Pharmacokinetic studies in mice with ICI D0490, a novel recombinant ricin A-chain immunotoxin

Calvete, J. A.; Newell, David R.; Charlton, C. J.; Wright, Andrew Martin

doi:10.1038/bjc.1993.243

Cited by 8 publications

(3 citation statements)

References 16 publications

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“…Thorpe, both in preclinical and clinical studies (14,17,18), showed that IT with a methyl-hindered linker had greater antitumor activity, when given in equimolar dose, than IT with unhindered linkers. Excellent in vivo stability as well as favorable pharmacokinetics were also shown in a colorectal tumor-directed immunotoxin (35). Other authors disagreed; studying the in vivo therapeutic efficacy of two anti CD7-saporin ITs constructed with either hindered (SMPT) or unhindered (SPDP) linkers, they were unable to show any significant difference (36).…”

Section: Discussionmentioning

confidence: 99%

“…Ethyl S-Acetyl-3-mercapto-3-phenylpropionthioimidate ester hydrochloride (Ph-AMPT, 10): yield 1.1 g (84%); mp 85 °C; 1 H NMR (CDCl 3 ) δ 7.6 (m, 5H, Ar-H), 4.9 (t, 1H, CH), 3.9-3.3 (m, 4H, -SCH 2 CH 3 , and CH 2 ), 2.5 (s, 3H, SAc), 1.6 (t, 3H, -SCH 2 CH 3 ); IR (KBr) cm -1 3300-2400 (NH), 1730 (CdO), 1610 (CdN), 1360 (SAc); MS-EI, m/z (relative intensity) 268 (M + , 8), 130 (100), 103 (40), 77 (35), 43 (38).…”

Section: -[(2-cyano-1-phenylethyl)dithio]benzamide (8)mentioning

confidence: 99%

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New Coupling Reagents for the Preparation of Disulfide Cross-Linked Conjugates with Increased Stability

Berlier¹,

Dosio²,

Brusa³

et al. 1997

Bioconjugate Chem.

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To improve the in vivo stability of disulfide-linked immunotoxins (ITs), a series of sterically hindered cross-linking reagents were designed and synthesized. These ligands are characterized by a thioimidate group linked to an S-acetyl thiol or a substituted aryldithio group. To select the reagent of choice, several aryldithio thioimidates, substituted with a methyl or a phenyl group adjacent to the disulfide, were analyzed in thiol-disulfide exchange reactions. Also analyzed were the following: (i) the stability and solubility of the linkers in aqueous solution, (ii) the rate of protein derivatization, and (iii) the steric hindrance due to methyl or phenyl group substituents toward cleavage of the disulfide bond by glutathione. Ethyl S-acetyl 3-mercaptobutyrothioimidate (M-AMPT) was chosen as reagent to prepare two types of stable disulfide-containing AR-3-gelonin conjugates (IT2 and IT3). IT2 was prepared by a 3-(4-carboxamidophenyldithio)propionthioimidate (CDPT)-derivatized antibody coupled to the M-AMPT-derivatized gelonin to afford a conjugate characterized by the presence of a methyl group adjacent to the sulfide bond. In the IT3 conjugate, an M-AMPT-derivatized toxin was coupled to the antibody thiolated with M-AMPT and then activated with Ellman's reagent (DNTB). The in vitro and in vivo stabilities of the three immunoconjugates were assayed, respectively, (i) by adding an excess of glutathione and monitoring protein release and (ii) by studying their pharmacokinetic behaviors. The specificity and cytotoxicity of all ITs were analyzed on target and unrelated cell lines, and no significant differences in activity were observed. IT3, consisting of a symmetrical dimethyl-substituted disulfide bond, was substantially more stable in vivo (t1/2 beta = 88.3 h) than the corresponding IT2, characterized by a disulfide-protected monomethyl substituent bond (t1/2 beta = 60.2 h) compared to the unhindered conjugate IT1 (t1/2 beta = 27.9 h). This family of cross-linking reagents therefore offers advantages, such as minimal perturbation of the protein structure and controlled reactivity due to the thioimidate moiety, as well as the capacity to yield immunotoxins possessing substantial stability in vivo.

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Section: Discussionmentioning

confidence: 99%

Section: -[(2-cyano-1-phenylethyl)dithio]benzamide (8)mentioning

confidence: 99%

New Coupling Reagents for the Preparation of Disulfide Cross-Linked Conjugates with Increased Stability

Berlier¹,

Dosio²,

Brusa³

et al. 1997

Bioconjugate Chem.

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“…In different B cell lines we investigated which factors contribute most to the cytotoxic activity of CD22-ricin A IT. For the production of the IT we used aglycosyl recombinant ricin A that already has been used for in vivo studies in mice [22].…”

Section: Discussionmentioning

confidence: 99%

Cytotoxic Potency of CD22‐Ricin A Depends on Intracellular Routing Rather than on the Number of Internalized Molecules

et al. 1995

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van Horssen PJ, van Oosterhout YJM, de Witte T, Preijers FWMB. Cytotoxic Potency of CD22-Ricin A Depends on Intracellular Routing Rather than on the Number of Internalized Molecules. Scand J Immunol 1995;41:559-65 Cytotoxicity of immunotoxins (ITs) varies considerably depending on factors like the capability of the target antigen to internalize IT molecules, intracellular processing and routing of the IT. We studied factors that may influence cytotoxicity of CD22-ricin A IT to several B cell lines. The antigen density varied from 5.9xlO3 to 6.0xlO4 molecules/cell. The ID 50, determined by protein synthesis inhibition, varied from 2.1xl0-12 to 3 .8 x l0 " n M IT in absence and from 2 .8 x l0 -14 M to 5 .2 x l0 " 12 M IT in presence of the cytotoxicity enhancer N H 4CI (6 mM). In absence as well as in presence of N H 4CI no correlation could be found between antigen density and ID 50. No relation was observed either with the rate of cytotoxicity. Even in cell lines with a low antigen density, such as KM3, protein synthesis was quickly inhibited. In order to investigate whether the cytotoxicity was dependent on the number of internalized molecules the kinetics of internalization and exocytosis of degraded 125I-labelled CD22 molecules were studied. After 24h the number of internalized CD 22 molecules was highest in Ramos (154,500), followed by Daudi (110,300) and KM3 (69,900). However, despite the higher internalization rate of Daudi the rate of cytotoxicity of 10" s M IT was comparable with KM3. N H 4CI did not influence the number of internalized molecules but postponed degradation of CD22. In conclusion, CD22-ricin A is a very potent and fast acting IT even for elimination of target cells that express low numbers of antigen. These results may have implication for treatment of different B cell malignancies with CD22-ricin A.

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Highly potent CD22-recombinant ricin A results in complete cure of disseminated malignant B-cell xenografts in SCID mice but fails to cure solid xenografts in nude mice

et al. 1996

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The highly specific cytotoxic action of ribosome-inactivating protein (RIP) containing immunotoxins (ITs) makes IT therapy a promising approach to eliminating residual malignant cells. We investigated the cytotoxicity of the IT CD22-recombinant ricin A to the B-cell line Ramos in vitro and in vivo. Cytotoxicity of CD22-recombinant ricin A in vitro was very high as expressed by the very low 50% inhibition dose (ID50) of 3.5 x 10(-11) M. Cytotoxicity was increased 7 times in the presence of the cytotoxicity enhancer NH4Cl. The ultimate kill of Ramos cells by CD22-recombinant ricin A was high (2.7-log kill) and was increased strongly in the presence of NH4Cl (4.2-log kill). Anti-tumor activity in vivo was investigated by i.v. treatment of solid s.c. Ramos xenografts in nude BALB/c mice. A single dose did not inhibit tumor growth. Treatment on 5 consecutive days resulted in evident tumor reduction. In one mouse, tumor could no longer be detected on the 6th day after starting treatment. However, after 8 days tumor volumes increased again. Antitumor activity was more pronounced in a disseminated tumor model in SCID mice. IT treatment (i.v.) 7 days after i.v. inoculation with Ramos cells resulted in cure of all mice. Non-specific toxicity was low. Alanine aminotransferase (ALAT) levels in serum were elevated temporarily. Serum values of gamma-glutamyl transferase (gamma-GT), bilirubin and creatinin did not change. Body weight was also transiently reduced. The LD50 in SCID mice after i.v. administration was high (0.626 mg IT per mouse). The clearance rate in SCID mice, as determined by ELISA, was biphasic.

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Pharmacokinetic studies in mice with ICI D0490, a novel recombinant ricin A-chain immunotoxin

Cited by 8 publications

References 16 publications

New Coupling Reagents for the Preparation of Disulfide Cross-Linked Conjugates with Increased Stability

New Coupling Reagents for the Preparation of Disulfide Cross-Linked Conjugates with Increased Stability

Cytotoxic Potency of CD22‐Ricin A Depends on Intracellular Routing Rather than on the Number of Internalized Molecules

Highly potent CD22-recombinant ricin A results in complete cure of disseminated malignant B-cell xenografts in SCID mice but fails to cure solid xenografts in nude mice

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