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From the *University of Kentucky College of Pharmacy; University of Kentucky College of Medicine; Division of Otolaryngology – Head & Neck Surgery, University of Kentucky A. B. Chandler Medical Center; Intranasal Therapeutics, Inc., Lexington, Kentucky.
Address correspondence and reprint requests to Daniel P. Wermeling, Pharm.D., University of Kentucky College of Pharmacy, 725 Rose Street, Lexington, Kentucky 40536. Address e-mail to dwermel{at}uky.edu.
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Abstract |
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 Top Abstract IntroductionMETHODSRESULTSDISCUSSIONREFERENCES |
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Introduction |
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TopAbstractIntroductionMETHODSRESULTSDISCUSSIONREFERENCES |
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The formulations used in the aforementioned trials are generally not appropriate for nasal administration. The formulations will be irritating because the aqueous solution is buffered to pH 3. Moreover, the formulae are too dilute for nasal administration. The adult nasal cavity can only receive and retain about 100–150 µL of liquid, requiring the dose of midazolam be solubilized within this volume. A nasal formula with a concentration of 2.5 mg per 100 µL is necessary to give a single spray per naris. Thus, the formulation methods must be considered when evaluating any estimates of the pharmacokinetics and pharmacodynamics in these reports. Given these findings, there appears to be an unmet medical need to develop an optimal midazolam formulation for nasal delivery.
The objectives of the study were to evaluate the bioavailability of a novel IN midazolam formulation and to compare the pharmacodynamic effects on psychomotor performance and subjective reporting of drug effect after 5 mg doses of midazolam via IN, IM, and IV routes of administration. The specific aims were to: 1) to obtain an IN bioavailability of more than or equal to 70% compared with an IV dose; 2) achieve maximum IN concentration within 10 min of administration; 3) observe sedative properties from IN administration within 10 min; and, 4) demonstrate a nonirritating, well-tolerated formula.
Absolute bioavailability of alternative drug delivery routes is frequently compared with an IV formula for a reference. Area under the concentration-time curve for each route is calculated. Bioavailability is simply the ratio of the IN to IV area under the curve, assuming the same dose was administered and clearance was constant. In this type of study it is generally assumed that clearance remains constant within each subject across test arm investigations.
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METHODS |
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A randomized, open-label, 3-way cross-over design was used. On three different occasions, separated by 1 wk, the subjects received a single dose of each of the following three treatments in random order, counterbalanced so that an equal number of subjects received each treatment first, second, or third:
The 25 mg/mL IN midazolam formulation was prepared aseptically, creating a sterile product, under Good Manufacturing Practices conditions in the University of Kentucky College of Pharmacy Center for Pharmaceutical Science and Technology. The IN formulation, a nonaqueous solution containing midazolam 25 mg/mL, polyethylene glycol 400, butylated hydroxytoluene, saccharin, and propylene glycol, provided 2.5 mg of midazolam in 0.1 mL spray from a modified version of a commercially available unit-dose spray pump (Pfeiffer of America, Princeton, NJ, unit dose system). Commercially available midazolam (Versed® Injection; Roche Laboratories, Nutley, NJ) was purchased for comparative IV and IM administration.
Before study drug administration, subjects gently blew their noses. A physician administered a spray to each naris and the subjects remained in a semi-recumbent position, with the head of the bed elevated at a 20°–40° angle for 30 min. The IV dose, 5 mg midazolam in 10 mL sterile saline solution, was administered by infusion over a period of 15 min in an antecubital vein of the contralateral arm for blood sampling.
Serial blood samples were obtained through an indwelling venous catheter according to the following schedule: 0 (pre-dose), 5, 10, 20, 30, and 45 min, and 1, 1.5, 2, 3, 4, 8, and 12 h after drug administration. Venous blood samples were collected in 10-mL heparinized Vacutainer® tubes. After collection, the blood was centrifuged at 4°C, and the plasma was transferred to polypropylene tubes. The plasma was stored at or below –20°C at the study site until shipped to AAI Development Services, Inc., Kansas City Facility in Shawnee, KS for midazolam assay.
Plasma samples were analyzed by AAI International, a Good Laboratory Practices compliant laboratory, using liquid chromatography/mass spectrometry/mass spectrometry (LC/MS/MS) to determine the concentrations of midazolam, 1-hydroxymidazolam, and the added internal standard triazolam-d4. Analytes were extracted from plasma using liquid phase extraction. They were separated using reverse phase high performance liquid chromatography on a 3-micron C18 column. The analytes were detected using a PE/Sciex API III+ LC/MS/MS system in multiple reaction monitoring (MRM) mode for the following precursor/products (m/z): midazolam, 326 and 291; 1-hydroxymidazolam, 342 and 203; and triazolam-d4, 347 and 312; with retention times of 1.75, 1.53, and 1.50 min, respectively. The lower limit of detection for midazolam and 1-hydroxymidazolam was 0.50 ng/mL. The method was linear over a concentration range of 0.50–500.0 ng/mL using a 0.5-mL sample volume. Overall accuracy (inter-batch) was 89.3%–106.0% for midazolam and 93.3%–108.0% for 1-hydroxymidazolam. The inter batch overall precision (%CV) was 2.9%–9.4% for midazolam and 3.4%–113.0% for 1-hydroxymidazolam. The specificity, linearity, sensitivity, accuracy, precision, and stability of the data were within the Food and Drug Administration guidelines for Bioanalytical Method Validation. Freeze/thaw stability (3 cycles) and long-term sample storage (13 mo, –20°C) were tested and were acceptable.
A physician was in attendance for at least 4 h after each dose, and subjects were observed throughout the study session by a research nurse. Vital signs (arterial blood pressure, heart rate, respiratory rate) were measured before and at 10, 20, 30, 45, 60, 75, 90, 105 min and 2, 3, 4, 6, 8, and 12 h after each dose. Continuous pulse oximetry monitoring was done for all subjects for the first 6 h and as clinically indicated thereafter. Any observation of oxygen saturation <90% was recorded as an adverse event. In addition to spontaneously reported subjective symptoms, which were allowed at any time, subjects were also questioned as to their adverse event experience each time that vital signs were recorded (12).
An otolaryngologist examined the nasal passages to evaluate development of local mucosal irritation, inflammation, bleeding, and excoriation or ulceration at screening, before dosing on each study day, at 2–4 h after each dose, and within 72 h after treatment.
Pharmacokinetic parameters were determined using standard noncompartmental methods (13) with log-linear least square regression analysis (weighting factor 1/Y) to determine the elimination rate constants (
Z) (WinNonlin version 3.2; Pharsight Corp., Palo Alto, CA). Time to and maximum plasma concentration (Tmax and Cmax), elimination half-life (t1/2), area under the plasma concentration-time curve from time zero to infinity (AUC0–
) were also calculated. The absolute bioavailability (F) for the IN and IM dosage forms was determined by the formula F = AUCIN,0–/AUCIV,0– for the IN dose and F = AUCIM,0–/AUCIV,0– for the IM dose.
The subjects completed assessments of drug-induced impairment including a Digit-Symbol Substitution Task (DSST), Visual Analog self-measures and the Stanford Sleepiness Scale (SSS) at 0 (1 h before dosing as a practice session), 10, 20, 30, and 45 min and 1, 1.5, 2, 3, 4, 6, 8, and 12 h (14,15).
The Visual Analog Scale (VAS) consisted of 10 statements ("Stimulated," "Sedated," "High," "Anxious," "Fatigued," "Hungry," "Headache," "Feel a drug effect," "Like the drug effect," and "Willing to take the drug again") that were presented sequentially above a 100-mm line labeled "not at all" on the left end and "extremely" on the right end.
For the SSS, subjects described their current level of sleepiness among the following options: "Feeling active and alert, wide awake," "Functioning at high level, but not peak, able to concentrate," "Relaxed, awake, not at full alertness, responsive," "A little foggy, not at peak, let down, " "Fogginess, beginning to lose interest in remaining awake, slowed down," "Sleepiness, prefer to be lying down, fighting sleep, woozy," or "Almost in reverie, sleep onset soon, lost struggle to remain awake" (15).
DSST performance was analyzed according to total trial rate, correct trial rate, incorrect trial rate, and percentage of trials that were correct. Ratings on the VAS were scored based on the number of discrete units between the subject’s rating and the left end-point on each 100-unit scale. When subjects failed to initiate the DSST or the SSS scales because they were asleep, they were assigned a "0" for total response rate and a "7" for the sleep rating. No other substitutions for missing values were possible given the nature of the other quantitative measures.
Dependent variables were analyzed as a function of route and time after dose. Analyses of peak effects, time to peak effects, and area under the curve for response (AUCR), using linear trapezoidal rules, were also evaluated. Separate AUCR analyses were completed between baseline and 4 h after dose (AUCR0–4; i.e., over the duration of peak effects) as well as between baseline and 12 h after dose (AUCR0–12, i.e., over the full time course).
Statistical analyses were performed using Proc GLM with PC SAS (version 6.12; SAS Institute, Cary, NC). An analysis of variance model with factors sequence, subject nested within sequence, treatment, and period, was performed for peak effects, time to peak effects, and AUCR. Gender and route were used to analyze peak effects, time to peak effects, and AUCR. For VAS analysis, given the number of missing values because of subjects being asleep, degrees of freedom were inconsistent across variables and conditions. The least square means for each treatment group and pairwise comparisons between treatment groups were presented. To assess the gender effect and gender-by-route interaction, an analysis of variance with factors gender, period, route, and gender-by-route interaction was performed.
Descriptive statistics, mean and standard deviation, were calculated for the pharmacokinetic parameters. The statistical tests were two-sided with a critical level of 0.05. An analysis of variance with factors sequence, subject nested within sequence, treatment, and period was performed for log-transformed AUC and Cmax. The carryover effect for the three treatments was analyzed using an analysis of variance of log-transformed AUC and Cmax. Analysis of variance with factors sequence, subject nested within sequence, treatment and period for sequence (P > 0.1) indicated that carryover effects were not significant. The difference in Tmax values between the IN and IM treatment was compared using an analysis of variance of rank-transformed Tmax.
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RESULTS |
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Mean midazolam plasma concentration versus time curve profiles (n = 12) over the first 2 h after IV, IM, and IN administration are shown in Figure 1. Midazolam was rapidly absorbed after IN administration, with concentrations reaching a peak in 2 individuals at 5 min and in 75% of the individuals in 
10 min (median Tmax = 10 min). Mean pharmacokinetic parameters are presented in Table 1. Cmax values after the IN dose were higher than those after the IM dose. A significantly shorter Tmax was observed for the IN formulation compared with the IM formulation (P = 0.0001). Levels of 1-hydroxymidazolam were very low, and as such, are not reported. The ratios of metabolite to parent AUCs were 0.16 to 0.22 for the 3 routes of administration.
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DSST
Differences in drug-induced psychomotor and cognitive impairment were observed across routes of administration. Subjects were awakened, when possible, to initiate performance tasks at all scheduled time points but were unable to complete all tasks on numerous occasions. Three subjects failed to complete performance tasks at 6 time points, as the result of sleepiness, after IM dose administration; 7 subjects failed to complete performance tasks at 13 time points after IN dose administration; and 11 subjects failed to complete performance tasks at 29 time points after IV dose administration. Figure 2 presents trial rate on the DSST as a function of time after dose administration. No gender or gender-by-route interactions were observed, except for AUCR0–4 (for gender, P = 0.0375). The carryover effect was not significant (P > 0.1) for all 8 DSST and parameters.
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Table 2 presents pharmacodynamic parameters for DSST Trial Rate. It was obvious that differences among routes of administration occurred during the first 30 min after drug administration. On all measures, the order of magnitude of effects were identical with IV producing larger effects with a faster onset of action than IN, which in turn produced larger effects with a faster onset than IM. Significant effects of route were obtained on AUCR0–4. Follow-up tests indicated a significant difference between the IV and IM routes only, with the IV route engendering significantly greater AUCR0–4 than the IM route (P = 0.002). No significant gender, route, or gender-by-route interactions were obtained on time to peak effect or AUCR0–12.
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Figure 3 presents the relationship between midazolam concentrations from each route of administration in relation to DSST trial rates. The data parallel conclusions from Figure 2 and 4 showing a rapid affect of each route with an IV>IN>IM orientation. A shallow clockwise hysteresis is present for each route of administration.
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Statistical comparisons for IM and IN to the IV route were precluded because of the relatively large number of missing values from the IV route. However, significant differences in ratings were observed across time for all VAS scales (P < 0.01), with the exception of Anxious. Differences in ratings of High were observed as a function of route (P < 0.05), with ratings after IN doses significantly larger than after IM doses. Route by time interactions were obtained on ratings of Sedated (P < 0.005), High (P < 0.0001), Headache (P < 0.05), and Feel Drug (P < 0.01). Simple effects analyses of these interactions indicated significant time effects during both the IN and IM routes, and significant differences between the IN and IM routes 10 (Sedated, High, Feel drug), 20 (High, Headache), and 60 (Headache) min after dose.
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DISCUSSION |
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TopAbstractIntroductionMETHODSRESULTSDISCUSSIONREFERENCES |
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Gudmundsdottir et al. (9) prepared a nonsterile aqueous solution, buffered to pH 4.2 and preserved with benzalkonium chloride, in which midazolam solubility was increased to 17 mg/mL by cyclodextrin complexation. Multiple sprays were necessary to deliver a 4- to 5-mg dose. The 28 mg/mL formulation used by Knoester et al. (11) had water and propylene glycol as co-solvents, buffered to pH 4, with the addition of 1% benzyl alcohol as an antimicrobial preservative (11). These investigators reported excellent pharmacokinetics, with a maximum concentration of 71 ng/mL achieved in 14 minutes on average, and an average bioavailability of 83%. These data were superior to those of Gudmundsdottir et al., who demonstrated a 64% bioavailability, with a maximum concentration of only 42 ng/mL achieved in 15.5 minutes (9). Both products caused nasal irritation, suggesting midazolam itself may be the primary cause.
The formulation used in our study was a 25 mg/mL solution using a nonaqueous co-solvent system of propylene glycol and PEG 400. The product is sterile, eliminating the use of irritating antimicrobial preservatives. A buffer system is not required in this format. Despite these formulation changes, the subjects still reported mild-to-moderate nasal and throat irritation, further suggesting the drug itself is the offending agent. Additional strategies are needed to manage irritation and bitter taste.
This formulation produced excellent pharmacokinetic and pharmacodynamic properties. Most notable was a median tmax of 10 minutes, faster than previous formulation reports, while achieving a peak level of 80 ng/mL, well above the 40 ng/mL threshold to induce sedation. The formulation demonstrated reproducible results across the subjects and a very small sd for each parameter as compared with previous reports.
Psychomotor impairment and sedation paralleled the pharmacokinetics producing impairment and sedation within 20 minutes. Pharmacodynamic analyses indicated clearly that all three routes of administration engendered performance impairment and subjective ratings of Sleep and VAS ratings of Fatigued, Sedated, High, Headache, and Feel Drug Effect. The duration of the effects occurred over a 3-hour interval after drug administration. The magnitude of the effects produced a ranking of IV>IN>IM on most parameters. Given similarities in the psychomotor recovery after the different routes of administration, it appears that route is not a major influence on recovery times, as the pharmacokinetic and pharmacodynamic profiles converged within approximately 1 hour in this study.
A limitation of this study was not obtaining a blood sample at 15 minutes after IN/IM administration and at the conclusion of the IV infusion. The deficiency has a potentially modest effect of over-estimating bioavailability of nasal administration. The data in aggregate suggest a good correlation between pharmacokinetic variables and pharmacodynamic response. The impact of this oversight is likely negligible in relation to the objectives of this pilot study.
In conclusion, this study clearly demonstrates that this IN midazolam formulation is rapidly and reliably absorbed. The formulation is worthy of further investigation as a therapeutic alternative for a convenient, noninvasive, and rapidly acting sedative.
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Footnotes |
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Supported, in part, by Intranasal Technology, Inc., Lexington, Kentucky.
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REFERENCES |
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TopAbstractIntroductionMETHODSRESULTSDISCUSSIONREFERENCES |
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