1 Investigative Treatment Division, National Cancer Center Research Institute East, 2 National Cancer Center Hospital, Tokyo; 3 Ibaraki Prefectural Central Hospital, Ibaraki; 4 Cancer Institute Hospital, Tokyo; 5 Mitsubishi Pharma Corporation, Tokyo, Japan
Received 6 August 2003; revised 3 October 2003; accepted 10 November 2003
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ABSTRACT |
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MCC-465 is an immunoliposome-encapsulated doxorubicin (DXR). The liposome is tagged with polyethylene glycol (PEG) and the F(ab')2 fragment of human monoclonal antibody GAH, which positively reacts to >90% of cancerous stomach tissues, but negatively to all normal tissues. In preclinical studies, MCC-465 showed superior cytotoxic activity against several human stomach cancer cells compared with DXR or DXR-incorporated PEG liposomes. The main purpose of this trial was to define the maximum tolerated dose (MTD), dose limiting toxicity (DLT), recommended phase II dose and pharmacokinetics (PK) of MCC-465.
Patients and methods:
Patients with metastatic or recurrent stomach cancer were eligible for entry. The initial dose was 6.5 mg/m2. MCC-465 was administered as a 1-h infusion every 3 weeks and the treatment continued for up to six cycles.
Results:
Twenty-three patients received a total of 62 cycles at the 6.545.5 mg/m2 dose level. DLTs were myelosuppression and appetite loss at the 45.5 mg/m2 dose level. Other toxicities were mild. Neither palmarplantar erythrodysesthesia nor cardiotoxicity was observed. Acute reactions related to infusion were observed commonly in 16 patients over the entire dose range. While no antitumor response was observed, stable disease (SD) was observed in 10 out of 18 evaluable patients. The pharmacokinetic study showed a similar AUC and Cmax to Doxil®.
Conclusion:
MCC-465 was well tolerated. The recommended dose for a phase II study of MCC-465, for a 3-week schedule, is considered to be 32.5 mg/m2 in an equivalent amount of DXR.
Key words: doxorubicin, drug delivery system, GAH, immunoliposome, MCC-465, pharmacokinetics
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Introduction |
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PEG-coated liposomes are stable, long-circulating drug carriers useful for delivering doxorubicin (DXR) to the sites of solid tumors. Compared with conventional liposomes, pegylated liposomes are less extensively taken up by the RES and remain in circulation for a long time [46]. The long-term circulation and the ability of pegylated liposomes to extravasate through leaky tumor vasculature results in localization of DXR in tumor tissue, probably due to the EPR effect. In a number of animal and human tumors, PEG liposomal DXR produced higher intratumor drug concentrations and better therapeutic responses than equivalent doses of non-pegylated liposome-encapsulated DXR or free DXR [7, 8].
MCC-465 is a newly formulated immunoliposome-encapsulated DXR (Figure 1). This liposome is chemically conjugated to PEG and the F(ab')2 fragment of the human monoclonal antibody, GAH, which recognizes a cell surface molecule on various types of cancer cells [9]. Therefore, this formulation should possess the ability of both active and passive targeting. So far, the antigen recognized by this antibody has not been successfully purified, the reason for which is assumed to be that the antibody may have recognized the epitope as the conformation of the antigen(s). This occurs because of the characteristics of the antibody, which reacts only to viable cells and native protein and not to denatured protein, thus making analysis by conventional methods based on protein chemistry useless.
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The antitumor activity of MCC-465 against GAH-positive human stomach cancer B37 cells was compared with that of GAH non-conjugated PEG liposomal DXR in vitro. The result clearly showed that MCC-465 was much more effective against B37 cells than GAH non-conjugated PEG liposomal DXR [9]. In nude mice, MCC-465 exhibited higher antitumor activity against several GAH-positive stomach cancers transplanted in the renal capsules of mice in comparison with free DXR or GAH non-conjugated PEG liposomal DXR [9]. Using a fluorescence-labeled liposome, it was revealed that GAH-tagged liposomes were extensively internalized, but GAH non-tagged liposomes were not [9]. Taking all the data together, we concluded that the GAH-conjugated immunoliposome was highly potent as a drug-targeting device, especially for human stomach cancer. Therefore, this phase I study was confined to patients with advanced gastric cancer.
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Patients and methods |
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Study drug and drug administration
MCC-465 was constructed from PEG (molecular weight 5 kDa on average), lipids, doxorubicin hydrochloride and F(ab')2/GAH. Lipids were consisted from dipalmitoylphosphatidylcholine, cholesterol and maleimidated cipalmitoylphosphatidyletahnolamine. The ratio of the conjugated F(ab')2/GAH, PEG, DXR and lipids was 1:4:5:50 (w/w/w/w), respectively. The mean size of MCC-465 was 143 nm [9].
MCC-465, which was manufactured under the good manufacturing practice regulations of the Ministry of Health, Labour and Welfare of Japan, was supplied by Mitsubishi Pharma Corporation (Osaka, Japan) in glass vials. Each vial contained lyophilized PEG immunoliposomes containing a total of 10 mg of doxorubicin hydrochloride. Appropriate amounts of MCC-465 dissolved in cold sterile saline for injection were diluted and adjusted with sterile saline up to 250 ml (or 500 ml when patients received >70 mg DXR equivalent dose/body). MCC-465 solution was infused intravenously for 60 min, or 120 min when the diluted volume was 500 ml, by an electric-driven pump with a fine filter F162 (Forte Grow Medical Co., Tochigi, Japan).
Dosage and dose escalation
The starting dose of MCC-465 was 6.5 mg/m2, which is equivalent to one-tenth of the LD10 in rats. MCC-465 was administered once every 3 weeks and the treatment was continued up to six cycles unless any severe adverse event or disease progression was observed. Dose escalation proceeded according to a accelerated titration method described previously [10]. Toxicity was graded from 1 to 4 using the criteria of the Japan Clinical Oncology Group. If grade 2 toxicity occurred during the first 21 days, the dose of the next level should be increased according to the modified Fibonacci method and toxicity was to be confirmed in at least three patients. Intrapatient dose escalation was not permitted. At the level at which dose-limiting toxicity (DLT) was observed, toxicity was confirmed in up to six patients. The maximum tolerated dose (MTD) was then defined as one level below that level at which three out of six patients experienced a DLT [11]. The recommended dose for a phase II trial was defined by the Efficacy and Safety Assessment Committee from the results of the safety and efficacy of this trial. DLT was defined as: (i) neutrophil count of <500/µl for >5 days or associated neutropenic fever of >38.5°C with infection; (ii) platelet count of <25 000/µl; and (iii) non-hematological toxicities except for nausea, vomiting and alopecia.
Pretreatment assessment and follow-up
At enrollment, patients were evaluated by a complete history and physical examination, performance status, complete blood cell count (CBC), blood chemistry, urinalysis, electrocardiogram (ECG), computed tomography or upper gastrointestinal series. Other exams were performed only in the presence of a clinical indication. Patients were monitored by physical examination every day up to the second administration, and at days 1, 2 and 3 and weekly thereafter by CBC and blood chemistry. ECG was recorded before and during treatment. Ultrasonic cardiography was repeated before every other administration. Human antihuman antibody (HAHA) was evaluated before every cycle. Tumor markers were also measured at the same time as HAHA.
Tumor response was evaluated according to the criteria of the Japan Society of Clinical Oncology. Complete response (CR) was defined as the disappearance of cancerous lesions and partial response (PR) required a >50% reduction in the sum of the bidimensional length of tumors on two points separated by at least 4 weeks. Stable disease (SD) was defined as a <50% reduction or <25% growth of lesions for at least 4 weeks. Progressive disease (PD) was defined as >25% of tumor growth, appearance of new malignant lesions or unequivocal worsening of other clinical evidence of malignancy. The Clinical Trial Coordinating Committee and the Efficacy and Safety Assessment Committee were organized to bridge between the three institutions at which the study was performed.
Sampling and storage
The measures recorded at the first cycle were as follows: (i) plasma concentrations of DXR (total DXR concentration); (ii) concentrations of DXR after gel filtration (liposomal-encapsulated DXR); (iii) concentration of DXR after ultrafiltration (free DXR); (iv) plasma concentrations of DXR metabolites; (v) urinary concentrations of DXR; and (vi) urinary concentrations of DXR metabolites. After the second cycle, only total DXR was measured. All the samples were chilled on ice during preparation and prepared using the appropriate method described below. Urine samples were stored in a refrigerator from the day before the drug administration to day 4. Prepared plasma and urinary samples were stored at 20°C except for encapsulated DXR samples, which were stored at 80°C.
Assay conditions
For the measurement of total DXR and metabolites [doxorubicinol (Dxol) and 7-deoxydoxorubicinol aglycon (7H-Dxol)] in plasma, 200 µl of boric acid buffer (pH 9.8) and 3 ml of chloroform/methanol (80:20 v/v) were added to 200 µl of human plasma and mixed. The separated organic phase was evaporated to dryness under a nitrogen stream. Residue was re-dissolved with the mobile phase and injected into a high-performance liquid chromatograph (HPLC) under the conditions described in HPLC conditions 1 in Table 1. For the measurement of encapsulated DXR by HPLC, plasma from each patient (60 µl) and the marker solution (60 µl) containing sufficient empty MCC-465 (no DXR), which is used as the marker for UV detection, were mixed. Then, 100 µl of the mixture was loaded onto a gel filtration column. The peak fraction of encapsulated DXR through gel filtration was collected, and 100 µl of the fractionated sample was mixed with 230 µl of methanol containing 0.15% trifluoroacetic acid. The mixture was injected into the HPLC under the condition described in HPLC conditions 2 in Table 1. For the free DXR measurement, 1 ml of freshly prepared plasma at each point was centrifuged immediately in Centrifree (Amicon Co., Ltd) at 2000 g at room temperature for 10 min. One hundred microliters of the collected sample were mixed with 250 µl of the mobile phase (described in Table 1) containing the internal standard. DXR and the metabolites in urine were measured using the same method as for plasma, except the urine volume was 400 µl.
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Results |
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Hematological toxicity
No significant myelosuppression was observed up to dose level 3. Grade 4 leucopenia was observed in one patient at level 5 (45.5 mg/m2). Grade 4 neutropenia was observed in three patients at the same level (Table 4). One of these three patients experienced grade 4 neutropenia that lasted for 5 days, which was defined as a DLT. Leucopenia and neutropenia started from day 48, and the median time to the nadir was 15 days.
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Antitumor activity
Although the antitumor activity was not the primary end point, 18 out of 23 patients were evaluable. No responses were observed definitely in these evaluable 18 patients. However, 10 patients had SD (median duration was 92.5 days, range 48135) (Table 6). Seven of these 10 received more than four cycles of treatment within at least 16 weeks, while these patients have received multiple (once as many as 5 cycles; median 2 cycles) prior chemotherapy cycles (Tables 25).
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Discussion |
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The present clinical data indicate that the PK parameters of MCC-465 differ from those of free doxorubicin, but were very similar to those of Doxil in humans [6]. These data show the stability of MCC-465 in the blood circulation. They also indicate that the conjugation of the F(ab')2 of GAH does not interfere with the stealth effect of the PEG liposomes.
The DLTs of MCC-465 were neutropenia and appetite loss. Unlike the findings from previous reports of phase I trials for a similar drug, Doxil, we did not experience any severe skin toxicity such as PPE or mucositis. The reason for the difference in toxicity between MCC-465 and Doxil is not known; however, it is speculated that the accumulation of MCC-465 in the skin is different from Doxil, or that Caucasian patients are more prone to skin damage by such stealth liposomal DXR in terms of skin toxicity compared with Japanese patients.
IRR was the most common adverse effect. Sixteen out of 23 treated patients showed a variety of symptoms. The major symptoms were fever, rigors, shivering, flushing, chest discomfort, back pain, red eye, itching, vomiting, feeling hot and numbness, which developed at the beginning of the infusion, while fever, rigors, shivering and hypertension were observed at the end of the infusion. Some patients showed two or more symptoms at the same time, but these symptoms were mild, and they disappeared during the infusion or within a few hours. Eight out of nine patients who had chill or shivering also experienced fever. Among these patients, four also had hypertension, and one developed grade 3 hypertension. Chlorpheniramine maleate was administered to four patients for shivering, itching or flushing, and an anti-hypertensive drug was administered to one patient. The other patients had no medication for IRR.
Other non-hematological toxicities were mild, and no malfunctioning was observed in the organs such as the liver and kidney. Although cardiac safety was not addressed specifically, since the maximum cumulative dose of doxorubicin in this phase I study was 195 mg/m2, no patients presented with cardiac toxicity symptoms or loss of function in terms of the left ventricular ejection fraction.
In this study, we needed a 3-week interval schedule in order to determine the safety, tolerability and PKs. No objective tumor response was seen. It is, however, worth noting that 10 SD patients were observed among our 18 evaluable patients, who belonged to a population with a very low probability of response because of extensive prior chemotherapy for gastric cancer.
Our study shows that the MTD of MCC-465 using the 3-week schedule (45.5 mg/m2) is slightly lower than the MTD of Doxil (50 mg/m2). This result may be due in part to the unstable condition of heavily treated patients with advanced gastric cancer. These data warrant further investigation of MCC-465, and the recommended dose for a phase II study with a 3-week administration protocol is considered to be 32.5 mg/m2.
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Acknowledgements |
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FOOTNOTES |
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