Cannabinoid Antagonist Drug Discrimination in Nonhuman Primates.

Despite a growing acceptance that withdrawal symptoms can emerge following discontinuation of cannabis products, especially in high-intake chronic users, there are no Food and Drug Administration (FDA)-approved treatment options. Drug development has been hampered by difficulties studying cannabis withdrawal in laboratory animals. One preclinical approach that has been effective in studying withdrawal from drugs in several pharmacological classes is antagonist drug discrimination. The present studies were designed to examine this paradigm in squirrel monkeys treated daily with the long-acting CB1 agonist AM2389 (0.01 mg/kg) and trained to discriminate the CB1 inverse agonist/antagonist rimonabant (0.3 mg/kg) from saline. The discriminative-stimulus effects of rimonabant were both dose and time dependent and, importantly, could be reproduced by discontinuation of agonist treatment. Antagonist substitution tests with the CB1 neutral antagonists AM4113 (0.03-0.3 mg/kg), AM6527 (0.03-1.0 mg/kg), and AM6545 (0.03-1.0 mg/kg) confirmed that the rimonabant discriminative stimulus also could be reproduced by CB1 antagonists lacking inverse agonist action. Agonist substitution tests with the phytocannabinoid ∆9-tetrahydrocannabinol (0.1-1.0 mg/kg), synthetic CB1 agonists nabilone (0.01-0.1 mg/kg), AM4054 (0.01-0.03 mg/kg), K2/Spice compound JWH-018 (0.03-0.3 mg/kg), FAAH-selective inhibitors AM3506 (0.3-5.6 mg/kg), URB597 (3.0-5.6 mg/kg), and nonselective FAAH/MGL inhibitor AM4302 (3.0-10.0 mg/kg) revealed that only agonists with CB1 affinity were able to reduce the rimonabant-like discriminative stimulus effects of withholding daily agonist treatment. Although the present studies did not document physiologic disturbances associated with withdrawal, the results are consistent with the view that the cannabinoid antagonist drug discrimination paradigm provides a useful screening procedure for examining the ability of candidate medications to attenuate the interoceptive stimuli provoked by cannabis discontinuation. SIGNIFICANCE STATEMENT: Despite a growing acceptance that withdrawal symptoms can emerge following the discontinuation of cannabis products, especially in high-intake chronic users, there are no FDA-approved pharmacotherapies to assist those seeking treatment. The present studies systematically examined cannabinoid antagonist drug discrimination, a preclinical animal model that is designed to appraise the ability of candidate medications to attenuate the interoceptive effects that accompany abrupt cannabis abstinence.

Mutagenicity and oral toxicity studies of Terminalia chebula.

The fruit of Terminalia chebula Retz. (T. chebula), which is a member of the Combfreetaceae family, is used widely in Asian countries as a traditional folk medicine, and its extract has been reported to be an anticancer, antidiabetic and anticaries agent. In our previous study, chebulic acid isolated from T. chebula extract was confirmed to show antioxidant activity and protective action against endothelial cell dysfunction. In order to support the safety-in-use of the ethyl acetate (EtOAc)-soluble portion of a T. chebula ethanol extract containing 29.4% chebulic acid content, the prepared portion was tested in an in vitro mutagenicity assay, and a single- and 14-day repeated dose oral toxicity study. In the bacterial mutation assay, up to 5000 µg/mL concentration of the EtOAc-soluble portion, the numbers of colonies did not increase whether with or without metabolic activation. In the oral toxicity study, the single oral dose of the extract at 2000 mg/kg did not produce mortality or abnormal lesions in the internal organs of rats. The results of a 14-day orally repeated dose showed that the EtOAc-soluble portion of T. chebula ethanol extracts gave no adverse effects at dosages of 2000 mg/kg in rats in the study.

Measuring the risk of torsades de pointes: electrocardiographic evaluation of PNU-142093 in conscious cynomolgus non-human primates using restraint and non-restraint procedures.

OBJECTIVE: Drug-induced torsades de pointes (TdP) arrhythmia is a serious public health concern that has significantly slowed the advancement of promising new therapeutic agents to the marketplace. Modeling for the potential to produce TdP has relied in part on the surrogate biomarker QT interval prolongation, measured in vivo in animals and in the clinic in man. This study was a comparison of the effects of PNU-142093, a selective 5HT1D-serotonin receptor agonist, on QT interval prolongation under restraint and non-restraint conditions in conscious cynomolgus non-human primates. METHODS: Lead II electrocardiograms (ECG) were collected following an oral single-dose (non-restraint conditions using radio-telemetry) and single- and multiple-doses for 14 days (restraint conditions using electrodes applied to the surface) at doses of 0, 5, 15, and 25 mg/kg. ECG were collected from non-restrained animals predose and for up to 5 hrs, and again at 7 hrs, postdose on 4 different days in a Latin-square crossover design; N=4/sex/dose level. ECG were collected from restrained animals on days 1, 7, and 13, predose and at approximately 4 hrs postdose; N=2/sex/group. RESULTS: Non-restrained animal heart rate ranged from 159+/-22.1 to 168+/-21.4 beats/minute when compared to restrained animal heart rate (ranging from 242+/-17.2 to 246+/-11.5 beats/minute), suggesting that non-restrained animals were under less stress. In non-restrained animals, PNU-142093 produced a non-dose related decrease in heart rate, associated with a dose-related increase in QT and QTc (QT interval corrected for changes in heart rate) intervals, which was accompanied by alterations in T-wave morphology (e.g., widening and notching of the T wave). In restrained non-human primates, PNU-142093 had no effect on heart rate or ECG morphology on any day of dosing and no statistically significant effect on QT or QTc intervals on days 1 or 7 of dosing. By day 13 there were statistically significant increases in QT and QTc intervals at 15 and 25 mg/kg. The increase in QTc interval in restrained animals on day 13 was 29+/-12 and 30+/-19 msec at 15 and 25 mg/kg/day, respectively, and that in non-restrained animals was 65+/-23 and 73+/-28 msec. DISCUSSION: These data demonstrate an ability to detect problematic drugs in conscious cynomolgus non-human primates using both restraint and non-restraint procedures. They further show that the sensitivity of these assays to identify this signal of cardiac risk is significantly improved under the condition of non-restraint.

Bimodal effect of humic acids on the LPS-induced TNF-alpha release from differentiated U937 cells.

Humic substances (HS) have been reported to possess anti-inflammatory as well as pro-inflammatory properties. The anti-inflammatory activity was demonstrated in the rat paw edema model and we found a preliminary explanation in the 5-lipoxygenase inhibitory effect of humic acids (HA). The pro-inflammatory activity is reflected by the production and release of pro-inflammatory cytokines in HA-treated neutrophilic granulocytes. With regard to the potential use of HA as antiviral and UV-protective agents it appears advisable to investigate the role of HS in the inflammation process in more detail. Hence we tested four different HS preparations - two naturally occurring HA from the Altteich peatland in Germany, one fulvic acid (FA) preparation from a Finnish spruce forest and a synthetic HA-like polymer (caffeic acid oxidation product, KOP) for their influence on the lipopolysaccharide (LPS)-induced TNF-alpha release in human U937 cells. In addition, the cytotoxicity of HS was determined. The results demonstrate a concentration-dependent bimodal effect of HA on the TNF-alpha release of differentiated LPS-stimulated U937 cells for both the natural black peat HA from the Altteich peatland and the HA-like polymer KOP. Low HA concentrations (10-80 microg/ml) enhanced the TNF-alpha release by up to threefold (pro-inflammatory activity), while HA concentrations >100 microg/ml reduced it about 10-fold (anti-inflammatory activity). FA failed to enhance TNF-alpha release, but reduced it at higher concentrations (>200 microg/ml) by the half. Brown water HA did not exert any significant effect on TNF-alpha release. No HS-stimulated TNF-alpha release was also observed in the absence of exogenously supplied LPS. This means that HS, unlike endotoxin, are no inflammation-causing agents for LPS-untreated cells. Differences in the effect of individual HS on TNF-alpha release are discussed in connection with the polyanionic character of HS, their molecular mass distribution and the hitherto imperfectly known chemical structure.

LGD-5552, an antiinflammatory glucocorticoid receptor ligand with reduced side effects, in vivo.

Treatment of inflammation is often accomplished through the use of glucocorticoids. However, their use is limited by side effects. We have examined the activity of a novel glucocorticoid receptor ligand that binds the receptor efficiently and strongly represses inflammatory gene expression. This compound has potent antiinflammatory activity in vivo and represses the transcription of the inflammatory cytokine monocyte chemoattractant protein-1 and induces the antiinflammatory cytokine IL-10. The compound demonstrates differential gene regulation, compared with commonly prescribed glucocorticoids, effectively inducing some genes and repressing others in a manner different from the glucocorticoid prednisolone. The separation between the antiinflammatory effects of LGD-5552 and the side effects commonly associated with glucocorticoid treatment suggest that this molecule differs significantly from prednisolone and other steroids and may provide a safer therapeutic window for inflammatory conditions now commonly treated with steroidal glucocorticoids.

The role of the new beta-blockers in treating cardiovascular disease.

The beta-adrenergic receptor blockers play an important role in the management of cardiovascular disease, including hypertension and chronic heart failure. However, concerns regarding safety and tolerability with currently available agents can limit their use. The beta-blockers vary with regard to several pharmacologic properties, including beta1/beta2 selectivity, intrinsic sympathomimetic activity, and, with the newest beta-blockers, vasodilation. These pharmacologic differences may result in clinically important differences in tolerability and hemodynamic properties. Nebivolol is a novel beta-blocker with both a greater degree of selectivity for beta1-adrenergic receptors than other agents in this class and an ability to stimulate endothelial nitric oxide production, leading to vasodilation and other potential clinical effects. Published randomized, controlled, multicenter studies with nebivolol have shown that once-daily treatment significantly reduces systolic and diastolic blood pressure in patients with mild-to-moderate hypertension, compared with placebo, in a dose-dependent manner, and is well tolerated, with an adverse event profile similar to that of placebo. When compared with other beta-blockers as well as with other antihypertensive classes of agents in head-to-head trials, nebivolol demonstrated similar antihypertensive efficacy and a lower incidence of adverse events. Nebivolol has also been shown to significantly reduce morbidity and mortality in a large population of elderly patients with chronic heart failure, independent of left ventricular ejection fraction. Nebivolol is currently available in Europe for the management of hypertension and is expected to be available soon in the United States.

A novel antiinflammatory maintains glucocorticoid efficacy with reduced side effects.

Glucocorticoids (GCs) are commonly used to treat inflammatory disease; unfortunately, the long-term use of these steroids leads to a large number of debilitating side effects. The antiinflammatory effects of GCs are a result of GC receptor (GR)-mediated inhibition of expression of proinflammatory genes as well as GR-mediated activation of antiinflammatory genes. Similarly, side effects are most likely due to both activated and repressed GR target genes in affected tissues. An as yet unachieved pharmaceutical goal is the development of a compound capable of separating detrimental side effects from antiinflammatory activity. We describe the discovery and characterization of AL-438, a GR ligand that exhibits an altered gene regulation profile, able to repress and activate only a subset of the genes normally regulated by GCs. When tested in vivo, AL-438 retains full antiinflammatory efficacy and potency comparable to steroids but its negative effects on bone metabolism and glucose control are reduced at equivalently antiinflammatory doses. The mechanism underlying this selective in vitro and in vivo activity may be the result of differential cofactor recruitment in response to ligand. AL-438 reduces the interaction between GR and peroxisomal proliferator-activated receptor gamma coactivator-1, a cofactor critical for steroid-mediated glucose up-regulation, while maintaining normal interactions with GR-interacting protein 1. This compound serves as a prototype for a unique, nonsteroidal alternative to conventional GCs in treating inflammatory disease.

Severe hepatotoxicity associated with the dietary supplement LipoKinetix.

BACKGROUND: LipoKinetix (Syntrax, Cape Girardeau, Missouri) is a dietary supplement marketed for weight loss. OBJECTIVE: To describe a possible causal association between LipoKinetix and hepatotoxicity. DESIGN: Case series. SETTING: Outpatient clinic, tertiary care hospital, and U.S. Food and Drug Administration databases. INTERVENTION: Routine medical and supportive care. MEASUREMENTS: Clinical and laboratory evaluation. RESULTS: All patients developed acute hepatotoxicity within 3 months of starting LipoKinetix. At presentation, symptoms and results of laboratory tests were characteristic of acute hepatitis. All patients recovered spontaneously after LipoKinetix use was discontinued. Three of the seven patients, including one who developed fulminant hepatic failure complicated by cerebral edema, were taking LipoKinetix alone at the time of presentation. Of the four patients who were taking multiple supplements, two resumed taking supplements other than LipoKinetix without incident. CONCLUSIONS: The use of LipoKinetix may be associated with hepatotoxicity. Despite extensive evaluations, no other cause for hepatotoxicity could be identified in the seven patients studied.

Repinotan (BAY x 3702): a 5HT1A agonist in traumatically brain injured patients.

Repinotan is a high-affinity, selective, full agonist of the 5HT1A-receptor subtype with neuroprotective properties. This paper presents the results of a randomized, double-blind, placebo-controlled study examining the safety and tolerability of three different doses of repinotan in patients with severe traumatic brain injury. Sixty patients were enrolled to receive repinotan (0.5, 1.25, or 2.50 mg/day) or placebo, by continuous i.v. infusion for 7 days. Repinotan treatment had no apparent adverse effects on intracranial pressure, hemodynamic parameters or laboratory parameters. No seizures occurred during treatment, and the incidence and severity of adverse events was as expected for this indication. No serious adverse events were considered related to drug treatment, with the possible exception of one case of inappropriate ADH secretion. No further safety concerns were raised during the 3 months following treatment. On a descriptive basis, the proportion of patients having good outcome or moderate disability (Glasgow Outcome Scale) was somewhat greater in repinotan-treated patients (60%) than in placebo (50%).

The role of humic substances in drinking water in Kashin-Beck disease in China.

We conducted in vitro and in vivo assays in a selenium-deficient system to determine if organic matter (mainly fulvic acid; FA) is involved in a free radical mechanism of action for Kashin-Beck disease. Cartilage cell culture experiments indicated that the oxy or hydroxy functional groups in FA may interfere with the cell membrane and result in enhancement of lipid peroxidation. Experiments with rats demonstrated that toxicity from FA was reduced when the hydroxy group was blocked. Induction of lipid peroxidation by FA in liver and blood of rats was similar to that exhibited by acetyl phenyl hydrazine. FA accumulated in bone and cartilage, where selenium rarely concentrates. In addition, selenium supplementation in rats' drinking water inhibited the generation of oxy-free radicals in bone. We hypothesized that FA in drinking water is an etiological factor of Kashin-Beck disease and that the mechanism of action involves the oxy and hydroxy groups in FA for the generation of free radicals. Selenium was confirmed to be a preventive factor for Kashin-Beck disease.

Treatment of chronic nonbacterial conjunctivitis with a cyclo-oxygenase inhibitor or a corticosteroid. Pranoprofen Study Group.

A multicenter, double-masked, parallel-group clinical trial was carried out in 151 patients with moderate to severe chronic conjunctivitis. The study compared the antiinflammatory efficacy and safety of pranoprofen 0.1%, a new cyclo-oxygenase inhibitor, with fluorometholone 0.1%, after topical doses four times a day for 15 days. The basal mean score for the signs and symptoms of inflammation, was significantly reduced (P < .001), with no significant difference between the two groups, at days 8 and 15. There was a statistically significant difference of approximately 1.0 mm Hg (P < .05) in the mean intraocular pressure between treatment, which was a decrease of 0.3 mm Hg with pranoprofen and an increase of 0.8 mm Hg with fluorometholone. One patient in the pranoprofen group had an adverse experience, compared to nine patients in the fluorometholone group (P < .03). Our data suggest that pranoprofen has efficacy equivalent to a moderate-potency corticosteroid with a better safety profile. It should be considered for the treatment of chronic conjunctivitis of presumed nonbacterial origin.

Selenium deficiency and fulvic acid supplementation induces fibrosis of cartilage and disturbs subchondral ossification in knee joints of mice: an animal model study of Kashin-Beck disease.

Kashin-Beck disease is an acquired, chronic and degenerative osteoarticular disorder. Selenium deficiency and fulvic acid in drinking water have been implicated in the cause of this disease. Pathologically, chondronecrosis of the growth plate and articular cartilage and subconsequent disturbance of ossification were observed in the joints. In this animal model study, mice were fed with a selenium deficient diet and fulvic acid supplemented drinking water for two generations. In undecalcified histological preparations of bone we carried out histological staining to detect mineralized and unmineralized bone and cartilage. The results revealed that selenium deficiency and fulvic acid supplementation induced degeneration of the articular cartilage in the knee joints of mice. Dynamic fluorescent labelling of ossification, enzyme histochemical detection of alkaline phosphatase activity in osteoblasts and a typical immunohistochemical localization of collagens type I and II indicated the development of fibrocartilage at the articular surface of knee joints, resembling the early stages of osteoarthrosis. This became obvious by disturbed development of the articular space and meniscus, markedly impaired formation of subchondral bone and early differentiation failure during enchondral ossification. This animal model provides an approach to study the molecular pathogenesis of Kashin-Beck disease.

Nonsteroidal estrogens of dietary origin: possible roles in hormone-dependent disease.

Equol, a nonsteroidal estrogen of dietary origin, was recently identified in human urine, and is excreted in amounts comparable to the classical steroidal estrogens. We confirm here that phytoestrogens which are abundant in dietary soya protein are converted by human gastrointestinal flora to this weak estrogen. After the ingestion of meals containing cooked soya protein the urinary excretion of equol in four of six subjects studied increased by up to 1000-fold and this compound was the major phenolic compound found in the urine. These data also indicate that some subjects are unable to either produce or excrete equol despite the challenge of a diet containing soya. In view of the increasing use of commercial soya products in the diet and the capacity of human bacterial flora to synthesize this weak estrogen from the abundance of phytoestrogens in soya, the potential relevance of these observations to the diseases implicating steroid hormones is discussed.