Physiologically based pharmacokinetic modelling of the three-step metabolism of pyrimidine using ¹³C-uracil as an in vivo probe

Abstract

Aims

Approximately 80% of uracil is excreted as β-alanine, ammonia and CO₂ via three sequential reactions. The activity of the first enzyme in this scheme, dihydropyrimidine dehydrogenase (DPD), is reported to be the key determinant of the cytotoxicity and side-effects of 5-fluorouracil. The aim of the present study was to re-evaluate the pharmacokinetics of uracil and its metabolites using a sensitive assay and based on a newly developed, physiologically based pharmacokinetic (PBPK) model.

Methods

[2–¹³C]Uracil was orally administrated to 12 healthy males at escalating doses of 50, 100 and 200 mg, and the concentrations of [2–¹³C]uracil, [2–¹³C]5,6-dihydrouracil and β-ureidopropionic acid (ureido-¹³C) in plasma and urine and ¹³CO₂ in breath were measured by liquid chromatography–tandem mass spectrometry and gas chromatograph–isotope ratio mass spectrometry, respectively.

Results

The pharmacokinetics of [2–¹³C]uracil were nonlinear. The elimination half-life of [2–¹³C]5,6-dihydrouracil was 0.9–1.4 h, whereas that of [2–¹³C]uracil was 0.2–0.3 h. The AUC of [2–¹³C]5,6-dihydrouracil was 1.9–3.1 times greater than that of [2–¹³C]uracil, whereas that of ureido-¹³C was 0.13–0.23 times smaller. The pharamacokinetics of ¹³CO₂ in expired air were linear and the recovery of ¹³CO₂ was approximately 80% of the dose. The renal clearance of [2–¹³C]uracil was negligible.

Conclusion

A PBPK model to describe ¹³CO₂ exhalation after orally administered [2–¹³C]uracil was successfully developed. Using [2–¹³C]uracil as a probe, this model could be useful in identifying DPD-deficient patients at risk of 5-fuorouracil toxicity.

Introduction

Pyrimidine and purine, which are present endogenously in nucleic acids, nucleotides and their derivatives, display a wide range of physiological functions. Since the catabolism and anabolism of pyrimidines are inextricably linked, their biological fate is complex. Uracil, a pyrimidine base, is metabolized to β-alanine, ammonia and CO₂ via three sequential reactions [1, 2] (Figure 1). Uracil is first reduced to dihydrouracil by dihydropyrimidine dehydrogenase (DPD), then hydrolysed to β-ureidopropionic acid by dihydropyrimidinase (DHPase), and finally decarbamoylated to β-alanine by β-ureidopropionase (UP). Of these enzymes, DPD is considered to represent the rate-limiting step [3, 4].

Figure 1.

The metabolism of uracil and 5-fluorouracil in humans

5-Fluorouracil (5-FU) is an anticancer agent in which the hydrogen atom at the C-5 position of uracil is substituted by  fluorine  (Figure 1).  Since  the  structure  of 5-FU is analogous to that of uracil, it is biotransformed to putative, biologically active metabolites, 5-fluoro-2′-deoxyuridine-5′-monophosphate or 5-fluorouridine-5′-triphosphate, by the same anabolic pathway as that of uracil [5, 6]. 5-FU is metabolized by conversion to biologically inactive metabolites by the same enzymes that metabolize uracil [7–10]. When 5-FU is given to patients with genetic DPD deficiency or those taking drugs which inhibit DPD activity, blood concentrations of the drug are markedly elevated, resulting in serious adverse effects [11–14]. The use of diagnostic methods to detect pyrimidine metabolic disorders [15–17] at the start of chemotherapeutic treatment would prevent the development of adverse effects with 5-FU and its prodrugs.

We previously developed a diagnostic product (UBIT®) for Helicobacter pylori infection which measures in vivo urease activity using expired ¹³CO₂ after oral administration of ¹³C-urea [18–20]. Extending this work, we have developed a new method for diagnosing pyrimidine metabolic disorders using [2–¹³C]uracil (¹³C-uracil), prepared by labelling the C-2 position of uracil with ¹³C, a stable isotope of ¹²C (Figure 1). We have already applied this method to dogs [21] to show that expired ¹³CO₂ is a good marker of hepatic DPD activity in the enzyme-deficient model. To gain further understanding of pyrimidine catabolism, the pharmacokinetics of ¹³C-uracil was studied following oral administration under fasting conditions to healthy subjects at escalating doses.

Methods

Subjects

The study protocol was approved by the Ethics Committee of Juntendo University Hospital, and written informed consent was obtained from each participant before enrolment. The subjects were 12 healthy Japanese males (21–57 years old; 56–90 kg) with normal pyrimidine metabolism as determined by measurement [22] of endogenous pyrimidine and dihydropirimidine in urine. Good general health was confirmed by 12-lead electrocardiogram, medical history and physical examination. The study was designed to evaluate the pharmacokinetic profile of ¹³C-uracil and its metabolites after single oral administration of ¹³C-uracil using an open label, single-centre, dose escalation design. The subjects were given ¹³C-uracil on three occasions at escalating doses of 50, 100 and 200 mg under fasted conditions. The subjects were given ¹³C-uracil as granules with 100 ml of water at approximately 09.00 h. Food was not permitted from 21.00 h on the evening before dosing to 4 h after dosing. Subjects were in a sitting position for 2 h postdose. The order of dosing was 50, 100 and 200 mg and the wash-out period was set at ≥ 5 days. Blood was taken immediately before and at 10, 20, 30, 40, 50, 60 and 90 min and 2, 4, 6, 8 and 12 h after dosing. Urine samples were collected before and at the periods of 0–2, 2–4, 4–8 and 8–12 h after dosing. Breath samples were collected in a bag (volume 300 ml) before and at 10, 20, 30, 40, 50, 60, 80 and 100 min and 2, 3, 4, 6, 8 and 12 h after dosing.

Chemicals

[2-¹³C]Uracil, [2-¹³C]5,6-dihydrouracil, β-ureidopropionic acid (ureido-¹³C), uracil (¹³C₄, ¹⁵N₂), 5,6-dihydrouracil (¹³C₄, ¹⁵N₂) and β-ureidopropionic acid (¹³C₄, ¹⁵N₂) were purchased from Cambridge Isotope Laboratories, Inc. (Andover, MA, USA). All other solvents and reagents were of the highest grade available.

Determination of ¹³C-uracil, ¹³C-DHU and ¹³C-UPA in plasma and urine by LC-MS/MS

Plasma concentrations of ¹³C-uracil and its metabolites (¹³C-DHU and ¹³C-UPA) were measured by LC-MS/MS (TSQ7000; Thermo Finnigan, San Jose, CA, USA). Isotope-labelled uracil (¹³C₄, ¹⁵N₂), 5,6-dihydrouracil (¹³C₄, ¹⁵N₂) and β-ureidopropionic acid (¹³C₄, ¹⁵N₂) were used as internal standards. ¹³C-Uracil and ¹³C-DHU were extracted from 0.5 ml of plasma using 4 ml of acetonitrile after the addition of 0.5 ml of a saturated aqueous ammonium sulphate solution. Samples were then centrifuged at 2200 g for 10 min. The organic layer was evaporated to dryness, the residue reconstituted in 200 µl of purified water, and a 50-µl aliquot was injected into LC-MS/MS. A Develosil RPAQUEAUS column (5 µm, 2.0 mm i.d. × 150 mm; Nomura Chemical Co., Ltd, Seto, Japan) and a mobile phase of water were used to separate the analytes. ¹³C-UPA was extracted from 0.2 ml of plasma by solid-phase extraction on silica after deproteinization. After evaporation of the eluate to dryness, the residue was reconstituted in 200 µl of purified water. This solution (30 µl) was injected onto the LC-MS/MS fitted with two columns in series, a Develosil RPAQUEAUS (5 µm, 2.0 mm i.d. × 150 mm) and a Capcell pak SCX UG80 (5 µm, 2.0 mm i.d. × 50 mm; Shiseido Co., Ltd, Tokyo, Japan). The mobile phase was an aqueous solution of 10 m m ammonium formate (pH 3.5). Protonated molecular ions [M+1]⁺ of the analytes including the internal standard, formed by atmospheric pressure chemical ionization, were fragmented, and the selected product ions were monitored (selected reaction monitoring). The calibration curves were linear over the ranges 5–250 ng ml⁻¹ for ¹³C-uracil and ¹³C-DHU and 50–2000 ng ml⁻¹ for ¹³C-UPA. The percent recoveries of isotope-labelled uracil (¹³C₄, ¹⁵N₂), 5,6-dihydrouracil (¹³C₄, ¹⁵N₂) and β-ureidopropionic acid (¹³C₄, ¹⁵N₂) from human plasma were 83–105%, 56–74% and 23–24%, respectively. The limit of quantification (LOQ), defined as the lowest concentration with a coefficient of variation (CV) of < 20% and accuracy within ± 20%, was 5 ng ml⁻¹ for ¹³C-uracil and ¹³C-DHU and 50 ng ml⁻¹ for ¹³C-UPA. Precision, estimated as CV, was < 15% and accuracy was within ± 15% for the analytes at all concentrations except the LOQ.

Urinary concentrations of each compound were measured using LC-MS/MS. ¹³C-Uracil and ¹³C-DHU were extracted from 0.2 ml of urine using 5 ml of ethyl acetate after the addition of 0.1 ml of a saturated aqueous ammonium sulphate solution. The samples were then centrifuged at 1800 g for 10 min. After the organic layer was evaporated to dryness, the residue was reconstituted in 200 µl of purified water and 30 µl of the solution was injected onto the LC-MS/MS. ¹³C-UPA was extracted from 0.1 ml of the urine by solid-phase extraction on silica after deproteinization. After evaporation of the solid-phase extraction eluate to dryness, the residue was reconstituted in 200 µl of purified water and 30 µl was injected onto the LC-MS/MS. The chromatographic conditions were similar to those for the plasma samples. The calibration curves for these analytes were linear over the range of 0.1–5 µg ml⁻¹, and the LOQ was 0.1 µg ml⁻¹. The percent recoveries of isotope-labelled uracil (¹³C₄, ¹⁵N₂), 5,6-dihydrouracil (¹³C₄, ¹⁵N₂) and β-ureidopropionic acid (¹³C₄, ¹⁵N₂) from human urine were 57–64%, 63–74% and 20–30%, respectively. Precision was < 15% and accuracy was within ± 15% at all concentrations except the LOQ.

Analysis of ¹³CO₂ in expired air by gas chromatography isotope ratio mass spectrometry (IRMS)

¹³CO₂ concentrations in expired air were determined using a gas chromatograph-IRMS (model ABCA-G; PDZ-Europa Ltd, Cheshire, UK). ¹³CO₂/¹²CO₂ ratios were expressed as δ¹³C value (permil, ‰) relative to the Pee Dee Belemnite Limestone standard, and changes in the δ¹³C value as Δ¹³C (‰) were compared with the baseline using the following equations:

$${\delta }^{\text{13}}\text{C}(‰)=\left[{\left({\text{CO}}_{\text{2}}{/}^{\text{12}}{\text{CO}}_{\text{2}}\right)}_{\text{sample}}-{\left({\text{CO}}_{\text{2}}{/}^{\text{12}}{\text{CO}}_{\text{2}}\right)}_{\text{PDB}}\right]/{\left({\text{CO}}_{\text{2}}{/}^{\text{12}}{\text{CO}}_{\text{2}}\right)}_{\text{PDB}}\times 1000$$

$${\Delta }^{\text{13}}{\text{C}}_{\text{t}}(‰){=\delta }^{\text{13}}{\text{C}}_{\text{t}}{-\delta }^{\text{13}}{\text{C}}_{\text{0}}$$

where Δ¹³C_t (‰) is the difference between respiratory δ¹³C_t measured at time t and baseline δ¹³C₀ following the administration of ¹³C-uracil.

Pharmacokinetic analysis

Pharmacokinetic parameters for ¹³C-uracil and its metabolites were calculated using noncompartmental pharmacokinetic analysis (WinNonlin Standard version 3.1; Pharsight Co., Mountain View, CA, USA). Maximum plasma concentration (C_max), time to C_max (t_max) and the area under the plasma concentration vs. time curve up to 12 h after administration (AUC_{12 h}) were determined. The apparent terminal-phase slope (λ_z) was estimated by linear regression of the semilogarithmic curve of plasma concentration vs. time. The terminal elimination half-life (t_½) was calculated as 0.693/λ_z. AUC_∞ was calculated by dividing the last measured concentration (C_last) by λ_z. The apparent total clearance (CL/F) is dose/AUC_∞, and the apparent volume of distribution (V_d/F) is CL/F/λ_z. The cumulative amount excreted into the urine for 12 h (Ae) was used to estimate the renal clearance (CL_R) from the expression Ae/AUC_{12 h}. The total amount of ¹³CO₂ recovered in the breath (m) was calculated from ¹³CO₂ excretion curves based on the method of Ghoos et al.[23], in which CO₂ production was assumed to be 300 mmol m⁻² h⁻¹.

Statistical analysis

The relationships between the dose and the pharmacokinetic parameters (C_max, AUC_t, and AUC_∞) were analysed by using a predictive power model based on the equation

$$\text{Parameter}=\text{A}.{\left(\text{Dose}\right)}^{\beta }$$

Statistical analysis was performed with SAS software, version 8.2 (SAS Institute Japan, Tokyo, Japan).

Model development

A physiologically based pharmacokinetic (PBPK) model model (Figure 2) was constructed to describe the time course of plasma concentrations of ¹³C-uracil and its metabolites, and ¹³CO₂ in expired air. This model incorporates Michaelis–Menten catabolic and first-order degradation processes. The differential equations for the PBPK model were as follows:

Figure 2.

A physiologically based pharmacokinetic model describing the concentration–time profiles for ¹³C-uracil, its metabolites and ¹³CO₂ in expired air

For ¹³C-uracil

$${\text{V}}_{\text{P}}\left({\text{dC}}_{\text{P}}/\text{dt}\right){=\text{Q}}_{\text{H}}{\text{C}}_{\text{h}}{/\text{K}}_{\text{P}}{-\text{Q}}_{\text{H}}{\text{C}}_{\text{P}}-{\text{C}}_{\text{P}}{\text{CL}}_{\text{R}}$$ (1)

$$V_{\text{h}}.\left(\text{d}{C}_{\text{h}}/\text{dt}\right)={\text{k}}_{\text{a}}.{\text{F}}_{\text{a}}.\text{Dose}{\text{.e}}^{{\text{-k}}_{\text{a}}\text{t}}-f_{\text{P}}.C_{\text{h}}.{\text{CL}}_{\text{int}}/K_P-Q_H.C_h/K_P+Q_H.C_P$$ (2)

$${\text{CL}}_{\text{int}}=V_{\max }/\left(K_m+f_{\text{P}}.C_{\text{h}}/K_P\right)$$ (3)

For ¹³C-DHU

$$V_{d\text{DHU}}.\left(\text{d}{C}_{\text{DHU}}/\text{dt}\right)=f_P.C_{\text{h}}/K_P.{\text{CL}}_{\text{int}}-C_{\text{DHU}}.V_{d\text{DHU}}.k_{e\text{DHU}}$$ (4)

For ¹³C-UPA

$$V_{d\text{UPA}}.\left(\text{d}{C}_{\text{UPA}}/\text{dt}\right)=C_{\text{DHU}}.V_{d\text{DHU}}.k_{e\text{DHU}}-C_{\text{UPA}}.V_{d\text{UPA}}.k_{e\text{UPA}}$$ (5)

For ¹³CO₂

$${\text{dX}}_{{\text{H}}^{\text{13}}{\text{CO3}}^{-}}{/\text{dt}=\text{C}}_{\text{UPA}}.{\text{V}}_{\text{dUPA}}.{\text{K}}_{\text{eUPA}}-{\text{K}}_{\text{e}}.{\text{X}}_{{\text{H}}^{\text{13}}{\text{CO3}}^{-}}$$ (6)

$${\Delta }^{13}\text{C}(‰)=\text{P1+P2}{\text{.X}}_{{\text{H}}^{\text{13}}{\text{CO3}}^{\text{-}}}$$ (7)

where V_h is the volume of the liver; V_p is the volume of distribution in rapidly equilibrating tissues, including the systemic plasma compartment of ¹³C-uracil; V_dDHU and V_dUPA are the pseudo-distribution volumes of ¹³C-DHU and ¹³C-UPA, respectively; C_h is the concentration of ¹³C-uracil in liver; C_p,C_DHU and C_UPA are the plasma concentrations of ¹³C-uracil, ¹³C-DHU and ¹³C-UPA, respectively; Q_H is the hepatic blood flow rate; K_p is the liver-to-blood concentration ratio of ¹³C-uracil; CL_R is the renal clearance of ¹³C-uracil; CL_int is intrinsic metabolic clearance; V_max and K_m are the maximum rate of ¹³C-uracil metabolism and the Michaelis–Menten constant, respectively; f_p is the unbound fraction of ¹³C-uracil in plasma; k_eDHU and k_eUPA are the degradation rate constants of ¹³C-DHU and ¹³C-UPA, respectively; X_H¹³_CO3- is the amount of H¹³CO₃⁻; k_e is the excretion rate constant of ¹³CO₂; and P1 and P2 are constants.

These equations are based on the following assumptions:

1. The gastrointestinal absorption of ¹³C-uracil follows a first-order process.

2. ¹³C-Uracil is eliminated by the liver and kidney, and is converted sequentially in the liver to ¹³C-DHU, ¹³C-UPA and H¹³CO₃⁻.

3. ¹³C-uracil is eliminated via a single irreversible and saturable Michaelis–Menten process.

4. The elimination of the metabolites of ¹³C-uracil follows first-order irreversible kinetics.

5. The elimination of ¹³C-DHU, ¹³C-UPA and H¹³CO₃⁻is described by a single-compartment model.

This assumption can be justified from the following findings: (i) a single-compartment model describes the pharmacokinetics of ¹³CO₂-H¹³CO₃⁻ after co-administration of sodium bicarbonate [24]; (ii) the kinetics of ¹³CO₂ after administration of ¹³C-compounds are best described by a one-compartment model [25–27].

Equation holds true because Δ¹³C (‰) is proportional to the amount of H¹³CO₃⁻ in the body [28].  The physiological data used, namely V_h = 1070 ml, Q_H = 1190 ml min⁻¹ and haematocrit value = 0.55, were obtained from the literature. K_p, f_p and F_a were set at 1.0 according to our preliminary experiments (data not shown), and CL_R at 120 ml min⁻¹ on the basis of our urinary excretion analysis.

The pharmacokinetic software SAAM II (SAAM Institute Inc., Seattle, WA, USA) was used for nonlinear least squares analysis to fit the parameters V_p, k_e, K_m and V_max to the set of plasma concentrations of ¹³C-uracil for dose-escalation experiments using equations. Using these fixed parameters, the parameters V_dDHU, V_dUPA, k_eDHU,k_eUPA, k_e, P1 and P2 were subsequently estimated by SAAM II using equations 4–7.

Results

Pharmacokinetics

Tables 1, 2 and 3 show the pharmacokinetic parameters for ¹³C-uracil, ¹³C-DHU and ¹³C-UPA in the plasma, respectively, and Tables 4 and 5 the urinary excretion and expiratory ¹³CO₂ excretion data. Figure 3 shows the concentration vs. time curves for ¹³C-uracil and its metabolites, and the Δ¹³C in the expired air vs. time curve.

Table 1. Pharmacokinetic parameters for ¹³C-uracil in plasma.

Dose	Cmax (µg ml−1)	AUC12 h (µg h ml−1)	AUC∝ (µg h ml−1)	tmax (h)	λz (1 h−1)	t1/2 (h)	CL/F (l h−1)	Vd/F (l)
50 mg	0.127	0.047	0.053	0.36	3.66	0.26	1082	466
	±0.083	±0.022	±0.021	±0.10	±1.71	±0.22	±410	±500
100 mg	0.534	0.165	0.170	0.39	5.25	0.21	772	296
	±0.430	±0.109	±0.107	±0.18	±2.92	±0.19	±368	±423
200 mg	1.205	0.545	0.567	0.54	2.91	0.32	464	296
	±0.899	±0.325	±0.312	±0.25	±1.19	±0.26	±265	±480

Values: mean ± SD (n = 12).

Table 2. Pharmacokinetic parameters for ¹³C-DHU in plasma.

Dose	Cmax (µg ml−1)	AUC12 h (µg h ml−1)	tmax (h)	λz (1 h−1)	t1/2 (h)
50 mg	0.102	0.144	0.49	0.68	1.10
	±0.050	±0.048	±0.15	±0.18	±0.34
100 mg	0.251	0.378	0.56	0.78	0.91
	±0.094	±0.135	±0.18	±0.11	±0.14
200 mg	0.551	1.054	0.93	0.60	1.37
	±0.255	±0.449	±0.50	±0.19	±0.74

Values: mean ± SD (n = 12).

Table 3. Pharmacokinetic parameters for ¹³C-DHU in plasma.

Dose	Cmax (µg ml−1)	AUC12 h (µg h ml−1)	tmax (h)	λz (1 h−1)	t1/2 (h)
50 mg	0.023	0.006	0.46	–	–
	±0.035	±0.014	±0.16	–	–
100 mg	0.076	0.034	0.50	1.17	0.87
	±0.040	±0.042	±0.19	±0.88	±0.61
200 mg	0.149	0.126	0.82	1.12	1.05
	±0.081	±0.105	±0.45	±0.67	±1.05

–, Not calculated. Values: mean ± SD (n = 12).

Table 4. Urinary excretion and kinetic parameters for ¹³C-uracil and its metabolites.

Substance	Parameter	50 mg	100 mg	200 mg
13C-uracil	Ae (mg)	0.35 ± 0.19	1.21 ± 0.80	4.52 ± 2.80
	CLR (l h−1)	6.8 ± 1.6	7.2 ± 2.1	7.7 ± 1.4
	Excretion (%/dose)	0.7 ± 0.4	1.2 ± 0.8	2.3 ± 1.4
13C-DHU	Ae (mg)	0.10 ± 0.05	0.28 ± 0.14	0.83 ± 0.44
	CLR (l h−1)	0.7 ± 0.2	0.7 ± 0.2	0.8 ± 0.3
	Excretion (%/dose)	0.2 ± 0.1	0.3 ± 0.1	0.4 ± 0.2
13C-UPA	Ae (mg)	0.21 ± 0.10	0.53 ± 0.31	1.33 ± 0.91
	CLR (l h−1)	17.1 ± 8.7	13.4 ± 6.3	9.1 ± 4.1
	Excretion (%/dose)	0.4 ± 0.2	0.5 ± 0.3	0.6 ± 0.4

¹³C-DHU: 5,6-dihydrouracil (2-¹³C)]¹³C-UPA,β-ureidopropionic acid (ureido-¹³C). Values: mean ± SD (n = 12).

Table 5. Expiratory excretion parameters for ¹³CO₂ (Δ¹³C).

Dose	Cmax (‰)	AUC12 h (‰ h)	AUC∝ (‰ h)	tmax (h)	λz (1 h−1)	t1/2 (h)	m (% dose−1)
50 mg	37.8	53.3	54.2	0.43	0.59	1.27	75.5
	±11.7	±5.3	±5.2	±0.15	±0.18	±0.36	±7.4
100 mg	67.9	106.4	107.8	0.54	0.56	1.41	75.9
	±17.3	±9.7	±9.9	±0.19	±0.21	±0.59	±3.3
200 mg	104.8	213.8	216.2	0.89	0.55	1.34	76.4
	±20.8	±25.0	±24.9	±0.44	±0.15	±0.37	±6.1

m, Amount of ¹³CO₂ recovered in the breath. Values: mean ± SD (n = 12).

Figure 3.

Plasma concentration–time curves for ¹³C-uracil and its metabolites, and Δ¹³C–time curves in expired air after oral administration of ¹³C-uracil at doses of (A) 50 mg, (B) 100 mg, and (C) 200 mg to 12 healthy males. (▴; ¹³C-uracil, Δ; ¹³C-DHU, ◊; ¹³C-UPA, ▪; ¹³CO₂, mean ± SD, n = 12)

¹³C-Uracil was absorbed rapidly after oral dosing to attain C_max within 0.54 h, and then declined rapidly in plasma, with a short half-life of less than 0.32 h. The major metabolite of ¹³C-uracil in plasma at all doses was ¹³C-DHU, with the relative ratios of the AUC_{12 h} of ¹³C-DHU to ¹³C-uracil at 50, 100 and 200 mg being 3.1, 2.3 and 1.9, respectively. ¹³C-UPA was a minor metabolite, and the relative ratios of the AUC_{12 h} of ¹³C-UPA to ¹³C-uracil at 50, 100 and 200 mg were 0.13, 0.21 and 0.23, respectively. Plasma concentrations of ¹³C-DHU were higher than those of ¹³C-uracil from 50 min after administration. At all doses, the elimination half-life of ¹³C-DHU was much longer than that of ¹³C-uracil. A predictive power model was used to evaluate the dose-proportionality of C_max, AUC_{12 h} and AUC_∞ values. None of these parameters was dose-proportional over the range of 50–200 mg [the 95% confidence interval (CI) for the slope of the regression line (β) did not include unity]. Nonlinearity in pharmacokinetics was clearly present in 10 out of the 12 subjects, although there was considerable interindividual variability between subjects.

The contribution of renal clearance to the total body clearance was negligible (Table 4).

The Δ¹³C of ¹³CO₂ in expired air vs. time curve was similar to that of ¹³C-DHU in plasma (Figure 3). The recovery of ¹³C in expired air was approximately 80% at each dose, which is in agreement with previous reports [8–10] on the deposition of 5-FU. C_max was found not to be dose-proportional over the range of 50–200 mg, but AUC_∞ and AUC_{12 h} were proportional to dose [the 95% CIs of the slopes (β) for these parameters were 0.93–1.06 and 0.94–1.06].

The predicted concentration–time courses of ¹³C-uracil, ¹³C-DHU, ¹³C-UPA and Δ¹³C (‰) are shown in Figure 4. Satisfactory agreement between the predicted curve and experimental data was obtained. The exception was the relationship for ¹³C-UPA, which had a higher limit of quantification (50 ng ml⁻¹) than with ¹³C-uracil and ¹³C-DHU (5 ng ml⁻¹), thus introducing some uncertainty into the data. The pharmacokinetic parameters estimated by nonlinear least squares regression are listed in Table 6. The clearance down each metabolic step was calculated as follows: intrinsic clearance (CL_int1) for the first step catalysed by DPD was assumed to be V_max/K_m, clearance (CL_int2) for the second step catalysed by DHPase to be V_dDHU·k_eDHU, and clearance (CL_int3) for the third step catalysed by UP to be V_dUPA·k_eUPA. We have assumed that, since ¹³C-DHU and ¹³C-UPA are generated sequentially only in the liver, their clearance represents intrinsic hepatic clearance. The rank order of metabolic clearances was CL_int3 > CL_int1 > CL_int2 (Table 6).

Figure 4.

Model fits for the mean plasma concentration–time data for ¹³C-uracil and its metabolites, and Δ¹³C in the expired air after oral administration of ¹³C-uracil at doases of (A) 50 mg, (B) 100 mg, and (C) 200 mg to 12 healthy males. (▴; ¹³C-uracil, Δ; ¹³C-DHU, ◊; ¹³C-UPA, ▪; ¹³CO₂). Solid lines represent the predicted values calculated by the PBPK model shown in Figure 2

Table 6. Parameters estimated from simultaneous fitting of mean data for ¹³C-uracil and its metabolites and ¹³CO₂ in expired air.

	Dose
Parameter	50 mg	100 mg	200 mg
Km (µg ml−1)	–	1.60*	–
Vmax (µg min−1)	–	33900*	–
Vp (l)	–	17.2*	–
ka (min−1)	–	0.259*	–
VdDHU (l)	–	408†	–
VdUPA (l)	–	10.0†	–
keDHU (min−1)	0.0119	0.0126	0.00948
keUPA (min−1)	4.58	5.00	2.54
ke (min−1)	0.151	0.0781	0.0578
P1	0.264	0.627	0.365
P2	0.00904	0.00458	0.00368
CLint1 (l min−1)‡	–	21.2	–
CLint2 (l min−1)§	4.86	5.14	3.87
CLint3 (l min−1)¶	45.8	50.0	25.4

–, Not calculated.

^*Calculated using plasma concentrations of ¹³C-uracil at the doses of 50, 100 and 200 mg by equations.

^†Calculated using plasma concentrations of ¹³C-uracil and its metabolites and ¹³CO₂ in expired air at the dose of 100 mg by equations.

^‡Calculated as V_max/K_m.

^§Calculated as V_dDHU·k_eDHU.

^¶Calculated as V_dUPA·k_eUPA.

Discussion

DPD is reported to be the rate-limiting enzyme in the metabolism of pyrimidine and its analogues under in vivo conditions [29–32]. However, this view has been disputed by others [2, 10, 33]. Wasternack [33] stated that ‘In most papers hitherto published, the first step in degradation has been considered as rate-limiting. However, recent results argue against this concept’ and ‘Under in vivo conditions little information is available because intermediates of degradation are not detectable.’ Daher et al.[10] reported that ‘all steps in the sequential 3-step reactions in pyrimidine metabolism have a potential to be rate-limiting and also it is still unclear which enzyme is rate-limiting’. In the present study, assuming that these discrepancies might be attributable problems in the analysis of pyrimidine and its metabolites in plasma or urine, we re-evaluated the pharmacokinetics of ¹³C-uracil and its metabolites using the high-resolution methods of LC-MS/MS and IRMS.

The results showed that ¹³C-uracil was reduced to ¹³C-DHU by DPD in the first catabolic step, which caused its rapid elimination from plasma. DHPase then mediated the conversion of ¹³C-DHU to ¹³C-UPA, but the relatively slow rate of this reaction meant that ¹³C-DHU remained in plasma for much longer than ¹³C-uracil. Subsequently, since ¹³C-UPA was rapidly biotransformed to H¹³CO₃⁻ by UP, the Δ¹³C in the expired air vs. time curves were similar to the plasma concentration vs. time curves of ¹³C-DHU.

We developed a PBPK model to describe the pharmacokinetics of uracil. PBPK models have been shown to be useful in quantitative evaluation of the metabolism and transport processes of drugs and endogenous substrates under physiological conditions [34–37]. The derived intrinsic clearances for each metabolic step are consistent with the rapid conversion of uracil to DHU, the slow biotransformation of DHU to UPA, and the rapid conversion of UPA to HCO₃⁻.

In contrast, Wasternack [33] suggested that the efflux of dihydropyrimidines from mitochondria into cytosol is rate-limiting owing to the cytosolic location of DPD. Thus, uncertainty remains regarding the rate-limiting step in the disposition of pyrimidines in vivo, which may involve factors such as the transportation of ¹³C-uracil and its metabolites into hepatocytes, the location of the three enzymes responsible for its metabolism, and coenzymes such as NADH, NADPH [38].

Sumi et al.[39] reported two cases of dihydropyrimidinuria among 21 200 infants in whom urinary pyrimidine and dihydropyrimidine concentrations were measured, and concluded that a defect in DHPase was a probable risk factor for an adverse response to 5-FU therapy. Hamajimaet al.[40] subsequently analysed the DHPase gene and demonstrated six types of gene mutation. In addition to these reports, 5-fluoro-5,6-dihydrouracil, the substrate of DHPase and the metabolite of 5-FU, is reported to have antitumour cytotoxic activity [41], suggesting its involvement in the toxicity of 5-FU. Given that the metabolic characteristics of 5-FU [42, 43] and uracil are similar, the present results for uracil should be applicable to 5-FU. Therefore, we came to the tentative hypothesis that the in vivo rate-limiting enzyme for 5-FU metabolism is not DPD but DHPase. This hypothesis is supported by a study [9] of [6-³H]5-FU that produced pharmacokinetic profiles of the drug and its metabolites comparable to our results for ¹³C-uracil. However, in DPD-deficient subjects, DPD is likely to be the rate-limiting enzyme for overall pyrimidine catabolism.

In conclusion, we investigated the metabolic fate of ¹³C-uracil, its metabolites and the end product ¹³CO₂ in expired air. Our PBPK model described the nonlinear pharmacokinetics of uracil and its metabolites well, and showed that of the three enzymes involved in pyrimidine degradation, DHPase is the least active in vivo in humans. Further study is required to show whether the analysis of ¹³CO₂ in expired air after administration of ¹³C-uracil will allow the identification of patients at risk of a severe adverse response to treatment with 5-FU.