Biomedical subjects
Publications and source records attributed to R Pauwels.
Neuropeptides and purines are possible autonomic neurotransmitters. Both tachykinins (neurokinin A, substance P) and adenosine cause acute airway narrowing by synergistic activation of airway mast cells and postganglionic vagal nerve endings.
Purines and neuropeptides may act as neurotransmitters of the local axon reflex in the airways. Nedocromil sodium has been shown to inhibit adenosine- and neurokinin A-induced bronchoconstriction both in the rat and in man. In this study we used the rat model for further investigation of the mechanism of action of nedocromil sodium in the adenosine- and neurokinin A-challenge. The animals were challenged intravenously, and immediately after maximal bronchoconstriction a bronchoalveolar lavage was performed and histamine was assayed in the supernatant of the lavage fluid. Nedocromil sodium significantly inhibited the adenosine- and the neurokinin A-induced increase in histamine concentration in the lavage fluid. Nedocromil sodium had no influence on the airway responsiveness to serotonin or carbachol. We therefore concluded that the inhibitory effect of nedocromil sodium on adenosine- and neurokinin A-induced bronchoconstriction can be explained by its activity on airway mast cells.
The first step in the replicative cycle of human immunodeficiency virus type 1 (HIV-1) is binding of the virions to the cellular CD4 receptor. This process may be considered as an important target for chemotherapeutic agents against acquired immune deficiency syndrome (AIDS). A method has now been devised whereby virion binding to the cell membrane was visualized by an indirect immunofluorescence assay using human anti-HIV-1 serum, rabbit anti-human-IG-F(ab')2-fluorescein isothiocyanate, and flow cytometry. Heparin, dextran sulfate, and pentosan polysulfate suppressed HIV-1 binding to MT-4 cells at concentrations that protected the cells against HIV-1 cytopathogenicity. Dextran and dermatan sulfate, two compounds that are inactive against HIV-1, had no inhibitory effect on the binding of HIV-1 to the cells. The potent and selective HIV-1 inhibitor azidothymidine (AZT) did not affect virus binding to the cells, whereas suramin partially blocked HIV-1 binding to the cells at concentrations that fully protected MT-4 cells against destruction of HIV-1. Our immunofluorescence assay thus demonstrated that suramin not only acts as an inhibitor of reverse transcriptase but also interferes with virus-cell binding. Also, Evans blue, an anionic dye structurally related to suramin, partially inhibited HIV-1 attachment to the cells. The present method permits a quantitative determination of the inhibitory effect of anti-HIV-1 agents on virion-cell binding.
Several cholic acid derivatives such as taurolithocholic acid, lithocholic acid 3-sulfate, taurolithocholic acid 3-sulfate, and glycolithocholic acid 3-sulfate were shown to inhibit selectively the replication of human immunodeficiency virus type 1 (HIV-1) in vitro. These compounds completely protected MT-4 cells against HIV-1-induced cytopathogenicity at a concentration of 100 micrograms/ml, whereas no toxicity for the host cells was observed at 200 micrograms/ml. They also inhibited HIV-1 antigen expression in HIV-1-infected CEM cells. The bile acids (cholic acid, deoxycholic acid, chenodeoxycholic acid, and lithocholic acid) did not show any inhibitory effect on HIV-1 replication at concentrations that were not toxic to the host (MT-4) cells. From a structure-function analysis of a number of cholic acid derivatives, the presence of either a sulfonate (as in the tauro conjugates) or a sulfate group as well as the "litho" configuration appeared to be necessary for the expression of anti-HIV-1 activity. The active cholic acid derivatives did not directly inactivate the virus particles at the concentrations that were not toxic to the host cells. Lithocholic acid 3-sulfate, taurolithocholic acid 3-sulfate, and glycolithocholic acid 3-sulfate, but not taurolithocholic acid, partially inhibited virus adsorption to MT-4 cells. These three compounds were also inhibitory to the reverse transcriptase activity associated with HIV-1.
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The novel 5-chloro-, 5-bromo-, and 5-iodo-derivatives of 3'-fluoro-2',3'-dideoxyuridine (FddUrd), designated FddCIUrd, FddBrUrd, and FddIUrd, respectively, have been synthesized and evaluated for their antiretrovirus activity against human immunodeficiency virus (HIV) and murine Moloney sarcoma virus. All three 5-halogeno-FddUrd analogues inhibited HIV-1 replication in MT4 cells with an effective dose (ED50) of about 0.2-0.4 microM. However, FddCIUrd was markedly more selective in its anti-HIV-1 activity than FddBrUrd or FddIUrd. The selectivity index of FddCIUrd was similar to that of 3'-azido-2',3'-dideoxythymidine (AZT) when evaluated in parallel (1408 and 1603, respectively). The FddUrd derivatives also had a marked inhibitory effect on HIV-2 replication in MT4 cells and HIV-1 induced antigen expression in HUT-78 cells. However, neither FddUrd nor its 5-halogeno derivatives were inhibitory to Moloney sarcoma virus-induced transformation of murine C3H cells. The anti-HIV-1 activity of FddUrd, FddCIUrd, FddBrUrd, and FddIUrd was reversed by the addition of thymidine and 2'-deoxycytidine. The 5-halogeno-FddUrd analogues had a markedly higher affinity for MT4 thymidine kinase than FddUrd (Ki/Km, 4.0-4.7, as compared with 302 for FddUrd).
Infection of human T4 lymphocyte MT-4 cells with human immunodeficiency virus (HIV) results in cell death 4-5 days after infection. We have now developed a highly sensitive and rapid procedure for estimating the cytopathic effect of HIV in MT-4 cells. This method is based on fluorometric as well as flow cytometric evaluation of HIV-infected MT-4 cultures. By the use of fluorescein diacetate (FDA), a non-fluorescent diacetyl fluorescein ester that becomes fluorescent upon hydrolysis by esterases present in the cytoplasm of viable cells, as few as 100-200 viable MT-4 cells can be accurately determined. Applying this new method to HIV-infected MT-4 cell cultures treated with differing concentrations of the potent anti-HIV agent azidothymidine (AZT), we obtained a virus-inhibitory dose-response comparable to those obtained by the conventional (labour-intensive and time-consuming) methods. The FDA-based cell viability assay appears particularly suited for the rapid, reliable and sensitive evaluation of potential anti-AIDS agents in cell culture.
Fuchsin acid, an anionic dye, is a selective inhibitor of human immunodeficiency virus type 1 (HIV-1) and type 2 (HIV-2) in vitro. Its 50% effective dose for inhibition of HIV-1-induced cytopathogenicity in MT-4 cells and HIV-1 antigen expression in HUT-78 cells is 42 and 16 microM, respectively. These values are comparable to those of suramin, the first compound shown to be a selective inhibitor of HIV-1. However, fuchsin acid is less cytotoxic than suramin. The selectivity index of fuchsin acid, based on the ratio of the 50% cytotoxic dose to the 50% effective dose, is greater than 74 in MT-4 cells and greater than 39 in HUT-78 cells. Fuchsin acid is a much weaker inhibitor of HIV-1 reverse transcriptase than are suramin and aurintricarboxylic acid. Fuchsin acid does not interfere with the adsorption of HIV-1 particles to MT-4 cells even at concentrations that completely block HIV-1 replication in MT-4 cells. The mechanism of action of fuchsin acid remains subject of further study.
The 3'-fluoro-and 3'-azido-substituted derivatives of 2',3'-dideoxythymidine (ddThd), 2',3'-dideoxyuridine (ddUrd), 2',3'-dideoxy-5-ethyluridine (ddEtUrd) and 2',3'-dideoxycytidine (ddCyd) have been synthesized and evaluated for their anti-retrovirus activity [against human immunodeficiency virus (HIV) and murine Moloney sarcoma virus (MSV)]. Based on their 50% effective doses the most potent inhibitors of HIV replication in human MT4 lymphocytes were: FddThd (0.001 microM), AzddThd (0.004 microM), FddUrd (0.04 microM) and AzddUrd (0.36 microM). Their selectivity indexes were 197, 5000, 500 and 677, respectively. In contrast, none of the 3'-substituted ddEtUrd derivatives had a marked antiviral effect. The 2',3'-dideoxynucleoside analogues showed poor, if any, substrate affinity for (bacterial) dThd phosphorylase. AzddThd and FddThd inhibited human dThd kinase to a much greater extent (Ki/Km: 0.66 and 3.4, respectively) than did AzddUrd or FddUrd (Ki/Km: 71 and 171, respectively). The Ki/Km values of FddCyd and AzddCyd for human dCyd kinase were about 60. Although phosphorylation is a prerequisite for the anti-retrovirus activity of the 2',3'-dideoxynucleoside derivatives, there is no close correlation between the anti-retrovirus potency of the 3'-fluoro- and 3'-azido-substituted ddUrd, ddThd, ddEtUrd and ddCyd derivatives and their affinity for dThd kinase or dCyd kinase.
A series of 5'-phosphorylated derivatives of 3'-azido-2',3'-dideoxythymidine (AzddThd), including AzddThd 5'-mono- and 5'-triphosphate, alpha, beta-methylene AzddThd-5'-diphosphate, alpha,beta-methylene AzddThd-5'-triphosphate, and beta,gamma-methylene AzddThd-5'-triphosphate, were evaluated for their cytostatic and anti-retrovirus properties, and their inhibitory effects on the reverse transcriptases of Moloney murine leukemia virus and human immunodeficiency virus. In contrast with the 5'-mono- and 5'-triphosphates of AzddThd, which showed cytostatic and anti-retrovirus activities comparable to those of AzddThd, the alpha,beta-methylene 5'-phosphonates of AzddThd were considerably less cytostatic and also much less inhibitory to cell transformation by Moloney murine sarcoma virus and cytopathogenicity of human immunodeficiency virus. The decreased biological activity of the phosphonate derivatives of AzddThd is most likely due to the resistance of these compounds to phosphorolytic attack by phosphodiesterases and phosphatases, and the reduced affinity for the retrovirus-associated reverse transcriptase.
Several 2',3'-dideoxyadenosine analogues with modifications in either the ribose or purine moiety were evaluated for their inhibitory effects on the replication of human immunodeficiency virus (HIV) in MT-4 cell cultures. The 2',3'-dideoxyriboside of 2,6-diaminopurine (ddDAPR) inhibited HIV antigen expression and HIV-induced cytopathogenicity at a 50% effective dose of 2.4-3.8 microM, as compared to 3-6 microM for 2',3'-dideoxyadenosine (ddAdo), whereas 50% inhibition of MT-4 cell viability was noted only at a concentration of 477 and 889 microM, respectively. Both ddDAPR and ddAdo were only weakly inhibitory to the proliferation of a number of T-lymphoblast and T-lymphocyte cell lines, pointing to the selectivity of these compounds as anti-HIV agents. In contrast to ddAdo, ddDAPR was found to be a poor substrate for adenosine deaminase, which may be advantageous from a chemotherapeutic viewpoint. Substitution of an azido or fluoro group at the 2' and 3'-position of the ribose moiety in either "up" or "down" configurations resulted in a decrease of the anti-HIV potency and selectivity of ddAdo. In addition to ddDAPR other purine-modified ddAdo analogues, i.e. several pyrrolo[2,3-d]pyrimidine 2',3'-dideoxynucleosides, were investigated for their anti-HIV activity, but none of these derivatives proved as potent or selective as ddDAPR.
Cell lines derived from different species show striking differences in their sensitivity to the cytostatic and anti-retrovirus activity, as well as the intracellular metabolism, of 3'-azido-2',3'-dideoxythymidine (AzddThd) and 2',3'-dideoxycytidine (ddCyd). AzddThd and ddCyd are considerably more cytostatic to human (i.e. Raji, Molt/4F, ATH8) cell lines than murine (i.e. L1210) cells. The intracellular levels of AzddThd 5'-triphosphate and ddCyd 5'-triphosphate formed do not seem related to the cytostatic effects achieved by these compounds. In human lymphoid (ATH8, Molt/4F) and caprine ovary (Tahr) cells AzddThd accumulates as its 5'-monophosphate (AzddTMP), whereas in murine leukemia (L1210) cells it is readily metabolized to the 5'-triphosphate (AzddTTP). The rapid conversion of AzddThd to AzddTTP in murine cells may explain why AzddThd has a pronounced activity against Moloney murine sarcoma virus (MSV)-induced transformation of murine C3H cells in vitro and MSV-induced tumor development in newborn NMRI mice in vivo. In contrast, ddCyd has not much activity in these murine assay systems, and this may seem related to the poor conversion of ddCyd to its 5'-triphosphate in murine cells. In human cells, however, ddCyd is more extensively phosphorylated to its 5'-triphosphate than in murine cells. When [3H]AzddThd and [3H]ddCyd were compared for their metabolism in ATH8 and Molt/4F cells, little [3H]AzddTTP was formed even after a 48-hr incubation period, whereas under the same conditions substantial levels of [3H]ddCTP built up gradually. Thus, much higher ddCTP than AzddTTP levels were achieved in human lymphoid cells, an observation that may be particularly relevant from a therapeutic viewpoint.
The effect of ketotifen on bronchial beta-adrenergic tachyphylaxis was studied in healthy volunteers. In a 2-week pilot study, 19 volunteers inhaled 300 micrograms of salbutamol, four times a day. Ten subjects became tachyphylactic to inhaled salbutamol, assessed by a dose-response curve, before and after the 2 weeks of treatment from a metered-dose inhaler. These 10 subjects were then involved in a double-blind, crossover study comparing the effect of ketotifen, 1 mg in the evening during 1 week, and 1 mg, twice daily, during 3 weeks, and placebo on the bronchial beta-adrenergic tachyphylaxis induced by the concomitant administration during the last 2 weeks of each treatment period of 300 micrograms of salbutamol, four times a day, from a metered-dose inhaler. The end of the pilot study preceded the double-blind study by at least 1 month. However, the salbutamol dose-response curve at the beginning of the double-blind study was still significantly decreased, compared to the dose-response curve before the pilot study. During placebo treatment, a further decrease in bronchial beta-adrenergic sensitivity was observed. Treatment with ketotifen prevented the induction of further beta-adrenergic tachyphylaxis. Each 2 weeks of treatment with inhaled salbutamol increased significantly the baseline specific airway conductance; therefore, based on the present data, it is impossible to determine whether ketotifen also restores the sensitivity of an already desensitized beta-adrenergic system in the airways.
A rapid, sensitive and automated assay procedure was developed for the in vitro evaluation of anti-HIV agents. An HTLV-I transformed T4-cell line, MT-4, which was previously shown by Koyanagi et al. (1985) to be highly susceptible to, and permissive for, HIV infection, served as the target cell line. Inhibition of the HIV-induced cytopathic effect was used as the end point. The viability of both HIV- and mock-infected cells was assessed spectrophotometrically via the in situ reduction of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT). The procedure was optimized as to make optimal use of multichannel pipettes, microprocessor-controlled dispensing and optical density reading. The absorbance ratio of the mock-infected control to the HIV-infected samples was about 20. This allowed an accurate determination of the 50% effective doses, as demonstrated for 3'-azido-2',3'-dideoxythymidine (AZT), 2',3'-dideoxycytidine (ddCyd), dextran sulfate and heparin. The technique significantly reduced labor time as compared to the trypan blue exclusion method, and permits the evaluation of large numbers of compounds for their anti-HIV activity.
Several sulfated oligo- or polysaccharides such as pentosan polysulfate, fucoidan, dextran sulfate, heparin and iota-, kappa- and lambda-carrageenans proved to be potent and selective inhibitors of human immunodeficiency virus type 1 (HIV-1) in vitro. The most potent anti-HIV-1 activity was recorded for the oligosaccharide pentosan polysulfate, its 50% antiviral effective dose (ED50) being 0.19 microgram/ml in MT-4 cells. It inhibited HIV-1 antigen expression in HUT-78 cells at an ED50 of 0.02 microgram/ml, and complete inhibition of HIV-1 antigen expression was obtained at a concentration of 4.0 micrograms/ml. No toxicity for MT-4 cells was observed with pentosan polysulfate at a concentration of 2500 micrograms/ml. The anticoagulant activity of pentosan polysulfate was more than ten-fold lower than that of heparin (14.4 and 177 U/mg, respectively). In fact, pentosan polysulfate achieved its anti-HIV-1 activity at a concentration that is 370-fold below its anticoagulant threshold (1 U). Pentosan polysulfate inhibits virus adsorption to the cells, as was demonstrated by monitoring the association of radiolabeled HIV-1 virions with MT-4 cells.
The combined effects of fractionated administration of the antiviral agent (E)-5-(2-bromovinyl)-2'-deoxyuridine (BVDU) and fractionated (4 injections at a 48-h interval) administration or continuous (7-day) infusion of 5-fluorouracil (FU) have been investigated in P388 leukemia-bearing mice. The LD50 of continuous FU infusion in normal mice was approximately 330 mg/kg, while that for the fractionated treatment was 160 mg/kg. When combined with BVDU (200 mumol/kg, 90 min before each FU injection; 4 injections at a 48-h interval), the LD50 of FU was decreased from 330 to 115 mg/kg and from 160 to 30 mg/kg, respectively. The 7-day continuous infusion of FU (up to a dose of 210 mg/kg) did not exert a therapeutic effect in P388-leukemic mice, and, similarly, combination of continuous FU infusion (at lower doses) with BVDU failed to increase the survival of the leukemic mice. If, however, FU was administered in fractionated doses, there was a dose-dependent increase in survival time of P388-leukemic mice, and simultaneous fractionated BVDU treatment allowed a 4-6-fold lower dose of FU to achieve the same increase in survival time, indicating that BVDU effectively increased the antitumor activity of FU in the leukemic mice.
In some animal species substance P and neurokinin A (NKA) cause bronchoconstriction by the release of acetylcholine from postganglionic cholinergic nerve endings. The aim of the present study was to investigate the effect of an anticholinergic drug, oxitropium bromide, on bronchoprovocation with NKA in asthmatics. Eleven mild asthmatics (mean % predicted FEV1 85.5) received on 2 separate days, double blind, in a randomised order, 400 mcg oxitropium bromide or placebo, 90 min before challenge with NKA. NKA was inhaled at 3 concentrations (10(-4), 3.10(-4) and 10(-3) M). Specific airways conductance (sGaw) and forced expiratory volume in 1 s (FEV1) were used as parameters of airway calibre. Compared to the placebo-aerosol, oxitropium bromide caused a significant increase in sGaw and FEV1. On the placebo-treatment day, NKA caused a concentration-dependent decrease in sGaw and FEV1. The percentage changes in sGaw and FEV1 on the oxitropium day were not statistically different from those occurring on the placebo day. We conclude that oxitropium bromide caused a significant bronchodilation but offered no significant protection against NKA-induced bronchoconstriction in mild asthmatic subjects.
A series of base-modified pyrimidine 3'-azido-2',3'-dideoxynucleosides and 3'-substituted purine and pyrimidine 2',3'-dideoxynucleosides have been synthesized and evaluated for their inhibitory activity against human immunodeficiency virus (HIV) replication in MT-4 cells. The following pyrimidine derivatives emerged as the most potent and/or selective inhibitors of HIV-induced cytopathogenicity (in order of decreasing selectivity: 3'-azido-3'-deoxythymidine (AZT), 3'-azido-2',3'-dideoxyuridine (AzddUrd), 3'-azido-2',3'-dideoxy-5-methylcytidine (AzddMeCyd), 3'-fluoro-ddUrd (FddUrd), 3'-fluoro-ddThd (FddThd), the N4-hydroxylated derivative of AzddMeCyd and the N4-methylated derivative of AzddMeCyd. Among the purine 2',3'-dideoxynucleosides, 3'-azido-2',3'-dideoxyguanosine (AzddGuo), 3'-fluoro-ddGuo (FddGuo), and 3'-fluoro-2,6-diaminopurine 2',3'-dideoxynucleoside (FddDAPR) were the most selective inhibitors of HIV replication.