Development of a theranostic agent for image-guided delivery of temozolomide to SSTR2 expressing cells
Introduction
Temozolomide (TMZ) is a DNA damaging agent that produces high response rates in neuroendocrine tumors (NETs) when the DNA repair enzyme, known as O6-methylguanine DNA methyltransferase (MGMT), is inactivated. When given at high doses, TMZ therapy can exhaust MGMT activity and its associated resistance mechanisms but also produces dose-limiting toxicities. Since nearly all NETs overexpress the somatostatin receptor subtype 2 (SSTR2), we hypothesized that a receptor-targeted TMZ analog could produce high intratumoral drug concentrations while avoiding systemic toxicity. Accordingly, we converted the clinically approved radiotracer 68Ga-DOTA-TOC into a radiolabeled peptide-drug conjugate (PDC) for SSTR2-targeted delivery of TMZ and report on receptor-binding properties, pharmacokinetics, and tissue biodistribution.
Methods
The PDC was synthesized by conjugating TMZ to a DOTA analog, and then adding the payload moiety to TOC on solid-phase. The resulting product, tumor-targeted TMZ (ttTMZ), was labeled with 67Ga and evaluated using radioligand assays to determine binding affinity, selectivity, and internalization properties. To evaluate DNA-damaging effects, an alkaline comet assay and ɣH2AX immunostaining were performed after exposure of the IMR-32 cells (SSTR2+) to the PDC in the presence or absence of octreotide blocking. To study the effect of the ttTMZ on the MGMT levels, IMR-32 cells (SSTR2+/MGMT+) were treated with increasing concentrations of ttTMZ, TMZ, and O6-Benzylguanine (O6BG; a pseudo-substrate for MGMT) and the cell lysate was analyzed by western blot. To investigate SSTR2 targeting in vivo, positron emission tomography (PET) was performed 1 h after injection of 68Ga-ttTMZ in mice that were bilaterally implanted with HCT116-WT (SSTR2-) and HCT116-SSTR2 (SSTR2+) cells. To further evaluate specificity and biodistribution at pharmacologically active drug concentrations, we performed a dose-escalation study with and measured biodistribution as injected activity per gram of tissue (%IA/g).
Results
ttTMZ was produced with (radio)chemical purity >90%. Cell-based experiments showed that binding of 67Ga-ttTMZ was similar to 67Ga-DOTA-TOC (Kd= 5.98 ± 0.96 nmol/L, and 4.68 ± 0.7 nmol/L, respectively). In HCT116-SSTR2 cells that highly overexpress SSTR2, the total radioactive uptake ranged from 9.57-12.67% for both 67Ga-ttTMZ and 67Ga-DOTA-TOC and decreased as a function of SSTR2 expression (1.37-2.75% and 1.19-2.72% in IMR-32 and NCI-H69 cells, respectively). Binding was reduced by nearly 90% when co-incubated with octreotide. Acid-wash experiments revealed tracer internalization that is consistent with the agonist properties of TOC. Mechanistic assays showed that ttTMZ caused DNA damage that was similar to TMZ, with the effect being significantly reduced (P<0.05) in the presence of octreotide blocking. Western blot analysis showed that ttTMZ reduces MGMT levels in a dose-dependent manner compared to untreated cells and similar to the control agents. PET/CT imaging demonstrated strong PDC uptake in SSTR2+ tumors at 1 h p.i., whereas receptor-negative tumors had no detectable signal. Dose escalation showed that receptor-mediated uptake was retained in the presence of a 5-fold increase in injected mass. Although examination of receptor-negative tumors and normal tissues revealed a dose-dependent increase in uptake, the amount of drug in those tissues (except for kidneys) was markedly lower than in SSTR2-expressing tumors at the corresponding dose level.
Conclusion
We developed a novel drug conjugate that selectively causes toxicity and MGMT depletion in SSTR2-expressing cells. Direct radiolabeling of ttTMZ provided quantitative evidence of SSTR2 mediated binding and biodistribution analysis in animal models. These findings demonstrate the utility of developing a radiolabeled drug conjugate and may guide optimization strategies.