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.

The effect of a low-protein diet and dietary supplementation of threonine on tyrosine and 2-(2-nitro-4-trifluoromethylbenzoyl) cyclohexane-1,3-dione-induced corneal lesions, the extent of tyrosinemia, and the activity of enzymes involved in tyrosine catabolism in the rat.

Rats fed a low-protein diet and administered 2-(2-nitro-4-trifluoromethylbenzoyl)cyclohexane-1,3-dione (NTBC) orally at 30 mumol/kg/day (10 mg/kg/day) or fed a low-protein diet containing 5 ppm NTBC develop lesions to the cornea of the eye within 3-8 days of exposure with an incidence of about 80%. This treatment also produces a marked inhibition of both hepatic and renal 4-hydroxyphenylpyruvate dioxygenase (HPPD) activity, an induction of hepatic but not renal tyrosine amino transferase activity, and a marked tyrosinemia in the plasma and aqueous humor. The extent of tyrosinemia and changes in the activity of tyrosine catabolic enzymes are similar to those reported for rats fed a normal protein diet and administered NTBC orally at 30 mumol/kg/day. However, the onset of corneal lesions occurs much earlier in rats fed a low-protein diet. The adverse ocular effects of NTBC can be alleviated by supplementing the low-protein diet with 1% w/w threonine. The protection afforded by threonine inclusion in the diet was not due to any amelioration in the extent of inhibition of hepatic HPPD activity or reduction in the extent of the tyrosinemia as measured 8 days after treatment. Rats fed L-tyrosine at 5% w/w in a low-protein diet rapidly develop lesions to the cornea of the eye, which are associated with a marked tyrosinemia, increased hepatic tyrosine aminotransferase activity, and about a 50% reduction in the activity of hepatic HPPD. The onset of corneal lesions produced by feeding a high tyrosine diet could be delayed, but not prevented, by inclusion of 1% w/w threonine in the low-protein diet. The basis for the beneficial effect of dietary supplementation of threonine in alleviating the corneal lesions produced by NTBC is unclear. However, our findings do illustrate that protein deficiency limits the ability of the rat to respond to a tyrosine load produced by inhibition of HPPD.