Effects of chronic ethanol administration on free radical defence in rat myocardium.

Cellular protection against free radical reactions was measured in myocardium from ethanol-fed rats using ethanol administration in drinking water as a model of moderate alcohol intoxication. The activities of Cu,Zn-superoxide dismutase (SOD) and glutathione-S-transferase were higher in ethanol-fed rats than in controls, whereas Mn-SOD, catalase and glutathione peroxidase activities were not altered by ethanol treatment. Myocardial zinc was higher and selenium concentration lower in ethanol-fed rats than in controls. Ethanol consumption, which failed to modify the myocardial vitamin E level, did not result in increased lipid peroxidation, but decreased cytosolic and membraneous protein thiols.


The reduction of 6-N-hydroxylaminopurine to adenine by xanthine oxidase.

The genotoxic and mutagenic compound 6-N-hydroxylaminopurine (HAP) can be detoxified in vitro by enzymatic N-reduction to adenine. This reaction is catalysed by both rat and rabbit liver cytosolic fractions. The formation of adenine was monitored using HPLC. Subcellular distribution of the activity, kinetic parameters and the influence of various cofactors and inhibitors were determined. The N-reduction required NADH or hypoxanthine or xanthine and was strongly inhibited by allopurinol. These observations suggested that the N-reductase activity is due to xanthine oxidase (EC 1.2.3.2). Moreover, the involvement of xanthine oxidase is supported by the observation that purified cow milk xanthine oxidase also catalysed this reaction. The N-reduction of HAP was inhibited only weakly by oxygen. In addition, the formation of adenine is catalysed by either the oxidase or dehydrogenase form of xanthine oxidase. Thus, this reaction should be significant for the in vivo detoxification of HAP.


Phenothiazines inhibit acetylcholinesterase by concentration-dependent-type kinetics. A study with trifluoperazine and perphenazine.

The properties of perphenazine (PPZ) and trifluoperazine (TFP) as fluorescent dyes were exploited to calculate their critical micellar concentrations. The relative fluorescence quantum yield of the two amphiphiles was dependent on their concentration, abruptly decreasing above 30-40 microM PPZ and 20-30 microM TFP. Evidence is presented that this phenomenon is driven by the formation of non-fluorescent drug aggregates. The type of inhibition kinetics displayed by PPZ and TFP on human erythrocyte acetylcholinesterase (AChE) was also dependent on drug concentration, turning from non-competitive to a "mixed" inhibition type at concentrations at which PPZ and TFP were demonstrated to undergo micelle formation. Results support the notion that phenothiazines may interact with AChE both as monomers and micellar aggregates, producing different inhibitory effects.


High-affinity binding of warfarin, salicylate and diazepam to natural mutants of human serum albumin modified in the C-terminal end.

High-affinity binding of warfarin, salicylate and diazepam to four natural mutants of human serum albumin was studied by equilibrium dialysis at pH 7.4. The mutants Alb Milano Fast and Alb Vanves possess single amino acid substitutions close to the C-terminus, namely 573 Lys-->Glu and 574 Lys-->Asn, respectively. By contrast, Alb Catania and Alb Venezia are chain termination mutants in which several amino acids have been changed or deleted. Binding of warfarin to the variants was lower than binding to normal (wild-type) albumin (Alb A). The association constants were 73% (Alb Milano Fast, Alb Vanves and Alb Catania) or 67% (Alb Venezia) of that determined for Alb A. The results obtained with salicylate were more dependent on the type of mutation. The constants were either comparable to the normal value (Alb Catania) or reduced to 64% (Alb Milano Fast), 71% (Alb Vanves) or 43% (Alb Venezia) of that value. Diazepam binding to the variants was normal, except for binding to Alb Venezia in which case the association constant was reduced to 76% of that calculated for Alb A. The results are in accordance with the view that warfarin, salicylate and diazepam bind to three different high-affinity sites. It is proposed that the sites for warfarin and salicylate are situated rather close to each other in domain II, and that these high-affinity sites are relatively susceptible to conformational changes of the protein. By contrast, the primary diazepam site is placed closer to, or within, domain III of albumin and seems to be less affected by conformational changes in the protein molecule.


Regulation by growth hormone and glucocorticoid of testosterone metabolism in long-term cultures of hepatocytes from male and female rats.

The activities of 2-, 6 beta-, 7 alpha- and 16 alpha-testosterone hydroxylase and 5 alpha-testosterone reductase were measured in intact hepatocytes from male and female rats cultured for 8 days in a modified Waymouth medium supplemented with 0.1 or 1.0 microM dexamethasone with or without addition of 1 microgram/mL growth hormone. During culture of hepatocytes from female rats the activity of the male-specific 16 alpha-testosterone hydroxylase increased. This increase was significantly inhibited at day 8 by 1 microM dexamethasone as well as by growth hormone. Furthermore, in cultures of hepatocytes from male rats, the activity of the constitutive 16 alpha-testosterone hydroxylase was decreased by 1 microM dexamethasone as well as by growth hormone. The induction of 6 beta-testosterone hydroxylase by dexamethasone was suppressed by growth hormone in hepatocytes from both male and female rats, while the 7 alpha-testosterone hydroxylase activity was unaffected by culture time, hormone additions and gender. The decrease in female-specific 5 alpha-reductase activity with culture time in hepatocytes from female rats was significantly attenuated by growth hormone at 0.1 microM dexamethasone. The effects of growth hormone on testosterone hydroxylase activities in hepatocyte cultures from male and female rats are in accordance with the concept of growth hormone as a "feminization signal". The results suggest that the glucocorticoid-dependent expression of the male constitutive 16 alpha-hydroxylase requires periods of low levels of growth hormone.


Plasma membrane as a site of redox activation of daunomycin in intact human erythrocytes. Quantitative evaluation of the hydrogen peroxide produced by the membrane with respect to the cytosol.

The relative importance in human red blood cells of the plasma membrane as a site of redox activation of anthracyclines as compared to hemoglobin was evaluated by assaying the H2O2 produced upon exposure to daunomycin. The method of H2O2-dependent irreversible inhibition of catalase (EC 1.11.1.6) activity by 3-amino-1,2,4-triazole was applied to intact erythrocytes, as well as to isolated membranes with added purified catalase. The results obtained indicate a secondary role in daunomycin activation for the plasma membrane from a quantitative point of view, although membrane pathways can be more harmful than cytosolic pathways, especially towards extracellular targets, when the high efficiency of the cytosolic antioxidative defences and the external location of the membrane activation site are considered.


Interaction of berbamine compound E6 and calmodulin-dependent myosin light chain kinase.

The interaction of the berbamine compound E6 and calmodulin (CaM)-dependent myosin light chain kinase (MLCK) has been studied. The experimental results showed that the inhibition of MLCK activity was increased with increasing amounts of E6 and was overcome completely by the addition of excessive CaM. The stimulatory activity of MLCK induced by CaM was gradually inhibited by the increasing concentrations of compound E6, showing that the inhibition of MLCK activity by compound E6 was concentration dependent; and the Ki was 0.95 microM. Compound E6 diminished the fluorescence intensity of dansyl-labeled CaM and the intensity was increased gradually by the addition of different amounts of CaM. Compound E6 had no effect on the activity of MLCK fragments produced by limited trypsinization, and it is a novel and considerably potent calmodulin antagonist.


Multiple effects of staurosporine, a kinase inhibitor, on thymocyte functions. Comparison with the effect of tyrosine kinase inhibitors.

The effects of staurosporine, a protein kinase inhibitor, on the signal transduction and proliferation of thymocytes were studied. Signal transduction in response to Concanavalin A (Con A) as well as Concanavalin A (Con A)-induced augmentation of [3H]inositol incorporation into phospholipids were inhibited by staurosporine (> or = 10(-8) M). Staurosporine inhibited thymocyte proliferation in response to Con A in the presence or absence of the phorbol ester, phorbol myristate acetate (TPA) (10 nM). This inhibition was observed regardless of whether staurosporine was added together with Con A or 3 hr later. High concentrations of staurosporine (> 10(-8) M) inhibited thymocyte proliferation induced by the calcium ionophore A23187 and the phorbol ester TPA, whereas lower concentrations of the inhibitor (< or = 10(-8) M) enhanced thymidine incorporation in response to these activators. This dual effect of staurosporine was also observed in the presence of the staurosporine-related kinase inhibitor, K252a. In contrast, the tyrosine kinase inhibitor, tyrphostin AG490, inhibited the response to A23187 and TPA at all concentrations of the inhibitor and no augmentation was seen. Interleukin 2 (IL-2)-driven mitogenesis in IL-2-dependent cells was also inhibited by staurosporine. We suggest that the inhibition of thymocyte proliferation by staurosporine results from inhibition of both protein kinase C and tyrosine kinase: the augmentation of the response to A23187 and TPA results from inhibition of protein kinase C. Inhibition of signal transduction as well as inhibition of IL-2-driven mitogenesis result from inhibition of tyrosine kinase.


Inhibition of EGR-1 and NF-kappa B gene expression by dexamethasone during phorbol ester-induced human monocytic differentiation.

The treatment of human myeloid leukemia cells (HL-60, U-937, THP-1) with 12-O-tetradecanoylphorbol-13-acetate (TPA) is associated with growth arrest and appearance of a differentiated monocytic phenotype. While previous studies have reported that the glucocorticoid dexamethasone blocks phenotypic characteristics of monocytic differentiation, we demonstrated in the present work that dexamethasone delays the effects of TPA on the loss of U-937 cell proliferation. We also demonstrated that this glucocorticoid inhibits TPA-induced increases in expression of the EGR-1 early response gene. The results of nuclear run-on assays and half-life experiments indicated that this effect of dexamethasone is regulated at the post-transcriptional level. Similar studies were performed for the NF-kappa B gene. While TPA treatment was associated with transient increases in NF-kappa B mRNA levels, this induction was blocked by dexamethasone. In contrast, dexamethasone had no significant effect on the activation of pre-existing NF-kappa B protein as determined in DNA-binding assays. Taken together, these findings suggest that the activated glucocorticoid receptor inhibits signaling pathways which include expression of the EGR-1 and NF-kappa B genes and that such effects may contribute to a block in TPA-induced monocytic differentiation.


Effects of chemical modification of nitrobenzylthioinosine on its binding to high-affinity membrane binding sites and inhibition of nucleoside transport.

Nitrobenzylthioinosine (NBTI) was systematically modified by attachment of substituents at the 2-, 5'-, 3'- and 2'-positions in order to assess the importance of these positions in the binding of NBTI to high-affinity membrane binding sites (Kd < or = 1 nM) and the inhibition of NBTI-sensitive, equilibrative nucleoside transport by mammalian cells. We determined the effect of the derivatives on the equilibrium binding of 1 nM [3H]NBTI to human erythrocytes and mouse P388 leukemia cells and on the inhibition of zero-trans influx of formycin B in P388 cells and equilibrium exchange of uridine in human erythrocytes. Placement of substituent groups at the 5'-position of NBTI had relatively little effect on its binding to high-affinity binding sites or its inhibition of nucleoside transport, regardless of the size of the substituent group (up to about 1000 kDa). All substituents at the 2-position considerably reduced the affinity of NBTI to membrane binding sites and its potency as an inhibitor of nucleoside transport, but some substituent groups reduced the affinity of binding more than the inhibition of nucleoside transport. The effect of the 2-substituents was not directly related to their size. Attachment of a succinate at the 3'- or 5'-position also reduced to a greater extent the binding of NBTI than its inhibition of nucleoside transport, which was relatively little affected. Attachment of succinates at both the 3' and 5'-positions almost completely abolished both binding to high-affinity sites and inhibition of nucleoside transport. Both functions of NBTI were abolished completely by the simultaneous blockage of the 2'- and 3'-positions. None of the NBTI derivatives significantly inhibited NBTI-resistant equilibrative formycin B transport in P388 and Novikoff rat hepatoma cells at concentrations of < or = 1 microM.


Inhibitory effect of an intracellular glutathione on delta 12-prostaglandin J2-induced protein syntheses in porcine aortic endothelial cells.

Delta 12-Prostaglandin (PG) J2 caused porcine aortic endothelial cells to synthesize a 31,000-dalton heme oxygenase and a 67,000-dalton protein (p67). Treatment of the cells with buthionine sulfoximine (BSO), an inhibitor of glutathione (GSH) synthesis, depleted intracellular GSH, and enhanced the induction of heme oxygenase and p67 syntheses by delta 12-PGJ2. In contrast, treatment with GSH increased the intracellular GSH level and reduced the induction. There was a reciprocal relationship between the level of intracellular GSH, and that of the induction of heme oxygenase and p67 syntheses by delta 12-PGJ2. An increase in the intracellular GSH level caused an increase in the ethyl acetate-unextractable form of delta 12-PGJ2 in the cytosol, but suppressed the accumulation of delta 12-PGJ2 in the nuclei. Furthermore, GSH strongly inhibited the in vitro binding of delta 12-PGJ2 to isolated nuclei, which is N-ethylmaleimide sensitive. Moreover, the induction of heme oxygenase and p67 syntheses by the thiol-reactive agents arsenite and diethylmaleate was also inhibited by GSH treatment and enhanced by BSO treatment. These results demonstrate that intracellular GSH suppresses delta 12-PGJ2-induced heme oxygenase and p67 syntheses by inhibiting the binding of delta 12-PGJ2 to nuclei.


Contributory role of lung pleura to release of anaphylactic mediators from guinea pig lung in response to ovalbumin or A23187.

Previous findings revealed greater contractile responses of guinea pig lung pleural surface strips to antigen or A23187 challenge than denuded lung parenchymal strips (lung strip devoid of any pleura). Moreover, we have identified a high density of mast cells distributed throughout the lung pleura. The present study examined mediators released from guinea pig lung pleural surface and denuded lung parenchyma fragments in response to immunologic challenge with ovalbumin (OA) or non-immunologic challenge with the ionophore A23187. Histamine levels were measured radioenzymatically; leukotrienes (LTs), prostaglandins (PGs) and thromboxane B2 (TXB2), a stable metabolite of thromboxane A2 (TXA2), were quantitated using an enzyme immunoassay. Histamine release reached a maximal level 3-5 min after OA challenge, whereas A23187-induced histamine release increased gradually in a time-dependent manner. Similar kinetics were observed in the release of LTs, PGs and TXA2. Pleural surface released a substantially (P < 0.05) greater amount of histamine to both challenges than denuded parenchyma. Moreover, histamine content in pleural surface was significantly (P < 0.05) higher than in denuded parenchyma. Pleural surface also released considerably (P < 0.05) more LTB4, LTC4, and LTE4 in response to OA and A23187 than denuded parenchyma. In contrast, pleural surface and denuded parenchyma released equivalent amounts of PGD2, PGE2, PGF2 alpha, and TXA2 in response to both challenges. The rank order of leukotriene release was LTC4 > LTE4 > LTB4, whereas that of prostanoid release was TXA2 >> PGD2 > or = PGF2 alpha >> PGE2. We conclude that pleural surface is the major source of histamine and leukotrienes released from guinea pig lung in vitro in response to OA and A23187, whereas both pleural surface and denuded parenchyma participate to the same extent in prostaglandin and TXA2 production after such challenges.


Dihydrolipoic acid--a universal antioxidant both in the membrane and in the aqueous phase. Reduction of peroxyl, ascorbyl and chromanoxyl radicals.

Thioctic (lipoic) acid is used as a therapeutic agent in a variety of diseases in which enhanced free radical peroxidation of membrane phospholipids has been shown to be a characteristic feature. It was suggested that the antioxidant properties of thioctic acid and its reduced form, dihydrolipoic acid, are at least in part responsible for the therapeutic potential. The reported results on the antioxidant efficiency of thioctic and dihydrolipoic acids obtained in oxidation models with complex multicomponent initiation systems are controversial. In the present work we used relatively simple oxidation systems to study the antioxidant effects of dihydrolipoic and thioctic acids based on their interactions with: (1) peroxyl radicals which are essential for the initiation of lipid peroxidation, (2) chromanoxyl radicals of vitamin E, and (3) ascorbyl radicals of vitamin C, the two major lipid- and water-soluble antioxidants, respectively. We demonstrated that: (1) dihydrolipoic acid (but not thioctic acid) was an efficient direct scavenger of peroxyl radicals generated in the aqueous phase by the water-soluble azoinitiator 2,2'-azobis(2-amidinopropane)-dihydrochloride, and in liposomes or in microsomal membranes by the lipid-soluble azoinitiator 2,2'-azobis(2,4-dimethylvaleronitrile); (2) both dihydrolipoic acid and thioctic acid did not interact directly with chromanoxyl radicals of vitamin E (or its synthetic homologues) generated in liposomes or in the membranes by three different ways: UV-irradiation, peroxyl radicals of 2,2'-azobis(2,4-dimethylvaleronitrile), or peroxyl radicals of linolenic acid formed by the lipoxygenase-catalyzed oxidation; and (3) dihydrolipoic acid (but not thioctic acid) reduced ascorbyl radicals (and dehydroascorbate) generated in the course of ascorbate oxidation by chromanoxyl radicals. This interaction resulted in ascorbate-mediated dihydrolipoic acid-dependent reduction of the vitamin E chromanoxyl radicals, i.e. vitamin E recycling. We conclude that dihydrolipoic acid may act as a strong direct chain-breaking antioxidant and may enhance the antioxidant potency of other antioxidants (ascorbate and vitamin E) in both the aqueous and the hydrophobic membraneous phases.


Induction of a pleiotropic response by phenobarbital and related compounds. Response in various inbred strains of rats, response in various species and the induction of aldehyde dehydrogenase in Copenhagen rats.

The ability of phenobarbital (PB) to induce a "pleiotropic response" which includes both cytochromes P450 (CYP) as well as other drug-metabolizing enzymes was investigated in mice, rabbits, hamsters, and various inbred strains of rats. PB induced similar drug-metabolizing enzymes (CYP2B, CYP3A, and epoxide hydrolase) in rats, mice, rabbits and hamsters. PB and two structural analogues (ethylphenylhydantoin and barbital) induced a variety of drug-metabolizing enzymes (CYP2B, CYP3A, CYP2A, epoxide hydrolase) in a series of inbred strains of rats. In contrast, levels of aldehyde dehydrogenase (ALDH) (propionaldehyde, NAD+) which were expressed constitutively in all strains of rats were induced by PB in only two of the eight strains (ACI, Copenhagen). Further investigations of ALDH induction by structurally diverse compounds in Copenhagen rats demonstrated a strong correlation between the induction of ALDH and other elements of the pleiotropic response (CYP2B, CYP3A, epoxide hydrolase). These results imply that induction of ALDH (propionaldehyde, NAD+) is associated with the PB pleiotropic response in Copenhagen rats.


Nonenzymic glycation of albumin by acyl glucuronides in vitro. Comparison of reactions with reducing sugars.

Acyl glucuronides are ubiquitous metabolites formed from acidic xenobiotics and endogenous compounds, such as bilirubin. Previous studies indicated that the covalent binding of acyl glucuronides to proteins occurs via an imine intermediate in a manner analogous to the glycation of proteins via reducing sugars. When glucuronic acid was incubated in solution with albumin, it formed 10 and 4 times more fluorescent, Maillard reaction products with albumin after 25 days than did glucose or fructose, respectively. However, radiolabeled glucuronic acid exhibited less covalent binding to albumin than either glucose or fructose. Circular dichroism measurements indicate that glucuronic acid is about 0.02% open chain form with exposure of the reactive aldehyde, whereas fructose and glucose have 2 and 0.0026% present in solution as the open chain; thus, differences in reactivity of the reducing sugars were not correlated with exposure of the free aldehyde. Methyl glucuronate formed little fluorescent product with albumin, suggesting that the C-6 carboxylate of glucuronic acid may facilitate the reactions after covalent binding that lead to the formation of fluorescent products. When acyl glucuronide metabolites of two previously marketed acidic drugs, zomepirac and suprofen, were incubated with albumin at a concentration of 2.5 mM, more fluorescent product was formed than by 500 mM glucose. Reversible binding of the acyl glucuronides to albumin was 60-90%, but almost zero for the free reducing sugars, which indicates that reversible binding may explain the enhanced reactivity of the acyl glucuronides in forming fluorescent products with albumin. These results indicate that acyl glucuronides are reactive metabolites that may cause significant glycation of proteins with glucuronic acid in vivo.


Effects of interleukin 1 alpha on the activities and gene expressions of the cytochrome P450IID subfamily.

The mechanism by which recombinant human interleukin 1 alpha (rhIL-1 alpha) inhibits the activities of drug-metabolizing enzymes of rat liver microsomes, especially debrisoquine monooxygenase and bufuralol monooxygenase (both cytochrome P450IID supported reactions), as well as other enzymes, was investigated by injecting IL-1 alpha into rats. rhIL-1 alpha suppressed the activities of various P450-linked monooxygenase systems such as aminopyrine N-demethylase, benzphetamine N-demethylase, and 7-ethoxycoumarin O-deethylase. It also suppressed the activities of debrisoquine monooxygenase and bufuralol monooxygenase. On the other hand, IL-1 alpha had little effect on the activity of p-nitroanisole N-demethylase. The suppression of debrisoquine monooxygenase and bufuralol monooxygenase activities was caused by a decrease in the amounts of immunoreactive P450IID protein and its mRNA. The reduction rates in the level of immunoreactive P450IID protein and its mRNA were comparable. These results suggest that at the mRNA level, the enzymatic activities of debrisoquine monooxygenase and bufuralol monooxygenase are down-regulated by IL-1 alpha.


Effect of lysosomotropic agents on the taurocholate-stimulated biliary excretion of horseradish peroxidase.

The effects of the lysosomotropic agents chloroquine and leupeptin on the taurocholate-stimulated biliary excretion of horseradish peroxidase (HRP) was studied in bile fistula rats. HRP (0.5 mg/100 g body wt) was injected into the portal vein during taurocholate (0.4 mumol/min/100 g body wt) or saline infusion. HRP appeared in bile showing both an early (approx. 5 min) and a late (approx. 25 min) excretion peak. The late peak, which represented about 95% of the total HRP excreted, is due to transcellular vesicular transport. The early peak is mainly due to paracellular leakage although a rapid vesicular transport also contributes. Taurocholate infusion significantly increased the biliary output of HRP (both peaks) and of the endogenous lysosomal enzyme acid phosphatase. Pretreatment with chloroquine or leupeptin inhibited the taurocholate-stimulated late excretion of HRP into bile, without affecting its early excretion. The lysosomotropic agents did not affect the biliary excretion of bile salts but significantly inhibited the taurocholate-stimulated biliary excretion of acid phosphatase. The results are consistent with a role of lysosomes in the taurocholate-stimulated major transcellular vesicular transport of HRP into bile.


[p26--a calcium binding protein from photoreceptor cells in the bovine retina: primary structure and expression in E. coli].

The primary structure of the bovine retinal calcium binding protein P26 has been determined by the parallel analysis of the protein and the corresponding cDNA. This protein is identical to recovering and shares 59% homology with visinin, a cone specific calcium binding protein from chicken retina. P26 was expressed in E. coli as a fusion protein and, after purification by affinity chromatography on IgG-Sepharose 6, cleaved off with enteropeptidase.


[Sequence-specific modification of nucleic acids by oligonucleotide derivative containing alkylating groups in the C-5-position of deoxyuridine].

A new type of alkylating derivatives of oligonucleotides with 4(N-methyl-N-2-chloroethylamino)benzyl (RCl) group at C-5 of deoxyuridine with a high extent of the target modification was prepared. The synthesized reagents d(ULNHRClCCACTT), where L = CH2 (Ia), CH2OCH2CH2 (Ib) and CH2NHCOCH2CH2 (Ic), proved to effectively (80-90%) modify the oligonucleotide d(TAAGTGGAGTTTGGC). The reagents (Ia) and (Ib) alkylate G6, G7 and G9 positions, while the reagent (Ic) modifies predominantly G9.


[Synthesis of an artificial gene coding for thymosin alpha1 and its expression in Escherichia coli as a hybrid with human tumor necrosis factor].

Chemical-enzymatic synthesis and cloning in Escherichia coli of an artificial gene encoding the immunoactive peptide thymosin alpha 1 have been carried out. Recombinant plasmids were constructed which contain fusion genes coding for hybrids of human tumour necrosis factor (TNF) and thymosin alpha 1 as N- or C-terminal part of the hybrid protein. In the C-terminal hybrid protein, TNF and thymosin alpha 1 are linked through a methionine residue, thus allowing for thymosin alpha 1 to be cleaved off the rest of the hybrid protein with cyanogen bromide. In case of the N-terminal hybrid protein, the linker between the thymosin alpha 1 and TNF sequences is the acid-labile dipeptide Asp-Pro. Expression of the hybrid genes in E. coli and properties of the recombinant proteins were studied. The N-terminal hybrid protein was secreted into periplasmic space, in contrast with the C-terminal hybrid protein, which formed insoluble aggregates inside bacterial cells. Procedures for the isolation of both hybrid proteins were developed. The N-terminal hybrid protein displayed full biological activity in the cytotoxic assay on the mouse fibroblast L-929 whereas the C-terminal hybrid protein proved to be much less active. Treatment of the hybrid protein TNF-thymosin alpha 1 with cyanogen bromide lead to a mixture of two polypeptides, from which thymosin alpha 1 was purified to homogeneity by simple chromatographic procedures.


[Expression of a synthetic gene for human interleukin-4 in E. coli cells. Preparation of a biologically active protein].

Expression E. coli plasmid were constructed in which the human interleukin-4 (hIL4) synthetic gene is controlled by tac promoter. The expression level of the gene depends on the distance between RBS and the initial codon ATG, with the maximal production in case of the nine base pair distance. The recombinant protein, accumulated in the inclusion bodies, was solubilized, renaturated, and purified to homogeneous, biologically active preparation, the yield being 2 mg/g wet cells.


[Synthesis of protected fragments of the capsular polysaccharide of Streptococcus pneumoniae, type 8].

Oxidation of acetates of allyl and 2-(benzyloxycarbonylamino)ethyl beta-cellobiosides (with OH-4' and OH-6' unprotected) with the Jones reagent followed by esterification (with diazomethane or phenyldiazomethane) gave corresponding uronates with OH-4' unsubstituted. Condensation of these glycosyl acceptors and benzylated derivatives of D-galactose or 4-O-(alpha-D-glucopyranosyl)-D-galactose led to the protected tri- and tetrasaccharide fragments of the capsular polysaccharide from Streptococcus pneumoniae type 8.


[Synthesis of oligodeoxyribonucleotides containing 5-fluoro-2'-deoxyribocytidine].

A synthesis of the phosphoroamidite derivative of 5-fluoro-2'-deoxycytidine which allows one to introduce the modified nucleoside residue into the 5'-position of the oligodeoxyribonucleotide by the standard solid phase phosphoroamidite method, has been carried out. Oligonucleotides with 5-fluoro-2'-deoxycytidine residues in various positions of the DNA strand are obtained by the combination of chemical and enzymatic syntheses.


[Folding of peptide chains during formation of the spatial structure of protein molecules is determined by the code of amino acid codon roots].

Basing on the analysis of a large number of protein sequences (Cserzo M., Simon I., 1989), the structure of the amino acid nearest neighbour pair whose occurrence has a maximal positive deviation from the mean statistical value, is shown to correspond in most cases to the code of the amino acid codon roots. It reveals particularly amino acid pairs in n and n+5 positions of polypeptide chains. Amino acids belonging to A/U family contribute mostly to the folding of peptide chains.


[Secretion of recombinant human epidermal growth factor into the periplasm in Escherichia coli cells].

Secretion vectors were constructed in which a synthetic gene of human epidermal growth factor (hEGF) joined with a gene coding for the leader peptide to one of the E. coli outer membrane major proteins (OmpF) is controlled by tac promoter. The increase of the hEGF yield was achieved by the multiplication of the gene copies. The hEGF in bacterial cells was secreted into periplasm. The recombinant protein was isolated by means of reverse phase chromatography as almost homogenous preparation (greater than 98%), the yield being 7 mg/l bacterial culture. The sequence of twenty-five N-terminal amino acid residues of the isolated hEGF coincided with that of the natural protein. The preparation proved to be biologically active.


[Structure of macrocyclic K+, Rb+-complexon of meso-valinomycin monohydrate, cyclo[-(D-Val-Hyi-Val-D-Hyi)3-].H2O, in a crystalline complex with dioxane by x-ray structural data].

The crystal structure of a valinomycin analogue, cyclo[-(D-Val-Hyi-Val-D-Hyi)3-]x(C60H102N6O18) crystallized with dioxane and water molecules, has been solved by X-ray direct methods. The conformation found is analogous to one established for free meso-valinomycin crystallized from other organic solvents. It is characterized by a centrosymmetric bracelet form, stabilized by six intramolecular 4----1 type hydrogen bonds between amide N-H and C = O groups. One water molecule is fixed asymmetrically by hydrogen bonds in the internal negatively charged cavity of the complexon. The meso-valinomycin molecule "bracelets" in the crystal form stacks alternatively with dioxane molecules.


[Substituted 6-aminonaphthalene-1-sulfamides as fluorogenic indicators groups for synthetic proteinase substrates].

Alkyl substituted 6-aminonaphthalene-1-sulphamides (ANSA), hydrobromides of substituted 6-(N alpha-benzyloxycarbonyl-L-arginyl)aminonaphthalene-1-sulphamides (Z-Arg-ANSA) and hydrobromides of 6-(benzyloxycarbonylglycylglycyl-L- arginyl)aminonaphthalene-1-sulphamides (Z-Gly-Gly-Arg-ANSA) are synthesized and their absorption and emission spectra measured. ANSA have an emission band at 470-480 nm, comparable or exceeding in intensity that of compounds used as fluorogenic leaving groups in peptide cleavage reactions. The bands of Z-Arg-ANSA and Z-Gly-Gly-Arg-ANSA are shifted to the short-wave side and do not overlap with ANSA's emission band. Reactions of Z-Arg-ANSA and Z-Gly-Gly-Arg-ANSA with trypsin were studied. The kinetic parameters (kcat and Km) of the reaction of Z-Arg-ANSA were found to depend on the nature and the number of substituents in the sulphamide. In the case of Z-Gly-Gly-Arg-ANSA, this dependence is negligible and kcat/Km exceeds by over ten times this parameter of Z-Arg-ANSA. ANSA can apparently be used in the synthesis of fluorogenic substrates of proteases.


[Synthesis of phosphono analogs of a phospholipid platelet activating factor].

Structural analogues of phospholipidic platelet activating factor, (2-acetoxy-3-octadecyloxy)propyl-1-phosphonocholine and (2-methoxy-3-octadecyloxy)propyl-1-phosphonocholine, were synthesized. High efficiency of the polymer-bound dibenzo-18-crown-6-ether as the O-alkylation catalyst was demonstrated. Reaction of allyl-octadecyl ether with methanol and iodine in the presence of zinc oxide was shown to give a mixture of 1-iodo-2-methoxy- and 1-methoxy-2-iodoprop-3-yl ethers of octadecanol in the 3:1 ratio.


[Study of the amino acid sequence of latroinsectotoxin from black widow spider venom].

The N-terminal amino acid sequence of alpha-latroinsectotoxin from the venom of Latrodectus mactans tredecimguttatus was determined. Then the toxin was digested by trypsin and total or partial amino acid sequences of twenty-six tryptic peptides were established. This resulted in the structural information needed for the construction of probes followed by the cloning of the alpha-latroinsectotoxin structural gene.


Low levels of somatomedin C in patients with the fibromyalgia syndrome. A possible link between sleep and muscle pain.

Fibromyalgia is a common syndrome of musculoskeletal pain and fatigue. Lacking distinctive tissue or laboratory correlations, it has often been considered a form of "psychogenic rheumatism." In the present study, the notion that the stage-4 sleep anomaly typically seen in the fibromyalgia syndrome may disrupt growth hormone secretion was tested. Because growth hormone has a very short half-life, serum levels of somatomedin C were measured; somatomedin C is the major mediator of growth hormone's anabolic actions and is a prerequisite for normal muscle homeostasis. Serum levels of somatomedin C were measured in 70 female fibromyalgia patients and 55 healthy controls, using a peptide-specific radioimmunoassay. Significantly lower levels of somatomedin C were observed in the fibromyalgia patients compared with controls (mean +/- SD 124.7 +/- 47 ng/ml versus 175.2 +/- 60 ng/ml; P = 0.000001). These results could not be explained by concomitant therapy or by weight, and in a subset of 21 patients in whom this was investigated, there was no correlation with various indices of disease activity. These findings indicate that there is a distinctive disruption of the growth hormone-somatomedin C neuroendocrine axis in a majority of fibromyalgia patients. It is hypothesized that this abnormality may explain the link between disturbed sleep and predisposition to muscle pain.


High-dose immunoglobulin therapy as an immunomodulatory treatment of rheumatoid arthritis.

To investigate the efficacy of high-dose intravenous immunoglobulin (IVIg) in the treatment of refractory rheumatoid arthritis (RA). Ten patients with active, severe RA that was unresponsive to first- and second-line agents were administered IVIg monthly, for 6 months. Following IVIg treatment, there was significant improvement in both subjective and objective parameters of disease activity in all 9 patients who completed the protocol. This improvement was noted to occur as early as after the second infusion of IVIg. After discontinuation of the treatment, all patients had a relapse of the disease within a few weeks. Since the reduction in clinical activity paralleled a decrease in the CD4+CDw29+:CD4+CD45RA+ cell ratio, some of the therapeutic benefits associated with IVIg may be due to a direct influence on the CD4+CD45RA+ subset. Although the possibility of carrying out further controlled studies on a larger scale is limited by the high cost of the treatment, IVIg appears to be an effective therapy for refractory RA.


Treatment of systemic sclerosis with recombinant interferon-gamma. A phase I/II clinical trial.

A phase I/II trial to examine the safety and efficacy of interferon-gamma (IFN gamma) therapy for patients with systemic sclerosis (SSc). An 18-week open-label study was performed. Eighteen patients with rapidly progressive SSc were enrolled, 14 of whom completed at least 16 weeks of the study. These 14 patients had a mean age of 40 years and had been diagnosed as having SSc an average of 10.1 months prior to study entry. Recombinant IFN gamma was injected intramuscularly 3 times weekly for 18 weeks. Six patients received a 0.1 mg/m2 dose, while 8 patients received a 0.5 mg/m2 dose. Patients who completed the 18-week trial were offered maintenance therapy at a dose of up to 0.5 mg/m2. The effects of IFN gamma on skin involvement were assessed by 2 methods: 1) evaluation of skin thickness, by scoring 15 zones according to a 0 (normal skin) to 3 (hidebound skin) scale; and 2) determination of the total body surface area involved, by using 2-dimensional body diagrams to indicate areas affected, and then having a second, "blinded," assessor calculate the area score with a planimeter. The mean skin thickness score decreased from a baseline of 25.9 to 19.1 (P < 0.03), and the mean area scores declined from 33.1 to 19.6 (P < 0.02) after 18 weeks of IFN gamma treatment. Ten patients had a > 25% decrease in area score. Five patients had a > or = 70% decrease in area score, and 3 of them have not experienced disease recurrence for 6 to 17 months after discontinuation of IFN gamma. Five patients withdrew before the study ended. Three of these patients developed renal crisis, which may reflect the severity of the SSc in the study group, although an adverse effect of IFN gamma in SSc cannot be excluded. IFN gamma was associated with a beneficial effect on the skin involvement in most of this series of patients with rapidly progressive SSc. A placebo-controlled study will be necessary to confirm these results.


Methotrexate treatment of recalcitrant childhood dermatomyositis.

To review the clinical course of 16 children with recalcitrant dermatomyositis (DM), who were treated with oral methotrexate (MTX) in addition to prednisone. Sixteen patients with recalcitrant DM who were treated with MTX in addition to prednisone were followed between 1984 and 1990. The patients' clinical responses to treatment, including alterations in muscle strength and muscle enzyme levels, changes in prednisone dosage, and development of toxicity or complications were reviewed retrospectively. All 12 patients who received MTX for at least 8 months regained normal muscle strength. In 11 of the 12, the prednisone dosage could eventually be tapered to < or = 5 mg/day. Complications during MTX treatment required discontinuation of MTX in 5 patients, and were unrelated to the cumulative dose of the drug. Active disease recurred in 5 patients in whom MTX had been discontinued after apparent clinical remission had been achieved. MTX, in combination with prednisone, is a useful adjunct in the treatment of recalcitrant childhood DM. However, recurrence of disease after withdrawal of MTX suggests that the drug may have a suppressive, rather than a remittive, effect.


Effects of interleukin-6 on the metabolism of connective tissue components in rheumatoid synovial fibroblasts.

High levels of interleukin-6 (IL-6) have been found in the synovial fluid of patients with rheumatoid arthritis (RA). We undertook the present study to investigate the role of IL-6 in this disease. We examined the effects of IL-6, in comparison with IL-1, on the biosynthesis of extracellular matrix macromolecules and of matrix-degrading proteinases in rheumatoid synovial fibroblasts. In rheumatoid synovial fibroblasts, IL-6 by itself enhanced the production of plasminogen activator, its inhibitor, and tissue inhibitor of metalloproteinases, whereas it did not modulate the biosynthesis of precursor of matrix metalloproteinase 1 (proMMP-1) (tissue collagenase), proMMP-3 (stromelysin), or connective tissue components. However, IL-1-induced production of proMMP-1 and proMMP-3 was preferentially augmented by IL-6. These results suggest that in RA, IL-6 may participate along with IL-1 in fine tuning of the catabolism of connective tissue components, by modulating the balance between connective tissue-degrading enzymes and their inhibitors.


Serum autoantibody to the nucleolar antigen PM-Scl. Clinical and immunogenetic associations.

The inflammatory myopathies are characterized by distinctive autoantibodies that are associated with certain clinical features and immunogenetic patterns. Anti-PM-Scl is one such antibody and is found in pure myositis, myositis in overlap, and systemic sclerosis (SSc). Our purpose was to describe the clinical and immunogenetic associations of the anti-PM-Scl antibody. Serum samples from 617 patients with various connective tissue diseases were screened for anti-PM-Scl antibody by indirect immunofluorescence and Ouchterlony double immunodiffusion. Patients with anti-PM-Scl were serologically typed for HLA-DR and DQ, and the genes encoding DQ alpha and DQ beta were characterized by hybridization of sequence-specific oligonucleotide to amplified genomic DNA. Twenty-three patients (4%) had serum anti-PM-Scl. Sixteen had either pure myositis or myositis in overlap, 6 had SSc alone, and 1 had SSc and rheumatoid arthritis. Twenty of the antibody-positive patients had serologic HLA typing performed; 15 (75%) were HLA-DR3 positive, and 17 (85%) expressed the DQw2 allele. None of the 5 DR3 negative patients shared a unique DR or DQ antigen with the DR3 positive patients, and further DNA analysis of 10 patients (4 of whom were DR3 negative) did not reveal any unique DQ alleles. Anti-PM-Scl identifies a subset of patients with myositis, SSc, or an overlap of the two disorders, and this antibody has a strong but not exclusive immunogenetic association with the HLA-DR3 antigen.


In vivo expression of stromelysin in synovium and cartilage of rabbits injected intraarticularly with interleukin-1 beta.

To examine the in vivo expression of the matrix metalloproteinase stromelysin in the synovium and articular cartilage of rabbits injected intraarticularly with recombinant human interleukin-1 beta (IL-1). The direct isolation of messenger RNA (mRNA) from articular cartilage without the prior isolation of chondrocytes is described. The in vivo expression of stromelysin was examined at the mRNA level by Northern blot analysis, and at the protein level by in situ immunolocalization and by enzyme-linked immunosorbent assay. In the synovium of IL-1-injected joints, stromelysin mRNA levels were highest at 4 hours and declined to background levels within 24 hours. In the cartilage of IL-1-injected joints, stromelysin mRNA was elevated at 4 hours and continued to increase until 8 hours, before declining. Stromelysin mRNA expression preceded a similar increase in stromelysin protein levels in both synovium and cartilage. Intraarticular injection of IL-1 induced the endogenous expression of stromelysin mRNA and protein in both synovium and cartilage. The kinetics of stromelysin expression correlated well with the accumulation of stromelysin and proteoglycan in synovial fluids. Therefore, the de novo synthesis of stromelysin in cartilage may have contributed to the loss of proteoglycan from that tissue.


Evidence for the presence of androgen receptors in the synovial tissue of rheumatoid arthritis patients and healthy controls.

To study the presence of androgen receptors in the synovial tissue of male and female patients with rheumatoid arthritis (RA) and matched healthy controls. Both site I (high affinity, low binding capacity) and site II (reduced affinity, higher binding capacity) androgen receptors were investigated in soluble and nuclear fractions of homogenized synovial samples, using the dextran-coated charcoal method. The finding of pure, high-affinity site I receptors in both fractions was considered indicative of androgen receptor positivity. In order to determine what type of synovial cell was positive for androgen receptors, cryosections of synovial tissues were immunostained with a specific monoclonal anti-androgen receptor antibody (MAb), using both immunofluorescence and immunoperoxidase techniques. Double immunostaining with this MAb and specific MAb directed toward different macrophage/granulocyte antigens was also performed. Remarkable differences were found between male and female controls: Most males were positive for androgen receptors, and most females were negative. The fetomolar content of androgen receptor in the nuclear fraction was fairly constant, but the soluble fraction showed a higher femtomolar concentration in female RA patients than in controls of either sex, as well as in male RA patients compared with female RA patients. The androgen receptor-positive cells in both RA and control synovial cryosections were found by immunostaining to be macrophage-like synoviocytes, and were also found to be HLA-DR positive. The immunosuppressive action exerted by androgens might, at least in part, be mediated through their interaction with macrophage-like synoviocytes functioning as antigen-processing and antigen-presenting cells in rheumatoid synovia.


Antibodies in rheumatoid synovial fluids bind to a restricted series of protein antigens in rheumatoid synovial tissue.

By searching the synovial fluid of patients with rheumatoid arthritis (RA) for antibodies that react to protein antigens in synovial tissue, we sought to identify putative antigens present in RA synovial tissue that might drive the pathologic immune response believed to be responsible for the joint inflammation. Synovial tissue was homogenized in sodium dodecyl sulfate polyacrylamide gel buffer, electrophoresed, and analyzed by immunoblotting. Antibodies from synovial fluids of patients with RA bound to several proteins in rheumatoid synovial tissues, including a series of low (27.5-, 29-, and 30-kd), middle (43- and 53-kd), and high (140-, 164-, and 182-kd) molecular weight proteins. Most of these antigens were also detected in normal synovial tissue, and the high molecular weight proteins were also present in normal dermal, muscle, and liver tissues. The low and middle molecular weight proteins were detected in some, but not all, of the other normal tissues and in Jurkat cell lysates. Antibodies to the low and high series of proteins were present in all rheumatoid synovial fluids tested, but were generally absent from synovial fluids from patients with other arthritic diseases. These results show that antibodies in synovial fluids consistently react to several proteins in RA and normal synovial tissues. These antigens are possibly the same antigens provoking the T cell response in RA; therefore, understanding the mechanism of the immune response against these proteins will likely lead to important insight into the etiology of RA.


Assessment of disease activity and impending flare in patients with systemic lupus erythematosus. Comparison of the use of complement split products and conventional measurements of complement.

To determine whether increased levels of the complement split products generated in the activation of the alternative or classical pathway accompany more severe disease activity in patients with systemic lupus erythematosus (SLE) and whether these measurements are useful in predicting flares of disease. Levels of Ba, Bb, SC5b-9, and C4d were measured in 380 plasma samples obtained from 86 SLE patients who were prospectively followed up for 15 months. In the 20 patients who had inactive disease at the initiation of the study, the mean values of all of the complement split products at entry were within the normal range. In the 47 patients with stable or moderate disease activity, levels of Ba were significantly increased, while the mean values for Bb, SC5b-9, and C4d did not differ significantly from those in patients with inactive disease. The mean entry value of each analyte was highest in the group of 19 patients who had the most severe disease activity at initial evaluation. Traditional measurements of complement, i.e., C3, C4, and CH50, followed similar trends, but did not discriminate between the 3 groups of patients as well as did measurements of the split products. Analysis of the disease course in the patients with inactive or stable/moderate disease revealed that an elevated level of C4d had the most sensitivity with regard to subsequent flare, while an elevated Bb level had the highest specificity and the greatest predictive value. These data suggest that elevated levels of complement split products, particularly products of alternative and terminal pathway activation, more accurately reflect disease activity than do conventional measurements of complement in SLE and may be useful in the prediction of impending disease flares.


Neuropathologic findings in multi-infarct dementia associated with anticardiolipin antibody. Evidence for endothelial injury as the primary event.

There are few reports describing histopathologic changes associated with the antiphospholipid antibody syndrome. We describe a patient with multi-infarct dementia and antiphospholipid antibody syndrome, in whom a brain biopsy was performed. Biopsy material from the left frontal cortex, including meninges, cortex, and underlying subcortical white matter, was investigated. Microscopic examination and special staining were performed. Microscopic examination showed lumenal occlusion by thrombi, and marked endothelial hyperplasia of small meningeal and cortical arterioles. These findings suggest that the pathogenesis of this cerebral vasculopathy is noninflammatory and is associated with reactive endothelial hyperplasia and thrombosis of small arterioles.


Presence of small proteoglycan fragments in normal and arthritic human cartilage.

To characterize the small proteoglycans decorin and biglycan in normal human patellar cartilage and in cartilage from individuals with chronic polyarthritis. Cartilage extracts were chromatographed on DEAE-Trisacryl and further separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis before and after enzymatic degradation of the glycosaminoglycan chains. Decorin and biglycan were visualized after Western blotting, using core protein-specific polyclonal and monoclonal antibodies. Core protein fragments of both proteoglycans were observed even in normal cartilage. In the case of decorin they amounted to up to 15% of the immunoreactive material, and up to 5% of the core protein was glycosaminoglycan free. The quantity of decorin core protein was reduced in arthritic cartilage, but the core protein fragments represented up to 45% of the immunoreactive material. Different zones of cartilage differed in their content of the fragments. Evidence for an increased proportion of biglycan fragments was not obtained. Chronic polyarthritis leads to increased degradation of small proteoglycans. A considerable proportion of decorin fragments is retained in the tissue. These alterations may have a negative influence on the mechanical stability of tissue.


Effect of intraarticular glycosaminoglycan polysulfate treatment on patellofemoral pain syndrome. A prospective, randomized double-blind trial comparing glycosaminoglycan polysulfate with placebo and quadriceps muscle exercises.

To compare the effects of intraarticular (IA) injections of glycosaminoglycan polysulfate (GAGPS) plus basic conservative treatment with the effects of placebo injections plus conservative treatment and with the effects of conservative treatment alone in patients with chronic patellofemoral pain syndrome (PFPS). We treated 53 patients who presented with chronic PFPS in 1 knee, according to 1 of the 3 protocols, in a prospective, randomized, double-blind study. Basic conservative treatment consisted of a 6-week program of quadriceps muscle exercise, elimination of symptom-producing activities, and oral doses of nonsteroidal antiinflammatory drugs. Physiologic saline served as placebo for injection. During the 6-week treatment period, 5 injections were given 1 week apart. Along with measurements of quadriceps strength, standardized subjective, functional, and clinical assessments were performed at presentation, after 6 weeks of treatment, and after 6 months. Results at 6 months indicated that IA injections of GAGPS or saline did not result in significant improvement beyond the good results achieved by the basic conservative treatment alone. More than two-thirds of the patients in each group experienced complete recovery. Neither the GAGPS injections nor the physiologic saline injections are more effective than conservative therapy in the treatment of chronic PFPS. Restoration of normal quadriceps muscle function to the affected knee seems to be crucial in treating PFPS.


Appendicular and vertebral bone mass in ankylosing spondylitis. A comparison of plain radiographs with single- and dual-photon absorptiometry and with quantitative computed tomography.

We assessed the prevalence, severity, and anatomic distribution of ankylosing spondylitis (AS)-related osteopenia (OP). We studied 70 patients (60 males, 10 premenopausal females) with AS (according to the New York criteria) to determine the frequency of OP. Bone mass was measured by plain radiographs of the spine, by single-photon absorptiometry (SPA) of the distal and midshaft of the radius on the nondominant side, by dual-photon absorptiometry (DPA) of the lumbar spine (L2-L4), and by quantitative computed tomography (QCT) of the lumbar spine. SPA values for the radius mass were normal in males and in females, both at the distal and midshaft sites. In contrast, spine radiographs showed diminished density of the vertebral bodies in 69% of the males and 50% of the premenopausal females. Two male patients had had a vertebral compression fracture, and one female patient had had two. DPA values for the spine mass were significantly diminished in the male patients compared with the controls, but not in the female patients. Males with less severe AS also had the largest reduction in lumbar bone mineral content. In patients with more severe disease, lumbar bone mineral content was not statistically different from that in controls. QCT of the lumbar spine performed in 10 patients disclosed low density of the trabecular bone of the vertebral bodies, more so in those with more severe AS and syndesmophyte formation and/or apophyseal joint fusion, which contrasts with the normal values on DPA in these patients. Male patients with AS have axial osteopenia. In those who have very severe AS with new bone formation, DPA demonstrates normal values as a result of two opposite trends: central osteopenia (as assessed from QCT) and peripheral new-bone formation, which transforms vertebral bodies into long bones. This could modify the mechanical resistance of the spine and account for the propensity for anteroposterior transvertebral and transdiscal fractures after trauma in AS.


Signal transduction in Sjögren's syndrome T cells. Abnormalities associated with a newly described human A-type retrovirus.

To study the effects of a novel A-type retrovirus, detected in cocultures of lip biopsy specimens from Sjögren's syndrome (SS) patients and a human T cell line, on the infected T cells. Interleukin-2 (IL-2) and IL-6 secretion were measured by bioassay and enzyme-linked immunosorbent assay, respectively, in the infected and noninfected cell lines. Surface antigen expression was determined by flow cytometry, using monoclonal antibodies. Protein kinase C (PKC) activity was measured using an enzyme assay kit, and calcium mobilization was assessed with a fluorescent probe. Infected cells expressed less CD4 and IL-6 receptor, but more HLA-DR, compared with noninfected cells. Infected cells also produced less IL-2 and displayed reduced PKC activation and calcium mobilization. A similar defect in calcium mobilization was detected in T cells from SS patients. These data suggest a possible involvement of the newly described retrovirus in T cell abnormalities.


Quantitative immunohistologic criteria are superior to the lymphocytic focus score criterion for the diagnosis of Sjögren's syndrome.

The aim of the present study was to evaluate the diagnostic value of quantitative immunohistologic criteria for Sjögren's syndrome (SS) in labial salivary gland biopsies. Quantitative immunohistologic examination was performed on labial salivary gland biopsy samples from 80 healthy controls, 32 patients with primary SS, 14 patients with secondary SS, 5 with "probable" SS, 36 with keratoconjunctivitis sicca (KCS) with a lymphocytic focus score less than 1 on the lip biopsy, and 18 with rheumatoid arthritis (RA) without clinical evidence of SS. This is the first study to show that immunohistologic criteria for SS, based on the percentages of IgA-containing and IgG-containing plasma cells, are able to 1) confirm the diagnosis of SS in labial salivary glands of KCS patients in the absence of grade IV lymphocytic adenitis; and 2) distinguish between a grade IV focal lymphocytic adenitis in the labial salivary glands of SS patients and of RA patients without SS. Quantitative immunohistologic criteria were shown to be much more sensitive and disease specific than the widely accepted grade IV lymphocytic adenitis criterion, which corresponds to a lymphocytic focus score greater than 1, and these criteria should be included in the international diagnostic criteria for Sjögren's syndrome.


Elevated serum levels of soluble interleukin-2 receptor in patients with Wegener's granulomatosis. Association with disease activity.

To investigate whether soluble interleukin-2 receptor (sIL-2R), a marker of T cell activation, could be a useful marker of disease activity in Wegener's granulomatosis (WG). Soluble IL-2R levels were determined by enzyme-linked immunosorbent assay. WG disease activity in 102 patients was assessed according to clinical features and levels of classic antineutrophil cytoplasmic antibody (c-ANCA) and C-reactive protein (CRP). Soluble IL-2R levels were higher in patients with generalized and active disease than in those with limited and inactive disease. In 25 patients with complete clinical remission, sIL-2R levels were significantly elevated, although levels of CRP and c-ANCA were normal. Eight of these 25 patients had disease relapses within 6 months. Levels of sIL-2R were significantly higher in patients who had relapses than in those who did not. Patients with clinically active WG but low c-ANCA or CRP levels had elevated levels of sIL-2R. Levels of sIL-2R correlate with disease activity in patients with WG, and may indicate imminent relapse.


Tryptophan metabolism via the kynurenine pathway in patients with the eosinophilia-myalgia syndrome.

To investigate the metabolism of L-tryptophan (LT) via the kynurenine pathway in patients with the eosinophilia-myalgia syndrome (EMS). Measurement of LT, L-kynurenine, and quinolinic acid in plasma and cerebrospinal fluid (CSF) from subjects with EMS, from asymptomatic users of LT, and from normal subjects. Plasma LT concentrations were lower in untreated EMS patients (n = 5) than in corticosteroid-treated EMS patients (n = 5; P less than 0.05) and in asymptomatic users of LT (n = 5; P less than 0.05). Untreated EMS patients, who had discontinued LT weeks to months prior to study, had significantly higher plasma levels of L-kynurenine and quinolinic acid than did corticosteroid-treated EMS patients (P less than 0.05), normal subjects (P less than 0.02), and asymptomatic users of LT (P less than 0.05). EMS patients also had significantly elevated levels of L-kynurenine (P less than 0.05) and quinolinic acid (P less than 0.001) in CSF compared with normal subjects. After a 1-gm oral dose of LT, untreated EMS patients (n = 4) showed lower peak levels of LT and accentuated synthesis of L-kynurenine and quinolinic acid, compared with these values in corticosteroid-treated EMS patients (n = 2), who responded like normal subjects (n = 5). These data demonstrate that during the active phase of EMS, LT metabolism via the kynurenine pathway was accentuated, probably secondary to induction of the enzyme indoleamine-2,3-dioxygenase. Ingestion of large amounts of LT (median daily dose 1.5 gm) resulted in high concentrations of kynurenine-pathway metabolites in blood and extrahepatic tissues, which was accentuated in EMS patients and which may have played a significant role in the pathogenesis of the disease.


IX 207-887 in rheumatoid arthritis. A double-blind placebo-controlled study.

To determine the efficacy and the safety of IX 207-887 treatment in rheumatoid arthritis. The IX compound [10-methoxy-4H-benzo(4,5)cyclohepta-(1,2-b)thiophene-4-yliden acetic acid] is effective in several animal models of rheumatoid arthritis and has a mechanism of action involving the inhibition of interleukin-1 release. A double-blind, controlled trial of 16 weeks' duration comparing placebo with IX at a daily dosage of 800 mg or 1,200 mg (20 patients/group) was conducted. Thirteen patients withdrew from the study, 3 because of lack of efficacy (all in the placebo group) and 10 because of side effects (1 in the placebo group [skin rash] and 9 in the IX groups [skin rash in 5, intestinal disturbances in 2, hepatitis in 1, meningitis in 1]). Intent-to-treat analysis showed a statistically significant difference in the variations of clinical and laboratory parameters between the 3 groups. Between-group comparisons showed an improvement in all these variables in the IX groups versus the placebo group. According to Paulus' criteria, 2 of the 20 placebo-treated patients (10%), 9 of the 20 IX 800 mg-treated patients (45%), and 11 of the 20 IX 1,200 mg-treated patients (55%) were considered responders (P = 0.008). The findings of this study suggest that the tolerability of IX is acceptable in rheumatoid arthritis patients, and that IX is an effective slow-acting drug for use in rheumatoid arthritis.


Effect of nebracetam on nicotinic and muscarinic acetylcholine receptors expressed in Xenopus oocyte by injecting exogenous mRNA.

Using voltage- and current-clamp methods the effects of nebracetam 4-aminomethyl-1-benzylpyrrolidine-2-one hemifumarate, WEB 1881 FU, CAS 118607-07-1), a new agent with nootropic property, on the nicotinic (nAChRs) and muscarinic acetylcholine receptors (mAChRs) were studied, which were expressed in Xenopus oocytes by injecting E. electricus mRNA and rat brain mRNA, respectively. Simultaneous application of nebracetam (0.03-2 mmol/l) with acetylcholine (ACh) (0.01-1 mmol/l) inhibited the ACh-responses of both nAChRs and mAChRs, whereas preapplication of these concentrations of nebracetam for 30 s to 1 min potentiated such inhibition. A simple competitive inhibition model for the effects of both drugs simultaneously applied yielded the inhibition constant, K1 of 0.419 and 0.212 mmol/l for nAChRs and mAChRs, respectively, indicating that the action on mAChRs is a little more potent than on nAChRs. Nebracetam induced a concentration-dependent slight increase in inward currents on mAChRs but not on nAChRs. It is suggested that the direct effects of nebracetam on nAChRs and mAChRS, which were induced only by a rather high concentration, as compared with the clinically expected plasma level, may be a contributing factor to the clinical effectiveness of the drug only if there is some critical change in the sensitivity to the drug.


Prophylactic effects of a new 1,5-benzothiazepine calcium antagonist on stroke in salt-loaded stroke-prone spontaneously hypertensive rats.

The influence of chronic treatment with clentiazem ((+)(2S,3S)-3-acetoxy-8-chloro-5-[2-(dimethylamino)ethyl]-2,3-dihydro- 2-(4-methoxyphenyl)-1,5-benzothiazepin-4 (5H)-one maleate, TA-3090), on blood pressure, incidence of stroke, stroke-related mortality and histological changes of the brain and other organs were examined in salt-loaded stroke-prone spontaneously hypertensive rats (SHRSP). Male SHRSP that were fed an 8% NaCl-containing diet began to die of a stroke 3 weeks after salt-loading, accompanied by decreases in body weight and food intake. Most of the rats (16 out of 18) died by the 8th week of salt-loading. Chronic treatment with clentiazem (300 or 1000 ppm) delayed the occurrence of stroke and death in a dose-related manner without any hypotensive action when measured by the tail-cuff method. However, examination of circadian changes in arterial blood pressure with implanted cannula under a freely-moving condition 3 weeks after salt-loading revealed that 1000 ppm clentiazem produced significant hypotension in the dark phase but not in the light phase. Histological studies (3 weeks after salt-loading) showed that 1000 ppm Clentiazem significantly suppressed the cerebral and renal damages, and vascular hypertrophy in all organs studied. Thus, clentiazem prevents stroke and also protects renal damage and vascular hypertrophy in salt-loaded SHRSP. The hypotensive effect and organ-protective action by clentiazem may be involved in its prophylactic action against stroke.


Pharmacological profile of the new antidepressant levoprotiline.

The pharmacological properties of a new antidepressant, levoprotiline ((-)-R-a-[(methylamino)methyl]-9,10-ethanoanthracene-9(10H)- ethanol hydrochloride, CGP 12103 A, CAS 76496-69-0) were investigated. 1. Central nervous system: Levoprotiline did not have any marked effects on the general behaviour of mice and rats at low doses, however it slightly suppressed the righting reflex and spontaneous motor activity of mice at higher doses. In rats, chewing behaviour and salivation were observed at higher doses. Levoprotiline had no effects on the traction test (mice) and inclined screen test (rats). Levoprotiline induced a slight drowsy EEG pattern, the effect being similar to that of typical antidepressants such as maprotiline or imipramine but the degree of potency of levoprotiline was less than that of the latter two drugs. Levoprotiline and maprotiline did not inhibit the arousal response induced by physostigmine, while imipramine clearly suppressed the response. 2. Respiratory and cardiovascular system: Heart rate decrease and QT prolongation were observed in anesthetized dogs. In an in vitro study, levoprotiline caused only slight stimulation of noradrenaline transmission in the isolated guinea pig atrium. 3. Smooth muscle: The effects of levoprotiline on the contractile response to histamine, acetylcholine and serotonin in the isolated guinea pig ileum were compared with those of maprotiline or imipramine. While levoprotiline potently inhibited the contractile response to histamine, its inhibition of acetylcholine-induced contraction was the weakest of the three compounds studied. No remarkable effect was observed in serotonin induced contractions.(ABSTRACT TRUNCATED AT 250 WORDS)


Variation of pharmacokinetics after oral administration of slow-release metoprolol tablets and pharmacogenetic considerations.

The maximum plasma concentrations (Cmax) after oral administration of 120 mg tablets of slow-release metoprolol (CAS 37350-58-6) to 75 Japanese healthy male volunteers and 15 arrhythmic patients were measured. Extensive and poor metabolizers after oral administrations of slow-release metoprolol tablets were classified by means of the frequency distribution of Cmax values. In addition, the frequency distribution of Cmax values after oral administration of slow-release metoprolol 120 mg tablets was compared with that of conventional metoprolol 40 mg tablets. 1. Mean +/- S.E. values of Cmax, tmax and AUC0-24 after oral administration of slow-release metoprolol tablets to 75 healthy male volunteers and 15 arrhythmic patients were 95.3 +/- 6.6 ng/ml, 4.4 +/- 0.2 h and 1000.4 +/- 70.9 ng.h/ml, respectively. 2. The number of poor metoprolol metabolizers after the oral administration of slow-release tablets in 75 healthy volunteers and 15 patients was estimated to be 2 subjects (2.2%). 3. The frequency distribution of Cmax values after oral administration of conventional metoprolol tablets was similar to that of slow-release metoprolol tablets. In addition, from the result of this study and that obtained in another study, in which the frequency of poor metoprolol metabolizers in British people has been examined, it is concluded that the frequency of poor metoprolol metabolizers varies between ethnic groups (2.2% in Japanese population and 11.3% in British population.


Synthesis, natriuretic, antikaliuretic and antimagnesiuretic properties of an acidic triamterene derivative.

2,4,7-Triamino-6-(4-methanesulfonamidophenyl) pteridine (RPH 3048) is a new acidic triamterene derivative. Relevant physico-chemical constants were determined (solubility at pH 7.4 = 3.7 mg/l; logP at pH 7.4 = 0.2) and pharmacokinetic as well as pharmacodynamic properties were investigated, using male Wistar rats. After intravenous application of the test substances urine was collected, its volume and electrolyte composition determined, and the urine recovery of the drugs was analysed. The comparison of RPH 3048 with triamterene (CAS 396-01-0) revealed almost equipotent natriuretic and potassium-retaining effects for both drugs and an additional relative magnesium-sparing activity of RPH 3048. The urine recovery of RPH 3048 after 6 h was higher (20.6%) than that of triamterene (12.9%). No metabolite of RPH 3048 could be detected in the urine whereas a triamterene metabolite was found. Due to its good solubility in alkaline medium RPH 3048 could be dissolved (at pH 11-12) and then administered intravenously together with a loop diuretic (furosemide). Urinary electrolyte excretion following administration of two different combinations of RPH 3048 and furosemide (combination A: 12.5 mumol/kg RPH 3048 and 25 mumol/kg furosemide; combination B: 25 mumol/kg RPH 3048 and 25 mumol/kg furosemide) was compared to urinary electrolyte excretion of a control group and a group only treated with furosemide (25 mumol/kg). The additional application of RPH 3048 reduced in both groups potassium and magnesium excretion to control level but did not compromise furosemide induced natriuresis. In contrast to earlier investigations these results suggest that it is possible to develop acidic triamterene derivatives with potent antikaliuretic effects.


Pharmacokinetics of azosemide in patients with T-drain after cholecystectomy.

In an open clinical trial the pharmacokinetics of orally administered azosemide (2-Chloro-5-(1H-tetrazol-5-yl)-4-[(2- thienylmethyl)amino] benzene sulfonamide. Luret, CAS 27589-33-9) was surveyed in a group of 10 patients with T-drain after cholecystectomy. The mean peak concentration was reached after 2.35 h (SE: 0.25 h) and was 474 ng/ml (142 ng/ml). The plasma elimination half-life was estimated to be 6.37 h (1.7 h). In 24 h 5.4% of the dose was excreted unchanged via urine and bile. Only a small fraction of the dose (0.53%) was recovered as glucuronide from urine and bile. Approximately 2% (0.74%) of the dose was excreted unchanged via bile. No other metabolites were detected. Plasma AUCO-24h was 3113 micrograms.h/l. The renal clearance of 27 ml/min (8.8 ml/min) was 3 times higher than the biliary clearance of 10 ml/min (2.4 ml/min). Azosemide is rapidly but incompletely absorbed and shows a longer half-life when compared with other loop diuretics. Enterohepatical circulation and first-pass effect seem to be less significant because low amount of azosemide is excreted via bile.


Inhibitory effects of propiverine hydrochloride on the agonist-induced or spontaneous contractions of various isolated muscle preparations.

Inhibitory effects of propiverine hydrochloride (P-4, CAS 60569-19-9), a new drug which reduces the frequency of micturition, were studied on the agonist-induced or spontaneous contractions of various isolated muscle preparations. P-4 (10(-6)-10(-4) mol/l) inhibited both the KCl-induced contractions of isolated guinea-pig urinary bladder and ileum, and its IC50 values (mol/l) were 1.8 +/- 0.3 x 10(-5) (urinary bladder) and 1.3 +/- 0.4 x 10(-5) (ileum), respectively. The spontaneous contractions of isolated guinea-pig atrium, rat uterus and rabbit duodenum were unaffected by P-4 at 10(-6) mol/l, but they were inhibited by the drug at 10(-5)-10(-4) mol/l. Its IC50 values (mol/l) were 10(-4) greater than (guinea-pig atrium), 1.3 +/- 0.3 x 10(-4) (rat uterus), and 1.0 +/- 0.5 x 10(-4) (rabbit duodenum), respectively. P-4 (10(-5)-10(-4) mol/l) inhibited the KCl-induced contraction of isolated rabbit aorta and the histamine-induced contraction of isolated guinea-pig tracheal chain. P-4 (10(-4) mol/l) also inhibited the noradrenaline-(norepinephrine) induced contractions of isolated guinea-pig urethra and vas deferens. In these isolated muscle preparations, the values of IC50 (greater than 10(-4) mol/l) could not be calculated. These results indicate that the inhibitory activities of P-4 on urinary bladder and ileum are 10 to 100 times more potent than those on other organs.


High pressure liquid chromatographic determination of the new non-steroidal anti-inflammatory agent butibufen.

High performance liquid chromatography (HPLC) method for the determination and validation of a new non-steroidal anti-inflammatory agent, butibufen (CAS 55837-18-8), microencapsulated butibufen and its pharmaceutical forms--tablets, sachets (microencapsulated butibufen with excipients), microemulsion and cream--is described. Acetonitrile proved to be the best solvent for the extraction of butibufen from its pharmaceutical forms. The results submitted in this paper suggest that the HPLC method for the quantitative determination of butibufen is linear, accurate, precise and sensitive.


Antitumor activity of some ruthenium derivatives in human colon cancer cell lines in vitro.

Six ruthenium derivatives were evaluated in vitro in two human colon cancer cell lines (SW707 and SW948) utilizing the microculture tetrazolium test (MTT) and cell counting with a Coulter Counter. The ruthenium compound sodium-(tetrachloroimidazoledimethylsulfoxideruthenate)- bisdimethylsulfoxide (Na(RuDMSOimCl4)) showed the best efficacy in inhibiting cell proliferation of both colon cancer cell lines followed by the other DMSO ruthenium compound sodium-(tetrachloroindazoledimethylsulfoxideruthenate) - bisdimethylsulfoxide (Na(RuDMSOIndCl4)), as demonstrated by IC50 values (80 and 90 micrograms/ml in SW707 and SW948 cell lines for Na(RuDMSOImCl4); 155 and 165 micrograms/ml in SW707 and SW948 cell lines for Na(RuDMSOIndCl4), respectively). Out of the ruthenium derivatives without DMSO, transindazolium - [tetrachlorobis(1H - indazole)ruthenate (III,N2)] (HInd[RuInd2Cl4(N2)]), was as active as its DMSO-containing congener whereas trans-imidazolium- [tetrachlorobisimidazoleruthenate)(III)], (HIm(RuIm2Cl4)) was less active, as shown by the IC50 values: (HIm (RuIm2Cl4) = 250 and 260 micrograms/ml in cell lines SW707 and SW948; HInd[RuInd2Cl4(N2)] = 110 and greater than 200 micrograms/ml in cell lines SW707 and SW948, respectively). The other ruthenium derivatives containing pyrazole and triazole as ligands (trans - pyrazolium (tetrachlorobispyrazoleruthenate) (III), PzH(RuPz2Cl4) and triazolium(tetrachlorobistriazoleruthenate) (III), TrH(RuTr2Cl4)) were active only at high concentrations that cannot be regarded as realistic in vivo, as shown by the respective IC50 values: (PzH(RuPz2Cl4) = 1056 and 750 micrograms/ml in cell lines SW707 and SW948; TrH(RuTr2Cl4) = 350 and 300 micrograms/ml in cell lines SW707 and SW948). The promising activity of ruthenium compounds with DMSO, indazole and imidazole as ligands should be evaluated in vivo for elucidating their possible role in the treatment of colorectal cancer.


Stereoselectivity in the disposition of chloroquine and desethylchloroquine in rabbits.

Stereoselective pharmacokinetic properties of chloroquine (CAS 54-05-7) and desethylchloroquine were investigated in rabbits by administration of the separate enantiomers according to a cross-over design. The terminal half-life was longer for (R)-chloroquine than for (S)-chloroquine. A striking difference was revealed in the concentrations of metabolites. The levels of (R)-desethylchloroquine were higher than those of (S)-desethylchloroquine, resulting in a statistically significant higher AUC for the (R)-metabolite than for the (S)-metabolite.


Influence of alpha-lipoic acid on intracellular glutathione in vitro and in vivo.

The influence of alpha-lipoic acid (CAS 62-46-4) on the amount of intracellular glutathione (GSH) was investigated in vitro and in vivo. Using murine neuroblastoma as well as melanoma cell lines in vitro, a dose-dependent increase of GSH content was observed. Dependent on the source of tumor cells the increase was 30-70% compared to untreated controls. Normal lung tissue of mice also revealed about 50% increase in glutathione upon treatment with lipoic acid. This corresponds with protection from irradiation damage in these in vitro studies. Survival rate of irradiated murine neuroblastoma was increased at doses of 100 micrograms lipoic acid/d from 2% to about 10%. In agreement with the in vitro studies, in vivo experiments with whole body irradiation (5 and 8 Gy) in mice revealed that the number of surviving animals was doubled at a dose of 16 mg lipoic acid/kg. Improvement of cell viability and irradiation protection by the physiological compound lipoic acid runs parallel with an increase of intracellular GSH/GSSG ratio.


In vitro and in vivo studies with flutrimazole, a new imidazole derivative with antifungal activity.

1-[(2-Fluorophenyl)(4-fluorophenyl)phenylmethyl]-1H-imidazole (flutrimazole, UR-4056, CAS 119006-77-8) is a new topical imidazole antifungal agent which displays potent broad-spectrum in vitro activity against dermatophytes, filamentous fungi and yeasts, saprophytic and pathogenic to animals and humans (MIC = 0.025-5.0 micrograms/ml). In most of these studies the activity of flutrimazole was comparable to that of clotrimazole and markedly higher than that of bifonazole (MIC differences of greater than or equal to 1 order of magnitude). Tested against Scopulariopsis brevicaulis (8 strains), both flutrimazole and clotrimazole exhibited fungistatic and fungicidal activity, and clotrimazole appeared something less active (MIC = 0.3-2.5 micrograms/ml) than flutrimazole (MIC = 0.15-0.6 micrograms/ml). In animal experiments, topical application of 1% and 2% flutrimazole, as a cream or solution, was highly effective in models of rat vaginal candidiasis and guinea-pig trichophytosis, giving a rate of cured or cured plus markedly improved animals higher than 80%. Flutrimazole shares the mode of action of other imidazole or triazole-containing antifungals, i.e. inhibition of fungal lanosterol 14 alpha-demethylase, as it strongly inhibits ergosterol biosynthesis in a cell-free homogenate of Candida albicans, with an IC50 value of 0.071 mumol/l.


Toxicity studies with flutrimazole.

Studies with 1-[(2-fluorophenyl)(4-fluorophenyl)phenylmethyl]-1H- imidazole (flutrimazole, CAS 119006-77-8), a new topical imidazole antifungal agent, have been carried out to investigate the acute toxicity of the active substance in mice and rats, as well as the acute ocular and dermal irritation in rabbits, the dermal tolerance after repeated dose (21 days) applications in rabbits, and the sensitising, photoallergic and phototoxic potential in guinea pigs using 1% flutrimazole cream. LD50 values after oral or intraperitoneal administration were greater than or equal to 1000 mg/kg in both mice and rats, which reveal a very low acute toxicity of flutrimazole. No differences were found between the excipient and 1% flutrimazole cream in the acute ocular and dermal irritation studies in rabbits, the irritation indexes being indicative of no lesions due to flutrimazole. Cumulative dermal irritation studies in rabbits showed an improved local tolerance of a skin cream containing 1% flutrimazole as compared to a commercial skin cream containing 1% clotrimazole. The irritation indexes were 1.2 and 3.7, respectively (p less than 0.01). The corresponding histophatological findings confirmed the better local tolerance of 1% flutrimazole cream. Furthermore, it has been found that flutrimazole cream lacks sensitising potential (Magnusson and Kligman test), is also devoid of phototoxic potential and does not induce photoallergic reactions in guinea pigs, these data being confirmed by histopathological studies. These results, together with the very slight systemic absorption rate of flutrimazole from the 1% topical drug form, clearly show that no restrictions should be taken in the use of the cream for reasons of systemic toxicity or dermal tolerance.


Percutaneous absorption and skin distribution of [14C]flutrimazole in mini-pigs.

The percutaneous absorption and skin distribution of a skin cream containing 1% 14C-labelled 1-[(fluorophenyl) (4-fluorophenyl) phenylmethyl]-1H-imidazole (flutrimazole, UR-4056, CAS 119006-77-8) was studied in minipigs. The same dose of flutrimazole was administered i.v. and topically (as a cream) on scarified skin according to a crossover protocol. Samples of urine and faeces were taken at various intervals after administration, and radioactivity was measured. The percentage of radioactivity accumulated in urine after topical and intravenous administration were 1.46% and 41.7%, respectively. In faeces, the percentage of radioactivity observed was 6.0% after intravenous administration, and none was detected after topical application. In order to study the distribution and penetration of [14C]flutrimazole, the cream was applied to intact and scarified skin. At various intervals after administration, skin samples were taken. The samples for the autoradiographic studies were cut transversely, and for the measurement of the levels of radioactivity at different skin depths, slices were cut parallel to the cutaneous layers. The results obtained indicate that [14C]flutrimazole penetrates quickly into the different epidermic layers and is retained mainly in the strata spinosum, granulosum and basale. The stratum basale possibly acts as a selective barrier preventing the penetration of the compound into the dermis. The percentage of radioactivity in the stratum corneum is lower than that detected in all the other epidermic layers taken together. The stratum corneum offers low resistance to penetration by the flutrimazole, which very probably crosses the epidermic strata by a transcellular route.


Pharmacokinetic study of [14C]flutrimazole after oral and intravenous administration in dogs. Comparison with clotrimazole.

This trial involved a comparative study using 6 Beagle dogs on the pharmacokinetics of 14C-labelled 1-[(2-fluorophenyl)(4-fluorophenyl)phenylmethyl]-1H-imidazole (flutrimazole, CAS 119006-77-8) and [14C]clotrimazole labelled in the imidazole ring. On the basis of a cross-over trial, each animal received a dose of 5 mg/kg (approx. 100 microCi) [14C]flutrimazole and [14C]clotrimazole, both intravenously and orally. The levels in plasma, urine and faeces of the total radioactivity, unchanged drug and the [14C]imidazole formed by metabolization of the unchanged drug were determined. Flutrimazole presented a biological half-life (t1/2) of 14.4 +/- 3.8 h and a clearance (Cl) of 6.7 +/- 0.8 l/h, while the values for clotrimazole were very different: t1/2 4.6 +/- 0.8 h and Cl: 13.6 +/- 1.0 l/h. After oral administration a fraction of absorbed dose (f) of 78 +/- 21% and bioavailability of 8.9 +/- 6.1% were calculated for flutrimazole. For clotrimazole, these were: 52 +/- 10% and 4.9 +/- 1.9%, respectively. Both drugs showed a significant first-pass effect, with 90% of the absorbed dose being metabolized before reaching the systemic circulation. The total recovery of radioactivity in faeces and urine 5 days after i.v. and oral administration was 58% and 68%, respectively, for [14C]flutrimazole, and 81% and 79% for [14C]clotrimazole. In both cases, most of the radioactivity was recovered in the faeces. The high radioactivity obtained in faeces after i.v. administration of both drugs confirms biliary elimination. For both flutrimazole and clotrimazole, less than 1% of the total recovered in the urine after i.v. administration was recovered as unchanged drug.(ABSTRACT TRUNCATED AT 250 WORDS)


Local and systemic tolerance of flutrimazole skin creams following single and repeated topical application in healthy volunteers.

A double-blind, randomized phase I study was performed in 21 healthy volunteers to evaluate the dermal tolerance of skin creams containing 1% and 2% 1-[(2-fluorophenyl)(4-fluorophenyl)phenylmethyl]-1H-imidazole (flutrimazole, UR-4056, CAS 119006-77-8) or only the excipient, versus a commercial skin cream containing 1% clotrimazole. The study was carried out using the patch-test procedure performed in three stages: 1. single application in the back skin; 2. induction period (usage test) using three skin areas on the volar side of the forearm of each subject, where skin cream samples were applied once a day for a period of three weeks; and 3. after a wash-out period of two weeks, challenge applications in the back and forearm skin. The systemic tolerance of the formulations was also tested. There was no evidence of allergic sensitization after the application of flutrimazole creams, or their excipients, with signs of mild and doubtful skin reactions being observed in few subjects with all formulations. Furthermore, no systemic side effects after topical administration were detected throughout the study.


Pharmacokinetic profile of [14C]flutrimazole following single topical application in normal and scarified skin of healthy volunteers.

14C-labelled 1-[(2-fluorophenyl)(4-fluorophenyl)phenylmethyl]-1H- imidazole (flutrimazole, UR-4056, CAS 119006-77-8) was administered topically (dermally) in three male volunteers to normal skin and scarified skin, respectively. Blood, urine and faeces were collected and radioassayed by liquid scintillation counting techniques. After topical application to healthy skin area, less than 1% of flutrimazole was absorbed percutaneously from a 1% cream and the amount absorbed was excreted with the urine. Following administration of the drug to scarified skin, the proportions absorbed were very similar to those measured after application to normal skin. About 0.5% of the administered dose is recovered in urine. The absorbed amount of drug leads to very low concentrations in the circulation, lower than a few ng/ml.


Rated performance, cardiovascular and quantitative EEG parameters during simulated instrument flight under the effect of terfenadine.

In a double-blind placebo-controlled randomized two-phase cross-over study comprising 10 (5/5) male subjects the effect of oral therapeutic doses of terfenadine (Teldane, CAS 50679-08-8) on rated performance, systolic and diastolic blood pressure, heart rate and the electroencephalogram of the subjects in an instrument flight (IF) procedure trainer was investigated. Terfenadine did not influence systolic and diastolic blood pressure, heart rate and the specific performance of subjects during instrument flight procedures. Under terfenadine the quantitative EEG (qEEG) showed more %alpha, especially %alpha 2 during rest and to a lesser extent during instrument flight procedures of the subjects. As a result the vigilance dynamics was increased. In the %beta band especially %beta 2 was increased only during instrument flight activities. The ratio theta/alpha was reduced during rest and during activities. Thus all these quantitative EEG indicators do not show a decrease in the level of vigilance nor are they indicative of signs of drowsiness during rest and during instrument flight activities under terfenadine. The results are discussed.


Quantification of dechelation of gadopentetate dimeglumine in rats.

An animal model was developed in order to estimate a potential in vivo dechelation of gadopentetate dimeglumine (Gd-DTPA; CAS 86050-77-3) and to provide information concerning the chemical form of long-term gadolinium not excreted. Excretion kinetics and biodistribution of 153Gd-DTPA and Gd-[14C]DTPA were studied in 2 groups of rats following the intravenous injection of 0.5 mmol/kg Gd-DTPA. Differences in the elimination and biodistribution profiles between the quantities of 153Gd and 14C radioactivity were taken as evidence for dechelation of Gd-DTPA within the body leading to a release of Gd ions. Statistically significant differences were observed in excretion and whole-body retention indicating an in vivo dechelation of less than 1% of the injected dose. 30% of the released Gd ions were excreted via urine and feces during the 21-day time interval. 21 days p.i., 70% of the released Gd ions remained in the body producing a significant 153Gd excess in liver, spleen, and the bones representing the principal target organs of released Gd ions after intravenous injection of Gd-DTPA. More than 96% of the released Gd ions found in the body 21 days p.i. were deposited in the bones. Liver and spleen showed minor contributions. In any case, the chemical form of the gadolinium present in liver, spleen, and the bones could predominantly be assigned to translocated Gd ions resulting from dechelation of Gd-DTPA.


Human recombinant interleukin-1 alpha and beta and articular tissue integrity. An in vivo study on hens and rats.

Human recombinant interleukin-1 alpha or beta was injected intraarticularly several times into the knee joints of rats and hens at different dosages (50-2000 ng) and intervals. No loss of cartilage detectable by width of the radiological joint space and no degenerative changes of subchondral bone could be observed during 16 weeks using X-ray analysis. The macroscopical examination of articular cartilage of hens after this period and of rats after one year showed no morphological cartilage damages. These data suggest that human recombinant Il-1 alpha and beta alone may not be sufficient to induce a progressing osteoarthritic process in these animals.


Behavioral characterization of the new potent nonselective dopamine agonist pergolide.

Pergolide (LY127809, CAS 66104-23-2), a non-selective dopamine agonist, was evaluated for broad behavioral properties in a wide range of pharmacological tests. The selective dopamine2(D2) agonist, bromocriptine, served as a reference standard for those tests where behavioral activity was noted with pergolide. Pergolide and bromocriptine were administered orally to mice at doses of 0.3-30 and 3-300 mg/kg, respectively. Both compounds produced biphasic effects on spontaneous activity, increased hexobarbital-induced sleep time, and lowered mouse body temperature. Qualitative changes with pergolide were observed with some mice showing hyporeactiveness, ptosis, slowed respiration and placing loss. Reserpine-induced hypothermia was reversed by pergolide with significant increases in the body temperature of reserpine-treated mice. However, a further reduction in the body temperature of reserpinized hypothermic mice was seen following bromocriptine administration. Acetic acid-induced writhing and performance on the rotarod were both impaired by higher doses of pergolide. Bromocriptine administration also reduced writhing at higher doses but did not alter performance on the rotarod. Pergolide had no effect on seizure activity as evaluated by electroshock, pentylenetetrazol (pentetrazol) or strychnine. Oxotremorine-induced tremors and salivation, grip strength, and tail-flick were not affected by pergolide. Neither pergolide nor bromocriptine altered established shuttle-avoidance behavior in rats at oral doses of 0.1 to 30 mg/kg. Behavioral assessment of pergolide in dogs was complicated by severe emetic responses at clinically relevant doses greater than 0.003 mg/kg. In summary, these data suggest that pergolide produces a behavioral profile which is characteristic of dopaminergics.(ABSTRACT TRUNCATED AT 250 WORDS)


General pharmacology of pergolide in animals. 2nd communication: gastrointestinal, renal and miscellaneous studies.

Pergolide mesylate ((8 beta)-8-[(methylthio)methyl]-6-propylergoline monomethanesulfonate, LY 127809, CAS 66104-23-2) is a novel and potent dopamine agonist marketed for treating the symptoms of Parkinson's disease. The potential secondary pharmacological effects of this agent on the gastrointestinal and renal systems, as well as effects on local anesthesia, hemolysis, platelet aggregation, circulating blood glucose, primary antibody production, and the acute inflammatory response were examined. Pergolide exhibited significant pharmacological effects in gastrointestinal, renal and anti-inflammatory tests at high oral doses. Pergolide was essentially inactive in blood hemolysis, platelet aggregation, primary antibody production and local anesthesia testing. In summary, these studies confirm the pharmacological selectivity of pergolide, and indicate a low potential for secondary pharmacological side effects upon the functions tested at clinically relevant doses.


Effects of mergocriptine on cerebral circulation and metabolism in cats.

Mergocriptine (CBM 36-733, CAS 81968-16-3) is an ergot alkaloid derivative and a dopaminergic agonist. Effects of mergocriptine on cerebral circulation and metabolism were examined by monitoring cerebral tissue oxygen and carbon dioxide tension (BrPO2, BrPCO2), cerebral blood flow (CBF) and blood pressure (BP) in 10 cats. Mergocriptine (10 micrograms/kg) infused into the carotid artery produced a significant increase in CBF during the administration followed by a decrease in BrPO2 in parallel with a significant decrease in BP.


Memantine in the treatment of mild to moderate dementia syndrome. A double-blind placebo-controlled study.

The efficacy and the tolerability of memantine (1-amino-3,5-dimethyladamantane hydrochloride, Akatinol Memantine, CAS 41100-52-1) were investigated in patients with mild to moderate dementia syndrome in a randomized two-centre placebo-controlled clinical study. The test substance was administered at a dose of 10 mg/d from day 1 to day 3 and then at a dose of 2 x 10 mg/d from day 4 to the end of treatment after 42 days. Altogether, 88 patients were recruited to the study; their average age was 71.5 years. The efficacy of memantine was judged on the basis of the baseline/6 week differences in the total sum scores of the Clinical Assessment Geriatric Scale (SCAG), the Gottfries-Bräne-Steen Scale (GBS), the SCAG and GBS subscales and the global assessment of the change in the patient's condition. The effects of memantine on performance were studied with the aid of psychomotor tests and a behaviour investigation relating to activities of daily living (ADL). The tolerability of memantine was assessed on the basis of the doctor's global assessment and of entries on structured documentation forms (DOTES/TWIS). Further safety parameters--in the form of clinicochemical tests and measurements of blood pressure and heart rate--were also monitored during the study. On both the psychopathological measurement level (SCAG, global assessment of the change in the patient's condition) and the behavioural level (GBS), confirmatory statistical analysis brought to light significant differences between memantine and placebo (p less than or equal to 0.05), these differences showing a superiority of memantine. The tolerability of memantine was good in the main. The observed adverse reactions were not serious and, except in 3 patients, were rated as causing little or no impairment. The present clinical study demonstrates the efficacy of memantine in patients suffering from mostly moderate dementia syndrome. Clinical and statistically relevant improvements in the dementia-induced disturbances were found on the both the psychopathological level (SCAG, CGI) and the behavioural level (GBS). On the performance level also, the ADL behaviour investigation detected a highly significant improvement in the quality of performance of instrumented activities of daily living under memantine. Also the time taken to carry out these tasks was significantly reduced in comparison with placebo.


Bioavailability of vinpocetine and interference of the time of application with food intake.

In a pilot study based on an open cross-over design involving four phases, the relative bioavailability of the eburnamenine derivative vinpocetine (CAS 42971-09-5) was investigated in 8 healthy volunteers in relation to different times of drug administration relative to food intake. The substance was applied orally as 10 mg film tablets. The areas under the plasma concentration-time curves (AUC) amounted to 27.3 +/- 18.1 ng.h/ml (fasting) and 42.8 +/- 27.4 up to 54.3 +/- 38.4 ng.h/ml (non-fasting, intake before and after meal, resp.). The relative bioavailability under non-fasting conditions was found to be approx. 60 to 100% higher than under fasting conditions.


Growth-promoting effect of a protein-free hemodialysate used in situations of hypoxia and for tissue repair as measured via stimulation of S6-kinase.

Solcoseryl is the low-molecular weight fraction of calf blood as manufactured by counterflow dialysis. This hemodialysate (HD) is in clinical use in situations involving hypoxia and for the normalization of tissue repair. The influence of the HD on ZR-75 cells was tested. These cells preferably express receptors for Epidermal Growth Factor (EGF)/Transforming Growth Factor alpha (TGF-alpha) or Somatomedin C (SMC = insulin like growth factor I resp. ILA-I) and react upon stimulation by enhancement of their S6-kinase activity, the latter being a prerequisite for growth. The functional presence of one or several of these peptide growth factors should therefore reflect in a stimulation of S6-kinase activity.


Synthesis and cardiovascular properties of furazanyl-1,4-dihydropyridines and of furoxanyl analogues.

A series of furazanyl- and furoxanyl-1,4-dihydropyridines has been synthesized and tested for their cardiovascular activity. The new compounds showed a modest activity in comparison with nifedipine and some selectivity for the myocardial muscle. The most active compound was diethyl 1,4-dihydro-2,6-dimethyl-4-(3-phenyl-4-furoxanyl)-3,5-pyridine dicarboxylate, 4d.