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© 1999 American Society for Clinical Oncology Phase I Clinical/Pharmacokinetic and Pharmacodynamic Trial of the c-raf-1 Antisense Oligonucleotide ISIS 5132 (CGP 69846A)From the Thomas Jefferson University, Kimmel Cancer Center, Philadelphia, PA; and ISIS Pharmaceuticals, Inc, Carlsbad, CA. Address reprint requests to James P. Stevenson, MD, University of Pennsylvania, Presbyterian Medical Center, Medical Arts Building, Suite 103, 51 North 39th St, Philadelphia, PA 19104.
PURPOSE: Raf-1 is a protein kinase that plays a broad role in oncogenic signaling and acts as a downstream effector of Ras in the mitogen-activated protein kinase pathway. The present study was designed to determine the maximum-tolerated dose (MTD), toxicity profile, pharmacokinetics, and antitumor activity of the c-raf-1 antisense oligodeoxynucleotide ISIS 5132 (CGP 69846A; ISIS Pharmaceuticals Inc, Carlsbad, CA). The effect of ISIS 5132 on c-raf-1 gene expression in peripheral-blood mononuclear cells (PBMCs) of treated patients was studied using a reverse transcriptase polymerase chain reaction assay. PATIENTS AND METHODS: Patients with refractory malignancies received ISIS 5132 as a 2-hour intravenous infusion three times weekly for 3 consecutive weeks. Pharmacokinetic sampling was performed during the first cycle in all patients; PBMCs for c-raf-1 mRNA analysis were collected at baseline and on days 3, 5, 8, and 15 of cycle 1 and on day 1 of each cycle thereafter.
RESULTS: Thirty-one patients received ISIS 5132 at one of nine dose levels ranging from 0.5 mg/kg to 6.0 mg/kg. Clinical toxicities included fever and fatigue, but these were not dose limiting. A clinically defined MTD was not reached. The harmonic mean half-life of ISIS 5132 was 59.8 minutes (range, 35.5 to 107.3 minutes). The area under the concentration-time curve increased linearly with dose, and mean plasma clearance was 1.86 mL/kg/min (range, 1.21 to 2.41 mL/kg/min). Two patients experienced prolonged stable disease lasting more than 7 months, which was associated with persistent reduction in c-raf-1 expression in PBMCs. Significant decreases in c-raf-1 expression were identified at time points after the baseline value (P < .05) at doses CONCLUSION: ISIS 5132 is well tolerated at doses up to 6.0 mg/kg when administered as a thrice weekly 2-hour infusion for 3 consecutive weeks. The pharmacokinetic behavior of the drug is reproducible, and suppression of target gene expression is observed in circulating PBMCs.
RECENT ADVANCES IN cancer biology have led to the identification of signaling proteins that may be rational therapeutic targets. Raf-1, a serine/threonine protein kinase encoded by c-raf-1, acts downstream of Ras in the MAP kinase signal transduction pathway1,2 (Fig 1). Mitogen-activated protein (MAP) kinase activation in response to growth factors (eg, epidermal growth factor) leads to the induction of transcription factors that promote expression of genes involved in cellular proliferation and differentiation.3 Mutated Ras and Raf-1 are constitutively active and have transforming potential in vitro4,5; ras and raf gene mutations have been identified in a range of human tumors, including codon 12 K-ras mutations in more than 80% of pancreatic adenocarcinomas.6,7 Raf-1 may play a broader role in tumorigenesis: it is activated independently of Ras by the antiapoptotic protein Bcl-28 and protein kinase C-alpha9 and promotes expression of the multidrug resistance gene mdr1.10 Considerable evidence to date suggests that disruption of Raf-1 signaling may therefore interfere with the maintenance of malignant phenotypes.
The specificity of Watson-Crick base pairing provides the basis for antisense therapeutics; the cellular delivery of oligonucleotides complementary to a specific mRNA sequence should permit mRNA binding and subsequent inhibition of gene expression through mRNA degradation.11 ISIS 5132 (CGP 69846A; ISIS Pharmaceuticals Inc, Carlsbad, CA) is a 20-base phosphorothioate antisense oligodeoxynucleotide (ODN) designed to hybridize to 3' untranslated sequences of c-raf-1 mRNA.12 ISIS 5132 decreased c-raf-1 mRNA levels and Raf-1 protein synthesis in A549 lung carcinoma cells in a sequence- and concentration-dependent fashion, and inhibited proliferation with a 50% inhibitory concentration of 100 nmol/L (range, 25 to 500 nmol/L).12 ISIS 5132 ODN shortened from the 3' end by 1, 2, 3, 4, or 5 bases showed reduced ability to inhibit c-raf-1 mRNA expression in A549 cells, losing activity in a length-dependent manner.13 Reduction of c-raf-1 mRNA expression in vivo occurred in mice bearing A549 human tumor xenografts within hours of ISIS 5132 therapy at a dose of 6 mg/kg/d. A mismatched control ODN had no effect on c-raf-1 mRNA expression in A549 tumors, which confirms a sequence-dependent target effect.12 The growth of implanted human MCF-7 breast carcinoma xenografts was significantly inhibited by ISIS 5132 at doses of 25 mg/kg/d.13 Pharmacokinetic studies of ISIS 5132 in animals indicated that plasma half-lives (30 to 85 minutes) and areas under the concentration-time curve (AUCs) were dose-dependent but not dose-linear after 2-hour intravenous infusion, as a consequence of tissue distribution of the ODN.14 Tissue distribution and metabolism were the two major routes of plasma clearance, and only 6% of ISIS 5132 or its metabolites was excreted into urine or feces. Intravenous ISIS 5132 doses of 100 mg/kg every other day produced fatal hepatotoxicity in mice but at lower doses did not. Hepatotoxicity was not observed in monkeys that received doses up to 10 mg/kg by the same route and schedule. Transient, asymptomatic prolongation of the activated partial thromboplastin time (aPTT) and activation of the alternative complement pathway were noted in monkeys at doses of 10 mg/kg and plasma concentrations of intact ODN greater than 35 µg/mL, but these were not observed at the 1.0 or 3.0 mg/kg dose levels. In animal studies, acute toxicities of ISIS 5132 (aPTT prolongation and complement activation) seemed to be related to plasma concentrations of ODN, whereas the subacute toxicities as a result of organ uptake were correlated with ODN plasma concentrations over time. These preclinical efficacy and toxicology studies suggest a large therapeutic window for ISIS 5132. Based on these data, we performed a phase I trial to determine the maximum-tolerated dose (MTD) of ISIS 5132 when administered as a 2-hour intravenous infusion three times a week in patients with refractory solid tumors. We describe the clinical and pharmacokinetic results of this trial. We demonstrate the down-regulation of c-raf-1 mRNA in the cells of treated patients, the results of which are presented in greater detail elsewhere (O'Dwyer et al, manuscript submitted for publication). These results define a regimen suitable for phase II testing in specific tumor types.
Patient Population Patients were accrued to this study between April 1996 and July 1997. Eligible patients with a histologic diagnosis of cancer for whom no effective therapy was available were required to be at least 18 years of age. An Eastern Cooperative Oncology Group (ECOG) performance status  2 and life expectancy  12 weeks were required. All patients were recovered from previous treatment, had measurable or nonmeasurable but assessable disease, and had received no therapy 28 days before screening. Eligibility requirements included the following: adequate bone marrow function (neutrophils 1,500/µL, hemoglobin 9.0 g/dL, and platelets 100,000/µL), serum creatinine less than 2.0 mg/dL, total bilirubin less than 2.0 mg/dL, aspartate aminotransferase less than two times upper limit of normal (< five times upper limit of normal in the presence of liver metastases), and no prolongation of the prothrombin time (PT) or aPTT. All patients received information regarding the purpose and conduct of this study and provided written informed consent in accordance with federal, state, and institutional guidelines. Pretreatment evaluation consisted of a history and physical examination, complete blood cell count, serum chemistries, electrolytes and creatinine, PT and aPTT, urinalysis, electrocardiogram, chest x-ray, and assessment of ECOG performance status. Blood counts and biochemical profiles were performed twice weekly during the first week, then once a week. Coagulation times and complement split products were measured before infusion of ISIS 5132 and at intervals during the infusion and up to 2 hours after the end of infusion in week 1 of the first treatment cycle and immediately pre- and post-infusion with the first dose in cycles 2 and 3. Toxicity during each cycle was assigned according to the World Health Organization toxicity grading scale. Lesions noted at baseline that were measured or evaluated by radiographic scan or x-ray were reviewed before each alternate course and evaluated for response according to standard criteria.15
Drug Administration
Study Design
Pharmacokinetic Sampling and Analysis Drug analysis was performed by capillary gel electrophoresis by Covance Laboratories (Madison, WI) using a previously described method (Leeds et al16) on aliquots of each sample of plasma and urine. Samples were prepared by strong anion-exchange solid-phase extraction followed by two desalting steps: elution from a reverse-phase solid-phase extraction column, then membrane dialysis. The preparation differed from the published method in that the strong anion-exchange solid-phase extraction loading buffer used for the present study contained 0.25 mol/L KCl in lieu of 0.5 mol/L KCl. A phosphorothioate ODN composed of 27 thymidine nucleotides (T27) was added to both plasma and urine as an internal standard. Capillary gel electrophoresis was performed with a Beckman P/ACE Model 5010 instrument (Beckman, Fullerton, CA) with a 27-cm column. ODN eluting from the column were detected by ultraviolet absorption at a wavelength of 260 nm. The linear range of concentrations of ODN detectable by this method is 10 nmol/L to 20 µmol/L in plasma and 2 nmol/L to 2 µmol/L in urine. Plasma and urine assays were validated to meet acceptance criteria for precision and reproducibility (% relative SD < 20%). Concentrations of full-length ISIS 5132 and of major metabolites (shortened ODN of 19, 18, and 17 nucleotides in length) were calculated. To do this, the corrected area (area under the ultraviolet absorbance curve divided by migration time) of the appropriate electropherogram peak was determined, divided by the corrected area of the internal standard peak, and multiplied by the known starting concentration of the internal standard added. An additional correction was made for differences between the extinction coefficient of ISIS 5132 and those of the internal standard or 19-mer, 18-mer, or 17-mer metabolites. In addition to plasma concentrations of intact ISIS 5132 and metabolites at various time points, pharmacokinetic parameters analyzed included half-life, AUC, clearance, and volume of distribution. Pharmacokinetic data were analyzed using descriptive statistics.
Measurement of c-raf-1 mRNA Expression in Peripheral-Blood Mononuclear Cells The c-raf-1 primer sequences were as follows: Raf(1) 5'-TCAGAGAAGCTCTGCTAAG-3', and Raf(2) 5'-CAATGCACTGGACACCTTA-3'. Beta-actin primer sequences were: BA(67) 5'-GCGGGAAATCGTGCGTGACATT-3', and BA(68) 5'-GATGGAGTTGAAGGTAGTTTCGTG-3' as described.17 cDNA synthesis was carried out with 2 µg of total RNA using Ready-to-Go You-Prime First-Strand Bead kits (Pharmacia Biotech, Uppsala, Sweden). PCR was carried out in Ready-to-Go PCR Bead kits (Pharmacia Biotech). Both sets of reactions were performed according to the manufacturer's instructions. The PCR reactions (25 µL total volume, containing 0.1 to 10 µL cDNA, 12.5 pmol of each of the c-raf-1 or beta-actin primers, and 1 µCi alpha-phosphorus-32 dCTP) were heated to 95°C for 5 minutes and then amplified for 28 to 36 cycles at 95°C for 1 minute, 55°C for 1 minute, and 72°C for 2 minutes. The products were loaded on 8% urea polyacrylamide gels. The gels were dried at 80°C for 1 hour under vacuum and exposed to film for several hours at -80°C. The band corresponding to c-raf-1 was either cut out and subjected to liquid scintillation counting or measured by densitometric scanning. Mean values of c-raf-1 expression on days 3, 5, 8, and 15 of ISIS 5132 therapy during the first cycle were compared with pretreatment expression using the Wilcoxon signed-rank test.
A total of 31 patients were entered onto this study; 29 were assessable for toxicity and response. Two patients received only one dose of ISIS 5132 and were not assessable: one patient voluntarily withdrew to pursue treatment elsewhere, whereas another patient experienced mental status changes 12 hours after his first dose. These symptoms were felt to be a result of a recent change in his outpatient narcotic regimen and not as a result of ISIS 5132 treatment. His mental status returned to baseline within 24 hours after holding the long-acting narcotic and he subsequently chose not to remain on the study. The 29 assessable patients received 65 cycles of ISIS 5132. There are no patients remaining on study. The demographic characteristics of the participants are listed in Table 1. The median age was 57 years, and all had good performance status. A broad range of tumor types was represented. All patients had received prior chemotherapy, and eight underwent previous irradiation.
Toxicity
Other laboratory toxicities included grade 2 elevation of the aPTT in three patients during ISIS 5132 infusion; in one patient, this persisted 2 hours postinfusion. There were no bleeding episodes associated with these transient elevations. All patients had C3a levels that were above the normal reference range at baseline. There was a trend in dose-related elevation of the activated complement components C3a but not Bb or C5a (Fig 2). These elevations were transient and unaccompanied by clinical signs or symptoms. The potential for complement activation prompted study closure at 6.0 mg/kg: dose escalation was halted because the observed maximum plasma concentrations (Cmax) of intact ISIS 5132 approached those associated with complement activation in primates.
Responses
ISIS 5132 Pharmacokinetics
Effect of ISIS 5132 on c-raf-1 mRNA Expression
The development of antisense technology and its application in the clinic represent a prototype of translational research. Demonstration of preclinical efficacy by a variety of ODNs targeted to specific gene products involved in oncogenesis or maintenance of the malignant phenotype has been followed by clinical studies in cancer patients, with promising results. The safety and tolerability of oligonucleotide therapy was first demonstrated during the 1970s in the initial trials of poly(ICLC), a double-stranded RNA construct and potent interferon inducer.19 The report of Bishop et al20 was the first in the literature of its kind: a phosphorothioate ODN designed to hybridize to p53 mRNA was administered in a phase I setting to patients with myelogenous leukemias and advanced myelodysplasia. The treatment was relatively nontoxic, and although there were no clinical responses, the authors demonstrated an inverse correlation between ODN AUC and leukemic cell production in bone marrow cultures of patient cells before and after treatment. Critical replies urging caution soon followed,21 as no specific antisense effect of the ODN on p53 mRNA was demonstrated. Gewirtz et al22 delivered a c-myb antisense ODN to 18 patients with refractory leukemias via a continuous intravenous infusion over 7 days with minimal side effects. The only response was observed in a patient with chronic myelogenous leukemia in blast crisis who reverted to chronic phase and survived for 14 months. Biologic end points were not reported in this interim analysis, leaving open the possibility that there was no antisense inhibition of c-myb expression. The small study of Webb et al23 was unique in that target gene effects were studied. Using flow cytometry, they documented a reduction in Bcl-2 protein expression in lymph node, blood, and bone marrow specimens that correlated with clinical benefit in two of nine patients with non-Hodgkin's lymphoma who were given a Bcl-2 ODN as a 2-week subcutaneous infusion. The principal toxicity attributed to the ODN in this phase I trial was inflammation at the infusion site. Initial results of phase I studies investigating different administration schedules with a protein kinase C-alpha ODN have also been reported, with clinical responses and tolerability observed.24,25 Thus early clinical development to date has demonstrated the safety and efficacy of ODNs targeted to a range of gene products. There has been little evidence presented to support antisense inhibition of gene expression as their mechanism of action. We report here that ISIS 5132, a phosphorothioate ODN designed to hybridize to c-raf-1 mRNA, is well tolerated when administered as an intermittent 2-hour infusion at doses up to 6.0 mg/kg. The principal toxicities we observed were low-grade fever and fatigue, as well as transient elevations of aPTT and changes in complement levels after infusion that had no clinical significance. None of these toxicities were dose limiting. Dose escalation was halted at 6.0 mg/kg as peak plasma concentrations neared those associated with activation of the alternative complement pathway in monkeys.14 Concerns about toxicities of ODNs resulting from tissue accumulation have been expressed but were not observed in the two patients on our study who received eight or more cycles of treatment. One patient was observed to have a progressive decline in platelet counts over 10 cycles of ISIS 5132. An antiplatelet antibody was detected during treatment; however, the clinical picture does not wholly support an antibody-mediated thrombocytopenia. A causative relationship between the antibody and the declining platelet counts has not been established. Alteration in immune function during ODN therapy should be considered, and preclinical studies with phosphorothioate ODNs have described polyclonal activation of murine and human B cells in vivo and in vitro.26 Krieg et al27 and Takamoto et al28 suggest that immunomodulatory effects may account for the observed preclinical efficacy of antisense ODNs associated with CpG motifs in the nucleotide sequence. However, in a phase I safety trial, Glover et al29 observed no humoral immune responses in patients treated with a phosphorothioate ODN targeting intercellular adhesion molecule-1, and Boggs et al30 have reported that ISIS 5132 did not stimulate murine-splenic natural killer-cell activity in vitro. Also, we observed no substantial intrapatient differences in Cmax achieved during cycles 1 and 2 in a limited number of patients treated on this study, which suggests that neutralizing antibodies to ISIS 5132 did not develop. Therefore, the available evidence does not support an immune mechanism for the antitumor and target effects of ISIS 5132 that we report here; future trials of other antisense ODNs will allow further clarification of this issue. The pharmacokinetic behavior of ISIS 5132 when administered on this schedule was consistent with preclinical observations. The linear increase in Cmax and AUC with increasing dose indicates no plasma accumulation as a result of saturable uptake at the doses and schedule studied. The large Vdss was consistent with broad tissue distribution of ISIS 5132. The antitumor effect of ISIS 5132 treatment that was observed in two patients who had progressive disease before study entry is encouraging. More encouraging was our ability to demonstrate substantial and reproducible reductions in PBMC c-raf-1 mRNA expression in multiple patients using RT-PCR. ISIS 5132 treatment produced effects on its intended target. The fact that reduced expression persisted in patients who continued to maintain radiographically stable disease with multiple cycles of therapy (and paralleled the decrease of a serum tumor marker in one patient) points toward an antisense mechanism of action. Nonantisense mechanisms for ODNs have been suggested in multiple preclinical studies31,32; however, these do not explain the inability of scrambled and mismatched ODNs to reproduce the activity of matched antisense ODNs in others.33 The effect of ISIS 5132 on c-raf-1 expression was not dose-dependent, as reductions were seen at all dose levels studied. Human A549 tumor xenograft studies in mice indicated that the effects of ISIS 5132 on gene expression after a dose of 6 mg/kg were greatest after repeated dosing over 13 days (data on file, ISIS Pharmaceuticals, Carlsbad, CA), but we have shown that intermittent dosing also achieves these effects. Another phase I trial of ISIS 5132 administration as a 21-day continuous infusion has been reported,34 and future studies will attempt to determine if the effects of ISIS 5132 are indeed schedule-dependent. The continued study of biologic end points in antisense trials will be vital in providing answers to the numerous questions of mechanism. Although the study of effects on tumor specimens is optimal, obtaining such specimens from patients participating in phase I trials is rarely feasible, and in future trials we will continue to use PBMCs as surrogate tissues in which biologic end points may be studied. Such a method will be more applicable to the patient populations to be studied in larger cooperative group trials. Phase II trials of ISIS 5132 in several solid tumor types are in progress, and phase I combination studies with cytotoxic agents have begun.
Supported in part by CA49820 from the National Cancer Institute, National Institutes of Health, and by a grant from ISIS Pharmaceuticals, Inc. We thank Richard Geary, PhD, for assistance with pharmacokinetic analysis.
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Glover JM, Leeds JM, Mant TG, et al: Phase I safety and pharmacokinetic profile of an intercellular adhesion molecule-1 antisense oligodeoxynucleotide (ISIS 2302). J Pharmacol Exp Ther 282:1173-1180, 1997 30. Boggs RT, McGraw K, Condon T, et al: Characterization and modulation of immune stimulation by modified oligonucleotides. Antisense Nucleic Acid Drug Dev 7:461-471, 1997[Medline] 31. Barton CM, Lemoine NR: Antisense nucleotides directed against p53 have antiproliferative effects unrelated to effects on p53 expression. Br J Cancer 71:429-437, 1995[Medline] 32. Smetsers TFCM, Linders EHP, van de Locht LTF, et al: An antisense Bcr-Abl phosphodiester-tailed methylphosphonate oligonucleotide reduces the growth of chronic myeloid leukaemia patient cells by a non-antisense mechanism. Br J Haematol 96:377-381, 1997[Medline]
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Monia BP, Sasmor H, Johnston JF, et al: Sequence-specific antitumor activity of a phosphorothioate oligodeoxyribonucleotide targeted to human C-raf kinase supports an antisense mechanism of action in vivo. Proc Natl Acad Sci U S A 93:15481-15484, 1996 34. Holmlund J, Nemunaitis J, Schiller J, et al: Phase I trial of C-raf antisense oligonucleotide ISIS 5132 (CGP 69846A) by 21-day continuous intravenous infusion (CIV) in patients with advanced cancer. Proc Am Soc Clin Oncol 17:210a, 1998 (abstr 811) Submitted November 5, 1998; accepted February 24, 1999.
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Copyright © 1999 by the American Society of Clinical Oncology, Online ISSN: 1527-7755. Print ISSN: 0732-183X
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ABSTRACT
INTRODUCTION
) and renal cell carcinoma (•) who experienced prolonged tumor growth inhibition with ISIS 5132 treatment.
activates Raf-1 by direct phosphorylation. Nature 364:249-252, 1993
