A Conditional Deletion of the NR1 Subunit of the NMDA Receptor in Adult Spinal Cord Dorsal Horn Reduces NMDA Currents and Injury-Induced Pain

To determine the importance of the NMDA receptor (NMDAR) in pain hypersensitivity after injury, the NMDAR1 (NR1) subunit was selectively deleted in the lumbar spinal cord of adult mice by the localized injection of an adenoassociated virus expressing Cre recombinase into floxed NR1 mice. NR1 subunit mRNA and dendritic protein are reduced by 80% in the area of the virus injection, and NMDA currents, but not AMPA currents, are reduced 86–88% in lamina II neurons. The spatial NR1 knock-out does not alter heat or cold paw-withdrawal latencies, mechanical threshold, or motor function. However, injury-induced pain produced by intraplantar formalin is reduced by 70%. Our results demonstrate conclusively that the postsynaptic NR1 receptor subunit in the lumbar dorsal horn of the spinal cord is required for central sensitization, the central facilitation of pain transmission produced by peripheral injury

The role of morphine-6-glucuronide (M6G) in pain control

Morphine-6beta-D-glucuronide (M6G) is an analgesically active metabolite of morphine, accounting for approximate to10% of the morphine dose when administered by systemic routes to humans. Although M6G is more hydrophilic than morphine, it crosses the blood-brain barrier, albeit relatively slowly. For this reason, it is generally thought that, after chronic dosing, M6G contributes significantly to the analgesic effects of systemically administered morphine. Owing to its polar nature, M6G is cleared from the systemic circulation primarily via renal elimination. As M6G accumulates in patients with renal impairment, there is an increased risk of M6G-induced respiratory depression in renal failure patients who are being dosed chronically with systemic morphine. Consistent with its analgesic and respiratory depressant properties, M6G binds to the p-opioid receptor in a naloxone-reversible manner. Although the affinity of M6G for the mu-opioid receptor is similar to or slightly less than that of morphine, preclinical studies in rodents show that M6G is one to two orders of magnitude more potent than morphine when administered by central routes. This major discrepancy between the markedly higher intrinsic antinociceptive potency of M6G relative to morphine, despite their similar p-opioid receptor binding affinities, is difficult to reconcile. It has been proposed that M6G mediates its pain-relieving effects through a novel 'M6G opioid receptor', while others have argued that M6G may have higher efficacy than morphine for transduction of intracellular events. When administered by parenteral routes to rodents, M6G's antinociceptive potency is no more than twofold higher than morphine. In humans, the analgesic efficacy and respiratory depressant potency of M6G relative to morphine have been assessed in a number of short-term studies involving the intrathecal or intravenous routes of administration. For example, in hip replacement patients, intrathecal M6G provided excellent postoperative analgesia but the occurrence of late respiratory depression in 10% of these patients raised serious concern about safety. In postoperative patients, intravenous M6G administered by means of patient-controlled analgesia (PCA), or bolus plus PCA, produced no analgesia in one study and limited analgesia in another. Similarly, there was a lack of significant analgesia in healthy volunteers who received intravenous M6G for the alleviation of experimental pain (carbon dioxide applied to the nasal mucosa). In contrast, satisfactory analgesia was produced by bolus doses of intravenous M6G administered to patients with cancer pain, and to healthy volunteers with experimentally-induced ischaemic, electrical or thermal (ice water) pain. Studies to date in healthy volunteers suggest that intravenous M6G may be a less potent respiratory depressant and have a lower propensity for producing nausea and vomiting than morphine. However, it is unclear whether equi-analgesic doses of M6G and morphine were compared. Clearly, more extensive short-term trials, together with studies involving chronic M6G administration, are necessary before the potential clinical utility of M6G as an analgesic drug in its own right can be determined

Pain pharmacology and analgesia

Pain is defined by the International Association for the Study of Pain as "an unpleasant sensory and emotional experience associated with actual or potential tissue damage, or described in terms of such damage". Pain may be described according to severity (mild, moderate, severe), duration (acute or chronic) or type (nociceptive, inflammatory, neuropathic). In the last two decades, considerable research directed at enhancing our collective understanding of the neurobiology of pain has revealed that persistent ongoing pain secondary to tissue inflammation or peripheral nerve injury is underpinned by considerable complexity and plasticity in the pain signaling system. Following tissue or peripheral nerve injury, there is sensitization of the somatosensory system so that innocuous stimuli are detected as painful (allodynia) or there is a heightened response to painful stimuli (hyperalgesia). Although a large number of "pain" targets for potential modulation by small molecules or biologics have been identified with several of these molecules now in preclinical or clinical development, these potential new pain medicines are yet to reach the clinic. Hence, pain is currently managed according to the World Health Organisations 3-step Analgesic Ladder. For mild pain, non-opioid analgesics such as paracetamol (acetaminophen) and nonsteroidal anti-inflammatory drugs are recommended, with adjuvants (e.g. antidepressants, anticonvulsants or anti-arrhythmics) added as required if pain has a neuropathic component. For moderate pain, weak opioids such as codeine and tramadol are added to non-opioids and/or adjuvants, as required. For moderate to severe pain, strong opioids are recommended with morphine as the drug of choice due to its ready availability worldwide at low cost. Strong opioids may be co-administered with non-opioids and with adjuvants, as required. In the next decade, a new generation of pain medicines is likely to reach the market, thereby expanding the armamentarium of drugs available to clinicians for the management of persistent on-going pain

Pharmacokinetics and effects of 17 beta-estradiol and progesterone implants in ovariectomized rats

For the pharmacokinetic evaluation of Silastic capsules, ovariectornized (OVX) rats were implanted subcutaneously with this dosage form containing 17 beta-estradiol (5, 10, 15, or 20% in cholesterol, where 5% 17 beta-estradiol equals 0.4 mg) or progesterone (20, 40, 110, or 220 mg of crystalline progesterone). The time-course of serum 17 beta-estradiol and progesterone released from these capsules in the OVX rat is characterized by an initial increase in serum hormone levels followed by a decline and then an apparent steady-state that persists from 7 to 24 days postimplant. Both hormones have large clearance values (total clearance is 97.7 L/day for 17 beta-estradiol and 20.9 L/clay for progesterone). For 17 beta-estradiol and progesterone only, 11% of the dose was released from the implant after 24 days. Thus, the Silastic membrane represents the rate controlling barrier for these hormones. The relationship between graded doses of 17 beta-estradiol or progesterone and serum concentration was linear. Neither tail flick latencies measured at 48, 52.5, and 55 degrees C nor the antinociceptive potency of morphine (ED50 values) were altered by continuous administration to steady-state of graded doses of 17 beta-estradiol or progesterone. We demonstrate how a dose-dependent analysis of some of the behavioral effects of 17 beta-estradiol or progesterone can be conducted at steady-state serum hormone concentrations. Perspective: we describe a method to obtain sustained serum levels of estrogen or progesterone and the consequences of these sustained hormone levels on acute thermal nociception and the antinociceptive response to morphine. This rat model of hormone replacement may provide insights into the role of these hormones in pathological pain states

Sex-related differences in antinociception and tolerance development following chronic intravenous infusion of morphine in the rat: modulatory role of testosterone via morphine clearance

This study investigated possible sex-related differences in levels of antinociception and the rate of development of tolerance to the antinociceptive effects following prolonged (48 h) intravenous (i.v.) morphine administration in the rat. Groups of adult intact male, castrated male, female, and testosterone-pretreated female Sprague-Dawley rats received prolonged (48 h) infusions of i.v. morphine (5 or 10 mg/day) plus intra-arterial (i.a.) saline or i.v. morphine (5 mg/day) plus i.a. chloramphenicol (300 mg/day). Antinociception was quantified using the hotplate test. Serum concentrations of morphine and morphine-3-glucuronide (M3G) were assayed using high performance liquid chromatography with electrochemical detection, whereas the serum testosterone concentrations were quantified using an enzyme-linked immunosorbent assay method. Consistent with our previous findings in intact male rats, prolonged coinfusion of chloramphenicol with morphine produced a marked increase in the extent and duration of morphine antinociception in all experimental groups. Additionally, female and castrated male rats developed tolerance more slowly than either intact male or testosterone-pretreated female rats, when coinfused with parenteral morphine plus chloramphenicol. However, mean levels of antinociception were not significantly correlated with either the mean serum morphine or M3G concentrations, but were significantly inversely correlated with the mean values of the M3G/morphine serum molar concentration ratio. Testosterone pretreatment of female rats for 1 week before chronic morphine infusion abolished antinociception and markedly reduced both the serum morphine and M3G concentrations. These latter findings imply that testosterone modulates antinociception evoked by prolonged morphine infusion in rats via a mechanism that appears to involve modulation of morphine metabolism

Tissue distribution of intravenously administrated poly-arginine peptide R18D in healthy male Sprague–Dawley rats

Aim: R18D is a poly-arginine peptide that has demonstrated neuroprotection in preclinical models of excitotoxicity, stroke, hypoxic-ischemic encephalopathy and traumatic brain injury. Here, we examined the peptide’s uptake in serum. Materials & methods: Healthy, male Sprague–Dawley rats were intravenously administered either 1000 nmol/kg R18D (D-enantiomer of R18) or approximately 2.5 nmol/kg (36 ± 9 MBq) [18F]R18D, for serum and organ tissue uptake, respectively. Serum samples underwent mass spectrometric analysis to detect unbound R18D peptide. Animals administered [18F]R18D were subjected to positron emission tomography imaging. Results & conclusion: Free R18D was detected at 5 min post-infusion in serum samples. [18F]R18D was rapidly distributed to the kidney (6–7%ID/g), and a small fraction localized to the brain (0.115–0.123%ID/g) over a 60-min acquisition period. Lay abstract R18D is a promising treatment for brain injury that has shown it can protect brain cells from damage in laboratory models of injury. These positive findings led us to examine what happens to R18D after administration in the body. Healthy, male rats were given 1000 nmol/kg R18D or approximately 2.5 nmol/kg (36 ± 9 MBq) [18F]R18D via an injection. Chemical and imaging analyses measured the amount of each compound. Free R18D was present in the blood 5 min after administration and was quickly absorbed by the kidney (6–7%ID/g) and brain (0.115–0.123%ID/g) within 60 min. Peptide-based therapies have been extensively explored due to their unique characteristics and potential for use following neurological injury. For example, cationic arginine-rich peptides (CARPs), such as the HIV-derived TAT, poly-arginine-9 and penetratin have been exploited in drug development for their cell-penetrating properties [1–3]. Not only are CARPs useful as carrier molecules for drug delivery, but they also possess intrinsic neuroprotective properties [4]. Studies from our laboratory have confirmed that the overall cationic change, and specifically the arginine content, are critical elements for CARP neuroprotective effects [5–7]. As a result of in vitro and in vivo neuroprotection studies using CARPs in different injury models, we have identified poly-arginine-18 (R18; 18-mer of arginine) as a leading therapeutic neuroprotective peptide [6]. Assessment of R18 and/or its D-enantiomer, R18D, in neuroprotection include studies using in vitro models of excitotoxicity and animal models of stroke, hypoxic-ischemic encephalopathy and traumatic brain injury [8–15]. However, despite the positive neuroprotection studies, the pharmacokinetic properties and systemic distribution of the R18 peptide have not been examined. Therefore, the aim of this study was to undertake a serum uptake and whole-body distribution study of R18 when administrated intravenously to healthy Sprague–Dawley rats. For the study, we decided to use R18D, as peptides synthesized with D-amino acids (i.e., D-arginine) are known to be more resistant to proteolytic degradation than peptides synthesized with naturally occurring L-amino acids