Synthesis, Pharmacological Evaluation, and Molecular Modeling of Lappaconitine-1,5-Benzodiazepine Hybrids.

Diterpenoid alkaloids, originating from the amination of natural tetracyclic diterpenes, have long interested scientists due to their medicinal uses and infamous toxicity which has limited the clinical application of the native compound. Alkaloid lappaconitine extracted from various Aconitum and Delphinium species has displayed extensive bioactivities and active ongoing research to reduce its adverse effects. A convenient route to construct hybrid molecules containing diterpenoid alkaloid lappaconitine and 3H-1,5-benzodiazepine fragments was proposed. The key stage involved the formation of 5'-alkynone-lappaconitines in situ by acyl Sonogashira coupling of 5'-ethynyllappaconitine, followed by cyclocondensation with o-phenylenediamine. New hybrid compounds showed low toxicity and outstanding analgesic activity in experimental pain models, which depended on the nature of the substituent in the benzodiazepine nucleus. An analogous dependence was also shown for the antiarrhythmic activity in the epinephrine arrhythmia test in vivo. Studies on the isolated atrium have shown that the mechanism of action of the new compounds is included the blockade of beta-adrenergic receptors and potassium channels. Molecular docking analysis was conducted to determine the binding potential of target molecules with the voltage-gated sodium channel NaV1.5. All obtained results provide a basis for future rational modifications of lappaconitine, reducing side effects, while retaining its therapeutic effects.

Structure-based discovery of nonopioid analgesics acting through the α2A-adrenergic receptor.

Because nonopioid analgesics are much sought after, we computationally docked more than 301 million virtual molecules against a validated pain target, the α2A-adrenergic receptor (α2AAR), seeking new α2AAR agonists chemotypes that lack the sedation conferred by known α2AAR drugs, such as dexmedetomidine. We identified 17 ligands with potencies as low as 12 nanomolar, many with partial agonism and preferential Gi and Go signaling. Experimental structures of α2AAR complexed with two of these agonists confirmed the docking predictions and templated further optimization. Several compounds, including the initial docking hit '9087 [mean effective concentration (EC50) of 52 nanomolar] and two analogs, '7075 and PS75 (EC50 4.1 and 4.8 nanomolar), exerted on-target analgesic activity in multiple in vivo pain models without sedation. These newly discovered agonists are interesting as therapeutic leads that lack the liabilities of opioids and the sedation of dexmedetomidine.

ɑO-Conotoxin GeXIVA isomers modulate N-type calcium (CaV 2.2) channels and inwardly-rectifying potassium (GIRK) channels via GABAB receptor activation.

αO-Conotoxin GeXIVA is a 28 amino acid peptide derived from the venom of the marine snail Conus generalis. The presence of four cysteine residues in the structure of GeXIVA allows it to have three different disulfide isomers, that is, the globular, ribbon or bead isomer. All three isomers are active at α9α10 nicotinic acetylcholine receptors, with the bead isomer, GeXIVA[1,2], being the most potent and exhibiting analgesic activity in animal models of neuropathic pain. The original report of GeXIVA activity failed to observe any effect of the isomers on high voltage-activated (HVA) calcium channel currents in rat dorsal root ganglion (DRG) neurons. In this study, we report, for the first time, the activity of globular GeXIVA[1,3] at G protein-coupled GABAB receptors (GABAB R) inhibiting HVA N-type calcium (Cav2.2) channels and reducing membrane excitability in mouse DRG neurons. The inhibition of HVA Ba2+ currents and neuroexcitability by GeXIVA[1,3] was partially reversed by the selective GABAB R antagonist CGP 55845. In transfected HEK293T cells co-expressing human GABAB R1 and R2 subunits and Cav2.2 channels, both GeXIVA[1,3] and GeXIVA[1,4] inhibited depolarization-activated Ba2+ currents mediated by Cav2.2 channels, whereas GeXIVA[1,2] had no effect. The effects of three cyclized GeXIVA[1,4] ribbon isomers were also tested, with cGeXIVA GAG being the most potent at human GABAB R-coupled Cav2.2 channels. Interestingly, globular GeXIVA[1,3] also reversibly potentiated inwardly-rectifying K+ currents mediated by human GIRK1/2 channels co-expressed with GABAB R in HEK293T cells. This study highlights GABAB R as a potentially important receptor target for the activity of αO-conotoxin GeXIVA to mediate analgesia.

Physicochemical Characterization and Evaluation of the Binding Effect of Acacia etbaica Schweinf Gum in Granule and Tablet Formulations.

This study is aimed at evaluating the binding effect of Acacia etbaica gum in granule and tablet formulations using paracetamol as a model drug. Some physicochemical properties of the purified gum such as pH, the presence of tannin and dextrin, solubility, viscosity, loss on drying, total ash value, water solubility index, swelling power, moisture sorption, and powder flow properties were investigated. Paracetamol granules were prepared using wet granulation method at 2%, 4%, 6%, and 8% w/w of the Acacia etbaica gum and compared with granules prepared with reference binders (PVP K-30 and Acacia BP) in similar concentrations. The granules were characterized for bulk and tapped densities, compressibility index and Hausner ratio, angle of repose, flow rate, and friability. Finally, the prepared granules were compressed into tablets and evaluated for different tablet characteristics: weight uniformity, thickness, diameter, crushing strength, tensile strength, friability, disintegration time, and in vitro release profile. The physicochemical characterization revealed that tannins and dextrin are absent in the gum, and the gum has acidic pH. Both the moisture content and total ash values were within the official limits. Furthermore, the gum was found to be soluble in cold and hot water but insoluble in organic solvent and exhibited a shear thickening viscosity profile and excellent flow properties with excellent compressibility. The granules prepared with the gum of Acacia etbaica and reference binders showed good particle size distribution and excellent flow and compressibility properties. All the prepared tablets passed pharmacopeial specifications with respect to their uniformity of weight, thickness, and disintegration time. Tablets formulated with Acacia etbaica gum and acacia BP meet the compendial specification for friability at binder concentrations more than 2%. Drug release properties of all the batches formulated with Acacia etbaica, PVP, and acacia BP complied with the pharmacopeial specification. It can be concluded that the gum of Acacia etbaica could be explored as an alternative excipient for its binder effect in granule and tablet formulations.

Structural Insights Accelerate the Discovery of Opioid Alternatives.

Opioids such as morphine and oxycodone are analgesics frequently prescribed for the treatment of moderate or severe pain. Unfortunately, these medications are associated with exceptionally high abuse potentials and often cause fatal side effects, mainly through the µ-opioid receptor (MOR). Efforts to discover novel, safer, and more efficacious analgesics targeting MOR have encountered challenges. In this review, we summarize alternative strategies and targets that could be used to develop safer nonopioid analgesics. A molecular understanding of G protein-coupled receptor activation and signaling has illuminated not only the complexities of receptor pharmacology but also the potential for pathway-selective agonists and allosteric modulators as safer medications. The availability of structures of pain-related receptors, in combination with high-throughput computational tools, has accelerated the discovery of multitarget ligands with promising pharmacological profiles. Emerging clinical evidence also supports the notion that drugs targeting peripheral opioid receptors have potential as improved analgesic agents.

Tracing the origin of paracetamol tablets by near-infrared, mid-infrared, and nuclear magnetic resonance spectroscopy using principal component analysis and linear discriminant analysis.

Most drugs are no longer produced in their own countries by the pharmaceutical companies, but by contract manufacturers or at manufacturing sites in countries that can produce more cheaply. This not only makes it difficult to trace them back but also leaves room for criminal organizations to fake them unnoticed. For these reasons, it is becoming increasingly difficult to determine the exact origin of drugs. The goal of this work was to investigate how exactly this is possible by using different spectroscopic methods like nuclear magnetic resonance and near- and mid-infrared spectroscopy in combination with multivariate data analysis. As an example, 56 out of 64 different paracetamol preparations, collected from 19 countries around the world, were chosen to investigate whether it is possible to determine the pharmaceutical company, manufacturing site, or country of origin. By means of suitable pre-processing of the spectra and the different information contained in each method, principal component analysis was able to evaluate manufacturing relationships between individual companies and to differentiate between production sites or formulations. Linear discriminant analysis showed different results depending on the spectral method and purpose. For all spectroscopic methods, it was found that the classification of the preparations to their manufacturer achieves better results than the classification to their pharmaceutical company. The best results were obtained with nuclear magnetic resonance and near-infrared data, with 94.6%/99.6% and 98.7/100% of the spectra of the preparations correctly assigned to their pharmaceutical company or manufacturer.

Microwave-Assisted Synthesis, Characterization and Modeling of CPO-27-Mg Metal-Organic Framework for Drug Delivery.

The coordination polymer CPO-27-Mg was rapidly synthesized under microwave irradiation. This material exhibits a sufficiently high drug loading towards aspirin (~8% wt.) and paracetamol (~14% wt.). The binding of these two molecules with the inner surface of the metal-organic framework was studied employing the Gaussian and Plane Wave approach of the Density Functional Theory. The structure of CPO-27-Mg persists after the adsorption of aspirin or paracetamol and their desorption energies, being quite high, decrease under solvent conditions.

Diagnosis of Agglomeration and Crystallinity of Active Pharmaceutical Ingredients in Over the Counter Headache Medication by Electrospray Laser Desorption Ionization Mass Spectrometry Imaging.

Agglomeration of active pharmaceutical ingredients (API) in tablets can lead to decreased bioavailability in some enabling formulations. In a previous study, we determined that crystalline APIs can be detected as agglomeration in tablets formulated with amorphous acetaminophen tablets. Multiple method advancements are presented to better resolve agglomeration caused by crystallinity in standard tablets. In this study, we also evaluate three "budget" over-the-counter headache medications (subsequently labeled as brands A, B, and C) for agglomeration of the three APIs in the formulation: Acetaminophen, aspirin, and caffeine. Electrospray laser desorption ionization mass spectrometry imaging (ELDI-MSI) was used to diagnose agglomeration in the tablets by creating molecular images and observing the spatial distributions of the APIs. Brand A had virtually no agglomeration or clustering of the active ingredients. Brand B had extensive clustering of aspirin and caffeine, but acetaminophen was observed in near equal abundance across the tablet. Brand C also had extensive clustering of aspirin and caffeine, and minor clustering of acetaminophen. These results show that agglomeration with active ingredients in over-the-counter tablets can be simultaneously detected using ELDI-MS imaging.

Zingiber officinale Roscoe rhizome extract alleviates neuropathic pain by inhibiting neuroinflammation in mice.

BACKGROUND: Current therapies for neuropathic pain are generally symptomatic and possess several side effects, limiting their prolonged usage. HYPOTHESIS/PURPOSE: Thus, it is urgent to develop novel and safe candidates for the management of this chronical condition. For this purpose, we investigated the analgesic effect of a standardized extract from Zingiber officinale Roscoe rhizomes (ZOE) obtained by CO2 supercritical extraction, in a mice model of peripheral neuropathy. We also explored the mechanism of action of ZOE and its main constituents using an in vitro model of neuroinflammation. METHODS: Peripheral mono-neuropathy was induced in mice, by spared nerve injury (SNI). The analgesic effect of ZOE after oral administration was assessed by measuring mechanical and thermal allodynia in SNI mice. The mechanism of action of ZOE and its main constituents were investigated using spinal cords samples and in an in vitro model of neuroinflammation by ELISA, western blotting and immunofluorescence techniques. RESULTS: Oral administration of ZOE 200 mg kg-1 ameliorated mechanical and thermal allodynia in SNI mice, with a rapid and a long-lasting effect. ZOE did not alter locomotor activity. In BV2 cells and spinal cord samples, ZOE, 6-gingerol and 6-shogaol reduced pERK levels, whereas ZOE and terpene fraction reduced HDAC1 protein levels, inhibited NF-κB signalling activation and decreased IL-1ß, TNF-α and IL-6 release. ZOE and each tested constituent had a positive effect on inflammation-impaired SH-SY5Y cell viability. CONCLUSIONS: The oral administration of ZOE attenuated SNI-induced neuropathic pain symptoms by reducing spinal neuroinflammation, suggesting ZOE as a novel and interesting candidate for the management of neuropathic pain.

Contribution of low-frequency Raman spectroscopy to determine the solubility curves of drugs in polymers: The case of paracetamol/PVP.

A new method for determining solubility lines of drugs in polymers, based on low-frequency Raman spectroscopy measurements, is described and the results obtained by this method are compared with those obtained using a more classical method based on differential scanning calorimetry investigations. This method was applied to the paracetamol/PVP system using molecular and crystalline dispersions (MCD) rather than usual physical mixtures to reach faster the equilibrium saturated states and make the determination of the solubility line more rapid.

Long-term stability of an infusion containing paracetamol, alizapride, ketorolac and tramadol in glass bottles at 5±3°C.

Background and objective: Infusion containing paracetamol, alizapride, ketorolac and tramadol is used after a general anaesthesia in order to limit pain, fever and nausea. Currently, these infusions are prepared according to demand in the anaesthesia unit, but the preparation in advance could improve quality of preparation and time management. The aim of this study was to investigate the long-term stability of this infusion in glass bottles at 5°C ± 3 °C. Method: Five bottles of infusion were stored at 5°C ± 3 °C for 60 days. A visual and microscope inspection were performed periodically to observe any particle appearance or colour change. pH and absorbance at three wavelengths were measured. The concentrations were measured by ultra-high performance liquid chromatography - diode array detection. Results: Multiple verifications were performed during the first 35 days and no crystal, impurity or colour change were observed. At the next time point (42nd day), crystals were visible to the naked eye. pH and absorbance at 350 nm and 550 nm were stable. A slight increase in the absorbance at 410 nm was observed during the study, suggesting that a degradation product could be formed and absorb at this wavelength. The infusion was considered chemically stable while the lower one-sided prediction limit at 95% remains superior to 90% of the initial concentration. Concentration measurements demonstrated that ketorolac and alizapride remained stable in the infusion for 35 days. The stability of tramadol was 28 days. However, degradation of paracetamol was much faster given that concentration has fallen below 90% of the initial concentration after 7 days. Conclusion: Infusion of paracetamol, alizapride, ketorolac and tramadol remains stable for 7 days in glass bottles at 5°C ± 3 °C and could be prepared in advance with these storage conditions.

Three-way analysis of pH-UV absorbance dataset for the determination of paracetamol and its pKa value in presence of excipients.

A three-way analysis method, parallel factor analysis (PARAFAC) model was applied to the pH-absorbance dataset for the simultaneous determination of paracetamol and its acid-base dissociation constant in presence of excipient interference in a syrup formulation without using chemical pretreatment or chromatographic separation step. The UV spectroscopic data matrices of calibration set, validation and unknown samples were obtained from the absorbance measurements at the five different pH media, considering conjugate acid/base properties of the related drug. Their pH-absorbance data matrices were arranged as a cubic data array (wavelength x sample x pH) (425x52x5). Three-way array of pH-absorbance dataset was decomposed into a trilinear set of spectral, pH and relative concentration profiles of paracetamol and excipients in the commercial syrup using PARAFAC model. In the PARAFAC implementation, paracetamol in the commercial syrup formulation and its pKa value were simultaneously predicted from the relative concentration and pH profiles, respectively. In the method validation step of this study, the performance of PARAFAC model was checked by analyzing the validation samples in terms of selectivity, sensitivity, accuracy and precision of the method. The determination results of paracetamol and its pKa value provided from PARAFAC application were compared to those obtained by a newly developed ultra-performance liquid chromatography (UPLC) method, in terms of simplicity, applicability, interpretability with low cost and short analysis time.

Crystallo-co-spray drying as a new approach to manufacturing of drug/excipient agglomerates: Impact of processing on the properties of paracetamol and lactose mixtures.

The novel process of crystallo-co-spray drying of paracetamol and α-lactose monohydrate was investigated by varying the spray dryer inlet temperature and inlet feed solvent composition. A crystalline agglomerate was obtained with no change to the physical structure of either component throughout both investigations and with possible interactions between the hydroxyl groups of the α-lactose monohydrate and the amide and hydroxyl groups of the paracetamol detected. The percentage soluble fraction of the components in the inlet feed had the largest influence on the morphology of the final dried agglomerate. Low excipient soluble fraction resulted in an increase in paracetamol surface coating and high excipient soluble fraction produced agglomerates of highly mixed components. The use of crystallo-co-spray drying can serve as a method of producing an agglomerated blend of crystalline co-spray dried components leading to possible opportunities for process intensification with the reduction of process steps such as blending, agglomeration, drying and milling into one single stage.

A Novel Acetaminophen Soft-Chew Formulation Produced via Hot-Melt Extrusion with In-line Near-Infrared Monitoring as a Process Analytical Technology Tool.

Patients who suffer from dysphagia have difficulty in swallowing hard tablets and capsules; hence, gelatin-based soft-chew dosages are used as an alternative and novel drug delivery approach to overcome this problem. However, the conventional method of producing gelatin-based soft-chew dosages has many potential issues. The objective of this study was to use glycerol and the hot-melt extrusion technique to address potential issues and optimize the formulation. Gelatin, acetaminophen, saccharin, xylitol, and sodium chloride and six different ratios of water and glycerol were used in the seven formulations. Extrusion process temperature of formulations 1-6 and formulation 7 were 90°C and 140°C, respectively. Near-infrared spectra were collected during extrusion to monitor quality consistency. Scanning electron microscopic images of the cross-section of the soft-chew dosages were recorded. Differential scanning calorimetry (DSC) was used to characterize the crystal states of each formulation. Texture profile analysis was used to evaluate the physical properties of the tablets. In vitro drug release characteristics were studied. A 45-day stability study was carried out to evaluate the stability of each formulation. Near-infrared spectra showed that formulations 1-6 were uniform while formulation 7 was not. From the DSC results, formulations 1 and 2 showed crystallinity of acetaminophen. Formulation 5 displayed the desired physical and chemical stability in texture profile analysis and in the in vitro drug release studies. By using glycerol and hot-melt extrusion, the potential issues of conventional methods were successfully addressed.

Bioisosteric Replacement of Amide Group with 1,2,3-Triazoles in Acetaminophen Addresses Reactive Oxygen Species-Mediated Hepatotoxic Insult in Wistar Albino Rats.

Acetaminophen (AP) is a popularly recommended over-the-counter analgesic-antipyretic in clinical use. However, the drug is handicapped by the occurrence of hepatotoxic insult following acute ingestion. Consequently, AP-induced hepatotoxicity is often implicated in accidental or suicidal overdose. In the current study, we investigated the potential of bioisosteric replacement of amide in AP with 1,2,3-triazoles in curbing AP-induced hepatotoxicity. The therapeutic utility of synthesized bioisosteres was established by careful tailoring and optimization of the synthetic methodology along with detailed toxicological testing of pharmacologically potent acetaminophen-triazole derivatives (APTDs). Along the same lines, we herein report a series of 17 novel APTDs synthesized via aromatic substitution using sodium azide, l-proline, and copper iodide followed by click reaction with substituted alkynes using copper sulfate and sodium ascorbate. Pharmacological evaluation of synthesized APTDs revealed that, out of the series of 17 compounds, 5a and 5e were found to be most efficacious in exerting anti-inflammatory, analgesic, and antipyretic activity in an animal model. Further toxicity studies documented that, in both acute and sub-acute toxicology, AP administration caused significant hepatotoxicity, which was found to be a consequence of ROS-mediated oxidative stress. Potent APTDs (5a and 5e), on the other hand, revealed no adverse event in both acute and sub-toxicological analyses. Median lethal dose (LD50) and no observed adverse effect level (NOAEL) values for 5a and 5e were found to be >1000 mg/kg and 2000 mg/kg, respectively. The human equivalent dose, defining the maximum safe concentration of a compound in a human's physiology, was found to be 27.68 mg/kg for the most potent APTDs (5a and 5e). Thus, it can be concluded that triazole incorporation into AP nucleus produced conjugates devoid of hepatotoxic manifestations, having the added advantage of anti-inflammatory efficacy along with analgesic and antipyretic potency.

Specialized pro-resolving lipid mediators: A new class of non-immunosuppressive and non-opioid analgesic drugs.

We now appreciate that the mechanism of resolution depends on an active and time-dependent biosynthetic shift from pro-inflammatory to pro-resolution mediators, the so-called specialized pro-resolving lipid mediators (SPMs). These SPMs are biosynthesized from the omega-3 fatty acids arachidonic acid (AA), eicosapentaenoic acid (EPA), docosapentaenoic acid (DPA), or docosahexaenoic acid (DHA). Despite effective for a fraction of patients with rheumatic diseases and neuropathic pain, current analgesic therapies such as biological agents, opioids, corticoids, and gabapentinoids cause unwanted side effects, such as immunosuppression, addiction, or induce analgesic tolerance. A growing body of evidence demonstrates that isolated SPMs show efficacy at very low doses and have been successively used as therapeutic drugs to treat pain and infection in experimental models showing no side effects. Moreover, SPMs work as immunoresolvents and some of them present long-lasting analgesic and anti-inflammatory effects (i.e. block pain without immunosuppressive effects). In this review, we focus on how SPMs block pain, infection and neuro-immune interactions and, therefore, emerge as a new class of non-immunosuppressive and non-opioid analgesic drugs.

Revealing Intermolecular Hydrogen Bonding Structure and Dynamics in a Deep Eutectic Pharmaceutical by Magic-Angle Spinning NMR Spectroscopy.

Liquid forms of pharmaceuticals (ionic liquids and deep eutectic solvents) offer a number of potential advantages over solid-state drugs; a key question is the role of intermolecular hydrogen bonding interactions in enabling membrane transport. Characterization is challenging since high sample viscosities, typical of liquid pharmaceutical formulations, hamper the use of conventional solution NMR at ambient temperature. Here, we report the application of magic-angle spinning (MAS) NMR spectroscopy to the deep eutectic pharmaceutical, lidocaine ibuprofen. Using variable temperature MAS NMR, the neat system, at a fixed molar ratio, can be studied over a wide range of temperatures, characterized by changing mobility, using a single experimental setup. Specific intermolecular hydrogen bonding interactions are identified by two-dimensional 1H-1H NOESY and ROESY MAS NMR experiments. Hydrogen-bonding dynamics are quantitatively determined by following the chemical exchange process between the labile protons by means of line-width analysis of variable temperature 1H MAS NMR spectra.

Automatic Dissolution Testing with High-Temporal Resolution for Both Immediate-Release and Fixed-Combination Drug Tablets.

Dissolution testing plays many important roles throughout the pharmaceutical industry, from the research and development of drug products to the control and evaluation of drug quality. However, it is a challenging task to perform both high-efficient separation and high-temporal detection to achieve accurate dissolution profile of each active ingredient dissolved from a drug tablet. In our study, we report a novel non-manual-operation method for performing the automatic dissolution testing of drug tablets, by combining a program-controlled sequential analysis and high-speed capillary electrophoresis for efficient separation of active ingredients. The feasibility of the method for dissolution testing of real drug tablets as well as the performance of the proposed system has been demonstrated. The accuracy of drug dissolution testing is ensured by the excellent repeatability of the sequential analysis, as well as the similarity of the evaluation of dissolution testing. Our study show that the proposed method is capable to achieve simultaneous dissolution testing of multiple ingredients, and the matrix interferences can be avoided. Therefore it is of potential valuable applications in various fields of pharmaceutical research and drug regulation.

Study of a cross-linked hydrogel of Karaya gum and Starch as a controlled drug delivery system.

A cross-linked hydrogel was synthesized using a hybrid backbone of karaya gum starch and grafted with polyacrylic acid. It showed a maximum swelling ratio (SR) of 30.5 g/g at pH 10 and was explored as an oral drug delivery carrier using paracetamol and aspirin as model drugs. In vitro release experiments revealed that maximum drug release at pH 7.4 in comparison to pH 1.2 (simulated intestinal vs gastric fluid) and neutral medium. The release profiles of these drugs showed no initial burst. It also showed good hemocompatibilty and non-cytotoxicity for its employment as a site specific drug delivery agent.