Terpenes from Cannabis sativa induce antinociception in a mouse model of chronic neuropathic pain via activation of adenosine A2A receptors.

ABSTRACT: Terpenes are small hydrocarbon compounds that impart aroma and taste to many plants, including Cannabis sativa. A number of studies have shown that terpenes can produce pain relief in various pain states in both humans and animals. However, these studies were methodologically limited and few established mechanisms of action. In our previous work, we showed that the terpenes geraniol, linalool, ß-pinene, α-humulene, and ß-caryophyllene produced cannabimimetic behavioral effects via multiple receptor targets. We thus expanded this work to explore the potential antinociception and mechanism of these Cannabis terpenes in a mouse model of chronic pain. We first tested for antinociception by injecting terpenes (200 mg/kg, IP) into male and female CD-1 mice with mouse models of chemotherapy-induced peripheral neuropathy (CIPN) or lipopolysaccharide-induced inflammatory pain, finding that the terpenes produced roughly equal antinociception to 10 mg/kg morphine or 3.2 mg/kg WIN55,212. We further found that none of the terpenes produced reward as measured by conditioned place preference, while low doses of terpene (100 mg/kg) combined with morphine (3.2 mg/kg) produced enhanced antinociception vs either alone. We then used the adenosine A2A receptor (A2AR) selective antagonist istradefylline (3.2 mg/kg, IP) and spinal cord-specific CRISPR knockdown of the A2AR to identify this receptor as the mechanism for terpene antinociception in CIPN. In vitro cAMP and binding studies and in silico modeling studies further suggested that the terpenes act as A2AR agonists. Together these studies identify Cannabis terpenes as potential therapeutics for chronic neuropathic pain and identify a receptor mechanism for this activity.

Terpenes from Cannabis sativa Induce Antinociception in Mouse Chronic Neuropathic Pain via Activation of Spinal Cord Adenosine A2A Receptors.

Terpenes are small hydrocarbon compounds that impart aroma and taste to many plants, including Cannabis sativa. A number of studies have shown that terpenes can produce pain relief in various pain states in both humans and animals. However, these studies were methodologically limited and few established mechanisms of action. In our previous work, we showed that the terpenes geraniol, linalool, ß-pinene, α-humulene, and ß-caryophyllene produced cannabimimetic behavioral effects via multiple receptor targets. We thus expanded this work to explore the efficacy and mechanism of these Cannabis terpenes in relieving chronic pain. We first tested for antinociceptive efficacy by injecting terpenes (200 mg/kg, IP) into male and female CD-1 mice with chemotherapy-induced peripheral neuropathy (CIPN) or lipopolysaccharide-induced inflammatory pain, finding that the terpenes produced roughly equal efficacy to 10 mg/kg morphine or 3.2 mg/kg WIN55,212. We further found that none of the terpenes produced reward as measured by conditioned place preference, while low doses of terpene (100 mg/kg) combined with morphine (3.2 mg/kg) produced enhanced antinociception vs. either alone. We then used the adenosine A2A receptor (A2AR) selective antagonist istradefylline (3.2 mg/kg, IP) and spinal cord-specific CRISPR knockdown of the A2AR to identify this receptor as the mechanism for terpene antinociception in CIPN. In vitro cAMP and binding studies and in silico modeling studies further suggested that the terpenes act as A2AR agonists. Together these studies identify Cannabis terpenes as potential therapeutics for chronic neuropathic pain, and identify a receptor mechanism in the spinal cord for this activity.

Structure-Based Design of Glycosylated Oxytocin Analogues with Improved Selectivity and Antinociceptive Activity.

Acute and chronic pain is often treated with opioids despite the negative side effects of constipation, physical dependence, respiratory depression, and overdose. The misuse of opioid analgesics has given rise to the opioid crisis/epidemic, and alternate nonaddictive analgesics are urgently needed. Oxytocin, a pituitary hormone, is an alternative to the small molecule treatments available and has been used as an analgesic as well as for the treatment and prevention of opioid use disorder (OUD). Clinical implementation is limited by its poor pharmacokinetic profile, a result of the labile disulfide bond between two cysteine residues in the native sequence. Stable brain penetrant oxytocin analogues have been synthesized by replacement of the disulfide bond with a stable lactam and glycosidation of the C-terminus. These analogues show exquisite selectivity for the oxytocin receptor and potent in vivo antinociception in mice following peripheral (i.v.) administration, supporting further study of their clinical potential.

Synthesis, Physicochemical Characterization, In Vitro 2D/3D Human Cell Culture, and In Vitro Aerosol Dispersion Performance of Advanced Spray Dried and Co-Spray Dried Angiotensin (1-7) Peptide and PNA5 with Trehalose as Microparticles/Nanoparticles for Targeted Respiratory Delivery as Dry Powder Inhalers.

The peptide hormone Angiotensin (1-7), Ang (1-7) or (Asp-Arg-Val-Tyr-Ile-His-Pro), is an essential component of the renin-angiotensin system (RAS) peripherally and is an agonist of the Mas receptor centrally. Activation of this receptor in the CNS stimulates various biological activities that make the Ang (1-7)/MAS axis a novel therapeutic approach for the treatment of many diseases. The related O-linked glycopeptide, Asp-Arg-Val-Tyr-Ile-His-Ser-(O-ß-D-Glc)-amide (PNA5), is a biousian revision of the native peptide hormone Ang (1-7) and shows enhanced stability in vivo and greater levels of brain penetration. We have synthesized the native Ang (1-7) peptide and the glycopeptide, PNA5, and have formulated them for targeted respiratory delivery as inhalable dry powders. Solid phase peptide synthesis (SPPS) successfully produced Ang (1-7) and PNA5. Measurements of solubility and lipophilicity of raw Ang (1-7) and raw PNA5 using experimental and computational approaches confirmed that both the peptide and glycopeptide have high-water solubility and are amphipathic. Advanced organic solution spray drying was used to engineer the particles and produce spray-dried powders (SD) of both the peptide and the glycopeptide, as well as co-spray-dried powders (co-SD) with the non-reducing sugar and pharmaceutical excipient, trehalose. The native peptide, glycopeptide, SD, and co-SD powders were comprehensively characterized, and exhibited distinct glass transitions (Tg) consistent with the amorphous glassy state formation with Tgs that are compatible with use in vivo. The homogeneous particles displayed small sizes in the nanometer size range and low residual water content in the solid-state. Excellent aerosol dispersion performance with a human DPI device was demonstrated. In vitro human cell viability assays showed that Ang (1-7) and PNA5 are biocompatible and safe for different human respiratory and brain cells.

Organic Solution Advanced Spray-Dried Microparticulate/Nanoparticulate Dry Powders of Lactomorphin for Respiratory Delivery: Physicochemical Characterization, In Vitro Aerosol Dispersion, and Cellular Studies.

The purpose of this study was to formulate Lactomorphin (MMP2200) in its pure state as spray-dried(SD) powders, and with the excipient Trehalose as co-spray-dried(co-SD) powders; for intranasal and deep lung administration with Dry Powder Inhalers (DPI). Lactomorphin is a glycopeptide which was developed for the control of moderate to severe pain. Particles were rationally designed and produced by advanced spray drying particle engineering in a closed mode from a dilute organic solution. Comprehensive physicochemical characterization using different analytical techniques was carried out to analyze the particle size, particle morphology, particle surface morphology, solid-state transitions, crystallinity/non-crystallinity, and residual water content. The particle chemical composition was confirmed using attenuated total reflectance-Fourier-transform infrared (ATR-FTIR), and Confocal Raman Microscopy (CRM) confirmed the particles' chemical homogeneity. The solubility and Partition coefficient (LogP) of Lactomorphin were determined by the analytical and computational methodology and revealed the hydrophilicity of Lactomorphin. A thermal degradation study was performed by exposing samples of solid-state Lactomorphin to a high temperature (62 °C) combined with zero relative humidity (RH) and to a high temperature (62 °C) combined with a high RH (75%) to evaluate the stability of Lactomorphin under these two different conditions. The solid-state processed particles exhibited excellent aerosol dispersion performance with an FDA-approved human DPI device to reach lower airways. The cell viability resazurin assay showed that Lactomorphin is safe up to 1000 µg/mL on nasal epithelium cells, lung cells, endothelial, and astrocyte brain cells.

Monoclonal 1- and 3-Phosphohistidine Antibodies: New Tools to Study Histidine Phosphorylation.

Histidine phosphorylation (pHis) is well studied in bacteria; however, its role in mammalian signaling remains largely unexplored due to the lack of pHis-specific antibodies and the lability of the phosphoramidate (P-N) bond. Both imidazole nitrogens can be phosphorylated, forming 1-phosphohistidine (1-pHis) or 3-phosphohistidine (3-pHis). We have developed monoclonal antibodies (mAbs) that specifically recognize 1-pHis or 3-pHis; they do not cross-react with phosphotyrosine or the other pHis isomer. Assays based on the isomer-specific autophosphorylation of NME1 and phosphoglycerate mutase were used with immunoblotting and sequencing IgG variable domains to screen, select, and characterize anti-1-pHis and anti-3-pHis mAbs. Their sequence independence was determined by blotting synthetic peptide arrays, and they have been tested for immunofluorescence staining and immunoaffinity purification, leading to putative identification of pHis-containing proteins. These reagents should be broadly useful for identification of pHis substrates and functional study of pHis using a variety of immunological, proteomic, and biological assays.

Novel factor Xa inhibitors based on a benzoic acid scaffold and incorporating a neutral P1 ligand.

A series of novel, highly potent, achiral factor Xa inhibitors based on a benzoic acid scaffold and containing a chlorophenethyl moiety directed towards the protease S1 pocket is described. A number of structural features, such as the requirements of the P1, P4 and ester-binding pocket ligands were explored with respect to inhibition of factor Xa. Compound 46 was found to be the most potent compound in a series of antithrombotic secondary assays.

Microwave-assisted solid-phase synthesis (MASS): parallel and combinatorial chemical library synthesis.

The use of microwave technology in solid-phase organic synthesis has attracted much attention in recent years. The combination of solid support, either as a medium for chemical synthesis or as a carrier for organic reagents, with microwave heating offers several advantages over conventional techniques. Rapid and elevated heating of reaction mixtures can induce the completion of chemical transformations in minutes while several hours or days may be required for the same chemistry under conventional conditions. With decreased time of exposure to high temperatures and lessened thermal degradation, microwave accelerated chemistries often deliver products of higher purity when compared to conventional heating techniques. Several chemical syntheses on solid-phase employing microwave irradiation have been reported in the literature. The reagents, solvents, and equipment selected for microwave-mediated synthesis are important contributors to the success of the chemical transformation. Owing to the timesavings in performing chemical synthesis under microwave irradiation, the technique has become an emerging partner in solid-phase organic synthesis.

Design and quantitative structure-activity relationship of 3-amidinobenzyl-1H-indole-2-carboxamides as potent, nonchiral, and selective inhibitors of blood coagulation factor Xa.

A series of 138 nonchiral 3-amidinobenzyl-1H-indole-2-carboxamides and analogues as inhibitors of the blood coagulation enzyme factor Xa (fXa) were designed, synthesized, and investigated by X-ray structure analysis and 3D quantitative structure-activity relationship (QSAR) studies (CoMFA, CoMSIA) in order to identify important protein-ligand interactions responsible for biological affinity and selectivity. Several compounds from this series are highly potent and selective inhibitors of this important enzyme linking extrinsic and intrinsic coagulation pathways. To rationalize biological affinity and to provide guidelines for further design, all compounds were docked into the factor Xa binding site. Those docking studies were based on X-ray structures of factor Xa in complex with literature-known inhibitors. It was possible to validate those binding modes by four X-ray crystal structures of representative ligands in factor Xa, while one ligand was additionally crystallized in trypsin to rationalize requirements for selective factor Xa inhibition. The 3D-QSAR models based on a superposition rule derived from these docking studies were validated using conventional and cross-validated r(2) values using the leave-one-out method and repeated analyses using two randomly chosen cross-validation groups plus randomization of biological activities. This led to consistent and highly predictive 3D-QSAR models with good correlation coefficients for both CoMFA and CoMSIA, which were found to correspond to experimentally determined factor Xa binding site topology in terms of steric, electrostatic, and hydrophobic complementarity. Subsets selected as smaller training sets using 2D fingerprints and maximum dissimilarity methods resulted in 3D-QSAR models with remarkable correlation coefficients and a high predictive power. The final quantitative SAR information agrees with all experimental data for the binding topology and thus provides reasonable activity predictions for novel factor Xa inhibitors.