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In the present study is described an immunoenzymatic assay for a new semi-synthetic immunomodulator consisting of a fraction derived from the strain ATCC 53966 of Corynebacterium granulosum to which were coupled polyaminoacid chains as a carrier to improve the oral bioavailability of the compound. This assay consists of having the specific antibody reacting with the compound and to determine the amount of antibody that has bound to the compound. For this the bound antibody was first reacted with the corresponding antispecies IgG antibody labelled with an enzyme and then the immobilized labelled antibody was revealed by a colorimetric reaction using a chromogenic substrate. The assay has further been validated by calculating the correlation factor linking the results obtained using the immuno-enzymatic assay to those obtained by determining with the aid of gas chromatography the concentration of muramic acid, a component characteristic of the compound structure.

The extranuclear endogenous mono-ADP-ribosylation of proteins in cellular fractions from retinas of control and diabetic rats was studied. At least six proteins were ADP-ribosylated in the crude extract, membrane and cytosolic fractions from control preparations, whereas in diabetic rats the number of labeled proteins and the extent of labeling were highly reduced. Treatment of diabetic animals with silybin, a flavonoid with ADP-ribosyltransferase inhibitory activity, did not affect hyperglycemia, but prevented the alterations of the extent of ADP-ribosylation of the 38 K cytosolic, 39 K, 40 K membrane and 39 K, 41 K and 42 K crude extract proteins. These data suggest a hyperactivity of extranuclear endogenous protein mono-ADP-ribosylation in the diabetic rat retina, and that treatment with silybin inhibits such enzyme activity, thus improving the extent of ADP-ribosylation. Sciatic nerve axonal transport of substance P was reduced markedly in diabetic rats, and inhibition of mono-ADP-ribosylation with silybin prevented such a loss in spite of high blood glucose levels. These results suggest that the abnormal endogenous ADP-ribosylation of proteins might play a role in the onset of diabetic peripheral neuropathy and its inhibition may represent a novel pharmacological approach to the treatment of diabetes complications.