Br. J. clin. Pharmac. (1991), 32, 69-76
ADONIS 030652519100141T
Dose-related analgesic effects of flupirtine T. HUMMEL, T. FRIEDMANN, E. PAULI, G. NIEBCH,1 H. 0. BORBE' & G. KOBAL Department of Pharmacology and Toxicology, University of Erlangen-Nurnberg, Universitatsstrasse 22, W-8520 Erlangen and 'Department of Biochemistry, ASTA Pharma AG, POB 100105, W-6000 Frankfurt/Main, Germany
1 Flupirtine is a novel and, in all probability, centrally acting, analgesic. The present investigation was conducted in order to investigate dose-related effects of perorally administered flupirtine in man, with special regard to specifically analgesic actions, employing a model based on pain-related chemosomatosensory evoked potentials and subjective intensity estimates of painful stimuli. 2 Plasma concentrations of flupirtine measured 2 h after dosing linearly increased as a function of the administered dose. 3 It was possible to reproduce our own previously obtained results, which established the analgesic action of 200 mg flupirtine administered perorally. 4 Intensity estimates linearly decreased as a function of the administered dose, whereas chemosomatosensory evoked potential amplitudes non-linearly changed in relation to the administered dose. 5 In the spontaneous EEG, a dose-dependent increment in the power-spectra was observed, and this mainly in the alpha- and beta-range.
Keywords flupirtine evoked potential electroencephalogram pain measurement central nervous system analgesimetry dose-response relationship
Introduction be the natural, specific stimuli for exciting the nociceptive system (Regan, 1989; Steen et al., 1990). The elicited responses (late nearfield evoked potentials; for review see Picton & Hillyard, 1988) are significantly correlated with subjective pain ratings (Kobal & Hummel, 1989). Huttunen et al. (1986) succeeded in localizing one of the generators of these late-nearfield evoked potentials in the somatosensory area SII. It can be assumed that nociceptive afferents are primarily projected here (Chaudler et al., 1985). In the past this model had been successfully employed in quantifying the action of several centrally acting analgesic drugs (Kobal et al., 1989 [fentanyl]; Kobal et al., 1990 [pentazocine]). Since previous studies had demonstrated (Kobal & Hummel, 1989b; Nickel & Zerrahn, 1987) that flupirtine causes a general increase in the power density of most frequency bands of the spontaneous electroencephalogram, it seemed expedient to assess simultaneously parameters of the subjects' vigilance and arousal. Possible changes in mood were also appraised in order to differentiate between specifically analgesic and nonspecific drug effects.
Flupirtine, a novel analgesic drug, has, in all probability, its site of action in the central nervous system (Bleyer et al., 1988; Carlsson & Jurna, 1987; Vaupel et al., 1989). However, the pharmacological effects are neither mediated by opioid nor by serotoninergic receptors (Nickel et al., 1985). New findings indicate that for the drug to be effective, the descending noradrenergic pain control system has to be intact (Szelenyi & Nickel, 1987; Szelenyi et al., 1989). However, the actual site of action of flupirtine is still unknown. The analgesic effects of flupirtine have already been demonstrated in several studies using clinical (Herrmann et al., 1987) and experimental pain models (Bromm et al., 1987; Kobal & Hummel, 1988b). In most of the studies only single doses of flupirtine have been investigated. Hence, the aim of the present study was to determine whether the effects of flupirtine on painrelated evoked potentials and on subjective pain ratings are dose-dependent. For this purpose an experimental pain measuring technique was used, by the aid of which short painful chemical (CO2) stimuli were applied to the nasal mucosa. These chemical stimuli are considered by some authors to
Correspondence: Dr T. Hummel, Department of Pharmacology and Toxicology, University of Erlangen-Nurnberg, Universitatsstrasse 22, W-8520 Erlangen, Germany
69
70
T. Hummel et al.
Methods
Subjects Twenty young healthy volunteers (10 male and 10 female subjects, between 22 and 35 years of age; mean age 24.9 years) participated in the experiments. Right-handedness had previously been ascertained (Edinburgh Inventory, Oldfield, 1971). The subjects were informed about the aim of the study and all possible side effects of the drug on trial. The Ethics Committee of the University of Erlangen-Nurnberg approved the study, which was performed in accordance with the Declaration of Helsinki/ Tokyo/Venice. The subjects' health was ascertained before and after the experiments by general clinical examination and by routine clinical biochemical investigations (25 different
compounds). Study design A double-blind, randomized, controlled, five-fold crossover study design was chosen. In an additional training session prior to the actual experiments subjects became acquainted with the experimental conditions and procedures. In each experiment the medication, either placebo, or 50, 100, 200 and 300 mg flupirtine, was administered orally in three identical white capsules. The subjects were requested to abstain from eating solid food for at least 8 h before the experiments started. One experiment consisted of two testing sessions. The first took place before, and the second 2 h after administration of the medication. Since a previous study (Kobal & Hummel, 1988a) had established the fact that the maximum analgesic effect of flupirtine, as well as its
maximum plasma concentration (Hlavica & Niebch, 1985) were observed 2 h after administration, the second measurements were obtained at exactly this time. Each testing session lasted for approximately 35 min. During this time, 45 painful carbon dioxide stimuli were delivered to the nasal mucosa via the left nostril. During the entire measuring sequence subjects were comfortably seated in an accoustically shielded, airconditioned chamber. White noise, of approximately 50 dB SPL, was used to mask switching clicks of the stimulator.
Painful carbon dioxide stimulation of the nasal mucosa For stimulation, painful carbon dioxide stimuli were delivered to the nasal mucosa. The presentation of the stimuli did not simultaneously activate mechano- or thermosensors in the nasal mucosa, because a specially devised stimulation technique was employed (for further details see Kobal, 1985; Kobal & Hummel, 1988a, 1989; Kobal et al., 1990). Stimulus duration was 200 ms, interstimulus intervals randomly varied between 40 and 50 s. Three concentrations of carbon dioxide (38, 45, and 52% v/v C02) were delivered in a pseudo-randomized sequence. Determination ofplasma concentrations Two hours after medication, i.e. around tmax of flupirtine maleate in man (Hlavica & Niebch, 1985), 10 ml blood
was drawn from a forearm vein and centrifuged (3500 rev min-'; 10 min). Plasma was removed and stored in a freezer until analyzed. The analysis of the flupirtine maleate plasma levels was carried out by ASTA Pharma AG, Frankfurt, FRG (Niebch et al., in preparation). The assay was an internal standard h.p.l.c. procedure with ether extraction/HCI back extraction and fluorescence detection. Its lower limit of quantitation was 25 ng ml-'. The accuracy amounted to ± 3-10%. The day-to-day precision was characterized by a coefficient of variation of ± 6%.
Estimates ofpainful intensities
After presentation of a painful stimulus, the subjects estimated the perceived intensity in relation to a standard stimulus which was always applied at the beginning of the first session (45% v/v CO2). They estimated the painful intensity by employing a visual analogue scale displayed on a computer monitor. The pain-related chemo-somatosensory evoked potential (CSSEP) The EEG was recorded from seven positions of the 10/20 system referenced to linked earlobes (Al + A2). Possible blink artifacts were monitored from an additional site (Fp2/Al + A2). The band pass of the system was between 0.2 and 70 Hz, and the sampling frequency of the stimulus linked EEG-segments of 2048 ms was 250 Hz at all recording sites. Recording started 540 ms before stimulus onset. The mean value of the pre-stimulus period served as baseline for amplitude measurements. After the analog-to-digital conversion, the EEG-segments were stored on disk cartridges of a computer (PDP11/ 23, DEC). Subsequently, these records were evaluated off-line using the averaging technique (for a review see Picton & Hillyard, 1988). All single responses contaminated by eye blinks or eye movements were discarded from the average, and averaged responses with a blink artifact greater than 40 p,V in the Fp2-lead (Fp2/A1 + A2) were excluded from further analyses. Averaging yielded pain-related late nearfield evoked potentials (Kobal et al., 1990). Their latencies were measured relative to stimulus onset. Three base-to-peak amplitudes (P1, Ni, P2), and two peak-to-peak amplitudes (P1/Ni, N1/P2) were also evaluated. The spontaneous EEG
Pre-stimulus segments of 4096 ms were recorded with a sampling frequency of 125 Hz using an additional computer system (CED 1401, Cambridge Electronic Devices). In this way the background activity just before onset of painful stimulation was determined. The timewindow of 4 s was chosen because it best allows to determine the background activity at the time of stimulation before changes in arousal due to painful stimulation have occurred. A time window of less than 4 s would not have been long enough to permit the recording of reliable power spectra (see Ba9ar 1984; Scharein et al., 1984). After examining the segments for eye blinks and motor artifacts, they were submitted to frequency analysis (Fast Fourier Transformation, FFT). The resulting power
Dose-related analgesic effects of flupirtine spectra were averaged and subsequently divided into seven frequency bands (delta 1-3.5 Hz, theta 3.5-8 Hz, alpha, 8-10.5 Hz, alpha2 10.5-13 Hz, beta1 13-18 Hz, beta2 18-21 Hz, beta3 21-30 Hz). The integrated power of these bands was used for further statistical evaluation (Kobal & Hummel, 1988b).
Tracking performance In order to be able to detect changes in the state of vigilance (and/or motoric coordination), subjects were requested to perform a simple task on a video screen: they had to keep a small square, which could be controlled by a joystick, inside a larger one, which unpredictably moved around. This 'Tracking Performance' was checked by counting how often and by measuring for how long the subjects had lost track of the independently moving square (Kobal et al., 1990). The task also helped in stabilizing the subjects' vigilance.
71
effects of flupirtine with those of placebo, differences between data obtained after and before administration were calculated and then submitted to t-tests for comparison. (c) Data were submitted to analysis of variance (MANOVA, repeated measurement design, with trial medication as 'within-subject-factor'). Trend-analyses were only employed after MANOVA had yielded differences (P < 0.1), in order to investigate dose dependent linear or non-linear changes. A significant dose-response relationship was defined when P < 0.05 was obtained in the trend-analysis. Data obtained after administration of 50 mg flupirtine were excluded from trend analyses because of the requirement of equidistant intervals between the dependent variables (0 (placebo), 100,200, 300 mg flupirtine). SPSS PC+ programs were employed for statistical evaluation of the data.
Cardiovascular parameters
Results
Heart rate and systolic and diastolic blood pressure (Riva-Rocchi) were recorded in the sitting subjects before and after administration of the medication, prior to the second part of the experiment.
Routine clinical biochemical investigations yielded no significant changes throughout the experiments. There was also no change in the general state of health of the subjects.
Adverse reactions
Plasma concentrations offlupirtine
Prior to the second part of the experiment, subjects were asked to report all physical or psychological effects of the medication that they might experience. Their reports were recorded verbatim. The subjective descriptions were categorized under headings such as 'tiredness' and 'drowsiness' (according to the WHO Adverse Reaction Terminology 1984) at the end of the experiments.
Due to technical problems, only 68 out of 80 blood samples were analyzed. Mean values of plasma concentrations were 447 ng ml-' (s.d. 224.4, n = 16) for 50 mg flupirtine, 872.5 ng ml-1 (s.d. 174.3, n = 19) for 100 mg, 1699.7 ng ml-' (s.d. 706.6, n = 16) for 200 mg, and 2216.5 ng ml-' (s.d. 465.0, n = 17) for 300 mg. A significant linear dose-dependent increment in plasma concentrations was observed (trend analysis: P < 0.001).
Statistical analyses Data were submitted to following statistical analyses: (a) t-tests were employed in order to establish differences between data obtained before and after administration of the trial medication. (b) In order to compare the
Estimates ofpainful intensities
Analyses of the data (Figure 1) obtained before and after administration of the trial drug revealed a significant
0
38% v/v C02 -20' 0
50 100
200
300
wU o
0
D wU
v/v C02 -^nI 45% 0 50 100
200
300
-20
52% v/v CO2 0
50 100
*
200
300
Flupirtine dose (mg) Figure 1 Estimates of painful intensities. Depicted are the differences between data obtained before and after medication. After stimulation with the highest concentrations of CO2, intensity estimates significantly decreased linearly as a function of the administered doses of flupirtine (trend analysis). * Linear trend (P < 0.05).
72
T. Hummel et al. CS6EP: grand mean (Cz) 0
-.
W
%. ....
.Ico.
(ms)
0UZ411U
(ms)
1tEZ4IJ
(ms)
1024-1W-: 1024110
(ms)
(mM)
Figure 2 Averaged CSSEPs (Cz) from all subjects and for all stimulus intensities. Narrow lines represent CSSEPs before, and bold lines CSSEPs after medication. The starting points of the averaged responses are synchronous to stimulus onsets. These grand means were only used for depiction purposes in this figure. Mean values and s.d.s of the data can be found in Table 1. before, - after.
decrease in the intensity estimates of the weakest (38% v/v C02) and the strongest (52% v/v C02) carbon dioxide stimuli (P < 0.05) after administration of the highest dose of flupirtine (300 mg). Comparisons between the observed differences (data obtained after drug administration minus data obtained before drug administration) and placebo revealed that intensity estimates decreased after administration of 50 mg and 300 mg flupirtine (45% v/v C02, P < 0.01) and of 300 mg flupirtine (38% v/v CO2, P < 0.05). Trend-analyses revealed a significant dose-response relationship (P < 0.05) in the intensity estimates of the strongst C02-stimuli (Figure 1). Pain-related chemo-somatosensory evoked potentials (CSSEP)
Figure 2 shows CSSEPs averaged across subjects (grand mean) recorded at Cz. Mean values and standard deviations of the CSSEPs to the strongest C02-stimulus are listed in Table 1. The grand means of the CSSEPs were only used to demonstrate overall effects in the evoked potentials. Statistics were calculated on the raw data. Data from four subjects had to be excluded from further evaluation on account of too many blink artifacts caused by the painful stimulation. Comparing data obtained before and after administration of 200 and 300 mg flupirtine significant decreases in amplitudes and increases in latencies (P < 0.05) became apparent. These changes were found at all recording sites and for all stimulus intensities. After administration of 50 mg and 100 mg flupirtine latencies of P1 and Ni increased. There were no differences after administration of placebo. When compared with placebo, for both of the stronger carbon dioxide concentrations a significant decrease (P < 0.05) in the amplitudes Ni (base-to-peak) and Ni/ P2 (peak-to-peak) was found (for Ni at C3, 50 and
200 mg; at Cz, 50, 100 and 200 mg; at C4, 200 mg; at Pz, 100 and 200 mg; for N1/P2 at F3, 50, 100 and 300 mg; at Fz, 50 and 200 mg; at F4, 50 mg; at C3, 200 mg; at Cz 50 mg; at C4, 50 and 200 mg; at Pz, 50 mg). This was most conspicuous after administration of 50 mg and 200 mg flupirtine (Figure 3). Data obtained after presentation of weak carbon dioxide stimuli showed no systematic changes (t-tests). Amplitudes of P1 (45% v/v C02, F3) and P2 (38% v/v C02, Fz) decreased linearly as a function of the administered dose of flupirtine (P < 0.05). Also, latencies of P1 significantly increased (45% v/v CO2, Pz). However, trend analyses revealed significant but non-linear changes in the base-to-peak amplitudes P1, Ni and P2 (P < 0.05). Often, these changes were found at parietocentral recording sites after presentation of the strongest C02-stimuli (38% v/v CO2 for P1 at Pz; 45% v/v CO2 for P1 at Pz; 52%/ v/v C02 for Ni at Cz, C4, Pz and for P2 at C3, Cz, Pz).
Frequency analysis of the spontaneous EEG A comparison of the data obtained before and after administration of flupirtine (t-tests) revealed that the power density in the delta-range, the alpha-range, and the entire beta-range increased after medication (P < 0.05), although little or no changes were found in the theta-range. Placebo, on the other hand, occasioned an increase in the power density in the range of 3.5 and 21 Hz (P < 0.05), and this mainly in the alpha1-range (P < 0.01). This finding was interpreted as a decrease in the subjects' arousal during the second part of the experimental session. The power density in the delta- and beta3-range remained the same. A comparison of changes observed after administration of flupirtine and after placebo (t-tests) yielded significant differences in both alpha- and in all three beta-ranges (P < 0.05). Although changes were observed at all
Dose-related analgesic effects of flupirtine Table 1 Parameters of chemosomatosensory evoked potentials at recording site Cz and pain ratings after stimulation with 52% v/v CO2 (amplitudes in ,uV, latencies in ms; EU = estimation units; s.d. n = 16)
Base-to-peak amplitudes Nl P1
Flupirtine' dose (mg) 0
before 2 h after
50 before 2 h after 100 before 2 h after
200 before 2 h after
300 before 2 h after
Peak latencies Ni P1
Peak-to-peak amplitudes P2 PIINI N1IP2
Intensity estimates EU P2
Mean s.d. Mean s.d.
-3.72 2.65 -4.33 3.55
14.09 7.58 9.65 6.01
-17.80 8.24 -19.86 7.03
17.80 7.01 13.98 5.93
31.89 8.92 29.51 6.54
242.00 34.29 245.75 42.21
326.25 31.93 326.50 33.68
526.75 66.35 529.00 54.82
105.82 33.17
Mean s.d. Mean s.d.
-4.74 3.88 -3.45 4.64
10.43 7.16 9.88 4.95
-18.29 8.33 -18.12 8.00
15.17 6.41 13.32 5.24
28.72 8.39 28.00 8.52
231.25 41.86 238.00 40.29
325.00 39.53 312.75 38.21
520.00 56.93 537.75 75.24
109.38 29.64 102.79 25.84
Mean s.d. Mean s.d.
-3.17 3.06 -3.83 3.41
12.56 6.81 11.71 6.18
-20.67 8.03 -17.00 6.93
15.73 7.55 15.53 5.43
33.23 8.73 28.70 7.26
240.50 48.77 231.25 25.77
326.50 37.55 315.75 36.65
518.00 47.55 538.50 70.17
113.78 26.17 115.13 29.32
Mean s.d. Mean s.d.
-3.20 2.39 -5.34 3.91
13.46 7.21 8.37 6.26
-18.44 5.68 -19.04 6.63
16.66 7.99 13.70 6.72
31.91 9.84 27.41 9.34
238.00 41.77 246.75 39.78
318.50 39.22 326.00 37.98
522.75 47.01 549.50 66.84
112.31 32.96 104.00 32.37
Mean s.d. Mean s.d.
-3.37 3.25 -4.19 2.46
11.49 4.49 8.73 4.06
-17.44 5.72 -17.16 4.90
14.86 4.46 12.93 3.77
28.93 7.03 25.90 6.93
236.25 35.67 240.00 28.58
326.25 30.84 340.50 41.39
523.00 44.36 548.25 56.41
111,24 29.28 96.73 21.19
I
0
-7 0
105.27 30.02
.
F3
Recording sites'
-+T
F4
Fz
iT
9
-7L C3
C4
Cz
I0 i Pz
Figure 3 Comparison of the action of placebo (0) and 200 mg flupirtine (O) on base-to-peak amplitude Ni. Depicted are the averaged differences between data obtained before and after medication. Significant differences were found between responses obtained after administration of placebo and 200 mg flupirtine at parieto-central recording sites. * P < 0.05.
73
74
T. Hummel et al. 70
-delta +
0
0
70
:alpha 2 *
-alpha 1 *
-theta
C D
0
:0C
0 0
0 70
beta 1 *
beta 3 *
beta 2 *
0~~~~
0
0
0
0
0
0
0
50
100
200
300
0
0
50 100
0
200
0
300
50 100
200
300
Flupirtine dose (mg) Figure 4 Frequency analysis of the spontaneous EEG at Pz. Depicted are mean differences between data obtained before and after medication. Dose-dependant significant linear increases in the power spectra (mainly in the alpha- and beta-bands) were obtained following flupirtine administration. t P < 0.05, * P < 0.001.
recording sites, the drug-induced increase in power density was most conspicuous at sites along the mid-line, i.e., Fz, Cz, and Pz (P < 0.05). Increases in the deltarange were found at the recording positions Cz, C4, and Pz (P < 0.05). In the theta-band, an increase in power density was only observed after administration of 50 mg flupirtine, when compared with placebo (Cz, C4). A significant dose-dependent linear relationship (trend analyses, P < 0.01) between these changes and doses of the trial drug was found in both alpha-ranges and all three beta-ranges at all recording sites. Flupirtine did not significantly affect the theta-range (Figure 4). In the delta-range, rising doses of flupirtine caused a linear increase in power density only at the parietal site (P < 0.05).
Tracking performance Comparing data obtained before and after medication, it was observed that the tracking performance improved after placebo (P < 0.05), and after administration of the lowest dose of flupirtine (50 mg). This improvement is a generally observed phenomenon in subjects who are tested in subsequent sessions (Kobal & Hummel, 1988b) and is probably due to the subjects becoming more skilled in the course of the experiments. In contrast, performance did not improve when higher doses of flupirtine were administered (Figure 5). However, there was no dose-dependent change in tracking performance.
92
2-S4I 0
50
100
200
300
Flupirtine (mg) Figure 5 Results of the tracking performances. Depicted are values obtained before (0) and 2 h after (@) administration of the medication. After administration of placebo and 50 mg flupirtine a significant increase in the tracking performances became evident. This improvement is a generally observed phenomenon in subjects who are tested in subsequent sessions. mean
*P
ADONIS 030652519100141T
Dose-related analgesic effects of flupirtine T. HUMMEL, T. FRIEDMANN, E. PAULI, G. NIEBCH,1 H. 0. BORBE' & G. KOBAL Department of Pharmacology and Toxicology, University of Erlangen-Nurnberg, Universitatsstrasse 22, W-8520 Erlangen and 'Department of Biochemistry, ASTA Pharma AG, POB 100105, W-6000 Frankfurt/Main, Germany
1 Flupirtine is a novel and, in all probability, centrally acting, analgesic. The present investigation was conducted in order to investigate dose-related effects of perorally administered flupirtine in man, with special regard to specifically analgesic actions, employing a model based on pain-related chemosomatosensory evoked potentials and subjective intensity estimates of painful stimuli. 2 Plasma concentrations of flupirtine measured 2 h after dosing linearly increased as a function of the administered dose. 3 It was possible to reproduce our own previously obtained results, which established the analgesic action of 200 mg flupirtine administered perorally. 4 Intensity estimates linearly decreased as a function of the administered dose, whereas chemosomatosensory evoked potential amplitudes non-linearly changed in relation to the administered dose. 5 In the spontaneous EEG, a dose-dependent increment in the power-spectra was observed, and this mainly in the alpha- and beta-range.
Keywords flupirtine evoked potential electroencephalogram pain measurement central nervous system analgesimetry dose-response relationship
Introduction be the natural, specific stimuli for exciting the nociceptive system (Regan, 1989; Steen et al., 1990). The elicited responses (late nearfield evoked potentials; for review see Picton & Hillyard, 1988) are significantly correlated with subjective pain ratings (Kobal & Hummel, 1989). Huttunen et al. (1986) succeeded in localizing one of the generators of these late-nearfield evoked potentials in the somatosensory area SII. It can be assumed that nociceptive afferents are primarily projected here (Chaudler et al., 1985). In the past this model had been successfully employed in quantifying the action of several centrally acting analgesic drugs (Kobal et al., 1989 [fentanyl]; Kobal et al., 1990 [pentazocine]). Since previous studies had demonstrated (Kobal & Hummel, 1989b; Nickel & Zerrahn, 1987) that flupirtine causes a general increase in the power density of most frequency bands of the spontaneous electroencephalogram, it seemed expedient to assess simultaneously parameters of the subjects' vigilance and arousal. Possible changes in mood were also appraised in order to differentiate between specifically analgesic and nonspecific drug effects.
Flupirtine, a novel analgesic drug, has, in all probability, its site of action in the central nervous system (Bleyer et al., 1988; Carlsson & Jurna, 1987; Vaupel et al., 1989). However, the pharmacological effects are neither mediated by opioid nor by serotoninergic receptors (Nickel et al., 1985). New findings indicate that for the drug to be effective, the descending noradrenergic pain control system has to be intact (Szelenyi & Nickel, 1987; Szelenyi et al., 1989). However, the actual site of action of flupirtine is still unknown. The analgesic effects of flupirtine have already been demonstrated in several studies using clinical (Herrmann et al., 1987) and experimental pain models (Bromm et al., 1987; Kobal & Hummel, 1988b). In most of the studies only single doses of flupirtine have been investigated. Hence, the aim of the present study was to determine whether the effects of flupirtine on painrelated evoked potentials and on subjective pain ratings are dose-dependent. For this purpose an experimental pain measuring technique was used, by the aid of which short painful chemical (CO2) stimuli were applied to the nasal mucosa. These chemical stimuli are considered by some authors to
Correspondence: Dr T. Hummel, Department of Pharmacology and Toxicology, University of Erlangen-Nurnberg, Universitatsstrasse 22, W-8520 Erlangen, Germany
69
70
T. Hummel et al.
Methods
Subjects Twenty young healthy volunteers (10 male and 10 female subjects, between 22 and 35 years of age; mean age 24.9 years) participated in the experiments. Right-handedness had previously been ascertained (Edinburgh Inventory, Oldfield, 1971). The subjects were informed about the aim of the study and all possible side effects of the drug on trial. The Ethics Committee of the University of Erlangen-Nurnberg approved the study, which was performed in accordance with the Declaration of Helsinki/ Tokyo/Venice. The subjects' health was ascertained before and after the experiments by general clinical examination and by routine clinical biochemical investigations (25 different
compounds). Study design A double-blind, randomized, controlled, five-fold crossover study design was chosen. In an additional training session prior to the actual experiments subjects became acquainted with the experimental conditions and procedures. In each experiment the medication, either placebo, or 50, 100, 200 and 300 mg flupirtine, was administered orally in three identical white capsules. The subjects were requested to abstain from eating solid food for at least 8 h before the experiments started. One experiment consisted of two testing sessions. The first took place before, and the second 2 h after administration of the medication. Since a previous study (Kobal & Hummel, 1988a) had established the fact that the maximum analgesic effect of flupirtine, as well as its
maximum plasma concentration (Hlavica & Niebch, 1985) were observed 2 h after administration, the second measurements were obtained at exactly this time. Each testing session lasted for approximately 35 min. During this time, 45 painful carbon dioxide stimuli were delivered to the nasal mucosa via the left nostril. During the entire measuring sequence subjects were comfortably seated in an accoustically shielded, airconditioned chamber. White noise, of approximately 50 dB SPL, was used to mask switching clicks of the stimulator.
Painful carbon dioxide stimulation of the nasal mucosa For stimulation, painful carbon dioxide stimuli were delivered to the nasal mucosa. The presentation of the stimuli did not simultaneously activate mechano- or thermosensors in the nasal mucosa, because a specially devised stimulation technique was employed (for further details see Kobal, 1985; Kobal & Hummel, 1988a, 1989; Kobal et al., 1990). Stimulus duration was 200 ms, interstimulus intervals randomly varied between 40 and 50 s. Three concentrations of carbon dioxide (38, 45, and 52% v/v C02) were delivered in a pseudo-randomized sequence. Determination ofplasma concentrations Two hours after medication, i.e. around tmax of flupirtine maleate in man (Hlavica & Niebch, 1985), 10 ml blood
was drawn from a forearm vein and centrifuged (3500 rev min-'; 10 min). Plasma was removed and stored in a freezer until analyzed. The analysis of the flupirtine maleate plasma levels was carried out by ASTA Pharma AG, Frankfurt, FRG (Niebch et al., in preparation). The assay was an internal standard h.p.l.c. procedure with ether extraction/HCI back extraction and fluorescence detection. Its lower limit of quantitation was 25 ng ml-'. The accuracy amounted to ± 3-10%. The day-to-day precision was characterized by a coefficient of variation of ± 6%.
Estimates ofpainful intensities
After presentation of a painful stimulus, the subjects estimated the perceived intensity in relation to a standard stimulus which was always applied at the beginning of the first session (45% v/v CO2). They estimated the painful intensity by employing a visual analogue scale displayed on a computer monitor. The pain-related chemo-somatosensory evoked potential (CSSEP) The EEG was recorded from seven positions of the 10/20 system referenced to linked earlobes (Al + A2). Possible blink artifacts were monitored from an additional site (Fp2/Al + A2). The band pass of the system was between 0.2 and 70 Hz, and the sampling frequency of the stimulus linked EEG-segments of 2048 ms was 250 Hz at all recording sites. Recording started 540 ms before stimulus onset. The mean value of the pre-stimulus period served as baseline for amplitude measurements. After the analog-to-digital conversion, the EEG-segments were stored on disk cartridges of a computer (PDP11/ 23, DEC). Subsequently, these records were evaluated off-line using the averaging technique (for a review see Picton & Hillyard, 1988). All single responses contaminated by eye blinks or eye movements were discarded from the average, and averaged responses with a blink artifact greater than 40 p,V in the Fp2-lead (Fp2/A1 + A2) were excluded from further analyses. Averaging yielded pain-related late nearfield evoked potentials (Kobal et al., 1990). Their latencies were measured relative to stimulus onset. Three base-to-peak amplitudes (P1, Ni, P2), and two peak-to-peak amplitudes (P1/Ni, N1/P2) were also evaluated. The spontaneous EEG
Pre-stimulus segments of 4096 ms were recorded with a sampling frequency of 125 Hz using an additional computer system (CED 1401, Cambridge Electronic Devices). In this way the background activity just before onset of painful stimulation was determined. The timewindow of 4 s was chosen because it best allows to determine the background activity at the time of stimulation before changes in arousal due to painful stimulation have occurred. A time window of less than 4 s would not have been long enough to permit the recording of reliable power spectra (see Ba9ar 1984; Scharein et al., 1984). After examining the segments for eye blinks and motor artifacts, they were submitted to frequency analysis (Fast Fourier Transformation, FFT). The resulting power
Dose-related analgesic effects of flupirtine spectra were averaged and subsequently divided into seven frequency bands (delta 1-3.5 Hz, theta 3.5-8 Hz, alpha, 8-10.5 Hz, alpha2 10.5-13 Hz, beta1 13-18 Hz, beta2 18-21 Hz, beta3 21-30 Hz). The integrated power of these bands was used for further statistical evaluation (Kobal & Hummel, 1988b).
Tracking performance In order to be able to detect changes in the state of vigilance (and/or motoric coordination), subjects were requested to perform a simple task on a video screen: they had to keep a small square, which could be controlled by a joystick, inside a larger one, which unpredictably moved around. This 'Tracking Performance' was checked by counting how often and by measuring for how long the subjects had lost track of the independently moving square (Kobal et al., 1990). The task also helped in stabilizing the subjects' vigilance.
71
effects of flupirtine with those of placebo, differences between data obtained after and before administration were calculated and then submitted to t-tests for comparison. (c) Data were submitted to analysis of variance (MANOVA, repeated measurement design, with trial medication as 'within-subject-factor'). Trend-analyses were only employed after MANOVA had yielded differences (P < 0.1), in order to investigate dose dependent linear or non-linear changes. A significant dose-response relationship was defined when P < 0.05 was obtained in the trend-analysis. Data obtained after administration of 50 mg flupirtine were excluded from trend analyses because of the requirement of equidistant intervals between the dependent variables (0 (placebo), 100,200, 300 mg flupirtine). SPSS PC+ programs were employed for statistical evaluation of the data.
Cardiovascular parameters
Results
Heart rate and systolic and diastolic blood pressure (Riva-Rocchi) were recorded in the sitting subjects before and after administration of the medication, prior to the second part of the experiment.
Routine clinical biochemical investigations yielded no significant changes throughout the experiments. There was also no change in the general state of health of the subjects.
Adverse reactions
Plasma concentrations offlupirtine
Prior to the second part of the experiment, subjects were asked to report all physical or psychological effects of the medication that they might experience. Their reports were recorded verbatim. The subjective descriptions were categorized under headings such as 'tiredness' and 'drowsiness' (according to the WHO Adverse Reaction Terminology 1984) at the end of the experiments.
Due to technical problems, only 68 out of 80 blood samples were analyzed. Mean values of plasma concentrations were 447 ng ml-' (s.d. 224.4, n = 16) for 50 mg flupirtine, 872.5 ng ml-1 (s.d. 174.3, n = 19) for 100 mg, 1699.7 ng ml-' (s.d. 706.6, n = 16) for 200 mg, and 2216.5 ng ml-' (s.d. 465.0, n = 17) for 300 mg. A significant linear dose-dependent increment in plasma concentrations was observed (trend analysis: P < 0.001).
Statistical analyses Data were submitted to following statistical analyses: (a) t-tests were employed in order to establish differences between data obtained before and after administration of the trial medication. (b) In order to compare the
Estimates ofpainful intensities
Analyses of the data (Figure 1) obtained before and after administration of the trial drug revealed a significant
0
38% v/v C02 -20' 0
50 100
200
300
wU o
0
D wU
v/v C02 -^nI 45% 0 50 100
200
300
-20
52% v/v CO2 0
50 100
*
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300
Flupirtine dose (mg) Figure 1 Estimates of painful intensities. Depicted are the differences between data obtained before and after medication. After stimulation with the highest concentrations of CO2, intensity estimates significantly decreased linearly as a function of the administered doses of flupirtine (trend analysis). * Linear trend (P < 0.05).
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T. Hummel et al. CS6EP: grand mean (Cz) 0
-.
W
%. ....
.Ico.
(ms)
0UZ411U
(ms)
1tEZ4IJ
(ms)
1024-1W-: 1024110
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Figure 2 Averaged CSSEPs (Cz) from all subjects and for all stimulus intensities. Narrow lines represent CSSEPs before, and bold lines CSSEPs after medication. The starting points of the averaged responses are synchronous to stimulus onsets. These grand means were only used for depiction purposes in this figure. Mean values and s.d.s of the data can be found in Table 1. before, - after.
decrease in the intensity estimates of the weakest (38% v/v C02) and the strongest (52% v/v C02) carbon dioxide stimuli (P < 0.05) after administration of the highest dose of flupirtine (300 mg). Comparisons between the observed differences (data obtained after drug administration minus data obtained before drug administration) and placebo revealed that intensity estimates decreased after administration of 50 mg and 300 mg flupirtine (45% v/v C02, P < 0.01) and of 300 mg flupirtine (38% v/v CO2, P < 0.05). Trend-analyses revealed a significant dose-response relationship (P < 0.05) in the intensity estimates of the strongst C02-stimuli (Figure 1). Pain-related chemo-somatosensory evoked potentials (CSSEP)
Figure 2 shows CSSEPs averaged across subjects (grand mean) recorded at Cz. Mean values and standard deviations of the CSSEPs to the strongest C02-stimulus are listed in Table 1. The grand means of the CSSEPs were only used to demonstrate overall effects in the evoked potentials. Statistics were calculated on the raw data. Data from four subjects had to be excluded from further evaluation on account of too many blink artifacts caused by the painful stimulation. Comparing data obtained before and after administration of 200 and 300 mg flupirtine significant decreases in amplitudes and increases in latencies (P < 0.05) became apparent. These changes were found at all recording sites and for all stimulus intensities. After administration of 50 mg and 100 mg flupirtine latencies of P1 and Ni increased. There were no differences after administration of placebo. When compared with placebo, for both of the stronger carbon dioxide concentrations a significant decrease (P < 0.05) in the amplitudes Ni (base-to-peak) and Ni/ P2 (peak-to-peak) was found (for Ni at C3, 50 and
200 mg; at Cz, 50, 100 and 200 mg; at C4, 200 mg; at Pz, 100 and 200 mg; for N1/P2 at F3, 50, 100 and 300 mg; at Fz, 50 and 200 mg; at F4, 50 mg; at C3, 200 mg; at Cz 50 mg; at C4, 50 and 200 mg; at Pz, 50 mg). This was most conspicuous after administration of 50 mg and 200 mg flupirtine (Figure 3). Data obtained after presentation of weak carbon dioxide stimuli showed no systematic changes (t-tests). Amplitudes of P1 (45% v/v C02, F3) and P2 (38% v/v C02, Fz) decreased linearly as a function of the administered dose of flupirtine (P < 0.05). Also, latencies of P1 significantly increased (45% v/v CO2, Pz). However, trend analyses revealed significant but non-linear changes in the base-to-peak amplitudes P1, Ni and P2 (P < 0.05). Often, these changes were found at parietocentral recording sites after presentation of the strongest C02-stimuli (38% v/v CO2 for P1 at Pz; 45% v/v CO2 for P1 at Pz; 52%/ v/v C02 for Ni at Cz, C4, Pz and for P2 at C3, Cz, Pz).
Frequency analysis of the spontaneous EEG A comparison of the data obtained before and after administration of flupirtine (t-tests) revealed that the power density in the delta-range, the alpha-range, and the entire beta-range increased after medication (P < 0.05), although little or no changes were found in the theta-range. Placebo, on the other hand, occasioned an increase in the power density in the range of 3.5 and 21 Hz (P < 0.05), and this mainly in the alpha1-range (P < 0.01). This finding was interpreted as a decrease in the subjects' arousal during the second part of the experimental session. The power density in the delta- and beta3-range remained the same. A comparison of changes observed after administration of flupirtine and after placebo (t-tests) yielded significant differences in both alpha- and in all three beta-ranges (P < 0.05). Although changes were observed at all
Dose-related analgesic effects of flupirtine Table 1 Parameters of chemosomatosensory evoked potentials at recording site Cz and pain ratings after stimulation with 52% v/v CO2 (amplitudes in ,uV, latencies in ms; EU = estimation units; s.d. n = 16)
Base-to-peak amplitudes Nl P1
Flupirtine' dose (mg) 0
before 2 h after
50 before 2 h after 100 before 2 h after
200 before 2 h after
300 before 2 h after
Peak latencies Ni P1
Peak-to-peak amplitudes P2 PIINI N1IP2
Intensity estimates EU P2
Mean s.d. Mean s.d.
-3.72 2.65 -4.33 3.55
14.09 7.58 9.65 6.01
-17.80 8.24 -19.86 7.03
17.80 7.01 13.98 5.93
31.89 8.92 29.51 6.54
242.00 34.29 245.75 42.21
326.25 31.93 326.50 33.68
526.75 66.35 529.00 54.82
105.82 33.17
Mean s.d. Mean s.d.
-4.74 3.88 -3.45 4.64
10.43 7.16 9.88 4.95
-18.29 8.33 -18.12 8.00
15.17 6.41 13.32 5.24
28.72 8.39 28.00 8.52
231.25 41.86 238.00 40.29
325.00 39.53 312.75 38.21
520.00 56.93 537.75 75.24
109.38 29.64 102.79 25.84
Mean s.d. Mean s.d.
-3.17 3.06 -3.83 3.41
12.56 6.81 11.71 6.18
-20.67 8.03 -17.00 6.93
15.73 7.55 15.53 5.43
33.23 8.73 28.70 7.26
240.50 48.77 231.25 25.77
326.50 37.55 315.75 36.65
518.00 47.55 538.50 70.17
113.78 26.17 115.13 29.32
Mean s.d. Mean s.d.
-3.20 2.39 -5.34 3.91
13.46 7.21 8.37 6.26
-18.44 5.68 -19.04 6.63
16.66 7.99 13.70 6.72
31.91 9.84 27.41 9.34
238.00 41.77 246.75 39.78
318.50 39.22 326.00 37.98
522.75 47.01 549.50 66.84
112.31 32.96 104.00 32.37
Mean s.d. Mean s.d.
-3.37 3.25 -4.19 2.46
11.49 4.49 8.73 4.06
-17.44 5.72 -17.16 4.90
14.86 4.46 12.93 3.77
28.93 7.03 25.90 6.93
236.25 35.67 240.00 28.58
326.25 30.84 340.50 41.39
523.00 44.36 548.25 56.41
111,24 29.28 96.73 21.19
I
0
-7 0
105.27 30.02
.
F3
Recording sites'
-+T
F4
Fz
iT
9
-7L C3
C4
Cz
I0 i Pz
Figure 3 Comparison of the action of placebo (0) and 200 mg flupirtine (O) on base-to-peak amplitude Ni. Depicted are the averaged differences between data obtained before and after medication. Significant differences were found between responses obtained after administration of placebo and 200 mg flupirtine at parieto-central recording sites. * P < 0.05.
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T. Hummel et al. 70
-delta +
0
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:alpha 2 *
-alpha 1 *
-theta
C D
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:0C
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0 70
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beta 2 *
0~~~~
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Flupirtine dose (mg) Figure 4 Frequency analysis of the spontaneous EEG at Pz. Depicted are mean differences between data obtained before and after medication. Dose-dependant significant linear increases in the power spectra (mainly in the alpha- and beta-bands) were obtained following flupirtine administration. t P < 0.05, * P < 0.001.
recording sites, the drug-induced increase in power density was most conspicuous at sites along the mid-line, i.e., Fz, Cz, and Pz (P < 0.05). Increases in the deltarange were found at the recording positions Cz, C4, and Pz (P < 0.05). In the theta-band, an increase in power density was only observed after administration of 50 mg flupirtine, when compared with placebo (Cz, C4). A significant dose-dependent linear relationship (trend analyses, P < 0.01) between these changes and doses of the trial drug was found in both alpha-ranges and all three beta-ranges at all recording sites. Flupirtine did not significantly affect the theta-range (Figure 4). In the delta-range, rising doses of flupirtine caused a linear increase in power density only at the parietal site (P < 0.05).
Tracking performance Comparing data obtained before and after medication, it was observed that the tracking performance improved after placebo (P < 0.05), and after administration of the lowest dose of flupirtine (50 mg). This improvement is a generally observed phenomenon in subjects who are tested in subsequent sessions (Kobal & Hummel, 1988b) and is probably due to the subjects becoming more skilled in the course of the experiments. In contrast, performance did not improve when higher doses of flupirtine were administered (Figure 5). However, there was no dose-dependent change in tracking performance.
92
2-S4I 0
50
100
200
300
Flupirtine (mg) Figure 5 Results of the tracking performances. Depicted are values obtained before (0) and 2 h after (@) administration of the medication. After administration of placebo and 50 mg flupirtine a significant increase in the tracking performances became evident. This improvement is a generally observed phenomenon in subjects who are tested in subsequent sessions. mean
*P
