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Home , Melphalan, Prednimustine

ANTICANCER RESEARCH 26: 2197-2204 (2006)

In Vitro Cytotoxic Effect of and Pilot Phase II Study in Hormone-refractory Prostate

PHILIPPE MOUGENOT1,2, FRANÇOISE BRESSOLLE1,2, STÉPHANE CULINE3, ISABELLE SOLASSOL1, SYLVAIN POUJOL1 and FRÉDERIC PINGUET1

1Onco-pharmacology Department, Pharmacy Service and 3Department of Medicine, Val d’Aurelle Anticancer Centre, parc Euromédecine, Montpellier; 2Clinical Pharmacokinetic Laboratory, University Montpellier I, Montpellier, France

Abstract. The objective of this study was to determine the considered the most adequate first-line treatment and impact of single versus sequential exposure to melphalan on controls symptoms in 80-90% of patients (1-3). The median the proliferation of an androgen-independent prostate cell line, duration of response is approximately 2-3 years. PC-3, and to report the results of a pilot phase II study. For is the next option for hormone-refractory exposure to a single bolus dose, the doses were added at the prostate cancer, but historically chemotherapy treatment start of the study and cell culture was continued for 96 h. For has produced relatively low response rates. sequential exposure, 1/9 of the dose was added every 1.5 h over Alkylating agents may be active against prostate cancer. 12 h, followed by cell culture for 84 h. Cell growth inhibition Single-agent studies of (4), nitrogen was determined by the MTT assay. The clinical study was mustard (4), (5), (6) carried out on 14 patients with advanced prostate cancer. and diazoquinone (7) have reported response rates of 10 to Melphalan was infused over a 24-h period. The sequential-dose 20%. Smith et al. (8) estimated the single-agent activity of schedule was more effective than the single-dose exposure, IC50 intravenous (over 30 min) melphalan in 27 hormone- values: 0.074 versus 0.77 Ìg/ml. Out of the 14 patients (42 resistant prostate cancer patients. No objective responses to courses) enrolled into the study, two patients were removed this regimen were observed by the authors; the median within the first 2 weeks because of rapid disease progression. survival was 11.5 months. However, a decrease of >50% in The toxicity profile did not differ greatly from that reported serum prostate-specific antigen (PSA) levels, that was after a 1-h infusion. Four PR and two SD were observed. The sustained for >6 weeks, was observed in two patients and median survival of the twelve patients was 23 weeks. one patient survived for more than 29 months. More Melphalan administered over a 24-h period to patients with recently, with estramustine and the androgen-independent prostate cancer appeared to provide / combination, approved by the some clinical benefits with manageable toxicity. Food and Drug Administration for use in this indication, have produced more promising palliative activity (9-11). Prostate cancer is the most common malignancy diagnosed Nevertheless, no treatment has been shown to prolong in men. However, the management of metastatic prostate survival. The drug resistance of prostate carcinoma would cancer remains a complex and difficult problem as there are be due to high levels of TNF-beta that protect tumors from limited treatment options and the prognosis continues to be cytotoxic cancer therapies (12). dismal in metastatic hormone-refractory prostate cancer. In Melphalan is an alkylating agent of the bischloroethylamine newly diagnosed metastatic disease, the suppression of type. It exerts a cytotoxic effect through the formation of androgenic activity, with or without anti-androgens, is inter-strand or intra-strand DNA cross-links or DNA-protein cross-links via the two chloroethyl groups of the molecule (13). Founded on the pharmacokinetic principles of phase- specific, plasma half-life and stability in solution, infusional Correspondence to: Dr. Françoise Bressolle, Ph.D., Laboratoire de schedules for chemotherapy administration represent a Pharmacocinétique Clinique, Faculté de Pharmacie, BP 14491, rational method for the delivery of many antineoplastic 34093 Montpellier Cedex 5, France. Tel: (33) 4 67 54 80 75, Fax: (33) 4 67 54 80 75, e-mail: [email protected] agents. For several antimitotic drugs, long-term continuous infusion increases the therapeutic activity or improves the Key Words: Melphalan, 24-hour continuous infusion, PC-3 cell line, therapeutic index by decreasing toxicity (14). Based on hormone-refractory prostate cancer, survival. pharmacokinetic considerations, melphalan is also a good

0250-7005/2006 $2.00+.40 2197 ANTICANCER RESEARCH 26: 2197-2204 (2006) candidate for continuous infusion; indeed, this drug has a centrifugation and washing with PBS IX, a nuclear suspension in short half-life, a small volume of distribution and a low protein PBS was obtained at a density of 200,000 cells/ml. DNA binding binding (15). However, due to its low stability in 5% dextrose was then performed with propidium iodide. The samples were analyzed using the EPIC XL Flow Cytometer (Coulter, Hialeah, and 0.9% sodium chloride, melphalan is usually administered FL, USA). Histogram analyses of the DNA distribution were by short-term infusion. However, a previous study showed that carried out using the «Multicycle» software (Coulter), which the stability of melphalan can be increased using 3% sodium allowed an accurate estimation of the S-phase. The coefficient of chloride (16). Consequently, it would be possible to consider variation was <2%. administering melphalan by continuous infusion. In a previous To evaluate the apoptotic capacity of the cell populations, in vitro study carried out on two human cell lines, one immunocytology (IC) was performed using the Bcl-2 anti-apoptotic myeloma and one ovary carcinoma (8226 and A2780, antibody (Bcl-2 oncoprotein, clone 124, Dakocytomation S.A., Trappes, France). For this, cells were prepared as described above. respectively), the antitumor activity of melphalan was higher Cytospin slides were obtained from 200 Ìl of the solution, fixed for sequential- than single-dose exposure (17). Based on with a methanol-acetone (50:50, v/v) fixative mixture at –20ÆC for results of a phase I trial, the maximum tolerated dose of 10 min and IC was carried out using the LSABR 2 peroxidase kit melphalan infused over a 24-h period was 30 mg/m2 (18). (Dakocytomationn S.A.). Simultaneously, both the hormonal The first objective of the present study was to determine receptor status of the cell lines, using an estrogen receptor the impact of single versus sequential exposure to melphalan antibody (clone 6F11, Dako, Glostrup, DK) and a on the proliferation of an androgen-independent prostate receptor antibody (PSR636, Dako), as well as the level of expression of the cerbB2-neu protein, using the CB11 monoclonal cell line, PC-3. The second objective was to report the antibody (Dako), were studied by IC. results of a phase II pilot study initiated to determine the To analyze DNA damage in the cell populations, the alkaline efficacy and toxicity of melphalan, administered over a 24-h Comet assay, a single-cell gel electrophoresis, was employed, period with an adapted individual dosing, in advanced according to a previously published method (19). Slide analysis was prostate cancer. done on a fluorescent microscope (Leica) with a cold triCCD camera, using SAMBA IPS Software, which enabled the “tail Patients and Methods moment” (TM) of a 50-cell comet to be determined.

Drug and chemicals. Melphalan (L-PAM) was kindly provided by Cell growth inhibition and viability. On day 0, the RPMI medium Glaxo-SmithKline (Paris, France). 3-(4,5-dimethylthiazol-2-yl)-2.5- was carefully removed from the flasks and the cells, in exponential- diphenyltetrazolium bromide (MTT) and phosphate-buffered phase growth, were harvested with 3 ml of trypsin-EDTA 1N saline pH 7.3 (PBS) were purchased from Sigma Chemical Co. (St. (Gibco Laboratories, Cergy Pontoise, France) for 5 min at 37ÆC, Louis, MO, USA). RPMI medium containing RPMI-1640, heat- were washed with 5 ml of RPMI medium and then centrifuged. inactived (56ÆC) fetal calf serum (FCS) and L-glutamine were The supernatant was removed and the pellet was resuspended in obtained from Gibco-BRL (Cergy-Pontoise, France). Isopropyl RPMI medium. The cells were counted then diluted in complete 3 alcohol and hydrochloric acid were of analytical grade (both from culture medium to a final density of 50x10 cells/ml. One hundred Merck, Darmstadt, Germany). Ìl of cell suspension (i.e., 5000 cells/well) were dispensed with a The complete culture medium consisted of RPMI medium pipette into each individual well of a lidded 96-well tissue culture supplemented with 10% FCS, 2 mM L-glutamine, 100 IU/ml plate (TPP, Trasadingen, Switzerland). The cells were re-incubated penicillin G sodium, 100 Ìg/ml streptomycin sulphate and 0.25 Ìg/ml at 37ÆC in a humidified atmosphere (about 17 h) to allow for amphotericin B. attachment. Melphalan was reconstituted in a water/ethanol/propylene-glycol Cell line and culture techniques. The experiments were conducted mixture (35:5:60, v/v), diluted in complete culture medium and with a human androgen-insensitive prostatic carcinoma cell line, added to the wells. For single-dose exposure, the doses of PC-3, obtained from the American Type Culture Collection melphalan were added only at the start of the study and the cell (Rockville, MD, USA). This cell line was cultured at 37ÆC, in flasks culture was continued for 96 h (i.e., 4-fold the doubling-time). For containing complete culture medium, under 95% relative humidity sequential exposure, 1/9 of the total dose was added at the in an atmosphere containing 5% carbon dioxide in air and was beginning of the study and every 1.5 h during the subsequent 12 h, routinely passaged when 90-95% confluent. Only exponentially- followed by cell culture for 84 h. The total dose in the two arms of growing viable cells were used for the study. The viability of the the study was the same. cells was assessed by their ability to exclude 0.5% trypan blue dye. Cell density in the culture flasks was determined by a Coulter MTT assay. Viable cell growth was determined by the MTT reduction counter (Model Z1, Hialeah, FL, USA). The doubling-time, under assay (20). At the end of the experiment, the supernatant of each well our experimental conditions, was approximately 28 h. was aspirated and 20 Ìl of MTT solution at 2 mg/ml in RPMI medium/PBS (3:2, v/v) were added to each well. The plates were Characteristics of the PC-3 cell line. Flow cytometry (FC) tests were incubated for 4 h at 37ÆC. This colorimetric assay is based on the used to determine the DNA content of the cultured cells and to ability of live and metabolically-unimpaired tumor-cell targets to subdivide the cell populations into different fractions. For reduce MTT to a blue formazan product. At the end of the this purpose, DNA cytometry was performed after trypsin incubation period, the supernatant was carefully aspirated and 100 Ìl treatment to remove cultured cells from their dishes. Following of a mixture of isopropyl alcohol and 1 M hydrochloric acid

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(96:4, v/v) were added to each well. Complete and homogeneous grade 4 thrombocytopenia; or ii) increased to achieve an AUC of solubilization of the MTT-formazan product was achieved after 2.5-3 mg ñ h/l for patients with grade 0 or 1 neutropenia or 10 min of incubation and vigorous shaking of the well contents. The thrombocytopenia; or iii) maintained in other cases. The absorbance was measured on a microculture plate reader (Dynatech chemotherapy was repeated every 28 days until evidence of MR 5000, Polylabo, Paris, France) at 570 nm. Thereafter, a linear progressive disease or intolerable toxicity developed. Radiation relationship was established between the MTT assay and the number therapy, given for pain control or for a severe or life-threatening of cells within the range of the experiments shown. The IC50 was condition, was taken as evidence of progressive disease sufficient defined as the concentration of drug which produced 50% cell growth to result in removal of the patient from the trial. All patients inhibition; 50% reduction of absorbance. received prophylactic anti-emetic premedication with 5HT3- Dose-response curves were fitted to the following four- receptor antagonists and corticoids before the melphalan infusion. parameter equation: S = a/(1+(D/c)m) plus background, where S is the surviving fraction and D the dose of drug. In this equation, (a) Toxicity and response evaluation. Toxicity was assessed weekly represents the difference between the maximum and the minimum according to the National Cancer Institute Common Toxicity response, (c) is the concentration needed to obtain 50% of the Criteria scale (version 2). Tumor response (23) was determined (m) maximum effect (if background = 0, then c = IC50) and is a according to the PSA levels. The PSA-defined partial response to slope factor. therapy was defined as a decline in the PSA level by >50% of the entry level, sustained for at least 8 weeks. Partial response in Patients and eligibility criteria. The study protocol was reviewed and measurable disease was defined as a 50% reduction in the products approved by the Board for the Protection of Persons subjected to of the diameters of all index lesions in patients with measurable Biomedical Research of Montpellier, France. The study was disease, sustained for at least 8 weeks. Stable disease was defined performed in accordance with the Declaration of Helsinki and with as stable PSA levels (+/– 10%) for at least 8 weeks. Progressive current European Community and U.S. Food and Drug disease was defined as three consecutive increases in PSA level by Administration guidelines for good clinical practice. Prior to entry, at least 1 ng/ml, the first value >25% above the nadir or baseline, all patients gave written informed consent to participate in the trial. measured at least 2 weeks apart; any increase greater than 25% in Fourteen patients with prostatic carcinoma were included in the the products of diameters of any measurable lesion; or the study, from March 2001 to December 2004. All the patients were appearance of an unequivocally new lesion. Whenever possible, followed-up by the Medical Oncology Service of the Anticancer patients received at least two infusions of melphalan before a Centre (Montpellier, France). The selection criteria for inclusion designation of progression was made. were: age more than 18 years, an expected survival of at least eight weeks, adequate , renal and hepatic functions and a Results performance status of 0-2 on the World Health Organization (WHO) scale. A chest X-ray, an abdominal/pelvic computerized PC-3 cell line characteristics. After a 96-h culture, PC-3 had tomography scan and a bone scan were obtained to document the a G1 major peak (62.2%) with a very high S-phase and a extent of the disease. The exclusion criteria were granulocytes lower than 1500/mm3, platelets lower than 100,000/mm3, bilirubin moderate G2 peak (5%). Expression of the Bcl-2 protein greater than 2.5 times the normal limit, transaminases (aspartate was moderate (mild to moderate labelling of 60% of the aminotransferase (AST) and alanine aminotransferase (ALT)) cells). No cells showed any level of expression of cerB2-neu greater than 3 times the upper normal limit or 5 times greater than protein. Using the Comet test, PC-3 was seen to exhibit a the upper limit in the presence of liver metastases, and significant high percentage of DNA degradation before any treatment: renal (creatinine clearance, CLCR, calculated according to 18% of the cells were strongly degraded; 32% showed mild Cockcroft and Gault (21) <30 ml/min), pulmonary (FEV1, FVC, to moderate lesions and 50% did not show any degradation. or DLCO <65% of the predicted values), or cardiac (isotopic left ventricular ejection fraction <50%) dysfunction. Influence of schedule on melphalan cytotoxicity. For this Treatment regimen. Melphalan was infused intravenously by an cytotoxic assay, the duration of the experiment was 96 h and automatic infusion pump over a 24-h period. The drug was the doses (expressed in Ìg/ml) were variable. Significant dissolved in four syringes of 60 ml 3% sodium chloride; each growth inhibition of the PC-3 cell line occurred (Figure 1). syringe was administered intravenously over 6 h through a central venous catheter. Before administration, the syringes were stored The sequential-dose schedule was more effective than the at +4ÆC (under these conditions melphalan was stable for 48 h) single-dose exposure; the IC50 values were 0.074±0.023 (16). The initial administered dose was 25 mg/m2. Drug Ìg/ml (n=9) and 0.77±0.25 Ìg/ml (n=9), respectively. The adjustments were then made using a population approach (22). IC50 ratio (single-dose exposure / sequential-dose exposure) The aim was to constrain the overall area under the plasma was equal to 10. concentration-time curve (AUC) to within 2-2.5 mg ñ h/l. This target AUC was chosen according to the first results obtained Patient population. The demographic, pretreatment and during the phase I study (18). Thus, according to the hematological toxicity encountered during the previous cycle, the disease history characteristics of all 14 treated patients are administered dose of the next course was: i) decreased to achieve detailed in Table I. The number of courses per patient an AUC of 1.5-2 mg ñ h/l for patients with intercurrent febrile ranged from one to five (two patients received one course, neutropenia or grade 4 neutropenia lasting more than 7 days or two patients received two courses, five patients received

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Figure 1. Growth inhibitory effects of melphalan against the PC-3 cell line. (▲) single dose exposure, (●) sequential dose exposure. Number of replicates: nine.

three courses, four patients received four courses and one Table I. Patient characteristics. patient received five courses). The median duration of hormonotherapy (monotherapy) before the start of Characteristics Number of patients chemotherapy was 38 months (12-88 months). Ten patients Number of patients 14 had received prior chemotherapy, including estramustine (six Metastatic state at diagnosis 7 patients), mitoxantrone (two patients), / Age (years) (three patients) and other (three patients had received drugs Median 67.5 under development; one patient had received adriamycin/ Range 55-80 cyclophosphamide as second-line then navelbine as third- Weight (kg) line; one patient had received docetaxel and one patient had Median 75 received /). Range 55-98 Performance status Out of the 14 patients (42 courses) enrolled into the WHO 0 2 study, two patients were removed within the first 2 weeks WHO 1 4 because of rapid disease progression, characterized by WHO 2 8 debilitating bone pain and fatigue. Thus, twelve patients Involved sites were assessable for toxicity, response and survival. For four Nodes 5 patients, the administered dose of the following courses was Lung 5 increased by 20% (one patient) or 40% (three patients), for Liver 4 Bone 14 five patients the administered dose remained stable over the Adrenal gland 2 treatment period, while for the other patients it was Radiotherapy 14 decreased by 20%. Line of chemotherapy First-line 4 Pharmacokinetic parameters. For all these patients, the Second-line 5 melphalan concentrations at steady state were higher than Third-line 2 Fourth-line 3 the IC50 value of 0.074 Ìg/ml. The mean clearance value was Serum PSA levels 13.8 l/h/m2 (range, 6.3-21.7 l/h/m2) and mean volume of 2 2 >ULN to ≤10 x ULN 2 distribution was 16.2 l/m (range, 7.0-36.5 l/m ). The area >10 x ULN to ≤100 x ULN 5 under the plasma concentration versus time curve averaged >100 x ULN 7 2.10 mg ñ h/l (range, 1.6 – 2.8 mg ñ h/l) and the elimination half-life was 0.90 h (range, 0.41-1.64 h). ULN, upper limit of normal.

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Table II. Hematological toxicitya.

Neutropenia Thrombocytopenia Anemia Course Number of (grade) (grade) (grade) patients 0 1 2 3 4 0 12 34 01234

1 124 2 2 2 2 7 10 22 62130 2 123 1 2 5 1 7 12 11 41412 3 102 1 2 4 1 3 15 01 41131 4 5 1 1 1 0 2 3 00 11 11210 5 1 0 0 0 0 1 0 01 00 00100 aToxicity was graded according to the National Cancer Institute Common Toxicity Criteria.

Toxicity. A total of 40 courses of chemotherapy were mesylate in combination with docetaxel (28) appeared superior analyzed. The hematological toxicity data are given in Table with a median duration of PSA response of 8 months. II. Grade 3-4 neutropenia was predominant, occurring in However, currently, the overall survival period of patients with 45% of the courses. Grade 3-4 thrombocytopenia occurred androgen-independent prostate cancer remains limited. in 22.5% of the courses, while grade 3-4 anemia occurred in The alkylating agents represent a chemically diverse 27.5% of the courses. Overall, three patients required group of compounds, with a broad spectrum of applications hospitalization for the management of treatment-related in cancer treatment. Infusional schedules for these agents side-effects because of febrile neutropenia. Two patients have an increasing application (29). Melphalan is commonly required a platelet and two patients a red blood cell administered orally or intravenously to treat a wide variety transfusion during therapy. Nausea and vomiting occurred of malignancies. Because it is relatively unstable in aqueous in 22.5% of courses (four patients). There were no solution, melphalan is not currently used in continuous treatment-associated deaths. infusion. In a previous study (16), the stability of melphalan was increased by using 3% sodium chloride, allowing for Response and survival. Twelve patients were assessable for continuous infusion administration over a 24-h period. response and survival. Four PR (33.3% of patients) were Melphalan is a short-lived agent (45 min) with a small observed with a median PSA response duration of 12 weeks distribution volume (20 l/m2) and low protein binding (17). (range, 8 to 20 weeks) and time to progression of 14 weeks Thus, this drug appears to be a good candidate for (range, 4 to 28 weeks). For three of these patients, the continuous infusion. In a recent paper, the antitumor administered dose was increased by 20% (one patient) or 40% activity of melphalan was investigated using two schedules (two patients) over the treatment period; they received three of exposure (single-dose versus sequential-dose exposure) in to five courses of chemotherapy. One patient with PR two human cancer cell lines (8226 and A2780) (17). This in received four courses of chemotherapy; the administered dose vitro study indicated that sequential exposure to melphalan was decreased by 20% for the second course then kept stable might be superior to single dose exposure. In the present for the following two courses. Two patients experienced SD study, the same protocol was used to compare the effects of for 12 and 20 weeks, respectively. The actuarial survival time the same dose of melphalan administered either as a single is presented in Figure 2. The median survival of the twelve dose or as sequential doses on an androgen-independent patients was 23 weeks (range, 12-52 weeks). prostate cell line, PC-3. The IC50 value was about 10 times lower for the sequential rather than for single-dose exposure Discussion (0.074 versus 0.77 Ìg/ml). These data indicate that sequential exposure to melphalan was more effective than Many chemotherapeutic agents, administered singly or in single-dose exposure using the same total dose. These combination, have been tested to treat androgen-independent results could be explained by: i) an increase in the prostate cancer (1, 11, 24, 25). Recently, the activity and intracellular concentration of melphalan; ii) the duration of toxicity of docetaxel alone (26, 27) and of imatinib mesylate in drug exposure; or iii) the mechanism of action of combination with docetaxel (28) have been evaluated. A PSA melphalan, which is not cell cycle-specific (17). decline by 50% maintained for 4 weeks was observed in 41% A phase II trial was then undertaken. Its goal was to gain (26) to 46% (27) of cases. The median times to progression preliminary insights into the activity of melphalan were 6.6 months (range, 3.2-18.2 months) and 20 weeks (range, administered over a 24-h period in this disease setting. A 12-87 weeks), respectively. The results obtained with imatinib preliminary phase I trial had shown that the maximum

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Figure 2. Survival times for twelve treated patients. tolerated dose was 30 mg/m2 (18). In the present study, In conclusion, melphalan administered over a 24-h period melphalan was given monthly as a single agent to 14 to patients with androgen-independent prostate cancer patients with advanced prostate cancer. Although the oral appeared to provide some clinical benefits with manageable form, commercially available in several countries, could toxicity. Other studies will allow confirmation of these offer greater flexibility in terms of schedule manipulation results. than infusion and could increase the quality of life, the intravenous form is widely used. Indeed, large inter- Acknowledgements individual variability in was observed following oral administration (20-80%) (15, 30); moreover, The authors gratefully acknowledge the support given by the "Ligue Nationale de Lutte contre le Cancer", Montpellier, France. the bioavailability of melphalan may be affected by food Special thanks are due to B. Hawkins, for his assistance in the intake and co-administered drugs (31, 32). preparation of this manuscript and to F. Malosse for his technical Two patients from this study ceased therapy within the assistance. first 2 weeks due to rapid disease progression. The area under the plasma concentration versus time curve averaged References 2.10 mg ñ h/l (1.6 – 2.8). The toxicity profile did not differ greatly from that reported after a 1-h infusion (33) and was 1 Culine S and Droz JP: Chemotherapy in advanced androgen- acceptable in such a heavily pretreated patient population. independent prostate cancer 1990-1999: a decade of progress? A PSA decline of 50%, maintained for 12 weeks, with a Ann Oncol 11: 1523-1530, 2000. median time to PSA progression of 14 weeks (range, 4 to 28 2 Robson M and Dawson N: How is androgen-dependent metastatic prostate cancer best treated? Hematol Oncol Clin weeks), was observed in 33.3% of cases. For three of these North Am 10: 727-747, 1996. four patients, the administered dose was increased by 20% 3 Goktas S and Crawford ED: Optimal hormonal therapy for (one patient) or 40% (two patients) over the treatment advanced prostatic carcinoma. Semin Oncol 26: 162-173, 1999. period. The median survival of the twelve patients was 23 4 Carter SK and Wasserman TH: The chemotherapy of urologic weeks (range, 12-52 weeks). cancer. Cancer 36: 729-747, 1975.

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J Clin Oncol melphalan using a 24-hour continuous infusion in patients with 11: 1768-1782, 1988. advanced malignancies. Clin Cancer Res 6: 57-63, 2000. 19 Olive PL, Wlodek D and Banath JP: DNA double-strand breaks measured in individual cells subjected to gel electrophoresis. Cancer Res 51: 4671-4676, 1991. 20 Skehan P, Storeng R and Scuderio G: New colorimetric cytotoxicity assay for anticancer-drug screening. J Natl Cancer Inst 82: 1107-1112, 1990. 21 Cockcroft DW and Gault MH: Prediction of creatinine Received January 30, 2006 clearance from serum creatinine. Nephron 16: 31, 1976. Accepted March 10, 2006

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