Raymond A. Firestone

Cathepsin B-sensitive dipeptide prodrugs. 1. A model study of structural requirements for efficient release of doxorubicin

A series of lysosomal protease-sensitive peptides attached to doxorubicin (DOX) was prepared as model substrates for internalizing anticancer immunoconjugates and potential antimetastasis prodrugs. Rates of cathepsin B-mediated release of free drug was measured for each, and human plasma stabilities for representative examples.

Cathepsin B-sensitive dipeptide prodrugs. 2. Models of anticancer drugs paclitaxel (Taxol®), mitomycin C and doxorubicin

Substrates containing doxorubicin (DOX), paclitaxel (taxol), and mitomycin C (MMC) attached to the cathepsin B-sensitive dipeptide Phe-Lys via a self-immolative spacer were prepared as model compounds for internalizing anticancer immunoconjugates. Cathepsin B-mediated release rates of free drug, rat liver lysosomal susceptibility and human plasma stability were measured for each.

Doxorubicin immunoconjugates containing bivalent, lysosomally-cleavable dipeptide linkages

Bivalent doxorubicin (DOX)-dipeptides (16a-c) were prepared and conjugated to the monoclonal antibody BR96. The dipeptides are cleaved by lysosomal proteases following internalization of the resulting immunoconjugates. Conjugate 18b demonstrated antigen-specific in vitro tumor cell killing activity (IC(50)=0.2 microM) that was equipotent to DOX with a near doubling of drug molecules/MAb. Size exclusion chromatography showed 18b to be a noncovalent dimer that was formed immediately upon conjugation.

Synthesis of an Immunoconjugate of Camptothecin

The first immunoconjugate of camptothecin has been synthesized wherein the drug is attached to the tumor-recognizing antibody BR96 via a Cathepsin B cleavable linker. Endocytosis of the immunoconjugate upon binding to the tumor cell followed by enzymatic cleavage of the linker inside the endosome ensures tumor-specific release of the drug. In this way, it is hoped that the dose-limiting side effects associated with camptothecin can be eliminated while the antitumor activity is preserved.

Synthesis and antitumor activity of the immunoconjugate BR96-Dox

Abstract BR96-Dox is an immunoconjugate (IC) in which doxorubicin (8 equivalents) is linked via an acid-labile hydrazone to the chimeric MAb BR96. It binds to a modified Le y Ag on tumor cells, which then internalize it via endocytosis into lysosomes. There, the acidic milieu hydrolyzes the hydrazone link, releasing free Dox. In vivo, it is more active and less toxic than untargeted Dox, producing complete remissions and many cures of subcutaneous human breast, lung and colon tumors, as well as disseminated lung tumors. In vivo, only BR96 + and not BR96 − tumors respond, and ICs with nonbinding Abs are inactive.

BR96 conjugates of highly potent anthracyclines.

The 6-maleimidocaproylhydrazone derivatives of highly potent antitumor agents 5-Diacetoxypentyldoxorubicin and Morpholinodoxorubicin were synthesized and conjugated to monoclonal antibody BR96 and control IgG. Immunoconjugate molar ratios were generally 7.5-8.5, and dimer aggregate levels were low. The linkers released parent drug at lysosomal pH 5, while they remained stable at neutral pH. BR96 conjugates were highly potent and antigen specific in vitro. The BR96-DAPDOX conjugate demonstrated an IC(50) of 0.03 micrometer and was at least 300-fold more potent than a non-binding IgG-DAPDOX control conjugate.

Volume of concert and heavy atom effects in Diels-Alder reaction mechanisms

Abstract The role of heavy atom effects (HAE) in Diels-Alder (DA) reactions is proposed in support of a stepwise-diradical mechanism. The presence of heavy atoms in diene or dienophile causes cycloadditions that are normally stereospecific in the dienophile to become nonstereospecific, because the initially-formed singlet diradical undergoes ISC to the triplet which can rotate but cannot ring close until a second ISC back to singlet. A concerted mechanism cannot accommodate these HAE. The dimerization of chloroprene, which had been analyzed via a dual mechanism based on activation-volume differences among the products, is now seen as unified in mechanism.

Synthesis and release of doxorubicin from a cephalosporin based prodrug by a β-lactamase-immunoconjugate

Abstract A cephalosporin based prodrug of doxorubicin has been synthesized which effciently releases doxorubicin in the presence of an immunoconjugate consisting of a β-lactamase-MAb.

The in vitro effects of three lysosomotropic detergents against three human tumor cell lines

Three lysosomotropic detergents, N-dodecylimidazole (NDI) 1, and two new analogues, serine dodecylamide (SDA) 2 and O-methyl serine dodecylamide (MSD) 3, were tested against three human tumor cell lines in vitro: HCT116, RCA and MCF-7. All three demonstrated dose-related cytotoxicity which, except for a few cases, was also dependent on incubation time, and visually resembled cell death by apoptosis.

Reversal of doxorubicin resistance and catalytic neutralization of lysosomes by a lipophilic imidazole

A number of lipophilic nitrogenous bases, designed to act as membrane-active, catalytic proton transfer agents, were tested for their ability to neutralize the acidity of lysosomes, a model for other acidic intracellular vesicles involved in drug sorting. The most successful of these, an imidazole 1, caused a 1.7 unit rise in lysosomal pH of RAW cells at 100 microM, compared to a 0.2 and 1.4 unit rise for ammonium chloride at 100 microM and 10 mM, respectively. Compound 1 also exhibited potent reversal of doxorubicin (DOX) resistance in the HCT116-VM46 cell line by a factor of 14 over the sensitive strain, and superior to that of widely used verapamil (VRP) by a factor of 1.75 at 20 microM. It also has antiviral properties, and potential applications in other lysosome-related areas such as immunotoxin potentiation and the control of bacterial toxins, immune response, prion replication, malaria and intralysosomal microorganisms.