J. Pineal Res.

2006; 41:337–343  2006 The Authors

Journal compilation  2006 Blackwell Munksgaard
Doi:10.1111/j.1600-079X.2006.00372.x
Journal of Pineal Research

Preclinical evaluation of pharmacokinetics and safety of melatonin

in propylene glycol for intravenous administration

Abstract: Melatonin is a highly effective treatment in different animal models Raymond Tak Fai Cheung1,
of excitotoxicity or ischemia/reperfusion injury. Due to a lack of George L. Tipoe2, Sidney Tam3,
patentability, commercial sponsors are not interested in funding clinical Edmond Shiu Kwan Ma4, Liang
evaluations of melatonin. Investigators may initiate small-scale clinical Yu Zou1 and Pui Shan Chan1
evaluation, and intravenous (i.v.) administration is appropriate in acute 1
Division of Neurology, University Department
stroke patients. Institutional Review Boards may require proper preclinical of Medicine, Faculty of Medicine, The
University of Hong Kong; 2Department of
evaluation of the preparation. In this pharmacokinetic and safety study, Anatomy, Faculty of Medicine, The University
melatonin in propylene glycol was evaluated in adult male Sprague–Dawley of Hong Kong; 3Division of Clinical
rats. Following a single i.v. injection at 5 or 15 mg/kg, plasma concentrations Biochemistry, Queen Mary Hospital, Pokfulam;
4
Department of Pathology, Hong Kong
of melatonin increased to 39 and 199 million pg/mL at 2 min and 128 000 Sanatorium & Hospital, Hong Kong
and 772 000 pg/mL at 120 min. Within 60 min of injection, the blood
pressure, heart rate and body temperature remained unaffected. Melatonin at
5 mg/kg did not influence the complete blood counts at 60 min, but
melatonin at 15 mg/kg had some effects on the differential white cell and
platelet counts. Melatonin at 5 or 15 mg/kg slightly elevated some liver
Key words: biochemistry, hematology,
enzymes at 60 min of injection, and melatonin at higher dose also elevated histology, melatonin, pharmacokinetics, rats
plasma creatinine and lactate dehydrogenase levels. At 24 hr after
Address reprint requests to Raymond T.F.
completion of six daily injections of melatonin, there was a 5.5% reduction in Cheung, Division of Neurology, University
body weight. Gross postmortem examination and histological examination Department of Medicine, Queen Mary Hospi-
tal, The University of Hong Kong, Pokfulam,
of the brain, kidney, liver and spleen did not reveal any evidence of toxicity.
Hong Kong.
In conclusion, melatonin in propylene glycol markedly elevates plasma levels E-mail: rtcheung@hkucc.hku.hk
of melatonin with no serious toxicity. This preparation should be further Received May 8, 2006;
evaluated in human patients. accepted June 28, 2006.

artery occlusion and Rose Bengal photothrombotic stroke

Introduction
Stroke per se accounts for 10% of the world’s deaths.
Annually, there are 5.7 million stroke deaths [1]. In
addition to being the second or third leading cause of
death, stroke is a major cause of disability, imposing a great
demand for medical and welfare services. For example,
stroke-related medical costs and disability in 2005 amoun-
ted to $57 billion in United States alone [2]. The global
burden of stroke will increase further with aging of the
world’s population [3]. Effective prevention and acute
therapies are both important. At present, acute therapies
for ischemic stroke are confined to intravenous (i.v.)
thrombolysis commenced within 3 hr of onset and intra-
arterial thrombolysis initiated within 6 hr of onset as well
as neuroprotection using a nitrone-based free radical
scavenger, NXY059, commenced within 6 hr of stroke
onset [4–7].
Melatonin (N-acetyl-5-methoxy-tryptamine) is an indole-
amine product secreted by the pineal gland. It is also found
in unicellular organisms and plants. Melatonin has no
serious toxicity even at high concentrations [8]. There is
strong evidence from the literature that melatonin protects
against experimental cerebral ischemic and reperfusion
damage in rats and mice, leading to reduced infarct size and
improved functional outcome in both middle cerebral
models [8–12]. Melatonin diffuses through biological mem-
branes and distributes in subcellular compartments to exert
its multiple beneficial actions via effective scavenging of
reactive oxygen and nitrogen species, suppression of nitric
oxide synthase activity, inhibition of nuclear factor kappa B
activity, anti-inflammation, anti-apoptotic effects, amelior-
ation of mitochondrial dysfunction, induction of antioxi-
dative enzymes, and stimulation of neuroprotective
signaling pathways [9–11, 13, 14].
Development of a new drug takes several years and
requires hundreds of millions of dollars. Despite being a
safe compound with a great potential as an effective
neuroprotectant against stroke, industrial sponsors are
not interested in funding clinical evaluations of melatonin,
which are required by regulatory authorities such as the
Food and Drug Administration. This is due to a lack of
patentability of melatonin to protect the sponsor’s com-
mercial interest. Small-scale clinical evaluation initiated by
interested academic or clinical scientists is a possibility, and
proper preclinical evaluation of the melatonin preparation
is required by the Institutional Review Board. It is
appropriate to use an i.v. preparation of melatonin to
bypass gastrointestinal absorption and hepatic clearance in
unstable patients with acute stroke. The present study was
conducted in adult male Sprague–Dawley rats to evaluate

337
Cheung et al.
the pharmacokinetics and safety of an i.v. preparation of
melatonin in propylene glycol.
Materials and methods
Animals
All experiments were conducted according to the institu-
tional guidelines with the protocol approved by the
Committee on the Use of Live Animals in Teaching and
Research (The University of Hong Kong). Normally fed
adult male Sprague–Dawley rats were obtained from the
Laboratory Animal Unit (The University of Hong Kong),
where they were housed under diurnal lighting conditions
(12 hr light beginning at 07:00 hr). Following transfer to
our laboratory, which is located near the Unit, they were
maintained under diurnal lighting conditions for about
4 days before experimentation (12 hr light beginning at
06:00 hr).
Melatonin preparation
Melatonin (Sigma Chemical, St Louis, MO, USA) was
dissolved in 10% propylene glycol to achieve a final
concentration of 7.5 mg/mL containing 0.405% sodium
chloride in purified water. The preparation was kept in
brown bottles, and each bottle contained 20 mL. These
bottles were stored at 20–25C and protected from light.
Pharmacokinetic substudy
Sixteen rats were used. The rat was anesthetized in the
daytime with an intraperitoneal (i.p.) injection of sodium
pentobarbital (Rhone Merieux Company, Pinkenba,
Queensland, Australia) at 60 mg/kg. Additional doses at
20 mg/kg were administered as needed to ensure stable
anesthesia, but they were rarely given. Rectal temperature
(RT) was maintained between 36.5 and 37.5C throughout
anesthesia by using a rectal thermostat probe and a
thermostatically regulated heating pad placed underneath
the rat (FHC, Bowdoinham, ME, USA). The right femoral
vein was cannulated using a PE-50 polyethylene tubing for
injection of melatonin and blood collection at 0.5 mL each
time. Immediately after collection of the first blood
specimen, melatonin at 5 or 15 mg/kg was injected as an
i.v. bolus. To maintain the blood volume and prevent
clotting of the i.v. cannula, 0.5 mL of heparinized saline
was injected after each blood collection. Following the i.v.
bolus of melatonin, 0.5 mL of blood was collected at 2, 5,
10, 15, 30, 45, 60, 90 and 120 min and centrifuged at 500 g
for 20 min at 4C to obtain the plasma from the superna-
tant. The plasma was kept inside Eppendorf tubes and
stored at )70C until analysis. The rat was killed by
decapitation after collection of the last blood specimen.
Melatonin concentrations in the plasma specimens were
measured using an enzyme-linked immunosorbent assay kit
(melatonin ELISA kit RE54021; DRG Instruments GmbH,
Marburg, Germany) after extraction with chloroform
according to the manufacturer’s instructions [15]. As the
standards range from 3 to 350 pg/mL, appropriate dilu-
tions were made to the plasma specimens so that the
338
obtained optical density values would fall within the linear
range of the calibration curve (from 10 to 100 pg/mL).
Safety substudy
Sixteen rats were used. The rat was anesthetized in the
daytime with an i.p. injection of sodium pentobarbital at
60 mg/kg. The right femoral artery was cannulated using a
PE-50 polyethylene tubing to permit monitoring of arterial
blood pressure (ABP) and heart rate (HR) (Powerlab/16
Data Acquisition System; AD Instruments Pty. Ltd,
Mountain View, CA, USA) for about 60 min. In addition,
RT was monitored for the same period using a rectal
thermostat probe. The right femoral vein was cannulated
using a PE-50 polyethylene tubing for injection of the first
dose of melatonin and blood collection at 0.7 mL each
time. Immediately after collection of the first blood
specimen, melatonin at 5 or 15 mg/kg was injected as an
i.v. bolus. To maintain the blood volume and prevent
clotting of the i.v. cannula, 0.7 mL of heparinized saline
was injected after each blood collection. At 60 min after the
i.v. bolus of melatonin, 0.7 mL of blood was collected.
From each of the 0.7 mL blood specimens, 0.1 mL of blood
was kept in pediatric hematology specimen bottles coated
with ethylenediaminetetraacetic acid (EDTA). The remain-
ing blood specimens were centrifuged at 500 g for 20 min at
4C to obtain the plasma from the supernatant. The plasma
was kept inside Eppendorf tubes and stored at )70C until
biochemical analysis. After the second blood specimen was
collected, the right femoral arterial and venous cannulae
were removed, and the right femoral artery and vein were
tied with surgical sutures. The rat was observed to recover
from the anesthesia before returning to the cage with free
access to food and water. Right leg weakness was absent
despite ligation of the right femoral artery and vein.
Melatonin injections were repeated daily five more times
via the tail vein. Distress or pain was not observed in the
rat. At 24 hr after the sixth injection, the rat was weighed
again and then killed by decapitation.
A gross postmortem examination was performed. Fresh
tissue blocks from the brain, liver, spleen and kidney were
cut. Tissue blocks were immediately fixed in 10% phos-
phate-buffered formaldehyde for at least 72 hr. After
washing with water, tissue blocks were dehydrated in
increasing grades of ethanol, cleared in chloroform and
embedded in paraffin. Paraffin sections of 5 lm thickness
were rehydrated and stained with hematoxylin and eosin (H
& E). H & E stained sections were observed under light
microscopy.
Hemoglobin (HB), white cell counts (WBC) and platelet
counts (PLT) were performed in the whole blood stored in
EDTA-coated pediatric specimens bottles according to
standard laboratory procedures (Advia 120 Automated
Blood Cell Analyzer; Bayer Diagnostics, Tarrytown, NY,
USA). A Wright-stained peripheral blood film was pre-
pared and 200 white cells were manually examined to give
the polymorphonuclear cell counts (PMN), lymphocyte
counts (LYM), mononuclear cell counts (MN), eosinophil
counts (EOS) and basophil counts (BAS). For determin-
ation of reticulocyte counts (RET), 1% brilliant cresyl blue
was added to the peripheral blood at a volume ratio of 1:2
 Pharmacokinetics and safety of intravenous melatonin in rats

before incubation at 37C for 30 min. After this supravital 5 mg/kg

staining, a blood film was prepared and 1000 red cells were 1 X 108

concentration (pg/mL)
15 mg/kg
manually examined to give the RET in percentage.

Common biochemical tests were performed in the stored
plasma specimens. Glucose (GLU) was measured by a
hexokinase method automated on the Hitachi-747 analyzer
(Roche Diagnostics GmbH, Mannheim, Germany). Creat-
inine (CRE), total protein (TP), albumin (ALB), bilirubin
(BIL), alkaline phosphatase (ALP), alanine transaminase
(ALT), aspartate transaminase (AST), gamma glutamyl
transaminase (GGT) and lactate dehydrogenase (LDH)
were measured by the conventional enzyme-coupled color-
imetric assays on the Hitachi-747 analyzer.
Statistical analysis
Numerical data are expressed as mean ± S.E.M. Student’s
t-test was used in the safety substudy. Statistical compar-
isons were made in the followings: difference in body weight
between day 7 and baseline, percentage change in body
weight, difference in initial and subsequent values of
physiological parameters [mean ABP (mABP), HR, RT],
complete blood counts, and common biochemical tests. A
two-tailed P-value of 0.05 or less was taken to infer
statistical significance.
Results
In the pharmacokinetic substudy, the body weight of the
rats was about 550 g (Table 1). Following a single i.v.
injection at 5 and 15 mg/kg, plasma concentrations of
melatonin increased to 39 and 199 million pg/mL, respect-
ively, at 2 min (Fig. 1). The initial fall in plasma concen-
trations within the first 10–20 min was fast with a half-life
of 2–3 min. Thereafter the plasma half-life of melatonin
was about 20–30 min, and the plasma melatonin levels
remained high at 128 000 and 772 000 pg/mL at 120 min
after a single i.v. injection of melatonin at 5 and 15 mg/kg,
respectively.
In the safety substudy, six daily injections of melatonin at
5 or 15 mg/kg significantly reduced the body weight by
about 5.5% (Table 2; P < 0.05 when compared with 0%,
Student’s t-test). At 60 min after the injection, melatonin
did not affect the mABP, HR and RT; the data were not
available from one rat in each group because of temporary
breakdown of the Powerlab/16 Data Acquisition System.
Table 3 shows the results of complete blood counts and
Plasma melatonin
1 X 107
1,000,000
100,000
0 10 20 30 40 50 60 70 80 90 100 110 120
Time in minutes
Fig. 1. Plasma concentration of melatonin (in pg/mL) at 2, 5, 10,
15, 30, 45, 60, 90 and 120 min following an intravenous injection at
5 or 15 mg/kg.
differential WBC. An injection of melatonin at 5 mg/kg did
not significantly affect any of the complete blood counts.
Nevertheless, an injection of melatonin at 15 mg/kg signi-
ficantly increased PMN and reduced LYM, MN and PLT
(P < 0.05; Student’s t-test). Table 4 shows the results of
common biochemical tests. At 60 min after the injection,
melatonin at 5 mg/kg significantly increased TP and AST
(P < 0.05; Student’s t-test), and melatonin at 15 mg/kg
significantly increased CRE, AST and LDH (P < 0.05;
Student’s t-test). There was no other significant change in
the biochemical results. At 24 hr after the sixth daily
injection of melatonin, gross postmortem examination as
well as examination of the histological sections from the
brain, kidney, liver and spleen of each rat did not reveal any
evidence of organ toxicity (Fig. 2).
Discussion
Enhancement of the tolerance of cerebral tissue to ische-
mia/reperfusion injury has been an attractive concept for
several decades, and encouraging results were indeed
obtained in many published experimental studies. With
the exception of NXY059, a nitrone-based free radical
scavenger [7], clinical trials have so far failed to confirm any
safe and effective neuroprotectants. Many clinical trials
were prematurely stopped due to adverse effects of the
putative neuroprotectants, such as many N-methyl-d-
aspartate-type glutamate receptor antagonists and some
a-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid-
type glutamate receptor antagonists [16].

Table 1. Baseline body weight (in g) in

the pharmacokinetic (PK) substudy in Baseline body Day 7 body
which the adult male Sprague–Dawley Study groups (number of rats) weight weight % Change
rats received an intravenous injection of
PK substudy, 5 mg/kg (8) 560.9 ± 12.4 NA NA
melatonin at 5 or 15 mg/kg as well as
PK substudy, 15 mg/kg (8) 535.8 ± 11.4 NA NA
baseline and day 7 body weight (in g) and
Safety substudy, 5 mg/kg (8) 458.1 ± 7.4 433.1 ± 5.7* )5.4 ± 1.0**
relative change in body weight (in %) in
Safety substudy, 15 mg/kg (8) 484.0 ± 6.0 456.1 ± 6.5* )5.6 ± 0.6**
the safety substudy in which the rats
received six daily intravenous injections of NA, not available.
melatonin at 5 or 15 mg/kg There is significant reduction in body weight between baseline and day 7 in the rats received
six daily doses of melatonin at 5 or 15 mg/kg in the safety substudy (*P < 0.05, Student’s
t-test). The relative change in body weight is significantly different from 0% (**P < 0.05,
Student’s t-test); there is no significant difference in this parameter between the two different
doses of melatonin (P > 0.05, Student’s t-test).

339
Cheung et al.

Table 2. Mean arterial blood pressure (mABP, in mmHg), heart rate (HR, in beats per min) and rectal temperature (RT, in C) within
60 min after the first dose of melatonin in the safety substudy in which the adult male Sprague–Dawley rats received six daily intravenous
injections of melatonin at 5 or 15 mg/kg

Daily dose of melatonin Before first 15 min after 30 min after 45 min after 60 min after
(number of rats) Parameters injection injection injection injection injection

5 mg/kg (7) mABP 103.9 ± 3.0 110.6 ± 4.1 108.3 ± 6.5 117.2 ± 7.7 109.0 ± 6.2
HR 318.2 ± 12.7 328.1 ± 12.2 325.3 ± 17.3 346.7 ± 16.8 343.2 ± 14.4
RT 35.7 ± 0.4 36.5 ± 0.4 36.4 ± 0.2 36.5 ± 0.4 36.6 ± 0.3
15 mg/kg (7) mABP 104.0 ± 5.6 105.5 ± 7.5 100.4 ± 5.6 99.3 ± 2.5 100.6 ± 4.1
HR 322.1 ± 10.0 321.7 ± 12.6 335.0 ± 12.0 346.0 ± 13.0 348.9 ± 17.5
RT 36.0 ± 0.4 36.4 ± 0.3 36.7 ± 0.2 36.8 ± 0.2 36.6 ± 0.2

There is no significant change in mABP, HR or RT within 60 min after the first injection of melatonin at 5 or 15 mg/kg (P > 0.05; Student’s
t-test comparing the data obtained before the injection and those obtained at 15, 30, 45 or 60 min after the injection).

In contrast, melatonin is an endogenous product without
significant toxicity. Very large oral doses of melatonin at
300 mg daily have previously been used for 4 months in a
clinical study involving women. Melatonin treatment in
combination with progestin reportedly inhibits ovulation
without causing serious side effects [17]. As i.v. preparation is
more appropriate in unstable patients with acute stroke and
melatonin is not very soluble in water, the current melatonin
preparation provides a concentration of melatonin at
7.5 mg/mL in 10% propylene glycol as the solvent. To
provide a dose of melatonin at 5 or 15 mg/kg, the rat will
receive 0.067 or 0.2 g/kg of propylene glycol. The pharma-
cokinetics and safety of this melatonin preparation were
evaluated in this study because Institutional Review Boards
may request proper preclinical evaluation of the melatonin
preparation prior to consideration of clinical research
protocol testing this preparation in human subjects.
Additional groups of rats were not included to control
for the isolated effects of propylene glycol, the barbiturate
anesthesia, the surgical procedures, blood specimens col-
lection, and heparinized saline. These groups are unneces-
sary in the initial preclinical evaluation of this i.v.
preparation of melatonin. If the plasma levels of melatonin
fail to rise and/or there is evidence of serious toxicity, this
i.v. preparation of melatonin may not be suitable for
further evaluation in human patients unless the observed
toxicity is due to some of the above factors. Under this
circumstance, additional control groups become necessary.
Propylene glycol is commonly used as a safe vehicle for
novel compounds in drug discovery research [18]. In a
recent study in dogs, slow i.v. infusion of propylene glycol
for 7 days did not induce any significant hematological or
clinical biochemical abnormalities when assessed at 24 hr
and 7 days postinfusion [18]. In rodents, however, chronic
inhalation of propylene glycol at high dose (>3 g/kg) can
lead to reduced body weight, raised CRE and raised ALP
[19]. The doses of propylene glycol used in this study (0.067
or 0.2 g/kg) are much lower.
Results from the pharmacokinetic substudy confirm that
the i.v. preparation can markedly elevate the plasma level
of melatonin. At 2 min after a dose of 5 mg/kg, the plasma
concentration of melatonin increases to about 40 mil-
lion pg/mL; this is almost 200 million pg/mL at 2 min
after a dose of 15 mg/kg. The fast initial fall in plasma
concentrations within the first 10–20 min suggests rapid
redistribution of melatonin into certain organs and body
tissues. Afterwards, the plasma half-life of melatonin
becomes longer at about 20–30 min. As the daytime levels
of melatonin in the rat are around 10–20 pg/mL [20], the
plasma melatonin levels remain very high at 128 000 and
772 000 pg/mL at 120 min after a single i.v. injection of
melatonin at 5 and 15 mg/kg, respectively. Such plasma
pharmacokinetics plus selective concentration of melatonin
in the brain and various subcellular compartments of neural
cells [21] are consistent with the dramatic benefits of a single
dose of melatonin in experimental stroke models [22–30].
It is important to note that the melatonin ELISA kit has
an analytical linearity from 10 to 100 pg/mL. The levels
within the first 60 min of melatonin injection were obtained
from the plasma specimens after 105- to 106-folds of
dilution. Errors may have been introduced in the dilution
process. The levels obtained at 90 and 120 min postinjec-
tions, which were obtained after 1000-fold of dilution, are
more reliable; these levels are much higher than the daytime
physiological levels of melatonin in the rat.
The safety substudy confirms the absence of serious
toxicity with this melatonin preparation. Within 60 min
after the first injection of melatonin at 5 or 15 mg/kg, there
is no significant effect on the two cardiovascular parameters
and body temperature. Melatonin is postulated to have a
modulatory role in hemopoiesis and its rhythms [31, 32],
whereas information on the hematological effects of pro-
pylene glycol is lacking [33]. Previous studies in male albino
mice have shown that subcutaneous injection of melatonin
at 25 lg can increase or decrease the LYM but predom-
inantly increase the PMN [31, 32]. At 60 min after the first
injection of melatonin at 5 mg/kg, there is no significant
influence on the complete blood counts and differential
WBC. Melatonin at 15 mg/kg, however, reduced LYM,
MN and PLT but increased PMN. Thus, it is advisable to
monitor complete blood counts in future studies on human
patients. Platelets contain melatonin [33], and melatonin
has been found to be useful in the treatment of low PLT
[34]. This is in contrast to the present finding of reduced
PLT at 60 min after given melatonin at 15 mg/kg, and a
type I statistical error cannot be excluded.
Melatonin is known to protect the kidney and liver
against ischemic and toxic insults mediated by free radicals
[35]. At 60 min after the first injection of melatonin in
propylene glycol, there is a statistically significant but
clinically minor increase in AST but no significant change
in other liver indices; liver toxicity from propylene glycol is

340
 Table 3. Complete blood counts, including hemoglobin (HB, in g/dL), reticulocyte counts (RET, in %), white cell counts (WBC, in 109/L), polymorphonuclear cell counts (PMN, in 109/L),
lymphocyte counts (LYM, in 109/L), mononuclear cell counts (MN, in 109/L), eosinophil counts (EOS, in 109/L), basophil counts (BAS, in 109/L) and platelet counts (PLT, in 109/L), were
performed in whole blood collected before and at 60 min following the first of six daily injections of melatonin at 5 or 15 mg/kg in the safety substudy in adult male Sprague–Dawley rats

Study groups (number of rats) HB RET WBC PMN LYM MN EOS BAS PLT

Before melatonin (16) 15.0 ± 0.4 1.88 ± 0.36 9.41 ± 0.79 1.43 ± 0.18 7.61 ± 0.64 0.33 ± 0.06 0.04 ± 0.01 0.00 ± 0.00 976.3 ± 31.9
At 60 min, 5 mg/kg (8) 15.6 ± 0.6 2.75 ± 0.41 8.89 ± 0.47 1.89 ± 0.21 6.74 ± 0.41 0.20 ± 0.06 0.05 ± 0.02 0.01 ± 0.01 1029.4 ± 52.6
At 60 min, 15 mg/kg (8) 15.1 ± 0.3 1.78 ± 0.51 7.54 ± 0.73 2.40 ± 0.55* 4.97 ± 0.35* 0.13 ± 0.02* 0.05 ± 0.02 0.00 ± 0.00 848.3 ± 55.6*

Baseline data from rats received the two different doses of melatonin are grouped together.
When compared with the baseline results, an injection of melatonin at 5 mg/kg does not significantly affect any of the complete blood counts (P > 0.05; Student’s t-test). In contrast, an injection of
melatonin at 15 mg/kg significantly increases PMN and decreases LYM, MN and PLT (*P < 0.05; Student’s t-test).

Table 4. Common biochemical tests, including glucose (GLU, in mm), creatinine (CRE, in lm), total protein (TP, in g/L), albumin (ALB, in g/L), bilirubin (BIL, in lm), alkaline phosphatase (ALP,
in U/L), alanine transaminase (ALT, in U/L), aspartate transaminase (AST, in U/L), gamma glutamyl transaminase (GGT, in U/L) and lactate dehydrogenase (LDH, in U/L), were performed in
the plasma collected before and at 60 min following the first of six daily injections of melatonin at 5 or 15 mg/kg in the safety substudy in adult male Sprague–Dawley rats

Study groups (number of rats) GLU CRE TP ALB BIL ALP ALT AST GGT LDH

Before melatonin (16) 6.1 ± 0.3 26.5 ± 1.8 44.4 ± 1.1 24.7 ± 0.8 2.0 ± 0.3 16.0 ± 0.0 62.4 ± 1.2 5.1 ± 0.3 7.6 ± 0.1 629.1 ± 63.2
At 60 min, 5 mg/kg (8) 6.2 ± 0.3 29.6 ± 0.5 49.1 ± 2.2* 26.8 ± 1.2 1.9 ± 0.2 16.0 ± 0.0 65.5 ± 2.1 8.7 ± 1.4* 8.0 ± 0.0 873.9 ± 114.0
At 60 min, 15 mg/kg (8) 5.8 ± 0.4 37.1 ± 1.5* 45.4 ± 1.7 24.0 ± 1.0 2.1 ± 0.4 16.0 ± 0.0 66.0 ± 3.5 9.1 ± 1.2* 7.3 ± 0.2 1285.1 ± 224.7*

Baseline data from rats received the two different doses of melatonin are grouped together.
When compared with the baseline results, an injection of melatonin at 5 mg/kg significantly increases TP and AST (*P < 0.05; Student’s t-test), and an injection of melatonin at 15 mg/kg
significantly increases CRE, AST and LDH (*P < 0.05; Student’s t-test). Otherwise, there is no significant change in the biochemical results (P > 0.05; Student’s t-test).
Pharmacokinetics and safety of intravenous melatonin in rats

341
Cheung et al.
A B
C D
E F
G H
possible [19]. The increase in CRE with melatonin at
15 mg/kg only and in TP with melatonin at 5 mg/kg only
can be attributed to a number of factors, such as type I
statistical error, hypovolemia from repeated blood taking,
and toxicity from propylene glycol [19]. The latter is a likely
explanation for the dose-dependent raise in LDH.
Six daily i.v. injections of melatonin at 5 or 15 mg/kg
lead to a mild but significant reduction in body weight of
about 5.5%. This is probably due to propylene glycol [19].
In addition, repeated daily injections may induce stress in
the rat and adversely affect food and water intake. At 24 hr
after the sixth daily injections of melatonin at 5 or 15 mg/
kg, both gross postmortem examination and histological
examination of tissue sections from the brain, kidney, liver
342
Fig. 2. Histological sections obtained
from the brain (A and B), kidney (C and
D), liver (E and F) and spleen (G and H)
of adult male Sprague–Dawley rats killed
at 24 hr after the sixth daily injection of
melatonin at 5 (A, C, E and G) or 15 (B,
D, F and H) mg/kg (hematoxylin and
eosin, · 200).
and spleen do not reveal any evidence of organ toxicity.
These findings are reassuring for the melatonin preparation.
Morphological H&E examination is adequate in demon-
strating gross evidence of cell necrosis as an objective sign
of toxicity, but it is not a sensitive test for looking at cell
damage such as apoptosis.
In conclusion, i.v. administration of melatonin in 10%
propylene glycol markedly elevates plasma levels of melato-
nin for a relatively long period of time. The present results
indicate that this preparation does not produce serious
toxicity and suggest that the preparation should be further
evaluated in human patients. Nevertheless, it is advisable to
monitor the complete blood counts, common clinical bio-
chemical tests and body weight in the study subjects.

Acknowledgments
The preparation of melatonin in propylene glycol used in
this study was a free gift of Professor Shiu Fun Pang, CK
Life Science International Holdings Inc., Hong Kong. This
work was supported by a grant (10203847) from the
Committee of Research and Conference Grants (The
University of Hong Kong, Hong Kong).
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