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THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS
Copyright © 1997 by The American Society for Pharmacology and Experimental Therapeutics
JPET 281:1013–1029, 1997
Vol. 281, No. 3
Printed in U.S.A.
Differential Reinforcement of Low Rate Performance,
Pharmacokinetics and Pharmacokinetic-Pharmacodynamic
Modeling: Independent Interaction of Alprazolam and Caffeine1
CHYAN E. LAU, YUNXIA WANG and JOHN L. FALK
Department of Psychology, Rutgers University, New Brunswick, New Jersey
Accepted for publication February 20, 1997
BZs are safe and widely prescribed for the chronic treatment of epilepsy, movement and panic disorders. They are
given for the acute and subchronic treatment of insomnia,
agitated psychosis and in surgery as effective preanesthetic
and anesthetic agents (Martin and Haefely, 1995). However,
interactions can occur with combinations of BZs and central
nervous system stimulants (e.g., caffeine, cocaine), where one
agent is prescribed and the other is ingested by choice (Boulenger et al., 1984; Charney et al., 1985). BZs exert their
effects through the GABA-BZ receptor complex (Haefely et
al., 1985). It is generally recognized that the antagonism of
adenosine receptors at least partly underlies the pharmacological effects of low doses of MXs, whereas phosphodiesterase inhibition and calcium mobilization become more signifReceived for publication October 24, 1996.
1
This work was supported by Grants R 37 # DA03117 and K05 DA00142
from the National Institute on Drug Abuse.
near the end of a session. The interaction was PK linked and
mainly not distinguishable from independence as indicated by
the Pöch dose-response curve method and the integration of
PK and pharmacodynamics. The sigmoid maximal effect-link
pharmacodynamic model indicated that caffeine did not alter
the concentration at half of the maximal effect value of alprazolam and suggested that the interaction is not competitive, but
independent. Although the nature of the benzodiazepine-methylxanthine interaction has been controversial in other behavioral studies, as is the role of PK in determining behavior, this
and our previous study make it evident that the interaction is
independent not only across doses and routes of administration, but also with respect to two indices of differential reinforcement of low rate performance.
icant at higher doses (Choi et al., 1988; Daly, 1993; Snyder et
al., 1981). Several studies have indicated that caffeine competes for binding at BZ sites, and conversely, that BZ may
interact with adenosine receptors, although with low affinity
in both cases, raising the question of the physiological relevance for these kinds of interactions (Bruns et al., 1983;
Marangos et al., 1979; Weir and Hruska, 1983). Thus, the
interactions between caffeine and the GABA-BZ system remain poorly defined.
Additive (Beer et al., 1972; Coffin and Spealman, 1985;
Valentine and Spealman, 1983), antagonistic (Kaplan et al.,
1990; Polc et al., 1981; Rush et al., 1994; Tang et al., 1989),
functional antagonistic (Baldwin and File, 1989; Roache and
Griffiths, 1987) and synergistic (Falk and Lau, 1991; Katims
et al., 1983; Lau and Falk, 1991) interactions have been
reported after concurrent BZ and MX administration using
various kinds of behavioral paradigms in animals and hu-
ABBREVIATIONS: AUC, area under the curve; AUMC, area under the first moment curve; BZ, benzodiazepine; Cmax, the maximum concentration;
Cl, clearance; cpt, compartment; DMX, dimethylxanthine; DRC, dose-response curve; DRL, differential reinforcement of low rate; E0, the effect
when alprazolam concentration is zero; Emax, the maximal effect; F, absolute bioavailability; HPLC, high performance liquid chromatography; IC50,
the concentration at half of the maximal effect; IRT, inter-response time; keo, the rate constant out of the effect compartment; MX, methylxanthine;
N, the slope factor of the sigmoid effect curve; PB, percent baseline; PK, pharmacokinetics; PD, pharmacodynamics; Tmax, the time at which Cmax
occurred; Vc, volume of distribution of the central compartment; Vss, volume of distribution at steady state.
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ABSTRACT
To investigate the interaction between alprazolam and caffeine,
performance on a differential reinforcement of low-rate behavior schedule and the respective pharmacokinetics (PK) were
explored in concurrent studies. Alprazolam PK was not altered
by caffeine, but alprazolam retarded caffeine absorption indirectly, as inferred by the lack of i.v. drug administration PK
interaction, thereby decreasing serum methylxanthine concentrations. Inasmuch as alprazolam was more potent and shortlived than caffeine in decreasing the reinforcement rate (consonant with their respective t1/2 values, 0.44 and 3.1 hr), the
alprazolam/caffeine potency ratio decreased across the session time, which determined the expression of the combined
effects. Thus, the decreased methylxanthine level yielded
slightly less disruption in performance for the observed combined effect, compared to the expected calculated effect, only
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Lau et al.
synergistic nor antagonistic, characterized the combined effects of alprazolam and caffeine by the i.p. route using reinforcement rate as the PD measure (Lau and Wang, 1996). In
that study, PK interaction was also characterized by using
tail-tip blood samples between 15 and 180 min. It was concluded that the PK of alprazolam, caffeine and their combination were predictive of the resultant behavior-time profiles. The differences in potency and PK between the two
drugs accounted for the expressions of the combined effects.
The PK of alprazolam was not altered by the presence of
caffeine, but the PK of caffeine was affected by alprazolam.
Inasmuch as the PK drug interaction was not evaluated by
the i.v. route in that study, the effects of alprazolam on
caffeine PK were difficult to interpret.
Different types of interaction for BZ-MX sometimes were
obtained from the same laboratory with the use of different
behavioral measures or paradigms (De Angelis et al., 1982;
Ghoneim et al., 1986; Loke et al., 1985). Our study is an
expansion of the previous work on both behavior and PK,
which aims to validate the interaction of alprazolam and
caffeine by using: 1) different routes of administration; 2) not
one but two different kinds of response measures, reinforced
and nonreinforced, to investigate whether they were in conformity with each other; 3) blood samples from jugular vein
between 2 and 180 min after drug administration to characterize the respective PK; 4) the i.v. route to calculate the PK
parameters (e.g., volume of distribution, clearance, and bioavailability) and to define the pure PK drug interaction without having to consider drug absorption and 5) integration of
PK and PD to delineate the nature of BZ-MX interaction and
the predictive ability of the model.
Both alprazolam and caffeine are metabolized by the P-450
cytochrome enzyme system (Aldridge et al., 1977; von Moltke
et al., 1993). Factors (e.g., food restriction) affecting this
enzyme system will affect the PK of these agents and will
lead to PD changes (Lau et al., 1995; Lau et al., 1996; Sachan,
1982). Both alprazolam and caffeine are absorbed rapidly in
rats with an elimination half-life of 0.5 to 0.9 and 3 hr,
respectively (Lau and Wang, 1996; Lau et al., 1995; Owens et
al., 1991). In humans, food deprivation or restriction can
occur for cosmetic, health or economic reasons. In DRL behavior, a food-deprivation regimen is applied to animals to
implement a food-reinforced behavioral DRL performance
baseline. Thus, it is important to investigate the PK of alprazolam, caffeine and their combinations in food-limited rats,
especially because these drugs are metabolized by the P-450
cytochrome enzyme system. Furthermore, based on their
half-lives, a 3-hr session was used, a period necessary to
investigate the interaction at the onset, peak and disappearance of serum alprazolam concentration, while that of caffeine remained constant, so that we could achieve a better
understanding of the mechanisms of drug action and interaction.
Different routes of administration provided an opportunity
to examine the interaction of drug concentration-time profiles that might differ from that of the i.p. route, as PK
parameters are generally route dependent (e.g., absorption
rate constant, metabolite formation and bioavailability). In
our study, alprazolam and caffeine were given s.c. and p.o.,
respectively. There are considerations for choosing these
routes of administration. As in the case of midazolam (Lau et
al., 1996), we found the s.c. route to be the route of choice
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mans. One of the major reasons for these differences arose
from describing the combined effects qualitatively rather
than from using quantitative methods specifically developed
for that purpose.
Inasmuch as the pharmacological response often can be
predicted from the respective PK, it is rational to investigate
the role of PK on drug action and interaction before receptor
mechanisms. However, the predictability of PK during drug
interaction is not as simple and direct as it is when a drug is
given alone. One needs not only to analyze the resultant PK
changes of the parent drugs and their active metabolites
after concurrent drug administration but also consider the
potency relation of the two agents before inferring the role of
PK in drug interaction. Failure to explore the potency relation may account, in part, for the conflicting findings relating
PK to PD in BZ-MX combined effects (Ghoneim et al., 1986;
Henauer et al., 1983; Kaplan et al., 1990; Tuncok et al., 1994).
Differential reinforcement of low rate schedules (e.g., DRL
45-s) produce low rates of responding as only those responses
that occur after a minimum time interval ($45 sec) after a
previous response are reinforced. Responses that occur before
this time has elapsed are not reinforced, and they reset the
timing of the interval. DRL behavior reaches baseline performance after sufficient training, and the effects of drug
treatments can be compared to the performance baseline.
The DRL schedule contingency not only involves time discrimination but also requires an appropriate inhibition of
responding for reinforcement to occur, and involves other
memory, sensory and motor capacities (Kramer and Riling,
1970). It has been suggested that the effect of many kinds of
drug is to reduce the inhibition of behavior associated with
signals of punishment or nonreward in DRL behavior (Gray,
1981). DRL performance satisfies many of the criteria proposed as ideal for PD measurement (Dingemanse et al., 1988;
Laurijssens and Greenblatt, 1996). The fulfilling of a required, objectively defined, behavioral contingency by the
subject, rather than using a passive measure of an unconditioned drug effect (e.g., EEG recording), affords the DRL
method a distinct advantage. The performance measure is a
continuous process rather than one limited to temporally
discrete trials. Furthermore, it is sensitive to drug effects,
and the effects are reproducible, an important feature for
defining and evaluating drug interaction (Lau et al., 1996).
Finally, after drug administration, reinforced and nonreinforced responses, which generally exhibit decreases and increases, respectively, can be used to evaluate the combined
drug effects.
Recently, we used the DRC method proposed by Pöch and
his colleagues (Pöch, 1993, 1992; Pöch and Pancheva, 1995;
Pöch et al., 1990) to quantitatively analyze the combined
effects of alprazolam and caffeine by using 3-hr sessions of
DRL 45-sec performance (Lau and Wang, 1996). This method
permits the evaluation of the combined effects not only from
a phenomenologic (e.g., larger or smaller effect) but also from
a mechanistic (additivity or independence) point of view. The
assumptions used in the DRC method also can be applied to
behavior-time profiles to extend the results obtained from
DRC analyses. Values derived from the usual dose-response
analyses (e.g., potency ratio of these agents) can aid in predicting the outcome of the combined effect, whereas the response-time curve describes the ongoing interaction profile.
An independent or additive interaction, which was neither
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1997
BZ and MX: Independent Interaction
Materials and Methods
DRL Performance
Animals. Seven male, albino, Sprague-Dawley rats from HSD
(Indianapolis, IN) were used. They were housed individually in a
temperature-regulated room with a daily cycle of illumination from
7:00 A.M. to 7:00 P.M. They were reduced to 80% of their initial, adult
free-feeding body weights (mean 5 383 g; range: 380–388 g) over a
2-wk period by limiting daily food rations: 5 g for the first day, 10 g
for the next 5 days and a food supplement (range 14–16 g) to maintain their 80% body weights. Water was continuously available in
the living cages. Experiments were executed in accordance with the
Guide for the Care and Use of Laboratory Animals (National Institute of Health Publ. no. 85–23, revised 1985).
Drugs. Alprazolam was obtained from Upjohn Laboratories
(Kalamazoo, MI). Alprazolam (5 mg) was dissolved in 50 ml of 1.2 N
HCl and further diluted to working concentration with 0.9% NaCl
solution. Caffeine was purchased from Sigma Chemical Co. (St.
Louis, MO) and was dissolved in sodium benzoate (37.5 mg/ml)
solution. Alprazolam and caffeine were administered s.c. and p.o. by
gavage, respectively, in an injection volume of 1 ml/kg body weight.
Apparatus. Four operant Plexiglas chambers were used and have
been described previously (Lau and Wang, 1996). Each chamber,
equipped with a response lever and a stainless steel food-pellet
receptacle into which 45-mg dustless pellets (BioServ, Frenchtown,
NJ) could be delivered, was enclosed in a sound-attenuating shell
and was controlled by an IBM-type 486 X computer. Session contingencies were programmed and data recorded using QuickBasic.
Procedure. Animals were magazine trained on a noncontingent
random-time schedule initially for 15 min and responses on the lever
were shaped by successive approximation and reinforced when IRTs
were greater than 3 sec. The temporal requirement was slowly increased to an IRT of 45 sec over 10 to 20 sessions. A 3-hr operant
session was conducted daily. After performance had stabilized, a
drug-administration series began. The series consisted of: 1) Alprazolam dose-response determination (vehicle, 0.125, 0.4, 1.25, 4 and 7
mg/kg); 2) caffeine dose-response determination (vehicle, 5, 10, 20,
40, 80 and 120 mg/kg); 3) alprazolam-caffeine combinations: (a)
alprazolam 1 30 mg/kg caffeine, i.e., vehicle 1 vehicle; vehicle 1 30
mg/kg caffeine; 0.125 to 7 mg/kg alprazolam 1 30 mg/kg caffeine (b)
alprazolam 1 20 mg/kg caffeine, i.e., vehicle 1 20 mg/kg caffeine;
0.125 to 7 mg/kg alprazolam 1 20 mg/kg caffeine. At the end of each
combination series, the caffeine dose for that series (e.g., vehicle 1 20
mg/kg caffeine) was redetermined. Injections were given immediately before the start of a session and separated by 3 to 5 days.
Injections within each series were given in a quasirandom order.
Each drug series was separated by 10 noninjection sessions.
Data analyses. The IRT distributions after the administration of
vehicle, alprazolam, caffeine and alprazolam-caffeine combinations
were analyzed for 3-hr sessions, omitting the first 2 min, which was
treated as the settling time. Baseline IRT distributions for each
session that immediately preceded an injection also were analyzed.
Behavioral parameters were derived from the IRT distributions:
shorter (nonreinforced)-response rate, reinforcement rate, total response rate and efficiency. Total number of responses consisted of
responses with IRT $ 45 and , 45 sec, which are the reinforced and
nonreinforced responses, respectively. These responses were calculated as rates (responses per min). Efficiency was calculated as the
ratio of reinforcement rate to the total response rate. We have found
that both the reinforcement rate in the 45-to-55- and $45-sec bins
decreased equivalently as a function of dosage for alprazolam and
caffeine by the i.p. route. The 45- to 55-sec bin function required a
lower dose to reach Emax for both drugs, and consequently resulted in
smaller ED50 values (Lau and Wang, 1996). The 45- to 55-sec bin
function has been used successfully to characterize the alprazolamcaffeine interaction, and justification will be given in “Results” referring to figures 4A–C. Specific attention was given to the 45- to
55-sec bin data in this study, facilitating the comparison of our
results with those from the previous study.
Peak deviation analysis developed by Richards et al. (1993) was
used for characterizing effects of alprazolam and caffeine with the
parameters, peak area and peak location that determine the size and
the center of the IRT distribution peak, respectively. Reduction in
peak area indicates that the IRTs are more disrupted, suggesting a
decrease in temporal stimulus control.
DRCs for caffeine, alprazolam and alprazolam-caffeine combinations were constructed using a four-parameter, logistic function of
the following equation by the ALLFIT curve-fitting program written
for the IBM PC (DeLean et al., 1978, 1992):
y 5 @~a 2 d!/~1 1 ~x/c!b!# 1 d
where, y is the percent of baseline performance in 45- to 55-sec bin
and x is the drug dose administered. The four fitted parameters
were: a, the Emin, i.e., the % baseline performance when x 5 0; d, the
Emax,i.e., the % baseline performance for “infinite” dose; b, the slope
factor that determines the steepness of the curve; c, the ED50, i.e., the
dose resulting in a response halfway between a and d.
Characterization of alprazolam-caffeine interaction by
DRC method proposed by Pöch. Alprazolam-caffeine interactions
were evaluated by comparing the dose-response curves for alprazolam in the presence and absence of two caffeine doses following the
method of combined effects proposed by Pöch and his colleagues
(Pöch, 1993; Pöch et al., 1990) and have been described previously
(Lau and Wang, 1996). From the results of seven-animal medians,
rather than means, values of both observed and expected DRCs of
the DRL behavior were used for statistical evaluation of observed
versus expected frequencies by the x2 goodness-of-fit test.
There are two models in the Pöch DRC method, additivity and
independence. The additivity model of dose-additive combinations is
based on the assumption that alprazolam and caffeine act alike, i.e.,
that alprazolam acts like caffeine or vice versa. Then, the combination of alprazolam plus caffeine should behave as if it were a combination of alprazolam and alprazolam. The Pöch DRC method assumes that alprazolam and caffeine act at the same receptor. Briefly,
theoretical additive interactions for the DRC method can be derived
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owing to its high absolute bioavailability, as well as its dependability in producing consistent within-subject serum
concentration-time profiles for repeated doses, whereas for
caffeine, the oral route is used by humans for its consumption
with high bioavailability (Axelrod and Reichental, 1953).
Although DRL performance has been used extensively in
behavioral pharmacology to study the effects of various drugs
from different classes, it has not been used for PK-PD studies, except in our laboratory. We have found not only that the
DRL 45-sec reinforcement rate-time profiles correlated well
with serum alprazolam, caffeine and midazolam concentration-time profiles but also that bioavailability values derived
from those profiles mirrored those estimated from PK for
midazolam following i.v., s.c., i.p. and p.o. routes of administration (Lau and Wang, 1996; Lau et al., 1996). Integrating
PK and PD permits the investigation and possible prediction
of drug concentration-effect relations, which are sensitive to
variables such as drug interaction, aging and the disease
state. Examination of the alprazolam concentration-effect
relation in the presence and absence of caffeine can shed light
on the nature of the interaction. The competitive interaction
between flumazenil and midazolam was demonstrated in
humans (Breimer et al. 1991) and in rats (Mandema et al.
1991) with PK-PD modeling by parallel shifts in the concentration-electroencephalography effect relation of midazolam
with increasing flumazenil concentration.
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Lau et al.
PBalp1caff 5 PBalp 3 PBcaff
where, PBalp1caff, PBalp, PBcaff are the PB values for alprazolamcaffeine combination, alprazolam, and caffeine, respectively, assuming drugs decrease the reinforcement rate. For example, if PBalp 5
0.25, PBcaff 5 0.6, then PBalp1caff 5 0.25 3 0.6 5 0.15. Thus, the
fractions by which alprazolam and caffeine decrease the reinforcement rate are not altered in the combined effect if alprazolam and
caffeine act independently. In this example, alprazolam reduces the
reinforcement rate to 1/4 from 0.6 to 0.15 in the presence of caffeine
and from 1.0 to 0.25 in the absence of caffeine.
Median rather than mean values were used to construct doseresponse curves of these agents and their combinations, and x2
analyses were used to compare the observed combined effects to
theoretical values of independent and additive interactions. If the
combined effects are greater or smaller than the theoretical curves,
then synergism or antagonism occurred, respectively. In addition,
mean rather than median behavior-time profiles were constructed
with respect to the mean expected independent curves. Statistical
analyses for the comparison of behavior-time profiles were performed by repeated measures, two-way analyses of variance using
SigmaStat, followed by Newman-Keuls tests (Jandel, San Rafael,
CA).
Pharmacokinetics of Alprazolam, Caffeine and their
Combinations
Animals. Eight male, albino rats of the same strain were used under
the conditions and food-limitation regimen used above. The mean initial, adult free-feeding body weight was 388 g (range 380–391 g).
Drugs and reagents. Alprazolam, a-hydroxyalprazolam and
4-hydroxyalprazolam were obtained from Upjohn Laboratories,
Kalamazoo, MI. Caffeine, theobromine, paraxanthine, theophylline
and b-hydroxyethyltheophylline were purchased from Sigma Chemical Company Co., St. Louis, MO. Reagents were obtained from
standard commercial sources.
HPLC determination of alprazolam, caffeine and their metabolites and serum sampling. HPLC. Serum microsample HPLC
methods for determination of alprazolam, caffeine and their metabolites have been described previously (Jin and Lau, 1994; Lau and
Falk, 1991). Separation for both drugs was performed on Beckman
Ultrasphere C18 columns (5-mm particle size, 150 3 2 mm I.D.).
Programmable absorbance UV detectors 785A (Applied Biosystems
Instruments, Foster City, CA) were operated at 230 and 270 nm for
alprazolam and caffeine methods, respectively. The capacity factors
for demoxepam used as internal standard, 4-hydroxyalprazolam,
a-hydroxyalprazolam and alprazolam were 2.08, 2.73, 3.37 and 4.43,
respectively, whereas for theobromine, paraxanthine, theophylline,
b-hydroxyethyltheophylline (internal standard) and caffeine were
1.31, 2.52, 2.97, 3.73 and 6.45, respectively. There was no mutual
interference between these two agents or among their metabolites
with respect to the HPLC methods.
Catheterization. Right jugular vein cannulation was perfomed under sterile conditions and has been described earlier (Lau et al.,
1996). The proximal end of the silastic catheter was inserted into
jugular vein and the distal end of the catheter was threaded s.c. and
exited through a small incision in the back of the animal. The
catheter was flushed with 0.9% saline with 50 U of heparin and
sealed with fishing line when not in use.
Drug administration and blood sampling. Animals were allowed
to recover for at least 2 days from the jugular vein catheterization
before the drug administration series. Animals in group 1 (N 5 4)
initially received an i.v. dose of alprazolam (1.25 mg/kg) via the
jugular vein catheter as their first drug treatment, followed on other
days by s.c. alprazolam doses into the skin on the back of the neck in
the presence and absence of 20 mg/kg p.o. caffeine by gavage (1.25
mg/kg alprazolam; 20 mg/kg caffeine; 1.25 mg/kg alprazolam 1 20
mg/kg caffeine; 4 mg/kg alprazolam; 4 mg/kg alprazolam 1 20 mg/kg
caffeine; 7 mg/kg alprazolam; 7 mg/kg alprazolam 1 20 mg/kg caffeine). Animals then received p.o. doses of caffeine, 80, 10, 40 and 120
mg/kg. Animals in group 2 (N 5 4) were used to study PK interaction
between alprazolam and caffeine by the i.v. route. Three i.v. bolus
doses were administered in random order on different days: 1.25
mg/kg alprazolam, 10 mg/kg caffeine and their combination. Drug
doses were separated by 3 to 5 days for both groups of animals. Drugs
were given in a volume of 1 ml/kg body weight. Although two caffeine
doses (20 and 30 mg/kg) were used for the evaluation of the PD
interaction, only 20 mg/kg caffeine was used for the PK interaction
because of the limitation of catheter life. Alprazolam and caffeine
doses used for the i.v. route were chosen mainly to approximate the
respective, comparable serum concentration-time profiles of the extravascular routes of administration. This made possible comparisons between pairs of profiles despite the initial profile differences
during the absorption phase, owing to the diverse nature of the i.v.
and extravascular routes of administration. The use of 1.25 i.v.
mg/kg alprazolam dose was arbitrary as the bioavailability of s.c.
alprazolam is complete (table 1). However, the use of 10 mg/kg i.v.
caffeine dose was the one most appropriate producing a serum caffeine concentration-time profile corresponding to that of 20 mg/kg
p.o. caffeine for the time when DRL performance was evaluated.
Blood samples (100 ml) from the jugular catheter were obtained
after drug administration at 2, 5, 15, 30, 45, 60, 90, 120, 180, 240 and
360 min postinjection. To maintain the feeding regimen and also
avoid the effect of food on drug PK, especially for the oral route, drug
doses were given 6 hr before the feeding time. Thus, the daily food
supplements were given immediately after the last blood samples.
Data analyses. PK analysis was performed using SAAM II (SAAM
Institute, Seattle, WA, 1994). The data were described by an open
two-compartment model for alprazolam and fit to the following equation:
Cp5Ae2at 1 Be2bt
where, Cp is the total serum drug concentration at time t, the terms
A and B are the extrapolated zero intercepts, and a and b represent
the apparent first-order distribution and elimination rate constants,
respectively. The t1/2 for the distribution or elimination phase, and
Vc, were calculated by the following equations: t1/2 5 0.693/a or b and
Vc 5 dose/(A 1 B). For the s.c. route of alprazolam administration, an
absorption rate constant, ka, was also calculated. The PK parameters, Cl and Vss were calculated using noncompartmental methodology. The area under the serum drug concentration-time curve
(AUC0-`) and area under the first moment of the serum drug concentration-time curve (AUMC0-`) were calculated by the following
equations: AUC0-` 5 A/a 1 B/b; AUMC0-` 5 A/a^2 1 B/b^2. Total Cl
was then defined as dose/AUC0-` and Vss as dose 3 AUMC0-`/
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from the dose-response relation of alprazolam alone: the same effect
obtained with alprazolam alone can be expected at doses of alprazolam minus the x dose of alprazolam with which a fixed dose of
caffeine is equieffective. For example, observing that 10 mg/kg caffeine is equieffective with a dose of x mg/kg alprazolam, then the
expected effect of y mg/kg alprazolam in an additive curve would be
equivalent to a dose of (y–x) mg/kg alprazolam. Thus, the expected
additive curve can be constructed for all the dose levels of alprazolam
in the presence of a fixed dose of caffeine. By the same principle, the
calculated additive curve for the combined effects in a behaviortime profile also can be calculated from the DRCs at different time
periods.
The second model of the Pöch DRC method, independent action,
implies that the response to alprazolam is unaffected by the presence
of caffeine, i.e., the net effect of alprazolam is not altered by caffeine
and vice versa. It assumes that these two agents act at different
receptors. The combined effects of theoretical independent actions
are not simply the sum of the individual effects, and can be calculated for both the DRC and time course curves by setting DRL
baseline performance level at 1:
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1997
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BZ and MX: Independent Interaction
TABLE 1
Mean PK parameters (6S.D.) for alprazolam after i.v. 1.25 mg/kg administration and three s.c. doses of alprazolam in the presence
and absence of p.o. 20 mg/kg caffeine in rats (N 5 4)
Alprazolam 1 20 mg/kg Caffeine
Alprazolam
Route of administration
Dose (mg/kg)
Dose (mg)
Vc (liter/kg)
Cl (liter/hr/kg)
Vss (liter/kg)
a (min21)
t1/2a (min)
b (min21)
t1/2b (min)
AUC(0-`) (mg 3 min/ml)
Cmax (mg/ml)
1.65
6.152 6 1.094
3.847 6 0.8788
0.148
(0.03)
5.14
(0.9)
0.018
(0.003)
40.58
(6.9)
13.12
(2.4)
Tmax (min)
F%
s.c.
1.25
0.41
(0.01)
s.c.
4
1.32
(0.02)
s.c.
7
2.38
(0.06)
s.c.
1.25
0.42
(0.00)
s.c.
4
1.31
(0.01)
s.c.
7
2.36
(0.04)
0.148
(0.03)
5.14
(0.9)
0.021
(0.005)
38.04
(8.8)
0.184
(0.04)
13.04
(2.9)
0.29
(0.05)
10.0
(4.1)
98.1
(5.4)
0.148
(0.03)
5.14
(0.9)
0.017
(0.002)
43.29
(5.8)
0.243
(0.07)
44.62
(6.9)
0.78
(0.10)
12.5
(1.7)
115.3
(29.7)
0.148
(0.03)
5.14
(0.9)
0.016
(0.003)
44.90
(5.9)
0.146
(0.04)
93.95
(18.8)
1.22
(0.36)
18.8
(1.4)
128.4
(13.9)
0.148
(0.03)
5.14
(0.9)
0.027
(0.006)
29.10
(7.00)
0.194
(0.09)
10.14
(1.2)
0.27
(0.04)
8.8
(2.7)
80.2
(9.8)
0.148
(0.03)
5.14
(0.9)
0.021
(0.007)
41.72
(13.3)
0.219
(0.10)
46.24
(7.8)
0.81
(0.17)
12.5
(1.7)
113.4
(16.5)
0.148
(0.03)
5.14
(0.9)
0.013
(0.002)
56.89
(11.1)
0.228
(0.03)
89.72
(11.8)
1.31
(0.27)
13.8
(2.8)
125.1
(11.7)
AUC20-`. The values reported as the Cmax and Tmax are the actual
observed values. The F for s.c. alprazolam (1.25 mg/kg) can be calculated by the following formula:
F 5 @Di.v.(AUC0-`!s.c.] / @~AUC0-`!i.v.Ds.c.]
where, for the s.c. and i.v. routes, Ds.c. and Di.v. are the respective
doses; (AUC0-`)s.c. and (AUC0-`)i.v. are the respective AUCs.
Inasmuch as the half-life of caffeine is 3 hr (Lau et al., 1995) PK
analysis was not conducted. Statistical analyses for the comparison
of PK parameters and serum concentration-time profiles were performed by repeated measures, one-way and two-way analyses of
variance, respectively, followed by Newman-Keuls tests, where appropriate.
PK-PD Modeling: PK and DRL Performance
Data Analysis. Integration of PK and PD was based on the
relation between mean serum alprazolam concentration-time profiles for the three s.c. doses (1.25–7 mg/kg) in the presence and
absence of p.o. 20 mg/kg caffeine of group 1 in PK studies (N 5 4) and
the respective mean behavior-time profiles in PD studies (N 5 7).
PK-PD modeling was also performed by using SAAM II. The model
consists of two parts (fig. 1). The first was a classical PK model with
two or three compartments (cpts) with elimination occurring from
the central compartment to describe the PK of alprazolam by the i.v.
(cpts 1 and 2) or s.c. (cpts 1, 2 and 3) routes of administration,
respectively. The k(1, 2) and k(2, 1) were the intercompartmental
rate constants, and k(0, 1) was the elimination rate constant from
the central cpt.
Linked to the PK model is a PD model that relates the observed
concentration of alprazolam in the central PK cpt to the observed
effect. The effect-link model proposed by (Sheiner et al., 1979) was
used in this study. This model has a hypothetical effect compartment
connected to a PK model. Alprazolam was assumed to enter and
leave the effect compartment according to first-order kinetics with
rate constants, kle and keo, respectively. The central cpt of the PK
model is linked to the effect compartment by kle. The general assumption is that mass loss via kle is “negligible” (Sheiner et al.,
1979); however, to ensure no loss of mass to the effect site, a “dum-
Fig. 1. Diagrammatic representation of the integrated PK-PD model
used to describe the reinforcement rate in the 45- to 55-sec bin (effectlink model) after s.c. administration of a single dose of alprazolam. The
k(1, 2) and k(2, 1) are the inter-cpt rate constants, and k(0, 1) is the
elimination rate constant.
my” compartment was linked to the central compartment via the
rate constant -kle. The addition of this compartment did not increase
the complexity of the model, as the rate constant was fixed. Therefore, the effect cpt did not alter the serum concentration-time profile,
making an additional exponential term unnecessary in the PK
model. Under these assumptions the value of kle is unimportant, and
keo (i.e., the elimination rate constant of alprazolam from the effect
compartment) characterizes the equilibrium time between serum
concentrations and pharmacological effect. The time required to
reach 50% of the steady-state effect is the half-time of effect equilibrium and is calculated as 0.693/keo. The decrement in the mean %
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ka (min21)
i.v.
1.25
0.42
(0.00)
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Lau et al.
Vol. 281
baseline reinforcement rate produced by alprazolam in the 45- to
55-sec bin was related to the serum alprazolam concentration using
a modified sigmoid Emax model (Wald et al., 1991).
S
Effect 5 E0 1 2
CN
N
IC50
1 CN
D
Results
DRL Performance
Figure 2A–B, as an example, show the effects of alprazolam and caffeine on IRT distributions for the first 30 min of
the sessions. For baseline days and vehicle administration,
the highest response rate occurred in the 40- to 50-sec band.
Both alprazolam and caffeine decreased the reinforced, and
increased the nonreinforced response rate in a dose-related
fashion.
Figure 3A–F show an overview of DRL performance for the
3-hr session after vehicle and drug administration. Decreases
Fig. 2. Mean effects of (A) s.c. alprazolam (0 –7 mg/kg); (B) p.o. caffeine
(0 –120 mg/kg) on IRT distributions for 2 to 30 min after drug administration. All the responses were nonreinforced (,45 sec) before the first
arrow and reinforced ($45 sec) after the first arrow. Responses between the two arrows were the 45- to 55-sec bin responses.
Fig. 3. Mean (S.E.) % baseline dose-response curves of alprazolam and
caffeine for 3-hr sessions: (A) reinforcement rate in the $45- and 45- to
55-sec bins; (B) shorter-response rate; (C) total response rate; (D)
efficiency; (E) peak area and (F) peak location. % B, percent baseline.
in reinforcement rate in the 45- to 55-sec bin, and in bins
larger than 45 sec, were linear with respect to alprazolam
dose, whereas these functions for caffeine reached a plateau
at higher doses (fig. 3A). At higher doses, both alprazolam
and caffeine increased shorter IRTs (,45 sec); however, the
increases were more profound for caffeine than for alprazolam (fig. 3B). The opposing relation between the reinforced
and nonreinforced response rate after drug administration
resulted in a higher total response rate only at the 40-mg/kg
caffeine dose (fig. 3C). Consequently, efficiency for both drugs
was similar to the reinforcement-rate function across doses
(fig. 3D). For both alprazolam and caffeine, dose-response
relations for the peak area measure were similar to those in
the 45- to 55-sec bin (fig. 3E), whereas the center of the IRT
distribution peak shifted to the shorter IRTs as shown by the
peak location measure (fig. 3F).
Figure 4A shows that the mean behavioral performance
measures (reinforcement rate for both the responses .45 sec
and in the 45- to 55-sec bin, shorter response rate, total
response rate and efficiency) during baseline days were similar across the duration of the 3-hr sessions. The respective
performance measures for 1.25 mg/kg alprazolam and 20
mg/kg caffeine (one dose from each drug as examples) are
shown in Figures 4B and C, respectively. During baseline
days, the ratio of reinforcement rate in the 45- to 55-sec bin
to the total reinforcement rate was approximately 0.9 (e.g.,
0.69/0.79 5 0.87 for time point at 60 min, fig. 4A), which
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where E0 is the effect when alprazolam concentration is zero in the
presence and absence of caffeine, and IC50 is the concentration (C)
that decreased E0 to 50%. N determines the sigmoid shape of the
function and contributes to the steepness of the slope. This produces
a PD model that describes effects as a function of time. In PK-PD
modeling, the PK model is first defined by obtaining the PK parameters derived from the serum alprazolam concentration-time profiles,
and then are used as constants in the PD model, with the DRL
behavior-time profiles as input, to estimate the PD model parameters, E0 and IC50, as well as the value of keo. Assessment of the
goodness of fit of each model to experimental data was based on
correlation matrix, residual and weighted residual plots, visual
plots, and error in parameter estimation (S.D.) that is derived from
the covariance matrix provided by SAAM II.
1997
implied that 90% of the reinforced responses occurred in the
45- to 55-sec bin. For the 1.25-mg/kg dose, the ratios were
0.25 and 0.9 at 15 and 180 min, respectively (fig. 4B). The
smaller the ratio, the more IRTs occurred in the bins larger
than 55 sec. Although both alprazolam and caffeine decreased reinforcement rate, it is apparent that alprazolam
effects were short-lived, whereas they remained relatively
constant for caffeine across the session. For example, at time
point 150 min, the reinforcement rate in the 45- to 55-sec bin
was approximately at the baseline level for alprazolam (0.64
min21), whereas it remained low for caffeine (0.34 min21).
Reinforcement rate in the 45- to 55-sec bin was more sensitive to drug effects than the total reinforcement rate was,
especially during the phase when performance was returning
to baseline. It also required lower doses to reach Emax than
the total reinforcement rate measure did. Therefore, the 45to 55-sec bin was used to characterize the effects of drugs
when given alone and in combination to minimize the possibility of behavioral toxicity that might occur if higher doses
were necessary to perform the analysis. The highest efficiency occurred at the time when the shorter-response rate
was the lowest.
Inasmuch as effects of alprazolam were short-lived, DRCs
for alprazolam and caffeine in the 45- to 55-sec bin were
constructed by ALLFIT using four time periods (fig. 5A–D).
Performance attained a plateau for alprazolam for the first
two time periods, but they differed in Emax values, 13.08 and
4.9% for 2 to 30 and 31 to 60 min, respectively. The Emax
value in the second time period was used for the two later
1019
Fig. 5. Alprazolam and caffeine DRCs constructed by using median %
baseline reinforcement rate in the 45–55 s bin for four time periods: (A)
2 to 30 min; (B) 31 to 60 min; (C) 61 to 120 min; (D) 121 to 180 min.
time periods because the dose-response relation for alprazolam is unlikely to change across time after it had reached
Emax. DRCs of alprazolam shifted to the right across the four
time periods, whereas those curves remained similar for caffeine (fig. 5A–D). Thus, ED50 values for alprazolam changed
across the four time periods from 0.26, 0.5, 1.72 to 5.26 mg/kg
(i.e., 0.84, 1.62, 5.57 to 17.04 mmol/kg, respectively), whereas
for caffeine those values, 14.13, 18.04, 20.42 and 16.71 mg/kg
(i.e., 72.8, 92.9, 105.2 and 86.1 mmol/kg, respectively) remained relatively similar throughout the session. As a result,
the potency ratios of these two agents in terms of mmol/kg
changed during a session from 86 to 5. The slope values for
alprazolam and caffeine were similar, 1.82 and 1.86, respectively. For the first hour, the effect of alprazolam on DRL
performance plateaued at 4 mg/kg, but approximately linear
dose-response relations occurred for the second and third
hours, with a disappearance of effect for the lower alprazolam doses.
As described in “Methods,” for each drug combination series, s.c. saline 1 a fixed dose of p.o. caffeine (20 or 30 mg/kg)
was given not only in the beginning, but also at the end of a
series, and these points are also shown in figure 5A–D. There
were only minor variations observed for the 30-mg/kg caffeine dose in the time periods 31 to 60 and 61 to 120 min,
which implied that the effects of a fixed dose of caffeine did
not vary across a combination series. Thus, mean value of the
two treatments in the series of a given caffeine dose was used
to characterize the combined effects below.
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Fig. 4. Mean reinforcement rate-, efficiency-, total-, and shorter-response-rate-time profiles: (A) baseline; (B) 1.25 mg/kg alprazolam and
(C) 20 mg/kg caffeine. Reinforcement rate and efficiency measures on
left ordinate scales. Total respond and shorter-response rate measures
on right ordinate scales.
BZ and MX: Independent Interaction
1020
Lau et al.
to 55-sec bin for the three highest alprazolam doses (1.25–7
mg/kg) in the presence of 20 mg/kg caffeine were similar to
those occurring when alprazolam was given alone. Although,
in the presence of 30 mg/kg caffeine the combined effects
deviated from alprazolam effects after 60 min in a doserelated fashion and approached independence. However, for
the lowest alprazolam dose (0.125 mg/kg), the combined effects were closer to those of caffeine or independent effects
rather than to those of alprazolam. The mean expected additive curves are not shown for the combination series as
those curves were not separable from the independent
curves. The two drug combination series showed independent
interaction for all the time points across the 3-hr session
(figs. 7 and 8), except the time point at 180 min for 7 mg/kg
alprazolam 1 20 mg/kg caffeine; the decrement in reinforcement rate was less than the expected independent effect,
although it did not differ from the effect of alprazolam given
alone. These results demonstrate that comparing observed
combined effects to calculated expected curves is crucial for
characterizing drug interaction.
For the three higher doses of alprazolam (1.25–7 mg/kg) in
the presence and absence of caffeine (20 mg/kg p.o.), the
shorter-response rate decreased to baseline level after the
initial stimulation, but again increased in a dose-related
fashion in terms of its time to peak and the duration of the
peak (fig. 9). For example, the peak times were at 60, 90, and
150 min for 1.25, 4 and 7 mg/kg, respectively, and the peak
Fig. 6. Alprazolam DRCs in the presence
and absence of a fixed dose of caffeine
(20 or 30 mg/kg) constructed by using median % baseline reinforcement rate in the
45- to 55-sec bin for time period 31 to 60
min. (A) Theoretical expected additive and
independent curves shown as pairs for
each caffeine dose (10 –120 mg/kg); (B)
alprazolam 1 20 mg/kg caffeine and (C)
alprazolam 1 30 mg/kg caffeine. The observed values and the two expected
curves are shown for each combination
series with x2 statistics.
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Using the 31- to 60-min time period as an example, the
effects of alprazolam in the presence of two fixed doses of
caffeine (20 –30 mg/kg) in the reinforcement rate in the 45- to
55-sec bin were analyzed by the Pöch DRC method for the
combined effects (Fig. 6B–C). Both the expected independent
and additive curves for all the caffeine combinations (10, 20,
30, 40, 80 and 120 mg/kg) can be obtained simply and predicted from the DRCs of alprazolam and caffeine (fig. 5A–D)
as described in “Methods,” an advantage of the Pöch method,
and the expected curves are shown in sequence as pairs in
figure 6A. The combined effects of alprazolam in the presence
of two doses of caffeine did not differ from either the theoretical expected independent or additive curves as reflected by
the x2 statistics, although there were two observed median
values deviant from the expected (e.g., 1.25 mg/kg alprazolam 1 30 mg/kg caffeine). Thus, the combined effects were
neither synergistic nor antagonistic.
The mean performance of behavior-time profiles in the 45to 55-sec bin for caffeine, alprazolam in the presence and
absence of two fixed doses of caffeine (20 –30 mg/kg) and the
expected independent curves from 15 to 180 min, are shown
in figs. 7 and 8. The effects of vehicle administration (saline,
sodium benzoate and their combination) were close to baseline (100%) on the DRL behavior-time profiles except at the
15 min for the vehicle combinations. Each of these vehicle
treatments is shown in separate quadrants for a clear view
and to avoid repetition. Generally, the decrements in the 45-
Vol. 281
1997
BZ and MX: Independent Interaction
1021
durations progressively increased. In each case the second
peak lasted longer, but was less elevated, compared to the
first peak. Caffeine at 20 mg/kg produced a milder stimulation (150% of baseline) across the session except a 350%
increase was observed in shorter response rate at 5 min.
Thus, the pattern of effects for the shorter-response rate
differed for the two drugs. However, the presence of caffeine
did not alter the dynamics of the above two-peak phenomenon (P . .05).
Pharmacokinetics of Alprazolam, Caffeine and their
Combinations
Alprazolam PK by the s.c. route in the presence and
absence of 20 mg/kg p.o. caffeine. After i.v. administration,
alprazolam was eliminated according to a biphasic process. Alprazolam was rapidly distributed with a mean distribution t1/2a of
5.14 min, and was eliminated with a mean terminal elimination
t1/2b of 40.58 min (table 1). The Vc, Vss and clearance were 1.65
liter/kg, 3.85 liter/kg and 6.15 liter/hr/kg, respectively. Alprazolam
metabolites, the two oxidative metabolites, 4-hydroxyalprazolam
and a-hydroxyalprazolam, were not detectable.
Fig. 10A–B and table 1 show the concentration-time profiles and PK parameters of the three s.c. doses (1.25–7 mg/kg)
of alprazolam in the presence and absence of 20 mg/kg p.o.
caffeine, respectively. For the three alprazolam doses, caffeine did not alter the rate and extent of alprazolam absorption and elimination. The absorption of alprazolam was
rapid, as is evident in the values of Tmax, 10 to 20 min and the
large ka values. After reaching the peak concentrations,
there were rapid decreases in alprazolam serum concentration, followed by a slower decay, for the three alprazolam
doses (table 1). Alprazolam was short-lived with a t1/2 in the
range of 29.1 to 56.89 min for the three doses in the presence
and absence of caffeine. Furthermore, caffeine did not alter
the alprazolam AUC(0-`) values, and these were a linear
function of dose. The mean F% for alprazolam was close to
100% (80.2–128.4%).
The two metabolites, 4-hydroxyalprazolam and a-hydroxyalprazolam, were only detected in two animals after s.c. alprazo-
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Fig. 7. Mean (S.E.) % baseline reinforcement rate- (45- to 55-sec) time profiles
after 20 mg/kg caffeine alone, and alprazolam (0.125–7 mg/kg) in the presence
and absence of 20 mg/kg caffeine. Expected independent curves (S.E.) are
shown for each combination. Saline and
sodium benzoic acid are the vehicles for
alprazolam and caffeine, respectively.
*P , .05 relative to the respective independent curve.
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Lau et al.
Vol. 281
lam administration. For one animal, caffeine did not alter the
formation and elimination of the two metabolites (data not
shown). For the other animal, serum concentrations of the two
metabolites were markedly low at all the time points.
Oral caffeine PK in the presence and absence of
three s.c. doses of alprazolam (1.25–7 mg/kg). The serum
caffeine and its three DMX metabolites (theobromine, paraxanthine and theophylline) concentration-time profiles after
five doses of p.o. caffeine (10–120 mg/kg) are shown in Fig.
11A–D. For the doses of 10, 40 and 120 mg/kg caffeine, not all
the serum samples were obtained from jugular vein catheters. In two of the four animals, their jugular vein catheters
became occluded and blood could not be withdrawn after nine
blood-sampling series as described in “Materials and Methods” Thus, for these two animals, tail-tip blood samples were
used for determining the serum concentrations of caffeine
and the three DMXs. Blood samples of one animal in this
group, who had completed the blood sampling series, was
used to determine whether the values estimated from the
tail-tip samples were in accordance with those values obtained from the jugular vein samples by simultaneously collecting both samples at 5, 15, 30 and 60 min after 40 mg/kg
p.o. caffeine administration. Serum caffeine concentrations
at 5, 15, 30 and 60 min for tail-tip and jugular vein samples,
respectively, were 3.26 and 8.88 mg/ml; 13.21 and 15.91 mg/
ml; 17.8 and 19.62 mg/ml; 20.85 and 21.73 mg/ml, respectively. Serum caffeine concentration was much lower in tailtip than in the jugular vein sample at 5 min, but
progressively indifferent for the two samples with time. Similar results were found for the serum DMX concentrations.
Fig. 9. Mean (S.E.) % baseline shorter-response rate- (,45-sec-time
profiles after alprazolam (1.25–7 mg/kg) in the presence and absence of
20 mg/kg caffeine.
Thus, for these three caffeine doses (10, 40 and 120 mg/kg),
mean serum caffeine and DMX concentrations were only
calculated between 15 to 180 min.
Caffeine attained its Cmax after about 2 hr, and serum
caffeine concentrations remained close to Cmax values for the
6 hr measured after the five p.o. doses of caffeine (fig. 11A).
Serum caffeine concentrations showed a dose-related increase except for the 40- and 80-mg/kg doses. Caffeine metabolizes to the three DMXs, in equal amounts and the formation of the three DMXs continue to progress at the sixth
hour as shown in fig. 11B–D. Serum DMX concentrations
were lower for the 10-mg/kg caffeine dose, but reached plateaus for all the caffeine doses.
Figure 12 shows the serum caffeine and total DMX concentration-time profiles of 20 mg/kg caffeine in the presence and
absence of three doses of alprazolam (1.25–7 mg/kg). All
three alprazolam doses significantly decreased the serum
DMX concentrations by a two-way repeated measures analyses of variance (P , .05). However, only 4 mg/kg alprazolam
significantly decreased the serum caffeine concentrations,
although those values were markedly, but not statistically,
lower between 60 to 120 min in the presence of 1.25 and 7
mg/kg alprazolam doses. Furthermore, caffeine concentrations were noticeably lower in the presence of the two higher
alprazolam doses (4 and 7 mg/kg) between 3 to 6 hr compared
to 1.25-mg/kg dose.
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Fig. 8. Mean (S.E.) % baseline reinforcement rate- (45- to 55-sec) time
profiles after 30 mg/kg caffeine alone, and alprazolam (0.125–7 mg/kg)
in the presence and absence of 30 mg/kg caffeine. Saline, and sodium
benzoic acid are the vehicles for alprazolam and caffeine, respectively.
Expected independent curves (S.E.) are shown for each combination.
1997
PK interaction between alprazolam and caffeine by
the i.v. route. Both alprazolam and caffeine i.v. serum concentration-time profiles were not altered by concurrent administration of i.v. caffeine and alprazolam, respectively (fig.
13A–B). The PK parameters for alprazolam were not influenced by caffeine (table 2). After i.v. administration, alprazolam was eliminated according to a biphasic process and the
PK parameter values estimated from the concentration-time
profiles were similar to those for the group 1 (table 1). Caffeine PK parameters 6 alprazolam could not be determined
accurately using the data in figure 13B, as the t1/2 of caffeine
was much longer than that of alprazolam. Caffeine PK parameters listed in table 2 were obtained from a different
group of animals (N 5 4) under feeding conditions similar to
those used in this experiment (C.E. Lau, Y. Wang and F. Ma,
unpublished data). After i.v. administration, caffeine was
eliminated according to a monophasic process. Vc and Vss for
alprazolam were larger than those for caffeine. Alprazolam
clearance was markedly greater than for caffeine, 6.9 vs. 0.29
liter/hr/kg, which accounted for its shorter t1/2 compared to
caffeine (24.8 vs. 187 min). The two hydroxy metabolites of
alprazolam were not detectable by the i.v. route in the presence or absence of caffeine. Alprazolam also did not alter the
AUC(0–6 hr) values of caffeine or the three DMXs (table 2,
bottom panel).
PK-PD Modeling: PK and DRL performance
PK-PD model of serum alprazolam concentrationtime profiles in the presence and absence of caffeine
1023
Fig. 11. Mean (S.E.) serum concentration-time profiles after p.o. caffeine (10 –120 mg/kg) administration: (A) caffeine; (B) theobromine; (C)
paraxanthine and (D) theophylline.
(20 mg/kg, p.o.). Alprazolam metabolite concentrations
were either low or not detectable, and with their relative low
potency compared to the parent compound, these metabolites
were not included in the PD analysis. Alprazolam distribution and elimination characteristics were determined initially for the i.v. 1.25-mg/kg dose using the mean alprazolam
serum concentration-time profile. The bioavailability values
of the three s.c. alprazolam doses were complete using the
mean data. All the values of the intercompartmental rate
constants derived from the i.v. route describe the three s.c.
alprazolam doses 6 20 mg/kg caffeine profiles well, except
the elimination rate constant values from the central cpt
varied somewhat for the two higher alprazolam doses when
given alone, as shown in table 3. Figure 10A–B show the
mean observed and fitted serum alprazolam concentrationtime profiles of the three alprazolam doses 6 20 mg/kg caffeine using these PK parameters, respectively.
The integrated PK-PD model predicted that decreases in
reinforcement rate in the 45- to 55-sec bin occur within 5 min,
and reach maximum effect at approximately 15 to 45 min
after alprazolam (1.25–7 mg/kg) 6 20 mg/kg caffeine administration (fig. 14A–B). When alprazolam was given alone, the
DRL performance returned to baseline (101.1%, E0) in a
dose-related fashion (fig. 14A). For the combined effects, the
DRL performance did not return to baseline, but rather,
remained at the effect level produced by caffeine (65.5%, E0),
Figure 14B and table 3. The equilibration half-lives between
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Fig. 10. Mean (S.E.) serum alprazolam concentration-time profiles and
the fitted curves after PK modeling following administration: (A) alprazolam s.c. 1.25 to 7 mg/kg and (B) alprazolam s.c. 1.25 to 7 mg/kg 1
p.o 20 mg/kg caffeine.
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Lau et al.
Vol. 281
Fig. 12. Mean (S.E.) serum caffeine and total DMX concentration-time
profiles after p.o. 20 mg/kg caffeine in the presence and absence of
1.25 to 7 mg/kg alprazolam administration. *P , .05 relative to the
respective serum caffeine and DMX concentrations without alprazolam.
serum alprazolam concentration in the central and effect cpts
(t1/2keo) for alprazolam 6 20 mg/kg caffeine are 4.4 and 3.0
min, respectively. The IC50 values were similar for the two
dose regimens, 0.0201 and 0.0199 mg/ml for alprazolam alone
and alprazolam in the presence of 20 mg/kg caffeine, respectively. These results suggested that the interaction between
alprazolam and caffeine is not competitive, but independent.
Figure 14C shows that DRL performance can be predicted or
simulated for any alprazolam dose in the linear range administered alone or in combination with 20 mg/kg caffeine by
using the estimated PD parameters.
Relation between serum drug concentrations and
DRL performance. The data for the first 10 min for both
PK and PD (shorter-response and reinforcement rate in the
45- to 55-sec bin) measures were not used in these analyses
owing to the equilibration time required for serum alprazolam concentration to the effect cpts (fig. 14). Figure 15 shows
the relations between mean serum alprazolam or mean caffeine concentration (N 5 4 rats) and mean DRL performance
in the 45- to 55-sec bin (N 5 7 rats) as constructed using
ALLFIT. The 0.125- and 0.4-mg/kg serum alprazolam concentrations were obtained from simulation of the PK parameters (table 3, fig. 10A). The effects of alprazolam on DRL
performance were concentration related regardless of alprazolam doses (0.125–7 mg/kg). For example, for the 1.25mg/kg alprazolam dose, a full concentration-effect relation
(from E0 to Emax) was observed, whereas other doses exhibited only partial functions, i.e., high or low doses associated
with larger or smaller effects, respectively.
Unlike alprazolam, for caffeine no single dose possessed a
full concentration-effect relation. At the lowest dose (10 mg/
kg), acute tolerance occurred, i.e., low concentrations of caffeine produced larger effects than did the higher concentrations (open circles of the right curve in fig. 15). For the higher
caffeine doses (20–120 mg/kg), the concentration-effect relations remained approximately similar within a dose. However, construction of the caffeine concentration-effect function for 3-hr session length required all the doses (10–120
mg/kg). It was apparent that alprazolam was more potent
than caffeine in affecting the DRL performance as reflected
by their IC50 values, 0.0375 and 8.07 mg/ml, respectively.
The relation of the shorter-response rate to the serum
alprazolam concentration after alprazolam doses (1.25–7 mg/
kg) 6 p.o. 20 mg/kg caffeine are shown in fig. 16A–C. After
drug administration, the initial stimulation occurred approximately at the highest alprazolam concentration for each
dose (the right-most points), although it was less noticeable
because the first two time points, 5 and 10 min, were omitted.
The shorter-response rate increased again at 60, 90, and 150
min for 1.25, 4 and 7 mg/kg alprazolam doses, respectively,
with the respective serum alprazolam concentrations at
about 0.06, 0.1 and 0.13 mg/ml, respectively, regardless of the
presence of caffeine. For the highest dose (7 mg/kg), the
shorter-response rate remained plateaued 6 caffeine even at
the end of the session with serum alprazolam concentrations
in the range of 0.1 mg/ml (fig. 16C).
Downloaded from jpet.aspetjournals.org at ASPET Journals on May 5, 2017
Fig. 13. Mean (S.E.) serum concentration-time profiles for: (A) alprazolam (1.25 mg/kg alprazolam 6 10 mg/kg caffeine); (B) caffeine (10
mg/kg caffeine 6 1.25 mg/kg alprazolam) by the i.v. route.
1997
1025
BZ and MX: Independent Interaction
TABLE 2
Mean (6S.D.) PK parameters for alprazolam and mean (6 S.D.) AUC(0 – 6 h) values for caffeine and its three DMX metabolites after i.v.
1.25 mg/kg alprazolam 6 i.v. 10 mg/kg; mean (6S.D.) PK parameters for caffeine after i.v. 10 mg/kg caffeine dose
Vc (liter/kg)
Cl (liter/hr/kg)
Vss (liter/kg)
a (min21)
t1/2a (min)
b (min21)
t1/2b (min)
AUC(0–`) (mg 3 min/ml)
h)
Alp 1 Caff
1.18
(0.41)
6.00
(0.63)
2.23
(0.38)
0.20
(0.06)
4.21
(1.33)
0.03
(0.00)
24.83
(1.23)
12.78
(1.15)
Caffeine
0.89
(0.17)
0.29
(0.06)
0.005
(0.00)
187.06
(57.35)
2315.31
(524.30)
values for caffeine and its three DMXs in the presence and absence of alprazolam
i.v. 10 mg/kg Caffeine 1 i.v. 1.25 mg/kg alprazolam
i.v. 10 mg/kg Caffeine
AUC(0–6
hr)
(mg 3 min/ml)
Caff
Theob
Para
Theop
Caff
Theob
Para
Theop
2184.80
(162.69)
71.01
(5.84)
99.61
(14.36)
74.18
(7.83)
2249.30
(130.05)
73.64
(7.10)
103.02
(14.53)
75.52
(9.15)
TABLE 3
PK and PD parameters (S.D.) for alprazolam (1.25–7 mg/kg) in the presence and absence of p.o. 20 mg/kg caffeine; PD measure:
% baseline in the reinforcement rate in the 45 to 55 sec bin
Route Dose (mg/kg)
Alprazolam alone
k(1,2) (min21)
k(2,1) (min21)
k(0,1) (min21)
ka (min21)
Vc (liter/kg)
keo (min21)
t1/2keo min
IC50 (mg/ml)
E0 (% base-line performance)
N
i.v. 1.25
s.c. 1.25
s.c. 4
s.c. 7
0.0638 (60.008)
0.0936 (60.033)
0.0745 (60.016)
0.0638
0.0936
0.0745
0.0681(60.003)
0.0638
0.0936
0.0897
0.0681
0.0638
0.0936
0.0683
0.0681
0.0638
0.0936
0.0745
0.0691
0.0638
0.0936
0.0745
0.0691
1.30 (60.01)
Alprazolam 1 p.o. 20 mg/kg caffeine
k(1,2) (min21)
k(2,1) (min21)
k(0,1) (min21)
ka (min21)
keo (min21)
t1/2keo min
IC50 (mg/ml)
E0 (% base-line performance)
N
Discussion
This study investigated PK and PD interactions between
alprazolam and caffeine initiated by our previous research
(Lau and Wang, 1996), but extended the findings to additional routes of administration, doses, as well as two indices
of DRL performance. Two DRL performance measures, reinforced and nonreinforced response rates, not only yielded
similar conclusions with respect to drug interaction, but also
bore interesting differential relations to serum alprazolam
concentrations. Behavior-time profile is the method of choice
for studying this kind of drug action and interaction. It would
have been simpler to analyze the 3-hr session data in a
0.1567 (60.053)
4.42
0.0201 (60.009)
101.1 (626.9)
1.28 (60.19)
0.0638
0.0936
0.0745
0.0691
0.2347 (60.025)
3.0
0.0199 (60.002)
65.5 (63.46)
2.00 (60.17)
collapsed form to make inferences, but that would have omitted the dynamics of the on-going behavior and its relation to
PK. The bioavailabilities of s.c. alprazolam in the presence
and absence of caffeine were high, and were not determined
for the i.p. route in the previous study. The lack of PK
interaction between alprazolam and caffeine by the i.v. route
suggested that the effect of s.c. alprazolam on p.o. caffeine PK
was an indirect effect of caffeine absorption. PK models permit the prediction of serum alprazolam concentration for
other doses in the linear range, especially for lower doses
that yielded drug concentrations below analytical sensitivity.
Although the combined effects are not distinguishable in
terms of additivity or independence by using the Pöch DRC
Downloaded from jpet.aspetjournals.org at ASPET Journals on May 5, 2017
AUC(0 – 6
Alp
1.68
(0.26)
6.90
(0.99)
3.22
(0.32)
0.165
(0.03)
4.64
(0.76)
0.025
(0.00)
27.85
(1.76)
11.52
(1.26)
1026
Lau et al.
Vol. 281
Fig. 14. Mean % baseline reinforcement rate in the 45- to 55-sec bin
time profiles and the predicted curves after PK-PD modeling following
administration of alprazolam (1.25–7 mg/kg). (A) alprazolam alone; (B)
alprazolam 1 p.o. 20 mg/kg caffeine and (C) simulated effect-time
profiles for alprazolam doses 6 p.o. 20 mg/kg caffeine by using the
estimated PK-PD parameters.
Fig. 15. Mean % baseline reinforcement rate in the 45- to 55-sec bin
(N 5 7) vs. mean serum alprazolam or caffeine concentrations (N 5 4).
Lines are the curves fitted by ALLFIT.
method, independent interaction is suggested by PK-PD
modeling as reflected in the IC50 values (table 3).
To study drug interaction, drug effects need to be reproducible, otherwise tolerance and sensitization might be interpreted as antagonism and synergism, respectively. We
have found that within-subject variability in reinforcement
rate on DRL 45-sec was not different after two consecutive
s.c. doses of midazolam separated by 3 to 5 days (Lau et al.,
1996). The effects of alprazolam on DRL performance were
similar to those of midazolam, not only in reinforcement rate,
but also in shorter-response rate (unpublished data). Furthermore, effects of caffeine 1 saline at the beginning and
end of a combination series on reinforcement rate in the 45to 55 sec bin (fig. 5) and on shorter-response rate were approximately similar (data not shown), suggesting no tolerance or sensitization occurred as a result of the acute repeated caffeine administration. Thus, the observed combined
effects resulted from drug interaction.
The PK of s.c. alprazolam, p.o. caffeine and their combinations mainly mirrored the respective behavior-time profiles
of the reinforcement rate in the 45- to 55-sec bin in 3-hr
sessions. The onset of alprazolam action was rapid and its
duration of action short, although the effect of caffeine remained mainly constant throughout the session, except for
the 10-mg/kg dose that showed acute tolerance (fig. 15). As a
result, the potency ratio of these two drugs changed markedly during the session, which determined the expression of
the combined effects. The combined effects of alprazolam and
caffeine were not distinguishable in terms of additivity or
independence, neither were they synergistic nor antagonistic, as shown by the DRCs (fig. 6), which were in agreement
Downloaded from jpet.aspetjournals.org at ASPET Journals on May 5, 2017
Fig. 16. Mean (S.E.) % baseline shorter-response rate vs. alprazolam
serum concentrations for the three doses of alprazolam (1.25–7 mg/kg)
6 p.o. 20 mg/kg caffeine.
1997
1027
adverse side effects. With the use of PK-PD modeling, one
could incorporate both kinds of effects and link them to
alprazolam PK to predict therapeutic and adverse side effects. To our knowledge, no explicit PK-PD modeling has
been developed that attempts to describe these relations.
Our study along with the previous results (Lau and Wang,
1996) demonstrated that, to characterize the combined effects properly, it is important to compare the experimental
values to the calculated expected effects across time. The
interaction between BZ and MX reported in the literature
mainly has been characterized qualitatively rather than
quantitatively; this may partially account for the differences
found across studies. Several reports have sampled limited
portions of such general dose-combination and temporal
functions, yielding results consistent with the independent
actions expected from physiological (i.e., functional) antagonism, rather than the competitive antagonism or synergistic
actions sometimes inferred (Marrosu et al., 1985; Stirt, 1981;
Wangler and Kilpatrick, 1985). Furthermore, the use of different terms to characterize and interpret combined effects
complicates this issue. Terms such as “antagonism” and “additivity” sometimes are used to refer to mechanisms of action,
and at other times simply to indicate the direction of an
observed effect.
Both alprazolam and caffeine are rapidly absorbed from
extravascular routes (Arnaud, 1993; Lau and Wang, 1996)
and are highly lipophilic (Arnaud, 1993; Greenblatt and
Shader, 1987). There is a rapid equilibrium between drug
concentration in blood and at central sites of action for both
drugs, a factor important in the onset of drug action. The
equilibration half-lives between serum alprazolam concentration in the central and effect cpts (t1/2keo) for alprazolam 6 20 mg/kg caffeine are 4.4 and 3.0 min, respectively
(table 3). These values were similar to the value reported for
midazolam, 2.2 min (Breimer et al., 1991). No acute tolerance
in the reinforcement rate in the 45- to 55-sec bin was observed for alprazolam and caffeine, except for 10-mg/kg caffeine dose (fig. 15).
The PK of alprazolam was not altered by caffeine, whereas
the PK of caffeine was altered by alprazolam and resulted a
significantly decreased formation of its three active DMX
metabolites (fig. 12). Orally administered caffeine is absorbed from the small intestine and the stomach (Chvasta
and Cook, 1971). The acute PK interaction observed in our
study was not a metabolic one, as no interaction was observed by the i.v. route (fig. 13). Rather, alprazolam affected
factors influencing the absorption of caffeine, such as gastrointestinal motility (Fargeas et al., 1984). Therefore, this kind
of indirect interaction would not be avoided by using a BZ
that is metabolized by conjugation with glucuronic acid instead of the cytochrome P-450 enzyme system, e.g., lorazepam (Schillings et al., 1975). However, when alprazolam
and caffeine were given by the i.p. route, the decreased caffeine and DMXs AUC(0 –3 hr) observed in a previous study
might have resulted from a different mechanism (Lau and
Wang, 1996). After i.p. administration, both drugs pass from
the peritoneal cavity through intercellular gaps in the mesenteric wall and the surrounding capillaries into general
circulation. Alprazolam and caffeine might compete with
each other during the absorption phase. Nevertheless, the
decreases in serum caffeine and DMX concentrations accounted for the diminished action observed for the combined
Downloaded from jpet.aspetjournals.org at ASPET Journals on May 5, 2017
with the findings reported previously (Lau and Wang, 1996).
Similar results were obtained for most of the behavior-time
profiles (figs. 7 and 8), except on one occasion diminished
effects were observed only in the latter part of the session (at
time point 180 min for 7 mg/kg alprazolam 1 20 mg/kg
caffeine), which could be attributable to the lower serum
caffeine and DMX concentrations produced by alprazolam
(fig. 12). This was not surprising, for as the potency ratio of
alprazolam decreased across the session, caffeine and its
active metabolites became more dominate in the expression
of the combined effects. These results suggest that the interaction between alprazolam and caffeine occurs by PK, rather
than by a receptor mechanism. The conclusion is warranted
by the PK results and the comparison of the observed combined effects to the theoretical expected values, crucial operations for the characterization of combined effects.
The reason for using the reinforcement rate in the 45- to
55-sec bin instead of total reinforcement rate as an evaluation of DRL performance is evident in figure 4. Total responses are comprised of both the reinforced and nonreinforced responses, and closely parallel the shorter-response
rate (,45 sec) for both alprazolam and caffeine (fig. 4). Thus,
the shorter-response, rather than the total response rate was
chosen as the second measure to characterize the combined
effects. In the previous study, the effects of alprazolam 6
caffeine on shorter-response rate were not explored (Lau and
Wang, 1996). The shorter-response rate increased for the
higher doses after alprazolam and caffeine administration
(fig. 3), but differed in pattern for the two drugs. For 20
mg/kg caffeine, the shorter-response rate remained at baseline level after the initial stimulation (fig. 9). Conversely for
alprazolam, the shorter-response rate decreased to baseline
level after the initial stimulation, but increased again in a
dose-related fashion in terms of its time to peak and duration
of the peak (fig. 9). It is interesting that for the combination
series (1.25–7 mg/kg alprazolam 1 20 mg/kg caffeine), caffeine did not alter the dynamics of the shorter-response rate,
evidence of independence (figs. 9 and 16). Similar results also
were found for the combination series of 1.25 to 7 mg/kg
alprazolam 1 30 mg/kg caffeine (data not shown). These
results further demonstrated that alprazolam, but not caffeine, is more potent in determining the pattern of the shorter-response rate in the combined effects.
Alprazolam is the most widely prescribed BZ, and is used
as an anxiolytic, antipanic and antidepressant agent (Dawson et al., 1984; Fawcett and Kravitz 1982), but adverse side
effects of BZs have been increasingly recognized in recent
years clinically, e.g., early-morning insomnia and daytime
anxiety, tension or panic (Vgontzas et al., 1995). The occurrence of these two kinds of effects may depend upon the levels
of BZ concentration in the body. For example, hypnotic effects may be associated with higher, whereas early-morning
insomnia with the approach to lower BZ concentrations. The
second peak of the shorter-response rate for alprazolam was
related to the lower range of serum alprazolam concentrations (fig. 16). Inasmuch as alprazolam is shorter lived (t1/2 5
25–57 min) in rats than in humans (t1/2 6 –16 h) (Greenblatt
et al., 1983; Smith et al., 1984), the concentration-dependent
increases constituting the second peak in the shorter-response rate, and decreases in the reinforcement rate in the
45- to 55-sec bin, suggests this as the possible mechanism of
the two kinds of effects observed in humans, therapeutic and
BZ and MX: Independent Interaction
1028
Lau et al.
ject variability might reflect the diminished effects observed
only at 180 min for 7 mg/kg alprazolam 1 20 mg/kg caffeine
rather than for the other alprazolam dose combinations (1.25–4
mg/kg). However, if caffeine had altered alprazolam PK, then
the combined effects would have yielded a totally different perspective than the present one.
In summary, the present approach can be applied to any
drug-drug interaction study, and use other behavioral paradigms. In humans, serum monitoring is often done to maximize treatment effectiveness, but these data can function
only as an uncertain guide for animal behavioral research.
Alprazolam is a drug with a markedly different half-life in
rats and humans (Greenblatt and Wright, 1993; Jin and Lau,
1994; Owens et al., 1991). Similar species differences in halflives have been found for other BZs: diazepam (Hironaka et
al., 1984; Igari et al. 1982; Sethy et al., 1987) and flurazepam
(Lau et al., 1987). Without assessing the potency ratio of the
two agents across a session, comparing the combined effects
to the expected calculated effects and acquiring the parallel
PK data, our study would have been difficult to interpret.
Acknowledgments
The authors acknowledge the dedicated assistance of Ms. Fang Ma for
the catheterization of the jugular vein and HPLC analyses. We are grateful
to Dr. Anne Heatherington of Center for Bioengineering, University of
Washington, Seattle, WA, for her helpful suggestions in PK-PD modeling
and also thank Dr. B. E. Williams of the Upjohn Co., Kalamazoo, MI, for a
generous supply of alprazolam and its two metabolites.
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BZ and MX: Independent Interaction