Different in vitro metabolism of paclitaxel and docetaxel in humans, rats, pigs, and minipigs.

We investigated cytochrome P450 (P450)-catalyzed metabolism of the important cancer drugs paclitaxel and docetaxel in rat, pig, minipig, and human liver microsomes and cDNA-expressed P450 enzymes. In rat microsomes, paclitaxel was metabolized mainly to C3'-hydroxypaclitaxel (C3'-OHP) and to a lesser extent to C2-hydroxypaclitaxel (C2-OHP), di-hydroxypaclitaxel (di-OHP), and another unknown monohydroxylated paclitaxel. In pig and minipig microsomes, this unknown hydroxypaclitaxel was the main metabolite, whereas C3'-OHP was a minor product. In minipigs, C2-OHP was the next minor product. In human liver microsomes, 6 alpha-hydroxypaclitaxel (6 alpha-OHP) was the main metabolite, followed by C3'-OHP and C2-OHP. Among different cDNA-expressed human P450 enzymes (CYP1A2, 1B1, 2A6, 2C9, 2E1, and 3A4), only CYP3A4 enzyme formed C3'-OHP and C2-OHP. Docetaxel was metabolized in pig, minipig, rat, and human liver microsomes mainly to hydroxydocetaxel (OHDTX), whereas CYP3A-induced rat microsomes produced primarily diastereomeric hydroxyoxazolidinones. Human liver microsomes from 10 different individuals formed OHDTX at different rates correlated with CYP3A4 content. Troleandomycin as a selective inhibitor of CYP3A inhibited the formation of C3'-OHP, C2-OHP, and di-OHP, as well as the unknown OHP produced in rat, minipig, and pig microsomes. In human liver microsomes, troleandomycin inhibited C3'-OHP and C2-OHP formation, and a suitable inhibitor of human CYP2C8, fisetin, strongly inhibited the formation of 6 alpha-OHP, known to be catalyzed by human CYP2C8. In conclusion, the metabolism of docetaxel is the same in all four species, but metabolism of paclitaxel is different, and 6 alpha-OHP remains a uniquely human metabolite. Pigs and minipigs compared with each other formed the same metabolites of paclitaxel.     

Protein Aggregates Seem to Play a Key Role Among the Parameters Influencing the Antigenicity of Interferon Alpha (IFN-α) in Normal and Transgenic Mice

Purpose. During long-term treatment of various malignant or viral diseases with IFN- up to 20% of patients develop anti-IFN- antibodies for as yet unknown reasons. Methods. To address this issue, a mouse model using Balb/C mice was established and the relevance of several potentially anti-IFN- antibodies inducing factors was studied. Results. The model revealed that both a higher frequency of injections and a higher dosage of IFN- were more immunogenic and that the route of administration affected the antibody response to IFN-. The intrinsic immunostimulatory activity of IFN- itself also enhanced the immune response. IFN- protein aggregates (IFN--IFN- and human serum albumin (HSA)-IFN- aggregates), which were recently identified in all marketed IFN- products, were significantly more immunogenic than IFN- monomers. These aggregates broke the tolerance against human IFN- monomers in human IFN- transgenic mice. Conclusions. Based on these animal studies it is proposed that the immune response to IFN- in humans is most probably elicited by a combination of several factors among which IFN- protein aggregates seem to play a key role.     

DDT increases hepatic testosterone metabolism in rats

DDT and its metabolites are considered as endocrine disruptors able to promote hormone-dependent pathologies. We studied the effects of technical-grade DDT on hepatic testosterone metabolism and testosterone hydroxylase activity ratios in the rat. Male and female Wistar rats were treated by gavage with a single dose of technical-grade DDT (0, 0.1, 1, 10, and 100 mg/kg body weight) and killed 24 h later. Hepatic microsomes were incubated with [4-¹⁴C]-testosterone and the metabolites were separated by thin-layer chromatography and quantified by radio scanning. DDT increased testosterone biotransformation and modified the profile of metabolites produced in a sex-dependent manner. Males treated with a representative dose (10 mg/kg) produced relatively less androstenedione (AD), 2-hydroxytestosterone (OHT), and 16-OHT but higher 6-OHT whereas treated females produced less 7-OHT and AD but higher 6-OHT and 6-OHT than their respective controls. In both sexes DDT decreased the relative proportion of AD and increased that of 6-OHT suggesting that the androgen-saving pathway was affected. The testosterone 6-/15-OHT ratio, a proposed indicator of demasculinization, was increased in treated males. This effect was in agreement with the demasculinizing ability proposed for DDT. The effects on 6-/16-OHT and 6-dehydrotestosterone/16-OHT ratios followed a similar tendency, with the ratio 6-/16-OHT being the most sensitive marker. Interestingly, these ratios were reduced in treated females suggesting that technical-grade DDT shifted testosterone hydroxylations toward a more masculine pattern. Thus, technical-grade DDT altered the hepatic sexual dimorphism in testosterone metabolism and decreased the metabolic differences between male and female rats.     

Liver growth and mixed-function oxidase activity: Dose-dependent stimulatory and inhibitory effects of α-hexachlorocyclohexane

Summary -hexachlorocyclohexane (-HCH) elicits liver growth and stimulation of hepatic microsomal mixed-function oxidase. In the present study the extent of these changes was determined in rats 2, 4 and 6 days after treatment with doses of -HCH ranging from 1 to 600 mg/kg. Above the respective threshold doses liver mass, liver DNA, and the rate of aminopyrine demethylation increased in proportion to the log dose. Threshold doses were calculated to be 30 mg/kg for the increase of liver weight and DNA, and 5 mg/kg for the stimulation of aminopyrine demethylation.Liver mass and liver DNA continued to increase up to the highest dose used; in contrast, the rate of aminopyrine demethylation declined at doses exceeding 200 mg/kg. This decline seems to be due to inhibition by -HCH retained in the microsomal fraction: -HCH is a potent, apparently competitive inhibitor of aminopyrine demethylation, and gaschromatographic determinations revealed that the amount of -HCH retained in the microsomes is sufficient to produce considerable inhibition of the enzymatic reaction.Abbreviations -HCH -1,2,3,4,5,6-hexachlorocyclohexane, -benzene hexachloride - EL 241 [,-bis(p-chlorophenyl)-3-pyridinemethanol] - RLW relative liver weight = liver weight in percent of body weight     

Postjunctional α-adrenoceptors

Summary The postsynaptic -adrenoceptors in rat aorta and in pithed rat were investigated according to their sensitivity to nine -adrenergic agonists and to the selective antagonists yohimbine (₂) and prazosin (₁) and the non-selective one, phentolamine. In addition, in radioligand binding studies, the affinity and selectivity of the drugs were determined on rat cerebral cortex using [³H] yohimbine and [³H] prazosin.On rat aorta, prazosin is 1,000 times more potent than yohimbine against each -adrenoceptor agonist, whether ₁- or ₂-selective. Rat aorta probably contains only ₁-adrenoceptors.Pressor effects in pithed rats are mediated by post-junctional ₁- and ₂-adrenoceptors. The dose-response curve for -methylnorepinephrine in the presence of prazosin, using Hofstee's plots, revealed ₁- and ₂-adrenoceptors, respective proportions being 80.5 and 19.5%     

Pharmacologic differentiation between pre- and postjunctional α2-adrenoceptors by SK & F 104078

Summary Most ₂-adrenoceptor antagonists do not discriminate between pre- and postjunctional ₂-adrenoceptors, and this has led to the commonly held belief that pre- and postjunctional ₂-adrenoceptors may represent one homogeneous population of receptors. SK&F 104078 has been shown to be a potent antagonist at postjunctional ₂-adrenoceptors at concentrations that do not block prejunctional ₂-adrenoceptors. Thus, SK & F 104078 is a competitive postjunctional ₂-adrenoceptor antagonist in canine and rabbit saphenous veins, canine saphenous artery and human platelet with a dissociation constant of approximately 100 nmol/l. Conversely, SK & F 104078 is inactive as a prejunctional ₂-adrenoceptor antagonist in atria from dog, guinea pig, rabbit and rat, and in guinea-pig ileum at concentrations up to 10,000 nmol/l. Likewise, SK & F 104078 has the ability to block postjunctional arterial ₂-adrenoceptors in vivo in the pithed rat at doses that do not inhibit prejunctional ₂-adrenoceptors in the same model. The results suggest that pre- and postjunctional ₂-adrenoceptors may not represent one homogeneous class, but rather are discrete subtypes of the ₂-adrenoceptor that may be differentiated by SK & F 104078. Send offprint requests to R. R. Ruffolo, Jr.     

Resveratrol regulates cellular PKC α and δ to inhibit growth and induce apoptosis in gastric cancer cells

Summary Resveratrol, a dietary phytoalexin, has emerged as a promising chemopreventive agent due to its antiproliferative and pro-apoptotic action toward cancer cells and its ability to inhibit tumor growth in animals. Gastric adenocarcinoma cells respond to resveratrol treatment with suppression of DNA synthesis, activation of nitric oxide synthase, induction of apoptosis and inhibition of total PKC and PKC activity. Here we demonstrate that treatment of gastric adenocarcinoma SNU-1 cells with resveratrol results in time and concentration dependent accumulation of tumor suppressors p21^{(cip1/WAF -1)} and p53 and is preceded by loss of membrane-associated PKC protein and a concomitant increase in cytosolic PKC . Arrest of the cell cycle at transition of S to G₂/M phases correlates with the profile of ³H-thymidine incorporation and accumulation of p21^{(cip1/WAF -1)} and was temporally dependent on increase of p53. SNU-1 cells respond to resveratrol treatment with up-regulation of both Fas and Fas-L proteins, whereas in KATO-III cells, with deleted p53, only Fas-L is increased after resveratrol treatment. Although Fas and Fas-L proteins in SNU-1 cells and Fas-L in KATO-III cells were elevated within 24 h of cell treatment with low concentrations of resveratrol, significant apoptotic response at these concentrations was observed only after 48 h. Altogether, our findings indicate that resveratrol engages PKC and signals in gastric adenocarcinoma SNU-1 cells prior to up-regulation of antiproliferative and pro-apoptotic signals. The specific cell death signals engaged by resveratrol appear to be cell type dependent and suggest that resveratrol has chemopreventive potential even after mutational changes have occurred.     

Über den nachweis und die bedeutung von α-methylnoradrenalin im harn von hypertonikern bei verabreichung von α-methyldopa

Zusammenfassung An 13 Hypertonikern wurde die Harnausscheidung von -Methyldopa, -Methyldopamin und Katecholammen während einer Dauerbehandlung oder nach einer Einzeldosis von -Methyldopa untersucht. Die im Harn enthaltenen Substanzen werden durch Ionenaustausch- und Papierchromatographie voneinander getrennt und fluorometrisch oder biologisch bestimmt. — Von alien Patienten wurde außer freiem und konjugiertem -Methyldopa und -Methyldopamin auch -Methylnoradrenalin ausgeschieden. Gleichzeitig nahm die Ausscheidung von Noradrenalin gegenüber der Vor-und Nachperiode bei fast allen Patienten ab. Es kann ausgeschlossen werden, daß die bei der Behandlung durch die Nieren ausgeschiedenen Mengen von -Methyldopa, -Methyldopamin oder -Methylnoradrenalin die renale Ausscheidung von Noradrenalin herabsetzten. Die während der Behandlung ausgeschiedene Menge von Noradrenalin, Adrenalin und -Methylnoradrenalin zusammengenommen unterschied sich nicht von der Katecholamin-Ausscheidung vor und nach der Behandlung. — Das biosynthetisch vom Menschen gebildete -Methylnoradrenalin verhielt sich nach dem Ergebnis chemischer und pharmakologischer Tests wie Corbadrin, welches die Erythro-Konfiguration aufweist; -Methylnoradrenalin ließ sich eindeutig von dem Diastereomeren von Corbadrin differenzieren, welches die Threo-Konfiguration besitzt. Im Harn der Patienten konnte -Methyladrenalin nicht nachgewiesen werden. Gegen eine Beteiligung von -Methyladrenalin, welches nach Tierversuchen durch Erregung von adrenergen -Receptoren blutdrucksenkend wirkt, an der hypotensiven Wirkung von -Methyldopa spricht ferner, daß die Blutdruckabnahme nach -Methyldopa durch Propranolol nicht aufgehoben, sondern eher sogar verstärkt wurde. — Die systolische Blutdrucksenkung war sowohl der Abnahme der renalen Ausscheidung von Noradrenalin als auch dem Prozentsatz von -Methylnoradrenalin an der gesamten Katecholamin-Ausscheidung significant korreliert; auch im Zeitverlauf ergab sich bei drei Patienten nach einer Einzeldosis von -Methyldopa eine Korrelation zwischen hypotensivem Effekt einerseits und Abnahme der Noradrenalin- bzw. Zunahme der -Methylnoradrenalin-Ausscheidung andererseits. — Die Ergebnisse machen es wahrscheinlich, daß der früher in Tierversuchen nach Verabreichung von -Methyldopa nachgewiesene teilweise Ersatz des sympathischen Überträgerstoffs Noradrenalin durch -Methylnoradrenalin auch unter therapeutischer Dosierung am Menschen stattfindet. Die Korrelationen von hypotensiven Effekten und Veränderungen im Aminstoffwechsel lassen den SchluB zu, daß -Methyldopa den Blutdruek von Hypertonikern durch Freisetzung von -Methylnoradrenalin als falscher Überträgersubstanz und entsprechende Verminderung der Noradrenalin-Freisetzung aus sympathischen Nervenfasern herabsetzt.

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Detection and significance of -Methylnoradrenaline in the urine of hypertensive patients after administration of -Methyldopa

Summary In 13 hypertensive patients the urinary excretion of -methyldopa, -methyldopamine und catechol amines was analyzed either during continuous administration or after a single dose of -methyldopa. The compounds contained in the urine samples were separated by ion exchange and paper chromatography and determined fluorimetrically and biologically. — All patients excreted both free and conjugated -methyldopa, -methyldopamine, and small amounts of -methylnoradrenaline. Compared with the pre- and postdrug period, in nearly all patients the noradrenaline excretion was decreased while -methyldopa was administered. The experimental data obtained exclude the possibility that the noradrenaline excretion was diminished by the simultaneous excretion of -methylnoradrenaline or of excessive amounts of -methyldopa and -methyldopamine. The combined quantities of noradrenaline, adrenaline and -methylnoradrenaline excreted daily during administration of -methyldopa did not differ significantly from the amount of noradrenaline and adrenaline excreted daily before drug treatment was started and after it was discontinued. —Evidence was obtained that -methylnoradrenaline isolated from urine of patients given -methyldopa behaved chemically and biologically in a way similar to (–)-corbadrine (erythro-configuration) but differed markedly from the diastereomer of corbadrine (threo-configuration). Hence, biosynthesis of -methylnoradrenaline leads to the levorotatory erythro-isomer. — In animal experiments carried out previously -methyladrenaline was found as another metabolite of -methyldopa which decreased blood pressure by activation of adrenergic -receptors. The patients given -methyldopa did not excrete -methyladrenaline. The method employed was sensitive enough to detect amounts of -methyladrenaline less than 3 per cent of the -methylnoradrenaline present. Furthermore, involvement of a -adrenergic component in the response of the blood pressure to -methyldopa in hypertensive patients was made unlikely by the observation that propranolol did not antagonize but rather enhanced the hypotensive effect of -methyldopa. — There was a significant correlation between the fall of systolic blood pressure and both the decrease in urinary excretion of noradrenaline and the increase in the percentage of -methylnoradrenaline of the total catecholamine output. Likewise, the time course of the depressor response to a single dose of -methyldopa closely corresponded to the decrease in noradrenaline and the increase in -methylnoradrenaline excretion. Conversely, the return of the blood pressure to the initial level was reflected by an increase in noradrenaline and a decrease in -methylnoradrenaline excretion. — In animal experiments it was previously found that administration of -methyldopa caused a partial displacement of noradrenaline by -methylnoradrenaline which subsequently was released by sympathetic nerve stimulation. The present findings demonstrate that -methylnoradrenaline is formed in man as well. It is concluded that -methyldopa lowers the blood pressure of hypertensive patients by the release of -methylnoradrenaline as a false neurotransmitter and the concomitant decrease in noradrenaline liberation from sympathetic nerve fibres.

     

Subclassification of presynaptic α2-adrenoceptors: α2D-autoreceptors and α2D-adrenoceptors modulating release of acetylcholine in guinea-pig ileum

The study was designed to classify in terms of _2A, _2B, _2C and _2D the presynaptic ₂-autoreceptors, as well as the ₂-receptors modulating the release of acetylcholine, in the myenteric plexus-longitudinal muscle (MPLM) preparation of the guinea-pig ileum. A set of antagonists was chosen that was able to discriminate between the four subtypes. Small pieces of the MPLM preparation were preincubated with ³H-noradrenaline or ³H-choline and then superfused and stimulated electrically.The stimulation periods used (3H-noradrenaline: 3 trains of 20 pulses, 50 Hz, train interval 60 s; ³H-choline: single trains of 30 pulses, 0.2 Hz) did not lead to ₂-autoinhibition or inhibition of ³H-acetylcholine release by endogenous noradrenaline. The ₂-selective agonist 5-bromo6-(2-imidazolin-2-ylamino)-quinoxaline (UK 14,304) reduced the evoked overflow of tritium in both ³H-noradrenaline and ³H-choline experiments. Most (³H-noradrenaline) or all (³H-choline) of the 10 antagonists shifted the concentration-inhibition curves of UK 14,304 to the right. pK_d values of the antagonists were calculated from the shifts. pK_d values from ³H-noradrenaline experiments correlated with pK_d values from ³H-choline experiments (r = 0.981).It is concluded that ₂-autoreceptors and ₂-heteroreceptors modulating the release of acetylcholine in the MPLM preparation are of the same subtype. Comparison with antagonist affinities for prototypic native ₂ binding sites, binding sites in cells transfected with ₂ subtype genes, and previously classified presynaptic ₂-adrenoceptors — all taken from the literature — indicates that both are _2D. The results are consonant with the hypothesis that at least the majority of ₂-autoreceptors belong to the _2A/D branch of the ₂-adrenoceptor tree, across mammalian or at least across rodent and lagomorph species. The same may hold true for ₂-adrenoceptors on non-noradrenergic neurones.     

Evidence in favour of a selective α1-adrenoceptor blocking action of WB 4101 in vivo

Summary The -adrenoceptor blocking potency of WB 4101 at ₁- and ₂-adrenoceptors has been investigated in pithed rats.WB 4101 was approximately 97 times more potent at antagonizing the vasopressor responses produced by the selective ₁-adrenoceptor agonist phenylephrine, than those produced by the selective ₂-adrenoceptor agonist M-7.A dose of WB 4101 (3 mg/kg) that caused extensive blockade of vascular ₁-adrenoceptors, but little or no blockade of vascular ₂-adrenoceptors, exerted no significant blockade of the presynaptic ₂-adrenoceptors in the rat heart.The results support the view that WB 4101 is a highly selective antagonist at ₁-adrenoceptors in vivo.     

A Candidate for Cancer Gene Therapy: MIP-lα Gene Transfer to an Adenocarcinoma Cell Line Reduced T\imorigenicity and Induced Protective Immunity in Immunocompetent Mice

Purpose. To evaluate the possibility of cancer gene therapy by the gene delivery of chemokine, the effects of human macrophage inflammatory protein l (hu-MIP-l), murine-macrophage inflammatory protein l (mu-MIP-l), and human-interleukin 8 (hu-IL-8) on tumor progression and immunization were studied. Methods. Cachexia-inducing and highly tumorigenic adenocarcinoma cells (cell line colon 26, clone 20) were transfected with either a control plasmid, hu-MIP-l, mu-MIP-l, or hu-IL-8 expression vector. The production of hu-MIP-1 reached >1.5 ng/ml in vitro when transfectant cells were cultured at a cell density of 2 × 10⁵ cells in 7 ml for 3 days. Immunocompetent BALB/c mice were inoculated into the footpad with the tumor cells, and then primary tumor growth, morphological analyses, and tumor immunogenicity were studied. Results. The secretion of hu-MIP-l, mu-MIP-l, and hu-IL-8 did not affect the growth rate in vitro. Reduced tumorigenicities in vivo were observed in transfected cells with hu-MIP-l and mu-MIP-l. Morphologic observation of the site of inoculation of cells transfected with hu-MIP-l showed infiltration of macrophages and neutrophils on the 5th day after the inoculation. Mice that had rejected cells transfected with hu-MIP-l gene were immune to a subsequent challenge with the parental cells. Conclusions. The rejection of the cells depends on cytolysis and generates potent and long lasting antitumor immunity. These data suggest that tumor cells transfected with the MIP-l gene might be useful as an effective therapy for the treatment of certain tumors.     

Über die Wirkung von α-Methyl-Dopa auf den Brenzcatechinamingehalt von Meerschweinchenorganen

Zusammenfassung 1. Versuche über die Wirkung von -Methyl-Dopa auf den Brenzcatechinamingehalt von Meerschweinchenorganen haben ergeben, daß Noradrenalin im Herzen, sowie Dopamin und Noradrenalin im Gehirn durch äquimolare Mengen von -Methyl-Noradrenalin bzw. -Methyl-Dopamin ersetzt werden.2. Die Adrenalinverarmung der Nebennieren nach Vorbehandlung mit -Methyl-Dopa wird demgegenüber nur zu einem geringen Teil durch die methylierten Brenzcatechinamine -Methyl-Noradrenalin und -Methyl-Dopamin ausgeglichen. Ein weiteres Brenzcatechinamin konnte nicht nachgewiesen werden, so daß -Methyl-Adrenalin wahrscheinlich nicht gebildet wird.3. Im Meerschweinchenherzen gespeichertes -Methyl-Noradrenalin wird durch Reserpin in vivo schlechter freigesetzt als Noradrenalin.4. Das in isolierten Herzgranula (Sediment 100 000·g) gespeicherte -Methyl-Noradrenalin wird durch Segontin bei der Inkubation in vitro ebenfall schlechter freigesetzt als Noradrenalin aus normalen Herzgranula.5. Der Mechanismus der -Methyl-Dopa-Wirkung wird diskutiert.6. Es wird eine fluorimetrische Methode zur Bestimmung von -Methyl-Noradrenalin angegeben.

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Summary 1. The catecholamine content of guinea pig organs after administration of -methyldopa has been determined. The results show that the loss of noradrenaline from heart and that of noradrenaline and dopamine from brain is compensated by a stoichiometric uptake of -methylnoradrenaline and -methyldopamine respectively.2. On the contrary the loss of adrenaline from the suprarenal medulla induced by pretreatment with -methyldopa is not completely restored by the -methylated amines, -methyldopamine and -methylnoradrenaline. -methyladrenaline could not be detected even by paperchromatography.3. The release of the stored -methylnoradrenaline from guinea pig heart by reserpine is very small in comparison with that of noradrenaline.4. Furthermore only small amounts of the stored -methyl-noradrenaline are released from isolated granules of the guinea pig heart after incubation in vitro with segontin. Whereas the same amount of segontin depleted completely the noradrenaline content of the granules isolated from normal hearts.5. The mechanism of action of -methyldopa is discussed.6. A method for the fluorimetric determination of -methylnoradrenaline is described. It is at first separated from -methyldopamine, dopamine and histamine by column chromatography, condensed with o-phthaldialdehyd and the obtained fluorescense is measured by using an Aminco-Bowman spectrophotofluorimeter.

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Mit 6 Textabbildungen

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Herrn Dr. H. Blaschko zum 65. Geburtstag gewidmet.

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Über einen Teil der Ergebnisse wurde auf der Frühjahrstagung der Deutschen Pharmakologischen Ges. in Mainz, April 1964, berichtet. Naunyn-Schmiedebergs Arch. exp. Path. Pharmak. 247, 297 (1964).

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Ausgeführt mit Unterstützung der Deutschen Forschungsgemeinschaft.     

Synthesis and pharmacological study of some alkylamini esters and amides of α,α-diarylaliphatic acids

Conclusions Sixteen alkylamino esters and amides of ,-diphenyl--ethoxyacetic acid and ,-diphenylpropionic acid have been synthesized and investigated pharmacologically. All the compounds prepared possess spasmolytic and cholinolytic properties. Derivatives of ,-diphenyl--ethoxyacetic acid are also characterized by the presence of analgesic and anti-cough action. Loading the alcoholic or acid part of the molecule, or replacement of the ester bond by an amide bond, leads to weakening of activity.Translated from Khimiko-Farmatsevticheskii Zhurnal, No. 1, pp. 9–14, January, 1970.     

Antagonists that differentiate between α2A- and α2D-adrenoceptors

Four antagonists were examined for their ability to differentiate _2A from the orthologous _2Dadrenoceptors. The antagonists were (2S,12bS) 1, 3-di-methylspiro(1, 3, 4, 5, 6, 6, 7,12b-octahydro-2H-benzo[b]furo[2,3-a]quinolizine)-2,4-pyrimidin-2-one (MK 912), 2-[2-(methoxy-1, 4-benzodioxanyl)imidazoline (RX 821002), efaroxan and benoxathian. The ₂-autoreceptors in rabbit brain cortex were chosen as _2A- and the a₂-autoreceptors in guinea-pig brain cortex as _2D-adrenoceptors. Slices of the brain cortex were preincubated with ³H-noradrenaline and then superfused and stimulated electrically by brief pulse trains (4 pulses, 100 Hz) that led to little, if any, ₂-autoinhibition. 5-Bromo-6-(2-imidazolin-2-ylamino)quinoxaline (UK 14,304) was used as an ₂-adrenoceptor agonist.UK 14, 304 decreased the stimulation-evoked overflow of tritium. The antagonists shifted the concentration-inhibition curve of UK 14, 304 to the right in an apparently competitive manner. Dissociation constants of the antagonists were calculated from the shifts. MK 912, RX 821002 and efaroxan had markedly higher affinity for (guinea-pig) _2D-adrenoceptors (pK _d values 10.0, 9.7 and 9.1, respectively) than for (rabbit) _2A-adrenoceptors (pK _d 8.9, 8.2 and 7.6, respectively). Benoxathian had higher affinity for _2A- (pK _d 7.4) than for _2D-adrenoceptors (pK _d 6.9). Ratios calculated from the K _d values of the four compounds differentiated between _2A and _2D up to 100 fold. It is concluded that MK 912, RX 821002, efaroxan and benoxathian are antagonists with high power to differentiate _2A- from _2D-adrenoceptors.     

Stimulation of prostaglandin E2-synthesis by noradrenaline in primary cell cultures from rabbit splenic pulpa is mediated by atypical α-adrenoceptors

Summary In primary cell cultures originating from rabbit splenic pulpa the effects of various adrenoceptor agonists on prostaglandin (PG)-synthesis were studied. The cells-microscopically identified as fibroblasts-released PGs into the medium: especially PGE₂ besides small amounts of PGF₂ and PGD₂.Noradrenaline increased dose-dependently the amount of PGs released into the medium. Besides noradrenaline, only the catecholamines adrenaline and -methylnoradrenaline strongly activated PG-synthesis. Other -adrenoceptor agonists like the phenylethylamine and imidazoline derivatives were only weak agonists or completely ineffective. All adrenoceptor agonists without intrinsic activity in these cells antagonized the noradrenaline effect on PG-synthesis, the imidazolines being more potent antagonists than the phenylethylamines.The -adrenoceptor agonist isoprenaline stimulated PG-synthesis at high concentrations only. The effects of both noradrenaline and isoprenaline were inhibited by low concentrations of phentolamine and phenoxybenzamine, but not by propranolol. The preferential ₂-adrenoceptor antagonists yohimbine and rauwolscine were about 50 times more potent in blocking the noradrenaline effect on PG-synthesis than the more ₁-specific antagonist corynanthine. However, prazosin, another ₁-adrenoceptor antagonist, was about equipotent with yohimbine.It is concluded that noradrenaline elicits PG-synthesis in rabbit splenic fibroblasts via -adrenoceptor stimulation. The -adrenoceptor involved has properties which are different from those reported so far for ₁- or ₁-adrenoceptors.     

Alpha-naphthoflavone induces vasorelaxation through the induction of extracellular calcium influx and NO formation in endothelium

The effect of -naphthoflavone (-NF) on vascular function was studied in isolated ring segments of the rat thoracic aorta and in primary cultures of human umbilical vein endothelial cells (HUVECs). -NF induced concentration-dependent relaxation of the phenylephrine-precontracted aorta endothelium-dependently and -independently at lower and higher concentrations, respectively. The cGMP, but not cAMP, content was increased significantly in -NF-treated aorta. Pretreatment with N -nitro-l-arginine methyl ester (L-NAME) or methylene blue attenuated both -NF induced vasorelaxation and the increase of cGMP content significantly. The increase of cGMP content induced by -NF was also inhibited by chelating extracellular Ca²⁺ with EGTA. These results suggest that the endothelium-dependent vasorelaxation induced by -NF is mediated most probably through Ca²⁺-dependent activation of NO synthase and guanylyl cyclase. In HUVECs, -NF induced concentration-dependent formation of NO and Ca²⁺ influx. -NF-induced NO formation was abolished by removal of extracellular Ca²⁺ and by pretreatment with the Ca²⁺ channel blockers SKF 96365 and Ni²⁺, but not by the L-type Ca²⁺ channel blocker verapamil. The Ca²⁺ influx, as measured by ⁴⁵Ca²⁺ uptake, induced by -NF was also inhibited by SKF 96365 and Ni²⁺. Our data imply that -NF, at lower concentrations, induces endothelium-dependent vasorelaxation by promoting extracellular Ca²⁺ influx in endothelium and the activation of the NO-cGMP pathway.     

DOPA decarboxylase inhibition does not influence the diuretic and natriuretic response to exogenous α-atrial natriuretic peptide in man

Summary The role of dopamine synthesis in the renal actions of human -atrial natriuretic peptide (ANP) was investigated in six dehydrated volunteers using the DOPA decarboxylase inhibitor carbidopa.Each subject received oral placebo or carbidopa (100 mg) followed by an infusion of ANP 10 pmol · kg^–1 · min^–1 for 1 h. The responses to placebo alone and to carbidopa alone were investigated on separate occasions. ANP produced a similar increase in plasma immunoreactive ANP whether placebo or carbidopa pretreatment had been given. Urinary dopamine excretion was increased by ANP. Carbidopa pretreatment substantially attenuated this increase without affecting the natriuretic or water-diuretic response to ANP. Carbidopa also failed to alter the change in filtration fraction produced by ANP.The results suggest that increased synthesis of intrarenal dopamine is not required for the renal effects of ANP in man.     

Exploring the pharmacology of the pro-convulsant effects of α2-adrenoceptor antagonists in mice

The effects of selective and specific ₂-adrenoceptor antagonists on electroshock seizure threshold in mice were investigated. Idazoxan, at low doses, efaroxan, RX811059 and RX821002 significantly lowered seizure threshold. The ₁-agonist St 587 and the -agonist isoprenaline were also pro-convulsant. On the other hand the ₂-agonists clonidine and UK 14,304 produced small increases in seizure threshold. Anticonvulsant effects were also produced by low doses of the noradrenaline uptake inhibitor desipramine. This compound increases levels of noradrenaline in the synaptic cleft which could subsequently act at post-synaptic ₂-adrenoceptors. The pro-convulsant action of ₂-adrenoceptor antagonists could be explained in terms of two mechanisms: a) blockade of endogenous noradrenaline which may normally exert a tonic anti-convulsant influence on seizure threshold, through post-synaptic ₂-receptors and/or b) increased activation of ₁- and -adrenoceptors by elevated synaptic noradrenaline levels following blockade of pre-synaptic ₂-adrenoceptors. Of the ₂-antagonists tested, idazoxan was unusual in that high doses were not pro-convulsant; this difference may be explained by ₁-adrenoceptor mediated actions or be related to its recently described affinity at a non-adrenoceptor site — a function for which is currently unknown.     

An Eastern Cooperative Oncology Group phase I trial of all-trans-retinoic acid and interferon-alpha: E2Y92

The Eastern Cooperative Oncology Group conducted a Phase I trial to determine the maximally tolerated doses of combination therapy with alpha interferon (IFN-) and all-trans-retinoic acid (tRA). Fifty patients with incurable malignancies received IFN- administered subcutaneously three times weekly, and tRA administered by mouth at bedtime. Doses were escalated between patient groups, starting at tRA dose level of 45 mg/m² and 3 million units of IFN-.Major, dose-limiting toxicities were attributable to either the tRA (rash, chelitis) or IFN (constitutional symptoms), and were observed only at tRA dose levels of 224 mg/m² and 291 mg/m², or 6 million units of IFN-. The maximally tolerated dose level of 172.5 mg/m² of tRA and 3 million units of IFN- was well-tolerated, with no grade 3 or 4 toxicities attributable to therapy. One patient at the third dose level (75 mg/m² of tRA and 3 million units of IFN-) developed acute hepatic and renal failure and a metabolic encephalopathy of unclear etiology.We conclude that tRA and IFN- may be safely administered together at the maximally tolerated dose of tRA as a single agent without unexpected side effects. The recommended doses of IFN- and tRA for Phase II trials are 3 million units of IFN- and 172.5 mg/m² of tRA.     

Plasma concentration of α-methyldopa and sulphate conjugate after oral administration of methyldopa and intravenous administration of methyldopa and methyldopa hydrochloride ethyl ester

Summary The plasma concentrations of free -methyldopa and methyldopa sulphate conjugate were measured in 7 hypertensive patients with normal renal function following -methyldopa (1 g) orally. Five of these patients subsequently received -methyldopa ethyl ester (250 mg) (methyldopate) intravenously and two further patients received 250 mg of -methyldopa intravenously. After oral administration a large amount of total plasma -methyldopa was present as sulphate conjugate. There were wide interindividual differences in the ratio of free: conjugated -methyldopa in plasma (ratio at 4 hours ranged from 3.73 – 0.83) suggesting that individual differences in the extent of sulphate conjugation may occur. There was no close correlation between the degree of conjugation and the fall in arterial pressure. At all time intervals examined, plasma concentrations were higher following intravenous -methyldopa than -methyldopate. The plasma concentration of -methyldopa (free and esterified) 60 minutes after i.v. -methyldopate was 1.7±0.3 µg/ml wile at the same time after the same dose of methyldopa by the same route the mean concentration was 5.9 µg/ml. Although small amounts of sulphate conjugate were detected after i.v. -methyldopate, insignificant quantities of conjugate were found after i.v. -methyldopa. The average fall in mean arterial pressure was 27 mm Hg following i.v. -methyldopa but only 2.7 mm Hg following -methyldopate. These results suggest that sulphate conjugation of -methyldopa occurs in the gastrointestinal tract during absorption. Hydrolysis of -methyldopa ethyl ester does not appear to be instantaneous and pharmacokinetic differences between the ester and free -methyldopa have been demonstrated.