Therapeutic efficacy of arginine-rich exenatide on diabetic neuropathy in rats.

Diabetes mellitus is characterized by metabolic dysregulation associated with a number of health complications. More than 50% of patients with diabetes mellitus suffer from diabetic polyneuropathy, which involves the presence of peripheral nerve dysfunction symptoms. The aim of this study was to evaluate the potential of a new synthetic arginine-rich exendin-4 (Peptide D) in the treatment of complications caused by diabetes, including peripheral neuropathy, in rats. Diabetes was induced by administering streptozotocin (STZ). Three groups of diabetic rats were treated with Peptide D (0.1, 1, and 10 µg/kg). One group of diabetic rats was treated with Byetta® (1 µg/kg) for 80 days. Neuropathic pain development was assessed by tactile allodynia. STZ-treated rats showed an increased level of tactile allodynia unlike naïve animals. A histological study revealed that the diameter of the sciatic nerve fibers in STZ-treated rats was smaller than that of the naïve animals. An IHC study demonstrated decreased expression of myelin basic protein (MBP) in the sciatic nerve of diabetic rats compared to that in the naïve animals. Peptide D reduced the severity of tactile allodynia. This effect was more pronounced in the Peptide D treated groups than in the group treated with Byetta®. Peptide D and Byetta® treatment resulted in increased MBP expression in the sciatic nerve and increased diameter of myelinated nerve fibers. These findings suggest that poly-arginine peptides are promising agents for the treatment of peripheral polyneuropathies.

Marinobufagenin in Urine: A Potential Marker of Predisposition to Ethanol and a Target for Spironolactone.

BACKGROUND AND OBJECTIVE: Previously it was demonstrated that digitalis-like cardiotonic steroid, marinobufagenin (MBG), is implicated in the development of ethanol addiction in rats. We hypothesized that (i) levels of sodium pump ligand, MBG, would be negatively correlated with the amount of ethanol consumed by rats, and (ii) that spironolactone would oppose the MBG induced ethanol-seeking behavior and blood pressure in rats. METHODS: Voluntary consumption of 9% alcohol (vs. water) during 10 days period by 11 adult male Wistar rats was studied. Eight weeks after the beginning of the experiment, the animals were divided into two treatment subgroups: high alcohol drinkers (HAD, n=6, daily consumption of ethanol > 4 g/kg) and low alcohol drinkers (LAD, n=5, daily consumption of ethanol < 4 g/kg) rats. Spironolactone treatment (7 days) was started following 3-day habituation to intragastric vehicle administration. Consumption of ethanol and blood pressure were recorded daily. RESULTS: Urinary MBG excretion at baseline was 11.2±0.6 pmoles in HAD rats and 19.1±2.9 pmoles (p<0.05) in LAD rats, respectively. Seven days of spironolactone treatment was associated with reduction in ethanol intake (2.9 g/kg/24 hr), reduction in systolic blood pressure (5 mm Hg), and increase in sodium excretion (1 mmol/24 hr). CONCLUSION: Levels of MBG may be a predisposing factor to voluntary ethanol intake. Spironolactone, along with antihypertensive effect, decreases ethanol intake.

A new tridecapeptide with an octaarginine vector has analgesic therapeutic potential and prevents morphine-induced tolerance.

A growing body of evidence suggests that peptides may possess analgesic effects without tolerance development. The synthetic tetrapeptide Tyr-d-Arg-Phe-Gly-NH2 was modified with the inclusion of a (d-Arg)8 vector to prevent the action of endopeptidase and to increase the duration of the analgesic action of the tetrapeptide when administered orally. The aim of this study was to estimate the analgesic efficacy of the tetrapeptide with (d-Arg)8 (tridecapeptide, TDP) in experimental models of acute and chronic pain. The analgesic effects of TDP were estimated using a model of acute visceral pain in mice (writhing test) and a model of chronic neuropathic pain (chronic constriction injury (CCI) of the sciatic nerve) in rats. The intravenous administration of morphine (0.32-1mg/kg) and TDP (0.32-1.8mg/kg) produced significant dose-related antinociceptive effects in the writhing test. The potency of TDP after i.g. administration was lower than that after i.v. administration but comparable with that of i.g. morphine. In the CCI model, TDP (0.1, 1 and 10mg/kg, i.g.) induced marked analgesia with repeated administration without any signs of tolerance. The single administration of TDP after morphine treatment (7days) produced a significant analgesic effect in morphine-tolerant rats, indicating the absence of cross-tolerance between these two drugs. The combined administration of TDP and morphine resulted in the reduction of analgesic tolerance to morphine. The absence of cross-tolerance to morphine and the ability to prevent morphine tolerance allows this compound to be a prospective candidate for chronic pain therapy. In order to find the target receptors for TDP, a docking study was performed. It was found that the molecule can bind to the NMDA receptor using electrostatic, hydrogen bonding and hydrophobic interactions.

Estradiol lowers intracranial self-stimulation thresholds and enhances cocaine facilitation of intracranial self-stimulation in rats.

Women initiate cocaine use at a younger age and have more complications (e.g., higher rates of major or minor depression) related to cocaine use than men. It has been proposed that estrogens play an important role in these sex differences. The addictive potential of psychoactive drugs can be measured in rats via a rewarding intracranial self-stimulation (ICSS) procedure. The rate-independent method of ICSS allows researchers to assess the "pure" rewarding effect of cocaine without influence of nonspecific motor reactions. The present study aimed to estimate effects of estradiol and a combination of estradiol and cocaine on ICSS in ovariectomized female rats. 17-ß-estradiol (5µg/animal/day, 2 days) produced a long-lasting gradual lowering of the thresholds for ICSS. The ability of estradiol to decrease thresholds for ICSS has never been shown previously. Combination of 17-ß-estradiol and cocaine (5.0mg/kg, 5 days) produced a greater effect on ICSS thresholds than the effect of either compound alone. No tolerance or sensitization to cocaine developed during the study. Present findings suggest estradiol increases sensitivity of the brain reward system in rats, which may have an important implication in understanding sex differences in cocaine effects.

The anxiolytic and analgesic properties of fenobam, a potent mGlu5 receptor antagonist, in relation to the impairment of learning.

Fenobam [N-(3-chlorophenyl)-N'-(4,5-dihydro-1-methyl-4-oxo-1H-imidazole-2-yl)urea] was suggested to possess anxiolytic actions 30 years ago. Hoffmann-La Roche researchers recently reported that it is a selective and potent mGlu5 receptor antagonist, acting as a negative allosteric modulator. In the present study, we show that fenobam readily penetrates to the brain, reaching concentrations over 600 nM, clearly above the affinity for mGluR5 receptors. Fenobam (at 10, 30, and 100 mg/kg) did not affect horizontal locomotor activity in the open field test. Anxiolytic-like activity in the context freezing test was seen at 30 mg/kg, while fenobam was not active in the elevated plus maze test at the tested concentrations. Fenobam had antinociceptive actions in the formalin test at 10 and 30 mg/kg, but failed to attenuate mechanical allodynia in the chronic constriction injury model. Impairment of learning was revealed in the passive avoidance test at 30 mg/kg. Fenobam also impaired performance in both the Morris water maze and in the contextual fear conditioning test at the doses of 30 and 10 mg/kg, respectively. Prepulse inhibition, used as a model of psychomimetic activity, was not affected by fenobam at doses of up to 60 mg/kg. Our results indicate that the beneficial effects of fenobam occur in a similar dose range as the potential side-effects.

Comparison of the mGluR1 antagonist A-841720 in rat models of pain and cognition.

In the current study we compared the potency of the selective metabotropic glutamate receptor (mGluR1) antagonist A-841720 (7-Azepan-1-yl-4-dimethylamino-7H-9-thia-1,5,7-triaza-fluoren-8-one) in rodent models of pain with its effects in models of learning and memory, to obtain information regarding the therapeutic window of this compound. A-841720 significantly reduced formalin-induced behaviours and complete Freund's adjuvant (CFA)-induced tactile allodynia, starting at doses of 1 and 10 mg/kg, respectively. At the dose of 3 mg/kg, however, A-841720 significantly reduced the percentage of spontaneous alternations in a radial-maze task. In contextual-fear conditioning, A-841720, given at the dose of 10 mg/kg before acquisition, significantly reduced freezing behaviour tested 24 h later. In the same task, repeated treatment for 5 days did not reduce the impairing effect of the challenge dose, indicating a lack of tolerance development. In a passive-avoidance task, A-841720 at 10 mg/kg administered before acquisition, significantly reduced the latency to enter the dark box on the retention test. Given that complete Freund's adjuvant is a better measure of analgesia, these results indicate that the selective mGluR1 antagonist A-841720 has analgesic potential in a dose range at which it also produces memory impairments. This diminishes its therapeutic potential for the treatment of chronic pain.

mGlu1 receptor blockade attenuates cue- and nicotine-induced reinstatement of extinguished nicotine self-administration behavior in rats.

Glutamatergic neurotransmission is believed to be critically involved in the acquisition and maintenance of drug addiction. The present study evaluated the role of metabotropic glutamate (mGlu) 1 receptors in the reinstatement of nicotine-seeking behavior. Rats were trained to nose-poke to receive response-contingent intravenous infusions of nicotine (0.01 mg/kg/infusion, free base). Following the subsequent extinction phase, reinstatement tests were conducted in animals that were exposed either to response-contingent presentations of the nicotine-associated discrete light cues or to non-contingent nicotine priming injection (0.3mg/kg, s.c., salt) just prior to the test session. In a separate experiment, rats were subjected to the nearly identical response-reinstatement procedure but operant responding was established using food pellets instead of nicotine infusions. Pretreatment with the mGlu1 receptor antagonist EMQMCM (JNJ16567083, (3-ethyl-2-methyl-quinolin-6-yl)-(4-methoxy-cyclohexyl)-methanone methanesulfonate) significantly inhibited cue-induced reinstatement of nicotine-seeking behavior (5 and 10, but not 2.5 mg/kg). EMQMCM (5 mg/kg) also prevented nicotine priming-induced reinstatement of nicotine-seeking behavior. At the highest tested dose only (10 mg/kg), EMQMCM attenuated cue-induced reinstatement of food-seeking behavior. Taken together with the previous reports, the present findings further suggest that blockade of mGlu1 receptors may be beneficial for preventing relapse to tobacco smoking in nicotine-dependent individuals.

Effects of NAAG peptidase inhibitor 2-PMPA in model chronic pain - relation to brain concentration.

N-acetylated-alpha-linked-acidic peptidase (NAAG peptidase) converts N-acetyl-aspartyl-glutamate (NAAG, mGluR3 agonist) into N-acetyl-aspartate and glutamate. The NAAG peptidase inhibitor 2-PMPA (2-(phosphonomethyl)pentanedioic acid) had neuroprotective activity in an animal model of stroke and anti-allodynic activity in CCI model despite its uncertain ability to penetrate the blood-brain barrier. The NAAG concentration in brain ECF under basal conditions and its alteration in relation to the brain ECF concentration of 2-PMPA is unclear. We therefore assessed those brain concentrations after i.p. administration of 2-PMPA, using in vivo microdialysis combined with LC/MS/MS analysis. Administration of 2-PMPA (50mg/kg) produced a mean peak concentration of 2-PMPA of 29.66+/-8.1microM. This concentration is about 100,000 fold more than is needed for inhibition of NAAG peptidase, and indicates very good penetration to the brain. Application of 2-PMPA was followed by a linear increase of NAAG-concentration reaching a maximum of 2.89+/-0.42microM at the end of microdialysis. However, during the time the anti-allodynic effects of 2-PMPA were observed, the NAAG concentration in the ECF did not reach levels which are likely to have an impact on any known target. It appears therefore that the observed behavioural effects of 2-PMPA may not be mediated by NAAG nor, in turn, by mGluR3 receptors.

Effects of memantine on estrogen-dependent acute tolerance to the morphine analgesia in female rats.

Both human and animal studies suggest that there are sex differences in responding to noxious stimulation as well as in effects of opiate analgesic drugs. Development and/or expression of tolerance to opiate analgesia are also affected by the hormonal status of the experimental subjects. The present study aimed to compare acute tolerance to morphine in cycling and ovariectomized female rats and to evaluate the effects of N-methyl-D-aspartate (NMDA) receptor channel blocker memantine as well as 17-beta-estradiol on tolerance development using the tail-flick test. Acute tolerance to morphine analgesia was observed as a substantial reduction in the response to a test dose of morphine (10 mg/kg) given 6 h after the tolerance-inducing dose (10 mg/kg). Significant acute tolerance was observed in proestrous female rats and was prevented by memantine (3 or 10 mg/kg) treatment. Ovariectomized rats did not demonstrate tolerance to morphine analgesic effects but chronic estradiol administration (5 microg/day, 5 days) reinstated induction of tolerance. Both estrogen receptor modulator tamoxifen (5 mg/kg/day, 5 days) and memantine (3 mg/kg/day, 5 days) prevented estradiol-induced tolerance in ovariectomized rats. Thus, estrogens were found to play a key role in induction of acute tolerance to morphine antinociception. Estradiol-induced acute morphine tolerance may have NMDA receptor-dependent mechanisms.

Estrous cycle stage-dependent expression of acute tolerance to morphine analgesia in rats.

Both baseline pain sensitivity and the response to antinociceptive treatment are sensitive to an animal's sex and estrous cycle stage. Sex differences are also observed in the development of antinociceptive tolerance induced by repetitive exposure to opiate drugs such as morphine. Conventional tolerance study protocols do not assess the impact of the estrous cycle stage. The present study aimed to compare the development of acute tolerance to morphine-induced antinociception in male and female (cycling and ovariectomized) Wistar rats using the tail-flick test. Acute tolerance was induced by two consecutive subcutaneous injections of morphine (10 mg/kg) or saline separated by an interval of 6 h. It was found that rats pretreated with morphine were tolerant to the second morphine dose. Tolerance was most pronounced in proestrous female rats and, to a lesser degree, in male rats. It was absent in ovariectomized rats as well as during the estrus, metestrus and diestrus phases. Thus, the estrous cycle exerts dramatic effects on the induction of acute tolerance to morphine-induced antinociception. These results suggest that pain management strategies can be optimized through the use of sex- and estrous cycle-specific techniques.