Klaus Schiene

(–)-(1R,2R)-3-(3-Dimethylamino-1-ethyl-2-methyl-propyl)-phenol Hydrochloride (Tapentadol HCl): a Novel μ-Opioid Receptor Agonist/Norepinephrine Reuptake Inhibitor with Broad-Spectrum Analgesic Properties

(–)-(1R,2R)-3-(3-Dimethylamino-1-ethyl-2-methyl-propyl)-phenol hydrochloride (tapentadol HCl) is a novel μ-opioid receptor (MOR) agonist (Ki = 0.1 μM; relative efficacy compared with morphine 88% in a [35S]guanosine 5′-3-O-(thio)triphosphate binding assay) and NE reuptake inhibitor (Ki = 0.5 μM for synaptosomal reuptake inhibition). In vivo intracerebral microdialysis showed that tapentadol, in contrast to morphine, produces large increases in extracellular levels of NE (+450% at 10 mg/kg i.p.). Tapentadol exhibited analgesic effects in a wide range of animal models of acute and chronic pain [hot plate, tail-flick, writhing, Randall-Selitto, mustard oil colitis, chronic constriction injury (CCI), and spinal nerve ligation (SNL)], with ED50 values ranging from 8.2 to 13 mg/kg after i.p. administration in rats. Despite a 50-fold lower binding affinity to MOR, the analgesic potency of tapentadol was only two to three times lower than that of morphine, suggesting that the dual mode of action of tapentadol may result in an opiate-sparing effect. A role of NE in the analgesic efficacy of tapentadol was directly demonstrated in the SNL model, where the analgesic effect of tapentadol was strongly reduced by the α2-adrenoceptor antagonist yohimbine but only moderately attenuated by the MOR antagonist naloxone, whereas the opposite was seen for morphine. Tolerance development to the analgesic effect of tapentadol in the CCI model was twice as slow as that of morphine. It is suggested that the broad analgesic profile of tapentadol and its relative resistance to tolerance development may be due to a dual mode of action consisting of both MOR activation and NE reuptake inhibition.

Cebranopadol: a Novel Potent Analgesic Nociceptin/Orphanin FQ Peptide and Opioid Receptor Agonist

Cebranopadol (trans-6′-fluoro-4′,9′-dihydro-N,N-dimethyl-4-phenyl-spiro[cyclohexane-1,1′(3′H)-pyrano[3,4-b]indol]-4-amine) is a novel analgesic nociceptin/orphanin FQ peptide (NOP) and opioid receptor agonist [Ki (nM)/EC50 (nM)/relative efficacy (%): human NOP receptor 0.9/13.0/89; human mu-opioid peptide (MOP) receptor 0.7/1.2/104; human kappa-opioid peptide receptor 2.6/17/67; human delta-opioid peptide receptor 18/110/105]. Cebranopadol exhibits highly potent and efficacious antinociceptive and antihypersensitive effects in several rat models of acute and chronic pain (tail-flick, rheumatoid arthritis, bone cancer, spinal nerve ligation, diabetic neuropathy) with ED50 values of 0.5−5.6 µg/kg after intravenous and 25.1 µg/kg after oral administration. In comparison with selective MOP receptor agonists, cebranopadol was more potent in models of chronic neuropathic than acute nociceptive pain. Cebranopadol’s duration of action is long (up to 7 hours after intravenous 12 µg/kg; >9 hours after oral 55 µg/kg in the rat tail-flick test). The antihypersensitive activity of cebranopadol in the spinal nerve ligation model was partially reversed by pretreatment with the selective NOP receptor antagonist J-113397[1-[(3R,4R)-1-cyclooctylmethyl-3-hydroxymethyl-4-piperidyl]-3-ethyl-1,3-dihydro-2H-benzimidazol-2-one] or the opioid receptor antagonist naloxone, indicating that both NOP and opioid receptor agonism are involved in this activity. Development of analgesic tolerance in the chronic constriction injury model was clearly delayed compared with that from an equianalgesic dose of morphine (complete tolerance on day 26 versus day 11, respectively). Unlike morphine, cebranopadol did not disrupt motor coordination and respiration at doses within and exceeding the analgesic dose range. Cebranopadol, by its combination of agonism at NOP and opioid receptors, affords highly potent and efficacious analgesia in various pain models with a favorable side effect profile.

Antinociceptive effect of antisense oligonucleotides against the vanilloid receptor VR1/TRPV1

To examine the role of the vanilloid receptor TRPV1 in neuropathic pain, we assessed the effects of the receptor antagonist thioxo-BCTC and antisense oligonucleotides against the TRPV1 mRNA in a rat model of spinal nerve ligation. In order to identify accessible sites on the mRNA of TRPV1, the RNase H assay was used, leading to the successful identification of binding sites for antisense oligonucleotides. Cotransfection studies using Cos-7 cells were employed to identify the most effective antisense oligonucleotide efficiently inhibiting the expression of a fusion protein consisting of TRPV1 and the green fluorescent protein in a specific and concentration-dependent manner. In an in vivo rat model of spinal nerve ligation, intravenous application of the TRPV1 antagonist thioxo-BCTC reduced mechanical hypersensitivity yielding an ED(50) value of 10.6mg/kg. Intrathecal administration of the antisense oligonucleotide against TRPV1, but not the mismatch oligonucleotide or a vehicle control, reduced mechanical hypersensitivity in rats with spinal nerve ligation in a similar manner. Immunohistochemical analysis revealed neuropathy- and antisense-associated regulation of TRPV1 protein expression in spinal cord and dorsal root ganglia. Our data demonstrate comparative analgesic effects of a TRPV1 anatagonist and a rationally designed TRPV1 antisense oligonucleotide in a spinal nerve ligation model of neuropathic pain and thus, lend support to the validation of TRPV1 as a promising target for the treatment of neuropathic pain.

Investigation of TRPV1 loss-of-function phenotypes in transgenic shRNA expressing and knockout mice

The function of the transient receptor potential vanilloid 1 (TRPV1) cation channel was analyzed with RNA interference technologies and compared to TRPV1 knockout mice. Expression of shRNAs targeting TRPV1 in transgenic (tg) mice was proven by RNase protection assays, and TRPV1 downregulation was confirmed by reduced expression of TRPV1 mRNA and lack of receptor agonist binding in spinal cord membranes. Unexpectedly, TRPV3 mRNA expression was upregulated in shRNAtg but downregulated in knockout mice. Capsaicin-induced [Ca(2+)](i) changes in small diameter DRG neurons were significantly diminished in TRPV1 shRNAtg mice, and administration of capsaicin hardly induced hypothermia or nocifensive behaviour in vivo. Likewise, sensitivity towards noxious heat was reduced. Interestingly, spinal nerve injured TRPV1 knockout but not shRNAtg animals developed mechanical allodynia and hypersensitivity. The present study provides further evidence for the relevance of TRPV1 in neuropathic pain and characterizes RNA interference as valuable technique for drug target validation in pain research.

Antinociceptive and Antihyperalgesic Effects of Tapentadol in Animal Models of Inflammatory Pain

The novel analgesic tapentadol HCl [(−)-(1R,2R)-3-(3-dimethylamino)-1-ethyl-2-methyl-propyl)-phenol hydrochloride] combines μ-opioid receptor (MOR) agonism and noradrenaline reuptake inhibition (NRI) in a single molecule and shows a broad efficacy profile in various preclinical pain models. This study analyzed the analgesic activity of tapentadol in experimental inflammatory pain. Analgesia was evaluated in the formalin test (pain behavior, rat and mouse), carrageenan-induced mechanical hyperalgesia (paw-pressure test, rat), complete Freunds adjuvant (CFA)-induced paw inflammation (tactile hyperalgesia, rat), and CFA knee-joint arthritis (weight bearing, rat). Tapentadol showed antinociceptive activity in the rat and mouse formalin test with an efficacy of 88 and 86% and ED50 values of 9.7 and 11.3 mg/kg i.p., respectively. Tapentadol reduced mechanical hyperalgesia in carrageenan-induced acute inflammatory pain by 84% with an ED50 of 1.9 mg/kg i.v. In CFA-induced tactile hyperalgesia, tapentadol showed 71% efficacy with an ED50 of 9.8 mg/kg i.p. The decrease in weight bearing after CFA injection in one knee joint was reversed by tapentadol by 51% with an ED25 of 0.9 mg/kg i.v. Antagonism studies were performed with the MOR antagonist naloxone and the α2-noradrenergic receptor antagonist yohimbine in the carrageenan- and CFA-induced hyperalgesia model. In the CFA model, the serotonergic receptor antagonist ritanserin was also tested. The effect of tapentadol was partially blocked by naloxone and yohimbine and completely blocked by the combination of both, but it was not affected by ritanserin. In summary, tapentadol showed antinococeptive/antihyperalgesic analgesic activity in each model of acute and chronic inflammatory pain, and the antagonism experiments suggest that both MOR activation and NRI contribute to its analgesic effects.

The antiallodynic effect of NMDA antagonists in neuropathic pain outlasts the duration of the in vivo NMDA antagonism

Clinical reports have described a long-lasting relief in neuropathic pain patients treated with NMDA receptor antagonists; it is unclear, however, what mediates this effect. In this work, we have used two NMDA antagonists of different class to investigate if the antiallodynic effects in a rat neuropathy model can outlast their in vivo NMDA antagonism. Both the uncompetitive NMDA antagonist ketamine and the glycine(B) antagonist MRZ 2/576 inhibited neuronal responses to iontophoretic NMDA in anaesthetised rats with the time course consistent with their known pharmacokinetics (t(1/2) approximately 10-12min, similar in control and nerve-injured rats). Surprisingly, the antiallodynic effects of the same doses of the NMDA antagonists in the neuropathic pain model were long-lasting (>3h with ketamine, >24h with MRZ 2/576). The effect of ketamine was further prolonged (>24h) when combined with a short-acting opioid, fentanyl, which only produced a short effect ( approximately 40min) when given alone. The duration of centrally mediated side effects of ketamine and MRZ 2/576 was short, similar to the in vivo NMDA antagonism. We speculate that NMDA receptor blockade may down-regulate the central sensitisation triggered by nerve injury, resulting in a long-lasting antiallodynic effect. Development of short-acting NMDA antagonists could represent a strategy for improving their tolerability.

Synergistic antihypersensitive effects of pregabalin and tapentadol in a rat model of neuropathic pain

Neuropathic pain is a clinical condition which remains poorly treated and combinations of pregabalin, an antagonist of the α2δ-subunit of Ca(2+) channels, with tapentadol, a μ-opioid receptor agonist/noradrenaline reuptake inhibitor, or with classical opioids such as oxycodone and morphine might offer increased therapeutic potential. In the rat spinal nerve ligation model, a dose dependent increase in ipsilateral paw withdrawal thresholds was obtained using an electronic von Frey filament after IV administration of pregabalin (1-10mg/kg), tapentadol (0.316-10mg/kg), morphine (1-4.64 mg/kg) and oxycodone (0.316-3.16 mg/kg), with ED(50) values (maximal efficacy) of 4.21 (67%), 1.65 (94%), 1.70 (96%) and 0.63 mg/kg (100%), respectively. Equianalgesic dose combinations of pregabalin and tapentadol (dose ratio 2.5:1), morphine (2.5:1) or oxycodone (6.5:1) resulted in ED(50) values (maximal efficacy) of 0.83 (89%), 2.33 (97%) and 1.14 mg/kg (100%), respectively. The concept of dose-equivalence suggested an additive interaction of pregabalin and either oxycodone or morphine, while a synergistic interaction was obtained with pregabalin and tapentadol (demonstrated by isobolographic analysis). There was no increase in contralateral paw withdrawal thresholds and no locomotor impairment, as measured in the open field, for the combination of pregabalin and tapentadol; while a significant increase and impairment was demonstrated for the combinations of pregabalin and either morphine or oxycodone. Because combination of pregabalin and tapentadol resulted in a synergistic antihypersensitive activity, it is suggested that, beside the use of either drug alone, this drug combination may offer a beneficial treatment option for neuropathic pain.

Differential effects of morphine on the affective and the sensory component of carrageenan-induced nociception in the rat

&NA; Pain is generally considered to have a sensory and an affective component. Clinical research has suggested that morphine more potently attenuates the affective component as compared to the sensory component. Because preclinical nociception models typically focus on the sensory component of nociception, and do not assess the affective component, it is unclear whether this potency difference of morphine can also be found in preclinical models. We therefore adapted the place conditioning paradigm to investigate negative affect accompanying carrageenan‐induced (0.5% intraplantar) inflammatory nociception in rats. We found that carrageenan produced clear conditioned place aversion (CPA). Morphine (0.01–10 mg/kg i.p.) dose‐dependently reduced carrageenan‐induced CPA with a minimal effective dose (MED) of 0.03 mg/kg. Since morphine has a rewarding effect by itself, morphine‐induced conditioned place preference (CPP) was also investigated. Morphine induced CPP with a MED of 1 mg/kg, suggesting that the rewarding effect of morphine was not responsible for reducing carrageenan‐induced CPA. We also demonstrated that morphine reduced carrageenan‐induced mechanical nociception as assessed in the Randall Selitto paradigm with a MED of 1 mg/kg. It is concluded that the CPA model allows for an assessment of the negative affective component of carrageenan‐induced nociception. Moreover, morphine was able to reduce the affective component of nociception at doses that did not affect the sensory component of nociception, and this effect was not due to its rewarding properties. The fact that this finding mirrors the clinical situation validates the use of the CPA model for assessing the affective component of nociception.

Burrowing as a non‐reflex behavioural readout for analgesic action in a rat model of sub‐chronic knee joint inflammation

Innate responses against spontaneous pain are proposed to improve the predictive validity of preclinical analgesia models. Therefore, development and validation of novel readouts is necessary. To investigate whether innate rodent burrowing is a useful alternative behavioural readout for assessment of analgesic efficacy, a complete Freunds adjuvant (CFA)‐induced model of sub‐chronic inflammation was used to compare the effects of naproxen, ibuprofen and pregabalin in weight‐bearing (WB), open‐field (OF) and burrowing assays.

2-(3-fluoro-4-methylsulfonylaminophenyl)propanamides as potent transient receptor potential vanilloid 1 (TRPV1) antagonists: structure-activity relationships of 2-amino derivatives in the N-(6-trifluoromethylpyridin-3-ylmethyl) C-region.

A series of N-(2-amino-6-trifluoromethylpyridin-3-ylmethyl)-2-(3-fluoro-4-methylsulfonylaminophenyl)propanamides were designed combining previously identified pharmacophoric elements and evaluated as hTRPV1 antagonists. The SAR analysis indicated that specific hydrophobic interactions of the 2-amino substituents in the C-region of the ligand were critical for high hTRPV1 binding potency. In particular, compound 49S was an excellent TRPV1 antagonist (K(i(CAP)) = 0.2 nM; IC(50(pH)) = 6.3 nM) and was thus approximately 100- and 20-fold more potent, respectively, than the parent compounds 2 and 3 for capsaicin antagonism. Furthermore, it demonstrated strong analgesic activity in the rat neuropathic model superior to 2 with almost no side effects. Compound 49S antagonized capsaicin induced hypothermia in mice but showed TRPV1-related hyperthermia. The basis for the high potency of 49S compared to 2 is suggested by docking analysis with our hTRPV1 homology model in which the 4-methylpiperidinyl group in the C-region of 49S made additional hydrophobic interactions with the hydrophobic region.