SLC6A19 inhibition facilitates urinary neutral amino acid excretion and lowers plasma phenylalanine

Abstract

BACKGROUNDThe toxic accumulation of phenylalanine (Phe) in the brain underlies the neurological presentation of phenylketonuria (PKU). Solute carrier family 6 member 19 (SLC6A19) is the major transporter responsible for the (re)absorption of Phe in the kidney and intestine. Here, we describe the characterization of the first small molecule SLC6A19 inhibitor to enter clinical development for the treatment of PKU.METHODSC57Bl/6J WT and Pahenu2 mice were dosed with an inhibitor of SLC6A19 to investigate the effects on urinary amino acids and plasma Phe. In a phase 1 study, healthy human volunteers were dosed with JNT-517, an investigational oral inhibitor of SLC6A19. The primary objective of the study was safety. Secondary objectives included pharmacokinetic and pharmacodynamic studies.RESULTSInhibition of SLC6A19 increased the urinary excretion of Phe in a mouse model of PKU, thereby reducing plasma Phe levels. JNT-517, an investigational oral SLC6A19 inhibitor, was found to be safe and well tolerated and increased the urinary excretion of Phe in a phase 1 healthy volunteer study.CONCLUSIONSThese data indicate that pharmacological inhibition of SLC6A19 presents a promising approach to lower toxic elevated levels of amino acids found in PKU and related amino acid metabolism disorders by facilitating their renal elimination.TRIAL REGISTRATIONAustralian New Zealand Clinical Trials Registry (ANZCTR), ACTRN12622001222730.FUNDINGThe studies in this paper were funded by Jnana Therapeutics.

Keywords: Amino acid metabolism; Clinical trials; Metabolism; Transport.

Figure 1. JNT-517 and JN-170 are potent inhibitors of human and mouse SLC6A19.

(A) Potency of 2 distinct chemical series, series 1 (yellow) and series 2 (purple), against overexpressed human and mouse SLC6A19 in an isoleucine transport assay, expressed as pIC₅₀. (B) Structure of JNT-517. (C) Representative concentration-response curves of JNT-517 in an isoleucine transport assay in Flp-In T-Rex 293 cells cooverexpressing human (yellow) SLC6A19 and TMEM27 or their mouse (purple) orthologs. (D) Concentration-response curve of JNT-517 in a glutamine transport assay in intestinal epithelial cells expressing endogenous SLC6A19 isolated from human postmortem intestine. (E) Structure of JN-170. (F) Representative concentration-response curves of JNT-170 in an isoleucine transport assay in Flp-In T-Rex 293 cells cooverexpressing human (yellow) SLC6A19 and TMEM27 or their mouse (purple) orthologs. Error bars denote SD from 2 (C and F) or 4 (D) technical replicates in a single run. IC₅₀ values are geometric means of n = 4 (C) or 6 (F) experiments. For postmortem epithelial transport, IC₅₀ is the geometric mean of n = 5 experiments from 3 individual donors.

Figure 2. JN-170 causes neutral aminoaciduria in C57Bl/6 mice in a dose-dependent manner.

(A) JN-170 pharmacokinetics, (B) 12 hour urine amino acids after a single oral dose in male C57Bl/6 mice (n = 6). All data represent mean ± SD.

Figure 3. JN-170 lowers plasma Phe in Pah^enu2 mice mimicking classic PKU or hyperphenylalaninemia.

(A) JN-170 pharmacokinetics in male Pah^enu2 mice after a single oral dose (n = 6). (B) Changes in 12 hour urine amino acid excretion in male Pah^enu2 mice treated with JN-170 (n = 4–6 per dose) or vehicle (veh, n = 24) or in mice with heterozygous or homozygous loss of Slc6a19 (data adapted from (14)). Data are displayed as log FC. (C) 12-hour urine Phe excretion in mice treated with vehicle (n = 24) or JN-170 (n = 6); 1-way ANOVA with Tukey’s post hoc analysis. (D) Plasma Phe in male Pah^enu2 mice treated with vehicle (n = 24) or 50, 100, 200, or 250 mg/kg JN-170 (n = 6); 2-way ANOVA with Dunnett’s post hoc analysis to compare vehicle versus JN-170 groups (statistical analysis shown for comparison of vehicle to 250 mg/kg JN-170). (E–G) Plasma Phe in male Pah^enu2 mice fed defined amino acid diets to modulate basal plasma Phe levels. Mice were fed 0.75% Phe (E, n = 17–22), 0.45% Phe (F, n = 4–9) or 0.225% Phe (G, n = 11–17) for 2 weeks prior to administration of a single 200 mg/kg dose of JN-170 or vehicle; 2-way ANOVA with Šidák’s post hoc analysis.**P < 0.01, ***P < 0.001, ****P < 0.0001; All data represent mean ± SD.

Figure 4. CONSORT diagram of patient disposition.

(A) SAD cohort, (B) MAD cohort. D/C discontinued, PBO, placebo control.

Figure 5. JNT-517 shows dose-proportional plasma exposure in single and multiple ascending doses in healthy human volunteers.

(A) Pharmacokinetics of JNT-517 in plasma in SAD cohorts. (B) Day 1 and Day 14 pharmacokinetics of JNT-517 in plasma in MAD cohorts. Values represent mean ± SD for n = 5–6 individuals dosed with active compound per cohort. In vitro IC₅₀ and IC₇₅ values for JNT-517 adjusted for plasma protein binding indicated. BID, twice a day, QD, once daily.

Figure 6. JNT-517 increases urinary neutral amino acid excretion in healthy human volunteers, reproducing the Hartnup aminoaciduria signature caused by genetic loss of SLC6A19 function.

Pharmacodynamics of JNT-517 in SAD (A–C) and MAD (D–G) cohorts. Urine amino acids excreted over various time periods (0–24 hours, 0–8 h, 8-24 hours) are shown. (A) Fold changes of urinary amino acid concentrations (in mmol/mol creatinine) following a single dose of JNT-517, calculated relative to predose baseline in samples collected over 8 hours following compound administration from 10–170 mg. FCs represent average FC for all participants at each dose level. Hartnup amino acid data were adapted from the Utrecht cohort reported by Haijes et al. (11). (B) Total 24 hour Hartnup and non-Hartnup amino acids (in mg) excreted following single ascending doses (10–170 mg) of JNT-517. (C) Total amount of urinary Phe excreted over 24 hours following single ascending doses (10–170 mg) of JNT-517. (D) Total 24 hour Hartnup amino acids excreted on days 1, 7, and 14 in MAD cohorts (dose levels 25 mg BID, 75 mg BID, 150 mg QD). (E–G) Total urinary Phe excreted on days 1, 7, and 14 in multiple ascending dose cohorts 0–24 hours (E), 0–8 hours (F) and 8–24 hours (G) after dose. Hartnup AAs are the sum of Ala, Asn, Gln, Ile, Leu, Phe, Ser, Thr, Tyr, Val. Non-Hartnup AAs are the sum of Arg, Cys, Glu, Gly, Lys, Pro. Values represent mean ± SD for n = 5–6 individuals dosed with placebo or active compound per cohort. BID, twice daily; PBO, placebo; QD, once daily