Agents for measuring pyrimidine metabolizability

[0132] Example 1

[0133] in the 2nd place is 13 C-labeled carbon atom of uracil (2- 13 C-labeled uracil) was used as the isotope-labeled pyrimidine compound. Preparation of a preparation containing 2- 13 C-labeled uracil is used as the active ingredient in the form of an intravenous solution for determination of pyrimidine metabolizing capacity.

[0134] (1) Production of preparations for determination of pyrimidine metabolism capacity

[0135] Uracil-2- 13 C (Cambridge Isotope Laboratory) (300 mg) was dissolved in 24 ml of 0.1N NaOH/saline solution (measured from 200 ml of 0.1N NaOH/saline solution, which was diluted with normal saline to 2 ml of 10N NaOH / saline solution), the preparation for determination of pyrimidine metabolizing capacity was obtained in the form of intravenous injection, in (each 4 ml injection contains 50 mg uracil-2- 13 C).

[0136] (2) Preparation of (E)-5-(2-bromovinyl)-uracil preparation

[0137](E)-5-(2-Bromovinyl)-uracil is known to be an inhibitor of dihydropyrimidine dehydrogenase (DPD) (Cancer Research 46, 1094-1101, March 1986, pp. 1094-1101). (E)-5-(2-Bromovinyl)-uracil (250 mg) and gum arabic (2.25 g) were mixed together and the mixture was kneaded while adding a small amount of water to make a suspension of 45 ml (Contains 83.3 mg of (E)-5-(2-bromovinyl)-uracil per 15 ml of suspension).

[0138] (3) experiment

[0139] Fasted beagle dogs were used as experimental animals. In group 1 (n=3), the preparation prepared above for determining pyrimidine metabolizing ability was injected intravenously (50 mg/body) (test group). In 2 groups (n=3), the (E)-5-(2-bromovinyl)-uracil preparation (DPD inhibitor) (83.3 mg/body) was first orally administered, and after 1 hour, the pyrimidine metabolizing capacity was determined The preparation of the drug was injected intravenously (50 mg/body) in a similar manner to Group 1 (control group: animal model of uracil metabolic disorder). In both groups, breath was collected before and at 15, 30, 45, 60, 75, 90, 105, 120, 135, 150, 180, 210, and 240 minutes after formulation administration to determine pyrimidine metabolizing capacity . Collected exhaled exhaled 13 C labeled CO 2 The concentration was detected by GC-MS analyzer (ABCA-G, Europa Scientific).

[0140] figure 2 Shown are the results of the test group (-◆-) and the control group (-■-). In this figure, plotted on the ordinate is Δ 13 C value (‰), which is the delta between the breath samples collected before the administration of the formulation and the breath samples collected at each time after the administration of the formulation to determine the pyrimidine metabolizing capacity 13 C value (‰) (in exhalation 13 CO 2 / 12 CO 2 Concentration ratio), the abscissa plots the time to collect exhaled breath after formulation administration.

[0141] as figure 2 At a glance, in the test group, it is observed that 2- 13 C-labeled uracil is metabolized and released in exhaled 13 CO 2 discharge. On the other hand, in an animal model (control group) in which uracil disorder was artificially created by pre-administration of DPD inhibitors, excretion of uracil in exhalation was observed. 13 CO 2 Significantly lower. Thus, by checking the exhaled 13 CO 2 The discharge behavior is evidenced by the use of 2- 13 The preparation of the present invention with C-labeled uracil as an active ingredient (by means of intravenous injection) makes it possible to detect and evaluate the presence or absence of uracil metabolic disorders (decrease in the ability to metabolize uracil), which is caused by Caused by inhibition or reduction of DPD activity.

[0142] Example 2

[0143] The preparation for determining pyrimidine metabolizing ability was prepared in oral form, using the same 2- 13 C-labeled uracil was used as the active ingredient and its detection was useful for determining pyrimidine metabolizing capacity.

[0144] (1) Preparation of preparations for determination of pyrimidine metabolism capacity

[0145] 2- 13 C-labeled uracil (Cambridge Isotope Laboratory) (300 mg) was dissolved in 24 ml of 0.1N NaOH/saline solution (measured from 200 ml of 0.1N NaOH/saline solution, which was diluted with normal saline 2 ml of 10N NaOH/brine solution). Water was then added to obtain in oral form 60 ml of a preparation of determined pyrimidine metabolizing capacity (per 10 ml of oral solution containing 50 mg of 2- 13 C-labeled uracil).

[0146] (2) experiment

[0147] Fasted beagle dogs were used as experimental animals. In group 1 (n=3), the preparation prepared above for determining pyrimidine metabolizing ability was orally administered (50 mg/10 ml/body) (test group). In Group 2 (n=3), (E)-5-(2-bromovinyl)-uracil preparation (DPD inhibitor) prepared in the same manner as in Example 1(2) was orally administered ( 83.3 mg/15 ml/body), and 1 hour later, the preparation for determining pyrimidine metabolizing ability was orally administered (50 mg/10 ml/body) in the same manner as Group 1 (control group: animal model of uracil metabolic disorder). In both groups, after oral administration of the preparation for determination of pyrimidine metabolizing ability, 10 ml of water was forcibly orally administered with the test tube used in the administration of the preparation in order to wash away the preparation remaining in the test tube. In both groups, breath was collected before and at 15, 30, 45, 60, 75, 90, 105, 120, 135, 150, 180, 210, and 240 minutes after formulation administration to determine pyrimidine metabolizing capacity. Collected exhaled exhaled 13 C labeled CO 2 The concentration of was detected in the same manner as in Example 1, using a GC-MS analyzer (ABCA-G, Europa Scientific). image 3 Shown are the results of the test group (-◆-) and the control group (-■-). exist image 3 In, the definition of abscissa and ordinate is the same as in Example 1.

[0148] as image 3 At a glance, in the test group, when the formulation of the present invention was administered orally, it was observed that 2- 13 C-labeled uracil is metabolized and released in exhaled 13 CO 2 Excretion, similar to the case of intravenous administration (Example 1). On the other hand, in the animal model (control group) in which uracil disorder was artificially created by pre-administration of DPD inhibitors, the excreted 13 CO 2 Significantly reduced. Thus, by checking the exhaled 13 CO 2 The excretion behavior proves that the active ingredient contains 2- 13 The use of the preparation according to the invention of C-labeled uracil (in oral form) makes it possible to detect and evaluate the presence or absence of a disorder of uracil metabolism (decrease in the ability to metabolize uracil), which is caused by the activity of DPD caused by inhibition or reduction.

[0149] The results obtained in Example 1 and Example 2 demonstrate that the preparation prepared according to the present invention can be used to easily and accurately determine the presence or absence of pyrimidine metabolic disturbance (decreased or increased pyrimidine metabolizing ability) in vivo by using exhaled breath.

[0150] Example 3

[0151] 56.5 mg, 113.1 mg, 226.2 mg and 452.3 mg of 2- 13 C-labeled uracil (Cambridge Isotope Laboratory) to prepare solutions with concentrations of 10 μmol/ml, 20 μmol/ml, 40 μmol/ml and 80 μmol/ml. These solutions were forcibly administered orally to fasting male Beagle dogs (n=3) using oral test tubes for dogs at a dose of 1 ml/kg body weight (doses: 10, 20, 40 and 80 μmol/kg), after which water was administered in 2 ml Amounts per kg body weight are administered orally to dogs compulsorily. Exhaled air was collected before administration and at 15, 30, 45, 60, 75, 90, 105, 120, 135, 150, 180, 210, and 240 minutes after administration. 13 C labeled CO 2 concentration, and pharmacokinetic parameters (area under the curve (AUC) and maximum 13 C labeled CO 2 Concentration (Cmax)) obtained from exhaled 13 C labeled CO 2 Calculated from the concentration transition curve.

[0152] Figure 4 shown exhaling 13 C labeled CO 2 Concentration transition curve. Figure 5 and Image 6 Respectively represent the relationship between dose (μmol/kg body weight) and the area under the curve (AUC), as well as the dose in the expiratory breath and the maximum 13 C labeled CO 2 Concentration (Cmax) relationship.

[0153] Figure 4 Show exhaling 13 C labeled CO 2 Concentrations reach a maximum 15-30 minutes after application and then decline rapidly. like Figure 5 and Image 6 As shown, the graph of AUC and Cmax versus dose is a straight line almost passing through the origin, indicating a linear relationship between dose and AUC and dose and Cmax. This means exhaling 2- 13 The excretion kinetics of C-labeled uracil is linear at doses of 10-80 μmol/kg body weight.

[0154] When containing 2- 13 The preparation of the present invention of C-labeled uracil was orally administered to subjects whose pyrimidine metabolizing ability was unknown at a dose of 40 μmol/kg body weight, and the 13 C labeled CO 2 The largest measured concentration 13 C labeled CO 2 Concentration (Cmax), and found to be 50‰, from Image 6 It was confirmed that the pyrimidine metabolizing capacity in the subjects was reduced to about 1/2 of the normal pyrimidine metabolizing capacity. (exist Image 6 Among them, Cmax=50‰ corresponds to 2- 13 C-labeled uracil administered at 20 μmol/kg body weight).