Anticancer Effects of Spiperone in C57BL/6 Mice with Emphysema and Lung Carcinoma.

The antitumor and antimetastatic activity of dopamine D2 receptor antagonists spiperone was studied in C57BL/6 mice in a model of combined pathology (emphysema and lung cancer). Emphysema was induced by administration of LPS and cigarette smoke extract. Lung cancer was induced by injection of Lewis lung carcinoma cells into the lung. It has been shown that under conditions of combined lung pathology, spiperone prevents inflammatory infiltration and emphysematous expansion of the lungs and reduces the size of the primary tumor node, the number of metastases, and the area of the lungs affected by metastases. Spiperone reduces the number of cancer stem cells (CSCs) in the lungs and blood of mice with combined pathology. CSCs isolated from the lungs and blood of mice with combined pathology treated with spiperone had a significantly lower potential to form a tumorosphere in vitro than CSCs from untreated mice with emphysema and lung carcinoma. Thus, blockade of dopamine D2 receptors is a promising approach for correcting combined lung pathology and can be used in the development of a method for treating lung cancer in patients with emphysema.

Micellar Hyaluronidase and Spiperone as a Potential Treatment for Pulmonary Fibrosis.

Concentration of hyaluronic acid (HA) in the lungs increases in idiopathic pulmonary fibrosis (IPF). HA is involved in the organization of fibrin, fibronectin, and collagen. HA has been proposed to be a biomarker of fibrosis and a potential target for antifibrotic therapy. Hyaluronidase (HD) breaks down HA into fragments, but is a subject of rapid hydrolysis. A conjugate of poloxamer hyaluronidase (pHD) was prepared using protein immobilization with ionizing radiation. In a model of bleomycin-induced pulmonary fibrosis, pHD decreased the level of tissue IL-1ß and TGF-ß, prevented the infiltration of the lung parenchyma by CD16+ cells, and reduced perivascular and peribronchial inflammation. Simultaneously, a decrease in the concentrations of HA, hydroxyproline, collagen 1, total soluble collagen, and the area of connective tissue in the lungs was observed. The effects of pHD were significantly stronger compared to native HD which can be attributed to the higher stability of pHD. Additional spiperone administration increased the anti-inflammatory and antifibrotic effects of pHD and accelerated the regeneration of the damaged lung. The potentiating effects of spiperone can be explained by the disruption of the dopamine-induced mobilization and migration of fibroblast progenitor cells into the lungs and differentiation of lung mesenchymal stem cells (MSC) into cells of stromal lines. Thus, a combination of pHD and spiperone may represent a promising approach for the treatment of IPF and lung regeneration.

Blockade of Dopamine D2 Receptors as a Novel Approach to Stimulation of Notch1+ Endothelial Progenitor Cells and Angiogenesis in C57BL/6 Mice with Pulmonary Emphysema Induced by Proteases and Deficiency of α1-Antitrypsin.

We studied the effects of spiperone, a selective blocker of dopamine D2 receptors, on the model of pulmonary emphysema provoked by administration of elastase and D-galactosamine hydrochloride to female C57BL/6 mice and characterized by activation of proteases in the lungs and systemic deficiency of its inhibitor α1-antitrypsin. In this model, spiperone prevented the development of inflammatory reaction and reduced the area of emphysematous expanded alveolar tissue. The expression of angiogenic marker CD31 in the lungs increased under these conditions. Regeneration of the damaged microvascular bed under the action of spiperone resulted from recruiting of Notch1+ endothelial progenitor cells (CD45-CD31+CD34+) into the lungs and blockade of the inhibitory effect of dopamine on phosphorylation of VEGF-2 receptors in endothelial cells of different maturity. In addition, spiperone produced a protective effect on hepatocytes and restored the production and secretion of α1-antitrypsin by these cells.

Biotinylated-spiperone ligands for quantum dot labeling of the dopamine D2 receptor in live cell cultures.

We have synthesized 3 analogs of the dopamine D2 receptor (D2 DR) antagonist spiperone that can be conjugated to streptavidin-coated quantum dots via a pegylated biotin derivative. Using fluorescent imaging we demonstrate that substitution on the spiro position is tolerated, whilst the length and rigidity of a spacer arm attached to spiperone is important in controlling specific labeling as well as minimizing nonspecific labeling to cells and the surface of cell culture dishes. The ligand with the most rigid linker IDT772 (4) had the best binding profile and had high specific binding to D2 DR expressing HEK-293T cells with low nonspecific binding to plates and HEK-293T cells that lacked the D2 DR.

Time outdoors and the prevention of myopia.

Recent epidemiological evidence suggests that children who spend more time outdoors are less likely to be, or to become myopic, irrespective of how much near work they do, or whether their parents are myopic. It is currently uncertain if time outdoors also blocks progression of myopia. It has been suggested that the mechanism of the protective effect of time outdoors involves light-stimulated release of dopamine from the retina, since increased dopamine release appears to inhibit increased axial elongation, which is the structural basis of myopia. This hypothesis has been supported by animal experiments which have replicated the protective effects of bright light against the development of myopia under laboratory conditions, and have shown that the effect is, at least in part, mediated by dopamine, since the D2-dopamine antagonist spiperone reduces the protective effect. There are some inconsistencies in the evidence, most notably the limited inhibition by bright light under laboratory conditions of lens-induced myopia in monkeys, but other proposed mechanisms possibly associated with time outdoors such as relaxed accommodation, more uniform dioptric space, increased pupil constriction, exposure to UV light, changes in the spectral composition of visible light, or increased physical activity have little epidemiological or experimental support. Irrespective of the mechanisms involved, clinical trials are now underway to reduce the development of myopia in children by increasing the amount of time they spend outdoors. These trials would benefit from more precise definition of thresholds for protection in terms of intensity and duration of light exposures. These can be investigated in animal experiments in appropriate models, and can also be determined in epidemiological studies, although more precise measurement of exposures than those currently provided by questionnaires is desirable.

Approach to the specificity and selectivity between D2 and D3 receptors by mutagenesis and binding experiments part I: Expression and characterization of D2 and D3 receptor mutants.

D3/D2 sub-specificity is a complex problem to solve. Indeed, in the absence of easy structural biology of the G-protein coupled receptors, and despite key progresses in this area, the systematic knowledge of the ligand/receptor relationship is difficult to obtain. Due to these structural biology limitations concerning membrane proteins, we favored the use of directed mutagenesis to document a rational towards the discovery of markedly specific D3 ligands over D2 ligands together with basic binding experiments. Using our methodology of stable expression of receptors in HEK cells, we constructed the gene encoding for 24 mutants and 4 chimeras of either D2 or D3 receptors and expressed them stably. Those cell lines, expressing a single copy of one receptor mutant each, were stably constructed, selected, amplified and the membranes from them were prepared. Binding data at those receptors were obtained using standard binding conditions for D2 and D3 dopamine receptors. We generated 26 new molecules derived from D2 or D3 ligands. Using 8 reference compounds and those 26 molecules, we characterized their binding at those mutants and chimeras, exemplifying an approach to better understand the difference at the molecular level of the D2 and D3 receptors. Although all the individual results are presented and could be used for minute analyses, the present report does not discuss the differences between D2 and D3 data. It simply shows the feasibility of the approach and its potential.

An in silico approach unveils the potential of antiviral compounds in preclinical and clinical trials as SARS-CoV-2 omicron inhibitors.

The increased transmissibility and highly infectious nature of the new variant of concern (VOC) that is severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) Omicron and lack of effective therapy need the rapid discovery of therapeutic antivirals against it. The present investigation aimed to identify antiviral compounds that would be effective against SARS-CoV-2 Omicron. In this study, molecular docking experiments were carried out using the recently reported experimental structure of omicron spike protein in complex with human angiotensin-converting enzyme 2 (ACE2) and various antivirals in preclinical and clinical trial studies. Out of 36 tested compounds, Abemaciclib, Dasatinib and Spiperone are the three top-ranked molecules which scored binding energies of -10.08 kcal/mol, -10.06 kcal/mol and -9.54 kcal/mol respectively. Phe338, Asp339, and Asp364 are crucial omicron receptor residues involved in hydrogen bond interactions, while other residues were mostly involved in hydrophobic interactions with the lead molecules. The identified lead compounds also scored well in terms of drug-likeness. Molecular dynamics (MD) simulation, essential dynamics (ED) and entropic analysis indicate the ability of these molecules to modulate the activity of omicron spike protein. Therefore, Abemaciclib, Dasatinib and Spiperone are likely to be viable drug-candidate molecules that can block the interaction between the omicron spike protein and the host cellular receptor ACE2. Though our findings are compelling, more research into these molecules is needed before they can be employed as drugs to treat SARS-CoV-2 omicron infections.

Spiperone Stimulates Regeneration in Pulmonary Endothelium Damaged by Cigarette Smoke and Lipopolysaccharide.

BACKGROUND: Endothelial dysfunction and destruction of the pulmonary microcirculation are important pathogenic factors in chronic obstructive pulmonary disease (COPD). In COPD, bronchial obstruction is associated with endothelial dysfunction. Thus, new pharmacological treatment options aimed at restoring the pulmonary endothelium represent a clinical need in COPD therapy. Notch1 has been shown to protect cells against apoptosis, inflammation, and oxidative stress caused by cigarette smoke extract (CSE). Therefore, drug which effect on Notch1 may be a potential therapeutic target for COPD in the future. METHODS: In this study, we assessed the potential of spiperone to mediate regeneration of pulmonary endothelium in model of pulmonary emphysema induced by a CSE and lipopolysaccharide (LPS) in female C57BL/6 mice. RESULTS: Spiperone increased the number of capillaries as well as the expression of the CD31 in the alveolar tissue compared to the controls. Moreover, application of spiperone prevented alveolar wall destruction (DI), and reduced the area of emphysema. Lastly, we demonstrated that spiperone positively influenced mobilization and migration of endothelial progenitor cells (EPC, CD45-CD34+CD31+), CD309+-endothelial cells, and angiogenesis precursors (CD45-CD117+CD309+) into the lung. Spiperone administration significantly reduced the number Notch1 positive CD309+-endothelial cells and Notch1+ EPCs. CONCLUSION: Overall, our results suggest that spiperone mediates endothelial regeneration in an animal model of COPD. Thus, it could represent a novel therapeutic approach for treatment of emphysema associated with COPD.

Verification of the role of spiperone in the treatment of COPD through bioinformatics analysis.

BACKGROUND: Aim of this study is investigates the influence of spiperone on hydrolase activity pathway in chronic obstructive pulmonary disease (COPD). PATIENTS AND METHODS: Differentially expressed genes (DEGs) were calculated by the limma package from microarray data GSE20257, and analysed via gene set enrichment analysis (GSEA) for identifying COPD related pathways. The regulation of hydrolase activity pathway related drugs was predicted by connectivity Map analysis (CMap). Western blotting and reverse transcription quantitative polymerase chain reaction (RT-qPCR) were used to investigate the effect of spiperone on regulation of hydrolase activity pathway in vitro experiment. RESULTS: A total of 378 DEGs were identified by the limma package. GSEA suggested that the regulation of hydrolase activity pathway was involved in the development of COPD. CMap of hub genes of regulation of hydrolase activity pathwayshown the most significant compound was spiperone. Results of vitro experiment verify that cigarette smoke extract (CSE) can increase the expression of fibronectin 1 (FN1) and epidermal growth factor (EGF), coinsided with decrease the expression of chemokine (C-X3-C motif) ligand 1 (CX3CL1), chemokoine (C-C motif) ligand 20 (CCL20), complement component 3 (C3) and slithomolog 2 (SLIT2) in BESA-2B cells and U937 cells. Spiperone can reverse the effect of CSE in BESA-2B cells and U937 cells. CONCLUSION: Regulation of hydrolase activity pathway was involved in the occurrence of COPD, spiperone was a potential drug for the treatment of COPD by affecting the regulation of hydrolase activity pathway. This study had provided new insights into the potential pathogenesis and treatment of COPD.

Inhibition of type 4 cAMP-phosphodiesterases (PDE4s) in mice induces hypothermia via effects on behavioral and central autonomous thermoregulation.

Inhibitors of Type 4 cAMP-phosphodiesterases (PDE4s) exert a number of promising therapeutic benefits, including potent anti-inflammatory, memory- and cognition-enhancing, metabolic, and antineoplastic effects. We report here that treatment with a number of distinct PDE4 inhibitors, including Rolipram, Piclamilast, Roflumilast and RS25344, but not treatment with the PDE3-selective inhibitor Cilostamide, induces a rapid (10-30 min), substantial (-5 °C) and long-lasting (up to 5 h) decrease in core body temperature of C57BL/6 mice; thus, identifying a critical role of PDE4 also in the regulation of body temperature. As little as 0.04 mg/kg of the archetypal PDE4 inhibitor Rolipram induces hypothermia. As similar or higher doses of Rolipram were used in a majority of published animal studies, most of the reported findings are likely paralleled by, or potentially impacted by hypothermia induced by these drugs. We further show that PDE4 inhibition affects central body temperature regulation and acts by lowering the cold-defense balance point of behavioral (including posture and locomotion) and autonomous (including cutaneous tail vasodilation) cold-defense mechanisms. In line with the idea of an effect on central body temperature regulation, hypothermia is induced by moderate doses of various brain-penetrant PDE4 inhibitors, but not by similar doses of YM976, a PDE4 inhibitor that does not efficiently cross the blood-brain barrier. Finally, to begin delineating the mechanism of drug-induced hypothermia, we show that blockade of D2/3-type dopaminergic, but not ß-adrenergic, H1-histaminergic or opiate receptors, can alleviate PDE4 inhibitor-induced hypothermia. We thus propose that increased D2/3-type dopaminergic signaling, triggered by PDE4 inhibitor-induced and cAMP-mediated dopamine release in the thermoregulatory centers of the hypothalamus, is a significant contributor to PDE4 inhibitor-induced hypothermia.

Spiperone: Tritium labelling at high specific activity.

An efficient method is described to tritiate spiperone at high specific activity.

Serotonin-2A homodimers are needed for signalling via both phospholipase A2 and phospholipase C in transfected CHO cells.

Different ligands differentially activate phospholipase A2 (PLA2) and phospholipase C (PLC) signalling pathways that are coupled to the serotonin 2A (5-HT2A) receptor, a class-A G-protein coupled receptor (GPCR). The serotonin 5-HT2A receptor has been shown to be expressed as a homodimer displaying some ligands negative cooperativity between protomers in the PLA2 signalling pathway. We hypothesized that the homodimeric complex is the minimum functional unit required for activation of the PLA2 and PLC pathways by the serotonin 5-HT2A receptor. To investigate this hypothesis, we partially blocked the serotonin 5-HT2A receptors with ritanserin and measured PLA2 and PLC activity simultaneously. We subsequently added the competitive antagonist spiperone to release the inactivator through a crosstalk mechanism and thus allow the dimer to return to a reactive state. Partial inactivation of the homodimer by ritanserin binding decreased the activity of the receptor by 59±13% and 70±4% in the PLA2 and PLC pathways respectively (P<0.001), with no difference in the potency of the serotonin (5-HT) was observed. The subsequent binding of spiperone released ritanserin due to the crosstalk between protomers and recovery of the receptor activity to 74±7% and 72±4%. Negative cooperativity between protomers in the dimer was maintained during arachidonic acid (AA) release after blocking ritanserin, as indicated by the biphasic inhibition curves for clozapine over 1µM serotonin (5-HT) in these conditions. These findings provide evidence that serotonin 5-HT2A receptors must be expressed as homodimers in order to activate both the PLA2 and PLC signalling pathways.

Brain regions and monoaminergic neurotransmitters that are involved in mouse ambulatory activity promoted by bupropion.

Bupropion (BUP), a substituted phenyl-ethylamine, has been utilized for the treatment of depression and for smoking cessation, however, one concern is that BUP may increase a risk of psychosis similar to other substituted phenyl-ethylamine amphetamine (AMPH) and methamphetamine (MetAMPH). BUP promotes ambulation in mice and causes behavioral sensitization on the ambulation-promoting effect when repeatedly administered as well as AMPH and MetAMPH. The present study aimed to elucidate brain regions and monoaminergic neurotransmitters that are involved in the ambulation-promoting effect of BUP. c-Fos-like immunoreactivity (c-Fos-IR) mapping in brain in combination with measuring ambulatory activity was conducted to determine brain region(s) that is involved in the ambulatory effect of BUP. Three kinds of statistical analyses for c-Fos-IR in 24 brain regions consistently showed that c-Fos-IR in the Caudate putamen (CPu) is positively correlated with the ambulatory response to BUP. In addition, multiple regression analysis indicated that the ambulatory response is a function of c-Fos-IR not only in the CPu but also in the lateral septum nucleus (LS), median raphe nucleus (MnR), lateral globus pallidus (LGP), medial globus pallidus (MGP), locus coeruleus (LC) and ventral hypothalamic nucleus (VMH). Effects of BUP on monoaminergic neurotransmitters in the CPu were examined using in vivo microdialysis method, as the pharmacological experiments indicated that monoaminergic neurotransmitters, dopamine (DA) in particular, mediate the ambulatory response to BUP. Response of DA in the CPu to BUP was parallel to the ambulatory response, showing that DA in the CPu is involved in the ambulatory response to BUP. The present study also suggests that other brain regions such as the LC, the origin nucleus of norepinephrine (NE) neurons, and another neurotransmitter NE may also play some roles for the ambulatory response to BUP, however, further studies are needed to elucidate the roles.

Use of radiolabeled antagonist assays for assessing agonism at D2 and D3 dopamine receptors: comparison with functional GTPγS assays.

BACKGROUND: Cell-based drug screening assays are essential tools for drug discovery and development targeting G protein-coupled receptors, which include dopamine D3 receptors. D3 is notorious for its poor coupling to G protein in most heterologous cell lines, and therefore D3 agonist-stimulated binding of [(35)S]GTPγS to G protein cannot be observed in many "non-functional" D3 expressing cell lines. NEW METHOD: The present work explores the use of an alternate method for assessing agonist activity, consisting of measuring the difference in agonist competition between [(3)H]spiperone bound to low-affinity states of the receptor and that with radioligand bound to high-affinity states (GTP shift assay). COMPARISON WITH EXISTING METHOD: The current study describes the determination of GTP shifts in [(3)H]spiperone binding assays for the assessment of agonists' potencies (at D2 and D3) and efficacies (at D3). Compared with GTPγ(35)S binding assays, the new method removes the cumbersome need of functional D3 cell lines and limited project duration due to short half-life of isotope (35)S. CONCLUSION: The new method allows the estimation of potency (D2 and D3) and efficacy (D3) at the level of receptor and G protein activation in a simple fashion from shifts in monophasic-inhibition curves. Moreover, it does not require [(35)S]GTPγS binding assays with functional D3 cells. This method will have wide applicability for D3-selective agonist screening. It may also be useful for other GPCRs circumventing the need for functional assays and offering the ability to detect agonist activity regardless of the particular signaling pathway.

The influence of different cellular environments on PET radioligand binding: an application to D2/3-dopamine receptor imaging.

Various D2/3 receptor PET radioligands are sensitive to endogenous dopamine release in vivo. The Occupancy Model is generally used to interpret changes in binding observed in in vivo competition binding studies; an Internalisation Hypothesis may also contribute to these changes in signal. Extension of in vivo competition imaging to other receptor systems has been relatively unsuccessful. A greater understanding of the cellular processes underlying signal changes following endogenous neurotransmitter release may help translate this imaging paradigm to other receptor systems. To investigate the Internalisation Hypothesis we assessed the effects of different cellular environments, representative of those experienced by a receptor following agonist-induced internalisation, on the binding of three D2/3 PET ligands with previously reported sensitivities to endogenous dopamine in vivo, namely [3H]spiperone, [3H]raclopride and [3H]PhNO. Furthermore, we determined the contribution of each cellular compartment to total striatal binding for these D2/3 ligands. These studies suggest that sensitivity to endogenous dopamine release in vivo is related to a decrease in affinity in the endosomal environment compared with those found at the cell surface. In agreement with these findings we also demonstrate that ∼25% of total striatal binding for [3H]spiperone originates from sub-cellular, microsomal receptors, whereas for [3H]raclopride and [3H]PhNO, this fraction is lower, representing ∼14% and 17%, respectively. This pharmacological approach is fully translatable to other receptor systems. Assessment of affinity shifts in different cellular compartments may play a crucial role for understanding if a radioligand is sensitive to endogenous release in vivo, for not just the D2/3, but other receptor systems.

Functional binding assays for estimation of the intrinsic efficacy of ligands at the 5-HT1A receptor: application for screening drug candidates.

INTRODUCTION: Determination of the intrinsic efficacy of ligands at the 5-HT1A receptor is important for selecting drug candidates, e.g. in the case of schizophrenia where partial agonism is a favorable property shared by different atypical antipsychotics. METHODS: Using seven ligands with different intrinsic efficacies and rat hippocampus synaptosomes, we compared critically three "functional" binding assays based on the ternary complex model that considers that the activated conformation of the receptor is the one coupled to G-protein. RESULTS: The Ki ratio method, based on the difference of affinity of the competing drug when using an antagonist vs. an agonist as radioligand, discriminated the ligands according to their intrinsic efficacies, with values from 77 for the full agonist 5-hydroxytryptamine to 0.09 for the inverse agonist WAY 100,635. The GTP-shift method, based on the decrease of affinity observed with agonists when GTP is added to the competition binding assay, was equally effective in classifying the drugs according to their intrinsic efficacy. The lower sensibility of the GTP-shift assay was investigated and explained by the different ionic conditions used in the two assays and the way competition curves were analyzed. Albeit more direct, the assay based on agonist-stimulated [(35)S]-GTPγS binding to G proteins was more expensive and of greater variability in our hands. DISCUSSION: We conclude that the GTP-shift procedure described herein for 5-HT1A receptors may expedite drug discovery efforts by predicting at the same time the affinity and intrinsic efficacy of ligands through a simple, rapid and economic ligand binding assay.

Induction of ovarian growth in Aegla platensis (Crustacea, Aeglidae) by means of neuroregulators incorporated to food

The freshwater crab Aegla platensis was used as a model to induce ovarian growth by adding different neuroregulators to a pellet food formulation. Added compounds were the dopaminergic inhibitor spiperone or the enkephalinergic inhibitor naloxone, both of them at a dose of 10-8 mol/animal. Animals were fed on the enriched pellets twice a week. After 7 wk, the gonadosomatic index (GI) was calculated as (gonad fresh weight / body fresh weight) x 100. GI significantly increased only for those females fed on spiperone pellets, compared to a control group receiving pellets with no compound added. During the assayed period, spiperone would be reverting the arrest exerted by dopamine on the neuroendocrine stimulation of ovarian growth. On the other hand, for both spiperone and naloxone a higher GI was correlated to a higher lipid content of both gonads and/or hepatopancreas, suggesting an increased energetic demand in accordance with an active investment in reproduction. Rev. Biol. Trop. 56 (3): 1201-1207. Epub 2008 September 30.

Se utilizó al anomuro de agua dulce Aegla platensis como modelo para inducir el crecimiento ovárico mediante el agregado de diferentes neuroreguladores a una formulación de alimento pelleteado. Los compuestos agregados fueron el inhibidor dopaminergico spiperona ó el inhibidor encefalinérgico naloxone, ambos a una dosis de 10-8 moles/animal. Los animales fueron alimentados dos veces a la semana con pellets enriquecidos con alguno de los neuroreguladores. Luego de 7 semanas, se calculó el índice gonadomático (IG) como (peso gonadal fresco/ peso corporal fresco) x 100. El IG mostró un incremento significativamente sólo en aquellas hembras alimentadas con pellets enriquecidos con spiperona, en comparación con un grupo control que recibió pellets sin agregado alguno. Durante el período ensayado, la spiperona estaría revirtiendo el arresto ejercido por la dopamina sobre la estimulación neuroendocrina del crecimiento ovárico. Por otro lado, para ambos grupos experimentales (spiperona y naloxone), un mayor valor de IG estuvo correlacionado a un mayor incremento del contenido de lípidos tanto en gonadas como en hepatopáncreas, sugiriendo una demanda energética incrementada en relación con una activa inversión en reproducción.