Leu-enkephalin actions on avoidance conditioning are mediated by a peripheral opioid mechanism

Leu-enkephalin (100 micrograms/kg, i.p.) administered to mice 5 min before training in a one way active avoidance task significantly reduced the number of avoidances observed in the peptide treated animals. This impairing action of Leu-enkephalin was partially attenuated by methylnaloxonium (naloxonium), a quarternary form of naloxone with a limited ability to penetrate the blood brain barrier. Passive immunization (i.v.) of mice with a Leu-enkephalin antiserum 4 hrs before training produced an effect on avoidance conditioning that was the opposite to that observed with Leu-enkephalin alone. That is, passive immunization increased the number of avoidances observed in the treated mice. The results suggest that Leu-enkephalin actions on avoidance conditioning are mediated by a peripheral opioid mechanism, that leu-enkephalin may have a primary site of action outside the blood brain barrier, and that peripheral Leu-enkephalin systems may normally operate to influence conditioned avoidance behavior.     

Optimizing action of synthetic peptide Selank on active avoidance conditioning test in rats

The action of a synthetic peptide Selank on learning and memory in active avoidance conditioning test was studied in rats with initially low learning ability and normal animals. The peptide was administered repeatedly 15 min prior the training session (4 days). The effect of Selank (300 micrograms/kg) was compared to that of Pyracetam (400 mg/kg). Selank was found to significantly improve learning in rats with low level ability even after a single administration on the first day of training session. The effect progressively increased with repeated Selank treatment: the total numbers of reactions and correct reactions increased, and the number of errors decreased (p < 0.05). In normal rats, the effect was maximal on the third day of treatment and training, i.e., after the completed initial consolidation. Some distinguishing features were revealed in the dynamics of activatory effects of Selank and Pyracetam. These data together with the evidence for ansiolytic effect of Selank show that this drug is promising for optimization of mnestic functions under conditions of high emotional stress.     

Intestine may be a major site of action for the apoA-I mimetic peptide 4F whether administered subcutaneously or orally

To determine if the dose of peptide administered or the plasma level was more important, doses of 0.15, 0.45, 4.5, or 45 mg/kg/day of the peptide D-4F were administered orally or subcutaneously (SQ) to apoliptotein (apo)E null mice. Plasma levels of peptide were ～1,000-fold higher when administered SQ compared with orally. Regardless of the route of administration, doses of 4.5 and 45 mg/kg significantly reduced plasma serum amyloid A (SAA) levels and the HDL inflammatory index (P < 0.0001); doses of 0.15 or 0.45 mg/kg did not. A dose of 45 mg/kg/day administered to apoE null mice on a Western diet reduced aortic atherosclerosis by ～50% (P < 0.0009) whether administered orally or SQ and also significantly reduced plasma levels of SAA (P < 0.002) and lysophosphatidic acid (P < 0.0009). Remarkably, for each dose administered, the concentration and amount of peptide in the feces was similar regardless of whether the peptide was administered orally or SQ. We conclude: i) the dose of 4F administered and not the plasma level achieved determines efficacy; ii) the intestine may be a major site of action for the peptide regardless of the route of administration.     

Effects of MSH/ACTH 4–10 on the classically-conditioned rabbit nictitating membrane

Subjects were conditioned/extinguished under four experimental conditions using either MSH/ACTH 4–10 (A) or diluent (D): D/D, D/A, A/D, and A/A. The major question investigated was whether or not the peptide has an effect on this classically-conditioned behavior similar to that reported for instrumental conditioning paradigms. The results indicated that it does not. An effect was seen on performance, not on learning or attentional processes. Animals treated with the peptide performed more poorly (i.e., displayed fewer conditioned responses) during both acquisition and extinction. In addition, there was an apparent residual effect of the peptide that lasted 24 but not 48 hours.     

A model for the study of the oral administration of peptide hormones.

The intragastric administration of lysine vasopressin (LVP) to rats is used as a model to study the biological activity of orally administered peptide hormones. Using a modification of the antidiuretic assay of Sawyer, LVP given by stomach tube caused a significant antidiuresis that was dose dependent in doses of 300 to 2000 mU. The simultaneous administration of the protease inhibitor, Trasylol, increased the antidiuretic effect of LVP. The synthetic peptide (1-deamino, 4 valine)-8-D-arginine-vasopressin also caused a dose-dependent prolonged and significant antidiuresis. No pressor effect was observed after intragastric administration of LVP in doses up to 40 U/rat. We are now using this model to test other procedures for enhancing the activity of lysine vasopressin administered in the gastrointestinal tract such as encapsulation into liposomes. The information gained with vasopressin will then be applied to insulin with the ultimate goal of making oral administration practical.     

Differential effects of dopamine D1 and D2 receptor antagonists on conditioned orienting behavior in the rat

Brain dopamine (DA) systems are known to be important in regulation of behavior conditioned to appetitive stimuli. Nevertheless, despite a large body of evidence showing behavioral deficits in the operant conditioning paradigm produced by DA receptor blockade, there have been relatively few studies directly assessing behavioral changes in classical conditioning paradigm under this drug treatment. By employing an appetitive Pavlovian conditioning task, the present work investigated the effects of selective D1 and D2 receptor antagonists on the expression and acquisition of the conditioned orienting response (COR) and food-cup approach. SCH23390 (0, 0.05, and 0.10 mg/kg) and raclopride (0, 0.1, and 0.2 mg/kg) were administered via an intra-peritoneal route in a between-group design. Data from Experiment 1 showed that both SCH23390 and raclopride suppressed expression of the COR and food-cup approach, but only the impairment produced by raclopride reached a significant level. In Experiment 2, with SCH23390 being administered during the acquisition phase, the suppressed COR was completely restored in a subsequent (24 h later) drug-free session. In contrast, the suppressed COR in raclopride-pretreated groups was only partially restored. These findings support the view that the DA system plays a role in the neural substrates underlying this appetitive conditioning. In addition, D2 receptors are more likely involved in the modulation of learning process of the COR than D1 receptors.     

Participation of the dopaminergic brain system in effects of glucocorticoid hormones

Following the conditioning with dexamethasone, a dose-dependent place preference in non-preferred compartment was observed on the second test day in male Wistar rats. Amphetamine in subthreshold dose exerted no effect if administered alone and induced a place preference in an unbiased paradigm after pre-treatment with dexamethasone. Administration of D2-dopamine receptors' antagonist sulpiride 30 min prior to dexamethasone conditioning completely blocked the acquisition of the place preference. The D1-dopamine receptors' antagonist SCH23390 exerted no effect on the place conditioning. The findings suggest that the D2-dopamine receptors take part in conditioned place preference with dexamethasone.     

Oral activity of the growth hormone releasing peptide His-D-Trp-Ala-Trp-D-Phe-Lys-NH2 in rats, dogs and monkeys

The purpose of this study was to evaluate the growth hormone (GH) releasing activity of orally administered His-D-Trp-Ala-Trp-D-Phe-Lys-NH2 (GHRP-6, SK&F 110679) in rats, dogs and monkeys. Rats were administered GHRP-6 orally by gavage or parenterally through femoral artery catheters. Blood was collected before and after GHRP-6 administration for estimation of plasma GH and comparison of GH changes resulting from enteral and parenteral administration of the peptide. GHRP-6 was administered to dogs intravenously (i.v.) through cephalic vein catheters, intragastrically (i.g.) through esophagostomy tubes or intraduodenally (i.d.) through vascular access ports, and blood was collected before and after peptide administration for estimation of plasma GH. Cynomolgus monkeys were administered GHRP-6 i.g., and blood was collected from abdominal aorta for estimation of changes in plasma GH. Enteral activity of GHRP-6 was observed in all 3 species tested. In rats, ED50's for enteral and parenteral administration of GHRP-6 were 4 mg/kg and 28 micrograms/kg, respectively. Thus in rats, enterally administered GHRP-6 was 0.7% as bioactive as the parenterally administered peptide. In dogs GHRP-6 was slightly less potent than in rats, with ED50's for i.g. and i.v. administration approximately 15 mg/kg and 125 micrograms/kg, respectively. However, enteral potency of GHRP-6 in dogs was 0.8% of parenteral potency, and thus, comparable to that in rats. Additionally, comparison of plasma GH levels following i.g. vs i.d. administration in dogs suggested greater activity by the i.d. route. Monkeys were the species most sensitive to enterally administered GHRP-6, with plasma GH increased in those receiving i.g. doses as low as 0.3 mg/kg and an ED50 of 0.75 mg/kg compared to 4 and 15 mg/kg in rats and dogs, respectively. The results of this study demonstrate that GHRP-6 releases GH when administered directly into the gastrointestinal tract. Although enteral activity is approximately 1% of parenteral activity, GHRP-6 is potent, especially in primates which require relatively low doses to provoke GH release. These data suggest that orally active GHRP-6 may provide a practical therapeutic alternative to parenterally administered peptides such as GHRH, especially if enteral activity is enhanced with appropriate formulation.     

Participation of the genetic apparatus of the hippocampal and neocortical cells in the mechanisms of action of desglycine-arginine vasopressin on learning and memory processes in rats

The paper deals with the influence of synthetic vasopressin analogue--desglycine-arginine vasopressin (DG-AVP) on the content of RNA and fractional composition of chromatin proteins in the tissue of neocortex and hippocampus of intact white rats and after establishing of two-way avoidance reflex. Administration of the peptide alone significantly increased RNA content in hippocampal tissue, injection of the peptide 10 min before conditioning did not lead to significant changes in RNA quantity as compared to that in animals in which the conditioned reflex was established against the background of saline administration. In neocortical tissue neither learning itself nor administration of DG-AVP alone was accompanied by significant changes in RNA content, while learning against the background of peptide injection significantly increased RNA in that structure. In hippocampal and neocortical tissues quantitative changes were observed in certain fractions of chromatin proteins in all animal groups studied.     

Long-term inhibition of ovulation by a GnRH-antagonist at low dose level

Ac-D-Trp1,3, D-Cpa2, D-Lys6, D-Ala10-GnRH has been prepared by solid phase synthesis. The peptide was found to completely inhibit ovulation when administered on proestrus day in a dose of 1.5 microgram/rat, s.c. The peptide completely inhibited ovulation for a period corresponding to three to four cycles when administered daily in a dose of 5 micrograms/rat, s.c. and caused 70% inhibition of ovulation in a dose of 3 micrograms/rat.     

Application of the trimethylsilyl trifluoromethanesulfonate deprotecting procedure for the synthesis of porcine peptide YY (PYY)

A 36-residue peptide amide corresponding to the entire amino acid sequence of porcine peptide YY (PYY) was synthesized by assembling eight peptide fragments of established purity, followed by hard acid deprotection with 1M trimethylsilyl trifluoromethanesulfonate in trifluoroacetic acid. beta-Cycloheptylaspartate, Asp(OChp), was employed to minimize the base-catalyzed succinimide formation. When administered to dogs, synthetic PYY was active as natural peptide in its effects on exocrine pancreatic secretion and pancreatic tissue blood flow.     

Impact of cytokine administration on the generation of antitumor reactivity in patients with metastatic melanoma receiving a peptide vaccine.

Patients with metastatic melanoma were immunized with an immunodominant peptide derived from the gp100 melanoma-melanocyte differentiation Ag that was modified to increase binding to HLA-A+0201. A total of 10 of 11 patients who received the g209-2M peptide alone developed precursors reactive with the native g209 peptide, compared with only 5 of 16 patients who received g209-2M peptide plus IL-2 (p2 = 0.005). Peptide reactivity closely correlated with the recognition of HLA-A+0201 melanoma cells (p < 0. 001). The decrease in immune reactivity when peptide was administered with IL-2 appeared specific for the immunizing peptide, since reactivity to an influenza peptide resulting from prior exposure was not affected. Preexisting antitumor precursors did not decrease when peptide plus IL-2 was administered. The administration of GM-CSF or IL-12 also resulted in a decrease in circulating precursors compared with the administration of peptide alone, though not as great a decrease as that seen with IL-2. Immunization with peptide plus IL-2 did, however, appear to have clinical impact since 6 of the 16 patients (38%) that received peptide plus IL-2 had objective cancer regressions. It thus appeared possible that immunization with peptide plus IL-2 resulted in sequestering or apoptotic destruction of newly activated immune cells at the tumor site. These represent the first detailed studies of the impact of immunization with tumor peptides in conjunction with a variety of cytokines in patients with metastatic cancer.     

Differential effects of amylin on memory processing using peripheral and central routes of administration.

Amylin is a peptide hormone secreted from the beta cells of the pancreatic islets. Amylin was administered peripherally or centrally following weak or strong training on footshock avoidance conditioning in a T-maze. Under conditions of weak training, amylin improved memory retention in a dose-dependent manner. Under conditions of strong training, it impaired retention over the same dose range. Central administration of amylin in mice given strong training impaired retention but had no effect on the retention of mice given weak training. These findings suggest that the mechanisms of action by which amylin altered memory processing are different for peripheral and central administration. Peripherally secreted amylin may play a role in the amnesia seen in diabetes and the memory enhancement following glucose administration.     

Potentiation of the Antihypertensive Activity of Orally Administered Ovokinin,a Vasorelaxing Peptide Derived from Ovalbumin,by Emulsification in Egg Phosphatidylcholine

Ovokinin, a vasorelaxing octapeptide derived from ovalbumin, significantly lowered the systolic blood pressure of spontaneously hypertensive rats (SHR) when orally administered as an emulsion in 30% egg yolk at a dose of 25 mg/kg, this effect being larger than that of the peptide administered as a solution at a dose of 100 mg/kg. Egg phospholipid, especially phosphatidylcholine, showed essentially the same effect as egg yolk. However, egg neutral lipid was ineffective. Soybean phospholipid was less effective than egg phospholipid in potentiating the antihypertensive activity of ovokinin.     

Synthesis of porcine neuropeptide Y(NPY) in solution

The porcine neuropeptide Y (NPY), a 36-residue peptide amide, was synthesized by assembling six peptide fragments followed by thioanisole-mediated deprotection with trifluoromethanesulfonic acid in trifluoroacetic acid. beta-Cycloheptyl aspartate, Asp(OChp), was employed to suppress base-catalyzed succinimide formation. When administered to dogs, the purified peptide (10 micrograms/kg) caused prolonged increase of systemic arterial blood pressure and decreased pancreatic blood flow.     

A neuropeptide FF agonist blocks the acquisition of conditioned place preference to morphine in C57Bl/6J mice

Neuropeptide FF behaves as an opioid-modulating peptide that seems to be involved in morphine tolerance and physical dependence. Nevertheless, the effects of neuropeptide FF agonists on the rewarding properties of morphine remain unknown. C57BL6 mice were conditioned in an unbiased balanced paradigm of conditioned place preference to study the effect of i.c.v. injections of 1DMe (D-Tyr1(NMe)Phe3]NPFF), a stable agonist of the neuropeptide FF system, on the acquisition of place conditioning by morphine or alcohol (ethanol). Morphine (10 mg/kg, i.p.) or ethanol (2 g/kg, i.p.) induced a significant place preference. Injection of 1DMe (1-20 nmol), given 10 min before the i.p. injection of the reinforcing drug during conditioning, inhibited the rewarding effect of morphine but had no effect on the rewarding effect of ethanol. However, a single injection of 1DMe given just before place preference testing was unable to inhibit the rewarding effects of morphine. By itself, 1DMe was inactive but an aversive effect of this agonist could be evidenced if the experimental procedure was biased. These results suggest that neuropeptide FF, injected during conditioning, should influence the development of rewarding effects of morphine and reinforce the hypothesis of strong inhibitory interactions between neuropeptide FF and opioids.     

Inhibition of insulin release by galanin and gastrin-releasing peptide in the anaesthetized rat

The present study was designed to determine the effects of intravenously administered galanin or gastrin-releasing peptide (GRP) on glucose- and/or glucose-dependent insulinotropic peptide (GIP)-stimulated insulin release in the anaesthetized rat. Galanin inhibited glucose-stimulated insulin responses in a dose-related manner. Galanin also inhibited insulin release in response to glucose administered with GIP; this effect was due largely to inhibition of the glucose-stimulated component since galanin did not inhibit GIP-stimulated insulin release. Galanin also inhibited insulin responses to ingestion of a mixed meal. GRP inhibited glucose-stimulated insulin responses, and the insulin responses to glucose plus GIP; unlike galanin, GRP inhibited both glucose- and GIP-stimulated insulin release. GRP also inhibited insulin release following ingestion of a mixed meal. The results suggest a possible modulatory role for these neuropeptides in regulation of insulin secretion.     

Cognitive disturbances in the cuprizone model of multiple sclerosis

Cognitive problems frequently accompany neurological manifestations of multiple sclerosis (MS). However, during screening of preclinical candidates, assessments of behaviour in mouse models of MS typically focus on locomotor activity. In the present study, we analysed cognitive behaviour of 9 to 10-week-old female C57Bl/6J mice orally administered with the toxin cuprizone that induces demyelination, a characteristic feature of MS. Animals received 400 mg/kg cuprizone daily for 2 or 4 weeks, and their performance was compared with that of vehicle-treated mice. Cuprizone-treated animals showed multiple deficits in short touchscreen-based operant tasks: they responded more slowly to visual stimuli, rewards and made more errors in a simple rule-learning task. In contextual/cued fear conditioning experiments, cuprizone-treated mice showed significantly lower levels of contextual freezing than vehicle-treated mice. Diffusion tensor imaging showed treatment-dependent changes in fractional anisotropy as well as in axial and mean diffusivities in different white matter areas. Lower values of fractional anisotropy and axial diffusivity in cuprizone-treated mice indicated developing demyelination and/or axonal damage. Several diffusion tensor imaging measurements correlated with learning parameters. Our results show that translational touchscreen operant tests and fear conditioning paradigms can reliably detect cognitive consequences of cuprizone treatment. The suggested experimental approach enables screening novel MS drug candidates in longitudinal experiments for their ability to improve pathological changes in brain structure and reverse cognitive deficits.     

Anticoagulant activity of a peptide boronic acid thrombin inhibitor by various routes of administration in rats.

The peptide boronic acid analog Ac-(D)Phe-Pro-boroArg-OH (I) is a potent and selective inhibitor of thrombin. The objective of this study was to determine whether I is active orally or when administered by alternative transmucosal routes. The measured effect was the time for clotting of plasma after initiation with thrombin. With this assay there was a narrow window from no measurable effect to the maximal effect, a clotting time greater than 300 seconds. Intravenous I at a 0.15 mg/kg dose in rats, a nasal 0.45 mg/kg dose, and 3 mg/kg doses administered orally, colonically, or rectally all produced maximal effects. Therefore, although bioavailability cannot be estimated, it is demonstrated that this peptide analog was absorbed by each of these routes.