We evaluated the in vivo biodistribution of indium- and yttrium-labeled second-generation polyamidoamine dendrimer (PAMAM) conjugated with 2-(p-isothiocyanatobenzyl)-6-methyl-diethylenetriaminepentaacetic acid (1B4M), a derivative of DTPA. In addition, we conjugated PAMAM-1B4M to humanized anti-Tac IgG (HuTac) and evaluated its in vitro and in vivo properties. PAMAM-1B4M was labeled with 111In at 37-48 MBq/mg (1.0-1.3 mCi/mg) or with 88Y at 3.7-4.8 MBq/mg (0.1-0. 13 mCi/mg), and an aliquot of radiolabeled conjugate was saturated with the corresponding stable yttrium or indium. Nontumor-bearing nude mice were injected intravenously with 55.5-66.6 kBq (1.5-1.8 microCi) of 88Y-labeled PAMAM-1B4M or with 185 kBq (5 microCi) of 111In-labeled PAMAM-1B4M. The mice were then sacrificed at 15 min, 90 min, 1 day, and 4 days postinjection. Then the PAMAM-1B4M was conjugated with HuTac and labeled with 111In at 111-259 MBq/mg (3-7 mCi/mg). Another preparation of 111In-labeled HuTac-PAMAM-1B4M was saturated with stable indium. Immunoreactivity of both preparations and biodistribution in normal mice 1 h after injection and in ATAC4 and A431 tumor-bearing mice 18 h after injection were evaluated and compared with those of 111In-labeled 1B4M-HuTac. We noted significantly higher accumulations (p< 0.05) of 111In-labeled and 88Y-labeled unsaturated PAMAM-1B4M than saturated preparations in the liver, kidney, spleen, and bone at most time points. The whole-body clearance times of unsaturated preparations were significantly slower than those of saturated preparations at all time points, with the exception of 168 h for 111In-labeled PAMAM-1B4M. The saturated preparation of 111In-labeled HuTac-PAMAM-1B4M showed lower hepatic uptake (27 +/- 2%ID/g) than the unsaturated (32 +/- 2%ID/g), but greater than the HuTac-1B4M control (10 +/- 0%ID/g). The splenic uptake showed 15 +/- 1, 38 +/- 5, and 8 +/- 1%ID/g for the saturated, unsaturated, and control, respectively. The biodistribution of the dendrimer conjugated HuTac in normal organs of tumor-bearing mice was similar to nontumor-bearing mice. Specific tumor (ATAC4) uptake was higher than that in nonspecific tumor (A431). In conclusion, we evaluated the biodistribution of radiolabeled PAMAM-1B4M. We noted high accumulation in the liver, kidney, and spleen, which significantly decreased when the chelates were saturated with the stable element. A similar phenomenon was observed between unsaturated and saturated 111In-labeled HuTac-PAMAM-1B4M, indicating that the PAMAM dendrimer had a detrimental effect on biodistribution.

Overexpression of cAMP-dependent protein kinase (PKA) type I isozyme is associated with cell proliferation and neoplastic transformation. The presence of PKA on the external surface of LS-174T human colon carcinoma cells has been shown. Here, we show that cancer cells of various cell types excrete PKA into the conditioned medium. This extracellular PKA (ECPKA) is present in active, free catalytic subunit (C subunit) form, and its activity is specifically inhibited by PKA inhibitory protein, PKI. Overexpression of the C␣ or RI␣ subunit gene of PKA in an expression vector, which upregulates intracellular PKA type I, markedly up-regulates ECPKA expression. In contrast, overexpression of the RII␤ subunit, which eliminates PKA type I, up-regulates PKA type II, and reverts the transformed phenotype, down-regulates ECPKA. A mutation in the C␣ gene that prevents myristylation allows the intracellular PKA up-regulation but blocks the ECPKA increase, suggesting that the NH 2-terminal myristyl group of C␣ is required for the ECPKA expression. In serum of cancer patients, the ECPKA expression is up-regulated 10-fold as compared with normal serum. These results indicate that the ECPKA expression is an ordered cellular response of a living cell to actively exclude excess intracellular PKA molecules from the cell. This phenomenon is up-regulated in tumor cells and has an inverse relationship with the hormone dependency of breast cancer. Thus, the extracellular PKA may serve as a potential diagnostic and prognostic marker for cancer.

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