An oxycodone conjugate vaccine elicits drug-specific antibodies that reduce oxycodone distribution to brain and hot-plate analgesia.

Opioid conjugate vaccines have shown promise in attenuating the behavioral effects of heroin or morphine in animals. The goal of this study was to extend this approach to oxycodone (OXY), a commonly abused prescription opioid. Haptens were generated by adding tetraglycine (Gly)(4) or hemisuccinate (HS) linkers at the 6-position of OXY. Immunization of rats with OXY(Gly)(4) conjugated to the carrier proteins bovine serum albumin (BSA) or keyhole limpet hemocyanin (KLH) produced high-titer antibodies to OXY and its metabolite oxymorphone with substantially lower affinities for other structurally related opioid agonists and antagonists. There was no measurable binding of antibody by the (Gly)(4) linker alone or off-target opioids methadone and buprenorphine. OXY(HS) conjugates were less immunogenic despite achieving protein haptenation ratios comparable to OXY(Gly)(4)-BSA. In rats given a single intravenous dose of OXY, immunization with OXY(Gly)(4)-KLH increased OXY protein binding and retention in serum while decreasing its unbound (free) concentration in plasma and distribution to brain. Vaccine efficacy correlated with serum antibody titers, and it was greatest in rats given the lowest OXY dose (0.05 mg/kg) but was significant even after a larger OXY dose (0.5 mg/kg), equivalent to the high end of the therapeutic range in humans. These effects of OXY(Gly)(4)-KLH on drug disposition were comparable to those of nicotine or cocaine vaccines that are in clinical trials as addiction treatments. Immunization with OXY(Gly)(4)-KLH also reduced OXY analgesia in a thermal nociception test. These data support further study of vaccination with the OXY(Gly)(4)-KLH immunogen as a potential treatment option for OXY abuse or addiction.

Chloroxymorphamine, and opioid receptor site-directed alkylating agent having narcotic agonist activity.

Chloroxymorphamine, the 6beta-N,N-bis(2-chloroethyl) derivative of oxymorphone, is a potent nonequilibrium narcotic agonist in the longitudinal muscle preparation of guinea pig ileum. The corresponding naltrexone analog,chlornaltrexamine, is a potent nonequilibrium antagonist of morphine. These receptor sitedirected alkylating agents possess considerable potenial as pharmacologic and biochemical probes of apoid receptors.

Methadone conformation and opioid activity.

The inactive methadone analog threo-5-methylmethadone has a solid-state conformation in which the nitrogen is antiperiplanar to the tertiary carbon C(4). Since threo-5-methylmethadone exhibits no opioid agonism either in vivo or in vitro, methadone analogs probably do not have this conformation when bound to an opioid receptor. The potent agonist (-)-erythro-5-methylmethadone has a solid-state conformation in which the nitrogen atom is rotated back toward the phenyl rings on the quarternary carbon, suggesting that this unusual conformation is the active one.

The FEMA GRAS assessment of alpha,beta-unsaturated aldehydes and related substances used as flavor ingredients.

This publication is the 12th in a series of safety evaluations performed by the Expert Panel of the Flavor and Extract Manufacturers Association (FEMA). In 1993, the Panel initiated a comprehensive program to re-evaluate the safety of more than 1700 GRAS flavoring substances under conditions of intended use. Since then, the number of flavoring substances has grown to more than 2200 chemically-defined substances. Elements that are fundamental to the safety evaluation of flavor ingredients include exposure, structural analogy, metabolism, toxicodynamics and toxicology. Scientific data relevant to the safety evaluation for the use of aliphatic, linear alpha,beta-unsaturated aldehydes and structurally related substances as flavoring ingredients are evaluated. The group of substances was reaffirmed as GRAS (GRASr) based, in part, on their self-limiting properties as flavoring substances in food; their low level of flavor use; the rapid absorption and metabolism of low in vivo concentrations by well-recognized biochemical pathways; adequate metabolic detoxication at much higher levels of exposure in humans and animals; the wide margins of safety between the conservative estimates of intake and the no-observed-adverse effect levels determined from subchronic and chronic studies. While some of the compounds described here have exhibited positive in vitro genotoxicity results, evidence of in vivo genotoxicity and carcinogenicity occurs only under conditions in which animals are repeatedly and directly exposed to high irritating concentrations of the aldehyde. These conditions are not relevant to humans who consume alpha,beta-unsaturated aldehydes as flavor ingredients at low concentrations distributed in a food or beverage matrix.

The FEMA GRAS assessment of aromatic substituted secondary alcohols, ketones, and related esters used as flavor ingredients.

This publication is the 11th in a series of safety evaluations performed by the Expert Panel of the Flavor and Extract Manufacturers Association (FEMA). In 1993, the Panel initiated a comprehensive program to re-evaluate the safety of more than 1700 GRAS flavoring substances under conditions of intended use. The list of GRAS substances has now grown to more than 2100 substances. Elements that are fundamental to the safety evaluation of flavor ingredients include exposure, structural analogy, metabolism, pharmacokinetics and toxicology. Flavor ingredients are evaluated individually and in the context of the available scientific information on the group of structurally related substances. In this monograph, a detailed interpretation is presented on the renal carcinogenic potential of the aromatic secondary alcohol alpha-methylbenzyl alcohol, aromatic ketone benzophenone, and corresponding alcohol benzhydrol. The relevance of these effects to the flavor use of these substances is also discussed. The group of aromatic substituted secondary alcohols, ketones, and related esters was reaffirmed as GRAS (GRASr) based, in part, on their rapid absorption, metabolic detoxication, and excretion in humans and other animals; their low level of flavor use; the wide margins of safety between the conservative estimates of intake and the no-observed-adverse effect levels determined from subchronic and chronic studies and the lack of significant genotoxic and mutagenic potential.

Bivalent ligands and the message-address concept in the design of selective opioid receptor antagonists.

Metabolically stable receptor antagonists that are subtype selective are indispensable pharmacological tools. In this article, Philip Portoghese describes the bivalent ligand approach to drug design which has resulted in the development of several highly selective non-peptide opioid receptor antagonists, such as the kappa-selective norbinaltorphimine and the delta-selective naltrindole. Models used resemble Schwyzer's message-address concept which originally described the recognition elements of peptide hormones; their success augurs well for the possibility of altering antagonist selectivity in a predictable fashion by simulating a portion of the address peptide component with a rigid nonpeptide moiety.

The FEMA GRAS assessment of phenethyl alcohol, aldehyde, acid, and related acetals and esters used as flavor ingredients.

This publication is the ninth in a series of safety evaluations performed by the Expert Panel of the Flavor and Extract Manufacturers Association (FEMA). In 1993, the Panel initiated a comprehensive program to re-evaluate the safety of more than 1700 GRAS flavoring substances under conditions of intended use. Elements that are fundamental to the safety evaluation of flavor ingredients include exposure, structural analogy, metabolism, pharmacokinetics and toxicology. Flavor ingredients are evaluated individually and in the context of the available scientific information on the group of structurally related substances. Scientific data relevant to the safety evaluation of the use of phenethyl alcohol, aldehyde, acid, and related acetals and esters as flavoring ingredients is evaluated. The group of phenethylalcohol, aldehyde, acid, and related acetals and esters was reaffirmed as GRAS (GRASr) based, in part, on their self-limiting properties as flavoring substances in food, their rapid absorption, metabolic detoxication, and excretion in humans and other animals, their low level of flavor use, the wide margins of safety between the conservative estimates of intake and the no-observed-adverse effect levels determined from subchronic and chronic studies and the lack of significant genotoxic and mutagenic potential. This evidence of safety is supported by the fact that the intake of phenethyl alcohol, aldehyde, acid, and related acetals and esters as natural components of traditional foods is greater than their intake as intentionally added flavoring substances.

The FEMA GRAS assessment of benzyl derivatives used as flavor ingredients.

This publication is the eighth in a series of safety evaluations performed by the Expert Panel of the Flavor and Extract Manufacturers Association (FEMA). In 1993, the panel initiated a comprehensive program to re-evaluate the safety of more than 1700 GRAS flavoring substances under conditions of intended use. Elements that are fundamental to the safety evaluation of flavor ingredients include exposure, structural analogy, metabolism, pharmacokinetics and toxicology. Flavor ingredients are evaluated individually and in the context of the available scientific information on the group of structurally related substances. Scientific data relevant to the safety evaluation of the use of benzyl derivatives as flavoring ingredients is evaluated. The group of benzyl derivatives was reaffirmed as GRAS (GRASr) based, in part, on their self-limiting properties as flavoring substances in food; their rapid absorption, metabolic detoxication, and excretion in humans and other animals, their low level of flavor use, the wide margins of safety between the conservative estimates of intake and the no-observed-adverse effect levels determined from subchronic and chronic studies and the lack of significant genotoxic and mutagenic potential. This evidence of safety is supported by the fact that the intake of benzyl derivatives as natural components of traditional foods is greater than their intake as intentionally added flavoring substances.

The FEMA GRAS assessment of hydroxy- and alkoxy-substituted benzyl derivatives used as flavor ingredients.

This publication is the ninth in a series of safety evaluations performed by the Expert Panel of the Flavor and Extract Manufacturers Association (FEMA). In 1993, the Panel initiated a comprehensive program to re-evaluate the safety of more than 1700 GRAS flavoring substances under conditions of intended use. Elements that are fundamental to the safety evaluation of flavor ingredients include exposure, structural analogy, metabolism, pharmacokinetics and toxicology. Flavor ingredients are evaluated individually and in the context of the available scientific information on the group of structurally related substances. Scientific data relevant to the safety evaluation of the use of hydroxy- and alkoxy-substituted benzyl derivatives as flavoring ingredients is evaluated. The group of hydroxy- and alkoxy-benzyl derivatives was reaffirmed as GRAS (GRASr) based, in part, on their self-limiting properties as flavoring substances in food; their rapid absorption, metabolic detoxication, and excretion in humans and other animals; their low level of flavor use; the wide margins of safety between the conservative estimates of intake and the no-observed-adverse effect levels determined from subchronic and chronic studies and the lack of significant genotoxic and mutagenic potential. This evidence of safety is supported by the fact that the intake of hydroxy- and alkoxy-substituted benzyl derivatives as natural components of traditional foods is greater than their intake as intentionally added flavoring substances.

A procedure for the safety evaluation of natural flavor complexes used as ingredients in food: essential oils.

A scientifically based guide has been developed to evaluate the safety of naturally occurring mixtures, particularly essential oils, for their intended use as flavor ingredients. The approach relies on the complete chemical characterization of the essential oil and the variability of the composition of the oil in the product intended for commerce. Being products of common plant biochemical pathways, the chemically identified constituents are organized according to a limited number of well-established chemical groups called congeneric groups. The safety of the intake of the each congeneric group from consumption of the essential oil is evaluated in the context of data on absorption, metabolism, and toxicology of members of the congeneric group. The intake of the group of unidentified constituents is evaluated in the context of the consumption of the essential oil as a food, a highly conservative toxicologic threshold, and toxicity data on the essential oil or an essential oil of similar chemotaxonomy. The flexibility of the guide is reflected in the fact that high intake of major congeneric groups of low toxicologic concern will be evaluated along with low intake of minor congeneric groups of significant toxicological concern (i.e., higher structural class). The guide also provides a comprehensive evaluation of all congeneric groups and constituents that account for the majority of the composition of the essential oil. The overall objective of the guide is to organize and prioritize the chemical constituents of an essential oil in order that no reasonably possible significant risk associated with the intake of essential oil goes unevaluated. The guide is, however, not intended to be a rigid checklist. The Flavor and Extract Manufacturers Association (FEMA) Expert Panel will continue to evaluate each essential oil on a case by case basis applying their scientific judgment to insure that each natural flavor complex is exhaustively evaluated.

Morphine promotes the growth of HIV-1 in human peripheral blood mononuclear cell cocultures.

Because morphine has been shown to alter the function of human T lymphocytes and monocytes, we postulated that morphine would promote the growth of HIV-1 in these cells. To test this hypothesis, a coculture assay was used consisting of phytohemagglutinin (PHA)-activated peripheral blood mononuclear cells (PBMC) from normal donors and PBMC which had been infected with a viral isolate from an asymptomatic patient, HIV-1AT. The growth of HIV-1AT, as reflected by the concentration of p24 antigen in coculture supernatants, was markedly increased in cocultures that contained morphine. A bell-shaped dose-response curve was observed with three- to fourfold increased growth at a morphine concentration of 10(-12) M. Augmentation of HIV-1AT growth by morphine required an interaction with the PHA-activated donor PBMC. Furthermore, potentiation of HIV-1AT growth by morphine was stereospecific and was antagonized by naloxone and beta-funaltrexamine indicating involvement of an opiate receptor mechanism. These findings provide an additional explanation of how opiates could act as a cofactor in the pathogenesis of HIV-1 in intravenous drug users.

Inhibition of substance P release from spinal cord tissue after pretreatment with capsaicin does not mediate the antinociceptive effect of capsaicin in adult mice.

Substance P (SP) is released from primary afferent fibers in response to nociceptive stimuli. Capsaicin, which produces an initial hyperalgesic response followed by persistent antinociception, also elicits release of SP from primary afferent fibers. Capsaicin pretreatment decreases the content and release of SP from primary afferent fibers. This effect on SP has been hypothesized to mediate the antinociceptive effect of capsaicin. To test this hypothesis, mice were injected intrathecally (i.t.) with antinociceptive doses of capsaicin or SP(1-7) before superfusion of spinal cord tissue with 3 microM capsaicin 24, 48, 96 or 168 h later. N-terminal metabolic fragments of SP that accumulate after capsaicin-induced SP release and are involved in the antinociceptive effect of capsaicin, were also tested. Like capsaicin SP(1-3), SP(1-4) and SP(1-7) were each antinociceptive when injected 24 h before nociceptive testing. However, at this time there was no decrease in capsaicin-evoked release of SP in tissue from capsaicin- and SP(1-7)-pretreated animals compared to those injected with vehicle. In contrast, capsaicin-evoked SP release decreased significantly in tissue from mice pretreated with capsaicin or SP(1-7) 48 h prior to testing. D-Substance P(1-7), which prevents antinociception, blocked capsaicin- and SP(1-7)-induced decreases in SP release, indicating that these effects are mediated by SP N-terminal activity. Total spinal cord content of SP did not differ amongst treatment groups. These data indicate that antinociception does not appear to depend on decreases in SP release or content as antinociception precedes decreases in SP release.

Selective opioid antagonist effects on opioid-induced inhibition of release of norepinephrine in guinea pig cortex.

Opioid agonists with selectivity for mu, delta and kappa-receptors have each been shown to inhibit the K+-stimulated release of [3H]norepinephrine (NE) from slices of guinea pig cortex maintained in vitro. In order to provide further evidence that each of these types of opioid receptor can regulate the release of NE in this tissue, experiments with receptor-type selective opioid antagonists have been conducted. In initial experiments, the selectivity of the antagonists for specific types of opioid receptors in the cortex of the guinea pig in an incubation medium of the same composition as that used for release studies was confirmed. The delta-receptor selective antagonist, ICI 174,864, prevented the inhibitory actions of the delta-selective agonist, [D-Pen2,D-Pen5]enkephalin (DPDPE), but had little effect on the inhibitory actions of the mu-selective agonist, Tyr-D-Ala-Gly-MePhe-Gly-ol (DAMGO), or the kappa-selective agonist, U-50,488H. In contrast, the kappa-selective antagonist, nor-binaltorphimine (nor-BNI) prevented the inhibitory actions of U-50,488H, but had little effect on the inhibitory actions of DPDPE or DAMGO. The greater potency of the partially mu-selective antagonist, naloxone, in reversing the effects of DAMGO relative to those of DPDPE or U-50,488H was confirmed. These results support the conclusion that mu- delta- and kappa-opioid receptors each exert a negative regulatory effect on the stimulated release of NE in the cortex of the guinea pig.

Endomorphin-1 potentiates HIV-1 expression in human brain cell cultures: implication of an atypical mu-opioid receptor.

Endogneous delta and kappa opioid peptides possess a variety of immunomodulatory properties, and kappa-opioid receptor ligands recently were shown to suppress the expression of human immunodeficiency virus type 1 (HIV-1) in microglial cells, the resident macrophages of the brain. To determine whether the newly discovered endogenous mu-opioid receptor ligands endomorphin-1 and -2 would affect HIV-1 replication, these peptides were added to acutely infected brain cell cultures. Endomorphin-1 potentiated viral expression, in a bell-shaped dose-response manner with maximal enhancement approximately equal to 35% at 10(-10) M, in both mixed glial/neuronal cell and purified microglial cell cultures. Endomorphin-1's amplifying effect was blocked by pretreatment of brain cells with either the mu-opioid receptor selective antagonist beta-funaltrexamine or the G protein inhibitor pertussis toxin. However, the classical mu receptor agonists morphine and DAMGO (Tyr-d-Ala-Gly-N-Me-Phe-Gly-ol) had no effect on viral expression or on endomorphin-1's amplifying effect. Taken together, these findings suggest that in this in vitro model of HIV-1 brain infection, endomorphin-1 potentiates viral expression via activation of an atypical mu-selective opioid receptor. They also provide evidence, for the first time, that an endogenous mu-opioid peptide has neuroimmunomodulatory activity.

Edward E. Smissman-Bristol-Myers Squibb Award Address. The role of concepts in structure-activity relationship studies of opioid ligands.

Various concepts have served as the basis for the development of models to explore the relationship between molecular structure and biological activity. In this presentation I have outlined five concepts that have been useful in our investigation of opioid receptor multiplicity and in the design of selective opioid receptor antagonists. The first of these, the multiple binding modality concept, led to our application of four other concepts in the development of opioid receptor probes. Some of these probes are now standard tools in opioid research. These include the mu-selective affinity label beta-FNA (10), the kappa opioid receptor antagonist norBNI (15), and the delta opioid antagonist NTI (20). These highly selective antagonists have advantages over the universal opioid antagonists naloxone and naltrexone because they are of value in probing the interaction of endogenous opioid peptides with opioid receptor types. Additionally, they are useful in evaluating the selectivity of new opioid agonists. Also, selective opioid antagonists have potential clinical applications in the treatment of a variety of disorders where endogenous opioids play a modulatory role. These include constipation, immune function, drug addiction, and alcoholism, to name only a few.

An approach to the design of receptor-type-selective non-peptide antagonists of peptidergic receptors: delta opioid antagonists.

Approaches to the design of peptidomimetic ligands are currently of great interest because of the discovery of an increasing number of endogenous peptides that modulate physiological processes. The inherent lability of peptides and their poor oral absorption have made peptidomimetics attractive targets for drug development. In this presentation I have discussed the design of a novel series of delta-selective opioid antagonists based on the message-address concept. The opioid peptides can be viewed to contain two elements: an essential message component that is recognized by the receptor subsite responsible for the signal transduction process and an address element that is recognized by a subsite that is unique to a single receptor type and functions to enhance binding to the site. Since the tyramine moiety in opiate structures is known to be important for activity, an identical element in Tyr1 of the opioid peptides can be viewed as the message. A key moiety of the delta address was considered to be the phenyl group of Phe4. Combining the universal opioid antagonist naltrexone (5) with a strategically located address mimic afforded naltrindole (6, NTI), the first nonpeptide delta opioid receptor antagonist.

Synthesis and evaluation of melphalan-containing N,N-dialkylenkephalin analogues as irreversible antagonists of the delta opioid receptor.

N,N-Dialkylated leucine enkephalin analogues containing melphalan (Mel) in place of Phe4 were synthesized as potentially irreversible antagonists of the delta opioid receptor. These compounds, along with the corresponding Phe4 peptides, were tested for both agonist and antagonist activity in the GPI and MVD smooth muscle preparations. All but two of the eight compounds showed antagonist activity at 1 microM against [D-Ala2,D-Leu5]enkephalin (DADLE) in the MVD when tested under reversible conditions; in all cases the Mel4 peptide had lower activity against DADLE than did the corresponding Phe4 peptide. At higher concentrations (10 microM) the two active Mel4 analogues, (benzyl)2Tyr-Gly-Gly-Mel-Leu (2a) and (allyl)2Tyr-Aib-Aib-Mel-Leu (3a), both showed weak irreversible antagonism at the delta receptor. Compound 2a was a selective irreversible delta opioid antagonist while 3a was an irreversible antagonist at both the mu and delta opioid receptors.

N,N-dialkylated leucine enkephalins as potential delta opioid receptor antagonists.

A series of N,N-dialkylated leucine enkephalins were prepared in order to study the effect of substitution on antagonist activity at the delta opioid receptor. The target peptides 1-7 were evaluated in the mouse vas deferens (MVD) and guinea pig ileum (GPI) at 1 microM. All of the compounds except [N,N-di-2-phenethyl,Leu5]enkephalin (7) showed antagonist activity in the MVD against the delta receptor agonist [D-Ala2,D-Leu5]enkephalin. The most potent congener, [N,N-dibenzyl,Leu5]enkephalin (3), was 2.5-fold more potent than [N,N-diallyl,Leu5]enkephalin (1). None of the compounds at 1 microM showed any antagonist activity against agonists for other receptor types. The N,N-di-2-phenethyl (7) and N,N-dioctyl (6) analogues showed significant agonist activity at 1 microM in the MVD.

Pyrrolomorphinans as delta opioid receptor antagonists. The role of steric hindrance in conferring selectivity.

A series of 2',3'-disubstituted pyrrolomorphinans (5a-i) were synthesized to determine the role of steric hindrance at mu and kappa receptors in promoting delta opioid receptor antagonist selectivity. In smooth muscle preparations, five members of the series (5a-c,e,f) possessed Ke values in the range 2-15 nM and were delta selective. Since the unsubstituted analogue 4 possessed delta antagonist potency of similar magnitude, but was not delta selective, it is suggested that the 2',3'-substitution confers delta selectivity by hindering the interaction of the pharmacophore at mu and kappa receptors, while not affecting delta receptors.

Synthesis and evaluation of 1- and 2-substituted fentanyl analogues for opioid activity.

We synthesized fentanyl analogues that possess key groups common to the opioid peptides to investigate whether or not these two classes of compounds interact with common subsites on opioid receptors. The design of the analogues was based on the possibility of structural analogy between the two aromatic rings of fentanyl and the Tyr1 and Phe4 residues of the opioid peptides. The synthesized compounds showed very weak or no opioid activity as tested in the electrically stimulated longitudinal muscle of the guinea pig ileum or mouse vas deferens preparations. These results, together with those of reported studies, suggest that fentanyl and the opioid peptides interact with different subsites on either mu or sigma receptors. Studies using the irreversible mu opioid receptor antagonist, beta-funaltrexamine, indicate that fentanyl interacts preferentially with mu opioid receptors.