Effect of blood microsampling (50 μL) on toxicological assessment in rats treated with tacrine, a drug known to have adverse effects that increase neutrophils

Abstract

To promote the 3Rs in toxicological assessment, the recommendation for blood microsampling in toxicokinetic evaluation is noted in the ICH Harmonized Guideline S3A Q&As. However, there are only a few articles reporting the practical application of microsampling in the toxicological assessment with toxic drugs. In this study, we investigate the effect of microsampling on toxicological assessment in rats treated with tacrine, which is known to have toxic effects that induce an increase in neutrophils and behavioral abnormalities. Thirty female Sprague-Dawley rats were divided into microsampling (MS) and non-microsampling (non-MS) groups, and orally administered tacrine once daily at dose levels of 0 (vehicle only), 3 and 10 mg/kg bw for 28 days (each group: n=5). In the MS group, blood samples (50 μL/time point) were collected at 6 time points on day 1 and 7 time points on day 28 to 29. All the animals underwent necropsy on day 29. By comparing the results of toxicological and toxicokinetic analysis between the MS and non-MS groups, we validated effects of microsampling for toxicological assessment. Although increase in neutrophils and repeated stereotypic behaviors were observed as toxic effects in the rats administered tacrine, we could not find any difference between the MS and non-MS groups, and also found that microsampling did not affect any other data from toxicological and toxicokinetic analysis. In conclusion, blood microsampling appeared to be a feasible technique for the toxicity study of tacrine and was considered to be applicable in the toxicity study of even drugs with toxic effects on hematological parameters, such as an increase in neutrophils.

INTRODUCTION

Toxicokinetic evaluation in a non-clinical study estimates the systemic exposure of a drug candidate and provides important information to assess the relationship between safety and exposure in the development of a new drug (ICH, 1994). In toxicokinetic assessment, blood samples are taken from experimental animals at regular intervals after administration of a drug candidate, and exposure is estimated from blood concentration of the test drug. In rodents which are commonly used in toxicity studies, since the amount of blood that can be collected from the circulation is limited, a satellite group is usually established for toxicokinetic evaluation. In case of toxicokinetic study in rats, approximately 200 μL of blood is collected per time point for exposure estimation.

Recently, due to advances in the physicochemical and analytical technology, it has become possible to determine the drug concentrations in minute amounts of blood samples with high reliability and reproducibility. The development of such measurement techniques makes it feasible to incorporate blood microsampling into a toxicity study and blood microsampling is being positively introduced into toxicokinetic evaluation. Microsampling is thought to potentially contribute to the 3Rs in a non-clinical study by reducing animal distress through a decrease in total amount of blood sample per individual and by reducing the number of animals used in toxicokinetic evaluation or by reducing/abolishing set of a satellite group. In addition, as another advantage of microsampling, it is expected that data for safety and exposure estimation can be obtained from the same animal, allowing a direct evaluation of the relationship between safety and exposure.

In order to promote blood microsampling in toxicological assessment, we have previously investigated the effect of blood microsampling on toxicological and/or toxicokinetic parameters in rats commonly used in non-clinical toxicity studies under a research project organized by the Japan Agency for Medical Research and Development (AMED). We demonstrated that microsampling from the jugular and tail veins was unlikely to interfere with toxicological parameters in a 4-week repeated dose rat toxicity study and also showed that (Yokoyama et al., 2020), regardless of whether blood was collected from the jugular or tail vein, any significant effects of blood microsampling on toxicokinetic parameters were not observed and that this technique was feasible with no apparent differences between multiple centers (Hattori et al., 2020). Furthermore, the effect of blood microsampling on toxicological parameters has been studied in toxicity assessments of drugs known to have adverse effects, and 4-week repeated dose rat toxicity studies of some drugs, including phenacetin (Ohtsuka et al., 2022), methapyrilene hydrochloride (Hattori et al., 2023), azathioprine (Tanaka et al., 2023), indicated that microsampling had no influence on most toxicological parameters. The results showed that under the conditions of a 4-week repeated dose toxicity study in rats, microsampling does not affect the toxicity evaluation. On the other hand, although the fluctuation ranges were not large, small changes were observed in white blood cell parameters (especially neutrophils) (Hattori et al., 2023; Tanaka et al., 2023), and considering the number of animals in this validation study, it is possible that these changes may affect the toxicity evaluation. Therefore, careful consideration is required in these evaluations when applying microsampling.

In this study, therefore we selected tacrine, a cholinesterase inhibitor known to adversely induce an increase in neutrophil count and repetitive stereotypic behaviors (self-biting and gnawing) in repeated dose study and conducted a 4-week repeated dose rat toxicity study of tacrine in condition with or without microsampling. Through consideration about the results of this toxicity study, we evaluated the effect of blood microsampling on toxicological parameters in 4-week repeated dose rat toxicity study and verified whether blood microsampling is applicable to toxicity study of a drug which has adverse effect on neutrophils.

MATERIALS AND METHODS

Organization and animal

The animal experiments were conducted at the Research Institute for Bioscience Products and Fine Chemicals, Ajinomoto Co., Inc. The procedures for animal experiment were approved by the Ethics Committee of the Ajinomoto Co., Inc. based on the Guideline for Animal Care and Use of the Experimental Facility. The concentration of tacrine in plasma samples was measured at the National Institute of Health Sciences.

Female rats are assumed to be more susceptible to the effect of blood sampling, since their total amount of circulating blood is relatively small (Hattori et al., 2020; Diehl et al., 2001). Therefore, female rats (Crl:CD(SD)) aged 5 weeks were purchased from Jackson Laboratory Japan, Inc. (Kanagawa, Japan) and used in this study. The animals were allowed free access to the rat chow CRF-1 (Oriental Yeast Co., Ltd., Tokyo, Japan) and drinking water (tap water), and were maintained at a temperature of 20-26°C and at a relative humidity of 30-70% with a flow of 10-15 air changes per hour. Room lighting was controlled to provide a 12-hr light cycle.

Study protocol

A 4-week repeated oral dosing study in rats was conducted in reference to the general toxicity study guideline (ICH S4) and toxicokinetic evaluation guideline (ICH S3A). After a quarantine and acclimation period of approximately one week, the animals were assigned to each group (n=5) by stratified randomization based on body weight. Tacrine treatment started at 6 weeks of age.

Administration

We decided the administration dose of tacrine at 3 mg/kg body weight (bw) and 10 mg/kg bw, which were known to be the no observed adverse effect level (NOAEL) and the lowest observed adverse effect level (LOAEL), respectively (Igarashi et al., 2015). Tacrine was shown to induce an increase in neutrophils and repetitive stereotypic behaviors at the LOAEL dose. Tacrine (Sigma-Aldrich, St. Louis, MO, USA) was suspended in 0.5% methylcellulose solution (FUJIFILM Wako Pure Chemical Co., Ltd., Osaka, Japan) at concentrations of 0.6 and 2 mg/mL. The prepared solutions were orally administered by gavage once daily for 4 weeks at a volume of 5 mL/kg bw.

Observation and examinations

Thirty female Sprague-Dawley rats were divided into 6 groups (n=5): vehicle (non-microsampling (MS)), 3 mg/kg bw tacrine (non-MS), 10 mg/kg bw tacrine (non-MS), vehicle (MS), 3 mg/kg bw tacrine (MS) and 10 mg/kg bw tacrine (MS). The following observations, examinations and analyses were performed.

General observation, body weight, food and water consumption

To see whether repetitive stereotypic behaviors such as self-biting and gnawing occurred, rats were monitored for 15 min each day after tacrine administration. Body weight and food consumption were assessed at least once a week, and water consumption was measured once during the 4^th week of tacrine treatment.

Ophthalmology, hematology, blood chemistry and urinalysis

Ophthalmological examinations were carried out on all animals before the treatment and once during the 4^th week of tacrine treatment. After macroscopic observation, tropicamide-phenylephrine ophthalmic solution (Midrin® P ophthalmic solution, Santen Pharmaceutical Co., Ltd., Osaka, Japan) was instilled, and the anterior segment and intermediate optic body were observed using a head-mounted binocular indirect ophthalmoscope (Omega 200, Heine Optotechnik GmbH & Co., Gilching, Germany) and a portable slit lamp (Kowa SL-15, Kowa Co., Ltd., Tokyo, Japan), and the fundus was observed using a head-mounted binocular indirect ophthalmoscope.

Urinalyses were carried out using fresh urine and 24-hr accumulate urine collected during the 4^th week of tacrine treatment. Parameters measured in a urinalysis included urine volume, specific gravity, pH, glucose, protein, occult blood, ketone bodies, bilirubin, urobilinogen and electrolytes (Na, K, and Cl). Measurements were performed using an automatic urine analyzer (Clinitek Advantus, Siemens Healthcare Diagnostics, K.K., Tokyo, Japan), Multistix test paper (Siemens Healthcare Diagnostics, K.K., Tokyo, Japan), an automatic electrolyte analyzer (EA07, A&T Corporation, Kanagawa, Japan) and urine specific gravity refractometer (UG-D, Atago Co., Ltd., Tokyo, Japan).

After overnight fasting (for 18-hr) the day before necropsy, blood samples were collected from the caudal vena cava under the anesthesia with isoflurane, and then all animals were exsanguinated to death.

Hematological analyses were carried out on the day of necropsy. Parameters measured in a hematological analysis included red blood cell (RBC) count, hematocrit (HCT), hemoglobin (HGB), mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), mean corpuscular hemoglobin concentration (MCHC), platelet count (PLT), white blood cell count (WBC), neutrophils count and ratio (NEUT), lymphocytes count and ratio (LYM), monocytes count and ratio (MONO), basophils count and ratio (BASO), eosinophils count and ratio (EOS), large unstained cells count and ratio (LUC), reticulocytes count and ratio (RETIC), fibrinogen (FBG), prothrombin time (PT), and activated partial thromboplastin time (APTT). The measurements were performed using a comprehensive hematology analyzer (ADVIA2120, Siemens Healthcare Diagnostics, K.K.), and an automatic blood coagulation analyzer (CS2000i, Sysmex Corporation, Hyogo, Japan).

Parameters measured in blood biochemical analysis included aspartate aminotransferase (AST), alanine aminotransferase (ALT), alkaline phosphatase (ALP), lactate dehydrogenase (LDH), creatine phosphokinase (CPK), glucose, total cholesterol, triglyceride, blood urea nitrogen (BUN), creatinine (CRE), total bilirubin, phospholipid, total protein, albumin, calcium (Ca), inorganic phosphorus (Inorganic P), Na, K, Cl, and albumin/globulin ratio (A/G ratio). Measurements were performed using a clinical chemistry autoanalyzer (TBA-120FR, Canon Medical Systems Corporation, Tochigi, Japan) and an automatic electrophoresis analyzer (Epalyzer 2 Junior, Helena Laboratories Corporation, Saitama, Japan).

Organ weight, necropsy and histopathology

After the exsanguination, following macroscopic examination of various organs throughout the body, the weights of the brain, pituitary gland, submandibular gland, thymus, heart, liver, spleen, kidneys, adrenal glands and ovaries were measured, and the body weight ratio was calculated based on the body weight on the day of necropsy. The weights of bilateral organs, including the kidneys, adrenal glands and ovaries were measured separately on the right and left sides, and their combined values were used for evaluation. The brain, liver, kidneys, heart, lungs, thymus, spleen, cervical lymph nodes, mesenteric lymph nodes, bone marrow (sternum), spinal cord and organs with macroscopic abnormalities were fixed in 10% neutral buffered formalin. Tissue specimens prepared from these organs were stained with hematoxylin and eosin (HE), and subjected to histopathological examination under a light microscope.

Blood microsampling to presume toxicokinetics of tacrine

On day 1 and day 27 of tacrine treatment, the blood microsamplings were conducted using heparinized Lo-Dose 3/10mL syringes and 29G injection needles (Nippon Becton Dickinson Co., Ltd., Tokyo, Japan). In the microsampling groups (vehicle, 3 mg/kg bw tacrine, and 10 mg/kg bw tacrine), 50 μL of blood was collected from all animals at each time point. No blood samples were collected from the corresponding non-microsampling groups. On day 1 of tacrine treatment, blood samples (50 μL) were collected from the jugular vein of rats without anesthesia at 0.5, 1, 2, 4, 8 and 24 hr after tacrine administration. On day 27 of tacrine treatment, blood samples (50 μL) were collected from the jugular vein of rats without anesthesia at 0, 0.5, 1, 2, 4, 8 and 24 hr after tacrine administration. Plasma samples were prepared for toxicokinetic analysis from the collected blood samples.

Toxicokinetic analysis

Tacrine concentrations in plasma samples were determined by a reversed-phase liquid chromatography-spectrometry system. We used a modified sample extraction method reported by Ponnayyan Sulochana et al. (2016). Briefly, 10 μL of plasma samples was mixed with 5 μL of 500 ng/mL phenacetin as internal standard. The sample mixtures were extracted with 800 μL of ethyl acetate; the mixture was vortexed for 5 min, followed by centrifugation for 10 min at 10,000 rpm. The organic layer (750 μL) was transferred to other tubes and evaporated to dryness at 40°C with nitrogen spray for 10 min. The residue was reconstituted in 40 μL of 0.2% formic acid in acetonitrile. After filtration using 0.2 μm PTFE membrane filter and 10 μL of mixture was injected into the ultra-high performance liquid chromatography (UHPLC) system. Chromatographic separation was performed using Ultimate 3000 UHPLC system (ThermoFisher Scientific, Waltham, MA USA) with ZORBAX SB-C18 (1.8 μm, 2.1 × 100 mm) column (Agilent Technologies, Santa Clara, CA, USA) maintained at 40°C. Mobile phase was 0.2% formic acid in acetonitrile, and the flow rate was 0.4 mL/min. Detection of tacrine and IS were performed using TSQ Vantage Triple-Stage Quadrupole Mass Spectrometer (ThermoFisher Scientific, Waltham, MA, USA) in positive multiple reaction monitoring mode. The range of calibration curve was made from 1 to 500 ng/mL and its linearity was R² > 0.98. The limit of detection and limit of quantification were 1 ng/mL and 5 ng/mL, respectively. The intra- and inter-assay relative errors were < 9% and < 6%, respectively. The intra- and inter-assay coefficient variations were < 3% and < 8%, respectively.

The maximum concentration of the plasma tacrine in each treated rat was determined from the time-course fluctuation obtained from the LC/MS data and the mean maximum drug concentration (C_max) was calculated for each treated group. The time when the blood tacrine concentrations reached the maximum level was estimated in each treated rat and the mean value was calculated as a time to the maximum plasma concentration (T_max) for each treated group. The area under the concentration-time curve (AUC_0-24hr) was determined from the fluctuation data of the blood tacrine levels, and the mean value for each treated group was calculated using the trapezoidal method.

Statistics and data analysis

Data were presented as means and standard deviations on body weight, food consumption, water consumption, organ weight (absolute and relative weight), and hematology, blood chemistry and urinalysis, and were statistically analyzed by Bartlett’s test for homogeneity of variance (two-tailed test, judged significant if p < 0.01). When the variance was homogeneous, significant differences in the means between the control group and tacrine treatment groups were analyzed by Dunnett’s multiple comparison test (two-tailed test, judged significant if p < 0.05). If the variances were not homogeneous, the average of the ranks between the control group and tacrine treatment groups was analyzed by Steel’s multiple comparison test (two-tailed test, judged significant if p < 0.05). Furthermore, statistical analysis was not performed on the observed symptom data.

RESULTS

Clinical observation

Data on repetitive stereotypic behaviors such as self-biting and gnawing are presented in Table 1. All animals survived to the end of the study. Six days after tacrine treatment, the stereotypic behaviors of both self-biting and gnawing were observed in the group that received 10 mg/kg bw of tacrine. Fourteen days after tacrine treatment, the stereotypic behaviors occurred in the group that received 3 mg/kg bw of tacrine. Regardless of whether animals received tacrine or not, no notable differences in the occurrence of stereotypic behaviors were observed between the non-MS and MS groups.

Table 1. Stereotypic behaviors (self-biting and gnawing) data for rats administered tacrine for 28 days.

Body weight, food and water consumption

The data on body weight and food consumption are presented in Table 2. All animals grew with age and, regardless of whether animals received tacrine or not, no notable differences in body weight and weight gain were observed between the non-MS and MS groups. There were also no differences in water consumption among all six groups.

Table 2. Body weight and food consumption data for rats administered tacrine for 28 days.

Ophthalmology and clinical pathology

Urinalysis and ophthalmological examination showed no notable alterations in all six group or differences among all six groups (data not shown).

Data on hematological analysis and blood biochemical analysis are presented in Table 3 and Table 4. In the rats given tacrine at the dose of 10 mg/kg bw, a non-significant but slight increase in neutrophils was observed, compared with the vehicle groups. There were no differences between the MS and the non-MS groups, although increasing trends in neutrophil counts and percentage were observed in MS groups compared with non-MS groups. In the 3 mg/kg bw tacrine (MS) group, PT was increased compared to the vehicle (MS) group, and fasting blood glucose level was also increased in the 3 mg/kg tacrine (non-MS) group compared to the vehicle (non-MS) group. These effects did not show a dose-dependency, so we decided that these events were incidental. T-Bil was significantly decreased in the 10 mg/kg bw tacrine (MS) group compared to the vehicle (MS) group. However, since the variance of the alteration was within the background data range (mean ± 2SD) of the rats used in this study, this decrease in total bilirubin was considered to be due to physiological variation and not an effect caused by tacrine.

Table 3. Hematology data for rats administered tacrine for 28 days.

Table 4. Blood biochemistry data for rats administered tacrine for 28 days.

Organ weight, necropsy and histopathology

The weight data of the organs are presented in Table 5. There are no differences in organ weight data among all six groups.

Table 5. Organ weight data for rats administered tacrine for 28 days.

Macroscopic examination revealed hemorrhagic lesions at the cervical sampling site in the MS groups (Table 6). No other abnormalities were found. Histopathological examination of the cervical sampling site revealed muscle fiber degradation was observed in the 3 of 5 rats in the vehicle (MS) group and all rats in the tacrine (MS) groups, and cellular infiltration in 4 of 5 rats in the vehicle (MS) group and all in the tacrine (MS) group (Table 7). Other pathological findings, due to their low frequency and subtle property, were considered to be within the range of physiological variability and not attributable to tacrine or blood micro-sampling (data not shown).

Table 6. Necropsy findings in rats administered tacrine for 28 days.

Table 7. Histopathological examination data of rats administered tacrine for 28 days.

Toxicokinetic analysis

Toxicokinetic data are presented in Table 8. It was observed that C_max and AUC_0-24hr were increased in a dose-dependent manner of tacrine. C_max and AUC_0-24hr obtained on day 27 of tacrine treatment were higher than those obtained on day 1 of the treatment. The toxicokinetic data in this study are similar to the data from humans treated with tacrine (Cutler et al., 1990). The mechanistic reason for the apparent accumulation of tacrine in Day 27 is unclear.

Table 8. Toxicokinetic parameters for tacrine in rat plasma

DISCUSSION

In this study, we investigated whether blood microsampling affects toxicological parameters in 4-week repeated dose rat toxicity study of tacrine, a cholinesterase inhibitor known. In this toxicity study, irrespective of blood microsampling treatment, no deaths were observed. No significant alterations in body weight, food consumption, organ weight, urinalysis, ophthalmology and hematology were observed in tacrine administration, and we also found that blood microsampling had no influences on body weight, food consumption, organ weight, urinalysis, ophthalmology and hematology. Hematological tests showed a slight increase in neutrophil count as a side effect of tacrine, but the difference was not significant. Furthermore, the increase in neutrophil count observed in the MS group was similar to that in the non-MS group. Furthermore, no differences in the onset or frequency of repetitive stereotypic behaviors occurrences were found between the MS and non-MS groups, indicating blood microsampling did not also affect behavioral abnormalities caused by tacrine.

In macroscopic examination, hemorrhagic lesions and inflammatory reactions were found at the cervical sampling site in the MS groups. In the review article on the impact of blood microsampling, it is noted that the multicenter studies have confirmed that the hemorrhagic lesions did not affect toxicological parameters (Takahashi et al., 2024). Since no influences of the blood microsampling were also found in our study, including hematological parameters, we considered that, if the amount of bleeding from the sampling site is slight, the influences of hemorrhage on the toxicological parameters is negligible.

Focusing on the changes in neutrophils, in this study, both neutrophil counts and percentages in the MS group tended to increase compared to the non-MS group, and that the baseline level was elevated due to microsampling (Neutrophil count (103/µL) (non-MS → MS); vehicle: 0.69 ± 0.39 → 0.99 ± 0.38, 3 mg/kg bw tacrine: 0.77 ± 0.37 → 0.98 ± 0.38, 10 mg/kg bw tacrine: 13.8 ± 7.4 → 15.9 ± 4.7). At the high dose (LOAEL), although there was no significant difference, neutrophil counts in both groups tended to increase, which is thought to be a change due to the effects of tacrine. In our previous study (Tanaka et al., 2023), both neutrophil counts and percentages also tended to increase in the MS group compared to the non-MS group, and that the baseline level is elevated due to MS, as with tacrine in this study. In contrast to this study, at the high dose, although there was no significant difference, neutrophil counts in both groups tended to decrease, which is thought to be a change due to the effects of azathioprine. These baseline changes in the MS group was suggested to be very mild inflammatory reaction due to invasive effects of sampling such as bleeding at the blood collection site.

Blood microsampling is becoming positively introduced in toxicokinetic evaluation, but this technique is not yet widespread enough. This is due to the concern that the total amount of the blood collected by microsampling is reasonably large and may impact the toxicological assessments. To address this concern, sparse sampling method is proposed to be adopted for toxicokinetic evaluation ICH. (2017). Generally, in toxicity study applying microsampling, the blood samples for toxicokinetic evaluation are not collected from all the animals in the experimental group (only 3 to 4 animals are used for microsampling). In sparse sampling method, to reduce the load of blood samplings, the animals in each experimental group are allocated for blood sampling at different time points and the load of blood samplings is evenly distributed among the experimental animals. In this study, as in previous research (Tanaka et al., 2023), blood samples were taken from each animal at 6-7 time points, with 50 μL of blood collected each time. At the 24-hr post-administration blood collection point, approximately 300-350 μL of blood in total was collected from the same animal, which did not have an impact on this rat study. However, we believe that reducing the number of blood collections will also reduce the total blood volume collected and minimize the impact, which would be needed especially for mice (or child animal) study bearing lower blood volumes. In other words, by combining sparse sampling and microsampling, it is possible to reduce the total amount of blood collected from each individual, thereby reducing the burden on the animals. One problem is that if the sparse blood sampling method is used for the toxicokinetic estimation of drugs, it will not be possible to evaluate the direct association between the safety and exposure since the data for safety and exposure assessment cannot be obtained from one same animal. Despite the remaining obstacles, microsampling can still contribute to the 3Rs by reducing animal distress and by abolishing set of a satellite group for the toxicokinetic evaluation. We expect that the obstacles to introduce blood microsampling in the safety assessment of drugs will be removed one by one through the solution of issues, such as incorporation of the sparse sampling method and that microsampling will become more widely use in the future.

In conclusion, we have shown that blood microsampling did not affect the toxicological parameters in the toxicity study of tacrine, and our study result indicates that blood microsampling may also be applicable to the toxicological assessment of drugs which have toxicologically adverse effect on neutrophils. However, few studies have yet reported application and feasibility of blood microsampling, and further studies are needed to validate this novel technique.

Funding

This study was supported in part by AMED under Grant nos. JP20ak0101073j0004 and JP21ak0101073j0005.

Conflict of interest

The authors declare that there is no conflict of interest.

Data availability

The data in this study are included in the article/supplementary materials. Contact the corresponding author(s) directly to request the underlying data.

Author contributions

Conceptualization: Norimichi Hattori, Yusuke Shibui, Yoichi Tanaka, Yoshiro Saito

Funding acquisition: Yoshiro Saito

Investigation: Norimichi Hattori, Yusuke Shibui, Yoichi Tanaka, Yoshiro Saito

Supervision: Norimichi Hattori, Yusuke Shibui, Yoichi Tanaka, Yoshiro Saito

Visualization: Norimichi Hattori, Yusuke Shibui, Yoichi Tanaka, Yoshiro Saito

Writing – original draft: Norimichi Hattori, Yusuke Shibui, Yoichi Tanaka, Yoshiro Saito.

Writing – review & editing: Norimichi Hattori, Yusuke Shibui, Yoichi Tanaka, Yoshiro Saito

Ethical approval and consent to participate

Not applicable.

Patient consent for publication

Not applicable.

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