Ligand-directed bias of G protein signaling at the dopamine D2 receptor.

G-protein-coupled receptors (GPCRs) represent the largest family of drug targets. Upon activation, GPCRs signal primarily via a diverse set of heterotrimeric G proteins. Most GPCRs can couple to several different G protein subtypes. However, how drugs act at GPCRs contributing to the selectivity of G protein recognition is poorly understood. Here, we examined the G protein selectivity profile of the dopamine D2 receptor (D2), a GPCR targeted by antipsychotic drugs. We show that D2 discriminates between six individual members of the Gi/o family, and its profile of functional selectivity is remarkably different across its ligands, which all engaged D2 with a distinct G protein coupling pattern. Using structural modeling, receptor mutagenesis, and pharmacological evaluation, we identified residues in the D2 binding pocket that shape these ligand-directed biases. We further provide pharmacogenomic evidence that natural variants in D2 differentially affect its G protein biases in response to different ligands.

A Simple HPLC/DAD Method Validation for the Quantification of Malondialdehyde in Rodent's Brain.

In the present study, a HPLC/DAD method was set up to allow for the determination and quantification of malondialdehyde (MDA) in the brain of rodents (rats). Chromatographic separation was achieved on Supelcosil LC-18 (3 µm) SUPELCO Column 3.3 cm × 4.6 mm and Supelco Column Saver 0.5 µm filter by using a mobile phase acetonitrile (A) and phosphate buffer (20 mM, pH = 6) (B). Isocratic elution was 14% for (A) and 86% for (B). The injection volume (loop mode) was 100 µL with an analysis time of 1.5 min. Flow rate was set at 1 mL/min. The eluted compound was detected at 532 nm by a DAD detector by keeping the column oven at room temperature. The results indicated that the method has good linearity in the range of 0.2-20 µg/g. Both intra- and inter-day precision, expressed as RSD, were ≤15% and the accuracies ranged between ±15%. The lower limit of quantification (LLOQ), stability, and robustness were evaluated and satisfied the validation criteria. The method was successfully applied in a study of chronic toxicology following different treatment regimens with haloperidol and metformin.

De novo design with deep generative models based on 3D similarity scoring.

We have demonstrated the utility of a 3D shape and pharmacophore similarity scoring component in molecular design with a deep generative model trained with reinforcement learning. Using Dopamine receptor type 2 (DRD2) as an example and its antagonist haloperidol 1 as a starting point in a ligand based design context, we have shown in a retrospective study that a 3D similarity enabled generative model can discover new leads in the absence of any other information. It can be efficiently used for scaffold hopping and generation of novel series. 3D similarity based models were compared against 2D QSAR based, indicating a significant degree of orthogonality of the generated outputs and with the former having a more diverse output. In addition, when the two scoring components are combined together for training of the generative model, it results in more efficient exploration of desirable chemical space compared to the individual components.

Non-invasive intranasal administration route directly to the brain using dendrimer nanoplatforms: An opportunity to develop new CNS drugs.

There are several routes of administration to the brain, including intraparenchymal, intraventricular, and subarachnoid injections. The blood-brain barrier (BBB) impedes the permeation and access of most drugs to the central nervous system (CNS), and consequently, many neurological diseases remain undertreated. For past decades, to circumvent this effect, several nanocarriers have been developed to deliver drugs to the brain. Importantly, intranasal (IN) administration can allow direct delivery of drugs into the brain through the anatomical connection between the nasal cavity and brain without crossing the BBB. In this regard, dendrimers may possess great potential to deliver drugs to the brain by IN administration, bypassing the BBB and reducing systemic exposure and side effects, to treat diseases of the CNS. In this original concise review, we highlighted the few examples advocated regarding the use of dendrimers to deliver CNS drugs directly via IN. This review highlighed the few examples of the association of dendrimer encapsulating drugs (e.g., small compounds: haloperidol and paeonol; macromolecular compounds: dextran, insulin and calcitonin; and siRNA) using IN administration. Good efficiencies were observed. In addition, we will present the in vivo effects of PAMAM dendrimers after IN administration, globally, showing no general toxicity.

Insights into the biased activity of dextromethorphan and haloperidol towards SARS-CoV-2 NSP6: in silico binding mechanistic analysis.

The outbreak of novel coronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) virus continually led to infect a large population worldwide. SARS-CoV-2 utilizes its NSP6 and Orf9c proteins to interact with sigma receptors that are implicated in lipid remodeling and ER stress response, to infect cells. The drugs targeting the sigma receptors, sigma-1 and sigma-2, have emerged as effective candidates to reduce viral infectivity, and some of them are in clinical trials against COVID-19. The antipsychotic drug, haloperidol, exerts remarkable antiviral activity, but, at the same time, the sigma-1 benzomorphan agonist, dextromethorphan, showed pro-viral activity. To explore the potential mechanisms of biased binding and activity of the two drugs, haloperidol and dextromethorphan towards NSP6, we herein utilized molecular docking-based molecular dynamics simulation studies. Our extensive analysis of the protein-drug interactions, structural and conformational dynamics, residual frustrations, and molecular switches of NSP6-drug complexes indicates that dextromethorphan binding leads to structural destabilization and increase in conformational dynamics and energetic frustrations. On the other hand, the strong binding of haloperidol leads to minimal structural and dynamical perturbations to NSP6. Thus, the structural insights of stronger binding affinity and favorable molecular interactions of haloperidol towards viral NSP6 suggests that haloperidol can be potentially explored as a candidate drug against COVID-19. KEY MESSAGES: •Inhibitors of sigma receptors are considered as potent drugs against COVID-19. •Antipsychotic drug, haloperidol, binds strongly to NSP6 and induces the minimal changes in structure and dynamics of NSP6. •Dextromethorphan, agonist of sigma receptors, binding leads to overall destabilization of NSP6. •These two drugs bind with NSP6 differently and also induce differences in the structural and conformational changes that explain their different mechanisms of action. •Haloperidol can be explored as a candidate drug against COVID-19.

Electromembrane extraction of chlorprothixene, haloperidol and risperidone from whole blood and urine.

Separation of antipsychotic drugs from whole blood and urine is of great importance for clinic and forensic laboratories. In this work, chlorprothixene, haloperidol and risperidone representing the first and second generations of antipsychotic drugs were studied. Among them, chlorprothixene and risperidone were investigated for the first time by electromembrane extraction (EME). After the screening, 2-nitrophenyl octyl ether (NPOE) was used as the supported liquid membrane (SLM). The EME performance for spiked water (pH 2), whole blood and urine was tested and optimized individually. Using NPOE and 60 V, efficient EME was achieved from urine and whole blood with trifluoroacetic acid as the acceptor solution. The equilibrium time required for EME was dependent on the sample matrices. The steady-state of EME was reached in 30 min and 20 min for whole blood and urine, respectively. At steady-state, the EME recoveries of the targets from different sample matrices were satisfactory, and were in the range of 74%-100%. The proposed EME approach combined with liquid chromatography-tandem mass spectrometry (LC-MS/MS) was evaluated using whole blood and urine. The obtained linearity was 1-200 ng mL-1, and the coefficient of determination (R2) was ≥ 0.9853 for haloperidol and ≥ 0.9936 for chlorprothixene and risperidone. The limit of detection (LOD) and accuracy for all the targets ranged from 0.2-0.6 ng mL-1 and 102%-110%, respectively, and the repeatability at low (1 ng mL-1), medium (10 ng mL-1) and high (200 ng mL-1) concentration was ≤ 12% (RSD). Finally, the validated approach was successfully used to determine chlorprothixene, risperidone and haloperidol in whole blood and urine from rats, which were treated with chlorprothixene, risperidone and haloperidol at low therapeutic dose, respectively.

Altered dopamine D3 receptor gene expression in MAM model of schizophrenia is reversed by peripubertal cannabidiol treatment.

Gestational methylazoxymethanol acetate (MAM) treatment produces offspring with adult phenotype relevant to schizophrenia, including positive- and negative-like symptoms, cognitive deficits, dopaminergic dysfunction, structural and functional abnormalities. Here we show that adult rats prenatally treated with MAM at gestational day 17 display significant increase in dopamine D3 receptor (D3) mRNA expression in prefrontal cortex (PFC), hippocampus and nucleus accumbens, accompanied by increased expression of dopamine D2 receptor (D2) mRNA exclusively in the PFC. Furthermore, a significant change in the blood perfusion at the level of the circle of Willis and hippocampus, paralleled by the enlargement of lateral ventricles, was also detected by magnetic resonance imaging (MRI) techniques. Peripubertal treatment with the non-euphoric phytocannabinoid cannabidiol (30 mg/kg) from postnatal day (PND) 19 to PND 39 was able to reverse in MAM exposed rats: i) the up-regulation of the dopamine D3 receptor mRNA (only partially prevented by haloperidol 0.6 mg/kg/day); and ii) the regional blood flow changes in MAM exposed rats. Molecular modelling predicted that cannabidiol could bind preferentially to dopamine D3 receptor, where it may act as a partial agonist according to conformation of ionic-lock, which is highly conserved in GPCRs. In summary, our results demonstrate that the mRNA expression of both dopamine D2 and D3 receptors is altered in the MAM model; however only the transcript levels of D3 are affected by cannabidiol treatment, likely suggesting that this gene might not only contribute to the schizophrenia symptoms but also represent an unexplored target for the antipsychotic activity of cannabidiol.

Markov State Model Analysis of Haloperidol Binding to the D3 Dopamine Receptor.

We have developed Markov state models (MSMs) and hidden Markov models (HMMs) that describe the binding of haloperidol to the D3 dopamine receptor. Haloperidol is an antipsychotic drug that binds with nanomolar affinity to the D3 dopamine receptor, where it functions as an inverse agonist. The models were constructed using an adaptive sampling approach from 519 individual molecular dynamics simulations totaling 122 µs of simulated time and encompass the entire drug binding process. They reveal short-lived metastable bound states and two distinct long-lived bound conformations that cannot be separated in affinity using our current methodology. This work extends the use of MSMs and HMMs to study ligand binding, which thus far has been limited to simpler systems.

Percutaneous Absorption of Lorazepam, Diphenhydramine Hydrochloride, and Haloperidol from ABH Gel.

The objective of this project was to study the percutaneous absorption of lorazepam, diphenhydramine hydrochloride, and haloperidol from a topical Pluronic lecithin organogel, also known as ABH gel, across the porcine ear skin and verify its suitability for topical application. ABH gel was prepared using lecithin in isopropyl palmitate solution (1:1) as an oil phase and 20% w/v Poloxamer 407 solution as an aqueous phase. The gel was characterized for pH, viscosity, drug content, and thermal behavior. A robust high-performance liquid chromatography method was developed and validated for simultaneous analysis of lorazepam, diphenhydramine hydrochloride, and haloperidol. The percutaneous absorption of lorazepam, diphenhydramine hydrochloride, and haloperidol from ABH gel was carried out using Franz cells across the Strat-M membrane and pig ear skin. The pH of ABH gel was found to be 5.66 ± 0.13. The retention time of diphenhydramine hydrochloride, haloperidol, and lorazepam was found to be 5.2 minutes, 7.8 minutes, and 18.9 minutes, respectively. The ABH gel was found to be stable for up to 30 days. Theoretical steady state plasma concentrations (CSS) of diphenhydramine hydrochloride, haloperidol, and lorazepam calculated from flux values were found to be 1.6 ng/mL, 0.13 ng/mL, and 2.30 ng/mL, respectively. The theoretical CSS of diphenhydramine hydrochloride, haloperidol, and lorazepam were much lower than required therapeutic concentrations for antiemetic activity to relieve chemotherapy-induced nausea and vomiting. From the percutaneous absorption data, it was evident that ABH gel failed to achieve required systemic levels of lorazepam, diphenhydramine hydrochloride, and haloperidol following topical application.

Structure-Kinetic Profiling of Haloperidol Analogues at the Human Dopamine D2 Receptor.

Haloperidol is a typical antipsychotic drug (APD) associated with an increased risk of extrapyramidal side effects (EPSs) and hyperprolactinemia relative to atypical APDs such as clozapine. Both drugs are dopamine D2 receptor (D2R) antagonists, with contrasting kinetic profiles. Haloperidol displays fast association/slow dissociation at the D2R, whereas clozapine exhibits relatively slow association/fast dissociation. Recently, we have provided evidence that slow dissociation from the D2R predicts hyperprolactinemia, whereas fast association predicts EPS. Unfortunately, clozapine can cause severe side effects independent of its D2R action. Our results suggest an optimal kinetic profile for D2R antagonist APDs that avoids EPS. To begin exploring this hypothesis, we conducted a structure-kinetic relationship study of haloperidol and revealed that subtle structural modifications dramatically change binding kinetic rate constants, affording compounds with a clozapine-like kinetic profile. Thus, optimization of these kinetic parameters may allow development of novel APDs based on the haloperidol scaffold with improved side-effect profiles.

An In Vitro Study of Aromatic Stacking of Drug Molecules.

In this paper, drug-drug chemical interactions between two different aromatic compounds were studied by mass spectrometry. Specifically, we examined non-covalent complexes (NCX) between paclitaxel, a chemotherapeutic compound, and medications widely used in palliative care for depression, psychosis, and anxiety. It is unknown whether psychotropic medications directly interact with paclitaxel. Here, we use a simple and rapid electrospray ionization mass spectrometry in vitro assay, which has been predictive in the case of neuropeptides, to measure the relative strength of non-covalent interactions. This chemical interaction is most likely due to the overlap of aromatic rings of π-orbitals between paclitaxel and five commonly used medications: diazepam, clonozepam, sertraline, fluoxetine, and haloperidol. Molecular modeling illustrates that differences in the stability of the NCXs are likely due to the distance between the aromatic rings present in both the paclitaxel and antidepressant medications. Graphical Abstract.

New analogs of SYA013 as sigma-2 ligands with anticancer activity.

Our previous study has revealed 4-(4-(4-chlorophenyl)-1,4-diazepan-1-yl)-1-(4-fluorophenyl)butan-1-one·2HCl (SYA013) 1 as a sigma ligand with moderate selectivity for the sigma-2 receptor. Given the overexpression of sigma receptors in solid tumors and reports of sigma ligands with anticancer activities, we selected 1 for evaluation in several solid tumor cell lines. In addition, we have synthesized new analogs of 1 and now report that several of them bind preferentially at the sigma-2 receptor and have shown inhibition of several cancer cell lines including MDA-MB-231, MDA-MB-486, A549, PC-3, MIA PaCa-2 and Panc-1 cells. In particular, compounds 1 and 12 have demonstrated sub-micromolar activity against the Panc-1 cell line. It has also been observed that several of these compounds demonstrate selective toxicity toward cancer cells, when compared to normal cells.

SYA 013 analogs as moderately selective sigma-2 (σ2) ligands: Structure-affinity relationship studies.

Several lines of evidence suggest that selective sigma-2 (σ2) ligands might be useful for the treatment of solid tumors. However, very few selective σ2 ligands have been identified. This study was aimed at identifying new selective σ2 receptor ligands using a previously identified agent, SYA 013 as a lead. Four groups, homopiperazine, piperazine, tropane and selected oxime analogs of the homopiperazines were identified, synthesized and subsequently screened at the σ1 and σ2 receptors. The results demonstrate that these scaffolds can be modified to obtain selective σ2 receptor ligands. 1-(5-Chloropyridin-2-yl)-4-(3-((4-fluorophenyl)thio)propyl)-1,4-diazepane, 7 and 3-(4-chlorophenyl)-8-(3-((2-fluorophenyl)thio)propyl)-8-azabicyclo[3.2.1]octan-3-ol, 21 were identified as the highest binding affinity ligands (σ2Ki = 2.2 nM) and (4-(4-(5-chloropyridin-2-yl)-1,4-diazepan-1-yl)-1-(4-fluorophenyl)-butan-1-one oxime, 22 as a high affinity and the most selective ligand for the σ2 receptor (σ1Ki/σ2Ki = 41.8).

Enhancement of Transdermal Delivery of Haloperidol via Spanlastic Dispersions: Entrapment Efficiency vs. Particle Size.

Haloperidol (Hal) is a well-known typical antipsychotic. Hepatic first pass metabolism leads to its limited oral bioavailability. This study aimed at enhancing transdermal delivery of Hal via spanlastic formulae. Hal-loaded spanlastics of Span®60 and an edge activator (EA) were successfully prepared by ethanol injection method according to a 31.41 full factorial design. In this design, independent variables were X1, EA type, and X2, Span®60 to EA ratio. Y1, percentage entrapment efficiency (EE%); Y2, particle size (PS); Y3, deformability index (DI); and Y4, percentage drug released after 4h (Q4h), were chosen as dependent variables. The Fourier-transform infrared spectral analysis showed no considerable chemical interaction between Hal and the used excipients. Both factors affected significantly all the responses except DI. Desirability of each prepared formula was calculated based on maximizing EE% and Q4h and minimizing PS. Formula F6, with X1, Tween®80, and X2, 8:2, had the highest desirability value followed by F7, with X1, Tween®80, and X2, 6:4, and both were chosen as selected formulae (SF) for further investigation. F6 (having more entrapped Hal), F7 (of smaller PS), and Hal solution in propylene glycol were subjected to ex vivo permeation test through newborn rat skin. Both formulae showed marked enhancement in drug permeation compared with drug solution. The significantly higher Q36h and J36h of F7 from F6 may indicate that the smaller particle size aided more than higher entrapment in achieving a higher permeation for Hal of 3.5±0.2µg/cm2.h. These results are promising for further investigation of this formula.

A Thieno[2,3- d]pyrimidine Scaffold Is a Novel Negative Allosteric Modulator of the Dopamine D2 Receptor.

Recently, a novel negative allosteric modulator (NAM) of the D2-like dopamine receptors 1 was identified through virtual ligand screening. This ligand comprises a thieno[2,3- d]pyrimidine scaffold that does not feature in known dopaminergic ligands. Herein, we provide pharmacological validation of an allosteric mode of action for 1, revealing that it is a NAM of dopamine efficacy and identify the structural determinants of this allostery. We find that key structural moieties are important for functional affinity and negative cooperativity, while functionalization of the thienopyrimidine at the 5- and 6-positions results in analogues with divergent cooperativity profiles. Successive compound iterations have yielded analogues exhibiting a 10-fold improvement in functional affinity, as well as enhanced negative cooperativity with dopamine affinity and efficacy. Furthermore, our study reveals a fragment-like core that maintains low µM affinity and robust negative cooperativity with markedly improved ligand efficiency.

Fragmentation studies of selected drugs utilized in palliative care.

The results of research on selected drugs used in palliative care are presented, including fentanyl, tramadol, metoclopramide, hyoscine butylbromide, midazolam, haloperidol, levomepromazine and clonazepam. Interpretation of their ESI mass spectra obtained by the use of a triple quadrupole linear ion trap mass spectrometer is given. As a result, fragmentation pathways described in the literature are complemented and presented with more details. On their basis, transitions for quantitative analysis are selected and chromatographic conditions for the determination of the palliative care drugs are proposed as well. These results enable future studies on palliative care drugs in elderly patients including both their quantitation in body fluids and easier identification of their metabolites.

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.

ß-Cyclodextrin-based ternary complexes of haloperidol and organic acids: the effect of organic acids on the drug solubility enhancement.

Haloperidol (HALO) is a weak base with very low aqueous solubility that is used as an antipsychotic drug. This study aimed to improve its solubility by forming HALO/ß-cyclodextrin (ß-CD)-based ternary complexes with organic acids. The solubility of HALO/ß-CD binary and HALO/ß-CD/organic acid ternary complexes in different media (i.e. citrate buffer pH 3 and 6) was explored. The stoichiometric ratio between the drug and ß-CD was 1:1 in all complexes formed. The solubility of HALO/ß-CD binary complexes significantly increased in citrate buffer pH 3 compared with citrate buffer pH 6. For the ternary complexes, HALO/ß-CD/tartaric acid and HALO/ß-CD/lactic acid in citrate buffer pH 3 increased HALO solubility compared with HALO/ß-CD/succinic acid due to their higher unionized species. The highest stability constant and complexation efficiency values in citrate buffer pH 3 were shown by the ternary complexes with lactic acid followed by tartaric acid and succinic acid, respectively. Results indicated that lactic acid provided the greatest binding strength and solubilization efficiency for the complex.

Penetration enhancer-containing spanlastics (PECSs) for transdermal delivery of haloperidol: in vitro characterization, ex vivo permeation and in vivo biodistribution studies.

Haloperidol (Hal) is one of the widely used antipsychotic drugs. When orally administered, it suffers from low bioavailability due to hepatic first pass metabolism. This study aimed at developing Hal-loaded penetration enhancer-containing spanlastics (PECSs) to increase transdermal permeation of Hal with sustained release. PECSs were successfully prepared using ethanol injection method showing reasonable values of percentage entrapment efficiency, particle size, polydispersity index and zeta potential. The statistical analysis of the ex vivo permeation parameters led to the choice of F1L - made of Span® 60 and Tween® 80 at the weight ratio of 4:1 along with 1% w/v Labrasol® - as the selected formula (SF). SF was formulated into a hydrogel by using 2.5% w/v of HPMC K4M. The hydrogel exhibited good in vitro characteristics. Also, it retained its physical and chemical stability for one month in the refrigerator. The radiolabeling of SF showed a maximum yield by mixing of 100 µl of diluted formula with 50 µl saline having 200 MBq of 99mTc and containing 13.6 mg of reducing agent (NaBH4) and volume completed to 300 µl by saline at pH 10 for 10 min as reaction time. The biodistribution study showed that the transdermal 99mTc-SF hydrogel exhibited a more sustained release pattern and longer circulation duration with pulsatile behavior in the blood and higher brain levels than the oral 99mTc-SF dispersion. So, transdermal hydrogel of SF may be considered a promising sustained release formula for Hal maintenance therapy with reduced dose size and less frequent administration than oral formula.

Formulation of 3D Printed Tablet for Rapid Drug Release by Fused Deposition Modeling: Screening Polymers for Drug Release, Drug-Polymer Miscibility and Printability.

The primary aim of this study was to identify pharmaceutically acceptable amorphous polymers for producing 3D printed tablets of a model drug, haloperidol, for rapid release by fused deposition modeling. Filaments for 3D printing were prepared by hot melt extrusion at 150°C with 10% and 20% w/w of haloperidol using Kollidon® VA64, Kollicoat® IR, Affinsiol™15 cP, and HPMCAS either individually or as binary blends (Kollidon® VA64 + Affinisol™ 15 cP, 1:1; Kollidon® VA64 + HPMCAS, 1:1). Dissolution of crushed extrudates was studied at pH 2 and 6.8, and formulations demonstrating rapid dissolution rates were then analyzed for drug-polymer, polymer-polymer and drug-polymer-polymer miscibility by film casting. Polymer-polymer (1:1) and drug-polymer-polymer (1:5:5 and 2:5:5) mixtures were found to be miscible. Tablets with 100% and 60% infill were printed using MakerBot printer at 210°C, and dissolution tests of tablets were conducted at pH 2 and 6.8. Extruded filaments of Kollidon® VA64-Affinisol™ 15 cP mixtures were flexible and had optimum mechanical strength for 3D printing. Tablets containing 10% drug with 60% and 100% infill showed complete drug release at pH 2 in 45 and 120 min, respectively. Relatively high dissolution rates were also observed at pH 6.8. The 1:1-mixture of Kollidon® VA64 and Affinisol™15 cP was thus identified as a suitable polymer system for 3D printing and rapid drug release.