Articles
`
`Low-Dose Pharmacokinetics and Oral Bioavailability of Dichloroacetate in
`Naive and GSTζ-Depleted Rats
`Shakil A. Saghir* and Irvin R. Schultz
`Battelle Pacific Northwest National Laboratory, Richland, Washington, USA
`
`We studied the pharmacokinetics of dichloroacetate (DCA) in naive rats and rats depleted of glu-
`tathione S-transferase-zeta (GSTζ), at doses approaching human daily exposure levels. We also
`compared in vitro metabolism of DCA by rat and human liver cytosol. Jugular vein-cannulated
`male Fischer-344 rats received graded doses of DCA ranging from 0.05 to 20 mg/kg (intra-
`venously or by gavage), and we collected time-course blood samples from the cannulas. GSTζ
`activity was depleted by exposing rats to 0.2 g/L DCA in drinking water for 7 days before initia-
`tion of pharmacokinetic studies. Elimination of DCA by naive rats was so rapid that only 1–20
`mg/kg intravenous and 5 and 20 mg/kg gavage doses provided plasma concentrations above the
`method detection limit of 6 ng/mL. GSTζ depletion slowed DCA elimination from plasma,
`allowing kinetic analysis of doses as low as 0.05 mg/kg. DCA elimination was strongly dose
`dependent in the naive rats, with total body clearance declining with increasing dose. In the
`GSTζ-depleted rats, the pharmacokinetics became linear at doses ≤ 1 mg/kg. Virtually all of the
`dose was eliminated through metabolic clearance; the rate of urinary elimination was < 1
`mL/hr/kg. At higher oral doses (≥ 5 mg/kg in GSTζ-depleted and 20 mg/kg in naive rats), sec-
`ondary peaks in the plasma concentration appeared long after the completion of the initial
`absorption phase. Oral bioavailability of DCA was 0–13% in naive and 14–75% in GSTζ-
`depleted rats. Oral bioavailability of DCA in humans through consumption of drinking water was
`predicted to be very low and < 1%. The use of the GSTζ-depleted rat as a model for assessing the
`kinetics of DCA in humans is supported by the similarity in pharmacokinetic parameter estimates
`and rate of in vitro metabolism of DCA by human and GSTζ-depleted rat liver cytosol. Key
`words: animal study, dichloroacetic acid, drinking water disinfection by-products, halogenated
`acetic acids, human risk assessment, human in vitro metabolism, low-dose pharmacokinetics, oral
`bioavailability, rat in vitro metabolism, toxicology. Environ Health Perspect 110:757–763 (2002).
`[Online 13 June 2002]
`http://ehpnet1.niehs.nih.gov/docs/2002/110p757-763saghir/abstract.html
`
`Dichloroacetate (DCA) is a drinking water
`disinfectant by-product commonly identified
`in municipal water supplies. Concentrations
`of DCA in finished drinking water have been
`reported as high as 133 µg/L (1), although
`concentrations < 25 µg/L are more common
`(2,3). DCA is a metabolite of certain chlori-
`nated industrial solvents and of several phar-
`maceuticals (4). DCA has also been used for
`decades as an investigational drug to treat
`numerous cardiovascular and metabolic dis-
`orders in humans, for example, diabetes,
`hypercholesterolemia, and amelioration of
`lactic acid during liver transplantation (4–6).
`Recently, DCA has been used in clinical tri-
`als to treat congenital or acquired lactic
`acidosis in children (5,7). Human exposure
`to DCA ranges from ~1 to 4 µg/kg/day
`through consumption of drinking water and
`up to 50 mg/kg/day from the use of DCA as
`a therapeutic drug (4).
`DCA is rapidly and completely absorbed
`from the gastrointestinal (GI) tract and is
`extensively metabolized both in rodents and
`in humans, with glyoxylate, oxalate, glyco-
`late, and CO2 being the major metabolites
`(8,9). Only a small percentage (< 3%) of the
`dose is excreted as the parent compound
`(8–10). DCA metabolism occurs primarily in
`
`the liver (11), mediated through a recently
`characterized class of glutathione S-trans-
`ferase, GSTζ (GSTZ1–1) (12). DCA is also
`a mechanism-based inhibitor of GSTζ, and
`prolonged exposure to DCA in rodents
`causes both reduction in metabolism and
`depletion of immunoreactive GSTζ protein
`levels from the liver (9,13–15). Thus, in
`repeated dosings of DCA, the first dose is
`always cleared more rapidly than are subse-
`quent doses because of the inactivation of
`GSTζ. This has prevented measurement of
`oral bioavailability of DCA using a crossover
`experimental design because the second dose
`is always eliminated more slowly regardless of
`the route of administration (16).
`DCA has been associated with a number
`of toxic effects in animals exposed to high
`doses, including testicular abnormalities,
`birth defects, and liver cancer (17–20).
`Consumption of chlorinated water has been
`linked to increased risk for certain cancers in
`humans (21) without any specific correlation
`with DCA or other haloacetates. DCA pre-
`sents an interesting dilemma for risk asses-
`sors because it has a history of safe use as a
`therapeutic, but it has created regulatory
`concern because of its carcinogenicity in ani-
`mals. The U.S. Environmental Protection
`
`Agency (EPA) has classified DCA as a likely
`human carcinogen based on the hepatocar-
`cinogenic effects observed in rodents (22).
`Because of the prevalence of halogenated
`acetic acids in finished drinking water and
`their possible link to human cancer, the U.S.
`EPA has set standards permitting a com-
`bined total of 60 µg/L of five common halo-
`genated acetic acids (HAA5) in drinking
`water. The goal of the U.S. EPA is the vir-
`tual elimination of DCA from drinking
`water under stage I regulations (22).
`Previous pharmacokinetic studies of
`DCA have focused on therapeutic (i.e., mil-
`ligram per kilogram) doses. Also, the effects
`of GSTζ depletion on DCA disposition have
`not been studied in detail. Therefore, we
`designed this study to determine the phar-
`macokinetics and oral bioavailability of
`DCA in cohorts of naive and GSTζ -
`depleted rats using a range of doses down to
`50 µg/kg. We also compared the in vitro
`metabolism of DCA in human liver cytosol
`with cytosol obtained from naive and GSTζ-
`depleted rats. We made this comparison to
`aid in determining the appropriateness of
`using the GSTζ-depleted rat model for
`understanding low-dose pharmacokinetics of
`haloacetates in humans.
`Materials and Methods
`Chemicals. We purchased DCA (> 99%
`pure as free acid) from Fluka Chemical
`Corp. (Milwaukee, WI). Reagent-grade
`methyl-tert-butyl ether (MTBE) was pur-
`chased from Fisher Scientific (Pittsburgh,
`PA). We prepared diazomethane from
`
`Address correspondence to I.R. Schultz, Battelle
`MSL, 1529 West Sequim Bay Road, Sequim, WA
`98382 USA. Telephone: (360) 681-4566. Fax:
`(360) 681-3681. E-mail: ir_schultz@pnl.gov
`*Current address: Dow Chemical Company,
`Toxicology and Environmental Research and
`Consulting, Midland, MI, USA.
`We thank G. Muñiz, Y. Rivera, E. Robershotte,
`and N. Flintoff for their help in various parts of the
`study.
`Research described in this article has been funded
`wholly or in part by the U.S. Environmental
`Protection Agency through STAR grant R82594.
`The article has not been subjected to the U.S. EPA’s
`required peer and policy review and therefore does
`not necessarily reflect the views of the U.S. EPA,
`and no official endorsement should be inferred.
`This work was presented in part at the 40th
`annual meeting of the Society of Toxicology, San
`Francisco, CA, 25–29 March, 2001.
`Received 17 October 2001; accepted 22 January
`2002.
`
`Environmental Health Perspectives • VOLUME 110 | NUMBER 8 | August 2002
`
`757
`
`ALKERMES EXHIBIT 2032
`Amneal Pharmaceuticals LLC v. Alkermes Pharma Ireland Limited
`IPR2018-00943
`
`Page 1 of 7
`
`

`

`Articles • Saghir and Schultz
`
`N-methyl-N-nitrosoguanidine following
`Aldrich Technical Information Bulletin AL
`121 (23). All other chemicals were of the
`purest grade available and were obtained
`from standard sources. All dosing solutions
`were prepared in 0.9% (w/v) NaCl and pH
`adjusted to 7.0 with NaOH.
`Animals and treatment. The Institutional
`Animal Care and Use Committee of Battelle,
`Pacific Northwest National Laboratory
`approved the animal care and experimental
`protocols, and animal care and treatment was
`conducted in accordance with their estab-
`lished guidelines.
`For pharmacokinetic experiments, we
`purchased 8- to 10-week-old male Fischer
`344 rats (185 ± 29 g body weight, mean ±
`SD; n = 41 naive rats) fitted with a jugular
`vein cannula from Taconic Laboratories
`(Germantown, NY). We also purchased 6
`noncannulated rats from Charles River
`Laboratories (Raleigh, NC) for the prepara-
`tion of liver cytosol. We housed jugular
`vein–cannulated rats individually, whereas we
`housed three noncannulated rats per cage.
`Each cage contained wood-chip bedding and
`stainless steel wire tops, and rats were housed
`under standard conditions (22°C, 40–60%
`relative humidity, 12-hr light/dark cycle). We
`allowed rats a minimum of 48 hr for recovery
`from transport before use in experiments.
`Initially, rats were provided with deion-
`ized water and Purina rat chow (St Louis,
`MO) ad libitum. We used deionized water
`throughout the experiments to avoid
`unwanted exposure to haloacetates, which
`can be present in drinking water sources and
`may cause some inactivation of GSTζ .
`Animals were fasted overnight before the
`administration of DCA. We dosed naive ani-
`mals intravenously (iv) or by gavage
`(4–6/dose group) with 1, 5, or 20 mg/kg
`DCA and housed them in polycarbonate
`metabolism cages. After the initial dosing
`experiments, we then provided the same
`individual rats with 0.2 g/L DCA in their
`drinking water for 7 days to deplete/inacti-
`vate GSTζ activity (henceforth GSTζ
`depleted). We also pretreated one group of
`three noncannulated rats for 7 days with 0.2
`g/L DCA in their drinking water to deplete
`GSTζ. We then switched the GSTζ-depleted
`animals to non-DCA–fortified water
`overnight (16 hr) to allow residual DCA to
`be cleared from the body. This treatment
`protocol was previously shown to reduce
`GSTζ activity by > 90% in rat liver cytosol
`(24), and the experimental results presented
`in this study further confirm this finding. We
`then dosed GSTζ-depleted rats (4–6 per dose
`group) iv with 0.05, 0.25, 1, 5, or 20 mg/kg
`or gavaged them with 0.25, 1, 5, or 20
`mg/kg DCA. We gavaged two additional
`GSTζ-depleted rats with 100 mg/kg DCA to
`
`estimate oral bioavailability at this highest
`dose using earlier reported iv data of
`Gonzalez-Leon et al. (9). We administered
`dosing solutions at a volume of 1 mL/kg.
`Sample collection and analysis. We col-
`lected serial blood samples (0.075–0.125 mL)
`from individual rats through the jugular vein
`cannula using a 1-mL syringe coated with
`sodium heparin. After each blood sample, we
`flushed the cannula with ~0.2 mL of
`heparinized saline (40 U/mL heparin). We
`obtained plasma by centrifugation, mixed it
`with 0.2 mL of ice-cold 0.1 M sodium acetate
`buffer (pH 5.2), and stored the plasma at
`–20°C until analysis. We determined actual
`plasma volumes gravimetrically using tared
`vials and assuming plasma density of 1.0. A
`typical blood sampling schedule after iv dos-
`ing was 0, 3, 6, 10, 15, 20, 25, and 30 min
`and variously thereafter up to 24 hr, depend-
`ing on the dose and pretreatment. For orally
`dosed animals, we added an additional 1-min
`sample. Sampling continued until plasma
`concentrations were expected to have declined
`below the method detection limit (MDL) for
`DCA. We calculated the MDL as described
`by Glaser et al. (25) using nine replicate
`plasma samples. The MDL for DCA in naive
`and GSTζ-depleted rat plasma was 6 and 10
`ng/mL, respectively. The MDL for plasma
`removed from GSTζ rats was slightly higher
`because of elevated background levels. We
`collected urine from each rat for 24 hr, mixed
`an aliquot with sodium acetate buffer, and
`stored the samples at –20°C until analysis.
`For experiments measuring diurnal
`changes in plasma DCA levels, we provided
`four rats with 0.2 g/L DCA water and col-
`lected time-course blood samples up to 24
`hr, after which we gave animals non-DCA
`water and collected the last blood samples 11
`hr later. We allowed animals used in the
`kinetic experiments an additional 5 hr on
`non-DCA water (16 hr total depuration
`before administering DCA). This additional
`time was to ensure that residual DCA con-
`centration in plasma approached back-
`ground values without significant resynthesis
`of the GSTζ enzyme (26). We verified resid-
`ual concentration after depuration by mea-
`suring DCA levels in plasma collected before
`dosing (time 0) and comparing them with
`levels in non-DCA treated rats.
`We analyzed all plasma samples antici-
`pated to contain > 100 ng DCA using a pre-
`viously described method (27). Briefly, we
`added 0.025 mL (0.2 µg) internal standard
`(dibromoacetic acid) to samples (plasma and
`urine), acidified them by adding 0.025 mL
`50% sulfuric acid (v/v), and extracted in vari-
`ous volumes (0.2–1.0 mL) of MTBE
`depending on the dose and sampling time.
`We extracted samples anticipated to contain
`< 100 ng DCA in 0.2 mL MTBE. We then
`
`concentrated the extracted DCA by reducing
`the volume of MTBE to 0.01–0.02 mL
`under a gentle stream of N2. We converted
`the free acid to the methyl ester by adding
`0.01–0.02 mL ethereal diazomethane (previ-
`ously diluted 1:10 with MTBE). We then
`analyzed samples by gas chromatography
`with electron-capture detection (Hewlett-
`Packard 5890-Series II, Avondale, PA). The
`additional preconcentration step increased
`the MDL for DCA by 50- to 75-fold when
`compared to our previous method. We deter-
`mined stability of DCA in urine by fortifying
`freshly collected urine from a naive rat with
`DCA (10 µg); the fortified samples were
`either stored at –20°C or left at room tem-
`perature for 24 hr. We then analyzed DCA
`as described above and compared the results.
`Degradation of DCA in urine was negligible;
`> 90% could be recovered from urinary sam-
`ples left at room temperature for 24 hr.
`Kinetic analysis. The methods we used
`to analyze the concentration–time profiles of
`DCA were similar to those used by Schultz
`et al. (27). Briefly, we analyzed the individ-
`ual plasma profiles after both iv and oral
`administration by noncompartmental meth-
`ods to obtain estimates of total body clear-
`ance, apparent volume of distribution at
`steady state (Clb, Vss, for iv doses only), and
`the mean residence time (MRT) using the
`standard equations for these parameters that
`are incorporated into the WinNonlin
`program (Pharsight Corp., Cary, NC).
`WinNonlin calculates the area under the
`curve (AUC0→∞) by the linear trapezoidal
`method with the terminal portion of the
`curve extrapolated from time 0 to infinity by
`Cp,t/β, where Cp,t is the concentration of
`DCA in plasma at the last observation and β
`is the slope of the terminal phase determined
`by linear regression. WinNonlin calculated
`the elimination half-life (t1/2,β) as 0.693/β.
`We calculated renal clearance as Clr =
`Χu0→24/AUC0→24, where Χu0→24 is the total
`amount of DCA recovered in the urine after
`24 hr. We also report the observed peak
`plasma concentration of DCA (Cmax) and the
`time of its occurrence (Tmax) after oral dos-
`ing. We calculated the oral bioavailability
`from the ratios of the average values for
`AUC0→∞ for the oral and iv doses, and calcu-
`lated the mean absorption time (MAT) as the
`difference between the MRToral and MRTiv.
`Preparation of liver cytosol. We prepared
`rat liver cytosol from male F-344 rats (8–10
`weeks old; n = 3 naive and 3 GSTζ depleted)
`by differential centrifugation as described by
`Okita and Okita (28). We purchased two
`human liver sections and a pooled S-9 frac-
`tion obtained from 10 donors from the
`International Institute for the Advancement
`of Medicine (Exton, PA). The human liver
`section designated Human 1 was from a
`
`758
`
`VOLUME 110 | NUMBER 8 | August 2002 • Environmental Health Perspectives
`
`Page 2 of 7
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`

`

`Articles • Kinetics of DCA in naive and GSTζ-depleted rats
`
`blood flow (18%) to the liver (33). For
`humans, we assumed total cardiac output to
`be 312 L/hr and liver blood flow to be 22%
`of this value [from Astrand (34) and
`Williams and Leggett (35), respectively].
`The calculated Qh for a 70 kg human was
`1.01 L/hr/kg. The unbound fraction for
`DCA in rat plasma was 0.94 ± 0.07 (27) and
`we assumed it to be the same for humans.
`Statistics. We assessed significant differ-
`ences between pharmacokinetic parameter
`estimates from the different treatment groups
`by Student’s t-test. We also performed analy-
`sis of variance on the individual Clb values to
`determine if they were significantly different.
`We considered a p-value of ≤ 0.05 to be sta-
`tistically significant.
`Results
`Plasma DCA levels while receiving DCA
`treatment. To study the pharmacokinetics of
`DCA in rats with reduced metabolism, we
`exposed animals to DCA (0.2 g/L) in drink-
`ing water for 7 days to effectively deplete the
`GSTζ enzyme. Periodically, we monitored
`consumption of water and plasma DCA lev-
`els to verify concentrations during the expo-
`sure and residual levels after 16 hr of
`washout. The average consumption of water
`by rats was 80 mL/kg/day, corresponding to
`a daily DCA dose of around 16 mg/kg.
`Figure 1 shows the diurnal plasma levels of
`DCA in rats provided with 0.2 g/L DCA-
`fortified water. DCA plasma levels were
`much higher during the dark cycle. We
`
`observed peak plasma levels at 500 hr (1 hr
`before lights on), which declined thereafter.
`The levels of DCA in plasma started to
`climb again 2 hr before the lights were
`turned off, corresponding to the increased
`activity (drinking/eating). We found the
`minimum plasma concentration of DCA at
`1600 hr. During an 11-hr depuration period
`after removal of DCA-fortified drinking
`water, plasma levels of DCA rapidly dropped
`to 10 ± 2 ng/mL (Figure 1). We provided
`animals used in kinetic experiments an addi-
`tional 5 hr of washout (16 hr total depura-
`tion) that allowed DCA plasma levels to fall
`below 10 ng/mL.
`Intravenous administration. Figure 2
`shows the mean (± SE) plasma concentra-
`tion–time profiles. The decline of DCA from
`plasma of naive rats was so rapid that the low-
`est dose that could be used for kinetic analysis
`was 1 mg/kg, which had a plasma elimination
`half-life of approximately 4 min (Figure 2A,
`Table 1). In contrast, elimination of DCA
`from the plasma of GSTζ-depleted rats was
`much slower, allowing kinetic analysis of doses
`as low as 0.05 mg/kg (Figure 2B inset). Visual
`inspection of the plasma concentration–time
`profiles and the pharmacokinetic parameters
`presented in Table 1 for the naive rats indicate
`that DCA declined from plasma in a monoex-
`ponential manner. Decline of DCA from
`plasma of the GSTζ-depleted rats became
`biexponential at the higher doses (Figure 2B).
`Table 1 summarizes the kinetic analysis
`of DCA for the naive and GSTζ-depleted
`
`Light /dark cycle
`
`DCA removed
`
`20
`
`10
`
`1
`
`0.1
`
`Concentration in plasma (µg/mL)
`
`0.01
`
`DCA in water
`
`2400
`
`400
`
`800
`
`1200
`
`1600
`Clock time
`
`2000
`
`2400
`
`400
`
`800
`
`Figure 1. Diurnal plasma concentration profile of DCA in rats administered 0.2 g/L DCA in drinking water
`and its elimination upon removal of the DCA-fortified water. Each point indicates mean ± SE (n = 4).
`
`69-year-old white male (body weight 90 kg;
`height, 1.78 m) who died of cardiopul-
`monary arrest; the Human 2 liver section was
`from a 68-year-old white female (body
`weight 73 kg; height 1.63 m) who died of a
`brain stem infarction. The pooled S9 desig-
`nated Human Pooled was prepared from
`liver tissue obtained from 10 white male
`donors of 8, 40, 40, 48, 51, 52, 53, 58, 63,
`and 64 years of age, who died of cardiovascu-
`lar disease, brain hemorrhage, anoxia, anoxia,
`head injury, anoxia, stroke, head injury,
`anoxia, and head injury, respectively. We
`prepared cytosol from the liver sections as
`described for rats by Okita and Okita (28)
`and from pooled human S-9 by centrifuga-
`tion at 100,000 × g for 1 hr. Liver sections
`had been perfused with University of
`Wisconsin medium and contained viable
`hepatocytes. We stored aliquots of cytosol at
`–70°C until use, and determined protein
`concentrations as described by Bradford (29).
`DCA depletion in cytosol and determi-
`nation of intrinsic metabolic clearance. We
`measured the depletion of added DCA using
`rat and human hepatic cytosol. Incubation
`mixtures consisted of 1–4 mg/mL protein,
`0.1 M phosphate buffer (pH 7.4), and 1.4
`mM glutathione in a final incubation vol-
`ume of 3 mL. We preincubated solutions for
`2 min at 37°C in a shaking water bath and
`started the reaction by adding 0.025 mg
`DCA prepared in 0.1 M phosphate buffer
`(pH 7.4). We removed a 0.15-mL aliquot
`from each incubate at various times (0.2–60
`min) and added it to a mixture of 0.25 mL
`0.1 M sodium acetate and 0.05 mL of 50%
`H2SO4 to stop the reactions. We added
`internal standard to each aliquot and
`extracted and analyzed DCA as described
`above. We plotted the loss of DCA against
`time and calculated AUC as described above.
`We calculated the intrinsic metabolic clear-
`ance (Clint) by dividing the initial amounts
`of DCA (at time 0.2 min) in the incubation
`medium with that of the AUC values (30).
`We scaled up the Clint to a whole animal/
`human by calculating the amount of cytoso-
`lic protein per gram of liver for rat and
`humans. For rats, we used measured liver and
`body weights. For humans, we assumed liver
`to be 2.5% of the body weight as reported by
`Davies and Morris (31). We calculated the
`hepatic clearance as
`
`×
`×
`
`Cl
`Cl
`
`,
`
`)int
`
`[1]
`
`=
`
`h
`
`×
`(
`+
`
`Q
`Q
`
`u
`
`f
`f
`
`Cl
`h
`
`h
`u
`int
`where Qh is the liver blood flow and fu is the
`unbound fraction of DCA in plasma. We
`assumed the total cardiac output for F-344
`rats was 17.38 L/hr/kg [from Hachamovitch
`et al. (32)]. We calculated the Qh to be 3.13
`L/hr/kg by adjusting for the percentage
`
`Environmental Health Perspectives • VOLUME 110 | NUMBER 8 | August 2002
`
`759
`
`Page 3 of 7
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`

`

`disproportionate increase in the AUC0→∞
`between the doses (Table 2). Maximum
`plasma concentrations were reached within
`5–10 min in naive and 8–45 min in GSTζ-
`depleted rats (Table 2, Figure 3). In the
`GSTζ-depleted rats, peak plasma concentra-
`tions (Cmax) were 4- to 6-fold higher than in
`the naive rats. The higher Cmax and longer
`MRT in GSTζ -depleted rats was also
`reflected by a 22- to 56-fold increase in the
`AUC0→∞ (Table 2). The MAT in both
`naive and GSTζ-depleted rats was increased
`in a dose dependent manner.
`The oral bioavailability of DCA was sig-
`nificantly reduced in naive rats. At doses of 5
`and 20 mg/kg, bioavailability was only 10%
`and 13%, respectively. At a higher dose of
`100 mg/kg, the oral bioavailability reached
`81% (Table 2). Bioavailability at 1 mg/kg
`could not be calculated because of the lack
`
`of detectable concentrations of DCA in
`plasma. In GSTζ-depleted rats, the oral
`bioavailability was 14%, 29%, 31%, and
`75% at the 0.25, 1, 5, and 20 mg/kg doses,
`respectively, and became 100% at 100
`mg/kg (Table 2).
`Correlation between dose and kinetic
`parameters. Figure 4 presents the correlation
`between dose and percent oral bioavailabil-
`ity. The relationship between dose and oral
`bioavailability of DCA for the GSTζ-
`depleted rats was best defined by saturable
`mechanism using a hyperbolic distribution
`with a correlation coefficient (r 2) of 0.90
`(Figure 4). The relationship between dose
`and oral bioavailability for the naive rats was
`less clear, and Figure 4 shows only the
`observed data.
`Cytosolic metabolism of DCA. The results
`of in vitro experiments using liver cytosol
`
`A
`
`100
`
`B
`
`1.0
`
`0.1
`
`0.01
`
`0.0
`
`0.5
`Time (hr)
`
`1.0
`
`10
`
`1
`
`0.1
`
`Plasma concentration (µg/mL)
`
`0.01
`
`0.005
`
`20 mg/kg
`5 mg/kg
`1 mg/kg
`0.25 mg/kg
`0.05 mg/kg
`
`100
`
`10
`
`1
`
`0.1
`
`Plasma concentration (µg/mL)
`
`0.01
`
`0.005
`
`0.0
`
`0.5
`
`1.5
`
`2.0
`
`0
`
`2
`
`4
`
`8
`
`10
`
`12
`
`6
`1.0
`Time (hr)
`Time (hr)
`Figure 2. The plasma concentration–time profiles of DCA after iv administration of a series of doses of
`1–20 mg/kg to naive (A) and 0.05–20 mg/kg to GSTζ-depleted rats (B). Data shown are mean ± SE (n =
`4–6). Error bars that fit within the data point are not shown. To avoid crowding of data points on the y-
`axis, the line was slightly shifted to the left.
`
`Table 1. Pharmacokinetic parameters of DCA after iv administration of a range of doses in naive and
`GSTζ-depleted adult male F-344 rats.
`Dose
`AUC0→∞
`(mg/kg)
`(µg/mL/hr)
`Naive rats
`1
`5
`20
`100b,c
`GSTζ-depleted rats
`0.05
`0.25
`1
`5
`20
`100c
`NR, not reported.
`aClrenal was < 0.7 mL/hr with the exception of 100 mg/kg (2.9 ± 0.5 mL/hr for naive and 8.9 ± 3.3 mL/hr for GSTζ-depleted)
`doses. bData from Schultz et al. (27). cData from Gonzalez-Leon et al. (9). dNot significantly different from each other: p ≥ 0.4.
`
`Vss
`(mL/kg)
`
`Clb
`(mL/hr/kg)a
`
`MRT
`(hr)
`
`t 1/2
`(hr)
`
`0.07 ± 0.01
`0.08 ± 0.01
`0.14 ± 0.01
`2.10 ± 0.86
`
`0.23 ± 0.05
`0.18 ± 0.03
`0.19 ± 0.04
`0.64 ± 0.04
`3.45 ± 0.09
`NR
`
`0.07 ± 0.001
`0.08 ± 0.003
`0.15 ± 0.01
`2.4 ± 0.15
`
`0.19 ± 0.05
`0.17 ± 0.02
`0.20 ± 0.05
`0.50 ± 0.03
`1.81 ± 0.09
`10.8 ± 2.0
`
`No.
`
`5
`6
`5
`5
`
`4
`6
`5
`4
`4
`6
`
`0.15 ± 0.01
`1.24 ± 0.05
`13.8 ± 0.85
`433 ± 233
`
`0.04 ± 0.01
`0.15 ± 0.03
`0.61 ± 0.02
`8.21 ± 0.50
`136.6 ± 3.4
`2,410 ± 406
`
`508 ± 68.6
`415 ± 47.2
`223 ± 111.0
`618 ± 319.0
`
`277 ± 33.4
`454 ± 57.3
`261 ± 13.6
`392 ± 31.4
`513 ± 18.5
`582 ± 146
`
`6,554 ± 356
`5,265 ± 636
`1,571 ± 97
`267 ± 105
`
`1,326 ± 342d
`1,816 ± 288d
`1,640 ± 57d
`614 ± 39
`168 ± 22
`43 ± 8
`
`Articles • Saghir and Schultz
`
`rats. In general, the kinetics of DCA were sim-
`ilar to previous descriptions (9,27): rapid elim-
`ination by the naive rats with GSTζ depletion
`causing an increase in t1/2,β and MRT and a
`decrease in the total body clearance (Clb).
`DCA was essentially eliminated through
`metabolism by both naive and GSTζ-depleted
`rats. The renal clearance of DCA accounted
`for < 1% of the total body clearance at most
`doses (Table 1). The steady-state volume of
`distribution (Vss) did not appear to be affected
`by GSTζ depletion or with dose, ranging non-
`systematically between 223 and 618 mL/kg
`(Table 1). The main pharmacokinetic parame-
`ter that was affected by treatment and dose
`was Clb. In naive rats, we observed nonlinear
`kinetics throughout the dosing regimen (Table
`1). In GSTζ-depleted rats, however, the phar-
`macokinetics became linear at doses < 1
`mg/kg; Clb was not different (p ≥ 0.4) at these
`lower doses (Table 1).
`Oral administration. The average
`plasma concentration–time profiles of DCA
`after gavage dosing are presented in Figure 3
`and a summary of the pharmacokinetic para-
`meters is presented in Table 2. DCA was
`rapidly absorbed after oral dosing and
`detected in plasma within 1 min after dosing
`(Figure 3). In naive rats, the greater capacity
`for metabolism limited the doses that could
`be used. Pilot experiments using a dose of 1
`mg/kg failed to detect plasma concentrations
`above the MDL (6 ng/mL) because DCA
`was apparently completely metabolized
`before reaching the general circulation
`(Table 2). The decline in the plasma concen-
`tration of DCA after the initial peak
`appeared to be monoexponential at lower
`doses (5 mg/kg in the naive and ≤ 1 mg/kg
`in the GSTζ-depleted rats; Figure 3). At
`higher doses (20 mg/kg in naive rats and ≥ 5
`mg/kg in GSTζ-depleted rats), DCA dis-
`played complex plasma concentration–time
`profiles, with secondary plasma peaks
`appearing between 4–8 hr after dosing, long
`after completion of the initial absorption
`phase (Figure 3). This observation is consis-
`tent with a previous report of the absorption
`of DCA in naive rats gavaged with a 100
`mg/kg dose (27). In GSTζ-depleted rats, the
`secondary plasma peak was less apparent at
`the highest gavaged dose of 20 mg/kg
`(Figure 3B). This observation implies that a
`complex dose–response relationship exists in
`GSTζ-depleted rats between the oral dose
`and appearance of the secondary plasma
`peaks, with both very low and high doses
`displaying a less pronounced secondary peak.
`This relationship may also apply to naive
`rats, although the high dose needed to
`obscure the secondary peak is apparently
`> 100 mg/kg.
`The complex plasma concentration–time
`profile at the higher doses contributed to the
`
`760
`
`VOLUME 110 | NUMBER 8 | August 2002 • Environmental Health Perspectives
`
`Page 4 of 7
`
`

`

`Articles • Kinetics of DCA in naive and GSTζ-depleted rats
`
`were consistent with in vivo kinetic analysis;
`that is, GSTζ depletion significantly
`decreases DCA metabolism/elimination. The
`rate of DCA metabolism by naive rat cytosol
`was significantly faster (p < 0.01) than that in
`the GSTζ-depleted rats (Figure 5). The
`intrinsic metabolic clearance (Clint) of DCA
`by the human liver cytosol closely resembled
`that in GSTζ-depleted rats (Figure 5) and
`was not statistically different (p > 0.3). Table
`3 presents the predicted hepatic clearance
`(Clh) and extraction efficiency (Ess) of DCA
`by rats and humans. We derived these pre-
`dicted values using in vitro Clint of DCA by
`the rat and human liver cytosol. The Clh and
`Ess of DCA by naive rats was 3-fold higher
`than that by the GSTζ-depleted rats. We
`predict humans to have somewhat lower Clh
`and similar E ss of DCA compared with
`GSTζ-depleted rats (Table 3).
`
`Discussion
`The results of this study demonstrate that
`elimination of DCA in naive rats exhibits
`nonlinear behavior at all doses that allowed
`pharmacokinetic analysis. The Clb continued
`to increase at lower iv doses and exceeded 6.5
`L/hr/kg at the 1 mg/kg dose. The cardiac out-
`put in F-344 rats of body size comparable to
`those used in this study has been reported to
`be 17.38 L/hr/kg (32). Therefore, the clear-
`ance of DCA in naive rats at low doses is at
`least 38% of cardiac output, which would
`exceed liver blood flow and implies extensive
`extrahepatic elimination of DCA occurs. In
`contrast, the pharmacokinetics of DCA in
`GSTζ-depleted rats becomes linear at doses
`≤ 1 mg/kg (Table 1); the Clb ranged between
`1.33 and 1.82 L/hr/kg after iv doses of
`0.05–1 mg/kg. Assuming liver blood flow is
`3.13L/hr/kg (i.e., fraction of cardiac output to
`
`B
`
`0.1
`
`0.01
`0.0
`
`0.5
`Time (hr)
`
`1.0
`
`30
`
`10
`
`1
`
`0.1
`
`Plasma concentration (µg/mL)
`
`0.01
`
`0.005
`
`A
`
`20 mg/kg
`5 mg/kg
`1 mg/kg
`0.25 mg/kg
`
`30
`
`10
`
`1
`
`0.1
`
`Plasma concentration (µg/mL)
`
`0.01
`
`0.005
`
`0
`
`2
`
`6
`
`8
`
`0
`
`4
`
`8
`
`16
`
`20
`
`24
`
`12
`4
`Time (hr)
`Time (hr)
`Figure 3. The plasma concentration–time profiles of DCA after oral administration of 5 and 20 mg/kg to
`naive rats (A) and a series of doses ranging from 0.25 to 20 mg/kg to GSTζ-depleted rats (B). Data shown
`are mean ± SE (n = 4–6). Error bars that fit within the data point are not shown. To avoid crowding of data
`points on the y-axis, the line was slightly shifted to the left.
`
`the liver is 0.18) (33) and liver metabolism
`accounts for the bulk of DCA elimination (in
`GSTζ-depleted rats), DCA clearance appears
`to correspond to 42–58% of liver blood flow.
`This finding indicates that DCA is moder-
`ately extracted by liver under linear kinetics
`by GSTζ-depleted rats. At higher doses,
`metabolism becomes saturated, and liver
`extraction decreases.
`The complex plasma concentration–time
`profiles of DCA observed after some oral
`doses (Figure 3) agreed with those in an ear-
`lier report (27). The extent of the secondary
`peaks appears to be reduced at lower doses
`and was absent after doses that only pro-
`duced detectable levels of DCA until 4 hr
`after dosing (5 and 20 mg/kg in this study).
`
`Naive
`GST ζ-depleted
`
`100
`
`90
`
`80
`
`70
`
`60
`
`50
`
`40
`
`30
`
`20
`
`10
`
`0
`
`Oral bioavailability (%)
`
`75
`
`100
`
`0
`
`25
`
`50
`Dose (mg/kg)
`Figure 4. Correlation of dose and oral bioavailability
`of DCA for the naive and GSTζ-depleted rats.
`Y = 106.8x ÷ (8.32 + x). r2 = 0.90. To avoid crowding
`of data points on the y-axis, the line was slightly
`shifted to the left.
`
`Human 1 Human 2 Human
`pooled
`
`*
`
`3.5
`
`3.0
`
`2.5
`
`2.0
`
`1.5
`
`1.0
`
`0.5
`
`Intrinsic metabolic clearance
`
`(mL/hr/mg protein)
`
`0.0
`
`Naive rat
`
`GST ζ-
`depleted
`rat
`Figure 5. Intrinsic metabolic clearance of DCA
`calculated from the progress metabolism experi-
`ment using liver cytosol from naive and GSTζ-
`depleted rats (n = 3). Human hepatic cytosol was
`from two donors or pooled from 10 individuals. For
`rats, each bar represents the mean ± SE (n = 3);
`for humans, each bar represents the mean ± SE of
`three experiments. Error bars that fit within the
`data point are not shown.
`*p < 0.01.
`
`Table 2. Pharmacokinetic parameters of DCA after oral administration of a range of doses in naive and
`GSTζ-depleted adult male F-344 rats.a
`Dose
`AUC0→∞
`(mg/kg)
`(µg/mL/hr)
`Naive rats
`1
`5
`20
`50b
`100c
`GSTζ-depleted rats
`0.25
`1
`5
`20
`100
`
`Tmax
`(hr)
`
`Cmax
`(µg)
`
`MRT
`(hr)
`
`MAT Bioavailability
`(hr)
`(%)
`
`BD
`0.12 ± 0.01
`1.82 ± 0.10
`11.7 ± 1.68
`218 ± 74.5
`
`0.02 ± 0.01
`0.18 ± 0.03
`2.58 ± 1.05
`103 ± 14.0
`2,730
`
`BD
`0.09 ± 0.02
`0.17 ± 0.01
`0.27 ± 0.04
`8.0
`
`0.13 ± 0.01
`0.12 ± 0.02
`0.25 ± 0.03
`0.75 ± 0.25
`3.32
`
`BD
`0.36 ± 0.07
`2.91 ± 0.24
`9.29 ± 1.87
`27.4
`
`0.08 ± 0.01
`0.26 ± 0.01
`1.66 ± 0.31
`17.0 ± 1.90
`151
`
`0
`0.28 ± 0.04
`1.70 ± 0.49
`NR
`6.70 ± 1.44
`
`0.39 ± 0.03
`1.05 ± 0.07
`1.80 ± 0.19
`4.63 ± 0.69
`12.5
`
`0
`0.20
`1.56
`NR
`4.5
`
`0.19
`0.86
`1.16
`1.18
`ND
`
`0
`9.68
`13.20
`ND
`80.93
`
`14.0
`29.4
`31.4
`75.0
`100
`
`No.
`
`3
`6
`6
`4
`5
`
`6
`4
`4
`4
`2
`
`Abbreviations: BD, below detection (< MDL); MAT, mean, absorption, time; ND, not determined; NR