Selective cancer therapy by extracellular activation of a highly potent glycosidic duocarmycin analogue.

Conventional cancer chemotherapy is limited by systemic toxicity and poor selectivity. Tumor-selective activation of glucuronide prodrugs by beta-glucuronidase in the tumor microenvironment in a monotherapeutic approach is one promising way to increase cancer selectivity. Here we examined the cellular requirement for enzymatic activation as well as the in vivo toxicity and antitumor activity of a glucuronide prodrug of a potent duocarmycin analogue that is active at low picomolar concentrations. Prodrug activation by intracellular and extracellular beta-glucuronidase was investigated by measuring prodrug 2 cytotoxicity against human cancer cell lines that displayed different endogenous levels of beta-glucuronidase, as well as against beta-glucuronidase-deficient fibroblasts and newly established beta-glucuronidase knockdown cancer lines. In all cases, glucuronide prodrug 2 was 1000-5000 times less cytotoxic than the parent duocarmycin analogue regardless of intracellular levels of beta-glucuronidase. By contrast, cancer cells that displayed tethered beta-glucuronidase on their plasma membrane were 80-fold more sensitive to glucuronide prodrug 2, demonstrating that prodrug activation depended primarily on extracellular rather than intracellular beta-glucuronidase activity. Glucuronide prodrug 2 (2.5 mg/kg) displayed greater antitumor activity and less systemic toxicity in vivo than the clinically used drug carboplatin (50 mg/kg) to mice bearing human lung cancer xenografts. Intratumoral injection of an adenoviral vector expressing membrane-tethered beta-glucuronidase dramatically enhanced the in vivo antitumor activity of prodrug 2. Our data provide evidence that increasing extracellular beta-glucuronidase activity in the tumor microenvironment can boost the therapeutic index of a highly potent glucuronide prodrug.

Photoactivatable prodrugs of highly potent duocarmycin analogues for a selective cancer therapy.

A main problem of common cancer chemotherapy is the occurrence of severe side effects caused by insufficient selectivity of the applied drugs. A possible concept to overcome this limitation is light-driven prodrug monotherapy. The synthesis as well as photochemical and biological evaluation of new photoactivatable prodrugs is described. Best results were obtained with prodrug (S,S)-7a. The photochemical labile protecting groups in (S,S)-7a can easily be removed by irradiation with UV-A light in 30 min with a power of only 2 J cm(-2). The determination of the in vitro cytotoxicity by using an HTCFA-test reveals a QIC(50) value of 8200 and the prodrug is more than two million times less cytotoxic than the corresponding seco-drug (-)-(S,S)-5 with an IC(50) value of about 110 fM. The big therapeutic window makes (S,S)-7a very suitable for its use in selective cancer therapy.

Prodrugs for targeted tumor therapies: recent developments in ADEPT, GDEPT and PMT.

The treatment of cancer with common anti-proliferative agents generally suffers from an insufficient differentiation between normal and malignant cells which results in extensive side effects. To enhance the efficacy and reduce the normal tissue toxicity of anticancer drugs, numerous selective tumor therapies have emerged including the highly promising approaches ADEPT (Antibody-Directed Enzyme Prodrug Therapy), GDEPT (Gene-Directed Enzyme Prodrug Therapy) and PMT (Prodrug Monotherapy). These allow a selective release of cytotoxic agents from non-toxic prodrugs at the tumor site either by targeted antibody-enzyme conjugates, enzyme encoding genes or by exploiting physiological and metabolic aberrations in cancerous tissue. Herein, recent developments in the design and biological evaluation of prodrugs for use in ADEPT, GDEPT and PMT are reviewed. As a highlight, a series of novel glycosidic prodrugs based on the natural antibiotics CC-1065 and the duocarmycins will be discussed which show a therapeutic window of up to one million. Notably, the corresponding drugs have tremendously high cytotoxicities with IC(50) values of down to 110 fM.

Synthesis and biological evaluation of prodrugs based on the natural antibiotic duocarmycin for use in ADEPT and PMT.

Chemotherapy of malign tumors is usually associated with serious side effects as common anticancer drugs lack selectivity. An approach to deal with this problem is the antibody-directed enzyme prodrug therapy (ADEPT) and the prodrug monotherapy (PMT). Herein, the synthesis and biological evaluation of new glycosidic prodrugs suitable for both concepts are described. All prodrugs but one are stable in human serum and show QIC(50) values (IC(50) of prodrug/IC(50) of prodrug in the presence of the appropriate glycohydrolase) of up to 6500. This is the best value found so far for compounds interacting with DNA.

Synthesis of the first spacer containing prodrug of a duocarmycin analogue and determination of its biological activity.

The synthesis of the first spacer containing, duocarmycin analogue prodrug was realised, its biological properties evaluated and compared to its counterpart prodrug without a spacer unit. The synthesis comprises the manufacture of the new acetylated derivatives and of two double spacer systems, their activation and coupling to the pharmacophoric seco-drug (+)-. Unprecedented biological results were found as the new prodrug showed a fairly low QIC(50) value of 20, but on the other hand a high stability and very low DNA alkylation efficiency. These findings indicate a changed cytostatic mode of action induced by the self-immolative spacer moiety which was employed.

Duocarmycin-based prodrugs for cancer prodrug monotherapy.

The synthesis and biological evaluation of novel prodrugs based on the cytotoxic antibiotic duocarmycin SA (1) for a selective treatment of cancer using a prodrug monotherapy (PMT) are described. Transformation of the phenol 8 with the glucuronic acid benzyl ester trichloroacetimidate 9b followed by reaction with DMAI x HCl (10) gives the glucuronide 11b, which is deprotected to afford the desired prodrug 4a containing a glucuronic acid moiety. In addition, the prodrug 4b with a glucuronic methyl ester unit is prepared. The cytotoxicity of the glucuronides is determined using a HTCFA-assay with IC(50) values of 610 nM for 4a and 3300 nM for 4b. In the presence of beta-glucuronidase, 4a expresses an IC(50) value of 0.9 nM and 4b of 2.1 nM resulting in QIC(50) values of about 700 for 4a and 1600 for 4b.