The Amount of Zeaxanthin Epoxidase But Not the Amount of Violaxanthin De-Epoxidase Is a Critical Determinant of Zeaxanthin Accumulation in Arabidopsis thaliana and Nicotiana tabacum.

The generation of violaxanthin (Vx) de-epoxidase (VDE), photosystem II subunit S (PsbS) and zeaxanthin (Zx) epoxidase (ZEP) (VPZ) lines, which simultaneously overexpress VDE, PsbS and ZEP, has been successfully used to accelerate the kinetics of the induction and relaxation of non-photochemical quenching (NPQ). Here, we studied the impact of the overexpression of VDE and ZEP on the conversion of the xanthophyll cycle pigments in VPZ lines of Arabidopsis thaliana and Nicotiana tabacum. The protein amount of both VDE and ZEP was determined to be increased to about 3- to 5-fold levels of wild-type (WT) plants for both species. Compared to WT plants, the conversion of Vx to Zx, and hence VDE activity, was only marginally accelerated in VPZ lines, whereas the conversion of Zx to Vx, and thus ZEP activity, was strongly increased in VPZ lines. This indicates that the amount of ZEP but not the amount of VDE is a critical determinant of the equilibrium of the de-epoxidation state of xanthophyll cycle pigments under saturating light conditions. Comparing the two steps of epoxidation, particularly the second step (antheraxanthin to Vx) was found to be accelerated in VPZ lines, implying that the intermediate Ax is released into the membrane during epoxidation by ZEP.

Dried blood spot analysis of donepezil in support of a GLP 3-month dose-range finding study in rats.

Donepezil hydrochloride is a reversible acetyl cholinesterase inhibitor approved for Alzheimer disease treatment. As an alternate therapy, a donepezil hydrochloride transdermal patch is in development. Recommended nonclinical safety studies include a 3-month Good Laboratory Practice (GLP) dose-range finding (DRF) study prior to conducting the 2-year dermal carcinogenicity study in rats. Demonstration of systemic exposure is necessary to interpret the in vivo data. Previous nonclinical reports supporting oral dosing have utilized liquid chromatography tandem mass spectrometry (LC/MS/MS) to quantify donepezil concentrations in plasma. Smaller species with limited blood volumes do not allow serial sampling to derive the full pharmacokinetic profile from a single animal. Therefore, the option of another analytical method requiring decreased sample volumes is desirable as it would decrease the required number of animals while obtaining the complete profile. The dried blood spot (DBS) technique allows drug level measurement from a few microliters; however, the method is still not widely utilized in GLP studies. Because donepezil plasma levels are known by the oral route, DBS was used to bridge the previous oral data and to support a 13-week GLP DRF study for repeated topical application in rats, comparing oral administration with 4 topical formulations. The DBS method was validated and demonstrated robustness and reproducibility for application to the DRF study. The assay results were comparable to a previously reported plasma LC/MS/MS assay-derived pharmacokinetic profile and provided justification for selection of the topical formulation and dose levels for the subsequent dermal carcinogenicity study.