1 Ottawa Regional Cancer Centre, Ottawa, ON, Canada; 2 Johns Hopkins Oncology Center, Baltimore, MD, USA; 3 National Cancer Institute of Canada Clinical Trials Group, Kingston, ON, Canada; 4 MGI Pharma, Bloomington, MN, USA; 5 MethylGene, Inc., Montreal, QC, Canada
Received 8 January 2003; revised 9 February 2003; accepted 28 February 2003
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Abstract |
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Hypermethylation and inactivation of tumor suppressor genes by the enzyme DNA methyltransferase may lead to neoplastic transformation. MG98, a phosphorothioate antisense oligodeoxynucleotide that is a specific inhibitor of mRNA for human DNA methyltransferase 1 (DNMT1), was evaluated in a phase I study.
Patients and methods:
MG98 was given as a 2 h i.v. infusion twice weekly three weeks out of every four to patients with solid tumors. Pharmacokinetic evaluation was performed on days 1 and 15 of cycle 1 and mRNA expression of DNMT1 was measured in peripheral blood mononuclear cells (PBMCs).
Results:
Nineteen patients were entered onto the study. A total of 74 cycles (range 118 cycles) were administered at dose levels from 40 to 480 mg/m2. Dose limiting toxicity was seen in two of three patients at 480 mg/m2 and consisted of a constellation of fever, chills, fatigue and, in one case, confusion beginning within 6 h after the first infusion. Other toxic effects included fatigue, anorexia, nausea, vomiting and diarrhea, reversible elevations in transaminases and partial thromboplastin time. Pharmacokinetic evaluation showed Cmax and AUC to be dose proportional with low inter- and intra-patient variability. No consistent changes in DNMT1 mRNA expression were noted in PBMCs. One partial response was documented in a patient with renal cell carcinoma treated at 80 mg/m2.
Conclusions:
The recommended dose of MG98 was 360 mg/m2 given by 2 h infusion twice a week for three weeks out of every four. Phase II trials using this dose and schedule are underway.
Key words: anti-sense oligodeoxynucleotide, DNA methyltransferase I, MG98, phase I
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Introduction |
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As described in a recent review [2], the process of oncogenesis involves numerous genetic and epigenetic changes in the cell. Among the changes that may occur are alterations of tumor suppressor genes such as p53 and the retinoblastoma gene [3]. Since DNA methylation patterns are aberrant in tumor cells [4], and since hypermethylation has been noted in a number of tumor suppressor loci and genes in cancer cells [5], it has been hypothesized that DNA methylation may be important in tumor progression [4, 6]. This hypothesis is supported by observations that DNA methyltransferase activity is often increased in tumor cell lines [7] and induction of abnormal expression of DNA methyltransferase via gene transfer may result in cellular transformation [8]. DNA methylation patterns are established during development by the de novo methyltransferase enzymes DNMT3a and DNMT3b, and maintained by DNMT1 [9]. DNMT1 is the preferred target to reverse established aberrant methylation in cancer cells as it encodes cellular maintenance methyltransferase activity. However, recent studies suggest that the de novo methyltransferase DNMT3b may also play a role in human cancer [10, 11].
Antisense oligonucleotides are synthetic nucleic acids that are designed to inhibit the translation of specific mRNAs by binding to a target region within the mRNA via WatsonCrick base pairing [12]. MG98, a second-generation phosphorothioate antisense oligodeoxynucleotide, is a specific inhibitor of human DNMT1 mRNA. It produces a dose-dependent reduction in cellular DNMT1 protein levels, resulting in the reactivation of aberrantly silenced tumor suppressor genes in human cancer cells [13]. In cell lines, MG98 has IC50 values from 5070 nM. In human tumor xenografts models it caused dose-dependent growth delay and tumor regression when compared with mismatch control oligodeoxynucleotides [14]. More frequent exposure was associated with greater antitumour effects.
MG98 was generally well tolerated in animals. Reversible prolongation of partial thromboplastin time and slightly elevated transaminases, bloodureanitrogen and creatinine were seen in rats [14]. In monkeys, histopathological changes were found in the liver and kidneys, but it was unclear whether these were due to MG98 or to exposure to natural parasites and bacteria [14]. MG98 binds extensively to human plasma proteins. When 35S-labeled MG98 was administered to rats, its major route of excretion was renal, and radioactivity levels in plasma, blood and tissues were measurable up to 30 days post-dose. Pharmacokinetic parameters were linear with dose in both monkeys and rats, and did not change over multiple cycles of drug administration [14].
The National Cancer Institute of Canada Clinical Trials Group (NCIC CTG) undertook two phase I trials of MG98 to determine the recommended dose of MG98 for further evaluation. The first study involved a continuous infusion schedule [15] and the second study, reported here, gave a 2-h intravenous infusion twice per week, three weeks out of every four. It was felt that this latter schedule was of interest to evaluate since the therapeutic index of antisense oligonucleotides in some instances appears better with an intermittent schedule than with a continuous high-dose schedule [16].
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Patients and methods |
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Study design
MG98 (supplied by MethylGene, Inc., Montreal, Canada) was administered by i.v. infusion over 2 h twice weekly (Monday and Thursday or Tuesday and Friday) for three weeks out of every four. The starting dose of MG98 was 40 mg/m2/injection, one-sixth of the highest dose tested in monkeys [14]. Planned dose levels were 40, 80, 160, 240, 360 and 480 mg/m2/injection, with one to six patients per dose level. Dose escalations were permitted in individual patients provided they had completed at least one full cycle with no grade >2 MG98-related toxicity, and provided that accrual had been completed for the higher dose level without dose-limiting toxicity. At least one patient was to be entered per dose level. Once any patient experienced grade 2 toxicity, a minimum of three patients was required at that and all subsequent dose levels. If one of three patients on a dose level experienced dose-limiting toxicity, then a further three patients were to be enrolled, for a total of six. If two or more of three or two or more of six patients experienced dose-limiting toxicity, then that dose was considered to be the maximum tolerated dose (MTD). The next lowest dose from the MTD was to be considered the recommended dose for future phase II studies using this schedule. The protocol permitted expansion of this dose level to further assess its safety profile.
The definition of dose-limiting toxicity (DLT) was the cycle one occurrences of one or more of: absolute granulocyte count <0.5 x 109/l for 5 days, febrile neutropenia or grade
3 neutropenic infection, platelets <50 x 109/l or thrombocytopenic bleeding, sustained grade
3 PTT, grade
3 non-hematological toxicity, or any toxic effect resulting in the patient missing two or more doses per cycle.
Patient evaluation
Patients were examined and interviewed for symptoms and toxicity at least once every 4 weeks. Toxicities were evaluated using the NCI Common Toxicity Criteria (NCI CTC), version 2.0. A complete blood count, differential and platelet count was performed twice weekly for cycles 1 and 2, and weekly thereafter. Serum biochemistry was evaluated weekly. Urinalyses were carried out weekly for cycles 1 and 2, then day 1 each cycle. Appropriate radiological exams to assess known disease were repeated every 8 weeks and, if bidimensionally measurable disease was present at baseline, response was assessed according to NCIC CTG (WHO based) criteria. An electrocardiogram was done when patients went off protocol treatment.
Planned dose alterations
Individual doses of MG98 were to be held if any of the following events were documented at the time of planned treatment: platelets <50 x 109/l, granulocytes <0.5 x 109/l, febrile neutropenia or grade 3 neutropenic infection, PTT grade
2, grade 4 nausea or vomiting despite antiemetics, or any other major organ toxicity grade
3 (except alopecia). Doses were to be held until the toxicity was grade
1, and then the patient was to be treated at the next lowest dose level. The patient was to go off treatment if there was no recovery after 2 weeks. Day 1 of a new cycle was delayed by 1 week if platelets were <100 x 109/l or granulocytes were <1.5 x 109/l, or if the PTT was grade
2 at the time of retreatment.
Pharmacokinetics
All participating patients underwent pharmacokinetic evaluation. On days 1 and 15 of cycle 1, 5 ml of blood were collected into vacutainer tubes containing EDTA 10 min prior to the start of MG98 infusion, then at 1, 2 h (just prior to the end of drug infusion), and at 4, 5, 6 and 8 h after the start of the infusion. Blood samples for pharmacokinetics were also collected 10 min prior to the start of MG98 infusion on day 8, cycle 1, and days 1 and 15 for cycle 2, and every second cycle thereafter. Blood samples were centrifuged at 2500 r.p.m. for 10 min at 4°C, and plasma was separated and stored in two 2 ml tubes at 20°C until assay.
MG98 assay
Plasma samples were assayed for full-length MG98 by ion exchange high-performance liquid chromatography (HPLC) with ultraviolet detection, using a method adapted and validated by Anapharm, Inc. (Ste Foy, Quebec,
Canada). In brief, MG98 was extracted from an aliquot of human EDTA plasma using a filtration procedure. Quantitation of MG98 was performed by peak area method using a weighted (1/C) linear regression to determine the concentration of MG98 in plasma samples. The method was found to be suitable for the determination of MG98 in human plasma over the range of 0.210 µg/ml. The lower limit of detection was 0.050 µg/ml. The precision determined by coefficients of variation (CV%) and accuracy measured of percentage of nominal value at the lower and upper limits of quantitations were 2.81% and 103%, respectively, and 1.29% and 100%, respectively. The between-run precision and accuracy of low, medium and high quality control samples ranged between 1.55% and 2.04%, and 98.1% and 98.8%, respectively. CV% within-run precision and accuracy of lower and higher limits of quantitation and low, medium and high quality control samples ranged between 0.32% and 3.04%, and 95.6% and 98.4%, respectively. MG98 was found to be stable in human EDTA plasma for 624 days at 20°C storage conditions.
Pharmacokinetic analysis
Pharmacokinetic analysis was performed using WinNonlinTM 3.2 (Pharsight Corp., CA, USA) by non-compartmental methods. Cmax was the maximal observed plasma concentration. Elimination rate constant (Kel) was determined from the terminal slope of the plasma concentrationtime data using ln-linear regression; terminal elimination half-life (T) was calculated as (ln2)/Kel. Area under the plasma concentrationtime curve from time zero to the last non-zero concentration, or Ct (AUC0t) was calculated by the linear trapezoidal rule and area under the concentrationtime curve from time zero to infinity (AUC0) was calculated as AUC0t + (Ct/Kel). Total body clearance (CLp) was calculated as dose/AUC0
and volume of distribution at steady-state (Vdss) was calculated as MRT x CLp, where mean residence time (MRT) was calculated as (AUMC
/AUC
)
/2, and
is time of infusion. The area under the first moment curve (AUMC) was measured using the linear trapezoidal rule and extrapolated to infinity as: AUMClast + t x Ct /Kel + Ct/(Kel)2.
Complement activation
On days 1 and 15 of cycle 1, 7 ml of blood were collected 10 min before the start of the MG98 infusion (time 0), then at 2 and 4 h for measurement of complement factor Bb. Blood samples were collected in EDTA-containing tubes, inverted 10 times, and immediately placed on ice until centrifuged. Plasma was separated by centrifugation within 1 h of sample collection at 3000 r.p.m. for 15 min at 4°C. The resulting plasma samples were frozen at 70°C until analysis. All specimens taken for the measurement of complement factor Bb were analyzed by a central laboratory (Specialty Laboratories, Santa Monica, CA, USA) for consistency and standardization.
DNA methyltransferase in peripheral blood mononuclear cells
Patients had 20 ml of blood collected into green-topped heparin-coated Vacutainer tubes for harvesting of peripheral blood mononuclear cells (PBMCs) 10 min prior to the start of MG98 i.v. infusion on days 1, 8 and 15 of cycle 1, days 1 and 15 of cycle 2, and every other cycle thereafter. Specimens were kept at controlled ambient temperature until processing (24 h later).
PBMC mRNA analysis
PBMCs were isolated on Lymphoprep gradients (Nycomed) and total RNA was isolated from PBMC using RNeasy extraction kit (Qiagen) according to the manufacturers instructions. The level of DNMT1 mRNA relative to ß-actin was measured from total RNA by RTPCR according to the following method. For primer annealing of the RT step, 1 µg total RNA was mixed with 500 ng of oligo(dT)1218 (Gibco) in 0.2 ml thin-walled tubes, heated at 65°C for 10 min, then chilled on ice. Reverse transcription was performed with 50 U Expand RT (Roche) in the provided buffer supplemented with 10 mM DTT, 1 mM each dNTP, and 20 U RNasin and incubated at 42°C for 60 min. The RT product was neither heated nor frozen, but stored at 4°C until used for PCR. A no-RT control, where the enzyme was omitted, was run for each blood sample.
PCR was performed using 2 µl of the RT reaction and Expand LT DNA Polymerase (Roche), following the manufacturers recommendations. The final reaction contained 500 µM each dNTP, 600 nM each primer, a total of 3 mM MgCl2 and 2.5 U enzyme, in the provided buffer 3. Each cycle consisted of a denaturation step at 94°C for 10 s, followed by annealing at 60°C for 30 s, and by extension at 68°C for 2 min, with a 20-s increment per cycle after the first 10 cycles. A preliminary time-course PCR was performed with DNMT1 and actin primers to determine the number of cycles within which the reaction is in the linear range. For quantitative reactions, DNMT1 and actin were amplified together in a multiplex reaction where the active primers are added for the 17 cycles. The PCR product was resolved on 1% agarose gel, visualized by ethidium bromide, and quantified by alpha-imager. Each sample was measured a minimum of three times, from three independent RT reactions.
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Results |
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Fever and chills were also noted in occasional patients at lower doses, but were less severe and not part of an acute syndrome as described. The recommended dose of MG98 for phase II studies is 360 mg/m2 when MG98 is administered as a 2-h i.v. infusion on days 1, 4, 8, 11, 15 and 18 of a 28 day cycle.
Other toxic effects
Other toxic effects seen with MG98 are shown in Tables 3, 4 and 5. Most patients experienced fatigue, which was grade 3 in severity in nine patients at some point during their treatment. In two cases the severe fatigue was documented during the same cycle that DLT was noted at 480 mg/m2 and represented part of the symptom complex of the DLT. In the remainder it was not considered dose limiting, either because it occurred later than cycle 1 (four patients: fatigue grade 3 was seen in cycles 4, 5, 17 and 18), was considered unrelated to therapy (one patient) or was seen in patients with grade 2 fatigue at baseline and thus the degree of fatigue attributable to drug was uncertain. Gastrointestinal toxicity (nausea, vomiting and diarrhea) seemed to be dose related, and was generally limited to grade 1 or 2. The solitary episode of grade 3 diarrhea occurred at 480 mg/m2 during the patients second cycle of therapy. Despite having no dose reduction, the patient did not have diarrhea of grade >1 thereafter. Laboratory evidence of reversible dose-related hepatic toxicity was also common. One patient with grade 3 elevation in bilirubin had a blocked stent, not drug toxicity as the underlying cause. When first versus worst cycle were compared (Table 3A and 3B), there was a suggestion that transaminase increases became more common and severe with repeated dosing, although numbers are small. Creatinine rises were documented in six cases: five were grade 12 and one was grade 3. MG98 may have been responsible for the rise in creatinine in this latter patient since no other cause could be found and following discontinuation of treatment, the creatinine level gradually fell.
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Two patients had events late in their treatment that were considered to be possible hypersensitivity reactions. The first patient developed rash, dizziness and hypotension after being dose escalated to 160 mg/m2 from a lower dose. His next dose was reduced to 80 mg/m2 given with antihistamines and dexamethasone. He experienced no recurrence of this reaction. A second patient re-escalated to 160 mg/m2 following a dose reduction for PTT experienced chills and fever after day 1, cycle 7. While receiving day 15, cycle 7, he developed severe bronchospasm, hypoxia, cyanosis, rigors and tachycardia. The reaction resolved with administration of oxygen and diphenhydramine. This patient was not retreated with MG98.
Complement activation
There was no clinical evidence of complement activation. Two patients treated with MG98 at 160 mg/m2 had minor elevations of complement factor Bb levels of 1.62.4 µg/ml. In all other patients, Bb factor levels were <1.6 µg/ml (normal).
Response
Fourteen patients were evaluable for response. One patient with renal cell carcinoma previously treated with retinoid plus interferon and an investigational alkylating agent had a partial response in lung metastases lasting 9 months. One patient with renal cell cancer and one with colorectal carcinoma had stable disease for 3.7 and 2.6 months, respectively. The remainder of patients with measurable disease had a best response of disease progression.
Pharmacokinetics
Plasma MG98 concentration-time profiles are shown in Figure 1. Plasma concentrations declined monoexponentially after termination of infusion when plotted as log concentration versus time (not shown). Pharmacokinetic parameters are presented in Table 6 and Figure 2. The AUC0 and Cmax both increased linearly with dose. The Vdss was low, approximating that of blood volume. There was a trend towards slightly lower CLp and longer T with higher MG98 doses. This may have been due to patient factors or the small numbers in each group. Mean elimination phase half-life was short and ranged from 0.9 to 2.6 h. Pharmacokinetic data from patients receiving the same dose on days 1 and 15 showed low inter- and intra-patient variability and no evidence of drug accumulation.
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DNMT1 mRNA expression
Changes in PBMC DNMT1 mRNA are presented in Figure 3A and B. Eighteen patients had baseline and follow-up measurements and were included in this analysis. No consistent changes in DNMT1 mRNA expression were noted in PBMCs. No dose-related trend was seen.
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Discussion |
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Whether or not MG98 proves active in any tumor type as a single agent, it could be of interest to test in combination with other anticancer agents. In addition to arising from overexpression of various characteristics, chemotherapy resistance may also be due to underexpression of some factors [17]. Underexpression of a characteristic may arise as a result of hypermethylation of the gene responsible. For example, resistance to cisplatin may be conferred by hypermethylation of the hMLH1 gene [18]. Hence, reversal of gene hypermethylation might reduce resistance to some chemotherapy agents.
The mechanism by which MG98 caused acute infusion reactions is unclear. Other antisense oligonucleotides may activate complement [19], but there was little evidence of complement activation in patients in our study. The risk of these reactions appeared to be dose-related and dose reduction prevented their recurrence. The timing of the reactions plus the observation of the increased exposure to the drug in the single patient at 360 mg/m2 who experienced these effects makes it probable that the event is exposure related.
No consistent changes in DNMT1 mRNA expression were noted in PBMCs. The degree of variability in DNMT1 expression in PBMCs is unknown, and it is unclear whether the expression in these cells is reflective of changes in tumor tissues. Unfortunately, the validity of using PBMC as a surrogate for tumor tissue levels of DNMT1 could not be assessed in murine models in advance of human studies. In addition, mRNA measurements were done at times of MG98 trough levels in order to determine whether a sustained decrease in message could be detected. Measurements taken 2448 h after the completion of the infusion may have been better suited to explore the impact of the drug and drug dose on DNMT1 mRNA expression. In some of the ongoing phase II studies, an attempt is being made to evaluate changes in tumor cell DNMT1expression following MG98 treatment.
After 2-h i.v. infusion of MG98, plasma concentration declined mono-exponentially. MG98 AUC0 and Cmax increased linearly with dose and the CLp and T remained unchanged on days 1 and 15. The CLp of MG98 was low, as was the Vdss, suggesting that MG98 does not distribute extensively into tissues. The impact of this characteristic on its efficacy is unclear, but direct measures of target effect in tumor tissue would be the preferred approach to evaluating this.
Since the phase II program of MG98 includes one study with renal cell carcinoma, it will be of interest to continue to explore the relationships between toxicity, nephrectomy and pharmacokinetics. Two of four patients with nephrectomy in this phase I trial had lower clearance and higher Cmax and AUC values than non-nephrectomized patients treated at the same doses. Since many patients with renal cell carcinoma will have had previous nephrectomy, this population will be important to follow closely for toxicity and the effect of drug on creatinine levels.
In summary, MG98 was generally well tolerated when given as a 2-h infusion on 2 days per week for three weeks out of four. Phase II studies in head and neck cancer, and renal cell carcinoma have recently been completed, and studies in other tumor types are underway.
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Acknowledgements |
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Footnotes |
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References |
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