|

Received: 11 April 2017    Accepted: 11 December 2017

DOI: 10.1111/jvp.12484

ORIGINAL ARTICLE

Pharmacokinetic profiles of the active metamizole metabolites

after four different routes of administration in healthy dogs

M. Giorgi1  | B. Łebkowska-Wieruszewska2 | A. Lisowski3 | H. Owen4 | 

A. Poapolathep5 | T. W. Kim6  | V. De Vito7

1
Department of Veterinary
Sciences, University of Pisa, Pisa, Italy Metamizole (MT), an analgesic and antipyretic drug, is rapidly hydrolyzed to the active
2
Department of Pharmacology, University of primary metabolite 4-­methylaminoantipyrine (MAA) and relatively active secondary
Life Sciences, Lublin, Poland metabolite 4-­aminoantipyrine (AA). The aim of this study was to assess the pharma-
3
Department of Animal Hygiene and
cokinetic profiles of MAA and AA after dose of 25 mg/kg MT by intravenous (i.v.), in-
Environment, University of Life Sciences,
Lublin, Poland tramuscular (i.m.), oral (p.o.), and rectal (RC) routes in dogs. Six dogs were randomly
4
Department of Veterinary allocated to an open, single-­dose, four-­treatment, four-­phase, unpaired, crossover
Sciences, University of Queensland, Gatton,
Australia
study design. Blood was collected at predetermined times within 24 hr, and plasma
5
Department of Pharmacology, Faculty of was analyzed by a validated HPLC-­UV method. Plasma concentrations of MAA and AA
Veterinary Medicine, Kasetsart University, after i.v., i.m., p.o., and RC administrations of MT were detectable from 5 (i.v. and i.m.)
Bangkok, Thailand
6
or 30 (p.o. and RC) min to 24 hr in all dogs. The highest concentrations of MAA were
College of Veterinary Medicine, Chungnam
National University, Daejeon, South Korea found in the i.v., then i.m., p.o., and RC groups. Plasma concentrations of AA were simi-
7
Department of Veterinary lar for i.v., i.m., and RC, and the concentrations were approximately double those in the
Medicine, University of Sassari, Sassari, Italy
PO groups. The AUCEV/IV ratio for MAA was 0.75 ± 0.11, 0.59 ± 0.08, and 0.32 ± 0.05,
Correspondence for i.m., p.o., and RC, respectively. The AUCEV/IV ratio for AA was 1.21 ± 0.33,
Mario Giorgi, Department of Veterinary
Sciences, University of Pisa, Pisa, Italy.
2.17 ± 0.62, and 1.08 ± 0.19, for i.m., p.o., and RC, respectively. Although further stud-
Email: mario.giorgi@unipi.it ies are needed, rectal administration seems to be the least suitable route of adminis-

Funding information
tration for MT in the dog.
Ex 60% University of Pisa 2015

1 |  INTRODUCTION
Metamizole (MT), or dipyrone, is one of the more effective nonopioid
analgesics available for the treatment of pain and fever in both human
and veterinary medicine (Baumgartner et al., 2009). It works as an ef-
ficacious COX-­3 inhibitor but also provides weak COX-­1 and COX-­2
inhibition (Botting, 2000; Chandrasekharan et al., 2002). In partial ex-
planation for this action, MT has been shown to interact with both the
N-­methyl-­D-­aspartate receptor and the opioid system; however, the
exact mechanism by which MT produces its analgesic action remains
unclear (Khodai, 2008; Tortorici, Vasquez, & Vanegas, 1996). Recent
studies have demonstrated that its analgesic action could partially be
due to the interaction with the cannabinoid receptors (Crunfli, Vilela, &
Giusti-­Paiva, 2015; dos Santos et al., 2014). MT is available in human
and veterinary markets in several countries (South America, Asia,
and European countries) but has been withdrawn in other countries
(Australia, USA, Sweden, Japan, Iran, and UK) because of safety con-
cerns in humans. Although MT is known to be a relatively safe drug
compared to other nonopioid analgesics, there are some reports, al-
though not unanimously accepted, suggesting that after long-­term
treatment, MT might cause hematopoietic pathology, agranulocyto-
sis, leukopenia, and even aplastic anemia in humans (Basak, Drozd-­
Sokołowska, & Wiktor-­Jedrzejczak, 2010; Bigal, Bordini, & Speciali,
2002; Garcıa-­Martınez et al., 2003; Hedenmalm & Spigset, 2002;
Imagawa et al., 2011). However, pharmacovigilance veterinary reports
indicate that the incidence of adverse reactions in the target species
is negligible (Committee for Veterinary Medicinal Products, 2003). For
the veterinary market, MT is labeled for use in porcine, bovine, equine,
and canine species. It is administered parentally in the dose range of
20–50 mg/kg (package leaflet, Biovetalgin, Biowet, Drwalew, Poland) or
orally 40–50 mg metamizole sodium/kg body weight (package leaflet,
Metapyrin oral 100%, Serumwerk Bernburg AG, Bernburg, Germany).

J vet Pharmacol Therap. 2018;1–9. © 2018 John Wiley & Sons Ltd |  1
wileyonlinelibrary.com/journal/jvp  
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2       GIORGI et al.
Metamizole is a prodrug which, in an aqueous environment,
spontaneously breaks down into numerous metabolic products
(Levy, Zylber-­Katz, & Rosenkranz, 1995; Vlahov, Badian, Verho, &
Bacracheva, 1990) . In humans, MT is rapidly hydrolyzed to the
main metabolite 4-­methylaminoantipyrine (MAA). MAA is then me-
tabolized to 4-­formylaminoantipyrine (FAA; an end-­metabolite) and
to 4-­aminoantipyrine (AA) (Levy et al., 1995). AA is further metabo-
lized to 4-­acetylaminoantipyrine (AAA) (Figure 1) (Levy et al., 1995;
Rogosch et al., 2012; Vlahov et al., 1990). MAA and AA are both active
metabolites (Vlahov et al., 1990; Weithmann & Alpermann, 1985). In
recent years, MT has experienced a revival for pain management in
human and veterinary medicine, with pharmacokinetic studies being
performed in food-­producing animals and pharmacodynamics studies
on dogs (Aupanun et al., 2016; Burmańczuk, Kowalski, Giorgi, Owen,
& Grabowski, 2016; Giorgi et al., 2015; Giorgi et al., 2017; Imagawa
et al., 2011; Kalchofner Guerrero et al., 2015; Schütter, Tünsmeyer, &
Kästner, 2016; Teixeira et al., 2013; Zanuzzo et al., 2015).
To the best of the authors’ knowledge, only two reports pres-
ent limited data on the plasma concentration vs. time of MAA and
AA after MT oral administration in dogs (Kalchofner Guerrero et al.,
2015; Loescher, 1993). This study was carried out to compare the
pharmacokinetic profiles of MAA and AA after intravenous (i.v.), in-
tramuscular (i.m.), oral (p.o.), and rectal (RC) administrations of MT
in healthy dogs.
2 | MATERIALS AND METHODS
2.1 | Animal experiments
The animal study was carried out in accordance with the European
law (Directive 2010/63/EU). Six adult, intact female Labradors, aged
between 3–6 years and weighing 36–42 kg, were used in the study.
The dogs were determined to be clinically healthy based on physi-
cal examination, serum chemistry, and hematological analyses. Food
was withheld for 10 hr prior to the beginning of the experiment while
water was given ad libitum. Feed was available from 4 hr after treat-
ment. Animal health was evaluated daily by trained personnel (up to
1 week after completion of the study) for visible adverse effects. Two
F I G U R E   1   Metabolic pathway of
metamizole (MT) reported in humans
(Geisslinger, Böcker, & Levy, 1996)
GIORGI et al.
weeks after completion of the experiment, the dogs underwent a
health check for any abnormalities.
Dogs were randomly assigned to four-­treatment groups (research
randomizer software [www.randomizer.org]), using an open, single-­
dose, four-­treatment, four-­phase, unpaired, crossover design (4 × 4
Latin-­square). During the first phase, each subject in group 1 (n = 2)
was administered with a single dose of 25 mg/kg MT (Biovetalgin,
injectable solution 500 mg/ml, BioWet, Drwalew, Poland) injected
i.v. into the jugular vein at a 1 ml/min injection rate. Group 2 (n = 1)
received an i.m. injection, at the same dose as the i.v. injection, in-
jected in the middle quadrant of the buttock muscle (Biovetalgin,
injectable solution 500 mg/ml, BioWet, Drwalew, Poland). Group
3 (n = 2) received the same dose (25 mg/kg) orally, in an immediate
release (human) oral formulation (Pyralgina–Zakłady farmaceutyczne
Polpharma S.A. 500 mg/tablet, Poland). The doses were prepared
by weighing and partitioning (by shaving) the marketed drug. Group
4 (n = 1) was given MT at 25 mg/kg via the RC route (suppositories,
Pyralgina, 750 mg, Zakłady farmaceutyczne Polpharma, Poland). The
suppositories were dipped in cold water (10°C) for half an hour prior
to use in order to facilitate their division and insertion. The dogs were
kept in individual cages and monitored for defecation for at least 2 hr
after insertion of the suppositories to ensure complete absorption of
drug. Animals were given the suppository while in a prone position.
The dose was determined based on package leaflet recommendations
(injectable) and on an earlier study showing that 25 mg/kg was an ef-
fective dose in dogs (Imagawa et al., 2011).
The washout period between the four phases (to ensure com-
plete metabolism and excretion of MAA and AA) was 1 week. The
groups were rotated until the crossover study was completed. When
the study was completed each animal had received MT by all formu-
lations. Thirty minutes before the commencement of the study to
facilitate blood sampling, an 18-­gauge catheter was placed into the
medial saphenous vein. Blood samples (2 ml) were collected into lith-
ium heparin tubes at 5, 15, 30, and 45 min and 1, 1.5, 2, 4, 6, 8, 10,
24, 36, and 48 hr after drug administration. Tubes were immediately
placed on ice and centrifuged at 2,200 g for 5 min within 30 min. The
collected plasma was stored at −20°C until analysis, within 30 days
of collection.
2.2 | Standard compounds
Pure AA and MAA analytical standard (>99.0% purity) were obtained
from Sigma-­Aldrich (St. Louis, MO, USA) and Toronto Research
Chemicals (Toronto, Canada), respectively. The Internal Standard (IS)
was metoclopramide (powder > 99.0% purity) supplied by Sigma-­
Aldrich (St. Louis, MO, USA). Dog control plasma samples were kindly
supplied by the canine blood bank at the University of Pisa.
2.3 | HPLC-­UV analysis
The analytical method was approached based on previously de-
scribed methods with slight modifications (Domínguez-­Ramírez
et al., 2012; Giorgi et al., 2015). The HPLC system was an LC Jasco
|
      3
(Como, Italy) consisting of a quaternary gradient system (PU 2089
PLUS), coupled with an ultraviolet detector (Jasco UV-­975) set at
254 nm. The column was a Luna C18(2) (250 mm × 4.6 mm inner
diameter, 5-­μ particle size [Phenomenex, Bologna, Italy]) preceded
by a security guard column with the same stationary phase (C18(2)
[Phenomenex, Bologna, Italy]). The HPLC system was kept at
25°C. The analytes were eluted in isocratic mode with a mobile
phase (ammonium acetate 20 mm:acetonitrile, pH 5, 80:20, v/v) at
a flow rate of 1 ml/min.
2.4 | Sample extraction
Sample extraction was carried out in a 15 ml polypropylene vial. IS
(100 μl, 25 μg/ml) was added to 0.5 ml of plasma. After 30 s vortexing,
sodium hydroxide (0.1 ml, 1 N) was added and the sample vortexed
again. Ethyl acetate:methylene chloride (4 ml, 3:7, v/v) was added,
and the sample was vortexed (30 s), shaken (60 osc/min, 10 min), and
centrifuged at 10,956 g (rotor radius 5 cm) for 10 min at 10°C. Three
ml of the organic layer was collected in a new 15 ml screw cap vial.
The organic phase was evaporated under a gentle stream of nitrogen
(40°C) and reconstituted with 100 μl of mobile phase. Fifty μl of this
solution was injected onto the HPLC.
2.5 | Method revalidation
The HPLC method was revalidated using control dog plasma. In brief,
MAA and AA were linear in the range of 100–5000 ng/ml. LOD was
30 ng/ml, and LOQ was 50 ng/ml. When the metabolite concentrations
in the samples exceeded the upper limit of the range, they were re-­
analyzed after appropriate dilution. The intraday and interday precision
coefficient of variations were lower than 5.9 and 6.7, and 8.1% and 7.9%
for MAA and AA, respectively. The relative error of accuracy in within-­
day and between-­day assays was lower than 4.7 and 5.3, and 6.0%
and 7.1% for MAA and AA, respectively. The recoveries of the extrac-
tion procedure were between 93.5% and 96.7% for MAA and between
95.1% and 98.0% for AA.
2.6 | Pharmacokinetic analysis and
statistical analysis
The pharmacokinetic calculations were carried out using WinNonlin v
5.3.1 (Pharsight Corp). The curve fit was performed by a noncompart-
mental analysis. The percent of AUC that was extrapolated to infinity
(AUCExtrap%) was always <20% in all the subjects. The extraction ratio
was estimated by dividing total clearance for each animal after i.v. ad-
ministration by cardiac output, where cardiac output was estimated as
180 ×  body weight (kg)−0.19 (Toutain & Bousquet-­Mélou, 2004). The
results are presented as mean ± standard deviation.
To make comparisons across treatments, the different parameters
were first tested for normal distribution and variance homogeneity.
Data were compared with the ANOVA or the nonparametric Wilcoxon
test, depending on whether the data passed a normality test. A p value
less than .05 was considered statistically significant.
 4       | GIORGI et al.

3 |  RESULTS
No behavioral changes or alterations in health parameters were ob-
served in the i.v., i.m., or RC groups of animals during or after (up
to 7 days) the drug administration. Some salivation (self-­limiting)
was noted in three subjects in the p.o. group 15–30 min after drug
administration.
Plasma concentrations of MAA and AA after i.v., i.m., p.o., and RC
administrations of MT were detectable from 5 (i.v., i.m.) or 30 (p.o., RC)
min to 24 hr in all dogs (Figure 2). Figure 3 reports the average plasma
concentration of MAA after the four routes of administration. Except
for the initial phase, the i.v. and i.m. concentrations vs. time profiles
were similar. The p.o. and RC concentrations vs. time profiles were also
similar in shape but not in MAA concentration.
The highest concentrations of MAA were found in the
i.v., > i.m. > p.o. > RC group (Figure 3). The concentrations of MAA
were higher than that of AA in the i.v. and i.m. groups (Figure 2 a, b).
In contrast, the concentration of AA exceeded that of MAA 6 hr after
MT administration in the p.o. and RC groups (Figure 2 c, d). It is note-
worthy that the average plasma concentration of AA in the p.o. group

25

(a)
20
Concentration (µg/ml)

15

10

0
0 6 12 18 24
Time (hr)
15

12 (b)
Concentration (µg/ml)

0
0 6 12 18 24
Time (hr)
8

6 (c)
Concentration (µg/ml)

0
0 6 12 18 24
Time (hr)
6

(d)
4
Concentration (µg/ml)

F I G U R E   2   Mean plasma concentrations

2 of 4-­methylaminoantipyrine ( , MAA) and
4-­aminoantypyrine ( , AA) vs. time curves
following intravenous (a), intramuscular
0 (b), oral (c), and rectal (d) dose of 25 mg⁄kg
0 6 12 18 24 metamizole (MT) in healthy dogs (n = 6).
Time (hr) Bars represent the standard deviations
GIORGI et al.
F I G U R E   3   Mean plasma concentrations of 4-­methylaminoantipyrine (MAA) vs. time curves following intravenous ( ), and intramuscular
( ), oral ( ), and rectal ( ) dose of 25 mg⁄kg metamizole (MT) in healthy dogs (n = 6). Bars represent the standard deviations. The panel
focus on the relevant area of the curve where the IC50s (–) for the human COX enzymes are located
was higher (double) than that of AA in the other treatment groups
(Figure 4).
The main pharmacokinetic parameters of MAA are presented in
Table 1. The ratios calculated based on the relative AUC0-last values
AUCIM MAA/AUCIV MAA, AUCPO MAA/AUCIV MAA, and AUCRC MAA/AUCIV
MAA were 0.75 ± 0.11, 0.59 ± 0.08, and 0.32 ± 0.05, respectively.
The main pharmacokinetic parameters of AA are presented in
Table 2. The ratios calculated based on the relative AUC0-last values
AUCIM AA/AUCIV AA, AUCPO AA/AUCIV AA, and AUCRC AA/AUCIV AA were
1.21 ± 0.33, 2.17 ± 0.62, and 1.08 ± 0.19, respectively.
4 | DISCUSSION
Metamizole has been used to treat pain and fever for almost a cen-
tury in some countries, while in others, it is completely unknown
Concentraon (µg/ml)
F I G U R E   4   Mean plasma concentrations
of 4-­aminoantypyrine (AA) vs. time curves
following intravenous ( ), intramuscular
( ), oral ( ), and rectal ( ) dose of
25 mg⁄kg metamizole (MT) in healthy
dogs (n = 6). Bars represent the standard
deviations
|
      5
or forgotten (Nikolova et al., 2012). MT acts as an effective pain-­
relieving, antipyretic, and spasmolytic agent (Levy et al., 1995). It does
not possess the contraindications or restrictions usually observed
with opioids or NSAIDs (Avellaneda et al., 2000; Baumgartner et al.,
2009; Edwards, Meseguer, Faura, Moore, & McQuay, 2010; Kemal,
Sahin, & Apan, 2007; Zukowski & Kotfis, 2009). The analgesic efficacy
of MT in humans is abundantly reported in the literature (Avellaneda
et al., 2000; Edwards et al., 2010; Kemal et al., 2007; Korkmaz Dilmen
et al., 2010; Olson, Sunshine, Zighelboim, & Lange, 1999; Zukowski &
Kotfis, 2009).
In contrast, the evidence is not as strong in veterinary medicine.
Some data regarding clinical and side effects of MT in horses, rab-
bits, rats, and dogs have been reported, and there is addition data
in relation to the pharmacokinetic profile of the metabolite MAA in
horses, donkeys, pigs, rats, dogs, cats, and sheep (Aupanun et al., 2016;
Baumgartner et al., 2009; Burmańczuk et al., 2016; Christ, Kellner,
3
1
0
0 6 12 18 24
Time (hr)
 |
6       GIORGI et al.

T A B L E   1   Main pharmacokinetic parameters of 4-­methylaminoantipyrine (MAA) following single intravenous (i.v.), intramuscular (i.m.), oral
(p.o), and rectal (RC) administrations of metamizole (MT) (25 mg/kg) in healthy dogs (n = 6)

Parameter Unit i.v. (Mean ± SD) i.m. (Mean ± SD) p.o. (Mean ± SD) RC (Mean ± SD)

2
R .92 ± .08 .94 ± .06 .90 ± .08 .88 ± .13
λz 1/hr 0.13 ± 0.04 0.11 ± 0.02 0.12 ± 0.02 0.12 ± 0.03
T1/2 λz# hr 5.94 ± 2.54 5.92 ± 1.04 5.59 ± 0.96 5.87 ± 1.44
Tmax hr 0.083bcd ± 0.000 0.625acd ± 0.130 1.910ab ± 0.200 1.910ab ± 0.200
Cmax μg/ml 21.80bcd ± 2.45 12.57acd ± 2.27 5.27abd ± 0.56 2.72abc ± 0.35
bcd acd abd
AUC0-last hr/μg/ml 45.34  ± 9.64 33.33  ± 4.83 26.62  ± 4.69 15.27abc ± 3.44
AUC0-∞ hr/μg/ml 46.79bcd ± 9.15 34.76acd ± 4.82 28.01abd ± 5.53 16.18abc ± 3.39
bcd acd abd
Vz/F ml/kg 4,946.33  ± 988.21 6,250.45  ± 1,409.44 7,449.71  ± 2,218.28 13,776.34abc ± 4,839.61
CL/F ml/hr/kg 552.43bcd ± 98.34 731.14acd ± 103.47 920.93abd ± 178.96 1,605.66abc ± 356.24
2 cd cd abd
AUMC0-∞ hr /μg/ml 246.57  ± 31.01 201.92  ± 53.035 202.93  ± 80.20 131.28abc ± 27.74
MRT0–∞ hr 5.37 cd ± 0.81 5.93cd ± 1.44 7.06ab ± 1.28 8.13ab ± 1.08
AUCEV/IV ratio % / 75.2cd ± 11.75 59.6bd ± 8.04 32.78ab ± 5.08

R2, correlation coefficient of the terminal portion of the curve; λz, terminal phase rate constant; T1/2λz, terminal half-­life; Tmax, time of peak; Cmax, peak
plasma concentration; Vz/F, apparent volume of distribution; CL/F, apparent clearance; AUC0-∞, area under the plasma concentration–time curve;
AUMC0-∞, area under the first moment curve; MRT0-∞, mean resident time; AUCEV/IV ratio, AUCextravascular/AUCIV.
Superscript letters indicate the statistically different values among the treatment groups.
#
Harmonic mean.

T A B L E   2   Main pharmacokinetic parameters of 4-­aminoantypyrine (AA) following single intravenous (i.v.), intramuscular (i.m.), oral (p.o.), and
rectal (RC) administrations of metamizole (MT) (25 mg/kg) in healthy dogs (n = 6)

Parameter Unit i.v. (Mean ± SD) i.m. (Mean ± SD) p.o. (Mean ± SD) RC (Mean ± SD)

2
R .97 ± .02 .98 ± .02 .98 ± .01 .98 ± .03
λz 1/hr 0.09 ± 0.03 0.08 ± 0.03 0.12 ± 0.04 0.10 ± 0.02
T1/2 λz# hr 8.05 ± 2.56 8.75 ± 2.58 6.12abd ± 2.08 7.25 ± 1.86
Tmax hr 5.33 ± 1.63 5.58 ± 2.49 6.33 ± 1.96 5.21 ± 1.67
Cmax μg/ml 1.29 ± 0.21 1.36 ± 0.38 2.64abd ± 0.44 1.43 ± 0.18
abd
AUC0-last hr/μg/ml 17.97 ± 2.91 21.28 ± 4.80 37.92  ± 7.60 19.08 ± 2.22
AUC0-∞ hr/μg/ml 21.08 ± 4.34 23.03 ± 7.31 42.37abd ± 11.78 21.87 ± 2.75
Vz/F ml/kg 13,855.67 ± 3,678.36 12,394.27 ± 3,898.61 5,146.44abd ± 393.45 11,978.01 ± 2,827.06
CL/F ml/hr/kg 1,224.03 ± 228.74 1,014.77 ± 242.02 628.43abd ± 168.32 1,156.87 ± 132.41
2 abd
AUMC0-∞ hr /μg/ml 273.97 ± 117.74 377.07 ± 169.98 489.45  ± 252.05 266.36 ± 69.61
MRT0–∞ hr 12.62 ± 3.13 14.07 ± 3.12 10.99abd ± 2.49 12.09 ± 2.73
bd
AUC EV/IV ratio % / 121.26 ± 33.59 217.55  ± 62.29 108.08 ± 19.93
2
R , correlation coefficient of the terminal portion of the curve; λz, terminal phase rate constant; T1/2λz, terminal half-­life; Tmax, time of peak; Cmax, peak
plasma concentration; Vz/F, apparent volume of distribution; CL/F, apparent clearance; AUC0-∞, area under the plasma concentration–time curve;
AUMC0-∞, area under the first moment curve; MRT0-∞, mean resident time; AUCEV/IV ratio, AUCextravascular/AUCIV.
Superscript letters indicate the statistically different values among the treatment groups.
#
Harmonic mean.

Ross, Rupp, & Schwarz, 1973; Flôr, Yazbek, Ida, & Fantoni, 2013; Giorgi
et al., 2015, 2017; Imagawa et al., 2011; Klaus, Schlingloff, Kleinitz,
Böttcher, & Hapke, 1997; Lebkowska-­Wieruszewska et al., 2017;
Loescher, 1993; Roelvink, Goossens, Kalsbeek, & Wensing, 1991;
Silva-­Moreno, Lopez-­Munoz, & Cruz, 2009; Teixeira et al., 2013;
Zanuzzo et al., 2015).
Due to MT’s striking pharmacological effect, low cost and safety
profile, there has been growing interest in its use in the veterinary field
over the past decade. Although MT is widely used in clinical practice,
pharmacokinetic–pharmacodynamic profiles of MT or its active me-
tabolites are limited or absent. In canine species, a single study only
reports basic pharmacokinetics of MAA and AA after a single 50 mg/
kg p.o. administration of MT (Kalchofner Guerrero et al., 2015).
The pharmacokinetic profiles of MAA after i.v. and i.m. MT ad-
ministrations were comparable. The substantial differences detected
in Cmax values were ascribable to the routes of administration of MT.
The complete/abrupt input of MT into the vascular compartment (i.v.
injection) may have produced, in the initial minutes, a quicker drug
GIORGI et al.
transformation (MT to MAA) increasing the Cmax of MAA, compared
to the i.m. group where an absorption phase is expected. MAA for-
mation depends on a nonenzymatic, pH-­dependent hydrolysis of MT
and is immediate once MT reaches the circulation (Ergun, Frattarelli,
& Aranda, 2004). Blood pH can accelerate its generation: the lower
the pH, the faster the hydrolysis (Ergun et al., 2004). Animals did not
show any abnormal signs following i.v. administration despite the im-
mediate peak of MAA concentration. The differences in Tmax values
may also be triggered by the absorption phase. The MAA AUC values
(i.v. vs. i.m.) showed that the exposition to the active metabolite over
time is statistically different but still high (75%) after i.m. administra-
tion of MT.
The half-­life (t1/2λz) reported for MAA in the present study
was similar to that previously reported in dogs (4–5 hr) and horses
(4.85 hr), higher to that reported in sheep (1.45–3 hr) and donkeys
(1.81 hr) and lower than that reported in piglets (8.57 hr) (Aupanun
et al., 2016; Burmańczuk et al., 2016; Giorgi et al., 2015; Klaus et al.,
1997; Loescher, 1993). The reasons for this variation might be due to
diverse factors such as differences in administration route, animal spe-
cies, age and pathophysiological conditions of animals, and sensitivity
of the analytical method.
Assuming that after i.v. administration of MT, all the drug is con-
verted to MAA, Vd/F, and CL/F can be considered as absolute values.
Vd/F is large, a finding consistent with MAA being detected in cere-
brospinal fluid and CL/F shows an extraction ratio of 6%–9% (Cohen,
Zylber-­Katz, Caraco, Granit, & Levy, 1998). This finding suggests that
the contribution of the liver and kidney (the main clearance organs) in
the clearance of MAA is limited in the dog. This is also in line with the
small amount of AA produced.
The Cmax value of MAA after p.o. administration of MT, if normal-
ized for the dose, was comparable to that shown in a previous study
in dogs (Kalchofner Guerrero et al., 2015). However, although the
Kalchofner Guerrero study does not report the PK parameters, the
elimination phase of MAA seems slower than that in the present study.
It is likely due to the different MT formulation used: immediate release
(present study) vs. new slow-­release formulation (Kalchofner Guerrero
et al., 2015).
The pharmacokinetic trend of MAA after RC administration of MT
mirrors that of the p.o. group but with lower concentrations. RC ad-
ministration of MT resulted in the lowest Cmax of MAA (2.72 μg/ml)
and AUC RC/IV ratio (32.78%) among the formulations tested. A specific
concern with RC administration of MT, and a likely reason for the low
MAA concentrations detected in the present study, is the chance for
sequestration of drug in fecal matter, a general drawback of RC route
for drug administration. This phenomenon might explain the low AUC
ratio obtained by this administration route. Previous studies reported
that RC administration of suppository marketed for humans showed
reduced bioavailability in dogs (De Vito et al., 2015; Giorgi, Del Carlo,
Saccomanni, Łebkowska-­Wieruszewska, & Kowalski, 2009) .
It is reported that the analgesic effect of MT correlates with
plasma concentration of its two active metabolites (Nikolova et al.,
2012). Both metabolites have been shown to inhibit human COX-­1
and COX-­2 with an IC50 of 2.55 μm and 4.65 μm and 8.2-­ to 9-­fold
|
      7
higher IC50 values for AA (Hinz et al., 2007). If these minimal effective
concentrations (MEC) apply to dogs, plasma concentration of MAA,
but not AA, was above the COX-­1/2 relevant IC50 values for a range of
time correlated to the route of drug administration (Figure 3). Hence,
only MAA might generate a therapeutic effect in the dog. The dif-
ferences in time MAA plasma concentrations were above the MECs
among i.v., i.m., and p.o. groups are small and might not be clinically
relevant. In contrast, the RC group showed a reduced time over the
MEC suggesting that this route of administration might be the least
effective among those tested in the dog.
The pharmacokinetic trends detected for the AA after i.v., i.m., and
RC administrations of MT were similar. In contrast, the p.o. groups
showed plasma concentrations and AUC0-last parameters double those
of the other groups. The explanation for this phenomenon is uncer-
tain, but a similar difference in AA plasma concentrations has already
been observed in horses after i.m. and i.v. administration of MT (Giorgi
et al., 2015) and could be due to first pass metabolism to AA in the
enteral route. As reported above, in humans, the analgesic effect of
MT correlates with the concentration of MAA and AA, which differ
with regard to their time of onset (MAA < AA) and terminal half-­life
(MAA and AA, 4–5 and 5–8 hr, respectively) (Nikolova et al., 2012).
MAA is about 50 times more active than MT as an inhibitor of COX-­3
enzymes, while AA is less active than MT (Nikolova et al., 2012). It is
important to specify that the metabolites which generate the anal-
gesic effect in dogs are still unknown. If it is assumed that analgesic
effect is only attributable to MAA and AA metabolites as in humans,
the impact of AA to overall therapeutic analgesic effect might be neg-
ligible due to both its weak activity and low plasma concentrations
(below the MECs). Hence, it might be supposed that MAA is the main
metabolite responsible for the overall analgesic effect. However, addi-
tional studies are essential, to evaluate whether the metabolic pattern
known in humans matches that in dogs. In addition, pharmacodynamic
studies determining the drug effectiveness in different types of pain
are required.
This is the first study reporting the biopharmaceutics of MT and its
active metabolites MAA and AA after i.v., i.m., p.o., and RC administra-
tions of MT in dogs. The MAA pharmacokinetic profiles were different
with each route of administration, while the AA pharmacokinetic pro-
files were similar for i.v., i.m., and RC administrations. Even though addi-
tional studies are warranted to understand the metabolism of MT along
with its safety profile, rectal administration appears to be the least ef-
ficient route of administration for MT in the dog. However, to prepare
the desired doses for p.o. and RC administration, it was assumed that
the drug was completely uniform in its distribution in the formulations.
This was not analytically confirmed. It is likely therefore that the true
doses administered could have varied from the calculated doses.
AC KNOW L ED G M ENTS
The study was carried out using funds from University of Pisa
(Athenaeum ex 60%, 2015). No external funding was used for prepa-
ration of the manuscript. Authors thank Dr A Di Salvo (University of
Perugia) for the critical editing of the manuscript.
 |
8       GIORGI et al.
CO NFLI CT OF I NTE RE S T
None of the authors have any financial or personal relationships that
could inappropriately influence or bias the content of the manuscript.
AUTHOR’S CONTRIBUTI O N
MG and BLW conceived of the presented idea. MG and TWK devel-
oped the theory and performed the computations. AL, BLW, and HO
carried out the animal study. VDV and AP analyzed the samples. All
authors discussed the results and contributed to the interpretation of
them. MG, HO, and TWK took the lead in writing the manuscript. All
authors read and approved the final manuscript.
O RCI D
M. Giorgi  http://orcid.org/0000-0003-3657-4703
T. W. Kim  http://orcid.org/0000-0002-5239-3833
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How to cite this article: Giorgi M, Łebkowska-Wieruszewska B,
Lisowski A, et al. Pharmacokinetic profiles of the active
metamizole metabolites after four different routes of
administration in healthy dogs. J vet Pharmacol Therap.
2018;00:1–9. https://doi.org/10.1111/jvp.12484