Modification of E. coli L-asparaginase with polyethylene glycol: Disappearance of binding ability to anti-asparaginase serum

Ashihara, Yoshihiro; Kono, Toshihiro; Yamazaki, Shojiro; Inada, Yuji

doi:10.1016/0006-291x(78)91002-1

Cited by 117 publications

(57 citation statements)

References 14 publications

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“…The low resistance of the modified streptokinase against trypsin compared with the modified preparations by polyethylene glycol in 13-51 is probably due to the lower degrees of the modification of amino groups in lysine residue, which are sites of cleavage with trypsin, in streptokinase (23.5%) than those of the modification of amino groups in lysine residues in catalase (43%) [4] and L-asparaginase (79%) [5]. The preparation of a modified streptokinase obtained here, which has no antigenicity against its antibody and retains activity, may be effective for treating patients who have antibodies formed by injection of streptokinase.…”

Section: Resultsmentioning

confidence: 99%

Preparation of polyethylene glycol‐modified streptokinase with disappearance of binding ability towards anti‐serum and retention of activity

1982

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Section: Resultsmentioning

confidence: 99%

Preparation of polyethylene glycol‐modified streptokinase with disappearance of binding ability towards anti‐serum and retention of activity

1982

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“…Conjugation typically has low yield (10-15%) (Svensson et al, 1994;Melton et al, 1996) and results in reduced enzyme activity. Ashihara et al (1978) showed that the activity of PEG-L-asparaginase conjugates decreased dramatically with increasing PEG molecular weight and in proportion to the degree of PEG substitution. The semi-random aminolysis method used to bind HPMA copolymer precursor to cathepsin B resulted in conjugates with a high yield (30-35% protein conjugation) and also conjugates with retained enzyme activity in vitro against both the low molecular weight and macromolecular substrates (20-25%) (Figure 3).…”

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confidence: 99%

PDEPT: polymer-directed enzyme prodrug therapy

2001

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Summary Polymer-directed enzyme prodrug therapy (PDEPT) is a novel two-step antitumour approach using a combination of a polymeric prodrug and polymer-enzyme conjugate to generate cytotoxic drug selectively at the tumour site. In this study the polymeric prodrug N-(2-hydroxypropyl) methacrylamide (HPMA) copolymer-Gly-Phe-Leu-Gly-doxorubicin conjugate PK1 (currently under Phase II clinical evaluation) was selected as the model prodrug, and HPMA copolymer-cathepsin B as a model for the activating enzyme conjugate. Following polymer conjugation (yield of 30-35%) HPMA copolymer-cathepsin B retained ~20-25% enzymatic activity in vitro. To investigate pharmacokinetics in vivo, 125 I-labelled HPMA copolymer-cathepsin B was administered intravenously (i.v.) to B16F10 tumour-bearing mice. HPMA copolymercathespin B exhibited a longer plasma half-life (free cathepsin B t 1/2α = 2.8 h; bound cathepsin B t 1/2α = 3.2 h) and a 4.2-fold increase in tumour accumulation compared to the free enzyme. When PK1 (10 mg kg -1 dox-equiv.) was injected i.v. into C57 mice bearing subcutaneously (s.c.) palpable B16F10 tumours followed after 5 h by HPMA copolymer-cathepsin B there was a rapid increase in the rate of dox release within the tumour (3.6-fold increase in the AUC compared to that seen for PK1 alone). When PK1 and the PDEPT combination were used to treat established B16F10 melanoma tumour (single dose; 10 mg kg -1 dox-equiv.), the antitumour activity (T/C%) seen for the combination PDEPT was 168% compared to 152% seen for PK1 alone, and 144% for free dox. Also, the PDEPT combination showed activity against a COR-L23 xenograft whereas PK1 did not. PDEPT has certain advantages compared to ADEPT and GDEPT. The relatively short plasma residence time of the polymeric prodrug allows subsequent administration of polymer-enzyme without fear of prodrug activation in the circulation and polymer-enzyme conjugates have reduced immunogenicity. This study proves the concept of PDEPT and further optimisation is warranted. PDEPT concept using PK1 and HPMA copolymer-cathepsin B as a model combination. PK1 has proven ability to target solid tumours by the EPR effect (Seymour et al, 1994) with concomitant renal elimination resulting in low blood levels within 1-5 h in animals and man (Seymour et al, 1990;Vasey et al, 1999). HPMA copolymer-cathepsin B ( Figure 2B) was selected for PK1 activation as the PK1 Gly-Phe-Leu-Gly polymer-dox linker was designed to permit intralysosomal dox liberation due to action of the lysosomal cysteine proteases (Duncan et al, 1984). First it was necessary to prepare an HPMA copolymer-cathepsin B conjugate that would retain sufficient enzyme activity after conjugation. Activity was monitored in vitro using a low molecular weight substrate N-Benzoyl-Phe-Val-Arg-p-nitroanilide hydrochloride (Bz-Phe-Val-Arg-NAp) and the polymeric substrate PK1. The biodistribution of 125 I-labelled HPMA copolymer-cathepsin B and 125 I-labelled cathepsin B was assessed in mice bearing subcutaneous (s.c.) B16F10 tumours and this model wa...

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“…The activity of L-asparaginase modified with PNP-PEG was much higher (modification, 30%; residual activity, 65%) than that of the enzyme modified to the same degree with CN-PEG (modification, 25%; residual activity, 25%) or CNbisPEG (modification, 35%; residual activity, 30%) [4,16]. In the case of further modification, the residual activity of L-asparaginase modified with PNP-PEG was higher (modification, 55%; residual activity, 33%) than that of CN-PEG (modification, 52%; residual activity, -mil%) or that of CN-bisPEG (modification, 57%; residual activity, 11%) [4,16].…”

Section: Discussionmentioning

confidence: 91%

“…For reduction of its immunogenicity, an enzyme is often covalently coated with monomethoxypolyethylene glycol (PEG) [1,2] . Moreover, several proteins modified with PEG show enhanced resistance against proteolytic digestion and thus an extended circulation life in the blood [3][4][5]. Especially PEG-modified L-asparaginase from E. coli [6,7] was developed for therapy of lymphoblastic leukemia, and similarly modified adenosine deaminase from bovine intestine [8] was prepared for therapy of immunodeficiency diseases.…”

mentioning

confidence: 99%

A Simple and Efficient Method for Preparation of Monomethoxypolyethylene Glycol Activated with p-Nitrophenylchloroformate and Its Application to Modification of L-Asparaginase.

Kito

Miron

Wilchek

et al. 1996

J. Clin. Biochem. Nutr.

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Modification of E. coli L-asparaginase with polyethylene glycol: Disappearance of binding ability to anti-asparaginase serum

Cited by 117 publications

References 14 publications

Preparation of polyethylene glycol‐modified streptokinase with disappearance of binding ability towards anti‐serum and retention of activity

Preparation of polyethylene glycol‐modified streptokinase with disappearance of binding ability towards anti‐serum and retention of activity

PDEPT: polymer-directed enzyme prodrug therapy

A Simple and Efficient Method for Preparation of Monomethoxypolyethylene Glycol Activated with p-Nitrophenylchloroformate and Its Application to Modification of L-Asparaginase.

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