PEPTIDES AS INHIBITORS OF FIBROTIC MATRIX ACCUMULATION

Abstract

The present invention relates to peptides that inhibit overproduction and/or excess accumulation of extracellular matrix in an organ or tissue. The inventive peptides have the general sequence Xa-Leu-Gln-Gly-Xb (SEQ ID NO: 1), wherein Xa is selected from Pro-Gly, Gly and Ac-Gly and Xb is selected from Glu and Glu-NH.sub.2, and are able of inhibit overproduction and excess accumulation of extracellular matrix in an organ or tissue both as linear peptides and as cyclic peptides. In particular the peptides disclosed herein can be used for treating fibrotic conditions characterized by an excess accumulation of extracellular matrix such as liver fibrosis, cirrhosis of the liver, lung fibrosis, chronic respiratory failure, cardiac fibrosis, ischemic heart disease, heart failure, diabetic nephropathy, glomerulonephritis, myelofibrosis, and various types of cancers such as breast cancer, uterus cancer, prostate cancer, pancreas cancer, colon cancer, skin cancer, blood cell cancers, cancers of the central nervous system, fibroids, fibroma, fibroadenomas and fibrosarcomas.

Claims

1. A peptide consisting of the general sequence Xa-Leu-Gln-Gly-Xb (SEQ ID NO: 1), wherein Xa is selected from Pro-Gly, Gly and Ac-Gly and Xb is selected from Glu and Glu-NH.sub.2, and the pharmaceutically acceptable salts thereof.

2. The peptide according to claim 1, wherein Xa is selected from Gly and Ac-Gly and Xb is selected from Glu and Glu-NH.sub.2, and the pharmaceutically acceptable salts thereof.

3. The peptide according to claim 1, wherein Xa is Ac-Gly and Xb is Glu (SEQ ID NO: 3) or Xa is Gly and Xb is Glu-NH.sub.2 (SEQ ID NO: 4).

4. The peptide according to claim 1, wherein Xa is Gly and Xb is Glu and Glu binds to Gly to form the cyclic peptide (SEQ ID NO: 5): ##STR00040##

5. The peptide according to claim 1, wherein Xa is Pro-Gly and Xb is Glu and Pro binds to Glu to form the cyclic peptide (SEQ ID NO: 6): ##STR00041##

6. A pharmaceutical composition comprising the peptide Xa-Leu-Gln-Gly-Xb (SEQ ID NO: 1) according to claim 1, together with at least one pharmaceutically acceptable vehicle, excipient and/or diluent.

7. The pharmaceutical composition according to claim 6, wherein Xa is selected from Gly and Ac-Gly and Xb is selected from Glu and Glu-NH.sub.2, and the pharmaceutically acceptable salts thereof.

8. The pharmaceutical composition according to claim 6, wherein Xa is Ac-Gly and Xb is Glu (SEQ ID NO: 3) or Xa is Gly and Xb is Glu-NH.sub.2 (SEQ ID NO: 4).

9. The pharmaceutical composition according to claim 6, wherein the peptide is ##STR00042##

10. The pharmaceutical composition according to claim 6, wherein the peptide is ##STR00043##

11. A method for the treatment of a fibrotic condition characterized by an excess accumulation of extracellular matrix in a tissue or an organ, comprising administering to a patient a therapeutically effective amount of a peptide consisting of the general sequence Xa-Leu-Gln-Gly-Xb (SEQ ID NO: 1), 15 wherein Xa is selected from Pro-Gly, Gly and Ac-Gly and Xb is selected from Glu and Glu-NH.sub.2, and/or the pharmaceutically acceptable salt thereof, wherein the accumulation of extracellular matrix in said tissue or organ is reduced from the level existing at the time of treatment.

12. The method according to claim 11, wherein the fibrotic condition is selected from the group consisting of liver fibrosis, cirrhosis of the liver, lung fibrosis, chronic respiratory failure, cardiac fibrosis, ischemic heart disease, heart failure, diabetic nephropathy, glomerulonephritis, myelofibrosis, breast cancer, uterus cancer, prostate cancer, pancreas cancer, colon cancer, skin cancer, blood cell cancers, cancers of the central nervous system, fibroids, fibroma, fibroadenomas and fibrosarcomas.

13. The method according to claim 11, wherein the peptide is Xa-Leu-Gln-Gly-Xb (SEQ ID NO: 1), wherein Xa is Ac-Gly and Xb is Glu (SEQ ID NO: 3) or Xa is Gly and Xb is Glu-NH.sub.2 (SEQ ID NO: 4).

14. The method according to claim 11, wherein the peptide is ##STR00044##

15. The method according to claim 11, wherein the peptide is ##STR00045##

16. The method according to claim 12, wherein the peptide is Xa-Leu-Gln-Gly-Xb (SEQ ID NO: 1), wherein Xa is Ac-Gly and Xb is Glu (SEQ ID NO: 3) or Xa is Gly and Xb is Glu-NH.sub.2 (SEQ ID NO: 4).

17. The method according to claim 12, wherein the peptide is ##STR00046##

18. The method according to claim 12, wherein the peptide is ##STR00047##

Description

DESCRIPTION OF THE FIGURES

[0118] FIG. 1 Prevention of fibrosis progression by proline-containing cyclic and amidated linear peptides. Mice were injected for 6 weeks with CCl.sub.4 in order to induce liver fibrosis. Starting on day 32 the mice received daily intraperitoneal injections of 25 mg/kg/mouse/day of the peptides in 0.9% NaCl for a total of 10 days. CCl.sub.4-treated mice also received NaCl 0.9%. The healthy control group (CT) only received 0.9% NaCl. N=4/ 10/ 8/ 5/10/ 8/ 11/ 7 in two experiments. The following peptides were tested on CCl.sub.4-treated mice: cyclic Pro-Gly-Leu-Gln-Gly-Glu (Cyclic PGLQGE), and compared to two controls: cyclic Pro-Gly-Leu-Asn-Gly-Glu (Cyclic CT1+P: cyclic PGLNGE) and cyclic Pro-Gly-Leu-Hyp-Gly-Glu (Cyclic CT2+P: cyclic PGLOGE). Linear Gly-Leu-Gln-Gly-Glu-NH.sub.2 (Linear GLQGE-NH.sub.2) was also tested and compared to two controls: linear Gly-Leu-Asn-Gly-Glu-NH.sub.2 (Linear CT1-NH.sub.2: linear GLNGE-NH.sub.2) and linear Gly-Leu-Hyp-Gly-Glu-NH.sub.2 (Linear CT2-NH.sub.2: linear GLOGE-NH.sub.2). *p<0.05. Evaluated by t-test. Data presented as mean±SEM.

[0119] FIG. 2 Prevention of fibrosis progression by cyclic peptides and acetylated linear peptides. Mice were injected for 6 weeks with CCl.sub.4 in order to induce liver fibrosis. Starting on day 32 the mice received daily intraperitoneal injections of 25 mg/kg/mouse/day of the peptides in 0.9% NaCl for a total of 10 days. CCl.sub.4-treated mice also received NaCl 0.9%. The healthy control group (CT) only received 0.9% NaCl. N=5/ 18/ 9/ 9/ 6/ 5/ 4/ 4 in two experiments. The following peptides were tested on CCl.sub.4-treated mice: cyclic Gly-Leu-Gln-Gly-Glu (Cyclic GLQGE), and compared to two controls: cyclic Gly-Leu-Asn-Gly-Glu (Cyclic CT1: cyclic GLNGE) and cyclic Gly-Leu-Hyp-Gly-Glu (Cyclic CT2: cyclic GLOGE). Linear Ac-Gly-Leu-Gln-Gly-Glu (Linear Ac-GLQGE) was also tested and compared to two controls: linear Ac-Gly-Leu-Asn-Gly-Glu (Linear Ac-CT1: linear Ac-GLNGE) and linear Ac-Gly-Leu-Hyp-Gly-Glu (Linear Ac-CT2: linear Ac-GLOGE). *p<0.05, **p<0.005. Evaluated by t-test. Data presented as mean±SEM.

[0120] FIG. 3 Prevention of cancer growth by cyclic GLQGE in form of hydrochloride salt (cyclic GLQGE HCl salt). B16 melanoma cancer cells were injected subcutaneously in mice. On day 7, injection with 1 mg of the peptide or 0.9% NaCl subcutaneously was performed. On day 12 mice were euthanized and tumors removed and weighed. N=11/11 mice. Data presented as mean±SEM.

[0121] FIG. 4 Prevention of fibrosis progression by the cyclic peptide Gly-Leu-Gln-Gly-Glu (acetate salt). Lung fibrosis was induced in 6-week-old male C57bl/6 mice using intratracheal bleomycin instillation at a dose of 0.005 units in 50 μl on day 0. Starting on day 11, cyclic Gly-Leu-Gln-Gly-Glu (cyclic GLQGE) was injected subcutaneously at a dose of 1 mg/mouse/day in 0.9% NaCl for a total of 10 days. The control group only received daily injections of 0.9% NaCl. The number of animals in the four groups was N=14/10/10/9 in three experiments. The cyclic peptide Gly-Leu-Gln-Gly-Glu (Cyclic GLQGE) (acetate salt) was tested and compared to cyclic Ac-Gly-Leu-Asn-Gly-Glu (Cyclic GLNGE) (acetate salt) and to cyclic Ac-Gly-Leu-Hyp-Gly-Glu (Cyclic GLOGE) (acetate salt), *p<0.05. Evaluated by t-test. Data are presented as mean±SEM.

[0122] FIG. 5 Prevention of breast cancer growth by cyclic GLQGE in form of hydrochloride salt (cyclic GLQGE HCl salt). Cells from the breast cancer cell line MDA-MB-231 were injected intratibially in mice. Starting on day 30, injection with 0.1 mg/mouse/day of the cyclic peptide Gly-Leu-Gln-Gly-Glu (cyclic GLQGE HCl salt) for 10 days or 0.9% NaCl subcutaneously was performed. On day 40, the size of the tumour was evaluated by bioluminescence imaging (Bioluminescence imaging was performed by detecting photon signal 5 minutes after D-luciferin injection (150 mg/kg) using an “IVIS Lumina II” imaging system. The resulting images were analysed using the software “Living Image”). RLU=relative light units. The number of animals in each group was N=14/7 mice, in two experiments. *p<0.05 as evaluated by t-test. Data are presented as mean±SEM.

[0123] The following examples are included to demonstrate preferred embodiments of the invention. It should be appreciated by those of skill in the art that the techniques disclosed in the examples which follow represent techniques discovered by the inventor to function well in the practice of the invention, and thus can be considered to constitute preferred modes for its practice. However, those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments which are disclosed and still obtain a like or similar result without departing from the spirit and scope of the invention.

[0124] Further modifications and alternative embodiments of various aspects of the invention will be apparent to those skilled in the art in view of this description. Accordingly, this description is to be construed as illustrative only and is for the purpose of teaching those skilled in the art the general manner of carrying out the invention. It is to be understood that the forms of the invention shown and described herein are to be taken as examples of embodiments. Elements and materials may be substituted for those illustrated and described herein, parts and processes may be reversed, and certain features of the invention may be utilized independently, all as would be apparent to one skilled in the art after having the benefit of this description of the invention. Changes may be made in the elements described herein without departing from the spirit and scope of the invention as described in the following claims.

EXAMPLES

Example 1: Peptide Synthesis

[0125] The peptide Gly-Leu-Gln-Gly-Glu (GLQGE) was synthesized in linear form as Gly-Leu-Gln-Gly-Glu-NH.sub.2 (also named linear GLQGE-NH.sub.2) and in linear form as acetate-Gly-Leu-Gln-Gly-Glu (also named linear Ac-GLQGE) and in cyclic form as cyclic Gly-Leu-Gln-Gly-Glu (also named cyclic GLQGE without C-terminal amide and without N-terminal acetate). The peptide Pro-Gly-Leu-Gln-Gly-Glu (also named cyclic PGLQGE) was only synthesized in cyclic form. Both control peptides Gly-Leu-Asn-Gly-Glu (also named as Linear CT1 (Linear GLNGE)) and Gly-Leu-Hyp-Gly-Glu (also named as Linear CT2 (Linear GLOGE)) were synthesized in linear form with C-terminal amidation (GLNGE-NH.sub.2 and GLOGE-NH.sub.2) and with N-terminal acetylation (Ac-GLNGE and Ac-GLOGE) and also in cyclic form with and without proline (cyclic GLNGE or cyclic PGLNGE as well as cyclic GLOGE or cyclic PGLOGE). The designation of all peptides used in the present application are listed in Table 1.

[0126] The linear peptides were synthesized on an ABI 433 peptide synthesizer (Life Technologies) using standard Fmoc (N-(9-fluorenyl)methoxycarbonyl) chemistry on Rink amide resin (Merck KGaA). Peptide purification was by RP-HPLC. Purity and identity of the peptides were verified by RP-HPLC and ESI-TOF mass spectrometry. Cyclic peptide Gly-Leu-Gln-Gly-Glu was synthesized where Glu binds to Gly directly (head to tail cyclization) and H.sub.2O is removed or in the presence of proline. The cyclic peptide with proline in the ring was synthesized as fully protected peptides on TCP resin (Intavis Bioanalytical Instruments AG) and cyclized using propylphosphonic anhydride. The cyclic peptides without proline were synthesized using the liquid phase synthesis. All the groups were protected by protective groups, leaving only the N-terminal amino group and C-terminal carboxyl group. After the ring was formed in the liquid phase, the protective groups were removed. In this case, no TCP resin, and no propylphosphonic anhydride were used.

[0127] Table 1 reports the peptides used in the present invention and the corresponding SEQ ID NOs in the sequence listing.

TABLE-US-00001 TABLE 1  SEQ ID Peptide sequence Form Designation NO Xa-Leu-Gln-Gly-Xb — general sequence 1 Gly-Leu-Gln-Gly-Glu linear Linear GLQGE 2 Ac-Gly-Leu-Gln-Gly-Glu linear Linear Ac-GLQGE 3 Gly-Leu-Gln-Gly-Glu-NH.sub.2 linear Linear GLQGE-NH.sub.2 4 Gly-Leu-Gln-Gly-Glu cyclic Cyclic GLQGE 5 Pro-Gly-Leu-Gln-Gly-Glu cyclic Cyclic PGLQGE 6 Gly-Leu-Asn-Gly-Glu linear Linear CT1 (GLNGE) 7 Ac-Gly-Leu-Asn-Gly-Glu linear Linear Ac-CT1 (Ac-GLNGE) 8 Gly-Leu-Asn-Gly-Glu-NH.sub.2 linear Linear CT1-NH.sub.2 (GLNGE-NH.sub.2) 9 Gly-Leu-Asn-Gly-Glu cyclic Cyclic CT1 (Cyclic GLNGE) 10 Pro-Gly-Leu-Asn-Gly-Glu cyclic Cyclic CT1+P (Cyclic PGLNGE) 11 Gly-Leu-Hyp-Gly-Glu linear Linear CT2 (GLOGE) 12 Ac-Gly-Leu-Hyp-Gly-Glu linear Linear Ac-CT2 (Ac-GLOGE) 13 Gly-Leu-Hyp-Gly-Glu-NH.sub.2 linear Linear CT2-NH.sub.2 (GLOGE-NH.sub.2) 14 Gly-Leu-Hyp-Gly-Glu cyclic Cyclic CT2 (Cyclic GLOGE) 15 Pro-Gly-Leu-Hyp-Gly-Glu cyclic Cyclic CT2+P (Cyclic PGLOGE) 16

[0128] Linear GLQGE (Gly-Leu-Gln-Gly-Glu)

##STR00025##

[0129] Linear Ac-GLQGE (Ac-Gly-Leu-Gln-Gly-Glu)

##STR00026##

[0130] Linear GLQGE-NH.sub.2 (Gly-Leu-Gln-Gly-Glu-NH.sub.2)

##STR00027##

[0131] Cyclic GLQGE

##STR00028##

[0132] Cyclic PGLQGE

##STR00029##

[0133] Linear CT1 (Gly-Leu-Asn-Gly-Glu)

##STR00030##

[0134] Linear CT1 -NH.sub.2 (Gly-Leu-Asn-Gly-Glu-NH.sub.2)

##STR00031##

[0135] Linear Ac-CT1 (Ac-Gly-Leu-Asn-Gly-Glu)

##STR00032##

[0136] Cyclic CT1 (Gly-Leu-Asn-Gly-Glu)

##STR00033##

[0137] Cyclic CT1+P (Pro-Gly-Leu-Asn-Gly-Glu)

##STR00034##

[0138] Linear CT2 (Gly-Leu-Hyp-Gly-Glu)

##STR00035##

[0139] Linear CT2-NH.sub.2 (Gly-Leu-Hyp-Gly-Glu-NH.sub.2)

##STR00036##

[0140] Linear Ac-CT2 (Ac-Gly-Leu-Hyp-Gly-Glu)

##STR00037##

[0141] Cyclic CT2 (Gly-Leu-Hyp-Gly-Glu)

##STR00038##

[0142] Cyclic CT2+P (Pro-Gly-Leu-Hyp-Gly-Glu)

##STR00039##

Example 2: Effect of Cyclic Peptides with Proline and Amidated Linear Peptides on Chemically Induced Liver Fibrosis in Mice

[0143] Mice were injected for 6 weeks with CCl.sub.4 in order to induce liver fibrosis. Starting on day 32 the mice received daily intraperitoneal injections of the peptides at a final dose of 25 mg/kg/mouse/day diluted in NaCl 0.9% for a total of 10 days. In these experiments, the following peptides were tested: cyclic peptide Pro-Gly-Leu-Gln-Gly-Glu, cyclic peptide Pro-Gly-Leu-Asn-Gly-Glu, cyclic peptide Pro-Gly-Leu-Hyp-Gly-Glu, linear peptide Gly-Leu-Gln-Gly-Glu-NH.sub.2, linear peptide Gly-Leu-Asn-Gly-Glu-NH.sub.2, linear peptide Gly-Leu-Hyp-Gly-Glu-NH.sub.2.

[0144] Results showed that the treatment with CCl.sub.4 significantly induced collagen production in the liver (marker of matrix accumulation), and the cyclic peptide Pro-Gly-Leu-Gln-Gly-Glu was able to significantly reduce collagen accumulation. Also the linear peptide GLQGE-NH.sub.2 was able to significantly reduce CCl.sub.4-induced collagen accumulation. In contrast neither the cyclic forms of the control peptides (with Pro: Pro-Gly-Leu-Asn-Gly-Glu or Pro-Gly-Leu-Hyp-Gly-Glu) nor the linear forms of the control peptides Gly-Leu-Asn-Gly-Glu-NH.sub.2 and Gly-Leu-Hyp-Gly-Glu-NH.sub.2 were able to reduce collagen amount in the liver. Thus, the peptides of sequence Gly-Leu-Gln-Gly-Glu in both linear and cyclic form are able to inhibit excess accumulation of extracellular matrix, and could be useful to prevent fibrosis progression (FIG. 1).

Example 3: Effect of Cyclic Peptides (Without Proline) and Acetylated Linear Peptides on Chemically Induced Liver Fibrosis in Mice

[0145] Mice were injected for 6 weeks with CCl.sub.4 in order to induce liver fibrosis. Starting on day 32 the mice received daily intraperitoneal injections of the peptides at a final dose of 25 mg/kg/mouse/day diluted in NaCl 0.9% for a total of 10 days. In these experiments, the following peptides were tested: cyclic peptide Gly-Leu-Gln-Gly-Glu, cyclic peptide Gly-Leu-Asn-Gly-Glu, cyclic peptide Gly-Leu-Hyp-Gly-Glu, linear peptide Ac-Gly-Leu-Gln-Gly-Glu, linear peptide Ac-Gly-Leu-Asn-Gly-Glu, linear peptide Ac-Gly-Leu-Hyp-Gly-Glu.

[0146] Results showed that the treatment with CCl.sub.4 significantly induced collagen production in the liver (marker of matrix accumulation), and the cyclic peptide Gly-Leu-Gln-Gly-Glu was able to significantly reduce collagen accumulation. Also the linear peptide Ac-Gly-Leu-Gln-Gly-Glu was able to significantly reduce CCl.sub.4-induced collagen accumulation. In contrast neither the cyclic forms nor the linear forms of the peptides Ac-Gly-Leu-Asn-Gly-Glu and Ac-Gly-Leu-Hyp-Gly-Glu were able to reduce collagen amount in the liver. Thus, the peptides of sequence Gly-Leu-Gln-Gly-Glu in both linear and cyclic form are able to inhibit excess accumulation of extracellular matrix, and could be useful to prevent fibrosis progression.

[0147] Moreover, the cyclic peptide Gly-Leu-Gln-Gly-Glu was more efficient in reducing collagen accumulation also in comparison to the cyclic peptide with proline Pro-Gly-Leu-Gln-Gly-Glu (compare the values on the Y-axis of FIGS. 1 and 2, the difference is statistically significant, p<0.001).

Example 4: Effect of Hydrochloride Salt of the Cyclic Peptide Gly-Leu-Gln-Gly-Glu (Cyclic GLQGE) on Cancer in Mice

[0148] The cyclic peptide Gly-Leu-Gln-Gly-Glu (cyclic GLQGE) in form of hydrochloride salt was tested for its ability in prevention of cancer growth in a melanoma model in mice. B16 melanoma cancer cells (10.sup.6 cells) were injected subcutaneously in mice. On day 7, injection with 1 mg of the cyclic GLQGE peptide or 0.9% NaCl (control mice) subcutaneously was performed. On day 12 mice were euthanized and tumours removed and weighed. N=11/11 mice. Results are shown on FIG. 3 as mean±SEM. Analysis of tumor weight in control and treated mice showed that the cyclic peptide Gly-Leu-Gln-Gly-Glu (HCl salt) was significantly able to diminish cancer size of approximately 72% as compared to control mice. Thus, the cyclic peptide Gly-Leu-Gln-Gly-Glu represents a promising therapeutic drug for use in the treatment of cancer.

Example 5: Effect of the Cyclic Peptide Gly-Leu-Gln-Gly-Glu (Cyclic GLQGE) in Form of Acetate Salt on Chemically Induced Lung Fibrosis in Mice

[0149] The cyclic peptide Gly-Leu-Gln-Gly-Glu (cyclic GLQGE acetate salt) was tested for its ability in prevention of lung fibrosis in mice. Lung fibrosis was induced in 6-week-old male C57bl/6 mice using intratracheal bleomycin instillation at a dose of 0.005 units in 50 μl 0.9% NaCl on day 0. Starting on day 11, cyclic Gly-Leu-Gln-Gly-Glu (cyclic GLQGE) or the control cyclic Ac-Gly-Leu-Asn-Gly-Glu (Cyclic GLNGE) or the control cyclic Gly-Hyp-Asn-Gly-Glu (Cylic GLQGE) (all three in form of acetate salt) was injected subcutaneously at a dose of 1 mg/mouse/day for 10 days. Results showed that administration of cyclic Gly-Leu-Gln-Gly-Glu (acetate salt) significantly diminished the total amount of collagen in the lung compared to mice that received bleomycin alone (p<0.05) or with the control peptides cyclic Gly-Leu-Asn-Gly-Glu (Cylic GLNGE acetate salt) or cyclic Gly-Hyp-Asn-Gly-Glu (Cyclic GLQGE acetate salt) (FIG. 4).

Example 6: Effect of the Cyclic Peptide Gly-Leu-Gln-Gly-Glu (HCl Salt) on Breast Cancer Model in Mice

[0150] The hydrochloride salt of cyclic peptide Gly-Leu-Gln-Gly-Glu (cyclic GLQGE) was tested for its ability in prevention of breast cancer in mice. A bone lesion of metastatic breast cancer was induced by injecting cells from the breast cancer cell line MDA-MB-231 intratibially in CD1 nude mice. Starting on day 30, injection with 0.1 mg of the cyclic peptide Gly-Leu-Gln-Gly-Glu (Cyclic GLQGE HCl salt) or 0.9% NaCl subcutaneously was performed daily for 10 days. On day 40, the size of the tumor was evaluated by bioluminescence imaging (Bioluminescence imaging was performed by detecting photon signal 5 minutes after D-luciferin injection (150 mg/kg) using an “IVIS Lumina II” imaging system. The resulting images were analyzed using the software “Living Image”). The number of animals in the control and treated group was N=14/7 mice in two experiments. Results showed that administration of cyclic Gly-Leu-Gln-Gly-Glu (HCl salt) significantly (p<0.05) diminished the size of the tumor compared to mice that received 0.9% NaCl (FIG. 5).