Isamu Itoh

The conjugation of RGDS peptide with CM-chitin augments the peptide-mediated inhibition of tumor metastasis

Abstract A water soluble 6- O -carboxymethyl chitin (CM-chitin) containing cell adhesive Arg-Gly-Asp-Ser (RGDS) sequence, i.e. CM-chitin-RGDS conjugate was synthesized, and the inhibitory effects of this compound on lung or liver metastasis of lung-metastatic B16-BL6 melanoma or liver-metastatic L5178Y-ML25 lymphoma cells in mice was examined. CM-chitin-RGDS showed the inhibitory effects on lung metastasis of melanoma cells in a dose-dependent manner (ranging from 100 to 1000 μg) and on liver metastasis of lymphoma cells. A mixture of CM-chitin and RGDS peptide or CM-chitin alone did not show any inhibitory effect on experimental lung metastasis as compared with the conjugate CM-chitin-RGDS on a molar basis. GRGDS peptide, however, required a higher dose (3000 μg) to obtain a sufficiently antimetastatic effect. The in-vitro tumor invasion study showed that CM-chitin-RGDS was apparently more effective for the inhibition of tumor cell penetration into reconstituted basement membrane Matrigel than RGDS or the mixture of RGDS and CM-chitin on a molar basis. Intermittent i.v. administration of CM-chitin-RGDS after the inoculation of B16-BL6 cells caused significant inhibition of spontaneous lung metastasis produced by intrafootpad injection of tumor cells as compared with the multiple administration of RGDS, CM-chitin or untreated control. These results demonstrate the importance of the conjugation of RGDS peptide with CM-chitin as a polymeric carrier for the increased therapeutic potential to cancer metastasis, thus implying a possibility that RGDS-polymer conjugation may lead to the prolongation of antimetastatic action of RGDS peptide in vivo .

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.

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.

N-methyl-2-dimethylaminoacetohydroxamic acid as a new reagent for the selective cleavage of active esters under neutral conditions

Abstract A new reagent, N-methyl-2-dimethylaminoacetohydroxamic acid 3 was developed for the selective cleavage of active esters under neutral conditions. The kinetic studies and the applications of 3 are described.

Inhibition of tumor metastasis by a synthetic polymer containing a cell-adhesive RGDS peptide

A water soluble polymer containing cell adhesive Arg-Gly-Asp- Ser (RGDS) sequence, i.e., poly(carboxyethylmethacrylamide-RGDS) conjugate [poly(CEMA-RGDS)], was synthesized. Poly(CEMA-RGDS) inhibited lung metastasis of B16-BL6 melanoma cells in a dose-dependent manner (20-1000 μg) and liver metastasis of L5178YML25 lymphoma cells. A mixture of poly- (CEMA) and RGDS peptide or poly(CEMA) alone did not show any inhibitory effect on lung metastasis. The Gly-Arg-Gly-Asp-Ser (GRGDS) peptide required high doses (3000 μ g) to obtain a sufficient antimetastatic effect. An in vitro study showed that poly(CEMA-RGDS) as well as RGDS + poly(CEMA) gave similar inhibition of B16-BL6 cell invasion into reconstituted basement mem brane Matrigel. Intermittent i.v. administration of poly(CEMA-RGDS) after in oculation with B16-BL6 cells caused significant inhibition of spontaneous lung metastasis as compared with the multiple administration of RGDS, poly- (CEMA) or untreated control. These results demonstrate that the conjugation

Regioselective Modification of Lysine's Amino Groups by Proteases

The regioselectivity of six serine proteases for the amino groups of lysine was investigated. alpha-Chymotrypsin showed a preference for the alpha-amino group, although the selectivity can be varied 10-fold depending on the reaction medium. Subtilisin Carlsberg and other bacterial proteases were highly specific for the epsilon-amino group, regardless of the reaction medium: they were used as catalysts for the preparative synthesis of isopeptides in anhydrous tert-amyl alcohol.