New insights into tetrahydrobiopterin pharmacodynamics from Pahenu1/2, a mouse model for compound heterozygous tetrahydrobiopterin-responsive phenylalanine hydroxylase deficiency

Abstract

Phenylketonuria (PKU), an autosomal recessive disease with phenylalanine hydroxylase (PAH) deficiency, was recently shown to be a protein misfolding disease with loss-of-function. It can be treated by oral application of the natural PAH cofactor tetrahydrobiopterin (BH(4)) that acts as a pharmacological chaperone and rescues enzyme function in vivo. Here we identified Pah(enu1/2) bearing a mild and a severe mutation (V106A/F363S) as a new mouse model for compound heterozygous mild PKU. Although BH(4) treatment has become established in clinical routine, there is substantial lack of knowledge with regard to BH(4) pharmacodynamics and the effect of the genotype on the response to treatment with the natural cofactor. To address these questions we applied an elaborate methodological setup analyzing: (i) blood phenylalanine elimination, (ii) blood phenylalanine/tyrosine ratios, and (iii) kinetics of in vivo phenylalanine oxidation using (13)C-phenylalanine breath tests. We compared pharmacodynamics in wild-type, Pah(enu1/1), and Pah(enu1/2) mice and observed crucial differences in terms of effect size as well as effect kinetics and dose response. Results from in vivo experiments were substantiated in vitro after overexpression of wild-type, V106A, and F263S in COS-7 cells. Pharmacokinetics did not differ between Pah(enu1/1) and Pah(enu1/2) indicating that the differences in pharmacodynamics were not induced by divergent pharmacokinetic behavior of BH(4). In conclusion, our findings show a significant impact of the genotype on the response to BH(4) in PAH deficient mice. This may lead to important consequences concerning the diagnostic and therapeutic management of patients with PAH deficiency underscoring the need for individualized procedures addressing pharmacodynamic aspects.

The mechanism of BH4-responsive hyperphenylalaninemia-As it occurs in the ENU1/2 genetic mouse model

“…1C). The values of Phe in blood and PAH protein content and activity compare well to those previously reported for this animal model [Lagler et al, 2010; Sarkissian et al, 2000]. Meanwhile, a novel finding of our ENU1/2 mouse investigation is that the PAH protein content and activity increase about 1.6‐fold upon l ‐Phe challenge, while no such effect was measured for the wild‐type controls (Supp.…”

Early prediction of phenotypic severity in Citrullinemia Type 1

“…This observation might owe to the overexpression strategy of equimolar transfection of both (homozygous/compound heterozygous) ASS1 expression vectors, which as a consequence most likely leads to an equimolar expression of the (mutated) proteins (encoded by both alleles), mimicking the endogenous situation in individuals with CTLN1. The utilized COS‐7 cell line has been used as overexpression system for the investigation of protein (dys‐) functions for more than three decades including the quantification of residual activities of enzymes of intermediary metabolism . Coupled enzyme assays have been successfully established and are widely used for the reliable determination of activities of glycolytic or TCA cycle enzymes as well as respiratory chain complexes, which allow the real‐time quantification of activity rates by exploiting redox‐reactions of redox‐active coenzymes such as NAD or FAD .…”

Up to date knowledge on different treatment strategies for phenylketonuria

“…No adverse-effects have been reported at these higher doses, but also little additional clinical benefit. It must be noted that animal studies revealed that compound heterozygous mice carrying both a mild and a severe mutation in the PAH gene, Pah enu1/2 , show an inhibitory effect at doses above 20 mg/kg/day, whereas this was not true in mice carrying a mild mutation in the homozygous state, Pah enu1/1 [61]. …”

“…Most patients are treated with single daily dosing, but dividing the total amount in 2–3 daily doses has been suggested to be beneficial for some patients in order to avoid daily Phe level fluctuations [57]. Again, this idea is supported by animal studies, where the in vivo effect of BH4 was significantly shorter in the compound heterozygous mouse Pah enu1/2 than in the homozygous mild phenotype Pah enu1/1 [61]. Taking the tablets with food, rather than fasting, has also been proven beneficial [62].…”