Inhibition of para‐Hydroxyphenylpyruvate Dioxygenase by Analogues of the Herbicide Nitisinone As a Strategy to Decrease Homogentisic Acid Levels, the Causative Agent of Alkaptonuria

Abstract

Alkaptonuria (AKU) is a rare multisystem metabolic disease caused by deficient activity of homogentisate 1,2-dioxygenase (HGD), which leads to the accumulation of homogentisic acid (HGA). Currently, there is no treatment for AKU. The sole drug with some beneficial effects is the herbicide nitisinone (1), an inhibitor of p-hydroxyphenylpyruvate dioxygenase (4-HPPD). 1 has been used as a life-saving drug in infants with type I tyrosinemia despite severe side effects due to the buildup of tyrosine. Four clinical trials of nitisinone to treat AKU have shown that 1 consistently decreases HGA levels, but also caused the accumulation of tyrosine in blood serum. Moreover, the human preclinical toxicological data for 1 are incomplete. In this work, we performed pharmacodynamics and toxicological evaluations of 1, providing the first report of LD50 values in human cells. Intracellular tyrosinemia was also evaluated. Three additional 4-HPPD inhibitors with a more favorable profile than that of 1 in terms of IC50, LD50, and tyrosine accumulation were also identified among commercially available compounds. These may be promising starting points for the development of new therapeutic strategies for the treatment of AKU.

Nitisinone: a review

“…13 A long-term LD 50 value lower than the short-term LD 50 was reported to support the hypothesis that the toxicity of nitisinone was linked to its mode of action. 13,[22][23][24][25] Nitisinone and hereditary tyrosinemia type 1…”

“…18 Administration of a single oral dose of [14C]-nitisinone in rats (0.3 µmol/kg [0.1 mg/kg] or 30 µmol/kg [10 mg/kg]) and in mice (30 µmol/kg [10 mg/kg]) led to selective retention of radiolabeled nitisinone in the liver (>90% associated reversibly with the cytosol fraction), in kidneys, and to a lesser extent in the Harderian gland. 13,18 Radioactivity was detected up to 7 days in these organs.…”

“…12 Studies on the rat liver cytosol extracts by Ellis and Laschi revealed an effective time-dependent reversible inhibition of liver HPPD with median inhibitory concentrations (IC 50 ) of 40 and 173 nM and a very slow dissociation of the NTBCenzyme complex. 6,13 A single oral dose of 0.1 mg/kg nitisinone produced complete inhibition of hepatic HPPD within 1 hour of administration and only 40%-50% of the enzyme returned normal 4 days after dosing. Thus, the interaction of nitisinone with HPPD is characterized by a rapid inhibition step to form an enzyme-inhibitor complex that can dissociate slowly with recovery of enzyme activity.…”

“…3 Human fibroblast cultures treated with nitisinone also revealed clearly that Tyr accumulation during administration of nitisinone appears to be dose-dependent. 13 Tyr accumulation in ocular fluids can lead to eye lesions both in human and in some animals. 14,18,22 Administration of a dose of 10 mg/kg/day to mice for 6 weeks increased plasma Tyr level to 650 nmol/L and to 2,300 nmol/L in aqueous humor without any eye lesions.…”

“…14,18,22 The first report of nitisinone lethal dose 50% (LD 50 ) values on human primary fibroblasts following both acute (24 hour) and chronic (144 hour) treatment revealed that the short-term LD 50 was considered to be >1 µM, while long-term LD 50 was about 1 µM. 13 A long-term LD 50 value lower than the short-term LD 50 was reported to support the hypothesis that the toxicity of nitisinone was linked to its mode of action. 13,[22][23][24][25] Nitisinone and hereditary tyrosinemia type 1…”