Protective Effects of Dilazep and its Derivative K-7259 on the Haemolysis Induced by Amphiphiles in Rat Erythrocytes

The effects of dilazep and K-7259, a dilazep derivative, on the haemolysis (as evidenced by release of haemoglobin) induced by palmitoyl-l -carnitine (PAL-CAR) or palmitoyl 1-α-lysophosphatidylcholine (PAL-LPC) have been determined in rat erythrocytes. At concentrations above the critical micelle concentration both PAL-CAR and PAL-LPC induced haemolysis; the concentrations of PAL-CAR and PAL-LPC producing 50% haemolysis were approximately 13 and 14 μm , respectively. The 50% haemolysis induced by PAL-CAR or PAL-LPC was attenuated by dilazep (1, 10 or 100 μm ) but not at the highest concentration used (1 mm ). K-7259 attenuated the 50% haemolysis induced by PAL-CAR or PAL-LPC at concentrations ranging from 1 μm to 1 mm . Similarly, dilazep (1 to 100 μm ) and K-7259 (1 μm to 1 mm ) significantly or insignificantly attenuated the 25% and 75% haemolysis induced by PAL-CAR or PAL-LPC. Neither dilazep nor K-7259 affected micelle formation by PAL-CAR or PAL-LPC, nor, at concentrations of 1 and 10 μm , did they attenuate the haemolysis induced by osmotic imbalance (hypotonic haemolysis). These results suggest that both dilazep and K-7259 protect the erythrocyte membrane from the damage induced by PAL-CAR or PAL-LPC. The protective effects of dilazep and K-7259 are mediated by some mechanism other than prevention of micelle formation or protection of the erythrocyte membrane against osmotic imbalance.     

Clinical use of antiviral drugs

Remarkable progress has been made in antiviral chemotherapy. Six approved antiviral drugs are now available for the treatment of various viral infections. Trifluridine, idoxuridine and vidarabine are all effective in patients with herpes keratitis; trifluridine is preferred due to its low toxicity. Acyclovir is the drug of choice in patients with infections due to herpes simplex viruses, including genital herpes, herpes encephalitis, and neonatal herpes, and infections due to varicella-zoster virus. Amantadine is the only drug currently available for prophylaxis and treatment of influenza A, but an investigational drug, rimantadine, appears to be equally effective and less toxic than amantadine. Ribavirin is the most recently approved antiviral agent for the treatment of respiratory syncytial virus infections. Numerous antiviral drugs are being studied in patients with acquired immunodeficiency syndrome. Although currently available drugs have improved our ability to manage a variety of viral illnesses, much needs to be learned about specific dosage guidelines based on the studies of pharmacokinetics, pharmacodynamics, potential adverse effects and viral resistance, and the role of combination therapy to optimize therapy.     

Interaction of ticlopidine with the erythrocyte membrane

The membrane effects of ticlopidine on the erythrocyte membrane were explored by the spin label method at the proteic and phospholipidic levels. This spectroscopic study was completed by polyacrylamide gel electrophoresis of proteins, measurement of the protection against haemolysis and observation of the erythrocyte shape changes induced by the drug. Two types of effects have been observed. At concentrations higher than 5 × 10^?4 M, ticlopidine is a weak denaturating agent of the membrane proteins. At concentrations of pharmacological interest, the main effect of the drug is a protection against hypotonic haemolysis, and an increase in the fluidity of the membrane phospholipidic core. This last result could explain in part the interesting pharmacological effect of ticlopidine on various circulatory troubles.     

具抗病毒活性的金刚烷胺衍生物的研究进展

目的 介绍M2离子通道抑制剂金刚烷胺衍生物的研究进展,为设计新的抗流感病毒药物提供相关依据和信息。方法 对近年来关具抗流感病毒活性金刚烷胺衍生物、类似物的相关文献进行综述。结果 国内外课题组主要集中在金刚烷的1位和2位修饰,合成一系列金刚烷胺衍生物或结构类似物,并进行抗流感病毒活性测试。结论 部分金刚烷胺衍生物具有较好的抗流感病毒活性,其中一些化合物的活性优于金刚烷胺和金刚乙胺,这些结果对设计新的抗流感病毒药物具有较好的参考价值。     

Respiratory viral infections in the elderly

Viral respiratory infections represent a significant challenge for those interested in improving the health of the elderly. Influenza continues to result in a large burden of excess morbidity and mortality. Two effective measures, inactivated influenza vaccine, and the antiviral drugs rimantadine and amantadine, are currently available for control of this disease. Inactivated vaccine should be given yearly to all of those over the age of 65, as well as younger individuals with high-risk medical conditions and individuals delivering care to such persons. Live, intranasally administered attenuated influenza vaccines are also in development, and may be useful in combination with inactivated vaccine in the elderly. The antiviral drugs amantadine and rimantadine are effective in the treatment and prevention of influenza A, although rimantadine is associated with fewer side-effects. Recently, the inhaled neuraminidase inhibitor zanamivir, which is active against both influenza A and B viruses, was licensed for use in uncomplicated influenza. The role of this drug in treatment and prevention of influenza in the elderly remains to be determined. Additional neuraminidase inhibitors are also being developed. In addition, to influenza, respiratory infections with respiratory syncytial virus, parainfluenza virus, rhinovirus, and coronavirus have been identified as potential problems in the elderly. With increasing attention, it is probable that the impact of these infections in this age group will be more extensively documented. Understanding of the immunology and pathogenesis of these infections in elderly adults is in its infancy, and considerable additional work will need to be performed towards development of effective control measures.     

Influenza

The currently available antiviral drugs rimantadine and amantadine are effective only for influenza A viruses. Another class of influenza antiviral drugs is the neuraminidase inhibitors, which selectively inhibit both influenza A and B viruses. Recent studies have found the neuraminidase inhibitors zanamivir and oseltamivir to be 67 - 82% effective in preventing laboratory-confirmed infection when administered as prophylaxis during the influenza season. As treatment, they reduce the duration of illness by 1 - 1.5 days when started within 36 - 48 h of illness onset. The reported adverse effects of these drugs are minimal, and unlike amantadine and rimantadine, the drugs do not appear to affect the central nervous system. Poor oral bioavailability and rapid renal clearance limit the use of zanamivir to inhalation and concern has been raised about its use in asthmatics. The sialic acid analogue, GS4071, has been shown to be a potent inhibitor of neuraminidase activity and is shown to be effective in controlling influenza, and its prodrug form - GS4104 (oseltamivir) can be given orally. Direct comparison of zanamivir and oseltamivir, their use for prophylaxis and treatment in high-risk groups, and evaluation of their cost effectiveness are all required before they enter routine clinical practice.     

Influenza

The currently available antiviral drugs rimantadine and amantadine are effective only for influenza A viruses. Another class of influenza antiviral drugs is the neuraminidase inhibitors, which selectively inhibit both influenza A and B viruses. Recent studies have found the neuraminidase inhibitors zanamivir and oseltamivir to be 67-82% effective in preventing laboratory-confirmed infection when administered as prophylaxis during the influenza season. As treatment, they reduce the duration of illness by 1-1.5 days when started within 36-48 h of illness onset. The reported adverse effects of these drugs are minimal, and unlike amantadine and rimantadine, the drugs do not appear to affect the central nervous system. Poor oral bioavailability and rapid renal clearance limit the use of zanamivir to inhalation and concern has been raised about its use in asthmatics. The sialic acid analogue, GS4071, has been shown to be a potent inhibitor of neuraminidase activity and is shown to be effective in controlling influenza, and its prodrug form--GS4104 (oseltamivir) can be given orally. Direct comparison of zanamivir and oseltamivir, their use for prophylaxis and treatment in high-risk groups, and evaluation of their cost effectiveness are all required before they enter routine clinical practice.     

Evaluation of metabolic parameters in vitro as a model for testing the cytotoxicity of antiviral drugs

A new test system based on in vitro assessment of the cellular viability and/or metabolism is proposed, which offers an informative approach to the screening of antiviral drugs. Cytotoxic effects of the antiviral drugs rimantadine and polyrem were studied on the cultures of some mammalian cells. The results of short-term (2 h) exposures with the drugs tested were close to their LD50 values in mammals, which makes the proposed test system promising for the assessment of drug toxicity in vivo for the whole organism. A study of the state of cell metabolism after a long-term (48 h) exposure showed that the system of endocytosis is more sensitive than other indices with respect to antiviral drugs. Is was demonstrated on the cell level that the binding of drugs into polymeric complexes can decrease the degree of its cytotoxicity: the toxicity of polyrem (a polymeric complex of rimantadine) was lower as compared to that of the equimolar concentration of rimantadine or a mixture of rimantadine with a polymeric carrier.     

Combating influenza with antiviral therapy in the pediatric population

Influenza viruses are accountable for annual epidemics worldwide that result in significant morbidity and mortality. In preschool and school-aged children, prospective surveillance of influenza demonstrates yearly infection rates of 15-42%. Children can easily transmit the virus to other children, to employees in day-care and school settings, and to family members. Two classes of antiviral drugs, the adamantine derivatives (amantadine, rimantadine) and neuraminidase inhibitors (zanamivir and oseltamivir), have been approved for treatment and prophylaxis of influenza in the pediatric population. Duration of clinical symptoms decreases and daily activities are resumed sooner when therapy is begun within 48 hours of the onset of influenza symptoms. Mechanism of action, adverse effects, and development of resistant variants differ between the two drug classes. To our knowledge, head-to-head clinical trials between the classes and involving the neuraminidase inhibitors are nonexistent. Antiviral agents do not replace the annual influenza vaccine, and clinical trials indicate that amantadine, rimantadine, zanamivir, and oseltamivir are safe and effective for administration in the pediatric population.     

Stimulation of suicidal erythrocyte death by amantadine

Amantadine is an effective drug for treatment of both, Parkinson's disease and viral infections. Side effects of amantadine include anemia, which may limit its therapeutic use. The cause of amantatine induced anemia is ill defined. At least in theory, the anemia could partially result from suicidal erythrocyte death or eryptosis, which accelerates the clearance of circulating erythrocytes. Eryptosis is characterized by cell shrinkage and cell membrane scrambling leading to phosphatidylserine exposure at the cell surface. Triggers of erythrocyte membrane scrambling include an increase of cytosolic Ca2+ concentration ([Ca2+]i) resulting from activation of Ca2+-permeable cation channels. The present study has been performed to test for an effect of amantadine on eryptosis. Erythrocytes from healthy volunteers were exposed to amantadine and annexin V binding (disclosing phosphatidylserine exposure), forward scatter (reflecting cell volume), and Fluo3-dependent fluorescence (reflecting [Ca2+]i) were determined by flow cytometry. Exposure of erythrocytes to amantadine (> or =0.2 microg/ml) increased [Ca2+]i and triggered annexin V binding, and increased forward scatter. The effect on annexin V binding was virtually abolished in the absence of extracellular Ca2+. The present observations disclose mechanisms presumably contributing to amantadine induced anemia.     

Synthesis, antiretroviral and antioxidant evaluation of a series of new benzo[b]furan derivatives

The antiretroviral and anti-oxidant profile of a series of new C-2 and C-7 substituted benzo[b]furans was explored by employing well established antiviral and antioxidant protocols. The most potent antioxidant compound tested was analog 7, which bears an OH at C-7 and a benzoyl group at C-2. In the influenza A type H3N2 virus screens analog 8a was almost five-fold more active than its counterparts and equipotent to rimantadine and amantadine. In the influenza B screening all of the new compounds tested were at least ten-fold more active than the control drug amantadine. The anti-HIV screening, using acutely infected MT-4 cells, showed that compound 8f (n = 4), was fifteen-fold more active than its monomer congeners, 8a and 8c, d and almost five-fold more potent than monomer 8b and dimer 8f (n = 3).     

Dansylcadaverine and rimantadine inhibition of phagocytosis, PAF-acether release, and phosphatidylcholine synthesis in human polymorphonuclear leukocytes

The effect of dansylcadaverine, amantadine, and rimantadine on phagocytosis, release of PAF-acether, phospholipid methylation, and phosphatidylcholine formation by the cholinephosphotransferase pathway was assessed in human peripheral polymorphonuclear leukocytes. All of the drugs induced a dose-dependent and reversible inhibition in the uptake of complement-coated zymosan particles as well as a reduction in the release of PAF-acether. Simultaneously, a marked reduction in the formation of phosphatidylcholine was observed. This was also dose-dependent and reversible, and showed this order of potency: dansylcadaverine greater than rimantadine greater than amantadine. Dansylcadaverine reduced phospholipid methylation to a lesser extent than the cholinephosphotransferase pathway. These data show that drugs that block receptor-mediated endocytosis inhibit the response of the human polymorphonuclear cell and suggest that this action may be mediated by their actions on phospholipid metabolism.     

Interactions of surface-active alkyltrimethylammonium salts with the erythrocyte membrane.

The interactions of ahomologue series of surface-active alky Itrimethylammonium salts (C₁₀-C₂₀) with the rat erythrocyte membrane were studied. The surfactants were found to have a biphasic effect on the erythrocyte membrane. At low concentrations they protected or stabilized erythrocytes against hypotonic haemolysis, but at higher concentrations they caused rapid haemolysis. The stabilizing and lytic effect increased with an increase in length of the alkyl chain to maximum activity at about C₁₆. It is suggested that laminar-micellar transitions in the lipid bilayer of the membrane are responsible for the lytic activity of the surfactants. Micellar regions in the lipid bilayer abolish the ability of the membrane to prevent the free exchange of ions, and haemolysis of the cell results from a secondary osmotic effect. The stabilizing effect, on the other hand, is proposed to stem from an expansion of the membrane caused by a fluidizing effect of the surfactants on the lipid bilayer. Binding studies with the C₁₆ homologue revealed that at a concentration causing 50 per cent haemolysis in an isotonic solution there are about 780,000 molecules bound per μm² of the erythrocyte membrane. At a concentration giving 50 per cent protection against hypotonic haemolysis, the number of molecules bound per μm² of the erythrocyte membrane was estimated to be 190,000.     

Development of antivirals against influenza

Morbidity and mortality due to influenza virus infections remain a major problem throughout the world. Yearly, medical costs and loss of productivity resulting from influenza infection are estimated to be in the range of 12 dollars bn in the USA. The predicted increases in the elderly and immune-deficient populations will make influenza an even greater threat in the future. Despite the availability of vaccines, they have been least effective in these high-risk populations. Coupled with the requirement for routine revaccination, the need for effective antiviral agents is illustrated. The currently approved drugs, amantadine, rimantadine and ribavirin (in some countries), have limitations. They are only inhibitory against influenza A viruses, are prone to adverse reactions and quickly give rise to resistant virus. This review examines current drug therapies, antivirals in development and possible future opportunities for anti-influenza drugs.     

Decreased viscosity of human erythrocyte suspension induced by chlorpromazine and isoxsuprine

The effects of two cationic drugs, chlorpromazine and isoxsuprine, on the suspension viscosity of human erythrocytes were studied. Chlorpromazine and isoxsuprine reduced the suspension viscosity, the former being more effective than the latter (i.e. a concentration of isoxsuprine about fifteen times higher than of chlorpromazine was needed to obtain similar effects). The cells transformed to stomatocytes and then to spherostomatocytes, depending on the drug concentration. The decrease in suspension viscosity paralleled the appearance of spherostomatocytes. Membrane fluidity, monitored by the motion of fatty acid spin labels, was increased by chlorpromazine at high concentrations, but was unaffected by isoxsuprine. Quantitative data of drug uptake by erythrocytes and ghosts revealed that the drugs were incorporated into both membrane and cytoplasm (partly bound to hemoglobin, as shown by equilibrium dialysis). The above drug effects appeared to depend primarily on the drug concentration in the membrane. In short, the transformation to spherostomatocytes, induced by drug incorporation into the erythrocyte membrane, was essential for the decrease in suspension viscosity.     

New small-molecule drug design strategies for fighting resistant influenza A

Influenza A virus is the major cause of seasonal or pandemic flu worldwide. Two main treatment strategies–vaccination and small molecule anti-influenza drugs are currently available. As an effective vaccine usually takes at least 6 months to develop, anti-influenza small molecule drugs are more effective for the first line of protection against the virus during an epidemic outbreak, especially in the early stage. Two major classes of anti-influenza drugs currently available are admantane-based M2 protein blockers (amantadine and rimantadine) and neuraminidase (NA) inhibitors (oseltamivir, zanamivir, and peramivir). However, the continuous evolvement of influenza A virus and the rapid emergence of resistance to current drugs, particularly to amantadine, rimantadine, and oseltamivir, have raised an urgent need for developing new anti-influenza drugs against resistant forms of influenza A virus. In this review, we first give a brief introduction of the molecular mechanisms behind resistance, and then discuss new strategies in small-molecule drug development to overcome influenza A virus resistance targeting mutant M2 proteins and neuraminidases, and other viral proteins not associated with current drugs.KEY WORDS: Influenza A virus, Drug discovery, Resistance, M2 ion channel, Neuraminidase     

Amantadine-induced lipidosis. A cytological and physicochemical study

The purpose of this study was to test whether or not the antiviral drug amantadine induces the structural features of lipidosis in intact animals (rats) and cultured cells, and to investigate the interactions between amantadine and phospholipids. Chlorphentermine was used as reference compound. When subchronically fed to rats at a daily dosage of approximately 180 mg/kg, amantadine induced ultrastructural symptoms of generalized lipidosis, the degree of which was, however, by far less marked than that previously reported for chlorphentermine. This was paralleled by the findings on cell cultures (rat peritoneal macrophages), where the lipidosis-inducing potency of amantadine was approximately 10-fold lower than that of chlorphentermine. As to drug-phospholipid interactions, amantadine had less marked effects than chlorphentermine upon the phase transition characteristics of phosphatidylcholine and phosphatidic acid; furthermore, amantadine was approximately 10-fold less potent than chlorphentermine in displacing Ca from phosphatidylserine monolayers. The present study has revealed a parallel between the comparatively low lipidosis-inducing efficacy inherent to amantadine and the comparatively low tendency to interact with phospholipids. It is suggested that the cage-like structure of the amantadine molecule hinders an effective intercalation of the drug into phospholipid aggregates, and that this is an essential factor responsible for the low inherent efficacy of amantadine with respect to lipidosis induction.     

Influenza viruses: basic biology and potential drug targets

Influenza A and influenza B viruses are continuing causes of morbidity and mortality on an annual basis. Influenza A viruses have historically caused periodic pandemics in the human population, sometimes with devastating consequences, such as in 1918. Fears of a new pandemic have increased in recent years because of continuing outbreaks of highly pathogenic H5N1 avian influenza viruses in birds with occasional, but often lethal infection of humans. Despite their importance as human pathogens, the antiviral drugs approved to treat influenza virus infections are currently limited to two targets, the viral neuraminidase and the viral ion channel, M2. The use of the M2 inhibitors amantadine and rimantadine is further limited by the propensity of these drugs to select for drug resistant variants. However, the replication cycle of influenza viruses has been intensively studied and is receiving increased attention. New opportunities exist to develop novel antiviral strategies targeting these viruses.     

Recent advances in antiviral therapy

Virus replication is described, and the clinical trials and indications for amantadine, rimantadine, vidarabine, vidarabine phosphate, acyclovir, ribavirin, and other promising antiviral agents are reviewed. Amantadine and rimantadine are useful for the treatment and prophylaxis of viral influenza A infections. Vidarabine is a second-line agent and is effective for the treatment of herpes simplex encephalitis, neonatal herpes simplex types 1 and 2, and varicella-zoster infections. Vidarabine phosphate (also known as vidarabine monophosphate) has a similar spectrum of activity and can be administered in smaller volumes than vidarabine. Acyclovir has demonstrated clinical efficacy for chickenpox, shingles (herpes zoster), genital herpes, and other herpes simplex infections. Acyclovir is also useful for the suppression of herpes infections. Systemically administered ribavirin is indicated for the treatment of Lassa fever. Aerosol ribavirin is effective for the treatment of respiratory syncytial virus pneumonia in children and infants and influenza A infections in adults. Only acyclovir, amantadine, ribavirin, and vidarabine are used in clinical practice. Vidarabine phosphate and investigational agents such as rimantadine, ganciclovir (DHPG, BW B759U), phosphonoformate, and bromovinyl-deoxyuridine (BVDU) need further investigation.     

Effect of tetracaine on membrane-bound acetylcholinesterase activity and anilinonaphthalene sulphonate-induced membrane fluorescence

Tetracaine (25-300 microM) reversibly inhibits (in vitro) AChE activity of rat brain synaptosome (4.4-100%) and erythrocyte membrane (3.9-65.2%) in a concentration dependent manner. IC50 values of tetracaine for AChE of synaptosome and erythrocyte membrane are 88 and 200 microM respectively. At sub-inhibitory concentrations (less than or equal to 10 microM) tetracaine activates (8.7-23%) AChE of synaptosome but not of erythrocyte membrane. Lineweaver-Burk plots indicate that tetracaine-induced inhibition of AChE is competitive in nature and Ki value decreases on increasing the concentration (greater than 100 microM) of tetracaine in both synaptosome and erythrocyte membrane. Tetracaine (25-500 microM) produces a concentration dependent increase in 1-anilino-8-naphthalene sulphonate (ANS)-induced relative fluorescence (F470) of both synaptosomal (6.5-102%) and erythrocyte membrane (2.4-53.3%) without shifting their emission maxima (470 nm). Further it is also noted that the quantum yield of F470 of both the membranes increases with the increase of tetracaine concentrations (100-500 microM). These results suggest that the interaction of tetracaine with both the enzyme and its lipid microenvironment may be the cause of inhibition of membrane-bound AChE activity.