A randomized, double-blind, placebo-controlled study of daily cannabidiol for the treatment of canine osteoarthritis pain.

ABSTRACT: Over the last 2 decades, affirmative diagnoses of osteoarthritis (OA) in the United States have tripled due to increasing rates of obesity and an aging population. Hemp-derived cannabidiol (CBD) is the major nontetrahydrocannabinol component of cannabis and has been promoted as a potential treatment for a wide variety of disparate inflammatory conditions. Here, we evaluated CBD for its ability to modulate the production of proinflammatory cytokines in vitro and in murine models of induced inflammation and further validated the ability of a liposomal formulation to increase bioavailability in mice and in humans. Subsequently, the therapeutic potential of both naked and liposomally encapsulated CBD was explored in a 4-week, randomized placebo-controlled, double-blinded study in a spontaneous canine model of OA. In vitro and in mouse models, CBD significantly attenuated the production of proinflammatory cytokines IL-6 and TNF-α while elevating levels of anti-inflammatory IL-10. In the veterinary study, CBD significantly decreased pain and increased mobility in a dose-dependent fashion among animals with an affirmative diagnosis of OA. Liposomal CBD (20 mg/day) was as effective as the highest dose of nonliposomal CBD (50 mg/day) in improving clinical outcomes. Hematocrit, comprehensive metabolic profile, and clinical chemistry indicated no significant detrimental impact of CBD administration over the 4-week analysis period. This study supports the safety and therapeutic potential of hemp-derived CBD for relieving arthritic pain and suggests follow-up investigations in humans are warranted.

Liposomal-encapsulated Ascorbic Acid: Influence on Vitamin C Bioavailability and Capacity to Protect Against Ischemia-Reperfusion Injury.

Intravenous administration of vitamin C has been shown to decrease oxidative stress and, in some instances, improve physiological function in adult humans. Oral vitamin C administration is typically less effective than intravenous, due in part to inferior vitamin C bioavailability. The purpose of this study was to determine the efficacy of oral delivery of vitamin C encapsulated in liposomes. On 4 separate randomly ordered occasions, 11 men and women were administered an oral placebo, or 4 g of vitamin C via oral, oral liposomal, or intravenous delivery. The data indicate that oral delivery of 4 g of vitamin C encapsulated in liposomes (1) produces circulating concentrations of vitamin C that are greater than unencapsulated oral but less than intravenous administration and (2) provides protection from ischemia-reperfusion-mediated oxidative stress that is similar to the protection provided by unencapsulated oral and intravenous administrations.

The putidaredoxin reductase-putidaredoxin electron transfer complex: theoretical and experimental studies.

Interaction and electron transfer between putidaredoxin reductase (Pdr) and putidaredoxin (Pdx) from Pseudomonas putida was studied by molecular modeling, mutagenesis, and stopped flow techniques. Based on the crystal structures of Pdr and Pdx, a complex between the proteins was generated using computer graphics methods. In the model, Pdx is docked above the isoalloxazine ring of FAD of Pdr with the distance between the flavin and [2Fe-2S] of 14.6 A. This mode of interaction allows Pdx to easily adjust and optimize orientation of its cofactor relative to Pdr. The key residues of Pdx located at the center, Asp(38) and Trp(106), and at the edge of the protein-protein interface, Tyr(33) and Arg(66), were mutated to test the Pdr-Pdx computer model. The Y33F, Y33A, D38N, D38A, R66A, R66E, W106F, W106A, and Delta106 mutations did not affect assembly of the [2Fe-2S] cluster and resulted in a marginal change in the redox potential of Pdx. The electron-accepting ability of Delta106 Pdx was similar to that of the wild-type protein, whereas electron transfer rates from Pdr to other mutants were diminished to various degrees with the smallest and largest effects on the kinetic parameters of the Pdr-to-Pdx electron transfer reaction caused by the Trp(106) and Tyr(33)/Arg(66) substitutions, respectively. Compared with wild-type Pdx, the binding affinity of all studied mutants to Pdr was significantly higher. Experimental results were in agreement with theoretical predictions and suggest that: (i) Pdr-Pdx complex formation is mainly driven by steric complementarity, (ii) bulky side chains of Tyr(33), Arg(66), and Trp(106) prevent tight binding of oxidized Pdx and facilitate dissociation of the reduced iron-sulfur protein from Pdr, and (iii) transfer of an electron from FAD to [2Fe-2S] can occur with various orientations between the cofactors through multiple electron transfer pathways that do not involve Trp(106) but are likely to include Asp(38) and Cys(39).

High-temperature electrocatalysis using thermophilic P450 CYP119: dehalogenation of CCl4 to CH4.

The use of a thermophilic cytochrome P450, CYP119, in electrocatalytic dehalogenations of C1 halocarbon solvents is studied. Temperature stable enzyme-modified electrodes were constructed using sol-gel and polymeric surfactant approaches. CYP119 deposited in a dimethyldidodecylammonium poly(p-styrene sulfonate) (DDAPSS) film has good retention of electrochemical activity up to 80 degrees C. At potentials approaching the FeII/I couple, the CYP119/DDAPSS films demonstrate high catalytic dehalogenations of the C1 chloromethanes CCl4, CHCl3, and CH2Cl2. Product analysis identified mixtures of sequentially dechlorinated products up to methane; no evidence for radical-coupled products was observed. The yield of methane from the CYP119-catalyzed reduction of CCl4 is increased 35-fold from 25 degrees C to 55 degrees C. In combination with the lack of C2 products, the facility of an overall eight-electron reductive dehalogenation suggests that the substrate is constrained within the protein during electrocatalytic turnover.

Electrocatalytic reductions of nitrite, nitric oxide, and nitrous oxide by thermophilic cytochrome P450 CYP119 in film-modified electrodes and an analytical comparison of its catalytic activities with myoglobin.

Previous investigations of nitrite and nitric oxide reduction by myoglobin in surfactant film modified electrodes characterized several distinct steps in the denitrification pathway, including isolation of a nitroxyl adduct similar to that proposed in the P450nor catalytic cycle. To investigate the effect of the axial ligand on these biomimetic reductions, we report here a comparison of the electrocatalytic activity of myoglobin (Mb) with a thermophilic cytochrome P450 CYP119. Electrocatalytic nitrite reduction by CYP119 is very similar to that by Mb: two catalytic waves at analogous potentials are observed, the first corresponding to the reduction of nitric oxide, the second to the production of ammonia. CYP119 is a much more selective catalyst, giving almost exclusively ammonia during the initial half-hour of reductive electrolysis of nitrite. More careful investigations of specific steps in the catalytic cycle show comparable rates of nitrite dehydration and almost identical potentials and lifetimes for ferrous nitroxyl intermediate (Fe(II)-NO(-)) in CYP119 and Mb. The catalytic efficiency of nitric oxide reduction is reduced for CYP119 as compared to Mb, attributable to both a lower affinity of the protein for NO and a decreased rate of N-N coupling. Isotopic labeling studies show ammonia incorporation into nitrous oxide produced during nitrite reduction, as has been termed co-denitrification for certain bacterial and fungal nitrite reductases. Mb has a much higher co-denitrification activity than CYP119. Conversely, CYP119 is shown to be slightly more efficient at the two-electron reduction of N(2)O to N(2). These results suggest that thiolate ligation does not significantly alter the catalytic reactivity, but the dramatic difference in product distribution may suggest an important role for protein stability in the selectivity of biocatalysts.