From toxicological problem to therapeutic use: the discovery of the mode of action of 2-(2-nitro-4-trifluoromethylbenzoyl)-1,3-cyclohexanedione (NTBC), its toxicology and development as a drug.

NTBC is a triketone with herbicidal activity that has been shown to have a novel mode of action by inhibiting the enzyme 4-hydroxyphenylpyruvate dioxygenase in plants. Early studies on the toxicity of this compound found that rats treated with NTBC developed corneal lesions. Investigations aimed at understanding the mechanistic basis for the ocular toxicity discovered that the rats developed tyrosinaemia and excreted large amounts of 4-hydroxyphenylpyruvate and 4-hydroxyphenyllactate, owing to inhibition of the hepatic enzyme 4-hydroxyphenylpyruvate dioxygenase. The corneal lesions resemble those seen when rats are fed a diet supplemented with tyrosine, leading us to conclude that the ocular toxicity seen with NTBC is a consequence of a marked and sustained tyrosinaemia. Studies in collaboration with Professor Sven Lindstedt showed that NTBC was a potent inhibitor of purified human liver 4-hydroxyphenylpyruvate dioxygenase. This interaction lead to the concept of using NTBC to treat patients with tyrosinaemia type 1, to block or reduce the formation of toxic metabolites such as succinylacetoacetate in the liver. Zeneca Agrochemicals and Zeneca Pharmaceuticals made NTBC available for clinical use and, with the approval of the Swedish Medical Products Agency, a seriously ill child with an acute form of tyrosinaemia type 1 was successfully treated in February 1991. Subsequently, other children with this inborn error of metabolism in Sweden and other countries have been treated with NTBC. The drug is now available to those in need via Swedish Orphan AB.

Tissue distribution of 2-(2-nitro-4-trifluoromethylbenzoyl)cyclohexane-1-3-dione (NTBC): effect on enzymes involved in tyrosine catabolism and relevance to ocular toxicity in the rat.

Administration of a single oral dose of 2-(2-nitro-4-trifluoromethylbenzoyl)cyclohexane-1,3-dione (NTBC) to rats produced a marked tyrosinemia in the plasma and aqueous humor. The tyrosinemia was both time- and dose-dependent with the duration being more marked at the higher doses. The dose-response curve was very steep with a single dose of 1.5 micromol NTBC/kg (0.5 mg/kg) and above producing maximal concentrations of tyrosine in plasma of about 2500 nmol/ml and in aqueous humor of about 3500-4000 nmol/ml at 24 hr after dosing. Analysis of the key hepatic enzymes involved in tyrosine catabolism showed that 4-hydroxyphenylpyruvate dioxygenase (HPPD) was markedly inhibited soon after dosing at either 0.3 or 30 micromol/kg (0.1 or 10 mg/kg) NTBC and that the activity recovered very slowly. In response to the tyrosinemia, the activity of tyrosine aminotransferase (TAT) in the liver was induced about twofold, while the activity of homogentisic acid oxidase (HGO) was not affected. Daily oral administration of NTBC for 6 weeks induced lesions to the cornea of the eye, with a dose of 0.3 micromol/kg/day producing about a 38% incidence and a higher dose of 30 micromol/kg/day a 75% incidence. Administration of a single oral dose of [14C]NTBC at either 0.3 or 30 micromol/kg led to selective retention of radiolabel in the liver and to a lesser extent in the kidneys and the Harderian gland. Concentrations of radioactivity in the liver and kidneys remained constant over 4 days and after the lower NTBC dose were about 2 nmol/g wet wt and 0.9 nmol/g wet wt, respectively. Subcellular fractionation of the liver showed that the majority of the radiolabel, >90%, was associated reversibly with the cytosol fraction. No retention of radiolabel was detected in the cornea, the site of toxicity. Our studies indicate that NTBC binds to protein in rat liver cytosol, inhibits the hepatic cytosolic enzyme HPPD, and causes a marked and sustained tyrosinemia. We suggest that this marked and sustained ocular tyrosinemia produced by NTBC in the rat is responsible for the corneal lesions since similar corneal lesions are produced by feeding rats a high tyrosine diet.