Journal of Pharmacological and Toxicological Methods 49 (2004) 57 – 64
`
`www.elsevier.com/locate/jpharmtox
`
`Original article
`
`An automated blood sampler for simultaneous sampling of systemic
`blood and brain microdialysates for drug absorption, distribution,
`metabolism, and elimination studies
`
`P. Chandrani Gunaratna*, Peter T. Kissinger, Candice B. Kissinger, James F. Gitzen
`
`Bioanalytical Systems, 2701 Kent Avenue, West Lafayette, IN 47906-1382, USA
`
`Received 17 June 2003; accepted 11 July 2003
`
`Abstract
`
`Introduction: A major problem in preclinical drug development where blood sampling from small animals is a routine practice is the time
`and labor involved in the serial sampling of small blood volumes from small animals such as rats for the duration of pharmacokinetic/
`pharmacodynamic (PK/PD) studies. The traditional method of manually drawing blood from the animal requires the animal to be anesthetized
`or restrained with some device, both of which cause stress to the animal. Methods: An automated blood sampler (ABS) was developed to
`simultaneously collect blood and brain microdialysate samples at preprogrammed time points from awake and freely moving animals. The
`samples are delivered to fraction collectors and stored at 4 jC until use. The lost blood volume during collection is replaced with sterile saline
`to prevent fluid loss from the animal. In addition, the system is capable of collecting urine and feces for metabolism studies and monitoring the
`animal activity for behavioral studies. In the present study, blood samples were collected for 24 h after dosing rats orally with a 5 mg/kg dose
`of olanzapine (OLAN). Brain dialysates were collected for the same duration from a microdialysis probe implanted in the striatum. Results:
`The pharmacokinetic parameters, obtained after an oral dose, are in good agreement with reported values in literature. The pharmacodynamic
`information obtained from brain dialysates data show that OLAN elevates the concentration of dopamine (DA) in the brain and remains in the
`brain even after it is cleared from the plasma. Discussion: The ABS described here is a very useful tool in drug development to accelerate the
`pace of preclinical in vivo studies and to simultaneously provide pharmacodynamic and physiological information.
`D 2003 Elsevier Inc. All rights reserved.
`
`Keywords: Automated blood sampler; Pharmacokinetics; Pharmacodynamics; Olanzapine; Microdialysis; Neurotransmitters
`
`1. Introduction
`
`Combinatorial chemistry and high throughput screening
`have contributed to the enormous number of drug candi-
`dates now entering preclinical mammalian studies. In vitro
`screening is largely used to weed out the likely failures and
`narrow the pool of drug candidates in early stages of drug
`discovery. Integrated in vivo studies are needed to under-
`stand the pharmacology and toxicity of the selected drug
`candidates for further development. Obtaining absorption,
`distribution, metabolism, and elimination (ADME) param-
`eters from small animals is essential early on in preclinical
`studies to avoid future failures. Because the pharmacolog-
`ical effect of a drug is directly influenced by the amount of
`drug at the target site, when developing drugs for the brain
`
`* Corresponding author. Tel.: +1-765-497-5824; fax: +1-765-497-1102.
`E-mail address: prema@bioanalytical.com (P.C. Gunaratna).
`
`1056-8719/$ – see front matter D 2003 Elsevier Inc. All rights reserved.
`doi:10.1016/S1056-8719(03)00058-3
`
`and central nervous system (CNS), it is also important to
`study the distribution of the drug in the brain.
`Significant advances have been made for in vitro screen-
`ing, sample preparation, and analysis using robotic work-
`stations in the 96-well format. On the other hand,
`automation of in vivo techniques remains far behind and
`is highly labor intensive.
`In preclinical drug development, blood sampling from
`small animals is a routine practice. One snag in drug
`discovery pipeline is the time and labor involved in the
`serial sampling of small blood volumes from small animals
`such as rats for the duration of pharmacokinetic/pharmaco-
`dynamic (PK/PD) studies. The traditional method of man-
`ually drawing blood from the animal requires the animal to
`be anesthetized or restrained with some device, both of
`which cause stress to the animal. It is known that stress
`affects the pharmacokinetics by reducing the absorption and
`altering the metabolism (Jamali & Kunz-Dober, 1999).
`Stress delays gastric emptying and slow the absorption of
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`drugs through the gastrointestinal tract (Lee & Sarna, 1997).
`Another drawback of manual blood collection is the re-
`quirement of trained personnel and manual labor.
`
`Here, we describe the use of an automated blood
`sampling (ABS) system, shown in Fig. 1, for serial blood
`sampling from awake and freely moving rats for pharma-
`
`Fig. 1. Schematic illustration of the blood sampling process in the ABS. Heparinized saline is used as a motive force to transfer blood through the system.
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`59
`
`cokinetic studies. We have used the same system to
`simultaneously sample brain microdialysates from the
`same animal. The ABS system is capable of collecting
`blood, urine, and feces while monitoring the animal
`activity during the experiment. The system collects blood
`in sealed, refrigerated vials, which are directly transferable
`to a 96-well plate. Urine is collected in chilled scintillation
`vials. Feces are collected separately on a stainless steel
`screen. The automated sampler withdraws blood from a
`freely moving animal according to a programmed sched-
`ule, stores the samples in an integrated refrigerated fraction
`collector, and replaces the withdrawn volumes with phys-
`iological saline.
`The ABS is equipped with a sensor assembly, which
`consists of a left sensor to sense the clockwise movements
`and a right sensor to sense the counterclockwise movements
`of the animal in the cage. The animal activity software
`records the direction and the duration of the sensor move-
`ments. All the features and the operation of the system are
`described in detail elsewhere (Bohs, Cregor, Gunaratna, &
`Kissinger, 2000; He, Kramp, Ramos, & Bakhtiar, 2001;
`Peters et al., 2000).
`Microdialysis is a well-utilized technique in neuroscien-
`ces to sample the extracellular space in brain to obtain
`information on neurochemicals. This technique is becom-
`ing a valuable tool to study the drug distribution in brain
`and blood – brain barrier penetration by drug molecules
`(Deguchi & Morimoto, 2001; De Lange & Danhof,
`2002). These studies are essential for development of
`drugs that would be used for the treatment of neurological
`and psychological diseases. A well-studied antipsychotic
`drug, olanzapine (OLAN), was selected to demonstrate the
`utility of this approach for both pharmacokinetic and
`microdialysis sampling.
`OLAN is well absorbed and reaches peak concentrations
`in f 6 h following an oral dose in humans. It is eliminated
`extensively by first-pass metabolism, with f 40% of the
`dose metabolized before reaching the systemic circulation.
`The major circulating metabolites are 10-N-glucuronide and
`4V-N-desmethylolanzapine (NDMO) (Kassahun et al., 1997;
`Mattiuz et al., 1997).
`
`2. Methods
`
`2.1. Materials
`
`OLAN, its major metabolite NDMO, and the internal
`standard LY17022 were all generous donations from Eli
`Lilly and Company (Indianapolis, IN, USA). Dopamine
`(DA), 3,4-dihydroxyphenylacetic acid (DOPAC), homova-
`nillic acid (HVA), 5-hydroxyindoleacetic acid (5-HIAA),
`and serotonin (5-HT) were all purchased from Sigma (St.
`Louis, MO, USA). Distilled, deionized water was generated
`from NANO Pure ultrapure water system (Barnstead/Ther-
`molyne, Dubuque, IA, USA).
`
`2.2. Apparatus
`
`Liquid chromatography/mass spectrometry (LCMS)
`studies were carried out using a Finnigan LCQ Deca ion-
`trap mass spectrometer with electrospray ionization (ESI)
`(ThermoFinnigan, San Jose, CA, USA) equipped with a
`BAS (West Lafayette, IN) PM-80 gradient pump and a BAS
`Sample Sentinel autosampler with a 20-Al injection loop
`(Bioanalytical Systems, West Lafayette, IN, USA). For
`neurotransmitter assay, a LC/electrochemistry (LCEC) sys-
`tem equipped with a BAS PM-92e pump and a BAS Epsilon
`dual-channel electrochemical detector with ChromaGraph
`software was used. Serial blood sampling from rats was
`accomplished by the BAS Culex automated blood sampler.
`
`2.3. Brain microdialysis
`
`The Internal Animal Care and Use Committee (IACUC)
`approved all the experimental procedures using animals.
`Guidelines in the Public Health Service Policy on Humane
`Care and Use of Laboratory Animals (NIH Publication,
`revised 1986) were followed.
`Adult Sprague – Dawley rats were anesthetized with ket-
`amine/xylazine (90 mg/kg + 10 mg/kg) by intraperitoneal
`injection. A brain probe guide cannula (MD-2250, BAS)
`was stereotaxically implanted in the cortex or in the stria-
`tum. After at least 3-day postsurgery recovery period, a
`BAS brain microdialysis probe (MD-2204, BAS) was
`inserted into the guide cannula 24 h before sampling was
`to begin. The probe was perfused using a BAS syringe
`pump with Ringer’s solution (147-mM Na + , 2.0-mM Ca2 + ,
`
`, pH 6.0) at a 1 Al/min flow rate.
`4-mm K + , 155-mM Cl
`Two 30-min blank dialysates were collected before dosing
`the animal. After dosing, dialysates were collected at 30-min
`intervals for the first 7 h and then at 1-h intervals for the
`duration of blood sampling. Dialysates were collected into
`300-Al vials with an equal volume of 0.2-M acetic acid to
`prevent degradation of neurotransmitters. Dialysates were
`split in two fractions. One fraction was assayed for OLAN
`and the other was assayed for neurotransmitters to obtain
`pharmacodynamic data.
`
`2.4. Pharmacokinetic studies
`
`The jugular vein of the same animal implanted with the
`brain guide cannula was cannulated and a catheter was
`implanted for blood collection. During the 24-h postsurgery
`recovery period in the Culex ABS, the catheter patency was
`maintained automatically by frequently flushing the catheter
`with sterile saline. After collecting two blank blood samples,
`the animal was dosed orally with 5 mg/kg of OLAN
`dissolved in 0.1-M HCl and then adjusted to pH 6 with
`0.1-N NaOH. Blood samples (100 Al) were collected at 5
`min, 15 min, 30 min, 45 min, 60 min, 90 min, 2 h, 4 h, 6 h,
`8 h, 12 h, and 24 h after dosing. The samples were diluted
`twofold with an equal volume of saline during the collec-
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`tion. Animal rotational behavior was monitored during the
`blood collection and for another 3 – 4 h after the 24-h sample.
`Noncompartmental pharmacokinetic parameters were calcu-
`lated using PK Solutions 2.0 (Summit Research Services,
`Montrose, CO, USA).
`
`2.5. Sample extraction
`
`Blood samples were centrifuged and plasma was sepa-
`rated. After adding 5 Al of a 1-Ag/ml solution of the internal
`standard to a 100-Al plasma sample (50 ng/ml
`in final
`volume), 400 Al of 2% NH4OH in ethyl acetate was added
`to precipitate protein. The sample was then vortexed for 2
`min and centrifuged for 10 min. The supernatant (400 Al)
`was dried under N2 and reconstituted in 100 Al of 0.1-M
`ammonium acetate/ACN (50:50 v/v). Dialysate samples
`were assayed without further sample treatment.
`
`a Ag/AgCl reference electrode. Dialysate samples (5.3 Al)
`were directly injected to the LC system using an autosam-
`pler. The compounds were separated isocratically at room
`
`temperature on a microbore ODS column (150  1.0 mm, 3
`Am) (BAS Unijet) using a mobile phase containing 25-mM
`NaH2PO4, 20-mM citric acid, 60-mM NaCl, 10-mM diethyl-
`amine hydrochloride, 0.5-mM octyl sodium sulfate, and
`0.03-mM Na2EDTA (pH 3.2) mixed with acetonitrile and
`dimethyl acetoamide in the ratio of 91.9:5:3.1 (v/v) at a flow
`rate of 100 Al. Neurotransmitter stock solutions, DA,
`DOPAC, HVA, 5-HIAA, and 5-HT (1 mg/ml each) were
`prepared in 0.2-M acetic acid. Calibration standards were
`prepared by appropriately diluting the stock solutions in 0.2-
`M acetic acid to give DA/DOPAC/HVA/5-HIAA/HT con-
`centrations ranging from 0.5/1.0/0.5/1.0/1.0 to 10.0/20.0/
`10.0/20.0/20.0 ng/ml.
`
`2.6. OLAN and metabolite assay
`
`3. Results
`
`OLAN and NDMO were assayed by LCMS using
`isocratic conditions. The mobile phase consisted of 40%
`0.1-M ammonium acetate/50% ACN and 10% methanol. A
`
`YMC basic column (150  4.6 mm, 5 Am) was used for
`the isocratic separation at a flow rate of 0.8 ml/min at
`ambient temperature. Samples (10 Al) were injected man-
`ually. The MS was operated in positive ESI mode. Nitro-
`gen was used as both sheath and auxiliary gas at a pressure
`of 80 and 20 arbitrary units, respectively. The spray
`voltage was set at 5.0 kV and the capillary temperature
`was at 300 jC. Helium was used as the target gas for
`collision-induced dissociation. Positive ion LC/MS/MS
`chromatogram was obtained by monitoring the daughter
`ions of NDMO (MH + 299.0, 38%) at m/z 230.2, OLAN
`(MH + 313.2, 38%) at m/z 256.4, and internal standard
`(MH + 327.2, 36%) at m/z 270.3.
`Stock solutions of OLAN and internal standard were
`prepared by dissolving 1.0 mg of each compound in
`methanol. Standard NDMO solutions (10 Ag/ml) were used
`as received from Lilly Laboratories. Working solutions were
`made by appropriately diluting stock solutions in ACN/
`NH4AC (50:50 v/v) mixture. Plasma standards for the
`calibration curves were prepared by adding appropriate
`aliquots of diluted OLAN and NDMO standards to rat
`plasma to give appropriate concentrations (1, 5, 10, 25,
`50, and 100 ng/ml). These standard samples were extracted
`after adding the internal standard (equal to 50 ng/ml) by the
`same method as described previously for pharmacokinetic
`samples. The working solutions and calibration standards
`were prepared fresh daily.
`
`2.7. Neurotransmitter assay
`
`Neurotransmitters in the dialysates, mainly DA, DOPAC,
`HVA, 5-HIAA, and 5-HT, were detected by LCEC on glassy
`carbon electrodes at two potentials, at 750 and 650 mV with
`
`3.1. Pharmacokinetics
`
`A marked difference in the animal’s behavior was ob-
`served after the oral administration of OLAN to the rat. The
`animal became catatonic a short time after the drug admin-
`istration and remained inactive for several hours. During
`this time, the animal showed no interest in food or water and
`there was no urine output. The animal regained its normal
`behavior about 8 h after dosing. The sensor movements
`recorded by the system shown in Fig. 2 are in agreement
`with the visual observations. All four data sets show an
`increase in activity shortly after drug administration fol-
`lowed by a inactive period. More information on the animal
`behavior can be obtained by examining the number of
`sensor activations and their duration times (Gunaratna,
`Cregor, & Kissinger, 2000).
`The ABS is designed to collect whole blood. If desired,
`anticlotting agents can be deposited in the sampling vial
`prior to placing whole blood to avoid coagulation. In these
`studies, the blood samples were collected with an equal
`volume of heparinized saline. Plasma concentration – time
`profiles obtained after administrating the animals with a
`single 5-mg/kg oral dose are shown in Fig. 3. The non-
`compartmental mean pharmacokinetic parameters listed in
`Table 1 are comparable with values reported in literature
`(Richelson & Souder, 2000).
`
`3.2. Brain microdialysis
`
`A microdialysis probe is a sampling device that contains
`a specific length of a semipermeable dialysis membrane. An
`implanted probe samples the extracellular fluid around the
`implanted tissue site. Small unbound drug or neurotrans-
`mitter molecules diffuse through the dialysis membrane into
`the perfusing medium, which is isotonic to the extracellular
`fluid. The percentage recovery of the analyte is dependent
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`61
`
`Fig. 2. Animal behavior was monitored after oral administration of 2-mg OLAN for a period of >27 h. Each bar represents sensor data within a 40-min
`segment, starting with the initiation of behavioral monitoring 41 min before dosing. The sun/moon symbols and the light/dark arrows indicate the time of day
`when these activity measurements were made.
`
`on membrane length, perfusing flow rate, and nature of the
`analyte. In these experiments, the probes implanted in the
`rat striatum contained a 4-mm length of membrane. In vitro
`percentage recovery for both OLAN and NDMO through
`the brain probe was found to be < 10%.
`Microdialysis samples collected from the same animal
`during the pharmacokinetic study were assayed for both
`OLAN and the metabolite NDMO and for major neuro-
`
`Fig. 3. Mean plasma OLAN concentration – time profiles (n = 6) of OLAN
`and NDMO after administering an oral dose of 5 mg/kg to Sprague –
`Dawley male rats.
`
`transmitters present in the brain. OLAN and NDMO profiles
`in the brain (Fig. 4) show that OLAN peaks in the brain
`about 2 h after an oral dose. OLAN levels remain elevated
`in the brain long after the plasma levels started to decline.
`It is known that antipsychotic drugs such as OLAN bind
`to dopaminergic receptors and increase the turnover of DA
`in the brain, which increases the amounts of HVA and
`DOPAC, primary metabolites of DA (Aravagiri, Teper, &
`Marde, 1999). A typical chromatogram of a brain dialysate
`is shown in Fig. 5. In this study, the neurotransmitter DA
`was barely detectable in the dialysates, whereas DOPAC
`(Fig. 6) and HVA (Fig. 7) were present in large amounts,
`indicating the increased release of DA in rat brain due to
`OLAN. The levels of DOPAC and HVA increased after
`OLAN administration and started to decline after about 10
`h, long after the OLAN is cleared from plasma. In contrast,
`the levels of 5-HIAA, a metabolite produced by the deam-
`ination of 5-HT by monoamine oxidase, remained un-
`
`Table 1
`Noncompartmental pharmacokinetic parameters (n = 6) for OLAN
`169 F 31 ng/ml
`0.8 F 0.2 h
`316 F 27 ng h/ml
`14.35 F 2.1 l
`
`Cmax
`tmax
`AUCl
`Vd
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`Fig. 4. OLAN and NDMO distribution in brain following an oral dose of 5 mg/kg to Sprague – Dawley male rats (n = 6).
`
`Fig. 5. A chromatogram of a mixture of neurotransmitter standards and a brain dialysate sample at 4-h time point after administering an oral dose of
`5-mg/kg OLAN.
`
`Fig. 6. DOPAC concentration in rat brain dialysates after administration of a
`5-mg/kg oral dose of OLAN (n = 6). DOPAC levels remain elevated for >10
`h, indicating the increased activity of dopaminergic neurons by OLAN.
`
`Fig. 7. Levels of HVA, a metabolite of DA, in rat brain dialysates after
`administration of a 5-mg/kg oral dose of OLAN (n = 6).
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`63
`
`obtained by microdialysis sampling provide near real-time
`information.
`In conclusion, we have successfully demonstrated the
`utility of a new ABS for simultaneous PK/PD and blood –
`brain barrier studies of drugs. Because the animals are freely
`moving and not stressed by human handling or by anesthe-
`sia,
`the PK/PD studies conducted are less subjected to
`experimental artifact and would be more reliable and
`accurate. Furthermore, near real-time information on other
`physiological processes can be obtained from the same
`animal simultaneously without additional manpower. The
`system can be integrated with other studies involving
`animals such as behavior monitoring and electrophysiology.
`Because the ABS can sample four animals at a time, two
`systems can be used to collect blood samples from eight
`animals onto the rows of a 96-well plate to facilitate high
`throughput analysis. The ABS developed here bridges the
`gap between the in vitro screening phase and the analysis of
`in vivo samples to accelerate decision-making before costly
`chronic toxicology and clinical trials.
`
`Acknowledgements
`
`The authors thank Dr. Quin Zhou of Bioanalytical
`Systems for her technical assistance in neurotransmitter
`analysis and Meloney C. Cregor for her assistance in brain
`probe implantation.
`
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`Fig. 8. Levels of 5-HIAA, a metabolite of 5-HT, in rat brain dialysates after
`administration of a 5-mg/kg oral dose of OLAN (n = 6).
`
`changed in brain dialysates (Fig. 8). This is in agreement
`with the observation Scheepers, Gispen-de Wied, Westen-
`berg, and Kahn (2001) made that OLAN significantly
`increased HVA concentrations and the HVA/5-HIAA ratio
`but did not alter 5-HIAA concentrations. The data obtained
`from this microdialysis study show the prolonged effect of
`OLAN in the brain even after the drug is cleared from
`plasma.
`
`4. Discussion
`
`OLAN is a selective monoaminergic antagonist with
`high-affinity binding to serotonergic 5-HT2A/2C, dopaminer-
`gic DA1 – 4, muscarinic Ml – 5, histamine H1, and a1-adren-
`ergic receptors (Bymaster, Hemrick-Luecke, Perry, & Fuller,
`1996; Nyberg, Farde, & Halldin, 1997; Seeman, 2002). It
`has been reported that this drug’s antipsychotic activity is
`mediated through a combination of DA and 5-HT2 antago-
`nism (Kapur et al., 1998; Wadenberg, Soliman, VanderSpek,
`& Kapur, 2001). OLAN’s antagonism of histamine H1
`receptors may explain the somnolence observed in the
`animals with this drug (Meltzer, 1999).
`After the oral dose, OLAN absorbs quickly as evident by
`the short tmax and readily distributes into the tissues. The
`high volume of distribution (Vd) is an indication of the high
`tissue distribution of OLAN.
`Aravagiri et al. (1999) have studied the distribution of
`OLAN in various brain regions. After repeated oral admin-
`istration of OLAN for 15 days, the animals were sacrificed
`and brain tissue samples were collected for OLAN analysis.
`They reported that OLAN levels in brain regions varied
`widely. After a 6-mg/kg/day oral dose of OLAN, the authors
`reported that
`the OLAN concentrations in caudate and
`cerebellum were twofold to threefold lower than the whole
`brain concentrations. In our study, we have implanted the
`probe in the striatum region. By implanting probes in
`different regions of the brain, the distribution of OLAN
`can be studied in awake, freely moving animals. The data
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`8 of 8
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`Alkermes, Ex. 1035