Structure-Based Identification of Potent Natural Product Chemotypes as Cannabinoid Receptor 1 Inverse Agonists.

Natural products are an abundant source of potential drugs, and their diversity makes them a rich and viable prospective source of bioactive cannabinoid ligands. Cannabinoid receptor 1 (CB1) antagonists are clinically established and well documented as potential therapeutics for treating obesity, obesity-related cardiometabolic disorders, pain, and drug/substance abuse, but their associated CNS-mediated adverse effects hinder the development of potential new drugs and no such drug is currently on the market. This limitation amplifies the need for new agents with reduced or no CNS-mediated side effects. We are interested in the discovery of new natural product chemotypes as CB1 antagonists, which may serve as good starting points for further optimization towards the development of CB1 therapeutics. In search of new chemotypes as CB1 antagonists, we screened the in silico purchasable natural products subset of the ZINC12 database against our reported CB1 receptor model using the structure-based virtual screening (SBVS) approach. A total of 18 out of 192 top-scoring virtual hits, selected based on structural diversity and key protein⁻ligand interactions, were purchased and subjected to in vitro screening in competitive radioligand binding assays. The in vitro screening yielded seven compounds exhibiting >50% displacement at 10 µM concentration, and further binding affinity (Ki and IC50) and functional data revealed compound 16 as a potent and selective CB1 inverse agonist (Ki = 121 nM and EC50 = 128 nM) while three other compounds-2, 12, and 18-were potent but nonselective CB1 ligands with low micromolar binding affinity (Ki). In order to explore the structure⁻activity relationship for compound 16, we further purchased compounds with >80% similarity to compound 16, screened them for CB1 and CB2 activities, and found two potent compounds with sub-micromolar activities. Most importantly, these bioactive compounds represent structurally new natural product chemotypes in the area of cannabinoid research and could be considered for further structural optimization as CB1 ligands.

Development of a Δ9-Tetrahydrocannabinol Amino Acid-Dicarboxylate Prodrug With Improved Ocular Bioavailability.

Purpose: The aim of the present study was to evaluate the utility of the relatively hydrophilic Δ9-tetrahydrocannabinol (THC) prodrugs, mono and di-valine esters (THC-Val and THC-Val-Val) and the amino acid (valine)-dicarboxylic acid (hemisuccinate) ester (THC-Val-HS), with respect to ocular penetration and intraocular pressure (IOP) lowering activity. THC, timolol, and pilocarpine eye drops were used as controls. Methods: THC-Val, THC-Val-Val, and THC-Val-HS were synthesized and chemically characterized. Aqueous solubility and in vitro transcorneal permeability of THC and the prodrugs, in the presence of various surfactants and cyclodextrins, were determined. Two formulations were evaluated for therapeutic activity in the α-chymotrypsin induced rabbit glaucoma model, and the results were compared against controls comprising of THC emulsion and marketed timolol maleate and pilocarpine eye drops. Results: THC-Val-HS demonstrated markedly improved solubility (96-fold) and in vitro permeability compared to THC. Selected formulations containing THC-Val-HS effectively delivered THC to the anterior segment ocular tissues in the anesthetized rabbits: 62.1 ng/100 µL of aqueous humor (AH) and 51.4 ng/50 mg of iris ciliary bodies (IC) (total THC). The duration and extent of IOP lowering induced by THC-Val-HS was 1 hour longer and 10% greater, respectively, than that obtained with THC and was comparable with the pilocarpine eye drops. Timolol ophthalmic drops, however, exhibited a longer duration of activity. Both THC and THC-Val-HS were detected in the ocular tissues following multiple dosing of THC-Val-HS in conscious animals. The concentration of THC in the iris-ciliary bodies at the 60- and 120-minute time points (53 and 57.4 ng/50 mg) were significantly greater than that of THC-Val-HS (24.2 and 11.3 ng/50 mg). Moreover, at the two time points studied, the concentration of THC was observed to increase or stay relatively constant, whereas THC-Val-HS concentration decreased by at least 50%. A similar trend was observed in the retina-choroid tissues. Conclusions: A combination of prodrug derivatization and formulation development approaches significantly improved the penetration of THC into the anterior segment of the eye following topical application. Enhanced ocular penetration resulted in significantly improved IOP-lowering activity.

In vitro opioid receptor affinity and in vivo behavioral studies of Nelumbo nucifera flower.

ETHNOPHARMACOLOGICAL RELEVANCE: Nelumbo nucifera Geartn., known as sacred lotus, has been used traditionally in South East Asia as a traditional medicine for various CNS disorders including stress, fever, depression, insomnia, and cognitive conditions. AIM OF THE STUDY: To investigate the in vitro cannabinoid and opioid receptor binding affinities, and in vivo behavioral actions of Nelumbo flower extracts and to isolate the potential compounds to treat CNS associated disorders. MATERIALS AND METHODS: The white and pink flowers of N. nucifera were extracted with 95% EtOH, followed by acid-base partitioning using CHCl3 to give acidic and basic partitions. These partitions were subjected to Centrifugal Preparative TLC (CPTLC) to yield benzyltetrahydroisoquinoline (BTIQ) alkaloids and long chain fatty acids, identified by physical and spectroscopic methods. In addition, EtOH extracts and partitions were analyzed for chemical markers by UHPLC/MS and GC/MS. In vitro neuropharmacological effects were evaluated by cannabinoid (CB1 and CB2) and opioid [delta (δ), kappa (ĸ), and mu (µ)] competitive radioligand binding and GTPγS functional assays. The in vivo behavioral effect was studied through the use of the mouse tetrad assay at 10, 30, 75 and 100mg/kg/ip doses that revealed the effect on locomotion, catalepsy, body temperature, and nociception of acidic and basic CHCl3 partitions, fractions, and compounds. RESULTS: Three aporphines, nuciferine (1), N-nor-nuciferine (2), asimilobine (3), and five BTIQs, armepavine (4), O-methylcoclaurine (5), N-methylcoclaurine (6), coclaurine (7), neferine (10), and a mixture of linoleic and palmitic acids (LA and PA), were identified and evaluated for cannabinoid and opioid receptor displacement activities. Compounds 5-7 showed binding affinities for the ĸ opioid receptor with equilibrium dissociation constant (Ki) values of 3.5 ± 0.3, 0.9 ± 0.1, 2.2 ± 0.2 µM, respectively. Compound 10 displayed affinities for δ-and µ- opioid receptors with Ki values of 0.7 ± 0.1 and 1.8 ± 0.2 µM, respectively, and was determined to be a weak δ agonist by GTPγS functional assay. The mixture of LA and PA (1:1) showed an affinity for δ opioid receptor with a Ki value of 9.2 ± 1.1 µM. The acidic and basic CHCl3 partitions, compounds 1 and 7, and 5-7 mixture were subjected to the tetrad assay, of which the acidic partition displayed decreased locomotion and increased catalepsy, antinociception, and hypothermia in animal at doses of 75-100 mg/kg/ip, and also showed clonic-tonic seizures upon touch at 100mg/kg. CONCLUSION: Bioassay-guided isolation revealed compounds 5-7, 10, and the mixture of LA and PA displayed various degrees of opioid receptor radioligand displacement affinities. The in vivo tetrad assay of acidic CHCl3 partition, enriched with aporphines 1 and 2, displayed actions on all four points of behavioral parameters. It can be concluded that the in vivo mild canabimimetic-type effect observed for the CHCl3 partition is likely mediated through other CNS mechanisms since the extracts, partitions, and isolated compounds had no affinity for the in vitro CB1 and CB2 receptors. This work, along with traditional use and the reported bioactivities of the BTIQ alkaloids, suggested further studies on N. nucifera are needed to understand the roles that the extracts and/or individual compounds might contribute to the behavioral effects.

Flavonoids from Perovskia atriplicifolia and Their in Vitro Displacement of the Respective Radioligands for Human Opioid and Cannabinoid Receptors.

Bioassay-guided fractionation of the leaves of Perovskia atriplicifolia (Russian sage) resulted in the isolation of four previously known flavonoid derivatives, 5-hydroxy-6,7,3',4'-tetramethoxyflavone (1), 5,7-dihydroxy-6,3',4'-trimethoxyflavone (2), 5-hydroxy-6,7,4'-trimethoxyflavone (3), and 5,7-dihydroxy-6,4'-dimethoxyflavone (4). Compounds 1, 3, and 4 showed displacement of the radioligand for the cloned human δ opioid receptor with Ki values ranging from 3.1 to 26.0 µM. In addition, the binding mode of the compounds in the active site of the δ opioid receptor was investigated through molecular modeling algorithms. This study may have implications in better understanding non-nitrogenous δ opioid receptor ligands.

Targeted injection of a biocomposite material alters macrophage and fibroblast phenotype and function following myocardial infarction: relation to left ventricular remodeling.

A treatment target for progressive left ventricular (LV) remodeling prevention following myocardial infarction (MI) is to affect structural changes directly within the MI region. One approach is through targeted injection of biocomposite materials, such as calcium hydroxyapatite microspheres (CHAM), into the MI region. In this study, the effects of CHAM injections upon key cell types responsible for the MI remodeling process, the macrophage and fibroblast, were examined. MI was induced in adult pigs before randomization to CHAM injections (20 targeted 0.1-ml injections within MI region) or saline. At 7 or 21 days post-MI (n = 6/time point per group), cardiac magnetic resonance imaging was performed, followed by macrophage and fibroblast isolation. Isolated macrophage profiles for monocyte chemotactic macrophage inflammatory protein-1 as measured by real-time polymerase chain reaction increased at 7 days post-MI in the CHAM group compared with MI only (16.3 ± 6.6 versus 1.7 ± 0.6 cycle times values, P < 0.05), and were similar by 21 days post-MI. Temporal changes in fibroblast function and smooth muscle actin (SMA) expression relative to referent control (n = 5) occurred with MI. CHAM induced increases in fibroblast proliferation, migration, and SMA expression-indicative of fibroblast transformation. By 21 days, CHAM reduced LV dilation (diastolic volume: 75 ± 2 versus 97 ± 4 ml) and increased function (ejection fraction: 48 ± 2% versus 38 ± 2%) compared with MI only (both P < 0.05). This study identified that effects on macrophage and fibroblast differentiation occurred with injection of biocomposite material within the MI, which translated into reduced adverse LV remodeling. These unique findings demonstrate that biomaterial injections impart biologic effects upon the MI remodeling process over any biophysical effects.

Flavanones from Miconia prasina.

A glycosidic flavanone miconioside C (1) has been isolated from the methanolic extract of the stems of Miconia prasina, together with 7-O-ß-D-apiofuranosyl-(1→6)-ß-D-glucopyranosylmatteucinol (2), miconioside B (3), matteucinol (4), farrerol (5) and desmethoxymatteucinol (6). Their structures were mainly established by extensive NMR studies (1H NMR, 13C NMR, DEPT, 1H-1H COSY, HSQC, HMBC) and mass spectrometry. The compounds 1- 3 were evaluated for in vitro binding assays using cannabinoid receptors (CB1 and CB2).

Injectable and bioresponsive hydrogels for on-demand matrix metalloproteinase inhibition.

Inhibitors of matrix metalloproteinases (MMPs) have been extensively explored to treat pathologies where excessive MMP activity contributes to adverse tissue remodelling. Although MMP inhibition remains a relevant therapeutic target, MMP inhibitors have not translated to clinical application owing to the dose-limiting side effects following systemic administration of the drugs. Here, we describe the synthesis of a polysaccharide-based hydrogel that can be locally injected into tissues and releases a recombinant tissue inhibitor of MMPs (rTIMP-3) in response to MMP activity. Specifically, rTIMP-3 is sequestered in the hydrogels through electrostatic interactions and is released as crosslinks are degraded by active MMPs. Targeted delivery of the hydrogel/rTIMP-3 construct to regions of MMP overexpression following a myocardial infarction significantly reduced MMP activity and attenuated adverse left ventricular remodelling in a porcine model of myocardial infarction. Our findings demonstrate that local, on-demand MMP inhibition is achievable through the use of an injectable and bioresponsive hydrogel.

Local hydrogel release of recombinant TIMP-3 attenuates adverse left ventricular remodeling after experimental myocardial infarction.

An imbalance between matrix metalloproteinases (MMPs) and tissue inhibitors of MMPs (TIMPs) contributes to the left ventricle (LV) remodeling that occurs after myocardial infarction (MI). However, translation of these observations into a clinically relevant, therapeutic strategy remains to be established. The present study investigated targeted TIMP augmentation through regional injection of a degradable hyaluronic acid hydrogel containing recombinant TIMP-3 (rTIMP-3) in a large animal model. MI was induced in pigs by coronary ligation. Animals were then randomized to receive targeted hydrogel/rTIMP-3, hydrogel alone, or saline injection and followed for 14 days. Instrumented pigs with no MI induction served as referent controls. Multimodal imaging (fluoroscopy/echocardiography/magnetic resonance imaging) revealed that LV ejection fraction was improved, LV dilation was reduced, and MI expansion was attenuated in the animals treated with rTIMP-3 compared to all other controls. A marked reduction in proinflammatory cytokines and increased smooth muscle actin content indicative of myofibroblast proliferation occurred in the MI region with hydrogel/rTIMP-3 injections. These results provide the first proof of concept that regional sustained delivery of an MMP inhibitor can effectively interrupt adverse post-MI remodeling.