Naloxegol, an opioid antagonist with reduced CNS penetration: Mode-of-action and human relevance for rat testicular tumours.

Naloxegol is an opioid antagonist which has been developed for the treatment of patients with opioid induced constipation. In the nonclinical safety program naloxegol was shown to have a very benign toxicity profile. In the rat, but not the mouse, 2-year carcinogenicity study a change in tumour pattern with an increase in testicular Leydig cell tumours (LCT) was observed after dosing at high (supra-pharmacological) concentrations. To establish the basis of the increase in LCT and to assess its potential relevance to humans, studies to exclude and potentially identify mode-of-action (MoA) were performed. A genotoxic mechanism was ruled out following negative results in the Ames, mouse lymphoma, and micronucleus assays. An effect on androgen metabolism was excluded since the treatment of rats with naloxegol for 14days did not result in any induction of CYP protein levels. It was demonstrated that administration of centrally restricted opioid antagonists naloxegol or methylnaltrexone at high doses induced an increase in LH release with no clear increase in testosterone, in contrast to the centrally acting opioid antagonist naloxone, which showed marked increases in both LH and testosterone. LCT due to increased LH stimulation is common in rats but not documented in humans. Collectively, the lack of genotoxicity signal, the lack of androgen effect, the increase in LH secretion in rats, which is no considered to be relevant for LCT formation in humans, and high margins to clinical exposures, the observed increase in LCT in the rat is not expected to be clinically relevant.

Chronic peripheral administration of kappa-opioid receptor antagonist advances puberty onset associated with acceleration of pulsatile luteinizing hormone secretion in female rats.

Puberty in mammals is timed by an increase in gonadotropin-releasing hormone (GnRH) secretion. Previous studies have shown involvement of the two neuropeptides, kisspeptin and neurokinin B (NKB), in controlling puberty onset. Little is known about the role of the other key neuropeptide, dynorphin, in controlling puberty onset, although these three neuropeptides colocalize in the arcuate kisspeptin neurons. The arcuate kisspeptin neuron, which is also referred to as the KNDy neuron, has recently been considered to play a role as an intrinsic source of the GnRH pulse generator. The present study aimed to determine if attenuation of inhibitory dynorphin-kappa-opioid receptor (KOR) signaling triggers the initiation of puberty in normal developing female rats. The present study also determined if stimulatory NKB-neurokinin 3 receptor (NK3R) signaling advances puberty onset. Female Wistar-Imamichi rats were weaned and intraperitoneally implanted with osmotic minipumps filled with nor-binaltorphimine (nor-BNI), a KOR antagonist, or senktide, a NK3R agonist, at 20 days of age. Fourteen days of intraperitoneal infusion of nor-BNI or senktide advanced puberty onset, manifested as vaginal opening and the first vaginal estrus in female rats. Frequent blood sampling showed that nor-BNI significantly increased luteinizing hormone (LH) pulse frequency at 29 days of age compared with vehicle-treated controls. Senktide tended to increase this frequency, but its effect was not statistically significant. The present results suggest that the inhibitory input of dynorphin-KOR signaling plays a role in the prepubertal restraint of GnRH/LH secretion in normal developing female rats and that attenuation of dynorphin-KOR signaling and increase in NKB-NK3R signaling trigger the onset of puberty in female rats.

Naltrexone and cyclazocine. A controlled treatment study.

The induction side effects of cyclazocine and naltrexone were compared in double-blind placebo-controlled studies involving 40 patients (20 for each drug). These studies were carried out with a twice-a-day dosage regimen. Naltrexone produced fewer side effects than cyclazocine. Naltrexone side effects fell to levels indistinguishable from those of placebo in the "induction after placebo" phase. In contrast, cyclazocine "induction after placebo" produced an even higher level of side effects than found in its induction. In no case was naltrexone discontinued because of side effects. On the other hand, three of 20 cyclazocine-treated patients discontinued the drug because of distressing side effects. No toxicity was noted with either agent. The controlled data reported supports the clinical impression that naltrexone produces fewer induction side effects than cyclazocine.

Effects of atomoxetine on attention and impulsivity in the five-choice serial reaction time task in rats with lesions of dorsal noradrenergic ascending bundle.

Atomoxetine, a noradrenaline reuptake inhibitor (NRI), which is a non-stimulating medicine that is used for the treatment of patients with attention deficit hyperactivity disorder (ADHD), has been found to be effective in reducing behavioral impulsivity in rodents, but its efficacy in a dorsal noradrenergic ascending bundle (DNAB)-lesioned condition has not been examined. The present study aimed to investigate the effects of DNAB lesions on attention and impulsive control in the five-choice serial reaction time task (5-CSRTT) in rats treated with atomoxetine. The drug-induced changes in noradrenaline efflux in the medial prefrontal cortex were also measured. 5-CSRTT-trained rats were included in one of the following groups: N-(2-chloroethyl)-N-ethyl-2-bromobenzylamine (DSP-4)/Atomoxetine, Sham/Atomoxetine, DSP-4/Saline, or Sham/Saline. Acute atomoxetine (0.3 mg/kg) was administered 14 days after the DSP-4 regime. The behavioral testing included manipulations of the inter-trial interval (ITI), stimulation duration and food satiety. In vivo microdialysis of the noradrenaline efflux in the medial prefrontal cortex and the expression of the noradrenaline transporter (NAT) in the DNAB areas were examined. Atomoxetine reduced impulsivity and perseveration in the long-ITI condition with no effects on any other variables. This phenomenon was not influenced by DSP-4 pre-treatment. The DNAB-lesioned rats had lower noradrenaline efflux in the medial prefrontal cortex. DSP-4 caused no change in NAT expression in the DNAB areas. These findings suggested that noradrenaline reuptake may not be exclusively responsible for the atomoxetine effects in adjusting impulsivity. The role of DNAB should also be considered, particularly in conditions requiring greater behavioral inhibition.

Naltrexone and dysphoria: a double-blind placebo controlled trial.

Naltrexone hydrochloride reportedly produces frequent dysphoria. This has led to speculation regarding the role of endorphins in the etiology of depression. Thirty-six subjects completed an 8-week trial of naltrexone or placebo with frequent mood assessments. No significant differences on POMS scales were noted for either subject group. One subject was discontinued from the study because of a severe dysphoric reaction. Naltrexone does not appear to significantly alter mood over a 2-month time course in nonaddicted, healthy individuals. Subpopulations of patients under physiological or psychological stress may react to naltrexone with dysphoric symptoms.

The role of the National Institute on Drug Abuse in the development of naltrexone.

Naltrexone was administered to 114 opiate-addicted business executives and 15 opiate-addicted physicians within a highly structured abstinence-oriented aftercare program following an inpatient detoxification program that used clonidine. The majority of patients successfully completed 6 months of naltrexone treatment without missed visits or drug-positive urines, and were still opiate-free at 12- to 18-months follow-up. The most successful outcomes were achieved by those who had entered treatment with unequivocal job jeopardy. Our findings suggest that naltrexone can be an extremely useful adjunct in the treatment of opiate addiction when administered to employed, motivated patients within a highly structured therapeutic program.

Physiologic and morphologic changes and incidence of neoplasms in mice and rats fed naltrexone HCl for 24 months.

Short-term (acute oral LD50 and 90-day oral subchronic) studies in mice and long-term (24 months) carcinogenesis bioassays were performed in B6C3F1 mice and Fischer 344 rats given naltrexone. The oral LD50 was approximately 1500 mg/kg; convulsions, hypopnea, and cardiac failure were dose-related. Naltrexone mixed with feed over 90 days did not evoke definitive signs of gross toxicity, and histopathology was unrelated to drug treatment. Similar drug/feed admixtures given for 24 months to mice or rats did not disturb behavior. In mice, naltrexone reduced growth rates 5-10% and food intake 9-19%, but survival rates were 70-82% for treated mice and controls. The frequency and location of predominant tumors were similar in treated and untreated mice. In the rat, the same dosages had little effect on growth or food intake. The majority of all sacrificed rats had neoplasms. Neither neoplasms nor nonneoplastic lesions in mice or rats were associated with drug treatment. It is concluded that naltrexone is not a carcinogen.

Reproductive toxicity and teratology evaluations of naltrexone.

Reproductive toxicology and teratology studies of naltrexone are reviewed. Naltrexone produced behavioral changes in rats at doses below those which affected body weight. Excitatory signs and increased production of seminal plugs occurred in male rats. Prolonged administration to female rats resulted in excitatory signs and impaired maternal activity. Estrus cycling and fertility were decreased in female rats at doses that depressed body weight gain. Higher doses given to pregnant rats for shorter periods of time did not impair fertility or produce embryo or fetal toxicity. In rabbits, there was no evidence of behavioral changes. The highest dosage administered produced transient weight depression and possibly increased resorption. These data are consistent with a report of transient changes in some normal men given single doses of naltrexone. These effects may be mediated via hypothalamic and pituitary mechanisms involved in the control of luteinizing hormone levels.

Activation of the zinc finger encoding gene krox-20 in adult rat brain: comparison with zif268.

Zif268 and krox-20 are transcription regulatory factors that contain highly homologous zinc finger DNA-binding domains. Recent studies have demonstrated that zif268 expression is rapidly regulated in brain by neuronal stimulation. We now report that, like zif268, krox-20 is rapidly and transiently activated by electroconvulsive shock treatment (ECT), D1 dopamine receptor activation, and opiate withdrawal. These studies indicate that, as found for the leucine zipper family of transcription factors, multiple members of the zinc finger family of transcription factors are induced by neuronal stimulation.

A kappa opioid antagonist blocks sensitization in a rodent model of Parkinson's disease.

Treatment of Parkinson's disease with levodopa is associated with fluctuations in motor function and dyskinesias, which may in part depend upon the mode of levodopa treatment. In rats with unilateral 6-hydroxydopamine lesions, intermittent levodopa results in sensitization to apomorphine-induced rotation, associated with massive ipsilateral increases in nigral dynorphin. We assessed the effects of nigral infusion of the selective kappa opioid antagonist nor-binaltorphomine (nor-BNI) in this model. Nor-BNI reduced apomorphine-induced rotation in animals receiving intermittent levodopa to a level comparable with that seen in animals treated with continuous levodopa or with saline. These data suggest that behavioural sensitization arising from intermittent levodopa therapy in a rodent model of parkinsonism depends upon increased expression of dynorphin in the striatonigral pathway and provides further insight into the mechanisms underlying motor fluctuations which develop during the treatment of Parkinson's disease.

Naltrexone induces melanocyte-stimulating hormone release and degenerative changes in pituitary innervation in amphibians.

Adult Rana pipiens (common frog) were treated with the long-acting opiate antagonist drug naltrexone hydrochloride to study potential effects of an opiate-receptor block on release of pro-opiomelanocortin (POMC) peptides from the pituitary intermediate lobe. The melanophore response of the animals was used to assess relative release of melanocyte-stimulating hormone (MSH). Animals were adapted to a light background, then injected with 20 or 50 mg/kg naltrexone for up to 3.5 days. All drug-treated animals showed increased release of MSH while on the light background while controls remained light-adapted. Ultrastructure of the pars intermedia of naltrexone-treated frogs revealed nerve fibers containing myelin figures, swelling of neurovesicles, and abnormal membrane components indicative of degenerative changes. The observations suggest that naltrexone has a damaging effect on pituitary innervation, and that MSH release may be the result of loss of neuroregulation from the normally inhibitory innervation. The findings are in agreement with our earlier studies in rats, which also demonstrated degenerative changes induced by naltrexone in intermediate lobe innervation. The long-acting antagonist may therefore have a "neurotoxic" effect after multiple injections or chronic use.

Antitussive effect of [Met5]enkephalin-Arg6-Phe7 in mice.

We examined the effect of [Met5]enkephalin-Arg6-Phe7 (MEAP) on the capsaicin-induced cough reflex in mice. Intracerebroventricular administration of MEAP significantly decreased the number of coughs in a dose-dependent manner. The antitussive effect of MEAP was blocked by nor-binaltorphimine, a selective kappa-opioid receptor antagonist. However, beta-funaltrexamine, a mu-opioid receptor antagonist, had no effect on the antitussive effect of MEAP. On the other hand, the antinociceptive effect of MEAP, as determined in the tail-flick test, was blocked by both nor-binaltorphimine and beta-funaltrexamine. Naltrindole, a delta-opioid receptor antagonist, had no effect on either the antitussive effect or the antinociceptive effect of MEAP. These data suggest that MEAP exerts its antitussive effect in mice through the stimulation of kappa-opioid receptors, whereas the antinociceptive effect of MEAP is mediated through the simulation of both kappa- and mu-opioid receptors.

Naltrindole, a selective delta-opioid receptor antagonist, potentiates the lethal effects of cocaine by a central mechanism of action.

The potentiation of the toxic and lethal effects of cocaine by the selective delta-opioid receptor antagonist naltrindole was explored in unrestrained, unanesthetized rats that received a continuous intravenous infusion of cocaine until death. The lethal dose of cocaine was lowered dose dependently in animals administered naltrindole intracisternally (3.0-30 microg), but not intravenously (30-300 microg). There was also a decrease in the lethal dose of cocaine following an injection of the nonselective opioid antagonist naltrexone, but not naloxone. However, the seizure-producing dose of cocaine was decreased dose dependently in rats that received naltrindole, regardless of the route of administration, naloxone, or naltrexone. In contrast, the effect of cocaine on heart rate was altered only by centrally administered naltrindole or intravenous naltrexone, with a dose of 30 microg naltrindole and 10 mg/kg naltrexone abolishing the bradycardic effect of cocaine. Despite this, neither naltrindole nor naltrexone changed the hypertensive effect of cocaine. Higher doses of naltrindole (100 microg i.c.) produced significant increases in heart rate and mean arterial pressure and were not tested in combination with cocaine. Because the lethal dose of cocaine was reduced only when naltrindole was administered intracisternally, the potentiation of the lethal effects of cocaine by naltrindole is through a central mechanism of action that may involve changes in cardiovascular function.

Comparison of naloxonazine and beta-funaltrexamine antagonism of mu 1 and mu 2 opioid actions.

beta-Funaltrexamine (beta-FNA) irreversibly blocks morphine analgesia, lethality and its inhibition of gastrointestinal transit, confirming that these actions involve mu receptors. In dose-response studies, beta-FNA antagonized all the actions with similar potencies (ID50 values of 12.1, 11.3 and 12.3 mg/kg, respectively). beta-FNA also reduced intra-cerebroventricular and intrathecal DAMGO analgesia equally well (ID50 values of 6.09 and 7.7 mg/kg, respectively). Naloxanazine blocked systemic morphine analgesia (ID50 value 9.5 mg/kg) and supraspinal DAMGO analgesia (ID50 value 6.1 mg/kg) as potently as beta-FNA. However, against spinal DAMGO analgesia, morphine's inhibition of gastro-intestinal transit or lethality, naloxonazine (ID50 values 38.8, 40.7 and 40.9 mg/kg, respectively) was significantly less active than beta-FNA (p less than 0.05). beta-FNA remains a valuable tool in the classification of mu opioid actions. Within the mu category, actions can be defined as either mu 1 (naloxonazine-sensitive) or mu 2 (naloxonazine-insensitive).

Influence of chronic prenatal and postnatal administration of naltrexone in locomotor activity induced by morphine in mice.

The influence of chronic pre- and postnatal naltrexone exposure on the sensitivity of offspring to the locomotor effects of morphine was investigated in C-57 Black mice. Pregnant mice were injected subcutaneously (sc) with either saline (0.1 ml/10 g) or naltrexone (10 mg/kg) twice daily during gestation and throughout lactation, 21 days postpartum. One, three and seven weeks after birth, male offspring were tested for locomotor activity. At 7 weeks of age, dose-response curves were obtained with morphine (10, 31.6, and 100 mg/kg) and amphetamine (0.31, 10 and 31.6 mg/kg) in naltrexone-pretreated and in saline-treated animals. Naltrexone exposure during gestation and lactation resulted in an augmented sensitivity of offspring to the locomotor activity-increasing effects of morphine. In these animals, the dose-response relationship for the effect of morphine on locomotor activity was displaced to the left about threefold. In contrast, naltrexone exposure did not alter the sensitivity of offspring to amphetamine. It was also found that offspring of naltrexone-treated animals have significantly greater spontaneous locomotor activity than that of the offspring of saline-treated mothers. The increased locomotor activity persisted for at least 4 weeks after the last injection of naltrexone. These findings indicate that chronic opioid receptor blockade during gestation and early postnatal development induces supersensitivity to the locomotor effects of morphine and is associated with long-lasting behavioral alterations.

Pharmacologic profile of NPC 168 (naltrexone phenyl oxime), a novel compound with activity at opioid receptors.

NPC 168 (naltrexone phenyl oxime) was synthesized as a novel opioid antagonist and evaluated in several in vitro and in vivo assays. NPC 168 inhibited binding to the mu, delta and kappa subtypes of the opioid receptor with nanomolar potencies. The potency of NPC 168 to antagonize morphine-induced analgesia was slightly less than that of naltrexone and nalmefene following either intraperitoneal (ED50 = 0.07 mg/kg) or oral (ED50 = 0.82 mg/kg) administration. The duration of action of NPC 168 was approximately 8 hr following subcutaneous administration, compared to 4 hr for nalmefene, to antagonize oxymorphonazine-induced analgesia. The long duration of action of NPC 168 was substantiated by pharmacokinetic data that demonstrated rapid uptake and slow clearance of NPC 168 from brain. NPC 168 (5, 10 and 20 mg/kg) also inhibited cumulative 6-hr food intake in rats that were deprived of food for 24 hr, but chronic administration of this compound to rats over a three-week period resulted in a marginal reduction in cumulative body weight gain. NPC 168 at doses of up to 10 mg/kg did not produce a conditioned taste aversion. However, NPC 168 was slightly more toxic than either naltrexone or nalmefene when administered parenterally, and as toxic as nalmefene when administered by the oral route. These data demonstrate that NPC 168 is a novel opioid antagonist with a longer duration of action than either naltrexone or nalmefene.

Effects of neonatal naltrexone on neurological and somatic development in rats of both genders.

The effects of a daily injection of the opioid antagonist naltrexone (NALTX, 1 mg/kg, s.c.) from birth to weaning on various parameters were investigated in male and female rats during postnatal development until adulthood. NALTX increased cerebellar DNA and protein content as well as cerebellar weight at 7 days. Eye opening was not affected by NALTX but it appeared advanced by 1 day in all groups compared to other studies, possibly due to a handling effect caused by the daily injection. Water and food consumption were augmented by NALTX during days 23-32 and days 55-70. Treated preweaning animals showed lower body growth rates than controls. However, NALTX caused a moderate increase in body weight measured during postweaning until adulthood. The effects of NALTX on the parameters evaluated (excepting the cerebellar measurements on day 7), although clearly statistically significant, were small in absolute terms. The preweaning opioid blockade caused by our NALTX treatment seems to affect more markedly the neural and behavioural development than the somatic growth. This work also provides potentially useful baseline data for the study of male and female rats during postnatal development.

Novel in situ gelling ocular films for the opioid growth factor-receptor antagonist-naltrexone hydrochloride: fabrication, mechanical properties, mucoadhesion, tolerability and stability studies.

Naltrexone hydrochloride (NTX) is an innovative drug used in ophthalmology for treatment of ocular surface diseases such as impaired corneal wound healing and severe dry eye. Poor chemical stability has been a major limitation for development of NTX in solution form. The aim of this study was to develop and characterise NTX in situ ocular films for enhanced chemical stability and improved ocular tolerability. The films were prepared from different amorphous polymers and characterised for physicochemical compatibility, moisture-sorption, surface pH, mechanical properties, sterilisability, surface morphology, mucoadhesion, in vitro release, conjunctival irritation and accelerated stability at 40°C/75% relative humidity for 3 months. Glycerin (GLY)-plasticised films exhibited significantly better mechanical properties, compared with polyethylene glycol (PEG) 400 and triethylcitrate (TEC)-plasticised formulations. Superior mucoadhesion was recorded for F7 and F9 plasticised with GLY and PEG 400, respectively. The stability of NTX was significantly enhanced more than 18-times, compared with the solution form. Combination of carboxymethylcellulose sodium (CMC) and sodium alginate (ALG) in a film formulation demonstrated minimal % moisture sorption, good mechanical properties, in vitro release, excellent chemical stability and minimal conjunctival irritation lending them as promising ocular formulations.

Conjunctival and corneal tolerability assessment of ocular naltrexone niosomes and their ingredients on the hen's egg chorioallantoic membrane and excised bovine cornea models.

This study aimed at combining the hen's egg test-chorioallantoic membrane (HET-CAM), bovine corneal opacity and permeability (BCOP) test and histological examination of excised corneas to evaluate the conjunctival and corneal toxicity of niosomes and their ingredients. Various surfactant/lipid combinations and concentrations (1-10%, w/v) were investigated for the ocular delivery of an ambitious drug (naltrexone hydrochloride) for treatment of diabetic keratopathy. Four niosomal formulations were investigated and found to be non irritant to the 10 days old HET-CAMs (an acceptable conjunctival model). Only one of the tested ingredients (sodium cholate - CH) showed moderate irritation, however such an effect was diminished when incorporated into niosomes. Corneal opacity and fluorescein permeability scores for the test substances correlated well with the HET-CAM test results. Corneal erosion and stromal thickness were found to be in agreement with the HET-CAM and BCOP results, which discriminated well between moderately and mildly irritant test substances. Corneal histological examination revealed toxicity signs included epithelial erosion, stromal condensation and stromal vacuolisation, which allowed better discrimination between strong and moderate irritants. It is concluded that the prepared niosomes possess good ocular tolerability and minimal ocular tissue irritation. They can be further investigated as ocular delivery systems using appropriate animal models.

Maternally administered sustained-release naltrexone in rats affects offspring neurochemistry and behaviour in adulthood.

Naltrexone is not recommended during pregnancy. However, sustained-release naltrexone implant use in humans has resulted in cases of inadvertent foetal exposure. Here, we used clinically relevant dosing to examine the effects of maternally administered sustained-release naltrexone on the rat brain by examining offspring at birth and in adulthood. Maternal treatment (naltrexone or placebo implant) started before conception and ceased during gestation, birth or weaning. Morphometry was assessed in offspring at birth and adulthood. Adult offspring were evaluated for differences in locomotor behaviour (basal and morphine-induced, 10 mg/kg, s.c.) and opioid neurochemistry, propensity to self-administer morphine and cue-induced drug-seeking after abstinence. Blood analysis confirmed offspring exposure to naltrexone during gestation, birth and weaning. Naltrexone exposure increased litter size and reduced offspring birth-weight but did not alter brain morphometry. Compared to placebo, basal motor activity of naltrexone-exposed adult offspring was lower, yet they showed enhanced development of psychomotor sensitization to morphine. Developmental naltrexone exposure was associated with resistance to morphine-induced down-regulation of striatal preproenkephalin mRNA expression in adulthood. Adult offspring also exhibited greater operant responding for morphine and, in addition, cue-induced drug-seeking was enhanced. Collectively, these data show pronounced effects of developmental naltrexone exposure, some of which persist into adulthood, highlighting the need for follow up of humans that were exposed to naltrexone in utero.