Development of radioiodine-labeled mequitazine for evaluation of hepatic CYP2D activity

Background: As drug-metabolizing enzyme activities are affected by a variety of factors, such as drug-drug interactions, a method to evaluate drug-metabolizing enzyme activities in real time is needed. In this study, we developed a novel SPECT imaging probe for evaluation of hepatic CYP2D activity. Methods: Iodine-123- and 125-labeled 4-iodobenzylmequitazine (123/125I-BMQ) was synthesized with high labeling and purity. CYP isozymes involved in the metabolism of 125I-BMQ in mouse liver microsomes were evaluated, and the utility of 123/125I-was assessed from biological distribution and SPECT imaging evaluation in normal and CYP2D-inhibited mice. Results: In vitro metabolite analysis using mouse liver microsomes showed that 125I-BMQ is specifically metabolized by CYP2D. Biological distribution and SPECT imaging of 123/125I-BMQ in normal mice showed that injection 123/125I-BMQ accumulated early in the liver and was excreted into the gallbladder and intestines. In CYP2D-inhibited mice, accumulation in the liver was increased, but accumulation in the gallbladder and intestines, the excretory organ, was delayed. Since only metabolites of 125I-BMQ are detected in bile, visualization and measuring of the accumulation of metabolites over time in the intestine, where bile is excreted, could predict the amount of metabolites produced in the body and evaluate CYP2D activity, which would be useful in determining the dosage of various drugs metabolized by CYP2D. Conclusion: 123/125I-BMQ is useful as a SPECT imaging probe for comprehensive and direct assessment of hepatic CYP2D activity in a minimally invasive and simple approach.


Introduction
After ingestion, drugs are converted to a hydrophilic form by cytochrome P450 (CYP) and other drug-metabolizing enzymes to facilitate excretion from the body (Ingelman-Sundberg et al., 1999;Rogers et al., 2002;Manikandan and Nagini, 2018).As the activity of drug-metabolizing enzymes varies between individuals, the effects of administered drugs can vary greatly as well (Singh et al., 2011;Reed and Backes, 2012;Ross et al., 2012).Individual-specific responses to drug administration are affected by many multifaceted and complex factors, such as the genotype with regard to drug-metabolizing enzymes, physiological factors (age, gender, body size, ethnicity), environmental factors (toxin exposure, diet, smoking), and even pathological conditions (liver and kidney dysfunction, diabetes, obesity, drug interactions), which can act alone or in combination to influence drug responses (Almazroo et al., 2017).Measuring the activity and capacity of drug-metabolizing enzymes in order to predict drug effects in individuals could be useful for selecting optimal drugs and determining optimal doses in personalized medicine, as well as for realtime measurement of individual drug responses.Although genotyping of CYPs is generally well established, the analyses have not been comprehensive, only evaluating genetic polymorphisms.A variety of approaches, such as serial blood, urine and breath sampling, have been used to comprehensively evaluate CYP activity (Michael et al., 2012;Matthaei et al., 2020).However, these approaches require specialized chemical analysers such as liquid chromatographs and mass spectrometers, which are not generally available in clinics and hospitals.A method for evaluating drug-metabolizing enzyme activities that is simple and applicable to many facilities is needed, and we have previously reported that SPECT imaging could be used to analyze drug metabolizing enzyme activities in mice and to evaluate the activities of hepatic carboxylesterase (Mizutani et al., 2018), CYP3A4 and 2D6 (Mizutani et al., 2022) by measuring the amounts of various radioactive metabolites accumulated in the gallbladder.The following conditions are considered important for SPECT imaging probes to evaluate drug-metabolising enzyme activity in the liver: 1) the SPECT imaging probe accumulate in metabolizing organs; 2) the SPECT imaging probe is metabolized by specific drug-metabolizing enzymes, and the metabolites are radioactive; 3) The radioactive metabolites are selectively transported from the metabolic organs to the excretory organs; and 4) the radioactive metabolites accumulate in excretory organs can be visualized and measured.In previous studies, 123/125 I-O-desmethylvenlafaxine was a SPECT imaging probe with the potential to directly and comprehensively detect and measure hepatic CYP3A4 and 2D6 activity (Mizutani et al., 2022).The new SPECT imaging probe in this study is based on the same concept as 123/125 I-Odesmethylvenlafaxine, but aims to specifically assess CYP2D6 activity only.Of the CYP isozymes, CYP2D6 contributes to the metabolism of approximately 13% of all prescription drugs (Williams et al., 2004).As many psychiatric prescription drugs, such as antidepressants and antipsychotics, are metabolized by CYP2D6, methods that allow estimation of CYP2D6 activity in each individual are very important an evidence-based medicine perspective.The purpose of this study was to evaluate hepatic CYP2D activity in mice as an initial study for future human application.

Metabolism of 125 I-BMQ in vitro
Animal studies were approved by the Animal Care Committee at Kanazawa University (AP-173851) and conducted in accordance with international standards for animal welfare and institutional guidelines.For preparation of pooled mouse liver microsomes (MLMs), three 6-week-old male ddY mice (Japan SLC, Tokyo, Japan) were euthanized under anesthesia using isoflurane.The liver of each mouse was removed and weighed.After adding 3 mL of Krebs-Ringer's phosphate buffer (pH 7.4) per gram of liver, the livers were pooled and homogenized using an ultrasonic homogenizer (SONIFIER250, Branson, MO, United States).The homogenate was centrifuged for 20 min at 9,000 g, and the supernatant was centrifuged for 60 min at 100,500 g.The resulting precipitate was suspended with Krebs-Ringer phosphate buffer and centrifuged for 60 min at 10,500 g.The resulting precipitate was obtained as microsomes, and the protein content was determined according to the bicinchoninic acid method (Smith et al., 1985).The pooled MLMs were stored at −80 °C.
To analyze CYP-mediated metabolism of 125 I-BMQ, nicotinamide adenine dinucleotide phosphate (NADPH) was used as an energy source for CYP.The NADPH-generating system created a CYP-activated state for 125 I-BMQ metabolism.Metabolism of 125 I-BMQ via CYP was carried out using the NADPH-generating system (0.5 mM β-NADP + , 5 mM MgCl 2 , 5 mM G6P, 1 U/mL G6PD), 100 mM sodium potassium phosphate buffer (pH 7.4), 50 μM EDTA disodium salt, and 1,000 μg protein/20 μL pooled MLMs in a final volume of 250 μL (NADPH [+]).The same mixture without the NADPH-generating system was used as control (NADPH [−]).Samples were incubated at 37 °C for 60 min with gentle shaking, and the reaction was stopped by adding 100 µL of ethanol.The samples were then centrifuged at 18,000 g for 5 min, and the supernatant of each sample was analyzed by thin layer chromatography (TLC).The supernatant was spotted onto a silica gel TLC plate 60 F 254 (Merck, Darmstadt, Germany), which was developed using methanol/acetic acid in a 100:1 ratio.After development and complete drying, the TLC plates were cut into 21 fractions, and the radioactivity associated with each fraction was measured using a γ-ray counter (AccuFLEX γ 7000, Aloka, Tokyo, Japan).The fractionation ratio of 125 I-BMQ, 125 I-BMQ, and other metabolites was calculated by dividing the radioactivity count for each fraction by the total radioactivity count.
A mixture consisting of 50 µL of NADPH-generating system (NADPH [+]), 50 µM disodium EDTA salt in 100 mM sodium potassium phosphate buffer (pH 7.4), 1,000 µg protein/20 µL pooled MLMs, and 25 µL of inhibitor in a final volume of 250 µL was used to examine the inhibition of 125 I-BMQ metabolism.Samples were incubated at 37 °C for 15 min with gentle shaking, and the reaction was stopped by addition of 100 µL of ethanol and centrifugation at 18,000 g for 5 min.The final supernatant was analyzed by TLC using methanol/acetic acid at a 100:1 ratio.The percentages of 125 I-BMQ metabolites in each sample were then calculated (n = 3).

Biological distribution of 125 I-BMQ in normal mice
125 I-BMQ was prepared to a specific radioactivity of 185 kBq/mL by adding saline.A total of 20 fasting 6-week-old male ddY mice were injected with 125 I-BMQ into the tail vein (18.5 kBq/100 µL/mouse), and after 2, 10, 30, 60, and 120 min, four mice each were euthanized under isoflurane, and blood, brain, thyroid, lung, heart, liver, gallbladder, stomach, pancreas, spleen, intestine, kidney, and urine tissue were collected.The tissue was weighted and the radioactivity was measured using a γ-ray counter to calculate the injected dose percent (%ID) or injected dose percent per gram of tissue (%ID/g).

Metabolism of 125 I-BMQ in vivo
125 I-BMQ was prepared to a specific radioactivity of 10 MBq/mL by adding saline and injected via tail vein into three mice (2.0 MBq/ 200 µL/mouse).After 30 min, each of the three mice was euthanized under isoflurane, and the gallbladder was removed.Radioactive products in the bile were analyzed by TLC using chloroform/ acetic acid/H 2 O at a ratio of 60:40:1.Bile was spotted directly onto the TLC plate.
2.6 Whole-body imaging of 123 I-BMQ in normal mice and CYP2D-inhibited mice 123 I-BMQ was labeled and purified according to the same method used for 125 I-BMQ.Whole-body SPECT imaging of normal mice was performed using a U-SPECT-II/CT system (MILabs, Utrecht, Netherlands).Two normal mice were injected with 14.5 MBq of 123 I-BMQ via the tail vein.Wholebody SPECT images were acquired under 2.0% isoflurane anesthesia every 10 min from 5 to 115 min after injection.The images were reconstructed using the filter-backed projection method, with 16 subsets and six iterations.The voxel size was set to 0.8 mm × 0.8 mm × 0.8 mm.Neither attenuation nor scatter correction was performed.Post-reconstruction smoothing filtering was applied using a Gaussian smooth 3D filter at 1.2 mm.Images were displayed using PMOD (ver.3.7).On SPECT images, volumes of interest were drawn for the liver, intestines (duodenum, small and large intestines), and kidney.Respective %ID values were calculated over time.
For whole-body SPECT imaging in CYP2D-inhibited mice, two mice were injected intraperitoneally with 30 mg/kg of paroxetine (MacLeod et al., 2017).At 60 min after paroxetine injection, 14.5 MBq of 123 I-BMQ was injected via the tail vein.Whole-body SPECT images were acquired under 2.0% isoflurane anesthesia every 10 min from 5 to 115 min after 123 I-BMQ injection.SPECT images of CYP2D-inhibited mice were analyzed according to the same method used for control mice.

Statistical analysis
All results represent the mean of at least three experiments, and data are expressed as the mean ± standard deviation.Data were analyzed using the F-test and Student's t-test, and p < 0.01 or <0.05 was considered statistically significant.HPLC analysis of 125 I-BMQ.Labeled 125 I-BMQ was detected separately from the raw material mequitazine.

Labeling of 125 I-BMQ
To identify 125 I-BMQ, 127 I-BMQ (cold-label) was used as a reference standard.Figure 2 shows HPLC chromatograms of mequitazine, 127 I-BMQ (cold-label), and 125 I-BMQ.The UV chromatogram confirmed the separation of mequitazine (retention time: 8 min) and 127 I-BMQ (retention time: 13.5 min), and the RI chromatogram showed that the main peak (retention time: 13.5 min) had the same retention time as 127 I-BMQ (In the HPLC system used in this study, the RI detector is located after the UV detector.The slight difference in retention times of the 125/127 I-BMQ peaks is due to the longer tube between the RI and UV detectors.)This confirmed that two-step 125 I labeling of mequitazine provided 125 I-BMQ with a labeling index of approximately 88%-91% and radiochemical purity >98%.

Metabolism of 125 I-BMQ in vitro
In this study, 125 I-BMQ and its metabolites were separated by TLC.The rate of flow (Rf) values in this analytical condition ranged from 0.15-0.30for 125 I-BMQ and 0.65-0.75for 125 I-NaI.In the metabolic reaction without NADPH (NADPH [−]), 125 I − was not detected and only 125 I-BMQ was detected.On the other hand, in the NADPH-mediated metabolic reaction (NADPH [+]), as in NADPH [−], 125 I − was not detected, 125 I-BMQ was detected, and other radioactive metabolites (Rf value: 0.05-0.10)were detected (Figure 3).With NADPH [+] conditions, the percentage of radioactive metabolite significantly increased from approximately 3%-25% with reaction time, and the percentage of 125 I-BMQ significantly decreased from approximately 93%-76% with reaction time.These results showed that the radioactive metabolite was an NADPH-mediated metabolite of 125 I-BMQ.
Figure 4 shows the percentages of radioactive metabolite produced from 125 I-BMQ.Under the condition with NADPH [−], paroxetine, quinidine, and mequitazine, the percentage of metabolites were 2.64%, 12.5%, 4.47%, and 1.34%, respectively significantly lower than under NADPH [+].Under the condition with 4-methylpyrazole and ketoconazole, the percentage of 125 I-BMQ was slightly lower, but not significantly.

Biological distribution of 125 I-BMQ in normal mice
Table 1 shows the biodistribution of 125 I-BMQ in normal mice.The injected 125 I-BMQ was rapidly distributed to tissues throughout the body.In the thyroid and stomach, radioactivity was consistently low.Radioactivity in the liver increased immediately after injection and then decreased gradually.In the gallbladder, intestine, and kidney, radioactivity increased gradually after injection.

Metabolism of 125 I-BMQ in vivo
The percentage of radioactive metabolites of 125 I-BMQ in mouse bile was analyzed by in vitro TLC.In this analytical conditions, the Rf values of 125 I − and 125 I-BMQ in bile were 0.45-0.55 and 0.65-0.75,respectively.In the bile of mice injected with 125 I-BMQ, no 125 I-BMQ was detected, only 125 I − and radioactive metabolite of 125 I-BMQ (Rf values; 0.00-0.35).The percentage of metabolites of 125 I-BMQ was >90% (Figure 5).

Whole-body imaging of 123 I-BMQ in normal mice and CYP2D-inhibited mice
Figure 6 shows SPECT images of 123 I-BMQ in normal mice and CYP2D-inhibited mice.In normal mice, radioactivity was found in the liver and intestine (Figure 6A, frame 1).Radioactivity in the liver  gradually decreased and that in the intestine gradually increased.Radioactivity excreted in the small intestine translocated to the large intestine (Figure 6A, frame 10).In CYP2D-inhibited mice, radioactivity was detected in the liver but not clearly in the intestine (Figure 6B, frame 1).The radioactivity in the intestine gradually increased, but remained on the duodenal side and did not translocated to the large intestine side (Figure 6B, frame 10).
Figure 7 shows the time-activity curves of 123 I-BMQ in the liver and intestines in SPECT images of normal and CYP2D-inhibited mice.CYP2D-inhibited mice accumulated more in the liver than control mice.Accumulation in the intestines was low in the early stages of 123 I-BMQ injection, but gradually accumulated to the same level as in control mice.

Discussion
In this study, we attempted to evaluate hepatic CYP2D activity by SPECT imaging using 123 I-BMQ.In general, SPECT imaging probe used in nuclear medicine are intended to evaluate their a %ID/organ was calculated from %ID and measured organ weights.%ID/g means percent injected dose per gram tissue and mean ± standard deviation obtained from three mice.Whole-body images obtained approximately 10, 50, and 100 min after injection of 14.5 MBq of 123 I-BMQ in normal mice (A) and CYP2Dinhibited mice (B). 123I-BMQ accumulated in the liver, gallbladder, small intestines, and kidney.
function in vivo, and the SPECT imaging probe themselves are not metabolized.Our previous study showed that 123 I-iomazenil was metabolized by hepatic carboxylesterase, after which almost all of the radioactive metabolites were translocated to the gallbladder and urinary bladder.Thus, hepatic carboxylesterase activity could be measured by detecting and evaluating the radioactive metabolites of 123 I-iomazenil accumulated in the gallbladder and/or urinary bladder (Mizutani et al., 2018).Using the same technique, we also reported that the newly developed 123/125 I-Odesmethylvenlafaxine is useful for direct and comprehensive detection and measurement of hepatic CYP3A4 and 2D6 in a simple and minimally invasive approach (Mizutani et al., 2022).However, since 123/125 I-O-desmethylvenlafaxine can only evaluate the total activity of CYP3A4 and 2D6, we tried to develop a new SEPCT imaging probe that can evaluate only CYP2D6 activity.It is very difficult to prescribe psychiatric pharmacotherapy probe in clinical practice due to significant variability between individuals.CYP2D6 contributes to the clearance of many psychiatric drugs, and thus, the ability to accurately evaluate CYP2D6 activity in a minimally invasive and simple manner is critical for proper prescribing of medications.The metabolic pathway of mequitazine has already been reported and primarily involves conversion to S-oxidized and hydroxylated compounds (Figure 8).Hydroxylation is the major metabolic fate of mequitazine, and this conversion primarily involves CYP2D6 (Nakamura et al., 1998).Thus, mequitazine was selected as a substrate for CYP2D6 and used as a raw material for a new SPECT imaging probe that can measure hepatic CYP2D6 activity.A stable radioiodine labelling method for mequitazine was established in this study and 123/125 I-BMQ showed very high labelling yield and radiochemical purity.
To confirm the involvement of CYP in the in vitro metabolism of 125 I-BMQ, the effect of an NADPH-generating system was examined.Under the condition of NADPH [+], 125 I − , 125 I-BMQ, and a radioactive metabolite of 125 I-BMQ were detected.In contrast, only 125 I − and 125 I-BMQ were detected under the condition of NADPH [−].Thus, the radioactive metabolite of 125 I-BMQ was only produced under condition of NADPH-generating system supplied energy to CYP, indicating the involvement of CYP in the metabolism of 125 I-BMQ.To further identify the CYP isozymes involved in 125 I-BMQ metabolites, the effects of inhibitors specific for each CYP were examined in an in vitro 125 I-BMQ metabolism inhibition study.Paroxetine, quinidine, and mequitazine significantly inhibited the metabolism of 125 I-BMQ (42%, 80%, and 94% inhibition, respectively).Therefore, 125 I-BMQ is mainly metabolized by CYP2D.Not only the raw material mequitazine, but also 125 I-BMQ, was shown to be specifically metabolised by CYP2D, making 123 I-BMQ a candidate for a new SPECT imaging probe to evaluate mouse hepatic CYP2D and human hepatic CYP2D6 activity.
To directly detect and evaluate the activity of drug-metabolizing enzymes in metabolic drug clearance, it is necessary to image and measure the accumulation in the excretory organs.The biodistribution of 125 I-BMQ in mice showed high radioactivity in the liver, gall bladder, and kidney immediately after injection.The The time-activity curves of 123 I-BMQ in the liver and intestines in SPECT images of normal and CYP2D-inhibited mice.Mice were scanned from 5 to 115 min after 123 I-BMQ injection.Accumulation in the liver was increased and accumulation in the intestines was decreased in CYP2D-inhibited mice.Proposed in vitro major metabolic pathways of mequitazine in human liver microsomes.
radioactivity in the metabolic organ (liver) decreased with time, whereas the radioactivity in the excretory organs (gallbladder, intestines, and kidney) increased early.Furthermore, the low accumulation in the thyroid and stomach indicated that 125 I-BMQ is rarely metabolized to 125 I − and that the majority of the radioactivity detected in vivo in mice is 125 I-BMQ or a radioactive metabolite of 125 I-BMQ.To determine whether the radioactivity in the bile was 125 I-BMQ or a radioactive metabolite of 125 I-BMQ, the gallbladder was removed 60 min after the mice were injected with 125 I-BMQ.As a result, more than 90% of the radioactivity in the bile was radioactive metabolite, and 125 I − or 125 I-BMQ was not detected.Therefore, it is suggested that the radioactivity in the bile is the radioactive metabolite of 125 I-BMQ and that the amount of radioactivity in the bile increases or decreases according to the amount of radioactive metabolites produced.With regard to radioactive metabolite, no chemical structure was identified in this study, but TLC analysis confirmed that was clearly radioactive metabolite of CYP2D rather than 125 I − or 125 I-BMQ.In the concept of this study, the chemical structure of the radioactive metabolite is not important, but the selective excretion of CYP2Dspecific radioactive metabolite, and 125 I-BMQ fulfilled this requirement.
SPECT imaging of 123 I-BMQ was performed to visualize the accumulation of radioactive metabolite for the purpose of assessing CYP2D activity.Images of normal mice injected with 123 I-BMQ showed a decrease in accumulation in the liver over time, but increased accumulation in the intestines over time.In CYP2Dinhibited mice, accumulation in the liver was initially higher than in control mice, but accumulation in intestines was lower than in control mice.Loading paroxetine, a CYP2D6 inhibitor, inhibited or delayed the metabolism of 123 I-BMQ.Inhibition of CYP2D resulted in 123 I-BMQ being retained unmetabolized, accumulation in the liver was less likely to decrease, while accumulation in the intestines decreased due to reduced production of metabolite.Accumulation in the intestine remained in the duodenal region and did not migrate to the lower part of the intestine.Paroxetine, used as an inhibitor of CYP2D6, has gastrointestinal symptoms as a side effect, which may have affected the results.In a preliminary study, the effect of intraperitoneal administration of paroxetine on hepatobiliary excretion was investigated.After the hepatobiliary probe 99m Tc-Npyridoxyl-5-methyltryptophan ( 99m Tc-PMT) was administered to normal mice and biliary excretion function was obtained by SPECT dynamic imaging, the same mice were treated with paroxetine as CYP2D inhibitors and similar SPECT dynamic imaging was performed.The results showed that hepatobiliary excretion and excretion rate of 99m Tc-PMT were similar in normal and CYP2D-inhibited mice.This result showed that the use of paroxetine in CYP2D-inhibited mice did not affect the biliary excretory function itself.Therefore, the observed changes in distribution in this study reflected reduced metabolite production and delayed metabolic responses associated with decreased CYP2D activity, and CYP2D activity can be evaluated by imaging intestinal accumulation over time after 123 I-BMQ injection.
These results indicated that 123/125 I-BMQ meets four conditions: 1) 123/125 I-BMQ accumulated in the liver as the metabolic organ; 2) 123/125 I-BMQ was metabolized specifically by CYP2D, and the metabolite is radioactive; 3) the radioactive metabolite of 123/125 I-BMQ selectively translocated from the liver to the gallbladder and intestines; and 4) the excretory organs in which radioactive metabolite selectively accumulate could be visualized and measured.

Conclusion
By visualizing and measuring the accumulation over time in the intestine, where bile containing only 123/125 I-BMQ metabolites is excreted, it is possible to evaluate CYP2D activity from the decrease or delay in accumulation.Therefore, 123/125 I-BMQ is useful as a SPECT imaging probe for comprehensive and direct assessment of hepatic CYP2D activity in a minimally invasive and simple approach.

FIGURE 4
FIGURE 4 Effect of CYP inhibitors and mequitazine (a substrate of CYP2D6) on the metabolism of 125 I-BMQ.Under NADPH [−], paroxetine, quinidine and mequitazine conditions, the percentage of radioactive metabolite was significantly lower than under NADPH [+].

FIGURE 5 TLC
FIGURE 5 TLC analysis of radioactive metabolites in bile collected from mice after 125 I-BMQ injection.The bars indicate 125 I-NaI (white), 125 I-BMQ (gray) and bile content (black), respectively. 125I-BMQ was not detected in the bile and more than 90% of the radioactivity was metabolites of 125 I-BMQ.

TABLE 1
Biological distribution of 125 I-BMQ in normal mice.