Evaluation of follow-up of therapy with fenbendazole incorporated into stabilized liposomes and immunomodulator glucan in mice infected with Toxocara canis larvae.

Anthelmintic activity of benzimidazole carbamate anthelmintics is low against dormant Toxocara canis larvae during late infections in paratenic hosts. The present study was conducted to examine the efficacy of pure fenbendazole, or drug incorporated into sterically stabilized liposomes (SL-FBZ) administered to T. canis-infected mice alone and after its co-administration with the immunomodulator (1-->3)-beta-D-glucan against larvae localized in muscles and brains. Therapy with either drug forms (in total 250 mg/kg in 10 doses) commenced on day 28 post-infection (p.i.) and the efficacy of treatment, examined on day 30 after the last dose of drug, was the highest in groups of mice treated with SL-FBZ in combination with glucan (89.5+/-5.8% in the muscles, 66.1+/-8.1% in brains). During 56 days of follow-up after termination of therapy, serum levels of anti-TES IgG antibodies, circulating IgG-TES immune complexes (CIC) as well as IgG antibodies to the most immunogenic part of recombinant myosin antigen of T. canis larvae were investigated. In contrast to anti-TES IgG antibodies, levels of CIC and anti-myosin antibodies were in the linear correlation with the efficacy of treatments beginning from day 38 post-therapy. We also showed that the serum levels of CIC as well as anti-myosin IgG antibodies seem to be the suitable serological markers for the monitoring of progress in larval destruction and TES resorption from the tissues.

Praziquantel and liposomized glucan-treatment modulated liver fibrogenesis and mastocytosis in mice infected with Mesocestoides vogae (M. corti, Cestoda) tetrathyridia.

Beta-glucans are immunomodulators able to activate innate immunity and to potentiate acquired immune reactions. We investigated the impact of co-administration of liposomized beta-glucan on the larvicidal effect of the anthelmintic praziquantel (PZQ) in the livers and peritoneal cavities in mice infected with Mesocestoides vogae (M. corti). Also, within 2 weeks following therapy (up to day 29 p.i.) we examined collagen synthesis in the livers of mice by means of biochemical determination of hydroxyproline concentration, total mast cell counts and cell proliferative capacity using immunohistochemical and radiometrical methods. After co-administration of liposomized glucan (LG) and PZQ efficacy (%) was significantly higher than after treatment with either compound alone, particularly in the peritoneal cavity compared to the liver. In comparison with the control, more intense collagenesis was found in the B-liver parts (high intensity of infection) and lowering of collagen content in the A-parts (very weak infection). This effect was strongest after LG treatment and co-administration of PZQ abolished the pro-fibrotic effect of LG. In all groups, mast cell counts were higher in the B-liver parts than in the A-parts and the dynamics of mastocytosis was profoundly modulated following therapy. Whereas the effect of PZQ was only moderate, early and very strong onset was seen after LG treatment. Administration of PZQ suppressed LG induced-elevation of mast cells counts in both liver parts. Using DNA S-phase markers (BrdU and 3H-thymidine) the proliferative capacity was shown to be associated with several kinds of liver cells. Therapy significantly stimulated [3H]-thymidine incorporation (cell proliferation) only in the A-parts over that in control, the most after LG administration. In summary (i) the anthelmintic effect of PZQ could be enhanced after simultaneous administration of the immunomodulator beta-glucan entrapped in a liposomal carrier, (ii) intense mastocytosis seen after treatment with LG seems to have a direct role in the glucan's pro-fibrotic activity and can be abolished after co-administration of PZQ in a time-dependent manner, (iii) the pattern of cell proliferation indicates that in the case of PZQ treatment, the reparative processes of liver parenchyma are enhanced in an inverse correlation with the intensity of infection.

Effects of free and liposomized praziquantel on worm burden and antibody response in mice infected with Mesocestoides corti tetrathyridia.

The parasite burden in the liver and peritoneal cavity, and antibody levels directed to whole worm extract, have been monitored in serum from ICR-strain mice, infected orally with 55 tetrathyridia of Mesocestoides corti (Cestoda, Cyclophyllidea). The subcurative does (3x or 6x) of praziquantel (PZQ) (10 mg.kg-1 body weight) were administered to mice from day 14 post infection (p.i.) in two drug formulations: as PZQ suspended in Dorfman vehicle, or as PZQ incorporated in liposomes (lip.PZQ). The appearance of antibodies was time-dependent and correlated with the rate of reduction in numbers of tetrathyridia. PZQ in three and six doses caused the highest fall of parasite numbers in the liver on day 1 post therapy (p.t.). In the peritoneal cavity, a similar reduction in worm burden occurred but only after six doses of the drug. The worm count in the peritoneal cavity from groups of mice injected with lip.PZQ decreased most markedly on day 7 p.t., in the group treated with six doses of the drug. In the liver, the highest larvicidal effect, compared with the controls, was observed 6 days later (i.e. day 13 p.t.), following three doses of lip.PZQ. In all treated groups, two peak values of antitetrathyridial antibody levels were detected between days 1 and 13 p.t. (i.e. days 17 to 29 p.i.), after which there was a gradual but continuous increase in antibody tire.(ABSTRACT TRUNCATED AT 250 WORDS)

Treatment of Toxocara canis infections in mice with liposome-incorporated benzimidazole carbamates and immunomodulator glucan.

Benzimidazole carbamates (mebendazole, albendazole and fenbendazole) are the most commonly used anthelmintic drugs for the treatment of larval toxocariasis (Toxocara canis) in paratenic hosts. However, the bioavailability of these drugs for tissues is very low due to their extremely low solubility, resulting in the administration of relatively high doses over a long period. To overcome this problem, neutral, negatively or positively charged and stabilized liposome drug carriers were examined in the chronic phase of T. canis infections in mice each orally inoculated with 1000 eggs. Moreover, liposomized albendazole and fenbendazole were co-administered with liposomized immunomodulator glucan. The highest efficacy of both drugs, evaluated 4 weeks after treatment, was recorded after their subcutaneous administration (ten doses of 25 mg kg(-1)) in stabilized liposomes and intramuscular co-administration of liposomized glucan (two doses of 5 mg kg(-1)). Fenbendazole was more effective in muscles (91.5%) whereas albendazole was more effective in the brain (92.2%). Liposomes with incorporated benzimidazole carbamate anthelmintics provide sustained drug-release reservoirs and can considerably enhance drug efficacy. Moreover, despite suppression by T. canis antigens, stimulation of the immune system by the immunomodulator glucan potentiates the effects of these antiparasitic drugs.

Effect of praziquantel and liposome-incorporated praziquantel on peritoneal macrophage activation in mice infected with Mesocestoides corti tetrathyridia (Cestoda).

The activation of peritoneal macrophage effector functions after therapy with free PZQ and PZQ incorporated in liposomes (lip.PZQ) was studied in the Mesocestoides corti-mouse model system. Each drug formulation was administered to an infected group of mice in 6 daily doses from day 14 p.i. Phagocytic activity of macrophages increased significantly after the administration of both drug formulations, more after lip.PZQ with an earlier peak observed for PZQ (day 3) than for lip.PZQ (day 6). Empty liposomes had no significant effect. The average counts of ingested particles in phagocytosing cells were significantly higher only after lip.PZQ administration. The pattern of changes in phagocytic activity correlated with the reduction of parasite numbers in the peritoneal cavity, with the highest observed on day 6 after therapy with lip.PZQ. Phagocytosis of lip.PZQ in vivo stimulated significantly the respiratory burst in peritoneal macrophages, with the highest concentration of superoxide anions recorded on day 1 after the last dose, whereas therapy with PZQ itself did not increase this process significantly. The capacity for the respiratory burst declined in all groups with progressing infection. It is proposed that the phagocytic activity of peritoneal macrophages after therapy was stimulated indirectly as a consequence of activation of the specific immune response. The larvicidal effect of lip.PZQ on the tetrathyridia in the peritoneal cavity was synergistic with the phagocytic activity and might be the result of double action of drug and superoxide anions generated during the respiratory burst stimulated by this drug formulation.

Distribution of [3H]cholesterol-labelled liposomes with or without praziquantel in mice infected with Mesocestoides corti (Cestoda) tetrathyridia.

The anthelmintic drug praziquantel (PZQ) has a short half-life in the circulation, necessitating repeated daily administration of PZQ for the therapy of larval stages of cestodes. The effect of incorporation of PZQ into multilamellar liposomes on their biodistribution in Mesocestoides corti (syn. M. vogae) infected mice has been examined using [3H]cholesterol as a liposomal marker. Incorporation of PZQ significantly increased the average size of liposomes with 70.3% of [3H]lip.PZQ particles up to 1.9 microm, whereas higher portion of [3H]liposomes (66.3% of total) were of smaller (up to 1.3 microm). Both liposome preparations were given intraperitoneally to avoid rapid sequestration in the liver. There were significant differences between [3H]liposomes and [3H]lip.PZQ-associated radioactivity in peritoneal adherent cells, liver- and peritoneal larvae, liver, spleen and lymph nodes within 16 days of examination. The highest uptake (about 2-fold more [3H]lip.PZQ than [3H]liposomes from the total dose) was found in peritoneal cells on day 1 post therapy (p.t.) followed by a rapid decline. The kinetic of decline in these cells recovered on day 1 p.t. was studied also in vitro. Disappearance of the marker due to the breakdown of liposomes and efflux of lipids and PZQ from cells was slower for [3H]lip.PZQ in comparison with drug-free liposomes and was not completed after 4 days-incubation. Significantly increased levels of radioactivity, more in [3H]liposomes treated groups, were recorded in the liver- and peritoneal larvae between days 8-16 p.t. indicating re-utilization of cholesterol by the larvae. The data suggest that incorporation of PZQ into liposomes contributes to the enlargement of liposome average size and slows down their degradation in phagocytosing cells. In this respect, these cells could serve as the secondary circulating depots for PZQ releasing it slowly to the circulation.

A morphological study of the effects of liposomized albendazole on the muscle phase of Trichinella spiralis in mice.

The pathomorphological effects of daily and weekly doses of liposomized albendazole on Trichinella spiralis larvae have been examined and compared during the course of muscle infections in mice. Treatment with three and six daily doses of the drug given by intraperitoneal injection to mice resulted in marked pathological alterations to encapsulated larvae, mainly in the walls of the capsules. There was a 5% efficacy against T. spiralis in mice given three daily doses of albendazole, and 38% efficacy in mice given six daily doses of the drug. The same doses of drug, when administered weekly, did not reduce the numbers of parasite larvae, and the application of drug at 6-weekly intervals resulted in a 5% reduction in numbers.

Effects of free and liposomized praziquantel on the surface morphology and motility of Mesocestoides vogae tetrathyridia (syn. M. corti; Cestoda: Cyclophyllidea) in vitro.

The effects of in vitro exposure to praziquantel (PZQ), liposomized PZQ (lip.PZQ), and empty liposomes on the surface morphology and motility of Mesocestoides vogae tetrathyridia were investigated using scanning electron microscopy (SEM) and a motility apparatus. Examination of treated larvae showed an effect that was concentration- and time-dependent, involving morphological damage that was similar in character for all of the treated groups. The most marked effects were a flattening and elongation of the larval body accompanied by irregularities in the surface architecture involving the development of tegumental protuberances and depressions. Erosion of the surface microvillous layer occurred only after overnight incubation, being most pronounced after treatment with lip.PZQ. The motility index of treated tetrathyridia corresponded well to the SEM observations. The frequency of contractions was maximal in worms treated with free PZQ at 25 micrograms/ml in both regimens. However, after incubation with lip.PZQ the increase in motility was concentration-dependent and of a greater extent. Empty liposomes and lipid mixtures of the same concentration and composition resulted in increased motility in treated larvae as compared with controls. More extensive tegumental damage and higher motility of larvae occurred after lip.PZQ treatment, perhaps resulting from a synergistic action of the drug and its associated lipid.

Modulation of liver fibrosis and pathophysiological changes in mice infected with Mesocestoides corti (M. vogae) after administration of glucan and liposomized glucan in combination with vitamin C.

The effects of glucan and liposomized glucan, alone or co-administered with vitamin C, and empty liposomes on hepatic fibrosis in mice infected with Mesocestoides corti (M. vogae) tetrathyridia were studied. Preparations were administered every third day from day 7 to day 31 post-infection (p.i.), nine doses in total. Activities of alanine aminotransferase (ALT), aspartate aminotransferase (AST) and cholesterol levels were measured in sera collected on days 11, 15, 21, 28, 32, 42, 50 and 65 p.i. Liver fibrosis was studied on the same days by measuring hydroxyproline concentration, which is considered a marker for collagen content. Larvicidal effects of the glucan and liposome preparations were estimated on day 65 p.i. in the liver and peritoneal cavity. Glucan formulations significantly enhanced collagen content, most prominently after administration of liposomized glucan in combination with vitamin C. Activities of both enzymes and cholesterol levels were slightly modified after administration of glucan alone. Liposomized glucan with vitamin C significantly increased ALT and AST activity and cholesterol levels up to days 28-32 p.i., after which they plateaued or declined. The most pronounced decrease was after administration of liposomized glucan and vitamin C. The same pattern of biochemical parameters in serum was observed after administration of empty liposomes, however, collagen content was not modified significantly. Larval counts in the liver and the peritoneal cavity were significantly reduced after treatment with either glucan formulation, but were unaffected following treatment with empty liposomes. In summary, intense fibrosis in the liver of mice treated with liposomized glucan and vitamin C did not result in the most extensive parenchymal cell injury but, rather in the highest efficacy of treatment. Liposomal lipids were probably utilized in the reparation of the damaged parenchymal cells, while glucan stimulated phagocytic cells.