Early.Phase Postmortem Redistribution of the Enantiomers of ...

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Journal of Analytical Toxicology,Vol. 29, May/June2005

Early.Phase Postmortem Redistribution of the Enantiomers of Citalopram and Its Demethylated Metabolites in Rats Fredrik C. Kugelberg1,2,*, Maria Kingb~ck1, Bj6rn Carlsson1, and Henrik Druid 2 IDepartment of Clinical Pharmacology, Faculty of Health Sciences, Link@ing University, SE-581 85 Link@ing, Sweden and 2Departmentof Forensic Medicine, Karolinska Institutet, SE-171 77 Stockholm, Sweden

Abstract I The aim of this study was to investigate the early-phase postmortem redistribution of the enantiomers of citalopram (CIT) and its metabolites demethylcitalopram (DCIT) and didemethylcitalopram (DDCIT) in a rat model. Furthermore, we wanted to examine the role of the lungs as a reservoir of postmortem drug release and to investigate the influence of storage temperature (21~ vs. 4~ on postmortem changes. Rats were administered a single CIT dose of 100 mg/kg (s.c.), and heart blood and lung samples were collected antemortem and 15 rain postmortem for enantioselective high-performance liquid chromatographic analysis. About three times higher blood drug and metabolite levels were observed in the postmortem rats than in the antemortem rats (p < 0.0001). Refrigeration at 4~ did not prevent, but significantly reduced, the postmortem increase in heart blood CIT levels as compared to the concentrations in the rats stored at 21~ (p < 0.05). The lung drug concentrations were lower postmortem than antemortem (p < 0.05). The enantiomeric (S/R) concentration ratios of CIT and metabolites in blood and lungs were of similar magnitude before and after death. The parent-drug-to-metabolite ratios for CIT/DCIT were unchanged after death. In conclusion, this study shows that heart blood CIT and metabolite levels increase rapidly after death. Further, a fall in postmortem CIT concentrations in the lungs was observed, indicating that the lungs seemed to represent one major source of drug release during early-phase postmortem redistribution.

Introduction Intoxication by prescription drugs is a widely used suicide method (1).Furthermore, antidepressant drugs are among the most common drugs involved in fatal poisonings (2). In order to determine if a death is due to poisoning, it is necessary to assess the postmortem blood concentrations of the drugs detected in the light of autopsy findings and circumstances * Author to whom correspondence should be addressed. E-mail: [email protected].

surrounding death. Reference information on postmortem blood concentrations of different drugs is therefore useful. Such data do exist for many prescription drugs, including the newer antidepressants (3,4). One complicating factor is, however, the fact that the concentration of a drug in an autopsy blood sample may not necessarily reflect the in vivo concentration just before death. This difference can be explained by postmortem drug redistribution, a phenomenon that forensic pathologists and toxicologists have to consider when interpreting drug concentrations (5). To study this phenomenon in more detail, animal models can be used to estimate the magnitude of postmortem redistribution of a drug. Citalopram (CIT) is a widely used antidepressant belonging to the class of selective serotonin reuptake inhibitors (SSRIs) (6,7). CIT is a racemic mixture of the S-(+)-enantiomer (SCIT) and the R-(-)-enantiomer (R-CIT). S-CIT is associated with the SSRI effect both in vitro (8,9) and in vivo (10), and SCIT is now also marketed as a single enantiomer drug (escitalopram) (11). The major CIT metabolites, demethylcitalopram (DCIT) and didemethylcitalopram (DDCIT), are less potent than the parent compound with regard to SSRI properties (12). In recent years, several reports on CIT concentrations in human postmortem cases have become available (3,13-18). In contrast, the literature is scarce concerning controlled animal studies exploring the possible postmortem redistribution of CIT and its metabolites. However,we have recently reported a detailed study in which the enantiomeric disposition and redistribution of CIT and its metabolites were investigated after acute, chronic, and acute-on-chronic administration to rats (19). In that study, heart blood concentrations of CIT,DCIT,and DDCIT increased postmortem compared to antemortem, and most of the increase occurred within 60 rain of death. The aim of the present study was to follow up these previous findings by investigating possible postmortal changes occurring shortly after death. Therefore, the previously investigated acute CIT dose of 100 mg/kg was administered subcutaneously (s.c.) to rats, and the heart blood concentrations of the enantiomers of CIT, DCIT, and DDCIT were analyzed at sacrifice or 15 min after death. Furthermore, we wanted to examine the role of the

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lungs as a reservoir of postmortem drug release and if the postmortem redistribution was different at room temperature (21~ compared with a cold environment (4~

Experimental Animals Twenty-one male Sprague-Dawley rats (Taconic M&B A/S, Ry, Denmark) weighing 217-322 g were used. All animals had free access to standard laboratory pelleted chow containing 14.5% crude protein (R70; Lactamin AB, Vadstena, Sweden) and tap water ad libitum. All rats were housed in groups of two animals in macrolone cages under climate-controlled conditions for regular indoor temperature and humidity. The rats were kept in a constant 12:12 h light-dark cycle synchronous with daylight (lights on at 8.00 a.m.). All experiments were performed in strict accordance with the guidelines and with the consent of the Animal Ethics Committee, Link6ping, Sweden (Permit No. 10-02).

Drugs and chemicals Citalopram HBr (CIT) (H. Lundbeck A/S, Copenhagen-Valby, Denmark) was dissolved in a mixture of 0.9% NaCI and propylene glycol (40:60, v/v) at a concentration such that the rats received 3 mL/kg s.c. All reagents used were of the highest purity commercially available. Experimental procedure Three experimental groups denoted as antemortem controls (n = 7), postmortem 4~ (n = 7), and postmortem 21~ (n = 7) were investigated separately. All rats received a single s.c. injection of 100 mg/kg of CIT under a brief anesthesia with halothane (Fluothane| Zeneca, Ltd., Macclesfield Cheshire, U.K.). Three hours after the drug administration, the postmortem rats were sacrificed with CO2 and left lying on their backs for 15 min at either room temperature (21~ or at 4~ in a refrigerator. After clamping inferior vena cava above the diaphragm, postmortem heart blood samples (0.3-0.5 mL) were

drawn from the right atrium by a polypropylene catheter connected to a syringe. The antemortem control rats were sacrificed under halothane anesthesia by collection of heart blood (0.5 mL) followedby decapitation. Aftercollection of the blood samples, the left lung was removed from each of the rats and stored in -70~ until the preparation for the drug analysis. The lung tissue samples were then weighed and homogenized in 2 mL Milli-Q| water (Millipore AB, Stockholm, Sweden) using a sonifier (Sonics Vibra-Cell VC 130, Chemical Instruments AB, Lidingi3,Sweden) and centrifuged at 3000 rpm for 15 rain. The lung supernatants, as well as the heart blood samples, were stored at -70~ until drug analysis. Enantioselective analysis of citalopram and its demethylated metabolites The concentrations of the S- and R-enantiomers of CIT, DCIT, and DDCIT were determined using HPLC with fluorescence detection (20-23). The extraction of the whole blood samples was carried out according to a previously described plasma method (24) with some modifications (18). The internal standard of choice was a chlorinated version of CIT (Lu 10-202-O; H. Lundbeck A/S). Briefly, 20 I~Lof the internal standard (2.5 IJg/mL) was added to 0.1-0.5 g of blood and diluted with 3 mL acetonitrile/0.025M phosphate buffer (pH 11.5) (5:95, v/v) in a glass tube. After vortex mixing and sonification for 5 min, the solution was centrifuged for 10 min at 3000 rpm. After conditioning of the solid-phase extraction columns, the centrifuged samples were poured on to the columns, and thereafter, the original extraction procedure was followed(24). The extraction of the lung samples was carried out according to a previously described plasma method (24) with some modifications (22,23). After elution and evaporation, the dried samples were redissolved in 100 ]at of methanol/]00mM citric acid triethylamine (pH 6.3) (55:45, v/v). A volume of 10-50 tJL was injected onto a Cyclobond12000 Ac 250 x 4.6-ram column (Astec,Whippany, NJ) with a Gynkotek Gina 50 autosampler (Dionex, Sunnyvale, CA). The mobile phase was delivered through a Gynkotek 480 pump (Dionex) at a flowrate of 0.8 mL/min. CIT, DCIT, and DDCITwere detected by excitation at 240 nm and emission at 300 nm using a Waters 474 fluorescence detector (Waters

Table I. Concentrations (S+R) and Enantiomeric Ratios (S/R) of Citalopram (CIT), Demethylcitalopram (DCIT), and Didemethylcitalopram (DDCIT) in Heart Blood (pg/g) and the Lungs (pg/g) following Single Administration of the Racemic CIT Dose 100 mg/kg to Rats Concentrations(@-R)* PMIt 0 min (Ctrl)

PMI 15 min (4~

Enantiomeric ratios (S/R)

PMI 15 min (21~

PMI 0 min (Ctrl)

PMI 15 min (4~

PMI 15 min (21~

Blood

CIT DCIT DDCIT

2.92 _+0.11 1.05 _+0.07 0.39 _+0.02

7.59 + 0.71 2.53 + 0.22 1.13 _+0.12

9.35 + 0.50 2.67 _+0.25 0.94 _+0.06

0.92 __.0.01 0.68 _+0.01 NC

0.92 _+0.01 0.54 _+0.01 NC

0.93 _+0.01 0.54 + 0.01 NC

Lungs

CIT DCIT DDCIT

66.0 -z-_5.71 29.1 _ 2.51 4.17 _ 0.23

48.0 • 4.85 24.2 +_3.11 3.81 _+0.45

54.6 __.3.53 26.7 + 2.81 3.58 + 0.26

0.97 _.+0.01 0.61 +_.0.02 NC

0.96 +_0.01 0.61 + 0.01 NC

0.97 _ 0.01 0.59 + 0.02 NC

* For DDCIT, only the R-enantiomer was detectable, and hence, the S/R DDCIT ratios could not be calculated (NC, not calculated). All values are means• SEM. t Abbreviations: PMI, postmortem interval and Ctrl, antemortem control rats.

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Corporation, Milford, MA).The detection signals were recorded and processed using the chromatography data system ChromeleonTM (Version 6.40, Dionex, Sunnyvale, CA). The limits of detection for the enantiomers of CIT and its metabolites were 0.002 ~g/g (S/N, 3:1), respectively. Analytical recoveries for CIT and metabolites in whole blood and lung tissue exceeded 85% and 75%, respectively.

Data analysis Data are expressed as means plus or minus the standard errors of the means (SEM). A probability of less than 5% (p < 0.05) was considered statistically significant. Possible differences between the groups were analyzed by one-factor analysis of variance (ANOVA).When the ANOVAreached statistical significance, Fisher's protected least significant difference (PLSD) post-hoc test was applied. All statistical analyses were performed using StatView| for Windows (version 5.0, SAS| Institute Inc., Cary, NC).

Results

Concentrations of the enantiomers of CIT,DCIT,and DDCIT are displayed in Table I and Figure 1. Both postmortem groups showed increased blood drug concentrations compared with

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15

15

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antemortem blood drug concentrations (p < 0.0001). In the rats stored at 4~ the ratio between postmortem and antemortem CIT concentrations was 2.6 at 15 rain after death. In the rats stored at 21~ the corresponding ratio was 3.2 at 15 min after death. Furthermore, the 21~ rats displayed higher postmortem blood drug concentrations than the 4~ rats (p < 0.05). In contrast to the blood results, the postmortem lung tissue concentrations of CIT were lower compared with the antemortem lung tissue concentrations of CIT,even if this difference only was statistically significant in the rats placed in the refrigerator at 4~ (p < 0.05). In the rats stored at 4~ and 21~ the ratios between postmortem and antemortem CIT concentrations were 0.73 and 0.83 at 15 rain after death, respectively. Similar to the CIT data, the postmortem blood metabolite concentrations were higher than the antemortem blood metabolite concentrations (p < 0.0001). Accordingly,the ratios between postmortem and antemortem blood concentrations of DCITand DDCITwere found in a similar range (i.e., 2.4-2.9) to that observed for CIT. The ratios between postmortem and antemortem lung concentrations of DCIT and DDCITwere in agreement with the corresponding ratios on CIT and found in the 0.83-0.92 interval. No statistical significant differences in blood and lung metabolite concentrations were observed between the two postmortem rat groups. Despite the differences in blood and lung CIT concentrations, the enantiomeric (S/R) concentrations ratios of CIT and DCITwere of similar magnitude antemortem and postmortem in both the blood and lung samples (Table I). However, the blood S/R ratios of DCIT were lower in the two postmortem groups as compared to the antemortem controls (0.54 vs. 0.68; p < 0.0001). In all three groups investigated, the parent drug/metabolite (P/M) ratios for CIT/DCITin blood and lung were around 3 and 2, respectively.

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Discussion Postmortem interval (min)

80- B

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I

70-

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605040" 3020100

0

15

15

4~

21~

Postmortem interval (min)

Figure 1. Concentrations (S+R) of citalopram (CIT) in heart blood (A) and in the lungs (B) following single administration of the racemic CIT dose 100 mg/kgto rats. Pleasenote the different scaleson the y-axis of the two figures. Values are means + SEM. *p < 0.05 and ***p < 0.001.

The major novel finding from the present study is that postmortem redistribution of CIT and its metabolites DCIT and DDCIT occurred shortly after death. This was evidenced by increased heart blood drug and metabolite concentrations already at 15 rain postmortem, and a corresponding fall in postmortem CIT concentrations in the lungs. The redistribution could not be prevented by refrigeration, although the changes were less pronounced at 4~ The experimental design was chosen to mimic a human acute overdose situation. Based on our previous results (25), the rats were killed 3 h after the injection of a single high/toxic dose of 100 mg/kg of CIT to cover the time point at which the maximal blood drug concentration appears. The achieved antemortem drug concentrations in these rats correspond with postmortem toxic concentrations found in humans (3,14). However, the CIT dose was obviously non-lethal in the rats of the present study because all of the rats survived until termination of the experiments. The use of CO2 asphyxiation is suggested to be suitable for producing death by an unrelated cause, and this method was 225

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applied for the postmortem rats. It has been reported that CO2 neither induces histopathological changes (26) nor affects the pH changes that occur postmortem (27). The observation of increased heart blood drug and metabolite levels already at 15 min postmortem indicates an early redistribution process in the present animal model. To our knowledge, no such early postmortem data of human autopsy cases are available, and it is therefore difficultto direct transfer our resuits to humans. However,the early change in heart blood drug concentrations of the present study is most probably also a true phenomenon in humans, but it is important to keep in mind the longer distance between organs in a human body, compared with a small animal like a rat, which can affect the time of the redistribution. The most likely explanation of the early change in postmortem drug concentrations adheres to a passive diffusion process from drug depots in solid organs such as the lungs. The capillary-rich lung, with its large amount of lipoproteins, has been suggested often to contain the highest concentration of a drug of any organ in the body (5). A common accumulation mechanism in the lungs has also been proposed for basic amines that are highly lipophilic and have PKavalues greater than 8.5 (28,29). Different basic drugs have been reported to accumulate in pulmonary alveolar macrophages and possibly in type II epithelial cells (30,31), and several reports are available showing particularly high lung levels of drugs in humans (32,33). Hence, the lungs represent an important source for postmortem drug redistribution, which can be explained by the large surface area of the alveoli, thin diffusion membrane, and high vascularization (34). Therefore, the lungs most probably contributed to the majority of the postmortem increase of heart blood drug concentrations in the present study, especially because CIT was administered s.c. and no functional drug depot was present in the gastrointestinal tract. The postmortem drug release from the lungs to heart blood was also evidenced by the fall in CIT concentration in the lungs, and this finding is in agreement with a rat study on amitriptyline (35). Considering the very high drug concentrations in the lungs, even a slight drop in the lung tissue levelwould explain a marked increase in heart blood drug concentrations. As CIT has both a high pKa value (9.5) and a high volume of distribution (21 L/kg in rats), the observation of an early rise in heart blood drug concentrations at 15 min postmortem was not surprising. Apart from the lungs, other possible sources of postmortem drug release could also exist such as the liver and the myocardium. Postmortem redistribution from the liver is complex, as it involves different mechanisms, but this process is apparently not as intense and early as redistribution from the lungs (36). Furthermore, because no drug depot was present in the gastrointestinal tract, the liver probably did not contribute to the early postmortem increase of heart blood CIT concentrations. Postmortem redistribution from the myocardium into heart blood has been described for several antidepressant drugs such as amitriptyline, maprotiline, imipramine, and desipramine (32,37). However, this process is probably not very rapid, because it has been reported that basic drugs deposited in large amounts in the myocardium contributed little to increases in their concentrations in cardiac blood during the early postmortem period (38). Further, the authors concluded 226

that drugs in the lungs were redistributed rapidly, especially into the left cardiac chambers via the pulmonary venous blood (38). The literature is scarce concerning consistent data on postmortem CIT and metabolite concentrations in different organs from humans. However,some data exist on CIT concentrations in bile, kidney, liver, muscle, and vitreous humor from postmortem cases (14-17). Moreover, in a case report describing a multiple drug intoxication, brain levels of CIT were analyzed (39). In a recent study, we have reported that brain concentrations of CIT and metabolites were about the same antemortem and postmortem in rats following acute, chronic, or acute-onchronic administration (19). To our knowledge, the present study is the first report on postmortem concentrations of CIT, DCIT,and DDCIT in lungs. In contrast to the observation of no major variation in brain drug concentrations (19), the present lung drug concentrations were lower postmortem compared with antemortem. Furthermore, the lung CIT concentrations were approximately 10 times higher than the previously reported brain CIT concentrations after administration of the same acute dose of 100 mg/kg. Higher postmortem drug concentrations in the lungs as compared to in the brain have also been observed for other antidepressants such as amitriptyline, mianserin, and nortriptyline after administration to rats (35,40). The present study also investigated if a decrease in storage temperature, from 21~ to 4~ influenced the degree and pattern of the postmortem drug redistribution. The lowering of temperature resulted in some differencesin postmortem heart blood CIT concentrations, which were lower in the 4~ rats than in the 21~ rats. However, this difference was not evident for the heart blood levels of DCITand DDCIT,but on the other hand, the metabolite levels were also lowerthan the levels of the parent compound. Few studies are available that compare postmortem drug redistribution at different temperatures. However, using a human cadaver model, Pounder and Smith (41) reported that the postmortem ethanol diffusionfrom the stomach was markedly inhibited by refrigeration at 4~ Recent studies have provided information regarding the usefulness of enantioselective analysis of CITand its metabolites in postmortem blood samples (18,19). In addition, we have shown in an in vivo study that the blood S/R CIT ratios decreased with time after single-dose administration of 100 mg/kg to rats (25). This finding was also supported by Holmgren et al. (18) who reported that blood S/R CIT ratios close to 1.0 in human postmortem cases were associated with an intake of the drug only a few hours or less before death. Furthermore, our previous observation of similar S/R ratios and P/M ratios of CIT and metabolites before and after death (19) was confirmed in the present study. P/M ratios may be used for interpretation of the time of ingestion (42). Hence, an acute overdose and a chronic dosage may be associated with high and low P/M ratios, respectively.The unchanged postmortem P/M ratios indicate that postmortem redistribution do not interfere with the use of P/M ratios in the interpretation of autopsy samples. In conclusion, the present animal study demonstrated increased postmortem heart blood concentrations of CIT and metabolites within 15 min after death. Furthermore, a fall in postmortem CIT concentrations in the lungs was observed, in-

Journal of Analytical Toxicology,Vol. 29, May/June2005

dicating that the lungs seemed to represent one major source of drug release during early-phase postmortem redistribution. We also conclude that refrigeration may to some extent reduce the postmortem redistribution of CIT. Finally, given the observed changes in concentrations of CIT and its metabolites, the S/R ratios and the P/M ratios seem to be unchanged, and may thus still be used to improve the interpretation of postmortem toxicological results.

Acknowledgment This work was supported by grants from the National Board of Forensic Medicine in Sweden and the Forensic Science Center of Link6ping University. The generous supply of citalopram by H. Lundbeck A/S, Copenhagen-Valby, Denmark, is gratefully acknowledged.

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Manuscript received April 23, 2004; revision received July 14, 2004.