In recent years, animal welfare has been attracting worldwide attention. In the field of pharmaceutical development, there has been a concerted effort to adhere to the principles of 3Rs: Replacement (utilizing alternatives to animal use wherever possible), Reduction (minimizing the number of animals used), and Refinement (reducing pain and suffering to animals to the greatest extent possible). In addition, the FDA Modernization Act 2.0, a new law that eliminates the requirement for animal use in new drug development, has been enacted in the U.S., and it is expected that the number of animal procedures will be gradually reduced in the future. However, it is not clear whether all animal procedures can be eliminated.

A method that has been attracting attention is microsampling (MS), which involves collecting a very small amount of blood. This technique is defined by the ICH S3A Q&As and typically refers to blood collection volumes of 50 µL or less. One of the reasons for the introduction of MS in nonclinical toxicity assessment is the increased analytical sensitivity of instruments such as mass spectrometers. This advancement has made it possible to measure drug concentrations using smaller blood samples.

At present, in general toxicity studies using rodents in nonclinical studies, it is common to set satellite animals specifically for blood sampling in addition to the main test animals for toxicity evaluation. On the other hand, by using MS to collect samples from the main test animals for toxicokinetic (TK) evaluation, it becomes possible to analyze the relationship between toxicity evaluation values and TK values for each individual animal. This approach also contributes to 3Rs by reducing or eliminating the need for satellite animals and minimizing the amount of blood collected. Furthermore, by reducing the number of animals used, the amount of test substance required can be minimized, which is expected to lower development costs.

Advantages of Microsampling

As an example, this is a study design introducing microsampling (MS) to a rat 2-week repeated dose toxicity study. Typically, 5 males and 5 females are used in each toxicity evaluation group, and 4 males and 4 females are used in each TK satellite group. When MS is applied, blood is collected from the toxicity evaluation group, eliminating the need for animals in the TK satellite group. As a result, the amount of test substance used can be reduced by 44%.

Six-week-old female Crl:CD(SD) rats were divided into two groups: the MS group, in which MS blood was drawn from the subclavian vein using microsampling, and the non-blood sampling group, in which no blood was drawn. Azathioprine (immunosuppressant) was administered orally to each group at doses of 0, 3, and 10 mg/kg/day for 4 weeks. Blood samples (50 µL/time point) were collected for TK measurement at 6 time points following the first administration and at 7 time points following the final administration. General condition observation, weight and food intake measurements, and urinalysis were performed during the administration period. On the day following the TK blood sampling, 24 hours after the final administration (Day 30), hematological test, blood biochemistry test, necropsy, organ weight measurement, and histopathological examination were performed. The results obtained were compared between the azathioprine group and the control group, and between the same dose in the MS and non-blood sampling groups. TK measurements were also performed using samples collected from the MS group at the first and final administrations.

*1: Control (0.5 w/v% MC)

As effects of azathioprine administration, lower WBC, lymphocyte, eosinophil, and monocyte levels were observed in the MS and non-blood sampling groups. There was a statistically significant difference in erythrocyte system parameters (red circles in the figure below) in the MS group compared to the non-blood sampling group, but the values were within the range of the institutional background data. In other words, there were no apparent differences between the same dose in the MS and non-blood sampling groups.

The effects of azathioprine administration were found to be similar between the MS and non-blood sampling groups. In addition, compared to the non-blood sampling group, there were statistically significant lower levels of total protein and albumin in the 10 mg/kg MS group, statistically significant lower levels of A/G and albumin and statistically significant higher level of α1 globulin in the 3 mg/kg group, and statistically significant higher level of calcium in the control group (green circles in the figure below), but these changes were within the range of institutional background data.

Effects of azathioprine administration included lower thymus weight, smaller thymus size, and decreased lymphocytes in the thymic cortex in the MS and non-blood sampling groups. There were no changes observed only in the MS group.

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

Since azathioprine is rapidly metabolized in vivo to 6-mercaptopurine, we measured the concentration of the metabolite 6-mercaptopurine. Plasma 6-mercaptopurine concentrations increased in a dose-dependent manner and were not affected by repeated administration. Plasma samples collected via MS were suitable for an accurate TK measurement.

At first administration

At final administration

The effects of azathioprine administration were similar in hematology and blood biochemistry tests and pathological examination in the MS and non-blood sampling groups. Comparisons between the same doses in the MS and non-blood sampling groups showed no apparent differences. These results show that the use of MS for general toxicity studies in rats allows for an accurate assessment of the effects of azathioprine.

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