The dopamine 3 receptor as a candidate biomarker and therapeutic for opioid use disorder.

Here, we present recent studies suggesting that specific DRD3 single nucleotide polymorphisms (SNPs, e.g. rs324029 and rs2654754) might serve as prognostic biomarkers for opioid use disorder (OUD). Additionally, preclinical studies with novel dopamine 3 receptor (D3R) partial agonists and antagonists have been evaluated as candidate OUD therapeutics and have shown a reduced risk of cardiovascular toxicity compared with the original D3R antagonist. From these findings, we argue that DRD3 SNPs could serve as a diagnostic tool for assessing OUD risk and that more research is warranted examining the D3R as a safe and effective therapeutic target for treating OUD.

Genome-wide analyses reveal shared genetic architecture and novel risk loci between opioid use disorder and general cognitive ability.

BACKGROUND: Opioid use disorder (OUD), a serious health burden worldwide, is associated with lower cognitive function. Recent studies have demonstrated a negative genetic correlation between OUD and general cognitive ability (COG), indicating a shared genetic basis. However, the specific genetic variants involved, and the underlying molecular mechanisms remain poorly understood. Here, we aimed to quantify and identify the genetic basis underlying OUD and COG. METHODS: We quantified the extent of genetic overlap between OUD and COG using a bivariate causal mixture model (MiXeR) and identified specific genetic loci applying conditional/conjunctional FDR. Finally, we investigated biological function and expression of implicated genes using available resources. RESULTS: We estimated that ~94% of OUD variants (4.8k out of 5.1k variants) also influence COG. We identified three novel OUD risk loci and one locus shared between OUD and COG. Loci identified implicated biological substrates in the basal ganglia. CONCLUSION: We provide new insights into the complex genetic risk architecture of OUD and its genetic relationship with COG.

Single nuclei transcriptomics in human and non-human primate striatum in opioid use disorder.

In brain, the striatum is a heterogenous region involved in reward and goal-directed behaviors. Striatal dysfunction is linked to psychiatric disorders, including opioid use disorder (OUD). Striatal subregions are divided based on neuroanatomy, each with unique roles in OUD. In OUD, the dorsal striatum is involved in altered reward processing, formation of habits, and development of negative affect during withdrawal. Using single nuclei RNA-sequencing, we identified both canonical (e.g., dopamine receptor subtype) and less abundant cell populations (e.g., interneurons) in human dorsal striatum. Pathways related to neurodegeneration, interferon response, and DNA damage were significantly enriched in striatal neurons of individuals with OUD. DNA damage markers were also elevated in striatal neurons of opioid-exposed rhesus macaques. Sex-specific molecular differences in glial cell subtypes associated with chronic stress were found in OUD, particularly female individuals. Together, we describe different cell types in human dorsal striatum and identify cell type-specific alterations in OUD.

Fentanyl dysregulates neuroinflammation and disrupts blood-brain barrier integrity in HIV-1 Tat transgenic mice.

Opioid overdose deaths have dramatically increased by 781% from 1999 to 2021. In the setting of HIV, opioid drug abuse exacerbates neurotoxic effects of HIV in the brain, as opioids enhance viral replication, promote neuronal dysfunction and injury, and dysregulate an already compromised inflammatory response. Despite the rise in fentanyl abuse and the close association between opioid abuse and HIV infection, the interactive comorbidity between fentanyl abuse and HIV has yet to be examined in vivo. The HIV-1 Tat-transgenic mouse model was used to understand the interactive effects between fentanyl and HIV. Tat is an essential protein produced during HIV that drives the transcription of new virions and exerts neurotoxic effects within the brain. The Tat-transgenic mouse model uses a glial fibrillary acidic protein (GFAP)-driven tetracycline promoter which limits Tat production to the brain and this model is well used for examining mechanisms related to neuroHIV. After 7 days of fentanyl exposure, brains were harvested. Tight junction proteins, the vascular cell adhesion molecule, and platelet-derived growth factor receptor-ß were measured to examine the integrity of the blood brain barrier. The immune response was assessed using a mouse-specific multiplex chemokine assay. For the first time in vivo, we demonstrate that fentanyl by itself can severely disrupt the blood-brain barrier and dysregulate the immune response. In addition, we reveal associations between inflammatory markers and tight junction proteins at the blood-brain barrier.

A consideration of CYP2D6 genetic variations in the Ghanaian population as a potential 'culprit' for the tramadol 'abuse crisis'.

BACKGROUND: Cytochrome P450 2D6 is involved in the metabolism of several important medicines including opioids. Variations in CYP2D6 have been implicated in drug response and according to the Clinical Pharmacogenetics Implementation Consortium Guideline (CPIC) for CYP2D6, dosing for CYP2D6 substrates should be based on variants carried by individuals. Although CYP2D6 variations in Ghana had been previously recorded, not all variants have been reported in the Ghanaian population. In this exploratory study we set to investigate certain unreported variations in the Ghanaian population in addition to the previously reported ones and use that to understand the tramadol 'abuse' crisis that is currently being experienced in Ghana. METHODS: This study employed a convenience sampling approach to include 106 unrelated participants who were recruited as part of the PHARMABIOME project. We successfully genotyped 106 samples using Iplex GOLD SNP genotyping protocol after extracting DNA from these individuals. Allele and diplotype frequencies were undertaken by counting from observed genotypes. Comparison of alleles reported from various studies were done. RESULTS: Unreported alleles such as *3, *9 and *41 which are classified as no function and decreased function were observed in our study cohort. In addition, variants such as (*1, *2, *4, *5, *10, *17 and *29 were observed with different frequencies. Our study showed 26% representation of intermediate metabolizers (IM) and 2% poor metabolizers (PM) in the study population. CONCLUSION: The implications for informal sector workers who use tramadol for recreational purposes, is that IMs and PMs will overdose as they may have reduced analgesic effects which will translate into increased risks of unforeseen adverse events. We therefore propose that CYP2D6 should be considered in opioid dosage while making use of these observed variations to implement new approaches to tackle the tramadol 'abuse crisis' in Ghana.

Opioid trail: Tracking contributions to opioid use disorder from host genetics to the gut microbiome.

Opioid use disorder (OUD) is a worldwide public health crisis with few effective treatment options. Traditional genetics and neuroscience approaches have provided knowledge about biological mechanisms that contribute to OUD-related phenotypes, but the complexity and magnitude of effects in the brain and body remain poorly understood. The gut-brain axis has emerged as a promising target for future therapeutics for several psychiatric conditions, so characterizing the relationship between host genetics and the gut microbiome in the context of OUD will be essential for development of novel treatments. In this review, we describe evidence that interactions between host genetics, the gut microbiome, and immune signaling likely play a key role in mediating opioid-related phenotypes. Studies in humans and model organisms consistently demonstrated that genetic background is a major determinant of gut microbiome composition. Furthermore, the gut microbiome is susceptible to environmental influences such as opioid exposure. Additional work focused on gene by microbiome interactions will be necessary to gain improved understanding of their effects on OUD-related behaviors.

Pharmacogenetics of Biochemically Verified Abstinence in an Opioid Agonist Therapy Randomized Clinical Trial of Methadone and Buprenorphine/Naloxone.

Methadone and buprenorphine/naloxone are opioid agonist therapies for opioid use disorder treatment. Genetic factors contribute to individual differences in opioid response; however, little is known regarding genetic associations with clinical outcomes in people receiving opioid agonist therapies. Participants diagnosed with opioid use disorder, principally consisting of prescription opioids (licit or illicit), were randomized to methadone or buprenorphine/naloxone for 24 weeks of daily treatment (NCT03033732). Urine was collected at 12 biweekly study visits and analyzed for non-treatment opioids. Variants in genes involved in methadone metabolism (CYP2B6, CYP2C19, and CYP3A4), buprenorphine metabolism (CYP3A4 and UGT2B7), and µ-opioid receptor function (OPRM1) were genotyped and analyzed for their association with the number of non-treatment opioid-free urine screens. Primary analyses focused on the last 12 weeks (6 study visits, post-titration) of treatment among those reporting White ethnicity. Additional sensitivity and exploratory analyses were performed. Among methadone-treated participants (n = 52), the OPRM1 rs1799971 AA genotype (vs. G-genotypes, i.e., having one or two G alleles) was associated with greater opioid-free urine screens (incidence rate ratio = 5.24, 95% confidence interval (CI) = 2.43-11.26, P = 0.000023); longitudinal analyses showed a significant genotype-by-time interaction over the full 24 weeks (12 study visits, ß = -0.28, 95% CI = -0.45 to -0.11, P = 0.0015). Exploratory analyses suggest an OPRM1 rs1799971 genotype effect on retention. No evidence of association was found between other genetic variants, including in metabolic variants, and non-treatment opioid-free urine screens in the methadone or buprenorphine/naloxone arms. Those with the OPRM1 rs1799971 G-genotypes may have a poorer response to methadone maintenance treatment, an effect that persisted through 24 weeks of treatment.

Neural Network Connectivity Following Opioid Dependence is Altered by a Common Genetic Variant in the µ-Opioid Receptor, OPRM1 A118G.

Opioid use disorder is a chronic, relapsing disease associated with persistent changes in brain plasticity. A common single nucleotide polymorphism (SNP) in the µ-opioid receptor gene, OPRM1 A118G, is associated with altered vulnerability to opioid addiction. Reconfiguration of neuronal connectivity may explain dependence risk in individuals with this SNP. Mice with the equivalent Oprm1 variant, A112G, demonstrate sex-specific alterations in the rewarding properties of morphine and heroin. To determine whether this SNP influences network-level changes in neuronal activity, we compared FOS expression in male and female mice that were opioid-naive or opioid-dependent. Network analyses identified significant differences between the AA and GG Oprm1 genotypes. Based on several graph theory metrics, including small-world analysis and degree centrality, we show that GG females in the opioid-dependent state exhibit distinct patterns of connectivity compared to other groups of the same genotype. Using a network control theory approach, we identified key cortical brain regions that drive the transition between opioid-naive and opioid-dependent brain states; however, these regions are less influential in GG females leading to sixfold higher average minimum energy needed to transition from the acute to the dependent state. In addition, we found that the opioid-dependent brain state is significantly less stable in GG females compared to other groups. Collectively, our findings demonstrate sex- and genotype-specific modifications in local, mesoscale, and global properties of functional brain networks following opioid exposure and provide a framework for identifying genotype differences in specific brain regions that play a role in opioid dependence.

A genome-wide association, polygenic risk score and sex study on opioid use disorder treatment outcomes.

Opioid use disorder continues to be a health concern with a high rate of opioid related deaths occurring worldwide. Medication Assisted Treatments (MAT) have been shown to reduce opioid withdrawal, cravings and opioid use, however variability exists in individual's treatment outcomes. Sex-specific differences have been reported in opioid use patterns, polysubstance use and health and social functioning. Candidate gene studies investigating methadone dose as an outcome have identified several candidate genes and only five genome-wide associations studies have been conducted for MAT outcomes. This study aimed to identify genetic variants associated with MAT outcomes through genome-wide association study (GWAS) and test the association between genetic variants previously associated with methadone dose through a polygenic risk score (PRS). Study outcomes include: continued opioid use, relapse, methadone dose and opioid overdose. No genome-wide significance SNPs or sex-specific results were identified. The PRS identified statistically significant results (p < 0.05) for the outcome of methadone dose (R2 = 3.45 × 10-3). No other PRS was statistically significant. This study provides evidence for association between a PRS and methadone dose. More research on the PRS to increase the variance explained is needed before it can be used as a tool to help identify a suitable methadone dose within this population.

A novel tetra-primer ARMS-PCR for genotyping of the OPRM1 gene rs1799971 variant associated with opioid use disorders.

OBJECTIVES: A SNV is a single nucleotide change that can occur at any point in the genome. SNVs are the most common genetic variants that occur in the human genome, and a number of SNVs have been found to be associated with human traits and disease. Researchers genotype SNVs using TaqMan technology, DNA microarray, MALDI-TOF mass spectrometry, and automated sequencing, which are expensive and time-consuming. The OPRM1 gene rs1799971 (A118G) has been identified for its association with Opioid use disorder (OUD). The present study focused on developing a single step identification test using Tetra-Primer Amplification Refractory Mutation System-PCR (T-ARMS-PCR) to detect the presence of SNV OPRM1 rs1799971 (A118G). This study was performed to optimize the protocol for the designed four primers and validate it using a total of 52 buccal samples from volunteers who are currently under rehabilitation for the drug abuse disorder. RESULTS: Utilizing 52 DNA samples, a novel T-ARMS-PCR assay was successfully developed, tested, and validated. The products of the T-ARMS PCR for rs1799971 contained 395 bp as the control band, 186 bp as G allele (variant) and 257 bp as A allele (wild type), which were observed in the gel image. The genotype frequencies for the OPRM1 gene rs1799971 (A118G) were 44% (22/52) of homozygous variant type (GG), 28.9% (15/52) of homozygous wild type (AA) and 28.9% (15/22) of heterozygous (AG). The G allele frequency was 56.7% and A allele frequency was 43.3%.

Methylation and expression quantitative trait loci rs1799971 in the OPRM1 gene and rs4654327 in the OPRD1 gene are associated with opioid use disorder.

Opioid use disorder (OUD) is a chronic and relapsing brain disease that results in significant mortality worldwide. Genetic factors are estimated to contribute to 40%-60% of the liability, with polymorphisms of opioid receptor genes implicated in this disorder. However, the mechanisms underlying these associations are not yet fully understood. In the present study, we first examined the methylation levels in the promoter region of the OPRM1, OPRD1, and OPRK1 genes in 111 healthy controls (HCs) and 120 patients with OUD, and genotyped three tag SNPs in these genes. Correlations between these SNPs and the methylation levels of the CpG sites and expression levels of the genes were analyzed. After identifying the mQTLs and eQTLs, we determined the associations between the mQTLs/eQTLs and susceptibility to and characteristics of OUD in 930 HCs and 801 patients with OUD. Our results demonstrated that SNPs rs1799971 in the OPRM1 gene and rs4654327 in the OPRD1 gene were both mQTLs and eQTLs. We observed unique correlations between mQTLs and methylation levels of several CpG sites in the OUD group compared to the HC group. Interestingly, both the two mQTLs and eQTLs were associated with the susceptibility to OUD. In conclusion, we suppose that mQTLs and eQTLs in genes may underlie the associations between certain risk genetic polymorphisms and OUD. These mQTLs and eQTLs could potentially serve as promising biomarkers for better management of opioid misuse.

Single nucleus transcriptomics of ventral midbrain identifies glial activation associated with chronic opioid use disorder.

Dynamic interactions of neurons and glia in the ventral midbrain mediate reward and addiction behavior. We studied gene expression in 212,713 ventral midbrain single nuclei from 95 individuals with history of opioid misuse, and individuals without drug exposure. Chronic exposure to opioids was not associated with change in proportions of glial and neuronal subtypes, however glial transcriptomes were broadly altered, involving 9.5 - 6.2% of expressed genes within microglia, oligodendrocytes, and astrocytes. Genes associated with activation of the immune response including interferon, NFkB signaling, and cell motility pathways were upregulated, contrasting with down-regulated expression of synaptic signaling and plasticity genes in ventral midbrain non-dopaminergic neurons. Ventral midbrain transcriptomic reprogramming in the context of chronic opioid exposure included 325 genes that previous genome-wide studies had linked to risk of substance use traits in the broader population, thereby pointing to heritable risk architectures in the genomic organization of the brain's reward circuitry.

Predictors of treatment retention and survival among methadone-maintained patients: A possible role for a functional delta opioid receptor gene variant.

BACKGROUND: Variants in the delta opioid receptor gene, OPRD1, were associated with opioid use disorder and response to treatment. The study goal was to assess whether OPRD1 variants predict survival and retention in methadone maintenance treatment (MMT). METHODS: Retention and survival time since admission (June 1993 - June 2022) until leaving treatment (for retention), or at the end of follow-up (Dec 2022) (for retention and survival) were analyzed in 488 patients. Vital data was taken from a national registry. Predictors were estimated using Kaplan-Meier and Cox regression models. RESULTS: Longer retention and survival were found for carriers of the T allele of SNP rs204076. This SNP is associated with OPRD1 expression in cortex (GTEx). Carriers of the T allele (n = 251) survived longer compared to non-carriers (24.7 vs. 20.2 years, p = 0.005) and had longer retention (11.2 vs. 8.8 years, p = 0.04). Multivariate analysis identified the T allele as an independent predictor of longer survival time (p = 0.003) and retention (p = 0.009). Additional predictors for survival were no benzodiazepine use after one year in MMT, no hepatitis C, <20 years of opioid usage, and admission at age < 30. Additional predictors for longer retention were no use of other drugs except opioids on admission, and no drugs at one year, as well as methadone dose ≥ 100mg/d at one year and axis I & II DSM-5 psychiatric diagnosis. CONCLUSIONS: The OPRD1 SNP rs204076 and non-genetic predictors contribute to survival time and retention in MMT patients.

Neonatal opioid toxicity: opioid withdrawal (abstinence) syndrome with emphasis on pharmacogenomics and respiratory depression.

The increasing use of opioids in pregnant women has led to an alarming rise in the number of cases of neonates with drug-induced withdrawal symptoms known as neonatal opioid withdrawal syndrome (NOWS). NOWS is a toxic heterogeneous condition with many neurologic, autonomic, and gastrointestinal symptoms including poor feeding, irritability, tachycardia, hypertension, respiratory defects, tremors, hyperthermia, and weight loss. Paradoxically, for the management of NOWS, low doses of morphine, methadone, or buprenorphine are administered. NOWS is a polygenic disorder supported by studies of genomic variation in opioid-related genes. Single-nucleotide polymorphisms (SNPs) in CYP2B6 are associated with variations in NOWS infant responses to methadone and SNPs in the OPRM1, ABCB1, and COMT genes are associated with need for treatment and length of hospital stay. Epigenetic gene changes showing higher methylation levels in infants and mothers have been associated with more pharmacologic treatment in the case of newborns, and for mothers, longer infant hospital stays. Respiratory disturbances associated with NOWS are not well characterized. Little is known about the effects of opioids on developing neonatal respiratory control and respiratory distress (RD), a potential problem for survival of the neonate. In a rat model to test the effect of maternal opioids on the developing respiratory network and neonatal breathing, maternal-derived methadone increased apneas and lessened RD in neonates at postnatal (P) days P0 and P1. From P3, breathing normalized with age suggesting reorganization of respiratory rhythm-generating circuits at a time when the preBötC becomes the dominant inspiratory rhythm generator. In medullary slices containing the preBötC, maternal opioid treatment plus exposure to exogenous opioids showed respiratory activity was maintained in younger but not older neonates. Thus, maternal opioids blunt centrally controlled respiratory frequency responses to exogenous opioids in an age-dependent manner. In the absence of maternal opioid treatment, exogenous opioids abolished burst frequencies at all ages. Prenatal opioid exposure in children stunts growth rate and development while studies of behavior and cognitive ability reveal poor performances. In adults, high rates of attention deficit disorder, hyperactivity, substance abuse, and poor performances in intelligence and memory tests have been reported.

Profiling neuronal methylome and hydroxymethylome of opioid use disorder in the human orbitofrontal cortex.

Opioid use disorder (OUD) is influenced by genetic and environmental factors. While recent research suggests epigenetic disturbances in OUD, this is mostly limited to DNA methylation (5mC). DNA hydroxymethylation (5hmC) has been widely understudied. We conducted a multi-omics profiling of OUD in a male cohort, integrating neuronal-specific 5mC and 5hmC as well as gene expression profiles from human postmortem orbitofrontal cortex (OUD = 12; non-OUD = 26). Single locus methylomic analysis and co-methylation analysis showed a higher number of OUD-associated genes and gene networks for 5hmC compared to 5mC; these were enriched for GPCR, Wnt, neurogenesis, and opioid signaling. 5hmC marks also showed a higher correlation with gene expression patterns and enriched for GWAS of psychiatric traits. Drug interaction analysis revealed interactions with opioid-related drugs, some used as OUD treatments. Our multi-omics findings suggest an important role of 5hmC and reveal loci epigenetically dysregulated in OFC neurons of individuals with OUD.

Genetics of cannabis use in opioid use disorder: A genome-wide association and polygenic risk score study.

BACKGROUND: Individuals with an Opioid Use Disorder (OUD) have increased rates of cannabis use in comparison to the general population. Research on the short- and long-term impacts of cannabis use in OUD patients has been inconclusive. A genetic component may contribute to cannabis cravings. AIMS: Identify genetic variants associated with cannabis use through Genome-wide Association Study (GWAS) methods and investigate a Polygenic Risk Score (PRS). In addition, we aim to identify any sex differences in effect size for genetic variants reaching or nearing genome-wide significance in the GWAS. METHODS: The study outcomes of interest were: regular cannabis use (yes/no) (n = 2616), heaviness of cannabis use (n = 1293) and cannabis cravings (n = 836). Logistic and linear regressions were preformed, respectively, to test the association between genetic variants and each outcome, regular cannabis use and heaviness of cannabis use. GWAS summary statistics from a recent large meta-GWAS investigating cannabis use disorder were used to conduct PRS's. Findings are limited to a European ancestry sample. RESULTS: No genome-wide significant associations were found. Rs1813412 (chromosome 17) for regular cannabis use and rs62378502 (chromosome 5) for heaviness of cannabis use were approaching genome-wide significance. Both these SNPs were nominally significant (p<0.05) within males and females, however sex did not modify the association. The PRS identified statistically significant association with cannabis cravings. The variance explained by all PRSs were less than 1.02x10-2. CONCLUSION: This study provides promising results in understanding the genetic contribution to cannabis use in individuals living with OUD.

OPRD1 rs569356 polymorphism has an effect on plasma norbuprenorphine levels and dose/kg-normalized norbuprenorphine values in individuals with opioid use disorder.

This study aimed to determine the effects of nine OPRM1, OPRD1 and OPRK1 polymorphisms on plasma BUP and norbuprenorphine (norBUP) concentrations and various treatment responses in a sample of 122 patients receiving BUP/naloxone. Plasma concentrations of BUP and norBUP were detected by LC-MS/MS. PCR-RFLP method was used to genotype polymorphisms. OPRD1 rs569356 GG had significantly lower plasma norBUP concentration (p = 0.018), dose- (p = 0.049) and dose/kg-normalized norBUP values (p = 0.036) compared with AA. Craving and withdrawal symptoms were significantly higher in OPRD1 rs569356 AG+GG relative to AA. There was a statistically significant difference between the OPRD1 rs678849 genotypes in the intensity of anxiety (13.5 for CT+TT and 7.5 for TT). OPRM1 rs648893 TT (18.8 ± 10.8) was significantly different to CC+CT (14.82 ± 11.3; p = 0.049) in view of the intensity of depression. This current study provides the first data on a prominent effect of the OPRD1 rs569356 variation on BUP pharmacology due to its metabolite norBUP.

Molecular and Epigenetic Aspects of Opioid Receptors in Drug Addiction and Pain Management in Sport.

Opioids are substances derived from opium (natural opioids). In its raw state, opium is a gummy latex extracted from Papaver somniferum. The use of opioids and their negative health consequences among people who use drugs have been studied. Today, opioids are still the most commonly used and effective analgesic treatments for severe pain, but their use and abuse causes detrimental side effects for health, including addiction, thus impacting the user's quality of life and causing overdose. The mesocorticolimbic dopaminergic circuitry represents the brain circuit mediating both natural rewards and the rewarding aspects of nearly all drugs of abuse, including opioids. Hence, understanding how opioids affect the function of dopaminergic circuitry may be useful for better knowledge of the process and to develop effective therapeutic strategies in addiction. The aim of this review was to summarize the main features of opioids and opioid receptors and focus on the molecular and upcoming epigenetic mechanisms leading to opioid addiction. Since synthetic opioids can be effective for pain management, their ability to induce addiction in athletes, with the risk of incurring doping, is also discussed.

Association of OPRK1 rs963549 and rs997917 polymorphisms with opioid use disorder and related phenotypes.

Aim: To evaluate the association between OPRK1 rs963549 and rs997917 and opioid use disorder (OUD) and related phenotypes. Methods: A sample of 208 individuals with (n = 100) and without (n = 108) OUD were enrolled. OPRK1 rs963549 and rs997917 were analyzed by PCR-RFLP. Craving, opioid withdrawal and the intensity of depressive and anxiety symptoms were measured by the appropriate scales. Results: OPRK1 rs963549 variation showed a trend of association with decreased opioid withdrawal. No significant associations were found between OPRK1 rs963549 and rs997917 polymorphisms and craving, depression or anxiety symptoms. Neither single OPRK1 SNPs nor OPRK1 haplotypes were associated with OUD. Conclusion: Our results could be useful for treatment failures of individuals who experience greater opioid withdrawal due to their OPRK1 rs963549 genotypes.