The Synthesis of a Prodrug of Doxorubicin Designed to Provide Reduced Systemic Toxicity and Greater Target Efficacy

Abstract

Doxorubicin (Dox) can provide some stabilization in prostate cancer; however, its use is limited because of systemic toxicities, primarily cardiotoxicity and immunosuppression. The administration of a prodrug of doxorubicin, designed to permit selective activation by the tumor, would reduce general systemic exposure to the active drug and would thereby increase the therapeutic index. Prostate specific antigen (PSA) is a serine protease with chymotrypsin-like activity that is a member of the kallikrein gene family. PSA's putative physiological role is the liquefaction of semen by virtue of its ability to cleave the seminal fluid proteins semenogelins I and II. Serum PSA levels have been found to correlate well with the number of malignant prostate cells. The use of a prodrug which is cleaved by the enzyme PSA in the prostate should in principle produce high localized concentrations of the cytotoxic agent at the tumor site while limiting systemic exposure to the active drug. Cleavage maps following PSA treatment of human semenogelin were constructed. Systematic modification of the amino acid residues flanking the primary cleavage site led to the synthesis of a series of short peptides which were efficiently hydrolyzed by PSA. Subsequent coupling of selected peptides to doxorubicin provided a series of doxorubicin-peptide conjugates which were evaluated in vitro and in vivo as targeted prodrugs for PSA-secreting tumor cells. From these studies we selected Glutaryl-Hyp-Ala-Ser-Chg-Gln-Ser-Leu-Dox, 27, as the peptide-doxorubicin conjugate with the best profile of physical and biological properties. Compound 27 has a greater than 20-fold selectivity against human prostate PSA-secreting LNCaP cells relative to the non-PSA-secreting DuPRO cell line. In nude mouse xenograft studies, 27 reduced PSA levels by 95% and tumor weight by 87% at a dose below its MTD. Both doxorubicin and Leu-Dox (13) were ineffective in reducing circulating PSA and tumor burden at their maximum tolerated doses. On the basis of these results, we selected 27 for further study to assess its ability to inhibit human prostate cancer cell growth and tumorigenesis.

Detection and quantification of enzymatically active prostate-specific antigenin vivo

“…A series of oligopeptide cytotoxic drug conjugates containing Gln-Ser have shown PSA-targeted pro-drug activities. 8 Thus, similar peptide sequences were prepared for the fluorogenic PSA FAST agents in which both the C and N termini were attached to a lysine molecule modified at the ε-amino group by an NIR fluorophore. The NIR-modified peptides were nonfluorescent due to the proximity of the two fluorophores.…”

“…A nine amino acid oligopeptide substrate based on a known PSAcleavable tetrapeptide sequence, Chg-Gln-Ser-Ser, 8 was purchased as the trifluoroacetic acid salt from Tufts Core Facility, Boston, MA. VivoTag-S 750 was obtained from PerkinElmer, Inc. (Waltham, MA).…”

Hydrazone, Amide, Carbamate, Macromolecular and Other Prodrugs of Doxorubicin

“…Garsky et al [41] reported that DOX had a limited utility in prostate cancer due to systemic toxicities, primarily cardiotoxicity and myelosuppresion. The administration of a prodrug of DOX designed to permit selective activation by the tumor, would reduce general systemic exposure to the DOX.…”

“…Glutaryl-Hyp-Ala-Ser-Chg-Gln-SerLeu-DOX (Fig. 10), as the peptide-DOX conjugate was described as 20-fold selective against human prostate [41]. A peptide prodrug of DOX (Leu-Dox) transformed into DOX in tumor cells by cathepsin-like enzymes and exhibited superior antitumor activity than DOX.…”

Prodrug chemotherapeutics bypass p-glycoprotein resistance and kill tumors in vivo with high efficacy and target-dependent selectivity

“…Several approaches have been developed in order to deliver specifically Doxorubicin to tumor cells using monoclonal antibodies (mAbs) or small peptides as carrier molecules (Arap et al, 1998;Nagy et al, 1998;King et al, 1999;Stan et al, 1999;Garsky et al, 2001;Langer et al, 2001). Targeted chemotherapy couples a drug to a carrier molecule (ligand), and depends on the ability of the ligand to bind selectively to the target tumor cells in such a way that it may increase the drug concentration near the tumor while decreasing systemic toxicity.…”