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© 2000 American Society for Clinical Oncology Phase I and Pharmacokinetic Study of Farnesyl Protein Transferase Inhibitor R115777 in Advanced CancerFrom the Medicine Branch, Division of Clinical Sciences, National Cancer Institute; Clinical Center, National Institutes of Health; and National Eye Institute, Bethesda; SAIC-Frederick, Frederick, MD; Janssen Research Institute, Titusville, NJ; Janssen Research Foundation, Beerse, Belgium; Ortho-Clinical Diagnostics, Rochester, NY; and Johnson and Johnson Research, Sydney, Australia. Address reprint requests to Jo Anne Zujewski, MD, Medicine Branch, Division of Clinical Sciences, National Cancer Institute, 9000 Rockville Pike, Bethesda, MD; email zujewski{at}nih.gov
PURPOSE: To determine the maximum-tolerated dose, toxicities, and pharmacokinetic profile of the farnesyl protein transferase inhibitor R115777 when administered orally bid for 5 days every 2 weeks. PATIENTS AND METHODS: Twenty-seven patients with a median age of 58 years received 85 cycles of R115777 using an intrapatient and interpatient dose escalation schema. Drug was administered orally at escalating doses as a solution (25 to 850 mg bid) or as pellet capsules (500 to 1300 mg bid). Pharmacokinetics were assessed after the first dose and the last dose administered during cycle 1. RESULTS: Dose-limiting toxicity of grade 3 neuropathy was observed in one patient and grade 2 fatigue (decrease in two performance status levels) was seen in four of six patients treated with 1,300 mg bid. The most frequent clinical grade 2 or 3 adverse events in any cycle included nausea, vomiting, headache, fatigue, anemia, and hypotension. Myelosuppression was mild and infrequent. Peak plasma concentrations of R115777 were achieved within 0.5 to 4 hours after oral drug administration. The elimination of R115777 from plasma was biphasic, with sequential half-lives of about 5 hours and 16 hours. There was little drug accumulation after bid dosing, and steady-state concentrations were achieved within 2 to 3 days. The pharmacokinetics were dose proportional in the 25 to 325 mg/dose range for the oral solution. Urinary excretion of unchanged R115777 was less than 0.1% of the oral dose. One patient with metastatic colon cancer treated at the 500-mg bid dose had a 46% decrease in carcinoembryonic antigen levels, improvement in cough, and radiographically stable disease for 5 months. CONCLUSION: R115777 is bioavailable after oral administration and has an acceptable toxicity profile. Based upon pharmacokinetic data, the recommended dose for phase II trials is 500 mg orally bid (total daily dose, 1,000 mg) for 5 consecutive days followed by 9 days of rest. Studies of continuous dosing and studies of R115777 in combination with chemotherapy are ongoing.
THERAPIES DIRECTED against specific molecular targets offer the promise of increased antitumor efficacy with decreased toxicity. The ras proto-oncogene encodes a 21-kd guanosine triphosphate–binding protein Ras, which is a critical component in cellular signal transduction associated with cell proliferation, differentiation, and other pleiotropic responses.1 Activating, oncogenic, point mutations in codons 12, 13, and 61 of the ras gene have been observed in approximately 30% of adult human solid tumors, including pancreas, lung, colon, bladder, and other tumors.1-11 The wild-type Ras protein may also contribute to the growth of tumors that are driven by the aberrant activation of growth factor receptors and other tyrosine-specific protein kinases.12-16 To function in signal transduction and malignant transformation, Ras must localize to the plasma membrane.17-20 Lacking membrane-binding domains, newly synthesized Ras requires sequential posttranslational enzymatic processing before membrane attachment. The initial and rate-limiting step involves the covalent attachment of a 15-carbon farnesyl moiety via a thioether bond to a single cysteine positioned exactly four amino acids from the carboxyl terminus.21 This reaction is catalyzed by the enzyme farnesyl protein transferase. The C-terminal recognition sequence has become known as a CAAX motif to indicate the requirement for a cysteine followed by two neutral amino acids (A) with a C-terminal serine or methionine for recognition by farnesyl protein transferase. Farnesylation is followed by cleavage of the three terminal amino acids by a CAAX protease.22 The resulting C-terminal farnesylcysteine moiety is further carboxy-O-methylated to create the proper hydrophobicity or molecular recognition features to allow plasma membrane localization within cells.22 The delineation and purification of enzymes involved in Ras processing created the opportunity to downregulate ras function in tumor cells by preventing proper localization of the protein. The demonstration that activated, oncogenic Ras lacking the c-terminal cysteine lost cell-transforming activity and the description of simple CAAX tetrapeptide inhibitors of the enzyme farnesyl protein transferase focused drug discovery efforts on this posttranslational step.21,23 Initial reports on the cellular effects of farnesyl protein transferase inhibitors that were CAAX peptidomimetics suggested that this class of agent selectively reversed the ras-transformed phenotype in cell lines bearing ras mutations.24-26 These findings were very promising because polymerase chain reaction (PCR)–based DNA diagnostics were available that would allow the detection of ras mutations and possibly the preselection of patients who would be the best candidates for this ras-targeted therapy.27 However, subsequent preclinical studies have shown the pharmacology of farnesyl protein transferase inhibitors to be more complex. First, farnesyl protein transferase inhibitors, including R115777, have shown antiproliferative effects in vitro and antitumor effects in vivo in cell lines with wild-type ras.28-30 The effects of this class of agent are clearly not dependent upon the presence of mutant Ras, although the compounds are highly effective in cell lines transformed by mutant ras also.25,31-34 Also, it was reported that the K-ras isoform of Ras had a much higher affinity for farnesyl protein transferase than the H-ras or N-ras isoform.35 Inhibitors that were competitive for the Ras substrate-binding site of the enzyme were much less effective in blocking K-ras farnesylation in cell-free systems. An additional complicating issue was introduced by the observation that the CVIM CAAX motif of the K-ras peptide allowed the molecule to be either farnesylated or geranylgeranylated by geranylgeranyl protein transferase type 1. Geranylgeranyl protein transferase type 1 is quite similar to farnesyl protein transferase but attaches a 20-carbon geranylgeranyl isoprenoid moiety to substrate proteins bearing a CAAX motif with a terminal leucine.36 In intact cell lines bearing K-ras mutations, alternative processing of K-ras by the geranylgeranyl protein transferase type 1 pathway was shown to produce resistance to some farnesyl protein transferase inhibitors.37,38 The results suggested that farnesyl protein transferase inhibitors might be of no practical use in the human tumor setting because K-ras mutations account for the vast majority of ras mutations in human tumors. However, it has been clearly established that tumors with mutant K-ras respond to this class of agent both in vitro and in vivo.39,40 Ironically, as the present clinical studies of R115777 and other compounds are being reported, the biochemical basis for the antitumor responses obtained in preclinical models is under intense reevaluation. An emerging hypothesis involving Rho B accounts for some of the discrepancies discussed previously. Like K-ras, Rho B can be either farnesylated or geranylgeranylated with isolated enzymes or in intact cells.41,42 Farnesylated Rho B seems to cooperate in expression of the transformed phenotype downstream of Ras through modulation of cytoskeletal proteins.43 Expression of constructs of Rho B that can only be geranylgeranylated seems to produce antiproliferative and antitransforming effects that are similar to the effects of farnesyl protein transferase inhibitors.44 Thus, farnesyl protein transferase inhibitors may produce antitumor effects by altering the balance of farnesylated and geranylgeranylated Rho B in cells. Although the role of Ras in the antitumor effects of protein farnesyl transferase inhibitors remains ambiguous in preclinical studies, it will be important to assess ras gene status in patients entering onto studies of this class of compound. Although the existing data preclude the ras gene mutations as an entry criterion for treatment with a farnesyl protein transferase inhibitor, clinical studies may ultimately find a correlation between oncogene status and responses to these compounds. The genetic instability and complexity of human tumor cell lines used for laboratory studies may not be appropriate to the characterization of newer therapies with specific molecular targets. Regardless of the mechanism, it is clear that this class of compound produces antitumor effects in standard preclinical tumor models, including human tumor xenografts as well as transgenic oncomouse models.32,44 The crux of modern cancer research is the translation of preclinical antitumor effects into effective clinical therapy. The first step of this translation are the phase I safety and pharmacokinetic evaluations that allow selection of dose and dose schedules for further evaluation. Presented herein are the phase I data for the first farnesyl protein transferase inhibitor to be evaluated in clinical trials, R115777 (Fig 1). R115777 is a substituted quinolone that is a competitive inhibitor of the CAAX peptide-binding site of farnesyl protein transferase.45 The molecule is an extremely potent inhibitor of farnesylation with isolated enzyme inhibition of lamin B1 (50% inhibitory concentration [IC50], 0.8 nmol/L) and K-ras peptide (IC50, 7.9 nmol/L).40 R115777 is also a potent inhibitor of proliferation of intact cell lines. The IC50 required to inhibit H-ras transformed fibroblasts is 1.7 nmol/L, and the IC50 required to inhibit pancreatic and colon cancer cell lines bearing K-ras mutations ranges from 16 to 22 nmol/L.40 Preclinical studies have demonstrated that R115777 has antitumor effects in murine xenograft models using H-ras–transformed fibroblasts46 and pancreatic and colon cell lines bearing K-ras mutations.30 No gross toxicity to the tumor-bearing host has been observed at effective doses of this compound. After oral administration of R115777 to male Wistar rats and male Beagle dogs, plasma concentrations of R115777 declined with a terminal half-life of less than 1.7 hours and 2.1 hours, respectively. The absolute oral bioavailability was 9% and 66% in the rat and dog, respectively (Janssen Research Foundation, Beerse, Belgium, unpublished observations).
Patient Eligibility Patients eligible for this trial had to meet the following criteria: pathologic confirmation of advanced cancer; no available therapy proven to improve survival; last dose of radiation therapy or chemotherapy at least 4 weeks before study entry (6 weeks for nitrosoureas or mitomycin); at least 18 years of age; Zubrod performance status of 0 or 1; adequate hepatic function (normal bilirubin, transaminase levels less than two times the upper limit of normal); normal creatinine levels (0.9 to 1.4 mg/dL for males and 0.7 to 1.3 mg/dL for females); and adequate bone marrow function (absolute neutrophil count >1,500/µL and platelet count >100,000/µL). Pregnant patients and lactating mothers were ineligible, as were patients with the following characteristics: extensive prior radiation therapy (> 25% of bone marrow reserve); previous bone marrow transplantation or high-dose chemotherapy with bone marrow or stem-cell rescue; untreated CNS metastases; concurrent radiation therapy, chemotherapy, hormonal therapy, or immunotherapy; coexisting medical or psychiatric conditions that were likely to interfere with study procedures; or known allergy to imidazole drugs. All patients were required to provide written informed consent according to National Cancer Institute institutional review board guidelines.
Patient Evaluations
Treatment Plan
During cycle 1, in order to accommodate 24-hour pharmacokinetic sampling after first-dose administration, a single dose was administered on the first day, followed by bid administration for the next 4 days (days 2 through 5). The final (10th) dose of the cycle was administered on the sixth day. The schema permitted intrapatient dose escalation, thereby reducing the number of patients who would be treated at potentially subtherapeutic doses.49,50 Intrapatient dose escalation to the next dose level was allowed during the second and subsequent treatment cycles provided that nonhematologic toxicity was less than grade 1 in severity, hematologic toxicity was less than grade 2 in severity in the preceding cycle, and no treatment delays were necessary. Three patients were enrolled at each dose level and observed for at least 14 days before additional patients were entered at the next dose level. If no dose-limiting toxicity was observed in three of three patients at a single dose level, additional patients were entered at the next higher dose level. If dose-limiting toxicity was observed in one patient, additional patients up to a total of six were entered at the same dose level. If two patients developed dose-limiting toxicity at a single dose level, the maximum-tolerated dose was determined to have been exceeded and accrual ceased at that dose level. The first cycle at a new dose level was considered for dose-limiting toxicity (whether entering trial at that dose level or escalating to that dose level). Subsequently, up to a total of six patients could be entered at one dose level below. The maximum-tolerated dose was defined as the highest dose level at which no more than one of six patients experienced a dose-limiting toxicity that could reasonably be attributed to the study drug.
Toxicity
Antitumor Response
Drug Administration
Pharmacokinetics To determine the pharmacokinetic parameters that could predict the occurrence of certain adverse events related to the intake of R115777, the following evaluation was performed. The pharmacokinetic parameters Cmax, AUC12sd (day 1), and AUC12ss (day 6) were tested as predictors for the occurrence of nausea, vomiting, diarrhea, and fatigue. A logistic regression analysis was performed by fitting a generalized linear model with a binary link to the data. The logistic regression model allows the binary data to be converted into a continuous relationship between measures of drug exposure and the probability of developing a certain adverse event. Adverse events were coded as binary response variables (yes or no) without taking into account the severity. The model was parameterized via (i) the predictor value corresponding to 50% probability of having a certain adverse event (P50) and (ii) the sigmoidicity parameter, which reflects the steepness of the probability versus predictor curve (n). The best estimates of parameters and their SEs were obtained via a bootstrap analysis. The number of bootstrap replications was 1,000. The S-PLUS package (Probability, Statistics, & Information, Seattle, WA) was used throughout the analysis.
Flow Cytometric Analysis
Analysis of Ras and Prenyl Protein Processing
ras Mutation Analysis
Patient Characteristics Twenty-seven patients were treated in this phase I study. Patient characteristics are listed in Table 2.
Adverse Events Table 3 includes all adverse events observed during cycle 1 of therapy considered possibly, probably, or very likely related to R115777. Dose-limiting toxicity was observed at the 1,300-mg dose level in one patient who had a prior history of mild peripheral neuropathy attributed to paclitaxel chemotherapy. During cycle 1, she developed severe burning in her lower extremities, oral cavity, and vaginal area. The pain required opioid analgesics and resolved within 24 hours after withholding of the drug. There were no signs of stomatitis or vaginitis on physical examination. The same patient experienced similar but less severe symptoms during her next treatment cycle at a reduced dose (800 mg bid); however, severe (grade 3) symptoms recurred during her third cycle of therapy at 800 mg bid.
Although not defined as dose-limiting, clinically significant fatigue was observed in patients treated at the higher dose levels (800 mg and 1,300 mg bid). With National Cancer Institute of Canada criteria, grade 2 fatigue (two-level decrease in performance status) was observed in one of three patients who received 1,300 mg bid during the first cycle of therapy and in four of six patients treated at 1,300 mg bid during any cycle (Table 4).
One patient developed a grade 2 increase in his serum creatinine level during his second treatment cycle. The patient’s baseline creatinine level was 1.1 mg/dL. He received the first cycle of R115777 at 800 mg bid without a significant change in his serum creatinine level. During cycle 2, in which R115777 was administered at 1,300 mg bid, his creatinine level increased to 3.3 mg/dL on day 6. His creatinine level had normalized by day 30. He received cycle 3 at the 800-mg bid dose without event. Evaluation of urine sediment during cycle 2 was remarkable for renal epithelial cells consistent with an acute tubular injury. Proteinuria was not significant. Other causes of renal dysfunction (eg, contrast dye administration, nonsteroidal analgesics, hypotension) and predisposing factors for renal dysfunction were excluded. Eight additional patients were noted to have increased creatinine levels in this study. In five of these eight patients, grade 1 creatinine elevation was noted and considered at least possibly related to R115777 (three patients at the 1,300-mg bid dose level, one patient at the 800-mg bid dose level, and one patient at the 200-mg bid dose level). In one of these five patients, examination of the urinary sediment was also consistent with acute tubular injury during the first cycle at 1,300 mg bid and during a subsequent cycle at 800 mg bid. In three patients, other causes were thought more likely to account for the creatinine elevation (obstruction due to malignant disease in two patients and an inferior vena cava thrombosis in one patient). Another prominent adverse event was nausea and vomiting. At dose levels 1 through 6, an oral liquid formulation was used. This liquid had an unpleasant taste, and nausea and vomiting were frequently reported. Although the capsule formulation was tolerated better than the liquid formulation, at the highest dose levels, the capsule formulation was also associated with grade 1 and 2 nausea and vomiting. Twenty of 27 patients required antiemetic therapy. The choice of antiemetic was made at the discretion of the prescribing physician. Drugs used included ondansetron, granisitron, prochlorperazine, metoclopromide, lorezepam, and promethazine. One patient with a baseline history of migraines treated with 125 mg bid experienced a grade 3 headache during her first cycle of therapy. This headache was similar in character but more severe than her prestudy headaches. She was able to continue treatment without subsequent events. Minimal hematopoietic toxicity was observed in this trial. One patient treated with 50 mg bid experienced grade 3 neutropenia. This patient had multiple prior therapies for breast cancer, including radiation. A review of her complete blood counts obtained before study drug administration demonstrated intermittent grade 3 neutropenia. This patient continued to receive study drug at the same dose level with resolution of her neutropenia. A second patient with a baseline platelet count of 103,000/µL developed grade 2 thrombocytopenia (72,000/µL) during cycle 1 of R115777 at the 1,300-mg bid dose level. This patient also experienced grade 3 peripheral neuropathy requiring a dose reduction. She was able to continue therapy without delay at the 800-mg bid dose level with resolution of her thrombocytopenia and no subsequent recurrences of thrombocytopenia. Eight patients required RBC transfusions during this trial. All patients requiring blood transfusions had received prior therapy for their advanced cancer and had multiple blood samples drawn for pharmacokinetic studies and toxicity monitoring. Several farnesylated proteins are important in maintenance of retinal cytoarchitecture and photoreceptor structure48; therefore, all patients were carefully evaluated for ophthalmologic abnormalities. No abnormalities were noted in D-15 color vision and contrast sensitivity testing. Two patients had small unilateral visual field defects while on therapy. In one patient, the visual field defect resolved during continued R115777 therapy. In the second patient, a possible defect in the same area was noted at baseline that became more apparent after initiation of therapy. Both patients were asymptomatic. Retinal examinations were remarkable for the development of abnormalities during drug administration in four patients, including cotton wool spots and small retinal hemorrhages (two patients), small hemorrhage (one patient), and Roth’s spots (one patient). These four patients also had a history of diabetes, hypertension, or anemia. All patients were asymptomatic, and the ophthalmologic findings were thought to be consistent with those observable in a chronically ill population. No new or worsening cataracts were noted in this trial. Several serious adverse events were observed during this trial that were not considered related to the study drug. One patient with history of pulmonary embolism developed an inferior vena cava clot at the site of an inferior vena cava filter. He was taken off study and treated with anticoagulant therapy. One patient with melanoma and a prior history of brain metastasis treated with radiation therapy experienced an unwitnessed seizure. Subsequent magnetic resonance imaging revealed new and enlarged brain metastases. One patient with a history of hypertension experienced an episode of confusion. Imaging studies were consistent with a new small cerebral hemorrhage thought secondary to hypertension. One patient developed a small pericardial effusion and atrial arrythmia thought to be related to progressive malignant disease.
Pharmacokinetics
Plots of the individual values of Cmax and AUC12h, evaluated after the first dose on day 1 and the last dose on day 6, versus the administered dose of R115777 (Fig 3) indicate a consistent dose-proportional increase in the 25 to 325-mg dose range for the oral solution. For the capsule, a dose-proportional increase was observed in the 500- to 1,300-mg dose range after the first dose on day 1. However, at day 6, AUC12h and Cmax seemed to increase less than dose proportional. Since vomiting occurred only during one out of 11 assessments for the pharmacokinetics of the 800- and 1,300-mg doses, it is not likely that the deviation from dose proportionality is the result of drug loss from emesis. The data also suggest that the bioavailability of the capsules is less than that of the oral solution. Furthermore, the data suggest substantial interindividual variability in the oral bioavailability of R115777.
The urinary excretion of unchanged R115777 (n = 15) was negligible, as less than 0.1% of the administered oral dose was excreted in the urine as unchanged drug. In addition, 16.5 ± 12.2% (mean ± SD) of the administered dose was excreted in the urine as the glucuronide conjugate of R115777. The estimates of the logistic regression model parameters, as listed in Table 6, related frequently observed adverse events with pharmacokinetic parameters. According to the model, fatigue was the only response for which the probability of occurrence could be predicted reliably on the basis of Cmax and AUCs. The AUC12h(ss) corresponding to a 50% probability to develop fatigue was estimated at 4,210 ± 1,390 ng · h/mL (mean ± SD). The model prediction for nausea had a borderline significance. Vomiting and diarrhea could not be related to any of the pharmacokinetic parameters.
Flow Cytometric Analysis The effect of farnesyltransferase inhibitors on T-, B-, and natural killer–cell populations was assessed by flow cytometry. To assess changes in the threshold for activation of the cells, the expression of early and late activation markers (CD69 and HLA-DR, respectively) and CD45 isoform markers of naive and activated/memory subpopulations was examined in CD4 populations. Finally, atypical CD8 populations expressing CD57 and lacking in CD28 expression expand after chemotherapy or transplantation, in human immunodeficiency virus and in the extreme elderly, in a process that may reflect terminal differentiation of chronically activated cells.53,54 These CD8 subpopulations were therefore assessed. Flow cytometric analyses were performed at five time points: pretreatment (cycle 1, day 1), end of the first drug treatment (cycle 1, day 6), end of the first cycle (cycle 1, day 14), end of the second cycle (cycle 2, day 14), and end of the third cycle (cycle 3, day 14). All 27 patients were assessed before the start of therapy, 21 were assessed through the end of two cycles of treatment and recovery, 16 were assessed through three cycles, and two were observed after four and six cycles. The absolute numbers (cells/µL) of all peripheral-blood lymphocyte populations assessed (CD4, CD8, total CD3, B, and natural killer cells) decreased during the first 5-day treatment period (P[CD4] = .047, P[CD8] = .01, P [natural killer] = .001, P [B] = .36) but recovered to pretreatment levels by the end of cycle 1 or cycle 2. T-cell subsets and B-cell populations at the end of the third cycle (cycle 3, day 14) were reduced 30% to 35% compared with pretreatment levels (P[CD4] = .001, P[CD8] = .001, P[natural killer] = .08, P[B] = .007) but remained within normal adult ranges. The reduction in numbers persisted in the two individuals observed for longer periods. The drop in T and B cells was associated primarily with a decrease in the overall frequency of lymphocytes from an average of 21% ± 2.3% at baseline (cycle 1, day 1) to 16.6% ± 2.3% at cycle 3, day 14; this corresponded to an average decrease in the total number of lymphocytes from 1,454 ± 166 cells/µL to 1,026 ± 114 cells/µL. The total WBC count did not change significantly (7.3 ± 0.71 x 103/µL v 7.1 ± 0.77 x 103/µL) at these time points. Thus, although a 14-day period was sufficient for recovery of lymphocyte levels after the first two cycles, it was not sufficient for the third. The percentages of T-cell subsets and B and natural killer cells within the total lymphocyte population remained remarkably constant throughout the study. Furthermore, the frequency of expression of activation markers (HLA-DR), of naive and memory phenotypes in CD4 cells, and of atypical chronically activated CD8 cells remained consistent within each patient. This lack of changes in subpopulations of T cells would be consistent with either altered trafficking of lymphocytes within the peripheral blood or with a nonspecific loss of lymphocyte populations.
Analysis of Ras and Prenyl Protein Processing
Antitumor Response
This phase I trial attempted to determine the maximum-tolerated dose of R115777 when administered orally twice daily for 5 consecutive days followed by 7 to 9 days of rest. The maximum-tolerated dose as defined in the protocol was not reached and dosing was terminated at the highest dose level (1,300 mg bid; total daily dose, 2,600 mg). Only one dose-limiting toxicity was observed in one of six patients who received R115777 1,300 mg bid. This patient developed grade 3 peripheral neuropathy, described as a painful burning sensation in the extremities, oral cavity, and vaginal area. Although not defined as dose limiting, grade 2 fatigue (decrease in two performance status levels) was observed in four of six patients at the 1,300-mg bid dose level and two of nine patients at the 800-mg bid dose level. Grade 1 to 2 increases in serum creatinine levels and urinary findings consistent with acute tubular injury were noted in two patients treated with the 1,300-mg dose, which suggests that R115777 may be nephrotoxic at high doses. Therefore, we recommend that patients maintain adequate hydration during R115777 therapy and that concurrent treatment with agents known to cause renal tubular injury be avoided. Minimal hematopoietic toxicity was observed in this trial, possibly due to the interrupted schedule. Pharmacokinetic studies demonstrate that R115777 is orally bioavailable with plasma concentrations reaching those necessary for an antitumor effect in preclinical studies. Dose-proportional pharmacokinetics in the 25- to 325-mg dose range were noted for the oral solution throughout the 5-day dosing regimen. For the capsule, dose proportionality could be demonstrated in the 500- to 1,300-mg dose range after the first dose on day 1 but not after the last dose on day 6. Furthermore, the data suggest that the bioavailability of the capsules is less than that of the oral solution. However, because of the limited data and the high interindividual variability, more data are needed to investigate these observations. The pharmacokinetics of R115777 will be further explored in the drug development of R115777 to allow correlation of pharmacokinetic parameters with patient characteristics, disease state, liver function, and concomitant medication using population pharmacokinetic analysis techniques. Also, other drug formulations and the effect of food will be evaluated. Determination of the therapeutic level will allow assessment of the importance of the interindividual pharmacokinetic variability. No objective tumor responses were observed in this phase I trial, although one patient with colon cancer metastatic to lungs experienced an improvement in her cough and decreased carcinoembryonic antigen levels. ras mutation analysis was performed on tumor specimens of patients participating in this trial. Three of 23 tumor specimens tested were positive for a ras mutation. The low frequency of ras mutations in this study can be attributed to the small sample size and patient selection factors. We also had other trials open at our institution for patients with known ras mutations, which may also have been a contributing factor. The results of phase II studies will be necessary to correlate ras mutation status with clinical response. Although the protocol-defined maximum-tolerated dose was not achieved in this trial, analysis of toxicity data from all cycles and the pharmacokinetic data suggest that 500 mg bid for 5 days every 14 days is an appropriate dose for phase II studies. Pharmacokinetic studies demonstrate that 500 mg orally twice daily achieves plasma concentrations correlating with an antitumor effect in preclinical studies. The most frequent clinically significant adverse event related to R115777 was fatigue. Two of seven patients treated with 800 mg bid and four of six patients treated at 1,300 mg bid reported grade 2 worsening of performance status. All patients had advanced cancer and virtually all of them had received multiple previous therapies that could have contributed to declines in performance status. Nonetheless, the association of increasing frequency of significant performance status reduction with doses greater than 500 mg bid is reasonably strong. Clinical applications of this schedule might be in combination with cytotoxic chemotherapy, especially in light of the finding that cisplatin and paclitaxel have been demonstrated to have additive to synergistic effects when combined with farnesyl protein transferase inhibitors.56 It remains to be seen whether the dose and schedule defined in this trial has utility as a chronic single-agent therapy, because only two patients received R115777 for at least 2 months. Further evaluation of this schedule can be addressed further in phase II studies with defined patient populations. Further support for phase II testing with R115777 at the 500-mg orally, twice-daily schedule comes from pharmacokinetic data showing a possible deviation from dose proportionality for the oral bioavailability of R115777 at doses greater than 500 mg after repeated dosing. Our initial attempts to develop a surrogate biochemical correlate to monitor farnesyl protein transferase inhibition in peripheral-blood lymphocytes failed to detect changes that have been seen in cell culture studies. It remains to be determined whether the problem is associated with technical limitations of the assay or was due to the lack of protein turnover in the normally quiescent lymphocyte compartment. In addition to the role mutant Ras may play in proliferation and apoptotic resistance in fibroblastic and epithelial malignancies,57 Ras proteins also play a central role in both the activation58 and apoptotic pathways59 of normal T and natural killer cells. Maintenance of homeostasis in T-cell populations in adults involves a complex interplay of low-level proliferation of peripheral cells, replenishment by maturation of new naive cells from the thymus, and loss of cells by apoptosis.60-63 The effect of inhibition of farnesyl protein transferase on T-cell homeostasis is unknown but an important concern in a chronically administered treatment. Natural killer cells, in contrast to T cells, are relatively short-lived, but little is known of the mechanisms regulating natural killer homeostasis. For these reasons, the effect of farnesyl protein transferase inhibitors on T-, B-, and natural killer–cell populations was assessed by flow cytometry. A small decrease in the total number of lymphocytes was noted at the end of the third cycle of therapy in this trial. Although this decrease was not of clinical significance, these data suggest the need for monitoring of the lymphocyte populations in long-term or continuous-treatment trials with R115777. This study indicates that R115777 is orally bioavailable with an acceptable safety profile. This first clinical trial with a farnesyl protein transferase inhibitor suggests further investigations are warranted. Major challenges with this and other agents that are considered cytostatic are dose and schedule selection and sequencing in combination with other cancer treatments. Early clinical trials of these and other similar compounds should continue exploratory studies of potential novel surrogate end points of drug effect64 (for example, levels of farnesylated proteins). These studies will help select optimal dosing schedules for definitive trials that would assess time to progression and, ultimately, overall survival. It may be necessary to administer farnesyl protein transferase inhibitors chronically or in combination with other therapies for maximum clinical benefit. A phase I study of chronic dosing with R115777 suggests that myelosuppression occurs at significantly lower doses than has been observed in this phase I trial using an interrupted schedule.65 The different toxicity profiles seen with the intermittent versus chronic dosing schedules have implications for sequencing with other agents that cause myelosuppression. Preclinical data with various farnesyl protein transferase inhibitors demonstrate synergy or additive effects with the traditional chemotherapy agents56 and radiation therapy.66 At the same time, preclinical data continue to underscore that ras (mutated or wild-type) is not the sole target of farnesyl transferase inhibition.67 The fact that ras mutation status does not predict preclinical anticancer activity28 suggests that phase II trials should target a wide variety of malignancies (including lung, breast, colon, ovarian, and hematologic malignancies), regardless of the incidence of ras mutations. Studies of continuous dosing and studies of R115777 in combination with other antineoplastic agents are ongoing.
Supported in part with federal funds from the National Cancer Institute, National Institutes of Health, under contract no. NO1-CO-56000. The authors acknowledge Tanya Applegate, Caroline Fuery, Natalie Robert, Michele Steinmann, Jianbo Sun, and Jackie Toner for their contributions to the ras mutation analysis; Louise R. Finch, Christine Maloney, Annie Lennon-Gold, and Sylvia Avery for their research assistance; and David Venzon for the statistical review of the manuscript.
The contents of this publication do not necessarily reflect the views or policies of the Department of Health and Human Services, nor does the mention of trade names, commercial products, or organization imply endorsement by the United States government.
1. Barbacid M: ras genes. Annu Rev Biochem 56:779-827, 1987[Medline] 2. Grunewald K, Lyons J, Frohlich A, et al: High frequency of Ki-ras codon 12 mutations in pancreatic adenocarcinomas. Cancer 43:1037-1041, 1989 3. Forrester K, Almoguera C, Han K, et al: Detection of high incidence of K-ras oncogenes during human colon tumorigenesis. Nature 327:298-303, 1987[Medline] 4. Vogelstein B, Fearon ER, Hamilton SR, et al: Genetic alterations during colorectal-tumor development. N Engl J Med 319:525-532, 1988[Abstract]
5.
Reynolds SH, Anna CK, Brown KC, et al: Activated protooncogenes in human lung tumors from smokers. Proc Natl Acad Sci USA 88:1085-1089, 1991
6.
Knowles MA, Williamson M: Mutation of H-ras is infrequent in bladder cancer: Confirmation by single-strand conformation polymorphism analysis, designed restriction fragment length polymorphisms, and direct sequencing. Cancer Res 53:133-139, 1993 7. Almoguera C, Shibata D, Forrester K, et al: Most human carcinomas of the exocrine pancreas contain mutant c-K-ras genes. Cell 53:549-554, 1988[Medline] 8. Bos JL, Fearon ER, Hamilton SR, et al: Prevalence of ras gene mutations in human colorectal cancers. Nature 327:293-297, 1987[Medline]
9.
Mills NE, Fishman CL, Rom WN, et al: Increased prevalence of K-ras oncogene mutations in lung adenocarcinoma. Cancer Res 55:1444-1447, 1995
10.
Bos JL: ras oncogenes in human cancer: A review. Cancer Res 49:4682-4689, 1989 [published erratum appears in Cancer Res 50:1352, 1990. 11. Moriyama N, Umeda T, Akaza H, et al: Expression of ras p21 oncogene product on human bladder tumors. Urol Int 44:260-263, 1989[Medline] 12. Clark GJ, Der CJ: Aberrant function of the Ras signal transduction pathway in human breast cancer. Breast Cancer Res Treat 35:133-144, 1995[Medline] 13. Janes PW, Daly RJ, deFazio A, et al: Activation of the Ras signalling pathway in human breast cancer cells overexpressing erbB-2. Oncogene 9:3601-3608, 1994[Medline]
14.
Patton SE, Martin ML, Nelsen LL, et al: Activation of the ras-mitogen-activated protein kinase pathway and phosphorylation of ets-2 at position threonine 72 in human ovarian cancer cell lines. Cancer Res 58:2253-2259, 1998 15. Feldkamp M, Lau N, Guha A: Astrocytomas are growth-inhibited by farnesyl transferase inhibitors through a combination of anti-proliferative and antiangiogenic activities. Proc Am Assoc Cancer Res 39:318, 1998 (abstr) 16. Guha A, Feldkamp MM, Lau N, et al: Proliferation of human malignant astrocytomas is dependent on Ras activation. Oncogene 15:2755-2765, 1997[Medline] 17. Kato K, Der CJ, Buss JE: Prenoids and palmitate: Lipids that control the biological activity of Ras proteins. Semin Cancer Biol 3:179-188, 1992[Medline]
18.
Casey PJ, Solski PA, Der CJ, et al: p21ras is modified by a farnesyl isoprenoid. Proc Natl Acad Sci USA 86:8323-8327, 1989 19. Der CJ, Cox AD: Isoprenoid modification and plasma membrane association: Critical factors for ras oncogenicity. Cancer Cells 3:331-340, 1991[Medline]
20.
Jackson JH, Cochrane CG, Bourne JR, et al: Farnesol modification of Kirsten-ras exon 4B protein is essential for transformation. Proc Natl Acad Sci USA 87:3042-3046, 1990 21. Reiss Y, Goldstein JL, Seabra MC, et al: Inhibition of purified p21ras farnesyl: protein transferase by Cys-AAX tetrapeptides. Cell 62:81-88, 1990[Medline] 22. Gutierrez L, Magee AI, Marshall CJ, et al: Post-translational processing of p21ras is two-step and involves carboxyl-methylation and carboxy-terminal proteolysis. EMBO J 8:1093-1098, 1989[Medline]
23.
Kato K, Cox AD, Hisaka MM, et al: Isoprenoid addition to Ras protein is the critical modification for its membrane association and transforming activity. Proc Natl Acad Sci USA 89:6403-6407, 1992
24.
Kohl NE, Mosser SD, deSolms SJ, et al: Selective inhibition of ras-dependent transformation by a farnesyltransferase inhibitor. Science 260:1934-1937, 1993 (see comments)
25.
James GL, Goldstein JL, Brown MS, et al: Benzodiazepine peptidomimetics: Potent inhibitors of Ras farnesylation in animal cells. Science 260:1937-1942, 1993 (see comments)
26.
Garcia AM, Rowell C, Ackermann K, et al: Peptidomimetic inhibitors of Ras farnesylation and function in whole cells. Biol Chem 268:18415-18418, 1993
27.
Ward R, Hawkins N, O’Grady R, et al: Restriction endonuclease-mediated selective polymerase chain reaction: A novel assay for the detection of K-ras mutations in clinical samples. Am J Pathol 153:373-379, 1998 28. Todd AV, Applegate TL, Fuery CJ, et al: Farnesyl transferase inhibitor (FTI): Effect of ras activation. Proc Am Assoc Cancer Res 39:317, 1998 (abstr)
29.
Sepp-Lorenzino L, Ma Z, Rands E, et al: A peptidomimetic inhibitor of farnesyl: protein transferase blocks the anchorage-dependent and -independent growth of human tumor cell lines. Cancer Res 55:5302-5309, 1995 30. Smets G, Xhonneux B, Cornelissen F, et al: R115777, a selective farnesyl protein transferase inhibitor (FTI), induces anti-angiogenic, apoptotic and anti-proliferative activity in CAPAN-2 and LoVo tumor xenografts. Proc Am Assoc Cancer Res 39:318, 1998 (abstr)
31.
Kohl NE, Wilson FR, Mosser SD, et al: Protein farnesyltransferase inhibitors block the growth of ras-dependent tumors in nude mice. Proc Natl Acad Sci USA 91:9141-9145, 1994 32. 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 (see comments)[Medline]
33.
Mangues R, Corral T, Kohl NE, et al: Antitumor effect of a farnesyl protein transferase inhibitor in mammary and lymphoid tumors overexpressing N-ras in transgenic mice. Cancer Res 58:1253-1259, 1998
34.
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
35.
James GL, Goldstein JL, Brown MS: Polylysine and CVIM sequences of K-RasB dictate specificity of prenylation and confer resistance to benzodiazepine peptidomimetic in vitro. Biol Chem 270:6221-6226, 1995 36. Yokoyama K, McGeady P, Gelb MH: Mammalian protein geranylgeranyltransferase-I: Substrate specificity, kinetic mechanism, metal requirements, and affinity labeling. Biochemistry 34:1344-1354, 1995 (published erratum appears in Biochemistry 34:14270), 1995[Medline]
37.
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
38.
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
39.
Liu M, Bryant MS, Chen J, et al: Antitumor activity of SCH 66336, an orally bioavailable tricyclic inhibitor of farnesyl protein transferase, in human tumor xenograft models and wap-ras transgenic mice. Cancer Res 58:4947-4956, 1998 40. End D, Skrzat S, Devine A, et al: R115777, a novel imidazole farnesyl protein transferase inhibitor (FTI): Biochemical and cellular effects in H-ras and K-ras dominant systems. Proc Am Assoc Cancer Res 39:270, 1998
41.
Armstrong SA, Hannah VC, Goldstein JL, et al: CAAX geranylgeranyl transferase transfers farnesyl as efficiently as geranylgeranyl to RhoB. J Biol Chem 270:7864-7868, 1995
42.
Lebowitz PF, Casey PJ, Prendergast GC, et al: Farnesyltransferase inhibitors alter the prenylation and growth-stimulating function of RhoB. J Biol Chem 272:15591-15594, 1997 43. Tapon N, Hall A: Rho, Rac and Cdc42 GTPases regulate the organization of the actin cytoskeleton. Curr Opin Cell Biol 9:86-92, 1997[Medline]
44.
Du W, Liebowitz P, Prendergast G: Cell growth inhibition by farnesyltransferase inhibitors is mediated by gain of gernylgeranylated Rho B. Mol Cell Biol 19:1831-1840, 1999 45. Venet M, Angibaud P, Sanz G, et al: Synthesis and in-vitro structure-activity relationships of imidazolyl-2-quinolinones as farnesyl protein transferase inhibitors (FTI). Proc Am Assoc Cancer Res 39:318, 1998 46. Skrzat S, Angibaud P, Venet M, et al: R115777, a novel imidazole farnesyl protein transferase inhibitor (FTI) with potent oral antitumor activity. Proc Am Assoc Cancer Res 39:316, 1998 (abstr) 47. Miyake Y, Horiguchi M, Tomita N, et al: Occult macular dystrophy. Am J Ophthalmol 122:644-653, 1996[Medline]
48.
Pittler SJ, Fliesler SJ, Fisher PL, et al: In vivo requirement of protein prenylation for maintenance of retinal cytoarchitecture and photoreceptor structure. J Cell Biol 130:431-439, 1995
49.
Christian MC, Korn EL: The limited precision of phase I trials. J Natl Cancer Inst 86:1662-1663, 1994 (editorial; comment)
50.
Simon R, Freidlin B, Rubinstein L, et al: Accelerated titration designs for phase I clinical trials in oncology. J Natl Cancer Inst 89:1138-1147, 1997 51. Gibaldi M, Perier D: Pharmacokinetics. New York, NY,Marcel Dekker, Inc (1982) 52. Yan N, Ricca C, Fletcher J, et al: Farnesyltransferase inhibitors block the neurofibromatosis type I (NF1) malignant phenotype. Cancer Res 55:3569-3575, 1995 |
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ABSTRACT
INTRODUCTION
