ラットを用いたアザチオプリン反復投与毒性試験における毒性評価に及ぼすマイクロサンプリング法の影響

P-225〇川口 瞬1，高橋 一彰1，船越 武1，中井 恵子1，山本 大1，岡村 隆之1，田中 庸一2，斎藤 嘉朗2, 中川 宗洋1 1株式会社ＬＳＩＭ安全科学研究所, 2国立医薬品食品衛生研究所

Effect of microsampling method on toxicity evaluation of azathioprine in repeated dose toxicity studies in rats
〇Shun Kawaguchi1, Kazuaki Takahashi1, Takeshi Funakoshi1, Keiko Nakai1, Dai Yamamoto1, Takayuki Okamura1, Yoichi Tanaka2, Yoshiaki Saitou2, Munehiro Nakagawa1
1LSIM Safety Institute Corporation 2National Institute of Health Sciences

Blood chemistry

Background and Purpose
Microsampling (MS) method is an useful technique used in toxicokinetics (TK) studies in which a verysmall amount of blood (less than 50 µL) is collected to determine the concentrations of a drug and its
metabolites. The high sensitivity of analytical instruments has made it possible to measure with small
amounts of samples, allowing TK analysis in the main study group of the toxicity study. Therefore, thistechnique can contribute to reducing the number of animals used.
On the other hand, there have not been enough reports on the effect of MS method on toxicity evaluation.
We have presented the effects of differences of the device and blood collection site on the evaluation,and the results comparing the MS method with the conventional method with drugs that induce
hepatotoxicity and myelotoxicity. It has been suggested that the stress on animals caused by the MS
method may affect the evaluation of immune system. Therefore, we investigated the effects of the MS
method on repeated-dose toxicity evaluation using azathioprine with reports on immunotoxicity.

Materials and Methods[Animals]
Crl:CD(SD) rats (Female), 6 weeks old[Dosing frequency]
Once daily, 4 weeks[Test article]
Azathioprine (Vehicle: 0.5 w/v% Methyl Cellulose; MC)[Microsampling method] About 50 µL/points from subclavian vein without anesthesia
Device: BD Lo-DoseTM Insulin syringe 29G(Becton, Dickinson and Company)[Examination items]
Clinical signs, Body weight, Food consumption, Urinalysis (Clinitek advantus, TBA-2000FR), Hematology (CA-500,XT-2000iV), Blood chemistry (Epalyzer2, TBA-2000FR), Pathology: Organ weight (*), Necropsy, Histopathology(Liver*, spleen*, kidney*, heart*, lung/bronchus*, thymus*, femur/bone marrow, mandibular lymph
node, mesenteric lymph node), TK analysis (Analyte: 6-mercaptopurine [azathioprine metabolite],
Points: Before dosing (only final dosing), 0.5, 1, 2, 4, 8 and 24 hours after dosing)[Statistical analysis]
The Data on body weight, food consumption, hematology, blood chemistry, urinalysis, and organ
weights were statistically analyzed as follows. The safety study system (tsPharma Labsite, Fujitsu
Limited) was used for the analysis.
1. The effects of azathioprine administrationThey were analyzed by comparing 0 mg/kg (control) with 3 and 10 mg/kg in the MS group and the nonblood sampling group. The Dunnet or Steel test was performed.
2. The effects of the MS method
They were analyzed by comparing the same doses between the non-blood sampling group and the MSgroup. The t-test or Welch's t-test was performed.
[Group composition]

V.S. Control：#P<0.05, ##P<0.01

V.S. Non-blood sampling group：*P<0.05, **P<0.01(V.S. Control)↓: Decrease, ↑: Increase

10 mg/kg: Total Cholesterol↑, Phospholipid↑, Ca↑, Total protein↑,Albumin↑, and α1 Globulin↑
3 mg/kg: Phospholipid↑, A/G↑, and Albumin↑➡The effects of azathioprine administration were seen in the MS group as well as in the nonblood sampling group. Therefore, no effects on the toxicity evaluation by the MS method werenoted.
↓: Decrease, ↑: Increase(V.S. Non-blood sampling group)

10 mg/kg: Total protein↓, Albumin↓, 3 mg/kg: A/G↓, Albumin↓, α1 Globulin↑, 0 mg/kg: Ca↑➡Attenuation of the effects of azathioprine caused by the MS method was noted; however, it
was within the range of the background data and considered to have no effects on theevaluation of azathioprine toxicity. (Green circle)

Pathological examination*: Control (0.5 w/v% MC)

V.S. Control：#P<0.05, ##P<0.01(V.S. Control)

Low thymus weight, macroscopic smallness, and decreased lymphocytes in thymic cortex werenoted.

Result➡The effects of azathioprine administration were seen in the MS group as well as in the nonblood sampling group. Therefore, no effects on the toxicity evaluation by the MS method werenoted.

Clinical signs, Body weight, Food consumption, Urinalysis
No effects on toxicity evaluation by the MS method were observed.

(V.S. Non-blood sampling group)➡No effects on toxicity evaluation by the MS method were noted.

Hematology

TK analysis【Initial dosing】

V.S. Control：#P<0.05, ##P<0.01

Plasma 6-mercaptopurine concentration increased with increasing dose. Moreover, no effects on
the exposure level were observed after repeated administration.

V.S. Non-blood sampling group：*P<0.05, **P<0.01(V.S. Control)↓: Decrease

10 mg/kg: RBC↓, HGB↓, HCT↓, WBC↓, Lymphocyte↓, Eosinophil↓, and Monocyte↓3 mg/kg: Eosinophil↓ and Monocyte↓➡The effects of azathioprine administration were seen in the MS group as well as in the nonblood sampling group. Therefore, no effects on the toxicity evaluation by the MS method
were noted.
(V.S. Non-blood sampling group)【Final dosing】↓: Decrease

0, 3 and 10 mg/kg: RBC↓, HGB↓, and HCT↓
➡Significant differences in erythrocyte system parameters caused by the MS method werenoted; however, these changes were within the range of the background data and considered
to have minor effects on the evaluation of azathioprine toxicity. (Red circle)

Conclusion
1. The results of this study suggest that the effect of the MS method in the evaluationof azathioprine toxicity is limited to minor changes within the background data andthat the MS method can be used for evaluation of immune system parameters.
2. The MS method was found to affect erythrocyte parameters as a previous report1).
It was deemed necessary to take into account toxicological characteristics of test
articles when using the MS method for repeated-dose toxicity studies.

Reference1) Yui Akagawa, Toxicity Evaluation in Repeated-Dose Toxicity Studies in Rats and Effects of Microsampling Methods
on Pharmacokinetics. Poster presented at: The 45th Annual Meeting of the Japanese Society of Toxicology, July 18,
2018; Osaka.