Combined effect of alcohol and cannabis on simulated driving.

RATIONALE: With alcohol and cannabis remaining the most commonly detected drugs in seriously and fatally injured drivers, there is a need to understand their combined effects on driving. OBJECTIVES: The present study examined the effects of combinations of smoked cannabis (12.5% THC) and alcohol (target BrAC 0.08%) on simulated driving performance, subjective drug effects, cardiovascular measures, and self-reported perception of driving ability. METHODS: In this within-subjects, double-blind, double-dummy, placebo-controlled, randomized clinical trial, cannabis users (1-7 days/week) aged 19-29 years attended four drug administration sessions in which simulated driving, subjective effects, cardiovascular measures, and whole blood THC and metabolite concentrations were assessed following placebo alcohol and placebo cannabis (<0.1% THC), alcohol and placebo cannabis, placebo alcohol and active cannabis, and alcohol and active cannabis. RESULTS: Standard deviation of lateral position in the combined condition was significantly different from the placebo condition (p < 0.001). Standard deviation of lateral position was also significantly different from alcohol and cannabis alone conditions in the single task overall drive (p = 0.029 and p = 0.032, respectively), from the alcohol alone condition in the dual task overall drive (p = 0.022) and the cannabis alone condition in the dual task straightaway drive (p = 0.002). Compared to the placebo condition, the combined and alcohol conditions significantly increased reaction time. Subjective effects in the combined condition were significantly greater than with either of the drugs alone at some time points, particularly later in the session. A driving ability questionnaire showed that participants seemed unaware of their level of impairment. CONCLUSION: Combinations of alcohol and cannabis increased weaving and reaction time, and tended to produce greater subjective effects compared to placebo and the single drug conditions suggesting a potential additive effect. The fact that participants were unaware of this increased effect has important implications for driving safety.

Acute and residual effects of smoked cannabis: Impact on driving speed and lateral control, heart rate, and self-reported drug effects.

BACKGROUND: Although driving under the influence of cannabis is increasingly common among young adults, little is known about residual effects on driver behavior. This study examined acute and residual effects of smoked cannabis on simulated driving performance of young cannabis users. METHODS: In this double-blind, placebo-controlled, parallel-group randomized clinical trial, cannabis users (1-4 days/week) aged 19-25 years were randomized with a 2:1 allocation ratio to receive active (12.5% THC) or placebo (0.009% THC) cannabis in a single 750 mg cigarette. A median split (based on whole-blood THC concentrations at the time of driving) was used to divide the active group into low and high THC groups. Our primary outcome was simulated driving performance, assessed 30 min and 24 and 48 h after smoking. Secondary outcomes included blood THC concentrations, subjective drug effects, and heart rate. RESULTS: Ninety-six participants were randomized, and 91 were included in the final analysis (30 high THC, 31 low THC, 30 placebo). Mean speed (but not lateral control) significantly differed between groups 30 min after smoking cannabis (p ≤ 0.02); low and high THC groups decreased their speed compared to placebo. Heart rate, VAS drug effect and drug high increased significantly immediately after smoking cannabis and declined steadily after that. There was little evidence of residual effects in any of the measures. CONCLUSION: Acutely, cannabis caused decreased speed, increased heart rate, and increases in VAS drug effect and drug high. There was no evidence of residual effects on these measures over the two days following cannabis administration.

Coming off cannabis: a cognitive and magnetic resonance imaging study in patients with multiple sclerosis.

Cognitive dysfunction affects 40-80% of patients with multiple sclerosis. Smoking cannabis may add to these deficits. It is unclear whether coming off cannabis results in cognitive improvement. To address this question, 40 patients with multiple sclerosis who started using cannabis after the onset of multiple sclerosis and who used it for at least 4 days a week over many years were divided by odd-even number selection into two groups: cannabis continuation and cannabis withdrawal. Assessments took place at baseline and after 28 days and included serial versions of the Brief Repeatable Neuropsychological Battery for multiple sclerosis containing tests of verbal and visual memory, processing speed and executive function; structural and functional MRI, the latter entailing a compatible version of the Symbol Digit Modalities Test; urine for cannabinoid metabolites to detect compliance with abstinence. Only those participants deemed globally impaired at baseline (failure on at least two cognitive domains) were enrolled. The results revealed that the two groups were well matched demographically and neurologically. One subject was removed from the withdrawal group because of failed abstinence. Urine analysis revealed the cannabinoid consumed was predominantly tetrahydrocannabinol (THC). There were no baseline between group cognitive differences, but by Day 28 the withdrawal group performed significantly better on every cognitive index (P < 0.0001 for all). Significant within group differences were present for every test over time, but only in the abstinent group (P < 0.0001 for all tests). There were no between group baseline or Day 28 differences in structural MRI indices (global atrophy, total T1 and T2 lesion volume). At index assessment the two groups had a similar performance on the functional MRI-compatible Symbol Digit Modalities Test and there were no group differences in brain activation. However, by Day 28, the withdrawal group completed more trials correctly (P < 0.012) and had a faster reaction time (P < 0.002), associated with significantly increased activation in brain regions known to be associated with performance of the test (bilateral inferior frontal gyri, caudate and declive/cerebellum, P < 0.001 for all regions). These results reveal that patients with multiple sclerosis who are frequent, long-term cannabis users can show significant improvements in memory, processing speed and executive function after 28 days of drug abstinence. The absence of similar improvements in a matched multiple sclerosis group that remained on cannabis shows that beneficial cognitive change after stopping cannabis is not solely attributable to the effects of practice.

Nicotine metabolite ratio (3-hydroxycotinine/cotinine) in plasma and urine by different analytical methods and laboratories: implications for clinical implementation.

BACKGROUND: The highly genetically variable enzyme CYP2A6 metabolizes nicotine to cotinine (COT) and COT to trans-3'-hydroxycotinine (3HC). The nicotine metabolite ratio (NMR, 3HC/COT) is commonly used as a biomarker of CYP2A6 enzymatic activity, rate of nicotine metabolism, and total nicotine clearance; NMR is associated with numerous smoking phenotypes, including smoking cessation. Our objective was to investigate the impact of different measurement methods, at different sites, on plasma and urinary NMR measures from ad libitum smokers. METHODS: Plasma (n = 35) and urine (n = 35) samples were sent to eight different laboratories, which used similar and different methods of COT and 3HC measurements to derive the NMR. We used Bland-Altman analysis to assess agreement, and Pearson correlations to evaluate associations, between NMR measured by different methods. RESULTS: Measures of plasma NMR were in strong agreement between methods according to Bland-Altman analysis (ratios, 0.82-1.16) and were highly correlated (all Pearson r > 0.96, P < 0.0001). Measures of urinary NMR were in relatively weaker agreement (ratios 0.62-1.71) and less strongly correlated (Pearson r values of 0.66-0.98, P < 0.0001) between different methods. Plasma and urinary COT and 3HC concentrations, while weaker than NMR, also showed good agreement in plasma, which was better than that in urine, as was observed for NMR. CONCLUSIONS: Plasma is a very reliable biologic source for the determination of NMR, robust to differences in these analytical protocols or assessment site. IMPACT: Together this indicates a reduced need for differential interpretation of plasma NMR results based on the approach used, allowing for direct comparison of different studies.

The heat shock protein 90 inhibitor IPI-504 induces KIT degradation, tumor shrinkage, and cell proliferation arrest in xenograft models of gastrointestinal stromal tumors.

The activity of the receptor tyrosine kinase KIT is crucial for gastrointestinal stromal tumor (GIST) growth and survival. Imatinib and sunitinib are very effective in advanced GIST, but have no curative potential. The observation that heat shock protein 90 (HSP90) inhibition results in KIT degradation prompted us to assess the efficacy of the HSP90 inhibitor retaspimycin hydrochloride (IPI-504) alone or in combination with imatinib or sunitinib in two GIST xenografts with distinctive KIT mutations. Nude mice were grafted with human GIST carrying KIT exon 13 (GIST-882; n = 59) or exon 11 (GIST-PSW; n = 44) mutations and dosed with imatinib (50 mg/kg twice daily), sunitinib (40 mg/kg once daily), IPI-504 (100 mg/kg 3 times per week), IPI-504 + imatinib, or IPI-504 + sunitinib. We evaluated tumor volume, proliferation and apoptosis, KIT expression and activation, as well as adverse events during treatment. Treatment with IPI-504 alone resulted in tumor regression, proliferation arrest, and induction of tumor necrosis. We documented downregulation of KIT and its signaling cascade in IPI-504-treated animals. Treatment effects were enhanced by combining IPI-504 with imatinib or sunitinib. On histologic examination, liver damage was frequently observed in animals exposed to combination treatments. In conclusion, IPI-504 shows consistent antitumor activity and induces KIT downregulation in GIST, as a single agent, and is more potent in combination with imatinib or sunitinib. The sequence of drug administration in the combination arms warrants further studies.

High efficacy of panobinostat towards human gastrointestinal stromal tumors in a xenograft mouse model.

PURPOSE: Histone deacetylase inhibitors have emerged as potent anticancer compounds. Using a nude-mouse xenograft model, for the first time we evaluated the response of human gastrointestinal stromal tumors (GIST) carrying different oncogenic KIT mutations to panobinostat (LBH589), administered single or in combination with imatinib. EXPERIMENTAL DESIGN: We grafted the human GIST882 cell line with KIT exon 13 mutation and two biopsies from patients radiologically progressing under imatinib showing KIT exon11 and KIT exon9 mutations, respectively. Our study included 4 groups: A (n = 9; control), B (n = 10; panobinostat 10 mg/kg daily, i.p.), C (n = 9; imatinib 150 mg/kg bidaily, p.o), and D (n = 8; combination panobinostat-imatinib, same dose/schedule as above). Treatment lasted 12 days. Tumor size was measured regularly using standard variables. Histopathological assessment was by H&E, and immunohistochemically with KIT, cleaved caspase-3, Ki-67, and histone acetylation staining. RESULTS: Overall, GIST xenografts responded rapidly to panobinostat as indicated by tumor regression, necrosis, hemorrhages, fibrosis, and/or myxoid degeneration, remarkable apoptosis, and substantial decline of cell proliferation. H3 and H4 acetylation increased significantly from control level in all treated groups. The combination of panobinostat and imatinib further enhanced most of the assessed parameters. CONCLUSIONS: We show for the first time potential therapeutic activity of panobinostat in human GISTs, in vivo. Our results warrant further exploration of histone deacetylase inhibitors for the treatment of advanced GISTs. Our study is also the first one on human GIST mouse xenografts established using patient biopsies.

Clinical impact of trabectedin (ecteinascidin-743) in advanced/metastatic soft tissue sarcoma.

BACKGROUND: Patients with advanced or metastatic non-gastrointestinal stromal tumour soft tissue sarcoma (STS) whose disease progresses during or after chemotherapy with doxorubicin or ifosfamide have few options and very limited life expectancy. In this setting, the DNA and transcription interacting agent trabectedin (ecteinascidin-743), isolated originally from the tunicate Ecteinascidia turbinata, has encouraging activity and is now approved in the European Union. OBJECTIVE: To review evidence for the efficacy of trabectedin in STSs. METHODS: This review includes material known to the authors through preclinical and clinical work with trabectedin, and information from relevant papers and abstracts. RESULTS: Pooled analysis of Phase II studies suggests that around 50% of STS patients, failing conventional chemotherapy, experienced long lasting tumour control (either objective response or stabilization of disease) when treated with trabectedin. Twenty-nine per cent of patients were alive at 2 years, and median overall survival was 10.3 months. Leiomyosarcomas and liposarcomas appear particularly sensitive to the drug. In myxoid and round-cell liposarcomas trabectedin seems exceptionally active. A link between specific translocations underlying this disease and the drug's mechanism of action is being explored. Trabectedin is also active in synovial, ewing sarcoma and other translocation-related STSs. Trabectedin is not cardio- or neurotoxic. The neutropenia and hepatic toxicity that occur are non-cumulative, reversible, and lessened by steroid premedication. The lack of cumulative toxicities could make trabectedin appropriate for prolonged treatment. CONCLUSION: The potential of trabectedin should be further explored in STSs in general and in specific subtypes, both in combination with other cytotoxic agents and with modulators of intracellular signalling.

Trabectedin (ET-743): evaluation of its use in advanced soft-tissue sarcoma.

Trabectedin (ET-743; Yondelis) is a novel DNA-binding agent, originally derived from the marine tunicate, Ecteinascidia turbinata, and now produced synthetically. The efficacy of trabectedin in patients with advanced soft-tissue sarcoma has been demonstrated in three Phase II studies involving 189 previously treated patients. A pooled analysis of data from these studies showed that trabectedin induced tumor control (objective responses plus disease stabilization) in approximately 50% of patients; median overall survival was 10.3 months and progression-free survival at 6 months was 19.8%, with 29.3% of patients alive at 2 years. Responses were achieved in patients who were resistant to both doxorubicin and ifosfamide. Trabectedin is generally well tolerated, with adverse events being non cumulative, reversible and manageable. Unlike other commonly used cytotoxic agents, trabectedin is not associated with cardiotoxicity or neurotoxicity and alopecia is rare. Trabectedin is an interesting new anticancer agent that offers much promise for the treatment of advanced soft-tissue sarcoma.

Imatinib for the treatment of patients with unresectable or metastatic malignant KIT-positive gastrointestinal stromal tumours: an open-label Belgian trial.

BACKGROUND: Gastrointestinal stromal tumours (GIST) are the most common mesenchymal tumours of the gastrointestinal tract. They are defined immunohistologically as KIT positive tumours. The only effective treatment for malignant GIST was surgery until 2000. Imatinib mesylate (STI571, Glivec) has shown substantial anticancer activity in patients with metastatic or unresectable GIST. PATIENTS AND METHODS: 57 patients who were diagnosed with unresectable or metastatic malignant GIST were entered into this study. The patients were given 400 mg Glivec orally once daily. The dose could be increased to 600 mg orally once daily and then to 400 mg twice daily if tumour progression was noticed. Daily treatment was interrupted or dose was decreased only in the case of limiting toxicities. We evaluated the tumour response and the safety of the drug. RESULTS: 85% of GIST patients showed a partial response or stable disease after 8 weeks of treatment with imatinib. The main side effects were nausea, vomiting, anorexia, skin rash, periorbital oedema and diarrhea. CONCLUSION: This study confirms that imatinib is an active agent against malignant GIST with manageable toxicities.

Modulation of purinergic signaling by NPP-type ectophosphodiesterases.

Extracellular nucleotides can elicit a wide array of cellular responses by binding to specific purinergic receptors. The level of ectonucleotides is dynamically controlled by their release from cells, synthesis by ectonucleoside diphosphokinases and ectoadenylate kinases, and hydrolysis by ectonucleotidases. One of the four structurally unrelated families of ectonucleotidases is represented by the NPP-type ectophosphodiesterases. Three of the seven members of the NPP family, namely NPP1-3, are known to hydrolyze nucleotides. The enzymatic action of NPP1-3 (in)directly results in the termination of nucleotide signaling, the salvage of nucleotides and/or the generation of new messengers like ADP, adenosine or pyrophosphate. NPP2 is unique in that it hydrolyzes both nucleotides and lysophospholipids and, thereby, generates products that could synergistically promote cell motility. We review here the enzymatic properties of NPPs and analyze current evidence that links their nucleotide-hydrolyzing capability to epithelial and neural functions, the immune response and cell motility.

NPP-type ectophosphodiesterases: unity in diversity.

Nucleotide pyrophosphatase/phosphodiesterase (NPP)-type ectophosphodiesterases are found at the cell surface as type-I or type-II transmembrane proteins, but are also found extracellularly as secreted or shedded enzymes. They hydrolyze pyrophosphate or phosphodiester bonds in a variety of extracellular compounds including nucleotides, (lyso)phospholipids and choline phosphate esters. Despite their structurally related catalytic domain, each enzyme has well-defined substrate specificity. Catalysis by NPPs affects processes as diverse as cell proliferation and motility, angiogenesis, bone mineralization and digestion. In addition, there is emerging evidence for non-catalytic functions of NPPs in cell signaling. NPP-type ectophosphodiesterases are also implicated in the pathophysiology of cancer, insulin resistance and calcification diseases, and they hold great promise as easily accessible therapeutic targets.

Proteolytic maturation and activation of autotaxin (NPP2), a secreted metastasis-enhancing lysophospholipase D.

Autotaxin (NPP2) is an extracellular protein that is upregulated in various malignancies, including breast and lung cancer. It potently stimulates cell proliferation, cell motility and angiogenesis, which is accounted for by its intrinsic lysophospholipase-D activity that generates the lipid mediators lysophosphatidic acid and sphingosine-1-phosphate. Based on its structural similarities with the better characterized nucleotide pyrophosphatase/phosphodiesterase NPP1, it has always been assumed that NPP2 is also synthesized as a type-II integral membrane protein and that extracellular NPP2 is generated from this membrane precursor. We show here, however, using domain swapping and mutagenesis experiments as well as N-terminal protein sequencing, that NPP2 is actually synthesized as a pre-pro-enzyme and that the proteolytically processed protein is secreted. Following the removal of a 27-residue signal peptide by the signal peptidase, NPP2 is subsequently cleaved by proprotein convertases (PCs). The removal of an N-terminal octapeptide by PCs is associated with an enhanced activity of NPP2 as a lysophospholipase D. These novel insights in the maturation of NPP2 have also implications for the development of NPP2 inhibitors as potential anti-cancer agents.

Substrate-specifying determinants of the nucleotide pyrophosphatases/phosphodiesterases NPP1 and NPP2.

The nucleotide pyrophosphatases/phosphodiesterases NPP1 and NPP2/autotaxin are structurally related eukaryotic ecto-enzymes, but display a very different substrate specificity. NPP1 releases nucleoside 5'-monophosphates from various nucleotides, whereas NPP2 mainly functions as a lysophospholipase D. We have used a domain-swapping approach to map substrate-specifying determinants of NPP1 and NPP2. The catalytic domain of NPP1 fused to the N- and C-terminal domains of NPP2 was hyperactive as a nucleotide phosphodiesterase, but did not show any lysophospholipase D activity. In contrast, chimaeras of the catalytic domain of NPP2 and the N- and/or C-terminal domains of NPP1 were completely inactive. These data indicate that the catalytic domain as well as both extremities of NPP2 contain lysophospholipid-specifying sequences. Within the catalytic domain of NPP1 and NPP2, we have mapped residues close to the catalytic site that determine the activities towards nucleotides and lysophospholipids. We also show that the conserved Gly/Phe-Xaa-Gly-Xaa-Xaa-Gly (G/FXGXXG) motif near the catalytic site is required for metal binding, but is not involved in substrate-specification. Our data suggest that the distinct activities of NPP1 and NPP2 stem from multiple differences throughout the polypeptide chain.