Pharmacology & Pharmacy, 2011, 2, 180-188
doi:10.4236/pp.2011.23026 Published Online July 2011 (http://www.scirp.org/journal/pp)
Intranasal Delivery of Two Benzodiazepines,
Midazolam and Diazepam, by a Microemulsion
System
Shafir Botner, Amnon C. Sintov*
Department of Biomedical Engineering, Faculty of Engineering Sciences, Ben Gurion University of the Negev, Beer Sheva, Israel.
Received January 2nd, 2011; revised May 4th, 2011; accepted June 23th, 2011.
ABSTRACT
Nasal application of benzodiazepines might be an alternative to intravenous administration in acute clinical situations
such as seizures emergencies. However, irritation and pain as well as symptoms like teary eyes, dizziness, discomfort,
nasal drainage and bad taste usually accompany subject received midazolam and diazepam via the nasal route. The
purpose of this study was to evaluate the use of a new alcohol-free microemulsion system as a carrier for diazepam or
midazolam given intranasally. Midazolam (base) or diazepam was solubilized in the microemulsion to obtain a high
drug concentration of 25 mg/g (2.5% by weight), to provide 2.5 mg drug in 100 µl spray (d ≈ 1.00 g/ml). The nasal ab-
sorption of both drugs from the same microemulsion formulation (containing 20% aqueous phase) was found to be
fairly rapid after administration of 0.4 mg/kg to rabbits. The absolute bioavailability of diazepam after intranasal ad-
ministration using this formulation was 33.45% ± 12.36% and the tmax was 18.33 ± 23.09 min, which was twice longer
than the tmax obtained after midazolam administration, 9.25 ± 6.75 min. The pharmacokinetic parameters of midazolam
in W/O (20% water) microemulsion and their comparison with midazolam in O/W (50% water) microemulsion have
shown that both formulations resulted in a relatively short time to reach the peak plasma level (tmax), that is, 9.25 ± 6.75
min and 6.75 ± 5.67 min, respectively. However, the peak plasma levels (Cmax) and the absolute bioavailability (FA) of
midazolam were significantly higher after administration of the W/O formulation than those obtained after application
of O/W formulation, i.e., 46.62 ± 17.38 µg/ml vs. 15.44 ± 4.00 µg/ml, and 35.19 % ± 11.83% vs. 19.83% ± 16.32%, re-
spectively. Our results suggest that the new microemulsion system may be useful for getting rapid-onset of midazolam
and diazepam following intranasal administration, resulting in reasonable peak plasma levels and bioavailability, but
most importantly, providing a high measure of tolerability and comfort.
Keywords: Microemulsion, Intranasal Drug Delivery, Benzodiazepines, Nasal Spray, Diazepam, Midazolam
1. Introduction
Benzodiazepines are a group of psychoactive drugs with
clinical effects like sedation, hypnotic, anxiolytic, anti-
convulsant, muscle relaxant and amnesia. These thera-
peutic properties make benzodiazepines useful in treating
anxiety, insomnia, agitation, seizures, muscle spasms,
alcohol withdrawal syndrome and as a premedication for
various medical or dental procedures. Benzodiazepines
have been the first line treatment of seizures, which are
an emergency medical situation [1]. While untreated
prolonged seizures increase the risk of mortality, mor-
bidity and permanent brain damage, an early and rapid
termination of the seizures by benzodiazepines is needed.
Benzodiazepines exert their anticonvulsant effect by in-
teracting with γ-aminobutric acid (GABA) receptors at
the benzodiazepine binding site and allosterically modi-
fying GABAA receptor current to enhance inhibition [2-
4].
Traditionally, intravenous and especially rectal diaze-
pam (DZP), a highly lipophilic benzodiazepine, has been
used as front line therapy. However, diazepam via
intravenous and rectal routes have several drawbacks [5-
8]: 1) The establishment of an intravenous access is not
practical in an emergency situation when the patient is
not in a hospital. A highly qualified, trained medical
person is required for this procedure; 2) The use of rectal
diazepam results in variable plasma levels and fails to
terminate 30% of seizures [9]. It is also socially embar-
rassing, and although difficult to administer during con-

Intranasal Delivery of Two Benzodiazepines, Midazolam and Diazepam, by a Microemulsion System 181
vulsions can be used only in children and in a few cases
in adolescents; 3) it is highly lipophilic and therefore has
a large volume of distribution. Repeated doses are some-
time needed and its accumulation may lead to compli-
cations such as bradypnea and even respiratory arrest
[10,11]. Diazepam is also available in tablets; however,
the oral route is not accessible during seizures and cannot
be used. Thus, an alternative fast-acting benzodiazepine
delivery route, such as the nasal route, is needed. By the
alternative route, diazepam should be easily administered
by the surrounding people or caregivers and could dra-
matically improve the management of out-of-hospital
seizures as well as the patient recovery. The intranasal
administration of diazepam was evaluated in several
studies, in which supersaturated formulations were used
with solvents such as a glycofurol/water mixture [7,12],
glycofurol/polyethylene glycol 200 [13,14], propylene
glycol/ethanol [15], and polyethylene glycol 300 [16].
Human studies [7,12], which were conducted on human
volunteers reported that subjects rated nasal diazepam as
causing considerable pain immediately following admini-
stration. In addition, discomfort, nasal drainage and wat-
ery eyes were also reported.
Midazolam hydrochloride (MDZ-HCl) is a short act-
ing, water soluble benzodiazepine. Its effectiveness on
the CNS is dependent on the dose, route of administra-
tion, and whether it is used concomitantly with other
medications. Midazolam has also been used for rapid an-
esthesia at emergency setting and as an agent for seda-
tion prior to medical procedures. Because of its high
solubility in aqueous solutions, MDZ-HCl can be used
intravenously, intramuscularly, buccally, and intranasally.
Although midazolam is marketed only in injectable and
oral syrup formulations, there is increased interest in its
administration via the nasal route and it is indeed the
most extensively studied nasal benzodiazepine [17]. A
survey research [18] among anesthesiologists showed
that the most commonly used (>80%) sedative premedi-
cant in children was midazolam, 8% of which have prac-
ticed intranasal midazolam, apparently in “off-label” use,
to premedicate pediatric patients preoperatively. The
interest in intranasal drug delivery arises from the unique
advantages presented by the nasal cavity such as: 1) A
large surface area available for drug deposition and ab-
sorption, 2) The nasal epithelium is thin and highly vas-
cularized, 3) Absorbed substances are transported di-
rectly into the systemic circulation thereby avoiding the
first pass metabolic effect, and 4) In some cases, drug
can be absorbed directly into the CNS by passing the
tight blood brain barrier [19]. Intranasal midazolam has
already been explored during the last decade and a
number of clinical works revealed the potential of its
administration via the nasal route [9-11,17,20-23]. Nev-
ertheless, there are still issues waiting to be resolved.
Most studies reported the use of various dilutions of a
commercial midazolam, dripped by syringe into patient's
nostrils. This use of aqueous solutions (usually employed
for injections) is not optimal for intranasal administration
from two main reasons: a) The acidic pH (pH ≈ 3) is too
low for the nasal mucosal membrane and is therefore a
potential irritant, and b) The solutions are too diluted to
provide a considerably small volume for human nostril,
namely, 100 - 150 µl of liquid at a time. Optimal for-
mulation should contain at least 2.5 mg of midazolam in
100 µl solution. Although more concentrated MDZ-HCl
solutions [17,21,26] resulted in a relatively high bio-
availability in healthy volunteers, the researchers reported
that irritation (“burning” sensation) and pain occurred in
all subject received midazolam, as well as symptoms like
teary eyes, dizziness, and bad taste.
In the present paper, we propose the use of a new mi-
croemulsion that in pre-clinical studies, seemed to have
solved the problem of irritability following regular nasal
benzodiazepine administrations. We have studied and
compared the pharmacokinetic characteristics of diaze-
pam and midazolam applied to rabbits in a microemul-
sion formulation via the nasal route. The new microe-
mulsion system did not contain alcohols or other irritant
chemicals and is generally recognized as safe (GRAS)
compounds. The purpose of this work was to evaluate
the use of the new microemulsion formulation as a car-
rier for diazepam or midazolam given via the nasal route.
2. Materials and Methods
2.1. Materials
Midazolam (base) and diazepam were kindly donated by
Rafa Laboratories Ltd., Jerusalem, Israel. Commercial
midazolam HCl solution for injection (Midolam amps., 5
mg/ml or 0.5% wt/v, Rafa Laboratories, Israel) was pur-
chased from a local pharmacy. Commercial diazepam
solution for injection (Assival amps., 10 mg/2ml, Teva
Group, Israel) was also purchased from a local pharmacy.
Glyceryl oleate was obtained from Uniqema, Brombor-
ough Pool, The Wirral, UK. Labrasol was obtained from
Gattefosse, France. Isopropyl palmitate (IPP) and pro-
pylene carbonate were purchased from Aldrich (Sigma-
Aldrich Inc., St. Louis, MO). Acepromazine (10 mg/ml
acepromazine maleate) was used from PromAce In-
jectable, Fort Dodge-Animal Health (Iowa, USA).
2.2. Preparation of Microemulsions
Generally, microemulsions were prepared by mixing La-
brasol, glyceryl oleate (surfactants) and isopropyl palmi-
tate (oil) with propylene carbonate (co-surfactant) and
water. Appropriate quantities of midazolam (base) or di-
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182 Intranasal Delivery of Two Benzodiazepines, Midazolam and Diazepam, by a Microemulsion System
azepam were then solubilized in the microemulsion to
reach a final concentration of 2.5% (wt/wt) of the desired
drug. The monophasic formulations were formed after a
short stirring at room temperature. The cosurfactant-
surfactants (CoS/S) weight ratio was 1:5, and the surfac-
tants; ratio was 1:3.
2.3. Construction of Phase Diagrams
Pseudo-ternary phase diagrams of oil, distilled water, and
co-surfactant (CoS)—surfactants (S) mixtures were con-
structed at fixed CoS/S weight ratios. The weight ratio of
the two surfactants, glyceryl oleate to Labrasol derivative,
were fixed and kept constant. Phase diagrams were ob-
tained by visual inspection of mixtures of the ingredients,
which were pre-weighed into glass vials, titrated with
water and stirred well at room temperature. As a con-
venient method, the construction of the phase diagrams
were done by drawing “water dilution lines” representing
an increase of water content while decreasing CoS-S and
oil levels. The water was titrated along dilution lines
drawn from the water apex to the opposite surfactant side
of the triangle. The line was arbitrarily denoted as the
value of the line intersection with the surfactant scale
(e.g., DL87 means line representing a surfactant-to-oil
ratio of 87:13). In case turbidity appeared followed by a
phase separation, the samples were considered as bi-
phasic. In case monophasic, clear and transparent mix-
tures were visualized, the samples were marked as points
in the phase diagram. The area covered by these points
was considered as the microemulsion region of exis-
tence.
2.4. Dynamic Light Scattering (DLS)
The hydrodynamic diameter spectrum of microemulsion
nano-droplets was collected using CGS-3 Compact Go-
niometer System (ALV GmbH, Langen, Germany). The
laser power was 20 mW at the He-Ne laser line (632.8
nm). Correlograms were calculated by ALV/LSE 5003
correlator, which were collected at 60˚, during 10 s for
20 times, at 25˚C. Measurements were performed at per-
manent angle of 60˚. The droplet size was calculated
using the Stokes-Einstein relationship, and the analysis
was based on regularization method as described by
Provencher [24].
2.5. Pharmacokinetic Study
All animal procedures were performed in accordance
with protocols reviewed and approved by the Institu-
tional & Use Committee, Ben Gurion University of the
Negev, which complies with the Israeli Law of Human
Care and Use of Laboratory Animals. New Zealand
white rabbits (HsdIf: NZW males, 2.0 - 3.5 kg body
weight, Harlan, Jerusalem) were used in the experiments.
The rabbits were housed individually with free access to
food and water. A 12 h light/12 h dark cycle was held to
keep a normal circadian rhythm in the animals. Nasal
formulations or intravenous drug dosage forms were ad-
ministered in a randomized cross-over design with a
wash-out period of at least four days. After the animals
had been tranquillized with 0.5 ml acepromazine, Ven-
flon™ cannula (22 G, Poly Medicure Ltd., Faridabad,
India) was inserted into the main artery of the rabbit ear.
Each rabbit was weighed and the drug (MDZ or DZP)
was nasally or intravenously administered. Microemul-
sion containing 2.5% wt/wt (=25 mg/g) midazolam or
diazepam was administered at a 0.4 mg/kg dose by nasal
spraying (approx. 100 µl of microemulsion containing
2.5 mg of drug, approx. 50 µl in each nostril). For the
purpose of comparison, MDZ at the same dosage was
also applied in macro-emulsion formulation (same for-
mulation without co-surfactant) and in a mixture of oil
and surfactants (same formulation without an aqueous
phase), all based on the same microemulsion’s compo-
nents and their ratios. Commercial midazolam solution
for injection (5 mg/ml) had first been diluted in sterile
saline solution (×5) before administered intravenously at
a 0.2 mg/kg dose (approx. 0.5 ml solution). Commercial
diazepam solution for injection (5 mg/ml) had first been
×5 diluted in propylene glycol then administered intra-
venously at a 0.2 mg/kg dose (approx. 0.5 ml solution).
The exact application volume was determined according
to the individual body weight. Spraying technique was
developed by using a 100 µl syringe connected to MAD
Nasal Drug Delivery Device (MAD 320, Wolfe Tory
Medical, Inc., Salt Lake City, UT). Blood samples were
collected at 0, 2, 5, 15, 30, 45, 60, 90, 120, and 180 min-
utes after application in heparin-containing tubes. Plasma
was obtained after centrifugation at 10,000 rpm for 10
minutes, and stored at –20˚C until analyzed for MDZ or
DZP. Plasma drug concentrations were determined using
LC /MS/MS method pre-developed in our laboratory.
2.6. Plasma Drug Determination
A LC/MS/MS analysis was performed using a Reprosil
C18-AQ 5 µm column (100 × 2 mm) (Dr. Maisch, Ger-
many), equipped with a C18 guard column. The mobile
phase consisted of a 33.3:66.7 v/v mixture of 1mM am-
monium acetate buffer (eluent A) and methanol-acetoni-
trile (20:80 v/v) (eluent B). The flow rate was 0.3 ml/min
at ambient temperature. Detection was performed using
an API 2000 instrument (MDX SCIEX, Concord, On-
tario, Canada). The API 2000 ES source was tuned by
infusing a standard solution of drug (1 µg/ml in methanol)
into the source at a flow rate of 10 µL/min. The optimal
parameters were: source temperature 550˚C, decluster-
ing potential 96 eV, focusing potential 370 eV, entrance
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Intranasal Delivery of Two Benzodiazepines, Midazolam and Diazepam, by a Microemulsion System 183
potential 12 eV, collision energy 37 eV, and collision
cell exit potential (CXP) 4 eV. The spectrometer was
used in the MS/MS mode with MRM of fragmentation
reactions selected for each drug. Positive ion mode was
used, and selected-ion monitoring was accomplished at
m/z 326 for MDZ and m/z 285 for DZP. Quantitative
on-line HPLC–ESI–MS/MS analyses were performed
using an Analyst Software system interfaced to an Ap-
plied Biosystems API2000 instrument (Foster City, CA,
USA).
2.7. Data Analysis
All pharmacokinetic parameters, i.e., peak plasma level
(Cmax), time to reach peak plasma level (tmax), elimination
terminal slope (λz), half-life of elimination (t1/2), were
obtained after analysis of the individual time-plasma
concentrations by WinNonlin Professional software ver-
sion 5.2.1 (Pharsight Corporation, Mountain View, CA),
using a non-compartmental model. The area under the
plasma concentration of midazolam or diazepam versus
time curve (AUC0→∞) was calculated using the linear
trapezoidal rule and extrapolated to infinity by adding
the last measurement of plasma concentration divided by
the terminal slope (Clast/λz). The nasal bioavailability of
midazolam and diazepam was relative to intravenous
administration done in the same animal normalized to
dose.
3. Results and Discussion
We first examined the immediate response of four con-
scious rabbits to midazolam in microemulsion formula-
tion (2.5%) compared to a plain aqueous solution of mi-
dazolam hydrochloride (0.5%). In a cross-over method,
aliquots (500 µl) of each formulation were sprayed into
one nostril and the animals were carefully observed. All
animals responded to the aqueous solution application in
a wild behavior, mainly by shaking their heads and mak-
ing sounds of distress. This response was ceased after a
few minutes. In addition, a temporary swelling was ob-
served around the nostril, where the drug had been ap-
plied. The swelling lasted for about 30 - 60 minutes. In
comparison, no visual sign of an irritative response was
noted after a similar volume application of a 5-fold
higher concentration of the drug given in the microemul-
sion.
To characterize the microemulsion system we con-
structed a phase diagram and measured the droplet size
of the inner phase. By constructing a phase diagram it is
easier to determine the range of concentrations and the
ratios of components in the existence region of microe-
mulsion. A pseudo-ternary phase diagram at a CoS/S
weight ratio of 1:5 is shown in Figure 1. As seen, in
compositions containing more than 10% oil, the maximal
Figure 1. Pseudo-ternary phase diagram of a microemul-
sion system (shaded area) made of isopropyl palmitate (oil),
glyceryl oleate and Labrasol (as surfactants at a 1:3 w/w
ratio), propylene carbonate (co-surfactant) and water. The
co-surfactant/surfactant ratio was 1:5.
water solubilization capacity of this microemulsion sys-
tem is 50%. Decreasing water content below 50% en-
ables an incorporation of more isopropyl palmitate into
the microemulsion. The average droplet size of drug-
unloaded microemulsion containing 20% water (surfac-
tants-to-oil ratio = 87:13) was 2 nm in diameter (99.7%
of total droplets). Our previous studies using this mi-
croemulsion system showed that loading of drug mole-
cules and even of protein drugs into the nano-droplets
did not much change the average droplet size [25].
The drug (midazolam base or diazepam) was solubi-
lized in the microemulsion to obtain a final concentration
of 25 mg/g (2.5% by weight), to provide 2.5 mg drug in
100 µl spray (d ≈ 1.00 g/ml). The microemulsion formu-
lation of choice used for the pharmacokinetic studies
contained 20% aqueous phase for the nasal delivery of
both midazolam and diazepam. As presented in Table 1,
a formulation containing 20% aqueous phase was chosen
due to an achievement of a higher bioavailability com-
pared with a formulation containing 50% aqueous phase.
The influence of the quantity of the water phase on drug
absorption may be due to changes occurring in the inter-
facial membrane's characteristics of the system, such as
micellar inversion (W/O to O/W and vice versa) and a
possible change in the number of surfactant layers in
which the drug is entrapped. Figure 2 illustrates sche-
matically how inversion from O/W to W/O microemul-
ion can affect the drug accessibility to free diffusion s
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Intranasal Delivery of Two Benzodiazepines, Midazolam and Diazepam, by a Microemulsion System 184
Table 1. Mean pharmacokinetic parameters of midazolam after IV and IN administration to rabbits (total of 8 animals).
Study 1, n = 4 (paired) Study 2, n = 4 (paired)
PK parameter IV solution* 0.2 mg/kgIN microemulsion with
20% water 0.4 mg/kgIV solution** 0.2 mg/kg IN microemulsion with 50% water 0.4 mg/kg
Cmax (μg/ml) 188.80 (±79.63) 46.62 (±17.38) 195.87 (±37.43) 15.44 (±4.00)
tmax (min) 0 9.25 (±6.75) 0 6.75 (±5.67)

z (min–1) 0.0207 (±0.0035) 0.0169 (±0.0041) 0.0394 (±0.0145) 0.0338 (±0.0158)
Elimination t1/2 (min) 34.19 (±5.88) 43.15 (±11.63) 19.57 (±7.24) 28.32 (±22.86)
AUC0–∞ (μg·min·ml–1) 3499 (±991) 2494 (±1098) 2050 (±334) 789 (±607)
AUC0–∞/dose (μg·min·ml–1·D–1) 17.50 (±4.95) 6.23 (±2.74) 10.25 (±1.67) 1.97 (±1.51)
FAa (%) ---- 35.19 (±11.83) ---- 19.83 (±16.32)
*IV reference group for the IN group which received 20% water-containing microemulsion; **IV reference group for the IN group which received 50% wa-
ter-containing microemulsion; aFA% = absolute bioavailability = (AUC0–∞
IN × Dose IV) × 100/(AUC0–∞ IV × Dose IN).
mulations resulted in a relatively short time to reach the
peak plasma level (tmax), 9.25 ± 6.75 min and 6.75 ± 5.67
min (t-test, p > 0.05), respectively. In contrast, the peak
plasma levels (Cmax) and the absolute bioavailability (FA)
of MDZ were significantly higher after administration of
the W/O formulation than those obtained after applica-
tion of O/W formulation, i.e., 46.62 ± 17.38 g/ml vs.
15.44 ± 4.00 µg/ml, and 35.19% ± 11.83% vs. 19.83% ±
16.32%, respectively (p < 0.05). It is to be noted that the
average elimination half-life (t1/2) of MDZ in rabbits of
Study 1 (Table 1) after IV administration was statisti-
cally different compared with the average value obtained
after IV administration to rabbits of study 2, i.e., 34.19 ±
5.88 min vs. 19.57 ± 7.24 min (t-test, p < 0.05), respec-
tively. This difference was probably due to the relatively
lower body weight of the animals in study 2. However,
there was no statistically significant change in the half-
lives obtained after IN administration to the same ani-
mals in each study. In addition, no statistical difference
was noted between half-lives of MDZ after IN admini-
strations in both studies. Table 2 presents the pharma-
cokinetic parameters of diazepam in a study involved
three rabbits which received both IV and IN administra-
tions. Figures 3 and 4 show the pharmacokinetic profiles
of 0.4 mg/kg doses of MDZ (Figure 3) and DZP (Figure
4) applied intranasally to rabbits as compared to IV ad-
ministrations of each drug at 0.2 mg/kg doses. The nasal
administration of both drugs was carried out by the same
microemulsion vehicle containing 20% aqueous phase
and 25 mg/ml drug concentration. As shown in Table 2,
the absolute bioavailability of DZP after IN administra-
tion using this formulation was 33.45% ± 12.36% and
the tmax was 18.33 ± 23.09 min, which was twice longer
than the tmax obtained after MDZ administration (9.25 ±
6.75 min). The tmax values obtained in this study for di-
azepam is in agreement with Gizurarson, et al. [13], who
achieved peak levels after 18 ± 11 min in healthy hu-
mans. The difference between tmax values of DZP and
Figure 2. Schematic illustration (not to scale) of possible
packing of midazolam in the nano-droplet’s membrane; up:
W/O microemulsion; bottom: O/W microemulsion.
from the interfacial membrane to the outer phase. The
pharmacokinetic parameters of midazolam in W/O (20%
water) microemulsion (Study 1) and their comparison
with midazolam in O/W (50% water) microemulsion
(Study 2) is presented in Table 1. As shown, both for-

Intranasal Delivery of Two Benzodiazepines, Midazolam and Diazepam, by a Microemulsion System 185
Figure 3. Pharmacokinetic profiles of midazolam after in-
travenous (solution, 0.2 mg/kg) and intranasal (microemul-
sion, 0.4 mg/kg) administrations to rabbits (n = 4).
Figure 4. Pharmacokinetic profiles of diazepam after intra-
venous (solution, 0.2 mg/kg) and intranasal (microemulsion,
0.4 mg/kg) administrations to rabbits (n = 3).
Table 2. Mean pharmacokinetic parameters of diazepam
after IV and IN administration to rabbits (n = 3).
PK parameter IV solution
0.2 mg/kg
IN microemulsion
0.4 mg/kg
Cmax (μg/ml) 18.63 (±3.76) 8.40 (±3.00)
tmax (min) 0 18.33 (±23.09)
z (min–1) 0.0478 (±0.0124) 0.0334 (±0.0171)
Elimination t1/2 (min) 15.15 (±3.80) 27.32 (±19.46)
AUC0–∞ (μg·min·ml–1) 383 (±93) 270 (±143)
AUC0–∞/dose (μg·min·ml–1·D–1) 1.91 (±0.46) 0.67 (±0.36)
FAa (%) ---- 33.45 (±12.63)
aFA% = absolute bioavailability = (AUC0–∞
IN × Dose IV) × 100/(AUC0–∞ IV ×
Dose IN).
MDZ in our study was also noted by Ivaturi, et al. [7],
who reported that tmax values in healthy volunteers after
intranasal administrations were 28.8 min and 21.6 min
for DZP and MDZ, respectively. Both benzodiazepines
had comparable half-lives of elimination (t1/2), that is,
43.15 ± 11.63 min (MDZ) vs. 27.32 ± 19.46 min (DZP)
after IN administration. Unlike t1/2 values obtained in
rabbits in the present study, the values obtained in hu-
mans according to the literature are significantly higher
for MDZ and especially for DZP. Furthermore, the ad-
ministration of DZP (IV and IN) to humans had a longer
half-life than administration of MDZ. According to Iva-
turi, et al. [7,12], the terminal half-life of DZP was 59.1
h [7] and 48.3 h [12], while that obtained for MDZ was
0.9 h after IV administration of 5 mg of each drug.
Wermeling, et al. [17] reported a half-life of 3.14 h after
5 mg IV dose of MDZ to healthy volunteers, and
Haschke, et al. [26] reported a half-life of 1.89 h after 1
mg IV dose of MDZ to human volunteers. Interestingly,
t1/2 values obtained in rats were much closer to our data
than those obtained in humans, 55.4 min and 105.5 min
for MDZ and DZP, respectively [27]. In light of the
pharmacokinetic data obtained in our study, it can be
concluded that: a) there is a species difference between
rabbits (and rodents) and humans with regard to the
elimination rate of benzodiazepines, but there is no ap-
parent difference in the absorption process, resulting in a
very short onset of time in both MDZ and DZP admini-
strations; b) the longer time to reach peak plasma level
(tmax) of DZP relative to MDZ may explain the clinical
advantage in the use of midazolam over diazepam in the
treatment of acute seizures [11].
The microemulsion system of this study provided a
high drug concentration of 25 mg/g and (or even 50 mg/g)
compared with the 5 mg/ml MDZ-HCl and DZP in plain
solutions. We have also noted that a W/O macro-emul-
sion and a mixture of the oil and the surfactants, which
had been formulated with the same components’ ratio,
provided high drug concentrations as well. Therefore, a
separate study aimed to evaluate the pharmacokinetics of
nasal MDZ using these formulations was carried out. The
pharmacokinetic parameters are summarized in Table 3
and the plasma levels—time curves are illustrated in
Figure 5. As shown, the resulted data were not much
different from those obtained after IN administration of
microemulsion. It should be emphasized that W/O ma-
cro-emulsion formulation contained all ingredients of the
20% water-containing microemulsion except the co-sur-
factant. Surfactants mixture includes all ingredients ex-
cept water. The ratio between the including ingredients
were kept constant. This study has revealed that the sur-
factants’ combination is more essential in solubilizing
and carrying MDZ through the nasal mucosal membrane
rather than the nanoparticulate structure of the formula-
tion. It is postulated, therefore, that MDZ and DZP per-
meate into the systemic circulation via the nasal route by
a mechanism involving entrapment within the micellar
layers of the surfactants followed by a release into the
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186 Intranasal Delivery of Two Benzodiazepines, Midazolam and Diazepam, by a Microemulsion System
Table 3. Mean pharmacokinetic parameters of midazolam
after IN administration of two non-microemulsion formula-
tions to rabbits (n = 4).
PK parameter IN Surfactant
mixture 0.4 mg/kg
IN Macroemulsion
0.4 mg/kg
Cmax (μg/ml) 46.20 (±23.07) 33.12 (±13.53)
tmax (min) 3.50 (±1.73) 5.00 (±0.0)
z (min–1) 0.0149 (±0.0076) 0.0204 (±0.0133
Elimination t1/2 (min) 55.02 (±23.57) 52.32 (±40.02)
AUC0–∞ (μg·min·ml–1) 3154 (±1246) 2736 (±919)
AUC0–∞/dose
(μg·min·ml–1·D–1) 7.88 (±3.11) 6.84 (±2.30)
FAa (%) 44.00 (±7.97) 40.46 (±16.23)
aFA% = absolute bioavailability = (AUC0–∞
IN × Dose IV) × 100/(AUC0–∞ IV ×
Dose IN).
Figure 5. Pharmacokinetic profiles of midazolam after in-
tranasal (0.4 mg/kg) administrations of three formulations
to rabbits (n = 4)—a comparison between microemulsion,
macroemulsion (both containing 20% aqueous phase) and
surfactants mixture (without water). All formulations con-
tain same components' ratios and 25 mg/g (2.5% by weight)
of the drug.
mucosa and transport. The possible entrapment and the
surfactant-accompanying drug diffusion may explain
why this method of nasal administration is apparently
non-irritable.
4. Conclusions
Very few studies have reported the use of microemulsion
for intranasal delivery of benzodiazepines. Just to make a
point but not to limit, using PubMed with key words
“intranasal”, “microemulsion” and “diazepam”, only
three reports were found [28-30], while the combination
of the two former keys with ‘midazolam’ yielded no
publications. In comparison, over 200 studies on intra-
nasal delivery of benzodiazepines have been published.
With no intention of course to devaluate the current
knowledge in the area, it may indicate that the potential
of microemulsion technology has not yet been exploited
enough for intranasal benzodiazepines. There is indeed a
wide recognition that intranasal treatment is more avai-
lable and easily administered even by the patient, in
managing of seizure emergencies. However, patient
compliance and tolerability are the major drawback in
the development of intranasal midazolam and diazepam.
In this paper, we present a new microemulsion that could
be used as a safe and effective intranasal drug delivery
system for midazolam or diazepam. An alcohol-free mi-
croemulsion formulation of a unique surfactant structure
that does not also require an acidic pH to dissolve mida-
zolam has been shown to have a potential of decreasing
epithelial irritation while achieving the desired thera-
peutic effect. Apart from preventing nasal irritation, the
microemulsion possesses two major advantages over
plain solutions, one is a high solubilization capacity for
MDZ base and DZP that exceeds their aqueous solubility
and thus allows reduction in the application volume (to
about 100 - 150 µg/ human nostril). The other advantage
is that both benzodiazepines can be rapidly absorbed
after nasal administration of the microemulsion to meet
the emergency treatment purpose. The absolute bio-
availability of MDZ and DZP in rabbits after application
of the nasal microemulsion were 35.19% (±11.83%) and
33.45% (±12.63%), respectively. Although a consider-
able amount of drug is absorbed, the absorption rate is
probably more important than the bioavailability in acute
medical treatment, as already noted by Lindhardt, et al.
[31]. The systemic absorption and tolerability of the mi-
croemulsion formulation in man remain to be established
in future clinical studies.
5. Acknowledgments
The authors are grateful for the practical and professional
assistance of Mr. Igor Krymberk and Ms. Lillia Shapiro
at the Laboratory of Biopharmaceutics at the E.D. Berg-
mann Campus, Ben Gurion University of the Negev.
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