Antiviral activity of low-dose alovudine in antiretroviral-experienced patients: results from a 4-week randomized, double-blind, placebo-controlled dose-ranging trial.

BACKGROUND: Alovudine inhibits replication of highly nucleoside reverse transcriptase inhibitor (NRTI)-resistant HIV strains in vitro. However, dose-dependent safety concerns resulted in its initial development being halted. Recently, a 4-week course of alovudine 7.5 mg/day added to a stavudine-free failing regimen yielded a significant decrease in viral load by -1.88 log(10) HIV-1 RNA copies/mL. The magnitude of the reduction in viral load suggested that lower doses might still be effective while offering adequate safety during long-term use. OBJECTIVE: To determine whether lower dosages of alovudine still provide significant antiviral activity in patients with broad NRTI resistance. METHODS: A randomized, double-blind, placebo-controlled trial investigating three doses of alovudine (0.5, 1 and 2 mg) or placebo added for 4 weeks to a failing regimen in patients with evidence of NRTI-resistant HIV strains [>or=2 thymidine-associated mutations (TAMs)]. The primary endpoint was the mean viral load reduction between baseline and week 4. RESULTS: Seventy-two patients were enrolled in the study: 21, 13, 18 and 20 in the placebo and 0.5, 1 and 2 mg arms, respectively. Baseline median CD4 count and viral load were 298 cells/microL (range 44-692 cells/microL) and 3.9 log(10) copies/mL (range 2.5-5.2 log(10) copies/mL), respectively. Baseline viral isolates harboured a median of four TAMs. Alovudine was added to a median four-drug failing regimen. At week 4, compared with placebo, mean viral load changes were -0.42 log(10) [95% confidence interval (CI) -0.67 to -0.18] and -0.30 log(10) (-0.55 to -0.06) in the 2 and 1 mg arms, respectively. There was no significant change in CD4 cell count. Alovudine was well tolerated. CONCLUSION: A 4-week course of alovudine 2 mg/day provided a modest but significant viral load reduction in patients harbouring viruses with a median of four TAMs.

Herpes simplex virus type 1 infection and glucocorticoid treatment regulate viral yield, glucocorticoid receptor and NF-kappaB levels.

The interplay between the endocrine and immune systems has come into focus in recent years with the insight that endocrine parameters may affect susceptibility to both auto-immune and infectious diseases. Our interest in immunoendocrine regulation led us to investigate the effects of glucocorticoids on Herpes simplex virus type 1 (HSV-1) infections. Glucocorticoids used to treat inflammatory conditions are not yet recommended for HSV-1 therapy, since they have been reported to prolong viral shedding both in vivo and in vitro. Here we report that glucocorticoids did not alter the viral yield in human gingival fibroblast (HGF) cell culture when glucocorticoid treatment and viral infection occured simultaneously, but the viral yield increased when cells were treated with the glucocorticoid dexamethasone (dex) prior to viral infection. We found that viral infection in our primary cell system increased NF-kappaB levels and DNA binding. In addition, the amount of glucocorticoid receptor (GR) increased following viral infection, and HSV-1 infection as such could induce glucocorticoid-driven transcription of a reporter gene in human embryo kidney (HEK) 293 cells stably transfected with GR. Dex treatment did not affect HSV-1-induced binding of p65 to an NF-kappaB element in an electrophoretic mobility shift assay, and acyclovir was still efficient as an anti-viral drug in the presence of dex. Further studies of the observed effects of HSV-1 infection and glucocorticoid treatment on GR and NF-kappaB regulation could give insights into the immunoendocrine mechanisms important for defence and therapy against viral infections.

Double-blind, randomized, placebo-controlled study of topical 5% acyclovir-1% hydrocortisone cream (ME-609) for treatment of UV radiation-induced herpes labialis.

Immunopathology is recognized as an important component of infectious disease manifestations, and corticosteroids have been used as an adjunct to antimicrobial therapy for a variety of conditions. Antiviral therapy of herpes labialis has been shown to result in only a small reduction in the time to healing and the duration of pain. To determine if topical application of a combination product containing 5% acyclovir and 1% hydrocortisone (ME-609) could provide benefit to herpes labialis patients, 380 immunocompetent adults with a history of herpes labialis were exposed to experimental UV radiation (UVR) to induce a recurrence. On day 2, just before the appearance of the majority of lesions ("delayed" lesions), subjects were randomized to receive active medication or vehicle control six times per day for 5 days. Overall, 120 of 380 patients developed delayed classical lesions, of whom 50 of 190 (26%) had been treated with ME-609 and 70 of 190 (37%) had received placebo (a reduction of 29% [P = 0.02]). Healing time, measured as the time to normal skin, was reduced by treatment with ME-609 (9.0 days for treated patients versus 10.1 days for the controls [P = 0.04]). There was a trend toward a reduction in the maximum lesion size in the ME-609 recipients compared to that in the controls (43 versus 60 mm(2), respectively [P = 0.07]). The treatment had no effect on lesion pain, but ME-609 treatment reduced the number of patients with moderate or severe tenderness. Compared to treatment with a placebo, treatment with the combination antiviral-immunomodulatory cream provided benefit to patients with experimental UVR-induced herpes labialis, reducing classical lesion incidence, healing time, lesion size, and lesion tenderness. ME-609 is a novel product that merits further evaluation as a treatment for cold sores.

Variations in the cytomegalovirus DNA polymerase and phosphotransferase genes in relation to foscarnet and ganciclovir sensitivity.

BACKGROUND: Identification of human cytomegalovirus (CMV) genome variation is important for understanding mutations associated with drug resistance. OBJECTIVES: To investigate the CMV resistance to foscarnet (PFA) and ganciclovir (GCV) in patients treated with antiviral drugs and to identify the DNA polymerase (UL54) and phosphotransferase (UL97) gene mutations inducing resistance. STUDY DESIGN: Antiviral susceptibility of CMV strains/isolates for PFA and GCV was compared by plaque reduction assay and in situ ELISA. UL54 and UL97 gene mutations were identified by sequencing. Growth phenotype of two CMV recombinants with mutations in UL54 was studied. RESULTS: Six of seven GCV resistant strains had alterations within the UL97. Five of them also had alterations in the UL54 (F412C, L802M or K513E), previously shown to induce GCV resistance. Seven isolates had no or reduced susceptibility to PFA, which had alterations in the UL54 (D588N, E756K, V781I or L802M). By in vitro mutagenesis, it was shown that a mutation at codon D588N of UL54 conferred 9-fold reduced susceptibility to PFA, while a mutation at codon V781I induced 4-fold reduced susceptibility to PFA and GCV. Both recombinants showed the same kinetics of protein expression (IE, E, and L antigen) and virus yields as the CMV Towne strain. CONCLUSIONS: The recombinants containing alterations within the UL54 (D588N and V781I) showed a reduced susceptibility to antiviral drugs but no change in the replication rate compared to the CMV Towne.

Herpes simplex virus zosteriform lesions with adoptive transfer of immune cells: a murine model which mimics human recurrent disease.

Existing murine models for cutaneous herpes simplex virus type 1 (HSV-1) infection have limited relevance to recurrent disease in humans, since the infection is usually primary rather than reactivated and infection occurs in the absence of an established immune response. To obtain a reproducible model to study the effects of topical antiviral therapy on recurrent disease we have adapted a mouse model which employs zosteriform spread of HSV-1 in the presence of adoptive transfer of immunity (ATI) which mimics human recrudescent lesions. Mice were infected with HSV-1 by scarification at the lateroventral line of the neck; 2 days later, the mice received adoptive transfer of immune cells from the cervical lymph nodes of syngeneic mice that had been infected in the ear pinna with the same strain of virus 7 days earlier. ATI resulted in a heightened inflammatory response in the target tissues for virus replication. Virus was cleared more quickly from the infected tissues in comparison with mice similarly inoculated without ATI, however, the intensity and duration of the inflammation was greater. The model was then used to test the effect of a topical formulation of foscarnet. The results presented demonstrate that the ATI model can provide useful data concerning the efficacy of topical antiviral chemotherapy in man.

Combinations of antiviral and anti-inflammatory preparations for the topical treatment of herpes simplex virus assessed using a murine zosteriform infection model.

Recently we have reported a zosteriform murine infection model which employs the adoptive transfer of immune cells (ATI) to recipient infected mice to produce a disease that mimics human recurrent herpes simplex virus (HSV) disease. Mice were infected with HSV-1 by scarification at the lateroventral line of the neck; 2 days later, the mice received immune cells from HSV-1-infected syngeneic mice. Although virus was cleared more quickly from the target tissues of virus replication in recipient mice, ATI resulted in a heightened inflammatory response and delayed healing. This model was used to test the effects of topical formulations containing foscarnet and/or the anti-inflammatory agent, hydrocortisone. Virus clearance and clinical signs, including ear thickness and zosteriform spread of lesions, were studied. Treatment with 3% foscarnet accelerated virus clearance but had little effect on clinical parameters. By contrast, 0.5% hydrocortisone increased the titre and extended the presence of infectious virus for at least 6 days, although the reduction in clinical signs was greater than that obtained with topical foscarnet. Foscarnet in combination with hydrocortisone produced a marked reduction in clinical signs while virus replication was reduced. These results are discussed in relation to the inflammation and discomfort experienced by patients and a possible role for anti-inflammatory formulations in the treatment of HSV reactivation episodes in man.

Synergistic inhibition of human immunodeficiency virus isolates (including 3'-azido-3'-deoxythymidine-resistant isolates) by foscarnet in combination with 2',3'-dideoxyinosine or 2',3'-dideoxycytidine.

The combinations of foscarnet plus 2',3'-dideoxyinosine and foscarnet plus 2',3'-dideoxycytidine synergistically inhibit the replication of human immunodeficiency virus type 1 isolates, including two 3'-azido-3'-deoxythymidine-resistant isolates. The combination of 2',3'-dideoxyinosine plus 2',3'-dideoxycytidine showed additive inhibition of the majority of the human immunodeficiency virus isolates tested. All three combinations showed pronounced antagonistic cytotoxicity and thus were less toxic to the growth of peripheral blood mononuclear cells than the separate drugs.

G- and GM-CSF in oncology and oncological haematology.

Administration of G- and GM-CSF increases the neutrophil counts in a number of clinical situations. GM-CSF shows the additional effect of increasing the number of monocytes and eosinophil granulocytes. Both G- and GM-CSF affect of neutrophil functions, in the case of GM-CSF there are some potentially negative effects on neutrophil migration and adhesiveness. The clinical relevance of the various effects on mature haematopoietic cells is not fully understood. Clinical data with G-CSF treatment indicate that increased levels of neutrophil granulocytes following cytotoxic chemotherapy may translate into clinical benefit such as a decreased rate of neutropenic infection and an increased cytotoxic chemotherapy dose even though the data are conflicting and the risk of "laboratory cosmetics" is apparent. Regarding treatment with GM-CSF following chemotherapy, the clinical benefit is unclear. The clinical benefit of GM-CSF-induced monocytes and eosinophils is unknown. G- and GM-CSF accelerates neutrophil recovery following autologous or allogeneic BMT. The influence on neutropenic infections is, however, less impressive. Pretreatment with G- or GM-CSF increases the yield of peripheral stem cell harvest, thereby reducing the number of leukaphereses needed. Transplantation of G- and GM-CSF primed autologous peripheral stem cells tends to reduce the period of post-transplant cytopenia, particularly thrombocytopenia, in comparison with traditional ABMT. In patients with MDS, G- and GM-CSF appear to increase the number of neutrophil granulocytes and there is some evidence that patients with severe infectious problems will benefit from this treatment. However, little influence was seen on the main clinical problems with these patients, which are anaemia and thrombocytopenia. In conclusion, G- and GM-CSF are two different proteins with different properties in vivo and in vitro. GM-CSF has, compared with G-CSF, more complex pharmacological effects and a more trouble-some side-effect profile. Early clinical development indicates that both compounds have a substantial influence on the levels of certain blood cells. Whether the increases in different blood cells translate into long-term clinical benefit for greater patient groups is the focus of ongoing research. The effects of G- and GM-CSF may be potentiated by other cytokines, an area which is presently being explored.

Assay of intracellular thymidylate synthetase activity and inhibition by 5-fluoro-2'-deoxyuridine in lymphocytes.

Thymidylate synthetase catalyses the formation of thymidine monophosphate from deoxyuridine monophosphate. Purified thymidylate synthetase can be assayed radiochemically using labelled deoxyuridine monophosphate as substrate, but cells are impervious to deoxyuridine monophosphate and so intracellular thymidylate synthetase activity cannot be assayed in this way. In this paper we describe the assay of intracellular thymidylate synthetase activity in intact cells using labelled 2'-deoxyuridine. The assay showed linear kinetics with respect to time, concentration of 2'-deoxyuridine, and cell concentration. 5-fluoro-2'-deoxyuridine inhibited intracellular thymidylate synthetase activity measured with this assay by 50% at 5 nM. Cell growth was inhibited by 50% at 6 nM 5-fluoro-2'-deoxyuridine. The assay was specific for thymidylate synthetase and enabled measurement of thymidylate synthetase activity in situ in intact cells.

Comparison of different tests of fencing performance.

Three different procedures to analyse fencing performances were tested. The test subjects were ten world class epée fencers from the Swedish national team. The procedures involved measurements of reaction time and response time to different stimuli. Test 1 measured the lunging performance as a response to a light. Tests 2 and 3 measured more complex fencing movements as response to a more fencing-like starting procedure. The results showed that test 3 but not tests 1 and 2 could differentiate between world class fencers and beginners (p less than 0.006). The reaction time in test 3 correlated significantly (p less than 0.01) with competition success within the group of world class fencers.

The mechanism of action of the anti-herpes virus compound 2,3-dimethyl-6(2-dimethylaminoethyl)-6H-indolo-(2,3-b)quinoxaline.

The compound 2,3-dimethyl-6(2-dimethylaminoethyl)6H-indolo-(2,3-b)quinoxaline (B-220) has been shown to exhibit potent antiviral activity against herpes simplex virus type 1 (HSV-1), varicella-zoster virus (VZV) and cytomegalovirus (CMV). The mechanism of antiviral action of B-220 against HSV-1 has been studied; from the results it appears that B-220 binds by intercalation into the DNA helix and then disturbs steps that are vital for viral uncoating.

Synergistic combinations and peptides in the inhibition of human immunodeficiency virus.

Various combinations of inhibitors of HIV reverse transcriptase were tested for inhibition of HIV replication in order to reveal any potential synergism or antagonism. PFA, a pyrophosphate analogue, gave synergistic inhibition of HIV replication in combination with both of the thymidine analogues AZT and FLT. The combination of PFA and AZT-TP gave only additive or weakly synergistic inhibition in a reverse transcriptase enzyme assay. The combination of AZT and FLT also gave synergistic inhibition of HIV replication, whilst the combination of AZT-TP and FLT-TP gave only additive or weakly synergistic inhibition of reverse transcriptase. Thus, the synergy does not arise from effects on reverse transcriptase alone but must be owing to other, cellular factors, such as effects on nucleoside metabolism or metabolism of the analogues. The results are consistent with the hypothesis that AZT may have an alternative mechanism of inhibition other than inhibition of reverse transcriptase. The diminished cytotoxicity observed in addition to the synergistic inhibition makes these combinations attractive from the point of view of combination chemotherapy. The inhibition of HIV replication by peptides from various parts of the V3 region of gp120 whose sequences were homologous with the tryptase inhibitor trypstatin was tested. Inhibitory activity was displayed by two peptides containing cysteine in their sequence. Antibodies to two peptides containing the two conserved cysteine residues from opposite sides of the neutralizing loop of gp120 were previously associated with protection from vertical transmission of HIV. The V3 region thus seems to be important for the function of gp120 and the transmission of HIV.

Inhibition of human herpesvirus 6 replication by 9-[4-hydroxy-2-(hydroxymethyl)butyl]guanine (2HM-HBG) and other antiviral compounds.

The in vitro susceptibilities of human herpesvirus 6 to foscarnet; the guanosine analogs acyclovir, ganciclovir, and two isomers of 9-[4-hydroxy-2-(hydroxymethyl)butyl]guanine; and the thymidine analogs 3'-azido-3'-deoxythymidine and 1-beta-D-arabinofuranosyl-E-5-(2-bromovinyl)uracil were investigated. All compounds except 3'-azido-3'-deoxythymidine and 1-beta-D-arabinofuranosyl-E-5-(2-bromovinyl)uracil inhibited human herpesvirus 6 replication. The highest in vitro selectivity was obtained for 9-[4-hydroxy-2-(hydroxymethyl)butyl]guanine.

Synergistic inhibition of human immunodeficiency virus replication in vitro by combinations of 3'-azido-3'-deoxythymidine and 3'-fluoro-3'-deoxythymidine.

The antiviral activity against human immunodeficiency virus type 1 of the two structurally related thymidine analogs azidothymidine and fluorothymidine, both alone and in combination, was tested. Fluorothymidine was tenfold more active than azidothymidine. The selectivity indices of the two compounds were similar. The combination of azidothymidine and fluorothymidine showed clearly synergistic antiviral activity, and diminished cytotoxicity. The inhibition of reverse transcriptase from human immunodeficiency virus type 1 by the triphosphates of azidothymidine and fluorothymidine, both alone and in combination was also tested. Azidothymidine triphosphate was a fourfold stronger inhibitor than fluorothymidine triphosphate. The combination of the two showed only additive (and not synergistic) effects upon reverse transcriptase. The combination of azidothymidine and fluorothymidine showed both synergistic antiviral activity and diminished cytotoxicity, and may therefore represent a promising therapeutic strategy. The additive (and not synergistic) inhibition of reverse transcriptase by the combination of the triphosphates indicates that in cell culture additional factors other than inhibition of the reverse transcriptase by the triphosphates influence the antiviral activity of the combination. Such factors might include effects upon normal nucleoside metabolism or metabolism of the analogs. Alternatively, one of the nucleosides might have an additional mechanism of action besides inhibition of the reverse transcriptase.

Improved sample preparation method for high-performance liquid chromatography of deoxyribonucleoside triphosphates from cell culture extracts.

The accurate determination of deoxyribonucleoside triphosphates in cells is difficult owing to the high concentrations of interfering ribonucleoside triphosphates. The latter can be degraded to their respective bases by periodate oxidation of cell extracts. However, the large amount of bases so produced can interfere with subsequent high-performance liquid chromatographic (HPLC) analysis. The use of a weak ion-exchange cartridge to partially purify and concentrate deoxyribonucleoside triphosphates in periodate-treated cell extracts, prior to HPLC, thus allowing accurate determination is described. The recovery of the deoxyribonucleoside triphosphates is greater than 95%, and greater than 90% of the interfering bases are removed.

Physiological and morphological characteristics of world class fencers.

Physiological and morphological characteristics of world class épée fencers were analysed. The results showed that épée fencers have a high maximal aerobic power and high maximal isometric and dynamic strength. The movement pattern of épée fencing results in an asymmetry of the body. Thus, weapon hand isometric elbow flexion and forward leg isometric and dynamic muscle strength were higher than the contralateral extremity. Finally, forward leg muscle mass--evaluated from computed tomography--was higher while the muscle fiber composition was not different from the contralateral leg.

Structure-activity relationships of fluorinated nucleoside analogs and their synergistic effect in combination with phosphonoformate against human immunodeficiency virus type 1.

One hundred nucleoside analogs with fluorine substitutions at various positions on the pentose ring were evaluated for inhibitory activity against human immunodeficiency virus type 1 (HIV-1). Nine compounds emerged as inhibitors of HIV-1 replication, with various degrees of selectivity; the most active of these was 3'-fluoro-3'-deoxythymidine, followed by 5'-amino-3'-fluoro-3'-deoxyadenosine. Substitution of fluorine at the 2'-deoxy or 3'-deoxy position resulted in increased antiviral activity of the thymidine analogs, whereas the activity of adenosine or cytidine analogs was not increased by fluorination at either position. The most potent inhibitor, 3'-fluoro-3'-deoxythymidine, was shown to give synergistic inhibition of HIV-1 replication in combination with the PPi analog phosphonoformate.

Inhibition of human immunodeficiency virus in vitro by combinations of 3'-azido-3'-deoxythymidine and foscarnet.

We describe a synergistic effect of combinations of foscarnet and 3'-azido-3'-deoxythymidine against human immunodeficiency virus type 1 multiplication in cell culture, an additive effect of foscarnet and 3'-azido-3'-deoxythymidine triphosphate against human immunodeficiency virus type 1 reverse transcriptase, and a low toxicity in cell culture of combinations of the two drugs.