Assays immunomodulating assay

After establishment of the drug exposure paradigm, the potential of these compounds to produce immunomodulation was examined in a panel of in vitro assays. These assays were carefully selected to yield the maximal amount of information on various immune regulatory and effector mechanisms. The rationale for the use of each assay and its execution are described below. 

Albers, R. et al., The use of reporter antigens in the popliteal lymph node assay to assess immunomodulation by chemicals. Toxicol. Appl. Pharmacol., 143, 102, 1997. 

Gutting, B.W. et al., A comparison of the direct and reporter antigen popliteal lymph node assay for the detection of immunomodulation by low molecular weight compounds. Toxicol. Sci., 51, 71, 1999. 

The other limitation of the CFU assay in either format is that it remains an in vitro environment where distant interactions, like liver metabolism (either detoxifying the compounds or producing toxic metabolites) or immunomodulation, are absent. Investigating the meciianism of toxicity in animals for some carefully chosen compounds after being ciiaracTerized in the two previous assays, can provide valuable information for the whole series, allow further refinement of the in vitro assays (e.g., addition of S9 for metabolism) and give an early indication of which biomarkers could be used in later GLP studies. 

The inhibitory effects of berbamine and ISO natural products on the cytotoxic activity of polymorphonuclear leukocytes (PMN) was investigated. The research employed the effects of natural products on the PMN activation by the antitumor immunomodulator TAK (a linear P-1,3-D-glucan from Alcaligenes faecalis var. myxogenes) using a PMN cytotoxicity assay system. Berbamine was found to inhibit the activation of PMN activation by TAK, showing an IDJ0 33 pg/ml. From this, and related studies, it was postulated that berbamine may impair the NADPH oxidase system in the plasma membrane of PMN [199]. 

Figure 3.3.1-2 Application of the TDAR assay in immunotoxicity assessment. When evaluating unintended immunosuppression, the TDAR assay may be conducted when evidence of immunotoxicity is seen in repeated-dose toxicity studies. In these instances, the assay should be conducted prior to Phase 3 or earlier, depending on factors such as the severity of the findings and the intended patient population. The TDAR assay also could be used early in the drug development process to screen for immunotoxicity potential. This approach may be useful particularly to help de-risk unintended off-target immunomodulation or when a novel target/mechanisms may alter immune function.

Immunomodulation not only relates to a series of physiologic and pathologic phenomena, but also it can be estimated by related reaction cells, such as mast cells, DCs and NK cells, and level of cell factors such as interleukin, interferon, tumor necrosis factor, and transforming growth factor. Based on those facts, the rat mast cell and rabbit aortic assay [105], dendritic cell assay [106], lymphoid organ assay [107], IFN-y assay [108], anti-rHuEPO NAb assay [109], and chemotaxic assay [110] are established. 

In the past 20 years hundreds of Chinese traditional and herbal medicines with immunomodulating functions have been screened, and their active components studied. Through these studies, six diterpene lactone epoxide compounds, with antiinflammatory and immunosuppressive actions, were isolated from T. wilfordii. Zheng et al. [183] assayed the half-effective dose (ED50), the therapeutic index (TI) and a certain safety factor (CSF) using croton oil induced ear swelling and hemolysin-antibody formation mouse models and found that triptolide (79), tripdiolide (80), triptonide (81), tripchlorolide (92), and triptolidenol (93) possessed both antiinflammatory and immunosuppressive activities, while triptriolide (94) showed antiinflammatory action only. Fig. (28). The results of the TI and CSF of both activities clearly demonstrated that the antiinflammatory and immunosuppressive compounds in T. wilfordii are pluralistic and the differences in these compounds can be used as a base for the evaluation and selection of them. 

Specialized Immunomodulation Fc effector function Cytotoxicity assay with... 

Wagner H, Jurcic K (1991) Assays for immunomodulation and effects on mediators of inflammation. Methods in Plant Biochemistry 6 195-217... 

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