Lieve Naesens

Update on Human Herpesvirus 6 Biology, Clinical Features, and Therapy

SUMMARY Human herpesvirus 6 (HHV-6) is a betaherpesvirus that is closely related to human cytomegalovirus. It was discovered in 1986, and HHV-6 literature has expanded considerably in the past 10 years. We here present an up-to-date and complete overview of the recent developments concerning HHV-6 biological features, clinical associations, and therapeutic approaches. HHV-6 gene expression regulation and gene products have been systematically characterized, and the multiple interactions between HHV-6 and the host immune system have been explored. Moreover, the discovery of the cellular receptor for HHV-6, CD46, has shed a new light on HHV-6 cell tropism. Furthermore, the in vitro interactions between HHV-6 and other viruses, particularly human immunodeficiency virus, and their relevance for the in vivo situation are discussed, as well as the transactivating capacities of several HHV-6 proteins. The insight into the clinical spectrum of HHV-6 is still evolving and, apart from being recognized as a major pathogen in transplant recipients (as exemplified by the rising number of prospective clinical studies), its role in central nervous system disease has become increasingly apparent. Finally, we present an overview of therapeutic options for HHV-6 therapy (including modes of action and resistance mechanisms).

Treatment of Severe Laryngeal Papillomatosis With Intralesional Injections of Cidofovir ((S)-1-(3-Hydroxy-2-Phosphonylmethoxypropyl) Cytosine)

Respiratory papillomatosis is a rare and often severe disease, usually localized in the larynx. It may cause respiratory distress and even life‐threatening obstruction of the airways. Treatment is generally based on the evaporation of the lesions with a CO2 laser, but microsurgery, cytotoxic and/or cytostatic drugs, interferons, and vaccines are also used. Cidofovir [(S)‐1‐(3‐hydroxy‐2‐phosphonylmethoxypropyl)cytosine] (HPMPC) was shown to suppress the growth of tumors induced by rabbit papillomavirus as well as human papillomavirus (HPV). The efficacy of cidofovir was assessed in 17 patients with severe respiratory papillomatosis. Cidofovir at a concentration of 2.5 mg/ml was injected directly in the different laryngeal papillomatous lesions during microlaryngoscopy under general anesthesia. Biopsies were taken before the treatment was started both for anatomopathology and viral typing. HPMPC kinetics in serum was monitored in three patients, the drug levels being determined by high‐performance liquid chromatography. Complete disappearance of the papillomatosis was observed in 14 patients. Four patients relapsed and were successfully treated again with cidofovir. Of the three remaining patients, one progressed while under treatment with cidofovir, after an initial marked response. One patient had a partial remission and remained stable for more than 1 year after the last injection. He had a very aggressive and extensive disease originally. Finally, one patient was lost to follow‐up after four injections. Intratumoral injections of cidofovir for the treatment of severe laryngeal papillomatosis is a powerful new therapeutic approach for this disease. Treatment was well tolerated, and no significant side effects were noted. J. Med. Virol. 54:219– 225, 1998.

HPMPC (cidofovir), PMEA (adefovir) and Related Acyclic Nucleoside Phosphonate Analogues: A Review of their Pharmacology and Clinical Potential in the Treatment of Viral Infections:

The acyclic nucleoside phosphonate (ANP) analogues are broad-spectrum antiviral agents, with potent and selective antiviral activity in vitro and in vivo. The prototype compounds are: (S)-1-(3-hydroxy-2-phosphonylmethoxypropyl)cytosine (HPMPC, cidofovir), which is active against a wide variety of DNA viruses; 9-(2-phosphonylmethoxyethyl)adenine (PMEA, adefovir), which is active against retro-, herpes- and hepadnaviruses, and (R)-9-(2-phosphonylmethoxypropyl) adenine (PMPA), which is active against retro- and hepadnaviruses. The antiviral action of the ANP analogues is based on a specific interaction of the active diphosphorylated metabolite with the viral DNA polymerase. The long intracellular half-life of the active metabolite accounts for the optimal efficacy in infrequent dosing schedules. The potential of HPMPC as a broad-spectrum anti-DNA virus agent, as originally observed in vitro and in vivo, has been confirmed in clinical trials. HPMPC has recently been commercially released in the USA for the treatment of cytomegalovirus retinitis in AIDS patients. In addition, topical systemic HPMPC is being (or will be) explored for use against other herpesviruses (i.e. herpes simplex virus, Epstein-Barr virus, or varicella-zoster virus), by adenoviruses, or by human papilloma- or polyomaviruses. Intravenous HPMPC is associated with dose-dependent nephrotoxicity, that should be counteracted by prehydration and concomitant administration of probenecid, and by the application of an infrequent dosing schedule. The oral prodrug of PMEA, bis(pivaloyloxymethyl)-PMEA, is currently being evaluated in patients infected with human immunodeficiency virus (HIV) or hepatitis B virus. Finally, preclinical data on the efficacy of PMPA in animal retrovirus models point to its potential usefulness against HIV infections, when given either prophylactically or therapeutically in the treatment of established HIV infections.

Physicochemical characterization of solid dispersions of the antiviral agent UC-781 with polyethylene glycol 6000 and Gelucire 44/14

The purpose of this study was to prepare and characterize solid dispersions of the antiviral thiocarboxanilide UC-781 with PEG 6000 and Gelucire 44/14 with the intention of improving its dissolution properties. The solid dispersions were prepared by the fusion method. Evaluation of the properties of the dispersions was performed using dissolution studies, differential scanning calorimetry, Fourier-transform infrared spectroscopy and X-ray powder diffraction. To investigate the possible formation of solid solutions of the drug in the carriers, the lattice spacings [d] of PEG 6000 and Gelucire 44/14 were determined in different concentrations of UC-781. The results obtained showed that the rate of dissolution of UC-781 was considerably improved when formulated in solid dispersions with PEG 6000 and Gelucire 44/14 as compared to pure UC-781. From the phase diagrams of PEG 6000 and Gelucire 44/14 it could be noted that up to approximately 25% w/w of the drug was dissolved in the liquid phase in the case of PEG 6000 and Gelucire 44/14. The data from the X-ray diffraction showed that the drug was still detectable in the solid state below a concentration of 5% w/w in the presence of PEG 6000 and Gelucire 44/14, while no significant changes in the lattice spacings of PEG 6000 or Gelucire 44/14 were observed. Therefore, the possibility of UC-781 to form solid solutions with the carriers under investigation was ruled out. The results from infrared spectroscopy together with those from X-ray diffraction and differential scanning calorimetry showed the absence of well-defined drug-polymer interactions.

Clinical features and treatment of adenovirus infections.

Adenoviruses (Ads) are common opportunistic pathogens that are rarely associated with severe clinical symptoms in healthy individuals. In contrast, in patients with compromised immunity, Ad infections often result in disseminated and potentially life‐threatening disease. Among these are AIDS patients, individuals with hereditary immunodeficiencies and recipients of solid organ or haematopoietic stem cell transplants (HSCT) who receive immunosuppressive therapy. The latter account for the largest number of severe Ad infections. There is currently no formally approved antiviral therapy for the treatment of severe Ad keratoconjunctivitis and life‐threatening Ad infections in immunocompromised patients. Here we update current knowledge on Ad biology, the clinical features observed in different patient groups and specific immune responses towards Ad infections. In addition, we review current and future treatment options, including: (i) the antiviral drugs cidofovir, ribavirin and new investigational compounds, as evaluated in the clinic or in relevant animal models, as well as (ii) novel immunotherapeutic strategies. Copyright

Adjuvant low-dose cidofovir therapy for BK polyomavirus interstitial nephritis in renal transplant recipients.

BK virus interstitial nephritis (BKVIN) is a serious complication after kidney grafting, necessitating drastic reduction of immunosuppressive therapy in order to enable viral clearance. Despite these measures, progressive graft dysfunction and graft loss occur in the majority of recipients.

Antiviral treatment is more effective than smallpox vaccination upon lethal monkeypox virus infection

There is concern that variola virus, the aetiological agent of smallpox, may be used as a biological weapon. For this reason several countries are now stockpiling (vaccinia virus-based) smallpox vaccine. Although the preventive use of smallpox vaccination has been well documented, little is known about its efficacy when used after exposure to the virus. Here we compare the effectiveness of (1) post-exposure smallpox vaccination and (2) antiviral treatment with either cidofovir (also called HPMPC or Vistide) or with a related acyclic nucleoside phosphonate analogue (HPMPO–DAPy) after lethal intratracheal infection of cynomolgus monkeys (Macaca fascicularis) with monkeypox virus (MPXV). MPXV causes a disease similar to human smallpox and this animal model can be used to measure differences in the protective efficacies of classical and new-generation candidate smallpox vaccines. We show that initiation of antiviral treatment 24 h after lethal intratracheal MPXV infection, using either of the antiviral agents and applying various systemic treatment regimens, resulted in significantly reduced mortality and reduced numbers of cutaneous monkeypox lesions. In contrast, when monkeys were vaccinated 24 h after MPXV infection, using a standard human dose of a currently recommended smallpox vaccine (Elstree-RIVM), no significant reduction in mortality was observed. When antiviral therapy was terminated 13 days after infection, all surviving animals had virus-specific serum antibodies and antiviral T lymphocytes. These data show that adequate preparedness for a biological threat involving smallpox should include the possibility of treating exposed individuals with antiviral compounds such as cidofovir or other selective anti-poxvirus drugs.

Antiadenovirus Activities of Several Classes of Nucleoside and Nucleotide Analogues

ABSTRACT The absence of any formally licensed antiadenovirus drugs and the increasing incidence of life-threatening adenovirus infections in immunosuppressed patients warrant the development of effective antiadenovirus compounds. A detailed study was performed on the antiadenovirus activities of several classes of nucleoside and nucleotide analogues in human embryonic lung fibroblast cells. The antiadenovirus activities were evaluated by three methods, viz., evaluating the adenoviral cytopathic effect, monitoring cell viability by a colorimetric assay, and real-time PCR quantitation of viral DNA as a direct parameter for virus replication. The most active and selective compounds were the acyclic nucleoside phosphonate analogues cidofovir, its adenine analogue (S)-9-(3-hydroxy-2-phosphonylmethoxypropyl)adenine [(S)-HPMPA], and the new derivative (S)-2,4-diamino-6-[3-hydroxy-2-(phosphonomethoxy)propoxy]pyrimidine [(S)-HPMPO-DAPy]; the N7-substituted acyclic derivative 2-amino-7-(1,3-dihydroxy-2-propoxymethyl)purine (S-2242); and the 2′,3′-dideoxynucleoside analogues zalcitabine and alovudine. No antiadenovirus activity was observed for the antiviral drugs ribavirin, foscarnet, acyclovir, penciclovir, and brivudin, while ganciclovir displayed modest activity. However, in human osteosarcoma cells transfected with herpes simplex virus thymidine kinase, ganciclovir demonstrated highly potent antiadenovirus activity, suggesting that the efficacy of ganciclovir against adenovirus is limited by inefficient phosphorylation in adenovirus-infected cells, rather than by insufficient inhibition at the viral DNA polymerase level. Collectively, our antiviral data show that the adenovirus DNA polymerase exhibits sensitivity to a relatively broad spectrum of inhibitors and should be studied further as an antiviral target in antiadenovirus drug development programs.

Novel Inhibitors of Influenza Virus Fusion: Structure-Activity Relationship and Interaction with the Viral Hemagglutinin

ABSTRACT A new class of N-(1-thia-4-azaspiro[4.5]decan-4-yl)carboxamide inhibitors of influenza virus hemagglutinin (HA)-mediated membrane fusion that has a narrow and defined structure-activity relationship was identified. In Madin-Darby canine kidney (MDCK) cells infected with different strains of human influenza virus A/H3N2, the lead compound, 4c, displayed a 50% effective concentration of 3 to 23 μM and an antiviral selectivity index of 10. No activity was observed for A/H1N1, A/H5N1, A/H7N2, and B viruses. The activity of 4c was reduced considerably when added 30 min or later postinfection, indicating that 4c inhibits an early step in virus replication. 4c and its congeners inhibited influenza A/H3N2 virus-induced erythrocyte hemolysis at low pH. 4c-resistant virus mutants, selected in MDCK cells, contained either a single D112N change in the HA2 subunit of the viral HA or a combination of three substitutions, i.e., R220S (in HA1) and E57K (in HA2) and an A-T substitution at position 43 or 96 of HA2. The mutants showed efficiency for receptor binding and replication similar to that of wild-type virus yet displayed an increased pH of erythrocyte hemolysis. In polykaryon assays with cells expressing single-mutant HA proteins, the E57K, A96T, and D112N mutations resulted in 4c resistance, and the HA proteins containing R220S, A96T, and D112N mutations displayed an increased fusion pH. Molecular modeling identified a binding cavity for 4c involving arginine-54 and glutamic acid-57 in the HA2 subunit. Our studies with the new fusion inhibitor 4c confirm the importance of this HA region in the development of influenza virus fusion inhibitors.

Role of MRP4 and MRP5 in biology and chemotherapy

Nucleotide efflux (especially cyclic nucleotides) from a variety of mammalian tissues, bacteria, and lower eukaryotes has been studied for several decades. However, the molecular identity of these nucleotide efflux transporters remained elusive, despite extensive knowledge of their kinetic properties and inhibitor profiles. Identification of the subfamily of adenosine triphosphate (ATP) binding cassette transporters, multidrug resistance protein (MRP) subfamily, permitted rapid advances because some recently identified MRP family members transport modified nucleotide analogs (ie, chemotherapeutic agents). We first identified, MRP4, based on its ability to efflux antiretroviral compounds, such as azidothymidine monophosphate (AZT-MP) and 9-(2-phosphonyl methoxyethyl) adenine (PMEA), in drug-resistant and also in transfected cell lines. MRP5, a close structural homologue of MRP4 also transported PMEA. MRP4 and MRP5 confer resistance to cytotoxic thiopurine nucleotides, and we demonstrate MRP4 expression varies among acute lymphoblastic leukemias, suggesting this as a factor in response to chemotherapy with these agents. The ability of MRP4 and MRP5 to transport 3,5-cyclic adenosine monophosphate (cAMP) and 3,5-cyclic guanosine monophosphate (cGMP) suggests they may play a biological role in cellular signaling by these nucleotides. Finally, we propose that MRP4 may also play a role in hepatic bile acid homeostasis because loss of the main bile acid efflux transporter, sister of P-glycoprotein (SPGP) aka bile-salt export pump (BSEP), leads to a strong compensatory upregulation in MRP4 expression. Cumulatively, these studies reveal that the ATP-binding cassette (ABC) transporters MRP4 and MRP5 have a unique role in biology and in chemotherapeutic response.