This Article Abstract Full Text (PDF) Purchase Article View Shopping Cart Alert me when this article is cited Alert me if a correction is posted Services Email this article to a colleague Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Save to my personal folders Download to citation manager Rights & Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by DiPaola, R. S. Articles by Yao, S.-L. Search for Related Content PubMed PubMed Citation Articles by DiPaola, R. S. Articles by Yao, S.-L. Social Bookmarking                            What's this?

Characterization of a Novel Prostate-Specific Antigen–Activated Peptide-Doxorubicin Conjugate in Patients With Prostate Cancer

From the Department of Medicine, University of Medicine and Dentistry of New Jersey/Robert Wood Johnson Medical School, and The Cancer Institute of New Jersey and the Dean and Betty Gallo Prostate Cancer Center, New Brunswick; Merck Research Laboratories, Rahway, NJ; University of Alabama Comprehensive Cancer Center, Birmingham, AL; US Oncology, Inc, Dallas, TX; and Merck Research Laboratories, West Point, PA.

Address reprint requests to Robert S. DiPaola, MD, The Cancer Institute of New Jersey, 195 Little Albany St, New Brunswick, NJ 08901; email: dipaolrs{at}umdnj.edu

	ABSTRACT

TOP ABSTRACT INTRODUCTION PATIENTS AND METHODS RESULTS DISCUSSION REFERENCES

 
PURPOSE: To evaluate safety and pharmacokinetics (PK), and determine the recommended dose for efficacy studies, of L-377202, a novel peptide conjugate of doxorubicin (Dox) that releases the active metabolites leucine-doxorubicin (Leu-Dox) and Dox on cleavage by membrane-bound prostate-specific antigen (PSA).

PATIENTS AND METHODS: Nineteen patients with advanced hormone-refractory prostate cancer were treated intravenously with 71 cycles of L-377202 at escalating dose levels of 20 (n = 1), 40 (n = 3), 80 (n = 4), 160 (n = 3), 225 (n = 6), and 315 mg/m² (n = 2) once every 3 weeks. Toxicity, response, and PK of L-377202 were assessed.

RESULTS: L-377202 was well tolerated. Dose-limiting grade 4 neutropenia was noted in two of two patients administered 315 mg/m² (both patients were able to resume therapy at 225 mg/m²). The recommended dose for efficacy studies was 225 mg/m², which induced grade 4 neutropenia in one of six patients. PK studies demonstrated that L-377202 was metabolized to Leu-Dox and Dox. PK were linear; after administration of single doses of 225 mg/m², the mean area under the concentration-time profiles of L-377202, Leu-Dox, and Dox were 6 µmol·L/h, 4 µmol·L/h, and 1 µmol·L/h, and peak concentrations were 14 µmol/L, 5 µmol/L, and 120 nmol/L, respectively. At 225 and 315 mg/m², five patients completed at least three cycles of therapy; two patients had a greater than 75% decrease in PSA, and one patient had a stabilized PSA. No response was noted at dose levels less than 225 mg/m².

CONCLUSION: This is the first study of selective drug delivery in humans using a novel PSA-activated agent. L-377202 was cleaved to produce detectable levels of the active metabolites Leu-Dox and Dox. L-377202 was well tolerated and established a safe dose level for further study.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION PATIENTS AND METHODS RESULTS DISCUSSION REFERENCES

 
ONE IN SIX MEN IN the United States will be diagnosed with prostate cancer during his lifetime, making prostate cancer the second most common cause of cancer death among men.¹ Chemotherapy is only temporarily effective in patients with hormone-refractory prostate cancer (HRPC).² Although many of these chemotherapeutic agents are highly effective in cell culture, similar dose and delivery to tumor cells in humans is often limited by dose-limiting systemic toxicities. In order to overcome some of these limitations, a strategy was devised to selectively kill prostate cancer cells using traditional cytotoxic chemotherapeutic agents.³ This new strategy, which is dependent on the production of prostate-specific antigen (PSA) by prostate cancer cells, may selectively enhance drug delivery to cancerous cells, thus potentially improving efficacy.³

Secretion of PSA, a specific serine protease, is essentially limited to normal and malignant cells of the prostate.⁴ As a serine protease, PSA is responsible for liquefaction of semen through selective cleavage of semenogelin at specific amino acid sequences.⁵ PSA enzymatic activity is predominantly maintained locally at prostate cells; proteolytic activity in the systemic circulation is limited by the covalent complexes formed with proteins such as alpha₂-macroglobulin and alpha₁-antichymotrypsin.⁶ Thus, PSA enzymatic activity is centered in the microenvironment where PSA is secreted. We developed a novel agent that is dependent on enzymatically functional PSA for activity and may selectively increase drug accumulation in prostatic tissue, thereby resulting in augmented prostate cancer cell-specific cytotoxicity.

L-377202 (Merck Research Laboratories, West Point, PA) is a conjugate consisting of the peptide N-glutaryl-(4-hydroxyprolyl)AlaSer-cyclohexylglycyl-GlnSerLeu-CO₂H covalently linked to the aminoglycoside portion of doxorubicin (Dox); 40% of the mass of L-377202 is attributable to Dox. PSA cleaves L-377202 at the Gln-Ser amide and the action of aminopeptidases then produce the biologically active forms leucine-doxorubicin (Leu-Dox) and Dox. Previous in vitro and in vivo studies demonstrated that L-377202 cleavage is PSA specific and that augmented cytotoxic activity is obtained in the presence of PSA.³ For example, in vitro activity against PSA-producing human prostate cancer cells was at least 20-fold greater than activity against non–PSA-producing prostate cancer cells; the difference approached 100-fold for comparisons with non–PSA-producing normal cells.³ Furthermore, studies in a nude mouse xenograft model demonstrated that L-377202 reduced PSA-producing LNCaP tumor weights by 90% compared with Dox, which reduced tumor weights by only 6%.³ In nude mouse DUPro-1 xenografts that do not produce PSA, both L-377202 and Dox equally reduced tumor weights by 33%. The concentration of doxorubicin in tumor from L-377202 administration was greater than from doxorubicin administration, suggesting that L-377202 could be administered at a convenient patient schedule, similar to clinical studies showing activity of Dox or mitoxantrone every 21 days.³ In conjunction with preclinical data that demonstrated that up to fivefold differences in Dox accumulation were present in tumor tissue compared with normal tissues such as the heart, these promising data prompted us to assess and determine, for the first time, the general safety, tolerability, and maximum-tolerated dose (MTD) of L-377202 in humans.

	PATIENTS AND METHODS

TOP ABSTRACT INTRODUCTION PATIENTS AND METHODS RESULTS DISCUSSION REFERENCES

 
Clinical Protocol and Treatment Plan
Eligible patients all had histologically documented androgen-independent prostate cancer. Additional requirements for participation included an Eastern Cooperative Oncology Group performance status 2, life expectancy greater than 3 months, PSA 20 ng/mL, left ventricular ejection fraction 45%, radiation to 25% of total bone marrow, absolute neutrophil count 1,500/mm³, platelet count 100,000/mm³, creatinine 1.5 mg/dL, and bilirubin 1.5 mg/dL. Patients could have received multiple prior chemotherapeutic regimens provided that total Dox or mitoxantrone doses did not exceed 180 or 40 mg/m², respectively. Antiandrogen or hormonal therapy was permitted provided that no changes were made within 8 weeks of L-377202 administration or during the study. Written informed consent was obtained from all participants and the protocol was approved at the respective institutional review boards of all participating centers.

The initial dose of L-377202 was 20 mg/m², or one tenth of the dose that resulted in the death of 10% of treated mice.⁷ Doses were doubled until one patient experienced an episode of grade 2 toxicity (other than nausea, vomiting, fatigue, alopecia, alkaline phosphatase changes, fever without neutropenia, or local reactions), after which smaller (40%) increments were used. After the first dose level, cohorts of three patients each were planned. Four patients were enrolled at the 80-mg/m² cohort, because two patients were started at this dose simultaneously. The MTD was the dose level at which two of six patients experienced a dose-limiting toxicity (DLT), which was defined as either grade 4 neutropenia, grade 3 nonhematologic toxicity, or irreversible toxicity (National Cancer Institute common toxicity criteria version 1). Patients who received at least two cycles of treatment were eligible to continue treatment if there was evidence of stable or responding disease.

Dose Adjustments
Patients could receive additional cycles of therapy if there was clinical evidence of benefit (stable/decreasing PSA, assessable disease, or pain). For patients failing to recover from drug-related toxicity, subsequent cycles could be delayed by up to 2 weeks. Other adjustments included a 25% dose reduction for grade 4 neutropenia longer than 1 week’s duration or treatment delay longer than 2 weeks. Doses were reduced 50% for bilirubin levels of 1.5 to 3.0 mg/dL and 75% for bilirubin levels of 3.1 to 5.0 mg/dL; greater elevations resulted in discontinuation from the study. In general, 25% dose reductions were instituted for grade 3 drug-related toxicities (eg, grade 3 nausea/vomiting refractory to antiemetics or grade 3 diarrhea) except in cases of allergy and neurotoxicity, where such toxicities would have resulted in discontinuation.

Treatment
L-377202 was supplied as a lyophilized powder in vials to which 20.6 mL of 0.9% sodium chloride for injection was added to produce an 8-mg/mL solution. The reconstituted solution was further diluted into a 100-mL 0.9% sodium chloride solution and then intravenously infused over a 30-minute period once every 3 weeks. Corticosteroid use was prohibited during the study, but prochlorperazine or granisetron could be used as premedications.

Monitoring
Histories, physical examinations, complete blood counts, serum chemistry profiles, prothrombin time/activated partial thromboplastin time, urinalyses, and 12-lead ECGs were obtained serially during each cycle. Both radionuclide angiocardiograms (eg, multigated angiograms) and echocardiograms were obtained at baseline and, initially, after every other cycle of therapy. After patients received cumulative L-377202 doses greater than 400 mg/m², radionuclide angiocardiograms and echocardiograms were obtained after every cycle of treatment. A significant decline in left ventricular ejection fraction was defined as one that declined to less than 45% or had declined by 20 absolute percentage points from baseline. Computed tomography or magnetic resonance imaging scans and bone scans were performed to assess disease status at baseline and after every two courses of treatment. Clinical complete response was defined as the disappearance of all measurable and assessable lesions and partial response was a 50% reduction in the sum of the products of perpendicular diameters of all measurable lesions and the absence of any new lesions; both required two separate assessments separated by at least 4 weeks. An increase of 25% in the sum of the products of perpendicular diameters of all measurable lesions or the appearance of new lesions constituted progressive disease, whereas stable disease was defined as disease that failed to fulfill criteria for a partial response, complete response, or progressive disease. Biochemical (PSA) responses were characterized according to changes from baseline (> 75% decrease or > 50% decrease). PSA assays were performed by Medical Research Laboratories (Highland Heights, KY).

Pharmacokinetics
During the first cycle of treatment, blood samples were obtained at predose and at 0.5, 0.75, 1, 1.5, 2, 2.5, 3.5, 4.5, 6.5, 9.5, 24, and 48 hours after initiation of the L-377202 infusion. Seven-milliliter blood samples, protected from light, were collected in tubes containing 35 mg of EDTA, placed immediately on ice, and centrifuged at 2,200 x g for 10 minutes at 0° to 5°C. The plasma portion was removed and frozen on dry ice or at -70°C. After addition of internal standard, C-8 solid phase extraction using 50-mg, 1-mL columns (Varian, Harbor City, CA) was performed to remove endogenous interfering compounds. Concentrations of L-377202, Leu-Dox, and Dox were determined by high-pressure liquid chromatography using an Agilent 1090 chromatograph (Agilent Technologies, Palo Alto, CA) connected to a fluorescence detector (LC-240; Perkin-Elmer, Norwalk, CT) and a three-part mobile phase composed of ammonium acetate 10 mmol/L (pH 4.5) in 24% acetonitrile (phase A), ammonium acetate 10 mmol/L (pH 5.0) in 31% acetonitrile (phase B), and 90% acetonitrile (phase C). A C-8 reverse-phase column was used for the analysis (5-micron Beta Basic, 3.2 x 100 mm; Keystone, Bellfont, PA) at a flow rate of 1.0 mL/min and temperature of 35°C. Mobile phases A, B, and C were pumped from 0 to 2.5, 2.5 to 7.5, and 7.5 to 8.5 minutes after injection, respectively, and the column was re-equilibrated in phase A from 8.5 to 11.5 minutes. The excitation wavelength was 480 nm and the emission wavelength was 560 nm.

Weighted (1/x) linear regression analysis of peak height ratios of L-377202, Leu-Dox, and Dox was used to generate data for calibration curves and to calculate the sample results. Plasma concentrations were converted to a molar basis using molecular weights of 1,396.5 for L-377202, 657.7 for Leu-Dox, and 543.5 for Dox. The plasma calibration curves were linear over the concentration range of 25 to 2,500 ng/mL (L-377202 17.9 to 1,790 nmol/L and Leu-Dox 38.0 to 3,800 nmol/L) and 12.5 to 1,250 ng/mL (Dox 23.0 to 2,300 nmol/L). Mean accuracy for plasma standards (n = 5) ranged from 86.6% to 105.8%, with intraday coefficients of variation from 2.2% to 6.9%. High-, middle-, and low-concentration plasma quality control samples were prepared before the start of the clinical pharmacokinetics (PK) studies, aliquoted, frozen at -70°C, and assayed along with the clinical samples. Interday coefficients of variation for 30 sets of quality control samples assayed over the course of 24 months were all less than 10%. Mean recoveries across the range of the standard line were 81.3%, 72.7%, 73.7%, and 87.3% for L-377202, Dox, Leu-Dox, and internal standard, respectively. The freeze-thaw stability of L-377202, Leu-Dox, and Dox in control human plasma was demonstrated for three different concentrations through four freeze-thaw cycles. Assay selectivity was evaluated using five different lots of human control plasma as well as plasma samples from patients enrolled onto this study.

For each plasma concentration-time profile, area under the concentration-time profile (AUC) was calculated from 0 to the last quantifiable point by a linear-log trapezoidal method. Peak plasma concentration, and time to peak plasma concentration were determined by inspection. For L-377202, plasma clearance was calculated as dose/AUC and apparent steady-state volume of distribution was calculated as dose x AUMC/AUC², where AUMC is the area under the first moment curve (time-concentration product v time) (Table 1). For doses where there were sufficient data above the assay limit of quantitation, the terminal rate constant for Dox was estimated by unweighted nonlinear regression using a monoexponential decay function. Dox AUC from the last quantifiable point to infinity was extrapolated as the quotient of the last quantifiable concentration and the terminal rate constant. Dox half-life was computed as the quotient of ln² and the terminal rate constant. Terminal rate constants, and therefore half-life, could not be determined for L-377202 and Leu-Dox because the observed declines in plasma concentration were generally not log-linear; more sophisticated modeling could not be applied because of a lack of additional sampling points.

	RESULTS

TOP ABSTRACT INTRODUCTION PATIENTS AND METHODS RESULTS DISCUSSION REFERENCES

Patient PK
L-377202 concentrations peaked at the end of infusion and then declined rapidly (Table 1). Leu-Dox plasma concentrations also peaked at the end of infusion and then declined rapidly, although at a somewhat slower rate (Fig 1). Dox plasma concentrations peaked later and at much lower concentrations but were sustained longer because of its long half-life. At the 225-mg/m² dosing level, the mean AUC of L-377202, Leu-Dox, and Dox were 6 µmol·h/L, 4 µmol·h/L, and 1 µmol·h/L, respectively; peak concentrations were 14 µmol/L, 5 µmol/L, and 120 nmol/L, respectively (Table 5).

View larger version (21K): [in this window] [in a new window]   Fig 1. Pharmacokinetics of L-377202, Leu-Dox, and Dox. The mean plasma concentrations (nM) of L-377202, Leu-Dox, and Dox are shown over 24 hours after administration of L-377202 225 mg/m2 in five patients.

	DISCUSSION

TOP ABSTRACT INTRODUCTION PATIENTS AND METHODS RESULTS DISCUSSION REFERENCES

Preclinical studies demonstrated the feasibility of targeted drug delivery in prostate cancer and selective delivery of Dox to tumor cells compared with non–PSA-producing tumor and normal cells.^3,8 L-377202 had increased cytotoxicity against PSA-producing xenografts in animal models compared with Dox, Leu-Dox, or a noncleavable leader peptide conjugated to Dox.³ L-377202 was less cytotoxic than Dox to cells in culture or xenografts that do not secrete PSA.³ Additionally, after L-377202 administration in nude xenograft mice, increased concentrations of Dox were found in tumor compared with normal tissues such as myocardium. Similar tumor selectivity has been reported by Denmeade et al⁸; a seven-amino-acid peptide coupled to the primary amine of doxorubicin in human prostate cancer cells had increased cytotoxicity in PSA-producing LNCaP cells compared with no cytotoxicity in non–PSA-producing TSU cells.

In the current clinical study, L-377202 was metabolized to the active metabolites Leu-Dox and Dox. Although the differential Dox delivery to PSA-producing tumor cells could not be measured in patients, the finding of a high MTD was encouraging; L-377202 was better tolerated at a dose of 225 mg/m² (equivalent to Dox 90 mg/m² on a molar basis) than expected for Dox alone, on the basis of historical data.^9-12

In preclinical studies, a substantial therapeutic index was demonstrated and molar equivalent doses eight- to nine-fold higher could be administered to xenograft-bearing nude mice without additional toxicity.³ In this human study, we were only able to administer approximately 1.5-fold more doxorubicin on a molar basis compared with conventional doxorubicin. The reasons for this are unclear, as it appears that the PK obtained in humans is not dissimilar to that observed in preclinical studies. Possible explanations for this finding could include tumor heterogeneity in humans compared with preclinical models and differences in tumor distribution (solitary masses are common in xenograft models, whereas humans more often present with disseminated disease, perhaps resulting in more nonspecific exposure). Nonetheless, a 1.3-fold molar increase in the amount of Dox administered in these preclinical models resulted in a 4.9-fold increase in mean PSA percentage reduction from baseline, suggesting that even a modest increase in administered dose can have a substantial impact.³ Larger clinical trials will have to be performed in order to determine if this observation holds true in humans, and to determine whether dosing of targeted therapies should be determined according to tumor burden.

In summary, the present study determined the safety and recommended phase II dose of L-377202 in patients with advanced HRPC. We also found that L-377202 was cleaved to the active metabolites Leu-Dox and Dox. Further studies are necessary to determine whether L-377202 is active against HRPC and if the active metabolites were specifically delivered to tumor compared with normal cells.

	ACKNOWLEDGMENTS

 
Supported by a grant from Merck Research Laboratories, Rahway, NJ.

	REFERENCES

TOP ABSTRACT INTRODUCTION PATIENTS AND METHODS RESULTS DISCUSSION REFERENCES

 
1. Stewart AK, Bland KI, McGinnis LS, et al: Clinical highlights from the National Cancer Data Base, 2000. CA Cancer J Clin 50: 171-183, 2000[Abstract]

2. Roth BJ: Androgen-independent prostate cancer: Not so chemorefractory after all. Semin Oncol 26: 43-50, 1999 (suppl 18)

3. DeFeo-Jones D, Garsky VM, Wong BK, et al: A peptide-doxorubicin prodrug activated by prostate specific antigen selectively kills prostate tumor cells positive for prostate specific antigen in vivo. Nat Med 6: 1248-1252, 2000[CrossRef][Medline]

4. Levesque M, Yu H, D’Costa M, et al: Prostate specific antigen expression by various tumors. J Clin Lab Anal 9: 123-128, 1995[Medline]

5. Akiyama K, Nakamura T, Iwanaza S, et al: The chymotrypsin like activity of human prostate specific antigen, gamma-seminoprotein. FEBS Lett 235: 168-172, 1987

6. Christensson A, Laurell C, Lilja H: Enzymatic activity of PSA and its reactions with extracellular serine proteinase inhibitors. Eur J Biochem 194: 755-763, 1990[Medline]

7. Johansen PB: Doxorubicin pharmacokinetics after intravenous and intraperitoneal administration in the nude mouse. Cancer Chemother Pharmacol 5: 267-270, 1981[CrossRef][Medline]

8. Denmeade SR, Nagy A, Gao J, et al: Enzymatic activation of a doxorubicin-peptide prodrug by PSA. Cancer Res 58: 2537-2540, 1998[Abstract/Free Full Text]

9. Torti FM, Carter SK: The chemotherapy of prostatic adenocarcinoma. Ann Intern Med 92: 681-689, 1980

10. Jain D: Cardiotoxicity of doxorubicin and other anthracycline derivatives. J Nucl Cardiol 7: 53-62, 2000[CrossRef][Medline]

11. Tannock IF, Osoba D, Stockler MR, et al: Chemotherapy with mitoxantrone plus prednisone or prednisone alone for symptomatic hormone-resistant prostate cancer: A Canadian randomized trial with palliative end points. J Clin Oncol 14: 1756-1764, 1996[Abstract/Free Full Text]

12. Kantoff PW, Halabi S, Conaway M, et al: Hydrocortisone with and without mitoxantrone in men with hormone-refractory prostate cancer: Results of the cancer and leukemia group B 9182 study. J Clin Oncol 17: 2506-2513, 1999[Abstract/Free Full Text]

Submitted July 2, 2001; accepted December 14, 2001.

CiteULike    Complore    Connotea    Del.icio.us    Digg    Facebook    Reddit    Technorati    Twitter    What's this?

This article has been cited by other articles:

				C. F. Albright, N. Graciani, W. Han, E. Yue, R. Stein, Z. Lai, M. Diamond, R. Dowling, L. Grimminger, S.-Y. Zhang, et al. Matrix metalloproteinase-activated doxorubicin prodrugs inhibit HT1080 xenograft growth better than doxorubicin with less toxicity Mol. Cancer Ther., May 1, 2005; 4(5): 751 - 760. [Abstract] [Full Text] [PDF]

				G. Minotti, P. Menna, E. Salvatorelli, G. Cairo, and L. Gianni Anthracyclines: Molecular Advances and Pharmacologic Developments in Antitumor Activity and Cardiotoxicity Pharmacol. Rev., June 1, 2004; 56(2): 185 - 229. [Abstract] [Full Text] [PDF]

				C. Pan, P. M. Cardarelli, M. H. Nieder, L. B. Pickford, S. Gangwar, D. J. King, G. T. Yarranton, D. Buckman, W. Roscoe, F. Zhou, et al. CD10 Is a Key Enzyme Involved in the Activation of Tumor-activated Peptide Prodrug CPI-0004Na and Novel Analogues: Implications for the Design of Novel Peptide Prodrugs for the Therapy of CD10+ Tumors Cancer Res., September 1, 2003; 63(17): 5526 - 5531. [Abstract] [Full Text] [PDF]

				S. R. Denmeade, C. M. Jakobsen, S. Janssen, S. R. Khan, E. S. Garrett, H. Lilja, S. B. Christensen, and J. T. Isaacs Prostate-Specific Antigen-Activated Thapsigargin Prodrug as Targeted Therapy for Prostate Cancer J Natl Cancer Inst, July 2, 2003; 95(13): 990 - 1000. [Abstract] [Full Text] [PDF]

				S. Goodin, K. V. Rao, and R. S. DiPaola State-of-the-Art Treatment of Metastatic Hormone-Refractory Prostate Cancer Oncologist, August 1, 2002; 7(4): 360 - 370. [Abstract] [Full Text] [PDF]

This Article Abstract Full Text (PDF) Purchase Article View Shopping Cart Alert me when this article is cited Alert me if a correction is posted Services Email this article to a colleague Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Save to my personal folders Download to citation manager Rights & Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by DiPaola, R. S. Articles by Yao, S.-L. Search for Related Content PubMed PubMed Citation Articles by DiPaola, R. S. Articles by Yao, S.-L. Social Bookmarking                            What's this?