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Evaluate a precision medicine approach to confirm a tentative diagnosis of fatal malignant hyperthermia (MH) in isoflurane-anesthetized pet dogs by identifying novel risk variants in known MH susceptibility genes. Retrospective case series. A male Pit Bull mix aged 7 years (case #1), a male Golden Retriever aged 12 months (case #2) and the dam and sire of case #2. Available case histories and medical records were reviewed. Missense variants in MH susceptibility genes RYR-1 , CACNA1S and STAC3 (case #2 only) were identified by next-generation sequencing of DNA from each case and the parents of case #2 with confirmation by Sanger sequencing. The pathogenicity of variants was evaluated by multiple in silico approaches. Both cases demonstrated clinical signs during isoflurane anesthesia consistent with volatile anesthetic-induced MH, including tachypnea, tachycardia, severe hyperthermia and muscle rigidity. Despite whole body cooling and other treatments, both dogs died after cardiac arrest within 15 minutes of detecting hyperthermia. Gene sequencing identified novel missense RYR-1 variants in case #1 (p.Gly2375Arg) and case #2 (p.Pro152Leu). Both variants were likely pathogenic based on multiple criteria, including gene location, amino acid alteration and population allele frequency. The case #1 variant was identical to a known human diagnostic MH variant (p.Gly2375Arg). Neither parent of case #2 had the case #2 variant, indicating this variant was not inherited, but arose de novo in a germ cell of either parent or early in embryogenesis. Whole genome sequence analysis confirmed parentage. Two missense variants were identified in CACNA1S. Both variants were considered nonpathogenic. No variants were identified in STAC3. Like humans, MH susceptibility in dogs is associated with different rare variants located in pathogenic hotspots in the RYR-1 gene. Next-generation sequencing is a useful tool to assist in the definitive diagnosis of MH in dogs. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

CYP2D15 is a major drug metabolizing P450 in canine liver. Like the human orthologue (CYP2D6), this enzyme is highly polymorphic with at least five common nonsynonymous variants reported that result in amino acid changes, including p.Ile109Val, p.Leu115Phe, p.Gly186Ser, p.Ile250Phe and p.Ile307Val. Furthermore, a mRNA splice variant of CYP2D15 has been found in canine liver that lacks the exon 3 gene region resulting in an inactive enzyme. The objective of this study was to evaluate whether any of these amino acid variants or the exon 3 deletion mRNA variant (exon3‐delta) was associated with differences in CYP2D15‐selective activities or protein content in a bank of canine livers. Livers were obtained from 25 Beagles and 34 dogs of various other breeds. CYP2D15‐selective activities measured included dextromethorphan o‐demethylation and tramadol o‐demethylation. Reverse transcription PCR showed that 76% of livers (44/58) expressed both exon3‐delta and normally spliced CYP2D15 RNA, while the remaining 24% (14/58) expressed only normally spliced RNA. The presence of exon3‐delta was not correlated with CYP2D15 activities or protein content. Compared with wild‐type livers, Beagle dog livers heterozygous for the p.Ile109Val and p.Gly186Ser variants showed from 40 to 50% reductions in median enzyme activities, while heterozygous p.Gly186Ser livers were associated with a 41% reduction in median CYP2D15 protein content (p <.05; Dunn's test). In the entire liver bank, livers homozygous for p.Ile109Val were also associated with a 40% reduction in median dextromethorphan O‐demethylation activities versus wild‐type livers (p <.05). These results identify several nonsynonymous CYP2D15 gene variants associated with variable CYP2D15 metabolism in canine liver. [ABSTRACT FROM AUTHOR]

We previously showed that (+)‐tramadol is metabolized in dog liver to (+)‐M1 exclusively by CYP2D15 and to (+)‐M2 by multiple CYPs, but primarily CYP2B11. However, (+)‐M1 and (+)‐M2 are further metabolized in dogs to (+)‐M5, which is the major metabolite found in dog plasma and urine. In this study, we identified canine CYPs involved in metabolizing (+)‐M1 and (+)‐M2 using recombinant enzymes, untreated dog liver microsomes (DLMs), inhibitor‐treated DLMs, and DLMs from CYP inducer‐treated dogs. A canine P‐glycoprotein expressing cell line was also used to evaluate whether (+)‐tramadol, (+)‐M1, (+)‐M2, or (+)‐M5 are substrates of canine P‐glycoprotein, thereby limiting their distribution into the central nervous system. (+)‐M5 was largely formed from (+)‐M1 by recombinant CYP2C21 with minor contributions from CYP2C41 and CYP2B11. (+)‐M5 formation in DLMs from (+)‐M1 was potently inhibited by sulfaphenazole (CYP2C inhibitor) and chloramphenicol (CYP2B11 inhibitor) and was greatly increased in DLMs from phenobarbital‐treated dogs. (+)‐M5 was formed from (+)‐M2 predominantly by CYP2D15. (+)‐M5 formation from (+)‐M1 in DLMs was potently inhibited by quinidine (CYP2D inhibitor) but had only a minor impact from all CYP inducers tested. Intrinsic clearance estimates showed over 50 times higher values for (+)‐M5 formation from (+)‐M2 compared with (+)‐M1 in DLMs. This was largely attributed to the higher enzyme affinity (lower Km) for (+)‐M2 compared with (+)‐M1 as substrate. (+)‐tramadol, (+)‐M1, (+)‐M2, or (+)‐M5 were not p‐glycoprotein substrates. This study provides a clearer picture of the role of individual CYPs in the complex metabolism of tramadol in dogs. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

This correlational study investigated the relationship between being in a committed relationship and academic performance in college females. The study involved 60 female undergraduate participants from a small, private, Catholic women's college in northeastern New Jersey. It was hypothesized that college females who were in a supportive committed relationship would have significantly higher GPAs than students who were in unsupportive relationships. The Psychosocial Intimacy Questionnaire (PIQ, Tesch, 1985) was used to measure intimacy, specifically romantic love, supportiveness, and communication ease in a participant's committed relationship. Participants self-reported current GPA and answered questions regarding course failure and academic probation. [ABSTRACT FROM AUTHOR]

Provides information on a study that examined a resource-sharing model for optimal call admission control of multiple classes of customers without waiting room. Significance of asynchronous transfer mode in the interest in resource-sharing models with different resource requirements; Methodology; Results and discussion.

A series of allenenes bearing an additional 11-oxo function is subjected to either conditions A) or B) containing a gold catalyst. [ABSTRACT FROM AUTHOR]

Phytocannabinoid‐rich hemp extracts containing cannabidiol (CBD) and cannabidiolic acid (CBDA) are increasingly being used to treat various disorders in dogs. The objectives of this study were to obtain preliminary information regarding the in vitro metabolism of these compounds and their capacity to inhibit canine cytochrome P450 (CYP)‐mediated drug metabolism and canine P‐glycoprotein‐mediated transport. Pure CBD and CBDA, and hemp extracts enriched for CBD and for CBDA were evaluated. Substrate depletion assays using pooled dog liver microsomes showed CYP cofactor‐dependent depletion of CBD (but not CBDA) and UDP‐glucuronosytransferase cofactor‐dependent depletion of CBDA (but not CBD) indicating major roles for CYP and UDP‐glucuronosytransferase in the metabolism of these phytocannabinoids, respectively. Further studies using recombinant canine CYPs demonstrated substantial CBD depletion by the major hepatic P450 enzymes CYP1A2 and CYP2C21. These results were confirmed by showing increased CBD depletion by liver microsomes from dogs treated with a known CYP1A2 inducer (β‐naphthoflavone) and with a known CYP2C21 inducer (phenobarbital). Cannabinoid‐drug inhibition experiments showed inhibition (IC50 = 4.6–8.1 μM) of tramadol metabolism via CYP2B11‐mediated N‐demethylation (CBD and CBDA) and CYP2D15‐mediated O‐demethylation (CBDA only) by dog liver microsomes. CBD and CBDA did not inhibit CYP3A12‐mediated midazolam 1′‐hydroxylation (IC50 > 10 μM). CBD and CBDA were not substrates or competitive inhibitors of canine P‐glycoprotein. Results for cannabinoid‐enriched hemp extracts were identical to those for pure cannabinoids. These in vitro studies indicate the potential for cannabinoid–drug interactions involving certain CYPs (but not P‐glycoprotein). Confirmatory in vivo studies are warranted. [ABSTRACT FROM AUTHOR]

Additionally a computational study of the regioselectivity is presented. [ABSTRACT FROM AUTHOR]