Richard I.H. Wang

A method of identifying narcotic analgesics in human urine after therapeutic doses.

Abstract Alkaloids were separated from urine in 15 min with an Amberlite XAD-2 resin column, and the methanol eluate was chromatographed on 2 thin-layer silica gel glass microfiber sheets to establish a pattern of spots for each narcotic analgesic and its metabolites. Ten milliliters of urine applied to a column of Amberlite XAD-2 resin (1 × 6 cm) was flushed with an equal volume of water. The adsorbed drug and its metabolites were eluted with methanol nonselectively into the same fraction as some highly colored urinary pigments. The latter served as a guide to collect the alkaloid-containing fraction. Sixty microliters of the fraction were spotted on 2 Gelman SG sheets; these chromatograms were developed in 2 solvent systems: I ( n -butanol-acetic acid-water, 35:3:10) and IV (CHCl 3 saturated with ammonium hydroxide). The chromatograms were sprayed with iodoplatinate reagent, and unknowns were identified by matching their pattern of spots (drug and metabolites) with the pattern for urine standards run on the same chromatograms. From patients receiving no medication and others receiving therapeutic doses of narcotic analgesics 140 coded urine samples were collected. These unknowns were not decoded until after completion of the analysis. Six (out of the 7) control samples from untreated patients were correctly demonstrated to contain no alkaloids. The remaining 134 of the 140 samples were shown to be positive to iodoplatinate. Of these 134, 88 were identified correctly to be 27 meperidine, 29 codeine, 2 morphine, 1 levorphanol, 27 pentazocine, and 2 dihydromorphinone samples. In the remainder, 13 could not be assigned to a specific narcotic analgesic, and 33 were misidentified. Both these types of errors arose largely from paucity in number of alkaloid spots for matching with the urine standards. In spite of these shortcomings, the ease, speed, high sensitivity for detection and possibility for differential identification of narcotic analgesics make this method highly practical.

Unidirectional non-cross tolerance to etorphine in morphine-tolerant mice and role of the blood-brain barrier

Abstract A unidirectional non-cross tolerance phenomenon to etorphine was observed under conditions of maximal tolerance development to morphine. Using a morphine pellet (75 mg morphine) implantation technique, a 30-fold increase in the analgesic ED50 for subcutaneously (sc) administered morphine was observed. When 30-fold morphine-tolerant animals were examined for cross tolerance to sc etorphine, no change in etorphines analgesic ED50 was observed. The removal of the tolerance-inducing morphine pellet prior to analgesia testing resulted in the expression of cross tolerance to etorphine. Further characterization of the phenomenon was provided by the reverse experiment in which a large amount of cross tolerance to sc morphine but little tolerance to sc etorphine was observed after etorphine pellet implantation. No change occurred in the analgesic ED50 for intracerebroventricular (icv) administration of etorphine or morphine after the animals were implanted with morphine pellets. These observations implicated the blood-brain barrier as a probable site for the differential expression of narcotic tolerance.

Narcotic Antagonist Activity of Several Metabolites of Naloxone and Naltrexone Tested in Morphine Dependent Mice

Summary A rabbit liver enzyme system was used to produce the 6β-OH reduced metabolites of naloxone and naltrexone. GC analysis indicated the presence of some 6α-0H metabolite in these samples. The narcotic antagonist activity of these 6β-OH metabolite samples were compared to nalox-one, naltrexone and standard 6α-OH nalox-one (EN 2265A) and 6α-OH naltrexone (EN 2260A) using the jumping response of morphine pellet implanted mice. For the nal-oxone series, the potencies were: Naloxone > EN 2265A > 6β-OH naloxone. For the naltrexone series: Naltrexone > EN 2260A > 6β-OH naltrexone. The low potency of the reduced metabolites and the rapid onset of action of the parent compounds militate against the formation of these metabolites contributing substantially to the overall narcotic antagonist action of the parent compounds. The authors wish to acknowledge the excellent technical assistance of Miss Ann Klecker. Dr. Harold Blumberg of Endo Laboratories generously provided the naloxone, naltrexone, EN 2265 and EN 2260.

Disulfiram and erythrocyte aldehyde dehydrogenase inhibition.

During disulfiram therapy erythrocyte aldehyde dehydrogenase (ALDH) was fully inhibited. The time for total loss of erythrocyte ALDH activity ranged from 36 to 120 hr. In contrast to the 85% recovery of in vitro disulfiram‐inhibited ALDH activity, this in vivo disulfiram‐ALDH inhibition could not be reversed by mercaptoethanol. It is proposed that the in vivo and in vitro mechanisms of ALDH inhibition by disulfiram differ. Erythrocyte ALDH activity can be readily monitored to determine patient compliance and is an accessible model for investigations of in vivo mechanisms of drug inhibition. Because the disulfiram‐inhibited erythrocyte ALDH is not regenerated until new erythrocytes are made (120 days), a significant portion of the extrahepatic acetaldehyde metabolic capacity remains inhibited for long periods after disulfiram is discontinued. Thus, the recidivistic patient who discontinues disulfiram and waits several days (to regenerate liver ALDH activity) before drinking will be exposed to even higher ethanol‐derived blood acetaldehyde levels than usual, which may induce further alcohol‐associated organ damage and alcohol dependence.

Mediation of reduced nicotinamide adenine dinucleotide phosphate dependent reduction of cytochrome c by morphine

Abstract The ability of morphine and other analgesics to mediate a transfer of electrons from reduced nicotinamide adenine dinucleotide phosphate (NADPH) to cytochrome c was investigated. This phenomenon was first reported by Wang and Bain ( J. Pharmac. exp. Ther. , 108 , 354, 1953), and we have confirmed their findings and provided further insight into this unusual action of morphine. This electron transfer was studied by following the reduction of cytochrome c and it was found that: (1) in order to mediate the transfer of electrons, the compound studied must be a morphinan with a free phenolic hydroxyl group in the 3 position and an oxygen bridge between positions 4 and 5; (2) substitution or rearrangement of other functional groups on the morphinan molecule did not greatly affect its ability to mediate the electron transfer; (3) by optimizing reaction condition (pH and purification of the morphine), we observed measurable rates of cytochrome c reduction (0·002 A/min) at morphine concentrations as low as 4·2 × 10 −7 M; and (4) preliminary evidence indicates that morphine acts catalytically in mediating the electron transfer. Since narcotic analgesics of the opioid type such as methadone, pentazocine and 3-hydroxy- N -methyl morphinan did not mediate the electron transfer, it appears that no correlation exists between this electron transferring action and the mechanism of analgesia in general. This does not, however, preclude that a correlation could exist between this electron transferring action and some other aspect of the pharmacological action of opiates.

Occurrence of corneal opacities in rats after acute administration of l-α-acetylmethadol

Abstract The ability of l-α-acetylmethadol (LAAM) to induce ocular opacities was studied in rats. Between the third and fifth days after subcutaneous or oral administration of a single dose of LAAM, a dose-dependent incidence of opacification of the anterior segments of rat eyes was observed. Observation of the eyes of LAAM-treated rats, using a dissection microscope and a slit beam biomicroscope, indicated that the opacities were localized to the cornea and the depth of corneal involvement varied from patches of grayish epithelium to full-thickness involvement. Light microscopy of eye sections from rats given LAAM, 20 mg/kg orally, revealed corneal changes ranging from thickening and loss of regularity of epithelial cell layers to stromal vascularization and spindle cell infiltration. The sc and oral ED-50 for LAAM-induced corneal opacities was found to be 4.7 (4.2–5.3) and 12.6 (9.8–16.1) mg/kg, respectively. This is similar to or lower than the ED-50 for pharmacological effects of LAAM such as analgesia and mydriasis. The mechanism(s) by which LAAM initiates the corneal opacities is unknown. However, in rats made tolerant to morphine by implantation of a morphine pellet, we observed a threefold tolerance to LAAM-induced corneal opacities, suggesting a central nervous system mechanism in the development of the observed corneal changes.

Enzymatic conversion of morphine to pseudomorphine.

Abstract The interaction of morphine with an enzymatic oxidation-reduction system was investigated. It has been reported that morphine is converted to a highly water-soluble metabolite by incubation with horseradish peroxidase (HRP), albumin and H 2 O 2 (Misra and Mitchell, Experientia 27, 1442 (1971)). After incubating [ 14 C]morphine with HRP and H 2 O 2 , we detected two radioactive compounds with t.l.c. One compound corresponded to morphine. Using spectral analysis, the other, 14 C-labeled compound, was identified as pseudomorphine, a dimerized oxidation product of two molecules of morphine. Using compounds structurally related to morphine, it was determined that: (1) a free phenolic hydroxyl group in position 3 was necessary for the enzymatic oxidation of morphine to pseudomorphine; (2) a carbonyl group in position 6 of the morphinan ring prevents the formation of the dimer; and (3) substitution of other functional groups on the morphine molecule did not affect the peroxidase-catalyzed dimerization. By optimizing reaction conditions, measurable rates of peroxidase-catalyzed pseudomorphine formation were observed at morphine concentrations as low as 5 × 10 −7 M. The nature of peroxidase enzymes and the phenolic character of morphine suggest that the formation of pseudomorphine proceeds through a morphine-free radical intermediate.

Effects of acute and chronic morphine treatment on methadone analgesia and metabolism

Morphine sulfate (5 mg/kg s.c.) given 30 min prior to administration of methadone prolonged methadone analgesia and increased the brain level of methadone measured 60, 120 and 180 min after administration of methadone. Rats rendered tolerant to morphine analgesia by subcutaneous implantation of two pellets, each containing 75 mg of morphine base, for 1-3 days showed cross-tolerance to methadone analgesia regardless of the presence or absence of morphine pellets. Decreases in the brain concentrations of methadone measured at 60 and 120 min time points accompanied the decreased analgesia. Neither acute nor chronic morphine pretreatment affects the biotransformation of methadone. The results suggest that the cross-tolerance to methadone analgesia seen in chronic morphine-implanted rats was partly associated with a decrease in the brain concentration of methadone occurring by a mechanism not directly related to a change in the biotransformation of methadone. In view of the known inhibitory effect of chronic morphine pretreatment on drug metabolism, our findings might demonstrate a unique phenomenon between morphine and methadone.

Enhancement of etorphine brain concentrations and changes in etorphine-naloxone pA2; values in morphine-pretreated mice

Abstract We had shown earlier that pretreatment of mice with a single dose of morphine sulfate enhanced the concentration of naloxone in the brain, and, therefore, the effect of this pretreatment on brain disposition of the narcotic agonist etorphine was examined for similar effects. Three hours after pretreatment with morphine, [ 3 H]etorphine was administered subcutaneously, and its brain concentrations were determined as a function of time. Etorphine brain concentrations were higher in morphinethan in saline-pretreated animals at 5, 15, 20 and 30 min. This enhancement of brain concentrations was not associated with a change in the analgesic ED 50 for etorphine. Morphine pretreatment in mice has been reported by others to increase the affinity of the antagonist receptor site for naloxone, as demonstrated by an increase in the in vivo apparent pA 2 value for a morphine-naloxone interaction. In the present study, the morphine pretreatment decreased the etorphine-naloxone apparent pA 2 value in the direction opposite to that observed for the morphine-naloxone interaction. The results are discussed relative to a morphine-induced change in the disposition of etorphine in the brain, or to a morphine-induced alteration in morphine, etorphine and naloxone interactions at agonist and antagonist binding sites.

Adverse reactions to sulfobromophthalein sodium. Report of 2 cases.

SummaryReports on fatal and nonfatal reactions to sulfobromophthalein (BSP) continue to appear in the medical literature, though infrequently. In this paper, a review of these reports and 3 additional cases are presented. To avoid BSP reaction, the allergic history of the patient and his response to previous administration of BSP should be obtained. BSP should be administered slowly to avoid thrombophlebitis, and other precautionary measures, before, during, and after BSP injection, should be observed.Reports on fatal and nonfatal reactions to sulfobromophthalein (BSP) continue to appear in the medical literature, though infrequently. In this paper, a review of these reports and 3 additional cases are presented. To avoid BSP reaction, the allergic history of the patient and his response to previous administration of BSP should be obtained. BSP should be administered slowly to avoid thrombophlebitis, and other precautionary measures, before, during, and after BSP injection, should be observed.