Phase II trial of branched peginterferon-{alpha} 2a (40 kDa) for patients with advanced renal cell carcinoma

R. J. Motzer1,+, A. Rakhit2, J. Thompson3, H. Gurney4,5, P. Selby4,5, R. Figlin6, S. Negrier7, S. Ernst8, M. Siebels9, M. Ginsberg1, K. Rittweger2 and L. Hooftman2

1 Memorial Sloan-Kettering Cancer Center, New York, NY; 2 Hoffmann-La Roche Inc., Nutley, NJ; 3 University of Washington Medical Center, Seattle, WA, USA; 4 Westmead Hospital, NSW, Australia; 5 St James University Hospital, Leeds, UK; 6 UCLA Medical Center, Los Angeles, CA, USA; 7 Centre Leon Berard, Lyon, France; 8 Tom Baker Cancer Center, Calgary, Alberta, Canada; 9 Universitaetsklinikum Grosshadern, Munich, Germany

Received 29 January 2002; revised 18 March 2002; accepted 10 April 2002


    Abstract
 Top
 Abstract
 Introduction
 Patients and methods
 Results
 Discussion
 References
 
Background:

Peginterferon-{alpha} 2a (40 kDa), PEGASYSTM (PEG-IFN), is a modified form of recombinant human interferon (IFN)-{alpha} 2a with sustained absorption and prolonged half-life after subcutaneous administration. A phase II trial was conducted in previously untreated patients with advanced renal cell carcinoma (RCC) to assess efficacy, toxicity and pharmacokinetic profile.

Patients and methods:

Forty previously untreated patients with advanced RCC were enrolled on this multicenter trial. The median age was 60 years and 63% had prior nephrectomy. PEG-IFN was administered at a dose of 450 µg/week on a weekly basis by subcutaneous injection. Serial venous blood samples were drawn to assess concentrations of PEG-IFN.

Results:

Five (13%) patients achieved a major response (four partial and one complete). The median time to progression was 3.8 months, and 63% of patients were alive at 1 year. The toxicity profile was mostly mild to moderate in intensity. Toxicity higher than grade 2 included neutropenia (six patients), fatigue/asthenia (four patients), nausea/vomiting (three patients) and elevated hepatic transaminase concentrations (four patients). Serum drug levels were studied in all patients; mean Cmax at week 1 was 19 ng/ml, and levels were sustained at close to peak over 1 week. With chronic dosing, drug concentration was increased 3-fold, and steady state was achieved in 5–9 weeks.

Conclusions:

The sustained maintenance of serum levels of PEG-IFN allows once-weekly dosing. The efficacy and tolerability profile was qualitatively similar to standard IFN-{alpha}, and adverse events were mostly mild to moderate in nature.

Key words: interferon-{alpha}, pegylated, renal cell carcinoma


    Introduction
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 Abstract
 Introduction
 Patients and methods
 Results
 Discussion
 References
 
Interferon (IFN)-{alpha} has a low antitumor effect against renal cell carcinoma (RCC) [1], and a modest survival benefit was shown in two phase III trials comparing IFN-{alpha} with vinblastine or medroxyprogesterone [2, 3]. Two preparations, recombinant IFN-{alpha} 2a (Roferon®; Hoffmann-LaRoche) and recombinant IFN-{alpha} 2b (Intron®; Schering Plough Laboratories), are commercially available and represent standard preparations used in clinical practice. However, both of the commercially available IFN-{alpha} preparations are characterized by a relatively short plasma half-life and require frequent dosing.

Peginterferon-{alpha} 2a (40 kDa), PegasysTM (PEG-IFN), is recombinant IFN-{alpha} modified by the covalent attachment of a 40 kDa branched methoxy polyethylene glycol molecule. PEG-IFN has sustained absorption, a slower rate of clearance, and a longer half-life than unmodified IFN. The pharmacokinetic profile allows weekly dosing. A phase I dose-escalation study of PEG-IFN was conducted in previously untreated patients with advanced RCC [4]. Twenty-seven patients were enrolled, and five (19%) achieved a partial response [4]. The maximum tolerated dose was defined as 540 µg/week, with dose-limiting toxicities of fatigue and elevated hepatic transaminase [4]. Six patients were treated at a dose of 450 µg/week without dose-limiting toxicity, and this was the dose used in the phase II study reported here. Serial venous blood samples were drawn to assess concentrations of PEG-IFN and two immunological surrogates, neopterin and 2',5'-oligoadenylate synthetase (OAS) [5, 6].


    Patients and methods
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 Abstract
 Introduction
 Patients and methods
 Results
 Discussion
 References
 
Patients
Between July 1999 and November 1999, 40 patients with advanced RCC entered this multicenter phase II trial approved by the Institutional Review Board at eight participating centers. Eligibility criteria included: age >=18 years; informed consent; measurable disease; Karnofsky performance status >=80%; and adequate hematological (white blood cell count >=3000 cells/mm3, granulocytes >=1500 cells/mm3, platelet count >=100 000 cells/mm3 and hemoglobin >=10 g/dl), renal (serum bilirubin <=1.5 times normal, transaminase levels and alkaline phosphatase <=2.5 times normal) and hepatic (serum creatinine concentration of <=1.5 times normal) function.

Exclusion criteria included brain metastases, history of psychiatric disabilities, history of seizure requiring anticonvulsant therapy, clinically significant cardiac abnormalities, or poor medical condition because of non-malignant organ or systemic disease. Prior chemotherapy, immunotherapy or hormonal therapy for RCC was not allowed.

Dose and treatment schedule
PEG-IFN was supplied by Hoffmann-La Roche, Inc. (Nutley, NJ, USA) as a ready-to-use solution in single-dose glass vials. PEG-IFN was administered by subcutaneous injection at a dose of 450 µg once weekly for 24 weeks. Each cycle lasted 28 days and consisted of outpatient subcutaneous injections on days 1, 8, 15 and 22 with no rest between cycles. Based on assessment of tumor size following cycle two, patients with stable disease or a major tumor response (complete or partial) received additional cycles of therapy until evidence of progression or unacceptable toxicity.

Dose modification was made according to toxicity. Treatment was withheld for grade 3 or 4 non-hematological toxicity, then restarted at a 90-µg lower dose when toxicity improved to grade 1 or better, or discontinued at the discretion of the treating physician in the case of grade 4 toxicity. Treatment was withheld for grade 3 or 4 neutropenia or thrombocytopenia, and then restarted at the next lowest dose level when toxicity improved. For grade 2 non-hematological toxicity, treatment could be withheld until improvement to grade 1 or better, then restarted at the same dose.

Patients’ physical examination, complete blood count and serum chemistry were monitored. Each patient had a reassessment of measurable disease after 8 weeks of treatment, then every 8 weeks thereafter. Response was assessed according to World Health Organization (WHO) criteria and toxicity according to Common Toxicity Criteria.

Pharmacodynamic and pharmacokinetic studies
Serial venous blood samples were drawn to assess concentrations of PEG-IFN and two immunological surrogates, neopterin and 2',5'-OAS [5, 6]. Sera were stored at –20°C until assay. Serum samples were obtained prior to treatment, and 6, 48, 72, 96, 120, 144 and 168 h following injection on weeks 1 and 9 of treatment. Serum samples were also obtained prior to treatment on day one of weeks 5, 10, 13, 17, 21 and 25.

The assay used to measure serum PEG-IFN was developed at Hoffmann-La Roche and has been described previously [4]. In brief, it is a quantitative sandwich enzyme-linked immunosorbent assay (ELISA) using two distinct mouse monoclonal antihuman (anti-Hu) IFN-{alpha} antibodies that recognize different epitopes of IFN-{alpha} 2a. In a one-step immunoreaction, PEG-IFN is bound to the peroxidase-conjugated anti-Hu-IFN-{alpha} antibody. Unbound complexes are removed by washing, and tetramethylbenzidine substrate solution is added. Conversion of tetramethylbenzidine by peroxidase to a colored product allows the reaction to be followed spectrophotometrically, as the absorbance at 450 nm is directly correlated to the concentration of PEG-IFN in the sample.

The analytical sensitivity is 125 pg/ml of PEG-IFN in human serum. The intra-assay variation of the quality control samples is between 0.8% and 12%, while the inter-assay variation was between 4.5% and 18.3%.

The serum concentration of neopterin was measured using a commercial radioimmunoassay (RIA) kit (HENNING test"; BRAHMS Diagnostica GmbH, Berlin, Germany) consisting of a single incubation step and using a double antibody phase separation technique. The method has been described previously [4]. The limit of quantitation is 3 nm using a 50-µl sample. The intra-assay and between-assay variabilities ranged from 1.8% to 6.5%.

The OAS enzyme activity in serum samples was measured by RIA with 125I-labeled tracer and a second antibody used as the bound/free separating agent (Eiken Chemical Co., Tokyo, Japan). The method has been described previously [4]. The limit of quantitation in serum was 240 pM/h. The intra-assay variability ranged between 3.6% and 11.7%. The samples were stable for at least 2 months at –20°C and >6 months at –70°C.

Measurement of serum antibody to PEG-IFN
Anti-IFN-{alpha} antibodies were measured in serum at baseline and 4 weeks after the end of treatment, using a one-step immunoreaction ELISA method developed at Hoffmann-La Roche, performed as described previously [4]. The sensitivity limit of the assay is 0.24 U/ml with a measurement range of 0.24 to 10.5 U/ml. Inter-assay variability (CV%) was 6.5% to 14%. The recovery of spiked anti-IFN-{alpha} antibody standard in human sera was >94%.

Serum concentration (or activity) versus time data was analyzed for PEG-IFN, neopterin and 2',5'-OAS by non-compartmental pharmacokinetic methods. The highest observed concentration and the corresponding sampling time were defined as Cmax and Tmax, respectively. The area under the concentration-versus-time curve (AUC0–{tau}) was calculated by use of the trapezoidal rule over the dosing interval of 1 week.


    Results
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 Abstract
 Introduction
 Patients and methods
 Results
 Discussion
 References
 
Patient characteristics
Forty patients were treated with PEG-IFN (Table 1). The median age was 60 years. Twenty-five patients (62%) had a prior nephrectomy. Assignment to Memorial Sloan-Kettering Cancer Center (MSKCC) risk group based on pretreatment clinical features [7] showed that 11 (27.5%) had favorable-risk clinical features.


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Table 1. Patient characteristics
 
Response and survival
Five of 40 evaluable patients (12.5%) achieved a major response (Table 2). One patient, who had undergone a nephrectomy 2 months prior to study entry, achieved a complete response following resolution of retroperitoneal adenopathy and is disease-free at 15+ months following start of therapy. The remaining four patients achieved a partial response, resulting in progression-free durations of 5.9, 13.1, 15.4 and 19+ months.


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Table 2. Characteristics of five patients with major response
 
Best response in the remaining patients was stable disease for 20 (50%) and progression for 13 (32.5%) patients; all except one patient have since progressed. One patient with best response of stable disease continues treatment and remains progression-free at 17.8+ months.

The proportion of patients alive at 12 months is 63% (Figure 1). The median progression-free survival is 3.8 months [95% confidence interval (CI) 2.9–5.6 months].



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Figure 1. Kaplan–Meier graph of overall survival of 40 patients (13 alive).

 
Treatment administered and toxicity
Over the course of treatment, the side-effect profile was mostly mild to moderate in intensity. The toxicities most frequently reported, regardless of intensity or relationship to treatment, included fatigue, fever, nausea, headache and rigors. Treatment-related grade 3 or 4 toxicities included neutropenia (six patients), lymphopenia (seven patients), thrombocytopenia (one patient), elevated serum transaminase concentrations (SGOT, SGPT) (four patients), fatigue/asthenia (four patients), nausea and/or vomiting (three patients), anorexia/weight loss (one patient), depression (one patient), rigors (one patient), and night sweats (one patient) (Table 3).


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Table 3. Toxicity (n = 40, possible or probable relationship to treatment)
 
Over the initial study period of 24 weeks, seven patients (18%) required dose attenuation. During the entire study, nine (23%) patients required dose reductions to 360 µg/week (eight patients) or 270 µg/week (one patient). Five patients (13%) had two or more consecutive doses withheld without dose reduction. The median time from start of treatment to dose attenuation was 2.8 months (range 0.7–15.4 months). Three patients were removed from the study because of adverse events. These included one patient who developed hyperbilirubinemia and one patient who developed gastrointestinal hemorrhage, both related to progression of disease. The third patient was removed from the study because of treatment-related toxicity (lethargy) following 17 months of treatment.

Pharmacokinetics
Serum concentration of PEG-IFN increased rapidly after the subcutaneous injection with measurable concentrations appearing at 6 h, the first time-point of determination (Table 4; Figure 2). Maximum serum concentrations (Cmax) appeared as early as 48 h and were sustained at close to peak from day 3 (48 h) to day 8 (168 h), the time of next dose. The mean (±SE) Cmax and Tmax after first dose of 450 µg administered subcutaneously were 19 (± 2) ng/ml and 95 (± 7) h, respectively.


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Table 4. Summary of serum concentrations of PEG-IFN and two associated immunological surrogates (neopterin and 2',5'-OAS) following 450 µg/week subcutaneous administration of PEG-IFN
 


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Figure 2. Mean PEG-IFN concentration versus time: weeks 1 and 9.

 
Serum concentrations of PEG-IFN increased by ~3-fold with chronic dosing (Figure 3). Trough serum levels were found to reach a steady state starting at approximately week 5. Serum concentration was followed over 6 months of treatment in individual patients and was found to remain stable on long-term therapy. The peak serum concentration (Cmax) and area under the serum concentration–time curve (AUC) at weeks 1 and 9 were: Cmax 19 compared with 54 ng/ml, and AUC 2215 compared with 6946 ng·h/ml. The average steady-state pre-dose serum concentration at 450 µg/week dosing was 40 (± 2) ng/ml.



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Figure 3. Pre-dose levels of PEG-IFN on chronic dosing. Mean data were collected from patients who maintained a 450 µg dose every week.

 
Anti-IFN-{alpha} antibodies were measured in serum at baseline and during treatment-free follow-up using an ELISA method. No significant antibody levels were observed in any of the patients studied.

Pharmacodynamics
Significant induction of both neopterin and 2',5'-OAS was observed in all patients studied (Table 4). Maximum induction was observed starting at ~48 h and maintained during the rest of the dosing interval. Immunological induction was maintained on chronic dosing over 6 months, with little accumulation of serum markers after week 1. Serum immunological markers did not appear to increase further on multiple dosing compared with that observed at week 1.


    Discussion
 Top
 Abstract
 Introduction
 Patients and methods
 Results
 Discussion
 References
 
The serum half-life of IFN-{alpha} ranges from 3 to 5 h [8], requiring frequent (three to seven times a week) dosing in order to maintain adequate serum concentrations of IFN-{alpha} and resulting in a large fluctuation in serum concentration during therapy. PEG-IFN was developed with an improved pharmacological profile and is being studied for the treatment of hepatitis C and IFN-{alpha}-responsive malignancies. Two randomized trials showed PEG-IFN to be more effective than IFN-{alpha} and well tolerated in treatment of patients with hepatitis C [9, 10]. IFN-{alpha} has a low but reproducible response proportion against metastatic RCC, and a modest survival benefit for this therapy against metastatic RCC has been cited in two recent phase III trials comparing IFN-{alpha} with vinblastine or medroxyprogesterone [2, 3]. The current trial was conducted to assess the efficacy, toxicity and pharmacologic profile of PEG-IFN given at 450 µg/week, the dose recommended from the phase I trial, to previously untreated patients with advanced RCC [4].

Antitumor activity was demonstrated by the complete and partial responses observed among patients treated both on this trial and the phase I trial [4]. The low degree of antitumor activity was similar to that with standard preparations. In a summary of the published literature, the overall response proportion to IFN-{alpha} in phase II trials was 14% (95% CI 12% to 17%) in 648 patients [11]. Combined results from the phase I and II trials of PEG-IFN in patients with RCC showed a 14.9% response proportion in 67 patients (95% CI 12.5% to 18.5%).

Over the course of treatment, the side-effect profile was mostly mild to moderate in intensity. Adverse events included fatigue, fever, neutropenia and elevated hepatic transaminase concentrations. The toxicity profile of PEG-IFN was consistent with standard IFN-{alpha} therapy, treatment was generally well tolerated and there did not appear to be cumulative toxicity with chronic dosing.

The maximum serum concentration of PEG-IFN was reached at ~48 h following subcutaneous administration of PEG-IFN. Serum concentration remained close to peak level for the remaining 5 days of the dosing interval, providing a close to sustained-release delivery of PEG-IFN over a 1-week period. On repeated administration, serum predose levels of PEG-IFN increased by nearly 3-fold with multiple dosing. The predose levels reached a steady state between weeks 5 and 9, and serum levels were then maintained for over 6 months when monitored in patients who continued on treatment with stable disease or a complete or partial response. In this setting, there was no further accumulation in PEG-IFN concentrations with long-term dosing.

In summary, the sustained maintenance of serum levels of PEG-IFN allows once-weekly dosing of this modified IFN. The efficacy and tolerability profile was qualitatively similar to standard IFN-{alpha}, and adverse events were mostly mild to moderate in nature.


    Acknowledgements
 
The authors would like to thank Lucy Dantis and Patricia Fischer for nursing support, and Carol Pearce for her review of the manuscript.


    Footnotes
 
+ Correspondence to: Dr R. J. Motzer, Memorial Sloan-Kettering Cancer Center, 1275 York Avenue, New York, NY 10021, USA. Tel: +1-646-422-4312; Fax: +1-212-988-0806; E-mail: motzerr{at}mskcc.org Back


    References
 Top
 Abstract
 Introduction
 Patients and methods
 Results
 Discussion
 References
 
1. Motzer RJ, Russo P. Systemic therapy for renal cell carcinoma. J Urol 2000; 163: 408–417.[ISI][Medline]

2. Pyrhonen S, Salminen E, Ruutu M et al. Prospective randomized trial of interferon alfa-2a plus vinblastine versus vinblastine alone in patients with advanced renal cell cancer. J Clin Oncol 1999; 17: 2859–2867.[Abstract/Free Full Text]

3. Medical Research Council and collaborators. Interferon-alpha and survival in metastatic renal carcinoma: early results of a randomised controlled trial. Lancet 1999; 353: 14–17.[ISI][Medline]

4. Motzer RJ, Rakhit A, Ginsberg M et al. Phase I trial of 40 kDa branched pegylated interferon alfa-2a for patients with advanced renal cell carcinoma. J Clin Oncol 2001; 19: 1312–1319.[Abstract/Free Full Text]

5. Huber C, Batchelor JR, Fuchs D et al. Immune response-associated production of neopterin: release from macrophages primarily under control of interferon-gamma. J Exp Med 1984; 160: 310–316.[Abstract]

6. Samuel C. Antiviral actions of interferon. Virology 1991; 183: 1–11.[ISI][Medline]

7. Motzer R, Bacik J, Murphy BA et al. Interferon-{alpha} as a comparative treatment for clinical trials of new therapies against renal cell carcinoma. J Clin Oncol 2002; 20: 297–301.[Abstract/Free Full Text]

8. Wills R, Dennis S, Spiegel HE et al. Interferon kinetics and adverse reactions after intravenous, intramuscular, and subcutaneous injection. Clin Pharmacol Ther 1984; 35: 722–727.[ISI][Medline]

9. Zeuzem S, Feinman SV, Rasenack J et al. Peginterferon alfa-2a in patients with chronic hepatitis C. N Engl J Med 2000; 343: 1666–1672.[Abstract/Free Full Text]

10. Heathcote ESM, Cooksley G, Dusheiko G et al. Peginterferon alfa-2a in patients with chronic hepatitis C and cirhosis. N Engl J Med 2000; 343: 1673–1680.[Abstract/Free Full Text]

11. Motzer RJ, Berg WL. Role of interferon in metastatic renal cell carcinoma. In Bukowski RM, Novick AC (eds): Renal Cell Carcinoma: Molecular Biology, Immunology, and Clinical Management, 1st edition. Totowa, NJ: Humana Press 2000; pp. 319–330.