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Cancer Therapy: Clinical |
Authors' Affiliations: British Columbia Cancer Agency, Vancouver Centre; University Health Network, Princess Margaret Hospital; Juravinski Cancer Centre at Hamilton Health Sciences; and the National Cancer Institute of Canada-Clinical Trials Group, Vancouver, British Columbia, Canada
Requests for reprints: Kim N. Chi, British Columbia Cancer Agency, 600 West 10th Avenue, Vancouver, British Columbia, Canada V5Z 4E6. Phone: 604-877-6000; Fax: 604-877-0585; E-mail: kchi{at}bccancer.bc.ca.
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Abstract |
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Experimental Design: Patients with cancers known from the literature to express clusterin were eligible. OGX-011 was given by 2-h i.v. infusion starting at 40 mg weekly after loading doses on days 1, 3, and 5. Docetaxel was given i.v. 30 mg/m2 weekly for 5 of 6 weeks (schedule A) or 75 mg/m2 every 3 weeks (schedule B). All patients had serial samples of peripheral blood mononuclear cells and serum assessed for clusterin expression.
Results: Forty patients were enrolled to eight cohorts. OGX-011 could be given at the full biologically effective single-agent dose of 640 mg with both docetaxel schedules. Toxic effects were primarily myelosuppression, fatigue, hair loss, gastrointestinal effects (expected docetaxel effects), as well as dose-related chills and fever (expected OGX-011 effects). OGX-011 AUC and Cmax increased proportionally with no apparent effect on docetaxel pharmacokinetics. At the end of cycle 1, serum clusterin showed mean decreases of 34% and 38% (range, 15-99%) at the 640-mg dose levels.
Conclusions: OGX-011 can be given at a biologically effective dose with standard doses of docetaxel. Phase II trials of combined OGX-011 and chemotherapy are ongoing in patients with prostate, breast, and lung cancers.
OGX-011 is a second-generation phosphorothioate antisense oligonucleotide that is complementary to the clusterin mRNA translation initiation site and strongly inhibits clusterin expression in in vitro and in vivo laboratory models (17). In addition to a phosphorothioate backbone, OGX-011 incorporates a 2'-methoxyethyl modification to the ribose moiety on the flanking four nucleotides on either end of the molecule. Unmodified phosphorothioate antisense oligonucleotides have relatively short serum and tissue half lives (<2 and 4 h, respectively) and only small amounts of full-length antisense oligonucleotide can be detected in tissues after 24 h. Consequently, clinical trials of unmodified phosphorothioate antisense oligonucleotides generally used continuous or frequent i.v. infusions of these agents. By contrast, second-generation phosphorothioate antisense agents such as OGX-011 form duplexes with RNA with a higher affinity, which results in improved potency, and are more resistant to nucleases, resulting in prolonged tissue half life in vivo and, therefore, allowing for the use of more convenient intermittent dosing schedules in the clinic (17). Second-generation antisense oligonucleotides have also been reported to be less toxic and to cause less nonspecific immune stimulation than unmodified phosphorothioate antisense oligonucleotides (18), potentially allowing for delivery of higher doses. In preclinical efficacy studies, OGX-011 was shown to significantly enhance the therapeutic effect of hormone therapy, chemotherapy, and radiation therapy in a variety of tumor models including prostate, breast, non–small cell lung, bladder, and kidney (19). No clinical signs of toxicity were observed in animal toxicity studies at doses of up to 50 mg/kg in mice or of up to 10 mg/kg in monkeys. The primary toxicities were alterations in liver function in the form of elevated transaminase in mice at a dose of 50 mg/kg, immune stimulation in the form of lymphohistiocytic cell infiltrates in mice, and minor evidence of complement activation related to peak concentration in monkeys at 10 mg/kg.
The first-in-man phase I study with OGX-011 used a neoadjuvant design to identify effective biological dosing (20). Patients with localized prostate cancer and high-risk features were enrolled on the dose escalation study and treated with OGX-011 for 1 month followed by prostatectomy. Prostatectomy specimens were then used to evaluate for clusterin expression for both inter-patient and intra-patient comparisons to baseline. In this way, changes in expression of clusterin could be correlated to the dose of drug received and drug levels within the prostate tissue itself could be determined. Treatment was well tolerated and OGX-011 produced significant dose-dependent effects on suppression of clusterin expression and on apoptotic rates in normal and tumor tissues. With this design and the use of these pharmacokinetic and pharmacodynamic end points, an effective biological dose of 640 mg was established for OGX-011 based on its ability to suppress clusterin mRNA by >90%.
Because the development of OGX-011 was to be in combination therapy and given that several cancers that overexpress clusterin are also docetaxel sensitive (11, 13, 21), OGX-011 plus docetaxel was a logical combination to be evaluated in a phase I combination study. Moreover, preclinical efficacy studies supported the combination. Thus, the phase I trial reported here was carried out to establish the recommended phase II dose of the combination of OGX-011 given with standard doses of docetaxel in two schedules commonly used at the time of study inception. Because clusterin is detectable in serum, serial samples were taken at baseline and while patients were on treatment to assess treatment-related changes in clusterin levels as a potential pharmacodynamic marker for biological activity of OGX-011.
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Patients and Methods |
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12 weeks; and be of age >18 years. There was no limit on the number of prior chemotherapy regimens allowed for patients enrolled onto the weekly docetaxel schedule; however, those enrolled to the docetaxel every-3-week schedule were to have had 
2 prior regimens. For all patients, no chemotherapy or radiotherapy within 4 weeks before study entry was permitted and patients had to have recovered from all toxicity (Common Toxicity Criteria version 2.0 grade 1) except for alopecia. Patients had to have adequate hematologic (absolute granulocytes 1.5 x 109/L and platelets 100 x 109/L), hepatic (normal bilirubin and aspartate aminotransferase/alanine aminotransferase 1.5 x upper limit of normal), and kidney (serum creatinine 2 x upper limit of normal) functions and normal coagulation parameters. Exclusion criteria were as follows: uncontrolled pain, anticoagulant therapy, known bleeding disorder, central nervous system metastases, prior history of serious allergic reaction to a taxane, preexisting peripheral neuropathy grade 2, pregnant or lactating women, or other serious illness or medical condition. All patients had to be able to provide written informed consent. The study was approved by the institutional review boards of all participating centers. Treatment plan. Each course of treatment was composed of OGX-011 (supplied by OncoGenex Technologies, Inc.) delivered as a 2-h infusion weekly, with three loading doses during week 1 of cycle 1. A fixed dose escalation plan was used for OGX-011 starting at 40 mg, unadjusted for body weight or surface area. Docetaxel (supplied by Sanofi-Aventis) was initially delivered as a 30 mg/m2 1-h infusion weekly for 5 of 6 weeks beginning on day 1 immediately following completion of OGX-011 infusion (schedule A with each cycle considered as 6 weeks). After a recommended phase II dose of OGX-011 had been determined with weekly docetaxel, patients were accrued to additional dose levels to determine a recommended phase II dose of OGX-011 in combination with docetaxel 75 mg/m2 given on an every-3-week schedule (schedule B with each cycle considered as 3 weeks). The loading doses for OGX-011 with the 3-weekly docetaxel schedule were given on week –1, cycle 1 (i.e., 1 week before start of docetaxel). All patients received standard dexamethasone prophylaxis with each docetaxel infusion. Patients could receive up to four cycles on the weekly docetaxel or eight cycles on the 3-weekly docetaxel schedule in the absence of unacceptable toxicity or disease progression.
Study design. Planned dose levels of OGX-011 were (schedule A) 40, 80, 160, 320, 480, and 640 mg per dose. Consideration would be given to escalating to 800 mg (higher than the recommended single-agent dose) only if toxicity permitted. Schedule B was initiated after the recommended dose level for schedule A was reached at one level below that recommended. Because 640 mg was recommended in schedule A, only two dose levels of OGX-011 were planned in schedule B: 480 and 640 mg per dose.
Dose-limiting toxicity (DLT) was defined as the occurrence of one or more of the following: grade 3 or 4 nonhematologic toxicity (except unpremedicated nausea or vomiting); thrombocytopenia <25 x 109/L or grade 3 thrombocytopenia associated with bleeding; grade 4 neutropenia lasting for 3 days (weekly docetaxel) or 5 days (3-weekly docetaxel); febrile neutropenia, international normalized ratio, or partial thromboplastin time elevation of grade 3 with associated bleeding; or missing 2 OGX-011 or weekly docetaxel doses due to toxicity. DLT was assessed during the first cycle for decisions on dose escalation to the next level in a new patient cohort. The maximum tolerated dose was defined as the dose level in which 2 or 3 or 2 of 6 patients experience DLT, and the recommended phase II dose as one level below the maximum tolerated dose. If 640 mg of OGX-011 could be delivered safely, further escalation would be undertaken only if no DLTs had been observed at that dose and if pharmacokinetic or other data warranted it; otherwise, 640 mg was to be the recommended dose of OGX-011. One patient was entered at the first dose level, and a minimum of three patients entered to subsequent dose levels with expansion in the case of DLT. Patients were assessed pre-study and during treatment with history and physical exam, hematology, coagulation tests (international normalized ratio and partial thromboplastin time), serum chemistry, tumor markers as clinically appropriate, and radiologic evaluation for tumor measurements.
Pharmacokinetics. Plasma samples were obtained to determine the single-dose and multiple-dose pharmacokinetic profiles of full-length intact OGX-011. With the weekly docetaxel schedule, on days 1 and 36, 2 mL of plasma were collected in EDTA tubes pre-dose and then after start of infusion at 1, 2, 2.25, 2.5, 3, 4, 6, and 9 h, and with the day 36 OGX-011 infusion additional samples at 12, 24, 48, 96, and 168 h. Trough and peak plasma samples were taken pre-dose and at end of infusion on days 3, 5, 8, 15, 22, and 29. With the 3-weekly docetaxel schedule, on days –7 and 1, plasma samples were taken pre-dose and then after start of infusion at 1, 2, 2.25, 2.5, 3, 4, 6, and 9 h, and with the day 1 OGX-011 infusion additional samples at 12, 24, and 48 h. Trough and peak plasma samples were taken pre-dose and at end of infusion on days –5, –3, 8, 15, and 22. Plasma samples were analyzed for OGX-011 using a validated ELISA/cutting method (20).
Analytic determination of docetaxel was conducted using a validated high-performance liquid chromatography-mass spectrometry assay. A limited sampling schedule was used for the weekly docetaxel schedule with 7-mL heparinized blood samples taken on days 1, 8, and 29 pre-dose and then after start of infusion at 1 h (end of infusion) and 4 h. For the 3-weekly docetaxel schedule, samples were collected pre-dose and then after the start of infusion at 1, 2, 4, 6, 8, 22, and 46 h.
Noncompartmental pharmacokinetic analysis was done on the plasma concentration data including assessment of the Tmax (time to reach peak plasma concentration), Cmax (observed peak plasma concentration), area under the plasma concentration versus time curve (AUC), k (plasma distribution rate constant), t1/2 (plasma distribution half life), Vz (apparent volume of distribution), and CL (total clearance from plasma) using WinNonlin (version 3.2, Pharsight, Inc.) computer software.
Clusterin expression levels in peripheral blood mononuclear cells and serum. Three (schedule A) or two (schedule B) serial baseline blood samples, separated by at least 24 h, were collected over 7 days for peripheral blood mononuclear cells (10 mL; CPT-Citrate, Becton-Dickinson) and serum clusterin level determination (7 mL) before the first OGX-011 infusion and then before the OGX-011 infusions on days 8, 15, and 22. Additional samples were taken on the weekly docetaxel schedule on days 29, 36, and 43. Quantitative real-time PCR for clusterin was done from RNA extracted from peripheral blood mononuclear cells as previously described (20). Serum clusterin was assessed by an ELISA. Ninety-six-well polystyrene ELISA plates were coated with 100-ng pAb-sc6419 (Santa Cruz Biotechnology, Inc.) in 35 mmol/L NaHCO3, 15 mmol/L Na2CO3, and 3 mmol/L NaN3 (pH 9.6) overnight at 4°C and blocked with 5% w/v bovine serum albumin, 0.1% v/v Tween 20 in PBS (pH 7.4) followed by three washes with 0.1% v/v Tween 20 in PBS. Thawed serum samples were diluted 1:10 in 1% w/v bovine serum albumin, 0.1% v/v Tween 20 in PBS, and 50 µL of diluted serum were added to each well. Serum-loaded plates were then incubated for 2 h, washed four times with 0.1% v/v Tween 20 in PBS, blocked for 2 h with blocking solution, and incubated with 25 ng/well mAb-41D (Upstate) diluted in 1% w/v bovine serum albumin and 0.1% v/v Tween 20 in PBS for 2 h. Plates were then washed thrice with 0.1% v/v Tween 20 in PBS, blocked for 2 h, and then incubated with 10 ng of horseradish peroxidase–conjugated antimouse IgG antibody per well (diluted in 1% w/v bovine serum albumin, 0.1% v/v Tween 20 in PBS). After three washes with 0.1% v/v Tween 20 in PBS, plates were incubated with 50 µL of substrate reagent (R&D systems) for 15 min and the reaction was terminated by the addition of 1 mol/L H2SO4. Plates were read at 450 nm in Power Wave Plate Reader. Intra-assay coefficient of variation was 11.1% (n = 88). Results are reported as a percentage value of the first baseline sample.
Statistical analysis. The Cochran-Armitage trend test (22) was used to test the significance of association between increasing dose level and toxicity incidences, and the Jonckheere's trend test (23) was used to test the significance of the association between increasing dose level and the plasma pharmacokinetic parameters for each schedule. The relationship between change of serum clusterin levels from baseline and pharmacokinetic parameters was tested from a linear regression model. All P values are two sided and were not adjusted for the number of parameters evaluated.
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Results |
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Adverse events and DLT
Schedule A (OGX-011 and weekly docetaxel). Adverse events that were considered related (i.e., possibly, probably, or definitely related) to protocol therapy are listed in Table 3
. Nonhematologic toxicities were minimal in the first three dose levels except for one patient at dose level 3A (OGX-011 160 mg) who experienced grade 3 diarrhea, nausea, vomiting, and dehydration. One patient at dose level 6A (OGX-011 640 mg) experienced a DLT in the form of grade 3 dyspnea, which was considered secondary to pleural effusion related to docetaxel. Fatigue occurred at all dose levels. The incidence of diarrhea, anorexia, nausea, and alopecia was higher for higher dose levels but none of these showed a statistically significant trend. Hematologic toxicity was primarily grade 1 or 2 as expected with a weekly schedule of docetaxel, and there was no significant worsening with dose escalation. Biochemical toxicity was also grade 1 or 2 only, predominantly in the form of elevation of liver enzyme tests. As has previously been described, after the first two infusions of OGX-011, grade 1 and 2 fever and rigors occurred several hours after completion of the infusion, were self-limited to a few hours duration, and did not recur with subsequent infusions. This was most pronounced at the 640-mg dose level with 42% and 57% of patients experiencing fever and rigors, respectively.
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Pharmacokinetics
For patients on schedule A, mean plasma half life for OGX-011 ranged from 0.66 to 2.27 h across dose groups with a significant trend of increasing half life as a function of dose (P < 0.0001). There was no apparent increase toward a plateau following repeated dosing either with Cmax or the average concentration observed during infusion. There was a proportional and predictable increase in Cmax and AUC values (P < 0.0001 for both parameters; Fig. 1
). Clearance and the apparent volume of distribution of OGX-011 were dose dependent with average values decreasing and increasing, respectively, as a function of dose (P = 0.002 and 0.0003, respectively). Overall, there was no sign of plasma accumulation from the repetitive dosing. For schedule B, there was no significant effect of increasing OGX-011 dose on docetaxel plasma levels (weekly docetaxel schedule; data not shown) and pharmacokinetic parameters (P > 0.1 for all parameters; Table 4
), which were similar to contemporarily reported data (24).
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values (P = 0.03; Fig. 2B and C). Peripheral blood mononuclear cells were also assessed for clusterin by quantitative real-time PCR; however, there was a wide variation in the intra-subject baseline values (median coefficient of variation of 76% with a range from 9% to 134% for all patients in schedule A and 49% with a range from 0.6% to 141% for all patients in schedule B), making any posttreatment effect interpretation unreliable.
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50% from baseline, confirmed by a second value; ref. 25); one patient had an unconfirmed PSA response; and two patients had unconfirmed PSA decreases of 29% and 30%. |
Discussion |
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TopAbstractPatients and MethodsResultsDiscussionReferences |
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In this study, OGX-011 at a biologically active dose was shown to be feasible when combined with standard doses of docetaxel. Toxicity of the combination was mild or moderate for the most part, with toxicities being qualitatively and quantitatively in keeping with what would be expected with docetaxel alone, although it did seem that gastrointestinal side effects such as diarrhea and mucositis may be increased at higher doses of OGX-011. Gastrointestinal toxicity has been observed with other antisense therapeutics and may be a potential class effect; however, this increased toxicity may also be indicative of target effect of OGX-011 because it has been postulated that clusterin plays a role in protection of the mucosal barrier (26). Adverse events associated with OGX-011 specifically have previously been described and are similar to those observed with other antisense molecules, including elevation in hepatic transaminases, fever, rigors, and fatigue. These adverse effects are generally considered non–sequence specific and related to the polyanionic nature of these compounds and their effects on cytokine release. Consistent with other antisense-chemotherapy combinations, there was no apparent effect of OGX-011 on the pharmacokinetic behavior of docetaxel. Although toxicity criteria for identifying maximum tolerated dose were not met, dose escalation of OGX-011 beyond 640 mg was not undertaken because of the observation of a 33% DLT rate in the expanded cohort with this dose and the 3-weekly schedule of docetaxel, which was to be taken forward in phase II trials. As well, the 640-mg dose had already been identified as the recommended single-agent phase II dose based on its biological activity (20).
Clusterin is produced from multiple tissues and is detectable in the serum. We observed low intra-patient variability in the paired pretreatment serum clusterin samples followed by an on-treatment dose-dependent and exposure-dependent decrease by a mean of 34% to 38%. The low intra-patient variability in the baseline samples lends greater confidence in concluding that these data support the biological activity of OGX-011, particularly at the recommended dose and schedule; however, whether this reduction translates into any clinical significance remains to be seen. Although the 640-mg dose of OGX-011 has been associated with a significant biological effect on clusterin expression in tumor tissue, the correlation between serum levels of clusterin and levels in tumor tissue has not been established as of yet. In addition, the effect of dexamethasone and docetaxel administration may independently have an effect on serum clusterin, and the design of the current study was not able to address these uncertainties. A quantitative, validated ELISA assay for serum clusterin will be used in the currently ongoing phase II clinical trials to address these questions. This includes a phase II trial evaluating serum and tissue levels of clusterin in all patients after OGX-011 administration and a randomized phase II study of docetaxel and corticosteroids with and without OGX-011 where serial samples of serum for clusterin protein will be correlated with baseline factors and outcomes, tumor tissue clusterin expression, and treatment received.
In conclusion, OGX-011 is a novel antisense oligonucleotide targeting clusterin with favorable pharmacokinetic properties and clinically demonstrable biological activity that can be combined with standard dose docetaxel chemotherapy. Phase II trials with OGX-011 in combination with chemotherapy are under way in patients with prostate, breast, and lung cancers.
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Footnotes |
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The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Note: Presented at the 41st Annual Meeting of the American Society of Clinical Oncology, Orlando, Floridda, May 2005.
Received 5/30/07; revised 8/20/07; accepted 10/10/07.
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References |
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in LNCaP cells by overexpression of sulfated glycoprotein-2 (clusterin). Cancer Res 1995;55:2431–7.This article has been cited by other articles:
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  O. Chayka, D. Corvetta, M. Dews, A. E. Caccamo, I. Piotrowska, G. Santilli, S. Gibson, N. J. Sebire, N. Himoudi, M. D. Hogarty, et al. Clusterin, a Haploinsufficient Tumor Suppressor Gene in Neuroblastomas J Natl Cancer Inst, May 6, 2009; 101(9): 663 - 677. [Abstract] [Full Text] [PDF]
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 E. Dean, D. Jodrell, K. Connolly, S. Danson, J. Jolivet, J. Durkin, S. Morris, D. Jowle, T. Ward, J. Cummings, et al. Phase I Trial of AEG35156 Administered as a 7-Day and 3-Day Continuous Intravenous Infusion in Patients With Advanced Refractory Cancer J. Clin. Oncol., April 1, 2009; 27(10): 1660 - 1666. [Abstract] [Full Text] [PDF]
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 I. P. Trougakos, J. Y. Djeu, E. S. Gonos, and D. A. Boothman Advances and Challenges in Basic and Translational Research on Clusterin Cancer Res., January 15, 2009; 69(2): 403 - 406. [Full Text] [PDF]
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 S. Chia, S. Dent, S. Ellard, P. M. Ellis, T. Vandenberg, K. Gelmon, J. Powers, W. Walsh, L. Seymour, and E. A. Eisenhauer Phase II Trial of OGX-011 in Combination with Docetaxel in Metastatic Breast Cancer Clin. Cancer Res., January 15, 2009; 15(2): 708 - 713. [Abstract] [Full Text] [PDF]
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