Tumor selective effect of RS-1541 (palmitoyl-rhizoxin) in M5076 sarcoma and host tissues in vivo.

RS-1541 is a 13-O-palmitoyl derivative of rhizoxin, an inhibitor of tubulin polymerization. After intravenous administration of RS-1541 to mice bearing M5076 sarcoma, the maximal inhibitory effect of RS-1541 on DNA synthesis in the tumor was observed 24 h after administration, in agreement with the Cmax of rhizoxin produced from RS-1541, but not with the Cmax of RS-1541. The inhibitory effect after RS-1541 was much higher than that after rhizoxin itself. In the spleen, thymus and bone marrow, DNA synthesis was strongly inhibited by rhizoxin but not by RS-1541. After administration of RS-1541, no significant amounts of rhizoxin were detected in the tissues, except for the tumor. In acute toxicity tests, RS-1541 appeared to be less toxic than rhizoxin. These results indicate that RS-1541 possesses a high tumor-selective effect compared with rhizoxin, because of the selective production of rhizoxin in the tumor after administration of RS-1541.

Plasma lipoproteins as targeting carriers to tumour tissues after administration of a lipophilic agent to mice.

We synthesized 14C-warfarin hexadecyl ether (14C-WHE) by addition of a palmityl moiety to the hydroxyl group at the 4-position of 14C-warfarin, a compound known to bind to serum albumin. 14C-WHE preferentially bound to the lipoproteins, low-density lipoprotein (LDL) and high-density lipoprotein (HDL), in mouse plasma both in vitro and in vivo. 14C-Warfarin mainly concentrated in the liver immediately after intravenous administration to mice bearing M5076 sarcoma, and was found at only low concentrations in other tissues including the tumour. 14C-WHE highly distributed to the tumour, adrenal, and spleen, as well as the liver. These tissues coincided with those in which human 125I-LDL was vigorously incorporated. The results indicate that chemical modification of an agent, giving it high lipophilicity, will enable it to bind to lipoproteins after intravenous administration. These modifications raise the possibility of lipoproteins as endogenous targeting carriers into tumour cells, which have high LDL-receptor activity.

Contribution of serum lipoproteins as carriers of antitumour agent RS-1541 (palmitoyl rhizoxin) in mice.

The tumour uptake as well as the anti-tumour activity of RS-1541 (palmitoyl rhizoxin), a potent antineoplastic agent, were investigated in mice bearing M5076 sarcoma. After intravenous administration, 14C-RS-1541 preferentially bound to the lipoproteins, to which 14C-rhizoxin did not bind. 14C-RS-1541 showed persisting high concentrations of radioactivity in the plasma (T 1/2 alpha, 4.9 h). The uptake of radioactivity by the tumour was second to those by the liver and spleen, and several times greater than those by the other tissues. Selective and sustained uptake by the tumour was also demonstrated by whole-body autoradiography. A considerable amount of rhizoxin was detected only in the tumour after administration of 14C-RS-1541, and the area under the tissue-concentration-time curve (AUCt) and the mean residence time (MRT) of rhizoxin in the tumour were much higher than those after administration of 14C-rhizoxin itself. The rhizoxin formation in the tumour was significantly reduced by chloroquine, a lysosomal enzyme inhibitor. RS-1541 showed a higher therapeutic activity than rhizoxin. At a 4 mg kg-1 dose, the maximum growth inhibition was 92% for RS-1541 and 41% for rhizoxin. These results indicate that RS-1541, but not rhizoxin, is taken up by the tumour via endocytosis, most likely via the low-density-lipoprotein receptor, after binding to lipoproteins. Thus, RS-1541 was considered to exhibit sustained high concentration in tumours and potent anti-tumour activity.

Downregulation of protein kinase C in guinea pig pancreatic acini: effects on secretion.

Pretreatment of guinea pig pancreatic acini with the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA) induced a time- and concentration-dependent down-regulation of protein kinase C. In control acini almost all of the protein kinase C activity was present in a cytosolic fraction. Incubation with TPA initially shifted protein kinase C activity to a particular fraction which then disappeared over the following 24-h incubation with TPA. To study the role of protein kinase C in stimulus-secretion coupling, acini were pretreated with TPA and then amylase release was studied in response to various secretagogues. Preincubation of acini with TPA led to a time- and concentration-dependent decrease in TPA-stimulated amylase release that correlated with protein kinase C downregulation. Preincubation of acini with 1 microM TPA for 24 h, resulting in complete loss of protein kinase C activity, abolished the secretory effect of subsequently added TPA. By contrast, the secretory effects of cholecystokinin octapeptide (CCK-8) and carbamylcholine chloride (CCh) were only inhibited by 44 and 34%, respectively, and amylase release stimulated by the Ca2+ ionophore A23187 and an adenosine 3',5'-cyclic monophosphate-mediated agonist, vasoactive intestinal peptide, was unaffected. Dose-response curves for CCK-8- or CCh-stimulated amylase release in TPA-pretreated acini revealed attenuation of both maximal efficacy and sensitivity. However, the CCh-stimulated intracellular Ca2+ increase as determined by use of the fluorescent probe fura-2 was not affected by the long-term TPA pretreatment of acini. This study strongly suggests that both protein kinase C and intracellular Ca2+ play a significant role in CCK-8- and CCh-stimulated amylase release.