Preclinical Assessment of the Abuse Potential of Purified Botanical Cannabidiol: Self-Administration, Drug Discrimination, and Physical Dependence.

Cannabidiol (CBD) is a constituent of the cannabis plant with a diverse array of pharmacological activities as well as potential therapeutic uses. An oral formulation of CBD (Epidiolex in the US; Epidyolex in Europe) is approved for treating seizures associated with rare and severe forms of epilepsy. These studies, which supported the approval of the medication, investigated abuse-related effects of CBD in rats and nonhuman primates (NHPs) using drug self-administration, drug discrimination, and physical dependence procedures and characterized its pharmacokinetics. In NHPs (n = 5) that self-administered midazolam (0.01 or 0.032 mg/kg/infusion), CBD (0.1-3.2 mg/kg/infusion) failed to maintain responding above vehicle levels. CBD maintained very modest levels of self-administration in rats (n = 7-8) that self-administered heroin (0.015 mg/kg/infusion) and did not increase drug-lever responding, up to a dose of 150 mg/kg (by mouth), in rats (n = 6) trained to discriminate 0.5 mg/kg (i.p.) midazolam. In juvenile (5-6 weeks old) and adult (10-11 weeks old) male and female rats, discontinuation of chronic treatment (twice daily for 20 days) with an oral formulation of CBD (20 or 100 mg/kg, by mouth) did not reliably produce signs of withdrawal. Pharmacokinetic studies confirmed that the dosing regimens used in these studies resulted in therapeutically relevant plasma levels. Taken together, the lack of reliable self-administration, the failure to increase drug-lever responding in rats trained to discriminate midazolam, and the absence of withdrawal signs upon discontinuation of chronic treatment indicate that CBD has very low abuse potential and is unlikely to produce physical dependence. SIGNIFICANCE STATEMENT: Legalization of cannabis across the United States and elsewhere has led to intense investigation into the safety and therapeutic potential of cannabis and its constituent materials, including cannabidiol (CBD). Results of these preclinical abuse potential studies on CBD indicate no rewarding properties, physical dependence potential, or similarity to a benzodiazepine. Together with data from in vitro pharmacology and human abuse potential studies, the abuse potential of Epidiolex in humans is likely to be negligible.

A contribution to the discussion on the safety of air weapons.

Firearms legislation in the UK stems from the Firearms Act 1968 with its definition of a firearm as a lethal barrelled weapon of any description. The Act allows certain exceptions to be held without licence, most notably air weapons although these are limited by The Firearms (Dangerous Air Weapons) Rules 1969 and related regulations to below 12ft lb (16.3J) for air rifles and below 6ft lb (8.1J) for air pistols. Despite this there are occasional fatalities, typically 1 or 2 each year in the UK, from legally owned air weapons. In the USA there are over 20,000 visits each year to emergency departments due to injuries from air weapons and paintball guns. Despite this, limited research appears to have been carried out into the safety of air weapons and the present study tries to address this. Fresh samples of animal tissue were obtained from an abattoir or butcher and were embedded in ballistic gelatin. Pig heart, lung, liver and shoulder were used. By firing pellets into gelatin alone and into the combination of the gelatin and animal tissue it was possible to compare gelatin as a model for these tissues. The depth of penetration was similar but the residual track appeared to remain more open in the animal tissue. Pellets penetrated completely through the organ, with total penetration of gelatin and organ being typically around 10-15cm. Samples of pig, cow and chicken skin were placed in contact with the gelatin or embedded in the gelatin to simulate the effect of skin on penetration into a body. Chicken skin had no effect, pig skin stopped the pellet and cow skin was perforated by the pellet. If cow skin was embedded in the gelatin there was little effect on the total amount of penetration, but cow skin on the front surface of the gelatin reduced penetration by about 30%. Computed tomography was used to examine the pellet track and to calculate the volume of damage produced. However, due to the similar densities of gelatin and organ a technique had to be developed to differentiate phases. A barium salt paste was applied to outer surfaces and iodine solution or barium nitrate solution containing red food colouring was injected into the pellet track to enhance the contrast of the track. The track through the gelatin tended to enclose itself whereas the track through the organ remained more open, presumably due to the inhomogeneity of the fibrous nature of the tissue. Pellets were also fired at construction materials (wood, plasterboard and brick) and computed tomography used to determine the volume of damage created. Pellets perforated single layers of wood and plasterboard and would embed in a second layer. However, if the two layers were in contact the pellet did not penetrate the first layer. An air rifle pellet could therefore perforate house construction materials, although the resultant kinetic energy would be low and further damage would be limited. Some of the possible physical parameters are discussed that might help predict the degree of damage caused, but from this study it is not possible to define a limit which could be proposed as safe.

Decontamination of transvaginal ultrasound probes: review of national practice and need for national guidelines.

AIM: To determine the national practice of transvaginal ultrasound (TVUS) probe decontamination in English hospitals and to develop recommendations for guidance. MATERIALS AND METHODS: A literature review was undertaken to clarify best practice and evaluate methods of decontamination of TVUS probes. A questionnaire was developed to ascertain TVUS probe decontamination programmes in current use within English hospitals. This was sent to ultrasound leads of 100 English hospitals; 68 hospitals responded. RESULTS: There is a wide variation in TVUS probe decontamination across English hospitals. Although the majority of respondents (87%, 59/68) reported having clear and practical written guidelines for TVUS decontamination, the frequency, methods, and types of decontamination solutions utilized were widely variable and none meet the standards required to achieve high-level disinfection. CONCLUSION: While the decontamination of other endoluminal medical devices (e.g., flexible endoscopes) is well defined and regulated, the decontamination of TVUS probes has no such guidance. There appears to be incomplete understanding of the level of risk posed by TVUS probes, and in some cases, this has resulted in highly questionable practices regarding TVUS hygiene. There is an urgent need to develop evidence-based national guidance for TVUS probe decontamination.

Thiocarbamate sulfoxides: potent, selective, and biodegradable herbicides.

Sulfoxidation of thiocarbamates yields a new class of chemicals having increased herbicidal activity along with greater tolerance of corn and soybeans in greenhouse tests. However, their thermal stability is not favorable. These sulfoxides are intermediates in the mammalian metabolism of thiocarbamates, being formed by liver microsomal oxidases and cleaved in a system consisting of glutathione and a soluble enzyme from liver.

Shock-induced figure-of-eight reentry in the isolated rabbit heart.

The patterns of transmembrane potential on the whole heart during and immediately after fibrillation-inducing shocks are unknown. To study arrhythmia induction, we recorded transmembrane activity from the anterior and posterior epicardial surface of the isolated rabbit heart simultaneously using 2 charge-coupled device cameras (32,512 pixels, 480 frames/second). Isolated hearts were paced from the apex at a cycle length of 250 ms. Two shock coils positioned inside the right ventricle (-) and atop the left atrium (+) delivered shocks at 3 strengths (0.75, 1.5, and 2.25 A) and 6 coupling intervals (130 to 230 ms). The patterns of depolarization and repolarization were similar, as is evident in the uniformity of action potential duration at 75% repolarization (131.4¿8.3 ms). At short coupling intervals (<180 ms), shocks hyperpolarized a large portion of the ventricles and produced a pair of counterrotating waves, one on each side of the heart. The first beat after the shock was reentrant in 90% of short coupling interval episodes. At long coupling intervals (>180 ms), increasingly stronger shocks depolarized an increasingly larger portion of the heart. The first beat after the shock was reentrant in 18% of long coupling interval episodes. Arrhythmias were most often induced at short coupling intervals (98%) than at long coupling intervals (35%). The effect and outcome of the shock were related to the refractory state of the heart at the time of the shock. Hyperpolarization occurred at short coupling intervals, whereas depolarization occurred at long coupling intervals. Consistent with the "critical point" hypothesis, increasing shock strength and coupling interval moved the location where reentry formed (away from the shock electrode and pacing electrode, respectively).

Mechanism of ventricular defibrillation for near-defibrillation threshold shocks: a whole-heart optical mapping study in swine.

BACKGROUND: To study the mechanism by which shocks succeed (SDF) or fail (FDF) to defibrillate, global cardiac activation and recovery and their relationship to defibrillation outcome were investigated for shock strengths with approximately equal SDF and FDF outcomes (DFT(50)). METHODS AND RESULTS: In 6 isolated pig hearts, dual-camera video imaging was used to record optically from approximately 8000 sites on the anterior and posterior ventricular surfaces before and after 10 DFT(50) biphasic shocks. The interval between the shock and the last ventricular fibrillation activation preceding the shock (coupling interval, CI) and the time from shock onset to 90% repolarization of the immediate postshock action potential (RT(90)) were determined at all sites. Of 60 shocks, 31 were SDF. The CI (59+/-7 versus 52+/-6 ms) and RT(90) (108+/-19 versus 88+/-8 ms) were significantly longer for SDF than FDF episodes. Spatial dispersions of CI (36+/-5 versus 34+/-3 ms) and RT(90) (40+/-16 versus 40+/-8 ms) were not significantly different for SDF versus FDF episodes. The first global activation cycle appeared focally on the left ventricular apical epicardium 78+/-32 ms after the shock. CONCLUSIONS: For near-threshold shocks, defibrillation outcome correlates with the electrical state of the heart at the time of the shock and on RT. Global dispersion of RT was similar in both SDF and FDF episodes, suggesting that it is not crucial in determining defibrillation outcome after DFT(50) shocks.

Optical mapping of drug-induced polymorphic arrhythmias and torsade de pointes in the isolated rabbit heart.

OBJECTIVES: This study sought to 1) test the hypothesis that in the setting of bradycardia and drug-induced action potential prolongation, multiple foci of early afterdepolarizations (EADs) result in beat to beat changes in the origin and direction of the excitation wave front and are responsible for polymorphic arrhythmias; and 2) determine whether EADs may initiate nonstationary reentry, giving rise to the typical torsade de pointes (TDP) pattern. BACKGROUND: In the past, it has been difficult to associate EADs or reentry with the undulating electrocardiographic (ECG) patterns of TDP. METHODS: A voltage-sensitive dye was used for high resolution video imaging of electrical waves on the epicardial and endocardial surface of the Langendorff-perfused rabbit heart. ECG and monophasic action potentials from the right septal region were also recorded. Bradycardia was induced by ablation of the atrioventricular node. RESULTS: Perfusion of low potassium chloride Tyrode solution plus quinidine led to prolongation of the action potential and the QT interval. Eventually, EADs and triggered activity ensued, giving rise to intermittent episodes of polymorphic arrhythmia. In one experiment, triggered activity was followed by a long episode of vortex-like reentry with an ECG pattern characteristic of TDP. However, in most experiments, focal activity of varying origins and propagation patterns was observed. Triggered responses also showed varying degrees of local block. Similar results were obtained with E-4031. Burst pacing both at control conditions and in the presence of quinidine consistently led to vortex-like reentry whose ECG pattern resembled TDP. However, the cycle length of the arrhythmia with quinidine was longer than that for control ([mean +/- SEM] 194 +/- 12 vs. 132 +/- 8 ms, p < 0.03). CONCLUSIONS: Drug-induced polymorphic ventricular arrhythmias may result from beat to beat changes in wave propagation patterns initiated by EADs or EAD-induced nonstationary reentrant activity. In contrast, burst pacing-induced polymorphic tachycardia in the presence or absence of drugs is the result of nonstationary reentrant activity.

Numbers and dispersion of repopulating hematopoietic cell clones in radiation chimeras as functions of injected cell dose.

Lethally irradiated mice were repopulated with low (10(5)), medium (10(6)) or high (10(7)) doses of congenic bone marrow cells. Marrow donors were heterozygous for the X-chromosome-encoded allozyme marker phosphoglycerate kinase (PGK-1). A second allozyme marker, phosphoglucose isomerase (GPI-1), distinguished between donor and radioresistant host cells. Use of these markers allowed the numbers and dispersion of repopulating hematopoietic clones to be estimated by binomial statistics. The number of major repopulating clones was related to the injected cell dose in a linear fashion, the inferred frequency of clonogenic cells in donor bone marrow being about 1:40,000. In high-dose recipients, the clones grew locally, with little or no dispersion between bones. Low-dose recipients, in contrast, carried widely dispersed clones; these tended to become reduced in number with increasing time after repopulation. Most of the (few) bone marrow clones present in low-dose recipients were also present in the thymus. In contrast, only about 10% of bone marrow clones in high-dose recipients were substantially represented in the thymus at any one time--about 16 clones in each lobe.

Dynamics of wavelets and their role in atrial fibrillation in the isolated sheep heart.

BACKGROUND: The multiple wavelet hypothesis is the most commonly accepted mechanism underlying atrial fibrillation (AF). However, high frequency periodic activity has recently been suggested to underlie atrial fibrillation in the isolated sheep heart. We hypothesized that in this model, multiple wavelets during AF are generated by fibrillatory conduction away from periodic sources and by themselves may not be essential for AF maintenance. METHODS AND RESULTS: We have used a new method of phase mapping that enables identification of phase singularities (PSs), which flank individual wavelets during sustained AF. The approach enabled characterization of the initiation, termination, and lifespan of wavelets formed as a result of wavebreaks, which are created by the interaction of wave fronts with functional and anatomical obstacles in their path. AF was induced in six Langendorff-perfused sheep hearts in the presence of acetylcholine. High resolution video imaging was utilized in the presence of a voltage sensitive dye; two-dimensional phase maps were constructed from optical recordings. The major results were as follows: (1) the critical inter-PS/wavelet distance for the formation of rotors was 4 mm, (2) the spatial distribution of wavelets/PSs was non-random. (3) the lifespan of PSs/wavelets was short; 98% of PSs/wavelets existed for < 1 rotation, and (4) the mean number of waves that entered our mapping field (15.7 +/- 1.6) exceeded the mean number of waves that exited it (9.7 +/- 1.5; P < 0.001). CONCLUSIONS: Our results strongly suggest that multiple wavelets may result from breakup of high frequency organized waves in the isolated Langendorff-perfused sheep heart, and as such are not a robust mechanism for the maintenance of AF in our model.

Right angle light scatter: a necessary parameter in flow cytofluorimetric analysis of human peripheral blood mononuclear cells.

The techniques of flow cytometry are being increasingly used to classify lymphocytes according to their surface antigens. Accurate analyses depend on the exclusion of non-lymphocytic contaminant cells. Using a fluorescence-activated cell sorter (FACS IV), virtually complete separation of human lymphocytes from monocytes and granulocytes can be achieved by means of wide-angle (90 degrees) light scatter. The technique described is simple and suitable for routine use. Used in conjunction with fluorescein-labelled monoclonal antibodies and logarithmic amplification of the fluorescence signals, it improves the accuracy of lymphocyte subset analysis. Its use is particularly necessary in the study of subjects such as renal allograft recipients, who have a high or variable contaminant cell:lymphocyte ratio.

Behavioural, physiological and morphological analysis of a line of apolipoprotein E knockout mouse.

Using apolipoprotein E knockout mice derived from the Maeda source [Piedrahita J. A. et al. (1992) Proc. natn. Acad Sci. US.A. 89, 4471 4475], we have studied the influence of apolipoprotein E gene deletion on normal CNS function by neurological tests and water maze learning, hippocampal ultrastructure assessed by quantitative immunocytochemistry and electron microscopy, CNS plasticity, i.e. hippocampal long-term potentiation and amygdaloid kindling, and CNS repair, i.e. synaptic recovery in the hippocampus following deafferentation. In each study there was little difference between the apolipoprotein E knockout mice and wild-type controls of similar age and genetic background. Apolipoprotein E knockout mice aged eight months demonstrated accurate spatial learning and normal neurological function. Synaptophysin and microtubule-associated protein 2 immunohistochemistry and electron microscopic analysis of these animals revealed that the hippocampal synaptic and dendritic densities were similar between genotypes. The induction and maintenance of kindled seizures and hippocampal long-term potentiation were indistinguishable between groups. Finally, unilateral entorhinal cortex lesions produced a marked loss of hippocampal synaptophysin immunoreactivity in both groups and a marked up-regulation of apolipoprotein E in the wild-type group. Both apolipoprotein E knockout and wild-type groups showed immunohistochemical evidence of reactive synaptogenesis, although the apolipoprotein E knockout group may have initially shown greater synaptic loss. It is suggested that either apolipoprotein E is of no importance in the maintenance of synaptic integrity and in processes of CNS plasticity and repair, or more likely, alternative (apolipo)proteins may compensate for the loss of apolipoprotein E in the knockout animals.

Electrophysiological characterisation of the dentate gyrus in five inbred strains of mouse.

Transgenic or knockout mouse models provide the opportunity to study the function of disease-related or novel genes. However, a confounding factor in all such research is the genetic and phenotypic variation of the mouse strain used to construct the models. A trait which is frequently studied in transgenic models of neurological disorders is synaptic transmission and plasticity of the dentate gyrus of the hippocampus. Consequently, we have investigated the variation in this trait across five strains of mouse (129 Ola, C3H, C57 albino, DBA/2, and FVB/N), in vivo. 129 Ola mice were found to have significantly larger maximal evoked EPSP slope and population spike amplitudes compared to the other strains. No differences across strains were found in paired-pulse facilitation of EPSP slope, a measure of pre-synaptic short-term plasticity. DBA/2 mice showed significantly reduced paired-pulse inhibition of population spike, a measure of poly-synaptic inhibitory feedback within the dentate gyrus. Potentiation of EPSP and population spike, following tetanic stimulation of the perforant path, was observed in all strains. However, DBA/2 mice showed a deficit in the maintenance of potentiation over 1 h, which confirms a previous report [S. Matsuyama, U. Namgung, A. Routtenberg, Long-term potentiation persistence greater in C57BL/6 than DBA/2 mice: predicted on basis of protein kinase C levels and learning performance, Brain Res. 763 (1997) 127-130]. These results show that electrophysiological traits do vary significantly across mouse strains, and that the selection of the strain may have a significant impact on results. Furthermore, since production of a transgenic or knock-out mouse frequently requires cross-breeding, care should be taken in establishing the contribution of parent strains to the final phenotype, as well as the potential interaction with the phenotype arising from the knock-out or transgene.

Pharmacokinetics of CuDIPS in mice.

Pharmacokinetic evaluation of Cu(II)2(3,5-diisopropylsalicylate)4(H2O)2 (CuDIPS), a copper complex with anticancer activity in mice, showed rapid absorption into the circulation after subcutaneous (SC) injection of a 0.50mumol, 0.75 mumol or 1.0 mumol dose. A direct relationship was observed between peak plasma copper concentration (one or two hours) and dose. Rate of plasma release also appeared to be dose related. The lowest dose of CuDIPS had no effect on plasma zinc levels; higher doses produced significant increases, but only at one hour. Rapid absorption into the blood (apparent peak in concentration, Tmax, at 0.5 hours) was also found in studies to determine distribution of 67Cu after SC injection of 67Cu and 14C double-labeled CuDIPS (50 mumol/kg body weight). Distribution to other tissues and organs occurred less rapidly with apparent peak 67Cu concentrations at three hours after administration in femur (bone marrow) and intestine, and at six hours in spleen and thymus.

Two case reports of breast carcinoma associated with prolactinoma.

Two cases of breast carcinoma associated with prolactinoma are presented. Literature review reveals only five previous case reports of this association. Both of our cases occurred in women, aged 55 and 34. Both were typical of the reported cases in that they had long histories of amenorrhea before diagnosis of prolactinomas and breast carcinomas. One patient had a three and a half year history of atypical ductal hyperplasia and a prominent intraduct component in the invasive tumor. Both had axillary lymph node metastases. The significance of the association of breast carcinoma with prolactinoma is discussed. Whereas studies in animals have shown prolactin to be an initiator and promoter of breast carcinoma, studies in humans have been inconclusive. Some studies have shown raised levels of prolactin in patients with breast carcinoma and their daughters, while others have not. The paucity of case reports linking breast carcinoma and prolactinoma may indicate that the association is mere coincidence, but studies evaluating the relationship between breast carcinoma and all forms of hyperprolactinemia need to be conducted before a causal link is dismissed. Prolactin may act as a cofactor with, for example, estrogen or stress, to induce breast carcinoma.

Influence of nematophagous fungi, earthworms and dung burial on development of the free-living stages of Ostertagia (Teladorsagia) circumcincta in New Zealand.

Biological options for nematode parasite control are being sought, as the long-term efficacy of conventional anthelmintics comes increasingly under threat from drug-resistant parasites. Three biological methods with the potential to reduce pasture contamination by parasitic nematode larvae were examined: (a) killing of larvae developing in dung by nematophagous fungi; (b) removal of dung through earthworm ingestion; (c) burial of dung in soil as might occur through the action of dung beetles. Field trials with the test bio-control agents were carried out in autumn and spring by adding dung from sheep infected with Ostertagia (Teladorsagia) circumcincta to pots of ryegrass/white clover. The factorial treatment structure included five fungal treatments (individual applications of Duddingtonia flagrans, Monacrosporium gephyropagum and Harposporium helicoides, a combination of all the three fungi together and an untreated control), two dung burial treatments (dung buried or deposited on the soil surface) and two earthworm treatments (earthworms present or absent). D. flagrans and H. helicoides, individually or in combination, reduced recovery of infective stage larvae in experiment 1, while only H. helicoides reduced recovery in experiment 2. In both the experiments, dung burial increased the total number of larvae recovered, while the number of infective larvae were reduced by the action of earthworms. Increased recovery following burial, along with the fact that larvae moved rapidly from soil onto herbage, suggests that soil may provide a protective reservoir for infective larvae infesting herbage.

Bioavailable copper complexes offer a physiologic approach to treatment of chronic diseases.

Copper (II)2(3,5-Diisopropylsalicylate)4(H2O)2 has been found to have antiinflammatory, antiulcer, anticonvulsant, anticancer, anticarcinogenic, antimutagenic, and radiation recovery activities and it prevents reperfusion injury. To study pharmacokinetic parameters accounting for these pharmacological effects the double labeled 67Cu(II)2(carboxy-14C-3,5-diisopropylsalicylate)4 complex was synthesized and used to obtain these parameters. Treatment of mice with 1 mumol of this complex revealed that 67Cu was distributed to blood, liver, kidney, intestine, lung, thymus, femur, muscle, spleen, brain, urine, and feces within 0.5 hr and patterned changes in 67Cu content of these tissues and excreta were found throughout the 96 hr term of this study.

Ventricular fibrillation and atrial fibrillation are two different beasts.

Although the mechanisms of fibrillation are no doubt multi-faceted, the geometry of the heart may play a major role in the dynamics of wave propagation during fibrillation [A. T. Winfree, Science 266, 1003-1006 (1994)]. The ventricles are thick chambers made up of sheets of parallel muscle fibers with the direction of fibers rotating across the ventricular walls (rotational anisotropy). The thick walls of the ventricles allow reentry to develop transmurally, provided the wavelength is sufficiently small. Depending on the kinetics of heart cells, the dynamics of rotating waves in three dimensions may be fundamentally different than in two dimensions, leading to destabilization of reentry and ventricular fibrillation (VF) in thick ventricles. The atria have an intricate geometry comprised of a thin sheet of cardiac tissue attached to a very complex network of pectinate muscles. The branching geometry of the pectinate muscles may lead to destabilization of two-dimensional reentry via "long-distance" electrical connections giving rise to atrial fibrillation (AF). Therefore, although fibrillation occurs via complex three-dimensional wave propagation in the ventricles and the atria, the underlying mechanisms and factors that sustain VF and AF are probably different.(c) 1998 American Institute of Physics.

Improving the assessment of heart toxicity for all new drugs through translational regulatory science.

Fourteen drugs have been removed from the market worldwide because they cause torsade de pointes. Most drugs that cause torsade can be identified by assessing whether they block the human ether à gogo related gene (hERG) potassium channel and prolong the QT interval on the electrocardiogram. In response, regulatory agencies require new drugs to undergo "thorough QT" studies. However, some drugs block hERG potassium channels and prolong QT with minimal torsade risk because they also block calcium and/or sodium channels. Through analysis of clinical and preclinical data from 34 studies submitted to the US Food and Drug Administration and by computer simulations, we demonstrate that by dividing the QT interval into its components of depolarization (QRS), early repolarization (J-Tpeak), and late repolarization (Tpeak-Tend), along with atrioventricular conduction delay (PR), it may be possible to determine which hERG potassium channel blockers also have calcium and/or sodium channel blocking activity. This translational regulatory science approach may enable innovative drugs that otherwise would have been labeled unsafe to come to market.