Sulfhydryl selective reactions were explored to conjugate oligomers of a peptidomimetic integrin
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3 antagonist,4-[2-(3,4,5,6-tetrahydropyrimidine-2-ylamino)ethyloxy]
benzoyl-2-(
S)-aminoethylsulfonylamino-
![](/images/gifchars/<font color=)
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body (MoA
b) to increase integrin
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3 receptor-
binding avidity. To generate sulfhydryl groups,
N-succinimidyl-
S-acetylthioacetate (SATA) was conjugated to
both MoA
b and IA. Sulfhydryl groups were thengenerated upon the deacetylation of the protecting acetyl group from the
S-acetylthioacetato (ATA) moiety ofMoA
b-(ATA)
n or IA-ATA with 0.02 M hydroxylamine in the presence of 1 mM EDTA at pH 7.2. The majorfocus was on optimizing the reaction concentrations, molar ratios, and reaction pH to conjugate high levels ofIA-(A-SH) to MoA
b-(A-SH)
n without causing the inter- and intramolecular cross-lin
king of MoA
b. Stepwisereactions of MoA
b-(A-SH)
n (15
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M MoA
b) with a homo
bifunctional cross-linker, 1,8-
bis(maleimido)diethyleneglycol (BM[PEO]
2) at a >50× molar excess to the -SH, followed
by the reaction of the purified product MoA
b-(A-
S-succinimidomaleimido-[PEO]
2)
n with IA-(A-SH) at pH 7.2 afforded monomeric MoA
b-(A-
S-succinimido-[PEO]
2-succinimido-
S-A-IA)
n with <10% high molecular weight oligomeric MoA
b. Monomeric MoA
b-(A-
S-S-[PEO]
2-S-
S-A-IA)
10 (MoA
b-IA
10) radiola
beled with
111In using 2-(
p-isothiocyanato
benzyl)cyclohexyl-DTPAand with
125I using the Iodogen method showed >70%
binda
bility to 0.4
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M
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3. When injected iv to nudemice with the receptor-positive M21 tumor, MoA
b-IA
10 radiola
beled with
both
111In and
125I accumulated rapidlyand was retained in the tumor for a 44 h period while the radioactivity cleared rapidly from the
blood, there
byresulting in increasing tumor-to-
blood ratios over time. The tumor uptake was similar
between the
125I la
bel andthe
111In la
bel for a 44 h period. In contrast, the
blood radioactivity was lower,
but liver and other organ uptakeswere much higher for the
111In la
bel than for the
125I. The
111In la
bel produced higher tumor-to-
blood ratios
butmuch lower tumor-to-organ ratios than the
125I. The rapid
blood clearance, a short peak tumor uptake time, anda low peak tumor uptake value with prolonged tumor retention of this macromolecule appear to support a hypothesisthat MoA
b-IA
10 primarily
binds to
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3 receptors on angiogenic vessels,
but not on the tumor. This hypothesiswas su
bstantiated
by the fluorescence microscopic analysis of FITC-MoA
b-IA
10, which showed that FITC-MoA
b-IA
10 outlined neovasculatures
but not tumor cells at 4 and 21 h ex vivo. Additional proof was o
bservedwhen
blood vessels outlined with rhodamine-lectin, which specifically
binds to
blood vessels, were superimposa
bleon neovasculatures outlined with FITC-MoA
b-IA
10.