In silico drug repurposing against SARS-CoV-2 using an integrative transcriptomic profiling approach: Hydrocortisone and Benzhydrocodone as potential drug candidates against COVID-19.

COVID-19 pathogenesis is mainly attributed to dysregulated antiviral immune response, the prominent hallmark of COVID-19. As no established drugs are available against SARS-CoV-2 and developing new ones would be a big challenge, repurposing of existing drugs holds promise against COVID-19. Here, we used a signature-based strategy to delve into cellular responses to SARS-CoV-2 infection in order to identify potential host contributors in COVID-19 pathogenesis and to find repurposable drugs using in silico approaches. We scrutinized transcriptomic profile of various human alveolar cell sources infected with SARS-CoV-2 to determine up-regulated genes specific to COVID-19. Enrichment analysis revealed that the up-regulated genes were involved mainly in viral infectious disease, immune system, and signal transduction pathways. Analysis of protein-protein interaction network and COVID-19 molecular pathway resulted in identifying several anti-viral proteins as well as 11 host pro-viral proteins, ADAR, HBEGF, MMP9, USP18, JUN, FOS, IRF2, ICAM1, IFI35, CASP1, and STAT3. Finally, molecular docking of up-regulated proteins and all FDA-approved drugs revealed that both Hydrocortisone and Benzhydrocodone possess high binding affinity for all pro-viral proteins. The suggested repurposed drugs should be subject to complementary in vitro and in vivo experiments in order to be evaluated in detail prior to clinical studies in potential management of COVID-19.

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.

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.

Prescription Opioids. IV: Disposition of Hydrocodone in Oral Fluid and Blood Following Single-Dose Administration.

The Substance Abuse and Mental Health Services Administration (SAMHSA) is currently evaluating hydrocodone (HC) for inclusion in the Mandatory Guidelines for Federal Workplace Drug Testing Programs. This study evaluated the time course of HC, norhydrocodone (NHC), dihydrocodeine (DHC) and hydromorphone (HM) in paired oral fluid and whole blood specimens by liquid chromatography-tandem mass spectrometry (limit of quantitation = 1 ng/mL of oral fluid, 5 ng/mL of blood) over a 52-h period. A single dose of HC bitartrate, 20 mg, was administered to 12 subjects. Analyte prevalence was as follows: oral fluid, HC > NHC > DHC; and blood, HC > NHC. HM was not detected in any specimen. HC was frequently detected within 15 min in oral fluid and 30 min in blood. Mean oral fluid to blood (OF : BL) ratios and correlations were 3.2 for HC (r = 0.73) and 0.7 for NHC (r = 0.42). The period of detection for oral fluid exceeded blood at all evaluated thresholds. At a 1-ng/mL threshold for oral fluid, mean detection time was 30 h for HC and 18 h for NHC and DHC. This description of HC and metabolite disposition in oral fluid following single-dose administration provides valuable interpretive guidance of HC test results.

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.

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.

Quantitative method for analysis of hydrocodone, hydromorphone and norhydrocodone in human plasma by liquid chromatography-tandem mass spectrometry.

A selective, sensitive and accurate high-performance liquid chromatography-tandem mass spectrometry (LC-MS-MS) method for the quantitation of hydrocodone, hydromorphone and norhydrocodone in human plasma was developed. The internal standard stock solution comprised of hydrocodone-d6, hydromorphone-d6 and norhydrocodone-d3 was added to 0.5 mL plasma samples. Samples were extracted using a copolymeric sorbent (mixed mode) solid phase extraction (SPE) column. Chromatographic separation was carried out using a reversed-phase C18 analytical column with a gradient mobile phase consisting of solvent A=5% acetonitrile with 0.1% formic acid and solvent B=100% acetonitrile. MS analysis was performed using positive electrospray ionization (ESI) in multiple reaction monitoring (MRM) mode. Linearity was established over the range 1-100 ng/mL with correlation coefficients ≥0.998 for all three analytes. The coefficient of variation (CV) of intra-day samples was ≤5.6% at 10 ng/mL. The precision of inter-day (6 days) samples resulted in CVs ≤8.1% at concentrations tested at 2.5, 10 and 25 ng/mL for all three analytes. The lower limit of quantification (LOQ) was 1.0 ng/mL with signal-to-noise (S/N) ratio >10, the limit of detection (LOD) was 0.25 ng/mL with S/N ratio >3 for the drug and its metabolites. Dilution effects, extraction recovery, stability, interference, carryover and ion suppression were also evaluated. This method was successfully applied to human subject plasma samples in support of a hydrocodone pharmacokinetic study.

The GC-MS detection and characterization of neopine resulting from opium use and codeine metabolism and its potential as an opiate-product-use marker.

Neopine, a minor opium alkaloid and an isomer of codeine (also known as beta-codeine), has been detected in both the urine of opium users and pharmaceutical codeine users. The characterization of neopine was achieved by comparison of the mass spectra and GC retention times of the trimethylsilyl derivative. The presence of neopine in the urine of pharmaceutical codeine users was attributed to the metabolism of codeine through a double bond migration in ring C, from the 7-8 to the 8-14 position. The potential use of the alkaloid as a confirmation marker of opium and/or pharmaceutical codeine use and the ability to differentiate these from heroin use has been discussed.

Engineering novel biocatalytic routes for production of semisynthetic opiate drugs.

The morphine alkaloids and their semisynthetic derivatives provide a diverse range of important pharmaceutical drugs. Current production of semisynthetic opiate drugs is by chemical means from naturally occurring morphine, codeine and thebaine. Although various microbial transformations of morphine alkaloids have been identified since the 1960s, more recently there has been considerable effort devoted to engineering biocatalytic routes for producing these important compounds. Such biocatalytic routes are attractive, as they would provide an alternative to the chemical production processes which suffer from limited supply of precursors, often low yields and toxic wastes. The biotransformation of morphine and codeine to the potent analgesic hydromorphone and the mild analgesic/antitussive hydrocodone, respectively, by recombinant Escherichia coli has been demonstrated and the problems encountered when engineering such a system will be discussed.

Nitrocinnamoyl and chlorocinnamoyl derivatives of dihydrocodeinone: in vivo and in vitro characterization of mu-selective agonist and antagonist activity.

Two 14beta-p-nitrocinnamoyl derivatives of dihydrocodeinone, 14beta-(p-nitrocinnamoylamino)-7,8-dihydrocodeinone (CACO) and N-cyclopropylmethylnor-14beta-(p-nitrocinnamoylamino)- 7, 8-dihydrocodeinone (N-CPM-CACO), and the corresponding chlorocinnamoylamino analogs, 14beta-(p-chlorocinnamoylamino)-7, 8-dihydrocodeinone (CAM) and N-cyclopropylmethylnor-14beta-(p-chlorocinnamoylamino) -7, 8-dihydrocodeinone (MC-CAM), were tested in opioid receptor binding assays and the mouse tail-flick test to characterize the opioid affinity, selectivity, and antinociceptive properties of these compounds. In competition binding assays, all four compounds bound to the mu opioid receptor with high affinity. When bovine striatal membranes were incubated with any of the four dihydrocodeinones, binding to the mu receptor was inhibited in a concentration-dependent, wash-resistant manner. Saturation binding experiments demonstrated that the wash-resistant inhibition of mu binding was due to a decrease in the Bmax value for the binding of the mu-selective peptide [3H][D-Ala2, MePhe4,Gly(ol)5] enkephalin and not a change in the Kd value, suggesting an irreversible interaction of the compounds with the mu receptor. In the mouse 55 degrees C warm water tail-flick test, both CACO and N-CPM-CACO acted as short-term mu-selective agonists when administered by i. c.v. injection, whereas CAM and MC-CAM produced no measurable antinociception at doses up to 30 nmol. Pretreatment of mice for 24 h with any of the four dihydrocodeinone derivatives produced a dose-dependent antagonism of antinociception mediated by the mu but not the delta or kappa receptors. Long-term antagonism of morphine-induced antinociception lasted for at least 48 h after i.c. v. administration. Finally, shifts in the morphine dose-response lines after 24-h pretreatment with the four dihydrocodeinone compounds suggest that the nitrocinnamoylamino derivatives may produce a greater magnitude long-term antagonism of morphine-induced antinociception than the chlorocinnamoylamino analogs.

Dihydrocodeinone-hydrazone, dihydrocodeinone-oxime, naloxone-3-OMe-oxime, and clocinnamox fail to irreversibly inhibit opioid kappa receptor binding.

Previous work from our lab identified two subtypes of the opioid kappa receptor. Whereas the kappa1 receptor can be labeled by [3H]U69,593 (5 alpha,7 alpha,8 beta-(-)- N-methyl-N-[7-(1-pyrrolidinyl)-1-oxaspiro(4,5)dec-8-yl]-phenyl- benzeneacetamide), the kappa2 receptor can be labeled by [125I]OXY (6 beta-125iodo-3,14-dihydroxy-17-cyclopropylmethyl-4,5 alpha-epoxymorphinan). Other data demonstrate that [125I]IOXY, like [3H]bremazocine, labels two populations of kappa2 receptors in guinea pig brain: kappa2a and kappa2b binding sites. In the present study, we tested the hypothesis that certain dihydrocodeinone and oxicodone derivatives, which have been shown to irreversibly block low affinity [3H]naloxone binding sites, would also bind irreversibly to opioid kappa receptor subtypes. We also tested the novel irreversible mu receptor antagonist, clocinnamox (14 beta-(p-chlorocinnamoylamino)-7,8-dihydro-N-cyclopropylmethylno rmorphinone mesylate). Wash-resistant inhibition (WRI) assays were conducted to detect apparent irreversible inhibition. The proportion of WRI attributable to inhibition of receptor binding, termed receptor inhibition (RI), was calculated by the equation: RI = WRI (wash-resistant inhibition) - SI (supernatant inhibition or inhibition attributable to residual drug.) Dihydrocodeinone-hydrazone, dihydrocodeinone-oxime and naloxone-3-OMe-oxime failed to produce any wash-resistant inhibition of kappa receptor binding. In contrast, preincubating guinea pig membranes with 1 microM clocinnamox produced a substantial degree of wash-resistant inhibition (greater than 90%) at kappa1 and kappa2 binding sites. However, as indicated by supernatant inhibition values of 70% to 90%, there was a large amount of residual clocinnamox which remained despite the use of an extensive washing procedure.(ABSTRACT TRUNCATED AT 250 WORDS)

[Effect of conorphone on the dimensions of the myocardial infarct and the ultrastructural changes in the adjacent tissue]. / Efecto de la conorfona sobre las dimensiones del infarto del miocardio y las alteraciones ultraestrcuturales del tejido adyacente.

Among contemporary problems of cardiovascular therapy, pain management in the cardiac patient is an important issue. Correct selection of a suitable analgesic drug is important in the acute phase of myocardial infarction because pain relief prevents adrenergic response which aggravates unbalance between myocardial oxygen supply and demands. Until today morphine has been the best choice, nevertheless its side effects and limited availability demands pharmacological alternatives. Meperidine has been used for this purpose although its anticholinergic and narcotic profiles do not fulfill the therapeutic requirements. Conorphone is a codeine derivative which retains analgesic potency without other narcotic side effects and which presents an antagonistic profile for the remainder of opiate effects. Preliminary studies demonstrate that conorphone lacks cardiovascular side effects. Conorphone also shows antiarrhythmic effects in experimental models such as in rat in which arrhythmias were induced by coronary ligation. In this paper a morphologic analysis with electron microscopy was done on adjacent myocardial tissue next to an area of infarct. Experimental myocardial infarction was induced by ligation of descendant branch of left coronary artery, four hours prior to rat sacrifice and suitable samples for ultrastructure study were obtained. The experiments demonstrate that a group of rats that received 3.1 mg/kg of conorphone had less expressions of cellular damage when compared with similar myocardial areas of a control group of rats.

Comparison of conorphone, a mixed agonist-antagonist analgesic, to codeine for postoperative dental pain.

The analgesic efficacy of two doses of conorphone (20 and 40 mg), a mixed agonist-antagonist analgesic, were compared to two doses of codeine for postoperative pain in the oral surgery model. Each subject received 2 of the 4 possible treatment at two separate sessions in an incomplete block, single crossover design. Both doses of conorphone and the 60 mg dose of codeine were superior to 30 mg of codeine for the various indices of analgesic activity. The 40 mg dose of conorphone resulted in a high incidence of side effects (25/30 subjects) such as drowsiness, dizziness, nausea and vomiting. The low dose of conorphone resulted in side effects similar to 60 mg of codeine with the exception of a greater incidence of drowsiness. These data suggest that while 40 mg of conorphone may not be well tolerated clinically, 20 mg of conorphone may be an alternative to 60 mg of codeine for postoperative pain.

Disposition and metabolism of codorphone in the rat, dog, and man.

The disposition and metabolism of codorphone, 17-cyclopropyl-methyl-4,5 alpha-8 beta-ethyl-3-methoxymorphinan-6-one (I), a new narcotic antagonist, analgesic agent, have been studied in the rat, dog, and man. Rats and dogs were given single 100- and 50-mg/kg po doses, respectively, of I-3H; human volunteers received single 10- to 30-mg doses of unlabeled I po. The compound appeared to be well absorbed in the three species. In rats the highest levels of radioactivity were in liver, adrenals, kidneys, spleen, and lungs. Excretion was primarily fecal in rats and dogs. In man about 50% of the dose appeared in the 24-hr urine. I was about 95% metabolized by each species. The major metabolites in rats resulted from 3- and/or 17-dealkylation. Metabolism in dogs was characterized primarily by 17-dealkylation. The major pathways of I metabolism in man were 17-dealkylation and 6-reduction. In the three species significant glucuronic acid conjugation of metabolites occurred.

Methodology for the identification of the urinary metabolites of the analgesic-narcotic antagonist, codorphone, by gas chromatography mass spectrometry.

Methodology is presented for the identification of codorphone and its metabolites in urine samples using gas chromatography mass spectrometry. The procedure focuses on the clean-up of biological samples and a derivatization technique suitable for these samples. Sep-Pak C-18 cartridges were employed in the clean-up procedure permitting the biological sample to be derivatized in a relatively small volume of reagents. The derivatization procedure incorporated a one-step trimethylsilyloxime reaction to prevent enol formation while simultaneously derivatizing free hydroxyl groups with the excess trimethylsilylimidazole present in the reaction mixture. This was followed by the addition of BSTFA directly to this reaction mixture to complete derivatization of any metabolites possessing dealkylation of the nitrogen. Using this derivatization scheme, synthetic metabolites were analyzed by gas chromatography mass spectrometry, and their mass spectra were characterized emphasizing the diagnostic fragment ions observed in the spectra. To illustrate the usefulness of this methodology, a urine sample obtained from a dog that had been dosed with codorphone was analyzed by gas chromatography mass spectrometry, and the metabolites were identified by comparison to the mass spectra of the synthetic derivatives.

Analgesic narcotic antagonists. 8. 7 alpha-Alkyl-4,5 alpha-epoxymorphinan-6-ones.

The preparation of a series of 7 alpha-alkylated dihydrocodeinones is described. N-(Cyclopropylmethyl) (P series) or N-(cyclobutylmethyl) (B series) 7 alpha-methyl (a series) or 7 alpha, 8 beta-dimethyl (b series) substituted dihydronorcodeinones (7) were prepared from the appropriately substituted N-(cycloalkylmethyl)-4-hydroxymorphinan-6-ones (5) by dibromination, 4,5-epoxy ring closure, and catalytic debromination. Treatment of 7 with BBr3 gave low yields of the corresponding 3-phenols 8. Alternatively, reaction of dihydrocodeinone (10) with dimethylformamide dimethyl acetal gave the 7-[(dimethylamino)methylene] adduct 11, which was hydrogenated to 7 alpha-methyl- (12) or 7 alpha-(hydroxymethyl)dihydrocodeinone (13). Treatment of 11 with lithium reagents, followed by hydrogenation, gave a mixture of 7 alpha-alkyl (15c-f) compounds and the corresponding 4,5-epoxy-cleaved products 16. Reaction of 11 with alpha-ethoxyvinyllithium gave intermediate 17, which on hydrolysis and hydrogenation yielded the 6,7-furyl (18) or pyrroyl (19) derivative. N-(Cycloalkylmethyl)-14-hydroxydihydronorcodeinones 23P,B reacted with dimethylformamide dimethyl acetal to give 25P,B, which were hydrogenated to the 7 alpha-methyl compounds 26P,B and O-demethylated to give 27P,B. The 7 alpha-methyl-N-methyl compounds were about equipotent with dihydrocodeinone. Derivatives with larger alkyl groups were less potent. Corresponding N-(cycloalkylmethyl) compounds did not show strong mixed agonist-narcotic antagonist activity.