Consecutive alternating administration as an effective anti-coxsackievirus B3 in vivo treatment scheme.

Although chemotherapy is generally indicated for treatment of enterovirus infections, antivirals are currently not used in clinical practice. The use of monotherapy is the main reason for this unfavourable state. This is related to the fact that enterovirus progeny consist of innumerable quasispecies, allowing the virus to develop drug resistance quickly. Here, we present a consecutive alternating administration (CAA) treatment scheme for combining enterovirus inhibitors. Applying the CAA approach with a combination of pleconaril (capsid binder), guanidine HCl (viral 2C inhibitor), and oxoglaucine (PI4KB inhibitor) (PGO) was found to be effective in the treatment of newborn mice infected with a massive inoculum (20 MLD50) of the coxsackievirus B3 cardiotropic Woodruff or neurotropic Nancy strain. In addition to preventing drug resistance, the CAA approach resulted in the parallel development of increased susceptibility to the compounds in the PGO combination. These observations demonstrate the therapeutic potential of the CAA approach for treatment of enterovirus infections.

Effects of double combinations of enterovirus replication inhibitors against Coxsackie B viruses.

The effects of double combinations of enterovirus (EV) replication inhibitors against Coxsackieviruses B1 (neurotropic Connecticut-5 strain) and B3 (cardiotropic Woodruff and neurotropic Nancy strains) were tested in cell culture experiments. Compounds with different mechanisms of action were studied: pleconaril, guanidine.HCl, MDL-860 and oxoglaucine. A three-dimensional method was applied for determining the character of the combined effect. The study determined several synergistic double combinations: guanidine.HCL + pleconaril or MDL-860 against Coxsackievirus B1; MDL-860 + each of the other EV replication inhibitors and guanidine.HCl + pleconaril against the cardiotropic Woodruff strain of Coxsackievirus B3; MDL-860 + oxoglaucine against the neurotropic Nancy strain of Coxsackievirus B3. No increased cytotoxicity was manifested in any of the tested double combinations. Keywords: antivirals; combination activity; Coxsackieviruses.

Anti-enteroviral activity of new MDL-860 analogues: Synthesis, in vitro/in vivo studies and QSAR analysis.

A series of 60 nitrobenzonitrile analogues of the anti-viral agent MDL-860 were synthesized (50 of which are new) and evaluated for their activity against three types of enteroviruses (coxsackievirus B1, coxsackievirus B3 and poliovirus 1). Among them, six diaryl ethers (20e, 27e, 28e, 29e, 33e and 35e) demonstrated high in vitro activity (SI > 50) towards at least one of the tested viruses and very low cytotoxicity against human cells. Compound 27e possesses the broadest spectrum of activity towards all tested viruses in the same way as MDL-860 does. The most active derivatives (27e, 29e and 35e) against coxsackievirus B1 were tested in vivo in newborn mice experimentally infected with 20 MLD50 of coxsackievirus B1. Compound 29e showed promising in vivo activity (protection index 26% and 4 days lengthening of mean survival time). QSAR analysis of the substituent effects on the in vitro cytotoxicity (CC50) and anti-viral activity of the nitrobenzonitrile derivatives was carried out and adequate QSAR models for the anti-viral activity of the compounds against poliovirus 1 and coxsackievirus B1 were constructed.

Tailoring acyclovir prodrugs with enhanced antiviral activity: rational design, synthesis, human plasma stability and in vitro evaluation.

Bile acid prodrugs have served as a viable strategy for refining the pharmaceutical profile of parent drugs through utilizing bile acid transporters. A series of three ester prodrugs of the antiherpetic drug acyclovir (ACV) with the bile acids cholic, chenodeoxycholic and deoxycholic were synthesized and evaluated along with valacyclovir for their in vitro antiviral activity against herpes simplex viruses type 1 and type 2 (HSV-1, HSV-2). The in vitro antiviral activity of the three bile acid prodrugs was also evaluated against Epstein-Barr virus (EBV). Plasma stability assays, utilizing ultra-high performance liquid chromatography coupled with tandem mass spectrometry, in vitro cytotoxicity and inhibitory experiments were conducted in order to establish the biological profile of ACV prodrugs. The antiviral assays demonstrated that ACV-cholate had slightly better antiviral activity than ACV against HSV-1, while it presented an eight-fold higher activity with respect to ACV against HSV-2. ACV-chenodeoxycholate presented a six-fold higher antiviral activity against HSV-2 with respect to ACV. Concerning EBV, the highest antiviral effect was demonstrated by ACV-chenodeoxycholate. Human plasma stability assays revealed that ACV-deoxycholate was more stable than the other two prodrugs. These results suggest that decorating the core structure of ACV with bile acids could deliver prodrugs with amplified antiviral activity.

Synthesis and anti-enterovirus activity of new analogues of MDL-860.

A series of twelve novel compounds, analogues of antiviral agent MDL-860 were synthesized and their antiviral activity was evaluated in vitro against enteroviruses poliovirus 1 (PV1), Coxsackieviruses B1 (CVB1) and Coxsackieviruses B3 (CVB3). Compounds 14, 24 and 25 manifested strong antiviral effects against CVB1 and PV1 (SI values of 405 and 118 for CVB1 and PV1 respectively). In contrast to the wide anti-enteroviral activity of MDL-860, these three compounds were inactive against CVB3. Compounds 14, 24 and 25 along with MDL-860 were tested in vivo in mice infected with CVB1. Marked protective effects of compounds 14 and 24 were established, PI values of 50% and 33.3%, respectively. In addition, almost all of the tested compounds manifested very low toxicity.

Virus inactivation under the photodynamic effect of phthalocyanine zinc(II) complexes.

Various metal phthalocyanines have been studied for their capacity for photodynamic effects on viruses. Two newly synthesized water-soluble phthalocyanine Zn(II) complexes with different charges, cationic methylpyridyloxy-substituted Zn(II)- phthalocyanine (ZnPcMe) and anionic sulfophenoxy-substituted Zn(II)-phthalocyanine (ZnPcS), were used for photoinactivation of two DNA-containing enveloped viruses (herpes simplex virus type 1 and vaccinia virus), two RNA-containing enveloped viruses (bovine viral diarrhea virus and Newcastle disease virus) and two nude viruses (the enterovirus Coxsackie B1, a RNA-containing virus, and human adenovirus 5, a DNA virus). These two differently charged phthalocyanine complexes showed an identical marked virucidal effect against herpes simplex virus type 1, which was one and the same at an irradiation lasting 5 or 20 min (Δlog=3.0 and 4.0, respectively). Towards vaccinia virus this effect was lower, Δlog=1.8 under the effect of ZnPcMe and 2.0 for ZnPcS. Bovine viral diarrhea virus manifested a moderate sensitivity to ZnPcMe (Δlog=1.8) and a pronounced one to ZnPcS at 5- and 20-min irradiation (Δlog=5.8 and 5.3, respectively). The complexes were unable to inactivate Newcastle disease virus, Coxsackievirus B1 and human adenovirus type 5.

Formyl Peptide Receptor 2 Plays a Deleterious Role During Influenza A Virus Infections.

BACKGROUND: The pathogenesis of influenza A virus (IAV) infections is a multifactorial process that includes the replication capacity of the virus and a harmful inflammatory response to infection. Formyl peptide receptor 2 (FPR2) emerges as a central receptor in inflammatory processes controlling resolution of acute inflammation. Its role in virus pathogenesis has not been investigated yet. METHODS: We used pharmacologic approaches to investigate the role of FPR2 during IAV infection in vitro and in vivo. RESULTS: In vitro, FPR2 expressed on A549 cells was activated by IAV, which harbors its ligand, annexin A1, in its envelope. FPR2 activation by IAV promoted viral replication through an extracellular-regulated kinase (ERK)-dependent pathway. In vivo, activating FPR2 by administering the agonist WKYMVm-NH2 decreased survival and increased viral replication and inflammation after IAV infection. This effect was abolished by treating the mice with U0126, a specific ERK pathway inhibitor, showing that, in vivo, the deleterious role of FPR2 also occurs through an ERK-dependent pathway. In contrast, administration of the FPR2 antagonist WRW4 protected mice from lethal IAV infections. CONCLUSIONS: These data show that viral replication and IAV pathogenesis depend on FPR2 signaling and suggest that FPR2 may be a promising novel strategy to treat influenza.

Phosphatidylinositol 4-kinase III beta is the target of oxoglaucine and pachypodol (Ro 09-0179) for their anti-poliovirus activities, and is located at upstream of the target step of brefeldin A.

In recent years, phosphatidylinositol 4-kinase III beta (PI4KB) has emerged as a conserved target of anti-picornavirus compounds. In the present study, PI4KB was identified as the direct target of the plant-derived anti-picornavirus compounds, oxoglaucine and pachypodol (also known as Ro 09-0179). PI4KB was also identified as the target via which pachypodol interferes with brefeldin A (BFA)-induced Golgi disassembly in non-infected cells. Oxysterol-binding protein (OSBP) inhibitor also has interfering activity against BFA. It seems that this interference is not essential for the anti-poliovirus (PV) activities of BFA and PI4KB/OSBP inhibitors. BFA inhibited early to late phase PV replication (0 to 6 hr postinfection) as well as PI4KB inhibitor, but with some delay compared to guanidine hydrochloride treatment. In contrast with PI4KB/OSBP inhibitors, BFA inhibited viral nascent RNA synthesis, suggesting that BFA targets some step of viral RNA synthesis located downstream of the PI4KB/OSBP pathway in PV replication. Our results suggest that PI4KB is a major target of anti-picornavirus compounds identified in vitro for their anti-picornavirus activities and for some uncharacterized biological phenomena caused by these compounds, and that BFA and PI4KB/OSBP inhibitors synergistically repress PV replication by targeting distinct steps in viral RNA replication.

HPV prevalence and type distribution in women with normal or abnormal Pap smear in Bulgaria.

Human papillomavirus (HPV) is a well-known pathogen for lower genital tract neoplasias, yet little is known regarding HPV prevalence in Bulgaria. The aim of this study was to investigate the prevalence of HPV DNA and to determine HPV types distribution among women with normal and abnormal cytology. Cervical smears with different cytological diagnoses were collected from 355 Bulgarian patients. The cohort of patients selected is the biggest ever studied in this country. Using the Roche Linear Array HPV Genotyping Test, papillomavirus DNA was found in 217 out of the 355 samples, 164 of which had only one and 53 had more than one HPV type. The distribution of the viruses tested in 355 samples was as follows: (i) the most common type was HPV 16, which was found in 61 samples; (ii) the next most frequent HPV type was HPV 33, found in 14 of the samples. A high prevalence of HPV infection was observed in this study. As HPV infection has a high correlation with cervical cancer, this study emphasizes the need for both primary prevention of cervical cancer with HPV vaccines as well as secondary prevention with screening. Currently, two HPV vaccines are included in the National immunization schedule in Bulgaria. Thus, new clinical studies will benefit from patient stratification by the presence or absence of HPV, and by designing separate clinical trials specifically for HPV associated cancers.

The combination of pleconaril, rupintrivir, and remdesivir efficiently inhibits enterovirus infections in vitro, delaying the development of drug-resistant virus variants.

Enteroviruses are a significant global health concern, causing a spectrum of diseases from the common cold to more severe conditions like hand-foot-and-mouth disease, meningitis, myocarditis, pancreatitis, and poliomyelitis. Current treatment options for these infections are limited, underscoring the urgent need for effective therapeutic strategies. To find better treatment option we analyzed toxicity and efficacy of 12 known broad-spectrum anti-enterovirals both individually and in combinations against different enteroviruses in vitro. We identified several novel, synergistic two-drug and three-drug combinations that demonstrated significant inhibition of enterovirus infections in vitro. Specifically, the triple-drug combination of pleconaril, rupintrivir, and remdesivir exhibited remarkable efficacy against echovirus (EV) 1, EV6, EV11, and coxsackievirus (CV) B5, in human lung epithelial A549 cells. This combination surpassed the effectiveness of single-agent or dual-drug treatments, as evidenced by its ability to protect A549 cells from EV1-induced cytotoxicity across seven passages. Additionally, this triple-drug cocktail showed potent antiviral activity against EV-A71 in human intestinal organoids. Thus, our findings highlight the therapeutic potential of the pleconaril-rupintrivir-remdesivir combination as a broad-spectrum treatment option against a range of enterovirus infections. The study also paves the way towards development of strategic antiviral drug combinations with virus family coverage and high-resistance barriers.

Whole genome methylation array analysis reveals new aspects in Balkan endemic nephropathy etiology.

BACKGROUND: Balkan endemic nephropathy (BEN) represents a chronic progressive interstitial nephritis in striking correlation with uroepithelial tumours of the upper urinary tract. The disease has endemic distribution in the Danube river regions in several Balkan countries.DNA methylation is a primary epigenetic modification that is involved in major processes such as cancer, genomic imprinting, gene silencing, etc. The significance of CpG island methylation status in normal development, cell differentiation and gene expression is widely recognized, although still stays poorly understood. METHODS: We performed whole genome DNA methylation array analysis on DNA pool samples from peripheral blood from 159 affected individuals and 170 healthy individuals. This technique allowed us to determine the methylation status of 27 627 CpG islands throughout the whole genome in healthy controls and BEN patients. Thus we obtained the methylation profile of BEN patients from Bulgarian and Serbian endemic regions. RESULTS: Using specifically developed software we compared the methylation profiles of BEN patients and corresponding controls and revealed the differently methylated regions. We then compared the DMRs between all patient-control pairs to determine common changes in the epigenetic profiles.SEC61G, IL17RA, HDAC11 proved to be differently methylated throughout all patient-control pairs. The CpG islands of all 3 genes were hypomethylated compared to controls. This suggests that dysregulation of these genes involved in immunological response could be a common mechanism in BEN pathogenesis in both endemic regions and in both genders. CONCLUSION: Our data propose a new hypothesis that immunologic dysregulation has a place in BEN etiopathogenesis.

Effect of castalagin against HSV-1 infection in newborn mice.

We tested in vivo anti-herpetic effect of castalagin, an ellagitannin compound, extracted from pedunculate oak (Quercus robur). Previous investigations found that castalagin possesses a strong inhibitory effect in vitro against HSV-1/2 equal to acyclovir (ACV). It is also effective against ACV-resistant mutants and shows a synergistic effect with ACV. We study castalagin's activity towards HSV-1 infection in newborn mice. Acute toxicity determination in mice showed LD50 value of 295 mg/kg. Prolonged toxicity was also constructed. Castalagin manifested a marked activity against HSV-1 (LD90/0.02 ml) administered in 7-day course at 0.02 ml s.c. doses of 7.5 or 10 mg/kg (PI 57-58%). ACV course demonstrated a marked activity at 20 mg/kg. The selectivity ratio LD50/ED50 (295/7.5) could be accepted as ≥ 33.

Bulgarians vs the other European populations: a mitochondrial DNA perspective.

To define the matrilineal relationships between Bulgarians and other European populations, we have evaluated the mitochondrial DNA (mtDNA) variation in a sample of 855 Bulgarian subjects from the mtDNA perspective. The molecular survey was performed by sequencing ∼750 bp of the control region, which resulted in 557 different haplotypes, and by a subsequent restriction fragment length polymorphism analysis to confirm haplogroup/subhaplogroup affiliation. The classification was carried out according to the most updated criteria as reported by van Oven and Kayser (Hum Mutat 30:386-394, 2009), allowing the identification of 45 mitochondrial clades. The observed pattern of mtDNA variation indicates that the Bulgarian mitochondrial pool is geographically homogeneous across the country, and that is characterized by an overall extremely high frequency of western Eurasian lineages. In the principal component analysis, Bulgarians locate in an intermediate position between Eastern European and Mediterranean populations, which is in agreement with historical events. Thus, while the Mediterranean legacy could be attributed to the Thracians, indigenous people that firstly inhabited the Balkans, the Eastern contribution is likely due to the Proto-Bulgarians originating from the Middle East and to the Slavs migrating from northeast Europe.

Chemical and antiviral study on alkaloids from Papaver pseudocanescens M. Pop.

The phytochemical investigation of the aerial parts of Papaver pseudocanescens M. Pop. of Mongolian origin resulted in the isolation and structural elucidation of 8 alkaloids of the isoquinoline and promorphinane type. 8,14-Dihydroamurine, 8,14-dihydroflavinantine, and flavinantine are promorphinanes. Alborine, mecambridine, and mecambridine methohydroxide are retroprotoberberines. Amurensinine is an isopavine alkaloid and O-methylarmepavine is a benzylisoquinoline alkaloid. O-Methylarmepavine is a new alkaloid for the genus Papaver. Promorphinane-type alkaloids have been found for the first time in the species. All structures were established by physical and spectral analysis. As a first attempt to describe some of the biological activities of these alkaloids, the antiviral effect was tested against the in vitro replication of several viruses which belong to different taxonomic groups and represent significant human pathogens. Based on the results, the conclusion could be drawn that particular alkaloids from P. pseudocanescens possess selective antiviral effects against the replication of poliovirus 1 and human rhinovirus 14, two viruses from the Enterovirus genus of the Picornaviridae family.

Disoxaril mutants of Coxsackievirus B1: phenotypic characteristics and analysis of the target VP1 gene.

Disoxaril inhibits enterovirus replication by binding to the hydrophobic pocket within the VP1 coat protein, thus stabilizing the virion and blocking its uncoating. Disoxaril-resistant (RES) mutants of the Coxsackievirus B1 (CVB1/RES) were derived from the wild disoxaril-sensitive (SOF) strain (CVB1/SOF) using a selection approach. A disoxaril-dependent (DEP) mutant (CVB1/DEP) was obtained following nine consecutive passages of the disoxaril-resistant mutant in the presence of disoxaril. Phenotypic characteristics of the disoxaril mutants were investigated. A timing-of-addition study of the CVB1/DEP replication demonstrated that in the absence of disoxaril the virus particle assembly stopped. VP1 RNA sequences of disoxaril mutants were compared with the existing Gen Bank CVB1 reference structure. The amino acid sequence of a large VP1 196-258 peptide (disoxaril-binding region) of CVB1/RES was significantly different from that of the CVB1/SOF. Crucially important changes in CVB1/RES were two point mutations, M213H and F237L, both in the ligand-binding pocket. The sequence analysis of the CVB1/DEP showed some reversion to CVB1/SOF. The amino acid sequences of the three VP1 proteins are presented.

Oncolytic rat parvovirus H-1PV, a candidate for the treatment of human lymphoma: In vitro and in vivo studies.

The incidence of lymphomas developing in both immunocompetent and immunosuppressed patients continues to steadily increase worldwide. Current chemotherapy and immunotherapy approaches have several limitations, such as severe side toxicity and selection of resistant cell variants. Autonomous parvoviruses (PVs), in particular the rat parvovirus H-1PV, have emerged as promising anticancer agents. Although it is apathogenic in humans, H-1PV has been shown to infect and suppress various rat and human tumors in animal models. In this study, we demonstrate the capacity of H-1PV for efficiently killing, through necrosis, cell cultures originating from Burkitt's lymphoma (BL), while sparing normal B lymphocytes. The cytotoxic effect was generally accompanied by a productive H-1PV infection. Remarkably, parvovirus-based monotherapy efficiently suppressed established BL at an advanced stage in a severe combined immunodeficient (SCID) mouse model of the disease. The data show for the first time that an oncolytic parvovirus deserves further consideration as a potential tool for the treatment of some non-Hodgkin B-cell lymphomas, including those resistant to apoptosis induction by rituximab.

New analogues of acyclovir--synthesis and biological activity.

New acyclovir esters with peptidomimetics were synthesized and evaluated in vitro for their antiviral activity against the replication of Herpes simplex virus type 1 (HSV-1) and type 2 (HSV-2). The influence of peptidomimetics containing oxazole and thiazolyl-thiazole moieties on the antiviral activity is also reported. The esters were synthesized using the coupling reagents N-ethyl-N'-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) and N,N-dimethyl-4-aminopyridine (DMAP) as a catalyst.

Effect of Consecutive Alternating Administration of a Triple Combination of Anti-Enteroviral Compounds in Mice Infected with Coxsackievirus B3.

A novel approach for treatment of enterovirus infections was characterized. Application of treatment course of consecutive alternating administration (CAA) of triple combination of enterovirus replication inhibitors in experimental infections (20 MLD50) with coxsackievirus B3 (CVB3) strains in newborn mice is presented. It was established that in infection with cardiotropic Woodruff strain the combination of pleconaril, МDL-860 and oxoglaucine (PMO) subjected to the CAA scheme, a significant protective effect was observed. Monotherapeutic courses as well as simultaneously daily applied PMO were without effect. Analogous data were observed at experimental infection with the neurotriopic Nancy strain of CVB3. Following IC50 values of virus samples taken every day from target organs of infected animals during the whole period of study, a drug-resistance was established in monotherapy with compounds-partners in the PMO combination. At courses by the treatment scheme CAA of PMO development of drug-resistance was not established, but an increased susceptibility to the effect of the inhibitor-components in the combination was proven. Toxicity of PMO applied via the CAA scheme and in the monotherapeutic courses in both healthy and CVB3 infected animals was recorded. All data obtained prove the potential of the CAA treatment scheme for development of effective chemotherapy of enterovirus infections.

Oseltamivir and S-Adenosyl-L-Methionine Combination as Effective Therapeutic Strategy for Suppression of Oxidative Damage in Lung Caused by Influenza Virus Infection in Mice.

BACKGROUND AND OBJECTIVES: The pathogenesis of influenza infection is associated with two general processes in the body: (a) lung damage based on virus replication; (b) overproduction of free radicals, antioxidant deficiency, and development of oxidative stress. To attack these aspects of flu pathogenesis, we explored the combined effect of the antiviral agent oseltamivir, and s-adenosyl-l-methionine (SAM) as a precursor of the endogenous antioxidant glutathione, in mice infected with influenza virus. METHODS: After inoculation of albino mice with 10 MLD50 of influenza virus A/Aichi/2/68 (H3N2), oseltamivir was applied twice a day, for five days post-infection in doses of 1.25 and 2.5 mg/kg. SAM was administered once a day for 10 days, starting 5 days before infection in doses of 50, 100 and 150 mg/kg. RESULTS: Monotherapy with SAM did not influence the markers of oxidative stress in the lung. Combination of SAM 50 mg/kg and oseltamivir 2.5 mg/kg affected best the virological parameters - viral titer, protection index, and mean survival time, as well as the biochemical markers of oxidative stress. INTERPRETATION AND CONCLUSIONS: Combining of SAM and oseltamivir in a dose of 1/4 of optimal therapeutic could be considered as a perspective therapy of influenza viral infection.

Antiviral, Cytotoxic and Antioxidant Effects of Tanacetum Vulgare L. Crude Extract In Vitro.

INTRODUCTION: Due to the high prevalence of viral infections having no specific treatment and the constant emergence of resistant viral strains, searching for effective antiviral compounds is crucial. The present study explores in vitro the antiviral activity of ethanolic extract from aerial parts of. AIM: The aim of the current study was to evaluate antiviral activity of ethanolic extract from herbaceous plant. MATERIALS AND METHODS: The crude aqueous ethanolic extract from aerial parts of. RESULTS: The results show that the extract has the lowest toxicity on the MDBK cell line and similar cytotoxicity in Hep-2, whereas in the MDCK cells it has more than twice the highest toxicity. Testing the antiviral activity of. CONCLUSION: The crude extract from aerial parts of the medicinal plant.