Coenzyme Q10 supplementation alleviates pain in pregabalin-treated fibromyalgia patients via reducing brain activity and mitochondrial dysfunction

Although coenzyme Q10 (CoQ10) supplementation has shown to reduce pain levels in chronic pain, the effects of CoQ10 supplementation on pain, anxiety, brain activity, mitochondrial oxidative stress, antioxidants, and inflammation in pregabalin-treated fibromyalgia (FM) patients have not clearly elucidated. We hypothesised that CoQ10 supplementation reduced pain better than pregabalin alone via reducing brain activity, mitochondrial oxidative stress, inflammation, and increasing antioxidant levels in pregabalin-treated FM patients. A double-blind randomised placebo-controlled trial was conducted. Eleven FM patients were enrolled with 2 weeks wash-out then randomly allocated to 2 treatment groups; pregabalin with CoQ10 or pregabalin with placebo for 40 d. Then, patients in CoQ10 group were switched to placebo, and patients in placebo group were switched to CoQ10 for another 40 d. Pain pressure threshold (PPT), FM questionnaire, anxiety, and pain score were examined. Peripheral blood mononuclear cells (PBMCs) were isolated to investigate mitochondrial oxidative stress and inflammation at day 0, 40, and 80. The level of antioxidants and brain positron emission tomography (PET) scan were also determined at these time points. Pregabalin alone reduced pain and anxiety via decreasing brain activity compared with their baseline. However, it did not affect mitochondrial oxidative stress and inflammation. Supplementation with CoQ10 effectively reduced greater pain, anxiety and brain activity, mitochondrial oxidative stress, and inflammation. CoQ10 also increased a reduced glutathione levels and superoxide dismutase (SOD) levels in FM patients. These findings provide new evidence that CoQ10 supplementation provides further benefit for relieving pain sensation in pregabalin-treated FM patients, possibly via improving mitochondrial function, reducing inflammation, and decreasing brain activity.     

Development of a robust nitrilase by fragment swapping and semi-rational design for efficient biosynthesis of pregabalin precursor

Protein engineering is a powerful tool for improving the properties of enzymes. However, large changes in enzyme properties are still challenging for traditional evolution strategies because they usually require multiple amino acid substitutions. In this study, a feasible evolution approach by a combination of fragment swapping and semi-rational design was developed for the engineering of nitrilase. A chimera BaNIT harboring 12 amino acid substitutions was obtained using nitrilase from Arabis alpine (AaNIT) and Brassica rapa (BrNIT) as parent enzymes, which exhibited higher enantioselectivity and activity toward isobutylsuccinonitrile for the biosynthesis of pregabalin precursor. The semi-rational design was executed on BaNIT to further generate variant BaNIT/L223Q/H263D/Q279E with the concurrent improvement of activity, enantioselectivity, and solubility. The robust nitrilase displayed a 5.4-fold increase in whole-cell activity and the enantiomeric ratio (E) increased from 180 to higher than 300. Molecular dynamics simulation and molecular docking demonstrated that the substitution of residues on the A and C surface contributed to the conformation alteration of nitrilase, leading to the simultaneous enhancement of enzyme properties. The results obtained not only successfully engineered the nitrilase with great industrial potential for the production of pregabalin precursor, but also provided a new perspective for the development of novel industrially important enzymes.     

Drugging the undruggable: gabapentin,pregabalin and the calcium channel α2δ subunit

In the post-genomic era, the idea of using the sequence of a protein to determine its potential role as a drug target has gained currency. The goal of this approach to drug discovery is to use the sequence of a protein that is known to bind a specific ligand or drug, along with the known structure of the ligand binding site, to predict other similar proteins that are also “druggable”. Gabapentin (Neurontin) and pregabalin (Lyrica) are drugs currently in the clinic that were developed based on the hypothesis that generating non-hydrolyzable analogs of GABA would lead to the development of antiepileptic agents. While these compounds are indeed good anticonvulsants, their activity is surprisingly not due to activity in the GABAergic system. By purifying the protein to which gabapentin bound, and determining its identity as the α₂δ₁ subunit of voltage gated calcium channels, it was possible to make progress in developing new compounds with similar activities to gabapentin, including pregabalin. The recognition of the α₂δ₁ subunit as the receptor for these drugs also meant that related proteins, such as α₂δ₃, may be interesting targets for novel pain therapeutics.     

Proteomic analysis to identify early molecular targets of pregabalin in C6 glial cells

Pregabalin is a lipophilic amino acid derivative of γ‐amino butyric acid that displays anticonvulsant and analgesic activities against neuropathic pain. Although a role for glial cells as an important player in pain control and also as a new target for pain medicine has been suggested, the effect of pregabalin on glial cells has not been elucidated. In the present study, we have investigated the action of pregabalin on the glial cell proteome. To identify immediate early protein targets of pregabalin in glial cells, a differential proteomics approach in C6 rat glioma cells treated with pregabalin was used. Seven proteins that sensitively reacted to pregabalin treatment were identified using two‐dimensional gel electrophoresis and MALDI–TOF‐MS (matrix‐assisted laser‐desorption ionization–time‐of‐flight MS). The calcium‐ion‐binding chaperone, calreticulin, and the oxidative response protein, DJ‐1, were up‐regulated after pregabalin treatment. Hsp (heat‐shock protein)‐90‐β, cytoskeleton protein actin and myosin also showed quantitative expression profile differences. Functionally relevant to the proteome result, immediate actin depolymerization was observed after treatment with pregabalin. These results suggest a previously undefined role of pregabalin in the regulation of chaperone activity and cytoskeleton remodelling in glial cells.     

BK Potassium Channels Suppress Cavα2δ Subunit Function to Reduce Inflammatory and Neuropathic Pain

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Embryo‐Fetal Developmental Toxicity Studies with Pregabalin in Mice and Rabbits

Pregabalin was evaluated for potential developmental toxicity in mice and rabbits. Pregabalin was administered once daily by oral gavage to female albino mice (500, 1250, or 2500 mg/kg) and New Zealand White rabbits (250, 500, or 1250 mg/kg) during organogenesis (gestation day 6 through 15 [mice] or 6 through 20 [rabbits]). Fetuses were evaluated for viability, growth, and morphological development. Pregabalin administration to mice did not induce maternal or developmental toxicity at doses up to 2500 mg/kg, which was associated with a maternal plasma exposure (AUC_0–24) of 3790 μg?hr/ml, ≥30 times the expected human exposure at the maximum recommended daily dose (MRD; 600 mg/day). In rabbits, treatment‐related clinical signs occurred at all doses (AUC_0–24 of 1397, 2023, and 4803 μg?hr/ml at 250, 500, and 1250 mg/kg, respectively). Maternal toxicity was evident at all doses and included ataxia, hypoactivity, and cool to touch. In addition, abortion and females euthanized moribund with total resorption occurred at 1250 mg/kg. There were no treatment‐related malformations at any dose. At 1250 mg/kg, compared with study and historical controls, the percentage of fetuses with retarded ossification was significantly increased and the mean number of ossification sites was decreased, which correlated with decreased fetal and placental weights, consistent with in utero growth retardation. Therefore, the no‐effect dose for developmental toxicity in rabbits was 500 mg/kg, which produced systemic exposure approximately 16‐times human exposure at the MRD. These findings indicate that pregabalin, at the highest dose tested, was not teratogenic in mice or rabbits     

A New Enantioselective Synthesis of the Anticonvulsant Drug Pregabalin (Lyrica) Based on a Hydrolytic Kinetic Resolution Method

A practical and efficient enantioselective synthesis of the anticonvulsant drug pregabalin is described for the first time using Jacobsen's hydrolytic kinetic resolution of a terminal epoxide as a key step and a source of chirality. Chirality 25:965–969, 2013. © 2013 Wiley Periodicals, Inc.     

Simple and sensitive spectrofluorimetric method for the determination of pregabalin in capsules through derivatization with fluorescamine

A new, simple and sensitive spectrofluorimetric method has been developed for the determination of pregabalin (PG) in capsules. The method is based on the reaction between pregabalin and fluorescamine in borate buffer solution of pH 10 to give a highly fluorescent derivative that is measured at 487 nm after excitation at 390 nm. The different experimental parameters affecting the development and stability of the reaction product were carefully studied and optimized. The fluorescence intensity concentration plot was rectilinear over the range of 0.01–0.3 µg mL^?1 with a lower detection limit of 0.0017 µg mL^?1 and limit of quantitation of 0.005 µg mL^?1. The developed method was successfully applied to the analysis of the drug in its commercial capsules. The mean percentage recovery of PG in its capsule was 99.93±1.24 (n = 3). Statistical comparison of the results with those of the comparison method revealed good agreement and proved that there was no significant difference in the accuracy and precision of the two methods. A proposed reaction pathway was postulated. Copyright © 2010 John Wiley & Sons, Ltd.     

Pregabalin and gabapentin inhibit substance P-induced NF-kappaB activation in neuroblastoma and glioma cells

Pregabalin and gabapentin are lipophilic amino acid derivatives of gamma-amino butyric acid that show anticonvulsant and analgesic activity against neuropathic pain. In this study, we investigated their actions on substance P-induced NF-kappaB activation in human neuroblastoma and rat glioma cells. Pregabalin and gabapentin decreased substance P-induced NF-kappaB activation in these cells. These drugs also inhibited NF-kappaB activation in rat spinal dorsal root ganglia cells pre-treated in vitro with substance P. These results suggest a previously undefined role of pregabalin and gabapentin in the regulation of inflammation-related intracellular signaling in both neuronal and glial cells.