Search

The free movements of a small laboratory animal on the bottom of a cage, containing capacitive sensors, are measured as electrical signals which are then amplified, classified in a classification device and counted. With reference to the class of the highest sensitivity and by the choice of appropriate device parameters, signals are only counted during the time in which animal movements are below a level defined as rest. By this, the resting time can be determined which expresses the time in which animals are in the defined rest during the whole test time. The ED50, namely the doses after which the resting time increases by 50% during the first 10 min of the exploration phase of mice has been determined of diazepam, chlordiazepoxide, haloperidol, droperidol, fluphenazine, butaperazine, 7-fluorobutaperazine, and chlorpromazine and it was compared with the ED 50 determined in the contact bottom plates motimeter according to Knoll et al. in mice and in the catalepsy test according to Wirth et al. in rats. respectively. The method of resting time determination has proved as to be striking more sensitive (5- to 10-fold) than the other methods.

The original view of glucocorticoid effects as being divided into physiological and pharmacological ones is no longer acceptable, i.e. all glucocorticoid effects are apparently mediated by receptor occupation which triggers RNA protein synthesis. The concentrations needed for receptor-mediated effects are low, e.g. the KD value for dexamethasone is in the nanomolar range. The high-dose pulse glucocorticoid therapy results in blood concentrations much higher than those necessary for receptor saturation. This makes sense only when nonspecific effects may be expected to occur which necessitate concentrations higher than 10(-6) mol.l-1. In this paper the question of nonspecific glucocorticoid effects in adjuvant arthritis and carrageenin paw edema of rats was investigated using the glucocorticoid receptor antagonist RU 38486 and by injecting the RNA/protein synthesis inhibitors actinomycin D or cycloheximide. We did not find convincing evidence for nonspecific glucocorticoid mechanisms.

Experiments in rats suffering from primary acute adjuvant inflammation showed independent changes in serum acute phase protein concentration and macroscopic paw inflammation during antiinflammatory treatment: soybean trypsin inhibitor and horse-radish peroxidase caused antiinflammatory effects but simultaneously produced increased alpha 2 macroglobulin levels. On the other hand, cycloheximide significantly inhibited the increase of alpha 2 macroglobulin concentration in adjuvant inflammation, however, it had no antiinflammatory effect. All forms of treatment caused even some change in protein plasma levels of healthy rats which probably relates to an activation of cells producing interleukin-1, interleukin-6, and/or hepatocyte stimulating factor which trigger the synthesis of acute phase proteins in the liver. In inflamed rats, the snake venom batroxobin caused a significant decrease in the fibrinogen level whereas the paw swelling remained completely unaffected. Therefore, it seems to be doubtful whether acute phase proteins essentially contribute to the modulation of acute inflammatory reaction in primary rat adjuvant inflammation.

Rat tail thrombosis (RTT) induced by i.v. injection of kappa-carrageenin was not inhibited by p.o. or i.p. administration of the aglucon of rutin, quercetin, but it was slightly inhibited by rutoside-containing Venoruton. The RTT was significantly inhibited by didymlaevulinate, Helodym 88, whereas samarium-containing Phlogosol gave inconsistent results because of the solvent propylene glycol. The RTT model is suitable to detect substances of the flavonoid and lanthanide types with weak antithrombotic activity.

Cyclooxygenase inhibitors (diclofenac, indomethacin, piroxicam) does not or only hardly inhibit anaphylactic vasodepression, except acetylsalicylic acid which could act as an oxygen free radical scavenger. However, the dual blocker BW 755 c strongly improves vasodepression. Pure lipoxygenase inhibitors (nordihydroguajaretic acid, oxphaman) only moderately improve vasodepression, the leukotriene antagonist FPL 55,712 is hardly effective. Obviously, endogenous prostaglandins do not and leukotrienes do take little part in the pathogenesis of anaphylactic vasodepression in the mouse and rat.

The action of butaperazine was diminished and simultaneously prolonged by fluorination in the position 3 or 7 as evidenced by a number of tests (prolongation of the resting time, catalepsy, apomorphine and dexphenmetrazine antagonism, conditioned escape reflex, aggressivity, algesimetry) on the rat and the mouse. The same seems to apply, in principle, to promazine and prochlorperazine. In contrast, the action of 7-fluorperphenazine and its esters with adamantanic acid and decanoic acid was markedly increased, but not prolonged, as demonstrated by a test (conditioned escape reflex) on the rat. The potency of fluphenazine is reached, but not its duration of action. This finding still needs confirmation by other tests. The duration of action of the butyrylphenazine esters was scarcely longer than that of butyrylphenazine. Considerable irritation has been seen at the site of injection. The esters of long-chained fatty acids and fluperidol and trifluperidol seem to be more potent than the parent substances. There is obviously no prolongation of the duration of action. The activity of the haloperidol oxime ethers (and probably of the esters, too) is weak as compared to that of haloperidol. The duration of action is not prolonged.

The carrageenin edema of rat paw and the primary phase of rat adjuvant arthritis were significantly inhibited by carrageenin whereas the increase of skin capillary permeability induced by prostaglandins (PGs) and further irritants as well as the secondary phase of adjuvant arthritis were mostly not significantly affected. Inhibitory effects after single carrageenin injection do not seem to be mediated by the complement system because total complement activity remained unaffected.

There is no group proof of long acting antirheumatics (LAA) in laboratory animal models, and it is not to be expected without an identical rheumatoid arthritis model in animals and with regard to the heterogeneity of LAA. However, LAA are to be detected according to D-penicillamine-like, levamisol-like etc. actions, which can be disclosed in the adjuvant arthritis as well as in the B. pertussis-vaccine pleuritis in rats the latter model best by including parameters of inflammatory exudate cells. Modification of the models or of model parameters (BCG-sensibilization, PPD reaction, vasoreactivity, RNA content of exudate cells, SH groups, copper zinc) are hardly advantageous, contrarily to dosage. Other models, among them paw edemas, do not permit sufficient testing of LAA, even not the methyl-albumin mice paw edema. There is no problem of pharmacologically separating LAA actions from nonsteroidal or steroidal antiphlogistics actions.

Kappa-carrageenins cause disseminated intravascular coagulation with thrombosis of the tail in rats and mice. Frequency and extent of tail thrombosis were used for determining antithrombotic effects after systemic and local external administration of substances. Inhibitors of cyclooxygenase and thromboxane synthetase caused irregular inhibition of thrombosis after relatively high doses. The cyclooxygenase/lipoxygenase inhibitor BW755C was ineffective after 50 mg/kg. Hitherto, no effective substances could be found after external administration on the tail of mice. At present, no convincing explanation for thrombogenic activity of kappa-carrageenin can be given. The advantages of the thrombosis model are discussed.

First, it is shown that it is not yet possible to outline a generally valid receptor model for the non-steroidal anti-inflammatory agents. This is demonstrated by three examples of receptor models, namely by the model according to Shen, by the receptor concept of Appleton and Brown, and by the concept concerning chelate complex formation between NSA and heme iron of the cyclooxygenase complex according to Peterson et al. This means that the "custom-made" synthesis of NSA with, perhaps, quantitatively and qualitatively better properties is not yet possible. On the basis of this statement, QSAR investigations may be justified in order to optimize known NSA. In this paper, QSAR calculations on 21 fenamate derivatives are reported. For this, both the multivariate and the univariate Hansch Analysis were used. Altogether, 16 approaches to QSAR has been performed using 1 - 4 biological and 3 - 19 physico-chemical parameters, respectively, with 7 - 21 objects. In all cases a dominating influence of pi has been indicated. Other physico-chemical parameters seemed to be of less importance, e. g., sigma, Es, log xi MR, MV, parachor, and Verloop's steric constants. Possible new fenamate structures of higher biological activity are briefly discussed.

In the isolated perfused hind legs of rats with enemas induced by carrageenin, dextran or Freund's adjuvant in both paws, resting perfusion pressure was slightly increased whereas the vasopressor action of noradrenaline, lysine-vasopressin and prostaglandin F2 alpha, was decreased. Admixture of indomethacin (3 micrograms.ml-1) to the perfusion fluid led to a decrease of resting perfusion pressure whereas its influence on EAmax of noradrenaline was only weak under these conditions. Concomitantly, the contents of prostaglandin E-like substances in the perfusate decreased. Prostaglandin F2 alpha exhibits only weak vasopressor activity in isolated perfused hind legs of rats with carrageenin edema. Altogether, the effect of indomethacin on resting perfusion pressure as well as on EA and EAmax, resp., of agonists is difficulty to explain by its effect on arachidonic acid cascade. The pD2-value of noradrenaline (4.68 - 4.87) and lysine-vasopressin (6.02 - 6.04) was apparently not changed, at least not decreased, in the acute phase of inflammation indicating no impairment of receptor affinity of noradrenaline and vasopressin in inflammation. Blood vessel reaction is apparently influenced in inflammation mechanically by the increased tissue pressure as well as by molecular mechanisms consisting in the presence of inflammatory mediators and/or particularly in a reduced intrinsic sensitivity of the blood vessel muscle itself.

In anaphylactic paw edema the reactivity of blood vessels to norepinephrine in the isolated perfused hind legs of rats and mice is reduced. A vasoreactivity depressing factor was postulated and searched for. PAF-acether, histamine and lipoxygenase products were found as being possibly responsible for depression of the vascular reactivity.

1. Injections of carrageenin (1,25 mg/kg i.v.) from the 1st to the 3rd day and then each 2nd or 3rd day inhibited paw swelling in adjuvant arthritis of the rat during the time of treatment. Injections from the 11th to the 15th day were ineffective. The level of plasma kininogen was slightly decreased but the total complement serum level was significantly lowered. 2,5 and 3 mg carrageenin/kg respectively were toxic after repeated injections. After a single administration the levels of plasma kininogen and of total serum complement were decreased by 50% although paw swelling was not affected. 2. Pentosane polysulfoester (25 mg/kg i.v.) did not influence paw swelling despite daily administration from the 1st to the 17th day. Heparin (10 000 IE/kg i.v.) was likewise ineffective. 3. Single or repeated injections of compound 48/80 (0,125-0,5 mg/kg i.v.; 1-5 mg/kg i.p.; 3-6 mg/kg s.c.), reserpine (0,2 mg/kg i.p.), cyproheptadine (5 mg/kg i.v.), bromolysergic acid diethylamide (2 x 2 mg/kg i.v.) or metiamide (10 mg/kg i.v.) were without effect on paw swelling. Neither did compound 48/80 effect the complement serum level. 4. Daily administration of chloropromazine (4-10 mg/kg p.o.) or of promethazine (10-15 mg/kg s.c. or p.o.) inhibited paw swelling in the first phase of adjuvant arthritis but not in the second one. 5. The soybean trypsin inhibitor (15 mg/kg i.v.) inhibited paw swelling significantly up to the 4th day, the Kunitz inhibitor (25 000 E/kg i.v.) was ineffective. 6. The content of prostaglandin E of the inflamed paws was increased threefold in both phases of arthritis. The results are discussed with regard to the putative role of mediators of inflammation (histamine, serotonin, kinins, prostaglandins, lysosomal enzymes, lymphokines, complement).

The ear edema of the mouse induced by local application of arachidonic acid is suitable for in vivo differentiation of lipoxygenase (LOX) and cyclooxygenase (COX) inhibitors. LOX blockers inhibit initially and plateau-like during the first hour of the course of the edema, while COX-blockers do so mostly only initially. For practice we recommend the measurement about 60 min following edema provocation. The dual blocker of LOX and COX, BW 755 c, acts like a LOX inhibitor.

During 6-sulphanilamidoindazole (6-SAI) arthritis a significant increase of serum levels of the protease inhibitor alpha 2 macroglobulin (= alpha 2 acute phase globulin) and of Darcy's glycoprotein was found, whereas the content of female specific protein and of sulfhydryl groups was proved to be significantly decreased. Intravenous administration of 5 mg/kg soybean trypsin inhibitor, twice daily for 4 days, was without any inhibitory effect on paw swelling in 6-SAI arthritis but it caused increased serum levels of alpha 2 macroglobulin and Darcy's glycoprotein. Thus, spontaneous remission of 6-SAI arthritis occurring, despite continued administration of 6-SAI, seems hardly likely to be caused by acute-phase reactants, e.g. via protease inhibition. Evidence is presented which shows that spontaneous remission of 6-SAI arthritis could be due to local processes.

Infusion of lysolecithin (LPC; e.g. 88 microgram/ml for 0.5-1.0 min) did not significantly impair the vasopressor action of norepinephrine (NE), prostaglandin F2 alpha (PGF2 alpha) and extract of posterior pituitary (EPP) in the isolated perfused hind legs of rats. In other words, vascular smooth muscle behaves differently from the smooth muscle of the guinea-pig small intestine, since, in the latter, contractions evoked by acetylcholine, prostaglandins etc., are inhibited by LPC. Triton X 100 which, by comparison, was used as a detergent effective on the guinea-pig small intestine, depressed the vasopressor effect of NE, PGF2 alpha and EPP. LPC, at low concentrations (40 mumol/l), potentiated (15% max.) ADP-induced platelet aggregation (PA) in rat PRP but, at high concentrations, inhibited PA (IC50 = 390 mumol/l). 2-Hexadecylglycerophosphocholine and its short-chain 1-alkyl ethers, which are structurally related to platelet-activating factor, as well as some long-chain alkanol phosphocholine esters, were somewhat more active than LPC. Dipalmitoyllecithin (4-700 mumol/l) was without any effect.

Based on a standardized in-vitro method for quantifying the activity of prostaglandin-synthetase by means of coupling malondialdehyde with 2-thiobarbituric acid the possibility of using this method also as ex-vivo technique is described. By this, more favourable prerequisites to pharmacological valuation of the effects of potential antiinflammatory substances exists compared to the application of in-vitro results, only.

A method is described, in which small laboratory animals are freely moving in the field of analogous capacitive sensors. The electrical signals induced by animal movements which, according to amplitude and polarity, contain informations on intensity and direction of movements are conveyed to a classification device and classified according to well-defined classes. By measuring during a fixed time (e.g. 10 min) or for a previously determined number of signals (e.g. 2000) a distribution of class frequencies, the movement profile, is obtained. Parameters of movement profile of mice in the orientation phase and factors of influence are reported on. The method allows the continuously automatic registration of movement profiles.

Vasoprotective activity of COX inhibitors (diclofenac, piroxicam, indomethacin, aspirin) and of LOX inhibitors (nordihydroguaiaretic acid, propyl gallate, oxphaman) or of both (BW 755c) as well as of OFRS (mannitol, ethanol, thiourea, vitamin E) was investigated ex vivo in the isolated perfused hindlegs of the animals after in vivo u.v.-irradiation and antigen-provocation in mice and rats, respectively. The results show that vasodepression is mainly prevented by OFRS, antioxidants/LOX inhibitors and aspirin, but not by other COX inhibitors.

Metal complexing agents could exert antiinflammatory activity by inhibition of oxygen radical production, by inhibition of eicosanoid synthesis and by inhibition of metalloenzymes. We found a moderate antiedema activity of the iron (II)/iron (III)-chelator combination, o-phenanthroline/desferrioxamine, which could refer to an involvement of iron ions in the inflammatory reaction. The newly synthetized complexing agent ethylene-diimino-dibutyric acid caused weak antiedema and antivasodepressor effects which remain to be explained. Altogether, the in vivo effects of the metal chelators were rather weak. In vitro, only desferrioxamine produced a remarkable inhibition of two lipoxygenases.

Tail thrombosis (infarction) induced by injection of kappa-carrageenin (0.88 or 1.76 mg/kg i.v.) is increased by i.v. or i.p. serotonin (1-2 mg/kg) but is decreased by cyproheptadine and ketanserin (minimal effective dose less than 1 mg/kg) as well as by preceding serotonin depletion by comp. 48/80 or reserpine. The effect of exogenous serotonin is antagonized by ketanserin and cyproheptadine, respect. In the anaphylactoid reaction induced by dextran, only weak thrombosis increasing effects were seen, apparently, because of vasodilation in this reaction. Application of heat to the tail weakly decreased tail thrombosis, application of cold strikingly increased it. Vasodilating substances (nicotinate, 3-hydroxymethylpyridine, trapidil, dipyridamole, iloprost) as well as the thromboxane antagonist BM 13,177 were without significant effect on tail thrombosis, even if vasodilating substances were applicated in form of ointments on the tail. The participation of endogenous serotonin in carrageenin-induced tail thrombosis has further to be substantiated.

Catalase, peroxidase and superoxide dismutase were found to inhibit significantly carrageenin edema and the primary phase of adjuvant arthritis in rats after i.v. injection. Heat-inactivated enzymes were as effective as the native enzymes. None of 10 scavengers of oxygen radicals inhibited the adjuvant arthritis at any time. Accordingly, no evidence for a participation of oxygen radicals in the secondary arthritis phase could be found, whereas a role of oxygen radicals in the primary arthritis phase and in carrageenin edema cannot be ruled out.

In the movement profile analysis, movements of small laboratory animals, according to both intensity and direction (amplitude and polarity), are electronically measured by means of capacitive sensors. The electrical signals are amplified, classified into 13 classes in a classification device and depicted as frequency distribution. The obtained distribution corresponds to a Gaussian distribution if normal control mice are used (normal movement profile, MP). The normal MP is characteristically changed by drugs. By centrally depressing drugs (droperidol, chlorpromazine, diazepam, medazepam) distribution classes of weak movements are dose-dependently increased while classes of strong movements are decreased. Stimulating drugs (dexphenmetrazine, methamphetamine, caffeine) gave opposite changes of the MP. A certain time after a stimulating drug the change of the MP can turn to the MP of depressing drugs obviously as the result of exhaustion of the animal. After low doses of echinoramine I a MP with the characteristics of a depressing drug was found, after high doses the MP was that of a stimulating drug, perhaps as the result of subtoxic effects. After apomorphine (1, 5, and 10 mg/kg) no clear dose-dependence could be found. The movement profile analysis can be useful in the specified analysis of movements-influencing drugs.

The tail thrombosis after kappa-carrageenin injection in rats was significantly inhibited by heparin and phenprocoumon. Heparin was yet effective at doses as low as 150 IU/kg. The present data point to an involvement of blood coagulation in this new model. Injection of cobra venom factor with complement depletion caused no thrombosis inhibition. The differences between the present kappa-carrageenin induced thrombosis model and a disseminated intravascular coagulation after lambda-carrageenin are discussed.

Nonsteroidal antiinflammatory agents of several subgroups, regarding eicosanoid-producing enzyme inhibition, have been tested in vitro and in vivo. There was no overt correlation between cyclooxygenase and/or lipoxygenase inhibition in vitro and antiinflammatory effects. It is possible that eicosanoids do not play a key role as inflammatory mediators, i.e. that the course of the life-protecting inflammatory reaction is ensured by an ensemble of mediators, cellular events and further mechanisms of the whole living organism.

After adsorption-chromatographic separation (using aluminium oxide) and chemical conversion to the corresponding trihydroxyindole derivatives, epinephrine, levarterenol and dopamine were determined fluorometrically in urine from rats. In case of carrageenin oedema, as well as during the secondary phase of adjuvant arthritis, the excretion of epinephrine and levarterenol was increased as compared to that in control animals. This difference was statistically significant. These findings can possibly be connected with an inhibitory activity of the catecholamines in inflammatory processes; as to dopamine, it seems to be of minor importance.

The anti-inflammatory effect of dexamethasone in carrageenin rat paw oedema was significantly reduced or abolished by local injection of 0.5-2.5 mg of the antiglucocorticoid progesterone, or of 5 micrograms cycloheximide, or of 2.5 micrograms actinomycin D, into the oedematous area. The results point to indirect dexamethasone action via receptor occupation and de novo protein synthesis, but this view could not be undoubtedly confirmed by systemic administration of essentially higher doses of the substances. The advantages of local low dose antagonist administration versus systemic injection of higher doses are mainly the production of effective tissue levels, as well as avoidance of toxic and other side-effects.

Our problem was, can phenylbutazone influence zinc level in serum and paw tissue and is there any relationship to its antiinflammatory effect? Phenylbutazone and zinc concentration were photometrically determined in serum and in homogenated paw tissue of rats injected with Freund's adjuvant. The course of total serum zinc level is opposite to the course of paw swelling whereas zinc content of paw tissue increases with swelling in the first phase of arthritis. Phenylbutazone increases serum zinc level, and the increase is highest if phenylbutazone is given daily. Phenylbutazone slightly decreases paw zinc level, especially 5 and 12 hrs after administration, but only in the first phase of arthritis. Pharmacokinetics of phenylbutazone in serum is unaffected in arthritic rats whereas phenylbutazone content of paws is increased in the first and second phase of arthritis. When given alone zinc and phenylbutazone accumulate in the inflamed tissue, perhaps, in their plasma protein bound form. Phenylbutazone inhibits inflammation and concomitantly decreases invasion of proteins and protein-bound zinc into the paw. Possibly phenylbutazone inhibits inflammation by inhibition of zinc-containing dehydrogenases since such enzymes are inhibited in vitro by phenylbutazone.

When rats were pretreated by intraplantar or i.v. injection of various inflammatory mediators, the vasopressor effect of i.a. norepinephrine in the subsequently isolated perfused hindlegs of the rats was found to be partly depressed. This vasodepression could also be detected if mediators were directly co-perfused in the isolated hindlegs. The vasodepressor effect was strongest following histamine pretreatment and co-perfusion, respectively, whereas endotoxin 0111:B4, PAF-acether, PGE1, and LTD4 were less effective. Only weak or no vasodepression could be induced by bradykinin, serotonin, lysolecithin, and substance P (1-11). The significance of the results is discussed with respect to anaphylactic disorders.

After a survey of the literature on the antimicrobial activity of the bee product propolis, the authors discuss their own findings as compared to the chemotherapeutical agents streptomycin, oxytetracycline, chloramphenicol, nystatin, griseofulvin and sulphamerazine. According to the results obtained by testing 25 isolated constituents on Bacillus subtilis, Staphylococcus aureus, Candida albicans and Trichophyton mentagrophytes, the antimicrobial properties of this mixture of natural substances are mainly attributable to the flavonoids pinocembrin, galangin, pinobanksin, pinobanksin-3-acetate as well as to the p-coumaric acid benzyl ester and a caffeic acid ester mixture. None of the isolated substances was as potent as the antibiotics tested for the purpose of comparison. The relatively good antimycetic activity of the 5,7-dihydroxyflavanone pinocembrin seems noteworthy. Finally, possible mechanisms of the antimicrobial action of the flavonoids are discussed.

In a series of fourteen 1-benzoyl-3-methyl-pyrazole derivatives the antiinflammatory activity in the carrageenin edema of the rat paw was found to be in the potency range of that of aminophenazone. This holds also true of the analgesic, anticonvulsive, and anticholinergic activity of the compounds whereas antihistaminergic activity was 10-fold better and acute toxicity was less than that of aminophenazone. Using the MASCA model as a multivariate method in QSAR the regression matrix of the derivatives substituted in p-position of the benzoyl moiety has been calculated. According to that, all of the biological activities are mainly described in terms of pi, sigma, xi, and L. The increase of inflammatory activity could be expected by substitution of electro-positive substituents of short length (L) and little relative surface tension (xi). Concomitantly, analgesic and anticonvulsive activities could increase whereas anticholinergic and antihistaminergic activities are expected to decrease. The HANSCH analysis is surely to be preferred in investigations of the relationship between physicochemical parameters of compounds and a certain biological activity evoked by them. However, the multivariate MASCA model could be of some advantage in the optimization of chemical structure against the whole profile of biological activities in a series of compounds. Some problems of the MASCA model are briefly discussed.