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- Materials and methods
Disulfiram is a cocaine pharmacotherapy that may act through increasing serotonin, benefiting patients with genetically low serotonin transporter levels (5-HTTLPR, S′ allele carriers) and low serotonin synthesis (TPH2, A allele carriers). We stabilized 71 cocaine and opioid co-dependent patients on methadone for 2 weeks and randomized them into disulfiram and placebo groups for 10 weeks. We genotyped the SLC6A4 5-HTTLPR (rs4795541, rs25531) and TPH2 1125A>T (rs4290270) variants and evaluated their role in moderating disulfiram treatment for cocaine dependence. Cocaine-positive urines dropped from 78% to 54% for the disulfiram group and from 77% to 76% for the placebo group among the 5-HTTLPR S′ allele carriers (F = 16.2; df = 1,301; P < 0.0001). TPH2 A allele carriers responded better to disulfiram than placebo (F = 16.0; df = 1,223; P < 0.0001). Patients with both an S′ allele and a TPH2 A allele reduced cocaine urines from 71% to 53% on disulfiram and had no change on placebo (F = 21.6; df = 1,185; P < 0.00001).
Cocaine dependence leads to substantial social and economic impacts on the more than 1.5 million afflicted with this disease (Mclellan et al. 2000; Samsha 2011). Pharmacological approaches for cocaine dependence that target various neurobiological mechanisms have had limited success (Lima et al. 2002, 2003). However, disulfiram has shown promise for treating cocaine dependence including opioid-dependent cocaine abusers (Carroll 1993; Carroll et al. 1998, 2004; George et al. 2000; Kosten et al. 2012; Petrakis et al. 2000).
Disulfiram treatment increases serotonin's precursor tryptophan, its metabolite 5-hydroxyindole acetic acid and the hydroxylated product of tryptophan, 5-hydroxytryptophan (Fukumori et al. 1979, 1980; Nagendra et al. 1993; Nilsson & Tottmar 1989). Since cocaine's mechanism of action is through blockade of dopamine, norepinephrine and serotonin transporters (Han & Gu 2006), it is possible that disulfiram may be effective for the treatment of cocaine dependence, in part, through its action on serotonin.
Making serotonin available for postsynaptic activity requires tryptophan hydroxylase and the serotonin transporter. Both genes coding for these proteins have functional variants that may make lower levels of serotonin available in the synapse. Tryptophan hydroxylase is the rate-limiting enzyme in the production of serotonin (Cooper & Melcer 1961) and is expressed from TPH2 in the brain (Zill et al. 2007). A synonymous variant 1125A>T (rs4290270) in exon 9 of the TPH2 gene is a marker for allelic expression imbalance (Lim et al. 2007). The T allele is expressed at approximately twofold the levels of the A allele in heterozygous subjects. Individuals who are A allele carriers may produce less serotonin than those with a TT genotype.
The SLC6A4 gene encodes the serotonin transporter that controls serotonin reuptake and inactivation from the synapse. In the promoter of the serotonin transporter gene is a repeat variant (5-HTTLPR) of either a short (S) or a long (L) form (Lesch et al. 1996). Recently, a variant was identified within the repeat that alters transcriptional activity of the serotonin transporter gene. Combining this variant with the repeat yields a triallelic polymorphism that can be classified as a high transcriptional activity L′ or a low activity S′ allele (Hu et al. 2006).
We hypothesized that the increase in serotonin from disulfiram would have the greatest impact on patients who had low levels of both serotonin transporter (S′S′ or L′S′ genotypes) and serotonin biosynthesis (TPH2 AA or AT genotypes). We first examined patients who were 5-HTTLPR S′ allele carriers expecting that its associated low activity levels would not overcome disulfiram's increase in serotonin and lead to a better treatment response. To test this mechanism of increased serotonin as the basis for a pharmacogenetic association, we next examined patients having at least one low activity TPH2 A allele. Finally, we next examined patients having both the low activity 5-HTTLPR (S′S′ or L′S′) and the low activity TPH2 (AA or AT) genotypes. We expected these patients also to have a better therapeutic response to disulfiram than the remaining patients.
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- Materials and methods
We have found that those patients with specific genetic polymorphisms and genotype patterns of the serotonin transporter and tryptophan hydroxylase 2 genes are associated with better treatment outcomes during the pharmacotherapy of cocaine dependence with disulfiram. This supports our hypotheses that we developed since disulfiram raises serotonin levels (Fukumori et al. 1979,1980; Nagendra et al. 1993; Nilsson & Tottmar 1989). Subjects with at least one copy of the 5-HTTLPR S′ allele or at least one TPH2 A allele had decreased cocaine-positive urines compared to those without these alleles. The combination of both low transporter levels and low TPH2 activity appeared to have an additive effect with the overall effect as reflected in the F-statistic going from 12 for no genetic selection, to 16 for either the serotonin transporter or TPH2, to 24 when both polymorphisms were combined.
The mechanism for this improved outcome with genetic selection may involve serotonin availability in the synapse. The serotonin transporter controls synaptic serotonin levels by pumping serotonin back into the presynaptic neuron after release. This refreshes the synapse following action potential activity. In patients treated with disulfiram who have increased levels of serotonin transporter, as found in those subjects with the L′L′ genotype, the disulfiram-induced increase in serotonin may not be physiologically effective. This may be due to the presynaptic neuron having an adequate level of serotonin transporter to maintain a basal serotonin level that is minimally affected when serotonin is increased by disulfiram. In contrast, the serotonin transporter may not efficiently remove serotonin from the synapse in patients with low serotonin transporter levels, when serotonin is increased due to disulfiram treatment. Thus, synaptic serotonin levels are effectively increased and then can bind more effectively to serotonin receptors stimulating downstream events. For example, serotonin modulates the response to drug-paired cues through binding to its post-synaptic receptors, some of which are involved in behaviors related to cocaine and the priming effects of cocaine cues (Fletcher et al. 2002; Liu & Cunningham 2006; Nic Dhonnchadha et al. 2009).
The functional polymorphism that we studied in the promoter of the serotonin transporter gene has been associated with a number of psychiatric disorders and behaviors, although some of these findings have lacked replication (Risch et al. 2009). The serotonin transporter is expressed in the raphe and thalamus, and in cortical and limbic areas such as the cingulate cortex, and hippocampal pyramidal cells, brain regions that process emotional components of behavior (Zhuang et al. 2005). Hence, emotional regulation, anxiety, and impulsivity may be modified by specific alterations of serotonin function (Courtet et al. 2005). An increase in anxiety related traits has been found in healthy volunteers with an S allele (Lesch et al. 1996). It has been suggested in studies on coping ability that S allele carriers are more cognitively vulnerable to anxiety when making decisions (Szily et al. 2008). Amygdala activation while making decisions as measured by fMRI was found to be lower in those with an LALA genotype (Roiser et al. 2009). Thus, when taking disulfiram, S′ allele carriers may be more sensitive than L′L′ individuals to the anxiety provoking effects from decisions related to taking cocaine or anxiety induced by cocaine. A number of other studies have reported associations of response to therapy with the serotonin 5-HTTLPR genotype. For example, a meta-analysis of 15 studies of the response to selective reuptake inhibitors for depression found an association with the S allele (Serretti et al. 2007).
As our serotonin transporter hypothesis relied on serotonin levels to explain the role of the serotonin transporter in response to disulfiram treatment, we also evaluated the role of a functional variant in the TPH2 gene. We found that patients with the low activity TPH2 A allele responded to disulfiram treatment better than those patients without this allele. This may be due to disulfiram having a greater effect in raising serotonin levels in those with genetically low serotonin biosynthesis. This same variant in TPH2 also has been found to be associated with heroin addiction (Nielsen et al. 2008).
Cocaine directly inhibits reuptake of dopamine, norepinephrine and serotonin (Han & Gu 2006), and disulfiram can affect all three, including increasing serotonin levels. Cocaine administration to rats increases serotonin, as well as dopamine, within seconds (Broderick et al. 2004). In cultured serotonergic neurons, cocaine treatment has been shown to enhance surface expression of the serotonin transporter, whereas serotonin downregulated serotonin transporter expression (Kittler et al. 2010). In post-mortem brains, cocaine use was associated with increased serotonin transporter levels in the midbrain and striatum (Little et al. 1993a,1993b). Therefore, long-term cocaine use may accentuate these genetic differences in the efficacy of disulfiram, since these enhanced transporter levels will distinguish further the patients with the high activity serotonin variant. Our patients' long histories of cocaine use (mean of 13 years) did not allow us to examine shorter term cocaine users, which may be of interest in future studies of disulfiram.
This study has several limitations including its relatively small sample size. Replication using larger populations will be required to substantiate these findings. Other limitations of this study were that all our subjects were opioid dependent. We cannot speculate as to how this co-dependency would affect our findings. In addition, no subjects had current alcohol dependence, but many had alcohol abuse. While the 250 mg dose is unlikely to produce significant adverse reactions with one or two alcohol drinks in many individuals, a disulfiram-induced decrease in alcohol use might also drive some reduction in cocaine use. Baseline rates of alcohol abuse were unusually low in the TPH2 TT genotype patients, and these patients were relatively non-responsive to disulfiram. Finally, disulfiram may increase serotonin through a variety of mechanisms not directly studied here, including inhibition of aldehyde dehydrogenase (Vallari & Pietruszko 1982) and monoamine oxidase (Schurr et al. 1978), both of which metabolize serotonin. Variants in these genes may also be important in disulfiram pharmacotherapeutic action and should be investigated in future studies.