Enhanced Enzymatic Stability and Antitumor Activity of Daunorubicin-GnRH-III Bioconjugates Modified in Position 4

Abstract

Here, we report on the synthesis, enzymatic stability, and antitumor activity of novel bioconjugates containing the chemotherapeutic agent daunorubicin attached through an oxime bond to various gonadotropin-releasing hormone-III (GnRH-III) derivatives. In order to increase the enzymatic stability of the bioconjugates (in particular against chymotrypsin), (4)Ser was replaced by N-Me-Ser or Lys(Ac). A compound in which (4)Lys was not acetylated was also prepared, with the aim of investigating the influence of the free ε-amino group on the biochemical properties. The in vitro cytostatic effect of the bioconjugates was determined on MCF-7 human breast, HT-29 human colon, and LNCaP human prostate cancer cells by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. Their stability/degradation (1) in human serum, (2) in the presence of rat liver lysosomal homogenate, and (3) in the presence of digestive enzymes (trypsin, chymotrypsin, and pepsin) was analyzed by liquid chromatography in combination with mass spectrometry. The results showed that (1) all synthesized bioconjugates had in vitro cytostatic effect, (2) they were stable in human serum at least for 24 h, and (3) they were hydrolyzed in the presence of lysosomal homogenate. All compounds were stable in the presence of (1) pepsin and (2) trypsin (except for the (4)Lys containing bioconjugate). In the presence of chymotrypsin, all bioconjugates were digested; the degradation rate strongly depending on their structure. The bioconjugates in which (4)Ser was replaced by N-Me-Ser or Lys(Ac) had the highest enzymatic stability, making them potential candidates for oral administration. In vivo tumor growth inhibitory effect of two selected bioconjugates was evaluated on orthotopically developed C26 murine colon carcinoma bearing mice. The results indicated that the compound containing Lys(Ac) in position 4 had significantly higher antitumor activity than the parent bioconjugate.

Enhanced cellular uptake and in vitro antitumor activity of short-chain fatty acid acylated daunorubicin–GnRH-III bioconjugates

“…94.5%) concentration, respectively [15]. It has to be mentioned that [D-Lys 6 (Dau=Aoa)]-GnRH-I showed similar cellular uptake as the GnRH-III bioconjugates containing the butyrate isomers ( Fig.s 2C and 2D).…”

“…The results showed that the bioconjugate had significantly higher antitumor activity than the parent bioconjugate [15]. Furthermore, the concentration dependent cellular uptake of GnRH-III( 4 Lys(Ac), 8 Lys(Dau=Aoa)) on MCF-7 cells was significantly higher compared to the nonacetylated version [15].…”

“…The results showed that the bioconjugate had significantly higher antitumor activity than the parent bioconjugate [15]. Furthermore, the concentration dependent cellular uptake of GnRH-III( 4 Lys(Ac), 8 Lys(Dau=Aoa)) on MCF-7 cells was significantly higher compared to the nonacetylated version [15]. Taken together these previous findings and also considering the effect of SCFAs on tumor growth inhibition, it is assumed that SCFAs on the side chain of 4 Lys in GnRH-III( 4 Lys(X), 8 Lys(Dau=Aoa)) may increase not only the cellular uptake and enzymatic stability, but also the antitumor activity of the bioconjugates.…”

“…We have previously reported on a daunorubicin-GnRH-III bioconjugate in which 4 Ser was replaced by Lys(Ac) and daunorubicin was attached to 8 Lys through an oxime bond [15].…”

“…Moreover, the daunorubicin-GnRH-III bioconjugates must possess increased stability in the presence of digestive enzymes present in stomach and intestine in order to be suitable for oral administration. In our previous work, we reported that the bioconjugate GnRH-III( 4 Lys(Ac), 8 Lys(Dau=Aoa)) was stable in human serum and in the presence of pepsin and trypsin even after 24 h of incubation [15,22]. In the presence of α-chymotrypsin, the -3 Trp-4 Lys(Ac)-peptide bond was cleaved in the bioconjugate; however, the degradation rate was slower than in the case of -3 Trp- 4 Ser-in the parent bioconjugate.…”