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Hemoglobin alpha chain peptide fragments useful for inhibiting stem cell proliferation

a technology of hemoglobin alpha chain and peptides, which is applied in the direction of peptides, peptide/protein ingredients, peptide sources, etc., can solve the problems of no clinical use approval of this factor, bone marrow suppression or gastrointestinal toxicity,

Inactive Publication Date: 2010-02-18
WELLSTAT THERAPEUTICS
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  • Summary
  • Abstract
  • Description
  • Claims
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AI Technical Summary

Benefits of technology

The present invention relates to polypeptides that inhibit the growth and proliferation of stem cells. These polypeptides have specific properties, including low activity in a colony-forming spleen (CFU-S) assay, sensitivity to degradation by trypsin, and a molecular weight range of 10,000 to 100,000 daltons. The polypeptides are also more hydrophobic than other proteins such as MIP-1α and TGFβ, and are retained after centrifugation at 10,000 g for 30 minutes at 4°C. The invention provides pharmaceutical compositions containing the polypeptides for treatment of disorders where stem cell proliferation needs to be inhibited, such as immunosuppression caused by stem cell hyperproliferation. The polypeptides can also be used as an adjuvant before or together with vaccination to increase immune response. The invention also includes a method for purifying the polypeptides and a method for administering them to humans or animals.

Problems solved by technology

Purification of this factor from primary sources was not accomplished due to the difficulties inherent in an in vivo assay requiring large numbers of irradiated mice.
To date, none of these factors have been approved for clinical use.
The major toxicity associated with chemotherapy or radiation treatment is the destruction of normal proliferating cells which can result in bone marrow suppression or gastrointestinal toxicity.
The effectiveness of purging of hematopoietic cells with cytotoxic drugs in order to eliminate residual malignant cells is limited due to the toxicity of these compounds for normal hematopoietic cells and especially stem cells.
The major limitations of the method are both the low normal steady state frequency of stem cells in peripheral blood and their high cycle status after mobilization procedures with drugs or growth factors (e.g., cyclophosphamide, G-CSF, stem cell factor).
There are several limitations for the introduction of genes into human hematopoietic cells using either retrovirus vectors or physical techniques of gene transfer: (1) The low frequency of stem cells in hematopoietic tissues has necessitated the development of high efficiency gene transfer techniques; and (2) more rapidly cycling stem cells proved to be more susceptible to vector infection, but the increase of the infection frequency by stimulation of stem cell proliferation with growth factors produces negative effects on long term gene expression, because cells containing the transgenes are forced to differentiate irreversibly and lose their self-renewal.

Method used

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  • Hemoglobin alpha chain peptide fragments useful for inhibiting stem cell proliferation
  • Hemoglobin alpha chain peptide fragments useful for inhibiting stem cell proliferation
  • Hemoglobin alpha chain peptide fragments useful for inhibiting stem cell proliferation

Examples

Experimental program
Comparison scheme
Effect test

example 1

In Vivo Stem Cell Proliferation Inhibition Assay

[0117]For the detection of stem cells proliferation the number of CFU-S in S-phase of the cell cycle was measured by the 3H-Thymidine “suicide” method (Becker et al., Blood 26:296-308, 1965).

[0118]Immature hematopoietic progenitors—Colony Forming Units in spleen (CFU-S)—can be detected in vivo by forming macroscopic colonies in the spleens of lethally irradiated mice, 8-12 days after the intravenous injection of hematopoietic cells (Till & McCulloch, 1961).

[0119]For the standard CFU-S proliferation assay the method of 3H-Thymidine “suicide” is usually applied (Becker et al., 1965). The method is based on incorporation of radiolabelled Thymidine, (3H-Thymidine) a precursor of DNA into cells during DNA synthesis. The CFU-S which are in S-phase of the cycle at the time of testing, are killed by the high radioactivity and therefore not able to form colonies in spleen. Thus, the difference between the number of CFU-S formed by the injection...

example 2

In Vitro Stem Cell Proliferation Inhibition Assay

[0134]Using the following test system (Lord et al., in The Inhibitors of Hematopoiesis pp. 227-239, 1987) the direct effect of INPROL was shown. The multilineage factor (IL-3) dependent stem cell line, FDCP mix A4 (A4), was maintained in IMDM medium supplemented with 20% horse serum and 10% WEHI-3-conditioned medium as a source of colony-stimulating IL-3.

[0135]A tritiated Thymidine incorporation assay was used to measure proliferation: A4 cells (5×104 in 100 μl medium with 20% horse serum and 50% of WEHI-3 CM) were incubated at 37° C. in 5% CO2 for 16 hours.

[0136]pINPROL or the crude BME (fraction IV) were added at the start. Tritiated thymidine ((3H-Tdr) 3.7 KBq in 50 μl at 740 GBq / mmole) was then added to each group for a further 3 hours of incubation. The level of proliferation was measured by harvesting cells and the % inhibition calculated using the formula

%Inhibition=cpmwithoutINPROL-cpmwithINPROLcpmwithoutINPROL×(100%)

[0137]Inc...

example 3

Inhibition of CFU-S Proliferation by INPROL Injected In Vivo

Doses and the Duration of the Effect

[0138]The studies of the effect of INPROL injected in vivo revealed that INPROL can effectively block the recruitment of CFU-S into cycle, thus protecting those cells from the cytotoxic effect of further treatment, showing its potential for clinical use.

[0139]The experimental protocol had two goals: to check the effect of INPROL on CFU-S when injected in vivo and to define the effective duration of INPROL activity in relation to cycling stem cells.

[0140]To stimulate CFU-S proliferation, the injection of testosterone-propionate was used based on the effect mentioned above in Example 1.

[0141]Mice BDF1 were injected with TSP (10 mg / 100 g) on Day 0; 24 hours later mice of each experimental group (4 mice per group) received a single pINPROL injection at doses of 0 μg, 5 μg, 10 μg, and 15 μg / mouse i.p.

[0142]Twenty-four hours after pINPROL injection, mice were sacrificed and the percent of cycli...

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Abstract

Disclosed and claimed are methods for the isolation and use of stem cell inhibiting factors for regulating the abnormal stem cell cycle and for accelerating the post-chemotherapy peripheral blood cell recovery. Also disclosed and claimed are the inhibitors of stem cell proliferation.

Description

FIELD OF THE INVENTION[0001]The present invention relates to the use of inhibitors of stem cell proliferation for regulating stem cell cycle in the treatment of humans or animals having autoimmune diseases, aging, cancer, myelodysplasia, preleukemia, leukemia psoriasis or other diseases involving hyperproliferative conditions. The present invention also relates to a method of treatment for humans or animals anticipating or having undergone exposure to chemotherapeutic agents, other agents which damage cycling stem cells, or radiation exposure. Finally, the present invention relates to the improvement of the stem cell maintenance or expansion cultures for auto- and allo-transplantation procedures or for gene transfer.BACKGROUND OF THE INVENTION[0002]Most end-stage cells in renewing systems are short-lived and must be replaced continuously throughout life. For example, blood cells originate from a self-renewing population of multipotent hematopoietic stem cells (HSC). Hematopoietic st...

Claims

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Application Information

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Patent Type & Authority Applications(United States)
IPC IPC(8): C12N5/02A61K38/00A61K38/42C07K14/47C07K14/475C07K14/52C07K14/805
CPCA61K38/42C07K14/4703C07K14/475C07K14/52C07K14/805A61K2300/00
Inventor TSYRLOVA, IRENAWOLPE, STEPHEN D.
Owner WELLSTAT THERAPEUTICS
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