The Fabry disease-associated lipid Lyso-Gb3 enhances voltage-gated calcium currents in sensory neurons and causes pain.

Fabry disease is an X-linked lysosomal storage disorder characterised by accumulation of glycosphingolipids, and accompanied by clinical manifestations, such as cardiac disorders, renal failure, pain and peripheral neuropathy. Globotriaosylsphingosine (lyso-Gb3), a deacylated form of globotriaosylceramide (Gb3), has emerged as a marker of Fabry disease. We investigated the link between Gb3, lyso-Gb3 and pain. Plantar administration of lyso-Gb3 or Gb3 caused mechanical allodynia in healthy mice. In vitro application of 100nM lyso-Gb3 caused uptake of extracellular calcium in 10% of sensory neurons expressing nociceptor markers, rising to 40% of neurons at 1µM, a concentration that may occur in Fabry disease patients. Peak current densities of voltage-dependent Ca(2+) channels were substantially enhanced by application of 1µM lyso-Gb3. These studies suggest a direct role for lyso-Gb3 in the sensitisation of peripheral nociceptive neurons that may provide an opportunity for therapeutic intervention in the treatment of Fabry disease-associated pain.

Therapeutic potential of VEGF and VEGF-derived peptide in peripheral neuropathies.

Besides its prominent role in angiogenesis, the vascular endothelial growth factor (VEGF) also exerts important protective effects on neurons. In particular, mice expressing reduced levels of VEGF suffer from late-onset motor neuron degeneration, whereas VEGF delivery significantly delays motor neuron death in ALS mouse models, at least partly through neuroprotective effects. Additionally, VEGF protects dorsal root ganglion (DRG) neurons against paclitaxel-induced neurotoxicity. Here, we demonstrate that VEGF also protects DRG neurons against hyperglycemia-induced neuronal stress as a model of diabetes-induced peripheral neuropathy. Specifically, VEGF decreased expression of the stress-related gene activating transcription factor 3 (ATF3) in DRG neurons isolated from streptozotocin-induced diabetic mice (ex vivo) and in isolated DRG neurons exposed to high glucose concentrations (in vitro). In vivo, local VEGF application also protected against paclitaxel- and diabetes-induced neuropathies without causing side effects. A small synthetic VEGF mimicking pentadecapeptide (QK) exerted similar effects on DRG cultures: the peptide reduced ATF3 expression in vitro and ex vivo in paclitaxel- and hyperglycemia-induced models of neuropathy to a similar extent as the full-length recombinant VEGF protein. By using transgenic mice selectively overexpressing the VEGF receptor 2 in postnatal neurons, these neuroprotective effects were shown to be mediated through VEGF receptor 2. Overall, these results underscore the potential of VEGF and VEGF-derived peptides for the treatment of peripheral neuropathies.

LPA5 receptor plays a role in pain sensitivity, emotional exploration and reversal learning.

Lysophosphatidic acid (LPA) is a bioactive lipid acting on the nervous system through at least 6 different G protein-coupled receptors. In this study, we examined mice lacking the LPA5 receptor using an extensive battery of behavioral tests. LPA5-deficient mice showed decreased pain sensitivity in tail withdrawal, faster recovery in one inflammatory pain procedure (complete Freund's adjuvant-induced inflammation) and attenuated responses under specific neuropathic pain conditions. Notably, deletion of LPA5 also induced nocturnal hyperactivity and reduced anxiety in the mutant mice. Several exploratory tasks revealed signs of reduced anxiety in LPA5 knockout mice including increased visits to the arena center and reduced thigmotaxis in the open field, and more open arm entries in the elevated plus maze. Finally, LPA5 knockout mice also displayed marked reduction in social exploration, although several other tests indicated that these mice were able to respond normally to environmental stimuli. While learning and memory performance was not impaired in LPA5-deficient mice, we found differences, e.g., targeted swim strategy and reversal learning, as well as scheduled appetitive conditioning that might indicate differential motivational behavior. These results imply that LPA5 might be involved in both nociception and mechanisms of pain hypersensitivity, as well as in anxiety-related and motivational behaviors. These observations further support the proposed involvement of LPA signaling in psychopathology.

A longitudinal study of sensory biomarkers of progression in patients with diabetic peripheral neuropathy using skin biopsies.

We aimed to identify biomarkers in skin punch biopsies that could be used to monitor progression of diabetic peripheral neuropathy (DPN), and, in future studies, to assess the efficacy of agents that may reduce progression. Patients with DPN were studied with clinical assessments, skin biopsies, quantitative sensory testing (QST), histamine-induced skin flare, nerve conduction studies and contact heat-evoked potentials (CHEPS). Skin biopsies were performed on two visits with a 6 month interval (n=29 patients) to quantify intraepidermal (IENF) and subepidermal (SENF) nerve fibres immunoreactive for: protein gene product 9.5 (PGP9.5), a pan-neuronal marker; transient receptor potential cation channel vanilloid 1 (TRPV1), the heat and capsaicin receptor; and growth associated protein-43 (GAP-43), a marker of regenerating fibres. The IENF were counted along the length of four non-consecutive sections, and results were expressed as fibres per millimetre length of section. SENF were measured by image analysis, and the area of highlighted immunoreactivity was obtained as a percentage (% area) of the field scanned. QST, skin flare and CHEPS were also performed at the two visits. We found that IENF and SENF were significantly reduced for both PGP9.5 and TRPV1 between the first and second skin biopsy over 6months. The annual rate ± standard error of the mean of IENF loss was 3.76 ± 1.46 fibres/mm for PGP9.5, and 3.13 ± 0.58 fibres/mm for TRPV1. The other tests did not show significant changes. Strongly positive GAP-43 nerve fibres were found in deep dermis in the patients with diabetes, even in those with an absence of IENF. We conclude that PGP9.5 and TRPV1 IENF and SENF in skin biopsies are useful markers of progression in DPN, whereas GAP-43 SENF could potentially help detect nerve regeneration in severe neuropathy.

Mitochondrial abnormality in sensory, but not motor, axons in paclitaxel-evoked painful peripheral neuropathy in the rat.

The dose-limiting side effect of the anti-neoplastic agent, paclitaxel, is a chronic distal symmetrical peripheral neuropathy that produces sensory dysfunction (hypoesthesia and neuropathic pain) but little or no distal motor dysfunction. Similar peripheral neuropathies are seen with chemotherapeutics in the vinca alkaloid, platinum-complex, and proteasome inhibitor classes. Studies in rats suggest that the cause is a mitotoxic effect on axonal mitochondria. If so, then the absence of motor dysfunction may be due to mitotoxicity that affects sensory axons but spares motor axons. To investigate this, paclitaxel exposure levels in the dorsal root, ventral root, dorsal root ganglion, peripheral nerve, and spinal cord were measured, and the ultrastructure and the respiratory function of mitochondria in dorsal roots and ventral roots were compared. Sensory and motor axons in the roots and nerve had comparably low exposure to paclitaxel and exposure in the spinal cord was negligible. However, sensory neurons in the dorsal root ganglion had a very high and remarkably persistent (up to 10 days or more after the last injection) exposure to paclitaxel. Paclitaxel evoked a significant increase in the incidence of swollen and vacuolated mitochondria in the myelinated and unmyelinated sensory axons of the dorsal root (as seen previously in the peripheral nerve) but not in the motor axons of the ventral root. Stimulated mitochondrial respiration in the dorsal root was significantly depressed in paclitaxel-treated animals examined 2-4 weeks after the last injection, whereas respiration in the ventral root was normal. We conclude that the absence of motor dysfunction in paclitaxel-evoked peripheral neuropathy may be due to the absence of a mitotoxic effect in motor neuron axons, whereas the sensory dysfunction may be due to a mitotoxic effect resulting from the primary afferent neuron's cell body being exposed to high and persistent levels of paclitaxel.

A multiparametric assay for quantitative nerve regeneration evaluation.

We introduce an assay for the semi-automated quantification of nerve regeneration by image analysis. Digital images of histological sections of regenerated nerves are recorded using an automated inverted microscope and merged into high-resolution mosaic images representing the entire nerve. These are analysed by a dedicated image-processing package that computes nerve-specific features (e.g. nerve area, fibre count, myelinated area) and fibre-specific features (area, perimeter, myelin sheet thickness). The assay's performance and correlation of the automatically computed data with visually obtained data are determined on a set of 140 semithin sections from the distal part of a rat tibial nerve from four different experimental treatment groups (control, sham, sutured, cut) taken at seven different time points after surgery. Results show a high correlation between the manually and automatically derived data, and a high discriminative power towards treatment. Extra value is added by the large feature set. In conclusion, the assay is fast and offers data that currently can be obtained only by a combination of laborious and time-consuming tests.

Opioid tolerance and dependence: an inevitable consequence of chronic treatment?

Although opioids provide effective analgesia, largely unsubstantiated concerns about opioid-induced tolerance, physical dependence and addiction have limited their appropriate use. As a consequence, many patients receive inadequate treatment for both malignant and non-malignant pain. However, it has been shown that analgesic tolerance develops less frequently during chronic opioid administration in a clinical context than in animal experiments, and that instituting an appropriate dosing regimen can minimise withdrawal symptoms. Early studies had suggested that addiction might result from chronic opioid therapy, though more recent data indicate a low risk in patients with no history of drug abuse. New treatment regimens may also reduce the risk of tolerance, physical dependence and addiction. Long-acting preparations, such as transdermal fentanyl and possibly some forms of other slow release opioids, which maintain constant opioid concentrations in the plasma, minimise the occurrence of the 'between-dose' symptoms such as withdrawal and opioid-induced euphoria. This review discusses the development of tolerance, physical dependence and addiction during opioid therapy, and the influence of these factors on the choice of treatment.

Is the second phase of the formalin test useful to predict activity in chronic constriction injury models? A pharmacological comparison in different species.

This study presents data of several reference drugs in rats and gerbils for both the second phase of the formalin test and the cold allodynia in animals with a constriction injury of the sciatic nerve. A pharmacological validation of the formalin test and the CCI model in gerbils was performed. It was evaluated whether the second phase of the formalin test could be used as a pharmacological screening to predict outcome in the cold plate test in CCI animals. Male Sprague Dawley and Wistar rats and male gerbils were used for both tests. For the formalin test, animals were injected in the right hind paw (5% formalin rat: 0.05 microl; gerbil: 0.01 microl) and flinching and licking or biting were recorded. For CCI testing, a Bennett operation was performed on the left hind paw 7 days before testing. Cold plate allodynia was evaluated before and after drug treatment. In rats, a good correlation between both test conditions for morphine, fentanyl, MK-801 and flunarizine was found. Clonidine tends to have more activity in the second phase of the formalin test, whereas baclofen, tramadol, amitryptiline, ketamine and topiramate demonstrate to be more active in the cold plate. In gerbils, a good correlation between both test conditions for fentanyl and ketoprofen was found. Tramadol and CP-96345 tend to have more activity in the second phase of the formalin test, whereas morphine, SR-48968, SR-142801 and R116301 demonstrates to be more active in the cold plate test. In the present acute test conditions, there is a correlation in the pharmacological activity in rats and gerbils for the tested compounds a correlation between the second phase of the formalin test and the cold allodynia in CCI animals is found. Comparing to human data the screening drugs tested in this study show a correlation between animal and human studies in these specific circumstances. Further validation studies are needed to make these correlations clinical applicable.

Internal and external factors affecting the development of neuropathic pain in rodents. Is it all about pain?

It is important to know the factors that will influence animal models of neuropathic pain. A good reproducibility and predictability in different strains of animals for a given test increases the clinical relevance and possible targeting. An obligatory requirement for enabling comparisons of results of different origin is a meticulous definition of the specific sensitivities of a model for neuropathic pain and a description of the test conditions. Factors influencing neuropathic pain behavior can be subdivided in external and internal factors. The most important external factors are; timing of the measurement of pain after induction of neuropathy, circadian rhythms, seasonal influences, air humidity, influence of order of testing, diet, social variables, housing and manipulation, cage density, sexual activity, external stress factors, and influences of the experimenter. The internal factors are related to the type of animal, its genetic background, gender, age, and the presence of homeostatic adaptation mechanisms to specific situations or stress. In practice, the behavioral presentations to pain depend on the combination of genetic and environmental factors such as accepted social behavior. It also depends on the use of genetic manipulation of the animals such as in transgenic animals. These make the interpretation of data even more difficult. Differences of pain behavior between in- and outbred animals will be better understood by using modern analysis techniques. Substrains of animals with a high likelihood for developing neuropathic pain make the unraveling of specific pathophysiological mechanisms possible. Concerning the effect of stress on pain, it is important to differentiate between external and internal stress such as social coping behavior. The individual dealing with this stress is species sensitive, and depends on the genotype and the social learning. In the future, histo-immunological and genetic analysis will highlight similarities of the different pathophysiological mechanisms of pain between different species and human subjects. The final objective for the study of pain is to describe the genetics of the eliciting pain mechanisms in humans and to look for correlations with the knowledge from basic research. Therefore, it is necessary to know the genetic evolution of the different mechanisms in chronic pain. In order to be able to control the clinical predictability of a putative treatment the evolutionary pharmacogenomic structure of specific transmitters and receptors must be clarified.

Partial versus full agonists for opioid-mediated analgesia--focus on fentanyl and buprenorphine.

In contrast to other opioids, fentanyl and buprenorphine share a number of physicochemical properties that render both agents potentially suitable for transdermal delivery. However, there are significant differences between them in terms of their pharmacological profiles, as fentanyl is a full mu opioid receptor agonist capable of exerting a maximal response in certain tissues, while buprenorphine is a partial agonist unable to exert this maximum effect even at high doses. This review examines the hypothesis that partial opioid agonists would confer a number of benefits over full agonists, namely effective analgesia with a better tolerability and a lower propensity for addiction, with respect to fentanyl and buprenorphine. An attempt is also made to correlate clinical differences between these drugs with their respective agonist profiles and other differential pharmacokinetic/pharmacodynamic properties. Despite a dearth of directly comparative trials, the pharmacology of fentanyl and buprenorphine is well documented. Considerable data concerning buprenorphine suggest that the advantages initially espoused for partial opioid agonists are not borne out in clinical practice. Indeed, it may be postulated that full mu opioid agonists, particularly those with high selectivity and potency such as fentanyl, have a superior clinical profile and fulfill the above criteria more closely. Relative receptor binding, selectivity, potency and intrinsic efficacy of the opioids appear to be key determinants of their individual pharmacological profiles, contributing significantly to the heterogeneity of this class of analgesics.

Interactions of NMDA antagonists and an alpha 2 agonist with mu, delta and kappa opioids in an acute nociception assay.

Animal and clinical studies have reported potentiation of opioid antinociception by co-administration of alpha-2 adrenoceptor agonists such as clonidine and NMDA receptor antagonists such as ketamine and dextromethorphan. The aim of this study was to compare these clinically available compounds in combination with classical morphine, fentanyl-like opioids, the delta opioid agonist SNC80 and the kappa opioid agonist U50,488H. Using a mouse hot-plate test, dose-response relationships were first determined for all compounds individually and then for opioids co-administered with fixed doses of clonidine, ketamine or dextromethorphan. Clonidine was also evaluated in combination with ketamine and dextromethorphan. ED50 values were calculated from the proportion of animals reaching a fixed cut-off criterion of 30 s. To varying degrees, all compounds produced increases in response latencies over time. Dextromethorphan produced lower ED50 values for morphine, fentanyl and sufentanil but exerted no effect on SNC80 or U50,488H. Similarly, ketamine potentiated the antinociceptive efficacy of morphine and sufentanil but not SNC80 or U50,488H. By contrast, clonidine potentiated all opioids tested. In addition, the potency of clonidine was found to increase with co-administration of ketamine but not dextromethorphan. The strongest opioid sparing interactions occurred between clonidine and the lipophilic mu opioids fentanyl and sufentanil and the delta opioid SNC80. In summary, these results suggest an important role for lipophilic opioids in combination therapies particularly with clonidine as well as possible advantages of specific delta or kappa opioid combinations with alpha-2 agonists.

Role of 5-HT(2) receptors in the tryptamine-induced 5-HT syndrome in rats.

We distinguished the functions of the different 5-hydroxytryptamine-2 (5-HT(2)) receptor (5-HT(2)R) subtypes in the tryptamine-induced 5-HT syndrome in rats using (1) the 5-HT(2A)R antagonist R93274 (N-[(3-p-fluorophenyl-1-propyl)-4-methyl-4-piperidinyl]-4-amino-5-iodo-2-methoxybenzamide), the 5-HT(2A/C)R antagonist R99647 (2-(dimethylaminomethyl)2,3,3a,8-tetrahydrodibenzo[c,f]isoxazolo[2,3-a]azepine), the 5-HT(2B/C)R antagonist SB-242084 (6-chloro-5-methyl-1-[[2-[(2-methyl-3-pyridyl)oxy]-5-pyridyl]carbamoyl]-indoline), and several 5-HT(2)R antagonists (ketanserin, risperidone, pipamperone and mianserin); and (2) chronic 5-HT(2)R activation by 1-(2,5-dimethoxy-4-methylphenyl)-2-aminopropane (DOM). In contrast to SB-242084, the selective 5-HT(2A)R antagonist R93274 as well as the non-selective 5-HT(2A)R antagonists (R99647, ketanserin, risperidone, pipamperone and mianserin) significantly inhibited tryptamine-induced forepaw treading and tremors, and reversed peripherally mediated cyanosis into hyperaemia; only the 5-HT(2A/C)R antagonists R99647 and mianserin inhibited the tryptamine-induced hunched back. Intermittent DOM administration (intravenously every 48 h for 12 days) did not change the centrally mediated tryptamine-induced forepaw treading, tremors and hunched back at 1, 4 or 7 days after the last DOM pretreatment. The DOM-induced head twitch response, measured immediately after every DOM injection, was not affected. In contrast, peripherally mediated cyanosis was reversed into hyperaemia in 75, 11 and 20% of all pretreated rats at 1, 4 and 7 days, respectively, after the last DOM administration. Taken together, these finding suggest that central 5-HT(2A)Rs mediate tryptamine-induced forepaw treading and tremors, that peripheral 5-HT Rs mediate tryptamine-induced cyanosis, and that 5-HT(2A)Rs mediate tryptamine-induced hunched back. Peripheral 5-HT(2C)Rs are more sensitive to desensitization after intermittent treatment with an agonist than central 5-HT(2A)Rs.

Probing opioid receptor-ligand interactions by employment of indolizidin-9-one amino acid as a constrained Gly(2)-Gly(3) surrogate in a leucine-enkephalin mimic.

The relationship between the conformation and biological activity of Leu-enkephalin was studied using (2S,6R,8S)-9-oxo-8-N-(Boc)amino-1-azabicyclo[4.3.0]nonane-2-carboxylic acid [(2S,6R,8S)-1, I(9)AA] as a constrained Gly(2)-Gly(3) dipeptide surrogate. [I(9)AA](2,3)-Leu-enkephalin 12 was assembled using solid-phase peptide synthesis on Merrifield resin with TBTU as the coupling reagent. The in vitro assays indicated that [I(9)AA](2,3)-Leu-enkephalin 12 exhibited affinities for the mu- and delta-opioid receptors that were three orders of magnitude lower than that of Leu-enkephalin, as well as partial agonist character for both receptors. In in vivo assays for spinal analgesia, the indolizidinone analog 12 showed significantly enhanced duration of action, indicating an increased metabolic stability. Conformational analysis was performed using NMR and CD spectroscopy. The amide temperature coefficients and 3J(NH-CalphaH) coupling constants for 12 could not support a hydrogen-bonded beta-turn structure; however, its CD spectrum indicated a turn conformation. Incorporation of indolizidinone amino acid 1 into Leu-enkephalin thus provided additional support for the importance of a turn conformation for the biological activity of the native peptide.

Antagonistic effects of naloxone and naloxonazine on sufentanil-induced antinociception and respiratory depression in rats.

Several binding studies in rodent brain homogenates have revealed two distinct micro-opiate binding sites based on differences in binding affinity of several opiate peptides and opiate alkaloids. Naloxonazine (NLZ), which preferentially binds to the high affinity micro(1) sites, is often used to discriminate between pharmacological effects mediated by micro(1) and micro(2) binding sites. The present series of experiments were undertaken to compare the opioid antagonistic properties of naloxonazine and naloxone (NLX) (a non-selective micro(1)-antagonist) on intravenous (i.v.) and intrathecal (i.t.) sufentanil (SUF)-induced antinociception and respiratory depression. The opioid antagonists were given either intravenously at 5 min after SUF, or subcutaneously (s.c.) 24 h prior to the opioid. Intravenous NLX and NLZ reduced the i.v. and i. t. SUF-induced antinociception, hypercapnia and hypoxia when given directly after the opioid. There were no major differences in activity between both antagonists. Pretreatment with 30 mg/kg NLX did not reverse the i.v. or i.t. SUF-induced antinociception and respiratory depression. Subcutaneous pretreatment with doses up to 30 mg/kg NLX only partially antagonized the i.v. SUF-induced antinociception, while a complete reversal was present of the opioid-induced hypercapnia and hypoxia. With regard to i.t. SUF, doses up to 30 mg/kg NLZ were unable to reduce the antinociception. The respiratory depression was partially affected; with 30 mg/kg NLZ, the i.t. SUF-induced hypercapnia returned to baseline levels, whereas the SUF-induced hypoxia was only minimally affected. These results challenge the classical view of the selectivity of NLZ for the high affinity micro(1) binding sites. They further fail to conform an exclusive role for micro(2) receptor sites in the respiratory depression and spinal analgesia induced by a strong lipophilic opioid such as SUF in rats.

Inability of antipsychotics to antagonize the cueing properties of cocaine in rats.

In this study the possible antagonistic effects of five different antipsychotics on the discriminative stimulus properties of 10 mg/kg cocaine were evaluated by use of a two-lever food-reinforced drug discrimination procedure in rats. To do so, rats were treated with several doses of haloperidol, risperidone, seroquel, sertindole, and olanzapine, either at 60 or 120 min prior to testing. With all compounds tested, no substantial antagonism of the cocaine cue was observed. Only with haloperidol (maximum 60%), risperidone (maximal 20%), and olanzapine (maximal 20%) a partial antagonism without clearcut dose-response was observed. Clozapine, seroquel, and sertindole did not influence the discriminative stimulus properties of cocaine. These results indicate that antipsychotics with different pharmacological profiles are unable to antagonize more than partially the cueing properties of 10 mg/kg cocaine in rats, pointing to the unique underlying stimulus properties of this stimulant.

The effects of intravenous naltrindole and naltrindole 5'-isothiocyanate on sufentanil-induced respiratory depression and antinociception in rats.

Although the interactions between the mu- and the delta-opiate receptor subtypes are well documented with regard to supraspinal analgesia, less is known about the mutual interactions on respiratory depression. To clarify the functional interactions between both opiate receptor subtypes with regard to antinociception and respiratory depression, male Wistar rats were intravenously injected with 2.5 microg/kg of the mu-opiate agonist sufentanil and subsequently intravenously challenged with the delta antagonist naltrindole (NTI) or naltrindole 5'-isothiocyanate (5'-NTII), a delta-2 antagonist. Antinociception was measured by means of the tail-flick latency, and respiratory depression was evaluated by means of analysis of PaCO2, PaO2, and oxygen saturation. To quantify the antagonistic properties of NTI and 5'-NTII, mean areas under the curve were calculated for groups treated with sufentanil, control vehicle, and sufentanil plus a dose of the antagonists. NTI, but not 5'-NTII, antagonized the sufentanil-induced antinociception at 10 mg/kg NTI. Below this dose the effects were inconsistent. The sufentanil-induced hypercapnia and hypoxia were diminished with 10 mg/kg NTI or 5'-NTII. These data indicate that NTI antagonizes the sufentanil-induced antinociception and respiratory depression in rats. A dissociation between the antinociception and respiratory depression following intravenous sufentanil could be obtained with 10 mg/kg 5'-NTII pointing to different regulatory effects of opiate delta receptor subtypes on mu-opiate agonist-induced behavioral effects.

Comparison of the analgesic and intestinal effects of fentanyl and morphine in rats.

Clinical studies report a low incidence of intestinal side effects with transdermally administered fentanyl (TTS-fentanyl) in comparison with oral morphine. To support these clinical data, analgesic and intestinal effects of both opioids were compared in rats. After subcutaneous injection, analgesia in the tail withdrawal reaction test was obtained at a peak effect dose of 0.032 mg/kg with fentanyl and 8.0 mg/kg with morphine. This analgesic dose exceeded the ED50 for inhibition of castor oil-induced diarrhea only slightly (1.1 x) in the case of fentanyl (0.028 mg/kg) but markedly (36 x) in the case of morphine (0.22 mg/kg). To reverse completely the antidiarrheal effect of equivalent analgesic doses of the opioids (their ED50S for analgesia lasting 2 hours), much more naloxone was required in the case of morphine (5.4 mg/kg) than in the case of fentanyl (0.19 mg/kg). After oral administration, the difference between both opioids was less pronounced. Analgesia was obtained at 0.85 mg/kg with fentanyl and 32 mg/kg with morphine. This analgesic dose only slightly (1.7 x) exceeded the antidiarrheal dose in the case of fentanyl (0.49 mg/kg) but significantly (6.2 x) in the case of morphine (5.2 mg/ kg). To reverse completely the antidiarrheal effect of equivalent analgesic oral doses of the opioids (their ED50S for analgesia lasting 2 hours), more naloxone was required in the case of morphine (11 mg/kg) than in the case of fentanyl (2.0 mg/kg). Rapid penetration of fentanyl into the brain is thought to be responsible for small dissociation between the analgesic and intestinal effect of this lipophilic opioid. The present data provide preclinical evidence to support the relatively low incidence of intestinal side effects observed clinically with the use of TTS-fentanyl in comparison with orally administered morphine.

Effects of chlordiazepoxide on opioid-induced antinociception and respiratory depression in restrained rats.

This study investigates the influence of possible stress due to housing in Bolman cages on antinociception and on respiratory depression following opioid administration. To evaluate the functional role of this stressor and to modulate it, rats were subcutaneously pretreated with the anxiolytic chlordiazepoxide (CDP; 10 mg/kg) or saline (SAL) before the immobilization in the Bolman cages and before the intravenous administration of small doses of morphine (MOR), sufentanil (SUF), or vehicle (VEH). Antinociception, respiratory impairment and stress were evaluated by means of the tail-flick latency, blood gas analysis, and serum corticosterone (CS), adrenocorticotropic hormone (ACTH), and prolactin (PRL) determinations. The results demonstrated that 10 mg/kg CDP did not alter the antinociceptive effects of low doses of morphine and sufentanil. CDP pretreatment differentially affected the various blood gas parameters. Compared to vehicle pretreatment, there was a larger decrease in PaO2 following MOR and SUF in the CDP-pretreated rats. The effects were most pronounced at the lowest doses of both opioids. A CDP potentiation was also observed for the short-lasting raises in PaCO2 with the lowest concentrations of the opioids. At higher concentrations of the opioids, CDP was without any effect. With regard to the stress hormones, immobilization and an intravenous injection resulted in increases in CS and PRL in both CDP- and VEH-pretreated rats. ACTH did not change in these controls. SUF prevented the CS raises independent of a CDP pretreatment, while ACTH only increased in the SUF plus CDP groups, pointing to a stress-reducing effect of SUF. Also, MOR without CDP prevented the increases in CS, but the opioid intrinsically increased ACTH. These results indicate that restraint in Bolman cages in the present setup, with animals recovering for several hours in these cages after being equipped with an arterial catheter, is stressful but without any significant effect on the opioid-induced antinociception. Pretreatment with an anxiolytic benzodiazepine only minimally affected the outcome of the opioids on respiratory depression and pointed to a stress-reducing effect of low doses of the opioids, especially sufentanil.

Alpha 2-adrenoceptor agonists and stress-induced analgesia in rats: influence of stressors and methods of analysis.

The present experiments were designed to investigate the role of housing and handling conditions during testing, as well as data analysis, on the outcome of antinociceptive testing of alpha 2-adrenoceptor agonists, fentanyl, and a high dose of chlordiazepoxide in the tail withdrawal reaction test (TWR test) in rats. Dose-response curve data were obtained with fentanyl, clonidine, xylazine, dexmedetomidine, and 40.00 mg/kg chlordiazepoxide and were compared under normal TWR test conditions and during immobilization or immobilization with continuous painful stimulation. Data were analyzed in terms of all-or-none criteria as well as percentage maximum possible effect (%MPE) analysis over the total measurement period or at any specific time point during testing. The results indicate that stress, induced by immobilization and immobilization with long-term-applied paw pressure, unmasked possible antinociceptive properties of the various alpha 2-adrenoceptor agonists and potentiated the effects of fentanyl. Stress also unmasked the positive effects of benzodiazepines. The manner of data analysis was shown to significantly affect the outcome measured in stress and nonstress conditions. The MPE analysis, particularly at one time point, appeared much more sensitive than the all-or-none criteria. The data indicate that the housing and handling conditions of animals during testing, together with data analysis, may affect the outcome of different classes of compounds in the TWR test, and this knowledge may help control for false positive results.