Charlie Hughes

L-Canavanine, a Dietary Nitrogen Source for the Seed Predator Caryedes brasiliensis (Bruchidae).

Larvae of the bruchid beetle Caryedes brasiliensis (Bruchidae) develop entirely within the seed of the neotropical legume Dioclea megacarpa. The seed contains an appreciable concentration of L-canavanine, a potent antimetabolite and structural analog of L-arginine. This bruchid beetle uses the nitrogen stored in this toxic allelochemical as an effective dietary nitrogen source for amino acid biosynthesis.

A simple and highly sensitive spectrophotometric method for the determination of cyanide in equine blood.

An epidemiological association among black cherry trees (Prunus serotina), eastern tent caterpillars (Malacosoma americana), and the spring 2001 episode of mare reproductive loss syndrome in central Kentucky focused attention on the potential role of environmental cyanogens in the causes of this syndrome. To evaluate the role of cyanide (CN −) in this syndrome, a simple, rapid, and highly sensitive method for determination of low parts per billion concentrations of CN − in equine blood and other biological fluids was developed. The analytical method is an adaptation of methods commonly in use and involves the evolution and trapping of gaseous hydrogen cyanide followed by spectrophotometric determination by autoanalyzer. The limit of quantitation of this method is 2 ng/mL in equine blood, and the standard curve shows a linear relationship between CN − concentration and absorbance (r >. 99). The method throughput is high, up to 100 samples per day. Normal blood CN − concentrations in horses at pasture in Kentucky in October 2001 ranged from 3-18 ng/mL, whereas hay-fed horses showed blood CN − levels of 2-7 ng/mL in January 2002. Blood samples from a small number of cattle at pasture showed broadly similar blood CN − concentrations. Intravenous administration of sodium cyanide and oral administration of mandelonitrile and amygdalin yielded readily detectable increases in blood CN − concentrations. This method is sufficiently sensitive and specific to allow the determination of normal blood CN − levels in horses, as well as the seasonal and pasture-dependent variations. The method should also be suitable for investigation of the toxicokinetics and disposition of subacutely toxic doses of CN − and its precursor cyanogens in the horse as well as in other species.

Synthesis and detection of toltrazuril sulfone and its pharmacokinetics in horses following administration in dimethylsulfoxide.

Triazine-based antiprotozoal agents are known for their lipophylic characteristics and may therefore be expected to be well absorbed following oral administration. However, although an increase in lipid solubility generally increases the absorption of chemicals, extremely lipid-soluble chemicals may dissolve poorly in gastrointestinal (GI) fluids, and their corresponding absorption and bioavailability would be low. Also, if the compound is administered in solid form and is relatively insoluble in GI fluids, it is likely to have limited contact with the GI mucosa, and therefore, its rate of absorption will be low. Based on the above considerations, we sought a solvent with low or no toxicity that would maintain triazine agents in solution. As the oral route is most preferred for daily drug therapy, such a solvent would allow an increased rate of absorption following oral administration. In present study, it was demonstrated that dimethylsulfoxide (DMSO) increased the oral bioavailability of toltrazuril sulfone (Ponazuril) threefold, relative to oral administrations of toltrazuril sulfone suspended in water. The cross-over study of toltrazuril sulfone formulated in DMSO indicated that the absolute oral bioavailability of toltrazuril sulfone in DMSO is 71%. The high bioavailability of the DMSO-preparation suggests that its daily oral administration will routinely yield effective plasma and cerebral spinal fluid (CSF) concentrations in all horses treated. Also, this improved formulation would allow clinicians to administer loading doses of toltrazuril sulfone in acute cases of Equine Protozoal Myeloencephalitis. Another option would involve administration of toltrazuril sulfone in DMSO mixed with feed (1.23 kg daily dose) meeting the US Food and Drug Administration (FDA) recommendations for the levels of DMSO permissible in pharmaceutical preparations.

The Toxicokinetics of Cyanide and Mandelonitrile in the Horse and Their Relevance to the Mare Reproductive Loss Syndrome

The epidemiological association between black cherry trees and mare reproductive loss syndrome has focused attention on cyanide and environmental cyanogens. This article describes the toxicokinetics of cyanide in horses and the relationships between blood cyanide concentrations and potentially adverse responses to cyanide. To identify safe and humane blood concentration limits for cyanide experiments, mares were infused with increasing doses (1-12 mg/min) of sodium cyanide for 1 h. Infusion at 12 mg/min produced clinical signs of cyanide toxicity at 38 min; these signs included increased heart rate, weakness, lack of coordination, loss of muscle tone, and respiratory and behavioral distress. Peak blood cyanide concentrations were about 2500 ng/mL; the clinical and biochemical signs of distress reversed when infusion stopped. Four horses were infused with 1 mg/min of sodium cyanide for 1 h to evaluate the distribution and elimination kinetics of cyanide. Blood cyanide concentrations peaked at 1160 ng/mL and then declined rapidly, suggesting a two-compartment, open model. The distribution (alpha) phase half-life was 0.74 h, the terminal (beta phase) half-life was 16.16 h. The mean residence time was 12.4 h, the steady-state volume of distribution was 2.21 L/kg, and the mean systemic clearance was 0.182 L/h/kg. Partitioning studies showed that blood cyanide was about 98.5% associated with the red cell fraction. No clinical signs of cyanide intoxication or distress were observed during these infusion experiments. Mandelonitrile was next administered orally at 3 mg/kg to four horses. Cyanide was rapidly available from the orally administered mandelonitrile and the C max blood concentration of 1857 ng/mL was observed at 3 min after dosing; thereafter, blood cyanide again declined rapidly, reaching 100 ng/mL by 4 h postadministration. The mean oral bioavailability of cyanide from mandelonitrile was 57% ± 6.5 (SEM), and its apparent terminal half-life was 13 h ± 3 (SEM). No clinical signs of cyanide intoxication or distress were observed during these experiments. These data show that during acute exposure to higher doses of cyanide (~600 mg/horse; 2500 ng/mL of cyanide in blood), redistribution of cyanide rapidly terminated the acute toxic responses. Similarly, mandelonitrile rapidly delivered its cyanide content, and acute cyanide intoxications following mandelonitrile administration can also be terminated by redistribution. Rapid termination of cyanide intoxication by redistribution is consistent with and explains many of the clinical and biochemical characteristics of acute, high-dose cyanide toxicity. On the other hand, at lower concentrations (<100 ng/mL in blood), metabolic transformation of cyanide is likely the dominant mechanism of termination of action. This process is slow, with terminal half-lives ranging from 12-16 hours. The large volume of distribution and the long terminal-phase-elimination half-life of cyanide suggest different mechanisms for toxicities and termination of toxicities associated with low-level exposure to cyanide. If environmental exposure to cyanide is a factor in the cause of MRLS, then it is likely in the more subtle effects of low concentrations of cyanide on specific metabolic processes that the associations will be found.

Trimetoquinol: bronchodilator effects in horses with heaves following aerosolised and oral administration

REASON FOR PERFORMING STUDY The bronchodilator effects of trimetoquinol (TMQ) have been studied when administered i.v. or intratracheally, but not in an aerosolised form. OBJECTIVES To define the relationship between the therapeutic and adverse responses (therapeutic index) of TMQ when administered as an aerosol or by the oral route. METHODS Increasing doses of TMQ were administered to horses with heaves as an aerosol and by the oral route. Dose ranged 100-1000 microg/horse for aerosolised TMQ and from 6-60 microg/kg bwt for the oral route. Airway and cardiac effects were assessed by measurement of maximal change in pleural pressure (deltaPplmax) and heart rate (HR), respectively. Side effects of sweating, agitation and muscle trembling were scored subjectively. Duration of action of aerosolised (1000 pg/horse) and oral (6-60 microg/kg bwt) TMQ was evaluated over 6 h. RESULTS Aerosol administration of TMQ caused dose-dependent bronchodilation but did not change HR or cause other observable side effects. When 1000 microg/horse was administered via aerosol, TMQ produced a 2-phase bronchodilation; an immediate effect lasting up to 30 min and a second phase between 2 and 4 h. Oral TMQ was therapeutically ineffective. CONCLUSION Aerosol administration of TMQ is a safe and effective method of producing bronchodilation in horses.

Catalytic conversion of alcohols: XXVIII. Product selectivities for 2-methylcyclohexanol conversion with metal oxide catalysts

Abstract Metal oxides exhibit a range of selectivities (dehydration percentage, alkene distribution and alcohol isomerization) for the conversion of a 2-methylcyclohexanol isomer. For many metal oxide catalysts, trans -2-methylcyclohexanol produces a predominance of the less stable 3-methylcyclohexene isomer. The grouping of metal oxides based on the production of the less stable alkene isomers from 2-octanol is similar to that for trans -2-methylcyclohexanol. It is proposed that the same catalytic properties determine the selectivity for both reactants: for smaller metal cations the product selectivity is determined by steric crowding in the transition state, and for the larger cations the product selectivity is determined by the basicity of the oxygen anion and the relative acidity of the β-hydrogens that are eliminated to produce water.

Synthesis, purification, and chemical characterization of 20-dihydro-6-methylprednisone, an isomeric metabolite of methylprednisolone in the horse, for use as an analytical standard

Methylprednisolone acetate, pregna-1,4-diene-3,20-dione, 21(acetoxy)-11,17-dihydro-6-methyl-,(6α,11β)-, is a synthetic glucocorticoid pro-drug widely used in equine medicine. It is recognized as a therapeutic medication by many equine organizations, including the American Association of Equine Practitioners (AAEP), the Racing Medication and Testing Consortium (RMTC), the Association of Racing Commissioners International (ARCI), and numerous racing and other regulatory authorities in the United States and elsewhere. As such, methylprednisolone is widely used on horses in training, usually as the long-acting Depo-medrol formulation administered by intra-articular or intra-muscular injection. With respect to racing and other equine performance events, its use as a controlled therapeutic medication on horses in training is regulated by the testing of plasma and/or urine, but usually by application of a quantitative regulatory threshold in plasma. For example, the RMTC lists methylprednisolone as a ‘controlled therapeutic medication’ and sets forth that a 100pg/mL methylprednisolone threshold in plasma or serum corresponds to a seven-day ‘restricted administration time’ (withdrawal time) following intra-articular administration of a ‘100mg total in one intra-articular space’. Approximately 15 metabolites of methylprednisolone have been reported in mammalian systems, though several of them have not been fully characterized due to a lack of authentic reference standards. Of these metabolites, at least three isobaric compounds have been identified in rat urine and four in human urine. It was observed, after reviewing our liquid chromatography-tandemmass spectrometry (LC-MS/MS) drug screening data and the relevant scientific literature, that standard equine analytical procedures commonly used to screen for or analyze methylprednisolone may occasionally have difficulty distinguishing between parent methylprednisolone and isobaric equine metabolites. One isomeric compound which was found in plasma extracts in conjunction with methylprednisolone (Figure 1), after examining mass spectral data, was thought to be 20-dihydro-6-methylprednisone (DHMP), an isomeric metabolite of methylprednisolone previously reported though not unequivocally identified in equine urine. This metabolite has a close structural relationship to methylprednisolone (Figure 2), and has, presuming a similar chromatographic retention time, the potential to interfere with the forensic identification and quantitation of methylprednisolone in equine racing samples. Given these possibilities, an authentic research quality reference standard of DHMP is required to unequivocally determine the

Plasma and urinary concentrations of trimetoquinol by LC‐MS‐MS following intravenous and intra‐tracheal administration to horses with heaves

Trimetoquinol (TMQ) is a very potent and fast acting bronchodilator in horses with heaves. This study assessed the plasma and urinary concentrations of TMQ in horses with heaves following administration via the intravenous (IV, 0.2 microg/kg) and intra-tracheal (IT, 2 microg/kg) routes. TMQ was administered to six horses affected with heaves (RAO - Recurrent Airway Obstruction, used interchangeably) by the above routes and plasma and urine samples collected and stored at -20 degrees C until analyzed. Solid Phase Extraction (SPE) of TMQ was followed by highly sensitive ESI(+)-LC-MS-MS (ElectroSpray Ionization, positive mode - Liquid Chromatography - Mass Spectrometry - Mass Spectrometry); with a Limit of Detection (LOD) estimated at 1 pg/mL. Following IV administration, TMQ plasma levels peaked at 1 min at 707 pg/mL, and at 9 min at 306 pg/mL following IT administration. Our results show that TMQ plasma concentrations decline rapidly following IV administration, which is consistent with the fast onset and short duration of TMQ effect that was observed in our previous studies. On the other hand, IT administration showed a very unique plasma concentration pattern. From a regulatory standpoint, the current available TMQ ELISA kit was also used in an attempt to detect TMQ from the plasma and urine samples. We report that the ELISA kit was unable to detect TMQ from any of the samples generated in these studies.

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.