Agents and Actions, vol.29, 3/4 (1990)
0065-4299/90/040342-18$1.50+0.20/0 9 1990BirkhfiuserVerlag, Basel
Effects of pentazocine and acetylsalicylic acid on pain-rating, pain-related evoked potentials and vigilance in relationship to pharmakokinetic parameters G. Kobal, C. Hummel, B. Nuernberg and K. Brune Institut fiir Pharmakologieund Toxikologie,UniversitfitErlangen-Nfirnberg,Universitfitsstr.22, D-8520 Erlangen, BRD
Abstract
Achieving objective and quantitative measurement of experimental pain in human volunteers and establishing the impact of drugs remains a difficult task. This problem may be overcome by employing a method which allows the simultaneous measurement of pain ratings elicited by standardized stimulation of the nasal mucosa by carbon dioxide, together with pain-rdated chemo-somatosensory evoked potentials (CSSEP) and vigilance. We assessed the effect of pentazocine and acetylsalicylic acid on these parameters in 14 human volunteers and related the effects to the pharmacokinetic parameters of the drugs measured at the same time. Pentazocine was found to reduce the pain ratings as well as the amplitudes of the pain-related evoked potentials and to increase their latencies. Vigilance (measured by EEG power spectra and performance of a tracking task) was also significantly reduced. These effects were observed during the distribution phase and the first period of the terminal elimination phase of the drug. Acetylsalicylic acid had no significant effects on pain ratings, but reduced the amplitudes of the event-related potentials when compared to placebo controls. At the same time a slight, but significant, effect on vigilance (reduced performance of the tracking task) was observed. These effects could not be related to the presence of unmetabolized acetylsalicylic acid in the plasma. They appeared at later times when only salicylic acid was left. It is concluded that chemical stimuli of sufficient intensity produce pain which may be suppressed by opioid analgesics such as pentazocine. The effect of acetylsalicylic acid on this experimental pain did not reach significance for all measured parameters under the experimental conditions chosen. The changes in vigilance and in the amplitudes of pain-related chemo-somatosensory evoked potentials indicated as yet unknown CNS-effects of this non-steroidal anti-inflammatory drug.
Introduction
The value of new analgesics must ultimately be defined under clinical conditions. Animal experiments may serve for the selection of potentially effective new types of analgesics. However, the principal types of analgesics such as morphine, salicylates, pyrazole drugs and aniline derivatives were found by serendipity and not by systematic pharmacological research [1, 2]. It appears that the
wide gap between preliminary experimental data obtained in animals and human pain should be bridged by quantitative measurement of drug effects in human volunteers in order to predict the possible clinical suitability of any compounds. Many attempts for measuring pain and analgesic effects in volunteers using event-related potentials [3] have been reported (reviewed by Chudler and Dong [4] and Stowell [5]; [6-12]). Many of these studies suffer either from limited reproducibility
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Agents and Actions, vol. 29, 3/4 (1990)
due to changes in the tissue irritated by the noxious stimulus, or from the lack of adequate control measurements which take into account vigilance, adaptation, and learning effects. Finally, the lack of objective, quantifiable parameters of pain preclude correlations with the pharmacokinetic parameters of the drugs used. Here, we report on the use of a pain model [13, 14] which allows for the quantification of drug effects on pain rating and pain-related evoked potentials as well as vigilance in connection with the assessment of pharmacokinetic parameters. The aim of the present study was to find out, whether pain-related evoked potentials - being epiphenomena of nociceptive electrophysiological events in the brain - not only reflect the effects of centrally acting analgesics, as it was repeatedly reported [8, 11, 12, 15 22], but also the action of peripherally acting analgesics, such as acetylsalicylic acid (ASA). To demonstrate the sensitivity of the pain model chosen for this study (for alternative models see [23]), we compared the effects of the ASA with the effects of the centrally acting kappa-agonist/mu-antagonist pentazocine, a drug widely used in clinical applications. In the case of pentazocine we expected to find robust drug effects, but probably not to such an extent as we observed after administration of 0.5 mg fentanyl which totally eliminated the pain-related chemosensory evoked potentials [24].
Material and methods
In 14 volunteers (7 female, 7 male, between 23 and 38 years of age) the analgesic effects of acetylsalicylic acid and pentazocine were measured by means of cortical event-related potentials after painful stimulation, as well as by the subjective estimates of stimulus intensities. Changes in the subjects' vigilance were assessed by evaluating a "tracking task" which the subjects performed on a video screen and by computing power spectra of the background EEG activity.
Stimulus procedures For painful stimulation carbon dioxide (CO2) pulses were delivered to the left nostril of the subjects. In order to achieve a pure chemical stimulation, thus avoiding alterations of the mechanical or
thermal conditions at the respiratory nasal mucosa, CO 2 pulses of 200 msec duration were mixed in a constantly flowing air stream with controlled temperature (36.5 ~ and humidity (80% rel. humidity). The rise time of the stimulus' concentration pulses was below 20 msec, i.e., it was sufficiently steep to elicit late nearfield event-related potentials [14, 25, 26]. To avoid respiratory air flow in the nose during stimulation, subjects were trained to practise velopharyngeal closure, breathing through the mouth [25]. On each occasion the volunteer received a train of 80 painful stimuli with an interstimulus interval of 60 sec. Thus, one experimental session lasted 80 min. Two concentrations ("weak": 45% v/v and "strong": 52% v/v) of CO 2 were alternately presented. These two concentrations were chosen to get one type of stimulus which was close to the pain threshold and another one which was clearly above threshold even in case of drug administration.
Pain-related responses Pain-related evoked potentials were obtained by EEG recordings at the sites FP2, F3, Fz, F4, C3, Cz, C4 and Pz of the international 10/20 system referenced to A1 (bandpass 0.16-70 Hz; Siemens Mingograph). EEG-records were digitized with a sampling frequency of 250 Hz, and stored on magnetic media. Data were evaluated off-line with a PDP 1t/73 computer (Digital Equipment Corporation) by OFFLAB-programs [25]. 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 ~tV in the eye channel (Fp2/A1) were excluded from further analysis. The subjects estimated the intensity of the painful stimuli two seconds after stimulus presentation by employing a visual analogue scale displayed on a computer screen. This assessment of intensity estimates followed the technique of cross modality matching [27, 28] with prescribed modulus (first stimulus in the experiment of 52% v/v COz). In order to observe changes in the subjects' state of vigilance (and/or motoric coordination) they were requested to perform a simple task on a video screen: they had to keep a smaller square, which could be controlled by a joy-stick, inside a larger square, which unpredictably moved around. This
344 "tracking performance" was checked by counting how often and by measuring for how long they had lost track of the moving square. Additionally pre-stimulus periods of 4096 msec were sampled from the positions Fz, Cz and Pz. These data were submitted to a Fast Fourier Transformation. The averaged power spectra were divided into 7 frequency bands: delta (1-3.5 Hz), theta (3.5-7 Hz), alphaa (8-10 Hz), alpha 2 (1013 Hz), beta 1 (13-18 Hz), beta2 (18-21 Hz), and beta 3 (21-30 Hz). The areas under the curves in the range of the defined frequency bands were submitted to statistical analyses. Mean values were calculated for all parameters (amplitudes and latencies of event-related potentials, estimates of intensity, tracking performance, power spectra) over periods of 16 minutes. Thus, one experimental session was divided into five "Sections" (0, I, II, III, IV). Event-related potentials and intensity estimates were grouped in relation to the two concentrations of the painful stimuli. Since both strengths of stimuli were alternately presented every other minute, mean values were calculated over maximal 8 (in case of artifacts minimal 6) EEG-recordings, 8 intensity estimates, and 16 assessments of the tracking performance. The event-related potentials were quantified by measuring the latencies of the peaks P1 (240 msec), N I (340 msec), P2 (520 msec), and N2 (760 msec) and the peak-to-peak amplitudes P1/NI, N1/P2 and P2/N2 [26]. Approximately 150 msec of the latencies are due to transportation time through the mucus and epithelial layer at the respiratory nasal mucosa and the utilization time at the receptor sites [14].
Materials and methods ,for determination of blood plasma levels Acetylsalicylic lysinate (Aspisol| acetylsalicylic acid (ASA), salicylic acid (SA), pentazocine lactate (Fortral| and pentazocine were supplied by Bayer AG (Wuppertal, FRG) and Winthrop (NeuIsenburg, FRG) respectively. Levallorphan as the internal standard for a quantitation of pentazocine was supplied by Hoffmann-La Roche (Grenzach, FRG) in commercially available ampoules (Lorfan| Heparin was used as Liquemin | ampoules (2500 IU/5 ml); the destilled water was filtered (~3 0.45 gin). All other chemicals and reagents were of HPLC- or analytical grade.
Agents and Actions, vol. 29, 3/4 (1990) For quantification of acteylsalicylic acid (ASA) and salicylic acid (SA) an isocratic high performance liquid chromatographic ultraviolet-method was used, according to Amick and Mason [29], Nieder et al. [30], see also [31]. The detection limits for ASA and SA were found to be less than 1 gg/ml. In the concentration range of 0.5 to 200 gg/ml the chromatographic system showed linearity. Mean values were calculated of at least two prepared samples, of which each was measured twice. The mean recovery for ASA was 72.15% (r = 0.99993, y=0.74x-0.82; n = 18) and for SA 67.62% (r = 0.99980, y = 0.70x - 1.24; n = 18). The coefficients of variation for ASA were in the range between 1.5 and 2.5% and those for SA between 3.2 and 4.6%. A specific high-pressure liquid chromatographic method for determination of pentazocine and the salicylates was developed. In brief: The following chromatographic instruments were used: one Waters pump model M6000A (Waters Milford, MA, USA), a model Waters U-6-K injector, a Kontron SFM 23/B fluorimetric detector set at 278 nm for excitation and 324 nm for emission, a Shimadzu model CR3A integrator. Plasma specimens were obtained and separated on a 50 x 3.9 mm guard column filled with 30 pm ODS Nucleosil TM and a 250 x 3.9 mm column filled with 10 pm Nucleosil TM ODS. The mobile phase consisted of acetonitrile -0.05 M phosphatic acid (25:75 v/v) in an isocratic system. The flow rate was 2.0 ml/min at 21 ~ The retention times were 5 min for levallorphan and 7.5 min for pentazocine. The calculations of all plasma samples were obtained by using the internal method establishing the ratio with levallorphan as internal standard, as well as by employing the external standard method. The detection limit of this method was less than 2 ng/ml. In the concentration range of 2 to 1500 ng/ml, the chromatographic system showed linearity. The mean recovery for pentazocine was 98.3% (r = 0.999991, y = 0.98x + 0.35; n = 32). The coefficient of variation ranged between 2.7 and 4.9%. These data were obtained by calculating the mean of at least two analyses employing the internal standard method (levallorphan 50 ng/ml), as well as by calculations using the external standard method. The pharmacokinetic parameters for acetylsalicylic acid, salicylic acid and pentazocine were com-
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puted on a Wang APC computer using the program TOPFIT [32]. Several open two- and three compartment infusion models were used for the analysis of the plasma concentration-time data. All parameters describing the intraindividual bioavailability in both groups - morning and afternoon (see below) - were subjected to statistical analyses developed by Steinijans and Diletti [33]. The tests (t-test, ANOVA, Westlake, Wilcoxon and Pitman) were also included in the TOPFIT program package.
Test procedure The study corresponded to a single blind three way crossover design in which each experiment was run twice with a wash-out period of one week between experiments. An additional training session was included. The preparations tested were 1800 mg lysinemono-acetylsalicylate LAS (equivalent to 1000 mg acetylsalicylic acid), 50 mg pentazocine lactate (equivalent to 30 mg pentazocine), and placebo (10 ml Ringer's solution). Indwelling catheters were inserted into veins of both forearms. Except during injection 500 ml of physiological saline solution was continuously infused into the blood stream through the right catheter. Shortly before injection, the contents of two ampoules of Aspisol ~ were dissolved in 10 ml aqua pro injectionem. Switching a three-way valve, the solution was injected through the right catheter over a period of 2 rain. In the other two experimental conditions, either I ml of pentazocine lactate solution, or 10 ml of Ringer's solution were administered over a period of 3 rain. Since there was approximately 1 m tubing between injection point and the forearm of the subjects infusion times were markedly longer. Considering the flow rate of the infusion the actual injection time ranged between 5 and 10 minutes. The starting point of injection was 16 rain after on-set of pain stimulation (i.e. after "section 0"). Through the left catheter, blood samples (10 ml) were withdrawn prior to and 1, 2, 4, 8, 12, 15, 20, 30, 40, 60 min after injection. In the case of pentazocine, several additional blood samples were collected over a period of 480 rain, if possible. The first 2 ml of the samples were always rejected in order to remove residual heparin that was used to flush the catheter after sampling. Immediately after collection, plasma was harvested by centrifuga-
tion at 900 9 (0 ~ and subsequently, the resulting blood plasma was stored at - 6 0 ~ No more than 7 days elapsed before the samples were analyzed. The volunteers were instructed not to partake of solid food or any kind of beverages during a period of a least 6 hours before the experiments started. A screen prevented the subjects to notice manipulation of the infusion. In this way, their attention was not diverted and the drugs could be administered unobserved. White noise (70dB SPL) masked all sounds, including the noise of the switching device. An anaesthesiologist monitored blood pressure, pulse rate, and respiration during the entire experiment, as well as for a period of 6 h after i.v. administration. He also assessed the state of health of the subjects by routine examinations and took the anamneses prerequisite for the subjects' participation in the study.
Subjects The experiments were performed in accordance with the declaration of Helsinki/Tokyo/Venice and were approved by the Ethics committee of the University of Erlangen-Niirnberg. After having given their informed consent, 7 healthy female and 7 healthy male volunteers, between 23 and 38 years of age, participated in the experiments. Due to allergic reactions after the first experimental session with LAS medication, one subject was excluded from further participation. Three subjects were excluded because of unwanted effects of the drugs such as nausea and vomiting (after pentazocine administration) and one female subject withdrew from further participation because of pregnancy. As a result 6 female and 4 male subjects were in on the LAS experiments to the end and 4 female and 5 male subjects completed the pentazocine experiments. Each experiment was run twice, at 9 am and at 2 pro. Due to technical problems, 2 male and 2 female subjects participated in one LAS experiment only.
Statistical analysis Mean values of the data obtained from two experimental sessions were subjected to analyses of variance to determine the main effect: experimental "Sections" (0 = before; I - I V = after drug administration) separately for each electrode site and each medication.
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Since all experiments were run with the same volunteers, this factor was additionally included in the analysis (repeated measurements design). The performance of the "Box Tests" did not reveal variance-covariance-homogeneity [34], consequently we used the conservative test of F-fractions with FGN . . . . . t o t : 1 and F G D. . . . inator=N-I ( N = n u m ber of samples) described by Glaser [35]. Where significant results were obtained, multiple t-tests were used to further specify the differences in the measures. The results of these analyses used to test the effects of the administration of drugs and placebo will only be mentioned when necessary (e.g. in Fig. 2 and 3). In order to establish differences between the tested drugs and placebo, they were compared in pairs by employing analysis of variance for repeated measurements (pentazocine/placebo, LAS/placebo, and pentazocine/LAS) separately for the different recording positions. The factor "Section" was not evaluated this time, because the results were already known from the first analysis. This procedure was rendered necessary because of the disparity in the number of subjects in the two drug application groups due to dropouts. In this case, percentage values of the four measuring sections (I IV) after i.v, injection relative to measures obtained prior to medication (section 0) were used.
This also helped to eliminate the variance between different days of testing. In addition, correlations between the electrophysiological (amplitudes N1/P2 and latencies TP1, T N I of the 3 central sites of recording Cz, C3, and C4), psychophysical (intensity estimates, tracking performances) and pharmacokinetic parameters such as AUC (area under the plasma-concentration-time-course) and M R T (mean residence time) were established.
Results
Pain-related data
Pain-related cerebral potentials were recorded from all subjects after stimulation of the nasal mucosa by CO 2. Mean values and standard deviations were calculated for all groups of data (Table 1; in extracts). For the purpose of graphical depiction only, grand means of the event-related potentials were computed across all subjects. Latency differences between subjects were compensated by standardizing all event-related potentials to the mean latency between N I and P2. These data are shown in Fig. I where pain-related potentials from one single subject are also illustrated.
Single Subject
Grand Means
P n ozo ,n
Placebo
"
~
I pV'~ 0
1024 rnsec
0
1024 mser
Figure 1 Pain-related evoked potentials elicited by carbon dioxide stimulation of the nasal mucosa. Effects of pentazocine, Iysine-mono-acetysalicylate (LAS), and placebo. Example from one subject (left) and the grand mean over all subjects. Stimulus 52% v/v CO 2 .
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Table 1 Means and standard deviations of pain-related evoked potentials. Recording sites Fz and Cz (versus AI) (amplitudes: gV; latencies: msec) 0=before, l = 0 16 min after, l I = 17 32 min after, I I I - 3 3 - 4 8 rain after, I V = 4 9 - 6 4 rain after drug application.
Pen tazocine Tracking performance 0
I 87.6 9.2
II 76.9 21.1
72.1 24.5
III 69.3 23.7
IV 68.7 25.0
45% v/v CO 2 0
I
52% v/v CO 2 II
III
IV
0
I
II
III
IV
Intensity estimates 65.3 18.1
32.5 20.6
29.6 21.3
36.9 31.0
50.1 38.8
113.8 12.6
75.1 31.4
74.6 37.9
83.1 38.3
83.8 37.9
Amplitudes N I / P 2 Fz 19.2 4.9 Cz 27.1 6.1
14.6 6.5 20.1 6.9
12.4 5.4 17.8 7.5
12.8 4.2 16.2 8.0
11.9 5.2 16.6 8.4
22.6 5.7 34.5 6.5
17.3 7.7 25.0 9.8
15.2 9.5 22.7 10.4
17.4 7.7 24.2 10.0
16.8 9.0 23.8 9.6
327.9 25.5 329.0 24.2
342.8 33.7 340.3 37.7
374.1 44.0 373.0 52.1
349.2 40.0 345.4 27.5
306.6 39.2 306.3 29.6
342.8 45.7 317.0 34.1
343.7 70.2 340.1 53.8
336.1 51.8 335.0 47.7
371.9 58.9 366.1 52.7
II
lII
IV
II
lII
IV
Latencies TNI Fz 327.7 34.8 Cz 332.1 34.2
Lysine-rnono-acetylsalicylate Tracking performance 0
I 81.6 11.9
79.6 13.7
77.6 11.6
78.6 10.9
78.9 12.2 52% v/v CO 2
45% v/v CO 2 0
1
II
Ill
IV
0
I
Intensity estimates 68.1 8.5
50.3 14.7
55.8 17.3
60.0 19.2
62.6 19.9
109.4 23.3
98.9 21.2
102.8 26.6
107.7 29.1
103.6 28.3
Amplitudes N I / P 2 Fz 20.8 6.6 Cz 27.3 6.9
16.7 5.5 22.1 6.1
15.3 5.2 20.6 4.8
15.5 7.2 21.4 9.3
16.7 6.2 21.6 6.8
25.9 7.9 33.1 8.6
19.6 9.1 25.6 9.3
19.7 7.5 26.6 8.6
18.7 8.2 25.6 9.3
19.5 10.6 27.3 11.2
376.7 53.9 372.5 45.8
372.7 33.1 361.1 33.9
388.2 55.9 375.7 47.1
373.7 36.6 355.7 26.3
343.7 33.5 332.7 22.6
347.7 50.1 341.5 33.2
350.7 40.7 340.3 31.5
341.7 31.4 330.1 24.3
348.1 37.9 337.9 28.1
Latencies TN1 Fz 353.9 33.1 Cz 351.9 34.2
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Agents and Actions,vol. 29, 3/4 (1990)
Placebo Tracking performance
0 76.9 14.7
1 77.4 11.1
II 79.6 13.1
Ill 79.5 15.5
IV 82.4 10.7 52% v/v CO2
45% v/v CO2 0 Intensityestimates 75.7 17.4 Amplitudes N1/P2 Fz 22.2 7.6 Cz 30.5 9.4 Latencies TN 1 Fz 368.4 35.3 Cz 346.8 20.8
1
II
lII
IV
0
I
II
IlI
IV
60.5 25.6
63.3 27.3
69.4 28.3
67.2 28.6
116.9 10.8
117.7 16.8
116.5 22.2
115.3 22.0
113.8 26.7
20.3 10.4 22.8 12.3
20.6 7.7 25.3 8.6
18.5 8.5 25.1 8.9
16.0 5.3 22.2 5.3
24.2 9.2 36.1 10.3
23.9 9.0 33.2 9.8
22.2 7.1 31.0 9.6
21.6 6.9 29.4 6.9
20.3 7.3 27.1 7.1
380.8 39.2 329.2 118.4
374.4 41.6 366.6 35.3
378.8 48.7 377.2 44.1
393.0 44.9 371.8 39.1
359.6 44.0 345.2 32.8
362.0 37.2 351.2 25.5
362.0 50.2 356.6 34.8
362.0 47.0 344.8 24.7
380.8 65.5 365.0 54.2
Changes relative to measures obtained before intravenous administration are shown for the amplitudes N1/P2 in Fig. 2, for the latencies of N1 in Fig. 3, and for the intensity estimates and the tracking performances in Fig. 4. Table 2 surveys significant influences of the medication on the experimental parameters obtained by employing analyses of variance. Pentazocine versus placebo
Compared to placebo, the amplitude N I / P 2 of the C S S E P s (see Fig. 2, Table 2) significantly decreased in the central recording sites ( p < 0 . 0 5 ; p < 0 . 0 1 ) as well as in F3 and Fz (p
0065-4299/90/040342-18$1.50+0.20/0 9 1990BirkhfiuserVerlag, Basel
Effects of pentazocine and acetylsalicylic acid on pain-rating, pain-related evoked potentials and vigilance in relationship to pharmakokinetic parameters G. Kobal, C. Hummel, B. Nuernberg and K. Brune Institut fiir Pharmakologieund Toxikologie,UniversitfitErlangen-Nfirnberg,Universitfitsstr.22, D-8520 Erlangen, BRD
Abstract
Achieving objective and quantitative measurement of experimental pain in human volunteers and establishing the impact of drugs remains a difficult task. This problem may be overcome by employing a method which allows the simultaneous measurement of pain ratings elicited by standardized stimulation of the nasal mucosa by carbon dioxide, together with pain-rdated chemo-somatosensory evoked potentials (CSSEP) and vigilance. We assessed the effect of pentazocine and acetylsalicylic acid on these parameters in 14 human volunteers and related the effects to the pharmacokinetic parameters of the drugs measured at the same time. Pentazocine was found to reduce the pain ratings as well as the amplitudes of the pain-related evoked potentials and to increase their latencies. Vigilance (measured by EEG power spectra and performance of a tracking task) was also significantly reduced. These effects were observed during the distribution phase and the first period of the terminal elimination phase of the drug. Acetylsalicylic acid had no significant effects on pain ratings, but reduced the amplitudes of the event-related potentials when compared to placebo controls. At the same time a slight, but significant, effect on vigilance (reduced performance of the tracking task) was observed. These effects could not be related to the presence of unmetabolized acetylsalicylic acid in the plasma. They appeared at later times when only salicylic acid was left. It is concluded that chemical stimuli of sufficient intensity produce pain which may be suppressed by opioid analgesics such as pentazocine. The effect of acetylsalicylic acid on this experimental pain did not reach significance for all measured parameters under the experimental conditions chosen. The changes in vigilance and in the amplitudes of pain-related chemo-somatosensory evoked potentials indicated as yet unknown CNS-effects of this non-steroidal anti-inflammatory drug.
Introduction
The value of new analgesics must ultimately be defined under clinical conditions. Animal experiments may serve for the selection of potentially effective new types of analgesics. However, the principal types of analgesics such as morphine, salicylates, pyrazole drugs and aniline derivatives were found by serendipity and not by systematic pharmacological research [1, 2]. It appears that the
wide gap between preliminary experimental data obtained in animals and human pain should be bridged by quantitative measurement of drug effects in human volunteers in order to predict the possible clinical suitability of any compounds. Many attempts for measuring pain and analgesic effects in volunteers using event-related potentials [3] have been reported (reviewed by Chudler and Dong [4] and Stowell [5]; [6-12]). Many of these studies suffer either from limited reproducibility
343
Agents and Actions, vol. 29, 3/4 (1990)
due to changes in the tissue irritated by the noxious stimulus, or from the lack of adequate control measurements which take into account vigilance, adaptation, and learning effects. Finally, the lack of objective, quantifiable parameters of pain preclude correlations with the pharmacokinetic parameters of the drugs used. Here, we report on the use of a pain model [13, 14] which allows for the quantification of drug effects on pain rating and pain-related evoked potentials as well as vigilance in connection with the assessment of pharmacokinetic parameters. The aim of the present study was to find out, whether pain-related evoked potentials - being epiphenomena of nociceptive electrophysiological events in the brain - not only reflect the effects of centrally acting analgesics, as it was repeatedly reported [8, 11, 12, 15 22], but also the action of peripherally acting analgesics, such as acetylsalicylic acid (ASA). To demonstrate the sensitivity of the pain model chosen for this study (for alternative models see [23]), we compared the effects of the ASA with the effects of the centrally acting kappa-agonist/mu-antagonist pentazocine, a drug widely used in clinical applications. In the case of pentazocine we expected to find robust drug effects, but probably not to such an extent as we observed after administration of 0.5 mg fentanyl which totally eliminated the pain-related chemosensory evoked potentials [24].
Material and methods
In 14 volunteers (7 female, 7 male, between 23 and 38 years of age) the analgesic effects of acetylsalicylic acid and pentazocine were measured by means of cortical event-related potentials after painful stimulation, as well as by the subjective estimates of stimulus intensities. Changes in the subjects' vigilance were assessed by evaluating a "tracking task" which the subjects performed on a video screen and by computing power spectra of the background EEG activity.
Stimulus procedures For painful stimulation carbon dioxide (CO2) pulses were delivered to the left nostril of the subjects. In order to achieve a pure chemical stimulation, thus avoiding alterations of the mechanical or
thermal conditions at the respiratory nasal mucosa, CO 2 pulses of 200 msec duration were mixed in a constantly flowing air stream with controlled temperature (36.5 ~ and humidity (80% rel. humidity). The rise time of the stimulus' concentration pulses was below 20 msec, i.e., it was sufficiently steep to elicit late nearfield event-related potentials [14, 25, 26]. To avoid respiratory air flow in the nose during stimulation, subjects were trained to practise velopharyngeal closure, breathing through the mouth [25]. On each occasion the volunteer received a train of 80 painful stimuli with an interstimulus interval of 60 sec. Thus, one experimental session lasted 80 min. Two concentrations ("weak": 45% v/v and "strong": 52% v/v) of CO 2 were alternately presented. These two concentrations were chosen to get one type of stimulus which was close to the pain threshold and another one which was clearly above threshold even in case of drug administration.
Pain-related responses Pain-related evoked potentials were obtained by EEG recordings at the sites FP2, F3, Fz, F4, C3, Cz, C4 and Pz of the international 10/20 system referenced to A1 (bandpass 0.16-70 Hz; Siemens Mingograph). EEG-records were digitized with a sampling frequency of 250 Hz, and stored on magnetic media. Data were evaluated off-line with a PDP 1t/73 computer (Digital Equipment Corporation) by OFFLAB-programs [25]. 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 ~tV in the eye channel (Fp2/A1) were excluded from further analysis. The subjects estimated the intensity of the painful stimuli two seconds after stimulus presentation by employing a visual analogue scale displayed on a computer screen. This assessment of intensity estimates followed the technique of cross modality matching [27, 28] with prescribed modulus (first stimulus in the experiment of 52% v/v COz). In order to observe changes in the subjects' state of vigilance (and/or motoric coordination) they were requested to perform a simple task on a video screen: they had to keep a smaller square, which could be controlled by a joy-stick, inside a larger square, which unpredictably moved around. This
344 "tracking performance" was checked by counting how often and by measuring for how long they had lost track of the moving square. Additionally pre-stimulus periods of 4096 msec were sampled from the positions Fz, Cz and Pz. These data were submitted to a Fast Fourier Transformation. The averaged power spectra were divided into 7 frequency bands: delta (1-3.5 Hz), theta (3.5-7 Hz), alphaa (8-10 Hz), alpha 2 (1013 Hz), beta 1 (13-18 Hz), beta2 (18-21 Hz), and beta 3 (21-30 Hz). The areas under the curves in the range of the defined frequency bands were submitted to statistical analyses. Mean values were calculated for all parameters (amplitudes and latencies of event-related potentials, estimates of intensity, tracking performance, power spectra) over periods of 16 minutes. Thus, one experimental session was divided into five "Sections" (0, I, II, III, IV). Event-related potentials and intensity estimates were grouped in relation to the two concentrations of the painful stimuli. Since both strengths of stimuli were alternately presented every other minute, mean values were calculated over maximal 8 (in case of artifacts minimal 6) EEG-recordings, 8 intensity estimates, and 16 assessments of the tracking performance. The event-related potentials were quantified by measuring the latencies of the peaks P1 (240 msec), N I (340 msec), P2 (520 msec), and N2 (760 msec) and the peak-to-peak amplitudes P1/NI, N1/P2 and P2/N2 [26]. Approximately 150 msec of the latencies are due to transportation time through the mucus and epithelial layer at the respiratory nasal mucosa and the utilization time at the receptor sites [14].
Materials and methods ,for determination of blood plasma levels Acetylsalicylic lysinate (Aspisol| acetylsalicylic acid (ASA), salicylic acid (SA), pentazocine lactate (Fortral| and pentazocine were supplied by Bayer AG (Wuppertal, FRG) and Winthrop (NeuIsenburg, FRG) respectively. Levallorphan as the internal standard for a quantitation of pentazocine was supplied by Hoffmann-La Roche (Grenzach, FRG) in commercially available ampoules (Lorfan| Heparin was used as Liquemin | ampoules (2500 IU/5 ml); the destilled water was filtered (~3 0.45 gin). All other chemicals and reagents were of HPLC- or analytical grade.
Agents and Actions, vol. 29, 3/4 (1990) For quantification of acteylsalicylic acid (ASA) and salicylic acid (SA) an isocratic high performance liquid chromatographic ultraviolet-method was used, according to Amick and Mason [29], Nieder et al. [30], see also [31]. The detection limits for ASA and SA were found to be less than 1 gg/ml. In the concentration range of 0.5 to 200 gg/ml the chromatographic system showed linearity. Mean values were calculated of at least two prepared samples, of which each was measured twice. The mean recovery for ASA was 72.15% (r = 0.99993, y=0.74x-0.82; n = 18) and for SA 67.62% (r = 0.99980, y = 0.70x - 1.24; n = 18). The coefficients of variation for ASA were in the range between 1.5 and 2.5% and those for SA between 3.2 and 4.6%. A specific high-pressure liquid chromatographic method for determination of pentazocine and the salicylates was developed. In brief: The following chromatographic instruments were used: one Waters pump model M6000A (Waters Milford, MA, USA), a model Waters U-6-K injector, a Kontron SFM 23/B fluorimetric detector set at 278 nm for excitation and 324 nm for emission, a Shimadzu model CR3A integrator. Plasma specimens were obtained and separated on a 50 x 3.9 mm guard column filled with 30 pm ODS Nucleosil TM and a 250 x 3.9 mm column filled with 10 pm Nucleosil TM ODS. The mobile phase consisted of acetonitrile -0.05 M phosphatic acid (25:75 v/v) in an isocratic system. The flow rate was 2.0 ml/min at 21 ~ The retention times were 5 min for levallorphan and 7.5 min for pentazocine. The calculations of all plasma samples were obtained by using the internal method establishing the ratio with levallorphan as internal standard, as well as by employing the external standard method. The detection limit of this method was less than 2 ng/ml. In the concentration range of 2 to 1500 ng/ml, the chromatographic system showed linearity. The mean recovery for pentazocine was 98.3% (r = 0.999991, y = 0.98x + 0.35; n = 32). The coefficient of variation ranged between 2.7 and 4.9%. These data were obtained by calculating the mean of at least two analyses employing the internal standard method (levallorphan 50 ng/ml), as well as by calculations using the external standard method. The pharmacokinetic parameters for acetylsalicylic acid, salicylic acid and pentazocine were com-
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puted on a Wang APC computer using the program TOPFIT [32]. Several open two- and three compartment infusion models were used for the analysis of the plasma concentration-time data. All parameters describing the intraindividual bioavailability in both groups - morning and afternoon (see below) - were subjected to statistical analyses developed by Steinijans and Diletti [33]. The tests (t-test, ANOVA, Westlake, Wilcoxon and Pitman) were also included in the TOPFIT program package.
Test procedure The study corresponded to a single blind three way crossover design in which each experiment was run twice with a wash-out period of one week between experiments. An additional training session was included. The preparations tested were 1800 mg lysinemono-acetylsalicylate LAS (equivalent to 1000 mg acetylsalicylic acid), 50 mg pentazocine lactate (equivalent to 30 mg pentazocine), and placebo (10 ml Ringer's solution). Indwelling catheters were inserted into veins of both forearms. Except during injection 500 ml of physiological saline solution was continuously infused into the blood stream through the right catheter. Shortly before injection, the contents of two ampoules of Aspisol ~ were dissolved in 10 ml aqua pro injectionem. Switching a three-way valve, the solution was injected through the right catheter over a period of 2 rain. In the other two experimental conditions, either I ml of pentazocine lactate solution, or 10 ml of Ringer's solution were administered over a period of 3 rain. Since there was approximately 1 m tubing between injection point and the forearm of the subjects infusion times were markedly longer. Considering the flow rate of the infusion the actual injection time ranged between 5 and 10 minutes. The starting point of injection was 16 rain after on-set of pain stimulation (i.e. after "section 0"). Through the left catheter, blood samples (10 ml) were withdrawn prior to and 1, 2, 4, 8, 12, 15, 20, 30, 40, 60 min after injection. In the case of pentazocine, several additional blood samples were collected over a period of 480 rain, if possible. The first 2 ml of the samples were always rejected in order to remove residual heparin that was used to flush the catheter after sampling. Immediately after collection, plasma was harvested by centrifuga-
tion at 900 9 (0 ~ and subsequently, the resulting blood plasma was stored at - 6 0 ~ No more than 7 days elapsed before the samples were analyzed. The volunteers were instructed not to partake of solid food or any kind of beverages during a period of a least 6 hours before the experiments started. A screen prevented the subjects to notice manipulation of the infusion. In this way, their attention was not diverted and the drugs could be administered unobserved. White noise (70dB SPL) masked all sounds, including the noise of the switching device. An anaesthesiologist monitored blood pressure, pulse rate, and respiration during the entire experiment, as well as for a period of 6 h after i.v. administration. He also assessed the state of health of the subjects by routine examinations and took the anamneses prerequisite for the subjects' participation in the study.
Subjects The experiments were performed in accordance with the declaration of Helsinki/Tokyo/Venice and were approved by the Ethics committee of the University of Erlangen-Niirnberg. After having given their informed consent, 7 healthy female and 7 healthy male volunteers, between 23 and 38 years of age, participated in the experiments. Due to allergic reactions after the first experimental session with LAS medication, one subject was excluded from further participation. Three subjects were excluded because of unwanted effects of the drugs such as nausea and vomiting (after pentazocine administration) and one female subject withdrew from further participation because of pregnancy. As a result 6 female and 4 male subjects were in on the LAS experiments to the end and 4 female and 5 male subjects completed the pentazocine experiments. Each experiment was run twice, at 9 am and at 2 pro. Due to technical problems, 2 male and 2 female subjects participated in one LAS experiment only.
Statistical analysis Mean values of the data obtained from two experimental sessions were subjected to analyses of variance to determine the main effect: experimental "Sections" (0 = before; I - I V = after drug administration) separately for each electrode site and each medication.
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Since all experiments were run with the same volunteers, this factor was additionally included in the analysis (repeated measurements design). The performance of the "Box Tests" did not reveal variance-covariance-homogeneity [34], consequently we used the conservative test of F-fractions with FGN . . . . . t o t : 1 and F G D. . . . inator=N-I ( N = n u m ber of samples) described by Glaser [35]. Where significant results were obtained, multiple t-tests were used to further specify the differences in the measures. The results of these analyses used to test the effects of the administration of drugs and placebo will only be mentioned when necessary (e.g. in Fig. 2 and 3). In order to establish differences between the tested drugs and placebo, they were compared in pairs by employing analysis of variance for repeated measurements (pentazocine/placebo, LAS/placebo, and pentazocine/LAS) separately for the different recording positions. The factor "Section" was not evaluated this time, because the results were already known from the first analysis. This procedure was rendered necessary because of the disparity in the number of subjects in the two drug application groups due to dropouts. In this case, percentage values of the four measuring sections (I IV) after i.v, injection relative to measures obtained prior to medication (section 0) were used.
This also helped to eliminate the variance between different days of testing. In addition, correlations between the electrophysiological (amplitudes N1/P2 and latencies TP1, T N I of the 3 central sites of recording Cz, C3, and C4), psychophysical (intensity estimates, tracking performances) and pharmacokinetic parameters such as AUC (area under the plasma-concentration-time-course) and M R T (mean residence time) were established.
Results
Pain-related data
Pain-related cerebral potentials were recorded from all subjects after stimulation of the nasal mucosa by CO 2. Mean values and standard deviations were calculated for all groups of data (Table 1; in extracts). For the purpose of graphical depiction only, grand means of the event-related potentials were computed across all subjects. Latency differences between subjects were compensated by standardizing all event-related potentials to the mean latency between N I and P2. These data are shown in Fig. I where pain-related potentials from one single subject are also illustrated.
Single Subject
Grand Means
P n ozo ,n
Placebo
"
~
I pV'~ 0
1024 rnsec
0
1024 mser
Figure 1 Pain-related evoked potentials elicited by carbon dioxide stimulation of the nasal mucosa. Effects of pentazocine, Iysine-mono-acetysalicylate (LAS), and placebo. Example from one subject (left) and the grand mean over all subjects. Stimulus 52% v/v CO 2 .
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Table 1 Means and standard deviations of pain-related evoked potentials. Recording sites Fz and Cz (versus AI) (amplitudes: gV; latencies: msec) 0=before, l = 0 16 min after, l I = 17 32 min after, I I I - 3 3 - 4 8 rain after, I V = 4 9 - 6 4 rain after drug application.
Pen tazocine Tracking performance 0
I 87.6 9.2
II 76.9 21.1
72.1 24.5
III 69.3 23.7
IV 68.7 25.0
45% v/v CO 2 0
I
52% v/v CO 2 II
III
IV
0
I
II
III
IV
Intensity estimates 65.3 18.1
32.5 20.6
29.6 21.3
36.9 31.0
50.1 38.8
113.8 12.6
75.1 31.4
74.6 37.9
83.1 38.3
83.8 37.9
Amplitudes N I / P 2 Fz 19.2 4.9 Cz 27.1 6.1
14.6 6.5 20.1 6.9
12.4 5.4 17.8 7.5
12.8 4.2 16.2 8.0
11.9 5.2 16.6 8.4
22.6 5.7 34.5 6.5
17.3 7.7 25.0 9.8
15.2 9.5 22.7 10.4
17.4 7.7 24.2 10.0
16.8 9.0 23.8 9.6
327.9 25.5 329.0 24.2
342.8 33.7 340.3 37.7
374.1 44.0 373.0 52.1
349.2 40.0 345.4 27.5
306.6 39.2 306.3 29.6
342.8 45.7 317.0 34.1
343.7 70.2 340.1 53.8
336.1 51.8 335.0 47.7
371.9 58.9 366.1 52.7
II
lII
IV
II
lII
IV
Latencies TNI Fz 327.7 34.8 Cz 332.1 34.2
Lysine-rnono-acetylsalicylate Tracking performance 0
I 81.6 11.9
79.6 13.7
77.6 11.6
78.6 10.9
78.9 12.2 52% v/v CO 2
45% v/v CO 2 0
1
II
Ill
IV
0
I
Intensity estimates 68.1 8.5
50.3 14.7
55.8 17.3
60.0 19.2
62.6 19.9
109.4 23.3
98.9 21.2
102.8 26.6
107.7 29.1
103.6 28.3
Amplitudes N I / P 2 Fz 20.8 6.6 Cz 27.3 6.9
16.7 5.5 22.1 6.1
15.3 5.2 20.6 4.8
15.5 7.2 21.4 9.3
16.7 6.2 21.6 6.8
25.9 7.9 33.1 8.6
19.6 9.1 25.6 9.3
19.7 7.5 26.6 8.6
18.7 8.2 25.6 9.3
19.5 10.6 27.3 11.2
376.7 53.9 372.5 45.8
372.7 33.1 361.1 33.9
388.2 55.9 375.7 47.1
373.7 36.6 355.7 26.3
343.7 33.5 332.7 22.6
347.7 50.1 341.5 33.2
350.7 40.7 340.3 31.5
341.7 31.4 330.1 24.3
348.1 37.9 337.9 28.1
Latencies TN1 Fz 353.9 33.1 Cz 351.9 34.2
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Agents and Actions,vol. 29, 3/4 (1990)
Placebo Tracking performance
0 76.9 14.7
1 77.4 11.1
II 79.6 13.1
Ill 79.5 15.5
IV 82.4 10.7 52% v/v CO2
45% v/v CO2 0 Intensityestimates 75.7 17.4 Amplitudes N1/P2 Fz 22.2 7.6 Cz 30.5 9.4 Latencies TN 1 Fz 368.4 35.3 Cz 346.8 20.8
1
II
lII
IV
0
I
II
IlI
IV
60.5 25.6
63.3 27.3
69.4 28.3
67.2 28.6
116.9 10.8
117.7 16.8
116.5 22.2
115.3 22.0
113.8 26.7
20.3 10.4 22.8 12.3
20.6 7.7 25.3 8.6
18.5 8.5 25.1 8.9
16.0 5.3 22.2 5.3
24.2 9.2 36.1 10.3
23.9 9.0 33.2 9.8
22.2 7.1 31.0 9.6
21.6 6.9 29.4 6.9
20.3 7.3 27.1 7.1
380.8 39.2 329.2 118.4
374.4 41.6 366.6 35.3
378.8 48.7 377.2 44.1
393.0 44.9 371.8 39.1
359.6 44.0 345.2 32.8
362.0 37.2 351.2 25.5
362.0 50.2 356.6 34.8
362.0 47.0 344.8 24.7
380.8 65.5 365.0 54.2
Changes relative to measures obtained before intravenous administration are shown for the amplitudes N1/P2 in Fig. 2, for the latencies of N1 in Fig. 3, and for the intensity estimates and the tracking performances in Fig. 4. Table 2 surveys significant influences of the medication on the experimental parameters obtained by employing analyses of variance. Pentazocine versus placebo
Compared to placebo, the amplitude N I / P 2 of the C S S E P s (see Fig. 2, Table 2) significantly decreased in the central recording sites ( p < 0 . 0 5 ; p < 0 . 0 1 ) as well as in F3 and Fz (p
