ABSTRACT
N-acetyl-DL-leucine is an analogue of the alpha amino acid leucine with a chiral stereocenter. The active L-enantiomer of the racemate is currently under development for rare neurological disorders. Here we present evidence that a selective recognition of N-acetyl-L-leucine versus L-leucine by different uptake transporters significantly contributes to the therapeutic effects of N-acetyl-L-leucine. A previous study of the pharmacokinetics of racemic N-acetyl-DL-leucine and N-acetyl-L-leucine revealed D-L enantiomer competition and saturation kinetics, best explained by carrier-mediated uptake. The strategy we used was to first analyze the physicochemical properties associated with good oral bioavailable drugs and how these are alerted by N-acetylation by comparing N-acetyl-L-leucine with L-leucine. Using in silico computational chemistry we found that N-acetylation has a profound impact on certain physicochemical properties that can rationalize why N-acetyl-L-leucine is drug-like compared to L-leucine. Our calculations show that at physiological pH, L-leucine is a zwitterion, whereas N-acetyl-L-leucine is present as mainly an anion. Specifically, N-acetylation removes a charge from the nitrogen at physiological pH and N-acetyl-L-leucine is an anion that is then a substrate for the organic anion transporters. We examined N-acetyl-L-leucine uptake in human embryonic kidney cells overexpression candidate organic anion transporters (OAT) and pharmacological inhibitors. We found that N-acetyl-L-leucine is a translocated substrate for OAT1 and OAT3 with low affinity (Km ~10 mM). In contrast, L-leucine is known to be transported by the L-type Amino Acid Transporter (LAT) with high affinity (Km ~0.2 mM) and low capacity. The clinical consequence is that L-leucine uptake becomes saturated at 50-fold lower concentration than N-acetyl-L-leucine. These results demonstrate a mechanism of action that explains why N-acetyl-L-leucine is effective as a drug and L-leucine itself is not.
Competing Interest Statement
GCC is a cofounder, shareholder and consultant to IntraBio. MS is a shareholder to IntraBio, and consultant for Abbott, Actelion, AurisMedical, Heel, IntraBio and Sensorion; he has received speaker's honoraria from Abbott, Actelion, Auris Medical, Biogen, Eisai, Grunenthal, GSK, Henning Pharma, Interacoustics, Johnson & Johnson, MSD, Otometrics, Pierre-Fabre, TEVA, UCB. TBE received honoraria for lecturing from Actelion and Sanofi Genzyme. MF is a co-founder, shareholder, and Chairman of IntraBio. MCP is a shareholder of IntraBio, and has received consulting fees, honoraria and research grants from Actelion Pharmaceuticals Ltd. and Biomarin. FMP is a cofounder, shareholder, and consultant to IntraBio and consultant to Actelion and Orphazyme. AG is a cofounder, shareholder and consultant to IntraBio. IntraBio owns patents EP3359146 and EP3416631 (related to treatment of lysosomal storage disorders and neurodegenerative diseases with acetyl-Leucine and its analogues). IntraBio has pending patent applications EP19174007.5, EP3482754, PCT/US2018/056420, PCT/US2018/018420, PCT/IB2018/054676, PCT/IB2019/051214, PCT/IB2017/054928, PCT/GB2017/051090, PCT/IB2017/054929, USPTO 62/812,987, USPTO 62/842,296, USPTO 62/888,894, USPTO 62/895,144, USPTO 62/868,383, USPTO 62/931,003, USPTO 62/960,637, and PCT/IB2019/060525 relating to treatment of lysosomal storage disorders, neurodegenerative diseases and neurodegeneration with acetyl-leucine and its analogues.