Localization of occult inflammatory foci is frequently essential to the therapeutic management of critically ill patients. Current imaging procedures, including: CT, Ultrasound and MRI rely primarily on changes in tissue density or composition that occur late changes in the inflammatory process. Radionuclide procedures that have been used to localize early inflammatory processes require a minimum of 12 hours, and typically 24-48 hours from the time of injection to imaging. Clearly, a method of rapidly localizing sites of acute inflammation would be very helpful for patient management. Recently, we developed methods for preparing analogs of the leukocyte chemoattractant peptide, For-MLF, that can be radiolabeled for external imaging. In vitro, these compounds have bioactivity and neutrophil For-MLF receptor binding comparable to the native peptide. Preliminary studies in animals have demonstrated that these agents bind to leucocytes in vivo, clear from the circulation rapidly and localize at sites of E. coli infection to an extent sufficient to yield external images early after injection. By labeling the peptides with 99mTc at very high specific activity, imaging can be performed at doses of peptide that do not induce neutropenia in rats, rabbits or monkeys. Control experiments designed to establish that infection localization is mediated by peptide-For-MLF receptor interaction, demonstrated: 1) When compared with reagents that localize at inflammation by non-specific mechanisms (radiolabeled DTPA, RBC's and IgG), T/B ratios for 99mTc-labeled peptide were much greater; 2) Comparisons between a high affinity agonist, a very low affinity antagonist and a receptor antagonist demonstrated that T/B is clearly related to receptor affinity. These studies also established that receptor antagonists localize at sites of infection without the possibility of adverse effects; and 3) The infection localization of 99mTc labeled peptide could be blocked by coinjection of a receptor antagonist. The objective of the proposed research is to develop an optimal chemotactic peptide reagent for the rapid detection and localization of focal sites of infection. These studies will involve the synthesis of a series chemotactic peptide analogs with emphasis on: design of a high affinity antagonists or partial agonist; uncomplicated, rapid, efficient and high specific activity labeling with 99mTc, favorable biodistribution, and enhanced speed of localization and contrast between lesion and background. Each reagent will be studied in vitro to determine its affinity for the For-MLF receptor on leukocytes and its ability to stimulate the production of superoxide by granulocytes. Biodistribution will be determined in animals with infection/inflammation. The rate and intensity of localization at sites of inflammation will be correlated with the in vitro For-MLF receptor affinity and biological activity data. Tissue and cell fractionation techniques will be used to determine the kinetics of distribution and metabolism of the peptides and metabolites.
| Derian, C K; Solomon, H F; Higgins 3rd, J D et al. (1996) Selective inhibition of N-formylpeptide-induced neutrophil activation by carbamate-modified peptide analogues. Biochemistry 35:1265-9 |
