PSMA-Targeted Stably Linked “Dendrimer-Glutamate Urea-Methotrexate” as a Prostate Cancer Therapeutic

One of the important criteria for achieving efficient nanoparticle-based targeted drug delivery is that the drug is not prematurely released at off-target sites. Here we report the preclinical evaluation of a serum-stable dendrimer-based drug conjugate capable of actively targeting into prostate cancer (PC) cells, delivered through the prostate-specific membrane antigen (PSMA). Multiple molecules of PSMA-binding small molecule glutamate urea (GLA; targeting agent) and the drug methotrexate (MTX) were conjugated to generation 5 PAMAM dendrimer (G5) through Cu-free “click” chemistry. The GLA was conjugated through a stable amide bond, and the MTX was conjugated either through ester (Es)- or amide (Am)-coupling, to generate G5-GLA_<i>m</i>-(Es)­MTX_<i>n</i> and G5-GLA_<i>m</i>-(Am)­MTX_<i>n</i>, respectively. In serum-containing medium, free MTX was slowly released from “G5-GLA_<i>m</i>-(Es)­MTX_<i>n</i>”, with ∼8% MTX released from the dendrimer in 72 h, whereas the MTX on G5-GLA_<i>m</i>-(Am)­MTX_<i>n</i> was completely stable. The G5-GLA_<i>m</i>-(Am)­MTX_<i>n</i> bound and internalized into PSMA-expressing LNCaP cells, but not into PSMA-negative PC3 cells. The conjugate-inhibited recombinant dihydrofolate reductase and induced potent cytotoxicity in the LNCaP cells, but not in the PC3 cells. Similar to the action of free GLA, stable amide-linked dendrimer-GLA was capable of inhibiting the enzyme N-acetylated α-linked acidic dipeptidase (NAALADase) activity of PSMA. The G5-GLA_<i>m</i>-MTX_<i>n</i> may serve as a serum-stable nanoparticle conjugate to specifically and effectively target and treat PSMA-overexpressing prostate tumors

Polyvalent Dendrimer-Methotrexate as a Folate Receptor-Targeted Cancer Therapeutic

Our previous studies have demonstrated that a generation 5 dendrimer (G5) conjugated with both folic acid (FA) and methotrexate (MTX) has a higher chemotherapeutic index than MTX alone. Despite this, batch-to-batch inconsistencies in the number of FA and MTX molecules linked to each dendrimer led to conjugate batches with varying biological activity, especially when scaleup synthesis was attempted. Since the MTX is conjugated through an ester linkage, there were concerns that biological inconsistency could also result from serum esterase activity and differential bioavailability of the targeted conjugate. In order to resolve these problems, we undertook a novel approach to synthesize a polyvalent G5–MTX_<i>n</i> conjugate through click chemistry, attaching the MTX to the dendrimer through an esterase-stable amide linkage. Surface plasmon resonance binding studies show that a G5–MTX₁₀ conjugate synthesized in this manner binds to the FA receptor (FR) through polyvalent interaction showing 4300-fold higher affinity than free MTX. The conjugate inhibits dihydrofolate reductase, and induces cytotoxicity in FR-expressing KB cells through FR-specific cellular internalization. Thus, the polyvalent MTX on the dendrimer serves the dual role as a targeting molecule as well as a chemotherapeutic drug. The newly synthesized G5–MTX_<i>n</i> conjugate may serve as a FR-targeted chemotherapeutic with potential for cancer therapy