Valutazione dell’immunotossicità per il nuovo inibitore della tirosin-chinasi della milza R406 – Associazione Veterinari Umbri Liberi Professionisti

Spleen tyrosine kinase (Syk) is a novel pharmaceutical target in the treatment of allergic, autoimmune and neoplastic diseases. Previous studies have shown that Syk signaling plays a key role in regulating the lymphohematopoietic system. These observations prompted us to investigate whether Syk’s inhibition promoted immunotoxicity.
In a series of studies, rats were given orally R406 at doses up to and including 100 mg / kg / day (or its prodrug R788 at doses up to and including 100 mg / kg / day, reduced to 50 mg / day). ). kg / day for women after passing MTD), a strong Syk inhibitor, twice daily for 28 days.
Oltre alle valutazioni tossicologiche standard, sono stati eseguiti test di immunofenotipizzazione citometrica a flusso e di risposta immunitaria umorale misurando i livelli di anticorpi IgM e IgG anti – KLH . Altri studi di immunotossicità includevano tre modelli di resistenza dell’ospite in topi Balb/c femmine per determinare ulteriormente gli effetti di R406 sull’immunità innata e acquisita.
Dopo il trattamento con R406, negli studi sui ratti sono stati osservati gli effetti immunomodulatori attesi (ad es. diminuzione del peso del timo e della milza, perdita di cellule del midollo osseo e diminuzione della conta dei linfociti, inclusi i linfociti T e B). Questi cambiamenti si sono generalmente risolti dopo un periodo di recupero di 14 giorni senza trattamento . KLH challenge nei ratti non ha mostrato effetti avversi sulla risposta IgG o IgM . R788/406, somministrato per via orale a dosi fino a 80 mg/kg/die inclusi per 28 giorni, non ha influenzato la clearance batterica o virale nei modelli di resistenza murina rispettivamente di Listeria, streptococco o ospiti dell’influenza.
Ciò era correlato a precedenti test di funzionalità dei macrofagi e dei neutrofili in vitro (valutazione di ioni migratori, fagocitosi, scoppio ossidativo e attività battericida) che hanno mostrato che l’ R406 non ha influenzato negativamente la funzione dei macrofagi o dei neutrofili nelle risposte immunitarie innate. Presi insieme, questi risultati mostrano che l’ R406 ha un’immunotossicità funzionale minima nonostante i suoi effetti linfocitopenici, suggerendo che l’inibizione di Syk potrebbe non portare a effetti avversi inaccettabili basati sui meccanismi.

Valutazione dell’immunotossicità per il nuovo inibitore della tirosin-chinasi della milza R406.

- Cdna 8Spleen tirosin chinasi (Syk) è un nuovo target farmaceutico nel trattamento delle malattie allergiche, autoimmuni e cancerose. Precedenti studi hanno dimostrato che la segnalazione Syk svolge un ruolo chiave nella regolazione del sistema linfoematopoietico.
- Queste osservazioni ci hanno spinto a indagare se l’inibizione di Syk promuovesse l’immunotossicità. In una serie di studi , ai ratti è stato somministrato per via orale R406 a dosi fino a 100 mg/kg/die inclusi (o il suo profarmaco R788 a dosi fino a 100 mg/kg/die inclusi, ridotti a 50 mg/die). kg/die per le donne dopo aver superato la MTD), un forte inibitore Syk, due volte al giorno per 28 giorni.
- In addition to standard toxicological assessments , flow cytometric immunophenotyping and humoral immune response tests were performed by measuring levels of anti – KLH IgM and IgG antibodies . Other immunotoxicity studies included three host resistance models in female Balb / c mice to further determine the effects of R406 on innate and acquired immunity.
- After treatment with R406, the expected immunomodulatory effects (e.g. decreased thymus and spleen weight, loss of bone marrow cells and decreased lymphocyte counts, including T and B lymphocytes) were observed in rat studies. .
- These changes generally resolved during the 14-day treatment-free recovery period. KLH challenge in rats showed no adverse effects on IgG or IgM response . R788 / 406, administered orally at doses up to and including 80 mg / kg / day for 28 days, did not affect bacterial or viral clearance in mouse resistance models of Listeria, streptococcus or influenza hosts, respectively.
- This was related to previous in vitro macrophage and neutrophil function tests (evaluation of migratory ions, phagocytosis, oxidative burst and bactericidal activity) which showed that R406 did not adversely affect macrophage or neutrophil function in immune responses. innate.
- Taken together, these results show that R406 has minimal functional immunotoxicity despite its lymphocytopenic effects, suggesting that Syk inhibition may not lead to unacceptable mechanism-based adverse effects.

To test whether IL-10 acts as an autocrine or paracrine growth factor for Ly-1 B cells, we treated mice continuously from birth to 8 weeks of age with a rat monoclonal IgM antibody that specifically neutralizes IL-10. murine. Mice treated in this manner were deprived of peritoneal Ly-1 B cells, contained significantly reduced serum levels of immunoglobulin M, and were unable to generate significant in vivo antibody responses to intraperitoneal injection of alpha 1,3-dextran or phosphorylcholine, antigens , for which specific B cells are found in the subset of Ly-1 B cells.

In contrast, spleen B cells from conventional anti -IL-10-treated mice were normal with respect to total number, phenotype, and in vitro response to B cell mitogens and trinitrophenyl hemocyanin antigen (TNP- KLH ) from thymus-dependent keyhole patella. ).

The mechanism of Ly-1 B cell depletion appears to be related to increased levels of endogenous interferon gamma (IFN-gamma) in mice treated with anti-IL-10 because they co-administered anti-IFN-gamma sub anti – IL. – 10 neutralizing antibodies . alli restored the number of Ly-1 B cells residing in the peritoneum of these mice. These results suggest that IL-10 is a growth regulator of Ly-1 B cells and identify a procedure for specific depletion of Ly-1 B cells, thus allowing further evaluation of the role of these cells in the immune system.

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Kit incl. pH-electrode 201T-DHS and cond.-cell 2301T, solutions, standards, accessories in a case, with DHS-function
EC-21-multi-5	each	751 EUR
Rat Anti-KLH IgM with controls
KT-568	96 tests	778 EUR
BCA Optima Protein Assay kit with Pre-diluted BGG (bovine gamma globulin) standards and BCA Strip plates (5), 500 tests
BCAO-522	1 kit	270 EUR
Pre-diluted BGG (bovine gamma globulin) standards (1 set: 2000, 1000, 500, 125, 62.5, 31.25, and 0 ng/ml BGG) for BCA Optima Protein Assay kit
BCA-BGG-1	1 pk	196.8 EUR
Human Immunoglobulin M (IgM) Antibody Pair Kit (with Standard)
MBS2089859-10x96Tests	10x96Tests	1390 EUR
Human Immunoglobulin M (IgM) Antibody Pair Kit (with Standard)
MBS2089859-10x96TestsMBS2090685AbPairsSupportPack110x96Tests	10x96Tests+MBS2090685(AbPairsSupportPack1,10x96Tests)	1510 EUR
Human Immunoglobulin M (IgM) Antibody Pair Kit (with Standard)
MBS2089859-5x96Tests	5x96Tests	880 EUR
Human Immunoglobulin M (IgM) Antibody Pair Kit (with Standard)
MBS2089859-5x96TestsMBS2090685AbPairsSupportPack15x96Tests	5x96Tests+MBS2090685(AbPairsSupportPack1,5x96Tests)	955 EUR
Mouse Immunoglobulin M (IgM) Antibody Pair Kit (with Standard)
MBS2089860-10x96Tests	10x96Tests	1800 EUR
Mouse Immunoglobulin M (IgM) Antibody Pair Kit (with Standard)
MBS2089860-10x96TestsMBS2090685AbPairsSupportPack110x96Tests	10x96Tests+MBS2090685(AbPairsSupportPack1,10x96Tests)	1920 EUR
Mouse Immunoglobulin M (IgM) Antibody Pair Kit (with Standard)
MBS2089860-5x96Tests	5x96Tests	1130 EUR

These results suggest that IL-10 is a growth regulator of Ly-1 B cells and identify a procedure for specific depletion of Ly-1 B cells, thus allowing further evaluation of the role of these cells in the immune system.