Relations between the penetration, binding and average concentration of cytostatic drugs in human tumour spheroids.

A penetration assay based on freeze-drying and vapour fixation was applied to show the spatial distribution of non-bound and bound cytostatic drugs in cellular spheroids. Several studies have proposed that peripheral binding of drugs correlates with limited penetration. We showed that granular accumulation, mainly at the peripheral part of spheroids, might occur in parallel with good penetration. For example, this was the case in human glioma spheroids after incubation with Adriamycin for 15-30 min. Following treatment with actinomycin D, colon carcinoma spheroids exhibited rather good penetration but also showed granular accumulation mainly in their peripheral regions. Ara-C accumulated largely and homogeneously in the peripheral regions of colon carcinoma spheroids and this severely delayed penetration. It took about 1 h for ara-C in the central regions of the spheroids to reach the same concentration as in the culture medium. In contrast, ara-C easily penetrated glioma spheroids without accumulating noticeably at the periphery. Retention tests involving washing and further incubation in drug-free culture medium revealed that the areas demonstrating extensive accumulation most often retained the drug, indicating binding, whereas the concentration of drug in other areas decreased. The oil-centrifugation method, which was used for rapid separation of the spheroids from the drug-containing medium, showed that the average concentration of daunomycin in the spheroids exceeded that in the culture medium as early as after 15 min, by which time only limited penetration had occurred. We found that good penetration of ara-C correlated with a low average concentration in glioma spheroids, whereas limited penetration correlated with a high average concentration in colon carcinoma spheroids. The latter finding was attributable to the high accumulation of drug at the spheroid periphery. Thus, there was an inverse relationship between penetration and binding and between penetration and average drug concentration. It seemed that binding delayed or prevented penetration, whereas little, if any binding resulted in better penetration. Granular binding such as that observed Adriamycin and actinomycin D gave intermediately good penetration.

Homogeneous penetration but heterogeneous binding of antibodies to carcinoembryonic antigen in human colon carcinoma HT-29 spheroids.

The monoclonal antibodies 38S1, directed against the carcinoembryonic antigen (CEA), were tested for penetration and binding in human colon carcinoma HT-29 spheroids. Penetration was studied with a method which has not previously been used in immunological investigations. The method, which allows unbound substances to be visualized, is based on freeze drying, vapour fixation, dry sectioning and dry autoradiography. The antibodies penetrated easily and all parts of the HT-29 spheroids seemed to be reached within 15 min. The penetration was even faster than in control glioma U-118MG spheroids that did not express CEA. Binding of the 38S1 antibodies was demonstrated after processing with conventional histology and autoradiography. The binding in the HT-29 spheroids was, after a 1-h incubation period, extremely heterogeneous and occurred mainly in the peripheral parts. More cells were binding the antibodies after 8-h and 32-h incubations and these cells were arranged in peripheral clusters. No binding at all was seen in the CEA-negative glioma spheroids. The distribution of CEA antigens in monolayers and in frozen sections of spheroids of HT-29 cells was analysed with immunohistochemical staining using polyclonal CEA antibodies. The CEA antigens were heterogeneously distributed in both spheroids and monolayers and were as heterogenous as the binding of the monoclonal antibodies in the living spheroids. Thus, the heterogeneous binding in the living spheroids was not due to penetration barriers, but instead to the heterogeneity in the CEA antigen expression.

Radioprotective activity of interferon inducers.

The radioprotective activity of interferon inducers (tilorone, Acranil, poly I:C and LPS) was investigated against acute X-ray irradiation and prolonged 60Co-gamma rays irradiation. The endogenous spleen colony formation test and percentage of surviving mice 30 days after irradiation were used as indicators. All interferon inducers investigated proved to have radioprotective activity.

Interferon synthesis in mouse peritoneal cells damaged by X-irradiation.

NDV-induced interferon of peritoneal cells of irradiated (X-rays, 400 R) and control mice was investigated in vitro. Irradiation or treatment with hydroxyurea (10(-5) M) and mitomycin C (25 microng/ml) did not change interferon synthesis in spite of an 80--90% inhibition of 3H-thymidine incorporation. Increased doses of mitomycin C and treatment with actinomycin D and puromycin blocked interferon production. De novo interferon synthesis occurred in cells with damaged replicative activity of DNA caused by irradiation or by treatment with antimetabolites.

Antiviral activity and induction of interferon-like substance by quinacrine and acranil.

Several drugs with certain structural similarities (tricyclic ring system with dialkylaminoalkyl side chains) to tilorone, a potent interferon inducer, were screened for antiviral activity in vivo. Two acridine drugs, Acranil and quinacrine, were found to be effective, the former being almost as protective as tilorone and the latter less so. Both agents induced an interferon-like substance which could be detected in the serum of treated mice. The concentration of the inhibitory factor in the serum was highest after exposure to tilorone, followed in turn by Acranil and quinacrine, based on the administration of equal weights of drugs. Both tilorone and Acranil induced lower levels of circulating interferon-like substance in Balb/c mice than in other strains of mice. The serum factor induced by Acranil was shown to be stable at pH 2.