Birgit Krewer

Antibody‐Directed Enzyme Prodrug Therapy: A Promising Approach for a Selective Treatment of Cancer Based on Prodrugs and Monoclonal Antibodies

The antibody‐directed enzyme prodrug therapy allows a selective liberation of cytotoxic agents from non‐toxic prodrugs in cancerous tissue by targeted antibody–enzyme conjugates. We have developed a series of novel glycosidic prodrugs based on the natural antibiotic CC‐1065 and the duocarmycins, which are up to 4800 times less toxic than the drugs liberated from these prodrugs in the presence of the activating enzyme (e.g., β‐d‐galactosidase). Furthermore, the drugs show very high cytotoxicities with IC50 values of as low as 4.5 pm. In this report, we summarize our recent results on the development and biological evaluation of these novel third‐generation prodrugs with higher water solubility, higher difference in cytotoxicity between the prodrugs and the corresponding drugs and improved cytotoxicity of the drugs as compared with previous compounds.

Synthesis and biological studies of different duocarmycin based glycosidic prodrugs for their use in the antibody-directed enzyme prodrug therapy.

The synthesis and biological evaluation of novel prodrugs for use in the antibody directed enzyme prodrug therapy (ADEPT) of cancer based on the cytotoxic antibiotic duocarmycin SA (1) are described. In this approach, we investigated the influence of the sugar moiety of the glycosidic prodrug on the QIC(50) values as well as on the stability and the water solubility. The best result was found for prodrug 22 containing an alpha-mannoside moiety with a QIC(50) value of 4500.

Asymmetric synthesis and biological evaluation of glycosidic prodrugs for a selective cancer therapy.

A severe limitation in cancer therapy is the often insufficient differentiation between malign and benign tissue using known chemotherapeutics. One approach to decrease side effects is antibody‐directed enzyme prodrug therapy (ADEPT). We have developed new glycosidic prodrugs such as (−)‐(1S)‐26 b based on the antibiotic (+)‐duocarmycin SA ((+)‐1) with a QIC50 value of 3500 (QIC50=IC50 of prodrug/IC50 of prodrug+enzyme) and an IC50 value for the corresponding drug (prodrug+enzyme) of 16 pM. The asymmetric synthesis of the precursor (−)‐(1S)‐19 was performed by arylation of the enantiomerically pure epoxide (+)‐(S)‐29 (≥98 % ee).

Novel analogues of CC-1065 and the duocarmycins for the use in targeted tumour therapies.

In recent years, a series of new and highly cytotoxic analogues of CC-1065 and the duocarmycins have been developed that can be transformed into much less toxic prodrugs for the use in antibody-directed enzyme prodrug therapy (ADEPT), gene-directed enzyme prodrug therapy (GDEPT) and prodrug monotherapy (PMT) of cancer. In all these approaches, a relatively non-toxic prodrug is applied and subsequently converted selectively in the tumour tissue into a highly cytotoxic drug, thus reducing undesired side effects accompanying conventional chemotherapy. Here, the design and biological evaluation of prodrugs based on analogues of CC-1065 and the duocarmycins for the use in tumour selective cancer therapies is reviewed. The advantage of this approach is the excellent therapeutic index of some of the new prodrugs of over 5000 and the high cytotoxicity of the corresponding drugs with IC(50) values of as low as 16 pM (IC(50): concentration required for 50 % growth inhibition of target cells). In addition, a novel method for the correlation of the alkylation efficiency and the cytotoxicity based on mass spectrometry is described.

Enhanced tumor therapy using vaccinia virus strain GLV-1h68 in combination with a β-galactosidase-activatable prodrug seco -analog of duocarmycin SA

Breast cancer is the most common cause of cancer-related death worldwide, thus remaining a crucial health problem among women despite advances in conventional therapy. Therefore, new alternative strategies are needed for effective diagnosis and treatment. One approach is the use of oncolytic viruses for gene-directed enzyme prodrug therapy. Here, the lacZ-carrying vaccinia virus (VACV) strain GLV-1h68 was used in combination with a β-galactosidase-activatable prodrug derived from a seco-analog of the natural antibiotic duocarmycin SA. Tumor cell infection with the VACV strain GLV-1h68 led to production of β-galactosidase, essential for the conversion of the prodrug to the toxic compound. Furthermore, drug-dependent cell kill and induction of the intrinsic apoptosis pathway in tumor cells was also observed on combination therapy using the prodrug and the GLV-1h68 strain, despite the fact that VACV strains encode antiapoptotic proteins. Moreover, GI-101A breast cancer xenografts were effectively treated by the combination therapy. In conclusion, the combination of a β-galactosidase-activatable prodrug with a tumor-specific vaccinica virus strain encoding this enzyme, induced apoptosis in cultures of the human GI-101A breast cancer cells, in which a synergistic oncolytic effect was observed. Moreover, in vivo, additional prodrug treatment had beneficial effects on tumor regression in GLV-1h68-treated GI-101A-xenografted mice.

Determination of the Biological Activity and Structure Activity Relationships of Drugs Based on the Highly Cytotoxic Duocarmycins and CC-1065

The natural antibiotics CC‑1065 and the duocarmycins are highly cytotoxic compounds which however are not suitable for cancer therapy due to their general toxicity. We have developed glycosidic prodrugs of seco-analogues of these antibiotics for a selective cancer therapy using conjugates of glycohydrolases and tumour-selective monoclonal antibodies for the liberation of the drugs from the prodrugs predominantly at the tumour site. For the determination of structure activity relationships of the different seco-drugs, experiments addressing their interaction with synthetic DNA were performed. Using electrospray mass spectrometry and high performance liquid chromatography, the experiments revealed a correlation of the stability of these drugs with their cytotoxicity in cell culture investigations. Furthermore, it was shown that the drugs bind to AT-rich regions of double-stranded DNA and the more cytotoxic drugs induce DNA fragmentation at room temperature in several of the selected DNA double-strands. Finally, an explanation for the very high cytotoxicity of CC-1065, the duocarmycins and analogous drugs is given.

CD-Spectroscopy As a Powerful Tool for Investigating the Mode of Action of Unmodified Drugs in Live Cells

Circular dichroism (CD) spectroscopy is a well-known method for the analysis of chiral chemical compounds and is often used for studying the structure and interaction of proteins, DNA and bioactive compounds in solution. Here we demonstrate that CD spectroscopy is also a powerful tool for investigating the cellular uptake and mode of action of drugs in live cells. By means of CD spectroscopy, we identified DNA as the cellular target of several novel anticancer agents based on the highly cytotoxic natural antibiotic CC-1065. Furthermore, time-dependent changes in the CD spectra of drug-treated cells enabled us to rationalize differences in drug cytotoxicity. The anticancer agents rapidly penetrate the cell membrane and bind to cellular DNA as their intracellular target. Thereby, the formation of a reversible noncovalent complex with the DNA is followed by a covalent binding of the drugs to the DNA and the more toxic compounds show a higher stability and a lower alkylation rate. Since no drug manipulation is necessary for this kind of investigation and achiral compounds bound to chiral biomolecules may also show induced CD signals, CD spectroscopy of live cells is not limited to the study of analogues of CC-1065. Thus, it constitutes a general approach for studying the mode of action of bioactive compounds on the cellular and molecular level.

Synthesis of a Novel Pentagastrin‐Drug Conjugate for a Targeted Tumor Therapy

The synthesis of the novel pentagastrin seco-CBI conjugate 3, which is based on the highly cytotoxic antitumor antibiotic (+)-duocarmycin SA (1), is reported. A key step in the synthesis is the palladium-catalyzed carbonylation of aryl bromide 7 to give the benzyl ester 16, which is transformed into the new seco-CBI derivative 21 bearing a carboxylic acid ester moiety. Subsequent transformation of 21 into an activated ester followed by the introduction of beta-alanine and tetragastrin led to the new pentagastrin drug 3 that contains a peptide moiety for targeting cancer cells expressing CCK-B/gastrin receptors.

Investigation of the transformations of a novel anti-cancer agent combining HPLC, HPLC–MS and direct ESI–HRMS analyses

AbstractOne of the main problems of anti-cancer therapy is an insufficient differentiation between normal and malignant cells by the known anti-proliferant agents. The antibody-directed enzyme prodrug therapy is a promising approach for a selective treatment of cancer, in which a non-toxic prodrug is enzymatically converted into a highly cytotoxic drug at the surface of malignant cells by a targeted antibody–enzyme conjugate. The transformations and the stability of a very promising novel prodrug and its corresponding cytotoxic derivative were now investigated in detail by high-performance liquid chromatography (HPLC)–mass spectrometry (MS). In order to determine the time-dependent DNA alkylation efficiency and the sequence selectivity of the novel compounds, DNA binding studies using direct electrospray–Fourier transform ion cyclotron resonance–MS (ESI–FTICR–MS) have been performed. These measurements were accompanied by HPLC analyses followed by MS of the separated species to confirm the results of the direct ESI–FTICR–MS measurements. The sites of DNA alkylation could be identified unambiguously by the mass spectrometric fragmentation pattern of the alkylated oligodeoxynucleotides as well as by the results of HPLC followed by MS. A combination of all techniques applied led to a better understanding of the mode of action of the new therapeutics and might be used for an estimation of the cytotoxicity of different prodrugs and drugs since the alkylation efficiency correlates with the bioactivity of the compounds in cell culture investigations. After enzymatic cleavage of the sugar moiety, the untoxic prodrug is converted rapidly into the corresponding highly cytotoxic drug that alkylates DNA with high efficiency

Synthesis and Biological Evaluation of a Novel Pentagastrin- Toxin Conjugate Designed for a Targeted Prodrug Mono- therapy of Cancer

A novel carbamate prodrug 2 containing a pentagastrin moiety was synthesized. 2 was designed as a detoxified analogue of the highly cytotoxic natural antibiotic duocarmycin SA (1) for the use in a targeted prodrug monotherapy of cancers expressing cholecystokinin (CCK-B)/gastrin receptors. The synthesis of prodrug 2 was performed using a palladium-catalyzed carbonylation of bromide 6, followed by a radical cyclisation to give the pharmacophoric unit 10, coupling of 10 to the DNA-binding subunit 15 and transformation of the resulting seco-drug 3b into the carbamate 2 via addition of a pentagastrin moiety.