A peptide-neurotensin conjugate that crosses the blood-brain barrier induces pharmacological hypothermia associated with anticonvulsant, neuroprotective and anti-inflammatory properties following status epilepticus in mice

Reviewer #1 (Public Review):

In this manuscript, Ferhat and colleagues describe their study aimed at developing a blood-brain barrier (BBB) penetrant agent that could induce hypothermia and provide neuroprotection from the sequelae of status epilepticus (SE) in mice. Hypothermia is used clinically in an attempt to reduce neurological sequelae of injury and disease. Hypothermia can be effective, but physical means used to reduce core body temperature are associated with untoward effects. Pharmacological means to induce hypothermia could be as effective with fewer untoward complications. Intracerebroventricularly applied neurotensin can cause hypothermia; however, neurotensin applied peripherally is degraded and does not cross the BBB. Here the authors develop and characterize a neurotensin conjugate that can reach the brain, induce hypothermia, and reduce seizures, cognitive changes, and inflammatory changes associated with status epilepticus.

Strengths:

(1) In general, the study is well-reasoned, well-designed, and seemingly well-executed.

(2) Strong dose-response assessment of multiple neurotensin conjugates in mice.

(3) Solid assessment of binding affinity, in vitro stability in blood, and brain uptake of the conjugate.

(4) Appropriate inclusion of controls for SE and for drug injections. However, perhaps a vehicle control could have been employed.

(5) Multifaceted assessment of neurodegeneration, inflammation, and mossy fiber sprouting in the different groups.

(6) Inclusion of behavioral assessments.

(7) Evaluates NSTR1 receptor distribution in multiple ways; however, does not evaluate changes in receptor distribution or ping wo/w SE and/or various drugs.

(8) Demonstrates that this conjugate can induce hypothermia and have positive effects on the sequelae of SE. Could have a great impact on the application of pharmacologically-induced hypothermia as a neuroprotective measure in patients.

Weaknesses:

(1) The authors make the claim, repeatedly, that the hypothermia caused by the neurotensin conjugate is responsible for the effects they see; however, what they really show is that the conjugate causes hypothermia AND has favorable effects on the sequelae of SE. They need to discuss that they did not administer the conjugate without allowing the pharmacological hypothermia (e.g., by warming the animal, etc.).

(2) In the status epilepticus studies, it is unclear how or whether they monitored animals for the development of spontaneous seizures. Can the authors please describe this?

(3) They do not evaluate changes in receptor distribution or ping wo/w SE and/or various drugs.

(4) It is not clear why several different mouse strains were employed.

Reviewer #2 (Public Review):

Summary:

The authors generated analogs consisting of modified neurotensin (NT) peptides capable of binding to low-density lipoprotein (LDL) and NT receptors. Their lead analog was further evaluated for additional validation as a novel therapeutic. The putative mechanism of action for NT in its antiseizure activity is hypothermia, and as therapeutic hypothermia has been demonstrated in epilepsy, NT analogs may confer antiseizure activity and avoid the negative effects of induced hypothermia.

Strengths:

The authors demonstrate an innovative approach, i.e. using LDLR as a means of transport into the brain, that may extend to other compounds. They systematically validate their approach and its potential through binding, brain penetration, in vivo antiseizure efficacy, and neuroprotection studies.

Weaknesses:

Tolerability studies are warranted, given the mechanism of action and the potential narrow therapeutic index. In vivo studies were used to assess the efficacy of the peptide conjugate analogs in the mouse KA model. However, it would be beneficial to have shown tolerability in naïve animals to better understand the therapeutic potential of this approach.

Mice may be particularly sensitive to hypothermia. It would be beneficial to show similar effects in a rat model.