Electroencephalographic effects and serum concentrations
`after intranasal and intravenous administration of
`diazepam to healthy volunteers
`
`Karsten Lindhardt,1 Sveinbjo¨ rn Gizurarson,2,3 Sigurjo´ n B. Stefa´nsson,4
`David R. O` lafsson2 & Erik Bechgaard1
`1The Royal Danish School of Pharmacy, Department of Pharmaceutics, Universitetsparken 2, 2100 Copenhagen, Denmark, 2Lyfjathroun hf,
`Hafnarbudir, Geirsgata 9, 101 Reykjavik, 3Department of Pharmaceutics, University of Iceland, 107 Reykjavik and 4Landspı´talinn,
`University Hospital of Iceland, Department of Neurology, 121 Reykjavik, Iceland
`
`Aims To evaluate the electroencephalographic (EEG) effects, blood concentrations,
`vehicle irritation and dose-effect relationships for diazepam administered nasally.
`Methods The study had a cross-over design with eight healthy volunteers (one drop
`out). It consisted of four legs with four different administrations: intranasal (i.n.)
`placebo, 4 mg diazepam i.n., 7 mg diazepam i.n. and 5 mg intravenous (i.v.) dia-
`zepam. Polyethylene glycol 300 (PEG300) was used as a vehicle in the nasal
`formulations to solubilize a clinically relevant dose of diazepam. Changes in N100,
`P200 and P300 brain event-related potentials (ERP) elicited by auditory stimulation
`and electroencephalographic b-activity were used to assess effects on neurological
`activity.
`Results The mean [95% confidence intervals] differences between before and
`after drug administration values of P300-N100 amplitude differences were x0.9
`[x6.5, 4.7], x6.4 [x10.1, x2,7], x8.6 [x11.4, x5.8] and x9.6 [x12.1, x7.1]
`for placebo, 4 mg i.n., 7 mg i.n. and 5 mg i.v. diazepam, respectively, indicating
`statistically significant drug induced effects. The bioavailabilities of 4 and 7 mg i.n.
`formulations, were found to be similar, 45% [32, 58] and 42% [22, 62], respectively.
`Conclusion The present study indicates that it is possible to deliver a clinically effec-
`tive nasal dose of diazepam for the acute treatment of epilepsy, using PEG300 as a
`solubilizer.
`
`Keywords: benzodiazepine, diazepam, EEG, electroencephalography, ERP, event-
`related potential, intranasal, nasal, PEG300, polyethylene glycol 300
`
`Introduction
`
`The present study was carried out to assess the intranasal
`administration of diazepam as a potential alternative to
`intravenous and rectal dosing in the treatment of acute
`epileptic seizures. A nasal spray is beneficial when a rapid
`onset of effect (within seconds or minutes) is required.
`Animal experiments have shown that
`the intranasal
`administration of diazepam may induce effects within
`5 min. In rabbits, a peak serum concentration is obtained
`about 5 min after
`the administration [1]. Diazepam
`
`Correspondence: Dr Sveinbjo¨ rn Gizurarson, University of Iceland, Department
`of Pharmaceutics, Hofsvallagata 53, 107 Reykjavik, Iceland. Tel.: 00354 5112020;
`E-mail: sg@lyf.is
`
`Received 16 October 2000, accepted 22 June 2001.
`
`has poor water solubility, but polyethylene glycol 300
`(PEG300), a vehicle causing relatively little local irritation,
`has been found to solubilize an expected clinically relevant
`dose (4–10 mg) of diazepam in the limited volume
`necessary for nasal administration [2].
`In an earlier clinical study a nasal dose of 2 mg diazepam
`was administered by use of PEG300 as the solubilizing
`vehicle [2]. Within 30 min the nasal bioavailability was
`found to be about 37%. The neurological measurements in
`this study were rather crude, and comprised parameters
`such as memory tests and the ability to catch a ruler. The
`quantification of drug effects on attention and vigilance
`was based on questionnaires. The results of this study
`showed only minor drug effects, probably because the dose
`was too low. Therefore,
`it was decided to administer
`higher nasal doses of diazepam (4 and 7 mg).
`
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`K. Lindhardt et al.
`
`The electroencephalographic (EEG) effects of benzo-
`diazepines are well known. Changes after drug adminis-
`tration have been observed in event-related potentials
`(ERP) and beta-activity [3–9]. The brain generates elec-
`trical waves of various wavelengths creating a spectrum,
`which may be divided into several
`frequency bands.
`The most important bands are found in the frequency
`range 8 –12 and 12–35 Hz, named the alpha and beta-
`activity, respectively. An increase of beta-activity has been
`found to be a more sensitive measure of benzodiazepine
`effect than a decrease in alpha-activity [10].
`Exposing study subjects to target tones (e.g. 2000 Hz
`auditory stimuli) among neutral tones (e.g. 1000 Hz audi-
`tory stimuli) generates ERPs. The neutral tones occur five
`times more frequently than the target. A positive wave
`appears 300 ms (P300) after the target tone. This wave is
`generated from the sensory discrimination of the target
`tone among the neutral tones. The P300 potential has
`been found to be particularly useful in measuring the
`intracerebral effects of benzodiazepines [8]. After both
`neutral and target tones a negative wave appears after
`100 ms (N100), but only limited changes appear after
`benzodiazepine administration [3]. As well as being related
`to the sedative and cognitive effects of benzodiazepines
`[8], changes in EEG, particularly the increase in beta-
`activity, has been found to correlate with anticonvulsant
`effect [10].
`Unrug et al. [3] found that the decrease in the P300
`potential after a 10 mg oral dose of diazepam was
`most pronounced at the vertex electrode. The peak of
`the P300 potential is usually identified as the most positive
`point in the waveform range between 200 and 400 ms and
`a change in the latency of this peak may also be useful in
`evaluating changes in P300 caused by diazepam [8].
`Fink et al. [9] found a linear correlation between the
`increase in EEG and beta-activity and blood concentrations
`of diazepam after oral administration to healthy volunteers.
`More recent studies of the pharmacological effects of
`benzodiazepines in man have been of nonblinded design,
`and only 3 out of 18 were controlled, emphasizing the need
`for additional well designed studies in this field [11].
`The aims of the present study were (1) to provide
`information on the pharmacodynamic response to nasally
`administered diazepam formulated in a polyethylene glycol
`300 vehicle (2) to evaluate and optimize EEG methods for
`measuring the neurological effects of diazepam, and (3) to
`define any relationship between the effect of diazepam on
`the EEG and serum concentrations of the drug.
`
`Methods
`
`Three male and five female healthy Caucasians, weighing
`84t17 kg, and between 20 and 40 years of age were
`studied (one of whom dropped out). They were asked not
`
`to drink alcohol during the entire study period and none
`was taking regular medication. Subjects received both
`written and oral information before giving their written
`consent. The National Icelandic Ethics Committee and
`the Icelandic Health Department approved the study.
`The study had a double-blind, randomized, cross-over
`design. Eight subjects received on separate occasions (1)
`placebo intranasal (i.n.) administration of PEG300, (2)
`4 mg diazepam i.n. solubilized in PEG300 (4 mg i.n.),
`(3) 7 mg diazepam i.n. solubilized in PEG 300 (7 mg i.n.),
`and (4) 5 mg diazepam intravenously (i.v.) administered in
`a commercially available formulation (Stesolid Novum1).
`The latter and PEG300 were obtained from Dumex-
`Alpharma A/S (Copenhagen, Denmark) and Union
`Carbide (Charleston, U.S.A.), respectively. In order to
`improve the spray properties of the viscous formulation, it
`was necessary to modify a unit-dose device from Pfeiffer
`(Radolfzell, Germany). Each device was filled to spray
`75 ml in each nostril (two devices per nasal round).
`
`Blood sampling
`Venous blood samples were taken at x10, x2, 3, 5, 8, 11,
`15, 20, 30, 45 and 60 min after drug administration. A
`commercially available enzyme-immunoassay (EIA) kit
`from STC Technologies, Inc. (Bethlehem, U.S.A.) was
`used for the analysis of diazepam in serum. The measure-
`ments were performed on a HTS7000 microplate reader
`from Perkin-Elmer (Wellelsly, U.S.A.) with u.v. detec-
`tion at 450 nm. Samples were centrifuged at 3200 g for
`10 min and serum was transferred to 1 ml cryotubes from
`NUNC (Copenhagen, Denmark) and stored at x80u C
`until analysis. Standards of 1, 2, 5 and 20 ng mlx1 (n=9)
`were analysed on separate days and a mean coefficient of
`variation was found to be 10% (range 7–13). The lowest
`level of detection was about 0.1 ng mlx1. All samples had
`concentrations higher than 1 ng mlx1.
`
`ERP and b-activity
`
`The hardware and software systems used for the EEG
`recordings and analyses were from Neuroscan1 (Sterling,
`U.S.A.). Nineteen silver scalp electrodes (F1, F2, F7, F3,
`Fz, F4, F8, T3, C3, Cz, C4, T4, T5, P3, Pz, P4, T6, O1,
`O2) were placed on the head according to the inter-
`national 10–20 system [12]. Electrodes placed on the
`left and right mastoid process (electrodes A1 and A2)
`were connected together and used as a reference. Two
`electrodes, one above the other below the left eye, were
`used to monitor eye movements. The impedance of the
`electrodes was tested before the recording started.
`The subjects sat in a chair during the recording and with
`their eyes shut. They were asked to listen to auditory
`stimuli delivered through earplugs to both ears. The neutral
`
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`stimulus was a 1000 Hz tone occurring five times more
`frequently than the 2000 Hz target tone. A preliminary
`test was performed to ensure the subjects were able to
`hear and separate the stimuli. The interstimulus-interval
`was 2 s, tone duration was 100 ms, and rise and fall
`time was 5 ms. The subjects were asked to tap with
`their finger and count when a target tone appeared. If
`subjects fell asleep, as happened a few times, especially
`in the i.v. group, they were gently awoken by touching
`their hands.
`The EEG was sampled at 200 Hz after low pass filtering
`at 40 Hz and high pass filtering at 0.3 Hz. Each
`auditory stimulus
`triggered a sampling of EEG that
`started 100 ms before the stimulus and had a total duration
`of 1280 ms
`(256 sampling points). The EEG epoch
`triggered by each auditory stimulus was stored for further
`analysis.
`The EEG was recorded before and after the subjects had
`received medication. Each recording session lasted about
`15 min during which 400–500 stimuli were delivered. The
`recordings were analysed by averaging the EEG epochs to
`the neutral and the target stimuli, respectively. Epochs
`were rejected when amplitudes exceeded t100 mV either
`in the lead across the eye or at the Fz, Cz or Pz electrodes.
`The peak of the P300 potential was defined by the highest
`potential between 200 and 500 ms and was located in the
`average ERP for each individual before and after treat-
`ment. The peak of the N100 potential was defined by the
`lowest potential between 50 and 200 ms. The Cz elec-
`trode resulted in the most sensitive effect measurements
`and was therefore chosen for the ERP calculations. To
`assess changes in beta activity, the mean amplitude and
`relative power spectrum were obtained after Fourier
`transformation of each epoch. The mean amplitude of
`the b-activity within different frequency bands between
`16 and 35 Hz was calculated.
`
`Diazepam intranasally administered
`
`Questionnaires
`
`The questionnaires were answered as soon as possible
`after each measurement. Subjects were asked to score a
`prefabricated list of various possible irritant effects from
`each formulation.
`
`Data analysis
`
`The area under the serum concentration-time curve from
`0 to 60 min (AUC(0,60 min)) was calculated using the
`trapezoidal method. AUC from 0 to 2 min for intravenous
`administrations were determined by extrapolation to zero
`by using logarithmic regression analysis on the initial two
`concentrations. A two-factor ANOVA was used to compare
`differences between pharmacodynamic measurements
`obtained by subtracting values after drug administration
`from pre-dose values. P300, P300-N100 differences and
`b-activities were tested. A two-sample t-test (one-sided)
`was used in the statistical analysis to compare the various
`data sets.
`
`Results
`
`Mean serum concentration-time profiles of seven subjects
`are shown in Figure 1. The mean bioavailability, Cmax and
`tmax, [95% confidence interval], for the 4 and 7 mg i.n.
`diazepam formulations were found to be; 45% [32, 58] and
`42% [22, 62], 99 ng mlx1 [83, 115] and 179 ng mlx1
`[126, 232], 18 min [11, 25] and 42 min [25, 59], respec-
`tively. tmax was significantly (P<0.05) higher after 7 mg
`i.n. administration than after 4 mg. The slower absorp-
`tion from the 7 mg dose was substantiated by differences
`in the AUCi.n./AUCi.v. ratio of the drug between the two
`formulations at the early time points (Table 1).
`A two-factor ANOVA was used to compare P300,
`P300-N100 amplitude differences and b-activity effects
`
`20
`
`40
`
`Time (min)
`
`60
`
`523
`
`AQUESTIVE EXHIBIT 1025 page 0003
`
`10000
`
`1000
`
`100
`
`10
`
`0
`
`Serum diazepam concentration (ng ml–1)
`
`Figure 1 Mean (ts.d.) serum
`concentration-time profiles after
`intranasal (i.n.) administration of 4 (#)
`and 7 mg (&) diazepam and 5 mg (m)
`diazepam intravenous (i.v.),
`respectively, to seven healthy subjects.
`
`f 2001 Blackwell Science Ltd Br J Clin Pharmacol, 52, 521–527
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`

`

`K. Lindhardt et al.
`
`Table 1 Mean AUCi.n./AUCi.v.ratios (expressed as percentage) at various times after the administration of 4 and 7 mg diazepam to seven healthy
`subjects.
`
`AUCi.n./AUCi.v. (%)
`Time (min)
`
`I.n.dose
`
`4 mg
`7 mg
`P valueb
`
`3
`
`12a
`7a
`0.07
`
`5
`
`14
`8
`0.05
`
`8
`
`17
`12
`0.09
`
`11
`
`21
`15
`0.14
`
`15
`
`25
`19
`0.17
`
`20
`
`29
`22
`0.12
`
`30
`
`34
`27
`0.09
`
`45
`
`40
`34
`0.19
`
`60
`
`45
`42
`0.63
`
`aOne AUC value was left out because it was thought to be an outlier, being more than three times the standard deviation above all the other values.
`bP values from the comparison between the two doses.
`
`Table 4 Mean (ts.d., n=7) EEG amplitude (nV) values of differences
`in the b-frequency range (16–35 Hz) at the vertex electrode between
`before and after drug administration.
`
`Formulation
`
`Placebo
`4 mg i.n.
`7 mg i.n.
`5 mg i.v.
`
`Difference between before
`and after administration
`
`95% confidenceb
`intervals
`
`64t163
`189t232*
`139t68*
`222t120*
`
`[x57, 184]
`[17, 360]
`[89, 189]
`[133, 342]
`
`aP<0.05(*), P<0.01(**), P<0.001(***) vs placebo.
`bNo significant differences were found between formulations or
`subjects.
`
`15
`
`10
`
`P200 P300
`
`300
`
`700
`
`1100
`
`Time (ms)
`
`Potential mV
`
`05
`
`–100
`–5
`
`–10
`
`–15
`
`N100
`
`Figure 2 Mean values from seven healthy subjects of the
`ERPs at the vertex electrode elicited by frequent nontarget
`events (1000 Hz tones, thin line) and rare target events
`(2000 Hz tones, thick line), respectively. Note the P300 evoked
`by the rare tones.
`
`The differences [95% confidence intervals] between the
`before and after values for the P300-N100 amplitude
`differences were x0.9 [x6.5, 4.7], x6.4 [x10.1, x2,7],
`x8.6 [x11.4, x5.8] and x9.6 [x12.1, x7.1] for
`placebo, 4 mg i.n., 7 mg i.n. and 5 mg i.v. diazepam,
`respectively. The overall means of the ERPs (all subjects)
`elicited, respectively, by neutral and target stimuli before
`treatment are shown in Figure 2 and those elicited by
`the target stimuli after treatment in Figure 3.
`
`Table 2 Mean (ts.d., n=7) in P300 potential (mV) at the vertex
`electrode between before and after administration.
`
`Formulation
`
`Placebo
`4 mg i.n.
`7 mg i.n.
`5 mg i.v.
`
`Difference between before
`and after drug administration
`
`x0.8t2.2
`x2.8t3.1
`x5.0t2.4*,a
`x5.6t2.8**
`
`95% confidenceb
`intervals
`
`[x3.0, 1.4]
`[x5.9, 0.3]
`[x6.7, x3.2]
`[x8.4, x2.7]
`
`aP<0.05(*), P<0.01(**), P<0.001(***) vs placebo.
`bNo significant differences were found between formulations or
`subjects.
`
`Table 3 Mean (ts.d., n=7) changes in P300-N100 potential
`differences (mV) at the vertex electrode between before and after drug
`administration.
`
`Formulation
`
`Placebo
`4 mg i.n.
`7 mg i.n.
`5 mg i.v.
`
`Difference between before
`and after drug administration
`
`x0.9t7.5
`x6.4t5.0*
`x8.6t3.8**
`x9.6t3.4***
`
`95% confidenceb
`intervals
`
`[x6.5, 4.7]
`[x10.1, x2.7]
`[x11.4, x5.8]
`[x12.1, x7.1]
`
`aP<0.05(*), P<0.01(**), P<0.001(***) vs placebo.
`bSignificant differences were found between formulations, but not
`between subjects.
`
`before and after treatment (Tables 2– 4). No significant
`difference was found between subjects. However for the
`P300-N100 amplitude differences a significant drug effect
`(P<0.05) was found. A significant reduction in P300
`amplitude was observed, using a two-sample t-test,
`compared with placebo, after 7 mg i.n. (P<0.05) and
`the 5 mg i.v. administrations (P<0.01), but not after the
`4 mg i.n. administration (Table 2). Significant decreases
`compared with placebo was also found in the P300-N100
`amplitude differences (P<0.05) (P<0.01) and (P<0.001)
`for the 4 mg i.n., 7 mg i.n. and 5 mg i.v. formulations,
`respectively (Table 3). The corresponding significance
`values for the beta-activity were (P<0.05) (P<0.05) and
`(P<0.05), respectively (Table 4).
`
`524
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`AQUESTIVE EXHIBIT 1025 page 0004
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`

`Diazepam intranasally administered
`
`Mean differences [95% confidence intervals] on the
`P300-N100 measured between the placebo and drug
`treatment were x3.3 [x0.4, x6.1], x4.8 [x2.4, x7.2]
`and x5.8 [x3.9, x7.8]
`for
`the 4 and 7 mg i.n.
`formulations and the 5 mg i.v. formulation, respectively,
`all of which were statistically significant effect of diazepam
`was found for all
`formulations. The mean difference
`[95% confidence intervals] between 4 mg i.n and 7 mg
`i.n., 5 mg i.v., respectively, were x1.5 [x3.3, 0.3] and
`x2.6 [x4.1, x1.0],
`indicating statistical difference
`between 4 mg i.n. and 5 mg i.v. The mean difference
`[95% confidence] between 7 mg i.n. and 5 mg i.v. was
`x1.1 [x3.1, 1.0], indicating no statistical difference in
`the neurological effect between these two diazepam
`formulations.
`
`No shift in latency of the ERP components was found
`after diazepam administration, and therefore, the ERP data
`are based on changes in ERP amplitudes obtained at the
`vertex electrode.
`By averaging the ERP epochs evoked by target stimuli
`within each consecutive 2 min period (approximately 10
`epochs of rare stimuli), it was possible to determine how
`the P300-N100 difference changed with time for different
`formulations of the drug. In Figure 4, this is shown as
`change in the ratio relative to placebo of the P300-N100
`difference in drug treatment.
`The questionnaires revealed that the adverse effects
`following drug treatment were limited. Bitter taste after
`nasal administration, was the most frequently reported
`adverse event.
`
`300
`
`700
`
`1100
`
`Time (ms)
`
`0–2
`
`2–4
`
`4–6
`6–8
`Time intervals (min)
`
`8–10
`
`10–12
`
`048
`
`12
`
`–100
`–4
`
`–8
`
`–12
`
`Voltage (mV)
`
`1.0
`
`0.9
`
`0.8
`
`0.7
`
`0.6
`
`Ratio of P300-N100 relative to placebo
`
`Figure 3 Mean values from seven
`subjects of the ERPs at the vertex
`electrode after hearing a rare target
`event (2000 Hz tone) after
`administration of placebo (thick line),
`4 mg diazepam (thin line), 7 mg
`diazepam (dotted line) intranasally or
`5 mg diazepam (dashed line)
`intravenously. Note the negative peak
`at 100 ms (N100) and the positive
`peak at 300 ms (P300).
`
`Figure 4 Mean values from seven
`subjects of N100-P300 potential
`differences (obtained by averaging
`ERPs within each consecutive 2 min
`period) after administration of placebo
`intranasally, 4 mg diazepam intranasally
`(&), 7 mg diazepam intranasally (
`) or
`5 mg diazepam intravenously (
`).
`Values (range 0.6–1.0) are illustrated as
`ratios of the P300-N100 difference for
`drug relative to placebo.
`
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`

`K. Lindhardt et al.
`
`Discussion
`
`The present study indicates that it is possible provide a
`clinically effective nasal dose of diazepam using PEG300 as
`a solubilizer. This could provide an alternative route of
`administration in the treatment of epilepsy with benefits in
`acute situations.
`The most relevant components of the ERPs are the
`negative peak at 100 ms (N100), the positive peak at
`200 ms (P200) and the positive peak at 300 ms (P300),
`which are only observed after target stimuli. The data
`confirm previous suggestions that the P300-N100 ampli-
`tude possesses increased sensitivity to diazepam, relative to
`the use of P300 alone. This difference in sensitivity may be
`due to cancellation of base-line fluctuations, as both the
`negative and positive peaks would be equally affected by
`such fluctuations. The vertex electrode (Cz) was found to
`be the most sensitive scalp location for the evaluation of
`the effect of diazepam, which is consistent with literature
`reports [3, 9].
`The P300-N100 amplitude relative to placebo increased
`throughout
`the 12 min study period following i.v.
`diazepam, whereas the serum diazepam concentration
`shows a corresponding decrease to about one third of the
`initial concentration. This indicates that changes in the
`ERPs following diazepam administration do not directly
`reflect the blood concentration of the drug, as suggested
`previously [9]. A more likely explanation is that diazepam,
`which is a very lipophilic substance, progressively dis-
`tributes into the fatty tissue of the brain as the serum
`concentration falls, resulting in a delayed pharmaco-
`dynamic effect. The apparent rapid onset of effect from the
`nasal formulation can be explained by olfactory absorp-
`tion [13], where drug is delivered directly from the nasal
`cavity to the brain.
`A later drug absorption phase was evident for 7 mg i.n.,
`but not for the 4 mg formulation. This may be due to
`absorption from sites other than the nasal cavity,
`for
`example the buccal or the gastrointestinal tract. The rapid
`transfer (20 min) of drugs from the nasal cavity to the
`gastrointestinal tract may limit the absorption of dia-
`zepam, due to precipitation in the nose. This may be more
`pronounced for the 7 mg dose, explaining the higher
`initial clearance. As expected, the intravenous formula-
`tion results in very high initial drug concentration in the
`blood and can be considered as a positive control for the
`effects of diazepam on the EEG effect measurements.
`Adding a taste-adjusting agent in a possible future nasal
`formulation, for example orange, may reduce its bitter
`taste. However, this disadvantage may be insignificant
`compared with the benefit of the nasal formulation in view
`of the severity of the clinical indication.
`One subject was unwilling to continue in the study,
`partly due to nausea and vomiting occurring 4 –5 h after
`
`i.n. placebo administration. Intravenous administration
`made most subjects tired, which did not occur with the
`nasal formulations. For further evaluation of the nasal
`administration of diazepam to treat epilepsy, it may be
`appropriate to measure photosensitive epileptic responses
`because these may be considered as primary measures of
`anticonvulsant activity.
`The present study indicates that it is possible deliver a
`clinically effective nasal dose of diazepam using PEG300 as
`a solubilizer. The P300-N100 amplitude difference in the
`EEG was the preferred method for measuring the central
`nervous system effects of diazepam.
`
`We thank the head of the Department of Neurology, Landspı´talinn,
`University Hospital of Iceland Elias O´ lafsson. The study was
`supported by the Centre of Drug Delivery and Transport (a project
`grant from the Danish Medical Research Council) and by Nycomed
`Pharma A/S.
`
`References
`
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`and pharmacodynamic response after intranasal administra-
`tion of diazepam to rabbits. J Pharm Pharmacol 1997;
`49: 747–750.
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