Synthesis and biological evaluation of an albumin‐binding prodrug of doxorubicin that is cleaved by prostate‐specific antigen (PSA) in a PSA‐positive orthotopic prostate carcinoma model (LNCaP)

Abstract

The prostate-specific antigen (PSA) is a serine protease that is over-expressed in prostate carcinoma and represents a molecular target for selectively releasing an anticancer agent from a prodrug formulation. We have recently investigated a macromolecular prodrug strategy for improved cancer chemotherapy based on 2 features: (i) rapid and selective binding of thiol-reactive prodrugs to the cysteine-34 position of endogenous albumin after intravenous administration, and (ii) enzymatic release of the albuminbound drug at the tumor site (Mansour et al., Cancer Res 2003, 63, 4062-4066). In this work, we describe an albumin-binding prodrug, EMC-Arg-Ser-Ser-Tyr-Tyr-Ser-Arg-DOXO [EMC: e-Maleimidocaproic acid; DOXO 5 doxorubicin; X 5 amino acid] that is cleaved by PSA. Because of the incorporation of 2 arginine residues, the prodrug exhibited excellent water-solubility and was rapidly and selectively bound to endogenous albumin. Incubation studies with PSA and tumor homogenates from PSA-positive tumors (LNCaP) demonstrated that the albumin-bound form of the prodrug was efficiently cleaved by PSA at the P 1 -P 0 1 scissile bond releasing the doxorubicin dipeptide H-Ser-Arg-DOXO, which was further degraded to doxorubicin as the final cleavage product. In cell culture experiments, the prodrug was 100-fold less active against LNCaP cells than the free drug. In contrast, in a mouse model of human prostate cancer using luciferase transduced LNCaP cells orthotopically implanted in SCID mice, the prodrug showed enhanced antitumor efficacy when compared to doxorubicin. Doxorubicin treatment at a dose of 2 3 4 mg/kg caused significant weight loss and mortality (225%), and did not result in a significant antitumor response at the end of the experiment. The prodrug at 3 3 12 mg/kg doxorubicin equivalents, however, was well tolerated and induced a significant reduction in tumor size of 62% (625%, **p 5 0.003) as well as a decrease of the metastatic burden in the lungs as detected in luciferase assays (250%, SD 6 115%, *p 5 0.038). ' 2007 Wiley-Liss, Inc.Key words: doxorubicin; macromolecular prodrug; human serum albumin; PSA; orthotopic animal model; LNCaP; luciferase; in vivo bioluminescence Hormone refractory prostate cancer responds unfavorably to chemotherapy. The best results to date are achieved with Taxotere. 1 To improve prostate cancer therapy, tumor-specific delivery of anticancer agents to the primary tumor and metastases is a goal worth pursuing.For selectively releasing anticancer agents, the prostate-specific antigen (PSA) is especially attractive as a target protease because it is solely expressed in prostate tissue and prostate carcinoma in prostate cancer patients with high levels up to mg/g present in human prostate carcinoma. 2,3 PSA is a serine protease that belongs to the kallikrein gene family with chymotrypsin-like activity that is involved in the hydrolytic processing of semenogelins (cleavage of the semenal fluid proteins semenogelin I and II), which is required for liquefaction of seminal fluids. 2,3 Over...

In vivo evaluation of a novel albumin-binding prodrug of doxorubicin in an orthotopic mouse model of prostate cancer (LNCaP)

“…9,16 Male SCID (C.B-17/IcrHanHsd-Prkdc-scid) mice were Germany). To test novel antitumor agents in a setting that reflects the human disease better than subcutaneous models, we used our previously designed orthotopic model for prostate cancer using PSA-positive luciferasemarked LNCaP luciferase-neomycin tumor cells orthotopically implanted into the prostates of SCID mice.…”

“…To test novel antitumor agents in a setting that reflects the human disease better than subcutaneous models, we used our previously designed orthotopic model for prostate cancer using PSA-positive luciferasemarked LNCaP luciferase-neomycin tumor cells orthotopically implanted into the prostates of SCID mice. 9 Moreover, to optimize sensitivity, we used high luciferase-expressing LNCaP LLN cell pools, which were generated by transduction using a VSV-G-pseudotyped lentivirus.…”

“…1,2 The reasons why some metastatic sites were more responsive to PSA9 could be that the first two metastatic sites (the lung and lumbar spine) are major targets for metastatic LNCaP cells, and therefore have a relatively high average Figure 3 The antitumor and antimetastatic activity of PSA9 compared with doxorubicin and an untreated control group and the previous data with PSA5 in the orthotopic PSA-positive model (LNCaP). 9 The mean of the control group was set at 100%, and the other values were calculated accordingly. Students' t-test was used for statistical analysis.…”

“…[7][8][9] These prodrugs were developed with the aim of reducing toxicity and improving both the efficacy and the selectivity of chemotherapeutic agents for treating hormone-refractory prostate cancer.…”

“…9 PSA5 was shown to rapidly bind to the circulating albumin and was superior over doxorubicin in an orthotopic PSApositive model (LNCaP) with respect to antitumor efficacy on the primary tumor but showed some but not statistically significant activity only against lung metastases. 9 The major drawback of this prodrug was that although it was cleaved efficiently by PSA, it liberated the less active doxorubicin dipeptide H-SerArg-DOXO as the major cleavage product, which only showed marginal cleavage to H-Arg-DOXO or doxorubicin in tumor homogenates of PSA-positive LNCAP tumors.…”