BLOOD GLUCOSE LOWERING COMPOUND

Abstract

The present invention relates to the field of medicine and the treatment of diabetes or hyperglycemia within that field. More particularly, the invention relates to a compound that lowers blood glucose, pharmaceutical compositions containing such a compound, and therapeutic uses of such a compound.

Claims

1. A compound comprising an A chain and a B chain, wherein the amino acid sequence of the A chain is SEQ ID NO: 1 and the amino acid sequence of the B chain is SEQ ID NO: 2, and wherein the A and B chains contain a disulfide bond between the cysteine at position 7 of the A chain and the cysteine at position 7 of the B chain, a disulfide bond between the cysteine at position 20 of the A chain and the cysteine at position 19 of the B chain, and a disulfide bond between the cysteine at position 6 of the A chain and the cysteine at position 11 of the A chain.

2. A pharmaceutical composition comprising the compound of claim 1 and one or more pharmaceutically acceptable excipients.

3. A method of treating diabetes in a patient comprising administering to a patient in need thereof an effective amount of the compound of claim 1.

4. A method of treating diabetes in a patient comprising administering to a patient in need thereof an effective amount of the composition of claim 2.

5. A method of treating hyperglycemia in a patient comprising administering to a patient in need thereof an effective amount of the compound of claim 1.

6. A method of treating hyperglycemia in a patient comprising administering to a patient in need thereof an effective amount of the composition of claim 2.

7. The compound of claim 1 for use in therapy.

8. The compound of claim 1 for use in the treatment of diabetes.

9. The compound of claim 1 for use in the treatment of hyperglycemia.

10. The use of a compound of claim 1, in the manufacture of a medicament for the treatment of diabetes.

11. The use of a compound of claim 1, in the manufacture of a medicament for the treatment of hyperglycemia.

Description

EXAMPLE 1

Expression and Purification of Compound 330

[0020] A precursor protein for Compound 330 is expressed in B strain E. coli bacteria, strain BL21 from New England Biolabs, product #C2530H. The precursor protein is produced from a modified pBR322 plasmid, whose gene is regulated by a phoA promoter. Typical fermentations are run from 36-72 hours during which the protein is secreted into the media.

[0021] Medium is conditioned by lowering the pH with glacial acetic acid. At a pH of 4, many contaminating proteins, lipids, cellular debris, and other matter precipitate and are removed by centrifugation. This conditioned media is put through a depth filter prior to an ultrafiltration/diafiltration (“UFDF”) step designed to remove salt and exchange the protein into 5 mM acetate, 10 mM NaCl, pH 4. The precursor protein is captured at low pH on Big Bead Q resin, an anion exchanger with large pore sizes to allow the passage of fines and other debris from the fermentation. The charged resin is washed and the precursor protein is eluted by salt gradient.

[0022] The pool from the anion exchange is treated with trypsin and carboxipeptidase B to remove regions of the precursor protein, generating a two-chain insulin. The cleavage process is performed at pH 7.5, and 15° C., for 24 hours. The reaction is quenched by lowering the pH to 4 via acid addition. The resulting solution is concentrated and diafiltered by TFF into 5 mM Acetate, pH 4.

[0023] The compound is captured by a cation exchanger (Fast Flow S Sepharose) selected for its ability to bind the positively charged portion of the compound. The charged resin is washed and the compound is eluted by salt gradient. The pool from the cation exchange step is further purified by reversed phase chromatography. The compound is bound to a YMC basic C8 resin with 10 μm bead size in the presence of 0.16% phosphoric acid, and is eluted by an acetonitrile gradient. The final pool is prepared for storage by an overnight dialysis step, followed by concentration, and buffer exchange using spin concentrators to place it in a final buffer of 20 mM Tris, 135 mM NaCl, pH 7.5.

In Vitro Receptor Affinity

[0024] Binding affinities of proteins are determined in receptor binding assays performed on membranes prepared from stably-transfected 293EBNA cells (293HEK human embryonic kidney cells expressing EBNA-1) over-expressing human insulin receptor isoform A (hIR-A), stably transfected 293HEK cells over-expressing human insulin receptor isoform B (hIR-B) containing a C9 epitope tag at the C-terminus, or stably transfected 293HEK cells over-expressing the human IGF-1 receptor (hIGF-1R).

[0025] Receptor binding affinities (K.sub.i) are determined from a competitive radioligand binding assay using either human recombinant (3-[.sup.125I]-iodotyrosyl-A14)-insulin (2200 Ci/mmol, for hIR-A and hIR-B assays) or human recombinant [.sup.125I]-insulin-like growth factor-1 (1800 to 2600 Ci/mmol, for the hIGF-1R assay), (Perkin Elmer Life and Analytical Sciences). The assays are performed with a scintillation proximity assay (SPA) method using polyvinyltoluene (PVT) wheat germ agglutinin-coupled SPA beads (Perkin Elmer Life and Analytical Sciences). SPA Assay Buffer (50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 0.1% w/v fatty-acid free BSA) is used for all reagent preparations.

[0026] Serial dilutions of test samples are prepared in Assay Buffer using a Freedom/Evo robot (Tecan) and 50 μL of dilution added to 96-well white, clear-bottom microplates (Corning/Costar) with a TeMO robot (Tecan). Radioligand (50 μL), membranes (50 μL), and SPA beads (50 μL) are added using a Multi-drop (Thermo Scientific) instrument. Following 10-hour incubation at room temperature, radioactivity is determined using a Microbeta Trilux scintillation counter (Perkin Elmer Life and Analytical Sciences).

[0027] Values for test samples are calculated as percent relative to the activity of unlabeled human insulin or IGF-1 after correcting for non-specific binding. IC.sub.50 values are determined from 4-parameter logistic non-linear regression analysis (XLFit version 4.0, IDBS). If necessary, curve top or bottom parameters are set to 100 or 0, respectively. The affinity constant (K.sub.i) is calculated from the IC.sub.50 value based upon the equation, K.sub.i=IC.sub.50/(1+D /K.sub.d), where D equals the concentration of radio-ligand used in the experiment and K.sub.d equals the equilibrium binding affinity constant of the radioligand determined from saturation binding analysis (hIR-A=0.251 nM; hIR-B=0.205 nM; hIGF−1R=0.233 nM). Reported values for Ki are shown as geometric mean± the standard error of the mean (SEM), with the number of replicate determinations indicated by “n” (Table 1). A qualifier (>) indicates that the data does not reach 50% inhibition, compared to maximum binding, whereby the K.sub.i is calculated using the highest concentration of the compound tested in the assay and no standard error is calculated.

[0028] Compound 330 has binding affinity at both hIR-A and hIR-B and selectivity over hIGF-1R (Table 1).

TABLE-US-00001 TABLE 1 Human Insulin Receptor Isoform A or B (hIR-A or hIR-B) and Human Insulin-Like Growth Factor-1 Receptor (hIGF-1R) Binding Affinity Receptor Binding Affinity, K.sub.i, nM (SEM, n) Sample hIR-A hIR-B hIGF-1R Compound 330 4.53 (0.63, n = 10) 6.07 (0.60, n = 10) >3530 (n = 5) Human insulin 0.189 (0.006, n = 45) 0.233 (0.008, n = 45) 80.1 (5.8, n = 35) IGF-1 4.10 (0.26, n = 45) 50.8 (2.7, n = 45)  0.111 (0.008, n = 33)

Receptor Functional Activation

[0029] Functional activity is determined by ELISA quantitation of hIR-A, hIR-B, or hIGF-1R auto-phosphorylation. Stably-transfected human 293HEK cells that over express hIR-A, hIR-B, or hIGF-1R, each containing a C-terminal C9 epitope tag (TETSQVAPA), are treated at 37° C. for 1 hour with 3-fold serially diluted test compounds in serum-free medium (DMEM, high glucose with glutamine, 10 mM HEPES, pH 7.4, 1 mM sodium pyruvate, 0.8 mg/mL geneticin, 1% penicillin/streptomycin) supplemented with 0.1% fraction V-fatty acid-free BSA (Sigma-Aldrich (St. Louis, Mo., USA)). Cells are rinsed with ice cold PBS and lysed with ice cold NP40 buffer [1% NP-40 (IGEPAL CA-630), 150 mM NaCl, 50 mM TRIS, pH 7.4, 2 mM vanadate, and cOmplete™ protease inhibitors].

[0030] Tyrosine phosphorylation is determined using a sandwich ELISA by capture with anti-C9 monoclonal antibody (RHO 1D4 Antibody, University of British Columbia) and detection with anti-phosphotyrosine monoclonal 4G10®-horseradish peroxidase (4G10®-HRP) conjugate (EMD Millipore, Billerica, Mass., USA) and 3,3,5,5-tetramethylbenzidine (TMB) Pierce HRP substrate (Thermo Scientific, Rockford, Ill., USA). The absorbance is recorded at 450 nm using a PerkinElmer Envision plate reader. The absorbance values are normalized to the maximal response of control cells treated with human insulin (100 nM, for the hIR-A and hIR-B assays) or 10 nM IGF-1 (for the hIGF-1R assay). The data are analyzed using a 4-parameter (curve maximum, curve minimum, EC50, Hill slope) logistic (sigmoidal) nonlinear regression routine (XLFit version 4.0: Activity Base, IDBS). Functional potency is reported as the concentration eliciting a half-maximal response (EC50), with values shown as the geometric mean± the standard error of the mean (SEM), with the number of replicate determinations indicated by “n”.

[0031] Compound 330 is an agonist for hIR-A and hIR-B and is selective for insulin receptors compared to hIGF-1R (Table 2).

TABLE-US-00002 TABLE 2 Human Insulin Receptor (hIR-A and hIR-B) and Human Insulin-like Growth Factor-1 Receptor (hIGF-1R) Phosphorylation in 293 Cells Receptor Phosphorylation EC50, nM (SEM, n) Sample hIR-A hIR-B hIGF-1R Compound 39.2 34.8 (4.9, n = 13) 4270 (611, n = 3) 330 (5.8, n = 12) Human 2.65 2.28 (0.28, n = 13)  254 (29, n = 6) insulin (0.31, n = 12) IGF-1 91.5  253 (36, n = 4)  2.15 (0.22, n = 6) (12.1, n = 11)

Evaluation of In Vivo Potency in a Rat Model of Type 1 Diabetes

[0032] The effects of Compound 330 are investigated in a streptozotocin (STZ)-treated rat diabetes model. Male Sprague-Dawley rats, 400-425 gram body weight, are obtained from Harlan Labs, Indianapolis, Ind. After acclimation for approximately one week, the rats are anesthetized with isoflurane and given a single injection of streptozotocin (Zanosar®, item #89256, Teva Parenteral Medicines, 40 mg/kg IV). The rats are used in studies three days after injection of the streptozotocin; only animals with non-fasted blood glucose between 400-550mg/dl are used in these studies.

[0033] Rats are distributed into groups to provide comparable variance in blood glucose and body weight; rats are randomized using Block Randomized Allocation Tool. Blood glucose is measured using Accucheck Aviva glucometer (Roche). STZ-treated rats are given a single subcutaneous (SC) injection of Compound 330 or vehicle, Sterile Normal Saline (0.9% w/v sodium chloride solution). Blood samples for glucose measurements are collected by tail bleed. The animals have free access to food and water throughout the experiment. Plasma samples from these studies are sent for analysis of compound levels.

[0034] Blood glucose levels for Compound 330 are measured at three dosage levels, 30, 100, 300 nmol/kg in (STZ)-treated rats. Blood glucose is measured 0, 0.5, 1, 2, 4, 6, 8, 10, 12, 24, 36, 48, 72, and 96 hours after injection. Data shown are mean± (standard error mean) SEM (n=5). Statistical analysis was performed using JMP software, and treatment groups are compared to a vehicle control group (*P<0.05).

[0035] Compound 330 shows glucose lowering over the control group of up to 76% with a 100 nmol/kg dose at 8 hours and up to 89% with a 300 nmol/kg dose at 24 hours (Table 3).

TABLE-US-00003 TABLE 3 Blood Glucose Levels over Time after Administration of Compound 330 (mg/dL) Time Compound 330 Compound 330 (hours) 100 nmol/kg (SEM) 300 nmol/kg (SEM) Vehicle (SEM) 0 484 (28) 532 (13) 552 (6)  0.5 471 (52) 539 (18) 541 (7)  1 470 (50) 519 (8)  550 (14) 2 373 (55) 423 (30) 498 (12) 4 301 (53) 139 (24) 512 (30) 6 196 (46) 111 (41) 456 (13) 8 111 (17)  90 (15) 466 (23) 10 136 (18) 119 (12) 488 (31) 12 280 (61) 141 (25) 567 (20) 24 156 (48) 59 (6) 528 (8)  36 386 (76) 98 (7) 590 (10) 48 330 (66) 121 (26) 572 (13) 72 409 (58) 451 (22) 553 (13) 96 480 (51) 503 (9)  562 (10)

TABLE-US-00004 Sequences Polypeptide Sequence (SEQ ID NO: 1) GIVEQCCTSICSLYQLENYCGSGPAGGTSESATPESGPGSEPATSGSETP GTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGS PAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSPA GSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSESA TPESGPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPT STEEGTSTEPSEGSAPGTSTEPSEGSAPGSEPATSGSETPGTSESATPES GPGTSTEPSEGSAPG Polypeptide Sequence (SEQ ID NO: 2) FVNQHLCGSHLVEALYLVCGERGFFYTKPT Polypeptide Sequence (SEQ ID NO: 3) MKKNIAFLLASMFVFSIATNAYAGSPGTSTEPSEGSAPGTSESATPESGP GTSESATPESGPGGAPGSGSGSGGSGGIEGRFVNQHLCGSHLVEALYLVC GERGFFYTKPTRGGGGRGIVEQCCTSICSLYQLENYCGSGPAGGTSESAT PESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPE SGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSET PGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPG TSESATPESGPGTSESATPESGPGTSESATPESGPGSEPATSGSETPGSE PATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGSEPA TSGSETPGTSESATPESGPGTSTEPSEGSAPG