Metabolic clearance of select opioids and opioid antagonists using hepatic spheroids and recombinant cytochrome P450 enzymes.

The opioid crisis is a pressing public health issue, exacerbated by the emergence of more potent synthetic opioids, particularly fentanyl and its analogs. While competitive antagonists exist, their efficacy against synthetic opioids is largely unknown. Furthermore, due to the short durations of action of current antagonists, renarcotization remains a concern. In this study, metabolic activity was characterized for fentanyl-class opioids and common opioid antagonists using multiple in vitro systems, namely, cytochrome P450 (CYP) enzymes and hepatic spheroids, after which an in vitro-in vivo correlation was applied to convert in vitro metabolic activity to predictive in vivo intrinsic clearance. For all substrates, intrinsic hepatic metabolism was higher than the composite of CYP activities, due to fundamental differences between whole cells and single enzymatic reactions. Of the CYP isozymes investigated, 3A4 yielded the highest absolute and relative metabolism across all substrates, with largely negligible contributions from 2D6 and 2C19. Comparative analysis highlighted elevated lipophilicity and diminished CYP3A4 activity as potential considerations for the development of more efficacious opioid antagonists. Finally, antagonists with a high degree of molecular similarity exhibited comparable clearance, providing a basis for structure-metabolism relationships. Together, these results provide multiple screening criteria for early stage drug discovery involving opioid countermeasures.

Dopamine D2 receptors bidirectionally regulate striatal enkephalin expression: Implications for cocaine reward.

Low dopamine D2 receptor (D2R) availability in the striatum can predispose for cocaine abuse; though how low striatal D2Rs facilitate cocaine reward is unclear. Overexpression of D2Rs in striatal neurons or activation of D2Rs by acute cocaine suppresses striatal Penk mRNA. Conversely, low D2Rs in D2-striatal neurons increases striatal Penk mRNA and enkephalin peptide tone, an endogenous mu-opioid agonist. In brain slices, met-enkephalin and inhibition of enkephalin catabolism suppresses intra-striatal GABA transmission. Pairing cocaine with intra-accumbens met-enkephalin during place conditioning facilitates acquisition of preference, while mu-opioid receptor antagonist blocks preference in wild-type mice. We propose that heightened striatal enkephalin potentiates cocaine reward by suppressing intra-striatal GABA to enhance striatal output. Surprisingly, a mu-opioid receptor antagonist does not block cocaine preference in mice with low striatal D2Rs, implicating other opioid receptors. The bidirectional regulation of enkephalin by D2R activity and cocaine offers insights into mechanisms underlying the vulnerability for cocaine abuse.

Deficient mitochondrial respiration in astrocytes impairs trace fear conditioning and increases naloxone-precipitated aversion in morphine-dependent mice.

Mitochondria are abundant in the fine processes of astrocytes, however, potential roles for astrocyte mitochondria remain poorly understood. In the present study, we performed a systematic examination of the effects of abnormal oxidative phosphorylation in astrocytes on several mouse behaviors. Impaired astrocyte oxidative phosphorylation was produced by astrocyte-specific deletion of the nuclear mitochondrial gene, Cox10, that encodes an accessory protein of complex IV, the protoheme:heme-O-farnesyl transferase. As expected, conditional deletion of the Cox10 gene in mice (cKO mice) significantly reduced expression of COX10 and Cytochrome c oxidase subunit I (MTCO1) of Complex IV, resulting in decreased oxidative phosphorylation without significantly affecting glycolysis. No effects of the deletion were observed on locomotor activity, anxiety-like behavior, nociception, or spontaneous alternation. Cox10 cKO female mice exhibited mildly impaired novel object recognition, while Cox10 cKO male mice were moderately deficient in trace fear conditioning. No group-related changes were observed in conditional place preference (CPP) that assessed effects of morphine on reward. In contrast to CPP, Cox10 cKO mice demonstrated significantly increased aversive behaviors produced by naloxone-precipitated withdrawal following chronic exposure to morphine, that is, jumping and avoidance behavior as assessed by conditional place aversion (CPA). Our study suggests that astrocyte oxidative phosphorylation may contribute to behaviors associated with greater cognitive load and/or aversive and stressful conditions.

Interactive Mechanisms of Supraspinal Sites of Opioid Analgesic Action: A Festschrift to Dr. Gavril W. Pasternak.

Almost a half century of research has elaborated the discoveries of the central mechanisms governing the analgesic responses of opiates, including their receptors, endogenous peptides, genes and their putative spinal and supraspinal sites of action. One of the central tenets of "gate-control theories of pain" was the activation of descending supraspinal sites by opiate drugs and opioid peptides thereby controlling further noxious input. This review in the Special Issue dedicated to the research of Dr. Gavril Pasternak indicates his contributions to the understanding of supraspinal mediation of opioid analgesic action within the context of the large body of work over this period. This review will examine (a) the relevant supraspinal sites mediating opioid analgesia, (b) the opioid receptor subtypes and opioid peptides involved, (c) supraspinal site analgesic interactions and their underlying neurophysiology, (d) molecular (particularly AS) tools identifying opioid receptor actions, and (e) relevant physiological variables affecting site-specific opioid analgesia. This review will build on classic initial studies, specify the contributions that Gavril Pasternak and his colleagues did in this specific area, and follow through with studies up to the present.

Histone Deacetylase Inhibitors Prevented the Development of Morphine Tolerance by Decreasing IL6 Production and Upregulating µ-Opioid Receptors.

BACKGROUND: Morphine tolerance on long-term usage leads to chronic pain conditions. Preclinical studies demonstrated that the upregulation of HDACs is associated with a decrease in the sensitivity of µ-opioid receptors, which results in morphine tolerance. OBJECTIVE: The present study was designed to assess the influence of the selected known HDAC inhibitors (NMJ2 and NMJ3) on the pain tolerance induced by chronic administration of morphine in Balb/c mice. METHODS: In the present study, morphine was administered in incremental doses 1, 2, 3.6, 6.5, 11.2, 21, and 21 mg/kg daily for seven days to develop morphine tolerance. The nociceptive thresholds, analgesia, and tolerance were assessed 30 min after morphine administration alternatively from 1st day to 7th day using the hot plate and mechanical allodynia methods. RESULTS: The morphine control group showed a reduction in the Paw Withdrawal Threshold (PWT) and the percentage Maximum Possible Effects (MPEs). In contrast, the combination of SAHA and test drugs with morphine increased the PWT and MPEs as compared to the morphine alone group. Administration of morphine alone also showed an increase in the production of the pro-inflammatory mediator, IL-6, and down-regulation of the µ-opioid receptor in the brain tissues. Treatment with HDAC inhibitors, SAHA, and test drugs showed a reversal in these changes. CONCLUSION: Results indicated that HDAC inhibitors were involved in the prevention of morphine tolerance in normal mice by inhibiting pro-inflammatory marker production and by increasing the sensitivity of neurons towards morphine in producing an analgesic effect in morphine tolerated mice.

Potential of Nociceptin/Orphanin FQ Peptide Analogs for Drug Development.

Nociceptin receptor (NOP) belongs to the family of opioid receptors but was discovered and characterized much later than the so called classical opioid receptors, µ, δ and κ (or MOP, DOP and KOP, resp.). Nociceptin/orphanin FQ (N/OFQ) is the endogenous ligand of this receptor and it controls numerous important functions in the central nervous system and in the periphery, so its analogs may be developed as innovative drugs for the treatment of a variety of conditions and pathological states. Availability of potent and selective ligands with high affinity to NOP receptor is essential to fully understand the role of NOP-N/OFQ system in the body, which in turn may lead to designing novel therapeutics. Here, we have focused on reviewing the structure of potent peptide-based agonists, antagonists, biased analogs and bivalent ligands that target NOP receptor.

Methylnaltrexone crosses the blood-brain barrier and attenuates centrally-mediated behavioral effects of morphine and oxycodone in mice.

BACKGROUND: Antagonism of peripheral opioid receptors by methylnaltrexone (MNTX) was recently proposed as a potential mechanism to attenuate the development of opioid analgesic tolerance based on experiments conducted in mice. However, reports indicate that MNTX is demethylated to naltrexone (NTX) in mice, and NTX may subsequently cross the blood-brain barrier to antagonize centrally-mediated opioid effects. The goal of this study was to determine whether MNTX alters centrally-mediated behaviors elicited by the opioid analgesics, morphine and oxycodone, and to quantify concentrations of MNTX and NTX in blood and brain following their administration in mice. METHODS: Combinations of MNTX and morphine were tested under acute and chronic conditions in thermal nociceptive assays. Effects of MNTX and NTX pretreatment were assessed in an oxycodone discrimination operant procedure. Blood and brain concentrations of these antagonists were quantified after their administration using liquid chromatography-mass spectrometry. RESULTS: MNTX dose-dependently attenuated acute and chronic morphine antinociception. MNTX and NTX dose-dependently antagonized the discriminative stimulus effects of oxycodone. MNTX and NTX were detected in both blood and brain after administration of MNTX, confirming its demethylation and demonstrating that MNTX itself can cross the blood-brain barrier. CONCLUSIONS: These results provide converging behavioral and analytical evidence that MNTX administration in mice attenuates centrally-mediated effects produced by opioid analgesics and results in functional concentrations of MNTX and NTX in blood and brain. Collectively, these findings indicate that MNTX cannot be administered systemically in mice for making inferences that its effects are peripherally restricted.

Crosstalk between Opioid and Anti-Opioid Systems: An Overview and Its Possible Therapeutic Significance.

Opioid peptides and receptors are broadly expressed throughout peripheral and central nervous systems and have been the subject of intense long-term investigations. Such studies indicate that some endogenous neuropeptides, called anti-opioids, participate in a homeostatic system that tends to reduce the effects of endogenous and exogenous opioids. Anti-opioid properties have been attributed to various peptides, including melanocyte inhibiting factor (MIF)-related peptides, cholecystokinin (CCK), nociceptin/orphanin FQ (N/OFQ), and neuropeptide FF (NPFF). These peptides counteract some of the acute effects of opioids, and therefore, they are involved in the development of opioid tolerance and addiction. In this work, the anti-opioid profile of endogenous peptides was described, mainly taking into account their inhibitory influence on opioid-induced effects. However, the anti-opioid peptides demonstrated complex properties and could show opioid-like as well as anti-opioid effects. The aim of this review is to detail the phenomenon of crosstalk taking place between opioid and anti-opioid systems at the in vivo pharmacological level and to propose a cellular and molecular basis for these interactions. A better knowledge of these mechanisms has potential therapeutic interest for the control of opioid functions, notably for alleviating pain and/or for the treatment of opioid abuse.

Design, Synthesis and Functional Analysis of Cyclic Opioid Peptides with Dmt-Tic Pharmacophore.

The opioid receptors are members of the G-protein-coupled receptor (GPCR) family and are known to modulate a variety of biological functions, including pain perception. Despite considerable advances, the mechanisms by which opioid agonists and antagonists interact with their receptors and exert their effect are still not completely understood. In this report, six new hybrids of the Dmt-Tic pharmacophore and cyclic peptides, which were shown before to have a high affinity for the µ-opioid receptor (MOR) were synthesized and characterized pharmacologically in calcium mobilization functional assays. All obtained ligands turned out to be selective antagonists of the δ-opioid receptor (DOR) and did not activate or block the MOR. The three-dimensional structural determinants responsible for the DOR antagonist properties of these analogs were further investigated by docking studies. The results indicate that these compounds attach to the DOR in a slightly different orientation with respect to the Dmt-Tic pharmacophore than Dmt-TicΨ[CH2-NH]Phe-Phe-NH2 (DIPP-NH2[Ψ]), a prototypical DOR antagonist peptide. Key pharmacophoric contacts between the DOR and the ligands were maintained through an analogous spatial arrangement of pharmacophores, which could provide an explanation for the predicted high-affinity binding and the experimentally observed functional properties of the novel synthetic ligands.

Mannosylated glycoliposomes for the delivery of a peptide kappa opioid receptor antagonist to the brain.

Dynantin is a potent and selective synthetic polypeptide kappa opioid receptor antagonist which has potential antidepressant and anxiolytic-like therapeutic applications, however its clinical development has been hampered by plasma stability issues and poor penetration of the blood brain barrier. Targeted liposome delivery systems represent a promising and non-invasive approach to improving the delivery of therapeutic agents across the blood brain barrier. As part of our work focused on targeted drug delivery, we have developed a novel mannosylated liposome system. Herein, we investigate these glycoliposomes for the targeted delivery of dynantin to the central nervous system. Cholesterol was tested and optimized as a formulation excipient, where it improved particle stability as measured via particle size, entrapment and ex vivo plasma stability of dynantin. The in vitro PRESTO-TANGO assay system was used to confirm that glycoliposomal entrapment did not impact the affinity or activity of the peptide at its receptor. Finally, in vivo distribution studies in mice showed that the mannosylated glycoliposomes significantly improved delivery of dynantin to the brain. Overall, the results clearly demonstrate the potential of our glycoliposomes as a targeted delivery system for therapeutic agents of the central nervous system.

Synthesis and Structure-Activity Relationships of 5'-Aryl-14-alkoxypyridomorphinans: Identification of a µ Opioid Receptor Agonist/δ Opioid Receptor Antagonist Ligand with Systemic Antinociceptive Activity and Diminished Opioid Side Effects.

We previously identified a pyridomorphinan (6, SRI-22138) possessing a 4-chlorophenyl substituent at the 5'-position on the pyridine and a 3-phenylpropoxy at the 14-position of the morphinan as a mixed µ opioid receptor (MOR) agonist and δ/κ opioid receptor (DOR/KOR) antagonist with potent antinociceptive activity and diminished tolerance and dependence in rodents. Structural variations at the 5'- and 14-positions of this molecule gave insights into the structure-activity relationships for binding and functional activity. Subtle structural changes exerted significant influence, particularly on the ability of the compounds to function as agonists at the MOR. In vivo evaluation identified compound 20 (SRI-39067) as a MOR agonist/DOR antagonist that produced systemically active potent antinociceptive activity in tail-flick assay in mice, with diminished tolerance, dependence/withdrawal, reward liability, and respiratory depression versus morphine. These results support the hypothesis that mixed MOR agonist/DOR antagonist ligands may emerge as novel opioid analgesics with reduced side effects.

Design, synthesis and biological evaluation of N-hydroxy-aminobenzyloxyarylamide analogues as novel selective κ opioid receptor antagonists.

Aminobenzyloxyarylamide derivatives 1a-i and 2a-t were designed and synthesized as novel selective κ opioid receptor (KOR) antagonists. The benzoyl amide moiety of LY2456302 was changed into N-hydroxybenzamide and benzisoxazole-3(2H)-one to investigate whether it could increase the binding affinity or selectivity for KOR. All target compounds were evaluated in radioligand binding assays for opioid receptor binding affinity. These efforts led to the identification of compound 1c (κ Ki = 179.9 nM), which exhibited high affinity for KOR. Moreover, the selectivity of KOR over MOR and DOR increased nearly 2-fold and 7-fold, respectively, compared with (±)LY2456302.

Computational insights into the molecular mechanisms of differentiated allosteric modulation at the mu opioid receptor by structurally similar bitopic modulators.

Targeting the mu opioid receptor (MOR) by applying orthosteric ligands is the most frequently employed method to treat opioid use disorder (OUD). Unfortunately, most of MOR orthosteric ligands produce severe side effects, mainly due to their low selectivity over other opioid receptors. In contrast, some G protein-coupled receptor allosteric modulators have been reported to exhibit high subtype selectivity and can effectively modulate the potency and/or efficacy of orthosteric ligands. Recently, NAQ and its analog NCQ were identified as novel MOR bitopic modulators. Interestingly, NAQ and NCQ were similar in structure but exhibited different efficacy profiles to the MOR. NAQ exhibited an antagonism activity to the MOR while NCQ showed a partial agonism activity to the MOR. In the present study, molecular modeling methods were applied to explore the putative molecular mechanisms of their different functional profiles to the MOR. When NAQ binding with the inactive MOR, the 'address' portion of NAQ interacted with the MOR allosteric site but showed no significant allosteric modulation of the efficacy of the 'message' portion of NAQ. However, when NCQ binding with the inactive and active MOR, the 'address' portion of NCQ seemed to be able to positively modulate the efficacy of the 'message' portion of NCQ at varying levels. Evidentially, the substituents at the 1'- and 4'-positions of the isoquinoline ring of NCQ seemed to play a critical role in the modulatory function of the 'address' portion of NCQ. These findings will be invaluable to develop our next generation of MOR bitopic modulators with high affinity and subtype selectivity to potentially treat OUD.

Naltrexone and Its Noroxymorphone Minor Metabolite - A Case Report.

As part of substance use maintenance programs, individuals are monitored for sobriety through urine drug screens. A positive screen, and its confirmation and interpretation, can have devastating consequences, sometimes even leading to termination from the program and relapse. Naltrexone metabolism involves several steps and metabolites - one minor metabolite with very little mention in medical literature being noroxymorphone. This is also the final intermediate in the metabolic pathway of oxycodone; hence, detection is naturally presumed by clinicians to be attributed to oxycodone use. Through this case report, we alert clinicians that, depending on individual pharmacogenomics, it is possible to obtain a positive confirmation of this component alone (without any oxycodone pathway intermediates) with naltrexone administration.

Noradrenergic Mechanisms in Fentanyl-Mediated Rapid Death Explain Failure of Naloxone in the Opioid Crisis.

In December 2018, the Centers for Disease Control declared fentanyl the deadliest drug in America. Opioid overdose is the single greatest cause of death in the United States adult population (ages 18-50), and fentanyl and its analogs [fentanyl/fentanyl analogs (F/FAs)] are currently involved in >50% of these deaths. Anesthesiologists in the United States were introduced to fentanyl in the early 1970s when it revolutionized surgical anesthesia by combining profound analgesia with hemodynamic stability. However, they quickly had to master its unique side effect. F/FAs can produce profound rigidity in the diaphragm, chest wall and upper airway within an extremely narrow dosing range. This clinical effect was called wooden chest syndrome (WCS) by anesthesiologists and is not commonly known outside of anesthesiology or to clinicians or researchers in addiction research/medicine. WCS is almost routinely fatal without expert airway management. This review provides relevant clinical human pharmacology and animal data demonstrating that the significant increase in the number of F/FA-induced deaths may involve α-adrenergic and cholinergic receptor-mediated mechanical failure of the respiratory and cardiovascular systems with rapid development of rigidity and airway closure. Although morphine and its prodrug, heroin, can cause mild rigidity in abdominal muscles at high doses, neither presents with the distinct and rapid respiratory failure seen with F/FA-induced WCS, separating F/FA overdose from the slower onset of respiratory depression caused by morphine-derived alkaloids. This distinction has significant consequences for the design and implementation of new pharmacologic strategies to effectively prevent F/FA-induced death. SIGNIFICANCE STATEMENT: Deaths from fentanyl and F/FAs are increasing in spite of availability and awareness of the opioid reversal drug naloxone. This article reviews literature suggesting that naloxone may be ineffective against centrally mediated noradrenergic and cholinergic effects of F/FAs, which clinically manifest as severe muscle rigidity and airway compromise (e.g., wooden chest syndrome) that is rapid and distinct from respiratory depression seen with morphine-derived alkaloids. A physiologic model is proposed and implications for new drug development and treatment are discussed.

Attenuation of antidepressant and antisuicidal effects of ketamine by opioid receptor antagonism.

We recently reported that naltrexone blocks antidepressant effects of ketamine in humans, indicating that antidepressant effects of ketamine require opioid receptor activation. However, it is unknown if opioid receptors are also involved in ketamine's antisuicidality effects. Here, in a secondary analysis of our recent clinical trial, we test whether naltrexone attenuates antisuicidality effects of ketamine. Participants were pretreated with naltrexone or placebo prior to intravenous ketamine in a double-blinded crossover design. Suicidality was measured with the Hamilton Depression Rating Scale item 3, Montgomery-Åsberg Depression Rating Scale item 10, and Columbia Suicide Severity Rating Scale. In the 12 participants who completed naltrexone and placebo conditions, naltrexone attenuated the antisuicidality effects of ketamine on all three suicidality scales/subscales (linear mixed model, fixed pretreatment effect, p < 0.01). Results indicate that opioid receptor activation plays a significant role in the antisuicidality effects of ketamine.

Methocinnamox Produces Long-Lasting Antagonism of the Behavioral Effects of µ-Opioid Receptor Agonists but Not Prolonged Precipitated Withdrawal in Rats.

A novel µ-opioid receptor antagonist, methocinnamox (MCAM), attenuates some abuse-related and toxic effects of opioids. This study further characterized the pharmacology of MCAM in separate groups of rats using procedures to examine antinociception, gastrointestinal motility, and withdrawal in morphine-dependent rats. Antinociceptive effects of opioid receptor agonists were measured before and after MCAM (1-10 mg/kg) using warm water tail withdrawal and sensitivity to mechanical stimulation in inflamed paws (complete Freund's adjuvant). Before MCAM, morphine, fentanyl, and the κ-opioid receptor agonist spiradoline dose dependently increased tail-withdrawal latency from 50°C water; MCAM attenuated the antinociceptive effects of morphine and fentanyl, but not spiradoline. Morphine increased sensitivity to mechanical stimulation and decreased gastrointestinal motility, and MCAM blocked both effects. These antagonist effects of 10 mg/kg MCAM were persistent, lasting for 2 weeks or longer. Withdrawal emerged after discontinuation of morphine treatment or administration of 10 mg/kg MCAM or 17.8 mg/kg naloxone; other than the day of antagonist administration when withdrawal signs were greater in rats that received antagonist compared with rats that received vehicle, there was no difference among groups in directly observable withdrawal signs or decreased body weight. These results confirm that MCAM is a selective µ-opioid receptor antagonist with an exceptionally long duration of action, likely due to pseudoirreversible binding. Despite its sustained antagonist effects, the duration of withdrawal precipitated by MCAM is not different from that precipitated by naloxone, suggesting that the long duration of antagonism provided by MCAM could be particularly effective for treating opioid abuse and overdose. SIGNIFICANCE STATEMENT: The opioid receptor antagonist MCAM attenuates some abuse-related and toxic effects of opioids. This study demonstrates that MCAM selectively antagonizes multiple effects mediated by µ-opioid receptor agonists for 2 weeks or longer, and like naloxone, MCAM precipitates withdrawal in morphine-dependent rats. Despite this persistent antagonism, withdrawal signs precipitated by MCAM are not significantly different from signs precipitated by naloxone or occurring after discontinuation of morphine, suggesting that using MCAM for opioid abuse or overdose would not produce sustained withdrawal.

Non-Opioid Neurotransmitter Systems that Contribute to the Opioid Withdrawal Syndrome: A Review of Preclinical and Human Evidence.

Opioid misuse and abuse is a major international public health issue. Opioid use disorder (OUD) is largely maintained by a desire to suppress aversive opioid withdrawal symptoms. Opioid withdrawal in patients seeking abstinence from illicit or prescribed opioids is often managed by provision of a µ-opioid agonist/partial agonist in combination with concomitant medications. Concomitant medications are administered based on their ability to treat specific symptoms rather than a mechanistic understanding of the opioid withdrawal syndrome; however, their use has not been statistically associated with improved treatment outcomes. Understanding the central and/or peripheral mechanisms that underlie individual withdrawal symptom expression in humans will help promote medication development for opioid withdrawal management. To support focused examination of mechanistically supported concomitant medications, this review summarizes evidence from preclinical (N = 68) and human (N = 30) studies that administered drugs acting on the dopamine, serotonin, cannabinoid, orexin/hypocretin, and glutamate systems and reported outcomes related to opioid withdrawal. These studies provide evidence that each of these systems contribute to opioid withdrawal severity. The Food and Drug Administration has approved medications acting on these respective systems for other indications and research in this area could support the repurposing of these medications to enhance opioid withdrawal treatment. These data support a focused examination of mechanistically informed concomitant medications to help reduce opioid withdrawal severity and enhance the continuum of care available for persons with OUD.

Urine is superior to oral fluid for detecting buprenorphine compliance in patients undergoing treatment for opioid addiction.

BACKGROUND: Buprenorphine (BUP) is commonly used in opioid agonist medication-assisted treatment (OA-MAT). Oral fluid (OF) is an attractive option for compliance monitoring during OA-MAT as collections are observed and evidence suggests that OF is less likely to be adulterated than urine (UR). However, the clinical and analytical performance of each matrix for monitoring BUP compliance has not been well studied. METHODS: We collected 260 paired OF and UR specimens. Concentrations of buprenorphine (BUP) and norbuprenorphine (NBUP) were measured by liquid chromatography-tandem mass spectrometry (LC-MS/MS) in each matrix. The glucuronide metabolites and naloxone concentrations were also measured in UR by LC-MS/MS. Medications were reviewed and UR creatinine concentrations were determined. RESULTS: 147/260 specimens (57%) were positive for BUP and/or metabolites in one or both matrices. BUP and/or metabolites were more likely to be detected in UR (p < 0.001). 1 OF specimen and 12 UR specimens were considered adulterated/substituted. The majority of patients prescribed BUP were positive for BUP in UR (97%) as opposed to OF (78%). The detection of undisclosed use approximately doubled in UR. CONCLUSIONS: UR is the preferred matrix for detecting both buprenorphine compliance and undisclosed use. Clinicians should consider the ease of collection, risk of adulteration and detection of illicit drug use when deciding on an appropriate matrix. If OF testing is clinically necessary, UR should be measured in conjunction with OF at least periodically to avoid false negative BUP results.

Study on structural insight of the analysis of negative effects of opioids analgesics in naltrexone with TLR4 Mutations.

Chronic pain has been defined as the persistence that remained for more than three months. The extent of previous time duration with the normal time of natural healing phase becomes poor and results in reduced life quality and morbidity. Opioids are well recognized therapy for pain management and the clinical prescriptions based on opioids have been defined with increasing implicating behavior among patients suffering with chronic pain. The association between the pain and immunity has long been established since the involvement of interleukin-1ß (IL-1ß) in sickness that is considered with the induced hyperalgesia. In the context of pharmacodynamics Toll like receptors (TLRs) are involved in the negative effects of opioids as analgesics. The soluble factors released by immune cells as well as from the disruptive cells bind to TLRs. This binding leads the pre and post-synaptic ends on endothelial and microglial cells that exhibit the activation of complex inhibitory and excitatory process at the synapses site. In TLRs, TLR4 is mostly reported that is strongly associated in specifically in areas of T cells and macrophages. The current study is designed to investigate the structural insights of the opioids and TLR4 interactions by using computational approach in the aspect of recognizing the chemical combinatorial factors that are involved in the pain management. This study targets that how opioids interact with TLR4 and the process of chemical interaction that leads to negative effects of opioids at neuroimmune interface as well as to investigate the extent of particular naltrexone that mediates with the negative effects of opioids.