Phase separation in phosphatidylcholine bilayers as a predictor of inhibition of blood platelet aggregation by amantadines.

The ability of eleven amantadine derivatives to induce phase separation in dipalmitoyl phosphatidylcholine bilayers was studied by differential scanning calorimetry. The relative potency varied with the shape and size of the hydrocarbon cage. These agents also markedly inhibited blood platelet aggregation. The relative potencies of these compounds to induce phase separation showed a significant correlation (r = 0.70) with their platelet inhibitory activity suggesting that their pharmacologic action may be at the level of the platelet membrane. The effective concentration of the parent component amantadine is similar to its pharmacologic concentration suggesting its use as an anti-platelet drug.

Interaction between an amantadine analogue and the transmembrane portion of the influenza A M2 protein in liposomes probed by 1H NMR spectroscopy of the ligand.

1H NMR spectroscopy of a fluoroamantadine ligand was used to probe the pH dependence of binding to the transmembrane peptide fragment of the influenza A M2 proton channel (M2TM) incorporated into 1,2-dimyristoyl-sn-glycero-3-phosphocholine liposomes. Above pH 7.5, when M2TM bound the ligand, fluoroamantadine resonances became too broad to be detected. Fluoroamantadine interacted weakly with the liposomes, indicating it may first bind to the bilayer and then block target channels after diffusion across the membrane surface.

Adjuvant effect of liposomes and adamantylamide dipeptide on antigenicity of entrapped synthetic peptide derived from HIV-1 transmembrane region glycoprotein gp41.

The synthetic peptide antigen (Ag) (the primary structure Tyr-Leu-Lys-Asp-Gln-Gln-Leu-Leu-Gly-Ile-Trp-Gly-Cys-Ser-Gly-Lys-Leu-Ile- Cys-Thr derived from the envelope glycoprotein gp41 of the human immunodeficiency virus type 1 (HIV-1) and exerting specificity with all HIV-1-positive sera available in the Czech Republic (and also in a panel of 10,000 sera from WHO)) was conjugated with bovine serum albumin (BSA) and encapsulated into liposomes. Adjuvant activities of liposomes with various lipid compositions were compared with Freund's complete adjuvant (FCA) and with aluminium hydroxide (AL). The immune response to BSA-Ag liposomes with coentrapped adamantylamide dipeptide (AdDP) was comparable with that of FCA in terms of longevity and levels of specific antibodies in mouse sera.

Stimulation of nonspecific immunity, haemopoiesis and protection of mice against radiation injury by 1-adamantylamide-L-alanyl-D-isoglutamine incorporated in liposomes.

1-Adamantylamide-L-alanyl-D-isoglutamine (adamantylamide dipeptide (AdDP)) belongs to a group of desmuramyl muramyl peptide derivatives which are able to protect an organism from some viral infections. Encapsulation of AdDP to egg phosphatidyl choline liposomes and the targeting of this drug to lymphatic node macrophages via subcutaneous (s.c.) administration proved to be the efficient way to protect mice against irradiation when administered s.c., 24 h prior to lethal gamma-irradiation (long-term survival rate in the range of 40% compared with 0% in saline or free drug control). Parameters characteristic for the recovery of haemopoiesis in the bone marrow (number of granulocyte-macrophage haemopoietic progenitor cells, granulocyte-macrophage colony forming cells (GM-CFC)) were significantly improved in comparison with the controls and free drug on day 10 after 6.5 Gy irradiation. The haemopoietic effect was observed in the broad application time window (72 h before and 48 h after irradiation). Very high radioprotective effect of s.c. administered liposomal AdDP (L-AdDP) can be explained (together with induction of haemopoiesis) by the effective and long-lasting activation of nonspecific immunity, which withholds the onset of septicemia in early days after irradiation. Induction of nonspecific immunity was proven in Candida albicans infectious model. L-AdDP significantly increased both the survival time and score (about 40% survival compared with 0% in controls and free drug). In conclusion, L-AdDP could be therapeutically beneficial to moderate the haemopoietic damage (undesirable effect of radiotherapy or chemotherapy) and induce the non-specific immunity to support the antimicrobial treatment of immunocompromised patients.

Restoration of femoral GM-CFC progenitors in sublethally irradiated mice of various ages treated with liposomal adamantylamide dipeptide.

In this study we tested the stimulatory effect of adamantylamide-l-alanyl-d-isoglutamine (AdDP) or its liposomal formulation (L-AdDP) on recovery of the granulocyte-macrophage hemopoietic progenitor cells in the bone marrow of sublethally irradiated mice of various ages. Number of GM-CFC progenitors in femur on day 10 was used as a parameter reflecting the stimulatory activity. Mice (aged 3-5 month) pre-treated with AdDP or L-AdDP via s.c. route displayed enhanced recovery of the granulocyte-macrophage hemopoietic progenitor cells at the dose of 5.5 Gy. Overaged mice (2 years) responded to the treatment when the dose was increased to 6.5 Gy, while radiation doses below 5.5 Gy should be used to see the stimulation effect in young mice (6 weeks). Entrapment of AdDP into liposomes enhanced costimulatory activity of sera of treated mice and prolongated this activity at least for 30 h after stimulation, in comparison to the mice treated with free AdDP where the costimulatory activity was spanned only up to 12 h. In conclusion, L-AdDP represents a suitable formulation of AdDP that induced recovery of GM-CFC progenitors in the femur of irradiated mice of various ages. The stimulatory effect depends on the extent of injury to bone marrow hemopoietic microenvironments caused by various doses of gamma-irradiation.

[The effect of bromantane on the dopamin- and serotoninergic systems of the rat brain]. / Vliianie bromantana na dofamin- i serotoninergicheskie sistemy mozga krys.

The effect of acute and chronic administration (50 mg/kg, p.o.) of a new immunostimulator, bromantan exhibiting psychostimulant features on the content of NE, DA and 5-HT, and their metabolites are studied. Bromantan induced a significant increase in the 5-HT and 5-HIAA content in the frontal cortex and delayed an increase in their content in subcortical brain regions. A stable decrease in the 5-HT and 5-HIAA levels in the cerebellum is observed. The drug also affected the DA parameters of the brain thus suggesting an important role of dopaminergic system in the mechanism of pharmacological effects of the drug.

Crystal structure of the supramolecular linear polymer formed by the self-assembly of mono-6-deoxy-6-adamantylamide-beta-cyclodextrin.

Mono-6-deoxy-6-adamantylamide-beta-cyclodextrin-dimethylformamide-15H2O, C53H85NO35.C3H7NO.15H2O, crystallizes in the orthorhombic space group P2(1)2(1)2(1). The adamantyl group is inserted into the cyclodextrin cavity of the adjacent molecule, entering by the side of the secondary hydroxy rim, thus forming a supramolecular linear polymer by self-assembly. Adjacent macrocycles are linked into columns by hydrogen bonds involving the nearest glucose residues, and the structure is further stabilized by their involvement in hydrogen bonding with water molecules which reside in channels surrounding the polymer columns, thus acting as bridges between the cyclodextrin units. The centroid of the adamantyl group lies below the plane formed by the seven glycosidic O atoms of the host cyclodextrin, excluding water molecules from the secondary side of beta-cyclodextrin (beta-CD). Between the adamantyl group and the primary hydroxy rim of the cyclodextrin cavity lies a dimethylformamide molecule, which shields the hydrophobic adamantyl group from the primary hydroxy rim of its carrying beta-CD and excludes water molecules from the primary side of the beta-CD cavity.

Influences of tromantadine and nonoxinol 9 on the stability of red cell membrane.

The antiviral compound tromantadine (ViruMerz) and the detergent nonoxinol 9 have been investigated in their effects on biophysical parameters of red cell membranes. Up to a maximum ratio of 1 mol per 800 mol of phospholipids tromantadine enhances the membrane phase transition/separation break at 16-20 degrees C measured by 1-anilinonaphthalene-sulfonate (ANS) fluorescence. It also increases order parameters obtained from spin labeling experiments with 5-doxyl stearic acid. Nonoxinol 9 interacts with the membrane at a maximum ratio of 1 mol per 40 mol of phospholipids determined by UV spectrophotometry. The substance decreases the intensity of the above phase transition/separation break and the order parameters of the spin label 5-doxyl stearic acid. These experiments indicate that tromantadine probably stabilizes the membrane whereas nonoxynol 9 exhibits opposite effects. Combination of both compounds equalizes the influences of the single substances on the above biophysical parameters of red cell membrane with predominance of the nonoxinol 9 effect.

Intranasal vaccination with recombinant P6 protein and adamantylamide dipeptide as mucosal adjuvant confers efficient protection against otitis media and lung infection by nontypeable Haemophilus influenzae.

Nontypeable Haemophilus influenzae (NTHi) is a leading etiologic agent of otitis media in children and recurrent respiratory infections in patients with chronic obstructive pulmonary disease. The highly conserved outer membrane protein P6 constitutes a promising vaccine candidate antigen. However, the small amount of P6 produced by this fastidious microorganism renders large-scale production difficult. Controversial data also exist concerning the suitability of recombinant P6 (rP6) as a vaccine antigen. Therefore, we performed a comparative evaluation of the immunogenicity and efficacy of native P6 and rP6 in mice intranasally vaccinated with adamantylamide dipeptide (AdDP) as an adjuvant. High titers of P6-specific serum antibodies were elicited in mice vaccinated with either native P6 or rP6, which cross-recognized both antigens. However, rP6 stimulated stronger mucosal responses. Mice vaccinated with rP6 were protected against both pulmonary and middle-ear infections (P<.01). This demonstrates that rP6 plus AdDP constitutes a promising vaccine formulation against the most relevant forms of disease caused by NTHi.