CYP2D in the brain impacts oral hydrocodone analgesia in vivo.

Cytochrome P450 2D (CYP2D) metabolises many centrally-acting substrates including opioids. Hydrocodone, an opioid and CYP2D substrate, is metabolised to hydromorphone, an active metabolite. CYP2D in the brain is active in vivo and can alter drug response however, it is unknown whether metabolism by CYP2D in the brain alters oral hydrocodone induced analgesia. Propranolol, a selective CYP2D mechanism-based inhibitor, or vehicle, was administered into the right cerebral ventricle of male rats, (HAN Wistars, Envigo), 24 h before testing for analgesia from oral hydrocodone (or hydromorphone, a non-CYP2D substrate). Hydrocodone and its CYP2D-mediated metabolites were simultaneously quantified using a novel LC-MS/MS assay. After propranolol vs vehicle pretreatment, there was significantly higher analgesia from oral hydrocodone, and a significantly lower brain CYP2D metabolic ratio (an in vivo phenotype of brain CYP2D activity that was derived from the molar sum of hydromorphone and its metabolites divided by hydrocodone). The brain CYP2D metabolic ratio correlated significantly with analgesia. There was no pretreatment effect on plasma hydrocodone concentrations, elimination rates, or metabolic ratio (an in vivo phenotype for hepatic CYP2D activity). The liver CYP2D metabolic ratio did not correlate with analgesia. Propranolol pretreatment had no impact on analgesia from oral hydromorphone. These data suggest that inhibited CYP2D activity in brain, causing reduced metabolism of brain hydrocodone, resulted in higher analgesia from oral hydrocodone, despite hydrocodone having a lower µ-opioid receptor affinity than hydromorphone. Thus, variation in CYP2D in the brain may be an important source of interindividual differences in response to CYP2D substrates, including oral hydrocodone.

Quantification of Opioids in Urine Using an Aptamer-Based Free-Solution Assay.

The opioid epidemic continues in the United States. Many have been impacted by this epidemic, including neonates who exhibit Neonatal Abstinence Syndrome (NAS). Opioid diagnosis and NAS can be negatively impacted by limited testing options outside the hospital, due to poor assay performance, false-negatives, rapid drug clearance rates, and difficulty in obtaining enough specimen for testing. Here we report a small volume urine assay for oxycodone, hydrocodone, fentanyl, noroxycodone, norhydrocodone, and norfentanyl with excellent LODs and LOQs. The free-solution assay (FSA), coupled with high affinity DNA aptamer probes and a compensated interferometric reader (CIR), represents a potential solution for quantifying opioids rapidly, at high sensitivity, and noninvasively on small sample volumes. The mix-and-read test is 5- to 275-fold and 50- to 1250-fold more sensitive than LC-MS/MS and immunoassays, respectively. Using FSA, oxycodone, hydrocodone, fentanyl, and their urinary metabolites were quantified using 10 µL of urine at 28-81 pg/mL, with >95% specificity and excellent accuracy in ∼1 h. The assay sensitivity, small sample size requirement, and speed could enable opioid screening, particularly for neonates, and points to the potential for pharmacokinetic tracking.

Neopinone isomerase is involved in codeine and morphine biosynthesis in opium poppy.

The isomerization of neopinone to codeinone is a critical step in the biosynthesis of opiate alkaloids in opium poppy. Previously assumed to be spontaneous, the process is in fact catalyzed enzymatically by neopinone isomerase (NISO). Without NISO the primary metabolic products in the plant, in engineered microbes and in vitro are neopine and neomorphine, which are structural isomers of codeine and morphine, respectively. Inclusion of NISO in yeast strains engineered to convert thebaine to natural or semisynthetic opiates dramatically enhances formation of the desired products at the expense of neopine and neomorphine accumulation. Along with thebaine synthase, NISO is the second member of the pathogenesis-related 10 (PR10) protein family recently implicated in the enzymatic catalysis of a presumed spontaneous conversion in morphine biosynthesis.

Reversal of oxycodone and hydrocodone tolerance by diazepam.

The Centers for Disease Control has declared opioid abuse to be an epidemic. Overdose deaths are largely assumed to be the result of excessive opioid consumption. In many of these cases, however, opioid abusers are often polydrug abusers. Benzodiazepines are one of the most commonly co-abused substances and pose a significant risk to opioid users. In 2016, the FDA required boxed warnings - the FDA's strongest warning - for prescription opioid analgesics and benzodiazepines about the serious risks associated with using these medications at the same time. The point of our studies was to evaluate the interactions between these two classes of drugs. We investigated whether diazepam adds to the depressant effects of opioids or do they alter the levels of tolerance to opioids. In the present study, we have found that the antinociceptive tolerance that developed to repeated administration of oxycodone was reversed by an acute dose of diazepam. Antinociceptive tolerance to hydrocodone was also reversed by acute injection of diazepam; however, a fourfold higher dose of diazepam was required when compared to reversal of oxycodone-induced tolerance. These doses of diazepam did not potentiate the acute antinociceptive effect of either opioid. The same dose of diazepam that reversed oxycodone antinociceptive tolerance also reversed oxycodone locomotor tolerance while having no potentiating effects. These studies show that diazepam does not potentiate the acute effect of prescription opioids but reverses the tolerance developed after chronic administration of the drugs.

Detection of Hydrocodone and Morphine as Metabolites in Oral Fluid by LC-MS/MS in Patients Prescribed Codeine.

A retrospective analysis of oral fluid drug testing results using LC-MS/MS was performed to determine the prevalence rates in oral fluid for codeine (COD) and 3 COD metabolites-morphine (MOR), norhydrocodone (NHC), and hydrocodone (HCOD). Oral fluid samples were collected using Quantisal oral fluid collection device (Immunalysis Inc.) and submitted to Millennium Health, LLC for the routine drug analysis by LC-MS/MS. Consistent with previously published literature, COD was the primary analyte detected in oral fluid after the use of COD. In COD-positive samples, HCOD, MOR, and NHC were detected at rates of 68.4%, 18.4%, and 6.3%, respectively. Concentration ranges of these analytes were 1.0 to >2000 ng/mL for COD, 1.0-20.2 ng/mL for MOR, 1.0-740.0 ng/mL for HCOD, and 2.1-47.5 ng/mL for NHC. In contrast to urine, where HCOD is typically detected as a minor metabolite of COD, HCOD was the most commonly detected metabolite in oral fluid in samples testing positive for COD with reported prescriptions for COD. This observation suggests that care should be taken when interpreting HCOD positives in oral fluid results, and that the use of COD should be considered as one possible explanation for HCOD positives.

Normalizing Oral Fluid Hydrocodone Data Using Calculated Blood Volume.

Oral fluid testing to assist in the assessment of treatment adherence for chronic pain patients is attractive for a number of reasons. However, efforts focused on interpreting patient results have been modest when compared to urine drug testing. This work details a retrospective approach developed to transform and normalize oral fluid testing results to provide a historical picture of patient values in this important test fluid. Using this approach, a model was developed using data from 6,800 independent patients who were both prescribed hydrocodone and tested positive (with limitations: reporting cutoff < X < upper limit of quantitation) by liquid chromatography-mass spectrometry. Patient demographic data were used to calculate the relevant parameters (e.g., calculated blood volume (CBV)) used in the transformation and normalization of the oral fluid data. The crucial normalizing factor in oral fluids was found to be the CBV which parallels the use of creatinine to normalize drug concentration levels in urine and is consistent with the view that oral fluid samples reflect plasma concentrations of the respective drugs. The resulting near Gaussian distribution is dose independent and as such should be of value to physicians in quickly assessing whether their patient is consistent with this historical population in the broad terms of this model. While this comparison alone is not definitive for adherence with a treatment regimen, together with patient interviews, prescription history and other clinical criteria, it can add an idea of expected patient values from oral fluid testing.

Gender difference in prescription opioid abuse: A focus on oxycodone and hydrocodone.

Several data gathered in the last decade indicate an increase of abuse of prescription opioid drugs oxycodone (OXY) and hydrocodone (HYDRO) in women. However, to date there are no conclusive evidences investigating the gender-dependent abuse liability of prescription opioids. This study aims to supply a specific focus on women's data through a selective summary of the literature analyzing gender differences in the pharmacokinetic and pharmacodynamic dimension of OXY and HYDRO. Findings from this study suggest that the majority of OXY and HYDRO pharmacokinetic and pharmacodynamic effects do not differ according to gender, though confirming a significant difference in the incidence of adverse effects as demonstrated by the increased gastrointestinal adverse reactions in female subjects. Although the majority of recent clinical studies include an equal number of female and male subjects, the main outcome parameters do not relate specifically to gender differences. Due to the gender influence in activity of CYP3A4 and its crucial role in metabolism of both OXY than HYDRO, we suggest that assessing pharmacokinetic and pharmacodynamic interactions in clinical studies may be useful to clarify the effect of the higher CYP3A4 activity in female in relation to CYP2D6 genotype. Overall, considering the paucity of data regarding gender differences in European Union, this work highlights that impact of new abuse deterrent formulations should be assessed with a special focus on data concerning female subjects.

Total biosynthesis of opiates by stepwise fermentation using engineered Escherichia coli.

Opiates such as morphine and codeine are mainly obtained by extraction from opium poppies. Fermentative opiate production in microbes has also been investigated, and complete biosynthesis of opiates from a simple carbon source has recently been accomplished in yeast. Here we demonstrate that Escherichia coli serves as an efficient, robust and flexible platform for total opiate synthesis. Thebaine, the most important raw material in opioid preparations, is produced by stepwise culture of four engineered strains at yields of 2.1 mg l(-1) from glycerol, corresponding to a 300-fold increase from recently developed yeast systems. This improvement is presumably due to strong activity of enzymes related to thebaine synthesis from (R)-reticuline in E. coli. Furthermore, by adding two genes to the thebaine production system, we demonstrate the biosynthesis of hydrocodone, a clinically important opioid. Improvements in opiate production in this E. coli system represent a major step towards the development of alternative opiate production systems.

Complete biosynthesis of opioids in yeast.

Opioids are the primary drugs used in Western medicine for pain management and palliative care. Farming of opium poppies remains the sole source of these essential medicines, despite diverse market demands and uncertainty in crop yields due to weather, climate change, and pests. We engineered yeast to produce the selected opioid compounds thebaine and hydrocodone starting from sugar. All work was conducted in a laboratory that is permitted and secured for work with controlled substances. We combined enzyme discovery, enzyme engineering, and pathway and strain optimization to realize full opiate biosynthesis in yeast. The resulting opioid biosynthesis strains required the expression of 21 (thebaine) and 23 (hydrocodone) enzyme activities from plants, mammals, bacteria, and yeast itself. This is a proof of principle, and major hurdles remain before optimization and scale-up could be achieved. Open discussions of options for governing this technology are also needed in order to responsibly realize alternative supplies for these medically relevant compounds.

The effect of CYP2D6 drug-drug interactions on hydrocodone effectiveness.

OBJECTIVES: The hepatic cytochrome 2D6 (CYP2D6) is a saturable enzyme responsible for metabolism of approximately 25% of known pharmaceuticals. CYP interactions can alter the efficacy of prescribed medications. Hydrocodone is largely dependent on CYP2D6 metabolism for analgesia, ondansetron is inactivated by CYP2D6, and oxycodone analgesia is largely independent of CYP2D6. The objective was to determine if CYP2D6 medication coingestion decreases the effectiveness of hydrocodone. METHODS: This was a prospective observational study conducted in an academic U.S. emergency department (ED). Subjects were included if they had self-reported pain or nausea and were excluded if they were unable to speak English, were less than 18 years of age, had liver or renal failure, or carried diagnoses of chronic pain or cyclic vomiting. Detailed drug ingestion histories for the preceding 48 hours prior to the ED visit were obtained. The patient's pain and nausea were quantified using a 100-mm visual analog scale (VAS) at baseline prior to drug administration and following doses of hydrocodone, oxycodone, or ondansetron. We used a mixed model with random subject effect to determine the interaction between CYP2D6 drug ingestion and study drug effectiveness. Odds ratios (ORs) were calculated to compare clinically significant VAS changes between CYP2D6 users and nonusers. RESULTS: A total of 250 (49.8%) of the 502 subjects enrolled had taken at least one CYP2D6 substrate, inhibitor, or inducing pharmaceutical, supplement, or illicit drug in the 48 hours prior to ED presentation. CYP2D6 drug users were one-third as likely to respond to hydrocodone (OR = 0.33, 95% confidence interval [CI] = 0.1 to 0.8) and more than three times as likely as nonusers to respond to ondansetron (OR = 3.4, 95% CI = 1.3 to 9.1). There was no significant difference in oxycodone effectiveness between CYP2D6 users and nonusers (OR = 0.53, 95% CI = 0.3 to 1.1). CONCLUSIONS: CYP2D6 drug-drug interactions appear to change effectiveness of commonly prescribed drugs in the ED. Drug-drug interaction should be considered prior to prescribing CYP2D6 drugs.

A microbial biomanufacturing platform for natural and semisynthetic opioids.

Opiates and related molecules are medically essential, but their production via field cultivation of opium poppy Papaver somniferum leads to supply inefficiencies and insecurity. As an alternative production strategy, we developed baker's yeast Saccharomyces cerevisiae as a microbial host for the transformation of opiates. Yeast strains engineered to express heterologous genes from P. somniferum and bacterium Pseudomonas putida M10 convert thebaine to codeine, morphine, hydromorphone, hydrocodone and oxycodone. We discovered a new biosynthetic branch to neopine and neomorphine, which diverted pathway flux from morphine and other target products. We optimized strain titer and specificity by titrating gene copy number, enhancing cosubstrate supply, applying a spatial engineering strategy and performing high-density fermentation, which resulted in total opioid titers up to 131 mg/l. This work is an important step toward total biosynthesis of valuable benzylisoquinoline alkaloid drug molecules and demonstrates the potential for developing a sustainable and secure yeast biomanufacturing platform for opioids.

Observations on hydrocodone and its metabolites in oral fluid specimens of the pain population: comparison with urine.

OBJECTIVE: Hydrocodone undergoes metabolism via cytochrome P450 (CYP) 3A4 (N-demethylation) to norhydrocodone and via CYP2D6 (O-demethylation) to hydromorphone. Hydrocodone, hydromorphone, and norhydrocodone are excreted in urine and secreted in saliva. The goal was to characterize hydrocodone and its metabolites in oral fluid specimens of a pain population and compare to urine specimens. DESIGN: This retrospective analysis included more than 8,500 oral fluid specimens and more than 250,000 urine specimens collected between March and June 2012 that were sent to Millennium Laboratories (San Diego, CA) and analyzed for hydrocodone, hydromorphone, and norhydrocodone using liquid chromatography-tandem mass spectrometry. Statistical analyses and linear regressions were conducted using Microsoft Excel® 2010 and OriginPro v8.6. RESULTS: The median oral fluid concentrations of hydrocodone and norhydrocodone were 122 and 7.7 ng/mL, respectively. However, the oral fluid concentrations of hydromorphone were below detection in many specimens (<1 ng/mL). The positive detection rate of parent drug and metabolites in oral fluid (17-31 percent detection rates) was much lower than in urine (63-75 percent detection rates). The geometric median metabolic ratio (MR) of norhydrocodone to hydrocodone was 0.07 in oral fluid and 1.2 in urine. The observed hydrocodone oral fluid concentrations were approximately 10-fold greater than previously reported plasma concentrations. CONCLUSION: Oral fluid had a much lower norhydrocodone to hydrocodone MR compared to urine. Reference ranges for oral fluid drug concentrations should not be extrapolated from plasma ranges. The observed ranges of secreted hydrocodone and metabolite concentrations in oral fluid should help determine reference ranges for medication monitoring.

Urinary hydrocodone and metabolite distributions in pain patients.

Hydrocodone combined with acetaminophen is commonly used for moderate pain. Hydrocodone is metabolized by cytochrome P450 (CYP) 2D6 into hydromorphone and by CYP3A4 into norhydrocodone. This was a retrospective study evaluating hydrocodone, hydromorphone and norhydrocodone distributions in urine. Urine specimens (n = 76,924) were obtained from patients on chronic opioid therapy during their first or single visit and were analyzed by liquid chromatography-tandem mass spectrometry (LC-MS-MS). The patients were at least 16 years of age and had documented hydrocodone use via a medication list. There were 48,710 specimens that were positive for all three analytes. Mean hydrocodone, hydromorphone and norhydrocodone mole fractions (95% confidence interval) were 0.39 (0.38-0.39), 0.12 (0.11-0.12) and 0.49 (0.48-0.49), respectively. Hydromorphone fractions were lower in women compared with men (0.11 versus 0.13; P < 0.0001). Hydrocodone mole fractions were higher in the 65-year and older age group compared with the 16- to 39-year age group (0.4 versus 0.36; P ≤ 0.005). Concurrent use of a CYP2D6 and/or CYP3A4 inhibitor altered hydromorphone and norhydrocodone mole fractions, compared with the control group. Patient factors affect hydrocodone and metabolite mole fractions and suggest increased awareness of their contribution when attempting to interpret urine drug testing results.

Hydrocodone in postoperative personalized pain management: pro-drug or drug?

BACKGROUND: Genetic variations in enzymes that produce active metabolites from pro-drugs are well known. Such variability could account for some of the clinically observed differences in analgesia and side effects seen in postoperative patients. Using genotyping and quantitation of serum concentrations of hydrocodone and its metabolites, we sought to demonstrate the clinical effects of the metabolites of hydrocodone on pain relief. The objective of the current study was to determine whether CYP2D6 genotype and serum hydromorphone levels account for some of the variability in pain relief seen with hydrocodone in a cohort of women post-Cesarean section. METHODS: In 156 post-Cesarean section patients who received hydrocodone, we assessed serum opioid concentrations and CYP2D6 genotypes. Blood samples were collected at that time for genotyping and determination of concentrations of hydrocodone and metabolites by LC-MS/MS. Multivariate analysis was used to determine the relationship between CYP2D6 genotypes, pain relief, side effects, and serum concentrations of hydrocodone and hydromorphone. RESULTS: The CYP2D6 genotyping results indicated that 60% of subjects were extensive, 30% intermediate, 3% poor, and 7% ultra-rapid metabolizers. In the poor metabolizers, the mean plasma hydromorphone concentration was 8-fold lower when compared to that of ultra-rapid metabolizers. Hydromorphone, and not hydrocodone concentrations correlated with pain relief. CONCLUSIONS: This study shows that hydromorphone is generated at substantially different rates, dependent on CYP2D6 genotype. Pain relief correlated with plasma concentrations of hydromorphone, and not with hydrocodone. This suggests that pain relief will vary with CYP2D6 genotype. Inability to metabolize hydrocodone to hydromorphone as seen in the poor metabolizers should alert the clinician to consider alternative medications for managing pain postoperatively.

In vivo activity of norhydrocodone: an active metabolite of hydrocodone.

Hydrocodone is primarily metabolized to hydromorphone and norhydrocodone. Although hydromorphone is a known active metabolite of hydrocodone, the in vivo activity of norhydrocodone is not well documented. In the current study, the pharmacodynamics of norhydrocodone were evaluated and compared with hydrocodone and hydromorphone. Binding studies established that norhydrocodone, similar to hydrocodone and hydromorphone, is a µ-selective opioid ligand. In vivo analgesia studies (tail flick) demonstrated that, following subcutaneous, intrathecal, and intracerebroventricular administration, norhydrocodone produced analgesia. Following subcutaneous administration, norhydrocodone was ∼70-fold less potent, and hydromorphone was ∼5.4-fold more potent than hydrocodone in producing analgesia. Following intrathecal administration, norhydrocodone produced a shallow analgesia dose-response curve and maximal effect of 15-45%, whereas hydrocodone and hydromorphone produced dose-dependent analgesia. Intrathecal hydromorphone was ∼174-fold more potent than intrathecal hydrocodone. Following intracerebroventricular administration, norhydrocodone had similar potency to hydrocodone in producing analgesia, while hydromorphone was ∼96-fold more potent than hydrocodone. Analgesia induced by the three drugs following subcutaneous, intrathecal, and intracerebroventricular administration was antagonized by subcutaneous naltrexone, confirming that it is opioid receptor-mediated. Subcutaneous norhydrocodone-induced analgesia was completely blocked by intracerebroventricular naltrexone, indicating that norhydrocodone-induced analgesia is likely a supraspinal effect. Seizure activity was observed following intrathecal administration of all three drugs. Norhydrocodone and hydromorphone were ∼3.7 to 4.6-fold more potent than hydrocodone in inducing seizure activity. Naltrexone did not antagonize opioid-induced seizure activity, suggesting that seizures were not opioid receptor-mediated. Taken together, norhydrocodone is an active metabolite of hydrocodone and may contribute to therapeutic and toxic effects following hydrocodone administration.

Pharmacokinetics of hydrocodone and hydromorphone after oral hydrocodone in healthy Greyhound dogs.

The purpose of this study was to determine the pharmacokinetics of hydrocodone and its active metabolite hydromorphone in six healthy Greyhound dogs. Hydrocodone bitartrate was administered at a targeted dose of 0.5 mg/kg PO. Plasma concentrations of hydrocodone and hydromorphone were determined by liquid chromatography triple quadrupole mass spectrometry. The mean hydrocodone CMAX was 11.73 ng/mL at 0.74 h with a terminal half-life of 1.60 h. The mean hydromorphone CMAX was 5.2 ng/mL at 1.37 h with a terminal half-life of 3.07 h. Mean plasma hydromorphone concentrations exceeded 2 ng/mL from 0.5 to 8 h after hydrocodone administration. Further studies assessing the antinociceptive effects of oral hydrocodone are needed.

Update on hydrocodone metabolites in rats and dogs aided with a semi-automatic software for metabolite identification Mass-MetaSite.

1. There has been a lack of in vivo metabolite profiling update of hydrocodone since the original report on species differences was published in 1978. As such, the mechanism for its analgesic activity in different species has been ambiguous. To address safety concern from regulatory agencies, hydrocodone metabolite profiles in rats and dogs are updated herein aided by a newly developed software, Mass-MetaSite. 2. Samples collected from rats and dogs dosed orally with hydrocodone were analyzed with reversed phase liquid chromatography coupled with LTQ-Orbitrap. The exact mass measurement data collected with data-dependent acquisition methodology were analyzed both traditionally, using Xcalibur Qual Browser and MetWorks, and by Mass-MetaSite. 3. Profiling of hydrocodone metabolites in rat and dog plasma reflected previously reported species differences in circulating metabolites. While hydrocodone mainly underwent O-demethylation and ketone reduction in rats forming hydromorphone and reduced hydromorphone, which were then subsequently cleared via glucuronide conjugation, hydrocodone in dogs was cleared predominantly by N-demethylation and N-oxidation. 4. Given the success ratio of metabolite detection offered by Mass-MetaSite, the software will be able to aid chemists in early identification of drug metabolites from complex biomatrices.