Synthesis of Carboxymethylpullulan-Peptide-Doxorubicin Conjugates and Their Properties.

Abstract

The amino group of doxorubicin (DXR) was found to be bound to the carboxyl group of carboxymethylpullulan (CMPul) either directly or through tetrapeptide spacers, including Gly-Gly-Phe-Gly, Gly-Phe-Gly-Gly and Gly-Gly-Gly-Gly. These conjugates had DXR contents of 6.1-7.1%, with the degree of substitution of carboxymethyl groups being 0.6 per sugar moiety. These conjugates associate in phosphate-buffered saline (PBS) (pH 7.4), forming micelles with hydrophobic DXR inside and hydrophilic CMPul on the outside. The amounts of DXR released from the conjugates in the presence of rat liver lysosomal enzymes were determined by HPLC. The rate of the drug release differed among the conjugates tested. CMPul-DXR conjugate bound through Gly-Gly-Phe-Gly released 35% of its DXR over 24 h. On the other hand, CMPul-DXR conjugate without spacer released no free DXR. The antitumor effect of each conjugate in rats bearing Walker 256 was studied by monitoring the tumor weights after a single intravenous injection. Compared with DXR, CMPul-DXR conjugates bound through Gly-Gly-Phe-Gly and Gly-Phe-Gly-Gly spacers significantly suppressed the tumor growth, while CMPul-DXR conjugate bound through Gly-Gly-Gly-Gly showed less antitumor effect than DXR. CMPul-DXR conjugate bound through Gly-Gly-Gly-Gly showed less antitumor effect than DXR. CMPul-DXR conjugate without spacer showed no in vivo antitumor effect even at a dose equivalent to as much as 20 mg/kg of DXR.

Characteristics of Tissue Distribution of Various Polysaccharides as Drug Carriers: Influences of Molecular Weight and Anionic Charge on Tumor Targeting

“…30) CMCh was hydrolyzed with 2 M hydrochloric acid for 7 h at 100°C in a sealed tube and its N-acetyl content was determined using HPLC to measure acetic acid released from the N-acetyl-glucosamine. The DS of CM groups of CMChs were determined by the titration method used by Nogusa et al 5) The structural and physicochemical characteristics of CMChs are summarized in Fig. 1a.…”

“…Its DS of the CM groups was determined to be 0.5 by titration. 5) CMMG (DS 0.1) (DS: 0.1; 481 mg), CMMG(DS 0.2) (DS: 0.2; 503 mg) and CMMG (DS 0.3) (DS: 0.3; 524 mg) were prepared from 500 mg of MG with 20 ml each of aqueous solutions of 1.1, 1.8, and 2.5 g sodium hydroxide, respectively, and 1.5, 2.5, and 3.5 g of chloroacetic acid, respectively. CMMG (DS 0.8) (DS: 0.8; 530 mg) and CMMG (DS 1.0) (DS: 1.0; 260 mg) were prepared from 500 mg of CMMG (DS 0.5) and 250 mg of CMMG (DS 0.8) by re-carboxymethylation, respectively.…”

“…The GGFG-DXR was prepared based on Nogusa et al 5) In the presence of EEDQ in a solution of DMF-H 2 O (1 : 1), the GGFG-DXR was conjugated with polysaccharide carriers to produce the conjugates.…”

“…Macromolecules are well known to accumulate in the tumor tissue through a mechanism known as the "enhanced permeability and retention (EPR) effect. "1)The use of macromolecules for the delivery of doxorubicin (DXR) [2][3][4] to tumors was investigated in a previous study 5) and is believed to enhance efficacy and reduce systemic side effects. 6) Recently, the N-(2-hydroxypropyl) methacrylamide (HPMA) copolymer-DXR conjugate, called PK1, has entered clinical development in the UK 7,8) and its Phase 1 results 9) showed that by conjugating the drug with a macromolecule, its distribution properties could be altered depending on the properties of the carrier macromolecules.…”

Improved in vivo Antitumor Efficacy and Reduced Systemic Toxicity of Carboxymethylpullulan‐peptide‐doxorubicin Conjugates

“…Chemicals CMPul-FG-DXR was synthesized at the Drug Delivery System Institute, Ltd. (Noda, Chiba) using pullulan with a molecular weight of 65 000 (M w /M n =1.2) according to a procedure described elsewhere. 6,8) DXR was obtained from Kyowa Hakko Kogyo (Tokyo). The conjugate had a degree of substitution of carboxymethyl groups of 0.6 per glucose unit and a DXR content of 6.0%.…”