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Clinical Trials |
Ottawa Regional Cancer Centre, Ottawa, Ontario, K1H 8L6 Canada [M. C. C.]; Hamilton Regional Cancer Centre, Hamilton, Ontario, Canada [A. L. F.]; Princess Margaret Hospital, Toronto, Ontario, Canada [A. M. O.]; London Regional Cancer Centre, London, Ontario Canada [M. J. T.]; Cross Cancer Institute, Edmonton, Alberta, Canada [A T. F.]; Isis Pharmaceuticals, Carlsbad, California [J. T. H., R. S. G.]; and National Cancer Institute of Canada, Clinical Trials Group, Kingston, Ontario, K7L 3N6 Canada [L. W. M., E. A. E.]
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ABSTRACT |
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 Top ABSTRACT INTRODUCTIONPATIENTS AND METHODSRESULTSDISCUSSIONREFERENCES |
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; and (b) ISIS 5132, an antisense inhibitor of c-raf kinase in patients untreated previously with recurrent or metastatic colorectal carcinoma. Patients and Methods: All patients had colorectal adenocarcinoma with measurable disease and no prior chemotherapy for metastatic disease. Patients were randomized to receive either ISIS 3521 or ISIS 5132 at a dose of 2 mg/kg/day as a continuous i.v. infusion 21 of 28 days. Cycles were repeated as long as progression was not seen, and doses of both agents were modified according to toxic effects. A two-arm study design was used with each study arm considered independently. Steady-state blood levels of both antisense molecules were measured on days 8, 15, and 22 of the first cycle of therapy.
Results: Thirty-seven eligible patients were enrolled, and 32 were evaluable for response (17 receiving ISIS 3521 and 15 receiving ISIS 5132). No responses were noted. Four of the patients receiving ISIS 3521 had stable disease, and 5 patients receiving ISIS 5132 were stable.
Conclusion: Neither ISIS 5132 nor ISIS 3521given in the dose and schedule studied induced objective responses in untreated colorectal cancer patients.
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INTRODUCTION |
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TopABSTRACTINTRODUCTIONPATIENTS AND METHODSRESULTSDISCUSSIONREFERENCES |
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45% of patients will eventually die of metastatic disease (2)
.
The standard treatment for metastatic colorectal cancer has historically been 5-FU and folinic acid (Mayo Regimen), but a recent trial adding CPT-11 has demonstrated a modest increase in survival (3)
. Given the limited survival advantage and the toxicity of the treatment, there is a need to identify more active or less toxic systemic agents. The purpose of this study was to evaluate two new antisense oligonucleotides targeting PKC-3
(ISIS 3521) and c-raf kinase (ISIS 5132) in untreated patients with advanced colorectal cancer in a noncomparative randomized Phase II design.
The molecular target for antisense oligonucleotides is the mRNA, which codes for a specific protein. On hybridizing to the mRNA coding for a specific protein, antisense oligonucleotides reduce or inhibit the protein expression of a protein. Both ISIS 3521 and ISIS 5132 are antisense phosphorothioate oligodeoxynucleotides that hybridize to the 3' untranslated region of the human PKC- or c-raf kinase mRNA, respectively.
PKC- (an abundant isozyme of PKC) is universally expressed throughout tissues and is found in many transformed cell lines (4
, 5)
. There is also considerable evidence implicating PKC in the abnormal proliferation, which occurs during tumor promotion and carcinogenesis (6)
. Evidence from human malignancies also suggests that PKC plays an important role in the growth or progression of some tumors (4)
. Thus, PKC- is an attractive target for antitumor drug (7)
.
Raf kinases serve as central regulators of mitogenic signaling pathways by connecting upstream growth factor-mediated tyrosine kinase stimulation with downstream activation of serine threonine kinases (8) . There is substantial evidence to support the direct role of raf kinases in the development and maintenance of human malignancies; it is central to the mitogen-activated protein kinase signaling cascade (8) and is a downstream effector of ras protein function. Because ras mutations are present in a high proportion of human cancers, including colorectal cancers (9) , novel therapies directed against raf kinase may prove useful in the treatment of ras-dependent tumors (10, 11, 12, 13) .
Experimental antitumor activity was demonstrated for ISIS 3521 and 5132 in several in vivo model systems. Decrease in the targeted protein expression was noted with both drugs after incubation in A549 carcinoma cells in vitro. The IC50 for ISIS 3521 was 100 nM and, for ISIS 5132, 50–100 nM in this assay system. Preclinical data suggested prolonged administration of oligonucleotide resulted in superior efficacy, thus Phase I trials of both drugs were carried out with both 21-day infusion and 2 h three times weekly infusion schedules (14, 15, 16, 17)
.
In the Phase I trials, evidence of antitumor activity was observed with both drugs. Nemunaitis et al. (14) reported complete responses in 2 patients with low-grade non-Hodgkin’s lymphoma, who received ISIS 3521 by 2-h infusion thrice weekly. Yuen et al. (15) reported a partial response in ovarian cancer after treatment with ISIS 3521 by 21-day infusion. In the trials of ISIS 5132, although objective responses of measurable disease were not observed, O’Dwyer et al. (16) reported prolonged stable disease in patients with colon and renal carcinoma and reduction of c-raf-mRNA in the peripheral blood mononuclear cells of the majority of patients studied after thrice weekly 2-h infusion.
Both drugs were well tolerated in both schedules. The 21-day infusion schedule was chosen for further study to ensure more continuous tumor exposure to drug; it was also more convenient for patients because it required only once weekly clinic visits (rather than three times a week). The maximum tolerated dose of ISIS 5132 given by continuous infusion was not reached at 4 mg/kg/day. For ISIS 3521, fatigue and thrombocytopenia were observed at 3 mg/kg/day. The recommended Phase II dose for both drugs was 2 mg/kg/day. The steady-state plasma concentrations of full-length oligonucleotide observed at this dose level were comparable with the in vitro IC50s of both drugs for mRNA expression, protein expression, and proliferation.
This randomized noncomparative Phase II trial was done to determine tolerability and quantitative efficacy of ISIS 3521 and ISIS 5132 in patients with locally advanced or metastatic colorectal cancer.
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PATIENTS AND METHODS |
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TopABSTRACTINTRODUCTIONPATIENTS AND METHODSRESULTSDISCUSSIONREFERENCES |
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12 months before study entry. Patients had to have an Eastern Cooperative Oncology Group performance status of 0 or 1 and a life expectancy of 12 weeks. Patients must have been 18 years of age, and there was no upper age limit. Additional eligibility criteria included absolute granulocytes 1.5 x 109/liter, platelets 100 x 109/liter, serum creatinine 
2 x upper limit, a bilirubin of 2 times the upper normal limit, < 5 times the upper limit of normal, and a normal prothrombin time (or international normalized ratio) and partial thrombin time. Patients may have had radiation therapy but must have recovered from acute toxic effects before registration. At a minimum, one measurable lesion had to have been 2 x 2 cm on computed tomography scan or 1 x 1 cm on chest X-ray or physical exam. Initially, patient entry was restricted to those with relatively nonbulky disease (largest lesion 40 cm2) because there was a theoretical concern that these agents might take some time to have an effect on tumor mass and might be preceded by temporary increases in tumor size. If this consideration was true, then patients with bulky metastatic disease might not tolerate a period of growth before shrinkage. However, later in the trial, this requirement was eliminated, because it reduced substantially the number of eligible patients, and we felt that, if the drugs were to be useful, it was best tested in a more widely representative population of metastatic disease patients. Informed consent was obtained from all patients before the start of treatment, and the study was approved by the Research Ethics Board at each participating institution.
The patients were evaluated at baseline by physical examination, computed tomography scans, and chest X-rays (and other X-rays as clinically indicated). Patients were then randomized to receive either ISIS 3521 or ISIS 5132.
Therapy consisted of either ISIS 3521 or ISIS 5132 at a dose of 2 mg/kg/day as a continuous i.v. infusion for 21 days. This was repeated every 4 weeks. A central line or a peripherally inserted central venous catheter was used for administration of the assigned drug. Evaluation of response was performed every two cycles with the repetition of all initially positive radiology.
A complete response was defined as disappearance of all clinical and radiological evidence of tumor determined by two observations not <4 weeks apart. The patients must also have been free of all tumor-related symptoms. Partial response was defined as a 50% decrease in the overall sum of the products of measurable lesions determined by two observations not <4 weeks apart. No simultaneous increase in the size of any lesion or the appearance of any new lesion may have occurred, or the appearance of any new lesions may have occurred. Stable disease was defined as disease less than a partial response that is <50% decrease in the overall sum of the products of measurable lesions or progression less than progressive disease documented to be present for 6 weeks after the start of therapy. Progressive disease was defined as an unequivocal increase of 25% in the overall sum of the products of measurable lesions as compared with baseline. The appearance of new lesions constituted progressive disease. Response duration was measured from the time the measurement criteria were first met until disease progression was documented.
Toxicity was assessed before every course of therapy and graded using the National Cancer Institute of Canada Clinical Trials Group Expanded Common Toxicity Criteria. Doses were reduced for hematologic and nonhematologic toxic effects. The dose of the next cycle was decreased by 50% if either nadir granulocytes were <0.5 x 109/liter, the patient experienced febrile neutropenia, or if the platelet count was <25 x 109/liter. Nadir blood values had only to be achieved on one occasion for the dose reduction rules to come into play. Treatment was discontinued if there was no recovery of counts by 2 weeks’ time. Grade 4 nausea and vomiting, despite antiemetic therapy, and grade 3 major organ toxicity required a dose reduction of 50% for the next cycle.
Blood sampling to determine plasma levels of ISIS 3521 and ISIS 5132 was done predose and on days 8, 15, and 22 5–10 min before the pump was stopped in cycles 1 and 3. An aliquot of plasma (100 µl) for each sample was spiked with a known concentration of internal standard (T27, a 27-mer phosphorothioate oligodeoxythymidine) and extracted using solid phase extraction and analyzed by capillary electrophoresis (18)
. Extracted samples were analyzed by capillary gel electrophoresis using a Beckman capillary electrophoresis instrument (Beckman Instruments, Irvine, CA) with UV detection at 260 nm. The limit of quantitation for this assay is 0.10 µg/ml in plasma.
The sample size for each arm of the trial was based on a Fleming two-stage design using a null and alternate hypothesis of 5 and 20%, respectively. Fifteen evaluable patients were to be recruited to each arm, and responses were assessed. Randomization would continue to 30 evaluable patients in each arm if at least one objective response was seen in both arms. If only one arm had response(s) observed, the other would close, and accrual was to continue only in the arm with activity. If neither arm had at least one objective response, the trial was to close.
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RESULTS |
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TopABSTRACTINTRODUCTIONPATIENTS AND METHODSRESULTSDISCUSSIONREFERENCES |
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. Liver was the predominant site of metastatic disease (28 patients). Fourteen patients had one site of disease, 10 had two sites of disease, and 5 patients had three sites. Eight patients had 4 sites of disease. Fourteen patients had prior adjuvant chemotherapy, and 13 patients had prior radiotherapy.
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Toxicity.
Toxicity was graded according to National Cancer Institute of Canada Clinical Trials Group Expanded Common Toxicity Criteria. In general, both agents were well tolerated. Toxic effects are shown in Table 2
. There were no grade 4 or 5 toxic effects in either arm. The only episode of grade 3 hematologic toxicity was a single patient receiving ISIS 5132 with grade 3 thrombocytopenia. The most common nonhematologic toxicities were lethargy, anorexia, nausea, and vomiting. There were also 15 patients who had transient elevations of liver enzymes while receiving therapy. Fourteen were grade 1 or 2, and none were clinically significant. In some cases, these changes were attributable to worsening of pre-existing liver metastases, but 2 of 9 patients who had no evidence of liver metastases at baseline exhibited grade 1 elevation of aspartate aminotransferase attributed to the antisense therapy. Both of these patients were receiving ISIS 5132. Frequent monitoring of partial thromboplastin time and international normalized ratio while on study showed no evidence of meaningful changes in these parameters.
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. Overall, 55–70% of oligonucleotides was in the intact, 20-mer form. The remainder had undergone some degradation to shorter length oligonucleotide. The overall mean steady-state concentration of ISIS 3521 was 0.892 µg/ml (130 nM) and of ISIS 5132 was 0.783 µg/ml (116 nM).
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DISCUSSION |
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TopABSTRACTINTRODUCTIONPATIENTS AND METHODSRESULTSDISCUSSIONREFERENCES |
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antisense has meaningful clinical activity in this disease. There are several potential explanations for this lack of efficacy. It is possible that with the dose and/or schedule used, there were insufficient tumor concentrations of the antisense agents achieved to effectively inhibit target protein production. Although the mean steady-state plasma levels for both agents were above their respective IC50s for inhibition of mRNA expression, these levels may not have been achieved in the target tissue. Assays of tumor levels of drug might have been helpful in determining this, but serial tumor measures were not thought to be feasible when the trial was designed. It could be also argued that the 2-h, three-times-a-week schedule might have been better suited for Phase II evaluation because higher peak concentrations of drug can be achieved with a short infusion. However, all preclinical evidence suggested that continuous exposure to antisense was desirable, so the rationale for the 21-day infusion seems sound. Secondly, it is possible that adequate levels were in fact achieved, and either the targets themselves are not important in this malignancy or other intracellular events prevented the drugs from inhibiting protein production in an effective manner.
A number of other Phase II trials with these agents are under way, but as yet, none have been published. In addition, a Phase I/II trial in non-small cell lung cancer of ISIS 3521 in combination with carboplatin and paclitaxel has been reported recently (19) . Investigators found no evidence of additive toxicity when the antisense was combined with chemotherapy, and the regimen was promising with respect to measures of efficacy (response rates, time to progression, and survival). These results have led to a large randomized trial of this combination regimen versus paclitaxel + carboplatin in patients with advanced non-small cell lung cancer.
Other small inhibitors of the same molecular targets are undergoing evaluation at this time, and these studies may help determine whether these targets are relevant in colorectal cancer. However, based on our results, we feel that additional single agent studies with ISIS 3521 and ISIS 5132 at this dose and schedule in this population are not warranted. In fact, the results of the randomized combination study referred to above will be very important, not only in determining the role of the particular antisense molecule, but in contributing to our more general understanding of how such targeted therapies might be best deployed.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 Supported by Grants from the National Cancer Institute of Canada and Isis Pharmaceuticals. 
2 To whom requests for reprints should be addressed, at Ottawa Regional Cancer Centre, 501 Smyth Road, Ottawa, Ontario, K1H 8L6 Canada. Phone: 613-737-7700, extension 6762; Fax: 613-247-3511; E-mail: Christine.Cripps{at}orcc.on.ca
3 The abbreviation used is: PKC, protein kinase C.
Received 9/ 4/01; revised 3/ 7/02; accepted 3/18/02.
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REFERENCES |
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expression in human A-549 by antisense oligonucleotides inhibits induction of intracellular adhesion molecule 1 (ICAM-1) mRNA by phobol ester. J. Biol. Chem., 269: 16416-16424, 1994. in patients with advanced cancer. J. Clin. Oncol., 17: 3586-3595, 1999. (ISIS 3521/ISI 641A) in patients with cancer. Clin. Cancer Res., 5: 3357-3363, 1999., with carboplatin and paclitaxel in non-small cell lung cancer. Proc. Am. Soc. Clin. Oncol., 20: 309a 2001.This article has been cited by other articles:
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  R. H. El-Maraghi and E. A. Eisenhauer Review of Phase II Trial Designs Used in Studies of Molecular Targeted Agents: Outcomes and Predictors of Success in Phase III J. Clin. Oncol., March 10, 2008; 26(8): 1346 - 1354. [Abstract] [Full Text] [PDF]
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 M. Lowenberg, A. Verhaar, B. van den Blink, F. ten Kate, S. van Deventer, M. Peppelenbosch, and D. Hommes Specific Inhibition of c-Raf Activity by Semapimod Induces Clinical Remission in Severe Crohn's Disease J. Immunol., August 15, 2005; 175(4): 2293 - 2300. [Abstract] [Full Text] [PDF]
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S. J. Cohen, R. B. Cohen, and N. J. Meropol Targeting Signal Transduction Pathways in Colorectal Cancer--More Than Skin Deep J. Clin. Oncol., August 10, 2005; 23(23): 5374 - 5385. [Abstract] [Full Text] [PDF]
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K. Naoki, T.-H. Chen, W. G. Richards, D. J. Sugarbaker, and M. Meyerson Missense Mutations of the BRAF Gene in Human Lung Adenocarcinoma Cancer Res., December 1, 2002; 62(23): 7001 - 7003. [Abstract] [Full Text] [PDF]
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