NMR investigations of the role of the sugar moiety in glycosylated recombinant human granulocyte-colony-stimulating factor.

Human granulocyte-colony-stimulating factor (G-CSF) is a hematopoietic growth factor that plays a major role in the stimulation of the proliferation and maturation of granulocyte neutrophil cells. With the recent increased understanding of its biological properties in vivo together with available preparations of recombinant human G-CSF, this growth factor has become an essential agent for clinical applications. The presence of an O-linked carbohydrate chain at position 133 greatly improves the physical stability of the protein. To clarify the molecular basis for the stabilisation effect of saccharide moieties on human G-CSF the whole glycoprotein expressed in CHO cells has been investigated by means of two 1H-NMR-spectroscopy and two 1H-detected-heteronuclear 1H-13C experiments at natural abundance, and compared with the non-glycosylated form. The present NMR study reports assignments of 1H and 13C resonances of the bound saccharidic chain NeuNAc(alpha2-3)Gal(beta1-3)[NeuNAc(alpha2-6)]GalNAc, where NeuNAc represents N-acetylneuraminic acid, and demonstrates the alpha-anomeric configuration of the N-acetylgalactosamine-threonine linkage. It also provides results suggesting that the carbohydrate moiety reduces the local mobility around the glycosylation site, which could be responsible for the stabilising effect observed on the glycoprotein.

Comparison of the potency of glycosylated and nonglycosylated recombinant human granulocyte colony-stimulating factors in neutropenic and nonneutropenic CD rats.

Recent studies in human bone-marrow culture and healthy human volunteers suggest that lenograstim [glycosylated, recombinant human granulocyte colony-stimulating factor (rHuG-CSF) produced in Chinese hamster ovary (CHO) cells] has greater in vivo potency than filgrastim [nonglycosylated, methionine-extended recombinant human granulocyte colony-stimulating factor (rmetHuG-CSF) produced in Escherichia coli]. To confirm and extend these results we investigated the in vivo potency of both products in normal rats and neutropenic CD rats as an animal model of chemotherapy-induced neutropenia. In normal rats, groups of eight normal male CD rats received four subcutaneous doses of 10, 30, or 100 micrograms/kg filgrastim or lenograstim on days 1-4 of the study, whereas a control group received the vehicle. Blood samples were collected from each animal before treatment (day -5) and on days 2, 3, 5, 8, and 12 of the study for determination of red blood cell (RBC), platelet, white blood cell (WBC), and differential counts. rHuG-CSF and r-metHuG-CSF produced increased WBC counts, principally due to elevated absolute neutrophil counts (ANCs); on days 2, 3, and 5, all groups receiving rG-CSF had ANCs that increased in a progressive and dose-related manner. With the exception of a single value, mean ANCs obtained on days 2, 3, and 5 in lenograstim-treated groups were higher (statistically significant on day 3 at 30 and 100 micrograms/kg and on day 5 at 10, 30, and 100 micrograms/kg) than the respective values obtained in filgrastim-treated groups. No compound-related effect was noted in RBC or platelet parameters. Neutropenia was induced in male CD rats (12 animals/group) with a single intraperitoneal dose of 50 mg/kg cyclophosphamide (CPA) on day 0. On days 1-4, CPA-treated groups were treated with the vehicle (control) or with filgrastim or lenograstim at 30 or 100 micrograms/kg per day. An additional group was not treated with CPA and served as the absolute control group. Blood was collected from alternating subgroups on study day -5 (pretest) and on days 2, 3, 4, 5, 6, 8, 9, and 12 for determination of RBC, platelet, WBC, and differential counts. No major adverse in-life effect was noted in neutropenic rats. Maximal depression of WBCs and ANCs occurred on day 5, followed by recovery to normal values by days 9 (ANC) and 12 (WBC). On day 3 and days 5-9, rHuG-CSF- and metHuG-CSF-treated groups had marked and dose-related increases in WBCs as compared with CPA-treated controls, principally due to elevated ANCs. With the exception of a few values, mean ANC values obtained in lenograstim-treated groups were consistently higher than the respective values obtained in filgrastim-treated groups; the difference was statistically significant on day 3 (30-microgram/kg groups) and on days 6 and 8 (100-microgram/kg groups). In conclusion, treatment of normal and neutropenic CD rats with lenograstim resulted in a dose-related elevation of ANCs that was consistently and significantly higher than the response to identical doses of filgrastim. These results suggest that lenograstim, the glycosylated form of rG-CSF, has superior in vivo potency in normal and neutropenic animals as compared with filgrastim, the nonglycosylated form of rG-CSF.

Metastatic phenotype of murine tumor cells expressing different cooperating oncogenes.

Four murine cellular tumor models expressing various combinations of oncogenes (SV40 large T and v-Ha-ras, SV40 large T and v-src, SV40 large T and neu, adenovirus EIA and v-Ha-ras) induce sarcoma when they are inoculated s.c. into the DBA/2 syngenic mice. The metastatic patterns, distribution and fate of these tumor cells transplanted by two different routes into syngenic DBA/2 mice have been studied. All the tumor cell lines except EIA-ras, induce massive overt artificial metastases principally in the lung after i.v. injection. In s.c. tumor-bearing mice, a few resting cells colonize the lung as micrometastases. When removed from this tissue context and injected s.c. these cells regain their proliferative potential and grow as local tumors which again give rise to occult pulmonary micrometastases.

Mode of action of the antitumor compound girodazole (RP 49532A, NSC 627434).

Girodazole (RP 49532A) or 3-amino-1-[4-(2 amino-1H-imidazolyl]-propanol, 2HCl is an experimental antitumor compound which inhibits protein synthesis in cell cultures and in cell free systems. The compound has been evaluated for its capacity to inhibit specific assays of initiation, elongation and termination of protein synthesis. Girodazole inhibited the release of nascent peptides from polyribosomes in rabbit reticulocyte lysates indicating that the major effect of the compound is on the protein synthesis termination step.

Antitumor activity and mechanism of action of the marine compound girodazole.

Girodazole, a new marine compound has been isolated from the sponge Pseudaxinyssa cantharella. Girodazole is active in vivo on several murine grafted tumors including leukemias (P388, L1210, i.p./i.p.) and solid tumors (MA 16/C mammary adenocarcinoma, M5076 histiocytosarcoma, s.c./i.v.). In addition, girodazole has identical cytotoxic properties in vitro on P388 and P388/DOX cells and retains antitumor activity in vivo on P388/DOX. Girodazole has a unique chemical structure different from those of known anticancer agents and of new compounds undergoing clinical trials. Biochemical studies indicate that girodazole inhibits protein synthesis during the elongation/termination steps. Toxicological studies have been done in mice and in dogs and did not reveal any major toxic effect which could preclude administration in patients. Girodazole is now undergoing phase I clinical studies.

Synthesis of new (+-)-3,5-dihydroxypentyl nucleoside analogues from 1-amino-5-(benzyloxy)pentan-3-ol and their antiviral evaluation.

The synthesis and antiviral evaluation of a series of (+-)-3,5- dihydroxypentyl nucleoside analogues related to acyclic nucleoside antiviral agents are reported. All purine and pyrimidine nucleoside analogues described in this paper have been obtained from 1-amino-5-(benzyloxy)pentan-3-ol. A synthesis of this amine is reported from 1-(benzyloxy)but-3-ene after epoxidation and regiospecific diethylaluminum chloride catalyzed opening of the epoxide by trimethylsilyl cyanide. The compounds were tested in vitro in infected MRC5 and CEM cells. None of the compounds exhibited antiviral activity against HSV-1, HCMV, and HIV-1 with the exception of the guanine derivative 7, which inhibited the cytopathic effect of HSV-1 by 50% at 12.5 micrograms/mL.

Inhibition of simian virus 40 DNA replication in CV-1 cells by an oligodeoxynucleotide covalently linked to an intercalating agent.

An octathymidylate covalently linked via its 3'-end to an acridine derivative inhibited the cytopathic effect of Simian Virus SV40 on CV-1 cells in culture. Control experiments revealed that this effect was virus-specific and did not arise as a result of oligonucleotide degradation by nucleases. A photoactive probe was covalently attached to the 5'-end of the oligonucleotide-acridine conjugate. Upon UV-irradiation, photocrosslinking was shown to occur at the A. T-rich region within the viral origin of replication. A local triple helix can form at moderate salt concentrations with two octathymidylate-acridine conjugates bound to the octaadenylate sequence. Alternatively the octathymidylate-acridine conjugate can bind to the major groove of duplex DNA forming a local triple helix. Different mechanisms are discussed to explain the inhibition of viral DNA replication.

Protective activity of tetracycline analogs against the cytopathic effect of the human immunodeficiency viruses in CEM cells.

Tetracycline analogs were evaluated for anti-HIV activity in CEM cells; minocycline and doxycycline were the most active of these in inhibiting the virus-induced cytopathic effect between 7 and 14 days post-infection. The active concentrations (0.3-1.5 micrograms/ml) were devoid of toxicity in uninfected cultures. Virus production, however, was not inhibited, indicating a dissociation between protection against cell death and suppression of virus growth. These protected cells could be maintained in culture for 6-7 weeks, even in the absence of the compounds. After that period, virus production ceased and cells could then be cultivated for several months without loss of viability or reappearance of virus production. As HIV stocks produced in the presence of tetracycline analogs were unable to induce cell death, we suggest that the cytopathogenicity of HIV may be due in some cases to the presence of tetracycline-sensitive contaminating microorganisms.

Selective inhibition of tyrosine protein kinase by a synthetic multisubstrate analog.

Synthetic compounds were designed in an attempt to mimic the possible transition state of tyrosine protein kinases. One representative compound (RP 53801) inhibited the enzyme purified from RSV-transformed cells. A serine/threonine kinase (kinase C) was 45 fold less sensitive. The inhibition was competitive with respect to ATP and noncompetitive with respect to the phosphate acceptor poly glu4-tyr1. The degree of inhibition (IC50 = 22 microM) was however lower than that expected from a transition state analog. The compound was capable of reducing tyrosine protein kinase activity in intact cells with some selectivity at 100 microM.

Selective inhibition of the cytopathic effect of type A influenza viruses by oligodeoxynucleotides covalently linked to an intercalating agent.

Oligodeoxynucleotides covalently linked to an acridine derivative were targeted to part of the 3'-terminal sequence which is common to the eight RNAs of type A influenza viruses. The cytopathic effect of the virus on MDCK cells in culture was strongly decreased by a heptanucleotide covalently attached to the acridine ring. Control experiments using other oligonucleotide sequences showed that the effect was specific for the complementary sequence of the 3'-terminal region of the viral RNAs. The RNA transcriptase reaction of a type A virus was also selectively inhibited in vitro by the heptanucleotide-acridine conjugate. A type B influenza virus was used as a control. The common sequence at the 3' end of its eight viral RNAs is different from that of type A viruses. Three mismatches were expected with the heptanucleotide which was fully complementary to type A viral RNAs. This heptanucleotide had no effect on the cytopathic effect of a type B influenza virus. These results demonstrate that viral RNAs are specific targets for the oligonucleotide-acridine conjugate that inhibits the cytopathic effect of type A influenza viruses.

Inactivation of herpes simplex virus by protein components of bovine neutrophil granules.

From an acid extract of granules of bovine neutrophils we isolated fractions of cationic proteins, exhibiting significant anti-herpesvirus activity at concentrations which were devoid of cytotoxicity and of activity against a picornavirus (rhinovirus). The mechanism of action seems to involve a direct neutralization of the virions. Two antiviral peptides with an approximate MW 7500 were purified to homogeneity by reversed phase high-performance liquid chromatography. Proline and arginine accounted for about 43% and 26-27% of their amino acid residues. One of these peptides (IIIa2 beta) had an MIC of 2 microM.

Antirhinovirus compound 44,081 R.P. inhibits virus uncoating.

44,081 R.P., or 2-[(1,5,10,10a-tetrahydro-3H-thiazolo[3,4-b]isoquinolin- 3-ylidine)amino]-4-thiazole acetic acid, is a compound which selectively inhibits rhinovirus in cell cultures. The compound, which was unable to inactivate infectivity of virions, appeared to act prior to RNA and protein synthesis without affecting adsorption or penetration of virus in MRC5 cells. In contrast, it protected intracellular [5-3H]uridine-labeled virions against the effects of RNase treatment, indicating that inhibition of virus uncoating is the mode of action of 44 081 R.P.

(+/-)-2-Amino-3,4-dihydro-7-[2,3-dihydroxy-4-(hydroxymethyl)-1- cyclopentyl]-7H-pyrrolo[2,3-d]pyrimidin-4-ones: new carbocyclic analogues of 7-deazaguanosine with antiviral activity.

5-Allyl-2-amino-4,6-dihydroxypyrimidine (3) was chlorinated and ozonized to yield (2-amino-4,6-dichloro-pyrimidin-5-yl)acetaldehyde (5). Acetalization of 5 with ethanol afforded a new pyrimidine intermediate 6 which can lead to 2-amino-3,4-dihydro-7-alkyl-7H-pyrrolo[2,3-d]pyrimidin-4-ones and therefore to carbocyclic analogues of 7-deazaguanosine. The 7-substituent was a cyclopentyl analogue of the arabinofuranosyl moiety in 10a, lyxofuranosyl moiety in 10b, and ribofuranosyl moiety in 10c. Compounds 10a and 10b exhibited selective inhibitory activities against the multiplication of HSV1 and HSV2 in cell culture. Repeated administration of compound 10a at 10mg/kg ip to mice infected with HSV2 increased the number of survivors and lengthened significantly the mean survival time.

[Use of permeabilized cells in the study of inhibitory products of herpes virus replication]. / Utilisation de cellules perméabilisées dans l'étude de produits inhibiteurs de la replication du virus de l'herpès.

Cells made permeable by exposure to lysolecithin following infection by HSV-1 synthesize DNA (in greater amounts than non-infected cells) in the presence of the four deoxyribonucleoside-triphosphates (dNTPs) : dATP, dCTP, dGTP, and dTTP. DNA synthesis also occurs if dTTP is replaced by dT or dTMP, indicating activity of enzymes such as thymidine kinase, thymidylate kinase, deoxyribonucleoside-diphosphate kinase and ADN polymerase. Examination of DNA synthesis in permeabilized cells enables detection of antiviral activity of agents incapable of penetrating into intact cells and therefore ineffective in cell cultures. No detectable protein-tyrosine kinase activity was found in HSV-1 infected cells.

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.

[Comparative effects of 4 cephalosporins on the incorporation of tritiated diaminopimelic acid during bacterial growth]. / Effects comparés de quatre céphalosporines sur l'incorporation d'acide diaminopimélique tritié au cours de la croissance bactérienne.

When comparing antibiotic activities, it might be of interest to study parameters other than minimal inhibitory concentrations (MIC's). Specific activity of beta-lactams on bacterial cell wall makes it possible to determine radiolabelled diaminopimelic acid (DAP) incorporation in growing cultures. We have studied the effects of various concentrations of cefalotin , cefotaxime, latamoxef (moxalactam) and ceftiolene (42 980 RP) on DAP incorporation in 6 strains of E. coli, E. cloacae and S. aureus. Drug concentration which inhibits 90 % of radioactivity incorporation (CII 90) was found to vary from 0.1 X MIC to 3 X MIC. This fact suggests that beta-lactam action on cell wall synthesis and/or structure and MIC's are not always strictly correlated.

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.

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.