The rapid hydrolysis of chlordiazepoxide to demoxepam may affect the outcome of chronic osmotic minipump studies

Article abstract of DOI:10.1007/s00213-009-1752-8

Background: In chronic studies, the classical benzodiazepine chlordiazepoxide (CDP) is often the preferred drug because, unlike other benzodiazepines, it is soluble in water. However, rapid CDP hydrolysis in solution has been described. This would diminish plasma levels in chronic minipump studies and introduce the corelease of active compounds. Methods: Therefore, the present study aimed to explore the putative hydrolysis of CDP in aqueous solution over time and to identify the hydrolysis products. Moreover, we aimed to characterize the hydrolysis products for their in vitro ( ³H-flunitrazepam binding and oocyte electrophysiology) and in vivo (stress-induced hyperthermia paradigm) GABA_A receptor potency. Results: CDP in solution hydrolyzed to the ketone structure demoxepam which was confirmed using mass spectrometry. The hydrolysis was concentration dependent (first-order kinetics) and temperature dependent. CDP exerted greater potency compared to demoxepam in vitro (increased activity at GABA_A receptors containing α₁ subunits) and in vivo (stress-induced hyperthermia), although ³H-flunitrazepam binding was comparable. Conclusions: The classical benzodiazepine CDP is rapidly hydrolyzed in solution to the active compound demoxepam which possesses a reduced activity at the GABA_A receptor. Chronic studies that use CDP in aqueous solution should thus be interpreted with caution. It is therefore important to consider drug stability in chronic minipump applications.

Full text of DOI:10.1007/s00213-009-1752-8

Psychopharmacology (2010) 208:555–562
DOI 10.1007/s00213-009-1752-8
ORIGINAL INVESTIGATION
The rapid hydrolysis of chlordiazepoxide to demoxepam
may affect the outcome of chronic osmotic minipump studies
Christiaan H. Vinkers & Gerdien A. H. Korte-Bouws & Javier Sastre Toraño &
Naheed R. Mirza & Elsebet Ø. Nielsen & Philip K. Ahring & Gerhardus J. de Jong &
Berend Olivier
Received: 5 September 2009 /Accepted: 2 December 2009 /Published online: 13 January 2010
#
The Author(s) 2010. This article is published with open access at Springerlink.com
Results CDP in solution hydrolyzed to the ketone structure
demoxepam which was confirmed using mass spectrometry.
The hydrolysis was concentration dependent (first-order
kinetics) and temperature dependent. CDP exerted greater
potency compared to demoxepam in vitro (increased activity
at GABAA receptors containing α1 subunits) and in vivo
(stress-induced hyperthermia), although 3H-flunitrazepam
binding was comparable.
Conclusions The classical benzodiazepine CDP is rapidly
hydrolyzed in solution to the active compound demoxepam
which possesses a reduced activity at the GABAA receptor.
Chronic studies that use CDP in aqueous solution should
thus be interpreted with caution. It is therefore important to
consider drug stability in chronic minipump applications.
Abstract
Background In chronic studies, the classical benzodiazepine
chlordiazepoxide (CDP) is often the preferred drug because,
unlike other benzodiazepines, it is soluble in water. However,
rapid CDP hydrolysis in solution has been described. This
would diminish plasma levels in chronic minipump studies
and introduce the corelease of active compounds.
Methods Therefore, the present study aimed to explore the
putative hydrolysis of CDP in aqueous solution over time
and to identify the hydrolysis products. Moreover, we
aimed to characterize the hydrolysis products for their in
vitro (3H-flunitrazepam binding and oocyte electrophysiol-
ogy) and in vivo (stress-induced hyperthermia paradigm)
GABAA receptor potency.
:
:
.
.
.
C. H. Vinkers (*) G. A. H. Korte-Bouws B. Olivier
Department of Psychopharmacology,
Utrecht Institute for Pharmaceutical Sciences (UIPS),
Faculty of Science, Utrecht University,
Sorbonnelaan 16,
Keywords Benzodiazepine Chronic Degradation
GABAA receptor
Abbreviations
3584 CA Utrecht, The Netherlands
e-mail: c.h.vinkers@uu.nl
CDP Chlordiazepoxide
CID Collision-induced dissociation
MS
SIH
Mass spectrometry
Stress-induced hyperthermia
:
:
C. H. Vinkers G. A. H. Korte-Bouws B. Olivier
Rudolf Magnus Institute of Neuroscience, Utrecht University,
Utrecht, The Netherlands
SEM Standard error of the mean
:
J. Sastre Toraño G. J. de Jong
Department of Biomedical Analysis, Utrecht Institute
for Pharmaceutical Sciences (UIPS),
Utrecht University,
Introduction
Utrecht, The Netherlands
:
:
The GABAA receptor is the main inhibitory receptor in the
central nervous system known to be closely involved in
stress and anxiety processes. Classical (nonselective)
benzodiazepines are clinically relevant anxiolytic drugs
that act on GABAA receptors, allosterically enhancing the
inhibitory GABA actions by binding to α1, α2, α3, or α5
N. R. Mirza E. Ø. Nielsen P. K. Ahring
Department of Pharmacology, NeuroSearch A/S,
Ballerup, Denmark
B. Olivier
Department of Psychiatry, School of Medicine, Yale University,
New Haven, CT, USA

556
Psychopharmacology (2010) 208:555–562
subunits (Rudolph and Mohler 2006). Besides the preferred
anxiolytic action, benzodiazepines possess a wide variety of
adverse effects, including dependence, tolerance, withdraw-
al, sedation, and cognitive impairing effects. Recently, the
concept that distinct GABAA receptor α subtypes generate
specific (side) effects has led to an increasing body of
research studying the acute and chronic effects of classical
benzodiazepines (Crestani et al. 2001; Rudolph et al. 1999).
In clinical practice, benzodiazepines are usually pre-
scribed for a longer period (ranging from weeks, months to
even years). Animal studies that aim to elucidate the largely
unknown mechanisms underlying the chronic benzodiaze-
pine (side) effects either administer drugs manually or use
osmotic minipumps (hereafter referred to as “minipumps”).
The main advantage of osmotic minipumps is a continuous
drug release for a longer period whereas no animal handling
is needed during the entire delivery period. However, when
loaded into a minipump, a drug needs to be dissolved in
order to reach the desired controlled delivery. If a compound
cannot be dissolved or is unstable in solution, this may have
enormous consequences for the interpretation of minipump
studies (van der Zwaal et al. 2008).
When designing chronic minipump studies using classi-
cal benzodiazepines, chlordiazepoxide (CDP) is often the
preferred drug of choice (e.g., Kas et al. 2008; West and
Weiss 2005) because it—in contrast to most other classical
benzodiazepines—is soluble in water (1:10; Moffat et al.
2004). However, the rapid hydrolysis of CDP in aqueous
solution to the active ketone product demoxepam has been
described (Han et al. 1976). This would diminish CDP
plasma levels in minipump studies over time and introduce
an active second compound that would be coreleased from
the minipump. Therefore, the present study aimed to
explore the putative hydrolysis of the classical benzodiaz-
epine CDP in aqueous solution and in minipumps over time
and to identify the hydrolysis products using mass
spectrometry. Moreover, we characterized the hydrolysis
products for their in vitro (3H-flunitrazepam binding and
oocyte electrophysiology) and in vivo (stress-induced
hyperthermia (SIH) paradigm) GABAA receptor potency.
The present study indicates that, although the use of
minipumps present in chronic studies presents a valid
alternative, drug stability and release should always be
considered and, when needed, carefully be investigated.
2 mg/ml at a temperature of 37°C for 40 days. Subsequent-
ly, it was filtered through a 0.2-μm filter and stored at −80°C.
Frozen demoxepam aliquots were defrosted shortly before
experiments.
Identification of the chlordiazepoxide hydrolysis products
using LC–MS/MS
To confirm the identity of the CDP hydrolysis products, a
liquid chromatography–mass spectrometry (LC–MS)/MS
setup was used. Chlordiazepoxide HCl (throughout the
present study, CDP in the salt form was used) at a
concentration of 2 mg/ml was kept for 40 days at a
temperature of 37°C and subsequently stored at −80°C. The
solution was then analyzed using a liquid chromatography
system and an Agilent 1100 Series LC/MSD SL ion-trap
mass spectrometer equipped with an electrospray ionization
source (MS, Agilent Technologies). The mobile phase
consisted of 100 mM ammonium formate (pH 4.5)–
methanol (1:1, v/v), and the flow rate was 0.5 ml/min.
The MS was operated in positive ion mode, the electrospray
voltage was optimized and set to +3.5 kV, and lens voltages
were optimized for maximal signal intensity. The nebuliz-
ing gas pressure was adjusted to 50 psi (345 kPa), and the
flow of the drying gas to 10 l/min with a drying
temperature of 350°C. The scan range was 140–310 m/z,
and five scans were averaged for one spectrum. Collision-
induced dissociation (CID) with helium as collision gas was
performed on the protonated molecular ion ([M + H]+) of
the degradation product. The m/z values of the precursor
ion was manually selected, and the collision energy was set
to the instrument default value of 100% (1.00 V), resulting
in significant presence of fragmentation ions.
Stability of chlordiazepoxide in solution
To investigate the hydrolysis kinetics under various
concentrations and environmental temperature condi-
tions, the stability of a standard CDP solution was
studied using a 10 μg/ml CDP solution that was stored
in a thermostated autosampler at a temperature of 37°C.
These samples were analyzed over time in the next
45 days. Furthermore, the influence of environmental
temperature on CDP hydrolysis was studied by storing a
2-mg/ml CDP solution at various temperatures with
subsequent analysis over time. Concentration-dependent
hydrolysis was studied by storing CDP solutions at
three different concentrations (0.02, 0.2, and 2 mg/ml)
at 37°C during 35 days which were subsequently
analyzed. For all samples, CDP and putative hydrolysis
products were detected simultaneously by HPLC with
UV detection at a wavelength of 254 nm (Spectroflow
757 Kratos Analytical). The system consisted of a pump
Materials and methods
Drugs
Chlordiazepoxide HCl (Pharbita, Zaandam, The Nether-
lands) was freshly dissolved at each testing day. Demox-
epam was obtained by keeping CDP at a concentration of

Psychopharmacology (2010) 208:555–562
557
(SpectraSeries P100 Thermo Separation products), a
vacuum degasser (ERC-3113 Erma CR Inc., Tokyo,
Japan), an autosampler (SpectraSeries AS300 Thermo
Separation products), a column (Hypersil BDS
150 mm×4.6 mm×5 μm), and a detector. The mobile
phase consisted of 50 mM phosphoric acid (pH 6.5) in
50% methanol and was pumped at 0.5 ml/min. From
each sample, 100 μl was injected onto the column. The
chromatograms were recorded and analyzed using the
Atlast 2003 chromatography data handling system
(Thermo Election Corporation, Altrincham, UK). For
degradation plots, the peak area of demoxepam after
35 days was set at 100−(% CDP).
In vivo comparison of chlordiazepoxide and demoxepam
The SIH procedure
To screen for the anxiolytic and hypothermic potency of a
CDP and demoxepam, the SIH paradigm was applied. The
SIH paradigm uses the stress-induced increase in body
temperature which is considered to be a functional body
response in anticipation to physiological demands (prepa-
ration for fight or flight; Vinkers et al. 2008). In the SIH
paradigm, classical as well as subunit-selective benzodia-
zepines consistently reduce the SIH response as well as
basal body temperature levels at higher doses. In the
present study, the SIH tests were carried out according to
the standard procedures (Vinkers et al. 2009). Briefly, the
rectal basal body temperature (T1) is measured and
functions as a stressor as well and is followed 10 min later
by a second rectal temperature measurement T2 that reflects
the stress-induced body temperature. The difference (ΔT=
T2−T1) is the SIH response. A between-subject design was
used. Cages were randomly and evenly allocated over
daytimes (morning–afternoon). The temperature of mice
was measured by rectally inserting a thermistor probe by a
length of 2 cm. Digital temperature recordings were
obtained with an accuracy of 0.1°C using a Keithley
871A digital thermometer (NiCr–NiAl thermocouple). The
probe, dipped into silicon oil before inserting, was held in the
rectum until a stable rectal temperature had been obtained for
20 s. Animals were injected intraperitoneally with either
CDP, demoxepam, or vehicle (intraperitoneally, 10 ml/kg)
60 min before the first temperature measurement (T1). The
temperature was again measured 10 min later (T2).
Chlordiazepoxide delivery rates from osmotic minipumps
To confirm that chlordiazepoxide delivery would indeed
decline over time, three osmotic minipumps (Alzet, 2004
model, pumping rate 0.25 μg/h during 28 days) were filled
with a CDP solution (2 mg/ml). Osmotic minipumps were
put into separate tubes containing 7.0 ml of saline and were
kept at 37°C. Minipumps were transferred regularly to
novel saline tubes for 5 weeks. The concentrations CDP
and demoxepam were determined using an identical HPLC
with UV setup as above.
3
In vitro H-flunitrazepam binding and oocyte
electrophysiology
To assess the in vitro affinity and efficacy of CDP and
3
demoxepam for the GABAA receptor, in vitro H-flunitra-
zepam and oocyte electrophysiology experiments were
3
carried out. In vitro H-flunitrazepam binding was done as
described previously (Mirza et al. 2008). Shortly, aliquots
of 500 μl of rat cortical membranes were added to 25 μl of
test compound, and 25 μl (1 nM, final concentration) of
3H-flunitrazepam (88 Ci/mmol; GE Healthcare) was mixed
and incubated for 40 min at 2°C. Binding was terminated
by rapid filtration, and the amount of radioactivity on the filters
was determined by conventional liquid scintillation counting.
Electrophysiological responses from GABAA receptors
expressed in Xenopus laevis oocytes were measured using
the two-electrode voltage-clamp technique as described
previously (Mirza et al. 2008). Briefly, GABA was freshly
dissolved in OR2 in a concentration known to elicit EC5–
EC25 currents for a given GABAA receptor subtype
combination (0.5–5 μM), and this solution was used for
controls as well as a stock solution for dissolving demoxepam.
Modulatory effects of demoxepam were calculated by
comparing demoxepam traces to a prior GABA control trace.
To enable comparison of effects between individual oocytes,
demoxepam potentiations were normalized to a control
0.5-μM diazepam potentiation on the same oocyte.
Animals
For in vivo experiments, male mice (129Sv/EvTac) were
obtained from Taconic M&B, Ry, Denmark. Animals were
housed in Macrolon type 3 cages enriched with bedding and
nesting material under a 12:12 light/dark cycle (lights on from
0600 to 1800 hours) at controlled temperature (20 2°C) and
relative humidity (40–60%) with free access to standard food
pellets and tap water. Experiments were carried out with
approval of the ethical committee on animal experiments of
the Faculties of Sciences, Utrecht University, The Netherlands,
and in accordance with the Declaration of Helsinki.
Statistics
For each individual mouse, a basal temperature (T1), an end
temperature (T2), and the difference (SIH response=T2−T1)
was determined. Treatment effects were evaluated using a
one-way analysis of variance with explanatory factor
treatment. If the overall analysis of variance appeared

558
Psychopharmacology (2010) 208:555–562
significant, post hoc tests (Dunnet’s post hoc test) were
used to identify significant differences.
strictly followed first-order kinetics ([CDP ]t=[CDP]0×e−kt
with t being time and k being the first-order rate constant)
and was temperature dependent. At storage conditions
below 0°C, no apparent hydrolysis occurred (Fig. 3b).
Identical to the results at −25°C, storage conditions at −80°C
did not result in any hydrolysis (data not shown). In line with
first-order kinetics, CDP hydrolysis was concentration
dependent (Fig. 3c).
Results
Identification of the chlordiazepoxide hydrolysis products
using LC–MS/MS
The extracted ion chromatogram at m/z 300.1 showed the
presence of CDP (Fig. 1b). The first and only eluting
degradation product with m/z 287.1 was demoxepam, the
CDP hydrolysis product after oxidative deamination (Fig. 1a).
The typical mass spectra of CDP ([M+H]+=300.1) and the
hydrolyzed product demoxepam ([M+H]+=287.1), both with
a typical chloride isotopic distribution pattern, are presented
in Fig. 2. The identification of demoxepam (with a calculated
monoisotopic mass of 286.1) was performed by MS/MS of
m/z 287.1. The resulting fragments (Fig. 2c) showed the loss
of a hydroxyl (corresponding to a N-oxide benzodiazepine), a
–COH (corresponding to a benzodiazepine with carbonyl
group) and a phenyl group. The interpretation of the various
fragments of demoxepam is in line with literature (Nedved et
al. 1996; Risoli et al. 2007; Smyth et al. 2000).
Chlordiazepoxide delivery rates from osmotic minipumps
CDP release from the minipump (n=3) declined over time
(Fig. 4a, black symbols). After 28 days, only 25% of all
CDP was released from the minipumps. When the
cumulative CDP concentration over time was corrected
for its hydrolysis, drug release from the minipumps
followed the theoretical release profile over time (white
symbols), suggesting that CDP hydrolysis completely
accounted for the declined CDP release over time.
3
In vitro H-flunitrazepam binding and oocyte
electrophysiology
Chlordiazepoxide and demoxepam both displayed comparable
affinity for rat GABAA benzodiazepine binding sites labeled
by 3H-flunitrazepam (Table 1). However, demoxepam
showed less efficacy for α1β2γ2 GABAA receptors com-
pared to CDP, whereas efficacy at α3β2γ2 GABAA receptors
was comparable (Fig. 5)
Stability of chlordiazepoxide in solution
At 37°C, CDP was rapidly degraded into demoxepam with
a half-time of around 8.8 days (Fig. 3a). CDP hydrolysis
Fig. 1 Extracted ion traces of
demoxepam (a) and chlordiaz-
epoxide (b) obtained by LC–MS
of a 2-mg/ml chlordiazepoxide
solution kept for 35 days at
37°C

Psychopharmacology (2010) 208:555–562
559
Fig. 2 Mass spectra and molec-
ular structures of demoxepam
with [M+H]+ 287.1 (a) and
chlordiazepoxide with [M+H]+
300.1 (b) obtained by LC–MS
analysis of the mixture of
chlordiazepoxide and demoxe-
pam in the 2-mg/ml chlordiaz-
epoxide solution kept for
35 days at 37°C. The CID
mass spectrum of m/z 287.1
confirmed the presence of
demoxepam (c)
In vivo comparison of chlordiazepoxide and demoxepam
CDP and demoxepam overall reduced SIH (drug effect
temperature as well as the SIH response compared to
demoxepam. Demoxepam reached the anxiolytic and
hypothermic effects of CDP at higher doses.
F
5, 69=11.91, p<0.001) as well as basal body temperature
(drug effect F5, 69=9.46, p<0.001; Fig. 6). Post hoc
testing revealed that CDP lowered basal body temperature
at 10 mg/kg (p<0.05) as well as 20 mg/kg (p<0.001),
whereas demoxepam lowered basal body temperature only
at its highest dose of 40 mg/kg (p<0.05). Planned
comparisons showed that CDP at a dose of 20 mg/kg
Discussion
The present study shows that CDP in solution rapidly
hydrolyzes to the ketone structure demoxepam with a half-
time of around 9 days at 37°C, thus confirming earlier
reports on CDP hydrolysis (Han et al. 1976). Using mass
spectrometry, we confirmed that the single hydrolysis
product is indeed demoxepam, arising from oxidative
deamination of CDP (Figs. 1 and 2). The CDP hydrolysis
was concentration dependent (following first-order kinetics)
as well as temperature dependent (Fig. 3b, c). In osmotic
minipumps, hydrolysis to demoxepam was again apparent
had a stronger effect on basal body temperature (F1, 23
=
9.49, p<0.01) as well as SIH attenuation (F1, 23=6.35,
p<0.05) compared to demoxepam 20 mg/kg. When we
compared CDP 20 mg/kg to demoxepam 40 mg/kg, there
were no differences in basal body temperature (F1, 21
=
0.001, p=0.97, NS) nor SIH reduction (F1, 21=0.01,
p=0.98, NS). CDP thus more potently reduced basal body
Fig. 3 Chlordiazepoxide (2 mg/ml) rapidly hydrolyzes into demoxepam at 37°C (a). This hydrolysis rate is absent at lower temperatures (b) and
concentration dependent (c; compound after 35 days at 37°C)

560
Psychopharmacology (2010) 208:555–562
Fig. 4 Release of chlordiazepoxide (2 mg/ml) from osmotic mini-
pumps (n=3) over time. CDP release declines over time (black
symbols). When CDP hydrolysis is taken into account, CDP release
follows the theoretical curve (white symbols), indicating that it is CDP
hydrolysis that leads to declined CDP release
(Fig. 4). Demoxepam is already known as an active
metabolite of CDP with a half-life markedly longer than
that of CDP (Schwartz et al. 1971) and with steady-state
concentrations exceeding those of CDP (Roy-Byrne et al.
1996). In contrast to another study, we did not find a parallel
degradation of demoxepam to a 2-amino-5-chlorobenzophe-
none and a glycine derivative (Han et al. 1976), although we
cannot exclude the possibility that hydrolysis products were
present which were not eluted in time to be detected.
Using the stress-induced hyperthermia paradigm (Vinkers
et al. 2008), our data in mice suggest that CDP may exert
greater acute potency compared to demoxepam (Fig. 5).
Interestingly, a study that found significant dependence
after chronic CDP administration showed low CDP blood
levels and high metabolite levels, indicating that demox-
epam is indeed active (Chan et al. 1989). Another study in
subjects suffering from anxiety disorder found a significant
correlation between anxiety reduction and demoxepam
plasma levels but not CDP plasma levels, suggesting that
after chronic exposure, demoxepam may possess anxiolytic
effects that surpass those of CDP itself (Lin and Friedel
1979). Demoxepam binding data to oocyte GABAA
receptors containing α1 and α3 receptors showed that
demoxepam displayed lower efficacy at α1 containing
Fig. 5 Efficacy (compared to diazepam) of demoxepam (upper
figure) and chlordiazepoxide (lower figure) in oocytes expressing
GABAA receptors containing α1 and α3 subunits
3
GABAA receptors, even though in vitro H-flunitrazepam
binding was not different. Thus, the active metabolites
Table 1 Inhibition of 3H-flunitrazepam binding to rat cortical
membranes by chlordiazepoxide and demoxepam. Data are mean
SEM, n=3–4 experiments
Compound
3H-flunitrazepam binding (Ki, μM)
Fig. 6 In vivo efficacy of chlordiazepoxide (0–20 mg/kg, IP) and
demoxepam (0–40 mg/kg, IP) on the basal body temperature (a) and
the stress-induced hyperthermia response (b) in 129Sv mice (n=10–
12). *p<0.05 CDP compared to vehicle; #p<0.05 demoxepam
compared to vehicle
Chlordiazepoxide
Demoxepam
0.67 0.10
0.44 0.02

Psychopharmacology (2010) 208:555–562
561
(including demoxepam) may significantly contribute to the
various effects of CDP.
product demoxepam are hard to dissect and the ratio CDP/
demoxepam is not stable over time in solution, we advise
that chronic CDP applications in which CDP is dissolved
should be interpreted with caution. The possibility of
using more lipophilic classical benzodiazepines (e.g.,
diazepam) in minipumps with solid PEG-400 dispersions
may present a valid alternative (Verheyen et al. 2002).
Although we only studied the degradation of CDP, it is
conceivable that other drugs may also be subject to
degradation in chronic minipump studies, including
olanzapine (van der Zwaal et al. 2008), zopiclone
(Fernandez et al. 2004), and sulpiride (Abdelal et al.
2009). In general, the use of osmotic minipumps presents
a valid and attractive alternative to the labor-intensive
daily injections. However, the issue of drug stability and
release should always be carefully investigated before
initiating chronic minipump experiments.
The apparent hydrolysis of CDP in solution was
previously not taken into consideration in chronic experi-
ments that used osmotic minipumps. Results obtained from
chronic CDP administration using osmotic minipumps may
be (partially) ascribed to demoxepam or the combination of
both rather than to only CDP itself. Studies that have tested
dissolved CDP in osmotic minipumps have yielded variable
results. In one study, chronic minipump treatment with
CDP (10 mg/kg/day) prevented hyperalgesia in rats
subjected to a social-defeat procedure (Alexandre et al.
2006), and in another study, 7–14-day minipump treatment
with CDP (10 mg/kg/day) increased GABAA receptor
desensitization in rats, as well as tolerance in an elevated-
plus-maze test (Cash et al. 1997). One-week CDP mini-
pump treatment (5–10 mg/kg/day) in C57BL/6J mice
reduced sheltered feeding preference without altering motor
activity levels (Kas et al. 2008). In contrast, 14-day CDP
minipump treatment (2 mg/kg/day) did not prevent the
stress-induced decrease in swim-test struggling in rats
unlike chronic treatment with different antidepressant drugs
(West and Weiss 2005). Only in one study, plasma
concentrations of CDP and its metabolites were determined
after 14 days (Cash et al. 1997). The extent to which CDP
hydrolysis may have altered the outcomes in these studies
cannot be determined as the active product demoxepam was
formed. However, because demoxepam is less potent
compared to CDP, an underestimation of the CDP effects
is possible.
Acknowledgments The authors would like to thank Dr. J.H. van
den Berg for carefully reading the manuscript.
Conflicts of interest The authors declare no financial disclosures or
conflicts of interest.
Open Access This article is distributed under the terms of the
Creative Commons Attribution Noncommercial License which per-
mits any noncommercial use, distribution, and reproduction in any
medium, provided the original author(s) and source are credited.
An alternative to minipump administration is the
chronic application of repeated daily injections. However,
adequate plasma levels are not easily established, and
repetitive injections may affect experimental outcomes,
especially in stress-related research. In a study that
compared daily injections with minipumps, chronic
diazepam administration resulted in sedative tolerance
under both conditions, although tolerance to the anxio-
lytic effects was only present after the daily injections
(Fernandes et al. 1999). This suggests that both ways of
establishing chronic benzodiazepine exposure may not
yield identical results. To complicate things even further,
withdrawal anxiety from chronic diazepam treatment by
manual daily injections was found to depend on adminis-
tration route as it was present after subcutaneous but not
after intraperitoneal injections (Allison and Pratt 2006).
In conclusion, the present study indicates that the
classical benzodiazepine CDP is rapidly hydrolyzed in
solution to the active compound demoxepam. The fact that
CDP is readily soluble in water makes it an attractive
choice for minipump applications, but it immediately
poses the researcher for some challenges. As the exact
anxiolytic contributions of CDP and its active hydrolysis
References
Abdelal, A, El-Enany N et al (2009) Simultaneous determination of
sulpiride and its alkaline degradation product by second
derivative synchronous fluorescence spectroscopy. Talanta 80
(2):880–888
Alexandre C, Popa D, Fabre V, Bouali S, Venault P, Lesch KP, Hamon
M, Adrien J (2006) Early life blockade of 5-hydroxytryptamine
1A receptors normalizes sleep and depression-like behavior in
adult knock-out mice lacking the serotonin transporter. J Neuro-
sci 26:5554–5564
Allison C, Pratt JA (2006) Differential effects of two chronic
diazepam treatment regimes on withdrawal anxiety and AMPA
receptor characteristics. Neuropsychopharmacology 31:602–619
Cash DJ, Serfozo P, Allan AM (1997) Desensitization of a gamma-
aminobutyric acid type A receptor in rat is increased by chronic
treatment with chlordiazepoxide: a molecular mechanism of
dependence. J Pharmacol Exp Ther 283:704–711
Chan AW, Leong FW, Schanley DL, Langan MC, Penetrante ML
(1989) A liquid diet model of chlordiazepoxide dependence in
mice. Pharmacol Biochem Behav 34:839–845
Crestani F, Low K, Keist R, Mandelli M, Mohler H, Rudolph U
(2001) Molecular targets for the myorelaxant action of diazepam.
Mol Pharmacol 59:442–445
Fernandes C, Arnot MI, Irvine EE, Bateson AN, Martin IL, File SE
(1999) The effect of treatment regimen on the development of
tolerance to the sedative and anxiolytic effects of diazepam.
Psychopharmacology (Berl) 145:251–259

562
Psychopharmacology (2010) 208:555–562
Fernandez C, Gimenez F et al (2004) Degradation and racemization of
zopiclone enantiomers in plasma and partially aqueous solutions.
Chirality 7(4):267-271
Han WW, Yakatan GJ, Maness DD (1976) Kinetics and mechanisms
of hydrolysis of 1, 4-benzodiazepines I: chlordiazepoxide and
demoxepam. J Pharm Sci 65:1198–1204
Rudolph U, Mohler H (2006) GABA-based therapeutic approaches:
GABAA receptor subtype functions. Curr Opin Pharmacol 6:18–23
Rudolph U, Crestani F, Benke D, Brunig I, Benson JA, Fritschy JM,
Martin JR, Bluethmann H, Mohler H (1999) Benzodiazepine
actions mediated by specific gamma-aminobutyric acid(A)
receptor subtypes. Nature 401:796–800
Kas MJ, de Mooij-van Malsen AJ, Olivier B, Spruijt BM, van Ree JM
(2008) Differential genetic regulation of motor activity and
anxiety-related behaviors in mice using an automated home cage
task. Behav Neurosci 122:769–776
Lin KM, Friedel RO (1979) Relationship of plasma levels of
chlordiazepoxide and metabolites to clinical response. Am J
Psychiatry 136:18–23
Mirza NR, Larsen JS, Mathiasen C, Jacobsen TA, Munro G, Erichsen
HK, Nielsen AN, Troelsen KB, Nielsen EO, Ahring PK (2008)
NS11394 [3′-[5-(1-hydroxy-1-methyl-ethyl)-benzoimidazol-1-
yl]-biphenyl-2-carbonitr ile], a unique subtype-selective GABAA
receptor positive allosteric modulator: in vitro actions, pharma-
cokinetic properties and in vivo anxiolytic efficacy. J Pharmacol
Exp Ther 327:954–968
Schwartz MA, Postma E, Gaut Z (1971) Biological half-life of
chlordiazepoxide and its metabolite, demoxepam, in man. J
Pharm Sci 60:1500–1503
Smyth WF, McClean S, Ramachandran VN (2000) A study of the
electrospray ionisation of pharmacologically significant 1, 4-
benzodiazepines and their subsequent fragmentation using an
ion-trap mass spectrometer. Rapid Commun Mass Spectrom
14:2061–2069
van der Zwaal EM, Luijendijk MC, Adan RA, la Fleur SE (2008)
Olanzapine-induced weight gain: chronic infusion using osmotic
minipumps does not result in stable plasma levels due to degradation
of olanzapine in solution. Eur J Pharmacol 585:130–136
Verheyen S, Blaton N, Kinget R, Van den Mooter G (2002) Mechanism
of increased dissolution of diazepam and temazepam from
polyethylene glycol 6000 solid dispersions. Int J Pharm 249:45–58
Vinkers CH, van Bogaert MJ, Klanker M, Korte SM, Oosting R,
Hanania T, Hopkins SC, Olivier B, Groenink L (2008)
Translational aspects of pharmacological research into anxiety
disorders: the stress-induced hyperthermia (SIH) paradigm. Eur J
Pharmacol 585:407–425
Vinkers CH, Klanker M, Groenink L, Korte SM, Cook JM, Van Linn
ML, Hopkins SC, Olivier B (2009) Dissociating anxiolytic and
sedative effects of GABA(A)ergic drugs using temperature and
locomotor responses to acute stress. Psychopharmacology (Berl)
204:299–311
Moffat A, Osselton M, Widdop B (2004) Clarke’s analysis of drugs
and poisons. Pharmaceutical, London
Nedved ML, Habibi-Goudarzi S, Ganem B, Henion JD (1996)
Characterization of benzodiazepine “combinatorial” chemical
libraries by on-line immunoaffinity extraction, coupled column
HPLC-ion spray mass spectrometry-tandem mass spectrometry.
Anal Chem 68:4228–4236
Risoli A, Cheng JB, Verkerk UH, Zhao J, Ragno G, Hopkinson AC,
Siu KW (2007) Gas-phase fragmentation of protonated benzo-
diazepines. Rapid Commun Mass Spectrom 21:2273–2281
Roy-Byrne P, Wingerson DK, Radant A, Greenblatt DJ, Cowley DS
(1996) Reduced benzodiazepine sensitivity in patients with panic
disorder: comparison with patients with obsessive–compulsive
disorder and normal subjects. Am J Psychiatry 153:1444–1449
West CH, Weiss JM (2005) A selective test for antidepressant
treatments using rats bred for stress-induced reduction of motor
activity in the swim test. Psychopharmacology (Berl) 182:9–23