July 2006 Biol. Pharm. Bull.

29(7) 1355—1359 (2006) 1355

Pharmacodynamics and Potential Toxicity of Intranasally Administered

Dipyrone
Shirui MAO,* Shiling YANG, and Dianzhou BI
College of Pharmacy, Shenyang Pharmaceutical University; Wenhua Road 103, Shenyang, 110016, China.
Received October 19, 2005; accepted March 17, 2006

Dipyrone is a non-narcotic analgesic and antipyretic drug used in both pediatric and adult patients. Dipy-
rone solution can be used intranasally as an antipyretic agent for infants. However, dipyrone is not stable in liq-
uid state. Therefore, a stable dipyrone formulation was developed and the antipyretic effect of the formulation
was studied after intranasal administration in rabbits and rats, respectively. To guarantee dose accuracy in ani-
mal studies, effect of dose volume on the distribution of dipyrone solution in rabbit nasal cavities were studied,
using gentian violet as an indicator. Animal fever model and intranasal administration methods were established.
In addition, the potential toxicity of the dipyrone formulation was studied. It was shown that the nasal volume of
rabbits is large enough to hold 100 m l solution. After intranasal administration, improved pharmacodynamics
was obtained with the new developed dipyrone formulation compared to the normal dipyrone solution, and sig-
nificantly decreased body temperature was observed 10 min after dosing. The toxicity was negligible. In conclu-
sion, the dipyrone formulation is effective and safe for clinical medication.
Key words dipyrone; nasal mucosa; pharmacodynamics; toxicity

For systemic medication, drugs are traditionally adminis- state indicated by yellow color, which limited its extensive
tered by oral and parenteral routes. However, in many in- application.
stances, oral administration is unsuitable if a drug is signifi- In order to solve this problem, a stable dipyrone formula-
cantly degraded in the gastrointestinal tract or is considerably tion was developed based on the stability investigation and
metabolized by the first-pass effect in the liver. In addition, formulation optimization.7) Briefly, using a colorimetry
the parenteral route can be undesirable or impractical if a method, influence of pH, sorts of stabilizers on the stability
drug is intended for treatment of children diseases. An alter- of 20% dipyrone solution was studied. It was shown that
native route of administration would be certainly preferable. 20% dipyrone solution was more stable in the pH range of
Compared to other non-parenteral routes, such as buccal, 5.0—6.3 and addition of ethylene diamine tetraacetic acid
rectal, transdermal and vaginal, intranasal administration has (EDTA) and sodium hydrogen carbonate will increase the
many advantages,1,2) such as rapid absorption, comparatively stability further. The color of pure dipyrone solution changed
high bioavailability, patient compliance, bypass of first-pass to yellow 4—6 h after preparation due to oxidization. In con-
hepatic metabolism, bypass of the blood brain barrier (BBB) trast, for the optimized dipyrone formulation only slight yel-
and therefore targeting of the central nervous system (CNS), low appeared after 7 d storage at room temperature, which is
reducing systemic exposure and thus systemic side effects. sufficient for clinical use since the new developed dipyrone
Both macromolecules and small molecules are extensively formulation contains two components, soluble tablet and sol-
studied for nasal delivery.2) vent, and the solution can be prepared before use. The phar-
Dipyrone is a non-narcotic analgesic and antipyretic drug, macodynamics and potential toxicity of the newly developed
which is used in both pediatric and adult patients. It was in- dipyrone formulation were investigated in two animal models
troduced into clinical practice in 1922 and is still in use in after intranasal administration, providing further evidence for
many countries. Agranulocytosis, dipyrone’s most serious clinical application. No similar research was found in avail-
and potentially fatal adverse effect, has led to its withdrawal able literatures to the best of our knowledge. In addition, ef-
in several countries. However, this issue has been criticized fect of dose volume on the distribution of drug solution in
by many other authors.3) It was reported that agranulocytosis the nostril was studied to guarantee the dosing accuracy in
is subject to geographical variability, ratio with at risks rang- the animal experiment.
ing from 0.8—23.7.4) In many countries dipyrone is still
widely used in children and adults and even as an over-the- MATERIALS AND METHODS
counter (OTC) preparation.5) Due to its strong analgesic ef-
fect, available parenteral formulation and low cost, dipyrone Materials Dipyrone was purchased from Shandong Xin-
is widely used in Europe and South America. Recently, addi- hua Pharmaceutical Factory, China. Typhoid, paratyphoid
tional beneficial effects of dipyrone (vascular smooth muscle triple vaccines were purchased from Institute of Biological
relaxant, antiapoptotic, and anticonvulsant) have been re- Product, The Ministry of Health, Wuhan, China. The suspen-
ported and increased the interest in this compound.5) sion of beer yeast was a kind gift from Shenyang Beer Fac-
Currently dipyrone is included in the pharmacopoeias of tory. All other chemicals used were of analytical reagent
four countries, namely China, European, Japan and Poland. It grade or higher from commercial suppliers.
is noted in the Chinese pharmacopoeia that 20% dipyrone so- Animals Adult male New Zealand-derived white rabbits
lution can be used intranasally as an antipyretic agent for in- weighing 2.0—3.0 kg, male Wistar rats weighting 300
25 g,
fants. Its effectiveness and convenience have been demon- were purchased from the Animal Center of Shenyang Phar-
strated in clinical.6) However, dipyrone is not stable in liquid maceutical University. All animals were housed at a constant
∗ To whom correspondence should be addressed. e-mail: [email protected] © 2006 Pharmaceutical Society of Japan
1356 Vol. 29, No. 7

temperature of 20
5 °C, animal food and water were al- 1 min to prevent leakage of the solution out of the nostril.
lowed ad libitum. All experimental protocols described in When a single administration is not sufficient due to the large
this study were approved by the Ethics Review Committee dose, multiply dosing will be employed with an interval of
for Animal Experimentation of Shenyang Pharmaceutical 3 min. Placebo animals underwent the same procedures but
University. were given 0.9% NaCl or excipients solution. Rectal temper-
Distribution of Dipyrone Solution Four rabbits were re- atures were measured at 0, 10, 20, 30, 40, 60, 90, 120, 180,
strained in the rabbit boxes and the heads kept in a supine po- 240, 300 and 360 min after administration.
sition. Different volumes of dipyrone (20% with 0.2% gen- Similarly, rats with normal rectal temperature in the range
tian violet), 20, 50, 70 and 100 m l, were administered into the of 36.6—38.3 °C and the difference in two measurements
right and left nostrils via a polyethylene tube inserted into less than 0.2 °C were used. Rats fever model was established
10 mm. The rabbits were sacrificed by decapitation 5 min by subcutaneous injection of 15% beer yeast suspension
after dosing, and the mandible, brain and excess soft tissue 10 ml/kg at the backside. Rats with body temperature in-
removed. The nasal cavity was opened along the midline sep- crease 0.8 °C were divided into four groups randomly, that
tum and drug solution distribution was observed and photos is, excipents group, dipyrone small dose group (90 mg/kg),
were taken. Additionally, three other rabbits were used for dipyrone large dose group (180 mg/kg) and dipyrone control
elimination study. Briefly, the first rabbit was sacrificed im- group (180 mg/kg) (n10). The dose was selected based on
mediately after dosing 50 m l of drug solution in each nostril, preliminary experiments. The administration volume is 20 m l
while the other two were sacrificed 2 h after administration. each nostril. Rats were accustomed to the dosing procedure
The nasal cavity was opened as abovementioned and com- to prevent withdrawal and defense reactions that may lead to
pared with that of the first rabbit. inaccurate dosing. Other procedures are the same as de-
Preparation of Nasal Formulations All drug solutions scribed for rabbits.
were prepared in pyrogen-free glassware that was heated at Toxicity Investigation Acute Toxicity after Intranasal
115 °C for 5 h before use. All solutions were passed through Administration: Clinically, dipyrone dose for infants is 5—
0.22 m m Millipore bacterial filters prior to administration. 10 mg/kg for injection and 10—20 mg/kg for tablet. In order
The composition of the tested dipyrone formulation is as fol- to investigate the potential toxicity of intranasally adminis-
lows: 20% dipyrone, 3% polyvinyl pyrrolidone as viscosity- tered dipyrone, 1000 mg/kg, a dose 50 times higher than the
enhancing agent (as adhesive in the soluble tablet), 0.2% upper limit (20 mg/kg) of dipyrone tablet, was tested. Eight
sodium hydrogen carbonate as antioxidant and 0.05% adult male New Zealand-derived white rabbits weighing be-
(EDTA) as chelator. The pH was adjusted to 5.5. The control tween 2.0 and 2.5 kg were randomly divided into two groups,
is 20% dipyrone solution without any excipients. All solu- a test group and a control group. For the test group, dipyrone
tions were prepared freshly. was administered intranasally at the dose of 1000 mg/kg.
Establishment of Animal Models The antipyretic activ- 0.9% NaCl was administered in the same manner as a con-
ity of dipyrone after intranasal administration was tested in trol. After administration, the rabbits were observed for 7 d.
two different animal models, rabbits and rats respectively. Any changes of the animals, including weight, respiration,
New Zealand-derived white rabbits were fasted overnight circulation, central nervous system and the activity of ex-
and weighed before experiment. Only rabbits with body tem- tremities were noted.
perature in the range of 38.7—40.3 °C and the difference in Nasal Mucosa Irritation: The potential irritation of the new
two measurements less than 0.2 °C were used. The rectal dipyrone formulation to the nasal mucosa was investigated
temperature of each animal was allowed to stabilize for at after a single and multiple administrations. Nine adult male
least 90 min before experimentation. The fever model was New Zealand-derived white rabbits weighing between 2.0
established by injecting typhoid, paratyphoid triple vaccines and 2.5 kg were randomly divided into three groups, a single
into the marginal ear vein of rabbits at a dose of 1 mg/kg. administration group, a multiple dosing group and a control
Thirty rabbits, with temperature increase 0.5 °C after 2 h of group. In the single administration group, the dipyrone solu-
injection, were randomly divided into five groups of six, tion was administered intranasally at a dose of 150 mg/kg
namely, 0.9% NaCl group, excipients group (3% polyvinyl and the rabbits were decapsulated 24 h after administration.
pyrrolidone, 0.2% sodium hydrogen carbonate and 0.05% Nasal parts were removed and fixed with 10% formalin. In
EDTA), dipyrone small dose group (75 mg/kg, test formula- the multiple dosing groups, the rabbits were administered in-
tion), dipyrone large dose group (150 mg/kg, test formula- tranasally at a dose of 150 mg/kg for 7 d, each daily. The rab-
tion) and dipyrone control group (150 mg/kg). The dose was bits were decapsulated 24 h after the last administration. His-
selected based on preliminary experiments. Throughout the tological changes of nasal mucosa were examined as de-
experiment, rectal temperatures (Tco) were measured at pre- scribed in the literature8) and compared with the control
determined time intervals with a thermometer. Administra- group.
tion method: The rabbit was restrained in a wooden box dur- Statistical Data Analysis Statistical comparisons were
ing the whole experiment. All nasal preparations were ad- performed using the Student’s t test for two groups and a
ministered with the rabbit in a fixed standing position forcing one-way ANOVA for multiple groups. A value of p0.05
its head slightly backward. Fifty microliters of the tested so- was considered to be indicative of statistical significance.
lution was administered into each nostril via a polyethylene
tube inserted into 10 mm. Before administration, the tube RESULTS AND DISCUSSION
was inserted into the nostril two times to prevent the sneez-
ing reflex of the rabbits upon insertion. After intranasal ad- Effect of Dose Volume on the Distribution of Dipyrone
ministration the rabbit box was kept in a vertical position for Solution Due to the structural characteristics of the nasal
July 2006 1357

Fig. 1. Effect of Dose Volume on the Distribution of Dipyrone Solution in the Nasal Cavity of Rabbits
(A) 100 m l, (B) 70 m l, (C) 50 m l, (D) 20 m l. The dipyrone solution contained 0.2% gentian violet, and observation was made on gentian violet.

cavity and their role of transferring drugs from the nasal

membrane into systemic circulation, drug distribution in the
nasal cavity affects the efficiency of nasal absorption. Appli-
cation of a large volume of solution gives a good distribution
over the nasal cavity, whereas a few drops give only unsatis-
factory results.9) However, solution leakage should be taken
into consideration when large volume was used. In order to
find the best volume for animal experiment, drug distribution
in the nasal cavity and in particular the possibility of leakage
in the posterior region at relative large volume was investi-
gated using different volume of dipyrone solution. For the
ease of observation, 0.2% gentian violet was added into the
Fig. 2. Images Indicating Drug Substance Elimination from the Nasal
solution as an indicator. Mucosa
Effect of dose volume on the distribution of dipyrone (A) Decapitated immediately after administration of 50 m l dipyrone solution. (B) De-
using gentian violet as an indicator is shown in Fig. 1. The capitated 2 h after administration of 50 m l dipyrone solution. The dipyrone solution con-
deposition area increased gradually with increasing volume. tained 0.2% gentian violet, and observation was made on gentian violet.

It shows that the nasal surface is large enough to hold 100 m l

solution in each nostril with little leakage. When the volume
was less than 70 m l, all the solution was retained in the nasal
cavity. Fifty microliters, which is in accordance with the lit-
erature report,10) was used in the pharmacological study and
the dosing accuracy was guaranteed.
On the other hand, time dependent nasal distribution and
elimination was investigated 5 min and 2 h after administer-
ing 50 m l of test dipyrone solution containing 0.2% gentian
violet. The results are shown in Fig. 2. The purple color be-
came weaker 2 h later, implying that the solution was proba-
bly absorbed across the nasal mucosa, or eliminated by the
nasal cavity due to the mucociliary clearance.
Pharmacodynamics of Dipyrone after Intranasal Ad-
ministration To study the absorption of dipyrone via the
nasal mucosa, antipyretic studies were performed in non-sur-
gically modified conscious animals. No anesthetic agents
were used in this study, as they were reported to increase the
nasal absorption of drugs greatly.11) This is probably due to
Fig. 3. Time Course of Mean Rectal Temperature in Rabbits Randomized
the impairment of the mucociliary clearance in the deeply to Receive Saline, Excipients, and Dipyrone Solutions
anaesthetized rats, together with the lack of any possible loss Bars show standard errors. Statistically significant difference (∗ p0.01, ∗∗ p0.05)
due to drainage and mechanical removal. from saline or excipients groups; test 150 mg/kg versus control 150 mg/kg:
p0.05
(n6). Rabbits were treated with typhoid, paratyphoid triple vaccines in advance. Test
Antipyretic Effect to the Triple Vaccine Induced Rabbits: solution is 20% dipyrone with excipients (PVP, EDTA, NaHCO3). Control solution is
In this study, 50 m l of dipyrone solution was delivered into 20% dipyrone without any excipients.
each nostril and a total volume of 100 m l per rabbit was ad-
ministered. This administration protocol was selected be- spray or 9% obtained from the 1100 m l intranasal dose.
cause the site of drug deposition within the nose is not only Such findings indicate that an optimal bioavailability may be
dependent upon the dosage form but also the dose volume. obtained by administering an equal dose into each nostril.
Previous studies12) showed that the bioavailability of in- Pharmacological responses of tested dipyrone formula-
tranasal desmopressin from a 250 m l dose was 20%, which tions were determined after intranasal administration and
represented a marked increase over 11% found with 150 m l compared with that of the control, as shown in Fig. 3. No sig-
1358 Vol. 29, No. 7

viscosity-enhancing agents on the intranasal absorption of

drugs, reports from literatures were quite controversial.
Ikeda13) showed that the bioavailability of penicillamine in-
creased from 11.7 to 20% after adding 2% hydroxypropyl
cellulose (HPC) in the solution. Hussain14) showed that the
blood levels of propranolol increased remarkably after
adding 3% (w/v) methyl cellulose (MC) in the nasal solution.
However, Harris et al.15) indicated that viscosity-enhancing
agent could only decrease the drug diffusion rate, which re-
sults in a longer retention time and slower clearance along
the nasopharynx, but no significant improvement in bioavail-
ablity was demonstrated. Anyhow, our experiment did
demonstrate that intranasal absorption of dipyrone could be
enhanced by increasing the viscosity of the administered so-
lution. It was clear that the contribution of improved stability
Fig. 4. Time Course of Mean Rectal Temperature in Rats Randomized to couldn’t be excluded. However, since the solutions were pre-
Receive Saline, Excipients and Dipyrone Solutions pared freshly before the experiment, and drug content de-
Bars show standard errors. Statistically significant difference (∗ p0.01, ∗∗ p0.05) crease was less than 2%, therefore, it was not a main con-
from saline group; test 180 mg/kg versus control 180 mg/kg:
p0.05 (n10). Rats
were treated with 15% beer yeast suspension in advance. Test solution is 20% dipyrone tributing factor. We assume that the influence of viscosity-en-
with excipients (PVP, EDTA, NaHCO3). Control solution is 20% dipyrone without any hancing agent on the intranasal absorption of drugs is related
excipients.
to the properties of the drug substance, such as molecular
weight, lipophilicity. Dipyrone is a strong hydrophilic drug
nificant difference between excipients and 0.9% NaCl group with a low molecular weight (351.3). Its absorption mecha-
was found, implying that the excipients have no pharmaco- nism via the nasal mucosa is passive diffusion.16) Increasing
logical activity. Body temperature decreased significantly in viscosity prolonged the contact time with the nasal mucosa,
all the treatment groups (p0.05) and a dose–response rela- leading to enhanced absorption.
tionship was found. For the large dose dipyrone (150 mg/kg) On the other hand, it was noted that a significant rectal
group, body temperature began to decrease significantly temperature decrease was observed at 10 min after adminis-
10 min after dosing (p0.01) and the action lasted for 6 h. In tering large dose dipyrone in both animal models. In contrast,
contrast, considerably decreased temperature was found at for the dipyrone control group, a remarkable activity was
20 min for small dose dipyrone (75 mg/kg) (p0.05) group found at 10 min in rats (p0.01) and 1 h in rabbits (p0.05).
and it lasted for 5 h. Regarding to the dipyrone control group, This could probably associate with interanimal variability.
significant antipyretic effect was found at 1 h (p0.05) and Toxicity of the Dipyrone Nasal Formulation The effec-
the action lasted for 4 h. No significant difference between tiveness of dipyrone was demonstrated in animal experi-
dipyrone control and small dose groups was found (p0.05), ments. However, the safety of the formulation is of special
but they were statistically different with the large dose group importance before clinical application. Therefore, the acute
(p0.05). This indicates that at the same dose, dipyrone in toxicity of the dipyrone formulation was investigated with an
the new formulation has stronger pharmacological activity. extremely high dose. Fortunately, no abnormality was found
Antipyretic Effect to Beer Yeast Induced Rats: Similarly, during the 7 days’ observation. The hair color of the rabbits
intranasal administration of dipyrone (90 and 180 mg/kg) in- was glossy, the respiration was normal, and no apparent ex-
duced significant body temperature decrease in rats (p0.05) citement or restrain behavior was observed. All the activities
and the activity was dose dependent, as shown in Fig. 4. were normal without death. No significant weight difference
Compared to the 0.9% NaCl group, a remarkable body tem- was detected (p0.05).
perature decrease was observed at 10 min in dipyrone small, In addition, morphology of the nasal mucosa after irrita-
large dose groups (p0.01) and control group (p0.01). The tion investigation is shown in Fig. 5. In sections prepared
activity lasted for more than 6 h for the large dose group, from undosed animals, a continuous epithelial layer covered
compared to 5 h for the small dose and control groups. A sta- all surfaces of the rat nasal cavity (Fig. 5A). After a single
tistical difference was found between the small and large and multiply dosing, the nasal cavities were covered by an in-
dose groups (p0.05), the large dose and the control groups tact, undamaged epithelium layer (Figs. 1B, 1C), comparable
(p0.05). However, no significant difference was found be- to that of the undosed control (Fig. 5A), indicating that the
tween dipyrone small dose and control groups (p0.05), im- formulation has no or slight irritation to the nasal mucosa.
plying an enhanced activity of dipyrone in the new formula- In summary, these results showed that the new developed
tion, an identical conclusion drawn from the rabbit experi- dipyrone formulation was almost nontoxic and was suitable
ment. for clinical use.
Apparently, the above animal experiments demonstrated Absorption of Dipyrone across the Nasal Mucosa
that the pharmacodynamics of the tested dipyrone solution Pharmacodynamics studies showed that dipyrone had strong
was considerably stronger than that of the control at the same antipyretic effect after intranasal administration both in rab-
dose. This could probably be explained by the viscosity-en- bits and rats, implying that dipyrone was absorbed into the
hancing agent in the tested solution, which prolonged the re- systemic circulation via the nasal mucosa. This point was in
tention time of dipyrone on the nasal mucosa to some extent, agreement with our previous investigation performed with in
leading to enhanced absorption. Regarding the influence of situ nasal recirculation method.16) This technique involves
July 2006 1359

centration of the drug remaining in the perfusing solution

using a HPLC method. The absorption amount, absorption
regulation and absorption rate of dipyrone across rat mucosa
were investigated in detail previously.16) It proved that a large
amount of dipyrone was absorbed via the nasal mucosa and
the absorption mechanism was passive diffusion. The aver-
age absorption rate constant was 0.02219 min1. It has been
reported that the in situ nasal absorption studies could be
used to predict approximately in vivo nasal absorption of
drugs.17) This point was demonstrated here in this paper.

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