Cytomegalovirus Inhibition Research Articles

Poloxamer 188 – quercetin formulations amplify in vitro ganciclovir antiviral activity against cytomegalovirus

Treatment of human cytomegalovirus (CMV) infection requires long-term administration of nucleoside analog antivirals such as ganciclovir (GCV), a therapy frequently limited by GCV-induced toxicity. Here, combining GCV treatment with two bioactive excipients, poloxamer 188 and quercetin, was investigated in vitro to reduce GCV dosage. Quercetin is a natural flavonoid exhibiting antiviral activity against CMV by a mechanism distinct from GCV, but is poorly soluble, limiting its use as a therapeutic. To overcome this challenge, quercetin was co-formulated with poloxamer 188 (P188, Pluronic ® F68). Quercetin-P188 (QP188) formulations yielded only modest CMV viral inhibition, with a selectivity index of 11.4, contrasted with a GCV selectivity index of 95. More significantly, when coadministered with GCV, QP188 exhibited an additive or synergistic interaction in subtherapeutic ranges of GCV. Fluorescence microscopy revealed QP188 accumulation in fibroblast mitochondria, suggesting that the excipient may modulate mitochondrial processes relevant to CMV infection. GCV antiviral therapy augmented with poloxamer-solubilized quercetin may be a viable approach to maintain CMV inhibition while lowering GCV doses, translating to reduced associated toxicity.

Inhibition of human cytomegalovirus replication by artemisinins: effects mediated through cell cycle modulation.

Artemisinin-derived monomers and dimers inhibit human cytomegalovirus (CMV) replication in human foreskin fibroblasts (HFFs). The monomer artesunate (AS) inhibits CMV at micromolar concentrations, while dimers inhibit CMV replication at nanomolar concentrations, without increased toxicity in HFFs. We report on the variable anti-CMV activity of AS compared to the consistent and reproducible CMV inhibition by dimer 606 and ganciclovir (GCV). Investigation of this phenomenon revealed that the anti-CMV activity of AS correlated with HFFs synchronized to the G0/G1 stage of the cell cycle. In contact-inhibited serum-starved HFFs or cells arrested at early/late G1 with specific checkpoint regulators, AS and dimer 606 efficiently inhibited CMV replication. However, in cycling HFFs, in which CMV replication was productive, virus inhibition by AS was significantly reduced, but inhibition by dimer 606 and GCV was maintained. Cell cycle analysis in noninfected HFFs revealed that AS induced early G1 arrest, while dimer 606 partially blocked cell cycle progression. In infected HFFs, AS and dimer 606 prevented the progression of cell cycle toward the G1/S checkpoint. AS reduced the expression of cyclin-dependent kinases (CDK) 2, 4, and 6 in noninfected cycling HFFs, while the effect of dimer 606 on these CDKs was moderate. Neither compound affected CDK expression in noninfected contact-inhibited HFFs. In CMV-infected cells, AS activity correlated with reduced CDK2 levels. CMV inhibition by AS and dimer 606 also correlated with hypophosphorylation (activity) of the retinoblastoma protein (pRb). AS activity was strongly associated with pRb hypophosphorylation, while its reduced anti-CMV activity was marked by pRb phosphorylation. Roscovitine, a CDK2 inhibitor, antagonized the anti-CMV activities of AS and dimer 606. These data suggest that cell cycle modulation through CDKs and pRb might play a role in the anti-CMV activities of artemisinins. Proteins involved in this modulation may be identified and targeted for CMV inhibition.

In Vitro Combination of Anti-Cytomegalovirus Compounds Acting through Different Targets: Role of the Slope Parameter and Insights into Mechanisms of Action

Conventional therapy for human cytomegalovirus (CMV) relies on inhibition of the viral DNA polymerase. Ganciclovir (GCV) is the first-line therapy, but when GCV-resistant strains emerge, alternative therapies are extremely limited and are associated with significant toxicities. Combination of anti-CMV agents that act on different targets or stages of virus replication has not been well studied, mostly because of the limited number of anti-CMV agents. We report our investigation of combinations of agents that inhibit CMV by targeting the viral DNA polymerase, cellular kinases, or other cell/virus mechanisms yet to be discovered. The selected compounds differed by the slopes of their dose-response curve: compounds with a slope of 1 (GCV) representing one target or noncooperativity and compounds with high slopes indicating positive cooperativity. Analysis of anti-CMV drug combinations using the Bliss model (which accounts for the slope parameter) distinguished between combinations with synergistic, antagonistic, and additive activities. The combination of GCV and foscarnet was slightly synergistic; strong synergism was found when GCV was used with artemisinin-derived monomers or dimers or the MEK inhibitor U0126. The combination of GCV and cardiac glycosides (digoxin, digitoxin, and ouabain) was additive. The monomeric artemisinin artesunate was synergistic when combined with U0126 or the multikinase inhibitor sunitinib. However, the combination of artemisinin-derived dimers (molecular weights, 606 and 838) and U0126 or sunitinib was antagonistic. These results demonstrate that members of a specific drug class show similar patterns of combination with GCV and that the slope parameter plays an important role in the evaluation of drug combinations. Lastly, antagonism between different classes of CMV inhibitors may assist in target identification and improve the understanding of CMV inhibition by novel compounds.

Unique and Highly Selective Anticytomegalovirus Activities of Artemisinin-Derived Dimer Diphenyl Phosphate Stem from Combination of Dimer Unit and a Diphenyl Phosphate Moiety

We report that the artemisinin-derived dimer diphenyl phosphate (DPP; dimer 838) is the most selective inhibitor of human cytomegalovirus (CMV) replication among a series of artemisinin-derived monomers and dimers. Dimer 838 was also unique in being an irreversible CMV inhibitor. The peroxide unit within artemisinins' chemical structures is critical to their activities, and its absence results in loss of anti-CMV activities. Surprisingly, the deoxy dimer of 838 retained modest anti-CMV activity, suggesting that the DPP moiety of dimer 838 contributes to its anti-CMV activities. DPP alone did not inhibit CMV replication, but triphenyl phosphate (TPP) had modest CMV inhibition, although its selectivity index was low. Artemisinin DPP derivatives dimer 838 and monomer diphenyl phosphate (compound 558) showed stronger CMV inhibition and a higher selectivity index than their analogs lacking the DPP unit. An add-on and removal assay revealed that removing DPP derivatives (compounds 558 and 838) but not the non-DPP backbones (artesunate and compound 606) at 24 h postinfection (hpi) already resulted in dominant CMV inhibition. CMV inhibition was fully irreversible with 838 and partially irreversible with 558, while non-DPP artemisinins were reversible inhibitors. While all artemisinin derivatives and TPP reduced the expression of the CMV immediate early 2 (IE2), UL44, and pp65 proteins at or after 48 hpi, only TPP inhibited the expression of both IE1 and IE2. Combination of a non-DPP dimer (compound 606) with TPP was synergistic in CMV inhibition, while ganciclovir and TPP were additive. Although TPP shared structural similarity with monomer DPP (compound 558) and dimer DPP (compound 838), its pattern of CMV inhibition was significantly different from the patterns of the artemisinins. These findings demonstrate that the DPP group contributes to the unique activities of compound 838.

Artemisinin-Derived Dimer Diphenyl Phosphate Is an Irreversible Inhibitor of Human Cytomegalovirus Replication

We previously reported that among a series of artemisinin-derived monomers and dimers, dimer diphenyl phosphate (838) was the most potent inhibitor of human cytomegalovirus (CMV) replication. Our continued investigation of a prototypic artemisinin monomer (artesunate [AS]) and dimer (838) now reveals that both compounds have specific activity against CMV but do not inhibit lytic replication of human herpesvirus 1 or 2 or Epstein-Barr virus. AS and 838 inhibited CMV replication during the first 24 h of the virus replication cycle, earlier than the time of ganciclovir (GCV) activities and prior to DNA synthesis. Neither compound inhibited virus entry. Quantification of DNA replication and virus yield revealed a similar level of inhibition by GCV, but AS and 838 had a 10-fold-higher inhibition of virus yield than of DNA replication, suggesting that artemisinins could inhibit CMV through multiple steps: a predominant early inhibition and possibly an additional step following DNA replication. During the strong early CMV inhibition, the transcription of immediate-early genes was not significantly downregulated, and viral protein expression was reduced only after 48 h. AS and GCV were reversible CMV inhibitors, but the inhibition of CMV replication by 838 was irreversible. Combinations of GCV and 838 as well as GCV and AS were highly synergistic. Finally, treatment with 838, but not AS, prior to CMV infection demonstrated strong anti-CMV activity. These findings illustrate the unique activities of dimer 838, including early and irreversible CMV inhibition, possibly by tight binding to its target.

An Artemisinin-Derived Dimer Has Highly Potent Anti-Cytomegalovirus (CMV) and Anti-Cancer Activities

We recently reported that two artemisinin-derived dimers (dimer primary alcohol 606 and dimer sulfone 4-carbamate 832-4) are significantly more potent in inhibiting human cytomegalovirus (CMV) replication than artemisinin-derived monomers. In our continued evaluation of the activities of artemisinins in CMV inhibition, twelve artemisinin-derived dimers and five artemisinin-derived monomers were used. Dimers as a group were found to be potent inhibitors of CMV replication. Comparison of CMV inhibition and the slope parameter of dimers and monomers suggest that dimers are distinct in their anti-CMV activities. A deoxy dimer (574), lacking the endoperoxide bridge, did not have any effect on CMV replication, suggesting a role for the endoperoxide bridge in CMV inhibition. Differences in anti-CMV activity were observed among three structural analogs of dimer sulfone 4-carbamate 832-4 indicating that the exact placement and oxidation state of the sulfur atom may contribute to its anti-CMV activity. Of all tested dimers, artemisinin-derived diphenyl phosphate dimer 838 proved to be the most potent inhibitor of CMV replication, with a selectivity index of approximately 1500, compared to our previously reported dimer sulfone 4-carbamate 832-4 with a selectivity index of about 900. Diphenyl phosphate dimer 838 was highly active against a Ganciclovir-resistant CMV strain and was also the most active dimer in inhibition of cancer cell growth. Thus, diphenyl phosphate dimer 838 may represent a lead for development of a highly potent and safe anti-CMV compound.

Artemisinin-Derived Dimers Have Greatly Improved Anti-Cytomegalovirus Activity Compared to Artemisinin Monomers

BackgroundArtesunate, an artemisinin-derived monomer, was reported to inhibit Cytomegalovirus (CMV) replication. We aimed to compare the in-vitro anti-CMV activity of several artemisinin-derived monomers and newly synthesized artemisinin dimers.MethodsFour artemisinin monomers and two novel artemisinin-derived dimers were tested for anti-CMV activity in human fibroblasts infected with luciferase-tagged highly–passaged laboratory adapted strain (Towne), and a clinical CMV isolate. Compounds were evaluated for CMV inhibition and cytotoxicity.ResultsArtemisinin dimers effectively inhibited CMV replication in human foreskin fibroblasts and human embryonic lung fibroblasts (EC50 for dimer sulfone carbamate and dimer primary alcohol 0.06±0.00 µM and 0.15±0.02 µM respectively, in human foreskin fibroblasts) with no cytotxicity at concentrations required for complete CMV inhibition. All four artemisinin monomers (artemisinin, artesunate, artemether and artefanilide) shared a similar degree of CMV inhibition amongst themselves (in µM concentrations) which was significantly less than the inhibition achieved with artemisinin dimers (P<0.0001). Similar to monomers, inhibition of CMV with artemisinin dimers appeared early in the virus life cycle as reflected by decreased expression of the immediate early (IE1) protein.ConclusionsArtemisinin dimers are potent and non-cytotoxic inhibitors of CMV replication. These compounds should be studied as potential therapeutic agents for the treatment of CMV infection in humans.

Response of asymptomatic cytomegalovirus viraemia to oral ganciclovir 3 g/day or 6 g/day in HIV‐infected patients

Reactivation of cytomegalovirus (CMV) following immunosuppression may result in the development of CMV disease and is associated with an increased risk of death. CMV viraemia detected by the polymerase chain reaction (PCR) precedes CMV disease in HIV-infected patients and identifies individuals at high risk of disease. Pre-emptive ganciclovir (GCV) therapy in patients who have evidence of CMV viraemia is effective in preventing disease. An open study was conducted to assess the response of CMV viraemia to oral GCV at a dose of 3 or 6 g/day for 28 days. HIV RNA was measured to determine if CMV inhibition affected HIV viral load. Fourteen patients were studied, three of whom entered both phases of the study. None of the patients had evidence of CMV disease at the time of entry into the trial; two patients developed CMV retinitis after completion of the trial. Oral GCV at both 3 and 6 g/day caused a decrease in CMV viral load in individual patients. However, a rebound in CMV viral load occurred in patients receiving the 3-g/day dose. None of the patients receiving oral GCV 3 g/day became PCR negative after 21 days compared with six of eight patients receiving 6 g/day. Five of eight patients (63%) receiving GCV 6 g/day were concurrently taking protease inhibitors compared with two of nine (22%) receiving 3 g/day. Ten patients remained PCR negative throughout follow up. No change was found in HIV viral load during receipt of GCV at either dose. Thus, oral GCV is effective in reducing CMV viral load, but a dose of 3 g/day is insufficiently potent for pre-emptive therapy. J. Med. Virol. 59:323–328, 1999. © 1999 Wiley-Liss, Inc.

Response of asymptomatic cytomegalovirus viraemia to oral ganciclovir 3 g/day or 6 g/day in HIV-infected patients

Reactivation of cytomegalovirus (CMV) following immunosuppression may result in the development of CMV disease and is associated with an increased risk of death. CMV viraemia detected by the polymerase chain reaction (PCR) precedes CMV disease in HIV-infected patients and identifies individuals at high risk of disease. Pre-emptive ganciclovir (GCV) therapy in patients who have evidence of CMV viraemia is effective in preventing disease. An open study was conducted to assess the response of CMV viraemia to oral GCV at a dose of 3 or 6 g/day for 28 days. HIV RNA was measured to determine if CMV inhibition affected HIV viral load. Fourteen patients were studied, three of whom entered both phases of the study. None of the patients had evidence of CMV disease at the time of entry into the trial; two patients developed CMV retinitis after completion of the trial. Oral GCV at both 3 and 6 g/day caused a decrease in CMV viral load in individual patients. However, a rebound in CMV viral load occurred in patients receiving the 3-g/day dose. None of the patients receiving oral GCV 3 g/day became PCR negative after 21 days compared with six of eight patients receiving 6 g/day. Five of eight patients (63%) receiving GCV 6 g/day were concurrently taking protease inhibitors compared with two of nine (22%) receiving 3 g/day. Ten patients remained PCR negative throughout follow up. No change was found in HIV viral load during receipt of GCV at either dose. Thus, oral GCV is effective in reducing CMV viral load, but a dose of 3 g/day is insufficiently potent for pre-emptive therapy.