Synthesis and in vitro biochemical evaluation of oxime bond-linked daunorubicin–GnRH-III conjugates developed for targeted drug delivery

Abstract

Gonadotropin releasing hormone-III (GnRH-III), a native isoform of the human GnRH isolated from sea lamprey, specifically binds to GnRH receptors on cancer cells enabling its application as targeting moieties for anticancer drugs. Recently, we reported on the identification of a novel daunorubicin–GnRH-III conjugate (GnRH-III–[4Lys(Bu), 8Lys(Dau=Aoa)] with efficient in vitro and in vivo antitumor activity. To get a deeper insight into the mechanism of action of our lead compound, the cellular uptake was followed by confocal laser scanning microscopy. Hereby, the drug daunorubicin could be visualized in different subcellular compartments by following the localization of the drug in a time-dependent manner. Colocalization studies were carried out to prove the presence of the drug in lysosomes (early stage) and on its site of action (nuclei after 10 min). Additional flow cytometry studies demonstrated that the cellular uptake of the bioconjugate was inhibited in the presence of the competitive ligand triptorelin indicating a receptor-mediated pathway. For comparative purpose, six novel daunorubicin–GnRH-III bioconjugates have been synthesized and biochemically characterized in which 6Asp was replaced by D-Asp, D-Glu and D-Trp. In addition to the analysis of the in vitro cytostatic effect and cellular uptake, receptor binding studies with 125I-triptorelin as radiotracer and degradation of the GnRH-III conjugates in the presence of rat liver lysosomal homogenate have been performed. All derivatives showed high binding affinities to GnRH receptors and displayed in vitro cytostatic effects on HT-29 and MCF-7 cancer cells with IC50 values in a low micromolar range. Moreover, we found that the release of the active drug metabolite and the cellular uptake of the bioconjugates were strongly affected by the amino acid exchange which in turn had an impact on the antitumor activity of the bioconjugates.

Enhanced In Vitro Antitumor Activity of GnRH-III-Daunorubicin Bioconjugates Influenced by Sequence Modification

“…Ligand competition assays with radiolabeled triptorelin were performed to evaluate the binding affinity of K2 and 10 to GnRH-RI expressed on human pituitary and human prostate cancer cells as reported earlier [34,40,41,42]. Tissue samples of human prostate cancer cells were obtained from a patient at the time of initial surgical treatment and normal human pituitary tissue (anterior lobe) derived by autopsy.…”

“…Apart from that, triptorelin was radioiodinated using the chloramine-T method, followed by purification by RP-HPLC [34,40,41,44]. To determine the binding affinities of the nonradio-labeled GnRH-III bioconjugates to GnRH-RI, the displacement of [ 125 I]-GnRH-I-[ 6 d -Trp] was studied by an in vitro ligand competition assay [34,40,41,42]. Hence, membrane homogenates which contained 50–160 mg protein were incubated in duplicate or triplicate with 60–80,000 cpm [ 125 I]-GnRH-I-[ 6 d -Trp] and increasing concentration (1 pM–1 µM) of nonradioactive bioconjugates as competitive binders in 150 mL binding buffer.…”

In Vivo Tumor Growth Inhibition and Antiangiogenic Effect of Cyclic NGR Peptide-Daunorubicin Conjugates Developed for Targeted Drug Delivery

“…In the last decades, huge efforts have been made to develop new strategies for the improvement of drug delivery and penetration into tumors [1][2][3]. Targeted tumor therapy is one of the most promising approaches that may provide a real break-through in this field [4].…”

Synthesis and biochemical evaluation of GnRH-III-drugconjugates

“…The cytostatic effect was determined by Alamar blue® assay and compared to the control compoundsK1 and K2 (Scheme 1). Figure 1A) [2,3]. To interpret these results, we performed additional studies of P19-H in direct comparison with our lead compound K2.…”

“…Due to that, a variety of GnRH-III-drugconjugates have been designed and characterized in our laboratories, in which the anthracycline daunorubicin (Dau) was linked to GnRH-III via oxime bond by insertion of an aminooxyacetyl moiety [1]. To achieve an improved antitumoractivity, we synthesized a set of oxime-linked GnRH-III-Dau conjugates containing different unnaturalamino acids within the sequence and studied their in vitro anticancer activity [2,3]. The best compoundswere chosen for further biochemical evaluation and as targeting moiety of novel drug conjugates containing a self-immolative p-aminobenzyloxycarbonyl (PABC) spacer between a cathepsin B cleavable dipeptide (Val-Ala or Val-Cit) and the drug Dau or paclitaxel (PTX).…”