Safety of tapentadol compared with other opioids in chronic pain treatment: network meta-analysis of randomized controlled and withdrawal trials.

OBJECTIVE: To assess the relative safety of oral tapentadol PR and other opioid analgesics for moderate or severe chronic pain in adults, we conducted a systematic review and network meta-analysis (NMA). METHODS: A systematic review was conducted to identify randomized controlled trials (RCTs) and randomized withdrawal trials of tapentadol with other WHO stage II and III opioid analgesics in patients with moderate or severe chronic pain. Searches were conducted in MEDLINE, EMBASE, PubMed, Cochrane databases and trial registries. Feasibility assessment evaluated the trials' suitability for NMA. Outcomes assessed were overall AEs, overall serious adverse events, constipation, nausea, dizziness, somnolence, headache, and discontinuation due to AEs. Randomized withdrawal trials were analyzed separately to other RCTs. RESULTS: Searches conducted in April 2019 identified 16,604 records. Following screening and feasibility assessment, 29 RCTs and 19 randomized withdrawal trials were identified and included in the NMA. Consistent with existing research, evidence from RCTs suggested that tapentadol is associated with relatively lower odds of adverse events occurring than most active comparators. The withdrawal trial data were less clear, with higher uncertainty around the results, and results that appear to contradict the RCT evidence. There are a number of trial design factors that may be affecting these results. CONCLUSIONS: RCT evidence suggests that tapentadol can be a useful treatment option for patients suffering from chronic pain and in need of an opioid analgesic. Opioids should be prescribed by a qualified physician only after other analgesics have been considered, taking side effects and misuse risk into account.

Leukemic spleen cells are more potent than bone marrow-derived cells in a transgenic mouse model of CML.

Spleen size ranks among the most important risk factors in chronic myeloid leukemia (CML), but the pathogenic mechanisms of splenic hematopoiesis in CML remain poorly defined. Here, we studied the biology of Bcr-Abl positive leukemia-initiating cells in the spleen, using an inducible transgenic mouse model of CML. Disease kinetics showed greater increases of immature leukemic cells in spleen vs bone marrow (BM). To assess how Bcr-Abl alters the behavior of spleen-derived CML cells, we transplanted these cells either before ('pre-uninduced') or 44 days after ('pre-induced') expression of the oncogene. Mice transplanted with pre-induced spleen cells showed significantly increased neutrophilia and splenomegaly compared with mice receiving pre-uninduced spleen cells, suggesting that Bcr-Abl expression in the donors had increased splenic tumor burden. However, pre-induction also altered the biology of these cells, as shown by a striking increase in erythropoietic potential. These results differ from those of BM-derived CML stem cells where pre-induction of Bcr-Abl had previously been shown to decrease disease transplantability. Moreover, splenic cells were less sensitive to imatinib than BM cells. In conclusion, Bcr-Abl alters the biology of splenic leukemic stem cells by a cell-autonomous mechanism, but the disease phenotype is also influenced by the microenvironment of these cells.

Novel method for the study of receptor Ca2+ signalling exemplified by the NK1 receptor.

We have used a novel technology (NovoStar from BMG Labtechnologies) for the study of the Ca2+ signalling of the human tackykinin NK1 (hNK-I receptor). The NovoStar is a microplate reader based on fluorescence and luminescence. The instrument implements a robotic pipettor arm and two microplate carriers, typically one for samples and one for cells. The robotic pipettor arm can transfer sample (agonist or antagonist) from the sample plate or other liquid containers to the cell plate, facilitating the study of Ca2+ signalling to such a degree that the instrument can be used for Medium Throughput Screening (MTS). Using the NovoStar we have found the molecular pharmacology of the NK1 receptor to be comparable to that observed in classical signal transduction assays. Thus, we have observed an EC50 value of 3 nM for substance P induced Ca2+ response. This value corresponds well with previously published values for substance P induced IP and cAMP turnover. [1] Using the NovoStar technology we have studied the pharmacological profile of the well known non-peptide NKI receptor antagonists CP96,345 and SR140,333 [2,3] in respect of inhibition of the Ca2+ response induced by substance P. Interestingly, the antagonistic potency of the antagonists depended greatly on the experimental design, e.g., a dependency of timing in the addition of antagonists vs. agonist was noted. Also, metal-ion site engineered NK1 receptors [2] were tested for the ability of metal-ions to inhibit signalling. It is concluded that the NovoStar is a reliable tool for the study of receptor Ca2+ signalling, both as a research tool and as a MTS system.

Partial agonism through a zinc-Ion switch constructed between transmembrane domains III and VII in the tachykinin NK(1) receptor.

Partly due to lack of detailed knowledge of the molecular recognition of ligands the structural basis for partial versus full agonism is not known. In the beta(2)-adrenergic receptor the agonist binding site has previously been structurally and functionally exchanged with an activating metal-ion site located between AspIII:08-or a His residue introduced at this position in transmembrane domain (TM)-III-and a Cys residue substituted for AsnVII:06 in TM-VII. Here, this interhelical, bidentate metal-ion site is without loss of Zn(2+) affinity transferred to the tachykinin NK(1) receptor. In contrast to the similarly mutated beta(2)-adrenergic receptor, signal transduction-i.e., inositol phosphate turnover-could be stimulated by both Zn(2+) and by the natural agonist, Substance P in the mutated NK(1) receptor. The metal-ion acted as a 25% partial agonist through binding to the bidentate zinc switch located exactly one helical turn below the two previously identified interaction points for Substance P in, respectively, TM-III and -VII. The metal-ion chelator, phenantroline, which in the beta(2)-adrenergic receptor increased both the potency and the agonistic efficacy of Zn(2+) or Cu(2+) in complex with the chelator, also bound to the metal-ion site-engineered NK(1) receptor, but here the metal-ion chelator complex instead acted as a pure antagonist. It is concluded that signaling of even distantly related rhodopsin-like 7TM receptors can be activated through Zn(2+) coordination between metal-ion binding residues located at positions III:08 and VII:06. It is suggested that only partial agonism is obtained through this simple well defined metal-ion coordination due to lack of proper interactions with residues also in TM-VI.

Disulfide bridge engineering in the tachykinin NK1 receptor.

As in most other seven-transmembrane receptors, the central disulfide bridge from the extracellular end of TM-III to the middle of the second extracellular loop was essential for ligand binding in the NK1 receptor. However, introduction of "extra", single Cys residues in the second extracellular loop, at positions where disease-associated Cys substitutions impair receptor function in the vasopressin V2 receptor and in rhodopsin, did not cause mispairing with the Cys residues involved in this central disulfide bridge. Cys residues were introduced in the N-terminal extension and in the third extracellular loop, respectively, in such a way that disulfide bridge formation could be monitored by loss of substance P binding and breakage of the bridge could be monitored by gain of ligand binding. This disulfide bridge formed spontaneously in the whole population of receptors and could be titrated with low concentrations of reducing agent, dithiothreitol. Another putative disulfide bridge "switch" was constructed at the extracellular ends of TM-V and -VI, i.e., at positions where a high-affinity zinc site previously had been constructed with His substitutions. Disulfide bridge formation at this position, monitored by loss of binding of the nonpeptide antagonist [3H]LY303.870, occurred spontaneously only in a small fraction of the receptors. It is concluded that disulfide bridges form readily between Cys residues introduced appropriately in the N-terminal extension and the third extracellular loop, whereas they form with more difficulty between Cys residues placed at the extracellular ends of the transmembrane segments even at positions where high-affinity metal ion sites can be constructed with His residues.

Conversion of agonist site to metal-ion chelator site in the beta(2)-adrenergic receptor.

Previously metal-ion sites have been used as structural and functional probes in seven transmembrane receptors (7TM), but as yet all the engineered sites have been inactivating. Based on presumed agonist interaction points in transmembrane III (TM-III) and -VII of the beta(2)-adrenergic receptor, in this paper we construct an activating metal-ion site between the amine-binding Asp-113 in TM-III-or a His residue introduced at this position-and a Cys residue substituted for Asn-312 in TM-VII. No increase in constitutive activity was observed in the mutant receptors. Signal transduction was activated in the mutant receptors not by normal catecholamine ligands but instead either by free zinc ions or by zinc or copper ions in complex with small hydrophobic metal-ion chelators. Chelation of the metal ions by small hydrophobic chelators such as phenanthroline or bipyridine protected the cells from the toxic effect of, for example Cu(2+), and in several cases increased the affinity of the ions for the agonistic site. Wash-out experiments and structure-activity analysis indicated, that the high-affinity chelators and the metal ions bind and activate the mutant receptor as metal ion guided ligand complexes. Because of the well-understood binding geometry of the small metal ions, an important distance constraint has here been imposed between TM-III and -VII in the active, signaling conformation of 7TM receptors. It is suggested that atoxic metal-ion chelator complexes could possibly in the future be used as generic, pharmacologic tools to switch 7TM receptors with engineered metal-ion sites on or off at will.

Split-receptors in the tachykinin neurokinin-1 system--mutational analysis of intracellular loop 3.

Several membrane proteins have been functionally expressed from non-covalently coupled, contiguous segments especially with the split-site located between natural domains. Experiments using such 'split-proteins' were here performed in the tachykinin neurokinin-1 (NK1) receptor with co-expression of contiguous segments with split-sites positioned in various intracellular and extracellular loops. The construct where the split-site was located in intracellular loop 3 gave a reasonable expression level of substance-P-binding sites, i.e. 12% of wild-type expression. Of the other split-receptors tested, only the one with the split-site located just outside transmembrane (TM) segment-V gave any detectable substance P binding, which however only was 1% of the wild-type expression level. The construct with the split-site located in intracellular loop 3 bound all of the tested peptide agonists and non-peptide antagonists with normal affinity and was able to stimulate inositol phosphate turnover with a normal EC50 for substance P and an Emax according to the expression level. When intracellular loop 3 was either extended with 112 amino acid residues derived from the muscarine M2 receptor or, when major parts of the loop were deleted in the non-split NK1 receptor, the affinity for neither substance P nor for the prototype nonpeptide antagonist, CP96,345 was affected, yet an increase in EC50 for substance P was observed. Also in the split-receptor, most of intracellular loop 3 could be substituted or even deleted without affecting ligand affinity, although a decreased expression level was observed in constructs having major deletions. It is concluded, that the NK1 receptor is preferentially reconstituted by co-expression of a putative A-domain including TM-I-V and a B-domain including TM-VI and -VII. It is suggested that a number of rhodopsin-like 7TM receptors may function as two-domain structures based on the finding that a network of short loops has been highly conserved within each of the putative domains and, that these domains are separated by a relatively long and in respect of length poorly conserved loop, i.e. intracellular loop 3.

Steric hindrance mutagenesis versus alanine scan in mapping of ligand binding sites in the tachykinin NK1 receptor.

Residues in transmembrane domain (TM)-III, TM-V, TM-VI, and TM-VII believed to be facing the deep part of the presumed main ligand-binding pocket of the NK1 receptor were probed by alanine substitution and introduction of residues with larger and/or chemically distinct side chains. Unaltered or even improved binding affinity for four peptide agonists, substance P, substance P-O-methyl ester, eledoisin, and neurokinin A, as well as normal EC50 values for substance P in stimulating phosphatidylinositol turnover indicated that these mutations did not alter the overall functional integrity of the receptor. The alanine substitutions in general had only minor effects on nonpeptide antagonist binding. However, the introduction of the larger and polar aspartic acid and histidine residues at positions corresponding to the monoamine binding aspartic acid in TM-III of the beta 2-adrenoceptor (ProIII:08, Pro112 in the NK1 receptor) and to the presumed monoamine interacting "two serines" in TM-V (ThrV:09, Thr201; and IleV:12, Ile204) impaired by > 100-fold the binding of a group of nonpeptide antagonists, including CP96,345, CP99,994, RP67,580, RPR100,893, and CAM4092. In contrast, another group of nonpeptide antagonists, LY303,870, FK888, and SR140,333, were little or not at all affected by the space-filling substitutions. Two of these compounds, FK888 and LY303,870, were those most seriously affected (75-89-fold) by alanine substitution of PheVI:20 located in the upper part of the main ligand-binding crevice. Surprisingly, substitution of AlaIII:11 (Ala115), which is located in the middle of TM-III, conceivably pointing toward TM-VII, with a larger valine residue increased the affinity for all 13 ligands tested, presumably by creating a closer interhelical packing. It is concluded that the introduction of larger side chains at positions at which molecular models indicate that this is structurally allowed can be a powerful method of locating ligand-binding sites due to the considerable difference between positive and negative results. Such steric hindrance mutagenesis strongly indicates that one population of nonpeptide antagonists bind in the deep pocket of the main ligand-binding crevice of the NK1 receptor, whereas another group of nonpeptide antagonists, especially SR140,333, was surprisingly resistant to mutational mapping in this pocket.

Engineering of metal-ion sites as distance constraints in structural and functional analysis of 7TM receptors.

G-protein-coupled receptors with their seven transmembrane (7TM) segments constitute the largest superfamily of proteins known. Unfortunately, still only relatively low resolution structures derived from electron cryo-microscopy analysis of 2D crystals are available for these proteins. We have used artificially designed Zn(II) metal-ion binding sites to probe 7TM receptors structurally and functionally and to define some basic distance constraints for molecular modeling. In this way, the relative helical rotation and vertical translocation of transmembrane helices TM-II, TM-III, TM-V, and TM-VI of the tachykinin NK-1 receptor have been restricted. Collectively, these zinc sites constitute a basic network of distance constraints that limit the degrees of freedom of the interhelical contact faces in molecular models of 7TM receptors. The construction of artificially designed metal-ion sites is discussed also in the context of probes for conformational changes occurring during receptor activation.

Connectivity and orientation of the seven helical bundle in the tachykinin NK-1 receptor probed by zinc site engineering.

A high affinity, tridentate metal ion site has been constructed previously by His substitutions in an antagonist binding site located between transmembrane segment (TM)-V and TM-VI in the substance P NK-1 receptor. Here, an attempt is made to probe helix-helix interactions systematically in the NK-1 receptor by engineering of bis-His Zn(II) sites. His residues were introduced at selected positions individually and in combinations in the exterior segments of TM-II, III and V in both the wild-type background and after Ala substitution of naturally occurring His residues, and the increase in the affinity for Zn(II) was monitored in competition binding experiments with iodinated substance P or a tritiated non-peptide antagonist. In this way, two high affinity bis-His sites were constructed between position 193 in TM-V (Glu193, G1uV:01) and position 109 in TM-III (Asn1O9, AsnIII:05) as well as between the neighboring, naturally occurring His108 in TM-III (HisIII:04) and position 92 in TM-II (Tyr92, TyrII:24), respectively. Functionally, the coordination of zinc ions at these two sites blocked the receptor as it antagonized the substance P-induced increase in phosphatidylinositol turnover. It is concluded that the bis-His zinc sites from the central TM-III helix to TM-II and -V, respectively, together with the interconnected, previously constructed tridentate site between TM-V and -VI, constitute a basic network of distance constraints for the molecular models of receptors with seven transmembrane segments which, for example, strongly support an anti-clockwise orientation of the seven helical bundle as viewed from the extracellular space.

Construction of a high affinity zinc switch in the kappa-opioid receptor.

Very limited structural information is available concerning the superfamily of G-protein-coupled receptors with their seven-transmembrane segments. Recently a non-peptide antagonist site was structurally and functionally replaced by a metal ion site in the tachykinin NK-1 receptor. Here, this Zn(II) site is transferred to the kappa-opioid receptor by substituting two residues at the outer portion of transmembrane V (TM-V), Asp223 and Lys227, and one residue at the top of TM-VI, Ala298, with histidyl residues. The histidyl residues had no direct effect on the binding of either the non-peptide antagonist [3H]diprenorphine or the non-peptide agonist, [3H]CI977, just as these mutations/substitutions did not affect the apparent affinity of a series of other peptide and non-peptide ligands when tested in competition binding experiments. However, zinc ions in a dose-dependent manner prevented binding of both agonist and antagonist ligands with an apparent affinity for the metal ion, which gradually was built up to 10(-6) M. This represents an increase in affinity for the metal ion of about 1000-fold as compared with the wild-type kappa receptor and is specific for Zn(II) as the affinity for e.g. Cu(II) was almost unaffected. The direct transfer of this high affinity metal ion switch between two only distantly related receptors indicates a common overall arrangement of the seven-helix bundle among receptors of the rhodopsin family.

Conversion of antagonist-binding site to metal-ion site in the tachykinin NK-1 receptor.

Mutational analysis of the tachykinin NK-1 (refs 1-7), NK-2 (ref. 8) and angiotensin AT-1 (refs 9, 10) receptors indicates that non-peptide antagonists act through residues located between the seven transmembrane segments, whereas natural peptide agonists bind mainly to residues scattered in the exterior part of the receptor. The presumed contact points for the prototype NK-1 antagonist CP96,345 cluster on opposing faces of the outer portions of transmembrane helices V and VI (refs 1-5). Here we show that systematic introduction of histidyl residues at this antagonist-binding site in the human NK-1 receptor gradually converts it into a high-affinity metal-ion-binding site without affecting agonist binding. In a double mutant with histidine residues substituted at the top of transmembrane segments V and VI, respectively, Zn2+ inhibits binding of radiolabelled agonist peptide and efficiently blocks phosphoinositol turnover induced by substance P. We propose that Zn2+ and CP96,345 act as 'allosteric competitive' antagonists by stabilizing inactive conformations of the mutant and the wild-type receptor respectively. Introduction of metal-ion-binding sites could be used as a general tool in the structural and functional characterization of helix-helix interactions in G-protein-coupled receptors, as well as in other membrane proteins.