in the 1-hour and 24-hour infusion schedules, respectively. Expression of cleaved caspase 3 and 9 in tumor cells significantly increased after treatment with the two schedules. In the 1-hour infusion schedule, expression of cleaved caspases at 24- and 2-hour time points was significantly increased, being more prominent at the 24-hour time point, whereas in the 24-hour infusion schedule, the expression significantly increased at 48- and 24-hour time points, being more prominent at the 48-hour time point (data not shown). The induction of apoptosis appeared related to dose in the 1-hour infusion schedule and it was observed in all patients with tumor samples at doses 209 mg/m2. However, the induction of apoptosis was not found related to dose in the 24-hour infusion schedule.
DISCUSSION
In this study, BMS-214662 was developed in a weekly schedule, a dosing regimen more consistent with the preclinical activity of the compound. This phase I study was aimed at identifying the most optimal weekly schedule of BMS-214662 based on the toxicity profile, PK, and PD evaluation of FT inhibition and apoptotic induction in normal and tumor cells. We found that the toxicity profiles of the 1- and 24-hour infusion regimens were completely different. Additionally, the patterns of FT inhibition and apoptotic induction were markedly different with the two regimens.
With the 1-hour infusion schedule, DLTs consisted of nausea/vomiting, diarrhea, acute pancreatitis, hypotension, thrombocytopenia and increased serum creatinine. Transient and reversible grade 3 AST/ALT elevations and grade 3 to 4 leukocytopenia/neutropenia lasting 24 to 48 hours after the end of BMS-214662 infusion were also seen, but they did not appear to be of clinical significance. In considering PK and PD studies, FT inhibition completely mirrored the concentrations of BMS-214662 in plasma, with a profound but short-lived FT inhibition in PBMCs and tumor cells.
After completing evaluation of weekly 1-hour infusions, we proceeded to evaluate weekly 24-hour infusions for several reasons. First, the AUC reached with the 1-hour infusion at the RD (27 μM x h) was lower than the AUC predicted in preclinical models to achieve antitumor activity (77 μM x h). To the extent that toxicities might be related to Cmax rather than to AUC, it was anticipated that a higher exposure would be reached with a 24-hour infusion. Second, at the RD with the 1-hour infusion, FT inhibition in tumor cells was nearly complete, but brief ( 6 hours), probably related to the short half-life of the drug (around 3 hours). It was again predicted that a longer infusion would result in a more sustained FT inhibition. Third, in preclinical models, the therapeutic index of BMS-214662 was enhanced by prolonging the exposure time. In in vitro models, the concentration x time product (AUC) required to achieve an IC50 was disproportionately reduced as the exposure time increased from 6 to 24 hours: 100-fold for proliferating cells and 10-fold for quiescent cells. Similarly, in in vivo models, the minimal effective AUC was 29 μM x h when BMS-214662 was administered as a 24-hour infusion and 77 μM x h when it was administered as a 1-hour infusion.
The 24-hour infusion schedule at the highest dose levels resulted in unexpected renal impairment, likely due to acute tubular necrosis. However, at the RD, this schedule was better tolerated without any significant nausea, vomiting, diarrhea, leukocytopenia, or liver toxicity. The differences in the safety profile with the two schedules suggest that some toxicities are related to Cmax, whereas others are related to global exposure to the drug. It is likely that the liver and hematologic toxicities are related to Cmax since Cmax with 1-hour infusion at 209 to 278 mg/m2 was 18.3 to 19.1 μM, whereas the 24-hour infusion yielded a Cmax of 1.1 to 2.9 μM at 209 to 492 mg/m2 dose levels. In contrast, the renal toxicity is likely related to more prolonged exposure. AUC was 23.5 to 55.7 μM x h at the dose levels 209 to 492 mg/m2 with the 24-hour infusion and 27.2 to 32.6 μM x h at 209 to 278 mg/m2 in the 1-hour infusion. In addition to the most favorable safety profile, the 24-hour infusion is preferred over the 1-hour infusion for several reasons. First, delivery of BMS-214662 in a 24-hour infusion at the RD of 278 mg/m2 allows for clinical drug exposures 30 μM x h, consistent with those required preclinically for single-agent activity. Second, the 24-hour infusion provides a longer duration of FT inhibition than does the 1-hour infusion, and third, the 24-hour infusion yields higher degrees of apoptosis.
An important end point of our study was to determine which correlations existed between exposure to the study agent and inhibition of FT in the PBMCs and, of great interest, in the tumor. Although other studies with this43 and other FTIs52,53 had demonstrated a correlation between FTI dose and FT inhibition in PBMCs, and bone marrow cells and buccal mucosa cells, the degree of FT inhibition in the tumor cells with these agents was unknown. Therefore, we evaluated the relationship between the degree of FT inhibition and its timing dependency in the tumor and in selected surrogate tissues. We have demonstrated that there was a good correlation between FT inhibition related to BMS-214662 in PBMCs and normal and tumor tissue samples. As a consequence of this, we suggest that PBMCs can replace tumor tissue to determine the inhibition profile of FT activity related to BMS-214662.
In order to characterize target inhibition, we decided to determine the biologic effects of BMS-214662 in the tumor. Based on the preclinical studies suggesting that BMS-214662 had a profound proapoptotic effect, we studied apoptosis induction in tumor samples. We have clinically demonstrated in the serial tumor PD studies that BMS-214662 induced apoptosis but did not inhibit MAPK signaling. Other FTIs have also demonstrated antitumor activity and induction of apoptosis through Ras-independent pathways.20,24,54,55 The degree and duration of apoptosis was higher in the 24-hour infusion than in the 1-hour infusion schedule. Whereas in the 1-hour infusion, there was a clear correlation between dose and degree of apoptosis, we did not find such correlation in the 24-hour infusion schedule. We could not establish any correlation between the degree of observed apoptosis and clinical benefit and/or toxicity.
Normal tissues such as PBMCs and skin may be good surrogates for evaluating the exposure of the drug and kinetics of the target inhibition in a clinical model. However, tumor PD studies may better explore the biologic effects of a selected agent than normal surrogate tissues, as tumor cells often respond in a different way to targeted drugs than normal cells, also demonstrated with other compounds in the clinical setting, such as the EGFR inhibitors EMD72000SUP>56 and gefitinib.57,58 Therefore, the antineoplastic PD effect of a selected compound on the human tumor cells in the human host can only be evaluated when tumor biopsies are obtained before and during treatment. We and others support PD studies with tumor biopsies from patients enrolled in clinical trials with new targeted therapies.59,60 These studies can not only evaluate the biologic effect of the drug in the tumor, but they may also identify the genomic and proteomic profile of the population with highest chances to benefit from treatment.
BMS-214662 has been administered by other routes and schedules. With the intravenous formulation, BMS-214662 has been administered as 1-hour infusion every 3 weeks,43,61 and once weekly for 4 weeks, followed by 2 weeks of rest.62 BMS-214662 was also formulated in an oral capsule, but the severe gastrointestinal toxicities observed in the clinical setting63 prevented further development with this formulation. Different phase I studies are currently evaluating the synergistic effect of BMS-214662 in combination with cisplatin,64 paclitaxel,65 and both carboplatin and paclitaxel66 in the clinical setting.
In summary, this phase I study with weekly 1-hour and 24-hour infusions of BMS-214662 has shown that this agent can be administered with either infusion duration. However, based on the preclinical evaluation, the clinical safety profile and the PK and PD results, with a more sustained FT inhibition and apoptosis induction with a prolonged infusion, we recommend the 24-hour infusion schedule for further development of this agent.
Authors' Disclosures of Potential Conflicts of Interest
The following authors or their immediate family members have indicated a financial interest. No conflict exists for drugs or devices used in a study if they are not being evaluated as part of the investigation. Employment: Maurizio Voi, Bristol-Myers Squibb; Michael Cooper, Bristol-Myers Squibb; Anne Van Vreckem, Bristol-Myers Squibb; Veeraswamy Manne, Bristol-Myers Squibb; James A. Manning, Bristol-Myers Squibb; Carmen Garrido, Bristol-Myers Squibb. Consultant/Advisory Role: Jose Baselga, Bristol-Myers Squibb. Stock Ownership: Maurizio Voi, Bristol-Myers Squibb; Veeraswamy Manne, Bristol-Myers Squibb; James A. Manning, Bristol-Myers Squibb. For a detailed description of these categories, or for more information about ASCO's conflict of interest policy, please refer to the Author Disclosure Declaration and Disclosures of Potential Conflicts of Interest found in Information for Contributors in the front of each issue.
Acknowledgment
We thank Ramon Salazar for critical review of this manuscript.
NOTES
Presented in part at the 11th NCI-EORTC-AACR Symposium on New Drugs in Cancer Therapy, Amsterdam, Holland, November 7-10, 2000; at the 37th Annual Meeting of the American Society of Clinical Oncology, San Francisco, CA, May 12-15, 2001; and AACR-NCI-EORTC International Conference of Molecular Targets and Cancer Therapeutics, Miami, FL, October 29-November 2, 2001.
Terms in blue are defined in the glossary, found at the end of this issue and online at www.jco.org.
Authors' disclosures of potential conflicts of interest are found at the end of this article.
REFERENCES
Bos JL: ras oncogenes in human cancer: A review. Cancer Res 49:4682-4689, 1989
Barbacid M: ras genes. Annu Rev Biochem 56:779-827, 1987
Boguski MS, McCormick F: Proteins regulating Ras and its relatives. Nature 366:643-654, 1993
Albanell J, Rojo F, Sonnicchsen D, et al: Elevated farnesyltransferase activity in chemo-naive breast cancer tissue as compared to normal surrounding tissue. Proc Am Soc Cancer Res 42:489, 2001 (abstr 2634)
Rowinsky EK, Windle JJ, Von Hoff DD: Ras protein farnesyltransferase: A strategic target for anticancer therapeutic development. J Clin Oncol 17:3631-3652, 1999
Gibbs JB, Graham SL, Hartman GD, et al: Farnesyltransferase inhibitors versus Ras inhibitors. Curr Opin Chem Biol 1:197-203, 1997
Gibbs JB: Ras C-terminal processing enzymes–new drug targets Cell 65:1-4, 1991
Gibbs JB, Oliff A: The potential of farnesyltransferase inhibitors as cancer chemotherapeutics. Annu Rev Pharmacol Toxicol 37:143-166, 1997
Cox AD, Der CJ: Farnesyltransferase inhibitors and cancer treatment: Targeting simply Ras Biochim Biophys Acta 1333:F51-F71, 1997
Lerner EC, Hamilton AD, Sebti SM: Inhibition of Ras prenylation: A signaling target for novel anti-cancer drug design. Anticancer Drug Des 12:229-238, 1997
Tamanoi F: Inhibitors of Ras farnesyltransferases. Trends Biochem Sci 18:349-353, 1993
James GL, Goldstein JL, Brown MS, et al: Benzodiazepine peptidomimetics: potent inhibitors of Ras farnesylation in animal cells. Science 260:1937-1942, 1993
Kohl NE, Mosser SD, deSolms SJ, et al: Selective inhibition of ras-dependent transformation by a farnesyltransferase inhibitor. Science 260:1934-1937, 1993
Kohl NE, Omer CA, Conner MW, et al: Inhibition of farnesyltransferase induces regression of mammary and salivary carcinomas in ras transgenic mice. Nat Med 1:792-797, 1995
Nagasu T, Yoshimatsu K, Rowell C, et al: Inhibition of human tumor xenograft growth by treatment with the farnesyl transferase inhibitor B956. Cancer Res 55:5310-5314, 1995
Manne V, Yan N, Carboni JM, et al: Bisubstrate inhibitors of farnesyltransferase: A novel class of specific inhibitors of ras transformed cells. Oncogene 10:1763-1779, 1995
Bishop WR, Bond R, Petrin J, et al: Novel tricyclic inhibitors of farnesyl protein transferase. Biochemical characterization and inhibition of Ras modification in transfected Cos cells. J Biol Chem 270:30611-30618, 1995
Lebowitz PF, Prendergast GC: Non-Ras targets of farnesyltransferase inhibitors: Focus on Rho. Oncogene 17:1439-1445, 1998
Du W, Lebowitz PF, Prendergast GC: Cell growth inhibition by farnesyltransferase inhibitors is mediated by gain of geranylgeranylated RhoB. Mol Cell Biol 19:1831-1840, 1999
Du W, Liu A, Prendergast GC: Activation of the PI3'K-AKT pathway masks the proapoptotic effects of farnesyltransferase inhibitors. Cancer Res 59:4208-4212, 1999
Lebowitz PF, Davide JP, Prendergast GC: Evidence that farnesyltransferase inhibitors suppress Ras transformation by interfering with Rho activity. Mol Cell Biol 15:6613-6622, 1995
Lebowitz PF, Sakamuro D, Prendergast GC: Farnesyl transferase inhibitors induce apoptosis of Ras-transformed cells denied substratum attachment. Cancer Res 57:708-713, 1997
Law BK, Norgaard P, Moses HL: Farnesyltransferase inhibitor induces rapid growth arrest and blocks p70s6k activation by multiple stimuli. J Biol Chem 275:10796-10801, 2000
Jiang K, Coppola D, Crespo NC, et al: The phosphoinositide 3-OH kinase/AKT2 pathway as a critical target for farnesyltransferase inhibitor-induced apoptosis. Mol Cell Biol 20:139-148, 2000
Prendergast GC, Khosravi-Far R, Solski PA, et al: Critical role of Rho in cell transformation by oncogenic Ras. Oncogene 10:2289-2296, 1995
Prendergast GC, Davide JP, deSolms SJ, et al: Farnesyltransferase inhibition causes morphological reversion of ras-transformed cells by a complex mechanism that involves regulation of the actin cytoskeleton. Mol Cell Biol 14:4193-4202, 1994
Lerner EC, Zhang TT, Knowles DB, et al: Inhibition of the prenylation of K-Ras, but not H- or N-Ras, is highly resistant to CAAX peptidomimetics and requires both a farnesyltransferase and a geranylgeranyltransferase I inhibitor in human tumor cell lines. Oncogene 15:1283-1288, 1997
Whyte DB, Kirschmeier P, Hockenberry TN, et al: K- and N-Ras are geranylgeranylated in cells treated with farnesyl protein transferase inhibitors. J Biol Chem 272:14459-14464, 1997
Rowell CA, Kowalczyk JJ, Lewis MD, et al: Direct demonstration of geranylgeranylation and farnesylation of Ki-Ras in vivo. J Biol Chem 272:14093-14097, 1997
Sun J, Qian Y, Hamilton AD, et al: Ras CAAX peptidomimetic FTI 276 selectively blocks tumor growth in nude mice of a human lung carcinoma with K-Ras mutation and p53 deletion. Cancer Res 55:4243-4247, 1995
Suzuki N, Del Villar K, Tamanoi F: Farnesyltransferase inhibitors induce dramatic morphological changes of KNRK cells that are blocked by microtubule interfering agents. Proc Natl Acad Sci U S A 95:10499-10504, 1998
Suzuki N, Urano J, Tamanoi F: Farnesyltransferase inhibitors induce cytochrome c release and caspase 3 activation preferentially in transformed cells. Proc Natl Acad Sci U S A 95:15356-15361, 1998
Prendergast GC: Farnesyltransferase inhibitors: antineoplastic mechanism and clinical prospects. Curr Opin Cell Biol 12:166-173, 2000
Sebti SM, Hamilton AD: Inhibition of Ras prenylation: A novel approach to cancer chemotherapy. Pharmacol Ther 74:103-114, 1997
Lobell RB, Kohl NE: Pre-clinical development of farnesyltransferase inhibitors. Cancer Metastasis Rev 17:203-210, 1998
Ashar HR, Armstrong L, James LJ, et al: Biological effects and mechanism of action of farnesyl transferase inhibitors. Chem Res Toxicol 13:949-952, 2000
Fiordalisi JJ, Johnson RL, 2nd, Weinbaum CA, et al: High affinity for farnesyltransferase and alternative prenylation contribute individually to K-Ras4B resistance to farnesyltransferase inhibitors. J Biol Chem 278:41718-41727, 2003
Hunt JT, Ding CZ, Batorsky R, et al: Discovery of (R)-7-cyano-2,3,4, 5-tetrahydro-1-(1H-imidazol-4-ylmethyl)-3-(phenylmethyl)-4-(2-thienylsulfonyl)-1H-1,4-benzodiazepine (BMS-214662), a farnesyltransferase inhibitor with potent preclinical antitumor activity. J Med Chem 43:3587-3595, 2000
Manne V, Lee FY, Bol DK, et al: Apoptotic and cytostatic farnesyltransferase inhibitors have distinct pharmacology and efficacy profiles in tumor models. Cancer Res 64:3974-3980, 2004
Rose WC, Lee FY, Fairchild CR, et al: Preclinical antitumor activity of BMS-214662, a highly apoptotic and novel farnesyltransferase inhibitor. Cancer Res 61:7507-7517, 2001
Lee FY, Arico-Gray M, Camuso A, et al: The pro-apoptotic FT inhibitor BMS-214662 produced synergistic effects in combination with chemotherapy with antiproliferative cytotoxic agents. Proceedings of the 2001 AACR-NCI-EORTC International Conference:82, 2001 (abstr 401)
Davis RJ: Signal transduction by the JNK group of MAP kinases. Cell 103:239-252, 2000
Ryan DP, Eder JP, Jr., Puchlaski T, et al: Phase I clinical trial of the farnesyltransferase inhibitor BMS-214662 given as a 1-hour intravenous infusion in patients with advanced solid tumors. Clin Cancer Res 10:2222-2230, 2004
Lacey LF, Keene ON, Pritchard JF, et al: Common noncompartmental pharmacokinetic variables: Are they normally or log-normally distributed J Biopharm Stat 7:171-178, 1997
Mizuta E, Tsubotani A: Preparation of mean drug concentration–time curves in plasma. A study on the frequency distribution of pharmacokinetic parameters. Chem Pharm Bull (Tokyo) 33:1620-1632, 1985
Lam FC, Hung CT, Perrier DG: Estimation of variance for harmonic mean half-lives. J Pharm Sci 74:229-231, 1985
Manne V, Roberts D, Tobin A, et al: Identification and preliminary characterization of protein-cysteine farnesyltransferase. Proc Natl Acad Sci USA 87:7541-7545, 1990
Albanell J, Rojo F, Averbuch S, et al: Pharmacodynamic studies of the epidermal growth factor receptor inhibitor ZD1839 in skin from cancer patients: Histopathologic and molecular consequences of receptor inhibition. J Clin Oncol 20:110-124, 2002
Matar P, Rojo F, Cassia R, et al: Combined epidermal growth factor receptor targeting with the tyrosine kinase inhibitor gefitinib (ZD1839) and the monoclonal antibody cetuximab (IMC-C225): Superiority over single-agent receptor targeting. Clin Cancer Res 10:6487-6501, 2004
Rohn TT, Rissman RA, Davis MC, et al: Caspase-9 activation and caspase cleavage of tau in the Alzheimer's disease brain. Neurobiol Dis 11:341-354, 2002
Woenckhaus C, Giebel J, Failing K, et al: Expression of AP-2alpha, c-kit, and cleaved caspase-6 and -3 in naevi and malignant melanomas of the skin. A possible role for caspases in melanoma progression J Pathol 201:278-287, 2003
Adjei AA, Erlichman C, Davis JN, et al: A Phase I trial of the farnesyl transferase inhibitor SCH66336: evidence for biological and clinical activity. Cancer Res 60:1871-1877, 2000
Karp JE, Lancet JE, Kaufmann SH, et al: Clinical and biologic activity of the farnesyltransferase inhibitor R115777 in adults with refractory and relapsed acute leukemias: A phase 1 clinical-laboratory correlative trial. Blood 97:3361-3369, 2001
Chun KH, Lee HY, Hassan K, et al: Implication of protein kinase B/Akt and Bcl-2/Bcl-XL suppression by the farnesyl transferase inhibitor SCH66336 in apoptosis induction in squamous carcinoma cells. Cancer Res 63:4796-4800, 2003
Mizukami Y, Ura H, Obara T, et al: Requirement of c-jun N-terminal kinase for apoptotic cell death induced by farnesyltransferase inhibitor, farnesylamine, in human pancreatic cancer cells. Biochem Biophys Res Commun 288:198-204, 2001
Tabernero J, Rojo F, Jimenez E, et al: A phase I PK and serial tumor and skin pharmacodynamic (PD) study of weekly (q1w), every 2-week (q2w) or every 3-week (q3w) 1-hour infusion EMD72000, a humanized anti-epidermal growth factor receptor (EGFR) antibody, in patients (pt) with advanced tumors. Proc Am Soc Clin Oncol 22:192, 2003 (abstr 770)
Rojo F, Tabernero J, Van Cutsem E, et al: Pharmacodynamic studies of tumor biopsy specimens from patients with advanced gastric carcinoma undergoing treatment with gefitinib (ZD1839). Proc Am Soc Clin Oncol 22:191, 2003 (abstr 764)
Baselga J, Albanell J, Ruiz A, et al: Phase II and tumor pharmacodynamic study of gefitinib (ZD1839) in patients with advanced breast cancer. Proc Am Soc Clin Oncol 22:7, 2003 (abstr 24)
Baselga J: Skin as a surrogate tissue for pharmacodynamic end points: Is it deep enough Clin Cancer Res 9:2389-2390, 2003
Arteaga CL, Baselga J: Tyrosine kinase inhibitors: Why does the current process of clinical development not apply to them Cancer Cell 5:525-531, 2004
Sonnichsen D, Damle B, Manning J, et al: Pharmocokinetics (PK) and pharmacodynamics (PD) of the farnesyltransferase (FT) inhibitor BMS-214662 in patients with advanced solid tumors. Proc Am Soc Clin Oncol 19:178a, 2000 (abstr 691)
Kim KB, Shin DM, Summey CC, et al: Phase I study of farnesyl transferase inhibitor, BMS-214662 in solid tumors. Proc Am Soc Clin Oncol 20:79a, 2001 (abstr 313)
Camacho LH, Soignet SL, Pezzulli S, et al: Dose escalation of oral farnesyl transferase inhibitor (FTI) BMS-214662 in patients with solid tumors. Proc Am Soc Clin Oncol 20:79a, 2001 (abstr 311)
Mackay HJ, Hoekstra R, Eskens FA, et al: A phase I pharmacokinetic and pharmacodynamic study of the farnesyl transferase inhibitor BMS-214662 in combination with cisplatin in patients with advanced solid tumors. Clin Cancer Res 10:2636-2644, 2004
Bailey HH, Marnocha R, Arzoomanian R, et al: Phase I trial of weekly paclitaxel and BMS214662 in patients with advanced solid tumors. Proc Am Soc Clin Oncol 20:79a, 2001 (abstr 314)
Dy GK, Bruzek LM, Croghan GA, et al: A phase I trial of the farnesyltransferase(FT) inhibitor, BMS-214662 (B) in combination with paclitaxel (P) and carboplatin (C) in patients with advanced cancer. Proc Am Soc Clin Oncol 23:211, 2004 (abstr3066)(Josep Tabernero, Fredy Ro)