Abstract
The present invention relates to a conjugated molecule comprising a peptide displaying at least 0.1% activity of native glucagon-like peptide 1 (GLP-1) at the GLP-1 receptor, and an N-methyl-D-aspartate receptor (NMDAR) antagonist, the peptide being covalently bonded to the NMDAR antagonist either directly or through a chemical linker, the conjugated molecule for use in therapy, pharmaceutical composition comprising the conjugated molecule, a method of reducing body weight of a mammal comprising administering the conjugated molecule to the mammal, and a non-therapeutic method of reducing body weight of a mammal comprising orally administering the conjugated molecule to the mammal.
Claims
1. A conjugated molecule comprising a peptide displaying at least 0.1% activity of native glucagon-like peptide 1 (GLP-1) at the GLP-1 receptor, and an N-methyl-D-aspartate receptor (NMDAR) antagonist, the peptide being covalently bonded to the NMDAR antagonist either directly or through a chemical linker.
2. The conjugated molecule according to claim 1, wherein the NMDAR antagonist in its free form has a dissociation constant K.sub.d with an NMDA receptor in the range of about 0.5 nM to 1000 nM.
3. The conjugated molecule of claim 1, wherein the peptide is of the glucagon-superfamily.
4. The conjugated molecule according to claim 1, wherein the peptide has at least 80% amino acid sequence identity to SEQ ID NO:1.
5. The conjugated molecule according to claim 1, wherein the peptide consists of at least 10 amino acids and no more than 60 amino acids.
6. The conjugated molecule according to claim 1, wherein the NMDAR antagonist is covalently bonded at the C-terminal region of the peptide.
7. The conjugated molecule according to claim 1, wherein the NMDAR antagonist is covalently bonded to the peptide via a cleavable chemical linker, the cleavable chemical linker being selected from acid-cleavable linkers, enzyme-cleavable linkers, peptide-cleavable linkers, and linkers comprising a disulfide group.
8. The conjugated molecule according to claim 7, wherein the chemical linker has the formula R.sub.1-R.sub.3-S-S-R.sub.4-R.sub.5-O-CO-R.sub.2, wherein R.sub.1 is the peptide, R.sub.2 the NMDAR antagonist, R.sub.3 is optional and when present is selected from C(CH.sub.3).sub.2, CH.sub.2—CH.sub.2, or CH.sub.2, bonded to a side chain of the peptide or to a carbon atom of the backbone chain of the peptide, R.sub.4 is (CH.sub.2).sub.n or C.sub.6H.sub.4, R.sub.5 is optional and when present is selected from C(CH.sub.3).sub.2, CH.sub.2—CH.sub.2, or CH.sub.2, and n is 1, 2, 3 or 4.
9. The conjugated molecule according to claim 1, wherein the NMDAR antagonist is MK801, neramexane or memantine.
10. (canceled)
11. A method of treatment of obesity, binge eating disorder, insulin resistance, type 2 diabetes, dyslipidaemia, non-alcoholic steatohepatitis, or non-alcoholic fatty liver disease, comprising administering the conjugated molecule according to claim 1 to a subject.
12. A pharmaceutical composition comprising the conjugated molecule according to claim 1 or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable carrier.
13. A method of reducing body weight of a mammal comprising administering the conjugated molecule according to claim 1 to the mammal.
14. A non-therapeutic method of treatment of a mammal for reducing body weight, which method comprises orally administering to said mammal the conjugated molecule according to claim 1.
15. The non-therapeutic method of treatment of a mammal for reducing body weight according to claim 14, wherein the mammal has a non-pathogenic body mass index
Description
BRIEF DESCRIPTION OF FIGURES
[0064] The above, as well as additional objects, features, and advantages of the present invention is better understood through the following illustrative and non-limiting detailed description of embodiments of the present invention, with reference to the appended drawings, wherein:
[0065] FIG. 1 shows an example of a peptide and NMDAR antagonist conjugate,
[0066] FIG. 2 displays the mechanism by which MK801 is released from the conjugate of FIG. 1.
[0067] FIG. 3 shows the in vitro human plasma stability of three versions of the conjugate of FIGS. 1 and 2,
[0068] FIG. 4 shows the weight-lowering effect of a conjugate of a peptide of SEQ ID NO:1 and memantine (GLP-1 Cys40/Memantine),
[0069] FIG. 5 shows the effect of GLP-1 Cys40/Memantine on cumulative food intake in mice,
[0070] FIG. 6 shows the effect of GLP-1 Cys40/Memantine on daily food intake in mice,
[0071] FIG. 7 shows the effect of GLP-1 Cys40/Memantine on body composition in mice,
[0072] FIG. 8 shows the weight-lowering effect of a conjugate of a peptide of SEQ ID NO:1 and MK801 (GLP-1 Cys40/MK801),
[0073] FIG. 9 shows the effect of GLP-1 Cys40/MK801 conjugate on cumulative food intake in mice,
[0074] FIG. 10 shows the effect of GLP-1 Cys40/MK801 conjugate on daily food intake in mice,
[0075] FIG. 11 shows the effect of GLP-1 Cys40/MK801 conjugate on body composition in mice,
[0076] FIG. 12 shows the weight-lowering effect of a conjugate of a peptide of SEQ ID NO:1 and MK801 (GLP-1 Pen40/MK801), wherein the cysteine residue in SEQ ID NO:1 has been substituted by L-penicillamine,
[0077] FIG. 13 shows the effect of GLP-1 Pen40/MK801 conjugate on daily food intake in mice, and
[0078] FIG. 14 shows the effect of GLP-1 Pen40/MK801 conjugate on body weight in mice.
[0079] FIG. 15 shows a synthesis route of a chemical linker derivatized memantine.
[0080] FIG. 16 shows an example synthesis route for the conjugation of peptides and small molecules with amino groups.
[0081] FIG. 17 shows a synthetic route for synthesizing a chemical linker derivatized MK801.
[0082] FIG. 18 shows a conjugation reaction of a linker derivatized MK801 with a peptide (the peptide having an amino acid sequence given by SEQ ID NO:1).
[0083] FIG. 19 shows the synthetic route for chemical synthesis of a linker derivatized MK801.
[0084] FIG. 20 shows a reaction for conjugation of linker derivatized MK801 with a peptide having the amino acid sequence of SEQ ID NO:1 and having the Pen40 modification.
[0085] FIG. 21 shows the effect of different doses of GLP-1 Pen40/MK801 conjugate on body weight in mice.
[0086] FIG. 22 shows the effect of different doses of GLP-1 Pen40/MK801 conjugate on daily food intake in mice.
[0087] FIG. 23 shows the effect of different doses of GLP-1 Pen40/MK801 conjugate on blood glucose in mice after a compound tolerance test.
[0088] FIG. 24 shows the effect of active and inactive MK801 in a GLP-1 Pen40/MK801 conjugate on body weight in mice.
[0089] FIG. 25 shows the effect of active and inactive MK801 in a GLP-1 Pen40/MK801 conjugate on cumulative food intake in mice.
[0090] FIG. 26 shows the in vitro human plasma stability of active and inactive MK801 used for the conjugate GLP-1 Pen40/MK801.
[0091] FIG. 27 shows the effect of GLP-1/MK801 conjugate with different linkers on body weight in mice.
[0092] FIG. 28 shows the effect of GLP-1/MK801 conjugate with different linkers on cumulative food intake in mice.
[0093] FIG. 29 is the GLP-1/MK801 conjugate with one type of linker.
[0094] FIG. 30 is the GLP-1/MK801 conjugate with one type of linker.
[0095] FIG. 31 shows the effect of GLP-1 Pen40/MK801 conjugate on sucrose intake in mice.
[0096] FIG. 32 show the effect of GLP-1 Pen40/MK801 conjugate on blood glucose in db/db mice after a compound tolerance test.
[0097] FIG. 33 shows the effect of the co-agonist GIP/GLP-1/MK801 conjugate on body weight in mice.
[0098] FIG. 34 shows the effect of the co-agonist GIP/GLP-1/MK801 conjugate on cumulative food intake in mice.
[0099] FIG. 35 shows an amino acid sequence alignment between co-agonist GLP-1/GIP of SEQ ID NO: 9 and the GLP-1 peptide of SEQ ID NO: 1 used in the drug conjugates, wherein X.sub.1 is D-alanine, D-serine, alpha-aminoisobutyric acid, N-methyl-serine, glycine, or valine, and X.sub.2 is cysteine (hCys40/Cys40) or L-penicillamine (Pen40).
[0100] FIG. 36 shows the effect of different NDMAR antagonists conjugated with GLP-1 Pen40 on body weight in mice.
[0101] FIG. 37 shows the effect of different NDMAR antagonists conjugated with GLP-1 Pen40 on daily food intake in mice
[0102] FIG. 38 shows the effect of different NDMAR antagonists conjugated with GLP-1 Pen40 on cumulative food intake in mice.
DETAILED DESCRIPTION
[0103] FIG. 1 shows an example of a peptide and NMDAR antagonist conjugate 100, which consists of MK801 101 chemically appended to a C-terminal cysteine 102 of the peptide of SEQ ID NO:1 103 through a chemical linker 104, the chemical linker 104 comprising a disulfide group 105. A side chain 106 of the C-terminal cysteine 102, may optionally be derivatised, such that length n of the side chain 106 is 1 or 2 carbon atoms and/or R is hydrogen or methyl. A modification called hCys40 of the side chain 106 has length n=2 carbon atom and R=hydrogen. A modification called hCys40 of the side chain 106 has length n=1 carbon atom and R=methyl. Regular cysteine is called Cys40.
[0104] FIG. 2 displays the mechanism by which MK801 is released from the conjugate 100 of FIG. 1. The chemical linker 104 comprising a disulfide group 105 is self-immolative and may be reduced in a reducing environment (not shown) such as an intracellular environment to produce thiol groups, separating the peptide part of the conjugate 107 from the MK801 part 108 of the conjugate. On the MK801 part 108 of the molecule, a liberated nucleophilic thiol 109 undergoes spontaneous intramolecular cyclization to release MK801 as the native unmodified MK801 drug (free form of MK801).
[0105] FIG. 3 shows the in vitro human plasma stability of three versions of the conjugate 100 of FIGS. 1 and 2, each version having a different cysteine derivative or residue. The first version GLP-1 Pen40/MK801 has cysteine derivative Pen40, the second version GLP-1 hCys40/MK801 has the cysteine derivate hCys40, and the third version GLP-1 Cys40/MK801 has an unmodified cysteine Cys40. The plasma stability of each version is shown as percentage recovery over time. LCMS analysis (not shown) revealed that the major contribution to conjugate degradation originates from deconjugation of MK801 likely by disulfide exchange of the linker. Consequently, single substitution of the C-terminal cysteine 102 (hCys40/Cys40) to L-penicillamine (Pen40) drastically increased the plasma stability by decreasing the accessibility of the disulfide bond due to increased steric hindrance.
[0106] FIG. 4-14 display the results of the in vivo mice studies disclosed in example 8.
[0107] FIG. 4 shows the weight-lowering effect of a conjugate of a peptide of SEQ ID NO:1 and memantine chemically appended via the linker shown in FIGS. 1 and 2, wherein the cysteine residue is unmodified cysteine (GLP Cys40/Memantine) (40 nmol/kg) and equimolar doses of the peptide of SEQ ID NO:1 (GLP-1 Cys40) or memantine measured in body weight percentage (BW %) of diet induced (DIO) mice treated for 8 days. Data is expressed as mean±SEM and N is 8 per group. Both GLP-1 Cys40 and GLP-1 Cys40/Memantine resulted in a lowered BW % in the DIO mice, the latter conjugate resulting in approximately 7% BW % reduction after 8 days of treatment.
[0108] FIG. 5 shows the effect of GLP-1 Cys40/Memantine and equimolar doses of GLP-1 Cys40 or memantine on cumulative food intake (FI Cumulative, gram per day) in DIO mice treated for 8 days. Data is expressed as mean±SEM and N=8 per group. Over the course of the treatment, a lowered cumulative food intake was observed in mice treated with GLP-1 Cys40 and GLP-1 Cys40/Memantine compared to the control (vehicle) and to memantine.
[0109] FIG. 6 shows the effect of GLP-1 Cys40/Memantine (40 nmol/kg) or equimolar doses of GLP-1 Cys40 or memantine on daily food intake (FI daily, gram per day) in DIO mice treated for 8 days. Data is expressed as mean±SEM and N=8 per group. During the 8 days of treatment, GLP-1 Cys40 and GLP Cys40/Memantine showed in general a lowered daily food intake compared to the memantine-treated mice and the control group (vehicle, i.e. saline). At the end of the study, mice treated with GLP-1 showed only a slight reduction in food intake compared to the control group (vehicle).
[0110] FIG. 7 shows the effect of GLP-1 Cys40/Memantine (40 nmol/kg) or equimolar doses of GLP-1 Cys40 or memantine on body composition (Delta change, g), in terms of change in fat and lean body mass, in DIO mice treated for 8 days. Data is expressed as mean±SEM and N=8 per group. After 8 days, mice treated with memantine, GLP-1 Cys40, and GLP-1 Cys40/Memantine all displayed a reduction in fat body mass, while nearly no change was seen in lean body mass. GLP-1 Cys40/Memantine resulted in the highest change in fat body mass with approximately 4 g fat mass reduction observed in the mice treated with this conjugate.
[0111] FIG. 8 shows the weight-lowering effect (BW %) of GLP-1 Cys40/MK801 (100 nmol/kg) or equimolar doses of GLP-1 Cys40 or MK801 in DIO mice treated for 10 days. Data is expressed as mean±SEM and N=8 per group. While MK801 showed nearly no percentage change in body weight (BW), both GLP-1 Cys40 and GLP-1 Cys40/MK801 resulted in approximately 8 and 12% reduction in BW, respectfully, after 10 days of treatment.
[0112] FIG. 9 shows the effect of GLP-1 Cys40/MK801 (100 nmol/kg) or equimolar doses of GLP-1 Cys40 or MK801 on cumulative food intake (FI Cumulative) in DIO mice treated for 10 days. Data is expressed as mean±SEM, N=8 per group. Over the course of the 10 days of treatment, a lowered cumulative food intake was observed in mice treated with GLP-1 Cys40 and GLP-1 Cys40/MK801 compared to the control (vehicle) and to MK801. Best results were observed for GLP-1 Cys40/MK801-treated mice which had a cumulative food intake of approximately 13 g/day, which is approximately 10 g/day less than the vehicle-treated mice (approximately 23 g/day).
[0113] FIG. 10 shows the effect of GLP-1 Cys40/MK801 (100 nmol/kg) or equimolar doses of GLP-1 Cys40 or MK801 on daily food intake (FI daily) in DIO mice treated for 10 days. Data is expressed as mean±SEM, N=8 per group. In general, the daily food intake fluctuated at varying degrees during the 10-days treatment, however, a reduction in food intake was observed all 10 days in mice treated with GLP-1 Cys40/MK801 compared to the control group (vehicle).
[0114] FIG. 11 shows the effect of GLP-1 Cys40/MK801 (100 nmol/kg) or equimolar doses of GLP-1 Cys40 or MK801 on body composition (Delta change, g), in terms of change in fat and lean body mass, in DIO mice treated for 10 days. Data is expressed as mean±SEM and N=8 per group. After the 10-days treatment, the group of GLP-1 Cys40/MK801-treated mice displayed a reduction in both fat and lean body mass, with the change in fat mass (reduction of almost 5 g) being most prominent.
[0115] FIG. 12 shows the effect of GLP-1 Pen40/MK801 (100 nmol/kg) or equimolar doses of GLP-1 Cys40 or MK801 on body weight % of DIO mice treated for 5 days. Data is expressed as mean±SEM, N=8 per group. After 5 days of treatment, GLP-1 Pen40/MK801-treated mice showed approximately 15% body weight reduction. In comparison GLP-1 Cys40-treated mice showed approximately 4% body weight reduction.
[0116] FIG. 13 shows the effect of GLP-1 Pen40/MK801 (100 nmol/kg) or equimolar doses of GLP-1 Cys40 or MK801 on food intake (g/day) in DIO mice treated for 5 days. Data is expressed as mean±SEM, N=8 per group. Mice treated with GLP-1 Pen40/MK801 displayed an instant reduction in food intake compared to the control group (vehicle-treated mice). Furthermore, the lowered food intake was sustained at around 0.2-0.7 g/day during the 5-days treatment period.
[0117] FIG. 14 shows the effect of GLP-1 Pen40/MK801 (100 nmol/kg) or equimolar doses of GLP-1 Pen40 or GLP-1 Cys40 on body weight % in DIO mice treated for 5 days. Data is expressed as mean±SEM, N=7 per group. Mice treated with GLP-1 Pen40 or GLP-1 Cys40 displayed similar reductions in body weight % (approximately 6%), while the GLP-1 Pen40/MK801 showed approximately 12% reduction in body weight. Additionally, based on the slope of the curve, it would seem that a further reduction in body weight could be expected for the GLP-1 Pen40/MK801 if the treatment was extended. FIGS. 21 and 22 show the effect of different doses (50 nmol/kg and 100 nmol/kg) of GLP-1 Pen40/MK801 conjugate compared to a control group (Vehicle, i.e. saline) on body weight (BW %, FIG. 21) and daily food intake (Daily FI in grams, FIG. 22) in DIO mice treated for 5 days. Data is expressed as mean±SEM, N=5 to 6 per group. Over the course of the treatment, a lowered body weight and daily food intake was observed for mice treated with both doses (50 nmol/kg and 100 nmol/kg) compared to the control group, with the most significant reduction observed for mice subjected to daily subcutaneous injections of 100 nmol/kg of the conjugate.
[0118] FIG. 23 shows the effect of different doses (50 nmol/kg and 100 nmol/kg) of GLP-1 Pen40/MK801 conjugate compared to a control group (vehicle, i.e. saline) on blood glucose level (mmol/L) in DIO mice subjected to ipGTT on day 7 of the treatment course. The blood glucose levels were measured over a course of 120 minutes. Data is expressed as mean±SEM, N=5 to 6 per group. In general, both doses, i.e. 50 nmol/kg and 100 nmol/kg, of the conjugate result in a significantly lower initial increase and overall lower blood glucose levels compared to the control group.
[0119] FIGS. 24 and 25 show the effect of active and inactive MK801 conjugated with GLP-1 Pen40 compared to a control group (vehicle, i.e. saline) on body weight (Δ Body weight in %, FIG. 24) and cumulative food intake (Cumulative FI in grams, FIG. 25) in DIO mice treated for 7 days. Data is expressed as mean±SEM, N=8 per group. Over the course of the treatment, a lowered body weight and cumulative food intake was observed for mice treated with GLP-1 Pen40 conjugated with active MK801. The conjugate with inactive MK801 showed similar results to unconjugated GLP-1 Pen40. It is concluded that MK801 and GLP-1 have a synergistic effect in reducing body weight and cumulative food intake in mice.
[0120] FIG. 26 shows the in vitro human plasma stability of active and inactive MK801 versions of the conjugate GLP-1 Pen40/MK801 compared to a PBS control. The plasma stability of inactive and active MK801 is shown as percentage (%) recovery over time (hours). The two conjugates display nearly identical plasma stabilities independent of whether MK801 is active or inactive.
[0121] FIGS. 27 and 28 show the effect of GLP-1/MK801 conjugate (100 nmol/kg) with different linkers compared to a control group (vehicle, i.e. saline) on body weight (BW in %, FIG. 27) and cumulative food intake (Cumulative FI in grams, FIG. 28) in DIO mice for 7 days. Data is expressed as mean±SEM, N=5 to 6 per group. The structures of the GLP-1/MK801 conjugates with different linkers are shown in FIG. 20 (GLP-1 Pen40/MK801), FIG. 29 (GLP-1 Lys40-triazole-PEG4-Val-Cit-PAB-MK801)) and FIG. 30 (GLP-1 Cys40-mc-Val-Cit-PAB-MK801). Over the course of treatment, mice treated with conjugates of GLP-1/MK801 with different linkers showed similar reduction in cumulative food intake. The most significant reduction in body weight over the 7 days of treatment was observed for the group of mice treated with GLP-1 Pen40/MK801 conjugate (approximately 20% reduction).
[0122] FIG. 31 shows the effect of GLP-1 Pen40/MK801 (100 nmol/kg) and equimolar doses of GLP-1 Pen40, MK801 or semaglutide on sucrose intake (in %) compared to the control group (vehicle, saline injection) in DIO mice treated for 8 days. Data is expressed as mean±SEM, N=8 per group. The most significant reduction in sucrose intake as expressed in comparison to the control group (vehicle) was observed for mice treated with semaglutide and the GLP-1/MK801 conjugate. It was concluded that the conjugated molecules of the invention are effective in inducing a food reward and satiety effect in the treated mice.
[0123] FIG. 32 shows the effect of GLP-1 Pen40/MK801 conjugate (100 nmol/kg) and equimolar doses of MK801 or semaglutide on blood glucose (mmol/L) in db/db (diabetic) mice subjected to ipGTT on day 7 of the treatment course. The blood glucose levels were measured over a course of 24 hours. Data is expressed as mean±SEM, N=8 per group. Mice treated with either semaglutide or the conjugate GLP-1 Pen40/MK801 displayed overall lower blood glucose levels compared to the control group (vehicle) and it was concluded that the conjugated molecule of the invention is suitable for treatment of diabetic mice.
[0124] FIGS. 33 and 34 show the effect of co-agonist GIP/GLP-1 Pen40/MK801 conjugate (SEQ ID NO:9) (50 nmol/kg) and a equimolar dose of GLP-1/GIP on body weight (in %, FIG. 33) and cumulative food intake (Cumulative FI in grams, FIG. 34) compared to the control group (vehicle, i.e. saline injection) in DIO mice treated for 7 days. Data is expressed as mean±SEM, N=8 per group. The most significant effect was observed in mice treated with the GIP/GLP-1/MK801 conjugate which mice showed an overall reduction in body weight of approximately 25% compared to the control group and an approximately 3 g cumulative food intake compared to the 15 g cumulative food intake observed for the control group.
[0125] FIGS. 36 to 38 show the effect of different NDMAR antagonists, i.e. MK801, memantine and neramexane, conjugated with GLP-1 Pen40 (100 nmol/kg) on body weight (in %, FIG. 36), daily food intake (Food intake, gram per day, FIG. 37), and cumulative food intake (cumulative FI in grams, FIG. 38) compared to the control group (vehicle, i.e. saline) in DIO mice treated for 5 days. Data is expressed as mean±SEM, N=8 per group. Over the course of the treatment, mice treated with the GLP-1 Pen40 conjugated with either MK801, memantine or neramexane all displayed a significant reduction in body weight and reduced daily and cumulative food intakes compared to the control group. It is concluded that different NMDAR antagonist may be conjugated to the peptides of the invention to obtain the same beneficial effect on body weight and food intake in mice.
[0126] Conclusion
[0127] The presented data demonstrate that chemical conjugation of a GLP1 analogue and an NMDAR antagonist represents a novel medicinal strategy for effectively reversing obesity. Conjugates based on this strategy are superior in suppressing food intake and lowering body weight relative to the GLP-1 peptide control and are not flawed with adverse central effects of NMDAR antagonism.
Examples
Example 1: Preparation of Peptides and Peptide-NMDAR Antagonist Conjugates
[0128] Materials: All solvents and reagents were purchased from commercial sources and used without further purification. H-Rink amide ChemMatrix® resin was used for peptide elongation. Unless otherwise stated Fmoc-protected (9-fluorenylmethyl carbamate) amino acids were purchased from Iris-Biotech or Gyros Protein Technologies, and H-Rink amide ChemMatrix® resin, 35-100 mesh; loading of 0.40-0.60 mmol/g from Sigma Aldrich. The commercially available AP-Fmoc amino acid building blocks were purchased as the following sidechain protected analogs: Arg, Pmc; Asp, OtBu; Cys, Trt; Gln, Trt; His, Trt; Lys, Trt; Ser, tBu; and Trp, Boc (Pmc=2,2,5,7,8-pentamethylchoman-6-sulfonyl, OtBu=tert-butyl ester, Trt=trityl, Boc=tert-butyloxycarbonyl, and tBu=tert-butyl ether).
[0129] All peptides and conjugates of peptides and NMDAR antagonists were characterized by analytical reverse phase ultra-performance liquid chromatography (RP-UPLC) (Waters) and electrospray ionization liquid chromatography mass spectrometry (ESI-LCMS) coupled to a Agilent 6410 Triple Quadrupole Massfilter with a C18 column (Zorbax Eclipse, XBD-C18, 4.6×50 mm). The ESI-LCMS was eluting with a binary buffer system composed of H.sub.2O:MeCN:TFA (A: 95:5:0.1, B: 5:95:0.1) at a flow rate of 0.75 mL/min. Purities were determined by RP-UPLC equipped with a C18 column (Acquity UPLC BEH C18, 1.7 μm, 2.1×50 mm) eluting with a binary buffer system composed of H.sub.2O:MeCN:TFA (A: 95:5:0.1, B: 5:95:0.1) at a flow rate of 0.45 mL/min.
[0130] Automated peptide synthesis protocol for Fmoc-protection scheme: Peptides were prepared as their C-terminally amidated derivatives using a Prelude X, induction heating assisted, peptide synthesizer (Gyros Protein Technologies, Tucson, Ariz., USA) with 10 mL glass vessels. All reagents were freshly prepared as stock solutions in DMF: Fmoc-protected amino acid (0.2 M), HCTU (0.5 M), DIPEA (1.0 M) and piperidine (20% v/v). Peptide elongation was achieved by consecutive synthetic manipulations using the following protocol: Deprotection (2×2 min, RT, 300 rpm shaking) and coupling (2×5 min, 75° C., 300 rpm shaking, for Arg and His 2×5 min, 50° C., 300 rpm shaking). Peptides were prepared using double and triple couplings consisting of AA/HCTU/DIPEA (ratio 1:1.25:2.5) in 5-fold excess compared to the resin.
[0131] Peptide cleavage: The synthesised peptides were liberated from the peptidyl resin by addition of 1.5 mL cleavage cocktail (2.5% EDT, 2.5% H.sub.2O, 2.5% TIPS, 2.5% thioanisole in TFA) per 100 mg peptidyl resin followed by agitation for 2 hours. The crude peptides were precipitated in cold diethyl ether, centrifuged at 2500×g for 10 min at 4° C., re-dissolved in MeCN:H.sub.2O:TFA (ratio 1:1:0.01), filtered and lyophilized.
[0132] Purification: The crude peptide or conjugates of peptides and NMDAR antagonist was analyzed by RP-UPLC and ESI-LCMS or MALDI-TOF mass spectrometry prior to purification. Purifications were performed with a reverse-phase high-performance liquid chromatography (RP-HPLC) system (Waters) equipped with a reverse phase C18 column (Zorbax, 300 SB-C18, 21.2×250 mm) and eluting with a linear gradient (flow rate 20 mL/min) using a binary buffer system of H.sub.2O:MeCN:TFA (A: 95:5:0.1; B: 5:95:0.1). Fractions were collected at intervals of 0.3 minutes and characterized ESI-LCMS. Purity was determined by RP-UPLC at 214 nm, and fractions with purities >95% were pooled and lyophilized. The final lyophilized products were used in further experiments.
[0133] Conjugation protocol for assembly of conjugates of peptides and NMDAR antagonists: The pure peptide and the pure thiopyridyl-activated NMDAR antagonist conjugate was dissolved in a binary solvent system (A: DMF; 6 M Guanidine, 1.5 M Imidazole in H.sub.2O at pH=8) (ratio 7:1) and agitated for at least 2 hours. The crude reaction mixture was monitored by analytical RP-UPLC and ESI-LCMS. Upon completion, the reaction mixture was diluted with buffer A and buffer B and purified directly using RP-HPLC eluting with a linear gradient.
[0134] Desalting: All peptides were desalted prior to biological experiments. Desalting was performed by consecutively re-dissolving the peptide or the conjugate of a peptide and an NMDAR antagonist in dilute aqueous 0.01 M HCl followed by lyophilization, repeated 3 times. The purity of the peptide or the conjugate was monitored by RP-UPLC and ESI-LCMS before being used for in vivo or in vitro experiments.
Preparation of GLP-1 Cys40/Memantine (Cysteine Linked).
[0135] A GLP-1 peptide with the amino acid sequence of SEQ ID NO:1 was synthesized using the Fmoc protocol as described above and conjugated to a chemical linker derivatized memantine analog. Synthesis of chemical linker derivatized memantine was performed via the synthetic route shown in FIG. 15. The first step in the synthetic route took place in MeOH at room temperature for 2 hours. The second step was carried out in CH.sub.2Cl.sub.2 in the presence of pyridine at 0° C. for 2 hours. The third step was carried out in DMF in the presence of N,N-Diisopropylethylamine (DIPEA) at 55° C. for 5 days. The final step (conjugation) was performed in a 6M guanidine, 1.5M imidazole buffer at room temperature for 2 hours.
[0136] 2′-Pyridyldithio ethanol. In a dry round-bottomed flask equipped with a magnetic stirring bar and under N.sub.2 atmosphere, 2′-aldrithiol (4.71 g, 21.3 mmol, 3 equiv.) was dissolved in dry MeOH (20 mL), followed by dropwise addition of 2-mercaptoethanol (0.56 g, 7.1 mmol, 0.5 mL, 1 equiv.) via a syringe. The reaction was left for 2 hours at ambient temperature before concentrated in vacuo. The crude yellow oil was purified by silica gel flash chromatography (EtOAc:CH.sub.2Cl.sub.2, 2:8), affording 2′-Pyridyldithio ethanol as a clear oil (1.33 g, 100%). R.sub.f=0.48; .sup.1H NMR (600 MHz, Chloroform-d) δ 8.49 (d, J=5.0 Hz, 1H), 7.57 (td, J=7.7, 1.8 Hz, 1H), 7.44-7.36 (m, 1H), 7.16-7.11 (m, 1H), 5.32 (s, 1H), 3.88-3.73 (m, 2H), 3.01-2.89 (m, 2H); .sup.13C NMR (151 MHz, CDCl.sub.3) δ 159.31, 149.86, 137.00, 122.12, 121.57, 58.37, 42.83.
[0137] 4-nitrophenyl (2-(pyridin-2-yldisulfaneyl)ethyl) carbonate. To a dry round-bottomed flask equipped with a magnetic stirring bar and under N.sub.2-atmosphere, 2′-Pyridyldithio ethanol (1.33 g, 7.1 mmol, 1 equiv.) and dry pyridine (0.56 g, 8.5 mmol, 0.575 mL, 1.2 equiv.) was diluted in anhydrous CH.sub.2Cl.sub.2 (15 mL). The reaction mixture was cooled to 0° C. and nitrophenyl chloroformate (1.72 g, 8.5 mmol, 1.2 equiv.) was added in one portion. The reaction was stirred for 10 minutes, allowed to reach ambient temperature and left for 2 hours under stirring. The reaction was diluted to 50 mL and extracted with 3× H.sub.2O (30 mL) and brine (30 mL), dried over MgSO.sub.4, filtered and concentrated in vacuo. The crude oil was purified by silica gel flash chromatography (Heptanes:EtOAc, 2:1), affording 4-nitrophenyl (2-(pyridin-2-yldisulfaneyl)ethyl) carbonate as a clear viscous oil (2.21 g, 89%). R.sub.f=0.34; Purity >95% (HPLC), Rt=15.99 min; UPLC/MS (ESI): m/z calcd. for C.sub.14H.sub.12N.sub.2O.sub.5S.sub.2[M+H].sup.+=353.0, found 353.3 m/z; .sup.1H NMR (600 MHz, DMSO-d.sub.6) δ 8.47 (ddd, J=4.8, 1.9, 0.9 Hz, 1H), 8.35-8.26 (m, 2H), 7.84 (td, J=7.8, 1.8 Hz, 1H), 7.78 (dt, J=8.1, 1.1 Hz, 1H), 7.58-7.48 (m, 2H), 7.26 (ddd, J=7.3, 4.8, 1.1 Hz, 1H), 4.48 (t, J=6.0 Hz, 2H), 3.24 (t, J=6.1 Hz, 2H); .sup.13C NMR (151 MHz, DMSO) δ 158.65, 155.17, 151.75, 149.66, 145.18, 137.80, 125.40, 122.53, 121.40, 119.52, 66.54, 36.42.
[0138] 2-(pyridin-2-yldisulfaneyl)ethyl (3,5-dimethyladamantan-1-yl)carbamate In a dry round-bottomed flask equipped with a magnetic stirring bar and under N.sub.2, 4-nitrophenyl (2-(pyridin-2-yldisulfaneyl)ethyl) carbonate (707 mg, 2.00 mmol, 1 equiv.) and Memantine hydrochloride (650 mg, 3.00 mmol, 1.5 equiv.) were dissolved in dry DMF (20 mL) and dry DIPEA (260 mg, 6.00 mmol, 0.35 mL, 3 equiv.) was added via syringe. Memantine was not completely dissolved and upon addition of DIPEA, the reaction turned yellow immediately. The reaction was left for 5 days followed by heating to 80° C. The reaction was then transferred to a separatory funnel with EtOAc (50 mL) and washed exhaustively with 5×half. Sat brine (50 mL) and brine (50 mL) to remove DMF. The organic layer was subsequently extracted 5×1 M aqueous NaOH (50 mL) (Until the yellow color of the aqueous layer ceased), dried over MgSO.sub.4, filtered and concentrated in vacuo. The crude oil was purified by silica gel flash chromatography eluting with a gradient (Heptanes:EtOAc, 9:1 to 3:1), affording 2-(pyridin-2-yldisulfaneyl)ethyl (3,5-dimethyladamantan-1-yl)carbamate as a glassy viscous oil (540 mg, 54%). R.sub.f=0.26; Purity >95% (HPLC), R.sub.f=19.36 min; UPLC/MS (ESI): m/z calcd. for C.sub.20H.sub.28N.sub.2O.sub.2S.sub.2 [M+H].sup.+=393.2, found 393.4 m/z; .sup.1H NMR (600 MHz, DMSO-d.sub.6) δ 8.46 (ddd, J=4.8, 1.9, 0.9 Hz, 1H), 7.85-7.75 (m, 2H), 7.25 (ddd, J=7.2, 4.8, 1.2 Hz, 1H), 6.89 (s, 1H), 4.10 (t, J=6.4 Hz, 2H), 3.05 (t, J=6.3 Hz, 2H), 1.69-1.63 (m, 2H), 1.54-1.43 (m, 4H), 1.31-1.20 (m, 5H), 1.07 (s, 2H), 0.80 (s, 6H); .sup.13C NMR (151 MHz, DMSO) δ 159.04, 153.78, 149.55, 137.79, 121.21, 119.23, 60.80, 51.40, 50.18, 47.07, 42.22, 37.46, 31.84, 30.05, 29.46.
[0139] GLP-1 Cys40 and GLP-1 Cys40/Memantine was prepared using the protocols described above. RP-UPLC and ESI-LCMS analyses determined the purity to >95%.
[0140] Preparation of GLP-1 Pen40/Memantine (Penicillamine linked). Synthesis of chemical linker derivatized memantine was performed using the synthetic route disclosed in FIG. 15. GLP-1 Pen40 and memantine were conjugated by the chemical reaction shown in FIG. 16, which was carried out in 6M guanidine, 1.5M imidazole buffer at room temperature for 2 hours.
[0141] Preparation of GLP-1 Cys40/MK801 (Cysteine linked).
A peptide with the sequence of SEQ ID NO:1 was synthesized using the Fmoc protocol disclosed above and conjugated with a chemical linker derivatized MK801 analog. Synthesis of chemical linker derivatized MK801 was performed via the second synthetic route disclosed in FIG. 17. The chemical reaction was performed in DMF in the presence of DIPEA at 55° C. for 5 days. Linker derivatized MK801 was conjugated to GLP-1 Cys40 by the chemical reaction shown in FIG. 18. The reaction was performed in a 6M guanidine, 1.5M imidazole buffer at room temperature for 2 hours. 2-(pyridin-3-yldisulfaneyl)ethyl 5-methyl-10,11-dihydro-5H-5,10-epiminodibenzo[a,d][7]annulen e-12-carboxylate. In a flame-dried schlenk round-bottomed flask equipped with a magnetic stirring bar and under N.sub.2 atmosphere, MK801 hydrochloride 191 mg, 0.86 mmol, 1.2 equiv.) was dissolved in dry DMF (10 mL) followed by addition of 4-nitrophenyl (2-(pyridin-2-yldisulfaneyl)ethyl) carbonate (253 mg, 0.72 mmol, 1.0 equiv.). Subsequently, dry DIPEA (375 μL, 2.14 mmol, 3.0 equiv.) was added and the solution turned yellow. The reaction was heated to 55° C. in an oil-bath and stirred for 4 days—until UPLC-MS indicated full consumption of the starting material. The reaction was diluted with EtOAc (50 mL) and washed thoroughly with half sat. brine (5×60 mL), 0.5 M aq. NaOH (5×60 mL) and brine. The organic layer was collected, dried over MgSO.sub.4, filtered and concentrated in vacuo. Purification by preparative HPLC (eluting with isocratic 60% B, over 17 mL/min) followed by lyophilization afforded 11 as a clear solid (250.2 mg, 80.1%); Purity >95% (HPLC), Rt=18.17 min; UPLC/MS (ESI): m/z calcd. for C.sub.24H.sub.22N.sub.2O.sub.2S.sub.2 [M+H].sup.+=435.1, found 435.4; .sup.1H NMR (600 MHz, DMSO-d.sub.6) δ 8.41 (dt, J=4.8, 1.4 Hz, 1H), 7.68 (dt, J=7.9, 4.1 Hz, 2H), 7.45 (d, J=7.1 Hz, 1H), 7.38-7.31 (m, 1H), 7.25-7.15 (m, 4H), 7.15-7.06 (m, 2H), 7.01-6.87 (m, 1H), 5.38 (d, J=5.5 Hz, 1H), 4.27-4.13 (m, 2H), 3.59 (dd, J=17.3, 5.7 Hz, 1H), 3.10 (s, 2H), 2.67-2.58 (m, 1H), 2.20 (s, 3H); .sup.13C NMR (151 MHz, DMSO) δ 158.92, 149.56, 143.37, 139.04, 137.70, 131.78, 130.25, 127.42, 127.34, 127.31, 125.88, 122.12, 121.66, 121.20, 119.19, 65.33, 62.21, 59.20, 37.55.
[0142] GLP-1 Cys40/MK801 was prepared from 2-(pyridin yldisulfaneyl)ethyl 5-methyl-10,11-dihydro-5H-5,10-epiminodibenzo[a,d][7]annulen e-12-carboxylate and GLP-1 Cys40 using the protocol disclosed above. RP-UPLC and ESI-LCMS analyses confirmed the product and determined the purity to >95%.
[0143] Preparation of GLP-1 hCys40/MK801 (Homocysteine linked).
A peptide with the amino acid sequence of SEQ ID NO:1 and the hCys40 modification was synthesized using the Fmoc protocol disclosed above and conjugated with a chemical linker derivatized MK801 analog. The chemical synthesis of linker derivatized MK801 was performed via the synthetic route shown in FIG. 19, the chemical reaction being performed in 6M guanidine, 1.5M imidazole buffer at room temperature for 2 hours.
[0144] GLP-1 hCys40: A peptide with the amino acid sequence of SEQ ID NO:1 and the hCys40 modification was prepared using the protocol disclosed above. RP-UPLC and ESI-LCMS analyses determined the purity to >95%. GLP-1 hCys40/MK801 was prepared using the protocol disclosed above. RP-UPLC and ESI-LCMS analyses determined the purity to >95%.
[0145] Preparation of GLP-1 Pen40/MK801 (Penicillamine linked).
A GLP-1 peptide derivative was synthesized using the Fmoc protocol disclosed above and conjugated with a chemical linker derivatized MK801 analog. The chemical synthesis of the chemical linker derivatized MK801 was performed via the route disclosed in FIG. 16.
[0146] GLP-1 Pen40/MK801: The conjugate was prepared using the protocol disclosed above and by the chemical reaction shown in FIG. 20, the chemical reaction being performed in 6M guanidine, 1.5M imidazole buffer at room temperature for 2 hours. RP-UPLC and ESI-LCMS analyses determined the purity to >95%.
Example 2: Investigation of In Vitro Human Plasma Stability
[0147] In vitro human plasma stability assay: Peptide stabilities were determined using normal human plasma containing citrate phosphate dextrose (3H Biomedical, lot P22). The human plasma was pre-heated at 37° C. for 15 min. Subsequently, 360 μL human plasma was spiked with 40 μL of GLP-1 Pen40/MK801, GLP-1 hCys40/MK801, or GLP-1 Cys40/MK801 conjugate stock solution (1 mM, prepared by dilution with PBS buffer from a 10 mM peptide in DMSO stock) and incubated under light shaking at 37° C. Aliquots of 45 μL were collected at t=0 and 5 additional timepoints (depending on the stability of the conjugate) and pre-treated with urea buffer (50 μL, 30 min) at 0° C., following treatment with 20% trichloroacetic acid in acetone and incubation at −20° C. overnight. After centrifugation (13400 rpm, 30 min), the supernatant was filtered and analyzed by RP-UPLC at 214 nm and ESI-LCMS. The area under the curve (AUC) was determined and plotted using prism 8.0. The half-lives (T.sub.1/2) were determined by fitting the data to a one-phase decay equation. The data is represented as the mean of three individual experiments.
Example 3: In Vivo Pharmacology Studies in Diet-Induced Obesity (DIO) Mice
[0148] C57BL6J male mice, in the following referred to as diet-induced obesity (DIO) mice, were maintained on a high-fat diet (58% energy from fat) and had, for each study, an average body weight of more than 45 gram prior to study start. Mice were either housed individually or double-housed. The mice were maintained on a 12 h dark-light cycle at 21-23° C. Compounds were administered subcutaneously once daily (between 2 pm-5 pm) and food intake (FI) and body weight (BW) measured at the corresponding time. For body composition, measures of fat and lean mass were performed prior to the study (1-3 days prior to study start) and on the final day of the study using an MRI scanner (EchoMRI). The group of mice injected with a vehicle (saline) served as the control group.
Example 4: Sucrose Preference Test in Chow-Fed Mice
[0149] C57BL6J male mice were single housed in cages and maintained on a chow diet. Compounds were administered subcutaneously once daily at a dose of 100 nmol/kg for all compounds, except semaglutide which was administered at a dose of 10 nmol/kg. The group of mice injected with a vehicle (saline) served as the control group. 8 mice were included in each treatment group. All cages were equipped with two drinking bottles and the mice acclimatized for a minimum of five days prior to start of the study. Upon study start, the water bottles were replaced by one bottle containing water and one bottle containing an aqueous sucrose solution of 10% (w/v). The sucrose bottles were distributed equally as the left and right bottle to correct for side preferences. Sucrose water intake and water intake were measured after 24 hours by weighing the bottles.
