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Although activated spinal cord glia contribute importantly to neuropathic pain, how nerve injury activates glia remains controversial. It has recently been proposed, on the basis of genetic approaches, that toll-like receptor 4 (TLR4) may be a key receptor for initiating microglial activation following L5 spinal nerve injury. The present studies extend this idea pharmacologically by showing that TLR4 is key for maintaining neuropathic pain following sciatic nerve chronic constriction injury (CCI). Established neuropathic pain was reversed by intrathecally delivered TLR4 receptor antagonists derived from lipopolysaccharide. Additionally, (+)-naltrexone, (+)-naloxone, and (-)-naloxone, which we show here to be TLR4 antagonists in vitro on both stably transfected HEK293-TLR4 and microglial cell lines, suppressed neuropathic pain with complete reversal upon chronic infusion. Immunohistochemical analyses of spinal cords following chronic infusion revealed suppression of CCI-induced microglial activation by (+)-naloxone and (-)-naloxone, paralleling reversal of neuropathic pain. Together, these CCI data support the conclusion that neuron-to-glia signaling through TLR4 is important not only for initiating neuropathic pain, as suggested previously, but also for maintaining established neuropathic pain. Furthermore, these studies suggest that the novel TLR4 antagonists (+)-naloxone and (-)-naloxone can each fully reverse established neuropathic pain upon multi-day administration. This finding with (+)-naloxone is of potential clinical relevance. This is because (+)-naloxone is an antagonist that is inactive at the (-)-opioid selective receptors on neurons that produce analgesia. Thus, these data suggest that (+)-opioid antagonists such as (+)-naloxone may be useful clinically to suppress glial activation, yet (-)-opioid agonists suppress pain.

Aims: To evaluate individual variations in plasma concentrations over time in patients with naltrexone implants., Methods: Ten opioid-dependent patients received up to four implants. Plasma samples were collected regularly for the analyses of naltrexone and the metabolite beta-naltrexol., Results: The median naltrexone C(max) was 12.3 (range 5.8-22.1) ng ml(-1), the median T(max) was 1 day (range 3 h to 35 days), and the median length of time that plasma concentrations were above 1 ng ml(-1) was 55 (range 30-80) days. Two patients reported heroin use without experiencing any effect. Tissue reactions were recorded in two patients after repeated implantation., Conclusion: Marked individual and intraindividual variations in naltrexone concentrations were observed. Further studies should be performed to evaluate the need for therapeutic drug monitoring during naltrexone implant treatment.

Naltrexone and naloxone, being competitive antagonists of opioid receptors, have found therapeutic applications in medicine. The experiments with mutant receptors showed that many amino acid residues within transmembrane domains play an important role in binding these drugs. Using the site-directed mutagenesis technique, it was established that even single mutations (replacing single amino acid residues) can significantly modify the affinity of antagonists to receptors, sometimes even imparting agonist-like properties to the compounds studied. Chronic administration of naltrexone and naloxone leads to an increase in the density of opioid receptors and in the sensitivity to agonists. This hypersensitivity and overdose risk in heroin abusers after chromic naltrexone treatment are discussed.

We hypothesized that the selectivity profile of the rat mu-opioid receptor for opioid receptor-selective ligands is determined by the nature of the amino acid residues at highly divergent sites in the ligand-binding pocket. To determine which characteristics of these residues contribute to opioid receptor ligand selectivity, we made various mutant receptors that replaced the Lys(303) and Trp(318) residues near the extracellular interface of transmembrane domains VI and VII, respectively. Ligand binding determinations using transiently transfected monkey kidney epithelial (COS-1) cells show that Lys(303) mutations cause little change in the receptor binding profile, whereas the Trp(318) mutant receptors have considerably lower affinity for micro-opioid receptor-selective ligands and greatly increased affinity for delta-opioid receptor-selective ligands. The nature of these mutations show that this effect is not due to sterics or charge alone. [35S]guanosine-5'-O-(3-thio)-triphosphate ([35S]GTPgammaS) activity assays show that these residues may influence functional, as well as binding selection. We conclude that a primary role for Trp(318) is to form a basis for ligand selectivity.

Highly selective heterocyclic opioid ligands with potent delta-antagonist activity have been developed on the basis of the "message-address" concept. Using this strategy, benzofuran derivatives corresponding to the non-selective opioid antagonist, naloxone, and to the mu-opioid receptor selective agonists, oxymorphone and oxycodone, were synthesized. In vitro opioid receptor binding profiles and agonist/antagonist character of these compounds were determined in rat brain membrane preparations with highly selective radioligands. All three benzofuran derivatives displayed high affinities for the delta-opioid receptor, much less potency toward the mu-binding site, and were the least effective at the kappa-site. The results indicated that the addition of the bezofuran moiety to these fused ring opioids confers delta-receptor selectivity. The Na+ indices suggested a partial agonist character for oxymorphone- and oxycodone-benzofuran, and an antagonist character for naloxone-benzofuran. These compounds were capable of irreversible inhibition of opioid binding sites in a dose-dependent.

Healthy male volunteers (N = 24) participated in a four-way crossover study to compare the rate and extent of absorption of naltrexone after administration of 50 mg tablets as 50, 100, and 200 mg doses and a 10 mg/ml reference syrup. A high-performance liquid chromatographic method was employed to measure naltrexone and 6-beta-naltrexol in plasma and urine. Compared to the syrup, the 50 mg tablets were absorbed more slowly but equally well. There was excellent linearity between the administered dose and the area under the plasma concentration-time profile, as well as total urinary recovery of both drug and metabolite. The mean half-lives for naltrexone and beta-naltrexol were approximately 4 and 12 hours, respectively. The fraction of drug reaching the systemic circulation was estimated to be 5% of the administered dose because of extensive first-pass metabolism. Less than 1% of the dose was excreted in the urine as naltrexone after 48 hours, while 25% was recovered as unconjugated beta-naltrexol. The renal clearance of naltrexone and beta-naltrexol was approximately 127 ml/min and 283 ml/min, respectively. The total systemic clearance for naltrexone was approximately 94 L/hr.

A biodegradable sustained-release naltrexone bead preparation containing 70% naltrexone in a physical mixture with a copolymer of 90% lactic acid and 10% glycolic acid was evaluated in three male subjects. Each subject received a 10-mg iv dose of naltrexone HCl and a 63-mg dose by subcutaneous implantation of naltrexone beads. Kinetics of naltrexone estimated from the intravenous dose indicated a plasma clearance range of 3.1 to 3.4 l/min and a t 1/2 range of 1.7 to 3.7 hr. After bead implantation, average plasma naltrexone levels were maintained at 0.3 to 0.4 ng/ml and naltrexol levels were at 0.4 to 1.0 ng/ml for a period of approximately 1 mo, during which urine naltrexone and naltrexol levels were about 20 to 30 and 70 to 200 ng/ml. It was estimated that approximately 70% to 77% of the dose was absorbed after bead implantation. There were no serious adverse effects other than tissue irritation in two of the three subjects.

The diastereomeric 6-desoxy-6-spiro-alpha-methylene-gamma-butyrolactone derivatives of naltrexone (4a and 5a) and of oxymorphone (4b and 5b) were prepared from their parent ketones. Diastereomers 4a and 4b were obtained from the 3,14-diacetate derivatives of naltrexone (6a) and oxymorphone (6b) by reaction with the Reformatsky reagent prepared from methyl alpha-(bromomethyl)acrylate. Deacetylation with methanol completed the synthesis. Diastereomers 5a and 5b were obtained from oxiranes 8a and 8b, respectively. The oxiranes were allowed to react with the sodium salt of ethyl acetoacetate, followed by methenation and deprotection to complete the synthesis of 5a and 5b, respectively. Compound 5a was the most potent agent tested in competition against [3H]naltrexone in the opioid radioreceptor assay. At a concentration of 5 nM this compound produced a 50% inhibition of binding. The majority of this inhibition (30%) was irreversible, i.e., it remained even after extensive washing of the membrane preparation in the presence and absence of Na+. Naloxone protected against this irreversible effect. The data suggest a receptor nucleophile, perhaps a sulfhydryl group, is located where it can add to the alpha, beta-unsaturated carbonyl system of 5a.

In vitro binding studies have demonstrated the existence of multiple opiate receptor types. An additional site in the rat brain (termed the lambda site) is distinct from the established types by its selectivity for 4,5-epoxymorphinans (such as naloxone and morphine). While the lambda site displays a high affinity for naloxone in vivo and in vitro in fresh brain membrane homogenates, these sites rapidly convert in vitro to a state of low affinity. The regional distribution of the lambda site in the brain is strikingly different from that of the classic opiate receptor types.

The serum kinetics of 5 mg/kg of i. v. naloxone or naltrexone were studied in each of two groups of five dogs; serum samples were obtained from 2 min to 2 hr after injection. Serum concentrations were determined by radioimmunoassay. Serum levels of both naloxone and naltrexone fell rapidly; serum half-life during the elimination phase was 71.2 +/- 8.9 min (mean +/- S.E.) for naloxone and 85.1 +/- 9.0 min (mean +/- S.E.) for naltrexone. Although human and dog kinetics are similar for naloxone, naltrexone is long-acting in man, but is quickly dissipated in the dog.

A method designed to quantitate in vivo naltrexone release rates from sustained-release systems has been applied to the evaluation of seven different naltrexone delivery systems in the monkey. The method consists of two phases: a single intravenous bolus dose quantitation of each monkey's pharmacokinetic parameters coupled with a delivery system study in which plasma naltrexone levels are measured throughout the time period of sustained-release. In vivo release rates and the total amount released are then calculated. It should be noted that these determinations require the analysis of unchanged naltrexone in plasma as the only experimental measurement. Data from injectable naltrexone pamoate microcapsule delivery systems indicate that 1) when these microcapsules are suspended in an aqueous vehicle, a significant part of the dose is released very rapidly, yielding release rate-time data that parallel a non-sustained-release control; 2) this rapid release for the aqueous vehicle is followed by a slow release phase lasting to about 24 days for the subcutaneous route and to about 45 days for the intramuscular route; and 3) when these microcapsules are suspended in an oily vehicle there is no initial rapid release, substantial release rates are obtained for at least 60 days, and an average of 89% of the dose is calculated to have been released. Data from implantable naltrexone delivery systems show that 1) the Alza system most closely approximates a zero-order release rate-time profile; 2) the Battelle system provides a rapid initial release followed by a slowly declining release rate; 3) the Dynatech system is characterized by a more rapid initial release rate of 3-8% of the dose per day over the first 3-5 days followed by a rather constant 1-3% per day to about day 36; and 4) essentially complete recovery of the dose was obtained for the Battelle and Dynatech systems.

Naltrexone release rates from a controlled release delivery system have been quantitated over a time period greater than one month in the monkey. The method requires calibration of the pharmacokinetic parameters of each monkey utilizing an intravenous bolus dose and assay of unchanged naltrexone levels in plasma as a function of time after dosing. Also required are periodic plasma levels of unchanged naltrexone obtained subsequent to administration of the delivery system. Release rates are then calculated as well as the total amount released. Application of the methodology to a biodegradable copolymer naltrexone delivery system in three monkeys showed an initial release rate of 3-8% of the dose per day over the first 3-5 days followed by a slow, rather constant release rate of 1-3% per day from day 5 to the time of the last measurable plasma sample (36-43 days). Comparison of alternative calculation methods using both experimental and simulated plasma naltrexone data verified the accuracy of the release rate calculations. The sum of the calculated total amount of naltrexone released plus the assayed amount remaining in the delivery system after removal from the animal accounted for 91-94% of the administered dose in the two monkeys in which complete data were obtained.

Chlornaltrexamine (CNA) produces ultralong-lasting (3--6 days) narcotic antagonism in mice and persistent stereospecific binding to rat-brain homogenate. Protection studies in mice suggest that CNA mediates its narcotic antagonist effects by interacting with the same receptors that are occupied by naloxone. A single icv dose of CNA also has been found to inhibit the development of physical dependence in mice for at least 3 days. These studies suggest that CNA exerts its sustained effects by selective covalent association with opioid receptors.

We have previously reported that chronic administration of naloxone to SHR and WKY rats results in a significant increase in their systolic blood pressure relative to control animals. In the present study we show that chronic naloxone is also accompanied by a marked increase in the number of brain opiate receptors. Although the relative difference in blood pressure diminishes with increasing maturity, the elevation in brain opiate receptors remains in the treated animals. Mechanisms for these differential effects are discussed.

A specific high-pressure liquid chromatographic assay was developed for the measurement of the narcotic antagonist drug, naltrexone. When 10 mg of naltrexone . HCl was added to the perfusate of the isolated perfused rat liver, the concentration of the drug in the perfusate declined rapidly with an apparent half-life of about 9 min. At 30 min 90% of the unchanged drug had been removed from the perfusate; the liver at this time retained 47% of the total radioactivity, of which only 75 represented unchanged drug. The bile at 30 min contained 28% of the total radioactivity and by 120 min this had risen to 70%. Initially naltrexone glucuronide was the principal metabolite in the bile. After 60 min N-dealkylated naltrexone glucuronide was the principal metabolite in bile. A marked increase in bile flow was noted immediately after the addition of the drug to the perfusate. The choleretic effect was attributed to the osmotic effect of the metabolites of naltrexone in the bile.

The basis for using narcotic antagonists for the treatment of opiate addiction is discussed briefly, and the chemistry, pharmacology, pharmacokinetics, clinical efficacy, adverse effects, and dosage of naltrexone hydrochloride, an opiate antagonist drug, are reviewed. Naltrexone is rapidly and completely absorbed after oral administration but undergoes substantial first-pass extraction and metabolism by the liver. Naltrexone has a half-life of 3.9-10.3 hours and a slow terminal elimination-phase half-life of 96 hours. The major metabolite of naltrexone is 6-beta-naltrexol, which is present in plasma in greater concentrations than the parent drug. Problems in study design and patient adherence to treatment have made assessment of naltrexone's clinical efficacy difficult; however, studies have consistently demonstrated that patients who remained off opiates longer were those who took naltrexone longer. Factors associated with successful treatment outcomes include sustained therapy with naltrexone, participation in multidisciplinary programs of behavioral therapy and psychotherapy, and good family and social support systems. Gastrointestinal irritation and, in some studies, clinically insignificant increases in blood pressure, have been the most common adverse effects. Currently available data suggest that naltrexone is a very safe drug. The usual dosage of naltrexone hydrochloride is 50 mg orally once daily or 350 mg orally per week in three divided doses. Patients should be detoxified and opiate free for two to five days before initiation of naltrexone therapy. Naltrexone appears to be a useful adjunct to therapy in opiate addicts who are well motivated and who have strong psychological support systems.

It has been inferred that ethanol suppresses the secretion of luteinizing hormone (LH) in the male by depressing the release of LH-releasing hormone (LH-RH) from the hypothalamus. Direct support for this inference has been difficult to obtain, however, because of significant technical difficulties in measuring LH-RH release under in vivo conditions. To circumvent these problems, we made use of the opiate antagonist naloxone, as a neuroendocrine probe, to elicit the release of LH-RH under in vivo conditions. We found that ethanol was a potent suppressor of the increase in serum LH levels evoked by naloxone at extremely low blood ethanol concentrations ( less than 60 mg/dl). Furthermore, we observed that the antagonism between ethanol and naloxone appeared to be competitive in nature since a fixed dose of ethanol (1 g/kg, blood ethanol concentration 60 mg/dl) shifted the naloxone dose-response curve significantly to the right and high doses of the antagonist overcame ethanol's effects. Finally, we found that the interaction between ethanol and naloxone took place at the level of the hypothalamus. Our results, therefore, seem to provide the first in vivo evidence supporting the widely-held hypothesis that ethanol reduces serum LH levels by depressing the hypothalamically-medicated release of LH-RH. The mechanisms underlying ethanol's depression of naloxone-induced increases in the release of LH-RH are not fully understood at this time, but one prominent possibility is that ethanol enhances the synthesis or release of endogenous opioids which in turn override naloxone's effects.

Ethylketocyclazocine (1) has greater kappa/mu selectivity than cyclazocine in brain binding assays. 10-Ketonaltrexone (11) and 10-ketooxymorphone (10) were prepared from naltrexone 3-methyl ether and oxycodone, respectively. Bioassays in the myenteric plexus longitudinal muscle preparation of the guinea pig ileum and in the mouse vas deferens, in addition to brain binding assays, demonstrated that 10 and 11 were far less potent than naltrexone (2) and oxymorphone (3) at mu sites and also had little affinity for kappa and delta sites. It is concluded that introduction of the 10-keto group in naltrexone and oxymorphone diminished opioid effects at all binding sites.

A rapid quantitative method was developed to assay in urine naltrexone and its major urinary metabolite, beta-naltrexol. Following solvent extraction and elimination of interfering materials, the weakly basic drug, its metabolite and the internal standard (etorphine) were silylated and analyzed by gas-liquid chromatography. As little as 0.02 mug/ml of naltrexone and beta-naltrexol was detectable using a hydrogen flame ionization detector. Twenty-four-hour urine samples were analyzed from three subjects taking 180 mg naltrexone daily. The major urinary excretion product was beta-naltrexol which accounted for 48.6% of the administered dose. Only 5% of the administered dose excreted in the urine was naltrexone. Beta-naltrexol was excreted 70% as free drug and 30% conjugated while naltrexone was 90% conjugated.

Sensitive reversed-phase HPLC assays with electrochemical detection, developed to quantify naltrexone, 6 beta-naltrexol, and their conjugates in biological fluids, provided assay sensitivities of 2-14 ng/mL in plasma, urine, and bile. Plasma, urine, and bile were monitored in dogs after bolus administrations of 0.5 and 5.0 mg/kg iv naltrexone hydrochloride. Plasma-time data showed two sequential half-lives of 5 +/- 1 (SEM) and 47 +/- 5 min. Pharmacokinetics were dose-independent; total and renal clearances were 1043 +/- 98 mL/min and 72 +/- 11 mL/min, respectively, with a similar renal clearance (85 +/- 12) for the conjugate. The percentages of the dose excreted in the urine as naltrexone and its conjugate were 7 +/- 1% and 58 +/- 3%, respectively, with the remainder being excreted in the bile as conjugates. As much as 36% was collected as conjugate in the total bile of the bile-cannulated dog. There was no biliary secretion of unchanged naltrexone. The conjugate was apparently enterohepatically recirculated. 6 beta-Naltrexol is not a metabolite of naltrexone in dogs. Within the limits of analytical detection (2 ng/mL) neither 6 beta-naltrexol nor its conjugates appeared in any monitored biological fluids when such fluids were assayed quickly after sampling.

The evaluation of the drug release characteristic of four naltrexone delivery systems has been carried out together with the development of analytical techniques and an investigation of the metabolic profile of naltrexone. Pharmacologic evaluation of the four delivery systems in the mouse indicated significant analgesic antagonism for a period of from 16-22 days. Further evaluation of one of these systems by measurement of the rate of excretion of radioactivity after administration of radiolabelled naltrexone in the delivery system confirmed that significant release occurs for a time period of about 15 days. Electron capture gas-liquid chromatographic assays for naltrexone and naloxone in plasma or urine have been developed that yield linear calibration curves and are sensitive to one ng/ml. Studies on naltrexone disposition indicate that (a) binding to plasma proteins in several species varies from 20-26 per cent, (b) distribution of drug from blood is extremely rapid and extensive, (c) beta-naltrexol is a major metabolite of naltrexone in man, monkey and guinea pig among six species studied, whereas alpha-naltrexol is a minor metabolite in the monkey and guinea pig only, and (d) metabolic reduction of naltrexone occurs in the 100,000 x g supernatant of guinea pig liver. Pharmacokinetic studies of naltrexone in the dog and monkey indicate that the drug is rapidly distributed and eliminated, has a very large apparent volume of distribution and a total body clearance greater than the rate of liver blood flow.

Naltrexone was incubated with the 9000 X g supernatant of guinea pig liver in the presence of an NADPH-generating system to determine the relative amounts of the 6-keto reduction products, alpha- and beta-naltrexol, formed in vitro. After a 0.5-2 hour period both alpha- and beta-naltrexol were formed as detected by electron capture gas chromatography of pentafluoropropionic anhydride-derivatized extracts of the incubation mixture. The identity of alpha-naltrexol was confirmed by nuclear magnetic resonance, infrared and mass spectra, as well as by mixed melting point determination. The percentage of natrexol found as the alpha-epimer ranged from 36-81% and was concentration-dependent at substrate concentrations ranging from 0.0007-0.1 mM. The hepatic enzyme(s) responsible for the reduction of naltrexone were localized in the 105,000 X g supernatant fraction of guinea pig liver. In contrast to results obtained for the reduction of naltrexone using guinea pig liver, the 9000 X g and 105,000 X g supernatant fractions of monkey liver appeared to reduce naltrexone almost exclusively to beta-naltrexol. The 9000 X g supernatant of rat liver was less active than similar preparations from guinea pig or monkey liver; only a small amount of beta-naltrexol and no detectable alpha-naltrexol was formed after two hours of incubation. Incubation of the naltrexone metabolites, alpha- or beta-naltrexol, with the 9000 X g supernatant of guinea pig liver and incubation of alpha-naltrexol with guinea pig kidney slices yielded no evidence of interconversion of these metabolites.

Synthetic polypeptides consisting of copolymers of glutamic acid and leucine have been shown to be useful materials for the fabrication of practical, biodegradable delivery vehicles for narcotic antagonists. Model delivery vehicles in film form were prepared from copolymers containing 10 mole percent to 40 mole percent glutamic acid, and loaded with 10% to 40% naltrexone by weight. The naltrexone was found to be released by diffusion, exhibiting diffusion coefficients that varied as a function of the glutamic acid content and the initial naltrexone loading. A wide range in diffusion coefficients were achieved (0.31 x 10(-7) cm2/hr to 120 x 10(-7) cm2/hr), leading to release rates within practical ranges of interest for meeting the program goals. We have demonstrated that the polypeptides can be fabricated into dosage forms that are amenable to administration by trochar. For example, rods 0.4 mm to 0.8 mm in diameter containing as much as 40% naltrexone by weight were extruded using a simple compression mold and die arrangement. An in vitro evaluation of the rods showed that antagonist is released by diffusion at a continuously decreasing rate, a behavior similar to that observed with the film devices that were, nonetheless, capable of blocking an AD80 challenge of morphine sulfate in mice for more than 30 days. One of the most promising delivery vehicles that we have developed to date consists of a polypeptide tube filled with a naltrexone/polypeptide core. Preliminary experiments have shown that these devices may be capable of administering high, constant rates of release for prolonged periods of time. Additional work, however, is required to develop techniques for the preparation of reproducible delivery vehicles.

The metabolism and elimination of [15,16-3H2] naltrexone hydrochloride was studied in man following oral and intravenous administration. The same metabolites, although in varying proportions, were observed in both cases; conjugated naltrexone and nonconjugated and conjugated 6 beta-naltrexol were the major metabolites observed in plasma, urine, and feces. 2-Hydroxy-3-O-methyl-6 beta-naltrexol was found in minor quantities. Naltrexone was almost completely in minor quantities. Naltrexone was almost completely absorbed following in the urine and only 5% in the feces. A similar urinary excretion pattern was observed after intravenous administration of naltrexone. In early time periods after oral administration there was a rapid increase in free naltrexone plasma levels up to 1 hr and then gradually declined. A similar pattern was observed for conjugated naltrexone and nonconjugated and conjugated 6 beta-naltrexol. These metabolites were found at levels 4-6 times higher than the parent compound at all times sampled. After intravenous administration, nonconjugated naltrexone plasma levels dropped sharply and continuously. The major metabolites exhibited a pattern closely resembling that found for oral administration. Combined gas chromatography-mass spectrometry was used to validate the presence of naltrexone, 6 beta-naltrexol and 2-hydroxy-3-O-methyl-6 beta-naltrexol in urine. The structure of the latter was rigorously proven by 13C-NMR. No evidence for the presence of noroxymorphone or 3-O-methyl-6 beta-naltrexol could be obtained by gas chromatography-mass spectrometry. The metabolism of naltrexone administered subcutaneously was also determined in two subjects. Larger amounts of 2-hydroxy-3-O-methyl-6 beta-naltrexol were found in plasma than had been present after oral or intravenous administration.

Experimental data on 3H labelled naltrexone obtained from the Research Triangle Institute were analyzed. Both plasma and urine curves were resolved into their exponential components. Mean total clearance of unconjugated naltrexone was 3510 (300) ml/min, and its mean urinary clearance was 76.0 (6.8) ml/min. Fraction of dose excreted through urine was between 55 and 60%. Orally administered drug is completely absorbed into plasma. Plasma curve resulting from intravenous administration contained three exponentials: 1.9 (.5)/min, 0.18 (.07)/min and 0.00106 (.00008)/min. Distribution volumes were 7.6 (.7) liters, 60 (12) liters and 164 (16) liters. After oral administration there were two exponentials: 0.012 (.002/min and 0.00122 (.00009)/min. The faster exponential corresponds to gastrointestinal absorption with a half life of about 1 hour.

The metabolism and elimination of [15, 16,-3H2]naltrexone was studied in man after oral and intravenous administration. The same metabolites, although in varying proportions, were observed in both cases; conjugated naltrexone and conjugated and unconjugated 6 beta-naltrexol were the major metabolites observed in plasma, urine, and feces. 2-Hydroxy-3-O-methyl-6 beta-naltrexol was found in minor quantities. Naltrexone was almost completely absorbed after oral administration. After oral and intravenous administration of naltrexone, about 60% of the dose was recovered in the urine in 48 and 72 hr, respectively. The route of administration did not significantly affect urinary clearance values obtained for unconjugated or conjugated naltrexone and 6 beta-naltrexol. The route of administration significantly affected terminal plasma half-life values obtained for unconjugated naltrexone (2.7 hr, iv; 8.9 hr, oral), but had little effect on comparable values obtained for total drug, conjugated naltrexone, and unconjugated and conjugated 6 beta-naltrexol. Combined gas chromatography-mass spectrometry was used to validate the presence of naltrexone, 6 beta-naltrexol, and 2-hydroxy-3-O-methyl-6 beta-naltrexol in urine.

A new metabolite of naltrexone was isolated from urine and erythrocytes of patients taking 100 mg naltrexone daily. Its tentative structure of 2-hydroxy-3-methoxy naltrexol was derived by analysis of the purified compound using gas chromatography combined with mass spectrometry, high resolution mass spectrometry and infrared spectrometry. Long bioavailability of the new metabolite is indicated by its slower excretion rate into the urine than naltrexone and beta-naltrexol. The rather high concentration of the new metabolite in the erythrocytes and its solubility in non-polar solvents like benzene demonstrate the ability of this compound to penetrate membranes. These features of the new metabolite suggest that it may contribute to naltrexone's long time course of action.

[3H]-Methionine-enkephalin, [3H]-naltrexone and [3H]-dihydromorphine are specifically bound in homogenates of not only the brain and myenteric plexus of the guinea pig but also the vas deferens of the mouse. Brain has a ratio of methionine-enkephalin to dihydromorphine binding in favour of methionine-enkephalin binding and the myenteric plexus a ratio in favour of dihydromorphine binding, with the mouse vas deferens being intermediate.

The elimination of radioactivity after [15,16-3H]naltrexone administration was studied in rats and guinea pigs. An average of 42% of the dose was eliminated in urine and 55% in feces following administration of 1 mg/kg iv to each of three rats. Analysis of radioactivity in the excreta of one rat that received the same dose im yielded similar results. On the other hand, four guinea pigs that received 1 mg/kg iv excreted only 14% of the dose in feces and 84% in urine. Similar results were obtained following im administration to guinea pigs at 1 and 20 mg/kg doses. In guinea pig excreta, an average of 64% of the dose corresponded to naltrexone and conjugates, 19% to beta-naltrexol and conjugates, and 2% to alpha-naltrexol and conjugates. In urine, the radioactivity corresponding to alpha-naltrexol and naltrexone was present mainly in conjugated form, whereas apparent beta-naltrexol was mainly unconjugated. The radioactivity in feces corresponded principally to unconjugated naltrexone and beta-naltrexol.

Epimeric 6-amino derivatives of naloxone and naltrexone have been synthesized and the configuration at the C-6 chiral center was determined from NMR studies. All of the derivatives possess narcotic antagonist activity in mice, with each of the 6beta epimers having greater potency than the corresponding 6alpha epimers. In vitro binding experiments indicate that the affinities of these epimers parallel thier in vivo potencies. Slight antinociceptic properties were observed with three of the four compounds. The naloxone derivatives 3a and 3b appear to be attractive candidates for investigation as long-acting narcotic antagonists in view of their fourfold greater duration of action relative to the other antagonists (1, 2, 4a, and 4b).

Stereospecific binding of 3H-labeled [D-Ala2,D-Leu5]enkephalin is irreversibly inactivated by the sulfhydryl group alkylating agent N-ethylmaleimide. This inactivation, like that of opiate binding, exhibits pseudo-first-order kinetics with a half-inactivation time of 10-12 min. The presence of opiates or enkephalins during incubation with N-ethylmaleimide provides good protection. Ouantitative studies of protection demonstrate that naltrexone and morphine are 20 and 8 times, respectively, more effective in protecting the binding of [3H]naltrexone than that of [3H]enkephalin. [D-Ala2,Leu]Enkephalin and [D-Ala2,Met]enkephalin, however, are more effective (7 and 30 times, respectively) for the protection of 3H-labeled [D-Ala2,D-Leu5]enkephalin binding. These results provide strong evidence for the existence of two classes of opiate receptor in rat brain.

beta-Funaltrexamine (beta-FNA) bound to mouse brain membranes in a reversible and an irreversible (not removed by washing of the membrane) manner, and a portion of each type of binding was opioid-specific. Addition of 100 mM NaCl to the incubating medium enhanced the binding of beta-FNA to membranes. Using membranes preincubated with beta-FNA (1 microM) and then washed three times, the maximum number of binding sites available to [3H]morphine was markedly diminished whereas the affinity of morphine for binding sites was not significantly altered. The binding of [3H]naltrexone was also reduced markedly by beta-FNA pretreatment. In similarly pretreated membranes, the binding of [3H]methionine enkephalin [3H][D-Ala2,D-Leu5]enkephalin (DADLE) or [3H]ethylketazocine was reduced to a smaller extent. Using brain membranes from mice pretreated with a single subcutaneous injection of beta-FNA (100 mg/kg) 48 h prior to use, the binding of [3H]methionine enkephalin was unaffected whereas the number of binding sites available to [3H]morphine was significantly reduced. The inhibition by various ligands of the reversible binding of [3H] beta-FNA resembled the relative ability of the same ligands to inhibit the binding of [3H]ethylketazocine. It was concluded that the irreversible portion of the binding of beta-FNA demonstrates a selectivity for mu over delta binding sites, and that the reversible portion of the binding of beta-FNA demonstrates a selectivity for kappa binding sites over mu or delta binding sites. As such, the binding characteristics of beta-FNA are consistent with its profile in vivo and in isolated tissue studies in vitro.

The disposition of naltrexone during acute and chronic administration of 100-mg oral dose was studied in 4 subjects. Following an acute dose the mean (X) peak naltrexone plasma level was 43.6 +/- 29.9 ng/ml at 1 hr and for the major biotransformation product, beta-naltrexol, was 87.2 +/- 25.0 ng/ml at 2 hr. Twenty-four hours after the dose the X levels of naltrexone and beta-naltrexol declined to 2.1 +/- 0.47 and 17.6 +/- 5.0 ng/ml, respectively. Following chronic administration and X peak plasma levels of naltrexone and beta-naltrexol rose to 46.4 +/- 18.5 and 158.4 +/- 89.9 ng/ml at 1 hr, but by 24 hr both compounds declined to levels of the same order as in the acute state at 24 hr. Plasma levels of naltrexone and beta-naltrexol measured 24 hr after the daily doses of naltrexone throughout the study indicated that steady-state equilibrium was rapidly attained and that there was no accumulation of naltrexone and beta naltrexol in the plasma after chronic treatment on 100 mg oral doses. Biexponential kinetics were observed for naltrexone and beta-naltrexol in the first 24 hr. The half-life of naltrexone and beta-naltrexol decreased slightly from the acute to thechronic study from 10.3 +/- 3.3 to 9.7 +/- 1.1 hr and from 12.7 +/- 2.6 to 11.4 +/- 2.0 hr. The plasma levels of naltrexone declined slowly from 24 through 72 hr from 2.4 to 1.7 ng/ml, with an apparent half-life of 96 hr. The renal clearance data indicate that naltrexone is partially reabsorbed while beta naltrexol is actively secreted by the kidney. During acute and chronic naltrexone administration the mean fecal excretion was 2.1% and 3.6% while urinary excretion was 38% and 70% of the dose in a 24-hr period. Opiate antagonism to 25 mg heroin challenges was nearly complete through 48 hr after naltrexone. At 72 hr the objective responses reappeared to a greater extent than the subjective ones. Correlation coefficient (r) between naltrexone plasma levels and opiate antagonism was 0.91 and between individual half-life of naltrexone and opiate antagonism it was 0.99.

Two gas liquid chromatographic methods differing mainly in sensitivity are described for the quantitative determination of naltrexone (NT) and its metabolites in human biofluids. Flame ionization detection of the N,O-bis-(trimethylsilyl) trifluoroacetamide (BSTFA) derivatives provided sufficient separation and sensitivity for quantitative of the bases in urine. However, the thousand times lower levels in serum, red blood cells (RBC) and saliva necessitated the use of more sensitive electron capture detection methods of the pentafluoro derivatives of NT and its metabolites. The 2-hydroxy-3-methoxy-6 beta-naltrexol (HMN) and 6 beta-naltrexol (beta-OL) pentafluoro derivatives had nearly identical gas liquid chromatographic retention times in a number of stationary liquid phases. Thus, their separation had to be achieved prior to chromatography. Differential extraction was based on the different partition characteristics of HMN and 6 beta-naltrexol between aqueous and organic solvents. Applicability of the methods was tested using the biofluids of four subjects taking 2 x 200 mg naltrexone per day chronically. Blood, saliva and urine samples were collected at the same time (prior to drug administration), which was 16 and 24 hours after the doses. In the plasma the relative percentages of the bases were 73.5 beta-OL; 23.1 HMN and 3.4 NT. The same in the urine were 76.6 beta-OL, 14.4 HMN and 9.0 NT. The lipophilic nature of HMN and the hydrophilic property of beta-OL may have influenced their distribution into RBC and saliva. In the RBC 96.1% HMN and only trace amounts of beta-OL distributed and in saliva 92.3% of beta-OL and no HMN was found; the difference in both cases was made up by naltrexone to 100%.

Synthesis and opioid radioreceptor assay data on analogues closely related to 6-desoxy-6-spiro-alpha-methylene-gamma-lactone 5a, a compound with irreversible activity in this assay, are reported. Saturated lactones (7a,b), endocyclic alpha, beta-unsaturated gamma-lactones (8a,b and 9a), and 6 alpha,7 alpha-fused alpha-methylene-gamma-lactones (10a and 11a) were prepared. Related 6-desoxy-6-methylene 6 beta- and 6 alpha-oxides (12a,b and 13a) and glycidate esters 14a,b and 15a,b were also prepared with use of naltrexone (1a) and oxymorphone (1b) as starting material. Compounds in the N-cyclopropylmethyl (N-CPM) series were more potent than those in the N-Me series in displacing [3H]naltrexone in the opioid radioreceptor assay, usually by 2-16-fold in the absence of Na ion. The most potent N-CPM analogues were epoxides 12a and 13a and glycidate esters 14a and 15a, showing IC50's of 2-6 nM, similar to that of 5a. Of the N-Me analogues, 6 beta-oxide 12b was most active, with an IC50 of 8 nM in the absence of Na ion. For the N-CPM analogues, the Na ion ratios were generally less than 1, with two exceptions. The N-Me analogues showed expected larger Na ion effects of 7 or greater. None of the lactone analogues had irreversible effects when preincubated in the rat brain membrane preparation, even at 37 degrees C for 30 min, i.e., washing restored [3H]naltrexone binding to control levels. These results clearly show that the alpha-methylene-gamma-lactone moiety of 5a is required for irreversible effects, consistent with it serving as a conjugate addition acceptor of a nucleophilic group from a ligand at or near the receptor. The epoxides and glycidate esters also had no irreversible activity, indicating more electrophilic functional groups are needed and/or these electrophiles are not properly aligned to react with nucleophilic groups at or near the opioid receptor.

A carbon-13 nuclear magnetic resonance comparison of synthetic and biological samples was used to identify unequivocally 2-hydroxy-3-O-methyl-6beta-naltrexol as a minor naltrexone metabolite in humans. This study points up the increasing usefulness of natural abundance carbon-13 nuclear magnetic resonance techniques in metabolism studies.

The time-action of opiate antagonist activity of naltrexone was evaluated in detoxified ex-opiate addicts, using 25 mg intravenous heroin challenges. A 100 mg naltrexone dose provided 96% blockade at 24 hr, 86.5% blockade at 48 hr and 46.6% blockade at 72 hr. Following oral administration, naltrexone was rapidly and completely absorbed. Peak levels of naltrexone and its major metabolite 6 beta-naltrexol were reached 1 hr after the dose. The high 6 beta-naltrexol plasma concentrations only 1 hr after drug administration indicate a rapid biotransformation process, converting a large fraction of the dose to less active metabolites. Over 70% of the dose was excreted in the 24 hr urine and less than 0.5% in the feces. No change was observed in the rate of naltrexone disposition during chronic dosing vs. the acute study, indicating no metabolic induction. The rapid achievement of steady state naltrexone plasma levels eliminates the need of stepwise induction at the beginning of naltrexone treatment.

Reduction of naltrexone and naloxone with sodium borohydride gave a mixture (85:15) of the 6alpha- and 6beta-hydroxy epimers, alpha- and beta-naltrexol and alpha- and beta-naloxol, respectively. Each pair of epimers was separated by preparative thin-layer chromatography and the physical and spectral properties of each compound were determined. Previous assignments for the configuration of the epimers were verified. A semi-quantitative electron capture gas-liquid chromatographic method was devised for distinguishing either alpha- or beta-naltrexol in the presence of the other and in the presence of large amounts (at least 10-fold greater) of naltrexone. The method was used to determine the approximate weight ratio of beta-naltrexol to naltrexone present in enzymatically hydrolyzed urine samples. It was found that substantially greater quantities of beta-naltrexol and/or its conjugates were excreted in the urine of man, monkey, guinea pig and rabbit after administration of naltrexone, whereas very small quantities were excreted by the mouse, rat and dog. In contrast, just trace amounts of the 6alpha-hydroxy epimer, alpha-naltrexol, were detected in the urine of only 2 of the 7 species that had received naltrexone, i.e., monkey and guinea pig. After administration of 3H-15,16-naltrexone, 1 mg/kg, i.v. to the guinea pig, 25% of the radioactivity found following thin-layer chromatography of the extract of acid-hydrolyzed urine corresponded to beta-naltrexol. In gall bladder bile from the guinea pig, only conjugates of naltrexone and beta-naltrexol were found 2 hours after administration of naltrexone. Following administration of beta-naltrexol, 1 mg/kg, i.v. to guinea pigs only beta-naltrexol and/or its conjugates were detected in urine or bile. However, urine collected after administration of alpha-naltrexol, 1 mg/kg, i.v. to guinea pigs contained alpha-naltrexol and its conjugates, as well as a yet unidentified metabolite.

The enzyme systems involved in the reduction of the narcotic antagonists, naloxone and naltrexone, were studied from the liver of chicken and rabbit. These secies were chosen because the relative amounts of diastereoisomeric products formed by enzymatic reduction between these species are so different; naloxone and naltrexone are converted in the chicken to 6alpha-naloxol and 6alpha-naltrexol, respectively, and in the rabbit to 6beta-naloxol and 6beta-naltrexol, respectively. In the present study, the enzymes for carrying out this reduction were found to require NADPH and to occur in the soluble fraction of liver homogenates. These enzymes were precipitated at 60-70% ammonium sulfate saturation for the chicken and at 50-60% for the rabbit. Differential sensitivity to inhibitors could be shown. For example, the rabbit enzyme was more sensitive to inhibition by morphine than was the chicken enzyme. The reverse order of sensitivity was seen with ketamine. These differences in properties between the chicken and rabbit liver enzymes indicate that the alpha- and beta-hydroxy product stereoselectivity arises from two different enzymes carrying out the reduction.

The effects of naloxone pretreatment on opiate agonist-induced depressions in serum luteinizing hormone (LH) levels were examined in male rats. Our results demonstrated a pronounced enhancement of morphine's actions 6 hours after the administration of naloxone (0.5 mg/kg). This effect was characterized by a 10 fold reduction in the ED50 (1.26 mg/kg versus 0.13 mg/kg in saline- and naloxone-pretreated rats, respectively) and much greater depressions in serum LH levels at each dose of morphine. The actions of naloxone were not confined to morphine, since similar increased potencies were found for opioid agonists with selectivity for a variety of opioid receptor subtypes. Because naloxone did not alter the uptake of subsequently administered morphine into brain, our results cannot be explained on the basis of an increased availability of the agonist. Rather, it appears that naloxone pretreatment induces a change in the sensitivity of those receptors involved in the effects of opioid agonists on LH.

After injection of (15,16-3H)naltrexone (10 mg/kg s.c.) in male Wistar rats, peak concentrations of drug occurred in brain and plasma within 0.5 hr. Levels of naltrexone were sustained in brain between 2 and 24 hr and were barely detectable at 48 hr. Significant amounts of metabolities were present in brain and plasma at longer time periods. The t1/2 of naltrexone in brain and plasma were approximately 8.0 and 11.4 hr. respectively. The brain/plasma ratios of naltrexone at earlier times (0.5-1 hr) were higher than those at later times. The binding of naltrexone in vitro with rat plasma proteins in concentrations of 1-10 mug/ml ranged between 41 and 59% 6beta-Naltrexol was present in very small amounts in brain but not in plasma. In addition to 7,8-dihydro-14-hydroxynormophinone and 7,8-dihydro-14-hydroxynormophine, tentative evidence was obtained for three other metabolites of naltrexone in brain. These metabolites were also present in plasma in addition to free and conjugated naltrexone and its N-dealkylated metabolites.