U.S. patent application number 09/299473 was filed with the patent office on 2002-07-25 for method for stimulating hematopoiesis using tgf-alpha.
Invention is credited to FELKER, THOMAS S., PASKELL, STEFAN L., TWARDZIK, DANIEL R..
Application Number | 20020099008 09/299473 |
Document ID | / |
Family ID | 23154954 |
Filed Date | 2002-07-25 |
United States Patent
Application |
20020099008 |
Kind Code |
A1 |
TWARDZIK, DANIEL R. ; et
al. |
July 25, 2002 |
METHOD FOR STIMULATING HEMATOPOIESIS USING TGF-ALPHA
Abstract
There is disclosed a novel genus of small peptides, much smaller
than TGF.alpha., was discovered as having TGF.alpha. biological
activity and therefore are useful as pharmacologic agents for the
same indications as full length TGF.alpha. polypeptide. There is
further disclosed that TGF.alpha. and consequently the genus of
small peptides disclosed herein, was found to have therapeutic
activity to stimulate hematopoiesis in patients undergoing
cytotoxic cancer chemotherapy and to act as a cytoprotective agent
to protect a patient undergoing cancer cytotoxic therapy from
gastrointestinal (GI) side effects, such as mucositis and otherwise
support the barrier function of the GI tract when it is harmed by
cytotoxic therapy.
Inventors: |
TWARDZIK, DANIEL R.;
(BAINBRIDGE ISLAND, WA) ; FELKER, THOMAS S.;
(VASHON, WA) ; PASKELL, STEFAN L.; (BAINBRIDGE
ISLAND, WA) |
Correspondence
Address: |
LISA A. HAILE, PH.D.
GRAY CARY WARE & FREINDENRICH LLP
4365 EXECUTIVE DRIVE
SUITE 1600
SAN DIEGO
CA
92121
US
|
Family ID: |
23154954 |
Appl. No.: |
09/299473 |
Filed: |
April 26, 1999 |
Current U.S.
Class: |
514/7.9 ;
514/12.2; 514/8.9; 530/351 |
Current CPC
Class: |
A61P 31/12 20180101;
A61P 37/00 20180101; A61P 25/00 20180101; A61P 25/18 20180101; A61P
31/18 20180101; A61P 25/28 20180101; A61P 25/16 20180101; A61P
21/00 20180101; A61K 38/00 20130101; A61P 27/02 20180101; C07K
14/495 20130101; A61P 25/32 20180101; A61P 1/02 20180101; A61P 3/10
20180101; A61P 1/04 20180101; A61P 25/14 20180101; A61P 9/10
20180101; A61P 1/14 20180101; A61P 27/06 20180101; A61P 31/04
20180101; Y10S 930/12 20130101; A61P 35/00 20180101; A61P 43/00
20180101; A61P 7/00 20180101; A61P 31/22 20180101; A61P 19/00
20180101; A61K 48/00 20130101; A61P 39/02 20180101 |
Class at
Publication: |
514/12 ; 514/2;
530/351 |
International
Class: |
A61K 038/00; A01N
037/18; C07K 001/00; C07K 014/00; C07K 017/00 |
Claims
We claim:
1. A compound that acts as a TGF.alpha. mimetic, comprising at
least an 11-membered peptide compound from formula I:
N--X.sub.1a-Cys-His-Ser-X.su-
b.1b--X.sub.2--X.sub.1a--X.sub.1b--X.sub.1a--X.sub.3-Cys COOH I
wherein X.sub.1 is independently Val, Gly or Ala, wherein X.sub.2
is Try or Phe, wherein X.sub.3 is Arg or Lys, and wherein the two
Cys moieties form a disulfide bond to create an 11-amino acid loop
peptide.
2. The compound of claim 1 wherein at least one or more of the
following seven amino acids are added to the C terminus Cys moiety
from formula II:
--X.sub.4-His-X.sub.1c--X.sub.4--X.sub.5--X.sub.6--X.sub.1c II
wherein X.sub.4 is Glu or Asp, wherein X.sub.5 is Leu or Ile, and
wherein X.sub.6 is Asp or Glu.
3. The compound of claim 1 wherein X.sub.1a is Val, X.sub.1b is Gly
and X.sub.1c is Ala.
4. The compound of claim 2 wherein X.sub.2 is Tyr, and X.sub.3 is
Arg.
5. The compound of claim 2 wherein the loop peptide is 13 amino
acids in length, wherein X.sub.1a is Val, X.sub.1b is Gly, X.sub.1c
is Ala, and X.sub.4 is Gly.
6. A pharmaceutical composition comprising a loop peptide in a
pharmaceutically acceptable carrier, wherein the loop peptide
compound comprises at least an 11-membered peptide compound from
formula I:
N--X.sub.1a-Cys-His-Ser-X.sub.1b--X.sub.2--X.sub.1a--X.sub.1b--X.sub.1a---
X.sub.3-Cys COOH I wherein X.sub.1 is independently Val, Gly or
Ala, wherein X.sub.2 is Try or Phe, wherein X.sub.3 is Arg or Lys,
and wherein the two Cys moieties form a disulfide bond to create an
11-amino acid loop peptide.
7. The pharmaceutical composition of claim 6 wherein at least one
or more of the following seven amino acids are added to the C
terminus Cys moiety from formula II:
--X.sub.4-His-X.sub.1c--X.sub.4--X.sub.5--X.sub.6--X.sub- .1c II
wherein X.sub.4 is Glu or Asp, wherein X.sub.5 is Leu or Ile, and
wherein X.sub.6 is Asp or Glu.
8. The pharmaceutical composition of claim 6 wherein X.sub.1a is
Val, X.sub.1b is Gly and X.sub.1c is Ala.
9. The pharmaceutical composition of claim 7 wherein X.sub.2 is
Tyr, and X.sub.3 is Arg.
10. The pharmaceutical composition of claim 7 wherein the loop
peptide is 13 amino acids in length wherein X.sub.1a is Val,
X.sub.1b is Gly, X.sub.1c is Ala, and X.sub.4 is Gly.
11. A method for treating a neurodegenerative disease with an
pharmaceutically active loop peptide, wherein the loop peptide
comprises at least an 11-membered peptide compound from formula I:
N--X.sub.1a-Cys-His-Ser-X.sub.1b--X.sub.2--X.sub.1a--X.sub.1b--X.sub.1a---
X.sub.3-Cys COOH I wherein X.sub.1 is independently Val, Gly or
Ala, wherein X.sub.2 is Try or Phe, wherein X.sub.3 is Arg or Lys,
and wherein the two Cys moieties form a disulfide bond to create an
11-amino acid loop peptide.
12. The method for treating a neurodegenerative disease of claim 11
wherein at least one or more of the following seven amino acids are
added to the C terminus Cys moiety from formula II:
--X.sub.4-His-X.sub.1c--X.s- ub.4--X.sub.5--X.sub.6--X.sub.1c II
wherein X.sub.4 is Glu or Asp, wherein X.sub.5 is Leu or Ile, and
wherein X.sub.6 is Asp or Glu.
13. The method for treating a neurodegenerative disease of claim 11
wherein X.sub.1a is Val, X.sub.1b is Gly and X.sub.1c is Ala.
14. The method for treating a neurodegenerative disease of claim 12
wherein X.sub.2 is Tyr, and X.sub.3 is Arg.
15. The method for treating a neurodegenerative disease of claim 12
wherein the loop peptide is 13 amino acids in length wherein
X.sub.1a is Val, X.sub.1b is Gly, X.sub.1c is Ala, and X.sub.4 is
Gly.
16. A method for treating a CNS disease or disorder, wherein the
CNS disease or disorder is selected from the group consisting of
CNS ischemia, spinal cord injury, MS, and retinal injury,
comprising with an pharmaceutically active loop peptide, wherein
the loop peptide comprises at least an 11-membered peptide compound
from formula I:
N--X.sub.1a-Cys-His-Ser-X.sub.1b--X.sub.2--X.sub.1a--X.sub.1b--X.sub.1a---
X.sub.3-Cys COOH I wherein X.sub.1 is independently Val, Gly or
Ala, wherein X.sub.2 is Try or Phe, wherein X.sub.3 is Arg or Lys,
and wherein the two Cys moieties form a disulfide bond to create an
11-amino acid loop peptide.
17. The method for treating a CNS disease or disorder of claim 16
wherein at least one or more of the following seven amino acids are
added to the C terminus Cys moiety from formula II:
--X.sub.4-His-X.sub.1c--X.sub.4--X- .sub.5--X.sub.6--X.sub.1c II
wherein X.sub.4 is Glu or Asp, wherein X.sub.5 is Leu or Ile, and
wherein X.sub.6 is Asp or Glu.
18. The method for treating a CNS disease or disorder of claim 16
wherein X.sub.1a is Val, X.sub.1b is Gly and X.sub.1c is Ala.
19. The method for treating a CNS disease or disorder of claim 17
wherein X.sub.2 is Tyr, and X.sub.3 is Arg.
20. The method for treating a CNS disease or disorder of claim 17
wherein the loop peptide is 13 amino acids in length wherein
X.sub.1a is Val, X.sub.1b is Gly, X.sub.1c is Ala, and X.sub.4 is
Gly.
21. A method for augmenting hematopoiesis during cytotoxic or
immune-suppressing therapy, comprising administering a TGF.alpha.
polypeptide or a pharmaceutically active loop peptide, or both,
wherein the loop peptide comprises at least an 11-membered peptide
compound from formula I:
N--X.sub.1a-Cys-His-Ser-X.sub.1b--X.sub.2--X.sub.1a--X.sub.1b--
-X.sub.1a--X.sub.3-Cys COOH I wherein X.sub.1 is independently Val,
Gly or Ala, wherein X.sub.2 is Try or Phe, wherein X.sub.3 is Arg
or Lys, and wherein the two Cys moieties form a disulfide bond to
create an 11-amino acid loop peptide.
22. The method for augmenting hematopoiesis during cytotoxic or
immune-suppressing therapy of claim 21 wherein at least one or more
of the following seven amino acids are added to the C terminus Cys
moiety from formula II:
--X.sub.4-His-X.sub.1c--X.sub.4--X.sub.5--X.sub.6--X.sub- .1c II
wherein X.sub.4 is Glu or Asp, wherein X.sub.5 is Leu or Ile, and
wherein X.sub.6 is Asp or Glu.
23. The method for augmenting hematopoiesis during cytotoxic or
immune-suppressing therapy of claim 21 wherein X.sub.1a is Val,
X.sub.1b is Gly and X.sub.1c is Ala.
24. The method for augmenting hematopoiesis during cytotoxic or
immune-suppressing therapy of claim 22 wherein X.sub.2 is Tyr, and
X.sub.3 is Arg.
25. The method for augmenting hematopoiesis during cytotoxic or
immune-suppressing therapy of claim 22 wherein the loop peptide is
13 amino acids in length wherein X.sub.1a is Val, X.sub.1b is Gly,
X.sub.1c is Ala, and X.sub.4 is Gly.
26. The method for augmenting hematopoiesis during cytotoxic or
immune-suppressing therapy of claim 21 further comprising
administering a second hematopoietic growth factor agent to
stimulate more mature hematopoietic precursor cells, wherein the
second hematopoietic growth factor is selected from the group
consisting of erythropoietin, thrombopoietin, G-CSF (granulocyte
colony stimulating factor), and GM-CSF (granulocyte macrophage
colony stimulating factor).
27. A method for treating or preventing mucositis of the
gastrointestinal tract during cytotoxic or immune-suppressing
therapy, comprising administering a TGF.alpha. polypeptide or a
pharmaceutically active loop peptide, or both, wherein the loop
peptide comprises at least an 11-membered peptide compound from
formula I: N--X.sub.1a-Cys-His-Ser-X.su-
b.1b--X.sub.2--X.sub.1a--X.sub.1b--X.sub.1a--X.sub.3-Cys COOH I
wherein X.sub.1 is independently Val, Gly or Ala, wherein X.sub.2
is Try or Phe, wherein X.sub.3 is Arg or Lys, and wherein the two
Cys moieties form a disulfide bond to create an 11-amino acid loop
peptide.
28. The method for treating or preventing mucositis of the
gastrointestinal tract during cytotoxic or immune-suppressing
therapy of claim 27 wherein at least one or more of the following
seven amino acids are added to the C terminus Cys moiety from
formula II:
--X.sub.4-His-X.sub.1c--X.sub.4--X.sub.5--X.sub.6--X.sub.1c II
wherein X.sub.4 is Glu or Asp, wherein X.sub.5 is Leu or Ile, and
wherein X.sub.6 is Asp or Glu.
29. The method for treating or preventing mucositis of the
gastrointestinal tract during cytotoxic or immune-suppressing
therapy of claim 27 wherein X.sub.1a is Val, X.sub.1b is Gly and
X.sub.1c is Ala.
30. The method for treating or preventing mucositis of the
gastrointestinal tract during cytotoxic or immune-suppressing
therapy of claim 28 wherein X.sub.2 is Tyr, and X.sub.3 is Arg.
31. The method for treating or preventing mucositis of the
gastrointestinal tract during cytotoxic or immune-suppressing
therapy of claim 28 wherein the loop peptide is 13 amino acids in
length wherein X.sub.1a is Val, X.sub.1b is Gly, X.sub.1c is Ala,
and X.sub.4 is Gly.
32. A bifunctional compound that acts as a TGF.alpha. mimetic,
comprising a compound from formula III: Loop peptide
N-terminus-linker-cyclic C.sub.4H.sub.8N.sub.2-linker-Loop peptide
N-terminus III wherein the linker moiety is designed to link the
N-terminus of the Loop peptide to a nitrogen atom of the ring
C.sub.4H.sub.8N.sub.2 and wherein the "loop peptide" comprises at
least an 11-membered peptide compound from formula I:
N--X.sub.1a-Cys-His-Ser-X.sub.1b--X.sub.2--X.sub.1a--X.sub.1b--X.sub.1-
a--X.sub.3-Cys COOH I wherein X.sub.1 is independently Val, Gly or
Ala, wherein X.sub.2 is Try or Phe, wherein X.sub.3 is Arg or Lys,
and wherein the two Cys moieties form a disulfide bond to create an
11-amino acid loop peptide.
33. The bifunctional compound that acts as a TGF.alpha. mimetic of
claim 32 wherein at least one or more of the following seven amino
acids are added to the C terminus Cys moiety from formula II:
--X.sub.4-His-X.sub.1c--X.sub.4--X.sub.5--X.sub.6--X.sub.1c II
wherein X.sub.4 is Glu or Asp, wherein X.sub.5 is Leu or Ile, and
wherein X.sub.6 is Asp or Glu.
34. The bifunctional compound that acts as a TGF.alpha. mimetic of
claim 32 wherein X.sub.1a is Val, X.sub.1b is Gly and X.sub.1c is
Ala.
35. The bifunctional compound that acts as a TGF.alpha. mimetic of
claim 32 wherein the linker group is independently selected from
the group consisting of substituted or unsubstituted C.sub.1-6
alkyl, substituted or unsubstituted C.sub.2-6 alkenyl, substituted
or unsubstituted C.sub.1-6 alkoxy, xylenyl, wherein the
substitutions are selected from the group consisting of oxo,
epoxyl, hydroxyl, chloryl, bromyl, fluoryl, and amino.
36. The bifunctional compound that acts as a TGF.alpha. mimetic of
claim 33 wherein X.sub.2 is Tyr, and X.sub.3 is Arg.
37. The bifunctional compound that acts as a TGF.alpha. mimetic of
claim 33 wherein the loop peptide is 13 amino acids in length
wherein X.sub.1a is Val, X.sub.1b is Gly, X.sub.1c is Ala, and
X.sub.4 is Gly.
38. A method for treating inflammatory bowel disease, colitis, and
Chron's Disease of the gastrointestinal tract, comprising
administering a TGF.alpha. polypeptide or a pharmaceutically active
loop peptide, or both, wherein the loop peptide comprises at least
an 11-membered peptide compound from formula I:
N--X.sub.1a-Cys-His-Ser-X.sub.1b--X.sub.2--X.sub-
.1a--X.sub.1b--X.sub.1a--X.sub.3-Cys COOH I wherein X.sub.1 is
independently Val, Gly or Ala, wherein X.sub.2 is Try or Phe,
wherein X.sub.3 is Arg or Lys, and wherein the two Cys moieties
form a disulfide bond to create an 11-amino acid loop peptide.
39. The method for treating inflammatory bowel disease, colitis,
and Chron's Disease of the gastrointestinal tract of claim 38
wherein at least one or more of the following seven amino acids are
added to the C terminus Cys moiety from formula II:
--X.sub.4-His-X.sub.1c--X.sub.413 X.sub.5--X.sub.6--X.sub.1c II
wherein X.sub.4 is Glu or Asp, wherein X.sub.5 is Leu or Ile, and
wherein X.sub.6 is Asp or Glu.
40. The method for treating inflammatory bowel disease, colitis,
and Chron's Disease of the gastrointestinal tract of claim 38
wherein X.sub.1a is Val, X.sub.1b is Gly and X.sub.1c is Ala.
41. The method for treating inflammatory bowel disease, colitis,
and Chron's Disease of the gastrointestinal tract of claim 39
wherein X.sub.2 is Tyr, and X.sub.3 is Arg.
42. The method for treating inflammatory bowel disease, colitis,
and Chron's Disease of the gastrointestinal tract of claim 39
wherein the loop peptide is 13 amino acids in length wherein
X.sub.1a is Val, X.sub.1b is Gly, X.sub.1c is Ala, and X.sub.4 is
Gly.
43. A method for treating an inflammatory reaction of autoimmune
diseases, comprising administering a TGF.alpha. polypeptide or a
pharmaceutically active loop peptide, or both, wherein the loop
peptide comprises at least an 11-membered peptide compound from
formula I: N--X.sub.1a-Cys-His-Ser-X-
.sub.1b--X.sub.2--X.sub.1a--X.sub.1b--X.sub.1a--X.sub.3-Cys COOH I
wherein X.sub.1 is independently Val, Gly or Ala, wherein X.sub.2
is Try or Phe, wherein X.sub.3 is Arg or Lys, and wherein the two
Cys moieties form a disulfide bond to create an 11-amino acid loop
peptide.
44. The method for treating an inflammatory reaction of autoimmune
diseases of claim 43 wherein the autoimmune diseases are selected
from the group consisting of Type II (Juvenile) Diabetes,
rheumatoid arthritis, lupus, and multiple sclerosis.
45. The method for treating an inflammatory reaction of autoimmune
diseases of claim 43 wherein at least one or more of the following
seven amino acids are added to the C terminus Cys moiety from
formula II:
--X.sub.4-His-X.sub.1c--X.sub.4--X.sub.5--X.sub.6--X.sub.1c II
wherein X.sub.4 is Glu or Asp, wherein X.sub.5 is Leu or Ile, and
wherein X.sub.6 is Asp or Glu.
46. The method for treating an inflammatory reaction of autoimmune
diseases of claim 43 wherein X.sub.1a is Val, X.sub.1b is Gly and
X.sub.1c is Ala.
47. The method for treating an inflammatory reaction of autoimmune
diseases of claim 45 wherein X.sub.2 is Tyr, and X.sub.3 is
Arg.
48. The method for treating an inflammatory reaction of autoimmune
diseases of claim 45 wherein the loop peptide is 13 amino acids in
length wherein X.sub.1a is Val, X.sub.1b is Gly, X.sub.1c is Ala,
and X.sub.4 is Gly.
Description
TECHNICAL FIELD OF THE INVENTION
[0001] The present invention provides a novel peptide that is
derived from a loop or "lollipop" region of transforming growth
factor alpha (TGF-.alpha.) and is biologically active for causing
stem cells to proliferate and migrate. The present invention
further provides a method for augmenting hematopoiesis,
particularly trilineage hematopoiesis, and a method for suppressing
immune functioning associated with autoimmune diseases, and a
method for suppressing inflammatory responses mediated (in part) by
excessive histamine release, comprising administering an effective
amount of a TGF-.alpha. polypeptide or a fragment thereof, such as
the lollipop region. The present invention further provides a
method for treating or preventing mucositis and gastrointestinal
side effects in patients undergoing cancer treatment, comprising
administering an effective amount of a TGF-.alpha. polypeptide or a
fragment thereof, such as the lollipop region.
BACKGROUND OF THE INVENTION
[0002] There are several disease treatments that could
significantly benefit by having cells regenerate after injury or
lesion formation, particularly in the CNS, in the immune system and
in the gastrointestinal tract. The expression of growth factors and
their receptors in the pre-implanted human embryo and maternal
reproductive tract indicates that such factors influence growth and
differentiation of embryonic cells in an autocrine and paracrine
manner. Such growth factors are peptides that variously support
survival, proliferation, differentiation, size and function of
nerve cells and other lineages of cells. EGF (epidermal growth
factor) is the first member found of the EGF family and
characterized many years ago (Savage and Cohen, J. Biol. Chem.
247:7609-7611, 1972; and Savage et al., J. Biol. Chem.
247:7612-7621, 1972). Additional members of the EGF family have
been found and they include vaccinia virus growth factor (VGF;
Ventatesan et al., J. Virol. 44:637-646, 1982); myxomavirus growth
factor (MGF; Upton et al. J. Virol. 61:1271-1275, 1987), Shope
fibroma virus growth factor (SFGF; Chang et al., Mol. Cell. Biol.
7:535-540, 1987), amphiregulin (AR; Kimura et al., Nature
348:257-260, 1990), and heparin binding EGF-like factor (HB-EGF;
Higashiyama et al., Science 251:936-939, 1991). A common structural
feature of these polypeptides is the presence of six cysteine
residues that form three disulfide cross links that support a
conserved structure that binds to the EGF receptor.
[0003] Another member of the EGF family is TGF.alpha. and it also
binds to the EGF receptor (Todaro et al., Proc. Natl. Acad. Sci.
USA 77:5258-5262, 1980). TGFU stimulates the EGF receptor's
tyrosine kinase activity and has many cellular functions, such as
stimulating a mitogenic response in a wide variety of cell types.
TGF.alpha. and EGF mRNAs reach their highest levels and relative
abundance (compared to total RNA in the early postnatal period and
decrease thereafter, suggesting a role in embryonic development.
From a histological perspective, TGF.alpha. acts on numerous cell
types throughout the body. The active form of TGF.alpha. is derived
from a larger precursor and contains 50 amino acids. TGF.alpha.
shares only a 30% structural similarity with the 53-amino acid form
of EGF, but including conservation of all six cysteine residues.
TGF.alpha. is highly conserved among species. For example, the rat
and human polypeptides share about 90% homology as compared to a
70% homology as between the rat and human EGF polypeptide. The
amino acid sequence of TGF.alpha. is shown in SEQ ID NO. 1. The
sequence shows that a family consisting of vaccinia growth factor,
amphiregulin precursor, betacellulin precursor, heparin binding
EGF-like growth factor, epiregulin (rodent only), HUS 19878 and
schwannoma derived growth factor have similar sequence motifs and
can be considered as members of the same family based upon their
shared cysteine disulfide bond structures.
[0004] TGF.alpha. is an acid and heat stable polypeptide of about
5.6 kDa molecular weight. It is synthesized as a larger 30-35 kDa
molecular weight glycosylated and membrane-bound precursor protein
wherein the soluble 5.6 kDa active form is released following
specific cleavage by an elastase-like protease. TGF.alpha. binds
with high affinity in the nanomolar range and induces
autophosphorylation to transduce signal with the EGF receptor.
TGF.alpha. is 50 amino acids in length and has three disulfide
bonds to forms its tertiary configuration. All three disulfide
bonds must be present for activity. TGF.alpha. is stored in
precursor form in alpha granules of secretory cells. Moreover, the
primary amino acid sequence is highly conserved among various
species examined, such as more than 92% homology at the amino acid
level as between human and rat TGF.alpha. polypeptides.
[0005] TGF.alpha. has been investigated extensively and has
recently been identified as useful for treating a patient with a
neurological deficit. This mechanism is thought to stimulate
proliferation and migration of neural-origin stem cells to those
site or lesions in a deficit. For example, Parkinson's Disease is
characterized by resting tremor, rigidity, inability to initiate
movement (akinesia) and slowness of movement (bradykinesia). The
motor deficits are associated with progressive degeneration of the
dopaminergic innervation to the nucleus accumbens and degeneration
of noradrenergic cells of the locus ceruleus and serotonergic
neurons of the raphe. Up to 80% of nigral dopamine neurons can be
lost before significant motor deficits are manifest. TGF.alpha.
(full polypeptide) was shown, when infused into rat brains, was
useful for the treatment of neurodegenerative disorders.
Intracerebroventricular (ICV) or intrastriatal infusions of
TGF.alpha. induced neuronal stem cell proliferation, but
degenerating or damaged or otherwise abnormal cells needed to be
present to facilitate migration of the neuronal stem cells to a
site of injury on a scale sufficient to impact recovery from an
associated neurological deficit. Forebrain neural stem cells, that
give rise to migrating progenitor cells that affect treatment and
recovery from a neurological deficit disorder, are the migrating
cells that affect treatment recovery from a neural deficit disorder
(e.g., Parkinson's Disease, Huntington's Disease, Alzheimer's
Disease and the like).
[0006] Neural stem cells have been found in subependyma throughout
the adult rodent CNS (Ray et al. Soc. Neurosci. 22:394.5, 1996) and
in the subependyma of adult human forebrain (Kirschenbaum et al.,
Cerebral Cortex 4:576-589, 1994). Thus, the discovery that
TGF.alpha. stimulates proliferation of neural stem cells and
promotes migration to a site of injury or deficit has led to its
investigation for the treatment of a neurodegenerative disorder
(Alzheimer's Disease, Huntington's Disease and Parkinson's Disease)
or CNS traumatic injury (e.g., spinal chord injury), demyelinating
disease, CNS inflammatory disease, CNS autoimmune disease (e.g.,
multiple sclerosis) or CNS ischemic disease (e.g., stroke or brain
attack).
[0007] A CNS stem cell has the potential to differentiate into
neurons, astrocytes and to exhibit replication of itself to provide
a resource for self-renewal. Both neurons and glial cells seen to
be derived from a common fetal precursor cell. In the vertebrate
CNS, multipotential cells have been identified in vitro and in
vivo. Certain mitogens, such as TGF.alpha., can cause proliferation
of CNS mutipotential cells in vitro and this is the basis for a
procedure to harvest such cell, treat them ex vivo to stimulate
proliferation in culture and then readminister such cells.
Immunohistochemical analysis in the human brain supports the notion
that TGF.alpha. is widely distributed in neurons and glial cells
both during development and during adulthood. In mice genetically
altered to lack expression of functioning TGF.alpha., there was a
decrease in neural progenitor cell proliferation in forebrain
subependyma, providing evidence for TGF.alpha. as a proliferative
factor for neural progenitor cells.
[0008] TGF.alpha. is found mainly in various neurons of the CNS
during development and in the adult brain in the cerebral
neocortex, hippocampus and striatum. It is also found in a few
glial cells, primarily in the cerebral and cerebellar cortex areas.
Northern blot analyses showed that TGF.alpha. and not EGF
(epidermal growth factor) is the most abundant ligand that binds to
the EGF receptor in the brain. TGF.alpha. mRNA levels were 15-170
times higher than EGF in cerebellum and cerebral cortex. TGF.alpha.
also appears in germinal centers of the brain during neurogenesis
and gliogenesis in the developing brain. In the midbrain, the
distribution of TGF.alpha. overlaps with tyrosine hydroxylase mRNA
and fetal dopaminergic neurons. In culture, TGF.alpha. enhanced
survival and neurite outgrowth of neonatal rat dorsal ganglion
neurons (EGF did not) and survival and differentiation of CNS
neurons. TGF.alpha. induced proliferation of neural precursor cells
of the murine embryonic mesencephalon and further induced a
significant increase in the number of astroglia and microglia in
fetal rat medial septal cells. TGF.alpha. increased glutamic acid
decarboxylase activity and decreased choline actetyltransferase
activity. Thus, TGF.alpha. acted as a general neuronal survival
factor affecting both cholinergic and GABAergic neurons. In
addition, TGF.alpha. is a mitogen for pluripotent brain stem cells.
Forebrain subependyma contains nestin positive neural stem cells
and their progeny, which are constitutively proliferating
progenitor epithelial cells. A "knockout" mouse that was
genetically engineered to delete the gene for TGF.alpha. showed a
reduction in neuronal progenitor cells in the subependyma and a
reduction in neuronal progenitors that migrate to the olifactory
bulb. In vitro, TGF.alpha. promoted dopamine uptake in fetal rat
dopaminergic neurons in a dose-dependent and time-dependent manner.
TGF.alpha. selectively promoted dopaminergic cell survival,
enhanced neurite length, branch number and the soma area of
tyrosine hydroxylase immunopositive cells. The levels of TGF.alpha.
were elevated in ventricular cerebrospinal fluid in juvenile
parkinsonism and Parkinson's Disease and may represent a
compensatory response to neurodegeneration. Further, TGF.alpha.
prevented a striatal neuronal degeneration in an animal model of
Huntington's Disease.
[0009] The mucosal epithelium of the intestine is in a continually
dynamic state known as "epithelial renewal" in which
undifferentiated stem cells from a proliferative crypt zone divide,
differentiate and migrate to the luminal surface. Once terminally
differentiated, they are sloughed from the tips of the villi. The
turnover of the crypt-villus cell population is rapid and occurs
every 24-72 hours. Continuous exfoliation of the cells at the
villus tip is counterbalanced by ongoing proliferation in the crypt
so that net intestinal epithelial mass remains relatively constant.
The rapidly-proliferating epithelium of the gastrointestinal tract
is extremely sensitive to cytotoxic drugs that are widely used in
the chemotherapy of cancer. This "side effect" reduces the
tolerated dose of such drugs as it can cause a breakdown of the GI
barrier function and septic condition in a patient already
immuno-compromised. This can also lead to life-threatening
hemorrhage. Therefore, there is a need in the art for the
development of products and delivery systems that stimulate the
repair and rejuvenation of mucosal epithelium in the
gastrointestinal tract to provide benefit to patient receiving
chemotherapy and radiation therapy for cancer.
[0010] Therefore, there is a need in the art to find improved
TGF.alpha. mimetic agents that are more economical to produce and
are smaller (in terms of molecular weight). The present invention
was made to address such a need.
SUMMARY OF THE INVENTION
[0011] The present invention is based upon two basic discoveries
that have not been reported before in the extensive literature of
TGF.alpha. . Firstly, a novel genus of small peptides, much smaller
than TGF.alpha., was discovered as having TGF.alpha. biological
activity and therefore are useful as pharmacologic agents for the
same indications as full length TGF.alpha. polypeptide. Secondly,
TGF.alpha. and consequently the genus of small peptides disclosed
herein, was found to have therapeutic activity to stimulate
hematopoiesis in patients undergoing cytotoxic cancer chemotherapy
and to act as a cytoprotective agent to protect a patient
undergoing cancer cytotoxic therapy from gastrointestinal (GI) side
effects, such as mucositis and otherwise support the barrier
function of the GI tract when it is harmed by cytotoxic
therapy.
[0012] The present invention provides a compound that acts as a
TGF.alpha. mimetic, comprising at least an 11-membered peptide
compound from formula I:
N--X.sub.1a-Cys-His-Ser-X.sub.1b--X.sub.2--X.sub.1a--X.sub.1b--X.sub.1a--X-
.sub.3-Cys COOH I
[0013] wherein X.sub.1 is independently Val, Gly or Ala, wherein
X.sub.2 is Try or Phe, wherein X.sub.3 is Arg or Lys, and wherein
the two Cys moieties form a disulfide bond to create an 11-amino
acid loop peptide. Preferably, at least one or more of the
following seven amino acids are added to the C terminus Cys moiety
from formula II:
--X.sub.4-His-X.sub.1c--X.sub.4--X.sub.5--X.sub.6--X.sub.1c II
[0014] wherein X.sub.4 is Glu or Asp, wherein X.sub.5 is Leu or
Ile, and wherein X.sub.6 is Asp or Glu. Preferably, X.sub.1a is
Val, X.sub.1b is Gly and X.sub.1c is Ala. Preferably, X.sub.2 is
Tyr, and X.sub.3 is Arg. Most preferably, the loop peptide is 13
amino acids in length wherein X.sub.1a is Val, X.sub.1b is Gly,
X.sub.1c is Ala, and X.sub.4 is Gly.
[0015] The present invention further provides a pharmaceutical
composition comprising a loop peptide in a pharmaceutically
acceptable carrier, wherein the loop peptide compound comprises at
least an 11 -membered peptide compound from formula I:
N--X.sub.1a-Cys-His-Ser-X.sub.1b--X.sub.2--X.sub.1a--X.sub.1b--X.sub.1a--X-
.sub.3-Cys COOH I
[0016] wherein X.sub.1 is independently Val, Gly or Ala, wherein
X.sub.2 is Try or Phe, wherein X.sub.3 is Arg or Lys, and wherein
the two Cys moieties form a disulfide bond to create an 11-amino
acid loop peptide. Preferably, at least one or more of the
following seven amino acids are added to the C terminus Cys moiety
from formula II:
--X.sub.4-His-X.sub.1c--X.sub.4--X.sub.5--X.sub.6--X.sub.1c II
[0017] wherein X.sub.4 is Glu or Asp, wherein X.sub.5 is Leu or
Ile, and wherein X.sub.6 is Asp or Glu. Preferably, X.sub.1a is
Val, X.sub.1b is Gly and X.sub.1c is Ala. Preferably, X.sub.2 is
Tyr, and X.sub.3 is Arg. Most preferably, the loop peptide is 13
amino acids in length wherein X.sub.1a is Val, X.sub.1b is Gly,
X.sub.1c is Ala, and X.sub.4 is Gly.
[0018] The present invention further provides a method for treating
a neurodegenerative disease with an pharmaceutically active loop
peptide, wherein the loop peptide comprises at least an 11-membered
peptide compound from formula I:
N--X.sub.1a-Cys-His-Ser-X.sub.1b--X.sub.2--X.sub.1a--X.sub.1b--X.sub.1a--X-
.sub.3-Cys COOH I
[0019] wherein X.sub.1 is independently Val, Gly or Ala, wherein
X.sub.2 is Try or Phe, wherein X.sub.3 is Arg or Lys, and wherein
the two Cys moieties form a disulfide bond to create an 11-amino
acid loop peptide. Preferably, at least one or more of the
following seven amino acids are added to the C terminus Cys moiety
from formula II:
--X.sub.4-His-X.sub.1c--X.sub.4--X.sub.5--X.sub.6--X.sub.1c II
[0020] wherein X.sub.4 is Glu or Asp, wherein X.sub.5 is Leu or
Ile, and wherein X.sub.6 is Asp or Glu. Preferably, X.sub.1a is
Val, X.sub.1b is Gly and X.sub.1c is Ala. Preferably, X.sub.2 is
Tyr, and X.sub.3 is Arg. Most preferably, the loop peptide is 13
amino acids in length wherein X.sub.1a is Val, X.sub.1b is Gly,
X.sub.1c is Ala, and X.sub.4 is Gly.
[0021] The present invention further provides a method for treating
a CNS disease or disorder, wherein the CNS disease or disorder is
selected from the group consisting of CNS ischemia, spinal cord
injury, MS, and retinal injury, comprising with an pharmaceutically
active loop peptide, wherein the loop peptide comprises at least an
1 1-membered peptide compound from formula I:
N--X.sub.1a-Cys-His-Ser-X.sub.1b--X.sub.2--X.sub.1a--X.sub.1b--X.sub.1a--X-
.sub.3-Cys COOH I
[0022] wherein X.sub.1 is independently Val, Gly or Ala, wherein
X.sub.2 is Try or Phe, wherein X.sub.3 is Arg or Lys, and wherein
the two Cys moieties form a disulfide bond to create an 11-amino
acid loop peptide. Preferably, at least one or more of the
following seven amino acids are added to the C terminus Cys moiety
from formula II:
--X.sub.4-His-X.sub.1c--X.sub.4--X.sub.5--X.sub.6--X.sub.1c II
[0023] wherein X.sub.4 is Glu or Asp, wherein X.sub.5 is Leu or
Ile, and wherein X.sub.6 is Asp or Glu. Preferably, X.sub.1a is
Val, X.sub.1b is Gly and X.sub.1c is Ala. Preferably, X.sub.2 is
Tyr, and X.sub.3 is Arg. Most preferably, the loop peptide is 13
amino acids in length wherein X.sub.1a is Val, X.sub.1b is Gly,
X.sub.1c is Ala, and X.sub.4 is Gly.
[0024] The present invention further provides a method for
augmenting hematopoiesis during cytotoxic or immune-suppressing
therapy, comprising administering a TGF.alpha. polypeptide or a
pharmaceutically active loop peptide, or both, wherein the loop
peptide comprises at least an 11-membered peptide compound from
formula I:
N--X.sub.1a-Cys-His-Ser-X.sub.1b--X.sub.2--X.sub.1a--X.sub.1b--X.sub.1a--X-
.sub.3-Cys COOH I
[0025] wherein X.sub.1 is independently Val, Gly or Ala, wherein
X.sub.2 is Try or Phe, wherein X.sub.3 is Arg or Lys, and wherein
the two Cys moieties form a disulfide bond to create an 11-amino
acid loop peptide. Preferably, at least one or more of the
following seven amino acids are added to the C terminus Cys moiety
from formula II:
--X.sub.4-His-X.sub.1c--X.sub.4--X.sub.5--X.sub.6--X.sub.1c II
[0026] wherein X.sub.4 is Glu or Asp, wherein X.sub.5 is Leu or
Ile, and wherein X.sub.6 is Asp or Glu. Preferably, X.sub.1a is
Val, X.sub.1b is Gly and X.sub.1c is Ala. Preferably, X.sub.2 is
Tyr, and X.sub.3 is Arg. Most preferably, the loop peptide is 13
amino acids in length wherein X.sub.1a is Val, X.sub.1b is Gly,
X.sub.1c is Ala, and X.sub.4 is Gly. Preferably, the invention
further comprises administering a second hematopoietic growth
factor agent to stimulate more mature hematopoietic precursor
cells, wherein the second hematopoietic growth factor is selected
from the group consisting of erythropoietin, thrombopoietin, G-CSF
(granulocyte colony stimulating factor), and GM-CSF (granulocyte
macrophage colony stimulating factor).
[0027] The present invention further provides a method for treating
or preventing mucositis of the gastrointestinal tract during
cytotoxic or immune-suppressing therapy, comprising administering a
TGF.alpha. polypeptide or a pharmaceutically active loop peptide,
or both, wherein the loop peptide comprises at least an 11-membered
peptide compound from formula I:
N--X.sub.1a-Cys-His-Ser-X.sub.1b--X.sub.2--X.sub.1a--X.sub.1b--X.sub.1a--X-
.sub.3-Cys COOH I
[0028] wherein X.sub.1 is independently Val, Gly or Ala, wherein
X.sub.2 is Try or Phe, wherein X.sub.3 is Arg or Lys, and wherein
the two Cys moieties form a disulfide bond to create an 11-amino
acid loop peptide. Preferably, at least one or more of the
following seven amino acids are added to the C terminus Cys moiety
from formula II:
--X.sub.4-His-X.sub.1c--X.sub.4--X.sub.5--X.sub.6--X.sub.1c II
[0029] wherein X.sub.4 is Glu or Asp, wherein X.sub.5 is Leu or
Ile, and wherein X.sub.6 is Asp or Glu. Preferably, X.sub.1a is
Val, X.sub.1b is Gly and X.sub.1c is Ala. Preferably, X.sub.2 is
Tyr, and X.sub.3 is Arg. Most preferably, the loop peptide is 13
amino acids in length wherein X.sub.1a is Val, X.sub.1b is Gly,
X.sub.1c is Ala, and X.sub.4 is Gly.
[0030] The present invention further provides a bifunctional
compound that acts as a TGF.alpha. mimetic, comprising a compound
from formula III:
Loop peptide N-terminus-linker-cyclic
C.sub.4H.sub.8N.sub.2-linker-Loop peptide N-terminus III
[0031] wherein the linker moiety is designed to link the N-terminus
of the Loop peptide to a nitrogen atom of the ring
C.sub.4H.sub.8N.sub.2 and wherein the "loop peptide" comprises at
least an I1 -membered peptide compound from formula I:
N--X.sub.1a-Cys-His-Ser-X.sub.1b--X.sub.2--X.sub.1a--X.sub.1b--X.sub.1a--X-
.sub.3-Cys COOH I
[0032] wherein X.sub.1 is independently Val, Gly or Ala, wherein
X.sub.2 is Try or Phe, wherein X.sub.3 is Arg or Lys, and wherein
the two Cys moieties form a disulfide bond to create an 11-amino
acid loop peptide. Preferably, at least one or more of the
following seven amino acids are added to the C terminus Cys moiety
from formula II:
--X.sub.4-His-X.sub.1c--X.sub.4--X.sub.5--X.sub.6--X.sub.1c II
[0033] wherein X.sub.4 is Glu or Asp, wherein X.sub.5 is Leu or
Ile, and wherein X.sub.6 is Asp or Glu. Preferably, X.sub.1a is
Val, X.sub.1b is Gly and X.sub.1c is Ala. Preferably, the linker
group is independently selected from the group consisting of
substituted or unsubstituted C.sub.1-6 alkyl, substituted or
unsubstituted C.sub.2-6 alkenyl, substituted or unsubstituted
C.sub.1-6 alkoxy, xylenyl, wherein the substitutions are selected
from the group consisting of oxo, epoxyl, hydroxyl, chloryl,
bromyl, fluoryl, and amino Preferably, X.sub.2 is Tyr, and X.sub.3
is Arg. Most preferably, the loop peptide is 13 amino acids in
length wherein X.sub.1a is Val, X.sub.1b is Gly, X.sub.1c is Ala,
and X.sub.4 is Gly.
[0034] The present invention further provides a method for treating
inflammatory bowel disease, colitis, and Chron's Disease of the
gastrointestinal tract, comprising administering a TGF.alpha.
polypeptide or a pharmaceutically active loop peptide, or both,
wherein the loop peptide comprises at least an 11-membered peptide
compound from formula I:
N--X.sub.1a-Cys-His-Ser-X.sub.1b--X.sub.2--X.sub.1a--X.sub.1b--X.sub.1a--X-
.sub.3-Cys COOH I
[0035] wherein X.sub.1 is independently Val, Gly or Ala, wherein
X.sub.2 is Try or Phe, wherein X.sub.3 is Arg or Lys, and wherein
the two Cys moieties form a disulfide bond to create an 11-amino
acid loop peptide. Preferably, at least one or more of the
following seven amino acids are added to the C terminus Cys moiety
from formula II:
--X.sub.4-His-X.sub.1c--X.sub.4--X.sub.5--X.sub.6--X.sub.1c II
[0036] wherein X.sub.4 is Glu or Asp, wherein X.sub.5 is Leu or
Ile, and wherein X.sub.6 is Asp or Glu. Preferably, X.sub.1a is
Val, X.sub.1b is Gly and X.sub.1c is Ala. Preferably, X.sub.2 is
Tyr, and X.sub.3 is Arg. Most preferably, the loop peptide is 13
amino acids in length wherein X.sub.1a is Val, X.sub.1b is Gly,
X.sub.1c is Ala, and X.sub.4 is Gly.
[0037] The present invention further provides a method for treating
an inflammatory reaction of autoimmune diseases, comprising
administering a TGF.alpha. polypeptide or a pharmaceutically active
loop peptide, or both, wherein the loop peptide comprises at least
an 11-membered peptide compound from formula I:
N-X.sub.1a-Cys-His-Ser-X.sub.1b-X.sub.2-X.sub.1a-X.sub.1b-X.sub.1a-X.sub.3-
- Cys COOH I
[0038] wherein X.sub.1 is independently Val, Gly or Ala, wherein
X.sub.2 is Try or Phe, wherein X.sub.3 is Arg or Lys, and wherein
the two Cys moieties form a disulfide bond to create an 11-amino
acid loop peptide. Preferably, the autoimmune diseases are selected
from the group consisting of Type II (Juvenile) Diabetes,
rheumatoid arthritis, lupus, and multiple sclerosis. Preferably, at
least one or more of the following seven amino acids are added to
the C terminus Cys moiety from formula II:
--X.sub.4-His-X.sub.1c--X.sub.4--X.sub.5--X.sub.6--X.sub.1c II
[0039] wherein X.sub.4 is Glu or Asp, wherein X.sub.5 is Leu or
Ile, and wherein X.sub.6 is Asp or Glu. Preferably, X.sub.1a is
Val, X.sub.1b is Gly and X.sub.1c is Ala. Preferably, X.sub.2 is
Tyr, and X.sub.3 is Arg. Most preferably, the loop peptide is 13
amino acids in length wherein X.sub.1a is Val, X.sub.1b is Gly,
X.sub.1c is Ala, and X.sub.4 is Gly.
BRIEF DESCRIPTION OF THE DRAWINGS
[0040] FIG. 1 shows the structure of human TGF.alpha. polypeptide
and its 50 amino acids arranged into three loops.
[0041] FIG. 2 shows a graph comparing TGF.alpha. biological
activity of the three loop peptide regions of TGF.alpha. (see FIG.
1) wherein Loop A is amino acids 1-21 (starting at the N terminus),
Loop B is amino acids 16 to 32 and Loop C is amino acids 33 to 50.
Only Loop C showed significant TGF.alpha. activity as determined by
cell proliferation and in a dose response fashion.
[0042] FIG. 3 shows a graph of mouse spleen weights that were
treated with Cis Platinum (CP) at either 5 .mu.g/g or 10 .mu.g/g
and with TGF.alpha. at concentrations of 10 ng/g or 50 ng/g. These
data show that TGF.alpha. treatment caused a return to normal
spleen weights despite CP treatment that reduced spleen weights
significantly.
[0043] In FIG. 4, three panels of H&E-stained spleens are
shown. Specifically, the top panel shows a CP-treated mouse spleen
(10 .mu.g/g) showing apoptotic cells (densely stained with
fragments of nuclei) in the germinal center (GC). The T cells with
the central arterial area show the absence of a marginal zone and
much fewer erythrocytes and T cells in the perifolecular area
(arrows). In the middle panel, a normal mouse spleen is shown (no
CO and no TGF.alpha.) fixed in formalin showing an arteriole with T
cells areas (arrow). A primary follicle and a second follicle are
shown as containing a germinal center (GC). There is a presence of
an erythrocyte rich (pink) perifollicular zone surrounding both a T
cell and B cell compartments of white pulp. In the bottom panel, a
mouse spleen treated with CP (10 .mu.g/g) and TGF.alpha. (50 ng/g)
shows an increased number of T cells and erythrocytes in the
perifolicular zone (arrows). The T cell area contains lymph vessels
in relation to arterioles. A germinal center (GC) is within the
mantle zone.
[0044] In FIG. 5 there are three panels showing the histological
examination of mouse intestines. In the top panel, CP (single ip
dose of 10 .mu.g/g) treated intestine is cross-sectioned and shows
significant injury to the villi. Specifically, the villi are
necrotic and the crypts are in irregular shapes. The tips of the
crypts were losing their cellular integrity (arrows). In the middle
panel is a cross section of a normal mouse GI tract (no CP and no
TGF.alpha.) and shows a normal intestinal surface with villi having
long and slender mucosal projections with a core of lamina propria
covered by a luminal epithelial layer. A single row of intestinal
crypt is found at the base of the mucosa. These crypts that lie
between adjacent villi are surrounded by the same lamina propria
that form the villous cores. Both columnar absorptive cells and
globlet cells cover the villous surfaces. The globlet cells contain
apical clear vacuoles. The bottom panel shows a cross section of a
mouse intestine exposed both the CP (10 .mu.g/g) and TGF.alpha. (50
ng/g). The intestinal structure is very similar to the normal
intestinal structure. Specifically, the villus is long and slender.
Both absorptive cells and globlet cells are visible at the surface
of the villi. There is an abundant amount of globlet cells on the
surface.
[0045] In FIG. 6, there are three panels shown at 160.times.
magnification again corresponding to a CP-treated mouse in the top
panel, a normal mouse in the middle panel and a CP treated and
TGF.alpha. treated mouse in the bottom panel at the same doses as
indicated for FIG. 5. In the top panel are injured villi with tips
degenerating and necrotic (arrows). Red blood cells are observed in
the damaged villi (arrows). The crypts (C) are in irregular shape
and in various heights. The middle panel shows that the tips of the
villi (arrows) are smooth and the nuclei of the enterocytes are
observed throughout the villus. The crypts (C) are similar in
height and regular in shape. The bottom panel has villi (arrows)
appearing normal as in the middle panel. The crypts (C) also appear
to be normal.
[0046] FIG. 7 shows three panels but the top and middle panels are
CP (10 .mu.g/g) treated without TGF.alpha. and the bottom panel is
CP (10 .mu.g/g) and 50 ng/g of TGF.alpha.. The panels are shown at
higher magnification. In the top panel, the severely injured crypt
surface from CP treatment shows cellular destruction due to
necrosis. Red cells appear at the damaged surface to indicate
intestinal bleeding. In addition, the middle panel of a CP-treated
mouse shows a loss of brush border and very little of a glycocalyx
or fuzzy coat. The interspersed globlet cells appear fewer in
number (than normal) and are seen as necrotic. In the bottom panel,
the effect of TGF.alpha. treatment shows protection of the villa
surface (arrows). Specifically, the epithelial cells are normal
appearing with extended brush borders. The nuclei are very densely
stained and elongated.
[0047] The histological data is summarized in FIG. 8 that measured
average crypt height of the three groups of mice. TGF.alpha.
treatment (50 ng/g) was able to more-than-restore crypt height loss
from CP treatment.
[0048] In FIG. 9, the three panels at 160.times. magnification are
shown to correspond to normal intestine in the top panel, CP only
treated (10 .mu.g/g) in the middle panel and both CP (10 .mu.g/g)
and TGF.alpha. (50 ng/g) in the bottom panel. In the normal
intestine (top panel), each villus extends from the luminal surface
to the basal muscularis mucosal surface. Globlet cells are
scattered and predominate in the base of the villus (arrows)
whereas columnar absorptive cells line the luminal surface. In the
middle panel, the alcian blue staining method shows that the villi
contain a fewer number of globlet cells (than normal) (arrows). The
injured absorptive and globlet cells are degenerating at the tip of
the villi (arrows). Abundant secretory mucus material is stained in
the luminal surface (arrows). In the bottom panel, there is an
increased number of globlet cells scattered throughout the villi
(arrows). The intestinal villi are in normal form with elongation.
The majority of enterocytes are not alcian blue stained positive.
The luminal plasma membranes of the villi (arrows) are well
protected by TGF.alpha. treatment.
[0049] FIG. 10 shows that TGF.alpha. treatment not only increased
the number of globlet cells but increased the number from CP
treatment to a higher level than normal intestine.
[0050] FIG. 11 shows that TGF.alpha. treatment causes mast cells
residing in the intestinal mucosal tissue and lamina propria to
remain intact and thus not release histamine and other
pro-inflammatory molecules. The bottom panel, by contrast, shows
CP-treated mice who did not receive TGF.alpha. wherein there was a
degranulation of mast cells and subsequent induction of
inflammatory responses.
DETAILED DESCRIPTION OF THE INVENTION
[0051] Loop Peptide
[0052] TGF.alpha. is a polypeptide of 50 amino acids shown in FIG.
1. The TGF.alpha. polypeptide can be divided roughly into three
loop regions corresponding roughly (starting at the N terminus) to
amino acids 1-21, to amino acids 16-32, and to amino acids 33-50.
Each of the three foregoing loop regions was investigated for
TGF.alpha.-like biological activity, such as stimulation of
cellular proliferation as measured by .sup.3H thymidine
incorporation of stem cells. As shown in FIG. 2, only the Loop C
peptide (corresponding to amino acids 33-50) showed significant
TGF.alpha. biological activity and is therefore a TGF.alpha.
mimetic peptide. Therefore, in view of the fact that the loop
peptide exhibits TGF.alpha. biological activity, data obtained with
TGF.alpha. (50 amino acid polypeptide is predictive of activity of
the loop peptide and similar loop peptides embodied in the genus of
formula I with or without the addition of a "tail" region of
formula II). These data predict activity for the loop peptide when
activity is also shown for TGFU.
[0053] Pharmaceutical Composition and Formulations
[0054] The inventive pharmaceutical composition comprises a loop
peptide in a pharmaceutically acceptable carrier. The
pharmaceutically acceptable carrier is suitable for the particular
form of administration contemplated by the pharmaceutical
composition. The term "carrier" is designed to mean any and all
solvents, dispersion media, coatings, isotonic agents,
antibacterial and antifungal agents designed to preserve a
formulation from contamination, absorption agents and similar
agents that are compatible with pharmaceutical administration
irrespective of the route of administration.
[0055] The pharmaceutical formulations are made based upon the
intended routes of administration. Specifically, those formulations
that will be intended for a GI indication will likely be
administered orally. In view of the peptide bonds present, such
formulations will be made to pass through the stomach and protect
the active compound from the low pH conditions of the stomach
before there is a better chance for local activity in the villi of
the small intestine and large intestine. The loop peptide
formulations are intended for parenteral administration through
some form of injection or for use in ex vivo culture media.
Parenteral forms of administration include, for example,
intravenous, intradermal, intramuscular, intraperitoneal for GI
effects, injection directly into a target organ (e.g., brain) at
the appropriate location, application in a biodegradable matrix to
a site of CNS injury (e.g., spinal cord).
[0056] Solutions or suspensions useful in the pharmaceutical
compositions that contains peptide components include sterile
diluents such as water, saline, fixed oils, polyethylene glycols,
glycerine, propylene glycol, or other synthetic agents, plus an
antibacterial or antifungal agent for preservation, antioxidants,
chelating agents, buffer and agents that adjust tonicity for direct
organ injections. Forms of pharmaceutical compositions include, for
example, sterile aqueous solutions or dispersions and sterile
powders for the extemporaneous preparation of sterile injectable
solutions of dispersions. For intravenous injection or direct organ
or peritoneal injections, suitable carriers include, for example,
saline, bacteriostatic water, Cremophor, or phosphate buffered
saline. The composition is formulated to preserve stability, be
easily mixed and preserved against contamination. Isotonic agents,
such as sugars or polyalcohols (e.g., glucose, fructose, mannitol,
sorbitol and the like) or sodium chloride are used. Agents that
delay target organ absorption can also be used and these include,
for example, aluminum monostearate and gelatin.
[0057] Sterile injectable solutions can be prepared by
incorporating the active agent (see formula I, formula II, or
formula III and TGF.alpha.) in the required amount in an
appropriate solvent and then sterilizing, such as by sterile
filtration. Further, powders can be prepared by standard techniques
such as freeze drying or vacuum drying.
[0058] In another embodiment, the active agent is prepared with a
biodegradable carrier for sustained release characteristics for
either sustained release in the GI tract or for target organ
implantation (e.g., brain or spinal cord) with long term active
agent release characteristics to the intended site of activity
(such as a site of injury or neuronal degradation). Biodegradable
polymers include, for example, ethylene vinyl acetate,
polyanhydrides, polyglycolic acids, polylactic acids, collagen,
polyorthoesters, and poly acetic acid. Liposomal formulation can
also be used.
[0059] In addition, the active compound for the pharmaceutical
composition needs to also be synthesized. If the compound is from
formula I or formula II, a preferred means for synthesizing
peptides of 13-18 amino acids in length is by direct peptide
synthesis generally starting with the N-terminal amino acid and
adding amino acids in the C terminal direction. Such small peptides
can also be synthesized and later purified by standard recombinant
techniques, but peptide of 18 amino acids in length are better
synthesized from the amino acid building blocks directly.
TGF.alpha. has bee made using recombinant techniques and is
available as a laboratory reagent commercially. The bifunctional
compounds of formula III are best synthesized with each loop
peptide moiety synthesized and then added to the heterocyclic
nitrogen atom using standard heterocyclic addition synthesis
[0060] Loop Peptide Mimics TGF.alpha. Neuroactive Therapeutic
Activity
[0061] The neuroactive activity of the loop peptide is based upon
the discovery that the loop peptide exhibits TGF.alpha. biological
activity and can therefore stimulate CNS multipotent precursor
cells to divide and migrate through the brain. This activity
indicates that the loop peptide is effective to treat neurological
deficits caused by a wide variety of diseases and injuries that
each result in a neurological deficit in some specific area of the
brain or specific kind of neuron. These include degenerative
diseases, including the more common Alzheimer's Disease (AD),
Parkinson's Disease (PD), and Huntington's Disease (HD), and the
less common Pick's disease, progressive supranuclear palsy,
striatonigral degeneration, cortico-basal degeneration,
olivopontocerebellar atrophy, Leigh's disease, infantile
necrotizing encephalomyelopathy, Hunter's disease,
mucopolysaccharidosis, various leukodystrophies (such as Krabbe's
disease, Pelizaeus-Merzbacher disease and the like), amaurotic
(familial) idiocy, Kuf's disease, Spielmayer-Vogt disease, Tay
Sachs disease, Batten disease, Jansky-Bielschowsky disease, Reye's
disease, cerebral ataxia, chronic alcoholism, beriberi,
Hallervorden-Spatz syndrome, cerebellar degeneration, and the
like.
[0062] Further, injuries (traumatic or neurotoxic) that cause a
loss of neuronal function can be treated by the loop peptide. Such
injuries include, for example, gunshot wounds, injuries caused by
blunt force, penetration injuries, injuries caused by surgical
procedure (e.g., tumor removal, abscess removal, epilepsy lesion
removal) poisoning (e.g., carbon monoxide), shaken baby syndrome,
adverse reactions to medications, drug overdoses, and
post-traumatic encephalopathy. Ischemia can further cause CNS
injury due to disruption of blood flow or oxygen delivery that can
kill or injure neurons and glial cells. Such injuries can be
treated by administration of the loop peptide and include, for
example, injuries caused by stroke, anoxia, hypoxia, partial
drowning, myoclonus, severe smoke inhalation, dystonias, and
acquired hydrocephalus. Developmental disorders that can be treated
by the loop peptide include, for example, schizophrenia, certain
forms of severe mental retardation, cerebral palsey, congenital
hydrocephalus, severe autism, Downs Syndrome, LHRH/hypothalamic
disorder, and spina bifida. The loop peptide can be further used to
treat disorders affecting vision caused by the loss or failure of
retinal cells and include, for example, diabetic retinopathy,
serious retinal detachment (associated with glaucoma), traumatic
injury to the retina, retinal vascular occlusion, macular
degeneration, optic nerve atrophy and other retinal degenerative
diseases. Injuries to the spinal cord can be treated by the loop
peptide. Examples of spinal cord injuries are post-polio syndrome,
amyotrophic lateral sclerosis, traumatic injury, surgical injury,
and paralytic diseases. Demylinating autoimmune disorders can be
treated by administration of the loop peptide and include, for
example, multiple sclerosis. Lastly, the loop peptide can be used
to treat neurological deficits caused by infection of inflammatory
diseases, including, for example, Creutzfeldt-Jacob disease and
other slow virus infectious diseases of the CNS, AIDS
encephalopathy, post-encephalitic Parkinsonism, viral encephalitis,
bacterial meningitis and other CNS effects of infectious
diseases.
[0063] The loop peptide provides TGF.alpha. activity and therefor
the present method of treating neurological deficit and injury
disorders is based upon the biological activity of the loop peptide
of formula I, formula II and formula III and the data available for
TGF.alpha. that has been published.
[0064] Hematopoiesis
[0065] TGF.alpha. showed surprising activity in an in vivo model of
general hematopoiesis when administered in conjunction with a
potent cytotoxic agent Cis Platinum (CP). FIG. 3 shows a graph of
mouse spleen weights that were treated with CP at either 5 .mu.g/g
or 10 .mu.g/g and with TGF.alpha. at concentrations of 10 ng/g or
50 ng/g. These data show that TGF.alpha. treatment caused a return
to normal spleen weights despite CP treatment that reduced spleen
weights significantly. This in vivo experiment is a predictive
model for hematopoiesis in humans as CP is a cytotoxic agent
commonly used for cancer chemotherapy that is known to
significantly reduce trilineage hematopoietic cells. Hematopoietic
cells are red blood cell precursors, platelet precursors
(megakaryocytes), and immune (white) blood cell precursors of
various forms of T cells, B cells and macrophages. Moreover,
platelet counts were higher in those mice injected with TGF.alpha.
(and CP) as opposed to CP alone were such counts were significantly
reduced from normal.
[0066] The experiment procedure dosed those animals to be treated
with TGF.alpha. 4 hours prior to challenge with CP. A single dose
of CP was administered. Additional doses (as indicated) of
TGF.alpha. were made at 24 hours, 48 hours, 72 hours and 96 hours
after the CP dose. All doses were made by IP injection. Controls
were dosed with saline instead of either or both of CP and
TGF.alpha..
[0067] The animals were sacrificed about 4 hours after the last
TGF.alpha. (or saline) dose. Key organs were removed and spleens
were immediately weighed after a clean incision. All the relevant
organs were placed in formalin, transported for histopathological
analysis, mounted, sectioned, stained and observed. The results of
this histological analysis of the spleens for hematopoietic effect
and the GI tract (below) provide the surprising data of the effect
of TGF.alpha. activity.
[0068] In FIG. 4, three panels of H&E-stained spleens are
shown. Specifically, the top panel shows a CP-treated mouse spleen
(10 .mu.g/g) showing apoptotic cells (densely stained with
fragments of nuclei) in the germinal center (GC). The T cells with
the central arterial area show the absence of a marginal zone and
much fewer erythrocytes and T cells in the perifolecular area
(arrows). In the middle panel, a normal mouse spleen is shown (no
CO and no TGF.alpha.) fixed in formalin showing an arteriole with T
cells areas (arrow). A primary follicle and a second follicle are
shown as containing a germinal center (GC). There is a presence of
an erythrocyte rich (pink) perifollicular zone surrounding both a T
cell and B cell compartments of white pulp. In the bottom panel, a
mouse spleen treated with CP (10 .mu.g/g) and TGF.alpha. (50 ng/g)
shows an increased number of T cells and erythrocytes in the
perifolicular zone (arrows). The T cell area contains lymph vessels
in relation to arterioles. A germinal center (GC) is within the
mantle zone. These in vivo data in a predictive model of
hematopoiesis and confirmed by blinded histological analysis (the
histologist/pathologist was blinded as to the treatment history of
the coded tissues received) providing surprising evidence of the
utility of peptides having TGF.alpha. activity to augment
hematopoiesis following cytotoxic exposure. These data predict and
provide a reasonable correlation that TGF.alpha. and the peptides
of formula I, formula II and formula III are useful therapeutic
agents for augmenting hematopoiesis following or during cytotoxic
therapy, such as cancer treatment. Therefore, a useful method for
treating cancer is to combine either TGF.alpha. or a peptide from
formula I, formula II or formula III or combinations thereof with
cytotoxic treatment regimens to reduce dose-limiting side
effects.
[0069] Mucositis and Gastrointestinal Diseases
[0070] The small intestine comprises the duodenum, jejunum and
ileum. It is the principal site for absorption of digestive
products from the GI tract. Digestion begins in the stomach and is
completed in the small intestine in association with the absorptive
process. The intestinal mucosa surface is made up of numerous
finger-like projections called villi. In addition, mucosa between
the basis of the villi (crypts) is formed into the crypts.
[0071] TGF.alpha. or a peptide from formula I, formula II or
formula III or combinations thereof are also useful for treating
mucositis associated intestinal bleeding , dyspepsia associated
with cytotoxic therapy and for improving the barrier function of
the GI tract compromised by cytotoxic therapy. The in vivo
experiment with seven groups of mice described above for
hematopoietic effects noted in spleens also examined the GI tract
of these treated mice. In FIG. 5 there are three panels showing the
histological examination of mouse intestines. In the top panel, CP
(single ip dose of 10 .mu.g/g) treated intestine is cross-sectioned
and shows significant injury to the villi. Specifically, the villi
are necrotic and the crypts are in irregular shapes. The tips of
the crypts were losing their cellular integrity (arrows). In the
middle panel is a cross section of a normal mouse GI tract (no CP
and no TGF.alpha.) and shows a normal intestinal surface with villi
having long and slender mucosal projections with a core of lamina
propria covered by a luminal epithelial layer. A single row of
intestinal crypt is found at the base of the mucosa. These crypts
that lie between adjacent villi are surrounded by the same lamina
propria that form the villous cores. Both columnar absorptive cells
and globlet cells cover the villous surfaces. The globlet cells
contain apical clear vacuoles. The bottom panel shows a cross
section of a mouse intestine exposed both the CP (10 .mu.g/g) and
TGF.alpha. (50 ng/g). The intestinal structure is very similar to
the normal intestinal structure. Specifically, the villus is long
and slender. Both absorptive cells and globlet cells are visible at
the surface of the villi. There is an abundant amount of globlet
cells on the surface.
[0072] In FIG. 6, there are three panels shown at 160.times.
magnification again corresponding to a CP-treated mouse in the top
panel, a normal mouse in the middle panel and a CP treated and
TGF.alpha. treated mouse in the bottom panel at the same doses as
indicated for FIG. 5. In the top panel are injured villi with tips
degenerating and necrotic (arrows). Red blood cells are observed in
the damaged villi (arrows). The crypts (C) are in irregular shape
and in various heights. The middle panel shows that the tips of the
villi (arrows) are smooth and the nuclei of the enterocytes are
observed throughout the villus. The crypts (C) are similar in
height and regular in shape. The bottom panel has villi (arrows)
appearing normal as in the middle panel. The crypts (C) also appear
to be normal.
[0073] FIG. 7 shows three panels but the top and middle panels are
CP (10 .mu.g/g) treated without TGF.alpha. and the bottom panel is
CP (10 .mu.g/g) and 50 ng/g of TGF.alpha.. The panels are shown at
higher magnification. In the top panel, the severely injured crypt
surface from CP treatment shows cellular destruction due to
necrosis. Red cells appear at the damaged surface to indicate
intestinal bleeding. In addition, the middle panel of a CP-treated
mouse shows a loss of brush border and very little of a glycocalyx
or fuzzy coat. The interspersed globlet cells appear fewer in
number (than normal) and are seen as necrotic. In the bottom panel,
the effect of TGF.alpha. treatment shows protection of the villa
surface (arrows). Specifically, the epithelial cells are normal
appearing with extended brush borders. The nuclei are very densely
stained and elongated.
[0074] The histological data is summarized in FIG. 8 that measured
average crypt height of the three groups of mice. TGF.alpha.
treatment (50 ng/g) was able to more-than-restore crypt height loss
from CP treatment.
[0075] An alcian blue staining method permits differentiation of
two major cell types that are an absorptive cell and a globlet
cell. The globlet cell mucus is stained a greenish blue color while
the absorptive cells remain less stained. In FIG. 9, the three
panels at 160.times. magnification are shown to correspond to
normal intestine in the top panel, CP only treated (10 .mu.g/g) in
the middle panel and both CP (10 .mu.g/g) and TGF.alpha. (50 ng/g)
in the bottom panel. In the normal intestine (top panel), each
villus extends from the luminal surface to the basal muscularis
mucosal surface. Globlet cells are scattered and predominate in the
base of the villus (arrows) whereas columnar absorptive cells line
the luminal surface. In the middle panel, the alcian blue staining
method shows that the villi contain a fewer number of globlet cells
(than normal) (arrows). The injured absorptive and globlet cells
are degenerating at the tip of the villi (arrows). Abundant
secretory mucus material is stained in the luminal surface
(arrows). In the bottom panel, there is an increased number of
globlet cells scattered throughout the villi (arrows). The
intestinal villi are in normal form with elongation. The majority
of enterocytes are not alcian blue stained positive. The luminal
plasma membranes of the villi (arrows) are well protected by
TGF.alpha. treatment. The number of globlet cells were counted on
the average unit length of intestine. These data are shown in FIG.
10. TGF.alpha. treatment not only increased the number of globlet
cells but increased the number from CP treatment to a higher level
than normal intestine.
[0076] Accordingly, these data show the effects of TGF.alpha., and
the loop peptides from formula I, formula II and formula III having
therapeutic activity to treat or prevent mucositis associated with
cytotoxic therapy and for inflammatory bowel diseases. Moreover,
the histological effect showing that there was a prevention of mast
cell degranulation (FIG. 11), provides additional data supporting
the gastrointestinal applications for TGF.alpha., and the loop
peptide of formula I, formula II and formula III.
[0077] In addition, TGF.alpha. stimulated the proliferation of
select immune cells (particularly of the T cell lineage) after
administration to mice after immune-suppression of CP
administration. The stimulated immune cells were phenotypically
identified as CD4 positive T cells and double null CD4 negative CD8
negative T cell progenitors with characteristics of NK-1 cells.
Thus, TGF.alpha. regulated immune functions and in particular
defects in NK-1 cells. Therefore, these data predict that
TGF.alpha. and the loop peptide of formula I, formula II and
formula III will be effective in treating autoimmune diseases by
mitigating over-inflammatory reactions. The in vivo activity of
TGF.alpha. (FIG. 11) (and the loop peptide of formula I, formula II
and formula III) to stimulate early T cell progenitors on the NK-1
type results in the release of TH-2 cytokines and this down
regulates autoimmune phenomena. The stimulation of select immune
cells, in particular cells of a T cell lineage, was seen
consistently in the mice who received CP and TGF.alpha. (FIG. 11
for GI tract) in lymphoid tissue, Peyers Patches and the spleen.
Further, recruitment of help via CD4 cells in some cases boosts
immune system function in general.
[0078] In FIG. 11, TGF.alpha. administration prevented mast cell
degranulation and subsequent histamine release. This is a parallel
activity that is in addition to the gastrointestinal
anti-inflammatory activity and prevention of mucositis of
TGF.alpha. (and the loop peptide of formula I, formula II and
formula III) described herein.
Sequence CWU 1
1
4 1 50 PRT Homo sapiens 1 Val Val Ser His Phe Asn Lys Cys Pro Asp
Ser His Thr Gln Tyr Cys 1 5 10 15 Phe His Gly Thr Cys Arg Phe Leu
Val Gln Glu Glu Lys Pro Ala Cys 20 25 30 Val Cys His Ser Gly Tyr
Val Gly Val Arg Cys Glu His Ala Asp Leu 35 40 45 Asp Ala 50 2 18
PRT Homo sapiens 2 Val Cys His Ser Gly Tyr Val Gly Val Arg Cys Glu
His Ala Asp Leu 1 5 10 15 Asp Ala 3 11 PRT Artificial Sequence Loop
peptide 3 Xaa Cys His Ser Xaa Xaa Xaa Xaa Xaa Xaa Cys 1 5 10 4 7
PRT Artificial Sequence Loop peptide 4 Xaa His Xaa Xaa Xaa Xaa Xaa
1 5
* * * * *