Metabolizing enzyme toxicology assay chip (MetaChip) for high-throughput microscale toxicity analyses

Abstract

The clinical progression of new chemical entities to pharmaceuticals remains hindered by the relatively slow pace of technology development in toxicology and clinical safety evaluation, particularly in vitro approaches, that can be used in the preclinical and early clinical phases of drug development. To alleviate this bottle-neck, we have developed a metabolizing enzyme toxicology assay chip (MetaChip) that combines high-throughput P450 catalysis with cell-based screening on a microscale platform. The MetaChip concept is demonstrated by using sol-gel encapsulated P450s to activate the prodrug cyclophosphamide, which is the major constituent of the anticancer drug Cytoxan, as well as other compounds that are activated by P450 metabolism. The MetaChip provides a high-throughput microscale alternative to currently used in vitro methods for human metabolism and toxicology screening based on liver slices, cultured human hepatocytes, purified microsomal preparations, or isolated and purified P450s. This technology creates opportunities for rapid and inexpensive assessment of ADME/Tox (absorption, distribution, metabolism, excretion/toxicology) at very early phases of drug development, thereby enabling unsuitable candidates to be eliminated from consideration much earlier in the drug discovery process.

Fig. 1.

Schematic of MetaChip platform and microscopic photographs of sol-gel spots. Shown are 30-nl P450 sol-gel spots (A), 30-nl sol-gel spots with 60 nl of prodrug solution after being stamped by MCF7 cell monolayer (B), and MCF7 cell monolayer after removal from sol-gel array and staining (C).

Figure 3

Fig. 2.

Reactivity of CYP3A4 (circles) and CYP2B6 (triangles) in solution (filled symbols) and in sol-gels (open symbols) on CP. Fresh CYP3A4 was added at 6 h, as indicated by the arrow and the dots inside the symbols.

Fig. 3.

Microarray scanning pictures of the MCF7 cell monolayer at 635 nm (Left) and 532 nm (Right) for CP activation. Shown are control with sol-gel spots without a P450 isoform (A) and full system with CYP3A4 (B).

Fig. 4.

Comparison of cytotoxicity results for the MetaChip and solution-phase reactions. (A) Cytotoxicity of P450-activated CP (as represented by the percentage of dead cells) for solution and sol-gel incubations. Control incubations consisted of all system components, except for a P450 isoform. (B) Effect of CP concentration on the cytotoxicity of MCF7 breast cancer cells for: 3A4 solution (•), 3A4 sol-gel (○), 2B6 solution (▾), and 2B6 sol-gel (▿). (C) Effect of Tegafur concentration on the cytotoxicity of MCF7 breast cancer cells for: 1A2 solution (•), 1A2 sol-gel (○), 3A4 solution (▾), and 3A4 sol-gel (▿). (D) Effect of acetaminophen concentration on the cytotoxicity of MCF7 breast cancer cells for: 3A4 solution (•), 3A4 sol-gel (○), 2B6 solution (▾), and 2B6 sol-gel (▿). In B–D, images from the array scanner are presented. In each 6 × 6 array segment, the columns represent different concentrations of spotted compounds (from left to right: 10, 100, 200, 500, 1,000, and 2,000 μM), and the rows represent replicates.