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Postmortem memantine concentration in a non-intoxication case, and the possibility of postmortem redistribution: A case report
Accepted Manuscript Title: Postmortem memantine concentration in a non-intoxication case, and the possibility of postmortem redistribution: a case report Author: Sayaka Nagasawa Daisuke Yajima Suguru Torimitsu Fumiko Chiba Hirotaro Iwase PII: DOI: Reference:
S0379-0738(15)00359-X http://dx.doi.org/doi:10.1016/j.forsciint.2015.08.015 FSI 8129
To appear in:
FSI
Received date: Revised date: Accepted date:
24-5-2015 14-8-2015 23-8-2015
Please cite this article as: S. Nagasawa, D. Yajima, S. Torimitsu, F. Chiba, H. Iwase, Postmortem memantine concentration in a non-intoxication case, and the possibility of postmortem redistribution: a case report, Forensic Science International (2015), http://dx.doi.org/10.1016/j.forsciint.2015.08.015 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Highlights
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・Memantine was detected at high concentrations in a non-intoxication postmortem case.
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・C/P and L/P ratios of memantine indicate likelihood of postmortem redistribution.
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・Postmortem reduction in blood pH may also promote the PMR of memantine.
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Postmortem memantine concentration in a non-intoxication case, and the possibility of postmortem redistribution: a case report
Sayaka Nagasawa,
1)
Daisuke Yajima,
1,2)
Suguru Torimitsu,
Chiba,
1,2)
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Hirotaro Iwase.
of Legal Medicine, Graduate School of Medicine, Chiba University, Chiba,
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1) Department
1,2 )Fumiko
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1)
Japan, 1-8-1 Inohana, Chuo-ku, Chiba 260-8670, Japan
Department of Forensic Medicine, Graduate School of Medicine, The University of
an
2)
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Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan
Institution at which the work was performed:
Corresponding author:
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Department of Legal Medicine, Graduate School of Medicine, Chiba University
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Sayaka Nagasawa, Ph.D.
Assistant Professor, Department of Legal Medicine, Graduate School of Medicine, Chiba University
1-8-1 Inohana, Chuo-ku, Chiba 260-8670, Japan Tel: +81-43-226-2078
Fax: +81-43-226-2079
E-mail: [email protected]
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Abstract
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In this case study, we measured the concentration of memantine in the heart blood, peripheral blood, urine, liver, thigh muscle, and subcutaneous fat of a 64-year-old
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woman who was prescribed memantine for early-onset Alzheimer’s disease. She died in
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hospital after an altercation with her husband. Cause of death was clearly not drug
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intoxication or overdose, so we investigated the postmortem redistribution (PMR) of memantine in the various tissues and blood ratios of the postmortem samples.
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Memantine concentrations detected were 1.35 µg/mL in the peripheral blood, 3.95
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µg/mL in central blood, 2.09 µg/mL in the urine, 25.54 µg/g in the liver, 1.16 µg/g in the
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thigh muscle and 2.13 µg/g in the subcutaneous fat. In all samples, the concentrations
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were higher than the accepted therapeutic range (which is approximately 0.09–0.15 µg/mL). The central blood to peripheral blood (C/P) memantine ratio was 3.01 while the liver to peripheral blood (L/P) ratio was 19.5. It is documented that a C/P ratio exceeding 2 and L/P ratio exceeding 20 highlight a propensity for significant PMR. Although this is a single case study, our data suggest that memantine exhibits PMR. Additionally, a lowered pH was found in peripheral blood (pH=6.2) and central blood (pH=6.1). This postmortem reduction in blood pH may also promote the PMR of memantine. Because there is very little available postmortem toxicological data on
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memantine, our case study will serve as a foundation to assist in future forensic
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investigations.
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Keyword
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Memantine Postmortem redistribution
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Liver
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Non-intoxication
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Alzheimer's disease
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Introduction
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In recent years, an increasingly aging population has been observed in many countries and in Japan in particular. Because of this, the incidence of dementia and Alzheimer’s
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disease (AD) is also rising rapidly. In the past 10 years, at least 80 countries around the
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world have prescribed memantine as a safe and effective therapeutic medicine for AD,
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and it is predicted that the number of prescriptions will increase as the number of patients rises [1-3].
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Memantine binds to a receptor of the neurotransmitter glutamic acid and acts to
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prevent neuropathy caused by excess glutamic acid [4]. Compared with cholinesterase
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inhibitors (donepezil, galanthamine, rivastigmine), cardiac side effects of memantine
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are rare, but severe side effects such as convulsions, fainting, and loss of consciousness have been reported [5]. Emergency responses to overdose cases (unknown whether it was intentional or accidental) are also being reported [6, 7]. As the main symptom of AD patients is dementia, it is likely that more incidents of overdoses will occur in the future. However, there is very little reporting on findings of postmortem memantine concentrations [8]. Postmortem drug concentrations in blood may not always reflect antemortem concentrations because of postmortem changes (e.g. postmortem diffusion and
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postmortem redistribution) [9]. Therefore, the true assessment of postmortem blood
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levels is difficult. In this study, we measured the concentration of memantine and donepezil in heart blood, peripheral blood, urine, liver, thigh muscle, and subcutaneous
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fat from a woman whose cause of death was clearly not drug intoxication or an overdose
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of memantine. Furthermore, we investigated the postmortem redistribution of
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memantine from the various tissues and blood ratios obtained.
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Case Circumstances
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A 64-year-old woman was diagnosed with early-onset AD at 58, and had been
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prescribed memantine for a year, following the worsening of her symptoms. On the
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morning of the day before her death, she had an argument with her husband, who attempted to strangle her with an electrical cord. She was transported to the hospital, where she died 22 h later. Details of her last prescription were memantine 20 mg/day and donepezil 10 mg/day after breakfast, tiapride 25 mg/dose after each meal and risperidone 0.5 mg/dose as needed for agitation. These were all within acceptable therapeutic dose ranges. The autopsy examination showed findings typical of strangulation, including congestion of the face and head; both palpebral and bulbar submucosal hemorrhage;
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linear keratolysis and allochroism running across the neck in the left-right direction;
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bleeding in the various muscles of the neck; bleeding in the tongue muscle; and fractures accompanied by bleeding in the greater cornu of the left hyoid bone and the
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superior horn of the right thyroid cartilage. From these findings it was concluded that
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an external force had been applied to her neck, resulting in congestion of the facial
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surface of her head. Histological examination of various organs showed no obvious lesions, and the diagnosis was asphyxiation due to strangulation. Heart and femoral
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blood was screened for drugs, and memantine and donepezil were detected in both
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samples, and alcohol level was 0 mg/mL Heart blood pH was 6.1, and femoral blood was
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6.2. Postmortem interval was estimated to be 48 h.
Materials and Methods
Sample collection and storage
Peripheral blood was drawn from the right and left of the femoral vein, and stored in
cryotubes together (Nunc Cryo Tube, Milian, Geneva, Switzerland). Central blood was collected from the heart (mixed blood) and placed in identical tubes. The right lobe of liver, right thigh muscle and subcutaneous fat of the thigh were collected and stored in a falcon tube. Urine samples were collected and stored in cryotubes. Following the
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standard protocol of our laboratory, all samples were pretreated and analyzed
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immediately, with the remainder of these samples being placed in storage at −̯20 °C.
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Toxicological analyses
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Systematic toxicological analysis was performed on the postmortem samples to detect
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the presence of alcohol, prescribed drugs, and illegal drugs. Screening for routine drugs in blood and urine was performed with a 3200 QTRAP® LC/MS/MS system (AB Sciex,
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Foster City, CA, USA) using the Multiple Reaction Monitoring (MRM) method. For each
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blood and urine sample, 900 µL of ACN was added to 100 µL of sample. The mixture was
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vortexed for 30 s, sonicated for 5 min, and centrifuged at 850 × g for 15 min. For tissue
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samples, ACN was added to three times the volume of the sample. The mixture was homogenized and centrifuged at 850 × g for 15 min. The supernatant was retained for subsequent analysis.
Reagents and chemicals
Memantine hydrochloride and donepezil hydrochloride were purchased from Sigma-Aldrich (St Louis, MO, USA). Ammonium formate was purchased from WAKO (Tokyo, Japan). Methanol and acetonitrile (ACN) were purchased from Kanto Kagaku
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(Tokyo, Japan). All other chemicals were of analytical reagent grade.
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Control samples of blood were collected from healthy volunteers after obtaining their informed consent, and liver, thigh muscle and subcutaneous fat samples were taken
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from selected cadavers, all of whom had no history of drug abuse. These samples were
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screened for common drugs (including memantine) and alcohol, and all were confirmed
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negative.
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Preparation of stock and calibration solutions
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Memantine hydrochloride and donepezil hydrochloride stock solutions (1 mg/ml) were
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prepared in distilled water and methanol respectively, and stored frozen at −20°C.
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Standards for quantification of memantine in blood and urine were prepared in 1 mL drug-free blood and urine samples by spiking the samples with memantine at concentrations of 0.1, 0.5, 1, 2.5, and 5 µg/mL. Standards for quantification of donepezil (DPZ) in blood and urine were prepared in 1-mL drug-free blood and urine samples by spiking the samples with DPZ at concentrations of 0.1, 0.25, 0.5, 1, 5 and 7.5 µg/mL.. Standards for quantification of memantine and DPZ in liver, thigh muscle and subcutaneous fat were prepared in 1 g drug-free samples of each tissue by spiking the samples with memantine at 1, 5, 10, 20 and 30 µg/g, and with DPZ at 0.5, 1, 2.5, 5 and
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10 µg/g. Negative blood, urine, liver, thing muscle and subcutaneous fat controls were
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also prepared before each analysis performed.
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Extraction
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Biological sample extraction was performed using Focus™ columns (Lake Foster, CA,
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USA) following the methodology described in our previous study [10]. To each blood and urine sample, 300 µL of 1M acetate buffer and 2 mL of distilled water were added. The
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mixture was vortexed for 30 s, sonicated for 5 min, and centrifuged at 850 × g for 15 min.
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To each tissue sample, 300 µL of 1M acetate buffer and 2 mL of distilled water were
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added. The mixture was homogenized and centrifuged at 850 × g for 15 min.
LC/MS/MS conditions
Samples were analyzed using the Prominence UFLC system (Shimazu, Kyoto Japan),
fitted with an AB Sciex 4000 QTRAP® mass spectrometer detector. The data was acquired in positive ion electrospray ionization mode. For chromatography, a Phenomenex L-column (150 × 2.1 mm, 5.0 µM) was used for separation at a column temperature 40°C. The mobile phase comprising 95% 10 mM ammonium formate and 5% methanol, and 5% 10 mM ammonium formate and 95% methanol was used at a flow
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rate of 5.0 µL/min.
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Memantine and DPZ concentration were measured in samples from the central blood (heart) (C), peripheral blood (P), urine, liver (L), thigh muscle (T) and subcutaneous fat
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(S). The C/P ratio was calculated using concentrations in the central blood and the
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peripheral blood, the L/P ratio was calculated from the liver concentration and the
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peripheral blood concentration, the T/P ratio was calculated from the thigh muscle concentration and the peripheral blood concentration, and S/P ratio was calculated from
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the subcutaneous fat concentration and the peripheral blood concentration.
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Validation of the analytical methodology
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MRM mode was used for quantification of memantine and DPZ in the biological specimens. The ions at m/z 180.1 and 107.0, and 380.2 and 91.0, were used to quantify memantine and DPZ, respectively. Memantine and DPZ concentrations were calculated using a linear regression analysis of the calibrator responses from a five or six point calibration curve. All curves met the acceptance criteria of r2 ≥ 0.991. According to this data, the limit of detection (LOD), calculated based on a signal to noise ratio of 3:1, for memantine and DPZ were 0.01 and 0.003 ng/mL in blood and 3 and 0.02 ng/mL in liver, respectively. The limit of quantification (LOQ) based on a signal to noise ratio of 10:1
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was 0.05 and 0.01 ng/mL in blood and 8 and 0.05 ng/mL in liver, respectively. Fifteen
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replicates of blood at concentrations of both 0.1 µg and 5 µg for memantine and 0.1 ug and 7.5 µg for DPZ, and of liver at concentrations of both 1 µg and 30 µg for memantine,
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and 0.5 µg and 10 µg for DPZ were analyzed through the complete procedure and the
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relative standard deviation (RSD) was within the acceptable range (±20%). This
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analysis indicates that there is no obvious matrix effect in different tissue types.
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Quantification
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Memantine and DPZ concentration were measured in samples from the central blood
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(heart) (C), peripheral blood (P), urine, liver (L), thigh muscle (T) and subcutaneous fat
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(S). Quantification was done twice, once immediately after autopsy and once 6 months later. Measurement from the day of autopsy was a simple quantitation using our sample screening process. The second quantification was performed using the validated methodology and samples that were pretreated using the column extraction as described above.
The C/P ratio was calculated using concentrations in the central blood and the peripheral blood, the L/P ratio was calculated from the liver concentration and the peripheral blood concentration, the T/P ratio was calculated from the thigh muscle
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concentration and the peripheral blood concentration, and S/P ratio was calculated from
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the subcutaneous fat concentration and the peripheral blood concentration.
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Results and Discussion
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There was no significant difference between the quantitative value obtained on
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autopsy day and the quantitative value of after freezing the blood, urine and tissue samples for 6 months. This result indicates minimal degradation or bacterial activity in
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the samples in the first 6 months. Therefore we used the quantitation values from the
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6-month samples for our data analysis, and these values are shown in Table 1.
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The central blood to peripheral blood (C/P) ratio, and the ratios of various tissues to
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the peripheral blood are shown in Table 2. Memantine and DPZ were detected in highest concentrations in the liver; the liver/peripheral blood (L/P) ratio was highest, at 19.5 and 18.8 respectively. The C/P ratio and subcutaneous fat–peripheral blood (S/P) ratio both also exceeded 1 in memantine. L/P, C/P and thigh muscle-peripheral blood (T/P) ratio of DPZ were also exceeded 1. Previous studies conducted by memantine tablet manufacturers have helped to establish the expected therapeutic range of memantine plasma concentration. In a clinical trial of elderly patients (n=11) a single-dose administration of 20 mg memantine
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gave a resulting Cmax of 31.73 ± 4.5 ng/mL [5]. In another, AD patients were started on
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oral 5 mg once daily, with an increase of 5 mg each week, then maintained 20 mg for 24 weeks, and plasma concentrations remained stable at approximately 0.12 µg/mL, from 4
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weeks through to 24 weeks [5]. Taking into account all these results, the clinical plasma
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therapeutic range was calculated to be approximately 0.09 µg/mL to 0.15 µg/mL, with a
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toxicity level set at 0.3 µg/mL and over [11].
A woman who survived the acute ingestion of 2000 mg developed coma, hypertension,
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tachycardia, seizures and a blood memantine concentration was 1.2 µg/mL [7]. Few
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reports discuss the blood memantine level in postmortem samples. In one case it was
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reported that memantine was detected at 0.65 µg/mL from the peripheral blood (whole
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blood), 1.8 µg/mL from the heart blood (whole blood) and 6.1 µg/g from the liver, in the suspicious death of a 62-year-old man [8]. The authors concluded that these results were not indicative of an overdose of memantine and cause of death was chloroform intoxication [8]. In the present case, there was no evidence of an overdose but nevertheless the detected memantine concentration was 1.35 µg/mL in the whole peripheral blood, 3.95 µg/mL in the whole heart blood, and 25.54 µg/g from the liver. These values are much higher than the non-intoxication case that was previously reported.
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Because the cause of death in the present case was asphyxiation due to strangulation,
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it is clear that drug intoxication was not involved in her death, and therefore could not be the cause of the high memantine concentrations we detected. Furthermore, the
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autopsy and pathological examinations showed no lesions or injury to the liver, which is
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a metabolic organ, or to her kidneys, an excretory organ. Only two drugs were detected
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in the screening process, so drug interactions are thought not to have had an influence here. Other factors that increase the concentration of the drug antemortem were not
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found.
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Generally, with a large volume of distribution (VD) >3 L/kg, highly lipophilic drugs are
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said to be readily redistributed [12]. Memantine has a large volume distribution, at 9 to
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11 L/kg, and the 1-octanol/buffer solution is 0.32, meaning it is fat-soluble, so memantine is said to be a drug that readily redistributes after death. In previous reports, C/P>1 and L/P>10 were described as reference points at which postmortem redistribution happens [10, 13-18]. The memantine C/P in this study was 3.01, and L/P was 19.5, which are both high. In addition, donepezil was detected in these samples, and this has already been reported to undergo postmortem redistribution [10]. As shown in Table 2 the C/P ratio and L/P ratio were high, at 1.12 and 18.8 respectively, suggesting postmortem redistribution. Accordingly, it is possible that postmortem
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memantine concentration in blood was increased through the process of PMR.
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The details of the mechanism of PMR are not yet clear. Compared with acidic drugs and neutral drugs, basic drugs tend to accumulate more in the lungs [19]. Flecainide,
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which is a basic drug (pKa=9.36), is associated with a postmortem rise in heart blood
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levels, reportedly caused by a decrease in the blood pH after death [20]. Accordingly, it is
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inferred that basic drugs are accumulated in the lungs antemortem, and the post-mortem pH decrease is accompanied by leaking of drugs that are accumulated in
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the lungs out into the heart blood. This results in a higher concentration of the drug in
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the heart blood. The postmortem pH in heart blood and peripheral blood in the present
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case was 6.1 and 6.2, respectively, representing a pH decrease, and this suggests
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possible redistribution of memantine after death. However, the concentration in the lungs could not be measured in the present case, and these details could not be obtained by any other means. Investigating drug redistribution in the future will require measuring changes in drug concentration distribution in the lungs. Memantine was also detected at a high concentration in the subcutaneous fat, and the
S/P ratio also exceeded 1. This implies an accumulation in the subcutaneous fat, because the distribution coefficient of memantine in a 1-octanol/buffer solution is 0.32, meaning it is highly fat-soluble.
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In the present case, the postmortem redistribution was investigated from the C/P ratio and L/P ratio at only one point in time, so it was not possible to determine whether the
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drug concentrations changed with time. Postmortem redistribution is thought to depend
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on the postmortem interval, so to examine PMR in more detail, there is a need for
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concentration measurements at specific times after death. As there is no prior report of memantine intoxication cases, the threshold between intoxication and non-intoxication
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in the postmortem concentration of memantine is not known. Further postmortem
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concentrations in memantine overdose cases and intoxication cases will need to be
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collected to clarify the relationship between postmortem concentration and intoxication.
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In addition, side effects can occur even within therapeutic ranges, and it will always be necessary to make comprehensive judgments when investigating fatalities. This includes not only the drug levels in the blood, but also circumstantial evidence and autopsy results, especially in cases where the involvement of the drug in the cause of death cannot be completely ruled out.
Conclusion We have found that memantine can be detected at relatively high concentrations even
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in non-intoxication postmortem specimens. Although this is only a single case study, it
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suggests that the cause of these high C/P and L/P ratios may be the PMR of memantine. It is also though that the chemical characteristics of memantine can cause a decrease in
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pH at the death, promoting PMR. The results of this case serve as a foundation for the
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future interpretation of toxicological data related to both therapeutic and toxicological
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levels of memantine, and the expectation of PMR of this drug.
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References
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1. Reisberg, B., Doody, R., Stoffler, A., for the Memantine Study Group. Memantine in moderate-to-severe Alzheimer’s disease. N Engl J M. (2003) 348:1333-1341.
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2. Tariot, P.N., Farlow, M.R., Grossberg, G.T., for the Memantine Study Group.
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Memantine treatment in patients with moderate to severe Alzheimer’s disease
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already receiving donepezil: a randomized controlled trial. JAMA (2004) 291:317-324.
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3. Winblad, B., Jones, R.W., Wirth, Y. Memantine in moderate to severe Alzheimer’s
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(2007) 24:20-27.
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disease; a meta-analysis of randomized clinical trials. Dement Geriatr Cogn Disord.
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4. Araki, T., Wake, R., Miyaoka, T., Kawakami, K., Nagahama, M., Furuya, M., Limoa, E., Liaury, K., Hashioka, S., Murotani, K., Horiguchi, J. The effects of combined treatment of memantine and donepezil on Alzheimer’s disease patients and its
relationship with cerebral blood flow in the prefrontal area. Int J Geriatr Psychiatry (2014) 29:881-889.
5. Daiichi Sankyo Company. Memary (memantine hydrochloride tablets). Product insert: Summary Basis of Approval. Daiichi Sankyo Co Ltd, Tokyo. 2011 6. Physician’s Desk Reference, Thomson PDR, Montvale, New Jersey, 2007
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7. Cekmen, N., Bedel, P., Erdemli, O. A memantin HCL intoxication responsive to
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plasmapheresis therapy. Bratisl Lek Listy. (2011) 112:527-529. 8. Bynum, N., Poklis, J., Garside, D., Winecker, R. Postmortem memantine
D.S., Braithwaite,
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Estimating
antemortem
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9. Cook,
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concentrations. J Anal Toxicol. (2007) 31:233-236.
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concentrations from postmortem blood samples; the influence of postmortem redistribution. J Clin. Pathol. (2000) 53:282-285.
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10. Nagasawa, S., Torimitsu, S., Chiba, F., Kubo, Y., Yajima, D., Iwase, H. Donepezil
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distribution in postmortem cases and potential for redistribution. Forensic Sci Int.
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(2015) 13:132-138.
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11. Pounder, D.J. The nightmare of postmortem drug changes, Leg Med. (1993) 161-191. 12. Saar, E., Beyer, J., Gerostamoulos, D., Drummer, O.H. The time-dependent post-mortem redistribution of antipsychotic drugs. Forensic Sci Int. (2012) 222:223-227.
13. Han, E., Kim, E., Hong, H., Heong, S., Kim, J., In, S., Chung, H., Lee, S. Evaluation of postmortem redistribution phenomena for commonly encountered drugs. Forensic Sci Int. (2012) 219:265-271. 14. Mclntyre, I.M. Liver to peripheral blood concentration ratio (L/P) as a marker of
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postmortem drug redistribution: a literature review. Forensic Sci Med Pathol. (2014)
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10: 91-96. 15. Mclntyre, I.M., Sherrard, J., Lucas, J. Postmortem carisoprodol and meprobamate
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concentrations in blood and liver: lack of significant distribution. J Anal Tox. (2012)
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36:177-178.
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16. Moore, K.A., Cina, S.J., Jones, R., Selby, D.M., Levine, B., Smith, M.L. Tissue distribution of tramadol and metabolites in an overdose fatality. Am J Forensic Med
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Pathol. (1999) 20:98-100.
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17. Mclntyre, I.M., Mallett, P., Sertraline concentrations and postmortem redistribution.
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18. Mclntyre, I.M., Anderson, D.T. Postmortem fentanyl concentrations: a review. J Forensic Res. (2012), http: //dx.doi.org/10.4172/2157-7145.1000157
19. Yoshida, M., Kubunai, T., Aoyagi, K., Saito, H., Utsugi, T., Wierzba, K., Yamada, Y. Specific distribution of TOP-53 to the lung and lung-localized tumor is determined by its interaction with phospholipids. Clin Cancer Res. (2000) 11:4396-4401.
20. Yoshitome, K., Ishizu, H., Miyamoto, S. Postmortem acidification of blood/organs induces an increase in flecainide concentration in cardiac blood and the contribution of the lung to this increase. J Analytical Toxicology. (2010) 34:26-31.
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Table 1. Memantine and Donepezil concentrations detected in blood, urine and of second measurement.
Memantine
Donepezil 3.95
Peripheral blood (µg/mL)
1.31
Urine (µg/mL)
2.09
Liver (µg/g)
25.54
Thigh muscle (µg/g)
1.16
Subcutaneous fat (µg/g)
2.13
0.55 0.49 6.13 9.21 1.50 6.57
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Central blood (µg/mL)
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Matrix
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each tissue samples
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Donepezil 3.01
L/P ratio
19.50
T/P ratio
0.88
S/P ratio
1.63
1.12
18.80 2.28
13.4
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C/P ratio
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Memantine
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Table 2. Ratio of C/P and L/P in the blood and tissue samples of the deceased
C, Central blood; P, Peripheral blood; L, Liver; T, Thigh muscle ;
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S, subcutaneous fat
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S0379-0738(15)00359-X http://dx.doi.org/doi:10.1016/j.forsciint.2015.08.015 FSI 8129
To appear in:
FSI
Received date: Revised date: Accepted date:
24-5-2015 14-8-2015 23-8-2015
Please cite this article as: S. Nagasawa, D. Yajima, S. Torimitsu, F. Chiba, H. Iwase, Postmortem memantine concentration in a non-intoxication case, and the possibility of postmortem redistribution: a case report, Forensic Science International (2015), http://dx.doi.org/10.1016/j.forsciint.2015.08.015 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Highlights
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・Memantine was detected at high concentrations in a non-intoxication postmortem case.
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・C/P and L/P ratios of memantine indicate likelihood of postmortem redistribution.
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・Postmortem reduction in blood pH may also promote the PMR of memantine.
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Postmortem memantine concentration in a non-intoxication case, and the possibility of postmortem redistribution: a case report
Sayaka Nagasawa,
1)
Daisuke Yajima,
1,2)
Suguru Torimitsu,
Chiba,
1,2)
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Hirotaro Iwase.
of Legal Medicine, Graduate School of Medicine, Chiba University, Chiba,
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1) Department
1,2 )Fumiko
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1)
Japan, 1-8-1 Inohana, Chuo-ku, Chiba 260-8670, Japan
Department of Forensic Medicine, Graduate School of Medicine, The University of
an
2)
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Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan
Institution at which the work was performed:
Corresponding author:
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Department of Legal Medicine, Graduate School of Medicine, Chiba University
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Sayaka Nagasawa, Ph.D.
Assistant Professor, Department of Legal Medicine, Graduate School of Medicine, Chiba University
1-8-1 Inohana, Chuo-ku, Chiba 260-8670, Japan Tel: +81-43-226-2078
Fax: +81-43-226-2079
E-mail: [email protected]
Page 2 of 23
Abstract
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In this case study, we measured the concentration of memantine in the heart blood, peripheral blood, urine, liver, thigh muscle, and subcutaneous fat of a 64-year-old
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woman who was prescribed memantine for early-onset Alzheimer’s disease. She died in
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hospital after an altercation with her husband. Cause of death was clearly not drug
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intoxication or overdose, so we investigated the postmortem redistribution (PMR) of memantine in the various tissues and blood ratios of the postmortem samples.
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Memantine concentrations detected were 1.35 µg/mL in the peripheral blood, 3.95
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µg/mL in central blood, 2.09 µg/mL in the urine, 25.54 µg/g in the liver, 1.16 µg/g in the
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thigh muscle and 2.13 µg/g in the subcutaneous fat. In all samples, the concentrations
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were higher than the accepted therapeutic range (which is approximately 0.09–0.15 µg/mL). The central blood to peripheral blood (C/P) memantine ratio was 3.01 while the liver to peripheral blood (L/P) ratio was 19.5. It is documented that a C/P ratio exceeding 2 and L/P ratio exceeding 20 highlight a propensity for significant PMR. Although this is a single case study, our data suggest that memantine exhibits PMR. Additionally, a lowered pH was found in peripheral blood (pH=6.2) and central blood (pH=6.1). This postmortem reduction in blood pH may also promote the PMR of memantine. Because there is very little available postmortem toxicological data on
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memantine, our case study will serve as a foundation to assist in future forensic
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investigations.
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Keyword
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Memantine Postmortem redistribution
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Liver
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Non-intoxication
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Alzheimer's disease
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Introduction
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In recent years, an increasingly aging population has been observed in many countries and in Japan in particular. Because of this, the incidence of dementia and Alzheimer’s
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disease (AD) is also rising rapidly. In the past 10 years, at least 80 countries around the
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world have prescribed memantine as a safe and effective therapeutic medicine for AD,
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and it is predicted that the number of prescriptions will increase as the number of patients rises [1-3].
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Memantine binds to a receptor of the neurotransmitter glutamic acid and acts to
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prevent neuropathy caused by excess glutamic acid [4]. Compared with cholinesterase
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inhibitors (donepezil, galanthamine, rivastigmine), cardiac side effects of memantine
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are rare, but severe side effects such as convulsions, fainting, and loss of consciousness have been reported [5]. Emergency responses to overdose cases (unknown whether it was intentional or accidental) are also being reported [6, 7]. As the main symptom of AD patients is dementia, it is likely that more incidents of overdoses will occur in the future. However, there is very little reporting on findings of postmortem memantine concentrations [8]. Postmortem drug concentrations in blood may not always reflect antemortem concentrations because of postmortem changes (e.g. postmortem diffusion and
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postmortem redistribution) [9]. Therefore, the true assessment of postmortem blood
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levels is difficult. In this study, we measured the concentration of memantine and donepezil in heart blood, peripheral blood, urine, liver, thigh muscle, and subcutaneous
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fat from a woman whose cause of death was clearly not drug intoxication or an overdose
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of memantine. Furthermore, we investigated the postmortem redistribution of
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memantine from the various tissues and blood ratios obtained.
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Case Circumstances
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A 64-year-old woman was diagnosed with early-onset AD at 58, and had been
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prescribed memantine for a year, following the worsening of her symptoms. On the
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morning of the day before her death, she had an argument with her husband, who attempted to strangle her with an electrical cord. She was transported to the hospital, where she died 22 h later. Details of her last prescription were memantine 20 mg/day and donepezil 10 mg/day after breakfast, tiapride 25 mg/dose after each meal and risperidone 0.5 mg/dose as needed for agitation. These were all within acceptable therapeutic dose ranges. The autopsy examination showed findings typical of strangulation, including congestion of the face and head; both palpebral and bulbar submucosal hemorrhage;
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linear keratolysis and allochroism running across the neck in the left-right direction;
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bleeding in the various muscles of the neck; bleeding in the tongue muscle; and fractures accompanied by bleeding in the greater cornu of the left hyoid bone and the
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superior horn of the right thyroid cartilage. From these findings it was concluded that
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an external force had been applied to her neck, resulting in congestion of the facial
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surface of her head. Histological examination of various organs showed no obvious lesions, and the diagnosis was asphyxiation due to strangulation. Heart and femoral
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blood was screened for drugs, and memantine and donepezil were detected in both
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samples, and alcohol level was 0 mg/mL Heart blood pH was 6.1, and femoral blood was
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6.2. Postmortem interval was estimated to be 48 h.
Materials and Methods
Sample collection and storage
Peripheral blood was drawn from the right and left of the femoral vein, and stored in
cryotubes together (Nunc Cryo Tube, Milian, Geneva, Switzerland). Central blood was collected from the heart (mixed blood) and placed in identical tubes. The right lobe of liver, right thigh muscle and subcutaneous fat of the thigh were collected and stored in a falcon tube. Urine samples were collected and stored in cryotubes. Following the
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standard protocol of our laboratory, all samples were pretreated and analyzed
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immediately, with the remainder of these samples being placed in storage at −̯20 °C.
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Toxicological analyses
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Systematic toxicological analysis was performed on the postmortem samples to detect
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the presence of alcohol, prescribed drugs, and illegal drugs. Screening for routine drugs in blood and urine was performed with a 3200 QTRAP® LC/MS/MS system (AB Sciex,
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Foster City, CA, USA) using the Multiple Reaction Monitoring (MRM) method. For each
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blood and urine sample, 900 µL of ACN was added to 100 µL of sample. The mixture was
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vortexed for 30 s, sonicated for 5 min, and centrifuged at 850 × g for 15 min. For tissue
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samples, ACN was added to three times the volume of the sample. The mixture was homogenized and centrifuged at 850 × g for 15 min. The supernatant was retained for subsequent analysis.
Reagents and chemicals
Memantine hydrochloride and donepezil hydrochloride were purchased from Sigma-Aldrich (St Louis, MO, USA). Ammonium formate was purchased from WAKO (Tokyo, Japan). Methanol and acetonitrile (ACN) were purchased from Kanto Kagaku
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(Tokyo, Japan). All other chemicals were of analytical reagent grade.
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Control samples of blood were collected from healthy volunteers after obtaining their informed consent, and liver, thigh muscle and subcutaneous fat samples were taken
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from selected cadavers, all of whom had no history of drug abuse. These samples were
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screened for common drugs (including memantine) and alcohol, and all were confirmed
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negative.
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Preparation of stock and calibration solutions
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Memantine hydrochloride and donepezil hydrochloride stock solutions (1 mg/ml) were
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prepared in distilled water and methanol respectively, and stored frozen at −20°C.
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Standards for quantification of memantine in blood and urine were prepared in 1 mL drug-free blood and urine samples by spiking the samples with memantine at concentrations of 0.1, 0.5, 1, 2.5, and 5 µg/mL. Standards for quantification of donepezil (DPZ) in blood and urine were prepared in 1-mL drug-free blood and urine samples by spiking the samples with DPZ at concentrations of 0.1, 0.25, 0.5, 1, 5 and 7.5 µg/mL.. Standards for quantification of memantine and DPZ in liver, thigh muscle and subcutaneous fat were prepared in 1 g drug-free samples of each tissue by spiking the samples with memantine at 1, 5, 10, 20 and 30 µg/g, and with DPZ at 0.5, 1, 2.5, 5 and
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10 µg/g. Negative blood, urine, liver, thing muscle and subcutaneous fat controls were
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also prepared before each analysis performed.
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Extraction
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Biological sample extraction was performed using Focus™ columns (Lake Foster, CA,
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USA) following the methodology described in our previous study [10]. To each blood and urine sample, 300 µL of 1M acetate buffer and 2 mL of distilled water were added. The
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mixture was vortexed for 30 s, sonicated for 5 min, and centrifuged at 850 × g for 15 min.
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To each tissue sample, 300 µL of 1M acetate buffer and 2 mL of distilled water were
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added. The mixture was homogenized and centrifuged at 850 × g for 15 min.
LC/MS/MS conditions
Samples were analyzed using the Prominence UFLC system (Shimazu, Kyoto Japan),
fitted with an AB Sciex 4000 QTRAP® mass spectrometer detector. The data was acquired in positive ion electrospray ionization mode. For chromatography, a Phenomenex L-column (150 × 2.1 mm, 5.0 µM) was used for separation at a column temperature 40°C. The mobile phase comprising 95% 10 mM ammonium formate and 5% methanol, and 5% 10 mM ammonium formate and 95% methanol was used at a flow
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rate of 5.0 µL/min.
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Memantine and DPZ concentration were measured in samples from the central blood (heart) (C), peripheral blood (P), urine, liver (L), thigh muscle (T) and subcutaneous fat
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(S). The C/P ratio was calculated using concentrations in the central blood and the
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peripheral blood, the L/P ratio was calculated from the liver concentration and the
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peripheral blood concentration, the T/P ratio was calculated from the thigh muscle concentration and the peripheral blood concentration, and S/P ratio was calculated from
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the subcutaneous fat concentration and the peripheral blood concentration.
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Validation of the analytical methodology
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MRM mode was used for quantification of memantine and DPZ in the biological specimens. The ions at m/z 180.1 and 107.0, and 380.2 and 91.0, were used to quantify memantine and DPZ, respectively. Memantine and DPZ concentrations were calculated using a linear regression analysis of the calibrator responses from a five or six point calibration curve. All curves met the acceptance criteria of r2 ≥ 0.991. According to this data, the limit of detection (LOD), calculated based on a signal to noise ratio of 3:1, for memantine and DPZ were 0.01 and 0.003 ng/mL in blood and 3 and 0.02 ng/mL in liver, respectively. The limit of quantification (LOQ) based on a signal to noise ratio of 10:1
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was 0.05 and 0.01 ng/mL in blood and 8 and 0.05 ng/mL in liver, respectively. Fifteen
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replicates of blood at concentrations of both 0.1 µg and 5 µg for memantine and 0.1 ug and 7.5 µg for DPZ, and of liver at concentrations of both 1 µg and 30 µg for memantine,
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and 0.5 µg and 10 µg for DPZ were analyzed through the complete procedure and the
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relative standard deviation (RSD) was within the acceptable range (±20%). This
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analysis indicates that there is no obvious matrix effect in different tissue types.
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Quantification
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Memantine and DPZ concentration were measured in samples from the central blood
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(heart) (C), peripheral blood (P), urine, liver (L), thigh muscle (T) and subcutaneous fat
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(S). Quantification was done twice, once immediately after autopsy and once 6 months later. Measurement from the day of autopsy was a simple quantitation using our sample screening process. The second quantification was performed using the validated methodology and samples that were pretreated using the column extraction as described above.
The C/P ratio was calculated using concentrations in the central blood and the peripheral blood, the L/P ratio was calculated from the liver concentration and the peripheral blood concentration, the T/P ratio was calculated from the thigh muscle
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concentration and the peripheral blood concentration, and S/P ratio was calculated from
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the subcutaneous fat concentration and the peripheral blood concentration.
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Results and Discussion
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There was no significant difference between the quantitative value obtained on
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autopsy day and the quantitative value of after freezing the blood, urine and tissue samples for 6 months. This result indicates minimal degradation or bacterial activity in
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the samples in the first 6 months. Therefore we used the quantitation values from the
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6-month samples for our data analysis, and these values are shown in Table 1.
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The central blood to peripheral blood (C/P) ratio, and the ratios of various tissues to
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the peripheral blood are shown in Table 2. Memantine and DPZ were detected in highest concentrations in the liver; the liver/peripheral blood (L/P) ratio was highest, at 19.5 and 18.8 respectively. The C/P ratio and subcutaneous fat–peripheral blood (S/P) ratio both also exceeded 1 in memantine. L/P, C/P and thigh muscle-peripheral blood (T/P) ratio of DPZ were also exceeded 1. Previous studies conducted by memantine tablet manufacturers have helped to establish the expected therapeutic range of memantine plasma concentration. In a clinical trial of elderly patients (n=11) a single-dose administration of 20 mg memantine
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gave a resulting Cmax of 31.73 ± 4.5 ng/mL [5]. In another, AD patients were started on
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oral 5 mg once daily, with an increase of 5 mg each week, then maintained 20 mg for 24 weeks, and plasma concentrations remained stable at approximately 0.12 µg/mL, from 4
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weeks through to 24 weeks [5]. Taking into account all these results, the clinical plasma
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therapeutic range was calculated to be approximately 0.09 µg/mL to 0.15 µg/mL, with a
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toxicity level set at 0.3 µg/mL and over [11].
A woman who survived the acute ingestion of 2000 mg developed coma, hypertension,
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tachycardia, seizures and a blood memantine concentration was 1.2 µg/mL [7]. Few
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reports discuss the blood memantine level in postmortem samples. In one case it was
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reported that memantine was detected at 0.65 µg/mL from the peripheral blood (whole
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blood), 1.8 µg/mL from the heart blood (whole blood) and 6.1 µg/g from the liver, in the suspicious death of a 62-year-old man [8]. The authors concluded that these results were not indicative of an overdose of memantine and cause of death was chloroform intoxication [8]. In the present case, there was no evidence of an overdose but nevertheless the detected memantine concentration was 1.35 µg/mL in the whole peripheral blood, 3.95 µg/mL in the whole heart blood, and 25.54 µg/g from the liver. These values are much higher than the non-intoxication case that was previously reported.
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Because the cause of death in the present case was asphyxiation due to strangulation,
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it is clear that drug intoxication was not involved in her death, and therefore could not be the cause of the high memantine concentrations we detected. Furthermore, the
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autopsy and pathological examinations showed no lesions or injury to the liver, which is
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a metabolic organ, or to her kidneys, an excretory organ. Only two drugs were detected
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in the screening process, so drug interactions are thought not to have had an influence here. Other factors that increase the concentration of the drug antemortem were not
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found.
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Generally, with a large volume of distribution (VD) >3 L/kg, highly lipophilic drugs are
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said to be readily redistributed [12]. Memantine has a large volume distribution, at 9 to
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11 L/kg, and the 1-octanol/buffer solution is 0.32, meaning it is fat-soluble, so memantine is said to be a drug that readily redistributes after death. In previous reports, C/P>1 and L/P>10 were described as reference points at which postmortem redistribution happens [10, 13-18]. The memantine C/P in this study was 3.01, and L/P was 19.5, which are both high. In addition, donepezil was detected in these samples, and this has already been reported to undergo postmortem redistribution [10]. As shown in Table 2 the C/P ratio and L/P ratio were high, at 1.12 and 18.8 respectively, suggesting postmortem redistribution. Accordingly, it is possible that postmortem
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memantine concentration in blood was increased through the process of PMR.
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The details of the mechanism of PMR are not yet clear. Compared with acidic drugs and neutral drugs, basic drugs tend to accumulate more in the lungs [19]. Flecainide,
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which is a basic drug (pKa=9.36), is associated with a postmortem rise in heart blood
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levels, reportedly caused by a decrease in the blood pH after death [20]. Accordingly, it is
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inferred that basic drugs are accumulated in the lungs antemortem, and the post-mortem pH decrease is accompanied by leaking of drugs that are accumulated in
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the lungs out into the heart blood. This results in a higher concentration of the drug in
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the heart blood. The postmortem pH in heart blood and peripheral blood in the present
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case was 6.1 and 6.2, respectively, representing a pH decrease, and this suggests
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possible redistribution of memantine after death. However, the concentration in the lungs could not be measured in the present case, and these details could not be obtained by any other means. Investigating drug redistribution in the future will require measuring changes in drug concentration distribution in the lungs. Memantine was also detected at a high concentration in the subcutaneous fat, and the
S/P ratio also exceeded 1. This implies an accumulation in the subcutaneous fat, because the distribution coefficient of memantine in a 1-octanol/buffer solution is 0.32, meaning it is highly fat-soluble.
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In the present case, the postmortem redistribution was investigated from the C/P ratio and L/P ratio at only one point in time, so it was not possible to determine whether the
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drug concentrations changed with time. Postmortem redistribution is thought to depend
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on the postmortem interval, so to examine PMR in more detail, there is a need for
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concentration measurements at specific times after death. As there is no prior report of memantine intoxication cases, the threshold between intoxication and non-intoxication
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in the postmortem concentration of memantine is not known. Further postmortem
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concentrations in memantine overdose cases and intoxication cases will need to be
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collected to clarify the relationship between postmortem concentration and intoxication.
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In addition, side effects can occur even within therapeutic ranges, and it will always be necessary to make comprehensive judgments when investigating fatalities. This includes not only the drug levels in the blood, but also circumstantial evidence and autopsy results, especially in cases where the involvement of the drug in the cause of death cannot be completely ruled out.
Conclusion We have found that memantine can be detected at relatively high concentrations even
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in non-intoxication postmortem specimens. Although this is only a single case study, it
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suggests that the cause of these high C/P and L/P ratios may be the PMR of memantine. It is also though that the chemical characteristics of memantine can cause a decrease in
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pH at the death, promoting PMR. The results of this case serve as a foundation for the
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future interpretation of toxicological data related to both therapeutic and toxicological
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levels of memantine, and the expectation of PMR of this drug.
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References
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1. Reisberg, B., Doody, R., Stoffler, A., for the Memantine Study Group. Memantine in moderate-to-severe Alzheimer’s disease. N Engl J M. (2003) 348:1333-1341.
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2. Tariot, P.N., Farlow, M.R., Grossberg, G.T., for the Memantine Study Group.
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Memantine treatment in patients with moderate to severe Alzheimer’s disease
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already receiving donepezil: a randomized controlled trial. JAMA (2004) 291:317-324.
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3. Winblad, B., Jones, R.W., Wirth, Y. Memantine in moderate to severe Alzheimer’s
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4. Araki, T., Wake, R., Miyaoka, T., Kawakami, K., Nagahama, M., Furuya, M., Limoa, E., Liaury, K., Hashioka, S., Murotani, K., Horiguchi, J. The effects of combined treatment of memantine and donepezil on Alzheimer’s disease patients and its
relationship with cerebral blood flow in the prefrontal area. Int J Geriatr Psychiatry (2014) 29:881-889.
5. Daiichi Sankyo Company. Memary (memantine hydrochloride tablets). Product insert: Summary Basis of Approval. Daiichi Sankyo Co Ltd, Tokyo. 2011 6. Physician’s Desk Reference, Thomson PDR, Montvale, New Jersey, 2007
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7. Cekmen, N., Bedel, P., Erdemli, O. A memantin HCL intoxication responsive to
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plasmapheresis therapy. Bratisl Lek Listy. (2011) 112:527-529. 8. Bynum, N., Poklis, J., Garside, D., Winecker, R. Postmortem memantine
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concentrations from postmortem blood samples; the influence of postmortem redistribution. J Clin. Pathol. (2000) 53:282-285.
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10. Nagasawa, S., Torimitsu, S., Chiba, F., Kubo, Y., Yajima, D., Iwase, H. Donepezil
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distribution in postmortem cases and potential for redistribution. Forensic Sci Int.
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(2015) 13:132-138.
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11. Pounder, D.J. The nightmare of postmortem drug changes, Leg Med. (1993) 161-191. 12. Saar, E., Beyer, J., Gerostamoulos, D., Drummer, O.H. The time-dependent post-mortem redistribution of antipsychotic drugs. Forensic Sci Int. (2012) 222:223-227.
13. Han, E., Kim, E., Hong, H., Heong, S., Kim, J., In, S., Chung, H., Lee, S. Evaluation of postmortem redistribution phenomena for commonly encountered drugs. Forensic Sci Int. (2012) 219:265-271. 14. Mclntyre, I.M. Liver to peripheral blood concentration ratio (L/P) as a marker of
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postmortem drug redistribution: a literature review. Forensic Sci Med Pathol. (2014)
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10: 91-96. 15. Mclntyre, I.M., Sherrard, J., Lucas, J. Postmortem carisoprodol and meprobamate
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concentrations in blood and liver: lack of significant distribution. J Anal Tox. (2012)
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16. Moore, K.A., Cina, S.J., Jones, R., Selby, D.M., Levine, B., Smith, M.L. Tissue distribution of tramadol and metabolites in an overdose fatality. Am J Forensic Med
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Pathol. (1999) 20:98-100.
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17. Mclntyre, I.M., Mallett, P., Sertraline concentrations and postmortem redistribution.
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18. Mclntyre, I.M., Anderson, D.T. Postmortem fentanyl concentrations: a review. J Forensic Res. (2012), http: //dx.doi.org/10.4172/2157-7145.1000157
19. Yoshida, M., Kubunai, T., Aoyagi, K., Saito, H., Utsugi, T., Wierzba, K., Yamada, Y. Specific distribution of TOP-53 to the lung and lung-localized tumor is determined by its interaction with phospholipids. Clin Cancer Res. (2000) 11:4396-4401.
20. Yoshitome, K., Ishizu, H., Miyamoto, S. Postmortem acidification of blood/organs induces an increase in flecainide concentration in cardiac blood and the contribution of the lung to this increase. J Analytical Toxicology. (2010) 34:26-31.
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Table 1. Memantine and Donepezil concentrations detected in blood, urine and of second measurement.
Memantine
Donepezil 3.95
Peripheral blood (µg/mL)
1.31
Urine (µg/mL)
2.09
Liver (µg/g)
25.54
Thigh muscle (µg/g)
1.16
Subcutaneous fat (µg/g)
2.13
0.55 0.49 6.13 9.21 1.50 6.57
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Central blood (µg/mL)
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Matrix
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each tissue samples
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Donepezil 3.01
L/P ratio
19.50
T/P ratio
0.88
S/P ratio
1.63
1.12
18.80 2.28
13.4
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C/P ratio
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Memantine
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Table 2. Ratio of C/P and L/P in the blood and tissue samples of the deceased
C, Central blood; P, Peripheral blood; L, Liver; T, Thigh muscle ;
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S, subcutaneous fat
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