Targeting of the Cholecystokinin-2 Receptor with the Minigastrin Analog 177Lu-DOTA-PP-F11N: Does the Use of Protease Inhibitors Further Improve In Vivo Distribution?

Patients with metastatic medullary thyroid cancer (MTC) have limited systemic treatment options. The use of radiolabeled gastrin analogs targeting the cholecystokinin-2 receptor (CCK2R) is an attractive approach. However, their therapeutic efficacy is presumably decreased by their enzymatic degradation in vivo. We aimed to investigate whether the chemically stabilized analog 177Lu-DOTA-PP-F11N (177Lu-DOTA-(dGlu)6-Ala-Tyr-Gly-Trp-Nle-Asp-Phe-NH2) performs better than reference analogs with varying in vivo stability, namely 177Lu-DOTA-MG11 (177Lu-DOTA-dGlu-Ala-Tyr-Gly-Trp-Met-Asp-Phe-NH2) and 177Lu-DOTA-PP-F11 (177Lu-DOTA-(dGlu)6-Ala-Tyr-Gly-Trp-Met-Asp-Phe-NH2), and whether the use of protease inhibitors further improves CCKR2 targeting. First human data on 177Lu-DOTA-PP-F11N are also reported. Methods: In vitro stability of all analogs was assessed against a panel of extra- and intracellular endoproteases, whereas their in vitro evaluation was performed using the human MTC MZ-CRC-1 and the transfected A431-CCK2R(+) cell lines. Biodistribution without and with the protease inhibitors phosphoramidon and thiorphan was assessed 4 h after injection in MZ-CRC-1 and A431-CCK2R(+) dual xenografts. Autoradiography of 177Lu-DOTA-PP-F11N (without and with phosphoramidon) and NanoSPECT/CT were performed. SPECT/CT images of 177Lu-DOTA-PP-F11N in a metastatic MTC patient were also acquired. Results:natLu-DOTA-PP-F11N is less of a substrate for neprilysins than the other analogs, whereas intracellular cysteine proteases, such as cathepsin-L, might be involved in the degradation of gastrin analogs. The uptake of all radiotracers was higher in MZ-CRC-1 tumors than in A431-CCK2R(+), apparently because of the higher number of binding sites on MZ-CRC-1 cells. 177Lu-DOTA-PP-F11N had the same biodistribution as 177Lu-DOTA-PP-F11; however, uptake in the MZ-CRC-1 tumors was almost double (20.7 ± 1.71 vs. 11.2 ± 2.94 %IA [percentage injected activity]/g, P = 0.0002). Coadministration of phosphoramidon or thiorphan increases 177Lu-DOTA-MG11 uptake significantly in the CCK2R(+) tumors and stomach. Less profound was the effect on 177Lu-DOTA-PP-F11, whereas no influence or even reduction was observed for 177Lu-DOTA-PP-F11N (20.7 ± 1.71 vs. 15.6 ± 3.80 [with phosphoramidon] %IA/g, P < 0.05 in MZ-CRC-1 tumors). The first clinical data show high 177Lu-DOTA-PP-F11N accumulation in tumors, stomach, kidneys, and colon. Conclusion: The performance of 177Lu-DOTA-PP-F11N without protease inhibitors is as good as the performance of 177Lu-DOTA-MG11 in the presence of inhibitors. The human application of single compounds without unessential additives is preferable. Preliminary clinical data spotlight the stomach as a potential dose-limiting organ besides the kidneys.

The Intrinsic Pepsin Resistance of Interleukin-8 Can Be Explained from a Combined Bioinformatical and Experimental Approach.

Interleukin-8 (IL-8, CXCL8) is a neutrophil chemotactic factor belonging to the family of chemokines. IL-8 was shown to resist pepsin cleavage displaying its high resistance to this protease. However, the molecular mechanisms underlying this resistance are not fully understood. Using our in-house database containing the data on three-dimensional arrangements of secondary structure elements from the whole Protein Data Bank, we found a striking structural similarity between IL-8 and pepsin inhibitor-3. Such similarity could play a key role in understanding IL-8 resistance to the protease pepsin. To support this hypothesis, we applied pepsin assays confirming that intact IL-8 is not degraded by pepsin in comparison to IL-8 in a denaturated state. Applying 1H-15N Heteronuclear Single Quantum Coherence NMR measurements, we determined the putative regions at IL-8 that are potentially responsible for interactions with the pepsin. The results obtained in this work contribute to the understanding of the resistance of IL-8 to pepsin proteolysis in terms of its structural properties.

Peripheral nerve injury induces a transitory microglial reaction in the rat infralimbic cortex.

Undeniable evidence shows that microglia in the spinal cord undergo marked reactions following peripheral injuries. However, only rare studies have investigated the possible short and long term microglial reaction in brain regions following peripheral nerve injury and its interspecies specificities. In the present study we examined microglia in subdivisions of the prefrontal cortex in mice and rats, 7days and 42days after spared nerve injury (SNI) of the sciatic nerve. We show that a bilateral increase of microglial density takes place in the infralimbic cortex in rats 7days post-injury (sham vs. SNI, n=5: ipsilateral 35.4% increase of the median, p=0.0317; contralateral 24.9% increase of the median, p=0.0079), without any detectable change in the other investigated regions, namely the anterior cingulate, prelimbic and agranular insular cortices. In mice, no observable difference could be found in any region at both time points, neither using Iba-1 immunostaining nor with CX3CR1-eGFP animals. Our results indicate that a transitory, species-specific and highly regionalized microglial reaction takes place in the prefrontal cortex following peripheral nerve injury.