Ehrlich II –2nd World Conference on Magic Bullets

Estrogen Metabolites In The Control Of Osteosarcoma MARAN A, DADSETAN M, BROPHY CM, YASZEMSKI MJ Mayo Clinic,Rochester, MN, USA Background: Osteosarcoma primarily affects children and young adults. Although a combination of surgery and chemotherapy has improved the survival rates, a specific therapy is yet to be determined. We have demonstrated that an estrogen metabolite, 2-methoxyestradiol (2-ME) is effective in killing osteosarcoma cells (bone cancer cells) and induces interferon gene expression. The goals of this study are: a) To determine the role of interferon-regulated RNA dependent protein kinase (PKR) in osteosarcoma apoptosis; and b) To develop a controlled drug delivery method for 2-ME in osteosarcoma cells. Methods: 2-ME actions were investigated in MG63 human osteosarcoma cells by western blot analyses and cell proliferation assays. Oligo polyethyleneglycol fumarate (OPF) hydrogel was synthesized, mixed with 2-ME and cross linked using ultraviolet light. MG63 osteosarcoma cells were treated directly and with OPF hydrogel encapsulated 2-ME for a total period of 9 days. Results: 2-ME treatment induced PKR kinase expression, activity and phosphorylation of the endogenous substrate, eukaryotic initiation factor-2. Whereas, 17-estradiol, 4-hydroxyestradiol and 16-hydroxyestradiol did not induce cell death and had no effect on PKR protein. Ds RNA, an activator of PKR protein, increased cell death when osteosarcoma cells were co-treated with 2-ME. In contrast, PKR inhibitor 2-aminopurine blocked the 2-ME-induced cell death. The hydrogel-mediated 2-ME delivery study shows that on day 3, cell survival was significantly less by direct treatment than in the hydrogels, indicating that there is no “burst” release of 2-ME from the hydrogels. However, hydrogels with 2-ME encapsulated showed extended effects as there was only 40% cell survival on day 9. This is significantly lower compared to direct treatment (p<0.0001). Conclusions: Our results demonstrate that RNA-dependent protein kinase, PKR, through translational regulation contributes to proapoptotic action of 2-ME in osteosarcoma cells. In addition, OPF polymeric delivery system may prove to be very useful in sustained delivery of 2-ME and could be further explored for treating osteosarcoma and other cancers in vivo.

Driving Under Influence In France (2007) : Performance Of Accurate Emergency Forensic Procedures With Drug Urine Detection (Nal-Von Minden) Assessed By Blood GC/MS MARC B^1, LECLERC J^1,.ZANDER T^2, FUCHE C^3,.COLLOT D^3, LEROY N^3, GOND A^3 ^1Emergency Forensic Unit, Compiègne Hospital, France, ^2Nal von minden, Regensburg, Germany, ^3CTSI (Interior Security Technical Centre, Ministry of Interior, France) The SAM survey was conducted by the INRETS (France) to produce reliable epidemiological data concerning the role played by alcohol and drugs in fatal road accidents in France between 2001 and 2003. The high incidence (26%) of alcohol or drugs among the population of drivers from this study involved in fatal accidents has highlighted the importance for road safety of the consumption of these substances. By a February 2003 law, all drivers in France suspected to drive under the influence of a substance can undergo a urine test and, if that was not possible or the test proved positive, have a blood sample taken in order to test for drugs (cannabis, cocaine, opiates, amphetamines) either they are involved in any accident or not. The results are combined with the usual procedures of the police force, including the results of tests for alcohol levels. Drug screening through urinalysis is a widely accepted tool for rapid detection of potential drug use and therefore, a potentially useful method for detecting drug use in a variety of contexts such as the penal justice. Roadside drug testing was assessed in urine by means of Nal von Minden immuno-enzymatic drug urine analysis Ratisbonne tests with cut-offs as follows : 50 ng/ml for THC and metabolites, 300 ng/ml for opiates and cocaine metabolites, 1000 ng/ml for amphetamines. These levels are those defined by French road Code (Décret n° 2001-751). In order to obtain the most accurate results, emergency forensic physicians sampled and analysed urine of the drivers suspected of driving under influence by the police force, at any time, less than 30 minutes after police control. A whole number of 99 drivers, aged from 18 to 57 years, were examined and sampled in 2007 by the emergency forensic physicians of the Compiègne hospital (Picardy, France) to answer police or gendarmerie request. Fifteen drug urine testing and breath alcohol analysis out of 99 were under cut-offs. Cannabis alone headed the list of illicit drugs detected, with a prevalence of 63.6 % (THC metabolites ≥ 50 ng/ml in urine); it was mostly present in the under-38s and especially the under-28s. In 10 other cases, urine drug analysis retrieved THC metabolites with another drug in 9 out of 99 (9.09%) and with two other in one case 1/99 (1,01%). Alcohol positive breath analysis was associated with THC positive urine drug testing in 3/99 cases (3,03%) and found alone in 7 other cases (7,07%). When urine Nal von minden test proved positive, suspected drivers had a blood sample taken by the required emergency forensic physician in order to test for drugs (cannabis, cocaine, opiates, amphetamines) within the 10 minutes following the test reading. Extraction and quantification of Delta(9)-tetrahydrocannabinol (THC) and 11-nor-9-carboxy-Delta(9)-THC (THC-COOH) and for the detection of 11-hydroxy-Delta(9)-THC (11-OH THC) in whole blood was realised by validated gas chromatography-mass spectrometry (GC-MS) technique. Same method was used for opiates, amphetamine(s) and cocaine. Urine drug testing results were compared with blood mass spectrometry results and demonstrated 100% sensitivities on any drug class. Specificities reached 100% for amphetamine(s), cocaine and opiates. In all cases a urine positive test for THC metabolites was correlated to a positive blood GC/MS result either for THC, 11-OH THC or THC-COOH. Since THC-COOH is not an active metabolite and since French law considered positive results in blood only if ≥ 1 ng/ml for THC and 11-OH THC, 76/84 results (90.4%) were considered positive both in blood and urine for active THC metabolites. Mean alcohol blood level was 1.19 g/l ± 0.37 [0.52-1.8] and 1.57 g/l when associated to cannabis. Cannabis was respectively associated to alcohol in 3, to cocaine in 1, to opiates in 3 and to amphetamines in 2 cases. Three substances were found in one driver under influence. Mean THC blood level measured by GC/MS technique was 6.29 ± 4.32 ng/ml [1.01-19.83] and mean 11-OH THC level 2.48 ± 1.14 ng/ml [1.11-5.06]. These levels are much higher than those required by French road Code (≥ 1 ng/ml for THC or 11-OH THC] to assess the road traffic offence. Moreover, mean THC-COOH level measured by GC/MS, linked to the frequency or concentration of cannabis use, reached 48.61 ± 40.62 ng/ml [4.24-221.15], which highlights the fact that most of the drivers sampled positive were regular users of cannabis or users of concentrated forms of cannabis.

Is Cannabidiol (CBD) An Accurate Marker For Cannabis Use? Analysis Of 2007-2008 Blood GC/MS Analysis From Drivers Under Influence MARC B^1, LECLERC J^1, FUCHE C^2, COLLOT D^2, LEROY N^2, GOND A^2 ^1Emergency Forensic Unit, Compiègne Hospital, France, ^2CTSI (Interior Security Technical Centre, Ministry of Interior, France) Cannabinoids are the natural constituents of cannabis. The main of them are delta-9-tetrahydrocannabinol (∆-9THC), psychoactive agent, cannabinol (CBN) and cannabidiol (CBD). A single active dose of 9THC is estimated on 520 mg. ∆-9THC is rapidly metabolised. It is hydroxylated to an active metabolite, 11-hydroxy-delta9-tetrahydro-cannabinol (11-OH-THC), then oxidised to an inactive 11-nor-9-carboxy-delta9-tetrahydrocannabinol (THC-COOH), which is conjugated with glucuronic acid and predominantly excreted in the urine. Adverse effects such as anxiety, changes in sensory perception, impairment of memory and psychomotor performance, increased heart rate and changed blood pressure which may have serious consequences are common after a dose is taken that exceeds an individually variable threshold. The maximum effect persists for 4-6 h after administration despite of very low ∆-9 THC blood concentrations. ∆-9 THC plasma concentration declined to values of 2-3 ng/ml during 3-4 h after smoking. Such a low concentration of the active compound justifies the use of sensitive analytical methods for detection and determination of ∆-9 THC and its metabolites. The most effective techniques for ∆-9 THC and related compounds determination in biological material are chromatographic ones (gas and liquid) with mass spectrometric detection and different ionization modes. ∆-9 THC and its two metabolites (11-OH-THC and THC-COOH) are present in blood. Presence of delta(9)-tetrahydrocannabinol (THC), the major psychoactive constituent of cannabis and its various preparations, and its active metabolite 11-hydroxy-delta9-tetrahydro-cannabinol (11-OH-THC) is verified by analysis of blood by gas chromatography-mass spectrometry (GC-MS) of individuals apprehended for driving under the influence of drugs if their urine screened by enzyme immunoassay method was THC positive. Extraction and quantification of active métabolites THC and 11-OH THC by GC/MS is currently used for samples collected by emergency forensic physicians from individuals suspected of driving under influence, as soon as possible after the police control and drug intake. Following Emergency Forensic Medicine department guidelines, most of the drivers, if positive in urine, were sampled for blood at any time, less than 45 minutes after police control. Hybrid varieties of cannabis are known to contain a higher potency through higher than usual levels of the active ingredient THC as "skunk" or « madweed », an hybrid plant originating from Afghanistan, Morocco, Holland and Thailand, specifically bred to produce a very high level of THC. Where the standard cannabis can be expected to have a THC content of about 1% to 5%, skunk has been known to contain as much as 30% and skunk users have reported experiencing intense paranoia or cardiovascular complications. A study of UK street cannabis published in the Journal of Forensic Sciences suggested that cannabis resin has the average highest rates of cannabidiol, while 'skunk' and imported herbal cannabis (weed) have the lowest. The very high THC levels measured in blood samples sent to a reference laboratory attracted our attention. Extraction and quantification of cannabidiol (CBD), a natural constituent of cannabis, was realised by GC/MS, simultaneously to those of THC and 11-OH THC to verify some hypothesis concerning for example the time of cannabis intake, and the type of cannabis used. A serial of 160 samples collected in 2007 in people positive in urine for cannabis abuse was analysed. On one hand, mean level of THC was 4.64 ± 6.02 ng/ml [0.1-48.4], mean level of 11-OH THC was 2.46 ± 2.64 ng/ml [0.09-15.03]. On the other hand, mean level of CBD was 3.33 ± 4.37 ng/ml [0.06-36.29]. Mean difference between THC and CND dosages is 1.31 ng/ml, with a mean difference value of 15.1% ± 40%. Mean difference between THC and 11-OH THC dosages is 2.18 ng/ml, with a mean difference value of 19.1% ± 109%. Correlation curves give interesting results between CBD and THC and discrepancies mainly seem related to the use of skunk or weed, known to be CBD free. We believe that cannabidiol may be dosed as an accurate marker of type and quantity of cannabis used and may bring some information about some deaths by overdose of cannabis which are more and more frequently encountered in forensic medicine.

Anti-Ige Therapy In The Management Of Asthma: Present Use And Possible Future Applications MARCUS P NY College of Osteopathic Medicine, Old Westbury, NY, USA; St. Francis Hospital, Roslyn, NY, USA The treatment of asthma has evolved over the past 50 years and asthma is now commonly viewed as an inflammatory disorder of the airways. This has led to a shift in treatments from bronchodilators to anti-inflammatory agents. Despite the availability of effective therapies, there continues to be a need for additive therapies to improve asthma control. Inhaled corticosteroids are widely accepted as first-line therapy for almost all patients with persistent asthma. For patients with more severe asthma, additional agents are often needed to achieve asthma control. The development of omalizumab, a monoclonal antibody against IgE has led to improvement in asthma control and subsequently in the quality of life for many patients. It is widely accepted that approximately 60% of asthma is IgE mediated, with even higher numbers in children, and that therapy directed against IgE can result in significant reductions in asthma exacerbations. This would translate to a reduction in urgent care visits, including emergency department visits and hospitalizations. The recent guidelines for the management of asthma (GINA [Global Initiative for Asthma] and NHLBI [National Heart Lung and Blood Institute]) now stress the pivotal role of omalizumab in the treatment algorithms for these patients. Omalizumab, a monoclonal antibody against IgE was introduced into the United States in 2003 and has become part of the therapeutic armamentarium to improve asthma control. Three pivotal studies demonstrated efficacy in patients previously treated with inhaled corticosteroids alone and subsequently at least two studies have shown efficacy in patients treated with broader regimens that have included long-acting beta agonists and leukotriene modifiers. In addition to resulting in reductions of free IgE, the use of omalizumab has also resulted in down-regulation of the high-affinity IgE receptor and in the ability of dendritic cells to process allergens. At present, omalizumab is approved for the treatment of “difficult to treat” asthma in those age 12 years of age and older. It is anticipated that pediatric use will soon be recognized and perhaps future uses will include the administration of this compound at an earlier stage in an effort to act as a true disease modifier.