Non-physiological amino acid (NPAA) therapy targeting brain phenylalanine reduction: pilot studies in PAHENU2 mice

Abstract

Transport of large neutral amino acids (LNAA) across the blood brain barrier (BBB) is facilitated by the L-type amino acid transporter, LAT1. Peripheral accumulation of one LNAA (e.g., phenylalanine (phe) in PKU) is predicted to increase uptake of the offending amino acid to the detriment of others, resulting in disruption of brain amino acid homeostasis. We hypothesized that selected non-physiological amino acids (NPAAs) such as DL-norleucine (NL), 2-aminonorbornane (NB; 2-aminobicyclo-(2,1,1)-heptane-2-carboxylic acid), 2-aminoisobutyrate (AIB), and N-methyl-aminoisobutyrate (MAIB), acting as competitive inhibitors of various brain amino acid transporters, could reduce brain phe in Pah (enu2) mice, a relevant murine model of PKU. Oral feeding of 5 % NL, 5 % AIB, 0.5 % NB and 3 % MAIB reduced brain phe by 56 % (p < 0.01), -1 % (p = NS), 27 % (p < 0.05) and 14 % (p < 0.01), respectively, compared to untreated subjects. Significant effects on other LNAAs (tyrosine, methionine, branched chain amino acids) were also observed, however, with MAIB displaying the mildest effects. Of interest, MAIB represents an inhibitor of the system A (alanine) transporter that primarily traffics small amino acids and not LNAAs. Our studies represent the first in vivo use of these NPAAs in Pah (enu2) mice, and provide proof-of-principle for their further preclinical development, with the long-term objective of identifying NPAA combinations and concentrations that selectively restrict brain phe transport while minimally impacting other LNAAs and downstream intermediates.

Fig. 1

NPAAs evaluated in the current study; A, norleucine (NL); B, 2-aminoisobutyric acid (AIB); C, N-methyl-2-aminoisobutyric acid (MAIB); D, 2-aminonorbornane (NB). MAIB is a selective competitive inhibitor of the system A transport system (see Fig. 2). Structures were obtained from web resources (www.wikipedia.com; www.sigma-aldrich.com).

Fig. 2

Schematic diagram of amino acid transport systems at the blood brain barrier. The sodium dependence of each system is indicated. The putative location for NPAA inhibition is depicted in the center of the diagram. Transport systems are enclosed in rectangles, and transported amino acids are presented in single letter format. Note that the LNAA transporter represents the general L (LAT) system, having overlap with the L1 system. Figure adapted from Broer and Brookes (2001).

Fig. 3

Schematic of dopamine (DA) and serotonin (5-hydroxytryptamine; 5-HT) synthesis and metabolism. Abbreviations: Tyr, tyrosine; Trp, tryptophan; L-DOPA, L-dihydroxyphenylalanine; 5-HTP, 5-hydroxytryptophan; DOPAC, 3,4-dihydroxyphenylacetic acid; HVA, homovanillic acid; 3-MT, 3-methoxytyramine (a DA metabolite released into the synaptic cleft); 5-HIAA, 5-hydroxyindoleacetic acid.

Fig. 4

Selected brain LNAAs amino acids as a function of diet and genotype (T, heterozygous Pah^enu2 mice; M, Pah^enu2 mice; trp levels not shown). Amino acid levels shown as nmol/gram (gr) wet weight tissue. Parenthetical values represent the number of mice studied. Total branched chain amino acids (BCAA) represent the sum of ile, val and leu. Statistical analysis (one-way ANOVA with Tukey post-hoc) compared cohorts with and without NPAAs, and within genotype only (*p<0.05 compared to control, no drug intervention).

Fig. 5

Selected brain LNAAs as a function of dietary intervention with 3% MAIB. For abbreviations, see Fig. 4 legend. Statistical analysis employed a two-way t test, comparing dietary intervention only within the same genotype.

Fig. 6

S-adenosylmethionine (SAMe) and S-adenosylhomocysteine (SAH) levels as a function of dietary intervention. For abbreviations, see Fig. 4 legend. Statistical analysis (one way ANOVA with Tukey post-hoc) compared cohorts with and without NPAAs (for AIB, NL and NB), and within genotype only (*p<0.05 compared to control). Statistical analysis for MAIB intervention employed a two-way t test, comparing dietary intervention only within genotype.

Fig. 7

Monoamine neurotransmitters and metabolites (see Fig. 3) in brain extracts of Pah^enu2 and heterozygous Pah^enu2 mice as a function of diet. Statistical analyses and abbreviations as described in legends to Figs. 3 and 4. Not shown are DOPAC, HVA, and the ratio of 3-MT/DA (dopamine release), which showed no significant differences for either genotype with any diet, with the exception of MAIB (see Fig. 8).

Fig. 8

Monoamine neurotransmitters and associated metabolites in brain extract of Pah^enu2 and heterozygous Pah^enu2 mice as a function of 3% MAIB feeding. Abbreviations and statistical analyses as described in Figs. 3 and 5.