Daily variation of NTBC and its relation to succinylacetone in tyrosinemia type 1 patients comparing a single dose to two doses a day

Hereditary tyrosinemia type 1 (HT1; McKusick 276,700) is a rare, autosomal recessive metabolic disorder caused by a deficiency of fumarylacetoacetate hydrolase, the last enzyme in the tyrosine catabolism pathway. This deficiency results in accumulation of toxic metabolites, such as maleylacetoacetate, fumarylacetoacetate (FAA), succinylacetoacetate, and succinylacetone (SA). The accumulation of these toxic metabolites can cause liver failure, hepatocellular carcinoma (HCC), renal tubulopathy, cardiomyopathy, and porphyria-like-syndrome with neuropathy (van Spronsen et al 1994, Larochelle et al 2012, de Laet et al 2013). Life expectancy in HT1 patients is low when they are treated with a low phenylalanine and tyrosine diet only (van Spronsen et al 1994, de Laet et al 2013).

The course of the disease has changed remarkably since 1992, when it was observed that 2-(2- nitro-4-trifluormethyl-benzyl)-1,3-cyclohexanedione (NTBC) prevents the accumulation of toxic metabolites by blocking the tyrosine degradation at the level of 4-OH-phenylpyruvate dioxygenase, proximal from the primary enzymatic defect (Lindstedt et al 1992). This in turn prevents the formation of toxic products, leading to resolution of liver failure and porphyria-like-syndrome and a substantial reduction in HCC (Holme and Lindstedt 1998, Holme and Lindstedt 2000). Consequently, life expectancy in HT1 has considerably improved (Larochelle et al 2012). However, as NTBC blocks the tyrosine degradation pathway, tyrosine concentrations increase, so restriction of dietary tyrosine and phenylalanine is still necessary (Russo et al 2001, de Laet et al 2013).

Despite improved treatment with NTBC, previous reports indicate that HT1 patients are still at risk for developing hepatocellular carcinoma (van Spronsen et al 2005, Koelink et al 2006, van Ginkel et al 2015). This may be related to suboptimal treatment with NTBC. Accumulation of FAA causes the development of HCC (Jorquera and Tanguay 1997, Jorquera and Tanguay 1999). As SA can be used as a surrogate marker of toxicity in the liver, we should aim at the lowest NTBC dose associated with the lowest possible SA concentration in blood and urine (Grompe et al Grompe 2001, de Laet et al 2013, Mayorandan et al 2014).

The current dosing recommendation is twice a day as specified by the manufacturers (Orfadin [package insert] 2017). However, a single dose of NTBC per day was suggested in HT1 patients (at 1 mg/kg/day), based on the long half-life of 54 h observed in healthy adults and the finding that in HT1 patients NTBC concentrations tended to be stable for at least 24 h with a single dose regime (Hall et al 2001, Schlune et al 2012, de Laet et al 2013).

However, in practice, blood NTBC levels are reported to vary greatly between individuals (Schlune et al 2012, de Laet et al 2013) and within the same patients with time. As a consequence, target blood NTBC concentrations are not well established. Therefore, the research questions of this study were: (1) Do mean NTBC concentrations and daytime variation of NTBC differ between different NTBC dosing regimens? (2) What is the occurrence of elevated SA with different NTBC dosing regimens? Answering these research questions may help to suggest an optimal dosing regimen and to indicate a minimal NTBC concentration associated with the absence of toxic metabolites represented by SA.

Subject characteristics are given in Table 1. The subjects were divided into three groups depending on their moment of intake of NTBC. For the group who received NTBC once daily (N = 13), six subjects received NTBC at breakfast and seven subjects received NTBC at the evening meal. The subjects taking NTBC twice daily received their NTBC both at breakfast and at the evening meal. The total daily NTBC dose (in mg/kg/day) did not significantly differ between the three groups of subjects (subjects receiving NTBC in a single dose or in two doses) (p = 0.13). Despite that natural protein intake, total protein intake, and tyrosine concentrations seemed to differ, none reached statistical significance (p = 0.12, p = 0.13, and p = 0.13, respectively). Figure 1 shows the variation of NTBC during the day, comparing subjects who received a single dose of NTBC, at breakfast or evening meal, and subjects who divided their NTBC in two doses a day. Mixed model analyses showed a significant interaction between the timing of NTBC intake and the moment of blood sampling (p = 0.041), indicating that the pattern of NTBC concentrations during the day was different between the three different groups. Post hoc analyses showed that the group of subjects taking NTBC at breakfast had a significant variation of NTBC concentrations especially in the morning (p = 0.026). No significant variation in NTBC concentrations during the day was seen in both other groups (subjects who took two doses of NTBC and subjects who took a single dose of NTBC at the evening meal).

Figure 1 also shows the mean NTBC concentrations during the day comparing the three different groups of subjects. Mixed model analyses were done to compare NTBC concentrations of the three different subject groups at all four different timing moments of blood sampling during the day, but no significant differences were found. However, NTBC concentrations tended to be higher during the day when subjects received NTBC twice daily as also shown in Table 2. Of note, one patient lived a far distance from the clinical center. The blood spots were taken in a warm climate and transport took more time. This subject had very low levels of NTBC (mean = 16.8 ± 3.9) that deviated from the NTBC concentrations of other subjects who were treated in the same center and treated similarly (Table 2). When excluding this patient, the tendency toward differences in NTBC concentrations in the morning between the group taking NTBC at breakfast and the group taking NTBC twice daily became statistically significant (p = 0.016).

To further analyze the correlation between NTBC concentrations and the samples with quantitatively detectable SA, mean NTBC concentrations during the study period were calculated and related to the samples with quantitatively detectable SA. Figure 2a shows that if one sample in an individual patient showed a SA concentration above the detection limit of quantification, SA concentrations were usually quantifiable during a large part of the study. Correlational analyses revealed a significant negative correlation between the mean NTBC concentration during the study period and the number of samples with quantitatively detectable SA per patient (ρ = −0.514, p = 0.029). In addition, Fig. 2a shows that when mean NTBC concentrations during the study period were >35 μmol/L, no SA ≥ 0.6 μmol/L was found. Figure 2b shows that the mean NTBC concentrations during the study period were significantly higher in the subjects who did not show any quantitatively raised SA concentrations (mean = 35.6 ± 8.5) compared to subjects where SA was quantifiable at least once (mean = 26.5 ± 6.0) (p = 0.026).

With the dosing regimen of NTBC as advised by de Laet (de Laet et al 2013), increased SA concentrations are still found. This could possibly indicate a sub-optimal block, of course acknowledging the fact that not detecting SA may be due to technical reasons rather than the absence of SA. Therefore, the aims of this study were to investigate NTBC concentrations and its variation during the day and to study SA in relation to the NTBC dosage. By doing this, we investigated if a specific NTBC dosing regimen could be advised in HT1 patients. The main findings were: (1) NTBC concentrations varied during the day, particularly when NTBC was given once daily, and especially when taken at breakfast only. (2) NTBC concentrations tended to be higher and more stable in the patients receiving two doses of NTBC a day, but this did not reach statistical significance, and (3) NTBC concentrations were negatively correlated to SA. Quantitatively detectable SA was only found in patients who took one single dose of NTBC, especially when momentary NTBC concentrations were <44.3 μmol/L.

Before discussing the results in more detail, four methodological issues need to be addressed. Firstly, this study was an observational study and would have had a higher power if both treatment regimens were being tested in the same patients using a cross-over design. Secondly, the study was performed at patient’s homes. All blood spots were sent together after finishing the complete study and were analyzed at the University Medical Center Groningen and stored at −20 °C until analyzed. However, one of the patients receiving two daily doses of NTBC lived in a warm climate 3500 km from the center where he was treated, resulting in a time delay for receipt of blood spot samples. Although it was found that NTBC concentrations in blood spots were stable for at least a period of 1 month when stored at room temperature (La Marca et al 2012), our laboratory experience revealed that NTBC and SA concentrations are more stable when stored at low temperature. Further studies are needed to explore whether different storage conditions (like warm climate or a longer duration until analysis) influence NTBC and SA concentrations in dried blood spots. Thirdly, especially our findings on SA concentrations may be influenced by the limit of quantification rather than the exact SA concentration. Since most subjects had SA levels below limit of quantification using a lower limit of quantification for SA would have resulted in different findings. Fourth, although not statistically significant, total and natural protein intake in the group of patients taking NTBC once daily seemed to be higher compared to the group taking NTBC twice daily. Theoretically, higher protein intake could result in higher tyrosine concentrations and a higher flux through the tyrosine degradation pathway (Holme and Lindstedt 1998). However, tyrosine concentrations did not differ significantly as well.

Our results show a significant variation in NTBC concentrations during a 24-h period especially seen in patients who took their NTBC as a single dose at breakfast. In this group a drop of 21.3% in NTBC concentrations in < 20 h was observed between the pre-midday and pre-breakfast sample. This contrasts with previous studies that indicated NTBC had a stability of at least 24 h in HT1 patients, while in healthy adults even a half-life of 54 h was found (Schlune et al 2012, Hall et al 2001). The variation in NTBC concentrations in our sample at least suggests that biological availability was < 24 h and that this could have implications regarding the detection of SA, especially for subjects taking a single daily dose of NTBC.

When considering NTBC concentrations, our data indicate a tendency toward lower NTBC concentrations for the patients receiving NTBC once daily in comparison with receiving NTBC twice daily rather than a statistically significant difference. However, clear statistical significance was reached when the subject with the long transport time of samples was excluded as an apparent outlier probably due to differences in pre-analytical conditions. This is in contrast to previous studies, which failed to show any differences between dosing once, twice, and three times a day (Schlune et al 2012).

Both, momentary and mean NTBC concentrations during the study period were negatively correlated to SA. The difference between momentary and mean NTBC concentrations without quantitatively detectable SA is expected to be caused by the long half-life of SA in plasma (Holme and Lindstedt 1998). Quantitatively detectable SA possibly indicates a period of lower NTBC concentrations before the moment of blood sampling. Most importantly, in our study, SA was not found when momentary NTBC concentrations were above 44.3 μmol/L, which is in accordance with the results of Herebian et al (2009) who concluded that NTBC concentrations should be above 50 μmol/L (Herebian et al 2009).

Acknowledging the importance of maintaining SA concentrations as low as possible, our data seem to indicate that dosing NTBC twice daily might be better than once daily. Our data also suggest that higher NTBC concentrations are related to lower SA concentrations. Before stronger conclusions can be drawn, further studies with more data on the dosing of NTBC and related SA concentrations are needed. Therefore, it is mandatory to monitor NTBC and SA concentrations frequently, preferably by home blood spot sampling for which measurement can be performed in expert centers for this disease.