Biotransformations

Recently, we described three substances in bovine hypothalamus, adrenal, and rat brain recognized by antisera raised against morphine, and we identified one as morphine and another as codeine by GC/MS. We now report the identification of the third immunoreactive (ir) morphinan from bovine brain as 6-acetylmorphine by chemical conversion to morphine, GC/MS, and high-resolution mass measurement. 6-Acetylmorphine has not previously been described as a natural product in plants or animals, but it has long been known as the metabolite in part responsible for the biological properties of heroin. However, we have excluded slaughterhouse or laboratory contamination by any morphinan as well as derivation from the morphine in tissues during our procedures. 6-Acetylmorphine is known to be more potent than morphine in vivo chiefly by virtue of its greater penetration into the central nervous system. Should morphinans prove to have physiological functions in animals, the properties of 6-acetylmorphine make it ideal for fulfilling the role of a peripheral-to-central hormone.Early work suggesting the possibility of morphine biosynthesis in mammalian brain (1) stimulated investigation of mammalian tissues with RIAs for morphine (2) and led to the detection of immunoreactive (ir) substances (3-7). Recently, we described three ir morphinans from mammalian brain and adrenal and identified one as morphine and another as codeine by GC/MS (8); this was subsequently confirmed for rat central nervous system (9). The possibility that morphine and codeine are endogenous in mammals is suggested by our recent demonstration that mammalian liver (but not brain or adrenal) can synthesize the morphine ring structure (10), carrying out the same critical step as does the opium poppy (11,12). We now report the identification of the third ir morphinan, previously designated "peak 5" (8), as 6-acetylmorphine . MATERIALS AND METHODS Materials. [methyl-3H]Morphine was from New EnglandNuclear; [ring-3H]morphine was from Amersham; synthetic 3-acetylmorphine and 6-acetylmorphine standards were a gift from A. Allen (U.S. Drug Enforcement Administration). Other reagents were from Baker or Sigma.Morphine RIAs. Anti-morphine antisera 937 and S17 were provided by Syva (Palo Alto, CA). A detailed description of preparation of antisera, assay conditions, analysis of RIA data, and the differing specificities of the two RIAs has appeared elsewhere (7).Analytical Purification and Controls. For the experiments illustrated in Fig. 1 Fig. 2, partially purified peak 5 (see Fig. 1; 45 pmol of ir morphine equivalents, antiserum 937) was incubated in water, NH40H (1 M; pH 11.8), NaOH (3.5 mM;,pH 11.8), or HCO (1 M) (final vol, 200 ,ul; 20 hr; 23°C). After incubation, nonacidic samples were acidified to pH 1-2 by addition of HCO, and each sample was analyzed by HPLC-C. The ir peaks were collected and lyophilized. Samples were dissolved in 5 mM trifluoroacetic acid (500 al) containing an internal standard of [ring-3H]morphine (4 x 104 cpm; 14 Ci/mmol); half of ea...

Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

6-Acetylmorphine: a natural product present in mammalian brain.

Weitz¹,

Lowney²,

Faull³

et al. 1988

“…We found that about half the total association of levorphanol with brain tissue is of the nonsaturable kind (trapped and dissolved drug), nearly half is of the nonspecific saturable kind, and only about 2% is stereospecific. Consequently, it is clear that neither measurements of total "binding" (19,(22)(23)(24) nor even a direct comparison of the distributions of a D(-) narcotic and its L(+) enantiomer in brain tissue (25) could be relevant to the identification of receptor sites.…”

Section: Discussionmentioning

confidence: 99%

Stereospecific and Nonspecific Interactions of the Morphine Congener Levorphanol in Subcellular Fractions of Mouse Brain

Goldstein

Lowney

Pal

1971

362

A method is described for analyzing the association of the opiate narcotic levorphanol with brain tissue into three components: nonsaturable, saturable nonspecific, and saturable stereospecific. The method may be of general applicability for the study of the interaction of drugs with body tissues. In mouse brain the stereospecific binding of levorphanol represents only 2% of the total association of drug with tissue, and it was found only in certain membrane fractions. The material responsible for the stereospecific binding might be the opiate receptor.

“…Unlike most other benzomorphan opiates, phenazocine possesses an N-phenethyl group that confers a potency exceeding that of N-methyl benzomorphans of the a-series. The greater potency of these drugs compared to morphine is not adequately explained by pharmacodynamic or metabolic variations (9,10).…”

Section: Conformational Features Of Certain Potent Agonistsmentioning

confidence: 99%

The opiate receptor: a model explaining structure-activity relationships of opiate agonists and antagonists.

Feinberg¹,

Creese²,

Snyder³

1976

126

A model of the opiate receptor is proposed which explains structure-activity relationships of opiate drugs, including (l) the unique potency of certain opiates such as etonitazene, fentanyl, phenazocine, and oripavines; (ih) the role of N-allyl substituents in conferring antagonist properties; and (iii) chemical features that afford "pure" antagonists. The model indicates molecular mechanisms for interconversion of the opiate receptor between respective states that bind agonists or antagonists with high affinity. Unique pharmacological properties of many opiates are not readily explained by their chemical structures. Examples include (i) the great potency of opiates such as etonitazene, fentanyl, phenazocine, and etorphine (Fig. 1); (ii) the dramatic change from agonist to antagonist effects conferred by transforming an opiate N-methyl to an N-allyl or related substituent; and (iii) the chemical features determining that "pure" opiate antagonists such as naloxone lack analgesic and euphoric actions while "mixed" agonist-antagonists possess both.Studies on opiate receptor binding differentiate agonists and antagonists (1). The opiate receptor appears to exist in two interconvertible forms, one with uniquely high affinity for antagonists and the other with selective affinity for agonists (2). Sodium ions enhance antagonist and depress agonist binding presumably by regulating interconversion of the two opiate receptor states, while manganese selectively facilitates agonist binding (2,3). These influences may physiologically regulate interactions of the opiate receptor with the naturally occurring, morphine-like peptide, enkephalin (4-7).Here we suggest possible conformations of opiates at their receptor site which can explain similarities in pharmacological actions of compounds whose chemical structures appear, superficially, quite different. A model of opiate receptor function is proposed specifying conformational changes that underlie the interconversion of the two receptor states and explaining the pharmacological differences of opiate agonists and antagonists.