Author response:
The following is the authors’ response to the original reviews.
Public Reviews:
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.
Sham animals received saline 0.9% which is the vehicle control considering it was used to dilute the water-soluble VH-N412 molecule.
(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.).
We agree with Reviewer 1. We indeed hypothesize that it is principally the hypothermia induced by the NT conjugate that is responsible for the effects we observe. However, we do not exclude the possibility that the conjugate itself can have direct effects on the sequelae of SE. We tried to address this question with the in vitro experiments. Our results suggest that indeed, in the absence of hypothermia, the conjugate showed intrinsic neuroprotection of cultured hippocampal neurons challenged with excitotoxic agents such as NMDA or KA. Besides the description of these results in the “Results Section”, end of page 19 of the original manuscript, we had discussed them at the end of the “Discussion Section”, top of page 43 of the original manuscript.
In order to separate the hypothermia component from the potential direct neuroprotective effects of the NT conjugate, we did consider abolishing hypothermia in animals that were injected with the NT conjugate by warming them up. However, it is particularly difficult to increase in a well- controlled manner the body temperature of mice, in particular undergoing seizures, in a closed temperature-controlled chamber. In response to Reviewer 1 demand, we added a few sentences in the “Discussion Section”, page 45 of the revised version.
(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?
The KA model we used was originally discovered more than 30 years ago, developed and very well characterized and mastered in our laboratory by Ben-Ari (Ben-Ari et al., 1979). Most of KA-treated mice that developed SE after KA injection developed spontaneous seizures subsequent to a latent period of about 1 week as described in Figure 3A, Results Section page 11 and in the reference we had mentioned in the Materials and Methods Section, page 27 (Schauwecker and Steward, 1997).
We agree that information regarding the development of spontaneous seizures is missing. We added 2 references, Gröticke et al., 2008; Wu et al., 2021 in the Materials and Methods Section, page 28 of the revised version, that describe the occurrence of spontaneous seizures after KA administration in mice. We also now added the following information in the Materials and Methods Section, end of page 29: In order to study mice in the chronic stage of epilepsy with spontaneous seizures, they were observed daily (at least 3 hours per day) for general behavior and occurrence of SRS. These are highly reproducible in the mouse KA model, allowing for visual monitoring and scoring of epileptic activity. After 3 weeks, most animals exhibited SRS, with 2 to 3 seizures per day, similar to previous observations (Wu et al., 2021). The detection of at least one spontaneous seizure per day was used as criterion indicating the animals had reached chronic phase that can ultimately be confirmed by mossy fiber sprouting (see Figure 7).
(3) They do not evaluate changes in receptor distribution or ping wo/w SE and/or various drugs.
It is not clear to us what changes in receptor distribution need evaluation. We suppose the question concerns NTSR1 receptor. It would indeed be very interesting to compare NTSR1 in brain regions and different brain cells wo/w SE and/or various drugs, to assess receptor distribution or re-distribution, if any. However, addressing such a question is a project in itself that could not be addressed in the present study. Reviewer 1 also evokes ping wo/w SE and/or various drugs and if our understanding is correct, Reviewer 1 alludes to PING, Pyramidal Interneuronal Network γ (Dugladze et al., 2013, see reference below). Although we did not assess PING per se, we used multi-electrode arrays (MEA) on hippocampal brain slices stimulated wo/w KA to assess whether the VH-N412 conjugate could modulate pyramidal neuron activity. In order to respond to Reviewer 1 concern we added these data as Figure S2 with corresponding modifications in the Material and Methods Section (pages 34-35), in the Results Section (page 19) and in the Discussion Section page 43 of the revised version of our manuscript.
Dugladze T, Maziashvili N, Börgers C, Gurgenidze S, Häussler U, Winkelmann A, Haas CA, Meier JC, Vida I, Kopell NJ, Gloveli T. GABA(B) autoreceptor-mediated cell type-specific reduction of inhibition in epileptic mice. Proc Natl Acad Sci U S A. 2013 Sep 10;110(37):15073-8. doi: 10.1073/pnas.1313505110. Epub 2013 Aug 26. PMID: 23980149; PMCID: PMC3773756.
Bas du formulaire
(4) It is not clear why several different mouse strains were employed.
We used 2 mouse strains in our work as mentioned in the Materials and Methods Section, page 21. The conjugates we developed and hypothermia evaluation were initially tested on adult Swiss CD-1 males. For the KA model and for behavioral tests, adult male FVB/N mice were used because they are considered as reliable and well described mouse models of epilepsy, where seizures are associated with cell death (Schauwecker, 2003). This not the case for a number of mouse strains that demonstrate very heterogeneous behavior in SE and heterogeneous neuronal death, sprouting and neuroinflammation. The FVB/N are also well suited for behavioral tests.
In response to the Reviewer 1 demand, the following sentence has been introduced in the Results Section, page 11 and in the Materials and Methods Section, page 21 of the revised manuscript: We assessed our conjugates in a model of KA-induced seizures using adult male FVB/N mice. This mouse strain was selected as a reliable and well described mouse model of epilepsy, where seizures are associated with cell death and neuroinflammation (Schauwecker, 2003; Wu et al., 2021).
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.
Tolerability studies were performed, but the results were not presented in the first version of the manuscript. In order to comply with Reviewer 2 demand, we have added the following text in the Results section, page 11 of the revised version to describe our tolerability results.
Finally, tolerability studies were performed with the administration up to 20 and 40 mg/kg Eq. NT (i.e. 25.8 and 51.6 mg/kg of VH-N412) with n=3 for these doses. The rectal temperature of the animals did not fall below 32.5 to 33.2°C, similar to the temperature induced with the 4 mg/kg Eq. NT dose. We observed no mortality or notable clinical signs other than those associated with the rapid HT effect such as a decrease in locomotor activity. We thus report a very interesting therapeutic index since the maximal tolerated dose (MTD) was > 40 mg/kg Eq. NT, while the maximum effect is observed at a 10x lower dose of 4 mg/kg Eq. NT and an ED50 established at 0.69 mg/kg as shown in Figure 1G.
Mice may be particularly sensitive to hypothermia. It would be beneficial to show similar effects in a rat model.
We have tested our conjugate in mice, rats, and pigs, with in all cases nice dose response curves. We added a few words in the Discussion Section, page 38 of the revised version to mention that we can elicit hypothermia with our conjugates in the above-mentioned species.
Recommendations for the authors:
Reviewer #1 (Recommendations For The Authors):
(1) In Figures 4, 5, 6, 8, and 9, scale bars are needed on all panels.
We have looked carefully at the Figures. Scale bars are present on all Figures, as mentioned in the Legends of all Figures, but not necessarily on all panel pictures at the same magnification.
(2) The supplemental would seemingly be better moved into the main body of the manuscript.
In agreement with Reviewer 1 demand, we moved the Supplemental Figures into the main body of the manuscript, except for Figure S1, previously Figure S3, and the new Figure S2. Tables S1 to S5 remain as Supplemental files.
Reviewer #2 (Recommendations For The Authors):
Activation of LDLRs can have widespread effects in the CNS and peripherally. The authors should further discuss any beneficial or untoward effects of binding to LDL and activating LDLRs.
As mentioned in the Introduction and in a number of references where we describe the development of our family of LDLR peptide ligands (see below), we only selected peptide ligands that do not compete with LDL, one of the major ligands of the LDLR. We indeed showed that while LDL binds the ligand-binding domain of the LDLR, the peptide ligands we developed bind to the EGF-precursor homology domain of the receptor (See Malcor et al., 2008 below).
We have studied our peptide ligands in vitro and in vivo for more than 15 years and we have not observed beneficial or adverse effects. Actually, one of the members of our LDLR peptide family has been validated as a theragnostic agent and is in Phase 1 clinical trials for brain glioblastoma and pancreatic cancer. Hence, to our knowledge, the peptide ligand we describe in the present study shows no beneficial or untoward effects on LDL binding and activation of the LDLR. In response to Reviewer 2 recommendation, we added the following information and references in the Introduction Section, page 6 of the revised version of our manuscript: These peptides bind the EGF precursor homology domain of the LDLR and thus do not compete with LDL binding on the ligand-binding domain. To our knowledge, they have no beneficial or untoward effects on LDL binding and LDLR activity (Malcor et al., 2012; Jacquot et al., 2016; David et al., 2018; Varini et al., 2019; Acier et al., 2021, Yang et al., 2023; Broc et al., 2024).
Broc B, Varini K, Sonnette R, Pecqueux B, Benoist F, Masse M, Mechioukhi Y, Ferracci G, Temsamani J, Khrestchatisky M, Jacquot G, Lécorché P. LDLR-Mediated Targeting and Productive Uptake of siRNA-Peptide Ligand Conjugates In Vitro and In Vivo. Pharmaceutics. 2024 Apr 17;16(4):548. doi: 10.3390/pharmaceutics16040548. PMID: 38675209; PMCID: PMC11054735.
Yang X, Varini K, Godard M, Gassiot F, Sonnette R, Ferracci G, Pecqueux B, Monnier V, Charles L, Maria S, Hardy M, Ouari O, Khrestchatisky M, Lécorché P, Jacquot G, Bardelang D. Preparation and In Vitro Validation of a Cucurbit[7]uril-Peptide Conjugate Targeting the LDL Receptor. J Med Chem. 2023 Jul 13;66(13):8844-8857. doi: 10.1021/acs.jmedchem.3c00423. Epub 2023 Jun 20. PMID: 37339060.
Acier A, Godard M, Gassiot F, Finetti P, Rubis M, Nowak J, Bertucci F, Iovanna JL, Tomasini R, Lécorché P, Jacquot G, Khrestchatisky M, Temsamani J, Malicet C, Vasseur S, Guillaumond F. LDL receptor-peptide conjugate as in vivo tool for specific targeting of pancreatic ductal adenocarcinoma. Commun Biol. 2021 Aug 19;4(1):987. doi: 10.1038/s42003-021-02508-0. PMID: 34413441; PMCID: PMC8377056.
Varini K, Lécorché P, Sonnette R, Gassiot F, Broc B, Godard M, David M, Faucon A, Abouzid K, Ferracci G, Temsamani J, Khrestchatisky M, Jacquot G. Target engagement and intracellular delivery of mono- and bivalent LDL receptor- binding peptide-cargo conjugates: Implications for the rational design of new targeted drug therapies. J Control Release. 2019 Nov 28;314:141-161. doi: 10.1016/j.jconrel.2019.10.033. Epub 2019 Oct 20. PMID: 31644939.
David M, Lécorché P, Masse M, Faucon A, Abouzid K, Gaudin N, Varini K, Gassiot F, Ferracci G, Jacquot G, Vlieghe P, Khrestchatisky M. Identification and characterization of highly versatile peptide-vectors that bind non- competitively to the low-density lipoprotein receptor for in vivo targeting and delivery of small molecules and protein cargos. PLoS One. 2018 Feb 27;13(2):e0191052. doi: 10.1371/journal.pone.0191052. PMID: 29485998; PMCID: PMC5828360.
Molino Y, David M, Varini K, Jabès F, Gaudin N, Fortoul A, Bakloul K, Masse M, Bernard A, Drobecq L, Lécorché P, Temsamani J, Jacquot G, Khrestchatisky M. Use of LDL receptor-targeting peptide vectors for in vitro and in vivo cargo transport across the blood-brain barrier. FASEB J. 2017 May;31(5):1807-1827. doi: 10.1096/fj.201600827R. Epub 2017 Jan 20. PMID: 28108572.
Jacquot G, Lécorché P, Malcor JD, Laurencin M, Smirnova M, Varini K, Malicet C, Gassiot F, Abouzid K, Faucon A, David M, Gaudin N, Masse M, Ferracci G, Dive V, Cisternino S, Khrestchatisky M. Optimization and in Vivo Validation of Peptide Vectors Targeting the LDL Receptor. Mol Pharm. 2016 Dec 5;13(12):4094-4105. doi: 10.1021/acs.molpharmaceut.6b00687. Epub 2016 Oct 11. PMID: 27656777.
Malcor JD, Payrot N, David M, Faucon A, Abouzid K, Jacquot G, Floquet N, Debarbieux F, Rougon G, Martinez J, Khrestchatisky M, Vlieghe P, Lisowski V. Chemical optimization of new ligands of the low-density lipoprotein receptor as potential vectors for central nervous system targeting. J Med Chem. 2012 Mar 8;55(5):2227-41. doi: 10.1021/jm2014919. Epub 2012 Feb 14. PMID: 22257077.
As described above, the authors should also comment on the tolerability of these analogs.
Tolerability studies were performed, but the results were not presented in the first version of the manuscript. In order to comply with Reviewer 2 demand, we have added the following text in the Results section, page 11 of the revised version to describe our tolerability results.
Finally, tolerability studies were performed with the administration up to 20 and 40 mg/kg Eq. NT (i.e. 25.8 and 51.6 mg/kg of VH-N412) with n=3 for these doses. The rectal temperature of the animals did not fall below 32.5 to 33.2°C, similar to the temperature induced with the 4 mg/kg Eq. NT dose. We observed no mortality or notable clinical signs other than those associated with the rapid HT effect such as a decrease in locomotor activity. We thus report a very interesting therapeutic index since the maximal tolerated dose (MTD) was > 40 mg/kg Eq. NT, while the maximum effect is observed at a 10x lower dose of 4 mg/kg Eq. NT and an ED50 established at 0.69 mg/kg as shown in Figure 1G.
