TREATMENT OF HEPATIC AND CARDIOVASCULAR DISORDERS
20230050846 · 2023-02-16
Inventors
Cpc classification
C07K2319/31
CHEMISTRY; METALLURGY
A61K47/60
HUMAN NECESSITIES
A61P1/16
HUMAN NECESSITIES
C12N15/63
CHEMISTRY; METALLURGY
A61K47/542
HUMAN NECESSITIES
C07K2319/30
CHEMISTRY; METALLURGY
International classification
A61K47/60
HUMAN NECESSITIES
A61P1/16
HUMAN NECESSITIES
Abstract
The present invention relates to a peptide and its use as a medicament, in particular in the treatment of metabolic syndrome, non-alcoholic fatty liver disease (NAFLD), cardiovascular disease (CVD) or cholestatic liver disease.
Claims
1. A peptide comprising a peptide having at least 70% sequence identity to the amino acid sequence of SEQ ID NO: 1, wherein said peptide is a F1-ATPase activator.
2. The peptide according to claim 1, comprising the amino acid sequence of SEQ ID NO: 1, preferably having the amino acid sequence of SEQ ID NO: 1.
3. The peptide according to claim 1, wherein said peptide has a N-terminal acetylation and/or a C-terminal amidation.
4. The peptide according to claim 1, wherein said peptide is modified by attaching at least one long-lasting molecule at one or more amino acid residues of the amino acid sequence, preferably at the C-terminus of the amino acid.
5. The peptide according to claim 4, wherein said long-lasting molecule is selected from the group consisting of fatty acid, albumin, polyethylene glycol (PEG) and Fc portion of immunoglobulin G, preferably a fatty acid.
6. The peptide according to claim 1, wherein said peptide is modified by attaching one palmitic acid at the C-terminus of the amino acid sequence.
7. The peptide according to claim 1, wherein said peptide has the formula (I) or (II):
CH.sub.3CO-[peptide comprising a peptide having at least 70% SEQ ID NO: 1]-NH.sub.2 (I)
CH.sub.3CO-[peptide having at least 70% SEQ ID NO: 1]-K-[palmitoyl]-NH.sub.2 (II).
8. The peptide according to claim 1, wherein said peptide has the formula (I) or (II):
CH.sub.3CO-[SEQ ID NO: 1]-NH.sub.2 (I)
CH.sub.3CO-[SEQ ID NO: 1]-K-[palmitoyl]-NH.sub.2 (II).
9. A pharmaceutical composition comprising a therapeutically active amount of the peptide according to claim 1 and a pharmaceutically acceptable vehicle or carrier.
10. A method of treatment of a disease comprising administering to a subject in need thereof the peptide according to claim 1.
11. A method of treatment of metabolic syndrome, cardiovascular disease (CVD), non-alcoholic fatty liver disease (NAFLD) or cholestatic liver disease comprising administering to a subject in need thereof the peptide according to claim 1.
12. The method of treatment according to claim 11, wherein the NAFLD is non-alcoholic steatohepatitis (NASH).
13. A nucleotide sequence encoding the peptide according to claim 1.
14. A vector comprising the nucleotide sequence according to claim 13.
15. A cell comprising the nucleotide sequence according to claim 13.
16. A method of treatment of a disease comprising administering to a subject in need thereof the pharmaceutical composition according to claim 9.
17. A method of treatment of metabolic syndrome, cardiovascular disease (CVD), non-alcoholic fatty liver disease (NAFLD) or cholestatic liver disease comprising administering to a subject in need thereof the pharmaceutical composition according to claim 9.
18. The method of treatment according to claim 17, wherein the NAFLD is non-alcoholic steatohepatitis (NASH).
19. A cell comprising the vector according to claim 14.
Description
DESCRIPTION OF THE FIGURES
[0094]
[0095]
[0096]
[0097]
[0098]
[0099]
[0100]
[0101]
[0102]
[0103]
[0104]
[0105]
[0106]
EXAMPLES
Example 1: Preparation of the Peptides
[0107] The following peptides were produced by BachemAG (Bubendorf, Switzerland) with >90% purity in acetate salts and dissolved in Phosphate-Buffered Saline (PBS) solution before use: [0108] Formula (I): CH.sub.3CO-[SEQ ID NO: 1]-NH.sub.2, hereafter called “peptide of formula (I)” or “formula (I)” (represented in
[0113] The palmitic acid was introduced via coupling the preformed derivative Fmoc-K-[palmitoyl]-OH (represented in
[0114] Other labeled and unlabeled peptides SEQ ID NO: 3 (EAGGAFGK) and SEQ ID NO: 4 (EAGGAFG-[.sup.13C.sub.6, .sup.15N.sub.4]-K) were purchased from ThermoFisher Scientific with >90% purity and dissolved at 1 mM in 50% acetonitrile.
Example 2: Surface Plasmon Resonance Analysis of the Interaction of the Peptide of Formula (I) and the Peptide of Formula (II) with Human F1Fo-ATPase
[0115] Materials and Methods:
[0116] Surface plasmon resonance (SPR) assays. Binding studies based on SPR technology were performed on a BIAcore T200 optical biosensor instrument (GE Healthcare®, Uppsala, Sweden). The peptide of formula (I) with C-terminal 6×His-tag (Formula (I)-His-tag: CH.sub.3CO-[SEQ ID NO: 1]-HHHHHH) and the peptide of formula (II) with C-terminal biotin (Formula (II)-Biotin: CH.sub.3CO-[SEQ ID NO: 1]-K-[palmitoyl]-AEEAc-K-[biotinyl]-NH.sub.2) were custom-synthesized by BACHEM AG (Bubendorf, Switzerland) with >90% purity in trifluoroacetate salt. Human F1Fo-ATPase was purified from HepG2 cells by immunocapture using mouse monoclonal anti-ATP synthase antibody (12F4AD8AF8, #ab109867, Abcam) according to manufacturer's instructions.
[0117] Immobilization of the peptide of formula (I)-6His-tag was performed on a nitrilotriacetic acid (NTA) sensorchip in HBS-P+ buffer (10 mM Hepes pH 7.4, 150 mM NaCl, and 0.05% surfactant P20) (GE Healthcare). To saturate the NTA surface with Ni.sub.2.sup.+, flow cells (Fc) were loaded with 0.5 mM NiCl.sub.2 solution. The channel Fc1 was left empty and used as a reference surface for nonspecific binding measurements. Formula (I)-6×His was injected in the channel Fc2 at a flow-rate of 5 μL/min and stabilized by amine coupling (Laboratory guideline 29-0057-17 AB). The total amount of immobilized Formula (I)-His-tag was 300-350 resonance units (RU): final concentration 25 μg/mL.
[0118] Immobilization of C-term biotinylated peptide of Formula (II)-biotin was performed on streptavidin-coated (SA) sensor chip in HBS-EP buffer (10 mM HEPES [pH 7.4], 150 mM NaCl, 3 mM EDTA, 0.005% surfactant P20) (GE Healthcare). The channel Fc1 was left empty and used as a reference surface for nonspecific binding measurements. Formula (II)-biotin was injected in the channel Fc2 at a flow-rate of 5 μL/min. The total amount of immobilized Formula (II)-biotin was 350-380 RU: final concentration 100 ng/mL. For binding analyses, the F1F0 analyte (584 KDa) was injected sequentially over the immobilized peptides with increased concentrations ranging (3.125 nM-6.25 nM-12.5 nM-25 nM-50 nM) in a single cycle without regeneration of the sensorship between injections. A single-cycle kinetic (SCK) analysis allowed to determine association, dissociation, and affinity constants (Ka, Kd, and K.sub.D, respectively). Binding parameters were obtained by fitting the overlaid sensorgrams either with the 1:1 Langmuir binding model or with Steady State Constant Rmax model in the BIAevaluation software version 3.0.
[0119] Results:
[0120] The results are shown in
[0121] Conclusion:
[0122] A direct high affinity interaction was measured between F1Fo-ATPase and the peptide of formula (I), and between F1Fo-ATPase and the peptide of formula (II).
Example 3: Effect of Peptide of Formula (I) and Peptide of Formula (II) on the F1-ATPase Activity
[0123] Materials and Methods:
[0124] F1-ATPase Activity Assay.
[0125] The mature human IF1 protein (SEQ ID NO: 5) was chemically synthesized by GenScript (Piscataway, N.J., USA) at >80% purity. The peptides derived from the mature human IF1 sequence (IF1-1-60, IF1-10-56, IF1-10-47) were produced by BachemAG (Bubendorf, Switzerland) with >90% purity.
[0126] Human F1Fo-ATPase was purified from HepG2 cells by immunocapture using mouse monoclonal anti-ATP synthase antibody (12F4AD8AF8, #ab109867, Abcam) according to manufacturer's instructions.
[0127] Measurement of F1-ATPase activity was assayed as previously described [20]. Briefly, 10 μg of F1Fo-ATPase was prepared into 50 μL of activity assay buffer (10 mM HEPES, 150 mM NaCl, 5 mM KCl, 5 mM MgCl2, 0.5 mM phosphoenolpyruvate, 250 μM NADH, 100 μM ATP, 20 U lactate dehydrogenase, 120 U pyruvate kinase). The mixture was incubated at 37° C. for 30 min. Then the F1-ATPase activity was measured in a 96-well microplate by adding 5 μL of the mixture (1 μg of F1Fo-ATPase per point) per well to 200 μL of activity assay buffer at 37° C., and by adding 5 μL of buffer with or without peptide (1 μM each). The reduction in the absorbance of NADH was measured at 340 nm for 5 min with a Varioskan™ Flash Multimode Reader (Thermo Fisher Scientific). A slope was calculated for each well and the results expressed as a percentage of the control slope.
[0128] Results:
[0129] The results are presented in
[0130] Conclusion:
[0131] Unlike IF1 and other peptides derived from the IF1 sequence, the peptide of formula (I) and the peptide of formula (II) do not inhibit but stimulate the F1-ATPase activity. These peptides are therefore F1-ATPase activator.
Example 4: In Vitro Activity of the Peptide of Formula (I) and the Peptide of Formula (II): F1-ATPase Activation
[0132] Materials and Methods:
[0133] The human hepatocyte cell line HepG2 was obtained from the American Type Culture Collection (#HB-8065). HepG2 were cultured in Dulbecco's Modified Eagle's Medium (DMEM)—high glucose (D0822, Sigma-Aldrich) supplemented with 10% fetal bovine serum (10270098, Life technologies), 1% Penicillin-Streptomycin solution (P0781, Sigma-Aldrich). HepG2 cells were seeded on 24-well plates at 75,000 cells/well (Day 0). After 24 hours of growing, cells were serum starved for 24 h in order to synchronize cell cycles (Day 1) and then replaced additional 24 h in complete cell growth medium (Day 2). On day 3, cells were washed and equilibrated in fresh DMEM—high glucose without red phenol for 1 h (D1145, Sigma-Aldrich).
[0134] The cells were then treated 5 min with different concentration of peptide of formula (I) (0.1 to 5 μM), the peptide of formula (II) (1 μM), SCR (1 μM), SCR-K-16 (1 μM) or apoA-I (10 μg/mL) purified from human plasma [5].
[0135] Specific ecto-F1-ATPase activation was assessed in the presence of IF1 protein (1 μM), a natural inhibitor of F.sub.1-ATPase that interacts with the β-subunit to inhibit the ATP hydrolysis activity [1] [5].
[0136] Supernatants were then collected and centrifuged (10,000 g, 5 min, 4° C.) and processed for ADP and ATP measurement. For ADP measurement, ADP was converted into ATP in 150 mM NaCl, 5 mM KCl, 2 mM MgCl2, pH 7.5 buffer containing 0.5 mM phosphoenolpyruvate (PEP) and pyruvate kinase (PK, 6 U per point for 15 min at 37° C.). For ATP measurement, 100 μl of sample was analyzed using the ATP bioluminescence assay kit CLS II (Roche Diagnostics). Samples were added to the ATP assay mixture and luminescence was measured in a microplate reader Infinite F500 (Tecan, Switzerland) for 1000 ms. The ATP standard curve was produced in the same medium as the samples and in the 10.sup.−5 to 10.sup.−10 M concentration range. The ADP concentration was then calculated as the ATP concentration following ADP conversion minus the basal ATP concentration. Data are expressed as nanomoles of ADP produced.
[0137] Results:
[0138] The results are presented in
[0139] Conclusion:
[0140] The peptide of formula (I) and the peptide of formula (II) stimulated ecto-F1-ATPase activity in hepatocytes, and competed with the binding of IF1 to cell surface F1Fo-ATPase. These peptides are therefore good candidates for activating cell surface F1Fo-ATPase.
Example 5: In Vitro Activity: HDL Endocytosis by Hepatocytes
[0141] Materials and Methods:
[0142] HDL Endocytosis Assays.
[0143] HepG2 cells were seeded on 96-well plates at 50,000 cells/well. HDL.sub.3 (d 1.12-1.21) were isolated from plasma of healthy human donors [12] and referred to as HDL. HDL was fluorescently labeled with AlexaFluor®568 dye (A10238, Thermofisher Scientific) according the instructions of manufacturer. 1 h30 before the assay, the cells were serum starved for 1 h30 in order to stabilize nucleotide secretion. Cells were incubated with inhibitors (H49K, 1 μM) for 10 min before treatment with the different peptides (1 μM) or apoA-I (10 μg/ml) purified from human plasma [5]. 5 min after peptide treatment, endocytosis was initiated by 50 μg/mL of AlexaFluor568®-labelled HDL. The same experiment was performed with a 25-fold excess of unlabelled HDL (2.5 mg/mL) to determine the nonspecific fluorescence signal. After 25 min at 37° C., cells were then washed in serum-free DMEM and extracellular membrane-bound HDL was disassociated by incubating cells at 4° C. in serum-free DMEM for 90 min. Following washes, cells were lysate in NaOH 0.1M SDS 1% during 2 h, lysates were transferred in a black 96-wells plate and fluorescence was recorded at 568 nm (Varioscan flash). Fluorescence for each condition was substrate with the value obtained in unlabelled HDL condition, and results were expressed as the fold change as compared with the basal condition (untreated cells).
[0144] Results:
[0145] The results are presented in
[0146] Conclusion:
[0147] F1Fo-ATPase-mediated HDL endocytosis in hepatocytes was significantly increased when F1-ATPase activity is pharmacologically stimulated by the peptide of formula (I) and the peptide of formula (II). Given that HDL endocytosis in hepatocytes is one key last step of reverse cholesterol transport for excess cholesterol removal [6], the peptides of formula (I) and formula (II) are therefore good candidates to improve reverse cholesterol transport and excess cholesterol elimination from the body.
Example 6: In Vitro Activity: Endothelial Nitric Oxide (NO) Production
[0148] Materials and Methods:
[0149] Nitric Oxide Production.
[0150] Nitric oxide (NO) was detected using a DAF-FM-DA probe (D2321, Sigma-Aldrich) which forms fluorescent benzotriazole when it reacts with NO. HUVEC cells (PromoCell #C-12203) were seeded in 96-well plates (10,000 cells per well) and cultured in endothelial cell basal medium 2 (PromoCell #C-22211) supplemented with GM2 supplement Mix (PromoCell #C-39211), until 80-90% confluence. The medium was then replaced with M-199 without serum for 4 h, and the cells were incubated for 45 min with DAF-FM-DA (5 μM) diluted in PBS. Cells were treated with increasing concentrations of the different peptides or apoA-I (10 μM) purified from human plasma [5] or histamine (1 mM) as positive control. In another set of experiments, cells were incubated with inhibitors (IF1, 1 μM) for 10 min before treatment with peptides or apoA-I. The fluorescence was recorded for 30 min (λex=495 nm, λem=515 nm) with a Tecan Flash Multimode Reader (Thermo Fisher Scientific). Fluorescence for each condition was compared with the value obtained in untreated cells, and results were expressed as the fold change as compared with the basal condition (untreated cells).
[0151] Results:
[0152] The results are shown in
[0153] According to the protocol disclosed in [5], the peptide of formula (I) increased femoral artery blood flow in conscious wild-type C57B/L6J mice in a process that strictly depend on endothelial NO production (data not shown).
[0154] Conclusion:
[0155] In human endothelial cells, F1Fo-ATPase-mediated NO production by eNOS was significantly increased when F1-ATPase activity is pharmacologically stimulated by the peptide of formula (I) and the peptide of formula (II). Given that NO production by eNOS preserves vascular homeostasis [16] and maintains quiescent both hepatic stellate cells, involved in liver fibrosis, and Kupffer cells, involved in liver inflammation [8], the peptides of formula (I) and formula (II) are therefore good candidates for the treatment of metabolic syndrome, cardiovascular disease (CVD), non-alcoholic fatty liver disease (NAFLD) or cholestatic liver disease.
Example 7: In Vitro Activity: Hepatic Steatosis
[0156] Materials and Methods:
[0157] Preparation of Oleate and Palmitate Solution.
[0158] A solution containing 250 mM palmitate (P0500, Sigma-Aldrich) was first prepared in 0.1 M NaOH at 70 C for 30 min then diluted in DMEM low glucose (D5546, Sigma-Aldrich) containing 10% fatty acid-free BSA (A7030, Sigma-Aldrich) to yield a 10 mM palmitate solution and allowed to dissolve for 30 min at 37° C., filter sterilized and stored in glass vial at −20 C until use. This palmitate stock solution and ready to use oleate solution (03008, Sigma-Aldrich) were used to prepared a 0.5 mM solution at a 2:1 ratio of oleate to palmitate in complete culture medium containing DMEM low glucose, 10% fetal bovine serum, 1% Penicillin-Streptomycin and 1% fatty acid-free BSA.
[0159] In-Vitro Evaluation of Steatosis.
[0160] HepG2 cell were grown in 12-well plates to 60-70% confluence then exposed for 48 h to culture medium (DMEM low glucose, 10% fetal bovine serum, 1% Penicillin-Streptomycin and 1% fatty acid-free BSA) alone or containing 0.5 mM oleate/palmitate mixture (2:1) to induce steatosis. For the last 24 h of the 48 h period, cells were treated with 1 μM of the peptide of formula (I) or the peptide of formula (II).
[0161] Primary mouse hepatocytes were isolated as described in Example 3 from mice fed western-diet for 11-weeks (Envigo #TD.88137 containing 0.2% cholesterol, 42% kcal from fat, 34% sucrose by weight). Primary mouse hepatocytes were seeded in a 12-well plate at a density of 600,000 cells/well in growth medium and treated for 24 h with apoA-I (10 μg/mL), peptide of formula (I) (1 μM) or peptide of formula (II) (1 μM).
[0162] For measurement of intracellular triglyceride content, cells were washed with PBS and scraped in 5% NP-40 lysis buffer and heated for 10 min at 85° C. Triglycerides were then quantified using triglyceride commercial kit (Biolabo #87319). Values were normalized to protein concentration in cell lysates.
[0163] Results:
[0164] The results are presented in
[0165] Conclusion:
[0166] The peptide of formula (I) and the peptide of formula (II) did reduce steatosis in a model of steatotic human hepatocytes and in steatotic primary mouse hepatocytes. Given that steatotic hepatocytes are key drivers of the pathogenic process in NAFLD/NASH [11], the peptide of formula (I) and the peptide of formula (II) are therefore good candidates to prevent and treat non-alcoholic fatty liver disease (NAFLD) and non-alcoholic steatohepatitis (NASH).
Example 8: In Vitro Toxicity Assay (Hepatocytes)
[0167] Materials and Methods:
[0168] Cytotoxicity Assays.
[0169] HepG2 cells were seeded in a 96-well plate at a density of 10,000 cells/well in growth medium (DMEM high-glucose, 10% fetal bovine serum). The next day, growth medium was changed and the peptide of formula (I) was added once with ascending dose for 24 h, 48 or 72 h or for 48 h with repeating once in 24 h. Similarly, the peptide of formula (II) was added once with ascending dose for 24 h or 48 h or for 48 h with repeating once in 24 h. Cells were incubated for 4 h with 5 mg/L MTT and 100 μL of DMSO was added in the well. Absorbance was recorded at 570 and 660 nm using the microplate spectrophotometer system (Varioscan flash). Cell viability was calculated by subtracting the 570 nm absorbance to the background measured at 660 nm.
[0170] Results:
[0171] The results are shown in
[0172] Conclusion: The peptide of formula (I) and the peptide of formula (II) did not display any cellular toxicity over time, neither with ascending single or repeated doses.
Example 9: Stability of the Peptide of Formula (I) and the Peptide of Formula (II) in PBS, Plasma and Serum
[0173] Materials and Methods:
[0174] Reagents.
[0175] UPLC/MS-grade acetonitrile and water, phosphate buffer saline (PBS) and formic acid were purchased from Biosolve (Valkenswaard, Netherlands).
[0176] Stability Assays.
[0177] Peptide of Formula (I) or peptide of Formula (II) were prepared at a concentration of 200 μg/mL in PBS or mixed with EDTA plasma or serum from human (Etablissement Frangais du Sang, EFS) or mouse (C57BL/6J, cardiac puncture). Aliquots from PBS samples (40 μL) were incubated at 4° C. or 37° C. for 0, 1, 2 and 4 weeks. Aliquots from plasma and serum samples (50 μL) were incubated at 4° C. or 37° C. for 0, 1, 2, 4, 6, 12, 24 h.
[0178] Sample Analysis.
[0179] A mixed solution of peptides of formula (I) and (II) was constituted and serially diluted in PBS to obtain seven standard solutions, ranging from 200 μg/mL to 0.2 μg/mL. In parallel, a mixed solution of labelled peptides of Formula (I) and (II) (Thermo Scientific, Biopolymers Darmstadt, Germany) was prepared in PBS at 100 μg/mL. The mixed solution of labelled peptides (25 μL) was added to 25 μL of standard solutions as well as to PBS, plasma and serum samples. Acetonitrile (150 μL) was added to each sample to precipitate plasma/serum proteins. After centrifugation (10,000×g, 4° C., 10 min), the clear supernatants (150 μL) were dried under a gentle stream of nitrogen (45° C.), reconstituted with 10% acetonitrile containing 0.1% formic acid (100 μL), and injected into the liquid chromatography-high-resolution mass spectrometry (LC-HRMS) system. LC-HRMS analyses were performed on an H-Class UPLC system (Waters Corporation, Milford, Mass., USA) by injection of 10 μL of samples onto an Acquity® Peptide CSH C.sub.18 column (2.1 mm×150 mm, 1.7 μm; Waters Corporation) held at 60° C. The mobile phase was composed of 5% acetonitrile as solvent A and 100% acetonitrile as solvent B, each containing 0.1% formic acid. The elution was carried out using a gradient of solvent B in solvent A over 20 min at a constant flow rate of 250 μL/min. Mobile phase B was kept constant at 1% for 1 min, linearly increased from 1% to 80% for 15 min, kept constant for 1 min, returned to the initial condition over 1 min, and kept constant again for 2 min before the next injection. HRMS detection was performed by a Synapt G2 HRMS Q-TOF mass spectrometer equipped with a Z-Spray interface for electrospray ionization (Waters Corporation). The resolution mode was applied in a mass-to-charge (m/z) ratio ranging from 200 to 4,000 at a mass resolution of 25,000 Full Width Half Maximum in the positive ionization mode. Ionization parameters were as follow: capillary voltage of 3 kV, cone voltage of 30 V, desolvatation gas flow of 900 L/hr, source temperature of 120° C., desolvatation temperature of 450° C., Nitrogen as desolvatation gas. Data were collected in the continuum mode at a rate of four spectra per second. Leucine enkephalin solution prepared at 2 μg/mL in an acetonitrile/water (50/50, v/v) mixture was infused at a constant flow (10 μL/min) in the lock spray channel. A spectrum of 1 s was acquired every 20 s and allowed mass correction during experiments. Peptides were analyzed according to their major exact m/z (i 5 ppm, Table 1) and each peptide signal was normalized with that of its labelled internal standard. Peptide concentrations were calculated using calibration curves plotted from standard solutions (linear regression, 1/x weighted, origin excluded).
TABLE-US-00001 TABLE 1 Mass spectrometry parameters used for peptide detection by LC-HRMS. Major Charge Peptide Sequence m/z state Formula Acetate- 590.9703 6+ (1) RGAGSIREAGGAFGKREQAEEERYFRAQSRE-amide Formula Acetate-RGAGSI-[.sup.13C.sub.6,.sup.15N.sub.4]R- 592.6370 6+ (1) (IS) EAGGAFGKREQAEEERYF-[.sup.13C.sub.6, .sup.15N.sub.4]R-AQSRE- amide Formula Acetate- 652.1923 6+ (II) RGAGSIREAGGAFGKREQAEEERYFRAQSREK- Palmitoyl Formula Acetate-RGAGSI-[.sup.13C.sub.6, .sup.15N.sub.4]R- 653.8590 6+ (II) (IS) EAGGAFGKREQAEEERYF-[.sup.13C.sub.6, .sup.15n.sub.4]R-AQSREK- Palmitoyl IS: internal standard
[0180] Results:
[0181] The results are shown in
[0182] Conclusion: The peptide of formula (I) and the peptide of formula (II) can be stored in PBS for at least 4 weeks at 4° C. and up to 37° C., without being degraded. The peptide of formula (II) presents little degradation in human and mouse biological matrices, including at 37° C. and up to 24 h, and is thus more suitable than the peptide of formula (I) for chronic injection.
Example 10: Pharmacokinetics of the Peptide of Formula (I) and the Peptide of Formula (II) In Vivo
[0183] Material and Method:
[0184] LC-MS/MS Peptide Quantification.
[0185] The peptide of formula (I) and the peptide of formula (II) were analyzed in mouse EDTA plasma using a validated assay involving trypsin proteolysis and the subsequent analysis of a signature peptide (SEQ ID NO: 3) by liquid chromatography-tandem mass spectrometry (LC-MS/MS). The working solution of unlabeled peptide (SEQ ID NO: 3, 1 mM) was serially diluted in water to obtain 7 standard solutions ranging 0.05-5 μM. Plasma and standard samples (40 μL) were reduced, alkylated and trypsin-digested overnight using the ready-to-use solutions of the ProteinWorks™ eXpress kit (Waters Corporation, Milford, Mass., USA), according to the manufacturer's instructions (except trypsin incubation time optimized to 7 h). The working solution of the labeled proteotypic peptide ([SEQ ID NO: 4]-[.sup.13C.sub.6, .sup.15N.sub.4]-K, 1 mM) was used as internal standard (IS) and added to the digestion buffer to a final concentration of 0.5 μM. After digestion, samples were cleaned using 30 mg Oasis HLB 1 cc Cartridges (Waters Corporation). Cartridges were conditioned, equilibrated, loaded, washed and eluted with methanol (1 mL), water (1 mL), samples (˜200 μL), 5% methanol containing 0.1% TFA (1 mL) and 60% methanol containing 0.1% TFA (1 mL), respectively. Eluates were dried under a nitrogen stream, reconstituted with 100 μL of 10% acetonitrile containing 0.1% formic acid, and 10 μL were injected into the LC-MS/MS system. LC-MS/MS analyses were performed on a Xevo® TQD mass spectrometer with an electrospray (ESI) interface and an Acquity H-Class® UPLC™ device (Waters Corporation). Proteotypic peptides were separated over 9 min on an Acquity® BEH C.sub.18 column (2.1×100 mm, 1.7 μm, Waters Corporation) held at 60° C. with a linear gradient of mobile phase B (100% acetonitrile) in mobile phase A (5% acetonitrile), each containing 0.1% formic acid, and at a flow rate of 600 μL/min. Mobile phase B was linearly increased from 1% to 50% for 5 min, kept constant for 1 min, returned to the initial condition over 1 min, and kept constant for 2 min before the next injection. Proteotypic peptides were then detected by the mass spectrometer with the ESI interface operating in the positive ion mode (capillary voltage, 3 kV; desolvatation gas (N.sub.2) flow and temperature, 900 L/h and 400° C.; source temperature, 150° C.). The multiple reaction monitoring mode was applied for MS/MS detection (SEQ ID NO: 3, m/z 368.8.fwdarw.536.5, y.sub.6.sup.+; [SEQ ID NO: 4]-[.sup.13C.sub.6, .sup.15N.sub.4]-K, m/z 372.8.fwdarw.544.4, y.sub.6) with cone and collision voltages set at 20 and 14 V, respectively. Data acquisition and analyses were performed with MassLynx® and TargetLynx® software, respectively (version 4.1, Waters Corporation). Chromatographic peak area ratio between unlabeled peptide and IS constituted the detector responses. Standard solutions were used to plot calibration curves for peptide quantification. The linearity was expressed by the mean r.sup.2 which was greater than 0.998 (linear regression, 1/x weighting, origin excluded). Each sample was assayed three times and the coefficients of variation did not exceed 4.5%. The peptide of formula (I) and the peptide of formula (II) concentrations were expressed in μM assuming 1 mole of peptide equivalent to 1 mole of the peptide of formula (I) and the peptide of formula (II), respectively. Concentrations were then converted to their standard unit (ng/mL) assuming molecular weights of 3540 Da and 3917 Da for the peptide of formula (I) and the peptide of formula (II), respectively.
[0186] Animals.
[0187] Wild-type C57B/L6J male mice were purchased from Janvier Labs (Le Genest Saint Isle, France). Mice were caged in animal rooms under specific pathogen free conditions at the animal facility of Rangueil (Anexplo platform, US006, Toulouse, France) with a light/dark schedule of 12 h/12 h and were fed ad libitum with a normal chow diet (#V1535 R/M-H, Ssniff, Germany). All animal experimental procedures were conducted in accordance with institutional guidelines on animal experimentation approved by the local ethical committee of animal care and are conformed to the guidelines from Directive 2010/63/EU of the European Parliament on the protection of animals used for scientific purposes or the NIH guideline.
[0188] Pharmacokinetics (PK) Studies.
[0189] 8-weeks old C57B/L6J male mice weighting 24.5±1.3 g were used for the following pharmacokinetic studies. All animals were allowed free access of food and water during the experiments. The peptide of formula (I) was administrated at 25 mg/kg, either intravenously (i. v.) at the tail vein or subcutaneously (s.c.). The peptide of formula (II) was subcutaneously administered 25 mg/kg. Three different animals were used for each time point. After administration, intracardiac blood was collected at 0.03, 0.117, 0.25, 0.5, 0.75, 1, 1.5, 2, 4 h for the peptide of formula (I) and 0.03, 1, 4, 6, 8, 10, 12, 16, 20, 24, 30, 48 h for the peptide of formula (II). EDTA was used as the anticoagulant and plasma was separated by centrifugation at 4,000 rpm for 10 min at 4° C. Plasma samples were placed on wet ice and, within 1 hour after collection, were stored at −80° C. until analyzed by liquid chromatography-mass spectrometry/mass spectrometry (LC-MS/MS) for quantification. Table 2 reports the calculated pharmacokinetics parameters in plasma, namely distribution and elimination half-lives (t.sub.1/21bd1 and t.sub.1/21bdz), maximum concentration (C.sub.max), time to reach C.sub.max (T.sub.max), Area Under the Curve (AUC), total plasma clearance (CI), volume of distribution (Vd), mean residence time (MRT).
TABLE-US-00002 TABLE 2 Pharmacokinetics characteristics of the peptide of formula (I) and the peptide of formula (II) iv-peptide of sc-peptide of sc-peptide of formula (I) formula (I) formula (II) Dose, 25 25 25 mg/kg t.sub.1/2lbd1, .sup.h 0.06 (3.8 min) 0.19 (11.6 min) 2.09 (125.3 min) t.sub.1/2lbdz, .sup.h 0.26 (15.8 min) 0.22 (13.4 min) 12.54 (752.4 min) C.sub.max, mg/L n.a 8.29 25.60 T.sub.max, h n.a 0.25 (15 min) 4 (240 min) AUC, 13290 6136 211323 mg .Math. h/L Cl, L/h/kg 1.9 1.9 0.05 Vd, L/kg 0.7 0.5 1 MRT, h 0.14 (8.4 min) 0.55 (33 min) 5.81 (348.6 min)
[0190] Results:
[0191] The results are presented in
[0192] In comparison to the peptide of formula (I), the peptide of formula (II) administrated subcutaneously at 25 mg/L was less rapidly distributed and eliminated (
[0193] Conclusion:
[0194] The peptide of formula (II) displayed improved pharmacokinetics properties as compared to the peptide of formula (I).
Example 11: In Vivo Efficacy of the Peptide of Formula (I) and the Peptide of Formula (II) on Biliary Lipid Secretions
[0195] Materials and Methods:
[0196] Animals.
[0197] Wild-type C57B/L6J male mice were purchased from Janvier Labs (Le Genest Saint Isle, France). LDLR knock-out mice (males, C57B/L6J background) were obtained from The Jackson Laboratory (Bar Harbor, Me., USA). Mice were caged in animal rooms under specific pathogen free conditions at the animal facility of Rangueil (Anexplo platform, US006, Toulouse, France) with a light/dark schedule of 12 h/12 h and were fed ad libitum with a normal chow diet (#V1535 R/M-H, Ssniff, Germany). All animal experimental procedures were conducted in accordance with institutional guidelines on animal experimentation approved by the local ethical committee of animal care and are conformed to the guidelines from Directive 2010/63/EU of the European Parliament on the protection of animals used for scientific purposes or the NIH guideline.
[0198] Gallbladder Cannulation and Bile Collection.
[0199] Depending of experiments, 8 weeks old mice were intraperitoneally injected with PBS, the peptide of formula (I), the peptide of formula (II) and SCR. Details of peptide use, dose and mode of administration and time course are specified in the description of
[0200] Biliary Lipid Analyses.
[0201] For bile acid analysis, 1 μL of bile samples was diluted with 99 μL of milliQ water then incubated with the work reagent (6 mg NAD, 0.5 M hydrazine hydrate buffer, 0.05 M Na-pyrophosphate) for 4 min. The mix was then incubated with a start reagent (0.03 M Tris-EDTA; 0.3 U/mL 3-alpha-OH steroid dehydrogenase) and measured for 30 min, under excitation of 340/330 nm and emission of 440/420 nm. For phospholipid analysis, 1 μL of bile samples was diluted with 49 μL of milliQ water then incubated with the work reagent (100 mM MOPS, pH 8; 0.55 mM HVA; 20 mM CaCl; 11 U/mL Phospholipase-D; 1.66 U/mL Peroxidase; 0.1% Triton X-100) for 4 min. The mix was then incubated with a start reagent (1 M MOPS, pH 8, 50 U/mL Choline oxidase) and measured for 67.5 min, under excitation of 340/330 and emission of 440/40. For cholesterol analysis, 1 μL of bile samples was diluted with 29 μL of milliQ water then was incubated with the work reagent (100 mM MOPS, pH 8, 0.25 mM HVA; 0.1% Triton X-100) for 4 min. The mix was then incubated with a start reagent (0.1 M MOPS, pH 8, 0.06 U/mL cholesterol oxidase, 0.15 U/mL cholesterol esterase, 0.45 U/mL Peroxidase, 0.06 mM Taurocholate) and measured for 45 min, under excitation of 340/330 nm and emission of 440/420 nm. Secretion values of bile acids, phospholipids and cholesterol were calculated by multiplying concentration and bile flow values, and expressed as nmol/min/100 g body weight (BW).
[0202] Results:
[0203] The results are presented in
[0204] Conclusion:
[0205] The peptide of formula (I) and the peptide of formula (II) stimulated biliary flux and biliary secretion of cholesterol and bile acids in both wild-type and dyslipidemic mice. Hepatic excretion of cholesterol in bile, either as bile acids or cholesterol, represent the main pathway of removing excess cholesterol responsible for atherosclerosis development [6]. Also, downregulation of biliary flux and biliary lipid secretion contributes to hepatic lipotoxicity and has been documented in NASH [13] and cholestatic liver condition [17]. Thus, the peptides of formula (I) and formula (II) are good candidates to protect against the development of metabolic syndrome, cardiovascular disease (CVD), non-alcoholic fatty liver disease (NAFLD) or cholestatic liver disease.
Example 12: In Vivo Efficacy of the Peptide of Formula (II) on the Development of NASH Associated Hepatic Steatosis
[0206] Materials and Methods:
[0207] Animals.
[0208] Wild-type C57B/L6J male mice were purchased from Janvier Labs (Le Genest Saint Isle, France). Mice were caged in animal rooms under specific pathogen free conditions at the animal facility of Rangueil (Anexplo platform, US006, Toulouse, France) with a light/dark schedule of 12 h/12 h. At the initiation of the dietary intervention, all animals were 8 weeks old and were fed ad libitum with a normal chow diet (#V1535 R/M-H, Ssniff, Germany). All animal experimental procedures were conducted in accordance with institutional guidelines on animal experimentation approved by the local ethical committee of animal care and are conformed to the guidelines from Directive 2010/63/EU of the European Parliament on the protection of animals used for scientific purposes or the NIH guideline.
[0209] Mouse Model of Diet-Induced Hepatic Steatosis.
[0210] 8 weeks old mice were fed fed western-diet for 4-weeks (Envigo #TD.88137 containing 0.2% cholesterol, 42% kcal from fat, 34% sucrose by weight). For the 2 last weeks of the 4-week period, mice were daily intraperitoneally administrated at 1 mg/kg/day with the peptide of formula (I) or PBS (control group). Following the treatment period, mice were fasted overnight, anesthetized by intra-peritoneal injection of ketamine and xylazine hydrochloride then killed by exsanguination. Body weight, liver triglyceride content, plasma lipids and transaminases were determined at sacrifice.
[0211] Liver Triglyceride Content.
[0212] 100 mg of liver tissue were homogenized in 900 μL of phosphate buffer pH 7.4 until complete tissue lysis. Lipids were extracted by mixing 125 μL of lysates with 1 mL of CHCl.sub.3:MeOH (2:1). After centrifugation, the chloroform phase was evaporated under nitrogen flux, and the dried residue was solubilized in 200 μL of isopropanol. Triglycerides were measured using commercial kits based on GPO-PAP detection method (Biolabo SA, Maizy, France). Results were expressed as mg of triglycerides/g liver.
[0213] Analyses of Plasma Lipid and Transaminase Levels
[0214] Triglycerides and cholesterol levels were determined using commercial colorimetric kits (Biolabo SA, Maizy, France) based on CHOD-PAP and GPO-PAP detection methods, coupling enzymatic reaction and spectrophotometric detection of reaction end products. Alanine-aminotransferase (ALT) and aspartate-aminotransferase (AST) levels were determined using a COBAS-MIRA+ biochemical analyser (Anexplo facility, Toulouse, France).
[0215] Oral Glucose Tolerance Test (OGTT)
[0216] After 8 weeks of diet, mice were treated after an overnight fasting period with an oral gavage glucose load (3 mg/g body weight). Blood glucose levels were measured by tail vein sampling with portable glucometer (Accu-check, Roche) 30 min before oral glucose load and at 0, 15, 30, 45, 60, 90 and 120 min after oral glucose load. Plasma insulin concentration was determined 30 min before and 15 min after glucose loading in 5 μL of plasma using an ELISA kit (Mercodia, Uppsala, Sweden) according to the manufacturer's instructions.
[0217] Results:
[0218] The results are presented in
[0219] Concerning glucose metabolism, the peptide of formula (II) improved oral glucose tolerance (
[0220] The treatment with the peptide of formula (I) demonstrated benefits on NASH-associated steatosis and glucose metabolism. The peptide of formula (I) is therefore a good candidate to treat and reverse hepatic steatosis and to resolve dysregulation of glucose metabolism, particularly in non-alcoholic steatohepatitis (NASH).
Example 13: In Vivo Efficacy of the Peptide of Formula (I) on the Development of NASH Associated Hepatic Fibrosis
[0221] Materials and Methods:
[0222] Animals.
[0223] Wild-type C57B/L6J male mice were purchased from Janvier Labs (Le Genest Saint Isle, France). Mice were caged in animal rooms under specific pathogen free conditions at the animal facility of Rangueil (Anexplo platform, US006, Toulouse, France) with a light/dark schedule of 12 h/12 h. At the initiation of the dietary intervention, all animals were 8 weeks old and were fed ad libitum with a normal chow diet (#V1535 R/M-H, Ssniff, Germany). All animal experimental procedures were conducted in accordance with institutional guidelines on animal experimentation approved by the local ethical committee of animal care and are conformed to the guidelines from Directive 2010/63/EU of the European Parliament on the protection of animals used for scientific purposes or the NIH guideline.
[0224] Mouse Model of Diet-Induced NASH Associated Hepatic Fibrosis.
[0225] 8 weeks old mice were fed for 6 week a choline-deficient, L-amino acid-defined, high-fat diet (CDAHFD #A06071302, Research Diet, USA) consisting of 60 kcal % fat and 0.1% methionine by weight [11]. For the 2 last weeks of the 6-week period, a group of mice were implanted with an osmotic pump containing the peptide of formula (I). Briefly, 200 μL osmotic pump were filled with 10 mg/mL of the peptide of formula (I) in PBS and were implanted subcutaneously into the mice according to the manufacturer's instructions (Alzet®, model pump #2002), to insure the peptide of formula (I) release at an estimated rate of 0.5 μL/h, which corresponds to an estimated amount of delivery of 5 mg of the peptide of formula (I) per kilogram of body weight per day (5 mg/kg BW/day). The control group was composed of mice that underwent the same chirurgical procedure used for osmotic pump implantation (sham-operated mice).
[0226] Liver histology. Following the treatment period, mice were fasted overnight, anesthetized by intra-peritoneal injection of ketamine and xylazine hydrochloride then killed by exsanguination. A sample of the main liver lobe was fixed with paraformaldehyde, embedded in paraffin, and sliced into 5 μm sections, then deparaffinized, rehydrated. Fibrosis was assessed by Sirius Red staining. Briefly, sections were incubated for 10 min in 1% Sirius Red (Sigma-Aldrich) dissolved in saturated picric acid and then rinsed with distilled water. Sections were then dehydrated for 15 min with absolute ethanol and incubated with Histoclear® clearing agent (Euromedex, France) before mounting with Distyrene Plasticizer Xylene (DPX) and coverslipping. After staining, slides were scanned with a NanoZoomer 2.0 RS (Hamamatsu, Japan).
[0227] Hepatic Hydroxyproiine Quantification.
[0228] Hepatic hydroxyproline was determined by hydrolizing 80-140 mg liver in a 6N HCl solution, overnight, at 110 degrees Celcius. The samples were diluted in citric-acetate buffer and treated with Chloramine T (Sigma-Aldrich-Aldrich) and 4-(dimethyl)aminobenzaldehyde (Sigma-Aldrich-Aldrich). Absorbance was measured at 550 nm and the results are expressed as micrograms of hepatic hydroxyproline per mg tissue.
[0229] Results:
[0230] The results are presented in
[0231] Conclusion:
[0232] The treatment with the peptide of formula (I) demonstrated benefits on NASH-associated fibrosis. The peptide of formula (I) is therefore a good candidate to treat and reverse hepatic fibrosis, particularly in non-alcoholic steatohepatitis (NASH).
Sequence Listing
[0233]
TABLE-US-00003 SEQ ID NO: Description Sequence 1 Peptide according the RGAGSIREAGGAFGKREQAEEER invention YFRAQSRE 2 Scramble peptide, SCR GEAKSYAEKGEARGERGTKGEFR (not according to the IFKREATD invention) 3 Signature peptide EAGGAFGK (not according to the invention) 4 Signature peptide EAGGAFG (not according to the invention) 5 Mature human IF1 GSDQSENVDRGAGSIREAGGAFGK REQAEEERYFRAQSREQLAALKKH HEEEIVHHKKEIERLQKEIERHKQ KIKMLKHDD
REFERENCES
[0234] [1] Martinez et al. 2003. Nature 421; 75-79 [0235] [2] Jacquet et al. 2005 Cell Mol Life Sci 62; 2508-2515 [0236] [3] Fabre et al. Hepatology 52; 1477-1483 [0237] [4] Smith et al. Curr Opin Investig Drugs. 2010 September; 11(9): 989-996 [0238] [5] Cabou et al. 2019. Acta Physiol (Oxf).; 226(3):e13268 [0239] [6] Martinez et al. 2015. Atherosclerosis. January; 238(1):89-100 [0240] [7] Martinez et al. 2003. Nature 421; 75-79 [0241] [8] Iwakiri Y. et al. Trends Pharmacol Sc. 2015 August; 36(8):524-36 [0242] [9] Musso, G. et al. 2013. Prog. Lipid Res. 52, 175-191 [0243] [10] Min, H. K. et al. 2012. Cell Metab. 15, 665-674 [0244] [11] Matsumoto et al. 2013. Int. J. Exp. Path. 94, 93-103 [0245] [12] Havel R J et al. (1955) J. Clin. Invest. 34, 1345-1353 [0246] [13] Ioannou G. N. Trends in Endocrinology & Metabolism, February 2016, Vol. 27, No. 2 [0247] [14] Lichtenstein et al. Cardiovasc Res. 2015 May 1; 106(2):314-23 [0248] [15] Castaing-Berthou A et al. Cell Physiol Biochem. 2017; 42(2):579-593 [0249] [16] Farah C et al. Nat Rev Cardiol. 2018; 15(5):292-316. [0250] [17] Corpechot et al. Clin Res Hepatol Gastroenterol. 2012 [0251] [18] Pasut, G.; Veronese, F. M. (2012). “State of the art in PEGylation: The great versatility achieved after forty years of research”. Journal of Controlled Release. 161 (2): 461-472 [0252] [19] Rai, A K (2013) J. Bioenerg. Biomembr. 45, 569e579 [0253] [20] Cardouat et al. Biochim Biophys Acta Mol Cell Biol Lipids. 2017 September; 1862(9):832-841