especially with NMDA and mGluR5 receptors cannot sufficiently explain the anti relapse action of this drug.Figure 2.

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Received 28 February 2013; Revised 16 September 2013; Accepted 17 September 2013Accepted article preview online 30 September 2013; Advance online publication 15 January 2014Top of pageAbstractAlcoholism is one of the most prevalent neuropsychiatric diseases, having an enormous health and socioeconomic impact. Along with a few other medications, acamprosate (Campral (N acetylhomotaurinate)) is clinically used in many countries for relapse prevention. Although there is accumulated evidence suggesting that acamprosate interferes with the glutamate system, the molecular mode of action still remains undefined. Here we show that acamprosate does not interact with proposed glutamate receptor mechanisms. In particular, acamprosate does not interact with NMDA receptors or metabotropic glutamate receptor group I. In three different preclinical animal models of either excessive alcohol drinking, alcohol seeking, or relapse like drinking behavior, we demonstrate that N acetylhomotaurinate by itself is not an active psychotropic molecule. Hence, the sodium salt of N acetylhomotaurinate (i) is ineffective in alcohol preferring rats to reduce operant responding for ethanol, (ii) is ineffective in alcohol seeking rats in a cue induced reinstatement paradigm, (iii) and is ineffective in rats with an alcohol deprivation effect. Surprisingly, calcium salts produce acamprosate like effects in all three animal models. We conclude that calcium is the active moiety of acamprosate. Indeed, when translating these findings to the human situation, we found that patients with high plasma calcium levels due to acamprosate treatment showed better primary efficacy parameters such as time to relapse and cumulative abstinence. Excessive alcohol drinking is a leading risk factor for chronic non communicable diseases and in fact, is linked to more than 60 diseases, including cancers, cardiovascular diseases, liver cirrhosis, neuropsychiatric disorders, and fetal alcohol syndrome (Rehm et al, 2009). Consequently, alcohol use and abuse bring considerable costs to society; on a global scale, the annual costs are estimated to be 760 billion Euros. One further consequence of excessive alcohol use is that 76 million adults worldwide are alcohol dependent (Rehm et al, 2009). Food and Drug Administration (FDA) to treat alcohol dependence: disulfiram, oral naltrexone, a long lasting injectable naltrexone, and acamprosate (Litten et al, 2012). In Europe, nalmefene has been also approved in 2013 (Mann et al, 2013).Almost 30 years ago, the inhibiting effect of acamprosate (calcium bis(N acetylhomotaurinate) the entire text abbreviated as Ca AOTA) on alcohol consumption in laboratory animals was described (Boismare et al, 1984). This initial observation led to the clinical development of acamprosate (Campral) and nowadays this drug is currently used in many countries for relapse prevention in abstinent alcohol dependent patients. Acamprosate is a safe and well tolerated drug that does not affect craving (Umhau et al, 2011) but the risk to relapse. In a recent Cochrane Review R et al (2010) summarized 24 randomized controlled trials (RCTs) on acamprosate, and concluded that acamprosate significantly reduces the risk of any drinking with a relative risk (RR) of 0.86. A RR of 1 means that there is no difference between placebo and treatment, whereas a RR means that relapse occurs less frequently in the treatment group. Acamprosate has been used to treat alcohol dependence in over 1.5 million patients since its introduction in Europe in 1989 and is currently available in most European and Latin American countries, Australia, parts of Asia, and Africa (Mason and Heyser, 2010). In 2004, it was approved by the FDA for the maintenance of abstinence from alcohol in detoxified alcohol dependent patients. A recent survey found that acamprosate is now the most widely prescribed medication for the treatment of alcoholism in the United States of America (Mark et al, 2009).In the past three decades, huge research efforts sought to elucidate the molecular mode of action of acamprosate. Many molecular candidate targets have been described (Spanagel and Vengeliene, 2013; De Witte et al, 2005; Mann et al, 2008) but the only accumulated evidence was found with respect to an interaction with the glutamate system. In concert, these studies suggest that acamprosate attenuates hyperglutamatergic states that occur during early and protracted abstinence, possibly involving N methyl D aspartate (NMDA) receptors and metabotropic glutamate receptor 5 (mGluR5) (Spanagel and Vengeliene, 2013; De Witte et al, 2005; Mann et al, 2008; Rammes et al, 2001; Harris et al, 2002). A prominent theory in the alcohol research field posits that chronic alcohol consumption leads to glutamatergic dysfunction. As a consequence, exaggerated glutamate activity is observed during alcohol withdrawal and conditioned withdrawal responses. This hyperglutamatergic state may then drive alcohol seeking and relapse behavior (Tsai et al 1995; Spanagel and Kiefer 2008). Acamprosate dampens hyperglutamatergic activity in excessively ethanol drinking mice, thereby reducing alcohol intake (Mann et al 2008; Spanagel et al, 2005). In a recent double blind, placebo controlled study,
buy fake oakleys, which applied magnetic resonance spectroscopy acamprosate also reduced glutamate levels in the brains of detoxified alcohol dependent patients (Umhau et al, 2010). Although the effects of acamprosate on glutamate levels are well documented,
replica ray ban sunglasses, the molecular mode of action of this drug and the putative interaction between acamprosate and glutamate receptors remains unclear.Here we describe a series of experiments and come to the surprising conclusion that N acetylhomotaurinate by itself is not an active psychotropic molecule. Instead,
fake oakleys, calcium is the active moiety of acamprosate In the first set of experiments, we tested the putative interactions of acamprosate with NMDA receptors and mGluR5 (Mann et al, 2008; Harris et al, 2002; Madamba et al, 1996). From these experiments, we have to conclude that the proposed glutamate receptor interactions of acamprosate cannot sufficiently explain the anti relapse action of this drug. In comparative experiments, we then studied the effects of Ca AOTA vs sodium N acetylhomotaurinate (Na AOTA) in three preclinical models of either excessive alcohol drinking,
buy fake oakleys, alcohol seeking, or relapse like drinking behavior. The rationale for doing these comparative experiments stems from US patent 4,355,043 (1982) where the initial investigators of acamprosate described that various salts of N acetylhomotaurinate produce their effects according to the nature of the counter ion. This assertion has so far not been tested. From these animal experiments, we conclude that calcium exhibits anti relapse effects and seems to be the major active ingredient of acamprosate. We further translated these findings at the clinical level. Using a clinical sample of placebo vs acamprosate treated abstinent alcohol dependent patients, we measured calcium plasma concentrations and show that patients with high plasma calcium levels due to acamprosate treatment exhibit better primary efficacy parameters.Top of pageMATERIALS AND METHODSSix different studies were performed. All information is provided in the Supplementary Information.Study 1: Screening panel for the mode of action of acamprosate.Study 2: Testing different salt formulations of acamprosate in the ADE model.Study 3: Testing different salt forms of acamprosate in alcohol seeking rats in the cue induced reinstatement model.Study 4: Testing different salt forms of acamprosate in alcohol preferring iP rats.Study 5: Pharmacokinetic (PK) profiles of different salt forms of acamprosate.Study 6: Calcium plasma levels in placebo and acamprosate treated patients. To test these putative interactions, we applied an extensive screening panel. By expressing human NR1 and NR2B subunits in Xenopus laevis oocytes, we first tested agonist activity of acamprosate on the glycine and glutamate binding site of the NMDA receptor. The two subunit types expressed for our purposes (hNR1A and hNR2B) provide a combination that is thought to be predominantly present in the human forebrain (Scherzer et al, 1998; Kosinski et al, 1998). To determine NMDA receptor agonist activity, glycine and glutamate were substituted with Ca AOTA. Multiple concentrations of Ca AOTA were tested up to a maximal concentration of 1 Elicited currents were measured and compared with currents seen with saturating concentrations of glycine and glutamate. Even at a maximal concentration, no activation of the receptor was seen indicating that acamprosate does not appear to have any effect at the glycine agonist binding site nor at the glutamate binding site (Figure 1a and b). To determine NMDA receptor antagonist activity, standard Schild plot analyses were performed to assess whether or not acamprosate could shift the affinity of glycine and glutamate. Concentration response curves of glycine and glutamate were again prepared with varying concentrations of Ca AOTA (100, 500, and 1000 No shift in affinity was seen in concentration response curves for glycine or glutamate, indicating no apparent antagonist activity in our assay (Figure 1c and d). In addition, using acute brain slices from rats, electrically evoked NMDA receptor dependent excitatory post synaptic potentials (EPSPs) were measured in the nucleus accumbens. Modest changes in EPSP amplitude were observed in 50 of the cells following addition of Ca AOTA (Supplementary Figure 1) suggesting that acamprosate may have some effect on the quantum release of presynaptic glutamate.Figure 1.(a) Glycine site agonist screen. Glycine was substituted with acamprosate in the presence of excess co agonist glutamate. No activation of the receptor was seen indicating that acamprosate (Ca AOTA) does not appear to have any effect at the glycine agonist binding site up to a 1 concentration. (b) Glutamate site agonist screen. Glutamate was substituted with acamprosate in the presence of excess co agonist glycine. No activation of the receptor was seen indicating that acamprosate does not appear to have any effect at the glutamate agonist binding site up to a 1 concentration. (c) Glycine site antagonist screen of acamprosate. Concentration response curves of glycine show no shift in affinity when increasing concentrations of acamprosate is introduced, indicating no apparent glycine site antagonist activity. (d) Glutamate site antagonist screen of acamprosate. Concentration response curves of glutamate show no shift in affinity when increasing concentrations of acamprosate is introduced, indicating no apparent glutamate site antagonist activity. (e) Functional Ca2 flux assays illustrate dose dependent mGluR1 activation by quisqualate and antagonism of this response by AIDA and MPEP, respectively. (f) Shows the lack of effect of Ca AOTA (acamprosate) on the response to quisqualate.Full figure and legend (81K)Download Power Point slide (792 KB)Next, calcium flux assays were used to study acamprosate interaction with mGluR1 For these experiments, human mGluR1 and 5 were expressed in HEK 293 cells. The mGluR1 agonist quisqualate produced a robust dose dependent increase in the fluorescence signal that was completely inhibited by the mGluR1 antagonist AIDA as well as by MPEP (Figure 1e). Ca AOTA had no effect in this assay (Figure 1f). Then we performed patch clamp electrophysiology to measure effects of mGluR1 on resting membrane potential and spike number. Again, brain slices were prepared from 6 male rats. Using whole cell patch clamp, the effects of the mGluR1 agonist DHPG on resting membrane potential and spike frequency were measured. DHPG induced changes to the membrane potential were inhibited by the mGluR1 antagonist LY367385 whereas DHPG induced increases in action potentials were blunted by addition of the mGluR1 and 5 antagonists, LY367385 and MPEP, respectively. In contrast, Ca AOTA had no effect under these conditions on membrane potential or the number of action potentials (Figure 2). From these studies, we conclude that the proposed glutamate receptor interactions of acamprosate, especially with NMDA and mGluR5 receptors cannot sufficiently explain the anti relapse action of this drug.Figure 2.(a) Effects of DHPG on resting membrane potential (Vm change) in CA1 neurons of the hippocampal region in the presence of mGluR1 antagonists or acamprosate. DHPG alone (n increased the spike number by more than 3. Addition of LY367385 (n or MPEP (n significantly reduced the spike number in the presence of DHPG (P Ca AOTA (n had no effect on the number of spikes. The data are presented as means Indicates significant differences from vehicle control group, P Note: In the condition DHPG and DHPG we lost in each experiment one recording during spike induction.Full figure and legend (48K)Download Power Point slide (538 KB)Na AOTA Is Ineffective in Preclinical Animal Models but Calcium Salts Produce Acamprosate Like EffectsIn US patent 4,355,043 (1982), the initial investigators of acamprosate described that various salts of N acetylhomotaurinate produce their effects according to the nature of the counter ion. This assertion has so far not been tested. Therefore, in a comparative experiment, we studied the effects of Ca AOTA vs Na AOTA in the alcohol deprivation effect (ADE) model. This is a standard preclinical rat model that measures relapse like drinking behavior by monitoring the ADE (Spanagel, 2009). This model provides excellent face and construct validity (Vengeliene et al, 2009), and has shown predictive validity as well (Spanagel and Kiefer, 2008). In particular, acamprosate was found in previous studies to reduce the ADE (Spanagel et al, 1996a; Heyser et al, 1998; Lid et al, 2012). As described in these previous studies, the calcium salt of N acetylhomotaurinate (acamprosate) decreased the ADE in the present experiment. Hence, a two way repeated measures ANOVA displayed a significantly different alcohol intake during ADE days between vehicle and Ca AOTA treated animal groups (F(5,250) P In contrast, an equimolar concentration of the corresponding sodium salt had no effect on the ADE (P (Figure 3a and b). This surprising finding suggests that calcium is a critical component for the efficacy of acamprosate, and we hypothesized that calcium salts should produce acamprosate like effects on the ADE. Indeed,
ray ban sunglasses replica, equimolar concentrations of calcium derived either from calcium chloride or calcium gluconate reduced the ADE in a similar way as acamprosate two way repeated measures ANOVA revealed significantly lower alcohol intake during ADE days in both calcium chloride and calcium gluconate treated animal groups when compared with the control animal group (F(10,105) P (Figure 3c). We then tested a mixture of Na AOTA and calcium chloride, which restored the effect of acamprosate on the ADE (F(5,60) P (Figure 3d). Locomotor activity was monitored throughout the ADE measurements by the home cage E motion system. Ca AOTA, as observed in previous experiments by us, (Spanagel et al, 1996b) reduced home cage activity following the first injection. This effect was, however, absent in NA AOTA (Supplementary Figure 2). This set of experiments suggests that N acetylhomotaurinate is an inactive molecule and that calcium is the active moiety of acamprosate. This model refers to the resumption of extinguished lever pressing behavior after exposure of an animal to alcohol conditioned stimuli. Reinstatement of alcohol seeking is used to study the neurobiological and molecular basis of craving, as there appears to be a good correspondence between the events that induce alcohol seeking in laboratory animals and those that provoke craving in humans (Shaham et al, 2003). In a previous study, it was shown that acamprosate completely abolishes the cue induced reinstatement response (Bachteler et al, 2005). As described in this previous report, the calcium salt of N acetylhomotaurinate (acamprosate) abolished the reinstatement response but equimolar concentration of the corresponding sodium salt formulation of acamprosate had no effect (Figure 4). No significant effect on lever responding during the cue induced reinstatement sessions was observed in the Na AOTA treated group (Figure 4a and b). Responding on the inactive lever was not affected by either drug during both reinstatement sessions, indicating the absence of a nonspecific reduction in lever pressing behavior (Figure 4a and b). In summary, these results clearly support our conclusion from the ADE model that calcium is the active moiety of acamprosate. Inactive lever (inactive lever) was present throughout the experiment and was used as a measure of a sedative effect of the treatment. All animals were injected with either of the compounds 12 and 2 before reinstatement sessions. Alcohol preferring rats have been used for decades to study excessive alcohol consumption and the efficacy of putative pharmacological interventions (Spanagel and Kiefer, 2008; Bell et al, 2006). In particular, iP rats self administering alcohol show sensitivity to acamprosate treatment (Cowen et al, 2005). Accordingly, Ca AOTA and Na AOTA were tested on operant behavior under a fixed ratio schedule 3 (FR3) (Supplementary Figure 3). A repeated measure one way ANOVA indicated a main effect of treatment (F(3,31) P Post hoc pairwise multiple comparison revealed for saline vs Ca AOTA P and for saline vs calcium chloride P In a second group of animals, saline treatment was compared with Na AOTA and calcium chloride. Although statistics indicated a treatment effect (F(3,28) P post hoc analysis indicated that Na AOTA did not significantly reduce the numbers of ethanol rewards (saline vs Na AOTA, P whereas again calcium chloride produced a significant reduction in earned ethanol rewards (saline vs CaCl2, P This independent experiment further supports our conclusion that N acetylhomotaurinate by itself is not an active psychotropic molecule. Instead, calcium seems to be the active moiety of acamprosate.

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