Mass spectrometric methods for evaluation of voriconazole avian pharmacokinetics and the inhibition of its cytochrome P450-induced metabolism.

Invasive fungal aspergillosis is a leading cause of morbidity and mortality in many species including avian species such as common ravens (Corvus corax). Methods were developed for mass spectral determination of voriconazole in raven plasma as a means of determining pharmacokinetics of this antifungal agent. Without further development, GC/MS/MS (gas chromatography-tandem quadrupole mass spectrometry) proved to be inferior to LC/MS/MS (liquid chromatography-tandem quadrupole mass spectrometry) for measurement of voriconazole levels in treated raven plasma owing to numerous heat-induced breakdown products despite protection of voriconazole functional groups with trimethylsilyl moieties. LC/MS/MS measurement revealed in multi-dosing experiments that the ravens were capable of rapid or ultrarapid metabolism of voriconazole. This accounted for the animals' inability to raise the drug into the therapeutic range regardless of dosing regimen unless cytochrome P450 (CYP) inhibitors were included. Strategic selection of CYP inhibitors showed that of four selected compounds including cimetidine, enrofloxacin and omeprazole, only ciprofloxacin (Cipro) was able to maintain voriconazole levels in the therapeutic range until the end of the dosing period. The optimal method of administration involved maintenance doses of voriconazole at 6 mg/kg and ciprofloxacin at 20 mg/kg. Higher doses of voriconazole such as 18 mg/kg were also tenable without apparent induction of toxicity. Although most species employ CYP2C19 to metabolize voriconazole, it was necessary to speculate that voriconazole might be subject to metabolism by CYP1A2 in the ravens to explain the utility of ciprofloxacin, a previously unknown enzymatic route. Finally, despite its widespread catalog of CYP inhibitions including CYP1A2 and CYP2C19, cimetidine may be inadequate at enhancing voriconazole levels owing to its known effects on raising gastric pH, a result that may limit voriconazole solubility.

Ciprofloxacin enhances therapeutic levels of voriconazole through CYP450 inhibition in the common raven (Corvus corax), possibly improving efficacy against aspergillosis: a pilot study.

OBJECTIVE: To determine if a cytochrome (CYP) P450 enzyme inhibitor can maintain therapeutic plasma levels of voriconazole when administered orally. ANIMALS: 11 healthy, common ravens (Corvus corax). METHODS: Birds were randomly assigned to pilot study groups to receive voriconazole orally alone or combined with a CYP inhibitor. Pilot studies with 3 CYP inhibitors launched the main study using ciprofloxacin (20 mg/kg) followed 1 hour later by voriconazole (6 mg/kg) every 12 hours for 14 days. Plasma voriconazole concentrations were measured at various time points by HPLC-MS. The study period lasted from September 2016 to December 2020. RESULTS: The birds failed to maintain therapeutic plasma levels of voriconazole during multidose administration alone or following preadministration with various CYP inhibitors. For the 14-day study period, voriconazole reached a maximum plasma concentration of 2.99 µg/mL with a time-to-peak drug concentration of 1.2 hours following preadministration of ciprofloxacin. One bird was removed from the study due to lethargy, but the other birds completed the study without incident. CLINICAL RELEVANCE: Ciprofloxacin (20 mg/kg) followed by voriconazole (6 mg/kg) maintained the concentration of voriconazole within the recommended therapeutic range of 0.5 to 5 µg/mL without toxicity. Ciprofloxacin prevented the saturable metabolism of voriconazole and maintained these levels for the study duration. This drug combination could be used in the treatment of chronic aspergillosis in the common raven.

A review of current chemistry, pharmacology, and regulation of endogenous anabolic steroids testosterone, boldenone, and nandrolone in horses.

Anabolic androgenic steroids are synthetic substances related to the male sex hormones (androgens). These agents promote the growth of skeletal muscle (anabolic effects) and the development of male sexual characteristics (androgenic effects). Anabolic steroids have been illegally used for many years as performance-enhancing drugs in human, equine, and canine sports and as growth promoters in livestock reared to provide meat for human consumption. The analytical challenge to developing effective means of control within these fields has been exacerbated by the reported endogenous nature of some of these steroids. Anabolic steroids have been employed extensively in equine practice over the past 50 years. Their usefulness is largely dependent on subjective opinions, as only minimal studies investigating pharmacodynamics have been carried out in horses. Therefore, their use will vary markedly between practitioners depending on their personal experiences and pressures by trainers to use them. They form part of rational therapy in a variety of conditions. In addition to their use for increasing muscle mass, they are used to varying extents in the raising of yearlings and in the training and racing of horses with the view of improving performance. The use of these agents is prohibited in the horseracing industry by the Association of Racing Commissioners International (ARCI), International Federation of Horseracing Authorities (IFHA), and Fédération Equestre Internationale (FEI).

Total Carbon Dioxide (TCO2) Concentrations in Thoroughbred and Quarter Racehorses in Louisiana.

The TCO2 (total carbon dioxide) test is performed on the blood of racehorses as a means of combatting the practice of administering alkalizing agents. This study evaluated serum TCO2 concentrations and factors influencing concentration of TCO2 in Thoroughbred and Quarter Horses. The normality of data were evaluated with a Shapiro-Wilk test. Mann-Whitney tests and Kruskal-Wallis tests were used against different effects. When a fixed effect was detected, Dunn's post-hoc comparisons were performed. The median pre-race serum TCO2 concentration (32.20 mmol/L (interquartile range (IQR): 30.80-33.50)) was higher than that of post-race samples (26.70 mmol/L (IQR: 24.55-29.25)) (P < .0001). The median TCO2 concentrations in pre-race samples were different between Thoroughbred (32.40 mmol/L (IQR: 30.90-33.60)) and Quarter Horses (31.30 mmol/L (IQR: 30.00-32.50)) (P < .0001). The median pre-race TCO2 concentrations were 32.75 (IQR: 31.40-33.90), 31.40 (IQR: 29.80-32.80), 32.50 (IQR: 31.20-33.88), and 31.60 (IQR 30.00-32.70) mmol/L in racehorses at Fair Grounds, Louisiana Downs, Delta Downs, and Evangeline Downs racetracks, respectively (P < .0001). The total serum TCO2 concentrations in Thoroughbred and Quarter Horse racehorses were affected by seasonal temperature variation (P < .0001). A smaller sample size was available for post-race samples (n = 205) and Quarter Horse pre-race samples (n = 351). The results of this study indicated that the breed, seasonal temperature variation, pre-race or post-race sampling, and track location are strongly correlated to total TCO2 concentrations. It was not clear whether the statistically significant differences in TCO2 levels among racetracks in Louisiana were due to location of racetracks and/or seasonal temperature variation.

Simultaneous quantification of caffeine and its main metabolites by gas chromatography mass spectrometry in horse urine.

Caffeine is a naturally occurring alkaloid and it is metabolized to paraxanthine, theophylline and theobromine. Analysis of caffeine and its metabolites is challenging since the metabolites theophylline and paraxanthine generate similar product and precursor ions. In this study, a new method was developed for the simultaneous analysis of caffeine, paraxanthine, theobromine and theophylline in horse urine using gas chromatography-mass spectrometry (GC-MS). Urine samples were treated using solid-phase extraction followed by the elution with dichloromethane-isopropanol (90:10) after the pH was adjusted to 6, and then derivatization with N-methyl-N-trimethylsilyl-trifluoroacetamide-1% trimethylchlorosilane before analysis with GC-MS. Sample preparation and derivatization steps were optimized and the method permitted elution all of these analytes within 13 min. The method was fully validated according to Commission Decision, 2002/657/EC guidelines. The calibration curves were linear with a correlation coefficient of >0.99. Precision and accuracy were well within the 15% acceptance range and the method was robust. The validation results demonstrated that the method is highly reproducible, easily applicable and selective. The method was applied to urine samples collected from racehorses to demonstrate its applicability.

Plasma concentrations of diclazuril following oral administration of diclazuril and diclazuril sodium salt to cattle.

Diclazuril is a triazine-based antiprotozoal agent widely used in veterinary practice that may have clinical application in the treatment of bovine protozoal diseases. The present study reports on the bioavailability, pharmacokinetics, and metabolism of diclazuril and diclazuril sodium salt in cattle following administration of diclazuril suspended in water and by direct application of diclazuril sodium salt to the oral mucosa. Compared with diclazuril itself, the sodium salt formulation of diclazuril applied to the oral mucosa was rapidly and reliably absorbed. Plasma concentrations of diclazuril peaked at around 8 h after oral-mucosal administration of diclazuril sodium salt. On the contrary, application of diclazuril itself orally resulted in delayed and variable absorption. The mean bioavailability of diclazuril as pure powder was 42.5% relative to diclazuril sodium salt indicating approximately 2.5-fold increase in bioavailability of diclazuril as a sodium salt relative to diclazuril as a pure compound in cattle. The present study also reports finding of a previously unreported diclazuril metabolite at high concentrations in plasma especially after oral administration of diclazuril. Further studies, including synthesis and characterization of the novel described metabolite, are required to accurately determine aspects of the metabolism of diclazuril in cattle.

The medication violations in racehorses at Louisiana racetracks from 2016 to 2020.

INTRODUCTION/BACKGROUND: The number of publications for most common drug violations in racehorses is limited. This study reports the most common medication violations in racehorses at four major racetracks in Louisiana between 2016 and 2020. METHODS: During this 5-year period, 27,237 blood samples and 25,672 urine samples collected during the course of normal race meeting activities were analysed by initial screening procedure utilizing Liquid Chromatography Mass Spectrometry (LC-MS/MS). Following initial screening, suspect samples were subject to quantitative or semi- quantitative confirmation analysis by LC-MS/MS. RESULTS: The total number of violations reported was 534 (1.01% of the total number of specimens analysed). The total number of violations reported in Thoroughbred horses was 210 while the total number of violations reported in Quarter Horses was 324. The percentage of total violations was %0.59 for all the specimens analysed in Thoroughbred horses while this percentage was %1.9 for all the specimens analysed in Quarter Horses during this 5-year period. The most frequent violations included the overages (concentrations of permitted medications equal to or exceeding the set threshold) of clenbuterol (165 violations), non-steroidal anti-inflammatory drugs (NSAIDs) such as phenylbutazone (73 violations), combination of phenylbutazone with flunixin (45 violations) and muscle relaxant methocarbamol (40 violations). DISCUSSION/CONCLUSIONS: The total number of violations were relatively low during 5-year period, but wide varieties of medications with different pharmacological actions were confirmed in performance horses in Louisiana. The most frequently reported violations in Louisiana were for permitted therapeutic medications (clenbuterol, phenylbutazone, flunixin methocarbamol) with established threshold and/or withdrawal guidelines in racehorses.

Pharmacokinetic profiles of three dose rates of morphine sulfate following single intravenous, intramuscular, and subcutaneous administration in the goat.

This study aimed to evaluate pharmacokinetic profiles of morphine in goats following a single dose administered intravenously, intramuscularly, or subcutaneously at 0.1 mg/kg, 0.25 mg/kg, and 0.4 mg/kg. Study population included eight healthy adult goats in a randomized cross-over study. Serial plasma samples were collected and morphine was quantified using high-performance liquid chromatography/mass spectrometry. Data fit a two-compartment model following intravenous administration and a non-compartmental model following both intramuscular and subcutaneous administration. Plasma elimination half-life was 2.88 ± 1.13 h (0.1 mg/kg), 2.30 ± 0.49 h (0.25 mg/kg), and 2.67 ± 0.82 h (0.4 mg/kg) following IV morphine. Intramuscular Cmax values were 13.4 ± 2.77 ng/ml (0.1 mg/kg), 34 ± 11.50 ng/ml (0.25 mg/kg), and 68.9 ± 24.5 ng/ml (0.4 mg/kg). Intramuscular Tmax f(h) or IM dosing (in hrs) was 0.19 ± 0.14 (0.1 mg/kg), 0.24 ± 0.24 (0.25 mg/kg), and 0.21 ± 0.24 (0.4 mg/kg). Subcutaneous Cmax values were 9.88 ± 3.31 ng/ml (0.1 mg/kg), 28.5 ± 11.6 ng/ml (0.25 mg/kg), and 39.4 ± 14.3 ng/ml (0.4 mg/kg). Subcutaneous Tmax (h) values for SC dosing were 0.36 ± 0.21 (0.1 mg/kg), 0.31 ± 0.17 (0.25 mg/kg), and 0.4 ± 0.13 (0.4 mg/kg). Intramuscular bioavailability values were 153.77 ± 12.60% (0.4 mg/kg), 104.8 ± 25.12% (0.25 mg/kg), and 100.7 ± 29.57% (0.1 mg/kg). Subcutaneous bioavailability values were 130.58 ± 19.07% (0.4 mg/kg), 116.6 ± 27.03% (0.25 mg/kg), and 111.6 ± 23.24% (0.1 mg/kg). No adverse effects were observed. Assuming plasma concentration required to induce analgesia is 16 ± 9 ng/ml in goats, as demonstrated in humans, it is suggested to administer morphine intramuscularly at 0.4 mg/kg every 3-4 h or SC every 2-3 h. This is a speculative conclusion therefore further studies evaluating pharmacodynamics and plasma analgesic threshold in goats is recommended.

Liquid chromatography/tandem mass spectrometric analysis of penicillamine for its pharmacokinetic evaluation in dogs.

Copper storage disease occurs in multiple dog breeds and is one of the most common causes of chronic hepatitis in this species. The disease is caused by hereditary defects in copper metabolism in conjunction with high dietary copper levels. The progressive copper accumulation leads to hepatitis, cirrhosis, and eventually death if left untreated. Copper chelators are critical in modulating the effects of this disease. It is therefore of significant practicality to understand the pharmacokinetic (PK) parameters of chelating agents, particularly since they are oftentimes quite expensive. A liquid chromatography-tandem mass spectrometric (LC/MS/MS) method was developed to measure plasma levels of one of the most common chelators, d-penicillamine. The compound was discovered to exist in two forms, monomeric and dimeric, and various chemical derivatizations were tried to force the compound into one form or the other. Eventually, the simplest approach was individual determination of penicillamine and its dimer, with summation of the two quantities. This enabled determination of canine PK parameters for penicillamine based on comparison of oral and intravenous administration of the drug, including time to maximum drug level (Tmax), concentration at maximum (Cmax), clearance (Cls) and volume of distribution (Vdss). The drug was found to exist predominantly in the dimeric form in plasma, which is incapable of chelating copper owing to lack of free sulfhydryl groups and must therefore provide a storage form of the drug in equilibrium with its monomeric form in vivo. Mechanisms are discussed for the electrospray-induced fragmentation of penicillamine as well as of its dimer.

The effect of sodium bicarbonate and validation of beckman coulter AU680 analyzers for measuring total carbon dioxide (TCO2) concentrations in horse serum.

This study evaluated the usage of Beckman Coulter AU680 analyzers for measurement of TCO 2 in horse serum, and the effect of sodium bicarbonate administrations on serum TCO 2 levels in resting horses. Treatment of horses with sodium bicarbonate did not result in any adverse events. Mean TCO 2 concentration was significantly higher from 1 to 8 h in the sodium bicarbonate-treated horses compared to the untreated controls. Within an hour, administration of sodium bicarbonate increased the TCO 2 level from 31.5 ± -2.5 (SD) to 34.0 ± 2.65 (SD) mmol/L and at 2-8 h post-administration, the TCO 2 level was above the 36 mmol/L cut-off level. In all quality control analysis of Australian standard by Beckman Coulter AU680 analyzer, the instrument slightly over estimated the TCO 2 level but the values were in close agreement with mean TCO 2 level being 38.03 with ± 0.87 mmol/L (SD). Expanded uncertainty was calculated using different levels of confidence interval. Based on 99.5% confidence interval using 0.805% expanded uncertainty using mean measured concentration of 38.05 mmol/L, it was estimated that any race samples TCO 2 level higher than 38.5 mmol/L will be indicative of sodium bicarbonate administration using Beckman Coulter AU680 analyzer in Louisiana.

Characterization of long-term elution of platinum from carboplatin-impregnated calcium sulfate hemihydrate beads in vitro by two distinct sample collection methods.

OBJECTIVE To characterize long-term elution of platinum from carboplatin-impregnated calcium sulfate hemihydrate (CI-CSH) beads in vitro by comparing 2 distinct sample collection methods designed to mimic 2 in vivo environments. SAMPLES 162 CI-CSH beads containing 4.6 mg of carboplatin (2.4 mg of platinum/bead). PROCEDURES For method 1, which mimicked an in vivo environment with rapid and complete fluid exchange, each of 3 plastic 10-mL conical tubes contained 3 CI-CSH beads and 5 mL of PBS solution. Eluent samples were obtained by evacuation of all fluid at 1, 2, 3, 6, 9, and 12 hours and 1, 2, 3, 6, 9, 12, 15, 18, 22, 26, and 30 days. Five milliliters of fresh PBS solution was then added to each tube. For method 2, which mimicked an in vivo environment with no fluid exchange, each of 51 tubes (ie, 3 tubes/17 sample collection times) contained 3 CI-CSH beads and 5 mL of PBS solution. Eluent samples were obtained from the assigned tubes for each time point. All samples were analyzed for platinum content by inductively coupled plasma-mass spectrometry. RESULTS Platinum was released from CI-CSH beads for 22 to 30 days. Significant differences were found in platinum concentration and percentage of platinum eluted from CI-CSH beads over time for each method. Platinum concentrations and elution percentages in method 2 samples were significantly higher than those of method 1 samples, except for the first hour measurements. CONCLUSIONS AND CLINICAL RELEVANCE Sample collection methods 1 and 2 may provide estimates of the minimum and maximum platinum release, respectively, from CI-CSH beads in vivo.

Pharmacokinetics and derivation of an anticancer dosing regimen for the novel anti-cancer agent isobutyl-deoxynyboquinone (IB-DNQ), a NQO1 bioactivatable molecule, in the domestic felid species.

Isobutyl-deoxynyboquinone (IB-DNQ) is a selective substrate for NAD(P)H:quinone oxidoreductase (NQO1), an enzyme overexpressed in many solid tumors. Following activation by NQO1, IB-DNQ participates in a catalytic futile reduction/reoxidation cycle with consequent toxic reactive oxygen species generation within the tumor microenvironment. To elucidate the potential of IB-DNQ to serve as a novel anticancer agent, in vitro studies coupled with in vivo pharmacokinetic and toxicologic investigations in the domestic felid species were conducted to investigate the tractability of IB-DNQ as a translationally applicable anticancer agent. First, using feline oral squamous cell carcinoma (OSCC) as a comparative cancer model, expressions of NQO1 were characterized in not only human, but also feline OSCC tissue microarrays. Second, IB-DNQ mediated cytotoxicity in three immortalized feline OSCC cell lines were studied under dose-dependent and sequential exposure conditions. Third, the feasibility of administering IB-DNQ at doses predicted to achieve cytotoxic plasma concentrations and biologically relevant durations of exposure were investigated through pharmacokinetic and tolerability studies in healthy research felines. Intravenous administration of IB-DNQ at 1.0-2.0 mg/kg achieved peak plasma concentrations and durations of exposure reaching or exceeding predicted in vitro cytotoxic concentrations. Clinical adverse side effects including ptyalism and tachypnea exhibited during and post-IV infusion of IB-DNQ were transient and tolerable. Additionally, IB-DNQ administration did not produce acute or delayed-onset unacceptable hematologic, non-hematologic, or off-target oxidative toxicities. Collectively, the findings reported here within provide important safety and pharmacokinetic data to support the continued development of IB-DNQ as a novel anticancer strategy for NQO1 expressing cancers.

Elution of platinum from carboplatin-impregnated calcium sulfate hemihydrate beads in vitro.

OBJECTIVE To characterize the elution of platinum from carboplatin-impregnated calcium sulfate hemihydrate (CSH) beads in vitro. SAMPLE 60 carboplatin-impregnated CSH beads and 9 CSH beads without added carboplatin (controls). PROCEDURES Carboplatin-impregnated CSH beads (each containing 4.6 mg of carboplatin [2.4 mg of platinum]) were placed into separate 10-mL plastic tubes containing 5 mL of PBSS in groups of 1, 3, 6, or 10; 3 control beads were placed into a single tube of PBSS at the same volume. Experiments were conducted in triplicate at 37°C and a pH of 7.4 with constant agitation. Eluent samples were collected at 1, 2, 3, 6, 12, 24, and 72 hours. Samples were analyzed for platinum content by inductively coupled plasma-mass spectrometry. RESULTS The mean concentration of platinum released per carboplatin-impregnated bead over 72 hours was 445.3 mg/L. Cumulative concentrations of platinum eluted increased as the number of beads per tube increased. There was a significant difference in platinum concentrations over time, with values increasing over the first 12 hours and then declining for all tubes. There was also a significant difference in percentage of total incorporated platinum released into tubes with different numbers of beads: the percentage of eluted platinum was higher in tubes containing 1 or 3 beads than in those containing 6 or 10 beads. CONCLUSIONS AND CLINICAL RELEVANCE Carboplatin-impregnated CSH beads eluted platinum over 72 hours. Further studies are needed to determine whether implantation of carboplatin-impregnated CSH beads results in detectable levels of platinum systemically and whether the platinum concentrations eluted locally are toxic to tumor cells.

Tear film concentrations of doxycycline following oral administration in ophthalmologically normal dogs.

OBJECTIVE To determine tear film concentrations of doxycycline in ophthalmologically normal dogs following oral doxycycline administration. DESIGN Crossover study. ANIMALS 10 privately owned dolichocephalic or mesaticephalic dogs free of ophthalmic disease. PROCEDURES Dogs were randomly assigned to receive doxycycline hyclate first at 5 mg/kg (2.3 mg/lb) or 10 mg/kg (4.5 mg/lb), PO, every 12 hours for 5 days, beginning on day 1. Doxycycline was administered 1 hour prior to feeding. Tear samples were collected from days 1 through 10 approximately 3 hours after the morning dose was administered. Following a 3-week washout period, dogs received the alternative dose in the same conditions. Doxycycline concentration in tear samples from 1 eye (same eye used for both sessions) was measured via liquid chromatography-mass spectrometry and compared between the 2 doxycycline doses. RESULTS Doxycycline was detected in tear samples of all dogs from days 1 through 10 for both doxycycline doses. Median peak doxycycline concentrations for the 5 mg/kg and 10 mg/kg doses were 2.19 ng/mL on day 3 and 4.32 ng/mL on day 4, respectively. Concentrations differed significantly with time, but this difference was not influenced by dose, dose order, or eye. A significant positive correlation was identified between doxycycline concentration and body weight (r = 0.22). CONCLUSIONS AND CLINICAL RELEVANCE Detectable doxycycline concentrations were achieved in the tear film of ophthalmologically normal dogs following oral administration of doxycycline at 5 or 10 mg/kg, every 12 hours. Dose had no significant effect on tear film concentration of the drug.

Removal of Metabolic Liabilities Enables Development of Derivatives of Procaspase-Activating Compound 1 (PAC-1) with Improved Pharmacokinetics.

Procaspase-activating compound 1 (PAC-1) is an o-hydroxy-N-acylhydrazone that induces apoptosis in cancer cells by chelation of labile inhibitory zinc from procaspase-3. PAC-1 has been assessed in a wide variety of cell culture experiments and in vivo models of cancer, with promising results, and a phase 1 clinical trial in cancer patients has been initiated (NCT02355535). For certain applications, however, the in vivo half-life of PAC-1 could be limiting. Thus, with the goal of developing a compound with enhanced metabolic stability, a series of PAC-1 analogues were designed containing modifications that systematically block sites of metabolic vulnerability. Evaluation of the library of compounds identified four potentially superior candidates with comparable anticancer activity in cell culture, enhanced metabolic stability in liver microsomes, and improved tolerability in mice. In head-to-head experiments with PAC-1, pharmacokinetic evaluation in mice demonstrated extended elimination half-lives and greater area under the curve values for each of the four compounds, suggesting them as promising candidates for further development.

Alkylation and Carbamylation Effects of Lomustine and Its Major Metabolites and MGMT Expression in Canine Cells.

DNA Alkylation is thought to be the reason for the efficacy of lomustine while carbamylation has been implicated as the cause for the side effects seen with lomustine treatment such as hepatotoxicity. In the alkylation study we show that lomustine and its metabolites form similar levels of the DNA adducts N7 hydroxyethylguanine and O6 hydroxyethyldeoxyguanosine. In terms of carbamylation, lomustine showed greater extent of carbamylation in the canine hepatocytes and lymphoma cell lines. The DNA repair enzyme O6 methylguanine DNA methyltransferase (MGMT) causes resistance of tumor cells to bifunctional nitrosourea, like lomustine. There is no data available regarding MGMT expression/activity in canine cells or tissues. Our study shows that there is low MGMT activity in the canine lymphoid cell line 17-71 while the GL-1 cells did not show any detectable enzyme activity or mRNA expression. The MGMT enzyme activity measured in canine hepatocytes is about 250-350 fmol/mg protein as compared to about 90 fmol/mg protein in 17-71 cells. We also show that MGMT mRNA expression in 17-71 cells and canine hepatocytes positively correlates with its enzyme activity in these cells.

Dual small-molecule targeting of procaspase-3 dramatically enhances zymogen activation and anticancer activity.

Combination anticancer therapy typically consists of drugs that target different biochemical pathways or those that act on different targets in the same pathway. Here we demonstrate a new concept in combination therapy, that of enzyme activation with two compounds that hit the same biological target, but through different mechanisms. Combinations of procaspase-3 activators PAC-1 and 1541B show considerable synergy in activating procaspase-3 in vitro, stimulate rapid and dramatic maturation of procaspase-3 in multiple cancer cell lines, and powerfully induce caspase-dependent apoptotic death to a degree well exceeding the additive effect. In addition, the combination of PAC-1 and 1541B effectively reduces tumor burden in a murine lymphoma model at dosages for which the compounds alone have minimal or no effect. These data suggest the potential of PAC-1/1541B combinations for the treatment of cancer and, more broadly, demonstrate that differentially acting enzyme activators can potently synergize to give a significantly heightened biological effect.

Scopolamine in racing horses: trace identifications associated with dietary or environmental exposure.

Scopolamine (L-hyoscine) identifications, often in small-number clusters, have been reported worldwide in performance horses over the last 30 years. Scopolamine is an Association of Racing Commissioners International (ARCI) class 3, penalty class B, substance with potential to affect performance. As such, scopolamine identification(s) in race or performance horses can result in significant penalties for the connections of the horse(s). Reviewed here is the worldwide distribution of scopolamine containing plants (primarily Datura spp.), with estimates of their potential toxicity to horses through dietary and/or environmental exposure. Also reviewed are the basic pharmacology of scopolamine and its precursor, urinary concentrations following feedstuff exposure, and the probable pharmacological/forensic significance of such findings. Based on an overview of the world literature on scopolamine, the expected characteristics of inadvertent environmental exposure are also presented with a view to making clear the potential of scopolamine identifications, with or without atropine, as a direct and expected outcome of both the worldwide distribution of scopolamine-containing plants and the sensitivity of modern equine drug testing. It is of particular interest that only 2/30 reported post-event equine identifications of scopolamine have been associated with atropine, suggesting that failure to identify atropine is not a biomarker of pharmaceutical administration of scopolamine. Available quantitative information associated with scopolamine identifications is consistent with the 75 ng/mL regulatory threshold for scopolamine currently used in Louisiana racing in the USA and the 30 ng/mL reporting threshold in effect in European racing.

A clinician's guide to factors affecting withdrawal times for equine therapeutic medications.

Equine forensic science can now detect concentrations down to 25 femtograms/mL (parts per quadrillion, ppq) or less in blood and urine. As such, horsemen are increasingly at risk of inadvertent 'positives' due to therapeutic medication 'overages' or trace identifications of dietary or environmental substances. Reviewed here are the factors which determine detection times and 'withdrawal times' for substances administered to horses. Withdrawal times are affected by many factors, including dose, formulation, route and frequency of administration, bioavailability, plasma half-life, sensitivity of the analytical process, the testing matrix (plasma, urine, or other), and the environmental presence and/or persistence of administered substances. Of these factors only dose is known precisely. For any given administration, horse-to-horse differences in the volumes of distribution, systemic clearance, and terminal plasma elimination half-life of substances are major and totally uncontrollable factors driving horse-to-horse variability in withdrawal times. A further complication is that chemically stable medications administered to horses and eliminated in the urine inevitably become part of the environment of the horse. The presence of these substances in the equine environment is increasingly giving rise to trace identifications long after nominal administration of these substances has ceased. Because of the unknown and uncontrollable horse-to-horse variability in medication pharmacokinetics, any therapeutic medication administration to a horse by definition includes the possibility of an inadvertent medication overage. As such, the caveat that there are no guarantees in life most assuredly applies to advisories concerning equine therapeutic medication withdrawal times.