Pharmacokinetics of citicoline after intravenous and intramuscular administration in

dogs

Mohammad Abbaszadeh1, Rokhsana Rasooli2, Mohsen Shadi Mazdaghani*1, Hamid

Accepted Paper

Rajaian2, Hosein Ali Shamsaei3

1
Department of small animals internal medicine, Faculty of Veterinary Medicine, University
of Shiraz, Shiraz, Iran
2
Department of Pharmacology, Faculty of Veterinary Medicine, University of Shiraz, Shiraz,
Iran. rajaian@shirazu.ac.ir
3
Master of central laboratory, Faculty of Veterinary Medicine, University of Shiraz, Shiraz,
Iran. sh.mehrjan@gmail.com

Corresponding author: Mohsen Shadi Mazdaghani, Department of small animals internal

medicine, Faculty of Veterinary Medicine, University of Shiraz, Shiraz, Iran
shahingenius14@gmail.com, phone number: +989390046603

Acknowledgement:: We are indebted to the authorities and personnel of the central

laboratory of Shiraz University and Mr Esfandiary for providing animals.

Conflict of interest: The authors declare that there is no conflict of interest. Author A
declares that he has no conflict of interest. Author B declares that she has no conflict of
interest. Author C declares that he has no conflict of interest. Author D declares that he has
no conflict of interest. Author E declares that he has no conflict of interest.
This research was financially supported by grants of Shiraz University Research Council.
All applicable international, national, and/or institutional guidelines for the care and use of
animals were followed.

This article has been accepted for publication and undergone full peer review but has not been through the copyediting,
typesetting, pagination and proofreading process, which may lead to differences between this version and the final one.
Please cite this article as doi: 10.4081/vsd.2018.6945
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Abstract
Background: New studies have confirmed the role of citicoline in reversing different
pathological conditions in canine medicine, but dosing regimens of this drug has not yet been
investigated in dogs. Using a high-performance liquid chromatography (HPLC) system, this
study aims to quantify the levels of citicoline in dogs plasma after intravenous (IV) and
intramuscular (IM) administration.
Design and methods: The subjects of this study were 12 male, mixed breed healthy dogs that
were approximately 2 to 3 years old and had a body weight between 15 and 25 kg. Plasma
samples were extracted following protein precipitation. Samples were eluted from the column
Accepted Paper

at flow rate of 0.7ml min-1. Solvents were degassed. The PH of the mobile phase of HPLC
grade acetonitrile: water (20:80, v/v) was adjusted to 3.0 using 1% orthophosphoric acid.
Both sample and standard solutions were filtered through 0.22 µm membrane filter. A sample
volume of 20 microliter was injected to HPLC to obtain a standard curve.
Results: No clinical signs or drug adverse effect was noticed in animals during and after the
period of administration of citicoline. Measuring plasma concentration of citicoline sodium
after IV and IM administration showed biphasic plasma peaks which occured at 15 minutes
and 5 hours after the drug administration in dogs. Conclusions: Giving the drug once a day
with 30-50 mg/kg dosage or twice daily with 15-30 mg/kg dosage would cause their levels to
remain elevated for much of the day and doesn’t have any serious adverse effect.
Keywords: Citicoline, Intravascular, Intramuscular, Kinetic, Dog

Introduction
The rationale administration of a drug regimen requires basic information of
pharmacokinetics in the animal species, and this prepares optimum clinical efficacy.
Citicoline (cytidinediphosphocholine, CDP-choline) is a complex organic mononucleotide
composed of ribose, cytosine, pyrophosphate, and choline (1). It is a water soluble compound
which is a neuroprotective when administered exogenously. It is also an intermediate in
membrane phosphatide biosynthesis. In human, citicoline has more than 90% bioavailability,
and is rapidly absorbed. Less than 1% is excreted in faeces (2).

“Produced endogenously, citicoline serves as a choline donor in the metabolic pathways for
biosynthesis of acetylcholine and neuronal membrane phospholipids, mainly Phosphatidyl
choline. As an exogenous compound, citicoline is also a source for acetylcholine synthesis, a
key neurotransmitter and a member of the group of molecules that play important roles in
cellular metabolism known as nucleotides “(1).
It is demonstrated that when a citicoline is taken orally or intravenously, it is completely
absorbed into the blood circulation (3). Once absorbed, citicoline is widely distributed
throughout the body and passes from the blood–brain barrier and reaches the central nervous
system as a main target, where it is incorporated into phospholipids (4). Subsequently,
citicoline enhances the biosynthesis of structural phospholipids that form neuronal
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membranes, increases brain metabolism, and influences levels of multiple neurotransmitters

(5). stabilizing cell membrane, it can reduce ischemic injury by reducing free radical
generation (6). Besides citicoline has antiedematous effects and decreases the formation of
free radicals through suppressing the phospholipases (A1, A2, C–D) which provides the
systems with antioxidant protection (7). Citicoline also has been proved to increase
acetylcholine, norepinephrine and dopamine levels in the central nervous system (8),
denoting it may has beneficial effects in conditions like head trauma of varying severity,
cognitive disorders, Parkinson’s disease, glaucoma and strabismus (9). New studies have
confirmed the role of citicoline in reversing different pathological conditions in canine
medicine. Citicoline has only recently gained significant attention as a neuroprotective agent
Accepted Paper

in dogs and it has been found to improve learning and memory (6). In a preliminary study, it
has shown that use of citicoline prior to thiopental sodium anesthesia can improve brain
function by decreasing the duration of lack of response to corneal reflex and also increasing
the duration of analgesia (10). Appropriate dosage selection is much dependent on the
pharmacokinetics of citicoline. This study was the first attempt to determine the blood levels
of citicoline after IV and IM administration in different times in dogs.

Material and Methods

The subjects of this study were 12 male, mixed breed dogs that were approximately 2 to 3
years old and had a body weight between 15 and 25 kg. The animals were randomly divided
into two treatment groups; intravenous and intramuscular. Dogs were considered healthy
based on physical examination, complete blood count, serum biochemistry, and urine specific
gravity before initiation of the study. Each dog received core vaccinations and routine
deworming. All dogs were kept in an approved animal care facility for 2 weeks before and
during the trial. The study was approved by and ran according to the faculty of veterinary
medicine of Shiraz university guidelines.

Experimental chemicals and drugs:

The standard of citicoline (Sigma), HPLC grade water (Merck), acetonitrile (E. Merck
Germany), citicoline injection (250mg/2ml ampules, AlborzDaru©) were used in this study.
Experiment:

Six dogs received an intravenous injection of 50 mg.kg-1 of citicoline via cephalic vein. Six
additional dogs with the same weight range received an Intramuscular injection of 50 mg.kg-
1 of citicoline in the quadriceps muscle. Blood samples (5 mL) were collected from all dogs
in heparinized test tubes before and at 5, 15, 30, 60, 120, 180, 300 minustes and 24 hours
after i.v and i.m administration. Plasma was harvested by centrifugation and stored frozen in
sterile plastic vials until analysis.
HPLC:
The Knauer HPLC system consisted of Smartline pump (1000), equipped with a UV detector
2500 knauer set at 260 nm. The separation was carried out isocratically using a Knauer
reverse phase C18 column model (vertex plus 250*4.6 mm Eurospher 100-5) with guard
cartridge, column oven model CTO-6A knauer.The system was used at 20˚C . Samples were
eluted from the column at flow rate of 0.7ml min-1. Solvents were degassed under vaccum. A
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mobile phase of HPLC grade acetonitrile: water (20:80, v/v) was adjusted at pH 3.0 using 1%
orthophosphoric acid. Both sample and standard solutions were filtered through 0.22 µm
membrane filter. A sample volume of 20 microliter was injected to HPLC. Six standard
concentrations were injected to obtain the standard curve. The resultant concentrations of
0.125, 1.25, 12.5, 62.5, 125 and 625 microgram/ml were used for drawing the standard curve
(R2=0.9996) . Retention time was set at 3.0 ± 0.2 minutes (Figure 1). Plasma samples were
extracted by protein precipitation with methanol. Serum (100µl) was mixed with 400µl of
HPLC grade methanol, incubated for 3minutes at 4˚C before centrifuging at 5000 rpm for
6minutes. The clear phase was transferred to another polypropylene tube for injection to the
HPLC device.
Accepted Paper

Results :
No clinical signs or drug adverse effect was noticed during and after the period of
administration of citicoline.
Citicoline was detected in the plasma at different concentrations via I.V and I.M dosing in
dogs. Table 1 summarizes the mean concentration of citicoline after I.V and I.M
adminitration after different intervals.

Table 1. Blood kinetics of citicoline after intravenous and intramuscular

administration to male dogs. mean±SD.
IV, Intra Venus; IM, Intra Mascular.

Time 5 min 15 min 30 min 60 min 120 180 min 300 1440
min min min
IV 15.3 ± 18.7 ± 9.7 ± 12.9 ± 12.7 ± 14.6 ± 21.8 ± 10.7 ±
route 0.09 0.05 0.09 0.02 0.05 0.01 0.02 0.07

IM 6.1 ± 7.8 ± 7.1 ± 7.7 ± 11.6 ± 5.1 ± 11.6 ± 8.2 ±

route 0.04 0.09 0.07 0.06 0.06 0.04 0.06 0.02

Fluctuation in the plasma concentration of citicoline sodium after both IV and IM

administration showed that citicoline by both mentioned routes gave rise to two
chromatographic peaks in plasma concentration. After intravenous administration, the first
peak was at 15 minutes, and the second larger peak at 5 hours post-dosing (Figure 2).
Furthermore, the peak concentration of citicoline were at 2 hours and 5 hours post dosing
after intramuscular administration (Figure 3). These two peaks in concentration indicate that
absorption into the systemic circulation is not instantaneous. Other related pharmacokinetic
parameters weren’t measurable because of biphasic kinetics of this drug.
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Accepted Paper

Fig 1. Retention time of the standard citicoline solution.

12

10
Plasma Concentration

0
5 10 15 30 60 120 180 300 1440
minutes

Fig 2. Plasma citicoline concentrations after different intervals post IV administration

in dogs.
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20
18
16
Plasma Concentration

14
12
10
8
6
4
Accepted Paper

2
0
5 10 15 30 60 120 180 300 1440
minutes

Fig 3. Plasma citicoline concentrations after different intervals post IM administration in dogs.

Fig 3. Plasma citicoline concentrations after different intervals post IM administration in

dogs.
Figure 4 shows comparison of the citicoline concentrations in the blood of rats (11) and dogs
(current study) after its IV administration from zero to 24 hours. Although the method used
in pharmacokinetics of citicoline in rats differed from that of ours, a reasonable comparison
can be made between the concentrations of the drug in both species at different times post IV
administration. The two curves show somehow similar increase and decrease in concentration
and also the peak levels in both species occur at the same time.

14

12
Plasma Concentration

10

6 Rat

Dog
4

0
5 10 15 30 60 120 180 300 1440
minutes

Fig 4. Comparison of the Plasma citicoline concentrations of dogs afte IV

administration with rats in the first 24 hours.

Discussion :
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Estimation of the plasma concentrations of the drug via different administration routes at
various intervals, let us to determine specific dosing regimens. In the present study,
measuring plasma concentration of citicoline sodium after I.V and I.M administration showed
biphasic plasma peaks which occurred at 15 minutes and 5 hours after I.V and at 2 and 5
hours after I.M administration.
Literature survey revealed that two methods such as HPLC (12-14) and Spectrophotometric
(15-18) applied for the analysis of citicoline either as an individual drug in pure,
pharmaceutical forms or in combination with impurities as well as in biological fluids. To
achieve the highest precision in quantitative estimation of citicoline, here, a reversed phase
liquid chromatography method was developed and validated. Method validation in terms of
Accepted Paper

linearity, precision, accuracy, detection limit, quantification limit and robustness was
performed. Our findings revealed that the kinetics pattern of citicoline was very similar to
the human and rat after I.V administration. Pharmacokinetic studies using citicoline in
humans showed elimination occurs mainly via respiratory and urinary excretion, in two
phases mirroring the biphasic plasma peaks (1). The first peak in plasma concentration is
followed by a sharp decline, which then slows over the next 4-10 hours. In the second phase,
an initially rapid decline after the 24-hour plasma peak is similarly followed by a slower
elimination rate. Similarly, in dogs the initial peak and subsequent decline was observed.
Present results showed that citicoline concentrations curves in different times after either IV
or IM administration didn’t show conventional logarithmic manner, therefore we cannot
discuss about half time and many other kinetics indexes. Plasma citicoline levels in this
assessment showed a relative high concentration for 24 hours, which means slow elimination
of citicoline from the body. It may favor prolonged therapeutic or possible toxic actions of
the drug. Since no clinical signs or drug adverse effect was noticed during and after the
period of administration of citicoline, we can assume it is a safe drug for canine medicine
application. Drug safety and high plasma citicoline concentration persisted for a number of
hours, so giving the drug once a day with 30-50 mg/kg dosage or twice daily with 15-30
mg/kg dosage would cause their levels to remain elevated for much of the day and doesn’t
have any serious adverse effect. The current study is important for several reasons. Firstly,
our data provides the first evidence that citicholine could administrate in dogs with the
dosage regimen like human. Secondly, our data proposes a new method for assessment of
citicholine kinetics in dogs.
Conclusion:
The proposed method of analysis provided a sensitive and specific assay for citicoline
determination in dog plasma. The current data from citicoline kinetic in dogs and its safety
showed the possibility to utilize the dosage used in humans, in canine patients according to
the pharmacokinetics data.

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Accepted Paper

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