Prostacyclin-stimulating drugs: New prospects

SKF 525-A (proadifen), a well-known inhibitor of drug metabolism and cytochrome P-450 activity, stimulated the release of prostacyclin (PGI₂) from the rabbit aorta in vitro. The PGI₂-stimulating activity of SKF 525-A was characterized by specific structural requirements : activity was abolished by the deletion of the terminal propyl chain and increased by its elongation into an isobutyl chain; chlorination of the phenyl rings increased the potency. SKF 525-A increased the production of PGI₂ by cultured endothelial cells from bovine aorta and human umbilical vein, but had no effect on cultured smoooth muscle from the bovine aortic media. In human platelets, SKF 525-A inhibited prostaglandin and thromboxane production induced by A23187, thrombin and ADP. Simultaneous stimulation of endothelial PGI₂ and inhibition of platelet TxA₂ represents an original pharmacological profile : SKF 525-A might thus constitute the prototype of a new class of antiplatelet drugs.     

On the activity of γ-aminobutyric acid and glutamate transporters in chick embryonic neurons and rat synaptosomes

The uptake of radioactive -aminobutyric acid (GABA) andd-aspartate and the effect of SKF 89976-A, a non-substrate inhibitor of the GABA transporter, on this uptake have been investigated. Neuronal cultures from eight-day-old chick embryos grown for three or six days in vitro, were used as a model. For comparison, we also used the P₂-fraction from rat. Neuronal cultures grown for three and six days expressed high-affinity uptake systems for [³H]GABA and ford-[³H]aspartate with an increasing V_max during this period. The lipophilic non-substrate GABA uptake inhibitor, SKF 89976-A, inhibited transporter mediated uptake of GABA both in cell cultures from chicken, and in P₂-fractions from rat. The results also showed that SKF 89976-A was a poor inhibitor of the uptake ofd-aspartate. We found no non-saturable uptake ofd-aspartate.     

Inhibition of steroid-mediated induction of δ-aminolevulinic acid synthase by 2-diethylaminoethyl-2,2-diphenylvalerate HCl (SKF 525-A)

The inhibition of the steroid-mediated induction of δ-aminolevulinate synthase, the rate-limiting enzyme in hepatic porphyrin-heme biosynthesis, by 2-diethylaminoethyl-2,2-diphenylvalerate HCl (SKF 525-A) as studied in cultured chick embryo liver cells. The formation of porphyrins in response to cyproterone, a synthetic steroid, was inhibited in a time-dependent manner by SKF 525-A, an inhibitor of several drug metabolizing enzyme systems. This action is a result of an inhibitory effect of SKF 525-A on the cyproterone-mediated induction of δ-aminolevulinate synthase; SKF 525-A laso inhibited the induction of the enzyme by the naturally occurring 5β-H steroids, etiocholanolone and pregnanolone. Employing [³H]etiocholanolone, we provide evidence that this inhibition is not associated with either decreased uptake or an altered metabolism of the steroid. Moreover, approx. 4–6-fold more radioactivity was associated with [³H]etiocholanolone-treated cells cultured in the presence of SKF 525-A. Alternative mechanisms for the induction of δ-aminolevulinate synthase by steroids are proposed which do not require the interaction of steroid-receptor complex with the genome.     

Acute inhibition of microsomal drug metabolism by propoxyphene in narcotic tolerant/dependent mice

Propoxyphene (Darvon) was compared to SKF 525-A, a prototypical inhibitor of hepatic microsomal mixed function oxidases, to assess propoxyphene's potential to inhibit drug metabolism in morphine tolerant/dependent mice. $\text{In vitro}$, both propoxyphene (K_i = 3.5 × 10^?5M) and SKF 525-A (K_i = 4.3 × 10^?6M) inhibited the activity of aminopyrine N-demethylase competitively in hepatic microsomes from tolerant/dependent animals. Propoxyphene and SKF 525-A were weaker, noncompetitive inhibitors of aniline hydroxylase activity. $\text{In vivo}$, equimolar doses (0.24 mmoles/kg, i.p.) of each compound inhibited both of the above monooxygenases in the 10,000$\text{g}$ supernatant fractions of livers from the tolerant/ dependent animals. Propoxyphene was 40–50% as potent an inhibitor of these activities as SKF 525-A. A dose (300 mg/kg) of propoxyphene napsylate, shown to prevent narcotic abstinence signs with no observable toxicity in withdrawing mice, significantly prolonged the blood levels of injected pentobarbital and tripled pentobarbital sleeping time in these animals. When administered at 300 mg/kg chronically, propoxyphene napsylate acted as an inducer of its own metabolism. Propoxyphene napsylate, then, given acutely to narcotic tolerant/dependent mice, is a potent inhibitor of microsomal drug metabolizing capacity. Given chronically, it enhances this capability.     

Liver metabolic reactions: tertiary amine N-dealkylation, tertiary amine N-oxidation, N-oxide reduction, and N-oxide N-dealkylation. II. N,N-dimethylaniline

Rat liver microsomal preparations enzymatically catalyze the N-demethylation and N-oxidation of dimethylaniline as well as the N-demethylation of dimethylaniline-N-oxide. Both compounds were used as substrates and the formation of formaldehyde and N-oxide were determined.Both demethylation and N-oxidation of dimethylaniline are dependent on NADPH. This cofactor also increases the demethylation of dimethylaniline-N-oxide, although it is not an absolute requirement. Nicotinamide increases the rate of formation of formaldehyde and N-oxide from dimethylaniline by a factor of about 4 and decreases the N-oxide demethylation by the same factor. The cofactor optimum consists of NADPH, nicotinamide, and magnesium ions for the demethylation and N-oxidation of dimethylaniline, and of NADPH alone for the demethylation of its N-oxide. The kinetic constants of the three test reactions have been determined under these optimal cofactor requirements.Various agents strongly influence the rates of product formation of the three test reactions studied. SH-blocking agents, the chelating agent EGTA, as well as nicotinamide influence the rates of formaldehyde formation from dimethylaniline and N-oxide demethylation in an opposite way. This demonstrates that, in the tertiary amine demethylation of dimethylaniline, a C-oxidation pathway is operative in addition to an N-oxidation pathway with subsequent N-oxide demethylation. The following influences on the actual metabolic reactions could be deduced from the effects of agents on the test reactions: SKF 525-A inhibits and phenobarbital pretreatment stimulates N-oxide demethylation; EDTA inhibits both the latter reaction and N-oxidation; EGTA and nicotinamide stimulate C-oxidation and inhibit N-oxide demethylation; SH-blocking agents inhibit C-oxidation and stimulate both N-oxidation and N-oxide demethylation.Quantitative and qualitative species differences with respect to cofactor requirement and effect of SKF 525-A have been observed between rat and pig liver microsomes. In addition, profound differences in subcellular localization and metabolic rates between dimethylaniline and other substrates are known. Thus it is unlikely that the three metabolic reactions dealt with in this report are characteristic of tertiarr amine N-dealkylation in general.     

Antimalarial acridines: Synthesis,in vitro activity against P. falciparum and interaction with hematin

A series of acridine derivatives were synthesised and their in vitro antimalarial activity was evaluated against one chloroquine-susceptible strain (3D7) and three chloroquine-resistant strains (W2, Bre1 and FCR3) of Plasmodium falciparum. Structure–activity relationship showed that two positives charges as well as 6-chloro and 2-methoxy substituents on the acridine ring were required to exert a good antimalarial activity. The best compounds possessing these features inhibited the growth of the chloroquine-susceptible strain with an IC₅₀ ? 0.07 μM, close to that of chloroquine itself, and that of the three chloroquine-resistant strains better than chloroquine with IC₅₀ ? 0.3 μM. These acridine derivatives inhibited the formation of β-hematin, suggesting that, like CQ, they act on the haem crystallization process. Finally, in vitro cytotoxicity was also evaluated upon human KB cells, which showed that one of them 9-(6-ammonioethylamino)-6-chloro-2-methoxyacridinium dichloride 1 displayed a promising antimalarial activity in vitro with a quite good selectivity index versus mammalian cell on the CQ-susceptible strain and promising selectivity on other strains.     

Effect of drug metabolism inducer and inhibitor on O,O,S-trimethyl phosphorothioate-induced delayed toxicity in rats

Oral administration of O,O,S-trimethyl phosphorothioate (OOS), an impurity present in widely used organophosphorus insecticides, causes delayed toxicity in rats, i.e., death occurring as late as 28 days after the treatment. The signs of toxicity include body weight loss (maximum on day 3), red staining around the nose, mouth and eyes, and an increased level of lactate dehydrogenase (LDH) in bronchopulmonary lavage fluid accompanied by morphological alteration of non-ciliated bronchiolar epithelial Clara cells.Pretreatment with phenobarbital, piperonyl butoxide (2 h), SKF 525-A, or small multiple doses of OOS protected against the OOS-induced elevated level of bronchopulmonary lavage LDH, and the other signs of delayed toxicity including morphological alteration of Clara cells. These studies support the view that OOS-induced delayed toxicity is mediated by the cytochrome P-450 dependent metabolism of OOS, and the lung may be the major target organ of delayed toxicity produced by OOS.     

Activity of some bromomethylated aromatic hydrocarbons on the in vitro synthesis of DNA and RNA

The effect of a series of bromomethylated polycyclic hydrocarbons on in vitro DNA and RNA synthesis has been studied by measurement of the incorporation of [³H]-dTMP or [¹⁴C]-AMP into new chains. The inhibition of RNA synthesis was less than 12% for 9-bromomethylanthracene, 9-methyl-10-bromomethylanthracene and 12-bromomethylbenzo(a)acridine, and more than 37% for 7-bromomethylbenzo(a)anthracene, 7,12-dibromomethylbenzo(a)anthracene and 7-bromomethylbenzo(c)acridine. Analogous results were found for the inhibition of DNA synthesis, except for 7-bromomethylbenzo(c)acridine which had little effect. Apart from this exception a good correlation was found between the inhibitory action of the bromo derivatives and the carcinogenicity of the non-halogenated parent hydrocarbons.     

Inhibition of transporter mediated γ-aminobutyric acid (GABA) release by SKF 89976-A,a GABA uptake inhibitor,studied in a primary neuronal culture from chicken

The effect of SKF 89976-A, a lipophilic non-substrate inhibitor of the -aminobutyric acid (GABA) transporter, on the release of radioactive GABA andd-aspartate has been studied. Neuronal cultures from 8 day old chick embryos, grown for six days, served as a model. The cultures were incubated with [³H]d-aspartate and [¹⁴C] GABA with the subsequent addition of high or low concentrations of SKF 89976-A. Finally the cultures were exposed to differently composed media for either 30 or 300 seconds. The release was quantified, using liquid scintillation counting. The efflux of [³H]d-aspartate and [¹⁴C] GABA was increased by [K⁺] and time, and a minimum value was obtained at [Ca²⁺] 1.05 mM. The release of both [³H]d-aspartate and [¹⁴C] GABA was inhibited by SKF 89976-A. The obtained results indicate that transporter mediated processes are the major mechanisms of transmitter release in the investigated model.     

Molecular chaperone function of Arabidopsis thaliana phloem protein 2-A1, encodes a protein similar to phloem lectin

Although several phloem sap proteins have been identified from protein extracts of heat-treated Arabidopsis seedlings using FPLC gel filtration columns, many of the physiological roles played by these proteins remain to be elucidated. We functionally characterized a phloem protein 2-A1, which encodes a protein similar to phloem lectin. Using a bacterially expressed recombinant protein of AtPP2-A1, we found that it performs dual functions, showing both molecular chaperone activity and antifungal activity. mRNA expression of the AtPP2-1 gene was induced by diverse external stresses such as pathogens, and other signaling molecules, such as ethylene. These results suggest that the AtPP2-A1 molecular chaperone protein plays a critical role in the Arabidopsis defense system against diverse external stresses including fungal pathogenic attack and heat shock.     

Studies on the structure-fluorescence relationships of chromatin-bound 9-amino acridine derivatives

Microscopic assessment and microphotometric measurement of the fluorescence intensity of human metaphase chromosomes stained with 24 9-amino acridine derivatives differing in chemical structure, led to the following conclusions:

1. 1. With the -OCH₃ group as a substituted radical in the acridine core, the fluorescent intensity of the stain bonded with chromosomes increases. The best fluorochromes contain this group in position 5 or 7. The fluorescent intensity increases in the presence of a halogen atom in position 3, or with a second -OCH₃ group in position 4. These observations suggest that the rise in fluorescent intensity is caused by substitutes that increase the density of the π electrons in acridine rings.

2. 2. Weak fluorescent intensity is displayed by 9-amino acridine derivatives containing less than 3 -CH₂ groups in the side chain in position 9.

3. 3. Alkylizing the guanine of chromosomal DNA by acridine derivatives is not an indispensible condition for achieving well differentiated fluorescence of metaphase chromosomes.

     

Synthesis of novel 5-amino-1,3,4-thiadiazole-2-sulfonamide containing acridine sulfonamide/carboxamide compounds and investigation of their inhibition effects on human carbonic anhydrase I,II, IV and VII

Herein, we report that acridine intermediates 5 were obtained from the reduction of nitro acridine derivatives 4, which were synthesized via condensation of dimedone, p-nitrobenzaldehyde with 4-amino-N-(5-sulfamoyl-1,3,4-thiadiazol-2-yl)benzamide, respectively. Then acridine sulfonamide/carboxamide (7a–i) compounds were synthesized by reaction of amino acridine 5 with sulfonyl chlorides and carbamoyl chlorides. The new compounds were characterized by melting points, FT-IR, ¹H NMR, ¹³C NMR and HRMS analyzes. The evaluation of in vitro test of the synthesized compounds against hCA I, II, IV and VII showed that some of them are potent inhibitors. Among them, compound 7e showed the most potent activity against hCA II with a K_I of 7.9 nM.     

Prostacyclin-stimulating drugs: new prospects

SKF 525-A (proadifen), a well-known inhibitor of drug metabolism and cytochrome P-450 activity, stimulated the release of prostacyclin (PGI2) from the rabbit aorta in vitro. The PGI2-stimulating activity of SKF 525-A was characterized by specific structural requirements: activity was abolished by the deletion of the terminal propyl chain and increased by its elongation into an isobutyl chain; chlorination of the phenyl rings increased the potency. SKF 525-A increased the production of PGI2 by cultured endothelial cells from bovine aorta and human umbilical vein, but had no effect on cultured smooth muscle from the bovine aortic media. In human platelets, SKF 525-A inhibited prostaglandin and thromboxane production induced by A23187, thrombin and ADP. Simultaneous stimulation of endothelial PGI2 and inhibition of platelet TxA2 represents an original pharmacological profile: SKF 525-A might thus constitute the prototype of a new class of antiplatelet drugs.     

Action mechanism of vascular spasmolytics. 5. Drug-related relaxation and adenosine-3',5'-monophosphate content of isolated coronary arteries under conditions of fluoride-induced contracture]

Tetracaine and SKF 525-A as well as stimulators of adenylate cyclase and inhibitors of the nucleoside-3',5'-monophosphate phosphodiesterase have spasmolytic effects. At the fluoride-iuduced contracture -- a form of contracture independent of extracellular calcium -- tetracaine and SKF 525-A effect an additional increase in muscular tension. Their relaxing effect evidently presupposes a functioning calcium exchange. Drugs acting on the cAMP system have invariably spasmolytic effects at this contracture model, although they produced no change of the cAMP content under these conditions. An increase in total cAMP content is not obviously a necessary condition for the occurrence of drug-induced relaxation.     

Inhibition of soybean lipoxygenase by SKF 525-A and metyrapone

To determine whether agents which inhibit cytochrome P-450 enzymes also inhibit lipoxygenase, the effects of metyrapone and SKF 525-A were assessed on soybean lipoxygenase using a spectrophotometric technique which allows for measurement of both the rate and magnitude of product formation. Both SKF 525-A and metyrapone inhibited the rate of product formation and the final amount of product formed in 5 min incubations SKF 525-A was 5 to 6 times more potent than metyrapone, with the IC₅₀ for SKF 525-A 40 uM and for metyrapone between 150 and 200 uM as determined by the total product formation in 5 minutes. Analysis of the reduced product by HPLC confirmed that the substances monitored were those generated by the 15-lipoxygenase enzyme.     

The activation of diazinon by ganglia of the cockroach Periplaneta americana L. and its action on nerve conduction and cholinesterase activity

When sixth abdominal ganglia of the cockroach Periplaneta americana were irrigated continuously with diazinon solution in situ, its effects on nerve conduction and cholinesterase activity closely resembled those of diazoxon; spontaneous activity and after-discharge increased until conduction was blocked, which happened while some cholinesterase was still uninhibited. The symptoms were only slightly relieved by irrigating ganglia with saline. Though the LD50's of diazinon and diazoxon applied topically to adult male P. americana were similar (2.5 ± 0.33 and 4.5 ± 0.38 μig. per insect), diazoxon was about 300 times more active than diazinon against nerve function and cholinesterase activity in the sixth abdominal ganglion. This is probably because in the nerve preparations contact between the insecticide and the tissues surrounding the nerve cord, which in whole insects convert diazinon, a thionophosphate, into its phosphate analogue diazoxon, a more active anticholinesterase, was minimized. Indeed, taking into account the evidence of workers who previously compared in vitro the anticholinesterase activities of several thionophosphates with those of their phosphate analogues and found the phosphates much more active, the effect of diazinon on cholinesterase activity and nerve function in our experiments was unexpectedly great. By applying diazinon to nerve cords with SKF 525-A, a compound likely to prevent oxidation of diazinon to diazoxon, an attempt was therefore made to decide whether diazinon directly affected nerve conduction or whether the effect resulted either from its conversion to diazoxon within the nerve tissue or from impurities in the diazinon used. Results were inconclusive, for SKF 525-A (p-diethylaminoethyl diphenylpropylacetate hydrochloride) not only failed to prevent the inhibition of cholinesterase, but interfered with the action of both diazinon and diazoxon on nerve conduction, and itself affected nerve conduction when applied alone. The possibility that diazinon is itself a mild anticholinesterase was not excluded. SKF 525-A applied to sixth abdominal ganglia at 2 × 10^-4M blocked conduction from cereal nerves to giant fibres in 50–97 min. and at 4 × 10^-5M decreased the post-synaptic response; applied to giant fibres at 2 × 10^-4M it blocked conduction in 90–208 min. The effects of the larger concentration were not completely reversible. Although SKF 525-A has been widely used to study the metabolism of drugs, its direct effects on conduction in nerve axons seem not to have been noted previously.     

Novel synthetic acridine derivatives as potent DNA-binding and apoptosis-inducing antitumor agents

Acridine derivatives have been explored as DNA-binding anticancer agents. Some derivatives show undesired pharmacokinetic properties and new derivatives need to be explored. In this work, a series of novel acridine analogues were synthesized by modifying previously unexplored linkers between the acridine and benzene groups and their antiproliferative activity and the DNA-binding ability were evaluated. Among these derivatives, compound 5c demonstrated DNA-binding capability and topoisomerase I inhibitory activity. In K562 cell lines, 5c induced apoptosis through mitochondria-dependent intrinsic pathways. These data suggested that compound 5c and other acridine derivatives with modified linkers between the acridine and benzene groups might be potent DNA-binding agents.     

Genetic and Molecular Properties of an Infectious Antibiotic Resistance (R) Factor Isolated from Klebsiella

A Klebsiella strain of human origin that was resistant to ampicillin, chloramphenicol, kanamycin, neomycin, streptomycin, and tetracycline was found to have all of these resistances associated with a R factor and a satellite molecular species of deoxyribonucleic acid (DNA) with an average buoyant density of 1.710 in cesium chloride gradients. There was no evidence of the existence of DNA with other buoyant densities. The strain bears two separable mutations for chloramphenicol resistance, both of which are associated with the R factor (KR9). Exposure of the Klebsiella strain to acridine derivatives or to ethidium bromide (which was more efficient) resulted in partial losses of resistance accompanied by the disappearance of the satellite DNA peak or shifts in its density. The R factor and its component genes were conjugally transmitted across generic boundaries and maintained in new hosts with different efficiencies. The basis of this difference lies not only in the efficiency of conjugal transfer but also in the stability of the components after transfer. All of the resistance genes and the resistance transfer factor were cotransducible by phage Plkc from Escherichia coli. Partially resistant strains could be reconstituted to full resistance or to a recombined pattern of partial resistance by conjugation with donors having complementary resistance patterns. This recombination serves as an efficient mechanism for rescuing superinfecting genes that are otherwise intracellularly excluded. KR9 is an fi⁺ type of R factor which in the natural state does not appear to be as repressed in conjugal transfer as other R factors.     

Oxidation of dimethylamine and trimethylamine in methazotrophic yeasts by microsomal mono-oxygenases sensitive to carbon monoxide

Whole cells of Candida boidinii grown on di- or tri-methylamine as sole nitrogen source readily oxidized both amines. The oxidation was potently inhibited by carbon monoxide. Cell-free extracts required the presence of 20 μM FAD before mono-oxygenase activity with both amines could be demonstrated. NADH was a better electron donor than NADPH. Activity was present in cells grown on secondary and tertiary amines but not on primary amines, and was detected in a number of different yeasts. Enzyme activity could be sedimented at 187 000 x g, and was associated with NADPH-cytochrome c reductase activity. It is thus probably microsomal. Activity was inhibited by cyanide, mercaptoethanol, carbon monoxide and proadifen hydrochloride (SKF 525-A).     

Synthesis and antitumor activity of novel amsacrine analogs: The critical role of the acridine moiety in determining their biological activity

A new series of N-[4-(2′-oxo-2H-pyrano[2,3-b]quinolin-5′-ylamino)-phenyl]-methanesulfonamides was prepared and analyzed as novel amsacrine-like derivatives. Our preliminary biological evaluation has shown that the replacement of the acridine moiety with the analogous 2-oxo-2H-pyrano[2,3-b]quinoline system drastically reduced both their anticancer activity and their propency to intercalate into double stranded DNA.