Patent Application
20230406883
The present invention relates to compounds and their use in treatment of disorders mediated by tachykinin receptors, such as the tachykinin receptor 2.
The family of Tachykinin neuropeptide receptors consists of three G protein-coupled receptors (GPCRs): Tachykinin receptor (Tacr) 1, Tacr2 and Tacr3, also known as Neurokinin receptor 1-3, (NK1-3R). The endogenous ligands for tachykinin receptors are the neuropeptides Substance P (SP) being the preferred ligand for NK1R, Neurokinin A (NKA) the preferred ligand for NK2R, and Neurokinin B the preferred ligand for NK3R. Neither of the endogenous ligands are specific for their receptor. Each peptide ligand can, thus, cross-activate all members of the Tachykinin receptor family with a potency close to the potency of its preferred receptor. Upon activation, the Tachykinin receptors preferentially couple to Gq, generating an intracellular inositol trisphosphate (IP3) signaling response. All receptors can, in addition, also couple to Gs and induce cAMP accumulation, although with lower potency than Gq-activation.
Obesity, insulin resistance and type 2 diabetes are multifactorial diseases. The diseases are all interconnected, and the exact pathological mechanisms are unknown.
Obesity is widely accepted to be caused by an imbalance between energy intake and energy expenditure (EE). Thus, increased high caloric intake accompanied by inactivity is believed to be the main driver of obesity. In addition to the energy imbalance, high circulating insulin levels, as seen in insulin resistance, is believed to augment weight gain due to increased insulin-mediated nutrient storage.
Insulin resistance is a condition where cells of the body do not respond properly to the endocrine hormone insulin. The role of insulin is to allow the cells of the body to take up glucose to be used as energy fuel or for storage as fat. This means that when facing insulin resistance, the body is more likely to build up glucose in the blood leading to elevated blood glucose (hyperglycemia). As a result, the body produces more insulin trying to cope with the hyperglycemia, and therefore individuals with insulin resistance often produces more insulin compared to healthy individuals.
Diabetes is a disease where the body's insulin producing cells fail to meet the demand for insulin to regulate blood glucose. In general, diabetes can by stratified into three different types: Gestational diabetes, which is diabetes occurring during pregnancy. Type 1 diabetes, which is an autoimmune disorder where the beta-cells are destroyed and the individual is not able to produce insulin. Type 2 diabetes, the most common form of diabetes and caused by progressive beta-cell loss and insulin resistance. Beta-cell loss in type 2 diabetes is believed to be caused by beta-cell exhaustion, due to increased insulin demand, in combination cellular damage as a result of elevated blood glucose and circulating fatty acids.
Due to the interconnectivity of obesity, insulin resistance and diabetes, treatment that targets energy expenditure, i.e. by activating B/BAT, will have the potential to treat all three. Thus, increased energy expenditure has potential to treat obesity by decreasing energy stores, insulin resistance through decrease of fasting glucose, and diabetes by reducing fasting glucose per se and protect the beta-cells from exhaustion via reduced insulin demand.
Brown and beige adipose tissue (B/BAT) can be physiologically stimulated by cold exposure to significantly consume glucose and triglyceride-derived fatty acids from the blood and increase energy expenditure. Classically, brown and beige adipose tissue is activated upon stimulation of the Gs-coupled beta-adrenergic GPCRs, to elicit an intracellular cAMP response that activates lipolysis, glucose and lipid uptake from the periphery, and uncoupling of electron transport chain in the mitochondria by activating uncoupling protein 1. The uptake of lipids and glucose by activated brown and beige adipose tissue is superior to any other tissues, and activation of those tissues is therefore attractive for development of therapies for obesity, insulin resistance and diabetes.
Current attempts to activate B/BAT in humans have focused on the beta-adrenergic/cAMP pathway. This pathway has indeed proven capable of inducing substantial EE but with concomitant increases in unwanted side-effects such as heart rate, blood pressure and blood glucose (hyperglycemia).
In addition to B/BAT activation, the NK2R and ligand NKA is able to activate NK2Rs on visceral smooth muscle and stimulate contraction of colon and urinary bladder. The contractile activity of NK2R activation is conserved across species including rats, dogs, pigs, and humans.
Several studies in mouse, rat, dog and macaques have shown that NK2R agonists are gastrointestinal and bladder prokinetic agents causing dose-dependent smooth muscle contractions by activating NK2Rs located on smooth muscle cells. Emesis and hypotension are common side effects caused by NK1R cross activation and hence, development of NK2R specific agonists is desired to decrease side-effects in these therapies.
The present inventors have developed a series of compounds targeting the tachykinin/neurokinin receptor 2 (NK2R). NK2R is a member of the tachykinin G-protein coupled receptor (GPCR) family also containing tachykinin/neurokinin receptor 1 and 3 (NK1R and NK3R). The endogenous ligand for NK2R is neurokinin A (NKA), whereas substance P and neurokinin B are the endogenous ligands for NK1R and NK3R, respectively. NKA is a 10 amino acid, locally acting neuropeptide mainly produced in enterochromaffin cells and it is known to activate smooth muscle contraction. NK2R preferentially couples to Gq-proteins but can also recruit Gs and Gbeta-gamma and beta-arrestins. The primary organs of Tacr2 mRNA expression are adrenal glands (mice) and gastrointestinal tract (humans and mice).
The present inventors provide the synthesis of chemically stable agonists of NK2R as activators of energy expenditure for treatment of NK2R mediated disorders, such as a NK2R mediated disorder selected from the group consisting of: obesity, dysfunctional voiding, diabetes, such as type-II diabetes, and diabetes-related disorders.
In a first aspect, a compound according to formula (I) is provided:
(A)-(B) (I),
wherein;
(B) is a conjugated moiety of the general formula (II)
Fa-Lg (II),
In a second aspect, a pharmaceutical composition is provided comprising the compound as defined herein, and one or more pharmaceutically acceptable adjuvants, excipients, carriers, buffers and/or diluents.
In a third aspect, a compound is provided as defined herein for use as a medicament.
In a fourth aspect, a method for treating a disease in a subject is provided, comprising administering a compound as herein for treatment of a NK2R mediated disorder.
In a fifth aspect, a method for modulating the activity of NK2R is provided, comprising contacting NK2R with a compound as defined herein.
In a sixth aspect, a use of a compound as defined herein is provided for the manufacture of a medicament for the treatment of a metabolic disorder.
To facilitate the understanding of the following description, a number of definitions are presented in the following paragraphs.
The term “alkyl” whether used alone or as part of a substituent group, refers to straight and branched carbon chains having 1 to 8 carbon atoms, such as 1 to 6 carbon atoms.
Therefore, designated numbers of carbon atoms (e.g., C1-8) refer independently to the number of carbon atoms in an alkyl moiety or to the alkyl portion of a larger alkyl-containing substituent.
In substituent groups with multiple alkyl groups such as, (C1-6alkyl)2amino-, the C1-6alkyl groups of the dialkylamino may be the same or different. Alkyl, as defined herein may be substituted by one or more substituents such as a halogen or one or more halogens. In one embodiment, an alkyl is substituted by 1,2 or 3 fluorine atoms.
In one embodiment, an alkyl is substituted by a carboxy group (CO2), such as a carboxy methyl (CO2Me).
It is understood that substituents and substitution patterns on the compounds of the present invention can be selected by one of ordinary skill in the art to provide compounds that are chemically stable and that can be readily synthesized by techniques known in the art as well as those methods set forth herein.
The term “subject” refers to an animal, preferably a mammal, and most preferably a human.
Proteinogenic “amino acids” (AA) are named herein using either their 1-letter or 3-letter code according to the recommendations from IUPAC, see for example http-//www.chem.qmul.ac.uk/iupac/AminoAcid/. Capital letter abbreviations indicate L-amino acids, whereas lower case letter abbreviations indicate D-amino acids.
A series of non-proteinogenic amino acids are referred to herein. The names employed should be clear and understandable to the skilled person. meta-Tyrosine (m-Y) is 3-hydroxyphenylalanine. The structure of methoxinine (Mox) is shown below:
The amino acid, beta-alanine (bA) as used herein is also known as 3-aminopropanoic acid. The amino acid, N-methyl-Leucine is referred to as (NmLeu) herein.
A “terminal fatty acid” is a fatty acid wherein the carboxylic acid group is localized on a terminal carbon atom of the fatty chain. A “terminal C16-C20 fatty acid” is thus a fatty acid chain consisting of 16 to 20 carbon atoms, wherein the acid group is located terminally and the carbon atom of the carboxylic acid group is a terminal chain carbon.
Tachykinin Receptor Activity
Agonist-induced G-protein coupled receptor (GPCR) activation can be measured by an Inositol-1,4,5-Trisphosphate [3H] Radioreceptor Assay (IPs Assay) as described in Example 1. The IP3 assay takes advantage of the tachykinin receptors' ability to induce production of the inositol trisphosphate (IP3) second messenger upon agonist (ligand) binding on receptor expressing cells following an initial 3H-inositol labelling period. In effect this means that production of the second messenger IP3 as a measure of receptor activity can be assessed by counting 3H-activity.
An “NK2R agonist” may possess varying degrees of selectivity relative to activity at the NK1 receptor and/or NK3 receptor as measured in biological assays, such as the IP3 assay presented herein. A “selective NK2R agonist” is herein defined as a ligand that binds to or activates the NK2 receptor with at least about 10 times or greater potency than it binds to or activates the NK1 and/or NK3 receptors. It is not necessary that a molecule be considered selective in both binding and functional (activation) assays to be a selective NK2R agonist. Binding potency is routinely reported as the EC50, with a lower EC50 value equating with greater potency. Thus, a
selective NK2R agonist possesses an NK2R binding EC50 that is at least about 10 times or more lower than its NK1 and/or NK3 binding EC50. Potency to activate a receptor is also routinely reported as the Ki, with the lower Ki value equating with a greater potency.
In a preferred embodiment, the compound provided herein is a neurokinin receptor 2 (NK2R) agonist. In one embodiment, the compound is a selective neurokinin receptor 2 (NK2R) agonist.
In one embodiment, the compound has an EC50 towards human NK2R of 300 nM or less, such as 250 nm or less, such as 200 nm or less, such as 150 nM or less, such as 100 nM or less, such as 90 nM or less, such as 80 nM or less, such as 70 nM or less, such as 60 nM or less, such as 50 nM or less.
In one embodiment, the compound has an EC50 towards human NK2R of 50 nM or less, such as 40 nm or less, such as 30 nm or less, such as 20 nM or less, such as 15 nM or less, such as 14 nM or less, such as 13 nM or less, such as 12 nM or less, such as 11 nM or less, such as 10 nM or less.
In one embodiment, the compound has an EC50 towards human NK1R of at least 100 nM, such as at least 200 nM, such as at least 300 nM, such as at least 400 nM, such as at least 500 nM.
In one embodiment, the compound has an EC50 towards human NK3R of at least 100 nM, such as at least 200 nM, such as at least 300 nM, such as at least 400 nM, such as at least 500 nM.
Tachykinin Receptor Mediated Disorders
In one embodiment, a compound is provided as defined herein for use as a medicament. The present inventors provide the synthesis of chemically stable agonists of NK2R as activators of energy expenditure for treatment of metabolic disorders, such as obesity, as well as for treatment of dysfunctional voiding.
In one embodiment, a method for treating a disease in a subject is provided, comprising administering a compound as herein for treatment of a NK2R mediated disorder.
In one embodiment, the NK2R mediated disorder is selected from the group consisting of: obesity, dysfunctional voiding, diabetes, such as type-II diabetes, and diabetes-related disorders.
In one embodiment, the NK2R mediated disorder is a metabolic disorder. In one embodiment, the metabolic disorder is a diabetes-related disorder. In particular, the diabetes-related disorder is selected from the group consisting of: impaired insulin tolerance and impaired glucose tolerance.
Further, in one embodiment, a method for modulating the activity of NK2R is provided, comprising contacting NK2R with a compound as defined herein.
In one embodiment, a use of a compound as defined herein is provided for the manufacture of a medicament for the treatment of a metabolic disorder.
Peptides—(A)
In a first embodiment, a compound according to formula (I) is provided:
(A)-(B) (I),
wherein;
(A) is a peptide comprising an amino acid sequence of the general formula X1X2X3X4X5X6X7, wherein
(B) is a conjugated moiety of the general formula (II)
Fa-Lg (II),
wherein;
Fa is a C10-C20 fatty acid, optionally substituted with one or more carboxylic acid groups,
Lg is a linking group, which covalently links (B) to the peptide (A),
and wherein (B) is covalently linked to a terminal amino acid or to a non-terminal amino acid.
In a second embodiment, the compound is provided wherein the peptide (A) is of the general formula X1X2X3X4X5X6X7, wherein
In a third embodiment, the compound is provided wherein the peptide (A) is of the general formula X1X2X3X4X5X6X7, wherein
In one embodiment, the compound is provided wherein X2 is arginine (R).
In one embodiment, the compound is provided wherein X3 is tyrosine (Y). In one embodiment, the compound is provided wherein X3 is tyrosine (Y) and wherein X2 is arginine (R). In one embodiment, X3 is tyrosine (Y), X2 is arginine (R), and X5 is 2-aminoisobutyric acid (Aib).
In one embodiment, the compound is provided wherein X4 is threonine (T).
In one embodiment, the compound is provided wherein X5 is selected from the group consisting of: 2-aminoisobutyric acid (Aib) and serine (S).
In one embodiment, the compound is provided wherein X6 is N-methyl-leucine (Me-Leu).
In one embodiment, the compound is provided wherein X7 is methoxinine (Mox). In one embodiment, the compound is provided wherein X7 is methoxinine (Mox) and wherein X2 is arginine (R). In one embodiment, X7 is methoxinine (MOx), X2 is arginine (R), and X3 is tyrosine (Y).
Preferably, the peptide (A) is amidated on the C-terminus.
In one embodiment, the peptide (A) comprises from 7 to 15 amino acids, such as from 7 to 14 amino acids, such as from 7 to 13 amino acids, such as from 7 to 12 amino acids, such as from 7 to 11 amino acids, such as from 7 to 11 amino acids, such as from 7 to 10 amino acids, such as from 7 to 9 amino acids, such as from 7 to 8 amino acids, preferably wherein the peptide comprises 7 amino acids.
In one embodiment, the peptide (A) comprises no more than 15 amino acids, such as no more than 14 amino acids, such as no more than 13 amino acids, such as no more than 12 amino acids, such as no more than 11 amino acids, such as no more than 10 amino acids, such as no more than 9 amino acids, such as no more than 8 amino acids, such as no more than 7 amino acids.
In one embodiment, the peptide (A) consists of 7 amino acids of the general formula X1X2X3X4X5X6X7. The peptide is preferably amidated on the C-terminus.
In one particular embodiment, the compound is provided wherein
In one particular embodiment, the compound is provided wherein
In one embodiment, the compound consists of the sequence of any one of SEQ ID NO: 1 to SEQ ID NO: 57.
In one particular embodiment, the compound is:
In the context of the present disclosure and unless otherwise stated, when a bond is drawn from an atom to an amino acid abbreviated by its one or three letter code, the bond is connected to the α-amino group or to the carbonyl carbon of the amino acid's backbone. For example in the case of compound with ID 398, “Lys” is connected to its adjacent carbonyl via its α-amino group, and “Thr” is connected to “NH” via its backbone carbonyl carbon.
Conjugated Moieties—(B)
Conjugated moieties are also referred to as protractors herein. In one embodiment, the compound as defined herein is provided, wherein Lg is of formula (Lg-1),
wherein Z is a chain comprising from 18 to 23 atoms in the backbone selected from the group consisting of: C, O, and N;
and wherein R is selected from the group consisting of H, and C1-6 alkyl. A person of skill in the art knows that C, O, and N may be substituted with hydrogen according to the valence of the particular atom. The backbone may comprise one or more carbonyl groups, such as 1, 2, 3, or 4 carbonyl groups. The backbone may also comprise one or more carboxylic acid groups, such as 1 or 2. In some embodiments, Z comprises fragments of ethylene glycol interrupted by one or more amide functionalities. An example is shown in formula (B1), wherein the fatty acid Fa is of formula (Fa-1), R of Lg-1 is H, and the backbone of Z comprises 21 atoms selected from the group consisting of C, O, and N.
In one embodiment, the compound is provided, wherein Fa is a terminal C16-C20 fatty acid.
In one embodiment, the compound is provided wherein Fa is of formula (Fa-1),
wherein n is from 11 to 20, such as from 12 to 19, for example from 13 to 18, such as from 14 to 17, preferably wherein n is 15;
and wherein X is selected from the group consisting of —OH, —OC1-6, —NH2, —NHC1-6, and N(C1-6)2. In a particular embodiment, n is 15 and X is —OH.
In one embodiment, the compound is provided wherein Lg of the conjugated moiety does not comprise functional groups that are positively charged at pH=7.4. In one embodiment, Lg of the conjugated moiety does not comprise more than 1 functional group that is negatively charged at pH=7.4. In one embodiment, Lg of the conjugated moiety has a net neutral charge or −1 at pH=7.4.
In one preferred embodiment, the compound as defined herein is provided, wherein the conjugated moiety is of formula (B1);
In a particularly preferred embodiment, the compound as defined herein is provided wherein the conjugated moiety is of the formula below;
In one embodiment, the conjugated moiety (B) is covalently attached to the N-terminus of (A), optionally via an amide bond.
In one embodiment, the conjugated moiety (B) is covalently attached to the N-terminus of (A) via an amide bond with the N-terminal α-NH2 group.
Pharmaceutical Compositions
In one embodiment, a pharmaceutical composition is provided comprising the compound as defined herein, and one or more pharmaceutically acceptable adjuvants, excipients, carriers, buffers and/or diluents.
Items
1. A compound according to formula (I):
(A)-(B) (I),
Fa-Lg (II),
16. The compound according to any one of the preceding items, wherein the conjugated moiety (B) is covalently attached to the N-terminus of (A), optionally via an amide bond.
17. The compound according to any one of the preceding items, wherein the conjugated moiety (B) is covalently attached to the N-terminus of (A) via an amide bond with the N-terminal α-NH2 group.
18. The compound according to any one of the preceding items, wherein the peptide (A) is amidated on the C-terminus.
19. The compound according to any one of the preceding items, wherein the peptide (A) comprises from 7 to 15 amino acids, such as from 7 to 14 amino acids, such as from 7 to 13 amino acids, such as from 7 to 12 amino acids, such as from 7 to 11 amino acids, such as from 7 to 11 amino acids, such as from 7 to 10 amino acids, such as from 7 to 9 amino acids, such as from 7 to 8 amino acids, preferably wherein the peptide comprises 7 amino acids.
20. The compound according to any one of the preceding items, wherein the peptide (A) comprises no more than 15 amino acids, such as no more than 14 amino acids, such as no more than 13 amino acids, such as no more than 12 amino acids, such as no more than 11 amino acids, such as no more than 10 amino acids, such as no more than 9 amino acids, such as no more than 8 amino acids, such as no more than 7 amino acids.
21. The compound according to any one of the preceding items, wherein the peptide (A) consists of 7 amino acids of the general formula X1X2X3X4X5X6X7.
22. The compound according to any one of the preceding items, wherein
26. The compound according to any one of the preceding items, wherein the compound is a neurokinin receptor 2 (NK2R) agonist.
27. The compound according to any one of the preceding items, wherein the compound is a selective neurokinin receptor 2 (NK2R) agonist.
28. The compound according to any one of the preceding items, wherein the compound has an EC50 towards human NK2R of 300 nM or less, such as 250 nm or less, such as 200 nm or less, such as 150 nM or less, such as 100 nM or less, such as 90 nM or less, such as 80 nM or less, such as 70 nM or less, such as 60 nM or less, such as 50 nM or less.
29. The compound according to any one of the preceding items, wherein the compound has an EC50 towards human NK2R of 50 nM or less, such as 40 nm or less, such as 30 nm or less, such as 20 nM or less, such as 15 nM or less, such as 14 nM or less, such as 13 nM or less, such as 12 nM or less, such as 11 nM or less, such as 10 nM or less.
30. The compound according to any one of the preceding items, wherein the compound has an EC50 towards human NK1R of at least 100 nM, such as at least 200 nM, such as at least 300 nM, such as at least 400 nM, such as at least 500 nM.
31. The compound according to any one of the preceding items, wherein the compound has an EC50 towards human NK3R of at least 100 nM, such as at least 200 nM, such as at least 300 nM, such as at least 400 nM, such as at least 500 nM.
32. A pharmaceutical composition comprising the compound as defined in any one of the preceding items, and one or more pharmaceutically acceptable adjuvants, excipients, carriers, buffers and/or diluents.
33. A compound as defined in any one items 1 to 31 for use as a medicament.
34. A method for treating a disease in a subject comprising administering a compound as defined in any one items 1 to 31 for treatment of a NK2R mediated disorder.
35. The method according to any one of the preceding items, wherein the NK2R mediated disorder is selected from the group consisting of: obesity, dysfunctional voiding, diabetes, such as type-II diabetes, and diabetes-related disorders.
36. The method according to any one of the preceding items, wherein the NK2R mediated disorder is a metabolic disorder.
37. The method according to any one of the preceding items, wherein the metabolic disorder is a diabetes-related disorder.
38. The method according to item 35, wherein the diabetes-related disorder is selected from the group consisting of: impaired insulin tolerance and impaired glucose tolerance.
39. A method for modulating the activity of NK2R, comprising contacting NK2R with a compound as defined in any one items 1 to 31.
40. Use of a compound as defined in any one items 1 to 31 for the manufacture of a medicament for the treatment of a metabolic disorder.
Items II
1. A compound according to formula (I):
(A)-(B) (I),
Fa-Lg (II),
8. The compound according to any one of the preceding items, wherein the conjugated moiety (B) is covalently attached to the N-terminus of (A) via an amide bond with the N-terminal α-NH2 group.
9. The compound according to any one of the preceding items, wherein the peptide (A) is amidated on the C-terminus.
10. The compound according to any one of the preceding items, wherein the peptide (A) comprises from 7 to 15 amino acids, such as from 7 to 14 amino acids, such as from 7 to 13 amino acids, such as from 7 to 12 amino acids, such as from 7 to 11 amino acids, such as from 7 to 11 amino acids, such as from 7 to 10 amino acids, such as from 7 to 9 amino acids, such as from 7 to 8 amino acids, preferably wherein the peptide comprises 7 amino acids.
11. The compound according to any one of the preceding items, wherein the peptide (A) consists of 7 amino acids of the general formula X1X2X3X4X5X6X7.
12. The compound according to any one of the preceding items, wherein the compound consists of the sequence of any one of SEQ ID NO: 1 to SEQ ID NO: 57.
13. The compound according to any one of the preceding items, wherein
15. The compound according to any one of the preceding items, wherein the compound is a neurokinin receptor 2 (NK2R) agonist, such as a selective neurokinin receptor 2 (NK2R) agonist.
Materials:
Formic acid, LiCl, CaCl2, Tris-HCl, EDTA, HEPES, NaCl, Chloroquine, and ovalbumin (albumin from chicken eggs) (Sigma Aldrich). COS-7 monkey kidney cell line was obtained from ATCC. DMEM 1885, FBS, Penicillin/Streptomycin (P/S) and HBSS were from Thermo Scientific/Gibco. Clear Costar 96 wells Tissue Culture-treated plates and solid white 96-well plates from Corning. Polylysine Coated Yttrium Silicate SPA Beads (#RPNQ0010) and Myo-[2-3H(N)]-inositol—(#NET114A[005MC]) from Perkin Elmer. Inositol-1,4,5-Trisphosphate [3H] Radioreceptor Assay (IPs Assay) from Perkin Elmer. pcDNA3.1(+) containing coding sequences of human and mouse tachykinin receptor 1, 2, 3 mRNA were obtained from Genscript (custom order). Synthesized peptides diluted in saline+0.2% (w/v) ovalbumin.
mRNA IDs
Methods:
Agonist-induced G-protein coupled receptor (GPCR) activation was measured by an Inositol-1,4,5-Trisphosphate [3H] Radioreceptor Assay (IPs Assay). Assays were carried out using COS-7 cells transiently transfected by calcium phosphate transfection with a vector pcDNA3.1(+) encoding one of the indicated receptors (Genscript). Briefly, DNA mixed with CaCl2 (2 M) and TE-buffer (10 mM Tris-HCl, 1 mM EDTA, pH 7.5) was dropwise added to 2×HBS (50 mM HEPES, 280 mM NaCl, 1.5 mM NaH2PO4, pH 7.2) and incubated for 45 min at room temperature (22±2° C.). The mixture and a final concentration of 100 μM Chloroquine were added to the cells and left to incubate for 5 hours at 37° C. under standard cell culture conditions (10% CO2) before changing medium to fresh medium containing 5 μl/mL Myo-[2-3H(N)]-inositol (labelling medium).
The IP3 assay takes advantage of the tachykinin receptors' ability to induce production of the inositol trisphosphate (IP3) second messenger upon agonist (ligand) binding on receptor expressing cells following an initial 3H-inositol labelling period. In effect this means that production of the second messenger IP3 as a measure of receptor activity can be assessed by counting 3H-activity.
Assay solutions used: Wash buffer (HBSS), Assay buffer (HBSS+10 mM LiCl and 0,2% w/v ovalbumin), Lysis buffer (10 mM formic acid), and SPA YSI beads (12.5 mg/ml in H2O). The assay was performed the day after transfection. Briefly, labelling medium was aspirated, and plates were washed ×1 in wash buffer before adding 100 μl assay buffer, pre-incubated for 30 min followed by 120 min incubation with agonist, both at 37° C. After incubation, plates were immediately placed on ice and the incubation medium was aspirated and 40 μl of 10 mM formic acid per well was added. Plates were incubated for at least 30 min on ice. 60 μl (1 mg/well) SPA YSI beads/well was pipetted into a solid white 96 wells plate and 35 μl of the lysis solution was transferred to the plate before covering plates with seal cover and shaking for 10 min. (max speed). Centrifuge plates and leave for 8 hours at room temperature before counting the plates in a MicroBeta plate counter (Perkin Elmer).
Materials:
Formic acid, LiCl, CaCl2, Tris-HCl, EDTA, HEPES, NaCl, NaH2PO4, Chloroquine, ovalbumin (albumin from chicken eggs), and human serum albumin (HSA) (Sigma Aldrich). COS-7 monkey kidney cell line was obtained from ATCC. DMEM 1885, FBS, Penicillin/Streptomycin (P/S) and HBSS were from Thermo Scientific/Gibco. Clear Costar 96 wells Tissue Culture-treated plates and solid white 96-well plates from Corning. Polylysine Coated Yttrium Silicate SPA Beads (#RPNQ0010) and Myo-[2-3H(N)]-inositol—(#NET114A[005MC]) from Perkin Elmer. Inositol-1,4,5-Trisphosphate [3H] Radioreceptor Assay (IPs Assay) from Perkin Elmer. pcDNA3.1(+) containing coding sequences of human tachykinin receptor 1, 2, 3 mRNA were obtained from Genscript (custom order). Synthesized peptides diluted in saline+0.2% (w/v) ovalbumin or saline+1% (w/v) HSA.
Methods:
Agonist-induced G-protein coupled receptor (GPCR) activation was measured by an Inositol-1,4,5-Trisphosphate [3H] Radioreceptor Assay (IPs Assay). Assays were carried out using COS-7 cells transiently transfected by calcium phosphate transfection with a vector pcDNA3.1(+) encoding one of the indicated receptors (Genscript). Briefly, DNA mixed with CaCl2 (2 M) and TE-buffer (10 mM Tris-HCl, 1 mM EDTA, pH 7.5) was dropwise added to 2×HBS (50 mM HEPES, 280 mM NaCl, 1.5 mM NaH2PO4, pH 7.2) and incubated for 45 min at room temperature (22±2° C.). The mixture and a final concentration of 100 μM Chloroquine were added to the cells and left to incubate for 5 hours at 37° C. under standard cell culture conditions (10% CO2) before changing medium to fresh medium containing 5 μl/mL Myo-[2-3H(N)]-inositol (labeling medium).
The indirect HSA binding IP3 assay takes advantage of the tachykinin receptors' ability to induce production of the inositol trisphosphate (IP3) second messenger upon agonist (ligand) binding on receptor expressing cells following an initial 3H-inositol labeling period. The assay relies on the assumption that high peptide HSA binding will result in low receptor-mediated production of the second messenger IP3. Thus, the assay is an indirect assessment HSA binding.
Assay solutions used: Wash buffer (HBSS), Assay buffer 0.2% OvAlb (HBSS+10 mM LiCl and 0,2% w/v ovalbumin) or Assay buffer 1% HSA (HBSS+10 mM LiCl and 1% w/v HSA), Lysis buffer (10 mM formic acid), and SPA YSI beads (12.5 mg/ml in H2O). The assay was performed the day after transfection. Briefly, labelling medium was aspirated, and plates were washed ×1 in wash buffer before adding 100 μl assay buffer 0.2% OvAlb or assay buffer 1% HSA, pre-incubated for 30 min followed by 120 min incubation with agonist, both at 37° C. After incubation, plates were immediately placed on ice and the incubation medium was aspirated and 40 μl of 10 mM formic acid per well was added. Plates were incubated for at least 30 min on ice. 60 μl (1 mg/well) SPA YSI beads/well was pipetted into a solid white 96 wells plate and 35 μl of the lysis solution was transferred to the plate before covering plates with seal cover and shaking for 10 min. (max speed). Centrifuge plates and leave for 8 hours at room temperature before counting the plates in a MicroBeta plate counter (Perkin Elmer).
Materials:
CaCl2, Tris-HCl, EDTA, HEPES, NaCl, NaH2PO4, Chloroquine, ovalbumin (albumin from chicken eggs, MnCl2·4H2O, and Bacitracin from Sigma Aldrich. COS-7 monkey kidney cell line was obtained from ATCC. DMEM 1885, FBS, Penicillin/Streptomycin (P/S), HBSS, and 1M Tris/HCl were from Thermo Scientific/Gibco. White/Clear bottom 96 well plates from Costar. 3H-NKA (Novo Nordisk #NNC0392-0000-0497). Ultima Gold XR from Perkin Elmer.
pcDNA3.1(+) containing coding sequences of human and mouse tachykinin receptor 1, 2, 3 mRNA were obtained from Genscript (custom order). Synthesized peptides diluted in saline+0.2% (w/v) ovalbumin (OvAlb).
Methods:
Assays were carried out using COS-7 cells transiently transfected by calcium phosphate transfection with a vector pcDNA3.1(+) encoding one of the indicated receptors (Genscript). Briefly, DNA mixed with CaCl2 (2 M) and TE-buffer (10 mM Tris-HCl, 1 mM EDTA, pH 7.5) was dropwise added to 2×HBS (50 mM HEPES, 280 mM NaCl, 1.5 mM NaH2PO4, pH 7.2) and incubated for 45 min at room temperature (22±2° C.). The mixture and a final concentration of 100 μM Chloroquine were added to the cells and left to incubate for 5 hours at 37° C. under standard cell culture conditions (10% CO2) before changing medium to fresh maintenance medium.
The 3H-NKA binding assay measures peptide-receptor binding by a competitive principle of receptor binding between radioactively labelled (3H) NKA (tracer) and synthesized peptide ligands on live cells expressing the receptor of interest. Assay solutions used: TKR buffer (50 mM Tris/HCl pH 7.5, 5 mM MnCl2, and 150 mM NaCl), wash buffer (TKR buffer+0.2% w/v OvAlb), binding buffer (wash buffer+0.1 mg/ml Bacitracin), and tracer solution (binding buffer+˜15000 cpm/well 3H-tracer). The assay was performed on ice the day after transfection.
Briefly, maintenance medium was aspirated, and plates were washed ×1 in cold wash buffer before adding 100 μl cold binding buffer and placing at 4° C. to let plates cool. The cold plates were added indicated peptides (ligand) immediately followed by addition of cold tracer solution (˜15000 cpm/well). Plates were immediately moved to 4° C. and incubated for 4 hours. After incubation, binding was stopped by wash ×2 with cold wash buffer before adding 225 μl Ultima Gold XR. Plates were shaken at medium speed for approximately 30 min and left overnight at room temperature before counting the plates in a MicroBeta plate counter (Perkin Elmer).
Materials:
CaCl2, Tris-HCl, EDTA, HEPES, NaCl, Chloroquine, 3-isobutyl-1-methylxanthine (IBMX), and ovalbumin (albumin from chicken eggs) (Sigma Aldrich). COS-7 monkey kidney cell line was obtained from ATCC. DMEM 1885, FBS, Penicillin/Streptomycin (P/S) and HBSS were from Thermo Scientific/Gibco. Solid white 96 well plates from Corning. Hithunter cAMP assay for Biologics from Discover X. pcDNA3.1(+) containing coding sequences of human and mouse tachykinin receptor 1, 2, 3 mRNA were obtained from Genscript (custom order). Synthesized peptides diluted in saline+0.2% (w/v) ovalbumin.
Methods:
As a secondary measure of agonist-induced G-protein coupled receptor (GPCR) activation cyclin adenosine monophosphate (cAMP) was measured by the Hithunter cAMP-assay from DiscoverX. Assays were carried out using COS-7 cells transiently transfected by calcium phosphate transfection with a vector pcDNA3.1(+) encoding one of the indicated receptors (Genscript). Briefly, DNA mixed with CaCl2 (2 M) and TE-buffer (10 mM Tris-HCl, 1 mM EDTA, pH 7.5) was dropwise added to 2×HBS (50 mM HEPES, 280 mM NaCl, 1.5 mM NaH2PO4, pH 7.2) and incubated for 45 min at room temperature (22±2° C.). The mixture and a final concentration of 100 μM Chloroquine were added to the cells and left to incubate for 5 hours at 37° C. under standard cell culture conditions (10% CO2) before changing medium to fresh maintenance medium.
The cAMP-assay takes advantage of the tachykinin receptors' ability to induce production of the cAMP second messenger upon agonist (ligand) binding on receptor expressing cells. Production of cAMP stems from receptor coupling to Gs-protein although coupling to Gq-protein (IP3-production) is considered the primary signalling mechanism by tachykinin receptors.
The assay was performed the day after transfection. Briefly, maintenance medium was aspirated, and plates were washed ×1 in HBSS before adding assay buffer (HBSS+1 mM IBMX), pre-incubated for 30 min at 37° C. followed by 15 min incubation with agonist (ligand) at 37° C. After incubation, plates were subjected to cell lysis and anti-cAMP-antibody incubation as described by manufacturer. Luminescence was measured with EnVision Multimode Plate Reader from Perkin Elmer.
General Methods
The following relates to methods for synthesising resin bound peptides (SPPS methods, including methods for de-protection of amino acids, methods for cleaving the peptide from the resin, and for its purification), as well as methods for detecting and characterising the resulting peptide (LCMS and UPLC methods).
SPPS Method
The Fmoc-protected amino acid derivatives used were the standard recommended: Fmoc-Ala-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Asn(Trt)-OH, Fmoc-Asp(OtBu)-OH, Fmoc-Cys(Trt)-OH, Fmoc-Gln(Trt)-OH, Fmoc-Glu(OtBu)-OH, Fmoc-Gly-OH, Fmoc-His(Trt)-OH, Fmoc-Ile-OH, Fmoc-Leu-OH, Fmoc-Lys(BOC)-OH, Fmoc-Met-OH, Fmoc-Phe-OH, Fmoc-Pro-OH, Fmoc-Ser(tBu)-OH, Fmoc-Thr(tBu)-OH, Fmoc-Trp(BOC)-OH, Fmoc-Tyr(tBu)-OH, Fmoc-Val-OH and Fmoc-Lys(Mtt)-OH supplied from e.g. Anaspec, Bachem, Iris Biotech, or NovabioChem.
The N-terminal amino acid is Boc protected at the alpha amino group (e.g. Boc-Asp(OtBu)-OH for peptides with Asp at the N-terminus).
The introduction of the substituent on the epsilon-nitrogen of a lysine was achieved using a lysine protected with Mtt (Fmoc-Lys(Mtt)-OH). Suitably protected building blocks such as Fmoc-8-amino-3,6-dioxaoctanoic acid, and Fmoc-Glu-OtBu were used for the introduction of the substituent. Introduction of the fatty acid moiety was achieved using building blocks such as octadecanedioic acid mono-tert-butyl-ester.
SPPS was performed on a SymphonyX Solid Phase Peptide Synthesizer from Protein Technologies (Tucson, AZ 85714 U.S.A.) at 100-μmol, 150-μmol, 300 μmol or 450-μmol scale using 4, 6, 12, or 18-fold excess of Fmoc-amino acids (300 mM in DMF with 300 mM Oxyma Pure®) relative to resin loading. Fmoc-PAL AM resin (Novabiochem, loading e.g. 0.61 nmol/g), Rink Amide AM polystyrene resin (Novabiochem, loading e.g. 0.64 mmol/g), or 2-chlorotrityl chlorid resin (loading 1.42 mmol/g) was used as the solid support. Fmoc-deprotection was performed using 20% piperidine in DMF. Coupling was performed using 1:1:1:1 amino acid/(Oxyma Pure®)/DIC/collidine in DMF. DCM (1×1.5 ml) and DMF top washes (6×6 ml) were performed between deprotection and coupling steps. Coupling times were generally 60 minutes (ranging from 60 min to 8 hours). Some amino acids including, but not limited to Fmoc-Arg(Pbf)-OH, and Fmoc-Gly-OH were “double coupled”, meaning that after the first coupling (e.g. 60 min), the resin is drained and more reagents are added (amino acid, Oxyma Pure®, DIC, and collidine), and the mixture allowed to react again (e.g. 60 min). The Mt group was removed by first washing the resin with DCM (1×1 min) followed by suspending the resin in HFIP/DCM/TIS (75/23/2) (1×5 min). The resin was washed with DCM and suspended in HFIP/DCM/TIS (75/23/2) (2×25 min with a DCM wash in between) subsequently washed in sequence with DMF(1×), DCM(4×), DMF(2×), Piperidine/DMF (20:80), DMF(1×), DCM(1×), DMF(6×).
Cleavage from the Resin
After synthesis the resin was washed with DCM, and the peptide was cleaved from the resin by a 2-3-hour treatment with TFA/TIS/water (95/2.5/2.5) followed by precipitation with diethylether. The precipitate was washed with diethylether.
Purification
The crude peptide was dissolved in a suitable solvent mixture (such as e.g. 10/20/70 acetic acid/MeCN/water) and purified by reversed-phase preparative HPLC (Waters Prep) on a column containing C18-silica gel. Elution was performed with an increasing gradient of MeCN in water containing 0.1% TFA or with an increasing gradient of 80:20 MeCN:MQ-water in phosphatebuffer (20 mm Na2HPO4, 20 mm NaH2PO4, 10% MeCN in MQ at pH 7.2). Relevant fractions were analysed by a combination of UPLC, and LCMS methods, and the appropriate fractions were pooled and freeze dried.
Methods for Detection and Characterization
LCMS Methods
LCMS was performed on a setup consisting of Waters Acquity UPLC system and LCT Premier XE mass spectrometer from Micromass. The analysis was performed at room temperature by injecting an appropriate volume of the sample (preferably 2-10 μl) onto the column (Waters Acquity UPLC BEH, C-18, 1.7 μm, 2.1 mm×50 mm) which was eluted with a gradient of A, B (and D).
Method: LCMS34
Eluents: A: 0.1% Formic acid in MQ-water. B: 0.1% Formic acid in acetonitrile. Gradient: Linear 5%-95% acetonitrile during 4.0 min at 0.4 ml/min. Detection: 214 nm (analogue output from TUV (Tunable UV detector)) MS ionisation mode: API-ES (positive mode). Scan: 100-2000 amu (alternatively 500-2000 amu), step 0.1 amu.
Method: LCMS43
Eluents: A: MQ-water. B: acetonitrile D: 100 mm triethylammonium acetate in water: acetonitrile 1:1 (pH adjusted to 7.8 with Et3N+AcOH). Gradient: Linear 0%-97.5% acetonitrile+isocratic 2.5% D during 4.0 min at 0.4 ml/min. Detection: 214 nm (analogue output from TUV (Tunable UV detector)) MS ionisation mode: API-ES (negative mode). Scan: 100-2000 amu (alternatively 500-2000 amu), step 0.1 amu.
UPLC Methods
The reverse phase-analysis was performed using a Waters UPLC system fitted with a dual band detector. UV detections at 214 nm were collected using an ACQUITY UPLC BEH, C18, 1.7 um, 2.1 mm×150 mm column. The UPLC system was connected to two eluent reservoirs A and B.
Method: UPLC01:
Column temperature: 40° C. Eluents: A: 99.95% MQ-water, 0.05% TFA, B: 99.95% CH3CN, 0.05% TFA. The following linear gradient was used: 95% A, 5% B to 40% A, 60% B over 16 minutes at a flow-rate of 0.40 ml/min.
Method: UPLC02:
Column temperature: 40° C. Eluents: A: 99.95% MQ-water, 0.05% TFA, B: 99.95% CH3CN, 0.05% TFA. The following linear gradient was used: 95% A, 5% B to 5% A, 95% B over 16 minutes at a flow-rate of 0.40 ml/min.
Method: UPLC6
Column temperature: 60° C. Eluents: A: 0.02 m Na2SO4, 0.002 m Na2HPO4, 0.002 m NaHPO4, B: 70% CH3CN in MQ-water. Step gradient: 10-20% B over 3 minutes, then 20-50% B over 17 minutes, then 50-80% B over 1 minute. Step gradient run-time: 21 minutes at a flow-rate of 0.40 ml/min.
Method: UPLC61:
Column temperature: 60° C. Eluents: A: 0.02 m Na2SO4, 0.002 m Na2HPO4, 0.002 m NaHPO4, B: 70% CH3CN in MQ-water. Step gradient: 10-20% B over 3 minutes, then 20-80% B over 17 minutes, then 80-90% B over 1 minute. Step gradient run-time: 21 minutes at a flow-rate of 0.40 ml/min.
Position 4 (X1) Mutations
Selectivity and activation were measured by IP3-assay on human NK1-, NK2- and NK3Rs as described in Example 1. Gs-coupling was investigated by cAMP accumulation as described in Example 4. Binding was measured by competitive 3H-NKA binding as described in Example 3.
Results:
Summary
Position 5 (X2) Mutations
Selectivity and activation were measured by IP3-assay on human NK1-, NK2- and NK3Rs as described in Example 1.
Summary
Position 6 (X3) Mutations
Selectivity and activation were measured by IP3-assay on human NK1-, NK2- and NK3Rs as described in Example 1. Gs-coupling was investigated by cAMP accumulation as described in Example 4. Binding was measured by competitive 3H-NKA binding as described in Example 3.
Summary
Position 7 (X4) Mutations
Selectivity and activation were measured by IP3-assay on human NK1-, NK2- and NK3Rs as described in Example 1. Gs-coupling was investigated by cAMP accumulation as described in Example 4. Binding was measured by competitive 3H-NKA binding as described in Example 3.
Results
Summary
Position 8 (X5) Mutations
Selectivity and activation were measured by IP3-assay on human NK1-, NK2- and NK3Rs as described in Example 1. Binding was measured by competitive 3H-NKA binding as described in Example 3.
Summary
Position 9 (X6) Mutations
Selectivity and activation were measured by IP3-assay on human NK1-, NK2- and NK3Rs as described in Example 1.
Summary
Position 10 (X7) Mutations
Selectivity and activation were measured by IP3-assay on human NK1-, NK2- and NK3Rs as described in Example 1. Gs-coupling was investigated by cAMP accumulation as described in Example 4. Binding was measured by competitive 3H-NKA binding as described in Example 3.
Summary
Materials:
NaH2PO4·H2O, Na2HPO4·2H2O, propylene glycol, maintenance diet for rats and mice (regular chow, #1320, Altromin), C57BL/6NRj mice (Janvier Labs), high fat diet (HFD) with 60% energy from fat (#D12492, Research Diets Inc.), peptide analogues (Novo Nordisk), d-glucose (Sigma), insulin (Novo Nordisk), sterile saline solution (Apoteket), Tween-80 (Sigma), Loperamide (Sigma), glucometer and glucose strips (Bayer), and Promethion System (Sable Systems International) for assessment of metabolic and behavioral information.
Time of flight liquid chromatography mass spectrometry (TF-LC-MS): ethanol, methanol, acetonitrile, formic acid, milli-q-water, TurboFlow Cyclone column 0.5×50 mm, Aeris Peptide XB-C18 2.1×50 mm (3.6 μm), Thermo TSQ Altis triple quadrupole Mass spectrometer.
Metabolite identification liquid chromatography mass spectrometry (MetID-LC-MS): methanol, acetonitrile, formic acid, milli-q-water, Water Acquity UPLC Protein BEH C4 2.1×50 mm 300 Å (1.7 μm), Bruker MaXis QTOF.
In vivo buffer for peptide analogues: 8 mM phosphate and 240 mM propylene glycol, pH 8.2.
In vivo buffer for Loperamide: Saline supplemented with 1% (v/v) Tween-80.
Methods:
Animals were housed with access to maintenance diet from weaning till around 6-10 weeks of age. At any time, except from fasting, mice had ad libitum access to food and water with a 12-hour light-dark cycle and 22-24 degree Celsius temperature. All animal experiments were performed according to Danish Animal Inspectorate regulations. For studies using diet-induced obese mice, mice were fed a HFD for at least 20 weeks prior to experimentation. Specifically, for mice undergoing glucose and insulin tolerance tests, mice above 45 g were selected.
Indirect calorimetry was used to evaluate the ability of individual peptide analogues to dose-dependently increase energy expenditure (EE) we used metabolic cages and indirect calorimetry measured by the Promethion system. To this end, oxygen consumption was used as a surrogate measure for EE. Substrate preference (fat or carbohydrate) was evaluated using the respiratory exchange ratio (RER). In parallel, behavioral information such as walking distance and water and food intake were recorded.
Prior to experimentation, DIO mice were transferred to habituation cages for at least 10 days (5 days outside and at least 5 days in the Sable Systems gas analyzer module) to acclimatize. For all in vivo compound tests for EE evaluation, mice received subcutaneous injections between 2 and 4 pm.
Pharmacokinetics: To evaluate the in vivo half-life of individual peptide analogues we used wild type lean mice at around 10 weeks of age injected once with a peptide analogue. For each sample time point 3-4 mice were used and blood was drawn from the submandibular vein at indicated time points following injection. Prior to injection mice were given ad libitum access to standard chow diet. Mice were subcutaneously injected with 0.5 mg/kg peptide analogue in a volume of 2 ml/kg.
The amount of peptide analogue and metabolite(s) present in blood samples were measured by TF-LC-MS and MetID-LC-MS, respectively.
TF-LC-MS:
Sample preparation: One volume of plasma is precipitated with three volumes of ethanol (with internal standard). The mixture is centrifuged at 13000 g for 20 min. One volume of supernatant is diluted with two volumes of Milli-Q water (1% formic acid).
Calibration curve: peptide analogue was spiked into blank mouse plasma. Range: 0.5 to 2000 nM (linear 1/x2).
Chromatography, Mobile phase: Mobile phase A: 5% (50/50 methanol/acetonitrile)+95% Milli-Q+1% formic acid. Mobile phase B: 5% Milli-Q+95% (50/50 methanol/acetonitrile)+1% formic acid.
Columns: TurboFlow Cyclone 0.5×50 mm and Aeris Peptide XB-C18 2.1×50 mm (3.6 μm)
Mass spectrometry: Thermo TSQ Altis triple quadrupole, Positive electrospray ionisation mode, MRM-mode.
MetID-LC-MS:
Sample preparation: One volume of plasma is precipitated with three volumes of methanol. The mixture is centrifuged at 13000 g for 20 min. One volume of supernatant is diluted with two volumes of Milli-Q water (1% formic acid)
Calibration curve: peptide analogue was spiked into blank mouse plasma. Range: 20, 200 and 2000 nM (linear 1/x2)
Chromatography: Mobile phase A: 0.1% formic acid in Milli-Q water. Mobile phase B: 0.1% formic acid in acetonitrile.
Column: Water Acquity UPLC Protein BEH C4 2.1×50 mm 300 Å (1.7 μm)
Mass spectrometry: Bruker MaXis QTOF, Positive electrospray ionisation mode
Full scan (m/z from 300 to 1800) and MS/MS.
Energy expenditure screening: After habituation, mice were subcutaneously injected with 0.5 mg/kg peptide analogue in a volume of 2 ml/kg. Mice were injected q.a.d. (quaque altera die) and received a total of two injections. EE was evaluated and increase over vehicle was calculated as percent of mean oxygen consumption over a 30-hour period after injection. Prior to injection peptide analogues were dissolved to 0.25 mg/ml in in vivo buffer.
Energy expenditure and weight loss pharmacodynamics: To evaluate the ability of individual peptide analogues to dose-dependently increase EE by indirect calorimetry in DIO mice. Body weights were monitored from beginning of habituation until end of experiment. After habituation, mice were treated with daily injections of NKA(4-10) analogues at four different doses or vehicle by daily subcutaneous injections for nine days.
EE, body weight, food intake, water intake and walking distance were observed for all 9 days. EE increase over vehicle was calculated as percent of mean oxygen consumption over a 30-hour period after injection. Prior to injection peptide analogues were dissolved and diluted in in vivo buffer. In order to take into account, the different half-lives for the compounds tested, the doses for each compound were calculated based on the given compound's individual pk-profiles. Target AUCs for each compound were calculated relative to compound 305 as seen below in order to reach same AUC of tested compounds.
Insulin tolerance test: The effect on insulin tolerance was determined using intraperitoneal insulin tolerance test (ipITT) 24 hours after single subcutaneous injection of NK2R-selective analogue in DIO mice. At day of experimentation, mice were fasted two hours prior to receiving 1.5 U/kg insulin diluted in saline solution (0.2 mL/kg) by intraperitoneal injection. Change in glucose was monitored using glucometer.
Glucose tolerance test: The effect on glucose tolerance was determined using intraperitoneal glucose tolerance test (ipGTT) 24 hours after single subcutaneous injection of NK2R-selective analogue in DIO mice. At day of experimentation, mice were fasted four hours prior to receiving 1 g/kg glucose diluted in saline solution (0.1 mL/kg) by intraperitoneal injection. Change in glucose was monitored using glucometer.
Dysfunctional voiding test: To investigate the effect on dysfunctional voiding, constipation was induced in lean wild-type, male and female, mice using Loperamide (5 mg/kg). 30 minutes after gavage, mice were subcutaneously dosed with either vehicle, 130, 260 and for males also 325 nmol/kg of EB344. 6 hours post Loperamide gavage, mice were removed from the cage and number of feces pellets were counted.
Position and composition of “conjugated moiety” i.e. “protractor” was investigated on endogenous NKA and NK2R-selective NKA(4-10) analogues.
Protractor position was addressed in the N-terminal region of NKA(4-10) analogues.
Charge of linker between peptide backbone and fatty acid was investigated.
Fatty acid species was investigated.
Selectivity and activation were measured by IP3-assay on human NK1-, NK2- and NK3Rs as described in Example 1. Gs-coupling was investigated by cAMP accumulation as described in Example 4. Binding was measured by competitive 3H-NKA binding as described in Example 3. Albumin binding was determined by receptor activation, using IP3 assay, in presence or absence of 1% human serum albumin as described in example 2. Energy expenditure was measured in diet-induced obese mice housed in metabolic cages as described in example 8. Pharmacokinetics and exposure were measured in plasma from lean mice treated with compound as described in example 8.
Results
Protractor Position
Linker Charge
Summary
Based on the present example, it is concluded that a protractor constituting 2OEG-gammaGlu-C18 diacid positioned on N-terminal of NKA(4-10) analogues, as exemplified by compound 304, 305 and 344 is optimal for NK2R activation, selectivity, half-life and energy expenditure induction.
Effect of protracted NKA analogues on energy expenditure and weight loss in diet-induced obese (DIO) mice.
Energy expenditure was measured in diet-induced obese mice housed in metabolic cages as described in example 8. Pharmacokinetics and exposure were measured in plasma from lean mice treated with compound as described in example 8.
Results
Summary
The present example demonstrates that highly NK2R selective and long-lived compounds of the present disclosure, such as 344 and 383, are preferred for weight loss induction.
Effect of protracted highly NK2R-selective NKA(4-10) analogues on glucose and insulin tolerance in insulin resistant and pre-diabetic diet-induced obese (DIO) mice.
Glucose and insulin tolerance tests were performed on diet-induced obese mice as described in example 8.
Results
The results are shown in
Summary
Pharmacological NK2R activation improves glucose and insulin tolerance in diet-induced obese mice. The compounds of the present disclosure therefore have the potential to treat insulin resistance and diabetes.
Effect of protracted highly NK2R-selective NKA(4-10) analogues on Loperamide-induced dysfunctional voiding in lean wild-type mice Loperamide-induced constipation was used as a model for dysfunctional voiding, as described in example 8.
Results
The results are shown in
Summary
Pharmacological NK2R activation corrects dysfunctional voiding in mice. The compounds of the present disclosure therefore have the potential to treat dysfunctional voiding.
| Number | Date | Country | Kind |
|---|---|---|---|
| 20206509.0 | Nov 2020 | EP | regional |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/EP2021/081057 | 11/9/2021 | WO |
