GROWTH HORMONE ANTAGONIST AND ANTI-CANCER COMPOSITION COMBINATION THERAPY

Abstract

A composition for treating a disease or condition responsive to human growth hormone receptor antagonists, comprising a modified human growth hormone receptor antagonist; and an anti-cancer composition. A method for treating cancer using human growth hormone antagonists, comprising pre-screening a patient by analyzing a tumor biopsy to confirm the presence of cancer and the presence of certain predetermined factors indicative of responsiveness to human growth hormone antagonists; and treating the patient with an effective amount of a composition that includes a modified human growth hormone receptor antagonist and an anti-cancer composition.

Claims

1. A composition for treating a disease or condition responsive to human growth hormone receptor antagonists, comprising: (a) a modified human growth hormone receptor antagonist; and (b) an anti-cancer composition.

2. The composition of claim 1, wherein the disease or condition responsive to human growth hormone receptor antagonists is a cancer that expresses predetermined levels of growth hormone receptor (GHR); predetermined levels of prolactin receptor (PRLR); predetermined levels of both GHR and PRLR); predetermined levels of ATP-binding cassette (ABC)-transporters; or predetermined levels of epithelial to mesenchymal transition (EMT) mediators.

3. The composition of claim 1, wherein the disease or condition responsive to human growth hormone receptor antagonists is cancer, and wherein the cancer is breast cancer, central nervous system cancer, melanoma, non-small cell lung cancer, ovarian cancer, prostate cancer, renal cancer, pancreatic cancer, endometrial cancer, meningioma, colorectal cancer, colon cancer, neuroblastoma, stomach cancer, liver cancer, lymphoma, combinations thereof, or any other cancer expressing predetermined amounts of GHR, PRLR, ABC transporters, EMT mediators, or combinations thereof.

4. The composition of claim 1, wherein the modified human growth hormone receptor antagonist comprises: (a) human growth hormone receptor antagonist G120K wherein two amino acids of human growth hormone receptor antagonist G120K have been changed to cysteine, wherein the two amino acids changed to cysteine are T142 and H151; and (b) a polyethylene glycol molecule conjugated to each substituted cysteine in the human growth hormone receptor antagonist G120K-H151C-T142C, wherein the polyethylene glycol molecules conjugated to the two amino acids changed to cysteine are two 4.5 kDa branched polyethylene glycols each containing three carboxylate anions.

5. The composition of claim 4, wherein the human growth hormone receptor antagonist G120K has a DNA sequence of SEQ ID NO: 1, and an amino acid sequence of SEQ ID NO: 2.

6. The composition of claim 4, wherein the human growth hormone receptor antagonist G120K-H151C-T142C has a DNA sequence of SEQ ID NO: 3, and an amino acid sequence of SEQ ID NO: 4.

7. The composition of claim 4, wherein the following amino acid substitutions have been made: H18D, H21N, R167N, K168A, D171S, K172R, E174S, and I179T, and wherein these mutations are operative to prevent binding to a prolactin receptor.

8. The composition of claim 7, wherein the composition is adapted for the treatment of acromegaly.

9. The composition of claim 4, wherein the polyethylene glycol molecule is prepared by stepwise organic chemistry and is a substantially pure single compound, and wherein the polyethylene glycol molecule is a branched structure.

10. The composition of claim 4, wherein the polyethylene glycol molecule contains a maleimide group for conjugation to a free sulfhydryl group.

11. The composition of claim 1, wherein the anti-cancer composition is an alkylating agent; an antimetabolite; a plant alkaloid; an antitumor antibiotic; or combinations thereof.

12. The composition of claim 11, wherein the alkylating agent is chlorambucil, cyclophosphamide, thiotepa, busulfan, cisplatin, or combinations thereof.

13. The composition of claim 11, wherein the antimetabolite is gemcitabine, 5-fluorouracil, 6-mercaptopurine, cytarabine, or combinations thereof.

14. The composition of claim 11, wherein the plant alkaloid is vincristine, paclitaxel, etoposide, irinotecan, or combinations thereof.

15. The composition of claim 11, wherein the antitumor antibiotic is doxorubicin, dactinomycin, mitoxantrone, idarubicin, or combinations thereof.

16. The composition of claim 1, wherein the anti-cancer composition is a targeted therapy.

17. The composition of claim 16, wherein the targeted therapy includes vemurafenib.

18. A method for treating diseases or conditions responsive to human growth hormone receptor antagonists, comprising administering to a patient an effective amount of the composition of claim 1.

19. A composition for treating a disease or condition responsive to human growth hormone receptor antagonists, comprising: (a) a modified human growth hormone receptor antagonist, wherein the human growth hormone receptor antagonist comprises: (i) human growth hormone receptor antagonist G120K wherein two amino acids of human growth hormone receptor antagonist G120K have been changed to cysteine, wherein the two amino acids changed to cysteine are T142 and H151; and (ii) a polyethylene glycol molecule conjugated to each substituted cysteine in the human growth hormone receptor antagonist G120K-H151C-T142C, wherein the polyethylene glycol molecules conjugated to the two amino acids changed to cysteine are two 4.5 kDa branched polyethylene glycols each containing three carboxylate anions, wherein the polyethylene glycol molecule is prepared by step-wise organic chemistry and is a substantially pure single compound, and wherein the polyethylene glycol molecule is a branched structure; and (b) an anti-cancer composition.

20. The composition of claim 19, wherein the disease or condition responsive to human growth hormone receptor antagonists is a cancer that expresses predetermined levels of growth hormone receptor (GHR); predetermined levels of prolactin receptor (PRLR); predetermined levels of both GHR and PRLR); predetermined levels of ATP-binding cassette (ABC)-transporters; or predetermined levels of epithelial to mesenchymal transition (EMT) mediators.

21. The composition of claim 19, wherein the disease or condition responsive to human growth hormone receptor antagonists is cancer, and wherein the cancer is breast cancer, central nervous system cancer, melanoma, non-small cell lung cancer, ovarian cancer, prostate cancer, renal cancer, pancreatic cancer, endometrial cancer, meningioma, colorectal cancer, colon cancer, neuroblastoma, stomach cancer, liver cancer, lymphoma, combinations thereof, or any other cancer expressing predetermined amounts of GHR, PRLR, ABC transporters, EMT mediators, or combinations thereof.

22. The composition of claim 19, wherein the human growth hormone receptor antagonist G120K has a DNA sequence of SEQ ID NO: 1, and an amino acid sequence of SEQ ID NO: 2.

23. The composition of claim 19, wherein the human growth hormone receptor antagonist G120K-H151C-T142-C has a DNA sequence of SEQ ID NO: 3, and an amino acid sequence of SEQ ID NO: 4.

24. The composition of claim 19, wherein the following amino acid substitutions have been made: H18D, H21N, R167N, K168A, D171S, K172R, E174S, and I179T, and wherein these mutations are operative to prevent binding to a prolactin receptor.

25. The composition of claim 24, wherein the composition is adapted for the treatment of acromegaly.

26. The composition of claim 19, wherein the polyethylene glycol molecule contains a maleimide group for conjugation to a free sulfhydryl group.

27. The composition of claim 19, wherein the anti-cancer composition is an alkylating agent; an antimetabolite; a plant alkaloid; an antitumor antibiotic; or combinations thereof.

28. The composition of claim 27, wherein the alkylating agent is chlorambucil, cyclophosphamide, thiotepa, busulfan, cisplatin, or combinations thereof.

29. The composition of claim 27, wherein the antimetabolite is gemcitabine, 5-fluorouracil, 6-mercaptopurine, cytarabine, or combinations thereof.

30. The composition of claim 27, wherein the plant alkaloid is vincristine, paclitaxel, etoposide, irinotecan, or combinations thereof.

31. The composition of claim 27, wherein the antitumor antibiotic is doxorubicin, dactinomycin, mitoxantrone, idarubicin, or combinations thereof.

32. The composition of claim 19, wherein the anti-cancer composition is a targeted therapy.

33. The composition of claim 32, wherein the targeted therapy includes vemurafenib.

34. A method for treating diseases or conditions responsive to human growth hormone receptor antagonists, comprising administering to a patient an effective amount of the composition of claim 19.

35. A method for treating cancer using human growth hormone antagonists, comprising: (a) pre-screening a patient by analyzing a tumor biopsy to confirm the presence of cancer and the presence of certain predetermined factors indicative of responsiveness to human growth hormone antagonists; and (b) treating the patient with an effective amount of a composition that includes a modified human growth hormone receptor antagonist and an anti-cancer composition.

36. The method of claim 35, wherein the certain predetermined factors include predetermined levels of GHR, PRLR, ABC transporters, EMT mediators, insulin-like growth factor-1 (IGF-1); IFG binding protein-3 (IGFBP3), suppressor of cytokine signaling (SOCS_-1, -2, -3; and cytokine inducible SH2 containing protein (CISH).

37. The method of claim 35, wherein the cancer is breast cancer, central nervous system cancer, melanoma, non-small cell lung cancer, ovarian cancer, prostate cancer, renal cancer, pancreatic cancer, endometrial cancer, meningioma, colorectal cancer, colon cancer, neuroblastoma, stomach cancer, liver cancer, lymphoma, combinations thereof, or any other cancer expressing predetermined amounts of the predetermined factors.

38. The method of claim 35, wherein the modified human growth hormone receptor antagonist comprises: (a) human growth hormone receptor antagonist G120K wherein two amino acids of human growth hormone receptor antagonist G120K have been changed to cysteine, wherein the two amino acids changed to cysteine are T142 and H151; and (b) a polyethylene glycol molecule conjugated to each substituted cysteine in the human growth hormone receptor antagonist G120K-H151C-T142C, wherein the polyethylene glycol molecules conjugated to the two amino acids changed to cysteine are two 4.5 kDa branched polyethylene glycols each containing three carboxylate anions.

39. The method of claim 38, wherein the human growth hormone receptor antagonist G120K has a DNA sequence of SEQ ID NO: 1, and an amino acid sequence of SEQ ID NO: 2.

40. The method of claim 38, wherein the human growth hormone receptor antagonist G120K-H151C-T142C has a DNA sequence of SEQ ID NO: 3, and an amino acid sequence of SEQ ID NO: 4.

41. The method of claim 38, wherein the polyethylene glycol molecule is prepared by step-wise organic chemistry and is a substantially pure single compound, and wherein the polyethylene glycol molecule is a branched structure.

42. The method of claim 38, wherein the polyethylene glycol molecule contains a maleimide group for conjugation to a free sulfhydryl group.

43. The method of claim 35, wherein the anti-cancer composition is an alkylating agent; an antimetabolite; a plant alkaloid; an antitumor antibiotic; or combinations thereof.

44. The method of claim 43, wherein the alkylating agent is chlorambucil, cyclophosphamide, thiotepa, busulfan, cisplatin, or combinations thereof.

45. The method of claim 43, wherein the antimetabolite is gemcitabine, 5-fluorouracil, 6-mercaptopurine, cytarabine, or combinations thereof.

46. The method of claim 43, wherein the plant alkaloid is vincristine, paclitaxel, etoposide, irinotecan, or combinations thereof.

47. The method of claim 43, wherein the antitumor antibiotic is doxorubicin, dactinomycin, mitoxantrone, idarubicin, or combinations thereof.

48. The composition of claim 35, wherein the anti-cancer composition is a targeted therapy.

49. The composition of claim 48, wherein the targeted therapy includes vemurafenib.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0024] The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.

[0025] The accompanying figures, which are incorporated into and form a part of the specification, schematically illustrate one or more example implementations of the disclosed inventive subject matter and, together with the general description given above and detailed description given below, serve to explain the principles of the disclosed subject matter, and wherein:

[0026] FIG. 1 is a bar chart depicting the level of GHR mRNA expression in multiple cell lines from thirty-seven (37) cancer cell types, which are listed along the x-axis of the chart;

[0027] FIGS. 2A-2C are plots for two breast cancer subtypes (triple-negative and HER2-enriched breast cancers) showing the differences in patient overall survival (in months) for cancers with low and high growth hormone receptor (GHR) expression levels, wherein FIG. 2A includes data for subtype HER2 enriched (ER-PR-HER+) breast cancer (n=96), wherein FIG. 2B includes data for subtype triple-negative breast cancer (n=405), and wherein FIG. 2C includes data for subtype triple-negative breast cancer/chemotherapy-treated (n=227);

[0028] FIGS. 3A-3D are graphs showing the relationship between overall survival for breast cancer (BC) patients (ungrouped/all) with specific gene expression in the Cancer Genome Atlas (TCGA) breast cancer database, wherein data for patients with high GHR expression (FIG. 3A) or high PRLR expression (FIG. 3B) had a poorer survival compared to low GHR or low PRLR expression groups, and wherein in breast cancer patients with high expression of both GHR and PRLR (FIG. 3C), or with high expression of GH, GHR, and PRLR (FIG. 3D), survival was significantly poorer than the corresponding low expression cohorts of the same gene-sets (processed using GEPIA2);

[0029] FIG. 4A is a heatmap showing a Spearman correlation coefficient between GHR expression and ABC type multidrug transporter expression across forty (40) different cancer types from human patients in The Cancer Genome Atlas (TCGA) database, wherein the heatmap indicates a consistent positive (red) correlation between GHR expression and ABC type multidrug transporters.

[0030] FIG. 4B is a heatmap showing a Spearman correlation coefficient between GHR expression and known EMT markers expression across forty (40) different cancer types from human patients in the TCGA database, wherein the heatmap indicates a consistent positive (red) correlation between GHR and markers of EMT across majority of cancer types.

[0031] FIGS. 5A(1)-5A(6) show the effect of GHR antagonists on the expression of six ABC transporters in a melanoma cell line, wherein FIG. 5A(1) includes data for MALME-3M-ABCB1, FIG. 5A(2) includes data for MALME-3M-ABCB8, FIG. 5A(3) includes data for MALME-3M-ABCC-1, FIG. 5A(4) includes data for MALME-3M-ABCC2, FIG. 5A(5) includes data for MALME-3M-ABCG1, and FIG. 5A(6) includes data for MALME-3M-ABCG2;

[0032] FIGS. 5B(1)-5B(9) show the effect of GHR antagonists on the expression of nine EMT markers in a melanoma cell line, wherein FIG. 5B(1) includes data for MALME-3M-FGFBP1, FIG. 5B(2) includes data for MALME-3M-CDH1, FIG. 5B(3) includes data for MALME-3M-CLDN1, FIG. 5B(4) includes data for MALME-3M-ZEB1, FIG. 5B(5) includes data for MALME-3M-CDH2, FIG. 5B(6) includes data for MALME-3M-EPAS1, FIG. 5B(7) includes data for MALME-3M-SNAI1, FIG. 5B(8) includes data for MALME-3M-SNAI2, and FIG. 5B(9) includes data for MALME-3M-VIM;

[0033] FIGS. 6A-6D show the effect of GHR antagonists (Peg=pegvisomant, G=Compound G) on a basement membrane invasion assay (in vitro) (FIG. 6A) using three pancreatic cancer cell lines (n=3, * indicates p<0.05), wherein GH increases the tumor cell invasion rate while the GHR antagonists significantly suppress it, and wherein FIG. 6B includes data for invasion assay—BxPC3, FIG. 6C includes data for invasion assay—PANC1, and FIG. 6B includes data for invasion assay—LTPA;

[0034] FIGS. 7A, 7B, and 7C(1)-7C(8) show the effect of GHR antagonists [Peg=pegvisomant, G=Compound G] on drug efflux rate (FIG. 7A), percentage of drug retention (FIG. 7B), and fluorescent images after a fluorescent drug surrogate (DiOC2) has been loaded into pancreatic cancer cells using an in vitro multidrug efflux assay method (n=3, * indicates p<0.05) (FIGS. 7C(1)-7C(8)), wherein GH increases the tumor cell drug efflux rate (decreases drug retention in tumor cells) while the GHR antagonists significantly suppress it;

[0035] FIG. 8 shows the viability of pancreatic cancer cells when incubated in the presence anti-cancer drugs and either PBS, GH, GH+pegvisomant, or GH+Compound G, wherein the anti-cancer drugs used were doxorubicin (doxo), erlotinib (erlo), or gemcitabine (gemc), and the control was DMSO;

[0036] FIG. 9A is a graph showing the effect of different treatments on the growth (tumor volume) of human pancreatic cancer (PANC1) xenografts implanted in male nude mice, wherein the hGHR antagonists were pegvisomant (peg) and compound-G (G) treated at 10 mg/kg/day while gem20=gemcitabine was treated at 20 mg/kg every 3 days, wherein all treatments started at day-17 and were by intra-peritoneal injections, and wherein gemcitabine, pegvisomant, or compound-G by themselves were effective in decreasing tumor growth rate when used as monotherapy; however, combinations of gem20+peg or gem20+G were significantly more efficacious than gem20 or peg or G alone;

[0037] FIG. 9B is a graph showing the effect of different treatments on the growth (tumor volume) of human pancreatic cancer (PANC1) xenografts implanted in male nude mice, wherein the hGHR antagonist was compound-G (G) treated at 10 mg/kg/day while gem20=gemcitabine was treated at 20 mg/kg every 3 days, or gem80=gemcitabine was treated at 80 mg/kg every 3 days, wherein all treatments started at day 17 and were by intra-peritoneal injections, wherein both doses of gemcitabine or compound-G were effective in decreasing tumor growth rate when used as monotherapy; however, combinations of gem20+G or gem80+G were significantly more efficacious than gem20 or gem80 or G alone, and wherein Gem20+G was more efficacious than Gem20, while Gem80+G was more efficacious than Gem80 alone and Gem20+G was better than Gem80 alone; and

[0038] FIG. 10 is a graph showing the effect of different treatments on the growth (tumor volume) of human pancreatic cancer (PANC1) xenografts implanted in female nude mice, wherein the hGHR antagonist is compound-G (G) treated at 10 mg/kg/day while gem20=gemcitabine treated at 20 mg/kg every 3 days or gem80=gemcitabine treated at 80 mg/kg every 3 days, wherein all treatments started at day-17 and were by intra-peritoneal injections, and wherein both doses of gemcitabine or compound-G were effective in decreasing tumor growth rate when used as monotherapy; however, combinations of gem20+G or gem80+G were significantly more efficacious than gem20 or gem80 or G alone, wherein Gem20+G was more efficacious than Gem20 while Gem80+G was more efficacious than Gem80 alone and Gem20+G was as efficacious as Gem80 alone.

DETAILED DESCRIPTION

[0039] Example implementations are now described with reference to the Figures. Reference numerals are used throughout the detailed description to refer to the various elements and structures. Although the following detailed description contains many specifics for the purposes of illustration, a person of ordinary skill in the art will appreciate that many variations and alterations to the following details are within the scope of the disclosed inventive subject matter. Accordingly, the following implementations are set forth without any loss of generality to, and without imposing limitations upon, the claimed subject matter.

[0040] The following abbreviations, which are used throughout this application, have the following specific meanings. dPEG®-A refers to MAL-dPEG®.sub.12-Tris(-dPEG®.sub.24-acid).sub.3 (Quanta BioDesign #1145). Compound G or G refers to hGHR antagonist hGH-G120K having T142 and H151 changed to cysteine and having both added cysteines conjugated with dPEG®-A. Compound D or D refers to hGHR antagonist hGH-G120K having T142 changed to cysteine and having the added cysteine conjugated with a 40 kDa branched polyethylene glycol. Peg refers to pegvisomant and Dox refers to doxorubicin. The disclosed technology includes compositions and methods for treating cancer patients who are identified by expression of GHR, PRLR, selected ABC drug efflux pumps, selected EMT modulators, IGF-1, IGFBP3, SOCS-1, or CISH, wherein treatment comprises administering to the patient an effective dose of a chemotherapeutic drug combined with an effective dose of Compound G.

GHR Expression in Cancer Cell Lines

[0041] The effectiveness of an hGHR antagonist for cancer treatment, either by itself or in combination with a cancer drug, is related closely to the expression of the hGHR by a particular cancer. When the hGHR antagonist is also an antagonist of the PRLR, then the level of PRLR expression will also determine the susceptibility of a cancer to this treatment. It was previously observed that most of 60 cell lines from nine cancer types expressed high levels of either the hGHR, the PRLR, or both receptors [5]. Analysis of the levels of GHR mRNA expression from 37 cancer types (see FIG. 1), with between 4 and 131 cell lines included for each cancer, indicated that almost all of the cancer types had high hGHR expression for at least a subset of the individual cancers tested.

[0042] The level of hGHR expression across multiple human patient datasets correlates with decreased patient survival for HER2 enriched breast cancer and triple-negative breast cancer (see FIGS. 2A-2C). FIGS. 3A-3D illustrate that breast cancer patients with increased levels of both GHR+PRLR expression or GH+GHR+PRLR expression have lower percent survivals than high levels of GH or GHR or PRLR expression alone.

GHR Expression and the Expression of ABC Transporters and FMT Markers

[0043] FIG. 4A shows a correlation across 40 different cancer cell types between GHR expression and the expression of ABC transporters across all patients in the TCGA database. FIG. 4B shows the same correlation between GHR expression and the expression of EMT mediators. Because upregulation of ABC transporters and EMT mediators lead to chemotherapy resistance, these Figures shows that chemotherapy resistance is associated with GHR expression.

[0044] The effect of GHR antagonists on the expression of six ABC transporters in a melanoma cell line is shown in FIGS. 5A(1)-5A(6). When used as a monotherapy, Compound G significantly reduces the expression of four of the six ABC transporters. When GH or doxorubicin is present, Compound G also reduces the expression of four ABC transporters. When both GH and doxorubicin are added, Compound G greatly reduces the expression of all six ABC transporters. This observation is due to the fact that the expression of ABC transporters is enhanced in presence of GH or doxorubicin or both GH and doxorubicin.

[0045] The effect of hGHR antagonists on the expression of nine EMT markers in a melanoma cell line is shown in FIGS. 5B(1)-5B(9). When used as a monotherapy, Compound G significantly reduces the expression of multiple EMT mediators. When GH or doxorubicin or both are present, Compound G also reduces the expression of multiple EMT mediators, presumably because the expression of EMT mediators is significantly enhanced in presence of GH or doxorubicin or both GH and doxorubicin.

[0046] FIGS. 6A-6D show the effect of the hGHR antagonists Compound G and pegvisomant on a basement membrane invasion assay using three pancreatic cancer cell lines. This assay quantifies the ability of cells to migrate through a membrane, a property of cells that have transitioned from epithelial cells to mesenchymal cells. For all three cell lines, the addition of GH increases cell migration. This effect of GH is blocked by the addition of either pegvisomant or Compound G.

The Effect of hGHR Antagonists on Drug Efflux

[0047] The effect of hGHR antagonists on the drug efflux rate, using DiOC2 dye as a drug surrogate, from pancreatic cancer cells is shown in FIGS. 7A-7C. With reference to FIG. 7A, after 120 minutes, the efflux rate with GH addition is approximately four-fold greater than the efflux rate with no additives (PBS). In the presence of GH+Pegvisomant or GH+Compound G, the efflux rate is markedly suppressed compared with GH alone by a factor of approximately two. FIG. 7B, illustrates the effect of hGHR antagonists on the percentage of drug retention from pancreatic cancer cells. In the PBS control, approximately 85% of the drug is retained after 120 minutes. The retention after 120 minutes decreases to ˜54% in the presence of GH but increases to ˜83% in the presence of GH plus Compound G or GH plus Pegvisomant. Finally, FIGS. 7C(1)-7C(8) provides fluorescent images of a labeled drug after being loaded into pancreatic cancer cells and incubated for 0 minutes and 120 minutes in PBS (FIG. 7C(1)-FIG. 7C(2)), GH (FIG. 7C(3)-FIG. 7C(4)), GH+Pegvisomant (FIG. 7C(5)-FIG. 7C(6)), or GH+Compound G (FIG. 7C(7)-FIG. 7C(8)). In FIGS. 7C(1)-7C(8), the fluorescence of the cells at 0 minutes is different for the four different conditions, so the drug remaining after 120 minutes (fluorescence) was compared with its own 0 min control. It is clear from the images that the cells treated with GH+Compound G retain the greatest amount of drug.

[0048] FIG. 8 illustrates the viability of pancreatic cancer cells when incubated in the presence of anti-cancer drugs and either buffer (PBS), GH, GH+Pegvisomant, or GH+Compound G. The anti-cancer drugs are doxorubicin (doxo), erlotnib (erlo), or gemcitabine (gemc) and the control is DMSO, the vehicle for the anti-cancer drugs. In all cases, the addition of GH increases the cell viability. However, the addition of either Pegvisomant or Compound G to the GH reduces the viability, in all cases, to below that of PBS.

The Effect of GHR Antagonists+Anti-Cancer Drugs on Pancreatic Tumor Xenografts

[0049] Specific volumes of pancreatic tumor xenografts implanted in male nude mice after treatment with hGHR antagonists (10 mg/kg/day), gemcitabine (20 mg/kg/3-days) and combinations of the antagonists+gemcitabine are shown in FIG. 9A. Treatments started at day-17. Both of the hGHR antagonists (pegvisomant and Compound G) significantly decrease the tumor volume relative to that of the PBS control by day-30 (13 days after start of treatment). However, for both of these antagonists, the tumor volume continues to increase. Gemcitabine alone shows a greater reduction of tumor volume compared to the hGHR antagonists, but the absolute tumor volume appears to resume trending upwards by day-43. The combinations of gemcitabine+pegvisomant and gemcitabine+Compound G show the greatest reduction of tumor volume. After day-43 (26 days of treatment) there is no indication that the tumor volume has begun to increase using the combination treatments. The combination of Compound G+gemcitabine gave the greatest tumor volume reduction.

[0050] FIG. 9B shows the volume of pancreatic tumor xenografts implanted in male nude mice after treatment with 80 mg/kg/3-days gemcitabine and Compound G (10 mg/kg/day)+80 mg/kg/3-days gemcitabine. The plots for PBS, Compound G (10 mg/kg/day) alone, gemcitabine (20 mg/kg/3-days) alone, or Compound G (10 mg/kg/day)+gemcitabine (20 mg/kg/3-days), which are taken from FIG. 9A, are also included in FIG. 9B. Gemcitabine by itself at 80 mg/kg/3-days does not decrease the tumor volume significantly beyond that obtained with 20 mg/kg/3-day dose of the same drug and, for the 80 mg/kg/3-day regimen, the tumor volume appears to have plateaued by day-43 (26 days of treatment). The 80 mg/kg/3-days Gemcitabine+10 mg/kg/day Compound G caused the maximum tumor volume reduction of all conditions, and the tumor volume appears to be still decreasing at the final day of the study (day-43).

[0051] FIG. 10 shows the volume of pancreatic tumor xenografts implanted in female nude mice after treatment with either Compound-G (10 mg/kg/day) alone, or gemcitabine (20 or 80 mg/kg/3-days) alone, or Compound G (10 mg/kg/day)+20 mg/kg/3-days gemcitabine, or Compound G (10 mg/kg/day)+80 mg/kg/3-days gemcitabine. Treatment started at day-17. After 25 days of treatment (day-42 of study), the tumor reduction due to 20 mg/kg/3-days gemcitabine alone or Compound G (10 mg/kg/day) alone are almost equivalent but only the gemcitabine treated mice show tumor volumes that are trending higher, indicating onset of chemoresistance. The combination of Compound G (10 mg/kg/day)+20 mg/kg/3-days gemcitabine reduces the tumor volume growth drastically and the tumor volume does not appear to trend upwards through the end of the study (day-44). Gemcitabine at 80 mg/kg/3-days alone shows the same tumor reduction at day-44 as Compound G (10 mg/kg/day)+20 mg/kg/3-days gemcitabine. Compound G (10 mg/kg/day)+80 mg/kg/3-days gemcitabine shows the greatest inhibition of tumor growth, which appears to be still decreasing after 44 days (end of study), at which point 3 of 8 animals in the group were tumor-free.

Personalized Medicine/Precision Medicine Preliminary Diagnostic Test

[0052] One implementation of the disclosed technology includes a preliminary molecular analysis of a tumor biopsy sample to determine if a patient is a suitable candidate for treatment with the disclosed combination therapy. This analysis involves analyzing expression levels of a predetermined set genes where specific changes in the expression levels of these genes correlates with the biological activities affected by the disclosed combination therapy. More specifically, identification of elevated levels of expression of selected genes is used to identify patients that are proper candidates for treatment with the GHR antagonist plus cancer therapeutic agent.

[0053] Genes whose expression levels are key indicators of effective responsiveness to the disclosed GHR antagonist plus cancer therapy treatment include GHR, PRLR or both GHR and PRLR. Expression levels in a tumor biopsy are measured and quantified by performing a diagnostic test that measures levels of mRNA encoding these proteins that is expressed by the tumor cells. For example, the tumor biopsy sample could be processed to isolate mRNA which is then reverse transcribed into cDNA. The amount of cDNA derived from genes that encode these two receptors could then be measured using a variety of standard assays including qPCR analysis or gene chip analysis. Patients whose tumors express elevated levels of GHR, PRLR or both GHR and PRLR are potential candidates for receiving treatment with the disclosed combination therapy. Alternatively, the levels of these target proteins could be measured using techniques that directly measure the amount of these proteins present in the tumor. This approach includes the use of assays such as Western blots or ELISA assays.

[0054] Additional genes whose expression levels are key indicators of effective response to the disclosed hGHR antagonist plus cancer therapeutic combination therapy include a key set of ATP-binding cassette (ABC) drug efflux pumps; ABCB1, ABCB5, ABCB8, ABCC1, ABCC2, ABCG1 and ABCG2. As with the target receptors described above, elevated levels of expression of at least some of these proteins identifies patients for which the disclosed combination hGHR antagonist plus cancer therapeutic agent would be effective. The levels of expression of these key genes are determined using the analytical techniques described above on samples derived from patient biopsies.

[0055] In addition to the drug efflux pump proteins discussed herein, expression levels of a selected set of genes involved in promoting the adverse progression of cancer driven by the Epithelial to Mesenchymal Transition (EMT) can be measured. The set of key EMT modulators analyzed in a preliminary diagnostic analysis of a patient biopsy include CDH1, CDH2, SNAI1, SNAI2, TGFB1, TGFB2, TGFB3, TGFBR2, TWIST1, TWIST2, VIM, ZEB1 and ZEB2. Elevated levels of expression of these genes further identify patients that are candidates for effective treatment with the disclosed combination GHR antagonist plus cancer therapeutic agent. The levels of expression of these genes would be determined by the analytical methods described above.

[0056] In addition to the target receptors GHR and PRLR, Insulin Like Growth Factor 1 (IGF-1), Insulin Like Growth Factor Binding Protein 3 (IGFBP3), suppressor of cytokine signaling (SOCS)-1, -2, -3, and cytokine inducible SH2 containing protein (CISH) are important genes whose (RNA or protein) expression levels in the tumor biopsy (all the above) or serum (IGF1 and IGFBP3) can be used to identify patients who will respond effectively to treatment with the disclosed GHR antagonist. As with the target receptor proteins, the levels of these GH inducible downstream signaling factors are determined by gene expression analysis using mRNA gene expression techniques or, preferably, serum protein quantification techniques. IGF-1, IGFBP3, SOCS-1, -2, -3 and CISH are particularly useful for identifying patients that would be effectively treated by continuing administration of the GHR antagonist following completion of a combination therapy using GHR antagonist plus cancer chemotherapeutic agent.

[0057] All literature and similar material cited in this application, including, but not limited to, patents, patent applications, articles, books, treatises, and web pages, regardless of the format of such literature and similar materials, are expressly incorporated by reference in their entirety. Should one or more of the incorporated references and similar materials differs from or contradicts this application, including but not limited to defined terms, term usage, described techniques, or the like, this application controls.

[0058] As previously stated and as used herein, the singular forms “a,” “an,” and “the,” refer to both the singular as well as plural, unless the context clearly indicates otherwise. The term “comprising” as used herein is synonymous with “including,” “containing,” or “characterized by,” and is inclusive or open-ended and does not exclude additional, unrecited elements or method steps. Although many methods and materials similar or equivalent to those described herein can be used, particular suitable methods and materials are described herein. Unless context indicates otherwise, the recitations of numerical ranges by endpoints include all numbers subsumed within that range. Furthermore, references to “one implementation” are not intended to be interpreted as excluding the existence of additional implementations that also incorporate the recited features. Moreover, unless explicitly stated to the contrary, implementations “comprising” or “having” an element or a plurality of elements having a particular property may include additional elements whether or not they have that property.

[0059] The terms “substantially” and “about” used throughout this specification are used to describe and account for small fluctuations, such as due to variations in processing. For example, these terms can refer to less than or equal to ±5%, such as less than or equal to ±2%, such as less than or equal to ±1%, such as less than or equal to ±0.5%, such as less than or equal to ±0.2%, such as less than or equal to ±0.1%, such as less than or equal to ±0.05%, and/or 0%.

[0060] Underlined and/or italicized headings and subheadings are used for convenience only, do not limit the disclosed subject matter, and are not referred to in connection with the interpretation of the description of the disclosed subject matter. All structural and functional equivalents to the elements of the various implementations described throughout this disclosure that are known or later come to be known to those of ordinary skill in the art are expressly incorporated herein by reference and intended to be encompassed by the disclosed subject matter. Moreover, nothing disclosed herein is intended to be dedicated to the public regardless of whether such disclosure is explicitly recited in the above description.

[0061] There may be many alternate ways to implement the disclosed inventive subject matter. Various functions and elements described herein may be partitioned differently from those shown without departing from the scope of the disclosed inventive subject matter. Generic principles defined herein may be applied to other implementations. Different numbers of a given module or unit may be employed, a different type or types of a given module or unit may be employed, a given module or unit may be added, or a given module or unit may be omitted.

[0062] Regarding this disclosure, the term “a plurality of” refers to two or more than two. Unless otherwise clearly defined, orientation or positional relations indicated by terms such as “upper” and “lower” are based on the orientation or positional relations as shown in the figures, only for facilitating description of the present invention and simplifying the description, rather than indicating or implying that the referred devices or elements must be in a particular orientation or constructed or operated in the particular orientation, and therefore they should not be construed as limiting the present invention. The terms “connected”, “mounted”, “fixed”, etc. should be understood in a broad sense. For example, “connected” may be a fixed connection, a detachable connection, or an integral connection; a direct connection, or an indirect connection through an intermediate medium. For an ordinary skilled in the art, the specific meaning of the above terms in the present invention may be understood according to specific circumstances.

[0063] It should be appreciated that all combinations of the foregoing concepts and additional concepts discussed in greater detail herein (provided such concepts are not mutually inconsistent) are contemplated as being part of the disclosed inventive subject matter. In particular, all combinations of claimed subject matter appearing at the end of this disclosure are contemplated as being part of the inventive subject matter disclosed herein. While the disclosed inventive subject matter has been illustrated by the description of example implementations, and while the example implementations have been described in certain detail, there is no intention to restrict or in any way limit the scope of the appended claims to such detail. Additional advantages and modifications will readily appear to those skilled in the art. Therefore, the disclosed inventive subject matter in its broader aspects is not limited to any of the specific details, representative devices and methods, and/or illustrative examples shown and described. Accordingly, departures may be made from such details without departing from the spirit or scope of the general inventive concept.

[0064] The following references form part of the specification of the present application and each reference is incorporated by reference herein, in its entirety, for all purposes. [0065] 1. Pasut, G. and Veronese, M. (2012) State of the Art in Pegylation: The Great Versatility Achieved After Forty Years of Research. J. Controlled Release 161, 461-472. [0066] 2. Parveen, S. and Sahoo, S. K. Nanomedicine: Clinical Applications of Polyethylene Glycol Conjugated to Proteins and Drugs Clin. Pharmacokinet. 45, 965-988. [0067] 3. Alconcel, S. N. S., Baas, A. S. and Maynard, H. D. (2011) FDA-Approved Poly(ethylene glycol)-Protein Conjugate Drugs. Polymer Chemistry 2, 1442-1448. [0068] 4. Kling, J. (2013) Pegylation of Biologics: A Multipurpose Solution. Bioprocess International 11, 35-43. [0069] 5. Perry, J. K., Wu, Z.-S., Mertani, H. C., Zhu, T., and Lobie, P. E. (2017) “Tumour-Derived Human Growth Hormone as a Therapeutic Target in Oncology” Trends in Endocrinology and Metabolism 28: 587-596. [0070] 6. Basu, R., Qian, Y., and Kopchick, J. J. (2018) “Lessons from growth hormone receptor gene-disrupted mice: are there benefits of endocrine defects?” European Journal of Endocrinology 178: R155-R181. [0071] 7. Goffin, V. (2017) “Prolactin Receptor Targeting in Breast and Prostate Cancers: New Insights into an Old Challenge” Pharmacology and Therapeutics 179: 111-126. [0072] 8. Sustarsic, E. G., Junnila, R. K., and Kopchick, J. J. (2013) “Human Metastatic Melanoma Cell Lines Express High Levels of Growth Hormone Receptor and Respond to GH Treatment” Biochem Biophys Res Commun. 441: 144-150. [0073] 9. Bukowski, K., Kciuk, M., and Kontek, R. (2020) “Mechanisms of Multidrug Resistance in Cancer Chemotherapy” Int. J. Mol. Sci. 21, 3233 [0074] 10. Basu, R., and Kopchick, J. J. (2019) “The Effects of Growth Hormone on Therapy Resistance in Cancer” Cancer Drug Resistance 2: 827-846, [0075] 11. Wu, A. M. L., Dalvi, P., Lu, X., Yang, M., Riddick, D. S., et al. (2013) “Induction of multidrug resistance transporter ABCG2 by prolactin in human breast cancer cells” Molecular Pharmacology 83:377-88. [0076] 12. Neradugomma, N. K., Subramaniam, D., Tawfik, O. W., Goffin, V., Kumar, T. R., et al. (2014) “Prolactin signaling enhances colon cancer stemness by modulating Notch signaling in a Jak2-STAT3/ERK manner” Carcinogenesis 35:795-806 [0077] 13. Zatelli, M. C., Minoia, M., Mole, D., Cason, V., Tagliati, F., Margutti, A., Bondanelli, M., Ambrosio, M. R., and Uberti, E.d (2009) “Growth Hormone Excess Promotes Breast Cancer Chemoresistance” Journal of Clinical Endocrinology and Metabolism 94: 3931-3938. [0078] 14. Minoia, M., Gentilin, E., Mole, D., Rossi, M., Filieri, C., Tagliati, F., Baroni, A., Ambrosio, M. R., and Uberti, E.d, Zatelli, M. C. “Growth Hormone Receptor Blockade Inhibits Growth Hormone-Induced Chemoresistance by Restoring Cytotoxic-Induced Apoptosis in Breast Cancer Cells Independently of Estrogen Receptor Expression” Journal of Clinical Endocrinology and Metabolism 97: E907-E916.