1University of Chicago Medical Center, Chicago, IL; 2Ohio State University, Columbus, OH, USA
Received 3 September 2001; revised 6 November 2001; accepted 22 November 2001.
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Abstract |
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Chemorefractory small-cell lung cancer (SCLC) is defined as disease that progresses during primary therapy or within 3 months of completion of primary therapy. Patients with chemorefractory SCLC have a very poor prognosis, and no treatment has been shown to be of significant clinical benefit. Elevated expression of Bcl-2 is found in the majority of SCLCs and has been associated with therapeutic resistance. Suppression of Bcl-2 levels through the use of G3139, an antisense oligonucleotide complementary to the mRNA encoding Bcl-2, might increase the antitumor efficacy of cytotoxic therapy.
Patients and methods
Twelve patients with chemorefractory SCLC participated in this pilot trial of paclitaxel combined with G3139. G3139 was given by continuous i.v. infusion over 7 days at a fixed dose of 3 mg/kg/day. Paclitaxel dose was initially 175 mg/m2 on day 6, but was decreased to 150 mg/m2 due to myelosuppression observed in two of the three patients treated in the first dose cohort.
Results
The combination of paclitaxel at 150 mg/m2 and G3139 at 3 mg/kg/day was found to be feasible and well tolerated. No objective responses were observed, but two patients had stable disease, one remaining stable on therapy for >30 weeks. Plasma G3139 levels were determined, and were found to be highest in the patient with prolonged stable disease, suggesting that individual variation in metabolism and clearance of the antisense oligonucleotide may influence activity.
Conclusions
This study demonstrates that G3139 can be combined with paclitaxel in a cytotoxic dose range, and suggests that a similar combination be tested for activity in the context of chemoresponsive disease.
Key words: apoptosis, chemoresistance, phosphorothioate, translational inhibition
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Introduction |
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Paclitaxel has been reported to be an active agent in SCLC, with first line efficacy comparable to cisplatin [8, 9]. Smit et al. reported that single agent paclitaxel had a response rate of 29% in 24 patients, with residual SCLC within 3 months of primary therapy [10]. These patients may not have all met criteria of chemorefractory SCLC (residual disease versus progressive disease within 3 months). Although the response rate was encouraging, no complete responses were observed, and the median survival was ~100 days. This study suggested that while paclitaxel had activity in residual SCLC, the clinical impact of this activity was minimal.
The oncogene bcl-2 is expressed in 8390% of SCLC, and therefore represents a potential therapeutic target in this disease [1113]. Several studies involving both cell culture and animal models have suggested that overproduction of Bcl-2, or related inhibitors of cell death, can confer multidrug chemotherapeutic resistance [1422]. Conversely, suppression of Bcl-2 in tumors in which it is expressed may increase chemotherapeutic efficacy [23, 24].
G3139, or Genasense, is an 18-base antisense phosphorothioate oligonucleotide complementary to the bcl-2 mRNA in the region encoding the first six amino acids of Bcl-2 [25]. The mechanism of action of G3139, and other antisense oligonucleotides, is thought to depend on binding of the antisense oligonucleotide to the cognate sequence within the mRNA, resulting in inhibition of mRNA translation and RNase H-mediated mRNA degradation [26, 27]. Preclinical and clinical studies have demonstrated that i.v. administered G3139 can be taken up by tumor cells, and can result in reduction of Bcl-2 protein production [2832].
Preclinical studies have demonstrated synergy between G3139 and taxane therapy [33, 34]. In a murine xenograft model of human breast cancer cell lines that overexpress Bcl-2, synergistic cytotoxicity and durable tumor regression were noted in animals treated with docetaxel together with G3139, but not in animals treated with either agent alone, or animals treated with docetaxel and control mismatch oligonucleotides [34]. Recent data suggest that paclitaxel cytotoxicity may be mediated in part through phosphorylation and functional inhibition of Bcl-2, and that up-regulation of Bcl-2 expression may be a mechanism of resistance to paclitaxel [35, 36].
This trial was designed to evaluate the safety, feasibility and toxicity profile of the combination of paclitaxel and G3139 in patients with chemoresistant SCLC.
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Patients and methods |
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Drug administration
G3139, an 18-base phosphorothioate oligonucleotide (5'-TCTCCCAGCGTGCGCCAT-3') was administered by continuous i.v. administration through a 0.22 µm in-line filter using a portable volumetric infusion pump over 7 days (day 18). Paclitaxel was administered by a 3-h constant rate i.v. infusion on day 6 of each cycle. This schedule was chosen, based on protein kinetic and pharmacological considerations, to permit sufficient time for the G3139 infusion to maximally suppress Bcl-2 protein levels (Bcl-2 t1/2 = 1224 h) before paclitaxel administration on day 6, and to prolong Bcl-2 suppression for the 48 h after paclitaxel, during which time paclitaxel-induced apoptosis may be maximal. No premedication was used before initiation of G3139. Before paclitaxel administration, all patients were given 20 mg i.v. dexamethasone, 50 mg i.v. diphenhydramine, 20 mg i.v. famotidine and 24 mg oral ondansetron. Cycle length was 21 days, no therapy was planned for days 921.
Study design
This was a single arm study designed to establish toxicity, feasibility and a recommended dose for the combination of paclitaxel and G3139 in this patient population. G3139 was administered at a fixed dose of 3 mg/kg/day. The initial paclitaxel dose was 175 mg/m2. In the event of cycle 1 dose-limiting toxicity (DLT) in at least two of six patients, the paclitaxel dose was to be decreased in 25 mg/m2 increments in subsequent dose cohorts, until a dose was reached at which less than two of six patients experienced DLT. Patients not completing cycle 1 for reasons other than DLT were considered not evaluable for toxicity and were replaced to meet enrollment criteria. In the event of DLT, therapy was withheld pending resolution of toxicity. Therapy could then be resumed at the next lower dose level, at the discretion of the patient and the treating physician.
Toxicity and response evaluation
Toxicity was assessed using NCI Common Toxicity Criteria version 2.0. DLT was defined as any grade 3 or 4 toxicity with the exception of grade 3 neutropenia, alopecia, nausea or vomiting.
Response was evaluated by computerized tomography (CT) in the last week of every second cycle, using a direct comparison with a CT scan obtained in the 2 weeks before initiation of therapy. Standard two-dimensional measurement criteria were used to classify the response. Patients with stable disease were allowed to continue therapy.
Pharmacokinetic analysis
Intact plasma G3139 levels were determined by anion exchange high performance liquid chromatography in cycle 1 on day 6 (at the time of paclitaxel administration) and day 8 (before discontinuation of G3139), as previously described [32, 37]. A plasma sample was also obtained before initiation of therapy, as a negative control. This analysis was performed by Oread Inc. (Lawrence, KS, USA).
Paclitaxel pharmacokinetic analysis was not performed in this trial. It was predicted that if G3139 resulted in significant inhibition of paclitaxel clearance, this would be detected as increased paclitaxel-related toxicity, particularly peripheral neuropathy.
Western blotting and protein quantitation
Blood draws were performed on day 1, before initiation of treatment, and day 6, before paclitaxel administration. Peripheral blood mononuclear cells were isolated using Vacutainer CPT tubes (Becton-Dickinson; Franklin Lakes, NJ, USA), according to the manufacturers instructions. Protein extracts were prepared by resuspension in RIPA buffer (Roche; Indianapolis, IN, USA). Western blotting was performed by electrophoresis through a 14% polyacrylamide gel, followed by electroblotting to nitrocellulose. The blot was first probed with monoclonal anti-Bcl-2 clone 124 (DAKO; Carpinteria, CA, USA), and developed using an enhanced chemiluminescence (ECL) kit (Amersham/Pharmacia; Piscataway, NJ, USA) according to the manufacturers instructions. The blot was then stripped and re-probed using monoclonal anti-ß-actin clone AC-15 (Sigma; St Louis, MO, USA). Quantitative densitometry was assessed using the ChemiImager 5500 system (Alpha Innotech; San Leandro, CA, USA).
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Results |
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Toxicity
The initial dose level evaluated was paclitaxel 175 mg/m2 combined with G3139 3 mg/kg/day continuous i.v. infusion. Two of the first three patients treated at this dose level experienced dose-limiting hematological toxicity in cycle 1. One of these patients had completed a course of lumbosacral and pelvic radiation immediately before starting G3139, and was found to have grade 3 leukopenia and thrombocytopenia on day 6 of therapy, before administration of paclitaxel. The second of these patients experienced grade 3 leukopenia, neutropenia, thrombocytopenia and anemia on day 16 of cycle 1. Shortly thereafter she was hospitalized with a pulmonary embolus and became acutely short of breath, requiring intubation and mechanical ventilation. A chest X-ray revealed disease progression. She died 3 days later. The third patient treated with paclitaxel 175 mg/m2 experienced no leukopenia or neutropenia, and only grade 1 thrombocytopenia, but had disease progression after two cycles of therapy.
Subsequent patients were treated at a paclitaxel dose of 150 mg/m2, combined with G3139 of 3 mg/kg/day. Only one of eight patients at this dose level experienced any cycle 1 hematologic toxicity greater than grade 2, this patient experienced grade 3 leukopenia. Hematological toxicity for patients in all cycles combined is shown in Table 2.
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Previous work has also suggested that Bcl-2 protein levels are most consistently suppressed if plasma G3139 levels are >12 µg/ml [32, 37, 39]. Notably, the only patient on this study to have G3139 levels consistently >2 µg/ml was the patient with prolonged stable disease over 10 cycles (Figure 1). The other patient with stable disease up to cycle 6 also had relatively high plasma G3139 levels on both day 6 and 8 (averaging ~2 µg/ml).
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Discussion |
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The rationale behind the combination of paclitaxel and G3139 was that Bcl-2 is commonly expressed in SCLC and has been associated with resistance to taxanes. Preclinical data suggested that suppression of Bcl-2 by G3139 can synergize with taxane therapy against solid tumors in vivo [33, 34]. Despite strong preclinical support for this combination, objective responses were not seen. There are several factors that could be considered in explaining this outcome.
First, the paclitaxel dose may have been below a necessary threshold. A paclitaxel dose of 150 mg/m2 every 21 days may be considered relatively low by many investigators, and some prior studies have suggested a measurable benefit of increased paclitaxel dose intensity [41]. However, other studies have failed to support a clear doseresponse relationship for taxane therapy in lung cancer [42]. Therefore, although a suboptimal paclitaxel dose may have contributed to the lack of objective response, we believe this is likely to be a relatively minor factor.
Secondly, the G3139 dose level chosen may have been insufficient to suppress expression of the target gene bcl-2. Prior and ongoing studies of G3139 have suggested that consistent suppression of Bcl-2 protein levels may depend on achieving a threshold G3139 concentration. The observed levels on days 6 and 8, in most cases <2 µg/ml, are consistent with the suggestion that an escalated G3139 dose should be considered. The intriguing observation of levels 3 µg/ml only in the patient with prolonged stable disease further supports this hypothesis. Bcl-2 protein level was only moderately suppressed even in this patient, although peripheral blood Bcl-2 levels may be an inadequate reflection of intratumoral effects. Notably, the observed extent of Bcl-2 suppression in this patient was similar to the intermediate levels of suppression (60% of baseline) associated with evident chemosensitization in patients with melanoma treated with G3139 and dacarbazine [40].
Thirdly, the Bcl-2 antisense oligonucleotide may not have been taken up by tumor cells, and therefore may not have gained access to the relevant intracellular mRNA. Data from tumor biopsies of a more readily accessible solid tumor, melanoma, have demonstrated intratumoral suppression of Bcl-2 [40]. In the design of the current trial, biopsies were permitted to assess intratumoral effects in patients with superficial lesions. However, due to poor PS and a lack of safely accessible tumors, no tumor biopsies could be obtained. Establishing invasive tumor monitoring will be challenging in this patient population.
It has been noted that the absolute level of Bcl-2 may be less relevant than the ratio of Bcl-2 to Bax in determining apoptotic threshold [43]. Bax levels were not measured in the current trial and could have an impact on the efficacy of therapy. However, the rapid expansion of the known members of this complex gene family precludes a simple analysis of such a ratio; it is likely that the relative balance of the many active pro- and anti-apoptotic Bcl-2 family members contributes to apoptotic sensitivity. Within tumor types clearly associated with elevated Bcl-2 expression (follicular lymphoma, melanoma, small-cell lung carcinoma), both preclinical and clinical data suggest that suppression of the single target Bcl-2 can enhance both tumor cell killing and chemosensitivity [2325, 2831, 40].
Finally, the highly resistant nature of this subset of recurrent SCLC may not be amenable to chemosensitization by G3139. Antisense inhibition of Bcl-2 expression may increase therapeutic efficacy of cytotoxic agents given for chemoresponsive disease, as demonstrated in several preclinical models, without reversing the inherent therapeutic insensitivity of chemorefractory disease.
An ideal setting in which to evaluate the ability of G3139 to enhance responsiveness of chemosensitive SCLC would be in patients with previously untreated disease, where the response rate is high but response duration remains disappointingly short. In contrast to the heavily pretreated population here, in patients receiving first-line therapy it may also be possible to maintain a relatively high dose intensity of both the antisense oligonucleotide and the cytotoxic therapy. We have chosen to pursue this approach. Analysis of G3139 in combination with cytotoxic chemotherapy for patients with previously untreated SCLC is ongoing.
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Acknowledgements |
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Footnotes |
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