N-Succinyl-(beta-alanyl-L-leucyl-L-alanyl-L-leucyl)doxorubicin: an extracellularly tumor-activated prodrug devoid of intravenous acute toxicity.

Intravenous administration of N-(beta-alanyl-L-leucyl-L-alanyl-L-leucyl)doxorubicin (4) induces an acute toxic reaction, killing animals in a few minutes. This results from its positive charge at physiological pH combined with its propensity to form large aggregates in aqueous solutions. Negatively charged N-capped versions of 4 such as the succinyl derivative 5 can be administered by the iv route at more than 10 times the LD(50) of doxorubicin without inducing the acute toxic reaction, and they are active in vivo.

Extracellularly tumor-activated prodrugs for the selective chemotherapy of cancer: application to doxorubicin and preliminary in vitro and in vivo studies.

Oligopeptidic derivatives of anthracyclines unable to penetrate cells were prepared and screened for their stability in human blood and their reactivation by peptidases secreted by cancer cells. N-beta-alanyl-L-leucyl-L-alanyl-L-leucyl-doxorubicin was selected as a new candidate prodrug. The NH2-terminal beta-alanine allows a very good blood stability. A two-step activation by peptidases found in conditioned media of cancer cells ultimately yields N-L-leucyl-doxorubicin. In vitro, when MCF-7/6 cancer cells are exposed to the prodrug, they accumulate about 14 times more doxorubicin than MRC-5 normal fibroblasts, whereas when exposed to doxorubicin the uptake is slightly higher in fibroblasts than in MCF-7/6 cells. This increased specificity of the prodrug over doxorubicin was confirmed in cytotoxicity assays using the same cell types. In vivo, the prodrug proved about nine times less toxic than doxorubicin in the normal mouse and also much more efficient in two different experimental chemotherapy models of human breast tumors.

Chemical pathways of peptide degradation. VII. Solid state chemical instability of an aspartyl residue in a model hexapeptide.

The chemical stability of an Asp-hexapeptide (Val-Tyr-Pro-Asp-Gly-Ala) in lyophilized formulations was evaluated as a function of multiple formulation variables--specifically pH of the bulk solution, temperature, moisture content, and type of bulking agent (amorphous vs. crystalline). The disappearance of the starting Asp-hexapeptide in the solid state conformed to pseudo-first-order reversible kinetics. This type of degradation profile was accounted for by the product distribution. The factorial experimental design of this study allowed statistical analysis of the effects of individual formulation variable (main effects) as well as those of two-factor interactions on the degradation of the Asp-hexapeptide. Analysis of Variance (ANOVA) calculations of the main effects indicated that while the influence of pH of the starting solution was not statistically significant, residual moisture level, temperature, and, especially, type of bulking agent had a significant impact on the solid state chemical reactivity of the hexapeptide. Furthermore, depending on which type of excipient was used in the lyophilized formulations, residual moisture level and temperature could be important stability variables. These types of factorial experiments have proven to be useful in the rapid identification of significant formulation variables in a given system and, consequently, in optimization of formulations.

Chemical pathways of peptide degradation. VI. Effect of the primary sequence on the pathways of degradation of aspartyl residues in model hexapeptides.

The influence of the primary sequence on the degradation of Asp4 residues (e.g., formation of the cyclic imide and Asp-X and/or X-Asp amide bond hydrolysis) was investigated using Val-Tyr-Y-Asp-X-Ala hexapeptides. These reactions were proposed to involve cyclization, which would duly be sensitive to steric hindrance. The effects on the rates of individual degradation routes and product distribution under both acidic and alkaline conditions were assessed upon substitutions made on the C-terminal side (X) and on the N-terminal side (Y) of the Asp residue. As expected, the rate of intramolecular formation of cyclic imide and, thus, the product yield were most affected by the size of the amino acid on the C-terminal side of the Asp residue. However, such structural changes had little or no impact on the rate of Asp-X and Y-Asp amide bond hydrolysis. In the former case, the substituted site was one atom removed from the reaction site, accounting for the diminished steric effect observed. As for the latter, the site of substitution was not a participant in the reaction itself, and hence, the rate was unperturbed by this modification. Placing Ser and Val C terminally to the Asp residue prompted racemization and peptide bond hydrolysis to occur under alkaline conditions. N-Terminal substitution of Pro with Gly had no effect on the rate of isomerization via cyclic imide formation but greatly enhanced the rate of Y-Asp amide bond hydrolysis.

Solid state chemical instability of an asparaginyl residue in a model hexapeptide.

The chemical stability of an Asn-hexapeptide (Val-Tyr-Pro-Asn-Gly-Ala) in lyophilized formulations was evaluated as a function of the pH of the bulk solution, temperature, and residual moisture content in a factorial study. Degradation pathways and product distribution in solid state were determined and characterized. It was shown in this study that the pH of the starting solution had a significant effect on the rate of deamidation and product distribution. In general, better stability for the Asn-hexapeptide was achieved at a slightly acidic pH range (3-5) in the solid state. The effects of residual moisture level and temperature on peptide stability proved to be less significant. A statistically significant two-factor interaction indicated that the pH of formulation solution determined the extent to which the peptide stability depends on moisture level and temperature. In general, the degradation of the Asn-hexapeptide in the solid state was similar to that observed in solution, except for the observation that no isoAsp-hexapeptide was detected at pH 5.0 in the solid state, whereas this was the major degradation product in solution.

Chemical pathways of peptide degradation. IV. Pathways, kinetics, and mechanism of degradation of an aspartyl residue in a model hexapeptide.

In this study the hexapeptide Val-Tyr-Pro-Asp-Gly-Ala (Asp-hexapeptide) was used as a model to investigate the kinetics of aspartate degradation in aqueous solution. The apparent rate of degradation of the Asp-hexapeptide was determined as a function of pH, buffer concentration, and temperature. At very acidic pH levels (0.3, 1.1, 1.5, 2.0, and 3.0), the apparent rate of degradation followed pseudo-first-order kinetics. In this pH region, the Asp-hexapeptide predominantly underwent specific acid-catalyzed hydrolysis of the Asp-Gly amide bond (Asp-X hydrolysis) to form a tetrapeptide (Val-Tyr-Pro-Asp) and a dipeptide (Gly-Ala). In addition, parallel formation of a cyclic imide intermediate could be observed, although no iso-Asp-hexapeptide was detected. At pH 4.0 and 5.0, the Asp-hexapeptide simultaneously isomerized via the cyclic imide to form the iso-Asp-hexapeptide and underwent Asp-X hydrolysis to produce the cleavage products. The pH-rate profiles (pH 0.3-5.0) for the Asp-X hydrolysis and the formation of cyclic imide revealed that the degree of ionization of the carboxylic acid side chain of Asp residue significantly altered the rate of reaction, with the ionized form being more reactive than the unionized form. Little or no buffer catalysis was observed for either pathway. Solvent isotope experiments were used to probe the mechanism of the Asp-X hydrolysis reaction. At pH values above 6.0, the apparent rate of degradation of the Asp-hexapeptide followed pseudo-first-order reversible kinetics, with the iso-Asp-hexapeptide being the only observed product (isomerization).(ABSTRACT TRUNCATED AT 250 WORDS)