2-fluoro-5-maleimidobenzoic acid-linked albumin drug (MAD) delivery for selective systemic targeting of metastatic prostate cancer

Abstract

Background: The SH-group at Cys-34 of human serum albumin (HSA) is a unique and accessible functional group that can be exploited for efficient linkage of a maleimide containing cytotoxic drug derivative to albumin. The specific maleimide chemistry used for production of the maleimide-linked albumin drug (MAD) is critical, however, to minimize the plasma concentration of "free" cytotoxic drug spontaneously released from albumin carrier thus decreasing dose-limiting host toxicity while enhancing the plasma half-life from minutes to days (ie, pharmacokinetic effect) and tissue concentration of the MAD in the extracellular cellular fluid at sites of cancer (ie, EPR effect).

Methods: To accomplish this goal, a chemical synthesis was developed using 2-fluoro-5-maleimidobenzoic acid to stably link the potent cytotoxic chemically modified analogue of the naturally occurring sesquiterpene γ-lactone, thapsigargin, 8-O-(12-aminododecanoyl)-8-O-debutanoyl thapsigargin (12ADT), to Cys-34 of albumin to produce 12ADT-MAD.

Results: Using FITC-labeling, LC/MS analysis, and in vitro growth and clonogenic survival assays on a series of 6 human prostate cancer lines (LNCaP, LAPC-4, VCap, CWR22R_v 1, PC3, and Du145), we documented that 12ADT-MAD is endocytosed by prostate cancer cells where it is degraded into its amino acids liberating cysteinyl-maleimide-12ADT which is both chemically stable at the acidic pH of 5.5 present in the endosome while retaining its high killing ability (IC₅₀ 50 nM) via SERCA inhibition.

Conclusions: Based upon these positive in vitro validation results, the in vivo efficacy versus host toxicity of this 12-ADT-MAD approach is presently being evaluated against a series of patient derived androgen responsive and castration resistant human xenografts in immune-deficient mice.

Keywords: EPR effect; albumin based drug uptake; maleimide linkage; prostate cancer.

FIGURE 1

Molecular structure of human serum albumin (HSA) with an indication of its subdomains (IA, IB, IIA, IIB, IIIA, an IIIB), of the N and C termini, of Sudlow’s sites I and II and of the seven fatty acid binding sites (FA1 to FA7). The side chain residues of Cys-34 are shown as purple spheres.

FIGURE 2

Rationale for chemical instability of N-alkyl linked maleimide-drug coupling to albumin

FIGURE 3

Chemical scheme for synthesizing 2-fluoro-5-maleimidobenzoic acid (compound 3), 2-fluoro-5-maleimidobenzoic acid-12ADT (compound 4), and albumin coupling to produce 12ADT-MAD (compound 5)

FIGURE 4

Chemical scheme for synthesizing cysteinyl-2-fluoro-5-maleimidobenzoic acid-12ADT (compound 6)

FIGURE 5

A, Differential uptake by PC-3 human prostate cancer cells of 500nM FITC-HSA at 4 h vs 500 nM FITC-HSA-maleimide at 1 h. Blue fluorescence is due to DAPI staining of DNA and Green fluorescence is due to fluorescein-HSA-maleimide uptake. B, Dose-Response of LNCaP and PC-3 human prostate cancer cells to 5 day of fluorescein-HSA-maleimide as evaluate percent viability compared to untreated control cells.

FIGURE 6

Kinetics of the dose response toxicity of 12ADT-MAD to LNCaP and PC-3 human prostate cancer cells