Elsevier

Bioorganic & Medicinal Chemistry

Volume 7, Issue 9, September 1999, Pages 1775-1780
Bioorganic & Medicinal Chemistry

Melanocyte-directed enzyme prodrug therapy (MDEPT): development of a targeted treatment for malignant melanoma

https://doi.org/10.1016/S0968-0896(99)00126-1Get rights and content

Abstract

A novel prodrug rationally designed to function as a tyrosinase substrate has been synthesised to allow targeted treatment of malignant melanoma. This agent has been evaluated for tyrosinase-mediated drug release, and has been shown to act in the desired manner. Furthermore, differential cytotoxicity has been demonstrated in cell lines which express tyrosinase and those which do not.

Introduction

Disseminated melanoma is a highly metastatic malignancy which is usually fatal. Systemic chemotherapy is often the only recourse,1 but to date the results have been very disappointing and the lack of selective cytotoxicity often leads to intolerable side effects. With the increasing occurrence of this disease, there is a clear and urgent need for improved treatments with enhanced specificity.

Improvements in treatment may be attained by the development of a selective prodrug strategy that can specifically target the malignant cells. In an ideal case, the prodrug would be entirely nontoxic, but would be metabolised to liberate a toxic moiety solely at the desired site of action. In this way, not only can more toxic agents be utilised than would otherwise be possible, but systemic toxicity is also greatly reduced. This approach has already been exploited in cancer chemotherapy in the ADEPT (antibody-directed enzyme/prodrug therapy)2 and VDEPT (virus-directed enzyme/prodrug therapy)3 protocols, but these have some limitations. For example, in both cases, drug liberation and thus selective toxicity is rarely wholly site-specific, as the targeting vector (the antibody or virus) binds, to some extent, nonselectively.

Our approach differs from these earlier protocols in that our activating mechanism relies upon the enzyme tyrosinase, which is already present in melanoma cells. Indeed, the enzyme is uniquely associated with melanocytes,4 suggesting a highly focused drug delivery system. Thus, the attachment of a lethal drug to tyrosine (or a tyrosine analogue) would, we hoped, liberate the free drug at the tumour site, via the mechanism depicted in Scheme 1.

For this activation to occur, our proposed tyrosinase substrates have to fulfil certain requirements. Predominantly, the drug must exhibit a catechol or phenolic moiety, to permit tyrosinase oxidation and thus drug release.5, 6 Secondly, the drug linker must be stable until drug release is required, suggesting the use of a carbamate linkage or other such group.

To evaluate the release properties of such prodrugs in biological systems, we required the incorporation of a known cytotoxic agent into our tyrosinase substrates. Nitrogen mustards have been exploited in a number of previous prodrug investigations,7 and it thus seemed logical to exploit this moiety in our own approach. This led to a target structural motif 2 as shown in Scheme 2.

Section snippets

Synthesis of the prodrug

It was envisaged that our desired prodrug 2 could be formed from the attachment of the tyrosinase substrate to the activated mustard moiety p-nitrophenyl mustard carbonate (PNMC) Scheme 2, Scheme 3, as shown in Scheme 2.

The PNMC was formed using adaptations of literature protocol. Benzyloxyaniline hydrochloride 3 was first neutralised to liberate the free amine, and treatment with ethylene oxide gave facile access to the di-(hydroxyethyl)amino derivative 4.8 Conversion to the

Conclusions

The prodrug 2 was rationally designed to act as a substrate for tyrosinase, to allow selective delivery of a toxic agent to those cell lines expressing tyrosinase. The compound was readily prepared from PNMC Scheme 2, Scheme 3, and its ability to act as a tyrosinase substrate was assessed by GC-MS studies and oxygen consumption in oximetry experiments. Furthermore, cell line screening showed 2 to demonstrate increased cytotoxicity against tyrosinase-upregulated lines, compared with cell lines

Experimental

All NMR spectra were recorded on a Bruker WM250 or Jeol AX400 spectrometer, using CHCl3 as an internal standard unless stated otherwise (7.26 ppm for 1H NMR, 77.0 ppm for 13C NMR). 13C spectra were recorded using Distortionless Enhancement by Polarization Transfer. Mass spectra were recorded on a Fisons VG Autospec. Infra red spectra were recorded on a Perkin–Elmer Paragon 1000 FT-IR spectrometer. Melting points were determined using an Electrothermal digital melting point apparatus, and are

Acknowledgements

We gratefully acknowledge the BBSRC (Post-doctoral fellowship to A.M.J., grant no. 45/SBDO7534) and the Royal Society for their financial support of this work.

References (19)

  • Z. Yang et al.

    Enzyme Microb. Technol.

    (1993)
  • M. Artico et al.

    Biochem. Pharmacol.

    (1968)
  • K.T. Yasunobu et al.

    J. Biol. Chem.

    (1959)
  • D.B. Thomson et al.

    Mel. Res.

    (1993)
  • L.N. Jungheim et al.

    Chem. Rev.

    (1994)
  • B.E. Huber et al.

    Proc. Natl. Acad. Sci. USA

    (1991)
  • J. Pawalek et al.

    Nature

    (1980)
  • S.G. Burton et al.

    Biotechnol. and Bioeng.

    (1993)
  • C.J. Springer et al.

    J. Med. Chem.

    (1995)
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