Flupyrimin: A Novel Insecticide Acting at the Nicotinic Acetylcholine Receptors.

A novel chemotype insecticide flupyrimin (FLP) [N-[(E)-1-(6-chloro-3-pyridinylmethyl)pyridin-2(1H)-ylidene]-2,2,2-trifluoroacetamide], discovered by Meiji Seika Pharma, has unique biological properties, including outstanding potency to imidacloprid (IMI)-resistant rice pests together with superior safety toward pollinators. Intriguingly, FLP acts as a nicotinic antagonist in American cockroach neurons, and [3H]FLP binds to the multiple high-affinity binding components in house fly nicotinic acetylcholine (ACh) receptor (nAChR) preparation. One of the [3H]FLP receptors is identical to the IMI receptor, and the alternative is IMI-insensitive subtype. Furthermore, FLP is favorably safe to rats as predicted by the very low affinity to the rat α4ß2 nAChR. Structure-activity relationships of FLP analogues in terms of receptor potency, featuring the pyridinylidene and trifluoroacetyl pharmacophores, were examined, thereby establishing the FLP molecular recognition at the Aplysia californica ACh-binding protein, a suitable structural surrogate of the insect nAChR. These FLP pharmacophores account for the excellent receptor affinity, accordingly revealing differences in its binding mechanism from IMI.

Quantitative prediction of oral absorption of PEPT1 substrates based on in vitro uptake into Caco-2 cells.

The method for predicting the fraction absorbed (Fa) of the PEPT1 substrates was established based on the in vitro uptake into Caco-2 cells. Uptake of a drug into Caco-2 cells was measured, and the carrier-mediated initial uptake clearance (DeltaCL uptake) was calculated as the difference between the uptake clearance in the absence of glycyl-sarcosine (Gly-Sar) and that in the presence of 30 mM Gly-Sar. The DeltaCL uptake of each drug was then divided by that of cephradine to obtain DeltaCL*uptake, which was a normalized parameter to correct for inter-day and/or inter-cell variability. Then, cephradine (CED), cefixime (CFIX), and cefotiam (CTM) were selected as marker compounds having excellent, medium and poor absorption, respectively. The DeltaCL*uptake and Fa values for CED, CFIX and CTM were fitted to the equation derived from the complete radial mixing (CRM) model, and the scaling factor (A') was obtained. Using the A' value, Fa was predicted from the DeltaCL*uptake value of each drug. Good correlation was observed between the predicted and reported Fa values, which demonstrated that Fa of PEPT1 substrates can be predicted based on the in vitro uptake in Caco-2 cells.

Stereoselectivity of the reduced folate carrier in Caco-2 cells.

Stereoselectivity of the human reduced folate carrier (RFC1) was examined in Caco-2 cells using methotrexate (l-amethopterin or l-MTX) and its antipode (d-amethopterin or d-MTX) as model substrates. The initial uptake rate of folic acid (FA) was concentration dependent, with a K(m) value of approximately 0.6 microM. The Eadie-Hofstee plot of the RFC1-mediated FA uptake revealed a single component for FA uptake into Caco-2 cells, demonstrating that only RFC1 is involved in FA uptake. l-MTX inhibited FA uptake in a competitive manner with a K(i) value of approximately 2 microM, similar to the K(m) value of l-MTX. d-MTX also competitively inhibited FA uptake with a K(i) value being approximately 120 microM, indicating that the affinity of d-MTX is ca. 60-fold less than that of l-MTX. The stereoselectivity of human RFC1 observed in the present study was consistent not only with the stereoselectivity of rabbit RFC1 observed in rabbit intestinal brush border membrane vesicles but also with the reported differences in oral absorption of amethopterin enantiomers in humans.