Cannabichromene Racemization and Absolute Stereochemistry Based on a Cannabicyclol Analog.

Cannabichromene (CBC) is unusual among cannabinoids in having been described as both a racemic and a scalemic compound from natural Cannabis sources. Several explanations are available for this circumstance, including facile racemization. Cannabichromene was resolved chromatographically, and the enantiomer matching CBC from local Cannabis was identified. To preclude racemization, CBC was converted to cannabicyclol for further stereochemical analysis. This permitted the (R) absolute stereochemistry to be assigned to natural CBC based on chiroptical data for related natural products and the absolute configuration of a cannabicyclol analog determined by X-ray crystallography. The racemization of CBC was found to be rather slow in the laboratory, but handling practices for natural cannabis products can be inferred to promote the process.

Activation of CB2 Receptors by (-)-Cannabichromene but Not (+)-Cannabichromene.

Introduction: Cannabichromene (CBC) is a minor constituent of cannabis that is a selective cannabinoid CB2 receptor agonist and activator of TRPA1. To date, it has not been shown whether (-)-CBC, (+)-CBC, or both can mediate these effects. In this study, we investigate the activity of the CBC enantiomers at CB1, CB2, and Transient receptor potential ankyrin 1 (TRPA1) receptors in vitro. Materials and Methods: CBC enantiomers were purified from synthetic CBC by chiral chromatography, and their optical activity was confirmed by spectroscopy. Human CB1 and CB2 receptor activity was measured using a fluorescent assay of membrane potential in stably transfected AtT20 cells. TRPA1 activation was measured using a fluorescent assay of intracellular calcium in stably transfected HEK293 cells. Results: The (-)-CBC activated CB2 with an EC50 of 1.5 µM, to a maximum of 60% of (-)CP55940. (+)-CBC did not activate CB2 at concentrations up to 30 µM. Only 30 µM (-)-CBC produced detectable activation of CB1, (+)-CBC was inactive. Both (-)-CBC and (+)-CBC activated TRPA1; at 30 µM (-)-CBC produced an activation 50% of that of the reference agonist cinnamaldehyde (300 µM), 30 µM (+)-CBC activated TRPA1 to 38% of the cinnamaldehyde maximum. Discussion: It is unclear whether (-)-CBC is the sole or even the predominant enantiomer of CBC enzymatically synthesized in cannabis. This study shows that (-)-CBC is the active isomer at CB2 receptors, while both isomers activate TRPA1. The results suggest that medicinal preparations of CBC that target cannabinoid receptors would be most effective when (-)-CBC is the dominant isomer.

Identification of a lichen depside polyketide synthase gene by heterologous expression in Saccharomyces cerevisiae.

Lichen-forming fungi produce a variety of secondary metabolites including bioactive polyketides. Advances in DNA and RNA sequencing have led to a growing database of new lichen gene clusters encoding polyketide synthases (PKS) and associated ancillary activities. Definitive assignment of a PKS gene to a metabolic product has been challenging in the lichen field due to a lack of established gene knockout or heterologous gene expression systems. Here, we report the reconstitution of a non-reducing PKS gene from the lichen Pseudevernia furfuracea and successful heterologous expression of the synthetic lichen PKS gene in engineered Saccharomyces cerevisiae. We show that P. furfuracea PFUR17_02294 produces lecanoric acid, the depside dimer of orsellinic acid, at 360 mg/L in small-scale yeast cultures. Our results unequivocally identify PFUR17_02294 as a lecanoric acid synthase and establish that a single lichen PKS synthesizes two phenolic rings and joins them by an ester linkage to form the depside product.

Alpha1-antitrypsin single dose adjuvant therapy for acute otitis media.

OBJECTIVES: Proteases have been shown to play a role in the pathogenesis of otitis media. Inhibition of these proteases can improve treatment outcomes in certain conditions. The goal of this study was to determine if intratympanic administration of a single dose of the protease inhibitor, recombinant alpha 1-antitrypsin (rAAT), can facilitate resolution of acute otitis media (AOM) in the chinchilla. METHODS AND MEASURES: Pneumococcus was injected into both middle ears of 12 chinchillas. After 3 to 4 days, middle ears were cultured, systemic antibiotics were initiated, and rAAT or its vehicle was administered into the middle ears of all animals. Serial tympanic membrane (TM) scoring, tympanometry, and auditory-evoked brain stem response testing were performed. Animals were sacrificed at varying timepoints and temporal bones studied for objective measures of OM. RESULTS: Although not reaching statistical significance, there was a trend to more rapid resolution of AOM in rAAT-treated ears. Tympanometry, auditory thresholds, and quantitative histologic parameters did not differ between rAAT and vehicle treated ears. CONCLUSIONS: A single dose of intratympanic rAAT likely does not facilitate the resolution of antibiotic-treated pneumococcal AOM in the chinchilla model. Serial administration of this protease inhibitor may be necessary to see a significant treatment effect.

Inflammatory proteases in chronic otitis externa.

OBJECTIVES: Proteases are known to contribute to the pathogenesis of chronic inflammatory skin conditions such as atopic dermatitis and psoriasis. Inhibition of these proteases has shown promise in the treatment of these skin conditions. The purpose of this study was to measure the matrix metalloproteinases (MMP) and human neutrophil elastase (HNE) activities in chronic otitis externa (COE) and to determine whether administration of protease inhibitors recombinant alpha 1-antitrypsin (rAAT) and ilomastat might reduce these protease activities. STUDY DESIGN: Prospective and ex vivo. METHODS: Twenty-five ear canals with COE and 34 with no pathology (i.e., controls) were debrided and filled with saline. After a tragal pump and 1 to 2 minutes, the washes were collected and analyzed for MMP and HNE activities and the inhibitory activity of rAAT and ilomastat on these proteases, respectively. RESULTS: MMP and HNE levels were significantly higher (P = .0057 and .0112) in ears with COE than normal ears. MMP activity greater than 3 mAU/minute was observed in 30% of COE and 0% of controls (P = .0270). HNE activity greater than 3 mAU/minute was found in 77% of COE versus 7% of controls (P < .0001). Ilomastat and rAAT inhibited 60% of MMP and 98% of HNE activity, respectively, in COE ears. CONCLUSIONS: Elevated levels of proteases found in COE, MMP, and HNE may be inhibited with ilomastat and rAAT. The therapeutic potential of these protease inhibitors warrants investigation.

Protease inhibitors alpha1-antitrypsin and ilomastat are not ototoxic in the chinchilla.

OBJECTIVES: Proteases of both the serine and the metalloprotease families have been shown to play a role in the pathogenesis of otitis media. Inhibitors of proteases from each of these families have been shown to beneficially impact disease progression in a number of related chronic inflammatory conditions. The purpose of this study was to assess the safety of protease inhibitors when instilled into the middle ear, with a view to their potential use in the treatment of human otitis media. STUDY DESIGN: Prospective, randomized, controlled trial in the chinchilla model. METHODS: After completing baseline auditory testing and bilateral transpalatal obstruction of the Eustachian tube, chinchillas received weekly transbullar injections of protease inhibitor (alpha1-antitrypsin, ilomastat, or both), vehicle, or saline. After 1 month, hearing was tested and the animals were sacrificed. Temporal bone histopathologic examination was performed. RESULTS: All treatment groups demonstrated a statistically insignificant average loss in long-term hearing (0 dB) for all measures using clicks and tones (P >.15 for all conditions). All treatment groups were statistically insignificantly different from one another (P =.5625). Histopathologic examination revealed no significant inner ear changes. CONCLUSIONS: Protease inhibitors that are currently under study in animal models and humans for the treatment of inflammatory diseases that are related to imbalances between protease and protease inhibitor have no significant toxic effect on the inner ear of chinchillas. These findings support the safety of further clinical trials using these inhibitors to treat middle ear inflammation.

Alpha 1-antitrypsin and ilomastat inhibit inflammatory proteases present in human middle ear effusions.

OBJECTIVES: Proteases of both the serine and metalloproteinase families have been shown to play a role in the pathogenesis of otitis media (OM). Inhibitors of proteases from each of these families have been shown to beneficially impact disease progression in a number of related chronic inflammatory conditions, but their use has not been studied in OM. The purpose of this study was to assess the activity of the protease inhibitors recombinant alpha 1-antitrypsin (rAAT) and ilomastat on inflammatory proteases present in human middle ear effusions (MEEs), with a view to their potential utility in the treatment of OM. STUDY DESIGN: Prospective and ex vivo. METHODS: MEEs were collected from 100 patients presenting for middle ear surgery, most commonly tympanostomy tube placement or treatment of acute posttympanostomy otorrhea (APTO). MEEs were analyzed for the presence of matrix metalloproteinases (MMPs) and human neutrophil elastase (HNE) and the inhibitory activity of rAAT and ilomastat on these proteases, respectively. RESULTS: MMP levels were highest in APTO, and HNE was highest in chronic suppurative OM and APTO. High levels of MMP and HNE (>3 mAU/min) were found in 52% and 37% of MEEs, respectively. Ilomastat and rAAT demonstrated significant inhibition of MMP and HNE activity (>30% reduction), respectively, in 80% and 82% of MEEs with high levels of activity. CONCLUSIONS: Proteases are commonly found in OM. Ilomastat and rAAT are potent inhibitors of proteases in MEEs across a wide range of OM in humans. Investigation into the potential therapeutic benefits of these protease inhibitors is warranted.

Inhaled recombinant alpha 1-antitrypsin ameliorates cigarette smoke-induced emphysema in the mouse.

In alpha 1-antitrypsin deficiency in humans, inadequately regulated activity of serine protease activity is responsible for the chronic lung tissue degeneration and irreversible loss of pulmonary function seen in those individuals with emphysema. Typically, disease symptoms in this patient population are exacerbated by cigarette smoke. Here we show that inhaled recombinant alpha 1-antitrypsin (rAAT) can provide significant protection against the development of emphysema in cigarette smoke-treated mice. As has been reported previously, cigarette smoke was seen to increase significantly the recruitment of neutrophils and macrophages into the lungs of these animals, leading to concomitant alveolar airspace enlargement and emphysema. In smoking animals treated for 6 months with inhaled rAAT, effects on lavage levels of neutrophils and macrophages were only moderate when compared with untreated animals. Furthermore, neutralizing antibodies to rAAT were generated in all rAAT-treated animals. Despite this, however, reductions in airspace enlargement of up to 73% were observed. These findings demonstrate that delivery of rAAT directly to the lungs of smoke-treated mice can inhibit lung tissue damage mediated by proteases, suggesting that rAAT inhalation therapy might represent a practical approach towards treating emphysema in humans, by modifying the course of the disease.

An inhaled matrix metalloprotease inhibitor prevents cigarette smoke-induced emphysema in the mouse.

Inadequately regulated proteolytic activity is responsible for the chronic lung tissue degeneration and irreversible loss of pulmonary function that define emphysema. In this study, we show that an inhaled broad-spectrum matrix metalloprotease inhibitor, ilomastat, can provide protection against the development of emphysema in cigarette smoke-treated mice. Control animals were exposed to daily cigarette smoke for 6 months. As has been reported previously, cigarette smoke was seen to increase significantly the recruitment of macrophages into the lungs of these animals, leading to concomitant alveolar airspace enlargement and emphysema. In animals treated daily with nebulized ilomastat for 6 months, lung macrophage levels were greatly reduced, and neutrophil accumulation was also inhibited. Corresponding reductions in airspace enlargement of up to 96% were observed. These striking observations suggest that delivery of ilomastat directly into the lungs of smoke-treated mice can not only inhibit lung tissue damage mediated by metalloproteases, but may also reduce that component of tissue degeneration mediated by excess neutrophil-derived products. Our data also suggest that the matrix metalloprotease inhibitors may represent a class of drugs that, when delivered by inhalation, could be used practically to treat cigarette smoking-related chronic obstructive pulmonary disease by modifying the course of the disease.