Toxicity of amantadine hydrochloride on cultured bovine cornea endothelial cells.

Amantadine hydrochloride (HCl) is commonly prescribed for treating influenza A virus infection and Parkinson's disease. Recently, several studies have indicated that the use of amantadine HCl is associated with corneal edema; however, the cytotoxic effect of amantadine HCl has not been investigated. In the present study, the effects of amantadine HCl on cell growth, proliferation, and apoptosis in bovine cornea endothelial cells, and in vitro endothelial permeability were examined. Results showed that lower doses of amantadine HCl do not affect cell growth (≤ 20 µΜ), whereas higher doses of amantadine HCl inhibits cell growth (≥ 50 µΜ), induces apoptosis (2000 µΜ), increases sub-G1 phase growth arrest (2000 µΜ), causes DNA damage (≥ 1000 µΜ), and induces endothelial hyperpermeability (≥ 1000 µΜ) in bovine cornea endothelial cells; additionally, we also found that amantadine HCl attenuates the proliferation (≥ 200 µΜ) and arrests cell cycle at G1 phase (≥ 200 µΜ) in bovine cornea endothelial cells. In the present study, we measured the cytotoxic doses of amantadine HCl on cornea endothelial cells, which might be applied in evaluating the association of corneal edema.

Leachables and cytotoxicity of root canal sealers.

This in vitro study aimed to detect leaching components from an epoxy resin- and a methacrylate-based endodontic sealer and correlate them to cytotoxicity induced by material extracts for up to 36 weeks. We qualitatively determined the substances released by aged AH Plus and RealSeal SE specimens at seven intervals between 0 and 36 weeks. Quantification was performed by ultra-performance liquid chromatography/mass spectrometry (UPLC/MS). We determined the viability of murine macrophage J774 cells after 24 h exposure to material extracts, at each interval, using a fluorescence staining/microscopy method. The leachables detected were 1-adamantylamine and bisphenol A diglycidyl ether from AH Plus and N-(p-tolyl) diethanolamine and caprolactone-2-(methacryloyloxy) ethyl ester from RealSeal SE. The largest UPLC/MS chromatogram peak areas of the leachables were detected within 72 h. Induction of cytotoxicity after exposure to AH Plus and RealSeal SE extracts coincided with leachant detected within the first 72 and 24 h, respectively. The clinical impact of the cytotoxicity due to resin-based endodontic sealers is unknown.

Electropharmacological effects of amantadine on cardiovascular system assessed with J-Tpeak and Tpeak-Tend analysis in the halothane-anesthetized beagle dogs.

Since amantadine-induced long QT syndrome has been clinically reported, we investigated its electropharmacological effects to estimate the extent of proarrhythmic risk by using the halothane-anesthetized beagle dogs (n = 4). Amantadine in doses of 0.1, 1 and 10 mg/kg was infused over 10 min with a pause of 20 min under the monitoring of multiple cardiovascular variables. J-Tpeak and Tpeak-Tend were separately measured on the lead II electrocardiogram to precisely analyze the net balance between inward and outward current modifications by amantadine. The low dose increased the ventricular contractile force, but suppressed the intraventricular conduction. The middle dose prolonged the QT interval besides enhancing the changes induced by the low dose. The high dose increased the mean blood pressure, left ventricular end-diastolic pressure and total peripheral resistance, and accelerated the atrioventricular nodal conduction, but decreased the cardiac output besides enhancing the changes induced by the middle dose. A reverse use-dependence was confirmed in the repolarization delay. Amantadine hardly affected the J-Tpeak, but prolonged the Tpeak-Tend. Amantadine can be considered to stimulate Ca(2+) channel but inhibit Na(+) and K(+) channels in the in situ heart. J-Tpeak and Tpeak-Tend analysis suggests that amantadine may possess modest risk for arrhythmia.

Cutaneous analgesia after subcutaneous injection of memantine and amantadine and their systemic toxicity in rats.

The purpose of the study is to find subcutaneous equianalgesic doses of memantine, amantadine and bupivacaine and use these doses to quantify the cardiovascular and central nervous system toxicity of these agents after intravenous administration. Memantine, amantadine and bupivacaine, a local anesthetic, in a dose-related fashion were determined for cutaneous analgesia by a block of the cutaneous trunci muscle reflex in rats, and equipotent doses were calculated. Following rapid intravenous infusion of equianalgesic bupivacaine, memantine, amantadine and saline (vehicle) in rats, we observed the onset time of seizure, apnea and impending death, and monitored mean arterial blood pressure and heart rate. Memantine and amantadine elicited dose-dependent cutaneous analgesia. At the 50% effective dose (ED(50)), the rank of potencies was bupivacaine [1.8 (1.7-2.0)]>memantine [19.1 (17.6-21.8)]>amantadine [36.1 (32.0-40.3)] (P<0.05). On ED(25), ED(50) and ED(75) basis, the duration caused by bupivacaine was similar to that caused by memantine or amantadine. At equianalgesic doses, the infusion time of memantine or amantadine required to induce seizure, impending death, and apnea was longer than that of bupivacaine during rapid intravenous infusion (P<0.01). The decreasing slope in mean arterial blood pressure and heart rate was slower with memantine and amantadine when compared with bupivacaine at equivalent doses (P<0.01). Our data showed that memantine and amantadine (i) were equal to bupivacaine at producing durations of cutaneous analgesia but (ii) were less likely than bupivacaine to cause cardiovascular and central nervous system toxicity.

An improved model to investigate the efficacy of antidyskinetic agents in hemiparkinsonian rats.

A number of experimental models of L-DOPA-induced dyskinesia have been proposed, but these models result in a low to medium rate of dyskinetic animals with mild to severe symptoms. The objective of this study was to combine a model of 6-OHDA-induced parkinsonism and of L-DOPA-induced dyskinesia in rats to establish a reliable preclinical model. Two stereotaxic injections of 6-OHDA were administered in the left striatum. This model led to 90-100% of rats with a marked contralateral circling behaviour, significant limb use asymmetry (20%), a decrease in ipsilateral striatal dopamine content (70%) and degeneration of dopamine neurons in the substantia nigra (70%). Chronic treatment with L-DOPA was administered for 35 days and consisted of three phases with incremental daily doses. The third phase resulted in 83-90% of rats developing severe abnormal involuntary movements (AIMs) which included limb and locomotive dyskinesia, axial dystonia and orolingual dyskinesia. Reproducibility of the model, criteria of strict blinding, placebo-controlled design, randomization of study subjects and pretrial determination of sample size were used to measure efficacy of amantadine and istradefylline and to validate the protocol design. Acute or subchronic post-treatment with amantadine reduced the severity of dyskinesia while istradefylline punctually attenuated AIMs. Our experimental conditions using gradual development of dyskinesia induced by increasing doses of L-DOPA resulted in a reliable model of L-DOPA-induced dyskinesia with a high rate of dyskinetic rats.

DNA damage in brain cells and behavioral deficits in mice after treatment with high doses of amantadine.

Amantadine (AMA) is an uncompetitive antagonist of the N-methyl-d-aspartate receptor, with clinical application, acting on treatment of influenza A virus and Parkinson's disease. It has been proposed that AMA can indirectly modulate dopaminergic transmission. In high doses, the central nervous system is its primary site of toxicity. To examine deleterious effects on CNS induced by AMA, this study evaluated possible neurobehavioral alterations induced by AMA such as stereotyped behavior, the effects on locomotion and memory and its possible genotoxic/mutagenic activities. Adult male CF-1 mice were treated with a systemic injection of AMA (15, 30 or 60 mg kg(-1) ) 20 min before behavioral tasks on open field and inhibitory avoidance. Higher AMA doses increased the latency to step-down inhibitory avoidance test in the training session in the inhibitory avoidance task. At 60 mg kg(-1) AMA induced impairing effects on locomotion and exploration and hence impaired habituation to a novel environment. Stereotyped behavior after each administration in a 3-day trial was observed, suggesting effects on dopaminergic system. Amantadine was not able to induce chromosomal mutagenesis or toxicity on bone marrow, as evaluated by the micronucleus assay. At the lowest dose tested, AMA did not induce DNA damage and it was unable to impair memory, locomotion, exploration or motivation in mice. However, higher AMA doses increased DNA damage in brain tissue, produced locomotor disturbances severe enough to preclude testing for learning and memory effects, and induced stereotypy, suggesting neurotoxicity.

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.

Interferon signal transduction of biphenyl dimethyl dicarboxylate/amantadine and anti-HBV activity in HepG2 2.2.15.

Biphenyl dimethyl dicarboxylate (DDB) is a hepatoprotectant, which is used as an adjuvant agent in a treatment for chronic hepatitis. Amantadine is an antiviral agent, which is utilized primarily in the treatment of influenza, but also, occasionally in the treatment of hepatitis C. In a previous study, we reported that DDB, coupled with amantadine, would exert an anti-HBV effect, via the induction of interferon-inducible gene expression in the HepG2 2.2.15 cell line. The primary objective of the present study was to determine whether or not DDB and/or amantadine exhibit anti-HBV properties, and what mechanisms of action might be involved in such properties. In our study, we were able to determine that DDB stimulates Jak/Stat signaling, and induces the expression of interferon alpha (IFN-alpha) stimulated genes, most notably 6-16 and ISG12. In addition, the antiviral effectors induced by IFN-alpha, PKR, OAS, and MxA, were regulated in the presence of DDB at its optimal concentration (250 microg/mL), to a degree commensurate with the degree of induction associated with the IFN-alpha treated group. Finally, we determined that the replication of pregenomic RNA and HBeAg was inhibited by DDB treatment, and this inhibition was maximized when coupled with the administration of amantadine (25 microg/mL). In conclusion, the results of this study demonstrated clearly that DDB, as well as the combination of DDB/amantadine, directly inhibited IFN-alpha signaling-mediated replication of HBV in infected hepatocytes, and thus may represent a novel treatment for chronic hepatitis B, which would be characterized principally by its improved safety over other treatment strategies.

Toxic effect of single treatment with bromantane on neurological status of experimental animals.

Neurotoxicological profile of actoprotector bromantane was studied on rats using S. Irwin's protocol of multi-test observation. The drug in doses of 30-300 mg/kg stimulated and in doses of 600-9,600 mg/kg suppressed behavioral activity. Spontaneous motor activity increased after single treatment with bromantane in doses of 30-300 mg/kg, did not change after treatment in doses of 600 mg/kg, and was inhibited after treatment in doses above 600 mg/kg. In doses of 300-600 mg/kg the drug reduced pain sensitivity threshold and in doses above 600 mg/kg elevated the pain threshold and tactile sensitivity and reaction to knock. Bromantane induced mydriasis in all studied doses; in doses above 10 g/kg the preparation induced blepharoptosis. In doses above 5 g/kg bromantane slightly increased respiration rate and depth (Kussmaul-like respiration). In some animals bromantane in high doses induced regurgitation, diarrhea, and polyuria. Rectal temperature decreased by 0.5-1 degrees C after virtually all doses. Behavioral effects of bromantane in doses of 30 and 600 mg/kg were associated with stimulation of the central dopamine and suppression of muscarinic and nicotinic cholinergic structures, n-cholinolytic effects of bromantane was more pronounced at a dose of 30 mg/kg than at a dose of 600 mg/kg.

Adamantylamide dipeptide as effective immunoadjuvant in rabbits and mice.

In the search for more potent and less toxic immunomodulators, adamantylamide dipeptide (AdDP) was synthesized by the covalent union of amantadine with the L-alanyl-D-isoglutamine residue of muramyldipeptide (MDP). The present experiments demonstrate the ability of AdDP, co-administered with a protein immunogen, to raise or enhance a humoral response in immunized animals. BALB/c mice were immunized either by the intraperitoneal (ip) or oral route with ovalbumin (Ova) alone or combined with either AdDP or CpG oligonucleotide (ODN-CpG), a proved adjuvant. A clear adjuvant dose-response relationship was observed on the increment of Ova-specific serum antibody titers when AdDP was used as adjuvant, irrespectively of the administration route. The IgG isotype analysis showed that AdDP promotes a consistent increment in IgG1 antibodies associated with a dominant Th2 response pattern. When administered by the oral route, AdDP was at least as efficient as ODN-CpG as adjuvant. Similar results were obtained in rabbits immunized by the oral route, suggesting that the adjuvanticity of AdDP is not restricted to the murine system. In conclusion, AdDP was shown to be a powerful and non-toxic adjuvant at both systemic and mucosal levels, which makes it a promising tool for vaccine development.

[Complex evaluation of the effect of bromantane on animal behavior]. / Kompleksnaia otsenka vliianiia bromantana na povedenie zhivotnykh.

The results of experimental investigation of the effect of bromantan on the behavioral activity of rats in the open-field test were treated by a combination of the discriminant and dispersion methods. The toxic effect of the drug on the neurogenic (neurologic) mechanisms conducting the behavior is evaluated.

[Effect of bromantane on the rat neurologic status in two month course]. / Vliianie bromantana na nevrologicheskii status krys pri dvukhmesiachnom vvedenii.

The oral administration of bromantan for two months on a toxic dose level produced a sex-dependent psychodysleptic action upon rats: the effective (30 mg/kg), intermediate (150 mg/kg), and toxic (600 mg/kg) doses reduced the motor activity in males, while not affecting (or increasing) this activity in females. The effective dose stimulated, and the toxic dose suppressed, the research activity and increased the number of grooming episodes, while ambiguously influencing the emotional state of rats. In the initial stage of treatment, bromantan causes hypothermia; in the second month, this effect is replaced by slight hyperthermia. Prolonged administration of a large dose of bromantan oppressed food uptake and slightly increased drink uptake. Upon the bromantan treatment, the body weight increased in females and decreased in males. The bromantan treatment course increased the muscle strength of rats; the operant activity was optimized during the first month of the course. The general physiological and behavioral characteristics of animals restored within two months after termination of the treatment course. During this period, the test animals exhibited no significant behavioral symptoms indicative of the drug dependence. The two-month treatment did not lead to the development of tolerance with respect to the optimizing drug action upon the physical and operant capacity.

[An acute toxicity study of bromantane]. / Issledovanie ostroi toksichnosti bromantana.

The toxicity of bromantan was evaluated by conventional acute tests (according to Belen'kii) and by the behavioral activity data (according to Irvin). A method of integral graphical representation of the behavioral activity data is suggested, according to which the results are plotted as a "dose trajectory." Using the dose trajectory constructed for bromantan, the levels of therapeutic, toxic, and lethal doses were calculated. It was established that catecholaminergic effects account for the mechanism of therapeutic action of bromantan, while cholinergic effects determine the drug action in toxic doses.

[Acute toxicity of bemithyl and bromithyl]. / Issledovanie ostroi toksichnosti bemitila i bromitila.

The experiments on rats showed for bemithyl LD50 = 581.48 (350.17-965.57) mg/kg and for bromithyl LD50 = 1750.30 (1463.07-2093.92) mg/kg (males) and 1584.29 (1280.46-1960.22) mg/kg (females). The therapeutic ratios are 4-6 for both drugs, while the toxicity index is 10-15 for bemithyl and 20 <196> 22 for bromithyl. It was established that ergotropic effects prevail in the toxicity of bemithyl administered in the 20-80 mg/kg dose range, while trophotropic effects are dominating at doses above 100 mg/kg. Bromithyl exhibits a dose-dependent trophotropic effect in the entire dose range.

[The effect of bromantane on the erythro- and leukocytic profile of the peripheral blood in rats]. / Vliianie bromantana na éritro- i leikotsitarnyi profil' perifericheskoi krovi.

The specific effect of bromantan on blood tissue is demonstrated in rats. In chronic injection in a dose of 30 mg/kg bromantan raised the level of hemoglobin and leukocytes. In doses of 150 and 600 mg/kg it increased at first (3 months) and then reduced the level of erythrocytes, hemoglobin, and leukocytes. Reversible poikilocytosis, granulocytosis, and agranulocytosis were encountered. Hyperchromatosis and hypertrophy of the hepatocytes were found in the tissues of the liver and hemosiderosis was discovered in the spleen. It is suggested that the blood tissue is a "target" in the toxic effect of bromantan.

[The characteristics of the neuropsychotropic activity of bromantane in laboratory animals]. / Kharakteristika neiropsikhotropnoi aktivnosti bromantana u laboratornykh zhivotnykh.

Bromantan [N-2-(para-(bromphenyl)-N-2-(aminoadamantan)] possesses psychostimulating activity in experiments on animals. With LD50 8100 mg/kg (mice, intraperitoneal injection) activates simple and complicated forms of behavior, induces EEG effects typical of psychostimulators, is an antagonist of substances with a deprivationg neuropsychotropic effect. Bromantan is characterized by a positive effect on mnemonic processes and on obtaining the results of complex operant activity in rats.

Amiodarone induces two different types of disorders in mouse alveolar macrophages.

It has been reported that amiodarone induces disorders of alveolar macrophages and pulmonary fibrosis, but the mechanism is not well-understood. This study was performed to elucidate the toxic mechanism from the standpoint of cellular function. Using alveolar macrophages obtained from a male Slc:ICR mouse, several injuries caused by amiodarone were compared to those caused by amantadine and mianserin as cationic amphiphilic drugs (CADs). As parameters for the drug effects, H(+)-ATPase and acid sphingomylinase activities, cellular pH, cytokine and prostaglandin releases, phagocytosis and neutral red uptake were measured. Amiodarone decreased H(+)-ATPase activity initially and subsequently increased cellular pH and decreased acid sphingomyelinase activity. These changes, which were also observed with amantadine and mianserin, were considered to be CAD-related. Amiodarone increased cytokine and prostaglandin releases and suppressed neutral red uptake and phagocytosis. These changes, being not induced by amantadine and mianserin, were considered to be specific for amiodarone. The above data suggest that amiodarone has two types of toxic effects on alveolar macrophages.

Evaluation of anti-influenza effects of camostat in mice infected with non-adapted human influenza viruses.

The anti-influenza effects of camostat, a serine protease inhibitor, on in vivo influenza infections were evaluated. Mice which received non-adapted human influenza viruses intranasally, developed a reproducible infection with very low mortality. The infection was readily detected by the recovery of the virus from an oropharyngeal swab. Five-week-old ICR mice received intraperitoneal injections of saline (control), amantadine (known positive drug), or camostat, after infection with influenza A/Taiwan/1/86 virus. Virus detection was performed on day 1, 2, 3, 5, and 7 of postinfection. Both camostat and amantadine were effective in ameliorating mouse influenza. On day 5, mice injected with camostat (45%) or amantadine (50%) showed a lower virus secreting rate than those receiving saline (90%). Additionally, camostat showed strong anti-influenza effects on an amantadine-resistant type A virus and a type B virus infection in vitro. The results show that blocking the hemagglutinin cleavage is an effective target for development of an anti-influenza agent. They also demonstrate that virus detection from the oropharynx of mice, infected with non-adapted virus, is a useful in vivo influenza model.