Relationship between ambient temperature at sampling and the interferon gamma test result for bovine tuberculosis in cattle.

Bovine tuberculosis (bTB) is a disease of significant economic and zoonotic importance, therefore, optimising tests for the identification of Mycobacterium bovis infected cattle is essential. The Interferon Gamma (IFN-γ) Release Assay (IGRA) can diagnose M. bovis infected cattle at an early stage, is easy to perform and can be used alongside skin tests for confirmatory purposes or to increase diagnostic sensitivity. It is known that IGRA performance is sensitive to environmental conditions under which samples are taken and transported. In this study, the association between the ambient temperature on the day of bleeding and the subsequent IGRA result for bTB was quantified using field samples from Northern Ireland (NI). Results of 106,434 IGRA results (2013-2018) were associated with temperature data extracted from weather stations near tested cattle herds. Model dependent variables were the levels of IFN-γ triggered by avian purified protein derivative (PPDa), M. bovis PPD (PPDb), their difference (PPD(b-a)) as well as the final binary outcome (positive or negative for M. bovis infection). IFN-γ levels after both PPDa and PPDb stimulation were lowest at the extremes of the temperature distribution for NI. The highest IGRA positive probability (above 6%) was found on days with moderate maximum temperatures (6-16 °C) or moderate minimum temperatures (4-7 °C). Adjustment for covariates did not lead to major changes in the model estimates. These data suggest that IGRA performance can be affected when samples are taken at high or low temperatures. Whilst it is difficult to exclude physiological factors, the data nonetheless supports the temperature control of samples from bleeding through to laboratory to help mitigate post-collection confounders.

Spatiotemporal analysis of prolonged and recurrent bovine tuberculosis breakdowns in Northern Irish cattle herds reveals a new infection hotspot.

Despite a state-led eradication programme, bovine tuberculosis (bTB) remains endemic in Northern Ireland (NI). Of particular concern are "chronic" prolonged and recurrent bTB breakdowns, which represent significant financial and administrative burdens. However, little is known regarding the spatiotemporal distribution of chronic breakdowns in NI. We therefore analysed both the spatial and spatiotemporal distributions of chronic bTB breakdowns between 2004 and 2014. Significantly positive values for Moran's Index of spatial autocorrelation were found, and Local Moran's I clustering was employed to assess for spatial associations in the number and prevalence of chronic bTB breakdowns across NI. Additional spatio-temporal analysis using SaTScan showed that the burden of chronic bTB infection tends to be found where bTB levels are already high. However, a novel hotspot was revealed wherein the prevalence of chronic breakdowns was higher than expected; this should be the subject of follow-up surveillance.

Structure-activity relationships defining the ACD-tricyclic cannabinoids: cannabinoid receptor binding and analgesic activity.

Previous studies of the structure-activity relationships (SAR) for binding of a series of AC-bicyclic cannabinoid structures to the cannabinoid receptors in rat brain (believed to comprise the CB1 subtype) demonstrated the importance of the A-ring aryl C-3 side chain and phenolic hydroxyl substituents, and elucidated the importance of a C-ring hydroxyalkyl substituent [Melvin et al. Mol. Pharmacol. 44, 1008-1015 (1993)]. The present investigation examines the SAR surrounding this region (D-ring) of the molecule that is not present in the structure of delta(9)-THC and other classical cannabinoid compounds. Both rigid fused ring benzo and cyclohexyl derivatives (creating the D-ring) retained binding affinity for the cannabinoid receptor. Extension of ketone or hydroxyl substituents from the C2 position of the D-ring resulted in a 3-fold increase in binding affinity over the unsubstituted structure. However, the fused ring structure is not critical for the interaction with the receptor in as much as opening the ring did not decrease the potency. Extension of the D-ring C-2 alcohol by one carbon in length resulted in a pair of structures, for which the greatest affinity for the CB1 receptor occurred for the hydroxymethyl group in the axial conformation [(+/-)-CP-55,244]. Upon resolution, the latter provided a pair of enantiomers: (-)-CP-55,244 was approximately 3-fold more potent than the racemic mixtures, and (+)-CP-55,244 failed to bind to the CB1 receptor with an IC50 below 1 mM. Opening of the D-ring of these structures resulted in a loss of binding affinity. This study demonstrates that the potency could be optimized in (-)-CP-55,244 for both binding to the CB1 receptor and the biological activity of analgesia. In addition, the rigid positioning of the hydroxypropyl moiety of CP-55,940 enforced by the decalin ring structure of CP-55,244 increased the enantioselectivity by greater than 100-fold. These data define the critical stereochemistry for a region of the nonclassical ACD-tricyclic cannabinoid structure that contributes a potential hydrogen bonding component to the ligand-receptor interaction mechanism. Inasmuch as this region of the molecule is not present on classical ABC-tricyclic cannabinoid compounds, these studies elucidate a unique agonist recognition site on the CB1 receptor.

Structure-activity relationships for cannabinoid receptor-binding and analgesic activity: studies of bicyclic cannabinoid analogs.

Cannabimimetic compounds, such as delta 9-tetrahydrocannabinol (delta 9-THC), evoke analgesia in addition to other behavioral responses in humans and animals. The cannabinoid receptor mediating this response has been characterized by its ability to bind the cannabinoid agonist [3H]CP-55,940 and to inhibit adenylyl cyclase via Gi. An investigation of structural requirements for antinociceptive activity of cannabinoid structures led to the development of a simple bicyclic cannabinoid agonist, CP-47,497, that possessed a spectrum of cannabinoid activities in animals that resembled that of delta 9-THC. The present investigation examines several series of CP-47,497 analogs for their binding affinity at the cannabinoid receptor and their ability to evoke analgesia in rodents. Analogs substituted at the C-3 alkyl side chain exhibited maximal affinity for the cannabinoid receptor with side chains of seven or eight carbons in length. Analgesic potency paralleled the receptor-binding affinity. The cyclohexyl ring was optimized as a six- or seven-membered ring structure for binding as well as analgesic activity. Cyclohexyl alkyl side chain extensions of up to four carbons in length had little influence on the affinity for the receptor or analgesic activity. Hydroxyalkyl side chains exhibited optimal binding affinity and antinociceptive activity at three or four carbon atoms in length; however, polar groups closer to the ring diminished binding to the receptor. The importance of the phenolic and cyclohexyl hydroxyl groups for binding affinity was demonstrated. In general, analgesic activity correlated well with the affinity of these analogs for the cannabinoid receptor. Exceptions could be explained by metabolic transformations likely to occur in vivo.

Nonclassical cannabinoid analgetics inhibit adenylate cyclase: development of a cannabinoid receptor model.

Extensive structure-activity relationship studies have demonstrated that specific requirements within the cannabinoid structure are necessary to produce potent analgesia. A three-point association between the agonist and the receptor mediating analgesia consists of: 1) the C ring hydroxyl, 2) the phenolic A ring hydroxyl, and 3) the A ring alkyl hydrophobic side chain. Potent tricyclic and bicyclic structures were synthesized as "nonclassical" cannabinoid analgetics that conform to this agonist-receptor three-point interaction model. At the cellular level, centrally active cannabinoid drugs inhibit adenylate cyclase activity in a neuroblastoma cell line. The structure-activity relationship profile for inhibition of adenylate cyclase in vitro was consistent with this same three-point association of agonists with the receptor. A correlation exists between the potency of drugs to produce analgesia in vivo and to inhibit adenylate cyclase in vitro. Enantio- and stereoselectivity were exhibited by the nonclassical cannabinoid compounds for both the analgetic response and the ability to inhibit adenylate cyclase. The magnitude of the enantioselective response was equal for both the biochemical and physiological endpoints. Based on the parallels in structure-activity relationships and the enantioselective effects, it is postulated that the receptor that is associated with the regulation of adenylate cyclase in vitro may be the same receptor as that mediating analgesia in vivo. A conceptualization of the cannabinoid analgetic receptor is presented.

Enhancement of brain [3H]flunitrazepam binding and analgesic activity of synthetic cannabimimetics.

Novel, synthetic cannabimimetics and delta 9-tetrahydrocannabinol were found to enhance the binding of [3H]flunitrazepam to mouse brain in vivo. This property, suggestive of facilitation of binding to benzodiazepine receptors, is consistent with the potentiation of the anticonvulsant activity of diazepam against pentylenetetrazol by these compounds. The relative potencies of delta 9-tetrahydrocannabinol and the new cannabimimetics for enhancing [3H]flunitrazepam binding in vivo could also be correlated with their relative analgesic efficacies. Similar pharmacological stereospecificity was displayed for both binding enhancement and analgesic effects. The following order of decreasing potency was observed: N-methyllevonantradol and (-)-CP-55,244 greater than levonantradol, canbisol, CP-42,096 and (-)-CP-55,940 greater than 9-beta-normethyl-9-beta-hydroxyhexahydrocannabinol, nabilone and CP-47,497 greater than delta 9-tetrahydrocannabinol. Dextronantradol, (+)-CP-55,940 and (+)-CP-55,244 were considerably less active than the respective (-)-enantiomers; cannabidiol was inactive. Extensive investigation of structure versus activity led to N-methyllevonantradol and the 3-(2-hydroxyphenyl)cyclohexanols derivative, (-)-CP-55,244, which are approximately 1000-fold more potent than delta 9-tetrahydrocannabinol.

A cannabinoid derived prototypical analgesic.

The synthesis and analgesic testing of 3-[4-(1,1-dimethylheptyl)-2-hydroxyphenyl]cyclohexanol (1) are described. Prior (SAR) studies led us to conclude that the pyran ring of 9-nor-9 beta-hydroxyhexahydrocannabinol (HHC) was not necessary for the expression of biological activity in this series of cannabinoids. Analysis of models and the use of molecular mechanics calculations suggested that a simpler compound, such as 1, would possess the biological activity of HHC. Compound 1 was prepared in nine steps from [3-(benzyloxy)phenyl]acetonitrile (2). Biological testing in five models of pain shows that compound 1 and morphine are equally potent as analgesics and demonstrates that the pyran ring of HHC is not necessary for biological activity. Further simplification of 1 was pursued by the synthesis of 4-[4-(1,1-dimethylheptyl)-2-hydroxyphenyl]-2-pentanol (17), but this derivative exhibits significantly reduced analgesic activity.

Cannabimimetic activity from CP-47,497, a derivative of 3-phenylcyclohexanol.

CP-47,497 (cis-3-[2-hydroxy-4(1,1-dimethylheptyl)phenyl]-cyclohexan-1-ol) is characterized as a cannabimimetic agent, with 3 to 28 times greater potency than delta 9-tetrahydrocannabinol delta 9-THC), based on the following. In common with delta 9-THC and other active structures closely related chemically to delta 9-THC, CP-47,497 exerts analgesic, motor depressant, anticonvulsant and hypothermic effects. It elicits vocalization in palpated rats and ataxia in dogs. In drug discrimination studies in rats, the stimulus properties of delta 9-THC (3.2 mg/kg i.p.) are generalized to CP-47,497, with an absolute threshold dose 3 to 14 times lower than the threshold dose of delta 9-THC itself, depending on route. Furthermore, rats are unable to discriminate between the stimulus properties of equated i.p. doses of delta 9-THC and CP-47,497 after prolonged training. Despite its potent behavioral effects, CP-47,497, like delta 9-THC, does not resemble standard antipsychotic, antidepressant, antianxiety or hypnotic drugs in simple drug interaction tests. Based on its pharmacology, CP-47,497 exemplifies a simplified structure capable of producing many effects in common with those of delta 9-THC and 9-normethyl-9 beta-OH-hexahydrocannabinol.

The use of high-pressure liquid chromatography with electrochemical detection for the assay of nantradol and its application to delta 9- tetrahydrocannabinol.

High-pressure liquid chromatography with electrochemical detection permits sensitive, selective and quantitative analysis of biological and pharmaceutical compounds containing electroactive functional groups. In support of pharmacological and clinical studies with nantradol, a potent, nonopioid analgetic, a specific HPLC/electrochemical assay for nantradol and related compounds in plasma was recently developed. In preliminary studies we have now extended this technique to the detection of delta 9- and 11-OH-delta 9- tetrahydrocannabinol. Results are presented which indicate that HPLC coupled to an electrochemical detector provides sensitivity for these compounds in plasma in the low ng/ml range.

Nantradol hydrochloride: pharmacokinetics and behavioral effects after acute and chronic treatment.

Studies with [3H]nantradol hydrochloride indicate that nantradol is extensively metabolized after oral administration. By using a liquid chromatography assay with electrochemical detection, specific for both nantradol and desacetylnantradol, only desacetylnantradol is detected in plasma of rats and dogs dosed with nantradol. In animal analgetic tests, desacetylnantradol exhibits activity at least equal to nantradol. Together, these findings and additional in vitro enzymatic studies suggest that desacetylnantradol is the analgetically active species in vivo. In rats and dogs, desacetylnantradol has an apparent half-life of about 2 hr. No evidence for drug accumulation or alteration of drug metabolizing enzymes is noted from desacetylnantradol plasma level determinations during 90 days of oral and 14 days of i.m. dosing in dogs or rats at doses of 0.125 to 0.5 mg/kg i.m. and 2.5 to 10 mg/kg p.o. The static ataxia test in dogs was used to assess behavioral tolerance after chronic dosing (90 days); unlike delta 9-tetrahydrocannabinol only modest tolerance development is observed.

Selective and potent analgetics derived from cannabinoids.

Based on the hypothesis that analgetic activity is a dissociable feature of the cannabinoid molecule, we examined modifications of the side chain, the phenolic moiety, and, most significantly, structures that lack the benzopyran functionality present in THC and (--)-9-nor-9 beta-hydroxyhexahydrocannabinol (HHC). A new grouping, the 1-methyl-4-phenylbutyloxy C-3 side chain, elaborates a unique lipopholic region. Replacement of the phenol substituent produced several derivatives which retain analgetic activity in the codeine potency range. Introduction of a weakly basic nitrogen at C-5 and deletion of the axial methyl group in the B ring, two structural changes forbidden by traditional cannabinoid SAR, resulted in a unique family of benzoquinolines with potent analgetic activity. The prototype of this series, levonantradol, exhibits potent and stereospecific analgetic and antiemetic activity.

Levonantradol: a role for central prostanoid mechanisms?

Although delta 9-tetrahydrocannabinol (THC) possesses many pharmacologic activities, attempts to define sites of biochemical action for the natural cannabinoids have been hampered by their low solubility, their low potency, and their relative lack of biologic selectivity. We have recently described a potent, cannabinoid-related analgetic, levonantradol, which acts stereospecifically in animals to produce analgesia qualitatively similar to morphine but at 1/9 to 1/34 the dose. While levonantradol does not act at or through the opiate receptor, the finding of one-way cross tolerance in animals suggests that morphine and levonantradol influence common nociceptive pathways. This report describes a striking structural homology between PGE1 and levonantradol as elucidated by x-ray and conformational studies. This observation is consistent with the generally recognized involvement of prostaglandins in pain and emesis and may have relevance to the site of levonantradol's analgetic actions. More importantly, it provides an ongoing, heuristic basis for exploring, in depth, the role of prostaglandins in the action of levonantradol and cannabinoids.

Potent analgetics derived from 9-nor-9 beta-hydroxyhexahydrocannabinol.

Based on the report of morphine-like analgetic activity of 9-nor-9 beta-hydroxyhexahydrocannabinol (HHC), we undertook a study of structural modifications of the C-3 side chain of HHC to optimize the analgetic activity. We ultimately examined four distinct classes of side chains: (1) alkyl (la-lc), (2) arylalkyl (ld-lh), (3) alkoxy (li-lj) and (4) arylalkyloxy (lk-lo). Three of these derivatives (lb, lf, ll) possessed analgetic activity 10X morphine. These studies demonstrate that the C-3 side chain of HHC can be modified in a structure-dependent fashion to yield potent, nonopioid analgetics. In addition, the effect of the 1 methyl-4-phenylbutyloxy side chain is unique among the side chains examined.

The analgetic properties of piroxicam in animals and correlation with experimentally determined plasma levels.

In animal antinociceptive tests responsive to non-steroidal anti-inflammatory drugs (NSAID), piroxicam is an extremely potent and effective analgetic at doses of 1--2 mg/kg p.o. In mice the plasma half-life and duration of analgetic action is short (t 1/2 = 1.7 h), unlike man, wherein piroxicam exhibits an exceptionally long duration of action (half-life 40--45 h). An excellent correlation is observed between plasma levels and analgetic activity in the writhing test in mice suggesting that piroxicam will exhibit potent and long-lasting analgetic activity in man.