Neurobehavioral Effects of Intranasal d-AmphetamineKelly, T.H., Babalonis, S., Emurian, C.S., Corbly, C.R., Martin, C.A., Wermeling, D.P., Joseph, J.E., and Lile, J.A.

University of KentuckyAbstract

AIMS: This study tested the hypotheses that the onset and magnitude of behavioral and cardiovascular effects of d-amphetamine would be enhanced following intranasal relative to oral administration and that regional changes in brain activation associated with neurobehavioral effects of intranasal d-amphetamine could be identified. METHODS: A double-blind, double-dummy, placebo-controlled, randomized design was first used to compare the effects of intranasal and oral d-amphetamine (0, 16, 24 and 32 mg/70 kg), followed by a double-blind placebo-controlled, randomized study to examine the time course of neurobehavioral effects of intranasal d-amphetamine in 6 healthy volunteers. Intranasal d-amphetamine solution was administered using a mucosal atomization device. Assessments were conducted before, and at regular intervals following drug administration, and included self-reported drug-effect questionnaires, cardiovascular indices, a psychomotor performance task, and two measures of impulsivity/reward seeking. Neurobehavioral effects were determined by fMRI measurement during completion of self-reported drug-effect questions. RESULTS: ANCOVA analyses indicated prototypical d-amphetamine-induced stimulant effects (e.g., increased subject ratings of Stimulated and Like Drug, elevated heart rate and blood pressure, and improved rate and accuracy on the DSST) irrespective of dose, but the onset of these effects was generally earlier following intranasal administration, with significant effects emerging at 15-30 minutes after intranasal dosing and 45-60 minutes after oral dosing. Comparable stimulant effects were also observed in the MRI environment, with brain activation in mesolimbic pathway regions of interest (i.e., nucleus accumbens, ventral tegmental area, cingulate gyrus and orbito-frontal cortex) varying as a function of dose. CONCLUSIONS: These investigations documented different time courses of behavioral effects associated with oral and intranasal d-amphetamine and identified brain regions activated by intranasal drug administration.

Supported by P20 RR015592, P50 DA 005312, K01 DA018772

Method: Intranasal and Oral d-AmphetamineSubjects: Six healthy adults (5 Caucasian males, 1 Caucasian female), ages 20 to 28, completed medical screening and gave written consent to participate. One subject reported using 5 tobacco cigarettes daily. No other substance use history was reported or identified via urinalysis throughout the study.

Design: A double-blind, double-dummy, placebo-controlled, randomized design was used to compare the effects of intranasal and oral d-amphetamine (0, 16, 24 and 32 mg/70 kg).

Procedure: Subjects completed two practice sessions to become familiarized with the behavioral

ResultsFigure 1 A significant interaction of dose and route (F’s3,330 = 3.7-8.8; p’s ≤ 0.01) and main effect of time (F’s3,330 = 3.8-12.0; p’s ≤ 0.001) was observed for subject ratings of Feel Drug and Like Drug, with the earliest significant self-reported effects occurring 30-min after administration of 16 and 32 mg/70 kg d-amphetamine. Following oral dosing, however, the earliest time point at which significant differences were found between d-amphetamine and placebo for subject ratings of Like Drug was 60 min after administration of the 32 mg/70 kg dose and 90 minutes after the 24 mg/70 kg dose. For the item Feel Drug, only the 24 mg/70 kg oral dose increased ratings starting 90 min after drug administration.Figure 2 A significant interaction of dose and route (F3,330 = 2.7; p ≤ 0.05) and dose and time (F3,330 = 2.5; p ≤ 0.001) was observed for heart rate (Figure 2, right panel), and the earliest time point at which a significant difference from placebo occurred was 15 min after administration of the 32 mg/70 kg dose. The earliest time point for oral d-amphetamine effects was 60 min for all doses. A significant main effect of dose (F3,330 = 24.2; p ≤ 0.001) and time (F3,330 = 7.0; p ≤ 0.001) were detected for systolic blood pressure. The earliest time point at which significant differences were observed occurred 30 min following administration of the 16 and 32 mg/70 kg dose of intranasal d-amphetamine and 45 min after the 32 mg/70 kg dose of oral d-amphetamine. Figure 3 Significant interactions of dose and route (F3,330 = 8.9; p ≤ 0.001) and dose and time (F3,330 = 2.0; p ≤ 0.05) were observed for trial completion rate (Figure 3, right panel). The earliest time point at which significant differences between active drug and placebo were detected occurring 60 min after drug administration (performance tasks were not presented at the 30 and 45 min assessments due to time limitations). A significant interaction of dose and route (F3,330 = 7.1; p ≤ 0.001) was observed for trial accuracy (Figure 3, left panel). All doses of d-amphetamine administered via both routes significantly improved trial accuracy, with the earliest significant time point occurring at 60 min. Figure 4 A significant interaction of dose and time (F’s17,85 = 2.3-3.8; p’s ≤ 0.01) was observed for subject ratings of Feel Drug, Like Drug, Take Again and Stimulated. Small magnitude increases were also observed on the Anxious scale, but these effects did not reach statistical significance (data not presented).Figure 5 The magnitude of the BOLD signal remained stable during the 30-min baseline was variable among subjects (data not shown). Change from baseline BOLD signal varied as a function of dose in the regions of interest, with d-amphetamine-induced changes in activation occurring in the right nucleus accumbens (suppression) and the right anterior cingulate gyrus (enhancement). d-Amphetamine effects were also observed in the ventral tegmental area and left interior orbital cortex (data not presented).

BackgroundIntranasal drug delivery is a useful method for both clinical practice and research. Drugs administered via the intranasal route have a faster onset of action and higher bioavailability compared to oral dosing, without the risk of injury and personal discomfort associated with routes requiring a needle stick, such as intravenous or intramuscular administration. The fast onset and high bioavailability of intranasal drug delivery, however, can also be associated with enhanced abuse potential. These studies characterized intranasal d-amphetamine by comparing dose-related behavioral effects of the drug when administered orally and intranasally, and by examining neural activation via BOLD signals in brain regions of interest during subjective reporting of drug effects. d-Amphetamine is a useful tool in human laboratory models because it can be safely administered to healthy subjects, and there do not appear to be any controlled studies on the effects of intranasal d-amphetamine in humans, despite reports that diverted prescriptions are used via this route.

Conclusions

1) d-Amphetamine engendered prototypical stimulant-like effects:

a) Verbal Reports of drug effectsb) Cardiovascular measuresc) Task performance

a) The time course for the behavioral and cardiovascular effects of intranasal d-amphetamine, which has not been published previously, exhibited statistically significant differences from placebo as early as 15-30 min, with the peak response occurring 1 hr after administration on average. Following oral administration, initial significant effects of d-amphetamine became apparent at 45-60 min, consistent with prior research. Area under the curve was greater for intranasal than oral routes of administration, suggesting enhanced drug bioavailability associated with intranasal administration. The magnitude of peak effects did not vary as a function of route of administration.

b) The time course of subject rated effects were replicated during the follow-up study neuroimaging study, with statistically significant differences from placebo emerging as early as 15 min post administration, and magnitude of effects increasing across the 45-minute session.

c) Brain activation in regions of interest also varied as a function of dose, with the time-course of effects in the right nucleus accumbens and anterior cingulate gyrus paralleling self-reported drug effects.

Figure 1: Dose- and time-response function for d-amphetamine administered by the intranasal (Top Panels) and oral (Bottom Panels) routes of administration for the items Like Drug (Left Panels) and Feel Drug (Right Panels) on a Visual Analog Scale. Error bars: 1 SEM. Filled symbols: significant difference from placebo.

completed immediately following drug administration (data not shown) indicated that subjects were unable to differentiate between active and placebo doses. Oral d-amphetamine consisted of generic d-amphetamine tablets over-encapsulated in size 0 gelatin capsules.

Data Analysis: Mixed-model ANCOVAs were conducted with route (PO vs IN), dose and time as factors, and baseline performance as a covariate.

Assessment Measures

Addiction Research Center Inventory (ARCI): The 49-item short form of the true-false ARCI provided reports of drug effects on five scales: Lysergic acid diethylamide (LSD), Amphetamine (A), Benzedrine Group (BG), Morphine-Benzedrine Group (MBG) and Pentobarbital, Chlorpromazine, Alcohol Group (PCAG).

Visual-Analog Rating Scales (VAS): Ratings of ‘Drug Effect,’ ‘Like Drug,’ ‘Stimulated,’ ‘Sedated,’ ‘Anxious,’ ‘Hungry,’ ‘Thirsty,’ and ‘High’ were obtained by placing marks on a 100-unit line anchored with "Not at all" on the left and "Extremely" on the right.

Adjective Rating Scale (ARS): The 16 items from the Stimulant subscale were rated using a numeric keypad to select among one of five response options: Not at All, A Little Bit, Moderately, Quite a Bit, and Extremely (scored numerically from 0 to 4, respectively; maximum score = 64).

Digit-Symbol Substitution Task (DSST – 1.5 minutes): Nine random 3-row by 3-column arrays of open and filled boxes (one filled box per row), labeled 1-9 from left to right, were displayed at the top of the computer monitor. A randomly generated number, between 1 and 9, was displayed in the center of the monitor, indicating which of the nine arrays should be reproduced on a given trial. Subjects reproduced the indicated array by pressing the buttons on a 3-row by 3-column keypad that corresponded to the positions of the filled boxes.

Cardiovascular Measures: Heart rate and blood pressure were recorded.

and cardiovascular measures and daily laboratory routine. During these sessions, the subjects also practiced administration of an intranasal drug solution (saline), but no active doses of d-amphetamine were administered. Subjects then completed eight experimental sessions, conducted Monday through Friday.

Daily Schedule: After successfully completing intake evaluations, including urine pregnancy and drug-use testing, subjects completed assessments 15 min before (i.e., baseline) and 15, 30, 45, 60, 90, 120 and 180 min after dose administration.

Drug: Intranasal d-amphetamine was delivered using a syringe capped with a mucosal atomization device (Wolfe Tory Medical, Inc.). An active placebo consisted of 100 mg/mL magnesium sulfate. Ratings of nasal cues

Method: Neuroimaging of Intranasal d-AmphetamineSubjects: Six healthy Caucasian females, ages 20 to 27, using oral birth control that included a placebo phase, completed medical screening and gave written consent to participate. No other substance use history was reported or identified via urinalysis throughout the study.

Design: A double-blind, placebo-controlled, randomized design was used to compare the effects of intranasal d-amphetamine (0, 32 mg).

Procedure: Subjects completed one practice session to become familiarized with the magnetic resonance imaging facility and the neuroimaging process, behavioral and cardiovascular measures and daily laboratory routine. During these sessions, the subjects also practiced administration of an intranasal drug solution (saline), but no active doses of d-amphetamine were administered. Subjects then completed two experimental sessions that were scheduled during the placebo phase of the oral birth control regimen when estradiol and progesterone levels were at their nadir.

Daily Schedule: After successfully completing intake evaluations, including urine pregnancy and drug-use testing, subjects complete assessments 15 min before (i.e., baseline) and 45 min after drug administration in the MRI facility while brain activation (BOLD signal) was measured.

Drug: Intranasal d-amphetamine was delivered using a syringe capped with a mucosal atomization device (Wolfe Tory Medical, Inc.). An active placebo consisted of 100 mg/mL magnesium sulfate.

Data Analysis: Mixed-model ANOVAs were conducted with dose (0 and 32 mg) and time as factors.

Assessment Measures

Visual-Analog Rating Scales (VAS): Ratings of ‘Like Drug,’ ‘Take Again,’ ‘Feel Drug,’ ‘Stimulated,’ and ‘Anxious’ were obtained by placing marks on a 100-unit line anchored with "Not at all" on the left and "Extremely" on the right.

Cardiovascular Measures: Heart rate and blood pressure were recorded.

Brain Activation: A Siemens Trio 3.0 Tesla magnet is used to collect functional brain images. A T2*-weighted gradient echo sequence was used with the following parameters: 29ms echo time, 64x64 matrix, 224x224-mm field of view, 40 3.5-mm axial, slices acquired in interleaved order, 3s repetition time. A 3D shim was performed before all EPI image acquisitions. High-resolution structural images (with 1mm cubic voxels) were acquired with a T1-weighted MPRAGE sequence (TE = 2.93 ms, TR = 2100 ms, 256x256 matrix, acquired in the sagittal direction). An 8-channel head coil was used to enhance signal resolution and optimize measurement of subcortical brain regions, including the nucleus accumbens. Using the FSL package, images in each participant’s time series was motion corrected with the MCFLIRT module. Images in the time series were spatially smoothed with a 3D Gaussian kernel (FWHM = 7.5 mm) and temporally smoothed using a high-pass filter. Subjects’ motion corrected and smoothed 4D EPI image was registered to the ICBM152 T1 template using the registration matrix created from the three step process which involved registering the average EPI volume to the MPRAGE volume and the MPRAGE volume to the ICBM152 T1 template,

Figure 2: Dose- and time-response function for d-amphetamine administered by the intranasal (Top Panels) and oral (Bottom Panels) routes of administration for systolic blood pressure (Left Panels) and heart rate (Right Panel). Error bars: 1 SEM. Filled symbols: significant difference from placebo.

Figure 3: Dose- and time-response function for d-amphetamine administered by the intranasal (Top Panels) and oral (Bottom Panels) routes of administration for correct (Left Panels) and total (Right Panel) trials on the DSST. Error bars: 1 SEM. Filled symbols: significant difference from placebo.

Figure 4: Dose- and time-response function for intranasal d-amphetamine administered for the items Like Drug, Take Again, Feel Drug, and Stimulated on a Visual Analog Scale.

Figure 5: Dose- and time- BOLD signal response function (change from baseline) for intranasal d-amphetamine in the right nucleus accumbens and anterior cingulate gyrus.

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using the FLIRT (Linear Image Registration Tool) module of the FSL package. Anatomical masks were then generated including nucleus accumbens, ventral tegmental area, cingulate gyrus and orbito-frontal cortex. These masks were used to extract each subjects’ individual time course in each region of interest (ROI).