Benzodiazepine antagonism by harmane and other beta-carbolines in vitro and in vivo

Harmane and other related beta-carbolines are putative endogenous ligands of the benzodiazepine receptor. Since the compounds are potent convulsants they may have agonist activities at the benzodiazepine receptor while the benzodiazepines may be antagonists. This hypothesis was proved by comparing the in vivo and in vitro antagonism of benzodiazepines by harmane and other beta-carbolines. Harmane is clearly a competitive inhibitor of benzodiazepine receptor binding in vitro. Moreover, harmane-induced convulsions can be inhibited reversibly by diazepam in a manner which is consistent with the assumption of competitive antagonism in vivo. For some beta-carboline derivatives a correlation was found between the affinity for the benzodiazepine receptor in vitro and the convulsive potency in vivo. Thus, the data reported suggest that harmane or other related beta-carbolines are putative endogenous agonists of the benzodiazepine receptor. This suggestion is further supported by the observation that diazepam is equally potent in inhibiting harmane- or picrotoxin-induced convulsions, indicating a convulsive mechanism within the GABA receptor-benzodiazepine receptor system.     

Pre- and postsynaptic alpha-adrenoceptor antagonism by neuroleptics in vivo

Different types of neuroleptics were studied for pre- and postsynaptic alpha-adrenoceptor antagonism in pithed rats using the blood pressure effect of clonidine as a measure of postsynaptic alpha-adrenoceptor activation and the depression of the heart rate response to electrical stimulation as a measure of presynaptic alpha-adrenoceptor activation. Yohimbine (0.1 mg/kg) was more active at pre- than postsynaptic alpha-adrenoceptors while the reverse was found with prazosin (0.02-5 mg/kg). Phentolamine (1-5 mg/kg) on the other hand was very active at both receptors. Cocaine 1-5 mg/kg had no effect on these responses indicating that noradrenaline uptake inhibition presumably does not interfere with the revaluation of the alpha-adrenoceptor antagonistic effect of the neuroleptics. Melperone, haloperidol, thioridazine and flupenthixol (0.15 mg/kg) were more selective antagonists at postsynaptic alpha-adrenoceptors than prazosin. Clozapine, chlorprothixene showed preferential presynaptic (0.15 mg/kg) were antagonists at both types of receptors. Chlorprothixene showed preferential presynaptic alpha-antagonism of high potency. Chlorprothixene was the only neuroleptic drug which like phentolamine (1-5 mg/kg) gave complete presynaptic alpha-antagonism. These results indicate widely different selectivity of neuroleptics for pre- and postsynaptic alpha-adrenoceptors on peripheral sympathetic nerves. It is suggested that neuroleptics with presynaptic alpha-adrenoceptor antagonism may enhance the activity of Beta-adrenergic systems indirectly both in peripheral organs like the heart, and within the central nervous system.     

Triazolodiazepines are potent antagonists of platelet activating factor (PAF) in vitro and in vivo

Summary The triazolodiazepines brotizolam, triazolam and alprazolam inhibited PAF-induced human platelet aggregation in vitro (IC₅₀ = 0.54, 7.6 and 13.7 M, respectively) but showed only a weak or no effect against other aggregating agents (ADP, adrenaline, collagen, serotonin, arachidonic acid). In comparison, flunitrazepam and diazepam, two diazepines without the triazole ring, showed IC₅₀-values of 42 and 260 M, respectively. Flunitrazepam does not possess the specificity shown by the other compounds. Brotizolam and triazolam also inhibited PAF-induced human neutrophil aggregation in vitro, with IC₅₀-values 0.21 and 6.6 M, respectively.In anaesthetized guinea pigs, brotizolam (2.5 to 10 mg/kg p.o. or 0.1 to 0.5 mg/kg i.v.) or triazolam (20 to 100 mg/kg p.o.) inhibited dose-dependently the intrathoracic accumulation and aggregation of ¹¹¹Indium labelled platelets induced by an i. v. infusion of PAF (30 ng/kg × min).Brotizolam at doses of 1 to 10 mg/kg p. o. and 0.1 to 0.5 mg/kg i. v. inhibited dose-dependently the reduction in tidal volume (bronchoconstriction), the systemic hypotension and the lethal effect due to i. v. PAF in guinea pigs. Triazolam inhibited these effects of PAF at doses of 50 to 200 mg/kg p.o.PAF-induced systemic hypotension in rats can be reversed by cumulative i. v. doses (0.05 to 1.0 mg/kg) of brotizolam.In conclusion, these results show that triazolodiazepines, like brotizolam and triazolam, are potent inhibitors of PAF-induced effects in vitro and in vivo. Send offprint requests to J. Casals-Stenzel at the above address     

Differences in results from in vivo and in vitro studies on the use-dependency of N-methylaspartate antagonism by MK-801 and other phencyclidine receptor ligands

We have used microelectrophoretic and intravenous administration of drugs to rat spinal cord neurones in vivo and bath application to rat cortical wedges in vitro to evaluate MK-801 and other phencyclidine (PCP) receptor ligands as N-methylaspartate (NMA) antagonists, paying particular regard to the possible use-dependent nature of their action. MK-801, 0.1-0.5 mg/kg, was a selective and long-lasting NMA antagonist. We were unable to demonstrate significant use-dependent onset of antagonism of NMA by any of the drugs in vivo. Recovery, however, for MK-801 was use-dependent. In vitro there was a gradation with MK-801 being very use-dependent, followed by (PCP), cyclazocine and ketamine, the last showing little or no use-dependence. Results of experiments modulating the in vitro environment suggest that a significant difference between the in vitro and in vivo systems was temperature. Raising the temperature of the wedge chamber from 23 to 33 degrees C reduced the use-dependence of MK-801, and lowering the temperature to 13 degrees C increased the use-dependence of PCP. The mechanism of action of PCP receptor ligands is discussed in the light of these results.     

Cancer chemopreventive activity of odorine and odorinol from Aglaia odorata.

In the course of our continuing search for novel cancer chemo-preventive agents from natural sources, we have carried out a primary screening in vitro assay of the compounds isolated from Aglaia odorata. Consequently, aminopyrrolidine-diamides, odorine and odorinol, were obtained as active constituents. These compounds exhibited potent anti-carcinogenic effects in a two-stage carcinogenesis test of mouse skin induced by 7,12-dimethylbenz[a]anthracene (DMBA) as an initiator and 12-O-tetradecanoylphorbol-13-acetate (TPA) as a promoter. Further, both compounds showed remarkable inhibitory effects in two-stage mouse skin carcinogenesis models induced by nitric oxide (NO) donors such as (+/-)-(E)-methyl-2-[(E)-hydroxyimino]-5-nitro-6-methoxy-3-hexenamide (NOR-1) or peroxynitrite as an initiator and TPA as a promoter. From these results, it was concluded that odorine and odorinol inhibited both the initiation and promotion stages of two-stage skin carcinogenesis.     

Platelet-activating factor (PAF) inhibitory profile of KO-286011 on blood platelets in vitro and in vivo

Summary A newly synthesised structural analogue of PAF, coded KO-286011 (1-O-hexadecyl-2-O-ethyl-racglycero-3-phosphoric acid 4-(N,N-dimethylamino)pyridinium butylester), was proved for its ability to inhibit PAF-mediated platelet responses in vitro and in vivo. The compound inhibited effectively the PAF-induced aggregation and secretion of human and rabbit platelets. In contrast, there was little influence on ADP-, collagen-, and arachidonic acid-triggered platelet responses. Schild-analysis of aggregation data ascertained in human platelet-rich plasma was consistent with a simple competitive antagonism and yielded a pA₂, of 6.44. Pro-aggregatory activity of KO-286011 was excluded turbidimetrically as well as by means of a single cell counting technique. [³H]PAF binding studies provided evidence that KO-286011 exerts its inhibitory action at the PAF-receptor level. A significant inhibition of the ex vivo PAF-induced platelet aggregation was found after i.v. administration of 0.5 mg/kg KO-286011 to rabbits. The effect was most pronounced 5 min after dosing the inhibitor and detectable over a period of 30 min. Intravenous administration of 10 and 25 μg/kg KO-286011 to guinea pigs prevented dose-dependently the PAF-induced formation of thromboxane A₂. The PAF-inhibitory action of KO-286011 was more potent than that of the ginkgolide BN 52021. Send offprint requests to G. Ostermann at the above address     

Effects of PAF and PAF antagonists on the shape of venous endothelial cells in vitro

Three platelet-activating factor (PAF) antagonists were tested for their ability to prevent or reduce PAF-induced shape changes of large vein endothelial cells in vitro. BN52021 had a significant protective action at concentrations of 1 microM and 0.1 microM, but at 100 microM had a damaging effect of its own. CV3988 (0.1 microM and 1 microM) and L652,731 (20 microM) did not reduce the responses to PAF, and at higher concentrations (CV3988 10 microM and 100 microM, L652,731 100 microM) both compounds alone caused significant changes of shape. BN52021 (0.1 microM) was also effective against leukotriene (LT) C4, at 1 microM against bradykinin and LTE4, and at 10 microM against LTD4 and the calcium ionophore A23187. BN52021 (10 microM) was ineffective against shape changes induced by histamine, prostaglandin (PG) E2 and lysophosphatidylcholine (LPC). Neither indomethacin (100 microM) nor verapamil (20 microM) altered the response to PAF. Using electron spin resonance (ESR) spectrometry it was shown that the damaging effects of LPC and CV3988 may be due partly to their detergent properties. It is suggested that the mechanism by which PAF alters the shape of large vein endothelial cells is primarily receptor mediated.     

Pharmacological differentiation by pertussis toxin of the in vivo acute responses to fMLP and PAF in guinea-pig lungs.

1. The effects of pertussis toxin on the N-formyl-L-methionyl-L-leucyl-L-phenylalanine (fMLP) and platelet-activating factor (PAF)-induced variations in pulmonary capillary albumin exchanges, blood volume, leucocyte or platelet sequestration were studied in the guinea-pig, by use of radioactive tracers. The effects of pertussis toxin on pulmonary insufflation pressure were studied in parallel. 2. The i.v. administration of fMLP and PAF to the guinea-pig was followed by bronchoconstriction, increased lung capillary albumin exchanges (vasopermeability) sequestration of leucocytes, leucopenia and reduction of blood volume (vasoconstriction). PAF also induced platelet sequestration in lungs and thrombocytopenia. 3. Pertussis toxin (10 micrograms kg-1, i.v., 72 h before the experiment) prevented all the studied fMLP-induced effects, but failed to modify PAF-induced bronchoconstriction, lung vasoconstriction, platelet sequestration, thrombocytopenia and the increased capillary vasopermeability. In the same conditions the lung leucocyte sequestration was not significantly affected when leucopenia was partially reduced. 4. It is suggested that the effects of fMLP, but not those of PAF, involve a Gi-like protein.     

Weakening of naloxone antagonism in vitro and in vivo with octreotide (SMS 201-995)

Octreotide, a potent long-acting somatostatin analogue, has been shown to have a wide range of physiological actions. We have demonstrated a somatostatin-like inhibition of the electrically evoked contractions of the mouse vas deferens and an opioid antagonistic property in the same tissue. In addition, pretreatment with octreotide reduced the potency of naloxone antagonism in vitro in the mouse vas deferens and in vivo in the mouse charcoal meal test. The latter effect suggests that the agonist receptor interactions are taking place on sites allosteric to the agonist site and that the conventionally accepted concept of opioid antagonism may have to be modified.     

Determination of in vivo hepatic extraction ratio from in vitro metabolism by rat hepatocytes.

Using isolated rat hepatocytes, the in vitro intrinsic hepatic clearance of three drugs, namely, antipyrine (AP), propranolol (P), and CGS 13429 was determined. The hepatic extraction ratios of AP, P, and CGS 13429 were then calculated to be 0.10, 0.90, and 0.88, respectively. To determine the in vivo hepatic extraction ratio in the rat, AP, P, or CGS 13429 was infused separately at a rate of 40, 20, and 20 micrograms/min.kg, via jugular and hepatic portal veins. The steady state plasma levels of AP, P, and CGS 13429 were 5.30 +/- 0.33 micrograms/ml, 608 +/- 76 and 380 +/- 46 ng/ml after jugular vein (iv) infusions, and 4.96 +/- 0.46 micrograms/ml, 162 +/- 26, and 148 +/- 23 ng/ml after hepatic portal vein (hpv) infusions, respectively. The blood to plasma ratios of AP, P, and CGS 13429 were 1.0, 0.78, and 1.0, respectively. The in vivo hepatic extraction ratio, calculated as (Css,iv--Css,hpv)/Css,iv from steady state blood levels following iv and hpv infusions for AP, P, and CGS 13429, were 0.065, 0.73, and 0.61, respectively. These results suggest that in vitro metabolism studies by hepatocytes may have potential application, during drug discovery, to distinguish drugs of low and high metabolic hepatic extraction ratio in vivo.     

In vitro and in vivo availability of hydrophilized phenytoin from capsules.

The effect of phenytoin hydrophilization on the liquid penetration rate into prepared plugs, on the disintegration time, on the in vitro release rate, and on in vivo absorption in humans was studied. Hydrophilization was performed by intensive mixing of the hydrophobic drug with a small amount of methylcellulose solution. Liquid penetration into the treated plugs was independent of the liquid wetting potency and extremely high compared to the pure drug plugs. Analogous results were obtained for the disintegration time and in vitro release rates from capsules loaded with pure and treated drug. A bioavailability study in seven healthy volunteers showed immediate absorption of the treated drug but a 1-hr absorption lag time for the pure drug.     

Comparison of in vitro and in vivo histamine methylation by tissues.

A method for determining the histamine-methylating enzyme (HME) using crude enzyme, and minute quantities of the substrate, was applied to tissues of mice, guinea-pigs and rats. Since high levels of endogenous histamine can affect the results, tissue homogenates were dialyzed prior to incubation. Findings were compared with in vivo data on methylating ability of individual tissues; most of this in vivo data is published but a new test of guinea-pig tissues was made using amodiaquine as an inhibitor. The correlation was good, better than that obtained by other procedures. It was observed that dialysis caused an increase in HME for some guinea-pig tissues, but a loss for some mouse tissues. Possible explanations are considered. Quinacrine N-mustard, a derivative of a known HME inhibitor, was tested in mice; it altered the distribution of injected 14C-histamine but showed no evidence of HME inhibition.     

Silica-induced apoptosis in vitro and in vivo.

Silica exposure results in an initially acute inflammatory response followed by chronic fibrotic change. The mechanism for the maintenance of silica-induced inflammation has not been understood yet. In silica-induced acute inflammation and chronic fibrosis, various mediators such as reactive oxygen species, cytokines and growth factors are released. And these substances are suggested to have the regulatory role for the inflammation and fibrosis by possessing the potential to influence apoptosis. To demonstrate the apoptosis as an underlying mechanism for the development of silicosis, in vitro and in vivo models were designed. In in vitro study, we evaluated that apoptotic cell fraction in silica (10, 50 microg/cm2)-treated A549 cells was significantly increased in comparison with control by FACS (fluorescein activated cell sorter). Also genomic DNA from silica (10, 50 microg/cm2)-treated A549 showed DNA ladder formation while control and 1 microg/cm2 groups didn't. In in vivo study, total cell numbers and apoptotic cell numbers of BAL (bronchoalveolar lavage) fluid from silica (10, 20, 40 mg/kg)-instilled rats were significantly higher than control group from 1 week. From these results, we concluded acute and chronic presence of apoptosis may contributes to silica-induced acute inflammation and chronic fibrosis.     

In vitro and in vivo inhibition of lysyl oxidase by aminopropionitriles.

Inhibition of lysyl oxidase (protein-lysine 6-oxidase, EC 1.4.3.13) decreases the rate of collagen and elastin cross-link formation and produces osteolathyrism in animals. Organic nitriles, including beta-aminopropionitrile (BAPN), have been shown to irreversibly inhibit lysyl oxidase in vitro. Both BAPN and 3,3'-iminodipropionitrile (IDPN) have been shown to produce osteolathyric changes when administered to animals. To date compounds that have been reported to inhibit this enzyme possess a primary amine functional group. In this study a series of primary and substituted aminopropionitriles was studied for their ability to inhibit lysyl oxidase activity both in vitro and in vivo. Our results show that of the compounds tested, BAPN was the most potent inhibitor of the enzyme. Reversible inhibition of lysyl oxidase in vitro was found with two secondary aminonitriles, IDPN and monomethylaminopropionitrile (MMAPN). There was no inhibition of enzyme activity associated with the tertiary compound 3,3'-dimethylaminopropionitrile (DMAPN) or propionitrile, a compound lacking an amine functional group. IDPN was found to produce a slight irreversible inhibition of the enzyme both in vitro and in vivo. Pretreatment of rats with pargyline, an inhibitor of monoamine oxidase, was found to increase the inhibitory potential of BAPN (p < or = .1). Pargyline pretreatment did not alter the inhibitory potential for any of the other aminonitriles tested. These results suggest that the presence of a primary amino functional group is not a strict requirement for inhibition of lysyl oxidase. In addition, reversible and irreversible mechanisms of inhibition may be involved in the production of osteolathyric changes associated with IDPN exposure.     

Enkephalin catabolism in vitro and in vivo.

Metabolism of an endogenous opiate like compound, enkephalin, was examined using high pressure liquid chromatography. The “opiate receptor” fraction of brain commonly used to measure enkephalin contains a large amount of amino-peptidase activity capable of metabolizing enkephalin but not β-endorphin. The regional distribution and kinetic parameters of this enzyme were examined. The release of tyrosine from enkephalin was at least 20 times faster than glycine or leucine. The enzyme can be inhibited by O-phenanthroline. When carrier free H enkephalin was injected intraventricularly, more than 90% was broken down within l min. The rapid metabolism of the enkephalins and related compounds should be prevented if meaningful assessments of relative potency are to be made.     

In vivo and in vitro toxicity of phospholipase C from Pseudomonas aeruginosa.

Pseudomonas aeruginosa produces phospholipase C (PLC), a heat-labile hemolysin. Histopathological analysis of PLC-treated mice revealed that the primary target organs involved in PLC-induced toxicity were the liver and kidney. Mice treated i.v. with PLC demonstrated significant tubular epithelial necrosis of the kidney with hematuria, while when given i.p. they exhibited hepatonecrosis with cellular infiltration. Splenomegaly was also a consistent finding. Results from in vitro studies indicate that PLC is toxic for mouse peritoneal cells and human leukocytes.