Toxicological and Safety Pharmacological Profiling of the Anti-Infective and Anti-Inflammatory Peptide Pep19-2.5
Abstract
:1. Introduction
2. Materials and Methods
2.1. Peptides
2.2. Stimulation of Mononuclear Cells (MNC) by LPS
2.3. Cytotoxicity Assays and Hemolysis
2.4. Hemolysis Assay
2.5. Chip-Based Cellular System (BIONAS)
2.6. Modelling of Peptide Structure
2.7. Electrophysiology
2.8. Irwin Test (Neurobehavioral Activity)
- Mortality: twice daily during the observation period
- Clinical observations: on the day of treatment before application
- Body weight development: on the day of delivery and on the day of treatment before application
- Modified Irwin test: at the time points pre-test (24 h prior to administration), 1 h, 4 h, 7 h, 24 h, 48 h, and 72 h after application.
2.9. Local Lymph Node Assay (LLNA)
- Test item concentrations were based on the results of the formulation evaluation and preliminary irritation/toxicity tests according to relevant guidelines (for example, S7A).
- The maximum attainable test concentration based on solubility was 5% (w/v) in aqueous 1% Pluronic®PE9200 (aqueous 1% Pluronic) using concentration series recommended by relevant guidelines
- Since no adverse effect of the test item was observed during the preliminary irritation/toxicity test up to this maximum concentration, Pep 19-2.5 was tested in the main test at 5%, 2.5%, 1%, and 0.5% (w/v) in aqueous 1% Pluronic
- Male (day 6: 208–259 g; day 1: 244–314 g) Wistar rats
- Five animals per group treated with one single i.v. dose of the respective formulation, application volume: 2 mL/kg
2.10. Repeated Dose Toxicology in Rats
2.11. Fluorescence Resonance Energy Transfer Spectroscopy (FRET)
3. Results
3.1. General In Vitro Toxicology
3.2. Toxicity in a Chip-Based System
3.3. Molecular Modeling of Pep19-2.5
3.4. Electrophysiology
3.5. Safety Pharmacology: Neurological Behaviour
3.6. Irwin Test
Statistical Analysis
3.7. Local Lymph Node Assay (LLNA)
- Application of substance on external surface of each ear (25 µL/ear) for 3 consecutive days
- On day 6, intravenous injection of tritiated methyl thymidine (3HTdR) in tail vein, and after about 6 h, sacrification and preparation of lymph node cell
- Cell proliferation in lymph node measured by incorporation of 3HTdR
3.8. Repeated Dose Toxicology in Rats
3.9. Origin of the Toxicology of the Pep19-2.5 Series
4. Discussion and Conclusions
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Acknowledgments
Conflicts of Interest
References
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HepG2 | Human Hepatom Cell Line | Cell Lines Service, Eppelheim Germany |
---|---|---|
LS-174T | Human colon adenocarcinoma cell line | Cell Lines Service |
Jurkat | Human acute lymphocytic leukemia cell line | Cell Lines Service |
PBMC | Human peripheral blood mononuclear cells [hPBMCs] | ProBioGen, Berlin Germany |
Group | No. of Animals | Treatment | Single Dose in [mg/kg] |
---|---|---|---|
Negative control | 6 males | 0.9% isotonic saline | - |
Positive control | 6 males | chlorpromazine | 3.00 |
Low dose | 6 males | Pep19-2.5 | 0.02 |
Mid dose | 6 males | Pep19-2.5 | 0.20 |
High dose | 6 males | Pep19-2.5 | 2.00 |
Peptide | Sequence | Molecular Weight |
---|---|---|
Pep19-2.5 | GCKKYRRFRWKFKGKFWFWG | 2711 |
Pep19-2.5AK | KKYRRFRWKFKGKFWFWCG | 2654 |
Pep19-Re1 | GKKYRWKFKGKFWFWG | 2149 |
Pep19-2.5M2 | GCKKYRRFRWKFK | 1802 |
Pep19-2.5M3 | GCKKYRRFRW | 1399 |
Pep19-2.5M4 | KFKGKFWFWG | 1330 |
Pep19-2.5M5 | YRRFRWKFKG | 1443 |
Pep19-2.5M6 | YRRFRWK | 1110 |
Aspidasept (Manual Patch-Clamp) (Rel. Rem. Current ± SD) | Aspidasept (QPatch) (Rel. Rem. Current ± SD) | Aspidasept (QPatch, 20 min Application) (Rel. Rem. Current) | |
---|---|---|---|
1 µg/mL | 1.00 ± 0.04 | 0.97 ± 0.02 | Not tested |
10 µg/mL | Beyond detection limit of the assay (no successful experiments (n = 7)) | 0.95 ± 0.02 | 0.92 |
100 µg/mL | Beyond detection limit of the assay (not tested) | Beyond detection limit of the assay (no successful experiments (n = 6)) | Not tested |
Group | No. of Animals | Treatment | Single Dose in [% (w/v)] |
---|---|---|---|
Negative control | 5 females | aqueous 1% Pluronic | - |
Low dose | 5 females | Pep19-2.5 in aq. 1% Pluronic | 0.5 |
Mid dose I | 5 females | Pep19-2.5 in aq. 1% Pluronic | 1.0 |
Mid dose II | 5 females | Pep19-2.5 in aq. 1% Pluronic | 2.5 |
High dose | 5 females | Pep19-2.5 in aq. 1% Pluronic | 5.0 |
Positive control | 5 females | α-Hexylcinnamaldehyde in AOO | 25 |
Neg. control of pos. control | 5 females | AOO (4:1 v/v acetone/olive oil) | - |
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Möller, C.; Heinbockel, L.; Garidel, P.; Gutsmann, T.; Mauss, K.; Weindl, G.; Fukuoka, S.; Loser, D.; Danker, T.; Brandenburg, K. Toxicological and Safety Pharmacological Profiling of the Anti-Infective and Anti-Inflammatory Peptide Pep19-2.5. Microorganisms 2022, 10, 2412. https://doi.org/10.3390/microorganisms10122412
Möller C, Heinbockel L, Garidel P, Gutsmann T, Mauss K, Weindl G, Fukuoka S, Loser D, Danker T, Brandenburg K. Toxicological and Safety Pharmacological Profiling of the Anti-Infective and Anti-Inflammatory Peptide Pep19-2.5. Microorganisms. 2022; 10(12):2412. https://doi.org/10.3390/microorganisms10122412
Chicago/Turabian StyleMöller, Clemens, Lena Heinbockel, Patrick Garidel, Thomas Gutsmann, Karl Mauss, Günther Weindl, Satoshi Fukuoka, Dominik Loser, Timm Danker, and Klaus Brandenburg. 2022. "Toxicological and Safety Pharmacological Profiling of the Anti-Infective and Anti-Inflammatory Peptide Pep19-2.5" Microorganisms 10, no. 12: 2412. https://doi.org/10.3390/microorganisms10122412