U.S. patent application number 09/839164 was filed with the patent office on 2002-07-25 for inhibitor of stem cell proliferation and uses thereof.
This patent application is currently assigned to Pro-Neuron, Inc.. Invention is credited to Kozlov, Vladimir, Tsyrlova, Irena.
Application Number | 20020098583 09/839164 |
Document ID | / |
Family ID | 21913839 |
Filed Date | 2002-07-25 |
United States Patent
Application |
20020098583 |
Kind Code |
A1 |
Kozlov, Vladimir ; et
al. |
July 25, 2002 |
Inhibitor of stem cell proliferation and uses thereof
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.
Inventors: |
Kozlov, Vladimir;
(Novosibirisk, RU) ; Tsyrlova, Irena;
(Gaithersburg, MD) |
Correspondence
Address: |
Nixon & Vanderhye P.C.
8th Floor
1100 N. Glebe Rd.
Arlington
VA
22201
US
|
Assignee: |
Pro-Neuron, Inc.
|
Family ID: |
21913839 |
Appl. No.: |
09/839164 |
Filed: |
April 23, 2001 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
09839164 |
Apr 23, 2001 |
|
|
|
08477668 |
Jun 7, 1995 |
|
|
|
08477668 |
Jun 7, 1995 |
|
|
|
08316424 |
Sep 30, 1994 |
|
|
|
08316424 |
Sep 30, 1994 |
|
|
|
PCT/US94/03349 |
Mar 29, 1994 |
|
|
|
PCT/US94/03349 |
Mar 29, 1994 |
|
|
|
08040942 |
Mar 31, 1993 |
|
|
|
Current U.S.
Class: |
435/366 ;
435/372; 514/13.4; 514/19.6; 514/7.9 |
Current CPC
Class: |
A61K 39/39 20130101;
C07K 14/475 20130101; A61P 37/04 20180101; A61P 35/00 20180101;
A61P 37/02 20180101; C07K 14/4703 20130101; A61K 38/42 20130101;
C07K 14/805 20130101; C07K 14/52 20130101; A61P 43/00 20180101;
A61P 35/02 20180101; A61K 38/42 20130101; A61K 2300/00
20130101 |
Class at
Publication: |
435/366 ; 514/6;
435/372 |
International
Class: |
A61K 038/42; C12N
005/08 |
Claims
What is claimed is:
1. A pharmaceutical composition comprising the alpha chain of
hemoglobin in a pharmaceutically acceptible carrier.
2. A pharmaceutical composition comprising the beta chain of
hemoglobin in a pharmaceutically acceptible carrier.
3. A pharmaceutical composition as in claim 1 further comprising
the beta chain of hemoglobin.
4. A pharmaceutical composition as in claim 1-3 in unit dosage
form.
5. A pharmaceutical composition as in claim 4 comprising 0.1 mgs.
to 6 gms. of the alpha and/or beta chain of hemoglobin.
6. A method of inhibiting stem cell proliferation comprising
contacting hematopoietic cells with a stem cell proliferation
inhibiting amount of INPROL.
7. A method as in claim 6 wherein said INPROL is selected from the
group consisting of the alpha chain of hemoglobin and the beta
chain of hemoglobin.
8. A method of stimulating the growth of B cells which comprises
contacting hematopoietic cells with a growth stimulating amount of
INPROL.
9. A method of treating cancer in a mammal suffering therefrom
comprising the steps of: a) administering radiotherapy or
chemotherapy, and b) administering a stem cell proliferation
inhibiting amount of INPROL
10. A method as in claim 9 wherein steps a and b are repeated one
or more times.
11. A method as in claim 9 wherein step a is conducted after step
b.
12. A method as in claim 9 wherein step b is conducted within 24
hours before or after step a.
13. A method for treating cancer in a mammal comprising: a)
removing bone marrow from said mammal, b) treating said bone marrow
ex vivo with INPROL, c) treating said bone marrow of step b with
chemotherapy or radiation, d) performing myeloablative treatment on
said mammal, and e) transplanting into said mammal the bone marrow
of step c.
14. A method as in claim 13 wherein said cancer is leukemia.
15. A method of inhibiting stem cell division in a mammal exposed
to an agent which damages or destroys stem cells undergoing
division comprising administering a stem cell proliferation
inhibiting amount of INPROL.
16. A method as in claim 15 wherein said agent is an antiviral
agent.
17. A method of maintaining mammalian hematopoietic stem cells ex
vivo comprising contacting hematopoietic cells with a stem cell
proliferation inhibiting amount of INPROL.
18. A method as in claim 17 wherein said hematopoietic cells are
selected from the group consisting of bone marrow cells, peripheral
blood cells and cord blood cells.
19. A method of treating a myeloproliferative or autoimmune disease
or epithelial stem cell hyperproliferation in a mammal suffering
therefrom comprising administering a hyperproliferative reducing
amount of INPROL.
20. A method as in claim 19 wherein said myeloproliferative disease
is a myelodysplastic syndrome.
21. A method for differentially protecting normal stem cells and
not cancer cells in a mammal from chemotherapy or radiation
comprising administering a stem cell protecting amount of
INPROL.
22. A method as in claim 21 wherein said INPROL is administered
after said normal stem cells are induced to proliferate by exposure
to a cytotoxic drug or radiation.
23. A method of vaccinating a mammal comprising administering
INPROL as an adjuvant before, during or after administration of a
vaccine.
24. A method of purifying an inhibitor of stem cell proliferation
substantially free from other proteinaceous material comprising the
following steps: a) isolating bone marrow and removing particulate
matter from an extract; b) heating said extract and removing
precipitate; c) acid precipitating said extract and collecting
precipitate; and d) isolating said inhibitor by reverse pahse
chromatography.
25. A purified inhibitor of stem cell proliferation wherein said
inhibitor is purified to apparent homogeneity by the method of
claim 24.
26. A method of treating a mammal having immunodepression caused by
stem cell hyperproliferation comprising administering to said
mammal an hyperproliferation reversing amount of INPROL.
Description
[0001] This application is a continuation-in-part application of
PCT/US94/03349 filed Mar. 29, 1994, which in turn is a
continuation-in-part application of U.S. Ser. No. 08/040924 filed
Mar. 31, 1993, both of which are hereby incorporated by
reference.
FIELD OF THE INVENTION
[0002] 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
[0003] 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). Because the hematopoietic stem
cells are necessary for the development of all of the mature cells
of the hematopoietic and immune systems, their survival is
essential in order to reestablish a fully functional host defense
system in subjects treated with chemotherapy or other agents.
[0004] Hematopoietic cell production is regulated by a series of
factors that stimulate growth and differentiation of hematopoietic
cells, some of which, for example erythropoietin and G-CSF, are
currently used in clinical practice. One part of the control
network which has not been extensively characterized, however, is
the feedback mechanism that forms the negative arm of the
regulatory process (Eaves et al. Blood 78:110-117, 1991).
[0005] Early studies by Lord and coworkers showed the existence of
a soluble protein factor in normal murine and porcine bone marrow
extracts, which was capable of reversibly inhibiting the cycling of
HSC (Lord et al, Br. J. Haem. 34:441-446, 1976). This inhibitory
activity (50-100 kD molecular weight) was designated stem cell
inhibitor (SCI).
[0006] 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. In an attempt to
overcome these problems Pragnell and co-workers developed an in
vitro assay for primitive hematopoietic cells (CFU-A) and screened
cell lines as a source of the inhibitory activity (see Graham et
al. Nature 344:442-444, 1990).
[0007] As earlier studies had identified macrophages as possible
sources for SCI (Lord et al. Blood Cells 6:581-593, 1980), a mouse
macrophage cell line, J774.2, was selected (Graham et al. Nature
344:442-144, 1990). The conditioned medium from this cell line was
used by Graham et al for purification; an inhibitory peptide was
isolated which proved to be identical to the previously described
cytokine macrophage inflammatory protein 1-alpha (MIP-1-alpha).
Thus, MIP-1-alpha was isolated from a cell line, not from primary
material. While Graham et al. observed that antibody to MIP-1-alpha
abrogated the activity of a crude bone marrow extract, other
workers have shown that other inhibitory activities are important
For example, Graham et al. (J. Exp. Med. 178:925-32, 1993) have
suggested that TGF.beta., not MIP-1.alpha., is a primary inhibitor
of hematopoietic stem cells. Further, Eaves et al. (PNAS
90:12015-19, 1993) have suggested that both MIP-1.alpha. and
TGF.beta. are present at sub optimal levels in normal bone marrow
and that inhibition requires a synergy between the two factors.
[0008] Other workers have described additional stem cell inhibitory
factors. Frindel and coworkers have isolated a tetrapeptide from
fetal calf marrow and from liver extracts which has stem cell
inhibitory activities (Lenfant et al., PNAS 86:779-782, 1989).
Paukovits et al. (Cancer Res. 50:328-332, 1990) have characterized
a pentapeptide which, in its monomeric form, is an inhibitor and,
in its dimeric form, is a stimulator of stem cell cycling. Other
factors have also been claimed to be inhibitory in various in vitro
systems (cf. Wright and Pragnell in Bailliere's Clinical
Haematology v.5, pp. 723-39, 1992 (Bailliere Tinadall, Paris)).
[0009] Tsyrlova et al., SU 1561261 A1, disclosed a purification
process for a stem cell proliferation inhibitor.
[0010] To date, none of these factors have been approved for
clinical use. However, the need exists for effective stem cell
inhibitors. 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. An effective stem cell inhibitor would
protect these cells and allow for the optimization of these
therapeutic regimens. Just as there is a proven need for a variety
of stimulatory cytokines (e.g., G-CSF, GM-CSF, erythropoietin,
IL-11) depending upon the clinical situation, so too it is likely
that a variety of inhibitory factors will be needed to address
divergent clinical needs.
[0011] Hemoglobin is a highly conserved tetrameric protein with
molecular weight of approximately 64,000 Daltons. It consists of
two alpha and two beta chains. Each chain binds a single molecule
of heme (ferroprotoporphyrin IX), an iron-containing prosthetic
group. Vertebrate alpha and beta chains were probably derived from
a single ancestral gene which duplicated and then diverged; the two
chains retain a large degree of sequence identity both between
themselves and between various vertebrates (see FIG. 16A). In
humans, the alpha chain cluster on chromosome 16 contains two alpha
genes (alpha.sub.1 and alpha.sub.2) which code for identical
polypeptides, as well as genes coding for other alpha-like chains:
zeta, theta and several non-transcribed pseudogenes (see FIG. 16B
for cDNA and amino acid sequences of human alpha chain). The beta
chain cluster on chromosome 11 consists of one beta chain gene and
several beta-like genes: delta, epsilon, G gamma and A gamma, as
well as at least two unexpressed pseudogenes (see FIG. 16C for cDNA
and amino acid sequences of human beta chain).
[0012] The expression of these genes varies during development. In
human hematopoiesis, which has been extensively characterized,
embryonic erythroblasts successively synthesize tetramers of two
zeta chains and two epsilon chains (Gower I), two alpha chains and
two epsilon chains (Gower II) or two zeta chains and two gamma
chains (Hb Portland). As embryogenesis proceeds, the predominant
form consists of fetal hemoglobin (Hb F) which is composed of two
alpha chains and two gamma chains. Adult hemoglobin (two alpha and
two beta chains) begins to be synthesized during the fetal period;
at birth approximately 50% of hemoglobin is of the adult form and
the transition is complete by about 6 months of age. The vast
majority of hemoglobin (approximately 97%) in the adult is of the
two alpha and two beta chain variety (Hb A) with small amounts of
Hb F or of delta chain (Hb A.sub.2) being detectable.
[0013] Heme has been extensively examined with regard to its
influences on hematopoiesis (see S. Sassa, Seminars Hemat.
25:312-20, 1988 and N. Abraham et al., Int. J. Cell Cloning
9:185-210, 1991 for reviews). Heme is required for the maturation
of erythroblasts; in vitro, hemin (chloroferroprotoporphyrin
IX--i.e., heme with an additional chloride ion) increases the
proliferation of CFU-gemm, BFU-E and CFU-E. Similarly, hemin
increases cellularity in long-term bone marrow cultures.
[0014] I. Chemotherapy and Radiotherapy of Cancer
[0015] Productive research on stimulatory growth factors has
resulted in the clinical use of a number of these factors
(erythropoietin, G-CSF, GM-CSF, etc.). These factors have reduced
the mortality and morbidity associated with chemotherapeutic and
radiation treatments. Further clinical benefits to patients who are
undergoing chemotherapy or radiation could be realized by an
alternative strategy of blocking entrance of stem cells into cell
cycle thereby protecting them from toxic side effects.
[0016] II. Bone Marrow Transplantation
[0017] Bone marrow transplantation (BMT) is a useful treatment for
a variety of hematological, autoimmune and malignant diseases. Ex
vivo manipulation of cells is currently being used to expand
primitive stem cells to a population suitable for transplantation.
Optimization of this procedure requires: (1) sufficient numbers of
stem cells able to maintain long term reconstitution of
hematopoiesis; (2) the depletion of graft versus host-inducing
T-lymphocytes and (3) the absence of residual malignant cells. This
procedure can be optimized by including a stem cell inhibitor(s)
for ex vivo expansion.
[0018] The effectiveness of purging of bone marrow 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. There is a need for
effective protection of normal cells during purging; protection can
be afforded by taking stem cells out of cycle with an effective
inhibitor.
[0019] III. Peripheral Stem Cell Harvesting
[0020] Peripheral blood stem cells (PBSC) offer a number of
potential advantages over bone marrow for autologous
transplantation. Patients without suitable marrow harvest sites due
to tumor involvement or previous radiotherapy can still undergo
PBSC collections. The use of blood stem cells eliminates the need
for general anesthesia and a surgical procedure in patients who
would not tolerate this well. The apheresis technology necessary to
collect blood cells is efficient and widely available at most major
medical centers. 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). An effective stem cell inhibitor would be useful to return
such cells to a quiescent state, thereby preventing their loss
through differentiation.
[0021] IV. Treatment of Hyperproliferative Disorders
[0022] A number of diseases are characterized by a
hyperproliferative state in which disregulated stem cells give rise
to an overproduction of end stage cells. Such disease states
include, but are not restricted to, psoriasis, in which there is an
overproduction of epidermal cells, and premalignant conditions in
the gastrointestinal tract characterized by the appearance of
intestinal polyps. A stem cell inhibitor would be useful in the
treatment of such conditions.
[0023] V. Gene Transfer
[0024] The ability to transfer genetic information into
hematopoietic cells is currently being utilized in clinical
settings. The bone marrow is a useful target for gene therapy
because of ease of access, extensive experience in manipulating and
treating this tissue ex vivo and because of the ability of blood
cells to permeate tissues. Furthermore, the correction of certain
human genetic defects may be possible by the insertion of a
functional gene into the primitive bone marrow stem cells of the
human hematopoietic system.
[0025] There are several limitations for the introduction of genes
into human hematopoietic cells using either retrovirus vector 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 the growth factors is
shown to produce negative effect on long term gene expression,
because cells containing the transgenes are forced to differentiate
irreversibly and lose their self-renewal. These problems can be
ameliorated by the use of a stem cell inhibitor to prevent
differentiation and loss of self-renewal.
SUMMARY OF THE INVENTION
[0026] The present invention relates to an inhibitor of stem cell
proliferation (INPROL) characterized by the following
properties:
[0027] (a) Specific activity (IC.sub.50) less than or equal to 20
ng/ml in a murine colony-forming spleen (CFU-S) assay (see Example
4),
[0028] (b) Molecular weight greater than 10,000 and less than
100,000 daltons (by ultrafiltration),
[0029] (c) Activity sensitive to degradation by trypsin,
[0030] (d) More hydrophobic than MIP-1.alpha. or TGF.beta. and
separable from both by reverse phase chromatography (cf. Example
12),
[0031] (e) Biological activity retained after heating for one hour
at 37.degree. C., 55.degree. C. or 75.degree. C. in aqueous
solution and
[0032] (f) Biological activity retained after precipitation with 1%
hydrochloric acid in acetone.
[0033] The present invention is further characterized and
distinguished from other candidate stem cell inhibitors (e.g.,
MIP-1.alpha., TGF.beta. and various oligopeptides) by its capacity
to achieve inhibition in an in vitro assay after a short
preincubation period (see Example 5).
[0034] The present invention also comprises pharmaceutical
compositions containing INPROL for treatment of a variety of
disorders.
[0035] The present invention provides a method of treating a
subject anticipating exposure to an agent capable of killing or
damaging stem cells by administering to that subject an effective
amount of a stem cell inhibitory composition. The stem cells
protected by this method may be hematopoietic stem cells ordinarily
present and dividing in the bone marrow. Alternatively, stem cells
may be epithelial, located for example, in the intestines or scalp
or other areas of the body or germ cells located in reproductive
organs. The method of this invention may be desirably employed on
humans, although animal treatment is also encompassed by this
method. As used herein, the terms "subject" or "patient" refer to
an animal, such as a mammal, including a human.
[0036] In another aspect, the invention provides a method for
protecting and restoring the hematopoietic, immune or other stem
cell systems of a patient undergoing chemotherapy, which includes
administering to the patient an effective amount of INPROL.
[0037] In still a further aspect, the present invention involves a
method for adjunctively treating any cancer, including those
characterized by solid tumors, by administering to a patient having
cancer an effective amount of INPROL to protect stem cells of the
bone marrow, gastrointestinal tract or other organs from the toxic
effects of chemotherapy or radiation therapy.
[0038] Yet another aspect of the present invention involves the
treatment of leukemia, comprising treating bone marrow cells having
proliferating leukemia cells therein with an effective amount of
INPROL to inhibit proliferation of normal stem cells, and treating
the bone marrow with a cytotoxic agent to destroy leukemia cells.
This method may be enhanced by the follow-up treatment of the bone
marrow with other agents that stimulate its proliferation; e.g.,
colony stimulating factors. In one embodiment this method is
performed in vivo. Alternatively, this method is also useful for ex
vivo purging and expansion of bone marrow cells for
transplantation.
[0039] In still a further aspect, the method involves treating a
subject having any disorder caused by proliferating stem cells.
Such disorders, such as psoriasis, myelodysplasia, some autoimmune
diseases, immuno-depression in aging, are treated by administering
to the subject an effective amount of INPROL to partially inhibit
proliferation of the stem cell in question.
[0040] The present invention provides a method for reversibly
protecting stem cells from damage from a cytotoxic agent capable of
killing or damaging stem cells. The method involves administering
to a subject anticipating exposure to such an agent an effective
amount of INPROL.
[0041] The present invention also provides:
[0042] An inhibitor of stem cell proliferation isolated from
porcine or other bone marrow by the following procedure (cf.
Example 12):
[0043] (a) Extraction of bone marrow and removal of particulate
matter through filtration,
[0044] (b) Heat treatment at 56.degree. C. for 40 minutes followed
by cooling in ice bath,
[0045] (c) Removal of precipitate by centrifugation at 10,000 g for
30 minutes at 4.degree. C.,
[0046] (d) Acid precipitation by addition of supernatant to 10
volumes of stirred ice-cold acetone containing 1% by volume
concentrated hydrochloric acid and incubation at 4.degree. C. for
16 hours,
[0047] (e) Isolation of precipitate by centrifugation at 20,000 g
for 30 minutes at 4.degree. C. and washing with cold acetone
followed by drying,
[0048] (f) Isolation by reverse phase chromatography and monitoring
activity by inhibition of colony formation by bone marrow cells
pretreated with 5-fluorouracil and incubated in the presence of
murine IL-3, as well as by absorption at 280 nm and by
SDS-PAGE.
[0049] The present invention also provides:
[0050] A method for purifying an inhibitor of stem cell
proliferation substantially free from other proteinaceous materials
comprising the preceding steps, as also described in more detail
below.
[0051] The present invention also provides:
[0052] A method of treatment for humans or animals wherein an
inhibitor of stem cell proliferation functions to ameliorate
immunosuppression caused by stem cell hyperproliferation.
[0053] The present invention also provides:
[0054] A method of treatment for humans or animals wherein said
inhibitor of stem cell proliferation is administered after the stem
cells are induced to proliferate by exposure to a cytotoxic drug or
irradiation procedure. Stem cells are normally quiescent but are
stimulated to enter cell cycle after chemotherapy. This renders
them more sensitive to a second administration of chemotherapy; the
current method protects them from this treatment.
[0055] The present invention also provides:
[0056] A method of treatment for humans or animals wherein said
inhibitor of stem cell proliferation is administered as an adjuvant
before or together with vaccination for the purpose of increasing
immune response.
[0057] The present invention also provides:
[0058] A method of treatment for humans or animals receiving
cytotoxic drugs or radiation treatment which comprises
administering an effective amount of the inhibitor of stem cell
proliferation to protect stem cells against damage.
[0059] The current invention describes an inhibitor of stem cells
(INPROL) which is different from those known in the art such as
MIP-1-alpha, TGF-beta, the tetrapeptide of Frindel and colleagues
or the pentapeptide of Paukovits and coworkers (cf., Wright &
Pragnell, 1992 (op cit)). INPROL has a molecular weight exceeding
10,000 daltons by ultrafiltration which distinguishes it from the
tetrapeptide as well as the pentapeptide. It is more hydrophobic
than MIP-1 alpha or TGF beta in reverse phase chromatography
systems, distinguishing it from those cytokines. Further, its mode
of action is different from that of any previously described
inhibitor in that it is active in an in vitro assay when used
during a preincubation period only. MIP-1-alpha for example, is not
effective when used during a preincubation period only (Example 5).
Further, INPROL is active in an assay measuring `high proliferative
potential cells" (HPP-PFC) whereas MIP-1-alpha is not (Example
6).
BRIEF DESCRIPTION OF THE DRAWINGS
[0060] FIGS. 1-4 show an SDS polyacrylamide gel run of the product
after each stage of purification.
[0061] FIG. 1--Lane 1 is chemotrypsinogen, Lane 2 is ovalbumin,
Lane 3 is BSA, Lane 4 is fractions, 30 kD, Lane 5 is fractions
30-50 kD and Lane 6 is fractions 50-100 kD.
[0062] FIG. 2--Lane 1 is after ammonium sulfate precipitation
(40-80%) and lanes 2-5 are DEAE fractions (Lane #2 represents the
active fraction).
[0063] FIG. 3--Lane 1 is the supernatant after ammonium sulfate
precipitation, Lane 2 is the active DEAE fraction, Lanes 3-5
represent gel filtration fractions (lane #5 represents the active
fraction)
[0064] FIG. 4--Lane 2 represents the final product.
[0065] FIG. 5 shows an HPLC chromatogram of the final
purification.
[0066] FIG. 6 shows tritiated thymidine incorporation (cpm) into
cells of the FDCP-mix line without (Control=100%) and with various
concentrations of INPROL. Data are normalized against the control
value.
[0067] FIG. 7 shows the percent of cells in the S phase of the cell
cycle after treatment of mice with testosterone propionate (TSP),
TSP plus INPROL, or vehicle (Control). Each group contained 25
animals (34 per time point).
[0068] FIG. 8 shows survival of mice treated with two doses of
5-FU, with or without INPROL treatment. Each group contained 30
animals.
[0069] FIG. 9 shows survival of irradiated mice, with and without
INPROL treatment. Each group contained 50 animals.
[0070] FIGS. 10A and 10B show regeneration of normal bone marrow
long term culture cells 1 week (10A) and 3 weeks (10B) after
treatment with Ara-C or Ara-C plus INPROL.
[0071] FIG. 11 shows survival of mice (75 per group) after lethal
irradiation and transplantation of 3.times.10.sup.4 bone marrow
cells after pre-incubation with medium (Control) or INPROL (25
ng/ml) for 4 hours. Survival was monitored for 30 days.
[0072] FIG. 12 shows CFU-GM number formed after 14 days in culture
by bone marrow cells from mice after lethal irradiation and
restoration with donor bone marrow cells preincubated with INPROL
or medium for 4 hours.
[0073] FIG. 13 shows suspension cells from lymphoid long-term
culture which were taken every week, washed out, and plated with
IL-7 (10 ng/ml) after preincubation with medium or INPROL for 4
hours.
[0074] FIG. 14 shows improved repopulating ability of leukemic
peripheral blood cells treated with INPROL. Long term culture
initiating cells (LTC-IC) were measured by plating adherent and
nonadherent LTC cells with and without INPROL, and scoring CFU-GM
on day 7. Data are normalized to control values.
[0075] FIG. 15A shows a C4 reverse phase chromatogram of purified
INPROL eluting at 53% acetonitrile. Lane 1 is the crude material,
Lane 2 is molecular weight markers and Lane 3 is the purified
material. FIG. 15B shows a C4 reverse phase chromatogram of MIP-1
alpha eluting at 43.9% acetonitrile. FIG. 15C shows an SDS-PAGE
chromatogram of the crude INPROL preparation and of the purified
preparation after reverse phase.
[0076] FIG. 16 shows hemoglobin sequences: FIG. 16A shows the cDNA
and amino acid sequences of human alpha hemoglobin and FIG. 16B
shows the cDNA and amino acid sequences of human beta hemoglobin.
Numbering is according to the amino acid. FIG. 16C shows an amino
acid sequence comparison of the alpha and beta chains of human,
murine and porcine hemoglobins.
[0077] FIG. 17 shows a comparison of the C4 reverse-phase HPLC
traces of INPROL (FIG. 17A) and of crystallized pig hemoglobin
(FIG. 17B).
[0078] FIG. 18 shows an SDS-PAGE gel of fractions from a C4 reverse
phase HPLC separation of crystallized pig hemoglobin. Lane 1 shows
molecular weight markers, Lane 2 shows Fractions 48-49, derived
from the first peak (at 47.11 min), Lane 3 shows fractions 50-51,
derived from the second peak (at 49.153 min), Lane 4 shows
fractions 54-55, derived from the third peak (at 52.25 min) and
Lane 5 shows fractions 56-57, derived from the fourth peak (at
53.613 minutes).
[0079] FIG. 19 shows a comparison of the 2-dimensional gel
electrophoreses of INPROL (FIG. 19A) and of purified pig alpha
hemoglobin (FIG. 19B).
[0080] FIG. 20 shows a comparison of the effects of purified pig
alpha hemoglobin, beta hemoglobin or INPROL in the FDCP-MIX
assay.
[0081] In order that the invention herein described may be more
fully understood, the following detailed description is set forth.
This description, while exemplary of the present invention, is not
to be construed as specifically limiting the invention and such
variations which would be within the purview of one skilled in this
art are to be considered to fall within the scope of this
invention.
DETAILED DESCRION OF THE PREFERRED EMBODIMENTS
[0082] INPROL reversibly inhibits division of stem cells.
Specifically, INPROL is effective in temporarily inhibiting cell
division of hematopoietic stem cells. Thus, the method of this
invention may be employed in alleviating the undesirable side
effects of chemotherapy on the patient's hematopoietic, myeloid and
immune systems by protecting stem cells from damage caused by
chemotherapeutic agents or radiation used to destroy cancer or
virally infected cells. In one embodiment of the invention, INPROL
is administered to the patient in a dosage sufficient to inhibit
stem cell division while the chemotherapeutic agent acts on
diseased cells. After the chemotherapeutic agent has performed its
function, the stem cells inhibited by INPROL will, without further
treatment, revert to dividing cells. If it-is desired to enhance
the regeneration of hematopoiesis, stimulatory growth factors or
cytokines may be used in addition.
[0083] As used herein, the term "INPROL" includes mammalian
proteins, purified as in the Examples, hemoglobin, the alpha chain
of hemoglobin (with or without the heme group), the beta chain of
hemoglobin (with or without the heme group), mixtures of alpha and
beta chains, and fragments or analogs of these proteins having the
ability to inhibit stem cell proliferation. The term "INPROL"
includes naturally occurring as well as non-naturally occurring
(e.g., recombinantly produced) forms of these proteins.
[0084] In a typical clinical situation, INPROL is administered to a
patient in a daily regimen by intravenous injection or infusion in
dosage unit form using, for example, 0.01 to 100 mg/kg,
advantageously 0.1 to 1.0 mg/kg, of INPROL administered, e.g., 4 to
60 hours prior to standard chemotherapy or radiation
treatments.
[0085] In another embodiment of the invention, pretreatment with
INPROL allows for increased doses of chemotherapeutic agents or of
radiation beyond doses normally tolerated in patients.
[0086] A large fraction of hematopoietic stem cells are normally
quiescent (non-cycling). However, as a compensatory response to
chemotherapy-induced hematopoietic damage, a larger proportion of
stem cells enter into cycling after chemotherapy, which makes them
particularly vulnerable to subsequent doses of cytotoxic
chemotherapy or therapeutic irradiation. By inhibiting cycling of
such stem cells, INPROL treatment permits earlier or more frequent
administration of subsequent doses of cytotoxic chemotherapy,
either at conventional or elevated doses.
[0087] In one embodiment of the invention, INPROL (0.1 mgs. to 6
gms--advantageously 1.0 to 60 mgs.) is administered about 24 hours
to 10 days after an initial dose of chemotherapy. After another 4
to 60 hours, advantageously 24 to 48 hours, another dose of
chemotherapy is administered. This cycle of alternating
chemotherapy and INPROL is continued according to therapeutic
benefit. Chemotherapy agents and protocols for administration are
selected according to suitability for particular tumor types in
standard clinical practice. Optionally, stimulatory growth factors
such as G-CSF, stem cell factor, are used after chemotherapy or
radiation treatment to further improve hematopoietic
reconstitution.
[0088] For ex vivo applications 0.1 ng to 100 ng/10.sup.6 cells/ml,
advantageously 20-50 ng/106 cells/ml, of INPROL are used.
[0089] In another embodiment of the invention, INPROL is employed
in a method for preparing autologous bone marrow for
transplantation. The marrow is treated ex vivo with an effective
amount of INPROL to inhibit stem cell division and then purged of
cancerous cells by administering to the marrow cultures an
effective amount of a chemotherapeutic agent or radiation.
Chemotherapy agents with specificity for cycling cells are
preferred. Marrow thus treated is reinjected into the autologous
donor. Optionally, the patient is treated with an agent known to
stimulate hematopoiesis to improve the hematopoietic reconstitution
of the patient.
[0090] In another embodiment of the invention, INPROL is employed
as an adjunctive therapy in the treatment of leukemia. For example,
in disease states where the leukemic cells do not respond to
INPROL, the leukemic bone marrow cells are treated ex vivo with
INPROL. The proliferation of normal stem cells is prevented by
administration of INPROL. Thus, during the time that the
proliferating leukemic cells are treated with a cell cycle-specific
cytotoxic agent, a population of normal stem cells is protected
from damage. Additionally, a stimulatory cytokine, such as IL-3 or
GM-CSF, is optionally administered to induce cycling in the
leukemic cells during drug or radiation treatment while the normal
stem cells are protected with INPROL. The patient is treated with
chemotherapy agents or radiation to destroy leukemic cells, and the
purged marrow is then transplanted back into the patient to
establish hematopoietic reconstitution.
[0091] Similarly, in another embodiment of the invention for
treatment of patients with serious viral infections that involve
blood cells or lymphocytes, such as HIV infection, bone marrow is
treated ex vivo with INPROL followed by antiviral agents, drugs
which destroy infected cells, or antibody-based systems for
removing infected cells. Following myeloablative antiviral or
myeloablative chemotherapy to eradicate viral host cells from the
patient, the INPROL-treated marrow cells are returned to the
patient.
[0092] In another embodiment of the invention, INPROL is employed
to treat disorders related to hyperproliferative stem cells. For
example, psoriasis is a disorder caused by hyperproliferating
epithelial cells of the skin and is sometimes treated with
cytotoxic drugs. Other pre-neoplastic lesions in which stem cell
proliferation is involved are also amenable to effective amounts of
INPROL employed to inhibit wholly or partially the proliferation of
the stem cells. For these uses, topical or transdermal delivery
compositions (e.g., ointments, lotions, gels or patches) containing
INPROL are employed where appropriate, as an alternative to
parenteral administration. In most cases of leukemia, the leukemia
progenitors are differentiated cell populations which are not
affected by INPROL and which are therefore treated by methods using
INPROL such as those described above. In cases where leukemia
progenitors are very primitive and are directly sensitive to
inhibition by INPROL, proliferation of leukemia cells is attenuated
by administration of effective amounts of INPROL.
[0093] Antibodies, monoclonal or polyclonal, are developed by
standard techniques to the INPROL polypeptides. These antibodies or
INPROL polypeptides are labeled with detectable labels of which
many types are known in the art. The labeled INPROL or anti-INPROL
antibodies are then employed as stem cell markers to identify and
isolate stem cells by administering them to a patient directly for
diagnostic purposes. Alternatively, these labeled polypeptides or
antibodies are employed ex vivo to identify stem cells in a bone
marrow preparation to enable their removal prior to purging
neoplastic cells in the marrow. In a similar manner, such labeled
polypeptides or antibodies are employed to isolate and identify
epithelial or other stem cells. In addition, such antibodies,
labeled or unlabeled, are used therapeutically through
neutralization of INPROL activity or diagnostically through
detection of circulating INPROL levels.
[0094] INPROL can be cloned from human gene or cDNA libraries for
expression of recombinant human INPROL using standard techniques.
For example, using sequence information obtained from the purified
protein, oligonucleotide probes are constructed which can be
labeled, e.g., with 32-phosphorus, and used to screen an
appropriate cDNA library (e.g., from bone marrow). Alternatively,
an expression library from an appropriate source (e.g., bone
marrow) is screened for cDNA's coding for INPROL using antibody or
using an appropriate functional assay (e.g., that described in
Example 2). Hemoglobin itself, as well as the individual alpha and
beta chains, have been cloned and expressed using methods known in
the state of the art (cf., Pagnier et al., Rev. Fr. Transfus.
Hemobiol. 35:407-15, 1992; Looker et al., Nature 356:258-60, 1992;
Methods in Enzymology vol. 231, 1994).
[0095] The present invention includes DNA sequences which include:
the incorporation of codons "preferred" for expression by selected
nonmammalian hosts: the provision of sites for cleavage by
restriction endonuclease enzymes; and the provision of additional
initial, terminal or intermediate DNA sequences which facilitate
construction of readily-expressed vectors or production or
purification of the alpha or beta chain of hemoglobin.
[0096] The present invention also provides DNA sequences coding for
polypeptide analogs or derivatives of hemoglobin alpha chain or
beta chain which differ from naturally-occurring forms in terms of
the identity or location of one or more amino acid residues (i.e.,
deletion analogs containing less than all of the residues
specified; substitution analogs, wherein one or more residues
specified are replaced by other residues; and addition analogs
wherein one or more amino acid residues is added to a terminal or
medial portion of the polypeptide) and which share some or all of
the properties of naturally-occurring forms.
[0097] In an advantageous embodiment, INPROL is the product of
prokaryotic or eukaryotic host expression (e.g., by bacterial,
yeast, higher plant, insect and mammalian cells in culture) of
exogenous DNA sequences obtained by genomic or cDNA cloning or by
gene synthesis. That is, in an advantageous embodiment, INPROL is
"recombinant INPROL". The product of expression in typical yeast
(e.g., Saccharomyces cerevisiae) or prokaryote (e.g., E. coli) host
cells are free of association with any mammalian proteins. The
products of expression in vertebrate (e.g., non-human mammalian
(e.g., COS or CHO) and avian) cells are free of association with
any human proteins. Depending upon the host employed, polypeptides
of the invention may be glycosylated or may be non-glycosylated.
Polypeptides of the invention optionally also include an initial
methionine amino acid residue (at position-1).
[0098] The present invention also embraces other products such as
polypeptide analogs of the alpha or beta chain of hemoglobin. Such
analogs include fragments of the alpha or beta chain of hemoglobin.
Following well known procedures, one can readily design and
manufacture genes coding for microbial expression of polypeptides
having primary conformations which differ from that herein
specified for in terms of the identity or location of one or more
residues (e.g., substitutions, terminal and intermediate additions
and deletions). Alternatively, modifications of cDNA and genomic
genes can be readily accomplished by well-known site-directed
mutagenesis techniques and employed to generate analogs and
derivatives of the alpha and beta chain of hemoglobin. Such
products share at least one of the biological properties of INPROL
but may differ in others. As examples, products of the invention
include the alpha or beta chain which is foreshortened by e.g.,
deletions; or those which are more stable to hydrolysis (and,
therefore, may have more pronounced or longer-lasting effects than
naturally-occurring); or which have been altered to delete or to
add one or more potential sites for O-glycosylation and/or
N-glycosylation or which have one or more cysteine residues deleted
or replaced by, e.g., alanine or serine residues and are more
easily isolated in active form from microbial systems; or which
have one or more tyrosine residues replaced by phenylalanine and
bind more or less readily to target proteins or to receptors on
target cells. Also comprehended are polypeptide fragments
duplicating only a part of the continuous amino acid sequence or
secondary conformations within the alpha or beta chain which
fragments may possess one property of INPROL (e.g., receptor
binding) and not others (e.g., stem cell inhibitory activity). It
is noteworthy that activity is not necessary for any one or more of
the products of the invention to have therapeutic utility (see,
Weiland et al., Blut 44:173-5, 1982) or utility in other contexts,
such as in assays of inhibitory factor antagonism. Competitive
antagonists are useful in cases of overproduction of stem cell
inhibitors or its receptor.
[0099] In addition, peptides derived from the protein sequence
which retain biological activity may be chemically synthesized
using standard methods.
[0100] Homologous or analogous versions of INPROL from other
species are employed in various veterinary uses, similar to the
therapeutic embodiments of the invention described above.
[0101] Further, INPROL acts on cycling stem cells by reversibly
placing them in an undividing "resting" state. When it is desirable
to stimulate the quiescent stem cells into division, e.g., after
treatment of a patient with cancer chemotherapy agents or
radiation, colony-stimulating factors and other hematopoietic
stimulants are administered to the subject. Examples of such
factors include but are not limited to: M-CSF, CSF-1, GM-CSF,
G-CSF, Megakaryocyte-CSF or other cytokines, such as IL-1, IL-2,
IL-3, IL-4, IL-5, IL-6, IL-7, IL9, IL-11, IL-12, IL-13, IL-14, or
erythropoietin.
[0102] INPROL polypeptides or active fragments having stem cell
inhibitory activity are purified or synthesized by conventional
chemical processes combined with appropriate bioassays for stem
cell inhibitory activity, as exemplified in the protocols described
below.
[0103] In one embodiment of the invention, a therapeutically
effective amount of the INPROL protein or a therapeutically
effective fragment thereof is employed in admixture with a
pharmaceutically acceptable carrier. This INPROL composition is
generally administered by parenteral injection or infusion.
Subcutaneous, intravenous, or intramuscular injection routes are
selected according to therapeutic effect achieved
[0104] When systemically administered, the therapeutic composition
for use in this invention is in the form of a pyrogen-free,
parenterally acceptable aqueous solution. Pharmaceutically
acceptable sterile protein solution, having due regard to pH,
isotonicity, stability, carrier proteins and the like, is within
the skill of the art.
[0105] Also comprehended by the invention are pharmaceutical
compositions comprising therapeutically effective amounts of
polypeptide products of the invention together with suitable
diluents, preservatives, solubilizers, emulsifiers, adjuvants
and/or carriers useful in INPROL therapy. A "therapeutically
effective amount" as used herein refers to that amount which
provides a therapeutic effect for a given condition and
administration regimen. Such compositions are liquids, gels,
ointments, or lyophilized or otherwise dried formulations and
include diluents of various buffer content (e.g., Tris-HCl,
acetate, phosphate), pH and ionic strength, additives such as
albumin or gelatin to prevent adsorption to surfaces, detergents
(e.g., Tween 20, Tween 80, Pluronic F68, bile acid salts),
solubilizing agents (e.g., glycerol, polyethylene glycol),
anti-oxidants (e.g., ascorbic acid, sodium metabisulfite),
preservatives (e.g., Thimerosal, benzyl alcohol, parabens), bulking
substances or tonicity modifiers (e.g., lactose, mannitol),
covalent attachment of polymers such as polyethylene glycol to the
protein, complexation with metal ions, or incorporation of the
material into or onto particulate preparations of polymeric
compounds such as polylactic acid, polyglycolic acid, hydrogels,
etc. or into liposomes, niosomes, microemulsions, micelles,
unilamellar or multilamellar vesicles, biodegradable injectable
microcapsules or microspheres, or protein matrices, erythrocyte
ghosts, spheroplasts, skin patches, or other known methods of
releasing or packaging pharmaceuticals. Such compositions will
influence the physical state, solubility, stability, rate of in
vivo release, and rate of in vivo clearance of INPROL. Controlled
or sustained release compositions include formulation in lipophilic
depots (e.g., fatty acids, waxes, oils). Also comprehended by the
invention are particulate compositions coated with polymers (e.g.,
poloxamers or poloxamines) and INPROL coupled to antibodies
directed against tissue-specific receptors, ligands or antigens or
coupled to ligands of tissue-specific receptors. Other embodiments
of the compositions of the invention incorporate particulate forms
of protective coatings, protease inhibitory factors or permeation
enhancers for various routes of administration, including
parenteral, pulmonary, nasal, topical (skin or mucosal) and oral.
In another embodiment, the composition containing INPROL is
administered topically or through a transdermal patch.
[0106] In one embodiment, the compositions of the subject invention
are packaged in sterile vials or ampoules in dosage unit form.
[0107] The invention also comprises compositions including one or
more additional factors such as chemotherapeutic agents (e.g., 5FU,
cytosine arabinoside, cyclophosphamide, cisplatin, carboplatin,
doxyrubicin, etoposide, taxol, alkylating agents), antiviral agents
(e.g., AZT, acyclovir), TNF, cytokines (e.g., interleukins),
antiproliferative drugs, antimetabolites, and drugs which interfere
with DNA metabolism
[0108] The dosage regimen involved in a method for treating the
subject anticipating exposure to such cytotoxic agents or for
treatment of hyperproliferating stem cells is determined by the
attending physician considering various factors which modify the
action of drugs; e.g., the condition, body weight, sex, and diet of
the patient, the severity of any infection, time of administration
and other clinical factors.
[0109] Following the subject's exposure to the cytotoxic agent or
radiation, the therapeutic method of the present invention
optionally employs administering to the subject one or more
lymphokines, colony stimulating factors or other cytokines,
hematopoietins, interleukins, or growth factors to generally
stimulate the growth and division of the stem cells (and their
descendants) inhibited by the prior treatment with INPROL. Such
therapeutic agents which encourage hematopoiesis include IL-1,
IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, Meg-CSF, M-CSF, CSF-1, GM-CSF,
G-CSF or erythropoietin. The dosages of these agents are selected
according to knowledge obtained in their use in clinical trials for
efficacy in promoting hematopoietic reconstitution after
chemotherapy or bone marrow transplant. These dosages would be
adjusted to compensate for variations in the physical condition of
the patient, and the amount and type of chemotherapeutic agent or
radiation to which the subject was exposed Progress of the reversal
of the inhibition of the stem cells caused by administration of
INPROL in the treated patient is monitored by conventional
methods.
[0110] In the treatment of leukemia, it is beneficial to administer
both INPROL to inhibit normal stem cell cycling and a stimulator of
leukemic cell growth, such as IL-3 or GM-CSF, simultaneously with
the cytotoxic drug treatment or during irradiation. By this
protocol, it is possible to achieve the greatest differences
between the cycling statuses and drug sensitivities of normal and
leukemic cells.
EXAMPLE 1
In Vivo Stem Cell Proliferation Inhibition Assay
[0111] For the detection of stem cells proliferation the number of
CFU-S in S-phase of cell cycle was measured by .sup.3H-Thymidine
`suicide" method (Becker et al., Blood 26:296-308, 1965).
[0112] 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).
[0113] For the standard CFU-S proliferation assay the method of
.sup.3H-Thymidine "suicide" is usually applied (Becker et al.,
1965). The method is based on incorporation of radiolabelled
Thymidine, (.sup.3H-Thymidine) a precursor of DNA into cells during
DNA synthesis. The CFU-S which are in S-phase of 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 of the cell sample
incubated without .sup.3H-Thymidine and the same cells incubated
with .sup.3H-Thymidine shows the percentage of the proliferating
CFU-S in the original sample.
[0114] The inhibitor testing can not be done with the bone marrow
stem cell population from unstimulated animals, as far as the
inhibitor only effects cycling CFU-S, which are as low as 7-10% of
the total CFU-S population in the bone marrow of normal mice.
[0115] To stimulate CFU-S proliferation, phenylhydrazine (PHZ), or
sublethal irradiation were used (Lord, 1976).
[0116] We have developed the method of using
testosterone-propionate (TSP) based on its stimulatory effect on
CFU-S cycling (Byron et al. Nature 228:1204, 1970) which simplified
the testing and did not cause any side effects. The TSP induced
stimulation of CFU-S proliferation within 20-24 hours after
injection and the effect could be seen for at least 7 days.
[0117] The procedure used for the screening of the fractions during
purification of the Inhibitor was as follows:
[0118] Mice: BDF.sub.1 or CBF.sub.1 mice strains were used
throughout all testing.
[0119] Donor mice were treated with a 10 mg/100 g dose of TSP by
intraperitoneal injection of 0.2 ml/mouse in order to induce 30-35%
of the CFU-S into S-phase.
[0120] Twenty-four hours later the bone marrow is to taken from the
femurs for the cell suspension preparation. Five to ten million
cells per ml are then incubated with different control and test
fractions for 3.5 hours at 37.degree. C. in water bath, with two
tubes for each group (one for hot (radioactive) and one for cold
(non-radioactive)).
[0121] After 3.5 hours, .sup.3H-Thymidine (1 mCi/ml, specific
activity 18-25 Ci/mmole) is added to each hot tube in a volume of
200 .mu.l per 1 ml of cell suspension; nothing is added to the cold
tubes. Incubation is continued for 30 more minutes at 37.degree.
C.
[0122] After the 30 minute incubation, the kill reaction is
terminated by adding 10 ml of cold (4.degree. C.) medium containing
400 .mu.g/ml nonradioactive Thymidine. Cells are washed extensively
(3 times).
[0123] Cells are resuspended and diluted to a desirable
concentration for the injections, usually 2-4.times.10.sup.4 cells
per mouse in 0.3-0.5 ml.
[0124] Recipient mice, 8-10 per group, are irradiated not later
than 6 hours before the injections.
[0125] Recipient spleens are harvested on day 9-12 and fixed in
Tellesnitsky's solution; the colonies are scored by eye score. The
percentage of cells in S-phase are calculated using the formula. 1
% S = a - b a 100 %
[0126] where a--CFU-S number with .sup.3H-Thymidine
[0127] where b--CFU-S number with .sup.3H-Thymidine
[0128] The test data of INPROL presented in Table 4 demonstrate
that cycling stem cells after treatment with INPROL become
resistant to the action of .sup.3H-Thymidine. The same is true for
the S-phase specific cytotoxic drug cytosine arabinoside and
hydroxyurea (data not shown). If the treated stem cells are then
washed with the cold media containing non-radioactive Thymidine,
the surviving stem cells proliferate in mouse spleens to form
colonies normally.
1TABLE 4 Inhibitory Activity Of INPROL On CFU-S Proliferation
During Four Hour Incubation With Bone Marrow Cells Percent CFU-S
Group -.sup.3H-TdR +.sup.3H-TdR Killed by .sup.3H-TdR No incubation
22.2 .+-. 2.0* 13.7 .+-. 2.4* 38.3 .+-. 1.7 4 Hour with 18.7 .+-.
3.0* 11.4 .+-. 1.3* 43.1 .+-. 1.4 Media 4 Hour with 21.2 .+-. 2.3*
20.7 .+-. 2.6* 2.1 .+-. 0.08 INPROL *CFU-S per 2 .times. 10.sup.4
cells
EXAMPLE 2
In Vitro Stem Cell Proliferation Inhibition Assay
[0129] 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.
[0130] A tritiated Thymidine incorporation assay was used to
measure proliferation: A4 cells (5.times.10.sup.4 in 100 .mu.l
medium with 20% horse serum and 50% of WEHI-3 CM) were incubated at
37.degree. C. in 5% CO.sub.2 for 16 hours.
[0131] INPROL or the crude BME (fraction IV) were added at the
start. Tritiated thymidine ((.sup.3H-Tdr) 3.7KBq in 50 .mu.l 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. 2 % Inhibition = cpm without INPROL - cpm with INPROL cpm
without INPROL .times. 100 %
[0132] Incorporation of tritiated thymidine (.sup.3H-Tdr) by
FDCPmix-A4 cells grown in the presence of graded doses of normal
bone marrow extract or INPROL is depicted on FIG. 6. It can be seen
that purified composition of INPROL is at least 1,000 times more
active than the starting material. Time of exposure (16 hours) is
an important factor for effective inhibition and shows the evidence
of the direct effect of INPROL on stem cells of A4 cell line.
EXAMPLE 3
Inhibition of CFU-S Proliferation by INPROL Injected in vivo: Doses
and the Duration of the Effect
[0133] 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.
[0134] The experimental protocol had two goals: to check 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.
[0135] To stimulate CFLT-S proliferation, the injection of
testosterone-propionate was used based on the effect mentioned
above in Example 1.
[0136] 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 SCPI injection at doses of 0 .mu.g, 5 .mu.g, 10
.mu.g, and 15 .mu.g/mouse i.p.
[0137] Twenty-four hours after SCPI injection, mice were sacrificed
and the percent of cycling CFU-S was measured by the assay
described in Example 1. TSP injection induced about 50% CFU-S into
cycling in comparison with 7% in untreated mice. INPROL in doses as
low as 2 .mu.g/mouse was able to inhibit TSP induced proliferation
down to the normal level.
[0138] For the duration of the effect evaluation, one group of mice
(21 mice per group) was injected with TSP only and another group
was injected both with TSP and INPROL (24 hours after TSP). The
CFU-S cycling was measured every 24 hours during a week by taking 3
donors from each group and measuring CFU-S cycle status in their
bone marrow by method described (see Example 1). Data presented in
FIG. 7 show that while the duration of the effect of TSP is at
least 7 days, a single injection of INPROL can place CFU-S into
quiescence and keep them out of cycle for no more than 48-72 hours.
Since the majority of chemotherapeutic agents used for cancer and
leukemia chemotherapy have a relatively short in vivo half-life,
usually less than 24 hours, the INPROL effect according to the data
obtained is maintained for longer than the effective time during
which the chemotherapeutic agents like cytosine arabinoside or
hydroxyurea are active in vivo. More importantly, for
chemotherapeutic and radiation treatments having longer intervals
(more than 24 hours and less than 96 hours) between the first
(non-damaging for the stem cells) and the second (damaging to the
CFU-S) treatments, a single injection of INPROL during the
intervals between the two applications of chemotherapeutic agent or
radiation should be sufficient. For several repeatable cycles of
cytotoxic therapy or radiation the same strategy could be applied
based on the duration of the INPROL effect.
EXAMPLE 4
Most Primitive Hematopoietic Stem Cells Stimulated to Cycle Rapidly
After Treatment With 5-FU are Protected by INPROL From the Second
5-FU Exposure
[0139] The drug 5-fluorouracil (5-FU) drastically reduces the
number of cells in the myeloid and lymphoid compartments. It is
usually thought of as being cell-cycle specific, targeting rapidly
proliferating cells, because incorporation of the nucleotide
analogue into DNA during S-phase of the cell cycle or before
results in cell death. The long-term survival and
immunohematopoietic reconstitution of the bone marrow of mice is
not affected by a single dose of 5-FU; however, it was demonstrated
(Harrison et al. Blood 78:1237-1240, 1991) that pluripotent
hematopoietic stem cells (PHSC) become vulnerable to a second dose
of 5-FU for a brief period about 3-5 days after the initial dose.
It can be explained that PHSC normally cycle too slowly for a
single dose of 5-FU to be effective and are stimulated into rapid
cycling by stimuli resulting from the initial 5-FU treatment. We
have proposed that PHSC can be returned to a slow cycle status by
INPROL and thus protected from the second 5-FU treatment.
[0140] The mice used in these experiments were BDFI male mice. A
stock solution of 5-FU (Sigma) was prepared in physiologic saline
at a concentration of 10 .mu.g/ml. Each treated mouse received 2 mg
of 5-FU per 10 g body weight via a tail vein at Day 0 of the
experiment; 24 hours later mice were injected with INPROL (10
.mu.g/100 g of body weight) intraperitoneally and on Day 3 were
injected with the second dose of 5-FU. The survival study was
conducted by monitoring the death of mice in experimental
(treatment with INPROL) and control groups of 30 mice each. The
survival curves are shown in FIG. 8.
EXAMPLE 5
Effects of Pre-incubation With INPROL vs. MIP 1 Alpha in Bone
Marrow Cells
[0141] The purpose of this experiment was to compare the inhibitory
effects of pre-incubation with INPROL and MIP-1-alpha on mouse bone
marrow cells in vitro.
[0142] The following procedure was used:
[0143] in vivo: BDF1 mice, 6-15 weeks of age, are injected with 200
mg/kg 5FU i.p. 48 hours before harvesting marrow from the
femurs.
[0144] in vitro: A single cell pooled suspension is counted and
5.times.10.sup.6 cells are incubated in a total of 2 mls with or
without INPROL or MIP-1 alpha, 5% horse serum. 1MDM media with
added L-glutamine, at 37.degree. C. and 5% CO.sub.2 for 4 hours.
The cells are then washed twice and recounted. They are plated in
methylcellulose in the following final conditions:
[0145] 0.8% methylcellulose
[0146] 25% horse serum
[0147] 20 ng/ml recombinant murine IL3
[0148] L-glutamine added
[0149] 5.times.10.sup.5 cells per ml
[0150] IMDM media
[0151] Plates were incubated for 11 days at 37.degree. C. and 5%
CO.sub.2 in 100% humidity. Colonies more than 50 cells were
counted.
2 Groups Colony Number Percent of Control Control 31.0 100% INPROL
21.25 68.5% MIP 1 alpha 35.25 114%
EXAMPLE 6
INPROL inhibits HPP-CFC proliferation
[0152] An in vitro assay for assessing murine reconstituting stem
cells and early precursors is the high proliferative potential
colony (HPP-PFC) assay; other related assays, e.g., CFU-A. CFU-GM,
CFU-E, and CFU-GEMM, detect progressively restricted progenitor
populations (M. Moore, Blood 177:2122-2128, 1991). This example
shows that pretreatment of cells with INPROL inhibits their
proliferation, whereas MIP-1.alpha. fails to do so under these
experimental conditions.
[0153] BDF1 mice were treated with 5-fluorouracil (200 mg/kg i.p.)
before their bone marrow was assayed for HPP-CFC numbers. Cells
were washed by centrifugation and incubated at densities of
10.sup.6 to 5.times.10.sup.6/ml in medium with either no added
agent (Controls), INPROL (25 ng/ml) or MIP-1.alpha. (200 ng/ml) for
4 hours. After incubation, cells were washed and plated in agar
(0.3%) with 30% FCS and combined conditioned medium from 5637 and
WEHI-3B cell lines (7.5% of each conditioned medium, as recommended
by Sharp et al., 1991). Plating concentration was 5.times.10.sup.4
cells/ml in 60 mm dishes. Colonies were scored on day 14 and the
results are indicated below.
3 Group HPP-CFU % of Control Control 15.5 .+-. 1.2 100% INPROL 8.3
.+-. 0.7 53.5% MIP-1 15.8 .+-. 0.9 101%
[0154] According to these results, MIP-1.alpha. did not inhibit
proliferation of the most immature precursors when present only
during the pre-incubation period. INPROL did effectively inhibit
proliferation under these conditions, indicating fundamental
differences between INPROL and MIP-1.alpha. in terms of biological
activity.
EXAMPLE 7
INPROL Therapy Effect on the Recovery from Radiation-induced Bone
Marrow Aplasia
[0155] Bone marrow aplasia is the primary limiting toxicity of
radiation cancer therapy. It has been demonstrated that some growth
factors (e.g., G-CSF, GM-CSF, erythropoietin) can accelerate
recovery from radiation-induced bone marrow aplasia. The concept of
protection by using an inhibitor of stem cell proliferation is a
different and complementary approach in coping with hematological
damage. To follow the treatment procedure developed earlier
(Examples 3, 4) a model of lethal irradiation of mice was
established. It is known in the art that mice receiving 9Gy of
cobalt 60 start dying after 10-14 days; by Day 30, mortality
approximates 50%. This lethal dose was used in our model by
splitting it into two subsequent applications of 4.5Gy each with an
interval 3 days between doses. Preliminary data showed that the
survival curve in that model was very close to that known for a
single irradiation with 9Gy; moreover the test for the CFU-S
proliferation showed that 24 hours after the first irradiation,
35-50% of CFU-S are induced to proliferate. Such cells may be
protected by a stem cell inhibitor delivered prior to the second
dose.
[0156] To examine this possibility, mice (50 mice/group) received
4.5Gy on Day 0. Twenty four hours later, one group received INPROL
(2 .mu.g/mouse i.p.) and another, control group was injected with
saline. The second dose of radiation (4.5 Gy) was given on Day
3.
[0157] FIG. 9 shows the increased survival after a single dose of
INPROL. The conditions of the model are clinically relevant for
treating any cancer, including those characterized by solid tumors;
such treatment would be administered to a patient having cancer by
delivering an effective dose of INPROL between two consecutive
dosages of radiation, thereby allowing greater dosages of radiation
to be employed for treatment of the cancer. It should also be
possible to extend this modality to chemotherapeutic agents.
EXAMPLE 8
INPROL Use for the Autologous Bone Marrow Transplantation
[0158] Bone marrow transplantation is the only known curative
therapy for several leukemias (CML, AML, and others). Ex vivo
conditioning of autologous BMT for infusion should provide
potential autologous sources of normal stem cells free of leukemic
contamination and able to repopulate the recipient's hematopoietic
system to allow aggressive and effective therapy.
[0159] 1. Long-term Bone Marrow Culture L1210 Leukemia Model for
the Study of INPROL Effect Preserving Normal Hematopoiesis During
Purging With AraC.
[0160] Long-Term Bone Marrow Cultures (LTBMC) were established
according to Toksoz et al. (Blood 55:931-936, 1980) and the
leukemic cell line L1210 was adopted to the LTBMC by co-cultivation
during 2 weeks. The simultaneous growth of normal and leukemic
progenitors occurred in these combined LTBMC/L1210 cultures,
similar to the situation in the bone marrow of a leukemic patient.
Discrimination between normal colony forming units CFU and leukemic
CFU was possible by growing them as agar colonies in the presence
or absence of the conditioned medium from WEHI-3 (a murine IL-3
producing cell line). Normal cells undergo apoptosis in the absence
of IL-3 whereas leukemic cells can form colonies in its absence.
Suspension cells from LTBMC-L1210 composition give approximately
150 colonies in presence of IL-3 (normal hematopoietic clones) and
70 colonies when growing without IL-3 (leukemic clones) per 50,000
cells plated.
[0161] The procedure of purging was as follows: on Day 0 all
suspension cells and media (10 ml/flask) were taken off the flasks
with LTBMC-L1210 and replace with 2 ml of media containing 200
.mu.g cytosine arabinoside (AraC) (Tsyrlova et al. in Leukemia:
Advances in Biology and Therapy v. 35, 1988); after 20 hours of
incubation, flasks were washed out and replaced with 2 ml of fresh
media alone (control group) or media containing INPROL at 25 ng/ml
for 4 hours. After this preincubation, cells were incubated again
with 100 .mu.g/flask AraC for 3 hours at 37.degree. C. Each group
contained 4 flasks. LTBMC-L1210 cultures were washed 3 times and
replaced with fresh LTBC media; they were maintained as before for
the regeneration studies for 34 weeks.
[0162] Data are presented in FIG. 10. There was no cell growth seen
in control cultures treated with AraC only, while in INPROL
protected flasks regeneration of hematopoiesis occurred much more
rapidly due to proliferation of progenitors from the adherent
layer. Moreover, the cells from the experimental group when plated
in agar grew only in the presence of IL-3 giving about 100 CFU per
50,000 cells; no leukemic cell growth was observed at least during
4 weeks. Thus, marrow treated ex vivo with an effective dose of
AraC in combination with INPROL can be purged of cancerous cells
while the stem cells are be protected. It should be possible to
extend this modality to other forms of chemotherapy or radiation
treatments.
[0163] 2. Marrow Repopulating Ability (MRA) and Thirty Days
Radioprotection are Increased by INPROL Treatment In Vitro.
[0164] MRA. the ability of cells to repopulate the bone marrow of
lethally irradiated mice, together with the ability to confer
radioprotection for 30 days, is a direct in vivo measurement of the
potential to rescue myelosuppressed animals (Visser et al. Blood
Cells 14:369-384, 1988).
[0165] For radioprotection studies BDF1 mice were irradiated with
9.5Gy and restored by transplantation of bone marrow from
testosterone-stimulated donors. One group of recipients was
restored by bone marrow cells preincubated for 4 hours with medium
(controls--group A) and another (group B) with 25 ng/ml INPROL.
Cells in both groups were washed and 30,000 cells per mouse were
transplanted into irradiated animals. The survival data are shown
(FIG. 11). The sum of 3 experiments is depicted, with controls
normalized to 100%. INPROL incubation increased the survival of
mice from 36.5% in control group up to 61.8% by Day 30.
[0166] The increase of MRA induced by preincubation with INPROL
could be one of the mechanisms in the improving of the
radioprotection. To examine this hypothesis, MRA was measured
according to Visser et al. (op. cit.). Briefly, the donor BDF1 mice
were pretreated with testosterone, their bone marrow was
preincubated with medium or INPROL for 4 hours and injected into
irradiated animals. On Day 13, the bone marrow cells from recipient
femurs were plated in agar in 3 different concentration (0.01,
0.05, 0.1 equivalent of a femur) in the presence of 20% of horse
serum and 10% of WEHI-CM. The number of Day 7 colonies represented
the MRA as far as the colony-forming cells in the bone marrow of
recipients at the time were the progenitors of the donor's immature
stem cells.
[0167] As can be seen on FIG. 12 MRA of preincubated with INPROL
cell population is greater than in control group (B).
EXAMPLE 9
Antihyperproliferative Effect of INPROL on Stem Cells can Chance
Their Differentiation Abnormalities.
[0168] Hyperproliferation of CFU-S is not only seen during
restoration from cytotoxic drugs or irradiation but also as a
consequence of normal aging, and is thought to be a major feature
in Myelodysplastic Syndrome (MDS). It is accompanied by the
differentiation disturbances such as a prevalence of the erythroid
differentiation while the differentiation along the granulocytic
pathway is reduced.
[0169] Bone marrow cells were incubated for 4 hours at 37.degree.
C. with 25 ng/ml of INROL or media (Control), washed and then
plated in agar with 20% of horse serum. 2U/ml Erythropoietin, and
10% WEHI-CM. The number of BFU-E and GM-CFU colonies were scored on
Day 7. Data presented in Table 5 are summarized from 3
experiments-4 animals per point were taken for each group; 4 dishes
were plated.
[0170] As is obvious from Table 5, the incubation of normal bone
marrow (NBM) from intact young animals (BDF.sub.1 8-12 weeks old)
with INPROL did not change the number or proportion of different
types of colonies. BDF.sub.1 donors pretreated with Testosterone
Propionate (TSP) showed the same increase in CFU-S proliferation as
was seen before (Example 1, 3, 4) a slight increase in the
erythroid progenitor number (BFU-E colonies) and a decrease in
GM-CFU, which were completely abrogated by the incubation with
INPROL. In addition, the abnormally high level of CFU-S
proliferation was returned to 10% of CFU-S in S-phase of cell
cycle. CFU-S hyperproliferation is known to be a feature of some
mouse strains susceptible to viral leukemia induction, for example
Balb/c mice (Table 5), and can also be observed in older animals
(Table 5). The same redistribution of committed progenitors seen in
TSP treated BDFI mice is observed in Balb/c and in older (23-25
month old) BDF1, which have in common the abnormally high level of
CFU-S proliferation. The correction of both the proliferation of
CFU-S and the differentiation was induced by the incubation with
INPROL. What is even more clinically relevant, the study showed
that the in vivo injection of INPROL (2 .mu.g/mouse) affected both
proliferation of CFU-S and the ratio of erythroid (BFU-E) and
GM-colonies (Table 5).
4TABLE 5 INPROL Effect On CFU-S Differentiation Into Committed
Progenitors BFU-E and CFU-GM Percent Donors CFU-S Of Bone Killed by
Marrow INPROL .sup.3HTdR BFU-E CFU-GM BDF.sub.1 - 12.0 .+-. 0.3
28.33 .+-. 1.91 46.22 .+-. 3.44 Young + 15.0 .+-. 1.3 22.00 .+-.
3.74 47.70 .+-. 3.72 BDF.sub.1 - 47.1 .+-. 1.9 43.75 .+-. 1.54 24.0
.+-. 1.33 Old + 11.4 .+-. 0.7 15.25 .+-. 1.45 44.0 .+-. 7.63
BDF.sub.1 - 53.2 .+-. 1.6 32.67 .+-. 2.44 15.71 .+-. 2.28
Stimulated + 7.2 .+-. 0.4 12.00 .+-. 1.83 35.50 .+-. 1.4 by TSP
Balb/C - 57.0 .+-. 1.9 47.60 .+-. 2.96 33.57 .+-. 3.45 + 23.0 .+-.
2.4 24.86 .+-. 2.53 70.60 .+-. 4.96
Example 10
Immunostimulatory Activity of INPROL
[0171] It has been observed that the incubation of the bone marrow
cells containing a high proportion of proliferating CFU-S with
INPROL not only changes the cycling of CFU-S, but also their
differentiation, switching the predominantly erythroid
differentiation in favor of granulocytic and lymphoid progenitors.
This property of INPROL is of importance due to the
immunosuppression side effects of cytotoxic chemotherapy or
radiotherapy, as well as the immunosuppression accompanying
hyperproliferative stem cell disorders and aging.
[0172] The example shows the direct effect of INPROL on the
differentiation of immature precursors from the Lymphoid Long Term
Culture (LLTC) established according to Wittlock & Witte (Ann.
Rev. Immun. 3:213-35, 1985) into pre-B progenitors, measured by the
formation of colonies in methylcellulose containing IL-7.
[0173] LLTC were established as described and fed with fresh
LLTC-media (Terry Fox Labs., Vancouver, Canada) twice a week.
Nonadherent cells were harvested once a week, washed free of
factors and incubated for 4 hours with 25 ng/ml INPROL or medium
alone for control. After the incubation, the cells were washed and
plated at a concentration of 10.sup.5 cells/ml in methylcellulose,
containing 30% FCS, and 10 ng/ml of IL-7. Data from 3 weeks are
shown in FIG. 13. The number of large pre-B colonies varied in
control, increasing with time, but preincubation with INPROL always
stimulated the growth of colonies 4 to 8 fold above the control
level. This demonstrates an immunostimulatory property of INPROL
which is of use in correcting immunodeficient states and in
increasing desired immune responses, e.g., to vaccination.
EXAMPLE 11
INPROL Improves Repopulating Ability of Stem Cells--Long Term
Culture Initiating Cells from Patient With CML
[0174] Chronic myeloid leukemia (CML) is a lethal malignant
disorder of the hematopoietic stem cell. Treatment of CML in the
chronic phase with single-agent chemotherapy, combination
chemotherapy, splenectomy, or splenic irradiation may control
clinical signs and symptoms, but does not significantly prolong
survival. As CML progresses from the chronic to the accelerated
stage, standard therapy is not effective. At present, bone marrow
transplantation (BMT) is the only known curative therapy for CML.
Therapy with unrelated donor BMT is difficult due to
histoincompatibility problems. Fewer than 40% of otherwise eligible
CML patients will have a suitably matched related donor; therefore
autologous transplantation is preferred. Ex vivo conditioning of
autologous BMT for infusion together with the ability to select
non-leukemic (Ph-negative) myeloid progenitors from Ph-positive
patients growing in Long Term Culture (LTC) suggest the potential
of autologous sources of normal stem cells to allow aggressive and
effective therapy of CML.
[0175] In the context of BMT, a hematopoietic stem cell may be
defined as one having the ability to generate mature blood cells
for extensive periods. We have used the human LTC system developed
by C. Eaves & A. Eaves both for quantitating stem cell numbers
and as a means to manipulate them for therapeutic use. This
involves seeding cells onto a pre-established, irradiated human
marrow adherent layer; these cultures are then maintained for 5
weeks. The end point is the total clonogenic cell content
(adherent+non-adherent) of the cultures harvested at the end of
this time. Clonogenic cell output under these conditions is
linearly related to the number of progenitors (Long Term Culture
Initiating Cells (LTC-IC)) initially added; the average output from
individual human LTC-IC is 4 clonogenic progenitors per LTC-IC. It
has been shown previously that when marrow from patients with CML
is placed under similar conditions, leukemic (Ph-positive)
clonogenic cells rapidly decline. By using quantitation of residual
normal LTC-IC, in patients with CML it is possible to select those
likely to benefit from intensive therapy supported by
transplantation of cultured autografts (Phillips et al., Bone
Marrow Transplantation 8:477-487. 1991).
[0176] The following procedure was used to examine the effect of
INPROL on the number of clonogenic cells (LTC-IC) among bone marrow
transplant cells established from the peripheral blood of a patient
with CML.
[0177] Cultures were initiated as long term cultures on
pre-irradiated stroma. The peripheral blood of a healthy donor was
used as the control. Peripheral blood cells from a CML patient were
preincubated with or without INPROL (25 ng/ml) for 4 hours, washed
and placed in the LTC-IC system for 5 weeks to determine the
control number of LTC-IC. For experiments, other, parallel cultures
were established for 10 days. The mixture of adherent and
non-adherent cells from cultures growing for 10 days was
preincubated with or without INPROL and placed on pre-established
feeders for an additional 8 weeks. The number of LTC-IC from each
experimental culture was estimated by plating both the adherent and
non-adherent cells in methylcellulose with the appropriate growth
factors (Terry Fox Laboratories, Vancouver, Canada) and counting
the resulting total number of colony forming cells. The LTC-IC
values obtained using this procedure were derived from assessment
of the total clonogenic cells (CFC) content using the formula:
# LTC-IC=#CFC/4
[0178] Data presented on FIG. 14 show that there was no loss in
LTC-IC during the first 10 days of culture initiated from the
healthy donor's bone marrow and approximately 30% of the number of
input LTC-IC were still present after 5 weeks in culture. The
number of the CML patient's LTC-IC was drastically reduced to about
8% during the 10 day period and did not recover during further
incubation, while the preincubation of cells with NPROL increased
the LTC-IC level to 30% of initial number and it was maintained
during 8 weeks.
[0179] Clinically relevant applications of INPROL predicted by
these preliminary data include their use in strategies for
selectively improving the normal stem cell content of fresh or
cultured marrow transplants, strategies for enhancing the
recruitment of residual normal stem cells in vivo also protocols
for transferring new genetic material into human marrow stem cells
for the further transplantation into patients.
EXAMPLE 12
A Method of Isolation of Immunoactive Inhibitor of Proliferation of
Stem Cells From Bone Marrow Preparations
[0180] The bone marrow was isolated from pigs' ribs. The ribs from
the pigs' carcasses were separated and cleaned from the muscle
fibers and fat, cut into pieces and the bone marrow was extracted
by a hydropress manufactured by the Biophyzpribor. The bone marrow
cells are separated by centrifugation in a centrifuge K-70 at 2,000
rpm for 20 minutes. The extract supernatant is then successively
subjected to ultrafiltration through Amicon USA membranes XM-100,
PM30, PM-50. According to the analysis by electrophoresis, the main
component of the product is albumin (see FIG. 1).
[0181] Biochemical Purification
[0182] The bone marrow extract and protein components of the
fractions were analyzed at every step of purification by gel
electrophoresis in 10% polyacrylamide, containing 0.1% sodium
dodecyl sulfate. Up to 7% of sodium dodecyl sulfate and up to
0.5-1% of mercaptoethanol were added to the samples which were
incubated for 5 minutes at 70.degree. C. prior to loading on the
gel.
[0183] The electrophoresis was conducted at 20Y cm of the gel for
five hours. Then the gel was stained in 0.25% Coomassie CBBC250 in
a mixture of ethanol:water:acetic acid 5:5:1 for one hour at
20.degree. C. and washed in several changes of 7% acetic acid. The
activity of the product was evaluated by the method of inhibition
of proliferation of stem hematopoietic cells (CFU-S). The method is
detailed hereafter.
[0184] Stage 1. Purification by Precipitation With Ammonium
Sulfate.
[0185] The activity was purified by precipitation with ammonium
sulfate at 25% with saturation of 40 to 80% which was selected
based on the results in Table 1.
5TABLE 1 Saturation (%) 0-40 40-60 60-80 80-100 Activity (%)
37.2-35.4 37.2-1.8 37.2-12.8 37.2-26.1 =1.8% =35.4% =24.4%
=11.1%
[0186] The amount of the preparation used for testing after each
step of purification was determined in accordance with the level of
purification and equivalent to the dose of 2.times.10.sup.-2 mg of
the initial product. Activity was determined by the formula:
Change=% Sa-% Sb
[0187] where
[0188] a is % S in control
[0189] b is % S after incubation with the test fraction.
[0190] The fraction was desalted in order to lower the
concentration of ammonium sulfate 20 times before each testing of
activity and before each following purification step.
[0191] Stage 2. The impure inhibitor from Stage 1 is applied after
desalting and fractionated utilizing ion exchange chromatography,
here DEAE 23 cellulose, and then eluted with a gradient of sodium
acetate buffer (pH 6.0).
[0192] The active fractions of inhibitor elute between 3-5 mM.
[0193] The volume of the column was 1 ml and speed of elution was 4
ml/hour. The detection was conducted by the chromatograph
Millicrome at 230 and 280 nm. Fraction 1 (see FIG. 2) which
exhibited the highest activity was isolated and eluted in 5 mM
sodium acetate buffer (see Table 2).
6TABLE 2 Fractions 1 2 3 4 5 Activity 46.3 - 0 = 46.3 - 14.1 = 46.3
- 42.1 = 46.3 - 19.6 = 46.3 - 45.1 = 46.3% 32.2% 4.2% 26.7%
1.2%
[0194] The electrophoresis data indicates that the main protein
contaminant--albumin (see FIG. 3) is removed from this fraction
which leads to an additional fourfold purification.
[0195] Stage 3. The partially purified inhibitor from Stage 2 is
applied directly to a G-75 Sephadex column.
[0196] The volume of the column is 20 ml (20.times.1), the elution
rate is 2 ml/hour. The elution buffer is 50 mM NaCl, 10 mM
Tris-HCl, pi 7.5. Detection was conducted on a chromatograph
Millichrome at 230 and 280 nm. Fraction 5 which had the highest
activity was isolated.
7TABLE 3 Fractions 1 2 3 4 5 Activity 42.2 - 19.1 = 42.2 - 35.2 =
42.2 - 21.5 = 42.2 - 38.8 = 42.2 - 0 = 23.1% 7.0% 20.7% 3.4%
42.2%
[0197] Stage 4. Reverse-phase chromatography (Pharmacia FPLC
System) utilizing Pro-REC columns is performed on an Ultrasfera
matrix. Protein is eluted using 0.1% trifluoracetic acid in an
acetonitrile gradient.
[0198] The homogeneity of a product with MW 16-17kD is equal to 90%
as was shown in analyzing the acrylamide/sodium dodecyl sulfate gel
(see FIG. 6). The result is represented in FIG. 4. Activity is
determined on fraction 5. The final yield of the product is 5%. As
a result, the total amount of protein with Mw 16 kD after the
purification is 650 ng/ml of the initial product. During the
purification process the product was submitted to heat incubation
at 70.degree. C. for several minutes but no loss of biological
activity was detected.
EXAMPLE 12B
Alternative method for Isolating Larger Quantities of INPROL
[0199] Initial Isolation
[0200] Ribs from fresh pig carcasses are cleaned of muscle fibers
and fat, then cut to pieces and soaked in phosphate-buffered saline
in the ratio 1:1 (weight/volume). The obtained mixture is crushed
by hydraulic press to separate bone marrow from solid bone
material.
[0201] The suspension of bone marrow cells is collected and
filtered free of solid particles through four layers of the
cheese-cloth. The filtrate is incubated at 56.degree. C. for 40
minutes, then cooled in an ice-bath to 4.degree. C. The resulting
precipitate is removed by centrifugation at 10,000 g for 30 minutes
at 4.degree. C. and discarded.
[0202] The clarified supernatant is added dropwise during 30
minutes to 10 volumes of stirred ice-cold acetone containing 1% by
volume of concentrated hydrochloric acid. The resulting mixture is
kept at 4.degree. C. for 16 hours for complete formation of the
precipitate. Then the precipitate is pelleted by centrifugation at
20,000 g for 30 minutes at 4.degree. C. This pellet is washed with
cold acetone and dried.
[0203] HPLC Purification
[0204] The pellet is dissolved in HPLC eluent buffer A containing
5% acetonitrile (MeCN) and 0.1% triflouroacetic acid (TFA) to final
protein concentration 8-10 mg/ml. This solution (0.5-0.6 ml) is
loaded onto 250.times.4.6 mm HPLC column packed with Polisil
ODS-300 (10 mcm) and equilibrated with the same buffer A.
[0205] The elution is accomplished by gradient of buffer B (90%
MeCN, 0.1% TFA) in buffer A at the flow rate of 1 ml/min according
to the following program:
8 Time, min % of B 0 0 4 0 5 25 25 90
[0206] An addition step of 100% B for 5 minutes is used to wash the
column prior to re-equilibration equilibration. Then the column is
equilibrated again for returning it to the initial state, and the
next portion of the protein solution may be loaded. A typical
chromatogram is shown in FIG. 5.
[0207] During the separation the column effluent is monitored at
280 nm for the detection of protein peaks. Fractions containing the
protein material are collected, separated peaks are pooled and
rotary evaporated at 30.degree. to dryness. The obtained residues
are dissolved in distilled water and assayed by bioactivity test
and by the SDS-PAGE (14% gel, reducing conditions). The peak
containing the active material is eluted between 70 and 80% of the
buffer B and contains the main protein band of 16 kD and the traces
of faster moving proteins as assayed by SDS-PAGE.
[0208] An analysis of the material obtained by collecting only the
second major HPLC peak is shown in FIG. 15 (A, B, and C). Material
containing both peaks (e.g., FIG. 5) will be referred to herein as
INPROL Preparation 1 and those consisting of only the second peak
will be referred to as INPROL Preparation 2.500 ug of this active,
purified INPROL Preparation 2 was loaded onto a C4 reverse phase
column (Vydac) and eluted using a linear gradient of 595%
acetonitrile in 0.1% trifluroacetic acid. The material eluted as a
single peak at 53% acetonitrile (FIG. 15A). When 250 .mu.g of MIP-1
alpha (R&D Systems), however, was run under identical
conditions, it eluted at 43.9 acetonitrile (note that earlier peaks
prior to 14% acetonitrile are artifactual and due to air bubbles in
the detector). Thus, INPROL is substantially more hydrophobic than
MIP-1 alpha under these conditions. TGF-beta is known to elute at
lower concentrations than that observed for INPROL under these
conditions (Miyazono et al. J. Biol. Chem. 263:6407-15, 1988).
[0209] A gel of the eluted INPROL material is shown in FIG. 15C.
Lane 1 is the crude material, Lane 2 is molecular weight markers
and Lane 3 is the purified material. There are two major bands, one
at approximately 14 kD and one at approximately 35 kD. It is
believed that the 35 kD band is a multimeric form of the 14 kD
band.
Example 13A
Active INPROL Preparations Contain Hemoglobin Beta Chain
[0210] INPROL was prepared as shown in FIG. 5 (i.e., INPROL
Preparation 1 (cf. Example 12B)). The material was run on SDS-PAGE
and transferred to nitrocelluose using standard techniques. The
material was subjected to N-terminal sequence analysis using an ABI
477A protein sequencer with 120A Online PTH-AA analyzer using
standard techniques. The following N-terminal sequence was
obtained:
[0211] VHLSAEEKEAVLGLWGKVNVDEV . . . .
[0212] Computer search of the protein databases reveal that this
sequence has identity with the N-terminal sequence of the beta
chain of porcine hemoglobin (cf. FIG. 16C).
EXAMPLE 13B
Active INPROL Preparations Contain Hemoglobin Alpha Chain
[0213] As shown in FIG. 15C, protein purified by collecting the
second major peak shown in FIG. 5 (i.e., INPROL Preparation 2)
resulted in two major bands corresponding to molecular weights of
approximately 15 K and 30 K, as well as several minor bands.
SDS-PAGE gels were transferred to nitrocellulose using standard
techniques and individual bands were excised and subjected to
N-terminal sequence analysis as in Example 13A. The following
N-terminal sequence was obtained for the 15 kD band:
[0214] VLSAADKANVKAAWGKVGGQ . . . .
[0215] The 30 kD band was subjected to limited proteolytic digest
and the following internal sequence was obtained: * *
FPHFNLSHGSDQVK . . . .
[0216] The first sequence shows identity with the N-terminal
sequence of porcine hemoglobin alpha chain whereas the second
sequence shows identity with residues 43-56 of the porcine
hemoglobin alpha chain (see FIG. 16C for a sequence comparison of
human, murine and porcine alpha and beta hemoglobin chains). Repeat
sequencing of these bands as well as of some of the minor bands
consistently yielded portions of the alpha globin sequence. Thus
the various bands observed in FIG. 15C represent either fragments
or aggregates of the porcine hemoglobin alpha chain.
[0217] In order to further compare INPROL to porcine hemoglobin,
twice crystallized porcine hemoglobin was obtained from Sigma and
subjected to reverse phase HPLC as described in Example 12B for
FIG. 15. As can be seen in FIG. 17, the HPLC chromatogram of intact
hemoglobin is similar to that seen for the INPROL Preparation 1.
Further, in a direct comparison, the INPROL Preparation 2 shown in
FIG. 17A (derived from the second peak of FIG. 5) is seen to
overlap with that of the second two peaks of porcine hemoglobin
(FIG. 17B), with retention times of 52.51 and 52.25 minutes for the
major peaks, respectively. It should be noted that heme co-migrates
with the first major peak in hemoglobin, in this case at 49.153
minutes; heme is therefore a component of INPROL Preparation 1 but
not of Preparation 2. This is confirmed by the lack of absorption
of this INPROL preparation at 575 nm, a wavelength diagnostic for
the presence of heme.
[0218] The predicted molecular weights of porcine hemoglobin alpha
and beta chains are 15038 Daltons and 16034 Daltons, respectively.
As can be seen in the SDS-PAGE chromatogram in FIG. 18, the first
two peaks are composed of the higher molecular weight chain and the
second two are composed of the lower molecular weight chain. Thus
the first two peaks represent hemoglobin beta chain and the second
two peaks represent hemoglobin alpha chain.
[0219] In order to further compare INPROL Preparation 2 and
hemoglobin alpha chain, 2-dimensional electrophoreses were
conducted (FIG. 19). As a first dimension, isoelectric focusing was
carried out in glass tubes using 2% pH 4-8 ampholines for 9600
volt-hours. Tropomyosin (MW 33 kD, pI 5.2) was used as an internal
standard; it's position is marked on the final 2D gel with an
arrow. The tube gel was equilibrated in buffer and sealed to the
top of a stacking gel on top of a 12.5% acrylamide slab gel. SDS
slab gel electrophoresis was carried out for 4 hours at 12.5
mA/gel. The gels were silver stained and dried.
[0220] A comparison of the 2D electrophoretic patterns revealed
only one or two minor spots that are different between HPLC
purified hemoglobin alpha chain and the INPROL Preparation 2.
Western analyses, using anti-porcine hemoglobin antibodies and
either 1D or 2D electrophoresis, confirm the presence of alpha
hemoglobin in the preparation. Thus the active INPROL Preparation
2, prepared according to Example 12B, is substantially porcine
hemoglobin alpha chain.
EXAMPLE 14
Hemoglobin Alpha Chain, Hemoglobin Beta Chain or Intact Hemoglobin
Exhibit Stem Cell Inhibitory Activity
[0221] To confirm that hemoglobin alpha chain has INPROL activity,
a suicide assay using bone marrow from testosterone-treated mice
was conducted using the methodology described in Example 1 using
material purified as in Example 12B. As shown in Table 1, 15% of
normal mouse bone marrow cells were killed as opposed to 36% in the
testosterone-treated animals. As expected, this indicated that
testosterone treatment increases the percentage of cells in cycle
(thus rendering them more susceptible to killing--cf. Example 1).
In sharp contrast, cells from testosterone-treated animals
incubated with either INPROL or purified hemoglobin alpha chain at
40 ng/ml showed a dramatic lowering of the percentage of cells in
cycle from 36% to 0% and to 7%, respectively. The higher dose of
200 ng was less effective for both proteins. As a positive control,
the previously characterized stem cell inhibitor MIP-la reduced
cycling to 13%.
[0222] As described in Example 2, INPROL inhibits the proliferation
of the murine stem cell line FDCP-MIX in a tritiated thymidine
uptake assay. FIG. 20 demonstrates that purified hemoglobin alpha
or beta chains are both active in this assay, with inhibitions seen
at <2ng/ml.
[0223] The foregoing provides evidence that the alpha chain of
porcine hemoglobin exhibits INPROL activity. Other data (e.g., FIG.
20) demonstrate that isolated beta chain, as well as intact
hemoglobin, are also active as stem cell inhibitors. Active
preparations also include mixtures of alpha and beta chains (e.g.,
FIG. 5).
[0224] The observations that isolated alpha globin chain and/or
isolated beta globin chain are active indicate that the activities
described here do not require an intact three-dimensional
hemoglobin structure. Fragments of alpha and beta chain are also
active as stem cell inhibitors.
9 TABLE 1 Treatment % Kill NBM.sup.1 15 TPBM.sup.2 36 INPROL 200
ng/ml 23 40 ng/ml 0 Hbg.sup.3 200 ng/ml 25 40 ng/ml 7 MIP-1a 200
ng/ml 13 .sup.1NBM = Normal Bone Marrow .sup.2TPBM = Bone marrow
from testosterone-treated mice .sup.3Hbg = C.sub.4 Reverse-phase
purified porcine hemoglobin alpha chain (derived from 2X
crystallized pig hemoglobin)
[0225] While the present invention has been described in terms of
preferred embodiments, it is understood that variations and
modifications will occur to those skilled in the art. Therefore, it
is intended that the appended claims cover all such equivalent
variations which come within the scope of the invention as claimed.
Sequence CWU 1
1
* * * * *