De novo TANGLED1 recruitment from the phragmoplast to aberrant cell plate fusion sites in maize.

Division plane positioning is crucial for proper growth and development in many organisms. In plants, the division plane is established before mitosis, by accumulation of a cytoskeletal structure called the preprophase band (PPB). The PPB is thought to be essential for recruitment of division site-localized proteins, which remain at the division site after the PPB disassembles. Here, we show that the division site-localized protein TANGLED1 (TAN1) is recruited independently of the PPB to the cell cortex by the plant cytokinetic machinery, the phragmoplast, from experiments using both the PPB-defective mutant discordia1 (dcd1) and chemical treatments that disrupt the phragmoplast in maize. TAN1 recruitment to de novo sites on the cortex is partially dependent on intact actin filaments and the myosin XI motor protein OPAQUE1 (O1). These data imply a yet unknown role for TAN1 and possibly other division site-localized proteins during the last stages of cell division when the phragmoplast touches the cell cortex to complete cytokinesis.

Validation of a bupropion, dextromethorphan, and omeprazole cocktail for simultaneous phenotyping of cytochrome P450 2B11, 2D15, and 3A12 activities in dogs.

OBJECTIVE: Develop a cytochrome P450 (CYP) phenotyping cocktail for dogs using specific substrates for hepatic P450 enzymes CYP2B11, CYP2D15, and CYP3A12 and determine whether alternative sampling methods (saliva and urine) or single time point samples could be used instead of multiple blood sampling. ANIMALS: 12 healthy client-owned dogs (8 females and 4 males) from February 2019 to May 2019. METHODS: In a randomized crossover study, dogs received oral administration of the probe drug bupropion (75 mg), dextromethorphan (30 mg), or omeprazole (40 mg) alone or as a 3-drug combination (Program in Individualized Medicine [PrIMe] cocktail) to evaluate simultaneous phenotyping of CYP2B11, CYP2D15, and CYP3A12. Pharmacokinetic profiles for the probe drugs and metabolites were determined using plasma, saliva, and urine. Dogs received probe drugs alone or combined. Pharmacokinetic profiles up to 6 hours postdose for the probe drugs and metabolites were determined using plasma, saliva, and urine. RESULTS: The PrIMe cocktail was well tolerated. There was no statistically significant interaction between the probe drugs when administered together. Single time point plasma metabolic ratios at 4 hours postdose for all probe drugs strongly correlated with the corresponding area under the plasma concentration-versus-time curve (AUC) ratios. Saliva AUC metabolic ratios for CYP3A12 and CYP2D15 and 6-hour urine for CYP2B11 and CYP2D15 were correlated with plasma AUC ratios. CONCLUSIONS: The PrIMe cocktail can be used for simultaneous CYP phenotyping using plasma 4-hour single time point sample metabolic ratios. Saliva and urine sampling are suitable for specific CYPs. CLINICAL RELEVANCE: The PrIMe cocktail has potential as a useful tool in dogs to detect clinically important CYP-mediated drug-drug interactions, identify novel pharmacogenes, determine the drug-metabolizing phenotype of individual dogs, aid in individualized dose selection, and evaluate the effects of various physiological states on drug metabolism.

De Novo TANGLED1 Recruitment to Aberrant Cell Plate Fusion Sites in Maize.

Division plane positioning is critical for proper growth and development in many organisms. In plants, the division plane is established before mitosis, by accumulation of a cytoskeletal structure called the preprophase band (PPB). The PPB is thought to be essential for recruitment of division site localized proteins, which remain at the division site after the PPB disassembles. Here, we show that a division site localized protein, TANGLED1 (TAN1), is recruited independently of the PPB to the cell cortex at sites, by the plant cytokinetic machinery, the phragmoplast. TAN1 recruitment to de novo sites on the cortex is partially dependent on intact actin filaments and the myosin XI motor protein OPAQUE1 (O1). These data imply a yet unknown role for TAN1 and possibly other division site localized proteins during the last stages of cell division when the phragmoplast touches the cell cortex to complete cytokinesis.

Pharmacogenomics of poor drug metabolism in greyhounds: Canine P450 oxidoreductase genetic variation, breed heterogeneity, and functional characterization.

Greyhounds metabolize cytochrome P450 (CYP) 2B11 substrates more slowly than other dog breeds. However, CYP2B11 gene variants associated with decreased CYP2B11 expression do not fully explain reduced CYP2B11 activity in this breed. P450 oxidoreductase (POR) is an essential redox partner for all CYPs. POR protein variants can enhance or repress CYP enzyme function in a CYP isoform and substrate dependent manner. The study objectives were to identify POR protein variants in greyhounds and determine their effect on coexpressed CYP2B11 and CYP2D15 enzyme function. Gene sequencing identified two missense variants (Glu315Gln and Asp570Glu) forming four alleles, POR-H1 (reference), POR-H2 (570Glu), POR-H3 (315Gln, 570Glu) and POR-H4 (315Gln). Out of 68 dog breeds surveyed, POR-H2 was widely distributed across multiple breeds, while POR-H3 was largely restricted to greyhounds and Scottish deerhounds (35% allele frequencies), and POR-H4 was rare. Three-dimensional protein structure modelling indicated significant effects of Glu315Gln (but not Asp570Glu) on protein flexibility through loss of a salt bridge between Glu315 and Arg519. Recombinant POR-H1 (reference) and each POR variant (H2-H4) were expressed alone or with CYP2B11 or CYP2D15 in insect cells. No substantial effects on POR protein expression or enzyme activity (cytochrome c reduction) were observed for any POR variant (versus POR-H1) when expressed alone or with CYP2B11 or CYP2D15. Furthermore, there were no effects on CYP2B11 or CYP2D15 protein expression, or on CYP2D15 enzyme kinetics by any POR variant (versus POR-H1). However, Vmax values for 7-benzyloxyresorufin, propofol and bupropion oxidation by CYP2B11 were significantly reduced by coexpression with POR-H3 (by 34-37%) and POR-H4 (by 65-72%) compared with POR-H1. Km values were unaffected. Our results indicate that the Glu315Gln mutation (common to POR-H3 and POR-H4) reduces CYP2B11 enzyme function without affecting at least one other major canine hepatic P450 (CYP2D15). Additional in vivo studies are warranted to confirm these findings.

Subcellular positioning during cell division and cell plate formation in maize.

Introduction: During proliferative plant cell division, the new cell wall, called the cell plate, is first built in the middle of the cell and then expands outward to complete cytokinesis. This dynamic process requires coordinated movement and arrangement of the cytoskeleton and organelles. Methods: Here we use live-cell markers to track the dynamic reorganization of microtubules, nuclei, endoplasmic reticulum, and endomembrane compartments during division and the formation of the cell plate in maize leaf epidermal cells. Results: The microtubule plus-end localized protein END BINDING1 (EB1) highlighted increasing microtubule dynamicity during mitosis to support rapid changes in microtubule structures. The localization of the cell-plate specific syntaxin KNOLLE, several RAB-GTPases, as well as two plasma membrane localized proteins was assessed after treatment with the cytokinesis-specific callose-deposition inhibitor Endosidin7 (ES7) and the microtubule-disrupting herbicide chlorpropham (CIPC). While ES7 caused cell plate defects in Arabidopsis thaliana, it did not alter callose accumulation, or disrupt cell plate formation in maize. In contrast, CIPC treatment of maize epidermal cells occasionally produced irregular cell plates that split or fragmented, but did not otherwise disrupt the accumulation of cell-plate localized proteins. Discussion: Together, these markers provide a robust suite of tools to examine subcellular trafficking and organellar organization during mitosis and cell plate formation in maize.

A KARRIKIN INSENSITIVE2 paralog in lettuce mediates highly sensitive germination responses to karrikinolide.

Karrikins (KARs) are chemicals in smoke that can enhance germination of many plants. Lettuce (Lactuca sativa) cv. Grand Rapids germinates in response to nanomolar karrikinolide (KAR1). Lettuce is much less responsive to KAR2 or a mixture of synthetic strigolactone analogs, rac-GR24. We investigated the molecular basis of selective and sensitive KAR1 perception in lettuce. The lettuce genome contains two copies of KARRIKIN INSENSITIVE2 (KAI2), which in Arabidopsis (Arabidopsis thaliana) encodes a receptor that is required for KAR responses. LsKAI2b is more highly expressed than LsKAI2a in dry achenes and during early stages of imbibition. Through cross-species complementation assays in Arabidopsis, we found that an LsKAI2b transgene confers robust responses to KAR1, but LsKAI2a does not. Therefore, LsKAI2b likely mediates KAR1 responses in lettuce. We compared homology models of KAI2 proteins from lettuce and a fire-follower, whispering bells (Emmenanthe penduliflora). This identified pocket residues 96, 124, 139, and 161 as candidates that influence the ligand specificity of KAI2. Further support for the importance of these residues was found through a broader comparison of pocket residues among 281 KAI2 proteins from 184 asterid species. Almost all KAI2 proteins had either Tyr or Phe identity at position 124. Genes encoding Y124-type KAI2 are more broadly distributed in asterids than in F124-type KAI2. Substitutions at residues 96, 124, 139, and 161 in Arabidopsis KAI2 produced a broad array of responses to KAR1, KAR2, and rac-GR24. This suggests that the diverse ligand preferences observed among KAI2 proteins in plants could have evolved through relatively few mutations.

KARRIKIN UP-REGULATED F-BOX 1 (KUF1) imposes negative feedback regulation of karrikin and KAI2 ligand metabolism in Arabidopsis thaliana.

SignificanceKarrikins are chemicals in smoke that stimulate regrowth of many plants after fire. However, karrikin responses are not limited to species from fire-prone environments and can affect growth after germination. Putatively, this is because karrikins mimic an unknown signal in plants, KAI2 ligand (KL). Karrikins likely require modification in plants to become bioactive. We identify a gene, KUF1, that appears to negatively regulate biosynthesis of KL and metabolism of a specific karrikin. KUF1 expression increases in response to karrikin or KL signaling, thus forming a negative feedback loop that limits further activation of the signaling pathway. This discovery will advance understanding of how karrikins are perceived and how smoke-activated germination evolved. It will also aid identification of the elusive KL.

Pharmacogenomics of poor drug metabolism in Greyhounds: Cytochrome P450 (CYP) 2B11 genetic variation, breed distribution, and functional characterization.

Greyhounds recover more slowly from certain injectable anesthetics than other dog breeds. Previous studies implicate cytochrome P450 (CYP) 2B11 as an important clearance mechanism for these drugs and suggest Greyhounds are deficient in CYP2B11. However, no CYP2B11 gene mutations have been identified that explain this deficiency in Greyhounds. The objectives of this study were to provide additional evidence for CYP2B11 deficiency in Greyhounds, determine the mechanisms underlying this deficiency, and identify CYP2B11 mutations that contribute to this phenotype in Greyhounds. Greyhound livers metabolized CYP2B11 substrates slower, possessed lower CYP2B11 protein abundance, but had similar or higher mRNA expression than other breeds. Gene resequencing identified three CYP2B11 haplotypes, H1 (reference), H2, and H3 that were differentiated by mutations in the gene 3'-untranslated region (3'-UTR). Compared with 63 other dog breeds, Greyhounds had the highest CYP2B11-H3 allele frequency, while CYP2B11-H2 was widely distributed across most breeds. Using 3'-UTR luciferase reporter constructs, CYP2B11-H3 showed markedly lower gene expression (over 70%) compared to CYP2B11-H1 while CYP2B11-H2 expression was intermediate. Truncated mRNA transcripts were observed in CYP2B11-H2 and CYP2B11-H3 but not CYP2B11-H1 transfected cells. Our results implicate CYP2B11 3'-UTR mutations as a cause of decreased CYP2B11 enzyme expression in Greyhounds through reduced translational efficiency.

Absolute Quantitation of Drug-Metabolizing Cytochrome P450 Enzymes and Accessory Proteins in Dog Liver Microsomes Using Label-Free Standard-Free Analysis Reveals Interbreed Variability.

Dogs are commonly used in human and veterinary pharmaceutical development. Physiologically based pharmacokinetic modeling using recombinant cytochrome P450 (CYP) enzymes requires accurate estimates of CYP abundance, particularly in liver. However, such estimates are currently available for only seven CYPs, which were determined in a limited number of livers from one dog breed (beagle). In this study, we used a label-free shotgun proteomics method to quantitate 11 CYPs (including four CYPs not previously measured), cytochrome P450 oxidoreductase, and cytochrome b5 in liver microsomes from 59 dogs representing four different breeds and mixed-breed dogs. Validation included showing correlation with CYP marker activities, immunoquantified protein, as well as CYP1A2 and CYP2C41 null allele genotypes. Abundance values largely agreed with those previously published. Average CYP abundance was highest (>120 pmol/mg protein) for CYP2D15 and CYP3A12; intermediate (40-89 pmol/mg) for CYP1A2, CYP2B11, CYP2E1, and CYP2C21; and lowest (<12 pmol/mg) for CYP2A13, CYP2A25, CYP2C41, CYP3A26, and CYP1A1. The CYP2C41 gene was detected in 12 of 58 (21%) livers. CYP2C41 protein abundance averaged 8.2 pmol/mg in those livers, and was highest (19 pmol/mg) in the only liver with two CYP2C41 gene copies. CYP1A2 protein was not detected in the only liver homozygous for the CYP1A2 stop codon mutation. Large breed-associated differences were observed for CYP2B11 (P < 0.0001; ANOVA) but not for other CYPs. Research hounds and Beagles had the highest CYP2B11 abundance; mixed-breed dogs and Chihuahua were intermediate; whereas greyhounds had the lowest abundance. These results provide the most comprehensive estimates to date of CYP abundance and variability in canine liver. SIGNIFICANCE STATEMENT: This work provides the most comprehensive quantitative analysis to date of the drug-metabolizing cytochrome P450 proteome in dogs that will serve as a valuable reference for physiologically based scaling and modeling used in drug development and research. This study also revealed high interindividual variation and dog breed-associated differences in drug-metabolizing cytochrome P450 expression that may be important for predicting drug disposition variability among a genetically diverse canine population.

Personalized medicine: going to the dogs?

Interindividual variation in drug response occurs in canine patients just as it does in human patients. Although canine pharmacogenetics still lags behind human pharmacogenetics, significant life-saving discoveries in the field have been made over the last 20 years, but much remains to be done. This article summarizes the available published data about the presence and impact of genetic polymorphisms on canine drug transporters, drug-metabolizing enzymes, drug receptors/targets, and plasma protein binding while comparing them to their human counterparts when applicable. In addition, precision medicine in cancer treatment as an application of canine pharmacogenetics and pertinent considerations for canine pharmacogenetics testing is reviewed. The field is poised to transition from single pharmacogene-based studies, pharmacogenetics, to pharmacogenomic-based studies to enhance our understanding of interindividual variation of drug response in dogs. Advances made in the field of canine pharmacogenetics will not only improve the health and well-being of dogs and dog breeds, but may provide insight into individual drug efficacy and toxicity in human patients as well.

Effects of low-level laser therapy on bone healing and signs of pain in dogs following tibial plateau leveling osteotomy.

OBJECTIVE To assess the effect of low-level laser therapy (LLLT) on markers of synovial inflammation and signs of pain, function, bone healing, and osteoarthritis following tibial plateau leveling osteotomy (TPLO) in dogs with spontaneous cranial cruciate ligament rupture (CCLR). ANIMALS 12 client-owned dogs with unilateral CCLR. PROCEDURES All dogs were instrumented with an accelerometer for 2 weeks before and 8 weeks after TPLO. Dogs were randomly assigned to receive LLLT (radiant exposure, 1.5 to 2.25 J/cm2; n = 6) or a control (red light; 6) treatment immediately before and at predetermined times for 8 weeks after TPLO. Owners completed a Canine Brief Pain Inventory weekly for 8 weeks after surgery. Each dog underwent a recheck appointment, which included physical and orthopedic examinations, force plate analysis, radiography and synoviocentesis of the affected joint, and evaluation of lameness and signs of pain, at 2, 4, and 8 weeks after surgery. Select markers of inflammation were quantified in synovial fluid samples. Variables were compared between the 2 groups. RESULTS For the control group, mean ground reaction forces were greater at 2 and 4 weeks after TPLO and owner-assigned pain scores were lower during weeks 1 through 5 after TPLO, compared with corresponding values for the LLLT group. CONCLUSIONS AND CLINICAL RELEVANCE Results suggested that the LLLT protocol used had no beneficial effects on signs of pain or pelvic limb function following TPLO. Further research is necessary to evaluate the effects of LLLT and to determine the optimum LLLT protocol for dogs with CCLR.

Disposition, Metabolism and Histone Deacetylase and Acetyltransferase Inhibition Activity of Tetrahydrocurcumin and Other Curcuminoids.

Tetrahydrocurcumin (THC), curcumin and calebin-A are curcuminoids found in turmeric (Curcuma longa). Curcuminoids have been established to have a variety of pharmacological activities and are used as natural health supplements. The purpose of this study was to identify the metabolism, excretion, antioxidant, anti-inflammatory and anticancer properties of these curcuminoids and to determine disposition of THC in rats after oral administration. We developed a UHPLC-MS/MS assay for THC in rat serum and urine. THC shows multiple redistribution phases with corresponding increases in urinary excretion rate. In-vitro antioxidant activity, histone deacetylase (HDAC) activity, histone acetyltransferase (HAT) activity and anti-inflammatory inhibitory activity were examined using commercial assay kits. Anticancer activity was determined in Sup-T1 lymphoma cells. Our results indicate THC was poorly absorbed after oral administration and primarily excreted via non-renal routes. All curcuminoids exhibited multiple pharmacological effects in vitro, including potent antioxidant activity as well as inhibition of CYP2C9, CYP3A4 and lipoxygenase activity without affecting the release of TNF-α. Unlike curcumin and calebin-A, THC did not inhibit HDAC1 and PCAF and displayed a weaker growth inhibition activity against Sup-T1 cells. We show evidence for the first time that curcumin and calebin-A inhibit HAT and PCAF, possibly through a Michael-addition mechanism.

Pharmacokinetic Analysis of an Oral Multicomponent Joint Dietary Supplement (Phycox®) in Dogs.

Despite the lack of safety, efficacy and pharmacokinetic (PK) studies, multicomponent dietary supplements (nutraceuticals) have become increasingly popular as primary or adjunct therapies for clinical osteoarthritis in veterinary medicine. Phycox® is a line of multicomponent joint support supplements marketed for joint health in dogs and horses. Many of the active constituents are recognized anti-inflammatory and antioxidant agents. Due to a lack of PK studies in the literature for the product, a pilot PK study of select constituents in Phycox® was performed in healthy dogs. Two novel methods of analysis were developed and validated for quantification of glucosamine and select polyphenols using liquid chromatography-tandem mass spectrometry. After a single oral (PO) administrated dose of Phycox®, a series of blood samples from dogs were collected for 24 h post-dose and analyzed for concentrations of glucosamine HCl, hesperetin, resveratrol and naringenin. Non-compartmental PK analyses were carried out. Glucosamine was detected up to 8 h post-dose with a Tmax of 2 h and Cmax of 9.69 µg/mL. The polyphenols were not found at detectable concentrations in serum samples. Co-administration of glucosamine in the Phycox® formulation may enhance the absorption of glucosamine as determined by comparison of glucosamine PK data in the literature.

Stereospecific pharmacokinetic characterization of liquiritigenin in the rat.

Liquiritigenin is a chiral flavonoid present in licorice and other medicinal plants. The nature of its biological fate with respect to the individual enantiomers has not been examined. In this study, we characterize, for the first time, the stereoselective pharmacokinetics of liquiritigenin. Liquiritigenin was intravenously (20 mg/kg) and orally (50 mg/kg) administered to male Sprague-Dawley rats (n = 4 per route of administration). Concentrations in serum and urine were characterized via stereospecific reversed-phase, isocratic HPLC method with UV detection. Serum concentrations were quantified but rapidly fell to undetectable levels. S-liquiritigenin showed a short half-life (0.25-0.54 h), while a better estimation of half-life (26-77 h) and other pharmacokinetic parameters was observed using urinary data. The flavonoid is predominantly excreted via non-renal routes (fe values of 0.16-3.46 %), and undergoes rapid and extensive phase II metabolism. Chiral differences in the chemical structure of the compound result in some pharmacokinetic differences. Serum concentrations rapidly declined, making modeling difficult. S-liquiritigenin showed an increased urinary half-life.

Pharmacological characterization of liquiritigenin, a chiral flavonoid in licorice.

Liquiritigenin is a chiral flavonoid present in plant based food, nutraceuticals, and traditional medicines. It is also an important ingredient present in licorice. The purpose of this study is to explore the pharmacological activity of racemic liquiritigenin utilizing several in vitro assays with relevant roles in colon cancer and diabetes. Where possible, the pure enantiomers were tested to identify the stereospecific contribution to the activity. In vitro antioxidant, anticancer, anti-inflammatory activities (cyclooxygenase inhibition), antidiabetic activities (alpha-amylase and alpha-glucosidase inhibition) as well as cytochrome P450 (CYP450) inhibitory activities were assessed. Racemic liquiritigenin demonstrated a dose-dependent inhibition of alpha-amylase enzyme whereas its pure enantiomers did not. Racemic liquiritigenin showed moderate antiproliferative activity on a HT-29 (human colorectal adenocarcinoma) cancer cell line that was dose-dependent and potent inhibitory effects on the cyclooxygenase-2 enzyme. The flavonoid did not inhibit the activity of cytochrome CYP2D6 over the concentration range studied but was a potent antioxidant. The current study demonstrated the importance of understanding the stereospecific pharmacological effects of liquiritigenin enantiomers in alpha-amylase inhibition.

Pre-Clinical Pharmacokinetic and Pharmacodynamic Characterization of Selected Chiral Flavonoids: Pinocembrin and Pinostrobin.

PURPOSE: Delineate the stereospecific pharmacokinetics and pharmacodynamics of the chiral flavonoids pinocembrin and pinostrobin. OBJECTIVE: Characterize for the first time the stereoselective pharmacokinetics of two flavonoids, pinocembrin and pinostrobin and their bioactivity in several in vitro assays with relevant roles in heart disease, colon cancer, and diabetes etiology and pathophysiology. METHODS: Chiral flavonoids were intravenously and orally administered to male Sprague-Dawley rats. Concentrations in serum and urine were characterized via stereospecific HPLC or LC/MS. Pure enantiomeric forms of each flavonoid were tested, where possible, to identify the stereospecific contribution to bioactivity in comparison to their racemates. RESULTS: Short half-lives (0.2-6 h) in serum were observed, while a better estimation of half-life (3-26 h) and other pharmacokinetic parameters were observed using urinary data. The flavonoids are predominantly excreted via non-renal routes (fe values of 0.3-4.6 %), and undergo rapid and extensive phase II metabolism. Chiral differences in the chemical structure of these compounds result in significant pharmacodynamic differences. CONCLUSION: The importance of understanding the stereospecific pharmacokinetics and pharmacodynamics of two chiral flavonoids were delineated.This article is open to POST-PUBLICATION REVIEW. Registered readers (see "For Readers") may comment by clicking on ABSTRACT on the issue's contents page.

Calebin A: Analytical Development for Pharmacokinetics Study, Elucidation of Pharmacological Activities and Content Analysis of Natural Health Products.

PURPOSE: To develop a bioanalytical assay using RP-HPLC to quantify the curcuminoid calebin A, to characterize its pharmacokinetics in rats after intravenous (IV) and oral (PO) administration, to identify its pharmacological activities and to evaluate its content in natural health products. METHODS: An RP-HPLC method was developed for the detection of calebin A. Separation was carried out using a Phenomenex® Kinetex® C18 column with UV detection at 339 nm. An isocratic mobile phase consisting of acetonitrile and water with 10 mM ammonium formate (pH 7.0) (40:60, v/v) at a flow rate of 0.8 mL/min was employed. Linear standard curves were established and applied in the pharmacokinetic study. Calebin A was administered to male Sprague-Dawley (CD) rats IV (20 mg/kg) or PO (500 mg/kg) (n=4/route of administration). Serum and urine samples were collected for 72 h post dose. In vitro antioxidant activity, anti-inflammatory activity (cyclooxygenase and lipoxygenase inhibition), dipeptidyl peptidase-4 (DPP-4) inhibition and cytochrome P450 inhibitory activties of calebin A were examined using commercial assay kits. Content analysis of calebin A in 14 natural health products advertised to contain turmeric were carried out using methanolic extraction. RESULTS: The HPLC method was successfully applied to a pharmacokinetic study of calebin A in rats. After IV and PO administration of calebin A, the compound was detected as the aglycone and a glucuronidated metabolite. Oral bioavailabitily was found to be ~0.5%, serum half-life was ~1-3 h. Calebin A appears to be primarily excreted via non-renal routes. Calebin A possessed concentration-dependent antioxidant activity and DPP-4 inhibition. Calebin A appears to be a non-selective cyclooxygenase inhibitor and also a poor lipoxygenase inhibitor. The curcuminoid displayed in vitro interactions with CYP2D6 and CYP1A2. Content analysis of 14 natural health products that claimed to contain turmeric showed that concentration of calebin A was inconsistent among the products. CONCLUSION: A successful assay was developed for the detection of calebin A using RP-HPLC. Preliminary pharmacokinetic studies indicate that an unoptimised formulation of calebin A has poor oral bioavailability. Calebin A exhibits various pharmacological activities. This article is open to POST-PUBLICATION REVIEW. Registered readers (see "For Readers") may comment by clicking on ABSTRACT on the issue's contents page.

Pharmacological effects of a C-phycocyanin-based multicomponent nutraceutical in an in-vitro canine chondrocyte model of osteoarthritis.

Multicomponent nutraceuticals are becoming increasingly popular treatments or adjunctive therapies for osteoarthritis in veterinary medicine despite lack of evidence of efficacy for many products. The objective of this study was to evaluate the anti-inflammatory and antioxidant activities of a commercially available C-phycocyanin-based nutraceutical and select constituent ingredients in an in-vitro model of canine osteoarthritis. Normal canine articular chondrocytes were used in an in-vitro model of osteoarthritis. Inflammatory conditions were induced using interleukin-1ß. The nutraceutical preparation as a whole, its individual constituents, as well as carprofen were evaluated at concentrations of 0 to 250 µg/mL for reduction of the following inflammatory mediators and indicators of catabolism of the extracellular matrix: prostaglandin E2 (PGE2), tumor necrosis factor-α (TFN-α), interleukin-6 (IL-6), metalloproteinase-3 (MMP-3), nitric oxide, and sulfated glycosaminoglycans (sGAGs). Validated, commercially available assay kits were used for quantitation of inflammatory mediators. The antioxidant capacities, as well as cyclooxygenase-1 (COX-1), cyclooxygenase-2 (COX-2), and lipoxygenase (LOX) inhibitory activities of the whole nutraceutical preparation and select constituents, were also assessed using validated commercially available assay kits. The antioxidant capacity of the nutraceutical and constituents was concentration-dependent. The nutraceutical and constituents appear to display anti-inflammatory activity primarily through the inhibition of COX-2. The nutraceutical displayed similar strength to carprofen in reducing TNF-α, IL-6, MMP-3, nitric oxide, and sGAGs at select concentration ranges. The C-phycocyanin (CPC)-based nutraceutical and constituents may be able to mediate 3 primary pathogenic mechanisms of osteoarthritis: inflammation, chondral degeneration, and oxidative stress in vitro. The nutraceutical may be clinically useful in veterinary medicine and its efficacy should be further investigated in vivo.

Les neutraceutiques à composés multiples deviennent de plus en plus populaires en médecine vétérinaire comme traitement ou thérapie alternative pour soigner l'ostéoarthrite, et ce malgré le manque d'évidence de l'efficacité de plusieurs produits. L'objectif de la présente étude était d'évaluer l'activité anti-inflammatoire et anti-oxydante d'un neutraceutique à base de C-phycocyanine disponible commercialement, et d'ingrédients constitutifs sélectionnés dans un modèle in vitro d'ostéoarthrite canine. Des chondrocytes articulaires canins normaux furent utilisés dans un modèle in vitro d'ostéoarthrite. Des conditions inflammatoires ont été induites en utilisant de l'interleukine-1ß. La préparation neutraceutique complète, ses constituants individuels, de même que du caprofène furent évalués à des concentrations de 0 à 250 µg/mL pour la réduction des médiateurs de l'inflammation suivants et des indicateurs du catabolisme de la matrice extracellulaire: prostaglandine E2 (PGE2), facteur-α nécrosant des tumeurs (TNF-α), interleukine-6 (IL-6), métalloprotéinase-3 (MMP-3), oxyde nitreux, et les glycosaminoglycans sulfatés (sGAGs). Des trousses commerciales validées furent utilisées pour la quantification des médiateurs de l'inflammation. Les capacités anti-oxydantes, de même que l'activité inhibitrice envers la cyclo-oxygénase 1 (COX-1), la cyclo-oxygénase-2 (COX-2), et la lipoxygénase (LOX) de la préparation neutraceutique complète et de constituants sélectionnés furent également évaluées au moyen de de trousses commerciales disponibles commercialement. La capacité anti-oxydante du neutraceutique et des constituants était dépendante de la concentration. Le neutraceutique et les constituants semblent manifester leur activité anti-inflammatoire principalement via l'inhibition de COX-2. Dans des écarts de concentrations sélectionnés, le neutraceutique a démontré une capacité similaire au carprofène en réduisant TNF-α, IL-6, MMP-3, oxyde nitreux, et sGAGs. Le neutraceutique à base de C-phycocyanine (CPC) et ses constituants peuvent être en mesure de médier trois mécanismes pathogéniques primaires de l'ostéo-arthrite: l'inflammation, la dégénération chondrale, et le stress oxydatif in vitro. Le neutraceutique pourrait être cliniquement utile en médecine vétérinaire et son efficacité devrait être investigué plus à fond in vivo.(Traduit par Docteur Serge Messier).

Enantiospecific pharmacokinetics of isoxanthohumol and its metabolite 8-prenylnaringenin in the rat.

SCOPE: Isoxanthohumol (IX) is a bioactive dietary prenylflavanone found in hops (Humulus lupulus L.), beer and nutraceuticals. IX is formed in vivo by xanthohumol and is a prodrug of 8-prenylnaringenin (8PN). IX and 8PN chirality has largely been ignored in the literature due to lack of enantiospecific bioanalytical methods. No single dose pharmacokinetic study of IX exists in the literature for any species. This study elucidates the enantiospecific pharmacokinetics of IX in rats and monitors the appearance of 8PN following intravenous and oral administration of ±IX. METHODS AND RESULTS: After intravenous (10 mg/kg) or oral (100 mg/kg) administration of ±IX to rats, serum, and urine were collected for 120 h and analyzed for IX and 8PN. Both were found as aglycones and glucuronide conjugates and displayed multiple peaking in serum suggestive of enterohepatic recycling. IX is primarily excreted through nonrenal routes. S-8PN was found excreted in the urine in greater amounts than R-8PN. Bioavailability was determined to be ∼4-5% for IX. CONCLUSION: Further enantiospecific pharmacokinetics of IX, subsequent 8PN and other metabolites are warranted along with continued enantiospecific bioactivity studies, especially in relation to gut microbial metabolism of IX and subsequent formation of 8PN.

Preclinical Pharmacokinetics and Pharmacodynamics and Content Analysis of Gnetol in Foodstuffs.

Studies were undertaken to evaluate the bioavailability in rats and content analysis of gnetol in Gnetum gnemon products reported to contain gnetol and to examine the pharmacological properties of gnetol in in vitro models including anti-inflammatory/analgesic, antidiabetic, anti-adipogenesis, and anticancer activity. Male Sprague-Dawley rats were cannulated and dosed either intravenously with gnetol (10 mg/kg) or orally (100 mg/kg). Various methanolic extractions of G. gnemon products were quantified. Gnetol's effect on cell viability in selected cell lines with or without inflammatory stimulus was assessed. α-Amylase and α-glucosidase inhibition was evaluated. Cyclooxygenase (COX)-1, COX-2, and histone deacetylase inhibition and adipogenesis inhibition were examined. After oral and intravenous administration, gnetol was detected in both serum and urine as the parent compound and as a glucuronidated metabolite. The bioavailability of gnetol was determined to be 6%. Gnetol is rapidly glucuronidated and is excreted in urine and via nonrenal routes. Gnetol was found to exist as an aglycone and as a glycoside in G. gnemon products. Gnetol showed concentration-dependent reductions in cell viability in cancer cell lines with greatest activity in colorectal cancer and potent COX-1, histone deacetylase, and weak COX-2 activities along with limited reduction in inflammation. Gnetol also possessed concentration-dependent alpha-amylase, alpha-glucosidase, and adipogenesis activities. Pretreatment of mice with gnetol was able to increase the latency period to response in analgesia models.