Drug repurposing of rimantadine for treatment of cancer.

Repurposing of approved drugs allows strong savings in time and investment. Rimantadine is an FDA-approved drug for prevention and treatment of influenza A infection. Patent US2021330605 describes the use of rimantadine, an adamantane derivative, for the treatment of melanoma, breast cancer and head and neck squamous cell carcinoma. Rimantadine inhibited proliferation of cell lines of melanoma, breast cancer, and head and neck squamous cell carcinoma, increased the survival of mice injected with cancer cell lines and restores the expression of MHC class I. Rimantadine has the potential to be used successfully in the treatment of head and neck squamous cell carcinoma.

Efficacy of Ion-Channel Inhibitors Amantadine, Memantine and Rimantadine for the Treatment of SARS-CoV-2 In Vitro.

We report the in vitro efficacy of ion-channel inhibitors amantadine, memantine and rimantadine against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). In VeroE6 cells, rimantadine was most potent followed by memantine and amantadine (50% effective concentrations: 36, 80 and 116 µM, respectively). Rimantadine also showed the highest selectivity index, followed by amantadine and memantine (17.3, 12.2 and 7.6, respectively). Similar results were observed in human hepatoma Huh7.5 and lung carcinoma A549-hACE2 cells. Inhibitors interacted in a similar antagonistic manner with remdesivir and had a similar barrier to viral escape. Rimantadine acted mainly at the viral post-entry level and partially at the viral entry level. Based on these results, rimantadine showed the most promise for treatment of SARS-CoV-2.

HPV16 E5 Mediates Resistance to PD-L1 Blockade and Can Be Targeted with Rimantadine in Head and Neck Cancer.

There is a critical need to understand mechanisms of resistance and to develop combinatorial strategies to improve responses to checkpoint blockade immunotherapy (CBI). Here, we uncover a novel mechanism by which the human papillomavirus (HPV) inhibits the activity of CBI in head and neck squamous cell carcinoma (HNSCC). Using orthotopic HNSCC models, we show that radiation combined with anti-PD-L1 immunotherapy significantly enhanced local control, CD8+ memory T cells, and induced preferential T-cell homing via modulation of vascular endothelial cells. However, the HPV E5 oncoprotein suppressed immune responses by downregulating expression of major histocompatibility complex and interfering with antigen presentation in murine models and patient tumors. Furthermore, tumors expressing HPV E5 were rendered entirely resistant to anti-PD-L1 immunotherapy, and patients with high expression of HPV16 E5 had worse survival. The antiviral E5 inhibitor rimantadine demonstrated remarkable single-agent antitumor activity. This is the first report that describes HPV E5 as a mediator of resistance to anti-PD-1/PD-L1 immunotherapy and demonstrates the antitumor activity of rimantadine. These results have broad clinical relevance beyond HNSCC to other HPV-associated malignancies and reveal a powerful mechanism of HPV-mediated immunosuppression, which can be exploited to improve response rates to checkpoint blockade. SIGNIFICANCE: This study identifies a novel mechanism of resistance to anti-PD-1/PD-L1 immunotherapy mediated by HPV E5, which can be exploited using the HPV E5 inhibitor rimantadine to improve outcomes for head and neck cancer patients. GRAPHICAL ABSTRACT: http://cancerres.aacrjournals.org/content/canres/80/4/732/F1.large.jpg.

Alkyl-imino sugars inhibit the pro-oncogenic ion channel function of human papillomavirus (HPV) E5.

Despite the availability of prophylactic vaccines the burden of human papillomavirus (HPV) associated malignancy remains high and there is a need to develop additional therapeutic strategies to complement vaccination. We have previously shown that the poorly characterised E5 oncoprotein forms a virus-coded ion channel or viroporin that was sensitive to the amantadine derivative rimantadine. We now demonstrate that alkylated imino sugars, which have antiviral activity against a number of viruses, inhibit E5 channel activity in vitro. Using molecular modelling we predict that imino sugars intercalate between E5 protomers to prevent channel oligomerisation. We explored the ability of these viroporin inhibitors to block E5-mediated activation of mitogenic signalling in keratinocytes. Treatment with either rimantadine or imino sugars prevented ERK-MAPK phosphorylation and reduced cyclin B1 expression in cells expressing E5 from a number of high-risk HPV types. Moreover, viroporin inhibitors also reduced ERK-MAPK activation and cyclin B1 expression in differentiating primary human keratinocytes containing high-risk HPV18. These observations provide evidence of a key role for E5 viroporin function during the HPV life cycle. Viroporin inhibitors could be utilised for stratified treatment of HPV associated tumours prior to virus integration, or as true antiviral therapies to eliminate virus prior to malignant transformation.

Amino acid derivatives of adamantane carbocycle are capable of inhibiting replication of highly virulent avian influenza A/H5N1 virus.

We studied the capacity amino acid derivatives of adamantane to inhibit replication of highly virulent avian influenza A/duck/Novosibirsk/56/05 (H5N1) virus in cultures of swine embryonic kidney cells. Amino acid derivatives of adamantane H-His-Rem and Ad(CH2-Ser-OMe)2 were characterized by lower toxicity than remantadine previously used in the treatment of influenza. Histidine-containing adamantane derivative (H-His-Rem) was the most effective and low-toxic inhibitor of influenza А/H5N1 virus replication and can be recommended for clinical trials to produce a preparation for the treatment and prevention of influenza.

Exploring the proton conductance and drug resistance of BM2 channel through molecular dynamics simulations and free energy calculations at different pH conditions.

BM2 channel plays an important role in the replication of influenza virus B. However, few studies attempt to investigate the mechanism of the proton conductance in BM2 channel, as well as the drug resistance of the BM2 channel. The first experimental structure of the BM2 protein channel has recently been solved, enabling us to theoretically study BM2 systems with different protonation states of histidine. By performing molecular dynamics simulations on the BM2 systems with different protonation states of four His19 residues, we provided our understanding of the structure, dynamics, and drug resistance of the BM2 channel. In general, the results of our study and other investigations both have demonstrated that whether the BM2 channel adopts an open or a closed form depends on the protonation state of His19. Meanwhile, we discovered that a drug (amantadine) was unable to enter into the center of the BM2 channel even at a low pH condition probably due to the number of hydrophilic residues of the BM2 channel. Finally, potentials of mean force (PMF) calculations were performed for the drug binding BM2 channel, energetically explaining why the BM2 channel exhibited drug resistance to two inhibitors of the AM2 channel, amantadine and rimantadine.

Prophylactic and therapeutic combination effects of rimantadine and oseltamivir against influenza virus A (H3N2) infection in mice.

The combined effect of rimantadine and oseltamivir in a prophylactic context (therapy beginning 4 h pre-virus infection) and therapeutic context (therapy started at 24 h post-viral inoculation) course on influenza H3N2 virus infection in mice was studied. In the prophylactic course 5 and 10 mg/kg/day rimantadine with 0.2 and 0.4 mg/kg/day (25:1 dose ratio) oseltamivir showed a protection index (PI) of 79.6% and 75%, respectively and a mean survival time (MST) of 13.1 and 12.9 days. The individual effects of the same doses ranged from 0% to 33.3% PI and 8.2 to 10.3 days MST, respectively. Lung virus titers were decreased 630-fold in the combination-treated groups as compared to monotherapy and placebo groups. The reduction of surface lung pathology in combination-treated groups demonstrated a protective effect for the combination of both antivirals. In the therapeutic course 5 and 10 mg/kg rimantadine combined with 0.2 and 0.4 mg/kg oseltamivir showed no beneficial effect. At higher dosage (0.8, 1.6, 3.2 mg/kg oseltamivir and 20, 40, 80 mg/kg rimantadine) preserving the 25:1 ratio, the resultant PI ranged from 57.6% to 80.5% and the MST was 12.8-13.4 days. Used alone at the same doses the compounds' protection varied between 10.7% and 71.8% PI, MST 9.8-12.8 days (8.7 days in PBS control). Compared to vehicle and individual treatment, a decrease in infectious viral titers of up to 1000-fold and other viral pneumonia parameters were also recorded. The therapeutic effect of the drugs' optimal effective doses combinations was characterized as synergistic. Survival of animals was 81.2-100% and MST was extended by 5-7 days compared to placebos. Monotherapy protection was from 9.1% to a maximum of 56.5%, MST being prolonged only by 1.3-4.2 days compared to 7.5 days in the PBS control group. Lung viral titers were decreased 1445-fold for the most efficacious combination groups and a significant reduction in lung parameters was observed. These data emphasize that prophylactic and therapeutic courses using a combination of oseltamivir and rimantadine have a significant protective effect in mice experimentally infected with drug-sensitive influenza virus A (H3N2).

[Information of the Center for Ecology and Epidemiology of Influenza, D. I. Ivanovsky Research Institute of Virology, Russian Academy of Medical Sciences, on the results of the 2009-2010 influenza and acute respiratory viral infection epidemic season (at week 40 of 2009 to week 22 of 2010) in the world and Russia].

The paper describes the specific features of the 2009-2010 epidemic season in Russia and the world, which are due to the wide spread of a new pandemic strain of influenza A(H1N1)v virus. There is an unusual early upsurge in the incidence of influenza and acute respiratory viral infection (ARVI) (in October-November 2009) with its peak at weeks 45 to 48 of the year with a succeeding reduction to the seasonal values by its end. The circulation of influenza B virus strains was recorded in February-April 2010, which was responsible for the higher epidemic thresholds of morbidity in a number of Russia's regions. A study of the antigenic properties of the strains defined their relationship to the reference strains A/California/07/2009 (H1N1)v and B/Brisbene/60/2008. There were strains with amino acid substitutions at position 222 of hemagglutinin in the population of pandemic influenza A(H1N1)v virus. The strains of the new pandemic influenza A(H1N1)v virus were resistant to remantadine and susceptible to oseltamivir, zanamivir, and arbidol. The influenza B virus strains were susceptible to oseltamivir, zanamivir, and arbidol. The proportion of pathogens of some ARVIs was as follows: parainfluenza viruses, 9.8%; adenoviruses, 5.5%; respiratory syncytial virus, 4.8%; and Mycoplasma pneumonia, 0.6%. There is evidence that there is a need for further monitoring of influenza viruses in Russia.

[Influence of rimantadine, ribavirine and triazavirine on influenza A virus replication in human monolayer and lymphoblastoid cell lines].

The influence of antivirals, such as rimantadine, ribavirine and triazavirine on influenza virus replication in human cell cultures was evaluated. All the antivirals inhibited viral nucleoprotein NP synthesis. The strongest effect was shown for ribavirine in lung carcinoma A-549 cells and endothelial ECV-304 cells. Hoechst-33258 staining revealed induction of apoptosis in all the cell lines. Rimantadine and ribavirine inhibited virus-induced apoptosis while ribavirine enhanced it. The effect was registered in monolayer cell cultures as well as in suspension cell cultures. The influence of the antiviral drugs on the virus-induced cell proliferation in the suspension cell cultures is also described.

Cys-loop receptor channel blockers also block GLIC.

The Gloeobacter ligand-gated ion channel (GLIC) is a bacterial homolog of vertebrate Cys-loop ligand-gated ion channels. Its pore-lining region in particular has a high sequence homology to these related proteins. Here we use electrophysiology to examine a range of compounds that block the channels of Cys-loop receptors to probe their pharmacological similarity with GLIC. The data reveal that a number of these compounds also block GLIC, although the pharmacological profile is distinct from these other proteins. The most potent compound was lindane, a GABA(A) receptor antagonist, with an IC50 of 0.2 µM. Docking studies indicated two potential binding sites for this ligand in the pore, at the 9' or between the 0' and 2' residues. Similar experiments with picrotoxinin (IC50 = 2.6 µM) and rimantadine (IC50 = 2.6 µM) reveal interactions with 2'Thr residues in the GLIC pore. These locations are strongly supported by mutagenesis data for picrotoxinin and lindane, which are less potent in a T2'S version of GLIC. Overall, our data show that the inhibitory profile of the GLIC pore has considerable overlap with those of Cys-loop receptors, but the GLIC pore has a unique pharmacology.

Flu channel drug resistance: a tale of two sites.

The M2 proteins of influenza A and B virus, AM2 and BM2, respectively, are transmembrane proteins that oligomerize in the viral membrane to form proton-selective channels. Proton conductance of the M2 proteins is required for viral replication; it is believed to equilibrate pH across the viral membrane during cell entry and across the trans-Golgi membrane of infected cells during viral maturation. In addition to the role of M2 in proton conductance, recent mutagenesis and structural studies suggest that the cytoplasmic domains of the M2 proteins also play a role in recruiting the matrix proteins to the cell surface during virus budding. As viral ion channels of minimalist architecture, the membrane-embedded channel domain of M2 has been a model system for investigating the mechanism of proton conduction. Moreover, as a proven drug target for the treatment of influenza A infection, M2 has been the subject of intense research for developing new anti-flu therapeutics. AM2 is the target of two anti-influenza A drugs, amantadine and rimantadine, both belonging to the adamantane class of compounds. However, resistance of influenza A to adamantane is now widespread due to mutations in the channel domain of AM2. This review summarizes the structure and function of both AM2 and BM2 channels, the mechanism of drug inhibition and drug resistance of AM2, as well as the development of new M2 inhibitors as potential anti-flu drugs.

Genotype-dependent sensitivity of hepatitis C virus to inhibitors of the p7 ion channel.

UNLABELLED: The hepatitis C virus (HCV) p7 protein plays a critical role during particle formation in cell culture and is required for virus replication in chimpanzees. The discovery that it displayed cation channel activity in vitro led to its classification within the "viroporin" family of virus-coded ion channel proteins, which includes the influenza A virus (IAV) M2 protein. Like M2, p7 was proposed as a potential target for much needed new HCV therapies, and this was supported by our finding that the M2 inhibitor, amantadine, blocked its activity in vitro. Since then, further compounds have been shown to inhibit p7 function but the relationship between inhibitory effects in vitro and efficacy against infectious virus is controversial. Here, we have sought to validate multiple p7 inhibitor compounds using a parallel approach combining the HCV infectious culture system and a rapid throughput in vitro assay for p7 function. We identify a genotype-dependent and subtype-dependent sensitivity of HCV to p7 inhibitors, in which results in cell culture largely mirror the sensitivity of recombinant protein in vitro; thus building separate sensitivity profiles for different p7 sequences. Inhibition of virus entry also occurred, suggesting that p7 may be a virion component. Second site effects on both cellular and viral processes were identified for several compounds in addition to their efficacy against p7 in vitro. Nevertheless, for some compounds antiviral effects were specific to a block of ion channel function. CONCLUSION: These data validate p7 inhibitors as prototype therapies for chronic HCV disease. (HEPATOLOGY 2008;48:1779-1790.).

[Antiviral action of some antioxidants/antihypoxants and their combinations with remantadine against human influenza A(H3N2) virus studied in in vitro models].

The possible antiviral activity of preparations with antioxidant and/or antihypoxant properties was studied on two in vitro models of influenza infection: (i) in cultures of chorio-allantoic membranes of chicken embryos and (ii) in MDCK cells. Preparations under study were hypoxene, reduced glutathione, dihydroquercetin, trolox, coenzyme Q10, and the enzymatic preparation of superoxide-dismutase (recsod). Preparations possessing combined antioxidant/antihypoxic and detoxicating properties (reduced glutathione and hypoxene) produced a significant antiviral effect and enhanced the antiviral effect of rimantadine. The antiviral effect of these preparations was manifested by a decrease in the production of viral particles and, to a more pronounced degree, by the inhibition of cytopathogenic action of virus on cultured cells, which was revealed in the tests for the activity of respiratory enzymes. In contrast to the compounds containing thio or sulfo groups, the antioxidants of "direct action" (free radical scavengers) - coenzyme Q 10, trolox, quercetin and the enzymatic preparation recsod did not show any pronounced protective effect and in some cases even enhanced the production of viral particles and decreased the antiviral action of rimantadine.

Hypothalamic growth hormone-releasing hormone (GHRH) deficiency: targeted ablation of GHRH neurons in mice using a viral ion channel transgene.

Animal and clinical models of GHRH excess suggest that GHRH provides an important trophic drive to pituitary somatotrophs. We have adopted a novel approach to silence or ablate GHRH neurons, using a modified H37A variant of the influenza virus M2 protein ((H37A)M2). In mammalian cells, (H37A)M2 forms a high conductance monovalent cation channel that can be blocked by the antiviral drug rimantadine. Transgenic mice with (H37A)M2 expression targeted to GHRH neurons developed postweaning dwarfism with hypothalamic GHRH transcripts detectable by RT-PCR but not by in situ hybridization and immunocytochemistry, suggesting that expression of (H37A)M2 had silenced or ablated virtually all the GHRH cells. GHRH-M2 mice showed marked anterior pituitary hypoplasia with GH deficiency, although GH cells were still present. GHRH-M2 mice were also deficient in prolactin but not TSH. Acute iv injections of GHRH in GHRH-M2 mice elicited a significant GH response, whereas injections of GHRP-6 did not. Twice daily injections of GHRH (100 microg/d) for 7 d in GHRH-M2 mice doubled their pituitary GH but not PRL contents. Rimantadine treatment failed to restore growth or pituitary GH contents. Our results show the importance of GHRH neurons for GH and prolactin production and normal growth.

Development of a novel influenza A antiviral assay.

Traditional methods used to monitor influenza infection typically require 2-5 days to perform, prompting a need for more rapid and quantitative methods for monitoring viral infection in 96-well formats. Such assays would find application in high-throughput screening for novel antiviral agents. A new method, based on branched DNA (bDNA) technology, is described for the specific detection of negative strand RNA of influenza A strains using a set of oligonucleotides designed for the A/PR/8/34 nucleoprotein (NP) transcript. By detecting the genomic strand, this signal amplification assay is appropriate for monitoring the kinetics of viral replication. Assay performance was monitored following infection of MDCK cells. The assay exhibited high reproducibility, good sensitivity over a range of multiplicity of infection and has a lower limit of detection of approximately 5 x 10 (5) RNA copies. Designed to quantitate the H1N1 strain A/PR/8/34, the assay also detects other influenza A subtypes, but not the evolutionarily more distant strain B/Yamagata/16/88. Validation as an antiviral assay was demonstrated with two influenza antivirals, zanamivir and rimantadine. The EC(50) values calculated following bDNA detection for zanamivir (265 nM) and rimantadine (9.4 microg/ml) in A/PR/8/34 infection correlate closely to data previously reported from visual CPE determinations, neutral red dye uptake and plaque assays, respectively. The advantages over the more time-consuming traditional assays suggest that the influenza bDNA assay is applicable to rapid screening of compound collections for antiviral activity.

Neuraminidase inhibitors in patients with underlying airways disease.

Influenza virus infection accounts for significant morbidity, mortality, and healthcare expenditures among persons worldwide. Approximately 20,000 to 40,000 people in the US die each year as a result of influenza. Individuals most susceptible to adverse outcomes include the elderly and those with asthma, chronic obstructive pulmonary disease (COPD), heart disease, renal failure, malignancy, or immunosuppression. Prior to the AIDS epidemic, underlying respiratory disease was the greatest risk factor for influenza-related hospitalization ranking third to heart disease and malignancy for risk of mortality. Although the influenza vaccine can help prevent pneumonia and hospitalization, it is limited by less than ideal immunization rates and the possibility of viral antigenic shifts that render the vaccine ineffective. Pharmacologic interventions play an important role in the management of influenza virus infection by shortening the duration of symptoms. The advent of the neuraminidase inhibitors (NAIs) zanamivir and oseltamivir has significantly affected the treatment of influenza. Unlike NAIs, the older therapeutic options amantadine and rimantadine may cause significant central nervous system adverse effects. In addition, amantadine and rimantadine are not active against influenza B viruses, whereas NAIs are active against both influenza A and B. Post-marketing surveillance of the NAIs has revealed that bronchospasm may occur in patients with underlying respiratory disease treated with the NAI zanamivir. Recent data suggest zanamivir is effective in patients with underlying respiratory disease, but the data are insufficient to elucidate the true risk of bronchospasm. Based on post-marketing reports, zanamivir should be used with caution in patients with asthma or COPD. Although oseltamivir has not been associated with any significant respiratory adverse effects, no data exist on the safety and efficacy of this NAI in patients with underlying respiratory disease.

Perspectives on antiviral use during pandemic influenza.

Antiviral agents could potentially play a major role in the initial response to pandemic influenza, particularly with the likelihood that an effective vaccine is unavailable, by reducing morbidity and mortality. The M2 inhibitors are partially effective for chemoprophylaxis of pandemic influenza and evidence from studies of interpandemic influenza indicate that the neuraminidase inhibitors would be effective in prevention. In addition to the symptom benefit observed with M2 inhibitor treatment, early therapeutic use of neuraminidase inhibitors has been shown to reduce the risk of lower respiratory complications. Clinical pharmacology and adverse drug effect profiles indicate that the neuraminidase inhibitors and rimantadine are preferable to amantadine with regard to the need for individual prescribing and tolerance monitoring. Transmission of drug-resistant virus could substantially limit the effectiveness of M2 inhibitors and the possibility exists for primary M2 inhibitor resistance in a pandemic strain. The frequency of resistance emergence is lower with neuraminidase inhibitors and mathematical modelling studies indicate that the reduced transmissibility of drug-resistant virus observed with neuraminidase inhibitor-resistant variants would lead to negligible community spread of such variants. Thus, there are antiviral drugs currently available that hold considerable promise for response to pandemic influenza before a vaccine is available, although considerable work remains in realizing this potential. Markedly increasing the quantity of available antiviral agents through mechanisms such as stockpiling, educating health care providers and the public and developing effective means of rapid distribution to those in need are essential in developing an effective response, but remain currently unresolved problems.

Amantadine and rimantadine have no direct inhibitory effects against hepatitis C viral protease, helicase, ATPase, polymerase, and internal ribosomal entry site-mediated translation.

Amantadine, a drug known to inhibit influenza A viral matrix (M2) protein function, was reported to be an effective treatment in some patients with chronic hepatitis C virus (HCV) infection. Sequence comparison shows no homology between M2 and any of the HCV proteins. The effects of amantadine and a related analogue, rimantadine, on viral protease, helicase, ATPase, RNA-dependent RNA polymerase, and HCV internal ribosomal entry site (IRES) translation were tested by established in vitro biochemical assays. No inhibition (>15%) of HCV protease, helicase, ATPase, and polymerase was observed with concentrations up to 400 microgram/mL. IRES-specific inhibition was not observed at clinically relevant concentrations, but both cap and IRES reporter genes were suppressed at higher levels, suggesting nonspecific translation inhibition. In conclusion, amantadine and rimantadine have no direct and specific inhibitory effects against HCV protease, helicase, ATPase, polymerase, and IRES in vitro.

[Polypeptides from influenza virus, obtained in the presence of high concentrations of rimantadine]. / Polipeptidy virusa grippa, poluchennogo v prisutstvii vysokikh kontsentratsii remantadina.

FPV/Rostock obtained in the presence of remantadine (25 micrograms/ml) is studied. This variant of the virus is characterized by a decreased amount of M1-protein. Its buoyant density in urograffin density gradient is 1.20 gamma/cm3 vs. 1.18 and 1.175 g/cm3 for initial FPV and remantadine-resistant FPV, respectively. Purification and concentration decreases the hemagglutinating activity of the virus 5-10 times more intensely than that of initial and remantadine-resistant FPV. On the other hand, the production of virus-specific proteins and intracellular distribution of hemagglutinin in this virus variant were not changed. The changes associated with a decrease of hemagglutinin and M1-protein content in virions are believed to manifest at a later stage preceding virion release from the cell.

Selective proton permeability and pH regulation of the influenza virus M2 channel expressed in mouse erythroleukaemia cells.

1. The M2 protein of influenza A virus is implicated in transmembrane pH regulation during infection. Whole-cell patch clamp of mouse erythroleukaemia cells expressing the M2 protein in the surface membrane showed a conductance due to M2 which was specifically blocked by the anti-influenza drug rimantadine. 2. The ion selectivity of the rimantadine-sensitive current through M2 was determined. Reversal potentials were close to equilibrium potentials for transmembrane pH gradients and not to those for Na+, K+ or Cl- concentration gradients. M2 permeability to Na+ relative to H+ was estimated to be less than 6 x 10(-7). 3. The M2 conductance increased as external pH decreased below 8.5 and approached saturation at an external pH of 4, effects attributable to increased permeability due to increased driving potential and to activation by low external pH. Both activation and permeation could be described by interaction of protons with sites on M2, with apparent dissociation constants of approximately 0.1 microM and 1 microM, respectively, under physiological conditions. 4. The M2 protein can transfer protons selectively across membranes with the H+ electrochemical gradient, properties consistent with its role in modifying virion and trans-Golgi pH during virus infection.