Screening for cerebroprotective agents using an in vivo model of cerebral reversible depolarization in awake rats.

The need to screen cerebroprotective compounds without anesthetic interference prompted the development of a model using hypoxic rats. In this model two outcome measures were used: (1) the time to reach isoelectric electroencephalogram (iEEG), caused by nitrogen gas inhalation in the test chamber, and (2) the time for behavioral recovery measuring the latency of restoration of the head-withdrawal reflex upon vibrissae stimulation. We report here data of blood chemistry, cerebral tissue oxygen measurements, a definition of a proposed scoring system, and the pharmacological results of RGH-2202. The findings with RGH-2202 are used here to show the utility of the screening method. Events during hypoxia: Arterial and venous pO(2), pCO(2), and pH, and brain tissue pO(2)significantly declined. Significant correlations were established among the pO(2)of cerebral tissue, blood, and the test chamber. RGH-2202 significantly and dose-dependently shortened the iEEG time; the compound's Effective Dose(30)was 227.8 mg kg(-1). Events during recovery: Immediately after the iEEG, when the atmosphere in the chamber was replaced with room air, the arterial, venous and brain tissue pO(2)increased above the control level and subsequently recovered to baseline levels. Behavioral recovery occurred before blood chemistry was otherwise normalized. RGH-2202 significantly and dose-dependently shortened the recovery time; the Effective Dose(30)was 8.71 mg kg(-1). The available data define and support the physiological basis of this practicable rat-screening model.

Cerebroprotective drugs shorten the hypoxia-induced onset of electrical silence in unanesthetized rats.

Pharmacological agents that delay the hypoxic arrest of neuronal electrical activity, as indicated by the suppression of electroencephalogram (EEG), have previously been thought to increase brain resistance to oxygen insufficiency. On the other hand, acceleration of the EEG suppression may offer some protection against severe hypoxia by reducing neuronal energy spending on electrogenesis. In unanesthetized rats we examined the effects of several antihypoxic drugs on the time of appearance of isoelectric EEG (tiEEG), caused by normobaric hypoxia. In addition, alterations in cerebral blood flow induced by hypoxia and by some drugs were monitored using polarographic techniques to determine if cerebrocirculatory changes play a significant role in the drug effects on tiEEG. We also assessed drug effects on behavioral recovery after hypoxia by measuring the latency of restoration of the head-withdrawal reflex upon vibrissae stimulation. Pentobarbital (30 and 60 mgkg(-1)i.p.), chloralhydrate (400 mgkg(-1)i.p.) flunarizine (50-100 mgkg(-1)p.o.), hydergine (3-50 mgkg(-1)p.o.), nicergoline (50 mgkg(-1)and 85 mgkg(-1)p.o.), sabeluzole (3 and 7.5 mgkg(-1)i.p.) and vincamine (80 mgkg(-1)p.o.) reduced tiEEG (mean 27.1 +/- 3.3 min prior to drugs). In contrast, idebenone (29-85 mgkg(-1)p.o.) and vinpocetine (29-85 mgkg(-1)p.o.) had no significant effects on tiEEG. The divergent effects on cerebral blood flow suggest an insignificant role for cerebrocirculatory changes in the drug-induced reduction of tiEEG during severe hypoxia. The drug effects on recovery of the head-withdrawal reflex (mean 4.2 +/- 1.3 min prior to drugs) varied from a delay (sabeluzole) to acceleration (flunarizine) with no correlation to the effects on tiEEG, suggesting that EEG criteria alone may not predict the course of functional recovery.

Classification of drugs based on visually evoked responses in rats.

Quantified parameters of photically evoked afterdischarge and EEG background activity of visual cortex of freely moving rats were used as variables to obtain a t profile for each drug dosage. Behavioural scores were also used. The determination of the dose/time-effect profile of a compound was based on normalization of data and on calculation of the first two factor values in comparison with pooled data of representatives of neuroleptic, anxiolytic, convulsant, petit mal anticonvulsant, antidepressant and psychostimulant drug classes. The compounds were investigated in different dosages and/or routes of administration. The model and procedure are validated for antipsychotic, anxiolytic and vigilance enhancer effect, and comprise the predictability of proconvulsive and petit mal anticonvulsive effects.

Inhibitory effect of hypoxic condition on acetylcholine release is partly due to the effect of adenosine released from the tissue.

Isolated longitudinal muscle strip with Auerbach's plexus attached was used to study the stimulation-evoked release of 3H-acetylcholine (3H-ACh) under normoxic and hypoxic conditions. Hypoxia reduced the release of ACh. Theophylline, a purinoceptor P1 antagonist and vinpocetine, an antiischemic compound partly reversed the effect of hypoxia. Unlike theophylline, the effect of vinpocetine was not mediated via adenosine action, since it failed to affect the presynaptic action of adenosine, and the effect of theophylline and vinpocetine was additive. When they were added together the effect of hypoxia was almost completely antagonized. Dipyridamole, an adenosine uptake inhibitor, potentiated the effect of hypoxia and the presynaptic inhibitory action of adenosine on ACh release. Evidence was obtained that the effect of hypoxia is at least partly due to adenosine formed from purine nucleotides.

Selective restoration of immunosuppressive effect of cytotoxic agents by thymopoietin fragments.

Swiss male mice were immunosuppressed by cyclophosphamide, cisplatinum, vincristine and methotrexate. The ability of the thymopoietin (TP) fragments TP-3, TP-4 and TP-5 to restore antibody production and phagocytosis was studied. Impaired antibody production after vincristine treatment was partially or totally restored by TP-3, TP-4 or TP-5. Only TP-3 or TP-5 interfered with the antibody-production-damaging effect of cisplatinum. The same effect of methotrexate could not be influenced by any of the TP fragments. The phagocytic capacity of peritoneal macrophages was reduced by vincristine, methotrexate and cyclophosphamide treatment. In this respect, TP-3 protected the function of macrophages against vincristine and cyclophosphamide treatment. TP-4 was active in the case of damage caused by vincristine and methotrexate, and TP-5 interfered with the phagocytosis-inhibiting effect of methotrexate. Each TP fragment seems to have a specific target orientation within the immune system. This also means that the proper TP fragment should always be chosen for combination therapy with various types of cytotoxic drugs.

Effects of thyrotropin-releasing hormone and its analogue L-6-ketopiperidine-2-carbonyl-leucyl-L-prolin-amide on behaviour and electroencephalogram.

A comparative dose-response investigation of thyrotropin-releasing hormone (TRH) and L-6-ketopiperidine-2-carbonyl-leucyl-L-proline-amide (RGH-2202) was carried out on rats and cats. RGH-2202 reduced the occurrence of photically evoked after-discharge in rats more significantly than TRH. Neither of the two compounds influenced the parameters of visually evoked potentials. Both compounds desynchronized the background electrocortical activity in rats. The amplitude of the acoustic startle reflex in rats was decreased dose-dependently only by RGH-2202, and this effect was nearly 3 times more potent than that of TRH. Both compounds increased the time spent awake, the latency to light sleep and the proportion of light sleep in a dose-dependent manner. The higher doses of TRH equalled the effects of the lower doses of RGH-2202. In cats the latency increasing effect of TRH on paradoxical sleep was about 30% less than that of RGH-2202; furthermore, the relative increase in the proportion of light sleep coupled with a corresponding decrease of deep sleep and paradoxical sleep was significant only in the case of RGH-2202. In a complex conditioned reflex situation in cats, the dose-dependent motivation decreasing effect on food intake and, as a consequence, on spontaneous motor activity was more pronounced in the case of TRH. The effective doses of RGH-2202 and TRH in rats were nearly equal, while in cats RGH-2202 showed about 1/10 of the potency of TRH. This finding suggests a considerably lower species variability of the effects of RGH-2202.(ABSTRACT TRUNCATED AT 250 WORDS)

Synthesis of angiotensin II antagonists with variations in position 5.

Six angiotensin II antagonists containing cyclohexylglycine (Chg) or cyclopentylglycine (Cpg) in position 5 were synthesized by stepwise elongation in solution, using the pentafluorophenyl ester method. The influence of substitution on the inhibitory properties of the analogues was studied in four different bioassays. [Sar1,Chg5,Lac8]AII proved to be the most potent antagonist with low intrinsic activity in both the in vitro and in vivo tests.

The effect of thymopoietin 32-34 (TP3) on suramin-induced inhibition on delayed type hypersensitivity in guinea pigs.

Suramin administration inhibits virus replication and produces impaired immunoregulation. Delayed type hypersensitivity (DTH)-reaction was inhibited by Suramin (200 mg/kg, i.p.) injection in guinea-pigs immunized with BCG vaccine. Suramin-inhibited DTH-reaction was restored to normal level after 4 days of treatment with thymopoietin 32-34 (TP 3) and TP5 (1 mg/kg, i.p.), TP3 greater than TP5. TP3 produced the strongest restoration when applied together with Suramin, but a single treatment with TP3 or TP5 oligopeptide inhibited the development of DTH-reaction. TP3 with Suramin treatment might be advantageous to eliminate defective side effects of Suramin on immune system and TP3 with indirect inducing action on IL-2, IFN-gamma production may be useful in therapy of AIDS patients.

Therapeutic possibilities of thymopoietin fragments (TP3 and TP4) based on experimental animal models.

The effects of thymic hormones are focused on the induction of T-cell subpopulations and restoration of the reactivity of an impaired immune system. TP3 and TP4 (corresponding to thymopoietin 32-34 and 32-35) exert a thymic hormone substitution effect. These peptides elicit dissimilar quantitative and qualitative effects. The aim of the present experiments was to investigate: (a) the effect of thymopoietin fragments in mice with unbalanced immune systems caused by experimental manipulation; and (b) the ratio of target cells after treatment. The distribution of Thy1, Lyt1, Lyt2 positivity was determined in a direct complement mediated cytotoxicity test. Autoantibody production was measured by Coombs' test. A count of Lewis Lung Tumour (LLT) metastases was made after two weeks of inoculation. Groups of mice were thymectomized and/or injected with cyclophosphamide (CY) (240 mg/kg) 96 h before tumour cell inoculation. The number of LLT metastases was decreased by treatment with peptides (TP3 = 72, TP4 = 97, TP5[thymopoietin 32-36] = 83.1 in %) and immunosuppression produced by CY was partly restored. After thymectomy, however, only TP3 treatment caused a decreasing effect (97.4%) on CY immunotoxicity independently of thymectomy. Inhibition of autoantibody production was detected with TP3 (5-6 weeks earlier than in mice treated with TP5). The ratio of Thy1+ and Lyt2+ cells was increased by treatment with TP3 and TP4, but the ratio of Lyt1+ cells was decreased by application of TP5. After TP3 treatment of nude mice the Lyt1+/Lyt2+ ratio increased both in bone marrow and spleen. No effect of TP4 was observed on Lyt 1+ cells, but the number of Lyt2+ increased in bone marrow.(ABSTRACT TRUNCATED AT 250 WORDS)

Immunoregulating peptides II. In vitro effects of TP5 analogs on E-rosette formation and cell division.

The effects of seventeen synthetic analogs of thymopentin (TP-5) have been studied in the active and azathioprine-inhibited E-rosette tests. Thymopentin was gradually shortened from the C terminus to peptides and single amino acids. Thymopoietin 32-34 (Arg-Lys-Asp-RGH-0205-TP-3) (II) and thymopoietin 32-35 (Arg-Lys-Asp-Val-RGH-0206-TP-4) (I) were the most active peptides. Dipeptide Arg-Lys produced significant stimulatory effect on azathioprine (ED75) inhibited E-receptor. Treatment of azathioprine (ED75)-inhibited E-rosette forming cells (ERFC) with arginine or especially lysine increased the number of ERFC. Some of TP-4 analogs decreased further the number of ERFC decreased by azathioprine ED30. These "suppressive" peptides as well as TP-3 caused a partial arrest of K 562 cell proliferation up to 96 hours. Results suggest that TP-5 is not the smallest active fragment of thymopoietins, since peptides (TP-3 and TP-4) exhibit similar or higher T-cell membrane activation on E-receptor. Arginine, lysine, and acidic aspartyl residue seem to be a necessary basic structure to produce a cumulative chemical signal on the activity of T-lymphocytes.

Stimulus-evoked efflux of GABA from preloaded slices of the rabbit oviduct.

The effect of electrical and chemical stimulation on the efflux of [3H]gamma-aminobutyric acid (GABA) from preloaded slices of the rabbit oviduct was examined. Electrical field stimulation significantly increased the outflow of [3H]GABA. This effect could not be prevented by tetrodotoxin or by the removal of Ca2+ from the medium. High K+ concentrations, veratrine and ethylenediamine also evoked a remarkable elevation in the efflux. The release induced by veratrine was completely abolished in a Ca2+-free medium or in the presence of tetrodotoxin, while the release evoked by high K+ or ethylenediamine was resistant to both conditions. These findings indicate that GABA can be released from the oviduct under the effect of depolarizing stimuli, raising the possibility of a physiological interaction between oviductual GABA and its receptors. The characteristics of oviductal GABA efflux differ from those of neuronal and glial GABA release.

Synthesis of thyrotropin-releasing hormone analogues. 2. Tripeptides structurally greatly differing from TRH with high central nervous system activity.

A new series of thyrotropin-releasing hormone (TRH) analogues, obtained by further modifications of our most potent central nervous system (CNS) stimulating neutral tripeptides at both termini, were synthesized by the pentafluorophenyl ester method and tested for CNS and thyrotropin (TSH) releasing activity. Replacement of pyroglutamic acid by pyro-2-aminoadipic acid, 2-oxoimidazolidine-4-carboxylic acid or gamma-butyrolactone-gamma-carboxylic acid and that of proline by pipecolic acid, thiazolidine-4-carboxylic acid, or homoproline in [Leu2]- and [Nva2]TRH led to tripeptides structurally widely different from TRH. In spite of this fact, 7 of the 17 analogues (1, 2, 8-10, 16, and 17) have stronger anticataleptic effect than TRH, with negligible or no hormonal potency. The highest CNS activity was achieved when pyroglutamic acid was replaced by pyro-2-aminoadipic acid at the N-terminus [pAad-Leu-Pro-NH2, 1 (RGH 2202), and pAad-Nva-Pro-NH2,2]. A novel synthesis of L-2-aminoadipic acid suitable for large-scale preparation is also described.

Antagonism of non-depolarising neuromuscular blockade by aminopyridines in cats.

The antagonism of pipecuronium- and vecuronium-induced neuromuscular blockade by 4-aminopyridine (4AP), 3,4-diaminopyridine (3,4AP) and 3-[(dimethylamino)-carbonyl] amino-4-aminopyridine (LF14) were studied in anaesthetized cats during constant infusion of the relaxants. Aminopyridines were administered cumulatively at steady state 90% block level. The ED50 values of 4AP, 3,4AP and LF14 were 243, 106 and 254 micrograms kg-1 in pipecuronium, and 232, 195 and 235 micrograms kg-1 in vecuronium blockade. It has been assumed that in cats the anticurare effect of aminopyridines is mainly a result of K+ channel blockade on motor nerve terminals which enhances the evoked release of acetylcholine.

The effect of TP-5 and its analogs on skin grafts in mice.

The immunostimulatory action of oligopeptides RGH-0205 (Arg-Lys-Asp), RGH-0206 (Arg-Lys-Asp-Val) and TP-5 (Arg-Lys-Asp-Val-Try) was measured using the B10LP to C57BL skin graft system and the determination of splenic T cell ratio. While thymectomy increased the period between skin grafting and rejection, each of the oligopeptides increased the number of splenic T cells and in some extent restored the rejection capacity of thymectomised C57Bl mice, presumably by restoration of thymic hormone function.

Comparative characterization of glutamate decarboxylase in crude homogenates of oviduct, ovary, and hypothalamus.

Some biochemical characteristics of L-glutamate decarboxylase (GAD) were compared using crude homogenates of the rat oviduct, ovary, and hypothalamus. As estimated by the measurement of CO2 production, the specific activities of oviductal and ovarian GAD were about 10 and 3% of the hypothalamic value, respectively. Stoichiometric formation of gamma-aminobutyric acid (GABA) and CO2 from L-glutamate could be observed in oviduct and hypothalamus, while in ovarian homogenates the production of CO2 was more than nine times that of GABA. Hypothalamic and tubal GAD showed similar time course, temperature dependence, and pH dependence. The dependence on protein concentration and on exogenous cofactor supply was also similar in these two tissues. The kinetic constant for L-glutamate as a substrate was nearly the same in oviduct (1.30 mM) and hypothalamus (1.64 mM). The responsiveness of tubal and hypothalamic GAD to various inhibitors was also similar. The present findings indicate that the oviductal and the hypothalamic GAD may be identical, and they suggest a possible GABAergic innervation of the Fallopian tube.

Synthesis of thyrotropin-releasing hormone analogues. 1. Complete dissociation of central nervous system effects from thyrotropin-releasing activity.

Twenty-four thyrotropin-releasing hormone (TRH) analogues containing mainly aliphatic amino acids in position 2 were synthesized and tested for central nervous system (CNS) and hormonal (TSH) activity. Application of the pentafluorophenyl ester method in the syntheses resulted in optimal yields and high purity of the products. The neutral tripeptides pGlu- Nva -Pro-NH2 (9), pGlu-Nle-Pro-NH2 (10), and pGlu-Leu-Pro-NH2 (3) with a three- or four-membered straight or branched alkyl side chain in the position of the central amino acid had 2.5 to 10 times stronger anticataleptic effect than TRH, demonstrating that the presence of histidine is not essential for the CNS activity. Analogue 9 exhibited tenfold anticataleptic activity as compared to TRH, and it was found to be fully inactive in the release of TSH.

GABAB receptor-mediated stimulation of the contractility of isolated rabbit oviduct.

The effects of gamma-aminobutyric acid (GABA) and related compounds were examined on longitudinal and circular muscle preparations isolated from oviducts of virgin rabbits. GABA and baclofen stimulated the spontaneous motility in each type of preparation, which action could not be antagonized by bicuculline, phentolamine, atropine or tetrodotoxin. Muscimol was virtually ineffective. Our results indicate the presence of GABAB receptors in the rabbit oviductal musculature which mediate the contractile response.

Gastrointestinal cytoprotection by indomethacin + sodium salicylate combination in rat.

Non-steroidal antiinflammatory agents are well known to cause gastrointestinal damage in many species including the rat and human. Pelsonin a combination of indomethacin and sodium salicylate (1:10 ratio) has a cytoprotective effect against acidic-alcohol induced gastric necrosis, and it does not induce intestinal ulceration. Pelsonin is capable of blocking the formation of intestinal ulcers induced by oral indomethacin 10 mg/kg (curative cytoprotection). The cytoprotective potency of Pelsonin is due to sodium salicylate.

High density of specific GABA binding sites in the human fallopian tube.

The concentration of gamma-aminobutyric acid (GABA) in the human Fallopian tube and the ability of a membrane preparation from the same organ to bind [3H]GABA specifically were examined. A GABA concentration of 177 nM/g frozen tissue was determined and a single population of high-affinity receptor binding sites has been identified in the human oviduct. The density of binding sites demonstrated was as high as in the brain. These results indicate a physiological significance of GABA in the human Fallopian tube.