Janet K. Coller

Pharmacogenetics of Opioids

Opioids are used for acute and chronic pain and dependency. They have a narrow therapeutic index and large interpatient variability in response. Genetic factors regulating their pharmacokinetics (metabolizing enzymes, transporters) and pharmacodynamics (receptors and signal transduction elements) are contributors to such variability. The polymorphic CYP2D6 regulates the O‐demethylation of codeine and other weak opioids to more potent metabolites with poor metabolizers having reduced antinociception in some cases. Some opioids are P‐glycoprotein substrates, whereas, ABCB1 genotypes inconsistently influence opioid pharmacodynamics and dosage requirements. Single‐nucleotide polymorphisms in the mu opioid receptor gene are associated with increasing morphine, but not methadone dosage requirements and altered efficacy of mu opioid agonists and antagonists. As knowledge regarding the interplay between genes affecting opioid pharmacokinetics including cerebral kinetics and pharmacodynamics increases, our understanding of the role of pharmacogenomics in mediating interpatient variability in efficacy and side effects to this important class of drugs will be better informed. Opioid drugs as a group have withstood the test of time in their ability to attenuate acute and chronic pain. Since the isolation of morphine in the early 1800s by Friedrich Sertürner, a large number of opioid drugs beginning with modification of the 4,5‐epoxymorphinan ring structure were developed in order to improve their therapeutic margin, including reducing dependence and tolerance, ultimately without success.

ABCB1 genetic variability and methadone dosage requirements in opioid‐dependent individuals

The most common treatment for opioid dependence is substitution therapy with another opioid such as methadone. The methadone dosage is individualized but highly variable, and program retention rates are low due in part to nonoptimal dosing resulting in withdrawal symptoms and further heroin craving and use. Methadone is a substrate for the P‐glycoprotein transporter, encoded by the ABCB1 gene, which regulates central nervous system exposure. This retrospective study aimed to investigate the influence of ABCB1 genetic variability on methadone dose requirements.

Role of active metabolites in the use of opioids

The opioid class of drugs, a large group, is mainly used for the treatment of acute and chronic persistent pain. All are eliminated from the body via metabolism involving principally CYP3A4 and the highly polymorphic CYP2D6, which markedly affects the drug’s function, and by conjugation reactions mainly by UGT2B7. In many cases, the resultant metabolites have the same pharmacological activity as the parent opioid; however in many cases, plasma metabolite concentrations are too low to make a meaningful contribution to the overall clinical effects of the parent drug. These metabolites are invariably more water soluble and require renal clearance as an important overall elimination pathway. Such metabolites have the potential to accumulate in the elderly and in those with declining renal function with resultant accumulation to a much greater extent than the parent opioid. The best known example is the accumulation of morphine-6-glucuronide from morphine. Some opioids have active metabolites but at different target sites. These are norpethidine, a neurotoxic agent, and nordextropropoxyphene, a cardiotoxic agent. Clinicians need to be aware that many opioids have active metabolites that will become therapeutically important, for example in cases of altered pathology, drug interactions and genetic polymorphisms of drug-metabolizing enzymes. Thus, dose individualisation and the avoidance of adverse effects of opioids due to the accumulation of active metabolites or lack of formation of active metabolites are important considerations when opioids are used.

Inhibiting the TLR4-MyD88 signalling cascade by genetic or pharmacological strategies reduces acute alcohol-induced sedation and motor impairment in mice.

BACKGROUND AND PURPOSE Emerging evidence implicates a role for toll‐like receptor 4 (TLR4) in the CNS effects of alcohol. The aim of the current study was to determine whether TLR4–MyD88‐dependent signalling is involved in the acute behavioural actions of alcohol and if alcohol can activate TLR4‐downstream MAPK and NF‐κB pathways.

Attenuation of microglial and IL-1 signaling protects mice from acute alcohol-induced sedation and/or motor impairment

Alcohol-induced proinflammatory central immune signaling has been implicated in the chronic neurotoxic actions of alcohol, although little work has examined if these non-neuronal actions contribute to the acute behavioral responses elicited by alcohol administration. The present study examined if acute alcohol-induced sedation (loss of righting reflex, sleep time test) and motor impairment (rotarod test) were influenced by acute alcohol-induced microglial-dependent central immune signaling. Inhibition of acute alcohol-induced central immune signaling, through the reduction of proinflammatory microglial activation with minocycline, or by blocking interleukin-1 (IL-1) receptor signaling using IL-1 receptor antagonist (IL-1ra), reduced acute alcohol-induced sedation in mice. Mice treated with IL-1ra recovered faster from acute alcohol-induced motor impairment than control animals. However, minocycline led to greater motor impairment induced by alcohol, implicating different mechanisms in alcohol-induced sedation and motor impairment. At a cellular level, IκBα protein levels in mixed hippocampal cells responded rapidly to alcohol in a time-dependent manner, and both minocycline and IL-1ra attenuated the elevated levels of IκBα protein by alcohol. Collectively these data suggest that alcohol is capable of rapid modification of proinflammatory immune signaling in the brain and this contributes significantly to the pharmacology of alcohol.

Naloxone-precipitated morphine withdrawal behavior and brain IL-1β expression: Comparison of different mouse strains

The development of opioid dependence involves classical neuronal opioid receptor activation and is due in part to engagement of glia causing a proinflammatory response. Such opioid-induced glial activation occurs, at least in part, through a non-classical opioid mechanism involving Toll-like-receptor 4 (TLR4). Among the immune factors released following the opioid-glia-TLR4 interaction, interleukin-1β (IL-1β) plays a prominent role. Previous animal behavioral studies have demonstrated significant heterogeneity of chronic morphine-induced tolerance and dependence between different mouse strains. The aim of this study was to investigate whether the heterogeneity of chronic opioid-induced IL-1β expression contributes to differences in opioid tolerance and withdrawal behaviors. Chronic morphine-induced tolerance and dependence were assessed in 3 inbred wild-type mouse strains (Balb/c, CBA, and C57BL/6) and 2 knockout strains (TLR4 and MyD88). Analysis of brain nuclei (medial prefrontal cortex, cortex, brain stem, hippocampus, and midbrain and diencephalon regions combined) revealed that, of inbred wild-type mice, there are significant main effects of morphine treatment on IL-1β expression in the brain regions analyzed (p<0.02 for all regions analyzed). A significant increase in hippocampal IL-1β expression was found in C57BL/6 mice after morphine treatment, whilst, a significant decrease was found in the mPFC region of wild-type Balb/c mice. Furthermore, the results of wild-type inbred strains demonstrated that the elevated hippocampal IL-1β expression is associated with withdrawal jumping behavior. Interestingly, knockout of TLR4, but not MyD88 protected against the development of analgesic tolerance. Gene sequence differences of IL - 1β and TLR4 genes alone did not explain the heterogeneity of dependence behavior between mouse strains. Together, these data further support the involvement of opioid-induced CNS immune signaling in dependence development. Moreover, this study demonstrated the advantages of utilizing multiple mouse strains and indicates that appropriate choice of mouse strains could enhance future research outcomes.

Association between the DRD2 A1 allele and response to methadone and buprenorphine maintenance treatments

The TaqI A polymorphism (A1) of the dopamine D2 receptor gene (DRD2), although not a specific predictor of opioid dependence, has been strongly associated with high levels of prior heroin use and poor treatment outcomes among methadone maintenance patients. The aims of this study were to confirm these findings via a retrospective analysis of A1 allele frequency in methadone (n = 46) and buprenorphine (n = 25) patients, and non‐opioid‐dependent controls (n = 95). Subjects were genotyped at the DRD2 TaqI A locus using PCR amplification followed by TaqI restriction enzyme digestion and gel electrophoresis. For methadone and buprenorphine subjects, heroin use (prior to treatment), treatment outcomes, and withdrawal occurrence were determined from comprehensive case notes. No significant differences in A1 allele frequency (%) were observed between: methadone (19.6%), buprenorphine (18.0%), and control (17.9%) groups (P > 0.7); successful and poor treatment outcome groups, methadone: 20.0% and 19.2%, respectively (P = 1.0); buprenorphine: 18.4% and 20.0%, respectively (P = 1.0). Also, there were no significant relationships between TaqI A genotype and prior heroin use (P = 0.47). However, among the successful methadone subjects, significantly fewer A1 allele carriers experienced withdrawal than non‐A1 carriers (P = 0.04). In conclusion, the DRD2 genotype effects did not affect opioid maintenance treatment outcomes. This suggests the need for a further prospective investigation into the role of the DRD2 A1 allele in heroin use and response to maintenance pharmacotherapies for opioid dependence.

The combined impact of CYP2C19 and CYP2B6 pharmacogenetics on cyclophosphamide bioactivation

AIMS The role of CYP pharmacogenetics in the bioactivation of cyclophosphamide is still controversial. Recent clinical studies have suggested a role for either CYP2C19 or CYP2B6. The aim of this study was to clarify the role of these pharmacogenes. METHODS We used a combined in vitro-in vivo approach to determine the role of these pharmacogenes in the bioactivation of the prodrug to 4-hydroxy cyclophosphamide (4-OHCP). Cyclophosphamide metabolism was determined in a human liver biobank (n= 14) and in patients receiving the drug for treatment of lupus nephritis (n= 16) RESULTS In livers of known CYP2C19 and CYP2B6 genotype and protein expression we observed that there was a combined role for both CYP2C19 and CYP2B6 in the bioactivation of cyclophosphamide in vitro. The presence of at least one loss of function (LoF) allele at either CYP2C19 or CYP2B6 resulted in a significant decrease in both V(max) (P= 0.028) and CL(int) (P= 0.0017) compared with livers with no LoF alleles. This dual genotype relationship was also observed in a preliminary clinical study, with patients who had ≥1 LoF allele at either CYP2C19 or CYP2B6 also displaying significantly (P= 0.0316) lower bioactivation of cyclophosphamide. The mean 4-OHCP : CP bioactivation ratio was 0.0014 (95% CI 0.0007, 0.002) compared with 0.0071 (95% CI 0.0001, 0.014) in patients with no LoF alleles at either of these genes. CONCLUSIONS The presence of ≥1 LoF allele(s) at either CYP2B6 or CYP2C19 appeared to result in decreased bioactivation of cyclophosphamide both in vitro and in patients. Further clinical studies to confirm this relationship are warranted.

Distribution of microsomal epoxide hydrolase in humans: an immunohistochemical study in normal tissues, and benign and malignant tumours.

Microsomal epoxide hydrolase is a biotransformation enzyme which is involved in the hydrolysis of various epoxides and epoxide intermediates. In the present study, its distribution was investigated in both normal human tissues and human tumours of different histogenetic origin using immunohistochemical techniques. In normal tissue, epithelial cells were more often and more intensely immunostained than mesenchymal cells. The main epithelial cell types expressing microsomal epoxide hydrolase were hepatocytes, acinus cells of the pancreas, and cells of salivary and adrenal glands. Immunostained cells of mesenchymal origin included monocytes, fibrocytes, fibroblasts, vessel endothelium, muscle cells, and cells of the reproductive system. Three patterns of expression were observed in tumour tissues: (1) moderate or strong in hepatocellular carcinomas, tumours of the adrenal gland, and theca-fibromas of the ovary; (2) inhomogeneous staining pattern of variable intensity in breast cancer, lung cancer, colorectal carcinomas, carcinoid tumours, and some tumours of mesenchymal origin; and (3) no expression in malignant melanomas, malignant lymphomas, and renal carcinomas. These data indicate that microsomal epoxide hydrolase expression is not restricted to tissue of any particular histogenetic origin. Nonetheless, immunohistochemical identification of microsomal epoxide hydrolase may be helpful in some well-defined histological settings, for example, confirmation of hepatocellular carcinoma.

oprm1 A118g genotype fails to predict the effectiveness of naltrexone treatment for alcohol dependence

Given the evidence from retrospective studies indicating that alcohol-dependent patients with homozygous or heterozygous A118G variant of the &mgr;-opioid receptor, OPRM1, gene have significantly better outcomes when treated with naltrexone; this study examined this prospectively in 100 alcohol-dependent participants prescribed naltrexone for 12 weeks and offered six sessions of cognitive-behavioral therapy or intervention. Comparisons were made among OPRM1 genotypic groups on several outcome measures. Naltrexone treatment produced significant decreases in self-reported and objective indicators of alcohol use and craving from baseline (P<0.0001 and 0.017, respectively), particularly during the first 2 months of treatment, with 68% completing the study. However, there was no evidence of a significant association between OPRM1 A118G genotype and treatment success on any of the outcome measures. Therefore, while naltrexone was an effective treatment for alcohol dependence, the OPRM1 A118G genotype was not a predictor of success.