University of Groningen. Transdermal delivery of anticholinergic bronchodilators Bosman, Ingrid Jolanda

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1 University of Groningen Tranermal delivery of anticholinergic bronchodilators Bosman, Ingrid Jolanda IMPORTANT NOTE: You are advised to consult the publisher's version (publisher's PDF) if you wish to cite from it. Please check the document version below. Document Version Publisher's PDF, also known as Version of record Publication date: 1996 Link to publication in University of Groningen/UMCG research database Citation for published version (APA): Bosman, I. J. (1996). Tranermal delivery of anticholinergic bronchodilators: methodological and clinical aspects. Groningen: s.n. Copyright Other than for strictly personal use, it is not permitted to download or to forward/distribute the text or part of it without the consent of the author(s) and/or copyright holder(s), unless the work is under an open content license (like Creative Commons). Take-down policy If you believe that this document breaches copyright please contact us providing details, and we will remove access to the work immediately and investigate your claim. Downloaded from the University of Groningen/UMCG research database (Pure): For technical reasons the number of authors shown on this cover page is limited to 10 maximum. Download date:

2 Chapter 7 A semi-automated solid-phase extraction and radioreceptor assay for the analysis of scopolamine in urine and plasma 7.0. Summary In a double-blind placebo-controlled cross-over study, we evaluated the therapeutic efficacy of tranermal scopolamine in ten patients with reversible airways obstruction. They received a patch (Scopoderm TTS) behind the ear for three days and samples of blood and urine were taken. A highly sensitive method was developed and validated to measure the low levels of free and total scopolamine in plasma and urine. The procedure consisted of a semi-automated solid-phase extraction followed by the analysis using radioreceptor assays. The measured plasma concentrations of free and total scopolamine were 43.6 pg/ml and pg/ml, respectively. In 24-hours urine 6.3 µg of free scopolamine and 83.4 µg total scopolamine was excreted. For urine samples of 1 ml, the limit of detection (LOD) of the assay is 550 pg/ml and the limit of quantitation (LOQ) is 610 pg/ml. For plasma samples of 1.5 ml, the LOD is 16 pg/ml and the LOQ is 38 pg/ml.

3 92 Chapter Introduction Scopolamine is a competitive inhibitor of the muscarinic receptors of acetylcholine. It is marketed as a tranermal drug delivery system (Scopoderm TTS) for the prevention of nausea and vomiting associated with motion sickness. Like other anticholinergics, it can also cause relaxation of the smooth muscle of the bronchi and bronchioles [1, 2]. The tranermal therapeutic system of scopolamine is a thin plaster (0.2 mm), comprising a reservoir which contains the drug in a mixture of mineral oil and polyisobutylene, held between a impermeable backing layer and a rate-controlling membrane. The unit contains 1.5 mg of scopolamine and is designed to deliver a total of 0.5 mg at an constant rate of approximately 5 µg/hour over a 3-day period [1]. On the epidermal side of the membrane is an adhesive layer containing approximately 140 µg as a priming dose to saturate certain binding sites within the skin and to accelerate achievement of steady state blood levels. Tranermal drug delivery by s of patches may offer benefits over conventional drug therapy by producing sustained, constant and controlled plasma concentrations over a prolonged period of time. This may result in a prolonged duration of action which improves patient compliance since frequent intake of the drug is no longer necessary [3]. For this reason, we evaluated the therapeutic efficacy of tranermal scopolamine in ten patients with reversible airways obstruction [4]. They received a patch (Scopoderm TTS) behind the ear for three days and samples of blood and urine were taken. In order to correlate the levels of scopolamine with therapeutic efficacy, a highly sensitive analytical method was needed to measure the plasma and urinary concentrations of scopolamine in these patients. Most described methods are sensitive enough to measure the concentrations in urine, however plasma levels have rarely been determined [1, 5, 6]. Since scopolamine is extensively metabolized in glucuronide and sulphate conjugates, we decided to quantitate scopolamine as well as the sum of scopolamine and metabolites. The analysis of scopolamine is performed by s of radioreceptor assay (RRA). The principle of RRA is based on the ability of a drug to compete with a radiolabelled ligand for a specific receptor binding site. The drug (scopolamine) exerts its pharmacological action through interaction with a given receptor (muscarinic). The drug can be analysed quantitatively when an aliquot of the sample is added to a solution that contains a fixed amount of receptor and a fixed amount of radiolabelled ligand ([ 3 H]N-methylscopolamine). The unknown quantity of scopolamine in urine and/or plasma can be calculated by determining the inhibition of labelled ligand binding and comparing this to the inhibition produced by known quantities of drug in calibration samples [7]. In principle metabolites which possess a binding affinity to the receptor, may be codetermined by the radioreceptor assay. However, no interferences of metabolites of scopolamine are to be expected [6, 8]. Therefore, the total amount of scopolamine (free plus conjugated) was determined by incubation of urine and

4 Analysis of scopolamine in urine and plasma 93 plasma samples with glusulase, which is a preparation of a ß-glucuronidase and arylsulfatase. In order to eliminate interferences of glusulase and matrix interferences, a semi-automated solid-phase extraction (SPE) was required. In this chapter, we describe the development and validation of a procedure to analyse free (unconjugated) and total scopolamine in urine and plasma. The procedure consists of a semi-automated solid-phase extraction with Si-columns using an ASPEC-system (Automatic Sample Preparation with Extraction Columns). Scopolamine was eluted with dichloromethane and the extracts were analysed with a radioreceptor assay (RRA) Materials and Methods Materials [N-methyl- 3 H]Scopolamine methyl chloride ([ 3 H]NMS, 80.4 Ci/mmol) was obtained from Du Pont NEN (Du Pont, Wilmington, DE, USA). Scopolamine hydrobromide trihydrate was obtained from Merck (Darmstadt, Germany). Glusulase was obtained from Calbiochem (La Jolla, CA, USA). Sodium hydroxide was obtained from Janssen Chimica (Beerse, Belgium). All other chemicals and solvents were of analytical grade and obtained from Merck (Darmstadt, Germany). Polyethylene tubes (4 ml and 12 ml) were obtained from Greiner (Alphen a/d Rijn, The Netherlands). The GF/B glassfibre filters were from Whatman (Maidstone, UK). Rialuma was used as scintillation liquid, obtained from Lumac (Olen, Belgium), in combination with mini-scintillation counting vials from Packard (Groningen, The Netherlands). Bond Elut extraction columns, type Si (silica gel), bed volume 2.8 ml were kindly donated by Varian (Sample Preparation Products, Harbor City, CA, USA). Heparinized human plasma was obtained from the local blood bank and stored at -20 C. Calf brains without cerebellum were obtained from the local slaughterhouse and stored at -80 C Preparation of solutions The 50 mm sodium phosphate buffer ph 7.4 (assay buffer) was prepared by dissolving 1.38 g NaH 2 PO 4.H 2 O and 7.12 g Na 2 HPO 4.2H 2 O in 1 l distilled water. The 50 mm sodium phosphate buffers ph 4.75 or ph 3.75 were prepared by the addition of 4 M hydrochloric acid to the assay buffer. The 50 mm sodium phosphate buffers ph 11.5 or were prepared by the addition of 4 M sodium hydroxide to the assay buffer. Glusulase solutions ph 4.75 or ph 3.75 were prepared by diluting the glusulase stock solution 75 times with 50 mm sodium phosphate buffer ph 4.75 or ph A scopolamine stock solution of 1x10-3 M was prepared in ethanol and stored at -20 C.

5 94 Chapter 7 Table 7.1. Overview of the analysis of scopolamine. INCUBATION WITH GLUSULASE *1 INCUBATION WITH GLUSULASE, ADAPTED METHOD EXTRACTION WITH THE ASPEC-SYSTEM CONDITIONING SAMPLE APPLICATION WASHING ELUTING EVAPORATION RECONSTITUTE RESIDUE RADIORECEPTOR ASSAY URINE 250 µl urine 750 µl glusulase solution (ph 4.75) incubation 10 hours 37 C 1 ml phosphate buffer (ph 11.5) 5 ml methanol 5 ml water 3 ml dichloromethane 2 ml phosphate buffer (ph 7.4) flow rate 3.0 ml/min 1000 µl urine *2 or 1600 µl incubated sample *1 flow rate 1.5 ml/min 1.5 ml phosphate buffer flow rate 3.0 ml/min 3 ml dichloromethane flow rate 1.5 ml/min vacuum concentrator 1000 µl phosphate buffer 100 µl of reconstituted residue 400 µl receptor suspension 50 µl [ 3 H]N-methylscopolamine PLASMA 250 µl plasma 750 µl glusulase solution (ph 3.75) incubation 10 hours 37 C 1 ml phosphate buffer (ph 11,25) 500 µl plasma 1500 µl glusulase solution (ph 3.75) incubation 10 hours 37 C 2 ml phosphate buffer (ph 11,25) 5 ml methanol 5 ml water 3 ml dichloromethane 2 ml phosphate buffer (ph 7.4) flow rate 3.0 ml/min 1500 µl plasma *2 or 1600 µl incubated sample *1 or 3600 µl incubated sample *1 (adapted) flow rate 1.5 ml/min 1.5 ml phosphate buffer flow rate 3.0 ml/min 3 ml dichloromethane flow rate 1.5 ml/min vacuum concentrator 100 µl phosphate buffer 100 µl reconstituted residue 100 µl receptor suspension 50 µl [ 3 H]N-methylscopolamine *1 To determine total (free plus conjugated) scopolamine. *2 To determine free (unconjugated) scopolamine.

6 Analysis of scopolamine in urine and plasma Preparation of receptor material Calf brains without cerebellum were homogenized in 6 volumes (w/v) icecold 0.32 M sucrose using a Teflon-glass Potter Elvejehem homogenizer (RW 20, Janke & Kunkel KG, IKA-Werk, Staufen i. Breisgau, Germany) at 1,200 rpm. The homogenate was centrifuged for 10 min at 1,000 x g and the resulting supernatant was centrifuged for 60 min at 100,000 x g (Beckman L8-55 ultracentrifuge, Beckman Instruments Nederland B.V., Mijdrecht, The Netherlands). The pellet was resuspended in the assay buffer and centrifuged 30 min at 100,000 x g. This washing was repeated and the final pellet was resuspended in 5 volumes (w/v) assay buffer. The entire procedure was carried out at 4 C. The obtained homogenate was frozen with liquid nitrogen and lyophilized for 24 hours (Hetosicc CD 52-1, lyophilizer, Heto, Birkerød, Denmark). The lyophilized preparation was stored at - 20 C. The optimum amount of lyophilized material for the radioreceptor assay of scopolamine in urine (3 mg tissue in 400 µl assay buffer) and in plasma (2 mg tissue in 100 µl) was experimentally determined Scopolamine analysis The procedure consists of a semi-automated solid-phase extraction followed by the analysis of scopolamine (free and total) using radioreceptor assays. To determine total scopolamine, the urine or plasma samples were pretreated with glusulase. An overview of the procedure is presented in Table 7.1. Instrumentation The extraction was performed with an ASPEC-system (Gilson Medical Electronics, Villiers le Bel, France). As shown in Figure 7.1, the ASPEC-system consists of three components: A Model 401 dilutor, a sample processor, and a set of racks and accessories to handle 3 ml SPE-columns and solvents. Incubation with glusulase Total scopolamine (free and conjugated) was measured after incubation of the urine or plasma samples with glusulase. The biological matrices were buffered with glusulase solution in order to obtain an incubation ph between 4.5 and 6.2. To 250 µl urine, 750 µl glusulase solution ph 4.75 was added and vortexed for 5 sec. The mixture was incubated for 10 hours at 37 C under constant shaking. The reaction was terminated by adding 1 ml phosphate buffer ph 11.5, to get a final ph of 7.4. For plasma samples (250 µl), a glusulase solution ph 3.75 (750 µl) was used and to terminate the incubation, phosphate buffer ph 11,25 (1 ml) was added to get a final ph of 7.4. Otherwise the procedure for plasma was the same as for urine. The incubated samples were vortexed and 1600 µl aliquots were used for the extraction. When the initial procedure for plasma appeared to be too insensitive to analyse the actual levels, an adaption was developed. To 500 µl plasma, 1500 µl glusulase

7 96 Chapter 7 Figure 7.1. The ASPEC-system (Automatic Sample Preparation with Extraction Columns), which consists of three main components: A Model 401 dilutor (A), a sample processor (B), and a set of racks and accessories to handle 3 ml SPE-columns and solvents (C1, C2, C3). solution ph 3.75 was added and vortexed for 5 sec. The mixture was incubated for 10 hours at 37 C and the reaction was terminated by adding 2 ml phosphate buffer ph 11.25, to get a final ph of 7.4. The incubated sample was vortexed and 3600 µl was used for the extraction. Extraction Bond Elut extraction columns, type Si (silica gel) were sealed with polypropylene caps and installed on the SPE-rack (Figure 7.1). The solvents and samples (urine or plasma) were placed in the solvent positions and sample rack, respectively. Polyethylene tubes (55 x 12 mm, 4 ml) were placed in the collection rack. The extraction was then performed by ASPEC in a sequential mode as follows. First, the column was preconditioned by subsequent washings with 5 ml methanol, 5 ml water, 3 ml dichloromethane and 2 ml assay buffer at a flow rate of 3.0 ml/min. The urine sample (1000 µl), plasma sample (1500 µl) or the incubated sample (1600 µl or 3600 µl for the adapted procedure) was dispensed onto the column and pushed through by positive pressure at a flow rate of 1.5 ml/min. To push the sample through the column completely, 1.5 ml of air was applied. After

8 Analysis of scopolamine in urine and plasma 97 washing the column with 1.5 ml assay buffer at a flow rate of 3.0 ml/min, the column was dried by passing through 6 ml of air. Scopolamine was then eluted into the collection tube by passing through 3 ml of dichloromethane at a flow rate of 1.5 ml/min and 1.5 ml of air was applied onto the column to push the dichloromethane through the column completely. Small droplets of water in the dichloromethane fraction were removed manually by s of a pipet. Dichloromethane was evaporated to dryness within 45 min, using a vacuum concentrator, at 1500 rpm and 40 C (Beun de Ronde, Abcoude, The Netherlands). The residue was reconstituted in 1000 µl (urine samples) or 100 µl (plasma samples) assay buffer by vortex mixing for 5 s. After standing for 1 hour, the tube was vortexed again for 5 s. To duplicate polyethylene tubes (12 ml), 100 µl of the urine extract was pipetted and used for the radioreceptor assay. However, the entire reconstituted plasma sample (100 µl) had to be used in the radioreceptor assay which t that for duplicate analyses two extractions were necessary. Radioreceptor assay (RRA) To each polyethylene tube (12 ml) with the 100 µl extraction sample, 400 µl receptor suspension (for urine samples) or 100 µl receptor suspension (for plasma samples) was added. The tubes were vortexed and incubated during 60 min at 0 C before 50 µl of [ 3 H]NMS solution (4x10-9 M) was added. The tubes were mixed again and incubated for another hour at 0 C. After the addition of 4 ml icecold assay buffer, the samples were immediately filtered through Whatman GF/B glassfibre filters under vacuum using a filtration apparatus (48S, University Centre for Pharmacy, Groningen, The Netherlands). The tubes were rinsed twice with 4 ml icecold assay buffer, which was also filtered. The total filtration and rinsing process, taking place in approximately 15 s, was carried out on each tube in turn. The filters were transferred into mini-scintillation vials and dispersed in 3.5 ml scintillation cocktail by shaking for 120 min. The vials were counted for 40,000 counts or 5 min in a liquid scintillation counter (Minaxi, Packard, Groningen, The Netherlands), whatever came first. Fifty µl of the used [ 3 H]NMS solution were added to 2 miniscintillation vials and counted as well Calibration curve From the scopolamine stock solution, appropriate dilutions were made freshly on the day of analysis in blank urine to provide concentrations ranging from 5x10-10 M to 2x10-7 M. After extraction, this resulted in final assay concentration ranging from 9.09x10-11 M to 3.64x10-8 M. In blank plasma, the concentrations were ranging from 1x10-11 M to 5x10-9 M giving final assay concentrations of 6x10-11 M to 3x10-8 M. The samples of the calibration curves were analysed in duplicate. The curves were fitted with the Ligand curve fitting program to calculate the parameters which characterize the calibration curves [9].

9 98 Chapter Quality control The assay of urine was validated with quality control samples which were prepared in blank urine, at 5x10-9 M, 1x10-8 M and 5x10-8 M, respectively. In blank plasma, quality control samples were prepared with concentrations of 1.25x10-10 M, 3x10-10 M and 1.25x10-9 M, respectively. The quality control samples were stored at -20 C. On the day of analysis, the quality control samples were extracted and analysed in duplicate. Incubated quality control samples (5x10-8 M and 1.25x10-9 M in urine and plasma, respectively) were codetermined to study the influence of glusulase on the assay. The adapted procedure for total scopolamine in plasma was validated with the quality control of 1.25x10-9 M. The obtained binding values (Bq) of the quality control samples were introduced in the fitted calibration curves and the concentrations in urine or plasma were calculated. For urine samples, inter-day data (between days) were assessed by duplicate analyses of the quality control samples on different days; intra-day data (within day) were determined by duplicate analyses of the quality control samples after 5 separate extractions on the same day. The quality control samples in plasma were analysed in duplicate on ten different days. Because for plasma samples duplicate analyses s duplicate extractions, intra- and inter-day data were calculated from the same samples using one-way ANOVA Patient samples Urinary and plasma levels of free and total scopolamine were determined in ten patients with reversible airways obstruction [4]. The patients received a scopolamine patch (Scopoderm TTS) or placebo behind the ear for 72 hours. The patches (placebo and Scopoderm TTS) were kindly provided by Ciba Geigy, Arnhem, The Netherlands. Every second patch day urine was collected during 24 hours and every third patch day at 9.00 a.m. blood was taken from the patients. Plasma was obtained by centrifugation of the blood sample (collected in a tube containing heparin) at 5,000 rpm for 11 min (Megafuge 1.0, Heraeus Sepatech, Germany). The urine and plasma samples were stored at -20 C until analysis. The same procedure as described for the quality control samples was performed to calculate the unknown amount of free and total scopolamine in urine and plasma of the patients.

10 Analysis of scopolamine in urine and plasma Results and Discussion Scopolamine analysis Radioreceptor assay For the analysis we used a radioreceptor assay (RRA), based on the competition between a radiolabelled ligand ([ 3 H]NMS) and unlabelled drug (scopolamine) for the binding to muscarinic receptors, because they are highly sensitive and selective [7]. Because of the potency of scopolamine, the concentrations of the drug in plasma and urine will be very low and difficult to detect. Therefore, we enhanced the sensitivity of the assay by applying a pre-incubation step of the drug with the receptor preparation at 0 C before the radiolabelled ligand was added [7, 10]. This resulted in a gain in sensitivity of a factor 2 compared to equilibrium conditions at 37 C. However, this gain was much smaller than a previously described gain of a factor 6.7 using [ 3 H]dexetimide as radiolabelled ligand [10]. Probably, the gain in sensitivity is not only dependent on the competitive drug but depends also on the radiolabelled drug. Besides a high sensitivity, another advantage of RRA is the selectivity towards compounds which produce a pharmacological effect via an interaction with receptors. This s that in principle metabolites may be codetermined when they have an affinity towards the receptor. In case of scopolamine, no interferences of metabolites are to be expected. The glucuronide and sulphate conjugates of scopolamine will have no affinity towards the muscarinic receptor because of their physico-chemical properties. Scheurlen et al. [6] showed that scopoline and scopine, possible degradation products of scopolamine, will have no interference with the assay. This was also observed in a previous study, testing the displacement of binding of [ 3 H]dexetimide to muscarinic receptors. The very low affinity constants of scopoline (4.1x10 6 M -1 ) and aposcopolamine (3.7x10 3 M -1 ), compared with 1.7x10 9 M -1 for scopolamine, showed that scopoline and aposcopolamine will not interfere with the assay of scopolamine. Ensing et al. [5] showed that direct radioreceptor assays of scopolamine in urine can be performed because of the relatively high urine concentrations after therapeutic dosing. However, using this direct assay, calibration curves had to be prepared for each patient in his/her own blank urine to compensate for the inhibition of binding caused by blank urine. Moreover, the addition of glusulase to urine samples, added to determine the total amount of scopolamine, also yielded some inhibition of binding. Although this inhibition seemed highly reproducible, extra calibration curves (to which glusulase had been added) had to be prepared in each patient s blank urine. For plasma, a sample clean-up and concentration step was considered unavoidable because plasma proteins disturb the sensitivity and accuracy of the assay, and because drug concentrations in plasma are much lower than in urine.

11 100 Chapter 7 Table 7.2. The calculated parameters which characterize the calibration curves. K 11 is the apparent affinity constant of [ 3 H]NMS (M -1 ); K 21 is the apparent affinity constant of scopolamine (M -1 ); R 1 is the receptor concentration (M); N 1 is percentage the non-specifically bound [ 3 H]NMS; MSQ is the sum of squares; IC 50 is the concentration of scopolamine which displaces 50% of [ 3 H]NMS (M). Parameters K 11 K 21 R 1 N 1 MSQ IC 50 (x10 9 ) (x10 9 ) (x10-9 ) (%) (x10-13 ) (x10-9 ) Urine (n=11) Plasma (n=10) Figure 7.2. Calibration curves for the inhibition of [ 3 H]N-methylscopolamine ([ 3 H]NMS) binding by scopolamine. = urine; = plasma.

12 Analysis of scopolamine in urine and plasma 101 Solid-phase extraction Solid-phase extraction (SPE) was applied as an effective sample pre-treatment to eliminate matrix interferences and the interferences of glusulase, and to concentrate plasma samples. The developed extraction procedure was based on the described sample preparation of N-0437 in plasma and urine with Si-columns [11], and earlier investigations about the extraction of scopolamine from urine. These manual procedures were adapted and automated using a Gilson automated extraction system ASPEC (Figure 7.1). Different flow rates, air volumes and eluting volumes were tested to improve the extraction procedure. Additionally, the ASPEC-system offers two possibilities to carry out extractions: A batch mode and a sequential mode. In the sequential mode, the ASPEC accomplishes the complete extraction process of the first sample, and then continues with the next sample, whereas in the batch mode all SPE-columns are treated batchwise. The sequential mode was used because higher recoveries and reproducibilities can be obtained compared with the batch mode [12] Validation of the assay Representative calibration curves of samples, prepared freshly on the day of analysis in blank urine or blank plasma, are shown in Figure 7.2. The parameters which characterize the calibration curves are given in Table 7.2. The relatively small variations in the calculated affinity constants of scopolamine (K 21 ) indicate the high reproducibility of the assay under non-equilibrium conditions. From the fitted calibration curves, the IC 50 value was calculated, which is defined as the concentration of ligand (scopolamine) which displaces 50% of the bound radioligand ([ 3 H]NMS). The IC 50 values in plasma were significantly higher in comparison with urine (Student t-test, p < 0.05). The differences in fitted parameters between the curves prepared in urine or plasma can be explained by differences in receptor concentrations and concentrations of the radiolabelled drug in the assay. The assay in plasma differed from the assay in urine because higher sensitivities were needed to analyse the low levels of scopolamine in plasma. Therefore, the incubation volume for the RRA of plasma was adapted to a volume of 250 µl, because the smaller the volume used, the greater the sensitivity [13]. With the thus obtained calibration curves, the samples used for the fitting were back-calculated to their original concentrations in urine or plasma, and the accuracy and precision data were established (Table 7.3 and 7.5). As expected, the precision became worse at the low and high end of the curves (Figure 7.3). For plasma, the precision exceeded the 15% coefficient of variation () at plasma concentrations lower than 5x10-11 M. For urine, precision data were below 15% at urine concentrations higher than 2x10-9 M. The higher values in plasma in comparison with urine can be explained by the difference in experimental set-up. In case of plasma samples, duplicate analyses t duplicate extractions whereas for urine samples only one extraction is needed to perform duplicate analyses.

13 102 Chapter 7 Table 7.3. Accuracy and precision of the back-calculated concentrations of the spiked calibration samples in urine (n=11). Added conc in urine (Log final assay conc) 2x10-9 M 5x10-9 M 1x10-8 M 2x10-8 M 5x10-8 M 1x10-7 M (-9.44) (-9.04) (-8.74) (-8.44) (-8.04) (-7.74) * acc *1 n=10. Table 7.4. Accuracy and precision of quality control samples prepared in urine. Quality control samples Without glusulase With glusulase Added conc in urine 5x10-9 M 1x10-8 M 5x10-8 M 5x10-8 M (Log final assay conc) (-9.04) (-8.74) (-8.04) (-8.74) Inter-day (n=9) acc Intra-day (n=5) acc

14 Analysis of scopolamine in urine and plasma 103 Table 7.5. Accuracy and precision of the back-calculated concentrations of the spiked calibration samples in plasma (n=10). Added conc in plasma (Log final assay conc) 5x10-11 M 1x10-10 M 2x10-10 M 5x10-10 M 1x10-9 M 2x10-9 M (-9.52) (-9.22) (-8.92) (-8.52) (-8.22) (-7.92) acc Table 7.6. Accuracy and precision of quality control samples prepared in plasma (n=10). Quality control samples Without glusulase With glusulase Added conc in plasma 1.25x10-10 M 3x10-10 M 1.25x10-9 M 1.25x10-9 M *1 (Log final assay conc) (-9.12) (-8.74) (-8.12) (-9.00) inter-day intra-day acc inter-day intra-day *1 n=9.

15 104 Chapter 7 Figure 7.3. Precision data of the back-calculated concentrations of the spiked calibration samples. = urine; = plasma. The method was validated with quality control samples without glusulase at three different concentrations (Table 7.4 and 7.6). The results show that the assay provides good accuracy, which should be within ± 15% of the actual value, and excellent precision, which should not exceed 15% [14]. Only the lowest quality control prepared in plasma deviates more than 15% of the actual value. However, at the limit of quantitation (LOQ), the value should be within 20% of the actual value and the precision should not exceed 20% [14]. This s that for plasma, the LOQ is close to 1.25x10-10 M, which corresponds to 38 pg/ml for plasma samples of 1.5 ml. The LOQ should be obtained independently from the calibration curve which s that at least five samples independent from the samples of the calibration curve should be determined [14]. Therefore, extra quality control samples were prepared in blank urine at 2x10-9 M. The calculated intra-day precision and accuracy, determined by duplicate analyses of five samples on the same day, were 8.3% and 113.3%, respectively. The inter-day data, obtained by duplicate analyses on five different days, show that a urine concentration of 2x10-9 M is near the LOQ: The precision was 25.6% and the accuracy 85.2%. Hence, the LOQ can be estimated to be about 610 pg/ml for urine samples of 1 ml.

16 Analysis of scopolamine in urine and plasma 105 Table 7.7. Free and total scopolamine (µg) in 24 hours-urine of 10 patients. Patient Free Phase 1 Free Phase 2 Total Phase 1 Total Phase n=10: Phase 1: Patients first received scopolamine, then placebo. Phase 2: Patients first received placebo, then scopolamine. All urines from patients that received placebo showed no detectable scopolamine concentrations. The LOQ must be differentiated from the limit of detection (LOD) which for RRA can be defined as the concentration of analyte at which the fraction bound label is significantly smaller than the fraction bound label in the absence of analyte [7]. This was determined experimentally by performing replicate measurements of the zero standard (amount of bound [ 3 H]NMS in absence of scopolamine). From these data the standard deviation () was calculated and the LOD was found at the intersection of the line representing the zero standard minus 2 with the composite calibration curve. For urine samples of 1 ml, the LOD of the assay was 550 pg/ml and for plasma samples of 1.5 ml, the LOD was 16 pg/ml. Incubated quality control samples were codetermined to study the influence of glusulase on the assay. The quality control data for plasma were obtained with the initial procedure. The presented data in Table 7.4 and 7.6 show that the incubated samples meet the above mentioned criteria (accuracy ± 15%, 15%). This s that a single calibration curve can be prepared to measure both free and total scopolamine in urine or plasma. However, the levels of total scopolamine in plasma samples of the patients were not detectable with the initial procedure.

17 106 Chapter 7 Table 7.8. Free and total scopolamine (pg/ml) in plasma of 10 patients. Patient Free Phase 1 Free Phase 2 Total Phase 1 Total Phase n=10: Phase 1: Patients first received scopolamine, then placebo. Phase 2: Patients first received placebo, then scopolamine. All plasma samples from patients that received placebo showed no detectable scopolamine concentrations. Therefore, the volume of the plasma samples was increased and the adapted procedure was validated with a quality control sample of 1.25x10-9 M scopolamine. The measured accuracy was 110.7% (± 0.4%) and the adapted procedure was found to be not statistically different from the initial procedure (Student t-test, p > 0.05) Patient samples Urine After application of Scopoderm TTS to the postauricular area of the patients, we found that an average of 6.3 µg (range µg) of free scopolamine and an average of 83.4 µg (range µg) of total scopolamine was excreted in 24- hours urine (Table 7.7). The measured urine concentrations (free and total) were all higher than the lowest quality control of 5x10-9 M (1.5 ng/ml). In the placebo phase no scopolamine was detected. The observed variations in scopolamine excretion corresponds with the four to sixfold variations in urine excretion rates reported before [15]. During steady state conditions approximately 120 µg of scopolamine is delivered to the patient in 24

18 Analysis of scopolamine in urine and plasma 107 hours. We found that of this dose, about 65% is excreted in 24-hours urine as glucuronide and sulphate conjugates and only 5% of the dose is excreted unchanged. These results are in line with earlier studies in which less than 10% of the administered dose was excreted unchanged [1, 6]. However, we found 70% of total scopolamine excreted in 24-hours urine, whereas Scheurlen et al. [6] found only 34% of the drug in the urine which may be due to the incubation procedure. Plasma Up till now, plasma concentrations of scopolamine have rarely been reported because a sensitive method for the measurement of such low levels was lacking [1]. We measured average plasma concentrations of free and total scopolamine of 43.6 pg/ml (range pg/ml) and pg/ml (range pg/ml), respectively (Table 7.8). Three of ten measured concentrations of free scopolamine were below the LOQ but above the LOD. With the adapted procedure, the plasma concentrations of total scopolamine were all above the LOQ of 7.5x10-10 M in the assay, which corresponds with 124 pg/ml in plasma used for the total scopolamine determination. In the placebo phase no scopolamine was detected. Muir et al. [16] reported a peak plasma concentration for free scopolamine of 0.42x10-9 M (127 pg/ml) in 4 volunteers after tranermal administration of a scopolamine patch with a priming dose of 200 µg and a release rate of 10 µg/hour. However, although the dose delivered was twice as high as in our study, the drug was detectable in only 4 of 12 volunteers. Recently, plasma concentrations in a group of 8 subjects were determined by a method which consisting of a preparative extraction step with C 18 columns, followed by an analytical quantitation step with a muscarinic radioreceptor assay [8]. The free plasma concentration was 134 pg/ml (range pg/ml) which is three times higher as the concentration we measured. If we assume that doubling of the dose results in two times higher concentrations, our results are comparable with the plasma concentration reported by Muir et al. [16], taking into account that they were unable to detect scopolamine in 8 of 12 volunteers. The method we described offers the advantage of assaying both free and total scopolamine which gives extra information on the bioavailability and kinetics of the drug Conclusions The procedure we developed and validated, consists of a semi-automated solidphase extraction followed by the analysis of free and total scopolamine with radioreceptor assay (RRA). Although the concentrations after tranermal drug delivery of scopolamine are very low, the assay is sufficiently sensitive to measure these levels. For urine samples of 1 ml, the limit of detection (LOD) of the assay is 550 pg/ml and the limit of quantitation (LOQ) is 610 pg/ml. For plasma samples of 1.5 ml, the LOD is 16 pg/ml and the LOQ is 38 pg/ml.

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