Didemnins: antiviral and antitumor depsipeptides from a caribbean tunicate.

Extracts of samples of a Caribbean tunicate (ascidian, sea squirt) of the family Didemnidae inhibit in vitro at low concentrations the growth of DNA and RNA viruses as well as L1210 leukemic cells. The active compounds isolated from the tunicate, didemnins A, B, and C, are depsipeptides, and didemnin B (a derivative of didemnin A) is the component active at the lowest concentration in inhibiting viral replication in vitro and P388 leukemia in vivo.

Effects of streptovaricins and their degradation products on infectivity of Rauscher leukemia virus.

The virucidal effects of streptovaricin (Sv) A, SvC, SvD, streptoval (Sval) C, Sval Fc, and streptovarone were evaluated by incubation of the drug with Rauscher leukemia virus (RLV) at 37 degrees C for 60 minutes prior to dillution and addition to cells (in vitro assay) or before ip injection into animals (in vivo assay). The in vitro and in vivo assays were plaque formation and splenomegaly, respectively. A dose-related effect was observed with all six compounds with the in vitro assay. On an equimolar basis, the Sv degradation products, i.e., Sval C, Sval Fc, and streptovarone were most inhibitory, followed by SvD; SvA and SvC were least active. At 0.0625 mumoles, the three Sv degradation products inactivated over 90% of the RLV. Similar results were obtained through the in vivo assay. At 0.06 mumoles, streptovarone, Sval C, and SvD showed 78,62, and 29% inhibition of splenomegaly, respectively; SvA and SvC were essentially inactive. A direct relationship was observed between inhibition on RNA-directed DNA polymrase of RLV by these compounds and their virucidal effects. No drug given at the time of injection, however, showed any significant effect on virus infective processes in vitro or in vivo. The reason for the lack of therapeutic effects of these compounds is discussed.

beta-D-Arabinofuran[1',2':4,5]oxazolo-1,3,5-triazine-5-N-methyl-4,6-dione and analogues, unusually specific immunosuppressive agents.

Sequential treatment of the protected beta-D-arabinofuran[1',2':4,5]-2-aminooxazoline (2) with methyl isocyanate and diimidazole carbonyl afforded the 2,2'-anhydro-beta-D-arabinofuranosyl nucleoside, 6. Deprotection and hydrolysis yielded the corresponding arabinoside. Although the anhydronucleoside exhibited in vitro antiviral activity against herpes simplex type 1, it exacerbated the infection in vivo. Further examination uncovered an in vitro inhibition of the induction of a cell-mediated immune response without cytotoxicity.

Unsaturated and carbocyclic nucleoside analogues: synthesis, antitumor, and antiviral activity.

A series of unsaturated analogues of nucleosides were prepared and their cytotoxic, antitumor, and antiviral activities were investigated. Alkylation of cytosine with (E)-1,4-dichloro-2-butene gave chloro derivative 2f, which was hydrolyzed to alcohol 2h. Cytosine, adenine, 2-amino-6-chloropurine, thymine, and (Z)-1,4-chloro-2-butene gave compounds 4c-f, which, after hydrolysis, afforded alcohols 4a, 4b, 4g, and 4h. Alkenes 4d and 4e were cyclized to heterocycles 12 and 13. Alkylation of 2,6-diaminopurine with 1,4-dichloro-2-butyne led to chloro derivative 6a, which was hydrolyzed to alcohol 6b. Allenic isomerization of 6b gave compound 5c. Chloro derivatives 2e-g, 4c-f, 5d, and 6c-e as well as pyrimidine oxacyclopentenes 9c and 9d are slow-acting inhibitors of murine leukemia L1210 of IC50 10-100 microM. The most active were analogues 4c, 4d, 4e, and 6e (IC50 10-20 microM). The corresponding hydroxy derivatives were less active of inactive. Inhibition of macromolecular synthesis with compounds 4c, 4d, 6e, 9c, and 9d follows the order: DNA greater than RNA greater than or equal to protein. Cytotoxic effects of 4c, 6e, and 9d are not reversed with any of the four basic ribonucleosides or 2'-deoxyribonucleosides. Inhibitory activity of cytosine derivative 9c is reversed with uridine and 2'-deoxyuridine but not with the corresponding cytosine nucleosides. Zone assays in several tumor cell lines show that active compounds are cytotoxic agents with little selectivity for tumor cells. Analogue 6c showed 16.7% ILS in leukemia P388/o implanted ip in mice at 510 and 1020 mg/kg, respectively. Cytallene (5b) and 6'beta-hydroxyaristeromycin (10) exhibited significant activity against Friend and Rauscher murine leukemia viruses. The rest of the hydroxy derivatives, with the exception of 4a, were moderately effective or inactive as antiviral agents. None of the chloro derivatives or oxacyclopentenes exhibited an antiviral effect at noncytotoxic concentrations. Z-Olefin 4b and 2-aminoadenallene (5c) are substrates for adenosine deaminase.

Pyrimidinones. 1. 2-Amino-5-halo-6-aryl-4(3H)-pyrimidinones. Interferon-inducing antiviral agents.

Interferon induction and antiviral activity was discovered with 2-amino-5-bromo-6-phenyl-4(3H)-pyrimidinone. An analogue study incorporating a series of 2-amino-5-substituted-6-arylpyrimidinones revealed that the most potent interferon inducers were mono- and difluorophenyl analogues. These same analogues were also potent antiviral agents against Semliki Forest virus and herpes simplex type 1. In addition the monomethoxyphenyl analogues were potent antiviral agents but weak interferon inducers. Relatively modest structural changes led to dramatic changes in bioactivity. There was a relatively poor correlation between levels of circulating interferons induced and systemic antiviral activity.

Bilayer stabilizing peptides and the inhibition of viral infection: antimeasles activity of carbobenzoxy-Ser-Leu-amide.

A number of carbobenzoxy-dipeptide-amides raise the bilayer to hexagonal phase transition temperature of dielaidoylphosphatidylethanolamine (stabilizes the bilayer). The potency of the peptides in stabilizing the bilayer phase is Z-Tyr-Leu-NH2 = Z-Gly-Phe-NH2 greater than Z-Ser-Leu-NH2 greater than Z-Gly-Leu-NH2 greater than Z-Gly-Gly-NH2. A linear correlation was found between the respective HPLC retention time parameter k' for the peptide and the slope of the bilayer stabilization curve determined with model membranes by differential scanning calorimetry. One dipeptide, Z-Ser-Leu-NH2, reduces measles virus cytopathic effect (CPE) in Vero cells. The mechanism by which this peptide reduces the CPE is not known, although some peptides which raise the bilayer to hexagonal phase transition temperature of phospholipids inhibit membrane fusion.

Influenza virus infection of hamsters. A model for evaluating antiviral drugs.

Inoculation of hamsters with influenza virus [A/PR/8/34 HON 1] produces an inapparent infection which can be monitored by virus titrations of nasal washes or of homogenates prepared from trachea or lung. Antibody can be detected in the serum within 7 days following virus inoculation. Hamsters previously infected were found to be resistant to challenge with the same virus. The utility of this model for evaluating anti-influenza drugs was demonstrated with two compounds. Calcium elenolate, a virucidal agent, reduced the virus titers of nasal washes when the drug was given as nose drops near the time of virus inoculation so as to affect high drug concentrations in the nasal passages. Virazole, an inhibitor of virus replication, reduced the virus titers of the nasal washes when multiple drug treatments were given as nose drops in an effort to provide drug during the time of virus replication. The model described may provide a useful means of evaluating potential antiviral during candidates inasmuch as the drug can be delivered directly into the nasal passages in a non-fatal influenza infection in a convenient laboratory animal.

Chemotherapy of genital herpes simplex virus type 2 infections of female hamsters.

The antiviral activity of 1-beta-d-arabinofuranosylcytosine (ara-C, cytarabine, Cytosar), 5-iodo-2'-deoxyuridine (IdUrd), 9-beta-d-arabinofuranosyladenine (ara-A), and disodium phosphonoacetate (PAA) have been compared in herpes simplex virus type 2 (HSV-2)-infected primary rabbit kidney cells and in female hamsters with genital HSV-2 infection. In vitro, ara-C and IdUrd were more active than ara-A, and PAA was least active. In female hamsters with genital HSV-2 infection, intravaginal treatment with PAA or ara-A was more effective than either ara-C or IdUrd. PAA was more active than ara-A when treatment was initiated early (1 h) after infection. The activity of PAA was greatly reduced if initiation of treatment was delayed for 24 h. Both PAA and ara-A reduced the virus titers of the vagina and protected hamsters from death when the drugs were given by either the intravaginal or subcutaneous route, with intravaginal treatment being more effective.

5,6-Dihydro-5-azathymidine: in vitro antiviral properties against human herpesviruses.

5,6-Dihydro-5-azathymidine (DHAdT), a novel water-soluble nucleoside antibiotic, inhibits herpes simplex virus type 1 (HSV-1) in appropriately infected cell cultures to a greater extent than herpes simplex virus type 2 (HSV-2). Vaccinia virus was less susceptible than HSV-2, and pseudorabies virus yields were not reduced at the concentrations studied. Plaque formation by varicella-zoster virus was suppressed by DHAdT. DHAdT was slightly toxic to cells at concentrations that were inhibitory for HSV-1 and varicella-zoster virus. Thymidine and deoxyuridine completely reversed the anti-HSV-1 activity of DHAdT, whereas deoxycytidine was partially effective. Compared with other nucleoside analogs with activity for HSV-1, DHAdT was less active than 5-iodo-2'-deoxyuridine or 1-beta-d-arabinofuranosylcytosine and nearly equipotent with 9-beta-d-arabinofuranosyladenine.

Genital infection of female hamsters with herpesvirus hominis type 2 (HVH-2).

Intravaginal inoculation of female hamsters (40-65 g) with HVH-2 (6 X 105 PFU) results in an infection of 40-67% of the animals. The illness is characterized by vaginitis with discharge, paralysis, and finally death. Washing the vagina with saline 30 min prior to virus inoculation enhances the infection so that 80-100% of the animals eventually die. Virus can be isolated from the vagina within 1-2 days after inoculation. Evidence of infection of the spinal cord is apparent by the third day whereas virus could not be isolated from the brain until Day 5. Attempts to demonstrate virus in whole blood, spleen, kidney, or liver homogenates prepared from infected animals were negative.

Effect of poly I: C on experimental respiratory infection in hamsters.

The interferon inducer double-stranded polyinosinic acid and polycytidylic acid (poly I:C) was studied in hamsters experimentally infected with parainfluenza 3 virus. Upper intranasal, deep intranasal, or intraperitoneal treatment of hamsters with poly I:C (100 mug/100- to 120-g animal) 24 hr before an upper respiratory infection significantly reduced the virus yields taken 28 hr after infection. Deep intranasal and intraperitoneal treatment with poly I:C greatly decreased the virus titers in the lungs, as measured 48 hr after a deep lung infection with parainfluenza 3 virus; however, the upper respiratory poly I:C treatment was ineffective.

Inactivation of myxoviruses by calcium elenolate.

Calcium elenolate inactivates all myxoviruses so far tested. The pH of the reaction mixture is less critical for myxovirus inactivation than that required for coxsackie A-21 virus; the myxoviruses are inactivated at a broad spectrum of pH with the maximum activity occurring at a pH below 7.0. The infectivity of the virus is more susceptible to the action of calcium elenolate than is either the neuraminidase activity or the hemagglutinin. The inactivation of Newcastle disease virus by calcium elenolate also destroys the ability of the virus to induce interferon formation in cell culture and in mice.

Antiviral activity of cytarabine in herpesvirus-infected rats.

Intranasal inoculation of herpesvirus (approximately 1.8 mean lethal doses [LD(50)] in 0.1 ml) into 105- to 115-g rats produces paralytic disease in 4 to 5 days and 80 to 100% mortality in 8 to 12 days. Cytarabine (ara-C) (40 to 320 mg/kg), administered subcutaneously to inoculated rats, delays the onset of paralysis and protects the animals from death. Drug treatments were given twice daily for 5 days. Beneficial drug effects were observed even when initiation of therapy was delayed for 3 days after virus inoculation. A dose-response relationship existed when therapy was initiated at 4 h after virus inoculation. However, when therapy was delayed for 3 days, it appeared that the highest drug level (320 mg/kg twice daily) was somewhat less effective than the lower doses (160 and 80 mg/kg twice daily). Virus could be detected in homogenates of brain beginning 3 days after inoculation, and the titer increased through 7 days. Ara-C treatment, initiated 4 h after inoculation, caused a delay in the appearance of virus, and a reduction in the titer in the brain homogenates. No virus was detected in blood serum, or in homogenates prepared from lung, kidney, thymus, or spleen of infected rats. The virus titration studies are in agreement with the illness and mortality produced by herpesvirus infection.

Activities of 5,6-dihydro-5-azathymidine against herpes simplex virus infections in mice.

5,6-Dihydro-5-azathymidine (DHAdT), a nucleoside antibiotic inhibitory for herpes simplex virus (HSV) in cell cultures (H. E. Renis, Antimicrob. Agents Chemother. 13: 613-617, 1978), was evaluated in mice with experimental HSV infections. DHAdT protected mice infected with HSV type 1 (HSV-1) when the virus was inoculated intravenously and the drug was given by subcutaneous or oral routes. The activity observed was dependent on the dose and schedule of treatment. Doses of 100 to 400 mg/kg given three to four times daily (at 4-h intervals) for 4 to 5 days gave greater protection than less frequent treatment for shorter time intervals. DHAdT treatment reduced the rate of isolation as well as the HSV-1 titers in homogenates prepared from spinal cords and brains, whereas the titers in kidney homogenates were only marginally affected. The above treatment regime with DHAdT afforded only partial protection to mice infected intracerebrally with HSV-1 or mice inoculated intravaginally with HSV-1 or HSV-2. The antiviral activity of DHAdT was reversed by the co-administration of thymidine. Under these conditions, DHAdT was not toxic in mice.

Antiviral melanization reaction of Heliothis virescens hemolymph against DNA and RNA viruses in vitro.

1. Antiviral activity of Heliothis virescens larval hemolymph was determined using a cytotoxicity/virus inhibition test (TClD50) done in Vero cell tissue cultures. Excellent antiviral activity was found especially against herpes simplex viruses-1 and -2 and also against vesicular stomatitis, parainfluenza-3, coxsackie B3 and sindbis viruses. 2. Prolonged incubation of herpes simplex virus-1 and vesicular stomatitis virus with hemolymph was virucidal and greatly reduced infectivity of the two viruses in tissue culture. 3. Antiviral activity was produced by both normal and immune (vaccinated larvae) cell-free hemolymphs. 4. Antiviral activity against herpes simplex virus-1 could be generated in vitro with hemolymph phenoloxidase or mushroom tyrosinase using four different substrates including tyrosine. 5. Activation of the insect melanization reaction by phenoloxidase was necessary for antiviral activity to occur.

Bropirimine-induced embryolethality after oral administration to the pregnant rat.

Oral bropirimine (an immunomodulator shown to induce interferon) was administered to timed-pregnant Sprague-Dawley rats in five experiments utilizing several different dosing schedules. Concentrations of 100, 200, and 400 mg/kg of bropirimine were used. Interferon levels were determined in maternal serum, spleen, and whole embryo extracts and uterine contents were evaluated for survival of the embryos. Maternal toxicity occurred in all experiments as evidenced by dose-related decreases in body weight during the first 24 hr postdosing. Hematoxicology analyses of maternal serum revealed significant decreases in urea nitrogen, potassium, and albumin, along with increases in aspartate transaminase, alanine transaminase, and total bilirubin, in bropirimine-treated dams as compared to the vehicle controls. In addition, the means for maternal thymus weight decreased while the means for spleen weight increased with increasing concentration of bropirimine. As compared to the vehicle controls, interferon titers were high in maternal serum, maternal spleen, and, to a lesser extent, whole embryos, 2 hr postdosing, but had decreased or were below detectable levels 24 hr postdosing. Embryolethality was pronounced (increases in pre- and postimplantational loss) after a single dose (Gestation Day 3, 4, 5, 8, 9, or 10) of bropirimine, as well as after 7 or 8 consecutive days (Gestation Days 6-12 or 6-13) of treatment. Although embryotoxicity never occurred in these experiments in the absence of pronounced maternal toxicity, the pregnant dams never died as the result of bropirimine treatment, whereas the embryos frequently failed to survive.