Method of treatment of hypoxia inducible factor (HIF)-related conditions

Abstract

The present invention relates to methods of treatment of hypoxia Inducible Factor (HIF)-related conditions, and in particular to methods of treatment of HIF-related conditions comprising the administration of a composition comprising transferrins.

Description

CROSS REFERENCE TO RELATED APPLICATIONS

This application Such neurodegenerative diseases include Alzheimer's disease, Parkinson's disease, Huntington's disease, and Amylotrophic Lateral Sclerosis. Since treatment of SH-SY5Y upregulates HIF, treatment of cells with Apo- or Holo-transferrin should provide a protective effect on cells subjected to substances known to induce neurodegeneration. FIG. 7 highlights data assessing whether majorities of Apo- and Holo-transferrin could protect SH-SY5Y cells from the toxic effects of the known neurodegenerative toxin oligomerized Abeta 1-42 peptide (FIG. 7). SH-SY5Y neuronal cells cultured in growth media were treated with 4 mg/mL Apo-transferrin or Holo-transferrin for 24 hrs under normal oxygen levels. After 24 hrs, cells were treated with oligomerized Abeta1-42 peptide for an additional 72 hours. Following treatment with oligomerized Abeta1-42, cells were subjected to a ApoGlo caspase 3/7 activation assay. Control cells, untreated cells, were set to a normalized value of 1. The average caspase induction, relative to control cells, and standard deviations are shown for each treatment condition. Interestingly, these data show that both majority ApoTf and HoloTf protect SH-SY5Y cells from Abeta induced toxicity. These data also further confirm lack of inherent toxicity with either ApoTf or HoloTf.

Example 8

Synergystic Effect with Small Molecule HIF Activators and ApoTf/HoloTf Mixtures

Transferrin may act synergistically with other HIF activating small molecules, such as other iron chelators or enzyme inhibitors. This could allow lower levels of these small molecules to be administered, eliciting fewer side effects but retaining high therapeutic levels. To determine whether Apotransferrin increases the potency of the iron chelator, DFO, and the phd2 inhibitor IOX2; SH-SY5Y neuronal cells cultured in serum free media were treated with 4 mg/mL of the indicated proteins in the presence or absence of small molecule drug under normal oxygen levels. The results of the experiment are shown in FIGS. 8A, 8B, and 8C. The data shown in FIG. 8A relates to treatment of cells with a combination of DFO or IOX2, at the indicated concentrations, plus 4 mg/mL protein. CoCl₂ was used as an experimental positive control. The data shown in FIG. 8B relates to treatment of cells with a combination of 10 uM M30 plus/minus 4 mg/mL protein. The data shown in FIG. 8C relates to treatment of cells with a combination of 200 uM DFO plus/minus 4 mg/mL protein. After 6 hrs intracellular proteins were harvested and tested for HIF1 alpha protein levels by ELISA. Data are shown in pg/mL with standard deviation.

Example 9

mRNA Expression Levels of Glut1 and VEGF in Response to Majority Apotransferrin and DFO or IOX2 Combinations

In addition, mRNA expression levels of Glut1 and VEGF in response to majority Apotransferrin and DFO or IOX2 combinations were determined. SH-SY5Y neuronal cells cultured in serum free media were treated with 4 mg/mL human serum albumin or majority Apotransferrin under normal oxygen levels. Where indicated, either 200 uM DFO or 1 uM IOX2 were co-treated with the HSA and majority Apotransferrin. After 6 hr treatments, intracellular mRNA was harvested and tested for Glut1 and VEGF expression levels by qPCR. Values are shown as Relative Gene Expression, with the target gene (Glut1 or VEGF) normalized for housekeeper (beta-actin) expression. Standard deviations are shown. FIGS. 9A and 9B show that Glut1 and VEGF mRNA levels increase synergistically and additively with the addition of both Apotransferrin and small molecule activators of the HIF pathway.

Example 10

Compositions of Majority ApoTf and Majority HoloTf Induce HIF1 Alpha Protein in Human Primary Kidney Cells

It is well-known in the art that many small molecules used for the treatment of conditions related or provoked by hypoxia are toxic and have numerous side effects, e.g. DFO. One of the most apparent side effects of said small molecules is kidney toxicity. Therefore, in order to assess whether transferrin and/or mixtures increase HIF1 alpha levels in primary kidney cells; human primary kidney cells, both primary human renal proximal tubule epithelial (RPTEC) or cortical epithelial cells (HRCE) were obtained. Primary human renal proximal tubule epithelial (RPTEC) or primary cortical epithelial (HRCE) cells cultured in serum free media were treated with 4 mg/mL majority Apo-transferrin, majority Holo-transferrin or various mixtures of each for 6 hrs under normal oxygen levels. After 6 hrs intracellular proteins were harvested and tested for HIF1 alpha protein levels by ELISA. FIGS. 10A and 10B reveal that HIF1 alpha levels are induced with transferrin composed of mixtures of Apo-transferrin and Holo-transferrin in RPTEC and HRCE, respectively.

Example 11

Viability of Human Primary Kidney Cells in the Presence of Transferrins or DFO, Including Caspase 3/7 Activation within Human Primary Kidney Cells in the Presence of Majority ApoTf or DFO

Considering the anticipated safety profile of a human plasma protein, toxicity of DFO and transferrins (majority Apo, majority Holo and mixtures) was assessed in primary human kidney cells. The renal proximal tubule epithelial (RPTEC) or cortical epithelial (HRCE) cells were treated with the indicated concentrations of majority ApoTf or DFO for 48 hours (FIG. 11A); and RPTEC or HRCE cells were treated for 72 hrs with 4 mg/mL of majority ApoTf, majority HoloTf, mixtures of transferrin (FIG. 11B). After 48 or 72 hours, cells were subjected to a Cell Titer Glow viability assay. Control cells, untreated cells, were set to a value of 100% viable. The average viability and standard deviations are shown for each treatment condition. FIGS. 11A and 11B show that while DFO had significant toxicity, none of the transferrin molecules showed any detrimental effects on these primary kidney cells.

In order to assess caspase 3/7 activation within human primary kidney cells in the presence of ApoTf or DFO; RPTE or HRC cells were treated with the indicated concentrations of ApoTf or DFO for 48 hours. After 48 hours, cells were subjected to a ApoGlo caspase 3/7 activation assay. Control cells, untreated cells, were set to a normalized value of 1. The average caspase activity, relative to control cells, and standard deviations are shown in FIG. 12 for each treatment condition.

Example 12

No Upregulation of HIF was Observed in Primary Human Hepatocytes or NCI-H1650, a Lung Cell Line

As detailed above, both plasma derived Apo-transferrin and Holo-transferrin increase the cellular levels of HIF-1alpha, in the human neuronal cell line SH-SY5Y. In addition to neuronal cells, liver and lung organ transplants may also benefit from induction of HIF signaling. Hence, in order to assess the same; effect of transferrins on HIF1alpha levels in primary hepatocytes and a lung cell line (NCI-H1650) was determined. The lung cell line NCI-H1650 or primary hepatocyte cells cultured in serum free media were treated with 4 mg/mL majority Apo-transferrin, majority Holo-transferrin or pd-Transferrin for 6 hrs under normal oxygen levels. After 6 hrs intracellular proteins were harvested and tested for HIF1alpha protein levels by ELISA. The data, as highlighted in FIGS. 13A and 13B, shows that HIF1 alpha levels are not induced with transferrin or mixtures of Apo-transferrin and Holo-transferrin in NCI-H1650 or primary hepatocytes.

Example 13

Viability of NCI-H1650 and Human Primary Hepatocytes in the Presence of Transferrins

Given the anticipated safety profile of a human plasma protein, toxicity of transferrins (majority Apo, majority Holo and pd-transferrin) in NCI-H1650 and primary human hepatocyte cells was assessed. The human lung cell line, NCI-H1650, and primary human hepatocytes were treated for 72 hours with 4 mg/mL of majority ApoTf, majority HoloTf, or pd-transferrin. After 72 hours, cells were subjected to a Cell Titer Glow viability assay. Control cells, untreated cells, were set to a value of 100% viable. The average viability and standard deviations are shown in FIGS. 14A and 14B for each treatment condition. The data shows that no toxicity was observed with compositions containing either majority HoloTf or majority ApoTf in lung cells, NCI-H1650, or primary hepatocytes.

CONCLUSIONS

The experiments performed in the human neuronal cell line SH-SY5Y showed that both plasma derived Apo-transferrin and Holo-transferrin increased the cellular levels of HIF-1α. The increase in HIF1 alpha levels occurred under both normoxic and hypoxic conditions. Administration of Apo-transferrin to cells under normal oxygen conditions raised the levels of HIF1 alpha to a similar level of that seen when cells were exposed to a hypoxic environment. Exposure of SH-SY5Y cells to Apo-transferrin in normoxic conditions for longer periods increased the level of HIF1 alpha to a greater extent than shorter time. The human serum albumin negative controls had no effect on HIF1-α levels.

Various mixtures of ApoTf and HoloTf all upregulated HIF1 alpha protein in SH-SY5Y neuronal cells and primary kidney cells.

No upregulation of HIF1 alpha was observed in primary human hepatocytes, or NCI-H1650, a lung cell line.

Various mixtures of ApoTf and HoloTf all upregulated HIF1alpha target genes in SH-SY5Y neuronal cells.

No toxicity was observed with compositions containing either majority HoloTf or majority ApoTf in any cell type (neuronal, lung, kidney or hepatocyte) or in vivo.

In vivo treatment of rats in a neurological stress model of ischemia-reperfusion showed that transferrin (composed of mostly ApoTf) protects rat cells from infarct.

Mixtures comprising mostly of ApoTf or HoloTf protected neuronal cells from the toxic effects of Abeta (1-42) oligomer.

Only mixtures composed of majority ApoTf had synergistic effects with M30 or DFO, and these synergistic activities only occurred in SH-SY5Y neuronal cells.

Claims

1. A method of treating a hypoxia Inducible Factor (HIF)-related pathological condition in a patient in need thereof, wherein the HIF-related pathological condition is Middle Cerebral Artery occlusion (MCAo), comprising administering to the patient a composition comprising a therapeutically effective amount of transferrin, and wherein the transferrin is a mixture of apo-transferrin and holo-transferrin in a ratio from 99% apo-Tf:1% Holo-Tf to 30% apo-Tf:70% Holo-Tf.

2. The method of claim 1, wherein the composition further comprises an iron chelator or prolyl hydroxylase domain-containing protein 2 (PHD2) enzyme inhibitor.

3. The method of claim 2, wherein the iron chelator is selected from the group consisting of M30, deferoxamine (DFO), Deferasirox, deferiprone, deferitrin, L1NAll, CP363, CP502 and Ethylenediaminetetraacetic acid (EDTA).

4. The method of claim 2, wherein the PHD2 enzyme inhibitor is selected from the group consisting of IOX2, IOX3 and dimethyloxallylglycine.

5. The method of claim 1, wherein the patient is a transplant recipient of an organ.

6. The method of claim 5, wherein the organ has been treated with the composition in preparation for the transplantation into the recipient.

7. The method of claim 6, where the composition further comprises an iron chelator or prolyl hydroxylase domain-containing protein 2 (PHD2) enzyme inhibitor.

8. The method of claim 1, wherein the condition is associated with ischemia or oxygen deprivation in the patient prior to surgery.

9. The method of claim 8, wherein the ischemia is due to cardiac arrest, thrombotic clots, traumatic injury or stroke.

10. The method of claim 1, wherein the condition is associated with interruption of blood flow during a surgical intervention in the patient.

11. The method of claim 1, wherein the transferrin is recombinant.

12. The method of claim 1, wherein the transferrin is modified by pegylation, glycosylation or polysialylation to extend its plasma half-life.

13. The method of claim 1, wherein the composition further comprises an iron chelator.

14. The method of claim 1, wherein the neurodegenerative disease is Parkinson's disease.

15. The method of claim 1, wherein the neurodegenerative disease is Alzheimer's disease.

16. The method of claim 1, wherein the neurodegenerative disease is Amvlotrophic Lateral Sclerosis.

17. The method of claim 1, wherein the transferrin is recombinant.

18. The method of claim 1, wherein the transferrin is modified by pegylation, glvcosvlation or polvsialvlation to extend its plasma half-life.

19. The method of claim 1, wherein the transferrin comprises an amino acid sequence with at least 80% identity to SEQ ID NO: 1.

20. The method of claim 1, wherein the transferrin comprises an amino acid sequence with at least 90% identity to SEQ ID NO: 1.

21. A method of treating a hypoxia Inducible Factor (HIF)-related pathological condition in a patient in need thereof, wherein the HIF-related pathological condition is a neurodegenerative disease selected from the group consisting of Alzheimer's disease, Parkinson's disease, Huntington's disease, and Amylotrophic Lateral Sclerosis, comprising administering to the patient a composition comprising a therapeutically effective amount of transferrin, wherein the transferrin comprises an amino acid sequence with at least 70% identity to SEQ ID NO: 1 and wherein the transferrin is a mixture of apo-transferrin and holo-transferrin in a ratio from 99% apo-Tf:1% Holo-Tf to 30% apo-Tf:70% Holo-Tf.

22. The method of claim 21, wherein the composition further comprises an iron chelator or prolyl hydroxylase domain-containing protein 2 (PHD2) enzyme inhibitor.

23. The method of claim 22, wherein the iron chelator is selected from the group consisting of M30, deferoxamine (DFO), Deferasirox, deferiprone, deferitrin, L1NAll, CP363, CP502 and Ethylenediaminetetraacetic acid (EDTA).

24. The method of claim 22, wherein the PHD2 enzyme inhibitor is selected from the group consisting of IOX2, IOX3 and dimethyloxallylglycine.

25. The method of claim 21, wherein the patient is a transplant recipient of an organ.

26. The method of claim 25, wherein the organ has been treated with the composition in preparation for the transplantation into the recipient.

27. The method of claim 26, where the composition further comprises an iron chelator or prolyl hydroxylase domain-containing protein 2 (PHD2) enzyme inhibitor.

28. The method of claim 21, wherein the condition is associated with ischemia or oxygen deprivation in the patient prior to surgery.

29. The method of claim 28, wherein the ischemia is due to cardiac arrest, thrombotic clots, traumatic injury or stroke.

30. The method of claim 21, wherein the condition is associated with interruption of blood flow during a surgical intervention in the patient.

31. The method of claim 21, wherein the transferrin is recombinant.

32. The method of claim 21, wherein the transferrin is modified by pegylation, glycosylation or polysialylation to extend its plasma half-life.

33. The method of claim 21, wherein the composition further comprises an iron chelator.

34. The method of claim 21, wherein the neurodegenerative disease is Parkinson's disease.

35. The method of claim 21, wherein the neurodegenerative disease is Alzheimer's disease.

36. The method of claim 21, wherein the neurodegenerative disease is Amylotrophic Lateral Sclerosis.

37. The method of claim 21, wherein the transferrin comprises an amino acid sequence with at least 80% identity to SEQ ID NO: 1.

38. The method of claim 21, wherein the transferrin comprises an amino acid sequence with at least 90% identity to SEQ ID NO: 1.