Stephen R. Edwards

Oxycodone and morphine have distinctly different pharmacological profiles: Radioligand binding and behavioural studies in two rat models of neuropathic pain

Abstract Previously, we reported that oxycodone is a putative κ‐opioid agonist based on studies where intracerebroventricular (i.c.v.) pre‐treatment of rats with the κ‐selective opioid antagonist, nor‐binaltorphimine (nor‐BNI), abolished i.c.v. oxycodone but not morphine antinociception, whereas pretreatment with i.c.v. naloxonazine (μ‐selective antagonist) produced the opposite effects. In the present study, we used behavioural experiments in rat models of mechanical and biochemical nerve injury together with radioligand binding to further examine the pharmacology of oxycodone. Following chronic constriction injury (CCI) of the sciatic nerve in rats, the antinociceptive effects of intrathecal (i.t.) oxycodone, but not i.t. morphine, were abolished by nor‐BNI. Marked differences were found in the antinociceptive properties of oxycodone and morphine in streptozotocin (STZ)‐diabetic rats. While the antinociceptive efficacy of morphine was abolished at 12 and 24 weeks post‐STZ administration, the antinociceptive efficacy of s.c. oxycodone was maintained over 24 weeks, albeit with an ∼3‐ to 4‐fold decrease in potency. In rat brain membranes irreversibly depleted of μ‐ and δ‐opioid binding sites, oxycodone displaced [3H]bremazocine (κ2‐selective in depleted membranes) binding with relatively high affinity whereas the selective μ‐ and δ‐opioid ligands, CTOP (d‐Phe‐Cys‐Tyr‐d‐Trp‐Orn‐Thr‐Pen‐Thr‐NH2) and DPDPE ([d‐Pen2,5]‐enkephalin), respectively, did not. In depleted brain membranes, the κ2b‐ligand, leu‐enkephalin, prevented oxycodone’s displacement of high‐affinity [3H]bremazocine binding, suggesting the notion that oxycodone is a κ2b‐opioid ligand. Collectively, the present findings provide further support for the notion that oxycodone and morphine produce antinociception through distinctly different opioid receptor populations. Oxycodone appears to act as a κ2b‐opioid agonist with a relatively low affinity for μ‐opioid receptors.

Small Molecule Angiotensin II Type 2 Receptor (AT2R) Antagonists as Novel Analgesics for Neuropathic Pain: Comparative Pharmacokinetics, Radioligand Binding, and Efficacy in Rats

OBJECTIVE Neuropathic pain is an area of unmet clinical need. The objective of this study was to define the pharmacokinetics, oral bioavailability, and efficacy in rats of small molecule antagonists of the angiotensin II type 2 receptor (AT₂R) for the relief of neuropathic pain. DESIGN AND METHODS Adult male Sprague-Dawley (SD) rats received single intravenous (1-10 mg/kg) or oral (5-10 mg/kg) bolus doses of EMA200, EMA300, EMA400 or EMA401 (S-enantiomer of EMA400). Blood samples were collected immediately pre-dose and at specified times over a 12- to 24-hour post-dosing period. Liquid chromatography tandem mass spectrometry was used to measure plasma drug concentrations. Efficacy was assessed in adult male SD rats with a unilateral chronic constriction injury (CCI) of the sciatic nerve. RESULTS After intravenous administration in rats, mean (±standard error of the mean) plasma clearance for EMA200, EMA300, EMA400, and EMA401 was 9.3, 6.1, 0.7, and 1.1 L/hour/kg, respectively. After oral dosing, the dose-normalized systemic exposures of EMA400 and EMA401 were 20- to 30-fold and 50- to 60-fold higher than that for EMA300 and EMA200, respectively. The oral bioavailability of EMA400 and EMA401 was similar at ∼30%, whereas it was only 5.9% and 7.1% for EMA200 and EMA300, respectively. In CCI rats, single intraperitoneal bolus doses of EMA200, EMA300, and EMA400 evoked dose-dependent pain relief. The pain relief potency rank order in CCI rats was EMA400 > EMA300 > EMA200 in agreement with the dose-normalized systemic exposure rank order in SD rats. CONCLUSION The small molecule AT₂R antagonist, EMA401, is in clinical development as a novel analgesic for the relief of neuropathic pain.

SEX DIFFERENCES IN THE PHARMACOKINETICS, OXIDATIVE METABOLISM AND ORAL BIOAVAILABILITY OF OXYCODONE IN THE SPRAGUE-DAWLEY RAT

1 The pharmacokinetics and oxidative metabolism of oxycodone were investigated following intravenous and oral administration in male and female Sprague‐Dawley (SD) rats. 2 High‐performance liquid chromatography (HPLC)–electrospray ionization (ESI)–tandem mass spectrometry (MS‐MS) was used to quantify plasma concentrations of oxycodone and its oxidative metabolites noroxycodone and oxymorphone following administration of single bolus intravenous (5 mg/kg) and oral (10 mg/kg) doses of oxycodone. 3 The mean (±SEM) clearance of intravenous oxycodone was significantly higher in male than female SD rats (4.9 ± 0.3 vs 3.1 ± 0.3 L/h per kg, respectively; P < 0.01). Mean areas under the plasma concentration versus time curves (AUC) for oxycodone were significantly higher in female than male SD rats following intravenous (~1.6‐fold; P < 0.01) and oral (~sevenfold; P < 0.005) administration. 4 The oral bioavailability of oxycodone was low (at 1.2 and 5.0%, respectively) in male and female SD rats, a finding consistent with high first‐pass metabolism. Noroxycodone : oxycodone AUC ratios were significantly higher in male than female SD rats after intravenous (~2.4‐fold; P < 0.005) and oral (~12‐fold; P < 0.005) administration. 5 Circulating oxymorphone concentrations remained very low following both routes of administration. Noroxycodone : oxymorphone AUC ratios were greater in male than female SD rats after intravenous (~13‐ and fivefold, respectively) and oral (~90‐ and sixfold, respectively) administration. 6 Sex differences were apparent in the pharmacokinetics, oxidative metabolism and oral bioavailability of oxycodone. Systemic exposure to oxycodone was greater in female compared with male SD rats, whereas systemic exposure to metabolically derived noroxycodone was higher in male than female SD rats. 7 Oral administration of oxycodone to the SD rat is a poor model of the human for the study of the pharmacodynamic effects of oxycodone.

Antinociception versus serum concentration relationships following acute administration of intravenous morphine in male and female Sprague-dawley rats: Differences between the tail flick and hot plate nociceptive tests

1 Antinociception versus serum morphine concentration relationships were defined in male and female Sprague‐Dawley rats administered single intravenous (i.v.) bolus doses of morphine, using the hot plate (2.1–14 mg/kg) and tail flick tests (1–8 mg/kg). 2 Serum concentrations of morphine and morphine‐3‐glucuronide (M3G), its major metabolite in the rat, were assayed using high‐performance liquid chromatography (HPLC) with electrochemical detection. 3 Significantly higher (P < 0.05) values of peak antinociception (approximately 1.7‐fold), as well as the extent and duration of antinociception (approximately fourfold), were observed in male compared with female rats administered 10 mg/kg morphine in the hot plate test. Although there were no significant sex‐related differences in the area under the serum morphine concentration versus time curve (AUC) at this dose, systemic exposure to M3G (M3G AUC) was significantly higher (approximately twofold; P < 0.05) in female than male rats. 4 In contrast with most previous studies investigating sex differences in morphine antinociception in rats, the antinociceptive effects of single i.v. doses of morphine (1–8 mg/kg) in the tail flick test did not differ significantly between male and female rats. 5 Morphine ED50 and EC50 values (95% confidence intervals) for antinociception in the hot plate test were significantly lower (P < 0.05) in male rats (ED50 8.4 mg/kg (7.6–9.2); EC50 1.8 nmol/L (1.5–2.1)) compared with female rats (ED50 10.6 mg/kg (9.1–12.0); EC50 3.7 nmol/L (3.4–4.1)). However, in the tail flick test, there was no significant difference between male and female rats in ED50 (1.8 (0.4–3.3) and 1.4 mg/kg (0.4–2.5), respectively) or EC50 (0.5 (0.3–0.6) and 0.4 nmol/L (0.2–0.5), respectively) values. 6 Supraspinal attenuation of morphine antinociception by M3G may account for these differences.

Enantioselectivity of thiopental distribution into the central neural tissue of rats: an interaction with halothane.

UNLABELLED Thiopental is a racemate. In this study, we examined whether thiopental total body clearance and its distribution into central nervous system (CNS) tissue of rats was enantioselective. Rats, either anesthetized with halothane or conscious and restrained, were infused to stepwise steady-state targets of 5, 10, and 20 microg/mL thiopental by computer-controlled infusions. Serial arterial plasma and steady-state samples of brain and spinal cord were assayed enantiospecifically for thiopental. In both groups, concurrent total and unbound plasma concentrations of S-thiopental were approximately 10%-20% higher than those of R-thiopental, corresponding to its higher clearance. CNS tissue concentrations of S-thiopental were approximately 20% higher than those of R-thiopental. Spinal cord to plasma distribution coefficients were approximately 2 x those in the brain, with relative distribution coefficients approximately 10% greater for R-thiopental in both tissues. Plasma concentrations and distribution coefficients of both enantiomers were approximately 10%-20% lower in the halothane-anesthetized group, with a slightly greater effect on R-thiopental distribution. We conclude that the total body clearance of R-thiopental > S-thiopental, that halothane enantioselectively reduces the relative uptake of R-thiopental into brain tissue, and that composition is important in determining the CNS tissue concentrations of thiopental. The reported higher potency of S-thiopental did not seem to be due to its greater distribution into CNS tissues. IMPLICATIONS Because thiopental is a mixture of two forms (termed R-and S-enantiomers), correct interpretation of its distribution into, and clearance from, the body requires knowledge about both enantiomers. In this study, performed in rats, we showed that the two enantiomers of thiopental differed significantly, with the R-enantiomer having the preferred profile.

Topical Application of a Novel Oxycodone Gel Formulation (Tocopheryl Phosphate Mixture) in a Rat Model of Peripheral Inflammatory Pain Produces Localized Pain Relief Without Significant Systemic Exposure

This study was designed to assess the analgesic efficacy and systemic exposure of oxycodone administered topically in a novel tocopheryl phosphate mixture (TPM) gel formulation, to the inflamed hindpaws in a rat model of inflammatory pain. Unilateral hindpaw inflammation was induced in male Sprague-Dawley rats by intraplantar (i.pl.) injection of Freunds complete adjuvant (FCA). Mechanical hyperalgesia and hindpaw inflammation were assessed by measuring paw pressure thresholds and hindpaw volume, respectively, just prior to i.pl. FCA and again 5-6 days later. The analgesic effects of oxycodone administered topically (1 mg in TPM gel) or by i.pl. injection (50 μg), were assessed. Systemic oxycodone exposure was assessed over an 8-h postdosing interval following topical application. Skin permeation of oxycodone from the gel formulation was assessed in vitro using Franz diffusion cells. Oxycodone administered topically or by i.pl. injection produced significant (p < 0.05) analgesia in the inflamed hindpaws. Systemic oxycodone exposure was insignificant after topical dosing. The in vitro cumulative skin permeation of oxycodone was linearly related to the amount applied. Topical TPM/oxycodone gel formulations have the potential to alleviate moderate to severe inflammatory pain conditions with minimal systemic exposure, thereby avoiding central nervous system (CNS)-mediated adverse effects associated with oral administration of opioid analgesics.

Comparative studies using the Morris water maze to assess spatial memory deficits in two transgenic mouse models of Alzheimer's disease

Evaluation of the efficacy of novel therapeutics for potential treatment of Alzheimers disease (AD) requires an animal model that develops age‐related cognitive deficits reproducibly between independent groups of investigators. Herein we assessed comparative temporal changes in spatial memory function in two commercially available transgenic mouse models of AD using the Morris water maze (MWM), incorporating both visible and hidden platform training. Individual cohorts of cDNA‐based ‘line 85’‐derived double‐transgenic mice coexpressing the ‘Swedish’ mutation of amyloid precursor protein (APPSwe) and the presenillin 1 (PS1) ‘dE9’ mutation were assessed in the MWM at mean ages of 3.6, 9.3 and 14.8 months. We found significant deficits in spatial memory retention in APPSwe/PS1dE9 mice aged 3.6 months and robust deficits in spatial memory acquisition and retention in APPSwe/PS1dE9 mice aged 9.3 months, with a further significant decline by age 14.8 months. β‐Amyloid deposits were present in brain sections by 7.25 months of age. In contrast, MWM studies with individual cohorts (aged 4–21 months) of single‐transgenic genomic‐based APPSwe mice expressing APPSwe on a yeast artificial chromosomal (YAC) construct showed no significant deficits in spatial memory acquisition until 21 months of age. There were no significant deficits in spatial memory retention up to 21 months of age and β‐amyloid deposits were not present in brain sections up to 24 months of age. These data, generated using comprehensive study designs, show that APPSwe/PS1dE9 but not APPSwe YAC mice appear to provide a suitably robust model of AD for efficacy assessment of novel AD treatments in development.

Comparative studies of the neuro-excitatory behavioural effects of morphine-3-glucuronide and dynorphin A(2-17) following spinal and supraspinal routes of administration.

Morphine-3-glucuronide (M3G) administered centrally produces dose-dependent neuro-excitatory behaviours in rodents via a predominantly non-opioid mechanism. The endogenous opioid peptide, dynorphin A (Dyn A) (1-17), is rapidly cleaved in vivo to the relatively more stable fragment Dyn A(2-17) which also produces excitatory behaviours in rodents via a non-opioid mechanism. This study investigated the possible contribution of Dyn A(2-17) to the neuro-excitatory behaviours evoked by supraspinally and spinally administered M3G in male Sprague-Dawley (SD) rats. Marked qualitative differences in behaviours were apparent following administration of M3G and Dyn A(2-17). Administration of 11 nmol i.c.v. doses of M3G produced intermittent myoclonic jerks, tonic-clonic convulsions, and ataxia, as well as postural changes, whereas i.c.v. Dyn A(2-17) at 15 nmol produced effects on body posture alone. Administration of 11 nmol i.t. doses of M3G produced intermittent explosive motor activity, and touch-evoked agitation, as well as postural changes, whereas i.t. Dyn A(2-17) at 15 nmol produced postural changes, touch-evoked agitation, and paralysis. Pre-treatment with Dyn A antiserum (200 microg) markedly attenuated total behavioural excitation following i.c.v. and i.t. administration of Dyn A(2-17) by approximately 94% and 78%, respectively. However, total behavioural excitation following i.c.v. and i.t. administration of M3G was less markedly attenuated (both approximately 27%) by pre-treatment with Dyn A antiserum, with reductions in tonic-clonic convulsions ( approximately 43%), explosive motor behaviour ( approximately 28%), and touch-evoked agitation ( approximately 22%). The present findings discount a major role for Dyn A in mediating the neuro-excitatory effects of M3G, although it may contribute to maintaining some individual neuro-excitatory behaviours.

Effects of a 5-HT3 antagonist on peripheral 5-hydroxytryptamine-induced anorexia

Since antagonizing peripheral 5-HT2receptors incompletely blocks the anorexia induced by systemically administered 5-HT, another 5-HT receptor subtype is implicated in this effect. To determine whether the 5-HT3 receptor is the additional type involved, the specific, peripheral 5-HT3 antagonist [[3-(1-Methyl-1H-mdol-3-yl)-1,2,4-oxadiazol-5-yl]methyl]trimethyl-ammonium iodide (MIO) was used. After an overnight fast, rats were injected with MIO followed 1 h later by 5 mg/kg of 5-HT. There was a dose-dependent potentiation by MIO of the 5-HT-induced anorexia that peaked at a dose of 2.5 mg/kg of MIO. However, MIO by itself failed to induce an anorexia Although the 5-HT3 receptor is implicated in 5-HT-induced anorexia, it is suggested that its activation counters the effects of 5-HT in the mediation of the anorexia.

Effects of chronic systemic administration of 5-HT on food intake and body weight in rats

The effects of chronic peripheral administration of 5-HT on food intake and body weight was investigated. In normal male Wistar rats, normal female Wistars, obese Zucker males, ovariectomised Wistar females, or normal Wistar males free fed a cafeteria diet, suppression of the creeping weight gain typical of control animals is observed. In females, this effect is not dependent on the local hormonal environment, because intact and ovariectomised females showed similar responses to treatment. One sex difference is that the weight suppressive effect in males is accompanied by an anorectic effect, whereas this anorectic effect is absent in females. Thus, although reduced food intake may partially explain the suppression of weight gain in males, in females it must be due to other, perhaps metabolic, effects. It is possible that these metabolic effects may also occur in males, suggesting one possible explanation of why the effect was typically larger in males than females.