Chloral hydrate, through biotransformation to dichloroacetate, inhibits maleylacetoacetate isomerase and tyrosine catabolism in humans.

BACKGROUND: Chloral hydrate (CH), a sedative and metabolite of the environmental contaminant trichloroethylene, is metabolized to trichloroacetic acid, trichloroethanol, and possibly dichloroacetate (DCA). DCA is further metabolized by glutathione transferase zeta 1 (GSTZ1), which is identical to maleylacetoacetate isomerase (MAAI), the penultimate enzyme in tyrosine catabolism. DCA inhibits its own metabolism through depletion/inactivation of GSTZ1/MAAI with repeated exposure, resulting in lower plasma clearance of the drug and the accumulation of the urinary biomarker maleylacetone (MA), a metabolite of tyrosine. It is unknown if GSTZ1/MAAI may participate in the metabolism of CH or any of its metabolites and, therefore, affect tyrosine catabolism. Stable isotopes were utilized to determine the biotransformation of CH, the kinetics of its major metabolites, and the influence, if any, of GSTZ1/MAAI. METHODS: Eight healthy volunteers (ages 21-40 years) received a dose of 1 g of CH (clinical dose) or 1.5 µg/kg (environmental) for five consecutive days. Plasma and urinary samples were analyzed by gas chromatography-mass spectrometry. RESULTS: Plasma DCA (1.2-2.4 µg/mL), metabolized from CH, was measured on the fifth day of the 1 g/day CH dosage but was undetectable in plasma at environmentally relevant doses. Pharmacokinetic measurements from CH metabolites did not differ between slow and fast GSTZ1 haplotypes. Urinary MA levels increased from undetectable to 0.2-0.7 µg/g creatinine with repeated CH clinical dose exposure. Kinetic modeling of a clinical dose of 25 mg/kg DCA administered after 5 days of 1 g/day CH closely resembled DCA kinetics obtained in previously naïve individuals. CONCLUSIONS: These data indicate that the amount of DCA produced from clinically relevant doses of CH, although insufficient to alter DCA kinetics, is sufficient to inhibit MAAI and tyrosine catabolism, as evidenced by the accumulation of urinary MA.

Pharmacokinetics of oral dichloroacetate in dogs.

We characterized the pharmacokinetics and dynamics of dichloroacetate (DCA), an investigational drug for mitochondrial diseases, pulmonary arterial hypertension, and cancer. Adult Beagle dogs were orally administered 6.25 mg/kg q12h DCA for 4 weeks. Plasma kinetics was determined after 1, 14, and 28 days. The activity and expression of glutathione transferase zeta 1 (GSTZ1), which biotransforms DCA to glyoxylate, were determined from liver biopsies at baseline and after 27 days. Dogs demonstrate much slower clearance and greater inhibition of DCA metabolism and GSTZ1 activity and expression than rodents and most humans. Indeed, the plasma kinetics of DCA in dogs is similar to humans with GSTZ1 polymorphisms that confer exceptionally slow plasma clearance. Dogs may be a useful model to further investigate the toxicokinetics and therapeutic potential of DCA.

Long-term safety of dichloroacetate in congenital lactic acidosis.

We followed 8 patients (4 males) with biochemically and/or molecular genetically proven deficiencies of the E1α subunit of the pyruvate dehydrogenase complex (PDC; 3 patients) or respiratory chain complexes I (1 patient), IV (3 patients) or I+IV (1 patient) who received oral dichloroacetate (DCA; 12.5 mg/kg/12 h) for 9.7 to 16.5 years. All subjects originally participated in randomized controlled trials of DCA and were continued on an open-label chronic safety study. Patients (1 adult) ranged in age from 3.5 to 40.2 years at the start of DCA administration and are currently aged 16.9 to 49.9 years (mean ± SD: 23.5 ± 10.9 years). Subjects were either normal or below normal body weight for age and gender. The 3 PDC deficient patients did not consume high fat (ketogenic) diets. DCA maintained normal blood lactate concentrations, even in PDC deficient children on essentially unrestricted diets. Hematological, electrolyte, renal and hepatic status remained stable. Nerve conduction either did not change or decreased modestly and led to reduction or temporary discontinuation of DCA in 3 patients, although symptomatic worsening of peripheral neuropathy did not occur. We conclude that chronic DCA administration is generally well-tolerated in patients with congenital causes of lactic acidosis and is effective in maintaining normal blood lactate levels, even in PDC-deficient children not consuming strict ketogenic diets.

Human polymorphisms in the glutathione transferase zeta 1/maleylacetoacetate isomerase gene influence the toxicokinetics of dichloroacetate.

Dichloroacetate (DCA), a chemical relevant to environmental science and allopathic medicine, is dehalogenated by the bifunctional enzyme glutathione transferase zeta (GSTz1)/maleylacetoacetate isomerase (MAAI), the penultimate enzyme in the phenylalanine/tyrosine catabolic pathway. The authors postulated that polymorphisms in GSTz1/MAAI modify the toxicokinetics of DCA. GSTz1/MAAI haplotype significantly affected the kinetics and biotransformation of 1,2-¹³C-DCA when it was administered at either environmentally (µg/kg/d) or clinically (mg/kg/d) relevant doses. GSTz1/MAAI haplotype also influenced the urinary accumulation of potentially toxic tyrosine metabolites. Atomic modeling revealed that GSTz1/MAAI variants associated with the slowest rates of DCA metabolism induced structural changes in the enzyme homodimer, predicting protein instability or abnormal protein-protein interactions. Knowledge of the GSTz1/MAAI haplotype can be used prospectively to identify individuals at potential risk of DCA's adverse side effects from environmental or clinical exposure or who may exhibit aberrant amino acid metabolism in response to dietary protein.

Peripheral neuropathy in rats exposed to dichloroacetate.

The use of dichloroacetate (DCA) for treating patients with mitochondrial diseases is limited by the induction of peripheral neuropathy. The mechanisms of DCA-induced neuropathy are not known. Oral DCA treatment (50-500 mg/kg per day for up to 16 weeks) induced tactile allodynia in both juvenile and adult rats; concurrent thermal hypoalgesia developed at higher doses. Both juvenile and adult rats treated with DCA developed nerve conduction slowing that was more pronounced in adult rats. No overt axonal or glial cell abnormalities were identified in peripheral nerves or spinal cord of any DCA-treated rat, but morphometric analysis identified a reduction of mean axonal caliber of peripheral nerve myelinated fibers. Dichloroacetate treatment also caused accumulation of oxidative stress markers in the nerves. These data indicate that behavioral, functional, and structural indices of peripheral neuropathy may be induced in both juvenile and adult rats treated with DCA at doses similar to those in clinical use. Dichloroacetate-induced peripheral neuropathy primarily afflicts axons and involves both metabolic and structural disorders. The DCA-treated rat may provide insight into the pathogenesis of this peripheral neuropathy and facilitate development of adjuvant therapeutics to prevent this disorder that currently restricts the clinical use of DCA.

The heme precursor delta-aminolevulinate blocks peripheral myelin formation.

Delta-aminolevulinic acid (delta-ALA) is a heme precursor implicated in neurological complications associated with porphyria and tyrosinemia type I. Delta-ALA is also elevated in the urine of animals and patients treated with the investigational drug dichloroacetate (DCA). We postulated that delta-ALA may be responsible, in part, for the peripheral neuropathy observed in subjects receiving DCA. To test this hypothesis, myelinating cocultures of Schwann cells and sensory neurons were exposed to delta-ALA (0.1-1 mM) and analyzed for the expression of neural proteins and lipids and markers of oxidative stress. Exposure of myelinating samples to delta-ALA is associated with a pronounced reduction in the levels of myelin-associated lipids and proteins, including myelin protein zero and peripheral myelin protein 22. We also observed an increase in protein carbonylation and the formation of hydroxynonenal and malondialdehyde after treatment with delta-ALA. Studies of isolated Schwann cells and neurons indicate that glial cells are more vulnerable to this pro-oxidant than neurons, based on a selective decrease in the expression of mitochondrial respiratory chain proteins in glial, but not in neuronal, cells. These results suggest that the neuropathic effects of delta-ALA are attributable, at least in part, to its pro-oxidant properties which damage myelinating Schwann cells.

Age-dependent kinetics and metabolism of dichloroacetate: possible relevance to toxicity.

Dichloroacetate (DCA) is an investigational drug for certain metabolic diseases. It is biotransformed principally by the zeta-1 family isoform of glutathione transferase (GSTz1), also known as maleylacetoacetate isomerase (MAAI), which catalyzes the penultimate step in tyrosine catabolism. DCA causes a reversible peripheral neuropathy in several species, including humans. However, recent clinical trials indicate that adults are considerably more susceptible to this adverse effect than children. We evaluated the kinetics and biotransformation of DCA and its effects on tyrosine metabolism in nine patients treated for 6 months with 25 mg/kg/day and in rats treated for 5 days with 50 mg/kg/day. We also measured the activity and expression of hepatic GSTz1/MAAI. Chronic administration of DCA causes a striking age-dependent decrease in its plasma clearance and an increase in its plasma half-life in patients and rats. Urinary excretion of unchanged DCA in rats increases with age, whereas oxalate, an end product of DCA metabolism, shows the opposite trend. Low concentrations of monochloroacetate (MCA), which is known to be neurotoxic, increase as a function of age in the urine of dosed rats. MCA was detectable in plasma only of older animals. Hepatic GSTz1/MAAI-specific activity was inhibited equally by DCA treatment among all age groups, whereas plasma and urinary levels of maleylacetone, a natural substrate for this enzyme, increased with age. We conclude that age is an important variable in the in vivo metabolism and elimination of DCA and that it may account, in part, for the neurotoxicity of this compound in humans and other species.

Controlled clinical trial of dichloroacetate for treatment of congenital lactic acidosis in children.

OBJECTIVE: Open-label studies indicate that oral dichloroacetate (DCA) may be effective in treating patients with congenital lactic acidosis. We tested this hypothesis by conducting the first double-blind, randomized, control trial of DCA in this disease. METHODS: Forty-three patients who ranged in age from 0.9 to 19 years were enrolled. All patients had persistent or intermittent hyperlactatemia, and most had severe psychomotor delay. Eleven patients had pyruvate dehydrogenase deficiency, 25 patients had 1 or more defects in enzymes of the respiratory chain, and 7 patients had a mutation in mitochondrial DNA. Patients were preconditioned on placebo for 6 months and then were randomly assigned to receive an additional 6 months of placebo or DCA, at a dose of 12.5 mg/kg every 12 hours. The primary outcome results were (1) a Global Assessment of Treatment Efficacy, which incorporated tests of neuromuscular and behavioral function and quality of life; (2) linear growth; (3) blood lactate concentration in the fasted state and after a carbohydrate meal; (4) frequency and severity of intercurrent illnesses and hospitalizations; and (5) safety, including tests of liver and peripheral nerve function. OUTCOME: There were no significant differences in Global Assessment of Treatment Efficacy scores, linear growth, or the frequency or severity of intercurrent illnesses. DCA significantly decreased the rise in blood lactate caused by carbohydrate feeding. Chronic DCA administration was associated with a fall in plasma clearance of the drug and with a rise in the urinary excretion of the tyrosine catabolite maleylacetone and the heme precursor delta-aminolevulinate. CONCLUSIONS: In this highly heterogeneous population of children with congenital lactic acidosis, oral DCA for 6 months was well tolerated and blunted the postprandial increase in circulating lactate. However, it did not improve neurologic or other measures of clinical outcome.

Evaluation of dichloroacetate treatment in a murine model of hereditary tyrosinemia type 1.

Hereditary tyrosinemia type 1 (HT1) is an autosomal recessive disease severely affecting liver and kidney and is caused by a deficiency in fumarylacetoacetate hydrolase (FAH). Administration of 2-(2-nitro-4-trifluoro-methylbenzyol)-1,3 cyclohexanedione (NTBC) improves the HT1 phenotype but some patients do not respond to NTBC therapy. The objective of the present study was to evaluate whether administration of dichloroacetate, an inhibitor of maleyl acetoacetate isomerase (MAAI) to FAH-knockout mice could prevent acute pathological injury caused by NTBC withdrawal. DCA (0.5 and 5g/L) was given in combination with a standard diet or with a tyrosine-restricted diet. With the low-tyrosine diet body weight loss and most of hepatic and renal injuries were prevented regardless the DCA dose. The administration of DCA with a standard diet did not prevent damage nor the oxidative stress response nor the AFP induction seen in FAH-knockout mice. DCA was shown to inhibit hepatic MAAI activity to 86% (0.5g/L) and 94% (5g/L) of untreated wild-type mice. Interestingly, FAH(-/-) mice deprived of NTBC (NTBC-OFF) and NTBC-treated FAH-knockout mice had similar low hepatic MAAI activity levels, corresponding to 10-20% of control. Thus the failure of DCA treatment in FAH(-/-) mice seems to be attributed to the residual MAAI activity, high enough to lead to FAA accumulation and HT1 phenotype.

Inhibition and recovery of rat hepatic glutathione S-transferase zeta and alteration of tyrosine metabolism following dichloroacetate exposure and withdrawal.

Dichloroacetate (DCA) is an investigational drug for certain metabolic disorders, a by-product of water chlorination and a metabolite of certain industrial solvents and drugs. DCA is biotransformed to glyoxylate by glutathione S-transferase zeta (GSTz1-1), which is identical to maleylacetoacetate isomerase, an enzyme of tyrosine catabolism. Clinically relevant doses of DCA (mg/kg/day) decrease the activity and expression of GSTz1-1, which alters tyrosine metabolism and may cause hepatic and neurological toxicity. The effect of environmental DCA doses (microg/kg/day) on tyrosine metabolism and GSTz1-1 is unknown, as is the time course of recovery from perturbation following subchronic DCA administration. Male Sprague-Dawley rats (200 g) were exposed to 0 microg, 2.5 microg, 250 microg, or 50 mg DCA/kg/day in drinking water for up to 12 weeks. Recovery was followed after the 8-week exposure. GSTz specific activity and protein expression (Western immunoblotting) were decreased in a dose-dependent manner by 12 weeks of exposure. Enzyme activity and expression decreased 95% after a 1-week administration of high-dose DCA. Eight weeks after cessation of high-dose DCA, GSTz activity had returned to control levels. At the 2.5 or 250 microg/kg/day doses, enzyme activity also decreased after 8 weeks' exposure and returned to control levels 1 week after DCA was withdrawn. Urinary excretion of the tyrosine catabolite maleylacetone increased from undetectable amounts in control rats to 60 to 75 microg/kg/24 h in animals exposed to 50 mg/kg/day DCA. The liver/body weight ratio increased in the high-dose group after 8 weeks of DCA. These studies demonstrate that short-term administration of DCA inhibits rat liver GSTz across the wide concentration range to which humans are exposed.

Unified gas chromatographic-mass spectrometric method for quantitating tyrosine metabolites in urine and plasma.

Tyrosine and many of its catabolites play significant roles in the in the toxicity associated with acquired and congenital forms of hypertyrosinemia. We now report a specific and sensitive GC/MS method for the simultaneous determination of tyrosine metabolites maleylacetone (MA), fumarylacetone (FA), succinylacetone (SA), fumarate and acetoacetate in urine and plasma. Tyrosine metabolites and an internal standard, 2-oxohexanoic acid (OHA), in urine or plasma samples were derivatized to their methyl esters with a 12% boron trifluoride-methanol complex (12%BF3-MeOH). The reaction mixture was extracted with methylene chloride and analyzed by GC/MS, using a selected ion monitoring (SIM) mode. The detection limits were in the range of 0.08-0.4 ng and the quantitation limits were 0.2-2 ng. Most of the intraday and interday coefficients of variation for three concentrations (low, medium and high) of the analytes were below 10%. Sensitivity and selectivity are superior to existing HPLC or enzymatic methods and derivatization of samples is simpler than the traditional silylation of organic acids used for analysis by GC/MS or derivatization to oximes, followed by silylation in the case of the ketoacids, such as SA. Furthermore, the current procedure can be performed in aqueous solution, which results in a high percentage yield without appreciable analyte degradation or formation of side products. Thus far, the method has been successfully applied in the analysis of over 5000 urine and plasma samples from humans and rodents.

Pharmacologic or genetic ablation of maleylacetoacetate isomerase increases levels of toxic tyrosine catabolites in rodents.

Dichloroacetate (DCA) is both an environmental contaminant and an investigational drug for diseases involving perturbed mitochondrial energetics. DCA is biotransformed to glyoxylate by maleylacetoacetate isomerase (MAAI). Previous studies have shown that DCA decreases MAAI activity in rat liver in a time- and dose-dependent manner and may target the protein for degradation in vivo. We now report that the MAAI protein is depleted in a time- and dose-dependent manner in the livers of Sprague-Dawley rats exposed to DCA. This decrease in protein expression is not mirrored by a decrease in the steady-state levels of MAAI mRNA, indicating that the depletion is exclusively a post-transcriptional event. We also investigated the pharmacokinetics of DCA in the recently developed MAAI knockout (MAAI-KO) mouse. MAAI-KO mice maintain high plasma and urine drug concentrations and do not biotransform DCA to monochloroacetate to a significant extent. Therefore, no alternative pathways for DCA clearance appear to exist in mice other than by MAAI-mediated biotransformation. DCA-nai;ve MAAI-KO mice accumulate very high levels of the tyrosine catabolites maleylacetone and succinylacetone, and DCA exposure did not significantly increase the levels of these compounds. MAAI-KO mice also have high levels of fumarylacetone and normal levels of fumarate. These results demonstrate that pharmacologic or genetic ablation of MAAI cause potentially toxic concentrations of tyrosine intermediates to accumulate in mice and perhaps in other species.

Population kinetics, efficacy, and safety of dichloroacetate for lactic acidosis due to severe malaria in children.

The authors conducted a randomized, double-blind, placebo-controlled trial of intravenous dichloroacetate (DCA) for the purpose of treating lactic acidosis in 124 West African children with severe Plasmodium falciparum malaria. Lactic acidosis independently predicts mortality in severe malaria, and DCA stimulates the oxidative removal of lactate in vivo. A single infusion of 50 mg/kg DCA was well tolerated. When administered at the same time as a dose of intravenous quinine, DCA significantly increased the initial rate and magnitude of fall in blood lactate levels and did not interfere with the plasma kinetics of quinine. The authors developed a novel population pharmacokinetic-pharmacodynamic indirect-response model for DCA that incorporated characteristics associated with disease reversal. The model describes the complex relationships among antimalarial treatment procedures, plasma DCA concentrations, and the drug's lactate-lowering effect. DCA significantly reduces the concentration of blood lactate, an independent predictor of mortality in malaria. Its prospective evaluation in affecting mortality in this disorder appears warranted.