Novel High Affinity Sigma-1 Receptor Ligands from Minimal Ensemble Docking-Based Virtual Screening.

Sigma-1 receptor (S1R) is an intracellular, multi-functional, ligand operated protein that also acts as a chaperone. It is considered as a pluripotent drug target in several pathologies. The publication of agonist and antagonist bound receptor structures has paved the way for receptor-based in silico drug design. However, recent studies on this subject payed no attention to the structural differences of agonist and antagonist binding. In this work, we have developed a new ensemble docking-based virtual screening protocol utilizing both agonist and antagonist bound S1R structures. This protocol was used to screen our in-house compound library. The S1R binding affinities of the 40 highest ranked compounds were measured in competitive radioligand binding assays and the sigma-2 receptor (S2R) affinities of the best S1R binders were also determined. This way three novel high affinity S1R ligands were identified and one of them exhibited a notable S1R/S2R selectivity.

Enhancement of Dissolution and Skin Permeability of Pentazocine by Proniosomes and Niosomal Gel.

Proniosomes (PN) are the dry water-soluble carrier systems that may enhance the oral bioavailability, stability, and topical permeability of therapeutic agents. The low solubility and low oral bioavailability due to extensive first pass metabolism make Pentazocine as an ideal candidate for oral and topical sustained release delivery. The present study was aimed to formulate the PNs by quick slurry method that are converted to niosomes (liquid dispersion) by hydration, and subsequently formulated to semisolid niosomal gel. The PNs were found in spherical shape in the SEM and stable in the physicochemical and thermal analysis (FTIR, TGA, and XRD). The quick slurry method produced high recovery (> 80% yield) and better flow properties (θ = 28.1-37.4°). After hydration, the niosomes exhibited desirable entrapment efficiency (44.45-76.23%), size (4.98-21.3 µm), and zeta potential (- 9.81 to - 21.53 mV). The in vitro drug release (T100%) was extended to more than three half-lives (2-4 h) and showed good fit to Fickian diffusion indicated by Korsmeyer-Peppas model (n = 0.136-0.365 and R2 = 0.9747-0.9954). The permeation of niosomal gel was significantly enhanced across rabbit skin compared to the pure drug-derived gel. Therefore, the PNs are found promising candidates for oral as dissolution enhancement and sustained release for oral and topical delivery of pentazocine for the management of cancer pain.

The sigma-1 receptors are present in monomeric and oligomeric forms in living cells in the presence and absence of ligands.

The sigma-1 receptor (S1R) is a 223-amino-acid membrane protein that resides in the endoplasmic reticulum and the plasma membrane of some mammalian cells. The S1R is regulated by various synthetic molecules including (+)-pentazocine, cocaine and haloperidol and endogenous molecules such as sphingosine, dimethyltryptamine and dehydroepiandrosterone. Ligand-regulated protein chaperone functions linked to oxidative stress and neurodegenerative disorders such as amyotrophic lateral sclerosis (ALS) and neuropathic pain have been attributed to the S1R. Several client proteins that interact with S1R have been identified including various types of ion channels and G-protein coupled receptors (GPCRs). When S1R constructs containing C-terminal monomeric GFP2 and YFP fusions were co-expressed in COS-7 cells and subjected to FRET spectrometry analysis, monomers, dimers and higher oligomeric forms of S1R were identified under non-liganded conditions. In the presence of the prototypic S1R agonist, (+)-pentazocine, however, monomers and dimers were the prevailing forms of S1R. The prototypic antagonist, haloperidol, on the other hand, favoured higher order S1R oligomers. These data, in sum, indicate that heterologously expressed S1Rs occur in vivo in COS-7 cells in multiple oligomeric forms and that S1R ligands alter these oligomeric structures. We suggest that the S1R oligomerization states may regulate its function(s).

Effects of (+)-pentazocine on the antinociceptive effects of (-)-pentazocine in mice.

Previous studies have shown that sigma-1 receptor chaperone (Sig-1R) ligands can regulate pain-related behaviors, and Sig-1R itself can regulate µ-opioid receptor functions as well as signal transduction. Even though (±)-pentazocine has been used clinically for the treatment of pain through opioid receptors, (+)-pentazocine is known to be a selective Sig-1R agonist. To the best of our knowledge, there is no information available regarding the involvement of Sig-1R agonistic action in the antinociceptive effects of (±)-pentazocine. Therefore, the present study was designed to investigate the effects of (+)-pentazocine on the antinociceptive effects of (-)-pentazocine in mice. Both and (-)-pentazocine induced biphasic antinociceptive effects as measured by the warm-plate test. The early phase, but not the delayed phase, of the antinociceptive effects induced by (-)-pentazocine, which are mediated by the activation of µ-opioid receptors, were suppressed by pretreatment with (+)-pentazocine. These results suggest that the innate antinociceptive action of (±)-pentazocine could be marginally reduced by the effects of (+)-pentazocine, but (+)-pentazocine can suppress the antinociceptive effects of (-)-pentazocine at certain time points.

Involvement of µ- and δ-opioid receptor function in the rewarding effect of (±)-pentazocine.

Most opioid receptor agonists have abuse potential, and the rewarding effects of opioids can be reduced in the presence of pain. While each of the enantiomers of pentazocine has a differential pharmacologic profile, (±)-pentazocine has been used clinically for the treatment of pain. However, little information is available regarding which components of pentazocine are associated with its rewarding effects, and whether the (±)-pentazocine-induced rewarding effects can be suppressed under pain. Therefore, the present study was performed to investigate the effects of pain on the acquisition of the rewarding effects of (±)-pentazocine, and to examine the mechanism of the rewarding effects of (±)-pentazocine using the conditioned place preference paradigm. (±)-Pentazocine and (-)-pentazocine, but not (+)-pentazocine, produced significant rewarding effects. Even though the rewarding effects induced by (±)-pentazocine were significantly suppressed under pain induced by formalin, accompanied by increase of preprodynorphin mRNA levels in the nucleus accumbens, a high dose of (±)-pentazocine produced significant rewarding effects under pain. In the normal condition, (±)-pentazocine-induced rewarding effects were blocked by a low dose of naloxone, whereas the rewarding effects induced by high doses of pentazocine under pain were suppressed by naltrindole (a δ-opioid receptor antagonist). Interestingly, (±)-pentazocine did not significantly affect dopamine levels in the nucleus accumbens. These findings suggest that the rewarding effects of (-)-pentazocine may contribute to the abuse potential of (±)-pentazocine through µ- as well as δ-opioid receptors, without robust activation of the mesolimbic dopaminergic system. We also found that neural adaptations can reduce the abuse potential of (±)-pentazocine under pain.

The sigma-1 receptor binds to the Nav1.5 voltage-gated Na+ channel with 4-fold symmetry.

The sigma-1 receptor (Sig1R) is up-regulated in many human tumors and plays a role in the control of cancer cell proliferation and invasiveness. At the molecular level, the Sig1R modulates the activity of various ion channels, apparently through a direct interaction. We have previously shown using atomic force microscopy imaging that the Sig1R binds to the trimeric acid-sensing ion channel 1A with 3-fold symmetry. Here, we investigated the interaction between the Sig1R and the Nav1.5 voltage-gated Na(+) channel, which has also been implicated in promoting the invasiveness of cancer cells. We show that the Sig1R and Nav1.5 can be co-isolated from co-transfected cells, consistent with an intimate association between the two proteins. Atomic force microscopy imaging of the co-isolated proteins revealed complexes in which Nav1.5 was decorated by Sig1Rs. Frequency distributions of angles between pairs of bound Sig1Rs had two peaks, at ∼90° and ∼180°, and the 90° peak was about twice the size of the 180° peak. These results demonstrate that the Sig1R binds to Nav1.5 with 4-fold symmetry. Hence, each set of six transmembrane regions in Nav1.5 likely constitutes a Sig1R binding site, suggesting that the Sig1R interacts with the transmembrane regions of its partners. Interestingly, two known Sig1R ligands, haloperidol and (+)-pentazocine, disrupted the Nav1.5/Sig1R interaction both in vitro and in living cells. Finally, we show that endogenously expressed Sig1R and Nav1.5 also functionally interact.

Statistically optimized pentazocine loaded microsphere for the sustained delivery application: Formulation and characterization.

Pentazocine (PTZ) is a narcotic analgesic used to manage moderate to severe, acute and chronic pains. In this study, PTZ loaded Ethyl cellulose microsphere has been formulated for sustained release and improved bioavailability of PTZ. These microspheres were fabricated by oil in water emulsion solvent evaporation technique. A three factorial, three levels Box-Behnken design was applied to investigate the influence of different formulation components and process variables on the formulation response using the numeric approach through the design expert® software. All the formulations were characterized for the morphology, different physicochemical properties and the results were supported with the ANOVA analysis, three dimensional contour graphs and regression equations. The maximum percentage yield was 98.67% with 98% entrapment of PTZ. The mean particle size of the formulations ranges from 50-148µm, which directly relates to the concentration of polymer and inversely proportional to the stirring speed. SEM revealed the spherical shape of PTZ microspheres with porous structures. These are physically, chemically and thermally stable as confirmed through Fourier transform infrared spectroscopy (FTIR), powder X-ray diffraction (PXRD) and thermal gravimetric (TG) analysis respectively. The microspheres provided a sustained release of the PTZ for more than 12 hours, following zero order with fickian and non fickian diffusion. The results indicate that prepared microspheres can be a potential drug delivery system (DDS) for the delivery of PTZ in the management of pains.

In vitro and in vivo evaluation of [125/123I]-2-[4-(2-iodophenyl)piperidino]cyclopentanol([125/123I]-OI5V) as a potential sigma-1 receptor ligand for SPECT.

INTRODUCTION: We investigated the characteristics of radio-iodinated 2-[4-(2-iodophenyl)piperidino]cyclopentanol (OI5V) as a single photon emission computed tomography (SPECT) ligand for mapping sigma-1 receptor (σ-1R), which plays an important role in stress remission in many organs. METHODS: OI5V was synthesized from o-bromobenzaldehyde in three steps. OI5V was evaluated for its affinity to VAChT, σ-1 and σ-2 receptor by in vitro competitive binding assays using rat tissues and radioligands, [3H]vesamicol, ( +)-[3H]pentazocine and [3H]DTG, respectively. [125/123I]OI5V was prepared from o-trimethylstannyl-cyclopentanevesamicol (OT5V) by the iododestannylation reaction under no-carrier-added conditions. In vivo biodistribution study of [125I]OI5V in blood, brain regions and major organs of rats was performed at 2, 10, 30 and 60 min post-injection. In vivo blocking study and ex vivo autoradiography were performed to assess the binding selectivity of [125I]OI5V for σ-1 receptor. SPECT-CT imaging study was performed using [123I]OI5V. RESULTS: OI5V demonstrated high selective binding affinity for σ-1R in vitro. In the biodistribution study, the blood-brain barrier (BBB) permeability of [125I]OI5V was high and the accumulation of [125I]OI5V in the rat cortex at 2 min post-injection exceeded 2.00%ID/g. In the in vivo blocking study, the accumulation of [125I]OI5V in the brain was significantly blocked by co-administration of 0.5 µmol of SA4503 and 1.0 µmol of pentazocine. Ex vivo autoradiography revealed that the regional brain accumulation of [125I]OI5V was similar to σ-1R-rich regions of the rat brain. SPECT images of [123I]OI5V in the rat brain reflected the distribution of sigma receptors in the brain. CONCLUSIONS: This study confirmed that [125/123I]OI5V selectively binds σ-1R in the rat brain in vivo. [123I]OI5V was suggested to be useful as a σ-1R ligand for SPECT.

Comparative study of dezocine, pentazocine and tapentadol on antinociception and physical dependence.

AIMS: Dezocine and pentazocine, widely prescribed in China for postoperative pain, were initially considered as mixed agonist/antagonist targeting µ-opioid receptors (MORs) and κ-opioid receptors (KORs). However, dezocine has been revealed to alleviate chronic neuropathic pain through MOR activation and norepinephrine reuptake inhibition (NRI). This study investigated dezocine- and pentazocine-induced antinociception and physical dependence development, compared to the typical MOR-NRI opioid tapentadol. MAIN METHODS: Calcium mobilization assay was conducted to assess the potency of the drugs while hot-plate test was performed to compare the antinociception. Physical dependence development was compared with morphine. KEY FINDINGS: Treatment with dezocine, pentazocine and tapentadol stimulated calcium mobilization in HEK293 cells stably expressed MORs but not KORs, whereas dezocine and pentazocine inhibited KOR activities. Subcutaneously injected dezocine-, tapentadol- and pentazocine-induced antinociception dose-dependently, in hot-plate test. Intrathecally injected MOR antagonist CTAP, norepinephrine depletor 6-OHDA and α2-adrenoceptor (α2-AR) antagonist yohimbine partially antagonized dezocine, pentazocine and tapentadol antinociception. Whereas specific KOR antagonist GNTI did not alter their antinociception, the putative inverse KOR agonist nor-BNI reduced dezocine and pentazocine antinociception. Moreover, combined CTAP and 6-OHDA or yohimbine blocked dezocine and tapentadol antinociception but displayed the same partial inhibition on pentazocine antinociception as CTAP alone. Furthermore, compared to morphine and pentazocine, long-term treatment with dezocine and tapentadol produced much less physical dependence-related withdrawal signs, which were restored by spinal 6-OHDA or yohimbine treatment. SIGNIFICANCE: Our findings illustrated that dezocine and tapentadol, but not pentazocine, exert remarkable antinociception in nociceptive pain with less abuse liability via dual mechanisms of MOR activation and NRI.

Development of matrix controlled transdermal delivery systems of pentazocine: In vitro/in vivo performance.

The present study aimed to develop hydroxypropyl methylcellulose based transdermal delivery of pentazocine. In formulations containing lower proportions of polymer, the drug released followed the Higuchi kinetics while, with an increase in polymer content, it followed the zero-order release kinetics. Release exponent (n) values imply that the release of pentazocine from matrices was non-Fickian. FT-IR, DSC and XRD studies indicated no interaction between drug and polymer.The in vitro dissolution rate constant, dissolution half-life and pharmacokinetic parameters (C(max), t(max), AUC(s), t(1/2), Kel, and MRT) were evaluated statistically by two-way ANOVA. A significant difference was observed between but not within the tested products. Statistically, a good correlation was found between per cent of drug absorbed from patches vs. C(max) and AUC(s). A good correlation was also observed when per cent drug released was correlated with the blood drug concentration obtained at the same time point. The results of this study indicate that the polymeric matrix films of pentazocine hold potential for transdermal drug delivery.

Enantiocontrolled synthesis of (-)-9-epi-pentazocine and (-)-aphanorphine.

We have developed novel asymmetric routes to (-)-9- epi-pentazocine and (-)-aphanorphine from a d-tyrosine derivative. The tricyclic frameworks of (-)-9- epi-pentazocine and (-)-aphanorphine were assembled stereoselectively via intramolecular Friedel-Crafts reaction of the corresponding bicyclic precursors, generated with titanium-promoted enyne cyclization and indium-initiated atom-transfer radical cyclization, respectively.

Structural basis for σ1 receptor ligand recognition.

The σ1 receptor is a poorly understood membrane protein expressed throughout the human body. Ligands targeting the σ1 receptor are in clinical trials for treatment of Alzheimer's disease, ischemic stroke, and neuropathic pain. However, relatively little is known regarding the σ1 receptor's molecular function. Here, we present crystal structures of human σ1 receptor bound to the antagonists haloperidol and NE-100, and the agonist (+)-pentazocine, at crystallographic resolutions of 3.1 Å, 2.9 Å, and 3.1 Å, respectively. These structures reveal a unique binding pose for the agonist. The structures and accompanying molecular dynamics (MD) simulations identify agonist-induced structural rearrangements in the receptor. Additionally, we show that ligand binding to σ1 is a multistep process that is rate limited by receptor conformational change. We used MD simulations to reconstruct a ligand binding pathway involving two major conformational changes. These data provide a framework for understanding the molecular basis for σ1 agonism.

Development of opioid formulations with limited diversion and abuse potential.

Non-medical abuse of prescription opioid medications is not a new phenomenon, but such use has been increasing in recent years. Various methods have been used and continue to be developed in an effort to limit diversion and abuse of opioid medications. A number of these methods will be described for opioid analgesic and addiction treatment formulations using relevant historical examples (e.g. propoxyphene, pentazocine, buprenorphine) as well as examples of formulations currently being considered or under development (e.g. oxycodone plus naltrexone, sustained-release buprenorphine). The focus, though not exclusively, will be on those formulations that represent a combination of an opioid agonist with an antagonist. These methods must take into consideration the pharmacokinetic profile of the agonist and antagonist, the expected primary route of abuse of the medication and the medication combination, the dose of medication that is likely to be abused, the availability of alternative drugs of abuse, and the population of potential abusers that is being targeted with the revised formulation.

(+)-cis-N-(para-, meta-, and ortho-substituted benzyl)-N-normetazocines: synthesis and binding affinity at the [3H]-(+)-pentazocine-labeled (sigma 1) site and quantitative structure-affinity relationship studies.

sigma 1 receptor ligands have potential pharmacological significance as antipsychotic drugs, as tools in the study of drug-induced motor function disorders, and as radiopharmaceutical imaging agents for the noninvasive imaging of malignant tumors in human subjects. A series of substituted N-benzyl-N-normetazocines were synthesized and their binding affinity at the sigma 1 receptor evaluated in order to examine the details of the structure--affinity relationships (SAR) of a previously determined high-affinity lead compound, (+)-cis-N-benzyl-N-normetazocine (Ki = 0.67 nM). Variation in the benzyl substituents of these compounds produced a 1590-fold range in affinity at the sigma 1 receptor from the unsubstituted benzyl analog to the lowest affinity p-tert-butylbenzyl analog (Ki = 1066 nM). The nanomolar binding affinity for the sigma 1 receptor of (+)-cis-N-(4-fluorobenzyl)-N-normetzocine suggests that this analog may be a useful PET imaging agent.

Synthesis and characterization of [125I]3'-(-)-iodopentazocine, a selective sigma 1 receptor ligand.

Pentazocine is a potent ligand at both opioid and sigma receptors, but with opposite stereoselectivities. Whereas (-)-pentazocine has high affinity for a number of opioid receptors, (+)-pentazocine labels sigma 1 receptors. Iodination of (-)-pentazocine at the 3'-position reverses its selectivity for opioid and sigma 1 receptors. 3'-(-)-Iodopentazocine competes at sigma 1 receptor binding sites with a Ki value of 8 nM, compared to approximately 40 nM for (-)-pentazocine. 3'-(-)-Iodopentazocine also has lost its affinity for opioid receptors. In contrast, iodination of (+)-pentazocine lowers its affinity at sigma 1 receptors. Synthesis of [125I]3'-(-)-iodopentazocine is readily performed with incorporations of up to 80%. Binding is of high affinity and shows the selectivity anticipated for a sigma 1 receptor-selective ligand. Exposing membranes prebound with [125I]3'-(-)-iodopentazocine to ultraviolet light can covalently couple the ligand into the membranes. Polyacrylamide gel electrophoresis reveals a major band at about 25 kDa and a minor one at about 20 kDa, indicating photolabeling of sigma 1 receptors with minor incorporation into sigma 2 sites.

Analysis of zopiclone (Imovane) in postmortem specimens by GC-MS and HPLC with diode-array detection.

A 26-year-old black female was found dead at home. Her mouth was covered with a fluid containing chalky particles. Empty strips of Imovane (zopiclone) and an empty bottle of Fortal (pentazocine) were also found. No urine was available at autopsy. Screening of postmortem blood and stomach contents with enzyme-multiplied immunoassay technique (EMIT) detected only caffeine. Further screening using routine high-performance liquid chromatography (HPLC) with diode-array detection and gas chromatography (GC) with mass spectrometric detection revealed the presence of large amounts of pentazocine in the blood and stomach contents. In the HPLC chromatogram, a second peak that was only partially resolved from the solvent front was observed. Thin-layer chromatography demonstrated the presence of zopiclone, but optimized HPLC and GC conditions had to be used for proper identification and quantitation. This case illustrates the fact that zopiclone can be easily overlooked during routine forensic screening.

Chitosan based pentazocine microspheres for intranasal systemic delivery: development and biopharmaceutical evaluation.

Bioadhesive chitosan microspheres (Ms) of pentazocine (Pz) for intranasal systemic delivery were prepared with the aim of avoiding the first pass effect, and thus improving the bioavailability and achieving sustained and controlled blood level profiles, as an alternative therapy to injection and to obtain improved therapeutic efficacy in the treatment of chronic pain such as cancer, trauma and post-operative pain, etc. The formulation variables were drug loading, polymer concentration, stirring rate during crosslinking and oils. The microspheres (Ms) were subjected to evaluation for physical characteristics, such as particle size, incorporation efficiency, swelling ability, in vitro bioadhesion, in vitro drug release characteristics and in vivo performance in rabbits. Application of in vitro data to various kinetic equations indicated matrix diffusion controlled drug delivery from chitosan Ms. Drug loading, polymer concentration and stirring speed influenced the drug release profiles significantly while oils had negligible effect. In vivo studies indicated significantly improved bioavailability of Pz from Ms with sustained and controlled blood level profiles as compared to i.v., oral and nasal administration of drug solution. Good correlation was observed between in vitro and in vivo data.

Asymmetric syntheses of (-)-pentazocine and (-)-eptazocine through an aza-Prins cyclization.

Two down more to go: The asymmetric syntheses of (-)-pentazocine and (-)-eptazocine are presented. The highlights of the syntheses are the construction of the core skeleton through an aza-Prins cyclization and intramolecular Friedel-Crafts reaction.

Neuroprotective effects of high affinity Σ1 receptor selective compounds.

We previously reported that the antipsychotic drug haloperidol, a multifunctional D2-like dopamine and sigma receptor subtype antagonist, has neuroprotective properties. In this study we further examined the association between neuroprotection and receptor antagonism by evaluating a panel of novel compounds with varying affinity at sigma and D2-like dopamine receptors. These compounds were evaluated using an in vitro cytotoxicity assay that utilizes a hippocampal-derived cell line, HT-22, in the presence or absence of varying concentrations (5 to 20 mM) of glutamate. While haloperidol was found to be a potent neuroprotective agent in this in vitro cell assay, the prototypic sigma 1 receptor agonist (+)-pentazocine was found not to be neuroprotective. Subsequently, the potency for the neuroprotection of HT-22 cells was evaluated for a) three SV series indoles which have nMolar affinity at D2-like receptors but varying affinity at sigma 1 receptor and b) two benzyl phenylacetamides sigma 1 receptor selective compounds which bind with low affinity at D2-like receptors but have nMolar affinity for the sigma 1 receptor. We observed that cytoprotection correlated with the affinity of the compounds for sigma 1 receptors. Based upon results from the HT-22 cell-based in vitro assay, two phenylacetamides, LS-127 and LS-137, were further evaluated in vivo using a transient middle cerebral artery occlusion (t-MCAO) model of stroke. At a dose of 100 µg/kg, both LS-127 and LS-137 attenuated infarct volume by approximately 50%. These studies provide further evidence that sigma 1 receptor selective compounds can provide neuroprotection in cytotoxic situations. These results also demonstrate that sigma 1 receptor selective benzyl phenylacetamides are candidate pharmacotherapeutic agents that could be used to minimize neuronal death after a stroke or head trauma.

Hollow fiber-based liquid-liquid-liquid microextraction combined with electrospray ionization-ion mobility spectrometry for the determination of pentazocine in biological samples.

A new method based on liquid-liquid-liquid microextraction combined with electrospray ionization-ion mobility spectrometry (LLLME-ESI-IMS) was used for the determination of pentazocine in urine and plasma samples. Experimental parameters which control the performance of LLLME, such as selection of composition of donor and acceptor phase, type of organic solvent, ionic strength of the sample, extraction temperature and extraction time were studied. The limit of detection and relative standard deviation of the method were 2 ng/mL and 5.3%, respectively. The linear calibration ranged from 10 to 500 ng/mL with r(2)=0.998. Pentazocine was successfully determined in urine and plasma samples without any significant matrix effect.