Synthesis and cytotoxicity on sensitive and doxorubicin-resistant cell lines of new pyrrolo[2,1-c][1,4]benzodiazepines related to anthramycin.

A new 7,8-methylenedioxy analogue (4) of (+)-porothramycin B (2) and its water-soluble sodium bisulfite derivative (15) have been synthesized in high yields and have been shown to exhibit high cytotoxic activities against several tumor cell lines. The new pyrrolo[2,1-c][1,4]benzodiazepine 4 was as effective against the resistant cell lines as against the doxorubicin-sensitive cell lines tested.

Cytotoxic activities of novel hexahydroindolizino[8,7-b]indole derivatives prepared by 1,3-dipolar cycloaddition reactions of 3,4-dihydro-beta-carboline ylides.

A series of 1-cyano and 2-cyanohexahydroindolizino[8,7-b]indole derivatives was prepared by 1,3-dipolar cycloaddition of acrylonitrile with ylides derived from 3,4-dihydro-beta-carboline and its 6-methoxy, 6-benzyloxy, 9-methyl and 9-benzyl analogues. The products, together with their reduced 1- or 2-aminomethyl derivatives, were evaluated for cytotoxic activity in L1210 cancer cells. Compounds derived from 6-benzyloxy or 9-benzyl-3,4-dihydro-beta-carboline were found to be the most active, with IC(50)'s in the 2-50 microM range. Of these, two compounds, the 1- and 2-cyano 8-benzyloxyindolizino[8,7-b]indole derivatives 20a and 20c, respectively, were found by cytometric flux analysis to stop cancer cell growth at the G(2)M and 8N (>G(2)M) stage of the cell cycle. These two compounds also showed no loss of cytotoxic activity in K562R cancer cells resistant to doxorubicin.

[The worldwide challenges of "new" or reemerging communicable diseases at the dawn of the 21st century]. / Les défis mondiaux des maladies infectieuses "nouvelles" ou résurgentes en ce début de XXIe siècle. Deuxième partie.

In the first part of this review, AIDS, prion diseases, Hantavirus and arbovirus infections, Ebola hemorrhagic fever, legionellosis, hepatitis C, enterotoxigenic Escherichia coli infections, Lyme disease, tuberculosis have provided alarming examples of emerging or reemerging infectious diseases. In this second part, the stress is placed on the reemergence of diphtheria and of serious streptococcal infections, on bartonelloses, Chlamydia infections, fungal infections, while malaria and cholera are still prevalent in several areas. The increasing resistance of too many pathogens to antimicrobial agents is a major source of concern, directly related to the challenge of nosocomial infections. An infectious cause has been demonstrated (or strongly suspected) for various diseases and the scope of infectiology keeps widening, while the threat of bioterrorism cannot be neglected. The causes of the emergence or reemergence of infectious diseases are multiple and diverse, often in direct relation with human activities (population migrations, changes in husbandry or farming practices, worldwide exchanges of goods and foods, inadequate uses of antibiotics) but also with climatic variations in several areas. The challenge represented by this unexpected comeback of infections to the forefront of human and animal pathology can only be met with a significant improvement of hygienic practices, cessation of certain dangerous behaviors and also, of course, with the development of novel antimicrobial molecules (acting on original targets) as well as of a whole series of new specific vaccines.

[The worldwide challenges of "new" or reemerging communicable diseases at the dawn of the 21st century]. / Les défis mondiaux des maladies infectieuses "nouvelles" ou résurgentes en ce début de XXIe siècle.

In spite of the very significant advances made during the 20 th century in the prevention and the treatment of communicable diseases, infections are still today, even in developed countries, a major cause of morbidity and mortality. New infectious diseases have emerged (AIDS, legionellosis, exterotoxigenic E. coli, Ebola fever), others have significantly reemerged (tuberculosis, diphtheria, Bartonella infections) or have seen their geographic distribution widen considerably (dengue, Hantavirus, West Nile Virus, Lyme disease). New and widespread hepatotropic viruses (mainly hepatitis C) have been identified, while the bacterial cause (Helicobacter pylori) of gastric ulcer was demonstrated. The second part of this review will deal with other examples of emerging or reemerging infections and with the problem of the increasing resistance of pathogens to antimicrobial agents. It will analyse the multiple causes of these various phenomena and describe the diverse strategies which should become available for the prevention and/or treatment of these numerous infectious diseases.

Synthesis and cytotoxicity of gossypol related compounds.

Gossypol, gossypolone, reduced gossypol and new Schiff's bases of racemic gossypol and gossypolone were extracted or synthesized. Their cytotoxic activities on KB human cancer cells were determined. Gossypolone and the ethylamine derivative of gossypolone were the most active compounds (IC(50) in the micromolar range in both cases). The cytotoxicity of gossypol and gossypolone was increased when the tests were performed in the absence of serum and decreased when catalase as well as mannitol were added to the culture medium.

Immunostimulating agents: what next? A review of their present and potential medical applications.

Many chemical entities, either from natural sources or prepared by synthesis, are known to exert stimulating activities on various functions of the immune system, such as antibody production, resistance to infections, rejection of malignant cells, etc. In this review, the origin, chemical structures and main activities of several immunostimulants are described, with special emphasis on their present or potential medical usefulness. An attempt is made to envisage the future of this type of pharmacological agents, excluding however from the presentation the endogenous modulators of the immune system (cytokines), the production and activities of which are influenced by the immunostimulants themselves.

Studies on photoinactivation by various phthalocyanines of a free or replicating non-enveloped virus.

The non-enveloped picornaviruses, which are particularly resistant to physicochemical inactivation, include the aetiological agents of poliomyelitis, hepatitis A and E and infectious common cold (rhinovirus). In this work we used human rhinovirus type 5 (RV-5) cultivated in VERO cells to study the photoinactivating effects of several phthalocyanines and naphthobenzoporphyrazines. Free RV-5 was photoinactivated by aluminium trisulphonated naphthobenzoporphyrazine at 5 x 10(-8) M concentration. This photosensitizer was also active on replicating virus when the infected VERO cells were treated with 5 x 10(-6) M concentration followed by a very short illumination period. On the other hand, the ZnPc(3-MeO-Py)4 phthalocyanine, which possesses four positive charges, does not photoinactivate free rhinovirus, but this molecule protects VERO cells against RV-5 infection when added to the cultures before virus inoculation, in the presence or absence of subsequent illumination, and may therefore be considered as an antiviral agent in itself.

[Current situation of synthetic immunostimulants]. / Situation actuelle des immunostimulants de synthèse.

In the novel field of immunopharmacology, one finds two main classes of potential drugs: immunosuppressive agents (autoimmune disorders, transplantations) and immunostimulants. The latter aim at stimulating the humoral and cell-mediated functions of the immune system, in order to treat various immunodeficiency states and to increase defenses against infectious agents and cancer cells. Some immunostimulants (the so-called adjuvants) enhance the immunogenicity of vaccines. Immunostimulants act directly on various cell populations of the immune system and modulate the production and activity of molecular mediators of the latter (cytokines). From a chemical perspective, one must distinguish between natural macromolecular immunostimulants obtained by extraction or genetic engineering and compounds prepared by chemical synthesis. In the latter class, we find many molecules the structures of which derive from those of natural substances (eucaryotic and procaryotic peptides) and a large number of purely synthetic compounds. The latter belong to a wide variety of chemical families and their mechanisms of action are not always perfectly elucidated. Presently, whereas natural macromolecular immunostimulants (bacterial extracts, cytokines, growth factors) are being increasingly used in human medicine, only a small number of synthetic immunostimulants are beyond the stage of preclinical evaluation or phase I clinical studies.

[Humoral and cellular immunity in influenza virus infection]. / Immunité humorale et immunité cellulaire dans l'infection par les virus de la grippe.

In man, humoral immunity against influenza viruses (induced by presently available vaccines) is exerted by local and circulating antibodies against two surface antigens of the virions: hemagglutinin (HA) and neuraminidase (NA). Within each major antigenic type of influenza virus (A, B and C) these antibodies are strictly specific of the HA and NA characterizing variable sub-types, which appear in the course of time from an epidemic to the next one. There exists however a cell-mediated facet of immunity: study of the experimental infection of laboratory mice with mouse-adapted viruses has shown that this cellular immunity is type-specific and therefore covers all the sub-types of a given type of virus. This heterologous immunity is primarily mediated by cytotoxic T lymphocytes (CTL) which recognize an internal nucleoprotein (NP) common to all the sub-types. These CTL are able to lyse influenza virus-infected cells and contribute to local production of interferon in the course of infection. Such mechanisms likely play a major role in the natural resistance of the host to this infection. Thanks to the molecular characterization of the NP protein, it is now possible to conceive the design of vaccines endowed with a wider spectrum than those presently used; in association with the latter, it should become possible in the future to stimulate efficiently both facets of anti-influenza immunity.

Identical photosensitizing activities of a sulfonated aluminium phthalocyanine on human erythroleukemia cell lines susceptible or resistant to the cytotoxic activity of doxorubicin.

In this paper we report that a human erythroleukemia cell line made 100 times more resistant than the parental line to the cytostatic activity of doxorubicin and spontaneously 500-1000 times more resistant to the cytostatic activity of an unrelated drug, namely taxol, exhibits on the other hand unchanged susceptibility to the photosensitizing activity of a sulfonated phthalocyanine.

[Chemoprophylaxis and chemotherapy of common colds caused by rhinoviruses: overview and outlook]. / Chimioprophylaxie et chimiothérapie des rhumes caués par les rhinovirus: bilan et perspectives.

Rhinoviruses are the main etiologic agents of infectious common colds, which represent about 40% of the acute respiratory infections in man. The antigenic diversity of rhinoviruses precludes any prevention by vaccination. Within the last 20 years, efforts have therefore concentrated on chemoprophylaxis or chemotherapy with antiviral agents. Interferons (alpha and beta) administered intranasally at high doses, exerted a significant prophylactic activity in volunteers inoculated with rhinoviruses or in the course of epidemics in families. By contrast, the therapeutic activity of interferons is almost nonexistent, which greatly limits their practical interest. Various synthetic chemicals exert a marked and selective inhibitory effect on the replication of various serotypes of rhinoviruses in cell cultures. In the absence of an animal model of rhinovirus infection, in vivo studies of these molecules have been performed in human volunteers, chiefly at the Common Cold Research Unit in Salisbury (Great Britain). 3 synthetic compounds (an imidazothiazole, a benzimidazole derivative and a piperazinyl-pyridazine) have exerted a significant prophylactic activity, especially marked with the latter. None of these compounds, however, was active when administered after the infectious challenge. The search for selective anti-rhinovirus compounds is still going on; it will probably be facilitated through a combination of structural studies of rhinovirus capsids (or of their cellular receptors) with computer-assisted design of synthetic molecules. The recent observation of the influence of psychological factors on the propensity of rhinovirus-infected subjects to develop clinically apparent common cold must be taken into account in future studies.

Natural and synthetic peptides (other than neuropeptides) endowed with immunomodulating activities.

A large number of peptides, in most cases of low or relatively low molecular weight, exert immunomodulating activities, i.e., they interact in vitro with various cell populations of the immune system and, in vivo, enhance or depress, according to the case, cell-mediated and humoral immune functions. Immunomodulating peptides include glycopeptides from the bacterial cell wall, natural acyloligopeptide ciclosporin, peptidic hormones from the thytide cyclosporine, peptidic hormones from the thymus, peptidic fragments of immunoglobulins and other plasma proteins, as well as peptides isolated from food proteins. An amazing diversity of structure exists among the various immunomodulating peptides. The molecular mechanisms of interaction between these peptides and the cells of the immune system remain, in most cases, to be elucidated. Possibilities of therapeutic applications exist for many of these immunomodulating peptides: one of them (ciclosporin) is widely used as an immunosuppressive drug, several others (glycopeptides, lipopeptides, tuftsin, thymic peptides) are under clinical investigation as immunostimulating and/or immunorestoring agents.

Fluoride effects on 31P NMR spectra of macrophages.

31P High resolution nuclear magnetic resonance studies have been carried out on the P388D1 tumoral cell line and the BCG elicited alveolar rabbit macrophages both in sedimented cells and in perfused agarose-embedded cells. When the cells were sufficiently oxygenated, the phosphorylated sugars and ATP concentrations attained high levels. The intensity of the peak representing phosphorylated sugars varied inversely with ATP level when macrophagic cells were treated by NaF. The identities of the phosphorylated sugars were revealed by 1H and 31P NMR studies of the P 388D1 cells perchloric extracts.

Synthetic immunostimulants (excluding sulfur-containing compounds).

Many different approaches have been used, over the last 15 years, for the design of potential immunostimulating drugs: Fractionation of crude natural substances (of eukaryotic or prokaryotic origin) already known to enhance immune functions, followed by chemical characterization and, in many cases, full synthesis of the active moiety: examples are provided by thymic hormones and the muramyldipeptide (MDP); Chemical modification of natural substances of known chemical structure in order to potentiate or change their biological activities or reduce their toxicity: murabutide, lipophilic MDP derivatives, lipopeptides (such as pimelautide), tuftsin analogs; Chemical synthesis (often without preconceived ideas about structure-activity relationship) of a great variety of molecules which are then screened in vitro and in vivo for immunopharmacological activity. The chemical structures and the biological profiles (in terms of possible primary cellular targets and mechanisms of immunostimulating activities) of representatives of class (b) and class (c) immunostimulants are reviewed in this paper.

Studies on 44 081 R.P., a new antirhinovirus compound, in cell cultures and in volunteers.

A synthetic compound, 2-[(1,5,10,10a-tetrahydro-3H-thiazolo[3,4b]isoquinolin-3-ylidene) amino]-4-thiazoleacetic acid (S), 44 081 R.P., inhibits the multiplication of rhinoviruses in cell cultures. Of the 69 rhinovirus strains and serotypes that have been studied, 39% were inhibited at a concentration of 7 micrograms/ml, far below that which affects cellular metabolism (250 micrograms/ml). Preliminary data indicate that the compound inhibits some early events of virus replication but that some cellular functions are also involved in its mechanism of action. Despite its antiviral activity in vitro, the compound, when self-administered intranasally as a 0.2% solution to volunteers from the day before to 5 days after inoculation with a human rhinovirus strain, had no significant effect on rhinorrhea, clinical score, or laboratory evidence of infection.

Immunostimulating hexapeptide from human casein: amino acid sequence, synthesis and biological properties.

A hexapeptide obtained from human casein by enzymatic digestion has been purified, sequenced and synthesized; its structure is: Val-Glu-Pro-Ile-Pro-Tyr. In vitro this hexapeptide stimulates the phagocytosis of opsonized sheep red blood cells by murine peritoneal macrophages. Administered intravenously to adult mice, it enhances the resistance to infection with Klebsiella pneumoniae.

Synergistic activities of type I (alpha, beta) and type II (gamma) murine interferons.

Type I (alpha, beta) and type II (gamma) murine interferons are able to potentiate each other with respect to the inhibition of encephalomyocarditis (EMC) virus and of herpes simplex virus type 1 (HSV-1) multiplication in a murine cell line (DBT). Examination of two double-stranded RNA-dependent enzymes in DBT cells, the 2-5A synthetase and the 67,000 MW protein phosphokinase indicates that mixed interferon preparations act synergistically at least with respect to an increase in the activity of the former enzyme. The results obtained with gamma interferons of different origin and of different specific activity suggest that interferon itself, rather than the lymphokines present in the interferon preparations, is responsible for the synergistic effect.

1H NMR studies of natural and synthetic immunostimulating peptides in aqueous solution.

The peptides LAla-DGlu (L-alanyl-D-glutamic acid), LAla-DGlu[LLA2pm(Gly)]NH2, i.e. N2-(L-alanyl-D-gamma-isoglutaminyl)-N6-glycyl-LL-2,6-diaminopimelic acid, and LAla-DGlu[LLA2pm(Gly)-D-Ala], i.e. N2-(L-alanyl-D-gamma-glutamyl)-N6-glycyl-LL-alpha-2,6-diaminopimelyl-D-alanine, extracted from a Streptomyces strain and their immunostimulating synthetic lauroyl derivatives were studied by 1H NMR spectroscopy at 270 MHz. The chemical shift analysis confirms (a) the sequence of these compounds, (b) the occurrence of a gamma bond between Glu (or GluNH2) and A2pm, (c) the presence of a carboxamide group on the backbone of the tetrapeptide LAla-DGlu[LLA2pm(Gly)]. Temperature and pH studies strongly suggest that the tetrapeptide and pentapeptide exist under several folded conformations characterized by a proximity between the NH3 group of Gly and the Glu (or GluNH2) residue. THe similarity between the chemical shifts of the corresponding protons in LAla-DGlu, lauroly-LAla-DGlu and muramyl-L:Ala-DGluNH2 shows that the coupling of the dipeptide with a lauroyl or a muramyl group does not induce significant changes in the structure of the peptide moiety. In the lipopeptide series the presence of the lauroyl chain leads to amphipathic substances which form, micelles in aqueous solution as shown by the large increase of the proton linewidths. The immunostimulating properties of this kind of peptide manifest themselves at very low concentration and only after their coupling with a sugar or lipid moiety. These features can be interpreted by the formation of mixed complexes between the immunoadjuvants and cell membrane constituents.

Immunostimulating substances from human casein.

Delipidated human casein was digested with trypsin and the enzymatic digest was fractionated on Sephadex G-50. The peptidic fractions were assayed for immunomodulating activity in two in vitro models. Fractions corresponding to molecular weights in the range of 2,000 +/- 600 were found to possess stimulating activity in these models.