In agreement with this idea, recent electrophysiological experiments have suggested that endocannabinoid compounds may serve as retrograde messengers in both hippocampus and cerebel- Ž lum Kreitzer and Regerh, 2001; Ohno-Shosaku et al., . 2001; Wilson and Nicoll, 2001 . Finally, the finding that anandamide and 2-arachidonylglycerol may be produced through divergent molecular mechanisms indicates that these two endocannabinoid lipids may exert their modulatory effects in separate and possibly complementary ways.Drugs that interfere with anandamide transport have both theoretical and practical interest. Theoretically, they may help uncover the functions of the endocannabinoid system, which remain largely uncharacterized. Practically, they may find applications in a range of therapeutic areas, such as pain, multiple sclerosis and motor disorders ŽPiomelli et al., 2000; Baker et al., 2000; Giuffrida et al., . 2000a . AM404—the first synthetic inhibitor of anandamide Ž . uptake Beltramo et al., 1997 —has been shown to poten- Ž tiate many effects elicited by anandamide in vitro Be- . Ž ltramo et al., 1997 and in vivo Beltramo et al., 1997; . Calignano et al., 1997a . Because of the inability of AM404 Ž to activate cannabinoid receptors Beltramo et al., 1997, . 2000 , the effects of this drug were suggested to result from the elevation of endogenous anandamide levels. However, this hypothesis was solely based on pharmacological evidence and was not supported by direct measurements of endogenous anandamide levels after AM404 administration. In the present study,pruning cannabis we show that systemic administration of AM404 causes an increase in the concentration of circulating anandamide.

This was accompanied by an elevation of AM404 levels in plasma, which reached a peak value of 123″22 pmolrml and remained as high as 70.5 pmolrml for at least 2 h after drug administration. These values are likely to reflect the amount of AM404 bound to plasma proteins. Indeed, we found that both AM404 and anandamide bind to a protein in plasma, which we identified as albumin by non-denaturing PAGE.The concentrations of AM404 at its sites of action, i.e., at the membranes of cells expressing the putative anandamide transporter, remain at present undetermined. To further investigate the biochemical effects of AM404 in vivo, we also monitored the plasma levels of palmitylethanolamide and oleylethanolamide, two fatty acylethanolamides that are produced through the same biosynthetic Ž mechanism of anandamide Di Marzo et al., 1994; Cadas . et al., 1997 , but do not serve as substrates for the anan- Ž . damide transporter Piomelli et al. 1999 . AM404 administration did not significantly affect the levels of palmitylethanolamide, but caused a slow increase of circulating oleylethanolamide statistically significant 120 min after AM404 injection. Since oleylethanolamide is not transported by anandamide carrier, a possible interpretation of this result is that AM404 may inhibit an as-yet uncharacterized transporter of oleylethanolamide. Alternatively, oleylethanolamide elevation may result from the interference of AM404 with anandamide amidohydrolase, of which oleylethanolamide represents a substrate. However, administration of the potent anandamide amidohydrolase blocker, Ž .Ž . palmitylsulfonyl fluoride AM374 Gifford et al., 1999 had no effect on circulating anandamide levels, although it significantly increased the levels of oleylethanolamide 30 Ž min after drug application A. Giuffrida, F. Nava, A. . Makriyannis and D. Piomelli, in preparation . Taken together, these results suggest that blockade of anandamide amidohydrolase activity is unlikely to participate in the elevation of anandamide in plasma, but may cause the accumulation of other fatty acid acylethanolamides.

In parallel with its ability to increase anandamide levels in plasma, AM404 also elicited a time-dependent inhibition of motor activity. The hypokinetic effect of AM404 is reminiscent of that observed after anandamide administra- Ž tion Fride and Mechoulam, 1993; Smith et al., 1994; . Romero et al., 1995 and was reversed by the cannabinoid CB receptor antagonist SR141716A. This drug prevents 1 Ž several responses of anandamide in rats Calignano et al., . Ž 1998 , mice Beltramo et al., 1997; Calignano et al., .Ž . 1997b and guinea pigs Calignano et al., 1997a . However, failure of SR141716A to reverse anandamide actions Ž . was also reported Adams et al., 1998 . The reason for these discrepancies are unresolved, and may be due to differences in species, dosage of the drug and experimental design. Since the motor inhibition produced by AM404 was achieved at a dose that also caused accumulation of anandamide in peripheral blood, and given the inability of Ž AM404 to activate cannabinoid receptors Beltramo et al., . 1997, 2000 , our results are consistent with the hypothesis that AM404 produced its behavioral effects by protecting endogenous anandamide from transport-mediated inactivation. Alternatively, the effects of AM404 may be ascribed to its ability to activate vanilloid VR1-type receptors ŽZygmunt et al., 2000; Smart and Jerman, 2000; Jerman et . Ž. al., 2000 . This possibility is unlikely for two reasons: 1 the effects of AM404 were prevented by the highly selective cannabinoid CB receptor antagonist, SR141716A, 1 which does not interfere with vanilloid VR1 receptor Ž . Ž. function Calignano et al., 2000 ; 2 injection of vanilloid agonists into the brain does not cause hypolocomotion Ž . Mezey et al., 2000 , the primary effect of AM404 ob- Ž served in the present and other studies Beltramo et al., . 2000; Gonzalez et al., 1999 . ´ Although the concentrations reached by anandamide in Ž . plasma approximately 10 nM are insufficient to activate cannabinoid receptors ŽK s50 nM and 1.6 mM for CB d 1 .Ž . and CB receptor, respectively Pertwee, 1997 , it is 2 reasonable to hypothesize that AM404 may cause anandamide to accumulate in brain tissue to an extent that is sufficient to cause biological effects. This would explain why the motor inhibition elicited by AM404 takes place before significant accumulation of anandamide in plasma is observed.

However, the sources of plasma anandamide following AM404 administration are still unknown. Indeed, anandamide production has been demonstrated not only within the central nervous system, but also in periph- Ž eral cells, such as macrophages and platelets Schmid et . al., 1997; Wagner et al., 1997 . Microdialysis studies aimed at measuring anandamide outflow in brain areas involved in motor control after AM404 administration should shed light in this important question.Heart failure is the fourth overall principal diagnosis and first among cardiovascular conditions as the reason for hospitalization in the U.S.Heart failure is a prevalent condition with several preventable etiologies including uncontrolled hypertension or ischemic heart disease.Behavioral risk factors such as tobacco, alcohol, and drug use are known to contribute to heart failure incidence.Alcohol, cocaine, and amphetamines have cardiotoxic effects. Drug overdose death rates in the U.S. are rising, especially in younger persons.The burden of active tobacco and substance use disorders among hospitalized heart failure patients in the U.S. has not been well described. Nationally representative administrative data facilitates understanding the burden of tobacco and substance use disorders among heart failure patients and its potential influence on health outcomes. Vulnerable populations,drying room including patients from racial/ethnic minorities or lower socioeconomic status, may be at increased risk of developing tobacco or substance use disorders for multiple reasons including social stressors, lack of economic opportunity, and community factors.Identifying heart failure patients with tobacco or substance use disorders is critical to developing treatment strategies to address observed cardiovascular health disparities. We describe the national burden of heart failure and comorbid tobacco or substance use disorder among hospitalized patients in the U.S. We used data from the 2014 National Inpatient Sample to describe diagnosis rates of tobacco and substance use disorders among hospitalized heart failure patients and examined demographic groups that may be at higher risk for these disorders.The NIS dataset provides hospital administrative data through the Agency for Healthcare Research and Quality’s Healthcare Cost and Utilization Project. It contains approximately 7 million weighted hospital discharges representing 35 million inpatient hospitalizations.The NIS unit of analysis is a discharge; therefore, readmissions are not identified. The sample is drawn from forty-four states and the District of Columbia, covering more than 96% of the U.S. population. A 20% stratified sample is obtained from 4,411 U.S. community hospitals. All insurance payer sources are included.Heart failure was defined by any International Classification of Diseases, Ninth Revision Clinical Modification code that mentioned a heart failure syndrome . A primary heart failure hospitalization was defined as any heart failure ICD-9-CM code used as the first listed discharge code, consistent with prior publications.Patients less than 18 years were excluded. Race/ethnicity was classified as white, black, Hispanic, Asian/Pacific Islander , or Native American as captured by administrative hospital data. Additional demographic factors included age, sex, payer source, geographic Census division, and median household income based on zip code. Substance use disorder was defined as any alcohol or drug use disorder, excluding tobacco, which was a separate outcome. Tobacco, alcohol, and drug use disorders were defined using Clinical Classifications Software and ICD-9-CM codes .Drug use disorder was sub-divided into cocaine, cannabis, opioid, amphetamine, psychotherapeutic , hallucinogen, and other use disorder categories.

Overall and for each tobacco and substance use disorder category, we estimated the national proportion of hospitalized heart failure patients and provided descriptive statistics for patient characteristics, select comorbidities, hospital length of stay, and inpatient mortality. We next stratified heart failure hospitalizations by sex and other demographic factors . For each stratum, we reported the percent of patients in each tobacco or substance use disorder category. Tobacco and substance use disorder rates were age-standardized using the 2000 US Standard Population, per Center for Disease Control and Prevention recommendations.To evaluate demographic factors associated with each comorbid tobacco or substance use disorder category, we used logistic regression models accounting for clustering and non-linear age-adjustment using multi-variable fractional polynomials.Selection of best-fit multivariable fractional polynomial models used a closed-test algorithm.This curvilinear adjustment was used to reduce residual confounding that may arise secondary to model misspecification using age as a single linear term.All estimation procedures were performed with appropriate NIS survey weights to account for sampling design, and all results are presented as the weighted national 2014hospitalized population. Analyses were performed in STATA 15.1 . Institutional IRB provided an exemption for this research.There were 989,080 heart failure hospitalizations in the U.S. in 2014 of which 15.5% had documented tobacco or substance use disorder. Tobacco use disorder was found in 12.1% , substance use disorder in 6.2% , alcohol use disorder in 3.5 % and drug use disorder in 3.5% . Both tobacco and substance use disorder were documented on 2.8% of heart failure hospitalizations, while both alcohol and drug use disorder were found in 0.7% . In the overall heart failure cohort, mean patient age was 72.0 , and females comprised almost half of the hospitalizations. The majority of heart failure hospitalizations were for patients age 65 or older , of white race/ethnicity , and with payer source of Medicare . The most common comorbidities were hypertension and coronary artery disease . Demographic patterns of the cohort with no tobacco or substance use disorder mirrored that of the overall heart failure cohort. Tobacco use disorder patients were younger than the overall heart failure cohort and 36.0% female. Tobacco use disorder was more common among males than females across demographic subcategories . Rates were highest for both sexes between ages 45 and 55 . Native American males had highest age-adjusted rates , while white and Native American females had highest age adjusted rates . Tobacco use disorder rates were highest in the East South Central region and for payer status of no charge , self-pay , or Medicaid . Rates of tobacco use disorder increased as median household income decreased. Heart failure hospitalizations with documented substance use disorder represented younger patients than the overall or tobacco use disorder cohorts and were 22.9% female . Substance use disorder diagnosis rates were highest for males 45 to 55 years of age and females <45 years of age . Native Americans had highest rates of substance use disorder when age-adjusted . Substance use disorder was highest for heart failure hospitalizations in the Pacific region, payer status of Medicaid, self-pay or no-charge, and for lower income quartiles. Alcohol use disorder was less common among female heart failure hospitalizations relative to tobacco and drug use disorder . Heart failure hospitalizations for those age 45 to 55 years had highest rates of alcohol use disorder .