Co-administration of morphine and oxycodone vaccines reduces the distribution of 6-monoacetylmorphine and oxycodone to brain in rats.

Opioid conjugate vaccines have shown promise in animal models as a potential treatment for opioid addiction. Individual vaccines are quite specific and each targets only a limited number of structurally similar opioids. Since opioid users can switch or transition between opioids, we studied a bivalent immunization strategy of combining 2 vaccines that could target several of the most commonly abused opioids; heroin, oxycodone and their active metabolites. Morphine (M) and oxycodone (OXY) haptens were conjugated to keyhole limpet hemocyanin (KLH) through tetraglycine (Gly)(4) linkers at the C6 position. Immunization of rats with M-KLH alone produced high titers of antibodies directed against heroin, 6-monoacetylmorphine (6-MAM) and morphine. Immunization with OXY-KLH produced high titers of antibodies against oxycodone and oxymorphone. Immunization with the bivalent vaccine produced consistently high antibody titers against both immunogens. Bivalent vaccine antibody titers against the individual immunogens were higher than with the monovalent vaccines alone owing, at least in part, to cross-reactivity of the antibodies. Administration of a single concurrent intravenous dose of 6-MAM and oxycodone to rats immunized with the bivalent vaccine increased 6-MAM, morphine and oxycodone retention in serum and reduced the distribution of 6-MAM and oxycodone to brain. Vaccine efficacy correlated with serum antibody titers for both monovalent vaccines, alone or in combination. Efficacy of the individual vaccines was not compromised by their combined use. Consistent with the enhanced titers in the bivalent group, a trend toward enhanced pharmacokinetic efficacy with the bivalent vaccine was observed. These data support the possibility of co-administering two or more opioid vaccines concurrently to target multiple abusable opioids without compromising the immunogenicity or efficacy of the individual components.

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.

The FEMA GRAS assessment of aliphatic and aromatic terpene hydrocarbons used as flavor ingredients.

This publication is the thirteenth 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 2600 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. Scientific data relevant to the safety evaluation of the use of aliphatic and aromatic terpene hydrocarbons as flavoring ingredients are evaluated. The group of aliphatic and aromatic terpene hydrocarbons 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 potential.

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.

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.

Safety evaluation of natural flavour complexes.

Natural flavour complexes (NFCs) are chemical mixtures obtained by applying physical separation methods to botanical sources. Many NFCs are derived from foods. In the present paper, a 12-step procedure for the safety evaluation of NFCs, 'the naturals paradigm', is discussed. This procedure, which is not intended to be viewed as a rigid check list, begins with a description of the chemical composition of the commercial product, followed by a review of the data on the history of dietary use. Next, each constituent of an NFC is assigned to one of 33 congeneric groups of structurally related substances and to one of three classes of toxic potential, each with its own exposure threshold of toxicological concern. The group of substances of unknown structure is placed in the class of greatest toxic potential. In subsequent steps, for each congeneric group the procedure determines the per capita intake, considers metabolic pathways and explores the need and availability of toxicological data. Additional toxicological and analytical data may be required for a comprehensive safety evaluation. The procedure concludes with an evaluation of the NFC in its entirety, also considering combined exposure to congeneric groups. The first experiences with the use of this procedure are very promising. Future safety evaluations of larger numbers of NFCs will indicate the usefulness of the system, either in its present form or in a form modified on the basis of experience.

Safety assessment of allylalkoxybenzene derivatives used as flavouring substances - methyl eugenol and estragole.

This publication is the seventh 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 flavouring substances under conditions of intended use. In this review, scientific data relevant to the safety evaluation of the allylalkoxybenzene derivatives methyl eugenol and estragole is critically evaluated by the FEMA Expert Panel. The hazard determination uses a mechanism-based approach in which production of the hepatotoxic sulfate conjugate of the 1'-hydroxy metabolite is used to interpret the pathological changes observed in different species of laboratory rodents in chronic and subchronic studies. In the risk evaluation, the effect of dose and metabolic activation on the production of the 1'-hydroxy metabolite in humans and laboratory animals is compared to assess the risk to humans from use of methyl eugenol and estragole as naturally occurring components of a traditional diet and as added flavouring substances. Both the qualitative and quantitative aspects of the molecular disposition of methyl eugenol and estragole and their associated toxicological sequelae have been relatively well defined from mammalian studies. Several studies have clearly established that the profiles of metabolism, metabolic activation, and covalent binding are dose dependent and that the relative importance diminishes markedly at low levels of exposure (i.e. these events are not linear with respect to dose). In particular, rodent studies show that these events are minimal probably in the dose range of 1-10 mg/kg body weight, which is approximately 100-1000 times the anticipated human exposure to these substances. For these reasons it is concluded that present exposure to methyl eugenol and estragole resulting from consumption of food, mainly spices and added as such, does not pose a significant cancer risk. Nevertheless, further studies are needed to define both the nature and implications of the dose-response curve in rats at low levels of exposure to methyl eugenol and estragole.

The FEMA GRAS assessment of pyrazine derivatives used as flavor ingredients. Flavor and Extract Manufacturers Association.

This is the fifth 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 taking into account the available scientific information on the group of structurally related substances. Scientific data relevant to the safety evaluation of the use of pyrazine derivatives as flavoring ingredients is evaluated.

The kappa-opioid antagonist GNTI reduces U50,488-, DAMGO-, and deprivation-induced feeding, but not butorphanol- and neuropeptide Y-induced feeding in rats.

Antagonists selective for either kappa- [e.g. nor-binaltorphimine (nor-BNI)] and mu- (e.g. beta-funaltrexamine) opioid receptors have previously been shown to reduce both kappa- and mu-opioid-induced feeding. In the present studies, the anorectic effects of GNTI, a newly synthesized antagonist selective for kappa-opioid receptors, were studied in rats. GNTI (0.032-0.32 nmol; i.c.v.), administered 15 min prior to food access, reduced feeding induced by the kappa-opioid agonist U50,488 (producing a 70% maximal decrease), the mu-opioid agonist DAMGO (90% maximal decrease), and 24 h acute food deprivation (60% maximal decrease). GNTI did not reduce the orexigenic effects of butorphanol, an agonist that binds to both kappa- and mu-opioid receptors, and neuropeptide Y (NPY). Taken together, these results suggest that GNTI is a potent anorectic agent and opioid antagonist in rats. Like nor-BNI, GNTI reduced feeding induced by both kappa- and mu-opioid agonists. However, unlike nor-BNI, GNTI did not alter the orexigenic effects of butorphanol or NPY. Given the selectivity of GNTI and its effectiveness in several of the present experiments, its potency, and its short duration of action compared to nor-BNI, GNTI may serve to be a useful tool to study behavioral effects mediated by kappa-opioid receptors.

Transformation of a kappa-opioid receptor antagonist to a kappa-agonist by transfer of a guanidinium group from the 5'- to 6'-position of naltrindole.

The importance of the indole scaffold of GNTI 3 in directing its address (5'-guanidinium group) to associate with the Glu297 residue of the kappa-opioid receptor was investigated by the synthesis and biological evaluation of its 4'- (4a), 6'- (4b), and 7'- (4c) regioisomers. The finding that only the 5'-regioisomer (GNTI) possessed potent kappa-opioid antagonist activity and high affinity at kappa-receptors illustrates the importance of the 5'-position in orienting the guanidinium group to the proper recognition locus (Glu 297) for potent kappa-antagonist activity. The discovery that the 6'-regioisomer of GNTI was a potent kappa-agonist, together with the results of site-directed mutagenesis studies that are consistent with association between the 6'-guanidinium group and Glu297, suggest that the transition from an inactive to an active state of the kappa-receptor involves a conformational change of TM6. We propose that association of the 6'-guanidinium group of 4b with Glu297 promotes axial rotational motion of transmembrane helix VI which leads to receptor activation via a conformational change of inner loop 3.

Affinity labels as tools for the identification of opioid receptor recognition sites.

Affinity labels have proven to be useful tools in opioid research. We review experiments carried out with the mu opioid receptor affinity label, beta-funaltrexamine (2), that support the concept of different recognition sites for mu opioid agonists and antagonists. The data are interpreted in the context of a dimeric receptor that contains two allosterically coupled binding sites: one that binds endogenous agonist, and the second that functions as an inhibitory modulator of agonism. It is proposed that exogenous antagonists bind selectively to the second site. The first of a new class of affinity labels, PGNA (5), that contains the phthaldehyde moiety attached to an opioid antagonist pharmacophore, is described. This class of ligands has been named 'reporter affinity labels' because covalent association leads to the formation of a fluorescent isoindole that is diagnostic for cross-linking of lysine and cysteine residues. PGNA binds opioid receptors covalently, as suggested by (a) irreversible binding to cloned opioid receptors, (b) irreversible opioid antagonism in the guinea pig ileum preparation, and (c) ultra-long opioid antagonism in mice. Since flow cytometry experiments revealed specific enhancement of fluorescence in cloned mu receptors after a 1 min exposure to 5, it is concluded that covalent binding has occurred via the formation of an isoindole, presumably by cross-linking neighboring lysine and cysteine residues in the vicinity of the receptor recognition site.

Pharmacological evidence for a 7-benzylidenenaltrexone-preferring opioid receptor mediating the inhibitory actions of peptidic delta- and mu-opioid agonists on neurogenic ion transport in porcine ileal mucosa.

The antidiarrheal and constipating effects of opiates are partly attributed to reductions in active anion secretion across the intestinal mucosa that are modulated by submucosal neurons. In this study, the opioid receptor mediating the actions of opioids on ion transport was characterized in mucosa-submucosa sheets from porcine ileum. Electrical transmural stimulation evoked transient increases in short-circuit current, an electrical measure of neurogenic ion transport, in this preparation. After serosal addition, the peptidic delta-opioid agonists [D-Ala(2)]-deltorphin II (pIC(50) = 8.4 +/- 0.7), [D-Ala(2),D-Leu(5)]-enkephalin (DADLE), [D-Pen(2),D-Pen(5)]-enkephalin (DPDPE), and [D-Ser(2),Leu(5),Thr(6)]-enkephalin (DSLET), and the mu-opioid agonists [D-Ala(2),N-methyl-Phe(4),Gly(5)-ol]-enkephalin (DAMGO) (pIC(50) = 8.0 +/- 0.1), endomorphin I, and PL-017 inhibited short-circuit current elevations. Nonpeptidic mu- or delta-opioid agonists (morphine, loperamide, and SNC80) and kappa-opioid agonists (U-50,488H and U-69,593) were <360-fold less potent than deltorphin II. At 100 nM, the delta(1)-opioid antagonist 7-benzylidenenaltrexone reduced the potencies of DPDPE and DAMGO by 13.5- and 15.5-fold, respectively; at an identical concentration naltriben, a delta(2)-opioid antagonist, or the mu-opioid antagonist D-Phe-Cys-Tyr-D-Trp-Orn-Thr-Pen-Thr-NH(2) (CTOP) reduced DPDPE potency by 4.1- and 3.4-fold, respectively, but had no significant effect on DAMGO potency. Using primary antisera directed toward cloned opioid receptors, delta-opioid receptor immunoreactivity was immunohistochemically localized in submucosal neurons and nerve fibers, but immunoreactivities to kappa- or mu-opioid receptors were not detected in the mucosa-submucosa. These results suggest that a novel 7-benzylidenenaltrexone-sensitive opioid receptor is expressed in submucosal neurons of the porcine ileum, which mediates the inhibitory effects of peptidic mu- and delta-opioid agonists on neurogenic ion transport.

Investigation of the selectivity of oxymorphone- and naltrexone-derived ligands via site-directed mutagenesis of opioid receptors: exploring the "address" recognition locus.

The delta-selective opioid antagonist naltrindole (NTI), as well as the kappa-selective opioid antagonists norbinaltorphimine (norBNI) and 5'-guanidinonaltrindole (GNTI), are derived from naltrexone, a universal opioid antagonist. Previous studies have indicated that extracellular loop III is the key region for discrimination by naltrexone-derived selective ligands between the delta, mu, and kappa opioid receptor types. It has been proposed that selective ligands could bind to all three receptor types if the appropriate portions of the extracellular loops were eliminated. To investigate this possibility, several single-point mutant opioid receptors have been generated with the aim of conferring enhanced affinity of selective ligands for their nonpreferred receptor types. Mutations were made in all three types of opioid receptors with the focus on two positions at the extracellular end of transmembrane regions (TM) VI and VII. It was found that the delta-selective NTI could bind both mu and kappa receptors with significantly enhanced affinity when an aromatic residue in TM VII was replaced with alanine (mu[W318A] and kappa[Y312A]). Similarly, kappa-selective antagonists, norBNI and GNTI, showed enhanced affinity for the mu[W318A] mutant and for both mu and delta receptors when a glutamate residue was incorporated into the extracellular end of TM VI (mu[K303E] and delta[W284E]). These results demonstrate that naltrexone-derived selective ligands achieve their selectivity via a combination of enhanced affinity of the address for a particular subsite along with loss of affinity due to steric interference at nonpreferred types. The results reveal key residues in the "address" recognition locus that contribute to the selectivity of opioid ligands and support the hypothesis that recognition of the naltrexone moiety is essentially the same for all three receptor types.