MC4R Agonist Efficacy in Subjects with MC4R Deficiencies and Impaired NFAT Signaling

Abstract

The invention relates to a Melanocortin-4 receptor (MC4R) agonist that exhibits greater induction of NFAT signaling compared to -MSH for use in the treatment and/or prevention of a medical condition associated with MC4R deficiency in a subject having an MC4R deficiency associated with impaired Nuclear factor of activated T-cells (NFAT) signaling. The present invention further relates to an in vitro method for the diagnosis, prognosis and/or assessment of likelihood of whether a subject with, or at risk of having and/or developing, a medical condition associated with MC4R deficiency, will respond to treatment with an MC4R agonist that exhibits greater induction of NFAT signaling compared to -MSH, the method comprising (i) providing a sample from said subject, and (ii) determining whether the subject has an MC4R deficiency associated with impaired NFAT signaling by assessing said sample, (iii) wherein the presence of an MC4R deficiency associated with impaired NFAT signaling is indicative that treatment with an MC4R agonist that exhibits greater induction of NFAT signaling compared to -MSH will be effective in said subject.

Claims

1. A method of treating and/or preventing a medical condition associated with MC4R deficiency, in a subject having an MC4R deficiency associated with impaired Nuclear factor of activated T-cells (NFAT) signaling, the method comprising administering to said subject a Melanocortin-4 receptor (MC4R) agonist that exhibits greater induction of NFAT signaling compared to -MSH.

2. The method according to claim 1, wherein the agonist: a. exhibits greater induction of NFAT signaling compared to the MC4R agonist LY2112688, b. preferentially induces G.sub.q signaling over G.sub.s signaling, and/or c. preferentially induces NFAT signaling over cAMP signaling, wherein the ratio of NFAT signaling to cAMP signaling is greater compared to the ratio obtained for -MSH or the MC4R agonist LY2112688.

3. The method according to claim 1, MC4R agonist for use as a medicament according to any one of the preceding claims, wherein the agonist is setmelanotide, RM511 or the -MSH analogue MC4-NN2-0453.

4. The method according to claim 1, wherein the MC4R deficiency in the subject is not associated with impaired G.sub.s signaling.

5. The method according to claim 1, wherein the MC4R deficiency is a genetic deficiency.

6. The method according to claim 5, wherein the MC4R genetic deficiency is a MC4R mutation at one or more of the following positions of the MC4R, namely T112, S77, V166, I170, A175, T178, I251 and N274, or a mutation selected from the list consisting of T112M, S77L, V166I, I170V, A175T, T178M, I251L and N274S.

7. The method according to claim 1, wherein the MC4R deficiency is an epigenetic deficiency.

8. The method according to claim 1, wherein the medical condition associated with MC4R deficiency is obesity (adiposis), a metabolic syndrome and/or hyperphagia.

9. The method according to claim 1, wherein the medical condition associated with MC4R deficiency is early onset obesity.

10. The method according to claim 1, comprising testing a sample obtained from the subject and determining whether the individual has an MC4R deficiency associated with impaired NFAT signaling, and providing treatment if the subject is identified as having said MC4R deficiency.

11. An in vitro method for the diagnosis, prognosis and/or assessment of likelihood of whether a subject with, or at risk of having and/or developing, a medical condition associated with MC4R deficiency, will respond to treatment with an MC4R agonist that exhibits greater induction of NFAT signaling compared to -MSH, the method comprising: providing a sample from said subject, and determining whether the subject has an MC4R deficiency associated with impaired NFAT signaling by assessing said sample, wherein the presence of an MC4R deficiency associated with impaired NFAT signaling is indicative that treatment with an MC4R agonist that exhibits greater induction of NFAT signaling compared to -MSH will be effective in said subject.

12. A method for determining whether a subject has an MC4R deficiency associated with impaired NFAT signaling, wherein said subject has, or is at risk of having and/or developing, a medical condition associated with MC4R deficiency, the method comprising providing a sample from said subject and determining whether the subject has an MC4R deficiency associated with impaired NFAT signaling by assessing said sample.

13. The method according to claim 11, wherein assessing the sample comprises assessing NFAT signaling in a reporter system comprising nucleic acid sequences corresponding to those determined in patient-specific MC4R genetic material.

14. The method according to claim 13, wherein reporter system comprises means for determining phospholipase C (PLC) activation.

15. The method according to claim 11, wherein assessing the sample comprises determining nucleic acid sequence characteristics of patient-specific MC4R genetic material and preferably incorporating nucleic acid sequences corresponding to those determined in patient-specific MC4R genetic material in a reporter system.

16. The method according to claim 11, wherein the subject is determined to have an MC4R deficiency associated with impaired NFAT signaling and the subject is treated with an MC4R agonist that exhibits greater induction of NFAT signaling compared to -MSH.

17. The method according to claim 12, wherein assessing the sample comprises assessing NFAT signaling in a reporter system comprising nucleic acid sequences corresponding to those determined in patient-specific MC4R genetic material.

18. The method according to claim 17, wherein reporter system comprises means for determining phospholipase C (PLC) activation.

19. The method according to claim 12, wherein assessing the sample comprises determining nucleic acid sequence characteristics of patient-specific MC4R genetic material and preferably incorporating nucleic acid sequences corresponding to those determined in patient-specific MC4R genetic material in a reporter system.

20. The method according to claim 12, wherein the subject is determined to have an MC4R deficiency associated with impaired NFAT signaling and the subject is treated with an MC4R agonist that exhibits greater induction of NFAT signaling compared to -MSH.

Description

BRIEF DESCRIPTION OF THE FIGURES

[0218] FIG. 1: Body weight course and hunger-score during Setmelanotide treatment.

[0219] FIG. 2: Weight course of LEPR patients before setmelanotide treatment start.

[0220] FIG. 3: Blood pressure and heart rate before and during setmelanotide treatment.

[0221] FIG. 4: Skin and hair color.

[0222] FIG. 5: Determination of setmelanotide, alpha-MSH and first generation MSH LY2112688 induced effect on cAMP formation.

[0223] FIG. 6: Determination of phospholipase C activation after setmelanotide, alpha-MSH and LY2112688 challenge.

[0224] FIG. 7: Controls for off-target effect of solvents for alpha-MSH, setmelanotide and LY2112688 and effects at MC3R.

[0225] FIG. 8: Structural models of MC4R-ligand complexes.

[0226] FIG. 9: Comparison between binding modes of setmelanotide and alpha-MSH at hMC4R.

[0227] FIG. 10: Structural homology model of alpha-MSH/hMC4R/Gprotein complex with highlighted positions of mutants reported to be like wild-type for Gs mediated signaling.

[0228] FIG. 11: Functional characterization of like wild-type MC4R variants in Gs and PLC signaling after alpha-MSH and setmelanotide challenge.

[0229] FIG. 12: Functional characterization of like wild-type MC4R variants in Gs and PLC signaling after alpha-MSH and setmelanotide challenge.

[0230] FIG. 13: MC4R deficiency associated with impaired NFAT.

[0231] FIG. 14: Investigation of NFAT signaling in mouse embryonic fibroblasts (MEF) deficient in Gq (MEF Dq).

[0232] FIG. 15: Test of further MC4R variants.

[0233] FIG. 16. Schematic illustration of the classification of melanocortin-4 receptor (MC4R) variants.

DETAILED DESCRIPTION OF THE FIGURES

[0234] FIG. 1. Weight course (colored line) and hunger-score (dotted grey line) after treatment start of two individuals with POMC deficiency (a,b) and three individuals with LEPR deficiency (c-e). The green area (a, b, d, e) represents off-drug periods or phases in which external factors (compromised compliance or changes of co-medication as hydrocortisone) led to increased hunger. In subject 2 with POMC deficiency a non-serious febrile infection made an increase of the hydrocortisone treatment necessary. Due to a misunderstanding, this planned temporary elevated dosage of hydrocortisone was not returned to her basal dose after a few days as planned; as a result, the hunger feelings started to increase followed by weight gain. After adaptation of the hydrocortisone dosage during her next visit the hunger feeling and weight decreased again. The green area for subject 3 with LEPR deficiency is described in the main body of the text.

[0235] FIG. 2. Weight course of the three included subjects with LEPR deficiency (a-c) before the study start. Standard percentiles were used.sup.27. All of them had a history of early onset obesity and severe hunger and were not abledespite tremendous effortto stabilize the body weight over a longer period of time. Subject 1 with LEPR deficiency (a) had a gastric banding operation, which led to a short interval of weight loss and subsequent weight regain. The dashed blue line visualized the weight gain months before the treatment start and is not adapted to the time scale of the y-axis. Additionally for subject 2 weight data are not available between 8 and 19 years of age.

[0236] FIG. 3. 24-hours ambulatory blood pressure monitoring before and during treatment with setmelanotide in three enrolled individuals with LEPR deficiency (a-c). Statistical analysis was performed using one-sided t-test analyzing before treatment data against each outlined week of treatment separately (***=p<0.001). Subject 1: before treatment n=67 individual measurements, week 13 n=68 individual measurements, week 27 n=54 individual measurements, week 61 n=51 individual measurements; subject 2: before treatment n=57 individual measurements, week 12 n=59 individual measurements, week 52 n=87 individual measurements; subject 3: before treatment n=54 individual measurements, week 13 n=33 individual measurements, week 34 n=22 individual measurements. In the box plots horizontal line inside each box indicates median and the top and bottom of the box indicate the interquartile range, The bars indicate the 5th and 95th percentiles and squares indicate outliers.

[0237] FIG. 4. Examples of changes in skin color during the MC4R agonist treatment before and after 52 treatment weeks in individual 2 with LEPR deficiency. Changes in skin color are evident at week 52. Additionally, in subject 2 with LEPR deficiency the treatment led additionally to a change of the hair color from red to brown.

[0238] FIG. 5. HEK293 cells were transiently transfected with MC4R and stimulated with indicated ligand concentrations. Results of seven independent experiments performed in triplicated were shown as raw data, data are means.e.m.. Statistical analysis was performed using a one-way ANOVA with Kruskal-Wallis test at a concentration of 10.sup.8 M. Setmelanotide was tested against alpha-MSH and LY2112688: **** for alpha-MSH, and ** for LY2112688, ** p 0.01, **** p 0.0001.

[0239] FIG. 6. All functional data are raw data of four independent experiments performed in triplicates and are given as means.e.m.. HEK293 cells were transiently transfected with MC4R and a reporter gene construct for determination of PLC activity in the absence (b-c) or presence of 50 ng/l PTX (f-h) or 100 nM AgRP (j-l). Setmelanotide is indicated as red triangle, alpha-MSH as grey circle and LY2112688 as green diamond. (a): Cartoon representation illustrating phospholipase C (PLC) activation after stimulation of MC4R with different ligands. (b-d): Statistical analysis was performed using a one-way ANOVA with Kruskal-Wallis test at a concentration of 10.sup.8M. Setmelanotide was tested against alpha-MSH (p 0.001) and LY2112688 (p 0.01). (e): Cartoon representation illustrating the effect of PTX on NFAT activation. In each graph the PTX effect on ligands is indicated in black by using the same symbols as the ligand (f-h). Statistical analysis of PTX-treated versus untreated was done by unpaired t-test with Welsh correction. (f): Setmelanotide: not significant (g): alpha-MSH: not significant. (h): LY2112688: significant (p 0.01). (i): Cartoon representation illustrating the effect of AgRP on displacing MC4R ligands. The effect of AgRP co-stimulation of each ligand is indicated in black by using the same symbol as the ligand (j-l). Statistical analysis was done for ligand stimulation versus ligand/AgRP co-stimulation. (j) Setmelanotide is not displaced by AgRP, n.s., (k): minor displacement of alpha-MSH by AgRP, n.s., (l): displacement of LY2112688 by AgRP, p 0.05. n.s.: not significant. Differences in raw activation of PLC are due to the fact that raw data are shown and rlu is dependent on several issues such as surrounding temperature, lot number and age of the substrate. In control experiments (see FIG. 6) with mock transfected cells, rlu activity never exceeded more than 5% of the activity observed in main experiments with the different ligands.

[0240] FIG. 7. HEK293 cells were transfected with empty vector (mock) and MC4R (a, b) or MC3R and TSHR (c). The result of four independent experiments performed in triplicates is shown as raw data, data represents means.e.m.. (a) mock transfected cells were stimulated with the solvent for alpha-MSH (PBS with 0.001% BSA) and the solvent for setmelanotide and LY2112688 (PBS with 0.1% DMSO). Statistical analysis was performed using a one-way ANOVA with Kruskal-Wallis test and reveled no statistical difference. MC4R transfected cells were stimulated with alpha-MSH (1 M) and the solvent for ligands. Basal activity of MC4R tested against mock transfection by unpaired t-test with Welsh correction and solvent effects at MC4R were tested against MC4R under basal condition by one-way ANOVA with Kruskal-Wallis test and revealed no statistical significance. (b) Identical set of experiments and statistical tests for activation for PLC as is indicated in (a). (c) HEK293 cells were cotransfected with MC3R or TSHR and a reporter gene for NFAT and PLC activation was tested. TSHR was stimulated with 100 mU/ml bTSH and served as control. MC3R was stimulated with indicated ligands (1 M). Statistical significance was tested by one-way ANOVA with Kruskal-Wallis test and revealed no difference.

[0241] FIG. 8. (a) The human hMC4R/alpha-MSH complex model (in surface representation) reveals that the peptidic agonist binds into a cleft between the extracellular loops (E1-3) and specific transmembrane helices (H). Amino acids covering the ligand binding pocket are colored pale-green. (b) Approximately twenty hMC4R amino acids (green lines) in the transmembrane region and in the E2 or E3 constituting the ligand binding pocket. Importantly, in alpha-MSH (amino acid sequence of alpha-MSH below the model-figure a central amino acid motif 6HFRW9 is involved in ligand recognition and induction of ligand effects.sup.28. This specific ligand motif is located between the helices and their extracellular ends. (c) The detailed interaction map for the MC4R/alpha-MSH complex shows a schematic overview of supposed interactions. (d) The surface representation of the MC4R/setmelanotide complex model highlights the general localization of this ligand bound to the hMC4R. Amino acids covering the supposed setmelanotide binding pocket are colored green. (e) The cyclic peptide agonist setmelanotide (amino acid sequence below the model-figure) interacts, comparable to alpha-MSH, with the MC4R by a supposed salt bridge between the ligand Arg6 in the central core and the negatively charged receptor residues Glu100 (TMH2), Asp122 and Asp126 (TMH3) in MC4R. (f) The interaction map between setmelanotide and MC4R shows a different interaction pattern as supposed for alpha-MSH at MC4R, however, there is partial overlap in participating MC4R amino acids such as Asp122, Glu100 or Phe284. Potential hydrogen bonds were analyzed using HBPLUS.sup.28 as implemented in the program LIGPLOT+1.45.sup.29. Residues with distances less than 3.9 were considered to be in van der Waals contact.

[0242] FIG. 9. The putative binding modes of (a) alpha-MSH and (b) setmelanotide at hMC4R reveal similarities and differences between both ligands (shown as Ca-backbone cartoon representation and clipped models). Of note, in contrast to -MSH, setmelanotide is a cyclic peptide with a disulfide bridge between two cysteines. A specific emphasis relays on the interactions and localization of the conserved ligand motif HFRW. The Arg6 (setmelanotide) and Arg8 (-MSH) interact with negatively charged side chains of Glu100 (TMH2) and Asp122/Asp126 in H3 of the receptor. Moreover, Trp9 in alpha-MSH and corresponding Trp7 in setmelanotide are both embedded by aromatic interactions with Phe261 in H6 and Phe284 in H7, in close proximity also to a activation related receptor residues.sup.24,28 Tyr268 in H6 and His283 in the H7-E3 transition. In the MC4R/setmelanotide complex additionally Phe280 participates in the interaction with Trp7. However, specifically the histidine (shown with red label) of the ligands is oriented differently in both docking poses. The histidine in setmelanotide is located between Asn123 (H3) and Phe184 (H4), but in the -MSH/MC4R complex the corresponding His6 is directed between Tyr268 and Ser191 of the receptor. Moreover, alpha-MSH Phe7 interacts with Cys130 in H3 and Phe184 in H4, whereby the corresponding Phe5 of setmelanotide is oriented towards H6 and interacts additionally with Phe261. It can be concluded that (i) both ligands potentially share specific interactions of the highly conserved motif HFRW with MC4R (shown as red filled translucent circle), on the other hand (ii) the corresponding ligand histidines and phenylalanines in this motif are different in detailed interactions with side chains in receptor helices 4-6 (shown as blue filled translucent rectangle).

[0243] FIG. 10. Wild-type amino acids of in this study experimentally tested MC4R mutants concerning their Gq and Gs signaling properties Table 5) are highlighted at a MC4R/ligand complex model extended by a heterotrimeric Gprotein molecule (surface representation, -subunit, -subunit, -subunit). These positions are located in helices (H) H2, H3 and are cumulated in H4. Moreover, we also exemplarily show further wild-type residues which were reported to be identified in obese patients and characterized in vitro as like wild-type mutations for cAMP accumulation. Of note, from the around 160 reported naturally occurring hMC4R single side chain variants approximately 30% were characterized so far like wild-type in Gs mediated signaling.

[0244] FIG. 11. HEK293 cells were transfected with MC4R wild-type and indicated MC4R variants and stimulated with indicated ligand concentrations. (a, b) cAMP accumulation after stimulation with alpha-MSH or setmelanotide. (c, d) MC4R mutants were tested for PLC signaling after co-transfection with NFAT reporter gene. Stimulation was performed with alpha-MSH (c) and setmelanotide (d). Raw data of four independent experiments are shown, data are means.e.m..

[0245] FIG. 12. HEK293 cells were transfected with MC4R wild-type and indicated MC4R variants and stimulated with indicated ligand concentrations. All MC4R mutants were tested for PLC signaling after co-transfection with NFAT reporter gene. Stimulation was performed with alpha-MSH (a) and setmelanotide (b). Raw data of four independent experiments are shown, data are means.e.m..

[0246] FIG. 13. MC4R deficiency associated with impaired NFAT. (A) cAMP accumulation after alpha-MSH stimulation, all variants perform essentially as MC4R-WT. (B) NFAT signaling after alpha-MSH stimulation, essentially nearly all variants show a loss of function. (C) NFAT signaling after setmelanotide stimulation, essentially nearly all variants show a rescue in NFAT signaling. The assays for A, B and C were performed independently, raw data of four independent experiments performed in triplicates are shown.

[0247] FIG. 14. Investigation of NFAT signaling in mouse embryonic fibroblasts (MEF) deficient in Gq (MEF Dq). MEF were transfected with MC4R and the NFAT reporter in the absence and presence of Gq and NFAT signaling was measured after challenge with alpha-MSH, setmelanotide, RM-511 and Ly (10-6M). One out of four independent experiments is shown.

[0248] FIG. 15. Test of further MC4R variants. Additional experiments were conducted with MC4R variants R7C, S36T, D37G, C40Y, V95I, V103I, S127L, S191T, M200del, F202L, C271R and E308L. For testing Gs signaling, dynamic formation of cAMP was measured after alpha-MSH challenge. Variants classified as Gs like wild-type as well as several Gs loss-of-function variants were included. (A) GloSensor data of 4 independent experiments performed in triplicates are given as area under the curve. This data represents cAMP accumulation after alpha-MSH treatment. (B) NFAT signaling was assessed after alpha-MSH (B) and setmelanotide (C) challenge was performed in one assay set, 4 times independently. The fold rescue of each variant after setmelanotide challenge was calculated (in C).

[0249] FIG. 16. Schematic illustration of the classification of melanocortin-4 receptor (MC4R) variants. The main signalling pathway of wildtype MC4R (dark grey folded protein schematic) is most likely via Gs/adenylyl cyclase activation (labelled Gs), thereby enhancing cAMP. In addition, other pathways can be activated following endogenous ligand stimulation. Unaffected signalling cascades are marked in dark grey (Gs labelled Gs; non-Gs signaling labelled as such); impaired cascades are marked in light grey. Class 1: MC4R variant (variant indicated as white dot) signalling is identical to the wild-type MC4R in all signalling pathways. Class 2: Gs signalling of MC4R variants is comparable with the wild-type MC4R; only signalling of other, non-Gs pathways is reduced (light grey). Class 3: Gs signalling and other signalling pathways of MC4R variants are decreased. Class 4: Only Gs signalling of MC4R variants is reduced; non-Gs signalling pathways are unaffected.

EXAMPLES

[0250] The invention is further described by the following examples. These are not intended to limit the scope of the invention, but represent preferred embodiments of aspects of the invention provided for greater illustration of the invention described herein.

[0251] Materials and Methods of the Examples

[0252] Study Protocol

[0253] The investigator-initiated phase 2 trial (sponsor: Charit Universittsmedizin Berlin, EudraCT No. 2014-002392-28, clinicaltrials.gov No. NCT02507492) started with a baseline phase in which a physical examination, measurement of height- and body weight, blood tests (including oral glucose tolerance test (OGTT), GnRH test, measurement of full blood count, serum lipids, liver and kidney function parameter, leptin, IGF-1), analysis of body composition (BodPod) and energy expenditure (indirect calorimetry, CareFusion, VMAX Encore system), careful dermatological examination and documentation of the skin and psychological evaluation was performed. This phase was followed by a dose-titration phase starting with a dosage of 0.5-mg setmelanotide s.c./q.d. Every two weeks the dosage was increased for 0.5 mg/q.d. depending on the weight course and tolerability accompanied by a careful monitoring of vital parameters including blood pressure. After 12-13 weeks following the treatment start study subjects could enter the extension phase if a reduction of the body weight was achieved of more than 10 kg. Within this phase the study subjects were regularly seen by the study doctor, at least every 3 months, and blood tests and analysis of vital signs and safety parameters were performed.

[0254] Functional Characterization of MC4R Ligands

[0255] Characterization of setmelanotide, first generation MSH analog LY2112688 and alpha-MSH was performed in HEK293 cells. HEK293 cells were authenticated by SNP analysis and analysis of mycoplasm infection was performed every four weeks. HEK293 cells for cAMP accumulation assays and PLC activation (NFAT-luc assays) were cultured in L-glutamine containing MEM Earle's media supplemented with 5% FBS (Biochrom AG, Berlin, Germany) and 1 non-essential amino acids (Biochrom AG, Berlin, Germany) at 37 C. with 5% CO.sub.2. For cAMP and NFAT-luc measurements, 1.5*10.sup.4 HEK293 cells per well were seeded in poly-L-lysine-coated (Biochrom AG, Berlin, Germany) 96-well plates (15,000 cells/well). For functional characterization of MC4R variants all variants were generated by standard mutagenesis techniques and verified by Sanger sequencing. Transient transfection of HEK293 cells with MC4R plasmid-DNA or co-transfection with NFAT reporter gene (0.45 ng/l of each plasmid) was performed 24 hours after seeding in supplement-free advanced MEM (Life technologies, Carlsbad, Calif.), using Metafectene (Biontex, Munich, Germany) according to the manufactures protocol. CHO-K1 cells stably expressing MC4R (Discoverx Corp, Fremont Calif., USA; Product code 05-0050E2 for cAMP Hunter eXpress cells, tested for mycoplasma every four weeks) were utilized to study the antagonism of setmelanotide and LY2112688 response by AGRP-83-132-amide (Anaspec (Fremont, Calif.). These cells were plated in a 96-well plate for the assay as per manufacturer's instructions.

[0256] Measurement of cAMP Accumulation

[0257] Gs signaling were determined by measuring cAMP accumulation. Forty-eight hours following transfection in HEK293 cells, stimulation was conducted with compounds diluted in a HEPES-buffered solution containing 1 mM 3-isobutyl-1-methylxanthine (IBMX, Sigma Aldrich, St. Louis, Mo.) to inhibit cAMP degradation by phosphodiesterases. Cells were incubated for 40 minutes with setmelanotide, LY2112688 and alpha-MSH in concentrations ranging from 10.sup.6M to 10.sup.12 M in order to stimulate adenylyl cyclase. Substance incubation was performed in triplicates and at 37 C. with 5% CO.sub.2 and was stopped by aspirating the medium. Cells were then lysed at 4 C. on a shaking platform with lysis buffer containing 5 nM HEPES, 0.1% BSA, 0.3% Tween20 and 1 mM IBMX. Intracellular cAMP accumulation was determined by a competitive immunoassay based on the AlphaScreen technology (Perkin-Elmer Life Science, Boston, Mass.) as previously described.sup.18.

[0258] Measurement of PLC by Luciferase Reporter Gene Assay

[0259] PLC activation was determined by NFAT-luc assays (Promega, Fitchburg, Wis.). This method requires the co-transfection of equal amounts (0.45 ng/L DNA of each plasmid) of MC4R and the reporter constructs pGL4.30[luc2P/NFAT-RE/Hygro] (Promega, Fitchburg, Wis.) containing a response element and firefly luciferase gene under the control of NFAT. Two days post-transfection, cells were incubated for six hours with ligands in supplement-free MEM at 37 C. with 5% CO.sub.2. In case of determination the effect of G activation of Gi/o, blocking with PTX (50 ng/ml) was done 18 hours before the experiment. In case of AgRP interfering with PLC activation, co-stimulation of ligands in the presence of 100 nM AgRP was performed. The reaction was terminated by aspirating of the media. Cells were lysed for 15 minutes on a shaking platform at room temperature using 1 passive lysis buffer (Promega, Fitchburg, Wis.). Measurement was conducted with automatic luciferase substrate injection of 40 L in a black 96-well plate using a Berthold Microplate Reader (Berthold Technologies GmbH & Co KG, Bad Wildbad, Germany).

[0260] Melanocortin-4 Receptor Structure Homology Modeling and Docking of Alpha-MSH and Setmelanotide

[0261] The hMC4R modeling was performed in its putative active state conformation. The hMC4R is characterized by several specific properties concerning the amino acid composition and related structural features, in particular: (i) a short second extracellular loop 2 (E2, constituted by four amino acids), (ii) a missing cysteine disulfide bridge between the E2 and transmembrane helix 3 (TMH3) that is highly conserved among GPCRs, (iii) a regular -helical conformation of TMHS because of a methionine instead of a proline that is conserved in class A GPCRs and induces a helical kink and bulge, (iv) a disulfide bridge in the third extracellular loop (E3).sup.19.

[0262] Our hMC4R homology model was built by using the solved crystal structure of the 2-adrenergic receptor (ADRB2)/Gs complex (PDB entry 3SN6.sup.20) as a template, because this receptor structure represents an active state conformation which complements to our purpose of docking agonistic peptides into the hMC4R model.

[0263] General modifications for template preparation were deletion of the extracellularly fused T4 lysozyme, removal of the ADRB2-ligand and of the interacting Gs molecule. Moreover, the ECL2 of the ADRB2 template was deleted, because of differences in length and amino acid composition compared to the sequence of the hMC4R E2. Gaps of missing residues in the loops (e.g. in 13) of the template structure were closed manually. Moreover, the upper part of the kinked helix 5 was removed and substituted by a regular a-helix, because crystal structures of few GPCRs like the S1PR1 (PDB entry 3V2Y.sup.21) have shown a regular a-helical conformation without a proline in TMHS. This secondary structure must be assumed also for the hMC4R with a methionine at this corresponding position. All structural modifications were performed with the software Sybyl X2.0 (Certara, N.J., US). The AMBER F99 force field was used for energy minimization and dynamic simulations.

[0264] Amino acids of this prepared receptor-template were substituted with residues of the hMC4R, followed by side chain minimization with constraint backbone atoms of the transmembrane helices until converging at a termination gradient of 0.1 kcal/mol*. The constraints were finally released in a second minimization step until converging at a termination gradient of 0.05 kcal/mol*. This first initial model was further refined by molecular dynamic simulations (30 OK, 2 ns) and energetic minimizations of the side chain orientations with constrained backbone atoms of helical parts until converging at a termination gradient of 0.1 kcal/mol*. The resulting model was energetically minimized without any constraint.

[0265] Ligand Models

[0266] The cyclic ligand setmelanotide was modeled based on the structurally determined oxytocin peptide (PDB entry 2MGO.sup.22), which is characterized by a disulfide bridge between two cysteines as also assumed for setmelanotide. Missing residues were inserted or added manually, setmelanotide specific residues were substituted, and the N- and C-terminal specific groups added. For alpha-MSH neither direct structural information nor determined structures of a homologous peptides are available. For this reason the oxytocin structure was also used as a fragmental template for the central part of alpha-MSH (sequence MEHFRWG). This procedure was used in accordance to the purpose to start the molecule dynamics based docking with a comparable structural conformation of the central HFRW motif in MSH (without a Cys-Cys disulfide bridge) and setmelanotide (with a Cys-Cys disulfide bridge). Missing residues at the alpha-MSH N- and C-terminus were added manually. The ligand structures were energetically minimized and used for docking into the hMC4R model.

[0267] Ligand/Receptor Complex-Assembling

[0268] Computational docking of the two ligands into MC4R was performed under consideration of an already known and evidenced receptor/ligand interaction pair. One of the most prominent interaction is experimentally evidenced between MSH amino acid Arg8 (in setmelanotide Arg6) and receptor amino acids Asp122 and Asp126 of TMH3 (reviewed in.sup.23). These complementary charged amino acids are essential for ligand/receptor recognition and ligand activity. Therefore, we used this supposed interaction as a functional structural constraint. We manually placed both ligands above the extracellular loops of the MC4R model and initiated molecular dynamic simulations (300 K, 3 ns) with a defined distant constraint of 2 between the side chains of MSH Arg8 (or Arg6 in setmelanotide, respectively) and MC4R Asp126. All backbone atoms of the receptor helices were constrained. The resulting models were energetically minimized, followed by a second dynamic simulation (2 ns) without any distance constraint. The resulting complex models were energetically minimized without any constraint.

[0269] Full Receptor/Ligand/Complex

[0270] The resulting hMC4R alpha-MSH complex was superimposed with the initial structural receptor template from the ADRB2/Gs complex (PDB entry 3SN6.sup.20). The heterotrimeric G-protein was substituted into the hMC4R complex model. Dynamic simulations (300 K, 2 ns) of the side chains and loop structures were used to optimize interactions and intra-molecular distances, whereby the backbone atoms of the receptor helices were constrained. The resulting model was energetically minimized without any structural constraint.

[0271] Data Presentation and Statistical Analysis

[0272] In most cases, we show results as raw data of pooled experiments performed in triplicated. The number of independent experiments is given in each figure legend. At minimum four experiments performed in triplicates were done. Data analysis was performed using GraphPad prism 6 software. Because raw data are shown the measurement of cAMP in nM or PLC in rlu may different between different sets experiments. The reason for this are e.g. changing room temperature and different charges of kits. Statistical analyses were performed using GraphPad Prism 6.0. The kind of statistical test in given in the figure legends. In general, we use a one-way ANOVA with Kruskal-Wallis test or unpaired t-test with Welsh correction as we do not expect normal distribution of values. In each figure legend it is indicated which data are tested against which data. Values were only excluded if they were identified as outliers based on a Grubbs test. Statistical significance was accepted at P<0.05. Data are reported as means.e.m.. For Schild's analysis agonist concentration response curves were fitted using GraphPad Prism 6 using the Gaddum/Schild EC50 shift to drive pA2 and Schild slope directly from the dose response using global non-linear regression.

Results of the Examples

Example 1: Body Weight Course and Hunger-Score During Setmelanotide Treatment

[0273] Individuals with POMC deficiency have been treated with setmelanotide for more than 2 years, resulting in profound reductions of hyperphagia and body-weight, without signs of serious adverse side effects (FIG. 1 a,b). We hypothesized that impaired activation of POMC neurons due to LEPR signaling deficiencies, based on LEPR gene mutations, might similarly contribute to a lack of MSH signaling. Thus MC4R agonist supplementation therapy might be of therapeutic benefit.

[0274] In previous studies we observed, that intraperitoneal injections of setmelanotide in leptin-receptor-deficient (db/db) mice potently reduced appetite when compared to wild-type mice (data not shown). This data highlighted a likely MSH deficiency due to the LEPR defects. We tested whether setmelanotide treatment of individuals with LEPR deficiency might similarly result in profound reductions of hunger and body-weight. Three individuals with confirmed LEPR homozygous loss-of-function mutations were enrolled in an investigator-initiated phase 2 trial (EudraCT number: 2014-002392-28; ClinicalTrials.gov number: NCT02507492). The subjects and their relatives gave informed consent and the study was performed according to the declaration of Helsinki (see Materials and Methods).

[0275] Subject 1 is a 23 years old male from France in which a homozygous c.2357T>C (p.L786P) LEPR mutation was identified. Despite intensive efforts at life style intervention weight gain persisted and this patient underwent bariatric surgery (gastric-banding-operation) at age 18. Although following surgery the patient lost 36 kg in 6 months, he started to regain weight with persistent hyperphagia (regain of 20 kg in the subsequent 24 months; FIG. 2a). At the time this subject was enrolled in the setmelanotide study his body-weight was stable (130.6 kg; 9 kg from pre-operative weight, 49 months post-surgery; BMI 39.9 .sup.kg/.sub.m.sup.2; BMI SDS: 3.57, height 181 cm). His global morning hunger-score measured 9/10, (Likert rating scale: 0 points=no hunger; 10 points=severe hunger). Following a careful dose escalation, prolonged treatment with a final dosage of 1.5 mg setmelanotide injected subcutaneously once per day (s.c./q.d.) notably reduced hyperphagia (from 9 to 1-2). Body weight was reduced by 21.6% (28.2 kg) after 26 weeks of treatment (FIG. 1d). On this dose, his weight has remained stable over further 35 weeks (25.1 kg [19.2%]) At this time he had been on treatment for 61 weeks. The treatment has been well tolerated.

[0276] The second adult individual (a 22 year old male from the United Kingdom), with a homozygous LEPR mutation (p.H684P), had a history of sustained weight gain since his first month of life.sup.7 (FIG. 2b). He had been unable to stabilize his body weight for any prolonged period of time; at time of enrollment, his weight was 122.1 kg (BMI 40.7 .sup.kg/.sub.m.sup.2, BMI SDS 3.26, height 173.3 cm) and his hunger-score showed severe hunger (initial score of 9.5 out of 10 points). He was marginally responsive to setmelanotide at the initial lower dosages, but his hunger-score decreased when his dose was increased from 1.5 to 2 mg s.c. per day (hunger-score 4). On this dose, he lost 9.6 kg by 17 weeks and 13.9 kg (11.4%) body-weight over 36 weeks (FIG. 1d). Despite good tolerance of treatment and marked weight loss the subject still felt dissatisfaction at the rate of weight loss and independently discontinued his setmelanotide administration for 2 weeks. During this time, he regained 5.2 kg and his hunger-score increased to severe hunger (hunger-score 9). The subject reported that he felt hungry every hour and was struggling to control his appetite. As a result, treatment was re-initiated at the 2 mg daily dose, and then was increased to 2.5 mg/q.d. with the goal of accelerating his weight loss; as a result, he demonstrated a significant reduction in hunger (hunger-score 2) and body-weight (FIG. 1e). He continues treatment with good tolerability.

[0277] The third subject is the 14-year-old adolescent sister of subject 1, carrying the same homozygous LEPR mutation (c.2357T>C, p.L786P). She had an initial body-weight, before treatment, of 120.6 kg (BMI 44.2 .sup.kg/.sub.m.sup.2; BMI SDS 3.65; height 165 cm) (FIG. 2c). Again, she consistently gained weight throughout her life. She lost 10.0 kg during the first 13 weeks when titrated to a maximum dose of 1.5 mg. Her hunger-score was reduced from 9 to 5 (FIG. 10. Before and during the treatment the subject showed adolescent behavior, which compromised compliance with the s.c./q.d. treatment regimen. This was manifested by a misunderstanding, which led her to perform the injections late in the afternoon and at an incorrect (lower) dosage of 1.0 mg setmelanotide. Since we have noted a dose-threshold for therapeutic benefit, and with a 10-12 hour half-life of s.c. setmelanotide injections we concluded that the combination of a lower dose and the maximum drug concentration occurring during the night most likely precipitated an interval of weight regain (FIG. 10. In support of this explanation, her hunger feeling was reduced during the night but started to increase during the day. Eventually, this dosing error was recognized and corrected. As a result, she continued on a dosage of 2.0 mg/q.d. setmelanotide, under careful monitoring, leading again to reduced hunger. Weight data are being accumulated.

[0278] Safety laboratory parameters and vital parameters including blood pressure did not change significantly in the three patients (Tables 1-3 and FIG. 3). All subjects tolerated the injections well. Setmelanotide treatment led to only mild adverse events (Table 4). As seen in prior trials, the treatment led to darkening of the skin and a change in hair-color (subject 2 from red to brown; FIG. 4); these effects reflect melanocortin-1-receptor activation by setmelanotide. Metabolic parameters, including pretreatment hyperinsulinemia, showed clear trends towards normalization, in parallel with the reduction of body weight (Tables 1-3).

[0279] These clinical data support the notion that treatment with setmelanotide results in a reduction of hyperphagia and body weight, without occurrence of severe side-effects and extends the therapeutic effects observed in individuals with POMC-deficiency to subjects with LEPR defects (mean weight loss 11.07 kg within the first 13 weeks). The severe weight-regain and marked recurrence of hunger, evident following treatment withdrawal in subject 2, and the effects of misdosing in subject 3 strongly support the consistent results of setmelanotide treatment when used optimally. These subjects with LEPR deficiency experienced profound and long-lasting reductions of body-weight for the first time in their lives as even a previous attempt to reduce weight by bariatric surgery had failed (subject 1). We anticipate that the weight loss observed will drive long-term clinical benefits, along with improved quality of life due to reduction in hunger and food craving. It is still unknown whether the magnitude of weight loss in individuals with LEPR deficiency will approach the effects seen following long-term treatment of subjects with POMC deficiency. These observations suggest that other individuals with monogenic obesity due to mutations in the LEPR gene.sup.8 and other genes (POMC) in the MC4R-pathway.sup.9,10 may benefit from setmelanotide treatment.

Example 2: Determination of Phospholipase C Activation after Setmelanotide, Alpha-MSH and LY2112688 Challenge

[0280] While the results of the clinical trial are striking we aim to elucidate the underlying molecular mechanisms to explain setmelanotide's clinical effects, which will be helpful for improving personalized treatment regimens for this drug and for the future development of new MC4R agonists. In addition, we aim to understand the different side effect profile of setmelanotide when compared to former first generation MC4R agonists (e.g. LY2112688). The main signaling pathway of MC4R described to date involves Gs/adenylyl cyclase.sup.11. Alternative signaling pathways have been proposed.sup.12. Several lines of evidence argue for additional MC4R-mediated intracellular responses; e.g. G of Gi/o and G.sub.q.sup.13. The importance of these findings was first highlighted after targeted inactivation of G.sub.q in the hypothalamic paraventricular nucleus of mice, the predominant site of MC4R-regulated body-weight, which resulted in increased food intake and body-weight.sup.14. We first measured the EC50 values of ligand-induced cAMP accumulation (Gs/adenylyl cyclase signaling) in MC4R expressing HEK293 cells, following exposure to increasing concentrations of setmelanotide, LY2112688 or alpha-MSH. In these studies, setmelanotide was more more potent in activating Gs signaling (EC.sub.50: 3.91.7*10.sup.9M) than alpha-MSH (EC.sub.50: 2.30.7*10.sup.8M) or LY2112688 (EC.sub.50: 1.40.4*10.sup.8M) (FIG. 5). We next investigated signaling mediated through phospholipase C (beta) (PLC) using a nuclear factor of activated T-cells (NFAT) reporter gene assay (FIG. 6a). In these experiments, we identified potency differences between setmelanotide and first generation MC4R agonists: Setmelanotide (FIG. 6b) was over 100-fold more potent to stimulate NFAT signaling (EC.sub.50: 5.91.8+10.sup.9M) compared to alpha-MSH (EC.sub.50: 4.82.6*10.sup.7M) or LY2112688 (EC.sub.50: 3.31.9*10.sup.7M) (FIG. 6c,d). This effect was specific for the MC4R as the MC3R did not mediate PLC via NFAT for either ligand (FIG. 7). Because activation of PLC might result either from G.sub.q or/and G of Gi/o, we measured NFAT reporter activity in the presence of pertussis toxin (PTX), which blocks Gi/o activation (FIG. 6e-h), establishing that the effects most likely resulted from setmelanotide specific G.sub.q activation. Taken together this data demonstrates a striking difference of MC4R activation by setmelanotide compared to alpha-MSH and a first-generation synthetic agonist LY2112688 with a higher efficacy via Gs/adenylyl cyclase signaling and a 100-fold higher activation of non-Gs, most likely G.sub.q signaling when compared to the other two ligands.

[0281] We also investigated whether setmelanotide might differentially impact the effects of the inverse agonist AgRP.sup.15 when compared to other MC4R agonists. We analyzed the effect of AgRP on activation of PLC via NFAT in HEK293 cells (FIG. 6i). Interestingly, setmelanotide activation of the MC4R could not be antagonized by 100 nM AgRP (FIG. 6j), which contrasts the ability of 100 nM AgRP to fully antagonize alpha-MSH (FIG. 6k) or LY2112688 (FIG. 6l). We conclude that MC4R activation by setmelanotide is only marginally inhibited by AgRP, where PLC signaling was not affected and Gs signaling was less affected when setmelanotide and LY2112688 are compared. The higher potency of setmelanotide to activate PLC/NFAT signaling compared to LY2112688 or alpha-MSH and its increased capacity to overcome AgRP antagonism compared to LY2112688 may explain the remarkable efficacy of setmelanotide for appetite control in individuals with severe hyperphagia. This is relevant as G.sub.q activation is mainly linked to body weight regulation while Gs activation is predicted to increase sympathetic tone and blood pressure.sup.14.

Example 3: Structural Models of MC4R-Ligand Complexes

[0282] To gain additional structural insights into the molecular mechanisms of alpha-MSH and setmelanotide agonist action at the MC4R, we built a three-dimensional MC4R model and docked both ligands within this model by using short-time molecular dynamic simulations where ligand binding mode differences could be predicted when compared between setmelanotide and LY2112688 (FIG. 8-10).

[0283] Our human (h) MC4R/alpha-MSH and hMC4R/setmelanotide complex models suggest that the peptidic agonists bind generally into a cleft between the extracellular loops (E1-3) and the transmembrane helices (TMHs or Hs) (FIGS. 8 and 9). Approximately twenty hMC4R amino acids constituting the ligand binding pockets. Specific parts of the N-terminus are also supposed to participate in alpha-MSH binding, e.g. by interactions of Lys33 or Asp37. However, these residues are not conserved among members of the MCR group, which may exclude a significant role for alpha-MSH binding.

[0284] Analyzing the docking complexes specific emphasis relays on the interactions and localization of the conserved ligand motif HFRW. For alpha-MSH this central amino acid motif (6HFRW9) is known to be involved in ligand recognition and induction of ligand effects and here supposed main interactions with the receptor are (-MSH/MC4R): Trp9/Phe261; Arg8/Glu100, Asp126, Asp122; Phe7/Phe184; and His6/Tyr268. Most of these interactions are also supposed for setmelanotide, e.g. the Arg6 (corresponds with Arg8 in -MSH) interact with negatively charged side chains of MC4R Glu100, Asp122 and Asp126. Moreover, Trp9 in alpha-MSH and corresponding Trp7 in setmelanotide are both embedded by aromatic interactions with Phe261 in H6 and Phe284 in H7, in close proximity also to a MC4R activation related residues 24,25 Tyr268 (H6) and His283 in the H7-E3 transition.

[0285] Despite partial overlap in the MC4R/alpha-MSH and MC4R/setmelanotide interaction pattern also differences can be observed, which might be reasoned by the fact that setmelanotide is a cyclic peptide with a disulfide bridge between two cysteines and in consequence is more rigid in the backbone compared to MSH. Specifically the ligand histidines in the ligand HFRW motifs are oriented differently in both docking poses. The histidine in setmelanotide is located between Asn123 (H3) and Phe184 (H4), but in the -MSH/MC4R complex the corresponding His6 is directed between Tyr268 and Ser191 of the receptor. Moreover, alpha-MSH Phe7 interacts with Cys130 in H3 and Phe184 in H4, whereby the corresponding Phe5 of setmelanotide is oriented towards H6 and interacts additionally with Phe261. Therefore, it can be postulated that despite shared interactions of both ligands with MC4R these differences in detailed interactions contribute to the diverse pharmacological profiles (FIG. 11).

Example 4: Functional Characterization of Like Wild-Type MC4R Variants in Gs and PLC Signaling after Alpha-MSH and Setmelanotide Challenge

[0286] Given the fact that cardiovascular-related adverse effects have not been described in individuals treated with setmelanotide, which contrasts the data obtained with LY2112688, it is tempting to speculate that setmelanotide's differential signaling profile and MC4R binding mode, may contribute to its safety profile. Based on this in vitro data and based on the previous findings in rodent models.sup.16 we have identified that MC4R signaling through non-Gs pathways is of importance for body weight regulation.

[0287] Most of the MC4R variants identified in humans so far have been characterized.sup.11,13 focusing on Gs signaling. As setmelanotide strongly activates PLC signaling via NFAT activation when compared to LY2112688, we re-analyzed MC4R genetic variants that were assumed to be like wild-type based on Gs activation, for a potential NFAT signaling defect. These MC4R variants with normal Gs signaling have been identified in subjects with severe obesity and in whom a mechanistic explanation for their obesity has not been available.

[0288] We identified that several of these MC4R variants, that were not affected in their cAMP response to alpha-MSH (FIG. 11a and Table 5), showed notably impaired alpha-MSH induced NFAT activation that did not allow the determination of EC50 values (FIG. 11c and Table 5). Strikingly, PLC signaling via NFAT for these variants was restored following setmelanotide challenge (FIG. 11d).

[0289] Therefore, it seemsin addition to individuals with MC4R deficiency with impaired Gs signaling in which setmelanotide treatment resulted in reductions of body weight.sup.10that a significant number of subjects with obesity who carry an MC4R mutation that was hitherto assessed as functionally wild-type, are affected by a pathogenic non-Gs MC4R signaling defect. Approximately 5% of subjects with early onset obesity are carriers of MC4R mutations but only 1.7% of them are carriers of loss-of function mutations impacting MC4R cell surface expression and/or G.sub.s signaling.sup.10,17. Treatment of these variants is now warranted, to establish the broader impact of non-Gs MC4R signaling in obesity.

Example 5: MC4R Deficiency Associated with Impaired NFAT

[0290] Characterization of MC4R variants with impaired NFAT signaling was carried out. As is shown in FIG. 13, setmelanotide rescues impaired NFAT signaling in cAMP-functional MC4R variants. A number of MC4R mutations have been identified, as mentioned above, that exhibit wild-type like characteristics. In other words, these MC4R variants do not respond effectively to treatment with alpha MSH. This is observed by the lack of significant response when assessing cAMP accumulation after alpha-MSH treatment (FIG. 13a). These MC4R variants refer to, without limitation, T11I, S77L, T112M, V166I, I170V, A175T, T178M, I251L and N274S.

[0291] However, when assessing NFAT signaling via PLC activation, these MC4R variants exhibit clearly reduced NFAT signaling compared to MC4R-WT (FIG. 13b). After alpha-MSH treatment the PLC activation is significantly reduced. Importantly, the treatment of these MC4R variants leads to rescue of this phenotype. As shown in FIG. 13c, setmelanotide treatment leads to an increase in NFAT signaling (as measured by PLC activation) of these variants, in the majority of cases bringing NFAT signaling back to levels comparable with WT.

Example 6: Investigation of NFAT Signaling in Mouse Embryonic Fibroblasts (MEF) Deficient in Gq (MEF Dq)

[0292] MEF were transfected with MC4R and the NFAT reporter in the absence and presence of Gq. NFAT signaling was measured after challenge with alpha-MSH, setmelanotide, RM-511 and Ly (10.sup.6M). As can be observed in FIG. 14, in the presence of Gq, NFAT signaling is strongly increased, indicating that MC4R coupling to Gq is involved in the NFAT signaling.

Example 7: Test of Further MC4R Variants

[0293] Additional experiments were conducted with MC4R variants R7C, S36T, D37G, C40Y, V95I, V103I, S127L, S191T, M200del, F202L, C271R and E308L. For testing Gs signaling, dynamic formation of cAMP was measured after alpha-MSH challenge. Variants classified as Gs like wild-type as well as several Gs loss-of-function variants were included.

[0294] As shown in FIG. 15A, cAMP accumulation after alpha-MSH treatment is essentially not affected in the like-WT MC4R variants. These variants continue to show WT-similar cAMP levels after alpha-MSH treatment. The loss-of-function variants show inhibited cAMP levels after alpha-MSH treatment. As shown in FIG. 15B, NFAT signaling was assessed after alpha-MSH treatment. The variants typically show reduced NFAT signaling, lower than WT levels. Treatment with alpha-MSH was unable to rescue the NFAT signaling back to WT levels. Setmelanotide challenge was performed and the fold rescue of each variant after setmelanotide challenge was calculated (FIG. 15C). As is shown in FIG. 15C, setmelanotide treatment leads to an increase in NFAT signaling (as measured by PLC activation) of these variants, in the majority of cases bringing NFAT signaling back to levels comparable with WT.

Conclusion of Examples

[0295] In summary, we demonstrate that setmelanotide treatment was well tolerated and led to significant amelioration of hunger and profound weight reduction in individuals with LEPR-deficiency. Our observations open new avenues to treat subjects with LEPR-deficiency, and additional patient populations with genetic deficiencies (MC4R, Leptin, POMC, PCSK1, MageI2, CPE) leading to impaired functioning of the MC4R-pathway.sup.11,12.

[0296] We provide novel molecular evidence of setmelanotide action on the MC4R showing a potential role of G.sub.q signaling. PLC/NFAT signaling and the ability to overcome AgRP antagonism by setmelanotide explains the improved efficacy of setmelanotide compared to former MC4R agonists.

[0297] Moreover, impaired PLC/NFAT signaling through a subset of MC4R variants plays an important role in the development of obesity. In addition to LEPR and POMC deficiency, MC4R variant carriers with impaired PLC signaling via NFAT, will benefit from setmelanotide treatment and other similar agents, similar to the proposal that some Gs impaired MC4R variants may respond disproportionally well to setmelanotide.sup.10. As pharmacotherapy treatment options for genetic defects of central weight regulation are lacking, setmelanotide-mediated neuropeptide replacement in the MC4R-pathway now leads to weight and appetite control for obese MC4R-mediated pathway-deficient patients, as described herein.

TABLES

[0298]

TABLE-US-00001 TABLE 1 Laboratory parameters subject 1. Laboratory safety parameter and oral glucose tolerance test results of subject 1 with LEPR deficiency. Laboratory values subject with LEPR deficiency 1: Pre-study After 13 weeks After 27 weeks After 61 weeks Reference value Cholesterol (mg/dl) 168 135 138 127 <200 HDL (mg/dl) 52 46 55 62 >45 LDL (mg/dl) 102 78 76 63 <130 Triglyceride (mg/dl) 57 53 40 36 <200 ALT (U/l) 20 19 10 28 <41 AP (U/l) 62 64 67 60 40-130 gamma-GT (U/l) 21 13 10 17 8-61 Cortisol (nmol/l) 199.8 319.4 243.2 287.0 Testosterone (g/l) 4.64 2.38 6.81 5.53 2.18-9.06 fT4 (ng/l) 12.48 11.7 10.64 13.04 9.3-17 TSH (mU/l) 0.59 0.99 0.88 0.52 0.27-4.2 HbA1c (%) 5.1 5.1 5.2 5.2 <6.0 Oral glucose tolerance test subject with LEPR deficiency 1: Pre-study After 13 weeks After 27 weeks After 61 weeks Time Blood Blood Blood Blood point glucose Insulin glucose Insulin glucose Insulin glucose Insulin (min) (mg/dl) (mU/l) (mg/dl) (mU/l) (mg/dl) (mU/l) (mg/dl) (mU/l) 0 75 6.09 76 69.74 73 3.72 71 1.89 30 164 119.8 108 58.03 131 9.23 113 39.80 60 147 89.92 152 92.55 74 56.24 161 106.00 90 130 67.02 128 66.44 64 30.25 136 65.10 120 114 46.02 117 64.44 101 42.94 104 29.40

TABLE-US-00002 TABLE 2 Laboratory parameters subject 2. Laboratory safety parameter and oral glucose tolerance test results of subject 2 with LEPR deficiency. Laboratory values subject with LEPR deficiency 2: After 12 After 52 Reference Pre-study weeks weeks value Cholesterol (mg/dl) 173 186 184 <200 HDL (mg/dl) 34 44 49 >45 LDL (mg/dl) 122 138 124 <130 Triglyceride (mg/dl) 141 123 103 <200 AP (U/l) 74 80 64 40-130 Cortisol (nmol/l) 184.8 364.4 216 Testosterone (g/l) 2.11 3.78 2.82 2.18-9.06 fT4 (ng/l) 11.49 10.55 9.73 9.3-17 TSH (mU/l) 1.27 2.43 1.85 0.27-4.2 HbA1c (%) 5.3 5.0 5.3 <6.0 Oral glucose tolerance test subject with LEPR deficiency 2: Pre-study After 12 weeks After 52 weeks Time Blood Blood Blood point glucose Insulin glucose Insulin glucose Insulin (min) (mg/dl) (mU/l) (mg/dl) (mU/l) (mg/dl) (mU/l) 0 74 15.25 77 17.29 73 9.12 30 131 128.5 109 95.59 152 11.23 60 153 126.8 121 75.20 137 69.40 90 136 115.4 161 102.20 126 100.40 120 129 131.2 145 93.11 114 107.40

TABLE-US-00003 TABLE 3 Laboratory parameters subject 3. Laboratory safety parameter and oral glucose tolerance test results of subject 3 with LEPR deficiency. Laboratory values subject with LEPR deficiency 3: After 13 After 34 Reference Pre-study weeks weeks values Cholesterol (mg/dl) 150 134 151 92-234 HDL (mg/dl) 34 34 43 >45 LDL (mg/dl) 99 92 102 <130 Triglyceride (mg/dl) 92 82 46 <200 AP (U/l) 24 na 67 <187 Cortisol (nmol/l) 206 175.1 266 48.0-579.0 Prolactin (g/l) 8.38 6.06 9.27 4.2-29.01 T4 (g/l) 79.5 75.8 81.7 59.1-132.0 fT4 (ng/l) 12.2 13.04 13.21 8.90-14.90 TSH (mU/l) 1.45 0.63 1.14 0.60-4.00 HbA1c (%) 5.3 5.0 5.4 <6.0 Oral glucose tolerance test subject with LEPR deficiency 3: Pre-study After 13 weeks After 34 weeks Time Blood Blood Blood point glucose Insulin glucose Insulin glucose Insulin (min) (mg/dl) (mU/l) (mg/dl) (mU/l) (mg/dl) (mU/l) 0 86 395.7 69 5.86 83 32.90 30 161 231.3 99 127.90 132 220.00 60 140 296.2 144 162.30 146 281.00 90 129 na 149 260.80 103 284.00 120 137 378.0 94 54.85 112 230.00

TABLE-US-00004 TABLE 4 Summary of adverse events. Adverse events (AEs), which have occurred during the treatment duration of the subjects with LEPR deficiency, are listed with information about frequency, severity and duration. In cases where more than one subject reported an AE, the duration for each individual is listed in days. The total duration of treatment reported in this AE table is ~574 days for subject 1, ~483 days for subject 2, and ~343 days for subject 3. Duration (days; Number of patients in listed for each AE description which AE occurred Severity patient) Reduced appetite 3 severe ongoing increased tanning 3 severe ongoing of the skin/nevi Skin folliculitis 1 mild ongoing Pain at injection 2 mild 3/8 site Induration/hematoma 2 mild 2/2 at injection site Bone & muscular 2 mild 2/4 pain Dry mouth 3 mild 2/7/ongoing Headache 3 mild 1/2/7 Nausea 1 mild 2 Abdominal pain 1 mild 1 SAE description none

TABLE-US-00005 TABLE 5 Functional characterization of like wild-type MC4R variants. MC4R variants that were classified as like wild-type concerning Gas/adenylyl cyclase signaling were evaluated for their capacity to induce NFAT signaling after alpha-MSH and setmelanotide challenge. HEK293 cells were transfected with indicated variants for determination of cAMP accumulation and co-transfected with NFAT reporter in case of PLC determination. Given data are results of four independent experiments performed in triplicates and are shown as mean s.e.m.. Comparison of variant-MC4R to wild-type MC4R stimulated with the same ligand was performed: a: * p < 0.05, b: ** p < 0.01 analyzed by one-way ANOVA with Kruskal-Wallis test, n.d.: not possible to determine with proper accuracy. Gs/cAMP accumalation alpha-MSH setmelanotide basal Emax at 10.sup.6 Emax at 10.sup.6 signaling [fold M [fold over M [fold over over wild-type wild-type wild-type mutation basal] basal] EC50 [nM] basal] EC50 [nM] wild-type 1 13.1 6.6 8.4 1.9 24 4.0 7.1 2.sup. T11I 0.83 0.05 12.3 1.5 8.5 0.9 19 1.3 8.7 2.8 S77L 0.99 0.03 11.2 1.1 10 2.7 16 0.5 3.4 0.2 T112M .sup.0.72 0.04.sup.b 10.6 0.8 8.3 1.6 18 1.5 43 15 V166I 0.79 0.08.sup.a 9.0 2.2 20 6.7 5.9 1.5 9.6 3.2 I170V 0.87 0.06 11.1 2.7 17 4.3 11 2.7 37 9.2 A175T 0.78 0.07 12.5 3.1 13 3.1 17 4.4 15 5.1 T178M 0.96 0.12 13.2 4.3 10 3.3 21 5.2 68 17 I225L .sup.0.76 0.09.sup.b 10.4 2.6 11 2.7 18 4.5 4.8 1.2 N274S 0.8 0.1.sup.a 11.4 2.8 7.6 1.9 14 3.4 43 11 wild-type 1 7.4 2.11 174 6.9 0.13 9.5 1.4 T11I 0.92 0.09 6.24 1.15 806 6.0 0.57 12 3.4 S77L 1.27 0.15 2.3 0.52 n.d. 6.3 1.13 8.8 0.4 T112M 1.4 0.11 3.29 0.55 n.d. 11.3 2.9.sup. 14 6 V166I 1.81 0.18.sup.a .sup.0.33 0.08.sup.b n.d. 3.3 0.79 n.d. I170V 1.22 0.13 1.89 0.4 n.d. 7.3 1.8 5.2 1.3 A175T 0.87 0.17 2.87 0.71 n.d. 11.7 2.9.sup. 26 6.6 T178M 1.10 0.21 2.87 0.71 n.d. 9.2 2.3 24 6 I225L 0.92 0.14 4.46 1.15 n.d. 7.5 1.9 18 4.3 N274S 1.29 0.22 1.65 0.41 n.d. 8.4 2.1 12 4

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