Tolerance and cross-tolerance to the antinociceptive effects of [D-Arg2]-dermorphin tetrapeptide analogue and morphine.

Rats were given repeated subcutaneous injections of [D-Arg2, Sar4]-dermorphin (1-4) [DAS-DER-(1-4)] and/or morphine over a period of 4 or 7 days. Antinociception was determined at 90 min for DAS-DER (1-4) and 30 min for morphine after each morning injection (9:00 a.m.), using the tail-flick and digit pinching tests in rats. Subcutaneous administration of DAS-DER (1-4) and morphine produced the development of antinociceptive tolerance, respectively. A marked tolerance to DAS-DER (1-4) and morphine was seen in rats made tolerant to morphine. However, administration of morphine produced no significant decrement in the antinociceptive activity in rats made tolerant to DAS-DER (1-4). These results suggest that the site of action of DAS-DER (1-4) may be more limited than that of morphine in the nociceptive pathways, for lack of its antinociceptive efficacy in morphine-tolerant rats.

Gastroprokinetic activity of nizatidine, a new H2-receptor antagonist, and its possible mechanism of action in dogs and rats.

We studied the anti-acetylcholinesterase (AChE) activity of a new H2-antagonist, nizatidine, in in vitro experiments and its gastroprokinetic action in the dog and rat in comparison with other H2-antagonists, neostigmine and cisapride. The IC50 of nizatidine for AChE was 6.7 x 10(-6) M, and this activity was reversible. The relative anti-AChE potency was in the following order: neostigmine > nizatidine > cimetidine >> famotidine. The inhibition of AChE by nizatidine was noncompetitive, with a Ki value of 7.4 x 10(-6) M. Gastrointestinal (GI) motility was examined during the interdigestive state in dogs with chronically implanted force transducers. Nizatidine (0.3-3 mg/kg, i.v.) significantly increased the motor index in a dose-dependent manner. It was of interest that the contractile response of the GI tract to nizatidine was similar to the interdigestive migrating contractions-like activity. At the doses used in this study, neither cimetidine nor famotidine had a significant effect on the motor index. Neostigmine at a higher dose of 0.06 mg/kg and cisapride at 0.3 mg/kg were found to stimulate GI contractions. Gastric emptying was determined in rats given phenol red as a liquid test meal. Nizatidine (3 mg/kg, i.p., or above) significantly increased gastric emptying, whereas the other H2-antagonists had no such effect. The ED50 and ED90 values of nizatidine for inhibition of gastric acid secretion were 0.18 and 3.22 mg/kg in dogs, and 2.94 and 19.6 mg/kg in rats, respectively. These findings suggest that nizatidine stimulates GI contractions and accelerates gastric emptying at gastric antisecretory doses.(ABSTRACT TRUNCATED AT 250 WORDS)

Antinociceptive mechanisms of [D-Arg2]-dermorphin tripeptide analogs.

These studies examined the antinociceptive effects and mechanisms of opioid activity of synthetic dermorphin tripeptide analogs containing D-Arg as the second amino acid, H-Tyr-D-Arg-Phe-NHCH3 and H-Tyr-D-Arg-Phe-N(CH3)2. Both tripeptides, administered i.c.v., i.t. and s.c. in mice, produced a potent and long-lasting antinociceptive activity as compared with morphine. In the guinea pig isolated ileum (GPI) assay, the IC50 value of both peptides was lower than that of morphine on the electrically induced contractions of the GPI. In radioligand binding studies with rat brain membrane synaptosomes, both tripeptides bound with high affinity to [3H]DAMGO-labeled mu-type sites, whereas they bound with no or negligible affinity to [3H]DPDPE-labeled delta sites and [3H]U-69593-labeled kappa sites. In the enzymatic degradation using high-performance liquid chromatography, both tripeptides showed good enzymatic stability after 25 hr of incubation with solubilized enzymes of mouse brain or spinal cord, in contrast to a rapid degradation of Met-enkephalin. The in vitro and in vivo pharmacological profile of [D-Arg2]-dermorphin tripeptide analogs demonstrates that they are potent and selective agonists at the mu opioid receptor. A high resistance of the tripeptides to enkephalin-degrading enzymes may largely contribute to their prolonged opioid activity.

[Effect of Z-103 on wound healing by dermal incision in guinea pigs].

We investigated the effects of Z-103, ZnSO4, L-carnosine and solcoseryl on wound healing by dermal incision in guinea pigs. The tensile strength, hydroxyproline contents and the value of angiogenesis (carmine contents) at the wounded site of dorsal skin were used as indices of wound healing. Z-103, given daily s.c., increased the tensile strength and hydroxyproline contents on day 4 after operation in a dose-dependent manner; in particular, the effect of 10 mg/kg of Z-103 was nearly equal to that of solcoseryl at 0.5 ml/animal. Moreover, Z-103 10 mg/kg increased the value of angiogenesis on day 3 after the operation. On the other hand, ZnSO4 and L-carnosine, components of Z-103, also similarly increased the tensile strength and hydroxyproline contents. These results suggest that Z-103 possessed an accelerative action on wound healing, and these effects may be due to the activity of its components, ZnSO4 and L-carnosine.

[Studies on the healing promoting action of Z-103 in chronic gastric ulcer models of rats].

We studied the healing promoting action of Z-103 on the chronic gastric ulcer induced by acetic acid (AAU) or Fe-ascorbic acid (FAU) in rats. The area of the gastric ulcers, hydroxyproline (Hyp) and DNA contents in the ulcer region were measured as an index of ulcer healing. The area of gastric ulcers was the largest on day 4 and thereafter gradually decreased, but the ulcers still remained at the 14th day. Hyp contents in the ulcer region decreased until the 7th day in both models, and then this level increased. Significant decrease in DNA contents in the ulcer region was observed on the 7th day only in FAU. In AAU and FAU, administration of Z-103 (3 mg/kg/day x 2, p.o.) resulted in a significant decrease in the area of gastric ulcers on the 14th day and a significant increase in Hyp contents in the ulcer region on the 7th day as compared with the control group. Z-103 increased the DNA contents in the ulcer region on the 4th day in AAU and on the 7th day in FAU. These results suggest that tissue destruction surrounding the ulcer region in AAU and FAU models might occur until the 4th or 7th day after operation, and that the acceleration of ulcer healing by Z-103 on these models may be facilitated by the wound healing action of this drug.

Exercise-entrained breathing and non-invasive determination of anaerobic threshold.

Incremental cycle exercise was performed by 17 subjects under two breathing conditions: spontaneous breathing (Non-ENT run) and deliberate entrainment of breathing frequency (f) to cycling rate at preferred coupling ratios up to possibly high work rates (ENT run). The purpose was to investigate the influence of entrainment on two non-invasive determinations of anaerobic threshold (AT): the break points of VE/VO2 (VAT) and VCO2 (V-slope AT) changes against VO2. Compared to the Non-ENT run, VAT in the ENT run was greater in 12 subjects entrained at higher f accompanied by relative hyperventilation at low work rates, while it was similar in five subjects entrained at lower f accompanied by relative hypoventilation. Relative hyperventilation and hypoventilation during the ENT run tended to elicit greater and lower VCO2, respectively, relative to VO2, as compared to the Non-ENT run. These different VCO2, responses between the two runs exerted little influence on the V-slope AT determination. It is concluded that breathing patterns and ventilatory response during incremental work tests can affect the VAT method but not the V-slope method.

Effects of successive doses of nizatidine, cimetidine and ranitidine on serum gastrin level and gastric acid secretion.

Nizatidine (N-[2-[[[2-[(dimethylamino)methyl]- 4-thiazolyl]methyl]thio]ethyl]-N'-methyl-2-nitro-1,1-ethenediamine , CAS 76963-41-2) is a new histamine H2-receptor antagonist which shows suppression of gastric acid secretion and antiulcer activity. In the present experiment, the effects of single s.c. administration of nizatidine, cimetidine and ranitidine on serum gastrin levels were studied in fasted rats. Nizatidine at 100 mg/kg increased serum gastrin level 3 h after administration, which however, returned to basal level 6 h after administration. Cimetidine and ranitidine at respective doses of 250 and 100 mg/kg markedly increased serum gastrin levels 3 and 6 h after administration. In a previous study, the suppressive effect of nizatidine on basal gastric acid secretion was 82.8% at a dose of 100 mg/kg s.c. in rat pylrus-ligated model. On the basis of these findings, changes in basal gastric acid secretion and serum gastrin level after withdrawal of nizatidine, cimetidine and ranitidine administered for 14 consecutive days were studied. One day after withdrawal, nizatidine at 100 mg/kg showed a tendency to increase the basal gastric acid secretion. However, 3 and 7 days after administration, almost no changes were obtained. Cimetidine at 250 mg/kg showed a tendency to increase the basal gastric acid secretion 7 days after withdrawal of the drug. Ranitidine at 100 mg/kg induced no changes in basal gastric acid secretion after withdrawal. No obvious influences of all drugs on serum gastrin level after withdrawals were obtained. These results indicate that consecutive administration of nizatidine may cause only a transient increase of gastric acid secretion but no hypergastrinaemia after its withdrawal.

Antinociceptive cross-tolerance between [D-Arg2]-dermorphin tetrapeptide analogs and morphine.

Cross-tolerance between [D-Arg2]-dermorphin tetrapeptide analogs and morphine with respect to antinociception was examined in the present set of experiments. Systemic administration of H-Tyr-D-Arg-Phe-Gly-NH2 (TDAPG-NH2), H-Tyr-D-Arg-Phe-beta-Ala-OH (TDAPA) or morphine over a period of 5 days produced the development of tolerance. In the cross-tolerance study, antinociception after subcutaneous (SC), intracerebroventricular (ICV) and intrathecal (IT) administrations of TDAPG-NH2 and TDAPA in morphine-tolerant mice was not significantly different from their respective effects in saline-pretreated control mice. A marked tolerance to SC- and ICV-administered morphine was seen in mice made tolerant to TDAPG-NH2 and TDAPA. However, IT administration of morphine produced no significant decrement in the antinociceptive activity in mice made tolerant to TDAPG-NH2 and TDAPA. These data indicate that [D-Arg2]-dermorphin tetrapeptide analogs can produce significant antinociception in morphine-tolerant mice.

N-terminal tetrapeptide of dermorphin and D-Arg-substituted tetrapeptides: inactivation process of the antinociceptive activity by peptidase.

Degradation products of the N-terminal tetrapeptide of dermorphin, H-Tyr-D-Ala-Phe-Gly-OH (ALPG) and D-Arg2-substituted tetrapeptide analogs of dermorphin, H-Tyr-D-Arg-Phe-Gly-OH (ARPG), H-Tyr-D-Arg-Phe-Gly-NH2 (TDAPG-NH2) and H-Tyr-D-Arg-Phe-beta-Ala-OH (TDAPA) by enkephalin degrading enzymes were studied by using reversed-phase high-performance liquid chromatography. After 5 and 25 hr incubations of the peptides with solubilized enzymes of mouse brain or spinal cord, liberation of the appreciable Tyr1 residue was observed in ALPG but not in ARPG, TDAPG-NH2 and TDAPA. When ARPG and TDAPG-NH2 were incubated with enzymes for 25 hr, a main degradation product was the N-terminal tripeptide produced from the hydrolysis of Phe3-Gly4 bond. Conversely, TDAPA did not produce the N-terminal tripeptide after 25 hr incubation with enzymes. In the enzyme assay, Tyr1-D-Arg2 bond of ARPG, TDAPG-NH2 and TDAPA was more stable than that of ALPG to the cleavage by aminopeptidase M (AP-M). Phe3-Gly4 bond of ALPG, ARPG and TDAPG-NH2 were easily hydrolyzed by carboxypeptidase Y (CP-Y) within 3 hr incubation, whereas the hydrolysis of Phe3-beta-Ala4 bond of TDAPA by CP-Y was not observed after 3 hr incubation. The present results and previous behavioural data suggest that a potent and prolonged antinociceptive activity of the D-Arg-substituted tetrapeptides is mainly attributed to the stability of Tyr1-D-Arg2 bond against aminopeptidase of peptidases.

A new class opioid peptide, [D-Arg2, beta-Ala4]-dermorphin tetrapeptide; physical dependence liability in mice.

Development of morphine-like physical dependence of the [D-Arg2, beta-Ala4]-dermorphin tetrapeptide (H-Tyr-D-Arg-Phe-beta-Ala-OH) has been evaluated and compared with the physical dependence liability of morphine or pentazocine. Degree of the physical dependence was assessed by the naloxone-precipitated jumping behaviour in mice after treatment of a single dose of each compound. The number of jumps and the time of latency to first jump were recorded in this experiment. Number of jumps in a group pretreated with the peptide showed less than that in morphine-treated group. In addition, latency to the appearance of the first jump in the peptide-treated mice was later than that in the morphine-treated group. The present results indicate that the physical dependence induced by [D-Arg2, beta-Ala4]-dermorphin tetrapeptide may be less marked than that produced by morphine. It is also interesting to note that the antinociceptive effect of this opioid peptide is more powerful and of longer duration than that induced by morphine or pentazocine.

Comparison of the antinociceptive effects of new [D-Arg2]-dermorphin tetrapeptide analogs and morphine in mice.

The antinociceptive effects of synthetic dermorphin tetrapeptide analogs containing D-Arg in position 2, H-Tyr-D-Arg-Phe-Gly-NH2 and H-Tyr-D-Arg-Phe-beta-Ala-OH, were measured in mice by the tail-pressure test. The antinociceptive effect produced by intracerebroventricular (ICV), intrathecal (IT) and subcutaneous (SC) administration of either peptide was greater than that produced by morphine. Oral (PO) administration of the peptides showed approximately the same antinociceptive potency as morphine. In addition, the antinociceptive effect produced by SC and PO administration of either peptide was of longer duration than morphine. Pretreatment with naloxone resulted in nearly complete antagonism of the antinociceptive effects produced by ICV and IT administration of either peptide or morphine. Dose ratios (ICV/IT) of H-Tyr-D-Arg-Phe-Gly-NH2 and H-Tyr-D-Arg-Phe-beta-Ala-OH, which were calculated from the AD50 (Antinociceptive Dose = 50% MPE) values, were 5.8 and 6.2, respectively, whereas that of morphine was only 1.46. These results suggest that the mechanisms of the antinociceptive effects of [D-Arg2]-dermorphin tetrapeptide analogs differ from morphine, and that these peptides may possess higher affinities than does morphine for opioid receptors in the spinal cord.

Antinociception and physical dependence produced by [D-Arg2] dermorphin tetrapeptide analogues and morphine in rats.

1. The antinociceptive effects of [D-Arg2] dermorphin tetrapeptide analogues, H-Tyr-D-Arg-Phe-Gly-NH2 and H-Tyr-D-Arg-Phe-beta-Ala-OH when administered subcutaneously (s.c.) in rats were measured by the tail-flick test. In addition, the appearance of typical withdrawal signs upon cessation of administration or on subsequent treatment with naloxone were measured after chronic administration of either peptide or morphine. 2. The dose of peptides and of morphine in the physical dependence test was determined from the AD50 to inhibit the tail-flick test in rats. Doses from 4 to 64 times the AD50 doses were employed in the s.c. administration schedules. 3. The intensity of the antinociception induced by either peptide was greater than that produced by morphine. Moreover, the antinociception induced by the peptides was of much longer duration than that produced by morphine. 4. Abrupt withdrawal after chronic administration of either peptide produced only slight loss of body weight. In contrast, morphine withdrawal produced sharp loss of body weight. 5. Naloxone precipitated withdrawal signs after chronic administration of either peptide were less intense than those after chronic morphine. 6. These results suggest that the antinociception produced by these peptides is more intense and of longer duration than that produced by morphine. It is also interesting to note that the physical dependence produced by these peptides is less marked than that produced by morphine.

Opioid activities of D-Arg2-substituted tetrapeptides.

The antinociceptive effects and mechanisms of action of H-Tyr-D-Ala-Phe-Gly-OH, H-Tyr-D-Arg-Phe-Gly-OH and H-Tyr-D-Arg-Phe-sarcosine(Sar)-OH have been investigated. The ED50 values of these peptides were 510.0, 8.2 and 2.0 pmol, respectively, when administered i.c.v. in the mouse tail-pressure test (dermorphin = 5.7 pmol and morphine = 1.2 nmol). These activities were remarkably potent and relatively long lasting. Their IC50 values were 676.8, 23.1 and 6.6 nM, respectively (dermorphin = 3.75 and morphine = 214.3 nM) in the guinea pig isolated ileum assay, and 138.50, 5.25 and 1.10 nM, respectively (dermorphin = 3.80 and morphine = 28.00 nM) in the radioreceptor assay utilizing [3H]naloxone as the opioid receptor ligand. In the evaluation of their inhibitory effects to enkephalin-degrading enzymes, the IC50 values of H-Tyr-D-Arg-Phe-Gly-OH, H-Tyr-D-Arg-Phe-Sar-OH and H-Tyr-D-Ala-Phe-Gly-OH were 5.4, 14.5 and more than 50.0 microM, respectively (bestatin = 0.1 microM) against aminopeptidase and 1.18, 1.40 and more than 50.0 microM, respectively (captopril = 0.38 and D-Phe-2S-, 3R-3-amino-2-hydroxy-4-phenylbutanoic acid = more than 100 microM) against the cleaving enzymes of enkephalin at its Gly3-Phe4 bond. The authors suggest that the marked antinociceptive potency of H-Tyr-D-Arg-Phe-Gly-OH and H-Tyr-D-Arg-Phe-Sar-OH is mainly due to high opioid receptor affinity. Their inhibitory effects on enkephalin-degrading enzymes and enzymatic stability also greatly contribute to their potent and long-lasting opioid activities.