Naoyuki Nishikawa

Liposomal Arg-Gly-Asp analogs effectively inhibit metastatic B16 melanoma colonization in murine lungs

Analogs of a synthetic peptide having the L-arginine-L-glycine-L-aspartic acid (RGD) sequence have been found to decrease metastatic colonization. To enhance the metastasis-suppressing efficacy of these analogs, we sought to stabilize these analogs and to prolong their circulation time by incorporating them into a liposomal formulation. Various structures of RGD analogs grafted to hydrophobic groups were synthesized and then incorporated into liposomes. Liposomes composed of distearoylphosphatidylcholine, cholesterol, dipalmitoylphosphatidylglycerol and appropriate RGD analogs were injected intravenously along with B16BL6 murine melanoma cells into mice. Liposomal RGD (0.6 mumol of the analog equivalent to ca. 200 micrograms RGD peptides) inhibited lung colonization up to 76%. This dose is an order of magnitude lower than that for comparable inhibition reported for free RGD. Multi-dose administration of liposomal RGD (0.15 mumol of the analog) also inhibited the spontaneous lung metastasis of cells from a primary tumor site of B16BL6 cells subcutaneously implanted into the footpad of mice. Taken together, our data indicate that liposomal RGD may serve as a useful anti-metastatic agent.

Inhibition of tumor metastasis by Arg-Gly-Asp-Ser (RGDS) peptide conjugated with sulfated chitin derivative, SCM-chitin-RGDS

We have synthesized a new compound in which Arg-Gly-Asp-Ser (RGDS) was conjugated with 6-0-sulfated and 6-O-carboxymethyl-chitin (SCM-chitin), i.e. SCM-chitin-RGDS, and tested the inhibitory effect on lung and liver metastases of three different types of tumors in mice. SCM-chitin-RGDS was more effective for the inhibition of liver metastasis of L5178Y-ML25 lymphoma and lung metastases of colon 26 M3.1 cells than SCM-chitin, RGDS or their mixture. GRGDS peptide, however, required a higher dose (3000 µg) to obtain a sufficiently antimetastatic effect. Intermittent i.v. administration of SCM-chitin-RGDS before or after the i.v. inoculation of L5178Y-ML25 cells caused significant inhibition of liver metastasis as compared with the multiple administration of RGDS, SCM-chitin or untreated control. Co-injection of lymphoma cells with SCM-chitin-RGDS or multiple treatment of SCM-chitin-RGDS after tumor inoculation showed significantly enhanced survival rate. SCM-chitin-RGDS also showed the spontaneous lung metastasis produced by intrafootpad injection of B16-BL6 melanoma cells by the multiple i.v. administrations. These results demonstrate that the conjugation of RGDS peptide with SCM-chitin led to augmentation of therapeutic potential to cancer metastasis, thus implying an importance of the conjugation of cell-adhesive RGDS peptide with structurally heparin-like SCM-chitin, which possesses binding ability to the heparin-binding domain of fibronectin or laminin and extremely low anticoagulant properties.

Application of liposomes for cancer metastasis

Abstract Metastasis is established by a complex cascade of activities, and adhesion of tumor cells to endothelia or to extracellular matrix is one of the critical steps in the metastatic cascade. Therefore, agents that suppress such interaction may serve as anti-metastatic drugs. We previously established a non-invasive method to determine metastatic tumor cell trafficking by use of positron emission tomography (PET). In this method, positron-labeled metastatic cells are injected into bloodstream to determine tumor cell biodistribution in real-time from immediately after injection in a living animal. Here, to elucidate the involvement of cellular surface adhesion molecules in metastatic process, we investigated the effect of liposomalized sialyl Lewis X (sLe X ) as well as l -arginine- l -glycine- l -aspartic acid (RGD)-related peptide on the trafficking of B16BL6 melanoma cells and on metastatic potential. The trafficking of B16BL6 cells after injection into the tail vein was highly affected by liposomal sLe X , but only little by RGD-related peptide, suggesting that the adhesion of metastatic cells to the target is initially mediated via selectin, and integrin-mediated adhesion may occur the later stages. Furthermore, liposomal sLe X suppressed experimental metastasis suggesting that adhesion via selectin is an important step for metastasis. Next, to enhance the metastasis-suppressing efficacy, liposomalizaton of RGD was attempted, since RGD-related peptides have been found to suppress metastasis. Various structures of RGD analogs grafted to hydrophobic groups were synthesized and then incorporated into liposomes. Some liposomalized RGD markedly inhibited lung colonization at the concentration of an order of magnitude lower than that for comparable inhibition reported for free RGD. The present study indicates that liposomal application is useful for both clarifying the mechanism of metastasis and the development of anti-metastatic pharmaceutics.

Oriented polypeptide monolayers by rapid spontaneous condensation of amphiphilic amino acid esters

Abstract Ampliphilic long alkyl derivatives of amino acids which bear moderately reactive phenyl ester groups were prepared. These amphiphiles underwent rapid self-condensation to produce polypeptides when they formed ordered monolayers on an aqueous surface, although the ampliphiles were fairly inactive in liquid solutions and in non-ordered solid films. The reaction rate of the condensation, as well as the molecular orientation in the product peptide monolayer were assessed based on IR spectroscopic analysis. Lateral progress of the self-condensation and the resultant morphologies of peptide monolayers were clearly observed by scanning electron microscopy (SEM) with a transferred monolayer on a Si substrate. SEM also revealed a marked effect of the chirality of the amino acid derivatives on the uniformity of the resultant peptide monolayers.

A new pseudo-peptide of Arg-Gly-Asp (RGD) with inhibitory effect on tumor metastasis and enzymatic degradation of extracellular matrix

A series of pseudo-peptide analogs of the Arg-Gly-Asp (RGD) sequence of fibronectin have been synthe-sized, and their anti-metastatic effects in mice and inhibitory effects on tumor cell invasion in vitro have been examined. The partially modified retro pseudo-peptide of RGD, Rrev-COCH2CO-D (FC-63), was more effective in inhibiting tumor metastasis than the original RGDS peptide. Replacement of the malonyl moiety of FC-63 with a carboxyethylene linkage (Rrev-COCH2CH2-D, FC-303 ) achieved more potent inhibition of lung metastasis of melanoma cells than FC-63. Among the analogs, FC-336, a p-xylylendiamine derivative having two FC-303 moieties, showed the most potent inhibitory effect on experimental lung metastasis produced by i.v. co-injection with B16-BL6 melanoma or colon 26 M3.1 cells in a dose-dependent manner. Multiple administrations of FC-336 after tumor inoculation also showed efficient therapeutic potency against spontaneous lung metastasis of B16-BL6 melanoma in mice. Furthermore, FC-336 effectively inhibited the invasion, migration and adhesion of tumor cells in vitro, but its inhibitory effects were not more than those of RGDS peptide. Zymography analysis revealed that FC-336 inhibited the degradation of gelatin substrate by matrix metalloproteinases (MMPs) produced by tumor cells, while the RGDS peptide did not affect the enzymatic degradation. These findings indicate that the pseudo-peptides of the RGD sequence, possessing the inhibitory property of the degradation by MMPs differently from original RGD-containing peptides, may be advantageous and useful in preventing tumor metastasis.

Synthesis and biological properties of partially modified retro and retro-inverso pseudo peptides of arg-gly-asp (RGD)

Abstract Partially modified retro and retro-inverso peptide analogs of Arg-Gly-Asp (RGD) were synthesized and examined their inhibitory effects of experimental lung metastasis of murine melanoma and adenosine 5′-diphosphate (ADP) induced platelet aggregation. The analogs showed efficient theraputic potency for the tumor metastasis but low inhibitory effect on ADP induced platelet aggregation.