Brain, Vol. 126, No. 5, 1092-1102,
May 2003
© 2003 Guarantors of Brain
doi: 10.1093/brain/awg115
Peripheral opioid analgesia in experimental human pain models
Pharmazentrum Frankfurt, Institut für Klinische Pharmakologie, Klinikum der Johann Wolfgang Goethe-Universität, Frankfurt am Main, Germany
Correspondence to: Irmgard Tegeder, MD, pharmazentrum frankfurt, Klinikum der Johann Wolfgang Goethe-Universität, Theodor Stern Kai 7, D-60590 Frankfurt am Main, Germany E-mail: tegeder{at}em.uni-frankfurt.de
Received September 20, 2002. Revised December 3, 2002. Second revision December 30, 2002. Accepted December 31, 2002.
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This placebo-controlled, double-blind crossover study assessed whether exclusive activation of peripheral opioid receptors results in significant pain reduction. To achieve opioid activity restricted to the periphery, we used a short-term (2 h) low dose infusion of morphine-6-ß-glucuronide (M6G) because M6G does not pass the blood–brain barrier during this time in amounts sufficient to induce CNS effects. The lack of central opioid effects of M6G was confirmed by a lack of change of the pupil size and absence of other opioid-related CNS effects. As a positive control, morphine was infused at a dosage that definitely produced CNS effects. This was evident by a rapid decrease of the pupil size and by other typical opioid-related side effects including nausea, vomiting, itchiness, hiccup and sedation. Three different pain models were employed to evaluate the analgesic effects: (i) cutaneous inflammatory hyperalgesia induced by briefly freezing a small skin area to –30°C (‘freeze lesion’); (ii) muscle hyperalgesia induced by a series of concentric and eccentric muscle contractions (DOMS model; delayed onset of muscle soreness); and (iii) pain induced by electrical current (5 Hz sinus stimuli of 0–10 mA). M6G significantly reduced cutaneous hyperalgesia in the ‘freeze lesion’ model as assessed with von Frey hairs. It also reduced muscle hyperalgesia in the DOMS model. Electrical pain, however, was not affected by M6G. Morphine was significantly more active in the ‘freeze lesion’ and DOMS model, and also significantly increased the electrical pain threshold and tolerance. Subcutaneous tissue concentrations of M6G and morphine as assessed with microdialysis were about half those of the respective plasma concentrations. The results of the study indicate that M6G has antihyperalgesic effects in inflammatory pain through activation of peripheral opioid receptors. Since this occurs at concentrations that do not cause central opioid effects, M6G might be useful as a peripheral opioid analgesic.
Keywords: morphine-6-glucuronide; pain; inflammation; peripheral opioid receptor
Abbreviations: DOMS= delayed onset of muscle soreness; M6G = morphine-6-glucuronide; VAS = visual analogue scale
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Introduction |
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For generations, opioids were believed to act solely on the CNS, producing analgesia by interaction with cerebral and spinal opioid receptors. This dogma was first challenged by an experimental study that recorded directly from the nasal mucosa the local elctrophysiological response to painful stimulation of nasal nociceptors (Kobal, 1985
). Pentazocine given prior to the painful stimulation decreased the amplitude of the pain-specific peripherally evoked negative mucosa potentials. Subsequent studies have focused on the contribution of peripheral opioid receptors to opioid analgesia. Local injection of opioids into peripheral inflamed tissues caused potent local naloxone-reversible antinociception in laboratory animals (Stein et al., 1988, 1989; Levine and Taiwo, 1989; Parsons et al., 1990; Kolesnikov et al., 1996; Perrot et al., 1999). In most of these studies, the opioid-induced peripheral antinociception occurred preferentially in inflamed tissue (Stein et al., 1988, 1989; Levine and Taiwo, 1989; Parsons et al., 1990; Kolesnikov et al., 1996; Perrot et al., 1999). Thus, it fits that the number of opioid receptors in peripheral tissue is normally low but rapidly increases when an inflammation comes into play (Hassan et al., 1993; Schafer et al., 1995; Stein et al., 1996). Constriction of the sciatic nerve abolished this peripheral opioid receptor upregulation, suggesting that opioid receptors are transported into the periphery by axonal transport (Hassan et al., 1993; Mousa et al., 2001). The physiological importance of peripheral opioid receptors was doubted until inflammation-attracted immune cells were identified as the source of endogenous opioid peptides. The release of these endogenous ligands allows for a communication between immune cells and nociceptors, and may constitute a mechanism of peripheral pain control (Stein et al., 1995; Schafer et al., 1996, 1997; Machelska et al., 1998, 2002; Rittner et al., 2001). Peripheral opioid analgesia might be employed in pain therapy. Injection of opioids into knee joints after knee surgery (Stein et al., 1991; Joshi et al., 1993a, b; McSwiney et al., 1993; Whitford et al., 1997) or local infiltration after dental surgery (Likar et al., 1998; Dionne et al., 2001) caused prolonged postoperative analgesia in humans. However, there are also reports that locally injected opioids failed to reduce postoperative pain (Gupta et al., 1993; Schulte-Steinberg et al., 1995; Aasbo et al., 1996; Rosseland et al., 1999; Motamed et al., 2000).
Systemically rather than locally administered opioids with exclusive or predominant peripheral action have been investigated rarely because most opioids cross the blood–brain barrier, and those that do not, such as loperamide, are not available for intravenous administration. Systemic administration would be more convenient than local administration in many cases. In the present study, we took advantage of special pharmacokinetic characteristics of morphine-6-ß-glucuronide (M6G) which is a major metabolite of morphine with potent opioid agonist activity (Shimomura et al., 1971; Paul et al., 1989; Frances et al., 1992). M6G is a very polar hydrophilic metabolite and therefore does not readily penetrate the blood–brain barrier (Bickel et al., 1996). We recently observed that during the first few hours of intravenous administration of M6G, the pupil size in healthy volunteers remains unchanged, suggesting the absence of CNS opioid effects that would be great enough to produce central analgesia (Lotsch et al., 2001; Skarke et al., 2003). This is caused by a slow transfer of M6G between plasma and CNS effect site, with a half-life of 6.4 h, as compared with 2.8 h for morphine (Lotsch et al., 2001). It was therefore possible to study exclusively the peripheral analgesic effects of M6G in the present study. To account for the differences in the number of peripheral opioid receptors between normal and inflamed tissues, we employed inflammatory and non-inflammatory pain models. The absence or presence of CNS opioid effects was monitored by means of pupil size measurements. Morphine served as a positive control. To check for the availability of M6G and morphine at peripheral sites, tissue concentrations were assessed by means of microdialysis.
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Subjects
Ten healthy, non-obese subjects (five men, five women; mean age 28 years, range 22–43 years), not taking any medication or drugs, were enrolled. All subjects gave written informed consent to participate in the study which was approved by the Ethics Committee of the Medical Faculty of the University of Frankfurt. We recently identified the A118G-single nucleotide polymorphism of the µ-opioid receptor gene to cause decreased M6G potency for pupil constriction in carriers of the mutated G118 allele (Lotsch et al., 2002
a). Therefore, we genotyped the subjects for that specific mutation in order to make sure that carriers of the mutated allele are not included more frequently than the average allele frequency of 12% in the population (Bergen et al., 1997). The method of genotyping is described elsewhere (Lotsch et al., 2002a). From the 10 subjects, two were heterozygous and one homozygous for the mutated G118 allele. This corresponds to an allelic frequency of 12.5%, which is exactly that reported for the general Caucasian population (Bergen et al., 1997).
Study design and medications
This was a randomized, placebo-controlled, double-blind two-way crossover study. M6G was administered as intravenous loading dose (short infusion of 12 min duration) followed by a constant rate infusion for 2 h. Based on previously obtained pharmacokinetic parameters (Lotsch et al., 2002b), the M6G dosage was adjusted to achieve M6G plasma concentrations of 500 ng/ml. In preliminary experiments, this concentration caused no CNS opioid effects, as indicated by a lack of pupil constriction. After 2 h, the infusion of M6G was discontinued and replaced with an infusion of morphine. The dosage of morphine was selected to reach plasma morphine concentrations of 100 ng/ml. In previous experiments, this morphine concentration was found to produce clear analgesic CNS effects in an electrical pain model (Skarke et al., 2003). After 4 h, the infusion of morphine was stopped and 1.6 mg of naloxone was infused over 30 min in order to observe the reversibility of the opioid effects on experimental pain and pupil size and in order to speed up the recovery of the subjects. In the next study, subjects received placebo/placebo infusions instead of M6G/morphine. Both investigators and subjects were blinded as to whether M6G/morphine or placebo/placebo was actually administered. However, since morphine was easily identified because of its adverse effects, subjects and investigators were actually blind only on the first study day. Half of the subjects were medical students or nurses who had accurate expectations concerning opioid effects. In addition, observers learned from pupil constriction whether morphine or placebo had been administered. A washout period between study days of at least 7 days was guaranteed.
M6G was prepared for intravenous administration in humans according to the German pharmacopoeia by Lipomed AG, Basel, Switzerland. The purity of M6G was at least 98.4% (Lot No: 57.1B13.1). Commercially available morphine hydrochloride was used (MSI, Mundipharma GmbH, Limburg/Lahn, Germany). Saline (0.9%) served as placebo.
Assessment of pain
Inflammatory hyperalgesia (freeze lesion)
At 22–24 h prior to testing, a cutaneous inflammation was induced by briefly freezing a small skin area at the anterior side of one forearm (Kilo et al., 1994). A copper cylinder of 15 mm diameter and 290 g weight was cooled with dry ice to –30°C and placed perpendicularly onto the skin for 8 s with no more pressure than that provided by its weight. For better thermal contact, a filter paper soaked with saline was placed between the skin and the copper cylinder. This procedure evoked a stinging or burning pain for 1–2 h (Kilo et al., 1994). Any spontaneous pain subsided within 1–2 h. However, the skin lesion remained reddened and sensitive to mechanical stimulation for a few days.
The threshold to mechanical pain stimuli was assessed by means of a punctuated stimulation using von Frey hairs of different strengths (0.008, 0.02, 0.04, 0.07, 0.16, 0.4, 0.6, 1, 1.4, 2, 4, 6, 8, 10, 15, 26, 60, 100, 180 and 300 g; North Coast Medical Inc., Morgan Hill, USA). They were placed perpendicularly onto the area of the freeze lesion and bent slightly to apply punctuated pressure. The stimuli were applied in a randomized order, and the subject’s task was to close the eyes and report whether pain was perceived. The mechanical pain threshold to stimulation with von Frey hairs was obtained by logistic regression of ‘yes/no’ answers for the different von Frey hairs. The pain threshold was assessed in a similar manner at the contralateral forearm. Thresholds to von Frey-evoked pain were tested before induction of the freeze lesion (‘before’), before starting medication (‘baseline’) and then half-hourly until the end of the experiment.
Muscle pain
Unaccustomed exercise incorporating eccentric muscle contractions was employed to induce delayed onset of muscle soreness (DOMS) in calf muscles, as has been described previously (Tegeder et al., 2002c). The DOMS exercise protocol consisted of two sets of 50 concentric/eccentric contractions of the calf muscles of one leg with a rest of 5 min in-between. The exercise was performed 22–26 h prior to medication. The efficacy of the DOMS exercise was assessed by analysis of plasma lactate levels before and after the exercise according to standard methods. The normal range of plasma lactate was 5.7–22 mg/dl. For pain measurements, muscle pain was stimulated by standing on tiptoes of one leg for 30 s, which requires a constriction of the affected calf muscles. The other leg was lifted and the subjects were allowed to hold on to a table to keep their balance. The pain intensity during this stimulation was then rated by means of a 10 cm visual analogue scale [(VAS), extending from ‘no pain’ to ‘intolerable pain’ with a precision of 1 mm]. The stimulation was repeated with the other leg. The sequence of the legs was chosen randomly. Muscle hyperalgesia was assessed before starting medication (‘baseline’) and then hourly up to the end of medication.
Electrical pain
The method has been described to be sensitive to quantify analgesic opioid effects (Angst et al., 2001). Pain threshold and tolerance following electric stimulation were assessed using a constant electrical current device (NEUROMETER® CPT, Neurotron Inc., Baltimore, MD, USA) that delivered a sine wave current at a frequency of 5 Hz. Two electrodes, connected by a 1.5 cm plastic band to facilitate positioning, were placed on the medial and lateral side of the distal phalangeal joint (middle finger of the left hand as default testing site). Skin impedance was reduced with an electrolytic paste (Prep-Paste, Neurotron Inc., Baltimore, MD, USA). The intensity of the current delivered via the electrodes was increased from 0 to 9.99 mA in steps of 0.2 mA/s. During testing, the subject continuously pressed an electrical switch until the electrical stimulation became painful or unbearable in the case of pain threshold or tolerance, respectively. By releasing the switch, the current was interrupted, and the threshold or tolerance was the intensity of the electrical current reached at that point. Each pain threshold or tolerance was the median of the values obtained in five successive repetitions of the measurements, separated by 
1 min. On each study day, two baseline measurements were obtained prior to the start of medication, followed by test sessions at half hourly intervals.
Assessment of pupil size
The pupil diameter was assessed to monitor the absence or presence of CNS opioid effects great enough to produce central analgesia. Pupil size was measured half hourly by means of a pupillograph (‘CIP’, Amtech GmbH, Weinheim, Germany) as described previously (Lotsch et al., 2001, 2002a). In brief, the subject’s head was placed on to an anatomically formed support which fixated the chin and forehead to ensure a constant pupil–sensor distance. In addition, the subjects focused their view onto a red dot on a wall 1.5 m behind the pupillograph during measurement. The investigator stood behind the device and used a 3.5 inch video display to control the correct positioning of the sensor by manoeuvring the device until the horizontal guideline was positioned correctly above the pupil diameter. The pupil diameter value was determined as the mean of five measurements (30 s rest period). The resolution of the device was 0.05 mm. Measurements took place in a dedicated room devoid of daylight but with some light effused by two computer screens, so that light intensity was kept lower than 14 Lux. Prior to each pupil diameter measurement, 3 min were allowed for pupil adjustment to semi-darkness.
Plasma concentrations of morphine and M6G
Blood samples (4 ml) were collected in potassium EDTA tubes before drug administration (baseline) and at 5, 10, 20, 30, 40 and 60 min, then every 30 min until the end of the opioid infusion, at 5, 10, 20, 30, 40 and 60 min after the infusion was stopped, and from then every 90 min until the end of the experiment, resulting in a total of 28–31 samples per subject. Plasma was separated within 15 min of blood collection and was stored with quality control samples at –25°C.
Tissue concentrations of morphine and M6G
The tissue concentrations of M6G and morphine immediately below the freeze lesion were assessed using in vivo microdialysis. This technique is based upon diffusion of small molecules through a semi-permeable membrane which is located at the tip of a microdialysis catheter. The catheter is perfused constantly with saline or other fluids so that molecules entering the catheter through the membrane are transported outward through the outlet tube and can be analysed in the dialysate. Dialysate concentrations are proportional to tissue concentrations. The microdialysis catheter (CMA 70, membrane length 30 mm, membrane diameter 0.6 mm, molecular weight cut-off 20 kDa, CMA, Stockholm, Sweden) was inserted into the subcutaneous space of the anterior forearm as described previously (Tegeder et al., 2002a), so that the membrane was localized underneath the freeze lesion. The catheter was perfused with 0.9% NaCl at a flow rate of 2 µl/min. Dialysates were collected in 20 min intervals.
In order to convert dialysate concentrations into tissue concentrations, the in vitro transfer rate was assessed as described (Tegeder et al., 2002b) for concentrations ranging from 12.5 to 1000 ng/ml. The recovery was calculated by linear regression analysis and was then used to convert dialysate concentrations into tissue concentrations. Tissue concentrations were plotted versus the midpoints of the dialysis intervals. These time points were corrected for the time needed to pass the dead space of the catheter shaft and outlet tube, which was 6 min.
Morphine and M6G analysis
Morphine and M6G concentrations in plasma and dialysate were assayed by liquid chromatography tandem mass spectrometry, with a lower limit of quantification of 1 ng/ml for morphine and 0.5 ng/ml for M6G. The coefficient of variation over the calibration range of 0.5–1250 ng/ml was <5%. The analytical method has been described in detail previously (Lotsch et al., 2001).
Statistics
Each study day comprised four treatment sessions: (i) before the start of the infusion (‘baseline’); (ii) the period of the first infusion (M6G or placebo); (iii) the period of the subsequent infusion (morphine or placebo); and (iv) the period of naloxone infusion. For the freeze lesion, an additional fifth session before the other sessions consisted of the measurements taken before induction of the freeze lesion (‘before inflammation’). The measurements obtained during these sessions were averaged separately for each session. Subsequently, these averages were submitted to multivariate ANOVA (analysis of variance) for repeated measures (within-subject factors ‘medication’, i.e. opioid or placebo, and ‘session’, i.e. the above-mentioned treatment sessions throughout the study day). Post hoc t tests with an 
-correction for multiple testing (according to Bonferroni) were employed to identify differences between the single treatment sessions. For the log-normally distributed data obtained with the von Frey hairs, the geometrical average was used rather than the arithmetical average, and log-transformed data were submitted to ANOVA. Separate analyses were performed for the side of the freeze lesion or DOMS and the respective control side. In the case of electrical pain, separate ANOVAs were performed for pain threshold and pain tolerance. Pupil sizes were analysed analogously. Statistics were done with SPSS 10.1.4 (SPSS Inc., Chicago, IL, USA). The -level was set at 0.05.
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Results |
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Pupil size
There were significant main ANOVA effects for both ‘medication’ and ‘session’ on the pupil size [‘medication’, F(1,9) = 201.0, P < 0.001; ‘session’, F(3,27) = 143.0; P < 0.001], and there was a significant interaction ‘medication by session’ [F(3,27) = 156.9, P < 0.001]. During the infusion of M6G, the pupil size did not decrease significantly (mean ± SD ‘baseline’ 6.78 ± 0.69 mm, ‘M6G’ 6.53 ± 0.65 mm, 95% confidence interval for differences –0.019 to 0.291) (Fig. 1). On the basis of a previous investigation of both pupil effects and analgesia of M6G (Skarke et al., 2003
), we can exclude that M6G had any CNS analgesic effect at this non-significant effect on the pupil diameter. The results of that study demonstrated that opioid effects on pupil size occur simultaneously with analgesic effects. However, since the variability of pupil size measurements is lower than that of pain measurements, the pupil size is the more sensitive parameter for central opioid effects. Thus, during the infusion of M6G, any analgesic effects could be attributed to peripheral opioid effects. In contrast, infusion of morphine resulted in maximum pupil constriction, indicating strong CNS effects (P < 0.001 for ‘morphine’ versus ‘baseline’). Naloxone administration reversed the pupil constriction only partially (mean ± SD for ‘morphine’ 2.9 ± 0.3 mm, ‘naloxone’ 4.8 ± 1.1 mm; P < 0.001 for ‘morphine’ versus ‘naloxone’). Because of the shorter half-life of naloxone compared with morphine, the pupil size again started to decrease after the naloxone infusion was stopped.
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Side effects
During infusion of M6G, no side effects were observed except for some reports of heavy legs and a warm feeling. When the subjects were asked to guess whether they were receiving placebo or M6G, only half of them guessed correctly, which is not more than chance. In contrast, morphine caused typical opioid-related side effects such as sedation, nausea, vomiting, visual disturbances, hiccup and itchiness.
Analgesic effects
Inflammatory hyperalgesia
The induction of the cutaneous inflammation caused a drop of the mechanical pain threshold in all subjects (Fig. 2A). In contrast, on the contralateral forearm, the pain threshold remained constant (Fig. 2B). Both ‘medication’ and ‘session’ had a significant main effect on the pain thresholds [‘medication’, F(1,9) 44.9, P < 0.001; ‘session’, F(4,36) = 46; P < 0.001], and there was a significant interaction ‘medication by session’ [F(4,36) = 54.1, P < 0.001]. At the start of the infusion (24 h after skin freezing), all subjects had an inflammatory hyperalgesia at the site of the freeze lesion, as indicated by the significant difference from the threshold before freezing (post hoc t tests: P < 0.001 for ‘before inflammation’ versus ‘baseline’). Hyperalgesia did not differ between study days (Fig. 2A). During infusion of M6G, the mechanical pain threshold at the site of the freeze lesion returned to the level before induction of the inflammation. This indicates that M6G exerted an antihyperalgesic effect (P = 0.003 for ‘M6G’ versus ‘baseline’). During morphine infusion, the pain threshold increased further. Thus, morphine not only reversed hyperalgesia but also had an additional analgesic effect (P < 0.006 for ‘morphine’ versus ‘before inflammation’). Morphine produced significantly greater analgesia than M6G (P = 0.001 for ‘morphine’ versus ‘M6G’). Its analgesic effects were completely reversed with naloxone (mean ± SD of ln threshold 3.0 ± 1.1 g for morphine, 1.3 ± 1.2 g for naloxone and 1.8 ± 1.1 g ‘before inflammation’). During placebo treatment, pain thresholds remained constant on the freeze lesion (Fig. 2A) as well as the control side (Fig. 2B). M6G caused a slight increase of the pain threshold on the control side (Fig. 2B). However, this increase did not reach statistical significance (P = 0.088 for ‘M6G’ versus ‘baseline’). In contrast, morphine had significant analgesic effects at the control side (P = 0.002 for ‘morphine’ versus ‘baseline’; Fig. 2B) which was completely reversed with naloxone. Naloxone had no effect on pain thresholds after placebo treatment.
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Muscle pain
The DOMS exercise caused an increase of plasma lactate levels from 12.0 ± 3.8 to 26.8 ± 8.4 mg/dl (mean ± SD) directly after the exercise, indicating considerable muscle strain. At 24 h, all subjects reported muscle pain while standing on tiptoes of the DOMS leg and lifting the control leg. The pain intensity at ‘baseline’ (before medication) was 40.6 ± 27.4% VAS and 36.4 ± 22.6% VAS (mean ± SD) before starting M6G/morphine or placebo/placebo, respectively (Fig. 3A). This difference between study days was not significant. Because of the high inter-individual variability of the ‘baseline’ pain intensity, the ‘percentage change’ of pain intensity during treatment was used for the statistical evaluation. Both ‘medication’ and ‘session’ had a significant main effect on the pain intensity [‘medication’, F(1,9) = 10.1, P = 0.011; ‘session’, F(3,27) = 7.7; P = 0.001], and there was a significant interaction ‘medication by session’ [F(3,27) = 7.4; P = 0.001]. Infusion of M6G caused a significant reduction of muscle pain by 37.8 ± 25.0% (post hoc analysis: P = 0.007 for ‘M6G’ versus ‘baseline’) (Fig. 3A). Morphine caused a reduction of pain intensity by 71.3 ± 25.2% (P = 0.001 for ‘morphine’ versus ‘baseline’). However, the difference between M6G and morphine was not statistically significant (P = 0.107, 95% confidence interval for differences –2.74 to 36.25). Naloxone completely reversed the analgesic effects of M6G/morphine (mean ± SD VAS for ‘naloxone’ 35.0 ± 23.4% versus ‘baseline’ 40.6 ± 27.4%) (Fig. 3A). Three subjects also reported some minor pain in the control leg that was reduced with M6G in one subject and with morphine in two subjects (Fig. 3B).
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Electrical pain
There were significant main effects of the ANOVA factors ‘medication’ and ‘session’ on both threshold and tolerance to electrical pain stimuli [threshold: ‘medication’, F(1,9) = 9.8, P < 0.05; ‘session’, F(3,27) = 11.8, P < 0.001; and tolerance: ‘medication’, F(1,9) = 9.5, P < 0.05; ‘session’, F(3,27) = 14.2, P < 0.001]. In addition, there were significant interactions ‘medication by session’ for both pain threshold [F(3,27) = 13.7, P < 0.001] and tolerance [F(3,27) = 13.4, P < 0.001] Electrical pain threshold and tolerance were not affected by infusion of M6G (mean ± SD threshold for ‘M6G’ 1.04 ± 0.45 mA versus ‘baseline’ 1.04 ± 0.42 mA; mean ± SD tolerance for ‘M6G’ 3.48 ± 2.05 mA versus ‘baseline’ 3.32 ± 2.56 mA). The 95% confidence intervals for differences between ‘M6G’ and ‘baseline’ were –0.17 to 0.09 for threshold and –0.743 to 0.613 for tolerance (Fig. 4A and B). In contrast, morphine treatment caused a significant increase in the electrical pain threshold (P = 0.01 for ‘morphine’ versus ‘baseline’; Fig. 4A) and tolerance (P = 0.001; Fig. 4B). The analgesic effect of morphine was completely reversed with naloxone. Infusion of placebo had no effect on electrical pain threshold or tolerance.
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Tissue and plasma concentrations of M6G and morphine
Mean plasma concentrations of M6G and morphine were very close to the intended concentrations of 500 ng/ml (509.9 ± 65.8 ng/ml; mean ± SD) and 100 ng/ml (106.2 ± 18.0 ng/ml), respectively (Fig. 5A), i.e. the target concentrations were hit. Mean tissue concentrations of M6G achieved approximately half of the plasma concentrations (211.3 ± 65.7 ng/ml). They increased slowly and reached a maximum only at the end of the M6G infusion. Similarly, morphine tissue concentrations were approximately half the morphine plasma concentrations (43.6 ± 13.5 ng/ml). They were relatively constant during the morphine infusion, without a slow increase as observed for M6G. The in vitro microdialysis recovery of M6G and morphine was 81.4 and 83.9%, respectively.
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Discussion |
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Peripheral opioid receptors have been suggested to be involved in analgesic effects of injected opioids (Kobal, 1985
; Stein, 1995) and endogenous opioid peptides secreted from immune cells at the site of inflammation (Stein, 1995). We show that the hydrophilic morphine metabolite, M6G significantly reduces inflammatory cutaneous hyperalgesia at plasma concentrations that do not cause CNS effects, as confirmed by a lack of change of the pupil size. In a previous study (Skarke et al., 2003), six times higher M6G doses than those used in the present study were needed to cause central opioid analgesic effects on electric pain. The onset of central analgesic and pupil-contracting effects was identical. However, because pupil size measurements show lower variability than pain measurements, pupil constriction was the more sensitive parameter and can therefore be considered an adequate method to assess central opioid effects in the present study. We simulated a 4 h time course of pupil size and pain tolerance on the basis of the pharmacokinetic/pharmacodynamic model obtained in the previous study (Skarke et al., 2003), using the plasma concentrations observed during the first 2 h of the present study, and two and five times these concentrations. With the actual plasma concentrations, both pupil size and pain tolerance started to leave the 95% confidence interval of the placebo observations at 3 h, i.e. well after the peripheral part of the study had ended. With double the plasma concentrations, pupil size would leave this confidence range shortly before 2 h, while pain tolerance would still be within the limit. With five times the plasma concentrations, pupil size and pain tolerance would stay within the limit until 1.5 h. Thus, our plasma concentration of 500 ng/ml was well below the range that would have caused CNS effects during 2 h (simulations not shown).
M6G also reduced muscle hyperalgesia in the DOMS model to a statistically significant degree. Although the exact mechanisms contributing to muscle pain in the DOMS model are unclear (Cleak and Eston, 1992; MacIntyre et al., 1995), it has been suggested that eccentric muscle contractions cause myofibrillar disruptions (Friden et al., 1981; Armstrong et al., 1983; Lieber et al., 1991) and a subsequent inflammatory reaction (Smith, 1991; Croisier et al., 1996; Smith et al., 2000) associated with muscle swelling (Tegeder et al., 2002c), cellular infiltrates (Jones et al., 1986; Round et al., 1987), increased plasma cytokine levels (Nosaka and Clarkson, 1996; Bruunsgaard et al., 1997; Rohde et al., 1997) and slightly elevated prostaglandin E2 levels in muscle tissue (Tegeder et al., 2002c). Hence, muscle hyperalgesia in the DOMS model is probably partly due to inflammation. In contrast, M6G had no significant effect on electrically evoked pain, which has no inflammatory component. This is in line with previous results that have shown that M6G does not affect pain induced by a specific stimulation of nasal nociceptors with short pulses of gaseous CO2 (Lotsch et al., 1997). Hence, our results suggest that inflammation facilitates peripherally mediated opioid analgesia. This agrees with previous suggestions based on experiments in laboratory animals that pointed toward an important role for inflammation-induced upregulation of peripheral opioid receptors in peripheral opioid analgesia (Stein et al., 1988, 1989; Levine and Taiwo, 1989; Parsons et al., 1990; Kolesnikov et al., 1996; Perrot et al., 1999).
Despite the disadvantage that morphine is easily recognized because of its side effects, we included morphine as a positive control to allow for a comparison between peripheral and peripheral plus central analgesic opioid effects. Although morphine surely affected the blindness of both subjects and investigators, blindness was generally maintained during M6G treatment, i.e. during the critical part of the experiments that accounted for recording of the peripheral opioid effects. In addition, contradictory results of previous studies concerning analgesic effects of M6G and our own experience of lack of M6G analgesia with relatively low M6G doses (Lotsch et al., 1997) prevented our expectations being biased towards M6G analgesia. Therefore, the identification of morphine probably had no impact on the results with M6G.
Our results might contribute to the explanation for the contradictory outcomes of human experiments on the analgesic effects of M6G. At intravenous doses of M6G that cause plasma concentrations close to the amounts of M6G which result from metabolism of analgesic doses of morphine, M6G exerted analgesic effects on ischaemic pain (Thompson et al., 1995; Penson et al., 2000) and cancer pain (Osborne et al., 1992). In contrast, M6G had no analgesic effects on pain evoked by short pulses of gaseous CO2 (Lotsch et al., 1997). The inflammatory component of these different kinds of pain has not been assessed in the context of these studies. It may be hypothesized that the different studies investigated pain associated with different degrees of inflammation, and the higher the inflammatory component was, the more efficient was M6G.
The results of the present study suggest the clinical use of systemically administered opioids that do not cross the blood–brain barrier at relevant amounts as analgesics in inflammatory pain. Opioid-induced CNS effects such as sedation, nausea and respiratory depression could be minimized with such compounds. Importantly, it has been demonstrated that peripheral opioid analgesia is less sensitive to the development of opioid tolerance (Stein et al., 1996; Tokuyama et al., 1998) than central opioid analgesia, suggesting that in the long-term treatment of chronic inflammatory pain, such as chronic arthritis, peripheral opioid receptors may become important targets for pain control. However, in the rat tail flick assay, antinociceptive effects of locally injected morphine were much more affected by the development of opioid tolerance than its effects after supraspinal or systemic delivery (Kolesnikov et al., 1996). Hence, it has to be assessed whether rapid tolerance towards peripheral opioid analgesia occurs in humans and whether M6G qualifies as a peripheral opioid analgesic. While a single low dose of M6G might be useful in this regard, M6G can hardly be recommended as a ‘peripheral opioid analgesic’ because higher doses of M6G and repeated doses are likely to cause CNS effects similar to those of morphine. Particularly in patients with renal failure in whom M6G will accumulate, a contribution of M6G to opioid toxicity after morphine administration has been discussed (Angst et al., 2000; Lotsch and Geisslinger, 2001).
In summary, we provide evidence for antihyperalgesic effects of M6G on pain with an inflammatory component. These effects are statistically significant with M6G plasma concentrations that did not cause any significant pupil constriction, making the peripheral mechanism of opioid analgesia highly probable. As long as M6G does not reach the CNS, its effects on non-inflammatory pain are small. The lack of typical opioid-mediated side effects with the dose of M6G that was efficient to reverse hyperalgesia suggests that systemically administered opioids that do not enter the CNS might be useful as peripheral analgesics in inflammatory pain.
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Acknowledgements |
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This study was supported by the Deutsche Forschungs gemeinschaft (DFG) (GE 695/1-1 and Lo 612/3–1).
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  M. E. Durieux Peripheral analgesic receptor systems. Br. J. Anaesth., September 1, 2006; 97(3): 273 - 274. [Full Text] [PDF]
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