Improved brain penetration of neurotensin(8–13) via blood-brain barrier shuttle conjugation underlies strong analgesia

The development of non-opioid analgesics remains a major therapeutic priority. Although peptide-based drugs offer high target specificity, their clinical utility for brain disorders is limited by poor blood-brain barrier (BBB) permeability. The neurotensin (NT) peptide elicits potent antinociception via activation of the seven-transmembrane receptors NTS1 and NTS2; however, its therapeutic potential is constrained by rapid metabolic degradation and inadequate brain penetration. Here, we report the functional characterization of An2-NT(8-13), a conjugate linking the minimal active NT fragment to the BBB-permeant shuttle peptide angiopep-2, which targets the LRP1 receptor highly expressed at the brain endothelium. An2-NT(8-13) exhibited improved plasma stability while retaining high affinity for both NTS1 and NTS2. PET-CT imaging demonstrated efficient BBB translocation of radiolabeled An2-NT(8-13) following systemic administration via an LRP1-dependent mechanism. Both intravenous and subcutaneous administration of An2-NT(8-13) produced robust antinociceptive effects in vivo, significantly reducing nocifensive behaviors in both phases of the formalin tonic pain model. In the chronic constriction injury model of neuropathic pain, intravenous treatment reversed mechanical allodynia, demonstrating efficacy in an opioid-resistant pain condition. An2-NT(8-13) did not induce marked hypotension or hypothermia following subcutaneous administration and did not cause constipation or respiratory depression. Finally, no biochemical or histopathological subacute toxicity or motor impairment was observed after repeated treatment over five consecutive days (0.25 mg/kg, i.v., twice daily). Together, these findings identify An2-NT(8-13) as a BBB-penetrant NT analog capable of producing effective central analgesia while minimizing NT- and opioid-associated adverse effects, supporting An2 conjugation as a promising strategy for developing NT-based non-opioid analgesics.