U.S. patent application number 09/852424 was filed with the patent office on 2002-10-24 for cxcr4 antagonist treatment of hematopoietic cells.
Invention is credited to Arab, Lakhdar, Cashman, Johanne, Clark-Lewis, Ian, Eaves, Connie J., Merzouk, Ahmed, Salari, Hassan, Saxena, Geeta, Tudan, Christopher R..
Application Number | 20020156034 09/852424 |
Document ID | / |
Family ID | 4165909 |
Filed Date | 2002-10-24 |
United States Patent
Application |
20020156034 |
Kind Code |
A1 |
Tudan, Christopher R. ; et
al. |
October 24, 2002 |
CXCR4 antagonist treatment of hematopoietic cells
Abstract
In accordance with various aspects of the invention, CXCR4
antagonists may be used to treat hematopoietic cells, such as
progenitor or stem cells, to promote the rate of cellular
multiplication, self-renewal, proliferation or expansion. CXCR4
antagonists may be used therapeutically to stimulate hematopoietic
stem/progenitor cell multiplication/self-renew- al.
Inventors: |
Tudan, Christopher R.;
(Vancouver, CA) ; Merzouk, Ahmed; (Richmond,
CA) ; Arab, Lakhdar; (Vancouver, CA) ; Saxena,
Geeta; (Vancouver, CA) ; Eaves, Connie J.;
(Vancouver, CA) ; Cashman, Johanne; (Vancouver,
CA) ; Clark-Lewis, Ian; (Vancouver, CA) ;
Salari, Hassan; (Delta, CA) |
Correspondence
Address: |
Bret E. Field
BOZICEVIC, FIELD & FRANCIS LLP
Suite 200
200 Middlefield Road
Menlo Park
CA
94025
US
|
Family ID: |
4165909 |
Appl. No.: |
09/852424 |
Filed: |
May 9, 2001 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
60205467 |
May 19, 2000 |
|
|
|
Current U.S.
Class: |
514/44R ;
424/93.21; 514/19.3; 514/19.8; 514/21.3; 514/7.9 |
Current CPC
Class: |
A61P 43/00 20180101;
C07K 14/4703 20130101; C12N 5/0647 20130101; A61K 48/00 20130101;
A61P 35/00 20180101; C12N 2501/21 20130101; C07K 14/522 20130101;
A61K 38/10 20130101; A61P 35/02 20180101; A61K 38/1709
20130101 |
Class at
Publication: |
514/44 ;
424/93.21; 514/12 |
International
Class: |
A61K 048/00; A61K
038/17 |
Foreign Application Data
Date |
Code |
Application Number |
May 9, 2000 |
CA |
2,305,787 |
Claims
What is claimed is:
1. A method of promoting the rate of hematopoietic cell
multiplication, comprising administering an effective amount of a
CXCR4 antagonist to hematopoietic cells.
2. The method of claim 1, wherein the hematopoietic cells are
hematopoietic stem or progenitor cells.
3. A method of increasing the circulation of hematopoietic cells in
a patient in need of such treatment, comprising administering to
the patient an effective amount of a CXCR4 antagonist to mobilize
the hematopoietic cells from a marrow locus to a peripheral blood
locus.
4. The method of claim 1, further comprising introducing a
heterologous gene into the hematopoietic cells for gene
therapy.
5. The method of claim 1, wherein the hematopoietic cells are ex
vivo.
6. The method of claim 1, wherein the hematopoietic cells are in
vivo.
7. The method of claim 1, wherein the hematopoietic cells are
selected from the group consisting of hematopoietic stem cells and
hematopoietic progenitor cells (including CFU-GEMM, BFU-E, CFU-Meg,
CFU-GM, CFU-M/DC CFU-Eo, CFU-Bas, Pro-B cells and lymphoid stem
cells), that are known to differentiate into mature myeloid and
lympoid blood cells, including erythrocytes, platelets,
neutrophils, monocytes, macrophages, dendritic cells (myeloid and
lymphoid related), eosinophils, basophils, mast cells, B cells. and
T cells.
8. The method of claim 1, wherein the CXCR4 antagonist comprises a
CXCR4 antagonist peptide.
9. The method of claim 8, wherein the CXCR4 antagonist peptide is
selected from the group consisting of:
14 KGVSLSYRCPCRFFESHVARANVKHLKILNTPNCALQIVARLKNNNRQ (SEQ ID No. 1)
VCIDPKLKWIQEYLEKALN; KGVSPSYRCPCRFFESHVARANVKHLKIL-
NTPNCALQIVARLKNNNRQ (SEQ ID No. 2) VCIDPKLKWIQEYLEKALN;
KGVSLPYRCPCRFFESHVARANVKHLKILNTPNCALQIVARLKNNNRQ (SEQ ID No. 3)
VCIDPKLKWIQEYLEKALN; KGVSLSPRCPCRFFESHVARANVKHLKILNTPNCA-
LQIVARLKNNNRQ (SEQ ID No. 4) VCIDPKLKWIQEYLEKALN;
KGVSLSYPCPCRFFESHVARANVKHLKILNTPNCALQIVARLKNNNRQ (SEQ ID No. 5)
VCIDPKLKWIQEYLEKALN; KGVSP*SYRGPCRFFESHVARANVKHLKILNTPNCA-
LQIVARLKNNNR (SEQ ID No. 6) QVCIDPKLKW1QEYLEKALN;
KGVSLP*YRCPCRFFESHVARANVKHLKILNTPNCALQIVARLKNNNR (SEQ ID No. 7)
QVCIDPKLKWIQEYLEKALN; KGVSLSP*RCPCRFFESHVARANVKHLKILNTPNC-
ALQIVARLKNNNR (SEQ ID No. 8) QVCIDPKLKWIQEYLEKALN;
KGVSLSYP*CPCRFFESHVARANVKHLKILNTPNCALQIVARLKNNNR (SEQ ID No. 9)
QVCIDPKLKWIQEYLEKALN; KGVSBtdYRCPCRFFESHVARANVKHLKILNTPNC-
ALQIVARLKNNNR (SEQ ID No. 10) QVCIDPKLKWIQEYLEKALN;
KGVSLBtdRCPCRFFESHVARANVKHLKILNTPNCALQIVARLKNNNR (SEQ ID No. 11)
QVCIDPKLKWIQEYLEKALN; KGVSLSBtdCPCRFFESHVARANVKHLKILNTPNC-
ALQIVARLKNNNR (SEQ ID No. 12) QVCIDPKLKWIQEYLEKALN;
wherein P*= 17with X=Ar, Ar--OH, alkyl and more and Btd= 18X=Alkyl,
Ar, Ar--OH and more
10. The method of claim 8, wherein the CXCR4 antagonist peptide is
selected from the group consisting of: a) KGVSLSYRCPCRFFESH b)
KGVSLSYRC
11. The method of claim 8, wherein the CXCR4 antagonist peptide is
selected from the group consisting of:
15 KGVSPSYRCPCRFFESH (SEQ ID No. 17) KGVSLPYRCPCRFFESH (SEQ ID No.
18) KGVSLSPRCPCRFFESH (SEQ ID No. 19) KGVSLSYPCPCRFFESH (SEQ ID No.
20) KGVSP*SYRCPCRFFESH (SEQ ID No. 21) KGVSLP*YRCPCRFFESH (SEQ ID
No. 22) KGVSLSP*RCPCRFFESH (SEQ ID No. 23) KGVSLSYP*CPCRFFESH (SEQ
ID No. 24) KGVSBtdYRCPCRFFESH (SEQ ID No. 25) KGVSLBtdRCPCRFFESH
(SEQ ID No. 26) KGVSLSBtdCPCRFFESH (SEQ ID No. 27) KGVSPSYRC (SEQ
ID No. 28) KGVSLPYRC (SEQ ID No. 29) KGVSLSPRC (SEQ ID No. 30)
KGVSLSYPC (SEQ ID No. 31) KGVSP*SYRC (SEQ ID No. 32) KGVSLP*YRC
(SEQ ID No. 33) KGVSLSP*RC (SEQ ID No. 34) KGVSLSYP*C (SEQ ID No.
35) KGVSBtdYRC (SEQ ID No. 36) KGVSLBtdRC (SEQ ID No. 37)
KGVSLSBtdC (SEQ ID No. 38)
wherein P*= 19with X=Ar, Ar--OH, alkyl and more and Btd= 20X=Alkyl,
Ar, Ar--OH and more
12. The method of claim 8, wherein the CXCR4 antagonist peptide is
selected from the group consisting of:
16 KGVSPSYRC KGVSLPYRC KGVSLSPRC KGVSLSYPC .vertline. .vertline.
.vertline. .vertline. KGVSPSYRC KGVSLPYRC KGVSLSPRC KGVSLSYPC
KGVSP*SYRC KGVSLP*YRC KGVSLSP*RC KGVSLSYP*C .vertline. .vertline.
.vertline. .vertline. KGVSP*SYRC KGVSLP*YRC KGVSLSP*RC KGVSLSYP*C
KGVSBtdYRC KGVSLBtdRC KGVSLSBtdC .vertline. .vertline. .vertline.
KGVSBtdYRC KGVSLBtdRC KGVSLSBtdC
21wherein P*= 22with X=Ar, Ar--OH, alkyl and more and Btd=
23X=Alkyl, Ar, Ar--OH and more
13. The method of claim 8, wherein the CXCR4 antagonist peptide is
selected from the group consisting of:
17 KGVSPSYR KGVSLPYR KGVSLSPR KGVSLSYP .vertline. .vertline.
.vertline. .vertline. X X X X .vertline. .vertline. .vertline.
.vertline. KGVSPSYR KGVSLPYR KGVSLSPR KGVSLSYP KGVSP*SYR KGVSLP*YR
KGVSLSP*R KGVSLSYP* .vertline. .vertline. .vertline. .vertline. X X
X X .vertline. .vertline. .vertline. .vertline. KGVSP*YR KGVSLP*YR
KGVSLSP*R KGVSLSYP* KGVSBtdYR KGVSLBtdR KGVSLSBtd .vertline.
.vertline. .vertline. X X X .vertline. .vertline. .vertline.
KGVSBtdYR KGVSLBtdR KGVSLSBtd
24wherein X is a natural or unnatural amino acid linker between
each of the arginines at position 8 in each sequencel; and, wherein
P*= 25with X=Ar, Ar--OH, alkyl and more and Btd= 26X=Alkyl, Ar,
Ar--OH and more
14. The method of claim 8, wherein the CXCR4 antagonist peptide is
selected from the group consisting of:
18 KGVSLSYRCPCRFF-G.sub.n-LKWIQEYLEKALN (SEQ No. 63)
KGVSLSYRCPCRFFESH-G.sub.n-LKWIQEYLEKALN (SEQ No. 64)
wherein n is an integer from 0 to 10.
15. The method of claim 8, wherein the CXCR4 antagonist peptide is
selected from the group consisting of:
19 KGVSLSYRCPCRFF-(CH.sub.2).sub.n-LKWIQEYLEKALN (SEQ No. 65)
KGVSLSYRCPCRFFESH-(CH.sub.2).sub.n-LKWIQEYLEKALN (SEQ No. 66)
where n is an integer from 1 to 20.
16. The method of claim 8, wherein the CXCR4 antagonist peptide is
selected from the group consisting of:
20 KGVSPSYRCPCRFF-GGGG-LKWIQEYLEKALN;
KGVSLPYRCPCRFF-GGGG-LKWIQEYLEKALN;
KGVSLSPRCPCRFF-GGGG-LKWIQEYLEKALN;
KGVSLSYPCPCRFF-GGGG-LKWIQEYLEKALN;
KGVSPSYRCPCRFFESH-GGGG-LKWIQEYLEKALN;
KGVSLPYRCPCRFFESH-GGGG-LKWIQEYLEKALN;
KGVSLSPRCPCRFFESH-GGGG-LKWIQEYLEKALN;
KGVSLSYPCPCRFFESH-GGGG-LKWIQEYLEKALN;
KGVSPSYRCPCRFF-(CH.sub.2).sub.n-LKWIQEYLEKALN;
KGVSLPYRCPCRFF-(CH.sub.2).sub.n-LKWIQEYLEKALN;
KGVSLSPRCPCRFF-(CH.sub.2).sub.n-LKWIQEYLEKALN;
KGVSLSYPCPCRFF-(CH.sub.2).sub.n-LKWIQEYLEKALN;
KGVSPSYRCPCRFFESH-(CH.sub.2).sub.n-LKWIQEYLEKALN;
KGVSLPYRCPCRFFESH-(CH.sub.2).sub.n-LKWIQEYLEKALN;
KGVSLSPRCPCRFFESH-(CH.sub.2).sub.n-LKWIQEYLEKALN;
KGVSLSYPCPCRFFESH-(CH.sub.2).sub.n-LKWIQEYLEKALN,
wherein n is an integer from 1 to 20.
17. The method of claim 8, wherein the CXCR4 antagonist peptide is
selected from the group consisting of:
21 KGVSP*SYRCPCRFF-GGGG-LKWIQEYLEKALN;
KGVSLP*YRCPCRFF-GGGG-LKWIQEYLEKALN;
KGVSLSP*RCPCRFF-GGGG-LKWIQEYLEKALN;
KGVSLSYP*CPCRFF-GCGG-LKWIQEYLEKALN;
KGVSP*SYRCPCRFFESH-GGGG-LKWIQEYLEKALN;
KGVSLP*YRCPCRFFESH-GGGG-LKWIQEYLEKALN;
KGVSLSP*RCPCRFFESH-GGGG-LKWIQEYLEKALN;
KGVSLSYP*CPCRFFESH-GGGG-LKWIQEYLEKALN;
KGVSP*SYRCPCRFF-(CH.sub.2).sub.n-LKWIQEYLEKALN;
KGVSLP*YRCPCRFF-(CH.sub.2).sub.n-LKWIQEYLEKALN;
KGVSLSP*RCPCRFF-(CH.sub.2).sub.n-LKWIQEYLEKALN;
KGVSLSYP*CPCRFF-(CH.sub.2).sub.n-LKWIQEYLEKALN;
KGVSP*SYRCPCRFFESH-(CH.sub.2).sub.n-LKWIQEYLEKALN;
KGVSLP*YRCPCRFFESH-(CH.sub.2).sub.n-LKWIQEYLEKALN;
KGVSLSP*RCPCRFFESH-(CH.sub.2).sub.n-LKWIQEYLEKALN;
KGVSLSYP*CPCRFFESH-(CH.sub.2).sub.n-LKWIQEYLEKALN;
KGVSBtdSYRCPCRFF-GGGG-LKWIQEYLEKALN;
KGVSLBtdRCPCRFF-GGGG-LKWIQEYLEKALN;
KGVSLSBtdCPCRFF-GGGG-LKWIQEYLEKALN;
KGVSBtdYRCPCRFFESH-GGGG-LKWIQEYLEKALN;
KGVSLBtdRCPCRFFESH-GGGG-LKWIQEYLEKALN;
KGVSLSBtdCPCRFFESH-GGGG-LkWIQEYLEKALN;
KGVSBtdYRCPCRFF-(CH.sub.2).sub.n-LKWIQEYLEKALN;
KGVSLBtdRCPCRFF-(CH.sub.2).sub.n-LKWIQEYLEKALN;
KGVSLSBtdCPCRFF-(CH.sub.2).sub.n-LKWIQEYLEKALN;
KGVSBtdYRCPCRFFESH-(CH.sub.2).sub.n-LKWIQEYLEKALN
KGVSLBtdRCPCRFFESH-(CH.sub.2).sub.n-LKWIQEYLEKALN;
KGVSLSBtdCPCRFFESH-(CH.sub.2).sub.n-LKWIQEYLEKALN,
wherein n is an integer from 0 to 20 and wherein P*= 27with X=Ar,
Ar--OH, alkyl and more and Btd= 28X=Alkyl, Ar, Ar--OH and more
18. The method of claim 8, wherein the CXCR4 antagonist peptide is
selected from the group consisting of: 29
19. A CXCR4 antagonist peptide selected from the group consisting
of: 30
20. The method of claim 8, wherein the CXCR4 antagonist peptide is
selected from the group consisting of:
22 KGVSLSYRCPCRFFGGGGSKPGVIFLTKRSRQV;
KGVSLSYRCPCRFF(CH.sub.2).sub.nSKPGVIFLTKRSRQV;
KGVSLSYRCPCRFFGGGGEEWVQKYVDDLELSA;
KGVSLSYRCPCRFF(CH.sub.2).sub.nEEWVQKYVDDLELSA,
where n is 0 or an integer between 1 and 20.
21. A method of treating a cancer in a patient in need of such
treatment comprising administering an effective amount of a CXCR4
antagonist to the patient to promote the rate of hematopoietic cell
multiplication.
22. A method of treating an autoimmune disease in a patient in need
of such treatment comprising administering an effective amount of a
CXCR4 antagonist to the patient to promote the rate of
hematopoietic cell multiplication.
Description
FIELD OF THE INVENTION
[0001] In one aspect, the invention relates to therapeutic uses of
chemokine receptor antagonists, including peptide antagonists of
CXC chemokine receptor 4 (CXCR4) for use in the treatment of
hematopoietic cells in vitro and in vivo. In another aspect, the
invention relates to novel CXCR4 antagonists which may be used in
the treatment of hematopoietic cells.
BACKGROUND OF THE INVENTION
[0002] Cytokines are soluble proteins secreted by a variety of
cells including monocytes or lymphocytes that regulate immune
responses. Chemokines are a superfamily of chemoattractant
proteins. Chemokines regulate a variety of biological responses and
they promote the recruitment of multiple lineages of leukocytes and
lymphocytes to a body organ tissue. Chemokines may be classified
into two families according to the relative position of the first
two cysteine residues in the protein. In one family, the first two
cysteines are separated by one amino acid residue, the CXC
chemokines, and in the other family the first two cysteines are
adjacent, the CC chemokines. Two minor subgroups contain only one
of the two cysteines (C) or have three amino acids between the
cysteines (CX.sub.3C). In humans, the genes of the CXC chemokines
are clustered on chromosome 4 (with the exception of SDF-1 gene,
which has been localized to chromosome 10) and those of the CC
chemokines on chromosome 17.
[0003] The molecular targets for chemokines are cell surface
receptors. One such receptor is CXC chemokine receptor 4 (CXCR4),
which is a 7 transmembrane protein, coupled to G1 and was
previously called LESTR (Loetscher, M., Geiser, T., O'Reilly, T.,
Zwahlen, R., Baggionlini, M., and Moser, B., (1994) J. Biol. Chem,
269, 232-237), HUMSTR (Federsppiel, B., Duncan, A. M. V., Delaney,
A., Schappert, K., Clark-Lewis, I., and Jirik, F. R. (1993)
Genomics 16, 707-712) and Fusin (Feng, Y., Broeder, C. C., Kennedy,
P. E., and Berger, E. A. (1996) HIV-1 entry cofactor: Functional
cDNA cloning of a seven-transmembrane G protein-coupled receptor,
Science 272, 872-877). CXCR4 is widely expressed on cells of
hemopoietic origin, and is a major co-receptor with CD4.sup.+ for
human immunodeficiency virus 1 (HIV-1) (Feng, Y., Broeder, C.C.,
Kennedy, P. E., and Berger, E. A. (1996) HIV-1 entry cofactor:
Functional cDNA cloning of a seven-transmembrane G protein-coupled
receptor, Science 272, 872-877).
[0004] Chemokines are thought to mediate their effect by binding to
seven-transmembrane G protein-coupled receptors, and to attract
leukocyte subsets to sites of inflammation (Baglionini et al.
(1998) Nature 392: 565-568). Many of the chemokines have been shown
to be constitutively expressed in lymphoid tissues, indicating that
they may have a homeostatic function in regulating lymphocyte
trafficking between and within lymphoid organs (Kim and Broxmeyer
(1999) J. Leuk. Biol. 56: 6-15).
[0005] Stromal cell derived factor one (SDF-1) is a member of the
CXC family of chemokines that has been found to be constitutively
secreted from the bone marrow stroma (Tashiro, (1993) Science 261,
600-602). The human and mouse SDF-1 predicted protein sequences are
approximately 92% identical. Stromal cell derived factor-1a
(SDF-1a) and stromal cell derived factor-1.beta. (SDF-1.beta.) are
closely related (together referred to herein as SDF-1). The native
amino acid sequences of SDF-1.alpha. and SDF-1.beta. are known, as
are the genomic sequences encoding these proteins (see U.S. Pat.
No. 5,563,048 issued Oct. 8, 1996, and U.S. Pat. No. 5,756,084
issued May 26, 1998). Identification of genomic clones has shown
that the alpha and beta isoforms are a consequence of alternative
splicing of a single gene. The alpha form is derived from exons 1-3
while the beta form contains an additional sequence from exon 4.
The entire human gene is approximately 10 Kb. SDF-1 was initially
characterized as a pre-B cell-stimulating factor and as a highly
efficient chemotactic factor for T cells and monocytes (Bieul et
al. (1996) J. Exp. Med. 184:1101 -1110).
[0006] Biological effects of SDF-1 may be mediated by the chemokine
receptor CXCR4 (also known as fusin or LESTR), which is expressed
on mononuclear leukocytes including hematopoietic stem cells. SDF-1
is thought to be the natural ligand for CXCR4, and CXCR4 is thought
to be the natural receptor for SDF-1 (Nagasawza et al. (1997) Proc.
Natl. Acad. Sci. USA 93:726-732). Genetic elimination of SDF-1 is
associated with parinatal lethality, including abnormalities in
cardiac development, B-cell lymphopoiesis, and bone marrow
myelopoiesis (Nagasawa et al. (1996) Nature 382:635-637).
[0007] SDF-1 is functionally distinct from other chemokines in that
it is reported to have a fundamental role in the trafficking,
export and homing of bone marrow progenitor cells (Aiuti, A., Webb,
I. J., Bleul, C., Springer, T., and Guierrez-Ramos, J. C., (1996)
J. Exp. Med. 185, 111-120 and Nagasawa, T., Hirota, S., Tachibana,
K., Takakura N., Nishikawa, S. -I., Kitamura, Y., Yoshida, N.,
Kikutani, H., and Kishimoto, T., (1996) Nature 382, 635-638). SDF-1
is also structurally distinct in that it has only about 22% amino
acid sequence identity with other CXC chemokines (Bleul, C. C.,
Fuhlbrigge, R. C., Casasnovas, J. M., Aiuti, A., and Springer, T.
A., (1996) J. Exp. Med. 184, 1101-1109). SDF-1 appears to be
produced constitutively by several cell types, and particularly
high levels are found in bone-marrow stromal cells (Shirozu, M.,
Nakano, T., Inazawa, J., Tashiro, K., Tada, H. Shinohara, T., and
Honjo, T., (1995) Genomics, 28, 495-500 and Bleul, C. C.,
Fuhlbrigge, R. C., Casasnovas, J. M., Aiuti, A., and Springer, T.
A., (1996) J. Exp. Med. 184, 1101-1109). A basic physiological role
for SDF-1 is implied by the high level of conservation of the SDF-1
sequence between species. In vitro, SDF-1 stimulates chemotaxis of
a wide range of cells including monocytes and bone marrow derived
progenitor cells (Aiuti, A., Webb, I. J., Bleul, C., Springer, T.,
and Guierrez-Ramos, J. C., (1996) J. Exp. Med. 185, 111-120 and
Bleul, C. C., Fuhlbrigge, R. C., Casasnovas, J. M., Aiuti, A., and
Springer, T. A., (1996) J. Exp. Med. 184, 1101-1109). SDF-1 also
stimulates a high percentage of resting and activated T-lymphocytes
(Bleul, C. C., Fuhlbrigge, R. C., Casasnovas, J. M., Aiuti, A., and
Springer, T. A., (1996) J. Exp. Med. 184,1101-1109 and Campbell, J.
J., Hendrick, J., Zlotnik, A., Siani, M. A., Thompson, D. A., and
Butcher, E. C., (1998) Science, 279 381-383).
[0008] Native SDF-1 has been demonstrated to induce the maturation
and activation of platelets (Hamada T. et al., J. Exp. Med. 188,
638-548 (1998); Hodohara K. et al., Blood 95, 769-775 (2000);
Kowalska M. A. et al., Blood 96, 50-57 (2000)), and CXCR4 is
expressed on the megakaryocytic lineage cells (CFUOMeg) (Wang J -F.
et al., Blood 92, 756-764 (1998)).
[0009] A variety of diseases require treatment with agents that are
preferentially cytotoxic to dividing cells. Cancer cells, for
example, may be targeted with cytotoxic doses of radiation or
chemotherapeutic agents. A significant side-effect of this approach
to cancer therapy is the pathological impact of such treatments on
rapidly dividing normal cells. These normal cells may for example
include hair follicles, mucosal cells and the hematopoietic cells,
such as primitive bone marrow progenitor cells and stem cells. The
indiscriminate destruction of hematopoietic stem, progenitor or
precursor cells can lead to a reduction in normal mature blood cell
counts, such as leukocytes, lymphocytes and red blood cells. A
major impact on mature cell numbers may be seen particularly with
neutrophils (neutropaenia) and platelets (thrombocytopenia), cells
which naturally have relatively short half-lives. A decrease in
leukocyte count, with concomitant loss of immune system function,
may increase a patient's risk of opportunistic infection.
Neutropaenia resulting from chemotherapy may for example occur
within two or three days of cytotoxic treatments, and may leave the
patient vulnerable to infection for up to 2 weeks until the
hematopoietic system has recovered sufficiently to regenerate
neutrophil counts. A reduced leukocyte count (leukopenia) and/or a
platelet count (granulocytopenia) as a result of cancer therapy may
become sufficiently serious that therapy must be interrupted to
allow the white blood cell count to rebuild. Interruption of cancer
therapy can in turn lead to survival of cancer cells, an increase
in the incidence of drug resistance in cancer cells, and ultimately
in cancer relapse. There is accordingly a need for therapeutic
agents and treatments, which facilitate the preservation of
hematopoietic progenitor or stem cells in patients subject to
treatment with cytotoxic agents. There is similarly a need for
therapeutic agents and treatments that facilitate the preservation
or regeneration (self-renewal) of hematopoietic cell populations in
cases where the number of such cells has been reduced due to
disease or to therapeutic treatments such as radiation and
chemotherapy.
[0010] Hematopoietic cells that are uncommitted to a final
differentiated cell type are identified herein as "progenitor"
cells. Hematopoietic progenitor cells possess the ability to
differentiate into a final cell type directly or indirectly through
a particular developmental lineage. Undifferentiated, pluripotent
progenitor cells that are not committed to any lineage are referred
to herein as "stem cells." All hematopoietic cells can in theory be
derived from a single stem cell, which is also able to perpetuate
the stem cell lineage as daughter cells become differentiated. The
isolation of populations of mammalian bone marrow cell populations
which are enriched to a greater or lesser extent in pluripotent
stem cells has been reported (see for example, C. Verfaillie et
al., J. Exp. Med., 172, 509 (1990), incorporated herein by
reference).
[0011] Bone marrow transplantation has been used in the treatment
of a variety of hematological, autoimmune and malignant diseases.
In conjunction with bone marrow transplantation, ex vivo
hematopoietic (bone marrow) cell culture may be used to expand the
population of hematopoietic cells, particularly progenitor or stem
cells, prior to reintroduction of such cells into a patient. In ex
vivo gene therapy, hematopoietic cells may be transformed in vitro
prior to reintroduction of the transformed cells into the patient.
In gene therapy, using conventional recombinant DNA techniques, a
selected nucliec acid, such as a gene, may be isolated, placed into
a vector, such as a viral vector, and the vector transfected into a
hematopoietic cell, to transform the cell, and the cell may in turn
express the product coded for by the gene. The cell then may then
be introduced into a patient. Hematopoietic stem cells were
initially identified as a prospective target for gene therapy (see
e.g., Wilson, J. M., et al., Proc. Natl. Acad. Sci 85: 3014-3018
(1988)). However, problems have been encountered in efficient
hematopoietic stem cell transfection (see Miller, A. D., Blood 76:
271-278 (1990)). There is accordingly a need for agents and methods
that facilitate the proliferation of hematopoietic cells in ex vivo
cell culture. There is also a need for agents that may be used to
facilitate the establishment and proliferation of engrafted
hematopoietic cells that have been transplanted into a patient.
[0012] The broad application of hematopoietic stem cell
transplantation therapy, however, may be limited by several
features. The acquisition of enough stem cells for clinical use may
require either a bone marrow harvest under general anesthesia or
peripheral blood leukapheresis; both are expensive and carry a risk
of morbidity. Grafts may contain only a limited number of useful
hematopoietic progenitors. Additionally, the kinetics of short-term
stem cell engraftment may be such that for the first 1-3 weeks
after infusion, these cells offer little hematopoietic support, and
therefor the recipients may remain profoundly myelosuppressed
during this time.
[0013] Hematopoietic stem cells are reportedly found in peripheral
blood of healthy persons. Their numbers however, may be
insufficient to permit collection of an adequate graft by standard
leukapheresis (Kessionger, A. et al., Bone Marrow Transplant 6,
643-646 (1989)). Fortunately, a variety of methods have been
discovered to increase the circulation of progenitor and stem cells
by "mobilizing" them from the marrow into the peripheral blood. For
autologous transplantation, hematopoietic stem/progenitor cells may
be mobilized into the peripheral blood (Lane T. A. Transfusion 36,
585-589 (1996)) during the rebound phase of the leukocytes after
transient leukopenia induced by myelosuppressive chemotherapy,
(Giralt S. et al., Blood, 89, 4531-4536 (1997) by hematopoietic
growth factors, or (Lasky L. C. et al., Transfusion 21, 247-260
(1981)) by a combination of both.
[0014] Hematopoietic stem cell mobilization into peripheral blood
has been used as a procedure following myelosuppressive
chemotherapy regimens to mobilize hematopoietic stem and progenitor
cells into the peripheral blood. Suggested treatment regimens for
mobilization may include cyclophosphamide alone, in single doses of
4-7 g/m2, or other agents such as Adriamycin (doxorubicin),
carboplatin, Taxol (paclitaxel), etoposide, ifosfamide,
daunorubicin, cytosine arabinosides 6-thioguanine, either alone or
in combination (Richman, C. M. et al., Blood 47, 1031-1039 (1976);
Stiff P. J. et al., Transfusion 23, 500-503 (1983); To L. B. et al.
Bone Marrow Transplant 9, 277-284 (1992)). Such a regiment may
induce a transient but profound myelosuppression in patients, with
white blood cell (WBC) counts in some cases dropping below 100
cells-mm.sup.3 7-14 days after chemotherapy. This may be followed
on day 10-21 by rapid reappearance of leukocytes in the peripheral
blood and frequently a "rebound" increase of the circulating
leukocytes above baseline levels. As the leukocyte count rises,
hematopoietic progenitor cells also begin to appear in the
peripheral blood and rapidly increase.
[0015] Hematopoietic stem cells (HSC) collected from mobilized
peripheral blood progenitor cells (PBPC) are increasingly used for
both autologous and allogeneic transplantation after myeloablative
or nonmyeloablative therapies (Lane T.A. Transfusion 36, 585-589
(1996)). Purported advantages of PBPC transplantation include rapid
and durable trilineage hematologic engraftment, improved tolerance
of the harvesting procedure (without general anesthesia), and
possibly diminished tumor contamination in the autologous setting
(Lasky L. C. et al., Transfusion 21, 247-260 (1981); Moss T. J. et
al, Blood 76,1879-1883)). Techniques for autologous mobilized PBPC
grafting may also be successful for allogeneic transplantation.
Early reports in animals and syngeneic transplants in humans
supported this hypothesis (Kessionger, A. et al., Bone Marrow
Transplant 6, 643-646 (1989)).
[0016] Many investigators have reported that PBPC mobilization
employing a combination of chemotherapy and followed by growth
factor (GM-CSF or G-CSF) administration is more effective than
either chemotherapy or growth factor alone (Siena S. et al., Blood
74, 1905-1914 (1989); Pettengel R. et al., Blood, 2239-2248 (1993);
Haas R. et al., Bone Marrow Transplant 9, 459-465 (1992); Ho A. D.
et al., Leukemia 7, 1738-1746 (1993)). The combination reportedly
results in a 50- to 75-fold increase in circulating CFU-GM and 10-
to 50-fold increase in CD34+cells (Pettengel R. et al., Blood,
2239-2248 (1993); Haas R. et al., Bone Marrow Transplant 9, 459-465
(1992); Ho A. D. et al., Leukemia 7,1738-1746 (1993)). Direct
comparisons show that chemotherapy and growth factors resulted in a
mean 3.5-fold greater peak number of circulating CFU-GM (range, 0
to 6.8 times greater verses chemotherapy or growth factor alone
(Siena S. et al., Blood 74, 1905-1914 (1989); Pettengel R. et al.,
Blood, 2239-2248 (1993); Haas R. et al., Bone Marrow Transplant 9,
459-465 (1992); Moskowitz C. H. et al. Clin. Cancer Res. 4, 311-316
(1998)).
[0017] It is reportedly possible to expand hematopoietic progenitor
cells in stroma-containing or nonstromal systems. Expansion systems
have reportedly shown increases in CFU_GM of more than 100-fold.
Enrichment of CD34+cells may be required before expansion in
nonstromal culture but may not be necessary in stroma-containing
systems. Early results of clinical trails are encouraging and have
been taken to demonstrate that the engraftment potential of the
expanded hematopoietic cells is not compromised by culture.
Expansion of cord blood-derived hematopoietic cells may be
especially important because of the limited number of cells that
can be collected. Successful expansion of primitive and committed
hematopoietic cells from cord blood may allow more extensive use in
clinical transplantation, particularly in adult patients. Other
possible applications of stem cell expansion include purging of
tumor cells; production of immune-competent cells, such as
dendritic cells and NK cells, and gene therapy.
[0018] Permanent marrow recovery after cytotoxic drug and radiation
therapy generally depends on the survival of hematopoietic stem
cells having long term reconstituting (LTR) potential. The major
dose limiting sequelae consequent to chemotherapy and/or radiation
therapy are typically neutropenia and thrombocytopenia. Protocols
involving dose intensification (i.e., to increase the log-kill of
the respective tumour therapy) or schedule compression may
exacerbate the degree and duration of myelosuppression associated
with the chemotherapy and/or radiation therapy. For instance, in
the adjuvant setting, repeated cycles of doxorubicin-based
treatment have been shown to produce cumulative and long-lasting
damage in the bone marrow progenitor cell populations (Lorhrman et
al., (1978) Br. J. Haematol. 40:369). The effects of short-term
hematopoietic cell damage resulting from chemotherapy has been
overcome to some extent by the concurrent use of G-CSF
(Neupogen.RTM.), used to accelerate the regeneration of neutrophils
(Le Chevalier (1994) Eur. J. Cancer 30A:410). This approach has
been met with limitations also, as it may be accompanied by
progressive thrombocytopenia and cumulative bone marrow damage as
reflected by a reduction in the quality of mobilized progenitor
cells over successive cycles of treatment. Because of the current
interest in chemotherapy dose intensification as a means of
improving tumour response rates and perhaps patient survival, the
necessity for alternative therapies to either improve or replace
current treatments to rescue the myeloablative effects of
chemotherapy and/or radiation therapy has escalated, and is
currently one of the major rate limiting factors for tumour therapy
dose escalations.
[0019] Transplanted peripheral blood stem cells (PBSC, or
autologous PBSC) may provide a rapid and sustained hematopoietic
recovery after the administration of high-dose chemotherapy or
radiation therapy in patients with hematological malignancies and
solid tumours. PBSC transplantation has become the preferred source
of stem cells for autologous transplantation because of the shorter
time to engraftment and the lack of a need for surgical procedures
such as are necessary for bone marrow harvesting (Demirer et al.
(1996) Stem Cells 14:106-116; Pettengel et al., (1992) Blood
82:2239-2248). Although the mechanism of stem cell release into the
peripheral blood from the bone marrow is not well understood,
agents that augment the mobilization of CD34.sup.+ cells may prove
to be effective in enhancing autologous PBSC transplantation. G-CSF
and GM-CSF are currently the most commonly used hematopoietic
growth factors for PBSC mobilization, although the mobilized
cellular profiles can differ significantly from patient to patient.
Therefore, other agents are required for this clinical
application.
[0020] It has been suggested that stem cell transplants for
autoimmune disease should be initiated using autologous or
allogenic grafts, where the former may be preferable since they may
bear less risk of complication (Burt and Taylor (1999) Stem Cells
17:366-372). Lymphocyte depletion has also been recommended, where
lymphocyte depletion is a form of purging autoreactive cells from
the graft. In practice, aggressive lymphocyte depletion of an
allograft can reportedly ameliorate alloreactivity (i.e.,
graft-versus-host disease (GVHD)) even without immunosuppressive
prophylaxis. Therefore, a lymphocyte-depleted autograft may prevent
recurrence of autoreactivity. As a consequence, any concurrent
therapy that may enhance the survival of the CFU-GEMM myeloid stem
cells, or BFU-E, CFU-Meg (CFU-MK) and CFU-GM myelomonocytic stem
cells may be beneficial in therapies for autoimmune diseases where
hematopoietic stem cells could be compromised.
[0021] Platelet activation in healthy subjects after G-CSF
administration has been reported. The effects were indicated by
increased platelet expression of P-selectin (Avenarius H. J. et
al., Int. J. Hematol. 58, 189-196 (1993), blood thromboxane B2, and
AT-III complex levels R. G-CSF reportedly enhances platelet
aggregation to collagen and adenosine diphosphate (Kuroiwa M. et
al., Int. J. Hematol. 63, 311-316 (1996)). There have, however,
been reports of arterial thrombosis in two patients with cancer who
were receiving G-CSF after chemotherapy (Shimoda K. et al., J.
Clin. Invest. 91, 1310-1313 (1993)), and concern has been expressed
regarding induction of a possible prethrombotic state in some
normal donors (Conti J. A. et al., Cancer 70, 2699-2707 (1992);
Kawachi Y. et al., Br. J. Haematol. 94, 413-416 (1996)) and such
risk was suggested in two cases (Anderlini P. et al., Blood 90,
903-908 (1997)).
[0022] Depressed platelet count after PBPC collection may occur in
healthy donors of allogeneic transplants. The decrease in platelet
counts during apheresis for autologous transplant recipient can
reportedly be substantial, especially for those heavily pretreated
patients mobilized with chemotherapy plus growth factor. Platelet
transfusion may be considered when the postapheresis count drops
below 20.000/mm3, although the threshold should be individualized
and depends on the status of the patient (inpatient vs.
outpatient), the history of platelet recovery after chemotherapy,
the amount of infused anticoagulant (hence the number of prior
apheresis sessions within the same mobilization and collection
series), and whether apheresis will be performed the next day. It
is possible to separate platelets from the PBPC product using a
low-speed centrifugation procedure. The platelets may by infused
fresh or cryopreserved for later infusion. (Schiffer C. A. et al.,
Ann N. Y. Acad. Sci. 411, 161-169 (1983)). The platelet
cryopreservation procedure, however, has not been universally
accepted. (Law P., Exp. Hematol. 10, 351-357 (1983)). Furthermore,
the PBPC product of patients with a low platelet count and who
require transfusion typically does not contain enough platelet to
warrant processing (Lane, unpublished observation (Lane T. A.
Transfusion 36, 585-589 (1996)).
[0023] Clinical trials using gene transfer into HSC have generally
relied on retrovirus-mediated gene transfer methods. Retroviruses
fill the need for stable and relatively efficient integration of
engineered genetic elements into the chromosomes of target T cells.
Other viral vector systems currently available, such as adenovirus
or adenovirus-associated viral vectors, or transfection methods,
such as lipofection, electroporation, calcium phosphate
precipitation, or bioballistics, may lack similar efficiency for
long-term expression of the transgenes in dividing HSC. Some
vectors may not enter the cells in sufficient numbers without
cytotoxicity and/or may not integrate stability into the
chromosomes with useful efficiency. In dividing cells, unintegrated
DNA is generally diluted and lost. Adenoviral vectors may also be
highly immunogenic.
[0024] Retrovirus-mediated gene transfer into murine hematopoietic
stem cells and reconstitution of syngeneic mice has demonstrated
persistence and functioning of the transgenes over extended period
of time (Kume et al. (1999) 69:227-233). Terminally differentiated
cells are relatively short-lived, except for memory B and T
lymphocytes, and a large number of blood cells are replaced daily.
Therefore, when long-term functional correction of blood cells by
gene transfer is required, the target cells may be hematopoietic
stem cells (Kume et al. (1999) 69:227-233). Compounds that can
maintain the survival and/or self-renewal (for example enhanced
number of cells in S-phase of the cell cycle) of the progenitor
stem cells may therefore increase the efficiency of the gene
transfer in that a greater population of hematopoietic stems cells
is available.
[0025] A number of proteins have been identified and may be
utilized clinically as inhibitors of hematopoietic progenitor cell
development and hematopoietic cell proliferation or multiplication.
These include recombinant-methionyl human G-CSF (Neupogen.RTM.,
Filgastim; Amgen), GM-CSF (Leukine.RTM., Sargramostim; Immunex),
erythropoietin (rhEPO, Epogen.RTM.; Amgen), thrombopoietin (rhTPO;
Genentech), interleukin-11 (rhIL-11, Neumega.RTM.; American Home
Products), Flt3 ligand (Mobista; Immunex), multilineage
hematopoietic factor (MARstem.TM.; Maret Pharm.), myelopoietin
(Leridistem; Searle), IL-3, myeloid progenitor inhibitory factor-1
(Mirostipen; Human Genome Sciences), stem cell factor (rhSCF,
Stemgen.RTM.; Amgen).
SUMMARY OF THE INVENTION
[0026] In accordance with various aspects of the invention, CXCR4
antagonists may be used to treat hematopoietic cells, for example
to increase the rate of hematopoietic stem or progenitor cellular
multiplication, self-renewal, expansion, proliferation, or
peripheralization. In various aspects, the invention relates to
methods of promoting the rate of hematopoietic cell multiplication,
which encompases processes that increase and/or maintain cellular
multiplication, self-renewal, expansion, proliferation or
peripheralization. This may for example be useful in some
embodiments for in vitro hematopoietic cell cultures used in bone
marrow transplantation, peripheral blood mobilization, or ex vivo
expansion. CXCR4 antagonists may also be used therapeutically to
stimulate hematopoietic cell multiplication, self-renewal,
expansion, proliferation or peripheralization in vivo, for example
in some embodiments involving human diseases such as a cancer or an
autoimmune disease. The hematopoietic cells targeted by the methods
of the invention may include hematopoietic progenitor or stem
cells.
[0027] In alternative embodiments, CXCR4 antagonists may be used to
treat a variety of hematopoietic cells, and such cells may be
isolated or may form only part of a treated cell population in vivo
or in vitro. Cells amenable to treatment with CXCR4 antagonists may
for example include cells in the hematopoietic lineage, beginning
with pluripotent stem cells, such as bone marrow stem or progenitor
cells, lymphoid stem or progenitor cells, myeloid stem cells,
CFU-GEMM cells (colony-forming-unit granulocyte, erythroid,
macrophage, megakaryocye), B stem cells, T stem cells, DC stem
cells, pre-B cells, prothymocytes, BFU-E cells (burst-forming
unit--erythroid), BFU-MK cells (burst-forming
unit--megakaryocytes), CFU-GM cells (colony-formng
unit--granulocyte-macrophage), CFU-bas cells (colony-forming
unit--basophil), CFU-Mast cells (colony forming unit--mast cell),
CFU-G cells (colony forming unit granulocyte), CFU-M/DC cells
(colony forming unit monocyte/dendritic cell), CFU-Eo cells (colony
forming unit eosinophil), CFU-E cells (colony forming unit
erythroid), CFU-MK cells (colony forming unit megakaryocyte),
myeloblasts, monoblasts, B-lymphoblasts, T-lymphoblasts,
proerythroblasts, neutrophillic myelocytes, promonocytes, or other
hematopoietic cells that differentiate to give rise to mature cells
such as macrophages, myeloid related dendritic cells, mast cells,
plasma cells, erythrocytes, platelets, neutrophils, monocytes,
eosinophils, basophils, B-cells, T-cells or lymphoid related
dendritic cells.
[0028] In some embodiments, the invention provides methods of
increasing the circulation of hematopoietic cells by mobilizing
them from the marrow to the peripheral blood comprising
administering an effective amount of a CXCR4 antagonist to
hematopoietic cells of a patient undergoing autologous mobilization
where hematopoietic stem/progenitor cells may be mobilized into the
peripheral blood (1) during the rebound phase of the leukocytes
and/or platelets after transient granulocytopenia and
thrombocytopenia induced by myelosuppressive chemotherapy, (2) by
hematopoietic growth factors, or (3) by a combination of both. Such
treatment may for example be carried out so as to be effective to
mobilize the hematopoietic cells from a marrow locus (i.e. a
location in the bone marrow) to a peripheral blood locus (i.e. a
location in the peripheral blood). Such treatments may for example
be undertaken in the context of or for the clinical procedure of
leukapheresis or apheresis. In alternative embodiments, CXCR4
antagonists may be used in ex vivo stem cell expansion to
supplement stem cell grafts with more mature precursors to shorten
or potentially prevent hematopoietic cell depletion, including
conditions such as pancytopenia, granulocytopenia,
thrombocytopenia, anemia or a combination thereof; to increase the
number of primitive progenitors to help ensure hematopoietic
support for multiple cycles of high-dose therapy; to obtain
sufficient number of stem cells from a single marrow aspirate or
apheresis procedure, thus reducing the need for large-scale
harvesting of marrow of multiple leukopheresis; to generate
sufficient cells from a single cord-blood unit to allow
reconstitution in an adult after high-dose chemotherapy; to purge
stem cell products of contaminating tumour cells; to generate large
volumes of immunologically active cells with antitumour activity to
be used in immunotherapeutic regimens or to increase the pool of
stem cells that could be targets for the delivery of gene
therapy.
[0029] In alternative embodiments, the invention provides methods
to enrich CD34+ progenitor cells which are utilized in bone marrow
(BM) and peripheral blood (PB) stem cell transplantation, wherein
the hematopoietic stem cell transplantation (HSCT) protocols may
for example be utilized for the purpose of treating the following
diseases (from Ball, E. D., Lister, J., and Law, P. Hematopoietic
Stem Cell Therapy, Chruchill Livingston (of Harcourt Inc.), New
York (2000)): Aplastic Anemia; Acute Lymphoblastic Anemia.; Acute
Myelogenous Leukemia; Myelodysplasia; Multiple Myeloma; Chronic
Lymphocytic Leukemia; Congenital Immunodeficiencies (such as
Autoimmune Lymphoproliferative disease, Wiscott-Aldrich Syndrome,
X-linked Lymphoproliferative disease, Chronic Granulamatous
disease, Kostmann Neutropenia, Leukocyte Adhesion Deficiency);
Metabolic Diseases (for instance those which have been HSCT
indicated such as Hurler Syndrome (MPS I/II), Sly Syndrome (MPS
VII), Chilhood onset cerebral X-adrenoleukodystrophy, Globard_cell
Leukodystrophy).
[0030] In some embodiments, peptide CXCR4 antagonists of the
invention may comprise an N-terminal portion derived from SDF-1,
covalently joined by a linker to a second N-terminal peptide,
containing or now modifications to mimic N-terminal beta-turning,
or C-terminal alpha-helices. The SDF-1 antagonist may also exist as
an N-terminal Dimer.
BRIEF DESCRIPTION OF THE FIGURES
[0031] FIG. 1: shows the effects of CXCR4 receptor binding of SDF-1
peptide antagonists in an .sup.1251-SDF-1 binding competition
assay. Full length SDF-1 antagonist and the indicated analogs
(competing ligands) were added to CEM cells in the presence of 4 nM
.sup.125O-SDF-1. CEM cells were assessed for .sup.125I-SDF-1
binding following 2 hr incubation. The results are expressed as
percentage of the maximal specific binding that was determined
without competing ligand.
[0032] FIG. 2: shows the effect of SDF-1 peptide antagonists
(defined in Examples) on the cycling of human progenitors from
fetal liver transplanted NOD/SCID mice. The cycling status of
mature and primitive colony forming cells (CFU-GM; colony forming
unit-granulocyte-monocyte precursor, BFU-E; burst forming
unit-erythroid precursor) in the suspension of CD34.sup.+ cells
isolated from the marrow of transplanted NOD/SCID mice was
determined by assessing the proportion of these progenitors that
were inactivated (killed) by short term (20 min) or overnight
(LTC-IC;Iong-term culture initiating cell) exposure of the cells to
20 .mu.g/ml of high specific activity .sup.3H-thymidine. Values
represent the mean +/-the S.D. of data from up to four experiments
with up to four mice per point in each. Since high specific
activity .sup.3H-thymidine affects proliferating cells, the higher
degree in cell death resulting from SDF-1 antagonist incubation
represents a significant enhancement in cell cycling
(self-renewal).
[0033] FIG. 3 shows the effect of SDF-1 peptide antagonists
(defined in Examples) on the engraftment of human cells in human
fetal liver transplanted NOD/SCID mice. A comparison of the number
of phenotypically defined hematopoietic cells detected in the long
bones (tibias and femurs) of mice four weeks after being
transplanted with 10.sup.7 light-density human fetal liver blood
cells and then administered with the indicated SDF-1 antagonists
(0.5 mg/kg) three times per week for two weeks before sacrifice.
Values represent the mean +/-one S.D. of results obtained from
three to seven individual mice in three experiments.
DETAILED DESCRIPTION OF THE INVENTION
[0034] In one aspect, the invention provides uses for CXCR4
antagonists derived from SDF-1 [P2G] in which glycine is
substituted for proline at amino acid position 2. The full (67
amino acid long) versions of this analogue, designated
SDF-1(1-67)[P2G], or SDF-1[P2G] (SEQ ID No. 1), is a potent CXCR4
receptor antagonist (Crump et al., (1997) EMBO J. 16(23):
6996-7007). SDF-1 binds to CXCR4 primarily via its N-terminus,
which appears flexible in the NMR studies of active N-terminal
peptides of SDF-1 (Elisseeva et al., J. Biol Chem (2000) 275(35)
26799-805). Residues 5-8, and to a less extent 11-14, form similar
structures that can be characterized as a beta-turn of the
beta-alpha R type. These structural motifs are likely to be
interconverting with other states, but the major conformation may
be important for recognition during receptor binding. The
importance of beta-turns of peptides and proteins may well be
crucial for receptor interactions that ultimately lead to
biological activity. In recognition of this, there have been
several efforts to `lock` peptides and proteins into beta-turn
configurations (Ripka, W. C. et al., Tetrahedron (1993) 49(17)
3593-3608 and Elseviers, M. et al., Biochem. Biophys. Res. Commun,
(1988) 154-515). The natural amino-acid proline is known to a
beta-turn inducer. In one aspect of this invention, versions of the
full length antagonist analogues in which proline (P) was
substituted into single position residues 5-8, designated(SEQ ID
No. 2-5. In the same scheme, replacement of the natural amino acid
proline by the so-called proline-amino acid chimera (P*) (Garland,
R. M. Tetrahedron (1993) 49(17) 3547-3558 and Raman, S. et al., J.
Org. Chem. (1996) 61(1) 202-208) in the full length anatagonist
gives rise to the designated analogues (SEQ ID No. 6-9). Another
mechanism of beta-turn induction/`locking` is the introduction of
the Bicyclic Turned Dipetide (Btd), as a beta-turn mimetic (Ukon
Nagai et al Tetrahedron (1993) 49(17) 3577-3592) in the sequence of
the full length anatagonist as for proline and proline schimera. In
this configuration, two successive amino acid are replaced at once
by the Btd molecule, which when inserted into the SDF-1 [P2G]
antagonist are designated as SEQ ID No. 10-12.
[0035] Sequences:
1 KGVSLSYRCPCRFFESHVARANVKHLKILNTPNCALQIVARLKNNNRQVCIDPKLKWI (SEQ
ID No.1) QEYLEKALN KGVSPSYRCPCRFFESHVARANVKHLKILN-
TPNCALQIVARLKNNNRQVCIDPKLKW (SEQ ID No.2) IQEYLEKALN
KGVSLPYRCPCRFFESHVARANVKHLKILNTPNCALQIVARLKNNNRQVCIDPKLKWI (SEQ ID
No.3) QEYLEKALN KGVSLSPRCPCRFFESHVARANVKHLKILNTPNCALQIVA-
RLKNNNRQVCIDPKLKWI (SEQ ID No.4) QEYLEKALN
KGVSLSYPCPCRFFESHVARANVKHLKILNTPNCALQIVARLKNNNRQVCIDPKLKWI (SEQ ID
No.5) QEYLEKALN KGVSP*SYRCPCRFFESHVARANVKHLKILNTPNCALQIV-
ARLKNNNRQVCIDPKLK (SEQ ID No.6) WIQEYLEKALN
KGVSLP*YRCPCRFFESHVARANVKHLKILNTPNCALQIVARLKNNNRQVCIDPKLK (SEQ ID
No.7) WIQEYLEKALN KGVSLSP*RCPCRFFESHVARANVKHLKILNTPNCALQIV-
ARLKNNNRQVCIDPKLK (SEQ ID No.8) WIQEYLEKALN
KGVSLSYP*CPCRFFESHVARANVKHLKILNTPNCALQIVARLKNNNRQVCIDPKLK (SEQ ID
No.9) WIQEYLEKALN KGVSBtdYRCPCRFFESHVARANVKHLKILNTPNCALQIV-
ARLKNNNRQVCIDPKLK (SEQ ID No.10) WIQEYLEKALN
KGVSLBtdRCPCRFFESHVARANVKHLKILNTPNCALQIVARLKNNNRQVCIDPKLK (SEQ ID
No.11) WIQEYLEKALN KGVSLSBtdCPCRFFESHVARANVKHLKILNTPNCALQ-
IVARLKNNNRQVCIDPKLKW (SEQ ID No.12) IQEYLEKALN
[0036] Where P*= 1
[0037] with X=Ar, Ar--OH, alkyl and more
[0038] and Btd= 2
[0039] X=Alkyl, Ar, Ar--OH and more
[0040] A variety of small SDF-1 peptide analogues may also be used
as CXCR4 antagonists, as disclosed in International Patent
Publications WO 00/09152 (published 24 February 2000) and WO
99/47158 (published Sep. 23, 1999), each of which is incorporated
herein by reference. One such peptide may be a monomer having the
following sequences; KGVSLSYRCPCRFFESH (SEQ ID No. 13); KGVSLSYRC
(SEQ ID No. 14), or dimer of amino acids 1-9 (within SEQ ID No.
13), in which the amino acid chains are joined by a disulphide bond
between each of the cysteines at position 9 in each sequence
(designated SDF-1 (1-9).sub.2[P2G] with the following sequence:
KGVSLSYRC-CRYSLSVPK (SEQ ID No. 15)). Other An alternative peptides
may for example be selected from the group consisting of peptides:
KGVSLSYR-X-RYSLSVPK (SEQ ID No. 16), that is a dimer of amino acids
1-8, in which the amino acid chains are joined by a linking moiety
X (X may be an amino acid like lysine; ornithine or any other
natural or unnatural amino acid serving as a linker between each of
the arginines at position 8 in each sequence (designated
SDF-1(1-8).sub.2[P2G]). Here again the notion of beta-turn mimetic
was applied either for monomer (SEQ ID No. 13) in this case the
following analogues were designated (SEQ ID No.17-27)
2 KGVSPSYRCPCRFFESH (SEQ ID No.17) KGVSLPYRCPCRFFESH (SEQ ID No.18)
KGVSLSPRCPCRFFESH (SEQ ID No.19) KGVSLSYPCPCRFFESH (SEQ ID No.20)
KGVSP*SYRCPCRFFESH (SEQ ID No.21) KGVSLP*YRCPCRFFESH (SEQ ID No.22)
KGVSLSP*RCPCRFFESH (SEQ ID No.23) KGVSLSYP*CPCRFFESH (SEQ ID No.24)
KGVSBtdYRCPCRFFESH (SEQ ID No.25) KGVSLBtdRCPCRFFESH (SEQ ID No.26)
KGVSLSBtdCPCRFFESH (SEQ ID No.27)
[0041] Similar modifications may be made to monomeric peptides of
the invention (SEQ ID No.14)
3 KGVSPSYRC (SEQ ID No.28) KGVSLPYRC (SEQ ID No.29) KGVSLSPRC (SEQ
ID No.30) KGVSLSYPC (SEQ ID No.31) KGVSP*SYRC (SEQ ID No.32)
KGVSLP*YRC (SEQ ID No.33) KGVSLSP*RC (SEQ ID No.34) KGVSLSYP*C (SEQ
ID No.35) KGVSBtdYRC (SEQ ID No.36) KGVSLBtdRC (SEQ ID No.37)
KGVSLSBtdC (SEQ ID No.38)
[0042] Alternative peptides based on SEQ ID No. 15 are as follows,
designated (SEQ ID Nos. 39-49)
4 3 4 5 6 7 8 9 10 11 12 13
[0043] In the same manner analogues based on the SEQ ID No. 16 are
as follows, designated SEQ ID Nos. 50-61). 14
[0044] where X may be an amino acid like lysine; ornithine or any
other natural or unnatural amino acid serving as a linker between
each of the arginines at position 8 in each sequence.
[0045] In some embodiments, the CXCR4 antagonists for use in the
invention may be substantially purified peptide fragments, modified
peptide fragments, analogues or pharmacologically acceptable salts
of either SDF-1.alpha. or SDF-1.beta.. SDF-1 derived peptide
antagonists of CXCR4 may be identified by known physiological
assays and a variety of synthetic techniques (such as disclosed in
Crump et al., 1997, The EMBO Journal 16(23) 6996-7007; and Heveker
et al., 1998, Current Biology 8(7): 369-376; each of which are
incorporated herein by reference). Such SDF-1 derived peptides may
include homologs of native SDF-1, such as naturally occurring
isoforms or genetic variants, or polypeptides having substantial
sequence similarity to SDF-1, such as 20%, 30%, 40%, 50%, 60%, 70%,
80%, 90%, 95% or 99% sequence identity to at least a portion of the
native SDF-1 sequence, the portion of native SDF-1 being any
contiguous sequence of 10, 20, 30, 40, 50 or more amino acids,
provided the peptides have CXCR4 antagonist activity. In some
embodiments, chemically similar amino acids may be substituted for
amino acids in the native SDF-1 sequence (to provide conservative
amino acid substitutions). In some embodiments, peptides having an
N-terminal LSY sequence motif within 10, or 7 amino acids of the
N-terminus, and/or an N-terminal RFFESH (SEQ ID No. 62) sequence
motif within 20 amino acids of the N-terminus may be used provided
they have CXCR4 antagonistic activity. One family of such peptide
antagonist candidates has an LSY motif at amino acids 5-7.
Alternative peptides further include the RFFESH (SEQ ID No. 62)
motif at amino acids 12-17. In alternative embodiments, the LSY
motif is located at positions 3-5 of a peptide. The invention also
provides peptide dimers having two amino acid sequences, which may
each have the foregoing sequence elements, attached by a disulfide
bridge within 20, or preferably within 10, amino acids of the N
terminus, linking cysteine residues or .alpha.-aminobutric acid
residues.
[0046] In other aspects, the invention relates to novel CXCR4
antagonists derived from SDF-1[P2G] and their use to increase the
rate of cellular multiplication and/or self-renweal of
hematopoietic stem/progenitor cells. The antagonist compounds of
the invention comprise an N-terminal portion of SDF-1[P2G]
covalently jointed by a linker to a second peptide. The N-terminal
portion may be any portion of the SDF-1 [P2G] N-terminus which
binds to CXCR4. The second peptide, which does not include an
N-terminal portion of SDF-1 [P2G], preferably enhances the
antagonistic effect of the compound and may be a C-terminal
fragment of SDF-1, for example any C-terminal fragment of any
chemokine that known to improve the activity by binding to GAG's.
(refer to Gabriele S. et al., Biochemistry (1999), 38:
12959-12968). SDF-1 Antagonists include an acid or amide peptide
analog having SDF-1 [P2G] N terminal amino acids 1-14 or 1-17
linked to C-terminal residues 55-67 by a four glycine linker:
5 KGVSLSYRCPCRFF-GGGG-LKWJQEYLEKALN (SEQ ID No.63)
KGVSLSYRCPCRFFESH-GGGG-LKWIQEYLEKALN (SEQ ID No.64)
[0047] where the number of glycines linking the N-terminal and
C-terminal amino acids may be varied, for example between 0 and 10,
and may be 4, 3 or 2 in selected embodiments. The size of the
linker may be adapted to correspond approximately to the distance
between C-terminal and N-terminal regions in the native folded
SDF-1 structure.
[0048] In other embodiments, a (CH.sub.2).sub.n linker may be used
to join the N-terminal and C-terminal amino acids:
6 KGVSLSYRCPCRFF-(CH.sub.2).sub.n-LKWIQEYLEKALN (SEQ ID No.65)
KGVSLSYRCPCRFFESH-(CH.sub.2).sub.n-LKWIQEYLEKALN (SEQ ID No.
66)
[0049] where n=1-20 or more. In such embodiments, the length of the
linker may be adapted to correspond to the distance between the N-
and C-terminal end of the full length SDF-1[P2G] polypeptide in its
native form (where the amino acids replaced by the corresponding
linker are present).
[0050] The N-terminal LSYR residues which form a beta-turn (see
Elisseeva et al., J. Bio. Chem. 275(35): 26799-26805) may be
modified, similarly as the full length SDF-1 [P2G] anatagonist, for
example, by substituting leucine (L); serine (S); tyrosine (y) and
arginine (R) with proline (P):
7 KGVSPSYRCPCRFF-GGGG-LKWIQEYLEKALN (SEQ ID No.67)
KGVSLPYRCPCRFF-GGGG-LKWIQEYLEKALN (SEQ ID No.68)
KGVSLSPRCPCRFF-GGGG-LKWIQEYLEKALN (SEQ ID No.69)
KGVSLSYPCPCRFF-GGGG-LKWIQEYLEKALN (SEQ ID No.70)
KGVSPSYRCPCRFFESH-GGGG-LKWIQEYLEKALN (SEQ ID No.71)
KGVSLPYRCPCRFFESH-GGGG-LKWIQEYLEKALN (SEQ ID No.72)
KGVSLSPRCPCRFFESH-GGGG-LKWIQEYLEKALN (SEQ ID No.73)
KGVSLSYPCPCRFFESH-GGGG-LKWlQEYLEKALN (SEQ ID No.74)
KGVSPSYRCPCRFF-(CH.sub.2).sub.n-LKWIQEYLEKALN (SEQ ID No.75)
KGVSLPYRCPCRFF-(CH.sub.2).sub.n-LKWIQEYLEKALN (SEQ ID No.76)
KGVSLSPRCPCRFF-(CH.sub.2).sub.n-LK- WIQEYLEKALN (SEQ ID No.77)
KGVSLSYPCPCRFF-(CH.sub.- 2).sub.n-LKWIQEYLEKALN (SEQ ID No.78)
KGVSPSYRCPCRFFESH-(CH.sub.2).sub.n-LKWIQEYLEKALN (SEQ ID No.79)
KGVSLPYRCPCRFFESH-(CH.sub.2).sub.n-LKWIQEYLEKALN (SEQ ID No.80)
KGVSLSPRCPCRFFESH-(CH.sub.2).sub.n-LKWIQEYLEK- ALN (SEQ ID No.81)
KGVSLSYPCPCRFFESH-(CH.sub.2).su- b.n-LKWIQEYLEKALN (SEQ ID
No.82)
[0051] where the number of glycines or n (of (CH.sub.2).sub.n)
correspond to the length of the linker conferred to four glycines,
or the distance between the N- and C-terminal end of the full
length SDF-1[P2G] polypeptide in its native form where the amino
acids replaced by the corresponding linker are present.
[0052] In other embodiments, leucine (L), Seine (S), tyrosine (Y)
or arginine (R) may be substituted with proline-amino acid chemira
(P*) (similar to Seq ID No. 6-9 for the full length SDF-1
antagonist):
8 KGVSP*SYRCPCRFF-GGGG-LKWIQEYLEKALN (SEQ ID No.83)
KGVSLP*YRCPCRFF-GGGG-LKWIQEYLEKALN (SEQ ID No.84)
KGVSLSP*RCPCRFF-GGGG-LKWIQEYLEKALN (SEQ ID No.85)
KGVSLSYP*CPCRFF-GGGG-LKWIQEYLEKALN (SEQ ID No.86)
KGVSP*SYRCPCRFFESH-GGGG-LKWIQEYLEKALN (SEQ ID No.87)
KGVSLP*YRCPCRFFESH-GGGG-LKWIQEYLEKALN (SEQ ID No.88)
KGVSLSP*RCPCRFFESH-GGGG-LKWIQEYLEKALN (SEQ ID No.89)
KGVSLSYP*CPCRFFESH-GGGG-LKWIQEYLEKALN (SEQ ID No.90)
KGVSP*SYRCPCRFF-(CH.sub.2).sub.n-LKWIQEYLEKALN (SEQ ID No.91)
KGVSLP*YRCPCRFF-(CH.sub.2).sub.n-L- KWIQEYLEKALN (SEQ ID No.92)
KGVSLSP*RCPCRFF-(CH.sub.2).sub.n-LKWIQEYLEKALN (SEQ ID No.93)
KGVSLSYP*CPCRFF-(CH.sub.2).sub.n-LKWIQEYLEKALN (SEQ ID No.94)
KGVSP*SYRCPCRFFESH-(CH.sub.2).sub.n-LKWIQEYLEKALN (SEQ ID No.95)
KGVSLP*YRCPCRFFESH-(CH.sub.2).sub.- n-LKWIQEYLEKALN (SEQ ID No.96)
KGVSLSP*RCPCRFFESH-(CH.sub.2).sub.n-LKWIQEYLEKALN (SEQ ID No.97)
KGVSLSYP*CPCRFFESH-(CH.sub.2).sub.n-LKWIQEYLEKALN (SEQ ID
No.98)
[0053] where the number of glycines or n (of (CH.sub.2).sub.n)
correspond to the length of the linker conferred to four glycines,
or the distance between the N- and C-terminal end of the full
length SDF-1[P2G] polypeptide in its native form where the amino
acids replaced by the corresponding linker are present.
[0054] In some embodiments, the peptidomimetics are of BTD (Bicyclo
Turned Dipeptide) as described previously for the full length SDf-1
antagonist (SEQ ID No. 99-110):
9 KGVSBtdYRCPCRFF-GGGG-LKWIQEYLEKALN (SEQ ID No. 99)
KGVSLBtdRCPCRFF-GGGG-LKWIQEYLEKALN (SEQ ID No. 100)
KGVSLSBtdCPCRFF-GGGG-LKWIQEYLEKALN (SEQ ID No. 101)
KGVSBtdYRCPCRFFESH-GGGG-LKWIQEYLEKALN (SEQ ID No. 102)
KGVSLBtdRCPCRFFESH-GGGG-LKWIQEYLEKALN (SEQ ID No. 103)
KGVSLSBtdCPCRFFESH-GGGG-LKWIQEYLEKALN (SEQ ID No. 104)
KGVSBtdYRCPCRFF-(CH.sub.2).sub.n-LKWIQEY- LEKALN (SEQ ID No. 105)
KGVSLBtdRCPCRFF-(CH.sub.2)- .sub.n-LKWIQEYLEKALN (SEQ ID No. 106)
KGVSLSBtdCPCRFF-(CH.sub.2).sub.n-LKWIQEYLEKALN (SEQ ID No. 107)
KGVSBtdYRCPCRFFESH-(CH.sub.2).sub.n-LKWIQEYLEKALN (SEQ ID No. 108)
KGVSLBtdRCPCRFFESH-(CH.sub.2).sub.n-LKWI- QEYLEKALN (SEQ ID No.
109) KGVSLSBtdCPCRFFESH-(CH.- sub.2).sub.n-LKWIQEYLEKALN (SEQ ID
No. 110)
[0055] where the number of glycines or n (of (CH.sub.2).sub.n)
correspond to the length of the linker conferred to four glycines,
or the distance between the N- and C-terminal end of the full
length SDF-1[P2G] polypeptide in its native form where the amino
acids replaced by the corresponding linker are present.
[0056] The SDF-1-derived CXCR4 antagonists of the invention may be
linear or cyclized. In some embodiments, the antagonists may be
cyclized at glutamic acid at position 24 with lysine at position 20
or 28 by removing the allylic group from both side chains of lysine
and glutamic acid using the palladium-(0) technique (as described
in Kates et al., (1993) Anal. Biochem. 212, 303-310): 1-Allyl
removal: A solution of tetrakis(triphenylphosphine)palladium(0) (3
fold excess) dissolved in 5% Acetic acid; 2.5% N-methylmorpholine
(NMM) in chloroform under argon. The solution is added to the
support-bound peptide previously removed from the column in a
reaction vial containing a small magnetic bar for gentle stirring.
The mixture is flushed with argon, sealed and stirred at room
temperature for 6 hours. The support-bound peptide is transferred
to a filter funnel, washed with a solution made of 0.5% sodium
diethyldithiocarbamate in dichloromethane (DMF) and then
dichloromethane. 0.2-Lactam formation is mediated by internal amide
bond formation between the lysine and glutamic acid. Cyclisation is
carried out manually in a peptide synthesis vial at room
temperature overnight with gentle agitation. The coupling agent is
7-azabenzotriazol-1 -yloxytris(pyrrolidino)phosphonium
hexafluorophosphate (PyAOP)/N-methylmorpholine (NMM) (3 fold
excess). (Jean-Rene Barbier et al., J.
[0057] Med Chem. (1997), 40: 1373-1380; ibid Biochemistry (2000),
39, 14522-14530). The following analogues were designated (SEQ ID
No.111-114). 15
[0058] In some embodiments, glutamic acid (E) at position 24 and
may be substituted with aspartic acid (D) and the aspartic acid
cyclized with lysine at position or 28 as described previously. In
other embodiments, lysine at position 20 or 28 may be substituted
with ornithine cyclized with either aspartic acid or glutamic acid
at position 24 as described previously. This kind of substitution
followed by cyclisation can be done with all analogues described
above (SEQ ID No. 67-1 10).
[0059] In other embodiments, lysine (K) at position 20 or 28 may be
substituted with ornithine (O) (SEQ ID No. 42 to 73) and ornithine
at position 20 or 28 cyclized with glutamic acid (or with
substituted aspartic acid (SEQ ID No. 74-89)) at position 24 as
described previously. Additionally, to form other cyclic rings,
lysine may be substituted by leucine (L), or other hydrophpobic
residues such as isoleucine (I), norleucine (NIe), valine (V),
alanine (A), tryptophan (W), or phenylalanine (F). Lysine may also
be substituted with methionine, however, methionine oxides and
forms a disulphide bond making the peptide synthesis and
purification more difficult.
[0060] CXCR4 antatognists of the present invention may further
include hydrid analogs comprising N-terminal amino acid residues of
SDF-1[P2G] and amino acid residues of MIP-1a that are associated
with GAG binding of the chemokine receptor, for example by
replacing the relevant SDF-1 GAG-binding sequence, which may not be
as specific as that of MlP-1.alpha. (see Gabriele S. et al.,
Biochemistry (1999) 38: 12959-12968 and Elisabeth M. et al.,
Virology (1999) 265, 354-364).
[0061] SDF-1 [P2G] (1-14)/MIP-1.alpha. (36-50) Hybrid Analog:
10 KGVSLSYRCPCRFFGGGGSKPGVIFLTKRSRQV (SEQ ID NO. 115)
KGVSLSYRCPCRFF(CH.sub.2).sub.n SKPGVIFLTKRSRQV (SEQ ID No. 116)
[0062] SDF-1 (1-14)/MIP-1.alpha.(55-70) Analog:
11 KGVSLSYRCPCRFFGGGGEEWVQKYVDDLELSA (SEQ ID No. 117)
KGVSLSYRCPCRFF(CH.sub.2).sub.nEEWVQKYVDDLELSA (SEQ ID No. 118)
[0063] where the number of glycines or n (of (CH.sub.2).sub.n)
correspond to the length of the linker conferred to four glycines,
or the distance between the N- and C-terminal end of the full
length SDF-1[P2G] polypeptide in its native form where the amino
acids replaced by the corresponding linker are present.
[0064] It is well known in the art that some modifications and
changes can be made in the structure of a polypeptide without
substantially altering the biological function of that peptide, to
obtain a biologically equivalent polypeptide. In one aspect of the
invention, SDF-1 derived peptide antagonists of CXCR4 may include
peptides that differ from a portion of the native SDF-1 sequence by
conservative amino acid substitutions. The present invention also
extends biologically equivalent peptides that differ from a portion
of the sequence of novel antagonists of the present invention by
conservative amino acid substitutions. As used herein, the term
"conserved amino acid substitutions" refers to the substitution of
one amino acid for another at a given location in the peptide,
where the substitution can be made without loss of function. In
making such changes, substitutions of like amino acid residues can
be made on the basis of relative similarity of side-chain
substituents, for example, their size, charge, hydrophobicity,
hydrophilicity, and the like, and such substitutions may be assayed
for their effect on the function of the peptide by routine
testing.
[0065] In some embodiments, conserved amino acid substitutions may
be made where an amino acid residue is substituted for another
having a similar hydrophilicity value (e.g., within a value of plus
or minus 2.0), where the following hydrophilicity values are
assigned to amino acid residues (as detailed in U.S. Pat. No.
4,554,101, incorporated herein by reference): Arg (+3.0); Lys
(+3.0); Asp (+3.0); Glu (+3.0); Ser (+0.3); Asn (+0.2); Gln (+0.2);
Gly (0); Pro (-0.5); Thr (-0.4); Ala (-0.5); His (-0.5); Cys
(-1.0); Met (-1.3); Val (-1.5); Leu (-1.8); lie (-1.8); Tyr (-2.3);
Phe (-2.5); and Trp (-3.4).
[0066] In alternative embodiments, conserved amino acid
substitutions may be made where an amino acid residue is
substituted for another having a similar hydropathic index (e.g.,
within a value of plus or minus 2.0). In such embodiments, each
amino acid residue may be assigned a hydropathic index on the basis
of its hydrophobicity and charge characteristics, as follows: lie
(+4.5); Val (+4.2); Leu (+3.8); Phe (+2.8); Cys (+2.5); Met (+1.9);
Ala (+1.8); Gly (-0.4); Thr (-0.7); Ser (-0.8); Trp (-0.9); Tyr
(-1.3); Pro (-1.6); His (-3.2); Glu (-3.5); Gln (-3.5); Asp (-3.5);
Asn (-3.5); Lys (-3.9); and Arg (-4.5).
[0067] In alternative embodiments, conserved amino acid
substitutions may be made where an amino acid residue is
substituted for another in the same class, where the amino acids
are divided into non-polar, acidic, basic and neutral classes, as
follows: non-polar: Ala, Val, Leu, lie, Phe, Trp, Pro, Met; acidic:
Asp, Glu; basic: Lys, Arg, His; neutral: Gly, Ser, Thr, Cys, Asn,
Gln, Tyr.
[0068] In some embodiments, CXCR4 antagonists are ligands that bind
to CXCR4 with sufficient affinity and in such a manner so as to
inhibit the effects of binding by an agonists, such as the natural
ligand SDF-1, such as SDF-1 -induced [Ca2+]i mobilization in cells.
Example of CXCR4 antagonist assays may for example be found in
International Patent Publications WO 00/09152 (published Feb. 24,
2000) and WO 99/47158 (published Sep. 23, 1999). In exemplary
assays for CXCR4 antagonist activity, fura-2,AM loaded THP-1 cells
may for example be incubated with putative antagonists, such as for
60 min prior to induction of [Ca2+]i mobilization by 10 nM SDF-1.
Antagonists will typically demonstrate a dose responsive inhibition
of SDF-1-induced [Ca2+]i mobilization.
[0069] Methods that may be utilized to determine whether a molecule
functions as a CXCR4 antagonists include, but are not limited to,
the following: Inhibition of the induction of SDF-1 receptor
mediated rise in free cytosolic Ca.sup.2+ concentration
([Ca.sup.2+]) in response to native SDF-1 (or agonist analogs of
SDF-1) (Loetscher P. et al., (1998) J. Biol. Chem. 273,
24966-24970), inhibition of SDF-1-induction of phosphoinositide-3
kinase or Protein Kinase C activity (Wang, J-F et al., (2000) Blood
95, 2505-2513), inhibition of SDF-1-induced migration of CD34.sup.+
hematopoietic stem cells in a two-chamber migration (transwell)
assay (Durig J. et al,. (2000) Leukemia 14, 1652-1660; Peled A. et
al., (2000) Blood 95, 3289-2396), inhibition of SDF-1 associated
transmigration of CD34.sup.+/CXCR4.sup.+ cells through vascular
endothelial cells in a cell chemotaxis assay, cell adhesion assay,
or real-time tracking of CD34.sup.+ cell migration in 3-D
extracellular matrix-like gel assays (Peled A. et al., (2000) Blood
95, 3289-2396), inhibition of SDF-1 associated chemotaxis of
marrow-derived B cell precursors (Duzzo M. et al., Eur. J. Immunol.
(1997) 27, 1788-1793), preventing CXCR4 signal transduction and
coreceptor function in mediating the entry of T- and dual-tropic
HIV isolates (Zhou N. et al., (2000) 39, 3782-3787), inhibition of
SDF-1 associated increases of CFU-GM, CGU-M or BFU-E colony
formation by peripheral blood Inc.sup.+ CD34.sup.+ progenitor cells
(Lataillade J-J. et al/. (2000) Blood 95, 756-768), or inhibition
of integrin-mediated adhesion of T cells to fibronectin and ICAM-1
(Buckley C. D et al., (2000) J. Immunology 165, 3423-3429). Where
it is necessary to assess the inhibition of SDF-1 associated
mechanisms in the aforementioned assays, various concentrations of
CXCR4 antagonist may be incubated under the appropriate
experimental conditions in the presence of SDF-1, in assays to
determine if the CXCR4 antagonist associated repression of the
respective mechanism results directly from inhibition of the CXCR4
receptor. ([Ca.sup.2+]) mobilization, chemotaxis assays or other
assays that measure the induction of CXCR4 are not limited to the
cell types indicated in the associated references, but may include
other cell types that demonstrate CXCR4 associated, and specific,
activation.
[0070] In alternative aspects, the invention provides uses for
CXCR4 antagonists that are identified as molecules that bind to
CXCR4 (whether reversible or irreversible) and are associated with
the repression of CXCR4 associated activity. Binding affinity of a
CXCR4 antagonists may for example be associated with ligand binding
assay dissociation constants (K.sub.D) in the range of a minimum of
1 pM, 10 pM, 100 pM, 1 uM, 10 uM or 100 uM up to a maximum of 1 mM,
or any value in any such range. CXCR4 antagonist associated KD
values may be determined through alternative approaches, such as
standard methods of radioligand binding assays, including High
Throughput Fluorescence Polarization, scintillation proximity
assays (SPA), and Flashplates.TM..RTM. (Allen et al., (2000) J.
Biomolecular Screening 5, 63-69), where the competing ligand is
native SDF-1. Alternatively, the affinity of a CXCR4 antagonist for
the SDF-1 receptor (CXCR4) may be ascertained through inhibition of
native SDF-1 binding to the CXCR4, where various concentrations of
the CXCR4 antagonist are added in the presence of SDF-1 and a
recombinant CXCR4 or a cell type that expresses an adequate
receptor titer.
[0071] In alternative embodiments, the present invention relates to
uses of small molecule non-peptide CXCR4 antagonists, such as a
naphthoic acid derivative designated herein as
3-hydroxy-2-naphthoic acid (CAS 92-70-6; molecular formula: 16
[0072] C11H8O3; molecular weight: 188.18):
[0073] In some embodiments, the invention provides pharmaceutical
compositions containing CXCR4 antagonists. In one embodiment, such
compositions include a CXCR4 antagonist compound in a
therapeutically or prophylactically effective amount sufficient to
alter bone marrow progenitor or stem cell growth, and a
pharmaceutically acceptable carrier. In another embodiment, the
composition includes a CXCR4 antagonist compound in a
therapeutically or prophylactically effective amount sufficient to
inhibit a cytotoxic effect of a cytotoxic agent, such as cytotoxic
agents used in chemotherapy or radiation treatment of cancer, and a
pharmaceutically acceptable carrier.
[0074] A "therapeutically effective amount" refers to an amount
effective, at dosages and for periods of time necessary, to achieve
the desired therapeutic result, such as reduction of bone marrow
progenitor or stem cell multiplication, or reduction or inhibition
of a cytotoxic effect of a cytotoxic agent. A therapeutically
effective amount of CXCR4 antagonist may vary according to factors
such as the disease state, age, sex, and weight of the individual,
and the ability of the CXCR4 antagonist to elicit a desired
response in the individual. Dosage regimens may be adjusted to
provide the optimum therapeutic response. A therapeutically
effective amount is also one in which any toxic or detrimental
effects of the CXCR4 antagonist are outweighed by the
therapeutically beneficial effects.
[0075] A "prophylactically effective amount" refers to an amount
effective, at dosages and for periods of time necessary, to achieve
the desired prophylactic result, such as preventing or inhibiting a
cytotoxic effect of a cytotoxic agent. Typically, a prophylactic
dose is used in subjects prior to or at an earlier stage of
disease, so that a prophylactically effective amount may be less
than a therapeutically effective amount.
[0076] In particular embodiments, a preferred range for
therapeutically or prophylactically effective amounts of CXCR4
antagonists may be 0.1 nM-0.1M, 0.1 nM-0.05M, 0.05 nM-15 .mu.M or
0.01 nM-100 .mu.M. It is to be noted that dosage values may vary
with the severity of the condition to be alleviated. For any
particular subject, specific dosage regimens may be adjusted over
time according to the individual need and the professional
judgement of the person administering or supervising the
administration of the compositions. Dosage ranges set forth herein
are exemplary only and do not limit the dosage ranges that may be
selected by medical practitioners.
[0077] The amount of active compound in the composition may vary
according to factors such as the disease state, age, sex, and
weight of the individual. Dosage regimens may be adjusted to
provide the optimum therapeutic response. For example, a single
bolus may be administered, several divided doses may be
administered over time or the dose may be proportionally reduced or
increased as indicated by the exigencies of the therapeutic
situation. It may be advantageous to formulate parenteral
compositions in dosage unit form for ease of administration and
uniformity of dosage. "Dosage unit form" as used herein refers to
physically discrete units suited as unitary dosages for subjects to
be treated; each unit containing a predetermined quantity of active
compound calculated to produce the desired therapeutic effect in
association with the required pharmaceutical carrier. The
specification for the dosage unit forms of the invention are
dictated by and directly dependent on (a) the unique
characteristics of the active compound and the particular
therapeutic effect to be achieved, and (b) the limitations inherent
in the art of compounding such an active compound for the treatment
of sensitivity in individuals.
[0078] As used herein "pharmaceutically acceptable carrier" or
"exipient" includes any and all solvents, dispersion media,
coatings, antibacterial and antifungal agents, isotonic and
absorption delaying agents, and the like that are physiologically
compatible. In one embodiment, the carrier is suitable for
parenteral administration. Alternatively, the carrier can be
suitable for intravenous, intraperitoneal, intramuscular,
sublingual or oral administration. Pharmaceutically acceptable
carriers include sterile aqueous solutions or dispersions and
sterile powders for the extemporaneous preparation of sterile
injectable solutions or dispersion. The use of such media and
agents for pharmaceutically active substances is well known in the
art. Except insofar as any conventional media or agent is
incompatible with the active compound, use thereof in the
pharmaceutical compositions of the invention is contemplated.
Supplementary active compounds can also be incorporated into the
compositions.
[0079] In some embodiments, CXCR4 agonists may be formulated in
pharmaceutical compositions with additional active ingredients, or
administered in methods of treatment in conjunction with treatment
with one or more additional medications, such as a medicament
selected from the following: recombinant-methionyl human G-CSF
(Neupogen, Filgastim; Amgen), GM-CSF (Leukine.RTM., Sargramostim;
Immunex), erythropoietin (rhEPO, Epogen.RTM.; Amgen),
thrombopoietin (rhTPO; Genentech), interleukin-11 (rhlL-11,
Neumega.RTM.; American Home Products), Flt3 ligand (Mobista;
Immunex), multilineage hematopoietic factor (MARstem.TM.; Maret
Pharm.), myelopoietin (Leridistem; Searle), IL-3, myeloid
progenitor inhibitory factor-1 (Mirostipen; Human Genome Sciences),
and stem cell factor (rhSCF, Stemgen.RTM.; Amgen).
[0080] Therapeutic compositions typically must be sterile and
stable under the conditions of manufacture and storage. The
composition can be formulated as a solution, microemulsion,
liposome, or other ordered structure suitable to high drug
concentration. The carrier can be a solvent or dispersion medium
containing, for example, water, ethanol, polyol (for example,
glycerol, propylene glycol, and liquid polyethylene glycol, and the
like), and suitable mixtures thereof. The proper fluidity can be
maintained, for example, by the use of a coating such as lecithin,
by the maintenance of the required particle size in the case of
dispersion and by the use of surfactants. In many cases, it will be
preferable to include isotonic agents, for example, sugars,
polyalcohols such as mannitol, sorbitol, or sodium chloride in the
composition. Prolonged absorption of the injectable compositions
can be brought about by including in the composition an agent which
delays absorption, for example, monostearate salts and gelatin.
Moreover, the CXCR4 antagonists may be administered in a time
release formulation, for example in a composition which includes a
slow release polymer. The active compounds can be prepared with
carriers that will protect the compound against rapid release, such
as a controlled release formulation, including implants and
microencapsulated delivery systems. Biodegradable, biocompatible
polymers can be used, such as ethylene vinyl acetate,
polyanhydrides, polyglycolic acid, collagen, polyorthoesters,
polylactic acid and polylactic, polyglycolic copolymers (PLG). Many
methods for the preparation of such formulations are patented or
generally known to those skilled in the art.
[0081] Sterile injectable solutions can be prepared by
incorporating the active compound in the required amount in an
appropriate solvent with one or a combination of ingredients
enumerated above, as required, followed by filtered sterilization.
Generally, dispersions are prepared by incorporating the active
compound into a sterile vehicle that contains a basic dispersion
medium and the required other ingredients from those enumerated
above. In the case of sterile powders for the preparation of
sterile injectable solutions, the preferred methods of preparation
are vacuum drying and freeze-drying which yields a powder of the
active ingredient plus any additional desired ingredient from a
previously sterile-filtered solution thereof. In accordance with an
alternative aspect of the invention, a CXCR4 antagonist may be
formulated with one or more additional compounds that enhance the
solubility of the CXCR4 antagonist. The invention also extends to
such derivatives of novel antagonists of the invention.
[0082] CXCR4 antagonist compounds of the invention may include
SDF-1 derivatives, such as C-terminal hydroxymethyl derivatives,
O-modified derivatives (e.g., C-terminal hydroxymethyl benzyl
ether), N-terminally modified derivatives including substituted
amides such as alkylamides and hydrazides and compounds in which a
C-terminal phenylalanine residue is replaced with a phenethylamide
analogue (e.g., Ser-Ile-phenethylamide as an analogue of the
tripeptide Ser-le-Phe). The invention also extends to such
derivatives of the novel antagonists of the invention.
[0083] Within a CXCR4 antagonist compound of the invention, a
peptidic structure (such as an SDF-1 derived peptide) maybe coupled
directly or indirectly to at least one modifying group. Such
modified peptides are also within the scope of the invention. The
term "modifying group" is intended to include structures that are
directly attached to the peptidic structure (e.g., by covalent
coupling), as well as those that are indirectly attached to the
peptidic structure (e.g., by a stable non-covalent association or
by covalent coupling to additional amino acid residues, or
mimetics, analogues or derivatives thereof, which may flank the
SDF-1 core peptidic structure). For example, the modifying group
can be coupled to the amino-terminus or carboxy-terminus of an
SDF-1 peptidic structure, or to a peptidic or peptidomimetic region
flanking the core domain. Alternatively, the modifying group can be
coupled to a side chain of at least one amino acid residue of a
SDF-1 peptidic structure, or to a peptidic or peptido-mimetic
region flanking the core domain (e.g., through the epsilon amino
group of a lysyl residue(s), through the carboxyl group of an
aspartic acid residue(s) or a glutamic acid residue(s), through a
hydroxy group of a tyrosyl residue(s), a serine residue(s) or a
threonine residue(s) or other suitable reactive group on an amino
acid side chain). Modifying groups covalently coupled to the
peptidic structure can be attached by means and using methods well
known in the art for linking chemical structures, including, for
example, amide, alkylamino, carbamate or urea bonds.
[0084] In some embodiments, the modifying group may comprise a
cyclic, heterocyclic or polycyclic group. The term "cyclic group",
as used herein, includes cyclic saturated or unsaturated (i.e.,
aromatic) group having from 3 to 10, 4 to 8, or 5 to 7 carbon
atoms. Exemplary cyclic groups include cyclopropyl, cyclobutyl,
cyclopentyl, cyclohexyl, and cyclooctyl. Cyclic groups may be
unsubstituted or substituted at one or more ring positions. A
cyclic group may for example be substituted with halogens, alkyls,
cycloalkyls, alkenyls, alkynyls, aryls, heterocycles, hydroxyls,
aminos, nitros, thiols amines, imines, amides, phosphonates,
phosphines, carbonyls, carboxyls, silyls, ethers, thioethers,
sulfonyls, sulfonates, selenoethers, ketones, aldehydes, esters,
--CF.sub.3, --CN.
[0085] The term "heterocyclic group" includes cyclic saturated,
unsaturated and aromatic groups having from 3 to 10, 4 to 8, or 5
to 7 carbon atoms, wherein the ring structure includes about one or
more heteroatoms. Heterocyclic groups include pyrrolidine, oxolane,
thiolane, imidazole, oxazole, piperidine, piperazine, morpholine.
The heterocyclic ring may be substituted at one or more positions
with such substituents as, for example, halogens, alkyls,
cycloalkyls, alkenyls, alkynyls, aryls, other heterocycles,
hydroxyl, amino, nitro, thiol, amines, imines, amides,
phosphonates, phosphines, carbonyls, carboxyls, silyls, ethers,
thioethers, sulfonyls, selenoethers, ketones, aldehydes, esters,
--CF.sub.3, --CN. Heterocycles may also be bridged or fused to
other cyclic groups as described below.
[0086] The term "polycyclic group" as used herein is intended to
refer to two or more saturated, unsaturated or aromatic cyclic
rings in which two or more carbons are common to two adjoining
rings, so that the rings are "fused rings". Rings that are joined
through non-adjacent atoms are termed "bridged" rings. Each of the
rings of the polycyclic group may be substituted with such
substituents as described above, as for example, halogens, alkyls,
cycloalkyls, alkenyls, alkynyls, hydroxyl, amino, nitro, thiol,
amines, imines, amides, phosphonates, phosphines, carbonyls,
carboxyls, silyls, ethers, thioethers, sulfonyls, selenoethers,
ketones, aldehydes, esters, --CF.sub.3, or --CN.
[0087] The term "alkyl" refers to the radical of saturated
aliphatic groups, including straight chain alkyl groups,
branched-chain alkyl groups, cycloalkyl (alicyclic) groups, alkyl
substituted cycloalkyl groups, and cycloalkyl substituted alkyl
groups. In some embodiments, a straight chain or branched chain
alkyl has 20 or fewer carbon atoms in its backbone
(C.sub.1-C.sub.20 for straight chain, C.sub.3-C.sub.20 for branched
chain), or 10 or fewer carbon atom. In some embodiments,
cycloalkyls may have from 4-10 carbon atoms in their ring
structure, such as 5, 6 or 7 carbon rings. Unless the number of
carbons is otherwise specified, "lower alkyl" as used herein means
an alkyl group, as defined above, having from one to ten carbon
atoms in its backbone structure. Likewise, "lower alkenyl" and
"lower alkynyl" have chain lengths of ten or less carbons.
[0088] The term "alkyl" (or "lower alkyl") as used throughout the
specification and claims is intended to include both "unsubstituted
alkyls" and "substituted alkyls", the latter of which refers to
alkyl moieties having substituents replacing a hydrogen on one or
more carbons of the hydrocarbon backbone. Such substituents can
include, for example, halogen, hydroxyl, carbonyl (such as
carboxyl, ketones (including alkylcarbonyl and arylcarbonyl
groups), and esters (including alkyloxycarbonyl and aryloxycarbonyl
groups)), thiocarbonyl, acyloxy, alkoxyl, phosphoryl, phosphonate,
phosphinate, amino, acylamino, amido, amidine, imino, cyano, nitro,
azido, sulfhydryl, alkylthio, sulfate, sulfonate, sulfamoyl,
sulfonamido, heterocyclyl, aralkyl, or an aromatic or
heteroaromatic moiety. The moieties substituted on the hydrocarbon
chain can themselves be substituted, if appropriate. For instance,
the substituents of a substituted alkyl may include substituted and
unsubstituted forms of aminos, azidos, iminos, amidos, phosphoryls
(including phosphonates and phosphinates), sulfonyls (including
sulfates, sulfonamidos, sulfamoyls and sulfonates), and silyl
groups, as well as ethers, alkylthios, carbonyls (including
ketones, aldehydes, carboxylates, and esters), --CF.sub.3, --CN and
the like. Exemplary substituted alkyls are described below.
Cycloalkyls can be further substituted with alkyls, alkenyls,
alkoxys, alkylthios, aminoalkyls, carbonyl-substituted alkyls,
--CF.sub.3, --CN, and the like.
[0089] The terms "alkenyl" and "alkynyl" refer to unsaturated
aliphatic groups analogous in length and possible substitution to
the alkyls described above, but that contain at least one double or
triple bond respectively.
[0090] The term "aralkyl", as used herein, refers to an alkyl or
alkylenyl group substituted with at least one aryl group. Exemplary
aralkyls include benzyl (i.e., phenylmethyl), 2-naphthylethyl,
2-(2-pyridyl)propyl, 5-dibenzosuberyl, and the like.
[0091] The term "alkylcarbonyl", as used herein, refers to
--C(O)-alkyl. Similarly, the term "arylcarbonyl" refers to
-C(O)-aryl. The term "alkyloxycarbonyl", as used herein, refers to
the group --C(O)--O-alkyl, and the term "aryloxycarbonyl" refers to
--C(O)--O-aryl. The term "acyloxy" refers to --O--C(O)--R.sub.7, in
which R.sub.7 is alkyl, alkenyl, alkynyl, aryl, aralkyl or
heterocyclyl.
[0092] The term "amino", as used herein, refers to
--N(R.sub..alpha.)(R.su- b..beta.), in which R.sub..alpha. and
R.sub..beta. are each independently hydrogen, alkyl, alkyenyl,
alkynyl, aralkyl, aryl, or in which R.sub..alpha. and R.sub..beta.
together with the nitrogen atom to which they are attached form a
ring having 4-8 atoms. Thus, the term "amino", as used herein,
includes unsubstituted, monosubstituted (e.g., monoalkylamino or
monoarylamino), and disubstitited (e.g., dialkylamino or
alkylarylamino) amino groups. The term "amido" refers to
--C(O)--N(R.sub.8)(R.sub.9), in which R.sub.8 and R.sub.9 are as
defined above. The term "acylamino" refers to
--N(R'.sub.8)C(O)--R.sub.7, in which R.sub.7 is as defined above
and R'.sub.8 is alkyl.
[0093] As used herein, the term "nitro" means --NO.sub.2; the term
"halogen" designates --F, --Cl, --Br or --I; the term "sulfhydryl"
means --SH; and the term "hydroxyl" means --OH.
[0094] The term "aryl" as used herein includes 5-, 6- and
7-membered aromatic groups that may include from zero to four
heteroatoms in the ring, for example, phenyl, pyrrolyl, furyl,
thiophenyl, imidazolyl, oxazole, thiazolyl, triazolyl, pyrazolyl,
pyridyl, pyrazinyl, pyridazinyl and pyrimidinyl, and the like.
Those aryl groups having heteroatoms in the ring structure may also
be referred to as "aryl heterocycles" or "heteroaromatics". The
aromatic ring can be substituted at one or more ring positions with
such substituents as described above, as for example, halogen,
azide, alkyl, aralkyl, alkenyl, alkynyl, cycloalkyl, hydroxyl,
amino, nitro, sulfhydryl, imino, amido, phosphonate, phosphinate,
carbonyl, carboxyl, silyl, ether, alkylthio, sulfonyl, sulfonamido,
ketone, aldehyde, ester, a heterocyclyl, an aromatic or
heteroaromatic moiety, --CF.sub.3, --CN, or the like. Aryl groups
can also be part of a polycyclic group. For example, aryl groups
include fused aromatic moieties such as naphthyl, anthracenyl,
quinolyl, indolyl, and the like.
[0095] Modifying groups may include groups comprising biotinyl
structures, fluorescein-containing groups, a
diethylene-triaminepentaacetyl group, a (-)-menthoxyacetyl group, a
N-acetylneuraminyl group, a cholyl structure or an iminiobiotinyl
group. A CXCR4 antagonist compound may be modified at its carboxy
terminus with a cholyl group according to methods known in the art
(see e.g., Wess, G. et al. (1993) Tetrahedron Letters, 34:817-822;
Wess, G. et al. (1992) Tetrahedron Letters 33:195-198; and Kramer,
W. et al. (1992) J. Biol. Chem. 267:18598-18604). Cholyl
derivatives and analogues may also be used as modifying groups. For
example, a preferred cholyl derivative is Aic
(3-(O-aminoethyl-iso)-choly- l), which has a free amino group that
can be used to further modify the CXCR4 antagonist compound. A
modifying group may be a "biotinyl structure", which includes
biotinyl groups and analogues and derivatives thereof (such as a
2-iminobiotinyl group). In another embodiment, the modifying group
may comprise a "fluorescein-containing group", such as a group
derived from reacting an SDF-1 derived peptidic structure with
5-(and 6-)-carboxyfluorescein, succinimidyl ester or fluorescein
isothiocyanate. In various other embodiments, the modifying
group(s) may comprise an N-acetylneuraminyl group, a
trans-4-cotininecarboxyl group, a 2-imino-1-imidazolidineacetyl
group, an (S)-(-)-indoline-2-carboxyl group, a (-)-menthoxyacetyl
group, a 2-norbornaneacetyl group, a -oxo-5-acenaphthenebutyryl, a
(-)-2-oxo-4-thiazolidinecarboxyl group, a tetrahydro-3-furoyl
group, a 2-iminobiotinyl group, a diethylenetriaminepentaacetyl
group, a 4-morpholinecarbonyl group, a 2-thiopheneacetyl group or a
2-thiophenesulfonyl group.
[0096] A CXCR4 antagonist compound of the invention may be further
modified to alter the specific properties of the compound while
retaining the desired functionality of the compound. For example,
in one embodiment, the compound may be modified to alter a
pharmacokinetic property of the compound, such as in vivo
stability, bioavailability or half-life. The compound may be
modified to label the compound with a detectable substance. The
compound may be modified to couple the compound to an additional
therapeutic moiety. To further chemically modify the compound, such
as to alter its pharmacokinetic properties, reactive groups can be
derivatized. For example, when the modifying group is attached to
the amino-terminal end of the SDF-1 core domain, the
carboxy-terminal end of the compound may be further modified.
Potential C-terminal modifications include those that reduce the
ability of the compound to act as a substrate for
carboxypeptidases. Examples of C-terminal modifiers include an
amide group, an ethylamide group and various non-natural amino
acids, such as D-amino acids and .beta.-alanine. Alternatively,
when the modifying group is attached to the carboxy-terminal end of
the aggregation core domain, the amino-terminal end of the compound
may be further modified, for example, to reduce the ability of the
compound to act as a substrate for aminopeptidases.
[0097] A CXCR4 antagonist compound can be further modified to label
the compound by reacting the compound with a detectable substance.
Suitable detectable substances include various enzymes, prosthetic
groups, fluorescent materials, luminescent materials and
radioactive materials. Examples of suitable enzymes include
horseradish peroxidase, alkaline phosphatase, beta-galactosidase,
or acetylcholinesterase; examples of suitable prosthetic group
complexes include streptavidin/biotin and avidin/biotin; examples
of suitable fluorescent materials include umbelliferone,
fluorescein, fluorescein isothiocyanate, rhodamine,
dichlorotriazinylamine fluorescein, dansyl chloride or
phycoerythrin; an example of a luminescent material includes
luminol; and examples of suitable radioactive material include
.sup.14C, .sup.123I, .sup.124I, .sup.125I, .sup.131I, .sup.99mTc,
.sup.35S or .sup.3H. A CXCR4 antagonist compound may be
radioactively labeled with .sup.14C, either by incorporation of
.sup.14C into the modifying group or one or more amino acid
structures in the CXCR4 antagonist compound. Labeled CXCR4
antagonist compounds may be used to assess the in vivo
pharmacokinetics of the compounds, as well as to detect disease
progression or propensity of a subject to develop a disease, for
example for diagnostic purposes. Tissue distribution CXCR4
receptors can be detected using a labeled CXCR4 antagonist compound
either in vivo or in an in vitro sample derived from a subject. For
use as an in vivo diagnostic agent, a CXCR4 antagonist compound of
the invention may be labeled with radioactive technetium or iodine.
A modifying group can be chosen that provides a site at which a
chelation group for the label can be introduced, such as the Aic
derivative of cholic acid, which has a free amino group. For
example, a phenylalanine residue within the SDF-1 sequence (such as
amino acid residue 13) may be substituted with radioactive
iodotyrosyl. Any of the various isotopes of radioactive iodine may
be incorporated to create a diagnostic agent. .sup.123I
(half-life=13.2 hours) may be used for whole body scintigraphy,
.sup.124I (half life=4 days) may be used for positron emission
tomography (PET), .sup.125I (half life=60 days) may be used for
metabolic turnover studies and .sup.131I (half life=8 days) may be
used for whole body counting and delayed low resolution imaging
studies.
[0098] In an alternative chemical modification, a CXCR4 antagonist
compound of the invention may be prepared in a "prodrug" form,
wherein the compound itself does not act as a CXCR4 antagonist, but
rather is capable of being transformed, upon metabolism in vivo,
into a CXCR4 antagonist compound as defined herein. For example, in
this type of compound, the modifying group can be present in a
prodrug form that is capable of being converted upon metabolism
into the form of an active CXCR4 antagonist. Such a prodrug form of
a modifying group is referred to herein as a "secondary modifying
group." A variety of strategies are known in the art for preparing
peptide prodrugs that limit metabolism in order to optimize
delivery of the active form of the peptide-based drug (see e.g.,
Moss, J. (1995) in Peptide-Based Drug Design: Controlling Transport
and Metabolism, Taylor, M. D. and Amidon, G. L. (eds), Chapter
18.
[0099] CXCR4 antagonist compounds of the invention may be prepared
by standard techniques known in the art. A peptide component of a
CXCR4 antagonist may be composed, at least in part, of a peptide
synthesized using standard techniques (such as those described in
Bodansky, M. Principles of Peptide Synthesis, Springer Verlag,
Berlin (1993); Grant, G. A. (ed.). Synthetic Peptides: A User's
Guide, W. H. Freeman and Company, New York (1992); or Clark-Lewis,
I., Dewald, B., Loetscher, M., Moser, B., and Baggiolini, M.,
(1994) J. Biol. Chem., 269, 16075-16081). Automated peptide
synthesizers are commercially available (e.g., Advanced ChemTech
Model 396; Milligen/Biosearch 9600). Peptides may be assayed for
CXCR4 antagonist activity in accordance with standard methods.
Peptides may be purified by HPLC and analyzed by mass spectrometry.
Peptides may be dimerized via a disulfide bridge formed by gentle
oxidation of the cysteines using 10% DMSO in water. Following HPLC
purification dimer formation may be verified, by mass spectrometry.
One or more modifying groups may be attached to a SDF-1 derived
peptidic component by standard methods, for example using methods
for reaction through an amino group (e.g., the alpha-amino group at
the amino-terminus of a peptide), a carboxyl group (e.g., at the
carboxy terminus of a peptide), a hydroxyl group (e.g., on a
tyrosine, serine or threonine residue) or other suitable reactive
group on an amino acid side chain (see e.g., Greene, T. W. and
Wuts, P. G. M. Protective Groups in Organic Synthesis, John Wiley
and Sons, Inc., New York (1991)).
[0100] In another aspect of the invention, CXCR4 antagonist
peptides may be prepared according to standard recombinant DNA
techniques using a nucleic acid molecule encoding the peptide. A
nucleotide sequence encoding the peptide may be determined using
the genetic code and an oligonucleotide molecule having this
nucleotide sequence may be synthesized by standard DNA synthesis
methods (e.g., using an automated DNA synthesizer). Alternatively,
a DNA molecule encoding a peptide compound may be derived from the
natural precursor protein gene or cDNA (e.g., using the polymerase
chain reaction (PCR) and/or restriction enzyme digestion) according
to standard molecular biology techniques.
[0101] The invention also provides an isolated nucleic acid
molecule comprising a nucleotide sequence encoding a peptide of the
invention. In some embodiments, the peptide may comprise an amino
acid sequence having at least one amino acid deletion compared to
native SDF-1. The term "nucleic acid molecule" is intended to
include DNA molecules and RNA molecules and may be single-stranded
or double-stranded. In alternative embodiments, the isolated
nucleic acid encodes a peptide wherein one or more amino acids are
deleted from the N-terminus, C-terminus and/or an internal site of
SDF-1.
[0102] To facilitate expression of a peptide compound in a host
cell by standard recombinant DNA techniques, the isolated nucleic
acid encoding the peptide may be incorporated into a recombinant
expression vector. Accordingly, the invention also provides
recombinant expression vectors comprising the nucleic acid
molecules of the invention. As used herein, the term "vector"
refers to a nucleic acid molecule capable of transporting another
nucleic acid to which it has been operatively linked. Vectors may
include circular double stranded DNA plasmids, viral vectors.
Certain vectors are capable of autonomous replication in a host
cell into which they are introduced (such as bacterial vectors
having a bacterial origin of replication and episomal mammalian
vectors). Other vectors (such as non-episomal mammalian vectors)
may be integrated into the genome of a host cell upon introduction
into the host cell, and thereby may be replicated along with the
host genome. Certain vectors may be capable of directing the
expression of genes to which they are operatively linked. Such
vectors are referred to herein as "recombinant expression vectors"
or "expression vectors".
[0103] In recombinant expression vectors of the invention, the
nucleotide sequence encoding a peptide may be operatively linked to
one or more regulatory sequences, selected on the basis of the host
cells to be used for expression. The terms "operatively linked" or
"operably" linked mean that the sequences encoding the peptide are
linked to the regulatory sequence(s) in a manner that allows for
expression of the peptide compound. The term "regulatory sequence"
includes promoters, enhancers, polyadenylation signals and other
expression control elements. Such regulatory sequences are
described, for example, in Goeddel; Gene Expression Technology:
Methods in Enzymology 185, Academic Press, San Diego, Calif. (1990)
(incorporated herein be reference). Regulatory sequences include
those that direct constitutive expression of a nucleotide sequence
in many types of host cell, those that direct expression of the
nucleotide sequence only in certain host cells (such as
tissue-specific regulatory sequences) and those that direct
expression in a regulatable manner (such as only in the presence of
an inducing agent). The design of the expression vector may depend
on such factors as the choice of the host cell to be transformed
and the level of expression of peptide compound desired.
[0104] The recombinant expression vectors of the invention may be
designed for expression of peptide compounds in prokaryotic or
eukaryotic cells. For example, peptide compounds may be expressed
in bacterial cells such as E. coli, insect cells (using baculovirus
expression vectors) yeast cells or mammalian cells. Suitable host
cells are discussed further in Goeddel, Gene Expression Technology:
Methods in Enzymology 185, Academic Press, San Diego, Calif.
(1990). Alternatively, the recombinant expression vector may be
transcribed and translated in vitro, for example using T7 promoter
regulatory sequences and T7 polymerase. Examples of vectors for
expression in yeast S. cerivisae include pYepSec1 (Baldari et al.,
(1987) EMBO J. 6:229-234), pMFa (Kurjan and Herskowitz, (1982) Cell
30:933-943), pJRY88 (Schultz et al., (1987) Gene 54:113-123), and
pYES2 (Invitrogen Corporation, San Diego, Calif.). Baculovirus
vectors available for expression of proteins or peptides in
cultured insect cells (e.g., Sf 9 cells) include the pAc series
(Smith et al., (1983) Mol. Cell. Biol. 3:2156-2165) and the pVL
series (Lucklow, V. A., and Summers, M. D., (1989) Virology
170:31-39). Examples of mammalian expression vectors include pCDM8
(Seed, B., (1987) Nature 329:840) and pMT2PC (Kaufman et al.
(1987), EMBO J. 6:187-195). When used in mammalian cells, the
expression vector's control functions are often provided by viral
regulatory elements. For example, commonly used promoters are
derived from polyoma, Adenovirus 2, cytomegalovirus and Simian
Virus 40.
[0105] In addition to regulatory control sequences, recombinant
expression vectors may contain additional nucleotide sequences,
such as a selectable marker gene to identify host cells that have
incorporated the vector. Selectable marker genes are well known in
the art. To facilitate secretion of the peptide compound from a
host cell, in particular mammalian host cells, the recombinant
expression vector preferably encodes a signal sequence operatively
linked to sequences encoding the amino-terminus of the peptide
compound, such that upon expression, the peptide compound is
synthesised with the signal sequence fused to its amino terminus.
This signal sequence directs the peptide compound into the
secretory pathway of the cell and is then cleaved, allowing for
release of the mature peptide compound (i.e., the peptide compound
without the signal sequence) from the host cell. Use of a signal
sequence to facilitate secretion of proteins or peptides from
mammalian host cells is well known in the art.
[0106] A recombinant expression vector comprising a nucleic acid
encoding a peptide compound may be introduced into a host cell to
produce the peptide compound in the host cell. Accordingly, the
invention also provides host cells containing the recombinant
expression vectors of the invention. The terms "host cell" and
"recombinant host cell" are used interchangeably herein. Such terms
refer not only to the particular subject cell but to the progeny or
potential progeny of surh a cell. Because certain modifications may
occur in succeeding generations due to either mutation or
environmental influences, such progeny may not, in fact, be
identical to the parent cell, but are still included within the
scope of the term as used herein. A host cell may be any
prokaryotic or eukaryotic cell. For example, a peptide compound may
be expressed in bacterial cells such as E. coli, insect cells,
yeast or mammalian cells. The peptide compound may be expressed in
vivo in a subject to the subject by gene therapy (discussed further
below).
[0107] Vector DNA can be introduced into prokaryotic or eukaryotic
cells via conventional transformation, transfection or infection
techniques. The terms "transformation", "transfection" or
"infection" refer to techniques for introducing foreign nucleic
acid into a host cell, including calcium phosphate or calcium
chloride co-precipitation, DEAE-dextran-mediated transfection,
lipofection, electroporation, microinjection and viral-mediated
infection. Suitable methods for transforming, transfecting or
infecting host cells can for example be found in Sambrook et al.
(Molecular Cloning: A Laboratory Manual, 2nd Edition, Cold Spring
Harbor Laboratory press (1989)), and other laboratory manuals.
Methods for introducing DNA into mammalian cells in vivo are also
known, and may be used to deliver the vector DNA of the invention
to a subject for gene therapy.
[0108] For stable transfection of mammalian cells, it is known
that, depending upon the expression vector and transfection
technique used, only a small fraction of cells may integrate the
foreign DNA into their genome. In order to identify and select
these integrants, a gene that encodes a selectable marker (such as
resistance to antibiotics) may be introduced into the host cells
along with the gene of interest. Preferred selectable markers
include those that confer resistance to drugs, such as G418,
hygromycin and methotrexate. Nucleic acids encoding a selectable
marker may be introduced into a host cell on the same vector as
that encoding the peptide compound or may be introduced on a
separate vector. Cells stably transfected with the introduced
nucleic acid may be identified by drug selection (cells that have
incorporated the selectable marker gene will survive, while the
other cells die).
[0109] A nucleic acid of the invention may be delivered to cells in
vivo using methods such as direct injection of DNA,
receptor-mediated DNA uptake or viral-mediated transfection. Direct
injection has been used to introduce naked DNA into cells in vivo
(see e.g., Acsadi et al. (1991) Nature 332:815-818; Wolff et al.
(1990) Science 247:1465-1468). A delivery apparatus (e.g., a "gene
gun") for injecting DNA into cells in vivo may be used. Such an
apparatus may be commercially available (e.g., from BioRad). Naked
DNA may also be introduced into cells by complexing the DNA to a
cation, such as polylysine, which is coupled to a ligand for a
cell-surfacc receptor (see for example Wu, G. and Wu, C. H. (1988)
J. Biol. Chem. 263:14621; Wilson el al. (1992) J. Biol. Chem.
267:963-967; and U.S. Pat. No. 5,166,320). Binding of the
DNA-ligand complex to the receptor may facilitate uptake of the DNA
by receptor-mediated endocytosis. A DNA-ligand complex linked to
adenovirus capsids which disrupt endosomes, thereby releasing
material into the cytoplasm, may be used to avoid degradation of
the complex by intracellular lysosomes (see for example Curiel el
al. (1991) Proc. Natl. Acad. Sci. USA 88:8850; Cristiano et al.
(1993) Proc. Natl. Acad. Sci. USA 90:2122-2126).
[0110] Defective retroviruses are well characterized for use in
gene transfer for gene therapy purposes (for reviews see Miller, A.
D. (1990) Blood 76:271, Kume et al. (1999) International. J.
Hematol. 69:227-233). Protocols for producing recombinant
retroviruses and for infecting cells in vitro or in vivo with such
viruses can be found in Current Protocols in Molecular Biology,
Ausubel, F. M. et al. (eds.) Greene Publishing Associates, (1989),
Sections 9.10-9.14 and other standard laboratory manuals. Examples
of suitable retroviruses include pLJ, pZIP, pWE and pEM, which are
well known to those skilled in the art. Examples of suitable
packaging virus lines include .p.psi.i.Crip, .p.psi.i.Cre,
.p.psi.i.2 and .p.psi.i.Am. Retroviruses have been used to
introduce a variety of genes into many different cell types,
including epithelial cells, endothelial cells, lymphocytes,
myoblasts, hepatocytes, bone marrow cells, in vitro and/or in vivo
(see for example Eglitis, et al. (1985) Science 230:1395-1398;
Danos and Mulligan (1988) Proc. Natl. Acad. Sci. USA 85:6460-6464;
Wilson et al. (1988) Proc. Natl. Acad. Sci. USA 85:3014-3018;
Armentano et al. (1990) Proc. Natl. Acad. Sci. USA 87:6141-6145;
Huber et al. (1991) Proc. Natl. Acad. Sci. USA 88:8039-8043; Ferry
et al. (1991) Proc. Natl. Acad. Sci. USA 88:8377-8381; Chowdhury et
al. (1991) Science 254:1802-1805; van Beusechem et al. (1992) Proc.
Natl. Acad. Sci. USA 89:7640-7644; Kay et al. (1992) Human Gene
Therapy 3:641-647; Dai et al. (1992) Proc. Natl. Acad. Sci. USA
89:10892-10895; Hwu et al. (1993) J. Immunol. 150:41044115; U.S.
Pat. No. 4,868,116; U.S. Pat. No. 4,980,286; PCT Application WO
89/07136; PCT Application WO 89/02468; PCT Application WO 89/05345;
and PCT Application WO 92/07573). In various embodiments, a genome
of a retrovirus that encodes and expresses a polypeptide compound
of the invention, may be utilized for the propagation and/or
survival of cells, such as hematopoietic progenitor stem cells, for
the purposes of maintaining and/or growing cells for the clinical
purposes of blood transfusion or engraftment, host conditioning or
applications relevant to chemotherapy, radiation therapy or
myeloablative therapy.
[0111] For use as a gene therapy vector, the genome of an
adenovirus may be manipulated so that it encodes and expresses a
peptide compound of the invention, but is inactivated in terms of
its ability to replicate in a normal lytic viral life cycle. See
for example Berkner et al. (1988) BioTechniques 6:616; Rosenfeld et
al. (1991) Science 252:431-434; and Rosenfeld et al. (1992) Cell
68:143-155. Suitable adenoviral vectors derived from the adenovirus
strain Ad type 5 dl324 or other strains of adenovirus (e.g., Ad2,
Ad3, Ad7 etc.) are well known to those skilled in the art.
Recombinant adenoviruses are advantageous in that they do not
require dividing cells to be effective gene delivery vehicles and
can be used to infect a wide variety of cell types, including
airway epithelium (Rosenfeld et al. (1992) cited supra),
endothelial cells (Lemarchand et al. (1992) Proc. Natl. Acad. Sci.
USA 89:6482-6486), hepatocytes (Herz and Gerard (1993) Proc. Natl.
Acad. Sci. USA 90:2812-2816) and muscle cells (Quantin el al.
(1992) Proc. Natl. Acad. Sci. USA 89:2581-2584). In various
embodiments, a genome of an adenovirus that encodes and expresses a
polypeptide compound of the invention, may be utilized for the
propagation and/or survival of cells, such as hematopoietic
progenitor stem cells, stromal cells, or mesenchymal cells, for the
purposes of maintaining and/or growing cells for the clinical
purposes of blood transfusion or engraftment, host conditioning or
applications relevant to chemotherapy, radiation therapy or
myeloablative therapy.
[0112] In some embodiments, adeno-associated virus (AAV) may be
used as a gene therapy vector for delivery of DNA for gene therapy
purposes. AAV is a naturally occurring defective virus that
requires another virus, such as an adenovirus or a herpes virus, as
a helper virus for efficient replication and a productive life
cycle (Muzyczka et al. Curr. Topics in Micro. and Immunol. (1992)
158:97-129). AAV may be used to integrate DNA into non-dividing
cells (see for example Flotte et al. (1992) Am. J. Respir. Cell.
Mol. Biol. 7:349-356; Samulski et al. (1989) J. Virol.
63:3822-3828; and McLaughlin et al. (1989) J. Virol. 62:1963-1973).
An MV vector such as that described in Tratschin et al. (1985) Mol.
Cell. Biol. 5:3251-3260 may be used to introduce DNA into cells
(see for example Hermonat et al. (1984) Proc. Natl. Acad. Sci. USA
81:6466-6470; Tratschin et al. (1985) Mol. Cell. Biol. 4:2072-2081;
Wondisford et al. (1988) Mol. Endocrinol. 2:32-39; Tratschin et al.
(1984) J. Virol. 51:611-619; and Flotte et al. (1993) J. Biol.
Chem. 268:3781-3790). In some embodiments, a genome of an MV that
encodes and expresses a polypeptide compound of the invention, may
be utilized for the propagation and/or survival of cells, such as
hematopoietic progenitor stem cells, stromal cells or mesenchymal
cells, for the purposes of maintaining and/or growing cells for the
clinical purposes of blood transfusion or engraftment, host
conditioning or applications relevant to chemotherapy, radiation
therapy or myeloablative therapy.
[0113] General methods for gene therapy are known in the art. See
for example, U.S. Pat. No. 5,399,346 by Anderson et al. A
biocompatible capsule for delivering genetic material is described
in PCT Publication WO 95/05452 by Baetge et al. Methods for
grafting genetically modified cells to treat central nervous system
disorders are described in U.S. Pat. No. 5,082,670 and in PCT
Publications WO 90/06757 and WO 93/10234, all by Gage et al.
Methods of gene transfer into hematopoietic cells have also
previously been reported (see Clapp, D. W., et al., Blood 78:
1132-1139 (1991); Anderson, Science 288:627-9 (2000); and,
Cavazzana-Calvo et al., Science 288:669-72 (2000), all of which are
incorporated herein by reference).
[0114] Cancers susceptible to treatment with CXCR4 antagonists in
accordance with various aspects of the invention may include both
primary and metastatic tumors, such as solid tumors, including
carcinomas of the breast, colon, rectum, oropharynx, hypopharynx,
esophagus, stomach, pancreas, liver, gall bladder and bile ducts,
small intestine, urinary tract (including kidney, bladder, and
urothelium), female genital tract (including cervix, uterus, and
ovaries as well as choriocarcinoma and gestational trophoblast
disease), male genital tract (including prostate, seminal vesicles,
testes, and germ cell tumors), endocrine glands (including the
thyroid, adrenal and pituitary glands), and skin, as well as
hemangiomas, melanomas, sarcomas (including those arising from bone
and soft tissues as well as Kaposi's sarcoma) and tumors of the
brain, nerves, eyes, and meninges (including astrocytomas, gliomas,
retinoblastomas, neuromas, neuroblastomas, Schwannomas, and
meningiomas). In some aspects of the invention, CXCR4 antagonists
may also serve in treating solid tumors arising from hematopoietic
malignancies such as leukemias (i.e., chloromas, plasmacytomas and
the plaques and tumors of mycosis fungoides and cutaneous T-cell
lymphoma/leukemia) as well as in the treatment of lymphoma (both
Hodgkin's and non-Hodgkin's lymphomas). In addition, CDCR4
antagonists may be therapeutic in the prevention of metastasis from
the tumors described above either when used alone or in combination
with cytotoxic agents such as radiotherapy or chemotherapeutic
agents (for instance refer to Zlotnik et al., Nature 410, 50-56,
2001).
[0115] In alternative aspects of the invention, CXCR4 antagonists
such as SDF-1 polypeptides and non-peptide small molecule
antagonists may target CD34.sup.+ cells to mediate release of
CD34.sup.+ cells to the peripheral blood. In these aspects of the
invention, CXCR4 antagonists may enhance circulating CD34.sup.+
cell proliferation and hematopoietic stem or progenitor cell
survival or levels, which may for example be useful in stem cell
transplantation or ex vivo expansion. Furthermore, CXCR4
antagonists may enhance hematopoietic stem or progenitor cell
mobilization.
[0116] In various aspects of the invention, CXCR4 antagonists may
be used in maintaining or augmenting the rate of hematopoietic cell
multiplication. Method of the invention may comprise administration
of an effective amount of CXCR4 antagonists to cells selected from
the group consisting of hematopoietic stem cells and hematopoietic
progenitor cells, stromal cells or mesenchymal cells. In
alternative embodiments, a therapeutically effective amount of the
CXCR4 antagonist may be administered to a patient in need of such
treatment. Patients in need of such treatments may include, for
example: patients having cancer, patients having an autoimmune
disease, patients requiring functional gene transfer into
hematopoietic stems cells, stromal cells or mesenchymal cells (such
as for the dysfunction of any tissue or organ into which a stem
cell may differentiate), patients requiring lymphocyte depletion,
patients requiring depletion of a blood cancer in the form of
purging autoreactive or cancerous cells using autologous or
aligenic grafts, or patients requiring autologous peripheral blood
stem cell transplantation. A patient in need of treatment in
accordance with the invention may also be receiving cytotoxic
treatments such as chemotherapy or radiation therapy. In some
embodiments, CXCR4 antagonists may be used in treatment to purge an
ex vivo hematopoietic stem cell culture of cancer cells with
cytotoxic treatment, while preserving the viability and
self-renewal of the hematopoietic progenitor or stem cells.
[0117] In alternative aspects the methods of treatment of the
invention may be utilized where a patient is undergoing
myelosuppressive treatment causing hematopoietic cell depletion,
including pancytopenia, granulocytopenia, thrombocytopenia, anemia
or a combination thereof. In further alternative embodiments, the
patient to be treated may be suffering from AIDS, and the treatment
may for example be effected to augment hematopoietic cell
counts.
[0118] Although various embodiments of the invention are disclosed
herein, many adaptations and modifications may be made within the
scope of the invention in accordance with the common general
knowledge of those skilled in this art. Such modifications include
the substitution of known equivalents for any aspect of the
invention in order to achieve the same result in substantially the
same way. Numeric ranges are inclusive of the numbers defining the
range. In the claims, the word "comprising" is used as an
open-ended term, substantially equivalent to the phrase "including,
but not limited to". The disclosed uses for various embodiments are
not necessarily obtained in all embodiments, and the invention may
be adapted by those skilled in the art to obtain alternative
utilities.
EXAMPLES
[0119] The following examples illustrate, but do not limit, the
present invention.
Example 1
[0120] FIG. 1 shows the results of CXCR4 receptor binding assay. To
obtain the results, antagonists (competing ligands) (20 M) were
added to 5.times.10.sup.6 CEM cell/ml in the presence of 4 nM
1251-SDF-1. CEM cells were assessed for 125I-SDF-1 binding
following 2 hr incubation. The results are expressed as percentages
of the maximal specific binding in the absence of a competing
ligand, and are the mean of three independent experiments. In FIG.
1, the antagonists tested were:
12 CTCE0012:
KGVSLSYRCPCRFFESHVARANVKHLKILNTPACALQIVARLKNNNRQVCIDPK- LKWI
QEYLEKALN-COOH CTCE9908: [KGVSLSYR].sub.2-K--CONH.sub.2 CTCE9907:
KGVSLSYRC(CONH.sub.2)-(CONH.sub.2)RYSLSVGK CTCE0014:
KGVSLSYRCPCRFF-GGGG- LKWIQEYLEKALN- COOH CTCE0018:
KGVSLSYRCPCRFF-GGGG- LKWIQEYLEKALN- CONH.sub.2 CTCE0019:
KGVSLSYRCPCRFF-GGGG- LKWIQEYLEKALN- CONH.sub.2 K20/E24
lactamization CTCE0020: KGVSLSYRCPCRFF-GGGG- LKWIQEYLEKALN-
CONH.sub.2 K28/E24 lactamization CTCE0016: KGVSLSYRCPCRFFESH-GGGG-
LKWIQEYLEKALN- COOH
Example 2
[0121] Table 1 shows the effect of CXCR4 antagonists on
hematopoietic cells, particularly primitive erythroide cells and
primitive granulocytes (hematopoietic progenitor cells), compared
to mature granulocytes. To obtain the data in Table 1, cells were
pre-incubated with each of the compounds or saline alone (as
control). The cells were then exposed to high dose
H.sup.3-thymidine, a cytotoxic agent. Rapidly dividing cells
accumulate proportionally more of the cytotoxic radioactive
thymidine and as a result are preferentially killed. The relative
proportion of cells killed by the thymidine treatment compared to
the control is indicative of the relative effectiveness of the
compounds in increasing cellular multiplication, i.e. increasing
the rate of cell cycle progression and DNA synthesis. A higher
proportion of killed cells compared to the control is indicative
that a compound increases cellular multiplication of the given cell
type.
13TABLE 1 Effect of CXCR4 Peptide Antagonists on the Cycling of
Bone Marrow Progenitor Cells Exposed to H.sup.3-Thymidine (% Cells
Killed). % Kill After 3H-Thymidine Treatment Dose (.mu.g/ml) BFU-E
CFU-GM None 10 3 +/- 2 3 +/- 3 SDF-1 (G2) 10 48 +/- 5 38 +/- 4
CTCE9907 50 39 +/ 7 28 +/- 6 CTCE9908 50 51 +/- 7 36 +/- 6 CTCE0012
10 60 +/- 8 44 +/- 4 CTCE0016 10 63 +/- 5 54 +/- 4 CTCE0017 50 57
+/- 3 52 +/- 6
[0122] In Table 1, SDF-1 (G2) is the peptide
KGVSLSYRCPCRFFESHVARANVKHLKIL-
NTPACALQIVARLKNNNRQVCIDPKLKWIQEYLEKALN-COOH, CTCE9907 is the
peptide [KGVSLSYRC-CONH.sub.2].sub.2, CTCE9908 is the peptide
[KGVSLSYR].sub.2K-CONH.sub.2, CTCE0012 is the peptide
KGVSLSYRCPCRFFESHVARANVKHLKILNTPACALQIVARLKNNNRQVCIDPKLKWIQEYLEKALN-COOH,
CTCE0016 is the peptide KGVSLSYRCPCRFFESH-GGGG-LKWIQEYLEKALN-COOH,
and CTCE0017 is the peptide
KGVSLSYRCPCRFF-GGGG-LKWIQEYLEKALN-CONH.sub.2
Example 3
[0123] FIG. 2 shows the efficacy of CXCR4 antagonists on enhancing
the proliferation of human progenitor cells in an in vivo
engraftment model.
[0124] In FIG. 2, the cycling status of mature and primitive colony
forming cells (CFU-GM; colony forming unit-granulocyte-monocyte
precursor, BFU-E; burst forming unit-erythroid precursor; LTC-IC,
long-term culture initiating cell) in the suspension of CD34 cells
isolated from the marrow of transplanted NOD/SCID mice was
determined by assessing the proportion of these progenitors that
were inactivated (killed) by short term (20 min) or overnight (16
hour) exposure of the cells to 20.mu.g/ml of high specific activity
.sup.3H-thymidine (values represent the mean +/-the S.D. of data
from up to four experiments with up to four mice per point in
each). Significant in the results is the observation that the SDF-1
peptide antagonists are effective at enhancing the proliferation of
"primitive" human progenitor cells, as measured by the reduction of
cells killed by exposure to high specific activity
.sup.3H-thymidine (which only affects proliferating cells).
[0125] In FIG. 2, the Control represents untreated cells, CTCE9907
is the peptide [KGVSLSYRC-CONH.sub.2].sub.2, CTCE9908 is the
peptide [KGVSLSYR].sub.2K-CONH.sub.2, CTCE0012 is the peptide
KGVSLSYRCPCRFFESHVARANVKHLKILNTPACALQIVARLKNNNRQVCIDPKLKWQEYLEKALN-COOH,
CTCE0016 is the peptide KGVSLSYRCPCRFFESH-GGGG-LKWIQEYLEKALN-COOH,
and CTCE0017 is the peptide
KGVSLSYRCPCRFF-GGGG-LKWIQEYLEKALN-CONH.sub.2
Example 4
[0126] FIG. 3. This example illustrates the effect of CXCR4 peptide
antagonists on the engraftment of human cells in human fetal liver
transplanted NODISCID mice. The frequency of the phenotypically
defined human hematopoietic cells detected in the long bones
(tibias and femurs) of mice was determined. Administration of 0.5
mg/kg of SDF-1 had no significant effect on the number of CD45/71,
CD19/20, or CD34 cells, nor on the CFC or LTC-IC. In addition, none
of the human cell types were detectably affected by this schedule
of CXCR4 agonist administration. This data indicates that SDF-1
peptide antagonists may effectively augment secondary engraftment
of human progenitor cells, and that these compounds are essentially
not toxic to the animals at the indicated doses.
[0127] In FIG. 3, the Control represents untreated cells, CTCE9907
is the peptide [KGVSLSYRC-CONH.sub.2].sub.2, CTCE9908 is the
peptide [KGVSLSYR].sub.2K-CONH.sub.2, CTCE0012 is the peptide
KGVSLSYRCPCRFFESHVARANVKHLKILNTPACALQIVARLKNNNRQVCIDPKLKWIQEYLEKALN-COOH,
CTCE0016 is the peptide KGVSLSYRCPCRFFESH-GGGG-LKWIQEYLEKALN-COOH,
and CTCE0017 is the peptide
KGVSLSYRCPCRFF-GGGG-LKWIQEYLEKALN-CONH.sub.2
* * * * *