Zanamivir: a rational approach to influenza B.

Influenza B viruses have co-circulated with the HIN1 and H3N2 subtypes of influenza A since 1977. Influenza A viruses are found in various animals, whereas influenza B viruses are probably restricted to humans. The lack of an animal reservoir means that the virus has no potential for genetic reassortment across species. In addition, influenza B viruses are more serologically homogeneous than influenza A viruses. Thus, the chance of influenza B causing a pandemic is much lower than that of influenza A. However, influenza B viruses are still a frequent cause of local disease outbreaks and epidemics as a result of antigenic drift. Any prophylactic or therapeutic measure must, therefore, be effective against both influenza A and B viruses. Zanamivir is the first widely approved neuraminidase inhibitor for the treatment of influenza. It is delivered directly to the primary site of viral replication, the respiratory tract, and is well tolerated and effective in the treatment of both influenza A and B. Data in prophylaxis are also encouraging. Zanamivir is the only drug proven to be clinically effective against both influenza A and B virus infections.

The synthesis and antiviral activity of 4-fluoro-1-beta-D-ribofuranosyl-1H-pyrazole-3-carboxamide.

A novel fluoropyrazole ribonucleoside has been shown to have significant anti-influenza activity in vitro. The compound is compared and contrasted with the structurally-related compound ribavirin in attempts to identify factors having significant bearing on the mode of action of both compounds.

Plasmodium falciparum lacks sialidase and trans-sialidase activity.

Sialic acid on the red cell surface plays a major role in invasion by the malaria parasite Plasmodium falciparum. The NeuAc(alpha 2,3) Gal motif on the O-linked tetrasaccharides of the red cell glycophorins is a recognition site for the parasite erythrocyte-binding antigen (EBA-175). Consequently, the interaction of P. falciparum and the red cell might share homology with that of the influenza virus. The cellular interactions of P. falciparum were examined for their sensitivity to 4-guanidino-2,3-didehydro-D-N-acetyl neuraminic acid (4-guanidino Neu5Ac2en), a potent inhibitor of influenza virus sialidase. Parasite invasion and subsequent development was unaffected by the sialidase inhibitor. The inhibitor did not affect rosette formation of parasite-infected erythrocytes with uninfected cells nor their cytoadherence to C32 melanoma cells. Furthermore, we were unable to confirm the presence of a previously reported parasite sialidase using sensitive fluorometric or haemagglutination assays, neither was any malarial trans-sialidase identified. We conclude that P. falciparum possesses neither sialidase nor trans-sialidase activity and that an inhibitor of influenza virus sialidase has no effect on important cellular interactions of this parasite.

Characterization of mutants of influenza A virus selected with the neuraminidase inhibitor 4-guanidino-Neu5Ac2en.

The development of viral resistance to the neuraminidase (NA) inhibitor, 4-guanidino-Neu5Ac2en, of influenza viruses was studied by serial passage of A/Turkey/Minnesota/833/80 (H4N2) in Madin-Darby canine kidney cells in the presence of increasing concentrations of inhibitor. Resistant mutants selected after eight passages, had a 10,000-fold reduction in sensitivity to the inhibitor in plaque assays, but their affinity (1/Kd) to the inhibitor was similar to that of the parental virus. Electron microscopic analysis revealed aggregation of the mutant virus at the cell surface in the presence of the inhibitor. Sequence analysis established that a substitution had occurred in the NA (Arg-249 to Lys) and in the HA2 subunit of the hemagglutinin (Gly-75 to Glu), in the vicinity of the proposed second sialic acid binding site. The change of residue 249 appears to be a chance mutation, for we were unable to reisolate this mutant, whereas subsequent experiments indicate changes in the hemagglutinin. After 13 passages of the parental virus, mutants that were resistant to the high concentrations of inhibitor tested were obtained. These viruses retained their drug-resistant phenotype even after five passages without the inhibitor. Electron microscopic analysis revealed no aggregation of virus on the surface of infected cells in the presence of the inhibitor. Sequence analysis of the NA gene from these drug-resistant mutants revealed an additional substitution of Glu to Ala at the conserved amino acid residue 119. This substitution is responsible for reducing the affinity of the inhibitor to the NA. Our findings suggest that the emergence of mutants resistant to 4-guanidine-Neu5Ac2en is a multistep process requiring prolonged exposure to the inhibitor.

Generation and characterization of variants of NWS/G70C influenza virus after in vitro passage in 4-amino-Neu5Ac2en and 4-guanidino-Neu5Ac2en.

The compounds 4-amino-Neu5Ac2en (5-acetylamino-2,6-anhydro-4-amino-3,4,5- trideoxy-D-glycerol-D-galacto-non-2-enoic acid) and 4-guanidino-Neu5Ac2en (5-acetylamino-2,6-anhydro-4-guanidino-3,4,5- trideoxy-D-glycerol-D-galacto-non-2-enoic acid), which selectively inhibit the influenza virus neuraminidase, have been tested in vitro for their ability to generate drug-resistant variants. NWS/G70C virus (H1N9) was cultured in each drug by limiting-dilution passaging. After five or six passages in either compound, there emerged viruses which had a reduced sensitivity to the inhibitors in cell culture. Variant viruses were up to 1,000-fold less sensitive in plaque assays, liquid culture, and a hemagglutination-elution assay. In addition, cross-resistance to both compounds was seen in all three assays. Some isolates demonstrated drug dependence with an increase in both size and number of plaques in a plaque assay and an increase in virus yield in liquid culture in the presence of inhibitors. No significant difference in neuraminidase enzyme activity was detected in vitro, and no sequence changes in the conserved sites of the neuraminidase were found. However, changes in conserved amino acids in the hemagglutinin were detected. These amino acids were associated with either the hemagglutinin receptor binding site, Thr-155, or the left edge of the receptor binding pocket, Val-223 and Arg-229. Hence, mutations at these sites could be expected to affect the affinity or specificity of the hemagglutinin binding. Compensating mutations resulting in a weakly binding hemagglutinin thus seem to be circumventing the inhibition of the neuraminidase by allowing the virus to be released from cells with less dependence on the neuraminidase.

Inhibition of replication of avian influenza viruses by the neuraminidase inhibitor 4-guanidino-2,4-dideoxy-2,3-dehydro-N-acetylneuraminic acid.

The sialidase inhibitor 4-guanidino-2,4-dideoxy-2,3-dehydro-N-acetylneuraminic acid (4-guanidino-Neu5Ac2en), designed with computer assistance and knowledge of the crystal structure of influenza virus neuraminidase, has shown antiviral effects in animal models of human influenza (M. von Itzstein et al., Nature, 363, 418-423, 1993). Here we demonstrate that the compound efficiently inhibits the enzyme activity of all nine subtypes of avian influenza A neuraminidase in vitro. When administered intranasally to chickens infected with lethal viruses, high doses of the compound (1000 micrograms/kg) protected 85% of birds harboring A/Chick/Victoria/1/85 (H7N7), a fowl plague virus, but not chickens infected with other highly virulent viruses of the N1, N2, or N3 subtype. This differential inhibitory effect was also seen in a plaque reduction assay with Madin-Darby canine kidney cells (MDCK), where 4-guanidino-Neu5Ac2en was more effective against A/Chick/Vic/85 (H7N7) than A/FPV/Rostock/34 (H7N1). In contrast to the substantial plaque reduction observed in MDCK cells, the drug failed to inhibit plaque formation in chicken embryo fibroblasts infected with either A/Chick/Vic/85 or A/FPV/Rostock/34, regardless of its concentration. The different levels of drug efficacy seen in two cell systems most likely reflect the location of virus budding and release in polarized versus nonpolarized cells, as well as the compound's mode of extracellular action.

The intracellular phosphorylation of (-)-2'-deoxy-3'-thiacytidine (3TC) and the incorporation of 3TC 5'-monophosphate into DNA by HIV-1 reverse transcriptase and human DNA polymerase gamma.

(-)-2'-deoxy-3'-thiacytidine (3TC) has been shown to be a potent, selective inhibitor of HIV replication in vitro, which requires phosphorylation to its 5'-triphosphate for antiviral activity. The intracellular concentration of 3TC 5'-triphosphate in phytohaemagglutinin (PHA)-stimulated peripheral blood lymphocytes (PBL) shows a linear dependence on the extracellular concentration of 3TC up to an extracellular 3TC concentration of 10 microM. At this extracellular concentration of 3TC, the resulting intracellular concentration of 3TC 5'-triphosphate is 5 microM. This value is similar to the inhibition constant (Ki) values for the competitive inhibition of human immunodeficiency virus type 1 (HIV-1) reverse transcriptase and human DNA polymerases (10-16 microM) by 3TC 5'-triphosphate. Since the concentration of 3TC producing 90% inhibition (IC90) of HIV replication in PBLs has been reported to be 76 nM, the antiviral activity of 3TC requires intracellular concentrations of 3TC 5'-triphosphate, which would result in very little inhibition of reverse transcriptase if its sole mode of action was competitive inhibition. This apparent discrepency may be explained by the ability of 3TC 5'-triphosphate to act as a substrate for reverse transcriptase. Primer extension assays have shown that 3TC 5'-triphosphate is a substrate for HIV-1 reverse transcriptase and DNA polymerase gamma, resulting in the incorporation of 3TC 5'-monophosphate into DNA. In the case of DNA polymerase gamma, the product of this reaction (i.e. double-stranded DNA with 3TC 5'-monophosphate incorporated at the 3'-terminus of the primer strand) is also a substrate for the 3'-5' exonuclease activity of this enzyme. This may explain the low levels of mitochondrial toxicity observed with 3TC.

4-Guanidino-Neu5Ac2en fails to protect chickens from infection with highly pathogenic avian influenza virus.

The effectiveness of the novel sialidase inhibitor 4-guanidino-Neu5Ac2en, which is highly effective in mouse and ferret models of influenza virus infection (von Itzstein et al. (1993) Nature 363, 418-423), has been assessed as a prophylactic agent in the prevention of infection of chickens with highly pathogenic avian influenza viruses. At best a small delay in the onset of pyrexia and death was observed with one strain of fowl plague virus, but not with two other strains. These results demonstrate that a locally acting drug may be ineffective if virus can escape from the site of inoculation and replicate elsewhere.

The kinetics of the acid-induced conformational change of influenza virus haemagglutinin can be followed using 1,1'-bis(4-anilino-5-naphthalenesulphonic acid).

1,1'-Bis(4-anilino-5-naphthalenesulphonic acid) (bis-ANS) has been shown by fluorescence spectroscopy to bind to bromelain-cleaved influenza haemagglutinin (BHA). The fluorescence intensity of 1.2 microM bis-ANS in the presence of BHA in its low-pH conformation is twenty-fold higher than in the presence of BHA in its neutral-pH conformation. The use of this probe provides a sensitive method for investigating the kinetics of the irreversible conformational change of BHA induced by low pH. At pH5.0 the reaction is described by a rapid burst followed by a double exponential increase in the fluorescence of bis-ANS, with rate constants of 5.2 +/- 0.9 x 10(-3) sec-1 and 6.7 +/- 1.9 x 10(-4) sec-1. This reaction is sensitive to the presence of tert-butylhydroquinone, an inhibitor of the conformational transition of BHA. The dependence of the reaction rate on pH indicates that the acid-induced conformational change is dependent upon the multiple protonation of the neutral-pH conformation of BHA.

Inhibition of influenza virus replication in mice by GG167 (4-guanidino-2,4-dideoxy-2,3-dehydro-N-acetylneuraminic acid) is consistent with extracellular activity of viral neuraminidase (sialidase).

We demonstrate the potent antiviral activity of a novel viral neuraminidase (sialidase) inhibitor, 4-guanidino-2,4-dideoxy-2,3-dehydro-N-acetylneuraminic acid (GG167), administered by the intranasal route in comparison with those of amantadine and ribavirin in experimental respiratory tract infections induced with influenza A and B viruses. In an extended study in which mice were infected (day 0) with influenza A/Singapore/1/57 virus, with treatments given prophylactically plus twice daily over days 0 to 3 and with mice observed to day 10, we show that intranasally administered GG167 at 0.4 and 0.01 mg/kg of body weight per dose reduced mortality, lung consolidation, and virus titers in the lung, with no virus growing back following the cessation of treatment. In other studies with influenza B/Victoria/102/85 virus in which infected mice were culled after the cessation of treatment, the calculated intranasal dose required to reduce virus titers in the lungs of treated animals to 10% of that seen in untreated controls (EDAUC10 [where AUC is area under the virus titer days curve]) was 0.085 mg/kg per dose. GG167 was inactive against influenza viruses A and B when given by the intraperitoneal or oral route (EDAUC10, > 100 mg/kg per dose). GG167 was metabolically stable, with an elimination half-life of 10 min following intravenous administration. While readily bioavailable by systemic routes, it was poorly bioavailable by the oral route. Its potent efficacy by the intranasal route but lack of efficacy by other routes, relative to those of amantadine and ribavirin, was explicable in terms of its in vitro activity, bioavailability, and pharmacokinetic properties and with the extracellular activity of viral sialidase.

Inhibition of the growth of influenza viruses in vitro by 4-guanidino-2,4-dideoxy-N-acetylneuraminic acid.

The sialidase inhibitor 4-guanidino-2,4-dideoxy-2,3-dehydro-N- acetylneuraminic acid was tested for growth inhibitory effects against a panel of avian influenza A viruses encompassing all nine neuraminidase subtypes. Growth in tissue culture of viruses from each subtype was inhibited by this compound at concentrations within a range previously found effective against human N1 and N2 viruses. This compound may prove a selective agent for the treatment (and prevention) of influenza virus infections.

Discovery and analysis of a series of C2-symmetric HIV-1 proteinase inhibitors derived from penicillin.

In order to identify a suitable peptide substrate for human immunodeficiency virus-1 (HIV-1) proteinase, a range of peptides from various cleavage sites within the gag-pol polyprotein were assayed by HPLC for specific cleavage. The peptide with the optimal combination of favorable kinetics and good solubility was based on the N-terminus cleavage site of HIV-1 proteinase (KQGTVSFNF*PQIT). The HPLC assay, using the above peptide, was developed into a rapid isocratic method in order to analyze inhibition kinetics. An assay suitable for high-throughput screening was developed using a radioactively labeled peptide with the same sequence, coupled to a solid phase. Using this assay, a C2-symmetric HIV-1 proteinase inhibitor derived from penicillin was discovered during random screening of a compound library. A chemical synthesis program developed this structure into a series of potent inhibitors. The lead structures were highly selective for HIV-1 proteinase with good antiviral activity in vitro against HIV and no cytotoxicity. The HPLC assay was used to demonstrate that these compounds are competitive tight-binding inhibitors of HIV-1 proteinase.

Effects of (-)-2'-deoxy-3'-thiacytidine (3TC) 5'-triphosphate on human immunodeficiency virus reverse transcriptase and mammalian DNA polymerases alpha, beta, and gamma.

(-)-2'-Deoxy-3'-thiacytidine (3TC) is a selective inhibitor of human immunodeficiency virus replication in vitro (J. A. V. Coates, N. Cammack, H. J. Jenkinson, A. J. Jowett, M. I. Jowett, B. A. Pearson, C. R. Penn, P. L. Rouse, K. C. Viner, and J. M. Cameron, Antimicrob. Agents Chemother. 36:733-739, 1992). The effect of 3TC 5'-triphosphate on both the RNA-dependent and DNA-dependent activities of human immunodeficiency virus type 1 reverse transcriptase and DNA polymerases alpha, beta, and gamma from HeLa cells was investigated. 3TC 5'-triphosphate is a competitive inhibitor (with respect to dCTP) of the RNA-dependent DNA polymerase activity (apparent Ki = 10.6 +/- 1.0 to 1.24 +/- 5.1 microM, depending on the template and primer used); the DNA-dependent DNA polymerase activity is 50% inhibited by a 3TC 5'-triphosphate concentration of 23.4 +/- 2.5 microM when dCTP is present at a concentration equal to its Km value. Chain elongation studies show that 3TC 5'-triphosphate is incorporated into newly synthesized DNA and that transcription is terminated in a manner identical to that found for ddCTP. The 50% inhibitory concentrations of 3TC 5'-triphosphate against DNA polymerases alpha, beta, and gamma at concentrations of dCTP equal to the Km were 175 +/- 31, 24.8 +/- 10.9, and 43.8 +/- 16.4 microM, respectively. More detailed kinetic studies with 3TC 5'-triphosphate and DNA polymerases beta and gamma are consistent with the fact that inhibition of these enzymes by 3TC 5'-triphosphate is competitive with respect to dCTP. The values of Ki were determined to be 18.7 microM for DNA polymerase beta and 15.8 +/- 0.8 microM for DNA polymerase gamma.

Cellular metabolism of (-) enantiomeric 2'-deoxy-3'-thiacytidine.

The metabolism of (-) enantiomeric 2'-deoxy-3'-thiacytidine (3TC) was examined in human immunodeficiency virus type 1 (HIV-1)-infected and mock-infected human cells. 3TC 5'-triphosphate levels accumulated comparably in HIV-1-infected and mock-infected phytohaemagglutinin-stimulated peripheral blood lymphocytes (PBL) and reached 40% or more of total intracellular 3TC metabolites after 4 hr. The rate of decay of 3TC triphosphate in HIV-1-infected and mock-infected PBL measured as a half-life (T1/2) ranged from 10.5 to 15.5 hr. 3TC did not significantly affect metabolism of deoxynucleotides in the U937 cell line, and was shown to be resistant to the action of human platelet pyrimidine nucleoside phosphorylase.

(-)-2'-deoxy-3'-thiacytidine is a potent, highly selective inhibitor of human immunodeficiency virus type 1 and type 2 replication in vitro.

The (-)-enantiomer of 2'-deoxy-3'-thiacytidine (3TC) was found to be a potent and selective inhibitor of human immunodeficiency virus types 1 (HIV-1) and 2 (HIV-2) in vitro. We determined its antiviral activity against a number of laboratory strains of HIV-1 and HIV-2 in a range of CD4-bearing lymphocyte cell lines (mean 50% inhibitory concentration [IC50] range, 4 nM to 0.67 microM). 3TC was also active against a range of HIV-1 strains in peripheral blood lymphocytes (mean IC50 range, 2.5 to 90 nM). The IC50 for cytotoxicity in seven lymphocyte cell cultures, including human peripheral blood lymphocytes, ranged from 0.5 to 6 mM. 3TC had no detectable antiviral activity against a range of other viruses or in cells chronically infected with HIV-1 or HIV-2. The effects of time of addition of the compound and varying the multiplicity of infection on the antiviral activity of 3TC were determined. The results showed that 3TC is a potent and selective inhibitor of HIV-1 and HIV-2 replication in vitro.

Megavoltage irradiation in a district general hospital remote from a main oncology centre: the Torbay experience reviewed.

A recent Royal College of Radiologists' report on the organization of clinical oncology services supports a greater consultant presence in district general hospitals but condemns satellite treatment facilities. However, new contract costs for treatment in remote main centres may induce 'client' purchasers or providers to re-examine the economics and benefits of a local facility. This article reviews the experience of a satellite facility, and suggests a minimum critical mass of capital and revenue below which such facilities are uneconomic, but above which savings can be expected. Comparison of outcomes for radical treatment overall with the local main centre and, for head and neck malignancy, with a large regional centre elsewhere, suggests no compromise of technical standards. Convenience for patients and carers is high and has resulted in a high uptake of services in the satellite-equipped district.

DNA chain termination activity and inhibition of human immunodeficiency virus reverse transcriptase by carbocyclic 2',3'-didehydro-2',3'-dideoxyguanosine triphosphate.

Carbocyclic 2',3'-didehydro-2',3'-dideoxyguanosine (carbovir, NSC 614846) is an anti-retroviral agent that may be useful in the treatment of AIDS. We have examined the ability of (-)-enantiomeric carbovir triphosphate to inhibit human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (EC 2.7.7.49). A comparison of inhibition kinetics was made with 3'-azido-2',3'-dideoxythymidine triphosphate and phosphonoformate. Inhibition of the reverse transcriptase was evaluated using poly(rA).oligo(dT)12-18, poly(rC).oligo(dG)12-18, or influenza virion RNA template with a specific oligodeoxynucleotide as primer. (-)-Carbovir 5'-triphosphate was shown to be a potent inhibitor of HIV-1 reverse transcriptase with an apparent Ki similar to that of 3'-azido-2',3'-dideoxythymidine triphosphate. Chain elongation studies utilizing an MS2 RNA template showed that (-)-carbovir 5'-triphosphate terminated transcription at positions identical to those where dideoxy-GTP terminated. This indicates that (-)-carbovir 5'-monophosphate is incorporated into the newly synthesized DNA and terminates transcription at that point. We conclude that (-)-carbovir 5'-triphosphate is a potent inhibitor of the HIV-1 reverse transcriptase enzyme and that (-)-carbovir most likely inhibits HIV by activity at the triphosphate level by a combination of direct competition for binding of the natural deoxynucleoside triphosphates to the reverse transcriptase and chain termination.