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Int J Pharm Biomed Res 2013, 4(1), 05-09

International Journal of
PHARMACEUTICAL
AND BIOMEDICAL
RESEARCH
ISSN No: 0976-0350

Research article

Pharmacokinetic study of phenytoin-caffeine combination

Widyati1, Zullies Ikawati2, Lukman Hakim2
1
2

Department of Pharmacy, Dr. Ramelan Navy Hospital, Surabaya, Indonesia

Faculty of Pharmacy, University of Gajah Mada, Yogyakarta, Indonesia

Received: 14 Dec 2012 / Revised: 25 Dec 2012 / Accepted: 30 Dec 2012 / Online publication: 19 Jan 2013

ABSTRACT
Background: Caffeine has been used as seizure drugs in Indonesian community for ages but lack of scientific evidence
supported. To probe anticonvulsant effect of caffeine in human, it has to be tested in addition to established anticonvulsant.
Aims: To investigate the pharmacokinetic profile of combination of phenytoin-caffeine, tolerated optimal dosing, side effects,
and interactions between phenytoin and caffeine. Method: Subjects were 22 adults who received phenytoin-caffeine capsules
with two different doses. Day 1 to 7 received a combination of phenytoin 5mg/kg + caffeine 2.5mg/kg. Day 8 to13 received
5mg/kg phenytoin only. Day 14 received a regimen of phenytoin 5mg/kg + caffeine 5mg/kg. Blood sampling was taken on
day 7 and 13 at 0, 2, 4, 8, 12, 24h after drug administration. Levels of phenytoin and caffeine were determined by HPLC
using a combination of methanol and phosphoric acid as eluent. Results: Caffeine addition to phenytoin result no significant
interaction in pharmacokinetics. Pharmacokinetics profile of phenytoin in phenytoin-caffeine as the following: (Clav) 0.2571
ml/min; volume of distribution (Vd) 0.644L/kg; elimination half-life (t) 34.8h; elimination rate constant (Ke) 0.018L/h.
Pharmacologic effects attributable to phenytoin were not augmented by caffeine addition. Study was ceased at day 14 due to
high incidence of dyspepsia when caffeine dose was increased to 5mg/kg. Conclusion: There was no pharmacokinetic
interaction between phenytoin with caffeine. The combination dose of phenytoin-caffeine that can be tolerated was phenytoin
5mg/kg + caffeine 2.5mg/kg. Combination of phenytoin-caffeine appeared safely as phenytoin only.
Key words: Drug interactions, Anticonvulsant, Michaelis-Menten, Drowsiness, Nausea

1. INTRODUCTION
Drug treatment of epilepsy has made remarkable strides,
with the introduction of 11 new antiepileptic drugs (AEDs)
since 1978: valproate, vigabatrin, tiagabine, lamotrigine,
oxcarbazepine,
felbamate,
topiramate,
gabapentin,
levetiracetam, zonisamide, and pregabalin. Improvement in
terms of clinical outcome, however, has fallen short of
expectations, with up to one third of patients continuing to
experience seizures or unacceptable medication-related side
effects in spite of efforts to identify optimal treatment
regimes with one or more drugs [1,2]. Failure of a single
agent in controlling seizures, requiring the addition of

*Corresponding Author. Tel: +62 81331383111 Fax:

Email: widyatiw@yahoo.com

2013 PharmSciDirect Publications. All rights reserved.

another agent or agents called add-on. It becomes

increasingly burdensome both in terms of patient compliance
and drug costs.
In connection with the seizure, coffee has become a myth
in Indonesian society as a drug for seizures for ages.
Empirically, caffeine has been prescribed in combination
with phenobarbital for years. Although, the aim of caffeine
addition to phenobarbital is to act as a stimulant which
antagonize drowsiness from phenobarbital, but this
combination generates a good seizure control. At present
time, caffeine is used as a CNS stimulant, smooth muscle
relaxants, cardiac muscle stimulant.
Some pre-clinical tests of caffeine in rats stimulated with
artificial seizures (electrical or chemical stimulation) result in
a controversy [3]. However the molecular actions of caffeine
result an increase of adenosine. Adenosine has been known to
have anticonvulsant effects due to blocking A2 and A3
receptor [4]. Adenosine receptor blockage resulted in the
stabilization of GABA-A receptors [5]. To be approved as the

Widyati et al, Int J Pharm Biomed Res 2013, 4(1), 05-09

2. MATERIALS AND METHODS

were separated, evaporated to dryness, reconstituted with

150L of 40%MeOH, and transferred to HPLC auto
sampling vial. An aliquot of 50 L was injected onto the
HPLC system. Calibration curves reporting peak area of
Phenytoin and caffeine /IS ratio as a function of each drug
concentrations were established in the range of
0.15-50g/mL, in the presence of 12.5g/mL of Tadalafil as
internal standard. Calibration curves were linear (r2>0.99) for
both caffeine and phenytoin and intercepts did not differ
significantly from zero. The intraday and interday coefficient
of variation (CVs) did not exceed 9%.

2.1. Patients

2.4. Pharmacokinetic studies

The research was conducted at Navy hospital in SurabayaIndonesia. The before and after study was carried out in
healthy subjects, comprised 22 healthy adults. All subjects
were recruited from Navy Hospital (hospital staff, resident
and post-graduate student who were practicing at the
hospital). Exclusion criteria included alcohol and coffee
consumption during study, severe adverse drug reaction and
poor compliance. An informed consent was signed by each
participant and they were personally interviewed for
information on drug effects, and compliance. Ethical
approval for the studies was granted by the research ethics
committees of Navy Hospital in Surabaya (No.
11/EC/KERS/2010)

The subjects were assigned to receive three consecutive

dosage regimens and blood sampling as follows:
Day 1-7: Phenytoin 5mg/kg + Caffeine 2.5mg/kg
Day 7: Blood sampling at 0, 2, 4, 8, 12, 24h after last dose
Day 8-13: Phenytoin 5mg/kg
Day 13: Blood sampling at 0, 2, 4, 8, 12, 24h after last dose
Day 14: Phenytoin 5mg/kg + Caffeine 5mg/kg
Blood samples were collected from the subjects on day 7
and 13 at different time points (0, 2, 4, 8, 12, 24h after last
dose). Serum was harvested from the above blood samples.
The serum samples (0.4mL) were then spiked with Tadalafil
(internal standard) to achieve serum concentration of
12.5g/mL. The solution was then extracted using a vortex
mixer. The organic extracts were separated, evaporated to
dryness, reconstituted with 150L of 40%MeOH, and
transferred to HPLC auto sampling vial. An aliquot of 50L
was injected onto the HPLC system.

first choice anticonvulsant, a new drug must have been use as

an add-on therapy for years with demonstrated excellent
effectiveness. Therefore, in this study caffeine will be tested
only as an add-on agent to first-line agents. Phenytoin was
selected as a first-line agent to study due to safe and did not
have Steven Johnson Syndrome as adverse reaction as in the
case of carbamazepin. Since the study had not been done in
humans, this study will concentrated in pharmacokinetic and
pharmacodynamic studies in healthy subjects.

2.2. Chromatographic apparatus and conditions

Serum concentration of phenytoin and caffeine were
determined by HPLC (Waters Associates, Milford, MA)
consisted of a solvent delivery system, auto sampler, and
ultraviolet detector. Phenytoin, caffeine and Tadalafil
(internal standard) were resolved on a Symmetry Waters
ODS analytical column (75mm 4.6mm i.d., 3.5m) in the
reversed-phase partition chromatographic condition. The
mobile phase composed of MeOH and H3PO4 solution (0.5%,
pH 3.0 0.05 adjusted with 85% H3PO4) in the ratio 40:60
v/v. The flow rate was 0.3mL/min and the analytes and
internal standard monitored at 230nm.
2.3. Preparation of standard curve
Stock standard solutions of Phenytoin, caffeine and
Tadalafil (internal standard) were prepared in 40% methanol,
protected from light and stored at 4C until used. Working
standard solutions were freshly obtained by diluting the stock
standard solutions with 40% methanol, during the analysis
day.
Aliquots of 0.4mL blank serum were spiked with the
working solutions of Phenytoin, caffeine and Tadalafil
(internal standard) to achieve serum concentration of 0.15-50
g/mL for Phenytoin and caffeine, each containing
12.5g/mL of Tadalafil (internal standard). The solution was
then extracted using a vortex mixer. The organic extracts

2.5. Analysis of pharmacokinetic data

The slope (beta) of the terminal log-linear phase of each
caffeine and phenytoin serum concentration vs. time curve
were determined by linear regression analysis, and used to
calculate elimination constant, the apparent volume of
distribution. Area under the curve (AUC) from zero until the
last detectable concentration was determined by the linear
trapezoidal method and extrapolated to infinity. Other
pharmacokinetic parameters were determined by calculation
using the following formulas [6].

Cav=
Css =

Vd =

FD o

[ AUC] 0 K
Do

Cl av = [ AUC]
0

Widyati et al, Int J Pharm Biomed Res 2013, 4(1), 05-09

2.6. Outcome measures

Table 1
Demographic profile

Outcome measures were serum level of phenytoin and

caffeine; pharmacological effects encountered during 14 days
of study.

Characteristics
Number of subjects
Age
Gender
Weight

Range
22
17 46 tahun
16 Male; 6 Female
41-92 kg

MeanSD
29.58.34
63.04 12.67

2.7. Statistical analysis

We carried out General Linear Model-Repeated Measures
test to compare the serum level of phenytoin before and after
caffeine administration. The serum levels then be calculated
using special formulas to generates pharmacokinetics profiles
such as K, volume of distribution, drug clearance, half life.
The pharmacological effects and their frequency were
described and determined. The difference in effects between
pre and post caffeine addition was assessed using Chi Square
test.
3. RESULTS
A total of 22 healthy adults included in this study
comprised of 6 women and 16 men, age 17-46 years, and
weight ranged from 41-92kg. Basic characteristic were
presented in Table 1. Serum phenytoin concentration before
and after caffeine deletion were not different. Although there
was increase in mean serum phenytoin after caffeine addition
consistent in every sampling time, however it was not
statistically significant (F: 0.907, P: 0.347, CI 95%). The
differences are visualized in Fig.1.

Mean phenytoin serum conc (g/mL)

14.0

Table 2
Mean serum concentrations of phenytoin and caffeine
Sampling time after Phenytoin only
the last dose (h)
Phenytoin level
(g/mL)
(Mean SD)
0
4.32 16.12
(10.39 3.36)
2
4.27 22.95
(10.58 5.36)
4
5.32 24.85
(10.52 5.03)
8
6.24 17.59
(10.65 3.38)
12
4.49 20.46
(9.24 3.89)
24
4.55 14.80
(9.84 4.05)

Phenytoin-caffeine combination
Phenytoin level
Caffeine level
(g/mL)
(g/mL)
(Mean SD)
(Mean SD)
3.89 20.48
-0.06 2.48
(10.64 3.83)
(0.59 0.69)
5.04 16.38
0.36 3.26
(10.82 3.74)
(1.12 0.77)
5.87 18.97
0.43 4.28
(12.55 4.07)
(1.20 0.99)
5.57 18.55
0.10 1.94
(11.19 3.06)
(0.58 0.53)
4.31 17.45
-0.03- 1.11
(9.32 3.45)
(0.32 0.34)
3.14 18.56
0.05 0.42
(8.90 3.58)
(0.14 0.09)

Table 3
Pharmacokinetic profile of phenytoins
Parameters
Ke (1/h)
Vd (L/kg)
Cl av (L/h)
t (h) at dose 5mg/kg
Cssmax
Cssmin

Phenytoin only
Phenytoin
0.0122
0.644
0.495
56.8
13.76
8.60

Phenytoin-caffeine combination
Phenytoin
Caffeine
0.018
0.10
0.58
0.92
0.646
1.56
34.8
6.81
14.01
1.35
8.73
1.235

12.0
10.0
8.0
6.0
4.0

Phenytoin
Phenytoin-Caffeine

2.0
0.0
0

12

24

Time (h)
Fig.1. Mean phenytoin serum concentration before and after caffeine
coadministration

followed Michaelis-Menten or saturable kinetics. Other

saturable kinetics parameters derived from previous study in
Indonesian people was Vmax 7.461.66 mg/kg/day [7].
Volume of distribution was unaffected by saturable
metabolism and was still related to clearance and half-life
using the same equation as for linear pharmacokinetics.
Clearance and half-life in non-linear pharmacokinetics were
dose or concentration-dependent, therefore clearance was
determined as mean body clearance. Mean while elimination
half-life was dependent on the Michaelis-Menten parameters
and concentration as described in the following formula.
Clav=

Phenytoin serum levels average in Table 2 demonstrated a

non linearity. Saturable kinetics also proved by result of Css
calculation which was above Km value for Indonesian
(2.351.52mg/L) [7]. The Css calculation assumed as either
linear or non-linear kinetics resulted 20.32mg/L and
9.17mg/L, consecutively. Therefore, phenytoin elimination

t1/2 =

=
.

(Km + Cp)

Pharmacokinetic calculation results were presented in

Table 3. Coadministration of phenytoin with caffeine
produced no pharmacokinetic parameter difference in

Widyati et al, Int J Pharm Biomed Res 2013, 4(1), 05-09

phenytoin, although parameter of both phenytoins correlated

better.
Pharmacological effect observation results were reported
in Table 4. The addition of caffeine reduced the level of
drowsiness, although the number of subjects who complain
sleepiness are almost similar. Interestingly, no subjects had
skin rash when they were on phenytoin-caffein, but
hypersensitivity sign from phenytoin were appeared in 3
subjects while they were on phenytoin only. The number of
hypersensitive subject was only 1 when they have phenytoincaffeine 5mg. Study was stopped after 7 subjects complain of
having gastric upset (dyspepsia) at day 1 on phenytoincaffeine 5mg/kg. From this fact, we concluded that only
phenytoin-caffeine 2.5mg/kg which can be tolerated.

Table 4
Pharmacologic effects
No
I

Drug regimen
Phenytoin 5mg +
Caffeine 2.5mg

Duration
7 days

II

Phenytoin 5mg

5 days

III

Phenytoin 5 mg +
Caffeine 5 mg

1 day

Pharmacological effect
Dizziness
Drowsiness
Dyspepsia
Nausea
Appetite increase
Hypersensitive
Dizziness
Drowsiness
Dyspepsia
Nausea
Appetite increase
Hypersensitive
Dizziness
Drowsiness
Dyspepsia
Nausea
Appetite increase
Hypersensitive

Frequency
9 (41%)
14 (64%)
2 (9%)
1(5%)
9(41%)
9 (41%)
15 (68%)
1 (5%)
0
8(36%)
3(14%)
0
1 (5%)
7(35%)
0
6(30%)
1(5%)

4. DISCUSSION
Coadministration of caffeine to phenytoin did not alter the
pharmacokinetic parameters of phenytoin. This mean
caffeine would not interact with phenytoin in
pharmacokinetics, maybe in pharmacodynamics, but this
need to be proved with other study. Caffeine addition to
phenytoin prolonged elimination half life and increased
volume of distribution of caffeine. These finding were
different to previous report which tested caffeine as a probe
to assess activity of hepatic cytochrom monooxygenase
system. It was found that phenytoin increased clearance of
caffeine from 1.5 to 3.6mL min/kg and reduced its half life
from 4.8 to 2.4h [8]. However in this study caffeine clearance
was not changed but elimination half life was increased to
6.8h. Those differences might be resulted from competition
in using CYP1A2 both for metabolizing caffeine and
catalyzing phenytoin to hydroxyphenytoin, then to
cathecol [9]. CYP 1A2 known as major substrates for

caffeine metabolism, but caffeine also acts as strong

inhibitors for the same isoenzyme [10]. As shown in Table 3,
caffeine metabolism was slow and resulted longer half life
compare to standar text and previous study, but phenytoin
was much faster. Another study combining caffeine with
fluvoxamine resulted in significant increase of caffeine serum
level [11]. This finding could not be compared to what we
found in our study, because we did not examine caffeine
only. In other studies, caffeine was added to lamotrigine,
oxcarbazepine, and tiagabine in mice and did not reduce anticonvulsant effect [3].
Phenytoin pharmacokinetics after caffeine deletion was
different to any standard text. One of the reasons was
investigations of the pharmacokinetics of phenytoin in the
standard text are largely represented by acute rather than
steady-state studies, which often use low doses that more
likely are associated with a shorter half-life. In addition, there
are few studies of patients in the oldest age category (i.e.,
older than 84 years), when drug metabolism may change
[12]. Moreover, pharmacogenetic influences on drug
metabolism are now widely appreciated, with polymorphisms
in the gene for CYP 2C9 and CYP 2C19 responsible for the
most interindividual variability in the handling of phenytoin
[9].
Dose of phenytoin 5mg/kg with or without caffeine
addition generated steady state concentration below
therapeutic range. This was caused by lower value of Km and
Vmax compare to standard text. These finding should attract
clinician attention to dose phenytoin higher than 5mg/kg in
order to achieve therapeutic range.
The pharmacological effect observed before and after
caffeine addition was reported no significant difference.
Therefore the addition of caffeine into phenytoin appeared
safely as phenytoin only. Although some subjects reported
less dizziness and drowsiness with phenytoin-caffeine but
still be counted as having both effects, because the number
presented in this study reflected frequency only not the level.
The caffeine dose tolerated in this study was 2.5mg/kg,
which was lower than other studies [11, 13-14] and might not
have been sufficient to produce adverse effects [15].
5. CONCLUSIONS
There was no pharmacokinetic interaction between
phenytoin with caffeine. The combination dose of phenytoincaffeine that can be tolerated was phenytoin 5mg/kg +
caffeine 2.5mg/kg. Combination of phenytoin-caffeine
appeared safely as phenytoin only.
ACKNOWLEDGMENT
We thankfully acknowledge the assistance from Centre
for Drug Research, Faculty of Pharmacy, University of
Surabaya, for drug level assay. The authors also wish to
acknowledge, Higher Commission of Education and Cultural
Ministry, for their research funding.

Widyati et al, Int J Pharm Biomed Res 2013, 4(1), 05-09

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