Marcin Mączyński

Restoration of immune system function is accelerated in immunocompromised mice by the B-cell-tropic isoxazole R-11

BACKGROUND Restoration of impaired immune response in immunocompromised patients is a crucial problem. In this study we evaluated the efficacy of isoxazole R-11 in reconstitution of the immune response in immunosuppressed mice. METHODS Mice were given a sublethal dose (250 mg/kg b.w.) of cyclophosphamide (CP). The cellular immune response to ovalbumin (OVA) and the humoral immune response to sheep erythrocytes (SRBC) were generated. R-11 was administered at repetitive, intraperitoneal doses (20 μg/mouse) until determination of the immune responses: 7 and 15 doses on alternate days for cellular and humoral immune response, respectively. For phenotypic studies R-11 was given per os, at a single dose of 20 μg/mouse. The ability of R-11 to affect interleukin- 6 (IL-6) production was determined in the whole human blood cell culture. RESULTS R-11 increased the content of CD19+ cells in the spleens and lymph nodes with a concomitant decrease of CD3+ and CD4+ cells. The compound significantly accelerated restoration of both cellular and humoral immune responses, elevated the numbers of circulating leukocytes and splenocytes and normalized the blood cell picture. Supplementary experiments showed that R-11 was not toxic with regard to human peripheral blood mononuclear cells (PBMC) and that it upregulated IL-6 production in blood cell culture stimulated with lipopolysaccharide (LPS). CONCLUSIONS We demonstrated that R-11 is likely a B-cell tropic agent which can restore both cellular and humoral immune responses in immunocompromised mice and may have a potential to be applied in therapy of immunocompromised patients.

In vitro immunomodulatory effects of 5-amino-3-methyl-4-isoxazolecarboxylic acid hydrazide on the cellular immune response

Abstract The aim of this study was to determine the immunomodulatory activity of 5-amino-3-methyl-4-isoxazolecarboxylic acid hydrazide in vitro. This compound was used for the synthesis of a series of 5-amino-3-methyl-4-isoxazolecarboxylic acid semicarbazides and thiosemicarbazides with documented immunotropic activity. The performed measurements assessed the cytotoxic effect of 5-amino-3-methyl-4-isoxazolecarboxylic acid hydrazide on the murine macrophages (cell line J774E.1) and lymphoblasts (cell line D10.G4.1), the influence of this compound on the proliferation of murine lymphocytes isolated from peripheral lymphatic organs and murine peritoneal macrophages stimulated with mitogens (concanavalin A(ConA), lipopolysaccharide (LPS), phytohemagglutinin A (PHA)). Moreover, the production of tumor necrosis factor (TNF)-α and interleukin (IL)-1β by the murine peritoneal macrophages stimulated with LPS from Escherichia coli was assessed. It was found that 5-amino-3-methyl-4-isoxazolecarboxylic acid hydrazide displayed no cytotoxic effects in the murine J774E.1 and D10.G4.1 cell lines in a wide range of concentrations (0.5–200 μg/ml). Furthermore, the compound stimulated proliferation of lymphocytes isolated from the spleen and mesenteric lymph nodes when used alone and in combination with mitogens (ConA and PHA). This effect was stronger in the nonstimulated cells, and it followed a dose–response relationship. The same phenomenon was observed for the proliferation of the murine peritoneal macrophages. The investigated hydrazide, at the highest used concentration of 150 μg/ml, increased the LPS-induced production of IL-1β and did not affect the level of TNF-α. These results confirmed the immunomodulatory properties of 5-amino-3-methyl-4-isoxazolecarboxylic acid hydrazide and indicated that this compound could be useful in further research aimed at finding novel functional drugs.

The effect of 5-amino-3-methyl-4-isoxazolecarboxylic acid hydrazide on lymphocyte subsets and humoral immune response in SRBC-immunized mice

Abstract 5-Amino-3-methyl-4-isoxazolecarboxylic acid hydrazide is a non-cytotoxic synthetic isoxazole derivative with considerable immunomodulatory properties demonstrated in in vitro experiments. The aim of this study was to investigate the influence of this compound, depending on the dosage and schedule of treatment, on lymphocyte subsets in non-immunized mice and humoral immune response in SRBC (sheep red blood cells)-immunized mice. An analysis of lymphocyte subsets was carried out by flow cytometry, using specific monoclonal antibodies stained with fluorescein isothiocyanate (FITC) or phycoerythrin (PE). In the SRBC-immunized mice, the influence of the compound on the humoral response was determined, depending on the time of administration relative to the antigen. The number of plaque forming cells (PFC) was determined by a local hemolysis technique in an agar gel. Total and 2-mercaptoethanol resistant serum agglutination titers were defined by active hemagglutination test carried out on microplates. The investigated hydrazide was able to modulate the percentage and absolute number of T lymphocyte subsets in the thymus, and T and B lymphocytes in the peripheral lymphatic organs. It also enhanced humoral immune response in SRBC-immunized mice by increasing the number of cells producing hemolytic anti-SRBC antibodies (PFC) and by augmenting the level of total and 2-mercaptoethanol resistant hemagglutinin. The present study showed modulatory effects of 5-amino-3-methyl-4-isoxazolecarboxylic acid hydrazide on lymphocyte subsets and humoral immune response in mice. This compound could be potentially useful for the treatment of autoimmune diseases, infections or as an adjuvant for boosting the efficacy of vaccines.

Anti-inflammatory properties of an isoxazole derivative – MZO-2

BACKGROUND A series of new isoxazole derivatives of expected immunosuppressive activities was synthesized. Following in vitro screening in the human cell models, the activity of MZO-2 compound (ethyl N-{4-[(2,4-dimethoxybenzyl)carbamoyl]-3-methylisoxazol-5-yl}acetimidate) in mouse in vivo models was evaluated. METHODS In vitro tests included evaluation of: peripheral blood mononuclear cells (PBMC) viability, phytohemagglutinin (PHA)-induced PBMC proliferation and lipopolysaccharide (LPS)-induced tumor necrosis factor α (TNF α) production in whole blood cell cultures. MZO-2 was studied in mice for its effects on: humoral immune response to sheep erythrocytes (SRBC), delayed type hypersensitivity (DTH) to ovalbumin (OVA), contact sensitivity to oxazolone and carrageenan-induced foot pad edema. In addition, the effect of MZO-2 on expression of caspases in Jurkat cells was determined. RESULTS The studied compounds exhibited differential, dose-dependent effects to suppress PHA-induced PBMC proliferation and a weak property to suppress LPS-induced production of TNF α. MZO-2 had no effect on the induction phase of the humoral immune response to SRBC in vitro and in vivo, but moderately suppressed the induction phase of DTH to OVA. Its inhibitory effect on carrageenan-induced paw inflammation was potent. Likewise, MZO-2, applied in ointment, was very effective in reducing ear edema and number of lymphocytes in draining lymph nodes of mice sensitized to oxazolone, comparably to tacrolimus, the reference drug. The expression of caspases 3, 8 and 9 in Jurkat cells was inhibited by the compound. CONCLUSION MZO-2, applied systemically or locally, may serve as a potential drug for amelioration of inflammatory processes.

Immune function in cyclophosphamide-treated mice is restored by the T-cell-tropic isoxazole derivative R-13

Abstract Reconstitution of the immune function in chemotherapy patients will lead to decreases in post-operative complications. A preliminary investigation showed that an isoxazole derivative R-13 (3,5-dimethyl-isoxazole[5,4-e]8H-triazepin-4-one) hydrochloride, given in a single oral dose to normal mice, induced significant increases in the content of CD4+ cells in the spleens and lymph nodes. That observation prompted the authors to assess the immune reconstituting effects of R-13 in mice pre-treated with cyclophosphamide (CP). Mice were given intraperitoneally (IP) a sublethal dose of CP (200 mg/kg) and then R-13 (as 20 µg IP doses, every 3 days post-CP treatment). Control mice, not treated with CP, received R-13 or the vehicle (DMSO in appropriate dilution). Blood leukocyte and splenocyte numbers, blood cell type levels, splenocyte spontaneous and ConA-induced proliferation, and delayed-type hypersensitivity (DTH) to ovalbumin (OVA) were investigated on day 15 post-CP treatment and five R-13 doses. The humoral immune response (antibody-forming cell development to sheep erythrocytes) was measured 30 days post-CP treatment and 10 R-13 doses. In CP-treated mice, five dosings with R-13 led to increases in numbers of splenocytes and blood leukocytes, as well as in spontaneous and ConA-induced splenocyte proliferation, relative to levels in mice that received only CP 15 days earlier. Blood analysis revealed decreases in neutrophil and eosinophil contents and an increased appearance of lymphocyte immature forms in all mice that received the R-13. Both cell-mediated responses to OVA and humoral immune response to sheep erythrocytes in CP-treated hosts were restored. Based on the data here, it is concluded that R-13 may be of potential value for reconstitution of the immune function of chemotherapy patients.

5‐amino‐3‐methyl‐4‐isoxazolecarboxylic acid hydrazide derivatives with in vitro immunomodulatory activities

Isoxazoles are an important class of compounds of potential therapeutic value. The aim of this study was to determine immunotropic effects of 5‐amino‐3‐methyl‐4‐isoxazolecarboxylic acid hydrazide derivatives on spontaneous and mitogen‐induced lymphocyte proliferation in young and old mice, cytokine production by peritoneal cells as well as possible mechanism of action in a model of Jurkat cells. Three‐month‐old and 13‐month‐old BALB/c mice were used as donors of the cells from a thymus, a spleen, mesenteric lymph nodes, and a peritoneal cavity. Spontaneous and concanavalin A or lipopolysaccharide (LPS)‐induced cell proliferation was measured by 3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyltetrazolium bromide colorimetric method. IL‐1β and TNF‐α production induced by LPS in macrophage‐enriched peritoneal cell cultures was measured by enzyme‐linked immunoassay. 5‐amino‐3‐methyl‐4‐isoxazolecarboxylic acid hydrazide, 01K (4‐phenyl‐1‐(5‐amino‐3‐methylisoxazole‐4‐carbonyl)‐thiosemicarbazide), and 06K (4‐(4‐chlorophenyl)‐1‐(5‐amino‐3‐methylisoxazole‐4‐carbonyl)‐thiosemicarbazide) exhibited regulatory activity in the proliferation tests. Prevailing stimulatory activity of the hydrazide and inhibitory activity of 01K and 06K was observed. Those effects were connected with different influence of the compounds on signaling proteins expression in Jurkat cells. The regulatory effects of the compounds on IL‐1β production were more profound than those on TNF‐α. Differences in the compound activity in young versus old mice were mainly restricted to 01K. Immunosuppressive isoxazole leflunomide and a stimulatory RM‐11 (1,7‐dimethyl‐8‐oxo‐1,2H‐isoxazole [5,4‐e]triazepine) were applied as reference drugs.

Immunoregulatory effects of 4‐(4‐chlorophenyl)‐1‐(5‐amino‐3‐methylisoxazole‐4‐carbonyl)‐thiosemicarbazide (06K) in non‐immunized and SRBC‐immunized mice

Immunoregulatory properties of 06K derivative (4‐(4‐chlorophenyl)‐1‐(5‐amino‐3‐methylisoxazole‐4‐carbonyl)‐thiosemicarbazide) in mouse in vivo models were investigated.

Synthesis, Immunosuppressive Properties, and Mechanism of Action of a New Isoxazole Derivative

This work describes the synthesis of a new series of isoxazole derivatives, their immunosuppressive properties, and the mechanism of action of a representative compound. A new series of N′-substituted derivatives of 5-amino-N,3-dimethyl-1,2-oxazole-4-carbohydrazide (MM1–MM10) was synthesized in reaction of 5-amino-N,3-dimethyl-1,2-oxazole-4-carbohydrazide with relevant carbonyl compounds. The isoxazole derivatives were tested in several in vitro models using human cells. The compounds inhibited phytohemagglutinin A (PHA)-induced proliferation of peripheral blood mononuclear cells (PBMCs) to various degrees. The toxicity of the compounds with regard to a reference A549 cell line was also differential. 5-amino-N′-(2,4-dihydroxyphenyl)methylidene-N,3-dimethyl-1,2-oxazole-4-carbohydrazide (MM3) compound was selected for further investigation because of its lack of toxicity and because it had the strongest antiproliferative activity. The compound was shown to inhibit lipopolysaccharide (LPS)-induced tumor necrosis factor (TNF α) production in human whole blood cell cultures. In the model of Jurkat cells, MM3 elicited strong increases in the expression of caspases, Fas, and NF-κB1, indicating that a proapoptotic action may account for its immunosuppressive action in the studied models.

Isoxazole Derivatives as Regulators of Immune Functions

In this review, we present reports on the immunoregulatory properties of isoxazole derivatives classified into several categories, such as immunosuppressive, anti-inflammatory, immunoregulatory, and immunostimulatory compounds. The compounds were tested in various models using resident cells from rodents and humans, cell lines, and experimental animal disease models corresponding to human clinical situations. Beneficial features of the described isoxazole derivatives include low toxicity and good bioactivity at low doses. In a majority of studies, the activities of investigated compounds were comparable or even higher than registered reference drugs. Whenever possible, a plausible mechanism of action of the investigated compounds and their potential therapeutic utility were proposed. Among the described compounds, particular attention was paid to the class of immune stimulators with a potential application in chemotherapy patients.