Biotransformation Capacity of Carboxylesterase in Skin and Keratinocytes for the Penta-Ethyl Ester Prodrug of DTPA

Abstract

The penta-ethyl ester prodrug of the chelating agent diethylene triamine pentaacetic acid (DTPA), referred to as C2E5, effectively accelerated clearance of americium after transdermal delivery. Carboxylesterases (CESs) play important roles in facilitating C2E5 hydrolysis. However, whether CESs in human skin hydrolyze C2E5 remains unknown. We evaluated the gene and protein expression of CESs in distinctive human epidermal cell lines: HEKa, HEKn, HaCaT, and A431. The substrates p-nitrophenyl acetate (pNPA) and 4-nitrophenyl valerate (4-NPV) were used to access esterase and CES activity. C2E5 hydrolysis was measured by radiometric high-performance liquid chromatography after incubation of [ 14 C]C2E5 with supernatant fractions after centrifugation at 9000g (S9) prepared from skin cell lines. CESspecific inhibitors were used to access metabolism in human skin S9 fractions with analysis by liquid chromatography-tandem mass spectrometry. We identified the human carboxylesterase 1 and 2 (CES1 and CES2) bands in a Western blot. The gene expression of these enzymes was supported by a real-time polymerase chain reaction (qPCR). pNPA and 4-NPV assays demonstrated esterase and CES activity in all the cell lines that were comparable to human skin S9 fractions. The prodrug C2E5 was hydrolyzed by skin S9 fractions, resulting in a primary metabolite, C2E4. In human skin S9 fractions, inhibition of C2E5 hydrolysis was greatest with a pan-CES inhibitor (benzil). CES1 inhibition (troglitazone) was greater than CES2 (loperamide), suggesting a primary metabolic role for CES1. These results indicate that human keratinocyte cell lines are useful for the evaluation of human cutaneous metabolism and absorption of ester-based prodrugs. However, keratinocytes from skin provide a small contribution to the overall metabolism of C2E5.

Prodrugs for Skin Delivery of Menahydroquinone-4, an Active Form of Vitamin K2(20), Could Overcome the Photoinstability and Phototoxicity of Vitamin K2(20)

“…Our results clearly show that MKH derivatives and MK-4 increase intracellular MKO levels in the keratinocyte cell line we used, indicating that they could be used to deliver MKH into keratinocytes, where it then acts as a cofactor for GGCX. We previously showed that MKH-DMG and MKH-SUC reconvert to MKH in a reaction catalyzed by carboxyl esterase [17], which is present in dermal cells [19,20,21], so accelerated intracellular reconversion to a parent drug is possible. Although UVA-irradiated MK-4 did not increase intracellular MKO, UVA-irradiated MKH derivatives were able to.…”

Enzyme-Instructed Assembly and Disassembly Processes for Targeting Downregulation in Cancer Cells

“…Cell viability tests indicated that 1-OMe-OP potently inhibits the cancer cells that downregulate CES (e.g., OVSAHO) but is innocuous to the cells that upregulate CES (e.g., HepG2), while those two cell lines exhibit comparable phosphatase activities. Control experiments (the addition of esterase inhibitors 20 ) confirmed that the action and expression level of CES are critical for selectively inhibiting the cancer cells. A dicarboxyl methyl ester analogue of 1-OMe-OP validated the generality of the concept.…”

“…To prove further that CES hydrolysis contributes to the low cytotoxicity of 1-OMe-OP against HepG2, we coincubated CES inhibitors and 1-OMe-OP with HepG2 (Figure 3B). The addition of troglitazone (a CES1 inhibitor 20 ) or loperamide (a CES2 inhibitor 22 ) reduced the IC 50 of 1-OMe-OP against HepG2 from 338 μM to 133 μM and 117 μM, respectively. BNPP (an inhibitor of both CES1 and CES2 23 ) lowered the IC 50 of 1-OMe-OP against HepG2 by almost an order of magnitude (from 338 μM to 43 μM).…”

Targeting Transient Receptor Potential Vanilloid 1 (TRPV1) Channel Softly: The Discovery of Passerini Adducts as a Topical Treatment for Inflammatory Skin Disorders

“…The significant difference in biotransformation capacity among the three skin cell lines is ascribable to the different levels of expression and activity between immortalized and primary cell cultures. 29 Furthermore, in order to evaluate the role of human carboxylesterases (hCEs) in the hydrolysis, the four selected compounds were incubated in the presence of recombinant hCE isoforms 1 and 2 (Table 3). Whereas nonspecific esterase activity has been demonstrated in human skin and cultured skin cells, investigations into the expression of specific CEs in skin were controversial in the literature.…”