In vitro profiling of opioid ligands using the cAMP formation inhibition assay and the ß-arrestin2 recruitment assay: No two ligands have the same profile.

Previously, we showed that no two of seven opioids administered by the intracerebroventricular route had the same potency rank order for evoking antinociception, constipation and respiratory depression in rats. To gain insight at the cellular level, this study was designed to systematically investigate the activity profiles of six commonly used opioid ligands using the forskolin-stimulated cAMP assay and a ß-arrestin2 recruitment assay in cultured HEK-293 cells transfected with MOP(µ), DOP(δ) or KOP(κ) receptors(-r). Morphine was a potent agonist at the MOP-r in the cAMP assay whereas it was a weak agonist at the KOP-r and DOP-r. Oxycodone had moderate efficacy and low potency at the MOP-r. Buprenorphine was a potent MOP-r and DOP-r agonist; its efficacy rank order was DOP > MOP > KOP. Fentanyl was a potent agonist at the MOP-r; its efficacy rank order was MOP > DOP > KOP. For DPDPE, its agonist efficacy was confined to the DOP-r, whereas for U69593, its efficacy rank order was KOP>> MOP. For the ß-arrestin2 assay, fentanyl had full efficacy at the MOP-r whereas morphine and oxycodone were weak with insignificant efficacy at DOP and KOP receptors. Buprenorphine did not recruit ß-arrestin2 at all three opioid-receptors. DPDPE and U69593 had full efficacy for ß-arrestin2 recruitment to the DOP-r and KOP-r respectively. Despite the low efficacy and potency of morphine, oxycodone and buprenorphine in recruiting ß-arrestin2 to the MOP-r herein, these opioids all evoked respiratory depression and constipation in rats. Together, our findings discount a key role for ß-arrestin2 recruitment at the MOP-r in evoking opioid-related side-effects.

Nitric oxide modulates µ-opioid receptor function in vitro.

Painful diabetic neuropathy (PDN) is a type of peripheral neuropathic pain that develops as a consequence of prolonged hyperglycaemia-induced injury to the long nerves. Apart from pain, PDN is also characterized by morphine hyposensitivity. Intriguingly, in streptozotocin (STZ)-induced diabetic rats exhibiting marked morphine hyposensitivity, dietary administration of the nitric oxide (NO) precursor, L-arginine at 1 g/d, progressively rescued morphine efficacy and potency over an 8-week treatment period. In earlier work, single bolus doses of the furoxan nitric oxide (NO) donor, PRG150 (3-methylfuroxan-4-carbaldehyde), evoked dose-dependent pain relief in STZ-diabetic rats but the efficacious doses were 3-4 orders of magnitude higher in advanced diabetes than that required in early STZ diabetes. Together, these findings suggested a role for NO in the modulation of µ-opioid (MOP) receptor signalling. Therefore, the present study was designed to assess a role for NO released from PRG150, in modulating MOP receptor function in vitro. Here, we show an absolute requirement for the MOP receptor, but not the δ-opioid (DOP) or the κ-opioid (KOP) receptor, to transduce the cellular effects of PRG150 on forskolin-stimulated cAMP responses in vitro. PRG150 did not interact with the classical naloxone-sensitive binding site of the MOP receptor, and its effects on cAMP responses in HEK-MOP cells were also naloxone-insensitive. Nevertheless, the inhibitory effects of PRG150 on forskolin-stimulated cAMP responses in HEK-MOP cells were dependent upon pertussis toxin (PTX)-sensitive Gi/o proteins as well as membrane lipid rafts and src kinase. Together, our findings implicate a role for NO in modulating MOP receptor function in vivo.

Longitudinal expression profiling of CD4+ and CD8+ cells in patients with active to quiescent giant cell arteritis.

BACKGROUND: Giant cell arteritis (GCA) is the most common form of vasculitis affecting elderly people. It is one of the few true ophthalmic emergencies but symptoms and signs are variable thereby making it a challenging disease to diagnose. A temporal artery biopsy is the gold standard to confirm GCA, but there are currently no specific biochemical markers to aid diagnosis. We aimed to identify a less invasive method to confirm the diagnosis of GCA, as well as to ascertain clinically relevant predictive biomarkers by studying the transcriptome of purified peripheral CD4+ and CD8+ T lymphocytes in patients with GCA. METHODS: We recruited 16 patients with histological evidence of GCA at the Royal Victorian Eye and Ear Hospital, Melbourne, Australia, and aimed to collect blood samples at six time points: acute phase, 2-3 weeks, 6-8 weeks, 3 months, 6 months and 12 months after clinical diagnosis. CD4+ and CD8+ T-cells were positively selected at each time point through magnetic-assisted cell sorting. RNA was extracted from all 195 collected samples for subsequent RNA sequencing. The expression profiles of patients were compared to those of 16 age-matched controls. RESULTS: Over the 12-month study period, polynomial modelling analyses identified 179 and 4 statistically significant transcripts with altered expression profiles (FDR < 0.05) between cases and controls in CD4+ and CD8+ populations, respectively. In CD8+ cells, two transcripts remained differentially expressed after 12 months; SGTB, associated with neuronal apoptosis, and FCGR3A, associatied with Takayasu arteritis. We detected genes that correlate with both symptoms and biochemical markers used for predicting long-term prognosis. 15 genes were shared across 3 phenotypes in CD4 and 16 across CD8 cells. In CD8, IL32 was common to 5 phenotypes including Polymyalgia Rheumatica, bilateral blindness and death within 12 months. CONCLUSIONS: This is the first longitudinal gene expression study undertaken to identify robust transcriptomic biomarkers of GCA. Our results show cell type-specific transcript expression profiles, novel gene-phenotype associations, and uncover important biological pathways for this disease. In the acute phase, the gene-phenotype relationships we have identified could provide insight to potential disease severity and as such guide in initiating appropriate patient management.

The furoxan nitric oxide donor, PRG150, evokes dose-dependent analgesia in a rat model of painful diabetic neuropathy.

Painful diabetic neuropathy (PDN) is a type of peripheral neuropathic pain that is often intractable. Elevated nitric oxide (NO) from neuronal and non-neuronal sources in the somatosensory system is implicated in the pathobiology of peripheral neuropathic pain. However, in diabetes, nitrergic nerve degeneration to deplete NO bioactivity appears causal in the pathogenesis of irreversible autonomic neuropathy, another long term complication of diabetes. Hence, this study hypothesized that progressive NO depletion may underpin the pathobiology of PDN and that NO donors may alleviate PDN. Diabetes was induced in rats with intravenous streptozotocin (STZ) at 70 mg/kg and confirmed if blood glucose levels (BGLs) on day 10 post-STZ were ≥15 mmol/L. Analgesic efficacy of subcutaneous (s.c.) bolus doses of the furoxan NO donor, PRG150 was assessed in the STZ-diabetic rat model of PDN at 10-, 14- and 24-weeks post-STZ relative to the sydnominine NO donor, SIN-1 and its prodrug, molsidomine. PRG150 produced dose-dependent analgesia in STZ-diabetic rats whereas SIN-1 and molsidomine evoked neuro-excitatory side-effects, but not analgesia. The 1000-fold larger doses of PRG150 needed to produce analgesia at 14- and 24-weeks (800 pmol/kg) c.f. 10-weeks (8 fmol/kg) post-STZ in rats, suggest that progressive NO depletion is also causal in PDN. Importantly, doses of PRG150 up to 10 000 fold higher than the analgesic dose did not produce hypotension in rats. The 50-fold greater release of NO by SIN-1 c.f. PRG150 in vitro, may underpin the neuro-excitatory rather than analgesic effects of SIN-1/molsidomine. PRG150 is worthy of further investigation as a potential novel analgesic for PDN.

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 Freund's 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.

Optimization and pharmacological characterization of a refined cisplatin-induced rat model of peripheral neuropathic pain.

Chemotherapy-induced peripheral neuropathy (CIPN) is the major dose-limiting side-effect of many front-line anticancer drugs. This study was designed to establish and pharmacologically characterize a refined rat model of cisplatin-induced CIPN. Adult male Sprague-Dawley rats received four (n=18) or five (n=18) single intraperitoneal bolus doses of cisplatin at 3 mg/kg, or saline (control group), once-weekly. Body weight and general health were assessed over a 49-day study period. von Frey filaments and the Hargreaves test were used to define the time course for the development of mechanical allodynia and thermal hypoalgesia in the hindpaws and for efficacy assessment of analgesic/adjuvant agents. The general health of rats administered four cisplatin doses was superior to that of rats administered five doses. Mechanical allodynia was fully developed (paw withdrawal thresholds≤6 g) in the bilateral hindpaws from day 32 to 49 for both cisplatin dosing regimens. They also showed significant thermal hypoalgesia in the bilateral hindpaws. In cisplatin-treated rats with paw withdrawal thresholds of up to 6 g, single bolus doses of gabapentin and morphine produced dose-dependent analgesia, whereas meloxicam and amitriptyline lacked efficacy. We have established and pharmacologically characterized a refined rat model of CIPN that is suitable for efficacy profiling of compounds from analgesic discovery programmes.

Endomorphin analogues with mixed µ-opioid (MOP) receptor agonism/δ-opioid (DOP) receptor antagonism and lacking ß-arrestin2 recruitment activity.

Analogues of endomorphin (Dmt-Pro-Xaa-Xaa-NH2) modified at position 4 or at positions 4 and 3, and tripeptides (Dmt-Pro-Xaa-NH2) modified at position 3, with various phenylalanine analogues (Xaa=Trp, 1-Nal, 2-Nal, Tmp, Dmp, Dmt) were synthesized and their effects on in vitro opioid activity were investigated. Most of the peptides exhibited high µ-opioid (MOP) receptor binding affinity (KiMOP=0.13-0.81nM), modest MOP-selectivity (Kiδ-opioid (DOP)/KiMOP=3.5-316), and potent functional MOP agonism (GPI, IC50=0.274-249nM) without DOP and κ-opioid (KOP) receptor agonism. Among them, compounds 7 (Dmt-Pro-Tmp-Tmp-NH2) and 9 (Dmt-Pro-1-Nal-NH2) were opioids with potent mixed MOP receptor agonism/DOP receptor antagonism and devoid of ß-arrestin2 recruitment activity. They may offer a unique template for the discovery of potent analgesics that produce less respiratory depression, less gastrointestinal dysfunction and that have a lower propensity to induce tolerance and dependence compared with morphine.

Analgesic efficacy of small-molecule angiotensin II type 2 receptor antagonists in a rat model of antiretroviral toxic polyneuropathy.

Individuals infected with the HIV and taking certain antiretroviral drugs to suppress viral replication have a high prevalence of neuropathic pain that is not alleviated by analgesic/adjuvant drugs that are often efficacious for the relief of other types of neuropathic pain. There is therefore a great need for new analgesics to alleviate the pain of antiretroviral toxic neuropathy (ATN). Small-molecule angiotensin II type 2 receptor (AT2R) antagonists, with ≥1000-fold selectivity over the angiotensin II type 1 receptor, produced analgesia in the chronic constriction injury of the sciatic nerve rat model of peripheral nerve trauma. Hence, the present study was designed to assess their analgesic efficacy in a rat model of ATN. The analgesic efficacy of small-molecule AT2R antagonists (EMA200 and EMA300) was assessed in a rat model of dideoxycytidine (ddC)-induced ATN. Single intraperitoneal bolus doses of EMA200 (0.3-10 mg/kg) induced dose-dependent analgesia in ddC-rats; the mean ED50 was 3.2 mg/kg. Twice-daily intraperitoneal administration of EMA300 at 30 mg/kg to ddC-rats for 3 days produced significant analgesia on days 2 and 3 of the treatment period. Therefore, small-molecule AT2R antagonists should be investigated further as novel analgesics for the relief of ATN.

Analgesic efficacy and mode of action of a selective small molecule angiotensin II type 2 receptor antagonist in a rat model of prostate cancer-induced bone pain.

OBJECTIVE: The pathobiology of prostate cancer (PCa)-induced bone pain (PCIBP) has both inflammatory and neuropathic components. Previously, we showed that small molecule angiotensin II type 2 receptor (AT2 R) antagonists with >1,000-fold selectivity over the angiotensin II type 1 receptor produced dose-dependent analgesia in a rat model of neuropathic pain. Here, we assessed the analgesic efficacy and mode of action of the AT2 R antagonist, EMA200, in a rat model of PCIBP. METHODS: At 14-21 days after unilateral intratibial injection of AT3B PCa cells, rats exhibiting hindpaw hypersensitivity received single intravenous bolus doses of EMA200 (0.3-10 mg/kg) or vehicle, and analgesic efficacy was assessed. The mode of action was investigated using immunohistochemical, Western blot, and/or molecular biological methods in lumbar dorsal root ganglia (DRGs) removed from drug-naïve and EMA200-treated PCIBP rats relative to sham-control rats. RESULTS: Intravenous bolus doses of EMA200 produced dose-dependent analgesia in PCIBP rats. Lumbar DRG levels of angiotensin II, nerve growth factor (NGF), tyrosine kinase A (TrkA), phospho-p38 mitogen-activated protein kinase (MAPK), and phospho-p44/p42 MAPK, but not the AT2 R, were increased significantly (P < 0.05) in PCIBP rats, c.f. the corresponding levels for sham controls. EMA200 produced analgesia in PCIBP rats by reducing elevated angiotensin II levels in the lumbar DRGs to attenuate augmented angiotensin II/AT2 R signaling. This in turn reduced augmented NGF/TrkA signaling in the lumbar DRGs. The net result was inhibition of p38 MAPK and p44/p42 MAPK activation. CONCLUSION: Small molecule AT2 R antagonists are worthy of further investigation as novel analgesics for relief of intractable PCIBP and other pain types where hyperalgesia worsens symptoms.

A small molecule angiotensin II type 2 receptor (AT2R) antagonist produces analgesia in a rat model of neuropathic pain by inhibition of p38 mitogen-activated protein kinase (MAPK) and p44/p42 MAPK activation in the dorsal root ganglia.

OBJECTIVE: There is an unmet clinical need for novel analgesics for neuropathic pain. This study was designed to elucidate the mechanism through which EMA300, a small molecule antagonist of the angiotensin II type 2 receptor (AT2R) with >1,000-fold selectivity over the angiotensin II type 1 receptor, produces analgesia in a rodent model of neuropathic pain. DESIGN AND METHODS: Groups of AT2R knockout, hemizygotes, and wild-type mice with a chronic constriction injury (CCI) of the sciatic nerve received single intraperitoneal (i.p.) bolus doses of EMA300 (100 or 300 mg/kg), and analgesic efficacy was assessed. Groups of control, sham-operated, and CCI rats were euthanized and perfusion fixed. Lumbar dorsal root ganglia (DRGs) were removed for investigation of the mechanism through which EMA300 alleviates neuropathic pain. RESULTS: EMA300 analgesia was abolished in AT2R knockout CCI mice with intermediate responses in the hemizygotes, affirming the AT2R as the target mediating EMA300 analgesia. In CCI rats, DRG immunofluorescence (IF) levels for angiotensin II, the main endogenous ligand of the AT2R, were increased ∼1.5-2.0-fold (P < 0.05) cf. sham-controls. Mean DRG IF levels for activated p38 (pp38) and activated p44/p42 (pp44/pp42) MAPK were also increased ∼1.5-2.0-fold (P < 0.05) cf. sham-controls. At the time of peak EMA300 analgesia in CCI rats, mean DRG levels of pp38 MAPK and pp44/pp42 MAPK (but not angiotensin II) were reduced to match the respective levels in sham-controls. CONCLUSION: Augmented angiotensin II/AT2R signaling in the DRGs of CCI rats is attenuated by EMA300 to block p38 MAPK and p44/p42 MAPK activation, a mechanism with clinical validity for alleviating neuropathic pain.

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.

Optimization and characterization of a rat model of prostate cancer-induced bone pain using behavioral, pharmacological, radiological, histological and immunohistochemical methods.

The major limitation of currently utilized rodent models of prostate cancer (PCa)-induced bone pain (PCIBP) involving intra-osseous injection of PCa cells, is their relatively short-term applicability due to progressive deterioration of animal health necessitating euthanasia. Here, we describe establishment of an optimized rat model of PCIBP where good animal health was maintained for at least 90-days following unilateral intra-tibial injection (ITI) of PCa cells. We have characterized this model using behavioral, pharmacological, radiological, histological and immunohistochemical methods. Our findings show that following unilateral ITI of 4×10(4) AT3B PCa cells (APCCs), there was temporal development of bilateral hindpaw hypersensitivity that was fully developed between days 14 and 21 post-ITI. Although there was apparent spontaneous reversal of bilateral hindpaw sensitivity that was maintained until at least day 90 post-ITI, administration of bolus doses of the opioid receptor antagonist, naloxone, rescued the pain phenotype in these animals. Hence, upregulation of endogenous opioid signaling mechanisms appears to underpin apparent spontaneous resolution of hindpaw hypersensitivity. Importantly, the histological and radiological assessments confirmed that tumor formation and development of osteosclerotic metastases was confined to the APCC-injected tibial bones. In our rat model of PCIBP, single bolus doses of morphine, gabapentin, meloxicam and amitriptyline produced dose-dependent relief of mechanical allodynia and thermal hyperalgesia in the bilateral hindpaws. The optimized rat model of PCIBP characterized herein has potential to provide new insights into the pathophysiological mechanisms associated with long-term (mal)adaptive pain due to advanced PCa-induced bony metastases and for screening novel compounds with potential for improved alleviation of this condition.

Peripherally acting novel lipo-endomorphin-1 peptides in neuropathic pain without producing constipation.

We previously described two novel analogues of endomorphin-1 (Tyr-Pro-Trp-Phe-NH2, 1), modified with an 8-carbon lipoamino acid (C8LAA) with or without replacement of Tyr(1) with 2,6-dimethyltyrosine (Dmt) at the N-terminus of the peptide (compounds 3 and 4, respectively). They were shown to be more stable and permeable, and acted as potent µ-opioid receptor agonists. In this study we report that the C8LAA modification resulted in successful systemic delivery of both analogues. They produced potent dose-dependent pain relief in a chronic constriction injury-rat model of neuropathic pain after intravenous administration with ED50 values obtained at 6.58 (±1.22) µmol/kg for 3 and 6.18 (±1.17) µmol/kg for 4. Using two different rat models of constipation that assess the effects of µ-opioid receptor agonists on stool hydration and gastro-intestinal motility, compound 3 produced insignificant constipation at 16 µmol/kg, whereas morphine elicited significant constipation at 2 µmol/kg. Compound 3 in contrast to morphine, did not attenuate the hypercapnic ventilatory response at 5 µmol/kg, a dose that fully alleviated hindpaw sensitivity at the time of peak effect in CCI-rats. This finding revealed the lack of respiratory depression effect at antinociceptive dose.

Lipo-endomorphin-1 derivatives with systemic activity against neuropathic pain without producing constipation.

To enhance the drug-like properties of the endogenous opioid peptide endomorphin-1 (1 = Tyr-Pro-Trp-Phe-NH(2)), the N-terminus of the peptide was modified with 2-aminodecanoic acid, resulting in compound 3. Tyr in compound 1 was replaced with 2,6-dimethyltyrosine yielding compound 2. Derivative 2 was also substituted with 2-aminodecanoic acid producing compound, 4. Lipoamino acid-modified derivatives showed improved metabolic stability and membrane permeability while maintaining high µ-opioid (MOP) receptor binding affinity and acting as a potent agonist. In vivo studies showed dose-dependent antinociceptive activity following intravenous (i.v.) administration of compounds 3 and 4 in a chronic constriction injury (CCI)-rat model of neuropathic pain with ED(50) values of 1.22 (± 0.93) and 0.99 (± 0.89) µmol/kg, respectively. Pre-treatment of animals with naloxone hydrochloride significantly attenuated the anti-neuropathic effects of compound 3, confirming the key role of opioid receptors in mediating antinociception. In contrast to morphine, no significant constipation was produced following i.v. administration of compound 3 at 16 µmol/kg. Furthermore, following chronic administration of equi-potent doses of compound 3 and morphine to rats, there was less antinociceptive tolerance for compound 3 compared with morphine.

Synthesis and biological evaluation of an orally active glycosylated endomorphin-1.

The endogenous opioid peptide endomorphin-1 (1) was modified by attachment of lactose to the N-terminus via a succinamic acid spacer to produce compound 2. The carbohydrate modification significantly improved the metabolic stability and membrane permeability of 2 while retaining µ-opioid receptor binding affinity and agonist activity. Analogue 2 produced dose-dependent antinociceptive activity following intravenous administration in a chronic constriction injury (CCI) rat model of neuropathic pain with an ED(50) of 8.3 (± 0.8) µmol/kg. The corresponding ED(50) for morphine was 2.6 (± 1.4) µmol/kg. Importantly, compound 2 produced dose-dependent pain relief after oral administration in CCI rats (ED(50) = 19.6 (± 1.2) µmol/kg), which was comparable with that of morphine (ED(50) = 20.7 (±3.6) µmol/kg). Antineuropathic effects of analogue 2 were significantly attenuated by pretreatment of animals with the opioid antagonist naloxone, confirming opioid receptor-mediated analgesia. In contrast to morphine, no significant constipation was produced by compound 2 after oral administration.

Insulin implants prevent the temporal development of mechanical allodynia and opioid hyposensitivity for 24-wks in streptozotocin (STZ)-diabetic Wistar rats.

OBJECTIVE: As the diabetes control and complications trial showed that intensive glycemic control in patients with Type 1 diabetes decreased the risk of development of long-term microvascular complications including painful diabetic neuropathy by approximately 60%, hyperglycemia was implicated as a causal factor in the etiology of this condition. Hence, the present study was designed as a 24-week longitudinal investigation of the extent to which the level of glycemic control in the streptozotocin (STZ)-diabetic rat model of Type 1 diabetes affects the development of mechanical allodynia and opioid hyposensitivity in these animals. RESULTS: Diabetes was fully developed (blood glucose levels ≥ 15 mM) in adult male Wistar rats by 7 days after intravenous STZ (75 mg/kg) administration. Mechanical allodynia developed in a temporal manner in the rat hindpaws, such that it was fully developed by 6 weeks and persisted for at least 24 weeks post-STZ administration. Morphine hyposensitivity also developed in a temporal manner in the same animals. By contrast, restoration and maintenance of euglycemia using insulin implants commencing at diabetes diagnosis on Day 7 post-streptozotocin administration, prevented development of both mechanical allodynia and opioid hyposensitivity in STZ-diabetic rats for the 24-week study duration. CONCLUSIONS: This study shows that long-term restoration of euglycemia over a 6-month period in STZ-diabetic rats prevents the hallmark symptoms of PDN including morphine hyposensitivity. CLINICAL RELEVANCE: Our findings are consistent with epidemiological data showing that tight glycemic control in patients with Type 1 diabetes markedly reduces the prevalence of PDN, further implicating persistent hyperglycemia as a pathogenic factor.

Longitudinal study of painful diabetic neuropathy in the Zucker diabetic fatty rat model of type 2 diabetes: impaired basal G-protein activity appears to underpin marked morphine hyposensitivity at 6 months.

OBJECTIVES: Epidemiological studies in patients with type 1 and type 2 diabetes show that hyperglycemia is associated with the development of long-term microvascular complications, including painful diabetic neuropathy (PDN). However, as the prevalence of type 2 diabetes in humans far exceeds that of type 1, the present study was undertaken as a 22-week longitudinal investigation commencing at 7 weeks of age, to assess the utility of the Zucker diabetic fatty (ZDF) rat model of type 2 diabetes for the study of PDN. DESIGN: Behavioral methods were used to characterize temporal changes in hindpaw sensitivity as well as morphine potency in these animals. The effect of long-term diabetes on µ-opioid receptor function and mRNA expression levels in the spinal cord was also assessed. RESULTS: Diabetes developed spontaneously in ZDF rats with marked hyperglycemia (blood glucose levels ≥15 mM) evident by 11 weeks of age, which was maintained until study completion at 29 weeks. In ZDF rats, there was progressive development of mechanical allodynia in the hindpaws such that it was fully developed by 6 months of age. Concurrently, there was temporal loss of opioid sensitivity in these animals such that marked morphine hyposensitivity was evident at 6 months. In the spinal cord, basal G-protein function was significantly impaired at 29 weeks of age, resulting in apparently reduced agonist-stimulated µ-opioid receptor function compared with the prediabetic state. CONCLUSIONS: Together, our findings suggest that impaired basal G-protein activity underpins morphine hyposensitivity in PDN. CLINICAL RELEVANCE: Clinical management of diabetic neuropathic pain has been challenging. This study provides a mechanistic explanation regarding the effectiveness, or lack thereof, of opioid analgesia in the treatment of diabetic neuropathic pain.

Rapid, opioid-sensitive mechanisms involved in transient receptor potential vanilloid 1 sensitization.

TRPV1 is a nociceptive, Ca2+-selective ion channel involved in the development of several painful conditions. Sensitization of TRPV1 responses by cAMP-dependent PKA crucially contributes to the development of inflammatory hyperalgesia. However, the pathways involved in potentiation of TRPV1 responses by cAMP-dependent PKA remain largely unknown. Using HEK cells stably expressing TRPV1 and the mu opioid receptor, we demonstrated that treatment with the adenylate cyclase activator forskolin significantly increased the multimeric TRPV1 species. Pretreatment with the mu opioid receptor agonist morphine reversed this increased TRPV1 multimerization. FRET analysis revealed that treatment with forskolin did not cause multimerization of pre-existing TRPV1 monomers on the plasma membrane and that intracellular pools of TRPV1 exist mostly as monomers in this model. This suggests that increased TRPV1 multimerization occurred from an intracellular store of inactive TRPV1 monomers. Treatment with forskolin also caused an increase in TRPV1 expression on the plasma membrane not resulting from increased TRPV1 expression, and this rapid TRPV1 translocation was inhibited by treatment with morphine. Thus, potentiation of TRPV1 responses by cAMP-dependent PKA involves plasma membrane insertion of functional TRPV1 multimers formed from an intracellular store of inactive TRPV1 monomers. This potentiation occurs rapidly and can be dynamically modulated by activation of the mu opioid receptor under conditions where cAMP levels are raised, such as with inflammation. Increased translocation and multimerization of TRPV1 channels provide a cellular mechanism for fine-tuning of nociceptive responses that allow for rapid modulation of TRPV1 responses independent of transcriptional changes.

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 (approximately 1.6-fold; P < 0.01) and oral (approximately 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 (approximately 2.4-fold; P < 0.005) and oral (approximately 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 (approximately 13- and fivefold, respectively) and oral (approximately 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.

Mechanisms involved in potentiation of transient receptor potential vanilloid 1 responses by ethanol.

The transient receptor potential vanilloid 1 or TRPV1 is a calcium-permeable ion channel that is activated by capsaicin, the active component of hot chilli peppers, and is involved in the development of inflammatory and neuropathic hyperalgesias. Ethanol can sensitise TRPV1-mediated responses, but the pathways contributing to the potentiation of TRPV1 by ethanol have not been clearly defined. Since the mu opioid receptor (MOP) agonist morphine can inhibit TRPV1 responses potentiated by cAMP-dependent protein kinase A (PKA), and ethanol-mediated modulation of other ion channels involves activation of PKA, we aimed to assess the contribution of MOP-sensitive pathways to the potentiation of TRPV1-mediated capsaicin responses by ethanol. Calcium responses elicited by the TRPV1 agonist capsaicin were potentiated by treatment with ethanol, but morphine was not able to inhibit ethanol-sensitised capsaicin responses. Indeed, cAMP-dependent PKA did not appear to contribute to potentiation of TRPV1 responses by ethanol, as the PKA inhibitor Rp-cAMPS did not inhibit ethanol-potentiated capsaicin responses. Similarly, treatment with specific PKC and PI3K inhibitors did not affect capsaicin responses in the presence of ethanol. However, treatment with wortmannin at concentrations reported to cause PIP2 depletion limited the ability of ethanol to sensitise TRPV1-mediated capsaicin responses. Among other plausible mechanisms, such as non-specific inhibition of kinases including mTOR, DNA-PK, MLCK, MAPK and polo-like kinases, this suggests that ethanol may affect the PIP2-TRPV1 interaction. This was confirmed by inhibition of ethanol-potentiation by the PLC inhibitor U73122. The results presented here suggest that morphine may be of limited use in inhibiting nociceptive TRPV1 responses that have been sensitised by exposure to ethanol.